The metabolism of beta-carbolines by cytochrome P-450 : effect of induction and influence of substrate structure
The metabolism of the beta-carbolines harmine, harmol and harman by C57/BL10 mouse liver microsomes has been investigated. Changes, induced by phenobarbitone (PB) or 3-methylcholanthrene (MC), in the apparent Michaelis-Menten enzyme kinetics for the metabolism of harmine and harmol, and in the metabolite profiles of harmine, harmol and harman, were studied. 1. A method for the differential extraction of harmine and its only previously reported metabolite, harmol, is described. As both harmine and harmol have identical excitation and fluorescence wavelengths, this differential extraction was an essential step in the fluorimetric quantitation of both harmine and harmol. By this method harmine metabolism was measured both as the overall rate of harmine disappearance (by unspecified reactions) and as the rate of O-demethylation (harmol formation). Marked changes in the apparent Michaelis-Menten kinetics of harmine were induced by PB and MC. Alteration of the metabolite profile was also induced by MC. With control (from untreated or olive oil pretreated mice) or PB-induced microsomes the overall metabolism of harmine proceeded by both high and low affinity reactions. Only the high affinity reaction occurred following MC pretreatment (app. Km =0.4 M) which was induced 31-fold to Vmax = 22 nmol min-1 mg protein-1. With control or PB-induced microsomes harmine was metabolised almost exclusively by 0-demethylation to harmol (3-fold induction by PB to Vmax = 9.8 nmol min-1 mg protein app. Km = 73 M and 14 M for control and PB-induced microsomes respectively). This was probably the low affinity reaction of overall harmine metabolism. With MC-induced microsomes harmine was also metabolised to two previously unidentified yellow compounds, designated and with harmol produced as a minor metabolite by a high affinity reaction (app. Km = 0.6 M). Control, PB-induced and MC-induced microsomes each metabolised harmol by a high affinity reaction (app Km = 1.9 - 0.6 M) to unidentified metabolite(s). Harmol metabolism was not induced by PB, but was markedly induced by MC (21-fold, Vmax = 11.0 nmol min-1mg protein-1). This partly explains the relative lack of net harmol formation after MC induction. 2. An HPLC system was developed for the separation of harmine and its metabolites. The rates of formation of Y1 and Y2 by MC-induced microsomes were measured using [3H]-labelled harmine as the substrate. The metabolites were extracted and then isolated by the HPLC system described above. The HPLC eluent was monitored by UV absorbance at 254nm. One ml fractions were collected and the radioactivity measured by scintillation counting. The collected radioactive fractions were identified as harmine and each of its metabolites by comparison with the RT, values of the UV absorbance peaks. Initial reaction rates for microsomal metabolism were calculated from the reaction progress curves and were found to be similar to the Vmax rates previously obtained by fluorimetry, ie. 24.0 nmol min-1 mg protein-1 for the overall rate of harmine disappearance and 4.0 nmole min-1 mg protein-1 for harmol formation. The initial rates of formation of and Y1 and Y2 were 11.0 nmol min-1 mg protein-1 and 3.8 nmol.min-1 mg protein-1 respectively. Incubation of [3H]-harmine with non-induced or PB- induced microsomes showed that Y1 and Y2 were also produced by these microsomes, although to a much lesser extent, at approximately 0.55 and 0.35 nmol min-1 mg protein-1, respectively. TLC separation of harmine and its metabolites provided visual confirmation,by means of spot size and fluorescence intensity,of the relative extents of formation of harmol, Y1 and Y2 by non-induced, PB-induced or MC-induced microsomes. The reaction progress curves suggested that Y1, as well as harmol, was metabolised further by MC-induced microsomes. The overall conclusion from this study was that sufficient information is not yet available to allow definitive structure-activity relationships to be drawn which would rationalize the metabolism of even a group of structurally similar compounds, eg. the beta-carbolines, by different isozymes of cytochrome P-450. Small structural differences in substrates can influence both the extent and molecular site of metabolism, which when considered together with the multiplicity of cytochrome P-450 isozymes, prevents an accurate prediction of metabolic reactivity and metabolite profiles.