Cytochrome P450 substrate specificities in rat, hamster and man
There are wide interspecies variations in the oxygenative cytochrome P450-dependent metabolism of foreign chemicals, and this has been shown to lead to species differences in chemical toxicity. This investigation studied a series of structurally similar substrates in rat, hamster and man. This approach may lead to a greater understanding of the characteristics of cytochrome P450 in different species in a way that relates to the chemical structure of substrates. The substrates investigated were phenoxazone (the parent compound), the benzyl ether (benzyloxyphenoxazone) and a homologous series of n-alkyl ethers of phenoxazone, with increasing alkoxy chain length from methoxy to octoxy. Preliminary results using the thin layer chromatographic (TLC) method showed that resorufin was a major metabolite formed during the metabolism of selected ethers of phenoxazone by microsomes from untreated male rats and hamsters, from rats and hamsters pretreated with PB and 3MC and from human liver. Further investigation using the HPLC method confirmed that resorufin was virtually the sole product of the metabolism of ethoxy-, pentoxy-, heptoxy- and benzyloxyphenoxazone by microsomes from untreated rats and mice, and PB and 3MC pretreated rats and mice. These observations indicated that 0-dealkylation was the major route of metabolism of the phenoxazone ethers by microsomes from these species. Having established that phenoxazone and its alkyl and aryl ethers were each metabolised to a common highly fluorescent phenolic metabolite, resorufin, the specific activity of hepatic microsomal resorufin formation was measured in male Sprague-Dawley rats, male and female Golden Syrian hamsters, and in man. Structure activity relationships (SAR) were constructed showing the effects of the presence (comparing phenoxazone with its alkyl and aryl ethers), length (C1-C8) and type (comparing the alkyl ethers with benzyloxyphenoxazone) of an ether side chain on the metabolic production of resorufin. The SAR were generally similar for humans, control rats and control hamsters, both in the overall effect of chain length and in the mean specific activity of resorufin production from each substrate. In microsomes from humans and control animals, the specific rate of resorufin formation declined with increasing chain length. The SAR were dramatically different in phenobarbitone (PB), 3-methylcholanthrene (3MC), Aroclor 1254, isosafrole and SKF-525A pretreated male rats compared to controls. Species differences in the responsiveness of rats and hamsters to PB, 3MC and isosafrole were observed. The effects of PB and 3MC pretreatment were found to be less marked in hamsters and were different from their effects on substrate metabolism in rat. In contrast, isosafrole pretreatment induced the microsomal metabolism of phenoxazone and its alkyl and aryl ethers to a greater extent in hamster than in rat. Sex differences were observed in the response of male and female hamsters to the inducers, betanaphthof1avone (BNF) and isosafrole. The SAR for 47 humans were similar among the majority of patients with neither sex or age related differences being observed. However, in two small groups of patients the SAR differed from the majority of patients. Heavy cigarette smokers (>30/day) showed an increased metabolism of the ethyl ether of phenoxazone (ethoxyresorufin). Three other patients had a high rate of metabolism of benzyloxyphenoxazone relative to ethoxyphenoxazone. This investigation showed that induction of different isozymes of cytochrome P450 in animals can be characterised by large changes that occur in the metabolism of selected members of a homologous series of phenoxazone ethers. By inference from these experiments in animals, differences observed in SAR between certain groups of patients were taken to be indicative of at least three different cytochrome P450 isozyme composition patterns in human hepatic microsomes: an "average pattern", a "heavy smoker" pattern and a "high benzyloxyphenoxazone debenzylation pattern." The "heavy smoker" pattern may reflect induction of cytochrome P448 type isozymes in human liver.