DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene

Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.     

Comparison of DNA adduct formation in mice fed coal tar or benzo[a]pyrene

Coal tar is a complex mixture containing hundreds of compounds,including the carcinogenic poly cyclic aromatic hydrocarbon,benzo[a]pyrene. In order to compare the metabolic activationof a single carcinogen versus a complex mixture containing thecarcinogen, we determined the DNA adduct profiles in B6C3F₁mice fed doses of coal tar or benzo[a]pyrene at concentrationscorresponding to the amount of benzo[a]pyrene found in the respectivecoal tar treatments. DNA adduct formation was quantified inliver, lungs and forestomach by ³²P-postlabeling and was foundto increase as a function of dose in each tissue with both coaltar and benzo[a]pyrene. In mice fed benzo[a]pyrene, a majoradduct was detected with the same elution characteristics byTLC and HPLC as the major adduct, 10ß-(deoxyguanosin-N²-yl)-7ß,8     

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.     

Rapid induction of uterine endometrial proliferative lesions in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) given a single intraperitoneal injection of N-ethyl-N-nitrosourea

In our previous study, uterine endometrial stromal sarcomas and atypical hyperplasias of the endometrial glands were induced in heterozygous p53 deficient mice (p53 (+/-) mice) of the CBA strain given a single dose of N-ethyl-N-nitrosourea (ENU). In order to clarify whether uterine tumors can be induced in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) that are very susceptible to genotoxic carcinogens, rasH2 mice and their wild-type littermates received an intraperitoneal injection of 120 or 0mg/kg body weight of ENU followed by no further treatment for 22 weeks. Eighteen and 94% of ENU-treated rasH2 mice had uterine endometrial adenocarcinomas and atypical hyperplasias, respectively. Other malignant and benign tumors such as lung alveolar/bronchiolar adenomas and carcinomas, forestomach squamous cell papillomas and carcinomas, splenic hemangiomas/sarcomas, skin papillomas, malignant lymphomas and harderian gland adenomas were also observed in ENU-treated rasH2 mice. The result in the present study suggests that female rasH2 mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine epithelial tumors.     

Carcinogenic effects in A/J mice of particulate of a coal tar paint used in potable water systems

Coal tar paints are among the products used as inside coatings for water pipes and storage tanks to retard corrosion in potable water supply systems. Four different formulations of these paints were tested in earlier work by this laboratory in the Ames mutagenesis and the mouse skin carcinogenesis bioassays. The paint most active in these assays were then tested in a particulate form in the lung adenoma assay with A/J mice. The paint was applied to clean glass plates, cured, collected and homogenized in 2% Emulphor. Doses of this coal tar suspension were administered by gavage at 1.0, 10.0 and 55.0 mg in 0.2 ml per mouse 3X weekly for 8 weeks. The total doses of coal tar paint were 24, 240, and 1320 mg/mouse. Benzo[a]pyrene (BaP), administered in a parallel schedule to a total dose of 6 mg/mouse, served as positive control. A negative control group received an equivalent volume of 2% Emulphor. Animals were killed at 9 months of age (8 months after first dose) and lung adenomas counted. A dose-related response, in the average number of lung tumors per mouse, was observed with the coal tar particulate. There were also squamous cell tumors of the forestomach in 42% of the mice receiving 55.0 mg coal tar paint per application.     

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.     

Increased plasma fibronectin concentrations in tumor bearing mice

Benzo[a]pyrene (BP) was administered intracolonically to ICR/Ha and C57Bl/6 female mice, 1 mg/mouse, once weekly for 14 weeks. Half of the mice received beta-naphthoflavone (beta-NF, a mixed-function oxygenase inducer) i.p. 24 h prior to the BP. No colonic neoplasms were found in any of the mice after 18 months. However, the BP treatment did cause a significant increase in numbers of primary lung tumors, forestomach papillomas, mammary carcinomas, and sarcomas in one or both strains, relative to controls, and the incidence of all of these except for the sarcomas was significantly reduced by treatment with beta-NF prior to BP. Overall, the beta-NF pretreatment reduced total incidence of neoplasms by about 30% in the ICR/Ha mice and by about 60% in the C57Bl/6 mice, and did not potentiate the action of the carcinogen in any organ.     

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.     

Investigations on the carcinogenic burden by air pollution in man. Intratracheal instillation studies with benzo[a]pyrene in a mixture of Tris buffer and saline in Syrian golden hamsters.

Syrian golden hamster received intratracheal instillations of 0.125, 0.25, 0.50 or 1.0 mg benzo[a]pyrene (B[A]P) in a mixture of Tris buffer and physiological saline once weekly for life. Papillary polyps, squamous cell papillomas and carcinomas developed in both the larynx and trachea. In addition, bronchiogenic adenomas, adenocarcinomas and squamous cell carcinomas were induced in the lung. The highest incidence of respiratory tract tumours (83%) was seen in hamsters receiving 0.25 mg B[a]P. The results of these investigations are statistically evaluated and discussed.     

Effects of organoselenium compounds on induction of mouse forestomach tumors by benzo(a)pyrene

The selenium analogues of three known inhibitors of chemical carcinogenesis were synthesized and the compounds were tested for their ability to inhibit the induction of forestomach tumors in mice by benzo(a)pyrene. Groups of female CD-1 mice were given NIH-07 diet, or NIH-07 diet to which one of the following test compounds had been added: p-methoxyphenol (30 mumol/g diet and 3.3 mumol/g diet); p-methoxybenzeneselenol (3.3 mumol/g diet); benzylthiocyanate (0.045 mumol/g diet); benzylselenocyanate (0.045 mumol/g diet); phenothiazine (3.8 mumol/g diet); and phenoselenazine (3.8 mumol/g diet). The test compounds were administered for 1 week prior to treatment with benzo(a)pyrene, during the 4 weeks of benzo(a)pyrene treatment, and for 1 week after benzo(a)pyrene treatment. Twelve weeks later the mice were sacrificed and forestomach tumors were counted and confirmed histologically as papillomas. p-Methoxyphenol was the most effective inhibitor and was the only one which significantly reduced both the percentage of tumor-bearing animals and the number of forestomach tumors per animal. At the 3.3-mumol/g diet, p-methoxyphenol reduced the number of tumors per animal from 3.3 to 0.8 (P less than 0.0003). p-Methoxybenzeneselenol reduced the number of tumors per animal from 3.3 to 2.0 (P less than 0.05). Benzylthiocyanate showed no significant inhibitory effect, but benzylselenocyanate reduced the number of tumors per animal from 3.3 to 1.7 (P less than 0.01). Phenothiazine significantly enhanced the number of tumors per animal from 3.3 to 6.5 (P less than 0.004). Phenoselenazine had no effect on tumor induction. The results of this study indicate that two synthetic organoselenium compounds, p-methoxybenzeneselenol and benzylselenocyanate, are effective inhibitors of mouse forestomach tumorigenesis induced by benzo(a)pyrene.     

7H-benzo[c]fluorene: a major DNA adduct-forming component of coal tar

Coal tar is a complex mixture that exhibits high carcinogenic potency in lungs of animals when administered in the diet. Studies have noted that lung tumor induction does not correlate with the benzo[a]pyrene content of coal tar, suggesting that other hydrocarbons may be involved in the observed tumorigenicity. Our previous studies have demonstrated that a major 'unknown' chemical-DNA adduct is formed in the lung of mice exposed to coal tar. We have used an in vitro rat microsomal activation system to generate the 'unknown' adduct with neat coal tar and fractions of coal tar obtained by chemical fractionation and HPLC. Chemical-DNA adduct formation was evaluated by (32)P-postlabeling using both multi-dimensional TLC and HPLC. GC-MS analysis of the coal tar fractions obtained from HPLC, which produced the 'unknown' adduct in vitro, demonstrated that the adducting hydrocarbon had a mass of 216. A careful evaluation of candidate hydrocarbons led to the conclusion that a benzofluorene derivative may be responsible for forming the 'unknown' chemical-DNA adduct. Comparative in vitro and in vivo studies on the adducting properties of all three isomers of benzofluorene indicated that 7H-benzo[c]fluorene is responsible for producing the 'unknown' adduct observed in the lung of mice ingesting coal tar. Animal feeding studies also demonstrated that 7H-benzo[c]fluorene formed considerably more lung DNA adducts than 11H-benzo[a]fluorene and 11H-benzo[b]fluorene. These data indicate that the four-ring polycyclic aromatic hydrocarbon 7H-benzo[c]fluorene, a hydrocarbon not previously shown to form DNA adducts in lung, is in fact a potent lung DNA adductor and is a candidate PAH for causing lung tumors in animals treated with coal tar.     

Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.     

Inhibitory effect of probucol on benzo[a]pyrene induced lung tumorigenesis

The effects of probucol, a clinically used cholesterol loweringand antioxidant drug, on benzo[a]pyrene (B[a]P) induced pulmonaryand forestomach tumorigenesis as well as induction of colonicaberrant crypt foci (ACF) in female A/J mice was investigated.Diet containing 1% probucol fed prior to, during and after 8bi-weekly 1 mg/mouse oral intubations of B[a]P reduced the numberof pulmonary adenomas by 52% (P < 0.001) and the number offorestomach tumors by 31%nt. The 0.06% probucol diet also resultedin decreased tumor formation but the differences did not reachstatistical significance. Both probucol diets significantlyreduced the numbers of large ACF, putative preneoplastic lesionsof the colon mucosa, but showed no effects on the total numbersof ACF. The results of this study suggest that probucol mayalso be useful as a chemopreventive agent, in addition to beinga cholesterol lowering and anti-atherogenic drug with low toxicity.     

Carcinogenicity of nitrosamines and methanesulphonate esters given intraperitoneally, in RF mice

Single injections of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), methyl methanesulfonate (MMS), and ethyl methanesulphonate (EMS) were given intraperitoneally to male RF mice at various doses. DEN induced lung adenomas, liver hepatomas and forestomach tumors (squamous-cell carcinomas and papillomas) at all doses, and Leydig-cell tumors of the testis at the highest dose given. DMN, while increasing lung tumor incidence, failed to induce liver tumors when given intraperitoneally —in contrast to high incidences obtained by oral administration. The reason for this difference is not obvious at this time. MMS and EMS were not carcinogenic as used in this study. None of these alkylating agents was leukemogenic.     

Comparative tumor initiating activity on mouse skin of 6-nitrobenzo[a]pyrene, 6-nitrochrysene, 3-nitroperylene, 1-nitropyrene and their parent hydrocarbons

6-Nitrobenzo[a]pyrene, 6-nitrochrysene, 3-nitroperylene, 1-nitropyrene, and the corresponding parent hydrocarbons were tested for tumor initiating activity on mouse skin with promotion by tetradecanoylphorbol acetate. The initiating doses of 6-nitrobenzo[a]pyrene and benzo[a]pyrene were 0.05 mg each; for all other compounds the initiating doses were 1.0 mg. 6-Nitrochrysene induced tumors in 60% of the mice (2.1 tumors per mouse), but was significantly less tumorigenic than chrysene. 3-Nitroperylene induced tumors in 42% of the mice (0.5 tumors per mouse) and was significantly more active than perylene. Neither 1-nitropyrene nor 6-nitrobenzo[a]pyrene exhibited significant tumorigenic activity in the concentrations tested.     

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene.

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.     

Evaluation of the antineoplastic activities of the riminophenazine agents clofazimine and b669 in tumor-bearing rats and mice

The riminophenazine agents clofazimine and its analogue B669 displayed anti-tumor activity at 30 mg/kg/day in benzo[a]pyrene (BP) induced sarcomas of mice as well as dimethylbenz-anthracene (DMBA)-induced rat mammary tumors. No hematological toxicity of these drugs at doses up to 60 mg/kg/day for one month was observed. This is the first study to document in vivo anti-neoplastic activities of clofazimine and B669.     

Forestomach carcinoma and hepatoma induced in mice and rats by N-3-methylbutyl-N-1-methylacetonylnitrosamine (MAMBNA)

MAMBNA is a new N-nitroso compound isolated from Fusarium moniliforme-inoculated corn meal. In the present studies the carcinogenicity of MAMBNA is shown by the induction of forestomach carcinomas and liver tumors in mice and rats following the gastric intubation of this compound. Among 42 mice of Kunming (KM) stock treated with MAMBNA (10-20 mg/week), 22 showed epithelial hyperplasia of the esophagus, 29 papillomas and 4 squamous carcinomas in the forestomach, and there were 6 liver adenomas and 3 hepatomas. One mouse had forestomach carcinoma and carcinoma in the liver. Most tumors developed in mice receiving the treatment for 136-317 days, with a total dose of 210-670 mg (Table 1). No tumor was found in 12 controls observed for 239-357 days. In the experiment with 29 Wistar strain rats, administration of MAMBNA (20-120 mg/week) resulted in 11 epithelial hyperplasias in the lower esophagus, 14 atypical hyperplasias and papillomas, and 11 squamous carcinomas in the forestomach. The earliest forestomach carcinoma appeared in a rat treated for 454 days, receiving 4480 mg of MAMBNA, and the other 10 carcinomas occurred in animals treated for 518-640 days. Hyperplasia of liver cells was noted in 4 rats and liver adenoma in 7 and hepatocellular carcinoma in 8. Most hepatomas developed in rats treated for 480-640 days, and 5 rats had both forestomach carcinoma and hepatoma (Table 2). In 11 untreated rats observed for 411-644 days only one forestomach papilloma was noted.     

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.     

Dietary selenium- and benzo[a]pyrene-induced respiratory tract tumours in hamsters

The modifying effect of selenium as sodium selenite on chemicallyinduced respiratory tract tumours was tested in Syrian goldenhamsters. Groups of 40 hamsters per sex (controls 60 per sex)were fed the following semisynthetic diets: control diet (0.1p.p.m. Se, low fat); high-Se diet (5 p.p.m.); high-fat diet(20% sunflower oil); or high-Se + high-fat diet. After an adaptationperiod of 30 days on the diets, respiratory tract tumours wereinduced by intratracheal instillation of benzo[a]pyrene attachedto ferric oxide. The experimental period was 429 days for malesand 374 days for females. Respiratory tract tumours includedmainly epidermoid papillomas, epidermoid carcinomas and combinedepidermoid and adenocarcinomas. Selenium included either ina low-fat or high-fat diet did not influence the tumour responsein the respiratory tract or in other organs. Neither was therea correlation between the serum or liver Se levels in the presenceof respiratory tract tumours. The tumour response in the respiratorytract and also in other organs was slightly enhanced by dietaryfat.