Actein contributes to black cohosh extract-induced genotoxicity in human TK6 cells

Black cohosh extract (BCE) is one of the most popular botanical products for relieving menopausal symptoms. However, recent studies indicate that BCE is not only ineffective for menopausal therapy but also induces genotoxicity through an aneugenic mode of action (MoA). In this study, the cytotoxicity of five constituents of BCE was evaluated in human lymphoblastoid TK6 cells. Among the five constituents, actein (up to 50 μM) showed the highest cytotoxicity and was thus selected for further genotoxicity evaluations. Actein caused DNA damage proportionally to concentration as evidenced by the phosphorylation of the histone protein H2A.X (γH2A.X) and resulted in chromosomal damage as measured by the increased percentage of micronuclei (%MN) in cells. In addition, actein activated DNA damage response (DDR) pathway through induction of p-ATM, p-Chk1, and p-Chk2, which subsequently induced cell cycle changes and apoptosis. Moreover, both BCE and actein increased intracellular reactive oxygen species (ROS) production, decreased glutathione levels, and activated the mitogen-activated protein kinases (MAPK) signaling pathway. N-acetylcysteine, a ROS scavenger, attenuated BCE- and actein-induced ROS production, apoptosis, and DNA damage. These findings indicate that BCE- and actein-induced genotoxicity is mediated, at least partially, through oxidative stress. Taken together, our data show that actein is likely one of the major contributors to BCE-induced genotoxicity.