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There is limited information on interactions between cannabinoids and many pharmacologically and toxicologically relevant targets in humans (e.g., receptors, ion channels, enzymes, and transporters). To address this data gap, seven cannabinoids were screened against a panel of 44 safety-related biological targets in competitive ligand binding or enzymatic activity assays. Diverse binding profiles were observed among the cannabinoids; however, colloidal aggregates were detected by dynamic light scattering and a detergent-sensitive enzyme inhibition assay. These aggregates may nonspecifically inhibit targets, yielding false positives. Although screening identified aggregates, additional testing is required to confirm cannabinoid aggregation in individual in vitro assays.

The worm Development and Activity Test (wDAT) measures C. elegans developmental milestone acquisition timing and stage-specific spontaneous locomotor activity (SLA). Previously, the wDAT identified developmental delays and SLA level changes in C. elegans with mammalian developmental toxicants arsenic, lead, and mercury. 5-fluorouracil (5FU), cyclophosphamide (CP), hydroxyurea (HU), and ribavirin (RV) are teratogens that also induce growth retardation in developing mammals. In at least some studies on each of these chemicals, fetal weight reductions were seen at mammalian exposures below those that had teratogenic effects, suggesting that screening for developmental delay in a small alternative whole-animal model could act as a general toxicity endpoint to identify chemicals for further testing for more specific adverse developmental outcomes. Consistent with mammalian developmental effects, 5FU, HU, and RV were associated with developmental delays with the wDAT. Exposures associated with developmental delay induced hypoactivity with 5FU and HU, but slight hyperactivity with RV. CP is a prodrug that requires bioactivation by cytochrome P450s for both therapeutic and toxic effects. CP tests as a false negative in several in vitro assays, and it was also a false negative with the wDAT. These results suggest that the wDAT has the potential to identify some developmental toxicants, and that a positive wDAT result with an unknown may warrant further testing in mammals. Further assessment with larger panels of positive and negative controls will help qualify the applicability and utility of this C. elegans wDAT assay within toxicity test batteries or weight of evidence approaches for developmental toxicity assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Consumer use of cannabidiol (CBD) is growing, but there are still data gaps regarding its possible adverse effects on reproduction and development. Multiple pathways and signaling cascades involved in organismal development and neuronal function, including endocannabinoid synthesis and signaling systems, are well conserved across phyla, suggesting that Caenorhabditis elegans can model the in vivo effects of exogenous cannabinoids. The effects in C. elegans on oxidative stress response (OxStrR), developmental timing, juvenile and adult spontaneous locomotor activity, reproductive output, and organismal CBD concentrations were assessed after exposure to purified CBD or a hemp extract suspended in 0.5% sesame oil emulsions. In C. elegans , this emulsion vehicle is equivalent to a high-fat diet (HFD). As in mammals, HFD was associated with oxidative-stress-related gene expression in C. elegans adults. CBD reduced HFD-induced OxStrR in transgenic adults and counteracted the hypoactivity observed in HFD-exposed wild-type adults. In C. elegans exposed to CBD from the onset of feeding, delays in later milestone acquisition were irreversible, while later juvenile locomotor activity effects were reversible after the removal of CBD exposure. CBD-induced reductions in mean juvenile population body size were cumulative when chronic exposures were initiated at parental reproductive maturity. Purified CBD was slightly more toxic than matched concentrations of CBD in hemp extract for all tested endpoints, and both were more toxic to juveniles than to adults. Dosimetry indicated that all adverse effect levels observed in C. elegans far exceeded recommended CBD dosages for humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Camacho, Welch, Ferguson, Sepehr, Vaught, Zhao, Fitzpatrick, Yourick, Sprando and Hunt.)

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 μM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction., (© 2024 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

Since the passage of the 2018 Agriculture Improvement Act (2018 Farm Bill), the number of products containing cannabis-derived compounds available to consumers have rapidly increased. Potential effects on liver function as a result from consumption of products containing cannabidiol (CBD), including hemp extracts, have been observed but the mechanisms for the effects are not fully understood. In this study, hepatocytes derived from human induced pluripotent stem cells (iPSCs) were used to evaluate potential hepatic effects of CBD and hemp extract at exposure concentrations ranging from 0.1 to 30 μM. Despite that a significant reduction in cell viability occurred only in the 30 μM group for both CBD and hemp extract, significant changes to cytochrome P450 activity, mitochondrial membrane potential, and lipid accumulation occurred within the concentration range of 0.1-3 μM for both CBD and hemp extract. Albumin and urea production, caspase 3/7 activity, and intracellular glutathione were significantly affected within the concentration range of 3-30 μM by CBD or hemp extract. These findings indicate that CBD and hemp extract can alter hepatic function and metabolism. The current study contributes data to help inform the evaluation of potential hepatotoxic effects of products containing cannabis-derived compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Cannabidiol (CBD) has been reported to induce hepatotoxicity in clinical trials and research studies; however, little is known about the safety of other nonintoxicating cannabinoids. New approach methodologies (NAMs) based on bioinformatic analysis of high-throughput transcriptomic data are gaining increasing importance in risk assessment and regulatory decision-making of data-poor chemicals. In the current study, we conducted a concentration response transcriptomic analysis of hemp extract and its four major constituent cannabinoids [CBD, cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN)] in hepatocytes derived from human induced pluripotent stem cells (iPSCs). Each compound impacted a distinctive combination of biological functions and pathways. However, all the cannabinoids impaired liver metabolism and caused oxidative stress in the cells. Benchmark concentration (BMC) analysis showed potencies in transcriptional activity of the cannabinoids were in the order of CBN > CBD > CBC > CBG, consistent with the order of their cytotoxicity IC 50 values. Patterns of transcriptomic changes induced by hemp extract and its median overall BMC were very similar to CBD but differed significantly from other cannabinoids, suggesting that potential adverse effects of hemp extract were largely due to its major constituent CBD. Lastly, transcriptomic point-of-departure (tPoD) values were determined for each of the compounds, with the value for CBD (0.106 µM) being concordant with a previously reported one derived from apical endpoints of clinical and animal studies. Taken together, the current study demonstrates the potential utility of transcriptomic BMC analysis as a NAM for hazard assessment of data-poor chemicals, improves our understanding of the possible health effects of hemp extract and its constituent cannabinoids, and provides important tPoD data that could contribute to inform human safety assessment of these cannabinoid compounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 μg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 μg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 μg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

We have adapted a semiautomated method for tracking Caenorhabditis elegans spontaneous locomotor activity into a quantifiable assay by developing a sophisticated method for analyzing the time course of measured activity. The 16-h worm Adult Activity Test (wAAT) can be used to measure C. elegans activity levels for efficient screening for pharmacological and toxicity-induced effects. As with any apical endpoint assay, the wAAT is mode of action agnostic, allowing for detection of effects from a broad spectrum of response pathways. With caffeine as a model mild stimulant, the wAAT showed transient hyperactivity followed by reversion to baseline. Mercury chloride (HgCl 2 ) produced an early dose-response hyperactivity phase followed by pronounced hypoactivity, a behavior pattern we have termed a toxicant "escape response." Methylmercury chloride (meHgCl) produced a similar pattern to HgCl 2 , but at much lower concentrations, a weaker hyperactivity response, and more pronounced hypoactivity. Sodium arsenite (NaAsO 2 ) and dimethylarsinic acid (DMA) induced hypoactivity at high concentrations. Acute toxicity, as measured by hypoactivity in C. elegans adults, was ranked: meHgCl > HgCl 2  > NaAsO 2  = DMA. Caffeine was not toxic with the wAAT at tested concentrations. Methods for conducting the wAAT are described, along with instructions for preparing C. elegans Habitation Medium, a liquid nutrient medium that allows for developmental timing equivalent to that found with C. elegans grown on agar with OP50 Escherichia coli feeder cultures. A de novo mathematical parametric model for adult C. elegans activity and the application of this model in ranking exposure toxicity are presented., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

Recently, there has been an increase in cannabis-derived products being marketed as foods, dietary supplements, and other consumer products. Cannabis contains over a hundred cannabinoids, many of which have unknown physiological effects. Since there are large numbers of cannabinoids, and many are not commercially available for in vitro testing, an in silico tool (Chemotargets Clarity software) was used to predict binding between 55 cannabinoids and 4,799 biological targets (enzymes, ion channels, receptors, and transporters). This tool relied on quantitative structure activity relationships (QSAR), structural similarity, and other approaches to predict binding. From this screening, 827 cannabinoid-target binding pairs were predicted, which included 143 unique targets. Many cannabinoids sharing core structures (cannabinoid "types") had similar binding profiles, whereas most cannabinoids containing carboxylic acid groups were similar without regards to their core structure. For some of the binding predictions (43), in vitro binding data were available, and they agreed well with in silico binding data (median fourfold difference in binding concentrations). Finally, clinical adverse effects associated with 22 predicted targets were identified from an online database (Clarivate Off-X), providing important insights on potential human health hazards. Overall, in silico biological target predictions are a rapid means to identify potential hazards due to cannabinoid-target interactions, and the data can be used to prioritize subsequent in vitro and in vivo testing., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Exposures to arsenic and mercury are known to pose significant threats to human health; however, the effects specific to organic vs. inorganic forms are not fully understood. Caenorhabditis elegans' ( C. elegans ) transparent cuticle, along with the conservation of key genetic pathways regulating developmental and reproductive toxicology (DART)-related processes such as germ stem cell renewal and differentiation, meiosis, and embryonic tissue differentiation and growth, support this model's potential to address the need for quicker and more dependable testing methods for DART hazard identification. Organic and inorganic forms of mercury and arsenic had different effects on reproductive-related endpoints in C. elegans , with methylmercury (meHgCl) having effects at lower concentrations than mercury chloride (HgCl 2 ), and sodium arsenite (NaAsO 2 ) having effects at lower concentrations than dimethylarsinic acid (DMA). Progeny to adult ratio changes and germline apoptosis were seen at concentrations that also affected gravid adult gross morphology. For both forms of arsenic tested, germline histone regulation was altered at concentrations below those that affected progeny/adult ratios, while concentrations for these two endpoints were similar for the mercury compounds. These C. elegans findings are consistent with corresponding mammalian data, where available, suggesting that small animal model test systems may help to fill critical data gaps by contributing to weight of evidence assessments.

Picamilon is an analogue of the neurotransmitter γ-aminobutyric acid (GABA), which is marketed as a nootropic claiming to enhance cognition. There is a lack of in silico, in vitro and in vivo data on the safety of picamilon. Therefore, to ascertain potential physiological effects of picamilon, it was screened against 50 safety-related biological targets (receptors, ion channels, enzymes and transporters) by in silico and in vitro methods. Using two in silico tools, picamilon was not predicted to bind to the targets. Similarly, picamilon exhibited weak or no binding to the targets when measured in vitro at 10 μM. Overall, this data shows that picamilon, although structurally similar to other GABA analogues, has a different biological target binding profile. Picamilon's lack of binding to the 50 targets fills important data gaps among GABA analogues, a group of structurally related substances found in drugs and other consumer products., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

There has been an increased public interest in developing consumer products containing nonintoxicating cannabinoids, such as cannabidiol (CBD) and cannabigerol (CBG). At the present time, there is limited information available on the pharmacokinetics of cannabinoids in humans. Since pharmacokinetic profiles are important in understanding the pharmacological and toxicological effects at the target sites, physiologically based pharmacokinetic (PBPK) modeling was used to predict the plasma and tissue concentrations of 17 cannabinoids in humans. PBPK models were established using measured (in vitro) and predicted (in silico) physicochemical and pharmacokinetic properties, such as water solubility and effective human jejunal permeability. Initially, PBPK models were established for CBD and the model performance was evaluated using reported clinical data after intravenous and oral administration. PBPK models were then developed for 16 additional cannabinoids including CBG, and the plasma and tissue concentrations were predicted after 30 mg oral administration. The pharmacokinetic profiles of the 16 cannabinoids were similar to CBD, and the plasma concentration and time profiles of CBD agreed well with clinical data in the literature. Although low exposure was predicted in the plasma (maximum plasma concentrations < 15 nM), the predicted tissue concentrations, especially the liver (maximum liver concentrations 70-183 nM), were higher after oral administration of 30 mg cannabinoids. These predicted plasma and tissue concentrations could be used to guide further in vitro and in vivo testing., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

Several cannabinoids (cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN) and cannabichromene (CBC)) and ethanol hemp extract are being used in primary human hepatocytes (PHH), Caenorhabditis elegans (C. elegans) and in vitro buccal membrane absorption models to elucidate their potential toxicological mechanisms, evaluate their oromucosal absorption, and to identify their metabolites. William's E medium, C. elegans habitation medium (CeHM), and HEPES-buffered hanks' balanced salt solution (HHBSS) are matrices used with these predictive test systems. Therefore, we developed and validated a sensitive fit-for-purpose ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantitation of CBDV, CBG, CBD, CBN, and CBC in extracellular matrices used with these models for the first time. The separation of the analytes was performed on a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 × 100 mm) protected with a Waters ACQUITY UPLC BEH C18 guard column (130 Å, 1.7 μm, 2.1 × 5 mm). Positive electrospray ionization and multiple reaction monitoring (MRM) modes were used. Under the developed experimental conditions, good linearities were obtained over the concentration range of 0.025-40 µg/ml with coefficients of determination (R 2 ) varying from 0.9953 to 0.9998. The intra-day precisions were between 0.5 and 9.6% with accuracies within ± 16.7%, and the inter-day precisions ranged from 0.6 to 13.1 % with accuracies within ± 13.7%. The method recoveries were between 85.8 and 105.1%. In addition, time-consuming sample preparation was avoided by applying a simple and efficient extraction procedure, which meets the need for potential large-scale routine analysis. The described method was successfully applied to quantitate the analytes in samples produced with different models as well as in ethanolic hemp extract., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Variability in supply, paucity of donors and cellular instability under in vitro conditions have limited the application of primary human hepatocytes (PHHs) to hepatotoxicity testing. Therefore, alternative sources have been sought for functional liver cells. Many of the earlier in vitro hepatotoxicity studies were carried out using hepatoma-derived cell lines. These cell lines have overcome some of the limitations of PHHs with regard to phenotypic stability and availability; however, they suffer from their own inherent limitations, such as the lack of drug-metabolizing functionality, which renders them inadequate for situations where toxic metabolite formation of the parent drug occurs. In the last decade we have witnessed a burgeoning interest of the research community in using hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) as in vitro hepatotoxicity models. HLCs offer the perspective of a defined and renewable supply of functional hepatocytes; more importantly, HLCs maintain their original donor genotype and afford donor diversity, thus opening new avenues to patient-specific toxicity testing. In this review, we first introduce various in vitro hepatotoxicity models, then focus on HLCs and their application in hepatotoxicity studies, and finally offer some perspectives on future developments of the field.

A variety of methods have been reported for the differentiation of hepatocyte-like cells (HLCs) from human induced pluripotent stem cells (iPSCs) using various growth factors or small molecules. However, direct comparison of the differentiation efficiency and the quality of the final HLCs between different methods has rarely been reported. To fill this data gap, we compared two hepatocyte differentiation methods, termed Method 1 and Method 2, and published the major findings in a research article entitled "Phenotypical, functional and transcriptomic comparison of two modified methods of hepatocyte differentiation from human induced pluripotent stem cells" (Li et al., 2022). The current data article describes the transcriptomic dataset comparing the two methods. HLCs were collected at early maturation (day 17) and late maturation (day 21) stages of the differentiation and total RNA were isolated. Global gene expression profiling of the HLCs was conducted using Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Primary human hepatocytes (PHHs) were also included for comparison. The microarray dataset has been deposited in the Gene Expression Omnibus of the National Center for Biotechnology Information with accession number GSE187011. Detailed interpretation and discussion of the data can be found in the corresponding research article (Li et al., 2022). This dataset is useful in providing a molecular basis for the differences observed between the two differentiation methods, offering new insights into gene regulations in hepatogenesis in vitro , and suggesting ways to further improve hepatocyte differentiation in order to obtain more mature HLCs for biomedical applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Exposures to mercury and arsenic are known to pose significant threats to human health. Effects specific to organic vs. inorganic forms of these toxic elements are less understood however, especially for organic dimethylarsinic acid (DMA), which has recently been detected in pups of rodent dams orally exposed to inorganic sodium (meta)arsenite (NaAsO2). Caenorhabditis elegans is a small animal alternative toxicity model. To fill data gaps on the effects of DMA relative to NaAsO2, C. elegans were exposed to these two compounds alongside more thoroughly researched inorganic mercury chloride (HgCl2) and organic methylmercury chloride (meHgCl). For timing of developmental milestone acquisition in C. elegans , meHgCl was 2 to 4-fold more toxic than HgCl2, and NaAsO2 was 20-fold more toxic than DMA, ranking the four compounds meHgCl > HgCl2 > NaAsO2 ≫ DMA for developmental toxicity. Methylmercury induced significant decreases in population locomotor activity levels in developing C. elegans . DMA was also associated with developmental hypoactivity, but at >100-fold higher concentrations than meHgCl. Transcriptional alterations in native genes were observed in wild type C. elegans adults exposed to concentrations equitoxic for developmental delay in juveniles. Both forms of arsenic induced genes involved in immune defense and oxidative stress response, while the two mercury species induced proportionally more genes involved in transcriptional regulation. A transgenic bioreporter for activation of conserved proteosome specific unfolded protein response was strongly activated by NaAsO2, but not DMA at tested concentrations. HgCl2 and meHgCl had opposite effects on a bioreporter for unfolded protein response in the endoplasmic reticulum. Presented experiments indicating low toxicity for DMA in C. elegans are consistent with human epidemiologic data correlating higher arsenic methylation capacity with resistance to arsenic toxicity. This work contributes to the understanding of the accuracy and fit-for-use categories for C. elegans toxicity screening and its usefulness to prioritize compounds of concern for further testing., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into hepatocytes could provide an unlimited source of liver cells, and therefore holds great promise for regenerative medicine, disease modeling, drug screening and toxicology studies. Various methods have been established during the past decade to differentiate human iPSCs into hepatocyte-like cells (HLCs) using growth factors and/or small molecules. However, direct comparison of the differentiation efficiency and the quality of the final HLCs between different methods has rarely been reported. In the current study, two hepatocyte differentiation methods were devised, termed Method 1 and 2, through modifying existing well-known hepatocyte differentiation strategies, and the resultant cells were compared phenotypically and functionally at different stages of hepatocyte differentiation. Compared to Method 1, higher differentiation efficiency and reproducibility were observed in Method 2, which generated highly homogeneous functional HLCs at the end of the differentiation process. The cells exhibited morphology closely resembling primary human hepatocytes and expressed high levels of hepatic protein markers. More importantly, these HLCs demonstrated several essential characteristics of mature hepatocytes, including major serum protein (albumin, fibronectin and α-1 antitrypsin) secretion, urea release, glycogen storage and inducible cytochrome P450 activity. Further transcriptomic comparison of the HLCs derived from the two methods identified 1,481 differentially expressed genes (DEGs); 290 Gene Ontology terms in the biological process category were enriched by these genes, which were further categorized into 34 functional classes. Pathway analysis of the DEGs identified several signaling pathways closely involved in hepatocyte differentiation of pluripotent stem cells, including 'signaling pathways regulating pluripotency of stem cells', 'Wnt signaling pathway', 'TGF-beta signaling pathway' and 'PI3K-Akt signaling pathway'. These results may provide a molecular basis for the differences observed between the two differentiation methods and suggest ways to further improve hepatocyte differentiation in order to obtain more mature HLCs for biomedical applications., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Li et al.)

Silver nanoparticles (AgNPs) have been increasingly used in a variety of consumer products over the last decades. However, their potential adverse effects have not been fully understood. In a previous study, we characterized transcriptomic changes in human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) in response to AgNP exposure. Here, we report findings of a follow-up proteomic study that evaluated alternations at the protein level in the same cell after being exposed to 10 μg/ml AgNPs for 24 h. In total, 6287 proteins were identified across two groups of samples (n = 3). Among these proteins, 665 were found to be differentially regulated (fold change ≥1.25, p < 0.01) between the AgNP-treated group and the untreated control group, including 264 upregulated and 401 downregulated. Bioinformatics analysis of the proteomics data, in side-by-side comparison to the transcriptomics data, confirms and substantiates previous findings on AgNP-induced alterations in metabolism, oxidative stress, inflammation, and potential association with cancer. A mechanism of action was proposed based on these results. Collectively, the findings of the current proteomic study are consistent with those of the previous transcriptomic study and further demonstrate the usefulness of iPSC-derived HLCs as an in vitro model for liver nanotoxicology., (Published by Elsevier Ltd.)

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (iPSCs) could provide an unlimited source of liver cells for regenerative medicine, disease modeling, drug screening, and toxicology studies. Here we describe a stepwise improved protocol that enables highly efficient, homogeneous, and reproducible differentiation of human iPSCs into functional hepatocytes through controlling all three stages of hepatocyte differentiation, starting from a single cell (non-colony) culture of iPSCs, through homogeneous definitive endoderm induction and highly efficient hepatic specification, and finally arriving at matured HLCs. The final population of cells exhibits morphology closely resembling that of primary human hepatocytes, and expresses specific hepatic markers as evidenced by immunocytochemical staining. More importantly, these HLCs demonstrate key functional characteristics of mature hepatocytes, including major serum protein (e.g., albumin, fibronectin, and alpha-1 antitrypsin) secretion, urea synthesis, glycogen storage, and inducible cytochrome P450 activity., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Human induced pluripotent stem cells (iPSCs) hold great promise for biomedical applications. However, establishment of new iPSC lines still presents many challenges. Here we describe a simple yet highly efficient two-step protocol for the isolation and purification of human iPSC lines. The first step adapts iPSCs to single cell culture and passaging, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Both steps utilize commercially available reagents. Using this protocol, iPSCs can be purified from cell preparations containing differentiated or unreprogrammed cells, or even be isolated directly from reprogramming vessels. The protocol could be adopted for high throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissue cells with donor diversity therefore promising to have widespread applications in regenerative medicine, disease modeling, drug discovery, and toxicity testing. Several somatic cell types have been utilized, with varying efficiencies, as source cells for the reprogramming of iPSCs. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation, and possess several advantages over other cell types. In this chapter, we describe a protocol that covers all steps of reprogramming iPSCs from blood-derived EPCs, including (1) isolation of mononuclear cells (MNCs) from blood samples, (2) derivation of EPCs from MNCs, and (3) generation of iPSCs from EPCs. The final step of reprogramming EPCs into iPSCs is achieved through ectopic expression of four transcription factors, OCT4, KLF4, SOX2, and c-MYC, using self-replicative RNA (srRNA) technology., (© 2021. Springer Science+Business Media, LLC.)

Anthraquinones exhibit various pharmacological activities (e.g., antioxidant and laxative) and are commonly found in consumer products including foods, dietary supplements, drugs, and traditional medicines. Despite their widespread use, there are limited data available on their toxicokinetic properties. Cytochrome P450 enzymes (CYPs) in the liver play major roles in metabolizing exogenous chemicals (e.g., pharmaceuticals, food ingredients, and environmental pollutants) and endogenous biomolecules (e.g., steroid hormones and cholesterol). Inhibition of CYP activities may lead to serious interactions among these compounds. Here, in silico (quantitative structure-activity relationship modeling) and in vitro (human recombinant enzymes and liver microsomes) methods were used to identify inhibitors of five major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) among 22 anthraquinones. First, in silico prediction and in vitro human recombinant enzyme assays were conducted for all compounds, and results showed that most of the anthraquinones were potent CYP1A2 inhibitors. Second, five selected anthraquinones (emodin, aloe-emodin, rhein, purpurin, and rubiadin) were further evaluated in human liver microsomes. Finally, plasma concentrations of the five anthraquinones in animal and humans were identified in the literature and compared to their in vitro inhibition potency (IC 50 values) towards CYP activities. Emodin, rhein, and aloe-emodin inhibited activities of multiple CYPs in human liver microsomes and potential in vivo inhibition may occur due to their high plasma concentrations. These in silico and in vitro results enabled rapid identification of potential CYP inhibitors and prioritized future in-depth studies., (© Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

The application of silver nanoparticles (AgNPs) in consumer products has been increasing rapidly over the past decades. Therefore, in vitro models capable of accurately predicting the toxicity of AgNPs are much needed. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) represent an attractive alternative in vitro hepatotoxicity model. Yet, the use of iPSC-derived HLCs (iPSC-HLCs) for the study of nanoparticle toxicity has not been reported so far. In the present study, transcriptomic changes induced by varying concentrations (5-25 μg/ml) of AgNPs were characterized in iPSC-HLCs after 24-h exposure. AgNPs caused concentration-dependent gene expression changes in iPSC-HLCs. At all the concentrations, members of the metallothionein (MT) and the heat shock protein (HSP) families were the dominating upregulated genes, suggesting that exposure to AgNPs induced oxidative stresses in iPSC-HLCs and as a result elicited cellular protective responses in the cells. Functional analysis showed that the differentially expressed genes (DEGs) were majorly involved in the biological processes of metabolism, response to stress, and cell organization and biogenesis. Ingenuity Pathway Analysis revealed that cancer was at the top of diseases and disorders associated with the DEGs at all concentrations. These results were in accordance with those reported previously on hepatoma cell lines and primary hepatocytes. Considering the advantages iPSC-HLCs have over other liver cell models in terms of unlimited supply, consistency in quality, sustainability of function in long-term culture, and, more importantly, affordability of donor specificity, the results of the current study suggest that iPSC-HLCs may serve as a better in vitro model for liver nanotoxicology.

A routine, selective and sensitive ultra-high performance liquid chromatography-electrospray ionisation tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the quantification of 3-monochloropropane-1,2-diol (3-MCPD) in Caco-2 cell transport buffer (FaSSIF-V2, the second version of a fasted state simulated intestinal fluid) and receiving buffer (HBSS, Hank's balanced salt solution). The method involves measuring deuterated 3-MCPD (3-MCPD- d5 ) as internal standard (IS) during the entire analytical procedure to obtain precise and accurate results. The separation was performed on a Poroshell 120 HILIC column (2.7  µ m, 3.0 × 50 mm) at a flow rate of 0.3 mL/min using water (containing 0.025% acetic acid) and acetonitrile (containing 0.025% acetic acid) as the mobile phases. Mass spectrometric detection was operated in dynamic multiple reaction monitoring (dMRM) in negative ion mode. The method exhibited high sensitivity. The limits of detection (LOD) for 3-MCPD in FaSSIF-V2 and HBSS were 0.012 and 0.014  µ mol/L, and the limits of quantification (LOQ) were 0.039 and 0.045  µ mol/L, respectively. Satisfactory results were observed for linearity ( R 2   > 0.999), intra-day precision ( RSD % <7.7% in FaSSIF-V2 and <6.6% in HBSS), inter-day precision ( RSD % <5.9% in FaSSIF-V2 and <5.6% in HBSS), accuracy (% error within ± 10%), and sample stability ( RSD % <7.7% and % error within ± 10%). The method has been successfully applied to quantify 3-MCPD in Caco-2 cell transport and receiving buffers. The results were in good agreement with those obtained with gas chromatography-tandem mass spectrometry (GC-MS).

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in some infant formula products and other foods in the United States. Although rodent studies have demonstrated that 3-MCPD and its palmitic esters have the potential to induce nephrotoxicity, our recent human cell culture studies using the human renal proximal tubule cell line HK-2 have not strongly supported this finding. Considering this disparity, we sought to examine whether changes in transporter gene expression on proximal tubule cells could be modulated by these compounds and allow us to glean mechanistic information on a possible indirect path to proximal tubule injury in vivo . If fundamental processes like water and solute transport could be disrupted by 3-MCPD compounds, then a new avenue of toxicity could be further explored in both infant and adult models. In our current study, we used HK-2 cells as an in vitro cellular model of human proximal tubule cells to investigate the effects of low (10 μM) and high (100 μM) 3-MCPD compound exposures to these cells for 24 hours (h) on the expression of 20 transporter genes that are known to be relevant to proximal tubules. Although we detected consistent upregulation of AQP1 expression at the RNA transcript level following HK-2 treatment with both low and high doses of several ester-bound 3-MCPD compounds, these increases were not associated with statistically significant elevations in their protein expression levels. Moreover, we observed a lack of modulation of other members of the AQP protein family that are known to be expressed by human proximal tubule cells. Overall, our study suggests the possibility that 3-MCPD-related nephrotoxicity could be associated with indirect modes of action relating to aquaporin homeostasis, but additional studies with other human-derived models would be pertinent to further explore these findings and to better understand transporter expression differences under different stages of proximal tubule development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

Background: Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level., Methods: HLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays., Results: HLCs derived using the SM protocol (HLC&#95;SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC&#95;SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC&#95;GF), and commercially available HLCs. Similar to HLC&#95;GF, HLC&#95;SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC&#95;SM compared to HLC&#95;GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs., Conclusions: Overall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo , we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD esters are contaminants present in a variety of processed foods, including infant formulas. Toxicological data are unavailable in humans, but rodent studies have demonstrated renal and testicular toxicity from 3-MCPD and 3-MCPD esters. There is evidence that 3-MCPD esters are hydrolyzed in the digestive system, releasing 3-MCPD that would be absorbed and induce damage. We assessed absorption and metabolism of 3-MCPD and three 3-MCPD monoesters, 1-oleoyl (1-Ol), 1-linoleoyl (1-Li) and 1-palmitoyl (1-Pa) commonly found in U.S. infant formula using differentiated Caco-2 cells. After 1-hour incubation, all three monoesters released free 3-MCPD and free fatty acids (FFA) into Caco-2 cell supernatants. Free 3-MCPD had a high apparent permeability (P app  = 30.36 ± 1.31 cm/s × 10 -6 ) suggesting that it is freely diffusible and highly absorbed by intestinal epithelium. 1-Li released 3-4-fold more 3-MCPD than 1-Ol and 1-Pa over 1 h, suggesting that this variable release rates might contribute to the overall in vivo exposure to 3-MCPD. None of the monoesters or FFA were detected in basolateral supernatants, suggesting that these compounds do not cross the intestinal wall without further transformation. In summary, this study provides relevant data to advance knowledge of in vivo intestinal absorption and metabolism of 3-MCPD monoesters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Anthraquinones are found in a variety of consumer products such as dietary supplements, traditional Chinese medicines, and drugs. Along with their widespread use, potential safety concerns have emerged, especially liver toxicity. Therefore, there is a need to conduct rapid and inexpensive safety assessment for anthraquinones due to a lack of animal and human toxicological data. Here, a combined in vitro cytotoxicity and in silico reverse dosimetry approach was adopted to consider the potential human liver toxicity of 16 anthraquinones and derivatives. First, cytotoxicity (EC 50 ) in two human liver cell lines (HepG2/C3A and HuH-7) was measured under two conditions (single and repeated dosing, 72 h). Second, toxic doses (D tox ) required to yield plasma steady-state concentrations (C ss ) equal to in vitro EC 50 values were predicted by reverse dosimetry simulation using a PBPK model. Finally, D tox was compared to literature-derived estimated daily intake (EDI) of anthraquinones to assess safety. Among the 16 anthraquinones, rhein was identified as a potential hepatotoxicant due to a combination of cytotoxicity, plasma concentration, and daily intake level. These in vitro and in silico findings provide preliminary data and guidance for further animal and clinical studies to confirm liver toxicity of anthraquinones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Chloropropanols are chemical contaminants that can be formed during industrial processing of foods, such as lipids used in commercially available infant and toddler formula in the USA. Many researchers have studied the most common chloropropanol contaminant, 3-monochloropropane-1,2-diol (3-MCPD), as well as its lipid ester derivatives. A plethora of toxicological outcomes have been described in vivo, including effects on the heart, nervous system, reproductive organs, and kidneys. To better understand the concordance of some of these effects to in vitro outcomes, we focused our research on using an in vitro cellular model to investigate whether the proximal tubule cells of the kidney would be vulnerable to the effects of free 3-MCPD and nine of its common esters in commercial formula. Using the established human kidney proximal tubule cell line, HK-2, we performed 24-h treatments using 3-MCPD and nine mono- or di-esters derived from palmitate, oleate, and linoleate. By directly exposing HK-2 cells at treatment doses ranging from 0 to 100 μM, we could evaluate their effects on cell viability, mitochondrial health, reactive oxygen species (ROS) production, and other endpoints of toxicity. Since chloropropanols reportedly inhibit cellular metabolism through interference with glycolysis, we also tested the extent of this mechanism. Overall, we found mild but statistically significant evidence of cytotoxicity at the highest tested treatment concentrations, which were also associated with mitochondrial dysfunction and transient perturbations in cellular metabolism. Based on these findings, further studies will be required to better understand the effects of these compounds under conditions that are more physiologically relevant to human infant and toddler proximal tubules in order to mimic their exposure to chloropropanol-containing foods.

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

Fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD) are chemical contaminants found in a wide range of edible oils that are thermally processed during industrial manufacturing of infant formula and other lipid-containing foods in the United States. Rodent studies have unequivocally demonstrated a plethora of in vivo toxicological effects including reproductive, neurological and renal dysfunction. To determine if similar effects are observed in human organ systems, in vitro studies using human cell lines are conducted to assess concordance of the data collected. One limitation to performing such in vitro research is the extremely high hydrophobicity of 3-MCPD esters; dissolving them into aqueous cell culture media is a tremendous challenge. To address this obstacle, we developed a simple protocol to circumvent the immiscibility of 3-MCPD esters and their corresponding free fatty acids into aqueous cell culture media in order to assess the effect of these esters on epithelial cells of kidney origin in vitro ., (© 2019 Published by Elsevier B.V.)

Due to the high cost and long duration of traditional testing methods for developmental neurotoxicity (DNT), only a small fraction of chemicals that humans are exposed to have been assessed for DNT activity. In order to ensure public safety, human-predictive methods for DNT detection that are faster and less resource intensive are urgently required. Using Caenorhabditis elegans, a novel worm Development and Activity test (wDAT) has been designed that uses a relatively inexpensive small-animal activity tracker and takes less than 4 days to complete. The wDAT was able to detect both developmental delay and hyperactivity for arsenic, lead, and mercury, heavy metals that are known human developmental neurotoxins and have been associated with hyperactivity in children. Lithium was also tested as a control developmental toxin that is not considered a mammalian neurotoxin. With the wDAT, lithium induced developmental delay but not hyperactivity. This initial assessment of a new assay for DNT detection indicates that the wDAT has potential for detecting at least some types of mammalian developmental neurotoxins. A planned 20-compound validation study will clarify the utility of the wDAT for predicitive toxicology., (Published by Elsevier Ltd.)

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings., (Published by Elsevier Ltd.)

Human-induced pluripotent stem cells (iPSCs) hold considerable promise for future biomedical applications. However, the generation, isolation, and establishment of an iPSC line still presents many challenges. In this study, we describe a simple yet highly efficient two-step method for the isolation, purification, and passaging of human iPSC lines that utilizes commercially available reagents. The first step adapts iPSCs to single cell culture and passage, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Using this method, we were able to purify iPSCs from cell preparations containing differentiated or unreprogrammed cells, and even to isolate iPSC lines directly from derivation plates. The iPSC lines generated by this method maintained their pluripotency and genomic stability, as demonstrated by trilineage differentiation and karyotype analysis. The method presented here could be adopted for high-throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited source of differentiated cells from individuals with specific genetic backgrounds. Using self-replicative RNA reprogramming technology, we generated nine iPSC lines from endothelial progenitor cells (EPCs) derived from blood samples of three different ethnicities: Black or African American, Latino or Hispanic, and Non-Hispanic White. The resulting iPSC lines showed normal karyotype in large part, expressed pluripotency marker genes, and spontaneously differentiated in vitro into the three germ layers. These iPSC lines offer the potential to generate tissues with ethnic diversity, and thus afford a valuable tool for ethnic-related toxicological applications., (Published by Elsevier B.V.)

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs) and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5-7days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications., (Published by Elsevier B.V.)

Effects of oral silver acetate exposure were assessed in P generation and F generation post-natal day 26 rats. Male and female Sprague Dawley rats (n = 20 each) were exposed to silver acetate at 0.4, 4.0 or 40.0 mg/kg bw in their drinking water for 10 weeks prior to and during mating. Females were exposed to silver acetate throughout gestation and lactation. Clinical signs, body weight, feed and fluid consumption were recorded regularly. Decreased mean daily fluid consumption was observed in male and female animals during the 10 week pre mating period and during gestation in the 40 mg/kg bw dose group. Decreased fertility was observed in the 40 mg/kg bw dose group. Decreased feed consumption was observed across all dose groups and decreased mean daily fluid consumption was observed in the 4.0 mg/kg dose group during lactation. Decreased implant numbers, mean numbers of pups born/litter and numbers of live pups born/litter was observed in the 40 mg/kg bw dose group. Pup weight was reduced on lactation days 0, 4 and 7 (males) and 4, 7 and 21 (females) in the 4.0 mg/kg bw dose group and in males at lactation day 21 (40 mg/kg bw dose group). Runting was observed in males (Lactation Day; LD 4) and female (LD 4 and 7) animals in the 4.0 mg/kg bw dose group. Reduced postnatal-day 26 pup weight was observed in male pups in the 40 mg/kg bw dose group and female pups in the 4.0 mg/kg bw dose group., (Published by Elsevier Ltd.)

The acute oral toxicity of diglycolic acid (DGA) was evaluated. Groups of female rats (n = 8 rats/group) received 28 consecutive daily single doses of 0.3, 1.0, 3.0, 10.0, 30.0, 100.0 or 300.0 mg DGA/kg body weight by gastric intubation. One group of animals served as vehicle control. Tissues and blood serum were collected at necropsy on day 29. Select organs were weighed and fixed in formalin for histopathological analysis. Animals from the 300 mg/kg bw dose group were removed from the study after 5 consecutive days of treatment as a consequence of adverse treatment related effects. The animals in the remaining treatment groups survived the exposure period. No adverse clinical signs were observed throughout the exposure period in the surviving animals. No significant differences from controls were observed for feed and fluid consumption or body weight gain in the surviving animals. Lesions were observed in the kidneys, liver, stomach, intestine, thymus, spleen and bone marrow in rats from the 300 mg/kg dose group and signs of renal tubular regeneration were observed only in the 100 mg/kg dose group. These results suggest that high levels of pure DGA would need to be consumed before renal and other forms of organ toxicity are observed., (Published by Elsevier Ltd.)

Diglycolic acid (DGA) is present in trace amounts in our food supply and is classified as an indirect food additive linked with the primary GRAS food additive carboxymethyl cellulose (CMC). Carboxymethyl starches are used as a filler/binder excipient in dietary supplement tablets and a thickening ingredient in many other processed foods. We sought to utilize the human proximal tubule HK-2 cell line as an in vitro cellular model system to evaluate its acute nephrotoxicity of DGA. We found that DGA was indeed toxic to HK-2 cells in all in vitro assays in our study, including a highly sensitive Luminex assay that measures levels of an in vitro biomarker of kidney-specific toxicity, Kidney Injury Molecule 1 (KIM-1). Interestingly, in vitro KIM-1 levels also correlated with in vivo KIM-1 levels in urine collected from rats treated with DGA by daily oral gavage. The use of in vitro and in vivo models towards understanding the effectiveness of an established in vitro system to predict in vivo outcomes would be particularly useful in rapidly screening compounds that are suspected to be unsafe to consumers. The merit of the HK-2 cell model in predicting human toxicity and accelerating the process of food toxicant screening would be especially important for regulatory purposes. Overall, our study not only revealed the value of HK-2 in vitro cell model for nephrotoxicity evaluation, but also uncovered some of the mechanistic aspects of the human proximal tubule injury that DGA may cause.

Paenibacillus alvei, a naturally occurring soil microorganism, may be used in the control and/or elimination of human/animal pathogens present on/within produce commodities associated with human consumption. The safety of oral exposure to P. alvei in male, nulliparous females, the pregnant dam and developing fetus was assessed. Adult male and female rats received a single oral dose (gavage) of P. alvei and tissues were collected at post exposure days 0, 3 and 14. To evaluate the effect of the test organism on fetal development, sperm positive female rats received the test organism every 3 days thereafter throughout gestation. As human exposure would be no more than 1 × 10 3  CFU/ml the following dose levels were evaluated in both study phases: 0 CFU/ml tryptic soy broth (negative control); 1 × 10 8  CFU/ml; 1 × 10 4  CFU/ml or 1 × 10 2  CFU/ml. Neither sex specific dose-related toxic effects (feed or fluid consumption, body weight gain, and histopathology) nor developmental/reproductive effects including the number of implantations, fetal viability, fetal weight, fetal length and effects on ossification centers were observed. The test organism did not cross the placenta and was not found in the amniotic fluid., (Published by Elsevier Ltd.)

Background: The widespread application of silver nanoparticles (AgNPs) and silver-containing products has raised public safety concerns about their adverse effects on human health and the environment. To date, in vitro toxic effects of AgNPs and ionic silver (Ag + ) on many somatic cell types are well established. However, no studies have been conducted hitherto to evaluate their effect on cellular transcriptome in embryonic stem cells (ESCs)., Results: The present study characterized transcriptomic changes induced by 5.0 µg/ml AgNPs during spontaneous differentiation of mouse ESCs, and compared them to those induced by Ag + under identical conditions. After 24 h exposure, 101 differentially expressed genes (DEGs) were identified in AgNP-treated cells, whereas 400 genes responded to Ag + . Despite the large differences in the numbers of DEGs, functional annotation and pathway analysis of the regulated genes revealed overall similarities between AgNPs and Ag + . In both cases, most of the functions and pathways impacted fell into two major categories, embryonic development and metabolism. Nevertheless, a number of canonical pathways related to cancer were found for Ag + but not for AgNPs. Conversely, it was noted that several members of the heat shock protein and the metallothionein families were upregulated by AgNPs but not Ag + , suggesting specific oxidative stress effect of AgNPs in ESCs. The effects of AgNPs on oxidative stress and downstream apoptosis were subsequently confirmed by flow cytometry analysis., Conclusions: Taken together, the results presented in the current study demonstrate that both AgNPs and Ag + caused transcriptomic changes that could potentially exert an adverse effect on development. Although transcriptomic responses to AgNPs and Ag + were substantially similar, AgNPs exerted specific effects on ESCs due to their nanosized particulate form.

Male and female rats (26-day old) were exposed to 0.0, 0.4, 4 or 40 mg/kg body weight silver acetate (AgAc) in drinking water for 10 weeks prior to and during mating. Sperm positive females remained within their dose groups and were exposed to AgAc during gestation and lactation. Splenic and thymic lymphocyte subsets from F1 generation PD (postnatal day) 4 and 26 pups were assessed by flow cytometry for changes in phenotypic markers. Spleens from PD4 pups had lower percentages of CD8 + lymphocytes in 4 and 40 mg/kg AgAc exposed groups and reduced Concanavalin A (Con A) response at all AgAc exposure groups. Splenic maturation increased in PD26 pups compared to PD4 pups. Con A and lipopolysaccharide (LPS) mediated splenic responses were lower in PD26 pups exposed to 40 mg/kg AgAc. Changes in PD 26 pup splenocyte phenotypic markers included lower TCR + cells at 4 and 40 mg/kg AgAc exposure and higher B cell population in the 40 mg/kg AgAc. PD26 pup splenic natural killer cell (NK) activity was higher in the 0.4 AgAc group and unchanged in 4 and 40 mg/kg AgAc groups. In conclusion, maternal exposure to AgAc had a significant impact on rat splenic development during the early lactation period., (Published by Elsevier Ltd.)

A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. "Dream herb," Calea zacatechichi , has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi for safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement.

Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

Adhatoda zeylanica is a dietary supplement ingredient present in several types of dietary supplements, including weight loss, respiratory relief, and immune regulating products. Due to its reported wide range of uses in folk medicine, it was hypothesized that it may have the potential to target multiple organs and lead to a range of toxicity features. As a preliminary evaluation of the safety of this herbal ingredient, an investigation into its effects on the kidney was sought. An in vitro study of its potential nephrotoxicity using the HK-2 human proximal tubule cell line in a variety of functional indicators was performed to capture both general forms of cellular toxicity as well as ones that are specific to proximal tubules. A. zeylanica was only capable of inducing detrimental short-term toxicity to HK-2 cells at relatively high treatment concentrations when exposed directly to the cells. The lack of acute and potent toxicity of A. zeylanica under our experimental conditions calls for further studies to better define its toxicant threshold and establish safe dosage levels.

The increased use of silver nanoparticles (AgNPs) in foods and cosmetics has raised public safety concerns. However, only limited knowledge exists on the effect of AgNPs on the cellular transcriptome. This study evaluated global gene expression profiles of human liver HepG2 cells exposed to 20 and 50 nm AgNPs for 4 and 24 h at 2.5 µg ml(-1) . Exposure to 20 nm AgNPs resulted in 811 altered genes after 4 h, but much less after 24 h. Exposure to 50 nm AgNPs showed minimal altered genes at both exposure times. The HepG2 cells responded to the toxic insult of AgNPs by transiently upregulating stress response genes such as metallothioneins and heat shock proteins. Functional analysis of the altered genes showed more than 20 major biological processes were affected, of which metabolism, development, cell differentiation and cell death were the most dominant categories. Several cellular pathways were also impacted by AgNP exposure, including the p53 signaling pathway and the NRF2-mediated oxidative stress response pathway, which may lead to increased oxidative stress and DNA damage in the cell and potentially result in genotoxicity and carcinogenicity. Together, these results indicate that HepG2 cells underwent a multitude of cellular processes in response to the toxic insult of AgNP exposure, and suggest that toxicogenomic characterization of human HepG2 cells could serve as an alternative model for assessing toxicities of NPs., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment., (Published by Elsevier Inc.)

Thalidomide is a potent developmental toxicant that induces a range of birth defects, notably severe limb malformations. To unravel the molecular mechanisms underpinning the teratogenic effects of thalidomide, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on the differentiation of mouse embryonic stem cells (mESCs), and published the major findings in a research article entitled "Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells" [1]. The data presented herein contains complementary information related to the aforementioned research article.