Caenorhabditis elegans is an alternative model organism for toxicology research that aligns with the 3Rs principle (Replace, Reduce, Refine) and new approaches to modernize toxicity testing strategies. This study developed and single-laboratory validated a sensitive ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for quantifying five cannabinoids- cannabidivarin, cannabigerol, cannabidiol, cannabinol, and cannabichromene- in juvenile and adult C. elegans matrices. Homogenized C. elegans samples were spiked with labeled internal standards and subjected to protein precipitation. The resulting supernatants were injected onto a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm), coupled to an Agilent 6460 Triple Quadrupole mass spectrometer detector, allowing for the detection and quantification of target analytes in a total run time of 14 min using a 50 µL sample volume. Single-laboratory method validation was performed using spiked quality control samples, consisting of six replicates at four concentrations of all cannabinoids in juvenile and adult C. elegans matrices, analyzed over three consecutive days. The validated method demonstrated linear regression calibration curves with R2 ≥ 0.99 across the concentration range of 0.1-7.5 µg/mL for all cannabinoids. The intra-day accuracy in juvenile and adult C. elegans was within 85-108% and 97-109% of the fortified concentration, respectively, with intra-day precision between 1.28-4.62% relative standard deviation (RSD) and 0.64-5.60% RSD. Similarly, the inter-day accuracy was within 86-110% and 97-106% of the fortified concentration, respectively, with the inter-day precision between 2.06-5.62% RSD and 1.42-10.9% RSD. This single laboratory validated UHPLC-ESI-MS/MS method provides accurate and reliable cannabinoid quantification and facilitates the translational utility of cannabinoid toxicity testing in C. elegans. Analytical method development to accurately detect chemicals within diverse tissue matrices supports dosimetry and toxicokinetic analyses, essential steps toward investigating food chemical safety.

A variety of health benefits have been claimed, but not scientifically confirmed, for cannabidiol (CBD) and other nonintoxicating cannabinoids in hemp extracts such as cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN). On the other hand, CBD has been reported to cause hepatotoxicity in clinical trials and research studies, while little is known about the safety of other cannabinoids. In the current study, we set out to understand the mechanism(s) of action of these cannabinoids, beneficial or adverse, through a comprehensive functional analysis of a recently published transcriptomic dataset. iPSC-derived hepatocytes were exposed to 40 µM CBC, 20 µM CBD, 40 µM CBG, and 10 µM CBN, respectively, for 24 h. Gene expression changes were profiled using Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Functional analysis was conducted using Ingenuity Pathway Analysis. Each compound impacted a unique list of canonical pathways, upstream regulators, diseases and biological functions, toxicity functions, and gene interaction networks with distinctive activation/inhibition patterns. Overall, the cannabinoids were predicted to affect metabolism and to have some beneficial effects on cardiovascular disease but adverse effects on the neural system. In addition, CBC and CBN, similar to but more potently than CBD, displayed liver toxicity and the potential to cause cancer while CBG protected from these adverse effects. This study provides a comprehensive comparison across the four cannabinoids and points to the directions for further research on the therapeutic effects and potential toxicities associated with long-term use of these cannabinoids.

BACKGROUND:Transcriptomic benchmark concentration (BMC) modeling provides quantitative toxicogenomic information that is increasingly being used in regulatory risk assessment of data poor chemicals. Over the past decade, RNA sequencing (RNA-seq) is gradually replacing microarray as the major platform for transcriptomic applications due to its higher precision, wider dynamic range, and capability of detecting novel transcripts. However, it is unclear whether RNA-seq offers substantial advantages over microarray for concentration response transcriptomic studies. RESULTS:We provide an updated comparison between microarray and RNA-seq using two cannabinoids, cannabichromene (CBC) and cannabinol (CBN), as case studies. The two platforms revealed similar overall gene expression patterns with regard to concentration for both CBC and CBN. However, in spite of the many varieties of non-coding RNA transcripts and larger numbers of differentially expressed genes (DEGs) with wider dynamic ranges identified by RNA-seq, the two platforms displayed equivalent performance in identifying functions and pathways impacted by compound exposure through gene set enrichment analysis (GSEA). Furthermore, transcriptomic point of departure (tPoD) values derived by the two platforms through BMC modeling were on the same levels for both CBC and CBN. CONCLUSIONS:Considering the relatively low cost, smaller data size, and better availability of software and public databases for data analysis and interpretation, microarray is still a viable method of choice for traditional transcriptomic applications such as mechanistic pathway identification and concentration response modeling.

Many weight loss products marketed as foods and dietary supplements are adulterated with structurally modified versions (analogues) of sibutramine, a weight loss drug withdrawn from the market due to adverse effects in the heart and nervous system. Unlike sibutramine, its analogues lack in vitro and in vivo safety data. Therefore, to identify potential health effects of sibutramine analogues, binding was predicted and measured between sibutramine analogues and a panel of 45 safety-related biological targets (e.g., receptors, ion channels, transporters, and enzymes) related to the heart, nervous system, and other organs. Target binding concentrations (Ki) were predicted in silico based upon quantitative structure-activity relationship (QSAR) models and measured in vitro by competitive ligand binding assays. The in vitro and in silico Ki values closely agreed for transporters of serotonin (SERT), norepinephrine (NET), and dopamine (DAT), which are linked to adverse health effects in the heart and nervous system. The chloro, homo, and desmethylsibutramine analogues exhibited similar binding profiles and particularly potent binding to SERT, NET and DAT (Ki, 9-403 nM). However, despite structural similarity among the compounds, benzyl and formyl analogues exhibited weaker binding to nearly all targets evaluated (Ki, 0.447 to >10 μM). Additionally, for selected analogues, target binding was predicted for metabolites; a majority of metabolites (70 %) exhibited similar binding potency (Ki within 10-fold) to their respective parent chemicals, suggesting they may also contribute to potential health effects. Overall, biological target binding profiles illustrate important structure-activity relationships among sibutramine analogues that can help identify potential adverse health effects.

Efficient new methods are needed to support initiatives to reduce, refine, and/or replace toxicity testing in vertebrates. 5-fluorouracil (5FU), hydroxyurea (HU), and ribavirin (RV) are mammalian teratogens. Skeletal, endocrine organ, and cardiac effects are often associated with teratogenesis, and a simple nematode like C. elegans lacks these systems. However, many genetic pathways required for mammalian morphogenesis have at least some conserved elements in this small, invertebrate model. The C. elegans lifecycle is 3 days. The effects of 5FU, HU, and RV on the C. elegans morphology were evaluated on day 4 post-initiation of the feeding after hatching for continuous and 24 h (early-only) developmental exposures. Continuous exposures to 5FU and HU induced increases in the incidences of abnormal gonadal structures that were significantly reduced in early-only exposure groups. The incidence of prolapse increased with continuous 5FU and HU exposures and was further increased in early-only exposure groups. Intestinal prolapse through the vulval muscle in C. elegans may be related to reported 5FU and HU effects on skeletal muscle and the gastrointestinal tract in mammals. Continuous RV exposures induced a phenotype lacking a uterus and gonad arms, as well as vulval anomalies that were largely, but not completely, reversed with early-only exposures, which is consistent with reported reversible reproductive tract anomalies after an RV exposure in mammals. These findings suggest that C. elegans can be used to detect the hazard risk from chemicals that adversely affect conserved pathways involved in organismal morphogenesis, but to determine the fit-for-purpose use of this model in chemical safety evaluations, further studies using larger and more diverse chemical test panels are needed.

Developmental delay and spontaneous locomotor activity changes, as well as the reversibility of these adverse effects are apical endpoints used in chemical safety evaluations. These endpoints were assessed at sublethal concentrations in C. elegans using 5-fluorouracil (5FU), hydroxyurea (HU), or ribavirin (RV), teratogens that are associated with reduced fetal growth in mammals. C. elegans develop from egg to egg-laying adult in about three days. Synchronized cohorts were exposed either continuously, or for 24 h (early-only) from first-feeding after hatching. Developmental delays were dose-responsive for all three chemicals in both exposure schemes. For 5FU and HU, developmental delays and hypoactivity levels were similar in continuous and early-only exposure groups, consistent with irreversible developmental effects. The observed hypoactivity in developing C. elegans may be related to reported 5FU-induced muscle impairment and HU-induced post-exposure effects on locomotion parameters in mammals. In contrast to 5FU- and HU-induced hypoactivity, RV was associated with a non-significant trend to slight hyperactivity in both exposure schemes. Continuous RV exposures induced delays to sequential developmental milestones that increased with exposure duration. RV-induced delays were significantly reduced but not eliminated in early-only exposure cohorts, consistent with cumulative RV effects on developmental progress. These findings suggest that C. elegans may be a useful model for detecting chemicals with irreversible, reversible, and/or cumulative effects on organismal development.

Cannabinoids are highly lipophilic constituents of the hemp plant, which is present in several products intended for consumption. While cannabidiol (CBD) effects to humans have been extensively investigated, there is limited information on other minor cannabinoids. CBD oral bioavailability is low but increases with food and high-fat intake. We used Caco-2 cells in vitro to assess intestinal absorption of five cannabinoids (CBD, CBC, CBG, CBN, and CBDV) present in a CBD-rich hemp extract. We used a fed-state simulated intestinal fluid (Fessif) for dissolution of cannabinoids. Cannabinoids did not alter Caco-2 monolayer integrity. Except for CBC, recovery of cannabinoids decreased significantly after 90-minute incubation, compared to 60-minute incubation. No measurable cannabinoids were identified in the bottom chambers. Recovery of CBD, CBC, CBG and CBN after incubation with hemp extract or cannabinoid mix containing 30 μM CBD was unchanged, but CBDV recovery decreased. With hemp extract or a mix containing 10 μM CBD, recovery of CBD and CBC did not change, CBG recovery was lower (80-82 %), and CBN and CBDV were unquantifiable. This study highlights the challenges of evaluating permeability of cannabinoids by Caco-2 cells to predict intestinal absorption, including the physicochemical properties of these compounds, incubation time and cell properties.

Interindividual differences in response to chemicals have been typically addressed through the use of a 10-fold default "uncertainty" factor. It was only recently that in vitro models emerged to quantitatively assess interindividual variability in the human population for specific chemicals. In the current study, we attempted to establish an in vitro model for assessing population variability in hepatotoxicity testing using a panel of hepatocytes derived from nine human induced pluripotent stem cell (iPSC) lines belonging to three different ethnic groups, Black or African American, Latino or Hispanic, and Non-Hispanic White. We demonstrated that the panel of iPSC-derived hepatocytes manifested diversity in hepatic function assays, in global and hepatic gene expressions, and in cytotoxic responses to four well know hepatotoxicants with distinct mechanisms of toxicity: acetaminophen, troglitazone, diglycolic acid, and usnic acid. However, due to the unavailability of model compounds with ethnicity specific toxicity as well as the small number of individuals in each ethnic group (n = 3), ethnic-specific effects were not observed using the model.

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.

Hemp extracts and consumer products containing cannabidiol (CBD) and/or other phytocannabinoids derived from hemp have entered the marketplace in recent years. CBD is an approved drug in the United States for the treatment of certain seizure disorders. While effects of CBD in the liver have been well characterized, data on the effects of other cannabinoids and hemp extracts in the liver and methods for studying these effects in vitro are limited. This study examined the hepatotoxic potential of CBD, CBD concentration-matched hemp extract, and cannabinol (CBN), at consumer-relevant concentrations determined by in silico modeling, in vitro using primary human hepatocytes. Primary human hepatocytes exposed to between 10-nM and 25-μM CBD, CBN, or hemp extract for 24 and 48 h were evaluated by measuring lactate dehydrogenase release, apoptosis, albumin secretion, urea secretion, and mitochondrial membrane potential. Cell viability was not significantly affected by CBD, CBN, or the hemp extract at any of the concentrations tested. Exposure to hemp extract induced a modest but statistically significant decrease in albumin secretion, urea secretion, and mitochondrial membrane potential at the highest concentration tested whereas CBD only induced a modest but statistically significant decrease in albumin secretion compared with vehicle control. Although this study addresses data gaps in the understanding of cannabinoid hepatoxicity in vitro, additional studies will be needed to determine how these results correlate with relevant consumer exposure and the biological effects of cannabinoids in human liver.

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 IC50 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.

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.

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.

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.

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 mu M. Despite that a significant reduction in cell viability occurred only in the 30 mu M 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 mu M 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 mu M 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 cannabisderived compounds.

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.

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.

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 (HgCl2) 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 HgCl2, but at much lower concentrations, a weaker hyperactivity response, and more pronounced hypoactivity. Sodium arsenite (NaAsO2) and dimethylarsinic acid (DMA) induced hypoactivity at high concentrations. Acute toxicity, as measured by hypoactivity in C. elegans adults, was ranked: meHgCl > HgCl2 > NaAsO2 = 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.

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.