Xenobiotic metabolism and transport in Caenorhabditis elegans.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFß, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Cannabis use in Parkinson's disease: Patient access to medical cannabis and physician perspective on product safety.

The rate of medical cannabis use has increased in parallel with the number of states legalizing its use. Parkinson's disease (PD) patients are of particular concern due to their higher cannabis use rate than in the general US population (25-40 % PD patient cannabis users vs. ∼18 % in the general population), as well as their susceptibility to environmental contaminants in cannabis, including pesticides, toxic elements, solvents, microbes, and mycotoxins. In order to address the complex nature of this industry, we examined the changes in PD-related qualifying conditions in the U.S. from 2019 to 2023. We also conducted an online survey to gain insight into the knowledge, risk perceptions, and opinions regarding medical cannabis and contamination issues from physicians who treated PD patients. The number of states including PD-related qualifying conditions increased over the past 5 years from 28 to 36 states. These conditions included PD (increasing from 14 to 16 states), muscle spasms (14 to 24), anxiety (1 to 5), and pain (17 to 35). State-by-state comparisons revealed high variability in the language used to describe the different qualifying conditions. Online surveys were sent out to 45 neurologists and movement disorder specialists who primarily treated PD patients. The response rate was 44 % from nine states (AZ, CA, FL, MA, MN, WI, PA, IL, and NM). When asked if they were aware of any contaminants in cannabis products, we found that 65 % of the physicians were unaware of any contaminants commonly found in cannabis and only 25 %, 15 %, and 15 % of them were aware of pesticide, toxic element, and solvent contaminants, respectively. In their free-text opinion response on the health impact of cannabis-borne contaminants, "long-term effect" (35 %) and "comorbidities and PD prognosis" (40 %) were identified as the two most common themes. These results point to the need for further regulatory deliberation regarding risks and susceptibility to cannabis contaminants. Additionally, education is needed to inform physicians on cannabis safety issues. Further research will identify the implementation strategies to reduce contaminant exposure and protect patient health.

Complementary biological and computational approaches identify distinct mechanisms of chlorpyrifos versus chlorpyrifos-oxon-induced dopaminergic neurotoxicity.

Organophosphate (OP) pesticides are widely used in agriculture. While acute cholinergic toxicity has been extensively studied, chronic effects on other neurons are less understood. Here, we demonstrated that the OP pesticide chlorpyrifos (CPF) and its oxon metabolite are dopaminergic neurotoxicants in Caenorhabditis elegans. CPF treatment led to inhibition of mitochondrial complex II, II + III, and V in rat liver mitochondria, while CPF-oxon did not (complex II + III and IV inhibition observed only at high doses). While the effect on C. elegans cholinergic behavior was mostly reversible with toxicant washout, dopamine-associated deficits persisted, suggesting dopaminergic neurotoxicity was irreversible. CPF reduced the mitochondrial content in a dose-dependent manner and the fat modulatory genes cyp-35A2 and cyp-35A3 were found to have a key role in CPF neurotoxicity. These findings were consistent with in vitro effects of CPF and CPF-oxon on nuclear receptor signaling and fatty acid/steroid metabolism observed in ToxCast assays. Two-way hierarchical analysis revealed in vitro effects on estrogen receptor, pregnane X receptor, and peroxisome proliferator-activated receptor gamma pathways as well as neurotoxicity of CPF, malathion, and diazinon, whereas these effects were not detected in malaoxon and diazoxon. Taken together, our study suggests that mitochondrial toxicity and metabolic effects of CPF, but not CPF-oxon, have a key role of CPF neurotoxicity in the low-dose, chronic exposure. Further mechanistic studies are needed to examine mitochondria as a common target for all OP pesticide parent compounds, because this has important implications on cumulative pesticide risk assessment.

Fungal and mycotoxin contaminants in cannabis and hemp flowers: implications for consumer health and directions for further research.

Medicinal and recreational uses of Cannabis sativa, commonly known as cannabis or hemp, has increased following its legalization in certain regions of the world. Cannabis and hemp plants interact with a community of microbes (i.e., the phytobiome), which can influence various aspects of the host plant. The fungal composition of the C. sativa phytobiome (i.e., mycobiome) currently consists of over 100 species of fungi, which includes phytopathogens, epiphytes, and endophytes, This mycobiome has often been understudied in research aimed at evaluating the safety of cannabis products for humans. Medical research has historically focused instead on substance use and medicinal uses of the plant. Because several components of the mycobiome are reported to produce toxic secondary metabolites (i.e., mycotoxins) that can potentially affect the health of humans and animals and initiate opportunistic infections in immunocompromised patients, there is a need to determine the potential health risks that these contaminants could pose for consumers. This review discusses the mycobiome of cannabis and hemp flowers with a focus on plant-infecting and toxigenic fungi that are most commonly found and are of potential concern (e.g., Aspergillus, Penicillium, Fusarium, and Mucor spp.). We review current regulations for molds and mycotoxins worldwide and review assessment methods including culture-based assays, liquid chromatography, immuno-based technologies, and emerging technologies for these contaminants. We also discuss approaches to reduce fungal contaminants on cannabis and hemp and identify future research needs for contaminant detection, data dissemination, and management approaches. These approaches are designed to yield safer products for all consumers.

Evaluation of a Behavioral Test for Sickness Behavior Associated with Fusarium Mycotoxin Ingestion in Female Beagle Dogs (Canis familiaris).

Animals exhibit behavioral changes during illness, including lethargy, anorexia, fever, adipsia, and anhedonia, which are believed to comprise an adaptive evolutionary strategy. Exploratory and social behaviors generally decrease during illness, but behavioral changes of dogs during illness have not been described. The objective of this study was to evaluate a novel canine behavior test during subclinical illness induced by dietary Fusarium mycotoxin. Twelve mature female beagle dogs received 3 treatment diets: a control diet (control), a diet formulated with grains contaminated with Fusarium mycotoxin (toxin), and the toxin diet together with a toxin binding agent (binder). All dogs received each diets for 14 d in a Latin square design with a 7-d washout period between diet trials. The test consisted of individually releasing dogs into the center aisle of the housing room for 4 min per day, during which interactions with familiar dogs in adjacent kennels were recorded by an observer outside the room who was blind to treatment groups. Total interactions, orientation, and attempted physical contact with other dogs were less frequent during the toxin and binder diet treatments. Conversely, frequencies of physical proximity and olfactory contact with familiar dogs in adjacent kennels were not associated with diet. In conclusion, induction of subclinical gastrointestinal illness influenced aspects of social interactions in beagle dogs. A clinical assessment sheet integrating these findings was developed to aid in early identification of subclinical illness in research dogs based on behavior.

The Desert Whale: the boom and bust of hemp in Arizona.

BACKGROUND: This paper examines the factors that led to the collapse of hemp grown for cannabidiol (CBD) in Arizona, the United States of America (USA), and particularly in Yuma County, which is a well-established agricultural area in the state. METHODS: This research uses a combination of mapping analysis along with a survey of hemp farmers to assess the reasons why the hemp industry collapsed as well as to foster solutions to these problems. RESULTS: In 2019, 5430 acres were sown with hemp seed in Arizona with 3890 acres inspected by the state to determine if they could be harvested. By 2021, there were only 156 acres planted, and only 128 of those acres were inspected by the state for compliance. (Crop mortality accounts for the difference between acres sown and acres inspected.) CONCLUSIONS: A lack of knowledge about the hemp life cycle greatly contributed to the failure of high CBD hemp crops in Arizona. Other problems included noncompliance with tetrahydrocannabinol limits, poor sources for seeds and inconsistent genetics of the hemp varieties sold to farmers, and diseases that hemp plants were susceptible to such as Pythium crown and root rot and beet curly top virus. Addressing these factors will go far in making hemp a profitable and widespread crop in Arizona. Additionally, hemp grown for other traditional uses (e.g., fiber or seed oil) as well as new applications (e.g., microgreens, hempcrete, and phytoremediation) offers other pathways for successful hemp agriculture in this state.

Caenorhabditis elegans Neurotoxicity Testing: Novel Applications in the Adverse Outcome Pathway Framework.

Neurological hazard assessment of industrial and pesticidal chemicals demands a substantial amount of time and resources. Caenorhabditis elegans is an established model organism in developmental biology and neuroscience. It presents an ideal test system with relatively fewer neurons (302 in hermaphrodites) versus higher-order species, a transparent body, short lifespan, making it easier to perform neurotoxic assessment in a time and cost-effective manner. Yet, no regulatory testing guidelines have been developed for C. elegans in the field of developmental and adult neurotoxicity. Here, we describe a set of morphological and behavioral assessment protocols to examine neurotoxicity in C. elegans with relevance to cholinergic and dopaminergic systems. We discuss the homology of human genes and associated proteins in these two signaling pathways and evaluate the morphological and behavioral endpoints of C. elegans in the context of published adverse outcome pathways of neurodegenerative diseases. We conclude that C. elegans neurotoxicity testing will not only be instrumental to eliminating mammalian testing in neurological hazard assessment but also lead to new knowledge and mechanistic validation in the adverse outcome pathway framework.

Comparison of State-Level Regulations for Cannabis Contaminants and Implications for Public Health.

BACKGROUND: The presence of contaminants in cannabis presents a potential health hazard to recreational users and susceptible patients with medical conditions. Because of the federally illegal status of cannabis, there are no unified regulatory guidelines mitigating the public health risk of cannabis contaminants. OBJECTIVE: To inform further research and provide solutions to the public health risk of cannabis contaminants at a national level, we examined the current landscape of state-level contaminant regulations, and cannabis contaminants of concern, as well as patient populations susceptible to contaminants. METHODS: We examined the regulatory documents for medical and recreational cannabis in all legalized U.S. jurisdictions and compiled a complete list of regulated contaminants, namely, pesticides, inorganics, solvents, microbes, and mycotoxins. We data mined the compliance testing records of 5,654 cured flower and 3,760 extract samples that accounted for ∼6% of California's legal cannabis production in 2020-2021. We also reviewed the publicly available medical cannabis use reports to tabulate the susceptible patient populations. RESULTS: As of 18 May 2022, 36 states and the District of Columbia listed a total of 679 cannabis contaminants as regulated in medical or recreational cannabis, including 551 pesticides, 74 solvents, 12 inorganics, 21 microbes, 5 mycotoxins, and 16 other contaminants. Different jurisdictions showed significant variations in regulated contaminants and action levels ranging up to four orders of magnitude. A failure rate of 2.3% was identified for flowers and 9.2% for extracts in the California samples. Insecticides and fungicides were the most prevalent categories of detected contaminants, with boscalid and chlorpyrifos being the most common. The contaminant concentrations fell below the regulatory action levels in many legalized jurisdictions, indicating a higher risk of contaminant exposure. Cannabis use reports indicated usage in several patient populations susceptible to contamination toxicity, including cancer (44,318) and seizure (21,195) patients. DISCUSSION: Although individual jurisdictions can implement their policies and regulations for legalized cannabis, this study demonstrates the urgent need to mitigate the public health risk of cannabis contamination by introducing national-level guidelines based on conventional risk assessment methodologies and knowledge of patients' susceptibility in medical use. https://doi.org/10.1289/EHP11206.

Regulatory Status of Pesticide Residues in Cannabis: Implications to Medical Use in Neurological Diseases.

Medical cannabis represents a potential route of pesticide exposure to susceptible populations. We compared the qualifying conditions for medical use and pesticide testing requirements of cannabis in 33 states and Washington, D.C. Movement disorders were the most common neurological category of qualifying conditions, including epilepsy, certain symptoms of multiple sclerosis, Parkinson's Disease, and any cause of symptoms leading to seizures or spasticity. Different approaches of pesticide regulation were implemented in cannabis and cannabis-derived products. Six states imposed the strictest U.S. EPA tolerances (i.e. maximum residue levels) for food commodities on up to 400 pesticidal active ingredients in cannabis, while pesticide testing was optional in three states. Dimethomorph showed the largest variation in action levels, ranging from 0.1 to 60 ppm in 5 states. We evaluated the potential connections between insecticides, cannabinoids, and seizure using the Comparative Toxicogenomics Database. Twenty-two insecticides, two cannabinoids, and 63 genes were associated with 674 computationally generated chemical-gene-phenotype-disease (CGPD) tetramer constructs. Notable functional clusters included oxidation-reduction process (183 CGPD-tetramers), synaptic signaling pathways (151), and neuropeptide hormone activity (46). Cholinergic, dopaminergic, and retrograde endocannabinoid signaling pathways were linked to 10 genetic variants of epilepsy patients. Further research is needed to assess human health risk of cannabinoids and pesticides in support of a national standard for cannabis pesticide regulations.

Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure.

We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6J/m(2)/day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m(2)/day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3h after exposure to 50 J/m(2) UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.

Examination of testicular gene expression patterns in Yorkshire pigs with high and low levels of boar taint.

Boar taint refers to the objectionable odor and flavor in meat of some uncastrated male pigs, which is primarily due to high levels of androstenone, a steroid produced in the testis, and 3-methylindole (skatole) which is produced by bacterial degradation of tryptophan in the intestinal tract. We determined testicular gene expression patterns of Yorkshire pigs with high and low levels of boar taint using swine DNA microarrays with two-color hybridization. The microarrays contained 19486 annotated probes; the expressions of 8719 genes were detected. Fifty-three genes were significantly up-regulated in the high boar taint group and four were significantly down-regulated (p < 0.05; fold change > +/-1.55). Gene ontology (GO) analysis short-listed 11 significant GO terms (p < 0.05), most of which are associated with steroid metabolism and mitochondrial components. Comparing the results of this study with published work on Duroc and Norwegian Landrace boars,(1) eleven genes (HSB17B4, FDX1, CYP11A1, DHRS4, PRDX1, CYB5, CYP17A1, FTL, IDI1, SULT2A1, and RDH12) were over-expressed in all three breeds with a high androstenone level. The current findings confirmed a number of candidate genes identified in previous functional studies and suggest several new genes differentially expressed with different levels of boar taint.

Mitochondria as a target of organophosphate and carbamate pesticides: Revisiting common mechanisms of action with new approach methodologies.

Mitochondrial toxicity has been proposed as a potential cause of developmental defects in humans. We evaluated 51 organophosphate and carbamate pesticides using the U.S. EPA ToxCast and Tox21 databases. Only a small number of them bind directly to cholinesterases in the parent form. The hydrophobicity of organophosphate pesticides is correlated significantly to TSPO binding affinity, mitochondrial membrane potential reduction in HepG2 cells, and developmental toxicity in Caenorhabditis elegans and Danio rerio (p < 0.05). Structural analysis suggests that in some cases the Krebs cycle is a potential target of organophosphate and carbamate exposure at early life stages. The results support the hypothesis that mitochondrial effects of some organophosphate pesticides-particularly those that require enzymatic activation to the oxon form-may augment the documented effects of disruption of acetylcholine signaling. This study provides a proof of concept for applying new approach methodologies to interrogate mechanisms of action for cumulative risk assessment.

Adverse outcome pathway of developmental neurotoxicity resulting from prenatal exposures to cannabis contaminated with organophosphate pesticide residues.

There is growing concern that increased use of medical and recreational cannabis may result in increased exposure to contaminants on the cannabis, such as pesticides. Several states are moving towards implementing robust regulation of the sales, cultivation, and manufacture of cannabis products. However, there are challenges with creating health-protective regulations in an industry that, to date, has been largely unregulated. The focus of this publication is a theoretical examination of what may happen when women are exposed pre-conceptually or during pregnancy to cannabis contaminated with pesticides. We propose an adverse outcome pathway of concomitant prenatal exposure to cannabinoids and the organophosphate pesticide chlorpyrifos by curating what we consider to be the key events at the molecular, cellular, and tissue levels that result in developmental neurotoxicity. The implications of this adverse outcome pathway underscore the need to elucidate the potential developmental neurotoxicity that may result from prenatal exposure to pesticide-contaminated cannabis.

Mycotoxins and the pet food industry: toxicological evidence and risk assessment.

Mycotoxin contamination in pet food poses a serious health threat to pets, causing an emotional and economical concern to the pet owners. Aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins and fusaric acid have been found in the ingredients and final products of pet food, resulting in both acute toxicity and chronic health problems in pets. Toxicological interaction among mycotoxins as a natural mixture further complicates the issue. The concepts of "risk assessment", using hazard identification, dose-response assessment, no observable adverse effect level (NOAEL), and lowest observed adverse effect level (LOAEL), should be applied to assess the risk and safety of mycotoxins in pet food, thereby instilling public confidence in the pet food industry.

Effects of foodborne Fusarium mycotoxins with and without a polymeric glucomannan mycotoxin adsorbent on food intake and nutrient digestibility, body weight, and physical and clinicopathologic variables of mature dogs.

OBJECTIVE: To investigate the effects of feeding cereal-based diets that are naturally contaminated with Fusarium mycotoxins to dogs and assess the efficacy of a polymeric glucomannan mycotoxin adsorbent (GMA) in prevention of Fusarium mycotoxicosis. ANIMALS: 12 mature female Beagles. PROCEDURES: Dogs received each of 3 cereal-based diets for 14 days. One diet was uncontaminated (control diet), and the other 2 contained contaminated grains; one of the contaminated diets also contained 0.2% GMA. Contaminants included deoxynivalenol, 15-acetyl deoxynivalenol, zearalenone, and fusaric acid. Food intake and nutrient digestibility, body weight, blood pressure, heart rate, and clinicopathologic variables of the dogs were assessed at intervals during the feeding periods. RESULTS: Food intake and body weight of dogs fed the contaminated diet without GMA were significantly decreased, compared with effects of the control diet. Reductions in blood pressure; heart rate; serum concentrations of total protein, globulin, and fibrinogen; and serum activities of alkaline phosphatase and amylase as well as increases in blood monocyte count and mean corpuscular volume were detected. Consumption of GMA did not ameliorate the effects of the Fusarium mycotoxins. For the GMA-contaminated diet, digestibility of carbohydrate, protein, and lipid was significantly higher than that associated with the control diet, possibly because of physiologic adaptation of the recipient dogs to reduced food intake. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that consumption of grains naturally contaminated with Fusarium mycotoxins can adversely affect dogs' feeding behaviors and metabolism. As a food additive, GMA was not effective in prevention of Fusarium mycotoxicosis in dogs.

Applying evolutionary genetics to developmental toxicology and risk assessment.

Evolutionary thinking continues to challenge our views on health and disease. Yet, there is a communication gap between evolutionary biologists and toxicologists in recognizing the connections among developmental pathways, high-throughput screening, and birth defects in humans. To increase our capability in identifying potential developmental toxicants in humans, we propose to apply evolutionary genetics to improve the experimental design and data interpretation with various in vitro and whole-organism models. We review five molecular systems of stress response and update 18 consensual cell-cell signaling pathways that are the hallmark for early development, organogenesis, and differentiation; and revisit the principles of teratology in light of recent advances in high-throughput screening, big data techniques, and systems toxicology. Multiscale systems modeling plays an integral role in the evolutionary approach to cross-species extrapolation. Phylogenetic analysis and comparative bioinformatics are both valuable tools in identifying and validating the molecular initiating events that account for adverse developmental outcomes in humans. The discordance of susceptibility between test species and humans (ontogeny) reflects their differences in evolutionary history (phylogeny). This synthesis not only can lead to novel applications in developmental toxicity and risk assessment, but also can pave the way for applying an evo-devo perspective to the study of developmental origins of health and disease.

Mycotoxins in pet food: a review on worldwide prevalence and preventative strategies.

Mycotoxins contaminate cereal grains worldwide, and their presence in pet food has been a potential health threat to companion animals. Aflatoxins, ochratoxin A, and Fusarium mycotoxins have been found in both raw ingredients and final products of pet food around the globe. Aflatoxin, a hepatotoxin and carcinogen, has caused several food poisoning outbreaks in dogs, and aflatoxin content is regulated in pet food in many countries. Ochratoxin A and Fusarium mycotoxins including trichothecenes, zearalenone, and fumonisins may have chronic effects on the health of companion animals. Grain processing, sampling error, analytical methods, conjugated mycotoxins, storage conditions, and synergistic interactions are common challenges faced by the pet food industry. Food-processing techniques such as sieving, washing, pearling, ozonation, and acid-based mold inhibition reduce the mycotoxin content of cereal grains. Dietary supplementation with large neutral amino acids, antioxidants, and omega-3 polysaturated fatty acids as well as inclusion of mycotoxin-sequestering agents and detoxifying microbes may ameliorate the harmful effects of mycotoxins in contaminated pet food.

Computational modeling and simulation of genital tubercle development.

Hypospadias is a developmental defect of urethral tube closure that has a complex etiology involving genetic and environmental factors, including anti-androgenic and estrogenic disrupting chemicals; however, little is known about the morphoregulatory consequences of androgen/estrogen balance during genital tubercle (GT) development. Computer models that predictively model sexual dimorphism of the GT may provide a useful resource to translate chemical-target bipartite networks and their developmental consequences across the human-relevant chemical universe. Here, we describe a multicellular agent-based model of genital tubercle (GT) development that simulates urethrogenesis from the sexually-indifferent urethral plate stage to urethral tube closure. The prototype model, constructed in CompuCell3D, recapitulates key aspects of GT morphogenesis controlled by SHH, FGF10, and androgen pathways through modulation of stochastic cell behaviors, including differential adhesion, motility, proliferation, and apoptosis. Proper urethral tube closure in the model was shown to depend quantitatively on SHH- and FGF10-induced effects on mesenchymal proliferation and epithelial apoptosis-both ultimately linked to androgen signaling. In the absence of androgen, GT development was feminized and with partial androgen deficiency, the model resolved with incomplete urethral tube closure, thereby providing an in silico platform for probabilistic prediction of hypospadias risk across combinations of minor perturbations to the GT system at various stages of embryonic development.

Systems Toxicology of Male Reproductive Development: Profiling 774 Chemicals for Molecular Targets and Adverse Outcomes.

BACKGROUND: Trends in male reproductive health have been reported for increased rates of testicular germ cell tumors, low semen quality, cryptorchidism, and hypospadias, which have been associated with prenatal environmental chemical exposure based on human and animal studies. OBJECTIVE: In the present study we aimed to identify significant correlations between environmental chemicals, molecular targets, and adverse outcomes across a broad chemical landscape with emphasis on developmental toxicity of the male reproductive system. METHODS: We used U.S. EPA's animal study database (ToxRefDB) and a comprehensive literature analysis to identify 774 chemicals that have been evaluated for adverse effects on male reproductive parameters, and then used U.S. EPA's in vitro high-throughput screening (HTS) database (ToxCastDB) to profile their bioactivity across approximately 800 molecular and cellular features. RESULTS: A phenotypic hierarchy of testicular atrophy, sperm effects, tumors, and malformations, a composite resembling the human testicular dysgenesis syndrome (TDS) hypothesis, was observed in 281 chemicals. A subset of 54 chemicals with male developmental consequences had in vitro bioactivity on molecular targets that could be condensed into 156 gene annotations in a bipartite network. CONCLUSION: Computational modeling of available in vivo and in vitro data for chemicals that produce adverse effects on male reproductive end points revealed a phenotypic hierarchy across animal studies consistent with the human TDS hypothesis. We confirmed the known role of estrogen and androgen signaling pathways in rodent TDS, and importantly, broadened the list of molecular targets to include retinoic acid signaling, vascular remodeling proteins, G-protein coupled receptors (GPCRs), and cytochrome P450s. CITATION: Leung MC, Phuong J, Baker NC, Sipes NS, Klinefelter GR, Martin MT, McLaurin KW, Setzer RW, Darney SP, Judson RS, Knudsen TB. 2016. Systems toxicology of male reproductive development: profiling 774 chemicals for molecular targets and adverse outcomes. Environ Health Perspect 124:1050-1061; http://dx.doi.org/10.1289/ehp.1510385.