Applications of the primary hepatocyte culture in drug metabolism and immunotoxicology

Abstract

Effects of dimethyl sulfoxide (DMSO), which is a common solvent to dissolve test compounds in mutagenesis and drug metabolism studies, on drug metabolism were investigated by using the primary culture of adult rat hepatocytes and the microsome isolated from rats. Addition of DMSO to the hepatocyte culture induced the P-450 II El-specific aniline hydroxylase activity and the P-450 I Al-specific ethoxyresorufin O-deethylase (EROD) activity dose- and time-dependently from 0,1\%(v/v) concentration. The P-450 II B-specific pentoxyresorufin O-dealkylase activity was also induced at 2\%(v/ v) concentration. To rnvestigate the mechanism of inductiuon of EROD activity by DMSO, Northern and dot blot analyses were performed by using an oligonucleotide probe, but no increase of P-450 I Al mRNA was found. When 1.0$\mu$M cycloheximide was treated to the hepatocyte culture, the induction of EROD activity by DMSO was disappeared, indicating that the induction was not mediated by the accumulation of P-450 I Al mRNA, but by the increase of P-450 I Al protein syntheses. In fact, the increase of P-450 I Al protein by DMSO was observed in microsomes isolated from the hepatocytes cultured with 2\% DMSO by using the ELISA and Western immunoblotting. On the other hand, when DMSO was added into the reaction mixtures for isozyme-specific monooxygenase reactions, the P-450 II El-specific p-nitrophenol hydroxylase (PNPH) activity was inhibited profoundly and dose-dependently from 5 mM concentration. Other monooxygenations were not affected at this concentraion. Demethylation of DMSO by itself was observed. The inhibition of PNPH by DMSO was mot related to NADPH P-450 reductase, but was a competitve inhibition with Ki value of 12.7 mM in microsomes from female rats and a weak mixed competitive-noncompetitive inhibition with Ki value of 32.3 mM in microsomes from male rats. Therefore, precautions should be taken of DMSO is a solvent to study drug metabolism and mutagenesis of chemicals, and...