&EPA
United Slates
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/AP-92/001f
August 1992
Workshop Review Draft
Chapter 6.
Carcinogenicity of
TCDD in Animals
Review
Draft
(Do Not
Cite or
Quote)
Notice
This document is a preliminary draft. It has not been formally released by EPA and should not
at this stage be construed to represent Agency policy. It is being circulated for comment on
its technical accuracy and policy implications.
-------
DRAFT EPA/600/AP-92/001f
DO NOT QUOTE OR CITE August 1992
Workshop Review Draft
Chapter 6. Carcinogenicity of TCDD in Animals
Health Assessment for
2,3,7,8-TetrachIorodibenzo-p-dioxin (TCDD)
and Related Compounds
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by the U.S.
Environmental Protection Agency and should not at this stage be construed to represent Agency
policy. It is being circulated for comment on its technical accuracy and policy implications.
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C.
.oS Printed on Recycled Paper
-------
DRAFT-DO NOT QUOTE OR CITE
DISCLAIMER
This document is a draft for review purposes only and does not constitute Agency policy.
Mention of trade names or commercial products does not constitute endorsement or recommendation
for use.
Please note that this chapter is a preliminary draft and as such represents work
in progress. The chapter is intended to be the basis for review and discussion at
a peer-review workshop. It will be revised subsequent to the workshop as
suggestions and contributions from the scientific community are incorporated.
ii m/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
CONTENTS
Tables , iv
Figures v
List of Abbreviations vi
Authors and Contributors , xi
6. CARONOGENieiTY OF TCDD IN ANIMALS 6-1
6.1. INTRODUCTION 6-1
6,2. ANIMAL BIOASSAYS FOR CANCER . , 6-3
6.2.1. Kociba Study 6-3
6.2.2. NTP Study (Osborne-Mendel Rats and B6C3F1 Mice) (NTP, 1982a) 6-6
6.2,3. Syrian Golden Hamster 6-7
6.2.4. B6C3 and B6C Mice (Delia Porta et al. 1987) 6-10
6.2.5. Carcinogenicity of Related Compound (NTP, 1980) 6-10
6.3. MECHANISMS OF TCDD-MEDIATED CARCINOOEMCITY . 6-11
6,4. INITIATION-PROMOTION STUDIES ! 6-13
6.4.1. Two-Stage Models in Rat Liver . 6-15
6.4,2. Rat Lung 6-18
6.4.3. Mouse Skin 6-20
6.5. BIOCHEMICAL RESPONSES 6-23
6.5.1. CYP1AI and 1A2 6-23
6.5.2. EGFR 6-28
6.5.3. UDPGT , 6-33
6.5.4. ER 6-34
6.5.5. Other Biochemical Endpoints 6-36
6.6, SUMMARY AND WEIGHT OF EVIDENCE FROM ANIMAL STUDIES 6-37
6,7. REFERENCES 6-38
iii 08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF TABLES
6-1 Sites for Increased Cancer in Animal Bioassays 6-4
6-2 Different Evaluations of Kociba Liver Tumor Data in Female Rats 6-5
6-3 Tumor Incidences in Male and Female Osborne-Mendel Rats Given TCDD by Gavage
for 2 Years 6-8
6-4 Tumor Incidences in Male and Female B6C3F1 Mice Given TCDD by Gavage for 2
Years 6-9
6-5 Preneoplastic Foci and Cell Proliferation After 30 Weeks of TCDD Tumor Promotion . . 6-17
6-6 Summary of Positive Tumor Promoting Studies on TCDD and CDFs 6-22
6-7 Classification of Members of the Ah Gene Battery 6-24
iv 08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF FIGURES
6-1 Schematic Representation of MuUistep Carcinogenesis Including
the Roles of Genetic Damage and Cell Proliferation 6-14
6-2 Operational Model of TCDD/Estrogen Interactions Relative to Tumor
Promotion in a Two-Stage Model of Hepatocarcinogenesis 6-19
6-3 Plausible Mechanism for the Role of EGF-Mediated Stimulation
of Mitotic Activity 6-30
08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF ABBREVIATIONS
ACTH Adrenoeorticotrophic hormone
Ah Aryt hydrocarbon
AHH Aryl hydrocarbon hydroxylase
ALT L-alanine aminotransferase
AST L-asparate aminotransferase
BDD Brominated dibenzo-p-dioxin
BDF Brominated dibenzofuran
BQF Bioconcentration factor
BOG Bovine gamma globulin
bw Body weight
eAMP Cyclic 3,5-adenosine monophosphate
CDD Chlorinated dibenzo-p-dioxin
cDNA Complementary DNA
CDF Chlorinated dibenzofuran
CNS Central nervous system
CTL Cytotoxic T lymphocyte
DCDD 2,7-DicMorodibenzo-p-dioxin
DHT Sct-Dihydrotestosterone
DMBA Dimethylbenzanihracene
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
vi 08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF ABBREVIATIONS (cont.)
ORE
DTG
DTH
ED*,
ECOD
EOF
EGFR
ER
EROD
EOF
FSH
GC-ECD
GC/MS
GOT
GnRH
GST
HVH
HAH
HCDD
HDL
HxCB
Dioxin-responsive enhancers
Delayed type hypersensitivity
Delayed-type hypersensitivity
Dose effective for 50% of recipients
7-Ethoxycoumarin-O-deethyIase
Epidermal growth factor
Epidermal growth factor receptor
Estrogen receptor
7-Ethoxyresurofin 0-deethylase
Enzyme altered foci
Follicle-stimulating hormone
Gas ehromatograph-electron capture detection
Gas chromatograpn/mass spectrometer
Gamma glutamyl transpeptidase
Gonadotropin-releasing hormone
Glutathione-S-transferase
Graft versus host
Halogenated aromatic hydrocarbons
Hexachlorodibenzo-p-dioxin
High density lipoprotein
Hexachlorobiphenyl
Vll
08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF ABBREVIATIONS (cont.)
HpCDD Heptachlorinated dibenzo-p-dioxin
HpCDF Heptachlorinated dibenzofuran
HPLC High performance liquid chromatography
HRGC/HRMS High resolution gas ehromatography/high resolution mass spectrometry
HxCDD Hexachlorinated dibenzo-p-dioxin
HxCDF Hexachlorinated dibenzofuran
I-TEF
LH
LDL
LPL
LOAEL
LOEL
MCDF
MFO
mRNA
MNNG
NADP
NADPH
NK
International TCDD-toxic-equivalency
Dose lethal to 50% of recipients (and all other subscripter dose levels)
Luteinizing hormone
Low density liproprotein
Lipoprotein lipase activity
Lowest-observable-adverse-effeet level
Lowest-observed-effect level
6-Methyl-l,3,8-trichlorodibenzofuran
Mixed function oxidase
Messenger RNA
#-methyl-./Y-nitrosoguanidffle
Nicotinamide adenine dinucteotide phosphate
Nicotinamide adenine dinucleotide phosphate (reduced form)
Natural killer
via
Q8/24/?2
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF ABBREVIATIONS (cont.)
NOAEL
NOEL
OCDD
OCDF
PAH
PB-Pk
PCB
OVX
PEL
PCQ
PeCDD
PeCDF
PEPCK
POT
PHA
PWM
ppm
PW
ppt
RNA
SAR
No-observable-adverse-effect level
No-observed-effect level
OctacMorodibenzo-p-dioxin
OetacMorodibenzofuran
Polyaromatic hydrocarbon
Physiologically based pharmacokinetic
Polychlorinated biphenyl
Ovariectomized
Peripheral blood lymphocytes
Quaterphenyl
Pentachlorinated dibenzo-p-dioxin
Pentachlorinated dibenzo-p-dioxin
Phosphopenol pyravate carboxykinase
Placental glutathione transferase
Phytohemagglutinin
Pokeweed mitogen
Parts per million
Parts per trillion
Ribonucleic acid
Structure-activity relationships
IX
08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
LIST OF ABBREVIATIONS (cont.)
SOOT
SOFT
SRBC
1*
TCAOB
TCI
TCDD
TEF
TGF
tPA
TNF
TNP-LPS
TSH
TTR
UDPGT
URO-D
VLDL
v/v
w/w
Serum glutamic oxaloacetic transaminase
Senun glutamic pyruvic transaminase
Sheep erythroeytes (red blood cells)
Half-time
Tetraehloroazoxybenzene
Tetrachlorobiphenyl
Tetraehlorodibenzo-p-dioxin
Toxic equivalency factors
Thyroid growth factor
Tissue plasminogen activator
Tumor necrosis factor
lipopolysaccharide
Thyroid stimulating hormone
Transthyretrin
UDP-glucuronosyltransferases
Uroporphyrinogen decarboxylase
Very low density lipoprotein
Volume per volume
Weight by weight
08/24/92
-------
DRAFT-DO NOT QUOTE OR CITE
AUTHORS AND CONTRIBUTORS
The Office of Health and Environmental Assessment (OHEA) within the Office of Research
and Development was responsible for the preparation of this chapter. The chapter was prepared
through Syracuse Research Coiporation under EPA Contract No. 68-CO-0043, Task 20, with Carol
Haynes, Environmental Criteria and Assessment Office in Cincinnati, OH, serving as Project Officer.
During the preparation of this chapter, EPA staff scientists provided reviews of the drafts as
well as coordinating internal and external reviews.
AUTHORS
George Lucier
National Institute of Environmental Health Sciences
Research Triangle Park, NC
EPA CHAPTER MANAGER
Charalingayya B. Hiremath
Office of Health and Environmental Assessment
Washington, DC
xi 08/24/92
-------
DRAFT—DO HOT QUOTE OR CITE
6. CARCINOGENICITY OP TCDD IN ANIMALS
6.1. INTRODUCTION
There is more scientific information relevant to the use of animal cancer
data for the estimation of human risks than was available in 1988, However, much
of the tumor incidence data in experimental animals was available in 1988 to
demonstrate that TCDD is a carcinogen at multiple sites in both sexes of rats and
mice. Some of the cancers occurred following low doses. Since 1988, TCDD has
been shown to be a carcinogen in hamsters and some of the tumor incidence data
in rat liver has b^en reevaluated during the last 3 years.
In the last few years there have been several studies which have impact on
the evaluation of cancer studies in experimental animals. For example, the
evidence is now considerably stronger that TCDD does not damage DNA directly
through the formation of DNA adducts. However, there are proposed mechanisms for
the possibility that TCDD might alter the DNA damaging potential of some
endogenous compounds including estrogens. In addition, there have been numerous
reports on TCDD-mediated modifications of growth factor pathways and cytokines
in experimental animals and cell systems. Some of the altered systems include
those for epidermal growth factor, transforming growth factor a, estrogen,
glucocorticoids, tumor necrosis factor a, interleukin 1 B, plasminogen inactiva-
ting factor and gastrin. Many of these pathways are involved in cell prolifera-
tion and differentiation and provide plausible avenues to research the mechanisms
responsible for the carcinogenic actions of TCDD. These effects are consistent
with the general accepted conclusion that TCDD acts as a tumor promoter in
multistage models for chemical carcinogenesis and is virtually devoid of
initiating activity in these models. It is important to note that "tumor
promotion" is an operational and not a mechanistic term and there are likely
multiple mechanisms of tumor promotion. Each of these mechanisms may be
fundamentally different from the other.
Over the last few years there has been growing consensus that most, if not
all, of TCDD's biochemical and toxic effects require interaction with the Ah
receptor. The properties of the Ah receptor and the mechanisms whereby this
receptor regulates gene expression will be evaluated in other chapters. However,
6-1 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
formation of the Ah receptor-TCDD complex is only the first of many steps
involved in the production of a biochemical and toxic effect. Although we are
gaining increasing detail of the subsequent steps, we know very little about some
components of the Ah receptor mediated responses. It is clear, however, that
cell specific factors other than the Ah receptor roust be involved in determining
tissue responses once TCDD binds the Ah receptor.
Evaluation of dose-response is one of the more issues that impact dioxin
risk assessments. The focus of the controversy centers on whether the effects
of dioxin would exhibit a threshold or not. It now appears that for some
responses there is a proportional relationship between receptor occupancy and
response which is evidenced by a linear relationship between target dose and
effect over a wide dose range. However, different dose response relationships
are seen for different responses so it is likely inappropriate to use a single
surrogate marker to estimate dioxin's risks. Furthermore, these data reveal
there is no unifying dose-response relationship for all Ah receptor mediated
events.
Another controversial area in risk assessment IB whether experimental animal
models are appropriate for estimating human risks. During the last few years
there has been increasing evidence that biochemical and toxic responses resulting
from human exposure to TCDD and its structural analogs appear to be similar to
responses in experimental animals. However, there is also increasing awareness
that interindividual variation in human responses to dioxin are a complicating
factor in risk assessment; it appears there are responsive and non-responsive
individuals to numerous environmental chemicals including TCDD.
Much of the controversy surrounding dioxin risk assessment reflects the
selection of methods; threshold or linear multistage. We now know considerably
more about mechanism of action of dioxin and this knowledge may permit the
construction of biologically-based models which removes some of the uncertainty
in current risk estimates. These approaches and recent advances on mechanisms
of tumor promotion and dose-response relationships for biochemical and biological
events relevant to the carcinogenic actions of dioxin will be discussed in more
detail in the following sections of the paper.
S-2 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
6.2. ANIMAL BIOASSAYS FOR CANCER
There have been seventeen long-term bioassays for carcinogen!city of TCDD
in several species. All seventeen produced positive results. It is clear that
TCDD is a multistage carcinogen in both sexes of rats and mice (Huff et al.»
1991; Zeise et al, 1990). It is also a carcinogen in the hamster which is
considered the most resistant species to the acute toxic effects of TCDD. The
seventeen studies are summarized in Table 6-1 including information on species,
sex, dose and tumor site. Some of the studies are especially relevant to risk
assessment. Detailed evaluations of these studies are given in the following
paragraphs.
6.2.1. Kociba study. The roost cited cancer bioassay for TCDD was published in
by Kociba et al, {1978}. It was a lifetime feeding study of male and female
Sprague-Dawley rats using doses of 1, 10 and 100 ng/kg/day. There were BO males
and 50 females in each group. Data derived from these studies provide the basis
for many of the risk assessments for TCDD. The most significant finding was an
increase in hepatocellular hyperplastic nodules and hepatocellular carcinomas in
female rats. The carcinomas were significantly elevated above the control
incidence at the 100 ng/kg/day dose, whereas increased incidences of hyperplastic
nodules were evident in the 10 ng/kg/day dose group. There have been two
revaluations of the Kociba slides of liver sections (Squire, 1985? Sauer, 1990).
The Squire review was requested by EPA as an independent review of the slides.
The Sauer review used 'diagnostic criteria for liver tumors described by Maronpot
et al., (1986). Liver tumor incidences for the three evaluations are compared
in Table 6-2. Although there are some quantitative differences in the
evaluations, the lowest detectable effect is consistently 10 ng/kg/day for liver
tumor incidence. In the 10 ng/kg/day dose group hyperplastic nodules of the
liver were observed in female rats (18 Kociba, 27 Squire). Two females had
carcinomas of the liver. In the recent reevaluation of liver lesions by Sauer
(1990), nine females were identified with hepatocellular adenomas and none with
carcinomas; thus only one-third of the previously observed tumors were confirmed.
There was no detectable increase in liver tumor incidences in male rats (Table
6-1) in any of the dose groups. The mechanism responsible for dioxin-mediated
6-3 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
TABLE 6-1
Sites for Increased Cancer in Animal Bioassays*
Species/Strain
Rats/Sprague-Dawley
Mice/Osborne-Mendel
Mice/B6C3Pl
Mice/B6CeFl
Syrian Golden
Sex
male
female
male
female
male
female
male/
female
male
Site
tongue
nasal turbinates/hard
lung
nasal turbinates/hard
liver
palate
palate
thyroid
adrenal cortex
liver
adrenal cortex
subcutaneous fibrosarcoma
liver
subcutaneous fibrosarcoma
liver
thyroid
thymic lymphomas
liver
facial skin carcinoma
*Source: Kociba et al.
Rao et al., 1988
, 1978; NTP, 1982; Delia Porta et al., 1987;
6-4
08/24/92
-------
DBAFT—DO NOT QUOTE OR CITE
TABLI 6-2
Dif.erent Evaluations of Kociba Liver Timor Data in Female Rats8'
Study
Kociba
Squire
Sauer
Tumor Type
hyperplastic nodule
hepatocellular carcinoma
hyperplsstic nodule;
hepatocellular carcinoma
hyperplastic nodule;
hepatocellulsr carcinoma
hepatocellular adenoma
hepatocellular carcinoma
hyperplastic nodule;
hepatocellular carcinoma
Control
8/86
p<0.001
1/86
p<0.001
9/86
p<0.001
16/68
p<0,001
2/S6
0/86
2/86
Dose (ng/kg/day)
1
3/50
p=0.8
0/50
3/50
p=o.r
8/50
p=0.?
1/50
0/50
1/50
10
18/50
p<0.001
2/50
p=0.3
20/50
p<0.001
27/50
p<0.001
9/50
0/50
9/50
100
23/50
p<0.001
11/50
p<0.001
34/50
p<0.001
33/47
p<0.001
14/50
4/50
18/50
"Source: Kociba et al., 1978
p-Values for Fisher's exact test are given beneath the incidence data for TCDD-treated animals;
Hantel-Haenszel trend test are given beneath the control incidences
6-5
08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
sex specificity for hepatocarcinogenesis in rats is not clear but may involve
estrogens and this is discussed in the section on tumor promotion.
Kociba et al. (1978) had reported that chemically-related preneoplastic or
neoplastic lesions were not found in the 1 ng/kg/day dose group. However, Squire
identified two male rats in the 1 ng/kg/day dose group with squamous cell
carcinoma of the nasal turbinates/hard palate and a separate male squamous cell
carcinoma of the tongue. These are both rare tumors for Sprague-Dawley rats and
these sites are targets for TCDD implying that the 1 ng/kg/day may not represent
a no observed effect level (NOEL).
In addition to the liver, tongue, nasal turbinates and hard palate,
increased lung tumor incidences were observed in female rats (seven Kociba, nine
Squire). The increase, at the high dose (100 ng/kg/day), was statistically
significant for keratinizing squamous cell carcinomas.
One of the more interesting findings in the Kociba bioassay was reduced
tumor incidences of the pituitary, uterus, mammary gland, pancreas and adrenals.
For example, carcinomas of the mammary gland occurred in 8/86 of the control
female rats whereas the incidence was 0/49 in the 1 ng/kg/day dose group.
However, the incidence of mammary gland carcinomas in the medium- and high-dose
groups was similar to that of control rats suggesting that protection against
breast cancer might be a low-dose effect. These findings coupled with the sex
specificity of TCDD induced liver tumors emphasizes that the carcinogenic actions
of TCDD involve a complex interaction of hormonal factors. Moreover, it appears
likely that cell specific factors modulate TCDD/hormone actions relevant to
cancer.
6.2.2. HTP Study (Osborne-Mendel Hats and B6C3P1 Mice) (NTP, 1982a). Groups
of 50 male rats, 50 female rats and 50 male mice received doses of 10, 50 or 500
ng/kg/week TCDD by gavage in two administrations each week for two years? groups
of 50 female mice were given 40, 200 or 2000 ng/kg/week. These exposures
correspond to average daily doses of 1.4, 7.1 or 71 ng/kg/day for rats and male
mice and to doses of 5.7, 28.6, or 286 ng/kg/day for female mice so the doses
were roughly similar to those used in the Kociba dietary study. There were no
statistically significant dose-related decreases in survival in any sex-species
group.
6-6 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Tumor data in the NTP bioassay are summarized in Tables 6-3 and 6-4. TCDD-
induced malignant liver tumors in the high-dose female rats and in male and
female mice. These can be considered to result from TCDD exposure since they are
relatively uncommon lesions in control Osborne-Mendel rats (male 1/2O8; female
3/208), are seen in female rats and mice of both sexes and their increasing
incidence with increasing dose is statistically significant (Cochran Armitage
trend test, p=0.004). Since liver tumors were increased in both sexes of mice,
this effect is not female specific as observed in rats. Interestingly, liver
tumor incidences were decreased in female rats in both the NTP and Kociba low
doses (not statistically significant compared to controls). For example, the
combined control incidence data were 11/161 compared to 4/99 (4%) in the low-dose
group. '
The incidences of thyroid gland (follicular cell) tumors were increased in
all three dosed groups in male rats. Because the responses in the two highest
dose groups are highly significant, the elevation of incidence in the lowest dose
group (Fisher exact p value=0.42) is considered to be caused by exposure to TCDD.
Thus, for this study the LOEL is 1.4 ng/kg/day and a NOEL was not achieved within
the specified dose range suggesting that thyroid tumor incidence may be the most
sensitive site for TCDD-mediated carcinogenesis.
TCDD induced neoplasms of the adrenal gland in high-dose female rats.
Fibrosarcomas of the subcutaneous tissue were significantly elevated in high-dose
female mice and possibly female rats. One additional tumor type, lymphomas, were
seen in high-dose female mice. Lung tumors were elevated in high-dose female
mice; the increase was not statistically significant when compared with concur-
rent controls but the increase was dose related (Cochran Armitage trend test
p=0.004).
Therefore, TCDD is a multisite carcinogen and it increased neoplasms in rats
and mice of both sexes. As in Kociba et al. (1978), liver tumors were observed
with greater frequency in treated female rats, but the male thyroid appears to
be the most sensitive (increased tumor incidence doses as low as 1.4 ng/kg/day).
6.2.3. Syrian Golden Hamster. Groups of 10-24 male Syrian Golden hamsters were
given two to six intraperitoneal or subcutaneous injections of TCDD over a 4-week
period at doses of 50 or 100 /jg/kg TCDD in dioxane (Rao et al., 1988). The
6-7 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
TABLE 6-3
Tumor Incidences in Male and Female Osborne-jKendel Rats Given TCDD by
Gavage for 2 Years*1*3
Target Organ/Tumor Type
Thyroid
follicular cell
adenoma
Liver
neoplastic nodule
Adrenal cortex
adenoma
Liver
neoplastic nodule
Adrenal cortex
adenoma or carcinoma
Subcut aneou s
fibrosarcoma
Sex
males
females
Dose (ng/kg/day)
0
1/69
p=0.006
0/74
p=0,Q05
6/72
p=0.26
5/75
p<0.001
11/73
p=0.014
0/75
~~
1.4
S/48
p=0,042
0/SO
— —
9/50
p=0.09
1/49
—
9/49
p=0.4
2/50
p=0.16
7.1
6/50
p=0.021
0/50
—
12/49
p=0.015
3/50
— —
5/49
—
3/50
p=0.06
71
10/50
p=0.001
3/50
p=0.06
9/49
p=0.09
12/49
p=0.006
14/46
p=0.039
4/49
p=0.023
aSource: NTP, 1982
"p-Values under the tumor incidence data of controls are
Armitage test for dose-related trend and p-values under
groups are from Fisher's exact trend test
from Cochran
TCDD-treated
6-8
08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
TABLE 6-4
Tumor Incidences in Male and Female B6C3F1 Mice Given TCDD by Gavage
for 2 Years*'"
Target Organ/Tumor Type
Liver
carcinoma
adenoma
Subcutaneous
f ibrosareoma
Liver
carcinoma
adenoma
Thyroid
follicular cell adenoma
Lymphoma
Sex
male
female
Dose (ng/kg/day)
0
8/73
p=O.OQ2
7/73
p=0.024
1/74
p=0.007
1/73
p=0 . 008
2/73
p=0.11
0/69
p=0.016
18/74
p=0,011
1.4
9/49
p=0.19
3/49
1/50
p=0.6
2/50
p=0.4
4/50
p=0.2
3/50
p=0.07
11/50
7.1
8/49
p=0.28
5/49
p=0.6
1/48
p=0.6
2/48
p=0.4
4/48
p=0.2
1/47
p=0.4
13/48
p=0.4
71
17/50
p=0.002
10/50
p=0 . 09
5/47
p=0,032
6/47
p=0.014
5/47
p=0,08
5/46
p=0 . 009
20/47
p=0 . 029
•source: NTP, 1982
"p-Values jor controls represents Cochran-Armitage trend test and
values for TCDD-treated groups derived from Fisher,s exact test.
6-9
08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
experiments were terminated after 12-13 months. The 100 jjg/kg groups (total dose
of 600 //g/kg) from both injection routes developed squamous cell carcinomas of
the skin in the facial region: 4/18 (22%) from the intraperitoneal injection and
3/14 (21%) from the subcutaneous injection. The lesions were large (1.5-3 cm)
with extensive necrosis and some metaetasized to the lung. The earliest
neoplasms were detectable 8 months after the initial injection. Similar lesions
were not seen in hamsters receiving two intraperitoneal injections of 100 pg/kg
TCDD or six subcutaneous injections of dioxane vehicle and none have been
reported over the past 10 years in this laboratory. An extensive study by Pour
et al. (1976) identified only one skin papilloma in S33 control Syrian hamsters.
This report demonstrates that the hamster, a non-responsive species (for acute
toxic effects) is susceptible to the carcinogenic actions of TCDD at doses well
below the maximum tolerated dose.
6.2.4. B6C3 and B6C aiice (Delia Porta et al. 1987). TCDD was administered
intraperitoneally in corn oil at doses of 0,1,30 and 60 ^g/kg to groups of 89-186
B6C3 and B6C mice of both sexes once weekly for 5 weeks starting at the day 10
of life, and the animals were observed until 78 weeks of age. Histopathological
observations were limited to the liver, kidney and organs with apparent or
suspected pathological changes. Thymic lymphomaa were induced at the 60 pg/kg
level in both sexes of both hybrids and at 30 yg/kg in all but female B6C3 mice.
Neoplasms of the liver occurred in male B6C3 at 30 /ag/kg and female B6C3 mice at
iO pg/kg. In a separate experiment, groups of 42-50 B6C3 mice were exposed to
0, 2.5 and 5.0 ^g/kg TCDD in corn oil by gavage once weekly for 52 weeks starting
at 6 weeks of age. The study was stopped at 110 weeks. Increased incidences of
liver tumors were related to TCDD exposure at both dose levels.
In summary, there is convincing evidence in the scientific literature that
TCDD is a potent multisite carcinogen in both sexes of several species and
carcinogenic effects have been observed at doses over two orders of magnitude
less than the maximum tolerated dose.
6.2.5. Carcinogenicity of Related Compounds (TOP, 1980)
A mixture of two isomers of HCDD (1,2,3,6,7,8 and 1,2,3,7,8,9) were given
by gavage twice weekly for 2 years to osborne-Mendel rats and B6C3P1 mice. The
doses of HCDD were 0, 1.25, 2.S or 5 pg/kg/week in rats and male mice. Doses for
6-10 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
female mice were 0, 2.5, 5 and 10 ^g/kg/week. There was no affect of administra-
tion of TCDD on survival of either aex of rats or mice (NIP, 1980). Results are
summarized in Table 6-4 and revealed that HCDD increased liver tumors in both
sexes of rats and mice although female rats seemed to be more sensitive than male
rats (significant increases detected in female rats in the 1.25 pg/kg/week dose
group? equivalent to 180 ng/kg/day). Therefore, HCDD is approximately 1/20 as
potent a liver carcinogen as TCDD.
Dermal application of the same HCDD mixture aa described above (NTP, 1982b)
were given to Swiss Webster mice for 104 weeks {thrice weekly). For the first
16 weeks, doses of 5 ng/application were used. Thereafter doses of 10 ng/appli-
cation were used. Ko HCDD-exposure-related carcinogenic responses were noted.
Dibenzo-p-dioxin given in the diet for 2 years at concentrations of 0, 5,000
and 10,000 ppm did not increase carcinogenic responses in Osborne-Mendel rats or
B6C3F1 mice (NCI, 1979a). DCDD in the diet of Osborne-Mendel rate for 110 weeks
or B6C3F1 mice for 90 weeks at levels of 0, 5,000 or 10,000 ppm did not increase
neoplasms in male or female rats or in female mice. In male mice, increased
incidences of lymphoma or hemangiosarcoma were observed in the low-dose group and
neoplasms of the liver were observed in both dose groups (NCI, 1979b). The more
highly chlorinated CDDs and CDFs have not been studied in long-term animal cancer
bioassays. Many of the CDDs and CDPs bioaecuraulate and exhibit toxicities
similar to those of TCDD and are considered as carcinogens (EPA Science Advisory
Board, 1989; CDHS, 1985).
6.3. MECHANISMS OF TCDD-MEDIATED CARCINOGENICITY
There is substantial evidence that TCDD is not a direct genotoxic agent.
Since "genotoxic" and "non-genotoxic" are controversial and often misused terms
it is prudent to describe accurately the scientific criteria used to call a
chemical "genotoxic" or "non-genotoxic" (1ARC, 1992). Some of the criteria for
designating TCDD a non-genotoxic agent are that it does not bind covalently to
DNA (does not form DNA adducts), is negative in short-term tests for genotoxicity
and is a potent promoter and weak initiator in multistage models for chemical
carcinogenesis. In a recent study (Turtletaub, 1990) using accelerator mass
spectrometry, DNA adducts were not detected in rodent tissue following exposure
to TCDD. This method is extraordinarily sensitive, being capable of detecting
6-11 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
one adduct in 10" normal nucleotides. For comparison, approximately one adduct
in 10° normal nucleotides are found in rodent tissues following carcinogenic
doses of benzo(a)pyrene (7,8 diol-9,10 epoxide deoxyguanoeine DMA adduct),
methylnitroeurea (0" methylguanine) or NNK (0" methylguanine).
Another criterion for designating TCDD a non-genotoxic carcinogen is that
numerous studies have demonstrated that TCDD is negative in the Salfflo,nel!a/Ames
test in the presence or absence of a MFO activating system. These negative
studies have encompassed 13 different bacteria strains with tests performed in
nine laboratories (Wassom et al., 1977,- Kociba, 1984; IARC, 1982; Giri, 1987; Shu
et al., 1987). NTP (1984) concluded that TCDD was non-mutagenic using its
battery of testa for genetic toxicity. Additionally, several scientific panels
have stated that false negatives for TCDD genetic toxicity are highly unlikely
(EPA Science Advisory Board, 1984). TCDD has been found to promote the transfor-
mation of C3H/10T1/2 cells; it was concluded that this response did not reflect
TCDD's ability to directly damage DNA (Abernethy et al., 1985), In human
populations accidentally or occupationally exposed to TCDD, there is no consis-
tent evidence for increased frequencies of chromosomal aberrations in workers
exposed to TCDD (Shu et al., 1987).
Although DNA is negative in genetic toxicity tests, recent reports have
demonstrated that TCDD (50-100 ^g/kg) induces single strand breaks in Sprague-
Dawley rats, presumably as a consequence of increased lipid peroxldation (Wahba
et al., 1988, 1989). In another set of studies, increased frequency of sister
chromatid exchanges were observed in lymphocytes of people exposed to PCDFs in
Taiwan when those lymphocytes are challenged with a-naphthoflavone (Lundgren et
al., 1986, 1988). The mechanism responsible for this effect is that the PCDFs
cause increased rates of metabolic activation of a-naphthoflavone to DNA reactive
metabolites. These findings are consistent with the idea that TCDD's ability to
induce drug-metabolizing enzymes (CYP1A1 and 1A2) may lead to increased rate of
formation of DNA reactive metabolites of some carcinogens, most notably the
polycyclic aromatic hydrocarbons and aromatic amines. However, there is evidence
that the opposite effect occurs in some cases since in vivo exposure to CYPlAl
inducers actually leads to a decrease in DNA adducts in target tissue following
6-12 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
in vivo exposure to PAHs such as benzo(a)pyrene (Cohen et al., 1979; Parkinson
and Hurwitz, 1991). It can reasonably be concluded that TCDD exposure may
increase the rate of DMA adduct formation for some carcinogens but decrease the
rate for others and that predictions should not be made without experimental data
on DNA adduct concentrations in control and TCDD-treated animals.
A final criterion for designating TCDD a non-genotoxic carcinogen is that
it is a potent tumor promoter and weak initiator in two-stage models for liver
(Pitot et al., 1980; Graham et al., 1988; Lucier et al., 1991; Clark et al.,
1991a; Flodstrom and Ahlborg, 1991) and skin (Poland et al,, 1982). These
findings will be discussed in more detail in the section on tumor promotion
including plausible mechanisms for the tumor promoting actions of TCDD such as
TCDD-mediated increases in cell proliferation rates of genetically-altered cells.
It is now accepted by the scientific community that most if not all of
TCDD's toxic and biochemical effects including tumor promotion are Ah receptor
dependent and that TCDD provides an example to evaluate the issues relevant to
risk assessment for receptor-mediated carcinogens. The steps involved in Ah
receptor-mediated events are reviewed in the chapter on Mechanisms (Whitlock).
6.4. INITIATION-PROMOTION STUDIES
The multistage nature of chemical carcinogenesis is being defined by an
increasing understanding of the discrete steps required to produce a genetically-
altered cell which is clonally-expanded and ultimately progresses to a tumor
{IARC, 1992; Barrett and Wiseman, 1987; Swenberg et al., 1987; Barrett, 1992)
(Figure 6-1). Briefly, the process involves damage to a specific site on DNA,
a round of cell replication to fix that damage into the genome, clonal expansion
of the genetically-altered cells (tumor promotion), followed by additional
genetic damage and rounds of cell replication (tumor progression). Figure 6-1
schematizes the multistage nature of cancer. Birth and death rates of
genetically-altered cells compared to normal cells is the centerpiece of risk
assessment models which recognize the multistage nature of chemical carcino-
genesis (Moolgavkar and Knudson, 1981; Portier, 1987). The roles of proto-
oncogene activation and tumor suppression genes have provided clues in attempts
to dissect out discrete steps in cancer. It is also clear that cell prolifera-
tion is an essential component of chemical carcinogeneais, for without it DMA
6-13 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITB
Initiation and Cell Proliferation
in Multistage Carcinogenesis
Ervnl
C*ll Proliferation
(tlonsJ
FXGWR1 6-1
Schematic representation of multiatep carcinogeneaifl including the roles of
genetic damage and cell proliferation. It i« important to note that several OKA
damaging steps and several cell proliferation steps are likely involved during
the complete process of chemical carcinogenesis.
6-14
08/24/92
-------
DRAFT—DO HOT QUOTE OR CITI
damage would not be fixed into the genome and clonal expansion of genetically-
altered cells would not occur.
Concurrent with our increased understanding of the mechanistic underpinnings
of chemical carcinogenesis, multistage models have been developed to identify the
particular stage or stages in which carcinogens act to increase tumor incidence.
There ia a wealth of information on liver initiation/promotion protocols in the
scientific literature {Pitot and Sirica, 1980; Farber, 1984,- Pitot and Campbell,
1987). These protocols frequently employ a single initiating dose of a chemical
which damages DNA, followed by enhancement of cell replication (partial hepatec-
tomy or cytotoxicity) to fix that damage into the genome {initiation) and then
chronic exposure to a chemical which produces clonal expansion of the geneti-
cally-altered cells (promotion). Increased tumor incidence is produced by
chemicals which act at either stage. It is important to note that "initiation"
and "promotion" are operational and not mechanistic terms since both stages are
likely comprised of multiple steps. Nevertheless, the protocols have provided
valuable information in our attempts to understand chemical carcinogenesis.
Detailed descriptions of initiation/promotion protocols in liver and skin are
provided elsewhere {Pitot and Campbell, 1987? Dragan et al,, 1991? Pitot et al.,
1987,- Parber, 1984,• Slaga et al., 1982,- Peraino et al., 1981; Ito et al., 1980).
6.4.1. Two-Stage Models in Rat Liver. Pitot et al. (1980) reported that TCDD
was a potent liver tumor promoter when rats were initiated with a single dose of
DIN followed by chronic TCDD exposure (0.14 and 1.4 j/g/kg aubcutaneously once
every 2 weeks for 7 months). These doses are equivalent to 10 and 100
ngTCDD/kg/ciay (the medium- and high-dose in the Kociba bioassay). Hiatological
evaluation revealed that five of seven animals which had received DSN and the
high TCDD dose had hepatocellular carcinomas. No liver tumors were evident in
rats receiving DEN only, DBN/low-dose TCDD or TCDD only (high or low-dose). EOF
in liver were also evaluated in this study and these are considered to represent
preneoplastic lesions since increases in EOF are associated with liver cancer in
rodents (Haronpot et al., 1989; Popp and Goldsworthy, 1989? Pitot et al., 1989?
Williams, 1989). The EOF data was consistent with the tumor data in that a large
proportion of the liver was occupied by the preneoplastic lesions (43%) in
animals receiving DEN and the high dose of TCDD. A much smaller portion of the
6-15 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITS
liver was occupied by EOF in the other groups. This work provides strong
evidence that: TGDD is a potent tumor promoter in liver with no detectable
initiating activity within the specified experimental framework.
A second set of studies (Graham et al., 1988; Lucier et al., 1991; Clark et
al., 1991a; Dragan et al., 1992) have confirmed and extended Pitot's findings
including data on the mechanistic basis for TCDD's tumor promoting effects in rat
liver. These studies also used DEN as the initiator and have demonstrated that
TCDD's liver tumor promoting actions are ovarian dependent. This finding is
consistent with two-year bioassays which showed that TCDD is a hepatocarcinogen
in female rats but not male rats. In the tumor promoting studies (Graham et al.,
1988? Lucier et al., 1991) DEN was used as the initiating agent and TCDD
(biweekly doses of 1.4 pgTCDD/kg equivalent to 100 ng/kg/day for 30 weeks) was
used as the promoter. There were four groups of intact female rats (controls,
TCDD only, DEN only and DEN+TCDD), The same four groups were also used following
ovariectomy. Data revealed that TCDD was a much weaker liver tumor promoter in
OVX rats (Table 6-5). For example, there were 387 GGT foci/cm in intact rats
compared to 80 in OVX rats in the DEN+TCDD groups. Corresponding differences
were evident in the proportion of liver occupied by GGT foci; 0.37% in DEN/TCDD
intact, rats compared to 0.08% in DEN + TCDD OVX rats. Few or no foci were found
in the control or TCDD only groups. PGT is being used increasingly as a
phenotypic marker of enzyme altered foci (Ito et al., 1989) and results with
this marker of preneoplasia were similar to those for GGT in that ovariectomy
protected against the liver tumor promoting actions of TCDD. The influence of
ovariectomy on liver tumor incidence was evaluated in m parallel experiment using
the same treatment groups in which TCDD was administered for 60 weeks. In the
intact DEN +• TCDD rats, liver tumor incidence was 13/37 with a total of 32 tumors
compared to 7/39 (11 total tumors) in DIN + TCDD OVX rats. Both hepatocellular
adenomas and carcinomas were evident along with a smaller incidence of hepato-
cholangiomas and hepatocholangiocarcinomas.
The mechanisms responsible for the protective effect of ovariectomy is not
clear but ovarian influences on liver TCDD retention does not seem to be
involved; liver TCDD concentrations were -20 ppb in both intact and OVX rate
6-16 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
TABLE 6-5
Preneoplastic Foci and Cell Proliferation After 30 Weeks of TCDD
Tumor Promotion8
GGT + foci/cm^
intact
OVX
GGT + foci {vol fraction}0
intact
OVX
BrdU-labeling index0
intact
OVX
s/c
6
0
0.01
0
0.3b
1.1
S/TCDD
5
0
0.01
0
6.0b
1.0
DEN/C
44
30
0.03
0.03
0.8
1.1
DEN/TCDD
38?b
80
0.37b
0.08
7.3b
0.7
aSourcei Clark et al., 1991
Significantly different from OVX
Percentage of hepatocytes undergoing replicative DNA synthesis in
1 week following 30 weeks of TCDD exposure
S/C = Controls? S/TCDD = TCDD only; DEN/C = DEN only no TCDD;
DEN/TCDD = DEN initiated and TCDD promoted
6-17
08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
(Lucier et al., 1991) which is similar to liver concentrations reported by Kociba
et al. (1978) using the same dose of TCDD (100 ng/kg/day) but for 2 years rather
than 60 weeks. One plausible mechanism may be related to cell proliferation
since TCDD did not stimulate cell proliferation rates in OVX rats whereas a mean
increase of 20-fold was apparent in intact rats receiving 100 ng TCDD/kg for 30
weeks (Table 6-5) (Lucier et al., 1991). There was considerable interindividual
variation in both cell proliferation rates and enzyme altered foci in the
DEN/TCDD groups. Comparisons of the two data seta revealed a strong positive
correlation between enzyme altered foci and cell proliferation, although the
importance of this finding is diminished by the fact that cell proliferation was
quantified in non-lesioned hepatocytes. The mechanism whereby ovarian hormones
and TCDD interact to produce cell proliferation in hepatocytes may involve growth
factor pathways. Consistent with this idea, TCDD produced a loss of plasma
membrane EGF receptor in intact rats but not OVX rats (Clark et al., 1991a). EGP
is thought to provide a mitogenic stimulus in hepatocytes and play a key role in
hepatocarcinogenesis (Vickers and Lucier, 1991; Velu, 1990? Shi and Yager, 1989;
Eckl et al., 1988). A schematic representation of a plausible mechanism for the
role of estrogen in TCDD-mediated liver cancer in rats is given in Figure 6-2.
Another possible mechanism for the influence of the ovaries is that TCDD
induces cytochrome F-4SO 1A2 which could lead to DNA reactive metabolites of 170-
estradiol, the naturally-occurring estrogen. P-4501A2 catalyzes the formation
of catechol estrogens which are considered by some to be DNA reactive precursors
(Metzler, 1984; Li and Li, 1990).
The CDFs and other CDDS are also liver tumor promoters. In a recent study
(Flodstrom and Ahlborg, 1991), enzyme-altered foci were increased in female
Sprague-Dawley rat livers by an initiating dose of DIN followed by TCDD,
1,2,3,7,8-pentachlorodibenzo-p-dioxin or 2,3,4,7,8-pentachlorodibenzofuran were
used as the promoting agent. Comparative potencies indicated that the two CDDs
were nearly eguipotent and the PCDF about I/10th as potent as TCDD. These
results are consistent with the idea that the hepatocarcinogenic actions of TCDD
and its structural analogs are Ah receptor dependent.
6.4.2. Rat. Lung, since the lung and respiratory tract may be target sites for
TCDD carcinogenesis in humans (Fingerhut et al., 1991), it is of interest to
6-18 OB/24/92
-------
POSSIBLE SEQUENCE OF EVENTS
INVOLVED IN ESTROGEN DEPENDENT
TCDD PROMOTION OF LIVER TUMORS
DEN
I
VO
INITIATED CELLS
TCDD + £2
CLONAL EXPANSION OF
INITIATED CELLS
Metabolic activation
of Ez produces
additional DMA damage
Ea and TCDD continue to
stimulate proliferation
of altered cells
TUMOR
PROGRESSION
8
I
•8
3
o
M
H
W
O
CO
FIGURE 6-2
Operational model of TCDD/estrogen interactions relative to tumor promotion in a two-stage model of
hepatocarcinogenesis. Clonal expansion of initiated cells may reflect stimulation of mitogenesis through
receptor-mediated events involving EGFR, ER and the Ah receptor.
VO
KJ
-------
DRAFT—DO NOT QUOTE OR CITE
evaluate whether TCDD is a tumor promoter in rodent lung. The only published
report on lung tumors used DEN as the initiating agent and TCDD (100 ng/kg/day
for 60 weeks) as the promoting agent (Clark et al.» 1991a). Both intact and OVX
rats were used and the results were surprising. In contrast to liver tumor
promotion, lung tumors were seen only in DEN/TCDD OVX rate (4/37). No lung
tumors were present in DEN/TCDD intact rats or in DEN only TCDD only, or control
rats with or without ovariectomy). The background incidence of lung tumors in
rats is very low so the lack of tumors in controls was not unexpected. The four
tumors in DEN/TCDD intact rats were comprised of two sguamous cell carcinomas and
two adenocarcinomas. These tumors were analyzed for the presence of activated
oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays
(Reynolds et al., 1992). A transforming gene of rat origin was detected in all
tumors but Southern blot analysis indicated that it is an unknown oncogene. This
unknown oncogene was not detected in the DEN/TCDD rat liver tumors.
The rodent tumorigenicity data provide clues to the complex hormonal
interactions that produce site specific carcinogenic actions of TCDD. Liver
tumors are ovarian dependent whereas the ovaries appear to protect against TCDD-
mediated tumor promotion in lung. Therefore, the rat tumor data is consistent
with the NIOSH study which revealed TCDD-related increases in respiratory tract
tumors but no statistically significant increases in liver tumors in a population
comprised mostly of men.
6.4.3. Mouse Skin. Initiation/promotion studies on skin have demonstrated that
TCDD is a potent tumor promoter in mouse skin as well as rat liver. Poland et
al. (1982) administered a single dermal initiating dose of MNNG to hairless mice
followed by twice weekly doses of TCDD (3.75, 7.5, 15 or 30 ng) or TPA (1 or 3
fig) for 20 weeks, TCDD promoted the development of papillomas at all doses and
the response was dose dependent (100% of the animals had tumors in the high-dose
TCDD group). Control animals or animals receiving MNNG or TCDD only exhibited
only a low incidence of tumors. These studies demonstrate that TCDD is at least
two orders of magnitude more potent an agent than TPA in mouse skin (Poland et
al., 1982). It appears that the skin tumor promoting actions of TCDD are Ah
receptor dependent. Moreover, tumorigenic responses segregate with the hr locus
6-20 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
and biochemical responses such as CYP1A1 induction can occur without earcino-
genesis (Poland and Knutaon, 1982; Poland et al., 1982).
Other studies have tested TCDD as an initiator and TPA as a promoter in CD-I
mice (DiGiovanni et al., 1977). Results revealed that TCDD had weak or no
initiating activity in this system. In order to better understand the possible
influence of TCDD-mediated induction of cytochrome P-450 on the carcinogenicity
of PAHs, TCDD was co-administered with benzo(a)pyrene or dimethylbenzathracene
to mice followed by promotion with TPA (Cohen et al.» 1979). Results revealed
that TCDD decreased tumor incidence of both PAHs compared to controls. However,
co-administration of TCDD with 3-methylcholanthrene to mice produced tumor
incidences similar to those produced by 3-methylcholanthrene alone (Kouri et al.,
1978). These results are consistent with the findings that TCDD induction of
drug metabolizing enzymes is associated with both metabolic activation as well
as deactivation of PAHs (Lucier et al., 1979).
The relative toxicity and tumor promoting capacity of two CDFs (2,3,4,7,8-
CDP and 1,2,3,4,7,8-CDP) has been investigated in hairless mice (Hebert et al.,
1990). These studies used a treatment protocol similar to that of Poland et al.
(1982) including the use of MNNG as the initiating agent and varying doses of
TCDD, 2,3,4,7,8-CDF or 1,2,3,4,7,8-CDP for 20 weeks. Proliferative lesions
(sguamous cell papilioma, squamous cell carcinoma or hyperproliferative nodules)
were quantified. Results demonstrated that 2,3,4,7,8-CDP was 0.2-0.4 times as
potent as TCDD and the 1,2,3,4,7,8-CDF was 0.08-0.16 times as potent as TCDD.
These data suggest that the tumor promoting potencies of structural analogs of
TCDD, like promotion of liver tumors, reflect relative binding properties to the
Ah receptor. However, this is an effect of chronic exposure so rates of
metabolism/clearance would obviously impact on correlations between Ah receptor
binding and tumo promotion.
Taken together, results on initiation/promotion protocols indicate that TCDD
is an extraordinarily potent promoter of liver and skin tumors (Pitot et al.,
1987) and they provide strong evidence that the carcinogenic actions are Ah
receptor-mediated. A summary of studies on tumor promotion by TCDD or the
polychlorinated biphenyls is given in Table 6-6. Plausible mechanisms of actions
6-21 08/24/92
-------
O>
K)
(O
TABLE 6-6
Summary of Positive Tumor Promoting STUDIES ON TCDD and CDFs
Species /Sex
Rat /female
Rat /female
Rat/female
Rat /female
Rat /female
Rat /female
Rat/female
Rat /female
Rat/ female
Mice/female hairless
Mice/ female hairless
Rat/ female (ovariectomized)
Rat /female (ovariectomized)
Initiator
DEN
DEN
DEN
DEN
DEN
DEN
DEN
DEN
DEN
MNNG
MNNG
DEN
DEN
Promoter
TCDD
TCDD
TCDD
TCDD
TCDD
TCDD
PCDFs
TCDD
TCDD
TCDD
PCDFs
TCDD
TCDD
Site
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Skin
Skin
Lung
Lung
Reference
Pitot et al, 1980
Graham et al, 1988
Lucier et al, 1991
Clark et al, 1991
Flodstrom et al, 1991
Flodstrom et al, 199 Ib
Flodstrom et al, 1991b
Dragan et al, 1992
Lucier et al, 1992
Poland et al, 1982
Hebert et al, 1990
Clark et al, 1991
Reynolds and Lucier, 1992
o
o
I
o
G
8
o
n
o
00
10
*»
to
(O
-------
DRAFT—DO NOT QUOTE OR CITE
responsible for the tumor promoting actions of TCDD and the impact of these
mechanisms on dose response relationships will be presented in the next section.
6.5. BIOCHEMICAL RESPONSES
There is an expanding list of effects that are produced by TCDD in experi-
mental animals and in cell systems. These effects include those which may alter
normal cell regulatory processes such as cell proliferation and differentiation,
metabolic capacity, and hormonal pathways. This section on biochemical responses
will summarize some of the changes produced by TCDD including discussion of (a)
possible relevance of the response to TCDD-tnediated cancer, (b) whether the
response is Ah-receptor mediated, (c) whether information is available on the
role of transcriptional activation, (d) dose-response relationships, and (e)
whether animal models are consistent with human responses. This chapter will not
attempt to evaluate all of the biochemical and molecular responses to TCDD but
will focus on the ones that are either the most relevant to carcinogenic
responses and/or have received the most study. The responses selected for
evaluation are cytochrome P-4501A1 (CYP1A1), cytochrome P-4501A2 (CYP1A2), EGFR,
ER, and UDPGT. Table 6-7 lists many of the biochemical changes produced by 1CDD
in in vivo and/or in vitro and some information on mechanisms of action.
6.5.1. CYP1A1 and 1A2. The most studied response to TCDD has been induction
of cytochrome P-450 isozymes (Whitlock, 1990; Silbergeld and Gasiewicz, 1989;
Poland and Knutson, 1982). The first reports of P-450 induction in vivo and in
vitro appeared in.1973 (Lucier et al., 1973; Greig and DeMatteis, 1973? Poland
and Glover, 1973) and hundreds of papers have been published on the subject since
that time. These papers have dealt with various aspects of TCDD-mediated
induction of P-450 such as isozyme specificity, time-course, structure-activity
relationships, molecular mechanisms of transcriptional activation of the CYPlAl
gene, identification of transcriptional activating factors, tissue and cell
specificity and dose-response relationships. The molecular mechanisms respon-
sible for enzyme induction are described in the chapter by Whitlock in this
volume.
The mechanistic relationship of CYP1A1 and 1A2 induction to cancer or any
other toxic endpoint following dioxin exposure has not been demonstrated, yet
considerable controversy exists on this subject (Roberts, 1991). Since CYP1A1
6-23 08/24/92
-------
TABLE 6-7
Classification of Members of the Ah Gene Battery*
Class
Gene/Product
Secreted
Protein
Activation of gene transcription; Ah receptor-mediated
CyplAI, Cytochroute p.,450
Gst-Ya, glutathione S-transferase
Nmo-1, menadione oxidoreductase
Activation of gene transcription, AhR agonist-mediated
Clone 1, unknown gene
Cyp1A2, cytochrome P^teQ
PAI-2, plasminogen activator inhibitor-2
T-ALDH, aldehyde deydrogenase
Induction of mRNA levels; AhR agonist-mediated
to
*>.
Clone 141, unknown gene
c-erb A related, hormone receptor
GST-Yb, glutathione S-transferase
GST-Yc
ahCG, human chorionic gonadotropin
IL-p, interteukin-1p
MDR-1, multidrug-resistance
Testosterone 7 a-hydroxylase
TGF-o, Transforming growth factor-a
a
o
I
o
|
Cd
O
O
Induction of enzyme activity; Ah receptor-mediated
ODC, ornithine decarboxylase
Ugt-1, UDP-glucuronyl transferase
EGFR, epidermal growth factor receptor
ER, estrogen receptor
Gastrin
TNF-flt. tumor necrosis factor-a
Induction of enzyme activity; AhR agonist-mediated
ALAS, (S-aminolevulinic acid synthetase
Aryl hydrocarbon binding protein
Choline kinase
60-kd microsomal esterase
Malic enzyme
Phospholipase A2
Protein kinase C
Enzyme pp60c~ .
tyrosine kinase
*Source: Sutter and Greenlee, 1991
o
oo
\D
-------
DRAFT—DO NOT QUOTE OR CITE
functions to catalyze the metabolic activation of many chemicals such as the
polycyclic aromatic hydrocarbons to DNA reactive metabolites, it has been
postulated that induction of CYP1A1 might enhance the carcinogenic actions from
a given exposure level to many PAHs. However, usually, preinduction of CYP1A1
diminishes the carcinogenic potency of PAHs such as 3-methylcholanthrene,
benzo(a)pyrene and dimethylbenzoanthracene if exposure to the inducing agent is
short term (Parkinson and Hurwitz, 1991; Wattenberg, 1985; Cohen et al., 1978;
Wattenberg, 1978; Miller et al., 1958). Induction also protects against the
carcinogenic actions of aflatoxin, diethylnitrosamine, arylamines and urethane.
Protection occurs at numerous cancer sites including liver and lung. Several
lines of evidence support the idea that enzyme induction is the mechanism
responsible for the protective effect. First, treatment of mice, deficient in
the Ah receptor, with inducers does not protect against PAH-mediated cancer
(Kouri et al., 1978). Second, the ability of inducing agents to protect against
cancer is positively correlated with their potency as inducing agents (Wattenberg
and Leong, 1970; Arcos et al., 1961). Third, the inducing agent must be
administered at least one day prior to treatment which allows sufficient time for
the inducer to produce elevated levels of CYP1A1 (Parkinson et al., 1983;
Wheatley, 1968).
The most probable mechanism responsible for the protective effect of enzyme
induction is that it leads to decreased concentrations of promutagenic DMA
adducts in target tissues. These findings appear to contradict the knowledge
that CYP1A1 is required for the metabolism of PAHs, aflatoxin and several other
carcinogens to DNA reactive arene oxides (Guengerich, 1988; Levin et al., 1982;
Conney et al., 1982). For example, the promutagenic DNA adduct of benzo(a)pyrene
appears to be 7,8-diol-9,10 epoxide metabolite adducted to deoxyguanosine, and
formation of this metabolite requires two separate actions of CYP1A1, The
contradiction can be resolved by analysis of the entire metabolic pathways for
chemical carcinogens whose potencies are decreased by pretreatment with inducing
agents. In addition to CYPlAl-mediated increases in metabolic activation, this
cytochrome also converts PAHs to inactive metabolites (Thakker et al., 1985;
Pelkonnen and Nebert, 1982). Moreover, induction of uridine diphosphoglucuronly-
transferase also occurs coordinately with CYP1A1 induction (Lucier et al., 1986).
6-25 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
This enzyme also detoxifies PftHs and many other carcinogens and facilitates their
excretion from the body (Thakker et al.» 1985; Nemoto and Oelboin, 1976).
Therefore, it appears that TCDD-mediated enzyme induction increases the rate of
detoxification of some carcinogens to a greater extent than it increases the rate
of formation of DNA damaging metabolites.
Although there is not clear mechanistic link between CYP1A1 induction and
cancer, it is important to note that many CY.P1A1 inducers are themselves
carcinogens when encountered in chronic dosing regimens BO the protective effect
of inducing agents is limited to short-term exposure. For example, benzo(a)-
pyrene, 3-methylcholanthrene and TCDD are CYP1A1 inducers and multisite carcino-
gens (Vanden Heuvel and Lucier, 1992? Levin et al., 1982,- Slaga et al., 1979;
Sims and Glover, 1974).
The relationship of CYP1A2 induction to the carcinogenic actions of other
compounds is less clear than it is for CYP1A1. For example, CYP1A2 catalyzes the
formation of catechol estrogens from 17j5-estradiol (Graham et al./ 1988). The
catechol estrogens are considered as possible toxic metabolites in that they
could lead to increased free radical damage to cellular roacromolecules such as
DMA (Lt and Li, 1990; Metzler, 1984). This mechanism could be in part, respon-
sible for the findings that TCDD is a hepatocarcinogen in female rats but not
male rats and that ovariectomy protects against the hepatocarcinogenic actions
of TCDD. Also consistent with the hepatocarcinogenicity data is the observation
that CY.P1A2 is induced in liver but not in extrahepatic organs with the possible
exception of the nasal mucosa (Goldstein and Linko, 1984). In contrast, CYP1A1
induction occurs in virtually every tissue of the body which is consistent with
the observation that the Ah receptor is found in a wide variety of cell types.
' There are a number of studies described in the scientific literature on dose
response relationships for TCDD'a effects on CYP1A1 and 1A2 (DeVito et al., 1991;
Lin et al., 1991aj Kedderis et al., 1991; Harris et al., 1990? Goldstein and
Safe, 1989; Abraham et al., 1988; Lucier et al., 1986; Vecchi et al., 1983;
Poland and Glover, 1980; Kitchin and Woods, 1979; Lucier, et al., 1973; Poland
and Glover, 1973). These studies include single and chronic dosing schedules
(Tritscher et al., 1992? Graham et al., 1988; Sloop and Lucier, 1987), time-
course evaluations and species comparisons. Dose response relationships have
6-26 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
been evaluated by quantitation of CYP1A1 and lA2-dependent enzyme activities,
mRNA levela by Northern blot analysis, quantitation of CYP1A1 and 1A2 protein by
radioinununoassay and also by intmunolocalization in tissue sections. All of the
above methods have yielded consistent results. The single dose ED^g for CYP1A1
or 1A2 induction is approximately 0.5-1.5 fig TCDD/kg in both rats and mice. In
a chronic exposure situation, the ED5Q is in the range of 5-10 ng/kg/day
(Tritscher et al., 1992). The limit of detection for enzyme induction varies
depending on the method used for quantitation; i.e. P-450 dependent enzyme
activities, mRNA, or protein. Recently, it was shown (VandenHeuvel et al., 1992}
that TCDD-mediated increases in CYP1 in mRNA were detectable following a single
dose of 0.1 ng/kg which produces a TCDD liver concentration equivalent to a
chronic dose of 2-5 pg/kg/day.
Evaluations of various data sets for TCDD-mediated dose response relation-
ships have revealed some interesting information. One way of analyzing data for
linearity or non-linearity of dose response for receptor-mediated events is the
Hill equation (Hayashi and Sakamoto, 1986). A Hill coefficient of 1 suggests a
linear relationship between exposure and dose throughout the experimental dose
range and would predict a proportional relationship between target tissue
concentration of TCDD and biological response at all dose levels. This would
imply that the response had no practical threshold or "no effect level." Hill
coefficients greater than 1 would indicate sublinearity in dose response, whereas
a Hill coefficient of less than 1 would indicate supralinearity for response in
the low-dose region. Analysis of both single exposure as well as chronic
exposure data for CYP1A1 and CYP1A2 induction in rat or mouse liver indicate a
Hill coefficient of slightly greater than 1 for CIP1A1 and slightly less than 1
for CYP1A2 (Portier et al., 1992,- Kohn et al., 1992). Although these analyses
involve an extrapolation beyond the range of experimental data, they are
consistent with the hypothesis that there is not a practical threshold for TCDD-
mediated induction of CYP1A1 and 1A2.
Immunological detection of induced CYP1A1 and 1A2 in liver sections obtained
from rats exposed chronically to TCDD suggest hepatocyte heterogeneity in
response to TCDD (Tritscher et al., 1992? Bars and Elcornbe, 1991), For example,
6-27 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
relatively low doses of TCDD (1 ng/kg/day) appear to induce maximally some cells
around the centrilobular region. Increasing doses of TCDD increase the number
of cells responding rather than the amount of induction in responding cells.
These data, which document cell differences in sensitivity to induction,
complicate evaluation of dose response relationships. For example, some hepato-
cytes appear to be maximally induced by low doses to TCDD whereas other hepato-
cytes exhibit no detectable P-45Q induction response by these same doses. As
discussed earlier a mechanistic link between P-450 induction and cancer has not
been established. Evaluation of P-450 induction and TCDD-mediated cell prolifer-
ation by immunocytochemical methods in rat liver reveal that cells which express
CXP1A1 and 1A2 are different from those exhibiting TCDD-mediated increases in DNA
replication (Lucier et al., 1992).
Placentas from Taiwanese women exposed to rice oil contaminated with
polychlorinated dibenzofurans (PCDFs) have markedly elevated levels of CYPlAl
{Lucier et al., 1987 j Wong et al., 1986). Comparison of these data with
induction data in rat liver suggest that humans are at least as sensitive as rats
to the enzyme inductive actions of TCDD and its structural analogs (Lucier,
1991). Consistent with this contention, the in vitro EC^Q for TCDD-mediated
induction of CYPlAl-dependent enzyme activities is approximately 1.5 nM when
using either rodent or human lymphocytes (Clark et al., 1992). However, binding
of TCDD to the Ah receptor occurs with a higher affinity in rat cellular
preparations compared to humans (Lorenzen and Okey, 1991; Okey, 1989). This
difference may be related to the greater lability of the human receptor during
tissue preparation and cell fractionation procedures (Manchester et al., 1987).
In any event, it does appear that humans contain a fully functional Ah receptor
(Cook and Greenlee, 1989) as evidenced by significant CYPlAl induction in tissues
from exposed humans and this response occurs with similar sensitivity as observed
in experimental animals.
6,5.2, EGFR. EGF is a potent mitogen and it stimulates the generation of
mitotic signals in both normal and neoplastic cells (Stoscheck and King, 1986;
Carpenter and Cohen, 1979). Several lines of evidence suggest that the EGF
receptor and its ligands, including transforming growth factor-a possess diverse
6-28 08/24/92
-------
DRAFT — DO NOT QUOTE OR CITE
functions relevant to cell transformation and tumorigenesis (Velu, 1990? Marti
et al., 1989; Mukku and Stancel, 1985). In fact, the mechanism of action for
several tumor promoters such as phenobarbital and the phorbol esters is thought
to involve the EOF receptor pathway (Stoscheck and King, 1986). A schematic
representation of the proposed mechanism for EGF- stimulated mitogeneaia is given
in Figure 6-3.
Several studies have shown that TCDD decreases the binding capacity of the
plasma membrane EOF receptor for its ligand without a change in K^ (Clark et al.,
1991a; Lin et al., 1991a; Abbot and Birnbaum, 1990; Astroff et al., 1990;
Sunahara et al., 1989; Hudson et al., 1985; Madhukar et al., 1984). One study
utilized a range of TCDD doses (3.5-125 ng/kg/day) for 30 weeks to evaluate the
effects of chronic TCDD exposure on EGF receptor in rat liver plasma membranes
(Sewall, 1992). There was a clear dose-response relationship for TCDD ' s effects
on the total binding capacity of the EGF receptor although TCDD did not produce
a change in binding affinity of the receptor. The maximal effect was a three-
fold decrease in the concentration of plasma membrane EGF receptor and the EDjQ
was -10/ng/kg/day based on administered dose and ~2 ppb TCDD based on liver TCDD
concentration. These values are similar to the ED for induction of CYP1A1 and
CYP1A2 for 30-week exposures. The dose-response data, like the data for CYP1A1
and CYP1A2 induction, was subjected to curve fitting analyses using the Hill
Equation (Portier et al., 1992). This analysis indicated that a Hill coefficient
of one provided the best fit suggesting that there is a linear relationship
between target tissue dose and the magnitude of response for effects on the IGF
receptor. Although, Hill analysis of dose response data for TCDD's effects on
the EGF receptor, CYP1A1 induction, and CYP1A2 induction are inconsistent with
the idea of a threshold, the lowest dose used in these experiments was 100
pg/kg/day so it is possible that dose-response relationships are different in the
very low-dose region (1-10 pg/kg/day) encountered as background human exposures.
Dose-response data on EGFR were compared to dose-response relationships for
TCDD-mediated increases in cell proliferation and growth of preneoplastic lesions
within the framework of a two stage model for hepatocarcinogenesis in rats
(Lucier et al., 1992). Results indicate that cell proliferation and the growth
6-29 08/24/92
-------
MITOGEN
(EGF)
COMMITTED STAT
•CH
RECEPTOR
BINDING '
DIMERIZATION
AUTOPHOSPHORYLATICN•
INTERNALIZATION
GENERATION OF
• SECOND
tESSENGERS
CELL DEATH
bJ
O
DMA REPLICATION
O
00
FIGURE 6-3
Plausible Mechanism for the Role of EGF-Mediated Stimulation of Mitotic Activity
D
O
I
1
M
O
M
M
VO
M
-------
DRAFT—DO NOT QUOTE OR CITE
of preneoplastic lesions are a less sensitive response to TCDD than is loss of
plasma membrane EGF receptor. Therefore, the E6F receptor may be involved in the
hepatocarcinogenic actions of TCDD but dose-response relationships for this
effect may be different from dose-response relationships for liver cancer in
rats. These data reflect the knowledge that several steps and/or several genes
are involved in the modulation of coordinated biological responses.
The mechanism by which TCDD alters EGF receptor binding capacity is not
fully understood although fCDD does not appear to decrease EGF receptor mRNA (Lin
et al., 1991aj Osborne et al., 1988). Using congenic mice, deficient in the high
affinity Ah receptor, TCDD's effects on the SGF receptor -were shown to require
the Ah receptor (Lin et al., 1991a). In control animals, the EGF receptor is
distributed on the surface of the plasma membrane and is comprised of an external
ligand binding domain, a transroembrane domain, and an intercellular domain (Velu,
1990; Carpenter, 1987). Ligands for the EGF receptor (EGF or TGF-a) in the
intracellular space bind the IGF receptor producing a conformational change which
stimulates the intercellular region to catalyze phosphorylation of the receptor
itself as well as other proteins involved in cell regulation. The process
results in internalisation of the receptor characterized by an increase in
cytosolic EGFR coupled with a decrease in membrane bound receptor. Effects of
TCDD and CDFs on the number of binding sites for the plasma membrane EGF receptor
are correlated with a concomitant decrease in EGF stimulated autophosphorylation
of the EGF receptor indicating that TCDD produces a true functional change in the
IGF receptor (Clark et al., 1991a; Sunahara et al,, 1989; Nelson et al., 1988;
Sunahara et al., 1988). It is important to note that addition of EGF to
hepatocytes or several cell lines in culture produces a loss of plasma membrane
EGF receptor coupled with a loss of EGF stimulated autophosphorylation (Velu,
1990; Carpenter, 1987). Therefore, TCDD produces an EGF receptor like response
consistent with the idea that TCDD enhances the generation of cellular mitotic
signals.
Although TCDD exposure mimics EGF actions in hepatocytes, TCDD itself does
not bind the EGF receptor. The most plausible mechanism for effects on the EGF
receptor involves the finding that TCDD induces production of TGF-a in hepato-
cytes as well as human keratinocytes (Choi et al., 1991). This response could
6-31 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
alter control of normal growth patterns since TGF-a binds the EGF receptor with
high affinity leading to enhanced production of mitogenic signals. Alterna-
tively, TCDD may affect EGF receptor transcription. In fact, TCDD has been shown
to decrease uterine EGF receptor mRNA levels (Astroff et al., 1990). Receptor
concentrations may also be altered by other events such as postranslational
glycosolation, increased lysosomal degradation or alterations in signal transduc-
tion pathways such as protein kinases (Madhukar et al., 1988). It is also
possible that TCDD alters phosphorylation of the EGP receptor by activation of
protein kinase c resulting in decreased binding capacity of the plasma membrane
EGF receptor. This effect occurs following exposure to the tumor promoter TPA
and is associated with decreased autophosphorylation rates and EGF receptor
internalization (Beguinot et al., 1985; Cochet et al., 1984), In any event,
TCDD-rnediated alterations in EGF receptor pathways may, in part, be responsible
for the tumor promoting actions of TCDD by enhancement of mitotic signals.
The effects on the EGF receptor system may be mediated by estrogen action
and it has been postulated that the estrogen and EGF receptor pathways are
integrated by "cross talk" mechanisms (Ignar Trowbridge et al., 1992; Astroff et
al., 1990). In vivo and in vitro studies have demonstrated that TCDD alters the
estrogen receptor {DeVito et al., 1992,- Lin et al., 1991a; Clark et al., 1991a;
Umbreit and Gallo, 1988? Romkes et al., 1987) and estrogens can, in turn, alter
EGF receptor binding and cellular distribution {Vickers and Lucier, 1991; Vickers
et al., 1989? Mukke and Stancel, 1985). Moreover, studies conducted within the
framework of a two-stage model for hepatocarcinogenesis have demonstrated that
TCDD-mediated decreases in plasma membrane EGF receptor are ovarian dependent
(Clark et al., 1991a; Sewall et al,, 1992). These studies concluded that ovarian
hormones are essential to the tumor promoting actions of TCDD in that TCDD does
not induce hepatocyte proliferation or stimulate the growth of preneoplastic
lesions in ovariectomized rats (see section on Initiation-Promotion studies).
There is evidence to indicate that TCDD and its structural analogs produce
the same effects on the EGF receptor in human cells and tissues as observed in
experimental animals. First, incubation of human keratinocytes with TCDD
decreases plasma membrane IGF receptor and this effect is associated with
increased synthesis of TGF-a (Choi et al., 1991; Hudson et al., 1985). Second,
6-32 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
placentas from humans exposed to rice oil contaminated with polychlorinated
dibenzof urans, exhibit markedly reduced IGF stimulated autophosphorylation of the
IGF receptor and this effect occurred with similar sensitivity as observed in
rats (Lucier, 1991 j Sunahara et al.» 1987). The magnitude of the effect on
autophosphorylation was positively correlated with decreased birth weight of the
offspring.
6.5.3. UDPGT. Several studies have shown that TCDD induces synthesis of at
least one isozyme of UDPGT (Lucier et al., 1973, 1974, 1986) by a mechanism which
requires the Ah receptor (Bock, 1991). The gene UGT-1 regulates synthesis of the
UDPGf isozyme which conjugates numerous substrates including 1-naphthol, p-
nitrophenol and thyroxine (Burchell et al,, 1991). This gene contains a TCDD
responsive element which permits transcriptional activation following binding of
the TCDD-Ah receptor complex. Other chemicals which bind the Ah receptor, such
as 3-methylcholanthrene and benzo(a)pyrene also induce UGT-1 (Bock, 1991).
UDPGTs are considered as a deactivation pathway for numerous environmental
chemicals and endogenous compounds such as steroid hormones by rendering them
water soluble and excretable as a consequence of the catalytic addition of a
glucuronide moiety (Tephly and Burchell, 1990). Therefore, induction of UDPGT
may, in part, be responsible for the finding that pretreatment with TCDD leads
to diminished DMA adducts for PAHs and decreased concentrations of some steroid
hormones.
Conjugation of thyroxine by UGT-1 leads to deactivation and elimination of
this thyroid hormone (Henry and Gasiewicz, 1987? Bastomsky, 1977). The decreased
levels of thyroxine, associated with UDPGT induction produces decreased feedback
inhibition of the pituitary gland which responds by secreting increased amounts
of TSH (Sanders et al., 1988; Barter and Klaassen, 1992). Several studies have
provided evidence that prolonged stimulation by TSH produces an oncogenic effect
on the thyroid (Hill et al., 1989). Interestingly, rat liver IGF receptor may,
in part, be regulated by thyroid hormones (Mukku, 1984). Increased incidence of
thyroid tumors is the most sensitive endpoint in cancer bioassays as evidenced
by a statistically significant increase at a dose of 1.4 ng/kg/day. Consistent
with this hypothesis, short-term rodent studies have shown that TCDD and other
inducers of hepatic UDPGT decreases thyroxine concentrations in blood which is
6-33 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
associated with increased levels of TSH (Barter and Klaassen, 1992} Henry and
Gasiewicz, 1987).
Dose response studies for TCDD's inductive effects on hepatic UDPGT in rats
have demonstrated that the single dose BDj0 is approximately 0.7 ^g/kg which is
similar to the 1059 for CYP1A1 induction (Lucier et al., 1986), Furthermore, the
shape of the dose response curve for both responses is similar. There is no data
on UDPGT induction in long-term studies. Since humans contain the dioxin
responsive UDPGT (UGT-1) (Burchell et al., 1991) and TCDD induces UDPGT in human
hepatocyte cell cultures it is reasonable to assume that TCDD and its structural
analogs would induce UDPGT in humans although laboratory data is needed to
validate this assumption.
6.5.4. ER. Several lines of evidence have demonstrated that interactions of
TCDD and estrogens are critical to some of the carcinogenic responses to TCDD.
Although the precise mechanisms of those interactions have not been established,
recent data indicate that TCDD effects on the ER and on estrogen metabolism are
involved. The mechanisms for TCDD/estrogen interactions appear to be tissue
specific. Of particular interest is the finding that TCDD increases liver tumor
incidence in rats and at the same time decreases tumor incidence in organs such
as the mammary gland, uterus and pituitary (Kociba et al., 1978). Threfore,
TCDD/estrogen interactions will be examined separately for liver and other
endocrine organs.
The liver contains a fully functional ER that possesses characteristics
similar to those identified for ER in mammary gland and uterus (Mastri and
Lucier, 1983; Powell-Jones et al., 1981| Eisenfeld et al., 1976). For example,
the liver exhibits high affinity binding for 17(l-estradiQl and other potent
estrogens, liver ER binding is specific for estrogens, the ligand receptor
complex interacts reveraibly with DNA, and this interaction leads to transcrip-
tional activation of estrogen responsive genes. Synthesis of hepatic ER, unlike
ER in other target tissues, is under pituitary control (Lucier et al., 1981).
Treatment of rats with a single dose of TCDD decreases binding capacity of the
hepatic ER and this effect is correlated with a decrease in ER protein
(Zacharewski et al., 1991, 1992; Harris et al., 1990b; Romkee and Safe, 1988;
6-34 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Romkes et al., 1987). TCDD also decreases rat hepatic ER in chronic exposure
experiments with a 3-fold decrease evident following a dose of 100 ng/kg/day for
30 weeks (Clark et al., 1991b>. TCDD also decreases hepatic ER binding in CB7B16
mice but a much higher dose is needed to produce this effect in congenic mice
deficient in the high affinity Ah receptor indicating that fCDD-mediated
decreases in ER are dependent on the Ah receptor (Lin et al., 1991). DOBS
response studies in mice demonstrate that the single dose ED*^ is -0.7 fig
TCDD/kg, similar to the ED«JQ for other biochemical endpoints such as CYP1A1
induction, loss of plasma membrane EGP receptor and induction of UDPGT. The
observation that TCDD decreases hepatic ER is in apparent contradiction to the
finding that TCDD increases hepatocyte proliferation since the 1R is thought to
produce mitogenic signals. However, quantitation of ER in control and TCDD-
treated rats was done using preparations from liver homogenates. Immunolocaliza-
tion studies are needed so that the relationship of ER concentrations to cell
proliferation in normal and preneoplastie cells can be more carefully evaluated.
In addition to effects on hepatic ER, TCDD may influence estrogen action in
another way. CYP1A2 efficiently catalyzes the conversion of estrogens to
catechol estrogens in liver (Graham et al., 1988; Dannan et al., 1986). CYP1A2
is not found in extrahepatic tissues, with the possible exception of the nasal
cavity, so catechol estrogen formation would be expected to occur only in liver.
Catechol estrogens have been postulated to possess macromolecule damaging
properties as a consequence of free radical generation (Li and Li, 1990; Metzler,
1984). Therefore, TCDD may increase the DNA damaging capacity of estrogens in
liver as a function of CYP1A2 induction. This effect may, in part, explain the
carcinogenic actions of TCDD in female rat liver and is consistent with the
knowledge that ovariectoray protects against the hepatocarcinogenic actions of
TCDD and that male rats are not susceptible to TCDD-induced liver tumors (Lucier
et al., 1991; Kociba et al., 1978). It is important to note that cancer is more
than a two-stage process and the stage specific actions of TCDD in multistage
cancer models are not known, although TCDD-mediated cell proliferation and
possible indirect genotoxic effects may be critical at more than one stage. A
6-35 08/24/92
-------
DRAFT—OO NOT QUOTE OR CITE
hypothetical mechanistic scheme for TCDD-mediated liver cancer is shown in
Figure 6-2.
The finding that chronic TCDD exposure decreases tumor incidences in
pituitary, mammary gland and uterus may also reflect TCDD'a effects on ER and
estrogen metabolism. As discussed above, TCDD decreases uterine ER concentra-
tions in cytosolic and nuclear fractions of rats and mice and these changes are
associated with diminished estrogen action in in vivo as well as in vitro
studies. TCDD also increases estrogen metabolism presumably as a consequence of
CYP1A2 in liver and UDPGT induction in liver and extrahepatic tissues (Shiverick
and Huther, 1982). Likewise, addition of TCDD to a breast cancer cell line
(MCF-7) results in increased estrogen degradation (Gierthy et al., 1988).
However, there are only small effects on seruin 17-p estradiol levels following
administration of TCDD to either rata or mice (Shiverick and Muther, 1983).
Therefore, the effect on serum estradiol is considerably less sensitive than
effects on the uterine receptor. This comparison has led investigators to
conclude that the antiestrogenic actions of dioxins are primarily caused by
effects on ER levels in reproductive tract tissues. Final evaluation on the role
of estrogen metabolism awaits data on concentrations of estrogens in responsive
cells of control and TCDD-treated rats which may be different from seruin
estradiol levels. In any event, it appears clear that TCDD does possess
antiestrogenic properties which are likely important to decreased tumor
incidences in some reproductive tract and endocrine organs. Numerous studies
have documented that the estrogen receptor is found in virtually every tissue of
the body although effects of TCDD on human estrogen receptor in vivo have not
been studied.
6.5.5. Other Biochemical Endpoint*. TCDD alters a number of other pathways
involved in regulation of cell differentiation and proliferation. The specific
relationships of these effects to multistage carcinogenesis is not known but the
broad array of effects on hormone systems, growth factor pathways, cytokines and
signal transduction components are consistent with the notion that TCDD is a
powerful growth dysregulator. It is also consistent with the findings that TCDD
alters cancer risks at a large number of sites possibly reflecting multiple
mechanaims of carcinogenicity. Biochemical/molecular/endocrine changes produced
6-36 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
by TCDD include the glucocorticoid receptor (Sunahara et al.f 1989), tyroaine
kinaae (Madhukar et al., 1988), gastrin (Mabley et al., 1990), interleukin IP
(Sutter et al., 1991), plasminogen activator inhibitor (Sutter et al., 1991),
tumor necrosis factor-a (Clark et al., 1991b), gonadotropin releasing hormone
"(Moore et al., 1989), testosterone (Moore et al., 1985), and LH (Mabley et al.,
1992). The importance of these responses to the carcinogenic process should not
be diminished by the lack of detail presented here. In every case studied, these
responses have been shown to be dependent on the Ah receptor.
6.6. SUMMARY AND WEIGHT OF EVIDENCE FROM ANIMAL STUDIES
There have be in 17 chronic studies designed to determine if f CDD is a
carcinogen in experimental animals. All of these studies have been positive and
demonstrate that TCDD is a multisite carcinogen, it is a carcinogen in both sexes
and in several species including the Syrian hamster, it is a carcinogen in sites
remote from the site of treatment and it increases cancer incidence at doses well
below the HTD. In two stage models for liver and skin cancer, it is clear that
TCDD is a potent promoting agent with weak or no initiating activity. This
finding is not surprising since TCDD does not form DMA adducts and it is negative
in short-term tests for genetic toxicity. The general consensus is that TCDD is
an example of receptor-mediated carcinogenesis in that (1) interaction with the
Ah receptor appears to be a necessary early step, (2) TCDD modifies a number of
receptor and hormone systems involved in cell growth and differentiation such as
the epidermal growth factor receptor and the estrogen receptor, and (3) hormones
exert a profound influence on the carcinogenic actions of TCDD. For example,
ovarian hormones are essential for the hepatocarcinogenic actions of TCDD in
rats, whereas TCDD promotion of lung tumors in rats appears to occur only in the
absence of ovarian hormones. Although tumor promotion data for the polychlori-
nated dibenzofurans and co-planar PCBs is limited, it appears that these
compounds are liver tumor promoters with potencies dependent on their binding
affinity to the Ah receptor.
Some of the central issues in the risk assessment of TCDD and its structural
analogs are (1) characterization of the shape of the dose response curve for
receptor-mediated events, (2) evaluation of the relevance of animal data in the
estimation of human risks, and (3) the health consequences of background
6-37 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
exposures (1-10 pg TEQ/kg/day) of dioxin and its structural analogs. In regards
to the shape of the dose response curve, it is clear from animal studies that
there are different dose response curves for different TCDD effects which is
consistent with the generally accepted dogma for steroid receptor-mediated
responses (Lucier, 1992). In general, the biochemical/molecular responses such
as cytochrome P-450 induction do not show evidence for a threshold although
unequivocal conclusions cannot be made and the mechanistic link, if any, between
biochemical responses and toxic effects have not been established. In fact,
coordinated biological responses such as TCDD-mediated cell proliferation and
growth of preneoplastic lesions (foci of cellular alteration in liver) appear to
be leas sensitive endpoints although evaluation of these responses is complicated
by a high degree of interindividual variations some animals do not exhibit any
increase in cell proliferation in response to chronic TCDD exposure.
The mechanistic basis for interindividual variation is unclear and this lack
of knowledge complicates approaches to estimate human risks from experimental
animal data. However, several studies indicate that, mostly, humans appear to
respond like experimental animals for biochemical and carcinogenic effects.
However, data from epidemiology studies are difficult to evaluate because the
carcinogenic effects, if any, resulting from background TCDD exposures are not
known, although biochemical effects such as cytochrome P-450 induction may be
produced by background exposures.
6.7. REFERENCES
Abbott, B. and L. Birnbaum. 1990. TCDD-induced altered expression of growth
factors may have a role in producing cleft palate and enhancing the incidence of
clefts after coadministration of retinoic acid and TCDD. Toxicol. Appl.
Pharmacol. 106: 418-432.
Abernethy, D.J., W.F. Greenlee, J.C. Huband and C.J. Boreiko. 1985. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2
cells. Carcinogenesis. 6s 651-653.
Abraham, K., R. Krowke and D. Neubert. 1988. Pharmacokinetics and biological
activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch. Toxicol. 62s 359-368.
6-38 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITI
Arcos, J. C., A.M. Conney and N.P. Buu-Hoi, 1961. Induction of mierosoraal
enzyme synthesis by polyeyclic aromatic hydrocarbons of different molecular
sizes. J. Biol. Chem. 236: 1291.
Astroff, B,, C. Rowlands, R. Dickerson S. and Safe. 1990. S. 2,3,7,8-Tetra-
chlorodibenzo-p-dioxin inhibition of 17p-estradiol-induced increases in rat
uterine epidermal growth factor receptor binding activity and gene expression.
Mol. Cell. Endocrin. 70: 247-252.
Barrett, J.C. 199?, Multistage carcinogenesis. Int Mechanisms of Carcino-
genesis in Risk Identification, H. Vanio, P.N. Hagee, D.B. McGregor and A,J.
McMichael, Ed. IARC, WHO, Lyon France.
Barrett, J.C. and R.W. Wiseman. 1987. Cellular and molecualar mechanisms of
multistep carcinogenesis: Relevance to carcinogen risk assessment. Environ.
Health Perspeet. 76s 65-70.
Bars, R.G. and C.R. Elcombe. 1991. Dose-dependent acinar induction of eyto-
chromes P450 in rat liver. Evidence for a differential mechanism of induction of
P4501A1 by p-naphthaflavone and dioxin. Biochem. J. 277s 577-580.
Barter, R.A. and C.D. Klaassen. 1992. ODP-glucuronosyltransferase inducers
reduce thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol.
Appl. Pharmacol. 113: 36-42.
Bastomaky, C.H. 1977. Enhanced thyroxine metabolism and high uptake goiters in
rats after a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology.
101: 292-296.
Beguinot, L., J.A. Hanover, S. Ito, M.D. Richert, M.C. Willingham and I. Pastan.
1985. Phorbol esters induce internalization without degradation of unoccupied
epidermal growth factor receptors. Proc. Natl. Acad. Sci. 82s 2774-2778.
6-39 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Bock, K.W. 1991, Roles of UDP-glucuronyltransferases in chemical carcino-
genesis. Crit. Rev. Biochem. Mol. Biol. 26(2)i 129-150.
Burchell, B., D.W. Nebert, D.R. Nelson et al. 1991. The UDP glucuronlytrans-
f erase gene super family: suggested nomenclature based on evolutionary divergence.
DNA Cell Biol. 10(7)5 487-494.
California Department of Health Services. 1985. Health effects of 2,3,7,8-
Tetrachlorodibenzo-p-dioxin and related compounds. Response to public comments.
California Scientific Review Panel Discussions. September, 19SS.
Carpenter, G. 1987. Receptors for epidermal growth factor and other polypeptide
mitogens. Ann. Rev. Biochem. 56: 881-914.
Carpenter, G. and S. Cohen. 1979. Epidermal growth factor. Ann. Rev. Biochem.
48s 193-216.
Choi, 1., D. ToBcano, J. Ryan, N. Reidel and W. Toscano. 1991. Dioxin induces
transforming growth factor-a in human keratinocytes. J. Biol. Chem. 266:
9591-9597.
Clark, G., A. Tritscher, R. Maronpot, J. Foley and G. Lucier, G. 1991a. Tumor
promotion by TCDD in female rats, in; Banbury Report 35: Biological Basis for
Risk Assessment of Dioxin and Related Compounds, M. Gallo, R. Scheuplein, and
K. Van Der Heijden, Ed. Cold Spring Harbor Laboratory, p. 389-404.
Clark, G.C., M.J. Taylor, A.M. Tritscher and G.W. tucier. 1991b. Tumor necrosis
factor involvement in 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated endotoxin
hypersensitivity in C57BL/6J mice congenic at the Ah locus. Toxicol. Appl.
Pharmacol. Ill: 422-431.
6-40 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Clark, 6., A. Tritscher, D. Bell and G. Lucier. 1992. Integrative approach for
evaluating species and interindividual differences in responsiveness to dioxine
and structural analogs. Environ. Health Perspect. (In press).
Cochet, C., G.N. Gill, J. Meisenhelder, J.A. Cooper and T. Hunter. 1984.
C-kinase phosphorylates the epidermal growth factor receptor and reduces its
epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol.
Cherti. 259: 2553-2558.
Cohen, G.M., W.M. Bracken, R.P, Iyer, D.L. Berry, J.K. Selkirk and T.J. Slaga.
1979. Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on
benzo[a]pyrene and 7,12-dimethylbenz[a]anthrene tumor initiation and its
relationship to DNA binding. Cancer Res. 39: 4027-4033.
Conney, A.H. 1982. Induction of microsomal emzymes by foreign chemicals and
carcinogenesis by polycyclic aromatic hydrocarbons: G.H.A. Clowes Memorial
Lecture. Cancer Res. 42: 4875-4917.
Cook, J.C. and W.F. Greenlee. 1989. Characterization of a specific binding
protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin in human thymic epithelial cells.
Mol. Pharmacol. 35: 713-719.
Dannan, G.A., D.J. Porubeck, S.D. Nelson, D.J. Waxman and F.P. Guengerich. 1986.
17p-Estradiol 2- and 4-hydroxylation catalyzed by developmental patterns, and
alterations in gonadectomy. Endocrinology. 118: 1952-1960.
Delia Porta, G., T.A. Dragani and G. sozzi. 1987. carcinogenic effects of
infantile and lone-term 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the
mouse, fumori. 73: 99-107.
6-41 OB/24/92
-------
DRAFT—DO HOT QUOTE OR CITE
DeVito, M.J., T. Thomas, E. Martin, T.H. Umbreit and M.A. Gallo. 1992.
Antiestrogenic action of 2,3,7,8-tetrachlorodibenzo-p-dloxins Tissue specific
regulation of estrogen receptor in CD1 mice. Toxicol. Appl. Pharmacol. 113:
284-292.
DeVito, M.J., T.H. Orabreit, T. Thomas and M.A. Gallo. 1991. An analogy between
the actions of the Ah receptor and the estrogen receptor for use in the biolog-
ical basis for risk assessment of dioxin. In; Banbury Report 35: Biological
Basis for Risk Assessment of Dioxin and Related Compounds, M.A. Gallo, R.J.
Scheuplein and K.A. Van Der Heijden, Ed. Cold Spring Harbor Laboratory.
p. 427-440.
DiGiovanni, J., A. Viaje, D.L. Berry, T. Slaga and M.R. Juchau. 1977. Tumor
initiating ability of TCDD and Aroclor 1254 in the two stage system of mouse skin
carcinogenesis. Bull. Environ. Contain. Toxicol. 18: 552-557.
Dragan, Y.P., T. Rizvi, Y.H. Xu et al. 1991. An initiation-promotion assay in
rat liver as a potential complement to the 2-year carcinogenesis bioassay. Fund.
Appl. Toxicol. 16: 525-547.
Dragan, Y.P., X. Xu, T.L. Goldsworthy, H.A. Campbell, R.R. Maronpot and
B.C. Pitot. 1992. Characterization of the promotion of altered hepatic foci by
2,3,7,8-tetrachlorodibenzo-p-dioxin in the female rat. Carcinogenesis. (In
press)
Eckl, P.M., S.A. Meyer, W.R. Whitcombe and R.L. Jirtle. 1988. Phenobarbital
reduces IGF receptors in the ability of physiological concentrations of calcium
to suppress hepatocyte proliferation. Carcinogenesis. 9j 479-483.
Eisenfeld, A.J., R. Aten, M.J. Weinberger and G. Haselbacher. 1976. Estrogen
receptor in the mammalian liver. Science. 191: 862-865.
6-42 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
EPA Science Advisory Board, 1984, Environmental Health Committee Transcripts,
November 29, 1984. Science Advisory Board, Washington, DC,
EPA Science Advisory Board 1989, Review of draft documents: A cancer risk-
specific dose estimate for 2,3,7,8-TCDD and Estimating risk exposure to 2,3,7,8-
TCDD. EPA SAB Ad Hoc Dioxin Panel, Washington, DC.
Farber, E. 1984. The multiple nature of cancer development. Cancer Res. 44:
4217-4223.
•r
Fingerhut, M.A., W.E. Halperin, D,A. Marlow et al. 1991. Cancer mortality in
workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. New Engl. J. Hed. 324:
212-218.
Flodatrom, S. and U.G. Ahlborg. 1991. Promotion of hepatocarcinogenesis in rats
by PCDDe and PCDFs. In; Banbury Report 35: Biological Basis for Risk Assessment
of Dioxin and Related Compounds, M.A. Gallo, R.J. Scheuplein and K.A. Van Der
Heijden, Ed. Cold Spring Harbor Laboratory, p. 405-414.
Gierthy, J.F., D.W. Lincoln, S.J. Kampick et al. 1988. Enhancement of 2- and
16-rs-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Biochem. Biophys, Res. Commun, 157: 515-520.
Giri, A.K. 1987, Mutagenic and genotoxic effects of 2,3,7,8-tetrachlorodibenzo-
p-dioxin: A review. Mutat. Res. 168: 241-248.
Goldstein, J.A. and P. Linko. 1984. Differential induction of two 2,3,7,8-
tetrachlorodibenzo-p-dioxin-indueible forms of cytochrome P-450 in extrahepatic
versus hepatic tissues. Mol. Pharmacol. 25: 185-191.
6-43 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Goldstein, J.A. and S. Safe. 1989. Mechanism of action and structure-activity
relationships for the chlorinated dibenzo-p-dioxine and related compounds. In;
Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins, and Related
Products, R.D. Kimbrough and A, A. Jensen, Ed. Elsevier, New York. p. 239-293.
Graham, M.J., G.W. Lucier, P. Linko, R.R. Maronpot and J.A. Goldstein. 1988.
Increases in cytochrome P-450 mediated 17p-estradiol 2-hydroxylase activity in
rat liver microsomes after both acute administration and subchronic administra-
tion of 2,3,7,8-tetrachlorodibenzo-jj-dioxin in a two-stage hepatocarcinogenesis
model. Carcinogenesis. 9(ll)s 1935-1941.
Greig, J.B. and F. DeMatteis. 1973. Effects of 2,3,7,8-Tetrachlorodibenzo-p-
dioxin on drug metabolism and hepatic microsomes of rats and mice. Environ.
Health Perspect. 5: 211-220.
Guengerich, F.P. 1988. Roles of cytochrorae P-450 enzymes in chemical carcino-
genesis and cancer chemotherapy. Cancer Rea. 48: 2946-2954.
Harris, M., T. Zacharewski, J. Piskorska-Pliszczynska, R. Rosengren and S, Safe.
1990a. Structure-dependent induction of aryl hydrocarbon hydroxylase activity
in C57BL/6 mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related congenerss
mechanistic studies. Toxicol. Appl. Pharmacol. 105: 243-253.
Harris, M., T. Zacharewski and S. Safe. 1990b. Effects of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin and related compounds on the occupied nuclear estrogen receptor
in MCF-7 human breast cancer cell. Cancer Res. 50: 3579-3584.
Hayashi, K. and N. Sakamoto. 1986. Dynamic analysis of enzyme systems. Japan
Scientific Societies Press, Tokyo.
6-44 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Hebert, C.D., M.w. Harris, M.R. Elwell and L.S. Birnbaum. 1990. Relative
toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), and 1,2,3,4,7,8-hexachloro-
dibenzofuran (HCDF) in hairless mice. Toxicol, Appl. Pharmacol. 102s 362-377.
Henry, B.C. and T.A. Gasiewicz. 1987. Changes in thyroid hormones and thyroxine
glucuronidatlon in hamsters compared with rats following treatment with 2,3,7,8-
tetraehlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 89 j 165-174.
Hill, R.N., L.S, Erdreieh, O.E. Paynter, P. ft. Roberts, S.L. Rosenthal and
C.F. Wilkinson. Thyroid follicular cell carcinogenesis. Fund. Appl. Toxicol.
12: 629-697.
Hudson, L.G., W.A. Toscano, Jr. and W.F. Greenlee. 198S. Regulation of
epidermal growth factor binding in human keratinocyte cell line by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 77s 251-259.
Huff, J.E., A.G. Salmon, N.K. Hooper and L. Zeise. 1991. Long-term carcino-
genesis studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin and hexachlorodibenzo-p-
dioxins. Cell Biol. and Toxicol. 7(1): 67-94.
Ignar-Trowtaridge, D.M., K.G. Nelson, M.C. Bidwell et al. 1992. Coupling of dual
signaling pathways? Epidermal growth factor action involves the estrogen
receptor. Proc. Natl. Acad. Sci. (In press).
IARC (International Agency for Research on Cancer). 1982. IARC Monographs on
the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Suppl. 4s
Chemicals, industrial processes, and industries associated with cancer in humans.
WHO, Lyon, France, p. 238-243.
IARC (International Agency for Research on Cancer). 1992. Mechanisms of
Carcinogenesis in Risk Identification. WHO, I.yon, France.
6-45 08/24/92
-------
DRAFT—DO NOf QUOTE OR CITE
Ito, N., M. Tatematsu, R. Hasegawa and H. Tsuda. 1989, Medium-term bioassay
system for detection of carcinogens and modifiers of hepatocarcinogenesis
utilizing the GST-P positive liver cell focus as an endpoint marker. Toxicologic
Pathol. 17(4 Part l)s 630-641.
Ito, N., M. Tatematsu, K. Nakanishi, R. Haaegawa et al. 1980. The effects of
various chemicals on the development of hyperplastic liver nodules in hepatecto-
mized rats treated with N-nitrosodiethylamine or N-2-fluorenlyacetamide. Jpn.
J. Cancer Res. 71s 832-842,
Kedderis, L.B., J.J. Diliberto, P. Linko, J,A, Goldstein and L.S. Birnbaum.
1991. Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachloro-
dibenzo-p-dioxin in the rat: Biliary excretion and induction of cytochromes
CYP1A1 and CTP1A2. Toxieol. Appl. Pharmacol. llli 163-172.
Kitchin, K.T. J.S. Woods. 1979. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.
Toxicol. Appl. Pharmacol. 47: 537-546.
Kociba, R.J., D.G. Keyes, J.E. Beyer et al. 1978. Results of a two-year chronic
toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats.
Toxicol. Appl. Pharmacol. 46: 279-303.
Kociba, R. 1984. Evaluation of the carcinogenic and rnutagenic potential of
2,3,7,8-TCDD and other chlorinated dioxins. In; A. Poland and R. Kimbrough
(eds.), Banbury Report 18: Biological Mechanisms of Dioxin Action. Cold Spring
Harbor, NYs Cold Spring Harbor Laboratory, p. 73-84.
Kohn, M.C., G.W. Lucier and C.J. Portier. 1992. A mechanistic model of effects
of dioxin on gene expression in the rat liver. Toxicol. Appl. Pharmacol. (To
be submitted).
6-46 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITI
Kouri, R.E., T.H. Rude, R. Joglekar et al. 1978. 2,3,7,8-letrachlorodibenzo-p-
dioxin as oocarcinogen causing 3-roethylcholanthrene-initiated subcutaneous tumors
in mice genetically "nonresponsive" at Ah locus. Cancer Res. 38s 2722-2783.
Levin, W., A. Wood, R. Chang et al. 1982, Oxidative metabolism of polycyclic
aromatic hydrocarbons to ultimate carcinogens. Drug Metab. Rev. 13: S55-580.
Lii J.J. and S.A. Li. 1990. Estrogen carcinogenesia in hamster tissues: a
critical review. Endocrine Rev. 11(4): 524-531.
Lin, F.H., G. Clark, L.S. Birnbaum, G.W. Lucier and J.A. Goldstein. 1991a.
Influence of the Ah locus on the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin
on the hepatic epidermal growth factor receptor. Mol. Pharmacol. 39i 307-313.
Lin, F., s. Stohs, L. Birnbaum, 6. Clark, G. Lucier and J. Goldstein. 1991b.
The effects of 2,3,7,8-tetrachlorodibenzo-j>-dioxin (TCDD) on the hepatic estrogen
and glucocorticoid receptors in congenic strains of Ah responsive and Ah non-
responsive C57BL/6J mice. Toxicol. Appl. Pharmacol. 108s 129-139.
Lorenzen, A. and A.B. Okey. 1991. Detection and characterization of Ah receptor
in tissue and cells from human tonsils. Toxicol. Appl. Pharmacol. 107s 203-214.
Lucier, G.W. 1991. Humans are a sensitive species to some of the biochemical
effects of structural analogs of dioxin. Environ. Toscicol. Chem. 10s 727-735.
Lucier, G.W. 1992. Receptor mediated carcinogenesis. In; Mechanisms of
Carcinogenesis in Risk Identification, H. Vanio, P.N. Magee, D.B. McGregor and
A.J. McMichael Ed. IARC, WHO, Lyon. p. 87-112,
Lucier, G.W., O.S. McDaniel, G.E.R. Hook, B. Fowler, B.R. Sonawane and E. Faeder.
1973. TCDD-Induced changes in rat liver microsomal enzymes. Environ. Health
Perspect. 5s 199-210.
6-47 OB/24/32
-------
DRAFT—DO NOT QUOTE OR CITE
Lucier, G.W., O,S. HcDaniel and G.E.R. Hook. 1974. Nature of the enhancement
of uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachloro-
dibenzo-p-dioxin in rats. Biochera. Pharmacol, 24: 325-334.
Lucier, G.W., E.M.K. Lui and C.A. Lamartiniere. 1979. Metabolic activation/
deactivation reactions during perinatal development. Environ. Health Perspect.
29; 7-16.
Lucier, G.W., S.R. Slaughter, C. Thompson, C.A. Lamartiniere and V, Powell-Jones.
1981. Selective actions of growth hormone on rat liver estrogen binding
proteins. Biochem. Biophys. Res. Commun. 103$ 872-879.
Lucier, G.W., R,C, Rumbaugh, Z. McCoy, R, Mass, D. Harvan and P. Albro. 1986.
Ingestion of soil contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
alters hepatic enzyme activities in rats. Fund. Appl. Toxicol. 6s 364-371.
Lucier, G.W., K.G. Nelson, R.B. Everson et al. 1987. Placental markers of human
exposure to polychlorinated biphenyls and polychlorinated dibenzofurans.
Environ. Health Perspect. 76: 79-87.
Lucier, G.W., A.M. Tritscher, T. Goldsworthy et al. 1991. Ovarian hormones
enhance TCDD-mediated increases in cell proliferation and preneoplastic foci in
a two stage model for hepatocarcinogenesis. Cancer Res. 51s 1391-1397.
Lucier, G.W., G. Clark, A. Tritscher, J. Foley and R. Maronpot. 1992.
Mechanisms of dioxin tumor promotion: Implications for risk assessment.
Chemosphere. (In press).
Lundgren, K., M. Andries, C. Thompson and G.W. Lucier. 1986. Dioxin treatment
of rats results in increased in vitro induction of sister chromatid exchanges by
alpha-naphthoflavone: an animal model for human exposure to halogenated
aromatics. Toxicol. Appl. Pharmacol. 85s 189-195.
6-48 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Lundgren, K., G.W. Collman, S. Wang-Wuu et al. 1988. Cytogenetic and chemical
detection of human exposure to polyhalogenated aromatic hydrocarbons. Environ.
Mol. Mutagen. Ills 1-11.
Mably, T.A., R.W. Moore, R.W. Goy and R.E. Peterson. 1992. In utero and
lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Toxicol. Appl. Pharmacol. 114: 108-117.
Mably, T.A., H.M. Theobald/ G.B. Ingall and R.E. Peterson. 1990. Hypergastrin-
emia is associated with decreased gastric acid secretion in 2,3,7,8-tetrachloro-
dibenzo-p-dioxin treated rats. Toxicol. Appl. Pharmacol. 106: 518-528.
Madhukar, B.V., D.W. Brewster and F. Matsumura, 1984. Effects of in vivo
administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on receptor binding of epidermal
growth factor in the hepatic plasma membrane of rat, guinea pig, mouse and
hamster. Proc. Natl. Acad. Sci. 81s 7407-7411.
Madhukar, B.V., K. Ibner, F. Matsutnura, D.W. Bombick, D.W. Brewater and
T. Kawamoto. 1988. 2»3,7»8-Tetrachlorodibenzo-p-dioxin causes an increase in
protein kinases associated with epidermal growth factor receptor in the hepatic
plasma membrane. J. Biochem. Toxicol. 3s 261-277.
Manchester, D.K., S.K. Gorson, C.L. Colas, E.A. Roberts and A.B. Okey. 1987, Ah
receptor in human placenta: Stabilization by molybdate and characterization of
binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methyleholanthrene, and
benzo[a]pyrene. Cancer Res. 47: 4861-4868.
Maronpot, R.R., C.A. Montgomery, G,A, Boorman and E.E. McConnell. 1986.
National Toxicology Program Nomenclature for hepatoproliferative lesions of rats.
Toxicol. Pathol. 14(2): 263-273.
6-49 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Maronpot, R.R., B.C. Pitot and C. Peraino. 1989. Use of rat liver altered focus
models for testing chemicals that have completed two-year carcinogenicity
studies. Toxicol. Pathol. 17(4 Part 1): 651-662.
Marti, M., S. Burwen and A. Jones. 1989. Biological effects of epidermal growth
factor, with emphasis on the gastrointestinal tract and livers an update,
Hepatology. 9: 126-139.
Mastri, C. and G» Lucier. 1983. Actions of hormonally active chemicals in the
liver. In; Endocrine Toxicology, J.A. Thomas, Ed. Raven Press, New York.
p. 335-355.
Metzler, M. 1984. Metabolism of stilbene estrogens and ateroidal estrogens in
relation to carcinogenicity. Arch. Toxicol, 55s 104-109.
Miller, E.C., J.A. Miller, R.R. Brown and J.C. MacDonald. 1958. On the protec-
tive action of certain polycyclic aromatic hydrocarbons against carcinogenesis
by aminoazo dyes and 2-acetylaminofluorene, Cancer Res. 18s 469.
Moolgavkar, S. and A. Knudson. 1981. Mutation and cancer: A model for human
carcinogenesis. J. Natl. Cancer Inst. 66s 1037-1052.
Moore, R.W., J.A. Parsons, R.C. Bookstaff and R.E. Peterson. 1989. Plasma
concentrations of pituitary hormones in 2,3,7,8-tetrachlorodibenzo-p-dioxin-
treated male rats. J. Biochem. Toxicol. 4: 165-172.
Moore, R.W., C.L. Potter, H.M. Theobald, J.A. Robinson and R.E. Peterson. 1985.
Androgenic deficiency in male rats treated with 2,3,7,8-tetrachlorodibenzo-p-
dioxin. Toxicol. Appl. Pharmacol. 79s 99-111.
Mukku, V.R. 1984. Regulation of epidermal growth factor receptor levels by
thyroid hormone. J. Biol. Chem. 259(10)t 6543-6547.
6-50 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Mukku, V. and 6. Stancel. 1985. Regulation of epidermal growth factor receptor
by estrogen. J. Biol. Chem. 260: 9820-9824.
NCI (National Cancer Institute). 1979a, Bioassay of dibenzo-p-dioxin for
possible carcinogenicity. National Institutes of Health, Bethesda, M.D. NCI
Tech. Rept. No. 122.
NCI (National Cancer Institute). 1979b. Bioassay of dichlorodibenzo-p-dioxin
for possible carcinogenicity. National Institutes of Health, Bethesda, M.D. NCI
Tech. Rept. No. 123.
NTP (National Toxicology Program). 1980. Bioassay of a mixture of 1,2,3,6,7,8-
hexachlorodibenzo-p-dioxin and l,2,3,7,8,9~hexachlorodibenzo-p~dioxin for
possible carcinogenicity (gavage study). U.S. DHHS, PHS, Research Triangle Park,
N.C. Tech. Rept. Ser, No. 198.
NTP (National Toxicology Program). 1982a. Bioassay of 2,3,7,8-tetraehloro-
dibenzo-p-dioxin for possible carcinogenicity (gavage study). U.S. DHHS, PHS,
Research Triangle Park, N.C. Tech. Rept. Ser. No, 201.
NTP (National Toxicology Program), 1982b. Bioassay of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin for possible carcinogenicity (dermal study). U.S. DHHS, PHS,
Research Triangle Park, N.C. Tech. Rept. Ser. No. 201.
NTP (National Toxicology Program). 1984, Report of the NTP Ad Hoc Panel on
Chemical Carcinogenesis Testing and Evaluation. Board of Scientific Counselors,
U.S. DHHS, PHS, Research Triangle Park, NC.
Nelson, K., A. Vickers, G. Sunahara and G. Lucier. 1988. Receptor and DNA
ploidy changes during promotion of rat liver carcinogenesis. In; Tumor
Promoters: Biological Approaches for Mechanistic Studies and Assay Systems.
Progress in Cancer Research and Therapy, Vol. 34, R, Langenbach, E. Elmore and
J. Barrett, Ed. Raven Press, New York. p. 387-405.
6-51 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Nemoto, N. and H.v, Gelboin. 1976. Enzymatic conjugation of benzo[a]pyrene
oxides, phenols and dihydrodiols with UDP-glucuronic acid. Biochem. Pharmacol.
25: 1221-1226.
Okey, A.B., M.S. Denison, R.D. Prokipcak, E.A. Roberts and P.A. Harper. 1989.
Receptors of polycyclic aromatic hydrocarbons. In; Biologie Prospective, M.M.
Galteau, G. Siest and J. Henny, Ed. John Libbey Eurotext, Paris, p. 605-610.
Osborne, R., J.c. Cook, K.M. Dold, L. Ross, K. Gaido and W.F. Greenlee. 1988.
TCDD receptors mechanisms of altered growth regulation in normal and transformed
human keratinocytes. In: Progress in Cancer Research and Therapy, Vol. 34,
R. Langenbach, J.C. Burrett and E. Elmore, Ed. Raven Press, New York.
p. 407-416.
Parkinson, A. and A. Hurwitz. 1991. Otneprazole and the induction of human
cytochrome P-450: A response to concerns about potential adverse effects.
Gastroenterology. 100{4)s 1157-1164.
Parkinson, A., P.S. Thomas, D.E. Ryan et al. 1983. Differential time course of
induction of rat liver microsomal cytochrome P450 isozymes and epoxide hydrolase
by Arolclor 1254. Arch. Biochem. Biophys. 225: 203-215.
Pelkonnen, O. and D.W. Nebert. 1982. Metabolism of polycyclic aromatic
hydrocarbons: etiologic role in carcinogenesis. Pharmacol. Rev. 34: 189-222.
Peraino, C., E.F. Staffeldt and V.A. Ludeman. 1981. Early appearance of histo-
chemically altered hepatocyte foci and liver tumors in female rats treated with
carcinogens 1 day after birth. Carcinogenesis. 2s 463-465.
Pitot, H.C. and H.A. Campbell. 1987. An approach to the determination of the
relative potencies of chemical agents during the stages of initiation and
promotion in multistage hepatocarcinogenesis in the rat. Environ. Health
Perspect. 76: 49-56.
6-52 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Pitot, H.C. and A.E. Sirica. 1980. The stages of initiation and promotion in
hepatocarcinogenesis. Biochim. Biophys. Acta. 60S: 191-215.
Pitot, H.C., T.Ii. Goldsworthy, H.A. Campbell and A. Poland. 1980. Quantitative
evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepato-
carcinogenesis from diethylnitrosamine. Cancer Res. 40: 3616-3620.
Pitot, H.C., T.L. Goldsworthy, S. Moran et al. 1987. A method to quantitate the
relative initiating and promoting potencies of hepatooarcinogenic agents in their
dose-response relationships to altered hepatic' foci. Carcinogenesis.
8: 1491-1499.
Pitot, B.C., H.A, Campbell, R.R. Maronpot et al. 1989. Critical parameters in
the quantitation of the stages of initiation, promotion, and progression in one
model of hepatocarcinogenesis in the rat. Toxicol. Pathol. 17(4 Part 1):
594-612.
Poland, A. and E. Glover. 1973. Studies on the mechanism of toxicity of
chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5: 245-252.
Poland, A. and J.C. Knutson. 1982. 2,3,7,8-Tetrachlorodibenzo-|j-dioxin and
related halogenated aromatic hydrocarbons: Examination of the mechanism of
toxicity. Ann. Rev. Pharmacol. Toxicol. 22: 517-554.
Poland, A., D, Palen and E. Glover. 1982. Tumor promotion by TCDD in skin of
HRS/J mice. Nature. 300(5889): 271-273.
Popp, J.A. and T.L. Goldsworthy. 1989. Defining foci of cellular alteration in
short-term and medium-term rat liver tumor models. Toxicol. Pathol. 17 J 4
Part 1): 561-568.
Portier, C.J. 1987. statistical properties of a two-stage model of carcino-
genesis. Environ. Health Perspect. 76: 125-132.
6-53 08/24/92
-------
DRAFT—DO NOT QUOTE OE CITE
Fortier, C., A. Tritscher, M. Kohn et al. 1992. Ligand/receptor binding for
2,3,7,8-TCDD: Implications for risk assessment. Fund. Appl. Toxicol. (In
press).
Pour, P., N. Kmoch, E. Greiser, U. Mohr, J. Althoff and A. Cardesa. 1976.
Spontaneous tumors and common diseases in two colonies of Syrian hamsters:
I. Incidence and sites. J. Natl. Cancer Inst. 56(5)s 931-935.
Powell-Jones, W., C. Thompson, S. Raeford and G.W. Lucier. 1981. Effect of
gonadeetomy on the ontogeny of estrogen-binding components in the rat liver
cytosol. Endocrinology. 109: 628-636.
Rao, M.S., V. Subbarao, J.D. Prasad and D.C. Scarpelli. 1988. Carcinogenicity
of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Syrian golden hamster. Carcino-
genesis. 9(9): 1677-1679.
Reynolds, S. and G. Lucier. 1992. Characterization of transforming genea in
lung tumors from rats exposed to a single dose of diethylnitrosamine and chronic
TCDD. Mol. Carcinogen. (To be submitted).
Roberts, L. 1991. Dioxin risks revisited. Science. 251: 624-626.
Romkea, M., J. Piskorska-Pllszczynska and S. Safe. 1987. Effects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin on hepatic and uterine estrogen receptor levels in
rats, foxicol. Appl. Pharmacol. 87: 306-314.
Romkes, H. and s. Safe. 1988. Comparative activities of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin and progesterone on antiestrogens in the female rat uterus.
Toxicol. Appl. Pharmacol. 92: 368-380.
6-54 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Sanders, J.E., D.A. Eigenberg, L.J. Bracht, W.R. Hang and J.J. Van Zwieten.
1988. Thyroid and liver trophic changes in rats secondary to liver microsomal
enzyme induction caused by an experimental leukotriene antagonist (L-649,923).
Toxicol. Appl. Pharmacol. 95: 378-387.
Sauer, R.M. 1990. 2,3,7,8~Tetraehlorodibenzo~p-dioxin in Sprague-Dawley rate.
Submitted to the Maine Scientific Advisory Panel by Pathco, Inc., Ijansville, MD.
March 13, 1990.
Sewall, C.H. and C.W. Lucier. 1992. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDDJ
mediated changes in the epidermal growth factor receptor in a two-stage model for
hepatocarcinogenesis in female rats. Carcinogenesis. (To be submitted),
Shi, Y.E. and J.D. lager. 1989. Effects of the liver tumor promoter ethinyl
estradiol on epidermal growth factor-induced DNA synthesis and epidermal growth
factor receptor levels in cultured rat hepatocytes. Cancer Res. 49: 3574-3580.
Shiverick, K.T. and T.F. Muther. 1982. Effects of 2,3,7,8-tetrachlorodibenzo-p-
dioxin on serum concentrations and the uterotrophic actions of exogenous eatrone
in rats. Toxicol. Appl. Pharmacol. 65s 170-176.
Shiverick, K.T. and T.F. Muther. 1983. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD) effects on hepatic microsomal steroid metabolism and serum estradiol of
pregnant rats. Biochem. Pharmacol. 32: 991-995.
Shu, H.P., D.J. Paustenbach and F.J. Murray. 1987. A critical evaluation of the
use of mutagenesis, carcinogenesis and tumor promotion data in a cancer risk
assessment of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Reg. Toxicol. Pharmacol.
7s 57-58.
Silbergeld, I.K. and T.A. Gasiewicz. 1989. Dioxins and the Ah receptor. Am.
J. Ind. Med. 16: 455-474.
6-55 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Sims, P. and P.L. Grover. 1974. Epoxides in polycyclic aromatic hydrocarbon
metabolism and carcinogenesis. Adv. Cancer Res. 20: 165-274.
Slaga, T.J., L. Becker, W.M. Bracken and C.E. Weeks. 1979. The effects of weak
or non-carcinogenic polycyclic hydrocarbons on 7,12-dimethyl benzja]anthracene
and benzo[aJpyrene skin tumor initiation. Cancer Lett. 7: 51-59.
Slaga, T.J., S.M. Fischer, C.I. Weeks, A.J.P. Klein-Szanto and J. Reiners. 1982.
Studies on the mechanisms involved in multistage carcinogenesis in mouse skin.
J. Cell. Biochem. 18: 99-119.
Sloop, T.C. and G.W. Lucier. 1987. Dose-dependent elevation of Ah receptor
binding by TCDD in rat liver. Toxicol. Appl. Pharmacol. 88: 329-337.
Stoacheck, c. and "L. King. 1986. Role of epidermal growth factor in carcino-
genesis. Cancer Res. 46: 1030-1037.
Sunaharm, G., G. Lucier, Z. McCoy, 1. Bresnick, E. Sanchez and K. Nelson. 1989.
Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated decreases in
dexamethasone binding to rat hepatic cytosolic glucocorticoid receptor. Mol.
Pharmacol. 36s 239-247.
Sunahara, G.I., K.G. Nelson, T.K. Wong and G.W. Lucier. 1988. Decreased human
birth weights after in utero exposure to PCBs and PCDFs are associated with
decreased placental EGF-stimulated receptor autophosphorylation capacity. Mol.
Pharmacol. 32s 572-578.
Sutter, T.R., K. Guzman, K.M. Dold and W.F. Greenlee. 1991. Targets for dioxin:
Genes for plasminogen activator inhibitor-2 and interleukin-lp. Science. 254:
415-417.
6-56 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Swenberg, J.A., F.C. Richardson, J.A. Bauoheron et al, 1987. High- to low-dose
extrapolation: Critical determinants involved in the dose response of carcino-
genic substances. Environ. Health Perspect. 76s 57-64.
Tephly, T. R. and B. Burchell. 1990. UDP-glucuronlytransferases? A family of
detoxifying enzymes. TIPS Rev. 11s 276-279.
Thakker, D.R., H. Yagi, W. Levin, A.W. Wood, A.H, Conney and D.M. Jerina. 1985.
Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens.
Ins Bioactivation of Foreign Compounds, M.W. Anders, Ed. Academic Press, Inc.,
New York. p. 177-242,
Tritscher, A.M., J.A. Goldstein, C.J. Portier, Z. McCoy, G.C. Clark and
G.W. Lucier. 1992. Dose-response relationships for chronic exposure to 2,3,7,8-
tetrachlorodibenzo-p-dioxin in a rat tumor promotion model: Quantification and
immunolocalization of CYP1A1 and CYP1A2 in the liver. Cancer Res. (In press).
Turteltaub, K.W., J.S. Pelton, B.L. Gledhill et al. 1990. Accelerator mass
spectrometry in biomedical dosimetryj Relationship between low-level exposure and
covalent binding of heterocyclic amine carcinogens to DNA. Proc. Natl. Acad.
Sci. 87s 5288-5292.
Umbreit, T.H. and M.A. Gallo. 1988. Physiological implications of estrogen
receptor modulation by 2,3,7,8,-tetraehlorodibenzo-p-dioxin. Toxicol. Lett.
42: 5-14.
U.S. EPA. 1985. Health Effects Assessment Document for Polychlorinated Dibenzo-
l»-dioxins. Prepared by the Office of Health and Environmental Assessment,
Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of
Emergency and Remedial Response, Washington, DC. EPA 600/8-84-014F.
6-57 08/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Vanden Heuvel, J.P. and G.W. Lucier. 1992. Environmental toxicology of poly-
chlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. Environ. Health
Perspect. (In press).
Vanden Heuvel, J.P., G.W. Lucier, G.C. Clark, A.M. Tritscher, W.F. Greenlee and
D.A. Bell. 1992. Use of reverse-transcription polymerase chain reaction to
quantitate mRNA for dioxin-responsive genes in the low-dose region in rat liver.
Chemosphere. (In press).
Vecchi, A., M. Sironi, M.A. Canegrati, M. Recchia and S. Garatini. 1983.
Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-fj-dioxin in strains of
mice with different susceptibility to induction of aryl hydrocarbon hydroxylase.
foxicol. Appl. Pharmacol. 68: 434-441.
Velu, T. 1990. Structure, function and transforming potential of the epidermal
growth factor receptor. Mol. Cell. Endocrin. 70: 205-216.
Vickers, A. and G. Lucier. 1991. Estrogen receptor, epidermal growth factor and
/
cellular ploidy in elutriated subtractions of hepatocytes during liver promotion
by 17a-ethinylestradiol in rats. Carcinogenesis. 12r 391-399.
Vickers, A.E.M., K. Kelson, Z. McCoy and G.W. Lucier. 1989. Changes in estrogen
receptor, DNA ploidy, and estrogen metabolism in rat hepatocytes during a two-
stage model for hepatocarcinogenesis using 17-ethinylestradiol as a promoting
agent. Cancer Res. 49: 6512-6520.
Wahba, Z.Z., T.A. Lawson, W.J. Murray and S.J. Stohs. 1989, Factors
influencing the induction of DNA single strand breaks in rats by 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD). Toxicology. 58: 57-69.
Wahba, Z.Z., T.A. Lawson and S.J. Stohs. 1988. Induction of hepatic DNA single
strand breaks in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cancer
Lett. 29: 281-286.
6-58 OB/24/92
-------
DRAFT—DO NOT QUOTE OR CITE
Wassotn, J.S., J.E. Huff, N. Loprieno. 1977. A review of the genetic toxicology
of chlorinated dibenzo-p-dioxins. Mutat. Res. 47: 141-160.
Wattenberg, L.W. and J.L. Leong. 1970. Inhibition of the carcinogenic action
of benzo(a)pyrene by flavones. Cancer Res. 30: 1922-1925.
Wattenberg, L.W. 1978. Inhibition of chemical carcinogenesis. J. Natl. Cancer
Innt. 60: 11-18.
Wattenberg, L.W. 1985* Chemoprevention of cancer. Cancer Res. 45: 108.
Wheatley, D.N. 1968. Enhancement and inhibtion of the induction by 7,12-
dimethylbenz(a)anthracene of mammary tumors in female Sprague-Dawley rats. Br.
J. Cancer. 22: 787-797.
Whitlock, Jr., J.P. 1990. Genetic and molecular aspects of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin action. Ann. Rev. Pharmacol. Toxicol* 30. 251-277.
Williams, G.M. 1989. The significance of chemically-induced hepatocellular
altered foci in rat liver and application to carcinogen detection. Toxicologic
Pathol. 17(4 Part 1): 663-674.
Wong, T.K., B.A. Domin, P.E. Bent, I.E. Blanto, M.W. Anderson and R.M. Philpot.
1986. Correlation of placental microsomal activities with protein detected by
antibodies to rabbit cytochrome P-450 isozyrae 6 in preparations from humans
exposed to polychlorinated biphenyls, quarterphenyls, and dibenzofurans. Cancer
Res. 46: 999-1004.
Zacharewski, T., M. Harris, L. Biegel, V. Morrison, M. Merchant and S. Safe.
1992. 6-Methyl-l,3,8-trichlorodibenzofuran (MCDFJ aa an antiestrogen in human
and rodent cancer cell lines: Evidence for the role of the Ah receptor. Toxicol.
Appl. Pharmacol. 113s 311-318.
6-59 08/24/92
-------
DRAfT—DO NOT QUOTI OR CITE
Zacharewski, T., M. Harris and S. Safe. 1991. Evidence for a possible mechanism
of action of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated decrease of nuclear
estrogen receptor levels in wild-type and mutant Hepa Iclc7 cells. Biochem.
Pharmacol. 41s 1931-1939.
Zeise, L., J.E. Huff, A.G. Salmon and H.K. Hooper. 1990. Human risks from
2,3,7,8-tetrachlorodibenzo-p-dioxinandhexachlorodibenzo-p-dioxino. Ins Advances
in Modern Environmental Toxicology, Vol. 17, pp. 293-342. Princeton Scientific
Publishing Co., Inc., Princeton, NJ.
6-60 08/24/92
------- |