Monobutyl phthalate (MBP) is the main metabolite of dibutyl phthalate (DBP) in vivo. MBP has a stable structure, can continuously accumulate in living organisms, and has the potentially to harm animal and human reproductive function. In the ovarian follicle microenvironment, MBP may lead to defects in follicular development and steroid production, abnormal meiotic maturation, impaired ovarian function and other reproductive deficits. In this study, SMART-seq was used to investigate the effects of MBP exposure on the in vitro maturation (IVM) and development of porcine oocytes. The results showed that differentially expressed genes after MBP exposure were enriched in the biological processes cytoskeleton, cell apoptosis, endoplasmic reticulum (ER) and mitochondria. Glycine (Gly) improved the developmental potential of porcine oocytes by regulating mitochondrial and ER function. The effect of Gly in protecting oocytes against MBP-induced damage was studied. The results showed that the addition of Gly significantly decreased the rate of MBP-induced spindle abnormalities, decreased the frequency of MBP-induced mitochondria-associated ER membrane (MAM) interactions, and downregulated the protein and gene expression of the linkage molecules Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) in the MAM. Additionally, treatment with Gly restored the distribution of the 1,4,5-triphosphate receptor 1 (IP₃R1) and voltage-dependent anion channel 1 (VDAC1), further decreasing the intracellular free calcium concentration ([Ca²⁺]ᵢ) levels and mitochondrial Ca²⁺ ([Ca²⁺]ₘ) , increasing the ER Ca²⁺ ([Ca²⁺]ER) levels, and thus significantly increasing the ER levels and mitochondrial membrane potential (ΔΨ m). Gly also decreased the levels of reactive oxygen species (ROS) and increased the levels of Glutathione (GSH), oocyte apoptosis-related indicators (Caspase-3 activity and Annexin V) and oocyte apoptosis-related genes (BAX, Caspase 3 and AIFM1). Our results suggest that Gly can ameliorate microtubule cytoskeleton abnormalities and improve oocyte maturation by reducing the defective mitochondrial–ER interactions caused by MBP exposure in vitro.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants (POPs) that have adverse effects on human health. However, the long-term health effects and potential mechanism of neonatal exposure to PCBs are still unclear. In this study, nursing male mice exposed to PCB138 at 0.5, 5, and 50 μg/kg body weight (bw) from postnatal day (PND) 3 to PND 21 exhibited increased serum uric acid levels and liver uric acid synthase activity at 210 days of age. We also found an increased kidney somatic index in the 50 μg/kg group and kidney fibrosis in the 5 and 50 μg/kg groups. Mechanistically, PCB138 induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which might have led to inflammatory responses, such as activation of the NF-κB (nuclear factor kappa-B) and NLRP3 (NOD-like receptor protein 3) pathways. The inflammatory response might regulate renal fibrosis and hypertrophy. In summary, this study reports a long-term effect of neonatal PCB exposure on uric acid metabolism and secondary nephrotoxicity and clarifies the underlying mechanism. Our work also indicates that early life pollutant exposure may be an important cause of diseases later in life.

Copper (Cu) is a vital micronutrient required for numerous fundamental biological processes, but excessive Cu poses potential detrimental effects on public and ecosystem health. However, the molecular details linking endoplasmic reticulum (ER) stress and apoptosis in duck renal tubular epithelial cells have not been fully elucidated. In this study, duck renal tubular epithelial cells exposed to Cu sulfate (CuSO₄) (0, 100 and 200 μM) and a PERK inhibitor (GSK2606414, GSK, 1 μM) for 12 h were used to investigate the crosstalk between ER stress and apoptosis under Cu exposure. Cell and ER morphological and functional characteristics, intracellular calcium (Ca²⁺) levels, apoptotic rates, ER stress and apoptosis-related mRNA and protein levels were examined. The results showed that excessive Cu could cause ER expansion and swelling, increase the expression levels of ER stress-associated genes (PERK, eIF2α, ATF4 and CHOP) and proteins (p-PERK and CHOP), induce intracellular Ca²⁺ overload, upregulate the expression levels of apoptosis-associated genes (Bax, Bak1, Caspase9 and Caspase3) and the cleaved-Caspase3 protein, downregulate Bcl-xl and Bcl2 mRNA levels and trigger apoptosis. PERK inhibitor treatment could ameliorate the above changed factors caused by Cu. In conclusion, these findings indicate that excessive Cu could trigger ER stress via activation of the PERK/ATF4/CHOP signaling pathway and that ER stress might aggravate Cu-induced apoptosis in duck renal tubular epithelial cells.

Known as a cause of food poisoning, Bacillus cereus (B. cereus) is widespread in nature. Cereulide, the heat-stable and acid-resistant emetic toxin which is produced by some B. cereus strains, is often associated with foodborne outbreaks, and causes acute emetic toxicity at high dosage exposure. However, the toxicological effect and underlying mechanism caused by chronic low-dose cereulide exposure require to be further addressed. In the study, based on mouse model, cereulide exposure (50 μg/kg body weight) for 28 days induced intestinal inflammation, gut microbiota dysbiosis and food intake reduction. According to the cell models, low dose cereulide exposure disrupted the intestinal barrier function and caused intestinal inflammation, which were resulted from endoplasmic reticulum (ER) stress IRE1/XBP1/CHOP pathway activation to induce cell apoptosis and inflammatory cytokines production. For gut microbiota, cereulide decreased the abundances of Lactobacillus and Oscillospira. Furthermore, cereulide disordered the metabolisms of gut microbiota, which exhibited the inhibitions of butyrate and tryptophan. Interestingly, cereulide exposure also inhibited the tryptophan hydroxylase to produce the serotonin in the gut and brain, which might lead to depression-like food intake reduction. Butyrate supplementation (100 mg/kg body weight) significantly reduced intestinal inflammation and serotonin biosynthesis suppression caused by cereulide in mice. In conclusion, chronic cereulide exposure induced ER stress to cause intestinal inflammation, gut microbiota dysbiosis and serotonin biosynthesis suppression. IRE1 could be the therapeutic target and butyrate supplementation is the potential prevention strategy.

Perfluorooctane sulfonate (PFOS) is one of the most widely used and distributed perfluorinated compounds proven to cause adverse health outcomes. Datasets of ecotoxico-genomics and proteomics have given greater insights for PFOS toxicological effect. However, the molecular mechanisms of hepatotoxicity of PFOS on post-translational modifications (PTMs) regulation, which is most relevant for regulating the activity of proteins, are not well elucidated. Protein glycosylation is one of the most ubiquitous PTMs associated with diverse cellular functions, which are critical towards the understanding of the multiple biological processes and toxic mechanisms exposed to PFOS. Here, we exploit the multilayered glycoproteomics to quantify the global protein expression levels, glycosylation sites, and glycoproteins in PFOS exposure and wild-type mouse livers. The identified 2439 proteins, 1292 glycosites, and 799 glycoproteins were displayed complex heterogeneity in PFOS exposure mouse livers. Quantification results reveal that 241 dysregulated proteins (fold change ≥ 2, p < 0.05) in PFOS exposure mouse livers were involved in the lipid and xenobiotic metabolism. While, 16 overexpressed glycoproteins were exclusively related to neutrophil degranulation, cellular responses to stress, protein processing in endoplasmic reticulum (ER). Moreover, the interactome and functional network analysis identified HP and HSP90AA1 as the potential glycoprotein biomarkers. These results provide unique insights into a deep understanding of the mechanisms of PFOS induced hepatotoxicity and liver disease. Our platform of multilayered glycoproteomics can be adapted to diverse ecotoxicological research.

The Pearl River Estuary (PRE) is the largest estuary in southern China and under high metal stress. In the present study, we employed an integrated method of transcriptomics and proteomics to investigate the ecotoxicological effects of trace metals on the Hong Kong oyster Crassostrea hongkongensis. Three oyster populations with distinct spatial distributions of metals were sampled, including the Control (Station QA, the lowest metal levels), the High Cd (Station JZ, the highest Cd), and the High Zn–Cu–Cr–Ni (Station LFS, with the highest levels of zinc, copper, chromium, and nickel). Dominant metals in oysters were differentiated by principal component analysis (PCA), and theirgene and protein profiles were studied using RNA-seq and iTRAQ techniques. Of the 2250 proteins identified at both protein and RNA levels, 70 proteins exhibited differential expressions in response to metal stress in oysters from the two contaminated stations. There were 8 proteins altered at both stations, with the potential effects on mitochondria and endoplasmic reticulum by Ag. The genotoxicity, including impaired DNA replication and transcription, was specifically observed in the High Cd oysters with the dominating influence of Cd. The structural components (cytoskeleton and chromosome-associated proteins) were impaired by the over-accumulated Cu, Zn, Cr, and Ni at Station LFS. However, enhanced tRNA biogenesis and exosome activity might help the oysters to alleviate the toxicities resulting from their exposure to these metals. Our study provided comprehensive information on the molecular changes in oysters at both protein and RNA levels in responding to multi-levels of trace metal stress.

We investigated the in vitro effects of pyriproxyfen on ionic balance in the testis of the zebrafish by measuring ⁴⁵Ca²⁺ influx. In vivo pyriproxyfen treatment was carried out to study oxidative stress, and conduct morphological analysis of the testis and liver. Whole testes were incubated in vitro with/without pyriproxyfen (10⁻¹², 10⁻⁹ or 10⁻⁶ M; 30 min) and ⁴⁵Ca²⁺ influx determined. To study pyriproxyfen’s mechanism of action, inhibitors/activators of ionic channels or pumps/exchangers, protein kinase inhibitors or a calcium chelator were added 15 min before the addition of ⁴⁵Ca²⁺ and pyriproxyfen. We evaluated the in vivo effects of 7 day exposure to waterborne pyriproxyfen (10⁻⁹ M) on reactive oxygen species (ROS) formation, lipid peroxidation, and reduced glutathione content (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and γ-glutamyltransferase (GGT) activity. Morphological analyses of the testis and liver were carried out after in vivo exposure of D. rerio to pyriproxyfen. Pyriproxyfen increased ⁴⁵Ca²⁺ influx by opening the voltage-dependent T-type channels (T-type VDCC), inhibiting sarco/endoplasmic reticulum ⁴⁵Ca²⁺-ATPase (SERCA) and the NCX exchanger (forward mode) and by mobilizing calcium from stores. The involvement of potassium channels and protein kinase C (PKC) was also demonstrated in pyriproxyfen-induced intracellular calcium elevation. In vivo pyriproxyfen treatment of D. rerio increased lipid peroxidation, decreased GSH content and increased GST activity in testes, in addition to increasing the number and size of spermatogonia cysts and inducing hepatocyte basophilia and dilation of blood vessels in the liver. The toxicity of pyriproxyfen is mediated by calcium overload, increased lipid peroxidation, and a diminished antioxidant capacity in the testis, due to GSH depletion, and altered spermatogenesis. The development of high basophilia in the liver suggests that pyriproxyfen may have estrogenic activity, possibly acting as an endocrine-disruptor. These findings indicate that these alterations may contribute to pyriproxyfen toxicity and spermatogenesis disruption.

The hazardous effects of herbicides are well known; however, their effects on the reproductive system remain unclear. In this study, we demonstrated the anti-proliferative effects of dinitramine (DN) on immature murine testicular cell lines (Leydig and Sertoli cells) mediated via endoplasmic reticulum (ER) stress-induced calcium dysregulation in the cytosol and mitochondria. The results demonstrated that the viability and proliferation of DN-treated TM3 and TM4 cells decreased significantly, even in the spheroid state. DN induced the apoptosis of TM3 and TM4 cells and decreased the expression of genes related to cell cycle progression. Treatment with DN increased the cytosolic and intramitochondrial levels of calcium by activating ER stress signals. DN activated the Erk/P38/Jnk Mapk pathway and inactivated the Pi3k/Akt pathway in murine testicular cells. Co-treatment with 2-aminoethoxydiphenyl borate (2-APB) mitigated DN-induced calcium upregulation in both testicular cell lines. Although 2-APB did not antagonize the anti-proliferative effect of DN in TM3 cells, treatment with 2-APB and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid restored the proliferation of DN-treated TM4 cells.

Glyphosate is the most widely used herbicide in the world. In recent years, many studies have demonstrated that exposure to glyphosate-based herbicides (GHBs) was related to the decrease of serum testosterone and the decline in semen quality. However, the molecular mechanism of glyphosate-induced testosterone synthesis disorders is still unclear. In the present study, the effects of glyphosate on testosterone secretion and the role of endoplasmic reticulum (ER) stress in the process were investigated in TM3 cells. The effects of glyphosate at different concentrations on the viability of TM3 cells were detected by CCK8 method. The effect of glyphosate exposure on testosterone secretion was determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of testosterone synthases and ER stress-related proteins were detected by Western blot and Immunofluorescence stain. Results showed that exposure to glyphosate at concentrations below 200 mg/L had no effect on cell viability, while the glyphosate above 0.5 mg/L could inhibit the testosterone secretion in TM3 cells. Treatment TM3 cells with glyphosate at 5 mg/L not only reduced the protein levels of testosterone synthase StAR and CYP17A1, inhibited testosterone secretion, but also increased the protein level of ER stress molecule Bip and promoted the phosphorylation of PERK and eIF2α. Pretreatment cells with PBA, an inhibitor of ER stress, alleviated glyphosate-induced increase in Bip, p-PERK and p-eIF2α protein levels, meanwhile rescuing glyphosate-induced testosterone synthesis disorders. When pretreatment with GSK2606414, a PERK inhibitor, the glyphosate-induced phosphorylation of PERK and eIF2α was blocked, and the glyphosate-inhibited testosterone synthesis and secretion was also restored. Overall, our findings suggest that glyphosate can interfere with the expression of StAR and CYP17A1 and inhibit testosterone synthesis and secretion via ER stress-mediated the activation of PERK/eIF2α signaling pathway in Leydig cells.

Environmental chemical exposures have been implicated as risk factors for the development of non-alcoholic fatty liver (NAFLD). Bisphenol S (BPS), widely used in multitudinous consumer products, could disrupt lipid metabolism in the liver. This study aimed at examining the hypothesis that long-term exposure to BPS promotes the development of liver fibrosis and inflammation by means of the application of a semi-static exposure experiment that exposed zebrafish to 1, 10, and 100 μg/L BPS from 3 h post fertilization to 120 day post fertilization. Results showed that the 120-d BPS exposure elevated plasma aspartate aminotransferase and alanine aminotransferase activities, increased triacylglycerol (TAG) and total cholesterol levels in male liver, and even induced hepatic apoptosis and fibrosis. Hepatic lipid accumulation observed in the 30-d BPS-exposed zebrafish was recovered after a 90-d depuration phase, thereby indicating that long-term BPS exposure promotes the progression of simple steatosis to non-alcoholic steatohepatitis. Furthermore, BPS exposure for 120-d promoted the synthesis of TAG and lipotoxic free fatty acids by elevating the transcription of srebp1, acc, fasn, and elovl6, induced endoplasmic reticulum (ER) stress with increasing expression levels of unfolded protein response (UPR) genes (perk, hsp5, atf4a, and ddit3), and then stimulated the expression of two key autophagy genes (atg3 and lc3) and inflammatory genes (il1b and tnfα). It is indicated that BPS can induce the development of steatohepatitis via the activation of the PERK-ATF4a pathway of the UPR. Data gathered suggest that environmental pollutants-induced ER stress with the activation of UPR can potentially trigger the NAFLD development in males. Overall, our study provided new sights into understanding of the adverse health effects of metabolism disrupting chemicals.