The objective of the present studies was to define the enzyme systems catalysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulphatoxymelatonin in rat hepatic postmitochondrial preparations and in precision-cut liver slices. Melatonin 6-hydroxylase activity was localized in microsomes and was supported by NADPH, but not NADH. Treatment of rats with beta-naphthoflavone more than tripled 6-sulphatoxymelatonin formation from melatonin, but gave rise only to a moderate increase (25%) in the sulphate conjugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, acetone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin generation when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline inhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin without any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with beta-naphthoflavone, and to a lesser extent phenobarbitone, elevated the levels of 6-sulphatoxymelatonin in the culture medium. No such increase was seen when slices from beta-naphthoflavone-treated rats were incubated with 6-hydroxymelatonin, whereas a modest increase was seen with slices from phenobarbitone-treated rats. Treatment of rats with acetone, dexamethasone or clofibrate failed to modulate the levels of 6-sulphatoxymelatonin generated from either melatonin or 6-hydroxymelatonin. Molecular modelling analysis revealed that melatonin had a high area/depth(2) ratio, displayed characteristics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, all the above data provide strong experimental evidence that CYP1A2 is an important catalyst of the 6-hydroxylation of melatonin.

Caffeine was administered to male Wistar albino rats for two weeks at three concentrations, namely 0.1, 0.2 and 0.3%, and hepatic cytochrome P450-dependent mixed-function oxidase determined. Caffeine administration gave rise to a marked, dose-dependent increase in the O-deethylation of ethoxyresorufin and, to a lesser extent, in the O-depentylation of pentoxyresorufin. Erythromycin N-demethylase, p-nitrophenol hydroxylase and lauric acid hydroxylase activities, as well as total cytochrome P450 content were unaffected by this treatment. Immunoblot analysis revealed that caffeine gave rise to a dose-dependent increase in the hepatic CYP1A2, and at the highest dose only, CYP2B apoprotein levels. Apoprotein levels of CYP3A and CYP2E1 were not modulated by the treatment with caffeine at all dose levels studied. Caffeine could not displace [3H]TCDD from the rat hepatic cytosolic Ah receptor. Computer analysis showed that caffeine is essentially a planar molecule with an area/depth ratio 4.8, characteristic of CYP1A substrates/inducers. Molecular modelling revealed that the caffeine molecule could orientate itself within the putative CYP1A2 active site so as to facilitate demethylation of the N-1, N-3 and N-7 positions. However, at physiological pH, the N-9 nitrogen atom is likely to be partially protonated, allowing it to participate in an electrostatic interaction with the negatively-charged glutamate 318-residue, favouring N-3 demethylation, the major pathway of metabolism in both humans and animals. In conclusion caffeine, being essentially planar, is an inducer of CYP1A2 in rat liver.

No abstract

The title compound is a strong carcinogen, similar in potency to benzo[a]pyrene in mouse skin assay. This paper describes a comparison of its in vitro metabolism by hepatic microsomal preparations from mouse, rat, rabbit, hamster, dog, monkey and man. Metabolites were isolated by preparative high pressure liquid chromatography from the ethyl acetate extractable material and their structures tentatively assigned on the basis of their retention times and ultraviolet spectra, when possible by direct comparison with authentic synthetic specimens. Mass spectrometry was then used to confirm these assignments. All these animals produce the same range of metabolites derived exclusively from oxidation at the benzo-ring A, the five-membered ring D, and at the 11-methyl group. However, the amounts of individual metabolites varied substantially. In particular all the animals yielded the proximate carcinogen 3,4-dihydroxy-11-methyl-3,4,15, 16-tetrahydrocyclopenta[a]phenanthren-17-one, from which it is reasoned that all might be susceptible to its carcinogenic action. A rationalization for the observed distribution of the metabolites is proposed on the basis of a molecular model of the active site of cytochrome P450 1A1, the oxidative enzyme involved.