Download HESI Project Committee on Dose

Methylene Chloride
a case study for
Dose-Dependent Transitions
Raymond M. David, Ph.D.
Eastman Kodak Company
©Eastman
Kodak Company, 2005
Overview

Methylene chloride (DCM) is a good case
study for risk assessment because
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it is a classic example of dose-dependent
transition in carcinogenesis
there are human data for metabolism around the
inflection point
example of species differences in metabolism and
genetic polymorphisms, which impact the
quantitation of risk
PBPK modeling has been used for species
extrapolation and risk assessment
Carcinogenic Potential


National Coffee Association (NCA) study –
0, 60, 125, 185, and 250 mg/kg body in
drinking water to F-344 rats and B6C3F1 mice
for 2 years
National Toxicology Program (NTP) study –
0, 2000, or 4000 ppm by inhalation to F-344
rats and B6C3F1 mice for 2 years
Tumor response
Dose
mg/kg/d
Tumor
Incidence
0
60
125
185
250
24/125
51/200
30/100
31/99
35/125
0
1582
3162
0
1582
3162
3/50
16/48
40/48
3/45
30/46
41/46
Organ
Sex/Strain/
Species
Study
Liver
Male
B6C3F1
NCA;
Serota et al.
Liver
Female
B6C3F1
NTP
Lung
Female
B6C3F1
NTP
Liver tumor response
90%
Percentage
80%
70%
60%
50%
40%
30%
20%
10%
0%
10
100
1000
log Dose equivalent (mg/kg)
10000
Tumor incidence summary
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Increased incidence of hepatocellular
adenomas and carcinomas were observed in
mice (one sex) at  125 mg/kg/d.
Increased incidence of lung tumors
(alveolar/bronchiolar adenomas) were
observed in mice exposed to airborne
concentrations of  2000 ppm.
F-344 rats showed no evidence of increased
liver or lung tumors.
Mode of action

At low concentrations, DCM is metabolized
primarily by cytochrome P450 (CYP).


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Kubic and Anders (1975) and Anders et al. (1977)
demonstrated that DCM was metabolized by CYP to
carbon monoxide.
Kim and Kim (1996) later identified CYP2E1 as the
isozyme associated with this pathway.
Kubic and Anders (1978) determined the Km (50.1
mM) and Vmax (5.4 nmol CO/mg prot/min).
McKenna et al. (1982) showed that CYP2E1 in
laboratory animals was saturated above
concentrations of 500 ppm.
Mode of action
CYP 2E1 catalyzed:
CH2Cl2  CHOHCl2  HCOCl  CO + CO2
formyl chloride
COHb
Mode of action

At higher concentrations, CYP pathway can be
saturated and GST pathway metabolizes DCM.

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Ahmed and Anders (1976) and Anders et al. (1977)
demonstrated that DCM was metabolized via a GST
pathway.
Gargas et al. (1986) proposed the current metabolic
scheme via GST pathway.
Blocki et al. (1994) showed that GST 5-5, a -class
GST (also known as T1-1 in humans), had the highest
specific activity for DCM (11,000 nmol/min/mg protein)
with a Km of 300 µM.
Mode of action
GST catalyzed:
CH2Cl2  GSCH2Cl
GSCH2OH  HCHO
chloromethylglutathione  GSCHO  HCOOH  CO2
MOA – supporting data

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Reynolds and Yee (1967) and Anders et al.
(1977) showed that 14C-DCM was bound to
tissue protein and lipid.
Casanova et al. (1992) demonstrated an
increase of DNA-protein cross-links (DPX) in
the liver and RNA-formaldehyde adducts
(RFA) in the lungs.
DNA adducts may also be formed directly
from the chloromethylglutathione
intermediate rather than formaldehyde
(Marsch et al., 2001, 2004).
MOA – supporting data
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
Graves et al. (1994) and Thier et al.
(1993) linked mutations observed only
in S. typhymurium strains TA1535 and
TA100 to nascent GST activity.
Other tests for genetic toxicity generally
negative.
Constructing the data set
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Metabolic parameters for different
species
Developing human data parameters
Developing a model

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Understanding the compartments
Physiological parameters in different
species
Species metabolic parameters
MFO pathway
Species
Km
Vmax
GST pathway
Km
Vmax
(mM)
(nmol/min/mg prot)
(mM)
(nmol/min/mg
prot)
mouse
1.84  0.33
15.90  1.10
137  21
118.2  14.4
rat
1.42  0.74
5.39  0.94
nd
nd
human
0.92 – 2.82
1.53 – 13.00
43.8 – 44.1
6.04 – 7.05
From Reitz et al., 1988. nd = not determined
Species metabolic parameters
Species Tissue
Mouse
Rat
Human
Liver
Lung
Liver
Lung
Liver
Lung
MFO
1.760 ±
0.732 ±
0.814 ±
0.111 ±
0.418 ±
0.0006 ±
0.115
0.115
0.118
0.035
0.157
0.0003
GST
5290 ± 430
727 ± 64
1380 ± 110
77 ± 5
1650 ± 480
78 ± 47
Specific activities from Lorenz et al. (1984) in nmol/min/mg protein as reported by Andersen et al. (1987)
Species metabolic parameters
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Glutathione transferase (GST T1-1) catalyzes
the conjugation of glutathione and DCM in
mice and humans.
The gene is polymorphic in humans
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Non-conjugators: GST T1 (–/–)
Low conjugators: GST T1 (+/–)
High conjugators: GST T1 (+/+)
Distribution of the null phenotype in humans
has been studied.
GSTT1 -/- Distribution
Group
% Population
% Homozygous
Asian
Caucasian
3.9
75.5
62
19.7
AfricanAmerican
12.2
21.8
MexicanAmerican
11.4
9.7
From El-Masri et al., 1999.
Human data sets
Exposure
levels (ppm)
Duration
hrs
100, 350
6
Number
of
subjects
6
Reference
50, 100,
150, 200
8
13
DiVincenzo et al.
(1986)
250, 500,
1000
2.5
14
Astrand et al.
(1975)
Andersen et al.
(1987)
Human data
35
30
25
20
15
10
OSHA Posterior
OSHA Prior
E
D
C
B
A
Individual
Values
(Jonsson et al.
(2001)
Clewell (1995)
Jonsson and
Johanson (2001)
Expert Elicitation
Individual Values (Sweeney et al., 2004)
13
12
11
10
9
8
7
6
5
4
3
0
2
5
1
Vmaxc/Km (/hr)
40
Population Values
PBPK modeling
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First interspecies extrapolation using
PBPK modeling was Andersen et al.
(1987).
Dose metric was blood, tissue, and
exhaled DCM.
Human metabolic values were mean
from subjects exposed to 100 or
350 ppm for 6 hours.
Andersen model
GST
CYP
Gas
Exchange
Lung
Metabolism
Blood
Richly perfused
Fat
Slowly perfused
Liver
GST
CYP
GI tract
PBPK models for DCM assessment
Citation
Remarks
Reitz et al., 1988
Deterministic approach. Andersen et al.
(1987) model updated with measured MFO
and GST rate constants.
Andersen et al., 1991
Deterministic approach. Blood compartment
added to describe carbon monoxide and
carboxyhemoglobin kinetics.
Dankovic et al., 1994
Deterministic approach. Mean values for
alveolar ventilation, cardiac ouput, and tissue
blood flow increased.
Casanova, et al., 1996
Deterministic approach. Liver DNA-protein
cross-links from formaldehyde used as the
dosimeter of effect.
PBPK models for DCM assessment
Citation
Remarks
Bois and Smith,
1995
Probabilistic (Bayesian) approach. Bone marrow
compartment added, variance in metabolic rate
constants increased.
Thomas et al.,
1996
Probabilistic (Bayesian) approach. Variability
from MFO induction, GST inhibition, and tissue
solubility included.
El-Masri et al.,
1999
Probabilistic (Bayesian) approach incorporating
GST-T1 polymorphisms and estimating DPX.
Jonsson and
Johanson, 2001
Probabilistic (Bayesian) approach. New fat and
muscle compartments. Includes population
estimates of glutathione transfersase T1 gene
frequencies
Changes in unit risk over time
Source
EPA 1985
Unit risk
(per µg/m3)
1.0  10-6
EPA 1991
4.7  10-7
El Masri et al., 1999
1.9  10-10
Jonsson and Johanson,
2001
1.9  10-10
DCM PBPK model results
Individual K2, Blood Carboxyhemoglobin
Blood Carboxyhemoglobin (percent)
6
5
50 ppm (model)
4
100 ppm (model)
200 ppm (model)
3
50 ppm data
100 ppm data
2
200 ppm data
1
0
0
10
20
30
Time (hr)
40
50
DCM PBPK model results
Individual K2, Exhaled Breath DCM
Exhaled breath DCM (ppm)
100
50 ppm (model)
100 ppm (model)
200 ppm (model)
10
50 ppm data
100 ppm data
200 ppm data
1
0.1
0
5
Time (hr)
10
DCM PBPK model results
Individual K2, Blood DCM
Blood DCM (mg/L)
10
50 ppm (model)
1
100 ppm (model)
200 ppm (model)
50 ppm data
100 ppm data
0.1
200 ppm data
0.01
0
2
4
6
Time (hr)
8
10
12
Sweeney model
GST
CYP
Gas
Exchange
Lung
Metabolism
Blood
Richly perfused
Fat
CYP
Slowly perfused
Liver
GST
CYP
GI tract
Updating the risk assessment
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Do the new human data and the model change
the calculated unit risk?
Perhaps --- the unit risk is 4.8 x 10-8 using the
Sweeney PBPK model compared with 4.7 x 10-7
used by the EPA.
Using probabilistic methodology and genetic
polymorphisms might also impact the unit risk
calculation.
Summary

DCM is a good example for quantitative risk
assessment because



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it demonstrates a dose-dependent transition from
non-carcinogenic pathway to carcinogenic
pathway
human data are available
genetic polymorphisms in human populations can
be factored into the assessment
PBPK models extrapolating from animal to humans
are available