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Circular RNAs (circRNAs), is a novel type of endogenous non-coding RNAs (ncRNAs) participated in the pathogenesis of many diseases. Beryllium is one of the carcinogenesis elements. However, the mechanism and function of circRNAs in human bronchial epithelial cells (16HBE) induced by beryllium sulfate (BeSO4) was rarely reported. Therefore, the high-throughput RNA sequencing analysis was performed to detect the circRNA profiles between control groups and BeSO4-induced groups. Furthermore, circRNA-miRNA-mRNA network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and PPI network analysis were used for bioinformatics analysis. CircRNA sequencing analysis revealed that 36 circRNAs were up-regulated and 35 circRNAs were down-regulated in the BeSO4-exposed groups. The selected circRNAs were verified by real-time fluorescent quantitative PCR (qRT-PCR). Hsa_circ_0004214 and hsa_circ_0003586 were validated to be up-regulated, hsa_circ_0047958, hsa_circ_0001944, and hsa_circ_0008982 were down-regulated. The circRNA-miRNA-mRNA network annotated the key signaling pathway including cellular senescence, TNF signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway, and Hippo signaling pathway. The PPI network indicated the most circRNAs might participate in the BeSO4 toxicity by acting as a sponge for the miR-663b through JAK–STAT signaling pathway. In summary, our study suggests that circRNAs may play roles in the mechanism of beryllium toxicity.

Background Permethrin is one of the pyrethroid insecticides, which is widely used in agriculture and public health. Although acute toxicity of the insecticide has been studied, the chronic toxicity upon the long-term exposure has not been clear yet. The purpose of the current study is to investigate the organ toxicities of permethrin following its long-term low-dose exposure. Methods Male Wistar rats were daily administrated orally with permethrin (75 mg/kg body weight/day, gavage) for 90 days, and then the samples of biofluids (blood and urine) and organs including liver and kidney were collected. The serum and urine samples were measured by biochemical assay and the tissues of kidney and liver were examined and analyzed by histopathological method. Results The results showed that no change was found in serum and urine biochemical parameters for the toxicity; however, significant changes including hyperchromatic nuclei swollen in the hepatic parenchymal cells and the swelling proximal tubules in the kidneys were observed in the tissue structures of liver and kidneys in the histopathological sections. Conclusion These results indicate that low-dose long-term exposure of permethrin can cause chronic toxicity with slight liver and kidney damage.

With the growth of the aging population, the prevalence of Alzheimer's disease (AD) has increased and influenced the work and daily life of AD patients, imposing a heavy burden on society and the patients' families. AD is a progressive disease with a long duration, and the pathogenesis is very complicated. Here, we found that alpha-lipoic acid (LA), an endogenous, naturally synthesized compound, could attenuate amyloid beta fragment (Aβ25-35 )-induced PC12 cell toxicity. Aβ25-35 treatment largely decreased the viability of PC12 cells, increased reactive oxygen species (ROS) levels, and increased the percentage of apoptotic cells, which were accompanied by changes in the expression of the apoptosis-related genes. Further, the Wnt pathway was inactivated, and the expression of Wnt pathway-related proteins such as Frizzled2, GSK3β, and phosphorylated GSK3β were dysregulated after Aβ25-35 treatment. LA efficiently attenuated Aβ25-35 -induced PC12 cell apoptosis and downregulated the phosphorylation-mediated degradation of β-catenin as well as GSK3β. Our results demonstrate that LA rescues Aβ25-35 -induced neurocytotoxicity through the Wnt-β-catenin pathway.

Background This study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells. Methods Wistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining. Results The results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos. Conclusions These findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

To screen the differentially expressed proteins (DEPs) in human bronchial epithelial cells (HBE) treated with atmospheric fine particulate matter (PMHBE cells were treated with PMOverall, 67 DEPs were screened in the Shenzhen sample-treated group, of which 46 were upregulated and 21 were downregulated. In total, 252 DEPs were screened in the Taiyuan sample-treated group, of which 134 were upregulated and 118 were downregulated. KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PMOur findings provide a valuable basis for further evaluation of PM

As an organophosphorus ester, tri-ortho-cresyl phosphate (TOCP) has been widely used in agriculture and industry. It is reported that TOCP can induce organophosphate-induced delayed neuropathy (OPIDN) in sensitive animal and human species. However, the exact molecular mechanisms underlying TOCP-induced neurotoxicity are still unknown. In this study, we found that TOCP could induce autophagy by activating protein kinase C alpha (PKCα) signaling in neuroblastoma SK-N-SH cells. PKCα activators could positively regulate TOCP-induced autophagy by increasing the expression levels of neighbor BRCA1 gene protein 1 (NBR1), LC3 and P62 autophagic receptor protein. Furthermore, PKCα activation impaired the ubiquitin-proteasome system (UPS), resulting in inhibition of proteasome activity and accumulation of ubiquitinated proteins. UPS dysfunction could stimulate autophagy to serve as a compensatory pathway, which contributed to the accumulation of the abnormally hyperphosphorylated tau proteins and degradation of impaired proteins of the MAP 2 and NF-H families in neurodegenerative disorders.

Carbamates and pyrethroids are widely used pesticides. However, their joint toxicity at low doses with long-term exposure remains unknown. Therefore, we investigated the subchronic joint hepatotoxicity of the two representative pesticides within these two classes, i.e., propoxur (PR) and permethrin (PE) in rats. The male Wistar rats were orally treated with three different doses of PR, PE and their mixtures for 90 consecutive days. Liver weight, serum clinical chemistry parameters and histopathological changes were measured to access the hepatotoxicity. In addition, oxidative stress markers in liver were measured using biochemical assays. The results showed that PR reduced liver weight and lead to prominent liver histological changes. Moreover, PR dose-dependently induced lipid peroxidation and reduced superoxide dismutase activity. In contrast, PE induced a relatively mild hepatotoxicity. Intriguingly, the mixture of PR and PE did not reduce liver weight or increase serum aspartate transaminase activity. In addition, the mixture did not reduce the antioxidant enzyme activity as PR did. Thus, these results showed that PR induced prominent hepatotoxicity with subchronic exposure, and there is a potential antagonistic interaction between PR and PE on the oxidative damage in liver of rats.

Background: Permethrin is a type of widely used pyrethroid pesticide. Although acute toxicity of permethrin has been well-characterised, the non-acute toxicity of permethrin upon long-term ...

As a major kind of carbamate insecticide, propoxur plays an important role in agriculture, veterinary medicine, and public health. The acute toxicity of propoxur is mainly neurotoxicity due to the inhibition of cholinesterase. However, little is known regarding the toxicity of propoxur upon long-term exposure at low dose. In this study, Wistar rats were orally administrated with low dose (4.25 mg/kg body weight/day) for consecutive 90 days. And the urine samples in rats treated with propoxur for 30, 60, and 90 days were collected and analyzed by employing 1H NMR-based metabolomics approach. We found that propoxur caused significant changes in the urine metabolites, including taurine, creatinine, citrate, succinate, dimethylamine, and trimethylamine-N-oxide. And the alteration of the metabolites was getting more difference compared with that of the control as the exposure time extending. The present study not only indicated that the changed metabolites could be used as biomarkers of propoxur-induced toxicity but also suggested that the time-course alteration of the urine metabolomic profiles could reflect the progressive development of the toxicity following propoxur exposure.

Neuropathy target esterase (NTE) is an endoplasmic reticulum membrane-associated phospholipase B, which is essential for embryonic and nervous system development. However, the regulation of NTE at the protein level had not been thoroughly investigated. Our previous study showed that NTE was degraded not only by the macroautophagy-lysosome pathway but also by the ubiquitin-proteasome pathway. Here we further reveal that androgen receptor-associated protein 54 (ARA54) regulated the ubiquitin-proteasome degradation of NTE. We find that deletion of the regulatory domain of NTE, which possesses a putative destruction box and thus is essential for its degradation by the proteasome, prevented its degradation by the proteasome. In addition, we demonstrate that ARA54, which has a RING finger domain and E3 ligase activity, interacts directly with NTE. Overexpression of ARA54 downregulates the protein level of NTE, and knockdown of ARA54 inhibits the degradation of NTE. The mutation in the RING domain of ARA54 blocks the degradation of NTE by ARA54, which indicates that the RING domain is essential for ARA54's E3 activity. These findings suggest that ARA54 acts as the ubiquitin ligase to regulate the ubiquitin-proteasome degradation of NTE.

Indoor air quality has great impact on the human health. An increasing number of studies have shown that printers could release particulate matters and pose adverse effects on indoor air quality. In this study, a thorough investigation was designed to assess the aerosol printer particle total number concentration (TNC) and size distribution in normal office environment, one copy center, and a clean chamber. Particle analyzers, SMPS, OPS, and CPC3007 were used to monitor the total printing process. In normal office environment, 37 laser printers out of all surveyed 55 printers were classified as high particle emitters. Comparing to laser printers, 5 inkjet printers showed no particle emission. Particle emission level in a copy center increased slightly with TNC elevating to about 2 times of the aerosol background. Simulating test in a clean chamber indicated that printer-emitted particles were dominated by particles in nanoscale (diameter of particle, D(p) < 100 nm). These particles in a sealed clean chamber attenuated so slowly that it still held at high level with the concentration of 1.5 x 10(4) particles/cm3 after printing for 2.5 hours. Our present results demonstrate that printers indeed release particulates which keeping at a high concentration level in the indoor environment. Special care should be taken to this kind of widely applied machines and effective controls of particle emission at printing processes are necessary.

The environmental pollution associated with electrical discharge machining is not yet clearly understood. Airborne exposure to nanoscale and respirable particles were investigated with regard to the aerosol characteristics of a wire electrical discharge machining (WEDM) workshop. The total number concentration of the aerosol was multimodal, with the highest peak maxima during the working hours of 10:00 am and 3:00 pm. The majority of the released particles were smaller than d = 100 nm, with the maximum amount sized 40 nm. A large quantity of metallic elements, including Fe, Al and Cu, were found in the aerosol particulates coming from WEDM processing. Furthermore, the aerosol particles exhibited higher cellular toxicity and ROS producing ability in human alveolar epithelial cells (16HBE) when compared to the atmospheric background. Our results indicate substantial hazards arising from exposure to polluted atmosphere of a WEDM workshop. Effective exposure controls and protections are thus strongly recommended.

Perfluorooctane sulfonate (PFOS), a new kind of persistent organic pollutant, is widely distributed in the environment and exists in various organisms, where it is also a neurotoxic compound. However, the potential mechanism of its neurotoxicity is still unclear. To examine the role of epigenetics in the neurotoxicity induced by PFOS, SK-N-SH cells were treated with different concentrations of PFOS or control medium (0.1% DMSO) for 48 h. The mRNA levels of DNA methyltransferases (DNMTs) and Brain-derived neurotrophic factor (BDNF), microRNA-16, microRNA-22, and microRNA-30a-5p were detected by Quantitative PCR (QPCR). Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the protein levels of BDNF, and a western blot was applied to analyze the protein levels of DNMTs. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the BDNF promoter I and IV. Results of MTT assays indicated that treatment with PFOS could lead to a significant decrease of cell viability, and the treated cells became shrunk. In addition, PFOS exposure decreased the expression of BDNF at mRNA and protein levels, increased the expression of microRNA-16, microRNA-22, microRNA-30a-5p, and decreased the expression of DNMT1 at mRNA and protein levels, but increased the expression of DNMT3b at mRNA and protein levels. Our results also demonstrate that PFOS exposure changes the methylation status of BDNF promoter I and IV. The findings of the present study suggest that methylation regulation of BDNF gene promoter and increases of BDNF-related-microRNA might underlie the mechanisms of PFOS-induced neurotoxicity.

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, can cause neurotoxicity such as organophosphorus ester-induced delayed neuropathy (OPIDN) in humans and sensitive animals. Moreover, it also affects the development of central nervous system and differentiation of neuronal cells. In this study, retinoic acid-induced differentiated human neuroblastoma SH-SY5Y cells are utilized to investigate the effects of TOCP on neurite outgrowth and the underlying mechanisms. We found that low concentrations of TOCP induced autophagy and inhibited neurite outgrowth in a dose-dependent manner with no effect on cell viability. The protein levels of high molecular weight neurofilament (NF-H), low molecular weight neurofilament (NF-L) and β-tubulin also decreased. Pretreatment cells with 3-methyladenine (3-MA), an autophagy inhibitor, not only inhibited the TOCP-induced autophagy, but also reversed the inhibition of neurite outgrowth and the degradation of NF-H, NF-L, and β-tubulin by TOCP. Taken together, these results indicated that TOCP treatment induced autophagy in differentiated SH-SY5Y cells, which lead to degradation of cytoskeletal components and inhibition of neurite outgrowth.

Anticholinesterase pesticides have been widely used in agricultural and domestic settings and can be detected in the environment after long-term use. Although the acute toxic effects of chlorpyrifos and carbaryl have been well described, little is known about the chronic toxicity of the pesticides mixture. To investigate their chronic neurotoxicity, Wistar rats were exposed to chlorpyrifos, carbaryl, and their mixture (MIX) for 90 consecutive days. The activities of serum cholinesterase (ChE) as well as acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in nerve tissues were determined. Furthermore, the histopathological examination was carried out. The results showed that ChE activity significantly decreased in all treated rats except the rats treated with low dose carbaryl. Treatment with middle- and high-dose chlorpyrifos and MIX in rats significantly inhibited AChE activity in the central nervous tissues, whereas treatment with carbaryl alone did not. In sciatic nerve, AChE activity was significantly inhibited by high-dose carbaryl and MIX, but not by chlorpyrifos alone. No significant NTE inhibition was observed in all treatment groups. Histopathological examination revealed that both chlorpyrifos and MIX treatment induced hippocampal damage. However, no obvious hippocampal damage was found in carbaryl-treated rats. Carbaryl and MIX, but not chlorpyrifos alone, induced pathological damage of sciatic nerve. Taken together, all of the results indicated that chlorpyrifos and carbaryl have different toxicological target tissues in nervous system and showed corresponding effects in the nervous tissues, which may reflect the different sensitivity of central and peripheral nervous tissues to different pesticides individually and in combination.

Previous studies have demonstrated that the anticholinesterase pesticides chlorpyrifos and carbaryl are neurotoxic to mammals. However, the toxicity of these pesticides to other organs and their potential interactive effects remain unclear. Our goal in this study was to assess the toxicities of ingestion of chlorpyrifos and carbaryl both separately, and in combination to non-nervous systems, especially the effect on urinary metabolic profiles, in rats. Chlorpyrifos, carbaryl and a mixture of these pesticides, were administered orally to Wistar rats for 90 consecutive days. Histopathological examination of liver and kidney and metabonomic analysis based on the urinary 1 H nuclear magnetic resonance spectra were used to investigate the toxic effects. The results showed that no histopathological changes were observed in the liver or kidney tissues, but metabonomic analysis revealed alternations in a number of urinary metabolites involving in the energy metabolism in liver mitochondria. Treatment of rats with chlorpyrifos alone led to an increase in creatine, glycine, dimethylglycine, dimethylamine, glutamine, succinate, alanine, lactate, and glucose. The categories of main differential urinary metabolites in carbaryl-treated rats were similar to those in chlorpyrifos-treated rats, whereas the changes were of varying degree. A combination of a low dose of chlorpyrifos and carbaryl resulted in an increase in the levels of main urinary metabolites compared to the controls, and the increase in signal intensity of the main metabolites was lower than that in the rats exposed to chlorpyrifos or carbaryl alone. All above results suggest that chronic exposure to chlorpyrifos and carbaryl alone, or in combination could cause disturbance of metabolic function in liver mitochondria and renal failure. Overall, we have shown that urine metabonomic analysis is non-invasive, sensitive, and relatively fast for assessing the individual or mutual effects following exposure to pesticides.

As a phospholipase B, neuropathy target esterase (NTE) is responsible for the conversion of phosphatidylcholine (PC) to glycerophosphocholine (GPC). We examined the role of cAMP in the regulation of NTE in mammalian cells. Endogenous NTE activity was increased by cAMP-elevating chemicals, including dibutyryl cAMP, forskolin and forskolin plus 1-isobutyl-3-methylxanthine (IBMX), but decreased by the adenyl cyclase inhibitor SQ22536 which can reduce intracellular cAMP levels. Exogenous GFP-tagged NTE activity was not affected by changes in intracellular cAMP. NTE protein levels were up-regulated by the cAMP-elevating reagents and down-regulated by the inhibitor. The effect of the adenyl cyclase activator forskolin on NTE protein and mRNA levels was blocked by pretreatment with the protein kinase A (PKA) activity inhibitor H89. In addition, we found that changes in GPC, but not PC, levels were correlated with cAMP induced changes in NTE activity. These results are the first evidence that cAMP/PKA signals regulate NTE expression and GPC content in mammalian cells.

Neuropathy target esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G1 and lowest at G2/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.

Neuropathy target esterase (NTE) is proven to act as a lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. In sensitive species, organophosphate (OP)–induced delayed neurotoxicity is initiated when NTE is inhibited by > 70% and then aged. It is hypothesized that homeostasis of phosphatidylcholine (PC) and/or lysophosphatidylcholine (LPC) in mice might be disrupted by the OPs since NTE and other phospholipases could be inhibited. To test this hypothesis, we treated mice using tri-o-cresyl phosphate (TOCP), which can inhibit and age NTE. Phenylmethylsulfonyl fluoride (PMSF), which inhibits NTE but cannot age, was used as a negative control. Effects on activity of NTE, LysoPLA, and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE in the brain, spinal cord, and sciatic nerve were examined. The results showed that the activities of NTE, NTELysoPLA, LysoPLA, NTE-PLB, and PLB were significantly inhibited in both TOCP- and PMSF-treated mice, and the inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient. The NTE inhibited by TOCP was of the aged type, while nearly all NTE inhibited by PMSF was of the unaged type. Although the GPC level was remarkedly decreased, no significant change of PC and LPC levels was observed. However, the inhibition of these enzymes in mice by TOCP exhibited different characteristics from the TOCP-treated hens that we previously reported, which indicates that these enzymes were inhibited and then recovered more rapidly in mice than in hens. All results suggest that PC and LPC homeostasis was not disrupted in mice after exposure to TOCP. Differences in inhibition of NTE, LysoPLA, and PLB activities by TOCP between mice and hens may elucidate why these two species display different signs after exposure to the same neuropathic OPs.

Aims Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in humans and some sensitive animals. NTE was recently identified as a novel phospholipase B that is anchored to the cytoplasmic side of the endoplasmic reticulum. However, little is known about the degradation of NTE. In this study, we have investigated the role of the macroautophagic-lysosomal pathway in NTE degradation in neuronal and non-neuronal cells. Main methods Macroautophagy inhibitors and activators were used to interrupt the lysosomal pathway, and NTE protein level was followed using western blotting analysis. A fluorescent microscopy assay was used to determine the co-localization of NTE and lysosomes. Key findings Western blotting analysis showed that the macroautophagy inhibitors 3-methyladenine and ammonium chloride increased the levels of a heterologously expressed NTE-GFP fusion protein as well as endogenous NTE. Starvation had the opposite effect. The role of macroautophagy in NTE degradation was further supported by the co-localization of exogenous NTE with lysosomes in starved COS7 cells. Furthermore, the contribution of NTE activity and protein domains to the degradation of NTE by macroautophagy was investigated, showing that both the transmembrane and regulatory domains played a role in the degradation of NTE and that the catalytic domain, and thus NTE activity, was not involved. Significance Our findings clearly demonstrate, for the first time, that the macroautophagy/lysosome pathway plays a role in controlling NTE quantity, providing a further understanding of the function of NTE.

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.

It has been known that tri- ortho -cresyl phosphate (TOCP) can induce delayed neurotoxicity in humans and sensitive animal species; however, it also has influence on the developing central nervous system or differentiating neuronal cells. In this study, the effects of TOCP on cell proliferation and cell cycle regulation and the mechanisms that contribute to this effect were investigated by using human neuroblastoma SH-SY5Y cell line. Treatment of the cells with TOCP suppressed cell proliferation and reduced cell viability in a dose- and time-dependent manner. Analysis of cell cycle profile indicated that TOCP blocked cell cycle progression by arresting the cell cycle at G 1 phase. The data of determination of cell cycle regulated molecules at mRNA and protein levels showed that TOCP decreased cyclin D1 and increased p21 expression, while did not affect the p53 and p27 levels. Thus, these results indicated that TOCP might induce potential neurodevelopmental toxicity, and a possible mechanism of this toxicity might be the disturbance of cell proliferation by disrupting cell cycle regulatory proteins cyclin D1 and p21 expression.

Patatin-domain containing proteins constitute a large family of enzymes including known lipases that hydrolyze triglycerides, diglycerides, and phospholipids, some of which still remain to be characterized. One of those is NTE-related esterase (NRE), which exhibits sequence and domain homology to neuropathy target esterase (NTE). A splice variant of the catalytic domain of mouse NRE (mNRECV) was identified in multiple adult tissues, including brain, kidney, liver and testis. Genomic organization showed that mNRECV gene lacked the 22nd exon of mouse NRE and the 14th exon termination site of mNRECV was behind of 5 bp with the comparison of the 34th exon of mNRE gene. Over-expression of mNREC and mNRECV in mammalian cells showed that they had similar phenyl valerate esterase activities, but different from human NTE esterase domain. Subcellular distribution of an enhanced green fluorescent protein-mNRECV fusion protein was mainly observed to colocalize with endoplasmic reticulum in the juxtanuclear area and a little in cytoplasm. Moreover, autophagy/lysosome pathway was found to be involved in the degradation of mNRECV protein by inhibition and induction of autophagy, as well as co-location of mNRECV-EGFP with lysosomes. The high identity between mNRECV and mNREC suggested that mouse NRE has similar characteristics.

Neuropathy target esterase (NTE) is recognized as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN). Adult hens are usually used as the animal model for experimental studies of OPIDN. However, the molecular cloning and characteristics of chicken NTE is unknown. On the basis of the predicted chicken NTE gene middle sequence and its 3′-end cDNA sequence, we cloned the gene sequence of chicken NTE activity domain (cNEST). The cloned cNEST gene is 1740 base pairs and encodes 579 amino acids, showing high identity with human and mouse NEST at amino acid level. The serine hydrolase signature motif GXSXG and the patatin domain were found in the cNEST sequence. Over-expression of cNEST tagged with enhanced green fluorescence protein (EGFP) in monkey kidney COS7 cells increased NTE activity significantly. The increased extent is similar to that in over-expression of hNEST cells. Moreover, over-expression of cNEST led to an accumulation of partial cNEST on the cytoplasmic surface of the endoplasmic reticulum. Partial cNEST located in the cytoplasm by comparing the distribution of cNEST and hNEST. After inhibition with different concentrations of mipafox for 60 min, the calculated I 50 value was 4.95 μM for COS7 cells over-expressing cNEST. These results firstly confirmed that the protein sequence, enzymatic activity, and cellular location of cNEST are very similar to that of hNEST at molecular level. The inhibition curve of mipafox on NTE activity of cNEST in mammalian cells was also reported here.

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.

Concerns have arisen about the health and environmental impacts of the increasing commercial use of silver nanoparticles (AgNPs). However, the toxic mechanisms and target tissues of AgNPs have not been fully defined. In this paper, we investigated the tissue toxicity of mice after intravenous administration of AgNPs at a single-dose of 0.2, 2 or 5 mg per kg (body weight), respectively. Biodistribution, endoplasmic reticulum stress, and oxidative stress were examined in mouse organs at eight hours after exposure. Stress markers, e.g. HSP70, BIP, p-IRE1, p-PERK, chop and xbp-1s proteins/genes, were significantly upregulated in a dose-dependent manner. In the liver, spleen, lung and kidney, high stress accompanied by apoptosis occurred. Low stress levels were observed in the heart and brain. Thus, it is proposed that the liver, spleen, lung and kidney are dominant target tissues of AgNP exposure. The lower stress and toxicity in the heart and brain were in agreement with lower AgNP accumulation. The present results demonstrated that AgNP exposure eventually resulted in permanent toxic damage by gradually imposing stress impacts on target organs. These findings highlight the potent applications of stress markers in future risk evaluation of silver nanoparticle toxicity.

Silver nanoparticles (AgNPs) attract considerable public attention both for their antimicrobial properties and their potential adverse effects. In the present study, endoplasmic reticulum (ER) stress was used as a sensitive and early biomarker to evaluate the toxic potential of AgNPs in three different human cell lines in vitro and in vivo in mice. In 16HBE cells, the activation of ER stress signaling pathway was observed by upregulated expression including xbp-1s, chop/DDIT3, TRIB3, ADM2, BIP, Caspase-12, ASNS and HERP at either the mRNA and/or protein levels. However, these changes were not observed in HUVECs or HepG2 cells. Furthermore, mice experiments showed that different tissues had various sensitivities to AgNPs following intratracheal instillation exposure. The lung, liver and kidney showed significant ER stress responses, however, only the lung and kidney exhibited apoptosis by TUNEL assay. The artery and tracheal tissues had lower ER stress and apoptosis after exposure. The lowest observable effect concentrations (LOEC) were proposed based on evaluation of AgNP induced ER stress response in cell and mouse models. In summary, preliminary evaluation of AgNP toxicity by monitoring the ER stress signaling pathway provides new insights toward the understanding the biological impacts of AgNPs. The adverse effects of exposure to AgNPs may be avoided by rational use within the safe dose.

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, 1 H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β -oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.

Background: NMR combined with pattern recognition was recently introduced as a new technique for rapid xenobiotic toxicity evaluation. In this article, metabolic changes in the biofluid of rats after 90-day oral treatment with propoxur, permethrin and a combination of these two pesticides were investigated. Results: Propoxur dosing induced increased urinary taurine, creatinine and glucose, whereas urinary lactate and acetate were increased in the highest permethrin dose group. Urinary acetate, alanine, lactate and trimethylamine levels were increased in the mixture group, accompanied by decreased urinary tricarboxylic acid cycle intermediates. In addition, the highest dose of the mixture displayed raised 3-D-hydroxybutyrate, acetate and lactate levels in the serum sample. Conclusion: Chronic exposure to a combination of propoxur and permethrin may induce hepatotoxicity and nephrotoxicity. An increase in acetate, alanine and formate in the urine could be a potentially sensitive biomarker of the chronic, combined effects of permethrin and propoxur.

Metabonomic analysis, clinical chemical analysis and histopathology were used to investigate the toxic effects of subchronic exposure to dichlorvos, deltamethrin, and a combination of these two pesticides, in rats. Weight loss, hind limb weakness and histopathological changes in kidney tissue were only observed in rats exposed to high doses of deltamethrin, or a combination of deltamethrin and dichlorvos. Urinary metabonomic analysis indicated that exposure to a mixture of dichlorvos and deltamethrin was followed by increases in urinary lactate, dimethylamine, N-glycoprotein (NAC) and glycine similar to those observed in rats treated with either dichlorvos or deltamethrin alone. Serum metabonomic analysis suggests that dichlorvos induced an increase in lactate and alanine and a decrease in dimethylglycine (DMG), NAC and very low- and low-density lipoprotein (VLDL/LDL). High levels of lactate and low levels of NAC and VLDL/LDL were observed in the deltamethrin treatment group. Treating rats with a mixture of dichlorvos and deltamethrin caused an increase in serum lactate, trimethylamine-N-oxide (TMAO), choline and alanine, with the highest levels of these metabolites observed in those that received the highest dose. Exposure to a mixture of dichlorvos and deltamethrin also resulted in a decrease in serum acetone, DMG, NAC, and VLDL/LDL. Changes in serum TMAO, alanine, choline and acetone in this treatment group were higher than in rats treated with either dichlorvos or deltamethrin. These results suggest that exposing rats to subchronic doses of dichlorvos, deltamethrin, or a combination of these pesticides, disrupted the energy metabolism of the liver and reduced kidney function.

Anticholinesterase pesticides have been widely used in agricultural and domestic settings and can be detected in the environment after long-term use. Although the acute toxic effects of chlorpyrifos and carbaryl have been well described, little is known about the chronic toxicity of the pesticides mixture. To investigate their chronic neurotoxicity, Wistar rats were exposed to chlorpyrifos, carbaryl, and their mixture (MIX) for 90 consecutive days. The activities of serum cholinesterase (ChE) as well as acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in nerve tissues were determined. Furthermore, the histopathological examination was carried out. The results showed that ChE activity significantly decreased in all treated rats except the rats treated with low dose carbaryl. Treatment with middle- and high-dose chlorpyrifos and MIX in rats significantly inhibited AChE activity in the central nervous tissues, whereas treatment with carbaryl alone did not. In sciatic nerve, AChE activity was significantly inhibited by high-dose carbaryl and MIX, but not by chlorpyrifos alone. No significant NTE inhibition was observed in all treatment groups. Histopathological examination revealed that both chlorpyrifos and MIX treatment induced hippocampal damage. However, no obvious hippocampal damage was found in carbaryl-treated rats. Carbaryl and MIX, but not chlorpyrifos alone, induced pathological damage of sciatic nerve. Taken together, all of the results indicated that chlorpyrifos and carbaryl have different toxicological target tissues in nervous system and showed corresponding effects in the nervous tissues, which may reflect the different sensitivity of central and peripheral nervous tissues to different pesticides individually and in combination.

Carbamate insecticide propoxur is widely used in agriculture and public health programs. To prevent adverse health effects arising from exposure to this insecticide, sensitive methods for detection of early stage organismal changes are necessary. We present here an integrative metabonomic approach to investigate toxic effects of pesticide in experimental animals. Results showed that propoxur even at low dose levels can induce oxidative stress, impair liver function, enhance ketogenesis and fatty acid β-oxidation, and increase glycolysis, which contribute to the hepatotoxocity. These findings highlight the applicability of (1)H NMR spectroscopy and multivariate statistics in elucidating the toxic effects of propoxur.

NTE-related esterase (NRE) is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE), which plays a role in energy metabolism. Here, we reported two alternative splicing variants of the murine NRE (mNRE) gene, termed mNREV1 and mNREV2. Genomic organization analysis indicated that 5' splice site of mNRE intron 33 was changed in both mNREV1 and mNREV2, and mNRE exon 21 was deleted in mNREV2. mNREV1 had the same protein domains with mNRE, while mNREV2 lacked the patatin domain in the C-terminal catalytic region. Green fluorescent protein-mNREV1 or mNREV2 fusion proteins localized to the endoplasmic reticulum. mNREV1 and mNRE exhibited equal hydrolytic activity to the substrate phenyl valerate, whereas mNREV2 did not have any catalytic activity. The expression profiles of mNRE and its splicing isoforms in white adipose tissue, cardiac muscle, skeletal muscle, and testis tissues were further analyzed by real time quantitative-PCR in fed and fasted states, which indicated that the major isoform of mNRE mRNA generated switched from mNREV2 to mNREV1 during fasting. Thus there was a nutritional regulation of mNRE expression at the mRNA levels via alternative splicing.

NTE-related esterase (NRE) is a novel endoplasmic reticulum-anchored lysophospholipase with high homology to neuropathy target esterase (NTE). However, little is known about the regulation of NRE protein. In the current study, we investigated the degradation pathways of mouse NRE (mNRE) in mammalian cells. Based on experiments with inhibitors and inducer of protein degradation pathways, we provide here the first evidence that mNRE is degraded by macroautophagy as well as by the proteasome. Moreover, the contribution of protein domains to the degradation of mNRE was investigated, which showed that the transmembrane and regulatory domain played a role in the degradation of mNRE by macroautophagy and the proteasome respectively. In contrast the C-terminal catalytic domain was not involved in both degradation pathways of mNRE. These findings showed for the first time that the degradation pathways in controlling mNRE quantity and may provide further insight into structure and regulation of mNRE.

Neuropathy target esterase has been shown to be a lysophospholipase in mouse. The authors investigate the effect of neuropathy target esterase inhibition in mouse nervous tissues in vitro on the homeostasis of phosphatidylcholine and lysophosphatidylcholine by treating the homogenates with tri-ortho-cresyl phosphate, paraoxon, paraoxon plus mipafox, and phenylmethylsulfonyl fluoride. The activity of neuropathy target esterase is significantly inhibited by phenylmethylsulfonyl fluoride and paraoxon plus mipafox but not by paraoxon alone. Tri-ortho-cresyl phosphate slightly but significantly inhibits neuropathy target esterase activity in brain. The levels of phosphatidylcholine and lysophosphatidylcholine in all 3 nervous tissues are not obviously altered after treatment with tri-ortho-cresyl phosphate, paraoxon, or paraoxon plus mipafox. However, phosphatidylcholine and lysophosphatidylcholine levels are clearly enhanced by phenylmethylsulfonyl fluoride. It is concluded that inhibition of neuropathy target esterase in mouse nervous tissues is not enough to disrupt the homeostasis of phosphatidylcholine and lysophosphatidylcholine and that the upregulation by phenylmethylsulfonyl fluoride may be the consequence of combined inhibition of neuropathy target esterase and other phospholipases.

Avermectins (AVMs) are macrocyclic lactone compounds that have been widely used as parasiticides in veterinary and human medicine and as pesticides in agriculture and horticulture. The multidrug resistance transporter, P-glycoprotein (P-gp), is associated with the efflux transport of AVMs and other drugs across the blood-brain and placental barrier, and plays an important role in attenuating the neurotoxicity and developmental toxicity of AVMs. In this study, the mouse neuroblastoma N2a cell line was used to investigate the neurotoxicity of two AVM derivatives: abamectin (ABM) and doramectin (DOR). We found that both these compounds caused significant dose-dependent inhibition of neurite growth in differentiating N2a cells. In addition, Western blotting analysis showed that ABM and DOR significantly inhibited the expression of not only P-gp but also the cytoskeletal proteins, beta-actin and beta-tubulin. This suggests ABM and DOR may inhibit neurite growth by down-regulating the expression of P-gp and cytoskeletal proteins. Furthermore, knockdown of P-gp expression by RNA interference in N2a cells reduced neurite growth even in the absence of ABM and DOR, and reduced it even more in the presence of low levels of these compounds. These results suggest that even subcytotoxic levels of ABM and DOR can be neurotoxic in differentiating cells and that this neurotoxicity may, at least in part, be the result of the down-regulation of P-gp and cytoskeletal proteins.

Chlorpyrifos (CPF) and carbaryl (CAR) have been widely used in agricultural and domestic settings. Previous studies have demonstrated that CPF and CAR are generally neurotoxic to mammals, whereas the toxicities of these pesticides to other organs and their potential interactive effects remain unclear. The purpose of this study assessed the alterations of histopathology, biochemical parameters, and metabolic profiles of serum in rats following the treatment with CPF and CAR alone or in combination. No histopathological changes were observed in the liver and kidney tissues. Biochemical analysis of blood showed that alanine aminotransferase and total bilirubin in serum increased slightly in CPF-treated rats as compared to controls. Metabonomic analysis revealed alternations in a number of metabolites involving the metabolism of glucose, free fatty acids, and amino acids in liver mitochondria. The treatment of rats with CPF alone resulted in a decrease in lactate, low- and very low-density lipoprotein (LDL/VLDL), dimethylglycine (DMG), and aspartate. This was accompanied by an increase in isoleucine and leucine, 3-hydroxybutyrate (3-HB), N-acetylglycoprotein (NAC), acetone, succinate, glutamine, choline, creatine, glucose, and amino acids in a dose-dependent manner. Similarly, treatment with a high dose of CAR alone led to a decrease in DMG, aspartate, LDL/VLDL, and dimethylamine and an increase in taurine, glucose, and amino acids. The levels of lactate and LDL/VLDL decreased, while those of 3-HB, NAC, acetone, succinate, and glutamine elevated in the group of rats treated with a mixture of CPF and CAR as compared to the groups of CPF or CAR alone. Our results suggest that subchronic exposure to CPF and CAR alone, or in combination, could cause a disturbance in energy and fatty acid metabolism in the liver mitochondria of rats. Overall, we have shown that analysis of metabolic profiles can make exceptional contributions to the understanding of the individual or mutual effects following exposure to a low dose of pesticides.

A mammalian family of lipid hydrolases, designated “patatin-like phospholipase domain containing (PNPLA)” recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) and adult hens are the animal model of OPIDN. However, little has been known about the sequence and characteristics of chicken NTE. Here, we firstly identified the full length cDNA of chicken NTE (cNTE), which contained an open reading frame of 3966 nucleotides encoding 1321 amino acids. Chicken NTE had two distinct regions, one was the regulatory domain (cNTER) and the other was the catalytic domain (cNEST). Over-expression of cNTER in mammalian cells did not show any NTE activity, whereas cNEST had NTE activity. Cells expressing cNTER tagged with green fluorescent protein (GFP) showed accumulation of cNTER-GFP in an endoplasmic reticulum-like localization pattern. In addition, macroautophagy and the proteasome pathways were found to be involved in the degradation of cNTER, but not cNEST. These results first showed that cNTE was an ER-anchored protein and degraded by macroautophagy as well as the proteasome.

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca 2+ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca 2+ level ([Ca 2+ ] i ) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca 2+ channel blocker, did not affect the LPC-induced Ca 2+ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca 2+ -free medium, LPC produced a significant [Ca 2+ ] i decrease in N2a cells, while it caused 64% of total [Ca 2+ ] i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca 2+ ] i increase was produced in each cell line through different mechanisms. In particular, the [Ca 2+ ] i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca 2+ channels as well as by Ca 2+ release from intracellular Ca 2+ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca 2+ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.

NTE-related esterase (NRE), conserved in mouse, rat and human, was a member of patatin-like phospholipases (PLPLA) with high homology to neuropathy target esterase (NTE). Little has been known about the characteristics of NRE and NRE functional esterase activity has yet not been defined. The C-terminal gene sequence of mouse NRE (mNREC) encoding 923-1,326 amino acid containing the patatin domain was first cloned and then expressed tagged with enhanced green fluorescence protein (EGFP) in mammalian cells. The results showed that mNREC had NTE esterase activity in mammalian cells. Overexpression of mNREC did not affect the esterase activity sensitive to paraoxon or resistant to both paraoxon and mipafox. mNREC was distributed in the cytoplasm in contrast to the distribution of human NTE esterase domain. The expression analysis of NRE gene in adult mouse tissues by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that there were higher levels of NRE mRNA in the brain and testis than in the liver and kidney, which was about 50% and 35% of that in the brain. These results firstly showed the tissue distribution of NRE gene in adult mouse and defined that NRE had functional esterase activity.

Neuropathy target esterase (NTE) was originally identified as the primary target site of those organophosphorus compounds that induce delayed neuropathy in human and some animals. Here we examined the role of protein kinase C (PKC) in the regulation of the NTE activity in mammalian cells. Six-hour exposure of human neuroblastoma SK-N-SH cell to a PKC activator phorbol 12-myristate 13-acetate (PMA) decreased the activity of NTE, and this effect was blocked by the PKC inhibitor staurosporine. These results suggest that PKC down-regulates the activity of NTE. NTE protein levels were down-regulated by PMA-stimulation as detected by Western blot analysis using the NTE-specific antibody, which resulted from down-regulation of NTE mRNA level as verified by real-time reverse transcription polymerase chain reaction (RT-PCR). However, there were no changes in the activity or protein levels of stable expression of NTE esterase activity domain (NEST) in SK-N-SH cells and transient expression of full-length NTE construct in COS7 cells driven by cytomegalovirus (CMV) promoter rather than by the cell's own one, despite the absence or presence of PMA stimulation. Together, these findings suggest that stimulation with PMA reduces the expression of NTE mRNA levels but does not affect the exogenous promoter-driven NTE expression in mammalian cells.