Mechanistic Evaluation of Black Cohosh Extract-Induced Genotoxicity in Human Cells.

Black cohosh extract (BCE) is marketed to women as an alternative to hormone replacement therapy for alleviating menopausal symptoms. Previous studies by the National Toxicology Program revealed that BCE induced micronuclei (MN) and a nonregenerative macrocytic anemia in rats and mice, likely caused by disruption of the folate metabolism pathway. Additional work using TK6 cells showed that BCE induced aneugenicity by destabilizing microtubules. In the present study, BCE-induced MN were confirmed in TK6 and HepG2 cells. We then evaluated BCE-induced DNA damage using the comet assay at multiple time points (0.5-24 h). Following a 0.5-h exposure, BCE induced significant, concentration-dependent increases in %tail DNA in TK6 cells only. Although DNA damage decreased in TK6 cells over time, likely due to repair, small but statistically significant levels of DNA damage were observed after 2 and 4 h exposures to 250 µg/ml BCE. A G1/S arrest in TK6 cells exposed to 125 µg/ml BCE (24 h) was accompanied by apoptosis and increased expression of γH2A.X, p-Chk1, p-Chk2, p53, and p21. Conditioning TK6 cells to physiological levels of folic acid (120 nM) did not increase the sensitivity of cells to BCE-induced DNA damage. BCE did not alter global DNA methylation in TK6 and HepG2 cells cultured in standard medium. Our results suggest that BCE induces acute DNA strand breaks which are quickly repaired in TK6 cells, whereas DNA damage seen at 4 and 24 h may reflect apoptosis. The present study supports that BCE is genotoxic mainly by inducing MN with an aneugenic mode of action.

Tissue-specific microRNA responses in rats treated with mutagenic and carcinogenic doses of aristolochic acid.

Aristolochic acid (AA) is an active component in herbal drugs derived from the Aristolochia species. Although these drugs have been used since antiquity, AA is both genotoxic and carcinogenic in animals and humans, resulting in kidney tumours in rats and upper urinary tract tumours in humans. In the present study, we conducted microarray analysis of microRNA (miRNA) expression in tissues from transgenic Big Blue rats that were treated for 12 weeks with 0.1-10mg/kg AA, using a protocol that previous studies indicate eventually results in kidney tumours and mutations in kidney and liver. Global analysis of miRNA expression of rats treated with 10 mg/kg AA indicated that 19 miRNAs were significantly dysregulated in the kidney, with most of the miRNAs related to carcinogenesis. Only one miRNA, miR-34a (a tumour suppressor), was differentially expressed in the liver. The expression of the two most responsive kidney miRNAs (miR-21, an oncomiR and miR-34a) was further examined in the kidney, liver and testis of rats exposed to 0, 0.1, 1.0 and 10mg/kg AA. Expression of miR-21 was up-regulated in the kidney only, while miR-34a was dose-dependently up-regulated in both the kidney and liver; the expression of miR-21 and miR-34a was unaltered by the AA treatment in the testis. Analysis of cII mutations in the testis of treated rats also was negative. Our results indicate that AA treatment of rats produced dysregulation of a large number of miRNAs in the tumour target tissue and that the up-regulation of miR-21 correlated with the carcinogenicity of AA while the up-regulation of miR-34a correlated with its mutagenicity.

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue transgenic rats.

The major constituents of isoflavones, daidzein (DZ) and genistein (GE) are known to interact with the alpha and beta estrogen receptors (ERalpha/beta) in several tissues including mammary. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E2) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue (BB) transgenic rats. The rats were fed control diet containing the isoflavones and E2 and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND 50. Animals were sacrificed at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E2 supplementation alone resulted in modest increases in the lacI MF that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lacI MFs in the mammary of both OVX and ovary intact (INT) rats and Hprt MFs in spleen lymphocytes (P

Endogenous estrogen status, but not genistein supplementation, modulates 7,12-dimethylbenz[a]anthracene-induced mutation in the liver cII gene of transgenic big blue rats.

A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an alternative to hormone replacement therapy. Although sex hormones have been implicated as an important risk factor for the development of hepatocellular carcinoma, there are limited data on the potential effects of the estrogens, including phytoestrogens, on chemical mutagenesis in liver. Because of the association between mutation induction and the carcinogenesis process, we investigated whether endogenous estrogen and supplemental genistein affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in rat liver. Intact and ovariectomized female Big Blue rats were treated with 80 mg DMBA/kg body weight. Some of the rats also received a supplement of 1,000 ppm genistein. Sixteen weeks after the carcinogen treatment, the rats were sacrificed, their livers were removed, and mutant frequencies (MFs) and types of mutations were determined in the liver cII gene. DMBA significantly increased the MFs in liver for both the intact and ovariectomized rats. While there was no significant difference in MF between the ovariectomized and intact control animals, the mutation induction by DMBA in the ovariectomized groups was significantly higher than that in the intact groups. Dietary genistein did not alter these responses. Molecular analysis of the mutants showed that DMBA induced chemical-specific types of mutations in the liver cII gene. These results suggest that endogenous ovarian hormones have an inhibitory effect on liver mutagenesis by DMBA, whereas dietary genistein does not modulate spontaneous or DMBA-induced mutagenesis in either intact or ovariectomized rats.