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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

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.

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.

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 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.

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.

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.