NBR1-mediated autophagic degradation of caspase 8 protects vascular endothelial cells against arsenite-induced apoptotic cell death.

Vascular endothelial cells play a critical role in maintaining the health of blood vessels, but dysfunction can lead to cardiovascular diseases. The impact of arsenite exposure on cardiovascular health is a significant concern due to its potential adverse effects. This study aims to explore how NBR1-mediated autophagy in vascular endothelial cells can protect against oxidative stress and apoptosis induced by arsenite. Initially, our observations revealed that arsenite exposure increased oxidative stress and triggered apoptotic cell death in human umbilical vein endothelial cells (HUVECs). However, treatment with the apoptosis inhibitor Z-VAD-FMK notably reduced arsenite-induced apoptosis. Additionally, arsenite activated the autophagy pathway and enhanced autophagic flux in HUVECs. Interestingly, inhibition of autophagy exacerbated arsenite-induced apoptotic cell death. Our findings also demonstrated the importance of autophagy receptor NBR1 in arsenite-induced cytotoxicity, as it facilitated the recruitment of caspase 8 to autophagosomes for degradation. The protective effect of NBR1 against arsenite-induced apoptosis was compromised when autophagy was inhibited using pharmacological inhibitors or through genetic knockdown of essential autophagy genes. Conversely, overexpression of NBR1 facilitated caspase 8 degradation and reduced apoptotic cell death in arsenite-treated HUVECs. In conclusion, our study highlights the vital role of NBR1-mediated autophagic degradation of caspase 8 in safeguarding vascular endothelial cells from arsenite-induced oxidative stress and apoptotic cell death. Targeting this pathway could offer a promising therapeutic approach to mitigate cardiovascular diseases associated with arsenite exposure.

Reproductive effects of pubertal exposure to neonicotinoid thiacloprid in immature male mice.

Thiacloprid (THIA) is a kind of neonicotinoid, a widely used insecticide class. Animal studies of adult and prenatal exposure to THIA have revealed deleterious effects on mammalian sperm fertility and embryonic development. A recent cross-sectional study linked higher THIA concentrations to delayed genitalia development stages in adolescent boys, suggesting that pubertal exposure to THIA may adversely affect reproductive development in immature males. Hence, this study aimed to investigate the effects of daily oral administration of THIA during puberty on the reproductive system of developing male mice. Young male C57 BL/6 J mice aged 21 days were administrated with THIA at concentrations of 10 (THIA-10), 50 (THIA-50) and 100 mg/kg (THIA-100) for 4 weeks by oral gavage. It is found that exposure to 100 mg/kg THIA diminished sexual behavior in immature male mice, caused a decrease in the spermatogenic cell layers and irregular arrangement of the seminiferous epithelium, and down-regulated the mRNA levels of spermatogenesis-related genes Ddx4, Scp3, Atg5, Crem, and Ki67, leading to an increase of sperm abnormality rate. In addition, THIA exposure at 50 and 100 mg/kg reduced the serum levels of testosterone and FSH, and decreased the expression levels of Star and Cyp11a1 related to testosterone biosynthesis. THIA exposure at 10 mg/kg did not produce any of the above significant changes. In conclusion, the high dose of THIA exposure impaired reproductive function in immature mice. It seems that THIA has no detrimental effects on the reproductive system of mice at low dose of 10 mg/kg.

Involvement of gut-brain communication in arsenite-induced neurobehavioral impairments in adult male mice.

Arsenite is a well-documented neurotoxic metalloid that widely distributes in the natural environment. However, it remains largely unclear how arsenite affects neurological function. Therefore, in this study, the healthy adult male mice were exposed to 0.5 mg/L and 5 mg/L arsenite through drinking water for 30 and 90 days, respectively. Our results showed that there was no significant alteration in the intestine and brain for 30 days exposure, but exposure to arsenite for 90 days significantly induced a reduction of locomotor activity and anxiety-like behavior, caused pathological damage and inflammatory responses in the brain and intestine. We also found that arsenite remarkably disrupted intestinal barrier integrity, decreased the levels of lysozyme and digestive enzymes. Intriguingly, chronic exposure to arsenite significantly changed the levels of gut-brain peptides. Taken together, this study provides meaningful insights that gut-brain communication may involve in the neurobehavioral impairments of arsenite.

Exposure to di (2-ethylhexyl) phthalate causes locomotor increase and anxiety-like behavior via induction of oxidative stress in brain.

Di (2-ethylhexyl) phthalate (DEHP) is one of the most prevalent xenoestrogen endocrine disruptor in daily life. A growing number of studies showed that DEHP could exhibit long-term adverse health effects on the human body, particularly in the liver, kidneys, heart and reproductive systems. However, the impact of oral intake of DEHP on the nervous system is extremely limited. In the present study, the adult C57BL/6J male mice were intragastrically administered with two dosages of DEHP for 35 days. The behavioral parameters were assessed using the elevated plus maze and open-field test. The mRNA expression levels of neuropeptides and the oxidative stress-associated proteins were detected by qPCR and western blot seperately. The histopathologic alterations of the brain were observed by H&E and Nissl staining. The results demonstrated that DEHP exposure could result in neurobehavioral impairments such as locomotor increase and anxiety-like behavior. Furthermore, pathological damages were clearly observed in the cerebral cortex and hippocampus, accompanied by a decrease in neuropeptides and an increase in oxidative stress, which were all positively correlated with the dose of DEHP. Together, these findings provide valuable clues into the DEHP-induced neurotoxicity.

Intestinal Microbiota-derived Propionic Acid Protects against Zinc Oxide Nanoparticle-induced Lung Injury.

With the rapid development of nanotechnology, the risks of accidental and/or occupational exposure to zinc oxide nanoparticles (ZnONPs) are increasing. Inhalation of ZnONPs induces metal fume fever in humans and acute lung injury (ALI) in animal models. Although the intestinal microbiota is considered an important modulator of various diseases, the role and mechanism of intestinal microbiota in the pathology of ZnONP-induced ALI are unclear. Herein, we established an intratracheal instillation of a ZnONP-induced ALI mouse model and found that the inhalation of ZnONPs caused ALI along with a perturbation of intestinal flora. Antibiotic cocktail treatment-mediated depletion of intestinal microbiota aggravated ZnONP-induced ALI, and in contrast, fecal microbiota transplantation-mediated restoration of intestinal microbiota exerted the opposite effects. A decrease in short-chain fatty acids, the intestinal microbiota-derived metabolites in the plasma-in particular, acetic acid and propionic acid-occurred after exposure to ZnONPs. It is important to note that supplementation with propionic acid, but not acetic acid, ameliorated ZnONP-induced ALI. We also showed that the source of inflammatory cytokines might partially be the infiltration of macrophages. Supplementation with propionic acid was found to act on macrophages through the receptor GPR43, because knockdown of GPR43 sharply reversed the protective effects of propionic acid during the ZnONP-induced inflammatory response and oxidative stress in both primary alveolar macrophages and RAW 264.7 macrophage cell lines. Altogether, a novel gut-lung axis mechanism is revealed in which intestinal microbiota and their derived metabolite propionic acid play protective roles against ZnONP-induced ALI and suggest that fecal microbiota transplantation and supplementation with propionic acid are potential remedy strategies.

Recombinant ACE2 protein protects against acute lung injury induced by SARS-CoV-2 spike RBD protein.

BACKGROUND: SARS-CoV-2 infection leads to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Both clinical data and animal experiments suggest that the renin-angiotensin system (RAS) is involved in the pathogenesis of SARS-CoV-2-induced ALI. Angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV-2 and a crucial negative regulator of RAS. Recombinant ACE2 protein (rACE2) has been demonstrated to play protective role against SARS-CoV and avian influenza-induced ALI, and more relevant, rACE2 inhibits SARS-CoV-2 proliferation in vitro. However, whether rACE2 protects against SARS-CoV-2-induced ALI in animal models and the underlying mechanisms have yet to be elucidated. METHODS AND RESULTS: Here, we demonstrated that the SARS-CoV-2 spike receptor-binding domain (RBD) protein aggravated lipopolysaccharide (LPS)-induced ALI in mice. SARS-CoV-2 spike RBD protein directly binds and downregulated ACE2, leading to an elevation in angiotensin (Ang) II. AngII further increased the NOX1/2 through AT1R, subsequently causing oxidative stress and uncontrolled inflammation and eventually resulting in ALI/ARDS. Importantly, rACE2 remarkably reversed SARS-CoV-2 spike RBD protein-induced ALI by directly binding SARS-CoV-2 spike RBD protein, cleaving AngI or cleaving AngII. CONCLUSION: This study is the first to prove that rACE2 plays a protective role against SARS-CoV-2 spike RBD protein-aggravated LPS-induced ALI in an animal model and illustrate the mechanism by which the ACE2-AngII-AT1R-NOX1/2 axis might contribute to SARS-CoV-2-induced ALI.

PINK1/TAX1BP1-directed mitophagy attenuates vascular endothelial injury induced by copper oxide nanoparticles.

Copper oxide nanoparticles (CuONPs) are widely used metal oxide NPs owing to their excellent physical-chemical properties. Circulation translocation of CuONPs after inhalation leads to vascular endothelial injury. Mitochondria, an important regulatory hub for maintaining cell functions, are signaling organelles in responses to NPs-induced injury. However, how mitochondrial dynamics (fission and fusion) and mitophagy (an autophagy process to degrade damaged mitochondria) are elaborately orchestrated to maintain mitochondrial homeostasis in CuONPs-induced vascular endothelial injury is still unclear. In this study, we demonstrated that CuONPs exposure disturbed mitochondrial dynamics through oxidative stress-dependent manner in vascular endothelial cells, as evidenced by the increase of mitochondrial fission and the accumulation of fragmented mitochondria. Inhibition of mitochondrial fission with Mdivi-1 aggravated CuONPs-induced mtROS production and cell death. Furthermore, we found that mitochondrial fission led to the activation of PINK1-mediated mitophagy, and pharmacological inhibition with wortmannin, chloroquine or genetical inhibition with siRNA-mediated knockdown of PINK1 profoundly repressed mitophagy, suggesting that the protective role of mitochondrial fission and PINK1-mediated mitophagy in CuONPs-induced toxicity. Intriguingly, we identified that TAX1BP1 was the primary receptor to link the ubiquitinated mitochondria with autophagosomes, since TAX1BP1 knockdown elevated mtROS production, decreased mitochondrial clearance and aggravated CuONPs-induced cells death. More importantly, we verified that urolithin A, a mitophagy activator, promoted mtROS clearance and the removal of damaged mitochondria induced by CuONPs exposure both in vitro and in vivo. Overall, our findings indicated that modulating mitophagy may be a therapeutic strategy for pathological vascular endothelial injury caused by NPs exposure.

Reciprocal regulation of NRF2 by autophagy and ubiquitin-proteasome modulates vascular endothelial injury induced by copper oxide nanoparticles.

NRF2 is the key antioxidant molecule to maintain redox homeostasis, however the intrinsic mechanisms of NRF2 activation in the context of nanoparticles (NPs) exposure remain unclear. In this study, we revealed that copper oxide NPs (CuONPs) exposure activated NRF2 pathway in vascular endothelial cells. NRF2 knockout remarkably aggravated oxidative stress, which were remarkably mitigated by ROS scavenger. We also demonstrated that KEAP1 (the negative regulator of NRF2) was not primarily involved in NRF2 activation in that KEAP1 knockdown did not significantly affect CuONPs-induced NRF2 activation. Notably, we demonstrated that autophagy promoted NRF2 activation as evidenced by that ATG5 knockout or autophagy inhibitors significantly blocked NRF2 pathway. Mechanically, CuONPs disturbed ubiquitin-proteasome pathway and consequently inhibited the proteasome-dependent degradation of NRF2. However, autophagy deficiency reciprocally promoted proteasome activity, leading to the acceleration of degradation of NRF2 via ubiquitin-proteasome pathway. In addition, the notion that the reciprocal regulation of NRF2 by autophagy and ubiquitin-proteasome was further proven in a CuONPs pulmonary exposure mice model. Together, this study uncovers a novel regulatory mechanism of NRF2 activation by protein degradation machineries in response to CuONPs exposure, which opens a novel intriguing scenario to uncover therapeutic strategies against NPs-induced vascular injury and disease.

Downregulation of beclin 1 restores arsenite-induced impaired autophagic flux by improving the lysosomal function in the brain.

Arsenite is a toxic metalloid that causes various adverse effects in the brain. However, the underlying mechanisms of arsenite-induced neurotoxicity remain poorly understood. In this study, both adult beclin 1+/+ and beclin 1+/- mice were employed to establish a model of chronic arsenite exposure by treating with arsenite via drinking water for 6 months. The results clearly demonstrated that exposure to arsenite profoundly caused damage to the cerebral cortex, induced autophagy and impaired autophagic flux in the cerebral cortex. Heterozygous disruption of beclin 1 in animals remarkably alleviated the neurotoxic effects of arsenite. To verify the results obtained in the animals, a permanent U251 cell line was used. After treating of cells with arsenite, similar phenomenon was also observed, showing the significant elevation in the expression levels of autophagy-related genes. Importantly, lysosomal dysfunction caused by arsenite was observed in vitro and in vivo. Either knockdown of beclin 1 in cells or heterozygous disruption of beclin 1 in animals remarkably alleviated the lysosomal dysfunction induced by arsenite. These findings indicate that downregulation of beclin 1 could restore arsenite-induced impaired autophagic flux possibly through improving lysosomal function, and correct that regulation of autophagy via beclin 1 would be an alternative approach for the treatment of arsenite neurotoxicity.

Polystyrene nanoparticles enhance the adverse effects of di-(2-ethylhexyl) phthalate on male reproductive system in mice.

Coexposure of nanoplastics (NPs) with other pollutants adsorbed from the surroundings has received extensive attention. Currently, the combined effects of NPs and plasticizers remain unclear. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer that has raised much concern owing to its ubiquitous pollution and endocrine-disrupting potential. This study aimed to investigate the toxic effects on the male reproductive system upon coexposure to NPs and DEHP. The C57BL/6J mice were orally administrated with polystyrene nanoparticles (PSNPs), DEHP or both for 35 days to evaluate their effects on sperm quality, histology of testes and epididymides, testicular transcriptomic characteristics as well as expression of some important genes in the epididymides. The low-dose PSNPs used here did not induce significant changes in sperm quality, while DEHP alone or cotreatment with DEHP and PSNPs caused notable impairment, mainly manifesting as decreased sperm quality and aberrant structure of the testis and epididymis. Moreover, enhanced toxic effects were found in the cotreatment group when compared with the individual DEHP treatment group, as manifested by more obvious alterations in the sperm parameters as well as histological changes in the testis and epididymis. Testicular transcriptomic analysis revealed differential regulation of genes involved in immune response, cytoplasmic pattern recognition receptor signaling pathways, protein ubiquitination, oxidative stress, necrotic cell death, ATP synthesis and the cellular respiratory chain. RT-qPCR verified that the expression patterns of Cenpb, Crisp1 and Mars were changed in testes, and genes relevant to epididymal function including Aqp9 and Octn2 were downregulated in epididymides, particularly in the cotreatment group. Collectively, our results emphasize that DEHP at an environmentally relevant dose can induce male reproductive toxicity, and PSNPs may aggravate the toxic effects.

Exposure to carbon black nanoparticles during pregnancy aggravates lipopolysaccharide-induced lung injury in offspring: an intergenerational effect.

Carbon black nanoparticles (CBNPs) are one of the most frequently used nanoparticles. Exposure to CBNPs during pregnancy (PrE to CBNPs) can directly induce inflammation, lung injury, and genotoxicity in dams and results in abnormalities in offspring. However, whether exposure to CBNPs during pregnancy enhances the susceptibility of offspring to environmental stimuli remains unknown. To address this issue, in this study, we intranasally treated pregnant mice with mock or CBNPs from gestational day (GD) 9 to GD18, and F1 and F2 offspring were normally obtained. By intratracheal instillation of mice with lipopolysaccharide (LPS) to trigger a classic animal model for acute lung injury, we intriguingly found that after LPS treatment, F1 and F2 offspring after exposure during pregnancy to CBNPs both exhibited more pronounced lung injury symptoms, including more degenerative histopathological changes, vascular leakage, elevated MPO activity, and activation of inflammation-related signaling transduction, compared with F1 and F2 offspring in the mock group, suggesting PrE to CBNPs would aggravate LPS-induced lung injury in offspring, and this effect was intergenerational. We also observed that PrE to CBNPs upregulated the mRNA expression of DNA methyltransferases (Dnmt) 1/3a/3b and DNA hypermethylation in both F1 and F2 offspring, which might partially account for the intergenerational effect. Together, our study demonstrates for the first time that PrE to CBNPs can enhance sensitivity to LPS in both F1 and F2 offspring, and this intergenerational effect may be related to DNA hypermethylation caused by CBNPs.

Knock-down of transcription factor skinhead-1 exacerbates arsenite-induced oxidative damage in Caenorhabditis elegans.

Transcription factor, skinhead-1 (skn-1) has been demonstrated to play central roles in regulation of oxidative damage. Arsenite is an oxidative damage inducer in the environment. However, the role of skn-1 in arsenite-induced oxidative damage remains unclear. Thus, in this study, by using RNAi feeding, different toxic responses of wild-type and skn-1 knockdown nematodes to arsenite were evaluated. Our results demonstrated that arsenite did not show any significant impacts on locomotory behaviors, but skn-1 knock-down worms were much more sensitive to arsenite treatment, manifested by an aggravated reduction of survival rate than that of wild-type nematodes. In arsenite-treated worms, down-regulation of skn-1 significantly exacerbated the arsenite-induced changed expressions of oxidative damage-related genes, xbp-1, apl-1 and trxr-2, but these regulated effects of skn-1 were not observed on spr-4 and sel-12 expressions under arsenite treatment. These findings together suggest that skn-1 may play a vital role in protection of C. elegans from arsenite-induced oxidative damage.

Pregnancy exposure of titanium dioxide nanoparticles causes intestinal dysbiosis and neurobehavioral impairments that are not significant postnatally but emerge in adulthood of offspring.

BACKGROUND: Pregnancy exposure to titanium dioxide nanoparticles (TiO2NPs) is a vital consideration due to their inadvertent ingestion from environmental contamination. The potential health effects of TiO2NPs on the neurodevelopmental process should be seriously concerned in health risk assessment, especially for the pregnant women who are susceptible to the neurodevelopmental toxicity of nano-sized particles. However, the available evidence of neurodevelopmental toxicity of TiO2NPs remains very limited. METHODS: In the present study, the pregnant mice were intragastric administered with 150 mg/kg TiO2NPs from gestational day (GD) 8 to 21, the maternal behaviors and neurodevelopment-related indicators in offspring were all assessed at different time points after delivery. The gut microbial community in both dams and their offspring were detected by using 16S ribosomal RNA (rRNA) gene sequencing. The gut-brain axis related indicators were also determined in the offspring. RESULTS: The results clearly demonstrated that exposure to TiO2NPs did not affect the maternal behaviors of pregnant mice, or cause the deficits on the developmental milestones and perturbations in the early postnatal development of offspring. Intriguingly, our data revealed that pregnancy exposure of TiO2NPs did not affect locomotor function, learning and memory ability and anxiety-like behavior in offspring at postnatal day (PD) 21, but resulted in obvious impairments on these neurobehaviors at PD49. Similar phenomena were obtained in the composition of gut microbial community, intestinal and brain pathological damage in offspring in adulthood. Moreover, the intestinal dysbiosis induced by TiO2NPs might be highly associated with the delayed appearance of neurobehavioral impairments in offspring, possibly occurring through disruption of gut-brain axis. CONCLUSIONS: This is the first report elucidated that pregnancy exposure to TiO2NPs caused delayed appearance of neurobehavioral impairments in offspring when they reached adulthood, although these perturbations did not happen at early life after delivery. These findings will provide valuable insights about neurodevelopmental toxicity of TiO2NPs, and call for comprehensive health risk assessment of TiO2NPs on the susceptible population, such as pregnant women.

Autophagy deficiency exacerbates acute lung injury induced by copper oxide nanoparticles.

Copper oxide nanoparticles (CuONPs) are one of the widely used metal nanoparticles in the industrial and commercial fields. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome and has been linked to nanoparticles-induced toxicity. In particular, the roles of autophagy in response to CuONPs have been explored in vitro, although the conclusions are controversial. To clarify the role of autophagy in CuONPs-induced acute lung injury, microtubule-associated protein 1 light chain 3 beta (Map1lc3b or lc3b) knockout mice and their corresponding wild type mice are applied. Our results showed that single-dose intratracheal instillation of CuONPs with dosages of 1.25, 2.5 or 5 mg/kg caused acute lung injury 3 days after treatment in a dose-dependent manner, as evidenced by deteriorative lung histopathology, more infiltration of macrophage cells, increased oxidative stress and copper ions. Loss of lc3b resulted in aggravated lung injury induced by CuONPs, which was probably due to the blockade of mitophagy and consequently the accumulation of aberrant mitochondria with overloaded copper ions. Our study provides the first in vivo evidence that autophagy deficiency exacerbates CuONPs-induced acute lung injury, and highlights that targeting autophagy is a meaningful strategy against CuONPs-associated respiratory toxicity.

Silicon dioxide nanoparticles induced neurobehavioral impairments by disrupting microbiota-gut-brain axis.

BACKGROUND: Silicon dioxide nanoparticles (SiO2NPs) are widely used as additive in the food industry with controversial health risk. Gut microbiota is a new and hot topic in the field of nanotoxicity. It also contributes a novel and insightful view to understand the potential health risk of food-grade SiO2NPs in children, who are susceptible to the toxic effects of nanoparticles. METHODS: In current study, the young mice were orally administrated with vehicle or SiO2NPs solution for 28 days. The effects of SiO2NPs on the gut microbiota were detected by 16S ribosomal RNA (rRNA) gene sequencing, and the neurobehavioral functions were evaluated by open field test and Morris water maze. The level of inflammation, tissue integrity of gut and the classical indicators involved in gut-brain, gut-liver and gut-lung axis were all assessed. RESULTS: Our results demonstrated that SiO2NPs significantly caused the spatial learning and memory impairments and locomotor inhibition. Although SiO2NPs did not trigger evident intestinal or neuronal inflammation, they remarkably damaged the tissue integrity. The microbial diversity within the gut was unexpectedly enhanced in SiO2NPs-treated mice, mainly manifested by the increased abundances of Firmicutes and Patescibacteria. Intriguingly, we demonstrated for the first time that the neurobehavioral impairments and brain damages induced by SiO2NPs might be distinctively associated with the disruption of gut-brain axis by specific chemical substances originated from gut, such as Vipr1 and Sstr2. Unapparent changes in liver or lung tissues further suggested the absence of gut-liver axis or gut-lung axis regulation upon oral SiO2NPs exposure. CONCLUSION: This study provides a novel idea that the SiO2NPs induced neurotoxic effects may occur through distinctive gut-brain axis, showing no significant impact on either gut-lung axis or gut-liver axis. These findings raise the exciting prospect that maintenance and coordination of gastrointestinal functions may be critical for protection against the neurotoxicity of infant foodborne SiO2NPs.

Titanium dioxide nanoparticles via oral exposure leads to adverse disturbance of gut microecology and locomotor activity in adult mice.

Titanium dioxide nanoparticles (TiO2NPs) have been widely used as food additives in daily life. However, the impact of oral intake of TiO2NPs on the nervous system is largely unknown. In this study, 7-week-old mice were treated with either vehicle or TiO2NPs suspension solution at 150 mg/kg by intragastric administration for 30 days. Our results demonstrated that oral exposure to TiO2NPs resulted in aberrant excitement of enteric neurons, although unapparent pathological changes were observed in gut. We also found the richness and evenness of gut microbiota were remarkably decreased and the gut microbial community compositions were significantly changed in the TiO2NP-treated group as compared with vehicle controls. Interestingly, oral exposure to TiO2NPs was capable to induce the inhibitory effects on locomotor activity, but it did not lead to significant change on the spatial learning and memory ability. We further revealed the mechanism that TiO2NPs could specifically cause locomotor dysfunction by elevating the excitement of enteric neuron, which might spread to brain via gut-brain communication by vagal pathway. However, inflammation response, enteric neurotransmitter 5-HT and major gut peptides might not be involved in this pathological process. Together, these findings provide valuable insights into the novel mechanism of TiO2NP-induced neurotoxicity. Understanding the microbiota-gut-brain axis will provide the foundation for potential therapeutic or prevention approaches against TiO2NP-induced gut and brain-related disorders.

Arsenite induces testicular oxidative stress in vivo and in vitro leading to ferroptosis.

Ferroptosis is a newly identified form of cell death characterized by accumulation of intracellular iron and requirement of lipid peroxidation. However, whether arsenite triggers testicular cell death via ferroptosis remains unclear. In this study, after administrating of adult male mice with 0.5, 5 and 50 mg/L arsenite for six months via drinking water, the results showed that arsenite caused the pathological changes in mouse testis and significantly reduced the number of sperm. Mitochondrial injuries were observed as the major ultrastructural damages induced by arsenite, and these damages were accompanied by the apparent mitochondrial oxidative damage in the testis, manifested by accumulation of iron, production of reactive oxygen species and lipid peroxidation products. We also demonstrated that arsenite significantly activated ferroptosis-related signal pathways in the mouse testis. To further verify the results obtained in the animal model, GC-2spd cells were employed as the in vitro culture system. Consistently, the results revealed arsenite remarkably triggered the ferroptotic cell death in vitro, and inhibition of ferroptosis by ferrostatin-1 could attenuate this adverse effect in cells. These findings together indicate that arsenite can trigger oxidative stress thus leading to testicular cell death by ferroptosis, suggesting that inhibition of ferroptosis would be a potential strategy for treatment of arsenite-related male reproductive toxicity.

Gas chromatography-mass spectrometry based metabolomics profile of hippocampus and cerebellum in mice after chronic arsenic exposure.

Intake of arsenic (As) via drinking water has been a serious threat to global public health. Though there are numerous reports of As neurotoxicity, its pathogenesis mechanisms remain vague especially its chronic effects on metabolic network. Hippocampus is a renowned area in relation to learning and memory, whilst recently, cerebellum is argued to be involved with process of cognition. Therefore, the study aimed to explore metabolomics alternations in these two areas after chronic As exposure, with the purpose of further illustrating details of As neurotoxicity. Twelve 3-week-old male C57BL/6J mice were divided into two groups, receiving deionized drinking water (control group) or 50 mg/L of sodium arsenite (via drinking water) for 24 weeks. Learning and memory abilities were tested by Morris water maze (MWM) test. Pathological and morphological changes of hippocampus and cerebellum were captured via transmission electron microscopy (TEM). Metabolic alterations were analyzed by gas chromatography-mass spectrometry (GC-MS). MWM test confirmed impairments of learning and memory abilities of mice after chronic As exposure. Metabolomics identifications indicated that tyrosine increased and aspartic acid (Asp) decreased simultaneously in both hippocampus and cerebellum. Intermediates (succinic acid) and indirect involved components of tricarboxylic acid cycle (proline, cysteine, and alanine) were found declined in cerebellum, indicating disordered energy metabolism. Our findings suggest that these metabolite alterations are related to As-induced disorders of amino acids and energy metabolism, which might therefore, play an important part in mechanisms of As neurotoxicity.

Arsenite-induced endoplasmic reticulum-dependent apoptosis through disturbance of calcium homeostasis in HBE cell line.

Calcium (Ca2+ ) is a ubiquitous cell signal responsible for multiple fundamental cellular functions, including apoptosis. Whether the homeostasis of Ca2+ is involved in arsenite-induced apoptosis remains unclear. In this study, we observed that arsenite significantly elevated the intracellular Ca2+ concentration in a dose- and time-dependent manner. By using the Ca2+ -ATPase inhibitor, thapsigargin, and the inositol 1,4,5- trisphosphate receptors (IP3Rs) inhibitor, heparin, we further confirmed that the disturbance of endoplasmic reticulum (ER) Ca2+ homeostasis caused Ca2+ overload in the cells. Moreover, loss of ER Ca2+ homeostasis also led to ER stress, mitochondrial dysfunction, and NF-κB activation. Importantly, pretreatment of cells with heparin remarkably attenuated the elevated cell apoptosis induced by arsenite, but inhibition of ER Ca2+ uptake with thapsigargin exacerbated arsenite-induced cell damage significantly. Together, we demonstrated for the first time that arsenite disturbed the Ca2+ homeostasis in ER, which subsequently led to ER stress, mitochondrial dysfunction, and NF-κB nuclear translocation, and thus consequently triggering cell apoptosis. Our findings indicate regulation of disrupted Ca2+ homeostasis in ER may be a potential strategy for prevention of arsenite toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 197-216, 2017.

Protection of Nrf2 against arsenite-induced oxidative damage is regulated by the cyclic guanosine monophosphate-protein kinase G signaling pathway.

Arsenite has been shown to induce a variety of oxidative damage in mammalian cells. However, the mechanisms underlying cellular responses to its adverse effects remain unknown. We previously showed that the level of Nrf2, a nuclear transcription factor significantly increased in arsenite-treated human bronchial epithelial (HBE) cells suggesting that Nrf2 is involved in responding to arsenite-induced oxidative damage. To explore how Nrf2 can impact arsenite-induced oxidative damage, in this study, we examined Nrf2 activation and its regulation upon cellular arsenite exposure as well as its effects on arsenite-induced oxidative damage in HBE cells. We found that Nrf2 mRNA and protein levels were significantly increased by arsenite in a dose- and time-dependent manner. Furthermore, we showed that over-expression of Nrf2 significantly reduced the level of arsenite-induced oxidative damage in HBE cells including DNA damage, chromosomal breakage, lipid peroxidation and depletion of antioxidants. This indicates a protective role of Nrf2 against arsenite toxicity. This was further supported by the fact that activation of Nrf2 by its agonists, tertiary butylhydroquinone (t-BHQ) and sulforaphane (SFN) resulted in the same protective effects against arsenite toxicity. Moreover, we demonstrated that arsenite-induced activation of Nrf2 was mediated by the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway. This is the first evidence showing that Nrf2 protects against arsenite-induced oxidative damage through the cGMP-PKG pathway. Our study suggests that activation of Nrf2 through the cGMP-PKG signaling pathway in HBE cells may be developed as a new strategy for prevention of arsenite toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2004-2020, 2017.