Induction of cytochrome P‐450 isozymes in rat hepatoma‐derived cell cultures

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Abstract
We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P‐450. One Reuber hepatoma‐derived line (Fu5‐C8), which under normal culture conditions produces no detectable cytochrome P‐450(MC) or cytochrome P‐450(PB)—the major cytochrome P‐450 isozymes induced by 3‐methylcholanthrene and phenobarbital, respectively‐ was tested for the ability to accumulate either cytochrome P‐450 isozyme in response to treatment with various xenobiotics. By immune‐precipitation from [35S]‐methionine‐labeled cell extracts, using monospecific anticyto‐chrome P‐450(MC) antybody or monoclonal anticytochrome P‐450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P‐450(MC) in response to 3‐methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P‐450(PB). RNA extracted from Fu5‐C8 cells directed the in vitro synthesis of immune‐precipitable cytochrome P‐450(MC) only after treatment of the cells with 3‐methylcholanthrene Kinetic analysis of the response of these cells to 3‐methylcholanthrene induction revealed detectable levels of immune‐precipitable cytochrome P‐450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P‐450(MC) and also cytochrome P‐450(PB) constitutively, as determined by specific immune‐precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3‐methylcholanthrene resulted in an induction of cytochrome P‐450(PB) or cytochrome F‐450(MC), respectively. Double‐labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P‐450(MC), but only a subset of cells synthesize cytochrome P‐450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P‐450(PB).