Strain differences of the ability to hydroxylate methotrexate in rats

doi:10.1016/S0742-8413(98)10134-2

Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology

Volume 122, Issue 3, March 1999, Pages 331-336

https://doi.org/10.1016/S0742-8413(98)10134-2 Get rights and content

Abstract

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, β-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.

Introduction

The antifolate agent methotrexate (MTX), 4-amino-N¹⁰-methylpteroglutamic acid, is widely used in the treatment of acute lymphocytic leukemia in children, as well as for other adult and childhood malignancies, including squamous carcinoma of the head and neck, lymphomas, osteosarcomas, and choriocarcinomas [1], [14], [16]. As an additional usage of MTX, it has become a standard therapy for the treatment of rheumatoid arthritis [8], [25]. MTX is metabolized to MTX polyglutamate [4], 2,4-diamino-N¹⁰-methylpteroic acid [6] and 7-hydroxymethotrexate (7-OH-MTX) [2], [3], [12], [20] in human and animal species (Fig. 1). 7-OH-MTX, a major metabolite of MTX, is cytotoxic [21], and has been demonstrated to affect cellular entry, polyglutamation, and efflux of the parent compound in vitro [7].

Most in vitro studies of the conversion of MTX to 7-OH-MTX have been carried out in rabbits. In this species, several tissues have significant capacity for 7-OH-MTX formation [19] and the liver seems to have a particularly high activity due to the action of aldehyde oxidase (EC 1.2.3.1) [9]. However, partially purified preparations of aldehyde oxidase from rat liver have only a limited capacity to hydroxylate MTX [10]. Yu et al. also suggested that aldehyde oxidase was not the predominant hydroxylating enzyme for MTX in the rat, based on the fact that the excretion ratio of 7-OH-MTX in the bile of rats dosed with MTX was not influenced by co-administration of an inhibitor of aldehyde oxidase [26]. The identity of the enzyme responsible for 7-hydroxylation of MTX in rat liver remains unclear.

Recently, we reported a significant variation of liver aldehyde oxidase activity in twelve strains of rats [23]. Investigation of strain differences in the hydroxylating activity toward MTX in rats could be a useful approach for examining the role of aldehyde oxidase in the metabolism of MTX in this species.

In this study, we investigated the variability of the hydroxylating activity toward MTX in liver cytosols from several strains of rats, and its relation to the aldehyde oxidase activities in these rats in order to clarify the role of the latter enzyme as a MTX hydroxylase.

Section snippets

Chemicals

Materials were obtained from the following sources: menadione, benzaldehyde, disulfiram, chlorpromazine hydrochloride and β-estradiol from Nacalai Tesque; (−)-methotrexate, oxypurinol and milk xanthine oxidase (x-1875) from Sigma Chemical; bovine serum albumin from Armour Pharmaceutical. MTX and 7-OH-MTX were kindly donated by Medical Research Laboratories, Lederle Japan.

Animals

Eight different strains of male rats (6–7 weeks old) were used. Slc:Wistar/ST, Slc:SD and F344/NSlc rats were obtained from

Time course and liver cytosol dependency of MTX hydroxylation

The time course of the hydroxylation of MTX to 7-OH-MTX by liver cytosols from rats (Sea:SD strain) was essentially linear for 3 h. However, the liver microsomes exhibited no hydroxylating activity toward MTX (Fig. 2A). When the cytosol was boiled, the activity was not observed (data not shown). The hydroxylase activities of Sea:SD rats increased linearly with increasing amount of liver cytosol up to 6 mg protein (0.4 ml). In contrast, very low activities were observed in Slc:Wistar/ST rats (

Strain differences of MTX-hydroxylase activity

This is the first report demonstrating strain differences of conversion of MTX to 7-OH-MTX in rats. When the activity for hydroxylation of MTX to 7-OH-MTX was assayed, marked variability was found in liver cytosols from eight strains of rats. The difference between the highest (Sea:SD) and lowest (WKA/Sea) activities was 104-fold. We have already reported that marked strain differences of aldehyde oxidase activity in rats were observed in an assay using benzaldehyde as a substrate. Among the

Acknowledgements

The authors wish to thank Medical Research Laboratories, Lederle Japan for the gift of methotrexate and 7-hydroxymethotrexate.

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Strain differences of the ability to hydroxylate methotrexate in rats

Abstract

Introduction

Section snippets

Chemicals

Animals

Time course and liver cytosol dependency of MTX hydroxylation

Strain differences of MTX-hydroxylase activity

Acknowledgements

J Biol Chem

Biochim Biophys Acta

Biochem Pharmacol

Cancer Lett

J Biochem

Arch Biochem Biophys

Blood