Alpha-naphthoflavone: an inhibitor of hydrocarbon cytotoxicity and microsomal hydroxylase.

Alpha-naphthoflavone inhibits the metabolism of 3,4-benzopyrene and 7,12-dimethylbenz(a)anthracene in hamster enlbryo cell cultures and protects the cells against the inhibition of cell multiplication by these carcinogens. Alphla-nalphthoflavone also inhibits the aryl hydrocarbon hydroxylase activity in homogenates of induced hamster embryo cells and in liver microsomes from rats previously treated with polycyclic aromatic hydrocarbons, but not in microsomes from control rats.

Glutathione S-transferase activity: enhancement by compounds inhibiting chemical carcinogenesis and by dietary constituents.

Benzyl isothiocyanate, beta-naphthoflavone, coumarin, alpha-angelicalactone, disulfiram, indole-3-carbinol and indole-3-acetonitrile induced increased glutathione (GSH) S-transferase activity in the liver and small intestine in female ICR/Ha mice. All seven compounds are inhibitors of chemical carcinogenesis. In additional work, several dietary constituents increased GSH S-transferase activity. Consumption of diets containing dried powdered preparations of brussels sprouts, cabbage, coffee beans, or tea leaves resulted in increased GSH S-transferase activity. Mice fed an unrefined diet (Purina Rat Chow) had a higher GSH S-transferase activity than those fed a semipurified diet. The results of the present study indicated that the composition of the diet can alter the activity of an important enzyme system having the capacity to detoxify chemical carcinogens.

Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds.

Benzyl isothiocyanate and phenethyl isothiocyanate, two compounds found in cruciferous plants, and phenyl isothiocyanate, a synthetic compound, all inhibit 7,12-dimethylbenz[a]anthracene (CMBA)-induced mammary tumor formation in female Sprague-Dawley rats when administered 4 hours prior to the DMBA. Comparable studies in which benzyl isothiocyanate was administered 24 hours before or 4 hours after DMBA showed almost complete loss of inhibition. Additions of benzyl isothiocyanate or phenethyl isothiocyanate to a diet containing CMBA inhibited formation of neoplasms of the forestomach and pulmonary adenomas in female ICR/Ha mice. Addition of benzyl isothiocyanate to a diet containing benzo[a]pyrene also inhibited carcinogenesis of the mouse forestomach due to this carcinogen. The finding of two additional anutrient dietary compounds which inhibit chemical carcinogenesis focuses on the possibility that dietary constituents of this nature may diminish the impact of exposures to chemical carcinogens.

Lack of involvement of 6-hydroxymethylation in benzo[a]pyrene skin tumor initiation in mice.

The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.

Effects of administration to mice of butylated hydroxyanisole by oral intubation on benzo[a]pyrene-induced pulmonary adenoma formation and metabolism of benzo[a]pyrene.

Administration of butylated hydroxyanisole (BHA) by oral intubation 4 hours before challenge with benzo[a]pyrene (BP) inhibited the formation of pulmonary adenomas in A/HeJ mice. Incubation of BP with liver microsomes from mice that received BHA 2,4, or 8 hours before being killed resulted in less binding of BP metabolites to added DNA than occurred with control microsomes. High-pressure liquid chromatography studies of the BP metabolite pattern produced by the incubation of BP with liver microsomes from mice given BHA by oral intubation showed a decrease in formation of BP-4,5-oxide and 9-hydroxybenzo[a]pyrene. In contrast, the formation of 3-hydroxybenzo[a]-pyrene was increased. The was increased. The short interval between the administration of BHA by oral intubation and the observed biochemical changes indicated that BHA could exert a direct effect on the microsomal metabolism of BP. These changes in metabolism of BP occurred under conditions of BHA administration that produced a decreased neoplastic response to this carcinogen.

Inhibition of promutagen activation by the antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) exhibited antimutagenic activity in the Salmonella typhimurium reversion test. Both BHA and BHT reduced reversion induced by chemicals requiring metabolic activation for effectiveness. However, they did not affect reversion induced by direct-acting mutagens. These results suggested that BHA and BHT may inhibit the metabolic activation processes and demonstrated that the S. typhimurium reversion test may be used to identify inhibitors of the neoplastic process.

Inhibition of macromolecular binding of benzo(a)pyrene and inhibition of neoplasia by disulfiram in the mouse forestomach.

Benzo[a]pyrene (BP) administered to female outbred ICR/Ha mice by oral intubation induced neoplasia in the forestomach after 30 weeks in more than 90% of treated animals. However, no tumors occurred at that site if 1% disulfiram was added to the experimental diet. This inhibition of tumor formation was paralleled by a large inhibition of macromolecular binding of [3H]BP and [14C]BP to RNA and protein in the forestomach. The inhibitory effect of specific binding was strongest in the RNA fraction isolated at 8,000 X g for 20 minutes (6 times that of the control) and weakest in the DNA fraction (no significant difference). The highest specific binding was measured in the RNA fraction isolated at 32,000 X g for 120 minutes and the lowest binding in the DNA fraction. The relative distribution of the BP binding was such that greater than 90% of the BP was bound to protein and less than 1% was bound to DNA. Of the total inhibition of BP binding to all the macromolecular species studied, that to protein constituted the largest component (95%). In contrast to the forestomach, no inhibitory effect of BP binding to DNA, RNA, or protein by disulfiram was found in the liver, which remained free of cancer under the experimental conditions employed.

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.

Inhibition of benz[a]pyrene-induced mammary carcinogenesis by retinyl acetate.

The administration of a 250-ppm retinyl acetate dietary supplement for various periods relative to intragastric administration of 50 mg benzo[a]pyrene (BP) significantly inhibited the induction of mammary cancers in virgin female inbred LEW/Mai rats. with day of BP administration taken as time 0, groups receiving the retinoid from weeks -2 to +1, +1 to +90, +20 to +90, and -2 to +90 showed a significant reduction in tumor response as compared to controls. The inhibition of carcinogenesis achieved by a +1 to +20 administration schedule was temporary; the tumor yield was suppressed initially but returned to control levels by week 60. Autoradiographic analysis of mammary glands from 50-day-old rats indicated that a 2-week exposure to supplemental retinyl acetate significantly reduced the mammary gland parenchymal cell labeling index in ductal, alveolar, and terminal end bud structures. Beginning the retinyl acetate supplement 1 week after the administration of BP significantly reduced the number of terminal ductal hyperplasias. The inhibition of carcinogenesis achieved by a short period of retinyl acetate administration before and during the period of carcinogen availability as well as the inhibition achieved by long-term postcarcinogen retinoid exposure may involve an antiproliferative effect on the rat mammary gland.

Effects of p-methoxyphenol and diet on carcinogen-induced neoplasia of the mouse forestomach.

Previously, p-methoxyphenol fed in the diet was found to be the most potent inhibitor of benzo(a)pyrene-induced neoplasia of the mouse forestomach of 18 phenols investigated. In the present study, the effects of p-methoxyphenol on the direct-acting carcinogen, beta-propiolactone (BPL), were determined. p-Methoxyphenol administered at 1 or 4 hr prior to BPL or fed in the diet markedly inhibited BPL-induced neoplasia of the mouse forestomach. Of 10 phenols tested by p.o. intubation, it was the only one that exerted a significant inhibitory activity. Thus far, p-methoxyphenol appears to be an effective inhibitor only when given prior to carcinogen administration. During these studies, it was found that the nature of the diet markedly altered the neoplastic response of the mouse forestomach to BPL but not to benzo(a)pyrene.

Inhibitory effects of phenolic compounds on benzo(a)pyrene-induced neoplasia.

The inhibitory effects of 18 synthetic phenolic compounds added to the diet on benzo(a)pyrene-induced neoplasia of the forestomach of female ICR/Ha mice have been determined. Seven of the compounds showed suppression of neoplasia. The most potent inhibitors were p-methoxyphenol, 2-tert-butyl-4-hydroxyanisole [the minor isomer of 2(3)-tert-butyl-4-hydroxyanisole] and 3,5-di-tert-butylcatechol. A second group of compounds with a weaker inhibitory activity consisted of 3,5-di-tert-butylphenol, 3-tert-butyl-4-hydroxyanisole [the major isomer of 2(3)-tert-butyl-4-hydroxyanisole], 2-tert-butylhydroquinone, and 2-tert-butylphenol. In additional experiments, three naturally occurring phenolic derivatives of cinnamic acid, i.e., o-hydroxycinnamic acid, 3,4-dihydroxycinnamic acid (caffeic acid), and 4-hydroxy-3-methyoxycinnamic acid (ferulic acid), were investigated. All three suppressed benzo(a)pyrene-induced neoplasia of the forestomach. Humans ingest a variety of phenols. Data as to the inhibitory capacities of members of this group of compounds are of importance for evaluating the role that they play in determining the reaction to exposure to chemical carcinogens.

Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by sodium cyanate.

The effects of sodium cyanate on the occurrence of 7,12-dimethylbenz(a)anthracene- and benzo(a)pyrene-induced neoplasia have been studied in rodents. Sodium cyanate added to the diet for 8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibits 7,12-dimethylbenz(a)anthracene-induced mammary neoplasia in female Sprague-Dawley rats. Likewise, sodium cyanate in the diet inhibits the occurrence of benzo(a)pyrene-induced neoplasia of the forestomach and lung of ICR/Ha mice. Inhibition of benzo(a)pyrene-induced pulmonary neoplasia in A/J mice also is brought about by this compound. The identification of sodium cyanate as an inhibitor of chemical carcinogenesis adds a new chemical structure to those found previously to have inhibitory properties.

Inhibition of benzo(a)pyrene-induced transformation of C3H/10T1/2 cells by allylisopropylacetamide and isopropylvaleramide.

Allylisopropylacetamide (AIA) and isopropylvaleramide (IVA) have been demonstrated previously to protect in vivo against the acute toxicity and adrenal necrotic effect of 7,12-dimethylbenz(a)anthracene. In the present study, the influence of these two amides on the in vitro transforming ability of two potent carcinogens, benzo(a)pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene, on C3H10T1/2 cells was investigated. Both AIA and IVA showed a dose-dependent inhibition of B(a)P-induced transformation of C3H10T1/2 cells when added simultaneously for 24 hr with the carcinogen. While pretreatment, simultaneous treatment, and posttreatment of the cells with AIA or IVA inhibited transformation, the 24-hr posttreatment was somewhat more effective. The protective effect did not appear to results from alterations in B(a)P metabolism inasmuch as aryl hydrocarbon hydroxylase activity and the metabolic products of B(a)P detected by high-pressure liquid chromatography were not changed by AIA or IVA treatment. Furthermore, AIA and IVA did not selectively kill chemically transformed C3H10T1/2 cells, as indicated by the absence of their effect on an established, chemically transformed cell line. AIA and IVA also inhibited 7,12-dimethylbenz(a)anthracene-induced transformation of C3H10T1/2 cells. These data suggest that AIA and IVA may be useful protective agents and that they presumably exert their protective effect at some stage between the activation of the carcinogen and the expression of the transformed phenotype.

Inhibitory effect of 3-hydroxybenzo(a)pyrene on the mutagenicity and tumorigenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The 12 isomeric phenols of benzo(a)pyrene were tested for their ability to inhibit the mutagenic activity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [B(a)P 7,8-diol-9,10-epoxide-2], an ultimate mutagenic and carcinogenic metabolite of benzo(a)pyrene. 3-Hydroxybenzo(a)pyrene [3-HO-B(a)P], a major metabolite of benzo(a)pyrene, was the most potent antagonist tested. Approximately 3 nmol of 3-HO-B(a)P, 14 nmol of 10-HO-B(a)P, and 5-8 nmol of 1-, 2-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, and 12-HO-B(a)P inhibited the mutagenic activity of 0.05 nmol of B(a)P 7,8-diol-9,10-epoxide-2 by 50% in Salmonella typhimurium strain TA 100. The importance of the phenolic group for antimutagenic activity was indicated by the lack of antimutagenic activity of benzo(a)pyrene itself. 3-HO-B(a)P also inhibited the mutagenic activity resulting from the metabolic activation of benzo(a)pyrene and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver microsomes. This inhibition may have resulted from an effect of 3-HO-B(a)P on the metabolic activation of these carcinogens and/or from a direct effect on the action of B(a)P 7,8-diol-9,10-epoxide-2. In a mammalian cell culture system utilizing Chinese hamster V79 cells, 3-HO-B(a)P (8 microM) inhibited the mutagenicity of B(a)P 7,8-diol-9,10-epoxide-2 (0.2 microM) by 50%. Although 3-HO-B(a)P was a potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo(a)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene in S. typhimurium strain TA 100, higher concentrations of 3-HO-B(a)P were needed to inhibit the mutagenicity of the chemically less reactive benzo(a)pyrene 4,5-oxide and the bay-region diol epoxides of benz(a)anthracene, chrysene, and benzo(c)phenanthrene. Both 3-HO-B(a)P and 10-HO-B(a)P accelerated the disappearance of B(a)P 7,8-diol-9,10-epoxide-2 from 1:9 dioxane-water solutions at pH 7 and 25 degrees C. 3-HO-B(a)P, the most effective antimutagen of the B(a)P phenols tested, was much more reactive with the diol epoxide than 10-HO-B(a)P, the least effective antimutagen. The rate constant for the reaction of 3-HO-B(a)P with the diol epoxide exhibited a nonlinear (greater than first-order) dependence on the concentration of the phenol. Evidence was obtained for covalent adduct formation between the diol epoxide and each of the two phenols.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative studies of the toxicity of benzo(a)pyrene to a mouse liver epithelial cell strain in culture.

The toxic effects of the carcinogen benzo(a)pyrene (BaP) were studied in a well-characterized epithelial cell strain NMuLi, derived from the livers of weanling Namru mice. These cells were extremely susceptible to the toxicity, 99% dying after a 6-day exposure to BaP, 5mug/ml. The toxic effects began between 11 and 24 hr postapplication of BaP to the cells and increased exponentially with the time of treatment. The toxicity was concentration dependent in cells treated for a specific time period. The survival curves were exponential and extrapolated to a survival fraction of 1.0. The toxic effects of BaP to logarithmically growing NMuLi were inhibited 40% by 7,8 benzoflavone, and the inhibition was concentration dependent. The 7,8-benzoflavone also inhibited aryl hydrocarbon hydroxylase (AHH) from NMuLi cell homogenates and microsomes by 99%. The concentration dependence for AHH inhibition by 7,8-benzoflavone paralleled its inhibition of cellular toxicity. The toxicity of BaP to these cells increased exponentially with the number of population doublings. Hence, the toxicity was 130 times greater in exponentially growing cells than in confluent cells. Levels of AHH, the enzyme that metabolizes BaP to its cytotoxic derivatives, were only 2.4 times higher in exponentially growing than in confluent cells, suggesting that cell division was responsible for the large differential toxicity. In addition, a toxic BaP metabolite was preferentially toxic to log-phase cells. The results indicate that the metabolism of BaP by AHH to produce cytotoxic metabolites, which may cause lesions that are expressed upon cell division, is responsible for the cytotoxicity of BaP to NMuLi.

The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase.

Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol.

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.

Synthesis and chemical carcinogen inhibitory activity of 2-tert-butyl-4-hydroxyanisole.

The title compound 1 was selectively synthesized in its pure isomeric form by means of the hydroxyl-protecting reagent dimethyl-tert-butylchlorosilane. Exclusive silylation occurred at the less hindered hydroxyl group of 3. Dimethyl sulfate methylation of 4 gave 5 in excellent yield. Compound 1 was then obtained by acid hydrolysis of 5. The two BHA isomers, 1 and 2, were tested on their inhibitory effects toward benzo[alpha]pyrene-induced neoplasia in the forestomach of the ICR/Ha mouse. Both isomers, when added to the diet, reduced the number of mice with tumors and the number of tumors per mouse. Isomer 1, which has the less hindered free hydroxyl group, showed higher inhibitory effect in the present experimental model.

Carcinogenic effects of intracolonic benzo[a]pyrene in beta-naphthoflavone-induced 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.

Benzo[alpha]pyrene antibody inhibition of benzo[alpha]pyrene-induced mutageneis.

An antibody to benzo[alpha]pyrene (BP) was prepared. The isolated antibody showed a specificity for BP and a low reactivity with another carcinogenic hydrocarbon, 7,12-dimethylbenz[alpha]anthracene (DMBA). The BP-antibody inhibited the in vitro cytotoxic and mutagenic activity of BP in both a rat embryo fibroblast- and a rat lung cell-mediated mutagenesis system. A possible correlation of these in vitro findings to the in vivo carcinogenesis situation is discussed.