Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by dietary soy sauce

We show that Japanese-style fermented soy sauce (shoyu) contains anticarcinogenic activity. Female ICR mice were fed a semipurified diet containing soy sauce (0-30%). Two weeks later a regimen consisting of 4 doses of benzo(a)pyrene (1 dose/week p.o. for 4 weeks) was begun to initiate forestomach neoplasia. Twenty-three weeks after the first intubation the animals were sacrificed, and forestomach neoplasms were counted and histologically confirmed. Soy sauce produced a significant dose-dependent reduction in forestomach neoplasms, which appeared to be maximal when soy sauce constituted 20% of the diet. Exposure to nitrite (0-500 ppm through drinking water) neither enhanced nor diminished the anticarcinogenic effect of the dietary soy sauce. Soy sauce was found to contain antioxidant activity which may be related to the observed anticarcinogenic effect. Contrary to expectations, mouse forestomach ornithine decarboxylase activity was induced by soy sauce. This appeared to be due at least in part to the relatively high sodium chloride content of soy sauce.     

Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid

Grilled ground beef contains factors that inhibit the initiation of mouse epidermal carcinogenesis by 7,12-dimethylbenz(a)anthracene. Previously we isolated an active principal and characterized it as an isomeric mixture of conjugated dienoic derivatives of linoleic acid (CLA). We now show that synthetic CLA inhibits the initiation of mouse forestomach tumorigenesis by benzo(a)pyrene. Four and 2 days prior to p.o. treatment with benzo(a)pyrene, female ICR mice were given (a) CLA in olive oil, (b) linoleic acid in olive oil, or (c) olive oil alone or plus 0.85% saline (control groups). Three days later the cycle was repeated for a total of 4 times. At 30 wk of age, the mice were sacrificed. In three independent experiments, mice treated with CLA developed only about half as many neoplasms/animal as mice in the control groups (P less than 0.025); in two of the experiments tumor incidence was also reduced (P less than 0.05). There were no significant differences in food intake or body weight among the groups. High-performance liquid chromatography/gas chromatography analysis established that, following intubation, only the c-9, t-11 CLA isomer was incorporated into forestomach phospholipids. In studies aimed at elucidating the mechanism of action, we found that CLA is an effective antioxidant. Under the conditions of the test CLA was more potent than alpha-tocopherol and almost as effective as butylated hydroxytoluene. These observations indicate that CLA might serve as an in situ defense mechanism against membrane attack by free radicals and may, at least in part, explain the anticarcinogenic properties of CLA.     

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.     

The role of antioxidant enzymes in benzo(a)pyrene-induced carcinogenesis

Chick embryo hepatocytes were cultured in the presence of benzo(a)pyrene in order to study the effects of this carcinogen on catalase, glutathione peroxidase and superoxide dismutase activity. The results demonstrate that benzo(a)pyrene is incapable of modifying the activity of these enzymes, even though it is taken up by cultured cells to form benzo(a)pyrene-DNA adducts. The effect of culturing, however, caused a marked reduction in the activity of these enzymes. The significance of these activity variations in benzo(a)pyrene in vitro carcinogenesis is discussed.     

Inhibition of benzo[a]pyrene-induced mouse forestomach neoplasia by a principal flavor component of Japanese-style fermented soy sauce.

A refined diet supplemented with Japanese-style fermented soy sauce (shoyu) inhibits benzo[a]pyrene-induced forestomach neoplasia in mice (Cancer Res., 51:2940-2942, 1991). In the present study, soy sauce was extracted with ethyl acetate. The soluble fraction contained flavor/aroma compounds and antioxidants, whereas amino-carbonyl compounds that impart color were concentrated in the ethyl acetate insoluble fraction. Both fractions inhibited benzo[a]pyrene-induced forestomach neoplasia in a protocol in which the test material was fed following exposure to the carcinogen. A principal flavor/aroma component of soy sauce, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone, was fed to mice following benzo[a]pyrene administration and found to inhibit the subsequent development of forestomach neoplasia. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone was effective when fed at 4 mg/kg body weight/day, indicating that it is a potent anticarcinogen.     

Melatonin suppresses benz(a)pyrene-induced carcinogenesis in mice

Skin tumors were induced in 3 groups of out-bred mice SHR by painting with 0.05% solution of benz(a)pyrene and 0.2ml acetone beginning from the age of 3 months. Each group included 40 mice; another 10 intact animals were in control. From day 2 on, several experimental animals received melatonin 2 and 20 mg/l with drinking water daily at nighttime. Mice were decapitated 24 weeks later. Among the parameters under study were frequency, multiplicity, size, morphological pattern, latent period of tumors and survival. Lipid peroxidation was evaluated on the basis of levels of malonic dialdehyde (MDA) and catalase in blood serum and tumor tissue. Tumor frequency in controls was 69.4%. That index in melatonin-treated mice fell as follows: 2 mg/l- 1.9 times (p<0.01); 20 mg/l - 2.2 times (p<0.05). Following melatonin 2 mg/l and 20 mg/l, the number of tumors per animal fell by 30.6% (p<0.05) and 27.4% (p<0.05), respectively; medium and maximum size of tumor decreased significantly too. There was no correlation between melatonin treatment and latent period duration. Melatonin 20 mg/l was followed by shorter survival after tumor development whereas 2 mg/l produced the opposite effect. Benz(a)pyrene boosted blood serum MDA by 190%, catalase - by 267% and 116% in tumor tissue as compared with untreated controls. Melatonin treatment supressed MDA and catalase levels in blood serum but not in tumor tissue. Relatively smaller doses exerted a more marked effect.     

Inhibition of benzo[a]pyrene-induced mutagenesis by (-)-epigallocatechin gallate in the lung of rpsL transgenic mice

Epigallocatechin gallate (EGCG) is a major water-soluble component of green tea. The antimutagenic activity of EGCG against benzo[a]pyrene (B[a]P)-induced mutations was assessed by using transgenic mice carrying the rpsL gene as a monitor of mutations. Seven-week-old male mice were given drinking water containing EGCG for 3 weeks. On day 7, mice were treated with a single i.p. injection of B[a]P (500 mg/kg body wt). Two weeks after the injection, the mutations in the rpsL gene were analyzed. B[a]P treatment resulted in an approximately 4-fold increase of mutation frequency at the rpsL gene in the lung. An approximately 60% reduction in the B[a]P-induced mutations in the lung was observed when mice were given EGCG at concentrations >0.005%. B[a]P-induced mutations mainly occurred at G:C basepairs in the several specific nucleotide sequences of the rpsL gene. These were AGG, CGG, CGT, TGG, TGC and GGT: all of them contained a guanine residue. Mutations seen similarly in the human Ki-ras codon 12 or p53 codons 157, 248, and 273 of lung tumor were also found in the rpsL gene, and the mutations were suppressed by the EGCG treatment. In conclusion, the antimutagenic effects of EGCG for B[a]P-induced mutagenesis in vivo suggest that drinking green tea may reduce the tumor-initiating potency of B[a]P in the lung.     

In vivo formation of benzo(alpha)pyrene diol epoxide-deoxyadenosine adducts in the skin of mice susceptible to benzo(alpha)pyrene-induced carcinogenesis

The hydrocarbon-deoxyribonucleoside adducts formed in mouse skin DNA have been determined following topical application of an initiating dose of benzo(a)pyrene to Swiss mice, a strain shown to be susceptible to benzo(a)pyrene-induced skin carcinogenesis. Several DNA-bound products were formed, of which the major one (60% of total adducts), in agreement with other workers' findings, was derived from reaction of (+/-) 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(alpha)pyrene (BDE) with the exocyclic aminogroup of deoxyguanosine. A further product (9-10% of total adducts), previously observed only after microsomal activation of benzo(a)pyrene, was observed and co-chromatographed with a further metabolite of 9-hydroxybenzo(alpha)pyrene bound to an uncharacterized base in the DNA. Two otherr products (2-3% of total adducts) were also found in the in vivo studies which co-chromatographed with BPDE-deoxyadenosine adducts and arose from cis and trans addition of the exocyclic amino group of deoxyadenosine to the 7R form, but not the 7S form, of BPDE. In contrast to this, the major in vitro deoxyadenosine-bound products, formed following reaction of BPDE with calf-thymus DNA, were derived from the 7S form of BPDE, suggesting either stereoselective formation or reaction of the 7R form of BPDE in mouse skin in vivo. Similar amounts of BPDE-deoxyguanosine and BPDE-deoxyadenosine adducts, as well as those derived from further metabolism of 9-hydroxybenzo(alpha)pyrene were formed in three strains of mice reported to have widely differing susceptibilities to polycyclic hydrocarbon-induced skin carcinogenesis. The relevance of these different hydrocarbon-DNA adducts to carcinogenesis requires further investigation.     

Chemoprevention of benzo(a)pyrene-induced lung tumors in mice by the farnesyltransferase inhibitor R115777.

PURPOSE: Inhibitors of farnesyltransferase (e.g., R115777) are being developed for therapy and prevention of various cancers. The efficacy of R115777 [Zarnestra; (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] to prevent the development of lung tumors in mice was determined. EXPERIMENTAL DESIGN: Female strain A mice (7-8 weeks of age) were given 100 mg/kg benzo(a)pyrene [B(a)P] by i.p. injection, and 4 or 14 weeks later, they were given 50 or 100 mg/kg R115777 by oral gavage 5 days/week. The mice were sacrificed 22 weeks after they received the B(a)P. RESULTS: Tumor multiplicity was 5.0 +/- 0.85, 4.5 +/- 0.52, 2.1 +/- 0.31, and 1.5 +/- 0.31 tumors/mouse in mice that received 0, 50, 100 (weeks 4-22), or 100 (weeks 14-22) mg/kg R115777. Thus, 100 mg/kg R115777 was similarly effective in preventing lung tumors when administered during the promotional phase of carcinogenesis [that is, either 4 or 14 weeks after B(a)P], whereas the lower dose of 50 mg/kg R115777 was ineffective. The proliferating cell nuclear antigen labeling index was also significantly reduced in lung tumors from mice treated with 100 mg/kg R115777 starting at 4 or 14 weeks. CONCLUSIONS: These results demonstrated that R115777 can prevent the development of lung tumors in the A/J mouse model, where tumors routinely have mutations in the Ki-Rasoncogene.     

Prevention of benzo(a)pyrene-induced mutagenicity by homogeneous epoxide hydratase.

Benzo(a)pyrene and benz(a) anthrancene which, in contrast to the K-region epoxides benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, are not mutagenic to Salmonella typhimurium TA 1537 in the absence of mammalian enzyme preparations, were activated by liver microsomes from C3H mice, which had not received any pretreatment, to mutagens reverting this tester strain to histidine prototrophy. Addition of epoxide hydratase inhibitors greatly increased this mutagenicity and addition of pure epoxide hydratase reduced it by more than 95% down to the range of spontaneous mutations as observed in absence of any added mutagen. This demonstrates than the metabolic pathway responsible for the mutagenicity of both polycyclic hydrocarbons observed in this system proceeds entirely via an epoxidation pathway and that the responsible metabolites are epoxides or species arising from them. Moreover, further metabolism by epoxide hydratase does not lead to produce contributing to the mutagenicity observed with the tester strain used. Finally, the epoxides relevant for the observed mutagenicity are substrates for epoxide hydratase; indeed, modest amounts of the pure enzyme can prevent the mutagenic effect.     

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.     

Studies of chemopreventive effects of myo-inositol on benzo(a)pyrene-induced neoplasia of the lung and forestomach of female A/J mice

There is a continuing effort at identifying chemopreventiveagents that might be useful in preventing cancer of the lung.In the present study, the effects of myo-inositol and dexamethasoneon benzo[a]pyrene (B[a]P)-induced pulmonary adenoma formationin female A/J mice was investigated. A diet containing 3% myo-inositolfed beginning 1 week after B[a]P administration reduced thenumber of pulmonary adenomas by 40% but did not prevent forestomachtumors, which also occur in this experimental model. Under thesame conditions, dexamethasone, 0.5µg/g diet, inhibitedpulmonary adenoma formation by 57% and also inhibited forestomachtumor formation to a similar extent. Feeding a diet containingboth myo-inositol and dexamethasone resulted in an additiveeffect on the inhibition of pulmonary adenoma formation. Thecombination of myo-inositol plus dexamethasone produced almostidentical inhibition of forestomach tumor formation to thatof dexamethasone alone. The results of the present study arepreliminary, but may provide a basis for future investigationinto strategies for chemoprevention of pulmonary neoplasia.     

Black tea polyphenols suppress cell proliferation and induce apoptosis during benzo(a)pyrene-induced lung carcinogenesis.

One of the most promising strategies for cancer prevention is chemoprevention by daily used food and beverages. Black tea, the most widely consumed beverage, is a source of compounds with antioxidative, antimicrobial, antimutagenic and anticarcinogenic properties. Lung cancer is the most common cause of cancer deaths in both men and women worldwide. Over one million people around the world are likely to be killed by lung cancer due to increased tobacco smoking and environmental pollutants, especially car exhausts. Therefore chemopreventive intervention using black tea and its active components may be a viable means to reduce lung cancer death. In the present investigation, we used benzo(a)pyrene (BP) to induce lung carcinogenesis in mice for the assessment of potential apoptosis-inducing and proliferation-suppressing effects of theaflavins and epigallocatechin gallate, active components of black tea. Hyperplasia, dysplasia and carcinoma in situ evident in the carcinogen control group on the 8th, 17th and 26th weeks respectively, were effectively reduced after treatment with theaflavins and epigallocatechin gallate. Significant reduction in number of proliferating cells and increased number of apoptotic cells was also found on the 8th, 17th and 26th week of treatment with theaflavins and epigallocatechin gallate in BP-exposed mice. Our observation suggests a promising role for black tea polyphenols in the prevention of lung cancer.     

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.     

Vitamin A (retinyl acetate) and benzo(alpha)pyrene-induced respiratory tract carcinogenesis in hamsters fed a commercial diet.

Male Syrian hamsters fed a commercial diet were given a series of 12 intratracheal instillations of 3 mg benzo (alpha) pyrene adherent to 3 mg Fe2O3 in 0.2 ml 0.15 M NaCl at weekly intervals. After the last instillation, the hamsters were randomly assigned to receive either 100, 1600, or 3300 (later reduced to 2400) mug retinyl acetate per week in divided intragastric doses. Hamsters in the 2400-mug retinyl acetate group had a significantly higher incidence of respiratory tract tumors than those in the group given 100 mug retinyl acetate per week. Liver vitamin A stores increased dramatically in the groups given 1600 and 2400 mug retinyl acetate and corresponded to the administration of retinyl acetate p.o. Serum vitamin A values were not consistently related to retinyl acetate administration or to hepatic stores of vitamin A.     

Inhibitory action of garlic oil on the initiation of benzo[a]pyrene-induced skin carcinogenesis in mice

When garlic oil was topically applied during the initiation phase of benzo[a]pyrene (B(a)P)-induced skin carcinogenesis in random bred adult female Swiss albino mice of two different substrains, there was a decline in the number of tumor-bearing mice as well as in the mean number of tumors per effective mouse.     

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.