Faculty Bibliography

The effects of a nine month administration of dietary: (1) 3H-1,2-dithiole-3-thione (D3T), (2) N-acetylcysteine (NAC), (3) antioxidant vitamin mix, (vitamin C+E), (4) free radical scavenger, amifostine, and (5) calorie restriction, (CR), on mutagenesis and lipid peroxidation in lung, kidney, spleen and liver of lacZ transgenic mice were examined. These agents/diets were chosen because they might inhibit certain proposed mechanisms of endogenous damage to DNA. The agents were added to a high fat, reduced antioxidant AIN-76 diet, to better approximate a Western style diet than the conventional AIN-76 diet. As the lacZ gene is not expressed, mutations in that gene are neutral, and simply accumulate over time. The mutant fractions in control mice increased about 50-100%. Most of the agents inhibited to various extents the age-related increase in mutagenesis in lung, kidney, and/or spleen, but no inhibition was observed in liver. There was no significant effect of age on lipid peroxidation levels in controls, possibly reflecting steady state turnover of lipid peroxidation products. Almost all of the treatments except D3T inhibited lipid peroxidation in most organs to different degrees. The vitamin C+E mix was the most effective at inhibiting lipid peroxidation, but a single most effective inhibitor of mutagenesis could not be discerned. Some associations were observed between the reduction in lipid peroxidation and the inhibition of mutagenesis. The results are consistent with a partial role for oxidative stress in the age-related increase in mutagenesis. These observations may have implications for chemoprevention of carcinogenesis.

Although butyl-hydroxybutyl-N-nitrosamine (BBN) is a powerful carcinogen that specifically causes bladder cancer in rodents, the reason for such a high degree of organ specificity is unclear. Mutagenesis induced by BBN in a chromosomally incorporated mutagenesis reporter gene was measured in bladder urothelial cells and smooth muscle cells of mice or rats and compared with mutagenesis in liver, kidney, ureter and forestomach. BBN was administered as a 0.05% solution in water for 2 weeks followed by 4 weeks of water to 6 male and female mice and 4 male rats. All animals were euthanized at 6 weeks. Since mutagenesis in the reporter gene is a surrogate for mutagenesis in general, we hypothesized that mutations also induced in critical growth control genes would contribute to the tumor formation induced by BBN. The mutant fractions in urothelial cells were: 257 +/- 25 mutants / 105 pfu for male mice, 276 +/- 43 for female mice and 54 +/- 6 for rats. In contrast, the mutant fractions in bladder smooth muscle cells were 8-9 fold lower than these values in all animals. In non-bladder tissues such as liver, kidney, ureter and forestomach, the mutant fractions were between 2 and 5 mutants / 105 pfu, a level comparable to the control groups without BBN treatment. Hence mutagenesis correlated extremely well with the organ specificity for cancer induction by BBN. Additionally, mutagenesis level was found to be significantly higher in mice than in rats, consistent with the fact that BBN is a much more potent bladder carcinogen in mice. Finally, no significant difference in mutagenicity of BBN between male and female mice was observed, suggesting that post-initiation processes may be responsible for the gender difference in bladder tumor susceptibility. BBN is a primary metabolite of the environmental carcinogen, dibutylnitrosamine and hence a potential human carcinogen. These results provide molecular explanations to the bewildering organ and species specificity of BBN, and indicate that BBN-induced mutagenesis in the urinary bladder represents a highly appropriate model for initiation of bladder cancer, and combined with other models can help elucidate distinct steps in bladder carcinogenesis

7,12-Dimethylbenzanthracene (DMBA) is a potent mammary carcinogen in rats. Combinations of non-toxic nutraceutical agents, administered at or near physiological levels, were investigated for their abilities to inhibit the mutagenicity of DMBA or DMBA-dihydrodiol (DMBAD, a primary metabolite and proximate mutagen of DMBA) in vitro, in rat mammary epithelial and fibroblast cells derived from a lacI (BigBlue) Fischer rat (McDiarmid, H.M., Douglas, G.R., Coomber, B.L., and Josephy, P.D. Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay. Mutat. Res., 497: 39-47, 2001). In the epithelial cells, DMBA was not appreciably mutagenic at concentrations up to 4 muM, but DMBAD, was significantly mutagenic at ten-fold lower concentrations. These results indicate that the epithelial cells can bioactivate the intermediate, DMBAD, but cannot effect the complete biotransformation of DMBA to its ultimate mutagenic metabolite, DMBA-dihydrodiolepoxide. In the fibroblast cell line, in contrast, DMBA was mutagenic at concentrations as low as 20 nM and DMBAD was even more potent than DMBA. Several combinations of nutraceuticals (dietary components providing health benefits) were tested for their abilities to inhibit mutagenesis in these cell lines; the concentrations tested were based on reported serum concentrations and these were used to establish 1x concentrations. The agents and their 1x concentrations were: resveratrol (Res), 2.4 muM; sulforaphane (Sul), 0.06 muM; antioxidant mix (alpha- and -tocopherol, 30 muM, plus vitamin C, 68 muM - VCE); alpha-lipoic acid (LA), 2 muM; epigallocatechin gallate (EGCG), 0.7 muM; and N-acetylcysteine (NAC), 12 muM. None of the agents or their combinations, tested at 1 - 3 x concentrations, showed any cytotoxicity. In both epithelial and fibroblast cells, Sul and Res alone slightly inhibited mutagenesis at 2x concentrations; no other agents had observable effects. All binary combinations of Res, LA, and Sul, at 2x concentrations, inhibited mutagenesis; the Res + Sul combination was particularly effective (ca. 50% inhibition). These results suggest a role for fibroblast cells in the bioactivation of carcinogens, implicating the microenvironment, and indicate that combinations of nutraceuticals can inhibit mutagenesis by polycyclic aromatic hydrocarbons

Head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy, represents a major international public health problem. The vast majority of HNSCC occurs in the oral cavity. Cancer of the mouth strikes about 30,000 Americans each year and less than 50% of patients survive 5 years after diagnosis. Tobacco and alcohol use are considered major causative agents. Current preclinical animal models to study oral carcinogenesis do not accurately mimic the biology of this disease in humans. We have developed a new mouse model by the application of the fjord region diol epoxide, (+)-anti-

As the annual breast cancer incidence in the US is about 200,000, prevention would be of enormous value. There are many leads from studies in isolated cell systems and experimental animals, but when individual putative protective agents have been tested in humans, results have been equivocal or negative. Levels generally present in foods, beverages or supplements may exert only weak protective effects, and combinations of potential chemopreventives may be necessary for effective cancer prevention. Here we investigated combinations of non-toxic nutraceutical agents dosed at or near physiological levels, for their abilities to modulate xenobiotic metabolism, and mutagenesis induced by the proximate rat mammary carcinogen, 7,12-dimethylbenzanthracene-dihydrodiol (DMBAD), in rat mammary epithelial cells. The agents and their 1x concentrations, were: resveratrol (Res), 2.4 muM; sulforaphane (Sul), 0.015 muM; antioxidant mix ({alpha}-, {gamma}-tocopherol, 30 muM, Vitamin C, 68 muM (VCE); {alpha}-lipoic acid (LA), 2 muM; epigallocatechin gallate, (EGCG) 0.7 muM; and N-acetylcysteine (NAC), 12 muM). All of the single agents were non-toxic at the 1x doses and had no statistically significant effect on cell proliferation. All combinations of the binary agents were non-toxic and several modestly inhibited cell proliferation: EGCG-NAC, 27% inhibition, EGCG-Res, 21% inhibition, EGCG-VCE, 26% inhibition. For protection against oxidative stress, inhibition of the oxidation of dihydrodichlorofluorescinediacetate (DHDCF) was monitored, and NAC and NAC+Res or EGCG produced small (10-20%) inhibition. The effects of the inhibitors on selected phase I and II gene expression were also examined. EGCG, Sul, VCE and Res alone induced NQO1, (often used as a marker of phase II, detoxification activity) expression and did not induce CYP1A1 or 1B1. In mutagenesis experiments most of the individual agents did not result in statistically significant inhibition of mutagenesis, but some led to minor inhibition (ca. 20%). We al!

The environmental pollutant 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. Our previous studies have shown that 6-NC is metabolized to trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1,2-DHD-6-NC) in rats and in several in vitro systems, including human breast tissue, and the latter is the proximate carcinogenic form in the rat mammary gland. Because optically active enantiomers of numerous polynuclear aromatic hydrocarbon (PAH) metabolites including chrysene have different biological activities, we hypothesized that the stereochemical course of 6-NC metabolism might play a significant role in the carcinogenic/mutagenic activities of the parent 6-NC. The goal of this study is to evaluate the effect of stereochemistry on the mutagenicity of 1,2-DHD-6-NC using the cII gene of lacI mammary epithelial cells in vitro. Resolution of (+/-)-1,2-DHD-6-NC was obtained by either nonchiral or chiral stationary phase HPLC methods. We determined that the ratio of (-)-[R,R]- and (+)-[S,S]-1,2-DHD-6-NC formed in the metabolism of 6-NC by rat liver microsomes is 88:12. The mutation fractions and mutation spectra of [R,R] and [S,S]-enantiomers were examined. Our results showed that the [R,R]-isomer is a significantly (p < 0.01) more potent mutagen than the [S,S]-isomer. The major types of mutation induced by the [R,R]-enantiomer are AT > GC, AT > TA, and GC > TA substitutions, and these are similar to those obtained from 6-NC in vivo in the mammary glands of rats treated with 6-NC. The mutation spectra of the [S,S]-isomer were similar to the [R,R]-isomer, but a higher percentage of AT > GC substitutions in the [R,R]-isomer was noted. On the basis of the results of the present study, we hypothesize that [R,R]-1,2-DHD-6-NC is the proximate carcinogen of 6-NC in the rat mammary gland in vivo and will test this hypothesis in a future study.

UV radiation (UVR) and exposure to tobacco smoke, a source of polycyclic aromatic hydrocarbons (PAH), have been linked to skin carcinogenesis. UVR-mediated activation of the aryl hydrocarbon receptor (AhR) stimulates the transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAH to genotoxic metabolites. We determined whether UVR exposure sensitized human keratinocytes to PAH-induced DNA adduct formation. UVR exposure induced CYP1A1 and CYP1B1 in HaCaT cells, an effect that was mimicked by photooxidized tryptophan (aTRP) and FICZ, a component of aTRP. UVR exposure or pretreatment with aTRP or FICZ also sensitized cells to benzo(a)pyrene (B[a]P)-induced DNA adduct formation. alphaNF, an AhR antagonist, suppressed UVR-, aTRP-, and FICZ-mediated induction of CYP1A1 and CYP1B1 and inhibited B[a]P-induced DNA adduct formation. Treatment with 17-AAG, an Hsp90 inhibitor, caused a marked decrease in levels of AhR; inhibited UVR-, aTRP-, and FICZ-mediated induction of CYP1A1 and CYP1B1; and blocked the sensitization of HaCaT cells to B[a]P-induced DNA adduct formation. FICZ has been suggested to be a physiologic ligand of the AhR that may have systemic effects. Hence, studies of FICZ were also carried out in MSK-Leuk1 cells, a model of oral leukoplakia. Pretreatment with alpha-naphthoflavone or 17-AAG blocked FICZ-mediated induction of CYP1A1 and CYP1B1, and suppressed the increased B[a]P-induced DNA adduct formation. Collectively, these results suggest that sunlight may activate AhR signaling and thereby sensitize cells to PAH-mediated DNA adduct formation. Antagonists of AhR signaling may have a role in the chemoprevention of photocarcinogenesis.

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.