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EPA/600/AP-92/001C
August 1992
Workshop Review Draft
Chapter 3. Acute, Subchronic, and Chronic Toxicity
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.
Printed on Recycled Paper
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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 recomnjendation
I
for use. i
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
a peer-review workshop. It will be revised subsequent to the workshop as
suggestions and contributions from the scientific community are incorporated.
at
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CONTENTS
Tables iv
List of Abbreviations v
Authors and Contributors .- x
3. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY 3-1
3.1. SCOPE AND LIMITATIONS 3-1
3.2. ACUTE TOXICITY 3-1
3.2.1. Signs and Symptoms of Toxicity 3-5
3.2.2. Studies In Vitro 3-8
3.2.3. Appraisal 3-9
3.3. SUBCHRONIC TOXICITY 3-9
3.3.1. Appraisal • 3-10
3.4. CHRONIC TOXICITY 3-11
3.4.1. Appraisal 3-13
3.5. SPECIFIC EFFECTS 3-13
3.5.1. Wasting syndrome 3-13
3.5.2. Hepatotoxicity 3-17
3.5.3. Epidermal Effects 3-20
3.5.4. Enzyme induction 3-21
3.5.6. Endocrine Effects 3-25
3.5.7. Vitamin A Storage 3-28
3.5.8. Lipid Peroxidation 3-31
3.6. MECHANISMS OF TOXICITY . 3-31
3.7. CONCLUSIONS 3-34
3.8. REFERENCES 3-36
in
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LIST OF TABLES
3.1
3.2
3.3
3-4
Acute Lethality of TCDD to Various Species and Substrains
Toxic Responses Following Exposure to 2,3,7,8-TCDD:
Species Differences
Studies on Chronic Exposure (Except for Studies on Cancer)
to TCDD in Laboratory Animals
Lowest Effect Levels for Biological Responses of 2,3,7,8-TCDD
in Experimental Animals
3-2
3-6
3-12
3-35
IV
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ACTH
Ah
AHH
ALT
AST
BDD
BDF
BCF
BGG
bw
cAMP
CDD
cDNA
CDF
CNS
CTL
DCDD
DHT
DMBA
DMSO
DNA
DRE
LIST OF ABBREVIATIONS
Adrenocorticotrophic hormone
Aryl hydrocarbon
a
Aryl hydrocarbon hydroxylase
L-alanine aminotransferase
L-asparate aminotransferase
Brominated dibenzo-p-dioxin
Brominated dibenzofuran
Bioconcentration factor
Bovine gamma globulin
Body weight
Cyclic 3,5-adenosine monophosphate
Chlorinated dibenzo-p-dioxin
Complementary DNA
Chlorinated dibenzofuran
Central nervous system
Cytotoxic T lymphocyte
2,7-Dichlorodibenzo-p-dioxin
5a-Dihydrotestosterone
Dimethylbenzanthracene
Dimethyl sulfoxide
Deoxyribonucleic acid
Dioxin-responsive enhancers
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DTG
DTK
ECOD
EOF
EGFR
ER
EROD
EOF
FSH
GC-ECD
GC/MS
GGT
GnRH
GST
HVH
HAH
HCDD
HDL
HxCB
HpCDD
LIST OF ABBREVIATIONS (cont.)
Delayed type hypersensitivity
Delayed-type hypersensitivity
Dose effective for 50% of recipients
7-Ethoxycoumarin-O-deethylase
Epidermal growth factor
Epidermal growth factor receptor
Estrogen receptor
7-Ethoxyresurofm 0-deethylase
Enzyme altered foci
Follicle-stimulating hormone
Gas chromatograph-electron capture detection
Gas chromatograph/mass spectrometer
Gamma glutamyl transpeptidase
Gonadotropin-releasing hormone
Glutathione-S-transferase
Graft versus host
Halogenated aromatic hydrocarbons
Hexachlorodibenzo-p-dioxin
High density lipoprotein
Hexachlorobiphenyl
Heptachlorinated dibenzo-p-dioxin
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LIST OF ABBREVIATIONS (cont.)
HpCDF
HPLC
HRGC/HRMS
HxCDD
HxCDF
Heptachlorinated dibenzofuran
High performance liquid chromatography
High resolution gas chromatography/high resolution mass spectrometry
Hexachlorinated dibenzo-p-dioxin
Hexachlorinated dibenzofuran
I-TEF
LH
LDL
LPL
LOAEL
LOEL
MCDF
MFO
mRNA
MNNG
NADP
NADPH
NK
NOAEL
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-effect level
Lowest-observed-effect level
6-Methyl-l,3,8-trichlorodibenzofuran
Mixed function oxidase
Messenger RNA
/V-methyl-N-nitrosoguanidine
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate (reduced form)
Natural killer
No-observable-adverse-effect level
vn
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NOEL
OCDD
OCDF
PAH
PB-Pk
PCB
OVX
PBL
PCQ
PeCDD
PcCDF
PEPCK
PGT
PHA
PWM
ppm
ppq
ppt
RNA
SAR
SCOT
LIST OF ABBREVIATIONS (cont.)
No-observed-effect level
Octachlorodibenzo-p-dioxin
Octachlorodibenzofuran
Polyaromatic hydrocarbon
Physiologically based pharmacokinetic
Polychlorinated biphenyl
Ovariectomized
Peripheral blood lymphocytes
Quaterphenyl
Pentachlorinated dibenzo-p-dioxin
Pentachlorinated dibenzo-p-dioxin
Phosphopenol pyruvate carboxykinase
Placental glutathione transferase
Phytohemagglutinin
Pokeweed mitogen
Parts per million
Parts per trillion
Ribonucleic acid
Structure-activity relationships
Serum glutamic oxaloacetic transaminase
Vlll
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LIST OF ABBREVIATIONS (cont.)
SGPT
SRBC
t*
TCAOB
TCB
TCDD
TEF
TGF
tPA
TNF
TNP-LPS
TSH
TTR
UDPGT
URO-D
VLDL
v/v
w/w
Serum glutamic pyruvic transaminase
Sheep erythrocytes (red blood cells)
Half-time
TetracWoroazoxybenzene
Tetrachlorobiphenyl
Tetrachlorodibenzo-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
IX
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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 Corporation under EPA Contract No. 68-CO-0043, Task 20, wijth Carol
Haynes, Environmental Criteria and Assessment Office in Cincinnati, OH, serving as Projept Officer.
During the preparation of this chapter, EPA staff scientists provided reviews of the! drafts as
well as coordinating internal and external reviews.
AUTHORS
Ulf G. Ahlborg
Karolinska Institute
Stockholm, Sweden
EPA CHAPTER MANAGER
Debdas Mukerjee
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, OH
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3. ACUTE, SUBCHRONIC AND CHRONIC TOXICITY
3.1. SCOPE AND LIMITATIONS
The acute, subchronic and chronic toxicology of the chlorinated dioxins,
dibenzofurans, biphenyl and related compounds has been reviewed extensively in
recent years [CDDs and CDFs, WHO/IPCS (1989), U.S. EPA (1984, 1985); PCBs and
PCTs, WHO/IPCS (1991); U.S. EPA (1990); PCBs, U.S. EPA (1990); and BDDs and BDFs,
U.S. EPA (1991)]. This chapter is intended to summarize our knowledge of the
toxicology of TCDD in the main, but includes references to other dioxin-like
compounds when relevant data are available. The chapter does not have the
intention to reference all published material but rather seeks to select various
data that are considered to be of importance to risk assessment. The chapter
covers experimental animal data. Immunotoxicity, reproductive/developmental
toxicity, carcinogenicity, toxicity to humans and epidemiology will all be dealt
with in separate chapters, nor will ecotoxicology be covered in this chapter.
3.2. ACUTE TOXICIXY
The range of doses of TCDD which are lethal to animals varies extensively
both with species and strain, as well as with sex, age and the route of
administration within a single strain (Table 3-1). Typically there is a delayed
toxicity, with the time to death after exposure, usually being several weeks.
However, deaths within the first week after exposure have been observed in guinea
pigs (Schwetz et al., 1973), rabbits (Schwetz et al., 1973) and Golden Syrian
hamsters (Olson et al., 1980). More than an 8000-fold difference exists between
the dose of TCDD reported to cause 50% lethality in male Hartley guinea pigs, the
most sensitive species tested (Schwetz et al., 1973), and the corresponding dose
in male Syrian Golden hamsters (Henck et al., 1981). Another animal with
extremely high sensitivity is the mink (Wustela vision) and for the male an LD50
value of only 4.2 /ug/kg has been calculated (Hochstein et al., 1988).
Among traditional experimental animals, the rat seems to be the second most
sensitive species, although there is a >300-fold variability in LD5Q values
between different strains. The Han/Wistar (Kupio) strain of rat has been shown
to be particularly resistant to TCDD exposure (Pohjanvirta and Tuomisto, 1987).
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of other
Among the five rats per dose group (0, 1500, 2000, 2500 or 3000 nq TCtiD/kg bw),
only one animal died within the 39-40 days observation period. Also,|the DBA/2
I
male mouse has been shown to have a high resistance to TCDD toxicity (Chapman and
i
Schiller, 1985). j
Data on sex-differences in sensitivity to the lethal effects ofj TCDD are
conflicting. Acute toxicity data which addresses the effect of age at!the point
of exposure to TCDD are scarce, and comparisons are hampered by the absence of
or the inadequacy of the information on the age and/or body weight of fhe tested
animals. Additionally, as demonstrated with other chemicals, the acutJ3 toxicity
may vary several-fold, depending on vehicle used or the presence
substances that affect uptake.
The differences in sensitivity towards TCDD among various strains of mice
have been claimed to depend on a genetic variability in the Ah Ifocus (see
Chapter 2). |
In two strains of male C57B/6J mice that differ only at the JAh locus,
Birnbaura et al. (1990) found LD50 values of 159 and 3351 pig/kg for the| wild-type
mice (Ahb/b) and the congenic mice (Ahd/d), respectively. The mean tittje to death
was 22 days and was independent of dose and genotype. Signs of toxicity were
similar in the two strains, and it was concluded that the spectrum o;f toxicity
is independent of the allele at the Ah locus. However, the relative dose needed
to bring about various acute responses is -8-24 times greater in congenic mice
homozygous for the "d" allele than for the wild-type mice carrying two copies of
the "b- gene. I
In contrast, the two strains of rats studied by Pohjanvirta et al. (1988)
[i.e., Long-Evans and Han/Wistar (Kuopio)] had intraperitoneal LD50 values of 10
and >3000 ng TCDD/kg, although no differences as regards the amojint or the
affinity of available Ah receptor could be found.
Geyer et al. (1990) utilized both their own and other data to determine a
correlation between total body fat content and the acute toxicity
species and strains of laboratory mammals. However, data from the
in various
Han/Wistar
(Kuopio) rats that are extremely resistant to TCDD-induced lethality (Pohjanvirta
and Tuomisto, 1987) were not included. They found a correlation of 0.834 and
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suggested that the reasons for this correlation is obvious that an increased
total body fat content represents an enhanced capacity to remove TCDD from the
systemic circulation. This factor may be important, but it almost certainly
doesn't explain all the interspecies differences.
In chickens, acute toxicity is characterized by clinical signs such as
dyspnea, reduced body weight gain, stunted growth, subcutaneous edema, pallor and
sudden death (chick edema disease). The disease first gained attention in 1957,
but the causal agents were not identified as CDDs until much later (Firestone,
1973). Chick edema occurred in birds given oral doses of 1 or 10 jjg TCDD/kg/day
or of 10 and 100 pg hexaCDD/kg/day, but it was not observed in chicks maintained
on a diet containing 0.1 or 0.5% OCDD (Schwetz et al., 1973).
3.2.1. Signs and Symptoms of Toxicity. TCDD affects a variety of organ systems
in different species. It should be noted that much of the comparative data base
is derived from high-dose effects. The liver is the organ primarily affected in
rodents and rabbits, while in guinea pigs, atrophy of the thymus and lymphatic
tissues seems to be most sensitive markers of toxicity (WHO/IPCS, 1989; U.S. EPA,
1984, 1985). It is not possible to specify a single organ whose dysfunction
accounts for the lethality. Dermal effects are prominent signs of toxicity in
subhuman primates, and changes in epithelial tissues dominate both cutaneously
and internally. This is most apparent in nonhuman primates, and the cutaneous
lesions closely mimic the chloracne and hyperkeratosis observed in humans. The
histopathological alterations observed in epithelial tissues include hyperplastic
and/or metaplastic alterations, as well as hypoplastic responses. The toxic
responses of various species to TCDD are summarized in Table 3-2 (WHO/IPCS,
1989).
Loss of body weight (wasting syndrome) is a characteristic sign observed in
most animals given a lethal dose of TCDD. The weight loss usually manifests
itself within a few days after exposure and results in a substantial reduction
of the adipose tissue (Peterson et al., 1984) and of the muscle tissue (Max and
Silbergeld, 1987) observed at autopsy. With sublethal doses of TCDD, a dose-
dependent decrease in body weight gain occurs.
The greatest species-specific differences in toxicity concern pathological
alterations in the liver. Lethal doses to guinea pigs do not result in liver
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damage, which IB comparable to the liver lesions described in rabbits and rats,
or to liver changes observed in mice (McConnell et al., 1978b; Moore et al.,
1979; Turner and Collins, 1983). In the hamster, manifest liver lesions do not
occur even after fatal doses; however, the ED^Q for increased hepatic weight is
only -15 ug/kg (Gasiewicz et al., 1986). Liver related enzyme activities in
serum are elevated in those animal species where liver damage is a prominent sign
of TCDD toxicity. In those animal species where hepatotoxicity is not as
apparent, such as monkeys and guinea pigs, these enzyme activities are nearly
normal.
Thymic atrophy has also been found in all animal species given lethal doses
of TCDD. Treatment of animals with TCDD inhibits the bone marrow hematopoiesis
in mice, both in vivo and in vitro, by directly altering the colony growth
efficiently of stem cells (Chastain and Pazdernik, 1985; Luster et al., 1980,
1985).
Among other signs and symptoms that have been demonstrated in various
species, the following should be noted: hepatic porphyria, hemorrhages in
various organs, testicular atrophy, reduced prostate weight, reduced uterine
weight, increased thyroid weight, lesions of the adrenal glands, inhibited bone
marrow hematopoiesis, decreased serum albumin and increased serum triglycerides
and free fatty acids). Details of all underlying studies for these observations
have been extensively reviewed (U.S. EPA, 1984, 1985; WHO/IPCS, 1989).
Effects on heart muscle have also been observed in guinea pigs and rats
(Brewster et al., 1987; Kelling et al., 1987; Canga et al., 1988). Five days
after a single dose of TCDD (10 jug/kg intraperitoneally). a significantly
decreased beta-adrenergic responsiveness was observed in the right ventricular
papillary muscle of guinea pig (Canga et al., 1988). In the TCDD-treated animals
a decrease in the positive inotropic effects of isoproterenol at 0.03-0.3 pM, but
not at 0.1-10 nM. was also demonstrated. Additionally, the responsiveness to
low-frequency stimulation and to increases in extracellular calcium were enhanced
in these animals. Based on these findings, the authors suggested that the heart
may be a major target for TCDD toxicity.
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In the monkey, several additional symptoms have been registeredj, such as
periorbital edema, conjunctivitis and thickening of the Meibomiarj glands,
followed by loss of the eyelashes, facial hair and nails (McConnelj et al.,
1978a). These are symptoms similar to those which have been observed in cases
of human intoxications (e.g., occupational exposure, the Seveso incident and the
Yusho and Yu-Cheng toxic oil intoxications (the latter involving exposure to PCBs
and CDFa; see chapter 1)
I
3.2.2. Studies In Vitro. Over 30 cell types, including primary cultures and
cello from established and transformed cell lines derived from various tissues
of at least six animal species, have been examined for their general cellular
responses to TCDD (Beatty et al., 1975; Knutson and Poland, 1980a; Hijra et al.,
1975; Yang et al., 1983a). The effects studied were changes in viability, growth
rate and morphology. Overall, there have been few or no effects documented.
However, other in vitro studies using more specific endpoints ojE toxicity
I
have clearly indicated effects of TCDD at comparatively low concentrations. Thus,
several studies have shown that TCDD affects cultured epidermal keratinocytes
through interactions with differentiation mechanisms and that this effect may be
regulated by the modulation of EOF binding to the cells (Hudson et ajl., 1986).
Additionally, in epithelial cells of human origin, TCDD has been shown to alter
differentiation (Hudson et al., 1985) and AHH EROD activity has been jhown to be
induced in vitro (see Section 3.5.4).
Wiebel et al. (1991) have identified a cell line (H4IIEC3-derived 5L
r
hepatoma cells) which responds with decreased proliferation at| low TCDD
concentrations. Thus, half-maximum inhibition of proliferation occurs at a
concentration of 0.1-0.3 nM, and the onset of the effect is faijrly rapid,
manifesting itself as early as 4-8 hours after treatment. Further sjudies have
also demonstrated that insensitive variants of this cell line were deficient in
cytochrome P-4501A1 activity and also lacked measurable amounts j of the Ah
receptor (GBttlicher et al., 1990). In addition, 3,3',4,4'-TCB also inhibited
proliferation in the sensitive cell-line, albeit at higher concentrations.
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3.2.3. Appraisal. The numerous studies of acute toxicity in various species
have demonstrated dramatic species- and strain-specific differences in sensi-
tivity. However, the spectrum of symptom is in general agreement, although
species differences exist.
Lethality is typically delayed by several weeks, and there is a pronounced
wasting syndrome in almost all laboratory animals. Studies in congenic mice
differing in their Ah responsiveness indicate that the sensitivity to acute
toxicity of TCDD segregates with the Ah locus. Furthermore, studies on other
CDDs, CDFs and coplanar PCBs demonstrate that the potency for inducing lethality
correlates with their ability to bind to the Ah receptor. In contrast, studies
in various other species, as well as in various strains of rats, have
demonstrated a wide range of sensitivities regardless of rather comparable levels
of the Ah receptor.
3.3. SUBCHRONIC TOXICITY
The available studies on the subchronic toxicity of TCDD have been reviewed
by the U.S. EPA (1984, 1985) and WHO/IPCS (1989). Overall, the signs and
symptoms observed are in agreement with those observed after administration of
single doses.
The study of Kociba et al. (1976) is of special interest as it has been used
for comparisons of the relative toxicities of other CDDs/CDFs (Pliiess et al.,
1988). Adult male and female Sprzigue-Dawley rats, in groups of 12, were given
0, 0.001, 0.01, 0.1 and 1.0 fig TCDD/kg bw by gavage 5 days/week for 13 weeks.
At the end of the treatment period, five rats of each sex were sacrificed for
histopathological examination. The remaining animals were continued for post-
exposure observation. The highest dose caused five deaths among the females,
three during the treatment period and two after, while two deaths occurred in
males in the post-treatment period. The rats given 0.01 ^ig TCDD/kg did not
differ from the controls except for a slight increase in the mean liver-to-body
weight ratio.
A 13-week dietary study in Sprague-Dawley rats given 1,2,3,4,8-PeCDF,
1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF or 1,2,3,6,7,8-HxCDF demonstrated that the
subchronic toxicity and the depletion of hepatic vitamin A reduction followed the
rank order of the ability of the compounds to bind to the Ah receptor or cause
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induction of AHH, for example (Pliiess et al., 1988; Hakansson et al.f 1990).
However, the direct comparisons of the effects are hampered by the differences
in toxicokinetic behavior of the compounds. Slightly different relationships
I
with regard to toxicity were obtained in a tumor promotion study, j where an
initial loading dose (aubcutaneous) of 2,3,4,7,8-PeCDF was given, followed by
repeated lower doses (subcutaneous) in order to obtain a steady-state concentra-
tion (Warn et al., 1991a). However, both of these studies support the assumption
that most signs and symptoms obtained may be mediated through the Ah!receptor.
In another atudy, groups of eight female Sprague-Dawley rata wete exposed
to 16 weekly oral doses of 0, 0.01, 0.1, 1.0 and 10.0 /ig TCDD/kg bw iji a study
primarily aimed at investigating TCDD-induced porphyria (Goldatein et al., 1982).
The no-effect dose for porphyria was 0.01 jig/kg/week.
Only two atudies of limited value have been performed in mice ([Harris et
al., 1973; Voa et al., 1973). Four weekly oral doses of 0.2, 1, 5 or 25 pg
TCDD/kg bw were given to male C57B1/6 mice in corn oil. No effects were noted
at 1 ng, which corresponds to -0.1 pg/kg bw/day.
In male and female Hartley guinea pigs, a 90-day feeding study ojf TCDD has
been performed by DeCaprio et al. (1986) where extenaive pathology, ihematology
and serum chemistry on aurviving animala were performed. The dieta contained 0,
2, 10, 76 or 430 ng TCDD/kg. The two loweat doaea, 2 and 10 ng/kp of diet,
produced no dose-related alteration. Baaed on thia atudy a no-observed-effeet
level of 0.6 ng TCDD/kg bw/day in guinea piga was eatimated. At tjhe highest
dose, severe body weight losaea and mortality were obaerved. No dcjae-related
mortality occurred at 76 ng/kg. j
A cumulative doae of 0.2 jig TCDD/kg bw, which waa divided into nine oral
signs of
Signs of
doses 3 times/week during days 20-40 of gestation, produced no clinic
toxicity in pregnant rheaua monkeya (Macaca mulatta) (McNulty, 1984)
toxicity such as body weight loss, epidermal changes and anemia occurred in thoae
monkeys who had received cumulative doses of 1.0 and 5.0 /jg TCDD/kg bw over the
same time period. j
3.3.1. Apprmi.ml. Utilizing the above data, subchronic NOAELs forj rats, mice
and guinea pigs are estimated 0.01 pg, 0.1 /jg and 0.6 ng TCDD/kg bw/day,
respectively. However, these studies cannot be directly compared; with each
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other. Furthermore, none of the studies have utilized initial loading doses, and
due to the long half-life of TCDD, steady states may not been reached in the
animals except at the very end of the study periods. Distribution between
tissues in the animals depends on both time of exposure and the dose levels (see
Chapter 1), which further complicates any comparisons.
Irrespective of this, the limited data available seem to indicate that signs
and symptoms of subchronic toxicity follow the same rank order as Ah receptor-
mediated effects, such as induction of AHH.
3.4. CHRONIC TOXICITY
The results of chronic toxicity studies performed on laboratory animals
exposed to TCDD are summarized in Table 3-3. Details have been reviewed by the
U.S. EPA (1984, 1985) and WHO/IPCS (1989).
The most important study in rats is the chronic study of Kociba et al.
(1978, 1979). Groups of 50 male and 50 female Sprague-Dawley rats were fed diets
providing daily doses of 0.001, 0.01 and 0.1 ng TCDD/kg bw for 2 years. Control
rats, 86 males and 86 females received diets containing the vehicle alone.
Increased mortality was observed in females given 0.1 /jg/kg/day, while increased
mortality was not observed in male rats at this dose, or in animals receiving
doses of 0.01 or 0.001>g/kg/day. From month 6 to the end of the study, the mean
body weights of males and females decreased at the highest dose and, to a lesser
degree, in females given 0.01 jug/kg/day. During the middle of the study, lower-
than-normal body weights were also occasionally recorded in the low-dose group,
although during the last quarter of the study, the body weights were comparable
to those of the controls.
Increased urinary coproporphyrin and uroporphyrin were noted in females, but
not in males, given TCDD at a dose rate of 0.01 and 0.1 fjg/kg/day. Analyses of
blood serum collected at terminal necropsy revealed increased enzyme activities
related to impaired liver function in female rats given 0.1 pg TCDD/kg/day.
Necropsy examination of the rats surviving TCDD exposure to the end of the study
revealed that liver effects in the liver constituted the most consistent
alteration in both males and females. Histopathological examination revealed
multiple, degenerative, inflammatory and necrotic changes in the liver that were
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DRAFT—DO NOT QUOTE OR CITE
more extensive in females. Multinucleated hepatpcytes and bile-duct hyperplasia
were also noted. Liver damage was dose-related, and no effect was observable at
the low-dose rate. The NOAEL was estimated to be 0,001 /ug/kg/day. At the end
of the study, the fat and liver concentration of TCDD at this dose was 540 ppt.
In male Swiss mice, weekly oral doses of 0, 0.007, 0.7 and 7.0 pg TCDD/kg
bw for 1 year resulted in amyloidosis and dermatitis (Toth et al., 1979). The
incidence of these lesions was 0 of 38, 5 of 44, 10 of 44 and 17 of 43 in the
control-, low-, medium- and high-dose groups, respectively. The LOAEL in this
study was estimated to be 0.001 pg/kg/day.
In the NTP (1980) gavage study in B6C3F1 male and female mice, no adverse
effects were seen at the lowest dose tested (i.e., 0.01 and 0.04 pg/kg bw/week
for males and females, respectively, corresponding to -1.4 and 6 ng/kg bw/day.
The limited studies (9-20 months) available in rhesus monkeys (Allen et al.,
1977; Barsotti et al., 1979; Schantz et al., 1979) have revealed signs and
symptoms similar to those recorded in more short-term studies. Adverse effects
were noted down to the lowest dose tested (i.e., -2-3 ng/kg bw/day for 20 months
(Schantz et al., 1979).
3.4.1. Appraisal. From the different long-term studies on TCDD, it can be
estimated that the NOAEL for the rat is 1 ng/kg bw/day, corresponding to a fat
and liver concentration (NOEL) of 540 ppt. For the male Swiss mouse, effects
(dermatitis and amyloidosis in 5 of 44 animals) were noted at the lowest dose
tested (i.e., the LOEL would be 1 ng/kg bw/day). However, in B6C3F1 mice, NOEL8
of 1.4 and 6 ng/kg/day were obtained for males and females, respectively. The
studies in the rhesus monkey cannot be used for such a determination. Adverse
effects have been observed at the lowest dose tested, -2-3 ng/kg body weight.
3.5. SPECIFIC EFFECTS
3.5.1. Hasting syndroae. TCDD at high doses (lethal or near lethal) causes a
starvation-like or wasting syndrome in several animal species. In young animals
or following a sublethal dose to adults, this response is manifested as a
cessation of weight gain. Animals exposed to near lethal or higher doses
characteristically lose weight rapidly. Numerous studies utilizing pair-feeding,
total parenteral nutrition and everted intestinal sacs have been performed to
elucidate the mechanisms behind the wasting syndrome (U.S. EPA, 1984, 1985;
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DRAFT—DO NOT QUOTE OR CITE
WHO/IPCS, 1989), but no single explanation has been obtained so far. No
generalized impairment of intestinal absorption seems to occur. PeterJon et al.
(1984) has suggested a model for the TCDD-induced wasting syndrome whiclj is based
on the assumption that body weight in rats is regulated around anj internal
standard or a hypothalamically-programmed set point. Thus, body weight at a
given age is constantly being compared to this set point value, and i|f differ-
ences occur, feed consumption is adjusted. When TCDD lowers this set point,
reduction in food consumption results as the rat attempts to reduce its weight
to a new lower level. This hypothesis has been tested in several experiments
under carefully controlled feeding conditions. Repeated studies have
demonstrated that reduction of feed intake due to increased food spillage is
sufficient to account for the loss of body weight in TCDD-treated Sprague-Dawley
rats. Additionally, TCDD-treated rats maintain and defend their reduced weight
level with the same precision as ad libitum fed control rats defend their normal
weight level (Seefeld and Peterson, 1983, 1984; Seefeld et al., 198^a,b); the
percentage of the daily feed intake that is absorbed by the gastrointestinal
tract of TCDD-treated and control rats is similar (Potter et al., 1986; Seefeld
i
and Peterson, 1984). Hypophagia was the major cause of adipose and Ijsan tissue
loss in male Fischer 344 rats, C57B1/6 mice and albino guinea pigs whbn exposed
to a calculated LD80 dose of TCDD. Body weight loss followed a similar time-
course in TCDD-treated and pair-fed control animals of all three speciejs (Kelling
et al., 1985). Thus, body weight loss appears to contribute to lethality in a
species- and strain-dependent fashion, but weight loss appears to pla^ a greater
role in causing death in Sprague-Dawley rats and guinea pigs than |Lt does in
Fischer 344 rats and C57B1/6 mice. Loss of body weight and loss of appetite are
alBO prominent signs of thyroid dysfunction. However, some data indicate that
the effect of TCDD on thyroid hormones cannot explain the TCDD-induced decrease
in body weight gain.
TCDD-induced wasting is always accompanied by the loss of adipose tissue.
The rate of fat storage is determined by LPL, which controls the serum level of
triglycerides. Brewster and Matsumura (1984) found in guinea pigs tjiat the LPL
activity was decreased to 20% of the value of ad libitum fed controls after
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1 day, and this effect persisted throughout the study (10 days). Thus, the
authors suggested that TCDD irreversibly reduces adipose LPL activity, thus
making the animals less capable to adapt to nutritional changes and needs.
In a series of studies on Wistar rats, Lakshman et al. (1988, 1989, 1991)
have demonstrated that single intraperitoneal injections of TCDD (from 1 jjg/kg)
caused a dose dependent inhibition of fatty acid synthesis in the liver and the
adipose tissue. The adipose tissue was found to be more sensitive than, the
liver. Furthermore, they also found an increased mobilization of depot fat into
the plasma compartment accompanied by an increase in plasma free-fatty acid
concentrations.
In vitro studies in isolated heart-mitochondria have indicated that a TCDD
concentration of 1.5 nmol/mg mitochondrial protein affects oxygen activation
associated with cell respiration. Superoxide radicals and ^2°2 were indicated
to be involved in the development of the effects observed (Nohl et al., 1989).
Loss of muscle tissue accompanied by a decreased glucocorticoid receptor-
binding capacity and an increased glutamine synthetase activity have been
observed in male Fisher 344N rats given a single oral TCDD dose of 100 pg/kg (Max
and Silbergeld 1987).
Another biochemical effect noted in TCDD-induced wasting is the ability of
TCDD to decrease hepatic vitamin A storage in animals (Thunberg et al., 1979;
Hikansson et al., 1989b, 1991). Vitamin A is necessary for growth, and vitamin
A deficiency will result in depressed body weight gain as well as in reduced food
intake. However, in contrast to TCDD-treated animals, the vitamin A deficient
animals continue to eat and grow, though body weight gain is less than normal
(Hayes, 1971).
That decreased feed intake could be a result of a direct TCDD effect on the
brain was initially indicated by Pohjanvirta et al. (1989), but this has been
contradicted by later studies (Stahl and Rozman, 1990). The administration of
TCDD at 50 pg/kg intraperitoneally to male Sprague-Dawley rats caused a
significant decrease in the serum concentration of prolactin detectable after
4 hours, compared to pair-fed vehicle controls and noninjected controls (Jones
et al., 1987). The rapid onset of this effect suggested that it may be mediated
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by a pathway other than through interaction with the Ah receptor. Further
studies have demonstrated that the effect of TCDD was reversed by pimozide, a
doparaine receptor antagonist, and that the rate constant of dopamine depletion
after a-methyl-p-tyrosine, as well as the turnover rate, were significantly
elevated in the median eminence. This suggested a hypothalamic site JDf action
of TCDD in their experiments (Russell et al., 1988). j
Changes in intermediary metabolism have been demonstrated in TCD^-treated
expariraental animals. Conflicting data on effects on serum glucose and hepatic
glucogen levels have been reported earlier (WHO/IPCS, 1989). Several recent
studies have suggested that the ultimate cause of death in some mammalian species
may be caused by a progressive hypoglycemia (Ebner et al., 1988; Gorski and
Rozman, 1987; Gorski et al., 1990). However, in the guinea pig, serum glucose
levels were not affected by treatment of the animals with TCDD (Gasiewicz and
Neal, 1979). Slight reductions in serum glucose levels were noted in both Long
Evans and Han/Wistar rats (Pohjanvirta et al., 1989). Rozman et al. (1990) have
suggested that the subchronic and chronic toxicities of TCDD are related to the
inhibition of key enzymes of gluconeogenesis. They demonstrated that the
induction of appetite suppression starts is preceded by the inhibition! of PEPCK,
which caused a reduction in gluconeogenesis. This was followed by a progressive
increase in plasma tryptophan levels which was suggested to cause a serotonin-
mediated reduction of the feed intake. In Sprague-Dawley rats, TCDD in doses of
25 and 125 pg caused a rapid decrease (50%) in PEPCK activity 2 days after
dosing, which was followed by a dose-dependent decrease in glucose-6-phosphatase
activity 4 or 8 days after exposure. Both appetite suppression and reduced PEPCK
activity occurred in the same dose range (Weber et al., 1991). TCpD-induced
impairments of carbohydrate synthesis have also been suggested by ptudies in
chick embryos (Lentnek et al., 1991).
Numerous studies have measured serum levels of free fatty acids, cholesterol
and triglycerides in various species after TCDD-treatment (WHO/IPCS, 1989), but
no pronounced Qualitative differences have been observed between jspecies or
j
strains of mice. j
The wasting syndrome thus seems to be a generalized effect, elicited in all
species and strains, but at various dosages (single or repeated administration).
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Specific studies have not been performed to elucidate if this syndrome is
elicited through the interaction of TCDD with the Ah receptor. However, strong
support for an Ah receptor-mediated mechanism comes from studies with other CDDs
and CDFs. The binding affinities of various CDDs and CDFs to the Ah receptor as
well as those of related PCBs have been shown to strongly correlate with their
potency of induction of the wasting syndrome in both rats and guinea pigs (Safe,
1990).
3.5.2. Hepatotoxicity. TCDD induces hyperplasia and hypertrophy of parenchymal
cells and, thus, hepatomegaly in all species investigated, even at sublethal
doses. There is, however, considerable variation in the extent and severity of
this lesion among the species tested. Other liver lesions are more species
specific. Lethality following the administration of TCDD cannot be explained by
these liver lesions alone, although they may be a contributing factor, at least
in the rat and rabbit. The morphological changes in the liver are accompanied
by impaired liver function, which is characterized by liver enzyme leakage,
increased microsomal monooxygenase activities, porphyria, impaired plasma
membrane function, hyperlipidemia and increased regenerative DMA-synthesis (U.S.
EPA, 1984, 1985; WHO/IPCS, 1989).
The hepatotoxic reaction in various strains of rats given lethal doses of
TCDD is characterized by degenerative and necrotic changes, with the appearance
of mononuclear cell infiltration, multinucleated giant hepatocytes, increased
numbers of mitotic figures and pleomorphism of cord cells, increase in the
hepatic smooth endoplasmatic reticulum and parenchymal cell necrosis. The
histological findings are accompanied by hyperbilirubinemia, hypercholesterol-
emia, hyperproteinemia, increased SCOT and SGPT activities, further indicating
damaged liver function (WHO/IPCS, 1989). These lesions may be severe enough to
be a contributing factor in death. The lesions observed after sublethal doses
are qualitatively almost identical to those after lethal doses.
Earlier studies in mice have found similar effects. Recently, Shen et al.
(1991) reported a comparative study on the hepatotoxicity of TCDD in Ah
responsive and nonresponsive mice (C57BL/6J and DBA/2J, respectively). C57BL/6J
mice given a single dose of 3 /jg/kg TCDD developed mild to moderate hepatic lipid
accumulation but no inflammation or necrosis. Severe fatty change, mild
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DRAFT — DO NOT QUOTE OR CITE
inflammation and necrosis occurred at 150
DBA/2 J mice given 30
developed hepatocellular necrosis and inflammation but no fatty change. Lipid
accumulation was only slight after 600 pg/kg. The authors concluded thjat the Ah
i
locus may be involved in determining the steatotic effects of TCDD. j
The guinea pig shows less severe morphological alteration in the l|iver than
in other species. Likewise, the hamster exhibits little or no liver dajmage even
after a fatal dose, but liver lesions have been observed after prolonged periods
following the administration of nonlethal doses. j
Several parameters relating to disturbed hepatic plasma membranae! function
have been studied (U.S. EPA, 1984, 1985; WHO/IPCS, 1989). ATPase activities were
depressed, and protein kinase C activity was increased in rats, but not] in guinea
pigs, treated with TCDD (Bombick et al., 1985). TCDD also induced a dejcrease in
the binding of EGF. j
The relative doses of TCDD needed to suppress EGF binding to 5^% of the
control level were 1, 14 and 32 pg/kg for the guinea pig, the Sprague-Dawley rat
and the Syrian Golden hamster, respectively (Madhukar et al., 1984). j A single
intraperitoneal dose of 115 pg TCDD/kg bw decreased the EGF binding by 93.1, 97.8
and 46.0% in C57B1/6, CBA and AKR mice, respectively, 10 days after I treatment
I
(Madhukar et al., 1984). !
Further studies on the interaction of TCDD with EGF have been performed in
congenic mice of the strain C57BL/6J (Lin et al., 1991a,b). The ED50 for the
TCDD-induced decrease in the maximum binding capacity of the EGF receptor was 10
times higher in the Ah-nonresponsive mice, compared to the Ah-responsive animals.
This study supports the hypothesis that the effects of TCDD on EGF receptor
ligand binding may be mediated by the Ah receptor. j
The effects of TCDD on biliary excretion of various compounds have also been
studied. Of special interest are studies on the excretion of ouabaih, a model
compound for neutral nonmetabolized substrates such as estradiol, progesterone
and cortisol, which was depressed in a dose-related manner by a single j oral dose
of TCDD in rats (Yang et al., 1977, 1983b). The available data suggest that the
hepatic membrane transport of ouabain may be selectively impaired by TCDD.
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Peterson et al. (1979a,b) have indicated that changes in ATPase activities are
not responsible for the reduced ouabain excretion.
TCDD administration stimulates the accumulation of porphyrins in the liver
and an increase in urinary porphyrin excretion. Indeed, during manifest
porphyria, accumulation of porphyrins occurs not only in the liver but also in
the kidney and spleen of rats (Goldstein et al., 1982).
Contradictory results on species variations have been published. It seems
clear that porphyria can be produced in both mice and rats but the condition is
always the result of subchronic or chronic administration. Exposure to single
doses has not been demonstrated to produce porphyria. The mechanism underlying
the induction of porphyria is not elucidated. Cantoni et al. (1981) exposed rats
orally to 0.01, 0.1 and 1 fjg TCDD/kg bw/week for 45 weeks and increased
copropporphyrin levels were observed at all dose levels. A marked porphyric
state appeared only at the highest dose tested, after 8 months of exposure.
TCDD is a potent inducer of rodent and murine ALA-synthetase, the initial
and rate-limiting enzyme involved in heme synthesis. However, increased ALA-
activity was not found in mice exposed to 25 pg TCDD/kg bw/week for 11 weeks,
despite porphyria being evident (Jones and Sweeny, 1980). Thus, the induction
of ALA-synthetase does not seem to be a necessary event in TCDD-induced
porphyria. A more likely suggestion is that decreased hepatic porphyrinogen
decarboxylase is the primary event in porphyria induced by halogenated aromatics
(Elder et al., 1976, 1978). TCDD depresses this enzyme activity in vivo in the
liver of mice (Cantoni et al., 1984a,b; Elder and Sheppard, 1982? Jones and
Sweeny, 1980), but not in vitro (Cantoni et al., 1984b).
A comparative study of TCDD-induced porphyria has not been conducted in
responsive and nonresponsive mice. However, in a study on Ah responsive (Ah")
and Ah nonresponsive (Ahd) C57BL/6J female mice, the urinary excretion of
porphyrins was studied after treatment of the animals with hexachlorobenzene for
<17 weeks (Hahn et al., 1988). After 15 weeks of treatment with 200 ppm
hexachlorobenzene in the diet, the excretion of porphyrins was 200 times higher
in the Ah** mice, compared to controls. In contrast, the Ah" mice only showed a
6-fold increase. Induction of P-450c(lAl) was observed only in Ahb mice, while
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i
induction of P-450d(lA2) was observed in both strains, but to a lesser Degree in
the Ahd mice.
3.5.3. Epidermal Effect*. Chloracne and associated dermatological changes are
widespread responses to TCDD in humans. However, this type of toaiicity is
expressed only in a limited number of animal species (i.e., rabbits, monkeys and
i
hairless mice).
In the rabbit ear bioassay, a total doses of 80 ng TCDD gave a chloracne-
genic response, while no response was obtained when the total dose applied to the
ear was 8 ng (Jones and Krizek, 1962; Schwetz et al., 1973). The application of
TCDD in various vehicles has been demonstrated to markedly decrease this response
(Poiger and Schlatter, 1980). The hairless mouse is a less sensitivejmodel for
chloracnegenic response than is the rabbit ear bioassay (Knutson and Poland,
1982; Puhvel et al., 1982). However, following repeated applications of -0.1 fjg
TCDD over several weeks, an acnegenic response was noted in the hairless mouse
strains, SkH;HRl and HRS/J. An acnegenic response was also caused b| repeated
applications of 2 mg 3,4,3',4'-TCB (Puhvel et al., 1982). Female HRS/J hairless
mice have also been used to test the dermal toxicity and skin tumor promoting
activity of TCDD, PeCDF and HxCDF (Hebert et al., 1990a). All of the tested
compounds induced coarse, thickened skin with occasional desquamation; these
effects were more severe after the application of PeCDF and HxCDF.
Keratinocytes, the principal cell type in the epidermis, have been utilized
as an in vitro model for studies of TCDD-induced hyperkeratosis both in human-
and animal-derived cell cultures. The response to TCDD is analogous to the
hyperkeratinization observed in vivo.
A TCDD-induced keratinization response in vitro was first demonjstrated in
a keratinocyte cell line derived from a mouse teratoma (XB cells). The
keratinization was dose-related (Knutson and Poland, 1980b). Late passage XB
cells (termed XBF cells) lost their ability to respond by keratinizjation upon
TCDD treatment. Both XB-cells (keratinization assay) and XBF-cells (flat-cell-
assay) have proven to be useful in in vitro bioassays to determine the "dioxin-
like" activities of both environmental samples and of pure isomers (Qierthy and
Crane, 1985a,b; Gierthy et al., 1984). j
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Several continuous lines of human keratinocytes, derived from neonatal
foreskin or squamous cell carcinomas, have been shown to respond to TCDD in nmol
concentrations with a variety of signs indicating alterations in the normal
differentiation program (WHO/IPCS, 1989). The responses include decreased DNA
synthesis, decreased number of proliferating basal cells, decreased binding of
EGF and an increase in the state of differentiation (Osborne and Greenlee, 1985;
Hudson et al., 1986). The responses were also obtained with TCDF but not with
2,4-diCDD (Osborne and Greenlee, 1985). TCDD has also been shown to inhibit
high-density growth arrest in human squamous carcinoma cell lines, and, indeed,
the minimum concentration for increases in cell proliferation was 0.1 nM in the
most sensitive cell line (SCC-15G). In studies on the same cell lines a
modulating effect of the transforming growth factor beta could not be
demonstrated (Hebert et al., 1990 b,c).
3.5.4. Enzyme induction. TCDD has repeatedly been found to increase the
activities of various enzymes. While observations of enzyme inhibition have also
been made, enzyme induction has been one of the most extensively studied
biochemical responses produced by TCDD. The MFO system is the most thoroughly
investigated, and AHH and EROD (as markers for CYP1A1 induction) are the most
frequently assayed enzyme activities. The induction of MFO activities might
potentiate the toxicity of other foreign compounds requiring metabolic
transformation by the MFO system before they can exert their toxic effect.
Furthermore, increased MFO activities might adversely affect important metabolic
conversions of endogenous compounds. TCDD has also been reported to affect a
variety of other enzymes (e.g., UDPGT and GST), which are multi-functional enzyme
systems involved in conjugating a wide variety of structures and play a key role
in biotransformation and detoxification of endogenous and exogenous compounds.
Several investigators have studied the relative potency of various
halogenated dioxins, dibensofurans and biphenyls to induce AHH and/or EROD
activities (Safe, 1990). An apparent structure-activity relationship was found
between the location of the halogen atoms on the dibenzo-p-dioxin molecule and
the ability to induce AHH activity both in vivo and in vitro. Isomers with
halogens at the four lateral ring positions produced a greater biological
response than those with halogens at three lateral ring positions, while two
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lateral halogen atoms seemed to be insufficient to produce a biological response.
Numerous studies have indicated that there is very good agreement between the Ah-
binding affinity of various CDDs, CDFs and related PCBs and their potency to
induce AHH, both in vivo and in vitro (Safe, 1990). Structure-activity studies
have also demonstrated clear correlation between the toxicity and induction
potency of a series of CDDs, CDFs and coplanar PCBs (Poland and Glover, 1973;
Safe, 1990). j
On a molecular basis, TCDD is the most potent MFO-inducing compound known,
and MFO induction seems to be the most sensitive biochemical response produced
by it. The measurement of the induction of AHH or EROD (mediated through CYP1A1)
are considered to be very sensitive markers of the TCDD-induced enzyme induction.
According to Kitchin and Woods (1979), induction in the rat takes place at doses
as low as 0.002 pg TCDD/kg bw. The NOEL for a single administration to rats
seems to be 1 ng/kg, while a single dose of 3 ng/kg causes a detectable induction
of AHH or EROD (Kitchin and Woods, 1979; Abraham et al., 1988).
Enzyme induction has also been observed in the offspring of variojs species
after prenatal and postnatal (milk) exposure to TCDD (Lucier et al., 1975; Korte
@t al., 1990; Warn et al., 1991b). j
The effect of TCDD on enzyme activities has been most frequently investi-
gated in the rat (WHO/IPCS, 1989). In the liver, TCDD has been shown to increase
both the contents of cytochrome P-4501A1 and cytochrome P-4501A2, as well as
other raicrosomal enzyme activities involved in the oxidative transformation and
conjugation of xenobiotics (e.g., aniline hydroxylase, AHH, biphenyl hydroxylase,
ECOD, EROD and UDPGT) (U.S. EPA 1984, 1985; WHO/IPCS 1989).
TCDD also affects some other hepatic enzymes not related to the MFO system,
including aldehyde dehydrogenase B-aminolevulinic acid synthetase, DT-diaphorase,
transglutaminase, ornithine decarboxylase, transaminases (ALT and AST), plasma
membrane ATPases, porphyrinogen carboxylase, prostaglandin synthetasje, enzymes
involved in testosterone metabolism and RNA polymerase (U.S. EPA, Ij984, 1985;
WHO/IPCS 1989).
Studies in different species have revealed that enzyme induction, due to
TCDD exposure is also both a species- and strain-specific phenomenon. Pdrjawirta
et al. (1988) studied enzyme induction in the Long-Evans and Han/Wistar (Kuopio)
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rat strains (LD50, -10 and >3000 /jg, respectively). Differences in the
inducibility of EROD, ECOD or ethylmorphine N-demethylase were not found, nor
were there any differences as regards the amount of available Ah receptor or the
amount of cytochromes P-450 in the hepatic microsomal fractions. Similarly,
differences regarding possible induction of UDPGT were absent (Pohjanvirta et
al., 1990).
Enzyme induction studies on mice have been performed mainly with strains
which are genetically different at the Ah locus, thus making them responsive or
nonresponsive to the induction of hepatic cytochrome P-4501A1 related enzyme
activities. Qualitatively and in general, the same responses can be obtained in
both strains, but there may be more than one order of magnitude difference as
regards the doses required to elicit a response. TCDD is thus 10-fold more
potent in inducing hepatic cytochrome P-4501A1 and the related AHH activity in
C57BL/6J mice (Ah-responsive) than in DBA/2 mice (Ah-nonresponsive) (Poland and
Knutson, 1982; Nebert, 1989).
The guinea pig, although it is the species most sensitive to the toxic
effects of TCDD, does not respond to the administration of TCDD with liver
toxicity nor with extensive enzyme induction. Indeed, even at lethal doses, the
induction of MFO is only very slight (Beatty and Neal, 1977; Hikansson et al.,
1992).
The data on enzyme induction in rabbits are rather limited and also somewhat
conflicting as regards increases in cytochromes P-450 (Hook et al., 1975; Liem
et al., 1980).
Similarly, hepatic enzyme induction has only been partially studied in
Syrian Golden hamsters. When hamsters were given a lethal dose of TCDD,
increased hepatic GST and glutathione reductase activities were found. The £050
values for the induction of hepatic ECOD and reduced NADP: menadione oxidoreduc-
tase activities and cytochrome P-450 content in male Syrian Golden hamsters were
1.0, 2.0 and 0.5 pg TCDD/kg bw, respectively (i.e., extremely low doses, compared
to doses that produce tissue damage and lethality in this species) (Gasiewicz et
al., 1986).
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In a comparative study on EROD induction in guinea pigs, rats, C57BL/6 and
DBA/2 mice, as well as Syrian Golden hamsters, the animals were given single
dose» that were intended to be equitoxic (i.e., 1, 40, 100, 400 and 400 pg
TCDD/kg, respectively) compared with the acute toxicity for the respective
species and strain. EROD induction was noted in all species exceptj for the
hamster. During the observation period (112 days), the-EROD induction dropped
to more or less normal values in all rats and mice, while the inductiop (albeit
low compared to the other species) was sustained for the whole peripd in the
I
guinea pig (Hakansson et al., 1992). j
The N-demethylation of caffeine has been applied as & noninvasive method for
studying enzyme induction in vivo. Studies on the marmoset monkey (Callithrix
jacchus) utilizing 14C-labeled caffeine and measuring 14CO2 exhalation by a
breath test has indicated a NOEL of 1 ng/kg and a LOEL of 3 ng/kg (Kruger et al.,
1990). Although the authors stated that the N-demethylation of caffeine probably
was P-4501A1 dependent, studies by Butler et al. (1989) indicate fhat this
j
reaction is dependent on cytochrome P-4501A2. j
in the chick embryo, both AHH and 6-aminolevulinic acid synthetase have
been reported to be extremely sensitive to the inductive effects of TCDD and
related compounds (Poland and Glover, 1973; BrunstrSm and Andersson, 1988;
BrunstrSra, 1990). \
Although TCDD is relatively nontoxic in cell cultures, it is a very potent
inducer of AHH or EROD activities in systems, including lymphocytes and primary
hepatocytes, as well as established and transformed cell lines. !
The ED50 values for AHH-induction by TCDD have been studied in 11
established cell lines and in fetal primary cultures from five animal species and
cultured human lymphocytes and ranged from 0.04 ng/mL medium in C57B1/6 mouse
fetal cultures and 0.08 ng/mL in the rat hepatoma H-4-II-E cell line to >66 ng/mL
in the HTC rat hepatoma cell line (Niwa et al., 1975). Several cultured human
cells or cell lines have been shown to be inducible for AHH activity by TCDD
[e.g., lymphocytes (Atlas et al., 1976), squamous cell carcinoma lines (Hudson
et al., 1983), breast carcinoma cell lines (Jaiswal et al., 1985) and ijymphoblas-
toid cells (Nagayama et al. 1985)]. j
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TCDD was demonstrated to be the most potent AHH inducer of 24 chlorinated
dibenzo-p-dioxin analogues (Bradlaw et al., 1980) in a rat hepatoma cell culture
(H-4-II-E), which is extremely sensitive to AHH induction. The EC50 values for
AHH- and EROD-induction in the same cell system varied over seven orders of
magnitude for 14 different CDDs, the most potent being TCDD and the least potent
being 2,3,6-triCDD (Mason et al., 1986).
The feasibility of using in vitro EROD induction to determine dioxin-like
activities of environmental samples has been demonstrated by several studies
(Zacharewski et al., 1989; Hanberg et al., 1991). However, in environmental
samples there exists a variety of compounds which bind to the Ah receptor. Some
of them might act as antagonists to the binding of CDDs and CDFs and, thus, give
an erroneous result. The synthetic compound, 6-methyl-l,3,8-trichlorodibenzo-
furan, has been shown to inhibit the binding of TCDD to the Ah receptor and to
antagonize the induction of both P-4501A1 and P-4501A2 in the rat (Astroff et
al., 1988).
3.5.5.1. Appraisal. Based on the data from Kitchin and Woods (1979),
Abraham et al. (1988) and Kruger et al. (1990), Neubert (1991) has calculated
NOEL values for enzyme induction in both rats and marmoset monkeys to a single
dose of 1 ng/kg bw. At this dose, the tissue concentrations for both species
were found to be 4 ppt for adipose tissue and 3 ppt for the liver. It is
interesting to note that the wide range of sensitivities towards the acute
toxicity of TCDD is also reflected in a wide range of sensitivities for enzyme
induction both in vivo and in vitro. However, it is evident that the guinea pig
is fairly insensitive to enzyme induction, while the hamster is highly sensitive
in this respect.
Finally, it is evident that the structure-activity relationships revealed
from in vitro testing correlate fairly well with in vivo studies within a given
species or strain.
3.5.6. Endocrine Effects. Alterations to endocrine regulation have been
suggested from human exposure to TCDD that resulted in hirsutism and chloracne.
Chronic exposure to TCDD causes impaired reproduction in experimental animals
possibly by interfering with the estrus cycle in combination with some steroid-
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i
like actions of TCDD. This has prompted studies on the interaction of TCDD with
steroid hormones and their receptors. |
Increased systemic levels of glucocorticoids may mimic some of the j symptoms
of TCDD-toxicity (e.g., involution of lymphoid tissues, edema and mobilization
of fatty acids from adipose tissues). Thus, TCDD has been suggested to increase
glucocorticoid activity through indirect effects on glucocorticoid receptors.
Poland et al. (1976) have demonstrated that cortisol and synthetic gltjicocorti-
coids did not bind to the TCDD receptor. j
Conflicting data have been reported on TCDD-induced levels of glxicocorti-
coids. However, significant changes to the liver cytosolic glucocorticoid
receptor were induced by TCDD at doses 10,000 lower in adrenal ectomizedl Sprague-
Dawley rats, compared to control rats (Sunahara et al., 1989). The data
furthermore indicate that it is the binding properties of the receptor, that are
affected rather than the amount of receptor protein. Studies in congenljc strains
of Ah responsive and Ah nonresponsive C57BL/6J female mice (Goldsteih et al.,
1990; Lin et al., 1991a,b) have also demonstrated that TCDD decreased trie maximum
binding capacity of the hepatic glucocorticoid receptor in both strains of mice
by -30%. Differences in dose-response curves between the different strains could
not be observed. These data suggests that this effect may be mediated by a
pathway different from that mediated by the Ah receptor. |
Steroids are endogenous substrates for the hepatic MFO system. TGDD, which
influences the activity of this enzyme system, may thus alter steroid metabolism
in vivo and, consequently, also the magnitude of steroid mediated functions.
Early studies also reported contradictory data on changes in steroid levels.
However, Umbreit and Gallo (1988) suggested that estrogen receptor modulation and
the animal's physiological response to this modulation can explain some of the
toxicity observed in TCDD-treated animals. The susceptibility of jdifferent
species to TCDD correlates, to some extent, with their steroid glucuronidation
capacity. Thus, hamsters have low steroid UDPGT activity while guinea! pigs have
a corresponding high activity. Another example is given by comparing the
Sprague-Dawley (S-D) and Gunn rat, the latter being defective in producing some
UDPGTs. The homozygous Gunn rat is 3-10 times more resistant to effects of TCDD
than is the S-D rat (Thunberg, 1984; Thunberg and Hakansson,-1983). However, the
i
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results of TCDD exposure in various species and strains are complex. In order
to counteract the TCDD-induced modulation of the estrogen receptor, the effects
observed will be dependent on the ability of the organism to synthesize and
excrete estrogens. Interactions of TCDD and related compounds with estrogen have
recently been reviewed by Safe et al. (1991).
The importance of estrogens as modulators of TCDD-induced toxicity has also
been demonstrated by Lucier et al. (1991), who found that the tumor-promoting
effects of TCDD could be effectively prevented by removing the ovaries from
female rats before exposure to TCDD. This finding agrees well with the results
obtained from the long-term bioassays that demonstrated liver tumors only in
female rats (Kociba et al., 1978; NTP, 1982).
In studies on coiigenic strains of Ah-responsive and Ah-nonresponsive
C57BL/6J female mice, a statistically significant difference in the responsive-
ness of the hepatic estrogen receptor was found, thus indicating that the Ah
receptor regulates the effects of TCDD on the binding of estrogen to the hepatic
estrogen receptor (Goldstein et al., 1990; Lin et al., 1991a,b).
TCDD-induced changes in levels or activities of testosterone or its
metabolites have been reported from several studies (Keys et al., 1985; Mittler
et al., 1984; Moore and Peterson, 1985). The data do not, however, allow for any
conclusions with regard to the possible relationship to receptor-mediated
toxicity. TCDD induces several enzymes related to testosterone metabolism, which
has suggested that the changes observed may be secondary to the induction of
various enzymes. Serum testosterone and dihydrotestosterone were found to be
dose-dependently depressed by TCDD treatment in male Sprague-Dawley rats, when
compared to pair-fed and ad libitum fed controls. The ED50 for this effect was
-15 /i/g/kg (Moore et al., 1985). It was further shown that testosterone
synthesis was decreased in the animals due to depressed production of pregneno-
lone by the testis (Kleeman et al., 1990). In the same strain of rats, a single
oral dose of TCDD of 100 pg/kg was found to cause a 55% decrease in testicular
cytochrome P-ASO^ activity but also to cause the inhibition of the mobilization
of cholesterol to cytochrome P-450^. The authors concluded that the latter
effect probably was responsible for the inhibition of testicular steroidogenesis
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T3 and TSH
(Moore et al., 1991). Despite this, the effects noted occur after exposure to
largo amounts of TCDD. In contrast, maternal exposure to TCDD has been shown to
affact the male reproductive system at much lower doses (i.e. the lowest dose
tested was 64 ng/kg) (Mably et al., 1991, 1992a,b,c) (see Chapter 5)|.
In-ovo exposure of white Leghorn chickens to TCDD in the dose range of
1-10,000 pmol/egg increased the cardiac release of prostaglandins (Quilley and
Rifkind, 1986). Studies on chick embryos have indicated that the TCDD-induced
induction of cytochromes P-450 species results in a major increase in the NADPH-
dependent metabolism of arachidonic acid (Rifkind et al., 1990). These effects
are thus clearly related to the receptor mediated enzyme induction.
Rather conflicting data have been published regarding TCDD-induced effects
on thyroid hormones (WHO/IPCS, 1989). The available data on serum T4,
levels are not sufficient to state whether or not TCDD-treated; rats are
functionally hypothyroidic, euthyroidic or hyperthyroidic. !
However, Brouwer (1987) has demonstrated that a "dioxin-like" |PCB (i.e.,
3,4,3',4'-TCB, through a rapidly produced metabolite, 5-OH-TCB) birds to TTR.
This binding causes interactions with the physiological functions of TTR and
i
thyroid hormone transport is severely affected. This finding may explain some
of the characteristic toxicological lesions found after PCB exposure.
I
3.5.7. Vitamin A Storage. Decreased hepatic vitamin A storages has been
reported in animals exposed to various chlorinated aromatic compounds. TCDD is
unique in its ability to reduce the vitamin A content of the liver, both
regarding the minute quantities needed to produce this effect and the persistence
of the effect. A single oral dose of 10 fig TCDD/kg bw decreased bot^i the total
amount and the concentration of vitamin A in the liver of adult male Sprague-
Dawley rats (Thunberg et al., 1979). The decrease was evident 4
dosing and progressed with time. After 8 weeks, the treated animals
liver vitamin A content corresponding to 33% of that of controls.. Decreased
dietary intake of vitamin A could not account for this difference. A significant
increase in the UDPGT activity was observed, suggestive of an increased excretion
of glucuronide conjugated vitamin A. However, no correlation between the UDPGT-
days after
had a total
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activity and the hepatic vitamin A reduction was seen when homozygous Gunn rats
lacking inducible UDPGT (Aitio et al., 1979) and heterozygous Gunn rats, with
inducible UDPGT, were treated with a single, oral dose of 10 /^g TCDD/kg bw
(Thunberg and Hakanseon, 1983).
In a study combining pair-feed restriction and a single TCDD treatment, it
was found that the decreases in liver reserves of vitamin A were not related to
a decreased intake of vitamin A via the diet (Hakansson et al., 1989a).
Puhvel et al. (1991) reported a comparative study in which congenic haired
(+/+) and hairless (hr/hr) HRS/J mice were fed a vitamin A-deficient diet and
treated topically with TCDD. The sensitivity to TCDD-induced cutaneous changes
was essentially 100 times higher in hairless mice than in haired mice (0.01 and
1.0 pg 3 times/week for 3 and 2 weeks, respectively). In the haired phenotype,
effects of vitamin A depletion by itself were not seen by cutaneous histology,
nor were any changes in cutaneous morphology attributable to TCDD observed. In
the hairless mice, however, vitamin A deficiency increased the keratinization of
dermal epithelial cysts and increased the sensitivity of these cysts to TCDD-
induced keratinization. Analysis of vitamin A demonstrated that TCDD-exposure
did not affect cutaneous levels of the vitamin but did significantly lower liver
levels of vitamin A. TCDD-induced body weight loss and atrophy of the thymus
glands was not affected by the vitamin A status in either strain.
In a study on tumor promotion by TCDD, utilizing the induction of enzyme
altered hepatic foci in the liver and performed on female S-D rats, Flodstrom et
al. (1991) found that vitamin A deficiency by itself enhanced foci development.
The effect of TCDD treatment was also markedly enhanced, as were other TCDD-
induced toxicities including thymus atrophy.
Several studies have been performed to elucidate the mechanism of TCDD-
vitamin A interaction. Hakansson et al. (1989c) and Hakansson and Hanberg (1989)
have demonstrated that TCDD specifically inhibits the storage of vitamin A in
liver stellate cells. Brouwer et al. (1989) demonstrated that a single dose of
TCDD (10 pg/kg) to female S-D rats reduced vitamin A in the liver, the lung, the
intestines and the adrenal glands while increasing its concentration in the
serum, the kidneys and the urine. They also found a 150% increase in the free
fraction of serum retinol binding protein. Taken together, all of these data in
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the rat indicate that TCDD induces an increased mobilization of vitamin A from
hepatic and extrahepatic storage sites into the serum which is accompanied by an
enhanced elimination of the vitamin via the kidney into the urine.
In a comparative study of TCDD toxicity in male S-D rats and Hartley guinea
pigs (Hakansson et al. (1989b), the animals were given single intraperitoneal
dose* of 40 and 0.5 /^g/kg bw, respectively (i.e., comparable fractions of their
respective LD50). In these species there were similar reductions in hepatic
vitamin A, while serum and renal vitamin A concentration were increased in the
rat, but unaffected in the guinea pig. Hepatic EROD activity wals markedly
increased in the rat but unchanged in the guinea pig. Furthermore, although rats
seemed to recover from the wasting, thymic atrophy and liver enlargement, and
resumed their ability to store vitamin A in the liver at 4-8 weeks after
exposure, no such trends for wasting and vitamin A storage were observed in
guinea pigs, even 16 weeks after exposure. A complementary study also included
alao C57BL/6 mice, DBA/2 mice and Syrian Golden hamsters (Hakanss|on et al.,
i
1991). The effects on TCDD-induced decrease of vitamin A in the liver and the
lung correlated reasonably well with other toxic symptoms observed in the
animals. On the other hand, studies on two strains of rats, Long-Evans and
Han/Wistar (the Han/Wistar being >300 times more resistant to TCDD toxicity)
could not demonstrate significant differences in the TCDD-induced changes in
vitamin A in the liver, the kidney, the testicles or the serum after a sublethal
dose (4 pg/kg) (Pohjanvirta et al., 1990). These findings show that the
correlations between TCDD-induced lethality and changes in vitamin A status found
j
among other species apply to these strains of rats. I
The interaction of 3,4,3•,4'-TCB with vitamin A has been studied by Brouwer
and Van der Berg (1983, 1984, 1986), Brouwer et al. (1985, 1986a,b) and Brouwer
(1987). The effects of TCB on vitamin A differs in many respects from those of
TCDD. TCB is rapidly converted in vivo into a polar 5-OH-TCB metabolite, and
consequence
and thyroid
The model
this metabolite binds with a relatively high affinity to TTR. As a
of this interaction, the physiological functions of TTR in retinoid
hormone transport are severely affected in TCB exposed animals.
proposed by Brouwer (1985) may explain some of the characteristic tpxicological
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lesions related to PCB exposure. This mechanism of action seems to be clearly
separated from the Ah receptor mediated toxicity of CDDs and CDFs. Hydroxylated
metabolites of TCDD have also been demonstrated to bind in a similar manner to
TTR (Lans et al., 1992). However, due to the very slow metabolism of TCDD (or
other 2,3,7,8-substituted CDDs/CDFs), this mechanism of toxicity probably plays
a very minor role in the toxicity.
Taken together, these data indicate that TCDD interferes with the storage
mechanism for vitamin A. As supplementation of dietary vitamin A seems to be
unable to counteract all of the observed toxic effects, this would imply either
that the effect on vitamin A storage is secondary to TCDD-toxicity or that the
cellular utilization of vitamin A is affected by TCDD. On the other hand, a
dioxin-like PCB such as 3,4,3' ,4'-TCB seems to deviate with regard to this
mechanism of action.
3.5.8. Lipid Poroxidation. Lipid peroxidation and oxidative stress have been
indicated as a factor that affects the acute toxicity of TCDD (WHO/IPCS, 1989;
Wahba et al., 1989a,b, 1990a,b; Pohjnavirta et al., 1989; Alsharif et al., 1990;
Stohs et al., 1990). Among the effects noted have been membrane lipid peroxida-
tion, decreased membrane fluidity and increased incidence of single strand breaks
in DNA. Studies relating these observations to the Ah receptor have not been
performed. However, when considering the available data on TCDD and lipid
peroxidation, it is not possible to attempt to define a relationship between
lipid peroxidation and TCDD-induced lethality.
3.6. MECHANISMS OF TOXICITY
Despite extensive research to elucidate the ultimate event(s) underlying the
toxic action of TCDD, definite information is not yet available. The toxicity
of TCDD apparently depends on the fact that the four lateral positions of the
molecule are occupied by chlorine. Toxicity decreases with decreasing lateral
substitution and increasing total chlorine substitution. TCDD toxicity involves
many different types of symptoms; these symptoms vary from species to species and
from tissue to tissue, both quantitatively and qualitatively. Furthermore, age
and sex related differences in sensitivity have been reported. A characteristic
of TCDD toxicity is also the delay required to manifest toxicity (from 2 weeks
to 2 months) which is seen in all species.
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Polymorphism in the Ah locus, which has been suggested to be the [structural
gene for the cytosolic receptor, seems to determine the sensitivity of
genetically different strains of mice to TCDD and congeners. Ah-fresponsive
strains of mice (e.g., C57B1/6) are characterized by high hepatic levels of the
TCDD-receptor protein, highly elevated levels of hepatic cytochrome P^SOlAl and
associated enzyme activities, in response to treatment with 3-MC, and sensitivity
to the ulcerative action of DMBA on the skin. Ah-nonresponsive mice (e.g.,
DBA/2) lack these characteristics. Based on these findings, several genetic
studies have been performed to elucidate the role of the receptor in TCDD-
toxicity. In contrast to 3-MC, TCDD induces AHH activity and several toxic
effects both in Ah-responsive and Ah-nonresponsive strains of mice. However, the
dose required to produce the effect in an Ah-nonresponsive strain :LS approxi-
mately 10-fold greater than that needed in a responsive strain. Thijs indicates
that the Ah-nonresponsive strain also contains the TCDD-recepto:: but this
receptor is defective (Okey and Vella, 1982). Data from studies of DBA/2 mice
I
given either single or multiple doses of TCDD (Jones and Sweeney, 1980; Smith et
al., 1981) suggest that the LD50 in this strain of mice is at least 5-fold
greater than the values recorded for the C57B1/6 and C57B1/10 strains (Jones and
Greig, 1975; Smith et al., 1981; Vos et al., 1974). TCDD-induced hepatic
porphyria has also been shown to segregate with the Ah locus in mice (Jones and
I
Sweeney, 1980). The correlative differences between the C57B1/6 and DBA/2
strains of mice, in terms of altered specific binding of TCDD and sensitivity to
this compound, may be unique and may not be applicable to other species
(Ganiewicz and Rucci, 1984). In a genetic crossing experiment between Long-Evans
and Han/Wistar rats (Pohjanvirta, 1990), it was demonstrated that the Fl
offspring were as resistant to TCDD toxicity as the Han/Wistar jrats (LD50,
I
>3000 /jg/kg). Further studies on the F2 generation indicated that the
distribution of resistant and susceptible phenotypes were consistent with
inheritance regulated by two (possibly three) autosomal genes displaying complete
dominance, independent segregation and an additive co-effect. Thus, in contrast
to the findings in mice, TCDD resistance seems to be a dominant trait in the rat.
Less convincing evidence for the model of a receptor-mediated toxicity of TCDD
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DRAFT—DO NOT QUOTE OR CITE
arise from studies of the toxicity, receptor levels and/or enzyme-induction of
TCDD in various species, tissues and cell cultures. Despite enormous variability
in the recorded LD50 values for guinea pig, rat, mouse, rabbit and hamster, the
amounts of and physical properties of the hepatic as well as extrahepatic
receptors are comparable in these species (Gasiewicz and Rucci, 1984; Poland and
Knutson, 1982). Furthermore, although the recorded LD50 values for TCDD vary
>100 times between the chick embryo, the C3H/HeN mice and the Sprague-Dawley rat,
the ED50 doses for AHH induction in these species are comparable (Poland and
Glover, 1974). Even in strains of rats with a difference of >300 times in LD50,
no differences in enzyme induction could be demonstrated (Pohjanvirta et al.,
1988). In the guinea pig, the most TCDD-susceptible species, AHH induction is
not a prominent symptom, even at lethal doses of TCDD. A number of cell types,
including primary cultures and established and transformed cell lines from
several species and tissues, are inducible for AHH activity, indicating the
presence of the receptor, yet toxicity is not expressed in these systems (Knutson
and Poland, 1980a). The available data thus suggest that the receptor for TCDD
may be a prerequisite but is not sufficient in itself for the mediation of
toxicity.
TCDD toxicity mimics in many respects endocrine imbalance, although evidence
indicating a direct involvement of hormones in the toxic action of TCDD does not
exist. However, the studies by Lucier et al. (1991) clearly indicate the
importance of interactions with estrogen regulation.
The most reliable and consistent symptom of TCDD toxicity among all
experimental animals is that of weight loss. The cause of the body weight loss
seems to be reduced food intake apparently occurring secondarily to a physiolog-
ical adjustment which reduces the body weight to a maintenance level lower than
normal. The physiological trigger for this body weight set point might be a
target for TCDD.
The ability of TCDD to impair vitamin A storage may be responsible for some
of the toxic effects produced by TCDD.
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3.7. CONCLUSIONS
From the complex picture that evolves from the above outlined cata, it is
amply evident that TCDD elicits a plethora of toxic responses, both after short
I
term and long-term exposure. The lowest doses (single or repeated) that have
been demonstrated to elicit various biological responses in certain animals have
been compiled in Table 3-4. The analysis of the various signs and symptoms that
occur in various species and strains may lead to the following conclusions:
When comparing species and strains it is amply evident that sometimes
there are enormous differences in the sensitivity to specific TCDD-
induced toxicities. This conclusion is valid for almost all the
responses studied. However, qualitatively there seems to be fairly good
agreement between the type of responses that can be recorded (i.e.,
almost all responses can be produced in every species and strain if the
right dose is chosen). In highly sensitive species (e.g., the guinea
pig), lethality may prevent a response occurring. \
Our present knowledge, however, rules out enzyme induction, as such, as
being the cause of toxicity and death. Although the toxicokinetics of
TCDD vary between species, these differences are not sufficient to
explain the variabilities in sensitivity to TCDD toxicity (see
Chapter 1). The available data indicate an involvement pf TCDD in
processes regulating cellular differentiation and/or division as well as
those controlling estrogen homeostasis. Alterations in the regulation
of such processes, which are not equally active in all cells throughout
the organism, would be expected to result in effects that vary among
tissues as well as among species. I
The overwhelming number of toxic responses to TCDD (including lethality)
typically show a delay in their appearance, which supports the assump-
tion that these responses are not the result of a direct insult from the
compound. j
The induction of hepatic cytochrome P-450 dependent monboxygenases
(mainly CYP1A1) is one of the hallmarks of TCDD exposure. This effect
has been demonstrated to be mediated through the interaction with a
specific protein called the Ah receptor. This process covers binding of
TCDD to the receptor followed by binding of the receptor-ligand complex
to DNA recognition sites leading to expression of specific genes and
translation of their protein products, which then mediate their
biological effects. !
Studies in congenic mice which are Ah responsive or Ah nonresponsive
have demonstrated that the majority of TCDD-induced toxic responses
segregate quantitatively with the Ah locus. However, the amount of Ah
receptor expressed in most laboratory species and strains is rather
comparable. The Ah receptor is thus unlikely to be the only determinant
of TCDD-induced toxicity. Rather, it has to be assumed that the species
and strain differences are confined to the latter parts of the receptor-
mediated chain of events, (i.e., binding of the receptor-ligand complex
to DNA and the subsequent expression of specific genes). Another
explanation may be that the binding affinity of the Ah receptor is
different or defective. In addition, some of the responses may be
secondary in the sense that they are caused by altered homeostasis of
endogenous compounds caused by the TCDD-induced increased activities of
various enzymes. !
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DRAFT—DO NOT QUOTE OR CITE |
I
It has repeatedly been reported that the current opinion is that all
known effects of TCDD are probably Ah receptor mediated (e.g|., Roberts,
1991). Except for the chain of events leading to the induction of
certain enzymes, clear evidence for such a conclusion is still lacking.
However, the studies in congenic mice in combination with jthe usually
rather strong correlation between enzyme induction and various other
TCDD-induced toxic responses makes the assumption rather likely.
Further support for the probability of a receptor-mediated! process is
provided by the very strong structure-activity relationship which has
been demonstrated between various CDDs/CDFs and a variety of toxic
responses.
I
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