Manipulation of the phenotype of immortalised rat hepatocytes by different culture configurations and by dimethyl sulphoxide

Abstract

The liver-specific phenotype of immortalised rat hepatocytes is not irretrievably lost as they age in culture but can be manipulated by modifying the culture environment. Testosterone metabolism was used to investigate the profile of cytochrome P450 isoenzymes present in two immortalised cell lines, P9 and LQC, and in primary cultures of rat hepatocytes, cultured on collagen films, gels and double gel cultures (sandwich configuration). The extent of testosterone metabolism, and the range of metabolites produced, was increased in immortalised cells by the presence of collagen as a substratum film or gel but survival was poorer and the range of metabolites was reduced in sandwich culture. In contrast, testosterone metabolism was retained in primary hepatocytes in sandwich cultures at a higher level than in collagen film or gel cultures. Expression of alpha class glutathione-S-transferases (GSTs) increased and that of GSTP1 decreased (changes which indicate a recovery of normal liver GST phenotype) when the medium of immortalised cell cultures was supplemented with dimethyl sulphoxide (DMSO). DMSO also improved ethoxyresorufin O-deethylation (EROD) and testosterone metabolism in immortalised cells. It also markedly inhibited proliferation, DNA, RNA and protein synthesis. Maximal testosterone metabolism was observed in immortalised cells cultured on collagen gels in the presence of 1% (v/v) DMSO. Development of a protocol for treating immortalised liver cells cultured on collagen gels with DMSO to switch between proliferation and differentiation may provide a convenient system expressing the xenobiotic metabolising enzymes required for in vitro toxicity testing.