Examination of immune parameters and host resistance mechanisms in B6C3F1 mice following adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Adult female B6C3F1 mice were given a single ip dose of 0, 01, 1.0, or 10.0 micrograms/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and examined for immune function and host resistance 7-10 d later. Exposure to TCDD resulted in a significant dose-related decrease in induction of both IgM and IgG antibody-forming cells. This suppression was noted for both T-dependent and T-independent antigens. TCDD at a dosage of 10 micrograms/kg was shown to suppress production of antibody to viral hemagglutinin. In contrast, TCDD exposure had no significant effect on natural killer cell function, production of interferon, or various parameters of macrophage function. Assessment of host resistance revealed a significant increase in susceptibility to fatal infection with influenza virus, but no significant alteration in susceptibility to infection with the bacterium Listeria monocytogenes.

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells.

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.

Selective immunosuppression following exposure to a novel anthraquinone, 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD).

The immune response of murine splenic lymphocytes was characterized following both in vivo and in vitro exposure to the novel and highly-substituted anthraquinone AEAD. Gross indicators of toxicity such as changes in body weight, lymphoid organ weights or lymphoid organ cellularity were unaffected. Similarly, the antibody plaque-forming cell response and natural killer cell function were unaltered. The most consistent effect noted was the selective suppression of the cytotoxic T-lymphocyte (CTL) response, which persisted for an extended period of time following in vivo exposure at concentrations of 0.75-3.0 micrograms/g. In contrast to the CTL suppression induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]-anthracene, AEAD-induced CTL suppression could not be restored by addition of exogenous interleukin-2. Studies are currently underway to further characterize AEAD-induced immunosuppression at the cellular and molecular levels.

Evaluation of the immunotoxicity of beta-hexachlorocyclohexane (beta-HCH).

beta-HCH, an isomeric contaminant formed during the manufacture of the insecticide lindane, is a persistent environmental and food chain pollutant which has been reported to exhibit estrogenic activity in rodents and in fish. To investigate potential toxic effects on the reproductive and immune systems, beta-HCH was fed to female B6C3F1 mice for 30 days. Mice exposed to 0, 100, or 300 mg of beta-HCH/kg of diet were evaluated for changes in ovarian and uterine histology, body weight, lymphoid organ weight and histology, splenic cellularity, antigen-specific IgM and IgG plaque-forming cells (PFC), proliferative responses to mitogens, natural killer (NK) cell activity, and induction of cytolytic T lymphocytes. The ovaries and endometrial epithelium exhibited normal architecture. No alterations were observed in body weight, lymphoid organ weight and histology, or splenic cellularity whereas significant changes were found in several immune functions at the 300 mg/kg dose. Proliferation of splenocytes to the mitogens LPS, PHA, and Con A was decreased by 39, 43, and 57%, respectively. T-lymphocyte-mediated cytolysis of tumor targets was decreased by 25% with a concurrent reduction of 45% in NK activity. There was no significant reduction in the number of IgM or IgG PFC in exposed animals. These data indicate that beta-HCH causes nonestrogenic immune function changes in the adult mouse without gross changes in lymphoid organ weight, histology, or cellularity.

Suppression of splenic lymphocyte function by 7,12-dimethylbenz[a]anthracene (DMBA) in vitro.

The effects of the immunosuppressive polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) were studied directly by in vitro exposure of splenic lymphocytes. On the basis of evidence from prior studies that DMBA immunotoxicity in vivo may not be dependent upon induction of the Ah locus in mice, splenocytes from Ah-responsive B6C3F1, Ah-nonresponsive DBA/2N, and in C57BL/6J Ah-congenic mice (responsive B6-Ah(b)Ah(d) and nonresponsive B6-Ah(d)Ah(d) were exposed to xenobiotic in culture. For some experiments, B6C3F1 mice were pretreated with 200 nmol 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) to induce Ah-associated enzymatic activity prior to in vitro splenocyte exposure to DMBA. Humoral immunity assessed as splenic antibody plaque-forming cells measured after a 5-day in vitro immunization to sheep erythrocytes (SRBC) was suppressed up to 99% by continuous exposure to 20 microM DMBA, and was comparable between control mice having basal levels of hepatic monooxygenase activity and Ah-induced mice (TCDD-treated) having elevated enzyme activity. Similarly, cytotoxic T-lymphocyte generation against P815 target cells was suppressed up to 88 and 86% in 40 microM DMBA-exposed splenocytes from Ah-induced and noninduced mice, respectively. The mixed lymphocyte responsiveness (MLR) of B6C3F1, DBA/2N, B6-Ah(b)Ah(d), and B6-Ah(d)Ah(d) splenocytes exposed in vitro to 40 microM DMBA was suppressed 54, 72, 51, and 29%, respectively. However, the degree of suppression was not significantly different between the strains. The secretion of interleukin 2 (IL2) was also suppressed in splenocytes from both strains exposed to 40 microM DMBA in vitro. Studies which included benzo[a]pyrene (BaP) as a control xenobiotic known to demonstrate Ah dependence showed that the MLR of splenic lymphocytes from Ah-congenic mice was comparably suppressed following 40 microM DMBA exposure, whereas exposure to 40 microM BaP resulted in suppression of the MLR only in B6-Ah(b)Ah(d) splenocytes. In addition, mitogen-stimulated proliferation was inhibited in both B6C3F1 and DBA/2N splenocytes exposed to 40 microM DMBA, whereas 40 microM BaP inhibited only B6C3F1 splenocyte proliferation to LPS. These data suggest that DMBA may act on immunocytes by mechanisms largely independent of the Ah locus and associated metabolic processes.

Persistent suppression of humoral immunity produced by 7,12-dimethylbenz(A)anthracene (DMBA) in B6C3F1 mice: correlation with changes in spleen cell surface markers detected by flow cytometry.

The purpose of these studies was to examine the effects of DMBA on subpopulations of splenocytes obtained from B6C3F1 mice, using cell surface markers defined by monoclonal antibodies and multiparameter flow cytometry. Changes were correlated with alterations in humoral immune function assessed in vitro. Mice were treated for 10 days during a 2 week period by subcutaneous (s.c.) injections of DMBA in corn oil at doses of 0.5, 5 and 10 micrograms/g/day (5-100 micrograms/g total dose). Four mice from each exposure group and an additional corn oil control group of mice were studied at 4 and 8 weeks following the last injection with DMBA. These studies demonstrated a dose-dependent decrease in the total number and percentage of spleen cells expressing B-cell markers (mu heavy chain, kappa light chain and 14.8 antigen) as well as T-cell markers (Thy 1.2, Lyt-1 and Lyt-2). The percentage of splenocytes expressing Mac-1 was increased by DMBA. Helper T-cells appeared to be a very sensitive population of spleen cells to DMBA exposure, as suggested by a decrease in the number and percentage of Lyt-1 positive cells recovered from the spleen 4 weeks after exposure to DMBA. DMBA produced a dose-dependent suppression of the in vitro primary humoral immune responses to SRBC, TNP-Ficoll and TNP-LPS. The fact that a functional suppression of humoral immunity was accompanied by a decrease in the number of mature B-cells and T-cells in the spleen suggests that cell surface markers may be useful indicators of immunotoxicity in animals receiving DMBA in sub-chronic studies.

Effects of 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and cyclosporin A on murine cytomegalovirus infection: studies of resistance mechanisms.

Susceptibility to murine cytomegalovirus (MCMV) was enhanced by treating B6C3F1 and CD-1 mice subcutaneously with 100 mg 7,12-dimethyl-benz[a]anthracene (DMBA)/kg fractionated over a 2 week period prior to sub-lethal infection. Virus-augmented natural killer cell (NKC) activity was depressed in B6C3F1 mice treated with 100 mg DMBA/kg, while serum interferon (IFN) levels were unaffected. Treatment with 50 mg DMBA/kg had no effect on susceptibility to virus or virus-augmented NKC activity. Susceptibility to MCMV was not affected by treating mice with 400 mg benzo[a]pyrene (B[a]P)/kg using the same exposure regimen. Virus-augmented NKC activity was suppressed in B[a]P-treated mice, but the magnitude of the suppression (18%) was much less than that for DMBA-treated mice (39%). Susceptibility to MCMV, virus-augmented NKC and IFN induction were not affected in mice treated intraperitoneally with 50 mg cyclosporin A (CSA)/kg/day for 5 days and infected on the 5th day of treatment. In contrast, enhanced susceptibility to MCMV and depressed NKC activity were observed in mice treated by the same exposure regimen on days 1-5 post infection. Susceptibility was not affected by CSA given on days 5-9 post infection. The data are useful not only because they show that DMBA and appropriately-timed CSA treatments suppress virus augmented NKC and enhance susceptibility to MCMV, but also because they help to define the relative importance of certain immune responses in defending against the infection, thus improving the usefulness of MCMV as a host resistance model for immunotoxicity testing. The data suggest that chemicals which depress NKC are likely to enhance susceptibility to MCMV, and conversely that effects on NKC should be suspected when chemical exposure enhances susceptibility to MCMV.

Immunosuppression following exposure to 7,12-dimethylbenz[a]anthracene (DMBA) in Ah-responsive and Ah-nonresponsive mice.

Recent reports suggest that the immunotoxicity of certain polycyclic aromatic hydrocarbons is associated with the Ah locus in mice. To test whether immunosuppression mediated by 7,12-dimethylbenz[a]anthracene (DMBA) is regulated by the Ah locus, several endpoints of immune function were measured in Ah-responsive B6C3F1 and Ah-nonresponsive DBA/2N and in Ah-congenic C57BL/6J (responsive B6-AhbAhd and nonresponsive B6-AhdAhd) mice dosed sc with up to 100 micrograms/g DMBA in corn oil. Some groups of B6C3F1 and DBA/2N mice were exposed to 100 micrograms/g benzo[a]pyrene (B(a)P) or 1 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for determination of hepatic microsomal monooxygenase activity. The body weights of all mice were unaffected by DMBA exposure, but thymus weights and spleen cellularity were decreased. Antibody plaque-forming cells (PFC) measured 4 days after iv sheep erythrocyte (SRBC) immunization were suppressed 99% in B6C3F1 and 96% in DBA/2 mice. Antibody PFC after in vitro immunization to SRBC were similarly suppressed 98% in both B6-AhbAhd and B6-AhdAhd Ah-congenic mice exposed to 100 micrograms/g DMBA. Responses to the T-cell mitogens concanavalin A and phytohemagglutinin were significantly suppressed in both B6C3F1 and DBA/2N strains, as was mitogenesis to bacterial lipopolysaccharide. The unidirectional mixed lymphocyte responses of the congenic strains were suppressed 76% in B6-AhbAhd and 85% in B6-AhdAhd, cytotoxic lymphocyte generation was suppressed 68% in B6-AhbAhd and 78% in B6-AhdAhd. The overall differences between immunosuppressive responses in splenocytes from B6-AhbAhd and B6-AhdAhd congenics were not significant. Induction of cytochrome P1-450, a marker of Ah responsiveness, was determined by 7-ethoxycoumarin O-deethylase monooxygenase activity in hepatic microsomes or splenocytes. This monooxygenase activity was not significantly increased in either B6C3F1 or DBA/2 mice exposed to DMBA, whereas B(a)P and TCDD exposure significantly induced enzyme activity in B6C3F1 hepatocytes. These data suggest that DMBA has an immunosuppressive action on murine splenocytes which is independent of the Ah locus and associated induction of cytochrome P1-450 xenobiotic-metabolizing enzymes.

The effect of formaldehyde exposure upon the mononuclear phagocyte system of mice.

The vapors of formaldehyde have been reported to represent a potential health hazard, resulting in an increased incidence of carcinomas of the nasal turbinates in experimental animals. To determine the potential role of alterations in the mononuclear phagocyte system (MPS) induced by inhalation of formaldehyde, we studied the systemic effects of exposure upon macrophages. Specifically, we examined the effects of formaldehyde exposure upon development of the MPS by use of an established system of quantitative objective markers, which characterizes and classifies populations of murine macrophages into several developmental stages. Exposure of mice to 15 ppm of formaldehyde for 6 hr daily for 3 weeks did not alter the number or impair the function of resident peritoneal macrophages, although this exposure increased (approximately twofold) competence for release of H2O2 from the macrophages. Furthermore, formaldehyde exposure did not alter the tumoricidal activation or differentiation of macrophages produced by the defined stimulant MVE-2. The data thus indicate that exposure of mice to formaldehyde can induce selective systemic alterations in the function of the MPS for H2O2 production, a change which has been shown in other studies to increase the frequency of mutagenesis.

Suppression of T-helper cell function in mice following exposure to the carcinogen 7,12-dimethylbenz[a]anthracene and its restoration by interleukin-2.

Previous studies in this laboratory have demonstrated that exposure of mice to the carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthrance (DMBA) results in suppression of cell-mediated immunity (CMI), specifically the ability to generate cytotoxic T cells. This is accompanied by an increased susceptibility to challenge with transplantable tumors. Our previous studies have demonstrated no appreciable change in the composition of splenic lymphocyte populations following exposure to DMBA, suggesting a modulation of lymphocyte function, probably at the level of the T lymphocyte. The purpose of this study was to examine the mechanism of DMBA-induced T lymphocyte dysfunction following DMBA exposure, and to determine whether CTL function could be reconstituted by the addition of untreated lymphocytes or lymphokines. Exposure to DMBA in vivo at doses of 50 and 100 micrograms/g and in vitro at doses of 20 and 40 microM suppressed the ability of splenic lymphocytes to generate cytotoxic T-lymphocytes (CTL). CTL-mediated lysis of allogeneic tumor target cells could be restored by the addition of 20% T cell-enriched naive lymphocytes and by 10% T-helper cell-enriched lymphocytes. DMBA suppressed splenocyte production of the lymphokine interleukin-2 (IL-2) in response to mitogenic or allogeneic stimulation by greater than 70% following in vitro exposure and greater than 45% following in vivo exposure. Although DMBA-exposed lymphocytes were impaired in their ability to produce IL-2, CTL responsiveness could be reconstituted by the addition of exogenous IL-2 (purified or recombinant DNA-produced). Complete restoration of CTL responsiveness by the addition of exogenous IL-2 suggests that the T-helper cell, rather than the T-cytotoxic cell, is the target for the DMBA-induced CMI lesion.

Effect of short-term inhalation exposure to 1,3-butadiene on murine immune functions.

Interest in 1,3-butadiene (BD) as a potential immunomodulator was prompted by reports of an increased incidence of neoplasia in humans exposed to BD during the manufacture of styrene-butadiene synthetic rubber, and by a recent study which demonstrated a high incidence of thymic lymphomas in B6C3F1 mice. B6C3F1 mice were exposed to 1250 ppm BD by inhalation 6 hr per day, 5 days per week, for 6 or 12 weeks. Immune function assays were selected to evaluate specific humoral and cell-mediated immunity and spontaneous cytotoxicity; lymphoid organ histopathology was also evaluated. A slight decrease in antibody plaque-forming cells (PFC) per spleen was observed in exposed mice, although PFC per 10(6) splenic lymphocytes was normal. Significant extramedullary hematopoiesis and erythroid hyperplasia was observed in spleens from exposed mice, and correlated with a twofold increase in thymidine incorporation in spontaneously proliferating splenocytes. No differences in proliferation to alloantigens were demonstrable between control and BD-exposed splenocytes. Mitogenesis by phytohemagglutinin, Concanavalin A, and lipo polysaccharide was suppressed in splenocytes from exposed mice, but may have been due to the cellular dilution effect of hematopoietic activity. Cytotoxic T-lymphocyte generation was suppressed after a 6-week exposure to BD, but was comparable to controls after 12 weeks of exposure. No differences in spontaneous cytotoxicity were observed between control and exposed mice. Overall, no persistent immunological defects were detectable after inhalation exposure to this tumorigenic agent.

Functions of mononuclear phagocytes in mice exposed to diethylstilbestrol: a model of aberrant macrophage development.

Administration of the synthetic estrogen diethylstilbestrol (DES) lowers the systemic resistance of mice to challenge with either tumor cells or the facultative intracellular parasite Listeria monocytogenes. To assess the potential role of impaired mononuclear phagocyte system (MPS) function in this depression of host resistance, we addressed the question of systemic perturbations of the MPS induced by administration of DES. A panel of objective quantitative markers which have been previously shown to identify and characterize macrophages in the several stages of development of activation was employed. DES perturbed the resident population of peritoneal macrophages by increasing their number approximately twofold and by enhancing their competence for phagocytosis, cytostasis of tumor cells, and secretion of plasminogen activator. When we examined the competence of the MPS in DES-treated mice to respond to challenge with activating stimuli, we found that DES systemically suppressed the development of macrophages, in response to either pyran copolymer or BCG, to develop tumoricidal function and to gain competence for secretion of reactive oxygen intermediates such as H2O2. Since these data suggested that DES inhibited the development of macrophages from a precursor stage (i.e., responsive macrophages) to activated macrophages in vivo, we tested this possibility directly by applying known activating signals in vitro to responsive macrophages. Responsive macrophages from DES-treated mice did not become activated in response to the application of two known potent activating signals (i.e., MAF + LPS). Taken together, the data indicate that DES systemically perturbs the MPS and does so by enhancing development of the early stages of maturation and suppressing subsequent development.

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice--II. Altered cell-mediated immunity and tumor resistance.

We have previously demonstrated that the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) produce a marked decrease in spleen weight, spleen and bone marrow cellularity and the number of IgM plaque forming cells generated in response to a T-dependent antigen. Exposure to DMBA, but not B[a]P, increased susceptibility to challenge with PYB6 tumor cells and Listeria monocytogenes suggesting that DMBA produces immune impairment involving cell-mediated immunity (CMI) and tumor resistance mechanisms. In this study, female B6C3F1 mice received total doses of 5, 50 and 100 micrograms DMBA/g of body weight in ten subcutaneous injections of 0.5, 5, or 10 micrograms/g over a 2 week period and CMI and tumoricidal functions were examined 3-5 days following the final injection of DMBA. DMBA exposed mice exhibited suppressed splenic cellularity (decreased 62%) and decreased numbers of resident peritoneal cells (down to 47% of control), although the proportion of T cell and T cell subsets, B cells and macrophages in spleens from exposed mice was not altered. Lymphocyte blastogenesis in response to mitogens was suppressed up to 49% with PHA, 48% with Con A and 76% with LPS. The response to alloantigens in unidirectional mixed lymphocyte culture was depressed as much as 73% following exposure to DMBA. Tumor cytolysis mediated by cytotoxic T cells (CTL) was impaired at doses of 50 and 100 micrograms DMBA/g body weight (88-95% suppressed respectively) as was natural killer cell (NK)-mediated tumor cytolysis (24% and 55% suppressed). Antibody-dependent cytotoxicity was significantly depressed in the highest exposure group. Peritoneal macrophage accumulation was decreased in DMBA-treated mice, but the macrophages present were pushed towards activation. The ability of DMBA-exposed mice to eliminate intravenously injected B16F10 tumor cells from the lungs was not impaired. Since NK- and M phi-mediated tumor cytotoxicity are thought to be primarily responsible for pulmonary elimination of B16F10 melanoma cells, the extent of NK suppression observed following DMBA exposure appeared to be insufficient to alter in vivo B16F10 pulmonary elimination. In contrast, the loss of the CTL tumoricidal response correlated with an increased frequency of tumors following challenge with PYB6 tumor cells.

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene.

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously been reported that the complete carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3-5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 micrograms/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3-5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.

Immunological studies in B6C3F1 mice following exposure to ethylene glycol monomethyl ether and its principal metabolite methoxyacetic acid.

Exposure to glycol ethers has been associated with adverse effects in laboratory animals including thymus atrophy and mild leukopenia. These effects may involve depletion of immunoresponsive cells. This study examined possible alterations in immune function and host resistance of B6C3F1 mice following exposure to ethylene glycol monomethyl ether (EGME) or its principal metabolite, methoxyacetic acid (MOAA). EGME and MOAA were administered by gavage to mice in 10 doses over a 2-week period at total dosages of 250, 500, and 1000 micrograms/g of body weight. Following exposure, immunopathology, humoral immunity, cell-mediated immunity, macrophage function, and host resistance to Listeria monocytogenes bacterial challenge were examined. A 48% reduction in thymus weight was observed at the intermediate and high doses of both chemicals. No significant alterations in immune function or host resistance to L. monocytogenes were observed in animals exposed to either EGME or MOAA.

Correlation of murine susceptibility to tumor, parasite and bacterial challenge with altered cell-mediated immunity following systemic exposure to the tumor promoter phorbol myristate acetate.

Phorbol myristate acetate (PMA), the most potent of the tumor promoting phorbol diesters, modulates function in several immunoresponsive cells following in vitro exposure. Since suppression of cellular mechanisms capable of limiting tumors and infections can adversely affect health, these experiments were designed to evaluate relevant components of cell-mediated immunity (CMI) following in vivo PMA exposure, and to determine the biological significance of any alterations utilizing assays of host resistance. Adult, female B6C3F1 mice were administered 0.2, 2.0, 20.0 or 40.0 micrograms PMA/g body weight subcutaneously over a two-week period. Mechanisms of cell-mediated host resistance were assessed by quantitating natural killer (NK), cytotoxic T-lymphocyte (CTL) and macrophage-mediated lysis of radiolabelled tumor target cells, and macrophage-induced cytostasis in tumor cell populations. Macrophages from PMA-treated mice were cytostatic to tumor cells, inhibiting up to 90% of growth in cultured tumor cells, but were not tumoricidal. Furthermore, pyran-elicited (primed) macrophages, which are activated to fully cytotoxic states by in vitro exposure to lipopolysaccharide, were inhibited in tumoricidal activation by in vivo PMA exposure. The induction of responsive but not cytotoxic macrophages by in vivo PMA exposure is consistent with the enhanced resistance to Listeria bacterial challenge, and increased susceptibility to B16F10 tumor and Trichinella parasitic challenges observed in these mice. Furthermore, previous reports of decreased in vitro NK activity following in vivo PMA exposure and present observations of correlative decreases of in vivo NK activity (55% decrease in mice exposed to 20 micrograms PMA/g) suggest an important role for NK activity in limiting in vivo B16F10 melanoma growth. CTL effector function was less susceptible to PMA-induced suppression than NK function at similar dosages, further supporting a predominant role of macrophages and NK cells or possibly other effector functions in the resistance to Listeria, Trichinella, or B16F10 challenge. Nevertheless, significant suppressive effects of PMA on CTL function at higher dosages cannot be excluded as contributing to altered host resistance to these agents. These studies demonstrate that in vivo exposure to PMA can modify cell-mediated mechanisms of host resistance with coincident alterations in the incidence of infections and tumors.

Mechanisms of dimethylbenzanthracene-induced immunotoxicity.

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.

Immunosuppression following 7,12-dimethylbenz[a]anthracene exposure in B6C3F1 mice. I. Effects on humoral immunity and host resistance.

It has previously been demonstrated that the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (B[a]P), suppresses the terminal step in B-cell differentiation, resulting in a decrease in antibody production to T-dependent and B-2 T-independent antigens. The purpose of this study was to ascertain if this effect was common to carcinogenic PAHs or specific for B[a]P. The PAH 7,12-dimethylbenz[a]anthracene (DMBA) was administered to B6C3F1 female mice by ten sc injections of 0.5, 5, or 10 micrograms/g over a 2-week period (i.e., total dose of 5, 50, and 100 micrograms/g). Immune function and host resistance assays were performed 3 to 5 days following the last injection. The 10 micrograms/g dosage resulted in a marked decrease in spleen weights and spleen and bone marrow cellularity, while thymus and body weights were not significantly altered. The ability to generate B-lymphocyte colonies in vitro from spleen precursor cells was also suppressed at the 10 micrograms/g dose. Exposure to DMBA at 5 micrograms/g or greater resulted in a reduction of up to 97% in the number of IgM plaque-forming cells in response to the T-dependent antigen sheep red blood cells (SRBC). The IgG response to SRBC was similarly depressed. The IgM response to the hapten-conjugated T-independent antigens trinitrophenyl-lipopolysaccharide (TNP-LPS) (specific for B-1 cells) and trinitrophenyl (TNP)-Ficoll (specific for B-2 cells) was also depressed (88 and 97%, respectively) at 10 micrograms/g. DMBA exposure resulted in an increased susceptibility to challenge with the PYB6 transplantable sarcoma and the bacterium Listeria monocytogenes, in contrast to B[a]P exposure, which had no effect on host resistance assays. Thus, DMBA, a more potent carcinogen than B[a]P, produces a more extensive B-cell suppression than B[a]P as well as alters host resistance to tumor and bacterial challenge.

Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde.

A series of immune function and host resistance parameters were examined in female B6C3F1 mice following a 21-day (6 hr/day) inhalation exposure to 15 ppm of formaldehyde (HCHO). Immune parameters examined included delayed hypersensitivity to keyhole limpet hemocyanin, antibody plaque-forming cell response to sheep erythrocytes (T-lymphocyte-dependent antigen) and TNP-Ficoll (T-lymphocyte-independent antigen), lymphoid organ weights and histopathology, routine hematology, bone marrow cellularity and CFU progenitor cell enumeration, lymphocyte subpopulation quantitation by cell surface markers, mitogen-induced lymphocyte blastogenesis, macrophage function parameters, and host resistance to challenge with the bacterium Listeria monocytogenes and transplantable tumor cells. Lymphoid organ weight, bone marrow cellularity, and hematology parameters were unchanged in HCHO exposed mice. Similarly, the percentage of T and B lymphocytes and their proliferative responses to mitogens were not significantly altered. Antibody (IgM) plaque-forming cell response following antigen challenge was unchanged. Macrophage function was normal although some evidence of enhanced H2O2 production associated with elevated bactericidal activity was observed in resident macrophages. Resistance to challenge with the bacteria Listeria monocytogenes was significantly enhanced, while resistance to tumor challenge remained unchanged. No evidence of immunosuppression following short-term exposure to HCHO was observed.