Direct suppression of in vitro antibody production by mouse spleen cells by the carcinogen benzo(a)pyrene but not by the noncarcinogenic congener benzo(e)pyrene.

The role of metabolic activation of benzo(a)pyrene B(a)P in mediating its suppression of humoral immune responsiveness of the female C57BL/6 X C3H F1 (hereafter called B6C3F1) mouse was addressed in these studies. The model was the in vitro antibody response by untreated splenic suspensions, to which was added directly either B(a)P or benzo(e)pyrene B(e)P. B(a)P suppressed the antibody response to DNP-Ficoll and sheep erythrocytes but not the polyclonal antibody response to LPS. This activity neither required nor was affected by addition of a metabolic activation system (i.e., S-9 crude liver homogenate from Aroclor-induced B6C3F1 mice) at 3 times the concentration (based on determination of protein content) which readily activated cyclophosphamide. Preliminary results with radiolabeled B(a)P verified that appreciable amounts of hydroxylated metabolites of B(a)P were obtained after only a 30-min preincubation. Therefore, production of the reactive metabolites of B(a)P which mediate its carcinogenicity are not essential for its immunosuppressive activity. The results, showing a parallel in the immunosuppressive profile of activity of B(a)P and the lack of immunosuppressive activity of B(e)P following in vivo and in vitro exposure, indicate that the in vitro antibody systems offer an ideal model system to characterize the PAHs.

Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

Effects of N-nitrosodimethylamine on cell-mediated immunity.

Dimethylnitrosamine (DMN) exposure altered the cell-mediated immune response of B6C3F1 adult female mice as assessed by several immunological assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice exhibited a depression in their lymphoproliferative response to the T-cell mitogens concanavalin A and phytohemagglutinin, and in their mixed lymphocyte response to mitomycin-treated DBA-2 spleen cells. The delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH), as measured by vascular permeability changes, was decreased by over 50% at the 5.0-mg/kg dose. When the DHR to KLH was measured by an influx of endogenously 125I-iododeoxyuridine (IUdR)-labelled monocytes, there was a 300% increase in the response of the 5.0-mg-DMN/kg group. Adoptive transfer studies using exogenously radiolabelled (51Cr) bone marrow cells from either vehicle- or DMN-treated (5 mg/kg) donors indicated a greater than 60% reduction in the DHR to KLH in DMN-treated mice (5.0 mg/kg level) regardless of the donor treatment. Animals exposed to DMN exhibited a decreased susceptibility to Listeria monocytogenes. The dichotomy in the results of the KLH DHR measured by monocyte influx and the increased resistance to the bacterial challenge were interpreted to reflect an effect on bone marrow. The numbers of granulocyte/monocyte stem cells were increased in a dose-related fashion in bone marrow from DMN-treated mice. The results indicate that DMN-treatment impairs cell-mediated immunity while increasing the number of bone marrow cells differentiating to form granulocytes or monocytes with an apparent enhancement in functional activity.

Effects of N-nitrosodimethylamine on tumor susceptibility.

Dimethylnitrosamine (DMN) exposure altered the activity of the macrophage and natural killer (NK) cell defense mechanisms against the B16F10 melanoma in B6C3F1 adult female mice as assessed by several immunologic assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice were injected (i.v.) with B16F10 melanoma. The incidence and number of lung nodules, determined 18 days after challenge, were decreased in the DMN-exposed animals. The initial observation indicated the mice exposed to 3 mg/kg DMN were afforded the greatest protection. However, when mice exposed to the highest dose of DMN were divided into subgroups of mice with or without ascites, there was a degree protection seen in the 5-mg/kg-treated mice without ascites that was comparable to that of the 3-mg/kg group. The development of ascites is an overt toxic effect reflecting damage to the liver and was frequently associated with exposure to 5 mg/kg DMN. Exposure to DMN produced only slight changes in the activity of splenic NK cells as determined by the cytotoxicity of 51Cr-labelled YAC-1 cells. The activity was significantly increased only in mice exposed to 3 mg/kg DMN and only at effector:target (E:T) ratios of 30:1 and 10:1. Interestingly, the activity of the NK cells was significantly decreased at all E:T ratios in mice exposed to 5 mg/kg that developed ascites. The number of peritoneal exudate cells was decreased, albeit nonsignificantly, in a dose-related fashion. The phagocytic activity, as measured by the uptake of fluorescent latex beads, was increased in a dose-related fashion with significance noted at the highest dose of DMN. The role of the macrophage in the increased resistance to the tumor challenge was assessed with bone marrow derived macrophages. The cytostatic activity versus B16F10 tumor cells, as measured by the uptake of 3H-thymidine, was markedly increased in the bone marrow derived macrophages from DMN (5mg/kg) mice when compared to vehicle controls. These results suggest that exposure to DMN alters bone marrow, particularly the differentiation of effector tumoricidal cells, which renders the host more resistant to metastatic tumor formation.

Identification of functional aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in murine splenocytes.

The objective of the present studies was to determine whether the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein are present and functional in B6C3F1 (C57BL/6 x C3H) mouse splenocytes. Northern analysis of poly(A) RNA isolated from splenocytes revealed transcripts of approximately 6.6 kb which hybridized to the AhR complementary DNA (cDNA) probe. Anti-AhR antibodies identified two major cytosolic forms of the AhR in splenocytes, approximately 95 and 104 kDa, corresponding to the codominately expressed Ahrb alleles in the B6C3F1 mice. Northern analysis utilizing an oligomer probe for ARNT identified three messenger RNA (mRNA) transcripts, approximately 5.6, 2.0, and 1.1 kb, in spleen which was consistent with the banding pattern observed in the B6C3F1 mouse liver. Western blotting confirmed the presence of the approximately 87 kDa ARNT protein in splenocytes. Protein quantitation by slot blot analysis demonstrated approximately 2.0-fold more AhR in liver than in splenocytes. Interestingly, ARNT was approximately 2.4-fold more abundant in splenocytes than in liver. Consistent with these results, comparison by quantitative reverse transcriptase-polymerase chain reaction analysis of AhR and ARNT transcripts in liver and splenocytes demonstrated approximately 2.3-fold more AhR transcripts in liver than in splenocytes and approximately 3.2-fold more ARNT transcripts in splenocytes than in liver. In addition, comparisons between AhR and ARNT transcripts isolated from the liver and splenocytes indicated a greater number of ARNT transcripts as compared with AhR in both preparations. TCDD treatment of splenocytes induced binding of the AhR nuclear complex to the dioxin-responsive enhancer (DRE) as detected by the electrophoretic mobility shift assay. These findings confirm that the AhR and ARNT are present in mouse splenocytes and are capable of binding to the DRE.

Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxycyclophosphamide.

Cyclophosphamide (CY) is metabolized to 4-hydroxy-CY which spontaneously breaks down to the reactive intermediates, phosphoramide mustard (PAM) and acrolein. The alkylating property of PAM is thought to mediate the anti-proliferative and cytotoxic actions of CY. Acrolein is known to bind sulfhydryl groups of cellular molecules and may contribute to the action of CY. However, the role of acrolein in the CY-induced immunosuppression remains unclear. The results of studies in which a hepatocyte co-culture system was used suggest that acrolein may play an important role in the cytotoxic action of CY, whereas those investigations using activated derivatives of CY indicate that acrolein is not an important factor. Thus, both approaches of CY exposure were utilized in the present study. Splenocytes of B6C3F1 mice were incubated with syngeneic isolated hepatocytes and CY or with 4-hydroperoxycyclophosphamide (4-HC) (which spontaneously decomposes to 4-hydroxy-CY). The in vitro antibody forming cell (AFC) response was found to be suppressed with both methods of exposure to CY metabolites. The addition of DNA to bind extracellular PAM was ineffective in preventing the suppression produced by hepatocyte-activated CY. However, it was also observed that DNA was able to attenuate the PAM-induced suppression. The sulfhydryl compounds 2-mercaptoethanesulfonate (MESNA) (15 microM) or reduced glutathione (GSH) (1 mM) inhibited the suppression of the AFC response of splenocytes incubated with CY and mouse hepatocytes. The suppression produced by 4-HC, however, was not affected by MESNA and only slightly inhibited by GSH. Similarly, the PAM-induced suppression was not affected by MESNA and slightly attenuated with GSH. In contrast, both MESNA and GSH were very effective in abrogating the acrolein-induced suppression, whereas DNA was ineffective. The findings of this study suggest that in the hepatocyte co-culture system, the immunosuppressive actions of CY are mediated by acrolein generated outside of the splenocyte, whereas the 4-HC induced suppression is not mediated by extracellular acrolein. Thus, this difference may explain the contradictory findings of previous studies that used different means of exposing cells to activated CY.

Role of the transfer of metabolites from hepatocytes to splenocytes in the suppression of in vitro antibody response by dimethylnitrosamine.

The metabolism and subsequent immunosuppressive effects of dimethylnitrosamine (DMN) were investigated in mixed cultures of mouse hepatocytes and mouse splenocytes. Hepatocytes were shown to activate DMN to an immunosuppressive form that caused the suppression of the in vitro antibody response to the T-dependent antigen, sheep erythrocytes (SRBC). A significant increase in the binding of DMN metabolites to trichloroacetic acid (TCA) precipitable material in splenocytes was induced when 94 microM [14C-methyl]DMN was added to the co-culture medium, indicating that reactive intermediates of DMN were transferred from hepatocytes to splenocytes and resulted in alkylation of macromolecules in splenocytes. The amount of [14C]DMN bound to TCA precipitable material in splenocytes increased in a time-dependent manner up to 4 hr of incubation. Aminoacetonitrile (AAN), a high-affinity DMN demethylase inhibitor, reversed the suppression by low concentrations of DMN (0.5 to 5 mM), but not by high concentrations of DMN (greater than 5 mM). AAN also inhibited the binding of [14C]DMN to both hepatocytes and splenocytes. These results suggest that reactive metabolites of DMN are released from hepatocytes and that the suppression of the antibody response by DMN is mediated via these reactive intermediates.

Role of metabolism in dimethylnitrosamine-induced immunosuppression: a review.

Dimethylnitrosamine (DMN) has been characterized as a potent hepatotoxin, carcinogen and mutagen. As described below, immunotoxicity should be added to its profile of activity. Although a broad spectrum of immune parameters is affected by DMN, humoral immunity is particularly sensitive. In order for DMN to produce its traditional profile of toxicity it requires metabolic activation to reactive intermediates which alkylate macromolecules. Similarly, DMN also must be metabolized to produce its immunological effects. However, as this review suggests, the metabolism of DMN to an intermediate capable of suppressing the humoral immune response may be qualitatively and/or quantitatively different from that which mediates hepatotoxicity and genotoxicity.

Importance of hepatocyte culture conditions in dimethylnitrosamine-induced suppression of antibody response in the mixed cultures of murine hepatocytes and splenocytes.

The role of the hepatocyte culture media in dimethylnitrosamine (DMN)-induced suppression of antibody responses by splenocytes against sheep erythrocytes (SRBCs) was investigated using the mixed culture system of murine hepatocytes and murine splenocytes. It was observed that hormone-supplemented complete media was required for hepatocyte cultures to optimally activate DMN to its immunosuppressive form(s). In the absence of the hormone supplement, the concentration of DMN required to produce a 50% suppression (IC50) was increased by over 10-fold (i.e., compare the IC50 in complete media of less than 0.5 mM to the IC50 in basal media of almost 10.0 mM). In contrast, the activation of cyclophosphamide (cytoxan, CTX), which was used in these studies as a comparative control, was not affected by the absence of the hormone supplement. These results indicate that the observed effect on the activation of DMN was not due to a generalized loss of metabolic capability of hepatocytes cultured without hormones. To examine the role of drug metabolizing capabilities of hepatocytes in the differential activation of DMN, we compared phase I and phase II enzyme activities of hepatocytes cultured for 24 h in either basal media or hormone-supplemented complete media. Our results indicated that there was a significant decrease of phase I monooxygenase activities of cultured hepatocytes when compared to freshly isolated hepatocytes. However, our results failed to show any difference in the activities of hepatocytes cultured in the two media. Most notably, there was no difference in the activity of either high- or low-affinity DMN demethylase, as measured by the generation of formaldehyde. We observed a similar profile with phase II conjugative capabilities, specifically sulfotransferase and glucuronyltransferase. These results indicate that the activation of DMN to its immunosuppressive form(s) can be modulated in the co-culture system by culturing hepatocytes under different conditions. Because we failed to show any differences in the metabolic capabilities of hepatocytes cultured under the two media conditions, the results suggest that the modulation of immunosuppressive activity may not be related to a change in the generation of the immunosuppressive metabolite(s).

Direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human tonsillar lymphocytes.

Murine lymphocyte function is quite sensitive to TCDD. However, in contrast to the murine model, the corresponding functional studies have not been undertaken with human lymphocytes. One laboratory has recently demonstrated that human tonsillar lymphocytes (HTL) possess the aryl hydrocarbon (Ah) receptor which mediates many of the effects of TCDD. This observation suggested that HTL may be sensitive to TCDD. In mitogen stimulated HTL, TCDD induced a dose-dependent increase in 7-ethoxyresorufin-O-deethylase (EROD) synthesis. Because we recently demonstrated that background proliferation in HTL and murine splenocytes was suppressed by TCDD, we purified human and murine B-cells into high density and low density populations. In low density human B-cells, TCDD suppressed background proliferation and IgM secretion from 0.3 to 30 nM. Interestingly, TCDD produced comparable effects on background proliferation and IgM secretion in purified low density murine B-cells. When low density human B-cells were stimulated with LPS and TRF, TCDD suppressed both proliferation and IgG secretion in a dose-dependent manner from 0.3 to 30 nM, although the suppression was modest when compared to the magnitude of suppression of the background responses. In contrast, TCDD did not alter background or stimulated proliferation in high density human B-cells. These results indicate that TCDD has a direct effect on human tonsillar lymphocyte activity and suggest that low density B-cells are a sensitive cellular target.

A review of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: 1991 update.

2,3,7,8-tetrachlorodibenzo-p-dioxin, more popularly called dioxin or TCDD and referred to in this review as 2,3,7,8-TCDD, is considered the prototype of the polychlorinated dibenzo-p-dioxins (PCDD). The PCDD are true contaminants and are formed primarily as byproducts in the manufacture of materials requiring the use of chlorinated phenols and during the combustion of chlorinated chemical products. From an environmental perspective, the PCDD have been most closely associated with the use of a number of phenolic herbicides, including Agent Orange, which is a 1:1 mixture of the butyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 2,3,7,8-TCDD and related PCDD are not produced commercially except in small amounts for research purposes and to date, have no known human benefit. 2,3,7,8-TCDD has been demonstrated to be the most potent and the most biologically active congener among the halogenated aromatic hydrocarbons (HAH), which include polychlorinated and polybrominated biphenyls (PCB and PBB, respectively) and the polychlorinated dibenzofurans (PCDF), in addition to the PCDD. An updated review on the effects of 2,3,7,8-TCDD on immunocompetence is timely from a number of perspectives. First, effects on immune function have been demonstrated to be among the earliest and most sensitive indicators of 2,3,7,8-TCDD-induced toxicity. Second, recent evidence indicates that exposure to 2,3,7,8-TCDD causes changes in innate immunity in addition to the changes in acquired immunity (i.e., which include effects on both cell-mediated and humoral immunity) previously shown to be associated with this chemical. Third, effects on immune function are almost universally observed among the animal species in which it has been evaluated, including some non-human primates. Fourth, effects of 2,3,7,8-TCDD on specific indicators of immune function have been correlated with changes in host resistance capabilities, which are often considered to be more holistic indicators of immunocompetence. Fifth, there are several reports which describe possible effects of 2,3,7,8-TCDD and related compounds (i.e., primarily PBB and PCB) on immune function in humans. It is important to emphasize at the onset that these studies have triggered much controversy, both political and scientific. However, it is equally important to speculate that at least part of the controversy associated with man's sensitivity to the immunological effects of 2,3,7,8-TCDD may be that the most appropriate approaches have heretofore not been applied. This possibility is discussed further in this review.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on glioma-specific cytotoxic T-cell activity in Fischer 344 rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent environmental immunomodulating agents identified so far. Historically, mice have been used to model mammalian immunobiology and most of the data gathered on the immunotoxicity of TCDD has been obtained from studies with mice. However, rats have been used more extensively in toxicological research to establish human risk assessment criteria. A need exists, therefore, to develop a database using the rat model in immunotoxicology so that complete animal toxicity studies can be conducted. We have treated female Fischer 344 rats with a single i.p. dose of 0.3, 3.0, or 30.0 micrograms/kg TCDD or corn oil vehicle and examined cytotoxic T-cell (CTL) activities 24 days following treatment. Syngeneic in vivo tumor-specific CTLs were generated that model cell-mediated immune reactions against neoplastically transformed self antigens. RT2, a virally-induced Fischer 344 rat glioma, and D74, a ethylnitrosurea-induced Fischer 344 rat glioma were used as targets. This immunological parameter was compared to body, thymic, and liver weights as well as liver ethoxyresorufin deethylase (EROD) activity on day 24 post-TCDD treatment. The results indicate that Fischer 344 rats are very sensitive to TCDD as indicated by severe thymic atrophy and EROD induction at all three doses. In contrast, CTL activity was only marginally affected by these same doses of TCDD with only a modest suppression noted at the highest dose. These results indicate that the CTL response in rats may not be useful in characterizing the effects of this xenobiotic on immunocompetence in the rat.

Role of hydrocortisone in dimethylnitrosamine-induced suppression of antibody response in the mixed culture of murine hepatocytes and splenocytes.

We have previously reported that the hormone-supplemented culture condition for primary hepatocytes is required in dimethylnitrosamine (DMN)-induced suppression of antibody response to sheep erythrocytes in the mixed cultures of murine hepatocytes and splenocytes. In the present investigation, the components of the hormone supplement were screened to identify the component(s) responsible for the increased ability of hepatocytes to activate DMN to its immunosuppressive form. The presence of hydrocortisone in the hepatocyte culture media had the primary role in DMN activation in the co-culture system. Other components of the hormone supplement showed slight or no effects. The effects of hydrocortisone were clearly confirmed through the dose-response study of both DMN and hydrocortisone. To characterize whether the effect of hydrocortisone is glucocorticoid-dependent we tested another potent glucocorticoid, dexamethasone (DEX), and determined if the activity by hydrocortisone could be reversed by RU 486. It was found that hepatocytes cultured in DEX-containing media could also activate DMN to its immunosuppressive form. However, the activity by hydrocortisone to increase DMN-induced immunosuppression was not reversed by RU 486. Furthermore, a possible direct interaction between DMN and hydrocortisone was ruled out. Finally, we transferred DMN-pre-treated culture supernatant from hepatocytes to spleen cell cultures, and found that the metabolite of DMN was very unstable, and that DMN-induced suppression of T-dependent antibody response was hepatocyte-dependent. The present results suggest that glucocorticoids, including hydrocortisone and DEX, in hepatocyte culture media can affect DMN-induced immunosuppression in the hepatocyte/splenocyte co-culture system via a pathway which does not appear to be related to the glucocorticoid receptor.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides.

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

Reduction by atropine of phencyclidine hypertension and apneusis.

The aim of this study was to assess the possible role of a central cholinergic component phencyclidine (PCP)-induced hypertension. Sprague-Dawley rats, lightly anesthetized with urethane, exhibited a dose related pressor response following 0.1-1.0 mg/kg PCP i.v. After i.v. atropine pretreatment, the PCP dose-response curve was shifted to the right, and the magnitude of the pressor responses was reduced by about 50%. In addition, atropine reduced the incidence of apneusis, but had no effect on the bradycardia that accompanied the pressor responses. Methylatropine (i.v.) did not reduce the PCP pressor responses, nor did it prevent the apneusis induced by PCP. These results suggest that in addition to its direct pressor effects the activation of central cholinergic systems contribute significantly to the cardiovascular and respiratory toxicity induced by PCP.

Direct exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases infectivity of human erythrocytes to a malarial parasite.

Direct exposure to 10 nM 2,3,7,8-TCDD caused a 75% increase and a 2-fold increase in the infectivity of isolated human erythrocytes to P. falciparum after 48 hours when the parasites were in an unsynchronized or synchronized state of growth, respectively. Treatment of human erythrocytes with 10 microM sodium orthovanadate (NaOV), an inhibitor of plasma membrane Ca-ATPase and phosphotyrosine phosphatase, decreased parasitemia by 30%. Co-treatment of RBCs with TCDD and NaOV completely blocked the TCDD-induced increase in parasitemia. Because erythrocytes are anucleated, these results are discussed as evidence for biochemical changes by TCDD without requiring the activation of gene products.

Recovery of dimethylnitrosamine-induced immunosuppression by pargyline in the mixed cultures of murine hepatocytes and splenocytes.

To investigate the role of monoamine oxidase (MAO) in dimethylnitrosamine (DMN)-induced suppression of the antibody response to sheep erythrocytes, the effect of an MAO inhibitor, pargyline, was studied in mixed cultures of murine hepatocytes and splenocytes. When pargyline was added simultaneously with DMN during the coculture, DMN-induced immunosuppression was clearly recovered dose-dependently. Cyclophosphamide was used as a comparative control in these studies. Surprisingly, pargyline also reversed cyclophosphamide-induced suppression of the antibody response in the coculture system. The results with cyclophosphamide were not consistent with a role by MAO, and suggested that pargyline may not be selective for MAO. To confirm our hypothesis, the ability of pargyline to inhibit three cytochrome P-450 (P-450) isozyme-specific monooxygenase activities in vitro was studied using mouse liver microsomes. Pargyline, under the same concentration ranges that we used in the coculture studies, clearly inhibited the P-450IIE1-specific p-nitrophenol hydroxylase activity and P-450IIB1-specific pentoxyresorufin O-dealkylase activity. Taken together, our present results indicate that pargyline inhibits P-450 activity and is not selective for MAO. Although further studies are required to confirm a possible role by MAO in DMN-induced immunosuppression, our results suggest that pargyline may recover DMN-induced immunosuppression by primarily inhibiting the ability of P-450 enzymes in hepatocytes to activate DMN to its immunosuppressive metabolite(s).

Pharmacological activity and toxicity of phencyclidine (PCP) and phenylcyclohexene (PC), a pyrolysis product.

Initial acute behavioral studies in mice indicated that phencyclidine (PCP) produced marked motor impairment as measured by the inverted screen technique with an ED50 value of 4.1 muMole/kg (i.v.). Phenylcyclohexene (PC) was considerably less active with an ED50 value of 325 muMole/kg (i.v.). PCP was also shown to be more lethal than PC as acute (24 hr; i.v. injection) LD50 values (muMoles/kg) in males were 57 and 448, and in females were /6 and 425, respectively. A greater acute lethality was also produced by PCP after i.p. and p.o. administration. Subchronic (14-day) exposure (i.p.) to PCP at doses up to approximately 40 percent of the acute LD50 value (123.6 muMole/kg, i.p., daily) was without significant effect on body and organ weights, hematology and clinical chemistry, and humoral and cell-mediated immunity. Higher doses of PCP were not possible because of acute lethality. Subchronic exposure to PC (63.4, 317, and 634.5 muMoles/kg; 4 percent, 20 percent and 40 percent of acute i.p. LD50 value, respectively) produced several marked effects. At the highest dose tested, body weight and thymus weight in both males and females, and liver weight in males were significantly decreased. The spleen weight of males exposed to 317 muMole/kg PC was also significantly decreased. Humoral immunity (production of antibody forming cells) was significantly inhibited in both males and females exposed to PC. In contrast, cell-mediated immunity (development of a delayed-type hypersensitivity response) was only significantly inhibited in females. As PCP has no measurable toxicity under these conditions and PC produced significant effects at relatively high doses, the results suggest that neither chemical is exceptionally toxic following subchronic exposure.

In vitro and in vivo evaluation of cis-methyl-phencyclidine (CIS-MPCP) as a potential antagonist of phencyclidine (PCP).

In four preparations/tests (isolated nerve, ventricular strip, rotarod, and mouse acute lethality), cis-N-phenyl-4-methyl-cyclohexyl piperidine (cis-MPCP) was consistently less active than PCP and trans-MPCP. As expected, cis-MPCP, at 10(-4)M, which did not depress the action potential evoked on frog sciatic nerves, reduced by half both the nerve block and prolongation of relative refractory period caused by PCP. However, cis-MPCP at 10(-6)M, which by itself had little effect, failed to reduce the positive inotropic effect of PCP on the field-stimulated rat ventricular strip. Cis-MPCP also failed to decrease the ataxic effect of 6 mg/kg PCP (ED80) in the mouse rotarod test. Finally, at a dose that was neither ataxic nor lethal to mice (20 mg/kg), cis-MPCP failed to reduce the 24-hour LD50 of PCP. These data suggest that the actions of PCP are mediated through a multiple receptor system.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on interleukin-4-mediated mechanisms of immunity.

Because of similarities in the independent actions of the pleiotropic cytokine, interleukin-4 (IL-4), and the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on murine B-lymphocytes suggested in earlier studies, we have investigated whether the immunosuppression mediated by direct exposure to TCDD in vitro is due to an IL-4-like biological activity. In particular, the ability of TCDD to mimic hallmark responses of B-cells to IL-4, such as upregulation of major histocompatibility complex (MHC) antigens of the class II type, increases in cell surface expression of the low affinity form of the Fc receptor for IgE (CD23) and induction of immunoglobulin class switching, was tested. At concentrations that readily suppress B-cell proliferative and antibody-forming cell responses, TCDD failed to demonstrate any of the activities of IL-4 observed in parallel cultures. Further, in experiments in which TCDD was preincubated with B-cells before addition of IL-4, no evidence of increased IL-4 activity was observed. Rather, TCDD preincubation resulted in decreased secretion of IgG1 and IgE in B-cell cultures stimulated to undergo immunoglobulin class switching by incubation with bacterial lipopolysaccharide (LPS) and IL-4. Because TCDD produced comparable suppression of IgM secretion induced by LPS alone (i.e., no IL-4), it appears that TCDD inhibits the formation of fully differentiated B-cells capable of secreting antibody and has no effects on class switching events per se. Coupled with previous reports from this and other laboratories, these observations indicate that TCDD is able to suppress secretion of several classes of immunoglobulin.