Cytotoxic and porphyrinogenic effects of diphenyl ethers in cultured rat hepatocytes: chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox

Abstract

We studied the cytotoxic and porphyrinogenic effects of four diphenyl ethers (DPEs), chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox, in rat hepatocytes cultured on Matrigel. Cytotoxicity was determined as a decrease in viability measured by the release of lactate dehydrogenase. Of the DPEs examined, CNP-amino was the most cytotoxic, with an LC 50 value of 0.36 m m (95% confidence interval, 0.33–0.40 m m). CNP also reduced the viability in a concentration-dependent manner at the concentrations of 0.50 m m or above. In contrast, no concentration-dependent decrease in viability was observed in the chlomethoxyfen- and bifenox-treated hepatocytes at the concentrations up to 1.0 m m. To identify the enzyme involved in the metabolic activation of CNP-amino, inhibition studies were carried out using SKF 525-A (0.050 m m) and methimazole (1.0 m m). SKF 525-A, a cytochrome P450 inhibitor, quickened the onset of cell killing by CNP-amino, while methimazole, an inhibitor of flavin-containing monooxygenase (FMO), partially suppressed the cytotoxicity of CNP-amino. These results suggest that FMO plays an important role in the cytotoxicity induced by CNP-amino, while cytochrome P450 participates in the detoxification, possibly via the ring-hydroxylation. The maximum porphyrin accumulation was observed at 0.13 m m for chlomethoxyfen (18-fold) and at 0.25 m m for CNP and bifenox (17- and 21-fold, respectively). In contrast to these DPEs, the porphyrinogenic effect of CNP-amino was weak, with the maximum accumulation at 0.13 m m (at least fivefold). The predominant species was protoporphyrin IX in all of the DPE-treated cultures. These results suggest that all of the DPEs examined, possibly including CNP-amino, inhibit protoporphyrinogen oxidase, resulting in the accumulation of protoporphyrin IX.