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INTRODUCTION TO THE REPORT OF THE "DIOXIN" UPDATE COMMITTEE
On July 1-2, 1986, a group of scientists met together in
Alexandria, VA, to discuss a set of scientific issues associated
with "dioxins", a family of chemically related compounds, some of
whose members have exhibited very high toxicity in standard
toxicology test systems. Dr. John A. Moore, Assistant
Administrator of the Office of Pesticides and Toxic Substances of
EPA convened this panel to determine their views on five specific
areas of "dioxin" toxicology and risk assessment:
a. Human health consequences
b. Immunotoxicity
c. Bioavailability
d. Mechanism of action
e. Appropriate risk assessment procedures
The Committee, chaired by Dr. Henrv Pitot of the McArdle
Laoboratory for Cancer Research at th-e University of Wisconsin,
was composed of individuals who were selected on the basis of
their demonstrated scientific competence and contributions to the
area of toxicity of "dioxins". In addition to Dr. Moore, there
were three observers from EPA present during the meeting. A list
of those present can be found at the end of the report.
In preparation for the meeting, five of the participants
were each asked to prepare a short background paper on one of the
five issues referred to above. The papers provided a juroping-off
point for a free, open discussion of the issues. Under the
guidance of the Chair, the participants reached consensus
Conclusions which form the substance of the Report for the
"Dioxin" Update Committee. Attached to the Conclusions are the
papers discussing each of the issue areas. Some of the papers
were re-drafted as a result of discussions at the meeting. In
addition, a sixth paper on Teratology and Reproduction was
prepared by Dr. Kimbrough at the request of the Committee.
This report has been received as information by the Agency
and will be considered, along with all other relevant
information, as a part of any Agency decisionmaking process.
The Office of Pesticides and Toxic Substances
August 28, 1986
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Dioxin Update Conference July 1 and 2, 1986
CONCLUSIONS
I. TOXICOLOGY - ANIMAL
1. TCDD administration involves morphologic changes including
necrosis and hyperplasia in several tissues including the thymus,
trachea, skin, liver and other tissues of several different animal
species. Neoplasia has been induced by the administration of
TCDD alone in the liver and respiratory tract of rats and in the
thyroid and liver of mice.
2. TCDD alters immune responses in animals in a negative and
very significant manner at a wide range of doses from nanomolar
to micromolar. There is no clear evidence for an adverse effect
on the immune system in humans. This may be due either to an
inherent resistance of the human to TCDD effects on the immune
system or to inadequacies of the observations carried out thus
far in the human including numbers of individuals observed, tech-
niques utilized, time between the exposure and the observation,
level of exposure, age of the cohort, lack of persistent effects
on the immune system, etc.
3. TCDD adainistration alters the expression of a number of genes
coding for enzymes of xenobiotic metabolism in all species studied.
Porphria is induced by TCDD in most species studied. Exposures
to the combination of halogenated aromatic hydrocarbons (mainly
hexachloro'cenzene) and TCDD have been reported to be porphyrogenic
in humans in two studies, but not in a number of other studies,
where exposure occurred primarily to TCDD. . „ .
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II. TOXICOLOGY - HUMAN
1. Chloracne is induced by significant acute or chronic exposure
to TCDD.
2. The epidemiologic evidence regarding TCDD exposure and cancer
in the human is contradictory. This situation is unusual in that
the data point either to a very high risk or a very low risk or
no risk resulting from exposures to mixtures of chorophenols and
phenoxy acid contaminated with TCDD. At present the epidemiologic
data are not persuasive regarding one interpretation over the
other, although the high risks from the Swedish studies cannot be
discounted.
3. Exposures to mixtures of halogenated aromatic hydrocarbons
which contained TCDD have been reported in two studies but not a
number of other studies to be porphyrogenic in the human.
4. Animal data suggest that the immune system of children would
be more susceptible than adults to CDDs and CDFs, since the
developing immune system appears to be more vulnerable. Unfortu-
nately, baseline data for the immune system of children is not
readily available, and the normal response in children of various
ages is not well defined. An unequivocal effect of TCDD on the
human immune system resulting in clinical illness has not been
demonstrated.
III. BIOAVAILABILITY AND PHARMACOKINETICS
1. The bioavaliability of TCDD in the environment is critical to
the estimation of human exposure. TCDD is very tightly bound to
some particulate materials while much less so to others where it
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may be easily removed. Reduced bioavailability of TCDD from soils
occurs when the soil has a high organic content and low concentra-
tion of lipid soluble solvents. The longer the TCDD is in/on the
soil the less the bioavailability. Host and dietary factors are
also involved in the bioavailability of TCDD. These include the
lipid content of the diet and the interaction of TCDD with entero-
cytes.
2. The bioavailability of TCDD in the environment can be deter-
mined by relatively simple chemical means as well as by biological
assay.
3. In several small laboratory animal species the acute oral
LDso varies by approximately 5000, and this large difference is
not attributable to the biological half-life (ti/2) of TCDD which
varies from 12 to 30 days. In contrast the limited data avilable
in some monkeys, cows, and humans suggest a much longer biological
ti/2- TCDD is retained in the organism and the predominate mode
of excretion is through bile following its metabolism by xenobiotic
enzymes. Most TCDD is deposited in adipose tissue.
IV. MECHANISM OF ACTION
1. The evidence to date is compatible with the fact that TCDD
action involves its interaction with an intracellular receptor
whose structure is coded for by the Ah locus in the rodent and
probably homologous genetic loci in other organisms. This evidence
involves both genetic studies and structure-functions correlation
investigations of the ligands including TCDD. However, the inter-
actions of TCDD with its receptor is by itself necessary but
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insufficient, to explain all mechanisms of TCDD toxicity, interac-
tion of TCDD with other receptors, and hormone functions in the
organism.
2. There is no evidence that TCDD or its metabolites alter the
structure of DNA, but TCDD .is carcinogenic in at least two rodent
species. It acts as a potent promoting agent .n at least two
different tissues in two different species, but there is no evi-
dence for initiation activity in any species.
3. In view of these conclusions, consideration of human risk
assessment should take into account a) species variation, b) a
corollary of reversible action, and c) the biological half-life
of TCDD and related compounds which become far more important
than in models assuming irreversible action.
V. HUMAN RISK
1. There is an apparent linear response to TCDD administration
with regard to tumor incidence in the female rat liver. Epidemio-
logical studies, which associate TCDD exposure with cancer, do
not have quantitative information concerning exposure and are
thus not useful for quantitative risk estimation.
2. TCDD is a potent promoting agent in the liver of rats and the
skin of hairless mice, with no evidence of initiating activity in
either system. Further, the carcinogenic effect of TCDU in life-
time rodent studies is consistent with its action as a promoting
agent only.
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3. Potential target organ systems such as the immune and repro-
ductive systems should be considered during risk assessment
analysis.
4. Mechanistic models should be used for quantitative risk esti-
mation for TCDD and related compounds. Such methods should con-
sider epidemiological data, sex-species susceptibility, the pro-
moting action of TCDD, and its pharmacokinetic properties in pre-
dicting risks for exposed populations.
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Review of the Epidemiologic Data
Regarding Oioxin and Cancer
Aaron Blair
National Cancer Institute
Several epidemiologic studies to evaluate cancer risks
associated with dioxin exposure are available for evaluation
and include cohort and case-control designs. Although most of
these studies followed standard epidemiologic procedures,
their limitations fall into two categories.
The case-control studies have limited information on
exposure assessment. Exposure to herbicides is estimated
from self-reported job history or from self-reports of contact
with herbicides. Athough this is a standard and useful
technique for assessing exposure, its accuracy is undoubtedly
lower than using job histories from employment files. Cohort
studies may do a better job of identifying persons exposed to
herbicides (the evidence that this is actually correct is
lacking), but they, are severely limited by small numbers.
Studies so far published are generally unable to focus directly
on exposure to dioxin and must rely on exposure to herbicides
as a surrogate for dioxin exposure.
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CASE CONTROL STUDIES
The approximately 5-fold risks for lymphoma and soft-tissue
among persons exposed to phenoxyacetic icids and chlorophenols
in the Swedish studies (1-3) have not been confirmed elsewhere.
New Zealand studies of soft-tissue sarcomas using cases and
controls from a cancer registry found no association with
agricultural activities or exposure to herbicides (4-5).
An interview study found a non-significant risk -:f 1.3 (6)
Case-control studies of lymphoma in the U.S. (7- ; and New
Zealand (9) have found elevated risks on the order of 1.5
to 2.0 for farmers. These studies, however, were based on
occupational information available on death certificates or
in tumor registry files and not from interview. A recently
completed interview study from New Zealand (10) found slight,
but non-magnificant , excesses (odds ratios = 1.4) of non-Hodgkins
lymphoma among persons having potential exposure to phenoxyacetic
acids and chlorophenols.
COHORT STUDIES
Applicators, industrial workers, and workers involved in
accidents have been evaluated. A study of approximately 2000
Finnish herbicide applicators found no deaths fr^m lymphoma
or soft-tissue sarcoma (11). There were, howeve:. only 26
deaths due to cancer. A study of cancer incider ? among
workers in the Danish phenoxy herbicide manufact ing industry
found five cases of soft-tissue • rcoma vs. 1.8 expected and
seven cases of lymphoma vs. 5.4 expected (12). T^.e plants
manufactured 2,4-D; 2,4,5-T ; 2,4-DP; and MCPA. Axelson et
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al (14) reported excess deaths from Lung and stomach cancer
anong Swedish railway workers with possible exposure to
herbicides, but no excesses for soft-tissue sarcoma or lymphomas.
The Ranch Hand study has so far reported six deaths from
cancer among the exoosed group and none were soft-tissue sarcoma
or lynphoma (15).
Several reports of cancer among workers exposed to dioxins
from industrial accidents are available and indicated on
excess of soft-tissue sarcoma. Fingerhut et al. (16) recently
reviewed the diagnosis and exposure of the reported cases and
indicated the four of the seven cases had employment where
exposure to 2,3,7,8 TCDD was likely and two of these had
pathologically confirmed soft-tissue sarcoma. These findings
underscore the difficulty in evaluating cancer risks, particularly
for soft-tissue sarcomas, and dioxin exposures when exposure
determination and diagnosis are difficult. Studies of the
mortality patterns among New York service men with and without
Vietnam experience found no significant association between
cancer and service in Vietnam (17,18).
Summary: The epidemiologic evidence regarding dioxin
exposure and cancer is contradictory. In fact the contradiction
is striking. On one hand we have the Scandinavian studies
where striking excesses of lyrphoma (5-fold) and soft-tissue
sarcomas (3-5-fold) occur and on the other hand studies from
Mew Zealand that find no risk or only a slight risk of these
tumors. The cohort studies lack sufficient power to adequately
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address the issue and may also suffer from diagnostic
difficulties. This situation is unusual in that the data
Point either to a very high risk or very low or no risk
resulting from dioxin or herbicide exposure. Vs it stands
now the epidemiologic data are not persuasive regarding one
interpretation over the other. The high relative risk seen
in the Swedish studies, however, cannot be dismissed.
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1. Hardell L et al., Br . J. Cancer 1079., 39:711-717.
2. Eriksson M et al., Br. J. Ind. Med. 1981; 38:27-33
3. Hardell L. et al., Br. J. Cancer 1981; 43:169-176.
4. Smith, A.H. et al., Comrunity Hlth Studies 1982; 6:114-119.
5. Smith A.H. et al., Chemosphere, 1983; 565-571.
6. Smith A.H. et al., JNCI 1984; 73:111-1117.
7. Cantor K., Int J Cancer 1982., 239-247.
8. Burmeister LF et al., Am J Epidemiol 1983; 118:72-77.
9. Pearce NE et al., Am J Epidemiol 1985., 121:225-237.
10. Pearce NE et al., Br J Tnd Med 1986., 75-83.
11. Riihimaki V et al. Scand J Work Envrion Hlth 1982., 8:37-42.
.270.
12. Lyng? E? B? J Cancer 1975., 52:259-
13. Axelson 0 et al. Scand J Work Environ Hlth 1980., 6:73-79.
14. EPA Health Assessment Document for Polychlorimated
Dichenza-p-dioxins , 1985.
15. Fingerhut M et al. Scand J Work Environ Hlth 1984;
10:299-303.
16. Lawrence C et al. Am J Public Hlth 75; 277-279.
18. Greenwald P et al. JNCI 73; 1107-1109.
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IMMUNOTOXICITY OF THE CHLORINATED DIBENZODIOXINS AND DIBENZOFURANS
Jack H. Dean, Ph.D. and Renate D. Kimbrough, Department of Cell Biology,
Chemical Industry Institute of Toxicology, Research Triangle Park, NC and
Center for Environmental Health, Center for Disease Control, Atlanta, GA
I. IMMUNOTOXICITY IN ANIMAL STUDIES
Laboratory animals exposed to the prototype chlorinated dibenzodioxin
(CDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), demonstrate severe thymus
atrophy (reviewed by McConnell, 1980; Vos et a I., 1980). Histologic
evaluation of the thymus reveals cortical lymphoid cell depletion similar to
cortisone-induced thymic atrophy. Depressed antibody responses, delayed-type
cutaneous hypersensitivity (DTH), graft-versus-host responses, and
lymphoprc iferative responses were observed at TCDD doses somewhat greater
than those inducing thymic atrophy (see review, Thomas and Faith, 1985).
Increased susceptibility to challenge with the bacteria Sal mono I la bern, but
not L i ster i a monocytogenes or Pseudorabies virus, was noted at dosages
inducing thymus atrophy and impaired immune function (Thigpen et al.( 1975).
Depressed antibody responses and DTH were also observed in guinea pigs
receiving cumulative dosages as low as 0.32 /tg/kg over an eight-week period
(Vos et al., 1973). Clark et al. (1983) observed depressed T-celI function
following exposure of adult mice to TCDD, which was associated with an
increase in suppressor T-lymphocyte expression and loss of. I-1ymphocyte
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mi
immun
cytotoxicity for tumor target cells. In other studies of adult mice exposure
to TCDD (Vecchi et a I., 1980; Dean and Lauer, 1984), depressed antibody
responses and some depression of lymphoproliferative responses of T-cells to
itogens were observed without significant alterations in eel I-mediated
ity (CMI), cytotoxicity for tumor cells, or altered susceptibility to
bacterial or tumor cell challenge. The suppressed antibody re oonse was
recently correlated with increased mortality following challenge with
Influenza virus (Lauer et a I., 1986).
In utero or perinatal exposure to TCDD during thymic organogenesis and
thymocyte differentiation in rodents produces a "wasting syndrome" which is
associated with depletion of T cell-dependent areas in the thymim,
leukopenia, lymph node atrophy, depressed bone marrow cellularity, and more
severe CMI suppression than that which occurs following adult exposure (Vos
and Moore, 1974; Faith and Moore, 1977; Luster et a I., 1980). In rodent
species, in utero exposure (via maternal dosing) appears to be necessary to
induce maximum immunosuppression (Vos et al., 1973; Luster et al., 1980).
Administration of TCDD j_n utero also results in decreased resistance of
offspring to bacterial and tumor cell challenge, which correlated with
altered CMI (Luster et a I., 1980) in these mice.
Currently it is believed that TCDD-induced immunosuppression is mediated
through a cytosolic receptor for TCDD. The TCDD receptor was originally
described by Poland and Clover (1976) in hepatic cytosol and subsequently in
thymic cytosol (Poland and Clover, 1980). Both genetic and structure-
activity data indicate that TCDD-induced thymic atrophy is mediated through
the TCDD cytosolic receptor protein since thymic atrophy segregates with the
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Ah locus, and halogenated congeners of TCDD that compete with [ H]-TCDD for
specific binding .sites in thymic cytosol fractions produce thymic atrophy in
v i vo (Poland and Glover, 1980). The target for immunotoxicity is thought to
principally be the thymic epithelial cells, as suggested by Clark et al.
(1983) and Greenlee et al. (19.85). Binding of TCDD to receptors in the
thymus may promote altered T-cell maturation and differentiation and may be
the molecular basis for the observed thymic atrophy and immunotoxicity.
Recent studies revealed that murine (Greenlee et a I., 1985) or human thymic
epithelium mono layers (Cook et a I., _1986) failed to support T-lymphocyte
precursor differentiation following exposure to TCDD. Since the endocrine
influence of thymic epithelium products (e.g., thymic hormones) in adult
animals and humans is poorly understood, immunosuppression observed in
rodents following adult exposure to TCDD may also involve toxicity to the
thymic epi the Ii urn.
A chlorinated dibenzofuran (CDF), 2,3,7,8-tetrachlorodibenzofuran
(TCDF), has been identified in various preparations of commercial Aroclors
(Vos et a I., 1970) and shares the same magnitude of toxicity as TCDD. The
similarity between TCDD and TCDF in chemical structure accounts for the
competition of TCDF for the putative TCDD cytosol receptor. One might
expect, therefore, that TCDF may also be immunotoxic. In animal studies,
TCDF produced severe thymic atrophy in most species studied (Moore et a I.,
1976) and suppressed lymphocyte responses to mitogens, delayed-type cutaneous
hypersensitivity to novel antigens, and lymphokine production (e.g., MIF) in
adult guinea pigs (Luster et a I., 1979).
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II. HUMAN EXPOSURE AND CLINICAL IMMUNOLOGY FINDINGS
Exposure of humans to TCDD is reported to produce toxic effects
including chloracne, biochemical alterations, and metabolic disturbances
(Suskind and Hertdzberg, 1984; Reggiani, 1980; Pazderova-VejIupkova et al.
1981; Moses et al., 1984), although few significant immune alterations have
been identified. Reports on the immune status of children exposed to TCDD in
Seveso, Italy, indicate that their immune capacity was normal to slightly
elevated (Reggiani, 1980; Sirchia, Personal Communication). Of the 344
school children residing in the TCDD-contaminated area, 20 children exhibited
severe chloracne (a classic sign of TCDO toxicity) were examined for
immunologicaI effects, although their serum immunoglobulin levels and
circulating complement levels were normal. Lymphoproliferative responses to
T- and B-celI mitogens were significantly elevated, a finding frequently
reported following low-level TCDD exposure in rodents, but whose biological
significance is undefined. In an unconfirmed study of British workers from a
chemical manufacturing plant who were accidentally exposed to CDDs, reduced
levels of serum IgO and IgA and depressed lymphocyte responses to T-
lymphocyte mitogens were observed (Ward, unpublished report). A correlation
was suggested between chloracne and altered immune status in this study.
The U.S. Air Force recently completed the preliminary evaluation of the
health and immune status of individuals involved in the aerosol use of TCDD-
contami nated defoliant Agent Orange in Vietnam (Ranchhand II study)
immunologic abnormalities were not apparent in these studies (Lathrop et al.
1984) .
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Likewise, Knutsen (1984) observed no statistically significant
alterations of CMI in residents of Times Beach, Missouri chronically exposed
to TCDD, although there were trends of decreased delayed-cutaneous
hypersensitivity in children and adult males, and decreased
lymphoproliferative responses to tetanus toxoid in children. In a recent
preliminary report the Center for Disease Control described a study of 154
individuals exposed to TCDD contaminated soil in a mobile home park in Gray
Summit, Missouri (e.g., TCDD values in soil ranged from 39 ppb to 1100 ppb)
revealed a significantly increased frequency of anergy (11.8. vs. 1.1%) and
^
relative anergy (35.3% vs. 11.8%) in skin tests to recall antigens. A non-
statistical ly significant increase in the frequencies of abnormal T-celI
subset values and functional responses were likewise observed (Hoffman «t
al., 1986). These findings suggest, that long-term exposure to 2,3,7,8-TCDD
is associated with depressed eel I-mediated immunity, although the effects
have not resulted in an excess of clinical illness. Furthermore, some biases
may have been introduced into the study whose impact can not be evaluated as
follows: 1) The four regular skin test readers did not read the DTH
response of 26 participants and the skin tests for these individuals were
read by 12 individuals. Because of the lack of standardized training among
these 12 readers, disproportionate mix of exposed and unexposed participants,
and potential for knowing subject exposure status these skin test results
were excluded from the analysis. 2) The frequency of anergy observed by two
of the four regular readers (readers 1 and 2) in unexposed participants was
15% and 40%, respectively, rates significantly higher than expected (P<.01)
when compared with published norms for a healthy population (0.2%). Skin
test results for all participants examined by these two readers were excluded
from subsequent analyses of DTH results. Results were therefore reported
-4-
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only for the 145 participants (54R of the total group, accounting for 395? of
the exposed group and 68% of the unexposed group) examined by the acceptable
readers. 3) There was a statistically significant difference between the
exposed and unexposed groups for the mean Hollingshead index score for the
head of the household (p<0.01) which is inversely related to socioeconomic
level, and the participants educational level (p<0.01). Educational and
socioeconomic levels were lower in the exposed group. Another concern in the
above mentioned study is that the multitest CMI assay system used to assess
delayed cutaneous reactivity to recall antigens produced less than the
expected frequency of reactivity previously reported in normal controls
(Kniker et al., 1984). It is presently not clear what if any impact these
factors may have had on the Missouri study, and the participants are being
vivalua.ted further.
Animal data suggest that children would be the most susceptible group to
CDD and CDF, since the developing immune system appears to be more
vulnerable. Unfortunately, baseline data for the immune system of children
is not readily available and the normal response in children of various ages
is not well defined. It is also not clear whether and how repeated doses
might effect the immune system or whether short-term exposure could result in
irreversible effects. Of greater importance is the fact that an unequivocal
effect of TCDO on the human immune system resulting in clinical illness has
not been demonstrated.
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III. RISK ASSESSMENT
Studies of the dose-response kinetics of CDDs and COFs in animals
suggest that immunotoxicity might represent one of the most sensitive
endpoints of toxicity, comparable to measurements of metabolic enzyme
induction. In studies by Clark et al. (1983), immunologicaI changes were
observed in mice exposed to ng amounts of TCDD, although these low exposure
effects have not been duplicated by independent laboratories. However, one
hundred-fold greater exposure levels have consistently produced broad-
spectrum immunosuppression in rodents. Hence, immunologic endpoints measured
in rodents could potentially be used for human risk assessment and
extrapolation,, a I though we I I-documented evidence does not exist to date for
any biologically significant immune abnormality or excessive illness produced
by exposure of humans to CDDs.
At this time overt toxicologicaI significance cannot be ascribed to the
immune effects reported in studies of human populations inadvertently exposed
to CDD since all immunologicaI changes observed have been minimal and a true
pattern of immune impairment and associated illness has not emerged.
Additional, we I I-control led clinical cohort studies with documented exposure
information will be. needed to determine if significant immune alterations are
present following exposure to CDDs and CDFs.
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IV. REFERENCES
Clark, D. A., Sweeney, G., Safe, S., Hancock, E., Kilburn, D.G., and Gauldie,
J. Cellular and genetic basis for suppression of cytotoxic T-celI generation
by haloaromatic hydrocarbons. Immunopharmacology 6:143-153, 1983.
Cook, J.C., Dodd, K.M., and Greenlee, W.F. Evidence that human thymic
epithelial (HuTE) cells are a target for 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). The lexicologist 6:172, 1986.
Dean, J.H. and Lauer, L . Immune logical effects following exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin: a review. In: Public Health Risk of
the Dioxins, Lowrance, W.W. (ed.). William Kaufmann, Los Altos, California
pp. 275-294, 1984.
Faith, R.E. and Moore, J.A. Impairment of thymus-dependent immune functions
by exposure of the developing immune system to 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD). Journal of Toxicology and Environmental Health 3:451-464,
1977.
Greenlee, W.F., Dold, K.M., Irons, R.D., and Osborne, R. Evidence for direct
ac - on of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epitheliun.
Toxicol. Appl . PharmacoK 79:112-120, 1985.
Hoffman, R.E., Stehr-Green, P.A., Webb, K.B., Evans, G., Knutsen, A.P.,
Schramm, W.F., Staake, J.L., Gibson, B.B., and Steinberg, K.K. Health
effects of long-term exposure to 2,3,7,8-tetrachIorodibenzo-p-dioxin. JAMA
255:2031-2038, 1986.
Kniker, W.T., Anderson, C.T., McBryde, J.L., Roumiantzeff, M., and Lesourd,
B. Multitest CMI for standardized measurement of delayed cutaneous
hypersensitivity and cell-mediated immunity: Normal values and proposed
scoring system for healthy adults in the U.S.A. Ann Allerg 52:75-82, 1984.
Knutsen, A.P. Immunologic effects of TCDD exposure in humans. Bull Environ
Contam Toxicol 33:673-681, 1984.
Lathrop, G.D., Wolfe, W.H., Albanese, R.A., and Moynahan, P.M. An
epidemic logic investigation of health effects in Air Force personnel
following exposure to herbicides. USAF, Brooks Air Force Base, Texas, 1984.
Lauer, L.D., House, R.V., Ward, E.C., Murray, M.J., Barbera, P.W., Fenters,
J.D., and Dean, J.H. Immune status following 2,3,7,8-tetrachlorodibenzo-p-
dioxin exposure in adult mice. I. Effects on humoral immunity and
susceptibility to influenza virus challenge. Fund. Appl. Toxicol.
(submitted, 1986).
Luster, M.I., Boorman, G.A., Dean, J.H., Harris, M.W., Luebke, R.W.,
Padarathsingh, M.L., and Moore, J.A. Examination of bone marrow, immunologic
parameters and host susceptibility following pro- and postnatal exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). International Journal of
Immunopharmacology 2:301-310, 1980.
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Luster, M.I., Faith, R.E., and Lawson, L.D. Effects of 2,3,7,8-
tetrachlorodibenzofuran (TCDF) on the immune system in guinea pigs. Drug
Chem. Toxicol. 2:49-60, 1979.
McConnelI, E.E. Acute and chronic toxicity, carcinogenesis. Reproduction,
Teratogenesis, and Mutagenesis in Animals. Elsevier/North-Holland Biomed.
Press, New York, pp. 241-266, 1980.
Moore, J.A., Gupta, B.N., and Vos, J.G. Toxicity of 2,3,7,8-
tetrachlorodibenzofuran—Preliminary --suits. In: Proc. Natl. Conf. on
Polychlorinated Biphenyls, Environmental Protection Agency, Washington, D.C
pp. 77-79, 1976.
Moses, M., Lilis, R., Crow, K.D., Thornton, J., Fis-hbein, A., Anderson,
H.A., and Selikoff, I.J. Health status of workers ^ th past exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin in the manuf ture of 2,4,5-
trichlorophenoxyacetic acid: Comparison of findings with and without
chloracne. American Journal of Industrial Medicine 5:161 182, 1984.
Pazderova-Vej lupkova, J., Nemcova, M., Pickova, J., Jira-sek, L., and Lukas,
E. The development and prognosis of chronic intoxication by
tetrachlorodibenzc-.p-dioxin in men. Arch Env Health 36:5-11, 1981.
Poland, A. and Glover, E. 2,3,7,8-tetrachlorodibenzo-p-cnoxin: Segregation
of toxicity with the Ah locus. Mol. Pharmacol. 17:86-94, 1980.
Poland, A. and Glover, E. Stereospecific, high affinity b nding of 2,3,7,8-
tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Bio . Chem. 251 936-
4945, 1976.
Reggiani, G. Acute human exposure to TCDD in Seveso, Italy. J. Toxicol.
Environ. Health 6:27-43, 1980.
Suskind, R.R. and Hertzberg, V.S. Human health effects of 2,4,5-T and its
toxic contaminants. JAMA 251(18):2372-2380, 1984.
Thigpen, J.E., Faith, R.E., McConnelI, E.E., and Moore, J.A. Increased
susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Infect. Immun. 12:1319-1324, 1975.
Thomas, P.T. and Faith, R.E. Adult and perinatal immunotoxicity induced by
halogenated aromatic hydrocarbons. In: Immunotoxicology and
Immunopharmacology, Dean, J.H., Luster, M.I., Munson, A.E., and Amos, H.E.
(eds.). Raven Press, New York, pp. 305-313, 1985.
Vecchi, A., Mantovani, A., Sironi, M., Luini, W., Spreafico, F., and
Garattini, S. The effect of acute administration of 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) on humoral antibody production and cell-
mediated activities in mice. Archives of Toxicology 4:163-165, 1980.
Vos, J.G., Faith, R.E., Luster, M.I. Immune alterations. In: Haloger.ated
biphenyls, terphenyls, napthalenes, dibenzodioxins and related products,
Kimbrough, R.D. (ed.). El sevier/North-HolI and, Amsterdam, pp. 241-266, 1980.
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Vos, J.G., Koeman, J.H., Van Der Maas, H.L., Ten Noever De Braaw, M.C., and
Oe Vos, R.H. Identification and toxicologicaI evaluation of chlorinated
dibenzofuran and chlorinated naphthalene in two commercial polychlorinated
biphenyls. Toxicology 8:625-673, 1970.
Vos, J.G. and Moore, J.A. Suppression of cellular immunity in rats and mice
by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin.
International Archives of Allergy and Applied Immunology 47:777-794, 1974.
Vos, J.G., Moore, J.A., and Zinkl, J.G. Effects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals.
Environ. Health Perspect. 5:149-162, 1973.
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Bioavailability of Dioxins from Complex Mixture
Dr. Michael ^. Gallo
The release, uptake, and biological effect of a xenobiotic
compound (or compounds) from a matrix encompasses the facets of
bioavailability. As would be expected, bioavailability can be
affected, either enhanced or inhibited, by agents or conditions
that alter release, uptake, metabolism and/or biological effects
of xenobiotics.
Since hazard is generally considered to be a function of
toxicity and exposure, it is important to understand bioavailability
to determine the extent of potential exposure after a compound or
mixture of compounds have been identified by chemical analysis.
Several studies have shown that in simple mixtures of 3 to 5
chlorinated solvents bioavailability is a function of lipid
solubility, concentration and the presence of solvents. In the
clinical setting the bioavailability of ionizable compounds is a
function of the pKa of the compound and the pH of the compartment
in which the compound is residing (example: aspirin in the stomach
as compared to aspirin in the small intestine). However, in complex
mixtures such as hazardous wastes, sewage sludge flyash, etc., it
is difficult, if not futile, to attempt to model toxicity of the
mixture based on the components of the mixture.
Recent studies in several laboratories have shown that the
bioavailability of 2 3,7,8,tetrachloro-p-dibenzodioxin (TCDD) from
environmental samples can vary from 85 percent to less than 0.1
percent depending on : 1) the matrix to which it is bound; 2) the
media from which it entered the environment; 3) the duration of the
binding to the environmental substrates; and 4) the presence of
other compounds in the mixture. There are probably other variables
that affect release from the matrices.
The biological effects of complex mixtures in the environment
have received relatively little attention from the toxicology
community. However, the extreme toxicity of 2,3,7,8-TCDD and the
apparent widespread distribution of this compound has led several
research teams to examine the toxicity of mixtures containing
2,3,7,8-TCDD, its congeners and analogs (Poiger and Schlatter,
1980; van der Berg et al., 1985; McConnell et al., 1984; Silkworth
et al., 1982; and Umbreit et al., 1985; 1986). • '
The studies of soil contamination ( McConnell et al., 1984;
and Umbreit et al., 1985; 1986) have clearly demonstrated that
2,3,7,3-TCDD is present in the environment as one of several
chlorinated hydrocarbons in complex mixtures. The use of 2,3,7,3-
TCDD as the model is important since it is a compound that has a
pathognomonic syndrome at very low doses in susceptible species,
and the analytical methods can detect 2,3,7,8-TCDO and its
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isomers at the picogram Level in rnost media. Soil analyses of
samples from Times Beach, Missouri (McConnell et al., 1994) and
Newark, New Jersey (Umbreit et al., 1985) have shown that contami-
nation at both sites contained several compounds in addition to
2,3,7,8-TCDD. For example, in Newark there were 53 chlorinated
dibenzofurans and dibenzodioxins at concentrations ranging from
<0.1 ppb to 4500 ppb (total of approximately 25,000 ppb), in
addition to chlorophenols, PCBs, PAHs and solvents such as methylene
chloride, xylene, toluene, benzene, etc. The total contamination
of the site was in the percent (parts per hundred) range. Similar
contaminants have been reported for the Times Beach area.
When bioassays were conducted with the contaminated soils
from Times Beach and Newark, at equivalent 2,3,7,3-TCDO doses, it
was shown that both soils induced aryl hydrocarbon hydroxylase
(AHH), a cytochrcme ?450 enzyme system that is the product of
activation of the r\h gene locus (Poland and Knutson. 1982) , in
rats, but only the soil from Times Beach induced the TCDO-syndrome
and death in Guinea pigs. Further research with the two soils
have shown that the TCDD and other compounds are readily extractable
(shaking in solvent and column chromatography) from Times Beach
samples, but only solvents and non-TCDD like compounds are extractable
by this method from the Newark soils To extract the 2,3,7,8-TCDD
and its analogs fron Newark soils one must use 48 to 72 hour
exhaustive Soxhlet extraction (Umbreit et al., 1986). The differences
in toxicity and extractability appear to be the result of soil
binding which in turn may be the result of duration of exposure
and the presence of other solvents and oils. Poiger and Schlatter
(1980) have reported that 2,3,7,8-TCDD has a greater substantivity
to carbon particles as a function of time, and van der Berg et
al..(1985) have made a similar observation regarding binding of
dioxins to flyash and soot. Gierthy et al., (1984) and Silkworth
et al., (1982) have shown a similar phenomena for PCBs and PC3
congeners when the compounds are adsorbed onto soot.
Bioavailability in these instances appears to be a function
of several interdependent variables making generalizations regarding
predictability difficult. However, what is obvious is that
several different analyses, varying in severity of extraction,
are necessary to predict potential bioavaliability (Gierthy et
al., 1984; and Umbreit et al., 1986).
An underlying concern of researchers and regulators is
whether it is proper in risk assessment to consider only the
presence of xenobiotics in a medium and the toxicity of these
xenobiotics (assuming exposure), or should the risk assessor
attempt to use bioavailability data to complete the exposure
assessment.
To elucidate the modifiers of bioavailability from complex
mixtures, research in the following areas is needed:
1.Interaction With Matrices
a) soils: Characterization of the soil(s). The soil in
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Newark appears to have a higher organic content than Times Beach
and binding sites appear to be more abundant in Newark soil.
A generalization that nay be possible is that matrices with
higher organic content .may have greater substantivity than a
similar matrix with lower organic content.
b) Flyash sediments and carbonaceous materials.
Poiqer and Schlatter (1980), and Rappe et al, (1985) have shown
that the presence of. these materials binds several organic
compounds but have a much greater affinity for chlorinated
dibenzodioxins and dibenzofurans.
c) Solvents: The presence of solvents or the continued
release of solvents at a site may aid in the percolation of
compounds through the soil and enhance binding to soil particles.
This phenomenon has been hypothesized as a possible explanation
for some of the soil binding of PCBs in Japan.
2. Alteration Of Biological Uptake
This is an extremely important aspect of bioavailability
because one or more compounds in "a mixture may alter the absorption
of the other compounds or the mixture may alter GI transit time,
which may affect absorption of several compounds including nutrients.
Analytical methods are now available to allow researchers to determine
differential uptake. Indeed, Bandiera et al., (1984) have recently
demonstrated that there is retention of specific chlorinated diben-
zofurans from complex mixtures of PCBs and PCDFs found at Yusho,
Japan.
3. The Role of Host Factors
Several host factors can modulate the bioavailability
of xenobiotics.
a) Dietary factors such as pattern of eating,
nutritional status and diet per se can alter bioavailability both
quantitatively and Qualitatively. The presence of plant
flavinoids, psoralens and fat soluble vitamins can alter the
absorption of environmental toxins (Hollander 1981). Many of
these toxic compounds are absorbed as trace nutrient lipids and
fat soluble vitamins (Hollander and Morgan, 1980), or they may be
incorporated into the lipid phase of micelles and be absorbed on
the villous surface (Patton, 1981).
b) The metabolic activity of the host's enterocytes can
alter the body, burden of toxic chemicals because at low doses
some of these compounds are metabolized in the enterocytes and
are not absorbed. Aliphatic hydrocarbons are readily metabolized
by ths enterocyte system while polycyclic compounds cross the gut
because they are not metabolized to any great extent by the
enterocytes (Kukis, 1984).
c) Selective uptake of lipophilic compounds from
complex mixtures can take place as these compounds traverse the
gut, (Xizelian (1982) has shown that lipophilic toxins can also be
resorbed or sequestered in the lumen during enterohepatic
circulation.
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Bioavai.labili.ty varies by routes of exposure. The
three routes: oral, dermal, and respiratory, vary in their
selectivity of uptake, rejection and storage. The
bioavailataility of a compound from a complex mixture will depend
on solubility, volatility, charge, concentration and other
compounds in the mixture. The variables of the oral route have
been previously discussed.
In liquid mixtures the chemicals that breakdown the
stratum corneum or dissolve the lipids in the skin can enhance
percutaneous absorption of selected compounds from a mixture.
Highland et al.,(1934) have shown that benzene in water can alter
the permeability of the skin and account for greater than half of
the total benzene exposure in a residential setting.
There are studies currently underway to determine if there
is selective percutaneous absorption of xenobiotics, particularly
dioxins, PCBs and benzofurans, from environmental samples.
In the lungs there is d-lfferential uptake of several
compounds. Volatile organics are taken up and are readily
transported to the blood while particulates are sequestered in
alveolar macrophages. There is little or no evidence of
selective respiratory uptake and retention of specific compounds
from complex mixtures.
4 Physical State of the Mixture
Bioavailability will depend on the physical state of
the mixture. Several investigators have studied the bioavailability
of dioxins and PCBs from liquid or semi-liquid media and have found
that there is reasonable agreement between theoretical and actual
biologic levels. However, in the studies of mixtures bound to
solid substrates there are marked differences from site to site.
Research efforts and resources should be used to determine
differential bioavailability from complex mixtures in different
physical states.
5. Receptor Binding or Alteration
If chemicals such as 2,3,7,8-TCDD, psoralens (Laskin
et al. 1985) and estrogens, etc., or others that are bound to
specific receptors are found in complex mixtures then there may
be differential and preferred uptake over other compounds in the
mixture that are crossing membranes by simple diffusion
mechanisms. Carrier mediated transport, a mechanism analogous to
receptor mediation, has been shown to transport seme toxio -
compounds across the gut as if they were natural ligands for the
carrier (Kukis, 1984). This area is seriously lacking in
research, particularly in the elucidation of transport of toxic
chemicals across the GI mucosa via carrier mechanisms for dietary
supplements.
6. Availability of Proper Biological Markers of Exposure
Proper biological markers of exposure can help the
investigators to determine if one or more of the compounds in a
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complex mixture have induced a toxic response in a host.
2,3,7,8-TCDD is a unique compound because of. the specific
syndrome which it induces in laboratory animals. It is because
of this syndrome that one can attempt to differentiate
bioavailability frcm mere presence in a ccnolex mixture.
7. Additivity, Synergism and Antaxonism
The study of interactions is a major focus of the
work with TCDD in complex mixtures. The interactions of
concern can also be cited as specific for bioavailability. As
stated above there are many interactions involving
bioavailability and multi-solvent exposure, particularly on the
skin. Other interactions have been demonstrated in the GI tract
and digestive processes. tJhile another group of interactions have
been shown to take place in the lung upon inhalation of mixtures
of volatile compounds.
Summary
The seven points suggest areas of new research where some
data currently exist. To better understand risk from
environmental contamination with TCDD we must have a better
knowledge of exposure. One of the largest factors in exposure is
bioavailability, yet there is little data in the literature
regarding bioavailability of dioxins and related toxic compounds
from complex environmental mixtures.
Michael A. Gallo PhD
UMDNJ.Rutgers Medical School
June 19.1986
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Mechanism of Action
Dr. Allan Poland
2,3,7,3-Tetrachlotrodibenzo-p-dioxin (TCDD) serves as the prototype for a
large series of halogenated aromatic hydrocarbons including CDD and CDF isomers
which share the following properties: 1) approximate isosterism, 2) the pro-
duqtion of a characteristic pattern of biochemical and histoloqic (i.e. toxic)
responses, and 3) an apparently similar mechanism of action.
Hie most studied of these bioloaic responses is the induction of cytochrome
Pl~450 (increased transcription of the pRNA for P^-450) and the associated
increase in Pi-450-mediated enzyme activity — e.g.., aryl hydrocarbon hydroxy-
lase (AHH) activity. This event appears to be very similar to the mechanism
of transcriptional activation by steroid hormones as detailed below: 1) TCDD
and related congeners show stereospecific, high affinity, saturable binding to
a soluble protein (referred to as the TCDD-binding protein or Ah receptor),
2) the ligand-receptor complex shows an increased affinity (compared to the
unliganded receptor) for nuclei or DMA, 3) the 5' regulatory seguence of cyto-
chrome P1450 has been cloned into a plasmid containing the chloramphenicol
acetyltransferase (CAT) gene, transfected into the wild type hepatoma cells
(containing the Ah receptor), and incubation with TCDD was shown to produce
CAT expression (Whitlock et al). These experiments strongly suggest, but do
not prove, that the TCDD-Ah receptor complex binds to specific DMA sequences
to initiate gene expression in a manner analogous to that shown for the gluco-
corticoid receptor.
Two independent lines of evidence suggest that all the biological effects
produced by TCDD and related compounds are mediated through their binding to
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the Ah receptor and the altered gene expression initiated by this drug complex.
For a large number of halogenated dibenzo-p-dioxin and dibenzofuran congeners,
the rank-ordered structure-activity relationship for receptor binding corres-
ponds very closely to that for biological activity (e.g. induction of hepatic
AHH activity, LD$Q in guinea pig, epidermal hyperplasia, cleft palate induction,
thymic involution, tumor promotion, immunosuppression). Secondly, among inbred
strains of mice there is a polymorphism in the genetic locus that determines
the Ah receptor (the Ah locus). Inbred stains hcmozygous for the Ah*3 allele
have a high affinity receptor and are sensitive to the effects of TCDD; while
inbred strains homozygous for the Ah^ allele have a lower affinity for the
receptor and are less sensitive to the effects of TCDD. In genetic crosses
between inbred strains or mice congenic for the Ah locus, a variety of toxic
responses produced by TCDD segregate with the Ah13 allele (e.g. cleft palate
formation, thymic involution, hepatic porphyria, epidermal hyperplasia and
metaplasia, hepatic tumor promotion).
However, while the Ah receptor appears to mediate the toxicity of TCDD,
the presence of the receptor in tissues does not assure the developnent of
toxicity. TCDD and congeners produce a variety of tissue specific histologic
lesions, many of which are confined to one or a few animals. For instance,
the skin of most mammals contains the receptor, and when challenged with these
compounds responds with the induction of AHH activity in all species tested,
but epidermal hyperplasia and hyperkeratosis and sebaceous gland metaplasia
(chloracne) is observed in only a few species. Thus the Ah receptor appears
to control two distinct and dissociable pleiotropic responses: 1) the induc-
tion of cytcchrome Pi450 and other enzymes in virtually all tissues in which
the receptor is present, and 2) induction of responses, most distinctively
proliterative and altered differentiation in epithelial tissues, which is
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restricted by tissue and animal species. The reason for this restricted exores-
sion is not known. The physiologic role of the receptor, if any, and an endo-
genous ligand for the Ah receptor are unknown. The gene products responsible
for toxicity, and the mediators, hormones, and second messengers involved are
largely unknown.
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Carcinogenicity - One manifestation of TCDD toxicity that has received much
attention is carcinogenicity. The chronic administration of TCDD and some
other halogenated aromatic hydrocarbons to rats and mice has been shown to
produce an increased incidence of tumors in the liver and other tissues.
Extensive testing has failed to show that TCDD is a mutagen. The maximum
level of covalent binding to rat liver DNA in vivo was estimated to be less
than 1 x 10-H moles of TCDD/mole of nucleotide, 4-5 orders of magnitude, lower
than most carcinogens. Thus, there is little evidence that TCDD is an initi-
ator; however, there is substantial evidence that TCDD and related compounds
act as tumor promoters, enhancing the neoplastic expression in otherwise initi-
ated cells. In two stage models of rat liver carcinogenesis and mouse skin
tumorigenesis, TCDD acted as a tumor promoter. In the latter model, the struc-
ture activity relationship among a limited number of congeners tested for tumor
promotion corresponded to that for receptor binding, suggesting this is a
receptor mediated event.
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Implications - The mechanises of toxicity of TCDD and related compounds, that
is the biochemical changes that result in specific tissue toxicities and death,
is largely unknown. However, there is substantial evidence that TCDD exerts
these events by stereospecific reversible binding to the Ah receptor which in
turn controls coordinate gene expression. Among animal species, the cause of
5000 fold variation in sensitivity to TCDD (LDsn) is unknown, but not attribut-
able in any significant degree to variation in receptor affinity or concentra-
tion, nor to the pharmacokinetics of the compound. Consideration of human
risk assessment should take into account the 1) large species variation, 2) that
the mechanism of action (receptor occupation) is reversible, and 3) a corrol-
lary of reversible action, that the biologic half life of the compounds become
far more important than in ...odels assuming irreversible action. Thus, assuming
a biologic half-life of TCDD of 4 yr, a chronic daily dose of 10 pg/kg/day
would produce a tissue cone, of 200 ng/kg (6xlO-in moles/kg) at an infinite
time (steady state).
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Risk Assessment
Dr. David H
Summary
The first issue one nust address is whether or not to conduct a
quantitative risk estimation or -.o apply one of the standard safety factor
aproaches. For chronic exposu -jf TCDD, experimental evidence suggests
that tha dose-response relatu* ip with regard to tumorigenesis and AHH
induction is linear.in the low- ;-jse region. Therefore, there is no
evidence to suggest that a threshold approach is appropriate. For acute
exposures, the experimental evidence indicates that TCDD functions solely
as a promoter in carcinogenesis and therefore, the affects nay Indeed be
irreversible. There is also evidence that the half-life of TCDD in man is
possibly of a reasonably lorrj period. As such, it is not clear where
irreversibi.lity ends and without this information, it is very difficult r.
make a convincing argument that a single acute exposure co TCDD is not
carcinogenic because of its promotional,..hence reversible properties. It
is important therefore, in attempting a quantitative risk -icimation, to
incorporate as much as possible the mechanisms of promote:.. ,
Once estimates have been made for the carcinogenic ef- -cts in rodents
after acute and chronic exposures, one ™ .:st deal with the ;blem of
extrapolating these effects to man. Thi ; is especially diL.:j.cult because
of the information concerning the large pecies variability with regard to
acute toxicity. Also, there is the sugcsstion that hormones are related to
carcinogenicity and as such, there may br. a strong sex effect in man.
Therefore, the incorporation of safety Lectors may be required in the
aninal-to-man extrapolation. Ideally, one would like to have some
quantitative information concerning exposure doses and carcinogenic effect
from epidemiological studies. This then, would allow some confident in a
species extrapolation. Unfortunately, the available epidemiolcgica.
studies do not provide any information concerning the doses of TCDD 'or
which man was exposed.
Dose Response
TCDD has been shown to be a tumor promoter in the skin of hairless
mice and the liver of rats. These models involved initiation with MNG
followed by promotion with TCDD or initiation with DEN (Pitot et. al, 1980,
Poland et. al, 1982). Although the studies did not have a full range of
exposure doses of the promoter, the available data on tumor incidence is
observed to be linear/ and therefore/ one is not able to argue for a
threshold value of TCDD's promoting effects based on the data generated in
these experiments. These studies were conducted on AHH induction of TCDD.
Two of these involved acute doses, (McConnell et. al, 1984) and the third
involved a chronic exposure (Lucier et. al/ 1986). All three of these
studies indicated a linear dose-response in the lower portion of the
dose-response curve relating AHH with TCDD administration.
Finally, NCI, MTP and Kociba conducted chronic roden . .noassays using
TCDD as s total carcinogen. In these studies, there was no evidence of
non-linear or threshold behavior with regard to lifetime tumor incidence
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induction. The data by Kociba in female rats has been used for
dose-response modeling with regard to liver ttrnors, which is the most
sensitive site in the most sensitive species. The data was ouite linear if
one adjusts the administered dose by either liver concentration of dioxin
or using AHH induction as a surrogate for dose, in either case, the risk
estimates ccne out to be about the same (sone Details are given in Portier
et. al, 1984).
Sex Differences
Data from the NTP 3ioassay and the Kociba Bioassay indicate that TCDD
is a hepatocarcinogen in female rats but not male rats. The mechanism
responsible for this observation is not clear. One possibility could
involve sex differences in the TCDD receptor. However, there is no
evidence of any significant sex differences in receptor properties.
Another possibility is that TCDD exposure "enhances the rate of metabolic
activation of endogenous hormones in hepatocytes. This could produce a
series of second hits. Some evidence in the literature as well as the
preliminary data (Lucier, unpublished) suggests that TCDD treatment of rats
increases the rate of metabolic activation of estrogens to species that
bind covalently to proteins. This finding is consistent with the
observations that TCDD is a hepatocarcinogen only in female rats.
Moreover, it has been observed that TCDD does not promote
hepatocarcinogenesis in ovariectomized female rats using the same
treatment protocols as Pitot's studies which revealed that TCDD is a potent
promoter of hepatocarcinogenesis in intact female rats. Evaluations of
these findings is complicated by the observation that the sex differences
in TCDD-mediated hepatocarcinogenesis is reversed in mice (OTP bioasssy).
However, sex differences in hepatic metabolic components are often opposite
in rats and mice.
Species Differences
Although wide species differences exists in acute toxicity for TCDD
and its structural analogs, there is insufficient data to evaluate species
variation in the carcinogenic potency of these compounds. It is known that
some other manifestations of TCDD exposure (AHH induction) do not exhibit
the same species variation as seen for acute toxicity. Moreover, there is
evidence that suggests that there is not a single unifying mechanism (such
as a single receptor system) for all the effects of TCDD. In other words,
one might expect qualitative differences in species sensitivity to TCDD and
its analogs for different toxic responses. Unfortunately, because of the
lack of quantitative exposure data in the epidemiological studies, it is
not possible to use the currently available epidemiological data to
validate estimates of human risk based on animal carcincqenesis studies.
Hopefully, data that will be obtained from the large cohort studies being
conducted by NIOSH and LAKC may offer some insight into this important issue.
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REFERENCES
Lucier, G.W.., Rumbaugh, R.C., McCoy, Z., Mass, R., Harvan, D., and
Albro, P.: Ingestion of Soil Contaminated with 2,3,7,8-Tetrachlorodibenzo-
p-dioxin (TCDDJ .Altars Hepatic Enzyma Activities in Pats. Fundamental and
Applied Toxicology, 6: 364-371, 1986.
McConnell, E.E., Lucier, G.W., Runbaugh, R.C., Albro, P.W., Harvan, D.J.,
Hass, J.R., and Harris, M.U.: Dioxin in Soil: Bioavailability After
Ingestion by Rats and Guinea Pigs. Science, 223: 1077-1079, 1984.
Pitot, Henry C., Goldsworthy, Thomas, Campbell, H.A., and Poland, Alan:
Quantitative Evaluation of the Promotion by 2,3,7,8-Tetrachlorodibenzo-p-
dioxin of Hepatocarcinogenesis from Disthylnitrosamine. Cancer Research,
40: 3616-3620, 1980.
Poland, Alan, Palan, David, and Glover, Edward: Tumor promotion by TCDD
in skin of HRS/J hairless mice. Nature, 300: 271-273, 1982.
Portier, Christopher J., Hoel, David G., Van Ryzin, John: Statistical
Analysis of the Carcinogenesis Siosssay Data Relating to the Risks from
Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin. From "Public Health Risks
of the Dioxin" proceedings of a symposium held on October 19-20, 1983
at The Rockefeller University, New York City. Edited by William w.
Lawrence. Published by William Kaufmann, Los Altos, California, 1984.
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Teratology and Reproduction
Studies with TCDD
Renate D. Kimbrough, M.D.
Animal studies
Teratogenic effects resulting from TCDD have primarily been reported in mice.
An increased frequency of cleft palate, along with an abnormality (dilatation)
of the central collection system of the kidney is seen. (Courtney and Moore
1971, Neubert and Dileman 1972, Moore et al. 1973, Smith et al. 1976). The
no-adverse-effect level (NOEL) for a teratogenic response in the mouse is 0.1
pg/kg/day (Smith et al. 1976). The rat has also been used in teratology
studies with TCDD. Results are listed in Table 1 (Courtney and Moore 1971,
Sparschu et al., 1971, Khera and Ruddick 1973). The no-adverse-effect level
for rat embryo fetotoxicity is in the range of 0.03-0.125 yg/kg/day.
Studies by Allen et al. (1977a and 1977b) showed substantial toxicity,
including alopecia, anemia, and death in eight monkeys fed diets containing
500 ppt (ng/kg) TCOD for up to 9.3 months. Breeding of the eight female
monkeys showing frank toxicity after 6 months of TCDD treatment resulted in
two pregnancies, one of which was aborted. Monkeys given diets containing 50
ppt (ng/kg) of TCDD had some slight toxicity, with four of seven pregnancies
terminating in abortion (Schantz et al. 1979).
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McNulty 1980 gave pregnant monkeys TCDD three times weekly for 3 wk during
gestation. At the dose of 0.24 yg/kg/day (^J 5000 ppt in diet), abortions
occurred in two of two monkeys that had severe toxicity resulting in death.
At an intermediate dose level of 0.048 yg/kg/day Aj1000 ppt in diet),
abortions occurred in three of four monkeys, with slight maternal toxicity.
At a dose level of approximately 0.0095 yg/kg/day (OJ 200 ppt in diet), the
abortion rate of 1/4 was comparable to the abortion rate of 3/11 for the
control group of monkeys.
Murray et al. (1979) reported the results of a 3-generation reproduction study
of rats maintained on diets containing 0, 0.1, 0.01, or 0.001 yg
TCDD/kg/day. No significant toxicity was noted in the f male or female
rats during the 90 days of TCDD treatment prior to mating. The high dose
level of O.lyg/kg/day caused decreased fertility and neonatal survival; an
intermediate dose level of 0.01 wg/kg/day caused decreased fertility and
other effects in the f and f but not f generations. At the dose
level of 0.001 yg/kg/day, there was no impairment of reproductive capacity
through the three consecutive generations.
Recent results suggest that the TCDD receptor may be related to the estrogen
receptor, and that TCDD has antiestrogenic effects which may be independent of
the Ah locus and AHH induction. Thus Gallo et al. (in press) found that
female weanling CS7B/6 mice treated with 6 wg/kg TCDD 3 times a week for one
month (total dose 72 wg/kg) had reduced relative uterine weights and
histopathological changes in the uterus. Weanling CD-I female mice were then
treated with estradiol (E ) subcutaneously daily for 2 weeks. Half the mice
also received 10 wg/kg TCDD in corn oil: acetone (9:1) by gavage 4 times
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during the second week. Control mice received either no E or not TCDD.
Mice were killed on day 15 and autopsided. Relative uterine weights increased
with increasing E doses; however, TCDD decreased this effect of E
markedly. Liver microsomes from these animals showed that cytochrome P -450
and P -450, and aryl hydrocarbon hydroxylase (AHH) induction by TCDD were
independent of E. dosage. Epoxide hydrolase was induced in TCUD treated
animals. Gels showed an E dose dependent decrease in a protein migrating
near epoxide hydrolase and P-450a in animals receiving both E. and TCDD.
Finally in bioassays it has been observed that sex hormone dependent tumors
are less frequent in TCDD exposed rodents (Kociba et al.).
Observations in human
At present it is not clear whether TCDD would affect reproduction in humans.
Over the years a number of anecdotal reports and reports which could not be
substantiated have appeared in the literature. Some of this information is
summarized by Reggiani (1980). No obvious effects on reproduction were noted
in Seveso, where TCDD exposure of the population living around a factory
occurred (Reggiani 1980).
In addition, a morbidity study was recently completed (Ranch Hand study) on
pilots who flew spraying missions in Vietnam and on other Air Force
personnel. These members of the military were exposed to Agent Orange, a
mixture of the herbicides 2,4-D and 2, 4,5-T. The 2,4,5-T was contaminated
with TCDD.
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Preliminary analysis of fertility and reproduction suggests a clustering of
birth anomalies of the skin in Ranch Handers' children. In addition, the
neonatal death rate (p = 0.02) was significantly increased for the Ranch Hand
group. Before their exposure in Southeast Asia, the Ranch Hand group had 20
newborn offspring who died, and the comparison group had 17. After their
service in Southeast Asia, however, the Ranch Hand group had 14 neonatal
deaths and the comparison group had only 3. Additional data analysis and
follow-up of the Ranch Handers may clarify the preliminary findings made in
this cross-sectional study. None of these findings, however, could be related
to herbicide exposure because no specific "dose-response effect" could be
shown (Lathrop et al. 1984).
The retrospective assessment of exposure in situations like Ranch Hand is
extremely difficult, even if the environment is well defined and the exposure
levels of a certain chemical are known. In an occupational situation, for
instance, two people in the same environment can, for a variety of reasons,
receive different doses. The reasons include variations in personal hygiene
and in the ability to metabolize and excrete chemicals. For example, some
investigators have found that workers who smoke have higher levels of
chemicals in their body fluids than their nonsmoking counterparts.
In a retrospective study such as this, when the last exposure to Agent Orange
was at least 12 years ago, at the time of the study it is difficult to assess
what effect other, later insults may have had on the subjects' health.
Furthermore, for purely statistical reasons, some differences will be found if
many endpoints are examined. None of the findings in the Ranch Hand study
have been confirmed in other studies.
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The Centers for Disease Control recently completed a case-control study to
determine if men who served in the U.S. military in Vietnam have been at an
increased risk of fathering babies with serious congenital malformations
(Erickson et al. 1984). Again, no striking findings were made, and it is not
really clear whether this study should be more appropriately classified as a
Vietnam experience study because of the great difficulties in appropriately
determining exposure to 2,3,7,8-TCDD in Agent Orange.
-------
References
1. Allen, J.R., Barsotti, D.A., and van Miller. J.P. Toxicol. Appl.
Pharmacol., 41, 177 (1977a).
2. Allen. J.R., Barsotti, D.A., van Miller, J.P., Abrahamson, L.J., and
Lalich, J.J. Food Cosmet. Toxicol., 15, 401 (1977b).
3. Courtney, K.D. and Moore, J.A. Toxicol. Appl. Pharmacol., 20, 396 (1971).
4. Erickson, J.D., Mulinare, J., McClain, P.W., Fitch, T.G., Levy, M.J.,
McClearn, A.B., Adams, M.J. JAMA 1984, 252, 903-12.
5. Gallo, M.A., Hesse, E.J., MacDonald, G.J., and Umbreit, T.H. Interactive
effects of estradiol and 2,3,7,8 tetrachlorodibenzo-p-dioxin on hepatic
cytochrome p-450 and mouse uterus. Toxicology letters 1986 in press.
6. Khera, K.S. and Ruddick, J.A. Polychlorodibenzo-p-dioxins: perinatal
effects and the dominant lethal test in Wistar rats, in
Chlorodioxins-Origin and Fate, Advances in Chemistry Series, no. 120
(Etcyl H. Blair, ed.), American Chemistry Society, Washington, D.C., 1973.
• » i
7. Kociba. R., Keyes, D.G., Beyer, J.E., Carson, R.M., Wade, E.E., Oittenber,
D.A., Kalnius, R.P., Frauson, L.F., Park, D.N., Barnard, S.D., Hummel,
R.A., and Humiston, C.G. Toxicol. Appl. Pharmacol. 46:279 (1978).
-------
8. Lathrop, G.D., Wolfe, W.H., Albanese, R.A., Moynahan, P.M. Brooks Air
Force Base, Texas: USAF School of Aerospace Medicine, Aerospace Medical
Division, 1984.
9. McNulty, W.P. Unpublished data submitted to U.S. EPA, 1980.
10. Moore, J.A., Gupta, B. N., Zinkl, J.G., and Vos, J.G. Environ. Health
Perspect., Exp. Issue no. 5, 81 (1973).
11. Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J.,
and Schwetz, B.A. Toxicol. Appl. Pharmacol., 50, 241 (1979).
12. Neubert, D. and Dillman, I. Naunyn Schmied. Arch. Pharmacol., 272, 243
(1972).
13. Reggiani, G. J. Toxicol. Environ. Health. 6:27-43, 1980.
14. Schantz, S.L., Barsotti, D.A., and Allen, J.R. Toxicol. Appl. Pharmacol.,
48, A180 (1979).
15. Smith, F.A., Schwetz, B.A., and Nitschke, K.D. Toxicol. Appl. Pharmacol.,
38, 517 (1976).
16. Sparschu, G.L., Dunn, F.L., and Rowe, V.K. Food Cosmet. Toxicol., 9, 405
(1971).
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Wistar
TABLE 1
Teratology Studies with TCDD in Rats
Strain
Sprague-
Dawley
CD
Embryotoxic
effects
Intestinal
hemorrhage
Kidney ab-
normality
NOE1
(pg/kg/day)
0.03
0.5
ED
(wg/kg/day)
0.125-8
0.5
Hemorrhage
0832q:CEH:OD:RKimbrough:sd
0.125
0.25-16
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"DIOXIN UPDATE" PARTICIPANTS
Chairman
Dr. Henry Pitot
McArdle Laboratory
University of Wisconsin
450 N. Randall Avenue
Madison, WI 53706
Tele: (608) 262-3247
Specialty Members
A. Human Health Consequences
Dr. Aaron Blair r
Occupational Study Section
National Cancer Institute
Landow Building
Room 4C16
Bethesda, MD 20892
Tele: 496-9093
B. Immunotoxicity
Dr. Jack Dean
CUT
P. O. Box 12137
Research Triangle Park, MC 27709
Tele: (919) 541-2070
C. Bioavailability
Dr. Michael Gallo
Department of Environment and
Community Medicine - UMDNJ
P. 0. Box 101
Rutgers University Medical School
Piscataway, NJ 08854
Tele: (201) 463-4773
D. Mechanism of Action
Dr. Allen Poland
McArdle Laboratory for Cancer Research
University of Wisconsin
450 N. Randall Avenue
Madison, WI 53706
tele: (608) 263-4959
E. Risk Assessment Methodology
Dr. David Hoel
NIEHS
P. 0. Box 12233
Research Triangle Park, NC 27709
Tele: FTS 629-3441
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-2-
General Members
1. Dr. John DoulL
University of Kansas
Kansas University Medical Center
Rm. L012 Breidenthal
39th and Rainbow Boulevard
Kansas City, KS 66103
Tele: (913) 588-7140
2. Dr. Renate Kimbrough
Center for Disease Control
1600 Clifton Road, N.E.
Atlanta, GA 30333
Tele: FTS 236-4625
3. Dr. Robert Meal
CUT
P. 0. Box 12137
Research Triangle Park, NC 27709
Tele: (919) 541-2070
Observers
1. Dr. Donald Barnes
Office of Pesticides and Toxic Substan (TS-788)
U.S. Environmental Protection Agency
401 K Street, S.W.
Washington, DC 20460
Tele: (202) 382-2897
2. Dr. Steven Bayard:
Office of Research and Development (RD-689)
U.S. Environmental Protection Agency
Washington, DC 20460
Tele: (202) 382-5722
3. Mr. David Cleverly
Office of Air Quality Planning and Standards
MD-12
U.S. Environmental Protection Agency
Research Triangle Park/ NC 27711
Tele: (FTS-8-629-5645
Convener
Dr. John A. Moore
Assistant Administrator
Office of Pesticides and Toxic Substances (TS-788)
U.S. Environmental Protection Agency
401 M Street/ S.W.
Washington/ DC 20460
Tele: (202) 382-2902
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