A cross-sectional clinical study in women to investigate possible genotoxicity and hematological abnormalities related to the use of black cohosh botanical dietary supplements.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

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.

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

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 BCE 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. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Evaluating Sufficient Similarity of Botanical Dietary Supplements: Combining Chemical and In Vitro Biological Data.

Botanical dietary supplements are complex mixtures with numerous potential sources of variation along the supply chain from raw plant material to the market. Approaches for determining sufficient similarity (ie, complex mixture read-across) may be required to extrapolate efficacy or safety data from a tested sample to other products containing the botanical ingredient(s) of interest. In this work, screening-level approaches for generating both chemical and biological-response profiles were used to evaluate the similarity of black cohosh (Actaea racemosa) and Echinacea purpurea samples to well-characterized National Toxicology Program (NTP) test articles. Data from nontargeted chemical analyses and gene expression of toxicologically important hepatic receptor pathways (aryl hydrocarbon receptor [AhR], constitutive androstane receptor [CAR], pregnane X receptor [PXR], farnesoid X receptor [FXR], and peroxisome proliferator-activated receptor alpha [PPARα]) in primary human hepatocyte cultures were used to determine similarity through hierarchical clustering. Although there were differences in chemical profiles across black cohosh samples, these differences were not reflected in the biological-response profiles. These findings highlight the complexity of biological-response dynamics that may not be reflected in chemical composition profiles. Thus, biological-response data could be used as the primary basis for determining similarity among black cohosh samples. Samples of E. purpurea displayed better correlation in similarity across chemical and biological-response measures. The general approaches described herein can be applied to complex mixtures with unidentified active constituents to determine when data from a tested mixture (eg, NTP test article) can be used for hazard identification of sufficiently similar mixtures, with the knowledge of toxicological targets informing assay selection when possible.

How similar is similar enough? A sufficient similarity case study with Ginkgo biloba extract.

Botanical dietary supplements are complex mixtures that can be highly variable in composition and quality, making safety evaluation difficult. A key challenge is determining how diverse products in the marketplace relate to chemically and toxicologically characterized reference samples (i.e., how similar must a product be in order to be well-represented by the tested reference sample?). Ginkgo biloba extract (GBE) was used as a case study to develop and evaluate approaches for determining sufficient similarity. Multiple GBE extracts were evaluated for chemical and biological-response similarity. Chemical similarity was assessed using untargeted and targeted chemistry approaches. Biological similarity was evaluated using in vitro liver models and short-term rodent studies. Statistical and data visualization methods were then used to make decisions about the similarity of products to the reference sample. A majority of the 26 GBE samples tested (62%) were consistently determined to be sufficiently similar to the reference sample, while 27% were different from the reference GBE, and 12% were either similar or different depending on the method used. This case study demonstrated that approaches to evaluate sufficient similarity allow for critical evaluation of complex mixtures so that safety data from the tested reference can be applied to untested materials.

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

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.