Major reviewInhibitory metabolite complex formation of methylenedioxymethamphetamine with rat and human cytochrome P450. Particular involvement of CYP 2D
Introduction
Methylenedioxymethamphetamine (MDMA or ecstasy) is a common recreational drug used at rave parties. Reports estimate that millions of young people use MDMA at least occasionally (Green et al., 1995; Grob et al., 1995; Schwartz and Miller, 1997). The toxicity of MDMA is well documented in animals and humans, and in some cases causes death (Berger et al., 1992; Coore, 1996; Aguirre et al., 1997). In addition to malignant hyperthermia and neurological effects, hepatotoxic effects of MDMA have also been reported (deMan et al., 1993; Khakoo et al., 1995; Fidler et al., 1996; Ellis et al., 1996; Hellinger et al., 1997). Liver function parameters spontaneously normalize upon withdrawal of the drug. The underlying mechanism of hepatitis has as yet to be clearly demonstrated by Khakoo et al. (1995).
Amphetamine derivatives are extensively metabolized into analogues of neurologically active amines such as dopamine by cytochromes P450 in rat and human liver (see Cho and Kumagai (1994) for a review). MDMA (Fig. 1) is oxidized at its methylenedioxy group to form catechol and the amino function can be demethylated into the corresponding primary amine. Such metabolites have been identified in the urine of rats given MDMA (Lim and Foltz, 1988; Ensslin et al., 1996). Amphetamine metabolites are involved in the observed toxicological effects (Cho and Kumagai, 1994; Tomkins et al., 1997; Carvalho et al., 1997). CYP 2B and 2D P450 isozymes are involved in amphetamine metabolism (Cho and Kumagai, 1994; Kumagai et al., 1994), as shown directly by metabolic studies (Cho and Kumagai 1994; Kumagai et al., 1994; Tucker et al., 1994; Lin et al., 1997) and by inhibitory effects (Tomkins et al., 1997; Wu et al., 1997). P450 2B and 2D are present in both liver and brain, and so reactive metabolites such as catechol are formed directly near the neuronal target. Both amphetamine and methylenedioxy compounds are precursors of stable inhibitory complexes with P450 iron which absorb at 455 nm (Franklin, 1977; Lindeke and Cho, 1982; Cho and Kumagai, 1994; Ortiz de Montellano and Correia, 1995). The amine function can be oxidized into a nitroso metabolite which readily reacts with Fe(II), and methylenedioxy can be metabolized into carbene, thus forming stable Fe(II) or Fe(III) complexes. These P450-metabolite complexes are responsible for the inhibition of enzymatic activities observed in vivo and in vitro in animals treated with tertiary amino compounds or methylenedioxy derivatives (Delaforge et al., 1985, Bensoussan et al., 1995).
Cytochrome P450 2D6 is a polymorphic member of the cytochrome P450 superfamily and is absent in about 10% of Caucasians (Guengerich, 1995). We now provide direct evidence of the formation of a stable MDMA inhibitory complex with rat and human cytochrome P450, the 2D isozymes being predominantly affected. This formation of a stable inhibitory complex may result in perturbed liver metabolism and thus in hepatotoxicity, as in the case of macrolide antibiotics (Mansuy and Delaforge, 1993). This also suggests potent risks upon concomitant ingestion with drugs mainly metabolized by CYP2D.
Section snippets
Materials
Methylenedioxymethamphetamine, NADPH, NADP, glucose 6 phosphate (Glc6P), glucose 6 phosphate dehydrogenase (Glc6PDH), 3-methylcholanthrene, phenobarbitone, were from Sigma. Dexamethasone, clofibrate were from Janssen Chemicals. Other chemicals were of the highest purity available.
Methods
Male Sprague–Dawley (SD) rats (Iffa Credo, St Germain l’Arbresle, France) and Dark-Agouti rats (Janvier, France) were treated as described previously (Bensoussan et al., 1995), and microsomes were stored at −80°C until use.
Yeast-expressed human P450s were produced in the presence of either yeast P450-reductase or human P450-reductase (Peyronneau et al., 1992; Pompon et al., 1995). The corresponding microsomes were prepared as described previously (Peyronneau et al., 1992). Human samples were
Spectral interactions
Addition of MDMA to hepatic microsomes from untreated rats led to the formation of type I spectral interactions (λmax 394 nm, λmin 417 nm) (Fig. 2a, Table 1). This spectral interaction was also observed for MDMA, but less markedly, with microsomes from SD rats treated with phenobarbital, dexamethasone, 3-methylcholanthrene, clofibrate or isoniazid. This strongly suggests that MDMA interacts preferentially with a P450 active site mainly present in untreated animals. The dissociation constant of
Conclusion
The formation of P450 metabolite complexes upon oxidation of drugs containing tertiary amine or methylenedioxy functions involves oxidation leading to inhibition of P450 catalytic activities. MDMA has the particularity of having on the same molecule both the tertiary amine and the methylenedioxy functions. It yielded significant amounts of 455 nm absorbing complexes with human and rat liver microsomes, arising mainly from the oxidation of the methylenedioxy function. This complex formation was
Acknowledgements
We thank Dr D. Pompon (Gif Sur Yvette) and Dr P. Beaune (Paris, France) for providing us with the W(R) yeast strains.
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