Toxicology and Applied Pharmacology

While distinct environmental exposures imprint unique mutational signatures on cancer genomes, the specific causal patterns for many known carcinogens remain uncharacterized in relevant human tissues. To address this gap, we developed a novel, physiologically relevant system that uses a combination of airway epithelial cells and whole genome sequencing to characterize mutational patterns induced by genotoxic carcinogens associated with lung cancer. After validating the platforms accuracy by successfully recapturing the known signature for Benzo(a)pyrene (BaP), we used this system to gain detailed insights into the types of mutations that occur with exposure to N-nitrosotris-(2-chloroethyl) urea (NTCU) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), genotoxic compounds that induce lung squamous cell carcinoma and lung adenocarcinoma in mouse models, respectively. Cells exposed to NTCU had significantly more somatic SNVs compared to control samples. An average of 82.3% of mutations in NTCU samples were attributed to a novel mutational signature distinct from those in the COSMIC database but highly correlated with recent in vivo mouse models. In contrast, NNK exposure did not demonstrate a distinct mutational pattern above background at both high and low concentrations. Ultimately, this in vitro system provides a robust platform to define causal links between environmental exposures and mutational patterns in lung cancer mutagenesis. Statement of SignificanceIn vitro exposure of N-nitrosotris-(2-chloroethyl) urea to airway epithelial cells revealed a distinct mutational signature.

The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

Acetaminophen (APAP) overdose is a leading cause of acute liver failure worldwide. The RNA-binding protein Human antigen R (HuR) is a multifunctional post-transcriptional regulator that plays a pivotal role in cellular stress responses, including those triggered by APAP toxicity. This study investigated the mechanisms by which HuR protects against APAP-induced hepatotoxicity in male mice. Hepatocyte-specific HuR-deficient (HuRHep-/-) male mice on a C57BL/6N background and wild-type (WT) littermates were treated with 200 mg/kg APAP, and liver tissues were collected at 2, 6, and 24 hours post-treatment. APAP administration increased hepatic HuR mRNA expression and induced HuR cleavage and the formation of a higher-molecular weight HuR-immunoreactive band, with the latter two correlating with injury severity. Compared with WT controls, HuRHep-/- mice exhibited markedly increased susceptibility to hepatotoxicity at both 2 and 6 hours. Metabolite profiling revealed altered APAP metabolism and reduced glutathione S-transferase (Gst) expression in HuRHep-/- livers, consistent with impaired APAP detoxification and increased APAP-protein adduct formation. Fourier-transform infrared (FTIR) spectroscopy further identified early biochemical differences between WT and HuRHep-/- livers as early as 2 hours after APAP exposure. Additionally, HuR deficiency resulted in pronounced mitochondrial structural abnormalities and dysfunction at 2 and 6 hours, accompanied by reduced expression of the mitochondrial fission and fusion proteins Drp1 and Mfn2, increased mitochondrial protein release, and enhanced hepatocyte death. Although pro-inflammatory cytokine levels were elevated in HuRHep-/- mice relative to WT controls at 24 hours, hepatocyte proliferation was similarly blunted in both genotypes, consistent with severe liver injury and delayed recovery. Collectively, these findings identify hepatocyte HuR as a critical regulator of xenobiotic metabolism and mitochondrial integrity and establish its essential role in early protection against APAP-induced hepatotoxicity in male mice.

Micro- and nanoplastics (MNPs) are increasingly detected in human tissues, yet their causal contribution to cardiovascular disease remains poorly understood. Here we show that oral exposure to polyethylene (PE) and polyvinyl chloride (PVC) -- the most abundant polymers found in human atheromas -- accelerates atherosclerosis in ApoE-/-mice through distinct, polymer-specific molecular mechanisms. While both polymers increased plaque burden and reduced contractile smooth muscle cell (SMC) markers, single-cell transcriptomic profiling revealed divergent phenotypic trajectories. PE exposure drives SMCs toward a chondromyocyte-like cell (CMC) state, characterized by upregulated osteogenic signaling and markedly increased vascular calcification. Conversely, PVC exposure promotes a fibromyocyte-like program associated with altered collagen metabolism and accelerated cell migration without enhancing calcification. These distinct SMC programs are reflected in the transcriptional signatures of symptomatic human carotid plaques, suggesting clinical relevance for polymer-specific vascular remodeling. Our findings establish a causal link between common environmental plastics and accelerated atherosclerosis, demonstrating that MNP-induced vascular risk is mediated by divergent SMC fate decisions. These results provide a mechanistic framework for assessing the cardiovascular impact of global plastic pollution and identifying potential therapeutic targets to mitigate MNP-associated vascular toxicity.

Development is a tightly regulated process that establishes body axes and orchestrates the spatial organization of tissues and organs. Although developmental programs contain inherent redundancies, they remain highly sensitive to environmental cues. Among environmental contaminants, per- and polyfluoroalkyl substances (PFAS), chemicals that resist degradation and bioaccumulate in the body, are of particular concern. These "forever chemicals" are widespread in our household products, including non-stick and waterproof materials, and drinking water remains a major source of exposure. PFAS accumulate in specific tissues and have been associated with developmental delays, childhood leukemia, and other adverse health outcomes, yet the cellular and molecular mechanisms by which they disrupt early development remain largely unknown. To address this, we employ zebrafish embryos as a New Approach Methodology (NAM) to investigate how perfluorooctanoic acid (PFOA), a prevalent environmental PFAS, alters early embryogenesis. Embryos were exposed to physiologically relevant low and high doses of PFOA and analyzed at 24 hours post-fertilization (hpf), a key stage of organogenesis. We also included a parental exposure group, in which adults were treated with PFOA and their offspring were collected to assess whether the effects of exposure were transmitted to the next generation. Developmental processes are inherently plastic, and we wanted to understand the extent to which PFOA impacts normal cellular processes as well as the redundancy in the system (different developmental signaling pathways) which ensures that an embryo develops properly. Towards this, we performed single-nucleus RNA sequencing at 24 hpf, and it revealed that neuronal and muscle tissue clusters are particularly sensitive to PFOA exposure. These molecular perturbations correspond with anxiety-like behavioral phenotypes we observed in the exposed larvae, linking early developmental disruptions to organism-level outcomes. Overall, our findings provide mechanistic insight into the way in which PFAS exposure alters development, disrupting gene expression patterns and chromatin organization in developing tissues, revealing how early molecular perturbations can give rise to long-term behavioral consequences. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/700343v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1fec69aorg.highwire.dtl.DTLVardef@1a0b3beorg.highwire.dtl.DTLVardef@17883e2org.highwire.dtl.DTLVardef@11a7571_HPS_FORMAT_FIGEXP M_FIG C_FIG

ImportanceGenetic factors only explain [~]50% of an individuals lifetime CAD risk. Workplace pollution exposure likely represents a significant, under-recognized, and modifiable contributor. Clarifying its independent effect - distinct from residential air pollution - could inform targeted prevention, clinical risk assessment, and policy strategies. ObjectiveTo quantify the independent association between workplace pollution exposure and incident CAD, rigorously adjusting for canonical risk factors, genetic risk, socioeconomic deprivation index, and residential air pollution. Design, Setting, and ParticipantsA prospective cohort analysis of 103,599 adults in the UK Biobank with complete baseline employment history and specialized workplace environment surveys. Participants with prior CAD, or missing genetic information, canonical risk factor data, or residential air pollution measurements, were excluded. ExposureCumulative, self-reported duration of exposure to workplace pollutants (including dust, smoke, exhaust, chemicals, asbestos, paints, and pesticides), summarized as a percentile measure. Main Outcomes and MeasuresIncident CAD. Associations were estimated using Cox proportional hazards models adjusted for demographics, comorbidities, CAD polygenic risk score, and residential air pollution metrics, accounting for competing death events. ResultsThe cohort (median age 64 years; 43% male) had high baseline prevalence of smoking (41%) and co-morbidities (hypertension 27%, hyperlipidemia 16%, diabetes 4.3%). Common exposures included smoke (56%), dust (39%), and chemicals (27%). Over a median 7.5-year follow-up, 4,327 CAD cases occurred. Compared to the low exposure group, high workplace pollution exposures showed a stronger unadjusted association with CAD (hazard ratio [HR], 1.51; 95% CI, 1.40-1.63) relative to residential air pollution (unadjusted HR, 1.23; 95% CI, 1.14-1.33). Importantly, in multivariable models, workplace pollution remained independently associated with higher CAD incidence (adjusted HR, 1.21; 95% CI, 1.03-1.41), robust to all adjustment. Significant specific drivers included paints, thinners or glues, diesel exhaust, fumes, asbestos, and dust. Conclusions and RelevanceWorkplace pollution is confirmed as a robust, independent risk factor for incident CAD. Its association is demonstrably stronger than residential air pollution and persists despite comprehensive adjustment for genetic predisposition and traditional cardiovascular risk factors. These findings underscore the need to strengthen worker protections, integrate occupational history into clinical risk assessment, and prioritize mechanistic research into non-lipid pollution pathways.

Perfluorooctane sulfonic acid (PFOS), a per- and polyfluoroalkyl substance (PFAS), is a widespread persistent environmental pollutant that has been implicated in various human health conditions, including infertility and cancer. Here, we investigate the effects of acute exposure to PFOS on human fallopian tube epithelial (FNE) cells that are essential for fertility and increasingly recognized as the origin site for high-grade serous ovarian cancer. We show that acute PFOS exposure changes morphology, arrests proliferation, impairs adhesion, and compromises epithelial integrity of FNE cells. Using transcriptomic profiling of FNE cells exposed to PFOS, we found increased expression of genes associated with stress-response signal transduction, including KRAS, and decreased expression of genes related to cholesterol transport and lipid homeostasis. We show that inhibition of MEK/ERK or cholesterol supplementation rescued changes in cell morphology. Further, we performed membrane fluidity measurements of cells exposed to PFOS and found elevated membrane disorder and fluidity. Our results are consistent with a model in which PFOS perturbs plasma membrane, activates stress-response signaling pathways, and impairs epithelial cell function. These studies establish a framework for understanding the effects of PFAS on cell physiology.

Phthalate plasticizers are contaminants of emerging concern that interfere with the synthesis, secretion, and transport of hormones and receptors, altering the immune response and energy balance. Phthalate metabolites have been detected in marine mammals globally, and while studies on phthalate toxicity in marine mammals are beginning to emerge, a comprehensive understanding of the cellular response to these compounds remains elusive. Here, we investigated the transcriptional and bioenergetic responses to mono-ethylhexyl phthalate (MEHP), the active metabolite of di(2-ethylhexyl) phthalate (DEHP), in primary dermal derived from northern elephant seals (Mirounga angustirostris), common dolphins (Delphinus delphis), and humans. MEHP exposure did not induce cytotoxicity in any species, but triggered distinct, species-specific changes in gene expression and mitochondrial metabolism. Human cells showed the greatest transcriptional response to MEHP, upregulating detoxification, antioxidant, and inflammatory genes, and downregulating lipid metabolism pathways. Although mitochondrial respiration declined only at the highest dose, sustained extracellular acidification rates and increased glycolytic gene expression indicate a metabolic shift toward glycolysis. In contrast, elephant seal cells upregulated antioxidant and immune genes while maintaining mitochondrial respiration until the highest MEHP dose, alongside increased expression of genes involved in oxidative phosphorylation, the TCA cycle, and mitochondrial dynamics, suggesting a delayed shift to glycolysis and a potential evolutionary adaptation to sustain mitochondrial function during energy-demanding conditions such as breath-hold diving. Dolphin cells exhibited fewer transcriptional changes, which were enriched for hormone signaling and mitotic pathways, and showed dose-dependent declines in both oxygen consumption and extracellular acidification rates, even at the lowest MEHP concentration, alongside upregulation of stress and hypoxia-related genes. Together, these findings highlight distinct cellular strategies for coping with phthalate exposure and likely species-specific susceptibility to toxicant-induced stress. This study provides new insights into how marine mammals respond to plastic-derived contaminants at the cellular level, reinforcing the need for species-specific ecotoxicological risk assessments.

Per- and polyfluoroalkyl substances (PFAS) are chemicals linked to obesity and metabolic dysfunction, but their role in bariatric surgery remains poorly understood. This prospective pilot study examined correlations between plasma PFAS concentrations, body composition, and glycemic measures in adults undergoing bariatric surgery. Thirty-two patients (91% female; 66% Black; mean age 43 years) were enrolled preoperatively; twenty-two completed follow-up at a mean 8.6 months post-surgery. Three PFAS (PFHxS, PFNA, and PFOS) were quantified by plasma liquid chromatography-mass spectrometry; body composition and insulin sensitivity were assessed by dual-energy X-ray absorptiometry and intravenous glucose tolerance testing. At baseline, higher plasma PFNA and PFOS concentrations tracked with lower total lean mass ({rho}s = -0.46 and -0.48, respectively) and lean mass index ({rho}s = -0.46 and -0.42), and PFNA was inversely correlated with body weight ({rho}s = -0.40). No baseline associations were observed with adiposity or glycemic indices. Postoperatively, PFHxS concentrations decreased (median = -1.103 ng/mL, p < 0.001), whereas PFNA and PFOS did not change. Average PFNA was positively correlated with postoperative changes in HOMA-IR ({rho}s = 0.51) and total lean mass ({rho}s = 0.49). No significant associations were observed for average PFHxS or PFOS. These findings suggest that PFNA and PFOS may be linked to reduced lean tissue at baseline, and that PFNA burden modestly tracks with attenuated metabolic and body composition recovery. In an ANCOVA, baseline PFNA was not significantly associated with postoperative HOMA-IR or total lean mass. Larger, longitudinal studies are needed to clarify how PFAS influence these associations.

Racial disparities in the incidence of, and mortality from, aggressive breast cancers are a pressing public health issue. Many factors have been investigated in these inequities; however, the role of toxicant exposures is not well characterized. We and others have identified substantial inequities in chemical biomarker concentrations by race. The goal of this study was to test the hypothesis that exposure to these chemicals is linked to biological changes relevant to aggressive breast cancers, such as dysregulation of the Hallmarks of Cancer. We used high throughput transcriptomic profiling of normal primary human breast epithelial cells from diverse donors (n=6) to test effects of 8 chemicals (cadmium, lead, arsenic, copper, PFNA, BPA, BPS, p,p-DDE) with documented exposure disparities by race/ethnicity across 3 concentrations (100nM, 1{micro}M, 10{micro}M). Across chemicals, we identified that pathways related to cell cycle regulation and protein secretion were commonly affected. Through bioinformatic estimation of cell type proportions, we found that metals like lead and cadmium induced cell-type shifts, consistent with the dysregulated cellular plasticity cancer hallmark. Lead and arsenic response genes were enriched for genes associated with poor breast cancer survival in the Cancer Genome Atlas. Integrating concentration-response modeling and chemical biomonitoring data, BPA, p,p-DDE, copper, and lead elicited expression changes at concentrations relevant to the US population. Finally, we identified substantial interindividual heterogeneity in response to organic compounds, but less so in metals. These findings highlight the value of high-throughput transcriptomics as a New Approach Methodology (NAM) in quantifying how common exposures may impact aggressive breast cancer associated biological processes.

Wildfires have become more frequent and intense worldwide. Wildfire emitted particulate matter (WFPM) can be more toxic than urban background PM due to its greater content of nanoscale size (WFPM0.1) and presence of more polar organic compounds, including polycyclic aromatic hydrocarbons (PAHs). While exposure to WFPM has been linked to cardiovascular and respiratory diseases, its impact on female reproduction remains elusive. Here, we used an in vivo mouse intratracheal exposure model and a 3D ovarian follicle culture system, together with molecular, transcriptomic, and computational approaches, to examine the female reproductive effects of lab-synthesized (LS-WFPM0.1) and real-world Canadian WFPM0.1 (C-WFPM0.1), collected from the New York City and New Jersey metropolitan area during the June 2023 wildfire events. Intratracheal exposure to environmentally relevant dose of LS-WFPM0.1 disrupted mouse estrous cycles and elevated serum concentrations of estradiol and testosterone. RT-qPCR and single-follicle RNA-sequencing (RNA-seq) analysis revealed altered steroidogenic genes, transcriptomic changes, and activation of aryl hydrocarbon receptor (AhR) in antral follicles from mice treated with LS-WFPM0.1. LS-WFPM0.1 consistently increased testosterone secretion and stimulated genes related to androgen synthesis and AhR in vitro. Single-follicle and single-oocyte RNA-seq analysis identified differentially expressed genes related to inflammation in somatic cells and mitochondrial respiratory chain in oocytes. Both C-WFPM0.1 and benzo[a]pyrene, a high-molecular-weight PAH, reproduced these ovarian defects. Mechanistically, AhR inhibition reversed hyperandrogenism induced by WFPM0.1. Together, our findings suggest that WFPM0.1, an increasingly pervasive environmental exposure, adversely impacts female reproductive functions by disrupting ovarian steroidogenesis and inducing hyperandrogenism through AhR activation, highlighting an urgent unmet need for further mechanistic studies and epidemiological investigations to define the reproductive risks of wildfire smoke exposure in human populations.

BackgroundFine particulate matter (PM2.5), airborne particles with an aerodynamic diameter [&le;]2.5 m that can penetrate deep into the lungs and enter the circulation, is increasingly recognized as a risk factor for chronic kidney disease (CKD) with long-term exposure. We previously demonstrated that high-dose PM2.5 exposure prior to ischemia-reperfusion injury (IRI) aggravates acute kidney injury (AKI). Here, we investigated how prolonged, low-concentration urban PM2.5 exposure (<15 {micro}g/m3) affects kidney repair after AKI. MethodsSix-week-old mice underwent bilateral IRI or sham surgery, followed by six months of exposure to either filtered air or ambient PM2.5 exposure in a unique exposome chamber. Kidneys were analyzed using pathomics, electron and super-resolution microscopy, immunohistochemistry, transcriptomics, and LC-MS lipidomics/metabolomics. Complementary in vitro hypoxia-reoxygenation and PM2.5 exposure experiments were performed in proximal tubular epithelial cells. ResultsLong-term PM2.5 exposure had minimal effects in sham-operated mice, including no significant changes in body weight or kidney function. Despite preserved kidney function, IRI+PM2.5 mice exhibited reduced weight gain, a marked expansion of the interstitial area, attributable to enhanced fibrosis and inflammatory responses, microvascular rarefaction, and endothelial-to-mesenchymal transition, consistent with maladaptive repair features. Proximal tubules displayed mitochondrial injury, glycolytic reprogramming, lipid accumulation, and a senescent phenotype. Energy Dispersive X-ray (EDX) microscopy confirmed PM2.5-derived elements within proximal tubules lysosomes, accompanied by lysosomal stress. Transcriptional signature-based drug screening identified nicotinamide as a compound capable of reversing PM2.5-induced metabolic alterations; in vitro validation confirmed restoration of mitochondrial function. ConclusionsTogether, these findings show that chronic post-AKI exposure to PM2.5 at levels currently considered safe by regulatory bodies drives maladaptive repair and accelerates CKD progression through mitochondrial dysfunction, lysosomal stress senescence in proximal tubules, due to local PM2.5 element accumulation. Translational StatementAcute kidney injury frequently progresses to chronic kidney disease due to maladaptive repair, yet environmental drivers of this transition remain underrecognized. Using a controlled exposome chamber, we demonstrate that chronic exposure to low, real-world concentrations of urban PM2.5 during post-ischemic recovery results in the accumulation of PM2.5-derived elements within proximal tubular lysosomes, leading to organelle dysfunction, metabolic reprogramming, lipid accumulation, and a senescence-like phenotype. Importantly, transcriptomics-based drug repurposing identified nicotinamide as a candidate compound capable of reversing metabolic dysfunction in injured proximal tubular cells subjected to hypoxia-reoxygenation and PM2.5 exposure, an effect validated in vitro.

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

Per- and polyfluoroalkyl substances (PFAS) are manmade chemicals that are persistent in the environment and have been linked to various physiological and neurobehavioral outcomes, including anxiety disorders. Trifluoroacetic acid (TFA), a short chain PFAS and the most common PFAS degradation product, is increasingly detected in water, soil, and human blood, raising significant concerns about its developmental toxicity. However, the impact of early-life TFA exposure on neurodevelopment and behavior remain insufficiently characterized. In this study, we employed Zebrafish (Danio rerio) embryos as a New Approach Methodology (NAM), to evaluate the development, behavior, and protein expression changes in response to early-life TFA exposure. Embryos were exposed to environmentally relevant low and high concentrations of TFA beginning at one-cell stage. Early developmental physiology was assessed by measuring viability, tail twitch response, hatching rates, and chorion diameters during embryogenesis. Anxiety-like behaviors were evaluated at 5- and 6-days post-fertilization using validated behavioral assays such as the Light-Dark Test and Startle Response. Each test evaluates distinct anxiety-related behaviors by measuring locomotor activity, thigmotaxis (wall preference), and stimulus reactivity, with anxious zebrafish larvae showing increased movement in light and greater wall preference. Then to identify molecular pathways underlying observed developmental phenotypes with TFA exposure, proteomic analyses were performed on embryos at 24- and 48-hours post-fertilization. Our results indicate that TFA exposure altered developmental physiology, evidenced by reduced chorion diameters, and lead to increased anxiety-like behaviors with larvae exhibiting thigmotaxis. These phenotypic changes were accompanied by detectable alterations in the embryonic proteome. Collectively, our findings provide insight into how short-chain PFAS exposure during critical windows of development may contribute to neurobehavioral dysfunction, highlighting potential risks relevant to inform public health policies and environmental regulations.

This study evaluates the impact of PFOA exposure on the metabolome and immune response to SARS-2 using a ferret model. Ferrets were separated into control or PFOA-exposed groups (10/mg/kg/day) and challenged with SARS-2. Longitudinal analyses encompassing clinical assessments, serological profiling, histopathology, and untargeted nuclear magnetic resonance (NMR) metabolomics revealed significant metabolic and immunological perturbations. We found prominent effects of PFOA exposure on metabolism, which resulted in altered metabolic responses to SARS-2 infection. PFOA exposure was also associated with impaired immune function, as evidenced by decreased serum IgG levels, increased viral loads, and prolonged peak infectivity. These findings underscore the consequences of PFOA exposure on host metabolism and immunity during infectious diseases.

Exposure of cells or tissues to chemical compounds can be analyzed through transcriptomic signatures, which can be used to classify chemical agents. This information can also enrich Adverse Outcome Pathways (AOP). Transcriptional signatures have generally been obtained using "bulk" analysis, by which the global gene expression pattern of an entire tissue is determined. Although this approach has been useful in toxicology, some information is lost, especially when tissues containing multiple cell types are considered. With the advent of single-cell transcriptomics (scRNA-seq), it is now possible to obtain higher resolution, cell type-specific responses in complex tissues. The aim of the present study was to evaluate the added value of scRNA-seq in analysis of the acute response of human bronchial epithelial cells grown at the air/liquid interface (ALI) to a known toxic compound, CdCl2, with well described transcriptional signatures of exposure. Fully differentiated mucocilliary epithelia obtained from three independent donors were exposed to 10 {micro}M CdCl2 and scRNA-seq analysis was performed on a total of 18255 cells to obtain cell type-specific signatures. Our results show that the contribution of each cell type to the overall transcriptomic bulk response varies. For example, the classical heavy metal detoxification response was only detected in multiciliated and secreting cells, while absent in basal cells. The data demonstrate that scRNA-seq provides high-resolution transcriptional signatures with unexpected features. This added information is likely to have implications for the refinement of AOPs and could serve as a basis for a new generation of tests in predictive toxicology.

BackgroundRising global temperatures and eutrophication are increasing the intensity and frequency of cyanobacterial harmful algal blooms that release toxins including microcystin-LR (MC-LR). MC-LR inhibits protein phosphatases in the human liver and brain, but its accumulation in the placenta is unclear. Placental transporter expression varies across pregnancy and is influenced by physiological cues, such as low oxygen concentrations which activate HIF1A, and trophoblast cell fusion forming syncytiotrophoblasts that engage CREB-driven transcription. This study examined whether MC-LR accumulates in placental cells, which transporters mediate uptake, and how these transporters are regulated by HIF1A and CREB. MethodsIntracellular accumulation of MC-LR (0.1-10 {micro}M, 3 hour) was measured in human cytotrophoblasts (JAR, BeWo) and extravillous trophoblasts (HTR-8/SVneo) by western blotting for MC-LR-adducted proteins. Organic anion transporting polypeptide (OATP) involvement was tested using cyclosporin A (10 {micro}M), an OATP inhibitor, before exposure to the OATP substrate or MC-LR. Cells were also cultured under 3%, 8%, or 20% O2 to induce hypoxic responses or treated with forskolin (a potent intracellular cAMP inducer) to stimulate cell fusion before MC-LR exposure. ResultsMC-LR accumulated in all three placenta cell lines in a concentration-dependent manner. Cyclosporin A reduced MC-LR uptake by 57% in JAR cells, confirming OATP-mediated transport. Low O2 increased OATP4A1 expression and function but reduced protein phosphatase expression, decreasing MC-LR-bound proteins by 52-72%. Forskolin increased OATP4A1 expression and enhanced MC-LR uptake >2.5-fold. ConclusionMC-LR enters placental trophoblasts via active OATP transport, likely OATP4A1, and uptake increases under hypoxia and trophoblast fusion.

Chronic exposure to pesticide mixtures through diet is common, yet their combined metabolic effects and interactions with dietary factors remain unclear. We identified four pesticides prevalent in human exposure (imazalil, thiabendazole, boscalid, lambda-cyhalothrin) and assessed their combined impacts on hepatic metabolism and metabolic homeostasis using human liver cells and male mice fed standard chow or western diets. We found that the pesticide mixture induced metabolic perturbations in human hepatocytes. In addition, the pesticide mixture altered hepatic gene expression in chow-fed mice and exacerbated western diet-induced glucose intolerance, fasting hyperglycemia, and insulin resistance without affecting body weight or liver steatosis. These findings reveal that dietary context influences the metabolic consequences of pesticide mixtures, highlighting the need to consider nutritional status when evaluating environmental contaminant risks. Our results suggest that pesticide mixtures at reference doses may contribute to metabolic dysregulation, particularly under obesogenic dietary conditions. Highlights- Four common pesticides in mixture disrupt metabolism in liver cells - Dietary exposure to this pesticide mixture alters hepatic gene expression in mice - The pesticide mixture exacerbates WD-induced disruptions in glucose homeostasis - Pesticides and diet interact in producing the metabolic effects of a pesticide mixture

Developmental exposure to organophosphate flame retardants (OPFRs) is a public health concern due to their endocrine-disrupting potential. We examined perinatal exposure to tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, and tricresyl phosphate in mice. Adult male and female offspring were assessed for memory and anxiety-like behavior. Dopamine and norepinephrine were quantified in the hippocampus and prefrontal cortex (PFC), and bulk RNA sequencing was conducted for the hippocampus. OPFR-treated females in high ovarian hormone states spent less time in the open field test (OFT) center, the Y-maze unknown arm, and with the displaced object in spatial object recognition (SOR) indicating increased anxiety-like behavior and impaired spatial memory. These females also illustrated improved memory on the short-term Barnes maze, and a trending improvement in the novel object recognition test. Females in low ovarian hormone states, demonstrated a trend in center OFT exploration. OPFR-treated males displayed disruption in memory in the SOR and the short- and long-term Barnes maze. Perinatal OPFR reduced hippocampal dopamine in males and altered prefrontal dopamine in females in a hormone-dependent manner. OPFR-treated females in high ovarian hormones states demonstrated a trending decrease in PFC norepinephrine. Perinatal OPFR treatment caused differential gene expression in 121 individual genes and alteration to functional modules related to RNA processing, cellular metabolism, and extracellular organization. Hormone status also affected gene OPFR-induced altered expression, with similarity between males and high ovarian hormone state females. Our findings suggest that perinatal OPFR exposure causes widespread, sex specific, and hormone dependent disruptions in behavior, neurochemistry, and gene expression in adulthood. HighlightsO_LIAnxiety-like behavior in OPFR-treated females varied with ovarian hormone status C_LIO_LIHigh ovarian hormone OPFR females showed task-dependent changes in memory C_LIO_LIMales displayed impaired spatial memory following perinatal OPFR treatment C_LIO_LIPerinatal OPFR modifies hippocampal and prefrontal dopamine and norepinephrine C_LIO_LIOPFR treatment altered individual gene and functional gene module expression C_LI

Doxorubicin (DOX) is an effective anti-cancer drug; however it can cause cardiotoxicity by inducing DNA double-strand breaks in cardiomyocytes. Cardiotoxicity can manifest immediately or years following treatment. Most human in vitro models of DOX-induced cardiotoxicity (DIC) focus on the acute effects of DOX treatment. To understand the long-term effects, we profiled the global gene expression response to DOX exposure over time. We treated iPSC-derived cardiomyocytes from six individuals with DOX for 24 hours and assayed responses after 0, 24 and 144 hours of recovery. DNA damage, determined by {gamma}H2AX expression, is induced following DOX treatment and is resolved by the final recovery timepoint. We identified both acute and chronic gene expression response signatures. The chronic signature, representing 501 genes, is enriched for p53 target genes and DNA damage response genes compared to acute response genes. P53 target genes are persistently activated, and DNA damage response genes are progressively downregulated over time. Our results suggest an altered cell state following repair of double-strand breaks that is distinct from pre-exposed cells. DOX response genes with persistent changes in expression can be applied to the design of toxicity biomarkers or therapeutic targets.