Toxicology and Applied Pharmacology

Lead (Pb) exposure is strongly associated with cardiovascular diseases, but the effects of developmental Pb exposure on the electrophysiological properties of human cardiomyocytes have not yet been investigated. Using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, we investigated the effects of human-relevant Pb exposure during ventricular cardiomyocyte differentiation on action potential properties and calcium dynamics in differentiated myocytes. We exposed 2 iPSC lines, from 1 male and 1 female donor, to Pb (0.5 or 5 micromolar) or control during only the first 8 days of differentiation, prior to functional maturation. Differentiation was carried out using an established protocol involving temporal modulation of Wnt signaling, followed by purification and maturation of contractile myocytes. We measured action potential properties and calcium dynamics using fluorescent dyes and optical mapping. We hypothesized that Pb exposure would adversely affect cardiomyocyte function, with effects persisting after cessation of exposure. In keeping with this hypothesis, we found significant Pb-induced changes in calcium (Ca) transients and action potential dynamics that were concentration and cell line specific and persisted well after exposure had ended. In the female cell line, beat frequency significantly increased with Pb exposure for both action potentials and Ca transients, while in the male cell line, beat frequency significantly decreased with Pb exposure for action potentials and increased for Ca transients. For voltage measurements, we observed significantly increased arrythmias with Pb exposure in both cell lines. Significant changes in repolarization and recovery time (for voltage and Ca, respectively) were present with Pb exposure in both cell lines. Gene expression of Ca transporters/channels and K channels were not affected, suggesting that persistent effects of Pb exposure on cardiomyocyte electrophysiology may occur through post-translational mechanisms. Overall, this study paves the way for future work to examine how exposure to Pb impacts long-term cardiac function through developmental programming.

Environmental exposure involves pollutant mixtures. Building on evidence that individual bisphenols alter zebrafish larval physiology and transcriptomic profiles at ng/L concentrations, we assessed the effects of specific mixtures of BPS, BPF, and BPAF without (M1) or with (M2) BPA. Experimentally, M1 and M2 induced 4.16% and 6.54% mortality in zebrafish larvae, despite the concentration addition model predicting no toxicity. Both mixtures reduced swimming-locomotor activities. These behaviors were accompanied by organ-level functional defects, as in vivo optical mapping revealed bradycardia and increased high-frequency (30-80 Hz) brain oscillations for both mixtures, while M2 additionally altered low-frequency (0.2-4 Hz) brain activity. The neurophysiological changes were accompanied by neurovascular remodeling, with in vivo two-photon microscopy revealing decreased vascular length and branching. Finally, transcriptomic analysis revealed common M1- and M2-induced alterations linked to pathways of vascular function, neuronal communication, and metabolism. M2 altered additional pathways of cardiac and neuronal signaling, consistent with the reported organ-physiology effects. We discuss the environmental relevance of mixture exposures and assess whether BPA intensifies mixture-induced responses.

Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor that plays a central role in metabolic adaptation by regulating gene expression programs involved in lipid utilization and energy homeostasis. Although the hepatic functions of PPAR and its target genes are well characterized, less is known about its transcriptional activity in extrahepatic tissues. Pemafibrate (K-877), a selective PPAR modulator (SPPARM), is a high-affinity PPAR ligand with improved potency and selectivity compared with conventional fibrates. Here, we investigated pemafibrate-induced tissue-specific transcriptional responses comparing wild type and whole-body PPAR-deficient littermate mice. Twelve-week-old male PPAR+/+ and PPAR-/- mice were treated daily with pemafibrate (0.1 mg/kg) or vehicle by oral gavage for 14 days. We performed genome-wide transcriptomic analyses in the liver, jejunum, kidney, soleus muscle, and BAT. Pemafibrate-induced transcriptional changes were entirely dependent on PPAR, as no differentially expressed genes were detected in PPAR-/- mice. The magnitude and nature of the response were tissue-specific, with the strongest transcriptional effects observed in the liver and jejunum, whereas the kidney, soleus muscle, and BAT showed limited responses. In the liver, pemafibrate primarily induced the expression of genes involved in fatty acid metabolism, mitochondrial function, and energy production. In the jejunum, pemafibrate activated lipid metabolic programs together with tissue-specific pathways related to cellular homeostasis. Collectively, our findings show that pemafibrate is a highly selective PPAR activator with a preferential transcriptional activity in the liver and intestine after oral administration. This study provides a resource for defining tissue-specific PPAR-responsive gene networks and expands our understanding of PPAR functions beyond the liver.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.

Tetrabromobisphenol A (TBBPA), is one the most widely produced brominated flame retardant, detected in human matrices including cord plasma, raising concern over its impact on embryonic development. Our previous work demonstrated that TBBPA disrupts craniofacial cartilage development in zebrafish; however, inhibition of bone morphogenetic protein (BMP) signaling did not rescue these defects, suggesting the involvement of alternative molecular mechanisms. Transcriptomic profiling revealed significant upregulation of aryl hydrocarbon receptor (Ahr) signaling genes, including cyp1a and cyp1c, as well as reactive oxygen species (ROS) responsive genes, such as nfe and gstp. Consistent with these findings, KEGG pathway enrichment analysis identified significant enrichment of pathways involved in xenobiotic metabolism, cytochrome P450-mediated metabolism, and molecular docking predicted stronger binding of TBBPA to Ahr2 than the Ahr2 agonist TCDD. Then we investigated the role of Ahr signaling and ROS in TBBPA-induced craniofacial cartilage defects using microinjection of Ahr2 translation-blocking morpholino and the antioxidant N-acetylcysteine (NAC), respectively. Immunohistochemistry confirmed concentration-dependent induction of Cyp1a and ROS at environmentally relevant concentrations (0.05;0.005 uM). Ahr2 knockdown using a translation-blocking morpholino rescued TBBPA-induced alterations in Cyp1a expression, ROS levels, DNA damage, the expression of chondrogenesis-related markers (Sox10 and Sox2), and epithelial to mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and Snail2). Furthermore, Ahr2 knockdown rescued several TBBPA-induced alterations in craniofacial cartilage parameters. Co-exposure of TBBPA with the antioxidant NAC rescued the ROS and DNA damage and rescued only ceratohyal cartilage length, whereas other cartilage parameters remained disrupted. These findings establish a causal role for the Ahr-Cyp1a axis in TBBPA-induced craniofacial cartilage developmental toxicity and identify ROS as an important downstream contributor, indicating that both ROS-dependent and ROS-independent Ahr mechanisms underlined the observed TBBPA induced craniofacial defects.

Environmental exposure to endocrine disrupting chemicals, like bisphenol-A (BPA), can impart detrimental effects on developing feto-placental unit, during pregnancy. Placenta remains a central player maintaining this feto-placental homeostasis for sustenance of a healthy pregnancy. Thus, the bisphenol-A mediated endocrine disruption affects the healthy functioning of placenta by altering key processes, such as tissue remodelling, angiogenesis, and metabolism. However, the underlying mechanism of BPA-altered ECM remodelling remains elusive. Therefore, in this study we investigated the BPA mediated changes in placental tissue remodelling using a bisphenol-A exposed murine model during pregnancy. The results reveal that, the phenotypic changes in feto-placental interface correlates with perturbed placental proteome in response to BPA. Further investigation highlights a S100a10-Annexin A2 axis mediated upregulation of tissue plasminogen activator (tPA), which drives altered extracellular matrix (ECM) degradation in placental decidua. This culminates into functional dysregulation in feto-placental axis, leading to reduced size of fetus and placenta. Therefore, this study provides novel insights of a S100a10-Annexin A2 axis associated mechanism for alteration of ECM remodelling in placental decidua due to BPA exposure, which may lead to toxicity related adverse pregnancy outcome.

Benzyl paraben (BzP) and butyl paraben (BuP) are used as preservatives and antimicrobial agents in cosmetics, pharmaceuticals, and foods. BzP and BuP have been detected in urine, placental tissue, and ovarian follicular fluid and are understudied with regards to their endocrine activity, raising concerns on their impact to the reproductive system. We tested the hypothesis that BzP and BuP are toxic to the ovary and behave similarly to other structurally similar endocrine disrupting chemicals, such as phthalates and terephthalates, by altering hormone levels and impacting cell growth and gene expression in vitro. Untreated mouse antral follicles were exposed to vehicle, 0.1, 1, 10, or 100 g/mL BzP, BuP, or the BuP analog monobutyl terephthalate (MBuTP) for 96 hours. The highest dose of BzP and BuP were cytotoxic to follicles, suggesting that medium chain length hydroxybenzoates are more cytotoxic to the ovary than corresponding terephthalic acids. BzP significantly increased levels of estradiol and testosterone compared to control. BuP decreased follicle growth and disrupted expression of cell cycle regulators, similar to phthalates. The peroxisome proliferator-activated receptor gamma (PPARg)-transcriptionally controlled gene Fabp4 was significantly upregulated by both BzP and BuP, whereas MBuTP upregulated all four PPAR target genes and decreased follicle growth. Overall, BzP, BuP, and MBuTP disrupted hormone levels, follicle growth, and cell cycle progression, suggesting similarities in toxicity to phthalates and potential endocrine disruption. Further study should be done on the reproductive toxicity and endocrine disrupting effects of chemicals similar in structure to phthalates to reduce exposure to endocrine disruptors and inform design of safer replacements.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with adverse female reproductive outcomes. However, most prior studies examined individual PFAS, despite that humans are exposed to complex PFAS mixtures. Here, we investigated the ovarian effects of a mixture of five commonly detected long-chain PFAS, including PFOS, PFOA, PFNA, PFDA, and PFHxS, using complementary in vivo mouse and in vitro 3D ovarian follicle culture models. Young adult female mice were exposed to a range of concentrations of the PFAS mixture (PFASmix) through daily drinking water for 8 weeks. PFASmix at high concentrations (PFASmix-high) disrupted mouse estrous cyclicity, decreased circulating estradiol, increased testosterone, and promoted follicular atresia. Antral follicles from exposed mice showed decreased Lhcgr and increased Amh expression. Although the number of ovulated oocytes was unchanged following superovulation, PFASmix-high reduced oocyte size and polar body extrusion and altered oocyte cytoskeletal characteristics. In the 3D follicle culture model, direct PFASmix exposure impaired follicle growth, ovulation, and oocyte maturation in a concentration-dependent manner. PFASmix-high suppressed the periovulatory expression of Areg, Ereg, Tnfaip6, and Adamts1 and impaired corpus luteal (CL) development and function, as evidenced by reduced CL spheroid growth and cell survival, decreased expression of Star, Cyp11a1, Hsd3b1, and Lhcgr, and reduced progesterone secretion. PFASmix-high exposure additionally altered the expression of multiple PPAR{gamma}-associated genes in cultured ovarian follicles. Benchmark dose modeling identified estrous cyclicity and follicle rupture as sensitive in vivo and in vitro endpoints, respectively. Together, these findings demonstrated that exposure to the mixture of long-chain PFAS disrupts multiple stages of ovarian function, from follicular development, oocyte maturation to ovulation and luteal development and function and provided mechanistic and quantitative evidence for the reproductive toxicity of PFAS mixtures.

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

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.

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.

Air pollution is a growing public health concern. The developing fetus is particularly vulnerable, with exposure during pregnancy linked to negative developmental health outcomes. Teratogenic studies rely on the use of model organisms, such as the chick embryo, a well- established model of human development. However, existing protocols for the exposure of chick embryos to gaseous and aerosol pollutants have financial and technical limitations. Here, we present a novel, non-invasive method for the long-term exposure of chicken embryos to a gaseous toxin, carbon monoxide (CO). Exposure is performed inside airtight incubation boxes, which can be used in a standard laboratory incubator. We demonstrate reliable dosing of precise internal CO concentrations up to 200ppm, using a simple volumetric approach. Following optimization of key incubation parameters, temperature and turning frequency, we determined the impact of the system on embryo viability and development. Closed box incubation caused minor developmental delay but had no effect on chick embryo viability. Internal oxygen concentrations remained above hypoxic levels. No significant effects of exposure up to 200ppm CO were observed on embryo viability, weight or developmental stage. In conclusion, we present a non-invasive, affordable, accessible and technically straightforward exposure method for air pollutant teratogenicity studies. This method can be applied to other model systems and organisms beyond the chick embryo as well as to other gaseous and aerosol toxins. Thus, the system offers a suitable platform for future research on teratogenic doses, mechanisms and effects of air pollutants.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Liver tumors are the most common carcinogenic outcome observed in two-year bioassays used for regulatory risk assessment of chemicals and pharmaceuticals. Because these traditional long-term studies are resource-intensive and not feasible for most chemicals, there is a critical need to develop alternative approaches that can identify potential rodent liver carcinogens in short-term studies (e.g., [&le;] 1 month). Biomarkers associated with molecular initiating events (MIEs) of early rodent liver tumorigenesis may enable the prediction of two-year outcomes from such studies. Here, we identified transcriptional changes associated with six key initiators of rat liver tumorigenesis: five transcription factors (TFs) associated with non-genotoxic modes of action (AhR, PXR, CAR, PPAR, and ER), and tumor suppressor protein p53, which is activated by diverse cellular stressors including genotoxicity. Three known chemical activators for each TF were administered orally once daily for five days at doses associated with tumorigenic responses in previous rat cancer bioassays. RNA-seq identified differentially expressed genes (DEGs) that were consistently regulated by at least two activators in wild-type but not in corresponding TF knockout animals. Gene set enrichment analysis of ranked differential expression results demonstrated enrichment of pathways reflecting established TF biology. When comparing the consensus DEGs for each TF to a library of chemically induced, liver-specific gene expression profiles, we found that in most cases the chemicals with the most similar profiles to the consensus lists were known to activate the corresponding TF. Collectively, these findings identified candidate gene sets that may have utility for monitoring the induction of carcinogenesis-associated MIEs in short-term studies.

Background/Objectives: Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, mitigates cellular stress by detoxifying reactive aldehydes produced during alcohol exposure and endogenous metabolic processes. ALDH2*2 polymorphism, which results in deficient catalytic activity, is prevalent in East Asian populations and is associated with an increased risk of cancer and other diseases. While the effects of ALDH2 deficiency on cellular stress responses are well documented, the specific metabolic dysregulations arising from this deficiency require further investigation. Methods; An ALDH2 knockout subline of SKBR-3 cells was established using CRISPR/Cas9 strategy. The metabolomic profiles of control (SKBR-3/C) and ALDH2 knockout (SKBR-3/ADKO) cells were evaluated via ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Results: ALDH2 knockout in SKBR-3 cells resulted in increased cellular stress, evidenced by elevated levels of 8-OHdG, reactive oxygen species (ROS), and malondialdehyde (MDA). Metabolomic analysis of the paired cell lines assigned a total of 5,033 signals, of which 281 were significantly different (fold change [&ge;] 2, p-value <0.05) between the two groups. Pathway analysis with MetaboAnalyst identified significant metabolic pathways associated with ALDH2 deficiency, particularly those involving arginine biosynthesis, and arginine and proline metabolism. ALDH2 deficiency reprogramed arginine metabolism toward polyamine pathways while suppressing the urea cycle. Network analysis further revealed alterations in amino acid and lipid metabolism in ALDH2-deficient cells. Conclusions; This study provides a novel insight into the role of ALDH2 in cancer metabolism and may have broader implication for cancer biology.

Carbaryl, a widely used carbamate pesticide, has been epidemiologically associated with genotoxic and carcinogenic outcomes in exposed human populations, including elevated cancer risk at several tissue sites and, in some agricultural communities, chronic tubulointerstitial kidney disease of unknown etiology (CKDu). However, the cellular mechanisms underlying these associations, and whether carbaryl acts uniformly across tissues, remain poorly defined. Screening a panel of cell lines of distinct species and lineage, we found that carbaryl elicits pronounced, dose-dependent cytotoxicity and cell cycle arrest selectively in a renal tubular epithelial line (IMCD3) and, to a lesser extent, a cervical epithelial line (HeLa), while a fibroblast line (NIH3T3) was unaffected. In IMCD3 cells, carbaryl induces S- and G2/M-phase arrest through direct binding to DNA and consequent DNA damage, triggers Bcl-2-mediated late apoptosis independently of reactive oxygen species, and drives irreversible mitotic arrest. Molecular dynamics simulations, cellular thermal shift assays, and kinase measurements converge on CDK1 as a direct target of carbaryl: carbaryl destabilises the CDK1 active site and inhibits its catalytic activity, whereas CDK2 is engaged through an opposing, rigidifying mode with no functional consequence. This asymmetry provides a coherent mechanistic basis for the combined S-phase and G2/M arrest observed. Together, these findings establish carbaryl as a genotoxic, cell-cycle-disrupting agent with tissue-selective potency acting directly on CDK1, and provide a cell-based mechanistic framework for interpreting its reported associations with renal and other tissue pathologies in exposed human populations.

Several studies have been conducted on human exposure to ultrafine particulate matter (UFPM), Black carbon (BC), and polystyrene nanoplastics (PS-NPs). However, it remains unclear whether different chemical types of environmental nanoparticles induce a similar mitochondrial stress response or a unique particle-specific response. In the present study, we examined the molecular mechanisms underlying nanoparticle-induced mitochondrial stress response and immunotoxicity using human peripheral blood mononuclear cells exposed to UFPM, BC, and PS-NPs under similar experimental conditions. Oxidative stress, mitochondrial adaptation, respiratory chain integrity, mitochondrial integrated stress response, inflammatory signaling, and systems-level interactions between molecules were analyzed through the evaluation of the expression of NRF2, HIF-1, PGC-1, TFAM, OMA1, DELE1, mitochondrial ND1, Complex I-V, NF-{kappa}B, TNF-, and NLRP3 and the use of principal component analysis, hierarchical clustering, and correlation networks. All three nanoparticles caused oxidative stress and mitochondrial dysfunction with different kinetics and mechanisms. UFPM mostly induced an acute antioxidant response and mitochondrial adaptation; BC led to chronic mitochondrial dysfunction, chronic activation of the OMA1-DELE1-mediated mitochondrial ISR pathway, and inflammation; while PS-NPs induced low but chronic mitochondrial adaptation along with mitochondrial biogenesis and stress responses. Our systems-level analysis showed that oxidative stress, mitochondrial adaptation, mitochondrial ISR, and inflammation represent a highly connected molecular network regardless of the physicochemical nature of the nanoparticles, with the OMA1- DELE1 axis being a key regulatory node connecting mitochondrial stress response and inflammation. Overall, we have found that mitochondrial stress response is a common mechanism underlying the toxicity of chemically different nanoparticles and have also revealed particle-specific stress-response dynamics responsible for the degree and persistence of cellular damage. The current work presents novel insights into the molecular mechanisms of nanoparticle-induced immunotoxicity and suggests OMA1, DELE1, NRF2, PGC-1, TFAM, ND1, and Complex I-V as potential biomarkers.

In utero alcohol exposure (IUE) can alter mammary development and increase mammary tumor susceptibility in experimental models. However, most previous studies have relied on carcinogen-induced or genetically tumor-prone models, leaving it unresolved whether prenatal alcohol exposure produces mammary developmental and molecular changes before the introduction of an oncogenic challenge. Here, we addressed this question using non-tumorigenic C57BL/6 mice. Pregnant dams received a Lieber-DeCarli liquid diet containing 1.7% (v/v) ethanol or an isocaloric control diet from gestational day 11 to 19, and mammary glands from female offspring were examined during puberty (postnatal day 40, PND40) and young adulthood (PND70). At PND40, IUE enhanced mammary ductal morphogenesis, increased terminal end bud numbers, and elevated epithelial proliferation. These developmental alterations persisted at PND70, with increased lateral branching and proliferative activity. IUE also altered mammary epithelial composition, expanding the basal/myoepithelial compartment and the CD24highCD49fhigh mammary repopulating unit/stem cell-enriched population. Mammary epithelial cells from IUE-exposed offspring exhibited increased colony-forming cells (CFCs), mammosphere formation, and 3D Matrigel growth, indicating enhanced stem/progenitor-associated activity. At the molecular level, IUE increased ER; expression and phosphorylation together with Cyclin D1, c-Myc, E2F1, and Bcl-2, and enhanced AKT, ERK, and STAT3 phosphorylation, IL-6 expression, and SOX2 at PND40. Importantly, major components of this signaling phenotype remained altered at PND70, including ER;-associated signaling, AKT/ERK activation, and IL-6/STAT3 signaling, whereas the pubertal increase in SOX2 was not sustained. Together, these findings demonstrate that prenatal alcohol exposure induces profound and persistent histomorphological, cellular, and signaling alterations in the mammary gland in the absence of a carcinogenic challenge or genetically introduced oncogenic driver. These results identify intrinsic developmental reprogramming of normal mammary tissue as a potential basis through which prenatal alcohol exposure may modify subsequent responses to tumor-promoting or other environmental challenges.

Omics methods have been envisioned to complement traditional toxicological testing for chemical risk assessment, in which identifying adverse effects is a critical step. However, the high dimensionality of transcriptomic data has historically led to reliance on context-dependent analysis. Liberality is a quantitative index that reduces genome-scale data dimensionality, with its changes reflecting underlying biological phenomena. In this study, we measured liberality in mouse liver RNA sequencing (RNA-Seq) datasets from studies in which mice were exposed to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 or 92 days, comparing dose-liberality relationships. Both 28- and 92-day TCDD treatments increased liberality but exhibited different dose-liberality relationships. Analysis of genes contributing to liberality revealed that longer exposure duration induced more extensive alterations in transcriptomic architecture. These findings suggest that liberality may serve as an unbiased metric to assess the extent of treatment-induced transcriptome perturbation.