Benzo(a)pyrene free-radicals formation in the presence of butylated hydroxyanisole and their possible importance in carcinogenesis.

By the method of UV irradiation of benzopyrene in the presence of butylated hydroxyanisole, the benzopyrene EPR spectra was investigated and the possible mechanism by which the carcinogenic activity of benzopyrene in the presence of butylated hydroxyanisole is lowered, was studied.     

Inhibitory effects of RRR-alpha-tocopheryl succinate on benzo(a)pyrene (B(a)P)-induced forestomach carcinogenesis in female mice

AIM: To study the inhibitory effects of VES (RRR-alpha-tocopheryl Succinate, VES),a derivative of natural Vitamin E, on benzo(a)pyrene(B(a)P)-induced forestomach tumor in female mice. METHODS: The model of B(a)P-induced forestomach tumor was established according to the methods of Wattenberg with slight modify-cations.One hundred and eighty female mice (6 weeks old) were divided into six groups equally; negative control (Succinic acid), vehicle control (Succinate+B(a)P),positive control(B(a)P), high VES(2.5 g/kg.b.w+B(a)P), low VES(1.25 g/kg.b.w+B(a)P)ig as well as VES by ip (20 mg/kg.b.w+B(a)P). Except the negative control group, the mice were administrated with B(a)P ig. and corresponding treatments for 4 weeks to study the anti-carcinogenetic effect of VES during the initiation period. The experiment lasted 29 weeks, in which the inhibitory effects of VES both on tumor incidence and tumor size were tested. RESULTS: The models of B(a)P-induced forestomach tumor in female mice were established successfully. Some were cauliflower-like, others looked like papilla, even a few were formed into the ulcer cavities.VES at 1.25 g/kg.b.w, 2.5 g/kg.b.w. by ig and 20 mg/kg.b.w. via ip could decrease the number of tumors per mouse (1.7 plus minus 0.41, 1.6 plus minus 0.34 and 1.1 +/- 0.43), being lower than that of B(a)P group (5.4 +/- 0.32, P<0.05). The tumor incidence was inhibited by 18.2%, 23.1% and 50.0%. VES at 1.25 g/kg.b.w., 2.5 g/kg.b.w. by ig and 20 mg/kg.b.w. via ip reduced the total volume of tumors per mouse (54.8 +/- 8.84, 28.4 8 +/- 8.32 and 23.9 8 +/- 16.05), being significantly lower than that of B(a)P group (150.2 8 +/- 20.93, P < 0.01). The inhibitory rates were 63.5%, 81.1% and 84.1%, respectively. CONCLUSION: VES has inhibitory effects on B(a)P-induced forestomach carcinogenesis in female mice, especially by ip and it may be a potential anti-cancer agent in vivo.     

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.     

Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells.

The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.     

Dose-dependent effect of trichloropropene oxide on benzo[a]pyrene carcinogenesis

Summary The epoxide hydratase inhibitor, 1,1,1-trichloro-2,3-propene oxide (TCPO) in combination with benzo[a]pyrene (B[a]P) was injected s.c. in ddN mice. The formation of fibrosarcoma by B[a]P was slightly accelerated at low dose of TCPO, and remarkably inhibited at high dose of TCPO. The correlation of carcinogenesis with B[a]P metabolism was discussed.This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture     

Effects of the antioxidant butylated hydroxytoluene (BHT) on mortality in BALB/c mice.

Butylated hydroxytoluene (BHT) was given in the feed to determine its effect on life span in genetically well-defined, barrier-derived BALB/c mice. Both sexes received 0.75% BHT for three different treatment periods: (A) 8 to 11 weeks of age; (B) for life, beginning at 11 weeks; (C) for life, beginning at 8 weeks of age. The control group (D) was untreated. All BHT treatment groups had mean survival times which exceeded that of controls. The order of survival was B greater than C greater than A greater than D (Males: 890, 832, 726, 684 days; Females: 875, 798, 759, 701 days). Most of the increases in mean survival time were related to a reduction in early deaths (350--600 days) in BHT-treated mice. The reason for the life-lengthening effect on BHT was not identified, but it may relate to alterations in specific disease incidences.     

Alteration of brain catecholamines during growth of benzo(a)pyrene induced murine fibrosarcoma.

Brain catecholamines (CA) were studied in discrete brain areas of benzo(a)pyrene (b(a)p) induced fibrosarcoma bearing mice. Dopamine (DA) and norepinephrine (NE) levels decreased significantly in different brain areas especially in corpus striatum and hypothalamus with the tumor progression, indicating an inverse relationship between brain DA and NE levels and tumor growth. Since impaired hormonal and immunological functions are manifestation of systemic alteration during tumor growth, it appears that during malignant growth an alteration of these brain CA may play an important role in the regulation of systemic alterations.     

Inhibition of benzo(a)pyrene Carcinogenesis in rats with vitamin C

Summary The s.c. injection of 10 mg benzo(a)pyrene dissolved in 1 ml tricaprylin induced in Wistar rats local malignant tumors, such as fibrosarcoma, rhabdomyosarcoma, and polymorph cell sarcoma. The growth of the tumors was relatively rapid, reaching weights of 140–155 g before rats died 142–168 days after the administration of the carcinogen. On the contrary, under the same experimental conditions, high doses of Vitamin C about 525 mg/day/rat administered orally in drinking water (total amount of Vitamin C 88 g/rat corresponding to 40% of their body weight) inhibited to a great extent the benzo(a)-pyrene carcinogenesis. Only one slowly growing rhabdomyosarcoma (13 g of weight) was developed showing characteristic damage of malignant cells and partial replacement of the neoplastic area with granuloma tissue. The significance of Vitamin C for cancer prevention and treatment is discussed.     

Acute hematotoxicity of oral benzo(a)pyrene: the role of the Ah locus

Our results show a marked acute hematotoxicity of oral benzo(a)pyrene (BaP) in D2 mice as well as the extreme resistance of BDF1 individuals to bone marrow toxicity induced by oral BaP. Continued oral BaP produced severe bone marrow depression in D2 mice affecting all myelopoietic lineages, but produced only moderate bone marrow depression in BDF1 mice affecting erythropoiesis only. Pluripotent hematopoietic stem cells were almost completely destroyed in D2 individuals, but only reduced to approximately 40% in BDF1 individuals after 7 days of BaP. D2 mice were killed by 13 days of continued oral BaP, but BDF1 mice were still alive and in good condition even after 19 days of continued oral BaP. Analysis of the bone marrow and peripheral blood changes showed that severe toxic chemical bone marrow depression in D2 mice by continued oral BaP cannot serve as an experimental model system of acute aplastic anemia.     

In vivo DNA adduct formation by benzo(a)pyrene in mouse and rat epidermal and dermal fibroblasts after topical application of an initiating dose of benzo(a)pyrene

In vivo adduct formation by benzo[a]pyrene (BP) has been compared in mouse and rat epidermal keratinocytes and dermal fibroblasts after topical application of an initiating dose of carcinogen. The BP-DNA adducts were analyzed by chromatography and acid hydrolysis of BP-deoxyribonucleoside adducts to BP-tetrols. BP was dissolved in acetone and applied, at similar doses per unit area (100 nmol/mouse and 240 nmol/rat), to 50-day-old Swiss mice and 35-day-old Wistar rats. Epidermal and dermal cells were isolated twenty four hours later. Reverse-phase HPLC of BP-deoxyribonucleoside adducts demonstrated the presence of three BP-deoxyribonucleosides adducts in mouse epidermal cells and one in mouse dermal cells. An unknown product (0.13 and 0.04 pmol/mg mouse epidermal and dermal cell DNA respectively) eluted before the BP-7,10/8,9-tetrol marker, at same relative position as 9-OH-BP-DNA adduct. The major adduct formed in mouse epidermal keratinocytes and dermal fibroblasts was dGuo modified by (+)-anti-BPDE and accounted for more than 70% of the adducts. Acid hydrolysis of the individual BP-DNA adducts was used to identify the BP-DNA adducts formed in mouse epidermal and dermal cells as anti- and syn-BPDE-dGuo. Twenty four hours after topical application of BP, the total levels of modified deoxyribonucleosides and (+)-BPDE-dGuo were 3 times greater in mouse epidermal cells than in dermal cells. The ratios of anti-BPDE to syn-BPDE was 17:1 and 12:1 in mouse epidermal and dermal cells DNA, respectively. This work provides the evidence that, at an initiating dose, 3H modified deoxyribonucleosides of rat epidermal keratinocytes and dermal fibroblasts are not detectable. This may be essential for the resistance of rat skin to the carcinogenic action of benzo[a]pyrene.     

Enzymatic conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene through a single enantiomer of r-7, t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene.

Benzo(a)pyrene is metabolically and stereospecifically converted by mixed-function oxidases of rat liver microsomes and epoxide hydratase (glycol hydro-lyase (epoxide-forming), EC 4.2.1.63)to the single enantiomer (-)r-7,t-8-dihydroxy-7,8-dihydrobenzol (A) pyrene. This enantiomer is further metabolized stereoselectively by the mixed-function oxidases to predominantly the diol-epoxide, r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzol(a)pyrene in which the 7-hydroxyl and the 9,10-epoxide are trans. Other unidentified metabolites are also formed from the r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Racemic r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)-pyrene is converted metabolically to both r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The diol-epoxides are unstable in aqueous medium, and their identification and characterization as r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene were accomplished by the identity of their tetrahydroxytetrahydrobenzo(a)pyrenes hydrolysis products with those of the authentic synthetic compounds with respect to mobility on high-pressure liquid chromatography and mass and ultraviolet absorption spectral analysis. The diol-epoxides were also reduced in the presence of NADPH to distinct trihydroxypentahydrobenzo(a)pyrenes. Since the synthetic racemic r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene is very highly mutagenic in mammalian cells, we suggest that it is the metabolically formed diol-epoxide that may be an ultimate carcinogenic form of benzo(a)pyrene.     

Transplacental and direct action of benzo(a)pyrene studied in organ cultures of embryonic lung tissue.

Transplacental effect of benzo(a)pyrene was studied in organ cultures of embryonic lung tissue explanted from mouse donors injected by the carcinogen. Hyperplastic alteration of epithelium, followed by adenomatous changes were seen in embryonic lung tissue cultures from donor mice injected by benzo(a)pyrene. No alteration was seen in control cultures in which the mice were injected by a non-carcinogenic hydrocarbon (pyrene). Besides the blastomogenic action, a growth-promoting effect of the benzo(a)pyrene was observed in the particular organ cultures of the embryonic lung tissue.     

(+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo (a)pyrene: a potent skin carcinogen when applied topically to mice.

(+/-)-trans-7,8-Dihydroxy-7,8-dihydrobenzo[a]-pyrene, a known metabolite of benzo [a]pyrene, has been tested for carcinogenic activity on mouse skin by topical application of 0.15 or 0.30 mumol every 2 weeks for 60 weeks. At the low dose (0.15 mumol), the compound was equipotent to the parent hydrocarbon, benzo[a]pyrene, and considerably more potent than its metabolic precursor, benzo[a]pyrene 7,8-oxide, in eliciting tumors, as determined by both the onset of tumors and the total number of animals developing carcinomas. Application of 7,8-epoxy-,8,9,10-tetrahydrobenzo[a]pyrene (0.30 mumol every 2 weeks), a compound related to the carcinogenic benzo[a]pyrene 7,8-oxide but with the double bond removed from the 9,10-position of the molecule, did not elicit any tumors. The above results indicate that the (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene is a more proximate carcinogen than benzo[a]pyrene 7,8-oxide and that the carcinogenicity of benzo[a]pyrene 7,8-oxide and (+/-)trans-7,8-digydrobenzo[a]pyrene may be due to metabolic conversion of these compounds to the highly reactive and mutagenic stereoisomers of 7,8-digydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.     

Identification of the major adducts formed by reaction of benzo(a)pyrene diol epoxide with DNA in vitro.

Covalent binding of the benzo[a]pyrene metabolite (+/-)7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[a]pyrene diol epoxide) and exocyclic amino groups of the respective purines.     

Inhibitory effect of small amounts of cellulose on colonic carcinogenesis with low-dose carcinogen

This study set out to evaluate the effects of dietary fiber on cancer development in the large bowel under in vivo experimental conditions as similar as possible to those under which this cancer develops in vivo in humans. Forty-eight Sprague-Dawley rats were divided into three groups that were fed either a nonfiber diet or a 3 g or 10 g/100 g cellulose diet in this experiment, and all groups received doses of a mild carcinogen, 1,2-dimethylhydrazine (5 mg/kg body weight) for 50 weeks. Following endoscopic observation of the large bowel, we found that the induction rates of tumor in the cellulose groups were significantly lower than that in the nonfiber diet group, both endoscopically and histologically. No differences were seen between the 3% and 10% cellulose groups in suppressing carcinogenesis. It is likely that the inhibitory effect of 3% cellulose could be confirmed only by a long-term experiment on carcinogenesis following the administration of a low dose of carcinogen.     

In vivo effect of piperine on serum and tissue glycoprotein levels in benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice

In recent years, considerable emphasis has been focused on identifying new cancer chemopreventive agents, which could be useful for the human population. Piperine is a pure, pungent alkaloid constituent of black and long peppers (Piper nigrum and Piper longum), that acts as an antioxidant and anticancer agent by its numerous macromolecules associated with them. In the present study, piperine was found to suppress benzo(a)pyrene (B(a)p) induced lung cancer in Swiss albino mice. In lung cancer bearing mice, altered levels of total protein and protein bound carbohydrate components (hexose, hexosamine and sialic acid) were observed in serum, lung and liver tissues. Dietary supplementation of piperine (50 mg/kg body weight) to B(a)p administered animals decreased the total protein and protein bound carbohydrate levels of lung cancer bearing animals in during initiation and post-initiation phases. Our data suggest that piperine may extend its chemopreventive effect through modulating the protein bound carbohydrate levels, as they are one of the indicators of tumorigenesis.     

Position-specific oxygenation of benzo(a)pyrene by different forms of purified cytochrome P-450 from rabbit liver.

High-pressure liquid chromatography was used to detect oxygenated products of benzo[a]pyrene formed in a reconstituted microsomal mixed-function oxidase system containing cytochrome P-450 (P-450LM), phospholipid, and NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4). Three cytochrome fractions purified from a single source, hepatic microsomes from phenobarbital-treated rabbits, were studied; the various forms of the cytochrome are designated by their relative electrophoretic mobilities. The total benzo[a]pyrene oxygenation rate was greatest for P-450LM1,7, intermediate for P-450LM2, and least for P-450LM4. The phenolic products were eluted in two peaks, A and B, that contained primarily 9-hydroxy- and 3-hydroxybenzo[a]pyrene, respectively. The ratio of peak A to peak B phenols was 0.11 for P-450LM2 and 0.45 for P-450LM4. Thus, the relative amounts of the various phenols formed by these two cytochrome fractions differ markedly. The positional specificity of the hydroxylation is also indicated by large differences in the fluorescence spectra of the phenolic products formed by the two cytochromes. P-450LM2 and P-450LM4 did not form benzo[a]pyrene dihydrodiols, thereby showing that benzo[a]pyrene oxide hydratase activity was absent from these purified preparations. Ninety percent of the phenols formed by P-450LM1,7 were eluted in peak B; the metabolites produced by this preparation also included dihydrodiols, thus indicating the presence of hydratase activity. The positional specificities of different forms of cytochrome P-450 may channel polycyclic aromatic hydrocarbon metabolism into the various activation and detoxification pathways and thereby help determine the cytotoxic and carcinogenic activity of these compounds.