Dietary N-acetyl-L-cysteine modulates benzo[a]pyrene-induced skin tumors in cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) mice

Epidemiologic studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence from clinical trials and laboratory data suggest that in some cases individual antioxidant supplements may actually exacerbate carcinogenesis. Our goal was to explore these paradoxical activities in a rodent model that possesses genotypic characteristics of human cancers. We selected the p53 haploinsufficient Tg.AC (v-Ha-ras) mouse as a model, because it contains an activated, carcinogeninducible ras oncogene and an inactivated p53 tumor suppressor gene, which are frequent genetic alterations in human cancers. These mice develop chemically induced benign and malignant skin tumors rapidly which can easily be quantified. Mice were fed basal diets with or without 3% N-acetyl-L-cysteine (NAC), a well-recognized antioxidant, prior to, during and after topical application of the carcinogen benzo[a]pyrene (64 microg/mouse) applied twice per week for 7 weeks. Tumor incidence exceeded 90% for both groups, and NAC did not reduce tumor latency. Mice fed NAC displayed a 43% reduction (P < 0.05) in tumor multiplicity and delayed the appearance of lesions (P < 0.05). Dietary NAC also significantly (P < 0.05) improved group survival by 5 weeks. Total tumor yields were reduced in both dietary groups but malignant spindle cell tumors (SCT) increased by 25% in NAC-fed mice. The v-Ha-ras oncogene and p53 protein products were clearly co-expressed in both benign and malignant lesions from both dietary groups. In summary, dietary supplementation with NAC was chemopreventive, but the marginal increase in SCT suggests a paradoxical effect.     

Dietary fibre, fat and beef modulation of colonic nuclear aberrations and microcapsule-trapped gastrointestinal metabolites of benzo[a]pyrene-treated C57/B6 mice consuming human diets.

Male C57/B6 mice were adapted to human diets of British origin that had 3-fold differences in either dietary fibre, fat or beef protein within the normal human range, and were then treated p.o. with 200 mg/kg benzo[a]pyrene (B[a]P) to induce colonic nuclear aberrations. [14C]B[a]P was included in the dose that followed 2 h after a gavage of magnetic PEI microcapsules. Untreated control groups were fed mouse chow or the baseline human diet which was low in all three dietary components (LLL). After the animals were killed at 24 h, large reductions (P less than 0.05) in colonic nuclear aberrations, and alterations to faecally excreted, microcapsule-trapped B[a]P metabolites were found for elevations of all three human diet components. Compared to untreated LLL control, B[a]P treatment gave an 8-fold increase in total nuclear aberrations, which was decreased 2- to 3-fold by increased fibre or fat. HPLC assay of B[a]P metabolites desorbed from microcapsules showed dietary fibre and beef protein to increase B[a]P diols and phenols but almost abolish B[a]P diones, consistent with a shift to enzymatic metabolism from non-specific oxidation. Increased fat considerably altered B[a]P metabolite disposition and microcapsule trapping, and comparison with microcapsules removed from colon contents indicated an altered enterohepatic circulation. Although it was not possible to attribute nuclear aberrations to individual B[a]P metabolites, a possible role of B[a]P diones seemed indicated, this being in line with previous microcapsule studies. These results show that microcapsules and human diets can be used in monitoring modulations of xenobiotic agents linked to mucosal chromosomal damage, with the eventual aim of human microcapsule biomonitoring.     

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.     

Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene.

The effects of topical administration of curcumin on the formation of benzo[a]pyrene (B[a]P)-DNA adducts and the tumorigenic activities of B[a]P and 7,12-dimethylbenz[a]anthracene (DMBA) in epidermis were evaluated in female CD-1 mice. Topical application of 3 or 10 mumol curcumin 5 min prior to the application of 20 nmol [3H]B[a]P inhibited the formation of [3H]B[a]P-DNA adducts in epidermis by 39 or 61% respectively. In a two-stage skin tumorigenesis model, topical application of 20 nmol B[a]P to the backs of mice once weekly for 10 weeks followed a week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks resulted in the formation of 7.1 skin tumors per mouse, and 100% of the mice had tumors. In a parallel group of mice, in which the animals were treated with 3 or 10 mumol curcumin 5 min prior to each application of B[a]P, the number of tumors per mouse was decreased by 58 or 62% respectively. The percentage of tumor-bearing mice was decreased by 18-25%. In an additional study, topical application of 3 or 10 mumol curcumin 5 min prior to each application of 2 nmol DMBA once weekly for 10 weeks followed a week later by promotion with 15 nmol TPA twice weekly for 15 weeks decreased the number of tumors per mouse by 37 or 41% respectively.     

Dietary benzo[a]pyrene intake and risk of colorectal adenoma.

We carried out a clinic-based case-control study specifically designed to address the hypothesis that dietary intake of polycyclic aromatic hydrocarbons (PAH) is associated with colorectal adenoma risk. We developed a food frequency questionnaire with detailed questions on meat-cooking methods and doneness levels and a benzo[a]pyrene (BaP) database (as a surrogate for total carcinogenic PAHs) based on the collection and analysis of a wide range of food samples. We estimated BaP intake derived from meat and from all foods to test its relationship with risk of colorectal adenomas. The median (10th and 90th percentiles) BaP intake in controls was 5 ng/d (0.2 and 66 ng/d) estimated from meat and 73 ng/d (35 and 140 ng/d) from all foods. In cases, median BaP intake was 17 ng/d (0.5 and 101 ng/d) from meat and 76 ng/d (44 and 163 ng/d) from all foods. Multivariate analysis was carried out on 146 cases and 228 controls. The odds ratios (95% confidence interval) for dietary BaP from meat with the first quintile as the reference group were 1.19 (0.51-2.80) for the second quintile, 1.71 (0.76-3.83) for the third quintile, 2.16 (0.96-4.86) for the fourth quintile, and 2.82 (1.24-6.43) for the fifth quintile (Ptrend=0.01). Increased risk of colorectal adenomas was more strongly associated with BaP intake estimated from all foods: 2.61 (1.08-6.29) for the second quintile, 4.21 (1.79-9.91) for the third quintile, 2.45 (0.98-6.12) for the fourth quintile, and 5.60 (2.20-14.20) for the fifth quintile (Ptrend=0.002). This study provides evidence that dietary BaP plays a role in colorectal adenoma etiology.     

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

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

Quantitation of benzo(a)pyrene metabolite: DNA adducts in selected hepatic and pulmonary cell types isolated from [3H]benzo(a)pyrene-treated rabbits

Benzo(a)pyrene metabolite: deoxyribonucleoside adducts were analyzed in hepatic and pulmonary cells isolated from rabbits 24 h after i.v. administration of [3H]BP (1 mg/kg; 50 mCi/kg). The major adduct in each of the cell types analyzed was (+)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine, but (+/-)-r-7, t-8-dihydroxy-c-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene: deoxyguanosine and very low levels of (-)-r-7, t-8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene -deoxyguanosine and an unidentified adduct were also observed. The level of the major adduct was similar in each of the isolated cell types and was at least as high in cells with very low cytochrome P-450-dependent monooxygenase activity (hepatic nonparenchymal cells and alveolar macrophages) as in those with higher activity (hepatocytes, alveolar type II cells, and Clara cells). The binding of benzo(a)pyrene metabolites to proteins was also determined, and again binding levels did not correlate with differences in cytochrome P-450 activity.     

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: II. Tissue specificity studies using comet assay.

Epidemiological data seem to be equivocal on the probable increase in cancer incidence in populations exposed to asbestos-fibre contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of oral asbestos exposure, the large surface area of the fibres, however, creates the possibility of cogenotoxic action with adsorbed water-borne organics. In our animal model, rats were gavaged with untreated UICC crocidolite and anthophyllite fibres and fibres that had been allowed to adsorb benzo[a]pyrene molecules from aqueous solutions. Peritoneal macrophages and intestine, parietal peritoneum and omentum samples were obtained from the animals after 24 h. The alkaline single-cell microgel electrophoresis assay (comet assay) was performed on cells isolated from the solid tissues. Tail moment was applied as a basis of evaluation following image analysis. Our results indicate high levels of DNA strand breaks in the cells prepared from the omentum and intestine. We could also demonstrate a significant potentiating effect of the adsorbed carcinogen on the induction of DNA damage in the omentum. The parietal peritoneum and macrophages were not involved in the early genotoxic alterations under study. Our results support the molecular model of asbestos cocarcinogenesis, including both asbestos-induced deletions and mutations caused by a mutagen carried by the same fibres.     

Topical treatment of mice with benzo[a]pyrene or parenteral administration of benzo[a]pyrene diol epoxide--DNA to rats results in faecal excretion of a putative benzo[a]pyrene diol epoxide--deoxyguanosine adduct

The administration of [³H]BPDE-DNA, whether by i.p. or i.v.injection, to male Wistar rats resulted in the majority of theradioactivity being recovered in the faeces. Excretion was rapid:within 24 h post-injection, 45% of the applied dose was recoveredin the faeces. H.p.l.c. analysis of radioactive material extractedfrom the faeces by methanol showed that it contained a singlecomponent which co-chromatographed with [³H]BPDE-dGuo and whichwas not affected by treatment with alkaline phosphatase, arylsulphatase or ß-glucuronidase. To determine if thisphenomenon occurs after topical application of BP to a targettissue, such as mouse skin, animals were treated with [³H]BPand their faeces collected. After an extensive extraction procedureinvolving differential solubility in organic solvents, SephadexLH-20 chromatography and h.p.l.c, a product was isolated frommice faeces which had characteristics consistent with a [³H]BPDE-dGuoadduct. These findings are discussed in relation to detectionof BPDE adducts in human populations.     

The effects of dietary corn oil on the metabolism and activation of benzo[a]pyrene by the benzo[a]pyrene metabolizing enzymes of the mouse

Male ICR Swiss mice, weighing 16 — 20 g, were fed ad libitumeither a fat-free diet or a diet containing 10% com oil. Afterthree weeks on these diets, the rates of benzo[a]pyrene (B[a]P)metabolite formation and metabolism to products which covalentlybind with macromolecules were compared using hepatic nucleiand microsomal preparations. The maximum activity of B[a]P hydroxylasehi microsomes from untreated animals was increased 50% by feedingthe com oil diet, however, B[a]P hydroxylase in microsomes from3-methylcholanthrene (3-MC)-treated mice was unaffected by diet.In animals treated with phenobarbital, B[a]P metabolism andB[a]P - DNA adduct formation were greater hi microsomes fromcorn oil fed mice compared to those fed the fat-free diet. Ata B[a]P concentration of 96 µM, microsomes from corn oilfed untreated mice produced 26% more extractable metabolitesand covalent binding to exogenous DNA was increased 46%. Atlower substrate concentrations (0.94–15.0 µM B[a]P),B[a]P-DNA and B[a]P -protein binding were 300 – 400% greaterwhen incubated with microsomes from corn oil fed mice than whenincubated with microsomes from mice fed fat-free diet. The apparentV_max's determined for the formation of each extractable metabolitewere increased 1.5 – 3.0 times by the corn oil diet. Hepaticnuclear B[a]P hydroxylase and nuclear activation of B[a]P toproducts which covalently bind to DNA in both non-induced and3-MC-pretreated animals fed the corn oil diet were greater thanthat observed in animals fed the fat-free diet. B[a]P hydroxylaseactivities in the lungs of these animals were unaltered by diet.     

Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice

The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.     

Dietary benzo[a]pyrene, alcohol drinking, and risk of breast cancer: a case-control study in Uruguay

In order to determine to the effect of benzo[a]pyrene (BaP) on breast cancer risk we conducted a case-control study in the time period 1996-2004. The study included 1,098 participants (460 cases and 638 controls). All the patients were drawn from the four major hospitals in Montevideo, Uruguay. Statistical analysis was performed using unconditional multiple logistic regression and the models included age, residence, urban/rural status, education, monthly income, body mass index, menopausal status, age at menarche, parity, smoking index, alcohol drinking, mate consumption, total energy, total vegetables and fruits, and BaP intake. The highest vs. the lowest quartile of BaP intake (OR 2.0, 95% CI 1.2-3.3) was significantly associated with breast cancer risk. Alcohol drinking was also directly associated with breast cancer risk (OR 1.63, 95% CI 1.19-2.23) and the joint effect of BaP and alcohol drinking showed an elevated risk of the disease (OR 3.32, 95% CI 2.17-5.06). The present study suggests that elevated consumption of BaP could play an important role in the etiology of breast cancer. This effect is enhanced by the intake of alcohol.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol in human mammary epithelial cells: feedback inhibition by 7-hydroxybenzo[a]pyrene

The metabolism of benzo[a]pyrene (B[a]P) and (-)-transbenzo[a]pyrene-7,8-dihydrodiol (B[a]P-diol) was compared in human mammary epithelial cells (HMEC) grown in serum-free medium, MCDB-170. Conversion of B[a]P-diol to the carcinogen (+)-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide (BPDE), as measured by analysis of their tetraol hydrolysis products, occurred much more efficiently in cultures incubated with [3H]B[a]P-diol than in cultures incubated with [3H]B[a]P. In cultures pretreated with unlabeled B[a]P (24 h, 400 nM), the conversion of [3H]-B[a]P-diol to [3H]tetraols is inhibited 49%, while the conversion of [3H]B[a]P to [3H]B[a]P-diol- is not affected. These observations led to the identification of a major B[a]P-derived metabolite as 7-hydroxybenzo[a]pyrene (B[a]P-7-ol), which was found to be an extremely potent and selective inhibitor of the conversion of B[a]P-diol to BPDE, with a KI estimated at 3-12 nM. Thus B[a]P activation in HMEC appears to be significantly limited by a feedback inhibition pathway induced by B[a]P-7-ol. The potency and selectivity of the B[a]P-7-ol-induced inhibition suggests that the diol to diolepoxide conversion is affected by a selective oxygenase in HMEC, rather than a non-enzymatic, peroxy radical-induced mechanism. B[a]P-7-ol should prove to be a valuable tool in the study of B[a]P carcinogenesis.     

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.     

Effect of temperature on benzo[a]pyrene-induced hyperplastic and neoplastic skin lesions in mice

The time required for tumor development and death in inbred C3H/HeJ mice treated with 1.0 or 0.1 mg benzo(a)pyrene (BP) twice weekly was shortest in mice kept in a cool environment (CE), intermediate in mice kept in a normal temperature environment (NE), and longest in mice kept in a warm environment (WE). Incidence of tumors (predominantly squamous carcinomas) and mortality was 100% by 36 weeks in all environments in the high-dose group. In the low-dose group, by 36 weeks tumor incidence was least in the WE compared with that in the NE and CE. Squamous carcinomas predominated in the NE and CE, but the incidence of fibromas and epithelial hyperplasias exceeded that of squamous carcinomas in the WE. Cutaneous epithelial hyperplasia, considered a preneoplastic change, was evaluated by light and electron microscopy at weekly intervals for 70 days. Increased epidermal thickness was induced by the toluene (T) vehicle alone, was significantly enhanced by BP treatment, and was more severe in mice in the CE and NE than in the WE. Although epithelial hyperplasia induced by BP was interpreted as a preneoplastic lesion, no ultrastructural differences were detected between T- and BP-treated mice in any environment. Results indicated that WE diminished the preneoplastic proliferative response of skin to BP and subsequently delayed the onset of squamous carcinomas induced by high doses of BP and reduced the incidence of squamous carcinomas at low doses of BP.     

Synthesis of a novel fluorinated benzo[a]pyrene: 4,5-difluorobenzo[a]pyrene

The synthesis of 4,5-difluorobenzo[a]pyrene, as a fluorinated probe to investigate the involvement of the K-region in the further metabolic activation of benzo[a]pyrene metabolites, is described. Benzo[a]pyrene-4,5-dione obtained from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone oxidation of cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene was fluorinated with dimethylaminosulfur trifluoride to give 4H,5H,4,4,5,5,-tetra-fluorobenzo[a]pyrene. Defluorination using lithium aluminum hydride in tetrahydrofuran gave 4,5,-difluorobenzo[a]pyrene.     

Binding of benzo[a]pyrene and intracellular transport of a bound electrophilic benzo[a]pyrene metabolite by lipoproteins

Human serum lipoproteins were isolated by means of size exclusionh.p.l.c. Non-covalent uptake of [³H]benzo[a]pyrene was quantitatedfor fractions collected from the effluent of a liquid chromatographicseparation of human serum, and was found to directly correlatewith the lipoprotein concentration. An electrophilic benzo[a]pyrenemetabolite, [³H]trans 7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene,non-covalently associated with low density lipoproteins wastransferred to human lymphocytes in vitro and bound acid-precipitablenucleic acids of the lymphocytes as a function of time. Benzo[a]-pyrenemetabolite binding to lymphocyte DNA was demonstrated by meansof CaCl density gradient analysis. Non-mitogen-stimulated lymphocytesexposed to very low concentrations of carcinogen in the presenceof low density lipo-protein demonstrated [³H]thymidine incorporation;without the concomitant addition of low density lipoproteinthe low concentrations of carcinogen did not stimulate [³H]thymidineincorporation.     

LacI mutation spectra following benzo[a]pyrene treatment of Big Blue mice

The mutation spectrum of the lacI gene from the liver of C57Bl6 Big Blue transgenic mice treated with benzo[a]pyrene (B[a]P) has been compared with the spectrum of spontaneous mutations observed in the liver of untreated Big Blue mice. Mice were treated with B[a]P for 3 days followed by a partial hepatectomy one day after the last injection. Liver tissue was removed for analysis at hepatectomy and, again, 3 days later at the time of sacrifice. Earlier, we reported that the lacI mutant frequency in these B[a]P-treated mice was elevated in the liver both at the time of hepatectomy and at sacrifice; however, a statistically significant increase in the mutant frequency was observed only at sacrifice. In this study, the DNA sequence spectra of lacI mutations observed in the liver of B[a]P-treated Big Blue mice at hepatectomy and at time of sacrifice were compared with each other and with the spectrum of spontaneous liver mutations. No differences were observed between the two B[a]P-treatment spectra. However, mutation frequencies of both GC-->TA and GC-->CG at the time of hepatectomy and at sacrifice were significantly elevated compared with the spontaneous frequency of these same transversions. Also, the frequency of AT-->TA transversions was significantly higher than the spontaneous frequency at the time of hepatectomy but not at sacrifice. The frequency of all other classes of mutations scored was not significantly different from the frequency of these same events in the spontaneous spectra. These data support the view that B[a]P treatment results in the induction of GC-->TA and GC-->CG transversions within 1 day of the last injection and they provide insights regarding the relative roles of benzo[a]pyrene-7,8-diol-9, 10-epoxide and radical cations of B[a]P in B[a]P-induced mutagenesis in vivo. Finally, these data provide evidence for B[a]P-induced mutagenesis under conditions where no statistical increase in mutant frequency could be shown.