Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue-derived cells in vitro.

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

Individual and combined hepatocytotoxicity of DDT and cadmium in vitro.

The organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) and heavy metal cadmium (Cd) are widespread environmental pollutants. They are persistent in the environment and can accumulate in organisms. Although the individual toxicity of DDT and Cd has been well documented, their combined toxicity is still not clear. Since liver is their common target, in this study, the individual and combined toxicity of DDT and Cd in human liver carcinoma HepG2 and human normal liver THLE-3 cell lines were investigated. The results showed that DDT and Cd inhibited the viability of HepG2 and THLE-3 cells dose-dependently and altered lysosomal morphology and function. Intracellular reactive oxygen species and lipid peroxidation levels were induced by DDT and Cd treatment. The combined cytotoxicity of DDT and Cd was greater than their individual cytotoxicity, and the interaction between Cd and DDT was additive on the inhibition of cell viability and lysosomal function of HepG2 cells. The interaction was antagonistic on the inhibition of cell viability of THLE-3 cells. These results may facilitate the evaluation of the cumulative risk of pesticides and heavy metal residues in the environment.

Lapatinib alleviates TOCP-induced axonal damage in the spinal cord of mouse.

Neuregulin-1 (NRG1), a family of EGF-like factors that activates ErbB receptors, can regulate the proliferation, migration, and myelinating of Schwann cells. We previously reported that NRG1/ErbB signal is responsible for organophosphate (OP)-induced delayed neuropathy (OPIDN) in hens, a susceptive animal model to neuropathic organophosphorous compounds. Our previous study discovered that a neuropathic OP, tri-o-cresyl phosphate (TOCP) activated NRG1/ErbB signaling pathway in both spinal cord and sciatic nerves of hens during the formation of OPIDN and lapatinib, a non-selective antagonist of ErbB1 and ErbB2 receptors, alleviated the toxicity. In this study, we intended to further look into the potential role of NRG1 in the pathogenesis of TOCP-induced axon damage in spinal cord and sciatic nerves and whether lapatinib could also rescue this damage in mice, an OPIDN-resistant animal model. The results revealed that no obvious toxic signs were observed after single TOCP exposure. However, slight histopathological wreck in lumbar spinal cord and sciatic nerves was found following TOCP intoxication, and the damage in sciatic nerves was characterized by axon degeneration of myelin sheath but not the loss of neural skeleton. Only histopathological damage induced by TOCP in spinal cord could be prevented by lapatinib. The translational expression of NRG1/ErbB signaling molecules was analyzed by both in vivo and in vitro studies. In general, NRG1/ErbB pathway was activated by TOCP while combined treatment with lapatinib attenuated TOCP-induced NRG1/ErbB signaling cascade. The results implied that NRG1/ErbB system may predominately play functional role in spinal cord (central nervous system) but not in sciatic nerves (peripheral nervous system) of mouse subjected to neurotoxic OP, which was confirmed by the study in vitro that lapatinib was not able to attenuate TOCP-induced neurotoxicity in rodent Schwann cell line RSC 96 cells.

Metabolomic biomarkers in urine of rats following long-term low-dose exposure of cadmium and/or chlorpyrifos.

Heavy metals and pesticides can be easily enriched in food chains and accumulated in organisms, thus pose significant threat to human health. However, their combined effects for long-term exposure at low dose has not been thoroughly investigated; especially there was no biofluid biomarker available to noninvasively diagnose the toxicosis of the combined exposure of the two chemicals at their low levels. In this study, we investigated the change of urine metabolites of rats with 90-day exposure to heavy metal cadmium (Cd) and/or organophosphorus pesticide chlorpyrifos (CPF) using gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. Our results showed that the interaction of Cd and CPF mainly displayed an antagonistic effect. We identified the panels of metabolite biomarkers in urine: benzoic acid and mannose were unique biomarkers for Cd exposure; creatinine and N-phenylacetyl glycine were unique biomarkers for CPF exposure; anthranilic acid, ribitol, and glucose were unique biomarkers for Cd plus CPF exposure. Our results suggest that 90-day exposure to Cd and/or CPF could cause a disturbance in energy and amino acid metabolism. And urine metabolomics analysis can help understand the toxicity of low dose exposure to mixed environmental chemicals.

Identification of metabolite biomarkers in serum of rats exposed to chlorpyrifos and cadmium.

Chlorpyrifos (CPF) and cadmium (Cd) are widespread environmental pollutants, which are often present in drinking water and foods. However, the combined effects of CPF and Cd were not entirely clear at present. There was also no biomarker available to diagnose the poisoning of the two chemicals at low dose for long-term exposures. In this study, we investigated the change of serum metabolites of rats with subchronic exposure to CPF, Cd, and CPF plus Cd using gas chromatography-mass spectrometer-based metabolomics approach. We performed a stepwise optimization algorithm based on receiver operating characteristic to identify serum metabolite biomarkers for toxic diagnosis of the chemicals at different doses after 90-day exposure. We found that aminomalonic acid was the biomarker for the toxicity of Cd alone administration, and serine and propanoic acid were unique biomarkers for the toxicities of CPF plus Cd administrations. Our results suggest that subchronic exposure to CPF and Cd alone, or in combination at their low doses, could cause disturbance of energy and amino acid metabolism. Overall, we have shown that analysis of serum metabolomics can make exceptional contributions to the understanding of the toxic effects following long-term low-dose exposure of the organophosphorus pesticide and heavy metal.

Disruption of Kidney Metabolism in Rats after Subchronic Combined Exposure to Low-Dose Cadmium and Chlorpyrifos.

Cadmium (Cd) and chlorpyrifos (CPF) often coexist in the environment and induce combined toxicity to organisms. Here we studied the combined nephrotoxicity of environmentally relevant low doses of Cd and CPF. We treated the mice for 90 days with different doses of Cd and CPF and their mixtures via oral gavage. Then histopathological evaluation and biochemical analysis for kidney tissues were carried out. The change of metabolites in kidney was detected by using a metabolomics approach using GC-MS. We found that Cd, CPF, and their mixtures caused oxidative damage as well as disturbance of renal amino acid metabolism. We identified potential metabolite biomarkers in kidney, which included acetic acid for CPF treatment, glycerol and carboxylic acid for Cd treatment, and l-ornithine for the mixture of CPF and Cd treatment, respectively. In addition, we found that Cd promoted the metabolism of CPF in kidney. This may contribute to the result that the toxicity of the mixtures was lower than the sum of the toxicities of Cd and CPF alone. In conclusion, our results indicated that CPF and Cd could disrupt the kidney metabolism in rats even when they were exposed to a very low dose of CPF and Cd.

Metabolomic analysis for combined hepatotoxicity of chlorpyrifos and cadmium in 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 90days. 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.

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.

Autophagy in Tri-o-cresyl Phosphate-Induced Delayed Neurotoxicity.

The widely used organophosphorus compound tri-o-cresyl phosphate (TOCP) elicits delayed neurotoxicity characterized by progressive axonal degeneration in the spinal cord and peripheral nerves. However, the precise mechanisms of TOCP-induced delayed neurotoxicity are not clear. Because autophagy has been linked to the pathogenesis of neurodegenerative diseases, we aimed to characterize autophagy in the progression of TOCP-induced delayed neurotoxicity. In vivo experiments using the adult hen animal model showed that autophagy in spinal cord axons and in sciatic nerves was markedly induced at the early preclinical stage of TOCP-induced delayed neurotoxicity; it was decreased as the delayed neurotoxicity progressed to the overt neuropathy stage. In cultured human neuroblastoma SH-SY5Y cells, TOCP reduced cell growth, and induced prominent autophagy. The autophagy inhibitor 3-methyladenine could attenuate TOCP-induced cytotoxicity, indicating that the autophagy is accountable for TOCP-induced neurotoxicity. In addition, we found that TOCP-induced Parkin translocation to mitochondria in SH-SY5Y cells, suggesting that autophagy may function to degrade mitochondria after TOCP exposure. These results suggest that autophagy may play an important role in the initiation and progression of axonal damage during TOCP-induced neurotoxicity.

Metabolomics analysis and biomarker identification for brains of rats exposed subchronically to the mixtures of low-dose cadmium and chlorpyrifos.

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.

In vitro and in vivo antitumor activity of scutebarbatine A on human lung carcinoma A549 cell lines.

During our systematic study on the anticancer activities of Scutellaria barbata, scutebarbatine A (SBT-A), one of the major alkaloids in S. barbata, was found to have antitumor effects on A549 cells. Thus, we designed the present study to investigate in detail the antitumor effects of SBT-A. The cytotoxic effect of SBT-A on A549 in vitro were determined by an MTT assay and evaluated by IC50 values. Furthermore, results of Hoechst 33258 and Annexin V/PI staining assays demonstrated that SBT-A had significant antitumor effects on A549 cells via apoptosis, in a concentration-dependent manner. What's more, the mechanism was explored by western blotting, and our study revealed that SBT-A can up-regulate the expressions of cytochrome c, caspase-3 and 9, and down-regulate the levels of Bcl-2 in A549 cells. Finally, the antitumor effects of SBT-A were evaluated in vivo by using transplanted tumor nude mice, and the results confirmed that SBT-A has a notable antitumor effect on A549 cancer via mitochondria-mediated apoptosis. Collectively, our results demonstrated that SBT-A showed significant antitumor effects on A549 cells in vivo and in vitro via mitochondria-mediated apoptosis by up-regulating expressions of caspase-3 and 9, and down-regulating Bcl-2.