Yi-Jun Wu

Metabolic profiles of serum from rats after subchronic exposure to chlorpyrifos and carbaryl.

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.

An NMR-based metabonomic investigation of the subacute effects of melamine in rats.

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 livers glucose, protein, and nitrogen metabolism.

Use of graphitic carbon black and primary secondary amine for determination of 17 organophosphorus pesticide residues in spinach.

Graphitized carbon black (GCB) and primary secondary amine (PSA) as dispersive-SPE sorbents were applied to optimize the method for the determination of 17 organophosphorus pesticides in spinach which contained so many pigments using GC with flame photometric detector (FPD). The sample was extracted with ACN, and an aliquot of the extract was concentrated to near dryness. Ethyl acetate or acetone was chosen as the dissolving solvent. Subsequently, dispersive-SPE was used for cleanup, and the type and quantity of sorbents (GCB, PSA and activated carbon) were tested in the experiments. The best results were when acetone was used to dissolve and 30 mg each of GCB and PSA for cleanup. In this condition, recoveries of pesticides analyzed were between 52-117% with RSD below 10%, and LOQ ranged from 10 to 20 microg/kg. This method was simple, effective and efficient, and can protect the GC system to some extent.

Induction of autophagy by TOCP in differentiated human neuroblastoma cells lead to degradation of cytoskeletal components and inhibition of neurite outgrowth.

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.

Avermectin induces P-glycoprotein expression in S2 cells via the calcium/calmodulin/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 ([Ca(2+)](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 Ca(2+) channel blocker, and niflumic acid, a chloride channel antagonist, significantly attenuated AVM-induced [Ca(2+)](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 [Ca(2+)](i), implying that P-gp and [Ca(2+)](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.

Effects of avermectins on neurite outgrowth in differentiating mouse neuroblastoma N2a cells.

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.

Combined subchronic toxicity of dichlorvos with malathion or pirimicarb in mice liver and serum: a metabonomic study.

Organophosphorus (OP) and carbamate (CM) pesticides are widely used in agriculture. These pesticides are highly toxic to humans and their residues in food pose potential threat to human health. In this study, we investigated the effect of subchronic low-level exposure of OPs (dichlorvos, DDVP; malathion, MAL), CM pirimicarb (PI), or their mixtures (DDVP+MAL, DDVP+PI) on mice liver. Metabonomic analysis based on (1)H nuclear magnetic resonance spectroscopy was carried out in combination with biochemical assays. Serum metabonomic analysis showed that levels of trimethylamine-N-oxide, lactate, acetone, very low- and low-density lipoprotein and 3-hydroxybutyrate changed after exposure to the pesticides. In the liver extracts, lactate, glucose, choline, glutathione, alanine, glutamine and isoleucine levels changed after the treatment by pesticides. Our results indicated that exposure to low dose DDVP, MAL and PI, either alone or in combination lead to alteration of liver glucose, fat and protein metabolism, energy metabolism and oxidative balance. This study also showed that metabonomics is of potential use in food toxicity study.

Dissipation and residue of dimethomorph in pepper and soil under field conditions

The dissipation and residual levels of dimethomorph in pepper and soil under field conditions were determined by gas chromatography with an electron capture detector (GC-ECD). The dissipation rates of dimethomorph were described using first-order kinetics and its half-life ranged from 1.7 to 3.8 days in pepper and 11.5-18.5 days in soil. At harvest time, the terminal residues of dimethomorph were below the EUs maximum residue limit (MRL, 0.5 mg kg⁻¹) in pepper when measured 7 days after the final application, which suggested that the use of this fungicide was safe for humans. The collected field samples were stable for up to two months when refrigerated at -20°C. The residues persistence varied among three geographically separated experimental fields, suggesting that it might be affected by climatic, soil properties and local microorganisms. These results will be helpful in setting MRL guidance for dimethomorph in pepper in China.

Neuropathy target esterase: an essential enzyme for neural development and axonal maintenance.

Neuropathy target esterase (NTE) is an endoplasmic reticulum-anchored protein conserved across species. The N-terminal regulatory region of NTE contains three cyclic nucleotide binding domains while the C-terminal catalytic domain has a patatin domain. The NTE gene is expressed in mouse early at embryonic day 7 and its expression is maintained throughout embryonic development. NTE protein is mainly distributed in the nervous system with a pattern that is more restricted to large neurons in older animals. NTE regulates phospholipid metabolism and is known to be a phospholipase B. Knockout of NTE is embryonic lethal in mice, indicating that NTE is essential for embryonic survival. Neuronal specific NTE knockouts survive to adulthood, but show vacuolation and neuronal loss characteristic of neurodegenerative diseases. Recently, mutations in human NTE have been shown to cause a hereditary spastic paraplegia called NTE-related motor neuron disorder, suggesting a critical role for NTE in the nervous system.

Joint toxicity of chlorpyrifos and cadmium on the oxidative stress and mitochondrial damage in neuronal cells.

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.