Stability of cytochrome P450 proteins in cultured precision-cut rat liver slices

Abstract

The objective of this study was to evaluate the stability of individual, xenobiotic-metabolising, cytochrome P450 proteins in precision-cut rat liver slices cultured for up to 72 h using the multiwell plate system. This was achieved using established diagnostic probes (O-dealkylation of methoxy-, ethoxy- and pentoxy-resorufin, testosterone 2α-hydroxylase, debrisoquine 4-hydroxylase, aniline p-hydroxylase and lauric acid hydroxylase) and immunologically using Western blotting. All cytochrome P450 activities declined in culture, the most rapid loss occurring at about 8–12 h of culture; in all cases no detectable activity was present in the 72-h cultured slices. Isoform-specific differences in the stability of various cytochrome P450 proteins were observed, with CYP2E1 being the most stable. When cytochrome P450 expression was determined immunologically, a different picture emerged. High levels of apoprotein were retained in the slices even when activity was very low. In the case of CYP2B, apoprotein levels even increased following the culture of hepatic slices. It is concluded, that for tissue slices to become an acceptable in vitro alternative system for long-term incubations, the culturing conditions must be improved to ensure that cytochrome P450 activities are better maintained.