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.

A Black Cohosh Extract Causes Hematologic and Biochemical Changes Consistent with a Functional Cobalamin Deficiency in Female B6C3F1/N Mice.

Black cohosh rhizome, available as a dietary supplement, is most commonly marketed as a remedy for dysmenorrhea and menopausal symptoms. A previous subchronic toxicity study of black cohosh dried ethanolic extract (BCE) in female mice revealed a dose-dependent ineffective erythropoiesis with a macrocytosis consistent with the condition known as megaloblastic anemia. The purpose of this study was to investigate potential mechanisms by which BCE induces these particular hematological changes. B6C3F1/N female mice (32/group) were exposed by gavage to vehicle or 1,000 mg/kg BCE for 92 days. Blood samples were analyzed for hematology, renal and hepatic clinical chemistry, serum folate and cobalamin, red blood cell (RBC) folate, and plasma homocysteine and methylmalonic acid (MMA). Folate levels were measured in liver and kidney. Hematological changes included decreased RBC count; increased mean corpuscular volume; and decreased reticulocyte, white blood cell, neutrophil, and lymphocyte counts. Blood smear evaluation revealed increased Howell-Jolly bodies and occasional basophilic stippling in treated animals. Plasma homocysteine and MMA concentrations were increased in treated animals. Under the conditions of our study, BCE administration caused hematological and clinical chemistry changes consistent with a functional cobalamin, and possibly folate, deficiency. Further studies are needed to elucidate the mechanism by which BCE causes increases in homocysteine and MMA.

Mechanism of hepatotoxicity due to black cohosh (Cimicifuga racemosa): histological, immunohistochemical and electron microscopy analysis of two liver biopsies with clinical correlation.

BACKGROUND: Consumption of herbal supplements in the developed world remains high. Cimicifuga racemosa (C. racemosa) extract, or black cohosh, is widely used as a hormone replacing and an anti-inflammatory agent, and has been shown to cause idiosyncratic hepatitis. The mechanism of acute liver injury in those cases is unclear. To date, hepatotoxic effects of C. racemosa have been studied mostly in vitro and in animal models. Data on human tissue is extremely limited, and mostly confined to histological findings of explanted livers. METHODS: We evaluated clinical data and examined surgical diagnostic liver biopsy specimens obtained from two female patients, who developed acute submassive liver necrosis, following consumption of C. racemosa. Both patients presented with acute elevation of liver enzymes, cholestasis, absence of reactivity to hepatitis A, B and C antibodies, and weak non-specific positivity for autoimmune serological markers. Initial histological interpretation of the biopsies, with focus on hepatic parenchyma and portal tracts, was done by light microscopy, followed by special stain series and immunohistochemical studies, including Cam 5.2, AE1/AE3, reticulin, α-actin, sirius red, and PAS with diastase. Areas of prominent lymphocytic infiltration of the periportal liver plate, observed microscopically, were further evaluated by electron microscopy (EM). 4HNE adduction study, an immunofluorescent assay, was performed to detect products of the oxidative damage and their localization in the liver parenchyma. RESULTS: Oxidative damage was evident by accumulation of 4HNE protein adducts in the cytoplasm of hepatocytes, secondary lysosomes and macrophages. We hypothesize that the adducted proteins, accumulated in the liver parenchyma, serve as autoantigens, which provoke an autoimmune response, and cause migration of lymphocytes to the affected regions. The formation of immunological synapses between hepatocytes and lymphocytes, predominantly T-lymphocytes, is demonstrated by electron microscopy. The autoimmune response induces piecemeal, or troxis necrosis of hepatocytes, a well described biological phenomenon, where lymphocytes gradually remove hepatocytes in a piecemeal fashion, slowly consuming them and leaving fragments of liver cells, or nubbins of anuclear cytoplasm of liver cell, at the interface between lymphocytes and hepatocytes. CONCLUSION: The pattern of pathological injury of liver cells in both patients, following consumption of black cohosh, is identical to troxis necrosis, seen during autoimmune hepatitis. Recognition of the possibility of the acute hepatic injury by the herbal supplement black cohosh is essential for early accurate diagnosis, and timely patient management.

Pregnane X receptor-mediated induction of Cyp3a by black cohosh.

Black cohosh (BC) has been widely applied for the treatment of menopausal symptoms. However, increasing concerns about herb-drug interactions demand the need for studies on the influence of BC on cytochrome 450. Cyp3a11 in liver was induced by 7-fold in wild-type mice treated with 500 mg/kg black cohosh for 28 days compared with the control group as assessed by quantitative real-time PCR; no difference was found in small intestine and kidney, suggesting that up-regulation of Cyp3a11 by black cohosh was liver-specific. Western blot, activity assays, and pharmacokinetic analyses established dose- and time-dependent induction of Cyp3a11. To determine the mechanism of Cyp3a11 induction, including the role of pregnane X receptor (PXR) in vivo and in vitro, respectively, in Pxr-null, PXR-humanized, and double transgenic CYP3A4/hPXR mice, cell-based luciferase assays were employed revealing that mouse PXR played a direct role in the induction of Cyp3a11; human PXR was not activated by black cohosh. Overall, these findings demonstrate that induction of Cyp3a11 is liver-specific and involved only mouse PXR, not the human counterpart. Thus, the incidence of herb-drug interaction in patients administered black cohosh may not be mediated by human PXR and CYP3A4.

Black cohosh increases metastatic mammary cancer in transgenic mice expressing c-erbB2.

Black cohosh is an herbal extract that is often used as an alternative to estrogen-based replacement therapies to treat hot flushes that frequently accompany the transition to menopause. Although cancer-free women as well as breast cancer patients and survivors use black cohosh to relieve vasomotor symptoms, there is limited information on its potential to influence breast cancer development or progression. Therefore, in this study, the effects of black cohosh on mammary tumorigenesis were investigated in the MMTV-neu mouse model due to its similarities to HER2(+) breast cancer, including stochastic development of mammary tumors, which frequently progress to metastatic disease. Using an adjusted dose for the mice to correlate to the recommended dose in women (40 mg/d), no differences were detected in the incidence or onset of mammary tumors in black cohosh-treated versus control females. The lack of effect on mammary tumor development suggests that black cohosh would not influence breast cancer risk if given to women before tumor formation. In contrast, black cohosh significantly increased the incidence of lung metastases in tumor-bearing animals compared with mice fed the isoflavone-free control diet. Additional studies will be needed to correlate these findings to women taking different black cohosh products at various times during breast cancer development; however, these results suggest caution for women using black cohosh, especially for extended periods of time. As metastatic progression is linked to patient survival, these data stress the importance of investigating how women's therapies influence all stages of mammary tumorigenesis, particularly for assessing their safety.

Hepatic effects of Cimicifuga racemosa extract in vivo and in vitro.

Extracts of Cimicifuga racemosa are used frequently for menopausal complaints. Cimicifuga is well tolerated but can occasionally cause liver injury. To assess hepatotoxicity of cimicifuga in more detail, ethanolic C. racemosa extract was administered orally to rats, and liver sections were analyzed by electron microscopy. Tests for cytotoxicity, mitochondrial toxicity and apoptosis/necrosis were performed using HepG2 cells. Mitochondrial toxicity was studied using isolated rat liver mitochondria. Microvesicular steatosis was found in rats treated with > 1,000 mg/kg [DOSAGE ERROR CORRECTED] body weight cimicifuga extract. In vitro, cytotoxicity was apparent at concentrations > or =75 microg/mL, and mitochondrial beta-oxidation was impaired at concentrations > or =10 microg/mL. The mitochondrial membrane potential was decreased at concentrations > or =100 microg/mL, and oxidative phosphorylation was impaired at concenq trations > or =300 microg/mL. The mechanism of cell death was predominantly apoptosis. C. racemosa exerts toxicity in vivo and in vitro, eventually resulting in apoptotic cell death. The results are compatible with idiosyncratic hepatotoxicity as observed in patients treated with cimicifuga extracts.

Posible hepatotoxicidad del rizoma de cimicífuga / No disponible

El uso de preparados de rizoma de cimicífuga se ha extendido en los últimos años, por su eficacia en el tratamiento de los síntomas asociados al climaterio. El hallazgo reciente de indicios sobre su posible toxicidad hepática, aunque con una frecuencia de aparición rara, ha llevado a las agencias de medicamentos a efectuar recomendaciones tanto a los pacientes como a los profesionales de la salud sobre la utilización de los mismos. De los más de 40 casos evaluados por la EMEA, sólamente cuatro muestran una secuencia temporal compatible con la administración del producto, aunque en ninguno de ellos se ha podido establecer con certeza una relación de causalidad. La evaluación se ha visto dificultada por la falta de una adecuada información médica, así como sobre la composición y la calidad de los preparados y sobre su forma de utilización (especialmente en productos no registrados), datos necesarios para establecer la posible relación con un determinado tipo de preparado, su dosificación, duración del tratamiento, o incluso si la causa se encuentra en una adulteración o falsificación

The use of black cohosh preparations has increased in the last years due to its efficacy for the treatment of climacteric symptoms. The recent reports of its possible hepatotoxicity, even considered rare, lead the medical agencies to make recommendations for patients as well as for health professionals on their use. Among more than 40 cases assessed by EMEA, only 4 showed a temporal sequence compatible with the use of the investigated product, although in none of them could be established a causality relationship with an entire certainty. The assessment has been difficulted by the lack of appropriate medical information, as well as on the composition and quality of the concerned products (especially for unlicensed ones), data that are needed for the establishment of a possible relationship with a specific type of preparation, its dose, the duration of the treatment, or even if any adulteration or falsification was involved

Cimicifuga racemosa L., Trifolium pratense L. e Vitex agnus-castus L.: a correspondência das indicações contida nas bulas dos fitoterápicos e o respaldo científico / Cimicifuga racemosa L., Trifolium pratense L. and Vitex agnus-castus L.: the degree of correspondence between instructions supplied with herbal medical preparations and relevant scientific data

É cada vez maior o número de mulheres que utilizam fitoterápicos como terapia alternativa para a menopausa, entretanto, na maioria das vezes esses produtos não possuem um mecanismo de ação conhecido, tão pouco eficácia clinica comprovada. O presente estudo objetiva avaliar a correspondência das indicações clínicas e os achados científicos de três produtos fitoterápicos de elevado índice de vendas na região de Piracicaba, destinados ao alívio dos sintomas da menopausa. Para isso foi realizado um levantamento dos fitoterápicos com maior venda numa farmácia pertencente a uma grande rede e lista das as plantas medicinais que compunham esses medicamentos. Posteriormente, verificou-se no Dicionário de Especialidades Farmacêuticas (DEF) 2004/05 todos fitoterápicos que tinham em sua composição pelo menos uma das três plantas medicinais selecionadas (Cimicifuga racemosa L., Trifolium pratense L. e Vitex agnus-castus L.) e feito um levantamento de como estavam registrados na ANVISA. Para análise da segurança do uso e eficácia dessas plantas medicinais fez-se uma busca em bases de dados científicos MEDLINE procurando trabalhos pré-clínicos de toxicidade e clínicos controlados randomizados que comprovassem a indicação terapêutica da planta. Dentre os resultados encontrados obteve-se que nenhum trabalho sobre toxicidade referia-se a segurança do uso da planta. Quanto a eficácia, das sete indicações propostas pelo fabricante do medicamento que contém a Cimicifugaracemosa L., somente uma das indicações se confirmava em estudo publicado; das nove indicações descritas na bula da Vitex agnus-castus L., duas se confirmavam através de pesquisas realizadas; e, das sete listadas para a Trifolium pratense L., uma foi comprovada.

Inhibitory effects of Cimicifuga heracleifolia extract on glutamate formation and glutamate dehydrogenase activity in cultured islets.

Hyperinsulinism-hyperammonemia syndrome is due either to hyperactivity of GDH or impaired inhibition of GDH by GTP. We have investigated the effect of Cimicifuga heracleifolia extract on the activities of glutamate dehydrogenase (GDH) in cultured rat islets. When the extract was present in the culture medium for 24 h prior to cell harvest, the Vmax of GDH was decreased by 45% with no significant change in Km. In addition, the concentration of alpha-ketoglutarate increased by approximately 39%, and glutamate decreased by 48%. Perfusion of islets with C. heracleifolia extract reduced insulin release by up to 47%. Although the relation between GDH activity and insulin release remains to be clarified, our results suggest that C. heracleifolia extract regulates insulin release by altering GDH activity in primary cultured islets and that this natural compound may be used to modulate GDH activity in patients with hyperinsulinism-hyperammonemia syndrome.