Advancing botanical safety: A strategy for selecting, sourcing, and characterizing botanicals for developing toxicological tools.

Increases in botanical use, encompassing herbal medicines and dietary supplements, have underlined a critical need for an advancement in safety assessment methodologies. However, botanicals present unique challenges for safety assessment due to their complex and variable composition arising from diverse growing conditions, processing methods, and plant varieties. Historically, botanicals have been largely evaluated based on their history of use information, based primarily on traditional use or dietary history. However, this presumption lacks comprehensive toxicological evaluation, demanding innovative and consistent assessment strategies. To address these challenges, the Botanical Safety Consortium (BSC) was formed as an international, cross-sector forum of experts to identify fit-for purpose assays that can be used to evaluate botanical safety. This global effort aims to assess botanical safety assessment methodologies, merging traditional knowledge with modern in vitro and in silico assays. The ultimate goal is to champion the development of toxicity tools for botanicals. This manuscript highlights: 1) BSC's strategy for botanical selection, sourcing, and preparation of extracts to be used in in vitro assays, and 2) the approach utilized to characterize botanical extracts, using green tea and Asian ginseng as examples, to build confidence for use in biological assays.

Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches.

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk. The National Toxicology Program (NTP) observed hepatotoxicity in its rodent studies of a commercially available, unfinished GbE product, but the application of these results to the broader GbE supplement market is unclear. Here, we use a combination of non-targeted and targeted chromatographic and spectrophotometric methods to obtain profiles of 24 commercially available finished GbE products and unfinished standardized and unstandardized extracts with and without hydrolysis, then used principal component analysis to group unfinished products according to their similarity to each other and to National Institute of Standards and Technology (NIST) standard reference materials (SRM), and the finished products. Unfinished products were grouped into those that were characteristic and uncharacteristic of standardized GbE. Our work demonstrates that different analytical approaches produced similar classifications of characteristic and uncharacteristic products in unhydrolyzed samples, but the distinctions largely disappeared once the samples were hydrolyzed. Using our approach, the NTP GbE was most similar to two unfinished GbE products classified as characteristic, finished products, and the NIST GbE SRM. We propose that a simple analysis for the presence, absence, or amounts of compounds unique to GbE in unhydrolyzed samples could be sufficient to determine a sample's authenticity.Graphical abstract.

Pretransplant Desensitization with Costimulation Blockade and Proteasome Inhibitor Reduces DSA and Delays Antibody-Mediated Rejection in Highly Sensitized Nonhuman Primate Kidney Transplant Recipients.

BACKGROUND: Patients with broad HLA sensitization have poor access to donor organs, high mortality while waiting for kidney transplant, and inferior graft survival. Although desensitization strategies permit transplantation via lowering of donor-specific antibodies, the B cell-response axis from germinal center activation to plasma cell differentiation remains intact. METHODS: To investigate targeting the germinal center response and plasma cells as a desensitization strategy, we sensitized maximally MHC-mismatched rhesus pairs with two sequential skin transplants. We administered a proteasome inhibitor (carfilzomib) and costimulation blockade agent (belatacept) to six animals weekly for 1 month; four controls received no treatment. We analyzed blood, lymph node, bone marrow cells, and serum before desensitization, after desensitization, and after kidney transplantation. RESULTS: The group receiving carfilzomib and belatacept exhibited significantly reduced levels of donor-specific antibodies (P=0.05) and bone marrow plasma cells (P=0.02) compared with controls, with a trend toward reduced lymph node T follicular helper cells (P=0.06). Compared with controls, carfilzomib- and belatacept-treated animals had significantly prolonged graft survival (P=0.02), and renal biopsy at 1 month showed significantly reduced antibody-mediated rejection scores (P=0.02). However, four of five animals with long-term graft survival showed gradual rebound of donor-specific antibodies and antibody-mediated rejection. CONCLUSIONS: Desensitization using proteasome inhibition and costimulation blockade reduces bone marrow plasma cells, disorganizes germinal center responses, reduces donor-specific antibody levels, and prolongs allograft survival in highly sensitized nonhuman primates. Most animals experienced antibody-mediated rejection with humoral-response rebound, suggesting desensitization must be maintained after transplantation using ongoing suppression of the B cell response.

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.

Thirdhand smoke beliefs and behaviors among healthcare professionals.

Thirdhand smoke (THS) refers to tobacco smoke contaminants and by-products that remain in the environment after a cigarette is extinguished. The purpose of this study was to assess beliefs and behaviors regarding THS among healthcare professionals, and to examine associations among smoking attitudes/beliefs, provider demographics, and THS beliefs and behaviors. Healthcare professionals (N = 204) at a comprehensive cancer center and affiliated general hospital in a northeastern urban area completed online questionnaires. About one third of the respondents had heard of THS before completing the survey, and more than two thirds of the sample believed that THS issues do not receive enough attention. Being female, likelihood of discussing THS with others, endorsing the belief that smoking affects the quality of parenting, and support for government action towards smoking bans were significantly associated with providers' belief that THS is harmful. Endorsing the belief that smoking affects the quality of parenting and belief that THS is harmful were significantly associated with the likelihood of discussing THS with others. Findings shed light on THS beliefs and behaviors of healthcare providers (a group of individuals that could be trained to educate and advise patients about THS) and inform recommendations for new tobacco policies and clinical guidelines for best practices in tobacco control and prevention.

The estrogenic content of rodent diets, bedding, cages, and water bottles and its effect on bisphenol A studies.

The lowest observed adverse effect level for bisphenol A (BPA) in mice and rats is currently poorly defined due to inconsistent study designs and results in published studies. The objectives of the current study were to (1) compare the estrogenic content of rodent diets, bedding, cages, and water bottles to evaluate their impact on the estrogenic activity of BPA and (2) review the literature on BPA to determine the most frequently reported diets, beddings, cages, and water bottles used in animal studies. Our literature review indicated that low-dose BPA animal studies have inconsistent results and that factors contributing to this inconsistency are the uses of high-phytoestrogen diets and the different routes of exposure. In 44% (76 of 172) of all reports, rodents were exposed to BPA via the subcutaneous route. Our literature review further indicated that the type of diet, bedding, caging, and water bottles used in BPA studies were not always reported. Only 37% (64 of 172) of the reports described the diet used. In light of these findings, we recommend the use of a diet containing low levels of phytoestrogen (less than 20 µg/g diet) and metabolizable energy (approximately 3.1 kcal/g diet) and estrogen-free bedding, cages, and water bottles for studies evaluating the estrogenic activity of endocrine-disrupting compounds such as BPA. The oral route of BPA exposure should be used when results are to be extrapolated to humans.

Mechanistic insights from the NTP studies of chromium.

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

Toxicology and carcinogenesis study of senna in C3B6.129F1-Trp53 tm1Brd N12 haploinsufficient mice.

Senna is a pod or leaf of Senna alexandrina P. Mill and is used as a stimulant laxative. In the large intestine, bacterial enzymes reduce sennosides to rhein-9-anthrone, the active form for the laxative effect. To determine the potential toxic effects of senna, a 5-week dose range finding study in the C57BL/6N mouse and a 40-week toxicology and carcinogenesis study in the C3B6.129F1-Trp53 (tm1Brd) N12 haploinsufficient (p53(+/-)) mouse were conducted. In the 5-week study, C57BL/6N mice were exposed to up to 10,000 ppm senna in feed. Increased incidences of epithelial hyperplasia of the cecum and colon were observed in males and females exposed to 5,000 or 10,000 ppm senna. These intestinal lesions were not considered to be of sufficient severity to cause mortality and, thus, in the p53(+/-) mouse 40-week study, the high dose of 10,000 ppm was selected. Significant increases in the incidences of epithelial hyperplasia of the colon and cecum were observed at 10,000 ppm in p53(+/-) males and females, and the incidence of hyperplasia of the colon was significantly increased at 3,000 ppm in females. In conclusion, the large intestine was the major target of senna-induced toxicity in both wild-type and the p53(+/-) mouse model. There was no neoplastic change when senna was administered to p53(+/-) mouse.

Acute hemorrhagic myocardial necrosis and sudden death of rats exposed to a combination of ephedrine and caffeine.

Because of possible side effects of herbal medicines containing ephedrine and guarana-derived caffeine, including increased risk of stroke, myocardial infarction, and sudden death, the Food and Drug Administration recently banned the sale of ephedra-containing products, specifically over-the-counter dietary supplements. We report cardiac in 7- and 14-week-old male F344 rats exposed by gavage to ephedrine(25 mg/kg) and caffeine (30 mg/kg) administered in combination for one or two days. The ephedrine-caffeine dosage was approximately 12- and 1.4-fold, respectively, above average human exposure, based on a mg/m2 body surface-area comparison. Several (5/7) of the exposed 14-week-old rats died or were sacrificed in extremis 4-5 h after the first dosing. In these hearts, changes were observed chiefly in the interventricular septum but also left and right ventricular walls. Massive interstitial hemorrhage, with degeneration of myofibers, occurred at the subendocardial myocardium of the left ventricle and interventricular septum. Immunostaining for cleaved caspase-3 and hyperphosphorylated H2A.X, a histone variant that becomes hyperphosphorylated during apoptosis, indicated multifocal generalized positive staining of degenerating myofibers and fragmenting nuclei, respectively. The Barbeito-Lopez trichrome stain revealed generalized patchy yellow myofibers consistent with degeneration and/or coagulative necrosis. In ephedrine-caffeine-treated animals terminated after the second dosing, foci of myocardial degeneration and necrosis were already infiltrated by mixed inflammatory cells. The myocardial necrosis may occur secondarily to intense diffuse vasoconstriction of the coronary arterial system with decreased myocardial perfusion. Our work shows the direct relationship between combined ephedrine and caffeine exposure and cardiac pathology.

Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids.

OBJECTIVES: To determine the relative quantities of two hepatotoxic pyrrolizidine alkaloids, symphytine and echimidine, in teas prepared from comfrey leaves (Symphytum officinale), and to determine the potential contribution of the N-oxide forms of these alkaloids to levels of the parent alkaloids. DESIGN: Comfrey leaves were purchased from three commercial sources and used to prepare tea in a manner consistent with the methods used by consumers. An extraction scheme was devised for extraction of the alkaloids, and a gas chromatographic method was developed to quantify the two major alkaloids, symphytine and echimidine. Recognising that the N-oxide derivatives of these alkaloids have also been identified in comfrey preparations, chemical reduction was applied to determine the total quantities of the alkaloids as free bases and as N-oxide derivatives. RESULTS: The concentration of symphytine and echimidine varied considerably between teas prepared from leaves purchased from the different vendors of plant material. Moreover, a much higher concentration of symphytine was found in the tea when steps were included to reduce N-oxides prior to analysis. The treatment of pure symphytine with hot water did not generate the N-oxide derivative de novo. CONCLUSIONS: Since the pyrrolizidine alkaloids are known to be hepatotoxic, consumption of herbal teas made from comfrey leaves may be ill-advised. The concentration of pyrrolizidine alkaloids in such teas may be underestimated substantially unless the concentration of N-oxides is taken into consideration.