Effects of potential dietary inhibitors of endogenous DNA damage on mutagenesis and lipid peroxidation in lacZ mice.

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.

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.

Effects of 1,4-phenylenebis(methylene)selenocyanate on mutagenesis and p53 protein expression in the tongue of lacI rats treated with 4-nitroquinoline-N-oxide.

Previously we showed that the organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC)(1) inhibits 4-nitroquinoline-N-oxide (4-NQO)-induced tongue tumorigenesis in Fisher rats. Here we investigate possible mechanisms of this inhibition by monitoring mutagenesis and p53 protein levels in lacI and conventional Fisher rats treated with: (1) a carcinogenic dose of 4-NQO for 10 weeks in drinking water, (2) 4-NQO+p-XSC (15 ppm as selenium), and (3) 4-NQO followed by p-XSC. For mutagenesis studies, rats were euthanized at 7, 12 or 23 weeks after the start of 4-NQO. For studies on p53 levels, rats were euthanized at 11, 15 and 23 weeks. Appropriate controls were also monitored. In the 4-NQO-alone groups, the mutant fraction (MF) in the cII gene in tongue increased at least 50x background level. The MF (in units of mutants/10(5) plaque forming units) for the 7, 12, and 23 weeks 4-NQO groups were respectively, 184 +/- 88, 237 +/- 105, and 329 +/- 110. Thus, mutagenesis increased with length of exposure and post-treatment time. p-XSC modestly (ca. 15-30%) inhibited mutagenesis under all conditions. The inhibition reached significance at the last time point. When p-XSC was administered after 4-NQO, the MF was also modestly reduced. In 4-NQO-alone animals, levels of p53 in tongue (determined by Western blotting) were 1, 1.5 and 2.4 control levels at 10, 15 and 23 weeks, respectively. In the p-XSC+4-NQO group, the enhancement in p53 levels by 4-NQO treatment was decreased about 90% at 15 weeks and 45% (P<0.05) at 23 weeks, and by slightly smaller percentages in corresponding post-treatment groups. p-XSC alone did not alter p53 levels. As p53 levels generally increase in response to DNA damage, these results suggest that p-XSC reduces 4-NQO-induced DNA damage, resulting in reduced 4-NQO-induced mutagenesis and carcinogenesis. However, the fact that p-XSC is also effective when administered after 4-NQO, suggests additional mechanisms of inhibition exist.

Mutagenic activity of 4-hydroxyestradiol, but not 2-hydroxyestradiol, in BB rat2 embryonic cells, and the mutational spectrum of 4-hydroxyestradiol.

Estrogens are hypothesized to contribute to breast cancer via estrogen receptor-mediated increases in cell proliferation and via genotoxic processes leading to mutations. In this latter process, estradiol (E(2)) is thought to be oxidized to 4-hydroxyestradiol and then to E(2)-3,4-quinone, which reacts with DNA leading to apurinic sites. These sites represent premutagenic lesions. Additionally, E(2)-3,4-quinone can undergo redox cycling with E(2)-3,4-hydroquinone, leading to the release of reactive oxygen species. Although there is evidence that estradiol and E(2)-3,4-quinone are carcinogenic or mutagenic in several systems, 4-hydroxyestradiol, a key intermediate in the proposed genotoxic pathway, has thus far been negative in mutagenesis assays. Another major metabolite of estradiol, 2-hydroxyestradiol, is essentially inactive in carcinogenicity or mutagenicity assays. Here, we report that when using multiple low-dose exposures 4-hydroxyestradiol is mutagenic in the cII assay in BB rat2 cells. Under similar conditions, 2-hydroxyestradiol is inactive. Furthermore, the mutational spectrum of 4-hydroxyestradiol contains a considerable proportion of mutations at A:T base pairs, consistent with the known ability of E(2)-3,4-quinone to form a significant fraction of DNA adducts at adenines. Thus, the results of this study support the proposal that estradiol can contribute to carcinogenesis via a genotoxic pathway.

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 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.

Mammary carcinogenesis and molecular analysis of in vivo cII gene mutations in the mammary tissue of female transgenic rats treated with the environmental pollutant 6-nitrochrysene.

We determined the mutant fractions (MF) and mutational specificities in the cII gene in histologically confirmed normal, non-involved and tumor mammary tissues of female transgenic (Big Blue F344 x Sprague-Dawley)F1 rats treated with the environmental pollutant 6-nitrochrysene (6-NC). At 30 days of age, three groups were set up for oral treatment with 6-NC dissolved in trioctanoin, or trioctanoin alone once a week for 8 weeks. Two dose levels of 6-NC (100 and 200 micromol/rat) were selected on the basis of our previous carcinogenicity bioassays with CD rats. The rats were decapitated 32 weeks after the last carcinogen dose. Both incidence and multiplicity of mammary adenocarcinomas were significantly elevated in the high dose (36%, 0.57, P < 0.01) group but at the low dose these outcomes (16%, 0.23, P < 0.1) were not significantly different from those of control rats (3%, 0.03). The MF in normal, non-involved and tumor tissues from the mammary glands of 6-NC-treated rats were comparable. At the high and low doses, respectively (4.8 +/- 2.0, 3.2 +/- 2.1) the MF of 6-NC-treated rats, were significantly higher (P < 0.05) than that observed in control rats (1.2 +/- 0.6). Control mutants consisted primarily of GC --> AT transitions, whereas 6-NC-induced mutants were comprised of several major classes of mutations with GC --> TA, GC --> CG, AT --> GC and AT --> TA as the most prevalent. Further studies indicated that the structures of 6-NC-DNA adducts in the mammary tissue are consistent with the mutational specificities. This is the first report that defines the relationship between carcinogenesis and mutagenesis, as well as between structures of 6-NC-DNA adducts and mutation characteristics in the target organ in vivo.