Black cohosh extracts and powders induce micronuclei, a biomarker of genetic damage, in human cells

Abstract

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15-1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416-426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Evidence for an Aneugenic Mechanism of Action for Micronucleus Induction by Black Cohosh Extract

“…MN and development of megaloblastic anemia can both be caused by disruption of the folate metabolism pathway. Although impairment of this pathway is primarily associated with chromosome breakage (clastogenicity), MoA studies using the MultiFlow DNA Damage assay in human TK6 cells classified two BCEs, the NTP BCE and a BC XRM, as having molecular signatures consistent with aneugenic activity (Smith‐Roe et al ), an unexpected observation that was replicated in this study.…”

“…The NTP investigated whether preparations made from cohosh roots possess genotoxic activity in general by testing the sample of BCE (“NTP BCE”) that was genotoxic in vivo , 10 BCE samples from different suppliers, a BC extract reference material (BC XRM), and reference material root powders from red, yellow, and Chinese cohoshes in the In Vitro MicroFlow® assay, which uses flow cytometry to quantify micronuclei (MN) in mammalian cell cultures (Bryce et al ). The 15 samples that were tested significantly increased MN in human lymphoblastoid TK6 cells, supporting the observations from the in vivo studies and suggesting that one or more genotoxic components may be common among cohosh root preparations (Smith‐Roe et al ). Although the NTP has not tested BCE products that are available for purchase by consumers (ie, “finished” products such as tablets or capsules), the NTP BCE has a chromatographic profile that is very similar to the BC XRM and a BCE product, Remifemin® (Mercado‐Feliciano et al ; Smith‐Roe et al ).…”

“…The 15 samples that were tested significantly increased MN in human lymphoblastoid TK6 cells, supporting the observations from the in vivo studies and suggesting that one or more genotoxic components may be common among cohosh root preparations (Smith‐Roe et al ). Although the NTP has not tested BCE products that are available for purchase by consumers (ie, “finished” products such as tablets or capsules), the NTP BCE has a chromatographic profile that is very similar to the BC XRM and a BCE product, Remifemin® (Mercado‐Feliciano et al ; Smith‐Roe et al ).…”

“…NTP BCE (lot number 3012872) was procured from PlusPharma (Vista, CA) and BC XRM (lot number ASB‐00030148‐005) was procured from ChromaDex (Irvine, CA). These samples of NTP BCE and BC XRM are the same materials that were tested by Smith‐Roe et al ().…”

“…Together with information on cytotoxicity at 24 h, the machine‐learning algorithms translate the collection of MultiFlow responses into predictions about predominant MoA—clastogenic, aneugenic, or non‐genotoxic (Bryce et al ; Bryce et al ; Bryce et al ; Dertinger et al ). The NTP BCE and BC XRM samples were characterized by the MultiFlow assay as having aneugenic activity (Smith‐Roe et al ).…”