Aflatoxin toxicity in cultured human epidermal cells: Stimulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aflatoxin B₁ (AFB₁) at 1 μg/ml was markedly toxic to human epidermal cells grown in the Rheinwald-Green 3T3 feeder layer system. At 0.1 μg/ml, the toxicity was barely evident, as assessed by colony expansion during a 2 week exposure, but it was dramatically stimulated by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was non-toxic alone. Neither AFB₁-dihydrodiol nor AFB₂ were toxic in the presence or absence of TCDD, indicating that metabolism to the 8,9-epoxide was responsible for the AFB₁ toxicity. Stimulation of AFB₁ epoxidation by TCDD was also indicated by the >20-fold increase in DNA adduct formation in cultures exposed to [¹⁴C]AFB₁ and TCDD for 4 days as compared to AFB₁ alone. Analysis of free metabolites in culture medium by reverse-phase HPLC revealed that confluent epidermal cultures metabolized AFB₁ to AFM₁, AFB_2a and aflatoxicol. In the presence of TCDD, the levels of AFM₁ were higher (14 versus 3% of dose) as were those of AFB_2a (3 versus 0.5% of dose), while aflatoxicol levels were lower (0.8 versus 2% of dose). In the absence of irradiated 3T3, the toxicities of AFB₁, AFB₂, AFM₁ and aflatoxicol to cells in serum-free medium (0.15 mM Ca²⁺) were similar to those in the feeder layer system. Although this moderately low calcium concentration appeared quite satisfactory for observing toxicity, the response was attenuated at a lower calcium concentration (0.09 mM Ca²⁺).