Induction of DT-diaphorase by anticarcinogenic sulfur compounds in mice.

The effects of the dietary administration of four anticarcinogenic sulfur compounds on the activity of DT-diaphorase, a protective enzyme in quinone and quinoneimine detoxification, have been investigated in female CD-1 mice. Bisethylxanthogen, disulfiram, sodium diethyldithiocarbamate, and benzylisothiocyanate, administered at 0.5% of the diet (by weight) for 14 days, each induced significant increases in DT-diaphorase specific activities in cytosol fractions of lung, kidney, urinary bladder, proximal small intestine, and colon. Cytosolic DT-diaphorase of the fore-stomach was elevated in response to bisethylxanthogen, disulfiram, and benzylisothiocyanate. The increases in cytosolic DT-diaphorase activities in organs of mice fed 0.5% bisethylxanthogen were similar in magnitude to those observed previously in response to 0.75% butylated hydroxyanisole. Liver cytosol DT-diaphorase specific activity was enhanced sevenfold by 0.5% bisethylxanthogen, twofold by 0.5% benzylisothiocyanate, and 2.6-fold by 1% disulfiram but was not significantly increased by disulfiram or sodium diethyldithiocarbamate at 0.5% of the diet. Diets containing 0.5% bisethylxanthogen or 0.5% benzylisothiocyanate also elevated microsomal DT-diaphorase specific activities in several organs. Even at the tenfold-lower concentration of 0.05% of the diet, bisethylxanthogen induced significant increases in DT-diaphorase specific activities in cytosol fractions of liver, lung, kidney, and small intestine and in liver and kidney microsomes. The protective function of DT-diaphorase in limiting free-radical formation and oxidative damage to cells suggests that the induction of this enzyme contributes to the anticarcinogenic effects of the four sulfur compounds studied.

Effects of disulfiram, diethyldithiocarbamate, bisethylxanthogen, and benzyl isothiocyanate on glutathione transferase activities in mouse organs.

Four sulfur compounds known to inhibit tumorigenic effects of chemical carcinogens were administered to female CD-1 mice at 0.5% of the diet for 14 days, and their effects on cytosolic glutathione transferase (EC 2.5.1.18) specific activities were examined in liver, lung, kidney, urinary bladder, forestomach, proximal small intestine, and colon. Disulfiram, sodium diethyldithiocarbamate, bisethylxanthogen, and benzyl isothiocyanate elevated glutathione transferase specific activities in most of the organs examined. The four sulfur compounds differed in the extents and organ specificities of their effects on these enzyme activities. In the liver, bisethylxanthogen and benzyl isothiocyanate increased glutathione transferase activities to at least 3 times control levels and caused differential increases in the isozyme patterns observed after isoelectric focusing of the cytosols. Bisethylxanthogen also increased immunoreactive glutathione transferase in liver cytosol. Recrystallized disulfiram was less effective in enhancing hepatic glutathione transferase activities than was commercial (97%) disulfiram. Among the six extrahepatic organs examined, the small intestine and the forestomach exhibited the greatest response of glutathione transferase activities to each of the four sulfur compounds. Benzyl isothiocyanate was most effective in these "portal of entry" organs but less effective than bisethylxanthogen in the other extrahepatic organs examined. Bisethylxanthogen elicited significant increases in glutathione transferase activities in liver, lung, and small intestine even when administered at 0.01% to 0.05% of the diet, suggesting that this compound may have considerable potential as an inhibitor of carcinogens susceptible to enzymatic conjugation with glutathione.

Kinetics of glutathione transferase, glutathione transferase messenger RNA, and reduced nicotinamide adenine dinucleotide (phosphate):quinone reductase induction by 2(3)-tert-butyl-4-hydroxyanisole in mice.

The mechanisms by which 2(3)-tert-butyl-4-hydroxyanisole (BHA) protects against chemical carcinogenesis and toxicity include enhancement of the activities of several detoxification enzymes. In previous studies, 14-day administration of BHA to female CD-1 mice at 0.75% of the diet led to large increases in cytosolic glutathione transferase (EC 2.5.1.18) and reduced nicotinamide adenine dinucleotide (phosphate) dehydrogenase (quinone) (EC 1.6.99.2) [NAD(P)H:quinone reductase; DT-diaphorase] specific activities in several tissues, and elevated hepatic glutathione transferase messenger RNA. In the present study, one day of dietary BHA significantly increased NAD(P)H:quinone reductase and glutathione transferase activities in the liver, kidney, and proximal small intestine, and NAD(P)H:quinone reductase activity in the forestomach and lung. In the proximal small intestine, glutathione transferase specific activities toward 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene rose to 2.6 and 8 times those of control, respectively, and NAD(P)H:quinone reductase specific activity doubled, within 1 day on the BHA diet. Six hr after a single p.o. dose of BHA (620 mg/kg), intestinal glutathione transferase specific activities were 30 to 50% above those of control mice. In liver, the kinetics of increase of glutathione transferase messenger RNA were in accord with increased synthesis as the mechanism of elevation of glutathione transferase activity in response to BHA. Although changes in mixed-function oxygenase activities have been reported to occur more rapidly, the kinetics of the response of glutathione transferase and NAD(P)H:quinone reductase specific activities to BHA indicates that nonoxidative detoxification potential is substantially enhanced within 24 hr or less after initiation of BHA administration.

Nitroreductases and glutathione transferases that act on 4-nitroquinoline 1-oxide and their differential induction by butylated hydroxyanisole in mice.

These studies concern the initial steps in 4-nitroquinoline 1-oxide (4NQO) metabolism in relation to mechanisms of anticarcinogenesis. Butylated hydroxyanisole (BHA) administration by a protocol known to inhibit the pulmonary tumorigenicity of 4NQO in A/HeJ mice enhanced hepatic and pulmonary activities for 4NQO metabolism by two major pathways, conjugative detoxification and nitroreductive activation. High-performance liquid chromatography analysis showed approximate doubling of two types of glutathione transferase subunits with 4NQO-conjugating activity in livers of BHA-treated mice. Similar increases were observed in hepatic 4NQO-conjugating activity and in Vmax, while Km for 4NQO was 39 to 43 microM. Pulmonary 4NQO-glutathione transferase activity increased 24 to 29%. DT diaphorase activity toward 4NQO was elevated 3.3-fold in livers and 2.7-fold in lungs of BHA-treated mice. However, the predominant 4NQO reductase of liver and lung was dicumarol resistant, had a strong preference for NADH, and showed little if any response to BHA. This Mr 200,000 enzyme, partially purified from livers of Swiss mice, exhibited the stoichiometry of 2-NADH/4NQO expected for reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide. Its high affinity for 4NQO (Km, 15 microM) signified a much greater influence on 4NQO metabolism than DT diaphorase (Km, 208 microM). The dicumarol-resistant 4NQO reductase differed from several known cytosolic nitroreductases. The results suggest that protection by BHA may result from alteration of the balance between 4NQO activation and conjugation.

Characterization of a cisplatin-resistant subline of murine RIF-1 cells and reversal of drug resistance by hyperthermia.

The development of tumor cell drug resistance is a major obstacle which often leads to failure of cancer chemotherapy. Therefore, reversing the cell drug resistance would have important implications in cancer treatment. We have developed a cisplatin-resistant mouse tumor cell line from the radiation induced fibrosarcoma (RIF-1) parental line; this line is named RIF/ptr1 versus the parental line RIF/pts1. It is shown that the formation of cisplatin-DNA interstrand cross-links is the same for both cell lines although the intracellular cisplatin concentrations of resistant line is significantly lower. The cytosolic activities of glutathione reductase, glutathione peroxidase, and DT-diaphorase were the same in two cell lines. However, the concentration of glutathione was significantly higher in the resistant line. The resistant line was shown to be more sensitive to the cytotoxicity of heat (43 degrees C) but the combination of heat and drug had the same tumoricidal effect for both cell lines. The addition of verapamil also had a similar effect on both cell lines. We conclude that the major difference between these two lines was the glutathione-related detoxification of platinum. Regardless of drug resistance, the combination of drug and heat can effectively kill both cell lines. Elevated glutathione in RIF/ptr1 cells may be associated both with enhanced heat sensitivity and drug resistance such that combined treatments with drug and heat were equally effective in killing cells of either line.

Conversion of 4-nitroquinoline 1-oxide (4NQO) to 4-hydroxyaminoquinoline 1-oxide by a dicumarol-resistant hepatic 4NQO nitroreductase in rats and mice.

The product formed from 4-nitroquinoline 1-oxide (4NQO), a potent carcinogen, by the action of mouse NADH:4NQO nitroreductase NR-1 was directly identified as 4-hydroxyaminoquinoline 1-oxide (4HAQO) by high performance liquid chromatography analyses in two systems. In liver cytosols from both male and female mice, NADH:4NQO nitroreductase was the predominant enzyme catalyzing the reduction of 4NQO. Rat liver cytosol catalyzed the conversion of 4NQO to either 4HAQO or a glutathione conjugate depending upon coenzyme or cosubstrate availability. Whereas NAD(P)H:quinone reductase (NAD(P)H:(quinone acceptor) oxidoreductase; DT diaphorase; EC 1.6.99.2) was the predominant 4NQO reductase present in liver cytosol from Sprague-Dawley rats, dicumarol-resistant NADH:4NQO nitroreductase specific activities were comparable with those of mouse liver cytosols. A 4NQO nitroreductase from rat liver cytosol was separated from NAD(P)H:quinone reductase chromatographically and shown to have a strong preference for NADH and to be insensitive to inhibition by dicumarol.

Inhibition of mouse glutathione transferases and glutathione peroxidase II by dicumarol and other ligands.

Dicumarol, often used as a specific inhibitor of DT diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase; EC 1.6.99.2), was found to potently inhibit GSH transferases (EC 2.5.1.18). Dicumarol exhibited an IC50 of 11 microM in inhibiting the conjugation of 1-chloro-2,4-dinitrobenzene (50 microM) by GSH transferase GT-8.7, the major hepatic class mu isoenzyme of CD-1 mice. The activities of GT-8.7 and of the class pi isoenzyme, GT-9.0, toward a carcinogenic substrate, 4-nitroquinoline 1-oxide (100 microM), were inhibited by dicumarol with IC50 values of 14 and 9 microM, respectively. Dicumarol also affected GSH peroxidase II activity, inhibiting the reduction of cumene hydroperoxide by GT-10.6, the predominant class alpha GSH transferase of mouse liver, with an IC50 of 14 microM. GSH peroxidase I (EC 1.11.1.9) and GSH peroxidase II activities were resolved by chromatography of liver and testis cytosols. While inhibiting GSH peroxidase II with IC50 of 9-10 microM, dicumarol did not affect the activity of the selenoenzyme, GSH peroxidase I. Whereas several other non-substrate ligands were more potent inhibitors of 1-chloro-2,4-dinitrobenzene conjugation, dicumarol effectively inhibited GSH transferase and GSH peroxidase II activities in the range of dicumarol concentrations frequently used for detection of DT diaphorase action. These results indicate that physiological consequences resulting from the use of supramicromolar concentrations of dicumarol should not be interpreted in terms of DT diaphorase inhibition alone.

Biochemical determinants of Adriamycin toxicity in mouse liver, heart and intestine.

Biochemical characteristics relevant to the differential susceptibilities of liver, heart, and intestine to acute Adriamycin toxicity were examined in female CD-1 mice with and without intravenous Adriamycin (dose range 23-30 mg/kg). The liver which, unlike heart and intestine, is relatively resistant to Adriamycin toxicity, had high levels of glutathione and glutathione peroxidase, and exhibited a sharp decline in non-protein thiol concentrations within 1-3 hr with rebound by 6 hr after Adriamycin. Covalent binding to Adriamycin or its metabolites could not account quantitatively for the loss of non-protein thiols, implicating an oxidative mechanism. No lipid peroxidation was observed in the liver, apparently due to effective utilization of antioxidant defenses. Adriamycin caused significant increases in cardiac lipid peroxides, indicative of oxidative tissue damage, which would be expected to exacerbate cardiotoxicity. However, non-protein thiol concentrations did not decrease in heart or in intestine in response to Adriamycin. Both heart and intestine had extremely low levels of glutathione peroxidase activity, which may limit glutathione utilization for protection against oxidative toxicity. The activity of DT diaphorase, which may have an activating role in Adriamycin metabolism, was high in heart and intestine and was induced 4-fold in liver in response to Adriamycin.

Elevation of quinone reductase activity by anticarcinogenic antioxidants.

NAD(P)H:quinone reductase exhibits broad specificity in the reduction of endogenous and exogenous quinones and quinone imines, such as those derived from polycyclic aromatic carcinogens, phenolic steroids, vitamin K, and numerous therapeutic drugs. This enzyme is found in several cell compartments and is widely distributed among tissues. In contrast to several other flavoprotein dehydrogenases, quinone reductase catalyzes obligatorily two electron reductions. Extensive studies by Huggins and by others have shown that the quinone reductase in liver and some other tissues of rats is inducible by various polycyclic hydrocarbons and aromatic amines, as well as by certain azo dyes. Huggins perceived that the relative effectiveness of such compounds in inducing quinone reductase correlated with their abilities to protect against toxicity and carcinogenesis. Certain antioxidants are also known to protect against the tumorigenic and toxic effects of carcinogens. Studies on the mechanisms underlying the protective effects of BHA, BHT, ethoxyquin, and disulfiram have revealed that these compounds alter the activity profiles of several enzymes which metabolize carcinogenic and toxic compounds. We have observed that quinone reductase specific activity is increased markedly in mouse liver and several extrahepatic tissues in response to dietary BHA, ethoxyquin, and disulfiram, whereas BHT has been shown by others to enhance this enzymatic activity in rat liver. These findings confirm and extend the correlation between the ability to elevate quinone reductase activity and to confer protection against carcinogenesis and toxicity. The broad specificity of quinone reductase, its apparent inability to catalyze one electron reductions of quinones, its widespread distribution, and its inducibility by a variety of structurally dissimilar protective compounds, suggest that quinone reductase may play a significant local protective role in various regions of the cell.

The conjugation of 4-nitroquinoline 1-oxide, a potent carcinogen, by mammalian glutathione transferases. 4-Nitroquinoline 1-oxide conjugation by human, rat and mouse liver cytosols, extrahepatic organs of mice and purified mouse glutathione transferase isoenzymes.

The conjugation of 4-nitroquinoline 1-oxide with GSH by human, rat and mouse liver cytosols, by purified mouse GSH transferases and by extrahepatic organ cytosols of male and female mice was investigated. 4-Nitroquinoline 1-oxide was as effectively conjugated by human liver cytosol as was 1-chloro-2,4-dinitrobenzene, at a substrate concentration of 0.1 mM. Mouse isoenzymes composed of Yb1 and Yf subunits exhibited high activity towards 4-nitroquinoline 1-oxide. Human, rat and mouse hepatic activities towards this substrate correlated with the hepatic isoenzyme compositions.

Nonheme iron proteins, XI. Some genetic aspects.

Ferredoxin was isolated from individual Leucaena glauca trees, and the distribution of amino acids at three of the four sites of sequence heterogeneity was determined. The results indicated that the most probable causes of the observed microheterogeneity are the presence of either nonallelic nuclear genes or differing chloroplast genes, and also ambiguity in translation of the genetic code.