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.

The progressive alteration of urine metabolomic profiles of rats following long-term and low-dose exposure to permethrin.

Background: Permethrin is a type of widely used pyrethroid pesticide. Although acute toxicity of permethrin has been well-characterised, the non-acute toxicity of permethrin upon long-term exposure at low dose has been seldom studied yet. The current study investigates the time-course change of the metabolomic profiles of urine following the low level long-term exposure of permethrin and identified biomarkers of the chronic toxicity of permethrin.Methods: Male Wistar rats were administrated orally with permethrin (75 mg/kg body weight/day, 1/20 LD50) daily for consecutive 90 days. The urine samples from day 30, day 60, and day 90 after the first dosing were collected and analysed by 1H NMR spectrometry. Serum biochemical analysis was also carried out.Results: Permethrin caused significant changes in the urine metabolites such as taurine, creatinine, acetate, lactate, dimethylamine, dimethylglycine, and trimethylamine-N-oxide. These biological markers indicated prominent kidney and liver toxicity induced by permethrin. However, there was no change in serum biochemical parameters for the toxicity, indicating that metabolomic approach was much more sensitive in detecting the chronic toxicity.Conclusion: The time-course alteration of metabolomic profiles of the urine based on 1H NMR reflects the progressive development of the chronic toxicity with the long-term low-level exposure of permethrin.

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.

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.

Clinical effects of 3-D printing-assisted personalized reconstructive surgery for blowout orbital fractures.

PURPOSE: One of the key challenges during orbital fracture reconstructive surgery, due to the complex anatomy of the orbit, is shaping and trimming the precise contour of the implants. The objectives of this study were to describe and evaluate the use of a three-dimensional (3-D) printing technique for personalized reconstructive surgery for repairing orbital fractures. METHODS: A total of 29 cases which had 3-D technique-assisted surgical reconstruction, and 27 cases which had traditional surgery, were retrospectively analyzed. Preoperative and postoperative CT images were measured using MIMICS software, and the contour of the fracture zone and the Medpor-titanium implant were analyzed and compared. The surgical duration was also compared between the two groups. RESULTS: There were statistically significant differences in the maximum width, depth and area between fracture zone and implant between the two groups, with the absolute value in the 3-D group markedly lower as compared to the control group. In addition, the difference in the medial-inferior wall angle between the surgical eye and healthy eye was also statistically significant between the groups. The average surgical duration in the 3-D group was substantially shorter than in the control group. Additionally, the postoperative clinical evaluation in the 3-D group was superior to that of the control group. CONCLUSION: The 3-D printing technique is of great value for predicting the precise fracture zone before, and during, personalized surgery, and can help surgeons achieve accurate anatomical reconstruction for repairs of blowout orbital fractures. Moreover, the simulated bone template produced by 3-D printing models allows for "true-to-original" orbital reconstruction, which can shorten the surgical duration and improve the accuracy and safety of the operation.

Cadmium and chlorpyrifos inhibit cellular immune response in spleen of rats.

Cadmium (Cd) and chlorpyrifos (CPF) are common pollutants coexisting in the environment, and both of them have been reported to have immunotoxicity to organisms. However, the joint effects of these two chemicals on the immune system are still unknown. In this study, we used CdCl2 and CPF to study their combined effects on immune functions in the spleen of rats. In in vivo experiments, SD rats were exposed to different doses of CdCl2 (0.7 and 6 mg kg-1 body weight/day) and CPF (1.7 and 15 mg kg-1 body weight/day) or their combinations for consecutive 28 days. The proliferation and cytokine production ability of the splenocytes isolated from the treated animals were assessed. In in vitro experiments, we used different concentrations of CdCl2 and CPF to treat concanavalin A (Con A)-induced splenocytes isolated from untreated rats. We found that the combination of CPF and high dose of CdCl2 had a synergistic inhibitory effect on production of IFN-γ by spleen cells induced by Con A. The in vitro results showed that two chemicals had different effects on the cell proliferation and cytokine production depending on the exposure doses and time. This result suggests that exposure to both CdCl2 and CPF at the environmentally-relevant low dose may be potentially more hazardous than exposure to each individual toxicant.

Neuroprotective Strategy in Retinal Degeneration: Suppressing ER Stress-Induced Cell Death via Inhibition of the mTOR Signal.

The retina is a specialized sensory organ, which is essential for light detection and visual formation in the human eye. Inherited retinal degenerations are a heterogeneous group of eye diseases that can eventually cause permanent vision loss. UPR (unfolded protein response) and ER (endoplasmic reticulum) stress plays an important role in the pathological mechanism of retinal degenerative diseases. mTOR (the mammalian target of rapamycin) kinase, as a signaling hub, controls many cellular processes, covering protein synthesis, RNA translation, ER stress, and apoptosis. Here, the hypothesis that inhibition of mTOR signaling suppresses ER stress-induced cell death in retinal degenerative disorders is discussed. This review surveys knowledge of the influence of mTOR signaling on ER stress arising from misfolded proteins and genetic mutations in retinal degenerative diseases and highlights potential neuroprotective strategies for treatment and therapeutic implications.

Neuropathy Target Esterase Is Degraded by the Ubiquitin-Proteasome Pathway with ARA54 as the Ubiquitin Ligase.

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.

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.

Redox status in liver of rats following subchronic exposure to the combination of low dose dichlorvos and deltamethrin.

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.

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.

Identification of human patatin-like phospholipase domain-containing protein 1 and a mutant in human cervical cancer HeLa cells.

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.

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 liver's glucose, protein, and nitrogen metabolism.

Orbital and eyelid diseases: The next breakthrough in artificial intelligence?

Orbital and eyelid disorders affect normal visual functions and facial appearance, and precise oculoplastic and reconstructive surgeries are crucial. Artificial intelligence (AI) network models exhibit a remarkable ability to analyze large sets of medical images to locate lesions. Currently, AI-based technology can automatically diagnose and grade orbital and eyelid diseases, such as thyroid-associated ophthalmopathy (TAO), as well as measure eyelid morphological parameters based on external ocular photographs to assist surgical strategies. The various types of imaging data for orbital and eyelid diseases provide a large amount of training data for network models, which might be the next breakthrough in AI-related research. This paper retrospectively summarizes different imaging data aspects addressed in AI-related research on orbital and eyelid diseases, and discusses the advantages and limitations of this research field.

Ivermectin inhibits tumor metastasis by regulating the Wnt/ß-catenin/integrin ß1/FAK signaling pathway.

Tumor metastasis is the major cause of cancer mortality; therefore, it is imperative to discover effective therapeutic drugs for anti-metastasis therapy. In the current study, we investigated whether ivermectin (IVM), an FDA-approved antiparasitic drug, could prevent cancer metastasis. Colorectal and breast cancer cell lines and a cancer cell-derived xenograft tumor metastasis model were used to investigate the anti-metastasis effect of IVM. Our results showed that IVM significantly inhibited the motility of cancer cells in vitro and tumor metastasis in vivo. Mechanistically, IVM suppressed the expressions of the migration-related proteins via inhibiting the activation of Wnt/ß-catenin/integrin ß1/FAK and the downstream signaling cascades. Our findings indicated that IVM was capable of suppressing tumor metastasis, which provided the rationale on exploring the potential clinical application of IVM in the prevention and treatment of cancer metastasis.

Long-term low-dose exposure of permethrin induces liver and kidney damage in rats.

BACKGROUND: Permethrin is one of the pyrethroid insecticides, which is widely used in agriculture and public health. Although acute toxicity of the insecticide has been studied, the chronic toxicity upon the long-term exposure has not been clear yet. The purpose of the current study is to investigate the organ toxicities of permethrin following its long-term low-dose exposure. METHODS: Male Wistar rats were daily administrated orally with permethrin (75 mg/kg body weight/day, gavage) for 90 days, and then the samples of biofluids (blood and urine) and organs including liver and kidney were collected. The serum and urine samples were measured by biochemical assay and the tissues of kidney and liver were examined and analyzed by histopathological method. RESULTS: The results showed that no change was found in serum and urine biochemical parameters for the toxicity; however, significant changes including hyperchromatic nuclei swollen in the hepatic parenchymal cells and the swelling proximal tubules in the kidneys were observed in the tissue structures of liver and kidneys in the histopathological sections. CONCLUSION: These results indicate that low-dose long-term exposure of permethrin can cause chronic toxicity with slight liver and kidney damage.

Paeoniflorin Derivative in Paeoniae Radix Aqueous Extract Suppresses Alpha-Toxin of Staphylococcus aureus.

The emergence and dissemination of bacterial infections is paralyzing our public health systems worldwide. Worse still, there are no effective antibiotics against bacterial toxins, which facilitate the infection. Natural herbs that target bacterial toxins may be a better choice for therapy of infectious diseases. However, most natural drugs present unknown compositions and unclear mechanisms. Here we demonstrated that the Chinese herb Paeoniae Radix aqueous extract (PRAE) could suppress alpha-toxin (α-toxin) of Staphylococcus aureus. We observed that the paeoniflorin derivative (PRAE-a) derivative in PRAE significantly abolished the hemolytic activity of S. aureus α-toxin. The analyses of high-performance liquid chromatography (HPLC), mass spectrometer (MS), Fourier transform infrared spectrometer (FTIR), and nuclear magnetic resonance (NMR) showed that PRAE-a was a glycoside compound with a paeoniflorin nucleus. We further found that PRAE-a disrupted the pore-forming ability of α-toxin by prevention of the dimer to heptamer. Therefore, PRAE-a proved to be an effective therapy for S. aureus lung infections in mice by inhibiting α-toxin. Collectively, these results highlighted that PRAE-a can be used as an antibacterial agent to attenuate S. aureus virulence by targeting α-toxin.

Experimental models and examination methods of retinal detachment.

Retinal detachment refers to the separation of the retinal neuroepithelium and pigment epithelium, usually involving the death of photoreceptor cells. Severe detachment may lead to permanent visual impairment if not treated properly and promptly. According to the underlying causes, retinal detachment falls into one of three categories: exudative retinal detachment, traction detachment, and rhegmatogenous retinal detachment. Like many other diseases, it is difficult to study the pathophysiology of retinal detachment directly in humans, because the human retinal tissues are precious, scarce and non-regenerative; thus, establishing experimental models that better mimic the disease is necessary. In this review, we summarize the existing models of the three categories of retinal detachment both in vivo and in vitro, along with an overview of their examination methods and the major strengths and weaknesses of each model.

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.

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.