Studies on the immunosuppressive effects of chemical carcinogens in hepatocyte-splenocyte coculture system

Abstract

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...