The effect of wheat sprout extract on benzo(a)pyrene and 7,2-dimethylbenz(a)anthracene activity.

Subcutaneous application of aqueous wheat sprout extract to mice resulted in a slight decrease of the ability of fraction S-9 from their skin to activate DMBA to metabolites mutagenic for S. typhimurium TA 98. Induction by benzo(a)pyrene of sperm abnormalities in mice was diminished after oral administration of the wheat sprout extract; however, even high doses of the extract did not completely abolish the effect of benzo(a)pyrene on spermatozoa. In the carcinogenicity studies, the wheat sprout extract, when applied to mouse skin during the initiation phase, enhanced fourfold the induction of papillomas by DMBA and shortened the period of latency from 9 to 5 weeks.

Nonmutagenicity of betel leaf and its antimutagenic action against environmental mutagens.

Betel leaf (Piper betel) water and acetone extract are nonmutagenic in S. typhimurium strains with and without S9 mix. Both the extracts suppress the mutagenicity of betel quid mutagens in a dose dependent manner. Moreover both the extracts of betel leaf reduce the mutagenicity of benzo(a)pyrene and dimethylbenzanthracene. Acetone extract is more potent than water extract in inhibiting mutagenicity of environmental mutagens.

Inhibition of the mutagenicity and metabolism of 6-methyl-benzo[a]pyrene and 6-hydroxymethyl-benzo[a]pyrene.

Previously reported inhibitors of benzo[a]pyrene (BaP) mutagenicity in Salmonella typhimurium strain TA98 were tested for their effectiveness against the mutagenicity of 6-methyl-benzo[a]pyrene (6-CH3-BaP), 6-hydroxymethyl-benzo[a]pyrene (6-CH2OH-BaP) and 6-acetoxymethyl-benzo[a]pyrene (6-CH3COOCH2-BaP). Dose-response curves obtained for phenothiazine (PTH), 2-chlorophenothiazine (2Cl-PTH), phenylisothiocyanate (PHN), phenethylisothiocyanate (PNE), trans-retinol (TR) and disulfiram (TETD) showed a variety of degrees of inhibition of mutagenicity. Additionally, glutathione (GSH) was found to inhibit the mutagenicity of 6-CH3COOCH2-BaP, and the mutagenicity of 6-CH2OH-BaP was enhanced by the addition of supplemental ATP, Na2SO4 and EDTA. Only 2Cl-PTH was equally as good an inhibitor of 6-CH3-BaP and BaP, reducing revertant colonies to less than 50% of control at 10 X BaP concentration. To probe the mechanism of inhibition, the effect of 2Cl-PTH on the binding of BaP and the 6-substituted benzo[a]pyrenes to cytochrome P-450 was investigated by difference spectroscopy. Also, the effect of 2Cl-PTH on the subsequent metabolism of 6-CH3-BaP and 6-CH2OH-BaP was investigated by rapid scan difference spectroscopy and high-performance liquid chromatographic separation of products. The results are consistent with a major mechanism of inhibition for 2Cl-PTH involving a competition for the cytochrome P-450 binding site.

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)

High-density lipoproteins decrease both DNA binding and mutagenicity of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in V79 Chinese hamster cells.

The effects of separate lipoproteins or of serum with high or low lipoprotein concentrations on formation of lipophilic carcinogen adducts with DNA and on mutagenicity of the carcinogen was investigated using V79 Chinese hamster lung cells. Binding of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to DNA and BPDE induction of 6-thioguanine (6-TG)-resistant mutants in V79 cells was significantly lower after 1 or 4 h when the medium was supplemented with purified HDL, and was lower after 1 h but not 4 h when the medium was supplemented with serum containing a high concentration of mixed lipoproteins (LP). Cells grown in medium without serum or LP supplementation exhibited the highest levels of both BPDE-DNA adduct formation and mutagenesis after 1 h. At 1 h, cells exposed to BPDE in LDL-supplemented medium showed decreased adduct formation and mutagenesis when compared to cells treated with BPDE in PBS-supplemented medium. After 4 h, cells treated with BPDE in LDL-supplemented medium gave the highest levels of adduct formation and the highest mutation frequency. These results suggest that both LDL and HDL effectively decrease the concentration of BPDE available to V79 cells exposed to the mutagen for short periods of time, resulting in decreased interaction of BPDE with DNA and decreased BPDE-associated mutagenesis, but that both BPDE-DNA adduct formation and mutagenesis increased as a function of increased exposure time in the presence of LDL. The results suggest that LDL, but not HDL, uptake by adsorptive endocytosis may be associated with potentiated entry of BPDE into V79 cells as a function of time.

Quantitative microspectrofluorimetry study of the "blocking effect" of 6-aminochrysene on benzo[a]pyrene metabolism using single living cells.

By microspectrofluorimetry on single living cells (murine fibroblasts 3T3), we have obtained monoexponential decreases of fluorescence intensity for benzo[a]pyrene (B[a]P) and 6-aminochrysene (6a-chrysene) metabolism. These kinetics are characteristics of B[a]P and 6a-chrysene metabolism and histograms can be drawn from the rate constants. We have studied the influence of 6a-chrysene on B[alpha]P metabolism. Using different methods of incubation, it has been observed that the presence of 6a-chrysene leads to modifications of the histogram profiles during B[a]P metabolism. Polycyclic aromatic hydrocarbons (PAH) are used to induce B[a]P metabolism. Whatever the experimental conditions we never detected such a phenomenon with 6a-chrysene. On the contrary we have observed an inhibition of B[a]P metabolism by 6a-chrysene, which can reach 80% of the aryl hydrocarbon hydroxylase (AHH) activity when 6a-chrysene remains constant in the cells. Compared with the results previously observed "in vitro" which presented 50% mean inhibition) we show that inhibition acts in an all-or-nothing mechanism at the cellular level.

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.

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.

Dietary carotenoids block photocarcinogenic enhancement by benzo (a)pyrene and inhibit its carcinogenesis in the dark.

The carotenoids beta-carotene (C) and canthaxanthine (CX), with and without pro-vitamin A activity, respectively, when perorally administered to mice, markedly prevent benzo(a)pyrene photocarcinogenic enhancement (BP-PCE), continue to block such BP-PCE and protect significantly against BP carcinogenesis in mice maintained in the dark. These results appear relevant to both the pathogenesis of chemical carcinogenesis and rational programs of skin cancer prevention in humans.

Anti-mutagenic chalcones: antagonizing the mutagenicity of benzo(a)pyrene on Salmonella typhimurium.

Several chalcone derivatives; e.g. Ro 09-0204, Ro 09-0323 and Ro 09-0501 were found to reduce markedly the revertant increase of Salmonella typhimurium TA100 by benzo(a)pyrene during the incubation with S-9 Mix. The antimutagenic activity was 100 - 700 times stronger than that of L-ascorbic acid. Effect on other mutagens, the structure activity relationship and the possible mechanism of action are briefly discussed.

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.

Prevention and reversal by a retinoid of 3,4-benzpyrene- and cigarette smoke condensate-induced hyperplasia and metaplasia of rodent respiratory epithelia in organ culture.

The influence of an aromatic analog of vitamin A, etretinate, on the effects of 3,4-benzpyrene and cigarette smoke condensate has been investigated in fetal mouse lung and neonatal rat tracheas grown in organ culture. In both tissues, 3,4-benzpyrene as well as cigarette smoke condensate induces a striking increase of epithelial mitosis within 12--14 days of treatment. The increase is associated with a loss of secretory activity and of ciliary function. These changes persist in the absence of benzpyrene or smoke condensate in explants transferred to control medium. Treatment with etretinate alone does not affect the normal epithelial growth rate or normal differentiation. If combined with either benzpyrene or smoke condensate, the aromatic compound inhibits the increase in cell division and prevents the loss of secretory activity or ciliary function. In explants pretreated with 3,4-benzpyrene or cigarette smoke condensate, etretinate reduces the carcinogen- or smoke condensate-induced increase in mitotic activity to normal levels and restores secretory differentiation and ciliary function. The mechanism of action involved in the anticarcinogenic activity of the retinoid is discussed.

Screening of antioxidants and other compounds for antimutagenic properties towards benzo[a]pyrene-induced mutagenicity in strain TA98 of Salmonella typhimurium.

Antioxidants and several other compounds, some of which are known to inhibit carcinogenicity, have been screened for their effectiveness as inhibitors of benzo[a]pyrene (BP) mutagenicity towards Salmonella typhimurium strain TA98 in the Ames test. A total of 32 compounds were tested. In the assay, metabolic activation of BP (8.2 nmoles/plate) was mediated by the S9 fraction from beta-naphthoflavone-induced rat livers. Among compounds which are known to inhibit carcinogenicity, retinol, phenothiazine, disulfiram, phenethylisothiocyanate and phenylisothiocyanate were the most effective inhibitors of BP mutagenicity, being effective at equimolar concentrations. Several other compounds showed inhibition at higher concentrations of antioxidant and the remainder showed little or no inhibition. Dose-response curves have been obtained for the 17 most active compounds. No general pattern of inhibition is obvious from our studies, inhibitors are not drawn ;from any single class of compounds, nor does a particular compound necessarily appear to inhibit more than one mutagen.

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 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.

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.

Inhibition of mutagenesis in Chinese hamster V-79 cells by antioxidants.

The mutagenicity of benzo(a)pyrene (BP) on Chinese hamster V-79 cells cocultivated with X-irradiated hamster embryo cells was inhibited by phenolic antioxidants, such as tert-butyl-4-hydroxyanisole (BHA) and butyl-hydroxytoluene (BHT), but not by disulfiram and its related compounds such as tetraethylthiuram disulfide, diethyldithiocarbamic acid and dimethyldithiocarbamic acid. The mutagenicity of BP on V-79 cells was reduced 44% by BHA and 25% by BHT. BHA inhibited BP-induced mutagenesis, but not N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF)-induced mutagenesis. BHA is suggested to inhibit the mutagenic action of BP by altering its metabolism.

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.