Myeloperoxidase (MPO) dysregulation contributes to cardiovascular disease, making MPO a promising therapeutic target. Mitiperstat, an irreversible 2-thioxanthine MPO inhibitor in clinical development, has been linked to delayed-onset maculopapular rash. This study investigated whether mitiperstat and its metabolites (M7, M9, M10, M12) activate human T-cells in vitro. De novo T-cell responses from healthy donors were assessed using a dendritic cell:T-cell co-culture assay, and T-cell clones (TCCs) were characterised for compound specificity, phenotype, function and cross-reactivity. Proliferation was measured by [3H]-thymidine incorporation, and cytokine/cytolytic molecule release (IFN-γ, IL-5, IL-13, granzyme-B) by ELISpot. Twenty-nine predominantly CD4+ TCCs were specific to mitiperstat, while forty-seven CD4+ and CD8+ TCCs responded to its metabolites. All drug-specific TCCs proliferated dose-dependently and secreted a heterogeneous Th1/Th2 cytokine and cytolytic profile. CD4+ and CD8+ TCC activation was attenuated by MHC-class II and MHC-class I blockade, respectively. Despite the irreversible on-target pharmacology of mitiperstat, nearly all responsive TCCs were activated via direct pharmacological interactions with proliferation occurring with drug or respective metabolite in the presence of glutaraldehyde-fixed antigen-presenting cells. In contrast, one TCC was activated with drug-pulsed antigen presenting cells. TCCs showed high specificity for their cognate compounds with no cross-reactivity. These findings demonstrate the intrinsic immunogenicity of mitiperstat and its metabolites and establish a framework for assessing and comparing T-cell reactivity of future MPO-targeting therapies.

In humans, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces chloracne, a skin condition that presents with acanthosis, hyperkeratosis, sebaceous gland (SG) atrophy (seboatrophy), and hamartomas. Although chloracne-like phenotypes have been reported in TCDD-treated mice, the underlying mechanisms remain poorly understood. Previous studies showed that TCDD-induced CYP1A1 protein is expressed in LRIG1+ progenitor cells in hair follicles, suggesting that TCDD targets specific cell populations within the pilosebaceous unit. To explore the effects of TCDD on the epidermis and pilosebaceous unit, we analyzed single-cell RNA expression in wild-type and Ahr-null mice at postnatal day 21 (P21) following in utero and lactational exposure. The results showed that TCDD preferentially induced the AHR target genes Cyp1a1 and Cyp1b1 in the lower infundibulum and subjacent junctional zone overlapping the LRIG1+ progenitor cell niche. TCDD also caused Ahr-dependent seboatrophy, accompanied by increased expression of Blimp1, a transcriptional repressor that regulates SG size. A second site of Cyp1a1 induction was the SG, where Cyp1a1 was markedly elevated in the basal proliferating cells and immature sebocytes. In a 3-day topical exposure study of early effects, TCDD produced a dose-dependent increase of Cyp1a1 expression in the SG that included the more differentiated sebocytes. This response was accompanied by expansion of the Scd1-positive area, elevated Nile Red lipid staining, and an increased number of Blimp1-high sebocytes, demonstrating that TCDD enhanced SG differentiation and lipid production in vivo. These changes preceded the onset of Ahr-dependent seboatrophy, providing new insight into the cellular and molecular events underlying chloracne pathogenesis.

We present an updated workflow for safety assessment of human systemic toxicity of cosmetic chemicals that is exposure-led and hypothesis driven and adapted from that proposed by the SEURAT-1 European program, taking account of day-to-day realities faced by cosmetic ingredient risk assessors. The workflow utilizes pre-existing legacy in vivo toxicity data for target chemicals and chemically and biologically suitable analogues. These are considered alongside human exposure data and are complemented by data provided by in silico and in vitro new-approach methodologies, when needed to build confidence in the safety decision or when legacy in vivo toxicity data are unavailable. The workflow specifies three consecutive Tiers. Tier 0 involves problem formulation and collection of existing data; if this provides sufficient certainty in a safety decision, further work is not needed. Tier 1 focuses on further hypothesis generation, plus data generation to explore (and potentially generate) hypotheses and quantify points of departure. Tier 2 encompasses further refinement activities that may be required if activities undertaken in Tiers 0 and 1 are insufficient. The workflow differs from existing frameworks by providing clear exit points, explicit integration of new approach methodologies-supported read-across and a practical structure for systemic toxicity decision making without new animal data generation. We have highlighted aspects that will benefit from further development and improvement - most notably, Tier 2 refinement. Case studies are presented that illustrate how the updated workflow is ready for practical use in cosmetic chemical safety assessments. We recommend its adoption in future cosmetic ingredient regulatory submissions.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, and impulsivity. Methylphenidate (MPH), a first-line medication, is often used long-term and not routinely discontinued in individuals planning to have children. Guided by the Paternal Origins of Health and Disease framework, in this study, we investigated intergenerational effects of paternal MPH exposure and reversibility of sperm epigenetic changes. Male BALB/c mice received MPH (15 mg/kg) or vehicle for 28 days and were mated with untreated females immediately after treatment (F0-T1) or after a 40-day washout period (F0-T2). Behavioral tests and brain gene expression analyses were conducted in the offspring (F1), and sperm small noncoding RNA (sncRNA) profiles were examined in F0 males. Male offspring in the F0-T1-MPH group showed increased open-arm entry into the elevated plus maze and reduced expression of Maoa, Maob, and Bdnf in the medial prefrontal cortex. These changes were absent in the offspring derived from the F0-T2-MPH group. Sperm sncRNA sequencing identified 5,684 differentially expressed fragments in F0-T1-MPH mice, including marked alterations in miRNA-derived fragments, such as miR-34c-5p and miR-30e-5p. The target genes of these altered miRNAs were enriched in ADHD-related pathways, including axon guidance and Rap1 signaling. After washout, the number of differentially expressed sncRNA fragments decreased to 665, whereas the number of altered miRNA-derived fragments decreased from 383 to 7. These findings suggest that paternal MPH exposure may influence offspring behavior and brain gene expression through sperm miRNA alterations and that these effects are largely reversible following drug withdrawal.

Perfluorooctanesulfonic acid (PFOS) is epidemiologically linked to gestational diabetes mellitus (GDM), but the mechanisms remain undefined. We hypothesized that PFOS exposure impairs pancreatic β-cell function through mitochondrial dysfunction and deficits in ATP/cAMP signaling. Pregnant and non-pregnant rats received PFOS (50 μg/mL in drinking water) for 17 days (gestation day [GD] 4-20 in pregnant dams). PFOS-exposed pregnant dams exhibited fasting hypoinsulinemia (65% decrease) with normal fasting glucose. Glucose tolerance was impaired (31% increase in glucose AUC), with blunted insulin secretion (52% decrease in insulin AUC). In contrast, non-pregnant PFOS-exposed rats showed no changes in fasting glucose or insulin but developed glucose intolerance (25% increase in glucose AUC) with only modest early insulin suppression, revealing greater vulnerability in pregnancy. Pancreatic islet morphology and insulin content remained unchanged in PFOS-exposed dams. Isolated islets from PFOS-exposed dams and INS-1 β-cells treated with PFOS (10-100 μM) showed reduced glucose-stimulated insulin secretion despite normal Ins1 and Ins2 expression and insulin content. In INS-1 cells, PFOS selectively reduced Complex II subunits, depleted ATP (32-39%) and cAMP (57-74%), and impaired insulin secretion under low and high glucose. Co-treatment with the mitochondria-targeted antioxidant MitoQ (0.1μM) fully restored ATP, cAMP, and insulin secretion to control levels. These findings demonstrate that PFOS exposure causes gestational glucose intolerance and β-cell secretory failure, accompanied by reduced mitochondrial Complex II subunit abundance and ATP/cAMP depletion. The complete rescue by MitoQ highlights oxidative mitochondrial dysfunction as a mechanistic contributor linking PFOS exposure and GDM risk.

Synthetic messenger RNA (mRNA) formulated in lipid nanoparticles (LNPs) is increasingly used for vaccines and therapeutics, yet the consequences of high-dose systemic exposure for bone marrow hematopoiesis remain poorly defined. We previously reported that intramuscular mRNA/LNP administration transiently alters circulating reticulocytes and bone marrow erythroid cellularity in mice; here, we investigated the mechanistic basis and cross-species generalizability of this hematopoietic bias in murine and non-human primate models. Animals received intramuscular LNP-formulated mRNA encoding SARS-CoV-2 spike protein or firefly luciferase at deliberately supra-clinical, upper-bound doses, and type I interferon signaling was interrogated by interferon-α/β receptor blockade. Bone marrow was assessed by histopathology, bulk and single-cell RNA sequencing, and trajectory analysis. LNP-formulated mRNA induced reversible erythroid-lineage suppression with concomitant granulocytic expansion, irrespective of the encoded antigen. These effects were absent after naked mRNA and only modestly induced by LNP alone, indicating that formulation of synthetic mRNA within the LNP was required to elicit the full phenotype. Single-cell analysis showed biased hematopoietic stem and progenitor cell differentiation toward granulopoiesis with broad induction of interferon-stimulated genes, and the bias resolved within 2 wk. Interferon-α/β receptor blockade mitigated both changes, demonstrating type I interferon dependence. High-dose LNP-formulated synthetic mRNA can therefore drive reversible, type I interferon-dependent hematopoietic lineage bias in preclinical animal models. This study identifies type I interferon signaling as the driver of a reversible, dose-dependent shift in bone marrow hematopoiesis under upper-bound exposure to LNP-formulated synthetic mRNA, informing preclinical safety evaluation of the rapidly expanding class of nucleic acid-based therapeutics.

Humans are exposed to complex mixtures of endocrine-disrupting chemicals (EDCs) in the real world. Most prior studies, however, focus on individual EDCs. Here, we investigated the ovarian impact of organic pollutant mixtures extracted from the Raritan River, a heavily industrialized watershed in central New Jersey, using a three-dimensional (3D) in vitro ovarian follicle culture system. Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) identified organic pollutant mixtures dominated by alkylphenols, pharmaceuticals, per- and polyfluoroalkyl substances (PFAS), and phthalates, together with previous reports demonstrating that the Raritan River also contains hydrophobic persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and dioxins. In vitro exposure to the Raritan River-derived EDC mixture did not affect follicle growth and ovulation but dose-dependently increased preovulatory testosterone (T) secretion while suppressing postovulatory progesterone (P4) production. Transcriptomic analysis, gene set enrichment analysis (GSEA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and in situ RNA hybridization revealed widespread reprogramming of follicular somatic cell gene expression, including altered steroidogenic pathways and activation of aryl hydrocarbon receptor (AhR) signaling. An established AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), reproduced the phenotype of EDC mixture-induced hyperandrogenism, which was reversed by a selective AhR inhibitor. Together, this study demonstrates that real-world EDC mixtures, particularly AhR-activating chemicals, disrupt ovarian steroidogenesis and induce hyperandrogenism, highlighting EDC mixtures as an underrecognized mechanism potentially linking aquatic pollution to impaired women's reproductive health.

Drug-induced liver injury (DILI) remains a major cause of preclinical and clinical attrition, reflecting persistent gaps in the predictive translation of preclinical safety data to human outcomes. To address these gaps, we established a refined multiparametric framework for early DILI risk prediction using a balanced reference set of 170 drugs with established clinical outcomes. Thirty physicochemical, mechanistic endpoints, and exposure-related features were systematically evaluated to identify high-specificity predictors of intrinsic DILI risk. Cytotoxicity in primary human hepatocytes and human liver microtissues, mitochondrial toxicity (i.e. mitotoxicity), and bile salt export pump inhibition were identified as top predictive features. Incorporation of clinical exposure data to derive margins of safety substantially improved assay performance, emphasizing the importance of exposure normalization. A multiparametric, flag-based hazard framework integrating ≥2 hazard flags achieved 98% specificity and a positive likelihood ratio of 18, providing a pragmatic approach for early compound triage and confidence in early decision-making. Random Forest models further enhanced predictive performance, identifying exposure, predicted lipophilicity (e.g. cLogD), mitotoxicity, cytotoxicity, and hepatic clearance as major contributors to DILI risk, with exposure-informed models achieving a balanced accuracy of 0.72. Application of the framework to contemporary Genentech portfolio molecules demonstrated translational utility in refining risk categorization, particularly for compounds with borderline or 0-1 hazard flags. Collectively, this work establishes a mechanistically informed and machine learning-enabled platform that combines interpretability, specificity, and practicality to guide safer molecule design in drug discovery and reduce hepatotoxicity-related attrition in clinical development.

As pervasive environmental contaminants, nanoplastics (NPs) have garnered increasing concern due to their potential health hazards. However, the impact of gestational and lactational exposure to NPs on offspring vascular structural injury remains unexplored. In this study, we used BALB/c mice to model gestational and lactational exposure to 100 nm polystyrene nanoplastics (PS-NPs) at doses of 0.1, 1, and 10 mg/l. Our findings revealed that PS-NPs exposure significantly reduced offspring body weight in both sexes. Histopathological analysis demonstrated that exposure induced multiorgan damage in offspring at postnatal day 21 (PND 21), with partial recovery by PND 90. Critically, PS-NPs exposure induced cardiovascular toxicity in offspring, which included acute vascular injuries (disordered elastic fibers and evident calcium salt deposition) at PND 21 that were largely resolved by PND 90, alongside more persistent issues of abnormal lipid deposition in the aortic root and dyslipidemia observed at both time points. Integrated biomarker response indices, principal component analysis, and hierarchical clustering confirmed dose-dependent toxicity in PS-NPs-exposed offspring. In vitro studies further showed that PS-NPs induced lipid accumulation in 3 major cardiovascular cell types: endothelial cells, smooth muscle cells, and macrophages. Our study firstly reveals that maternal PS-NPs exposure throughout gestation and lactation induces vascular injury and lipid deposition in mouse offspring, indicating that PS-NP pollution represents a critical environmental risk factor for offspring cardiovascular adverse outcomes later in life.

Potentially toxic metal(loid) contamination of agricultural environments may pose serious public health concerns, particularly for children. This study assessed contamination of agricultural soils, irrigation water, and commonly consumed vegetables in Kasese District, Uganda, an area influenced by mining and industrial activities. Agricultural soils (n = 9 composite samples), point-source irrigation water (n = 1), and vegetables (onion, cabbage, and amaranth; n = 36 composite samples) were analysed for 14 potentially toxic elements (PTEs) using Inductively Coupled Plasma Optical Emission Spectroscopy. Age-specific health risks for children were evaluated using the Agency for Toxic Substances and Disease Registry's Public Health Assessment Site Tool and complemented with Monte Carlo simulation (10,000 iterations) to account for exposure variability and uncertainty. Mean concentrations of arsenic (1.780-3.569 mg/kg), lead (2.017-9.670 mg/kg), mercury (0.025-0.700 mg/kg), nickel (0.523-3.655 mg/kg), and chromium (0.043-2.043 mg/kg) frequently exceeded recommended limits. Hazard index values were greater than one, indicating significant non-carcinogenic health risks. Deterministic cancer risk (6.4 × 10[-4]-2.1 × 10[-3]) and probabilistic estimates (5.79 × 10[-4]-3.86 × 10[-3]) exceeded acceptable priority screening thresholds (10[-6]-10[-4]), with arsenic and nickel as dominant contributors to probabilistic cancer-risk variability. However, the study is based on a single sampling campaign, and the exposure model relies partly on default parameters; therefore, the risk estimates should be interpreted as screening-level estimates rather than population-level incidence predictions. The findings support expanded spatial and seasonal monitoring, improved characterisation of dietary exposure and contaminant speciation, and targeted food-safety and agricultural risk-management interventions in the Mubuku irrigation scheme.

Benzene exposure is associated with increased cardiovascular disease (CVD) risk, yet the mechanisms linking benzene to vascular injury remain incompletely understood. Here, we investigated the effects of benzene and its reactive metabolite, trans, trans-muconaldehyde (MA), on endothelial activation and leukocyte recruitment, which are early events in atherogenesis. Wild-type and endothelial-specific heat shock protein A1B overexpressing mice were exposed to inhaled benzene (1 ppm), and leukocyte dynamics were assessed by intravital microscopy. Complementary studies in vitro examined MA-induced endothelial activation via leukocyte adhesion assays, RNA sequencing, pharmacological interventions, and small interfering RNA-mediated gene silencing. Benzene inhalation increased leukocyte rolling (16- to 21-fold) and adhesion (11- to 44-fold) in vivo with female mice exhibiting greater responses than males. In vitro, MA enhanced monocyte adhesion (1.7-fold) and transmigration (1.4-fold) and induced a conserved transcriptional program characterized by activation of oxidative stress, unfolded protein response, MAPK signaling, and heat shock pathways. MA stimulated phosphorylation of p38, JNK, and eIF2α, increased XBP1 splicing, elevated reactive oxygen species generation, and depleted glutathione. Alleviation of endoplasmic reticulum stress with 4-phenylbutyric acid attenuated MA-induced expression of HSPA1B, ATF3, and ICAM1. Conversely, HSPA1B silencing exacerbated endothelial activation and stress signaling, whereas endothelial-specific overexpression of HSPA1B significantly reduced benzene-induced leukocyte recruitment in vivo. Collectively, these findings identify proteotoxic stress as a central mechanism of benzene-induced vascular toxicity. Further, we demonstrate that activation of the heat shock pathway (HSF1-HSPA1B) serves as a protective response that limits endothelial inflammation. These results provide new mechanistic insight into how environmental benzene exposure may promote vascular injury and CVD in humans.

Di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP) are plasticizers used in many consumer products. Both phthalates alter estrous cyclicity, fertility indices, and folliculogenesis throughout the reproductive lifespan. Our companion paper showed that exposure to a phthalate mixture containing DEHP and DiNP altered the aryl hydrocarbon receptor (AHR) signaling pathway in the ovary. However, limited information was available on how DEHP and DiNP individually affect the AHR signaling pathway in the ovary throughout reproductive aging. Thus, we hypothesized that DEHP and DiNP individually affect the gene and protein expression and localization associated with the AHR pathway throughout the reproductive lifespan. We treated adult female CD-1 mice with corn oil control or doses (0.15 ppm, 1.5 ppm, or 1,500 ppm) of DEHP and DiNP in the diet for 1 to 12 mo. Sera were used to measure circulating estradiol (E2) concentrations. The AHR pathway was assessed using quantitative PCR (qPCR) for gene expression, RNAscope in situ hybridization for transcript localization, and immunohistochemistry for protein localization. Exposure to DEHP, but not DiNP, reduced serum E2 after 1 mo compared with control. DEHP exposure increased Cyp1a1 expression and CYP1B1 localization in the ovary after 6 mo compared with control. DiNP also increased Arnt expression and CYP1B1 localization in the ovary after 1 mo compared with control. DiNP exposure decreased Ahr and Cyp1a1 expression as well as CYP1A1 localization in the antral follicle after 12 mo compared with control. Overall, DEHP and DiNP both influence the ovarian AHR pathway, but at different time points and doses.

Phthalates are plasticizers and solvents used in numerous consumer products. Exposure to a phthalate mixture (Mix) alters estrous cyclicity, fertility indices, ovarian follicle dynamics, and steroidogenesis. In cultured antral follicles, phthalates alter the aryl hydrocarbon receptor (AHR) pathway, which is known for estradiol (E2) and xenobiotic metabolism. However, how phthalates affect the AHR pathway in the ovary in vivo and how this pathway changes throughout the reproductive lifespan are unknown. Thus, we hypothesized that expression and localization of genes and proteins in the AHR pathway change throughout the reproductive lifespan and in response to Mix exposure. We exposed adult female CD-1 mice to corn oil control or varying doses (0.15, 1.5, or 1,500 ppm) of Mix for 1 to 12 months. Sera and ovaries were collected at each time point. Sera were used to determine circulating E2 concentrations. The AHR pathway in the ovaries was examined using quantitative polymerase chain reaction for gene expression, RNAscope in situ hybridization for transcript localization, and immunohistochemistry for protein localization. The AHR pathway was altered throughout the reproductive lifespan, with increased Cyp1a1 expression and decreased CYP1B1 protein localization in the ovary at 13 months of age. Circulating E2 decreased throughout aging. Mix exposure altered Arnt and Ahr expression after 1 and 12 months, respectively. Additionally, Mix exposure increased CYP1A1 protein localization after 6 months, with areas of increased staining intensity in the ovary. Overall, the AHR pathway was altered with reproductive aging, and Mix exposure affected the pathway over time.

Ethanol (EtOH) exposure frequently occurs before pregnancy recognition, making early embryonic development particularly vulnerable to inadvertent alcohol exposure. This study evaluated the effects of binge-like EtOH exposure during early gestation on neurobehavioral, oxidative, and inflammatory outcomes in male and female rat offspring. Pregnant rats received EtOH (3 g/kg/day, intragastric gavage) on gestational days 6 to 8, whereas control animals received distilled water. Offspring were evaluated at postnatal day 23 (PN23) and postnatal day 42 (PN42) using the pole, open field, and rotarod tests. The cerebellum was collected for analysis of oxidative stress markers and cytokines. Data were analyzed by two-way ANOVA followed by Tukey's post hoc test (P < 0.05). EtOH exposure induced locomotor alterations at both PN23 and PN42, mainly characterized by reduced total distance traveled, accompanied by impaired balance and forced locomotion. In the cerebellum, both ages exhibited reduced glutathione levels and total antioxidant capacity, together with increased lipid peroxidation. Cytokine alterations displayed a temporal pattern, with reduced IL-1β and TNF-α levels at PN23 and increased levels at PN42, whereas IL-6 and IL-10 remained elevated at both time points. Females exhibited more pronounced impairments in spontaneous locomotion, particularly at PN42. These findings demonstrate that brief binge-like EtOH exposure during early gestation is sufficient to induce persistent motor dysfunction accompanied by cerebellar redox imbalance and cytokine dysregulation, with evidence of sex-dependent behavioral susceptibility.