Evidence for an Aneugenic Mechanism of Action for Micronucleus Induction by Black Cohosh Extract
Derek T. Bernacki, Steven M. Bryce, Jeffrey C. Bemis, Stephen D. Dertinger, Kristine L. Witt, Stephanie L. Smith-Roe
Environmental and Molecular Mutagenesis, (in press): First Published: 30 September 2019 DOI: https://doi.org/10.1002/em.22334
Black cohosh extract (BCE) is a popular botanical dietary supplement marketed to relieve symptoms of various gynecological ailments. Studies conducted by the National Toxicology Program (NTP) showed that BCE induces micronucleated erythrocytes in female rats and mice. Subsequently, the NTP showed that a variety of BCEs, including the sample that induced micronuclei (MN) in vivo(“NTP BCE”) had a similar effect in human TK6 cells. Further testing with the MultiFlow® DNA Damage Assay revealed that TK6 cells exposed to NTP BCE, as well as a black cohosh extract reference material (BC XRM), exhibited a signature consistent with aneugenic activity in TK6 cells. Results from experiments reported herein confirmed these in vitro observations with NTP BCE and BC XRM. We extended these studies to include a novel test system, the MultiFlow Aneugen Molecular Mechanism Assay. For these experiments, TK6 cells were exposed to NTP BCE and BC XRM over a range of concentrations in the presence of fluorescent Taxol (488 Taxol). After 4 h, nuclei from lysed cells were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3) and Ki-67. Whereas BCEs did not affect p-H3:Ki-67 ratios (a signature of aneugenic mitotic kinase inhibitors), 488 Taxol-associated fluorescence (a tubulin binder-sensitive endpoint) was affected. More specifically, 488 Taxol-associated fluorescence was reduced over the same concentration range that was previously observed to induce MN. These results provide direct evidence that BCEs destabilize microtubules in vitro, and this is the molecular mechanism responsible for the aneugenicity findings.