Studies on immunosuppressive effects of dimethylnitrosamine 2-acetylaminofluorene in vitro

Abstract

The immunosuppressive effects of dimethylnitrosamine (DMN) and 2-acetylaminofluorene (AAF) were investigated using various immunological parameters. DMN did not cause any suppression of in vitro antibody responses and mitogen-induced proliferation of splenocytes without metabolic activation or co-cultured with liver enzyme fractions (S-9). When the splenocytes were co-cultured with mouse hepatocytes along with DMN for 4hr, DMN markedly suppressed the response to SRBC, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS. The proliferative responses of splenocytes to B cell mitogen lipopolysaccharide (LPS) and T cell mitogen concanavalin A (ConA) were also inhibited by DMN after co-culture with hepatocytes. The suppression by DMN was related to the rocking speed during the 4hr co-culture period and was optimally produced when the cultures were not rocked. Addition of serum into the medium (10\% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response. However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the response by DMN, indicating that DNA represents the primary macromolecular target for the reactive intermediate of DMN. To elucidate the dependence of intact hepatocytes in vitro activation of DMN to immunosuppressive forms, S-9 fractions from both phenobarbital (PB)-and isopropanol (iso)-pretreated mice were used. These S-9 displaced significantly greater demethylase activity than uninduced S-9 fractions. However, when incubated with spleen cells, neither S-9 preparation was capable of activating DMN to a immunosuppressive forms. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. These results indicate that the in vitro activation of DMN to an immunosuppressive form is dependent on intact cells and on ...