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

Abstract

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.

Keywords: DNA damage response; TK6 cells; botanical extract; comet assay; in vitro genotoxicity; micronucleus assay.

Figure 1.

BCE-induced micronuclei formation in TK6 and HepG2 cells. TK6 (A) and HepG2 (B) cells were exposed to various concentrations of BCE for 24 h. Micronuclei formation was detected by the in vitro micronucleus (MN) assay. MN frequency (%MN) is presented as the percentage of micronuclei relative to intact nuclei (left y-axis and black bar). The cytotoxicity is presented as the percentage of relative survival (right y-axis and red line) from treated cells compared with the vehicle control. PC, positive control (50 ng/ml mitomycin C). *p < .05, **p < .01, and ***p < .001, compared with the vehicle control.

Figure 2.

Evaluation of BCE-induced cytotoxicity in TK6 and HepG2 cells. TK6 (A) and HepG2 cells (B) were exposed to 25–250 µg/ml and 50–500 µg/ml BCE, respectively and the relative cytotoxicity (% of control) was measured using the CellTiter-Glo luminescent cell viability (ATP) and CellTiter-Blue cell viability assays at 0.5, 1, 2, 4, and 24 h time points. The data are expressed as the mean ± SD from at least 3 independent experiments. *p < .05, **p < .01, and ***p < .001, compared with the vehicle control.

Figure 3.

Evaluation of BCE-induced DNA damage in TK6 and HepG2 cells. TK6 (A) and HepG2 cells (B) were exposed to 25–250 µg/ml and 50–500 µg/ml BCE, respectively and DNA damage (% tail DNA) was detected using the comet assay at 0.5, 1, 2, 4, and 24 h time points. PC, positive control (100 µM MMS) for 4 h. The data are expressed as the mean ± SD from at least 3 independent experiments. *p < .05, **p < .01, and ***p < .001, compared with the vehicle control.

Figure 4.

Effects of physiological folic acid on BCE-induced cytotoxicity and DNA damage in TK6 cells. TK6 cells were cultured in the standard (2268 nM folic acid [FA]) and low FA (120 nM) RPMI-1640 medium for 5 days. BCE-induced cytotoxicity and DNA damage were evaluated by the CellTiter-Blue cell viability assay (A) and the CometChip assay (B), respectively. Black bars represent TK6 cells cultured in the standard medium whereas gray bars represent TK6 cells cultured in FA 120 nM medium. The data are expressed as the mean ± SD from at least 3 independent experiments. *p < .05, **p < .01, and ***p < .001, compared with the vehicle control.

Figure 5.

Activation of molecular biomarkers in DNA damage response pathway by BCE in TK6 cells. A, Total cellular proteins of TK6 cells cultured in the standard FA (2268 nM) or low FA (120 nM) media were extracted following a 4-h or 24-h treatment with BCE. The expression levels of γH2A.X, H2A.X, p-Chk1, p-Chk2, p53, and p21 were detected by Western blotting analysis. GAPDH was used as a loading control. B, Intensity of γH2A.X was normalized to the intensity of H2A.X. Intensities of the bands on the other bar graphs were normalized to the amount of GAPDH. The bar graphs are the mean ± SD of fold changes compared with the vehicle control from at least 3 independent experiments. *p < .05, **p < .01, and ***p < .001 versus the vehicle control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-Chk1, phosphorylated checkpoint kinase1; p-Chk2, phosphorylated checkpoint kinase2.

Figure 6.

BCE-induced apoptosis in TK6 cells. TK6 cells were exposed to various concentrations (25–250 µg/ml) of BCE for 4 or 24 h. BCE-induced apoptosis was evaluated by ethidium monoazide staining from the MN assay (A), the TUNEL assay (B), caspase 3/7 enzyme activity assay (C), and the expression of protein levels of cleaved caspase 3 (c-Cas3) and PARP by Western blotting (D). GAPDH was used as a loading control. (E) The bar graphs represent the densitometric analysis of c-Cas3 and cleaved-PARP (c-PARP, second band at 89 kDa). Intensities of bands were normalized to the amount of GAPDH. The data are expressed as the mean ± SD from at least 3 independent experiments. *p < .05, **p < .01, and ***p < .001 versus the vehicle control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly (ADP-ribose) polymerase.

Figure 7.

BCE-induced cell cycle arrest in TK6 cells. A, DNA histograms representing cell cycle distribution following a 24-h treatment with BCE (25–125 µg/ml) in TK6 cells. B, The proportion of cells (%) in various stages (subG1, G1, S and G2/M) of the cell cycle were analyzed by measuring the nuclear DNA content of treated cells.

Figure 8.

Evaluation of BCE-induced global DNA methylation in TK6 and HepG2 cells. DNA methylation of long interspersed nucleotide elements (LINE) 1 was assessed in TK6 (A) and HepG2 cells (B) using the methylated DNA immunoprecipitation (MeDIP) assay combined with real-time quantitative PCR (qPCR) for LINE1 region 1 and region 2. The data are expressed as the mean ± SD from at least 3 independent experiments. The values were expressed as the percentage (%) of methylated DNA in total DNA.