Toxicology and Applied Pharmacology

Fetal exposure to endocrine-disrupting bisphenol A (BPA) showed a long-lasting programming effect on organ development and predisposed to the metabolic risk of adult diseases. However, limited data on developmental exposure to BPA-substitute bisphenol S (BPS) in predisposing liver metabolic disease is available. Here, the effects of BPS exposure were assessed on hepatic metabolism by examining adiposity and inflammation in the adipose and liver of the 90-day male offspring. Pregnant Wistar rats were exposed to BPA and BPS (0.0, 0.4, 4.0 {micro}g/kg bw) via gavage from gestational day 4 to 21. Prenatal BPS-exposed offspring exhibited a higher obesogenic effect than BPA, including changes in body weight, body fat, feed efficiency, and leptin signalling. The fasting blood glucose did not change, but BPS exposure elevated plasma corticosterone levels and adipocyte hypertrophy of the visceral adipose tissue (VAT) to a greater extent than BPA. Adipocyte hypertrophy was augmented by modulated expression of lipid uptake (PPAR{gamma}, FABP4), glucocorticoid (HSD11{beta}1), inflammation (IL6, IL1{beta}, CRP, COX2), oxidative stress (CHOP) and apoptotic (Caspase 3) mediators. Liver histology showed numerous lipid droplets, and hepatocyte ballooning, associated with upregulated expression of cholesterol, lipid biogenesis and glucocorticoid activators, indicating microvesicular steatosis in the prenatally BPS-exposed adult offspring. The upregulated PPAR, ADRP, and FGF21 expression and increased lipid peroxidation in the offsprings liver suggest metaflammation due to fetal exposure to BPS. Fetal BPS exposure demonstrated a more significant disruption in metabolism involving adiposity, liver fat, inflammation in excess, and predisposition to hepatic steatosis in the male offspring. HighlightsO_LIFetal BPS exposure exhibited enlarged and inflamed adipocytes more than BPA C_LIO_LIPrenatal BPS exposure induced excess lipid droplets & hepatocyte ballooning in liver C_LIO_LIIn utero exposure to BPS induces microvesicular steatosis in adult rats C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/543354v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@89c53org.highwire.dtl.DTLVardef@14f176borg.highwire.dtl.DTLVardef@18a934corg.highwire.dtl.DTLVardef@cc7af6_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectiveTo evaluate the synergistic effects created by fine particulate matter (PM2.5) and corticosteroid use on hospitalization and mortality in older adults at high-risk for cardiovascular thromboembolic events (CTEs). Design and SettingA retrospective cohort study using a US nationwide administrative healthcare claims database. ParticipantsA 50% random sample of participants with high-risk conditions for CTE from the 2008-2016 Medicare Fee-for-Service population. ExposuresCorticosteroid therapy and seasonal-average PM2.5. Main Outcome MeasuresIncidences of myocardial infarction or acute coronary syndrome, ischemic stroke or transient ischemic attack, heart failure, venous thromboembolism, atrial fibrillation, and all-cause mortality. We assessed additive interactions between PM2.5 and corticosteroids using estimates of the relative excess risk due to interaction (RERI) obtained using marginal structural models for causal inference. ResultsAmong the 1,936,786 individuals in the high CTE risk cohort (mean age 76.8, 40.0% male, 87.4% White), the mean PM2.5 exposure level was 8.3 {+/-} 2.4 g/m3 and 37.7% had at least one prescription for a systemic corticosteroid during follow-up. For all outcomes, we observed increases in risk associated with corticosteroid use and with increasing PM2.5 exposure. PM2.5 demonstrated a non-linear relationship with some outcomes. We also observed evidence of an interaction existing between corticosteroid use and PM2.5 for some CTEs. For an increase in PM2.5 from 8 g/m3 to 12 g/m3 (a policy-relevant change), the RERI of corticosteroid use and PM2.5 was significant for heart failure (15.6%, 95% CI: 4.0%-27.3%). Increasing PM2.5 from 5 g/m3 to 10 g/m3 yielded significant RERIs for incidences of heart failure (32.4; 95% CI: 14.9%-49.9%) and myocardial infarction/acute coronary syndromes (29.8%; 95% CI: 5.5%-54.0%). ConclusionPM2.5 and systemic corticosteroid use were independently associated with increases in CTE hospitalizations. We also found evidence of significant additive interactions between the two exposures for heart failure and myocardial infarction/acute coronary syndromes suggesting synergy between these two exposures. Strengths and Limitations of this StudyO_LIWe conduct analyses using robust causal inference and machine learning techniques and incorporate a large set of individual-level factors that are typically absent in environmental health analyses with large claims data sets. C_LIO_LIWe present statistics that evaluate the synergy between fine particulate matter and corticosteroid therapy on the additive scale, which is more relevant for assessing excess risks and informing policy. C_LIO_LIPatient medical history prior to receiving Medicare benefits is unknowable within a Fee-for-Service claims database, which may lead to exclusion of some high-risk individuals from the cohort. C_LIO_LIWe censor participants after the earlier of the end of first corticosteroid regimen or the first incidence of the outcome of interest, which makes the analyses statistically tractable but sacrifices some information in the data. C_LI

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.

BackgroundLong-term exposure to air pollution, even at levels below regulatory standards, has been associated with higher risk of cardiovascular-related mortality. Less is known about the association of air pollution and initial development of CVD in low-exposure settings in generally healthy human populations. ObjectiveIn the Canadian Alliance for Healthy Hearts and Minds Cohort Study (CAHHM), we aimed to investigate the association between low-level exposure to key air pollutants and subclinical carotid atherosclerosis in adults without known clinical CVD. To date, the association of ambient air pollution and atherosclerosis measured by magnetic resonance imaging (MRI) has not been studied. MethodsWe studied 6,645 Canadian adults recruited between 2014-2018 from the provinces of British Columbia, Alberta, Ontario, Quebec, and Nova Scotia, for whom average long-term exposures to nitrogen dioxide (NO2), ozone (O3), and fine particulate matter (PM2.5) were estimated for five years prior to the start of CAHHM recruitment, and who underwent MRI to assess carotid vessel wall volume (CWV). Linear mixed models were used to quantify associations between each air pollutant and CWV adjusting for individual-level and community-level risk factors for CVD. Secondary analyses included region-specific stratification and modeling the effect of one pollutant on CWV within low, medium, and high levels of a second pollutant to test for interactions. ResultsHigher PM2.5 was nominally associated with lower CWV (quintile 5: 893.3 mm3, quintile 1: 908.8 mm3; p-trend =0.05), but this was not robust in region-stratified analysis. Higher NO2 was associated with lower CWV (quintile 5: 889.5 mm3, quintile 1: 918.6 mm3; p-trend <.0001). Higher O3 was associated with higher CWV (quintile 5: 925.4 mm3, quintile 1: 899.7 mm3; p-trend =0.02). NO2 emerged as a consistent effect modifier of both PM2.5 and O3. ConclusionIn a cohort of generally healthy adults living in Canada, a country with relatively low levels of air pollution, exposure to NO2 was negatively associated, and O3 was positively associated with CWV as a measure of subclinical atherosclerosis by MRI, while associations to PM2.5 were inconsistent. The reasons for these associations warrant further study.

Aberrant signal transduction pathways can adversely derail developmental processes. One such process is embryonic eyelid closure that requires MAP3K1. Map3k1 knockout mice have defective eyelid closure and an autosomal recessive eye-open at birth phenotype. In utero exposure to dioxin, a persistent environmental toxicant, causes the same eye defect in Map3k1+/- hemizygous but not wild type pups. Here we explore the mechanisms of Map3k1 (gene) and dioxin (environment) interactions (GxE) in the tissue closure defect. We show that, acting through the AHR, dioxin activates EGFR signaling, which in turn depresses MAP3K1-dependent JNK activity. This effect of dioxin is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr, encoding the S1P receptors upstream of the MAP3K1-JNK pathway, potentiates dioxin toxicity. Our novel findings suggest that dioxin and genes of the AHR, EGFR and S1P-MAP3K1-JNK pathways constitute a multifactorial mechanism underlying tissue closure abnormalities. Summary statementThe crosstalk between a global environmental pollutant and the pre-existing genetic conditions is mediated through interactive signaling pathways, resulting in anatomical tissue closure abnormalities in development.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 {micro}g/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl2 exposed mice were 9081{+/-}900 vs. 1879{+/-} 293 at 6 h and 2966{+/-}463 vs. 1555{+/-}250 at 50 h post injection (ng/ml; X{+/-}1 SEM=3; p<0.01). Cl2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and to the therapeutic effects of hemopexin.

Adverse Outcome Pathways (AOPs) are designed to provide mechanistic insights into toxicological processes after exposure to a stressor and facilitate the replacement of animal studies with in vitro testing systems. Starting with a Molecular Initiating Event (MIE) and ending with an Adverse Outcome, the sequence of Key Events (KEs) spans across biological levels, but the majority of KEs in AOP-Wiki describe molecular and cellular processes. That suggests that established transcriptome-wide studies can be used to validate or measure a multitude of KEs simultaneously and be a goldmine of useful information about cellular responses. Currently, in toxicology, omics technologies are not widely applied in risk assessments of chemicals because of their complexity and lack of consensus on aspects such as standardization, analysis and interpretation. Given their value in hypothesis generation and ability to provide a vast amount of information about a biological response, the challenge lies in the acceptance of using transcriptomics data in regulatory risk assessments. We here introduce molecular AOPs to define the connections between KEs of the AOP-Wiki and curated biological pathways in WikiPathways, thereby providing a new method for the analysis and interpretation of transcriptomics data to identify KE activation. To study this, we performed case studies on liver steatosis and mitochondrial complex I inhibition, for which molecular AOPs were developed and public transcriptomics datasets were selected. Upon extension of the molecular AOP networks in Cytoscape, we mapped and analysed transcriptomics data, and calculated an enrichment score for individual KEs. Further interpretation of the data was done through the visualisation of the data on the specific molecular pathways. Two molecular AOPs were developed and KEs were linked to the appropriate molecular pathways, allowing a detailed exploration of molecular processes with the selected transcrip-tomics datasets. This has shown us that we can verify the activation of specific MIEs and KEs, and assess progression across the AOP in the steatosis case study through variables in exposure time and dose. These case studies have shown that transcriptomics data can be used for identifying the potential activation of KEs. However, it is also clear that extensive datasets are required to fully test the capabilities of molecular AOPs, and the process of linking molecular pathways and KEs can be challenging, not always allowing one-to-one mapping. While proven valuable to analyse and understand transcriptomic data, pathways linked to KEs appear to show inconsistent levels of activation and should be looked into and refined. More case studies are required to optimize the approaches used for the development and use of molecular AOPs with transcriptomics datasets.

Single doses of perfluoro-n-decanoic acid (PFDA) cause wasting, a progressive loss of 30 to 50% body weight, increasing liver/body weight ratios, and death within several weeks (Olson and Andersen, 1983). Repeat high doses of perfluorooctane sulfonate (PFOS) produce a subset of these responses in rats and monkeys. The mode of action (MOA) of these wasting-like syndromes is not clear, nor is it understood if these responses are limited to a subset of perfluoroacid substances (PFAS) or a common response to high dose exposure with a larger number of PFAS. To identify pathway perturbations in liver caused by PFAS, we analyzed published in vitro gene expression studies from human primary liver spheroids treated with various PFAS for treatment times up to 14 days (Rowan-Carroll et al., 2021). With treatment times of 10 to 14 days, longer-chain PFAS compounds, specifically PFOS, perfluorodecane sulfonate (PFDS) and higher doses of perfluorooctanoic acid (PFOA), downregulated large numbers of genes in pathways for steroid metabolism, fatty acid metabolism and biological oxidations. Shorter chain PFAS compounds upregulated genes in pathways for fatty acid metabolism. Although PFDA was more toxic and could only be examined at 1-day of treatment, it also downregulated genes for lipid metabolism, steroid metabolism, and biological oxidations. Shorter chain PFAS, both carboxylic and sulfonic acids, did not lead to downregulation of pathways for fatty acid or steroid metabolism. TCDD is also known to cause wasting responses in rodents and humans. In intact rats, high dose responses of longer chain PFAS produce downregulation of batteries of genes associated with fatty acid oxidation and lipogenesis similar to those seen with TCDD. Based on our results, when combined with other literature, we propose that the longer-chain PFAS impair lipogenic pathways through inhibitory interactions between PPAR{beta}, PPAR and PPAR{gamma}.

Maintenance of genome integrity is essential for normal human development, particularly during pre-gastrulation stages when rapid proliferation and intense transcriptional activity increase susceptibility to DNA damage. Environmental genotoxins such as benzo[a]pyrene (BaP), a widespread polycyclic aromatic hydrocarbon, and its reactive metabolite benzo[a]pyrene diol epoxide (BPDE) form bulky DNA adducts that interfere with replication and transcription, thereby posing significant risks to embryonic genome stability. To examine how genetic defects in DNA repair influence these effects, we assessed human induced pluripotent stem cells (iPSCs) carrying pathogenic mutations in ERCC6 (encoding the Cockayne syndrome B, CSB, protein), a key component of transcription-coupled nucleotide excision repair. Pathogenic ERCC6 mutations result in Cockayne syndrome- a severe neurodevelopmental disorder characterized by growth failure, premature aging, and multisystemic degeneration, thus underscoring the essential developmental functions of CSB. Exposure of healthy and CSB-deficient patient derived iPSCs to BPDE revealed impaired proliferation, persistent accumulation of DNA damage and defective checkpoint activation in CSB-deficient lines. Although the levels of key pluripotency-regulating proteins such as OCT4 and NANOG remained unaltered, we observed altered levels of SOX2 and p-SMAD1/5 signaling thus implying that unrepaired DNA damage can perturb developmental-associated signaling pathways and biological processes. Transcriptomic profiling revealed broad suppression of DNA repair and cell-cycle pathways together with activation of p53-, TNF-, and MAPK/JNK-mediated stress responses in CSB-deficient lines. Failure to induce anti-oxidant defenses, including SOD2 and IDO, further contributed to oxidative imbalance and incomplete apoptotic clearance. These findings demonstrate that CSB function is essential for coupling DNA repair with transcriptional recovery and redox homeostasis in pluripotent cells. Loss of CSB destabilizes the genome stability under genotoxic stress, providing a mechanistic basis for developmental toxicity of environmental polycyclic aromatic hydrocarbons and underscoring the importance of considering genetic susceptibility in developmental toxicology risk assessment.

Emerging studies find arteriolar dysfunction in offspring with in-utero electronic cigarette (Ecig) exposure, but the long-term effect on offsprings cerebrovascular vascular and neurocognitive health is poorly understood. Ecigs provides a unique opportunity to directly evaluate the contributions of inhaled nicotine from the vehicle e-liquid - which was not possible with traditional cigarettes. Moreover, many Ecigs have variable power settings, which can alter the toxicity of the aerosol cloud produced. We hypothesize maternal vaping at different wattages will have variable effects on cerebrovascular function in the offspring, and that these effects would be independent of nicotine. We used time-mated female Sprague-Dawley rats with Ecig exposure from gestation day (GD)2-21. We studied male and female offspring for vascular and neurocognitive function at 1-, 3-, 6- and 12-months of age. We found that, both sexes, offspring with in-utero exposure (at 5w and 30w Ecig conditions) exhibited impaired middle cerebral artery (MCA) reactivity. While the magnitude of impairment was greater at higher that lower watts, Ecig at 5-watts still exhibited significant impairments in MCA function (suggesting the harm threshold for blood vessels is very low). Vascular dysfunction was evident with or without nicotine in the e-liquid, but nicotine exposure resulted in short-term memory deficits, evidence of neuronal damage, and increased astrocyte interaction with endothelial cells in 6- and 12-month-old offspring. We also observed altered expression of clock genes and antioxidant signaling pathways, along with a decrease in sirtuin-1 expression, decreased ratio of beta-amyloid A 42/40 protein expression, and increased in NOX1, which are consistent with redox imbalance, neuroinflammation, and advancing cellular senescence. These preclinical data provide evidence suggesting that in utero exposure to Ecigs from maternal vaping can be expected to adversely affect the brain health of offspring in their adult life and that neurocognitive outcomes are worsened with exposure to nicotine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/638202v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1e7a7a1org.highwire.dtl.DTLVardef@1ae5bceorg.highwire.dtl.DTLVardef@d323eorg.highwire.dtl.DTLVardef@5db4af_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender

BackgroundAir pollution has several negative health effects. Particulate matter (PM) is a pollutant that is often linked to health adversities. PM2.5 (PM with an aerodynamic diameter of [&le;]2.5m) exposure has been associated with negative cardiovascular (CV) outcomes. However, the impact of PM10 (PM with an aerodynamic diameter of [&le;]10m) exposure is often overlooked due to its limited ability to pass the alveolar barrier. This study aims to assess the association between PM10 exposure and risk of myocardial infarction (MI) amongst adults ([&ge;]18 years of age) as this has been poorly studied. MethodsThe study protocol was published on PROSPERO (CRD42023409796) on March 31, 2023. Literature searches were conducted on 4 databases (OVID Medline, Embase, CINAHL, and Web of Science) for studies looking at associations between PM and MI. English studies from all time periods were assessed. Studies selected for review were time-series, case-crossover, and cohort studies which investigated the risk of MI as an outcome upon PM10 exposure. The quality of evidence was assessed using Cochranes GRADE approach. Data for different risk outcomes (risk ratio (RR), odds ratio (OR), hazard ratio (HR)) and 3 lags was meta-analyzed using an inverse variance statistical analysis using a random effects model. The pooled effect sizes and the 95% confidence intervals (CIs) were reported in forest plots. ResultsAmong the 1,099 studies identified, 41 were included for review and 23 were deemed eligible for meta-analysis. Our analysis revealed that there is an increased risk (OR=1.01; 95% CI:1.00 - 1.02) of MI with a 10 g/m3 increase in PM10 after a lag 0 and lag 1 delay. ConclusionsOur findings indicate that PM10 exposure is associated with an increased risk of MI. This can aid in informing environmental policy-making, personal-level preventative measures, and global public health action.

BackgroundEpidemiology has demonstrated a strong relationship between fine particulate matter (PM) exposure and cardiovascular disease. Whether PM aggravates myocardial ischemia/reperfusion (I/R) injury and its related mechanisms remain unclear. Our previous study showed that adipose stem cell-derived exosomes (ADSC-Exo) contain a large amount of miR-221/222. This study investigated the effects of PM exposure on I/R-induced cardiac injury through mitophagy and apoptosis, as well as the potential role of miR-221/222 in ADSC-Exo. MethodsWild-type, miR-221/222 knockout (miR-221/222 KO), and miR-221/222 overexpressed transgenic (miR-221/222 TG) mice were intratracheally injected with 100 g/kg PM for 24 h before I/R treatment. Ischemia was induced by temporarily occluding the left anterior descending (LAD) coronary artery with sutures for 30 min, followed by 3 h of reperfusion. In an in vitro model, H9c2 cells were exposed to 50 g/mL PM for 6 h and subjected to hypoxia (1% O2) at 37{degrees}C for 6 h, followed by 12 h reoxygenation. ResultsPM aggravates I/R (H/R)-induced cardiac injury by increasing ROS levels and causing mitochondrial dysfunction, leading to an increase in mitochondrial fission-related proteins like Drp1 and Mff, mitophagy-related proteins such as BNIP3 and LC3B, as well as apoptosis-related proteins like PUMA and p-p53 in vivo and in vitro studies. In comparison, transfection of ADSC-Exo and miR-221/222 mimics significantly reduced PM+I/R (H/R)-induced cardiac injury. Importantly, ADSC-Exo contains miR-221/222, which directly targets BNIP3, LC3B, and PUMA, decreasing their expression and ultimately reducing cardiomyocyte mitophagy and apoptosis. ConclusionsThe study showed that PM aggravates I/R or H/R-induced cardiac injury, and ADSC-Exo treatment significantly reduced this by regulating mitophagy and apoptosis through miR-221/222/BNIP3/LC3B/PUMA.

Polychlorinated biphenyls (PCBs) are ubiquitous and representative pollutants that pose great health risks. While cells responses to dioxin-like PCBs tend to be studied on a bulk scale, few studies have been made from a single-cell level. Here, by using single-cell RNA sequencing, we depicted a detailed landscape of hepatic nonparenchymal cells intricate responses to PCB126 exposure. A total of 13 clusters were identified. Notably, PCB126 exposure resulted in cell-type-specific gene expression profiles and genetic pathways. By analyzing genes related to aryl hydrocarbon receptors, we discovered that PCB126 induced the canonical genomic AhR pathway mainly in endothelial cells. In contrast, other cell types showed little induction. Enrichment pathway analysis indicated that immune cells changed their transcriptional patterns in response to PCB126. ScRNA-seq is a powerful tool to dissect underlying mechanisms of chemical toxicity regarding biological heterogeneity. Taken together, our study not only extends our current understanding of PCB126 toxicity, but also emphasizes the importance of in vivo cell heterogeneity in environmental toxicology.

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.

This study investigated the age and sex-dependent comorbidities contributing to coronary artery disease (CAD) within the UAE population. A cohort of 3,000 individuals was analyzed by integrating genetic data, environmental stressors (e.g., PM2.5 exposure), and demographic profiles to identify CAD and nine other diseases with heterogeneous comorbidity patterns. Key genetic markers, including APOE rs429358, PCSK9, and LPA were significantly associated with CAD risk, amplified by environmental exposure and metabolic conditions such as diabetes and obesity. Notably, APOE rs429358 carriers exposed to high PM2.5 levels exhibited a 2.8-fold increase in CAD risk (p < 0.001), emphasizing the synergistic effects of gene-environment interactions. Monte Carlo and Markov Chain Monte Carlo simulations validated the results, enabling the identification of high-risk genetic profiles across various environmental and demographic conditions. Kaplan-Meier survival analyses revealed accelerated disease progression in high-risk groups, whereas Principal Component Analysis and hierarchical clustering identified distinct genetic clusters stratified by age and sex. This study further identified demographic-specific disease subtypes with implications for public health strategies, such as addressing higher environmental susceptibility in males and targeted management of metabolic comorbidities in females such as obesity, diabetes, and stroke. These findings support precision medicine strategies tailored to regional populations, promoting targeted interventions to mitigate CAD risk. This study synthesizes observational findings and computational simulations to establish a comprehensive framework for elucidating the pathogenesis of coronary artery disease (CAD) and enhancing public health interventions in the United Arab Emirates (UAE). The actionable outcomes include the development of sex-specific health interventions and environmental policies to reduce CAD risk in high-susceptibility groups.

BackgroundGlobally nearly 20% of cardiovascular disease deaths were attributable to air pollution. Out-of-hospital cardiac arrest (OHCA) represents a major public health problem, therefore, the identification of novel OHCA triggers is of crucial relevance. The aim of the study was to evaluate the association between air pollution (short-, mid-and long-term exposure) and out-of-hospital cardiac arrest (OHCA) risk, during a 7 years-period from a highly polluted urban area with a high density of automated external defibrillators (AEDs). Methods and resultsOHCA were prospectively collected from the "Progetto Vita Database" between 01/01/2010 to 31/12/2017; day-by-day air pollution levels were extracted from the Environmental Protection Agency (ARPA) stations. Electrocardiograms of OHCA interventions were collected from the AEDs data cards. Day-by-day particulate matter (PM) 2.5 and 10, ozone (O3), carbon monoxide (CO) and nitrogen dioxide (NO2) levels were measured. A total of 880 OHCAs occurred in 748 days. A significantly increased in OHCA risk with the progressive increase in PM 2.5, PM 10, CO and NO2 levels was found. After adjustment for temperature and seasons, a 9% and 12% increase of OHCA risk for each 10 g/m3 increase of PM 10 (p< 0.0001) and PM 2.5 (p< 0.0001) levels was found. Air pollutants levels were associated with both asystole and shockable rhythm risk while no correlation was found with pulseless electrical activity. ConclusionsShort-term and mid-term exposure to PM 2.5 and PM 10 is independently associated with the risk of OHCA due to asystole or shockable rhythm.

Inhalation of ultrafine particles (UFP) mediates systemic vascular impairment which is, in part, driven by elevated rates of oxidant generation. One significant source of oxidant production in the vascular compartment is the purine catabolizing enzyme, xanthine oxidoreductase (XOR). However, mechanisms linking XOR and/or endothelial glycosaminoglycan (GAG)-sequestered XOR to vessel dysfunction allied to UFP inhalation remain underexplored. Based on known interactions between UFP and the liver, we hypothesized that exposure could lead to hepatic release of XOR to the circulation which subsequently contributes to vascular impairment. Utilizing our murine hepatocyte-specific XOR knockout (XORHep-/-) model (loss of function) in conjunction with reintroducing exogenous XOR (restoration of function) we demonstrate a specific role for liver-derived XOR in the pathogenesis of UFP-induced vascular impairment. Exposure of mice as well as in vitro exposure of hepatocytes to our model UFP, nano titanium dioxide (nTiO2) results in the upregulation and active release of XOR. Drinking water supplemented with the XOR inhibitor febuxostat or nitrite (NaNO2-) partially prevented nTiO2-induced impairment of vascular reactivity. Interestingly, nitrite appears to cause a down-regulation of hepatic XOR. XORHep-/- mice were partially protected against both impairment of endothelial dependent dilation and augmented angiotensin II constriction. To further demonstrate the role of circulating XOR in nTiO2-induced impairment of vessel reactivity, XORHep-/- mice had circulating XOR restored by i.v. injection prior to exposure, which eliminated the protection of the hepatic knockout. It is important to note that acute restoration of intraluminal XOR in isolated vessels did not alter endothelial-dependent dilation or angiotensin II constriction. As such, we interrogated potential downstream mediators of XOR effects on endothelial function and found a decrease in the repressive trimethylation of lysine 9 on histone 3. Together these findings demonstrate that circulating XOR is a key contributor to endothelial dysfunction caused by UFP exposure. However, the impairment is not acute in nature and might involve epigenetic-mediated alterations in gene expression.

bioRxiv has withdrawn this preprint because we no longer have confidence on the validity of the manuscript and the identity of the authors. Therefore, this work should not be cited as a reference for this project.

Effects of chronic heavy metal stress on hepatocellular pathophysiology remains ill-understood. Human livers are a long-term accumulative site for many toxic heavy metals (e.g., cadmium and arsenic) whose effects are unknown. In the current study, we studied effects of chronic, low-dose exposures of cadmium (CLEC) modulated by normoglycemic (5.6 mM) and hyperglycemic (15 mM) exposures, focusing on hepatocellular mitochondrial function. HepG2 and HUH7 cell lines were exposed to CLEC and glucose for 24 weeks, mimicking a chronic heavy metal exposure paradigm seen in normal and type II diabetic individuals. We observe that CLEC exposures significantly affect the long-term health of mitochondria, including decreased mitochondrial mass, increased superoxide production, and loss of mitochondrial membrane potential (MMP) in a CLEC and glucose-dependent manner. Furthermore, the Seahorse MitoStress assay revealed CLEC induced significant chronic oxidative stress. In particular, CLEC cells showed altered levels of basal and non-mitochondrial respiration, causing dysregulation in mitochondrial oxygen consumption rates (OCRs). Lastly, we identified significant impacts of CLEC and glucose exposures on the mitochondrial dynamics (fission/fusion) of the CLEC cells, which showed enhanced mitochondrial fragmentation and turnover rates. We also identified novel cell compensatory mechanisms that may mask the true extent of chronic Cd exposure induced damage in liver cells. CLEC and glucose work additively to damage hepatocellular mitochondrial function. New approach methodologies (NAMs), such as the current vitro toxicology study, establish the insidious effects of chronic heavy metal pollutant exposures on human hepatocellular function.

Environmental chemicals play a significant role in the development of metabolic disorders, especially when exposure occurs early in life. We have recently demonstrated that benzene exposure, at concentrations relevant to a cigarette smoke, induces a severe metabolic imbalance in a sex-specific manner affecting male but not female mice. However, the roles of benzene in the development of aberrant metabolic outcomes following gestational exposure, remain largely unexplored. In this study, we exposed pregnant C57BL/6JB dams to benzene at 50 ppm or filtered air for 5 days/week (6h/day from gestational day 1 to birth) and studied male and female offspring metabolic phenotypes in their adult life. While no changes in body weight or body composition were observed between groups, 4-month-old male and female offspring exhibited reduced parameters of energy homeostasis (VO2, VCO2, and heat production). However, only male offspring from benzene-exposed dams were glucose intolerant and insulin resistant at this age. By six months of age, both male and female offspring displayed glucose and insulin intolerance, associated with elevated expression of hepatic gluconeogenesis and inflammatory genes. Additionally, this effect was accompanied by elevated insulin secretion and increased beta-cell mass only in male offspring. Thus, gestational benzene exposure can reprogram offspring for increased susceptibility to the metabolic imbalance in adulthood with differential sensitivity between sexes.