Toxicology I: Principles & Mechanisms
Marine Mammal Toxicology
Spring 2004
Mark Hahn
Woods Hole Oceanographic Institution
Exposure
1. Absorption/route of entry
Dose
1. Distribution/toxicokinetics
2. Biotransformation
3. Excretion
Tissue concentration
1. Molecular mechanism
2. Pathogenesis
Effect (individual)
Approaches to studying toxicological mechanisms
in marine mammals
• Direct exposure?
• Semi-field studies (feeding studies)
• Extrapolation
• Biomarkers of exposure, effect, susceptibility
• Field associations (chemicals and effects)
• in vitro studies
- tissues and subcellular fractions
- cloned, in vitro expressed proteins
- tissue/cell culture
Dose-Response
• shapes of curves; thresholds
• timing of exposure and effects
(acute vs chronic)
(algal toxins versus POPs)
(exposure and effects separated in time)
• low-dose extrapolation
Distribution/toxicokinetics
• hydrophobicity and lipid content
• protein binding
• effect of physiological condition
(fasting, pregnancy)
• compartmental analysis
• physiologically based pharmacokinetic models
Biotransformation (Metabolism)
• Phase I (add functional group)
- cytochrome P-450s (CYP) (hydroxylation)
- flavin monooxygenases (N-, S-oxidation)
- esterases,hydrolases, dehydrogenases…
• Phase II (conjugation)
- glutathione transferases
(GSH = g-glu-cys-gly)
- sulfotransferases
- UDP-glucuronosyl transferases
- acetylases; methylases
Cytochrome P450 (CYP)
• multiple forms (57 in humans)
• mostly in endoplasmic reticulum (microsomal)
• hemoproteins
• require NADPH and O2
• tissue-, sex-, and stage- specific expression
• broad substrate specificity
(endogenous and xenobiotic)
• some inducible
• nomenclature (family-subfamily-gene: e.g. CYP1A1)
Human P450 enzymes
Family
# subfamilies
#
genes
substrates (examples)
inducers (examples)
1
2
3
PAH, non-ortho-PCB, E2,
xenobiotics
PAH, non-ortho-PCB, dioxins
2
11
16
ortho-PCB, barbiturates,
steroids, ethanol, xenobiotics
phenobarbital, ortho-PCB, DDT,
ethanol
3
1
4
steroids, xenobiotics
glucocorticoids, (PCBs)
4
6
12
fatty acids
phthalate esters, (PCBs)
5
1
1
7
2
2
8
2
2
11
2
3
cholesterol, steroid 11
17
1
1
steroids (pregnenolone 17)
19
1
1
testosterone
20
1
1
21
1
1
steroids (progesterone 21)
24
1
1
vit. D
26
3
3
retinoids
27
3
3
vit. D
39
1
1
OH-cholesterol
46
1
1
bile acids
51
1
1
lanosterol
TOTAL
57
cholesterol
estrogens
Regulation of CYP gene expression by soluble receptors
Transcription
factor
Dimerization
partner
AHR
ARNT
CAR
Examples of ligands
Genes
Regulated
Dioxins, non-ortho PCBs, some PAHs,
bilirubin, etc .
CYP1A, CYP1B
GST, UGT, NQO
RXR
Phenobarbital (PB), TCPOBOP,
chlorinated pesticides, ortho-PCBs,
androstanol/ androstenol (inhibits)
CYP2B, CYP3A
GST, ABC transporters
PXR
(SXR)
RXR
PB, ortho-PCBs, organochlorine
pesticides, dexamethasone,
pregnenalone, corticosterone, bile acids
(lithocholic acid)
CYP3A, CYP2B, CYP7A
(repression)
GST, ABC transporters
PPAR
RXR
Fibrate drugs, phthalate esters, linoleic
acid, arachidonic acid
CYP4A, CYP7A (repression),
CYP8B, LXR
LXR
RXR
Cholesterol; (24 S)- hydroxycholesterol
CYP7A, ABC transporters,
LXR
FXR
RXR
Bile acids, chenodeoxycholic acid
Represses CYP7A, CYP8B,
CYP27A
ER
ER
Structurally diverse xenoestrogens
CYP19
Reactions - PAH metabolism
EH
CYP1A1
DHD-DH
CYP1A1
Reactions - PCB metabolism
Differential susceptibility to biotransformation:
Preferential loss of 3,4-unsubstituted congeners
[CB ] to [CB-138] Ratio
x
1.5
2,2’,5,5’-TCB
1.0
0.5
0
0.5
1.0
1.5
CB-52
CB-70
2,2’,4,5,5’-PCB
CB-92
CB-101
CB-99
CB-99
CB-105
CB-110
CB-118
CB-118
CB-128
CB-138 ***
2,2’,4’,5,5’,6-HCB
CB-149
CB-153
CB-138 ***
CB-153
CB-156
CB-156
2,2’,3,4,4’,5’-HCB
2,2’,4,4’,5,5’-HCB
CB-180
Technical PCB mixture Clophen A50
Rob Letcher, Univ. of Windsor
PCB congeners in mink muscle
Reactions - PCB metabolism
OH-PCB
Formation
Pathways
O
Cl
Cl
Cl
HO
Epoxide opening
Cl
Cl
Cl
Cl
Cl
Cl
Cl
+
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
Cl
Cl
Cl
+
OH
SG
Cl
n
SH
-G
SG
Cl
Cl
Cl
SCysNAC
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
SCys
SH
Cl
Cl
Cl
Cl
MAP
Cl
Cl
OH
Cl
Cl
Cl
Cl
Cl
+
Cl
Cl
Cl
OH
Cl
OH
Cl
Cl
SG
Cl
Cl
-H2O
Cl
Cl
Epoxide opening
Cl
Cl
Cl
Cl
CB-101
Cl
1,2-shift
Cl
Cl
Cl
Cl
Direct insertion
Cl
Rob Letcher, Univ. of Windsor
Cl
Cl
n
Cl
Formation
Pathway for
Persistent
MeSO2-PCBs
OH
Cl
SCH3
Cl
Cl
Cl
Cl
SO2CH3
(-SO2 Me)
Reactions - PCB metabolism
OH-PCB
Formation
Pathways
O
Cl
Cl
Cl
HO
Epoxide opening
Cl
CYP2B
Cl
Cl
Cl
Cl
+
Cl
Cl
Cl
Cl
Cl
O
GST
Cl
Cl
Cl
Cl
Cl
Cl
Cl
+
OH
SG
Cl
n
SH
-G
Cl
Cl
Cl
SCysNAC
NAT
Cl
Cl
Cl
Cl
Cl
Cl
-lyase
MeT
Cl
Cl
Cl
Cl
SCys
SH
Cl
Cl
Cl
Cl
Cl
MAP
Cl
Cl
SG
OH
Cl
Cl
Cl
Cl
Cl
+
Cl
Cl
Cl
OH
Cl
OH
Cl
Cl
SG
Cl
Cl
-H2O
Cl
Cl
Epoxide opening
Cl
Cl
Cl
Cl
CB-101
Cl
1,2-shift
Cl
Cl
Cl
Cl
Direct insertion
Cl
Rob Letcher, Univ. of Windsor
Cl
n
Cl
Formation
Pathway for
Persistent
MeSO2-PCBs
Cl
Cl
OH
Cl
SCH3
Cl
CYP Cl
FMO
Cl
Cl
SO2CH3
(-SO2 Me)
OH-PCBs
OH
Clm
Cln
• Formed by CYP1A and CYP2B
• Less hydrophobic than parent PCBs
• Most readily excreted;
some persist in blood (m- and p-hydroxy w/ o-Cl)
• Poor substrates for conjugation
(glucuronidation and sulfation)
• Multiple effects
log P
- displace T4 from transthyretin
- inhibit sulfotransferase
(T4, E2, 3-OH-BaP)
- inhibit glucuronosyl transferase
(3-OH-BaP)
- agonists for estrogen receptors
8
PCB
6
Hydroxy PCB
4
2
1
2
3
4
5
6
7
8
Number of Chlorine Atoms
OH-PCBs as inhibitors of T4
transport by transthyretin (TTR)
Brouwer et al 1998
Methylsulfonyl-PCBs
• Formed by sequential enzymatic reactions
• Less hydrophobic than parent PCBs but still persistent
• Bioaccumulate and persist in tissues
(m- and p-MeSO2 w/ 2,5,(6)-Cl) (liver, lung > fat)
- likely role for CYP2B epoxidation as initial step
• adipose [MeSO2-PCB]/[PCB] = .01-.25
(highest in Baltic ringed and grey seal)
• Protein interactions
- uteroglobin (progesterone-binding protein)
- glucocorticoid receptor antagonist
- estrogen receptor antagonist?
• Induce CYP2B,C and CYP3A enzymes
Biotransformation in marine mammals
• What is the capacity for xenobiotic metabolism in MM?
Are there species differences in xenobiotic-metabolizing enzymes?
- diversity
- expression
- inducibility
- catalytic function (rates and specificity)
• Direct measurement of metabolites
• Inferences from contaminant patterns in MM tissues
• Direct assessment in vitro
- immunochemical detection
- in vitro catalytic assay (model substrates; correlations; ± inhibitors)
- cloning, expression, characterization
Biotransformation capacity inferred
from patterns of PCB congeners
(Dall’s porpoise vs human)
m-p unsub
(CYP2B)
o-m unsub
(CYP1A)
o-m
unsub
m-p
unsub
Tanabe et al (1988) Capacity and mode of PCB metabolism in marine mammals
2,2’,5,5’-TCB
2,3’,4,4’-TCB
Relative ratios (Rrel) vs food
for PCB congeners
harbor seal
0 m,p H
2 o Cl
0 m,p H
1 o Cl
(CYP1A)
otter
1 m,p H
2-3 o Cl
(CYP2B)
Boon et al (1997)
harbor porpoise
common dolphin
Immunochemical characterization of hepatic microsomal
cytochromes P450 in beluga
antibody to CYP forms
MAb fish 1A1
PAb rodent 1A1/2
PAb fish “2B”
PAb rat 2B1
MAb rat 2B1
PAb rabbit 2B4
PAb dog 2B11
PAb rat 2E1
PAb rat 2E1
band in beluga
hepatic microsomes
+
+(1)
+
+
+
+(2)
White, et al. (1994) Catalytic and immunochemical characterization of hepatic microsomal
cytochromes P450 in beluga whales (Delphinapterus leucas). Toxicol. Appl. Pharmacol. 126: 45-57.
Immunochemical detection of CYPs in marine mammals
Letcher, et al (1996) Immunoquantitation and
microsomal monooxygenase activities of hepatic
cytochromes P4501A and P4502B and chlorinated
hydrocarbon contaminant levels in polar bear
(Ursus maritimus). Toxicol Appl Pharmacol 137:
127-140.
CYPs in marine mammals
Immunochemical evidence and cDNA cloning
CYP1
CYP2
CYP3
CYP4
(+/-)
+
+
+
+
Cetacea – odontocetes
++ (1A1, 1B)
Cetacea – mysticetes
++(1A)
Pinnipeds
++(1A1, 1A2)
+
Mustelids
+
+
Sirenians
Ursids
++(1A)
++(2B)
Catalytic characterization of hepatic microsomal
cytochromes P450 in beluga
White, et al. (1994) Catalytic and immunochemical characterization of hepatic
microsomal cytochromes P450 in beluga whales (Delphinapterus leucas). Toxicol.
Appl. Pharmacol. 126: 45-57.
Rates of PCB metabolism by hepatic microsomes
(pmol/min/mg protein)
beluga
rat
rat
rat
(male)
(con)
(3MC)
(PB)
PCB-77 (3,3’,4,4’-TCB)
22
(low)
18-50
(low)
PCB-52 (2,2’,5,5’-TCB)
1.1
0-10
(low)
66-1450
White et al. (2000) Compar. Biochem Physiol. 126, 267
Fig. 9. (White et al. (2000)) Proposed pathways for the metabolism of 3,3',4,4'-TCB in beluga whale liver
microsomes. The thickness of the arrows reflects the significance of an indicated pathway. The 4-hydroxy3,3',4',5-TCB reflects a positional shift of a Cl.
StL
HB
R.J. Letcher, et al. (2000). Methylsulfone PCB and DDE metabolites in beluga whale (Delphinapterus
leucas) from the St. Lawrence river estuary and western Hudson Bay, Canada. Environ. Toxicol. Chem.
19(5), 1378-1388.
Molecular mechanisms of toxicity
• covalent binding to protein or DNA
• oxidative stress (e.g. via Reactive Oxygen Species)
- lipid peroxidation
- oxidative DNA damage
- oxidative damage to proteins (-SH)
• enzyme inhibition (e.g. OP pesticides & AChE)
• interference with ion channels
- e.g. saxitoxin, brevetoxin
• interference with receptor-dependent signaling
- membrane bound receptors (neurotransmitter)
- intracellular receptors (hormone)
Soluble receptors involved in xenobiotic effects
Receptor
Xenobiotic ligands
Target genes
Aryl hydrocarbon (Ah) receptor (AHR) ?
dioxins, PCBs, PAHs
CYP1A,B; GST; UGT
Constitutive androstane
receptor (CAR)
androstanes,
bile acids
barbiturates; PCBs
OAT, MRP
CYP2 (CYP3), UGT, GST,
Pregnane X receptor (PXR)
bile acids,
pregnenolone
organochlorine pesticides; CYP3; (CYP2); UGT
PCBs
Peroxisome-proliferatoractivated receptor (PPAR)
fatty acids
Farnesoid X Receptor (FXR)/
Liver X Receptor (LXR)
bile acids,
oxysterols
Retinoid receptors
(RAR, RXR)
Endogenous
ligands
retinoids
fibrates,phthalates
and metabolites
CYP7, ABC-A1
methoprene
Estrogen receptors (ER)
17--estradiol
OC pesticides;
alkylphenols; others
Androgen Receptors (AR)
testosterone
OC pesticides
glucocorticoids
MeSO2-PCBs
Glucocorticoid receptor (GR)
CYP4
CYP19, Vtg
(CYP3)
Definitions
• Receptor (P. Erlich, 1913; J.N. Langley, 1906)
A macromolecule with which a hormone, drug, or other
chemical interacts to produce a characteristic effect.
Two essential features:
– chemical recognition
– signal transduction
• Ligand: A chemical that exhibits specific binding to a
receptor.
Definitions
• Specific binding (SB): High-affinity, low capacity binding of ligand
to receptor
• Non-specific binding (NSB): Low-affinity, high capacity binding of
ligand to other proteins
• Agonist: A ligand that binds to a receptor, increasing the proportion
of receptors that are in an active form and thereby causing a
biological response.
• Antagonist: A ligand that binds to a receptor without producing a
biological response, but rather inhibits the action of an agonist.
• Partial agonist: An agonist that produces less than the maximal
response in a tissue, even when all receptors occupied. Partial
agonists have properties both of agonists and of antagonists.
Definitions
• Potency: The concentration or amount of a chemical
required to produce a defined effect. Location along the dose
axis of dose-response curve (property of ligand and tissue).
• Efficacy: The degree to which a ligand can produce a
response approaching the maximal response for that tissue
(property of ligand and tissue).
• Affinity: The tenacity with which a ligand binds to its receptor
(property of ligand).
• Intrinsic Efficacy: Biological effectiveness of the ligand when
bound to the receptor; e.g. ability to “activate” receptor once
bound (property of ligand).
Affinity, Efficacy, and Potency
Ligand
+
Receptor I
LigandReceptor I
AFFINITY
Kd
INTRINSIC
EFFICACY
LigandReceptor A
TISSUE
COUPLING
POTENCY
EC50
EFFICACY
KE
RESPONSE
Hestermann et al. 2000
nucleus
hsp90
AHR
pRb
Ara9
?
E2F
TCDD
ARNT
cell
cycle
proteasomal
degradation
nuclear
export
XRE
Co-act
BTF
cytoplasm
XRE
mRNA
TATA
e.g. CYP1A1
Evidence for role of Ah receptor
in effects of dioxins / planar PCBs
Genetics
• inbred strains of mice
(responsive and “non-responsive”)
Pharmacology
• Structure-activity relationships
for AHR binding and toxicity
Cell Biology
• Mouse hepatoma cell mutants
Molecular biology
• AHR-null mice
log ED50 for Thymic Atrophy in Rats
Structure-activity relationships
4
3
2
1
0
(1)
y = 1.119x + 8.374 r 2 = 0.642
(2)
(9)
(8)
(7)
(6)
(5)
(4)
log Kd for AHR binding
The toxic potencies of many halogenated aromatic hydrocarbons
are related to their AHR-binding affinities.
Data from Safe, S. (1990) CRC Crit. Rev. Toxicol. 21: 51-88.
3D Structure of PCBs:
Calculated Dihedral Angle
100
Dihedral Angle [°]
Cl
Cl
80
Cl
Cl
Cl
PCB 118
60
Cl
Cl
Cl
Cl
Cl
PCB 153
Cl
Cl
PCB 95
Cl
Cl
40
20
Cl
Cl
Cl
Cl
Cl
Cl
Cl
PCB 126
0
0
1
2
3
4
Number of ortho-Chlorine Atoms
Hans-Joachim Lehmler, Univ. of Iowa
post-AHR mechanisms of dioxin/PCB toxicity
• induction of CYP1A
(metabolism of endogenous compound; release of ROS)
• altered expression of other target genes
(cell proliferation/differentiation)
• recruitment of AHR away from endogenous function
• competition for factors required for other signaling
pathways (ARNT, coactivators; HIF, SIM)
• cross-talk with other signaling pathways
(estrogen, progesterone)
PAH vs PCB as agonists for the AHR
PAH
PCB
affinity
variable (high)
variable (high)
timing of activation
transient
sustained
clearance of ligand
rapid
some slow
nature of metabolites
reactive (electrophiles)
stable but bioactive
biomarkers
CYP1A (early)
CYP1A
protein or DNA adducts parent compounds
and metabolites
Mechanisms of toxicity of PCBs and their metabolites
Congener/metabolite
Molecular Target
Action
non-ortho and monoortho-PCBs
aryl hydrocarbon receptor
(AHR)
altered gene expression (CYP1A and
others); oxidative stress?
di-ortho PCBs
ryanodine receptor
altered calcium homeostasis,
neurotoxicity?
di-ortho PCBs
??
altered neurotransmitter metabolism
(dopamine & serotonin)
ortho PCBs
induction of CYP2B
(PCB-164: 2,3,3’,4’,5’,6-HCB)
constitutive androstane
receptor (CAR)
highly chlorinated PCBs
rodent PXR (agonists)
induction of CYP3A; varies by species
(PCB-184, -196, -153)
human SXR (antagonists)
OH-PCB
transthyretin (TTR)
(PCB-95: 2,2’,3,5,6-PCB)
inhibition of thyroid hormone transport and
retinoid homeostasis
(rodents > humans; TTR vs TBG)
OH-PCB
sulfotransferase,
glucuronosyl transferase
inhibition of sulfotransferase (E2 and T4 ,
3-OH-BaP)
PCB and OH-PCB
estrogen receptor (ER)
ER agonist or antagonist
methylSO2-PCB
uteroglobin
displacement of progesterone??
methylSO2-PCB
glucocorticoid receptor
GR antagonist
Toxic equivalency (TEQ)
approach using
toxic equivalency
factors (TEFs)
(AHR-dependent
effects only)
TCDD toxic equivalency (TEQ) approach
using toxic equivalency factors (TEFs)
chemical
type
2,3,7,8-TCDD
PCB-126
PCB-77
PCB-105
PCB-118
PCB-153
other PCB
non-ortho
non-ortho
mono-ortho
mono-ortho
di-ortho
conc
(ng/kg lw)
45
983
2,351
119,000
376,000
5,320,000
7,630,000
13,448,334
Total PCB
(ng/kg lw)
TEF
(mammals)
TEQ
(ng/kg lw)
1
0.1
0.0001
0.0001
0.0001
0
0
% of TEQ
45.00
98.30
0.24
11.90
37.60
0.00
0.00
% of [PCB]
23.31
50.92
0.12
6.16
19.48
0.00
0.00
193.04
Total TEQ
(ng TCDD-Eq/kg lw)
• Calculated TEQs versus Bioassay-derived TEQs
0.007
0.017
0.885
2.796
39.559
56.736
TEQ approach: Assumptions
• compounds act via common mechanism
• additivity (no synergism, antagonism)
• no differences in intrinsic efficacy (all full agonists)
• similar structure-activity relationships for endpoints of
concern and endpoints used to generate TEF values
• similar structure-activity relationships for species of
concern and species used to generate TEF values
Ross et al (2000)
Receptor-dependent mechanisms of toxicity
in marine mammals
• Species differences in receptor characteristics?
- diversity
- expression
- function (affinity, SAR, target genes)
Differential Sensitivity to Dioxin (2,3,7,8-TCDD)
Mammals
- laboratory species: 5000-fold variability (lethality)
- humans: ?
- marine mammals: ?
Birds: up to 1000-fold variability among species
Reptiles: ?
Amphibians
- anurans: 1000-fold less sensitive than fish
- other amphibians: ?
Bony fishes:
40-fold variability among species
Ligand-binding assays
• High affinity, low capacity binding
(Specific Binding)
Total [3H]-TCDD
Free (loosely bound)
Bound (Total)
Non-specific Specific
binding
binding
Analysis of AHR specific binding on sucrose density gradients
AHR + [3H]TCDD
AHR + [3H]TCDD + TCDF (100x)
10% sucrose
Total binding
Non-specific binding
30% sucrose
• Incubate
• Spin for 2 hours
• Fractionate
• Count
Fractions
Sucrose gradient analysis of
in vitro-expressed and tissue-derived AHR proteins
cloned, in vitro expressed
1600
1200
dpm
Beluga Liver Cytosol
Beluga AHR
1600
TB
1200
800
800
NSB
400
400
0
0
10
20
30
10
20
30
40
Mouse Liver Cytosol
2500
2000
dpm
0
0
40
Mouse AHR
2500
2000
1500
1500
1000
1000
500
0
tissue-derived
500
0
10
20
30
fraction number
40
0
0
10
20
30
40
fraction number
Jensen & Hahn (2001)
Saturation binding analysis of in vitro-expressed AHR proteins
beluga AHR
mouse AHR
pSP64belAHR
Kd = 0.34 nM
pSPORTmoAHR
2000
TB
DPM
DPM
Kd = 0.75 nM
1500
1000
SB
pSPORThuAHR
1000
K d = 1.23 nM
750
DPM
1500
human AHR
1000
500
500
500
250
NSB
0
0
1
2
3
4
5
6
0
0
0
Free TCDD (nM)
2
3
4
5
0
M
H
1
2
3
Fre e TCDD (nM)
Fre e TCDD (nM)
B
[35S]methioninelabeled proteins
1
UPL
4
5
Equilibrium Dissociation Constants (Kd)
for in vitro-expressed AHR proteins
mean Kd (n=4)
beluga AHR
0.43 ± 0.16 nM **
mouse AHR
0.68 ± 0.23 nM *
human AHR
1.63 ± 0.64 nM
*p<0.05 versus human AHR
**p<0.01 versus human AHR
Beluga express a high-affinity (low Kd) AHR
In vitro binding affinity vs. In vivo tissue burdens
KD for TCDD: 0.43 nM in vitro
TCDD-Eqs in liver of St. Lawrence beluga:
0.13 nM (adult male)
(Muir et al. 1996 Environ. Pollut.)
Result: 23% AHR occupancy
(% Maximum response depends on receptor concentration)
Jensen & Hahn (2001)
Relative Potencies or Toxic Equivalency Factors (TEFs)
for dioxin-like compounds in wildlife
TEF values
congener
PCDD/PCDF
IUPAC
#
2,3,7,8-TCDD
2,3,7,8-TCDF
rodent
1
0.1
marine
mammals
1
?
non-ortho PCB
3,3’,4,4’,5-PeCB
3,3’,4,4’,5,5’-HCB
3,4,4’,5-TCB
3,3,’4,4’-TCB
126
169
81
77
0.1
0.01
0.0001
0.0001
?
?
?
?
mono-ortho PCB
2,3,3’,4,4’-PeCB
2,3’4,4’,5-PeCB
2,3,3’,4,4’,5-HCB
105
118
156
0.0001
0.0001
0.0005
?
?
?
Source: van den Berg, et al. (1998) Environ. Health Persp. 106: 775-792.
Competitive binding
of PCB congeners using
in vitro expressed AHRs
and [3H]TCDD
Beluga AHR
TCDD
TCDF
126
169
77
81
105
118
156
128
1.0
0.8
0.6
0.4
0.2
0.0
-5 -4 -3 -2 -1
IC50: One-site competition
model
(Prism)
0
1
2
3
4
5
6
log[HAH] nM
Mouse AHR
KI: From IC50, [3H]TCDD
(Cheng and Prusoff)
TCDD
TCDF
126
169
77
81
105
118
156
128
1.0
0.8
0.6
0.4
0.2
0.0
-5 -4 -3 -2 -1
0
1
2
3
4
5
6
log[HAH] nM
Jensen & Hahn (2001)
Correlation between beluga and mouse AHR binding affinities
105
x=y
beluga KI (nM)
104
118
103
102
156
128
10-1
Di-ortho PCB
81
77
101
100
105
Mono-ortho PCBs
169
126
Non-ortho PCBs
TCDF
TCDD
10-2
10-1 100 101 102 103 104 105
mouse KI (nM)
PCDD/F
dpm/fraction
Harbor seal versus mouse AHR
A
2000
Harbor seal
1500
1000
500
0
0
dpm/fraction
10
15
20
B
2000
25
30
35
Mouse
1500
[3H]TCDDbinding
1000
500
0
0
5
10
15
20
25
C
2000
dpm/fraction
[35S]methioninelabeled proteins
5
30
35
UPL
1500
1000
500
0
0
5
10
15
20
25
30
35
Fraction
Kim & Hahn (2002)
Bound 3H-TCDD (fmol)
TB
Mouse AHR
SB
100
mouse AHR
KD = 1.70 ± 0.26 nM
50
NSB
0
0
2
4
6
8
10
[free 3H-TCDD] (nM)
Bound 3H-TCDD (fmol)
100
Seal AHR
75
TB
SB
50
25
seal AHR
KD = 0.93 ± 0.19 nM
NSB
0
0
2
4
6
8
10
[free 3H-TCDD] (nM)
Kim & Hahn (2002)
Trainer & Baden (1999) High affinity binding of red tide neurotoxins
to marine mammal brain. Aquat Toxicol. 46: 139-148.
Weight of evidence approach
for assessing impact of contaminants
on marine mammals
Epidemiological and observational studies
in wildlife species
Comparative mechanistic studies
Mechanistic studies in laboratory animals