Activation of the astrocytic Nrf2/ARE system ameliorates the formation of demyelinating lesions in a multiple sclerosis animal model.

Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219-2230.

Inhibition of tumor promotion by a biomimetic superoxide dismutase.

A low molecular weight, lipophilic, copper coordination complex with superoxide dismutase-mimetic activity inhibited biochemical and biological actions of a tumor promoter in mouse epidermis. Such inhibitory effects implicate reactive oxygen species in the tumor promotion process.

Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury.

The transcriptional factor Nrf2 has a unique role in various physiological stress conditions, but its contribution to ischemia/reperfusion injury has not been fully explored. Therefore, wildtype (WT) and Nrf2 knockout (Nrf2(-/-)) mice were subjected to 90-min occlusion of the middle cerebral artery (MCA) followed by 24-h reperfusion to elucidate Nrf2 contribution in protecting against ischemia/reperfusion injury. Infarct volume, represented as percent of hemispheric volume, was significantly (P<0.05) larger in Nrf2(-/-) mice than in WT mice (30.8+/-6.1 vs. 17.0+/-5.1%). Furthermore, neurological deficit was significantly greater in the Nrf2(-/-) mice. To examine whether neuronal protection was mediated by Nrf2, neurons were treated with various compounds to induce excitotoxic or oxidative stress. Translocation of Nrf2 into the nucleus was increased by the free-radical donor tert-butylhydroperoxide, but not by glutamate or N-methyl-D-aspartic acid (NMDA). In addition, a common Nrf2 inducer, tert-butylhydroquinone, significantly attenuated neuronal cell death induced by tert-butylhydroperoxide (83.6+/-1.6 vs. 62.0+/-7.7%) but not as substantially when excitotoxicity was induced by NMDA (91.9+/-1.6 vs. 79.3+/-3.3%) or glutamate (87.8+/-1.5 vs. 80.2+/-2.6%). The results suggest that Nrf2 reduces ischemic brain injury by protecting against oxidative stress.

Modulation of azaserine-induced pancreatic foci by phenolic antioxidants in rats.

Effects of the dietary phenolic antioxidants butylated hydroxyanisole [(BHA) CAS: 25013-16-5; (1,1-dimethylethyl)-4-methoxyphenol] and butylated hydroxytoluene [(BHT) CAS: 128-37-0; 2,6-di-tert-butyl-p-cresol] on pancreatic tumorigenesis were examined. Male LEW inbred rats were given injections of 30 mg azaserine [CAS: 115-02-6; diazoacetate (ester) serine] per kg body weight once a week for 3 weeks and maintained on either a control diet or 0.45% BHA- or 0.45% BHT-supplemented control diet throughout the initiation and post-initiation phases of the experiment. At 4 months post initiation, pancreatic tissue sections were quantitatively examined for the number and size of preneoplastic foci. BHT and BHA treatments reduced the number of acidophilic foci per pancreas by 32 and 48%, respectively, but were without effect on focal size. By contrast, basophilic foci were not subject to modulation by these antioxidants. A constellation of enzyme activities involved in carcinogen inactivation and known to be perturbed by antioxidant treatment was examined in liver and pancreas. The hepatic activities of glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione-S-transferases were markedly elevated while catalase and superoxide dismutase activities were unchanged. Glutathione peroxidase activity was diminished. In the pancreas, only glutathione peroxidase activity was affected, and it was reduced in both the BHA and BHT treatment groups. Although the pancreas is refractory to the enzyme inductive effects of these antioxidants, morphometric analysis of foci demonstrated chemoprevention by BHA and BHT of azaserine-induced foci. Whether this reduction reflected inhibition of an initiation, postinitiation , or a combination of effects was not known.

Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China.

BACKGROUND: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models. METHODS: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided. RESULTS: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682). CONCLUSIONS: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.

Inhibiton of phorbol ester-stimulated chemiluminescence in human polymorphonuclear leukocytes by retinoic acid and 5,6-epoxyretinoic acid.

Chemiluminescence (CL) is an index of both the generation of and reactions mediated by O2-. and 1O2. The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of CL by human polymorphonuclear leukocytes; treatment with TPA (100 ng/ml) provokes a CL response that peaks within five min and persists for over 30 min. The response is proportional to concentration over the range of one to 100 ng/ml. The ability of different phorbol diesters to stimulate both CL and O2-. production correlates with their relative activities as tumor promoters in vivo. Non-phorbol diester tumor promoters such as iodoacetic acid, anthralin, and tween 60 are inactive in this system. The TPA-mediated stimulation of CL can be inhibited by retinoids; cells preincubated for 15 min with 100 microM retinoic acid show only a marginal CL response to TPA. Addition of retinoic acid to resting polymorphonuclear leukocytes results in a transient burst of CL without concomitant O2-. release, observations indicative of an excitable substrate. A similar CL response is seen when retinoic acid is incubated with potassium superoxide in a cell-free system. 5,6-Epoxyretinoic acid, and even more effective inhibitor of TPA-stimulated CL than retinoic acid when added simultaneously with TPA, does not undergo these two CL reactions. Thus, it appears that retinoic acid may undergo oxidative activation to a species that exert enhanced antipromoter activities. Polymorphonuclear leukocytes provide a useful system for exploring the roles of reactive oxygen species in the mechanism of action of both TPA and retinoic acid.

Retinoic acid inhibition of the comitogenic action of mezerein and phorbol esters in bovine lymphocytes.

12-O-Tetradecanoylphorbol-13-acetate (TPA) is an effective comitogen in phytohemagglutinin-treated bovine lymphocytes. Concurrent addition of 10(-8) M TPA gives a greater than 6-fold increase in DNA synthesis over cultures treated with the lectin alone. The delayed addition of phorbol ester, relative to the start of the lectin treatment, eliminates this synergistic action. Structure-function studies show that the comitogenic activity of different phorbol diesters runs parallel to their tumor-promoting activity. A nontoxic level (50 micronM) of retinoic acid selectively antagonizes this synergistic effect of phorbol ester. This inhibitory action requires the near-concurrent addition of retinoic acid with TPA. In contrast, the TPA-mediated induction of RNA and protein synthesis is unaffected by retinolic acid. A number of natural and synthetic retinoids were evaluated; none were as inhibitory as was retinoic acid. Lymphocyte cultures appear to provide a useful system for exploring the mechanisms of action of both TPA and retinoic acid.

A role for protein kinase C-delta in the regulation of ornithine decarboxylase expression by oxidative stress.

The expression of genes that regulate cell growth, such as ornithine decarboxylase (ODC), can be modulated by oxidant tumor promoters. Treatment of murine papilloma PE cells with H2O2 led to a transient induction of ODC enzyme activity, which could be blocked by calphostin, a nonspecific inhibitor of protein kinase C (PKC). Peak activity (11-fold) occurred 5-6 h after treatment, followed by a rapid decline. The increase in ODC activity was associated with an elevation of both ODC mRNA (3-fold) and protein (7-fold). Direct involvement of PKC in the regulation of ODC by oxidants was determined by stable transfection of PE cells with a dominant-negative PKC-delta mutant. PKC-delta activity was completely inhibited in response to H2O2 in cells overexpressing mutant PKC-delta compared with cells transfected with a blank plasmid. Induction of ODC mRNA, protein, and activity was also completely inhibited in cells expressing the PKC-delta mutant after H2O2 treatment. Activation of an ODC promoter-luciferase reporter construct by H2O2 was attenuated in mutant cells compared with control cells, further confirming that ODC is regulated transcriptionally by PKC-delta. However, fold-increases in ODC mRNA and protein were much less than the increase in activity, suggesting that ODC may also undergo posttranscriptional regulation in the presence of oxidants. Taken together, these studies provide new insight into the regulation of ODC by oxidants and suggest that PKC-delta may play a critical role in this regulation.

Transcriptional control of glutathione S-transferase gene expression by the chemoprotective agent 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) in rat liver.

The substituted 1,2-dithiole-3-thione oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] protects against the acute and chronic toxicities of many xenobiotics, including aflatoxin B1, in rodents. These protective effects are mediated, in part, through elevation of glutathione S-transferase (GST) activities. Because studies by Coles et al. [Carcinogenesis (Lond.), 6: 693-697, 1985] suggested that the detoxication of aflatoxin through conjugation with glutathione is principally catalyzed by GST homodimer YaYa, we have investigated the regulation of the gene coding for the Ya subunit in the liver of F344 rats following dietary administration of oltipraz. Overall GST activity, as measured by conjugation with 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene, as well as the levels of GST Ya protein, was elevated 1.5-fold by 24 h and maximally (2.7- to 3.5-fold) and persistently after 5 days on a purified diet supplemented with 0.075% oltipraz. Steady state mRNA levels for GST subunit Ya, as quantified by slot blot analysis using rat liver GST complementary DNA clone pGTB38, were also elevated by 24 h, with a maximal elevation of 3-fold observed at 3 days. However, mRNA levels decreased thereafter, despite continued feeding of oltipraz. Northern blot analyses demonstrated that oltipraz did not alter the size of GST mRNA. Transcriptional activity of the GST Ya gene, as determined by nuclear run-off analysis, was increased 2-fold after 24-h feeding of oltipraz, was maximally induced 2.4-fold at 3 days, and returned to near control levels at 7 days, despite sustained feeding of oltipraz. Modulation of GST activity by oltipraz was not accompanied by changes in the methylation pattern at internal sites of the GST Ya gene. These results show that the initial induction of hepatic GST activity during oltipraz exposure correlates with changes in steady state levels of GST mRNA and rates of GST gene transcription; however, the continued elevation of GST enzymatic activities and GST Ya protein levels in the face of declining GST Ya mRNA levels and transcription rates suggests that additional mechanisms may be involved in regulating GST Ya expression by oltipraz.

Inhibition by 2(3)-tert-butyl-4-hydroxyanisole and other antioxidants of epidermal ornithine decarboxylase activity induced by 12-O-tetradecanoylphorbol-13-acetate.

The relationship between reactive oxygen and/or free radical species and tumor promotion was evaluated by investigating the inhibitory effects of 2(3)-tert-butyl-4-hydroxyanisole (BHA) and other antioxidants on the induction of ornithine decarboxylase (ODC) activity in mouse epidermis by a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice maintained on a diet containing 0.75% BHA for 8 days showed a 50% reduction in maximal ODC induction following treatment with TPA when compared to mice fed a control diet. Topical application of BHA (55 mumol) 30 min prior to TPA treatment (17 nmol) elicited an 80% inhibition of promoter-induced ODC activity. BHA was ineffective as an inhibitor when administered either 16 hr before or 2 hr after the promoter. The inhibition by BHA was dose dependent with a dose producing a 50% inhibition of ODC induction of 6 mumol. A structure-activity study with BHA analogues (2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl-1,4-dimethoxybenzene,tert-butylhydroquinone, 4-hydroxyanisole, p-hydroquinone, phenol, and 2-tert-butyl-phenol) showed that hydroxyl and tert-butyl substituents were important determinants of inhibitory activity. A spectrum of other antioxidants were also tested. Butylated hydroxytoluene was nearly equipotent to BHA; alpha-tocopherol, propyl gallate, and disulfiram were all less potent, and L-ascorbate was inactive. None of the antioxidants affected basal ODC activity in non-TPA-treated mice. Collectively, these results demonstrate an early and direct inhibition of TPA-induced ODC activity by lipophilic phenolic antioxidants and suggest a role for reactive oxygen and/or free radical species in tumor promotion.

Mitogen-activated protein kinase (MAPK) activation by butylated hydroxytoluene hydroperoxide: implications for cellular survival and tumor promotion.

The mitogen-activated protein kinase (MAPK) cascade plays an important role in carcinogenic development. Herein, we show that the skin tumor promoter butylated hydroxytoluene hydroperoxide (BHTOOH) stimulates a rapid and potent (14- to 20-fold) activation of extracellular signal-regulated kinase (ERK) in vivo and in cultured mouse keratinocytes. BHTOOH also moderately (5-fold) activated c-jun-N-terminal kinase, and 38-kDa MAPK-related protein in these same cells. N-acetylcysteine and o-phenanthroline abolished ERK activation by BHTOOH, consistent with a requirement for metal-dependent formation of reactive intermediates. Indeed, 4-CD3-BHTOOH, an analogue that generates less of the metabolite BHT-quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) and fewer tumors in vivo, accordingly exhibited diminished potency for activating ERK. ERK activation by BHTOOH was inhibited by suramin, and by expression of dominant-negative Ras-N-17 in PC12 cells, suggesting overlap between the pathways for BHTOOH and growth factor signaling. Induction of MAPK-dependent genes c-fos and MAPK phosphatase-1 by BHTOOH was also blocked by Ras-N-17 expression. Moreover, expression of Ras-N-17 or kinase-defective MAPK kinase (MEK) diminished cell survival following BHTOOH exposure. Similarly, pretreatment with suramin or the MEK inhibitor PD098059 also potentiated the toxicity of BHTOOH. On the other hand, expression of constitutively active MEK enhanced cell survival. Thus, we demonstrate that the MAPK cascade is critical to the cellular response to BHTOOH. This study suggests a functional role for MAPK activation in tumor promotion stimulated by oxidants and other agents.

Effects of retinoic acid and juvenile hormone on the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate.

The tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), a highly active comitogen in phytohemagglutinin-treated bovine lymphocytes, induces an 11-fold increase in ornithine decarboxylase activity over cultures treated with the lectin alone. This synergistic action of TPA could be antagonized by the simultaneous addition of the acyclic sesquiterpene, insect juvenile hormone III. Retinoic acid (vitamin A acid), an inhibitor of the tumor-promoting action of TPA in mice, was also an effective antagonist but required administration to lectin-activated lymphocytes 1 hr prior to TPA. These data suggest that metabolic activation of retinoic acid is required in order to exert its antagonistic action. Comparison of the responses in the lymphocytes and mouse skin suggests that the lymphocytes provide an excellent system for studying the molecular processes through which phorbol esters and retinoids influence the growth and differentiation of both normal and premalignant cells.

Molecular biomarkers for aflatoxins and their application to human cancer prevention.

The rapidly expanding understanding of the progressive processes of carcinogenesis provides opportunities for the identification of molecular biological markers reflecting events from exposure through clinical disease. These molecular biological markers can be classified into categories of markers of exposure reflecting the dose of toxic agents, markers of effect indicating a biological response to exposure, and markers of susceptibility providing information about the inherent sensitivity of individuals to the toxic agents. By definition some of these markers are chemical agent specific, such as a carcinogen-DNA or -protein adduct, while others are biological process specific, such as the altered expression of a gene. This article reviews the development and validation of molecular biomarkers of aflatoxins using experimental and human population studies. The development of molecular biomarkers for aflatoxins is based upon the extensive research database available about their metabolism, macromolecular adduct formation, and general mechanisms of action. The long-term goal of the research described in this paper is the application of aflatoxin biomarkers to the development of preventive interventions in human populations at high risk for liver cancer.

Effect of hepatocarcinogens on the binding of glucocorticoid-receptor complex in rat liver nuclei.

The effects of a number of carcinogens and hepatotoxins on the binding kinetics of the interactions of glucocorticoidcytosol receptor complex with nuclear acceptor sites in rat liver were investigated. Both the apparent sites in rat liver were investigated. Both the apparent concentration of nuclear binding sites and the Kd were significantly diminished following treatment of rats with sublethal doses of the carcinogens aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, thioacetamide, 3'-methyl-4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene, and 3-methylcholanthrene. Treatment with actinomycin D resulted in a slight reduction in the apparent concentration of nuclear acceptor sites but had no effect on the nuclear binding Kd. The hepatotoxic but noncarcinogenic analgesic, acetaminophen, as well as the weakly toxic aflatoxin B1 cognate, aflatoxin B2, were without effect on the kinetics or binding capacity of glucocorticoid-nuclear acceptor site interaction. These experiments suggest that chemically induced alteration of functional glucocorticoid binding sites on chromatin may be involved in the biochemical effects produced in liver by carcinogens of several chemical types. This experimental model may provide a useful approach for further elucidation of early events in carcinogenesis.

Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione S-transferases.

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] protects against chemical carcinogenesis in several animal models and is currently under evaluation as a possible chemopreventive agent in humans. Ideally, clinical chemopreventive interventions use dosing regimens that maximize efficacy while minimizing toxicity. Toward this end, the chemopreventive efficacy achieved by administration of intermittent doses of oltipraz was evaluated in rats. F344 rats were treated with oltipraz (0.5 mmol/kg, p.o.) once weekly, twice weekly, or daily over a 5-week period. After the first week, all rats were gavaged with 20 micrograms/kg of aflatoxin B1 for 28 consecutive days. Livers were analyzed 2 months after the last aflatoxin B1 dose, and the volume of liver occupied by glutathione S-transferase (GST)-P positive foci, a presumptive marker of neoplasia, was observed to be decreased > 95%, > 97%, or > 99% in livers of rats receiving once-, twice-weekly or daily oltipraz treatments, respectively. The chemopreventive actions of oltipraz have been associated with increases in the levels of phase 2 detoxifying enzymes, such as the glutathione S-transferase isozymes. Accordingly, GST conjugation activity measured with 1-chloro-2,4-dinitrobenzene as substrate increased 1.5-, 1.8-, or 2.4-fold for the once-weekly, twice-weekly or daily treatments, respectively, throughout a 7-day period. Quantitative HPLC analyses of GST subunits 24 h after 2 or 7 daily administrations of oltipraz showed that the levels of subunits Yb1, Yp, Yc2, and Ya2 were increased with maximum elevations of 5.6-, 11.1-, 6.4-, and 10.4-fold, respectively. In comparison, levels of subunits Yb2 and Yc1 were modestly elevated 1.8- to 2.6-fold, respectively, whereas subunit Ya1 was not induced. Remarkably, the levels of subunit Yp and Ya2 remained elevated approximately 2.3-fold 7 days after a single dose of oltipraz. In contrast, the levels of subunits Yb1 and Yc2 diminished to approximate control levels within 7 days after a single dose of oltipraz. GST mRNA levels for Ya, Yb, and Yp were measured by Northern blot analysis and were found to be elevated maximally to 13.7-, 13.5-, and 3.9-fold, respectively, after two daily oltipraz doses. Interestingly, GST Ya and Yb mRNA diminished to constitutive levels after 7 daily doses of oltipraz, with no corresponding decreases in GST subunit or activity levels. The levels of GST Ya and Yb mRNA decreased to constitutive levels within 4 days after a single oltipraz administration, whereas GST Yp mRNA levels remained elevated throughout the 7-day follow-up period.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones.

1,2-Dithiol-3-thiones, reported constituents of cruciferous vegetables, are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, and chemoprotective activities. The effects of dietary administration of a substituted 1,2-dithiol-3-thione, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione], a potent antischistosomal agent, on aflatoxin B1 (AFB1) metabolism, DNA adduct formation, and hepatic tumorigenesis were examined in male F344 rats. Rats were fed graded doses of oltipraz (0.01-0.1%) for 4 wk. During the second and third wk of oltipraz feeding rats were gavaged with 250 micrograms of AFB1/kg five times a wk. Rats were finally restored to control diet 1 wk after cessation of AFB1 dosing. At 4 months focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase activity. Treatment with oltipraz at all doses reduced by greater than 90% the volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Levels of AFB1 bound to hepatic DNA were reduced between 40 and 80% in animals fed increasing doses of dietary oltipraz (0.01-0.1%) for 1 wk prior to a single exposure to AFB1. Feeding of the higher levels of oltipraz led to marked increases in the specific activity of glutathione S-transferases, presumably serving to facilitate the detoxication of the ultimate electrophilic form of AFB1, the 8,9-oxide. At low dietary concentrations of oltipraz (0.01%), the only inductive effects seen were on the activities of selected cytochrome P-450 monooxygenases. Therefore, the protection afforded by oltipraz may be due to both the enhancement of electrophile detoxication pathways as well as modified oxidative metabolism of AFB1. In in vitro metabolism studies with hepatic post-mitochondrial supernatant, low-dose oltipraz pretreatment facilitated the oxidative production of aflatoxins P1 and Q1, but not M1, from AFB1. High-dose (0.1%) oltipraz pretreatment enhanced the primary metabolism of AFB1 to aflatoxins P1, M1, and Q1 as well as the formation of chloroform-insoluble metabolites. Feeding studies with a series of 1,2-dithiol-3-thione and 1,2-dithiol-3-one derivatives of oltipraz demonstrated that the inductive activity for cytochrome P-450-dependent monooxygenases and electrophile detoxication enzymes, such as glutathione S-transferases, could be readily separated by minor modifications of the 1,2-dithiol-3-thione structure. The unsubstituted 1,2-dithiol-3-thione nucleus strongly induced electrophile detoxication enzymes, but not the monooxygenases, and was the most effective inhibitor of the binding of AFB1 to hepatic DNA in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short-term molecular dosimetry.

Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation, and hepatic tumorigenesis in rats fed ethoxyquin: role of induction of glutathione S-transferases.

The effects of dietary administration of ethoxyquin (EQ) on aflatoxin B1 (AFB1) metabolism, DNA adduct formation and removal, and hepatic tumorigenesis were examined in male Fischer rats. Rats were fed a semipurified diet containing 0.4% EQ for 1 wk, gavaged with 250 micrograms of AFB1 per kg 5 times a wk during the next 2 wk, and, finally, restored to the control diet 1 wk after cessation of dosing. At 4 mo, focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase. Treatment with EQ reduced by greater than 95% both area and volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Utilizing the same multiple dosing protocol, patterns of covalent modifications of DNA by AFB1 were determined. EQ produced a dramatic reduction in the binding of AFB1 to hepatic DNA: 18-fold initially and 3-fold at the end of the dosing period. Although binding was detectable at 3 and 4 mo postdosing, no effect of EQ was observed, suggesting that these persistent adducts are not of primary relevance to AFB1 carcinogenesis. Analysis of nucleic acid bases by high-performance liquid chromatography revealed no qualitative differences in adduct species between treatment groups. The inhibitory effect of EQ on AFB1 binding to DNA and tumorigenesis appears related to induction of detoxication enzymes. Rats fed 0.4% EQ for 7 days showed a 5-fold increase in hepatic cytosolic glutathione S-transferase (GST)-specific activities. Multiple molecular forms of GST were induced, and concomitant elevations in messenger RNA levels coding for the synthesis of GST subunits were observed. Correspondingly, biliary elimination of AFB1-glutathione conjugate was increased 4.5-fold in animals on the EQ diet during the first 2 h following p.o. administration of 250 micrograms of AFB1 per kg. Thus, induction by EQ of enzymes important to AFB1 detoxication, such as GST, can lead to enhanced carcinogen elimination, as well as reductions of AFB1-DNA adduct formation and subsequent expression of preneoplastic lesions, and, ultimately, neoplasia.

Long-term association of N-(phosphonacetyl)-L-aspartate with bone.

By means of enzymatic and autoradiographic techniques, it has been demonstrated that, 24 hr after a single dose of the antitumor amino acid N-phosphonacetyl-L-aspartic acid (PALA), (400 mg/kg i.p.; 1.15 mmol/kg) to C57BL x DBA/2 F1 mice, the agent accumulates in bone to a concentration of approximately 400 microM; this is 3000 times greater than the Ki of PALA for its target enzyme, aspartate carbamoyltransferase. However, disproportionately low inhibition of enzyme activity was demonstrated in homogenates of bone from these recipients, suggesting that the drug was sequestered from its target in this tissue. Autoradiography of sections of femoral shafts from mice treated with 14C-labeled drug demonstrated that autoradiogram density due to [14C]PALA equivalents was confined to the bony matrix, with no label above background resolvable in bone marrow. Following in vivo administration of PALA (400 mg/kg i.p.), the half-life of the drug in the bone was approximately 23 days. In vitro, with equilibrium dialysis at pH 7.4, it was demonstrated that: (a) normal pulverized and decalcified bone bound PALA with capacities of 3.5 nmol/mg and 0.1 nmol/mg bone, respectively, at a PALA concentration of 5 mM; (b) binding of PALA to normal bone reached saturation at a concentration of 200 mM; and (c) PALA functions as a solubilizer of bone at concentrations above this. Since administration of PALA was shown to produce long-lasting inhibition of aspartate carbamoyltransferase in liver and tumor and since its ultimate half-life in the plasma of mice, following a single 400-mg/kg administration of the drug, is 8 days, it is suggested that bone serves as a reservoir from which PALA is released at a slow rate into plasma and other tissues.

Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.

The prostate has been identified as a target for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced carcinogenesis. Humans are exposed to PhIP through ingestion of well-done cooked meats, and there is evidence from epidemiological studies that implicates red meat consumption in prostate carcinogenesis. The alpha and pi class isoforms of glutathione S-transferases (GSTs) have been shown to inhibit adduction of activated PhIP metabolites to DNA in cell-free systems. In humans, silencing of GST pi(GSTP1) through CpG island hypermethylation is found in nearly all prostate carcinomas and is believed to be an early event in prostate carcinogenesis. We hypothesized that suppressed GSTP1 expression in prostate cells would increase their vulnerability to cytotoxicity and DNA adduct formation mediated by activated PhIP metabolites. To test this hypothesis, the human prostate adenocarcinoma cell line, LNCaP, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1 cells exposed to N-OH-PhIP, but not parent PhIP, for 24 h showed a dose-dependent decrease in cell viability. GSTP1-overexpressing cells had LC50s 30-40% higher than cells transfected with the vector alone. PhIP-DNA adducts isolated from LNCaP-derived cells and primary human prostate tissue cultures exposed to N-OH-PhIP were analyzed by liquid chromatography/electrospray ionization mass spectrometry. Primary cultures of human prostate tissue and LNCaP-GSTP1 cells had approximately 50% lower adduct levels than parental LNCaP and vector control cells. Bioactivation assays using LNCaP cytosols showed that enzymatic activation of N-OH-PhIP to a DNA binding species was dependent on ATP and could be inhibited by recombinant human GSTP1 in the presence of glutathione. This evidence confirms that N-OH-PhIP can be bioactivated to a DNA binding species in human prostate and human prostate-derived cells. These observations provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model system for studying the role of this enzyme in protection against N-OH-PhIP induced DNA damage in prostate carcinogenesis. Loss of GSTP1 expression in human prostate may, therefore, enhance its susceptibility to carcinogenic insult by compounds such as N-OH-PhIP. Conversely, induction of GSTs in early-stage prostate carcinogenesis may be a useful protective strategy.