Toxicological Sciences最新文献

Next-Generation Risk Assessment (NGRA) frameworks use New Approach Methodologies (NAMs) to support regulatory decisions without animal testing. While NAM-based approaches are well established for hazard and dose-response assessment, inter-individual variability is still typically addressed using default uncertainty factors for inter-individual variability. This study evaluated a NAM-based strategy to quantify chemical-specific variability using a human cell model. We hypothesized that integrating chemical-specific variability data into NGRA would yield more protective risk estimates. Using 131 human lymphoblastoid cell lines (LCLs) from four European and African subpopulations, we assessed differences in cytotoxic responses to 53 substances, including industrial chemicals, pharmaceuticals, pesticides, and consumer-use compounds. Concentration-response testing (0.3 nM-300 μM) data were analyzed using Bayesian modeling to calculate points of departure per cell line. Of the substances tested, 18 exhibited cytotoxic effects, enabling the derivation of chemical-specific variability factors. These factors were designated as toxicodynamic variability factors at the 5th percentile (TDVF05) because of the limited metabolic capacity of lymphoblast cell lines. The median TDVF05 was 3.8 (range 1-46), largely consistent with default assumptions. A genome-wide association study (GWAS) identified genomic loci, primarily containing transporter and metabolism genes, associated with variability in cytotoxicity, suggesting mechanistic bases for inter-individual differences. Overall, this study shows that human LCLs are a practical high-throughput in vitro model for quantifying inter-individual variability, strengthening confidence in NGRA risk predictions and supporting hypothesis generation on chemical-specific genetic and mechanistic drivers of human variability. However, cell-based systems have limited coverage of adverse effects and require careful alignment with in vivo dosimetry.

Seizures are a common reason for drugs to fail during development, but they are difficult to predict preclinically. Enoxacin, a fluoroquinolone, rarely causes convulsions as a monotherapy, but convulsions have been seen after combination therapy with fenbufen, a non-steroidal anti-inflammatory drug. The interaction between the drugs is thought to result from inhibition of GABAA receptors, which are not their primary targets. Here we show that, among 15 marketed fluoroquinolones and one active metabolite, six, including enoxacin, inhibited GABA-evoked depolarization in cells expressing human GABAA receptors when administered with felbinac, the active metabolite of fenbufen. Within these six except for a prodrug possessed a piperazinyl group at the seventh position of the quinolone ring. We also administered enoxacin or norfloxacin plus felbinac to rats and determined the cerebrospinal fluid concentrations of each drug when convulsions occurred. As we previously reported, an increase in network burst frequency recorded from primary cultured rat cortical neurons on microelectrode arrays is a risk marker for seizures, so we tested whether this assay could predict seizures induced by drug interactions between fluoroquinolones and felbinac. When co-administered with felbinac, only those fluoroquinolones that inhibited GABA currents in patch-clamp tests increased network burst frequency. Principal component analysis using 17 microelectrode array parameters supported that the mechanism of action was due to GABA antagonism in rodent neurons. Thus, the microelectrode array assay predicted seizure risks from the combination of enoxacin and the active metabolite of fenbufen and identified other fluoroquinolones with seizure risk potential.

Polystyrene nanoplastics (PS-NPs) are small particles derived from plastic degradation that have been detected in several human tissues. Phthalates are ubiquitous plasticizers used to increase flexibility in polymers which act as endocrine disruptors, impacting hormonal homeostasis. Considering that both pollutants have been detected in human follicular fluid, there is increasing concern regarding their potential effects on female reproductive health. This study evaluated the isolated and combined effects of environmentally relevant doses of PS-NPs and a phthalate metabolite mixture (MM) on antral follicle growth, hormone production, and the expression of genes involved in apoptosis, oxidative stress, steroidogenesis, and hormone receptor signaling. Antral follicles from adult CD-1 mice were cultured with vehicle control (DMSO and water), metabolite mixture (0.01, 0.1, 1, and 10 μg/mL), or PSNPs (5, 25, 50, and 100 μg/mL) or MM + PS-NPs (5 µg/mL PS-NPs + 0.01 µg/mL MM; 100 μg/mL PS-NPs + 10 μg/mL MM). Follicle growth was monitored every 24 h for 96 h. PS-NPs and MM were internalized by follicles and they inhibited follicle growth alone and in co-exposure. Both pollutants altered the expression of apoptosis-related (Casp3, Casp8, Bcl2) and oxidative stress-related (Cat, Nrf2, Gpx1) genes without significantly affecting steroid hormone levels. Co-exposure also reduced Esr2 and Ar expression, demonstrating more pronounced effects under low-dose combined exposure. Altogether, these findings indicate that environmentally relevant exposure to PS-NPs and phthalate mixtures impairs antral follicle growth and disrupts molecular pathways essential for ovarian function, highlighting potential pathways and the importance of understanding combined exposures in reproductive toxicity.

Wildfire smoke (WFS) exposures are becoming more common, and firefighters and community members are often exposed to WFS for days to weeks. Controlled studies assessing the effects of repeated exposure to WFS and woodsmoke (WS; as a surrogate for WFS) or compared acute versus repeated exposure have evaluated a limited number of timepoints and endpoints using rodent models. Here, we leveraged differentiated primary human bronchial epithelial cells (HBECs) to test the hypothesis that different molecular responses occur upon acute vs repeat exposures to WS. HBECs (n = 4 donors) were exposed to 22 µg/cm2 red oak WS condensate for an acute, 4-hour exposure, or repeat exposures of 4 hours/day, 3 days/week, across 2 weeks. Membrane permeability, cell viability, and transcriptional responses were measured at the end of each exposure paradigm, and secreted proteins were measured throughout the repeat exposure. Acute exposure significantly increased expression of genes involved in fibrosis and immune response, while repeated exposure significantly decreased expression of genes involved in tissue repair and remodeling. Secreted protein responses were similar to transcriptomic responses and demonstrated temporal variation in response to exposure. This study supports the feasibility of using HBECs to evaluate acute and repeat WS exposures and indicates differential responses from these exposures with direct relevance to pulmonary disease processes, including those involved in fibrosis, asthma, and chronic obstructive pulmonary disease (COPD). These findings highlight the need for future studies to better understand molecular responses to repeated smoke exposures.

Multi-omic investigations into environmental effects on health and disease are aided by inclusion of microbial microbiomes with assessment of mirobes producing metabolites that differentially modulate host organ functions. The gut microbiome is key because many environmental toxicants enter the body orally and may disrupt gut microbes that help digest food, as well as the microbiome-gut-brain axis, which produces regulatory metabolites with systemic effects. Environmental stressors may differentially alter brain development and function, even among identical twins, in that over time, there may be divergence due to epigenetic effects from the environment, including microbes within the microbiome. The diversity of microbiomes is presented as playing a key role in the influence of organs on each other, health, and the development of disorders. The gut microbes and their metabolites may cause mitochondria to produce less ATP and more reactive oxygen species (ROS). The metabolites produced by microbes during the digestion of foods can nourish or harm a person's cellular and molecular functions and vary depending on each person's exposome. The detrimental effects of environmental stressors are discussed, focusing on how altered levels of neuropeptides, neurotransmitters, and the inflammatory/anti-inflammatory balance affect health and disorders. During ATP production, dysfunctional mitochondria may produce more ROS, which can lead to inflammation and oxidative stress, causing cell damage and disrupting products needed for neuronal development, connections, and functions. The balance between inflammatory/anti-inflammatory biomarkers and metabolites and between oxidants/antioxidants is discussed in relation to some clinical connections; for example, the proportions of CD4 and CD8 T cells in HIV patients and the ROS-to-glutathione ratio in inflammatory bowel disease and septic patients. These imbalances are reviewed regarding brain development and functions leading to anxiety, depression, and dementia. The integration of multi-omics, dysbiosis, and mitochondrial dysfunction with a person's clinical evaluation is discussed to inform the formulation of prevention measures and therapeutic interventions regarding environmental effects on the microbiome-gut-brain axis and physical and mental health.

Humans are exposed to chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) that cause toxicity through activation of the aryl hydrocarbon receptor (AHR). There is inter-individual variation in sensitivity to the effects of AHR ligands, but it is not fully explained by variation in the AHR. A clue to the genetic mechanisms underlying differential sensitivity to AHR agonists has emerged from studies of Atlantic killifish (Fundulus heteroclitus) populations with evolved tolerance to PCBs, TCDD, and PAHs. Genomic studies of these populations identified AHR-interacting protein (AIP/Ara9/XAP2) as the strongest candidate resistance gene. However, the precise role of AIP in the mechanism of resistance is unknown. To understand the role of AIP in the toxicity of dioxin-like compounds in vivo, we used CRISPR-Cas9 to generate AIP loss-of-function alleles in killifish and zebrafish (Danio rerio). Homozygous mutant killifish and zebrafish die during larval development-by 30 and 12 days post-fertilization, respectively-while heterozygous mutants develop, survive, and reproduce normally. During embryonic and early larval stages, homozygous mutant zebrafish exhibit reduced sensitivity to embryotoxic effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) and TCDD. Gene expression profiling of aip-deficient larvae revealed hundreds of differentially expressed genes. PCB126 induced similar sets of well-known AHR-regulated genes in mutant and wild-type larvae, although with reduced magnitude overall in AIP mutants. This study highlights the important role of AIP in fish larval development and demonstrates that AIP status can influence the response of vertebrate embryos to dioxin-like compounds in vivo.

Chronic fluorosis can cause injury to the central nervous system. Since fat mass and obesity-associated protein (FTO) connected with demethylation of N6-methyladenosine (m6A) plays a crucial role in maintaining brain function, we examined whether FTO might help resist the neurotoxicity of fluoride. Sprague-Dawley rats with chronic fluorosis and cultured nerve cells exposed to fluoride with overexpression or knockdown of FTO were employed. Morris water maze test was employed to assess learning and memory. Expressions of FTO, m6A, postsynaptic density protein 95 (PSD95) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) and its subunit GluR2 mRNA stability, and apoptosis and reactive oxygen species (ROS) were determined by Western blotting, RT-PCR, and biochemical methods, respectively. The results showed that the impaired learning and memory of rats with chronic fluorosis, and the decreased FTO, PSD95 and AMPARs, the elevated m6A and the disrupted synapse morphology as well as apoptosis in their brains were determined. Similar abnormal changes were further confirmed in primary neurons and SH-SY5Y cells exposed to fluoride, accompanied by a significant decline of GluR2 and high ROS. Notably, overexpression of FTO reduced m6A, enhanced GluR2 and attenuated neurotoxicity induced by fluoride, while knockdown had the opposite effects. FTO can alleviate the neurotoxicity induced by fluoride, in which the mechanism may be involved in its regulating m6A to enhance expression of GluR2.

Glyphosate is an herbicide found worldwide in glyphosate-based formulations (GBFs). Although glyphosate appears to have a low toxicity profile for humans and mammals, conflicting reports exist regarding the risk for cancer in humans. US-EPA and European regulatory agencies have described glyphosate as unlikely to pose a carcinogenic hazard to humans. However, the International Agency for Research on Cancer (IARC) classified glyphosate as "probably carcinogenic to humans (Group 2A)", citing "mechanistic data provide strong evidence for genotoxicity and oxidative stress". Given these discrepancies, the Division of Translational Toxicology at NIEHS designed an experimental strategy to expand mechanistic evidence and address critical gaps within existing literature (e.g., mechanistic evaluations of glyphosate alongside GBFs, inclusion of context-defining positive controls). Cell morphology, viability, H2O2 and γH2AX formation were assayed in human keratinocytes (HaCaT), previously cited by IARC, and human hepatocytes (HepaRG™) to derive benchmark concentrations and fold-change response metrics. Our findings revealed glyphosate alone was weakly and inconsistently bioactive for oxidative stress and DNA damage when compared to positive controls. In contrast, most of the 13 GBFs evaluated were more clearly bioactive with no apparent correlation to varied glyphosate concentrations. Hierarchical clustering of biological responses revealed some bioactive GBFs to cluster near well-characterized positive controls for oxidative stress, while four GBFs clustered more similar to negative controls and glyphosate. Collectively, this study provides a robust dataset with context-defining results that advance our understanding of the hazard potential of GBFs while revealing glyphosate is likely not a primary driver of oxidative stress from GBF exposures.

While acetylcholinesterase (AChE) is the primary inhibitory target for organophosphate (OP) insecticides and chemical warfare nerve agents, research supports the concept that the wide range of systems affected by OPs are not solely dependent on the anticholinesterase activity of OPs but are attributable to the inhibition of other serine hydrolases. Oxime reactivators play an integral role in the treatment of acute OP exposure by returning function to OP-inhibited AChE. Our laboratory has synthesized and patented a platform of oxime reactivators (US Patent 9,227,937) that have provided central neuroprotection through returning function to OP-inhibited AChE in the brain and preservation of neuronal and glial structures from damage in a rat model; the current U.S. approved oxime, pralidoxime (2-PAM), does not provide central neuroprotection. Thus, returning function to other OP-inhibited serine hydrolases by these novel oximes could provide additional secondary neuroprotection. Rat brain proteins were studied using activity-based protein profiling (ABPP) for serine hydrolase targets of a highly relevant sarin surrogate, nitrophenyl isopropyl methylphosphonate (NIMP), alone or in combination with Oxime 20, our lead novel AChE reactivator. ABPP indicated that NIMP significantly inhibited 6 serine hydrolases and of these were the well-known serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), which are important in endocannabinoid signaling. Oxime 20 administration after NIMP treatment showed only limited remediation of the inhibition experienced by these targets. The impact of this study is the identification of secondary serine esterase targets that could be explored for esterase reactivating therapeutics for the treatment of OP poisoning.

Exposure to per- and polyfluoroalkyl substances (PFAS) elicits changes in various metabolic responses but few studies have evaluated whole-body metabolic changes. This study compared whole-body bioenergetics in adult female C57BL/6 mice orally dosed with 0 or 7.5 mg/kg perfluorooctanoic acid (PFOA) or fed a 60% high fat diet (HFD) for 24-hours or 15-days. After 24-hours, HFD mice showed increased oxygen consumption (VO2) and reduced active-phase respiratory exchange ratio (RER), indicating early lipid utilization. After 15 days, PFOA-exposed mice exhibited decreased resting RER and suppressed active VO2, suggesting impaired circadian metabolic activation. In contrast, HFD mice maintained elevated VO2 across both resting and active cycles and showed reduced RER variation between phases, indicating metabolic inflexibility. PFOA exposure also increased relative liver weights and ACOX-1 activity. HFD resulted in an increase in body weight and increase in fat mass without hepatomegaly. These results indicate that both exposure paradigms disrupt whole-body metabolism and circadian bioenergetics. PFOA induced pronounced peroxisomal proliferation and suppressed resting-cycle energy expenditure without weight gain, whereas HFD drove obesogenic effects with impaired substrate switching. These findings provide mechanistic insight into how environmental and dietary stressors alter metabolic rhythms.