Prominent role of DT-diaphorase as a cellular mechanism reducing chromium(VI) and reverting its mutagenicity

Rat liver postmitochondrial (S-12) fractions accounted for the bulk of the activity of whole cell homogenates in reducing chromium(VI) and accordingly in decreasing its mutagenicity. Both cytosolic (S-105) and microsomal fractions concurred to this process, which in all subcellular preparations tested was selectively induced by phenobarbital and especially by Aroclor 1254, but not by 3-methylcholanthrene. Cytosolic fractions were markedly more efficient in reducing chromium(VI) than microsomal fractions recovered from the same amount of tissue (liver or lung), although the latter preparations had a higher specific activity. The microsomal activity was exclusively NADPH dependent. A minor part of the cytosolic reduction was determined by nonenzymatic components, notably by some electron donors and chiefly by reduced glutathione, which proved to reduce chromium(VI) at physiological concentrations. However, also in cytosolic fractions, the most important contribution to chromium reduction was enzyme catalyzed, as shown by the following properties: thermolability; requirement for exogenous NADH or NADPH [supplied as such or in the form of a NADPH-generating system (S-9 mix)]; and saturation by chromium(VI). The likely involvement of DT-diaphorase in this metabolic process is supported by several findings, including its sharp pH dependence and its partial suppression by known inhibitors of this enzyme protein, such as p-chloromercuribenzoate, L-thyroxine, and dicumarol (which conversely did not counteract the metabolic deactivation of the other direct-acting mutagens 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine 2HCl and epichlorohydrin). Similarly, cytosolic reduction of chromium(VI) was partially inhibited by selective metabolic depletors of both coenzymes of DT-diaphorase, i.e., NADPH and NADH. Pretreatment of rats with enzyme inducers (phenobarbital and 3-methylcholanthrene) stimulated the activity of DT-diaphorase in liver cytosolic fractions. A dramatic stimulation (35 to 40 times over untreated controls) was produced by Aroclor 1254, which also coinduced the liver cytosolic activity of enzymes involved in the glucose 6-phosphate-dependent pathway of both nicotinamide-adenine-dinucleotide phosphate and glutathione reduction (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase). In the lung cytosol, a slight yet significant stimulation of some of these enzyme activities was determined by the daily intratracheal instillations of high doses of chromium(VI) itself for 4 weeks, a condition which has been found to enhance the pulmonary metabolism of this metal ion.     

Influence of DT diaphorase on the mutagenicity of organic and inorganic compounds

The metabolic activation or detoxification of mutagens and carcinogens of several chemical classes was investigated in the presence of various rat liver and lung subcellular fractions and of dicoumarol, a specific inhibitor of DT diaphorase activity. His- Salmonella typhimurium strains were used as targets of mutagenicity. Dicoumarol partially prevented the metabolic activation of some promutagens, such as the heterocyclic amines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, and a cigarette smoke condensate. Moreover, detailed experiments, also using purified enzyme, confirmed the participation of DT diaphorase in the metabolic reduction of 4-nitroquinoline N-oxide 4NQO and of hexavalent chromium [Cr(VI)] compounds. The results obtained provide evidence for broad involvement of DT diaphorase in the metabolism of both organic and inorganic mutagens and carcinogens. Moreover, they suggest a dual role of this enzyme, providing not only a cellular detoxifying system but also, with a few substrates, an activating mechanism.     

Pulmonary metabolism of mutagens and its relationship with lung cancer and smoking habits

The S-12 fractions of lung peripheral parenchyma obtained from 80 male individuals, aged 17-71 years, were assayed as blind samples for the ability either to convert promutagens into bacterial mutagens or to decrease the potency of direct-acting mutagens in the Ames reversion test. In this system, lung preparations were completely ineffective in activating an N-nitroso compound (i.e., N-nitrosomorpholine) and polycyclic aromatic hydrocarbons [i.e., 3-methylcholanthrene and benzo(a)pyrene] or their metabolites [i.e., 3-hydroxy-benzo(a)pyrene and benzo(a)pyrene-trans-7,8-diol]. They yielded a borderline and sporadic activation of a cigarette smoke condensate, and a weak but frequent activation of an aromatic amine (i.e., 2-aminofluorene), of a heterocyclic amine (i.e., 2-amino-3,4-dimethylimidazo[4,5-f] and of a diamide (i.e., cyclophosphamide). The pulmonary metabolism was more oriented in the sense of detoxification, as shown by the consistent decrease of potency of direct-acting mutagens, including a metal (i.e., sodium dichromate), an acridine and nitrogen mustard derivative (i.e., 2-methoxy-6-chloro-9-[3-(2-chloromethyl)aminopropylamino]acridine or ICR 191), an epoxide (i.e., epichlorohydrin) and an N-oxide (i.e., 4-nitroquinoline-N-oxide). As assessed by means of a numerical score quantifying the variation of mutagenicity, a marked interindividual variability (up to 20-fold) was detected in the ability of lung specimens to affect the mutagenicity of test compounds. Such variability was not significantly related to the protein concentration of S-12 fractions, nor to the age of the patients under scrutiny, who during hospitalization were on normal institutional diets and did not receive any special drug treatment. The only significant difference between 20 noncancer and 60 lung cancer patients, irrespective of the histological type, was a decreased activation of cyclophosphamide in the latter group. Probably due to the high prevalence of smokers among lung cancer patients, a significantly decreased activation of cyclophosphamide was also observed in the group of smokers. Smoking habits were associated with a stimulation of detoxifying mechanisms which, in agreement with the results of a previous study with human alveolar macrophages (F. L. Petrilli et al., J. Clin. Invest., 77:1917-1924, 1986), was significant in the case of sodium dichromate. Such effect was further enhanced by considering only individuals smoking during the last 24 h before collecting lung specimens, and under these conditions it became significant also for ICR 191.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of N-acetylcysteine on the mutagenicity of direct-acting compounds and procarcinogens

N-Acetylcysteine (NAC), reduced (GSH) and oxidized (GSSG) glutathionewere negative in the Ames test with 7 Salmonella strains, whileL-cysteine was activated by rat liver S-9 fractions to metabolitesmutagenic to strains TA102, TA97 and TA100. The mutagenic responsein S. typhimurium strains (TA1535, TA98, TA100, TA102) and thelevels of enzyme activities, responsible for NADP+ or GSSG reductionand for the utilization of NADPH or GSH in rat liver S-9 fractions,were investigated following in vitro preincubation of NAC withfour direct-acting mutagens and six procarcinogens. Treatmentwith this nucleophilic and reducing compound resulted in a dose-relateddecrease of the direct mutagenicity of epichlorohydrin, hydrogenperoxide and, sharply, of 4-nitroquinolino-N-oxide and sodiumdichromate. The mutagenicity of these compounds, both in theabsence and in the presence of NAC, was decreased by rat liverS-9 fractions and to some extent by lung S-9 fractions. A diphasiceffect was observed in the case of procarcinogens (cyclophosphamide,2-aminofluorene, cigarette smoke condensate, Trp-P-2, aflatoxinB₁ and benzo[a]pyrene), i.e., an enhancement of S-9 requiringmutagenicity at intermediate NAC doses, which could be ascribedto metabolic factors acting in vitro, and a loss of mutagenicityat high NAC doses, which could be ascribed to trapping of electrophilicmetabolites. Out of the five S-9 enzyme activities under study,i.e., glucose-6-phosphate dehydrogenase, 6-phosphogluconatedehydrogenase, malic enzyme, GSH peroxidase and GSSG reductase,only the last one showed significant changes following mutagenand/or NAC treatment.     

In vivo effects of N-acetylcysteine on glutathione metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds

N-acetylcysteine (NAC) was administered to rats in various combinationswith an enzyme inducer (Aroclor 1254) and with depletors ofreduced glutathione (GSH), i.e., diethyl maleate (DEM) and buthioninesulfoximine (BSO). NAC increased intracellular glutathione levelsin erythrocytes and in liver and lung cells, and replenishedits stores following depletion. It did not affect the concentrationsnor the spectral properties of cytochromes P-450 in hepaticand pulmonary microsomes, whereas it stimulated, especiallyin Aroclor-pre-treated animals, cytosolic enzyme activitiesinvolved in NADP reduction (glucose 6-phosphate dehydrogenaseand 6-phosphogluconate dehydrogenase), in glutathione reduction(GSSG-reductase) and in the reductive detoxication of xenobioticsby-passing formation of reactive oxygen species (DT-diaphorase).In vivo treatment with the drug enhanced detoxication by liverand lung S-12 fractions of direct-acting mutagens (ICR 191,epichlorohydrin, 4-nitroquinolino-N-oxide and dichromate) andcounteracted opposite effects triggered by administration ofGSH depletors. The metabolic activation of procarcinogens (aflatoxinB₁, 2-aminofluorene, cyclophosphamide, benzo[a]-pyrene, a tryptophanpyrolysate product and cigarette smoke condensate) was inhibitedby NAC in uninduced rats, while it was further stimulated inAroclor-pre-treated animals. Additional assays, performed alsowith other enzyme inducers (phenobarbital and 3-methylcholanthrene)suggested that the effect of NAC on the metabolic activationof procarcinogens depends on the balance between an increasedproduction of mutagenic metabolites (prevailing in induced animals)and their binding by intracellular thiols (prevailing undernormal conditions). Thus, due to its dual role as a nucleophileand as a SH donor, NAC appears to exert protective effects bymodulating glutathione metabolism and the biotransformationof mutagenic/carcinogenic compounds. This may have clinicalrelevance, since NAC is administered to individuals, such ascigarette smokers, who are more heavily exposed to GSH depletorsand to carcinogenic agents.     

Enhanced metabolic activation of chemical hepatocarcinogens in woodchucks infected with hepatitis B virus

The metabolism of chemical carcinogens was investigated in liverpreparations from 28 captive woodchucks (Marmotamonax). Of these,23 were naturally infected with the wood-chuck hepatitis virus(WHV), and eight also had primary hepatocellular carcinoma (PHC).Twenty-nine parameters were investigated in liver subcelhilarfractions, including cross-reactivity with HBsAg, and biochemicalparameters, such as -glutamyl transpeptidase, cytochrome P-450and mirosomal monooxygenases (aryl hydrocarbon hydroxylase,ethodycoumarin and ethoxyresorufin deethylases, amino-pyrineand dimethylnitrosamine demethylases, and testosterone 7-and16ß- and 6ß-hydroxylases), uridine 5'-diphos-phoglucuronosyltransferase, GSH and related enzymes (peroxidase, reductaseand S-transferase), as well as other cytosolic enzyme activities(glucose 6-phosphate and 6-phosphogluconate dehydrogenases,NADPH- and NADH-dependent diaphorases, and DT diaphorase). Inaddition, liver preparations were used in order to quantifythe metabolic activation into bacterial mutagens of five procarcinogens(aflatoxin B¹, the pyrolysis products Trp-P-2 and MelQ, 2-aminofluoreneand dimethylnitrosamine) and the decrease of potency of threedirect-acting mutagens (sodium di-chromate, ICR 191 and 4-nitroquinoline1-oxide). WHV infection produced a significant stimulation ofcarcinogen metabolism, as shown by the simultaneous change indetoxification parameters (GSH depletion) and activation indices(enhancement of microsomal monooxygenases and of pro-carcinogenactivation into mutagenic metabolites). There were no significantdifferences between WHV-positive samples from animals, withoutPHC and the noncancerous tissue of PHC-bearing animals, whereasa decrease of both activation and detoxification indices wasrecorded in the turmorous tissue. There was a considerable interindividualvariability among WHV carriers, which was tentatively ascribedto genetic factors. Pregnancy was the only known factor influencingthe results in WHV carriers. However, even by excluding pregnantanimals, the effects on carcinogen metabolism produced by WHVinfection were still statistically significant. These results,together with previous data obtained in humans, revealed thatmetabolic factors may play a role in the synergism between viralhepatitis and chemical hepato-carcinogens in the etiopathogenesisof PHC.     

Alterations in hepatic peroxidation mechanisms in thioacetamide-induced tumors in rats. Effect of a rhodium(III) complex.

A model of liver hyperplastic noduligenesis was induced in rats in vivo by long-term administration of thioacetamide (TAM; 100 mg/kg day i.p.). Three doses of 50 mg/kg of an antitumoral rhodium(III) complex were administered at 14, 9 and 5 days before the end of TAM treatment. Blood and liver were obtained from either TAM, Rh(III) complex or TAM plus Rh(III) complex-treated rats in order to determine the interaction of both (tumoral and antitumoral) substances with the biochemical pathways related to glutathione redox cycle, enzyme activities involved in the oxidative stress coupled to the NADPH/NADP pair and enzymes related to the mono-oxygenase P450 system. The results showed that TAM induced an imbalance between the activities of glutathione-coupled enzymes. Glutathione reductase activity increased along with the intoxication, while glutathione peroxidase activity decreased. Alterations in the activity of soluble glutathione peroxidase were parallel to those of catalase. These results, together with decreased activities of enzymes related to cytochrome P450 mono-oxygenase system, NADPH cytochrome P450 reductase and NADH cytochrome b5 reductase, suggest that liver cells are not protected against the peroxidative stress produced by chronic administration of TAM. The Rh(III) complex did not produce significant changes in the parameters assayed when administered alone. When this complex was administered to TAM-treated rats, significant restoration of the following activities was observed: those of NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and malic enzyme), that of glutathione reductase (NADPH-consuming enzyme), NADPH-cytochrome P450 reductase and total catalase. These results, together with others in previous studies, suggest that the altered liver function induced by chronic administration of TAM can be partially restored by this rhodium complex. The mechanisms by which this complex counteracts the TAM-induced changes have not yet been established.     

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.     

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.     

Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer

This review describes woodchucks chronically infected with the woodchuck hepatitis virus (WHV) as an animal model for hepatocarcinogenesis and treatment of primary liver cancer or hepatocellular carcinoma (HCC) induced by the hepatitis B virus (HBV). Since laboratory animal models susceptible to HBV infection are limited, woodchucks experimentally infected with WHV, a hepatitis virus closely related to HBV, are increasingly used to enhance our understanding of virus-host interactions, immune response, and liver disease progression. A correlation of severe liver pathogenesis with high-level viral replication and deficient antiviral immunity has been established, which are present during chronic infection after WHV inoculation of neonatal woodchucks for modeling vertical HBV transmission in humans. HCC in chronic carrier woodchucks develops 17 to 36 mo after neonatal WHV infection and involves liver tumors that are comparable in size, morphology, and molecular gene signature to those of HBV-infected patients. Accordingly, woodchucks with WHV-induced liver tumors have been used for the improvement of imaging and ablation techniques of human HCC. In addition, drug efficacy studies in woodchucks with chronic WHV infection have revealed that prolonged treatment with nucleos(t)ide analogs, alone or in combination with other compounds, minimizes the risk of liver disease progression to HCC. More recently, woodchucks have been utilized in the delineation of mechanisms involved in innate and adaptive immune responses against WHV during acute, self-limited and chronic infections. Therapeutic interventions based on modulating the deficient host antiviral immunity have been explored in woodchucks for inducing functional cure in HBV-infected patients and for reducing or even delaying associated liver disease sequelae, including the onset of HCC. Therefore, woodchucks with chronic WHV infection constitute a well-characterized, fully immunocompetent animal model for HBV-induced liver cancer and for preclinical evaluation of the safety and efficacy of new modalities, which are based on chemo, gene, and immune therapy, for the prevention and treatment of HCC in patients for which current treatment options are dismal.     

Effects of retinoids on metabolizing enzymes and on binding of benzo(a)pyrene to rat tissue DNA

Retinyl acetate, 13-cis-retinoic acid (13cisRA), and N-(4-hydroxyphenyl)-retinamide (4HPR) were assayed for their in vivo effects on hepatic levels of cytochrome P450, cytosolic glutathione-S-transferase, and quinone reductase. When given p.o. to Sprague-Dawley rats, all of the retinoids caused significant suppression in the levels of arylhydrocarbon hydroxylase, yet 13cisRA and 4HPR caused elevations in cytosolic levels of quinone reductase and glutathione-S-transferase, respectively. Scans of sodium dodecyl sulfate-polyacrylamide gels of microsomal proteins from the livers of retinoid-dosed animals showed changes in both the intensities and the number of stained bands. For microsomes from 13cisRA-dosed animals, there were additional changes in the absorption maximum of the carbon monoxide and octylamine difference spectra. There was, compared to controls, a 62% reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins from 13cisRA-dosed animals. Fluorography of the sodium dodecyl sulfate-polyacrylamide gels showed that the major reduction in metabolite binding occurred in the Mr 50,000 region of the gel. The reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins in vitro and the reduction in hepatic arylhydrocarbon hydroxylase levels correlated with a reduction in the in vivo binding of benzo(a)pyrene to rat liver DNA. Animals dosed for 7 days with 13cisRA, retinyl acetate, or 4HPR showed a 38, 27, and 40% reduction in binding of benzo(a)pyrene to liver DNA and a 29, 32, and 21% reduction in binding to stomach DNA, respectively, when the carcinogen was administered on the eighth day, and the tissues were harvested 24 h later. Binding to lung DNA was reduced by 23 and 11%, respectively, in the 13cisRA- and 4HPR-dosed rats. No differences were observed in binding to kidney. Thus, retinoids, by altering the metabolism of carcinogens, could influence the initiation stage of carcinogenesis.     

Comparative in vitro metabolism of aflatoxicol by liver preparations from animals and humans.

The metabolism of [14C]aflatoxicol by liver postmitochondrial and microsomal fractions from humans and eight other species was compared. A major metabolic pathway involves the dehydrogenation of aflatoxicol yielding aflatoxin B1. Human liver preparations were more active in this regard than preparations from any of the other species tested. The aflatoxicol dehydrogenase activity was mainly associated with the microsomal fraction and required a hydrogen acceptor (e.g., nicotinamide adenine dinucleotide phosphate), but was not inhibited by carbon monoxide, which implies that it was not dependent on the heme-containing microsomal drug-metabolizing system. It had a pH optimum of 8.0. Postmitochondrial liver fractions also oxidized aflatoxicol (and/or the aflatoxin B1 made from it) to at least five other metabolites that comigrated on thin-layer chromatography plates with authentic standards of aflatoxins Q1,P1,H1,M1, and B2a. None of these oxidative metabolites were formed in the presence of carbon monoxide. We also report on the in vitro reduction of aflatoxin B1 to aflatoxicol by the cytosol fractions from eight species. Most active in this regard were rabbit and trout preparations, while this activity was almost absent in the guinea pig. Preparations from humans and four other species were intermediate between these extremes.     

Renal reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase-mediated metabolism of the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The [14C]NFTA:glutathione or [3H]glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with [3H]glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-[4-(2-acetylaminothiazolyl)]-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.     

Augmentation of ethanol-induced enhancement of dimethylnitrosamine and diethylnitrosamine metabolism by lowered carbohydrate intake

Hepatic metabolism in vitro of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), 3,4-benzopyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was assayed using the liver of rats maintained for 3 weeks on liquid diets containing three levels of carbohydrate (CHO) with or without supplementation of ethanol (5 g/100 ml). Ethanol consumption increased the metabolism of DMN and DEN to different degrees depending on the diet consumed simultaneously. Lowered CHO intake, which by itself increased the metabolism, dose-dependently augmented the increase due to ethanol: the lower the CHO intake, the more remarkable was the enhancement caused by ethanol. Ethanol increased microsomal cytochrome P-450 content only when combined with low-CHO diets. It was confirmed by a Salmonella mutagenicity test that the enhancement of DMN and DEN metabolism was accompanied by increased capacity of the liver to activate these nitrosamines to mutagens. On the other hand, neither lowered CHO intake, nor ethanol consumption, nor their combination affected the metabolism of BP and DMBA. Ethanol combined with a diet deficient in CHO even suppressed the metabolism of BP.     

Sex differences in indomethacin-sensitive 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol

The 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidoreductase (EC 1.1.1.50) of rat liver cytosol is indistinguishable from trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC 1.3.1.20) (T.M. Penning, I. Mukharji, S. Barrows, and P. Talalay, Biochem. J., 222: 601-611, 1984) and has been implicated in the detoxification of ultimate carcinogens (H. R. Glatt et al., Science (Wash. DC), 215: 1507-1509, 1982). Using trans-1,2-dihydroxy-3,5-cyclohexadiene as a model substrate for trans-dihydrodiol proximate carcinogens, this study shows that the specific activity of 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidoreductase is 2-fold higher in the 40-75% ammonium sulfate fraction prepared from female rat liver cytosol than in similar fractions prepared from males. Comparable differences were also observed for the nicotinamide adenine dinucleotide-dependent oxidation of 5 alpha-androstan-3 alpha-ol-17-one. Chromatofocusing of these cytosolic fractions separated the bulk of the protein from the dehydrogenase, which eluted as a single peak at pH 5.4. Examination of the protein profiles indicates that twice as much protein coeluted with the enzyme from female rat liver cytosol, suggesting that induction is responsible for the sex difference in enzyme activity. These differences were abolished by ovariectomy, while administration of a single dose of estradiol 3-sulfate (100 micrograms) to ovariectomized rats restored enzyme activity to within 90% of normal female levels. These findings suggest that ovarian estrogen is a natural inducer of rat liver 3 alpha-hydroxysteroid:nicotinamide adenine dinucleotide (phosphate) oxidase reductase/trans-1,2-dihydrobenzene-1,2-diol dehydrogenase.     

Epithelial binding of 1,2-dibromoethane in the respiratory and upper alimentary tracts of mice and rats

The metabolism and binding of the volatile carcinogen 1,2-dibromo[14C]ethane (DBE) were studied in C57BL mice, Sprague-Dawley rats, and Fischer rats. As shown by the whole-body and light microscopic autoradiography with heated and/or extracted sections, a selective accumulation of metabolites occurred in a number of tissues, preferentially in the reported target tissues for DBE-induced lesions [i.e., in the nasal cavity, lung, forestomach, and liver (tumors) and the adrenal, testicle, liver, and kidney (nonneoplastic lesions)]. High levels of nonextractable metabolites were registered in the epithelia of the entire respiratory tract, the upper alimentary tract, the vagina, and the subepithelial glands of the olfactory mucosa. Lower levels of metabolites were observed in the liver, adrenal cortex, testicular interstitium, and kidney. Autoradiography of slices from various extrahepatic tissues incubated in vitro with DBE showed that most epithelia of the respiratory tract, upper alimentary tract, vagina, and the testicular interstitium have a marked ability to activate DBE to metabolites that become bound to the tissue. Further in vitro experiments, performed with S-1 fractions prepared from various tissues, indicated that the nasal mucosa was most active in transforming DBE to products which could not be extracted from the protein precipitate. It is proposed that tissue-selective metabolism and activation of DBE in the epithelia of the respiratory and upper alimentary tract are responsible for the observed DBE-induced lesions in these organ systems.     

Sulfate esters of hydroxymethyl-methyl-benz[a]anthracenes as active metabolites of 7,12-dimethylbenz[a]anthracene

7-Hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) and 12-hydroxymethyl-7-methylbenz[a]anthracene (12-HMBA), carcinogenic major metabolites of 7,12-dimethylbenz[a]anthracene (DMBA) in untreated rat liver, showed high mutagenic activities toward Salmonella typhimurium TA 98 after preincubation with a sulfotransferase-PAPS system consisting of ATP, sodium sulfate, and a post-mitochondrial fraction (S-9) or a soluble supernatant fraction (S-105) from untreated rat liver. The 7- and 12-HMBAs themselves induced His+ mutation in TA 98 only slightly after preincubation with S-9 in the presence of an NADPH-generating system. Mutagenicity of DMBA toward TA 98 after preincubation with S-9 in the presence of the NADPH-generating system was remarkably enhanced by the addition of ATP and sodium sulfate. The active metabolites, 7-HMBA sulfate and 12-HMBA sulfate, were isolated from these preincubation systems and identified by comparison with the corresponding synthetic specimens. The sulfuric acid ester conjugates were potent mutagens toward TA 98 in the absence of rat liver subcellular fractions. The conjugates bound covalently at significant rates to calf-thymus DNA as well as to S-105 proteins at 37 degrees and pH 7.4 through the 7- or 12-methylene carbon with concomitant loss of their sulfate group. In the presence of S-105, glutathione inhibited the mutagenicity of the metabolically formed or exogenously added 7- and 12-HMBA sulfates. The non-mutagenic glutathione conjugates were isolated from the incubation mixtures and identified as S-(12-methylbenz[a]anthracen-7-yl)methylglutathione from 7-HMBA or its sulfate and S-(7-methylbenz[a]anthracen-12-yl)methylglutathione from 12-HMBA or its sulfate.     

Hepatic drug-metabolizing enzymes in primary and secondary tumors of human liver

Significant increases in activities of epoxide hydrolase, UDP-glucuronosyltransferase, and glutathione S-transferase, and marked reductions in cytochrome P-450 mixed-function oxidase systems occur in hyperplastic nodules induced in rat liver by chemical mutagens. In contrast, activities of both oxidative (Phase I) and conjugative (Phase II) enzymes are decreased in hepatocellular carcinomas induced by peroxisome proliferators. The present work compares alterations induced by chemical mutagens or peroxisome proliferators with changes in enzyme activities that occur in primary and secondary hepatic tumors in man. The above activities, along with beta-glucuronidase and arylsulfatase, were measured in liver samples from 6 normal livers obtained at immediate autopsy, and liver specimens obtained by surgical biopsy from the following patients: 8 with hepatomas, 5 with nonmetastatic colorectal carcinomas, and 14 with metastatic colorectal carcinomas. Cytochromes P-450MP and P-450NF in addition to epoxide hydrolase were measured by immunoquantitation. Enzymes involved in conjugation reactions were either assayed fluorometrically (UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase) or spectrophotometrically (glutathione S-transferase) using umbelliferyl substrates or 1-chloro-2,4-dinitrobenzene. Secondary hepatic tumors showed no significant change in drug-metabolizing enzymes, in contrast to primary hepatomas, which displayed decreases in all of the measured drug metabolizing enzymes. Arylsulfatase was markedly depressed in primary hepatomas (14% of normal values). Thus, activities of drug-metabolizing enzymes in human primary tumors resemble those associated with altered hepatic foci induced by peroxisome proliferators such as ciprofibrate. The marked decreases in sulfatase that occurred in primary but not in secondary human tumors suggest that sulfation of endogenous compounds and xenobiotics may differ in patients with primary and secondary hepatic tumors.     

Elevated formation of nitrate and N-nitrosodimethylamine in woodchucks (Marmota monax) associated with chronic woodchuck hepatitis virus infection.

Nitrate balance and N-nitrosodimethylamine (NDMA) excretion were studied in woodchucks chronically infected with woodchuck hepatitis virus (WHV). Twenty-four-h urinary recovery of a bolus dose of [15N]nitrate was 54 +/- 12% in woodchucks. WHV-infected animals formed 3-fold more nitrate endogenously than did control animals (P less than 0.01). Treatment of WHV-infected animals with Escherichia coli lipopolysaccharide increased nitrate excretion 15-fold, while uninfected animals increased nitrate excretion 4-fold. The endogenous formation of NDMA was higher in WHV-infected woodchucks than in uninfected controls. After administration of L-[15N2]arginine, [15N]nitrate, and [15N]NDMA were detected in urine indicating that arginine is a precursor of biosynthesized nitrate and the hepatocarcinogen NDMA. NDMA probably results from the formation of nitrosating agents during the oxidation of arginine to oxides of nitrogen and citrulline. Woodchucks chronically infected with WHV develop hepatocellular carcinomas with high frequency. Our observations suggest an additional mechanism that may be involved in the pathogenesis of hepatocellular carcinoma associated with chronic WHV infection.