Contribution of CYP1A2 in the hepatic metabolism of melatonin: studies with isolated microsomal preparations and liver slices

The objective of the present studies was to define the enzyme systems catal ysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulph atoxymelatonin in rat hepatic postmitochondrial preparations and in precisi on-cut liver slices. Melatonin 6-hydroxylase activity was localized in micr osomes and was supported by NADPH, but not NADH. Treatment of rats with bet a -naphthoflavone more than tripled 6-sulphatoxymelatonin formation from me latonin, but gave rise only to a moderate increase (25%) in the sulphate co njugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, ace tone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin g eneration when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline i nhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin with out any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with bet a -naphthoflavone, and to a lesser extent phenobarbitone. elevated the leve ls of 6-sulphatoxymelatonin in the culture medium. No such increase was see n when slices from beta -naphthoflavone-treated rats were incubated with 6- hydroxymelatonin, whereas a modest increase was seen with slices from pheno barbitone-treated rats. Treatment of rats with acetone, dexamethasone or cl ofibrate railed to modulate the levels of 6-sulphatoxymelatonin generated f rom either melatonin or 6-hydroxymetatonin. Molecular modelling analysis re vealed that melatonin had a high area/depth(2) ratio, displayed characteris tics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, a ll the above data provide strong experimental evidence that CYP1A2 is an im portant catalyst of the 6-hydroxylation of melatonin.