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

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 LC50 value of 0.36 mm (95% confidence interval, 0.33–0.40 mm). CNP also reduced the viability in a concentration-dependent manner at the concentrations of 0.50 mm 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 mm. To identify the enzyme involved in the metabolic activation of CNP-amino, inhibition studies were carried out using SKF 525-A (0.050 mm) and methimazole (1.0 mm). 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 mm for chlomethoxyfen (18-fold) and at 0.25 mm 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 mm (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.