ATLA::Alternatives to Laboratory Animals

Volume 26, Number 2

In vitro studies on the metabolism and toxicity of aflatoxin B1 in primary cultures of black catfish (Ictalurus melas) hepatocytes.

ATLA 26, 225-239, March/April 1998

Michela Ferraris,1 Erminio Marafante1 and Manjit Dosanjh1,2

1ECVAM, JRC Environment Institute, 21020 Ispra (VA), Italy; 2Lawrence Berkeley Laboratories, University of California, Berkeley, CA 94720, USA

SUMMARY

The capacity of a primary hepatocyte culture of black catfish (Ictalurus melas) to metabolise toxic compounds was evaluated by using aflatoxin Bl (AFB1) as a xenobiotic model. Hepatocytes were isolated by a two-step liver perfusion procedure and cultured in a serum-free medium on both porous membranes and 96-well microplates coated with Matrigel®. A viability of 95-98% was obtained. The ability to maintain cytochrome P450 activities was examined by measuring ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities, at various culture times. The cytotoxic effects of AFB1 on catfish hepatocytes were examined by using a neutral red uptake assay on 96-well microplates. The concentration causing 50% inhibition of uptake (IC50) value was 3 µM. The AFB1 metabolites were analysed by HPLC with an ultraviolet diode array and fluorescent detectors. The results obtained show that catfish hepatocytes cultured on Matrigel retain their AFB1 metabolising activities for at least 6 days after seeding. This comparative study, using primary hepatocytes and isolated microsomes from black catfish, trout, and mice, confirms that the observed marked differences in toxic response following exposure to AFB1 in these species are likely to be related to their various metabolising capacities.

Keywords: aflatoxin B1 metabolism, black catfish, trout, EROD, microassays