investigation of the mechanisms of aromatic amine
TRANSCRIPT
INVESTIGATION OF THE MECHANISMS OF
AROMATIC AMINE-INDUCED IDIOSYNCRATIC
DRUG REACTIONS
by
Winnie Wing Yee Ng
A thesis submitted in conformity with the requirements for the degree of
DOCTOR OF PHILOSOPHY
Graduate Department of Pharmaceutical Sciences
Faculty of Pharmacy
University of Toronto
© Copyright by Winnie Wing Yee Ng, 2013
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Investigation of the Mechanisms of Aromatic Amine-Induced
Idiosyncratic Drug Reactions
Winnie Wing Yee Ng
Doctor of Philosophy
Department of Pharmaceutical Sciences
Faculty of Pharmacy
University of Toronto
2013
ABSTRACT
Drugs with the primary aromatic amine structure are notorious structural alerts in drug
development because of their association with a high incidence of idiosyncratic drug reactions
(IDRs). The toxicity of aromatic amines has been attributed to the formation of reactive
metabolites common to this functional group that can form antigenic substances and redox cycle
to induce oxidative stress. To date, there has not been a systematic study to investigate the
mechanisms of aromatic amine-induced IDRs. Therefore, in the first part of my thesis, the
danger hypothesis was tested through a global screen of hepatic gene expression in rats treated
acutely with the aromatic amine drugs sulfamethoxazole (SMX), dapsone (DDS), and
aminoglutethimide (AMG). Although one gene, Eiih, was up-regulated by all drugs tested and
could be a general biomarker to drugs that induce IDRs, few changes were found that are likely
to represent danger signals. To confirm this, a more comprehensive study was performed on the
liver with AMG after longer treatment intervals. Despite substantial changes in apoptotic and
mitochondrial pathways, there were few immune changes and no histological evidence of
damage. These findings suggest that the liver is not a direct target of aromatic amine drugs or, if
it is, the default response may be immune tolerance.
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The presence of myeloperoxidase in neutrophils and antigen presenting cells provides a
more plausible mechanism of aromatic amine toxicity through direct activation of the immune
system, and this was tested in the second part of my thesis research. A significant increase in
activated neutrophils was observed in AMG-treated rats, which was attributed to an increase in
granulocyte progenitors in the bone marrow. This effect is opposite to the agranulocytosis that is
observed with AMG clinically, but it may have important implications to the early effects
preceding the pathology. In the spleen and lymph nodes, T-cell proliferation was found with
simultaneous indications of immune modulation through differential intracellular cytokine
expression. Moreover, down-regulation of immune activation may be why the AMG-treated rats
do not develop hypersensitivity. Further investigations into tolerogenic mechanisms may have
broader implications into why IDRs only develop in a subset of patients.
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ACKNOWLEDGEMENTS
In addition to satisfying my scientific curiosity, my Ph.D. degree has also been a mental
test of creativity, endurance, and perseverance. Throughout this, I have been fortunate enough to
have amazing support to share both the highs and the lows. To all of you, I am forever grateful
for your patience, kindness, guidance, and unfaltering faith in me.
First and foremost I would like to thank my supervisor, Dr. Jack Uetrecht, for allowing
me the opportunity to experience the breadth of academic research in its entirety. It amazes me
how well-versed he is in so many fields, and his constant barrage of changing ideas has taught
me to think outside of the box both in science and as applied to life. I especially appreciate his
trust in my judgment, but what I admire most is his integrity and commitment to his research
and students; and I hope to carry these influences throughout my career and life. I would also
like to thank my advisory committee members Dr. Denis Grant, Peter O’Brien, and Dana
Philpott for their continued guidance and eagerness to help whenever needed.
My family is very important to me and I could not do without their support. I want to
thank my parents who taught me to always do more and expect more from life. Their constant
encouragement has become something I depend on. As stereotypical Asian parents, they had
always wanted a doctor in the family… I think they have something much better. I would also
like to acknowledge my sister and brother, Judy and Chris, who have always been so
understanding and supportive of my work.
I would like to thank my all of my lab mates both past and present who have experienced
and overcome the same hurdles in our lab. I especially want to thank Ervin Zhu, Xin Chen, Feng
Liu, and Ping Cai for their patience in teaching me, their words of wisdom, and their genuine
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friendship. Many scientists, exchange students, and interns have also passed through the lab
during my 5 years, in which I am grateful and privileged to have met and have left me with
significant influences. Lastly, I would like to thank Imir Metushi for his gracious support and
continuous belief in me, and for including me in his crazy ambitions.
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TABLE OF CONTENTS
ABSTRACT ...................................................................................................................................ii
ACKNOWLEDGEMENTS ......................................................................................................... iv
LIST OF THESIS PUBLICATIONS .........................................................................................xii
LIST OF ABBREVIATIONS ................................................................................................... xiii
LIST OF TABLES .....................................................................................................................xvii
LIST OF FIGURES ................................................................................................................. xviii
LIST OF APPENDICES ............................................................................................................ xxi
CHAPTER 1 INTRODUCTION .................................................................................................. 1
1.1 ADVERSE DRUG REACTIONS ..................................................................................... 2
1.2 IDIOSYNCRATIC DRUG REACTIONS ....................................................................... 5
1.2.1 Clinical Characteristics of IDRs ........................................................................... 6
1.2.2 Immune Involvement in IDRs .............................................................................. 7
1.2.3 Mechanisms of IDRs .............................................................................................. 9
1.2.4 Hapten Hypothesis ............................................................................................... 11
1.2.5 Danger Hypothesis ............................................................................................... 13
1.2.6 Pharmacological Interaction with Immune Receptors (P-I) Hypothesis........ 16
1.2.7 Other Hypotheses................................................................................................. 17
1.3 ORGAN-SPECIFIC IDRs............................................................................................... 19
1.3.1 Hepatotoxicity ...................................................................................................... 19
1.3.2 Haematotoxicity ................................................................................................... 21
1.3.3 Cutaneous Toxicity .............................................................................................. 22
1.3.4 Autoimmunity ...................................................................................................... 23
1.4 IMMUNE SYSTEM ........................................................................................................ 24
1.4.1 Tests for Characterizing Immune Changes ...................................................... 26
1.5 AROMATIC AMINE DRUGS ....................................................................................... 28
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1.5.1 Sulfamethoxazole (SMX) .................................................................................... 33
1.5.2 Dapsone (DDS) ..................................................................................................... 33
1.5.3 Aminoglutethimide (AMG) ................................................................................. 34
1.6 ANIMAL MODELS ........................................................................................................ 35
1.6.1 Nevirapine-Induced Skin Rash ........................................................................... 36
1.6.2 D-Penicillamine-Induced Autoimmunity .......................................................... 37
1.6.3 SMX-Induced Immunogenicity .......................................................................... 39
1.7 HAEMATOLOGICAL CONCEPTS RELEVANT TO IDRs ..................................... 41
1.7.1 White Blood Cells (WBCs) .................................................................................. 41
1.7.2 Neutrophils ........................................................................................................... 42
1.7.3 Neutrophil Activation .......................................................................................... 45
1.7.4 Neutrophil Regulation of Adaptive Immunity .................................................. 47
1.7.5 Agranulocytosis .................................................................................................... 48
1.7.6 Clozapine-Induced Agranulocytosis .................................................................. 49
1.7.7 AMG-Induced Agranulocytosis .......................................................................... 49
1.8 OVERVIEW OF THESIS RESEARCH ....................................................................... 51
CHAPTER 2 CHANGES IN GENE EXPRESSION INDUCED BY AROMATIC
AMINE DRUGS: TESTING THE DANGER HYPOTHESIS ........................................... 53
2.1 ABSTRACT ..................................................................................................................... 54
2.2 ABBREVIATIONS .......................................................................................................... 55
2.3 INTRODUCTION ........................................................................................................... 56
2.4 MATERIALS AND METHODS .................................................................................... 61
2.4.1 Reagents ................................................................................................................ 61
2.4.2 Animals ................................................................................................................. 61
2.4.3 Treatments............................................................................................................ 61
2.4.4 RNA Extraction ................................................................................................... 62
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2.4.5 Microarray analysis ............................................................................................. 63
2.4.6 Real-time (RT)-PCR of early insulin-induced hepatic gene (Eiih) ................. 63
2.4.7 Activity assays ...................................................................................................... 64
2.4.8 Statistical Analysis ............................................................................................... 64
2.5 RESULTS ......................................................................................................................... 65
2.5.1 Microarray quality .............................................................................................. 65
2.5.2 SMX-induced hepatic gene expression .............................................................. 65
2.5.3 DDS-induced hepatic gene expression ............................................................... 65
2.5.4 AMG-induced hepatic gene expression ............................................................. 66
2.5.5 Aromatic amine gene expression profile............................................................ 66
2.6 DISCUSSION ................................................................................................................... 74
CHAPTER 3 HEPATIC EFFECTS OF AMINOGLUTETHIMIDE: A MODEL
AROMATIC AMINE ............................................................................................................. 80
3.1 ABSTRACT ..................................................................................................................... 81
3.2 TABLE OF CONTENTS GRAPHIC ............................................................................ 81
3.3 ABBREVIATIONS .......................................................................................................... 82
3.4 INTRODUCTION ........................................................................................................... 83
3.5 MATERIALS AND METHODS .................................................................................... 86
3.5.1 Reagents ................................................................................................................ 86
3.5.2 Animals ................................................................................................................. 86
3.5.3 Treatment ............................................................................................................. 86
3.5.4 Histology ............................................................................................................... 87
3.5.5 Liver Enzymes ...................................................................................................... 87
3.5.6 RNA Extraction ................................................................................................... 89
3.5.7 PCR Arrays .......................................................................................................... 89
3.5.8 Statistical Analysis ............................................................................................... 89
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3.6 RESULTS ......................................................................................................................... 91
3.6.1 Effects of AMG on the Liver............................................................................... 91
3.6.2 AMG-Induced Hepatic Gene Changes .............................................................. 95
3.7 DISCUSSION ................................................................................................................. 102
CHAPTER 4 EFFECT OF AMINOGLUTETHIMIDE ON NEUTROPHILS IN
RATS: IMPLICATIONS FOR IDIOSYNCRATIC DRUG-INDUCED BLOOD
DYSCRASIAS ....................................................................................................................... 107
4.1 ABSTRACT ................................................................................................................... 108
4.2 TABLE OF CONTENTS GRAPHIC .......................................................................... 109
4.3 ABBREVIATIONS ........................................................................................................ 110
4.4 INTRODUCTION ......................................................................................................... 111
4.5 MATERIALS AND METHODS .................................................................................. 114
4.5.1 Chemicals and Reagents ................................................................................... 114
4.5.2 Antibodies ........................................................................................................... 114
4.5.3 Animals ............................................................................................................... 114
4.5.4 Treatments.......................................................................................................... 115
4.5.5 Measurement of AMG Blood Levels ................................................................ 115
4.5.6 Leukocyte Counts .............................................................................................. 115
4.5.7 Measurement of Cytokines ............................................................................... 116
4.5.8 Leukocyte Isolation from Peripheral Blood .................................................... 116
4.5.9 Measurement of New Neutrophil Release Using 5-bromo-2′-deoxyuridine
(BrdU) ................................................................................................................. 117
4.5.10 Bone Marrow Histology .................................................................................... 117
4.5.11 Characterization of Bone Marrow Cells ......................................................... 117
4.5.12 Investigation of Bone Marrow Progenitor Cells ............................................. 118
4.5.13 Determination of Neutrophil Apoptosis........................................................... 118
4.5.14 Determination of Neutrophil Activation .......................................................... 119
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4.5.15 Flow Cytometry Analysis .................................................................................. 119
4.5.16 Data Analysis ...................................................................................................... 119
4.6 RESULTS ....................................................................................................................... 120
4.6.1 Effect of AMG on Peripheral Blood Leukocyte Counts ................................ 120
4.6.2 Effect of AMG on Bone Marrow ...................................................................... 125
4.6.3 Effect of AMG on Neutrophils.......................................................................... 131
4.7 DISCUSSION ................................................................................................................. 135
CHAPTER 5 INVESTIGATION OF THE IMMUNE CHANGES INDUCED BY
AMINOGLUETHIMIDE ..................................................................................................... 140
5.1 ABSTRACT ................................................................................................................... 141
5.2 ABBREVIATIONS ........................................................................................................ 142
5.3 INTRODUCTION ......................................................................................................... 143
5.4 MATERIALS AND METHODS .................................................................................. 147
5.4.1 Chemicals and Reagents ................................................................................... 147
5.4.2 Antibodies ........................................................................................................... 147
5.4.3 Animals ............................................................................................................... 148
5.4.4 Treatments.......................................................................................................... 148
5.4.5 Measurement of Serum Cytokines ................................................................... 148
5.4.6 Detection of Cell Proliferation in Lymphoid Organs ..................................... 149
5.4.7 Phenotyping of Immune Cells........................................................................... 149
5.4.8 Characterization of an Immune Response through the Lymphocyte
Transformation Test (LTT). ............................................................................. 150
5.4.9 Flow Cytometry Analysis .................................................................................. 151
5.4.10 Data Analysis ...................................................................................................... 151
5.5 RESULTS ....................................................................................................................... 152
5.5.1 Systemic Immune Response .............................................................................. 152
5.5.2 Effect of AMG on Lymphoid Organs .............................................................. 155
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5.5.3 AMG-Induced Immune Cell Changes ............................................................. 159
5.6 DISCUSSION ................................................................................................................. 162
CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTION ............................................. 167
6.1 DISCUSSION AND CONCLUSIONS ......................................................................... 168
6.2 FUTURE DIRECTIONS .............................................................................................. 176
REFERENCES .......................................................................................................................... 178
APPENDIX 1 .............................................................................................................................. 211
APPENDIX 2 .............................................................................................................................. 215
APPENDIX 3 .............................................................................................................................. 221
APPENDIX 4 .............................................................................................................................. 223
APPENDICES REFERENCES ................................................................................................ 224
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LIST OF THESIS PUBLICATIONS
1. Ng, W., Lobach, A. R. M., Zhu, X., Chen, X., Liu, F., Metushi, I. G., Sharma, A., Li, J., Cai,
P., Ip, J., Novalen, M., Popovic, M., Zhang, X., Tanino, T., Nakagawa, T., Li, Y., and
Uetrecht, J. Animal models of idiosyncratic drug reactions. Adv. Pharmacol., 2012. 63: p.
81-135.
2. Ng, W., and Uetrecht, J. Changes in gene expression induced by aromatic amine drugs:
Testing the Danger Hypothesis. J. Immunotox., 2013. 10: p. 178-191.
3. Ng, W., Metushi, I.G., and Uetrecht, J. Hepatic effects of aminoglutethimide: a model
aromatic amine. (Submitted to J. Immunotox., 2013).
4. Ng, W., and Uetrecht, J. Effect of aminoglutethimide on neutrophils in rats: implications for
idiosyncratic drug-induced blood dyscrasias. Chem. Res. Toxicol., 2013. 26: p. 1272-1281.
Informa Healthcare and the American Chemical Society granted permission to include my
article in this thesis.
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LIST OF ABBREVIATIONS
ADRs, adverse drug reactions
ALT, alanine aminotransferases
AMG, aminoglutethimide
APCs, antigen presenting cells
APC, allophycocyanin
β2M, β2-microglobulin
BrdU, 5-bromo-2’-deoxyuridine
CFUs, colony forming units
Cxcl1, chemokine (C-X-C) 1
P450, cytochrome P450
DAMPs, damage-associated molecular patterns
DAPI, 4', 6-diamidino-2-phenylindole dihydrochloride
DDS, dapsone
DMSO, dimethyl sulfoxide
DRESS, drug reaction with eosinophilia and systemic symptoms
Dusp1, dual specificity phosphatase 1
Eiih, early insulin-induced hepatic gene
FACS, florescence-activated cell sorting
FBS, fetal bovine serum
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FDR, false discovery rate
FITC, fluorescein isothiocyanate
G-CSF, granulocyte-colony stimulating factor
GLDH, glutamate dehydrogenase
GSH, glutathione
GSH-S-Tr, glutathione-S-Transferase
H&E, hematoxylin and eosin
HIV, human immunodeficiency virus
HLA, human leukocyte antigen
IDILI, idiosyncratic drug-induced liver injury
IDRs, idiosyncratic drug reactions
IL, interleukin
IMDM, Isocove’s Modified Dulbecco’s Medium
LPS, lipopolysaccharide
LTT, lymphocyte transformation test
MHC, major histocompatibility complex
MPO, myeloperoxidase
NAT, N-acetylation
NK, natural killer
NOD-like receptors, nucleotide-binding oligomerization domain receptors
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PABA, para-aminobenzoic acid
PAMPs, pathogen-associated molecular patterns
PBMCs, peripheral blood mononuclear cells
PBS, phosphate buffered saline
PE, phycoerythrin
PerCP, peridinin chlorophyll
P-I hypothesis, pharmacological interaction with immune receptors hypothesis
PMA, phorbol myristate acetate
PRRs, pattern recognition receptors
RAGE, receptors for advanced glycosylation end products
RBCs, red blood cells
SDH, sorbitol dehydrogenase
SEM, standard error of the mean
Sgk, serum/glucocorticoid-regulated kinase 1
SMX, sulfamethoxazole
SMX-OH, hydroxyl metabolite of sulfamethoxazole
SMX-NO, nitroso metabolite of sulfamethoxazole
SOD2, superoxide dismutase 2
TLRs, Toll-like receptors
TNF-α, tumor necrosis factor alpha
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Txnrd, thioredoxin reductase
WBCs, white blood cells
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LIST OF TABLES
Table 1.1. Drugs associated with ADRs under restricted use and/or patient monitoring programs
in the US from 1990-2002 . ........................................................................................................... 3
Table 1.2. Drugs associated with IDRs . ....................................................................................... 7
Table 1.3. Potential danger signals .............................................................................................. 16
Table 1.4. Comparison of the characteristics between the innate and adaptive immunity. ......... 24
Table 1.5. Aromatic amine drugs and associated IDRs. .............................................................. 30
Table 1.6. Pharmacokinetic parameters of the aromatic amine drugs SMX, DDS, and AMG . . 32
Table 1.7. WBC composition in normal rats and humans . ......................................................... 42
Table 1.8. Components of neutrophil granules ........................................................................... 46
Table 2.1. Similar genes differentially regulated at least 1.4-fold change in the same direction in
all aromatic amine drugs tested 12 hr after treatment.................................................................. 67
Table 2.2. Keap1-Nrf2-ARE-regulated genes that were changed ≥ 1.4 fold by at least one of the
aromatic amine drugs (SMX, DDS, or AMG) tested 12 hr after treatment................................. 71
Table 3.1. Hepatic changes in gene expression induced by AMG in the apoptosis pathway.. ... 95
Table 3.2. Mitochondrial gene expression changes in the liver of AMG-treated rats. ................ 99
Table 3.3. AMG-induced hepatic gene changes in chemokines and receptors ......................... 100
Table 3.4. Hepatic gene changes induced by AMG in various immune pathways ................... 101
Table A1.1. SMX-induced hepatic gene changes at 12 hr……………………………………. 211
Table A1.2 DDS-induced hepatic gene changes at 12 hr…………………………………….. 212
Table A1.3. AMG-induced hepatic gene changes at 12 hr ……………………………………213
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LIST OF FIGURES
Figure 1.1. Mechanistic scheme for the hapten and danger hypotheses. ..................................... 11
Figure 2.2. Pharmacological interaction of drugs with immune receptors (PI) hypothesis. ....... 17
Figure 1.3. Scheme for the general metabolism of primary aromatic amine drugs..................... 30
Figure 1.4. Chemical structures of the aromatic amine drugs SMX, DDS, and AMG. .............. 32
Figure 1.5. Overview of haematopoiesis in the bone marrow focusing on granulocyte
progenitors. .................................................................................................................................. 44
Figure 1.6. Mechanistic scheme for the production of oxidants by neutrophils......................... 45
Figure 2.1. The Danger Hypothesis as applied to IDRs... ........................................................... 57
Figure 2.2. Metabolic scheme for the formation of reactive metabolites of aromatic amine drugs
..................................................................................................................................................... 58
Figure 2.3. Structures of the aromatic amine drugs used in this study and some associated IDRs
..................................................................................................................................................... 60
Figure 2.4. Eiih expression induced by aromatic amine drugs 12 hr after treatment.. ................ 68
Figure 2.5. Time-course of hepatic Eiih expression induced by AMG. ...................................... 69
Figure 2.6. Hepatic gene expression of Eiih induced by several other drugs associated with
IDRs. .......................................................................................................................................... 70
Figure 2.7. Hepatic Txnrd activity after treatment with aromatic amine drugs. ......................... 72
Figure 2.8. Time-course of hepatic GSH-S-Tr activity in AMG-treated rats .............................. 73
Figure 3.1. Histological changes in the liver of AMG-treated rats. ............................................ 92
Figure 3.2. Comparison of hepatocyte proliferation in control and AMG-treated animals……..93
Figure 3.3. AMG-induced changes in serum levels of liver enzymes……………..……………94
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Figure 3.4. Pathway analysis of significantly changed hepatic genes in the apoptotic pathway in
AMG-treated rats……………………………………………………………………………..…97
Figure 4.1. Structure of AMG and the formation of its reactive metabolites. ........................... 113
Figure 4.2. Blood levels of AMG in rats. .................................................................................. 121
Figure 4.3. Acute AMG-induced changes on peripheral blood neutrophils in rats given an oral
dose of 80 mg/kg/day.......................................................................................................................
Figure 4.4. Acute AMG-induced changes on peripheral blood neutrophils in rats given an oral
dose of 125 mg/kg/day............................................................................................................... 122
Figure 4.5. Changes in peripheral leukocyte cell counts induced by treatment of rats with AMG
at a dose of 125 mg/kg/day for 14 days. .................................................................................... 123
Figure 4.6. AMG-induced changes in neutrophil-associated cytokines in the blood of rats. .... 124
Figure 4.7. Effect of AMG on new neutrophil release from the bone marrow. ........................ 126
Figure 4.8. Bone marrow changes induced by AMG. ............................................................... 127
Figure 4.9. AMG-induced changes in bone marrow cells. ........................................................ 128
Figure 4.10. Effect of AMG on bone marrow granulocyte and macrophage CFUs .................. 130
Figure 4.11. AMG-induced changes in neutrophil apoptosis .................................................... 132
Figure 4.12. Changes in neutrophil CD62L expression induced by AMG. .............................. 133
Figure 5.1. Early changes in serum levels of Grow/KC, MCP-1, and IP-10 induced by AMG
treatment.. .................................................................................................................................. 153
Figure 5.2. Later changes in serum cytokines induced by AMG treatment. ............................. 154
Figure 5.3. Cell proliferation detected by Ki-67 staining induced in the white pulp of the spleen
after 14 days of AMG treatment. ............................................................................................... 156
Figure 5.4. Analysis of proliferating (Ki-67+) splenocytes after 14 days of AMG treatment. .. 157
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Figure 5.5. LTT using lymphocytes from the lymph nodes of AMG-treated rats. .................... 158
Figure 5.6. Early changes in the fraction of CD4+ T cells from the spleen and lymph nodes that
express IL-17 or IL-10 induced by AMG treatment.. ................................................................ 160
Figure 5.7. AMG-induced changes in macrophage subsets in lymph nodes after 14 days of
treatment .................................................................................................................................... 161
Figure A2.1. Metabolism of AMG by rat and mouse neutrophils.…………………………….218
Figure A2.2. Mass fragmentation of (A) AMG and (B) product of m/z 249.0.……………….219
Figure A2.3. Potential structures for m/z 249 hydroxylation of AMG…………………..……220
Figure A3.1. Peripheral blood changes induced by SMX and DDS. ………………………....221
Figure A4.1. Changes in peripheral blood WBCs induced by AMG in mice.………………...223
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LIST OF APPENDICES
Appendix 1: Supplementary material from Chapter 2: Changes in Gene Expression Induced by
Aromatic Amine Drugs… …………………………………………………………………...211
Appendix 2: In Vitro Metabolism of AMG by Neutrophils to a Hydroxylated Metabolite…215
Appendix 3: Effect of SMX and DDS on Peripheral Blood Neutrophils……………………221
Appendix 4: Haematological Effects of AMG in Mice……………………………………...223
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CHAPTER 1
INTRODUCTION
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1.1 ADVERSE DRUG REACTIONS
Adverse drug reactions (ADRs) are defined as an appreciably harmful or unpleasant
reaction, resulting from an intervention related to the use of a medicinal product, which predicts
hazard from future administration and warrants prevention or specific treatment, or alteration of
the dosage regimen, or withdrawl of the product [1]. ADRs are a significant issue of concern
because approximately 6% of hospital admissions are related to ADRs [2], and most ADRs go
unreported. The most commonly implicated drugs are non-steroidal anti-inflammatory drugs,
warfarin, diuretics, and ACE inhibitors; however; over half of the drugs on the market are
associated with serious ADRs, which may not be detected prior to market release [3]. ADRs
were ranked the fourth leading cause of death; trailing behind only heart disease, cancer, and
stroke; with an estimated 106 000 deaths from ADRs in 1994 in the United States alone [4].
Therefore, it is essential to better understand these reactions so that it is easier to prevent or treat
them.
In addition to the risk to patients, ADRs can lead to the attrition of prime drug candidates
resulting in significant financial losses and they play an important role in the risk of drug
development. In 2000, the price of developing a new drug was estimated to be over $400 million
US [5], although there is much debate and higher numbers have been projected. Thus
determining the safety of a drug as early as possible during the development pipeline is crucial to
decreasing the financial risks to a pharmaceutical company, and the development of preclinical
tests to detect ADRs prior to market release would have profound implications for drug
development. Approximately 3% of newly approved drugs were withdrawn from the market
from 1975-2000 due to safety issues [6], although some have been given restricted use rather
than withdrawn (Table 1.1).
3
Table 1.1. Drugs associated with ADRs under restricted use and/or patient monitoring
programs in the US from 1990-2002 [7].
Pharmacovigilance programs have been helpful in monitoring ADRs in the general population;
however, there are still significant limitations on the current ability to predict and detect ADRs
and to determine the offending drug culprit. This may, in part, be due to the varying clinical
characteristics of ADRs and the fact that many ADRs are not related to the pharmacology of the
drug and can be dependent on other factors such as underlying host pathologies or drug-drug
interactions. Thus, ADRs have been commonly classified based on their characteristics as
follows [adapted from 8]:
Type A (Augmented): Type A reactions represent 80% of ADRs and are usually dose-
related and due to an augmentation of the pharmacological effect
of the drug. These reactions are typically predictable and recovery
usually occurs upon drug withdrawl. Examples include
4
hemorrhaging with warfarin toxicity; hypotension with anti-
hypertensives.
Type B (Bizarre): Type B reactions are also known as idiosyncratic drug reactions.
These reactions are uncommon and unpredictable. They do not
have a simple dose-response nor are they related to the
pharmacology of the drug. Examples are sulfonamide
hypersensitivity; halothane-induced hepatitis.
Type C (Chemical): Type C reactions are usually due to, and can be predicted by, the
chemical structure of the drug. Examples include penicillin
hypersensitivity and acetaminophen hepatotoxicity.
Type D (Delayed): Type D reactions are delayed upon initiating drug therapy and can
occur as late as years after initiating the drug. Examples are
teratogenesis with thalidomide; tumorigenesis with carcinogens.
Type E (End-of-Treatment): Type E reactions are usually due to (sudden) withdrawl of
treatment. Examples include adrenal insufficiency with cortisol
withdrawl; seizures upon anticonvulsant withdrawl.
No drug is free from the risk of ADRs. Even if the drug is considered very safe, there is
usually some level of risk of developing ADRs. Since the majority of ADRs are Type A and can
be predicted, careful monitoring by both patient and medical practitioners can usually mitigate or
manage ADRs such that effective drugs with a high risk of ADRs can still have a positive
5
risk/benefit ratio. However, the more significant concerns are the ADRs that are unpredictable;
with these reactions often there is little prior warning before their development. Thus, Type B or
idiosyncratic drug reactions are the main focus of my thesis research.
1.2 IDIOSYNCRATIC DRUG REACTIONS
Idiosyncratic drug reactions (IDRs) are often referred to as hypersensitivity reactions;
however, the definition of IDRs that we use is an adverse reaction that occur uncommonly in the
population at doses that are therapeutically achievable, and do not involve the therapeutic
mechanisms of the drug [9]. The incidence of IDRs is quite low, only representing 6-10% of all
ADRs; yet, they are important to study because many drugs are able to induce IDRs, and a large
number of people take these drugs. These reactions can also be quite severe and even fatal with
the annual economic burden for hospital admissions due to IDRs in the United States estimated
to be between $275-600 million [10], and this does not include outpatient costs. Contrary to the
common perception, these reactions are dose dependent because IDRs are more likely to develop
with drugs that are given at high doses. In general, drugs given below 10 mg per day rarely lead
to the development of IDRs [11], and patients that are slowly titrated to a full dose can often be
desensitized to these reactions [12]. Moreover, there is always a dose at which no IDR will
occur, and the dose-response curve for IDRs is typically different from that of the curve for the
therapeutic effect [13], suggesting that the mechanism of IDRs differs from the therapeutic
effects of the drug.
Unlike Type A reactions, the major concern for IDRs is the inability to effectively predict
or detect which drugs will induce IDRs. Preclinical safety studies have not been effective at
6
detecting IDRs [14], and these reactions typically only surface in the general population after
market release. Thus the low incidence of IDRs hinders our ability to systematically study these
reactions and poses significant issues for the development of new drug candidates and the ability
to effectively treat patients. Despite the potential to develop serious reactions, many drugs
associated with IDRs are still very pharmacologically effective for the majority of the
population. Thus if factors could be identified that predispose certain individuals to IDRs, this
would have profound implications for the prediction and prevention of these reactions in both
new drug candidates and patient populations.
1.2.1 Clinical Characteristics of IDRs
The clinical manifestations of IDRs vary quite substantially, even between different
patients that take the same drug. In fact, one drug can induce a range of different IDRs, even
simultaneously, while one type of reaction can be induced by many different drugs (Table 1.2).
The varying clinical characteristics suggest that there are differences in the mechanisms that lead
to the initiation of IDRs. The most common organ systems affected by IDRs include the liver,
bone marrow, and skin. In general, there is a delay between initiating the drug therapy and the
onset of IDRs. Typically skin rashes occur the earliest, about 1-2 weeks after starting the drug
and can range from mild to severe cutaneous reactions such as Stevens-Johnson syndrome and
toxic epidermal necrolysis. Liver toxicity and blood dyscrasias usually take more time to develop
and typically occur approximately after 1-2 months after initiating drug treatment, and
autoimmune reactions can occur more than 1 year after starting drug treatment. Although the
delay in onset is common with most IDRs, there are rare exceptions in which the onset is quite
short such as in cases of telithromycin-induced hepatitis that can develop within a few days of
treatment [15]. Interestingly, when patients are re-challenged with drug, IDRs usually, but not
7
always, occur with a quicker onset. Drug reaction with eosinophilia and systemic symptoms
(DRESS) is one type of IDR. Underlying host factors such as sex, age, weight, and disease state
are also important risks factors that predispose individuals to IDRs, but are unlikely to
substantially increase the risk to IDRs [9].
Table 1.2. Drugs associated with IDRs [16].
1.2.2 Immune Involvement in IDRs
The majority of the clinical characteristics displayed by patients with IDRs suggest the
involvement of the immune system. Antigen recognition by T-cells in a primary response, and
subsequent clonal expansion requires time; this could account for the delayed onset of IDRs.
Moreover, a memory T-cell response could explain why the onset of reaction upon re-challenge
is quicker. Other common features of IDRs include fever, rash, and eosinophilia, and these
symptoms are typically taken as evidence of activation of an immune response. Perhaps the most
profound evidence of an immune response is the presence of anti-drug antibodies in the serum of
8
IDR patients. Antibody production requires immune activation through T- and B-cell dependent
processes to produce plasma cells to secrete drug-specific immunoglobulins. Drugs can also
induce the formation of auto-antibodies, as in the case of tienilic acid-induced hepatitis where
antibodies were found against the cytochrome P450 (P450), CYP2C9 [17]. However, despite
activation of the immune system, the presence of antibodies may or may not necessarily be
pathogenic, as was found with halothane hepatitis [18]. Furthermore, it is likely that these
biochemical changes may occur in the majority of patients, and are precursors to IDRs in which
there are some other additional factors that make only certain individuals more susceptible.
Genetic associations with IDRs have also been investigated and the most convincing of
these findings suggest the involvement of immune mechanisms. An increased odds of
developing diclofenac hepatotoxicity was observed in individuals with variant alleles for the
cytokines interleukin (IL)- 10 and IL-4 [19]. Genetic differences in human leukocyte antigen
(HLA) have also been associated with the risk that an individual will develop an IDR to certain
drugs. All patients with immunologically confirmed cases of abacavir hypersensitivity reactions
were positive for the HLA-B*5701 allele [20]. Moreover, the HLA-B*5801 allele was found in
all patients who developed severe cutaneous adverse reactions to allopurinol; however, this
seemed to be restricted to the Han Chinese, and HLA-B*5801 was observed in 15% of
allopurinol tolerant patients as well [21]. This suggests that although HLA associations can be
one determinant of whether an individual is at risk, there are likely other factors that are required
to initiate an IDR. Interestingly, polymorphisms in the T-cell repertoire have also been
associated with IDRs because the VB-11-ISGSY clonotype was found in carbamazepine-specific
CD8+ T-cells from 84% of patients with carbamazepine-induced Stevens-Johnson syndrome and
toxic epidermal necrolysis but not found in tolerant patients [22], and this, taken together with
9
HLA associations, may be strong determinants to whether individuals are predisposed to IDRs.
Thus, there is extensive clinical evidence to support the involvement of the immune system, and
most IDRs are proposed to be due to the initiation of an adaptive immune response [23].
1.2.3 Mechanisms of IDRs
In addition to immune-mediated mechanisms, metabolic idiosyncrasy has also been
proposed as a mechanism for the initiation of IDRs. Polymorphisms in drug metabolizing
enzymes could influence the disposition of a drug, specifically how much and to what chemical
species the body is exposed. A slow acetylator phenotype is a risk factor for the development of
severe reactions to sulfonamides [24], suggesting a shift from drug clearance to oxidation to
form a reactive metabolite. Similarly polymorphisms in P450 have also been implicated because
individuals with the genotype CYP2E1 c1/c1 were at a higher risk of isoniazid-induced
hepatotoxicity [25]. Although differences in drug metabolizing enzymes could contribute to the
development of IDRs, the increase in risk in individuals with a metabolic polymorphism is too
small to be responsible for the idiosyncratic nature of IDRs [26]. This is in contrast to the
increased risks observed with some HLA associations such as the odds ratio of 960 associated
with the HLA-B*5701 allele in abacavir hypersensitivity [27]. Thus, there is currently no IDR
that can be fully explained by differences in drug metabolizing enzymes [28].
In contrast, the differences in the clinical manifestations of IDRs can be rationalized by
the diversity of the immune response and variations in the antigens that the T-cell repertoire
recognizes. Immune mechanisms of IDRs also provide an explaination as to why most
individuals do not get IDRs and why some IDRs do not occur rapidly upon re-challenge. Most
patients taking a drug associated with idiosyncratic drug-induced liver injury will often
experience a mild and transient increase in transaminases; however, these levels will typically
10
return to normal and transaminases levels will only continue to increase in a few susceptible
individuals [29]. This suggests that in the majority of the population the default response is
adaptation or tolerance, and IDRs may result only when this tolerance is overcome. This can be
further illustrated with cases of aminoglutethimide-induced agranulocytosis that have resolved
on their own without discontinuing the drug [30]. Furthermore, some IDRs have a long time to
onset upon re-challenge, and this has been taken to be a lack of immune memory response.
However, this does not necessarily mean that an IDR is not immune-mediated. A lag in response
upon re-challenge is commonly observed in idiosyncratic autoimmune diseases, which in its self-
reactive nature may inhibit the anamnestic response by deleting immune memory cells, and this
may be typical of the drugs D-penicillamine and propylthiouracil that are associated with
idiosyncratic autoimmunity [28]. In light of the large body of clinical evidence to support the
involvement of the immune system, several immune mechanisms have been proposed for IDRs
including the hapten, danger, and pharmacological interaction of drugs with immune receptors
hypotheses.
11
Figure 1.1. Mechanistic scheme for the hapten and danger hypotheses.
1.2.4 Hapten Hypothesis
The theory behind the hapten hypothesis was first introduced by Landsteiner and Jacobs
in 1935 in studies of animal sensitization to simple chemicals [31]. The hapten hypothesis
proposes that small molecules less than 1000 Da are not immunogenic on their own but must
form stable conjugates with endogenous macromolecules in order to be recognized by the
immune system as “foreign” and elicit an immunogenic response. This concept has been
commonly illustrated with studies on the penicillin family of drugs. Penicillin is chemically
reactive in the absence of metabolism due to the ring strain in its beta-lactam ring, and it reacts
with lysine groups on proteins to form antigenic substances. Penicillin-protein conjugates were
found to induce skin rashes in guinea pigs and rabbits and also led to the production of drug-
12
specific antibodies [32]. In terms of IDRs, many drugs are not chemically reactive as the parent
molecule, but rather they are prohaptens that require metabolic bioactivation in vivo to gain
reactivity and covalently bind to proteins to become antigenic and induce an immune response.
This concept has been widely adapted in the pharmaceutical industry, and reactive metabolite
screens are generally routine tests in new drug development. Moreover, covalent binding of
drugs and/or reactive metabolites have been used to predict the potential of a drug to develop
IDRs, and some pharmaceutical companies have utilized this as a standard preclinical test for
new drug candidates. Typically the dose is correlated to the amount of covalent binding with an
in vivo threshold limit of 50 pmol drug equivalent/mg total liver protein [33]. However, there are
some exceptions in which drugs that form reactive metabolites are not associated with a
significant risk of IDRs. An example is olanzapine, which forms a reactive nitrenium ion but
does not lead to a high incidence of IDRs [34], presumably because the daily dose is low. Other
drugs such as ximelagatran and allopurinol are associated with IDRs but do not appear to form
reactive metabolites [9]. The magnitude of the binding may also not necessarily correlate to the
severity of the reaction [35]. In the case of metabolism to a very reactive metabolite that is a
suicide inhibitor; covalent binding less likely to cause toxicity because binding is limited to the
enzyme that formed the reactive metabolite. Covalent binding of the reactive metabolite of
tienilic acid was believed to be exclusively to P450 [36], and thus was thought to be relatively
harmless. However, tienilic acid was able to induce more changes in gene expression that were
representative of cell stress than sulfamethoxazole, which is a primary aromatic amine that can
be very metabolically reactive [37]. Moreover, tienilic acid was later found to bind to many
hepatic proteins [38], and the functionality of the protein to which the drug modifies may be an
important determinant of whether an IDR occurs. The localization of the covalently-modified
13
protein and whether it is organ specific may also factor into which system the IDR is targeted
[13]. In addition, the type of immune response induced by a drug may also depend on what
portion of the drug-modified protein the T-cell receptor recognizes. For example, an adaptive
immune response may arise from recognition of the drug portion of the adduct, whereas
autoimmunity may result from recognition of the protein portion [28]. Thus, reactive metabolites
and covalent binding are very important factors to consider in the mechanism of IDRs.
1.2.5 Danger Hypothesis
The Danger Hypothesis was first proposed by Matzinger in 1994 in response to
discrepancies in the field of immunology as to what and how an immune response is initiated
[39]. At that time, the self-nonself theory was generally accepted in which the immune system
responded only to substances that were “foreign”. These antigenic substances would be detected
by antigen presenting cells (APCs) and undergo uptake and processing before being presented to
T-cells; this represents signal 1. In addition to this, costimulatory molecules such as CD80 and
CD86 needed to be expressed on the surface of APCs to interact with receptors on the surface of
the T-cell in order to initiate an immune response, and this is known as signal 2. Janeway had
proposed that the expression of costimulatory molecules was regulated by pattern recognition
receptors (PRRs) on APCs that were activated by pathogen-associated molecular patterns
(PAMPs) in what is known as the infectious-nonself model that dictates immunity to pathogens
[40]. Examples of PRRs include the Toll-like receptors (TLRs), receptors for advanced
glycosylation end products (RAGE), and nucleotide-binding oligomerization domain receptors
(NOD-like receptors). However, it did not fully explain some discrepancies such as why some
autologous transplants are rejected, nor did it explain the effectiveness of non-bacterial adjuvants
such as alum [41]. Moreover, immunity is rarely directed against new proteins produced during
14
natural processes such as puberty, pregnancy, and aging, to which the body had never been
exposed. Thus, Matzinger proposed that the immune system does not react to foreign substances
but rather reacts to substances that are deemed dangerous. Similar to the infectious-nonself
model, PRRs on APCs would recognize so-called danger signals released from cells undergoing
damage or stress (known as damage-associated molecular patterns or DAMPs) and this would
lead to the upregulation of costimulatory molecules on APCs to stimulate an immune response.
In the absence of signal 2, T-helper cells would not be activated and this would lead to tolerance.
The postulates of the danger hypothesis can also be applied to the mechanisms of IDRs
[42]. Oxidative activation of drugs to reactive metabolites may induce cell stress or damage, and
this could lead to the release of danger signals and the upregulation of costimulatory molecules
on APCs to produce signal 2. In addition, covalent drug-proteins adducts could be antigenic and
act as signal 1. Moreover, the danger hypothesis is attractive because if danger signals were to be
identified for drugs associated with IDRs, they may provide a better mechanistic understanding
as to how these reactions are initiated, and could potentially be biomarkers for drugs predisposed
to causing these reactions.
However, danger signals are very vaguely defined with the only set characteristic being
their release under situations of cell stress or damage and their ability to activate APCs through
the upregulation of costimulatory molecules to initiate an immune response [43]. They can be
constitutive and released upon damage to the cellular membrane, or inducible and secreted upon
cell stress. They can also be a result of underlying host factors such as disease or invasive
procedures such as surgery. For example, human immunodeficiency virus (HIV)-infected
individuals have 10 times greater incidence of hypersensitivity reactions to co-trimoxazole than
patients who are not infected [44], and this is presumably due to a greater number of danger
15
signals associated with HIV infections. Various pathological factors have also been found to
increase sulfamethoxazole-protein adducts in human APCs [45], implying that danger signals
play a role in cell activation that may lead to an increase in drug metabolism and covalent
binding. However, in general inflammation inhibits the synthesis of metabolic enzymes and
decreases drug metabolism [46]. Post-translational modifications of proteins can also be danger
signals, such has been reported with the hyperacetylation or phosphorylation of high mobility
group box proteins [47]. Although many potential danger signals have been identified (Table
1.3); only a handful of studies have confirmed their role in the induction of an immune response.
Uric acid (monosodium urate) was found to upregulate CD86 on bone marrow-derived dendritic
cells from rodents and stimulated a CD8 T-cell response in vivo [48]. Interestingly, cell death
signals have also been implicated in immune activation because APCs only expressed
costimulatory molecules in the presence of necrotic splenocytes in a rodent model of
sulfamethoxazole immunogenicity [49].
The danger hypothesis also has the potential to answer questions as to what type of
immune response is initiated. In this respect, the immune response is thought to be initiated at the
site at which the damage or stress occurs; therefore, the injured tissue is responsible for dictating
what type of response is elicited [50]. This has important implications for IDRs, because for
drugs that require metabolic activation, the site of drug metabolism may be the primary site of
immune activation and a major target of IDRs. Although not initially acknowledged, the danger
hypothesis is now widely accepted and provides a mechanistic basis from which to model our
studies on how drugs may initiate immune-mediated IDRs.
16
Table 1.3. Potential danger signals. [Adapted from 42, 43, 51].
1.2.6 Pharmacological Interaction with Immune Receptors (P-I) Hypothesis
The P-I hypothesis was first introduced by Pichler based on observations in which drug-
specific T-cell clones from sulfamethoxazole hypersensitive individuals were activated by the
parent drug (in the absence of drug metabolism) and without uptake or processing by APCs [52].
Thus, the P-I hypothesis proposes that some drugs can interact directly with the major
histocompatibility complex (MHC) of APCs and the T-cell receptor in a labile, non-covalent,
MHC-restricted interaction to activate T-cells [53]. In addition, the underlying assumption of the
P-I hypothesis is that what the T-cell recognizes is what induced the initial primary response.
However, this is not always true because in a model of nevirapine-induced skin rash in which the
rash was induced by the 12-hydroxy-nevirapine metabolite; T-cells proliferated with a greater
response to the parent drug, nevirapine, even though they had never been previously exposed.
Furthermore, we know that it is actually a reactive sulfate metabolite that induces the rash, not
nevirapine [54]. Although the P-I hypothesis may be valid for some drugs such as lamotrigine, it
17
is more likely due to a cross-reactivity of the T-cell receptor on memory cells rather than direct
activation by the parent drug as a super antigen, and T cells specific for both sulfamethoxazole
and its nitroso metabolite have been found in naive individuals [55].
Figure 2.2.. Pharmacological interaction of drugs with immune receptors (PI) hypothesis.
1.2.7 Other Hypotheses
In terms of an immune response, the hapten and P-I hypotheses focus on signal 1;
whereas the danger hypothesis concentrates on the importance of signal 2. Thus, the above
hypotheses are not mutually exclusive, and it is likely that more than one or all of them are
simultaneously involved with the initiation of IDRs. The hapten and danger hypotheses are the
most logical for how a drug may elicit an immune response, and thus they have been the
backbone to my thesis research. In addition, other hypotheses have been proposed such as the
inflammagen hypothesis and mitochondrial toxicity; however, although they may play a role,
they do not involve the adaptive immune system, and the likelihood for these mechanisms to be
significant determinants of an IDR is quite low.
18
The inflammagen hypothesis was proposed by Roth and states that concomitant
inflammatory stress during drug therapy may be required for the development of IDRs [56]. In
studies focusing on idiosyncratic drug-induced liver injury (IDILI), co-treatment of rats with
lipopolysaccharide (LPS) as an inflammagen and with drugs such as ranitidine and trovafloxacin
have induced liver injury that is not present when drugs are administered without the
inflammatory stress [57, 58]. However, LPS is a strong immuno-stimulant, and the clinical
characteristics of this model are not representative of the IDILI that occurs in humans. In the
inflammagen model, liver injury occurs very quickly, within hours, and the injury is not
sustained. In contrast, the response to LPS is rapidly down-regulated repeated exposure and
therefore a sustained response is unlikely [59]. In addition, the inflammagen model is associated
with an infiltration of neutrophils into the liver [56], whereas the more common histology
observed in IDILI is infiltration of lymphocytes and eosinophils [60]. Some of the drugs tested,
such as ranitidine, are also quite safe; therefore, there is a high potential for false positives in the
inflammagen model. Thus, although it is likely that inflammatory stress may be a risk factor for
IDRs, the relative significance is unknown, and the inflammagen model, as it stands, is not a
valid representation of IDRs [61].
Mitochondrial injury has also been implicated in the mechanism of IDRs, specifically for
drug-induced liver toxicity [62]. Mitochondria are functionally very important to energy
production, lipid metabolism, and programmed cell death such that even subtle insults by a drug
may tip the balance towards toxicity. Mitochondrial toxicity has been studied in the context of
IDRs with heterozygous superoxide dismutase 2 deficient (SOD2+/-) mice. Superoxide
dismutase 2 (SOD2) is a mitochondrial associated enzyme involved with modulating cellular
oxidative stress, which can be quite high in the mitochondria because of the electron transport
19
chain. Heterozygous mice have impaired mitochondrial function and increased mitochondrial
oxidative stress [63]. Troglitazone treatment induced liver injury in SOD2+/- mice but not in
wildtype control [64]; however, despite similar clinical characteristics to IDILI in humans, such
as midzonal hepatic necrosis [65], the changes were very mild and were not reproducible in
independent studies [66]. Furthermore, if mitochondrial injury were to play a significant role in
the initiation of IDRs an increase in dose would likely lead to an IDR. Thus, although
mitochondrial toxicity could contribute to the initiation of IDRs, it does not seem to be a
dominant mediator.
1.3 ORGAN-SPECIFIC IDRs
1.3.1 Hepatotoxicity
Liver injury is a common reaction observed with drugs that are associated with IDRs, and
IDILI is a major cause for drug withdrawl [29]. The liver is a common target presumably
because of its high content of drug metabolizing enzymes such as the P450s, which have the
potential to form reactive metabolites that may lead to the initiation of IDRs. As such, the liver is
conventionally used as the primary organ for metabolism tests during new drug development.
The main forms of liver injury are either hepatocellular or cholestatic; however, the clinical
characteristics of IDILI tend to be more hepatocellular injury rather than cholestatic injury [13].
Hepatocellular injury typically involves hepatocyte cell death, and histologically there is an
infiltration of mononuclear cells and eosinophils [60]. Cholestatic injury is typically less severe
than hepatocellular injury, and it is characterized by an accumulation of bile acids that can be
toxic to the hepatocytes and potentially lead to the release of danger signals and an immune
response [67].
20
Although the time to onset of IDILI is typically 1-3 months after initiation of treatment,
there are some drugs that such as fluoroquinolones that develop liver injury after only a few days
of treatment [68]. Measurement of enzymes released from hepatocytes such as alanine
aminotransferases (ALT), sorbitol dehydrogenase (SDH), and glutamate dehydrogenase (GLDH)
have been used as biomarkers of hepatotoxicity, and elevated ALT levels are the gold standard
for hepatotoxicity [69]. Clinically, mild elevations in liver injury enzymes in patients that take
drugs that cause IDILI are common; however, the injury usually resolves on its own without
discontinuing the drug, suggesting an adaptive response, and only in a small percentage of
patients does serious IDILI occur [13].
Mechanistically, there is evidence to suggest that IDILI is caused by an adaptive immune
response [70]. Anti-drug and antinuclear antibodies are common with IDILI, and some
antibodies such as in cases of halothane hepatitis have been able to recognize liver-specific
antigens [71]. The liver is an ideal organ for immune activation because a proportion of the non-
parenchymal cells are lymphocytes [72]. In addition, innate cells are also present in the liver, e.g.
Kupffer cells, which are hepatic macrophages, as well as a large number of natural killer (NK) T-
cells [73]. There is evidence that innate immune cells can be protective and decrease liver injury
caused by directly toxic drugs such as acetaminophen [74]; specifically, depletion of Kupffer
cells increased the severity of acetaminophen-induced liver injury presumably due to a decrease
in release of regulatory cytokines and mediators [75] and the phagocytosis of dying cells which
decreases inflammation [76]. Thus, the liver is an important site for immune activation, and this
may be responsible for the hepatotoxicity associated with drugs that can induce IDRs.
21
1.3.2 Haematotoxicity
Blood cells are another common target of drugs that induce IDRs, and the most common
drug-induced blood dyscrasias are agranulocytosis, aplastic anemia, and thrombocytopenia [77].
Agranulocytosis is a severe drop in the granulocyte population primarily characterized by the
depletion of neutrophils, and drugs that can induce idiosyncratic agranulocytosis include
aminopyrine, clozapine, vesnarinone, amodiaquine, and aminoglutethimide. Aplastic anemia is
the depletion of hematopoietic cells in the bone marrow, and it is usually idiopathic rather than
caused by drugs [78]. Drugs that induce aplastic anemia include felbamate and remoxipride.
Thrombocytopenia is a decrease in platelets, and drugs associated with idiosyncratic
thrombocytopenia include heparin and quinidine.
Acute and predictable haematotoxicity usually occurs with anticancer drugs, which is an
extension of their therapeutic effect of killing cancer cells, although it may take some time to
develop because the target is dividing neutrophil precursor cells and agranulocytosis is not
present until mature cells are depleted. As with other IDRs, idiosyncratic drug-induced blood
dyscrasias are typically delayed in onset. Drugs can target the bone marrow in addition to the
blood cells themselves; therefore, studies on the mechanism of blood dyscrasias have often
focused on the progenitors and cellular reserve of the bone marrow. There is also evidence to
suggest that many idiosyncratic blood dyscrasias are immune-mediated, primarily through the
formation of drug-dependent antibodies. Aminopyrine-induced agranulocytosis was found to be
mediated by antibodies [79]. Heparin and quinidine have both been shown to produce antibodies
against platelets, and this has been implicated in their ability to induce thrombocytopenia [80].
Drug metabolism has also been implicated in idiosyncratic blood dyscrasias; however, in place
of P450s in hepatocytes, formation of reactive metabolites can occur through oxidizing enzymes
22
found within immune cells such as NADPH oxidase or myeloperoxidase (MPO) found within
neutrophils [81]. In activated neutrophils, clozapine was metabolized to a reactive nitrenium ion
that formed covalent adducts to cells [82], and vesnarinone was metabolized to the reactive
quinine iminium ion by activated neutrophils, but this was not observed in metabolism studies in
the liver [83]. Thus, the localized formation of reactive metabolites within immune cells may
play an important role in the development of idiosyncratic blood dyscrasias; however, other than
covalent binding, it is still not clear exactly how these reactive metabolites may elicit a response
leading to a blood dyscrasia. Because a large portion of my thesis research focuses on
idiosyncratic drug-induced agranulocytosis, a more detailed discussion on agranulocytosis will
be presented in later sections (see Section 1.7.5).
1.3.3 Cutaneous Toxicity
Many IDRs are manifested as skin rashes, presumably because the skin is a very
immunologically sensitive organ, and its immune function has been extensively reviewed [84].
Although their expression is low, many metabolizing enzymes such as P450s, flavin
monooxygenases, sulfotransferases, etc., have been found to be expressed in skin [85], and these
could potentially lead to localized bioactivation of drugs, which may play a role in the induction
of cutaneous reactions. Drug-induced skin rashes include maculopapular rashes, urticaria, fixed
drug eruptions, and acute generalized exanthematous pustulosis. Typically skin rashes manifest
fairly early, approximately 1-2 weeks after initiation of treatment. Drug reactions with
eosinophilia and systemic symptoms (DRESS) is included in this category, and it is characterized
by fever, skin rash, and some additional organ involvement such as hepatotoxicity,
haematotoxicity, lymphadenopathy, etc.; this is commonly considered to be a generalized
hypersensitivity reaction. Anticonvulsants such as carbamazepine are the most common drugs
23
implicated in causing DRESS [86]. Furthermore, some of these reactions can be quite severe;
among these are Stevens-Johnson syndrome and toxic epidermal necrolysis. Toxic epidermal
necrolysis is characterized by sloughing of the epidermis and mucositis, which may lead to fatal
infections. The fatality rate of toxic epidermal necrolysis is fairly high: approximately 30% [87].
However, the mortality rate of toxic epidermal necrolysis can be decreased to 4% with early
treatment in a burn centre [88]. Toxic epidermal necrolysis appears to be involve keratinocyte
apoptosis caused by activated CD8+ T cells [87]. The etiology of Stevens-Johnson is similar to
toxic epidermal necrolysis except the extent of skin involvement is less than 10%.
1.3.4 Autoimmunity
Autoimmune-type IDRs can resemble idiopathic systemic lupus erythematosus. Typically
the onset of autoimmunity occurs quite late, usually more than a year after starting the drug, and
patients tend to develop antinuclear antibodies. Drugs can also induce organ-specific
autoimmunity such as vasculitis in the skin or autoimmune hepatitis. Drugs associated with
idiosyncratic autoimmunity include carbamazepine, procainamide, minocycline, D-
penicillamine, propylthiouracil, and isoniazid. Anti-neutrophil cytoplasmic antibodies have been
found in drug-induced lupus [89], and this suggests a potential role for metabolic activation of
drugs within leukocytes in the induction of idiosyncratic drug-induced autoimmunity.
24
1.4 IMMUNE SYSTEM
The immune system is a complicated network of cells and lymphoid organs that work
together to defend the body against insults and maintain homeostasis. The immune system is
subdivided into two broad categories: the innate and adaptive immunity, and their main
characteristics are highlighted in Table 1.4.
Table 1.4. Comparison of the characteristics between the innate and adaptive immunity.
Innate immunity usually occurs quite early in response to a pathogen or injury and is
relatively non-specific. In contrast, adaptive immunity occurs later, and is antigen-specific based
on what the T-cell receptor recognizes. Although canonically the innate immune system is
activated first, APCs bridge the gap between the innate and adaptive immunity by taking up,
25
processing, and presenting antigens to the adaptive immune system [90]. The clinical
characteristics of IDRs suggest that most are due to an adaptive immune response [91]. In
addition, a fine balance and appropriate regulatory mechanisms must be maintained to ensure
that the appropriate immune response is elicited when needed, and that the response is resolved
when there is no longer a threat. T cells can be activated to elicit an immunogenic response by
antigenic substances that include: endogenous macromolecules sequestered within tissues
released through processes such as cell death, covalently-modified proteins, molecular mimicry
of endogenous macromolecules by immunogenic macromolecules, and epitope spreading. To
counteract T-cell activation, tolerogenic mechanisms have been developed to ensure that the
immune response remains in check. They include: clonal deletion of B-cells that produce auto-
reactive antibodies, clonal anergy of T-cells through inhibitory costimulatory molecules such as
CTLA-4 and PD1, and also T-regulatory cells that dampen the immune response. Inappropriate
responses to antigens may lead to susceptibility to pathogenic agents; however, inadequate
induction of immune tolerance may lead to self-reactivity and autoimmunity. In terms of IDRs, if
they are immune-mediated, the default response in the majority of the population seems to be
immune tolerance, and only in some individuals is tolerance overcome, which could explain the
rare incidence of IDRs [13]. The exact mechanisms of how immune tolerance is overcome are
still unknown and are currently being investigated.
The primary role of the immune system is to defend against insults; therefore, modulation
of the response is important, and it is only when these responses are uncontrolled that a
pathological response occurs. Thus, it is quite possible that the immune system may be activated
without observed pathology if it is able to efficiently deal with the offending agent. The
interactions of the immune system are quite complex, and a precise interplay between different
26
immune factors is required to maintain tight regulation of the response. Yet, if we could monitor
these changes and detect when the immune threshold is exceeded, this would be particularly
useful for predicting drug hypersensitivity.
1.4.1 Tests for Characterizing Immune Changes
The status of the immune system can be determined using various diagnostic tests. The
systemic immune response can be determined through the measurement of cytokine and
chemokine mediators in the serum; however, changes in expression levels that differ from basal
levels tend to be difficult to detect if the magnitude of the response to drug is low. In addition,
for optimal control of the immune response, the changes in these mediators may be localized to
the tissues involved. Therefore, identifying cellular changes at the level of the target organ or
lymphoid organs, such as the lymph node and spleen, may be better for detecting an immune
response. Characterizing the changes in immune cell populations can be done through
immunohistochemistry to determine the relative localization within the tissue. To quantify the
exact number of immune cell changes, florescence-activated cell sorting (FACS) analysis can be
used to detect the expression of activation markers on the cell surface or production of cytokines
and chemokines intracellularly. These methods have been employed throughout my research to
characterize the immune changes induced by aromatic amine drugs.
Determining the immune changes related to drug exposure provides an idea as to whether
there is an immune response; however, it does not determine whether the response is due to the
drug itself or if the immune is elicited in response to tissue damage. Detection of anti-drug
antibodies implies that the drug is recognized by the immune system as antigenic and is able to
initiate an immunogenic response to activate plasma cells to produce drug-specific antibodies.
These antibodies usually take a minimum of 2 weeks of drug treatment to be detected; however,
27
drug-modified protein is required in order to develop an assay to detect anti-drug antibodies, and
in most cases such antibodies have not been tested for. However, some ex vivo and in vitro
assays have been developed specifically to determine whether drugs can activate an adaptive
immune response.
The lymphocyte transformation test (LTT) is a diagnostic tool used to detect drug
hypersensitivity by determining whether lymphocytes can be activated by the drug in a
secondary response that is dependent on the formation of drug-specific memory immune cells
from a prior primary immune sensitization event. This is analogous to an ex vivo re-challenge to
test the drug culprit responsible for the reaction. Peripheral blood mononuclear cells (PBMCs)
from drug hypersensitive patients are taken and re-stimulated with the drug in question, and
lymphocyte proliferation is measured by the incorporation of 3H-thymidine or 5’-bromo-2’-
deoxyuridine to test the anamnestic response as a stimulation index that compares the response
of cells to the drug and the proliferation in the absence of drug. However, the LTT is limited in
sensitivity and has been found to be more useful in identifying certain types of drug
hypersensitivity reactions such as generalized exanthema and DRESS, than other reactions such
as blood dyscrasias [92]. Thus, a negative LTT may not necessarily indicate that a specific IDR
is not caused by a specific drug. The inconsistencies can also be a result of the immune response
directed not to the parent drug, but to the reactive metabolite bound to proteins, in which case
stimulation with a drug-modified protein would be required. Another disadvantage of the LTT is
that in order to detect a response, the individual must have had previous exposure and a robust
immunogenic episode, such that, although it can detect the drug responsible, it does not have
predictive value as to whether some individuals will develop a specific drug reaction without
28
prior exposure. Nevertheless, despite false negatives, if a patient has a positive LTT to a drug it
is very likely that if the patient is given the drug again they will have an IDR.
Recently, another assay has been reported to test immune system activation without the
necessity of prior drug exposure. Similar to the LTT, this method involves a cell culture drug
stimulation system; in short, naive T-cells are obtained from healthy individuals and co-
incubated with dendritic cells and the drug in question to simulate an initial priming event after
which T cell proliferation is measured. Using this system it was found that the nitroso
sulfamethoxazole metabolite (SMX-NO) was able to expand CD45RO+ memory T cells, and
these cells expressed the chemokine receptors CCR2, CCR3, and CXCR3 as well as the
cytokines IFN-γ, IL-5, and IL-13 [93]. Thus, this method may be more predictive of whether a
drug is able to elicit primary sensitization. Furthermore, this assay can be useful in predictive
testing of patients prior to treatment to determine their susceptibility instead of a confirmatory
test after the development of hypersensitivity. However, this assay is quite labour-intensive in
terms of its practicality, and more studies will have to be performed to determine its applicability
to other drugs that cause IDRs.
1.5 AROMATIC AMINE DRUGS
Drugs containing the primary aromatic amine moiety are notorious structural alerts in
drug development because they are almost always associated with a high incidence of IDRs
(Table 1.5). It has been assumed that a hypersensitivity reaction to one sulfonamide means that a
patient will have a hypersensitivity reaction to all sulfonamide drugs; however, this is a common
misconception because it appears that only sulfonamide drugs with a primary aromatic amine
29
moiety are associated with IDRs, and challenge of patients with a history of a hypersensitivity
reaction to an aromatic amine sulfonamide does not predict an IDR to sulfonamides that do not
contain the arylamine moiety [94]. Examples of drugs that are sulfonamides but do not contain
the aromatic amine moiety are the non-steroidal inflammatory, celecoxib, and the diuretic,
acetazolamide.
The toxicity of aromatic amine drugs is presumably attributed to their ability to form
reactive metabolites common to the group [94]. Nitrogen contains a lone pair of electrons that
makes it easily oxidized. Oxidation produces an electron deficient or electrophilic species that
can react with endogenous macromolecules such as proteins and DNA that are nucleophilic. In
addition, nitrogen-containing molecules can undergo redox cycling, and this may lead to
oxidative stress. These chemical characteristics may predispose aromatic amines to cause IDRs.
Moreover, the majority of aromatic amine drugs contain an electron withdrawing group in the
para position to the arylamine. This decreases the electron density of the aromatic ring and
makes it more difficult to oxidize the nitrogen. In addition, the electron withdrawing group also
prevents the formation of a reactive nitrenium ion, which is responsible for the toxicity of some
drugs such as clozapine [95], and also appears to play a role in the carcinogenic potential of more
electron rich aromatic amines [96].
30
Table 1.5. Aromatic amine drugs and associated IDRs.
*withdrawn from the market
Figure 1.3. Scheme for the general metabolism of primary aromatic amine drugs.
31
In the general metabolic scheme (Figure 1.3), primary aromatic amines are detoxified by
N-acetylation, and low expression of N-acetyltransferases could be a risk factor for toxicity to
sulfonamide drugs [24]. Aromatic amines can also undergo oxidation by enzymes such as P450
and myeloperoxidase to form a hydroxylamine metabolite. Loss of the hydroxide can lead to the
formation of the reactive nitrenium ion; however, the presence of the electron-withdrawing
group prevents this from occurring, and the hydroxylamine is not very reactive per se. However,
the hydroxylamine can also be further oxidized to a nitrosoamine, which is electrophilic and can
undergo nucleophilic attack by endogenous protein thiols to form sulfinamide adducts [97].
Under reducing conditions such as in the presence of ascorbic acid, the hydroxylamine is quite
stable, and it can distribute throughout the body as a precursor to the reactive nitrosoamine to
elicit distal tissue toxicity. In addition, the nitrosamine metabolite can be reduced back to the
parent drug by antioxidants such as ascorbic acid and glutathione, and this could potentially lead
to redox cycling. The consequence of redox cycling is usually an increase in oxidative stress in
the cell. Drugs containing an aromatic nitro group can also be reduced back to the same
oxidation products as the products of aromatic amine oxidation [94]. Thus, the metabolic
disposition of aromatic amine drugs provides mechanisms by which IDRs may be elicited, but it
is still not exactly known how. It is hypothesized that aromatic amines induce IDRs through
immune-mediated mechanisms through the formation of antigenic substances and the production
of danger signals by the induction of oxidative cell stress. Therefore, my thesis research has
focused on investigating the mechanisms of aromatic amine-induced IDRs in the context of
understanding the underlying immune mechanisms by specifically focusing on the aromatic
amine drugs sulfamethoxazole (SMX), dapsone (DDS), and aminoglutethimide (AMG).
32
Figure 1.4. Chemical structures of the aromatic amine drugs SMX, DDS, and AMG.
Table 1.6. Pharmacokinetic parameters of the aromatic amine drugs SMX, DDS, and AMG
[98].
33
1.5.1 Sulfamethoxazole (SMX)
SMX is a broad spectrum bacteriostatic sulfonamide antimicrobial used to treat infections
and as a prophylaxis for Pneumocystis pneumonia in HIV-infected individuals. It is usually given
in conjunction with trimethoprim which is marketed as co-trimoxazole under the trade names
Bactrim (Roche) and Septra (Glaxosmithkline). SMX is an analog of para-aminobenzoic acid
(PABA), and therefore its pharmacological mechanism of action is to inhibit folic acid synthesis
that is required by bacteria. It does so my mimicking PABA and inhibiting dihydropteroate
synthase. Typical IDRs associated with SMX include generalized hypersensitivity, a lupus-like
syndrome, and agranulocytosis. SMX is also one of the drugs associated with the highest
incidences of Stevens-Johnson syndrome and toxic epidermal necrolysis [99]. The incidence of
SMX hypersensitivity is approximately 3% in normal patients, whereas in HIV-infected
individuals the incidence increases ~10-fold [100]. Although their immune system is
compromised, the increase in hypersensitivity could potentially be due to an increase in danger
signals within these individuals. SMX-modified proteins have been found in humans treated with
SMX [101], and anti-SMX antibodies have also been detected [102].
1.5.2 Dapsone (DDS)
DDS was first discovered in the late 1930s as a sulfone derivative, and it is still currently
in use. DDS is structurally and functionally very similar to SMX. It is a sulfonamide analog of
PABA that blocks folate synthesis. In contrast to SMX, DDS also has some anti-inflammatory
properties [103, 104], and can also inhibit the oxidative burst of neutrophils [105]. It is also
indicated for the treatment of leprosy and dermatologic diseases [106]. In terms of IDRs, DDS
has a similar profile to SMX with the exception of a greater number of blood dyscrasias such as
hemolytic anemia, methemoglobinemia, and agranulocytosis [107].
34
1.5.3 Aminoglutethimide (AMG)
AMG is an analog of the sedative hypnotic, glutethimide, and it was first approved by the
Food and Drug Administration in the 1960’s as an anticonvulsant. However, AMG had poor
efficacy as an anticonvulsant, and it was found to induce adrenal suppression [108]. Further
studies found that it inhibited two main enzymes: desmolase (CYP11) and aromatase (CYP19).
CYP11 is the limiting enzyme for the production of steroid hormones, and aromatase converts
androgens to estrogens through successive hydroxylations. However, AMG has a higher affinity
for the aromatase enzyme such that it has been used for the treatment of hormone responsive
breast and prostate cancer. AMG is usually given in conjunction with hydrocortisone to
circumvent the effects on adrenal suppression, yet this effect has also been useful for treating
Cushing’s syndrome because of its ability to inhibit cortisol production.
AMG was marketed as Cytadren (Novartis) and has since been discontinued. Despite its
effectiveness in cancer treatment, AMG is not commonly used because of its propensity to
induce ADRs, and it has been replaced by the safer 3rd
generation aromatase inhibitors,
anastrozole and letrozole. Approximately 50% of patients taking AMG experience mild ADRs
such as lethargy and ataxia [109]; however, this is thought to be due to the high daily dosages of
drug (up to 1 gram per day), and a decreased toxicity profile has been observed with a decrease
in dose with similar efficacy [110]. AMG has been reported to induce smooth endoplasmic
reticulum proliferation and induce P450s in rats [111], and an upregulation of drug metabolism
could account for increased drug clearance. Interestingly, this can affect AMG itself, and the
half-life of AMG tends to decrease during the course of AMG treatment [112]. This could affect
the amount of parent drug vs. metabolite that the body is exposed to, and it may contribute to
drug tolerance. In terms of IDRs, AMG is associated with skin rashes, cholestasis, systemic
35
lupus erythematosus, and blood dyscrasias [109]. Of these reactions, agranulocytosis is the most
commonly reported, occurring in approximately 2% of treated patients [113]. Unfortunately,
other than scattered case-reports, very few studies have been performed on the mechanism of
AMG-induced IDRs. Although AMG contains an aromatic amine moiety, it is quite different
from SMX and DDS because it does not contain an electron withdrawing group; therefore, the
electron density within the aromatic ring is higher than that of SMX and dapsone, and AMG is
more easily oxidizable than the other two drugs. Thus, AMG was studied for its uniqueness to
compare drugs containing the same aromatic amine moiety, despite varying chemical structures,
to determine whether these drugs induce IDRs through similar mechanisms.
1.6 ANIMAL MODELS
IDRs are very difficult to study in humans because the incidence of a specific IDR is
typically very low, and there are many ethical issues associated with obtaining patient samples
and performing controlled experiments. Thus animal models represent a crucial tool to study the
mechanisms of IDRs in a system containing a complete immune system. However, the issue
remains that IDRs are just as idiosyncratic in animals as they are in humans, and treating animals
with a drug that may cause IDRs in humans rarely leads to an IDR in animals. The animal model
must also have similar characteristics as to what is observed in patients with an IDR. Animal
models of IDRs that are clinically different to what occurs in humans are not useful in
elucidating the mechanisms of these adverse reactions in humans. Thus development of valid
animal models is difficult. Our lab has attempted to develop various animal models, and the
majority of these attempts have been unsuccessful, even with various interventions to increase
susceptibility such as the depletion of antioxidants or attempts to stimulate the immune system
36
and overcome tolerance [61]. However, there are a few good animal models that we have been
able to use to rigorously test hypotheses related to the mechanisms of IDRs, and this could
potentially lead to the discovery of biomarkers or pre-clinical diagnostics that could be used to
predict whether a drug candidate is predisposed to causing IDRs. The following is a discussion
of specific animal models: nevirapine-induced skin rash and D-penicillamine-induced
autoimmunity, which have been extensively studied in our lab and are each important to
highlighting clinical differences in the characteristics of IDRs.
1.6.1 Nevirapine-Induced Skin Rash
Nevirapine is a non-nucleoside reverse transcriptase inhibitor used to treat HIV-1
infections and as a prophylaxis in pregnant women to prevent transmission of postnatal HIV
infections. In terms of IDRs, nevirapine is associated with hepatotoxicity, but the more serious
concern is skin rashes including Stevens-Johnson syndrome and toxic epidermal necrolysis,
which can be fatal [114]. An animal model of nevirapine-induced skin rash has been developed
in our lab in the female Brown Norway rat, and it has characteristics that are similar to what is
observed in patients [115]. Clinically, the time to onset is delayed, typically 1-3 weeks [114], and
the incidence tends to be higher in females than males [116].
Female Brown Norway rats treated with nevirapine (150 mg/kg/day) developed red ears
at about 7 days, and a skin rash by day 21. The rash was characterized by an inflammatory
infiltrate of CD4 and CD8 T cells as well as macrophages [115]. This rash was not observed in
males and was strain dependent because the incidence in female Sprague Dawley rats was only
20%. Re-challenged rats developed red ears within 24 hours, and splenocytes from re-challenged
animals were able to transfer susceptibility to naive animals, which provides strong evidence that
the reaction is immune-mediated. In addition, a decrease in CD4 T-cells was able to prevent the
37
skin rash in female Brown Norway rats [117], and this is similar to what is reported in humans
whereby a low CD4 T-cell count appears to be associated with a lower risk of skin rash [118].
In terms of mechanistic studies, hydroxylation of nevirapine to 12-OH-nevirapine is
required for the rash to occur in rats, and deuterium substitution at the 12 position of nevirapine
inhibited the skin rash [119]. However, 12-OH-nevirapine is not chemically reactive, and it is
sulfated to a reactive benzylic sulfate. This reactive sulfate has been shown to be responsible for
the skin rash because a topical sulfotransferase inhibitor prevented covalent binding and the rash
where it was applied [120]. Immunologically, lymphocytes from re-challenged nevirapine-
treated rats responded to nevirapine by expressing interferon gamma and IL-10 [54], and the
immunosuppressants, cyclosporine and tacrolimus, were also able to prevent the skin rash [117].
The IL-1β inflammasome pathway has been implicated in contact hypersensitivity [121], and this
pathway is implicated in the nevirapine-induced rash because the chemically reactive sulfate
conjugate induces IL-1β release by keratinocytes in vitro, and an anti-IL-1β antibody markedly
suppresses the rash. These promising results could lead to a biomarker to predict the risk that a
drug candidate would cause severe dermatologic reactions. Thus, the nevirapine-induced skin
rash in Brown Norway rats represents a valid animal model that can be used to test further
hypotheses and gain a better understanding of the metabolic pathway that leads to the rash and to
understand the biological mediators of these reactions.
1.6.2 D-Penicillamine-Induced Autoimmunity
D-penicillamine has been used for the treatment of rheumatoid arthritis and as a chelating
agent to treat Wilson’s Disease; however, it is associated with a high incidence of adverse
reactions [122], and it causes autoimmune disease in 2% of treated patients [123]. Clinical
characteristics include rash, gastrointestinal upset, stomatitis, proteinuria, leucopenia, and
38
thrombocytopenia. In addition, antinuclear antibodies against DNA have been reported in
patients treated with D-penicillamine [124].
An animal model of D-penicillamine-induced autoimmunity has been developed; it is
characterized by dermatitis, granulomatous and necrotic lesions, antinuclear antibodies, immune
complexes, and immunoglobulin G deposits on the glomerular basement membrane [125]. D-
penicillamine was also able to induce liver injury in the rats. Interestingly, these responses occur
after 3 weeks of treatment and were dose-dependent. Dosages less than 20 mg/kg produced little
response, in fact it induces tolerance to higher doses, and the higher dose of 50 mg/kg did not
increase the incidence of autoimmunity. The response to D-penicillamine is also strain
dependent, with an incidence of autoimmunity in approximately 70% of Brown Norway rats, but
no effect in Lewis or Sprague-Dawley rats. Using this model, our lab has been able to investigate
many of the immune mechanisms that may play a role in the initiation of IDRs.
D-penicillamine is reactive on its own without the need for prior drug metabolism. D-
penicillamine treatment in rats induced the infiltration of macrophages into the spleen prior to the
onset of reaction, and macrophage depletion decreased the incidence of autoimmunity [126].
Furthermore, D-penicillamine was found to covalently bind to aldehyde groups on macrophages
[127], and this interaction may mediate macrophage activation because the RAW264.7
macrophage cell line upregulated the cytokines TNF-α, IL-6, and IL-23 when treated with D-
penicillamine [128]. In addition, another inflammatory cell type, the T-helper 17 cell, was found
to be significantly upregulated by D-penicillamine treatment of rats, and Th17 cells appear to be
involved in the pathogenesis of D-penicillamine-induced autoimmunity [129].
39
Interestingly, the time to onset of reaction is not quicker upon re-challenge with D-
penicillamine in rats, and this characteristic has been taken to indicate a lack of an immune
mechanism. However, D-penicillamine-induced autoimmunity is obviously immune-mediated. A
lack of an anamnestic response has been observed with other drugs that can induce autoimmune
diseases, which may be due to a deletion of memory T-cells [28]. Additional evidence for an
immune response includes the transfer of tolerance from the splenocytes of a tolerant animal to a
naive animal [130], and the TLR activator poly (I:C) was able to increase the severity of the
autoimmune reaction [131]. Thus, D-penicillamine-induced autoimmunity represents a good
animal model with clinical characteristics similar to what occurs in humans, which we are able to
utilize to study the immune mechanisms of one IDR and to determine factors that modulate this
type of immune response.
1.6.3 SMX-Induced Immunogenicity
Dogs have been reported to develop sulfonamide hypersensitivity that is characterized by
fever, thrombocytopenia, hepatotoxicity, and neutropenia [132]. This has been commonly
attributed to the lack of N-acetyltransferase expression in dogs [133]. These changes are similar
to what occurs in humans who have sulfonamide hypersensitivity reactions, and therefore, this is
a good naturally occurring animal model. However, the use of dogs for experimental studies
raises significant ethical and practical issues, and the low incidence makes this model of limited
practical use. To date there are no practical animal models of aromatic amine-induced IDRs. In
light of the high incidence of SMX hypersensitivity found in patients, many studies have been
performed on the immunogenicity of SMX in rodents, and some of these findings have even
been translatable to patients.
40
In terms of the metabolic disposition, SMX incubated with rat liver microsomes only lead
to covalent adduct formation in the presence of an NADPH oxidizing system, while incubation
with the hydroxyl metabolite of SMX (SMX-OH) readily induced covalent binding [134]. The
nitroso metabolite of SMX (SMX-NO) was found to covalently bind preferentially to the APCs
rather than the T-cells in the spleen, and haptenation over a certain threshold led to cell death
[135]. These results suggest that the nitroso metabolite can form an antigenic substance and a
danger signal. Treatment of rats with the SMX-NO led to splenocyte proliferation in response to
the nitroso metabolite; however, treatment with SMX alone did not induce a response, and
splenocyte proliferation was only observed in the presence of a Freund’s adjuvant and only upon
re-stimulation with SMX-NO [136], which suggests that the nitroso metabolite is responsible for
the immunogenicity rather than the parent drug. Anti-SMX antibodies were found in rats after 4
weeks of treatment with SMX-NO, and to a lesser extent with SMX-OH, but not in rats treated
with SMX alone [137]. These studies provide evidence for the immunogenicity of the SMX
metabolites rather than the parent drug.
Mechanistically, treatment of normal human epidermal keratinocytes with the SMX-OH
led to up-regulation of danger signals such as heat shock protein 70, TNF-α, and IL-1β [138].
Stimulation of dendritic cells with SMX and SMX-NO induced the expression of the
costimulatory molecule CD40, and when mice were given an anti-CD40 antibody it prevented T-
cell activation [139]. This suggests the requirement of Signal 2 for the induction of an immune
response. In addition, adoptive transfer of dendritic cells treated with SMX and SMX-NO into
naive mice induced T-cell proliferation in response to SMX- and SMX-NO-derived metabolites
[49]. Despite evidence that SMX induces an immunogenic response, rodents treated with SMX
or SMX-NO do not develop hypersensitivity reactions; however, this model of SMX-induced
41
immunogenicity has been useful for understanding how IDRs may be initiated by reactive
metabolites. Perhaps what is more useful is that similar changes occur in rodents as have been
observed in humans, such as an increase in expression of CD40 on the dendritic cells of HIV-
infected individuals that are hypersensitive to SMX [139].
1.7 HAEMATOLOGICAL CONCEPTS RELEVANT TO IDRs
The following is a discussion of the haematological concepts relevant to understanding
my thesis research on the mechanisms of idiosyncratic drug-induced agranulocytosis.
1.7.1 White Blood Cells
Monitoring the changes in white blood cells (WBCs), especially in patients, can provide
an early indicator of whether a drug is more prone to haematological toxicity, and for some drugs
such as clozapine, monitoring the WBC differential has led to a decrease in drug-associated
fatalities [140]. Changes in the WBC differential can also provide insight into the response of the
bone marrow because it is the main site of haematopoiesis. The majority of the WBCs in humans
are neutrophils, whereas the lymphocytes only constitute 20-45% of the WBCs [141]. In
contrast, the relative numbers of WBCs in rodents differs from what is found in humans.
Specifically, in mice and rats, lymphocytes constitute approximately 69-90% of WBCs, whereas
neutrophils comprise only 5-30% [142]. Nevertheless, rodents contain the same types of WBCs
as found in humans, and therefore they can be used to study haematological toxicities. The WBC
differentials in humans and rats are presented in Table 1.7.
42
Table 1.7. WBC composition in normal rats and humans [142, 143].
1.7.2 Neutrophils
Neutrophils or polymorphonuclear cells are typically the first immune cells to be
recruited and activated in response to an inflammatory or immune insult. As such, they play an
important role in the development and resolution of an immune response, and they have
implications as to how drugs may lead to an IDR. Morphologically, neutrophils have a
distinctive hyper-segmented/multi-lobular nucleus, and the status of neutrophils can be
determined through the number of lobes of the nucleus. For example, a shift towards an increase
in immature neutrophils with fewer lobes is often described during bouts of neutrophilia [142].
The rate of neutrophil production is fairly high at approximately 1010
cells per day [144] to
compensate for their short circulating half-life of approximately 8-10 hours in humans [145] and
11 hours in mice [146]. Neutrophils originate from the bone marrow from pluripotent stem cells
that differentiate into the colony forming unit (CFU) - granulocyte-macrophage and subsequently
43
to CFU-granulocyte (Figure 1.5). Within the bone marrow the neutrophils are categorized into
the mitotic pool, which can maintain cell division and includes the myeloblasts, promyelocytes,
and myelocytes, and the post-mitotic or maturation pool consisting of the metamyelocytes,
immature band cells, and mature neutrophil cells that are released into the circulation. From the
time neutrophils enter the post-mitotic pool to their release from the bone marrow into the
peripheral blood usually takes about 7 days in humans [145]; however in mice it is faster (about
4 days) [146]. Granulocyte-colony stimulating factor (G-CSF) plays a major role in the
proliferation, maturation, release, survival, and activation of neutrophils [146]. Moreover, the
chemokine CXCL12 and its corresponding receptor CXCR4 are involved with regulating the
release of neutrophils from the bone marrow [147].
Neutrophils are a terminally differentiated cell type and are programmed to undergo
apoptosis readily. They are then scavenged through uptake by macrophages in the liver, spleen,
and bone marrow [144]. Phagocytosis of apoptotic neutrophils by macrophages leads to the
expression of anti-inflammatory mediators such as TGF-β and IL-10 [148]. In addition,
neutrophils can also release lipid mediators such as lipoxins, resolvins, and protectins that can
activate the reticuloendothelial system and down-regulate its own chemotactic and oxidative
functions to mediate the resolution of the inflammatory effects [149]. The requirement for the
meticulous control of neutrophils is presumably due to their ability to mount an intense
inflammatory response, which if left unchecked, could lead to excessive tissue damage.
44
Figure 1.5. Overview of haematopoiesis in the bone marrow focusing on granulocyte
progenitors.
45
1.7.3 Neutrophil Activation
When stimulated by inflammatory signals, neutrophils from the blood extravasate into
tissues through chemokine-receptor gradients for homing to the site of inflammation. Neutrophil
recruitment involves an initial capture and rolling of the neutrophil on the endothelium of the
blood vessel, and this is mediated by selectins such as CD62L and CD62P, L- and P-selectin,
respectively. However, in order to extravasate into tissues, the selectins must be shed, and
intracellular adhesion molecules such as β2-integrins must be up-regulated to facilitate firm
adhesion and transmigration from the endothelium into the tissue. The hallmark of neutrophil
activation is the respiratory burst (Figure 1.6), which can be activated by PRRs such as the TLRs
on neutrophils [150] and by cytokines such as TNF-α [151]. The respiratory burst begins with an
increase in oxygen uptake by neutrophils and the assembly of NADPH oxidase machinery on its
endosomal membrane. NADPH oxidase converts molecular oxygen to superoxide, which then is
converted to hydrogen peroxidase by superoxide dismutase. The enzyme myeloperoxidase
(MPO) then uses the hydrogen peroxide to oxidize chloride ions to hypochlorous acid, which is a
very reactive oxidant that can have microbicidal activities and can damage endogenous
macromolecules.
Figure 1.6. Mechanistic scheme for the production of oxidants by neutrophils.
46
Neutrophils also have other granules that contain enzymes that are involved with immunological
defense (Table 1.8); moreover, some of these enzymes such as MPO can be involved with the
oxidation of drugs, and this may play a role in the initiation of IDRs.
Table 1.8. Components of neutrophil granules [adapted from 149].
The major enzyme within neutrophils that has been extensively studied in terms of its
ability to metabolize drugs is MPO [152]. MPO is 150 kDa heme-containing enzyme that
contains a catalytic histidine site that can form the antimicrobial oxidant hypochlorous acid.
Similar to other peroxidases, it is also prone to oxidizing compounds with a phenol or aniline
moiety [153]. Myeloperoxidase makes up 5% of the neutrophil [154] and is found within the
azurophilic granules. Drugs associated with IDRs such as SMX, DDS, carbamazepine, and
ticlopidine have been found to be oxidized by MPO of activated neutrophils [155-157]. This
47
suggests that localized reactive metabolite formation within the immune cells may play a role in
the activation of an immune response and the induction of an IDR.
1.7.4 Neutrophil Regulation of Adaptive Immunity
Neutrophil function is not restricted to its role in innate defense. Recently, studies have
shown that neutrophils can significantly contribute to adaptive immunity through the expression
of an array of different cytokines including CXC- and CC-chemokines, pro- and anti-
inflammatory cytokines, immunoregulatory cytokines, colony-stimulating factors, angiogenic
factors, and TNF superfamily members [reviewed in 158]. Neutrophils can crosstalk to adaptive
cells and regulate their function. In the spleen, neutrophils were found to enhance the
immunoglobulin production of marginal zone B-cells by releasing the factors BAFF, APRIL, and
IL-21, and these phenotypically distinct neutrophils were defined as B-helper neutrophils [159].
In humans treated with endotoxin, a subset of neutrophils expressing
CD11cbright
/CD62Ldim
/CD11bbright
/CD16bright
were found to inhibit T-cell proliferation that was
dependent on Mac-1 (alpha M subunit of the integrin alpha-M beta-2) and the localized
production of hydrogen peroxide [160]. This has important implications for the induction of
immune tolerance in response to drugs that can cause IDRs. A similar group of cells, the
myeloid-derived suppressor cells have also been reported to inhibit T-cells and are potential
cancer targets because such cells appear to prevent the immune system from eliminating tumors.
These cells inhibit the immune response through mechanisms such as the production of arginase,
reactive oxygen species, inducible nitric oxide synthase, IL-10, and through the depletion of
cysteine [161]. Alternatively, neutrophil plasticity has also been described in tumour
immunology where N1 neutrophils were found to be more activated and inhibited tumour growth
through the release of proinflammatory mediators such as TNF-α and Fas. In contrast, N2
48
neutrophils were more immunosuppressive and pro-tumour growth; this plasticity was dependent
on TGF-β expression [162]. Thus, the neutrophil is a dynamic cell type; which could play a very
important role in the development of immunogenic responses and should be investigated in the
context of IDRs.
1.7.5 Agranulocytosis
Agranulocytosis is a blood dyscrasia characterized by a severe depression of
granulocytes, in particular neutrophils, below 0.5x109 cells/mL, which can be very serious and
even fatal. Chemotherapeutic drugs can induce agranulocytosis; however this is thought to be
mediated by its pharmacological effect of cytotoxicity. The most common non-chemotherapeutic
drugs that have been associated with agranulocytosis include clozapine, DDS, dipyrone,
methimazole, procainamide, and propylthiouracil [163]. The incidence of fatality due to
agranulocytosis is quite high, approximately 4-16% in clozapine-induced agranulocytosis [140],
and many patients develop complications due to infection and septicemia.
The onset of drug-induced agranulocytosis usually occurs more than 1 month after
initiating the drug [163]. Mechanistically, agranulocytosis is thought to be either due to direct
drug toxicity to the granulocyte progenitors in the bone marrow or an immune-mediated
mechanism [164]. In patients treated with aminopyrine, anti-neutrophil antibodies have been
found [165]. Peripheral destruction of neutrophils could lead to a compensatory increase in
production by the bone marrow that is eventually overwhelmed leading to agranulocytosis. Since
the clinical characteristics are relevant to understanding the changes observed in my thesis
research, the following is a discussion on clozapine- and AMG-induced agranulocytosis.
49
1.7.6 Clozapine-Induced Agranulocytosis
Clozapine is an atypical antipsychotic associated with agranulocytosis in 0.8% of
patients with a higher incidence in female patients and patients of increased age [166]. The onset
of agranulocytosis typically occurs after about 2 months of treatment [163], and patients’ WBC
differential must be monitored in order to be prescribed the drug [140]. An initial neutrophilia
has been reported in patients treated with clozapine, which only leads to agranulocytosis in some
patients [167]. Many patients have also been observed to have elevated expression of the
cytokines TNF-α, sIL-2R, IL-6, and G-CSF as early as after 1 week of treatment [168, 169].
Many studies have been performed to understand the metabolic disposition of clozapine
and its relationship to agranulocytosis. In vitro, neutrophil apoptosis was induced by the
nitrenium ion metabolite of clozapine [170]. Activated neutrophils metabolize clozapine to a
reactive nitrenium ion, and this metabolite covalently binds to neutrophils in patients [171].
Moreover, covalent binding was observed in the bone marrow upon treatment of rats with
clozapine [172]. Thus, although the exact mechanism is unknown, the reactive nitrenium
metabolite of clozapine seems to be involved in the induction of agranulocytosis, and localized
formation of the reactive metabolite may play an important role in the pathogenesis.
1.7.7 AMG-Induced Agranulocytosis
AMG-induced agranulocytosis has been reported in 1.9% of treated patients [113].
Unfortunately, much less is known about what occurs prior to the onset of agranulocytosis
because, in contrast to clozapine, patients are not typically monitored prior to the development of
agranulocytosis, and AMG is not commonly used in the clinic anymore because of the high risk
of ADRs. Previous case reports describe agranulocytosis as occurring within 4 weeks of starting
the AMG treatment, and the bone marrow aspirate was hypocellular; however, the cellularity of
50
the bone marrow usually returns to normal upon cessation of the drug [30]. Moreover, adaptation
has been described wherein AMG-induced agranulocytosis recovered on its own without
discontinuing drug. If the reaction is immune-mediated, this could potentially be immune
tolerance. Nevertheless, our knowledge of the clinical characteristics of AMG treatment remains
limited.
Few studies have focused on the mechanisms of AMG-induced agranulocytosis. An
animal model of AMG-induced leucopenia has been described in mice treated with AMG (50
mg/kg/day) in both B6C3F1 hybrids [173] and ICI-derived mice [174]. However, this was not
successfully replicated in CD1 mice [175], and it is characterized by a decrease in the total WBC
count, not just a change in the granulocyte population. Leucopenia occurred as early as 2 weeks
and was quicker in onset upon re-challenge [174]. In contrast, leucopenia was not observed in
Wistar rats treated with AMG at a dose of 50 mg/kg/day, suggesting that there are species
differences in the effect of AMG on WBCs [173]. This effect was also dependent on the
aromatic amine structure because glutethimide (an anticonvulsant analog of AMG without the
aromatic amine) did not produce similar changes in WBCs in mice [174]. In terms of the
metabolic disposition, AMG can be metabolized to a hydroxylamine metabolite [176], and the
subsequent formation of the reactive nitroso metabolite is presumably responsible for the
toxicity. Although there are few studies that focus on the ability of neutrophils to metabolize
AMG, MPO has been found to oxidize AMG in vitro into a MPO-protein free radical [177], and
this could be involved in the mechanism of agranulocytosis.
51
1.8 OVERVIEW OF THESIS RESEARCH
The objective of my thesis research has been to investigate the mechanisms of IDRs
caused by drugs containing the aromatic amine moiety, which is notorious for its ability to
induce IDRs. In doing so, the main hypothesis has been that aromatic amine drugs may induce
IDRs through immune mechanisms that involve their ability to form reactive species and
covalently bind to endogenous proteins. In addition, they are able to redox cycle which has the
potential to induce oxidative cell stress. However, because there is no valid animal model of
aromatic amine-induced IDRs, we were unable to fully test this hypothesis. Our laboratory has
made many attempts to develop models by introducing various immune interventions to see how
this may affect the immune response such as the severity of IDRs, but the majority of these
attempts have not been successful. Thus, although there is substantial indirect clinical evidence
for immune involvement, the main goal was to characterize the immune changes induced by
aromatic amine drugs, and perhaps this may lead to the discovery of biomarkers and to the
development of an animal model.
In the initial studies, the objective was to determine whether aromatic amines drugs
induced changes in the liver that could be danger signals through a screen of hepatic gene
expression. Given that most aromatic amine drugs are metabolized to common reactive
metabolites, then they should induce a similar pattern of injury and similar changes in gene
expression that could potentially be biomarkers to drugs predisposed to IDRs. To our knowledge,
this is the first systematic study of the mechanisms of IDRs induced by aromatic amine drugs in
terms of detecting similar gene expression profile changes. We also performed more detailed
studies with AMG to investigate the downstream effects of gene expression changes, in
comparison to physiological changes in the liver, to determine whether the liver is a target organ
52
of aromatic amine drugs. The second half of my thesis focuses on investigating the immune
effects induced by one representative aromatic amine drug, AMG. Since most aromatic amine
drugs are also associated with agranulocytosis, the effect of AMG on neutrophils was
investigated to gain a better understanding of aromatic amine-induced agranulocytosis. Studies
on the immune changes induced by AMG were also performed to gain mechanistic insight into
how immune responses may be induced by aromatic amine drugs. Brown Norway rats were
chosen because we have experience with this species, and we have developed several good
animal models with this species. Our findings suggest that the default response to aromatic
amine drugs may be tolerance and this may explain why only a small fraction of the population
is susceptible to IDRs.
53
CHAPTER 2
CHANGES IN GENE EXPRESSION INDUCED BY
AROMATIC AMINE DRUGS: TESTING THE DANGER
HYPOTHESIS
Winnie Ng and Jack Uetrecht
(Reproduced with permission from: Ng, W., and Uetrecht, J. Journal of Immunotoxicology,
2013. 10: p. 178-191. Copyright 2012 Informa Healthcare USA Inc. Informa Healthcare granted
permission to include my own article in this thesis).
54
2.1 ABSTRACT
Virtually all drugs that contain a primary aromatic amine are associated with a high
incidence of idiosyncratic drug reactions (IDRs), suggesting that this functional group has
biological effects that may be used as biomarkers to predict IDR risk. Most IDRs exhibit
evidence of immune involvement and the ability of aromatic amines to form reactive metabolites
and redox cycle may be responsible for initiation of an immune response through induction of
cell stress as postulated by the Danger Hypothesis. If true, danger signals could be biomarkers of
IDR risk. Our previous attempt to test the Danger Hypothesis found that sulfamethoxazole
(SMX), the only aromatic amine tested, was also the only drug not associated with an increase of
cell stress genes in mice. To ensure that these observations were not species specific and to
determine biomarkers of IDR risk common to aromatic amines, rats were treated with SMX and
two other aromatic amine drugs, dapsone (DDS) and aminoglutethimide (AMG), and hepatic
gene expression was determined using microarrays. As in mice, SMX induced minimal gene
changes in the rat and none indicated cell stress, whereas DDS and AMG induced several
changes including up-regulation of enzymes such as aldo-keto reductase, glutathione-S-
transferase, and aldehyde dehydrogenase, which may represent danger signals. Early insulin-
induced hepatic gene (Eiih) was up-regulated by all three drugs. Some mRNA changes were
observed in the Keap-1-Nrf2-ARE pathway; however, the pattern was significantly different for
each drug. Overall, the most salient finding was that the changes in the liver were minimal even
though aromatic amines cause a high incidence of IDRs. The liver generates a large number of
reactive species; however, the ability of aromatic amines to be bioactivated by cells of the
immune system may be why they cause a high incidence of IDRs.
55
2.2 ABBREVIATIONS
AMG, aminoglutethimide
2M, 2-microglobulin
P450, cytochrome P450
DDS, dapsone
Dusp1, dual specificity phosphatase 1
Eiih, early insulin-induced hepatic gene
FDR, false discovery rate
GSH-S-Tr, glutathione-S-Transferase
IDR, idiosyncratic drug reaction
SMX, sulfamethoxazole
SMX-OH, hydroxyl metabolite of sulfamethoxazole
SMX-NO, nitroso metabolite of sulfamethoxazole
Sgk, serum/glucocorticoid-regulated kinase 1
Txnrd, thioredoxin reductase
56
2.3 INTRODUCTION
Drugs that contain the primary aromatic amine functional group are almost always
associated with a high incidence of idiosyncratic drug reactions (IDRs) and this is considered a
structural alert for drug development [94]. The inability to predict which drug candidates will
cause such reactions, and the fact that they are usually not detected until very late in
development or after the drug has been marketed, markedly increases the risks associated with
the development of new/novel drugs.
The exact mechanisms of IDRs remain unclear; however, many lines of evidence suggest
that most IDRs are mediated by the adaptive immune system [23]. The Danger Hypothesis has
been proposed as a mechanism for the initiation of immune-mediated IDRs [42]. This hypothesis
posits that the tissues are responsible for initiating an immune response through the release of
danger signals in response to cell stress or damage [39]. This hypothesis compliments other
hypotheses such as the hapten hypothesis because it may result in activation of antigen-
presenting cells leading to costimulatory signals, referred to as signal 2, that are required for
activation of T-helper cells (Figure 2.1). In the context of IDRs, a drug, or more likely its
reactive metabolite, could induce cell stress or damage and this might contribute to the ability of
a drug to cause IDRs.
57
Figure 2.1. The Danger Hypothesis as applied to IDRs. The drug or its reactive metabolite
can form antigenic adducts with endogenous molecules that are presented to T-cells on major
histocompatibility complexes of antigen-presenting cells (Signal 1). Additionally, the drug or
reactive metabolite may induce cell stress or damage through processes such as redox cycling
and oxidative damage. This may lead to the release of danger signals to up-regulate co-
stimulatory molecules such as B7 and CD40 on antigen-presenting cells to activate T-cells
(Signal 2). Signals 1 and 2 are required concurrently to initiate an adaptive immune response and
certain drugs have the ability to induce both signals.
To date, many potential danger signals have been identified such as heat shock proteins,
high-mobility group protein B1, ATP, cytokines, and nuclear DNA [178]. However, it is difficult
to rigorously test whether the induction of danger signals is an important biomarker of IDR risk.
Danger signals vary widely in their characteristics; the only consistent feature being that they are
always endogenous molecules, either actively secreted or released due to destruction of cell
membrane integrity in the event of cell stress or damage. If the release of danger signals could be
used as a biomarker to predict the risk that a drug candidate would cause IDRs, it would have a
profound effect on the process of drug development.
58
The IDRs associated with aromatic amine drugs are thought to be due to the ease with
which they form reactive metabolites (Figure 2.2). Aromatic amines can be oxidized by
cytochrome P450s (P450s) and also myeloperoxidase to hydroxylamine metabolites, which are
not very reactive, but can auto-oxidize to electrophilic nitroso metabolites that can covalently
bind endogenous proteins. The nitroso metabolite can be easily reduced back to the parent amine
with antioxidants and this redox cycling could lead to oxidative stress. Thus, aromatic amine
drugs have the potential to form antigenic substances and to cause cell stress and damage, which
could produce danger signals and initiate immune-mediated IDRs.
Figure 2.2. Metabolic scheme for the formation of reactive metabolites of aromatic amine drugs.
Oxidation of aromatic amines to hydroxylamines can occur through enzymes such as P450s or
myeloperoxidase and this is prevented by N-acetylation. Hydroxylamines are not very reactive but can
auto-oxidize to electrophilic nitroso metabolites. The nitrosoamine can react with thiol-containing
nucleophiles such as glutathione or endogenous proteins to form sulfinamide adducts. Additionally, the
nitrosoamine can be reduced back to the parent drug leading to redox cycling and oxidative stress.
59
Sulfamethoxazole (SMX) is an aromatic amine antimicrobial that is widely used in
combination with trimethoprim for many indications including prophylaxis against and to treat
Pnuemocystis carinii pneumonia in HIV-infected individuals. SMX can cause a generalized
hypersensitivity reaction and is also associated with among the highest incidences of Stevens-
Johnson syndrome and toxic epidermal necrolysis, which can be very serious and even fatal
[179, 180].
Previously we tried to test the Danger Hypothesis with a study of changes in gene
expression induced by tienilic acid, which causes idiosyncratic liver injury. Its reactive
metabolite binds principally to P450 that is unlikely to cause much cell stress. However, it binds
to several other proteins and it induced several changes in gene expression in the liver that likely
represented danger signals [37]. In contrast, treatment of mice with SMX, which was meant as a
positive control because it can covalently bind to a wider variety of proteins and also induce
oxidative stress, did not lead to up-regulation of genes that were likely to represent danger
signals, and the few changes that were induced in the liver were mostly down-regulation of gene
expression [37]. It is possible that down-regulation of mRNAs represents a danger signal.
However, metabolism of SMX in mice is limited [181], and the metabolism of SMX in rats more
closely resembles its metabolism in humans [182]. Thus a global screen of hepatic gene
expression was performed to determine the effects of SMX in the rat to investigate whether it can
induce cell stress leading to the release of danger signals. In addition, two other aromatic amine
drugs associated with a high incidence of IDRs, dapsone (DDS) and aminoglutethimide (AMG;
Figure 2.3), were tested to determine whether similar expression profiles exist between aromatic
amine drugs that could potentially indicate a similar mechanism of action and be useful as
biomarkers for drugs with the aromatic amine functional group. As the major site of drug
60
metabolism and target for IDRs, the liver (or hepatocytes) are extensively used for reactive
metabolite and toxicity studies during drug development. Therefore, we chose the liver in which
to study changes in gene expression induced by aromatic amines; possible downstream effects of
these gene changes were also tested.
Figure 2.3. Structures of the aromatic amine drugs used in this study and some associated IDRs.
61
2.4 MATERIALS AND METHODS
2.4.1 Reagents
Sulfamethoxazole (SMX) and dapsone (DDS) were purchased from Sigma (Oakville,
Ontario). AMG was purchased from Toronto Research Chemical (North York, Ontario).
Nevirapine was obtained from Boehringer Ingelheim (Ridgefield, Connecticut). Clozapine was
provided by Novartis (Dorval, Quebec). RNeasy Mini kits were obtained from Qiagen (Missis-
sauga, Ontario). Gene chips were purchased from Affymetrix (Santa Clara, CA). Light Cycler
FastStart DNA Master SYBR Green I was purchased from Roche Applied Science (Laval,
Quebec). Primers were obtained from Integrated DNA Technologies (Coralville, IA).
2.4.2 Animals
Male Brown Norway rats (≈ 8-wk-old, 200-250 g) were purchased from Charles River
(Montreal, Quebec). Rats were housed under standard conditions (doubly housed in plastic
cages, standard rat chow, automatic watering, 12:12 hr light:dark cycle, and 22°C). Food and
water were provided ad libitum. Rats were acclimatized for one week before the start of
experiments. In other studies performed with penicillamine and nevirapine in our laboratory, the
Brown Norway rat was more susceptible to IDRs induced by these drugs (i.e., autoimmunity and
skin rash, respectively) than other strains; for this reason, this strain was selected for use in the
current study. The study protocol was approved by and performed in accordance with the
University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee.
2.4.3 Treatments
The treatment protocol was similar to Pacitto et al. with some modifications [37]. Each
treatment had 4 rats per group unless otherwise stated. The drugs were administered by oral
gavage at doses of 150 mg/kg (SMX), 20 mg/kg (DDS), and 80 mg/kg (AMG). These dosages
62
were chosen from previous rat studies [182-185] to mimic peak drug concentrations in the blood
of patients taking these drugs for therapy (≈ 37 µg/mL for 1 000 mg SMX [98], 1.7 µg/mL for
100 mg DDS [98], and 3 µg/mL for 250 mg AMG [186]).
Drugs were suspended in 0.5% methylcellulose due to the poor solubility of DDS and
AMG. For the microarray study, rats were euthanized by CO2 asphyxiation after 12 hr drug
treatment. Treatments for follow-up studies were performed as described previously using
several different time points, with the exception that for time points >24 hr, rats were given an
additional dose of drug every 24 hr. At necropsy, the liver was extracted using aseptic
techniques, immersed in RNAlater RNA Stabilization Reagent (Qiagen), and stored according to
manufacturer instructions at -80°C until RNA extraction. Liver tissue was also immersed in
phosphate buffered saline (PBS, pH 7.4) with protease inhibitor (Sigma) for activity assays. PBS
was used instead of lysis buffer to minimize possible interferences by the solvent with assay
matrices.
2.4.4 RNA Extraction
Liver tissue (≈20-30 mg) stored in RNAlater RNA Stabilization Reagent was homo-
genized with a rotor stator homogenizer (IKA Ultra-Turrax T25 S1, Janke & Kunel, Staufen,
Germany). Total RNA was extracted using an RNeasy Mini Kit (Qiagen) as per manufacturer
protocol. Extracted RNA was eluted in 40 µl of RNase-free water and stored at -80°C. The
quality and purity of the extracted RNA was initially measured using UV spectrophotometry
based on the A260/A280 ratio before further quality check through capillary electrophoresis using
the Agilent Bioanalyzer (Affymetrix).
63
2.4.5 Microarray analysis
Affymetrix RatGene 1.0 ST chips were used for detection of changes in gene expression.
Microarray processing of the liver samples was performed at the microarray facility in The
Centre for Applied Genomics (Hospital for Sick Children, Toronto, Ontario, Canada) based on
Affymetrix protocols. For microarray data, Expression Console software was used for quality
control analysis before fold-changes were determined using Partek software. The Robust Multi-
array Analysis method was used for background correction, which included Quantile Normaliza-
tion and Median Polish summarization. Data was log-base 2 transformed and fold-change values
were obtained for treatments as compared to the control group. One-way ANOVA analysis was
performed to determine the statistical significance based on differences between the treatment
and control mean intensities for each gene. Attempts were made to obtain more accurate p-values
by performing multiple test corrections using the false discovery rate (FDR).
2.4.6 Real-time (RT)-PCR of early insulin-induced hepatic gene (Eiih)
Extracted RNA samples were converted to cDNA using the Omniscript RT Kit (Qiagen),
with oligo(dT15) primers (Roche) and RNase inhibitor (Roche), as per the manufacturer’s
protocol. Quality was checked using the spectrophotometry method as previously described. For
each sample, 2 µg of RNA was converted to cDNA in a total reaction volume of 20 µl. Using
Light Cycler FastStart DNA Master SYBR Green 1 (Roche), PCR was performed using a Light
Cycler instrument (Roche) as per the following conditions: pre-incubation at 95°C for 10 min
and amplification for 45 cycles (denaturation at 95°C for 15 sec, annealing at 60°C for 5 sec, and
elongation at 72°C for 10 sec). Primers (Integrated DNA Technologies) for Eiih were as follows:
forward primer 5’-AGCTCTCCAGCTCTGGATTCTT-3’, reverse primer 5’-CACACCCAG-
AACAGAGTCTTAC-3’. 2-Microglobulin (2M) was used as the housekeeping gene and the
64
primers (Integrated DNA Technologies) were as follows: forward primer 5’-TCAGTTCCACCC-
ACCTCAGATAGA-3’, reverse primer 5’-TGTGAGCCAGGATGTAGAAAGAC-3’. Data was
analyzed using the RelQuant software and normalized to a calibrator control.
An additional two drugs, nevirapine and clozapine, were both tested in the rat for Eiih
expression using RT-PCR. The treatment protocol, tissue extraction, and PCR analysis were
similar to previously stated. Nevirapine and clozapine were administered at 150 mg/kg and 50
mg/kg, respectively, through oral gavage and samples were taken 6 and 12 hr after treatment.
2.4.7 Activity assays
Thioredoxin reductase (Txnrd) and glutathione-S-transferase (GSH-S-Tr) activity in the
liver was measured using a Txnrd assay kit from Cayman Chemical (Ann Arbour, MI) and a
GSH-S-Tr activity colorimetric assay kit from Abcam (Cambridge, MA), respectively, as per
manufacturer instructions. Assayed samples contained a total protein concentration of 4 mg/mL
in PBS with proteinase inhibitor. The levels of sensitivity of the Txnrd and GSH-S-Tr kits/assays
were 0.015 and 0.025 µmol/min/mL, respectively.
2.4.8 Statistical Analysis
Data obtained from PCR and activity assays were analyzed by ANOVA using GraphPad
Prism 5. Bonferroni post-tests were used to determine statistical significance between treatment
and control groups.
65
2.5 RESULTS
2.5.1 Microarray quality
The extracted rat liver RNA demonstrated good quality as demonstrated via UV spectro-
photometry and the Agilent Bioanalyzer methods. The A260/A280 ratios of the samples were in the
range of 1.96-2.29 indicating good RNA quality. Only one sample in the SMX treatment had a
RNA Integrity Number < 7, but was still sufficient for microarray processing. Expression
Console software revealed no outlier arrays and the Spearman Rank Correlation (r2) was > 0.982,
indicating a good correlation between signal values of different gene chips. Thus, all arrays were
used for microarray data analysis.
2.5.2 SMX-induced hepatic gene expression
SMX induced very few gene changes > 2-fold expression in the rat 12 hr after treatment
(see Appendix 1, Table A1.1). When FDR was applied for multiple test corrections, none of the
changes were significant (p < 0.05); of the gene changes that occurred, none seemed to explicitly
indicate cell stress or initiation of an immune response. Interestingly, genes involved with the
acute cell response were down-regulated including dual specificity phosphatase 1 (Dusp1) and
serum/glucocorticoid-regulated kinase (Sgk).
2.5.3 DDS-induced hepatic gene expression
In contrast to SMX, a greater number of genes were up-regulated > 2-fold with DDS
treatment in the rat (see Appendix 1, Table A1.2). However, only changes in six genes were
statistically significant (p < 0.05) with the FDR applied: aldo-keto reductase 7A3 (Akr7a3, 5.38-
fold), glutathione-S-transferase Yc2 subunit (Yc2, 3.18-fold), aldehyde oxidase 1 (Aox1, 1.63-
fold), ferritin light polypeptide (Ftl1, 1.44-fold), UDP glucosyltransferase 1A3 (Ugt1a3, 1.39-
fold), and glutathione reductase (Gsr, 1.38-fold). Up-regulation of genes such as Akr7a3, Yc2,
66
Ugt1a3, and Gsr could indicate cell stress due to their function in drug metabolism and
cytoprotection against oxidative stress. Again, Dusp1 and Sgk were among the most down-
regulated genes similar to what was observed with SMX.
2.5.4 AMG-induced hepatic gene expression
AMG induced the most gene changes of the aromatic amine drugs tested (see Appendix
1, Table A1.3). Among these changes, 552 genes were significantly different using the FDR. Of
these genes, quite a few were involved in antioxidant and drug metabolising pathways, including
P450 oxidoreductase (Por), aldehyde dehydrogenase 1A1 (Aldh1a1), UDP
glucuronosyltransferase 2B1 (Udpgtr2), epoxide hydrolase 1(Ephx1), aldo-keto reductase 7A3
(Akr7a3), glutathione-S-transferase Yc2 subunit (Yc2), thioredoxin reductase 1 (Txnrd1),
glutathione reductase (Gsr), and UDP glycosyltransferase 1A3 (Ugt1a3), which could indicate
that cell stress was induced. Additionally, several heat shock proteins were up-regulated such as
Hspca, Hsph1, Hspa1b, and Hspb8 that could be potential danger signals to initiate an immune
response. The pattern of decreased gene expression was also similar to the other aromatic amine
drugs, as Dusp1 and Sgk were among the most down-regulated.
2.5.5 Aromatic amine gene expression profile
Although the gene changes were different for each aromatic amine drug, in general,
aromatic amines induced greater down-regulated than up-regulated gene changes (Table 2.1).
67
Table 2.1. Similar genes differentially regulated at least 1.4-fold change in the same
direction in all aromatic amine drugs tested 12 hr after treatment.
Gene Function
Fold-change
SMX DDS AMG
Eiih (hepatic protein EIIH)
Unknown function; induced early in rats upon insulin treatment; may play role in carbohydrate metabolism through association with glucose
transporter 2 [187].
2.86 4.23 12.94*
Socs2
(suppressor of cytokine signaling 2)
Inhibits growth hormone activity; role in mediating the anti-inflammatory
effects of lipoxins [188].
1.48 2.08 1.41
Dusp1
(dual specificity
phosphatase 1)
Involved with dephosphorylation of tyrosine and serine/threonine residues;
role in signalling pathways due to interaction with p38, JNK, ERK;
inducible early response to LPS, hypoxia, heat shock, oxidative stress
[189].
-3.58 -3.01 -3.61
Sgk
(serum/glucocorticoid regulated kinase)
Serine/threonine protein kinase involved with cellular signalling pathways
of metabolism, proliferation, and differentiation in response to glucocorticoids and serum; induced transiently during early response to
stress [190].
-2.69 -3.72 -5.37*
G0s2
(G0/G1 switch gene 2)
Involved with mitochondrial induction of apoptosis and growth arrest in
human fibroblasts; target of retinoic acid and peroxisome proliferator-activated receptor agonists; may play role in adipogenesis [191, 192].
-2.11 -2.03 -1.59
Jun (Jun proto-oncogene)
Activated by JNK phosphorylation; interacts with c-Fos to form AP-1 transcription factor that regulates cell proliferation and apoptosis
pathways in response to stress [193].
-1.97 -2.49 -1.51
Cyr61
(cysteine rich protein 61)
Heparin binding angiogenesis inducer, involved with endothelial adhesion
and migration and tumour growth [194].
-1.87 -2.5 -1.94
Slc25a25
(solute carrier family 25,
member 25)
Regulates ATP homeostasis as a shuttle across inner mitochondrial
membrane; involved with maintaining metabolic efficiency [195].
-1.86 -2.67 -4.04*
Acat2 (acetyl-Coenzyme A
acetyltransferase 2)
Cytosolic acetyl-Coenzyme A acetyltransferase activity; involved with synthesis of cholesteryl esters and cholesterol absorption; mainly
localized to liver and intestines; may play a role in atherosclerosis [196].
-1.63 -1.63 -2.28
Slc34a2 (solute carrier family 34,
member 2)
Sodium-phosphate transporter found in intestines, kidney, and lung; may be involved with regulating phosphate levels in intestinal epithelium [197].
-1.59 -1.82 -3.02*
Sqle
(squalene epoxidase)
Involved in rate-limiting step of cholesterol biosynthesis; oxidizes squalene
to squalene epoxide; negatively regulated by cholesterol [198].
-1.51 -1.42 -1.72
Inhbe
(inhibin beta E) Member of TGF-β family; may induce apoptosis in hepatoma cells and
down-regulated in hepatocarcinogenesis [199].
-1.44 -1.51 -2.01*
Data expressed as fold-change between treated and controls. *p < 0.05 with FDR applied (n = 4).
68
Among these, hepatic protein EIIH (Eiih; aka early insulin-induced hepatic gene) was the
most highly up-regulated gene in all three drugs tested. Increased Eiih gene expression was
confirmed through RT-PCR for both DDS and AMG treatment, but not SMX (Figure 2.4).
Further time-course investigation using RT-PCR found that Eiih was expressed only acutely,
with the highest expression at 8 and 12 hr after AMG treatment (Figure 2.5). Other drugs known
to cause IDRs such as nevirapine and clozapine also increased expression of Eiih at early time
points in treated rats (Figure 2.6).
Figure 2.4. Eiih expression induced by aromatic amine drugs 12 hr after treatment. Among the
treatment groups (solid bars), AMG induced a significant increase in Eiih expression as compared to
control (open bars). DDS also induced an increase in Eiih; however, no change was observed for SMX.
Rats were given 150 mg SMX/kg, 20 mg DDS/kg, or 80 mg AMG/kg, and liver samples were taken for
RT-PCR analysis. Eiih expression was normalized to 2-microglobulin (B2M) as a housekeeping gene and
a calibrator control. RT-PCR was run in triplicate (n = 4; ***p < 0.001).
69
Figure 2.5. Time-course of hepatic Eiih expression induced by AMG. Eiih was expressed the greatest
8 and 12 hr after AMG treatment (solid bars) as compared to control (open bars). Rats were treated with
80 mg AMG/kg, and liver samples were taken for RT-PCR analysis. Eiih expression was normalized to
2-microglobulin (B2M) as a housekeeping gene and a calibrator control. RT-PCR was run in triplicate (n
= 2).
70
Figure 2.6. Hepatic gene expression of Eiih induced by several other drugs associated with
IDRs. Clozapine (CLZ), an atypical antipsychotic drug, induced an increase in Eiih 12 hr after
treatment of rats, and nevirapine (NVP), an anti-retroviral drug, increased Eiih expression at both 6
and 12 hr after treatment of rats. Data expressed as fold-change between treated and controls (n =
4; * p < 0.05, ** p < 0.01).
71
A greater number of similarities were also observed between DDS and AMG in terms of
genes regulated by the Keap1-Nrf2-ARE pathway (Table 2.2).
Table 2.2. Keap1-Nrf2-ARE-regulated genes that were changed ≥ 1.4 fold by at least one of
the aromatic amine drugs (SMX, DDS, or AMG) tested 12 hr after treatment.
Keap-1-Nrf2-ARE-Regulated Genes Fold-change
SMX DDS AMG
Xenobiotic Metabolism
Glucuronidation:
Udpgtr2 (UDP glucuronosyltransferase 2 family, polypeptide B1) 1.12 1.28 3.59*
Ugt1a3 (UDP glycosyltransferase 1 family, polypeptide A3) 1.09 1.39* 2.06*
Ugt2b4 (UDP glycosyltransferase 2 family, polypeptide B4) -1.09 1.10 1.45
Glutathione Conjugation:
Yc2 (glutathione S-transferase Yc2 subunit) 1.01 3.19* 2.23*
Gstm5 (glutathione S-transferase, mu 5) 1.08 1.53 1.14
Gsta2 (glutathione-S-transferase, alpha type2) -1.02 1.31 1.49*
Other:
Aldh1a1 (aldehyde dehydrogenase family 1, member A1) -1.23 2.33 4.15*
Ephx1 (epoxide hydrolase 1, microsomal) 1.02 1.71 2.33*
Nqo1 (NAD(P)H dehydrogenase, quinone 1) 1.13 1.57 1.53
Regulation of Cellular Redox/Antioxidant Status:
Txnrd1 (thioredoxin reductase 1) 1.16 1.40 2.23*
Gsr (glutathione reductase) 1.08 1.38* 2.06*
Energy Production:
Me1 (malic enzyme 1) 1.09 1.42 2.76*
Heme Catabolism:
Hmox1 (heme oxygenase (decycling) 1) 1.09 1.06 1.72
Data expressed as fold-change between treated and controls. *p < 0.05 with FDR applied (n = 4).
72
Further testing of downstream effects of these gene changes found that enzyme activity did not
always correlate with changes in mRNA expression. Greater Txnrd activity was found after
SMX treatment when Txnrd1 gene expression was low compared to that induced by DDS or
AMG, whereas gene expression of Txnrd1 was higher with the other two drugs, when Txnrd
activity was low (Figure 2.7a). Nevertheless, a time-course study with AMG found that Txnrd
activity was only increased at 48 hr after treatment (Figure 2.7b). GSH-S-Tr activity was also
tested, but no significant changes were observed except for a possible increase in activity 48 hr
after AMG treatment (Figure 2.8).
Figure 2.7. Hepatic Txnrd activity after treatment with aromatic amine drugs.
Thioredoxin reductase activity was increased at both 12 and 24 hr after SMX treatment (A)
but not with the other two aromatic amine drugs (n = 4; ***p < 0.001). However, a time-
course study (B) found that AMG appeared to increase thioredoxin reductase activity 48 hr
after treatment (n = 2). Rats were treated with 150 mg SMX/kg, 20 mg DDS/kg, or 80 mg
AMG/kg before liver samples were taken for activity assay. For time points > 24 hr, rats
were given an additional dose of drug every 24 hr.
73
Figure 2.8. Time-course of hepatic GSH-S-Tr activity in AMG-treated rats. No significant
change was observed in GST activity after treatment with 80 mg AMG/kg (squares) in the liver
as compared to controls (open circles). For time points > 24 hr, rats were given an additional
dose of drug every 24 hr (n = 2).
74
2.6 DISCUSSION
If biomarkers that predict the risk that a drug candidate will cause an unacceptable risk of
IDRs could be found it would have a profound effect on the process of drug development. The
liver is a logical place to look because it is the site of most reactive metabolite formation, and it
is the target of IDRs that are most likely to lead to drug withdrawl. Therefore, when drug
candidates are screened for reactive metabolite formation, this is done almost exclusively in the
liver or with hepatocytes. Accordingly, much of the screening for toxicities of drug candidates is
focused on the liver.
Drugs that are primary aromatic amines represent a good starting point because this
functional group is notorious for being associated with IDRs. Microarray technology represents
the easiest way to examine a wide variety of possible biomarkers. The initial goal of these
experiments was to verify whether the changes in hepatic gene expression induced by SMX were
similar to our previous study performed in mice and whether there were changes that could be
danger signals, biomarkers, or both. We chose here to focus on early timepoints because subtle
signs of cell stress, through changes in mRNA expression, would likely happen rapidly upon
drug treatment and thus be good predictors. In comparison, effects on host immune responses
would not be expected to occur - or to be detected - as early. Our findings indicate that, as in
mice, SMX induced minimal hepatic gene changes in the rat. Further, of these changes, none met
the strict criteria set here for denoting statistical significance.
It was surprising that there was a significant difference between the gene expression
profile of SMX and DDS because they are structurally very similar with similar pharmacology,
although DDS causes hemolytic anemia and methemoglobinemia and SMX does not [200];
which presumably reflects differences in the amount of redox cycling and distribution to red
75
cells [201]. AMG is a first generation aromatase inhibitor used to treat estrogen-responsive
breast and prostate cancer. AMG is structurally different from the other two drugs; in particular,
there is no electron-withdrawing group on the aromatic ring to decrease the electron density of
the aromatic amine and this should change its redox potential. AMG was included to determine
the extent of the involvement of the aromatic amine group in the induction of IDRs to determine
whether the mere presence of the moiety is enough to induce similar changes. Interestingly,
AMG induced the most significant number and magnitude of gene changes. This is not
surprising because AMG is associated with a variety of adverse reactions in ≈ 50% of patients
that take this drug, although only 1% of patients experience idiosyncratic hematologic toxicities
including thrombocytopenia, leucopenia, pancytopenia, and agranulocytosis [201]. To some
degree the similarities between DDS and AMG correlate with the fact that they are more prone
to induce blood dyscrasias and bone marrow toxicity than SMX [107, 202]. Covalent adducts of
SMX hydroxylamine have been found to localize on the surface of normal human epidermal
keratinocytes in vitro, whereas covalent adducts of DDS hydroxylamine were found to localize
intracellularly [203]. These distinct covalent binding patterns may play a role in the observed
differences and types of IDRs induced because intracellular adducts may have a greater ability to
induce a strong immune response.
DDS and AMG induced some genes that could be considered danger signals. Our
previous microarray studies on drugs including tienilic acid, carbamazepine, phenytoin, and D-
penicillamine have all shown induction of cell stress through the up-regulation of genes involved
with the Keap1-Nrf2-ARE pathway [37, 204, 205], and this may potentially be a sign of danger
to the immune system. The Keap1-Nrf2-ARE pathway is responsible for regulating the
transcription of a variety of genes involved with detoxification and cytoprotection. The up-
76
regulation of these genes suggests that cell stress occurred and there is an increased need to
modulate potential damage that could eventually lead to an immune response.
In the present study, the same general pattern of increased expression of Keap1-Nrf2-
ARE-regulated genes was observed, although mainly for DDS and AMG. Nevertheless, although
the changes induced by SMX were not statistically significant, the same Keap1-Nrf2-ARE-
regulated genes as DDS and AMG appeared to be slightly elevated. Txnrd is an enzyme
analogous to glutathione reductase that functions to provide reducing equivalents to thioredoxin
to act as an antioxidant, and it is crucial in maintaining cell redox status [206]. Surprisingly, it
was SMX that induced the greatest increase in Txnrd activity. Furthermore, there was a lag
period between gene induction and Txnrd activity during AMG treatment. This implies that gene
expression does not necessarily correlate with functional changes and that gene expression was
likely induced before protein expression, which may explain the inconsistent relationship
between gene and protein activity. This also raises an important issue as to whether gene changes
are meaningful on their own. The activities of certain proteins are governed by post-translational
modifications or by cytoplasmic “regulators”. In the canonical pathway, Nrf2 is kept inactivated
by Keap1 in the cytoplasm, and only when Keap1 undergoes proteolytic cleavage is Nrf2
released to migrate into the nucleus where it then binds to the antioxidant response element and
initiates transcription [207]. Thus, changes in gene expression of Nrf2 may not be useful because
activation is regulated post-translationally.
The acute cell response genes Sgk and Dusp1 were consistently the most down-regulated
with all aromatic amine drugs tested. Sgk, a serine/threonine protein kinase involved in
regulating cellular metabolism, proliferation, and differentiation, can be induced transiently by
77
glucocorticoids and serum [190]. Dusp1, a nuclear phosphatase involved in regulating cellular
signaling pathways, has been reported as inducible in early response to LPS, heat shock, and
oxidative stress [189]. Down-regulation of these genes opposes what would be expected upon
drug treatment; however, decreased Sgk expression upon tienilic acid treatment was attributed to
the short half-life of Sgk mRNA [37, 190]. It is also plausible that a lack of signal could alert the
cell to stress and initiate an immune response, although the exact mechanism is unclear.
Possibly the most interesting change was the increase in Eiih mRNA that was observed
with all three drugs, although we failed to confirm increases in Eiih expression with RT-PCR for
SMX. Additional testing of two other drugs associated with IDRs: clozapine and nevirapine,
found a rapid increase in Eiih expression. These other drugs are not primary aromatic amines,
and therefore it is possible that this is a more general biomarker of IDR risk. However, without
testing a variety of drugs including negative controls that are not associated with a significant
risk of IDRs it would be premature to conclude that this represents a useful biomarker of IDR
risk. Unfortunately, little is known about the function of this gene. To date, there has only been
one published study on Eiih that found it was induced early and acutely upon insulin treatment in
rats with expression mainly localized to the liver, intestine, and islets of Langerhan, and there is
some indication for its involvement in carbohydrate metabolism [187]. Its predicted protein
arrangement suggests that it has the potential to be secreted or membrane bound, which implies
that Eiih could possibly be a danger signal if secreted or act as a receptor-signalling complex on
cellular membranes.
Although there were changes in gene expression induced by these primary aromatic
amines, especially AMG, which could represent a danger signal, the most salient finding of this
78
study is that primary aromatic amines do not appear to cause many changes in gene expression in
the liver. Given that the primary aromatic amine is such a notorious structural alert and is readily
oxidized to reactive metabolites in the liver and can also redox cycle, this is surprising. SMX and
AMG frequently cause liver injury: the incidence of elevated γ-glutamyltransferase is greater
than 60% in AMG-treated patients [208]. However, SMX-induced liver injury is usually part of a
more generalized hypersensitivity IDR, and the more severe AMG-induced liver injury is more
commonly cholestatic rather than hepatocellular. The most likely explanation for these results is
that, although this functional group is readily oxidized to reactive metabolites in the liver, the
liver is well equipped to deal with reactive metabolites. For example, in the case of an animal
model of nevirapine-induced skin rash, there was more covalent binding in the liver than in the
skin, but there were many more significant changes in gene expression in the skin (>400,
unpublished observation, manuscript in preparation). It is likely that what makes the aromatic
amine functional group a structural alert is that it can also be oxidized by myeloperoxidase,
which is present in antigen presenting cells. The formation of reactive metabolites by antigen
presenting cells has been shown to lead to their activation [49], and this is likely to be a strong
stimulus for the induction of an immune response. Specifically, dendritic cells treated with SMX
and SMX-NO were found to increase expression of the co-stimulatory molecule CD40 [209].
Similarly, keratinocytes were found to up-regulate their expression of heat shock protein 70,
which could be a potential danger signal, upon treatment with the hydroxylamine metabolite of
SMX [210].
Even in the case of immune-mediated liver injury, it appears that activation of the
immune system outside of the liver is necessary in order to cause an immune response in the
liver that results in injury [211]. It is notable that all three of these drugs are associated with a
79
relatively high incidence of idiosyncratic agranulocytosis; this presumably involves oxidation of
the drug to reactive metabolites by myeloperoxidase, the major oxidizing enzyme in neutrophils.
This clinical picture is most consistent with activation of the immune system outside of the liver
with the immune response sometimes extending to the liver. Therefore, the present focus on the
liver by the pharmaceutical industry may miss signals that predict IDR risk, especially for drugs
that also cause IDRs outside of the liver. In particular, bioactivation of easily oxidized drugs,
such as those containing aromatic amines, by myeloperoxidase in neutrophils and antigen-
presenting cells may play an important role in the induction of many types of IDRs.
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CHAPTER 3
HEPATIC EFFECTS OF AMINOGLUTETHIMIDE: A MODEL
AROMATIC AMINE
Winnie Ng, Imir G. Metushi, and Jack Uetrecht
(Submitted to the Journal of Immunotoxicology for publication, 2013).
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3.1 ABSTRACT
Primary aromatic amine drugs are structural alerts in drug development because of their
association with a high incidence of idiosyncratic drug reactions (IDRs). Although the
mechanisms of IDRs remain unclear, there is a large amount of evidence to suggest that most are
immune-mediated. If biomarkers could be found that predict IDR risk, it would have a major
impact on drug development. Previous attempts to do this through screening of hepatic gene
expression profiles in rodents treated with aromatic amine drugs found limited changes. Of the
drugs studied, aminoglutethimide (AMG) induced the most changes, and this led to a more
comprehensive study of its effects on the liver. Brown Norway rats treated with AMG for up to
14 days showed hepatocyte hypertrophy and hyperplasia, and only a transient elevation of
glutamate dehydrogenase was observed. The transient nature of the liver injury is similar to the
adaptation observed in many patients treated with a drug that can cause idiosyncratic liver injury.
Pathway-specific PCR arrays found few AMG-induced gene changes associated with an immune
response, and of these changes, the majority were involved with innate immunity such as Tlr2,
Ticam2, CD14, and C3. AMG treatment did lead to significant changes in the apoptosis and
mitochondrial panel of genes. Although these changes may be precursors to IDRs, it is difficult
to discern their mechanistic importance because AMG-treated rats did not exhibit any obvious
liver pathology. Moreover, tolerogenic mechanisms could prevent liver injury, and that may be
why most people do not develop IDRs.
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3.2 ABBREVIATIONS
ALT, alanine aminotransferases;
AMG, aminoglutethimide;
DAB, 3’3 diaminobenzidine
GLDH, glutamate dehydrogenase;
H&E, hematoxylin and eosin
HLA, human leukocyte antigen
HRP, horseradish peroxidase
IDRs, idiosyncratic drug reactions;
PBS, phosphate buffered saline
TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling
83
3.3 INTRODUCTION
The primary aromatic amine functional group is a notorious structural alert in drug
development because drugs with this group are associated with a high incidence of idiosyncratic
drug reactions [IDRs; 94]. Despite the potential for toxicity, many aromatic amine drugs remain
pharmacologically very effective; therefore, understanding the mechanism of IDRs or
determining biomarkers associated with these reactions could provide a tremendous benefit to
drug development. Although the types of IDRs vary from drug to drug, and one drug can cause
more than one type of IDR, the toxicity of aromatic amine drugs is presumably due to their
ability to form common reactive metabolites that could potentially induce cellular damage and
lead to an adverse drug response [212].
Immune involvement has been implicated in the mechanisms of IDRs [13]; however, our
current understanding of IDRs is still far from complete. IDRs pose significant issues to drug
development because of their lack of predictability, and the inherent risks posed to patients once
the drug is out in the market. Ideally, the ability of drug candidates to induce IDRs would be
detected early in preclinical studies to decrease the enormous financial risk of drug attrition and
to ensure drug safety to patients. However, in reality, IDRs are much more difficult to predict,
largely due to our lack of understanding of the underlying mechanisms, and as a result, although
there have been strong human leukocyte antigen (HLA) gene associations; there are currently no
reliable tests to predict IDR risk. In addition, development of valid animal models of IDRs with
clinical characteristics that closely mimic what occurs in humans has proven to be a daunting
task despite many attempts at various immune interventions [61]. Analyzing changes in gene
expression of drugs that induce IDRs has been proposed as a method of determining biomarkers
84
to predict drugs predisposed to these reactions, and this has had some success in distinguishing
model hepatotoxins in vitro [213]. However, it is difficult to determine the relevancy of these
gene changes and how well these signatures would apply to predicting IDR risk for new drug
candidates.
In an attempt to understand the mechanisms underlying aromatic amine-induced IDRs,
microarray studies were previously performed in both mice and rats to look for common hepatic
gene changes induced acutely by aromatic amine drugs that could provide mechanistic clues or
act as potential biomarkers to identify drugs predisposed to causing IDRs. The rationale for
choosing early time points was that it is usually early cell injury, which may occur in most
patients and animals, that presumably stimulates an immune response and later presents as an
IDR in rare patients. It was also anticipated that the predictive value of these results would have
significant implications for the assessment of preclinical drug safety. However, in mice treated
with sulfamethoxazole, few gene changes were observed in the liver except for down-regulation
of the expression of a few genes [37]. Yet, it was uncertain whether these results were species- or
drug-specific. Additional testing in rats with three different aromatic amine drugs - including
sulfamethoxazole, dapsone, and aminoglutethimide - found few common gene changes, and
again the majority were down-regulated [212]. Among these drugs, aminoglutethimide induced
the most significant gene changes, and several were associated with the Keap-1-Nrf2-ARE
pathway, which suggests that some cellular stress occurred. This is consistent with gene changes
observed with other drugs associated with IDRs such as carbamazepine, phenytoin, tienilic acid,
and D-penicillamine [37, 204, 205]. Given that the liver is the conventional target of drug
metabolism studies because of its predominant function in drug metabolism, and aromatic
amines are readily oxidized to reactive metabolites, the paucity of changes in gene expression
85
was unexpected. Furthermore, given the postulates of the danger hypothesis, the liver would be
expected to experience more adverse effects because it is assumed to be the site where most
reactive metabolites would form and induce damage.
To ensure that any aromatic amine-induced changes in the liver were not overlooked
because of experimental limitations, a more comprehensive study combining physiological tests
with gene arrays at successive time-points was performed in the liver of rats to confirm whether
aromatic amine drugs affect the liver as a target organ. The aromatase inhibitor
aminoglutethimide (AMG) was used in these studies as a prototypical aromatic amine drug
because it induced the most changes in the previous microarray study and was more likely to
result in observable effects. AMG is primarily indicated for breast and prostate cancer treatment
and is associated with a variety of IDRs including cholestasis, agranulocytosis, lupus, and skin
rashes [214]. AMG is associated with a higher incidence of IDRs than sulfamethoxazole and
dapsone, the other two aromatic amines that were studied previously, and because of this it is not
currently used clinically. This higher incidence of IDRs with AMG may be a result of a higher
electron density which would facilitate the oxidation of the aromatic amine. Thus, the effect of
AMG on the liver was tested including pathway-specific gene expression to gain a more detailed
understanding of the mechanisms of aromatic amine-induced IDRs.
86
3.4 MATERIALS AND METHODS
3.4.1 Reagents
AMG was purchased from Toronto Research Chemical (North York, ON). Epitope
Unmasking Solution and Antibody Dilution Buffer were acquired from ProHisto (Columbia,
SC). 3’3 diaminobenzidine (DAB) was obtained from Vector Labs (Burlingame, CA). Mouse
anti-rat Ki-67 (clone: MIB-5), rabbit anti-mouse biotinylated IgG, and streptavidin-horseradish
peroxidase (HRP) was purchased from Dako (Burlington, ON). Mouse anti-rat CD8 (clone: OX-
8) and mouse anti-rat CD68 (clone: ED1) were obtained from Abcam (Cambridge, MA). Alanine
Aminotransferase (ALT) Liquid Stable Reagent was purchased from Thermo Scientific
(Middletown, VA). Glutamate dehydrogenase (GLDH) Assay Kit was from Randox Laboratories
Ltd. (Crumlin, UK). RNAlater RNA Stabilization Solution, Trizol Reagent, and RNase free
water were from Life Technologies (Burlington, ON). RNeasy Mini Kit was purchased from
Qiagen (Mississauga, ON). RT2 Profiler PCR Arrays and RT
2 First Strand Kit were obtained
from SABiosciences (Valencia, CA) and were supplied by Roche.
3.4.2 Animals
Male Brown Norway rats (≈ 9-wk-old, weighing 200-250 g) were purchased from
Charles River (Montreal, QC). Rats were housed at the Department of Comparative Medicine
(University of Toronto) under standard care conditions (i.e., doubly-housed, 12:12 hr light:dark
cycle, and 22°C room temperature). Standard rat chow and water were provided ad libitum. Rats
were given 1 wk to acclimatize prior to the start of experiments. The protocol was approved by
the University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee.
87
3.4.3 Treatment
AMG were suspended in 0.5% methylcellulose and administered by oral gavage at a dose
of 125 mg/kg/day. This dosage is an increase from that which was used in our previous study
[212] to ensure that maximal changes were induced, while maintaining the blood levels of AMG
within the range found in humans taking AMG therapeutically [≈ 4.7-32.4 µg/ml; 215]. Control
rats were given 0.5% methylcellulose vehicle only. Blood levels of AMG in rats were measured
in our previous studies and are within the range of AMG blood levels found in humans [216].
Control animals were given methylcellulose as a vehicle control. Four rats were tested for each
treatment group unless otherwise stated. Body weight and general health of rats were monitored
daily. At various time points (Day 0, 3, 7, and 14), blood samples isolated for use in the
protocols below. In addition, at Day 1, 7, and 14, subsets of rats were euthanized by CO2
asphyxiation and liver (as well as a final blood) samples isolated for use in the protocols below.
3.4.4 Histology
At necropsy, livers were excised and immediately immersed in 10% formalin solution.
H&E-stained and unstained paraffin-embedded slides were prepared at the Division of Pathology
at the Hospital for Sick Children (Toronto, ON). Immunohistochemistry was carried out using
standard procedures. Briefly, slides were de-paraffinized with xylene and rehydrated with serial
dilutions of ethanol. Heat-induced antigen retrieval was performed for 20 min (Ki-67 staining) or
incubated at 71°C overnight (for CD8 and CD68 staining) in Epitope Unmasking Solution.
Endogenous peroxidases were blocked with 3% hydrogen peroxide in phosphate-buffered saline
(PBS, pH 7.4) for 10 min. Washes were then performed using PBS-0.05% Tween-20 solution
and antibodies were diluted in Antibody Dilution Buffer. Slides were incubated with primary
antibodies against Ki-67, CD68, and CD8 at room temperature. For mouse anti-rat Ki-67
88
antibody, the dilution used was 1:25 and incubation time was 30 min. For mouse anti-rat CD68
and anti-rat CD8 antibodies, the dilution used was 1:100 and incubation time was 90 min. Rabbit
anti-mouse biotinylated IgG was used as a secondary antibody (1:250 dilution) and incubated for
30-40 min at room temperature. Streptavidin-HRP (1:500) was then added and the slide
incubated 15 min after which the signal was developed with DAB solution; haematoxylin was
used as counterstain. Slides were dehydrated with ethanol and xylene before coverslips were
attached with Cytoseal. Histological images were taken at the Microscopy Imaging Lab (Faculty
of Medicine, University of Toronto) on a Zeiss fluorescence microscope with deconvolution.
Immunohistochemical grading was performed by counting the number of cells/field of view; at
least two slices of tissue (3-6 mm2) were mounted on glass slides. Five areas from each slice
were manually counted under the microscope. All slides were read in a blinded manner.
3.4.5 Liver Enzymes
Serial samples of blood were taken from the tail vein of rats to determine liver enzyme
activities in the serum. Blood was processed within 1 hr of collection, and ALT and GLDH
activities were determined using ALT Liquid Stable Reagent and a GLDH Assay Kit,
respectively, according to manufacturer protocols. For GLDH, minor modifications were made
to the protocol as previously described [217], wherein 200 µL of Reagent 1 was premixed with 8
µL of Reagent 2, and 200 µL of this mixture was loaded into each well of a 96-well plate before
serum samples were added. All reagents were reconstituted per manufacturer specifications and
absorbance values at 340 and 405 nm monitored on a SpectraMax Plus384 (Molecular Devices,
Sunnyvale, CA) plate reader for at least 5 min at 25°C.
89
3.4.6 RNA Extraction
At necropsy, the livers were removed, rinsed in PBS, and immediately immersed in
RNAlater RNA Stabilization Solution to preserve RNA for extraction. Liver samples were then
homogenized with a IKA Ultra-Turrax T25 S1 rotor stator homogenizer (Janke and Kunel,
Staufen, Germany), and RNA was extracted using Trizol Reagent as per manufacturer protocols.
An additional RNA clean-up step was performed using an RNeasy Mini Kit. Ultimately,
extracted RNA was eluted in 40 µl RNase-free water and stored at -80°C. The quality and purity
of the extracted RNA was measured using UV spectrophotometry based on the A260/A280 ratio.
3.4.7 PCR Arrays
Extracted RNA was converted to cDNA using the RT2 First Strand Kit with 400 ng RNA
per sample, following manufacturer protocols. Six different panels of genes were analyzed using
the RT2 Profiler PCR Arrays, including: apoptosis (PARN-012), chemokines and receptors
(PARN-022), common cytokines (PARN-021), mitochondria (PARN-087), toll-like receptor
signaling (PARN-018), and Th17 and autoimmunity (PARN-073). However, limitations on the
number of plates prevented analysis of every gene panel at all time points (1, 7, 14 days);
specifically, only Day 14 data were collected for common cytokine, toll-like receptor signaling,
and Th17 response pathways. Array studies were performed according to manufacturer protocols
on 384-well plates that held four samples simultaneously. For each sample, cDNA converted
from 400 ng total RNA was loaded on the plate, and each plate had samples staggered between
AMG-treated and control animals. Arrays were run on an ABI7900HT real-time PCR instrument
(Applied Biosystems, Grand Island, NY) according to the SABiosciences protocols. A threshold
cycle value of 0.2 was assigned to each array plate for data normalization. Data was analyzed
using RT² Profiler PCR Array Data Analysis Template Version 3.2 (SABiosciences); lactate
90
dehydrogenase A and ribosomal protein P1 were used as housekeeping genes. Pathway analyses
were performed using Gene Network Central Pro software (SABiosciences).
3.4.8 Statistical Analysis
Data were analyzed with GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA)
using a Mann-Whitney U test or two-way analysis of variance (ANOVA), depending on the
constraints of the data being analyzed. PCR array data were analyzed with RT² Profiler PCR
Array Data Analysis Template v3.2 (SABiosciences) using Student’s t-test.
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3.5 RESULTS
3.5.1 Effects of AMG on the Liver
Rats tolerated the 125 mg/kg/day AMG dose well for a period of up to 14 days without
significant changes in body weight or evidence of distress. Histological analysis of the liver of
AMG-treated rats found enlarged hepatocytes in Zone 3 as compared to control rats (Figure
3.1A, B). In the representative H&E staining of the liver, the noticeable presence of red blood
cells in sinusoids of the control is likely an artifact from lack of perfusion prior to fixation rather
than a treatment effect. An increase in the liver weight of AMG-treated rats was also found, and
this was most significant after 7 days of treatment (Figure 3.1C). Although no data on liver
weight was collected at the 14-day endpoint, the histological findings suggest that the increase in
liver weight with AMG was sustained. In addition, there appeared to be a slight increase in cell
proliferation, as determined by the proliferation marker Ki-67, in the liver of AMG rats after 7
days compared to the untreated group (Figure 3.2); however, this did not reach statistical
significance.
Examination of H&E-stained slides did not reveal any evidence of liver injury (neither
hepatocellular nor cholestatic) in AMG-treated rats after 14 days, nor were immune cell
infiltrates detected in the liver. Staining for specific immune cells such as CD8 T-lymphocytes
and CD68 macrophages in the liver found no differences using immunohistological grading
between AMG-treated and control animals (data not shown).
In serum, few changes were observed in enzymes associated with liver injury in AMG-
treated rats. A subtle but transient increase in ALT was observed with AMG treatment at Day 3
(Figure 3.3A). A significant increase in GLDH was also observed with AMG at Day 3; however,
92
these changes were transient and returned back to baseline/control levels at Day 7 (Figure 3.3B).
No changes were observed in ALT or GLDH at Day 14 of AMG treatment (data not shown).
Figure 3.1. Histological changes in the liver of AMG-treated rats. Representative H&E
staining of (A) control and (B) AMG-treated (125 mg/kg/day, 14 days) rat. Evidence of Zone 3
hepatocyte hypertrophy was found in rats treated with AMG as compared to controls. 20X
magnification. (C) Rats treated with AMG had an increase in liver weight that was most
significant on Day 7 of treatment. Values shown are mean (± SEM) for each group (n = 4). *p <
0.05 compared to control (Mann Whitney U-test).
93
Figure 3.2. Comparison of hepatocyte proliferation in control and AMG-treated animals.
Representative Ki-67 staining in tissue from (A) control and (B) AMG-treated (125 mg/kg/day,
14 days) rats. 20X magnification. (C) A possible increase in Ki-67 was observed at Day 7 of
AMG treatment. Though some evidence of hepatocyte proliferation was found in the AMG-
treated rats, the main effect was hypertrophy rather than hyperplasia. Open bars = control rats;
solid bars = AMG-treated rats. Values shown are mean (± SEM) number of Ki-67-positively-
stained nuclei per 10X field of view (n=4). No results were statistically significant (two-way
ANOVA).
94
Figure 3.3. AMG-induced changes in serum levels of liver enzymes. (A) ALT and (B) GLDH
in rats treated with AMG (125 mg/kg/day) compared to control rat values. Open bars = control
group; solid bars = AMG-treated group. Values shown are mean (± SEM) (n=4). **p < 0.01 as
compared to control (two-way ANOVA).
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3.5.2 AMG-Induced Hepatic Gene Changes
Hepatic gene expression was determined at various time points of AMG treatment using
PCR arrays that were pathway-specific. The greatest number of AMG-induced differential
changes in gene expression was primarily found within the apoptotic panel of genes, but both
pro- and anti-apoptotic regulatory pathways were induced (Table 3.1). Although the majority of
changed genes were involved with the induction of apoptosis, significant up-regulation of anti-
apoptotic genes was also quite notable including Aven, Bag1, Bcl2l1, Birc3, and Nol3. At earlier
time points, a general down-regulation of apoptotic genes was observed, whereas up-regulation
of genes was mainly observed at the latter 14-day endpoint. A pathway analysis of the up-
regulated genes induced by AMG did not reveal any particular dominant signaling pathway,
although most genes were associated with Nfkb1 (Figure 3.4). ). In terms of downstream effects
of the gene changes, TUNEL staining for apoptotic cells in the liver did not show any differences
between AMG-treated and control rats (data not shown). This may be due to the activation of
genes in opposing apoptosis pathways, which may act to modulate apoptotic events.
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Table 3.1. Hepatic changes in gene expression induced by AMG in the apoptosis pathway.
Gene
Symbol
Gene Name Day 1 Day 7 Day 14
Aven Apoptosis, caspase activation inhibitor -1.16 -1.05 2.39*
Bad BCL2-associated agonist of cell death -2.01* -1.29 2.53
Bag1 BCL2-associated athanogene -1.10 1.13 2.43*
Bax Bcl2-associated X protein -1.25 1.03 2.28*
Bcl2l1 Bcl2-like 1 -2.95 -2.07 2.39*
Bcl2l2 Bcl2-like 2 -2.24* -1.09 1.14
Bid BH3 interacting domain death agonist -1.08 1.21 2.65*
Hrk Harakiri, BCL2 interacting protein (contains only BH3 domain) -3.26* -1.71 -1.27
Birc3 Baculoviral IAP repeat-containing 3 -2.03 1.28 3.42*
Birc5 Baculoviral IAP repeat-containing 5 1.12 2.35* -1.52
Casp1 Caspase 1 -1.13 -1.22 -3.71*
Casp7 Caspase 7 1.31 1.88* 6.51*
Casp9 Caspase 9, apoptosis-related cysteine peptidase -1.94 -1.26 2.67*
Cidea Cell death-inducing DFFA-like effector a 1.99 1.90* 5.85*
Dapk1 Death associated protein kinase 1 -3.46* -3.99 2.68
Dffa DNA fragmentation factor, alpha subunit -2.13* 1.16 1.37
Fadd Fas (TNFRSF6)-associated via death domain 1.03 1.09 2.62*
Ltbr Lymphotoxin beta receptor (TNFR superfamily, member 3) -1.06 1.12 5.55*
Nfkb1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 -2.17 -1.44 2.21*
Nol3 Nucleolar protein 3 (apoptosis repressor with CARD domain) 7.72* 11.61* 7.18*
Prlr Prolactin receptor -3.81* 1.91 3.15
Prok2 Prokineticin 2 1.11 3.36* -1.33
Tnfrsf1a Tumor necrosis factor receptor superfamily, member 1a 1.40 1.49 2.89*
Cd40 CD40 molecule, TNF receptor superfamily member 5 -1.13 1.08 -2.02*
Fas Fas (TNF receptor superfamily, member 6) -1.73 2.32* -1.07
Tnfsf10 Tumor necrosis factor (ligand) superfamily, member 10 -3.09* -1.12 1.10
Tnfsf12 Tumor necrosis factor ligand superfamily member 12 -1.58 -1.37 4.50*
Tradd TNFRSF1A-associated via death domain -1.29 -1.28 2.61*
Values are expressed as fold-change in AMG-treated compared to control rats (n = 4). *p < 0.05
compared to control.
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Figure 3.4. Pathway analysis of significantly changed hepatic genes in the apoptotic
pathway in AMG-treated rats. Only genes with changes >2-fold (up-regulation) after 14 days
of AMG treatment were included in analysis for tends in the apoptotic panel of genes. Arrows
indicate known associations and do not represent how genes are regulated by AMG. Green = up-
regulation; red = down-regulation; yellow = physical interaction. Analysis was performed using
Gene Network Central Pro software (SABiosciences).
98
Many mitochondria-associated genes were also significantly changed upon AMG
treatment (Table 3.2). Again, down-regulation of genes was primarily observed at the earlier
time points whereas up-regulation was observed after 14 days of AMG treatment. Of these
AMG-induced genes, many were associated with mitochondrial transportation such as the solute
carrier family 25 (Slc25) and translocase (Tomm34, Tomm40, Tspo, Timm17b) genes.
Mitochondria-associated apoptotic genes such as Aifm2, Bbc3, and Bid were also significantly
up-regulated with AMG treatment; the function of these genes suggested induction of apoptosis
via the intrinsic mitochondrial pathway.
In terms of changes in immune-associated genes, AMG induced fewer significant
changes than that observed for the other two pathways, and most of these changes were more
suggestive of changes in innate immunity. In the chemokine and receptor pathways there were
significant increases in C3, Ccl19, and Nfkb1 (Table 3.3); in contrast, a sustained down-
regulation was observed for Cxcl13 upon AMG treatment. Increases in the toll-like receptor Tlr2
and the adaptor molecule Ticam2 genes were also observed with an increase in Cd14 and
changes in the IL-1 pathway (Il1f5, IL1rn) upon AMG treatment (Table 3.4). Up-regulation of
Cd4 was the predominant change associated with adaptive immunity.
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Table 3.2. Mitochondrial gene expression changes in the liver of AMG-treated rats.
Gene
Symbol Gene Name 7 Days 14 Days
Aifm2 Apoptosis-inducing factor, mitochondrion-associated 2 1.52 2.54*
Bbc3 Bcl-2 binding component 3 2.67* 2.80*
Bid BH3 interacting domain death agonist 2.37* 2.00
LOC691853
Similar to COX10 homolog, cytochrome c oxidase assembly protein,
heme A: farnesyltransferase 1.53 -2.26*
Mfn1 Mitofusin 1 -1.63 2.21*
Mipep Mitochondrial intermediate peptidase 1.62* 2.05*
Msto1 Misato homolog 1 (Drosophila) 1.35 3.30*
Ppargc1a Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha -3.97* 2.51
Rnf135 Ring finger protein 135 1.17 2.38*
Sfn Stratifin 2.98* -1.44
Slc25a12 Solute carrier family 25 (mitochondrial carrier, Aralar), member 12 -1.26 2.64*
Slc25a13 Solute carrier family 25, member 13 (citrin) -1.23 2.20*
Slc25a19
Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier),
member 19 -1.13 3.61*
Slc25a23
Solute carrier family 25 (mitochondrial carrier; phosphate carrier),
member 23 1.13 2.91*
Slc25a25
Solute carrier family 25 (mitochondrial carrier, phosphate carrier),
member 25 -2.23* 2.08*
Slc25a30 Solute carrier family 25, member 30 -3.79* -1.29
Slc25a5
Solute carrier family 25 (mitochondrial carrier: adenine nucleotide
translocator) member 5 -1.13 2.00*
Tomm34 Translocase of outer mitochondrial membrane 34 1.33 2.06*
Tomm40 Translocase of outer mitochondrial membrane 40 homolog (yeast) -1.02 2.69*
Tspo Translocator protein 27.30 4.95*
Timm17b Translocase of inner mitochondrial membrane 17 homolog B (yeast) -1.31 2.43*
Values are expressed as fold-change in AMG-treated compared to control rats (n = 4). *p < 0.05
compared to control.
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Table 3.3. AMG-induced hepatic gene changes in chemokines and receptors.
Gene
Symbol
Gene Name Day 1 Day 7 Day 14
C3 Complement component 3 -1.08 1.90* 2.16*
Ccbp2 Chemokine binding protein 2 -3.54* -2.03 1.62
Ccl19 Chemokine (C-C motif) ligand 19 1.64* 2.24* 1.30
Ccl3 Chemokine (C-C motif) ligand 3 -4.53* 1.09 1.25
Ccr5 Chemokine (C-C motif) receptor 5 -5.08* -1.13 -2.03
Cxcr7 Chemokine (C-X-C motif) receptor 7 -1.75 -4.66* 3.46*
Cmtm3 CKLF-like MARVEL transmembrane domain containing 3 -2.47* -1.40 1.03
Cx3cl1 Chemokine (C-X3-C motif) ligand 1 1.19 2.06* -1.42
Ppbp Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) -2.11* 1.49 2.26*
Tymp Thymidine phosphorylase -3.87* -2.80* 1.64
Cxcl13 Chemokine (C-X-C motif) ligand 13 -5.05* -2.21 -5.61*
Mmp2 Matrix metallopeptidase 2 -2.11* -1.67 -1.99
Mmp7 Matrix metallopeptidase 7 1.41 2.32* -1.80
Nfkb1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 -2.21* 1.11 2.79*
Tlr2 Toll-like receptor 2 -3.76* -1.43 1.30
Tnf Tumor necrosis factor (TNF superfamily, member 2) -2.41* -1.05 1.08
Tnfrsf1a Tumor necrosis factor receptor superfamily, member 1a -1.16 1.11 2.57*
Trem1 Triggering receptor expressed on myeloid cells 1 1.32 2.42* -1.26
Values are expressed as fold-change in AMG-treated compared to control rats (n = 4). *p < 0.05
compared to control.
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Table 3.4. Hepatic gene changes induced by AMG in various immune pathways after 14
days of treatment.
Gene
Symbol
Gene Name p-value Fold-
Change
Common Cytokines Pathway
Bmp1 Bone morphogenetic protein 1 0.019 2.48
Il16 Interleukin 16 0.035 -2.89
Il1f5 Interleukin 1 family, member 5 (delta) 0.001 -2.38
Il1rn Interleukin 1 receptor antagonist 0.004 2.49
Ltb Lymphotoxin beta (TNF superfamily, member 3) 0.001 3.28
Tnfsf12 Tumor necrosis factor ligand superfamily member 12 0.019 2.83
Toll-Like Receptor Signalling Pathway
Cd14 CD14 molecule 0.005 6.48
Hmgb1 High mobility group box 1 0.032 -3.27
Irak1 Interleukin-1 receptor-associated kinase 1 0.008 3.23
Map2k3 Mitogen activated protein kinase kinase 3 0.047 2.86
Map4k4 Mitogen-activated protein kinase kinase kinase kinase 4 0.028 4.26
Nfkbil1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 0.017 -1.75
Ppara Peroxisome proliferator activated receptor alpha 0.006 3.55
Sarm1 Sterile alpha and TIR motif containing 1 0.032 5.43
Ticam2 Toll-like receptor adaptor molecule 2 0.022 4.14
Tlr2 Toll-like receptor 2 0.035 2.28
Tnfrsf1a Tumor necrosis factor receptor superfamily, member 1a 0.006 3.27
Th17 Response Pathway
Cd4 Cd4 molecule 0.015 4.39
Icam1 Intercellular adhesion molecule 1 0.005 3.04
Il17rb Interleukin 17 receptor B 0.009 5.57
Il18 Interleukin 18 0.008 -2.91
Nfatc2 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 0.004 3.41
Stat3 Signal transducer and activator of transcription 3 0.004 2.73
Stat5a Signal transducer and activator of transcription 5A 0.004 2.40
Stat6 Signal transducer and activator of transcription 6 0.016 3.17
Values are expressed as fold-change in AMG-treated compared to control rats (n = 4). *p < 0.05
compared to control.
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3.6 DISCUSSION
The current findings are consistent with our previous experiments in that aromatic amine
drugs do not appear to cause significant liver injury even though AMG treatment resulted in
more changes in hepatic mRNA expression that are consistent with liver injury than the other
aromatic amines that were tested. In retrospect, although AMG is associated with a very high
incidence of IDRs including cholestatic liver injury, the non-steroidal anti-inflammatory,
bromfenac, is the only primary aromatic amine drug to be withdrawn from the market because of
idiosyncratic hepatocellular liver injury [218]. Sulfamethoxazole often causes idiosyncratic liver
injury, but it is usually part of a more generalized hypersensitivity reaction than liver-specific
toxicity. Furthermore, because the liver is so extensively exposed to xenobiotics that can be
oxidized to reactive metabolites, the primary response of the liver to drug-modified proteins may
be tolerance [211]; thus, liver toxicity caused by aromatic amines may be more a consequence of
a generalized hypersensitivity reaction rather than direct liver injury.
It has been proposed that primary activation of T-lymphocytes in the liver leads to
immune suppression through CD8+ T-lymphocyte apoptosis - the liver has been referred to as a
T-lymphocyte graveyard [219] - whereas primary activation outside of the liver may lead to a
robust immune response in the liver [211]. This may be an appropriate response for an organ
with high xenobiotic exposure and reactive metabolite formation. Although inflammatory
infiltrates were not observed in the liver histologically, the transient increase in GLDH, which is
a marker of liver injury, may be a sign of mild injury followed by adaptation events. Thus, if
these AMG-induced changes in the liver are immune-mediated, then the response to AMG in the
liver in the majority of patients may be immune tolerance.
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Approximately 60% of AMG-treated patients experience elevations in γ-
glutamyltransferse [208], which may be an indicator of cholestatic liver injury. The more serious
form of idiosyncratic liver injury is hepatocellular rather than cholestatic [13]. Although
enzymes of cholestatic liver injury were not measured, there was no histological evidence of
cholestatic injury or bile accumulation in the AMG-treated rats. The hypertrophy and hyperplasia
of hepatocytes observed in the AMG-treated rats is consistent with the ability of AMG to induce
cytochrome P450 [215], and this is presumably the reason that the half-life of AMG decreases
with sustained AMG therapy [112].
The largest number of AMG-induced gene changes were within the apoptotic panel of
genes, and this suggests that apoptosis may be involved in AMG-induced IDRs. Apoptosis is a
dynamic process; as cells undergo apoptotic cell death they are cleared by phagocytic cells, and
if this process is not overwhelmed then apoptotic changes may not be observed. However, in the
context of IDRs, excessive apoptotic cell death that cannot be efficiently cleared by phagocytes
may lead to secondary necrosis and the release of danger signals that activate the immune system
[51].
Although at later time points, AMG up-regulated more pro-apoptotic than anti-apoptotic
genes, it was difficult to discern which of the pathways was more dominant. Interestingly, a
pathways analysis found many associations with Nfkb1, which is a part of the nuclear factor-
kappaB (NF-κB) family of transcription factors. NF-κB is involved in immune responses and
inflammation, which ranges from involvement in haematopoiesis and organogenesis to
transcription of cytokines and chemokines [220]. Furthermore, NF-κB can also regulate
programmed cell death, and it has a predominant pro-survival effect because of its ability to
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transcribe anti-apoptotic genes such as inhibitor of apoptosis proteins and anti-apoptotic Bcl-2
proteins [221]. This suggests that these changes may modulate the potential effects of the pro-
apoptotic gene expression. Consistent with this, TUNEL staining found no difference between
AMG-treated rats and control rats in the number of apoptotic cells in the liver. Since NF-κB is
regulated in the cytoplasm by the inhibitory protein, IκB, there are limitations in the
interpretation of changes in gene expression because the effect of NF-κB is dependent on
cytoplasmic activation [221]. There is also evidence to suggest that NF-B may play a role in
immune tolerance since lack of NF-B1 expression in dendritic cells activates CD8+ T-
lymphocytes [222], which could be an additional factor contributing to the tolerogenic response
in the liver. However, based on our results, it seems that AMG exposure in the liver induces
modest gene expression in apoptotic pathways that is balanced by anti-apoptotic gene changes
and prevents the induction of apoptosis.
Mitochondrial injury has been proposed to contribute to the mechanisms of IDRs as a
danger signal [13]. Given the importance of mitochondria in maintaining cellular energy and
regulating programmed cell death, it is not surprising that AMG also induced a significant
number of mitochondrial gene changes. Although the majority of mitochondrial gene changes
are involved with solute carriers and transporters, all of the apoptotic genes that were up-
regulated by AMG positively regulate the intrinsic apoptotic pathway, and these changes may
compliment the observed changes in the apoptosis pathways.
AMG induced fewer changes in immune-related genes at these early time points, and the
majority of these changes involved genes associated with the innate immune response. Although
they provide some evidence for the up-regulation of immune signaling pathways, the only
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conclusive evidence for activation of an adaptive response was an increase in Cd4 gene
expression. However, a histological increase in lymphocytes was not observed in the liver with
AMG. Thus, activation of the innate immune response upon AMG treatment may be sufficient to
deal with the drug insult without activating the adaptive immune response in the majority of
patients. Moreover, the innate immune system has been proposed to be protective, such as in
cases of hepatic inflammation induced by acetaminophen [74].
Similar to previous gene array studies of aromatic amine drugs, successive time point
testing found that, at least for aromatic amine drugs, the majority of genes are down-regulated at
early time points. The true implications for these findings are still unknown; however, there may
be some compensatory mechanisms in which a threshold needs to be crossed before gene
expression is elicited. As seen with these studies, the mere presence of changes in gene
expression does not necessarily correlate with pathology. Furthermore, we did not investigate
downstream protein expression or signaling pathways because our main goal was only to detect
early biomarkers to predict drug risk because, according to the danger hypothesis, it is early
injury of the target cells, in this case the hepatocytes, that would determine the immune response.
Overall, the implications of this study are that the effects of AMG on the liver are small,
and that the very high incidence of IDRs associated with this drug may originate from outside the
liver. The finding that immune cells can also bioactivate aromatic amines may be the key to their
ability to cause IDRs [49]. If this is true it also suggests that in addition to screening for reactive
metabolite formation in the liver, bioactivation by immune cells, especially macrophages, may
provide important biomarkers for detecting the potential of whether a drug candidate can cause
IDRs. Conversely, perhaps the primary response in the liver is adaptation and/or tolerance. Even
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though there was no pathological response, the changes in gene expression induced by AMG
could be mechanistic clues, and these changes may occur in most individuals, and it is only when
some additional factor is present that an IDR occurs. The most significant findings were that the
changes in the apoptosis and mitochondrial panel of genes may play a role in the mechanisms of
AMG-induced IDRs, potentially as danger signals. Further-more, because the predominant
immune changes were associated with innate immunity, it is quite possible that innate immune
cells are able to effectively deal with the injury caused by the drug and effectively down-regulate
any potential adaptive immune response. We have performed high through-put arrays with
multiple IDR-associated drugs and found differential changes suggestive of cellular stress and an
immune response at early-time points with most. The major exceptions are the primary aromatic
amine drugs, which emphasizes the uniqueness of this chemical moiety and the need for its
study.
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CHAPTER 4
EFFECT OF AMINOGLUTETHIMIDE ON
NEUTROPHILS IN RATS: IMPLICATIONS FOR
IDIOSYNCRATIC DRUG-INDUCED BLOOD
DYSCRASIAS
Winnie Ng and Jack Uetrecht
(Reproduced with permission from: Ng, W., and Uetrecht, J. Chemical Research in Toxicology,
2013. 26: p.1272-1281. Copyright 2013 American Chemical Society. The American Chemical
Society granted permission to include my own article in this thesis).
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4.1 ABSTRACT
Aminoglutethimide (AMG) is an aromatic amine aromatase inhibitor associated with a
high incidence of idiosyncratic blood dyscrasias, especially agranulocytosis. Animal models of
idiosyncratic drug reactions (IDRs) represent essential tools to study these reactions; however,
there is currently no valid model of idiosyncratic drug-induced agranulocytosis. Although AMG
does not cause agranulocytosis in most animals or humans, drugs associated with serious IDRs
generally cause a higher incidence of mild reactions that resolve despite continued treatment.
Therefore, the effects of AMG on neutrophils and bone marrow in rats were studied to
understand the mechanisms of more serious IDRs. An increase in peripheral blood neutrophils
occurred as early as 24 h after AMG treatment with minimal changes to the total leukocyte
count. Further investigation using 5-bromo-2’-deoxyuridine (BrdU) found an increased release
of neutrophils from the bone marrow. Histologically, this corresponded to an increase in myeloid
cells in the bone marrow, which was confirmed by differential staining with CD45 and CD71.
AMG treatment stimulated an increase in colony forming unit granulocyte-macrophage and
colony forming unit granulocyte ex vivo. There was also a marked increase in the number of
activated neutrophils in the circulation expressing the extravasation marker CD62L. These
findings indicate that AMG affects neutrophil production, release, and function. Similar effects
on neutrophil kinetics in clozapine-treated rats have previously been found, and transient
neutrophilia has been observed in patients taking other drugs associated with idiosyncratic
agranulocytosis; therefore, the changes observed with AMG may be biomarkers to predict the
risk that a drug will cause agranulocytosis.
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4.2 TABLE OF CONTENTS GRAPHIC
110
4.3 ABBREVIATIONS
AMG, aminoglutethimide
APC, allophycocyanin
BrdU, 5-bromo-2’-deoxyuridine
CFUs, colony forming units
Cxcl1, chemokine (C-X-C) 1
DAPI, 4',6-diamidino-2-phenylindole dihydrochloride
FACS, fluorescence-activated cell sorting
FBS, fetal bovine serum
FITC, fluorescein isothiocyanate
G-CSF, granulocyte colony stimulating factor
GSH, glutathione
H&E, hematoxylin and eosin
IDRs, idiosyncratic drug reactions
IMDM, Isocove’s Modified Dulbecco’s Medium
MPO, myeloperoxidase
NAT, N-acetylation
PE, phycoerythrin
PI, propidium iodide
RBCs, red blood cells
TNF-α, tumor necrosis factor alpha
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4.4 INTRODUCTION
Aminoglutethimide (AMG) is a first-generation aromatase inhibitor used primarily for
the treatment of hormone-responsive breast and prostate cancers. Structurally, AMG contains a
primary aromatic amine. This chemical moiety is almost always associated with a high incidence
of idiosyncratic drug reactions (IDRs); therefore, this is a notorious structural alert for drug
development [223]. The toxicity of aromatic amines is presumably due to the ease with which
they can be oxidized to reactive electrophilic metabolites, which can covalently bind to cellular
macromolecules and potentially induce damage [212]. However, the exact mechanisms of IDRs
are still poorly understood. More importantly, it is unknown why only a subset of patients
develops these reactions whereas most do not. Animal models, with similar pathogenesis as
occurs in humans, are essential for studying the underlying mechanisms of IDRs; yet, valid
animal models of IDRs are rare and quite difficult to develop [61].
AMG is associated with a wide range of IDRs including serious skin rashes, cholestasis,
and haematological toxicities [224]. Of these reactions, the most commonly reported are blood
dyscrasias such as agranulocytosis, which is characterized by a decrease in neutrophils to below
500/µL of blood; and if sustained for a prolonged period of time, usually results in a fatal
infection. Agranulocytosis occurs in approximately 1.9% of AMG-treated patients and typically
manifests after more than 4 weeks of treatment [113]. In these patients, bone marrow aspirates
are usually hypocellular with decreased myeloid cells, which return to normal after
discontinuation of the drug. However, the WBC counts sometimes return to normal without
cessation of AMG therapy [225], which suggests the induction of tolerogenic mechanisms that
may be why most patients do not develop agranulocytosis. In fact, it is a common characteristic
that agents that can induce serious IDRs always cause a much higher incidence of mild reactions
112
that often resolve despite continued treatment. This suggests that such drugs have biological
effects that are quite common and asymptomatic even if the severe reactions are rare.
Nevertheless, data is lacking on the effects in most patients, in terms of the early events leading
up to agranulocytosis, because blood monitoring of AMG-treated patients does not usually occur
before symptoms arise.
In general, aromatic amines can be oxidized to a N-hydroxylamine metabolite by
cytochrome P450 enzymes or myeloperoxidase, which is relatively stable and un-reactive.
However, the hydroxylamine can auto-oxidize to a nitrosoamine metabolite that is electrophilic
and reactive and can covalently bind to proteins. Compared to other aromatic amine-containing
drugs, such as the more common sulfonamides, AMG has a higher electron density, which
allows it to be even more easily oxidizable (Figure 4.1). N-hydroxyl-AMG has been detected in
both humans and mice [226, 227]. Neutrophils contain high concentrations of myeloperoxidase
[228]; therefore, it is quite likely that AMG can be oxidized by this cell type and lead to cell
damage. Alternatively, myeloperoxidase protein free radicals have been detected in vitro upon
incubation with AMG [177], which further suggests a role for oxidation by neutrophils in the
pathogenesis of agranulocytosis.
To date very few studies, if any, have focused on the effect of AMG on neutrophils, and a
true animal model of AMG-induced agranulocytosis has not yet been established. Leucopenia
was observed in both ICI-derived and B6C3F1 hybrid mice as early as after 2 weeks of AMG
treatment [174, 229]; however, these mouse strains are uncommon, and this model was not
reproducible in CD-1 mice [175]. Given our negative results in mice and our previous experience
113
with Brown Norway rats, we investigated the effects of AMG on neutrophils and bone marrow in
this rat strain.
Figure 4.1. Structure of AMG and the formation of its reactive metabolites. AMG can be
detoxified through N-acetylation (NAT). Alternatively, AMG can be oxidized by cytochrome
P450 (P450) or myeloperoxidase (MPO) to a hydroxylamine, which can spontaneously form a
highly electrophilic and reactive nitrosoamine that can covalently bind to protein thiols. The
nitrosoamine can also be reduced back to the parent drug by ascorbate or glutathione (GSH) and
redox cycle to cause oxidative stress.
114
4.5 MATERIALS AND METHODS
4.5.1 Chemicals and Reagents
AMG was purchased from Toronto Research Chemicals (North York, ON). Phenacetin,
dextran-500, BrdU, and 10% neutral buffered formalin were obtained from Sigma (Oakville,
ON). Heat-inactivated fetal bovine serum (FBS), Iscove’s Modified Dulbecco’s Medium
(IMDM), and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Life
Technologies (Burlington, ON).
4.5.2 Antibodies
Anti-rat granulocyte phycoerythrin (PE; clone: RP-1), 7-amino-actinomycin D (7-AAD),
anti-BrdU fluorescein isothiocyanate (FITC), annexin V FITC, propidium iodide (PI), anti-rat
CD18 FITC (clone: WT.3), and anti-rat CD11b allophycocyanin (APC; clone: WT.5) were
acquired from BD Biosciences (San Jose, CA). Anti-rat CD71 FITC (clone: OX-26), and anti-rat
CD62L FITC (clone: OX-85) were purchased from Cedarlane (Burlington, ON). Anti-rat CD45
PE (clone: OX-1) was obtained from BioLegend (San Diego, CA).
4.5.3 Animals
Male Brown Norway rats (176-200 g) were purchased from Charles River (Montreal,
QC). Rodents were housed doubly under standard conditions with automatic watering and a
12:12 h light/dark cycle at 22°C. Food was provided ad libitum, and all animals were given
standard rodent chow. Animals were acclimatized for one week before the start of experiments.
The experiment protocol was approved by and performed in accordance with the University of
Toronto Faculties of Medicine and Pharmacy Animal Care Committee.
115
4.5.4 Treatments
Rats were treated with 80, 125, or 160 mg/kg/day of AMG in 0.5% methylcellulose
through oral gavage. Control animals were given methylcellulose vehicle only. Each group had 4
rats each unless otherwise stated. The 80 mg/kg AMG dose was chosen as a starting dose based
on previous rat studies [230] to mimic the plasma concentrations found in patients taking
therapeutic doses of AMG [231].
4.5.5 Measurement of AMG Blood Levels
Blood levels of AMG were determined 1 h and 24 h after a single 80 or 160 mg/kg dose
of AMG, and analysis was performed similar to Adam et al. [232]. Briefly, blood was collected
from the tail vein of rats into heparinised tubes, and plasma was obtained by centrifugation. To
20 µl of plasma, 10 µl of phenacetin (10 µg/ml stock in methanol) was added as an internal
standard and 80 µl of methanol. The solution was vortexed vigorously for 30 s and then
incubated at -20 °C for 30 min. The sample was centrifuged at 16000g for 10 min, and the
supernatant was transferred to a clean tube and evaporated to dryness under nitrogen. Samples
were then reconstituted in 50 µl of mobile phase (42% methanol: 58% water) and 10 µl was
injected into the HPLC (Hewlett-Packard Series 1050) for analysis with an Ultracarb column (5
µ ODS (30) 100 x 2.0 mm; Phenomenex) at a flow rate of 0.2 ml/min. AMG was detected by UV
absorption at 245 nm, and a standard curve was produced to quantify AMG. The retention times
were 3.8 and 7.3 min for AMG and phenacetin, respectively, and a standard curve was produced
with a correlation coefficient greater than 0.99. The limit of detection was 0.96 µg/ml.
4.5.6 Leukocyte Counts
Blood was collected from the tail vein into EDTA-coated tubes. Whole blood was diluted
20-fold in Turks solution (Ricca Chemical Company, Arlington, TX) to lyse red blood cells
116
(RBCs), and the total WBC count was obtained manually using a haemocytometer. A 5 µl
aliquot of whole blood was also smeared on a slide, fixed, and Wright-Giemsa stained using
CAMCO Stain Pak (Cambridge Diagnostic Products Inc., Fort Lauderdale, FL) as per the
manufacturer’s instructions, and the WBC differential was determined manually under a light
microscope by characterizing 100 leukocytes per slide.
4.5.7 Measurement of Cytokines
Peripheral blood cytokines, chemokine (C-X-C) 1 (Cxcl1), and tumor necrosis factor
alpha (TNF-α) were measured using the Rat Quantikine ELISA kits (R&D Systems,
Minneapolis, MN) according to manufacturer’s protocol. Granulocyte colony stimulating factor
(G-CSF) was also measured in the peripheral blood with a Mouse Quantikine ELISA kit (R&D
Systems). Although the G-CSF ELISA was not specifically developed for use in rats, a BLAST
search shows greater than 80% gene sequence identity to rat G-CSF.
4.5.8 Leukocyte Isolation from Peripheral Blood
Collected whole blood was mixed with equal volumes of 3% dextran (prepared in saline)
and incubated for 18 min at room temperature. The straw-coloured upper layer was collected and
centrifuged at 350g for 5 min and the supernatant discarded. RBCs were lysed with red cell lysis
buffer by incubation for 10 min. Cells were washed twice in florescence-activated cell sorting
(FACS) buffer (5% FBS in PBS) before final resuspension in FACS buffer, and cell counting
was performed using trypan blue and a Countess Automated Cell Counter (Invitrogen, Life
Technologies).
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4.5.9 Measurement of New Neutrophil Release Using 5-bromo-2′-deoxyuridine
(BrdU).
Rats were treated with either methylcellulose vehicle control or AMG for 10 days. BrdU
is known to be carcinogenic and was handled using proper procedures for carcinogenic
substances as outlined by the University of Toronto Faculties of Medicine and Pharmacy Animal
Care Committee. On day 4 of treatment, BrdU (100 mg/kg dissolved in warm saline) was
injected intraperitoneally, and blood samples were taken from the tail vein to monitor neutrophil
incorporation of BrdU after leukocyte isolation using the FITC BrdU Flow Kit (BD Biosciences)
as per the manufacturer’s protocol. Anti-rat granulocyte PE antibody was included as a surface
marker for granulocytes. Cells were analyzed by BD FACSCalibur (BD Biosciences) at a flow
rate of no more than 400 events/s.
4.5.10 Bone Marrow Histology
The femurs of the rats was isolated using standard procedures and fixed in neutral
buffered formalin (Sigma) for 2-5 days. Tissues were submitted to the Toronto Centre for
Phenogenomics Histology Laboratory (Toronto, ON) for paraffin embedding and H&E stained
slide preparation. Microscopic pictures were taken at the Microscopy Imaging Lab (Faculty of
Medicine, University of Toronto) on a Zeiss fluorescence microscope with deconvolution.
4.5.11 Characterization of Bone Marrow Cells
The extracted rat femur was flushed with 5 ml of IMDM containing 10% FBS with a 22
gauge needle. Aggregates were broken up through repeated aspiration, and cells were passed
through a 40 µm filter to make a single cell suspension. Cells were centrifuged at 300g for 5 min
and resuspended in 5.0 ml of FACS buffer before cell counting as per above. Bone marrow cells
were characterized similar to the protocol of Saad et al [233]. For surface staining, 5 µl of anti-
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rat CD71 FITC and 1.25 µl of anti-rat CD45 PE antibodies were added to 1x106 cells in a total
volume of 100 µl and incubated in the dark for 30 min at 4°C. Cells were washed twice with
FACS buffer before cells were fixed with IC Fixation Buffer (eBioscience) for 20 min, washed,
and left overnight in FACS buffer. Cells were then re-fixed and permeablized with
Fixation/Permeablization Buffer (eBioscience) for 20 min, washed once in FACS buffer, and
then resuspended in 200 µl of staining buffer (100 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl2,
0.1% Tween20, 0.5 mM MgCl2 ). DAPI was added to the sample in a final concentration of 3
µM in 100 µl volume and incubated in the dark for 15 min at room temperature before samples
were analyzed immediately using flow cytometry.
4.5.12 Investigation of Bone Marrow Progenitor Cells
Bone marrow cells were extracted as previously described and resuspended in IMDM
containing 2% FBS. Cells were counted manually with a haemocytometer using Turk’s solution.
Progenitor cells in the bone marrow were studied in a colony-forming cell (CFC) assay for rat
cells using the MethoCult GF R3774 medium (StemCell Technologies, Vancouver, BC)
according to the manufacturer’s protocols. Briefly, cells were plated at 1.5x104 per 1.1 ml culture
in 35 mm dishes and incubated at 37 °C, 5% CO2, with ≥95% relative humidity for 10 days
before colony forming unit-granulocyte-macrophage (CFU-GM), colony forming unit-
granulocyte (CFU-G), and colony forming unit-macrophage (CFU-M) were manually assessed
under a light microscope. All colonies on the plate were counted, and each sample was
duplicated in a separate dish.
4.5.13 Determination of Neutrophil Apoptosis
Apoptosis was measured using the Annexin V Apoptosis Kit (BD Biosciences) as per the
manufacturer’s protocol. Specifically, isolated blood leukocytes were washed twice with PBS
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and then reconstituted at 2x105 cells per 100 µl binding buffer. Cells were then incubated with 5
µl each of annexin V FITC and PI for 15 min in the dark at room temperature. Binding buffer
(400 µl) was added to each sample to stop the reaction and cells were analyzed by flow
cytometry within 2 h.
4.5.14 Determination of Neutrophil Activation
Leukocytes were isolated as described previously and cell surface markers were stained
using standard flow cytometry procedures. Anti-rat CD62L FITC, anti-rat CD11b APC, anti-rat
CD18 FITC, and 7-AAD were incubated in the dark for 30 min at 4°C, washed twice in FACS
buffer, and then analyzed by flow cytometry.
4.5.15 Flow Cytometry Analysis
Unless otherwise stated, flow cytometry experiments were run at the Faculty of Medicine
Flow Cytometry Facility (University of Toronto) on the BD FACSCanto II (BD Biosciences)
using BD FACSDiva Software (BD Biosciences) with a flow rate of no more than 400 events/s.
Compensation was corrected using BD Compensation Beads (BD Biosciences). FlowJo Software
(Tree Star, Inc., Ashland, OR) was used for detailed analysis of the flow cytometry results.
4.5.16 Data Analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La
Jolla, CA). The Mann-Whitney U test or two-way ANOVA with Bonferroni post-tests were used
depending on the constraints of the data to determine statistical significance between AMG and
control groups. Error is represented as standard error of the mean (SEM).
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4.6 RESULTS
4.6.1 Effect of AMG on Peripheral Blood Leukocyte Counts
Plasma levels of AMG 1 h after a single gavage dose of 80 and 160 mg/ml AMG were
15.0 (± 2.0) µg/ml and 18.4 (± 2.1) µg/ml, respectively (Figure 4.2), which is within the
therapeutic range (4.7-32.4 µg/ml) found in humans during chronic AMG therapy [234]. Upon
AMG treatment at a dose of 80 mg/kg/day, an increase in peripheral blood neutrophils was
observed as early as 24 h and remained elevated at 48 h with minimal changes to the total WBC
count (Figure 4.3). Further investigation of the dose-response relationship found no clear
changes in the WBC differential of rats treated with 160 mg/kg/day of AMG (data not shown).
This was presumably due to direct toxicity because the rats did not tolerate the high dose of
AMG well, and treatment had to be withdrawn. However, at a lower dose of 125 mg/kg/day of
AMG, a similar elevation in peripheral blood neutrophils was found compared to the dose of 80
mg/kg/day; this was also significantly increased after 24 h (Figure 4.4) and sustained through 14
days of treatment (Figure 4.5). Again, changes in total WBC counts were minimal in the AMG-
treated animals because the neutrophil increase seemed to be compensated for by a slight
decrease in the lymphocyte count, most noticeable after 14 days AMG treatment (Figure 4.5C).
Because the most significant neutrophil response was observed at 125 mg/kg/day of AMG, this
dose was used for subsequent experiments. To provide further evidence for the effect of AMG on
neutrophils, blood cytokines associated with neutrophil production (G-CSF), chemotaxis
(Cxcl1), and activation (TNF-α) were measured; they were all found to be noticeably elevated
within a day of AMG treatment (Figure 4.6).
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Figure 4.2. Blood levels of AMG in rats. Blood was obtained from the tail vein of rats at 1 and
24 h after a single oral dose of 80 or 160 mg/kg of AMG. Values expressed are mean ± SEM for
each group (n = 4).
Figure 4.3. Acute AMG-induced changes on
peripheral blood neutrophils in rats given an
oral dose of 80 mg/kg/day. Blood was taken
from the tail vein of rats and the WBC
differential was determined by Wright-Giemsa
staining. (A) No major changes were observed
in the total WBC count; however, (B) an
increase in neutrophils was observed as early as
24 h after AMG treatment. Values expressed are
mean ± SEM for each group (n=4). None of the
results were statistically significant by Mann
Whitney U test.
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Figure 4.4. Acute AMG-induced changes on peripheral blood neutrophils in rats given an
oral dose of 125 mg/kg/day. Blood was taken from the tail vein of rats, and the WBC
differential was determined by Wright-Giemsa stain. (A) No differences were observed in the
total WBC count between AMG-treated and control rats; whereas, (B) significant increases in
peripheral blood neutrophils were observed after both 1 and 7 days of AMG treatment, which
seemed to be compensated for by (C) a slight decrease in peripheral blood lymphocytes. Open
bars represent the control group; solid bars represent the AMG-treated group. Values expressed
are mean ± SEM (n=4). *p<0.05 and ***p<0.001 compared to controls by two-way ANOVA.
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Figure 4.5. Changes in peripheral leukocyte cell counts induced by treatment of rats with
AMG at a dose of 125 mg/kg/day for 14 days. (A) No changes were observed in total WBC
between control and AMG-treated rats; whereas, (B) a noticeable increase in the neutrophil
population was found after both 7 and 14 days of AMG treatment and (C) a corresponding slight
decrease in lymphocyte counts. Open circles and dotted lines represent the control group; solid
squares and lines represent the AMG-treated group. Values expressed are mean ± SEM (n=4).
None of the results were significant by two-way ANOVA.
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Figure 4.6. AMG-induced changes in neutrophil-associated cytokines in the blood of rats.
(A) G-CSF, involved with stimulating neutrophil production, was significantly elevated after 7
days of AMG treatment. (B) The neutrophil chemoattractant Cxcl1 was noticeably increased
after both 1 and 7 days of AMG treatment, and (C) TNF-α, involved with neutrophil activation,
was up-regulated after both 1 and 7 days of AMG treatment. Values are expressed as mean ±
SEM (n=4). *p<0.05 and ***p<0.001 compared to baseline measurements by Mann Whitney U
test.
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4.6.2 Effect of AMG on Bone Marrow
Given that all neutrophils originate from bone marrow and BrdU is a thymidine analogue
that gets incorporated into newly formed DNA, BrdU was used to track new neutrophils released
from the bone marrow into the circulation by flow cytometry and double staining with an anti-rat
granulocyte antibody. Consistent with the increase in peripheral blood neutrophils, an increase in
newly formed neutrophils labeled with BrdU was observed in the AMG-treated rats with the
most significant changes occurring at 120 h post-BrdU injection (Figure 4.7). Furthermore, the
increase in newly formed neutrophils was sustained throughout AMG treatment, which suggests
that AMG prolongs the half-life of circulating neutrophils. H&E staining of bone marrow from
the femurs of rats found hypercellularity induced by AMG that was attributed to an increase in
the myeloid cells and was not observed in the controls (Figure 4.8). To confirm this, nucleated
cells of the bone marrow, detected with DAPI, were phenotyped by flow cytometry through
differential staining of CD45 and CD71 to identify changes in the myeloid, lymphoid, and
erythroid cell populations. A significant increase in the myeloid cell population was found with
AMG treatment, and this corresponded to a simultaneous decrease in the lymphoid cell
population (Figure 4.9). The myeloid to erythroid ratio of the bone marrow in AMG-treated rats
was also significantly elevated compared to controls (2.36 ± 0.31 vs. 1.48 ± 0.13, respectively,
p=0.03). To determine which myeloid cell populations were affected by AMG, a MethoCult
assay was performed ex vivo to evaluate the proliferative capacity of granulocyte and
macrophage colony forming units (CFUs) in response to AMG treatment. The effect of AMG
seemed to be more specific to granulocytes because there was an increase in the number of CFU-
granulocyte-macrophage (CFU-GM) and CFU-granulocyte (CFU-G) colonies, but there was not
much of a change in CFU-macrophage (CFU-M) colonies (Figure 4.10).
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Figure 4.7. Effect of AMG on new neutrophil release from the bone marrow. Rats were
treated with 125 mg/kg/day of AMG for 10 days. On the fourth day, 100 mg/kg BrdU was
injected i.p., and blood from the tail vein was monitored for changes in neutrophils by flow
cytometry. Neutrophils were gated by forward and side scatter, and newly formed neutrophils
were characterized as cells double positive for anti-granulocyte antibody and BrdU. Both the (A)
percent BrdU-stained neutrophils, and the (B) absolute number of newly formed neutrophils
were increased by AMG treatment. Open circles and the dotted line are from control animals;
solid squares and lines represent AMG-treated animals. Values expressed are mean ± SEM (n=5
control, n=6 AMG). *p<0.05 compared to control group by two-way ANOVA.
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Figure 4.8. Bone marrow changes induced by AMG. Representative H&E staining of bone
marrow from (A) control rat femur and (B) rat femur after 14 days of AMG at a dose of 125
mg/kg/day. 40x magnification.
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Figure 4.9. AMG-induced changes in bone marrow cells. Bone marrow cells were extracted
from rats treated for 14 days with 125 mg/kg/d of AMG and analyzed through FACS analysis.
Representative flow plots are shown for (A, B) control and (C, D) AMG-treated rats. (A, C)
From the nucleated cells, stained as DAPI positive, nucleated erythroid cells were defined as
CD71 positive, CD45 medium expression, whereas the myeloid and lymphoid cells were defined
as CD71 low, CD45 medium-high. (B, D) From this, myeloid and lymphoid cells were further
separated based on side scatter where myeloid cells were more granular than lymphoid cells. (E)
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No change was observed in the erythroid population between treated and control animals;
however, in the AMG-treated rats there was a significant increase in myeloid cells with a
simultaneous significant decrease in the lymphoid population. Open bars represent the control
group; solid bars represent the AMG-treated group. Values expressed are mean ± SEM (n=4).
**p<0.01 compared to the control group by two-way ANOVA.
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Figure 4.10. Effect of AMG on bone marrow granulocyte and macrophage CFUs. Bone
marrow cells were extracted from rats treated with 125 mg/kg/day AMG for 14 days. Cells were
then seeded into culture dishes at 1x105 cells per 1.1 ml MethoCult medium and incubated at
37°C, 5% CO2, and ≥95% humidity. After 10 days colonies were counted under a light
microscope and classified as either CFU-GM, CFU-G, or CFU-M. AMG significantly increased
the total number of CFUs and CFU-GM. An increase in the CFU-G was also observed with
AMG treatment; however, no change was observed in CFU-M. Open bars represent the control
group; solid bars represent the AMG-treated group. Samples were performed in duplicate.
Values expressed are mean ± SEM (n=4). *p<0.05 and **p<0.001 compared to control by two-
way ANOVA.
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4.6.3 Effect of AMG on Neutrophils
Since AMG induced a sustained increase in newly formed neutrophils in rats, further
investigation into the effect of AMG on neutrophil survival was warranted. Neutrophil apoptosis
was investigated through annexin V and PI staining using flow cytometry whereby neutrophils
were gated based on forward and side scatter, and cells that were annexin V positive/PI negative
were considered early apoptotic cells. It is interesting to note the increase in density of the
neutrophil population in the AMG-treated rats (Figure 4.11A, B), which corresponds to the
increase in blood neutrophils that was previously observed. Using this approach, AMG treatment
induced a significant reduction in the number of early apoptotic neutrophils compared to controls
(62.5% at 7 days treatment, p=0.01; and 55.6%, after 12 days of treatment, p=0.03) as shown in
representative flow plots (Figure 4.11C, D). In terms of activation, AMG significantly down-
regulated the expression of CD62L in neutrophils over time (Figure 4.12), while a simultaneous
increasing trend was observed in CD11b expression in neutrophils, most noticeable at day 7 and
12 (data not shown). However, no changes were found in CD18 expression (data not shown).
Both CD62L and CD11b are involved with the infiltration of leukocytes into tissues, and these
changes suggest neutrophil activation.
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Figure 4.11. AMG-induced changes in neutrophil apoptosis. Blood taken from the tail vein of
rats treated with AMG at a dose of 125 mg/kg/day was incubated with annexin V and PI to
determine the number of apoptotic cells. As compared to (A) control, representative flow plots
show (B) an increase in the percentage of neutrophils upon AMG treatment. From the neutrophil
population, (C) the percent annexin V positive/PI negative early apoptotic cells are high in the
control rats, whereas (D) there is a decrease in the number of apoptotic neutrophils in the AMG-
treated rats.
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Figure 4.12. Changes in neutrophil CD62L expression induced by AMG. Blood taken from
the tail vein of rats treated with 125 mg/kg/day of AMG were analysed by flow cytometry for
CD62L expression on neutrophils. Neutrophils were identified by their distinct forward and side
scatter characteristics as shown previously in Figure 12. Representative flow plots from (A)
control and (B) AMG-treated rats show that CD62 is expressed in neutrophils under normal
physiological conditions; however, upon AMG treatment there is an increase in the number of
neutrophils that have decreased CD62L expression, as shown by the increase in cells in the
CD62L low gate. (C) The mean florescence intensity (MFI) for the CD62L channel is
significantly decreased at both days 7 and 12 in the AMG-treated as compared to controls. (D) A
significant increase was also observed in the percentage of cells in the CD62L low gate in the
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AMG-treated at days 7 and 12. Open bars represent control; solid bars represent AMG-treated.
Values expressed are mean ± SEM (n=4). *p<0.05, **p<0.01 compared to control by two-way
ANOVA.
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4.7 DISCUSSION
To date, there have been no detailed studies on the effect of AMG on neutrophils despite
the many reports of AMG-induced agranulocytosis. The IDRs induced by AMG are not
surprising given the fact that it is an aromatic amine drug, and aromatic amines are readily
oxidized by neutrophil myeloperoxidase [235, 236]. In the current studies, the major finding was
that AMG increased the number of peripheral neutrophils. The effect on neutrophils was
observed as early as 24 h, which suggests that AMG mobilized the marginal zone neutrophils;
however, this increase was sustained by an increase in granulocyte production for at least 14
days. This change was compensated for by a slight decrease in peripheral blood lymphocytes
such that no significant changes were found in the total WBC count. In rodents, the neutrophils
make up only approximately 10-30% of the total WBCs in the blood [142], whereas in humans
the majority of peripheral WBCs are neutrophils, thus the compensatory effect of lymphocytes is
consistent to a subtle change.
The dosages of AMG given to rats in this study produced blood levels of AMG similar to
what was found in humans. However, the dosage of AMG used in human therapy is fairly high
(can be up to 1000 mg per day) and it would not be surprising if AMG can induce neutrophilia
similar to what occurs commonly during stress or inflammation. The stress response, specifically
corticosterone is known to increase neutrophils; however this effect is unlikely to be a significant
issue because AMG has been found to decrease corticosterone levels in the blood [237].
Moreover, inflammation may be a central feature to induce immune activation, and if these
reactions are immune-mediated, this may lead to an immune response to drugs associated with
agranulocytosis.
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It is interesting that the effect of AMG on neutrophils in rats is the opposite of the
agranulocytosis that is observed in some patients [225] and the leucopenia that occurs in mice
[174]. However, in general, AMG patients are not monitored until blood dyscrasias arise;
therefore, it is unknown whether patients experience earlier changes in their WBC differential. In
the previous mouse models, leucopenia was observed as early as after 2 weeks of treatment
[174], and it may be possible that if we had continued treatment for a much longer period of time
it would result in agranulocytosis. A previous study with AMG-treated rats found no changes in
WBC differential even after 3 weeks of treatment [229]; however, the dose was much lower (50
mg/kg/day) than the dose currently used. In addition, rats had more sustained levels of AMG in
the blood 24 h after a single dose than mice treated with similar doses (data not shown). For
these reasons, the rat may be a better model of AMG-induced blood dyscrasias and be more
useful as a tool for mechanistic studies.
Interestingly, AMG-induced changes in neutrophils share common characteristics to what
is observed in humans upon clozapine therapy. Clozapine is an atypical antipsychotic that is
associated with agranulocytosis in approximately 1% of patients, and the mechanism is thought
to be immune-mediated [77]. Moreover, clozapine patients experience an initial neutrophilia
before the onset of agranulocytosis [167] and increases in CD34+ hematopoietic stem and
progenitor cells has also been reported within 2 weeks of initiating therapy [238], suggesting the
mobilization of neutrophil precursors. A similar increase in neutrophils has been observed in
rabbits [239] and rats (unpublished observations) upon treatment with clozapine, and this is
consistent with the increase in neutrophils that we have observed in AMG-treated rats. Clozapine
also appears to decrease neutrophil half-life in rabbits [239], and it may be the lack of ability of
the bone marrow reserve to keep up with demand that leads to agranulocytosis. In contrast, the
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increased release of newly formed neutrophils from the bone marrow and the kinetic profile of
the BrdU-labelled neutrophils suggest that AMG may increase neutrophil survival and half-life.
Although further studies using annexin V and PI found a decrease in apoptotic neutrophils in the
AMG-treated rats, which is consistent with an increase in neutrophil survival, this change may be
caused by a greater number of younger neutrophils being released from the bone marrow that
express less phosphatidylserine. The basis for this observation may be the high levels of
apoptosis in the control rats (although partially due to the handling procedures) that decreases
upon AMG treatment concomitant with a greater number of neutrophils. Furthermore, in vitro
studies on neutrophil survival in the presence of AMG resulted in no difference in neutrophil
apoptosis from vehicle-treated (data not shown). Therefore, more detailed studies are required to
determine how AMG alters neutrophil survival, although the rapid turnover rate and clearance of
apoptotic neutrophils may make such studies difficult. In the bone marrow of the AMG-treated
rats there was also an increase in CFU-GM and CFU-G colonies, which are precursors to
neutrophils, and this is consistent with an increase in the production of progenitor cells from the
bone marrow.
In terms of cytokine expression, clozapine-induced fever is typically mediated by IL-6,
and patients characteristically express increased levels of TNF-α and sIL-2r that are exacerbated
by fever [169]. Clozapine has also been found to transiently increase the levels of G-CSF in
patients that was independent of the development of fever [168]. Thus, these changes in serum
cytokines upon clozapine treatment are common in the majority of patients and could be
biomarkers to identify drugs predisposed to causing agranulocytosis. In our study, an increase in
neutrophil-associated cytokines, G-CSF and TNF-α, was observed in the blood upon AMG
treatment in rats. The release of G-CSF stimulates the production of granulocytes, and it has
138
been given to patients to treat neutropenia. Moreover, TNF-α can stimulate the neutrophil
respiratory burst by activating NADPH oxidase [151]. Thus there seems to be similarities in the
expression of cytokines by both clozapine and AMG and these changes could be biomarkers to
identify drugs predisposed to causing agranulocytosis.
Although the rats, under our current experimental protocols, do not develop
agranulocytosis or show evidence of any other pathologies, the changes in the blood upon AMG
treatment may occur in the majority of treated patients and may also provide clues to the
mechanism of more serious IDRs. One common explanation for the lack of development of a
pathological response is the induction of immune tolerance. Evidence for this is provided by the
resolution of clozapine-induced agranulocytosis without discontinuing treatment [166].
Activated neutrophils may lead to an immune response that leads to agranulocytosis; however,
the usual response may be immune tolerance. From the current studies, AMG appears to
stimulate the bone marrow to release new neutrophils that are activated in terms of the
extravasation marker CD62L. In humans challenged with endotoxin, CD62Ldim
neutrophils were
found to be more hypersegmented than the CD62Lbright
neutrophils, and this population was also
able to suppress T-cell activation [160], which may be a mechanism of immune tolerance.
However, it is unknown how this is affected in AMG-induced IDRs, and more studies will be
required to determine the link between neutrophil activation and agranulocytosis. Another
limitation that we were unable to sufficiently address was the extent of the involvement of the
arylamine group of AMG to the effects observed on the neutrophils, as we were unable to obtain
glutethimide because it is classified as a controlled substance. Nevertheless, numerous studies
have shown additional effects of neutrophils on the adaptive immune system in addition to their
traditional role in innate immunity; specifically, their ability to interact and regulate a variety of
139
different immune cells [240]. Thus, it is imperative to consider the role of neutrophils when
investigating the initiation of IDRs.
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CHAPTER 5
INVESTIGATION OF THE IMMUNE CHANGES
INDUCED BY AMINOGLUTETHIMIDE
Winnie Ng, Imir G. Metushi, Libia Vega-Loyo, and Jack Uetrecht
141
5.1 ABSTRACT
Idiosyncratic drug reactions (IDRs) are a significant issue of concern primarily because
of their unpredictable nature. The mechanisms of IDRs are not fully understood, although most
IDRs exhibit characteristics that suggest they are immune-mediated. The primary aromatic
amines are associated with a high incidence of IDRs. This is presumably because they can be
easily oxidized to reactive metabolites that covalently bind to proteins, and they also redox cycle,
which could induce oxidative stress. Protein modification and cell stress may activate the
immune system, which in some patients may lead to an IDR. Previous studies of rodents treated
with aromatic amine drugs found few immune changes in the liver; however, aromatic amines
can also be oxidized in cells of the immune system, and this may be more important for the
induction of an immune response. The objective of this study was to determine the immune
response to one representative aromatic amine, aminoglutethimide (AMG) in rats. AMG
treatment increased serum levels of the chemotactic proteins Gro/KC, MCP-1, and IP-10.
Increased cell proliferation was observed in the white pulp of the spleen after 14 days of AMG
treatment, and this was attributed to an increased proliferation of CD4 T-cells. Interestingly, a
lymphocyte transformation test (LTT) using splenocytes was negative, but it was positive when
using lymphocytes from the auricular lymph nodes. There were also transient increases in
intracellular IL-17 and IL-10 in lymphocytes from the spleen and lymph nodes following AMG.
An increase in M2 macrophages was observed and this may be part of the immune response that
prevents a pathogenic immune response. In short, although AMG does not cause overt toxicity, it
induces an immune response that is consistent with the development of immune tolerance.
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5.2 ABBREVIATIONS
AMG, aminoglutethimide
PC, allophycocyanin
APCs, antigen presenting cells
DMSO, dimethyl sulfoxide
FACS, florescence-activated cell sorting
FITC, fluorescein isothiocyanate
H&E, hematoxylin and eosin
IDRs, idiosyncratic drug reactions
LTT, lymphocyte transformation test
MPO, myeloperoxidase
P450, cytochrome P450
PBS, phosphate buffered saline
PE, phycoerythrin
PerCP, peridinin chlorophyll
PMA, phorbol myristate acetate
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5.3 INTRODUCTION
Idiosyncratic drug reactions (IDRs) are of great concern to the pharmaceutical industry
primarily due to their unpredictable nature. Promising new drug candidates often have to be
withdrawn from the market or achieve a “black box” warning because of a serious IDR that was
not detected during clinical trials. If the risk that a drug candidate would cause serious IDRs
could be predicted, or if the patients at high risk could be identified it would have a profound
effect on drug development and therapy.
The mechanisms of IDRs have yet to be fully elucidated; however, there is increasing
evidence that most IDRs are caused by reactive metabolites and are immune-mediated [13]. The
delay in onset and rapid onset upon re-challenge, seen commonly with IDRs, provides indirect
evidence of a T-cell response that requires time for T-cell clonal expansion and a subsequent
memory response upon re-stimulation with the drug. Moreover, the formation of anti-drug and
anti-nuclear antibodies in some patients further implicates the involvement of the immune
system. Thus, the majority of IDRs are thought to be due to an adaptive immune response to
drug-modified proteins that can become antigenic or induce danger signals through the induction
of cellular stress [241].
Traditionally the liver has been a main focus of drug metabolism and toxicity studies
because it has high levels of metabolic enzymes such as the cytochrome P450s (P450s) that can
oxidize drugs to chemically reactive metabolites. Although idiosyncratic drug-induced liver
injury is a major cause for drug withdrawl [242], the relevancy of using liver tests as a universal
indicator of drug toxicity is questionable, especially with drugs for which the primary target of
toxicity is not the liver.
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Drug metabolizing enzymes such as P450s have been found in peripheral blood
mononuclear cells in humans [243]. Although this could potentially contribute to reactive
metabolite formation, their expression is presumably much less than in the liver. In contrast,
neutrophil activation was observed upon stimulation with prohaptenic chemical sensitizers, and
this effect was attributed to the presence of myeloperoxidase (MPO) in these cells that was not
present in the other cell types tested [244]. MPO is a heme-containing enzyme primarily
involved in the production of strong microbicidal oxidants, and it is found within the azurophilic
granules of a variety of immune cells including neutrophils, monocytes, and macrophages [245].
Oxidation of drugs to reactive metabolites by MPO has been implicated in the mechanisms of
IDRs [246]. Alternatively, there are many enzymes other than MPO that are found within
immune cells which may have the capability to oxidize drugs [149]. Thus, the localized
production of reactive metabolites and oxidative stress within immune cells may provide a
stronger stimulus for the initiation of an immune response.
The primary aromatic amine moiety is a notorious structural alert for drug development
because the majority of aromatic amine drugs are associated with a high incidence of IDRs [94].
Although aromatic amine drugs can induce liver injury, it is one of the less common reactions
induced by these drugs, and liver injury is more likely a result of a more generalized
hypersensitivity reaction. Studies in rodents to test the changes in hepatic gene expression
induced by several aromatic amine drugs: sulfamethoxazole (SMX), dapsone (DDS), and
aminoglutethimide (AMG), found few changes that were suggestive of liver injury or the
initiation of a cellular immune response in the liver [37, 212]. Additionally, a comprehensive
study of the liver to determine the effects of AMG, an aromatic amine aromatase inhibitor, found
few patho-histological changes, and only minor changes in genes involved with the innate
145
immune system (Ng et al.; submitted). Despite the capacity for the liver to mount an immune
response, in comparison to other organ systems, the dominant immune response in the liver is
immune tolerance [247].
Aromatic amines are easily oxidized and can be oxidized by enzymes other than P450s
such as MPO [248], and some aromatic amine drugs have been shown to be oxidized in vitro by
activated neutrophils to hydroxylated metabolites [235, 249]. Interestingly, the aromatic amine
anti-microbial SMX has been extensively studied in models of immunogenicity [250]. In
particular, antigen presenting cells (APCs) were found to metabolize SMX to reactive species,
and these drug-modified cells were able to induce an immunogenic response in lymphocytes
from SMX hypersensitive individuals [49]. Thus, testing the direct effect of aromatic amines on
immune cells may be a better approach to understanding the mechanisms of IDRs caused by
these drugs.
In general, the reactivity of aromatic amines is thought to be due to their ability to form
common reactive metabolites such as the highly electrophilic nitrosoamine. Since aromatic
amines can be metabolized to reactive species that could covalently bind to endogenous
molecules to produce antigenic substances and can also redox cycle to cause oxidative damage, it
is quite possible that aromatic amine drugs could induce IDRs through danger mechanisms
[251]. Given that aromatic amine drugs can be oxidized within immune cells, the objective of
this study was to investigate the role of the immune system in aromatic amine-induced IDRs by
characterizing the AMG-induced immune changes and determining how they may be involved in
initiating a cellular immune response. In these studies, AMG was used as a prototypical aromatic
146
amine drug because our previous experiments found that AMG induced the most biological
changes compared to other aromatic amine drugs tested.
147
5.4 MATERIALS AND METHODS
5.4.1 Chemicals and Reagents
AMG was purchased from Toronto Research Chemical (North York, ON).
Methylcellulose, dextran, 5-bromo-2′-deoxyuridine (BrdU), ethylenediaminetetraacetic acid
(EDTA), Tris-base, sodium chloride, calcium chloride, magnesium chloride, phorbol myristate
acetate (PMA), ionomycin, sodium bicarbonate, 10% neutral buffered formalin, xylene,
hematoxylin, and Cytoseal were obtained from Sigma-Aldrich (Oakville, ON). Phosphate
buffered saline (PBS), heat inactivated fetal bovine serum (FBS), RPMI-1640, 2-
mercaptoethanol, and trypan blue were purchased from Life Technologies (Burlington, ON).
Potassium bicarbonate, dimethylsulfoxide (DMSO), hydrogen peroxide, and Tween20 were
obtained from BioShop Canada Inc. (Burlington, ON). Ammonium chloride was purchased from
ACP Chemical (Montreal, QC). Methanol was acquired from Caledon Laboratory Chemicals
(Georgetown, ON). Ethanol was purchased from Commercial Alcohols (Brampton, ON). Saline
was obtained from Baxter (Mississauga, ON). Epitope Unmasking Solution and Antibody
Dilution buffer were obtained from ProHisto (Columbia, SC). Lymphoprep was obtained from
Axis-Shield (Oslo, Norway).
5.4.2 Antibodies
Anti-rat IL-10 phycoerythrin (PE; clone: JES5-16E3), anti-rat CD32 (FcγII; clone: D34-
485), 7-amino-actinomycin D (7-AAD), anti-BrdU fluroescein isothiocyanate (FITC), and
GolgiStop were acquired from BD Biosciences (San Jose, CA). Anti-rat CD4 FITC (clone:
W3/25), anti-rat NKRP1 PE (clone: 10-78), and anti-rat CD45RA/B FITC (clone:MRC OX-33)
were purchased from Cedarlane (Burlington, ON). Anti-rat CD3 Pacific Blue (clone: IF4), anti-
rat CD163 Alexa647 (clone: ED2), anti-rat CD68 (clone: ED1), and anti-rat CD11c Alexa647
148
(clone: 8A2) were obtained from AbD Serotec (Raleigh, NC). Anti-mouse/rat Ki-67 peridinin
chlorophyll (PerCP; clone: SolA15), anti-rat CD8a PECy7 (clone: OX8), anti-mouse/rat IL-17
allophycocyanin (APC; clone: ebio17B7), anti-rat MHCII APC (clone: HIS19), and anti-
mouse/rat TNF-α PECy7 (clone: TN3-19) were purchased from eBiosciences (San Diego, CA).
Anti-rat Ki-67 (clone MIB-5), rabbit anti-mouse biotinylated IgG, and streptavidin-HRP were
acquired from Dako (Burlington, ON).
5.4.3 Animals
Male Brown Norway rats (176-200 g) were purchased from Charles River (Montreal,
QC). Animals were housed under standard conditions with automatic watering, 12:12 hr
light/dark cycle and a temperature of 22 °C. Food and water were provided ad libitum and all
animals were given standard rodent chow. Animals were acclimatized for one week before the
start of experiments. The experiment protocol was approved by and performed in accordance
with the University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee.
5.4.4 Treatments
Rats were treated with AMG (125 mg/kg/day in 0.5% methylcellulose) through oral
gavage. Control animals were given methylcellulose vehicle only. Each group had 4 rats each
unless otherwise stated.
5.4.5 Measurement of Serum Cytokines
Blood was collected from the tail vein of rats. Samples were analyzed at the University
Health Network Microarray Centre (Toronto, ON) using a Rat Cytokine/Chemokine Kit
(MILLIPLEX MAP, Millipore) that contained a 23-Plex Premix bead set according to
149
manufacturer’s protocol. Data was read with a Luminex 100 instrument and analyzed using Bio-
Plex Manager 6.0 software (Bio-Rad Laboratories, Mississauga, ON).
5.4.6 Detection of Cell Proliferation in Lymphoid Organs
Spleen and auricular lymph node sections were excised from the rat and immediately
immersed in 10% formalin solution. H&E and unstained paraffin-embedded slides were prepared
by the Division of Pathology at the Hospital for Sick Kids (Toronto, Ontario).
Immunohistochemistry was carried out using standard procedures. Briefly, slides were
deparaffinised with xylene and serial dilutions of ethanol. Heat-induced antigen retrieval was
performed using Epitope Unmasking Solution (ProHisto, Columbia, SC) and boiling the slides
for 20 min. Endogenous peroxidases were blocked with 3% hydrogen peroxide in PBS for 10
min. Washes were performed using PBS-0.05% Tween20 solution and antibodies were diluted in
Antibody Dilution Buffer (ProHisto). The primary antibody for Ki-67 (1:25 dilution) was
incubated for 30 min. Rabbit anti-mouse biotinylated IgG (1:250 dilution) was used as a
secondary antibody for 30-40 min. Streptavidin-HRP (1:500 dilution) was incubated for 15 min
after which 3’3-diaminobenzidine (DAB, Burlingame, CA) was used for visualization and
haematoxylin was used as a counter-stain. Slides were dehydrated with ethanol and xylene
before slip covers were mounted with Cytoseal (Sigma). A Zeiss fluorescence microscope with
deconvolution was used to obtain histological pictures at the Microscopy Imaging Lab (Faculty
of Medicine, University of Toronto).
5.4.7 Phenotyping of Immune Cells
Spleen and lymph nodes were removed, placed in florescence-activated cell sorting
(FACS) buffer (5% FBS in PBS solution), and a single cell suspension was made using the blunt
end of a syringe through a 40 µm nylon mesh filter. Red blood cells were lysed with red blood
150
cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) after which FACS buffer was
added and cells were washed twice before cell counting using trypan blue and a Countess
Automated Cell Counter. Each sample consisted of 1.0 x 106 cells in a total volume of 100 µL
per well. Non-specific binding was blocked by the addition of anti-rat CD32 (FcγII Receptor)
antibody for 15 min. After several washes using FACS buffer, primary antibodies were
incubated for 30 min before washing and resuspension in FACs buffer for analysis.
For intracellular cytokines, 1.0x106 cells were stimulated for 4 h with in culture medium
containing RPMI 1640, 10% FBS, 50 ng/mL PMA, 1 µg/mL inomysin, and GolgiStop prior to
the staining of extracellular antibodies. Cells were then fixed and permeablized with Fix/Perm
solution (BD) for 15 min before the addition of intracellular antibodies in Permeabilization
Buffer for 1 h. After several washes cells were re-suspended in FACs buffer before analysis.
5.4.8 Characterization of an Immune Response through the Lymphocyte
Transformation Test (LTT).
After 14 days of AMG treatment, a single cell suspension of spleen and auricular lymph
node cells was prepared as previously described. Cells extracted from the lymph nodes were
used neat, whereas, in the spleen, lymphocytes were extracted using Lymphoprep (Axis-Shield)
according to manufacturer’s instructions. For each reaction, 1x106 cells were incubated with 10
µM BrdU and various concentrations of AMG (in 30 µL DMSO) in a final reaction volume of 1
mL culture medium (RPMI 1640, 10% FBS, antibiotics, 50 µM β-mercaptoethanol, 1 µg/mL
indomethacin). Cells were incubated for 72 h at 37°C with 5% CO2 after which the reaction was
stopped. Cells were washed with FACS buffer and BrdU was detected using the FITC BrdU
Flow Kit (BD Biosciences) as per the manufacturer’s protocol.
151
5.4.9 Flow Cytometry Analysis
Flow cytometry experiments were run at the Faculty of Medicine Flow Cytometry
Facility (University of Toronto) on the BD FACSCanto II (BD Biosciences) using BD
FACSDiva Software (BD Biosciences). The flow rate was no more than 400 events/s, and
FlowJo Software (Tree Star, Inc., Ashland, OR) was used for detailed analysis of the flow
cytometry output.
5.4.10 Data Analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La
Jolla, CA). Unless otherwise stated, ANOVA and Bonferroni post-test were used to determine
the statistical significance between AMG and control groups.
152
5.5 RESULTS
5.5.1 Systemic Immune Response
Rats did not develop any overt signs of an immune or toxic response to AMG treatment
up to 14 days. At early time-points an increasing trend in innate immune factors was observed in
the AMG-treated as compared to control (Figure 5.1), which suggests early immune changes
because Gro/KC, MCP-1, and IP-10 are associated with neutrophil, monocyte, and T-cell
chemotaxis, respectively. Although these results were not statistically significant, this could be a
type 2 error because there does look to be a difference between AMG-treated and control and
perhaps an increase in sample size would make this change more apparent. Treatment with AMG
for up to 14 days also induced innate cytokine changes (Figure 5.2). Again Gro/KC and MCP-1
were up-regulated; however, a significant increase in IL-5 was also observed with a concurrent
significant down-regulation of GM-CSF. IL-5 is a cytokine associated with immunoglobulin
production in B-cells, and its transient increase by AMG may suggest induction of a tolerogenic
mechanism. We have previously reported an increase in blood neutrophils in rats treated with
AMG [216]. Thus, it is possible that the down-regulation of GM-CSF in the current study may
represent a negative feedback mechanism to inhibit granulocyte and monocyte production and
maturation. Therefore the AMG-induced systemic changes are predominantly of an innate
nature.
153
A B
C
24 480
200
400
600
800
Control
AMG
Time (h)
Gro
/KC
(pg
/ml)
24 480
2000
4000
6000
Control
AMG
Time (h)
MC
P-1
(p
g/m
l)
24 480
50
100
150
200
250
Control
AMG
Time (h)
IP-1
0 (
pg
/ml)
Figure 5.1. Early changes in serum levels of Grow/KC, MCP-1, and IP-10 induced by AMG
treatment. Open bars represent control; solid bars represent AMG-treated. Values are expressed
as mean ± SEM (n=3 control, n=4 AMG). None of the results were statistically significant.
154
A B
0 7 140
1000
2000
3000
**
Control
AMG
Day
Gro
/KC
(p
g/m
l)
0 7 140
1000
2000
3000Control
AMG
Day
MC
P-1
(p
g/m
l)
C D
0 7 140
20
40
60
80
Day
*
Control
AMG
IL-5
(p
g/m
l)
0 7 140
50
100
150
200
*
Control
AMG
Day
GM
-CS
F (
pg
/ml)
Figure 5.2. Later changes in serum cytokines induced by AMG treatment. (A) Gro/KC, (B)
MCP-1, and (C) IL-5. Alternatively, AMG induced a significant decrease in GM-CSF. Open bars
represent control; solid bars represent AMG-treated rats. Values are the mean ± SEM (n=4
control, n=6 AMG). *p<0.05, **p<0.01 compared to control.
155
5.5.2 Effect of AMG on Lymphoid Organs
Cellular proliferation was measured in the auricular lymph nodes and the spleen through
the expression of the nuclear marker Ki-67. Although no changes in cellular proliferation were
observed in the auricular lymph nodes, a significant increase in Ki-67-stained cells was observed
in the spleen (Figure 5.3). The Ki-67 positive cells were clustered in the white pulp of the spleen
in AMG-treated rats, whereas in the control the proliferating cells were sparsely distributed
throughout the spleen. This suggests that AMG induces immune cell proliferation because the
white pulp is important for immune activation, and that is where the B- and T-cells are primarily
found within this organ. FACS analysis subsequently found that the proliferating cells were
CD4+ CD3
+ T-lymphocytes (Figure 5.4). It should be noted that, although the magnitude of the
changes between the methodologies vary quite noticeably, this is presumably due to the focal
nature of the proliferation, which was not as easily detected by analyzing the whole organ with
FACS. More importantly, the functional impact of these changes was investigated to determine
whether the proliferation of T-lymphocytes induced by AMG translated into an adaptive immune
response as measured by the lymphocyte transformation test (LTT). Despite the T-lymphocyte
proliferation found in the spleen, there was no change upon re-stimulation with AMG; however,
lymphocytes from the auricular lymph nodes did stimulate a slight increase in proliferation in
response to re-stimulation with AMG concentrations of 50 and 100 µg/mL (Figure 5.5),
suggesting a small secondary adaptive response to the drug; however, the magnitude of these
changes were too small to be certain.
156
Figure 5.3. Cell proliferation detected by Ki-67 staining induced in the white pulp of the
spleen after 14 days of AMG treatment. (A) control, (B) AMG-treated rat. 20x magnification.
A
B
157
A
B CD3+ CD45RAB
0
5
10
15Control
AMG
Percen
t K
i-67+
Cells in
Lym
ph
ocyte
Su
bp
op
ula
tio
n
CD4+ CD8+ NKRP1+0
5
1015
20
2560
70
80Control
AMG
Perc
en
t K
i-67
hig
h/C
D3+
C
CD11c NKRP1 CD68 CD1630
20
40
60
80
**Control
AMG
Perc
en
t K
i-67
hig
h C
ells
Figure 5.4. Analysis of proliferating (Ki-67+) splenocytes after 14 days of AMG treatment.
(A) Lymphocyte subpopulation (B) T-cell subpopulation and (C) innate cell subpopulations.
Open bars represent control, solid bars represent AMG-treated rats. Values are the mean ± SEM
(n=4). **p<0.01 as compared to control.
158
Control 1
6.25 12.5 25 50 1000.0
0.2
0.4
0.6
0.8
DMSO
AMG (ug/ml)
Brd
U (
Perc
en
t S
tain
ed
in
Lym
ph
ocyte
Gate
)
Control 2
6.25 12.5 25 50 1000.0
0.2
0.4
0.6
0.8
DMSO
AMG (ug/ml)
Brd
U (
Perc
en
t S
tain
ed
in
Lym
ph
ocyte
Gate
)
AMG 1
6.25 12.5 25 50 1000.0
0.2
0.4
0.6
0.8
DMSO
AMG (ug/ml)
Brd
U (
Perc
en
t S
tain
ed
in
Lym
ph
ocyte
Gate
)
AMG 2
6.25 12.5 25 50 1000.0
0.2
0.4
0.6
0.8
DMSO
AMG (ug/ml)
Brd
U (
Perc
en
t S
tain
ed
in
Lym
ph
ocyte
Gate
)
Figure 5.5. LTT using lymphocytes from the lymph nodes of AMG-treated rats. Rats were
treated with AMG (125 mg/kg/day) for 14 days before lymphocytes were extracted from the
lymph nodes. Ex vivo cell proliferation was measured using BrdU after 72 h re-stimulation with
various concentrations of AMG. Each graph represents one animal (n=2).
159
5.5.3 AMG-Induced Immune Cell Changes
AMG induced very subtle changes in the phenotype of immune cells despite various
attempts to characterize changes in immune cell subsets. No significant changes were found in
CD4-, CD8-, CD45RAB-, CD11c-, or NKRP1-positive cells at both early (24 h) and late (14
days) time-points in the spleen or lymph nodes upon AMG treatment (data not shown). However,
subtle changes were observed in intracellular cytokines. A slight elevation in both IL-17- and IL-
10-positive cells at early time-points was observed upon AMG treatment in the spleen and
auricular lymph nodes (Figure 5.6). Although not statistically significant, up-regulation of both
would suggest that there may be a modulating effect occurring fairly early upon treatment.
Furthermore, an increase was observed in the M2 alternative macrophage subset upon AMG
treatment, which may be modulating the immune response as well (Figure 5.7).
160
A B
Control 24 h AMG 48 h AMG0.0
0.2
0.4
0.6
0.8
IL-1
7+
(perc
en
t C
D3
+C
D4
+ c
ell
s)
*
Control 24 h AMG 48 hAMG0.0
0.1
0.2
0.3
0.4
0.5
*
IL-1
7+
(pe
rce
nt
CD
3+C
D4
+ c
ells)
C D
Control 24 h AMG 48 h AMG0.0
0.1
0.2
0.3
0.4
0.5
IL-1
0+
(Pe
rce
nt
CD
3+C
D4
+cells)
Control 24 h AMG 48 h AMG0.0
0.1
0.2
0.3
0.4
0.5
IL-1
0+
(Pe
rce
nt
CD
3+C
D4
+cells)
Figure 5.6. Early changes in the fraction of CD4+ T cells from the spleen and lymph nodes
that express IL-17 or IL-10 induced by AMG treatment. (A and C) cells from the spleen and
(B and D) cells from auricular lymph nodes. Values are mean ± SEM (n=4). *p<0.05 compared
to control.
161
M1 M2ab M2c0
5
10
15
20
25* Control
AMG
Perc
en
t L
N M
acro
ph
ag
es
Figure 5.7. AMG-induced changes in macrophage subsets in lymph nodes after 14 days of
treatment. For phenotyping, M1 was defined as MHCII+/CD68
-, M2ab was defined as
MHCII+/CD68
+, and M2c was defined as CD68
+/MHCII
-. Open bars represent control; solid bars
represent AMG-treated rats. Values are the mean ± SEM (n=4 control, n=6 AMG). *p<0.05
compared to control.
162
5.6 DISCUSSION
The fact that primary aromatic amines are associated with a high incidence of IDRs
independent of their therapeutic class presumably reflects the fact that they are readily oxidized
to reactive metabolites. The liver is the major site of drug metabolism and reactive metabolite
formation; however, with the exception of bromfenac, liver injury associated with aromatic
amines is usually part of a more general hypersensitivity reaction rather than being confined to
the liver. The liver contains high concentrations of P450s that can oxidize a wide variety of
xenobiotics to reactive metabolites including components of the diet; therefore, the liver also
possesses multiple protective mechanisms such as high levels of glutathione and detoxifying
enzymes. In addition, the major immune response to covalent binding in the liver appears to be
immune tolerance. Even when a xenobiotic is able to cause an immune response leading to liver
injury, it appears that activation of the immune system outside of the liver is required to
overcome immune tolerance in the liver [211].
The ease of aromatic amine oxidation also means that it can be oxidized by enzymes
other than P450s such as MPO [94]. The fact that neutrophils and their precursors contain high
levels of MPO [252] is presumably why most aromatic amines can cause agranulocytosis. APCs
also contain MPO and bioactivation of SMX has been demonstrated in APCs [49, 155].
AMG is a first generation aromatase inhibitor and is primarily indicated for the treatment
of breast and prostate cancer. Severe IDRs associated with AMG include: skin rashes,
cholestasis, blood dyscrasias (most notably agranulocytosis), and systemic lupus erythematosus
[253]. The more commonly reported AMG-induced IDRs are related to its effects on specific
immune cells such as the granulocyte progenitors and is delayed in onset [225]. Compared to
other aromatic amine drugs, AMG does not have an electron withdrawing group para to the
163
aromatic amine moiety, such that the electron density in the aromatic ring is higher and AMG
may be more susceptible to oxidation than other drugs with an aromatic amine moiety. In
preliminary studies, neutrophils have been found to be capable of oxidizing AMG to a
hydroxylated species in vitro (unpublished observations). Thus, AMG has the potential to elicit
immune effects to initiate IDRs.
Previously we had found that treatment of rats with AMG did not lead to liver injury;
however, in this study we found an immune response to AMG including proliferation of
leukocytes in the white pulp of the spleen and an increase in Gro/KC (CXCL1) and IL-5 after 7
days of treatment that returned to normal at 14 days. IL-5 is involved with immunoglobulin
production in B-cells; however it takes time for antibodies to be produced and it is unlikely that
an antibody response would be detected as early as 7 days. In contrast, elevated IL-5 has been
found in patients with eosinophil-associated drug hypersensitivity, and the increase in IL-5
preceded the elevation in eosinophils, which is known to be driven by IL-5, suggesting that it
could be a biomarker to predict the onset of drug hypersensitivity syndrome [254]. Interestingly,
GM-CSF is also associated with the activation of eosinophils and the successive decrease in GM-
CSF observed with AMG is consistent to a decrease in or modulation of a response. GM-CSF is
a cytokine involved primarily with granulopoiesis but is also associated with the activation of
granulocytes such as neutrophils and implicated in a variety of inflammatory diseases [255]. The
decrease of GM-CSF with AMG may also be a negative feedback mechanism to the increase in
granulocyte progenitors induced by AMG in the bone marrow that was previously observed
[216].
164
Immune cell infiltration was not observed within the lymphoid organs; however, a
significant increase in cellular proliferation was found in the white pulp of the spleen upon AMG
treatment, and this was attributed to the proliferation of CD4 T cells. Further investigation found
that these changes may potentially be more of a localized effect rather than an immune response
to drug treatment, as re-stimulation of splenocytes through the LTT found no proliferation. The
LTT is a diagnostic tool to determine drug hypersensitivity and is dependent on the formation of
memory lymphocytes after a subsequent primary immune sensitization. It is likely that the
immune response is to the reactive metabolite of AMG, rather than to the parent drug and the
immune response was insufficient to lead to a large number of memory T cells that cross react
with the parent drug. Unfortunately, a drug-modified protein is currently unavailable for testing,
and a negative LTT does not necessarily indicate lack of immune response. In contrast, no
cellular proliferation was observed in the auricular lymph nodes, yet these cells were able to
induce a subtle proliferative response to AMG upon secondary re-stimulation. The doses of
AMG that increased lymphocyte proliferation were approximately two times higher than the
blood levels of AMG found in patients [256], and only one animal had only a marginal
stimulation index at this concentration so the results are of questionable significance. An in vitro
method was recently reported to detect drug hypersensitivity in naive T cells co-cultured with
dendritic cells [93], and this method may be a better method to determine whether the drug is
able to elicit primary sensitization. Nevertheless, these immune changes may be below the
threshold to elicit a systemic immune response, which may be why rats do not experience
pathology.
In terms of a cell-specific response, a subtle up-regulation of IL-17- and probably IL-10-
producing CD4+ T cells was observed in the spleen at 24 h, as well as an increase in M2
165
macrophages in lymph nodes after 14 days. Early increases in both IL-17 and IL-10 upon AMG
treatment suggest modulation of an immune response. IL-17 is a proinflammatory cytokine that
can be released from Th17 cells to mediate inflammatory downstream effects [257], and has
been found to be increased as early as 2 h in mice with acetaminophen-induced liver injury,
which suggests that this cell type has innate functions in addition to its adaptive functions [258].
In contrast, IL-10 is an anti-inflammatory cytokine released from T-regulatory cells to dampen
immune responses such that early up-regulation by AMG may suppress immune activation.
Macrophages have also been implicated in the pathogenesis of drug-induced reactions in
both an inflammatory and protective capacity due to the heterogeneity of different macrophage
subsets. AMG induced an increase in the M2 macrophage subset, which may prevent a
pathological immune response. M2 alternatively activated macrophages were found to be
induced in the liver of acetaminophen-treated mice and depletion of these macrophages
prolonged acetaminophen-induced liver injury [259]. This suggests that macrophages may play a
protective role to prevent the initiation of a response, and this may be what is occurring with
AMG. In our studies, M2 macrophages were identified only by differential expression of CD68
and MHCII. Further investigation and more detailed phenotyping of these macrophages with M2
markers such as CCR2, arginase 1, Ym1, etc. will be required to determine more accurately what
role these macrophages play in AMG-induced IDRs.
From our findings, there was data to suggest that the main deterrent to AMG-induced
IDRs may be early immune modulating events that prevent immune activation. These changes
are consistent with an immune response that ends in immune tolerance. Given that AMG-
induced IDRs are idiosyncratic in humans it is not surprising that a clinically evident adverse
166
reaction did not occur in these rats but evidence of an immune response may be a biomarker to
predict which drug candidates are likely to cause IDRs in susceptible individuals.
167
CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTION
168
6.1 DISCUSSION AND CONCLUSIONS
As our understanding of the events that lead up to an IDR are limited, any mechanistic
clues as to how these reactions may lead to an immune response and subsequent pathology
would have profound implications on the prediction, prevention, detection, and treatment of
IDRs. In our studies, the approach has not been to focus on a specific type of organ injury, but
rather to concentrate on a group of drugs that are frequently associated with IDRs. The general
hypothesis of my thesis research is that aromatic amine drugs may induce IDRs through immune
mechanisms. However, currently there is no valid animal model of aromatic amine-induced IDRs
in which to fully test this hypothesis; animal models of IDRs are more difficult to develop than
merely administering the drug alone [61]. Nevertheless, monitoring the immune changes induced
by aromatic amine drugs may reveal how these drugs activate or suppress the immune system,
which may perhaps lead to the development of an animal model. In the case of the absence of a
reaction, this information may be very important to understanding why the majority of the
population are tolerant to IDRs, and identifying the factors that overcome this suppression will
be essential to understanding individual susceptibility to IDRs.
Our initial studies were an extension of previous findings in our lab that tested the danger
hypothesis in SMX-treated mice in which few hepatic gene changes were observed [37]. These
findings were surprising, because aromatic amines can be fairly easily oxidized to reactive
metabolites, and we were curious to see whether other aromatic amine drugs induced similar
changes and whether these changes were species-specific. However, even in Brown Norway rats
treated with SMX, the hepatic gene changes were minimal and varied quite significantly from
the other aromatic amine drugs tested: DDS and AMG. Since SMX and DDS are structurally
quite similar, it is disconcerting to see such differences. One explanation may have been the
169
differences in the dosages, although DDS was given at a much lower dose than SMX, and
dosages were designed to produce similar blood levels to those found in patients for each drug.
Another possible explanation may be the pharmacology of the drugs wherein DDS has a higher
volume of distribution and protein binding than SMX [98]. Variations in drug localization could
also account for these differences because SMX-OH was found to bind to the cell surface
whereas the N-hydroxyl DDS metabolite was found to form adducts intracellularly [203].
The similarities observed between DDS and AMG in the upregulation of genes involved
with the Keap1-Nrf2-ARE pathway are commonly found with drugs that induce IDRs [37, 204,
205]. Although, changes in genes regulated by the Keap1-Nrf2-ARE pathway are not direct
indicators of cellular stress, expression of these genes implies the presence of a cellular insult
that requires these genes to respond. We also focused on the downstream effects of protein
expression, and more importantly, protein functionality. However, our studies on thioredoxin
reductase and glutathione S-transferase activity found that gene changes do not necessarily
translate to functional changes. Thus, although gene changes could be biomarkers to predict
drugs associated with IDRs, focusing on protein pathways may be a better approach for
elucidating mechanisms.
Interestingly, despite the lack of many changes in gene expression, Eiih was up-regulated
at acute time points by all aromatic amine drugs tested and also by other drugs associated with
IDRs such as clozapine and nevirapine. Although the function of this gene is yet to be
determined, our findings show that it has the potential to act as a biomarker of drugs associated
with IDRs. However, due to the lack of commercially available reagents, we are unable to
investigate the mechanistic implications of Eiih expression further than testing several drugs
170
associated with IDRs and also testing for the lack of Eiih up-regulation in drugs not associated
with IDRs. Eiih was found to be up-regulated by insulin in rats [187], and it has been reported
that insulin plays a role in maintaining innate immunity [260]. Thus, Eiih could potentially be
involved in the immune response. It should also be noted that the change in expression of Eiih
induced by SMX using RT-PCR was not significant; therefore, a lack of Eiih response does not
demonstrate that a drug cannot cause IDRs. This illustrates the difficulty of finding good
biomarkers that are universally applicable. Nevertheless, this is the first systematic study of
drugs containing the primary aromatic amine moiety in the context of their common association
with IDRs through a global screen of hepatic gene expression. Not surprisingly, even for drugs
with the same chemical moiety and the potential to form common reactive metabolites, it seems
that most of the effects are drug-specific. Given the lack of commonality between drugs that are
chemically quite similar, finding universal tests to predict IDRs may not be possible, and it is
more likely that each drug elicits characteristic effects based upon where it is accumulated within
the body.
Our concern for the lack of significant changes in the liver induced by aromatic amine
drugs led to a comprehensive study in the liver to test the changes induced by AMG. AMG is
structurally and functionally significantly different from SMX and DDS, and it was chosen for
its uniqueness despite containing the aromatic amine moiety. Greater changes had been expected
with AMG because it induced the most significant changes in gene expression, and so it was
used as a prototypical aromatic amine drug. Even so, few physiological changes were found in
the liver with AMG despite monitoring up to 14 days. AMG has been reported to induce P450
and decrease its own half-life as well as that of other drugs given concurrently [176, 215];
therefore, it may be possible that increased AMG clearance led to a decrease in response. On the
171
other hand, increased metabolism could also lead to the generation of more reactive metabolites
leading to greater hepatocyte damage. However, no matter what mechanism, the liver seems to
be able to handle the insult because AMG did not induce liver injury at the time points tested.
In terms of gene expression, AMG induced differential gene changes associated with the
apoptotic and mitochondrial pathways, although there was no evidence for apoptosis as
determined by the TUNEL assay or mitochondrial injury in the form of microvesicular steatosis.
Despite this, these changes could be very important as danger signals to initiate an immune
response because APCs were only activated in the presence of cell death in a rodent model of
SMX-induced immunogenicity [49]. Apoptosis in the liver also has broader implications because
apoptotic hepatic leukocytes may mediate a tolerogenic effect whereas apoptosis of hepatocytes
may stimulate the immune system through the release of DAMPs. Unfortunately we were unable
to differentiate which cells undergo apoptosis because the gene expression of the liver was tested
as a whole rather than separating the hepatocytes from immune cells. Hepatic gene changes were
also observed in innate immune genes, which also suggests that innate immunity is able to
mitigate the toxic effects of the drug.
Interestingly, aromatic amine drugs rarely induce direct liver injury in humans, but rather
hepatotoxicity is usually due to indirect effects of generalized hypersensitivity. Although AMG
does induce symptoms of cholestasis [208], this type of liver injury is not usually life-
threatening. The basis for our focus on the liver as a target organ of aromatic amine drugs was
due to the abundance of metabolizing enzymes in the liver, and the ability of aromatic amines to
be oxidized to reactive metabolites; however, this seems to be disconnected from what is
172
clinically observed. In spite of this, studying the effects of aromatic amines in the liver has given
us another perspective and shifted our attention to metabolism by immune cells.
Although drug metabolism by immune cells does not make a significant contribution to
the clearance of most drugs, they do possess drug metabolizing enzymes. Neutrophils incubated
with AMG in the presence of ascorbic acid were able to oxidize AMG to a hydroxylated
metabolite (see Appendix 2). It is likely the hydroxylamine but we were unable to identify the
exact location of the oxidation because of a lack of an authentic N-hydroxyl-AMG standard;
however, this data suggest that enzymes within neutrophils are able to oxidize AMG.
The most profound and interesting findings for AMG were the elevation of peripheral
blood neutrophils in rats that was not observed with the other aromatic amine drugs, SMX and
DDS (see Appendix 3). Although it did seem that DDS induced an increase in neutrophils, this
was not statistically significant; however, this is consistent with DDS being associated with a
higher incidence of agranulocytosis than SMX. These changes are opposite to the idiosyncratic
agranulocytosis that is observed in patients treated with AMG [261] and to a mouse model of
AMG-induced leucopenia [262]. We have treated mice with AMG and found a similar decrease
in WBCs as previously reported (see Appendix 4); however, we believe that the rat may be a
more clinically relevant model of idiosyncratic drug-induced agranulocytosis. This is because
rats were found to have more sustained blood levels of AMG than mice (data not shown), and
often patients taking other drugs that are associated with idiosyncratic agranulocytosis such as
clozapine experience an initial neutrophilia that occurs before the onset of agranulocytosis [263].
Thus, we believe that the rat is a better model of the early changes in AMG-induced
agranulocytosis. The AMG studies ended at 14 days because mice were shown to develop
173
leucopenia as early as 14 days, and so it was assumed that this would be enough time to develop
some cellular response in the peripheral blood; yet it may be possible that if AMG treatment in
the rats were prolonged, the rats may eventually develop a decrease in neutrophils.
Although the kinetics vary slightly, the elevated neutrophils induced by AMG in rats are
similar to what is observed in clozapine-treated rats [264] and rabbits [265]. AMG induced an
increased release of neutrophils from the bone marrow; however, the BrdU-labeled neutrophils
appeared to have a longer half-life in the peripheral blood; this is in contrast to the decrease in
neutrophil half-life observed in clozapine-treated rabbits [265]. We were unable to perform a
more detailed study on neutrophil kinetics because of the small blood volume of rats; however,
we did find a decrease in apoptotic neutrophils upon AMG treatment using annexin V and PI.
This is likely confounded by a decreased expression of phosphatidylserine in newly formed
neutrophils and may not indicate an intrinsically prolonged survival of neutrophils. It is also
unknown whether AMG directly affects the neutrophils and leads to a compensatory production
of granulocyte progenitors in the bone marrow or whether AMG directly effects the bone
marrow. From these findings, it is more likely that AMG affects the bone marrow directly
because there did not seem to be a change in neutrophil clearance. In addition, no change was
observed in the total WBC count because the elevation in neutrophils was accompanied with a
decrease in lymphocytes; the exact implications of this are not known. Neutrophils in AMG-
treated rats also expressed lower levels of CD62L suggesting activation and extravasation into
tissue targets, and future effort should be put forth to monitor neutrophil trafficking in relation to
bone marrow neutrophil production to determine whether this is an important effect of AMG.
The effect of AMG on neutrophils is intriguing and more research should be invested into
174
determining how this may be related to drug-induced blood dyscrasias, specifically
agranulocytosis.
The majority of the immune changes induced by AMG in the rat were subtle, and even
the most profound immune change, cell proliferation in the spleen, did not lead to a positive
LTT. In isolation, this suggests that AMG may be mitogenic rather than immunostimulatory; yet,
the LTT is not entirely specific and depends essentially on the development of an immune
memory response, which may not be very strong because the rats were only treated for 14 days.
Thus, testing the ability of AMG to induce primary T cell sensitization may be a better approach.
Conversely, in the auricular lymph nodes, no cell proliferation was observed, but slight increases
in T cell proliferation were found upon re-stimulation at higher AMG concentrations. These
results are difficult to interpret because they suggest small localized immune reactions, which
may not be sufficient to stimulate an immune response that becomes pathological. However,
these small immune changes are mechanistically important because they are associated with an
early elevation of IL-17 and IL-10, which suggests a modulating effect that may prevent immune
activation. Throughout these studies there have been changes to suggest immune tolerance that
prevents the initiation of an IDR in the rats. However, it must be noted that although we loosely
call this immune tolerance, immunologists might disagree and call it immune suppression or
modulation of basal immune responses because we have not determined whether these changes
induced by AMG are in response to antigen through activation of the T cell receptor. Perhaps a
more useful approach would be in vitro studies to determine the effect of AMG on specific cell
types to determine whether immune cells are directly activated. In vivo studies are useful for
detecting an overall physiological response; however, it may mask some very important and
175
essential mechanisms, especially for drugs such as AMG in which the immune response is quite
mild.
Moving forward, it is anticipated that it will be difficult to continue studying AMG in the
context of IDRs because it is no longer commonly used, and obtaining clinical samples to
confirm whether these findings in rats are applicable to humans will be difficult. Although our
initial attempts to discover the factors common to aromatic amine drugs that are responsible for
inducing IDRs were not entirely successful, as the projects evolved, we have found various clues
as to how the body may be responding to aromatic amine drugs and how these changes may be
implicated in IDRs. This is especially true with AMG as our focus gradually shifted to it as a
prototypical aromatic amine drug and greater evidence was found to suggest immune modulation
as a mechanism to prevent the initiation of an immune-mediated IDR.
176
6.2 FUTURE DIRECTIONS
The current findings for AMG have provided a strong foundation for further investigation
of the mechanisms of aromatic amine-induced IDRs; however, the biggest deterrent for the
success of future studies is the lack of an antibody against AMG that could be used to detect
covalent binding. In fact, this has been a major limitation throughout my thesis research. The
generation of AMG-modified protein was attempted using several methods including through the
formation of a diazonium salt and through the targeting of the heterocyclic ring of AMG; both of
which were not successful. The major chemical limitation seems to be maintaining the stability
of the heterocyclic ring while introducing a reactive site required for protein conjugation.
Nevertheless, if a drug-modified protein were available, an anti-AMG antibody could be
generated. The antibody could then be used for covalent binding studies to determine exactly
where AMG covalently binds and which target organs to focus on. It would also allow for the
determination of which cells and where within the cell AMG binding is localized. In addition,
the AMG-modified protein would allow us to perform more detailed studies on the metabolism
of AMG and the involvement of the reactive metabolite in immune activation in assays such as
the LTT.
Another area of focus would be to determine whether AMG directly activates immune
cells and the functional consequences of such activation. These studies would primarily be
carried out in vitro before confirming the findings in vivo. The effect of AMG on neutrophils is
worth pursuing, and it is essential to determine whether other drugs associated with
agranulocytosis also induce similar changes, for example in CD62L expression. In doing so,
perhaps an expression profile could be developed for drug-induced agranulocytosis that could be
used to test new drug candidates. It is also important to determine the effect of the neutrophils in
177
the scheme of immune activation, and how it may be involved in an adaptive cellular response.
Previously it was reported that endotoxin-induced CD11cbright
/CD62Ldim
/CD11bbright
/CD16bright
neutrophils were able to inhibit T-cell proliferation in a mechanism dependent on the release of
hydrogen peroxide [160]. These results are quite interesting because the implications are that the
innate immune system may suppress a T cell response, and this protective effect may prevent the
initiation of IDRs and should be investigated as a potential mechanism for AMG.
It is also essential to determine whether a reactive metabolite of the drug is involved with
the immune response and whether this can lead to primary immune sensitization. Park et al. have
had been successful doing this with SMX-NO, in a method that involves co-culture of naive T-
cells with APCs pre-treated with drug [93], but they have not as yet determined whether other
drugs that induce IDRs may act through a similar mechanism. Thus it would be interesting to see
whether this is true for AMG and other aromatic amine drugs, and if it works, this would be
widely applicable to preclinical drug safety testing.
Lastly, development of an animal model of AMG-induced IDRs would provide an
invaluable tool to rigorously test new hypotheses. Given that IDRs do not occur in most patients
that take a drug like AMG it is not surprising that simply giving the drug to animals does not
produce an IDR. A better understanding of the immune effects of drugs, especially their effects
on immune tolerance, may make it easier to develop animal models that could be utilized to
study the detailed steps involved in the initiation of an IDR.
178
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211
APPENDIX 1
Supplementary Material from Chapter 2: Changes in Gene Expression Induced by
Aromatic Amine Drugs
Table A1.1. SMX-induced hepatic gene changes at 12 hr (fold-change between treated and
controls).
RefSeq Gene symbol Gene name Fold-
change
p-value
Up-regulation
NM_001009965 Eiih hepatic protein EIIH 2.86 0.0227
J03863 Sds serine dehydratase 2.15 0.0237 NM_021660 Ihpk2 inositol hexaphosphate kinase 2 1.91 0.0206
NM_030845 Cxcl1 chemokine (C-X-C motif) ligand 1 1.67 0.0476
NM_198776 MGC72973 hemoglobin, beta, adult major chain 1.67 0.1509 NM_022671 Onecut1 one cut homeobox 1 1.61 0.3665
NM_033234 Hbb hemoglobin, beta 1.54 0.1647
NM_001034125 Per1 period homolog 1 (Drosophila) 1.53 0.1517 NM_001013853 LOC287167 globin, alpha 1.52 0.1826
NM_001010946 LOC288741 huntingtin interactor protein E 1.51 0.0480
NM_012571 Got1 glutamate oxaloacetate transaminase 1 1.51 0.0035 NM_001007146 Tob2 transducer of ERBB2, 2 1.50 0.0456
Down-regulation
NM_017136 Sqle squalene epoxidase -1.51 0.0336 NM_017268 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 -1.52 0.0098
NM_080886 Sc4mol sterol-C4-methyl oxidase-like -1.55 0.0196
NM_057107 Acsl3 acyl-CoA synthetase long-chain family member 3 -1.57 0.0374 NM_013060 Id2 inhibitor of DNA binding 2 -1.58 0.0221
NM_053380 Slc34a2 solute carrier family 34 (sodium phosphate), member 2 -1.59 0.0076
NM_001012072 Ppp1r3c protein phosphatase 1, regulatory subunit 3C -1.61 0.0383 NM_001008352 Pmvk phosphomevalonate kinase -1.62 0.0108
NM_001006995 Acat2 acetyl-Coenzyme A acetyltransferase 2 -1.63 0.0259
NM_001106882 Unc5cl unc-5 homolog C (C. elegans)-like -1.66 0.0357 NM_019237 Pcolce procollagen C-endopeptidase enhancer protein -1.67 0.0075
NM_198738 Psat1 phosphoserine aminotransferase 1 -1.67 0.0772
NM_032063 Dll1 delta-like 1 (Drosophila) -1.68 0.0029 NM_212505 Ier3 immediate early response 3 -1.69 0.0022
NM_053328 Bhlhb2 basic helix-loop-helix domain containing, class B2 -1.72 0.0969
NM_001013071 Tm7sf2 transmembrane 7 superfamily member 2 -1.76 0.0139 NM_206950 Mig12 MID1 interacting G12-like protein -1.76 0.0010
NM_145677 Slc25a25 solute carrier family 25 (mitochondrial, phosphate), member
25
-1.86 0.0162
NM_001033066 Ddhd1 DDHD domain containing 1 -1.87 0.0323
NM_031327 Cyr61 cysteine rich protein 61 -1.87 0.0025 NM_021835 Jun jun proto-oncogene -1.97 0.0019
NM_001009632 G0s2 G0/G1 switch gene 2 -2.11 0.0201
NM_019232 Sgk serum/glucocorticoid regulated kinase 1 -2.70 0.0023 NM_053769 Dusp1 dual specificity phosphatase 1 -3.58 0.0020
Data shown are gene changes ≥ 1.50 fold expression. p-values are unadjusted (n = 4).
212
Table A1.2. DDS-induced hepatic gene changes at 12 hr (fold-change between treated and
controls).
RefSeq Gene symbol Gene name Fold-
change
p-value
Up-regulation
NM_013215 Akr7a3 aldo-keto reductase family 7, member A3 (aflatoxin aldehyde reductase)
5.38 2.89 x 10-7
NM_001009965 Eiih hepatic protein EIIH 4.23 0.0038
NM_001013181 Zbtb16 zinc finger and BTB domain containing 16 3.55 0.0154 NM_001009920 Yc2 glutathione S-transferase ,Yc2 subunit 3.19 7.52 x 10-7
NM_001012111 Lpin1 lipin 1 2.59 0.0200
NM_001014162 RGD1309578 acyl-CoA synthetase medium-chain family member 5 2.37 0.0329 NM_022407 Aldh1a1 aldehyde dehydrogenase family 1, member A1 2.33 0.0049
NM_058208 Socs2 suppressor of cytokine signaling 2 2.08 0.1430
NM_012603 Myc myelocytomatosis oncogene 2.00 0.0238 BC89763 Nr1i3 nuclear receptor subfamily 1, group I, member 3 1.95 0.0052
NM_145775 Nr1d1 nuclear receptor subfamily 1, group D, member 1 1.94 0.0885
NM_001025623 Vnn1 vanin 1 1.92 0.0957 NM_052798 Zfp354a zinc finger protein 354A 1.85 0.1089
NM_198776 MGC72973 hemoglobin, beta, adult major chain 1.80 0.1046
Down-regulation
NM_001012072 Ppp1r3c protein phosphatase 1, regulatory subunit 3C -1.81 0.0129
NM_053380 Slc34a2 solute carrier family 34 (sodium phosphate), member 2 -1.82 0.0014 NM_032063 Dll1 delta-like 1 (Drosophila) -1.85 0.0008
NM_031678 Per2 period homolog 2 (Drosophila) -1.86 0.0074
NM_012565 Gck glucokinase -1.89 0.0450 NM_001013175 Apol3 apolipoprotein L, 3 -1.90 0.0005
NM_053328 Bhlhb2 basic helix-loop-helix domain containing, class B2 -1.93 0.0500
NM_138912 Ppp1r3b protein phosphatase 1, regulatory subunit 3B -1.96 0.0038 NM_001009632 G0s2 G0/G1 switch gene 2 -2.03 0.0263
NM_019138 Cyp7b1 cytochrome P450, family 7, subfamily b, polypeptide 1 -2.05 0.0286
NM_012703 Thrsp thyroid hormone responsive protein -2.08 0.0023 NM_001003706 LOC360228 WDNM1 homolog -2.17 0.0022
NM_021835 Jun jun proto-oncogene -2.49 0.0002
NM_031327 Cyr61 cysteine rich protein 61 -2.50 0.0001
NM_001033066 Ddhd1 DDHD domain containing 1 -2.50 0.0040
NM_206950 Mig12 MID1 interacting protein 1 -2.59 1.06 x 10-5
NM_145677 Slc25a25 solute carrier family 25 (mitochondrial, phosphate), member 25
-2.67 0.0008
NM_053769 Dusp1 dual specificity phosphatase 1 -3.01 0.0052
NM_019232 Sgk serum/glucocorticoid regulated kinase 1 -3.72 0.0003
Data shown are gene changes ≥ 1.80-fold expression. p-values are unadjusted (n = 4).
213
Table A1.3. AMG-induced hepatic gene changes at 12 hr (fold-change between treated and
controls).
RefSeq Gene symbol Gene name Fold-
change
p-value
Up-regulation
NM_001009965 Eiih hepatic protein EIIH 12.94 0.0072 NM_145775 Nr1d1 nuclear receptor subfamily 1, group D, member 1 5.77 0.0291
NM_031576 Por P450 (cytochrome) oxidoreductase 4.78 0.0003
NM_133401 Abcb1a ATP-binding cassette, sub-family B, member 1A 4.50 0.0001 NM_133586 Ces2 carboxylesterase 2C 4.38 0.0051
NM_022407 Aldh1a1 aldehyde dehydrogenase family 1, member A1 4.15 0.0121
NM_001008321 Gadd45b growth arrest and DNA-damage-inducible, beta 3.81 0.0009 NM_001008363 Zfand2a zinc finger, AN1-type domain 2A 3.77 0.0076
NM_022927 Mid1 midline 1 3.71 0.0021
NM_001009474 Pir pirin (iron-binding nuclear protein) 3.67 2.98 x 10-5 NM_173295 Udpgtr2 UDP glucuronosyltransferase 2 family, polypeptide B1 3.59 0.0008
NM_144743 Ces6 carboxylesterase 2A 3.48 3.33 x 10-5
NM_013105 Cyp3a23 cytochrome P450, family 3, subfamily a, polypeptide 23/polypeptide 1
3.37 3.79 x 10-5
NM_144755 Trib3 tribbles homolog 3 (Drosophila) 2.79 0.0329
NM_012600 Me1 malic enzyme 1, NADP(+)-dependent, cytosolic 2.76 0.0076 NM_033352 Abcd2 ATP-binding cassette, sub-family D (ALD), member 2 2.57 0.0039
NM_001047090 Aph1b anterior pharynx defective 1b homolog (C. elegans) 2.56 0.0291 NM_175761 Hspca heat shock protein 90, alpha (cytosolic), class A member 1 2.52 0.0145
NM_013063 Parp1 poly (ADP-ribose) polymerase 1 2.46 0.0002
NM_001001509 Hdmcp solute carrier family 25, member 47 2.46 0.2365 NM_001007706 RGD1359191 tRNA methyltransferase 61 homolog A (S. cerevisiae) 2.40 0.1436
NM_001025623 Vnn1 vanin 1 2.35 0.3351
NM_173116 Sgpl1 sphingosine-1-phosphate lyase 1 2.34 0.0246 NM_001011901 Hsph1 heat shock 105/110 protein 1 2.34 0.0048
NM_001034090 Ephx1 epoxide hydrolase 1, microsomal 2.33 0.0009
NM_001109022 LOC368066 indolethylamine N-methyltransferase 2.32 0.1376 NM_013215 Akr7a3 aldo-keto reductase family 7, member A3 (aflatoxin
aldehyde reductase)
2.23 0.0305
NM_199103 Ikbkg inhibitor of kappa light polypeptide gene enhancer in B-
cells, kinase gamma
2.23 0.0006
NM_001009920 Yc2 glutathione S-transferase Yc2 subunit 2.23 0.0066
NM_031614 Txnrd1 thioredoxin reductase 1 2.23 0.0003 NM_131906 Slco1a4 solute carrier organic anion transporter family, member 1A2 2.22 0.0702
NM_001005384 Osmr oncostatin M receptor 2.21 0.0501
NM_212504 Hspa1b heat shock 70kD protein 1B 2.20 0.0130 NM_178091 Insig2 insulin induced gene 2 2.20 0.0018
M58040 Tfrc transferrin receptor 2.19 0.1851
NM_024127 Gadd45a growth arrest and DNA-damage-inducible, alpha 2.19 0.1613 NM_144752 Oas1b 2-5 oligoadenylate synthetase 1B 2.14 0.0020
NM_145091 Pdp2 pyruvate dehyrogenase phosphatase catalytic subunit 2 2.09 0.0115
NM_001007687 Cndp1 carnosine dipeptidase 1 (metallopeptidase M20 family) 2.08 0.0635 NM_001010946 LOC288741 huntingtin interactor protein E 2.08 0.0782
NM_053906 Gsr glutathione reductase 2.06 0.0000
NM_001013181 Zbtb16 zinc finger and BTB domain containing 16 2.06 0.5563 NM_201424 Ugt1a3 UDP glycosyltransferase 1 family polypeptide A3 2.06 2.86 x 10-5
AF082126 Ahr aryl hydrocarbon receptor 2.05 0.0733
NM_019283 Slc3a2 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2
2.05 0.0485
NM_001008301
Usp14
ubiquitin specific peptidase 14 (tRNA-guanine
transglycosylase) 2.04 0.0135 ENSRNOT00000056174 Usp18 ubiquitin specific peptidase 18 2.04 0.0300
NM_153312 Cyp3a2 cytochrome P450, family 3, subfamily a, polypeptide 2 2.03 0.0116
NM_001007690 Fbxo30 F-box protein 30 2.01 0.0246 NM_053612 Hspb8 heat shock protein B8 2.01 0.0036
Down-regulation
NM_031678 Per2 period homolog 2 (Drosophila) -2.00 0.1059 NM_001003706 LOC360228 WDNM1 homolog -2.00 0.1222
NM_001108118 Itga5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) -2.01 0.0205
NM_031815 Inhbe inhibin beta E -2.01 0.0132
214
NM_001011985 Mknk2 MAP kinase-interacting serine/threonine kinase 2 -2.02 0.0612
NM_080782 Cdkn1a cyclin-dependent kinase inhibitor 1A -2.03 0.3319
NM_198780 Pck1 phosphoenolpyruvate carboxykinase 1 (soulable) -2.05 0.0272 NM_012941 Cyp51 cytochrome P450, subfamily 51 -2.07 0.0346
NM_001109430 LOC680883 leucine-rich repeats and transmembrane domains 2 -2.07 0.0635
NM_012508 Atp2b2 ATPase, Ca++ transporting, plasma membrane 2 -2.08 0.0006 NM_001106873 LOC301113 solute carrier family 25 (mitochondrial; phosphate), member
23
-2.11 0.0257
NM_001034081 Paqr7 progestin and adipoQ receptor family member VII -2.14 0.0071 NM_178096 Nrep neuronal regeneration related protein -2.15 0.0273
ENSRNOT00000027756 Fads2 fatty acid desaturase 2 -2.20 0.0116
NM_001006995 Acat2 acetyl-Coenzyme A acetyltransferase 2 -2.28 0.0526 BC107921 RGD1309879 family with sequence similarity 89, member A -2.31 0.0121
NM_022392 Insig1 insulin induced gene 1 -2.32 0.0272
NM_012703 Thrsp thyroid hormone responsive -2.33 0.0441 NM_198790 Rgsl2h regulator of G-protein signaling like 2 homolog (mouse) -2.34 0.0385
NM_001033066 Ddhd1 DDHD domain containing 1 -2.41 0.1299
NM_001004096 Reg4 regenerating islet-derived family, member 4 -2.47 0.0026 NM_022392 Insig1 insulin induced gene 1 -2.50 0.0298
NM_022280 Lrat lecithin-retinol acyltransferase (phosphatidylcholine-retin -2.59 0.0212
U11038 Lox lysyl oxidase -2.70 0.3845 NM_001109912 Tsc22d1 TSC22 domain family, member 1 -2.72 0.0080
NM_013144 Igfbp1 insulin-like growth factor binding protein 1 -2.73 0.5886
ENSRNOT00000024658 Slc28a2 solute carrier family 28 (sodium-coupled nucleoside transporter), member 2
-2.80 0.0076
NM_053922 Acacb acetyl-Coenzyme A carboxylase beta -2.80 0.0197 NM_001008352 Pmvk phosphomevalonate kinase -2.83 0.0063
NM_053380 Slc34a2 solute carrier family 34 (sodium phosphate), member 2 -3.02 0.0025
NM_019138 Cyp7b1 cytochrome P450, family 7, subfamily b, polypeptide 1 -3.04 0.0815 NM_001012621 Slc13a4 solute carrier family 13 (sodium/sulfate symporters),
member 4
-3.15 0.0023
NM_139258 Bmf Bcl2 modifying factor -3.25 0.0123 NM_053769 Dusp1 dual specificity phosphatase 1 -3.61 0.0712
NM_145677 Slc25a25 solute carrier family 25 (mitochondrial, phosphate), member
25
-4.05 0.0076
NM_019232 Sgk serum/glucocorticoid regulated kinase 1 -5.37 0.0064
Data shown are gene changes ≥ 2-fold expression. p-values are adjusted using FDR (n = 4).
215
APPENDIX 2
In Vitro Metabolism of AMG by Neutrophils to a Hydroxylated Metabolite
A2.1 BACKGROUND
Aminoglutethimide (AMG) has been found to decrease the number of circulating
neutrophils in mice, but it has the opposite effect on neutrophils in rats. This discrepancy could
be caused by a species difference in the metabolism of AMG because N-hydroxyl AMG was
observed in the urine of humans and mice [1, 2], but not in rats [3]. However, lack of detection
of the N-hydroxyl AMG metabolite does not mean that it does not form, and localized formation
of reactive metabolites in immune cells, which may not make a significant contribution to
urinary metabolites, may provide a greater stimulus for immune activation. To determine the
metabolic capacity of oxidative enzymes within the neutrophil to directly metabolize AMG,
peripheral blood neutrophils from rats were isolated and incubated with AMG. For this study
neutrophils from mice were tested in addition to rat neutrophils to determine whether there was a
species difference in AMG metabolism to the N-hydroxyl metabolite. Ascorbic acid was added
to the incubation to prevent the oxidation of N-hydroxyl AMG to the nitroso AMG metabolite.
216
A2.2 MATERIALS AND METHODS
A2.2.1 Animals. Male Brown Norway rats (176-200 g) and male C57BL/6 mice (7-9
weeks old) were purchased from Charles River (Montreal, QC). Rodents were housed doubly
under standard conditions with automatic watering, 12:12 hr light/dark cycle at 22°C. Food and
water were provided ad libitum and all animals were given standard rodent chow. Animals were
acclimatized for one week before the start of experiments. The experiment protocol was
approved by and performed in accordance with the University of Toronto Faculties of Medicine
and Pharmacy Animal Care Committee.
A2.2.2 Neutrophil Isolation from Blood. For isolation of peripheral blood neutrophils,
blood (5 mL) was collected terminally, after CO2 asphyxiation, through the central vein from rats
or pooled from the venous sinus of mice into heparin coated syringes. Equal parts of 3% dextran
(in 0.9% NaCl) were mixed with the blood in 15 mL tubes and incubated at room temperature for
18 min. The straw-coloured upper layer was collected and centrifuged at 300g for 5 min. The
pellet was washed once more with Minimum Essential Media (MEM-α; Invitrogen, Life
Technologies) and the leukocytes were resuspended in a final volume of 2 mL MEM-α.
Isolated leukocytes were carefully overlaid on a Percoll gradient (80%, 65%, 55%; GE
Healthcare) and centrifuged at 400g for 30 min at 4°C. The supernatant was removed from the
65% layer and the neutrophils were collected from the 80/65% interface. The cells were washed
once with PBS and then lysed with 2 mL red blood cell (RBC) lysis buffer (155 mM NH4Cl, 10
mM KHCO3, and 0.1 mM EDTA) for 5 min and the reaction was stopped by adding 10 mL PBS.
Cells were washed again before resuspension in 2 mL Hank’s Balanced Salt Solution (HBSS)
217
and cell counting using trypan blue and Countess Automated Cell Counter (Invitrogen, Life
Technologies).
A2.2.3. AMG Metabolism by Activated Neutrophils. The protocol to determine the
metabolism of AMG by activated neutrophils was performed similar to Uetrecht et al. [4].
Briefly, a reaction mixture of 2.5x106 neutrophils (in 250 µL HBSS), 1 mM ascorbate, 40 ng/mL
phorbol 12-myristate 13-acetate (PMA; 5 µL in DMSO), and 100 µM AMG (10 µL solution in
DMSO) was made and incubated at 37 °C in a shaking water bath for 10 min. The mixture was
then centrifuged at 13000g for 2 min and 10 µL of the supernatant was directly injected into an
LC system (SLC-10A, Shimadzu) coupled to a MS detection with ionspray (API 3000, PE Sciex)
to look for AMG metabolites using an Ultracarb 5 µ ODS (30) 100x2.0 mm column
(Phenomenex, Torrance, CA), mobile phase of water-methanol (80:20, v/v), and a flow rate of
0.2 mL/min. The supernatant was analyzed by selective ion monitoring for specific m/z ratios
that corresponded to AMG (m/z 232.5-233.5) and the N-hydroxy-AMG (m/z 248.5-249.5)
metabolite. A separate total ion scan was also performed to determine whether other metabolic
products formed.
218
A2.3 RESULTS AND DISCUSSION
Using LC-MS in the selective ion monitoring (SIM) mode, peaks were observed for
AMG (m/z 233) and a hydroxylated AMG (m/z 249) both at a retention time of approximately
5.7-5.9 min in both rats and mice. The similarity in retention times is not surprising, as the
differences in polarity between AMG and a hydroxylated AMG metabolite are fairly small. A
separate total ion scan of the supernatant found no other substantial metabolic peaks.
Figure A2.1. Metabolism of AMG by rat and mouse neutrophils. Selected ion monitoring
was performed for AMG by scanning m/z 232.5-233.5 and for a hydroxylated AMG metabolite
scanning m/z 248.5-249.5.
219
The differences in the magnitude of the formation of the hydroxylated AMG metabolite
between rats and mice were very subtle with a slightly higher formation observed in the mice
than rat (Figure A2.1). Interestingly, neutrophils were also able to form the hydroxylated AMG
metabolite in the absence of PMA stimulation. Although, less N-hydroxyl AMG was expected to
form in the absence of PMA, it is likely that the low levels of N-hydroxyl AMG observed
without stimulation were due to some activation of neutrophils through the isolation process on a
Percoll gradient or metabolism by other oxidizing enzymes within neutrophils.
Figure A2.2. Mass fragmentation of (A) AMG and (B) product of m/z 249.0.
A
B
220
Further investigation into the hydroxylated metabolite through LC-MS/MS analysis
found that the fragmentation pattern was quite different from AMG (Figure A2.2). Nevertheless,
although these findings suggest that AMG has been hydroxylated by neutrophils, the exact
location of the hydroxyl group is unknown and hydroxylation of the heterocyclic ring is possible
as well (Figure A2.3). If the metabolic product is N-hydroxyl AMG we would expect to see the
loss of water and a m/z of 231. Although, we do see this fragment, the magnitude is low and we
would need to confirm; however, an authentic standard of N-OH AMG is not available.
Figure A2.3. Potential structures for m/z 249 hydroxylation of AMG.
These findings indicate that endogenous enzymes within rodent neutrophils are able to
oxidize AMG to a common metabolite associated with aromatic amine toxicity and the
differences between rats and mice in this metabolic capacity are quite subtle. Although we are
unable to confirm the identity of the metabolite, these findings may play a significant role in
AMG-induced agranulocytosis.
221
APPENDIX 3
Effect of SMX and DDS on Peripheral Blood Neutrophils
A
24 480
5000
10000
15000
20000Control
SMX
DDS
Time (h)
To
tal W
BC
s
(x 1
03
cell
s/m
l b
loo
d)
B
24 480
500
1000
1500
2000
2500Control
SMX
DDS
Time (h)
Neu
tro
ph
ils
(x10
3/
ml
blo
od
)
Figure A3.1 Peripheral blood changes induced by SMX and DDS. Male Brown Norway rats
were treated with 150 mg/kg/day SMX or 20 mg/kg/day DDS and blood was taken from the tail
vein to determine the WBC differential using Wright-Giemsa staining after 24 and 48 h. No
222
change was observed for both drugs in either (A) the total WBC count or (B) the number of
peripheral blood neutrophils. Values are mean ± SEM, n=4. None of the results are statistically
significant by two-way ANOVA.
223
APPENDIX 4
Haematological Effects of AMG in Mice
0 7 14 210
500
1000
1500
2000
4000
6000
8000
10000Total WBCs
Lymphocytes
Neutrophils
Days
Cells (
x10
3/m
l b
loo
d)
Figure A4.1 Changes in peripheral blood WBCs induced by AMG in mice. Male C57BL/6
mice were treated with 75 mg/kg/day AMG through oral gavage in 0.5% methylcellulose
vehicle. Blood was taken at various time-points through the venous sinus of mice and the WBC
differential was determined through Wright-Giemsa staining. A decreasing trend was observed in
the total WBCs and lymphocytes with the most dramatic decrease found in the neutrophils.
Values are mean ± SEM, n=3.
224
APPENDICES REFERENCES
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2. Seago, A., et al., Identification of hydroxylaminoglutethimide as induced urinary
metabolites of aminoglutethimide in C57BL/6 mice, in Biological Oxidation of Nitrogen
in Organic Molecules, J.W. Forrod and L.A. Damani, Editors. 1985, Ellis Horwood Ltd.:
Chichester. p. 149-1563.
3. Egger, H., et al., Metabolism of aminoglutethimide in the rat. Drug Met. Dispos., 1982.
10: p. 405-412.
4. Uetrecht, J., et al., Metabolism of dapsone to a hydroxylamine by human neutrophils and
mononuclear cells. J. Pharmacol. Exp. Ther., 1988. 245: p. 274-279.