Archives of Toxicology

The presence of N-nitrosamine drug substance-related impurities (NDSRIs) in pharmaceuticals represents a significant regulatory and safety challenge due to their classification as "cohort of concern" compounds. This paper describes the toxicological evaluation of N-Nitrosotrimetazidine (NTMZ), performed to refine the initial default acceptable intake (AI) limits of 18 to 26.5 ng/day established by regulatory authorities. The evaluation followed a tiered approach: NTMZ was first confirmed as mutagenic in vitro via the standard Ames test. To further investigate its genotoxic potential, two in vivo studies were conducted in Wistar and transgenic rats. Detection of DNA strand breaks in the liver and duodenum (comet assay) together with positive results in the cII mutation assay confirmed an in vivo mutagenic mode of action. Benchmark Dose (BMD) analysis of the transgenic rat data yielded a BMDL50 of 7 mg/kg/day in the male liver. To characterize long-term carcinogenic risk, a GLP-compliant 2-year carcinogenicity study was conducted in Wistar rats. Chronic exposure induced dose-dependent increases in liver tumors (hemangiosarcomas, hepatocellular carcinomas and adenomas) and intestinal tumors (adenomas and adenocarcinomas), leading to a Tumor Dose 50 (TD50) of 23 mg/kg/day in male rats. Benchmark dose analysis of tumor incidence identified a lowest BMDL10 of 2.6 mg/kg/day in females, which served as the basis for deriving an AI of 13 microg/day. This assessment demonstrates a strong predictive correlation between the BMD derived from the in vivo transgenic model, the BMDL10 and the final TD50 values obtained in the 2-year carcinogenicity study. These findings provided the scientific basis for establishing a conservative AI of 13 microg/person/day based on the BMDL10 and further support the regulatory acceptance and use of BMD-derived approaches for the evaluation of nitrosamine impurities.

Abstract Background. Acrylamide, a neurotoxicant in heated foods and smoke, is linked to occupational neuropathy, but evidence regarding chronic, low-level population exposure remains limited. We evaluated the association between acrylamide exposure biomarkers and peripheral neuropathy among U.S. adults. Methods. A total of 2,266 NHANES 2003-2004 participants (age >40) were analyzed. Exposure was assessed via hemoglobin adducts (HbAA/HbGA); neuropathy via monofilament testing >1 site). Survey-weighted logistic regression models adjusted for confounders. Sensitivity analyses included cubic splines, diabetes stratification, and multiple imputation. Results. Neuropathy prevalence was 15.5%. In adjusted models, neither adduct was associated with neuropathy (HbAA OR: 0.98, 95% CI: 0.82-1.17; HbGA OR: 0.91, 95% CI: 0.77-1.08). No dose-response gradient was observed. Expected risk factors (age, diabetes) showed strong associations, validating model sensitivity. The null result remained robust across sensitivity analyses, including a stricter outcome definition and multiple imputation (pooled OR: 0.97, 95% CI: 0.83-1.14). Conclusions. Acrylamide adducts were not associated with peripheral neuropathy in this national sample. General population levels (~55-70 pmol/g) lie well below established occupational no-observed-adverse-effect levels (~510 pmol/g) and clinical neuropathy thresholds (~6,000 pmol/g), providing a mechanistically coherent explanation for this null result.

Integrating high-throughput in vitro data into next-generation risk assessment (NGRA) workflows requires screening strategies that yield quantitative potency estimates and mechanistically interpretable biological signals. Transcriptomic and morphological profiling are increasingly adopted for early-stage hazard identification by enabling triage of substances for resource-intensive follow-up and prioritizing candidates most likely to present meaningful risk. In this study, we aimed to characterize biological concordance and uncertainty by quantifying how well high-throughput transcriptomics (HTTr) and Cell Painting PLUS (CPP) bioactivity profiles recover target-relevant biological signals in immortalized human renal proximal tubule epithelial RPTEC/TERT1 cells using 313 reference chemicals with high-confidence target annotations. Through quality control procedures and biological activity filters we yielded 142 reference chemicals spanning 66 different targets, which were systematically evaluated for biological concentration-responses by HTTr and CPP. HTTr was evaluated using TXG-MAPr-based qualitative and quantitative gene network activity analysis. HTTr showed the most prominent activity for targets that were highest expressed in RPTEC/TERT1 cells. Active chemical-pairs showed strong gene network activity correlation albeit with different potencies. Similarly, the highest transcriptomic concordance was observed for reference chemicals acting in the same pathway, such as EGFR/MEK or PI3K/AKT/mTOR. CPP often showed high sensitivity primarily at the organelle level providing limited statistical power for chemical grouping. Collectively, the results support HTTr and CPP as complementary early-tier assays within an in vitro weight-of-evidence safety testing framework. Although CPP is suitable as a cost-effective screening modality, HTTr offers higher mechanistic resolution for mode-of-action inference in high-throughput bioactivity screening and therefore remains necessary for high-confidence mechanistic interpretation.

People are widely exposed to the antimicrobial cetylpyridinium chloride (CPC) via consumer products, but CPC is a mitochondrial toxicant with potency comparable to that of canonical mitotoxicants. CPC is largely unregulated despite growing usage, bioavailability, and ability to cross the blood-brain barrier. Previously, we showed, in several cell types at non-cytotoxic and exposure-relevant doses, CPC inhibits ATP and OCR, endpoints of the electron transport chain (ETC). Mitochondrial toxicity is linked to multiple diseases (e.g., diabetes, Parkinsons, myalgic encephalomyelitis), but CPC has not been studied epidemiologically, and little mechanistic information is available. To determine why OCR and ATP are hampered by CPC, we hypothesized that CPC inhibits individual ETC components, cardiolipin, or TCA enzymes. Here, we show that, in primary human skin cells, an immune mast cell model, and isolated mitochondria, CPC apparently inhibits multiple ETC Complexes. Detailed investigation pinpointed the mechanism to the distal end of ETC: Complex III-cytochrome C-Complex IV. Using multiple approaches, we show that CPC does not directly inhibit any of the Complexes (not even Complex I as earlier reported), nor TCA enzymes, nor coenzyme Q. Yet, we found that CPC exhibits mitotoxicity as potent as cyanide. Anionic lipid cardiolipin attracts cytochrome C to the inner mitochondrial membrane so that it may shuttle electrons from Complex III to IV. Despite not altering levels of cardiolipin, CPC hinders cytochrome C by electrostatically interfering with cardiolipin. To aid epidemiology, risk analysis, and predictive toxicology, we have determined the precise biochemical mechanism of action of this ubiquitous compound.

2,4-Dinitrochlorobenzene (DNCB) is a well-characterized skin sensitizer that has been widely used in immunological and toxicological research and, historically, in clinical immunotherapy. Although it is a well-investigated chemical, this is the first study focusing on the dose response behavior at low-level concentrations. The aim was to reveal potential hormetic effects due to its known Nrf2 inducting activity. Therefore, THP-1 cells were treated with low doses of DNCB and two endpoints were evaluated for hormetic responses: metabolic activity using a resazurin-based assay and immune activation by measuring CD86 and CD54 expression using flow cytometry. The results showed a significant hormetic effect on the metabolic endpoint at the lower cell density for both analyzed time points, and a hormetic tendency at the higher cell density. Metabolic activity increased to approximately 125% of the control at 0.05 micromolar DNCB. For the immunological endpoint a slight decrease in CD86 and CD54 surface marker expression was observed, up to -16% and up to -12% compared to control at 0.5 micromolar DNCB. These findings highlight the importance of including low dose concentrations when characterizing chemical dose-response relationships and evaluating toxicological risk.

Hexafluoropropylene oxide dimer acid (GenX), a replacement for legacy per- and polyfluoroalkyl substances (PFAS), is increasingly detected in the environment, yet its chronic toxicity remains poorly characterized. Current safety assessments rely largely on short-term, high-dose studies that may not capture the biological consequences of long-term, low-dose exposure. To address this gap, we employed 3D human liver (HepG2/C3A) spheroids cultured in a continuously rotating bioreactor system (ClinoStar) to systematically evaluate dose- and time-dependent mRNA changes in response to GenX under environmentally relevant conditions. Spheroids were exposed to GenX (0.08-50 M, spanning environmentally relevant to mechanistically informative concentrations) for acute (4 days) and chronic (4 weeks) durations, followed by genome-wide TempO-Seq transcriptomic profiling and benchmark dose (BMD) modeling. GenX elicited pronounced non-monotonic mRNA changes in acute exposure conditions, with the greatest number of differentially expressed genes (DEGs) observed at an intermediate concentration (0.4 M). In contrast, chronic exposure exhibited a generally concentration-dependent increase in DEGs, except for the 10 M condition, indicating a more consistent dose-response relationship than acute exposure. Notably, acute and chronic exposures elicited qualitatively distinct mRNA changes with low concordance across matched concentrations, demonstrating that exposure duration was a major determinant of mRNA changes. Acute low-dose GenX exposure preferentially modulated mRNA encoding components of cell cycle-related pathways, whereas acute higher dose exposures suppress mRNA levels of the constituents of lipid metabolic pathways and increase expression of mRNA encoding proteins involved in stress- and toxicity-associated signaling. Chronic exposure revealed a different pattern of changes in mRNA expression not observed under acute exposure conditions, including suppression of cellular components involved in lipid-related pathways at the lowest concentration tested. At higher concentrations, mRNA levels of components of multiple metabolic pathways were altered. Benchmark dose modeling identified a significantly lower transcriptomic point of departure (tPOD) for chronic exposure as compared to acute exposure, suggesting increased cellular sensitivity to prolonged GenX exposure and supporting the relevance of chronic models for human exposure assessment. Collectively, these findings demonstrate that GenX elicits time-dependent and non-monotonic changes in mRNA levels of human liver (HepG2/C3A) spheroids, with distinct responses depending on the exposure duration and dose. This study, therefore, highlights the importance of incorporating chronic, human-relevant in vitro models and transcriptomic endpoints into PFAS risk assessment and suggests that conventional short-term assays may underestimate the biological impact of sustained low-dose exposure. Key message (Impact of the study)This study provides systematic comparisons of short term (4 day) versus longer term (4 weeks), environmentally relevant GenX exposure in human liver spheroids, revealing non-monotonic, time-dependent changes in mRNA levels encoding cellular components of lipid metabolism-related pathways with potential implications for appropriate dose and time exposure parameters for use in New Approach Methods to be applied in risk assessment.

Chemical exposures during pregnancy are linked to an increased risk of pregnancy complications that contribute significantly to maternal and infant morbidity and mortality and can lead to long term health consequences for both the mother and the offspring. The placenta, a central regulator of pregnancy health, is a direct target of environmental toxicants. Epidermal growth factor receptor (EGFR), highly expressed in the placenta, regulates proliferation, migration, invasion, fusion, and cellular bioenergetics. To identify compounds of environmental concern with potential for EGFR-disrupting activity, we optimized a high-throughput virtual screening protocol for the identification of EGFR inhibitors and achieved enrichment factors of EF1% = 10.09, EF5% = 3.86, and EF10% = 3.0 in a benchmarking dataset. We applied this protocol to screen the Collaborative Estrogen Receptor Activity Prediction Project database, finding that top-scoring compounds were enriched for aromatic and fused-ring chemical classes, including dyes. Kinase activity assays revealed that two out of thirteen selected compounds, Vat Red 32 and Reactive Red 136, inhibited EGFR kinase activity with micromolar IC50 values. Additionally, pose refinement with molecular dynamics simulations characterized the binding interactions of Reactive Red 136 within the EGFR kinase domain, and functional assays in HTR-8/SVneo placental trophoblast cells showed that Reactive Red 136, but not Vat Red 32, partially attenuated EGF-mediated cell migration despite both compounds inhibiting EGFR kinase activity. Together, this study has generated an enriched dataset of candidate environmental EGFR modulators, with experimental validation confirming enrichment for EGFR-disrupting activity among the selected compounds. These results provide a valuable resource for toxicological studies.

Transactive response DNA-binding protein 43 (TDP-43) aggregation and loss of function are hallmark features of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among other neurodegenerative diseases. Despite epidemiological evidence linking environmental exposures to neurodegeneration, few toxicants have been directly associated with neurodegeneration. Here, we performed a high-content imaging screen, using a library of over a thousand chemical compounds that are considered high risk for human exposure and identified 21 toxicants that drive TDP-43 aggregation. Among the top chemical hits, five belonged to the dithiocarbamate (DTC) class of thiol-reactive compounds including the agricultural pesticides thiram and ziram. Thiram directly promoted TDP-43 cysteine oxidation and intermolecular crosslinking, whereas ziram induced TDP-43 aggregation via zinc imbalance and enhanced oxidative stress, suggesting DTCs disrupt redox homeostasis. In primary neurons and human iPSC-derived neurons, DTCs led to TDP-43 aggregation and prominent splicing defects consistent with loss of TDP-43 function. In exposed zebrafish, DTCs impaired TDP-43 function and triggered widespread transcriptional changes reflected by perturbed stress response and metabolic signatures. By combining TDP-43 loss of function mutations with chemical exposures, we observed accelerated TDP-43 loss of function and chemical-induced aggregation, supporting a multiple hit mechanism driving TDP-43 dysfunction. Together, these findings identify DTCs, particularly those used as agricultural pesticides, as dominant modifiers of TDP-43 proteostasis and identify redox imbalance and zinc homeostasis as a central molecular mechanism linking toxicant exposure to TDP-43 proteinopathy.

Microcystin-LR (MC-LR), a cyanobacterial toxin produced during harmful algal blooms, is an increasing environmental and public health concern as the frequency and intensity of harmful algal blooms continue to rise globally. While the effects of MC-LR have been extensively studied in young organisms, much less is known about how aging influences susceptibility to cyanotoxin exposure. Here, we used the naturally short-lived turquoise killifish, Nothobranchius furzeri, to investigate transcriptional responses to low-level MC-LR exposure in a senescent vertebrate. Approximately 8-month-old GRZ killifish were exposed to a low dose of 0.5 g/L MC-LR, followed by whole-body RNA sequencing and sex-stratified differential expression analysis. Despite identical experimental conditions and exposure, males and females exhibited strikingly distinct transcriptional responses, with 313 differentially expressed genes (DEGs) in males and 263 in females and only 27 DEGs shared between the sexes. Among the shared responses, pck1, a key regulator of gluconeogenesis, was strongly downregulated in both sexes, accompanied by altered expression of genes associated with mitochondrial function, metabolic regulation, extracellular matrix remodeling, and genome maintenance. Males exhibited prominent remodeling of skeletal muscle and contractile programs, supported by enrichment of sarcomeric, myofilament, and contractile-fiber-associated genes. In contrast, females showed pronounced alterations in reproductive and metabolic programs, including vitellogenin- and zona pellucida-associated transcripts. Cell/tissue associated marker-module analysis further revealed distinct sex-dependent shifts in structural, neural, immune, metabolic, and reproductive transcriptional signatures. Together, these findings demonstrate that MC-LR elicits a broad but strongly sex-dependent transcriptional response in senescent N. furzeri, involving responses in multiple physiological systems. Our study identifies biological sex as an important determinant of cyanotoxin responses in an aging context and establishes naturally aged N. furzeri as a tractable vertebrate model for investigating interactions between environmental exposure and biological aging.

Background: In the United States, several states have restricted sales of flavoured tobacco products, including popular menthol- and mint-flavoured Oral Nicotine Pouches (ONP). In response, tobacco companies introduced "unflavoured" ONP containing odorless synthetic cooling agents. Since these, in turn, have become targets of legislative bans, the tobacco industry may seek out flavourants with other sensory effects to increase the appeal of "unflavoured" ONP. Methods: Online merchants were searched for "unflavored" ONP marketed to consumers in jurisdictions with flavour bans. Sensory effects of aqueous extracts from identified "heat", "spicy" and "unflavoured" ONP were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the human heat/chili pepper flavourant (capsaicinoid) receptor, hTRPV1. ONP were analyzed for capsaicinoids and sweeteners by Liquid Chromatography/Mass Spectrometry (LC/MS). Results: A new category of "heat" or "spicy" ONP was identified, including products marketed as "unflavoured". Extracts from all these ONP robustly activated TRPV1, with "unflavoured" Lucy Heat the most potent. Chemical analysis demonstrated that Lucy Heat contained the synthetic capsaicinoid nonivamide at high levels (~675 microgram/pouch), while others contained mixtures of capsaicinoids (5-25 microgram/pouch) combined with other characterizing flavours (tropical, fruit). All tested ONP contained sweeteners. Conclusions: The tobacco industry continues to probe regulatory loopholes by claiming that newly introduced capsaicinoid flavourants and sweeteners in ONP do not represent characterizing flavours. This is contradicted by industry and regulatory determinations assigning characterizing flavour properties to these additives. The toxicological health risks of repeated capsaicinoid exposures due to ONP use, in combination with nicotine and other constituents, need to be assessed.

Bone marrow chimeras are widely used to study immune development and function. As the field moves from cesium-137 (137Cs)-based irradiators to X-ray irradiators for safety reasons, it is essential to determine if there are differences in immune system reconstitution after irradiating mice with one of these two radiation sources. Here, we performed a comprehensive immunological comparison of mice lethally irradiated with 137Cs or one of two different X-ray platforms and reconstituted with congenic bone marrow. Mice received 12 Gy total body radiation in two 6 Gy sessions followed by intravenous transfer of donor hematopoietic stem cells and were analyzed eight weeks post-transplant. We assessed mouse survival, donor chimerism, immune cell subset distribution, and activation states across multiple organs (bone marrow, spleen, lymph nodes, liver, and lung). All groups exhibited comparable survival and high levels of donor chimerism, with expected organ-specific reconstitution patterns. Immune lineage distributions, CD4/CD8 ratios, and activation states did not differ by irradiation type. Host-derived radioresistant cells were also similar across all irradiation groups and were predominantly composed of T cells skewed toward an activated phenotype. Overall, our data show that X-ray irradiation with proper filters and energy levels (225 KVp and 320 KVp) can yield equivalent immunological outcomes, including immune reconstitution and activation states, as compared to the same radiation dose from 137Cs-based irradiation in bone marrow chimera models. These results support the continued adoption of X-ray irradiation systems in place of 137Cs for generating bone marrow chimeras to be used across a wide range of immunologic studies.

Gabapentin (GBP) and pregabalin (PGB) are widely used gabapentinoids. Previously, we have demonstrated, for the first time, that GBP and PGB modulate the nociceptive response to noxious heat in C. elegans at an optimal concentration. In the current study, we use C. elegans and paired thermal nociception assays with direct internal drug concentration measurements to characterize the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of both compounds. Neither drug altered baseline mobility or quadrant preference, confirming that behavioral effects reflected genuine antinociceptive action. Both GBP and PGB produced dose- and time-dependent reductions in thermal avoidance, with 500 uM exposures generating a biphasic, V-shaped time course in which suppression of thermal sensitivity deepened before partially reversing. This partial reversal occurred later with PGB than with GBP. Internal concentrations confirmed dose-dependent absorption and retention for both drugs, yet at 500 uM, internal drug levels remained elevated through 360 min even as behavioral avoidance recovered, indicating that the recovery limb reflects active counter-regulation rather than passive clearance, consistent with previously reported transcriptional and proteomic signatures. Exposure-response profiles were notably flat, suggesting a saturable pharmacodynamic ceiling. Molecular modeling revealed conserved electronic pharmacophores supporting shared alpha-2-delta engagement, alongside shape-descriptor differences that may contribute to divergent absorption kinetics. These findings position C. elegans as a valuable model for dissecting gabapentinoid PK/PD relationships. Beyond mechanistic insight, these findings support the continued investigation of C. elegans as a screening platform whose validation could help address the 3R (Replacement, Reduction, Refinement) principles guiding animal research.

Cigarette smoke-induced lung inflammation is a central driver of pulmonary diseases. The limited efficacy of current anti-inflammatory agents underscores the need for structurally novel therapeutics with distinct mechanisms. We recently demonstrated that AB-free kava, a flavokavains A/B-depleted formulation from Piper methysticum containing six major kavalactones, effectively suppresses cigarette smoke-induced lung inflammation in mice. This study aims to identify the bioactive constituent(s) and elucidate underlying mechanisms. These kavalactones revealed a clear structure-activity relationship in suppressing lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in macrophages with desmethoxyyangonin (DMY) as the most potent kavalactone whereas dihydrokavain (DHK, a structurally similar analog) with minimal activity. DMY also effectively reduced LPS-induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-) production while DHK was ineffective. Mechanistically, DMY, but not DHK, attenuated COX-2 induction and reduced phosphorylation of cAMP response element-binding protein (CREB). Pharmacological inhibition of protein kinase A (PKA) similarly reduced p-CREB, COX-2 and PGE2, supporting a PKA-dependent CREB/COX-2 signaling in mediating PGE2 suppression while these effects were independent of nuclear factor kappa B (NF-{kappa}B) and activator protein 1 (AP-1) signaling. Similar results were observed for DMY and DHK in attenuating cigarette smoke condensate-induced proinflammatory pathways and PGE2 production. Consistently, DMY demonstrated significant in vivo efficacy in suppressing cigarette smoke-induced lung inflammation while DHK was not effective. Interestingly, dihydromethysticin (DHM) demonstrated the greatest in vivo anti-inflammatory efficacy, although it only exhibited moderate in vitro potency, likely due to its superior bioavailability over DMY. Concordantly, cigarette smoke exposure elevated p-CREB and COX-2 expressions in mouse lungs, which were attenuated by AB-free kava and its bioactive kavalactones with the extent of suppression correlating with their in vivo anti-inflammatory efficacy. DHM effectively suppressed LPS-induced neutrophil accumulation in mouse lungs as well. Collectively, these studies identify bioactive kavalactones in AB-free kava that suppress cigarette smoke- and LPS-induced lung inflammation through the modulation of the PKA/CREB/COX-2 signaling axis, providing a foundation for developing structurally distinct anti-inflammatory agents, particularly targeting smoke-induced inflammation and associated pulmonary diseases.

Typical environmental exposures to the toxic class of chemicals known as polycyclic aromatic hydrocarbons (PAHs) involve complex mixtures; however, relatively few mechanistic toxicity studies have evaluated them as environmental mixtures, instead focusing on individual compounds or simple mixtures. In this study, we first derived Republic Sediment Extract (REPSE), a complex PAH mixture extracted from sediment at the Republic Creosoting site of the Elizabeth River in Norfolk, Virginia. After characterizing the PAH contents of REPSE, we evaluated its mechanisms of developmental neurotoxicity in three evolutionarily distinct taxa, leveraging the unique strengths of Atlantic killifish, zebrafish, and Caenorhabditis elegans as model species, with a focus on the Aryl hydrocarbon Receptor (AhR) pathway. Embryonic REPSE exposure caused induction of CYP1A in both fish species at sub-teratogenic concentrations, consistent with activation of the canonical AhR pathway. These sub-teratogenic exposures nevertheless induced neurotoxicity across both fish species, altering neurobehavioral phenotypes in fish, and induced dopaminergic neuronal damage in worms, again at non-teratogenic concentrations. To determine whether these effects were linked to canonical AhR response pathways, we examined killifish offspring from the pollution-adapted Republic Creosoting population, which exhibited characteristic recalcitrance to CYP1A induction, but remained susceptible to the neurobehavioral effects of REPSE. The induction of neuronal damage in worms provides orthogonal evidence for a non-AhR mechanism, because C. elegans AhR is not transcriptionally activated by PAHs as in vertebrates. Further probing of potential mechanisms underlying REPSE-induced neurotoxicity in worms revealed altered neuronal redox status (roGFP) and energy availability (ATP:ADP ratio). Collectively, our multispecies approach reveals conserved mechanisms of PAH mixture neurotoxicity, including effects that extend beyond canonical AhR signaling.

Long-term in vivo transcriptomic time courses are costly, limiting assessment of chronic molecular responses from short studies. We developed a pharmacokinetics-informed transcriptomic ordinary differential equation model (PKT-ODE) that links an oral pharmacokinetic profile and Hill drug-effect function to first-order turnover of co-expression modules. The model was fitted to rat liver responses to fenofibrate at three doses in Open TG-GATEs through day 8. At the held-out day-29 endpoint, PKT-ODE achieved Pearson r = 0.960 and mean squared error (MSE) = 0.148. In this dataset, these values achieved lower prediction error and higher correlation than four statistical baselines and validation-selected linear and multilayer-perceptron transition models. Literature-curated peroxisome proliferator-activated receptor target genes occurred only in modules with positive fitted drug effects. These results provide a proof of concept for pharmacokinetics-informed transcriptomic extrapolation; cross-compound, cross-organ and alternative-regimen performance remain to be tested.

Toxicants in the environment can significantly impact physiology. Environmental chemical exposures during early developmental stages disturb normal embryonic development and programming, and dramatically impact long-term health as individuals age. Female and male animals show distinct phenotypes when responding to a given chemical exposure. Here, through the TaRGET II (Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription) consortium, we systematically explored sex-specific transcriptomic and epigenomic alterations in response to various toxicants, including arsenic (As), lead (Pb), tributyltin (TBT), bisphenol A (BPA), di(2-ethylhexyl) phthalate (DEHP), dioxin (TCDD), and fine particulate matter (PM2.5), across three time points in mice exposed two weeks prior to conception through gestation and lactation. After being exposed to toxicants during the embryonic and early postnatal developmental stages, 1,025 omics datasets were generated from the liver and analyzed across three mouse life stages. We discovered a significant sex-biased molecular response to distinct exposures in the liver at both the transcriptomic and epigenetic levels, showing dynamic changes across mouse development and aging. The perturbed pathways and transcription factors in response to different chemical exposures in both sexes were further evaluated to measure the sex-specific impact of each toxic exposure in the liver. Overall, this study presents the most detailed investigation of sex-specific molecular signatures under the influence of developmental exposures to toxic substances.

Background & AimsCurrent nucleos(t)ide analogs efficiently suppress hepatitis B virus (HBV) replication but have limited effects on viral transcription from covalently closed circular DNA (cccDNA) and integrated HBV DNA. We aimed to identify clinically applicable compounds that directly inhibit HBV transcription by screening FDA-approved drugs. Approach & ResultsScreening of 1,470 FDA-approved compounds using an HBV enhancer I/X promoter reporter system identified vorapaxar and aripiprazole as potent inhibitors of viral promoter activity. Both compounds suppressed HBV replication in HBV-producing cells, HBV-infected HepG2-hNTCP cells, and primary human hepatocytes. Aripiprazole reduced hepatocyte nuclear factor 4 (HNF4) protein levels through an ERK/JNK-dependent pathway and inhibited HBV core promoter activity, whereas vorapaxar acted independently of HNF4. Both compounds suppressed enhancer I/X promoter activity through inhibition of STAT3 signaling. Vorapaxar inhibited PAR-1-mediated SRC, EGFR, and STAT3 activation, while aripiprazole suppressed SRC-STAT3 signaling independently of EGFR. PAR-1 activation enhanced HBV transcription, whereas PAR-1 knockdown reduced promoter activity and viral RNA expression. Both compounds also reduced HBV replication in human liver chimeric mice at clinically relevant exposure levels without apparent severe toxicity. ConclusionsVorapaxar and aripiprazole suppress HBV transcription and replication through distinct host signaling pathways. These findings identify PAR-1-STAT3 signaling as a previously unrecognized regulator of HBV transcription and suggest that host-targeting approaches may complement current therapies by suppressing viral gene expression from both cccDNA and integrated HBV DNA. Impact and implicationsCurrent nucleos(t)ide analogues effectively suppress HBV reverse transcription but have limited effects on viral transcription from cccDNA and integrated HBV DNA, highlighting the need for therapies targeting viral gene expression. We identify PAR-1- STAT3 signaling as a previously unrecognized regulator of HBV transcription and demonstrate that two clinically approved drugs, vorapaxar and aripiprazole, suppress HBV replication through distinct host signaling pathways. These findings are relevant to researchers developing host-targeting antivirals and to clinicians seeking complementary therapeutic strategies beyond current nucleos(t)ide analogue therapy. Although further clinical validation and combination studies are required, our results provide a rationale for repurposing approved drugs and for developing transcription-targeting therapies that may complement existing treatments for chronic hepatitis B. HighlightsO_LIVorapaxar and aripiprazole suppress HBV through distinct host pathways. C_LIO_LIBoth drugs inhibit HBV replication in vitro and in humanized liver mice. C_LIO_LIPAR-1 inhibition reduces HBV transcription by blocking SRC/EGFR/STAT3 signaling. C_LIO_LIPAR-1-STAT3 signaling is a novel regulator of HBV transcription. C_LIO_LIHost-targeting antiviral therapy complements current HBV treatment. C_LI

BackgroundZebrafish (Danio rerio) are a powerful vertebrate model for developmental toxicology and chemical safety assessment, yet large-scale transcriptomics in zebrafish remains limited by cost and data heterogeneity. Targeted transcriptomics offers a cost-effective alternative, but gene extrapolation methods tailored to zebrafish have not been systematically developed or evaluated. ObjectivesWhile the S1500+ platform is widely used for toxicogenomics research with rat, mouse, and human cell lines as model systems, its use in zebrafish has been limited due to data scarcity and lack of suitable bioinformatics approaches for analysis of such data. To that end, we sought to (i) curate a large zebrafish transcriptomic training data resource, and (ii) evaluate multiple machine learning strategies for reconstructing unmeasured transcriptome-wide expression profiles for data originating from the zebrafish-specific reduced representation gene set ("Zf S1500+"). MethodsWe assembled 14,924 zebrafish RNA-Seq samples covering 21,930 genes across 1,246 studies. Using the Zf S1500+ gene subset (3,062 genes), we trained and tested three extrapolation approaches: principal components regression (PCR), a locally weighted extension of PCR (PCR+), and a neural network mixture-of-experts model (NN-MoE). Model performance was assessed using mean absolute error (MAE), mean squared regression error (MSRE), and weighted variants of these metrics. ResultsExtrapolation performance using the baseline approach was strongly influenced by tissue and developmental context, with within-tissue models outperforming cross-tissue models. Errors were lowest when training and testing were conducted within the same tissue or between developmentally related tissues. Both PCR+ and NN-MoE improved upon the baseline PCR approach, with NN-MoE reducing average MAE by [~]20% and MSRE by [~]17%. Importantly, extrapolation remained reliable for the majority of genes, even when limiting output to high-confidence predictions using an empirical MAE threshold. ConclusionsWe demonstrate that targeted transcriptomics can be effectively extended to zebrafish, enabling robust transcriptome-wide extrapolation at reduced cost. The NN-MoE method provided the most substantial gains, highlighting the value of non-linear and ensemble modeling in heterogeneous datasets. These results establish a scalable framework for zebrafish toxicogenomics and suggest that accuracy will continue to improve with larger, better-annotated datasets, paving the way for broader application in chemical safety assessments.

Per-and polyfluoroalkyl substances (PFAS) constitute a diverse class of persistent synthetic chemicals utilized across industrial, medical, and consumer sectors that are pervasive global pollutants. Exposure to PFAS is linked to adverse impacts on both innate and adaptive immune systems. Human lactoferrin (hLF) is a key antimicrobial component of the developing innate immune system present in colostrum and breast milk. We hypothesized that hLF is a potential PFAS binding protein related to PFAS immunotoxicity. The results of thermal stability experiments indicated that all 11 tested PFAS bind and destabilize the structure of hLF. Notably PFBA, PFOS, HFPO-DA, and 6:2 FTSA decreased apo-hLF melting temperatures from 64oC to [≤] 37oC, suggesting that PFAS exposures destabilize the native hLF protein under physiological conditions. Relative binding affinities (Kd) ranged from 0.2-11 mM across tested PFAS. Molecular docking was used to confirm experimental binding affinities and identify molecular interactions involved with PFAS binding. Calculated Gibbs Free Energies of binding ranged from -4.4 to -8.8 kcal/mol. Together, these results demonstrate that PFAS bind hLF at affinities comparable to human serum albumin and other PFAS binding proteins, and that some PFAS can destabilize hLF protein structure at physiologically relevant temperatures and conditions.

We published recently that one of the main constituents of cannabis products, cannabidiol (CBD), is an efficacious negative allosteric modulator (NAM) of the mu opioid receptor (MOR1) (Bosquez-Berger et al., 2023). Here, we investigated how the presence of cannabidiol (CBD) is associated with fentanyl (FEN) binding across MOR1 conformations. We performed molecular dynamics simulations of systems containing FEN alone or FEN+CBD in three mouse MOR1 conformational backgrounds: active-like 5C1M, inactive-like 4DKL, and a modeled Morph50 intermediate between the 5C1M and 4DKL conformations. Three independently seeded 200 ns trajectories were analyzed per model and condition (18 trajectories total), with the trajectory treated as the independent unit. Across the matched 0-200 ns window, consensus CBD contacts and CBD-associated changes in FEN contacts were strongly state dependent. Corrected intracellular TM3 to TM6 analyses separated the expected active-like, intermediate, and inactive-like backgrounds but did not identify a CBD-associated shift that was consistent across both geometric definitions and all three replicates. Equal-weight replicate-composite density maps preserved both the shared ligand distributions and this between-trajectory variability. These descriptive results support receptor-state-dependent CBD, FEN, MOR1 interactions while emphasizing the limited inferential power of three trajectories per condition.