Suppression of the in vitro humoral immune response of mouse splenocytes by 7,12-dimethylbenz[ a]anthracene metabolites and inhibition of immunosuppression by α-naphthoflavone

Abstract

Exposure to 7,12-dimethylbenz[ a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10 −9 to 10 −5 m. DMBA and benzo[ a]pyrene (B[ a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to splenocyte cultures produced a concentration-dependent suppression of the PFC response, in which B[ a]P was found to be 17.5-fold more potent that DMBA. These results are in contrast to those found in vivo, where DMBA has been shown to be more potent than B[ a]P at suppressing humoral immunity. The 3,4-diol metabolite of DMBA produced a concentration-dependent suppression (10 −8 to 10 −5 m) of the in vitro PFC response and was found to be 65-fold more potent than the parent compound DMBA. In contrast, the 5,6-diol metabolite of DMBA had no effect on the PFC response or cell viability. Both the 3-OH-DMBA and 7-hydroxymethyl-12-methyl-benz[ a]anthracene (7-OHMe-12-Me-BA) metabolites were found to be immunosuppressive at concentrations of 10 −6 m. Furthermore, suppression by 7-OHMe-12-Me-BA was observed at concentrations as low as 10 −8 m. Immunosuppression by the 7-Me-12-OHMe-BA and the di-OHMe-BA metabolites was only observed at high (10 −5 m) concentrations. The cytochrome P450 inhibitor, α-naphthoflavone (ANF), was utilized to determine if cytochrome P450-mediated metabolism is involved in DMBA-induced suppression of the in vitro PFC response. ANF (10 −5 m) reversed the DMBA-induced immunosuppression seen at 10 −5 m and attenuated the immunosuppression at 3 × 10 −5 and 10 −4 m. The results of these studies demonstrate that several metabolites of DMBA which can be generated by the cytochrome P450-dependent monooxygenase systems are immunosuppressive in the in vitro PFC response assay. Furthermore, the cytochrome P450 inhibitor, ANF, was able to reverse DMBA-induced immunosuppression. However, since the degree of DMBA-induced and B[ a]P-induced suppression of humoral immunity in vitro does not correlate with that observed in vivo, DMBA metabolites generated within the spleen may not be responsible for or may only partially contribute to the immunosuppression observed in the whole animal.