Potentiation of benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis in mice by D,L-buthionine-S,R-sulfoximine-mediated tissue glutathione depletion

In vitro studies have suggested that the glutathione (GSH) S-transferase (GST)-catalyzed GSH conjugation is an important mechanism for the detoxification of (+)-anti-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated form of the widespread environmental pollutant benzo[a]pyrene (BP). However, in vivo experimental evidence for the importance of GSH/GST system in defense against carcinogenic effects of BP is lacking. We hypothesized that if GSH/GST were to play an important role in the detoxification of (+)-anti-BPDE, the tumorigenic activity of BP would be increased by depleting the levels of GSH, which is the required nucleophilic substrate for GST-catalyzed conjugation reactions. In the present study, we have tested the above hypothesis by determining the effect of D, L-buthionine-S,R-sulfoximine (BSO)-mediated tissue GSH depletion on BP-induced tumorigenesis of the lung and forestomach in female A/J mice. Treatment of mice with three i.p. injections of 2.5 mmol BSO/kg (12 h apart) plus 20 mM BSO in drinking water, resulted in a statistically significant reduction in hepatic, pulmonary and forestomach GSH levels. At the same time, BSO-administration caused a statistically significant increase in BP-induced pulmonary and forestomach tumor multiplicity. To the best of our knowledge, the present study is the first report that provides in vivo experimental evidence for the importance of GSH/GST system in cellular protection against carcinogenic effects of BP.     

Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis.

The potential antitumor effect of thymoquinone (TQ), the main constituent of the volatile oil of Nigella sativa seed, on fibrosarcoma induced by 20-methylcholanthrene (MC) in male Swiss albino mice was investigated in vivo and in vitro. Administration of TQ (0.01% in drinking water) I week before and after MC treatment significantly inhibited the tumor incidence and tumor burden by 43% and 34%, respectively, compared with the results in the group receiving MC alone. Moreover, TQ delayed the onset of MC-induced fibrosarcoma tumors that appeared at 12 weeks and produced less MC-induced mortality. Lipid peroxide accumulation, decreased glutathione (GSH) content, and decreased activities of glutathione S-transferase (GST) and quinone reductase (QR) were observed in the liver of MC-induced tumor-bearing mice. TQ alone showed a significant induction in the enzyme activities of hepatic GST and QR. Mice treated with TQ along with MC showed reduction in hepatic lipid peroxides and increased GSH content and increased enzyme activities of GST and QR as compared to results of the control group. The in vitro studies showed that TQ inhibited the survival of fibrosarcoma cells with IC50 of 15 microM. Conversely, TQ inhibited the incorporation of [3H] thymidine in fibrosarcoma cells with IC50 of microM. Our data indicate the potential of TQ as a powerful chemopreventive agent against MC-induced fibrosarcoma tumors. The possible modes of action of TQ may be through its antioxidant activity and interference with the DNA synthesis coupled with enhancement of detoxification processes.     

Effect of dietary tannic acid on epidermal, lung, and forestomach polycyclic aromatic hydrocarbon metabolism and tumorigenicity in Sencar mice

Tannic acid inhibits the mutagenicity of several polycyclic aromatic hydrocarbons (PAHs) and their bay-region diol-epoxides. Our prior studies have shown that when applied topically to Sencar mice, tannic acid caused substantial inhibition of epidermal PAH metabolism, subsequent PAH-DNA adduct formation, and PAH-induced skin tumorigenesis (H.Mukhtar et al., Cancer Res., 48:2361-2365, 1988, and references therein). In this study the effects of tannic acid supplementation in the diet (1%, w/w, in AIN-76 diet) of Sencar mice on benzo(a)pyrene (BP) metabolism and its subsequent DNA binding and tumorigenesis in lung and forestomach were evaluated. Animals receiving a tannic acid-containing diet showed diminished aryl hydrocarbon hydroxylase and 7-ethoxy-resorufin O-deethylase activities in the forestomach and lung. Elevated glutathione S-transferase and NAD(P)H:quinone reductase activities were observed in these tissues. Maximum effects occurred after 45 days of feeding. Administration of [3H]BP p.o. to animals resulted in lower covalent binding to DNA in forestomach and lung of animals receiving tannic acid-containing diet as compared to animals receiving AIN-76 control diet. Tumor induction studies in forestomach and lung revealed significant protection against BP-induced tumorigenesis in animals fed tannic acid-supplemented diet as compared to animals fed control diet. The mice fed tannic acid-supplemented diet developed 3.3 forestomach tumors/mouse compared to 5.2 tumors/mouse in animals receiving control diet. The numbers of pulmonary tumors per mouse in animals fed tannic acid-supplemented diet and control diet were 1.6 and 3.1, respectively. Topical application of 7,12-dimethylbenz(a)anthracene to animals fed tannic acid-supplemented diet did not result in significant protection against skin tumorigenesis. However, a slight delay in the onset of skin tumor formation occurred in tannic acid-fed animals when compared to animals receiving control diet. Our data suggest that dietary supplementation with tannic acid affords protection against BP-induced forestomach and lung tumorigenesis in rodents.     

Induction of glutathione S-transferase π as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model

There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase π (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10- tetrahydrobenzo(a)pyrene [(+)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP-induced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r = −0.89; p < 0.05) as well as in the forestomach (r = −0.97; p < 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model. Int. J. Cancer 73:897–902, 1997. © 1997 Wiley-Liss, Inc.     

Enhancement of glutathione S-transferase activity of the mouse forestomach by inhibitors of Benzo[a]pyrene-induced neoplasia of the forestomach

The effects of glutathione S-transferases (GST) activity and acid-soluble sulfhydryl levels of a number of compounds previously investigated for their capacity to inhibit benzo[a]pyrene (BP)-induced neoplasia of the mouse forestomach were determined. Five of the compounds studied increased the GST activity of the forestomach 78-182%. The five compounds were: p-methoxyphenol, 2-tert-butyl-4-hydroxyanisole, coumarin, alpha-angelicalactone, and benzyl isothiocyanate. With the exception of benzyl isothiocyanate. With the exception of benzyl isothiocyanate, these compounds also increased acid-soluble sulfhydryl levels, but to a lesser magnitude. All five chemicals inhibited BP-induced neoplasia of the forestomach. These data indicate that enhancement of the GST activity by an amount of about 75% or greater is associated with a reduced carcinogenic response of the forestomach to BP. The data also suggest that such enhancement might be used as a method of identifying compounds likely to inhibit BP and other carcinogens detoxified in a similar manner. Inasmuch as inhibition of the action of carcinogens may involve mechanisms other than detoxification by GST, the failure to increase GST activity substantially does not rule out the possibility of a compound being an inhibitor.     

Protection against N-nitrosodiethylamine and benzo[a]pyrene-induced forestomach and lung tumorigenesis in A/J mice by green tea

In recent years we and others have shown the cancer chemopreventiveeffects of green tea in several animal tumor models. In thisstudy we assessed the cancer chemopreventive effects of waterextract of green tea (WEGT) and the polyphenolic fraction (GTP)isolated from WEGT against N-nitrosodiethylamine (DEN)- andbenzo[a]pyrene (BP)-induced forestomach and lung tumorigenesisin A/J mice. The protective effects, both in forestomach andlungs, were evident by a decrease in number of tumors and thepercentage of mice with tumors when WEGT and GTP were fed toanimals during initiation, post-initiation and entire periodof tumorigenesis protocols. Oral feeding of 0.2% GTP in drinkingwater to mice afforded 68–82 and 39–66% protectionagainst DEN- and BP-induced forestomach tumorigenesis respectively.In case of pulmonary tumor multiplicity caused by DEN and BP,the protective effects of GTP were between 38–43 and 25–46%respectively. Similarly, oral feeding of 2.5% WEGT to mice alsoafforded 80–85 and 61–71% protection against DEN-and BP-induced forestomach tumorigenesis respectively. In caseof lung tumorigenesis, the protective effects of WEGT were 43–62and 25–51% respectively. Histological studies of forestomachtumors showed significantly lower squamous cell carcinoma countsin GTP- and WEGT-fed groups of mice compared to carcinogen alonetreated control group of mice. When pulmonary tumors were examinedhistologically, no adenocarcinomas were observed in GTP-andWEGT-fed groups of mice compared to 20% mice with adenocarcinomasin carcinogen alone treated control group. Oral feeding of GTPand WEGT in drinking water also showed significant enhancementin the activities of glutathione S-transferase and NADP(H):quinone reductase in liver, small bowel, stomach and lung. Theresults of this study suggest that green tea possesses chemopreventiveeffects against carcinogen-induced tumorigenesis in internalbody organs, and that the mechanism of such effects may involvethe enhancement of phase II and anti-oxidant enzyme systems.     

Glutathione S-transferases and glutathione in human head and neck cancer

Glutathione S-transferase (GST) enzyme activity, GST isoenzymecomposition and glutathione (GSH) concentration were assessedin normal and squamous cell carcinoma specimens of 14 patientswith oral or oropharyngeal cancer and 11 patients with laryngealcancer. Comparing malignant with normal oral/oropharyngeal tissues,no significant differences in GSH content, GST enzyme activityor isoenzyme composition were found. However, some tumours hadup to 3-fold increased GST enzyme activities and 11 malignantsamples over-expressed GST-     

Protective effects of trifluralin on benzo(a)pyrene-induced tumors in A/J mice

Trifluralin, a widely used herbicide, added to the diet before the p.o. administration of benzo(a)pyrene (BP) and fed continuously, significantly inhibited the induction of lung and forestomach tumors in female A/J mice. Dietary intake of trifluralin before the administration of BP resulted in a significant increase in glutathione in lung and forestomach but not in liver and glandular stomach. Trifluralin treatment also inhibited the binding of [3H]BP to liver and lung DNA, as well as to protein in the liver. Under these conditions, the protection against BP-induced lung tumors and perhaps forestomach tumors may be due to an elevation of tissue glutathione, resulting in a decreased binding of reactive metabolites of BP to macromolecules at these sites. The results indicate that trifluralin has a "blocking" effect in its inhibition of BP-induced tumors. Our studies show that trifluralin also inhibits chemical carcinogenesis in lung and forestomach when started in the diet 1 day after the administration of BP and fed continuously thereafter. In the case of lung, although maximum inhibition of tumors occurred when trifluralin was started 1 day after BP, there was significant protection at all time intervals (0 to 7 days) against lung tumors. The finding that trifluralin protects against BP tumorigenesis when started in the diet after the administration of the carcinogen clearly demonstrates that trifluralin also has a "suppressive" effect against BP-induced tumors.     

Neoplastic effects of oral administration of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and their inhibition by butylated hydroxyanisole.

The neoplastic effects of administration of benzo[a]pyrene (BP) and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) by oral intubation to noninbred female Ha:ICR mice have been determined. Under the experimental conditions, BP induced papillomas of the forestomach. BP 7,8-dihydrodiol also induced papillomas of the forestomach and was more potent than BP. In addition, administration of BP 7,8-dihydrodiol caused a large number of pulmonary adenomas and lymphomas. Butylated hydroxyanisole (BHA) added to the diet at a concentration of 5 mg/g inhibited BP-induced neoplasia of the forestomach. BHA also inhibited neoplasia of the forestomach, lungs, and lymphoid tissues that was caused by administration of BP 7,8-dihydrodiol. These data suggest that the inhibitory effect of BHA on BP carcinogenesis may entail events that occur subsequent to the formation of BP 7,8-dihydrodiol.     

The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

Walker cellsin vivo orin vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of k_cat= 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated fromE. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.     

Mechanism(s) of turmeric-mediated protective effects against benzo(a)pyrene-derived DNA adducts.

The effects of turmeric feeding before and after benzo(a)pyrene [B(a)P] exposure on the levels of B(a)P-derived DNA adducts were studied in tissues of Swiss mice employing (32)P-postlabelling analysis. A reduction in the levels of B(a)P-derived DNA adducts in liver, lung, and forestomach was observed in animals pre-treated with 0.2 or 1% turmeric diet and exposed to B(a)P by oral intubation when compared to animals receiving standard laboratory diet and B(a)P. The observed decrease was not due to dilution caused by nascent DNA synthesis. Comparative evaluation of levels of B(a)P-derived DNA adducts in tissues of animals shifted to 0.2 or 1% turmeric diet after 24 h of oral intubation of B(a)P with those continued on standard laboratory diet did not suggest enhanced disappearance/repair of B(a)P-derived DNA adducts due to exposure to turmeric. Further, pre-treatment of mice with 1% turmeric diet significantly reduced the B(a)P-induced increase in activity of cytochrome P450 (CYP450) isozymes CYP 1A1 and 1A2 in liver, lung, and forestomach of mice. In addition, hepatic glutathione S-transferase (GST) was found to be elevated in turmeric pre-treated mice. Thus turmeric-mediated decrease in induction of phase-I enzymes in liver, lung, and forestomach of mice and enhancement of hepatic GST appear to play an important role in reducing the B(a)P-induced DNA damage in target and non-target tissues.     

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.     

Differential induction of glutathione transferase isoenzymes of mice stomach by diallyl sulfide, a naturally occurring anticarcinogen

Diallyl sulfide (DAS), an organosulfur compound identified as the flavor component in garlic, has been shown to inhibit chemically induced neoplasia of forestomach and lung in mice. Even though the exact mechanism(s) of anti-neoplastic activity of DAS is not known, several independent studies suggest that this effect may, at least in part, be due to the elevation of glutathione-S-transferase (GST) activity. To gain further insight into the mechanism(s) of anti-carcinogenic activity of DAS, we have determined effect of orally administered DAS (25, 50 and 75 mumol) on levels of alpha, mu and pi class GSTs and glutathione (GSH) peroxidase and GSH reductase activities of female A/J mice stomach. Western blotting revealed presence of alpha, mu and pi class GSTs in mice stomach. A significant increase in all the three classes of GSTs was observed in the stomach of mice treated with DAS. Maximum increase in GST alpha and pi was evident by treating the animals with 75 mumol DAS whereas maximum induction of GST mu occurred after treating mice with 50 mumol DAS. GSH peroxidase activity towards t-butyl-hydroperoxide increased in a dose-dependent fashion in the mice stomach treated with DAS. Even though this activity towards hydrogen peroxide was similar in mice treated with 50 or 75 mumol DAS, these values were significantly higher than that of the control. GSH reductase was also elevated in the stomach of mice treated with 75 mumol DAS. These results suggest that DAS may exert anti-neoplastic effect by modulating GSH dependent detoxification enzymes.     

Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse

In the present study, eight organosulfur compounds from garlic and onions were studied for their inhibitory effects on benzo[a]pyrene (BP)-induced neoplasia of forestomach and lung of female A/J mice when administered 96 and 48 h prior to carcinogen challenge. These compounds had one, two or three linearly connected sulfur atoms. They included the four allyl group-containing derivatives: allyl methyl trisulfide (AMT), allyl methyl disulfide (AMD), diallyl trisulfide (DAT), and diallyl sulfide (DAS), and also four corresponding saturated compounds in which propyl groups were substituted for the allyl groups. All four allylic compounds inhibited BP-induced neoplasia of the forestomach. The saturated analogs were almost without inhibitory activity, indicating the importance of the allyl groups. DAT, which contains two allyl groups, was more potent than AMT, which contains only one allyl group, thus providing further evidence for the role of allyl groups in the inhibitory effects observed. DAS and AMD, but not DAT or AMT, inhibited pulmonary adenoma formation. The fact that in the lung the monosulfide and disulfide inhibited, but the trisulfide did not inhibit, indicates that the number of sulfur atoms in the molecule can control the organ sites at which protection against carcinogenesis will occur. All four allylic compounds induced increased glutathione S-transferase (GST) activity in the forestomach, but varied in their capacity to induce GST in lung, liver and small bowel. Their saturated analogs produced little or no induction. In evaluating relationships between diet and cancer, it would be useful to consider the possible role of garlic and onion organosulfur compounds as protective agents. In addition, further studies of this class of chemicals might lead to the identification and development of useful new chemopreventive compounds.     

Chemopreventive effect of Ginkgo biloba extract against benzo(a)pyrene-induced forestomach carcinogenesis in mice: amelioration of doxorubicin cardiotoxicity

Ginkgo biloba extract (EGb) is a natural product that possesses antioxidant and anticlastogenic properties. The current study was conducted to investigate the effect of EGb on benzo(a)pyrene (BP)-induced forestomach neoplasia, and to explore its possible beneficial effects against doxorubicin (Dox)-induced cardiotoxicity. Tumor was induced in female Swiss albino mice by oral administration of 1 mg BP, twice weekly for four weeks. EGb was given, at a daily oral dose of 150 mg kg(-1), two weeks before and during BP administration. Dox was given ip at a dose of 1.5 mg kg(-1), once weekly, for four weeks, during BP administration. EGb and Dox were given as combined or monotherapies. Results of the present investigation revealed that EGb blunted forestomach tumor multiplicity, as compared to control tumor bearing group. It also exhibited high activity to induce cytosolic glutathione S-transferase and glucose-6-phosphate dehydrogenase (G6PDH) in liver, as well as replenished hepatic glutathione that have been inhibited or depleted by tumorigenesis. Furthermore, it normalized nitric oxide (NO) serum level, without any observed alteration in neither the activity of liver microsomal NADPH-cytochrome P-450 reductase nor serum level of tumor necrosis factor-alpha (TNFalpha). Similar results have been obtained with Dox, but it failed to affect G6PDH activity, while increased serum TNFalpha and NO levels. The combined therapy did not add further to the anticarcinogenic effect of Dox, however it succeeded in ameliorating the deleterious effects of Dox on the heart; as evidenced by the reduction of cardiac lipoperoxidation, with modulation of Dox-induced pathological changes. Therefore, EGb confers a beneficial chemopreventive effect against BP-induced gastric carcinogenesis in mice, and possesses a salutary ameliorating potential on the cardiotoxic effects of Dox.     

Suppression of 7,12-dimethylbenz[alpha]anthracene-induced carcinogenesis and hypercholesterolaemia in rats by tocotrienol-rich fraction isolated from rice bran oil.

The anti-tumour and anti-cholesterol impacts of tocotrienol-rich fraction (TRF) were investigated in rats treated with the chemical carcinogen 7,12-dimethylbenz [alpha]anthracene (DMBA), which is known to induce mammary carcinogenesis and hypercholesterolaemia. DMBA administration to rats was associated with the appearance of multiple tumours on mammary glands after 6 months. Alkaline phosphatase (ALP) and glutathione-S-transferase (GST) are used as marker enzymes to monitor the severity of carcinogenesis. Although no tumours were visible on livers, hepatic ALP and GST activities of DMBA-treated rats were profoundly elevated in comparison to enzyme activities of normal control rats. Feeding of TRF (10 mg/kg body weight/day) for 6 months, isolated from rice bran oil (RBO), to DMBA-administered rats, reduced the severity and extent of neoplastic transformation in the mammary glands. Similarly, plasma and mammary ALP activities increased during carcinogenesis (95% and 43%, respectively), were significantly decreased in TRF-treated rats, whereas TRF mediated a further increase of 51% in hepatic ALP activity. TRF treatment to rats maintained low levels of GST activities in liver ( approximately 32%) and mammary glands ( approximately 21%), which is consistent with anti-carcinogenic properties of TRF. Administration of DMBA also caused a significant increase of 30% in plasma total cholesterol and 111% in LDL-cholesterol levels compared with normal control levels. Feeding of TRF to rats caused a significant decline of 30% in total cholesterol and 67% in LDL-cholesterol levels compared with the DMBA-administered rats. The experimental hypercholesterolaemia caused a significant increase in enzymatic activity (23%) and protein mass (28%) of hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase. Consistent with TRF-mediated reduction in plasma lipid levels, enzymatic activity and protein mass of HMG-CoA reductase was significantly reduced. These results indicate that TRF has potent anti-cancer and anti-cholesterol effects in rats.     

Differential effects of dietary BHA on hepatic enzyme activities and benzo[a]pyrene metabolism in male and female NMRI mice

The effect of dietary 2(3)-tert-butyl-4-hydroxy-anisole (BHA)alone and in combination with intraperitoneal injections of3-methylcholanthrene (MC), on hepatic enzyme activities andbenzo[a]pyrene (BP) metabolism was compared in male and femaleNMRI mice. In general, the characteristic induction patternfollowing dietary BHA administration in female mice could alsobe seen when male mice were used. The increase in epoxide hydrolaseand cytosolic glutathione S-transferase following BHA feedingwas however not as pronounced in males as in females. Also,MC treatment appeared to counteract the induction of GST activityby BHA in females but had no such effect in males. Liver microsomesfrom untreated male mice catalyzed the metabolism of BP lessefficiently than did microsomes from females. BHA treatmentincreased this activity in both sexes to a comparable extentand the overall activity was the same in males and females havingreceived MC. The pattern of BP metabolites was altered followingBHA treatment. In general, an increase in BP-4,5-diol and adecrease in 9-OH-BP was observed. This pattern was also noticedwhen microsomes from MC treated and MC + BHA treated animalswere compared. MC treatment alone increased the amount of BP-7,8-diol,the quinones and the phenols. The present report indicates thatseveral factors may contribute to the response to dietary BHAin mice. Whether this has any consequence in regard to thiscompound's anticarcinogenic effect remains to be elucidated.     

Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.     

Effect of butylated hydroxyanisole on the metabolism of benzo[a]-pyrene and the binding of metabolites to DNA, in vitro and in vivo, in the forestomach, lung, and liver of mice

The effects of butyldted hydroxyanisole (BHA) administrationon the amounts of benzo[a]pyrene (BP) metabolite-DNA adductsformed in vivo in the forestomach of A/HeJ mice were investigated48 h after oral administration of BP. BP was administered tomice in amounts known to result in BPInduced neoplasia in certaintissues. Analysis of deoxyribonudeosides by h.p.l.c. showedthat several BP metabolite-DNA adducts were formed in this tissue.The major identified adduct was the (±)-7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDEI) deoxyguanosine adduct. Addition of BHA to the diet inhibitedBPDE I-DNA adduct formation in the forestomach. The inhibitionof BPDE I-DNA adduct formation by BHA occurred under the sameexperimental conditions as does inhibition of tumor formationby this compound. These results in forestomach and previousresults in lung and forestomach demonstrated that inhibitionof the formation of the BPDE I-DNA adduct in the target tissueis a possible mechanism by which BHA inhibits BP-induced neoplasia.BP metabolism and DNA binding were also studied under in vitroconditions using microsomes prepared from forestomach, lung,and liver of A/HeJ mice. The amount of BPDE-DNA adduct formedin vitro is either equal to or lower than the amount of BP phenol-oxide-DNAadduct formed. BPDE I-DNA adduct formation was not significantlydifferent in incubations containing microsomes prepared fromBHA-treated or untreated mice. These results suggest that alterationsof the microsomal monooxygenases induced by BHA feeding arenot sufficient to account for the observed decreases in BPDE-DNAadduct formation in vivo. The monooxygenases were apparentlyaltered by BHA feeding as indicated by the substantial changesin the metabolic profile of BP and the decrease in the formationof the BP phenol-oxide-DNA adducts in the forestomach. The exclusionof glutathione transferases from the in vitro incubations couldaccount for the lack of effect of BHA treatment on BPDE-DNAadduct formation. BHA enhancement of ghitathione transferaseactivity has been postulated to play a role in the anticarcinogenicaction of BHA.     

Low glutathione and glutathione S-transferase levels in Barrett's esophagus as compared to normal esophageal epithelium.

Patients with Barrett's esophagus, wherein squamous epithelium has been replaced by columnar epithelium, have an increased risk for developing esophageal adenocarcinoma as compared to the general population. Glutathione S-transferase (GST), a family of detoxification enzymes consisting of class alpha, mu, pi, and theta isoforms, is involved in detoxification of carcinogens and low levels of these enzymes correlated with high cancer risk. We have now compared GST enzyme activity, GST isoenzyme composition and glutathione (GSH) content of Barrett's mucosa with that of adjacent normal squamous epithelium. Biopsy specimens of 98 patients with Barrett's esophagus were taken from both Barrett's and adjacent normal squamous epithelium. GST enzyme activity towards 1-chloro-2,4-dinitrobenzene was measured, and GST isoenzyme levels were determined by densitometrical analyses of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high-performance liquid chromatography after conjugation with monobromobimane. Wilcoxon's signed rank test and Spearman correlation analyses were used for statistical evaluation. As compared with adjacent normal squamous epithelium, GST enzyme activity in Barrett's epithelium was reduced by 35%, and GST mu, GST pi and GSH levels were reduced by 24%, 30%, and 63%, respectively. However, the minor GST alpha and GST theta levels were higher in Barrett's epithelium (by 625% and 33%, respectively). High levels of GSH and GSTs in general are correlated with protection against cellular or cytogenetic damage. The observed reduction in GSTs and GSH in Barrett's epithelium may therefore contribute to the increased cancer risk in this tissue.