ENVR 132/TOXC 142/BIOC142Biochemical & Molecular Toxicology

Induction of Metabolism by Toxicants

Instructor:Edward LeCluyse, Ph.D.

e-mail: edl@cellzdirect.com

Induction: Definitions and Principles

• The process of increasing the amount or theactivity of a protein.

• A homeostatic mechanism for regulating enzymeproduction in a barrier organ, such as the liver,intestine, kidney.

• In enzymology, an inducer usually combines withand deactivates/activates a regulatory proteinwhich leads to increased gene expression.

P450 Enzyme Induction

• Induction can cause marked increases in P450composition (>20-fold) and chemical clearance orbioactivation.

• As a result, induction can increase tolerance to sometoxicants while enhancing the toxicity of others.

• Induction can decrease the therapeutic effect of drugsby increasing the rate and pattern of metabolism.

• Xenobiotics are known to induce enzymes that play amajor or no role in their biotransformation (e.g.,omeprazole vs. ethanol).

Internal Exposure to Natural andMan-made Chemicals

• drugs

• industrial chemicals

• pesticides

• pollutants

• alkaloids

• cigarette smoke

• cruciferous vegetables(indole-3-carbinol)

• secondary plantmetabolites

• toxins produced bymolds, plants, andanimals

• pyrolysis products incooked food

Induction of Rat Liver P450 Enzymes byPrototypical Inducers In Vivo

CLO

PCN

PB

BNF

Inducer

10,693 ± 620489 ± 52CYP4A

12,693 ± 2,2552,460 ± 780CYP3A

1,460 ± 18023.8 ± 4.2CYP2B

3,320 ± 183152 ± 27CYP1A

Induced ActivityControl Activity

In vivo Induction in Male Rats

P450 Enzyme

CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6β-hydroxylation;CYP4A, lauric acid 12-hydroxylation.

Induction and Inhibition of P450 in Mice Treatedwith PB or SKF525A: [14C-methyl]aminopyrine

Unwanted Side-effects ofEnzyme Induction

• Failure of drug therapy

• Chemical tolerance with auto-induction

• Xenobiotic toxicity potentiated

• Chemical carcinogenesis potentiated

• Endogenous substrate metabolism perturbed

• Proliferation of cellular ER and peroxisomes

Effects of Inducers on Rodent LiverPhysiology and Function

Serr

um tr

iazo

lam

(ng

/ml)

Rifampin Effects on Triazolam Disposition

Villikka et al., Clin Pharmacol Ther 1997;61:8-14.

Rifampin Placebo

Acetaminophen Toxicity

ProteinAdducts,

GSHdepletion

Phase IIconjugates

Induced byEtOH andIsoniazid

Disruption of Thyroid Function

UGT1A

Types of P450 Inducers

• Many “prototypical” inducers of specificfamilies or subfamilies of P450 enzymes– CYP1A inducers: 3-MC, BNF, omeprazole, TCDD

– CYP3A inducers: rifampin, dexamethasone,troglitazone

– CYP2B inducers: phenobarbital, PCBs, phenytoin

– CYP2E1 inducers: ethanol, isoniazid

• Some overlap in “specificities” of inducers

• An inducer for one family of enzymes mayalso suppress another family (e.g., BNF)

Molecular Mechanisms of P450Enzyme Induction

Mechanisms of P450 Induction• Receptor-mediated transcriptional activation

– Receptor• A macromolecule with which a hormone, drug, or other

chemical interacts to produce a characteristic effect.

– Two key features:• chemical recognition• signal transduction

– Ligand: A chemical that exhibits specific binding toa receptor.

• mRNA stabilization

• Protein stabilization

Enzyme InductionGeneral mechanism of enzyme induction

protein

activity

mRNA

Gene transcription

Drug

Nuclear Receptor

DR cytosol DR nucleus

CYP1ACYP2BCYP3ACYP2C

P-gpMrp2

UGT1A1

Transcription factor

Dimerization partner

Examples of ligands

Genes Regulated

AHR ARNT Dioxins, non -ortho PCBs, some PAHs, bilirubin, etc.

CYP1A, CYP1B GST, UGT, NQO

CAR

RXR

Phenobarbital (PB), TCPOBOP, chlorinated pesticides, ortho -PCBs, androstanol / androstenol (inhibits)

CYP2B , CYP3A GST, ABC transporters

PXR (SXR)

RXR

PB, ortho-PCBs, organochlorine pesticides, dexamethasone, pregnenalone, corticosterone, bile acids (lithocholic acid)

CYP3A , CYP2B, CYP7A (repression) GST, ABC transporters

PPAR

RXR

Fibrate drugs, phthalate esters, linoleic acid, arachidonic acid

CYP4A , CYP7A (repression), CYP8B, LXR

LXR RXR Cholesterol; (24 S) - hydroxycholesterol CYP7A, ABC transporters, LXR

FXR RXR Bile acids, chenodeoxycholic acid Represses CYP7A, CYP8B , CYP27A

ER ER Structurally diverse xenoestrogens CYP19

Receptors Involved in the Regulation ofCYP Gene Expression

AhR Signaling Pathway

Cytoplasm Nucleus

9090

X

AhR

L

L

9090

X

L

9090

X

L

L

9090

X

L

or Arnt

From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA

AhR Signaling Pathway

XRE promoter gene(CYP1A1)

Translation

Increased expressionCYP1A1 protein

Increased expression ofother gene products

+

AhR/Arntheterodimer

mRNA

IC

+

Co-regulation of Target Genes by NR’s• Complementary roles of NR’s in protection against

xenobiotic exposure.

• Increased expression of the hepatic genes involved indrug metabolism and excretion (e.g., CYP’s, UGT’s,GST’s, transporters).

• These target genes represent redundant but distinctlayers of defense.

• There are distinct and overlapping species differencesin response to activators of NR’s.

Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002

Coordinate Regulation of P450’s, UGT’s andTransporters by PXR and CAR

UGT’s

MRP3

Amino Carboxy

AF-1 DBD LBD AF-2

Modulators interactwith some cofactors

Binding to responseelements of target genes

Ligand and coactivatorbinding pockets

Translocaseactivity

5’ 3’ 5’ 3’ 5’ 3’

Monomers RXR Heterodimers Homodimers

LBD

DBD

NR-LBD RXR-LBD

DBD DBD DBD DBD

NR-LBD NR-LBD

RORTLXERRNGFI-B

PXRCARPPARLXRFXRRAR

GRCOUP-TFHNF4Rev-ErbGCNF

5’ 3’

n

n

n

DRn

IRn

ERn

CYP2B Response elements

CYP2B6 TGTACT n=4 TGACCCCYP2b10 TGTACT n=4 TGACCTCYP2B1 TCTACT n=4 TGACCTCYP2B2 TGTACT n=5 TGACCT

NR1s

CYP2B6 TGGACT n=4 TGAACCCYP2b10 TCAACT n=4 TGACACCYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACAC

NR2s

NR3

CYP2B6 TGGACT n=4 TGACCC

CYP3A Response elements

CYP3A4 TGAACT n=3 TGACCC CYP3A2 TGACCT n=3 TGAGCT CYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACT

DRs

CYP3A4 TGAAAT n=6 GGTTCACYP3A4 TGAACT n=6 AGGTCACYP3A23 TTAACT n=6 AGGTCACYP3A5 TGAACT n=6 AGGTAACYP3A7 TTAACT n=6 AGGTCACYP3A7 TGAAAT n=6 AGTTCA

ERs

Other Genes

UGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCA rMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT

Similar Binding of PXR and CARto Promoter Response Elements

Goodwin et al., Mol. Pharmacol., 2001

Differential Binding of PXR andCAR to Other Promoter Regions

XREM-2B6 ER6-3A4

PXR + + + + + +CAR + + + + + +RXR + + + + + + + + + + + +

PXR/RXRCAR/RXR

CYP2B, CYP3A, CYP1A,CYP2A, CYP2Cs, OATP2UGT1A1, MRP2, SULT1A1

CYP1A, UGT1A1, SULT1A1

CAR RXR

XREM / PBREM

AhR ARNT

XRE / DRE

PXR RXR

XREM / PXRE

CYP3A, CYP2B, CYP2Cs,CYP7A, MDR1, MRP2,OATP2, GSTA-2, UGT1A1,AldHs, Carboxyesterase 2, 3

Aryl hydrocarbonReceptor

ConstitutiveAndrostane Receptor

Pregnane XReceptor

Molecular Basis for the SpeciesDifferences in Enzyme Induction

Rabbit

Human

Rat

0.1%

DM

SO

5µ

M P

CN

10µ

M R

ifam

pici

n

10µ

M S

R12

813

10µ

M D

TB

A

CYP3A6

CYP3A4

CYP3A23

Species Differences in the Regulationof CYP3A Enzymes

Species Differences in CYP2BInduction by PB

Species Differences in CYP1AInduction by Xenobiotics

CYP1A1/2 Activity in Rat Hepatocytes as a Function of Treatment with Drug 'X'

0

100

200

300

400

500

600

700

800

900

1000

Contro

l (0.1

% D

MSO)

3-M

C 1µM

Drug

'X' 0

.6µM

Drug

'X' 2µM

Drug

'X' 6µM

Drug

'X' 2

0µM

Phe

nace

tin

O-D

ealk

ylat

ion

(pm

ol/m

in/m

g)

CYP1A Activity in Dog Hepatocytes as a Function of Treatment with Drug 'X'

0

100

200

300

400

500

600

Contro

l (0.1

% D

MSO)

3-M

C 2µM

Drug

'X' 0

.6µM

Drug

'X' 2µM

Drug

'X' 6µM

Drug

'X' 2

0µM

Phe

nace

tin

O-D

ealk

ylat

ion

(pm

ol/m

in/m

g)

CYP1A2 Activity in Human Hepatocytes as a Function of Treatment with Drug 'X'

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Contro

l (0.1

% D

MSO)

3-MC 2µM

Drug '

X' 0.2µ

M

Drug '

X' 2µM

Drug '

X' 6µM

Drug '

X' 20µ

M

CY

P1A

2 A

ctiv

ity

(nm

ol/m

in/m

g)

Observations and Questions• Significant species differences are observed

in response to inducers.

• All major subfamilies of inducible CYP’s(CYP1A, CYP2B, CYP3A, CYP4A) exhibitthis behavior.

• What is the molecular basis of the species-specific responses?

• What is the significance of these differencesto predicting human toxicity?

PXR ExpressionPlasmid

PXR

RXR PXR

PXRE Reporter Gene

RXR

Drug

Reporter Plasmid

Transfection Assay for P450Enzyme Induction

CV-1HuH7 cell

Differential Activation of Human, Rabbit,and Rat PXR by CYP3A Inducers

PCN

rifampicin

lovastatin

clotrimazole

Normalized Reporter Activity

0 20 40 60 80 100 300 350 400

OHOHONNNMeNHOOOHOAcOMeOOHHO

NNCl

OOHOHOOH

HOOHCN

PXR Sequence Homology1 41 107 141 434

Human PXR1

Rat PXR11 38 104 138 431

Xenopus ONR11 37 102 136 386

Human VDR 1 24 89 122 427

LigandDNA

96

69

63 37

76

42

Mouse PXR11 38 104 138 431

96 76

Rabbit PXR11 41 107 141 434

8294 Variation in ligandbinding domainconsistent with invivo species differ-ences in responseto inducers

Amino Acid Differences in theLigand Binding Domain of PXR

(Zhang et al., Arch. Biochem. Biophys., 1999)

Ser187 Leu213 Asp266 Glu337 Ile417

Gly181 Leu206 Tyr263 His333hPXR

Phe184 Leu210 Asp263 Lys334 Ser414

Gly178 Arg203 Tyr260 Arg333mPXR

Val184 Val210 Glu263 Glu333 Thr414

Asp178 Ser203 His260 Arg333rPXR

Summary• Induction of metabolism is caused by many

structurally unrelated xenobiotics.• Induction occurs mainly by transcriptional

regulation of metabolizing enzymes andtransporters.

• Nuclear receptors mediate the inductionresponse by most xenobiotics.

• Amino acid differences in the ligand-binding domain of the receptors are mainlyresponsible for the species differences in theinduction of CYP450 enzymes.

Additional Reading• Parkinson, A.: Biotransformation of xenobiotics. In: Casarett and

Doull’s Toxicology. The Basic Science of Poisons. Sixth edition(edited by C.D. Klaassen). McGraw Hill, New York, 2001.

• Wang, H. and Negishi, M. (2003) Transcriptional regulation ofcytochrome p450 2B genes by nuclear receptors. Curr Drug Metab.4(6):515-25.

• Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L.,Sydowbackman, M., Ohlsson, R., Postlind, H., Blomquist, P. andBerkenstam, A. (1998) Identification of a human nuclear receptordefines a new signaling pathway for CYP3A induction. Proc. Natl.Acad. USA. 95:12208-12213.

• Blumberg, B., and Evans, R.M. (1998) Orphan nuclear receptors – newligands and new possibilities. Genes Dev. 12:3149-3155.

• Geick A., Eichelbaum M., and Burk O. (2001) Nuclear receptorresponse elements mediate induction of intestinal MDR1 by rifampin. JBiol Chem. 276(18):14581-14587.

Additional Reading• Goodwin B., Hodgson E., and Liddle C. (1999) The orphan human

pregnane X receptor mediates the transcriptional activation of CYP3A4by rifampicin through a distal enhancer module. Mol Pharmacol56:1329-1339.

• Honkakoski P. and Negishi M. (1998) Regulatory DNA elements ofphenobarbital-responsive cytochrome P450 CYP2B genes. J BiochemMol Toxicol 12:3-9.

• Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G.B., Hamilton, G. A.,McKee, D. D., Tomkinson, N. C. O., LeCluyse, E. L., Wilson, T. M.,Kliewer, S. A. and Moore, J. T. 2000. The pregnane X receptor, apromiscuous xenobiotic receptor that has diverged during evolution.Mol. Endocrinol. 14: 27-39.

• Wang, H., and LeCluyse E. L. 2003. Role of orphan nuclear receptorsin the regulation of drug metabolising enzymes. Clin. Pharmacokinet.42: 1331-1357.