•Mixtures of propoxur (PR) and permethrin (PE) induced oxidative stress in rat liver.•Lipids were sensitive to the oxidative damage by PR and PE.•PR and PE displayed antagonistic interaction on the induction of lipid peroxidation.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.

Pesticides and heavy metals are widespread environmental pollutants. Although the acute toxicity of organophosphorus pesticide chlorpyrifos (CPF) and toxic heavy metal cadmium (Cd) is well characterized, the combined toxicity of CPF and Cd, especially the hepatotoxicity of the two chemicals with long-term exposure at a low dose, remained unclear. In this study, we investigated the toxicity in the liver of rats upon subchronic exposure to CPF and Cd at environmentally relevant doses. Rats were given three different doses (1/135 LD50, 1/45 LD50 and 1/15 LD50) of CPF and Cd as well as their mixtures by oral gavage for 90 days. After treatment, the liver tissues were subjected to histopathological examination and biochemical analysis. Gas chromatography-mass spectrometry (GC–MS) was used to analyze the metabolomic changes in the rat liver upon CPF, Cd and their mixtures treatment. The results showed that CPF and Cd-induced oxidative damage and disrupted energy, amino acid, and fatty acid metabolism in the liver. Eleven biomarkers in liver were identified for CPF-, Cd-, and their mixture-treated rats. Three metabolites, i.e., butanedioic acid, myo-inositol, and urea, were identified as unique biomarkers for the mixture-treated rats. Moreover, we found that Cd could accelerate the metabolism of CPF in the liver when given together to the rats, which may lead to the potential antagonistic interaction between CPF and Cd. In conclusion, our results indicated that even at environmentally relevant doses, CPF and Cd could disrupt the liver metabolism. In addition, the accelerated metabolism of CPF by Cd may lead to their potential antagonistic interaction.

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.

Cadmium (Cd) and chlorpyrifos (CPF) are widespread harmful environmental pollutants with neurotoxicity to mammals. Although the exposure to Cd and CPF at the same time may pose a significant risk to human health, the subchronic combined neurotoxicity of these two chemicals at low levels in the brain is poorly understood. In this study, we treated rats with three doses (low, middle, and high) of Cd, CPF, or their mixture for 90 days. No obvious symptom was observed in the treated animals except those treated with high-dose CPF. Histological results showed that middle and high doses of the chemicals caused neuronal cell damage in brains. GC–MS-based metabonomics analysis revealed that energy and amino acid metabolism were disturbed in the brains of rats exposed to the two chemicals and their combinations even at low doses. We further identified the unique brain metabolite biomarkers for rats treated with Cd, CPF, or both. Two amino acids, tyrosine and l-leucine, were identified as the biomarkers for Cd and CPF treatment, respectively. In addition, a set of five unique biomarkers (1,2-propanediol-1-phosphate, d-gluconic acid, 9H-purine, serine, and 2-ketoisovaleric acid) was identified for the mixtures of Cd and CPF. Therefore, the metabolomics analysis is more sensitive than regular clinical observation and pathological examination for detecting the neurotoxicity of the individual and combined Cd and CPF at low levels. Overall, these results identified the unique biomarkers for Cd and CPF exposure, which provide new insights into the mechanism of their joint toxicity.

To effectively biodegrade organophosphorus pesticides residue in environment, we constructed a genetically engineered bacterium (GEB) which can not only emit red fluorescence but also degrade organophosphorus pesticides residue, and this GEB can commit suicide when required. Two genes with different functions were placed under the control of different promoters. One was the dual gene expression vector pL-DsRed–pL-OPH in which genes coding for DsRed and organophosphorus hydrolase were independently placed downstream of two pL promoters. These genes could be expressed freely as long as the GEB was alive. The other was the conditional suicide plasmid pDS containing two suicide cassettes designed to induce bacteria to commit suicide when they detect arabinose. The lethal gene used in the suicide plasmid was the nuclease gene of Serratia marcescens without the leader-coding sequence. This was put under the control of the T7 promoter. Applying this type of secure GEB could potentially be a less hazardous environmental strategy in degrading pesticides and contamination.

Tri-ortho-cresyl phosphate (TOCP) is an organophosphorus ester and has been widely used in industry. It is found that TOCP induced delayed neurotoxicity in humans and sensitive animal species. However, the mechanism of TOCP-induced neural cytotoxicity remains unclear. In this study, we studied whether autophagy is involved in TOCP-induced neural cytotoxicity in human neuroblastoma SH-SY5Y cells. We found that 0.5 and 1.0 mM TOCP treatment significantly increased the ectopic accumulation of microtubule-associated protein 1 light chain 3 (LC3)-immunopositive puncta, Beclin 1, and LC3-II/LC3-I levels in SH-SY5Y cells in a dose-dependent manner. Notably, by monodansylcadaverine staining method, we found abundant punctate fluorescent acidic vesicular organelles in TOCP-treated cells. Furthermore, ultrastructural observation under the transmission electron microscope indicated that the cytoplasm was occupied by autophagosomes in TOCP-treated SH-SY5Y cells. Thus, these results suggest that TOCP may induce autophagy, and autophagy may be involved in the development of TOCP-induced neural cytotoxicity.

Recently members of mammalian patatin-like phospholipase domain containing (PNPLA) protein family have attracted attention for their critical roles in diverse aspects of lipid metabolism and signal pathway. Until now little has been known about the characteristics of PNPLA1. Here, the full length coding cDNA sequence of human PNPLA1 (hPNPLA1) was cloned for the first time, which encoded a polypeptide with 532 amino acids containing the whole patatin domain. Tissue expression profiles analysis showed that low mRNA levels of hPNPLA1 existed in various tissues, except high expression in the digestive system, bone marrow and spleen. Subcellular distribution of hPNPLA1 tagged with green fluorescence protein mainly localized to lipid droplets. Furthermore, a nonsense mutation of PNPLA1 in human cervical cancer HeLa cells was identified. The hPNPLA1 mutant encoded a protein of 412 amino acids without the C-terminal domain and did not colocalize to lipid droplets, which suggested that the C-terminal region of hPNPLA1 affected lipid droplet binding. These results identified hPNPLA1 and a mutant in HeLa cells, and provided insights into the structure and function of PNPLA1.

The subacute toxic effects of 28 days of exposure to three dosages (250, 500, 1000 mg/kg/day) of melamine on Wistar rats were investigated using nuclear magnetic resonance spectra, histopathological examination, and biochemical analysis. Rats treated with melamine developed adverse health effects compared to the controls, including decrease in body weight and kidney damage. Blood biochemical analysis showed that the blood urea nitrogen and creatinine increased distinctly compared to the control group. Urinary metabonomic analysis indicated that melamine caused an increase in succinate and citrate. Serum metabonomic analysis showed that the lowest dose led to an increase in dimethylglycine, N-acetylglycoprotein (NAC), accompanied by a decrease in taurine and glucose. Rats treated with the highest dose developed high levels of serum choline and 3-hydroxybutyrate (3-HB) together with low lactate levels. Metabonomic analysis of liver tissue indicated that melamine caused an increase in NAC, choline, and creatine, accompanied by a decrease in lactate, trimethylamine-N-oxide, glutamate, and glucose. All three dosages resulted in an increase in glutamate, lactate, choline, glucose, and animo acids and a decrease in 3-HB and pyruvate in aqueous kidney extract. These results indicate that melamine not only caused renal disfunction but also disturbed the liver’s glucose, protein, and nitrogen metabolism.

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.

One way to reduce the potential risk of genetically engineered microorganisms (GEMs) to the environment is to use a containment system that does not interfere with the performance of the GEM until activated. Such a system can be created by inserting a suicide cassette consisting of a toxin-encoding gene controlled by an inducible promoter. We constructed a GEM that can degrade organophosphorus compounds, emit green fluorescence, and commit suicide when required by putting the genes that control these different functions under different promoters. The genes for enhanced green fluorescent protein (EGFP) and organophosphorus hydrolase (OPH) were cloned downstream of the lambda PL promoter in the plasmid pBV220. These genes could be expressed freely as long as the GEM was metabolizing because the repressor sequence cIts857 had been deleted. The extracellular nuclease gene of Serratia marcescens, without its leader-coding sequence, provided the suicide mechanism. This was put under the control of the T7 promoter to form a suicide cassette activated by the presence of an environmental signal, in this case, arabinose. To improve the reliability of this containment system, the suicide cassette was duplicated within the conditional suicide plasmid. The plasmid carrying the EGFP and OPH fusion genes and that containing the suicide cassette were compatible and coexisted in the same host.

A relatively simple method for the determination of azoxystrobin residues in grapes and soil using gas chromatography equipped with electron capture detector (GC-ECD) is described. Samples were extracted with acetone, and further partitioned with dichloromethane and petroleum ether. The extracts were then cleaned up in a glass clean-up column filled with active charcoal and silica gel, and eluted with dichloromethane/ethyl acetate (70:30, v/v). The eluate was collected and concentrated for GC-ECD analysis. The results showed good linearity (r2 = 0.9998) over the concentration range of 6.25–400 ng mL−1. The limits of detection (LOD) and quantification (LOQ) of azoxystrobin were 3 and 10 ng mL−1. Recovery from soil and grape samples was in the range of 83.52–107.36 and 82.21–107.31%, with corresponding relative standard deviations (RSD) of 5.21–9.11 and 4.53–5.90% for the three fortified levels. Inter- and intra-day RSDs were in the range of 0.87–6.76 and 2.01–5.46%. The accuracy and sensitivity of the GC-ECD method was independently confirmed by LC and GC-MS. It was demonstrated that the proposed method was simple and efficient, and particularly suitable for detecting azoxystrobin residues in grapes and soil.

Genetically engineered Escherichia coli, expressing the fusion protein of enhanced green fluorescent protein (EGFP) and carboxylesterase B1 (CarE B1), was successfully constructed by cloning the genes into the pET-28b vector and then transforming E. coli BL21 (DE3). Expression of the fusion protein was induced in E. coli BL21 (DE3) which could then degrade environmental pesticides and could be easily detected using fluorescence spectrophotometry or by the naked eye in daylight.

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.

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.Download full-size image

•Subchronic combined toxicity of dichlorvos (DDVP) and deltamethrin (DM) at low-levels was studied.•Oxidative stress was induced by DDVP, DM and their mixtures in rat liver.•Lipids were particularly sensitive to the oxidative damage by DDVP and DM.•The combined effect of DDVP and DM was similar to their individual effect on induction of oxidative stress.Organophosphates and pyrethroids are widely used pesticides with prominent toxicity to humans. However, their joint toxicity has not been thoroughly investigated. In this study, we investigated the oxidative damages induced by low dose dichlorvos (DDVP) and deltamethrin (DM), the representative organophosphate and pyrethroid, respectively, and their mixtures in the liver of rats for 90 consecutive days. Two oxidative stress markers, malondialdehyde (MDA) and protein carbonyl (PCO) levels, were measured to reflect the extent of lipid peroxidation and protein oxidation, respectively. DDVP, DM, and their mixtures induced levels of MDA and PCO dose-dependently, although no toxic signs and pathological changes of liver were found in the rats following 90-day exposure. DDVP and DM induced greater increase of MDA than PCO, which indicated that lipids were particularly sensitive to the oxidative damage. We found that DDVP, DM and their mixtures could inhibit the activity of two antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The effects of DM on SOD activity, lipid peroxidation and protein oxidation were greater than those of DDVP. The combined effect of DDVP and DM was lower than the sum of their individual effects. Thus the interaction between dichlorvos and deltamethrin may be antagonistic on the induction of oxidative stress in rat liver.Download full-size image

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 1H 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.Graphical abstractDownload full-size imageHighlights► This study was concerned with non-nervous system toxicities of pesticides. ► The combined effects of low dose of pesticides were assessed. ► Metabonomic analysis based on NMR spectra was used to evaluate the toxic effects. ► Chlorpyrifos and carbaryl cause disturbance of metabolic function in rats.

•Chlorpyrifos (CPF) and cadmium (Cd) induced oxidative damage in the rat brain.•Lipids and protein were sensitive to the oxidative damage of CPF and Cd.•The mixture of CPF and Cd had an antagonistic effect on lipid and protein oxidation.Pesticides and heavy metals can be easily biomagnified in food chains and bioaccumulated in individuals, thus pose significant threat to human health. However, their joint toxicity for long-term exposure at low dose has not been thoroughly investigated. In the present study, we investigated the oxidative damages in brain of rats exposed subchronically to organophosphorus pesticide chlorpyrifos (CPF) and heavy metal cadmium (Cd), and their mixtures at the environmentally relevant doses. Rats were given different doses of CPF and Cd by oral gavage for three months. After treatment, brain tissues were subjected for biochemical analysis. Mitochondrial damage and reactive oxidative species were also measured in neuroblastoma SH-SY5Y cells treated with CPF, Cd and their mixtures. The results showed that CPF and Cd generated protein and lipid peroxidation, disturbed the total antioxidant capability, and altered mitochondria ultrastructure in the brain. Lipids and proteins were sensitive to the oxidative damage induced by CPF and Cd. CPF and Cd decreased mitochondrial potential and induced reactive oxygen species in SH-SY5Y cells. However, the mixture did not display higher toxicity than the sum of that of the individual treatments. Thus, CPF and Cd could have a potential antagonistic interaction on the induction of oxidative stress.Download high-res image (226KB)Download full-size image

The effects of methamidophos and tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ-32P]ATP as phosphate donor. Tri-o-cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.

The residual level and dissipation rate of triazophos in wheat crops and the soil in which they were grown were determined by gas chromatography (GC). Maximum final residues of triazophos in wheat grain, stems and leaves, and soil were 1.865, 44.506, and 0.973 mg/kg, respectively. The mean half-life of triazophos in wheat plants (grain, stems, and leaves) was 5.22 days with a dissipation rate of 90% over 14 days. The half-life in soil was 7.93 days with a dissipation rate of 90% over 21 days. Dissipation rates in two geographically separated experimental fields differed, suggesting that this was affected by local soil characteristics and climate. Although residual levels of triazophos in wheat plants may pose risks to the health of humans and other animals, comparatively low residues in soil suggest that this pesticide may be otherwise environmentally safe.

•A direct relationship between P-glycoprotein (P-gp) and abamectin (ABM) resistance in Drosophila was observed.•High-level P-gp expression was found in blood–brain barrier of the ABM-resistant fly.•The ABM-induced P-gp expression level was increased via the dEGFR/dAkt pathways in ABM-resistant fly.Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC50 values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood–brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.Download high-res image (55KB)Download full-size image

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.

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.

In this study, we investigated whether two brain regions, the bed nucleus of the stria terminalis (BNST) and the basolateral amygdala (BLA), affected male rats’ (Rattus norvigicus) ability to innately discriminate between a predator odor (cat urine) and female rat urine. Muscimol, a GABAa receptor agonist, was bilaterally microinjected into either the BNST or BLA of rats through implanted stainless-steel guide cannulas to temporarily inactivate these brain nuclei. The behavioral responses of the treated rats to female rat urine and cat urine were then tested in an experimental arena. Compared to a saline infusion control, the injection of muscimol into the BNST strongly reversed the innate aversion of rats to cat urine but the injection of muscimol into the BLA had no effect. Furthermore, intra-BNST infusion of muscimol caused rats to be equally attracted to urine from cats and female rats but intra-BLA infusion did not stop rats manifesting fear on exposure to cat urine and exploratory behavior on exposure to female rat urine. We conclude that the BNST plays a more crucial role in modulating innate fear responses in rats than the BLA.Graphical abstractDownload high-res image (52KB)Download full-size imageHighlight► Cat urine odor induces fear behavior in rats. ► Injection muscimol into the BNST blocks the freezing induced by cat urine odor. ► The BNST, not BLA, is essential for innate fear responses in rats.

•Metabonomics was used to analyze the biofluids of subchronic pyrethroids-treated rats.•The two types of pyrethroids show similar metabolic profiling of the urine and serum.•Similar metabolic pathways in the rats are perturbed by the two types of pyrethroids.•The pyrethroids induced disturbance of the energy metabolism and lipid peroxidation.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, 1H 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.

•Avermectin (AVM) induced upregulation of P-glycoprotein (P-gp) level in S2 cells.•Avermectin caused elevation of intracellular calcium and chloride levels.•The increase of [Ca2+]i and [Cl−]i correlated with the AVM-induced P-gp expression.•Avermectin upregulated the expression level of Relish (NF-κB) protein.•Avermectin induced P-gp expression level through CaM/NF-κB pathway.Avermectin (AVM) is a macrocyclic lactone agent widely used as a nematicide, acaricide and insecticide in veterinary medicine and plant protection. P-glycoprotein (P-gp) is an ATP-dependent drug efflux pump for xenobiotic compounds, and is involved in multidrug resistance. To understand the development of AVM resistance in invertebrates, we investigated the mechanisms by which AVM affected P-gp expression in Drosophila S2 cells. We found that AVM induced upregulation of P-gp protein expression, increased P-gp ATPase activity and enhanced cellular efflux of the P-gp substrate rhodamine 123 from cells. Furthermore, we observed that AVM-induced expression of P-gp was due to elevation of intracellular calcium concentration ([Ca2+]i). This occurred both directly, by activating calcium ion channels, and indirectly, by activating chloride ion channels. These results are supported by our observations that verapamil, a Ca2+ channel blocker, and niflumic acid, a chloride channel antagonist, significantly attenuated AVM-induced [Ca2+]i elevation, thereby reducing P-gp expression. Inhibition of P-gp with anti-P-gp antibody or cyclosporine A (a P-gp inhibitor) reduced the AVM-induced elevation of [Ca2+]i, implying that P-gp and [Ca2+]i regulate each other. Finally, we found that trifluoperazine, a calmodulin inhibitor, and pyrrolidine dithiocarbamic acid, an NF-κB inhibitor, attenuated the AVM-induced expression of P-gp, suggesting that AVM induces P-gp protein expression via the calmodulin/Relish (NF-κB) signaling pathway.Download full-size image

Lysophosphatidylcholine (LPC), an important compound in the immune system, regulates a variety of biological processes. We examined and compared the effect of exogenous LPC on intracellular Ca2+ overload in human Jurkat CD4+ T lymphocytes and mouse CTLL-2 CD8+ T lymphocytes. LPC caused a dose-dependent intracellular Ca2+ level ([Ca2+]i) increase in both Jurkat and CTLL-2 lymphocytes. Pretreatment of cells for 5 min with 30 μM of ruthenium red, a potent ryanodine receptor inhibitor, reduced the LPC-induced Ca2+ response in both Jurkat and CTLL-2 T lymphocytes. Moreover, pretreatment of cells with 100 μM 2-APB for 15 min, a cell-permanent IP3 receptor inhibitor, reduced about two thirds of the LPC induced calcium response in both kinds of cells. However, preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, did not affect the LPC-induced [Ca2+]i increase in CTLL-2 lymphocytes but inhibited this in Jurkat lymphocytes by 26%. In Ca2+-free medium, LPC produced 75.8% of the total [Ca2+]i increase in CTLL-2 lymphocytes and 38% of the total [Ca2+]i increase in Jurkat lymphocytes. These data suggested that the LPC-induced [Ca2+]i increase in human Jurkat and mouse CTLL-2 cell lines occurs via different pathways.

AimsLysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. LPC could be deacylated to form glycerophosphocholine by neuropathy target esterase (NTE)/Swiss cheese protein (SWS). Although NTE/SWS is important in maintaining Ca2 + homeostasis, the role of LPC in regulating the intracellular calcium concentration ([Ca2 +]i) in Drosophila remains poorly understood. We aimed to study the mechanism of LPC-induced [Ca2 +]i changes in Drosophila S2 cells.Main methodsThe [Ca2 +]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM.Key findingsOur results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca2 +]i in the presence of external calcium ([Ca2 +]e). The LPC-induced [Ca2 +]i increase was reduced by 60.7% in the absence of [Ca2 +]e. Furthermore, the Ca2 + influx was inhibited by 37.3% after the cells were preincubated with an L-type Ca2 + channel blocker. In the Ca2 +-free medium, the LPC-induced [Ca2 +]i increase was completely blocked using an inositol triphosphate receptor (IP3R) inhibitor. However, a ryanodine receptor (RyR) inhibitor had no effect on the LPC-induced [Ca2 +]i increase.SignificanceThe LPC-induced [Ca2 +]i increase in S2 cells was dependent on both the release of Ca2 + stored in the endoplasmic reticulum and [Ca2 +]e influx. Both L-type Ca2 + channels and IP3R might be involved in this process. The LPC-induced [Ca2 +]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC.

This study investigated the role of the endoplasmic reticulum pathway in apoptosis induced by trichlorfon in SH-SY5Y human neuroblastoma cells. Flow cytometric analysis demonstrated that trichlorfon and its degradation product dichlorvos-induced apoptosis in a dose-dependent manner and Hoechst 33342 staining experiments revealed trichlorfon/dichlorvos-induced nucleus condensation. Western blot analysis indicated decreased expression of caspase-12 and increased activated caspase-12 in trichlorfon-treated cells compared to a control, suggesting that trichlorfon may induce apoptosis in SH-SY5Y partly via the endoplasmic reticulum. Intracellular Ca2+ level ([Ca2+]i) in SH-SY5Y cells increased after treatment with trichlorfon but was significantly reduced by pre-treatment with a combination of a calcium channel blocker, an inositol trisphosphate receptor inhibitor, and a ryanodine receptor inhibitor. Percent apoptosis and activated caspase-3 and caspase-12 decreased in pre-treated cells compared to those treated with trichlorfon alone. Trichlorfon-induced apoptosis was also inhibited by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). These results suggest that endoplasmic reticulum stress, which is related to calcium, may be involved in the cytotoxicity of trichlorfon.

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 G1 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.

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 I50 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.

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, β-actin and β-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.