AN IN-VITRO STUDY ON METHOTREXATE HYDROXYLATION IN RAT AND HUMAN LIVER

Methotrexate (MTX) was investigated for possible effect on the metabol ism of ethoxyresorufin, pentoxyresorufin and ethoxycoumarin, the model substrates of cytochrome P450. The investigation was carried out in l iver microsomes of rats pretreated with classical inducers of cytochro me P450 as well as in microsomes of two human livers. Furthermore, we measured the conversion of MTX (100 mu M) to its main metabolite, 7-hy droxymethotrexate (7-OHMTX), in microsomes and cytosolic fractions of rat and human livers. The inhibition of 7-OHMTX formation by menadion (inhibitor of aldehyde oxidase) and allopurinol (inhibitor of xanthine oxidase) was studied in the cytosol of rat and human livers. In both species, MTX in the concentration range 0.5-500 mu M exerted no inhibi tory effect on enzymatic activities associated with cytochrome P450. M oreover, we did not observe any measurable formation of 7-OHMTX in liv er microsomes. MTX was metabolized at a similar rate in the cytosol of rat and human liver. Allopurinol (100 mu M) reduced the rate of MTX h ydroxylation by 31.5% in the cytosol of human livers but had no effect in the rat. Menadion (100 mu M) decreased the rate of 7-OHMTX formati on in the cytosol of human and rat liver by 69% and 94%, respectively. Our results confirmed that MTX is oxidized by a soluble enzymatic sys tem in both the rat and human liver. In human tissues, both aldehyde o xidase and xanthine oxidase may play an important role in the metaboli sm of MTX. Depression of cytochrome P450 and related enzymatic activit ies observed in vivo cannot be explained by a direct inhibitory action of MTX on cytochrome P450.