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Biomarkers
Molecular Effects
Ecotoxicology
Biology 5868
2009
Levels of Biological Organization
Biosphere
Molecular
Cell
Tissue
Organ
Individual
Population
Community
Ecosystem
Landscape
Contaminant
Assessment
Exposure
Effects
Screening
Mechanistic
in vitro
in vivo
in silico
Contaminant Analysis
population
receptorgene
protein
celltissue
organism multi-generation
info
rmation
(ecolo
gic
al re
levance)
time complexity cost
low
high
low high(log scale)
Key Question what is the predictive ability of any given test
Biomarkers
Biomarker - ldquohellipa biochemical physiological morphological or
histological quality used to imply exposure to or effect of a toxicantrdquo
Exposure biomarkers - mechanism necessary or implied
Effects biomarkers - no mechanism necessary
Biomarker Types
Biomarker Tests
Tier I - screening
Tier II - mechanistic
Biomarker efficiency
E = efficiency of the biomonitoring methodology
i = population of interest
Ui = concentration at which the undesirable effect occurs
Bi = concentration at which the method can detect or predict
E = Ui
Bi
Ideal Biomarker CharacteristicsNewman amp Unger criteria for biochemicalmolecular biomarkers
1 Measurable before an adverse effect occurs at a higher level of
biological organization
2 Rapid inexpensive easy (Pragmatic importance)
3 Quality control quality assurance
4 Specific to a single toxicant or class of toxicants (Unlikely)
5 Concentration-effect relationship should exist
(Linear over exposure range if possible)
6 Applicable to a broad range of sentinel species (If these even exist)
7 Linkage of biomarker changes with some toxicant-related decrease
in individual fitness
8 System should be familiar incorporate qualities of organism that
influence biomarker (Asking a lot especially if using native species)
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Levels of Biological Organization
Biosphere
Molecular
Cell
Tissue
Organ
Individual
Population
Community
Ecosystem
Landscape
Contaminant
Assessment
Exposure
Effects
Screening
Mechanistic
in vitro
in vivo
in silico
Contaminant Analysis
population
receptorgene
protein
celltissue
organism multi-generation
info
rmation
(ecolo
gic
al re
levance)
time complexity cost
low
high
low high(log scale)
Key Question what is the predictive ability of any given test
Biomarkers
Biomarker - ldquohellipa biochemical physiological morphological or
histological quality used to imply exposure to or effect of a toxicantrdquo
Exposure biomarkers - mechanism necessary or implied
Effects biomarkers - no mechanism necessary
Biomarker Types
Biomarker Tests
Tier I - screening
Tier II - mechanistic
Biomarker efficiency
E = efficiency of the biomonitoring methodology
i = population of interest
Ui = concentration at which the undesirable effect occurs
Bi = concentration at which the method can detect or predict
E = Ui
Bi
Ideal Biomarker CharacteristicsNewman amp Unger criteria for biochemicalmolecular biomarkers
1 Measurable before an adverse effect occurs at a higher level of
biological organization
2 Rapid inexpensive easy (Pragmatic importance)
3 Quality control quality assurance
4 Specific to a single toxicant or class of toxicants (Unlikely)
5 Concentration-effect relationship should exist
(Linear over exposure range if possible)
6 Applicable to a broad range of sentinel species (If these even exist)
7 Linkage of biomarker changes with some toxicant-related decrease
in individual fitness
8 System should be familiar incorporate qualities of organism that
influence biomarker (Asking a lot especially if using native species)
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Contaminant Analysis
population
receptorgene
protein
celltissue
organism multi-generation
info
rmation
(ecolo
gic
al re
levance)
time complexity cost
low
high
low high(log scale)
Key Question what is the predictive ability of any given test
Biomarkers
Biomarker - ldquohellipa biochemical physiological morphological or
histological quality used to imply exposure to or effect of a toxicantrdquo
Exposure biomarkers - mechanism necessary or implied
Effects biomarkers - no mechanism necessary
Biomarker Types
Biomarker Tests
Tier I - screening
Tier II - mechanistic
Biomarker efficiency
E = efficiency of the biomonitoring methodology
i = population of interest
Ui = concentration at which the undesirable effect occurs
Bi = concentration at which the method can detect or predict
E = Ui
Bi
Ideal Biomarker CharacteristicsNewman amp Unger criteria for biochemicalmolecular biomarkers
1 Measurable before an adverse effect occurs at a higher level of
biological organization
2 Rapid inexpensive easy (Pragmatic importance)
3 Quality control quality assurance
4 Specific to a single toxicant or class of toxicants (Unlikely)
5 Concentration-effect relationship should exist
(Linear over exposure range if possible)
6 Applicable to a broad range of sentinel species (If these even exist)
7 Linkage of biomarker changes with some toxicant-related decrease
in individual fitness
8 System should be familiar incorporate qualities of organism that
influence biomarker (Asking a lot especially if using native species)
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Biomarkers
Biomarker - ldquohellipa biochemical physiological morphological or
histological quality used to imply exposure to or effect of a toxicantrdquo
Exposure biomarkers - mechanism necessary or implied
Effects biomarkers - no mechanism necessary
Biomarker Types
Biomarker Tests
Tier I - screening
Tier II - mechanistic
Biomarker efficiency
E = efficiency of the biomonitoring methodology
i = population of interest
Ui = concentration at which the undesirable effect occurs
Bi = concentration at which the method can detect or predict
E = Ui
Bi
Ideal Biomarker CharacteristicsNewman amp Unger criteria for biochemicalmolecular biomarkers
1 Measurable before an adverse effect occurs at a higher level of
biological organization
2 Rapid inexpensive easy (Pragmatic importance)
3 Quality control quality assurance
4 Specific to a single toxicant or class of toxicants (Unlikely)
5 Concentration-effect relationship should exist
(Linear over exposure range if possible)
6 Applicable to a broad range of sentinel species (If these even exist)
7 Linkage of biomarker changes with some toxicant-related decrease
in individual fitness
8 System should be familiar incorporate qualities of organism that
influence biomarker (Asking a lot especially if using native species)
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Ideal Biomarker CharacteristicsNewman amp Unger criteria for biochemicalmolecular biomarkers
1 Measurable before an adverse effect occurs at a higher level of
biological organization
2 Rapid inexpensive easy (Pragmatic importance)
3 Quality control quality assurance
4 Specific to a single toxicant or class of toxicants (Unlikely)
5 Concentration-effect relationship should exist
(Linear over exposure range if possible)
6 Applicable to a broad range of sentinel species (If these even exist)
7 Linkage of biomarker changes with some toxicant-related decrease
in individual fitness
8 System should be familiar incorporate qualities of organism that
influence biomarker (Asking a lot especially if using native species)
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Ideal Biomarker Characteristics2
(Melancon 2002 criteria)
1 Know specificity of response to contaminant presence of exposure
2 Some correlation of presence or magnitude of response to
amount of contaminant
3 Known temporal relationship between exposure and effect
4 Response should precede harm but ideally predict future harm
5 Samples should be readily obtainable
(in quantities needed for measurement)
6 Sampling should be nonlethal (ideally)
7 Assay method should be accurate and reproducible
8 Overall costs should be reasonable
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Biomarker SignalNoise Ratio
Signalnoise ratio eg
- immune system
- stress-response system
Bio
mark
er
Time (diurnal seasonal lifestage etc)
Stressor B = higher signalnoise
response to
xenobiotic
stressors
Stressor A = low signalnoise
response to
ldquonormalrdquo
stressors
normal stress response = noise
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Molecular Biomarkers
Me PCBs poryphyrin profiles
OCs considerable natural variationretinol profiles
EDCs sensitive indicatorvitellogenin ()
Me OCs PAHs wide variety many testsother serum enzymes
metals OCs wide variety many testsstress proteins
rodenticides anticoagulant proteins
lead Inhibition of ALAD alternative to Pb analysis
OCs PAHs MFO (P450)geneenzyme induction
multiple forms
cadmium metallothionein signalnoise issues
OPs carbamates AChE inhibition signalnoise issues
Contaminant CommentsBiomarker
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Heme Synthesis
porphyrin
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Lead PCB effects on
porphyrin synthesis
Glycine + Succinyl CoA
-Aminolevulinic Acid
Porphobilnogen
Uroporphyrinogen
Heptacarboxyporphyrinogen
Hexacarboxyporphyrinogen
Pentacarboxyporphyrinogen
Coprophyrinogen
Protoporyphyrinogen IX
Protoporpyrin IX
Hemeglobin
Hemoglobin
-aminolevulinic acid synthetase
-aminolevulinic acid dehydratase
uroporphyrinogen I synthetase
uroporphyriongen III cosythetase
uroporphyrinogen decarboxylase
coproporphyrinogen oxidase
protoporphyrinogen oxidase
ferrochelatase + Fe(II)
uroporphyrinogen decarboxylase
uroporphyrinogen decarboxylase
inhibited by lead
(ALAD-deficiency porphyria)
Pb
PCBs cause buildup of multiple porphyrins
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Ha M-H Choi J 2008 Chemosphere 711928-1936
NP - nonylphenol
BPA - bisphenol A diglycidyl ether
B[a]P - benzo[a]pyrene
CP ndash chlorpyriphos
PQ - paraquat dichloride
Pb - lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Induction of differential hemoglobin profiles in midges
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P) chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P
CP PQ Pb
Ha M-H Choi J 2008 Chemosphere 711928-1936
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
nonylphenol (NP) bisphenol A diglycidyl ether (BPA) benzo[a]pyrene (B[a]P)
chlorpyriphos (CP) paraquat dichloride (PQ) and lead(II)nitrate (Pb)
Hemoglobin Induction
NP BPA B[a]P CP PQ Pb
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Acetylcholinesterase Inhibitors1 Neurotransmitter acetylcholine must be broken down rapidly by the receiving neuron
- continued stimulation rarr uncoordinated muscle movement seizures and death
Axon
Acetylcholine
Action
Potential
Synapse Acetylcholinesterase
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Acetylcholinesterase
Acetylcholinesterase
Acetylcholine
glutamyl
serine
OH
CH3 C O N(CH3)C3CH2 CH2
O
+
O
CH3 C
O
HO N(CH3)C3CH2 CH2
+
Choline
+ H2O
CH3 C
CH
O
Acetic
acid
glutamyl
serine
serine
OH
glutamyl
2 Acetylcholinesterase (AChE) binds acetylcholine by donating proton from serine
- results in the release of the choline group
- acetyl group released and the serine reactivated by a proton donated by H2O
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
3 Organophosphates inhibit AChE by initially binding to serine at the active site
- allows irreversible binding of organophosphate and an active site glutamyl residue
- acetylcholine is prevented from entering the active site = no degradation
POCH3
OCH3
S
O2N O
Methyl Parathion
POCH2CH3
OCH2CH3
S
O
CH3C
H3C
CH3
N
N
Diazinon
Organophosphate Insecticides
O
O2N O POCH2CH3
OCH2CH3
O
Organophosphate
(eg Paraoxon)
+ O2N O
Acid leaving group
POCH2CH3
OCH2CH3
O
C
O-
O
OH
C
O
O
POCH2CH3
OCH2CH3
O
glutamyl
serine
OH
glutamyl
serine
glutamyl
serine
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Acetylcholinesterase Inhibition
Bervoets L et al 2009 Ecotox Environ Saf 72720-728
sites at increasing distance from
contaminant source
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Mixed Function Monooxygenase Induction
- 150 - 400 varieties
- 28 families multiple subfamilies
- nomenclature
- gene family = cyp
- family = 1 - 28
- subfamily = A - Z
- individuals = 1 hellip (s)
eg CYP1A1 P4501A1
CYP1 2 ndash drug steroid xenobiotic metabolism
CYP3 11 17 19 21 - steroid metabolism
CYPxhellip arachadonic acid thromboxine vitamin D retinoic acid etc
MFO CYP450s etc
eg RH + O2 + 2H+ + 2e- rarr ROH + H2O
(also oxidation reduction epoxidation dealkylation etc)
~ 8800 known sequences
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
CYP1A Activities
EROD - ethoxyresorfurin O-deethylase
O
N
OHOO
N
OC2H5O
AHH - aryl hydrocarbon hydroxylase
Benzo[a]pyrene
Benzo[a]pyrene diol epoxide
OH
O
OH
Note ndash CYP1A1 induced via activated aryl hydrocarbon (Ah) receptor
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
CYP450 Assays
PAHs
Carcinogenic
PAHs
EROD
BPMO
L Marsili et al 2009 Science of the Total Environment
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Starr et al Environ Res 108 (2008) 271-279
Permethrin
Metabolite Biomarkers
eg Pyrethroid metabolites
Esfenvalerate
Allethrin
Pyrethroid pesticides and their metabolites in vacuum cleaner dust collected from homes and day-care centers
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Metallothionein Induction
Metallothioneins (MT) - uptake internal compartmentalization sequestration
excretion of essential and non-essential metals
- normal homeostatic function
- inducible
- higher levels (in the presence of metals) = increased fitness
- small ubiquitous proteins (6-7 kDa) four major isoforms
~ 25 cysteine no aromatics or histidine
- well conserved Drosophila fish mouse human)
- transcriptional regulation via Zn-dependent transcription factor (MTF-1)
- induction by metals other than Zn not well understood via MTF-1
- MT gene also has glucocorticoid and antioxidant response element
- antibodies for specific metallothioneins
- cDNA probes
- MTF-1 binds to Metal Response Element (MRE)
- metals bind to sulfhydryl (thiol) group of cysteine
MT Detection
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Vitellogenin
Vitellogenin (VTG) - protein precursor of several yolk proteins eg phosvitin
lipovitellin in the eggs of various vertebrates and invertebrates
- sensitive assay in males and immature organisms
- not as sensitive in females (protein is normally present)
Western blots
ELISA
- Note - restricted to oviparous organisms
RT-QPCR (reverse transcriptase quantitative PCR)
- estrogen sensitive
- synthesized in the liver
VTG Detection
fathead minnow
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Stress ProteinsCellular stress response ndash ldquoorchestrated induction of key proteins that
form the basis for the cellrsquos protein protection and recycling systemrdquo
- inducible by a variety of ldquostressorsrdquo
- includes heat shock proteins (hsp) and others
- hsp 100 hsp90 hsp70 hsp60 low MW ubiquitin
- chaperones ndash protein folding (re-folding)
- ubiquitin ndash tags proteins destined for recycling
- constitutive expression stress 90 (high) stress 70 (low)
- LMW varies in stress inducibility
- highly conserved highly inducible
Stress protein detection
- protein fingerprinting
- transcriptional regulation by Heat Shock Factor (HSF)
- infection inflammation ethanol arsenic metals UV starvation hypoxia
- but both are inducible to higher concentrations by stress
- ldquosuiterdquo of stress response proteins may be functional
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Oxidative Stress and Antioxidant Response
Oxidative stress ndash damage to biomolecules from reactive oxygen species (ROS)
- aerobic metabolism ie mitochondrial e- transport
- photosynthetic e- transport
- phagocytosis
- catalysis eg prostaglandin synthase guanyl cyclase glucose oxidase
ROS also produced by
superoxide radical (O2-)
hydroxyl radical (OH)
eg free oxyradicals
eg molecular antioxidants
Antioxidant response
- tocopherol (vitamin E)
- ascorbic acid (vitamin C)
- retinaldehyde (vitamin A)
- glutathione (GSH)
- β-carotene
- catecholamines
- uric acid
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Antioxidant Response
Glutathione
peroxidase
Superoxide
dismutase
Catalase
4 O2-bull
2 O2
2 H2O2
2 H2O + O2
4 GSH
2 GSSG
4 H2O
Glutathione
reductase
NADP+
NADPH
or
Enzymatic antioxidants
Xenobiotics (eg PAH radicals) can produce free radicals after biotransformation
- interfere with antioxidant enzymes
- undergo redox cycling eg xenobiotic is reduced to a radical
produces oxygen radical recycles to original form
Inducible
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
DNA Modification
Genotoxicity ndash damage by a physical or chemical agent to genetic materials
chromosomes DNA RNA
- mutagens
- teratogens
- carcinogens
- clastogens (agents that cause chromosome damage)
adducts
(cisplatin)- free radicals can cause strand breakage
- oxyradicals oxidize bases
- adducts xenobiotics bind covalently to nucleotide
alters structure
- metals bind phosphates or bases eg
- Hg crosslinks DNA strands Cu displaces Mg on PO4
Mechanisms
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer
Genotoxicity Response
Assay methods
- chromosome staining
- flow cytometry (aneuploidy)
- 32P-labeling (adducts)
- comet assay (single cell DNA assay)
DNA repair mechanisms - highly active and efficient but can be overwhelmed
C Burd amp TK Archer