Identification of inducible mixed function oxidase system in rat glioma C6 cell line

The mixed function oxidase system consists of NADPH-cytochrome P450 reductase (P450 reductase) and various isoforms of cytochrome P450 (P450), which can catalyze the oxidation of a broad range of endogenous and exogenous compounds. In this study, we examined the rat glioma C6 cell line for the presence of P450 reductase and three isozymes of cytochrome P450, 1A1, 2B1, and 2B2, by reverse transcription followed by PCR (RT-PCR). Rat glioma C6 cells were treated with hepatic P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). Cytochromes P450 1A1, P450 2B1, and P450 2B2, and P450 reductase, were detected in all the different treatment groups. Restriction digestion was used to confirm the PCR fragments and the expected digestion products were obtained. The induction of P450 1A1 and 2B was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected by competitive PCR. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 spectra with absorption at 450 nm. Ethoxyresorufin O-deethylase activity (11.5 +/- 1.7 pmol/min/mg of microsomal protein) and pentoxyresorufin O-dealkylation activity (8.9 +/- 1.4 pmol/min/mg of microsomal protein) confirmed the induction of P450 1A and 2B at the protein level in response to BA or PB treatments, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active mixed function oxidase system that can be induced by hepatic P450 inducers.