The immunosuppressive effects of chemical carcinogens, dimethylnitrosamine (DMN), 2-aminofluorene(AF), 2-acetylamino-fluorene(AAF) and safrole were studied in vitro splenocyte culture using a variety of immunological parameters. Primary mouse and rat hepatocyte cultures and liver homogenates(S-9) were used to metabolize carcinogens which require metabolic activation to exert their bilogical activity. In vitro antibody responses to sheep erythrocytes(SRBC), DNP-Ficoll and lipopolysaccharide(LPS) and lymphoproliferation assay were used to assess immunological function of splenocytes.
DMN did not produce suppression of antibody response when it was added directly or metabolized by S-9. DMN produced suppression of antibody response only when it was incubated in mouse hepatocyte-splenocyte coculture. However, S-9 metabolized DMN to formaldehyde, one of proposed metabolite of DMN, at a rate of 50 times faster than hepatocytes.
In an attempt to elucidate the mechanism whereby primary hepatocytes, but not S-9, generate immunosuppressive metabolites of DMN, the production of DNA single strand breaks(SSB) in splenocytes was investigated with alkaline elution assay. Both hepatocytes and S-9 induced DNA SSB in splenocytes by DMN - minimum detectable doses with the two metabolic activation systems were 1 uM and 5 mM respectively. DNA elution profiles were linear in splenocytes cocultured with DMN and hepatocytes and convex in splenocytes incubated with DMN and S-9. Aminoacetonirile(AAN), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either metabolic activation system. Addition of exogenous calf thymus DNA to the hepatocyte coculture medium did not affect the production of DNA SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from liner to convex implying that the types of DNA damage was changed to alkali labile one. All of these experimental manipulations have been previously shown to block the immunosuppression by DMN is not related to DNA damage and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.
Experiments were designed to identify the cellular target and immunosuppressive metabolites of DMN in splenocytes. Splenocytes were separated to B-cells and T-cells, co-cultivated with hepatocytes in the presence of DMN and then reconstituted to B + T-cells for antibody response. Results showed that both B-and T-cells were affected by DMN. To study the nature of active intermediates of DMN in immunosuppression, we investigated the role by a Phase II generated intermediate, specifically a sulfate conjugate. Addition of sodium sulfate to the hepatocyte coculture system, at concentrations up to 10 mM, produced a dose related enhancement in the DMN-induced suppression of T-dependent antibody response. Addition of the sulfate conjugation inhibitors, salicylamide (SAM) produced a dose-related reversal of the DMN-induced immunosuppression. Both series of results were consistent with a role by a sulfate conjugate in DMN-induced immunosuppression. Structure activity relationship of nitrosamines, diethylnitrosamine(DEN), methylethylnitrosamine(MEN), and methylbutylnitrosamine(MBN), on the suppression of antibody response were studied. All of these nitrosamines required metabolic activation to produce suppression of antibody response and the ED50 was 0.5-1.0, 1.0, 20, and more than 20 mM for DMN, MBN, DEN and MEN respectively.
The immunosuppressive effects of 2-amino fluorene(AF) and 2-acetylaminofluorene(AAF) were studied in mouse splenocyte culture. Direct addition of AF and AAF to the splenocyte culture produced a dose-related suppression of the in vitro antibody response to sheep erythrocytes(SRBC), DNP-Ficoll, and lipopoly-saccharide(LPS). AF and AAF also produced suppression on lymphoproliferative responses to LPS and concanavalin A(Con A). The immunosuppressive effects of AF and AAF, however, diminished when AF and AAF incubated in the splenocyte-rat hepatocyte coculture system for 4 hr. When hepatocyte cultures were pretreated with SKF 525A, a cytochrome P-450 inhibitor, before coculture with spleen cell along with AF and AAF, a dose-related suppression of in vitro antibody response reappeared. Meanwhile, both AF and AAF produced dose related DNA SSB in spleen cells only if AF and AAF were treated to spleen cells cocultured with hepatocytes. These results indicate that the immunosuppression by AF and AAF is not mediated by the reactive metabolites implicated to DNA damage.
Safrole produced suppression of antibody response and mitogen-induced lymphoproliferation when it was added directly to the splenocyte culture. This suppression was not changed by coculture with rat hepatocytes or addition of sulfate conjugation cosubstrate, sodium sulfate, and sulfate conjugation inhibitor, SAM, to splenocyte culture. These results suggest that carcinogenic metabolite of safrole, 1-sulfooxysafrole might not be involved in safrole-induced immunosuppression.