Ding-Xin Long

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.

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.

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.

The homeostasis of phosphatidylcholine and lysophosphatidylcholine was not disrupted during tri-o-cresyl phosphate-induced delayed neurotoxicity in hens

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.

Subchronic neurotoxicity of chlorpyrifos, carbaryl, and their combination in rats

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.

1H NMR-based metabonomic analysis of the serum and urine of rats following subchronic exposure to dichlorvos, deltamethrin, or a combination of these two pesticides.

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.

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

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.

Growth inhibition and induction of G1 phase cell cycle arrest in neuroblastoma SH-SY5Y cell by tri-ortho-cresyl phosphate

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.

Applying biofluid metabonomic techniques to analyze the combined subchronic toxicity of propoxur and permethrin in rats

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.

1H NMR-based metabonomic profiling of rat serum and urine to characterize the subacute effects of carbamate insecticide propoxur

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 1H NMR spectroscopy and multivariate statistics in elucidating the toxic effects of propoxur.