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EP A/600/AP-92/OOU
August 1992
Workshop Review Draft
Chapter 4. Immunotoxic Effects
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 recommendation
for use.
Please note that this chapter is a preliminary draft and as such represents work
in progress. The chapter is intended to be the basis for review and discussion at
a peer-review workshop. It will be revised subsequent to the workshop as
suueestions and contributions from the scientific community are incorporated.
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CONTENTS
Tables iv
Figures • v
List of Abbreviations vi
Authors and Contributors xi
4. IMMUNOTOXICITY 4-1
4.1. INTRODUCTION 4-1
4.2. ROLE OF THE AH LOCUS IN HAH IMMUNOTOXICITY 4-3
4.3. TOXIC EQUIVALENCY FACTORS (TEFs) FOR IMMUNOTOXICITY 4-11
4.4. INTERACTIONS BETWEEN HAH 4-14
4.5. SENSITIVE TARGETS FOR HAH IMMUNOTOXICITY 4-14
4.6. INFLUENCE OF TCDD ON HOST RESISTANCE TO DISEASE 4-20
4.7. IN VITRO IMMUNOTOXIC EFFECTS OF HAH 4-23
4.8. INDIRECT MECHANISMS OF HAH IMMUNOTOXICITY 4-25
4.9. ROLE OF THE THYMUS IN HAH IMMUNOTOXICITY 4-26
4.10. IMMUNOTOXICITY FOLLOWING PRE/NEONATAL EXPOSURE TO HAH 4-27
4.11. IMMUNOTOXICITY OF HAH IN NON-HUMAN PRIMATES 4-30
4.12. IMMUNOTOXICITY OF HAH IN HUMANS 4-33
4.13. REFERENCES • 4-37
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LIST OF TABLES
4-1 TEFs Based on the IDX for Suppression of the PFC Response
to SRBC in Ah Responsive B6 Mice
4-2 TEFs Based on the IDjo for Suppression of Alloantigen-
Specific CTL Response in Ah Responsive B6 Mice . . .
4-3 Effect of Single Versus Multiple Dosing with TCDD on
Suppression of the Antibody Response to SRBC in
C57B1/6 Mice •
4-4 Influence of Route of Antigen Challenge on Suppression of
the Antibody Response to SRBC in C57B1/6 Mice
4-5 Immunotoxic Effects of TCDD in the Offspring Following
Pre/Neonatal Exposure to TCDD
4-7
4-10
4-12
4-13
4-28
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LIST OF FIGURES
4-1 Structure Dependent Immunotoxicity of Some CDD and CDF
Isomers
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ACTH
Ah
AHH
ALT
AST
BDD
BDF
BCF
BGG
bw
cAMP
CDD
cDNA
CDF
CNS
CTL
DCDD
DI1T
DMBA
DMSO
DNA
DRE
LIST OF ABBREVIATIONS
Adrenocorticotrophic hormone
Aryl hydrocarbon
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
Dimelhylbenzanthracene
Dimethyl sulfoxide
Deoxyribonucleic acid
Dioxin-responsive enhancers
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LIST OF ABBREVIATIONS (cont.)
DTG
DTH
ECOD
EGF
EGFR
ER
EROD
EOF
FSH
GC-ECD
GC/MS
GOT
GnRH
GST
HVH
HAH
HCDD
HDL
HxCB
HpCDD
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-Ethoxyresuroftn 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
IDso
I-TEF
LH
LDL
LPL
LOAEL
LOEL
MCDF
MFO
mRNA
MNNG
NADP
NADPH
NK
NOAEL
Heptachlorinated dibenzofuran
High performance liquid chromatography
High resolution gas chromatography/high resolution mass spectrometry
Hexachlorinated dibenzo-p-dioxin
Hexachlorinated dibenzofuran
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
W-methyl-Af-nitrosoguanidine
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate (reduced form)
Natural killer
No-observable-adverse-effect level
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LIST OF ABBREVIATIONS (cont.)
NOEL
OCDD
OCDF
PAH
PB-Pk
PCB
OVX
PEL
PCQ
PeCDD
PeCDF
PEPCK
PGT
PHA
PWM
ppm
ppq
ppt
RNA
SAR
SCOT
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
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LIST OF ABBREVIATIONS (cont.)
SGPT
SRBC
«»
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
Tetrachloroazoxybenzene
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
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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, with Carol
Haynes, Environmental Criteria and Assessment Office in Cincinnati, OH, serving as Project Officer.
During the preparation of this chapter, EPA staff scientists provided reviews of the drafts as
well as coordinating internal and external reviews.
AUTHORS
Nancy Kerkvliet
College of Veterinary Medicine
Oregon State University
Coryallis, OR
EPA CHAPTER MANAGER
Gary R. Burleson
Health and Environmental Research Laboratory
Research Triangle Park, NC
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4. IMMUNOTOXICITY
4.1. INTRODUCTION
Concern over the potential toxic effects of chemicals on the immune system
arises from the critical role that the immune system plays in maintaining health.
It is well recognized that suppressed immunological function can result in
increased incidence and severity of infectious diseases as well as some types of
cancer. Conversely, the inappropriate enhancement of immune function or the
generation of misdirected immune responses can precipitate or exacerbate the
development of allergic and autoimmune diseases. Thus, suppression as well as
enhancement of immune function are considered to represent potential immunotoxic
effects of chemicals.
The immune system consists of a complex network of cells and soluble
mediators that interact in a highly regulated manner to generate immune responses
of appropriate magnitude and duration. Consequently, comprehensive evaluation
of immunotoxicity must include specific assessments of multiple functional
parameters on a kinetic basis. In addition, because an immune response develops
in a time-dependent manner relative to antigen exposure, the immunotoxicity of
a chemical can be profoundly influenced by the timing of chemical exposure
relative to antigen challenge. Consideration of these levels of complexity
involved in immunotoxicology assessment are critical for interpretation of the
effects of chemical exposure on immune function.
Extensive evidence has accumulated over the past 20 years to demonstrate
that the immune system is a target for toxicity of TCDD and structurally related
HAH, including the CDFs, PCBs and PBBs. This evidence has derived from numerous
studies in various animal species, primarily rodents, but also guinea pigs,
rabbits, monkeys, marmosets and cattle. Epidemiological studies also provide
evidence for the immunotoxicity of HAH in humans. In animals, relatively high
doses of HAH produce lymphoid tissue depletion, except in the thymus where
cellular depletion occurs at lower doses. Alterations in specific immune
effector functions and increased susceptibility to infectious disease have been
identified at doses of TCDD well below those which cause lymphoid tissue
depletion. Both cell-mediated and humoral immune responses are suppressed
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following TCDD exposure, suggesting there are multiple cellular targets within
the immune system that are altered by TCDD. Evidence also suggests that the
immune system is indirectly targeted by TCDD-induced changes in nonlymphoid
tissues. In addition, in parallel with increased understanding of the cellular
and molecular mechanisms involved in immunity, studies on TCDD are beginning to
establish biochemical and molecular mechanisms of TCDD immunotoxicity. These
advances will be highlighted in this document.
There is an enormous literature based on descriptive studies on the
immunotoxic effects of TCDD and related HAH in laboratory animals. Unfortu-
nately, due to widely differing experimental designs, exposure protocols, and
immunologic assays used, it has been very difficult to define a "TCDD-induced
immunotoxic syndrome" in a single species, let alone across species. For
example, there is only one report that directly compared the effects of TCDD on
the immune system of rats, mice, and guinea pigs, and, even then, different
immunologic parameters were assessed and different antigens were used in the
different species (Vos et al., 1973). In that study, the DTH response to
tuberculin was evaluated in guinea pigs and rats for assessment of cell-mediated
immunity, while the GVH response was measured in mice. A decreased DTH response
to tuberculin was observed in guinea pigs following 8 weekly doses of 40 ng/kg
TCDD (total dose, 320 ng/kg), while the DTH response of rats to tuberculin was
unaffected by 6 weekly doses of 5 pg/kg TCDD (total dose, 30,000 ng/kg TCDD).
The GVH response in mice was suppressed by 4 weekly doses of 5 ^9/kg TCDD (total
dose, 20,000 ng/kg TCDD). The greater sensitivity of guinea pigs compared to
rats and mice to the immunosuppressive effects of TCDD is consistent with the
greater sensitivity of guinea pigs to other toxic effects of TCDD (McConnell et
al., 1978; Poland and Knutson, 1982). Although these results appear to suggest
that cell-mediated immunity in mice is more sensitive to TCDD than in rats, no
studies have directly compared rats and mice using the same antigens and
endpoints. In another study in mice, the DTH response to oxazolone was
suppressed by 4 weekly doses of 4 pg/Kg TCDD (total dose, 16,000 ng/kg), while
the DTH response to SRBC was unaffected by a 10-fold higher dose of TCDD (Clark
et al., 1981), illustrating that DTH responses to different antigens are not
equally sensitive to TCDD-induced suppression, even in the same species. When
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PCB and PBB studies are considered, variable effects on DTH and other immune
reactions are also apparent (Fraker, 1980; Vos and Van Driel-Grootenhuis, 1972;
Luster et al., 1980a; Thomas and Hinsdill, 1978). Because the exact basis for
the inter-study variability is not known, it would serve no useful purpose in
terms of risk assessment to catalogue all of the effects of TCDD and other HAH
on the immune system that have been reported. Several comprehensive reviews have
been published on the immunotoxic effects of HAH in general (Kerkvliet, 1984; Vos
and Luster, 1989), and TCDD in particular (Holsapple et al., 1991a,b). The
reader is also referred to the previous EPA TCDD-Risk assessment document,
Appendix E (Sonawane et al., 1988) for another perspective on TCDD immuno-
toxicity. The present document will not reiterate this extensive literature, but
rather, will emphasize more recent developments in the field of HAH immuno-
toxicity that may assist in the risk assessment process. Gaps in our knowledge
that require further research will also be identified.
4.2. ROLE OF THE AH LOCUS IN HAH IMMUNOTOXICITY
One of the most important advances in the study of HAH toxicity in recent
years has been the elucidation of a genetic basis for sensitivity to the toxicity
of these chemicals, which may ultimately provide a logical explanation for much
of the controversial data in the literature regarding HAH toxicity in different
species and in different tissues of the same species. In this regard, many of
the biochemical and toxic effects of HAH appear to be mediated via binding to an
intracellular protein known as the Ah or TCDD receptor, in a process similar to
steroid hormone receptor-mediated responses (Poland and Knutson, 1982; Cuthill
et al., 1988). Ah receptor activation follows stereospecific ligand binding;
interaction of the receptor-ligand complex with DREs in the genome induces the
transcription of the structural genes encoding mRNA for CYP1A1 enzyme activity
(i.e., cytochrome Pj450), as well as the expression of additional unidentified
genes, the products of which are hypothesized to mediate HAH toxicity (Whitlock,
1990). Differences in toxic potency between various HAH congeners generally
correlate with differences in Ah receptor binding affinities. The most toxic HAH
congeners are approximate stereoisomers of 2,3,7,8-TCDD and are halogen-
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substituted in at least three of the four lateral positions in the aromatic ring
system.
in mice, allelic variation at the Ah locus has been described (Poland et
al., 1987; Poland and Glover, 1990). The different alleles code for Ah receptors
that differ in their ability to bind TCDD, and thus help to explain the different
sensitivities of various inbred mouse strains to TCDD toxicity. Ahbb C57B1/6
(B6) mice represent the prototype "responsive" strain and are the most sensitive
to TCDD toxicity, while Ahdd DBA/2
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dioxin and furan isomers that contaminate technical grade pentachlorophenol. The
1,2,3,6,7,8-HxCDD, 1,2,3,4,6,7,8-HpCDD and 1,2,3,4,6,7,8-HpCDF isomers, which
bind the receptor, were all significantly immunosuppressive. The dose of each
isomer that produced 50% suppression of the antiSRBC response (1050) was 7.1, 85
and 208 pg/kg for HxCDD, HpCDD and HpCDF, respectively (Figure 4-1). The ID50
for TCDD was 0.65 pg/kg based on the data of Vecchi et al. (1980). In contrast,
OCDD, which does not bind the receptor, was not immunosuppressive at a dose as
high as 500 ^g/kg (Kerkvliet et al., 1985). More extensive structure-dependent
immunosuppressive activities of technical grade PCB mixtures (Davis and Safe,
1990), PCB congeners (Davis and Safe, 1989), and CDF congeners (Davis and Safe,
1988) have also been reported. Results of these studies using different HAH
congeners are summarized in Table 4-1.
The role of the Ah receptor in suppression of the antiSRBC response has
recently been verified in studies using B6 mice congenic at the Ah locus
(Kerkvliet et al., 1990a). As expected, congenic Ahdd-B6 mice were significantly
less sensitive to TCDD-induced immune suppression compared to wild-type Ah -B6
mice. Unexpectedly, however, the dose-response in congenic B6-Ahdd mice appeared
to be bimodal, with a portion of the response sensitive to suppression by low
doses of TCDD. Because of the bimodal response, the data did not permit
extrapolation of an ID50 dose in the congenic mice. The results were interpreted
to suggest potential non-Ah receptor mediated immunosuppressive effects. It
should be noted, however, that recent studies by Dr. Jay Silkworth using
re-derived congenic Ahdd-B6 mice have thus far not corroborated a bimodal dose-
response (Silkworth, personal communication). The issue of Ah receptor-
independent immunotoxicity will be discussed in detail in a subsequent section
of this document.
Ah receptor dependency of HAH immunotoxicity has also been demonstrated in
mice using other immunologic responses. For example, Kerkvliet et al. (1990a)
reported that the ID50 for suppression of the antibody response to TNP-LPS in
Ahbb B6 mice was 7.0 pg/kg compared to a significantly higher ID50 of 30 pg/kg
in congenic Ahdd B6 mice. Since the antibody response to TNP-LPS shows little
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.Anti-SRBC response (% of control)
100 --
80 --
60
40
20
0
ID,
BO
• TCDD
O HxCDD
A HpCDD
A HpCDF
• OCDD
0.65
7.1
85
208
>500
0.1
i i 11 in| 1—i t i i tu| 1—i i i i ni| 1—i i i i ii
10
Dose (/zg/kg)
100
FIGURE 4-1
Structure Dependent Immunotoxicity of Some CDD and CDF Isomers.
Immunotoxicity Assessed by Suppression of the Splenic
Antibody Response to SRBC
Source: Kerkvliet et al., 1985
DIOXINS.HEALTH ASSESSMENT
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TABLE 4-1
TEFs for CDDs, CDFs and PCBs on the Acute Single Dose ID50 fof Suppression of the RFC Response to SRBC
in Ah Responsive B6 Mice
Congener
2,3,7,8-TCDD
2,3,7,8-TCDD
2.3.7,8-TCDD
2,3,7,8-TCDD
1,2,3,6,7,8-HxCDD
1.2,3,4.6,7,8-HpCDD
OCOD
2,3,4,7,8-TCDF
2,3,7,8-TCDF
1,2.3,4,6,7.8-HpCDF
1,2,3, 7,9-PeCDF
1,3,6,8-TCDF
3.4,3',4'-TCB
2,3,4,5,3',4'-HxCB
2.3,4,5,3',4'-HxCB
2,4,3',4',5',6'-HxCB
2,3,4,3',5'-PeCB
2,3,4,5,3',5'-HxCB
2,4,2',4'-TCB
2.4,5,2',4'.6'-HxCB
2,4,6,2',4',6'-HxCB
2,4,5,2'4',5'-HxCB
1D60
0.74 fig/ kg
0.65 fig/kg
0.77 fig/kg
0.60 fig/kg
7.1 tig/kg
85.0 fig/ kg
>500 /ig/kg
1.0 itg/kg
4.3 >ig/kg
208 ng/kg
239 /19/kg
11 /ig/kg
28 mg/kgb
0.7 mg/kg
36 mg/kgb
43 mg/kg
65 mg/kg
72 mg/kg
>100 mg/kg
>360 mg/kg
>360 mg/kg
>360 mg/kg
TEF
1.0a
1.0a
1.0a
1.0a
0.1
8.2x10'3
>1.4x10'3
0.7
1.6X10"1
3.4x10"3
2.9x10"3
6.4x10"5
2.5x10'6
1.0x10"3
1.9x10'5
1.6x10"5
1.1x10"5
9.7x10"6
>7.0x10"6
>1.9x10"6
>1.9x10'6
>1.9x10"6
Reference
Kerkvliet and Brauner, 1990
Vecci et a I., 1980
Davis and Safe, 1988
Kerkvliet et al., 1990a
Kerkvliet et al., 1990s
Kerkvliet et al., 1985
Kerkvliet et al., 1985
Davis and Safe, 1988
Davis and Safe, 1988
Kerkvliet et al., 1985
Davis and Safe, 1988
Davis and Safe, 1988
Silkuorth and Grabstein,
1982
Davis and Safe, 1990
Silkuorth et al., 1984
Davis and Safe, 1990
Davis and Safe, 1990
Davis and Safe, 1990
Silkworth et al., 1984
Davis and Safe, 1990
Davis and Safe, 1990
Biegel et al., 1989
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Aroetor 1248
• Nil • •• ""
Aroclor 1242
"Based on mean IDBO of 0.7±0.07 jtg/kg
blntcrpolated from two data points
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requirement for macrophages or T helper cells (Jelinek and Lipsky, 1987), these
results suggest an Ah receptor dependent B cell response. In terms of cytotoxic
T cells, Clark et al. (1983) was first to report data suggesting that TCDD and
PCB isomers suppressed in vitro CTL responses of B6 and D2 mice through an Ah
receptor-dependent mechanism. Subsequently, Kerkvliet et al. (1990b) reported
that B6 mice congenic at the Ah locus showed Ah-dependent sensitivity to
suppression of the CTL response following exposure to either TCDD or
3,4,5,3',4',5'-HxCB. Furthermore, the potency of TCDD and of three HxCB
congeners to suppress the CTL response of Ahbb-B6 mice directly correlated with
their relative binding affinities for the Ah receptor (Table 4-2). The ID50 of
TCDD for suppression of the CTL response in B6 mice was 7.0 pg/kg .
In summary, the data relating HAH immunotoxicity, at least in part, to Ah
receptor-dependent events are convincing. However, it should be emphasized that
all of the data have been obtained from studies in inbred mice using an acute or
subacute exposure regimen. Except for thymic atrophy, structure-immunotoxicity
relationships in other species including rats have not been established, and the
availability of inbred strains of other species with defined Ah genotype are not
currently available. The importance of Ah receptor mediated events in chronic,
low-level HAH immunotoxicity also remains to be established. Morris et al.
(1992) have recently reported that the sensitivity of D2 mice to TCDD-induced
suppression of the antiSRBC response increased significantly when TCDD was
administered daily over two weeks rather than as an acute single dose.
Unfortunately, in these studies, the lowest dose of TCDD produced near-maximum
suppression of the antiSRBC response of B6C3F1 mice in the acute exposure model,
precluding the detection of any similar increase in sensitivity of the B6C3F1
mice to chronic dosing. In contrast to these findings, Vecchi et al. (1983)
reported that multiple exposures to TCDD (2 M9/kg for 5 weeks or 0.5 /jg/kg for
8 weeks) did not increase the sensitivity of D2 mice to suppression of the
antiSRBC response. Thus, the basis for any change in potency resulting from
^The dose of TCDD required to suppress the CTL response reported by Kerkvliet et al. (I990b) is significantly greater than that
reported by Clark et al. (1981), who reported CTL suppression following four weekly doses of 0.1 fig/kg TCDD. Clark et al (1983) also
reported that doses of TCDD as low as 4 ng/kg to B6 mice suppressed the in vitro generation of CTL and thai the suppression was Ah dependent.
The potency of TCDD described in the Clark el al. (1981, 1983) studies has not been corroborated by other laboratories.
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TABLE 4-2
TEF0 Based on the ID50 for Suppression of Alloantigen
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multiple treatment or chronic exposure to TCDD and the role of Ah receptor-
mediated events in the phenomenon remain to be elucidated.
4.3. TOXIC EQUIVALENCY FACTORS (TEFs) FOR IMMUNOTOXICITY
Based on the available data from mice, the majority of the immunotoxic
effects of HAH appear to be mediated via the Ah receptor. Thus, the toxicity of
different HAH congeners can be compared by calculating TEFs. TEFs based on
acute, single dose exposure (oral or intraperitoneal) of B6 mice to various HAH
for suppression of the antiSRBC response and the CTL response are presented in
Table 4-1 and 4-2, respectively. As shown in Table 4-1, the potency of TCDD to
suppress the antibody response to SRBC has been reported by several laboratories,
with remarkable agreement2 in the ID50 value of 0.7 jug/kg in B6 mice. The ID50
of B6C3F1 mice is similar (<1 /jg/kg) (House et al., 1990) or slightly higher (1.2
pg/kg) (Holsapple et al., 1986a) in comparison to B6 mice. This data thus
provides a well-defined base value to use in calculating TEFs for other HAH
congeners in the context of suppression of the antiSRBC response.
However, in contrast to the reproducible data on TCDD, the accuracy of the
derived TEFs for other HAH congeners shown in Table 4-1 is difficult to evaluate
since few congeners have been examined in more than one study. In the few cases
where the same congener has been evaluated independently, discrepancies in the
data exist. For example, both Davis and Safe (1990) and Silkworth et al. (1984)
evaluated the potency of the 2,3,4,5,3',4'-HxCB congener in the antiSRBC
response. Based on the ID50's from the two data sets, the respective TEFs differ
by almost two orders of magnitude (IxlO"3 versus 2xlO"5). When the same congener
was compared to TCDD for suppression of the CTL response, the TEF was 1x10
(Table 4-2). The basis for these discrepancies is unknown. Thus, the data base
^Several laboratories have reported that the antibody response to SRBC is sensitive to suppression following acute exposure to TCDD,
either inlraperitoneally or orally, at doses < I fig/kg. In contrast, Clark et al. (1981) reported thai 4 weekly ip doses of 10 but not 1 or 0.1
US/kg TCDD significantly suppressed the antiSRBC response in B6 mice. The chronic dosing protocol used by Clark does not readily explain
his decreased potency since Vecchi et al (1983) reported that five weekly doses of 2 fig/kg or eight weekly doses of 0.5 pg/kg TCDD signifi-
cantly suppressed the antiSRBC response. Likewise, when total doses of 0.2 or 1 ng/kg TCDD were given as a single dose or divided into five
daily doses, the divided dose produced slightly more suppression that the single dose (Table 4-3) (Kerkvliet and Deyo, unpublished data). In
addition, the route of antigen challenge [intravenous (used by Clark) versus inlraperiloneal (used by Vecchi)] does not appear to greatly influence
the degree of suppression of the anliSRBC response produced by TCDD (Table 4-4) (Kerkvliet and Deyo, unpublished data). Thus, the basis
for the discrepancies between the data of Clark el al. (1981, 1983) and other laboratories regarding the potency of TCDD to suppress the
antiSRBC response is unknown.
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TABLE 4-3
Effect of Single Versus Multiple Dosing with TCDD on Suppression
of the Antibody Response to SRBC in C57B1/6 Mice
Plaque-Forming Cells/106 Spleen Cells
(mean ± SD)
Total Dose
Single3
Multiple1
2460±657
3846±1618
0.2
18791445 (76)
2356±592 (61)
1.0
1293±285 (52)c
1143±208 (30)'
aTotal dose of TCDD given once two days prior to SRBC injection
blotal dose of TCDD divided into five equal doses administered on
days -7 to -2 prior to SRBC injection
cp<0.01
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TABLE 4-4
Influence of Route of Antigen Challenge on Suppression of the
Antibody Response to SRBC in C57B1/6 Mice3
Dose of TCDD
(^9/kg)
0
0.2
1.0
Plaque-Forming Cells/10b Spleen Cells
(mean ± SD)
Intravenous
1151±367
623±324 (54)
4661212 (40)b
Intraperitoneal
1812±872
1197±519 (66)
697±163 (38)b
a2.5xlO^ SRBC were injected intravenously or intraperitoneally
2 days after oral dosing with TCDD; plaque-forming cells were
measured 5 days later.
bp< 0.01
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for TEF comparisons using immunotoxicity data must be expanded considerably
before TEFs can be used with confidence in risk assessment.
4.4. INTERACTIONS BETWEEN HAH
If the immunotoxicity of TCDD and structurally related HAH depend on Ah-
receptor mediated mechanisms, then co-exposure to subsaturating levels of more
than one Ah receptor ligand should produce additive effects. An additive
interaction has been demonstrated in mice co-exposed to 1,2,3,6,7,8-HxCDD and
1,2,3,4,6,7,8-HpCDD, two relatively strong Ah receptor ligands (Kerkvliet et al.,
1985). On the other hand, Davis and Safe (1988, 1989) have reported that
co-exposure of mice to an immunotoxic dose of TCDD and a subimmunotoxic dose of
different commercial Aroclors or different PCB congeners resulted in partial
antagonism of TCDD suppression of the antiSRBC response. In limited studies, an
apparently similar antagonism was observed following co-exposure to 2,3,7,8-TCDF
(10 pg/kg) and TCDD (1.2 pg/kg) (Rizzardini et al., 1983). The mechanism for the
antagonism has not been fully elucidated, but the effects are consistent with
competition for binding at the Ah receptor, since the weaker agonist was
administered in excess compared to TCDD. Furthermore, Silkworth et al. (1988)
and Silkworth and O'Keefe (1992) have shown that the immunotoxicity of TCDD can
be modified by coexposure to other HAH present as co-contaminants of actual
environmental samples from Love Canal. Such interactions complicate hazard
assessment of mixtures based on TEFs and may preclude dependence on TEFs without
biological response evaluation for risk assessment.
4.5. SENSITIVE TARGETS FOR HAH IMMUNOTOXICITY
Despite considerable investigation, the cells that are altered by HAH
exposure leading to suppressed immune function have not been unequivocally
identified. The main reason for the lack of definitive progress in this area is
the conflicting data reported from different laboratories regarding the ability
of TCDD to suppress lymphocyte functions when examined "ex vivo" or in vitro.
As discussed in a subsequent section of this document, the in vitro effects of
TCDD are greatly influenced by the in vitro culture conditions, which may explain
the discrepancies in effects observed in different laboratories.
In contrast to in vitro studies, the in vivo immunotoxicity of TCDD,
expressed in terms of suppression of the antiSRBC response of B6 or B6C3F1 mice,
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is highly reproducible between laboratories. Since the magnitude of the antiSRBC
response depends on the concerted interactions of antigen-presenting cells (APC),
regulatory T cells (helper and suppressor), and B cells, this response has been
used most widely to evaluate target cell sensitivity to HAH. In addition, the
antibody response to SRBC can be modulated by many non-immunological factors,
including hormonal and nutritional variables, and HAH are known to affect
numerous endocrine and metabolic functions. These latter effects will be
apparent only in in vivo studies, while only direct effects of HAH on APC and
lymphocyte functions would be evident following in vitro exposure to HAH. To
date, direct in vitro effects of TCDD on purified B cell activity have been
reported (Holsapple et al., 1986a; Morris and Holsapple, 1991; Luster et al.,
1988), while direct effects on macrophages and T cells in vitro have not been
described. (The in vitro effects of TCDD will be discussed in more detail in a
subsequent section of this document.)
Kerkvliet and Brauner (1987) compared the sensitivity of antibody responses
to antigens that differ in their requirements for APC and T cells as an in vivo
approach to evaluate the cellular targets of 1,2,3,4,6,7,8-HpCDD humoral
immunotoxicity. The T-helper cell independent (TI) antigens, DNP-Ficoll and TNP-
LPS, were used in these studies. These TI antigens differ from each other in
their requirement for APC (higher for DNP-Ficoll) and their sensitivity to
regulatory (amplifier and suppressor) T cell influence (DNP-Ficoll is sensitive,
TNP-LPS is not) (Braley-Mullen, 1982). Obviously, all antibody responses require
B cell differentiation into antibody-secreting plasma cells. Although HpCDD
produced dose-dependent suppression of the antibody response to all three
antigens, sensitivity to suppression directly correlated with the sensitivity of
the response to T cell regulation. The ID50 values were 53, 127 and 516 pg/kg
for SRBC, DNP-Ficoll, and TNP-LPS, respectively. These results were interpreted
as follows: If one assumes that B cell function is targeted in the TNP-LPS
response, then regulatory T cells and/or APC may represent the more sensitive
target in the SRBC and DNP-Ficoll responses. The difference in sensitivity
between the SRBC and DNP-Ficoll responses suggest that the T helper cell may be
a particularly sensitive target. The differential sensitivity of the antibody
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responses to TNP-LPS versus SRBC has been corroborated in TCDD-treated mice
(House et al. 1990; Kerkvliet et al., 1990a). Thus, the exquisite ±n vivo
sensitivity of the antibody response to SRBC would appear to depend on the T cell
and/or APC components of the response rather than the B cell, unless the B cells
that respond to SRBC are different from the B cells that respond to TNP-LPS.
Currently, evidence for such a difference is lacking. However, this interpreta-
tion conflicts with the "ex vivo" data of Dooley and Holsapple (1988). Using
separated spleen T cells, B cells and adherent cells from vehicle- and TCDD-
treated mice, they reported that B cells from TCDD-treated mice were functionally
compromised in in vitro antibody responses but T cells and macrophages were not.
The basis for'these discrepant findings has not been established. However, it
is possible that the effects of TCDD on T cells are indirectly induced following
antigen exposure such that removal of the cells from the TCDD environment of the
host prior to antigen challenge would preclude detection of T cell dysfunction.
This interpretation is supported by the findings of Tomar and Kerkvliet (1991)
that spleen cells taken from TCDD-treated mice were not compromised in their
ability to reconstitute the antibody response of lethally irradiated mice. This
interpretation is also consistent with the reported lack of direct effects of
TCDD and other HAH on T cells in vitro (Clark et al., 1981; Kerkvliet and
Baecher-Steppan, 1988a).
While the direct effects of TCDD on T cells in vitro have not been
demonstrated, it is clear that functional T cell responses generated in vivo are
compromised following in vivo exposure. Nude mice that are congenitally T cell
deficient are significantly less sensitive to HpCDD-induced immunotoxicity when
compared to their T cell-competent littermates (Kerkvliet and Brauner, 1987)
Likewise, exposure to TCDD or HxCB suppresses the development of CTL activity
following alloantigen challenge (Kerkvliet et al., 1990b). Interestingly, the
sensitivity of the CTL response to suppression by TCDD is approximately the same
as the sensitivity of the antibody response to TNP-LPS (both have an ID50 of
approximately 7 /jg/kg in B6 mice) (Kerkvliet et al., 1990a,b). since the B cell
response to TNP-LPS and the CTL response to I-A" P815 alloantigen have little
requirement for antigen-presenting cells or classic CD4+ T helper cells, these
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results suggest that the differentiation of B cells and CD8+ CTLp to effector
cells have an equivalent "functional" sensitivity to TCDD.
The influence of TCDD exposure on regulatory T cell functions has been
addressed in a limited number of studies. Clark et al. (1981) first proposed
that T suppressor cells were induced by TCDD in the thymus that were responsible
for the suppressed CTL response. However, increased suppressor cell activity in
peripheral lymphoid tissue was not observed in mice exposed to TCDD (Dooley et
al., 1990) or 3,4,5,3',4',5'-HxCB (Kerkvliet and Baecher-Steppan 1988b). In
terms of T helper cell activity, Tomar and Kerkvliet (1991) reported that a dose
of 5 /jg/kg TCDD suppressed the in vivo generation of carrier-specific T helper
cells. Lundberg et al. (1990) reported that thymocytes from B6 mice treated with
TCDD (50 jjg/kg) were less capable of providing help for an in vitro antiSRBC
response. However, Clark et al. (1983) reported in ex vivo studies that T cells
from TCDD-treated mice produced normal levels of IL-2. The in vivo effect of
TCDD on the production of IL-2 as well as other lymphokiries important in the
development of an antibody response (e.g., IL-4, IL-5) have not been reported.
The influence of TCDD exposure on B cell function has been addressed
primarily in in vitro studies. The issue is difficult to address in vivo given
that most B cell responses (except perhaps anti-LPS responses) are dependent on
interactions with T cells and macrophages. In vitro studies have described the
direct effects of TCDD on the activation and differentiation of purified B cells
(Luster et al., 1988; Morris et al., 1991). These studies suggest that TCDD
inhibits the terminal differentiation of B cells via alteration of an early
activation event (Luster et al., 1988). Increased phosphorylation and tyrosine
kinase activity in TCDD-treated B cells may underlie this B cell dysfunction
(Kramer et al., 1987; Clark et al., 1991a).
Macrophage functions have also been examined following TCDD exposure and
generally found to be resistant to suppression by TCDD when assessed ex vivo.
Macrophage-mediated phagocytosis, macrophage-mediated tumor cell cytolysis or
cytostasis, oxidative reactions of neutrophils and macrophages, and NK (cell
activity) were not suppressed following TCDD exposure, with doses as high as 30
failing to suppress NK and macrophage functions (Vos et al., 1978;
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Mantovani et al., 1980). A potentially important exception is the reported
selective inhibition of phorbol ester-activated antitumor cytolytic and
cytostatic activity of neutrophils by TCDD (Ackermann et al., 1989).
On the other hand, it is interesting to note that the pathology associated
with TCDD toxicity often includes neutrophilia and an inflammatory response in
liver and skin characterized by activated macrophage and neutrophil accumulation
(Weissberg and Zinkl, 1973; Vos et al., 1973; Vos et al., 1974; Puhvel et al.,
1988). While these observations may simply reflect a normal inflammatory
response to tissue injury, there is some preliminary experimental evidence that
suggests inflammatory cells may be activated by TCDD exposure. For example,
Alsharif et al. (1990) recently reported that TCDD increased superoxide anion
production in rat peritoneal macrophages. In addition, it has been shown that
TCDD exposure results in an enhanced inflammatory response following SRBC
challenge (Kerkvliet and Brauner, 1990b). This effect of TCDD was characterized
by a 2-4 fold increase in the number of neutrophils and macrophages locally
infiltrating the intraperitoneal site of SRBC injection. However, the kinetics
of the cellular influx was not altered by TCDD. Likewise, the expression of
macrophage activation markers (I-A and F4/80) and the antigen-presenting function
of the peritoneal exudate cells was unaltered by TCDD. Thus, the effect of TCDD
appeared to reflect a quantitative rather than a qualitative change in the
inflammatory response. Importantly, TCDD-induced suppression of the antiSRBC
response could not be overcome by increasing the amount of antigen used for
sensitization, suggesting that enhanced antigen clearance/degradation by the
increased numbers of phagocytic cells (e.g., decreased antigen load) was not
responsible for the decreased antibody response in TCDD-treated mice. Thus, the
relationship, if any, between the inflammatory and immune effects of TCDD remain
to be elucidated.
One mechanism by which TCDD and related HAH may augment inflammatory
responses is via enhance production of inflammatory mediators such as interleukin
1 (IL-1) and TNF. Recent evidence suggests that the long-recognized hypersuscep-
tibility of TCDD- and PCB-treated animals to endotoxin (LPS) (Thomas and
Hinsdill, 1978; 1979; Vos et al., 1978; Loose et al., 1979) may be related to an
increased production of TNF and/or IL-6 in the chemically treated animals (Clark
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et al., 1991b; Taylor et al., 1990; Hoglen et al., 1992). The ability of
methylprednisolone to reverse the mortality associated with TCDD/endotoxin
treatment is also consistent with an inflammatory response (Rosenthal et al.,
1989). Similarly, increased inflammatory mediator production may underlie the
enhanced rat paw edema response to carrageenan and dextran in TCDD-treated rats
(Theobald et al., 1983; Katz et al., 1984). Limited, preliminary data is
available to indicate that the production of inflammatory mediators such as TNF
(Taylor et al., 1990; Clark et al., 1991b) and IL-6 (Hoglen et al., 1992) may be
increased in HAH-treated animals. Serum complement activity, on the other hand,
has been reported to be suppressed in dioxin-treated mice (White et al., 1986),
although enhanced activity was reported at the lowest exposure level when
1,2,3,6,7,8-hexachlorodioxin was tested. A primary effect of TCDD on IL-1 is
supported by the recent findings of Sutter et al. (1991) that the IL-lp gene
contains a ORE. Likewise, Steppan and Kerkvliet (1991) have reported that under
some exposure conditions TCDD increased the level of mRNA for IL-1 in TCDD-
treated IC21 cells, a macrophage cell line derived from B6 mice. On the other
hand. House et al. (1990) reported that inflammatory macrophages obtained from
TCDD-treated mice produced control levels of IL-1 when examined ex vivo. Thus,
the effect of TCDD on inflammatory mediator production may be a "priming effect"
and require co-exposure to antigen or LPS. The influence of TCDD on inflammatory
mediator production and action is an important area for further study.
Since the rapid influx of phagocytic cells to the site of pathogen invasion
is an important factor in host resistance to infection, the ability of TCDD to
augment the production of inflammatory chemoattractive mediators would imply that
TCDD exposure could result in enhanced host resistance. However, since TCDD
exposure is, at the same time, immunosuppressive, resulting in decreased specific
immune responses generated by T and B lymphocytes, the overall impact of TCDD
exposure on disease susceptibility will likely vary depending on the nature of
the pathogen and the major mode of host response to the specific infectious
agent. Such effects may in fact help to explain the disparate effects of TCDD
in different host resistance models that have been previously reported.
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4.6. INFLUENCE OF TCDD ON HOST RESISTANCE TO DISEASE
The ability of an animal to resist and/or control viral, bacterial,
parasitic and neoplastic diseases is determined by both nonspecific and specific
immunological functions. Decreased functional activity in any immunological
compartment may result in increased susceptibility to infectious and neoplastic
diseases. In terms of risk assessment, host resistance is often accorded the
"bottom line" in terms of relevant immunotoxic endpoints. Animal host resistance
models that mimic human disease are available and have been used to assess the
effect of TCDD on altered host resistance.
TCDD exposure increases susceptibility to challenge with the gram negative
bacterium Salmonella. TCDD was given per os at 0.5 to 20 ^g/kg once a week for
4 weeks to male 4-week-old C57Bl/6Jfh (J67) mice and challenged 2 days after the
fourth dose (when mice were 8 weeks old) with either Salmonella hern or
tferpesvirus suzs (also known as pseudorabies virus). Results with S. 2>ern
indicated there was an increased mortality at 1 ^g TCDD/kg (total dose of 4
pg/kg) and a reduced time to death after bacterial challenge with 5 »q TCDD/kg
(total doae of 20 /jg/kg) / In contrast, the same doses of TCDD did not alter the
time to death or the incidence of mortality following H. suis infection (Thigpen
et al., 1975). A TCDD feeding study by Hinsdill et al. (1980) also demonstrated
increased susceptibility of 7-week-old Swiss Webster outbred female mice to
Salmonella typhimurium var. Copenhagen. Mice were fed control feed or feed
containing 10, 50, or 100 ppb TCDD for 8 weeks, after which they were injected
intravenously with 103'5 S. typhimurium var. Copenhagen. Results indicated that
50 and 100 ppb TCDD increased mortality from Salmonella and shortened the time
to death while 10 ppb caused an increased bacteremia. Vos et al. (1978) reported
that TCDD resulted in a reduced resistance to endotoxin (Eschericnia coli O 127:B
8 lipopolysaccharide) and suggested that the increased susceptibility to
Salmonella caused by TCDD may be due to the lipopolysaccharide or endotoxin
present on this gram negative bacterium. Vos et al. (1978) demonstrated reduced
resistance to endotoxin with a single oral dose of 100 Ug TCDD/kg using 3- to
4-week-old outbred female mice and challenged with endotoxin 5 days later. Vos
et al. (1978) also reported enhanced mortality from the intravenous injection of
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endotoxin 2 days after the final oral dose of TCDD (1.5, 5, 15 or 50 pg/kg once
a week for 4 weeks) in 3- to 4-week-old male outbred Swiss mice. These studies
indicate a reduced resistance to endotoxin after single or multiple doses of
TCDD. Thomas and Hinsdill (1979), using S. typhimurium lipopolysaccharide,
demonstrated a reduced resistance to endotoxin in the offspring of female Swiss
Webster mice fed TCDD prior to mating, during gestation and between parturition
and weaning. Rosenthal et al.{1989) used female B6C3F1, DBA/2, as well as
congenic mice to demonstrate that acute doses of 50, 100 or 200 fjg TCDD per os
increased endotoxin-induced mortality in B6C3F1 mice, associated with hepatotox-
icity and decreased clearance of the endotoxin. D2 and Ah^ congenic mice were
relatively resistant to this effect implicating Ah receptor dependent mechanisms
in endotoxin hypersensitivity.
White et al.(1986) reported that Streptococcus pneumoniae, a gram positive
bacterium that does not contain endotoxin, caused increased mortality in 5- to
6-week-old female B6C3F1 mice after subchronic oral administration of TCDD
(1 pg/kg for 14 days) and challenged with S. pneumoniae intraperitoneally 1 day
after the last treatment. The 1,2,3,6,7,8-HCDD isomer also resulted in a
dose-dependent increase in susceptibility to S. pneumoniae.
Enhanced susceptibility to viral disease has also been reported after TCDD
administration. Clark et al. (1983) injected TCDD intraperitoneally once a week
for 4 weeks and challenged mice 7-22 days later with Herpes simplex type II
strain 33 virus. Mice receiving TCDD at 0.04, 0.4, or 4.0 pg/kg weekly (total
dose of 160, 1600 and 16,000 ng/k) all had significantly enhanced mortality to
Herpesvirus type II infection. House et al. (1990) also reported an enhanced
susceptibility to viral infection following low level single dose TCDD
administration intraperitoneally,, B6C3F1 female mice, 6-8 weeks of age, were
challenged with Influenza/A/Taiwan/1/64 (H2N2) virus 7-10 days following TCDD.
TCDD administration at 10, 1.0 and 0.1 pg/kg decreased resistance to virus.
TCDD exposure also results in more severe parasitic diseases. Tucker et al.
(1986) studied the effects of TCDD administration on Plasmodium yoelii 17 XNL,—
a nonlethal strain of malaria, in 6- to 8-week-old B6C3F1 female mice. A single
dose of TCDD at 5 pg/Kg or 10 pg/kg per os resulted in increased susceptibility
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to P. yoelii. The peak parasitemia was greater and of longer duration in
TCDD-treated animals than in controls, the difference being significant at 5
pg/kg on day 10 and at 10 /jg/kg on days 12 and 14.
Luster et al. (1980b) demonstrated enhanced growth of transplanted tumors
in mice treated with TCDD at doses of 1.0 or 5.0 pg/Kg in B6C3F1 mice. Mothers
were given TCDD by gavage at day 14 of gestation and again on days 1, 7 and 14
following birth; host resistance studies were performed 6-8 weeks after weaning.
This exposure protocol resulted in an increased incidence of PYB6 tumors in pups
from dams receiving repeated doses of 1.0 but not 5.0 ^g TCDD/Kg.
While it is clear that TCDD adversely affects numerous host resistance
models detailed above, the effects of TCDD on susceptibility to Listeria
nonocytogenes infections are ambiguous. The disparate results may reflect
different study designs including dose, route, single versus multiple administra-
tions, mouse strain, age or sex. However, it is clear that TCDD, under certain
conditions, results in increased susceptibility to Listeria. Hinsdill et al.
(1980) reported the increased susceptibility of 7-week-old Swiss Webster
outbred female mice to Listeria. Mice were fed control feed or feed containing
10 or 50 ppb TCDD for 8 weeks, after which they were injected intravenously with
105 iisteria. Results indicated that the 50 ppb diet increased bacteremia and
mortality. Luster et al. (1980b) used doses of 1.0 or 5.0 ug TCDD/Kg in B6C3F1
mice. Mothers were given TCDD by gavage at day 14 of gestation and again on days
1, 7 and 14 following birth and host resistance studies were performed 6-8 weeks
after weaning. This exposure protocol resulted in an increased susceptibility
to Listeria in pups from dams receiving repeated doses of 5.0 pg TCDD/Kg.
However, Vos et al. (1978) reported that oral administration of 50 pg TCDD/kg
once a week for 4 weeks to 3- to 4-week-old male Swiss mice followed by
intravenous challenge 4 days after the last dose with Listeria had no affect on
nonspecific phagocytosis and killing of Listeria. House et al. (1990) used
B6C3F1 female mice, 6-8 weeks of age, and challenged intravenously with Listeria
7-10 days following a single dose of TCDD at 10, 1.0 and 0.1 »g/kg. TCDD did not
enhance mortality to Listeria.
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In summary, results from host resistance studies provide evidence that
exposure to TCDD results in increased susceptibility to bacterial, viral,
parasitic and neoplastic disease. These effects are observed at low doses and
likely result from TCDD-induced suppression of immunological function. However,
it is interesting that the role of the Ah receptor has not been addressed in
terms of host resistance models except in studies on endotoxin hypersensitivity
by Rosenthal et al. (1989). Furthermore, the specific immunological functions
targeted by TCDD in each of the host resistance models remain to be fully
defined.
4.7. IN VITRO IMMUNOTOXIC EFFECTS OF HAH
Investigators in the field of TCDD immunotoxicity have long acknowledged the
difficulties in consistently demonstrating the immunotoxicity of TCDD when cells
from treated animals are tested ex vivo as well as when TCDD is added to culture
in vitro. While effects following in vitro and ex vivo exposure to TCDD on
lymphocyte functions have been reported (Tucker et al., 1986; Luster et al.,
1988; Dooley and Holsapple, 1988), other laboratories have failed to observe
suppression with in vitro-or ex vivo exposure to dioxin (Lundberg et al., 1990;
Clark et al., 1981; Kerkvliet and Baecher-Steppan, 1988b; Kerkvliet, unpublished
data). In addition, the effects of TCDD seen in vitro are sometimes inconsistent
with those observed after in vivo assessment of immunotoxicity. For example, the
rank order of sensitivity to suppression of T helper cell-dependent and T helper
cell-independent antibody responses seen in vivo (Kerkvliet and Brauner 1987;
1990a; House et al., 1990) is not seen in vitro (Holsapple et al., 1986a; Tucker
et al., 1986) suggesting different cellular targets may be affected following in
vitro exposure to TCDD. More importantly, some data suggest that suppression of
the in vitro antibody response may occur independent of the Ah receptor. Tucker
et al. (1986) and Holsapple et al. (1986a) reported that direct addition of TCDD
in vitro suppressed the antibody response to SRBC. However, based on the
response of cells from congenic mice as well as a limited structure-activity
study, the data of Tucker et al. (1986) supported an Ah receptor-dependent
suppression while the data of Holsapple et al. (1986a) did not. In the latter
study, the magnitude of suppression was comparable using cells from responsive
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B6C3F1 or congenio heterozygous (B6-Ahbd) mice compared to nonresponsive D2 or
homozygoua B6-Ahdd mice. In addition, they reported that the 2,7-dichloro-
dibenzo-p-dioxin congener which lacks affinity for the Ah receptor, was
equipotent with TCDD in suppressing the in vitro response.
in other studies, Davis and Safe (1991) directly compared the in vitro
Btructure-immunotoxicity relationships for a series of HAH congeners which show
>14,900 fold-difference in in vivo immunotoxic potency. Results of these studies
indicated that all of the congeners were equipotent in vitro and produced a
similar concentration-dependent suppression of the in vitro antiSRBC response
using cells from either B6 or D2 mice. Co-exposure to the Ah-receptor antagonist
a-napthoflavone antagonized the immunosuppression induced by either TCDD or
1,3,6,8-TCDF (a weak Ah receptor agonist). Collectively, the results supported
a mechanism of suppression in vitro that was independent of the Ah receptor.
The basis for these variable effects of TCDD in vitro are currently not
known. However, recent studies by Morris et al. (1991) demonstrated that the in
vitro effects of TCDD on the antiSRBC response were critically dependent on the
type and concentration of the serum used in the in vitro culture. Only 3 of 23
lots of serum were able to support a full dose-responsive suppression, and, in
serum-free cultures, TCDD caused a 15-fold enhancement of the antiSRBC response.
Thus, differences in media components used in in vitro cultures may account for
the different effects seen in vitro in different laboratories. Other factors
such as the TCDD carrier/solvent used, the calcium content of the media, or
procedures used for preparation of spleen cell suspensions may all contribute to
variable effects of TCDD in vitro.
The obvious question relates to the relevance of the in vitro findings to
the in vivo immunotoxicity. In this respect, it is important to note that the
concentrations of TCDD required for in vitro suppression of immune function
(1-30X10'9 M) of murine lymphocytes is several orders of magnitude higher than
the concentration found in lymphoid tissues following exposure in vivo to an
immunotoxic dose of TCDD (Neumann et al., 1992). The amount of TCDD associated
with isolated spleen cells obtained from mice 2 days following treatment with 5
pg/kg 3H-TCDD was 2xlO'15 M per 107 spleen cells. Importantly, as much as 50% of
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the radioactivity associated with whole spleen tissue was recovered in the
stromal and/or capsular material (i.e., splenic tissue that resisted passage
through the mesh screens used for preparation of spleen cell suspensions). These
findings suggest that the most potent effects of TCDD on immune function in vivo
may be induced indirectly by effects on nonlymphoid cells, or that based on the
delivered dose of TCDD, this molecule is more toxic than previously thought.
Alternatively, TCDD effects in vivo on nonlymphoid cells may amplify the direct
effects of TCDD on lymphoid tissue. Certainly, additional studies are needed to
elucidate the serum components that are permissive for suppression or enhancement
of immune responses in vitro and to determine their relevance to in vivo
conditions. Such studies are also likely to provide insight into the mechanisms
of TCDD interaction with lymphoid cells.
4.8. INDIRECT MECHANISMS OF HAH IMMUNOTOXICITY
The difficulty in demonstrating consistent, direct effects of TCDD in vitro
on lymphocytes, the dependence of -those effects on serum components, and the
requirement for high concentrations of TCDD are all consistent with an indirect
mechanism of TCDD on the immune system. One potentially important indirect
mechanism is via effects on the endocrine system. Several endocrine hormones
have been shown to regulate immune responses, including glucocorticoids, sex
steroids, thyroxine, growth hormone and prolactin. Importantly, TCDD and other
HAHs have been shown to alter the activity of all of these hormones (see chapter
on endocrine system, this document).
Kerkvliet et al. (1990b) reported that exposure of mice to 3,4,5,3',4',5'-
HxCB followed by injection of P815 allogeneic tumor cells induced a dose-
dependent elevation of serum corticosterone concentrations which correlated with
the dose-dependent suppression of the antiP815 CTL response. However, since
adrenalectomy or treatment with the glucocorticoid receptor antagonist RU38486
failed to protect mice from the immunosuppressive effect of HxCB (DeKrey et al.,
1990), a role for the elevated CS in the suppression of the CTL response seems
unlikely. Adrenalectomy and hypophysectomy also failed to prevent TCDD-induced
thymic atrophy in rats (Van Logten et al., 1980).
Using the P815 allogeneic tumor model, Kerkvliet and Baecher-Steppan (1988a)
reported that male mice were more sensitive than female mice to suppression of
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the CTL response by HxCB (Kerkvliet and Baecher-Steppan, 1988a). Castration of
male rats partially ameliorated the immunosuppressive effects of HxCB (DeKrey et
al., 1992), suggesting a role for testosterone in suppression of the CTL
response.
Pazdernik and Rozman (1985) suggest that thyroid hormones may play a role
in TCDD immunotoxicity based on the finding that radiothyroidectomy prevented the
suppression of the antiSRBC response in rats treated with TCDD. However, since
thyroidectomy alone suppressed immune function, the significance of the findings
require further study.
4.9. ROLE OF THE THYMUS IN HAH IMMUNOTOXICITY
Thyraic involution is one of the hallmarks of exposure to TGDD and related
HAH in all species examined. In mice, thymic involution occurs by an Ah receptor
dependent mechanism (Poland and Knutson, 1982). Because the thymus plays a
critical role in the ontogeny of T lymphocytes, thymic involution is often
referred to as an immunotoxic effect. However, while an intact thymus is crucial
to the developing immune system during the prenatal and early postnatal period
of rodents as well as during the prenatal period of humans, the physiological
role played by the thymus in adult life has not been established. In animal
models, adult thymectomy has little affect on the quantity or quality of
T lymphocytes, which have already matured and populated the secondary lymphoid
organs (Benjamini and Leskowitz, 1991). Likewise, in humans, childhood and adult
thymectomy produces no clearly identifiable adverse consequences in terms of
altered immune function, although some might argue that such studies have not
been done. Based on this knowledge, it is not surprising that a direct
relationship between the effects of TCDD on the thymus and immune suppression has
not been established in studies using adult animals. In fact, adult thymectomy
prior to HAH exposure did not modify TCDD- or HpCDD-induced suppression of the
antiSRBC response (Tucker et al., 1986; Kerkvliet and Brauner, 1987).
Furthermore, suppression of immune responses occurs at dose levels of HAH
significantly lower than those required to induce thymic atrophy (Vos et al.,
1978; Silkworth and Antrim, 1985; Holsapple et al., 1986b; Tucker et al., 1986;
Kerkvliet and Brauner, 1990a). Thus, it is clear that thymic involution does not
represent a surrogate marker for TCDD immunotoxicity in adult animals. On the
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other hand, it is possible that chronic exposure to TCDD resulting in a chronic
thymic atrophy may produce more delayed, subtle effects on immune function not
yet identified (Clarke and MacLennan, 1986).
In contrast to adult animals, congenital thymic aplasia or neonatal
thymectomy results in severe reduction in the number and function of T lympho-
cytes, and produces a potentially lethal wasting disease (Benjamini and
Leskowitz, 1991). Similarly, there is evidence from studies carried out in the
1970's that rodents exposed to TCDD or PCBs during the pre/neonatal period are
more sensitive to immune suppression compared to rodents exposed as adults, and
that the prenatal effects are more selective for cell-mediated immunity (Vos and
Moore, 1974; Faith et al., 1978; Luster et al., 1980b). TCDD has also been shown
to alter thymocyte differentiation in vitro in cell cultures (Greenlee et al.,
1985; Cook et al., 1987) and organ cultures (Dencker et al., 1985; d'Argy et al.,
1989) as well as in vivo following prenatal exposure to TCDD (Blaylock et al.,
1992). These observations suggest that altered thymic T cell maturation induced
by TCDD in the thymus may play an important role in the suppressed immune
function of prenatally exposed animals. However, since TCDD also influences B
cell development in the bursa of chick embryos (Nikolaidis et al., 1990) as well
as lymphocyte stem cells in the fetal liver and bone marrow of mice (Fine et al.,
1989; 1990), other mechanisms of immunotoxicity are also likely to be important.
4.10. IMMUNOTOXICITY FOLLOWING PRE/NEONATAL EXPOSURE TO HAH
The reported increase in susceptibility of very young animals to HAH
immunotoxicity necessitates a close examination of the available literature on
prenatal/neonatal immunotoxic effects. Several studies have examined immune
function in mice, rats and/or guinea pigs following exposure to TCDD or PCB
during fetal development (Vos et al., 1973; Vos and Moore, 1974; Thomas and
Hinsdill, 1979; Luster et al., 1980a).
The results of three major studies in which exposure of the progeny occurred
via placental transfer and lactation are summarized in Table 4-5. The most
sensitive indicator of TCDD immunotoxicity in these studies was an increase in
the growth of transplanted tumor cells in the offspring of B6C3F1 mice (Ah
responsive strain) treated with 1 pg/kg TCDD at 4 weekly intervals. (Total TCDD
dose to dam was 4 pg/kg; dose to offspring was not determined.) The offspring
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TABLE 4-5
Inmunotoxic Effects of TCDD in the Offspring Following Pre/Neonatal Exposure to TCDD
Protocol 1
s====— =========s==ss====r:
Study #1:
Pregnant B6 or B6C3F1
mice oiven 1, 2, 5 or 15
-7, 0, +7, +14 relative
to parturition on day 0
I
Study «1:
Pregnant Swiss mice fed
diets containing 1.0,
2.5 or 5.0 ppb TCOD for
7 weeks (4 weeks prior
I to and 3 weeks after
I birth)
Study #3:
Pregnant Fischer 344
rats given 1 or 5 /tg/kg
TCDD orally on days -3,
j 0, +7 and +14 relative
I to parturition on day 0
I
Endpoints I
==_=======— =========4=
PYB6 tumor incidence
allograft rejection time
body, thymus, spleen weight
bone marrow cellularity
Con A, PHA blastogenesis
mortality following Listeria
monocytogenes challenge
bone marrow CFU-S colonies
mortality
IPS blastogenesis
anti-SRBC serum liters
endo toxin
thymus weight
PFC response to SRBC
DTK response
anti-SRBC serum liters
LPS and Con A blastogenesis
Listeria- i nduced morta I i ty
allograft rejection time
PHA blastogenesis
DTH response
mortality following Listeria
challenge
body and thymus weight
anti-BGG serum liters
Effect LOAEL
increased
increased
decreased
decreased
decreased
decreased
decreased
increased
--
--
increased
decrease
decreased
decreased
--
--
--
increased
decreased
decreased
increased
decreased
--
1 /tg/kg x 4
2 /ig/kg x 4
5 /ig/kg x 4
5 /tg/kg K 4
5 /ig/kg x 4
5 /ig/kg x 4
5 /tg/kg x *
15 /ig/kg x 4
15 /ig/kg x 4
15 /tg/kg x 4
1.0 ppb diet8
2.5 ppb diet
5.0 ppb diet
5.0 ppb diet
>5.0 ppb diet
>5.0 ppb diet
>5.0 ppb diet
5 /ig/kg x 4
5 /ig/kg x 4
5 /tg/kg x 4
5 jig/kg x 4
5 /ig/kg x 4
>5 /ig/kg x 4
Reference
=====
Vos and
Moore, 1974;
Luster et
al.. 1980
Thomas and II
Hinsdill,
1979
Vos and
Moore,
19774; Failh
and Moore,
1977
I I
aA 1.0 ppb diet consumed by a 20 g mouse al a rale of 5 g/day = 0.25 ng TCOD/kg/day.
LOAEL = Lowesl-observed-adverse-effecl level; BOG = bovine gamma globulin; LPS = lipopolysaccharide;
PHA» phylohlmagglulinin; Con A = Concanavalin A; SRBC = sheep red blood eel s; DTH = delayed-lype
hypersensilivity; PFC = plaque-forming cell; CFU-S = colony-forming units-spleen
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of Swiss mice fed a diet containing 1 ppb TCDD for 7 weeks showed enhanced
mortality following endotoxin challenge, whi.le the plaque-forming cell response
to SRBC and delayed hypersensitivity response were suppressed in offspring of
mice fed 5.0 ppb TCDD diets. (Estimated daily dose to 20 g dam consuming 5 g of
5 ppb TCDD diet is equivalent to 1.25 jug/kg TCDD/day.) Rats appeared to be more
resistant to the immunotoxic effects of pre/neonatal exposure to TCDD based on
the finding that 5 but not 1 pg/kg TCDD given four times at weekly intervals
produced immunotoxicity in the offspring. Immunotoxic endpoints that were
unaffected by the highest exposure levels in these studies included blastogenesis
induced by LPS and serum antibody titers to SRBC and BQG.
Two recent studies have examined immune function in offspring of female mice
exposed to TCDD (Holladay et al., 1991) or PCB (Kanechlor 500) (Takagi et al.,
1987) but that were cross-fostered to unexposed lactating mice at birth. Thus,
exposure was limited to in utero exposure. (It is important to recognize that
rodents are born with an immature immune system that matures in the first few
weeks following birth. In contssat, the human immune system is considered to be
more mature at birth.) B6 mice exposed to 3.0 fig/kg TCDD on gestational days
6-14 gave birth to offspring that had significant thymic atrophy and hypoplasia
measured on gestational day 18 or on day 6 postnatally. The thymic effects were
no longer apparent by day 14. At 7-8 weeks postnatally, mitogen responses and
antibody plaque forming cell response to SRBC were unaltered while the CTL
response was significantly suppressed compared to controls (Holladay et al.,
1991). These results suggest a selectivity of prenatal TCDD on the CTL and not
the T helper cells involved in the antibody response to SRBC. In contrast to
these results, Takagi et al. (1987) exposed female C3H mice per os to 50 mg/kg
Kanechlor 500 twice per week for 4 weeks, at which time steady state tissue
levels were noted. The offspring derived from mating to unexposed males had an
unaltered antibody response to the T-independent antigen DNP-dextran. On the
other hand, carrier-primed T helper cell activity assessed by adoptive transfer
was significantly suppressed by PCB exposure when assessed 4 and 7 weeks after
birth, but fully recovered by 11 weeks. Together, these studies confirm prior
studies to indicate that T cell function is selectively altered by HAH when
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exposure IB prenatal. While both T helper cells and CTL Bhow altered function,
T helper cell activity may recover faster than CTL function.
Fine et al. (1990) reported on TCDD levels in offspring following maternal
treatment with TCDD (10 pg/kg) on gestational day 14. The fetal liver had the
highest concentration on gestational day 18 (235 fg/mg) which declined slightly
by postnatal day "6 to around 100 fg/mg. The concentration of TCDD in the thymus
on gestation day 18 was 140 fg/mg, which declined to 20 fg/mg on day 6 after
birth. (These thymic TCDD concentrations are equivalent to 60 to 425 pM assuming
1 kg of tissue is equivalent to 1 L of water.) TCDD concentrations in the spleen
remained constant at about 40 fg/mg during the same time frame, while bone marrow
concentrations were very low (-3 fg/mg). These concentrations of TCDD were
associated with thymic'atrophy (Fine et al., 1989) and significant reduction in
the ability of prothymocytes in liver and bone marrow to repopulate an irradiated
thymus (Fine et al., 1990).
4.11. IMMUNOIOXICITY OF HAH IN NON-HUMAN PRIMATES
A limited number of studies using nonhuman primates as surrogate models for
humans have been conducted to assess HAH immunotoxicity. Immunological effects
were described in Rhesus monkeys and their offspring chronically exposed to TCDD
at levels of 5 or 25 ppt for 4 years (Hong et al., 1989). In the mothers, the
total number of T cells increased in monkeys fed 25 ppt TCDD, with a selective
increase in CD8+ cells and a decrease in CD4 + cells. However, no significant
effect on T cell function was established when assessed as proliferation response
to mitogens, alloantigens, or xenoantigens. Natural killer cell activity and
production of antibodies to tetanus immunization were normal. In the offspring
of TCDD exposed dams examined 4 years after exposure, a significantly increased
antibody response to tetanus toxoid immunization was observed which correlated
with TCDD tissue levels. The body burden of TCDD in the offspring ranged from
a low of 290 ppt to a high of 1400 ppt. Interestingly, there was no strict
correlation between exposure levels and resulting body burden.
In other TCDD studies, a single injection of TCDD in marmosets (Callithrix
j'acchus) resulted in a delayed decrease in the percentage of CD4+ T cells and
CD20+ B cells in the blood and an increase in the percentage of CD8+ cells
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(Neubert et al., 1990). The total number of T cells was not significantly
altered by TCDD exposure. The CD4 + subset most affected was the CDw29~*~ "helper-
inducer" or "memory" subset, with significant effects observed after a TCDD dose
of 10 ng/kg. The NOEL for this effect was 3 ng/kg TCDD. Concomitant with
suppression of the CDw29 subset in TCDD treated animals, the percentage of
CD4+CD45RA+ cells increased. This subset has been classified as "suppressor-
inducer" or "naive" cells. The changes in the T cells subsets were intensified
following in vitro culture of the cells with mitogen (Neubert et al., 1991).
Interestingly, however, a recent study from the same laboratory reported that
chronic exposure of young marmosets to very low levels of TCDD (0.3 ng/kg/week
for 24 weeks) produced the opposite effect on the CD4+CDw29+ subset, resulting
in a significant increase in this population (Neubert et al., 1992). Concomi-
tantly, the CD4+CD45RA + subset decreased. Upon transfer of the animals to a
higher dose of TCDD (1.5 ng/kg/week) for 3 weeks, the enhancement effect was
reversed, and suppression of the CD4+CDw29+ subset was observed, with maximum
suppression after 6 weeks of exposure to the higher dose. In addition, the CD8+
CD56 + T cytotoxic T cell subset was transiently increased, but which normalized
even though TCDD dosing continued. After discontinuation of dosing, the
reduction in the percentage and absolute number of CD4 + CDw29 + cells persisted
for 5 weeks, reaching normal range 7 weeks later. These results led the authors'
to conclude that "extrapolations of the results obtained at higher doses to very
low exposures is not justified with respect to the effects induced by TCDD on the
immune system of marmosets."
The immunomodulatory effects of chronic low level PCB exposure in monkeys
has also been investigated. In early studies, Thomas and Hinsdill (1978)
reported that rhesus monkeys fed diets containing 2.5 or 5 mg/kg of Aroclor 1248
had significantly suppressed antibody response to SRBC but not to tetanus toxoid
(TT). These monkeys also had chloracne, alopecia and facial edema. Similarly,
exposure of cynomolgus monkeys to Aroclor 1254 (100 or 400 pg/kg/day) for 3
months suppressed antibody responses to SRBC but not TT (Truelove et al., 1982).
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Suppressive effects on antiSRBC responses were more severe in cynomolgus monkeys
when the PCB mixture contained PCDFs (Hori et al., 1982).
Tryphonas et al. (1989; 1991a,b) have recently reported results of studies
in rhesus monkeys exposed chronically to Aroclor 1254 (5-80 pg/kg/day) for 23 or
55 months. These exposures resulted in steady state blood PCB levels that ranged
from a mean low of 0.01±0.001 ppm in the 5 /jg/kg group to a mean high of
0.11*0.01 ppm in the 80 pg/kg group. The only consistently altered immune
parameter was the primary and anamnestic antibody responses to SRBC which were
suppressed in a dose-dependent manner. In contrast, the antibody response to
pneumococcus vaccine antigen measured at 55 months of exposure was not
significantly altered. At 23 months, the percentage of T helper cells in the
blood was significantly decreased in the 80 pg/kg group, and the percentage and
absolute number of T suppressor cells was increased; however, these effects were
not apparent at 55 months of exposure (Tryphonas, et al., 1991b). Lymphoprolif-
erative responses to PHA and Con A were not significantly altered at 23 months
but were dose-dependently suppressed at 55 months. Proliferation to alloantigens
was not significantly altered. Likewise, serum immunoglobulin and hydrocortisone
levels did not differ between treatment groups. After 55 months, the chemilumin-
escent response (time to peak) of monocytes was slower in PCB exposed cells.
Also noted at 55 months was a significant elevation in serum hemolytic complement
levels, a dose-related increase in natural killer cell activity, and a dose-
related increase in thymosin alpha-1 levels but not thymosin beta-4 levels
(Tryphonas et al., 1991a). Effects on interferon levels were inconsistent, and
TNF production was not altered.
The studies in nonhuman primates are important from the standpoint that the
antibody response to SRBC emerges as the only immunological parameter consis-
tently suppressed by HAH in several different animal species. Other immunolog-
ical endpoints such as total T cell numbers, percentages of T cell subsets,
lymphoproliferative responses, and DTH responses are inconsistently increased or
decreased in various studies. At the present time, it is not clear why the
antibody response to SRBC is most consistently altered by HAH exposure in
different species. The sensitivity of the antiSRBC response does not appear to
be due solely to the T-cell dependency of the response since antibody responses
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to other T-dependent antigen (e.g., TT, BGG) are not suppressed and may be
enhanced following HAH exposure. It is possible that the particulate nature of
the SRBC antigens is an important factor even though a mechanistic basis for this
is not readily apparent. The sensitivity of the technique used to quantify the
antibody response may also contribute to apparent increased sensitivity of the
SRBC model, which is most often measured as the PFC response rather than serum
antibody titers which are usually more variable. Nonetheless, the finding that
the SRBC response is also suppressed in nonhuman primates exposed to PCB lends
support to the use of the antiSRBC data generated in mice to calculate TEFs for
immunotoxicity.
4.12. IMMUNOTOXICITY OF HAH IN HUMANS
The immunotoxicity of TCDD and related HAH in humans has been the subject
of several studies derived from accidental and/or occupational exposures to PCBs,
PBBs, and TCDD. Immunological assessment was carried out on patients who
consumed acnegenic and hepatotoxic doses of PCDF-PCB contaminated rice oil in
Taiwan in 1979. Clinical symptoms were primarily related to increased frequency
of various kinds of infection, especially of the respiratory tract and skin (Lu
and Wu, 1985). Immunologic effects included decreased serum IgA and IgM but not
IgG, decreased percentage of T cells in blood related to decreased CD4+ T helper
cells and increased CD8+ T suppressor cells, and suppressed dermal delayed type
hypersensitivity responses to streptokinase/-streptodornase and tuberculin
antigens (Lu and Wu, 1985). The percentage of anergic patients increased and the
degree of induration decreased with increased PCB concentration in the blood.
In contrast, lymphoproliterative responses of PEL to PHA, PWM and tuberculin but
not Con A were significantly augmented in PCB-exposed patients. PCB concentra-
tions in the blood ranged from 3-1156 ppb with a mean of 89±6.9 ppb. The oil was
contaminated at PCB concentrations of 4.8-204.9 ppm with a mean of 52±39 ppm.
Immunotoxic effects were also described in Michigan dairy farmers exposed
to PBBs via contaminated dairy products and meat in 1973 (Bekesi et al., 1979).
Like PCB-exposed patients, the percentage and absolute numbers of T cells in
peripheral blood of PBB-exposed farmers were significantly reduced compared to
a control group. However, in contrast to PCB, lymphoproliferation responses to
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PHA, PWM and allogeneic leukocytes were significantly decreased in PBB exposed
persons. Also in contrast to PCB, skin testing using standard recall antigens
indicated that PBB-exposed Michigan dairy farmers had significantly increased
responses, particularly to Candida and varidase. Tissue levels of PBB in the
subjects were not determined in these studies.
Webb et al. (1989) reported the findings from immunologic assessment of 41
persons from Missouri with documented adipose tissue levels of TCDD resulting
from occupational, recreational or residential exposure. Of the participants,
16 had tissue TCDD levels <20 ppt, 13 had levels between 20 and 60 ppt and 12 had
levels >60 ppt. The highest level was 750 ppt. Data were analyzed by multiple
regression based on adipose tissue level and the clinical dependent variable.
Increased TCDD levels were correlated with an increased percentage and total
number of T lymphocytes. CD8+ and T11+ T cells accounted for the increase, while
CD4 + T cells were not altered in percent or number. Lymphoproliferative
responses to Con A, PHA, PWM or tetanus toxoid were unaltered as was the
cytotoxic T cell response. Serum IgA but not IgG was increased. No adverse
clinical disease was associated with TCDD levels in these subjects. Only 2 of
the 41 subjects reported a history of chloracne. These findings differ from
those reported for the Quail Run Mobile Home Park residents (tissue levels
unknown) in which decreased T cell numbers (T3, CD4 and Til) and suppressed cell-
mediated immunity was reported (Hoffman et al., 1986). However, subsequent
retesting of these anergic subjects failed to confirm the anergy (Evans et al.,
1988). On the other hand, when serum from some of these individuals was tested
for levels of the thymic peptide, thymosin alpha-1, the entire frequency
distribution for the TCDD-exposed group was shifted toward lower thymosin alpha-1
levels (Stehr-Green et al., 1989). The statistically significant difference
between the TCDD-exposed persons and controls remained after controlling for age,
sex, and socioeconomic status, with a trend of decreasing thymosin alpha-1 levels
with increasing number of years of residence in the TCDD-contaminated residential
area. The thymosin alpha-1 levels were not correlated with changes in other
immune system parameters nor with any increased incidence of clinically diagnosed
immune suppression. The decrease in thymosin alpha-1 levels in humans contrasts
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with the increase in thymosin alpha-1 seen in PCB treated monkeys (Tryphonas et
al., 1991b).
Finally, Mocarelli et al. (1986) reported studies on the immune status of
44 children, 20 of whom had chloracne, that were exposed to TCDD following an
explosion at a herbicide factory in Seveso, Italy. No abnormalities were found
in the following parameters: serum immunoglobulin concentrations, levels of
circulating complement, or lymphoproliterative responses to T and B cell
mitogens. Interestingly, in a study conducted 6 years after the explosion, a
different cohort of TCDD-exposed children exhibited a significant increase in
complement protein levels, which correlated with the incidence of chloracne, as
well as increased numbers of peripheral blood lymphocytes, and increased
lymphoproliterative responses (Tognoni and Bonaccorsi, 1982). However, no
specific health problems were correlated with dioxin exposure in these children.
It is readily apparent that no clear pattern of immunotoxicity to HAH
emerges from these studies in humans. In some cases T cell numbers increase; in
others, they decrease. The findings are not unlike the varied and often
conflicting reports found in the literature regarding animal studies of HAH
immunotoxicity. The basis for the lack of consistent, significant exposure-
related effects is unknown and may be dependent on several factors. Most notable
in this regard is the generic difficulties in assessing subclinical immunomodula-
tion, particularly in outbred human populations. Most immunological assays have
a very broad range of normal responses reducing the sensitivity to detect small
changes. Similarly, the assays used to examine immune function in humans exposed
to TCDD and related HAH have unfortunately been based to a greater extent on what
was clinically "doable" (e.g., mitogen responsiveness) rather than on assays that
have been shown to be sensitive to TCDD in animal studies. Thus, the lack of
consistent and/or significant immunotoxic effects in humans resulting from TCDD
exposure may be as much a function of the assays used as the immune status of the
cohort. In addition, few studies have examined the immune status of individuals
with known, documented exposure to HAH. Rather, cohorts based on presumption of
exposure have been studied. There is some evidence to suggest that the lack of
consistent, significant effects may sometimes be due to the inclusion of subjects
that had little or no actual exposure to TCDD (Webb et al., 1989). Likewise, the
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important role that Ah phenotype plays in TCDD immunotoxicity has not been
conaidered when addressing human sensitivity. Whether there are human
equivalents of murine Ahbb and Ahdd types is not known. Finally, in most
studies, the assessment of immune function in exposed populations was carried out
long after exposure to TCDD ceased. Thus, recovery from the immunotoxic effects
of TCDD may have occurred.
in summary, one might speculate that any future study to determine HAH
immunotoxicity in humans should evaluate their antibody response to SRBC.
However, it should be emphasized that even the relatively low exposure levels
that have been shown to suppress the antiSRBC response in nonhuman primates
resulted in blood and tissue PCB or TCDD levels that far exceed the levels
measured in humans in most studies published to date involving environmental
exposure. Thus, even the antiSRBC response may not have been sensitive enough
to demonstrate immune suppression in these cohorts. Given the current lack of
data correlating clinical immunological endpoints with immune status in humans
(except in cases of overt immune deficiencies), massive retrospective studies of
poorly defined exposure groups cannot be justified to try to "prove" that immune
modulation has occurred in these people. Rather, such efforts would be better
directed toward the establishment of a broad data base of normal values for the
clinical immunology endpoints that may be of use in immunotoxicity assessments.
in conjunction with this effort, research must focus on the definition of
sensitive endpoints (i.e., biomarkers) of immune dysfunction in humans so that,
in the future, emergency response teams could respond rapidly to accidental
exposures to assess the immunological status of the exposed persons. To validate
these biomarkers, there is a parallel need for animal research to identify TCDD-
sensitive immune endpoints in animals that can also be measured in humans in
order to establish in correlative changes in the biomarker and immune function.
in particular, it will be important to determine in animal models how well
changes in immune function in the lymphoid organs (e.g., spleen, lymph nodes)
correlate with changes in the expression of lymphocyte subset/activation markers
in peripheral blood. Until such correlations are established, the interpretation
of changes observed in subsets/activation markers in human peripheral blood
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lymphocytes in terms of health risk will be limited to speculation. Research
must also continue to develop well-defined animal models using multiple animal
species that will lead to an understanding of the underlying mechanisms of HAH
immunotoxicity. For example, there is a clear need to document Ah receptor
involvement in the immunotoxicity of TCDD and related HAH in species other than
mice. These studies need to go beyond descriptive immunotoxicity assessment to
determine the mechanistic basis for differences in species sensitivity to TCDD
immunotoxicity following both acute and chronic exposure. In the interim, the
available data base derived from well-controlled animal studies on HAH
immunotoxicity can be used for establishment of no effect levels and acceptable
exposure levels for human risk assessment of TCDD using the same procedures that
are used for other noncarcinogenic toxic endpoints. Because the antibody
response to SRBC has been shown to be dose-dependently suppressed by TCDD and
related HAH in several animal species, this data base is best suited for current
application to risk assessment. The validity of using TEFs to extrapolate from
one HAH to another, however, remains to be established.
4.13. REFERENCES
Ackermann, M.F., T.A. Gasiewicz, K.R. Lamm, D.R. Germolec and M.I. Luster. 1989.
Selective inhibition of polymorphonuclear neutrophil activity by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 101: 470-480.
Alsharif, N.Z., T. Lawson and S.J. Stohs. 1990. TCDD-induced production of
superoxide anion and DNA single strand breaks in peritoneal macrophages of rats.
The Toxicologist 10: 276.
d'Argy, R., J. Bergman and L. Dencker, L. 1989. Effects of immunosuppressive
chemicals on lymphoid development in foetal thymus organ cultures. Pharmacol.
Toxicol. 64: 33-38.
Bekesi, J.G., H.A. Anderson, J.P. Roboz et al. 1979. Immunologic dysfunction
among PBB-exposed Michigan dairy farmers. Ann. NY Acad. Sci 320: 717-728.
4-37
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Benjamin!, E. and S. LeskowitzS. 1991. Immunology; A Short Course, 2nd ed.
Wiley-Liss, Inc. New York, NY. p 26.
Biegel, L., M. Harris, D. Davis, R. Rosengren, L. Safe and S. Safe. 1989.
2,2',4,4',5,5'-Hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin
antagonist in C57B1/6 mice. Toxicol. Appl. Pharmacol. 97: 561-571.
Birnbaum, L.S., M.M. McDonald, P.C. Blair, A.M. Clark, and M.W. Harris. 1990.
Differential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6
mice congenic at the Ah locus. Fund. Appl. Toxicol. 15: 186-200.
Blaylock, B. L., S.D. Holladay, C.E. Comment, J.J. Heindel and M.I. Luster.
1992. Exposure to tetrachlorodibenzo-p-dioxin (TCDD) alters fetal thymocyte
maturation. Toxicol. Appl. Pharmacol. 112: 207-213.
Braley-Mullen, H. 1982. Differential effect of activated T amplifier cells on
B cells responding to thymus-independent type-1 and type-2 antigens. J. Immunol.
129: 484-489.
Clark, D.A., J. Gauldie, M.R. Szewczuk and G. Sweeney. 1981. Enhanced
auppressor cell activity as a mechanism of immunosuppression by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Proc. Exp. Biol. Med. 168: 290-299.
Clark, D.A., G. Sweeney, S. Safe, E. Hancock, D.G. Kilburn and J. Gauldie. 1983.
Cellular and genetic basis for suppression of cytotoxic T cell generation by
haloaromatic hydrocarbons. Immunopharmacology. 6: 143-153.
Clark, G.C., J.A. Blank, D.R. Germolec and M.I. Luster. 1991a. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin stimulation of tyrosine phosphorylation in B
lymphocytes: Potential role in immunosuppression. Molec. Pharmacol. 39:
495-501.
4-38
OB/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Clark, G.C., M.J. Taylor, A.M. Tritscher and G.W. Lucier. 1991b. Tumor necrosis
factor involvement in 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated endotoxin
hypersensitivity in C57B1/6 mice congenic at the Ah locus. Toxicol. Appl.
Pharmacol. Ill: 422-431.
Clarke, A.G. and K.A. MacLennan. 1986. The many facets of thymic involution.
Immunology Today. 7: 204-205.
Cook, J.C., K.M. Dold and W.F. Greenlee. 1987. An in vitro model for studying
the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to human thymus. Toxicol.
Appl. Pharmacolo 89: 256-268.
Cuthill, S., A. Wilhelmsson, G.G.F. Mason, M. Gillner, L. Poellinger and
J-A. Gustafsson. 1988. The dioxin receptor: A comparison with the glucocorti-
coid receptor. J. Steroid Biochem. 30: 277-280.
Davis, D. and S. Safe. 1988. Immunosuppressive activities of polychlorinated
dibenzofuran congeners: quantitative structure-activity relationships and
interactive effects. Toxicol. Appl. Pharmacol. 94: 141-149.
Davis, D. and S. Safe. 1989. Dose-response immunotoxicities of commercial
polychlorinated biphenyls (PCBs) and their interactions with 2,3,7,8-tetrachloro-
dibenzo-p-dioxin. Toxicol. Lett. 48: 35-43.
Davis, D. and S. Safe. 1990. Immunosuppressive activities of polychlorinated
biphenyls in C57B1/6N mice: Structure-activity relationships as Ah receptor
agonists and partial antagonists. Toxicology. 63: 97-111.
Davis, D. and S. Safe. 1991. Halogenated aryl hydrocarbon-induced suppression
of the in vitro plaque-forming cell response to sheep red blood cells is not
dependent on the Ah receptor. Immunopharmacology. 21: 183-190.
4-39
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
DeKrey, G.K., L.B. Steppan, J.A. Deyo and N.I. Kerkvliet. 1990. Adrenalectomy
(ADX) and 3,4,5,3',4',5'-hexachlorobiphenyl (HXCB) suppression of cytotoxic T
lymphocyte (CTL) response to P815 allogeneic tumor in C57B1/6 mice. The
Toxicologist. 10: 290. (Manuscript submitted)
DeKrey, G.K., J.A. Deyo and N.I. Kerkvliet. 1992. Castration (ODX) partially
alleviates the suppression of cytotoxic T lymphocyte (CTL) activity by
3,3',4,4',5,5'-hexachlorobiphenyl (HxCB). The Toxicologist. 12: 132.
(Manuscript submitted)
Dencker, L., E. Hassoun, R. D'Argy and G. Aim. 1985. Fetal thymus organ culture
aa an in vitro model for the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and
its congeners. Mol. Pharmacol. 28: 357-363
Dooley, R.K. and M.P. Holsapple. 1988. Elucidation of cellular targets
responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of
antibody responses: the role of the B lymphocyte. Immunopharmacology. 16:
167-180.
Dooley, R.K., D.L. Morris and M.P. Holsapple. 1990. Elucidation of cellular
targets responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression
of antibody repsonse: 2. Role of the T lymphocyte. Immunopharmacology. 19:
47-58.
Evans, R.G., K.B. Webb, A.P. Knutsen et al. 1988. A medical follow-up of the
health effects of long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Arch. Environ. Health. 43: 273-278.
Faith, R.E. and J.A. Moore. 1977. Impairment of thymus-dependent immune
functions by exposure of the developing immune system to 2,3,7,8-tetrachloro-
dibenzo-p-dioxin- J' Toxicol. Environ. Health. 3: 451-464.
4-40
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Faith, R.E., M.I. Luster and J.A. Moore. 1978. Chemical separation of helper
cell function and delayed hypersensitivity responses. Cell. Immunol. 40:
275-284.
Fine, J.S., T.A. Gasiewicz and A.E. Silverstone. 1989. Lymphocyte stem cell
alterations following perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Mol. Pharmacol. 35: 18-25.
Fine, J.S., T.A. Gasiewicz, N.C. Fiore and A.E. Silverstone. 1990. Prothymocyte
activity is reduced by perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.
J. Expt. Pharmacol. Ther. 255: 1-5.
Fraker, P.J. 1980. The antibody mediated and delayed type hypersensitivity
response of mice exposed to polybrominated biphenyls. Toxicol. Appl. Pharmacol.
53: 1-7.
Greenlee, W.F., K.M. Dold, R.D. Irons and R. Osborne. 1985. Evidence for direct
action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium.
Toxicol. Appl. Pharmacol. 79: 112-120.
Hinsdill, R.D., D.L. Couch and R.S. Speirs. 1980. Immunosuppression in mice
induced by dioxin (TCDD) in feed. J. Environ. Pathol. Toxicol. 4(2-3): 401-425.
Hoffman, R.E., P.A. Stehr-Green, K.B. Webb. 1986. Health effects of long-term
exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Am. Med. Assoc. 255:
2031-2038.
Hoglen, N., A. Swim, L. Robertson and S. Shedlofsky. 1992. Effects of
xenobiotics on serum tumor necrosis factor (TNF) and interleukin-6 (IL-6) release
after LPS in rats. The Toxicologist. 12: 290.
4-41
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Holladay, S.D., P. Lindstrom, B.L. Blaylock. 1991. Perinatal thymocyte antigen
expression and postnatal immune development, altered by gestational exposure to
tetrachlorodibenzo-p-dioxin (TCDD). Teratology. 44: 385-393.
Holsapple, M.P., R.K. Dooley, P.J. McNerney and J.A. McCay. 1986a. Direct
suppression of antibody responses by chlorinated dibenzodioxins in cultured
spleen cells from (C57B1/6 x C3H)F1 and DBA/2 mice. Immunopharmacol. 12:
175-186.
Holsapple, M.P., J.A. McCay and D.W. Barnes. 1986b. Immunosuppression without
liver induction by subchronic exposure to 2,7-dichlorodibenzo-p-dioxin in adult
femal B6C3F1 mice. Toxicol. Appl. Pharmacol. 83: 445-455.
Holsapple, M.P., D.L. Morris, S.C. Wood and N.K. Snyder. 1991a. 2,3,7,8-
tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: Possible
mechanisms. Ann. Rev. Pharmacol. Toxicol. 31: 73.
Holsapple, M.P., N.K. Snyder, S.C. Wood and D.L. Morris. 1991b. A review of
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: 1991
Update. Toxicology. (In press).
Hong, R., K. Taylor and R. Abonour. 1989. Immune abnormalities associated with
chronic TCDD exposure in Rhesus, chemosphere. 18: 313-320.
Hori, S.t H. Obana, T. Kashimoto et al. 1982. Effect of polychlorinated
biphenyls and polychlorinated quaterphenyls in cynomolgus monkey (tfacaca
fasicularis). Toxicology. 24: 123-139.
House, R.V., L.D. Lauer, M.J. Murray et al. 1990. Examination of immune
parameters and host resistance mechanisms in B6C3F1 mice following adult exposure
to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxicol. Environ. Health. 31:
203-215.
4-42
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Jelinek, D.F. and P.E. Lipsky. 1987. Regulation of human B lymphocyte
activation, proliferation, and differentiation. Adv. Immunol. 40: 1-59.
Katz, L.B., H.M. Theobald, R.C. Bookstaff and R.E. Peterson. 1984. Character-
ization of the enhanced paw edema response to carrageenan and dextran in 2,3,7,8-
tetrachlorodibenzo-p-dioxin-treated rats. J. Pharmacol. Exp. Therap. 230:
670-677.
Kerkvliet, N.I. n.d. Unpublished data.
Kerkvliet, N.I. 1984. Halogenated aromatic hydrocarbons (HAH) as immunotoxi-
cants. In; Chemical Regulation of Immunity in Veterinary Medicine: Progress in
Clinical and Biological Research, Vol. 161, M. Kende, J. Gainer and M. Chirigos,
Ed. Alan R. Liss, Inc., New York. p. 369-387.
Kerkvliet, N.I. and L. Baecher-Steppan. 1988a. Suppression of allograft
immunity by 3,4,5,3',4',5'-hexachlorobiphenyl: I. Effects of exposure on tumor
rejection and cytotoxic T cell activity. Immunopharmacology. 16: 1-12.
Kerkvliet, N.I. and L. Baecher-Steppan. 1988b. Suppression of allograft
immunity by 3,4,5,3',4',5*-hexachlorobiphenyl: II. Effects of exposure on mixed
lymphocyte reactivity in vitro and induction of suppressor cells. Immunopharma-
cology. 16: 13-23.
Kerkvliet, N.I. and J.A. Brauner. 1987. Mechanisms of 1,2,3,4,6,7,8-hepta-
chlorodibenzo-p-dioxin (HpCDD)-induced humoral immune suppression: Evidence of
primary defect in T cell regulation. Toxicol. Appl. Pharmacol. 87, 18-31.
Kerkvliet, N.I. and J.A. Brauner. 1990a. Flow cytometric analysis of lymphocyte
subpopulations in the spleen and thymus of mice exposed to an acute immuno-
suppressive dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Environ. Res. 52: 146-
164.
4-43
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Kerkvliet, N.I. and J.A. Brauner. 1990b. Functional analysis of antigen-
presenting cells following antigen challenge: Influence of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD). In: 29th Annual Meeting of the Society of Toxicology,
Miami Beach, FL, February 12-16, 1990. The Toxicologist. 10: 289.
Kerkvliet and Deyo. n.d. Unpublished data.
Kerkvliet, N.I., J.A. Brauner and J.P. Matlock. 1985. Humoral immunotoxicity
of polychlorinated diphenyl ethers, phenoxyphenols, dioxins and furans present
as contaminants of technical grade pentachlorophenol. Toxicology. 36: 307-324.
Kerkvliet, N.I., L.B. Steppan, J.A. Brauner et al. 1990a. Influence of the Ah
locus on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
immunotoxicity: evidence for Ah receptor-dependent and Ah receptor-independent
mechanisms of immunosuppression. Toxicol. Appl. Pharmacol. 105: 26-36.
Kerkvliet, N.I., L.B. Steppan, B.B. Smith, J.A. Youngberg, M.C. Henderson and
D.R. Buhler. 1990b. Role of the Ah locus in suppression of cytotoxic T
lymphocyte (CTL) activity by halogenated aromatic hydrocarbons (PCBs and TCDD):
structure-activity relationships and effects in C57B1/6 mice. Fund. Appl.
Toxicol. 14: 532-541.
Kramer, C.M., K.W. Johnson, R.K. Dooley and M.P. Holsapple. 1987. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein
kinase activity in murine B cells. Biochem. Biophys. Res. Commun. 145: 25-32.
Loose, L.D., J.B. Silkworth, S.P. Mudzinski, K.A. Pittman, K.F. Benitz and
W. Mueller. 1979. Environmental chemical-induced immune dysfunction.
Ecotoxicol. Environ. Saf. 2: 173-198.
Lu, Y.C and Y.C. Wu. 1985. clinical findings and immunological abnormalities
in Yu-Cheng patients. Environ. Health Perspec. 59: 17-29.
4-44
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Lubet-, R.A., B.N. Lemaire, D. Avery and R.E. Kouri. 1986. Induction of
immunotoxicity in mice by halogenated biphenyls. Arch. Toxicol. 59: 51-77.
Lundberg, K., L. Dencker and K.O. Gronvik. 1990. Effects of 2,3,7,8-tetra-
chlorodibenzo-p-dioxin (TCDD) treatment in vivo on thymocyte functions in mice
after activation in vitro. Int. J. Immunopharmacol. 12s 459-466.
Luster, M.I., G.A. Boorman, M.W. Harris and J.A. Moore. 1980a. Laboratory
studies on polybrominated biphenyl-induced immune alterations following low-level
chronic and pre-/postnatal exposure. Int. J. Immunopharmacol. 2: 69-80.
Luster, M.I., G.A. Boorman, J.H. Dean et al. 1980b. Examination of bone marrow,
immunologic parameters and host susceptibility following pre- and postnatal
exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Int. J. Immunopharmacol.
2: 301-310.
Luster, M.I., D.R. Germolec, G. Clark, G. Wiegand and G.J. Rosenthal. 1988.
Selective effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and corticosteroid on
in vitro lymphocyte maturation. J. Immunol. 140: 928-935.
Mantovani, A., A. Vecchi, W. Luini et al. 1980. Effect of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin on macrophage and natural killer cell mediated cytotoxicity in
mice. Biomedicine. 32: 200-204.
McConnell, E.E., J.A. Moore, J.K. Haseman and M.W. Harris. 1978. The
comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs.
Toxicol. Appl. Pharmacol. 44: 335-356.
Mocarelli, P., A. Marocchi, P. Brambilla, P. Gerthoux, D.S. Young and N. Mantel.
1986. Clinical laboratory manifestations of exposure to dioxin in children: A
six-year study of the effects of an environmental disaster near Seveso, Italy.
J. Am. Med. Assoc. 256: 2687-2695.
4-45
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Morris, D.L., S.D. Jordan and M.P. Holsapple. 1991. Effects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) on humoral immunity: I. Similarities to
Staphylococcus aureus Cowan Strain I (SAC) in the ±n vitro T-dependent antibody
response. Immunopharmacol. 21: 159-170.
Morris, D.L., N.K. Snyder, V. Gokani, R.E. Blair and M.P. Holsapple. 1992.
Enhanced suppression of humoral immunity in DBA/2 mice following subchronic
exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl.
Pharmacol. 112: 128-132.
Neubert, R., U. Jacob-Muller, R. Stahlmann, H. Helge and D. Neubert. 1990.
Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system:
1. Effects on peripheral lymphocyte subpopulations of a non-human primate
(Callithrix jacchus) after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Arch. Toxicol. 64: 345-359.
Neubert, R., U. Jacob-Muller, H. Helge, R. Stahlmann and D. Neubert. 1991.
Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system: 2. Jn
vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on lymphocytes of
venous blood from man and a non-human primate (Callithrix jacchus). Arch
Toxicol. 65: 213-219.
Neubert, R., G. Color, R. Stahlmann, H. Helge and D. Neubert. 1992. Polyhaloge-
nated dibenzo-p-dioxins and dibenzofurans and the immune system: 4. Effects of
multiple-dose treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus).
Arch Toxicol. 66: 250-259.
Neumann, C.M., L.B. Steppan and H.I. Kerkvliet. 1992. Distribution of 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) in splenic tissue of C57B1/6J mice. Drug
Metab. Disposition. 20: 467-469.
4-46
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Nikoulaidis, E., B. Brunstrom, L. Dencker and T. Veromaa. 1990. TCDD inhibits
the support of B-cell development by the Bursa of Fabricius. Pharmacol. Toxicol.
67: 22-26.
Pazdernik, T.L. and K.K. Rozman. 1985. Effect of thyroidectomy and thyroxine
on 2,3,7,8-tetrachlorodibenzo-p-dioxin induced inununotoxicity. Life Sci. 36:
695-703.
Poland, A. and E. Glover. 1990. Characterization and strain distribution
pattern of the murine Ah receptor specified by the Ah^ and Ah""-* alleles. Molec.
Pharmacol. 38: 306-312.
Poland, A. and J.C. Knutson. 1982. 2,3,7,8-tetrachlorodibenzo-p-dioxin and
related halogenated aromatic hydrocarbons: Examination of the mechanism of
toxicity. Ann. Rev. Pharmacol. Toxicol. 22: 517.
Poland, A., E. Glover and B.A Taylor. 1987. The murine Ah locus: A new allele
and mapping to chromosome 12. Molec. Pharmacol. 32: 471-478.
Puhvel, S.M. and M. Sakamoto. 1988. Effect of 2,3,7,8-tetrachlorodibenzo-p-
dioxin on murine skin. J. Investig. Dermatol. 90: 354-358.
Rizzardini, M., M. Romano, F. Tursi et al. 1983. lexicological evaluation of
urban waste incinerator emissions. Chemosphere. 12: 559-564.
Rosenthal, G.J., E. Lebetkin, J.E. Thigpen, R. Wilson, A.M. Tucker and
M.I. Luster. 1989. Characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin
induced endotoxin hypersensitivity: Association with hepatotoxicity. Toxicology.
56: 239-251.
Silkworth. n,d, Personal communication.
4-47
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Silkworth, J.B. and L. Antrim. 1985. Relationship between Ah receptro-mediated
polychlorinated biphenyl (PCB)-induced humoral immunosuppression and thymic
atrophy. J. Pharmacol. Exp. Ther. 235: 606-611.
Silkworth, J.B. and E.M. Grabstein. 1982. Polychlorinated biphenyl immunotox-
icity: dependence on isomer planarity and the Ah gene complex. Toxicol. Appl.
Pharmacol. 65: 109-115.
Silkworth, J.B. and P. O'Keefe. 1992. Immunotoxicity as a probe of TCDD
toxicity in a complex environmental mixture. The Toxicologist. 12: 238.
Silkworth, J.B., L. Antrim and L.S. Kaminsky. 1984. Correlations between
polychlorinated biphenyl immunotoxicity, the aromatic hydrocarbon locus, and
liver microsomal enzyme induction in C57B1/6 and DBA/2 mice. Toxicol. Appl.
Pharmacol. 75: 156-165.
Silkworth, J.B., G. Sack and D. Cutler. 1988. Immunotoxicity of 2,3,7,8-
tetrachlorodibenzo-p-dioxin in a complex environmental mixture from the Love
Canal. Fundam. Appl. Toxicol. 12: 303-312.
Sonawane, B.R., R.J. Smialowicz and R.W. Luebke. 1988. Immunotoxicity of
2,3,7,8-TCDD: Review, Issues, and Uncertainties. Appendix E. EPA Review Draft,
A Cancer Risk-specific dose estimate for 2,3,7,8-TCDD.
Stehr-Green, P.A., P.H. Naylor and R.E. Hoffman. 1989. Diminished thymosin
alpha-1 levels in persons exposed to 2,3,7,8-tetrachlorodizenzo-p-dioxin. J.
Toxicol. Environ. Health. 28: 285-295.
Steppan, L.B. and N.I. Kerkvliet. 1991. Influence of 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) on the production of inflammatory cytokine mRNA by C57B1/6
macrophages. The Toxicologist. 11: 35.
4-48
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Sutter, T.R., K. Guzman, K.M. Dold and W.F. Greenlee. 1991. Targets for dioxin:
genes for plasminogen activator inhibitor-2 and interleukin-lp. Science. 254:
415-418.
Takagi, Y., S. Aburada, T. Otake and N. Ikegami. 1987. Effect of polychlori-
nated biphenyls (PCBs accumulated in the dam's body on mouse filial immunocompe-
tence. Arch. Environ. Contam. Toxicol. 16: 375-381.
Taylor, M.J., G.C. Clark, Z.Z. Atkins, G. Lucier and M.I. Luster. 1990.
2,3,7,8-Tetrachlorodibenzo-p-dioxin increases the release of tumor necrosis
factor-alpha (TNF-a) and induces ethoxyresorufin-o-deethylase (EROD) activity in
rat Kupffer's cells (KCs). The Toxicologist. 10: 276.
Theobald, H.M., R.W. Moore, L.B. Katz, R.O. Peiper and R.E. Peterson. 1983.
Enhancement of carrageenan and dextran-induced edemas by 2,3,7,8-tetrachloro-
dibenzo-p-dioxin and related compounds. J. Pharmacol. Exp. Therap. 225: 576-
583.
Thigpen, J.E. R.E. Faith, E.E. McConnell and J.A. Moore. 1975. Increased
susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetra-
chlorodibenzo-p-dioxin. Infect. Immun. 12: 1319-1324.
Thomas, P.T. and R.D. Hinsdill. 1978. Effect of polychlorinated biphenyls on
the immune responses of rhesus monkeys and mice. Toxicol. Appl. Pharmacol. 44:
41-51.
Thomas, P.T. and R.D. Hinsdill. 1979. The effect of perinatal exposure to
tetrachlorodibenzo-p-dioxin on the immune response of young mice. Drug Chem.
Toxicol. 2: 77-98.
Tognoni, G. and A. Bonaccorsi. 1982. Epidemiological problems with TCDD (a
critical view). Drug Metab. Rev. 13: 447-469.
4-49
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Tomar, R.S. and N.I. Kerkvliet. 1991. Reduced T helper cell function in mice
exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Lett. 57:
55-64.
Truelove, J., D. Grant, J. Mes, H. Tryphonas, L. Tryphonas and 2. Zawidzka.
1982. Polychlorinated biphenyl toxicity in the pregnant cynomolgus monkey: A
pilot study. Arch. Environm. Contam. Toxicol. 11: 583-588.
Tryphonas, H., S. Hayward, L. O'Grady et al. 1989. Immunotoxicity studies of
PCS (Aroclor 1254) in the adult rhesus (Macaca mulatta) monkey: Preliminary
report. Int. J. Immunopharmacol. 11: 199-206.
Tryphonas, H., M.I. Luster, G. Schiffman et al. 1991a. Effect of chronic
exposure of PCB (Aroclor 1254) on specific and nonspecific immune parameters in
the rhesus (Macaca mulatta) monkey. Fund. Appl. Toxicol. (In press).
Tryphonas, H., M.I. Luster, K.L. White, Jr. et al. 1991b. Effects of PCB
(Aroclor 1254) on non-specific immune parameters in rhesus (Macaca mulatta)
monkeys. Int. J. Immunopharmacol. 13: 639-648.
Tucker, A.N., S.J. Vore and M.I. Luster. 1986. Suppression of B cell
differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol. 29:
372-377.
van Logten, M.J., B.N. Gupta, E.E. McConnell and J.A. Moore. 1980. Role'of the
endocrine system in the action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
the thymus. Toxicology. 15: 135-144.
Vecchi, A., A. Mantovani, M. Sironi, M. Luini, W. Cairo and S. Garattini. 1980.
Effect of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral
antibody production in mice. Chem. Biol. Interact. 30: 337-341.
4-50
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Vecchi, A., M. Sironi, M.A. Canegrati, M. Recchis and S. Garattini. 1983.
Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of
mice with different susceptibility to inductino of aryl hydrocarbon hydroxylase.
Toxicol. Appl. Pharmacol. 68: 434-441.
VOB, J.G. and M.I. Luster. 1989. Immune alterations. In: Halogenated
biphenyls, terphenyls, naphthalenes, dibenzodioxins and related products,
R.D. Kimbrough and S. Jensen, Ed. Elsevier Science Publishers B.V., The
Netherlands, p. 295-322.
Vos, J.G. and J.A. Moore. 1978. Suppression of cellular immunity in rats and
mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Arch.
Allergy. 47: 777-794.
Vos, J.G. and L. van Driel-Grootenhuis. 1972. PCB-induced suppression of the
humoral and cell-mediated immunity in guinea pigs. Int. Arch. Allergy. 47:
777-794. .
Vos, J.G., J.A. Moore and J.G. Zinkl. 1973. Effect of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health
Perspec. 5: 149-162.
Vos, J.G., J.A. Moore and J.G. Zinkl. 1974. Toxicity of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29: 229-241.
Vos, J.G., J.G. Kreeftenberg, H.W.B. Engel, A. Minderhoud and L.M. Van Noorle
Jansen. 1978. Studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immune
suppression and decreased resistance to infection: Endotoxin hypersensitivity,
serum zinc concentrations and effect of thymosin treatment. Toxicology. 9:
75-86.
4-51
08/06/92
-------
DRAFT—DO NOT QUOTE OR CITE
Webb, K.B., R.G. Evans, A.P. Knutsen. 1989. Medical evaluation of subjects with
known body levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Toxicol. Environ.
Health. 28: 183-193.
Weisaberg, J.B. and J.G. Zinkl. 1973. Effects of 2,3,7,8-tetrachlorodibenzo-p-
dioxin upon hemostasis and hematologic function in the rat. Environ. Health
Perspec. 5: 119-123.
White, K.L., H.H. Lysy, J.A. McCay and A.C. Anderson. 1986. Modulation of serum
complement levels following exposure to polychlorinated dibenzo-p-dioxins.
Toxicol. Appl. Pharmacol. 84: 209-219.
Whitlock, J.P. 1990. Genetic and molecular aspects of -2,3,7,8-tetrachloro-
dibenzo-p-dioxin action. Ann. Rev. Pharmacol. Toxicol. 30: 251-277.
4-52
08/06/92
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