Effects of E-Cigarette Aerosols with Varying Levels of Nicotine on Biomarkers of Oxidative Stress and Inflammation in Mice.

To provide insights into the cause of e-cigarette (e-cig) associated lung injury, we examined the effects of propylene glycol (PG) and glycerol (G), two common solvent carriers used to deliver nicotine/flavor, on markers of oxidative stress and inflammation in female B6C3F1 mice which had been used successfully in tobacco smoke (TS)-induced lung carcinogenesis. Mice exposed to air and TS were used as negative and positive controls, respectively. Using LC-MS/MS, we showed that PG/G alone, in the absence of nicotine, significantly increased the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG or its tautomer 8-oxodG), a biomarker of DNA oxidative damage, in lung and plasma of mice; moreover, addition of nicotine (12 and 24 mg/mL) in e-cig liquid appears to suppress the levels of 8-oxodG. Exposure to e-cig aerosols or TS induced nonsignificant increases of plasma C-reactive protein (CRP), a biomarker of inflammation; nonetheless, the levels of fibronectin (FN), a biomarker of tissue injury, were significantly increased by e-cig aerosols or TS. Although preliminary, our data showed that exposure to e-cig aerosols induced a higher score of lung injury than did control air or TS exposure. Our results indicate that the B6C3F1 mouse model may be suitable for an in-depth examination of the impact of e-cig on lung injury associated with oxidative stress and inflammation and this study adds to the growing evidence that the use of e-cig can lead to lung damage.

Effects of the Tobacco Carcinogens N'-Nitrosonornicotine and Dibenzo[a,l]pyrene Individually and in Combination on DNA Damage in Human Oral Leukoplakia and on Mutagenicity and Mutation Profiles in lacI Mouse Tongue.

In previous studies, we showed that the topical application of dibenzo[a,l]pyrene (DB[a,l]P), also known as dibenzo[def,p]chrysene, to the oral cavity of mice induced oral squamous cell carcinoma. We also showed that dA and dG adducts likely account for most of the mutagenic activity of DB[a,l]P in the oral tissues in vivo. Here we report for the first time that the oral treatment of lacI mice with a combination of tobacco smoke carcinogens, DB[a,l]P and N'-nitrosonornicotine (NNN), induces a higher fraction of mutations than expected from a simple sum of their induced individual mutation fractions, and a change in the mutational profile compared with that expected from the sum of the individual agents. The mutational profile of the combination of agents resembled that of the P53 gene in human head and neck cancers more than that of either of the individual agents, in that the percentage of the major class of mutations (GC > AT transitions) is similar to that seen in the P53 gene. A preliminary study was performed to understand the origin of the unexpected mutagenesis observations by measuring specific DNA adducts produced by both NNN and DB[a,l]P in human oral leukoplakia cells. No significant differences in the expected and observed major adduct levels from either agent were observed between individual or combined treatments, suggesting that additional adducts are important in mutagenesis induced by the mixture. Taken together, the above observations support the use of this animal model not only to investigate tobacco smoke-induced oral cancer but also to study chemoprevention.

Carcinogenesis of the Oral Cavity: Environmental Causes and Potential Prevention by Black Raspberry.

Worldwide, cancers of the oral cavity and pharynx comprise the sixth most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol, and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful, and survival rates have not improved despite advances in therapeutic approaches. Therefore, new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal, several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12-dimethylbenz[a]anthracene in hamster cheek pouch and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N'-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that (1) utilize environmental carcinogens, (2) reflect tumor heterogeneity, and (3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective, we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance the repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

Effects of Black Raspberry Extract and Berry Compounds on Repair of DNA Damage and Mutagenesis Induced by Chemical and Physical Agents in Human Oral Leukoplakia and Rat Oral Fibroblasts.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Mechanisms of oral carcinogenesis induced by dibenzo[a,l]pyrene: an environmental pollutant and a tobacco smoke constituent.

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

Mechanisms underlying the varied mammary carcinogenicity of the environmental pollutant 6-nitrochrysene and its metabolites (-)-[R,R]- and (+)-[S,S]-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene in the rat.

The mechanisms that can account for the remarkable mammary carcinogenicity of the environmental pollutant 6-nitrochrysene (6-NC) in the rat remain elusive. In our previous studies, we identified several 6-NC-derived DNA adducts in the rat mammary gland; one major adduct was derived from (±)-trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1,2-DHD-6-NC). In the present study, we resolved the racemic (±)-1,2-DHD-6-NC into (-)-[R,R]- and (+)-[S,S]-1,2-DHD-6-NC and compared their in vivo mutagenicity and carcinogenicity in the mammary glands of female transgenic (BigBlue F344 × Sprague-Dawley)F1 rats harboring lacI/cII and Sprague-Dawley rats, respectively. Both [R,R]- and [S,S]-isomers exerted similar mutagenicity and carcinogenicity but were less potent than 6-NC. Additional in vivo and in vitro studies were then performed to explore possible mechanisms that can explain the higher potency of 6-NC than 1,2-DHD-6-NC. Using ELISA, we found that neither 6-NC nor 1,2-DHD-6-NC increased the levels of several inflammatory cytokines in plasma obtained from rats 24 h after treatment. In MCF-7 cells, as determined by immunoblotting, the effects of 6-NC and 1,2-DHD-6-NC on protein expression (p53, Akt, p38, JNK, c-myc, bcl-2, PCNA, and ERß) were comparable; however, the expressions of AhR and ERα proteins were decreased by 6-NC but not 1,2-DHD-6-NC. The expression of both receptors was decreased in mammary tissues of rats treated with 6-NC. Our findings suggest that the differential effects of 6-NC and 1,2-DHD-6-NC on AhR and ERα could potentially account for the higher carcinogenicity of 6-NC in the rat mammary gland.

Mutagenesis and carcinogenesis induced by dibenzo[a,l]pyrene in the mouse oral cavity: a potential new model for oral cancer.

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.

Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis.

Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis.

Stereoselective metabolism of the environmental mammary carcinogen 6-nitrochrysene to trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene by aroclor 1254-treated rat liver microsomes and their comparative mutation profiles in a laci mammary epithelial cell line.

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.

E-cigarette Aerosol Condensate Enhances Metabolism of Benzo(a)pyrene to Genotoxic Products, and Induces CYP1A1 and CYP1B1, Likely by Activation of the Aryl Hydrocarbon Receptor.

E-cigarette aerosol contains lower levels of most known carcinogens than tobacco smoke, but many users of e-cigarettes are also smokers, and these individuals may be vulnerable to possible promoting and/or cocarcinogenic effects of e-cigarettes. We investigated the possibility that a condensate of e-cigarette aerosol (EAC) enhances the metabolism of the tobacco carcinogen, benzo(a)pyrene (BaP), to genotoxic products in a human oral keratinocyte cell line. Cells were pretreated with EAC from two popular e-cigs and then with BaP. Metabolism to its ultimate carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro B[a]P (BPDE), was assayed by measuring isomers of its spontaneous hydrolysis products, BaP tetrols. The pretreatment of cells with EAC enhanced the rate of BaP tetrol formation several fold. Pretreatment with the e-liquid resulted in a smaller enhancement. The treatment of cells with EAC induced CYP1A1/1B1 mRNA and protein. The enhancement of BaP tetrol formation was inhibited by the aryl hydrocarbon receptor (AhR) inhibitor, α-napthoflavone, indicating EAC likely induces CYP1A1/1B1 and enhances BaP metabolism by activating the AhR. To our knowledge, this is first report demonstrating that e-cigarettes can potentiate the genotoxic effects of a tobacco smoke carcinogen.

Effects of Black Raspberry on Dibenzo[a,l]Pyrene Diol Epoxide Induced DNA Adducts, Mutagenesis, and Tumorigenesis in the Mouse Oral Cavity.

We previously showed that metabolic activation of the environmental and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) to its active fjord region diol epoxide (DB[a,l]PDE) is required to induce DNA damage, mutagenesis, and squamous cell carcinoma (SCC) in the mouse oral cavity. In contrast to procarcinogens, which were employed previously to induce SCC, DB[a,l]PDE does not require metabolic activation to exert its biological effects, and thus, this study was initiated to examine, for the first time, whether black raspberry powder (BRB) inhibits postmetabolic processes, such as DNA damage, mutagenesis, and tumorigenesis. Prior to long-term chemoprevention studies, we initially examined the effect of BRB (5% added to AIN-93M diet) on DNA damage in B6C3F1 mice using LC/MS-MS and on mutagenesis in the lacI gene in the mouse oral cavity. We showed that BRB inhibited DB[a,l]PDE-induced DNA damage (P < 0.05) and mutagenesis (P = 0.053) in the oral cavity. Tumor incidence in the oral cavity (oral mucosa and tongue) of mice fed diet containing 5% BRB was significantly (P < 0.05) reduced from 93% to 66%. Specifically, the incidence of benign tumor was significantly (P < 0.001) reduced from 90% to 31% (62% to 28% in the oral cavity and 28% to 2% in the tongue), a nonsignificant reduction of malignant tumors from 52% to 45%. Our preclinical findings demonstrate for the first time that the chemopreventive efficacy of BRB can be extended to direct-acting carcinogens that do not require phase I enzymes and is not just limited to procarcinogens. Cancer Prev Res; 11(3); 157-64. ©2017 AACR.

Comparative mutational profiles of the environmental mammary carcinogen, 6-nitrochrysene and its metabolites in a lacI mammary epithelial cell line.

The dietary and environmental agent, 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. It is known to be metabolized by ring-oxidation, nitro-reduction and a combination of the two pathways. In order to determine the ultimate mutagenic metabolites, we have compared the previously determined mutational profile of 6-NC in rat mammary gland [T. Boyiri, et al. (2004) Carcinogenesis, 25, 637-643] with that of five of its known metabolites in the cII gene of lacI mammary epithelial cells in vitro. In vivo, 6-NC gives rise to three major mutations, AT > GC, AT > TA and GC > TA (in decreasing order) which comprise >70% of the mutations. The metabolite whose mutational profile was most similar to that of 6-NC in vivo was trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C) which arises from both ring-oxidation and nitro-reduction. However, metabolites arising from either ring-oxidation or nitro-reduction alone exhibited some similarities to mutational profile of 6-NC. These results, taken in conjunction with previous data showing that the major DNA adducts in mammary tissue of rats treated with 6-NC are products of the reaction of 1,2-DHD-6-NHOH-C with guanine and adenine, make a strong case that 1,2-DHD-6-NHOH-C is the ultimate genotoxic metabolite from 6-NC.

Effects of Black Raspberry Extract and Protocatechuic Acid on Carcinogen-DNA Adducts and Mutagenesis, and Oxidative Stress in Rat and Human Oral Cells.

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response-enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704-12. ©2016 AACR.

Comparative mutagenicities of bleomycin and ferric-nitrilotriacetate in lacZ mice.

Bleomycin and ferric nitrilotriacetate (Fe-NTA) give rise to reactive oxygen species (ROS). Bleomycin was mutagenic in lacZ mouse kidney, liver and lung, but Fe-NTA was non-mutagenic in kidney and lung and marginally mutagenic in liver. Fe-NTA-treatment led to an increase in 8-hydroxydeoxyguanosine levels in kidney and liver, while the corresponding levels in bleomycin-treated mice were if anything, lower than those for bleomycin. It appears that factors other than simply the ability to generate ROS, play a role in mutagenesis by these compounds.

Mutational spectrum of bleomycin in lacZ mouse kidney: a possible model for mutational spectrum of reactive oxygen species.

The mutational spectrum of bleomycin was compared with the spontaneous mutational spectrum in lacZ mouse kidney. Mice were treated with four 20 mg/kg of doses of bleomycin over a two-week period, leading to a mutant fraction several times greater than that of controls. The major class of bleomycin-induced mutations consisted of small deletions, in particular -1 deletions at AT base pairs and hot spots for deletions at 5'-GTC-3' sequences. Smaller, but significant fractions of GC > AT followed by GC > TA substitutions were also observed. In untreated mice, the major class of mutations consisted of GC > AT substitutions followed by GC > TA mutations, and a much smaller fraction of deletions. Other than the specificity of bleomycin for AT base pairs and the 5'-GTC-3' hotspots, the mutational spectrum of bleomycin in mice is similar to that reported for ionizing radiation. However, bleomycin initially mediates the formation of oxidized DNA via reduction of molecular oxygen, as opposed to the radiolysis of water. In this respect mutagenesis induced by bleomycin may be more similar to that induced by endogenous reactive oxygen species (ROS) than mutagenesis induced by ionizing radiation. If bleomycin-induced mutagenesis is an appropriate model for mutagenesis induced by ROS, then, based on the difference between the mutational spectrum of bleomycin and spontaneous mutagenesis, the latter appears not to result predominantly from ROS, at least in mouse kidney.

Effects of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and vitamin E on 4-nitroquinoline-N-oxide (4-NQO)-induced mutagenesis in lacZ mouse upper aerodigestive tissue.

The effects of dietary administration of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and Vitamin E on 4-nitroquinoline-N-oxide (4-NQO)-induced mutagenesis in lacZ mouse upper aerodigestive tissues were investigated. 4-NQO was a potent mutagen in tongue, other pooled oral tissues and esophagus when given in drinking water for 4 weeks at a concentration of 20 microg/ml [corrected]. The mutant fractions (MFs) in these tissues were: 144+/-73, 130+/-52 and 61+/-24 mutants/10(5), respectively. Background levels were 3.7+/-1.9 in tongue, 2.9+/-1.2 in esophagus and 2.4+/-1.0 in pooled oral tissue. Vitamin E at levels of 200 and 400 IU/kg diet led to no significant effects on mutagenesis although a small decrease in the MF was observed in all tissues at the higher dose. Dietary p-XSC at levels of 2.5 and 10 ppm selenium also resulted in no statistically significant effects on mutagenesis, but mutagenesis was somewhat reduced in esophagus and pooled oral tissue at the higher dose. However, the combination of the low doses of p-XSC and Vitamin E resulted in nearly a 40% decrease in mutagenesis in tongue and esophagus, and this decrease was statistically significant (P=0.008 and 0.023, respectively. No inhibition was observed using a combination of the higher doses of p-XSC and Vitamin E. These results lend support to the use of low doses of inhibitors of mutagenesis in combinations. The application of in vivo mutagenesis assays to the screening of chemopreventive agents enables investigators to evaluate potential inhibitors when given individually and in combinations on the initiation stage of carcinogenesis in a short-term in vivo bioassay.

Effects of 3H-1,2-dithiole-3-thione, 1,4-phenylenebis(methylene)selenocyanate, and selenium-enriched yeast individually and in combination on benzo[a]pyrene-induced mutagenesis in oral tissue and esophagus in lacZ mice.

We have studied the effects of three chemopreventive agents alone or in binary combinations on benzo[a]pyrene (BaP)-induced mutagenesis in the oral cavity and esophagus of lacZ mice using galE(-) selection. The mice were fed diets supplemented with 1,4-phenylenebis(methylene)selenocyanate (p-XSC) at 2.5 and 10 ppm Se, selenium-enriched yeast (SeY) at 2.5 and 10 ppm Se, and 3H-1,2-dithiole-3-thione (D3T) at 65 and 250 ppm, for 6 weeks. Two weeks after the start of the dietary regimen, mice were gavaged with five doses of 125 mg/kg BaP over 2 weeks, and the experiment was terminated 2 weeks later. Mutagenesis was measured in tongue, other pooled oral tissues (OTs), and esophagus. In mice treated with BaP alone, mutagenesis in the above tissues was in the range of 21-32 mutants/10(5)pfu (ca. 6-10 background levels for the corresponding tissues). p-XSC modestly inhibited mutagenesis (10-33% inhibition) in all tissues, but statistical significance was only observed at the low dose in esophagus, and pooled OT. SeY was not inhibitory alone. Greater inhibitory effects were observed with D3T, and inhibition was statistically significant at the high dose in tongue and esophagus (ca. 33%). Two combinations of low doses of the inhibitors were tested, and the D3T + SeY mix was most effective, leading to statistically significant inhibition in all three tissues (ca. 30-40% inhibition). The mixture D3T + p-XSC was of similar effectiveness as the low dose of D3T alone. This study combined with those previously done in our laboratory demonstrates effectiveness of D3T and to a lesser extent, p-XSC in the inhibition of mutagenesis, and provides support for the use of certain combinations of inhibitors as a means to increase effectiveness and reduce the dose of chemopreventive agents.