Neurodevelopmental exposure to chlorpyrifos (CPF) could increase risks for neurological disorders, such as autism spectrum disorder, cognitive impairment, or attention deficit hyperactivity disorder. The potential involvement of microglia reactive to inflammatory stimuli in these neurological disorders has been generally reported. However, the concrete effects and potential mechanisms of microglia dysfunction triggered by developmental CPF exposure remain unclear. Therefore, we established mouse and human embryonic microglial cells (HMC3 cell) models of developmental CPF exposure to evaluate the effects of developmental CPF exposure on neuroinflammation and underlying mechanisms. The results showed that developmental exposure to CPF enhanced the expression of Iba1 in hippocampus. CPF treatment increased inflammatory cytokines levels and TSPO expression in hippocampus and HMC3 cells. The levels of necroptosis and necroptosis-related signaling RIPK/MLKL were increased in hippocampus and HMC3 cells following CPF exposure. Furthermore, the expression of TLR4/TRIF signaling was increased in hippocampus and HMC3 cells subjected to CPF exposure. Notably, the increased levels of TLR4/TRIF signaling, RIPK/MLKL signaling, necroptosis and pro-inflammatory cytokines induced by CPF treatment were remarkably inhibited by TAK-242 (a specific TLR4 inhibitor). Additionally, the necroptosis and pro-inflammatory cytokines production induced by CPF treatment were significantly relieved by Nec-1 (a specific RIPK1 inhibitor). In general, the above results suggested that activated microglia in hippocampus subjected to developmental CPF exposure underwent RIPK1/MLKL-mediated necroptosis regulated by TLR4/TRIF signaling.

Microplastics (MPs), an emerging environmental pollutant, have been clarified to induce testicular disorder in mammals. And the current studies have delineated a correlation between gut microbiota and male reproduction. However, it's still unclear whether gut microbiota gets involved in MPs-induced reproductive toxicity. In this work, we constructed a mouse model drinking 5 μm polystyrene-MPs (PS-MPs) at the concentrations of 100 μg/L and 1000 μg/L for 90 days. Evident histological damage, spermatogenetic disorder and hormones synthesis inhibition were observed in PS-MPs exposed mice. With fecal microbiota transplantation (FMT) trial, the recipient mice exhibited gut microbial alteration, and the elevated abundance of Bacteroides and Prevotellaceae_UCG-001 were positively correlated with testicular disorder according to spearman correlation analysis. Mechanistically, increased proportion of pro-inflammatory bacteria may drive translocation of T helper 17 (Th17) cells, resulting in overproduced interleukin (IL)-17 A and downstream inflammatory response in both the mice exposed to PS-MPs and corresponding recipient mice. In summary, our findings revealed the critical role of gut microbiota in PS-MPs-induced reproductive toxicity, and tried to elucidate the underlying mechanism of gut microbial dysregulation-mediated IL-17 A signaling pathway. Furthermore, this study also provides the research basis for gut microbiota-targeted treatment of male infertility in the future.

Carbendazim (CBZ), a broad-spectrum pesticide frequently detected in fruits and vegetables, could trigger potential toxic risks to mammals. To facilitate the assessment of health risks, this study aimed to characterize the cytochrome P450 (CYPs)-mediated metabolism profiles of CBZ by a combined experimental and computational study. Our results demonstrated that CYPs-mediated region-selective hydroxylation was a major metabolism pathway for CBZ in liver microsomes from various species including rat, mouse, minipig, dog, rabbit, guinea pig, monkey, cow and human, and the metabolite was biosynthesized and well-characterized as 6-OH-CBZ. CYP1A displayed a predominant role in the region-selective hydroxylation of CBZ that could attenuate its toxicity through converting it into a less toxic metabolite. Meanwhile, five other common pesticides including chlorpyrifos-methyl, prochloraz, chlorfenapyr, chlorpyrifos, and chlorothalonil could significantly inhibit the region-selective hydroxylation of CBZ, and consequently remarkably increased CBZ exposure in vivo. Furthermore, computational study clarified the important contribution of the key amino acid residues Ser122, and Asp313 in CYP1A1, as well as Asp320 in CYP1A2 to the hydroxylation of CBZ through hydrogen bonds. These results would provide some useful information for the metabolic profiles of CBZ by mammalian CYPs, and shed new insights into CYP1A-mediated metabolic detoxification of CBZ and its health risk assessment.

N6-methyladenosine (m⁶A) mRNA methylation plays a role in various brain functions. Exposure to chronic constant light (CCL) has been reported to impair cognition, yet whether the underlying mechanism involves m⁶A remains unknown. In this study, mice exposed to CCL for 3 weeks show impaired cognitive behavior, which was associated with increased m⁶A level in hippocampus. Accordingly, the m⁶A demethylase FTO was inhibited while the methyltransferases METTL3, METTL14 and WTAP, as well as the reader protein YTHDF2, were elevated in the hippocampus of CCL-exposed mice. CCL exposure significantly activated hippocampal expression of circadian regulator cryptochrome 1 and 2 (CRY1 and 2). Meanwhile, hippocampal neurogenesis was impaired with suppression of BDNF/TrκB/ERK pathway. To further delineate the signaling pathway and the role of m⁶A, we altered the expression of CRY1/2 in hippocampus neuron cells. CRY1/2 overexpression inhibited FTO and increased m⁶A levels, while CRY1/2 knockdown led to opposite results. Luciferase reporter analysis further confirmed CRY1/2-induced FTO suppression. Furthermore, FTO knockdown increased m⁶A on 3′UTR of TrκB mRNA, and decreased TrκB mRNA stability and TrκB protein expression, in a YTHDF2-dependent manner. These results indicate that CCL-activated CRY1/2 causes transcriptional inhibition of FTO, which suppresses TrκB expression in hippocampus via m⁶A-dependent post-transcriptional regulation and contributes to impaired cognitive behavior in mice exposed to constant light.

Humans benefit from nuclear technologies but consequently experience nuclear disasters or side effects of iatrogenic radiation. Hematopoietic system injury first arises upon radiation exposure. As an intricate new layer of genetic control, the posttranscriptional m⁶A modification of RNA has recently come under investigation and has been demonstrated to play pivotal roles in multiple physiological and pathological processes. However, how the m⁶A methylome functions in the hematopoietic system after irradiation remains ambiguous. Here, we uncovered the time-varying epitranscriptome-wide m⁶A methylome and transcriptome alterations in γ-ray-exposed mouse bone marrow. 4 Gy γ-irradiation rapidly (5 min and 2 h) and severely impaired the mouse hematopoietic system, including spleen and thymus weight, blood components, tissue inflammation and malondialdehyde (MDA) levels. The m⁶A content and expression of m⁶A related enzymes were altered. Gamma-irradiation triggered dynamic and reversible m⁶A modification profiles and altered mRNA expression, where both m⁶A fold-enrichment and mRNA expression most followed the (5 min_up/2 h_down) pattern. The CDS enrichment region preferentially upregulated m⁶A peaks at 5 min. Moreover, the main GO and KEGG pathways were closely related to metabolism and the classical radiation response. Finally, m⁶A modifications correlated with transcriptional regulation of genes in multiple aspects. Blocking the expression of m⁶A demethylases FTO and ALKBH5 mitigated radiation hematopoietic toxicity. Together, our findings present the comprehensive landscape of mRNA m⁶A methylation in the mouse hematopoietic system in response to γ-irradiation, shedding light on the significance of m⁶A modifications in mammalian radiobiology. Regulation of the epitranscriptome may be exploited as a strategy against radiation damage.

Pendimethalin (PDM) is a dinitroaniline crop pesticide that is extensively utilized worldwide. However, the reproductive toxicity and cellular mechanisms of PDM have not been identified. Therefore, we elucidated the adverse effects of PDM on the reproductive system using mouse testicular Leydig and Sertoli cells (TM3 and TM4 cells, respectively). Our results demonstrated that PDM suppressed the viability and proliferation of TM3 and TM4 cells. Additionally, PDM induced cytosolic calcium upregulation and permeabilization of mitochondrial membrane potential in both TM3 and TM4 cells. We also verified that PDM activates the endoplasmic reticulum (ER) stress pathway and autophagy. Furthermore, we confirmed that activation of ER stress and autophagy were blocked by 2-aminoethoxydiphenyl borate (2-APB) treatment. Finally, we confirmed PDM-induced cell cycle arrest and apoptosis in TM3 and TM4 cells. Thus, we first demonstrated that PDM impedes the survival of testis cells, and further, their function.

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4′–OH–PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4′–OH–PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4′–OH–PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4′–OH–PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4′–OH–PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4′–OH–PPF alone. We conclude that 4′–OH–PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

The number of deaths from air pollution worldwide is estimated at 8.8 million per year, more than the number of deaths from smoking. Air pollutants, such as PM₂.₅, are known to induce respiratory and cardiovascular diseases by inducing oxidative stress. Thioredoxin (Trx) is a 12-kDa endogenous protein that exerts antioxidant activity by promoting dithiol disulfide exchange reactions. We previously synthesized human serum albumin-fused thioredoxin (HSA-Trx), which has a longer half-life in plasma compared with Trx, and demonstrated its efficacy against various diseases including respiratory diseases. Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice. Urban aerosols induced lung injury and inflammatory responses in ICR mice, but intravenous administration of HSA-Trx markedly inhibited these responses. We next analyzed reactive oxygen species (ROS) production in murine lungs using an in vivo imaging system. The results show that intratracheal administration of urban aerosols induced ROS production that was inhibited by intravenously administered HSA-Trx. Finally, we found that HSA-Trx inhibited the urban aerosol-induced increase in levels of neutrophilic extracellular trap (NET) indicators (i.e., double-stranded DNA, citrullinated histone H3, and neutrophil elastase) in bronchoalveolar lavage fluid (BALF). Together, these findings suggest that HSA-Trx prevents urban aerosol-induced acute lung injury by suppressing ROS production and neutrophilic inflammation. Thus, HSA-Trx may be a potential candidate drug for preventing the onset or exacerbation of lung injury caused by air pollutants.

Paraquat (PQ), a widely used herbicide and well-known oxidative stress inducer, has been linked to numerous neurodegenerative diseases, but the underlying mechanism(s) remains unknown. Circular RNAs (circRNAs) have recently been reported to be associated with oxidative stress in Parkinson's disease. Herein, we performed methylated RNA immunoprecipitation and RNA sequencing assays for mouse neuroblastoma (Neuro-2a) cells and successfully established a positive link between the alteration of circRNAs driven by m⁶A modification and PQ-induced oxidative stress. We observed oxidative stress and antioxidative stress present distinct m⁶A modification pattern of circRNAs as well as biological effect. Gene ontology and pathway analysis predicted that differentially m⁶A-methylated and expressed circRNAs are highly clustered in pathways associated with function and development of nervous system, including axon cargo transport, nervous system development, long-term potentiation, and neurotrophic signaling pathways. Moreover, we demonstrated that the alteration of m⁶A-methylated circRNAs upon PQ exposure could be partially reversed by N-acetylcysteine pretreatment. The mechanistic analysis further demonstrated that N-acetylcysteine pretreatment attenuated the decreased expression of target genes (UBC and PPP2CA) induced by PQ. These findings revealed distinct patterns of differentially m⁶A-modified circRNAs, indicating that m⁶A could participate in a specific regulatory network of circRNAs to modulate the expression of downstream genes in response to PQ-induced oxidative stress. In conclusion, our work established a link between m⁶A modification of circRNAs and PQ-induced oxidative stress, and further studies are required to explore the underlying molecular mechanisms associated with PQ-induced neurotoxicity.

As ubiquitous, persistent organic pollutants, polycyclic aromatic hydrocarbons (PAHs) have adverse impacts on human health. Phenanthrene (Phe) is one of the most abundant PAHs in the environment. However, the long-term effects of exposure to environmental level of Phe on the kidneys and the potential mechanisms are unclear. T helper (Th) cells, a subtype of CD4⁺ T cells that play a central role in the renal immune microenvironment. In this study, male mice were chronically exposed to 5, 50, and 500 ng/kg bw Phe every other day for total 210 days. Those results indicated that environmental Phe exposure caused kidney hypertrophy, injury and fibrosis in the mice. Chronic, long-term environmental level of Phe exposure did not significantly alter the innate immune response but induced adaptive immune response changes (Th1/Th2 related cytokines release), causing a type 1 immune response in the 5 ng/kg bw Phe group and a type 2 immune response in the high dose groups (50 and 500 ng/kg bw). This study provides novel insights into the roles of adaptive immune response in long-term PAH exposure-induced chronic kidney injury and fibrosis, which is beneficial for further understanding the potential health hazards of PAHs and providing new avenues for immune intervention strategies to alleviate PAHs toxicity.