The activities of some drug metabolizing enzymes in various / pathological states or the rat liver and during the development of primary hepatocellular carcinoma

PhD Thesis

This thesis reports the results of a study designed to compare the effect of different hepatocarcinogens - N,N-dimethyl- 4-phenylazoaniline (DAB) and diethylnitrosamine (DEN) - on the activities of representative microsomal and lysosomal enzymes involved in the metabolism of exogenous chemicals throughout the entire carcinogenic process in adult male rats. 1) The effect of feeding a low protein diet containing 0.06 % N,N-dimethyl-4-phenylazoaniline for 29 weeks on the activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic acid as substrate), N-oxidase (N,N-dimethylaniline as substrate), N-demethylase (DAB as substrate), cytochrome P-450, NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase were studied. Rapid decreases occured in the activity of the first 6 enzymes, reaching minimum values between 4 and 8 weeks. Activities then increased in all cases to control or nearly control levels. This rate of increase was least for cytochrome P-450. At 4 weeks azoreductase activi ty wi th 2- (4'-di (2"- bromopropyl )aminophenylazo )bf:llzoic acid (CBlO-252) as substrate was actually higher than in control rats. Early increases occured in the activities of betaglucuronidase and arylsulphatase A and the latter never dropped below the control level. An investigation was made of the differential effects of the dye feeding on some of the enzyme activities in the two major lobes and differences were found. The effect of phenobarbitone pretreatment on the DABfed rats was studied at 4 weeks interval. The activity of DAB-azoreductase and nitroreductase were increased throughII out the whole period, while the activities of the lysosomal enzymes.were decreased. After feeding DAB for ~.weeks the effect of phenobarbitone and 3-methylcholanthrene on the activities of_DAB-azoreductase, CBlO-252-azoreductase and components of the azoreductases - cytochrome P-450, NADPH-cytochrome c reductase, the carbon monoxide-sensitive pathway and the NADH-dependent pathway were studied. The activity of CBlO-252-azo- reductase was not induced by.phenobarbitone or 3-methylcholanthrene, and CO did not inhibit the reduction. Its reduction was dependent only slightly on NADH. CO caused a greater relative 'decrease in the ac tIvi ty of DAB-azoreductase in dye-fed anim&ls following phenobarbitone &nd 3-methylcholanthrene pretreatment, implying a greater role of cytochrome P-450 in dye-fed rats. 2) The effect of feeding DEN (qiethylnitrosamine) by stomach tube for 32 weeks on the activities of DAB-azoreductase, nitroreductase, NADPH-cytochrome c reductase, cytochrome P-450, CBlO-252-azoreductase, beta-glucuronidase and arylsulphatase A was studied. Decreases occured in the activities of the first 4 enzymes reaching a minimum between 4 and 7 weeks, similar to the DAB-feeding. The activities returned to normal or nearly normal level before decreases in the activities were observed at the end of the experiments. The activity of CBlO-252-azoreductase showed increased level after 4 weeks and remained above the control level throughout the experiment. Increase in the activity of the lysosomal enzymes in the "lysosomal!! as well as the "microsomal" fractions was observed, the increase being deIII layed in the microsomal fraction. The effect of phenobarbitone pretreatment on the activities of the enzymes in the diethylnitrosamine-fed rats was studied at 4 weeks interval. The activities of the lysosomal enzymes showed decreased levels, whereas DAB-azoreductase, cytochrome P-450 and NADPH-cytochrome c reductase all showed slightly increased activity. 3) The effect of experimental cirrhosis and hepatitis ,; on the activities of some of the enzymes was investigated. The activities of the drug metabolizing enzymes showed a decrease~in both pathological states, whereas the activity of the lysosomal enzymes was increased. CBIO-252-azoreductase showed increased activity in hepatitis and decreased activity in cirrhosis. 4) The effect of a single large dose of a liver carcinogen - N,N-dimethyl-4-phenylazoaniline, urethane and 2-acetylaminoflourene - on the activities of DAB- and CBIO-252 azoreductase, NADPH-cytochrome c reductase and the level of cytochrome P-450 has been studied. 5) It has been demonstrated, "that the system involved in the reduction of 2-(4'-di(2"-bromopropyl)aminophenylazo) benzoic acid (CBIO-252) is localized mainly in the 108000 x g supernatant fraction of the rat liver homogenate, and that it is also present in other organs, particularly in the spleen. N,N-dimethyl-4-phenylazoaniline (DAB)-azoreductase is present almost entirely in the microsomes and high activity is found only in the liver. The pH maxima for CBIO-252-azoreductase and DAB-azoreductase were 6.2 and 6.9, respectively. Methylred-azoreductase had in most respects properties simiIV lar to CBIO-252-azoreductase. The use of enzyme inhibitors and other additives showed that CBIO-252-azoreductase was not identical with xanthine oxidase or dihydrofolate reductase. Its activity was not affected by carbon monoxide, phenobarbitone or 3/methylcholanthrene pretreatment. Enhancement of the activity by ferrous ions and flavin-adenine-dinucleotide indicates, that_at least part of the reduction system could involve a flavoprotein with FAD as the prosthetic group. CBIO-252- and methylred-azoreductases were inhibited by menadione (vitamin K ), cyanide ion and propylgallate. A diaphorase pre~aration from pig heart was able to reduce both CBIO-252 and methylred with a NADPH-generating system as well as a NADH-gener.s.ting system. The properties listed above and dependence of enzyme activitity on NADPH and NADH indicate a similarity to DT-diaphorase (NAD(P)H dehydrogenase). Non-enzymatic reduction of CBIO-252 by reduced glutathione and ascorbic acid was also demonstrated. The involvement of different enzyme systems in the reduction of N,N-dimethyl-4-phenylazoniline on the one hand and 2-(4'-di(2"-bromopropyl)aminophenylazo)benzoic acid on the other, are suggested to be due to the presence of the carboxylic group in the latter compound.