Some characteristics of two azoreductase systems in rat liver. Relevance to the activity of 2-[4'-di(2″-bromopropyl)-aminophenylazo]benzoic acid (CB10-252), a compound possessing latent cytotoxic activity

Abstract

The system involved in the reduction of 2-[4'-di(2″-bromopropyl)aminophenylazo]benzoic acid (CB10-252), an agent designed for treating primary liver cell cancer, has been demonstrated to be localised mainly in the 108 000 × g supernatant fraction of rat liver homogenate. It is also present in other organs particularly in the spleen. DAB-azoreductase as shown previously is present almost entirely in the microsomal fraction and is found in high concentration only in liver. The pH maxima for CB10-252-azoreductase and DAB-azoreductase were 6.2 and 6.9, respectively. Methylred-azoreductase had properties in most respects similar to CBlO-252-azoreductase implying the importance of the 2'-car☐yl group in determining substrate specificity.

The use of enzyme inhibitors and other additives showed that CB10-252 was not xanthine oxidase or dihydrofolate reductase. Its activity was not affected by carbon monoxide, phenobarbitone (PB), or 3-methylcholanthrene (MC) pretreatment. Enhancement of the activity by ferrous ions and FAD indicated that at least part of the reduction system could involve a flavoprotein with FAD as the prosthetic group.

The activity of CB10-252-azoreductase and methylred-azoreductase was reduced by menadione (vitamin K3), cyanide and propylgallate.

A diaphorase preparation from pig heart reduced both CB10-252 and methylred with both NADPH- and NADH-generating systems.

The properties listed above and dependency of enzyme activity on both NADH and NADPH indicate a similarity of CB-10-252 and methylred-azoreductases to DT-diaphorase (NAD(P)H dehydrogenase).

Non-enzymatic reduction of CB10-252 was achieved by both glutathione and ascorbic acid in high concentration.

The involvement of different enzyme systems in the reduction of DAB on the one hand and CB10-252 and methylred (MeRed) on the other, are probably due largely to the presence of the 2'-car☐ylic group and its ability to form a hydrogen bond with the β-nitrogen atom of the azo linkage. Abbreviations

CB10-252, 2-[4'-di(2″-bromopropyl)aminophenylazolbenzoic acid; pCMB, p-chloromercuric benzoate; DAB, N,N-dimethyl-4-phenylazoaniline; DMSO, dimethylsulfoxide; Lilly 008-163-163, 2,4-dichloro-6-phenylphenoxy-ethyl diethylamine hydrobromide; MC, 3-methylcholanthrene; MeRed, 2-(4'-N,N-dimethylaminophenylazo)benzoic acid; PB, phenobarbitone; SKF 525-A, 2-diethylaminoethyl-2,2-diphenyl valerate hydrochloride; TCA, trichloroacetic acid