A Simple Bioassay for Evaluating Immunotoxic Properties of Chemicals by Use of in Vitro Antibody Production System

Antibody production is one of biomarkers sensitive to chemical toxicity. A simple assay system for evaluating toxic effect of chemicals on antibody production was developed using pokeweed mitogen (PWM)-induced IgM production by spleen cell cultures obtained from BALB/c mice. It was evaluated with reference immunomodulating chemicals. Cultured spleen cells significantly and day-dependently produced IgM after PWM stimulation. Test chemicals were added to the cultures simultaneousely with PWM. At 4 day-culture, medium IgM level and cell proliferation were determined by ELISA method and WST-8 method for mitochondrial dehydrogenase activity, respectively. Results showed that PWM-induced IgM production was dose-dependently suppressed by cyclosporin A, a typical immunosuppressor, with more susceptible inhibition of cell proliferation. On the contrary, it was increased by lead nitrate, an immunoenhancer, around 100 μM with little change in the cell proliferation. Moreover some estrogen-related compounds such as estradiol, ethynylestradiol, diethylstilbestrol and bisphenol A dissolved in dimethyl sulfoxide were applied to the assay system. Those below 1 μM affect neither the IgM production nor the cell proliferation in spleen cell cultures irrespective of mouse sex. Further for detecting active metabolites, the preincubation of a chemical with S9 mix was coupled with the assay system. The availability of the procedure was confirmed by inhibitory effect of cyclophosphamide after preincubation with S9 mix, which had no effect without S9 mix. Thus the newly developed in vitro IgM production system by mouse spleen cells is effective for evaluating toxic or modulating effects of chemicals on antibody production.