动物模型 z-bo 2020-09-03 400

　　Introduction: To overcome the limitations associated with traditional biological substrates, new methods based on alternative substrates (such as hair) have been proposed. This paper reviews several publications and reports on the potential for the detection of organic pollutants from various chemicals in the hair, which reflect individual environmental or occupational exposures. Hair samples can be collected in a non-invasive manner and easily stored. The main advantage is that it can represent several months of exposure time, depending on the length of the sample. Unlike body fluids such as urine and blood, the concentration of chemicals found in hair is not affected by short-term changes. Concentration corresponds to the average exposure level of an individual and is the most relevant information related to the study of biological effects. This study investigated the relationship between exposure levels and the concentration of pesticides produced and their metabolites in the hair. We used mice during the study and mixed 19 pesticides in 8 different doses within 90 days. The relationship between the naked concentration and the concentration of chemicals in the hair collected at the end of the experiment and the concentration of plasma and urine collected from the same animal were compared. We also evaluated the difference in pesticide concentration between different exposure groups, and the potential to determine the animal's exposure level based on the pesticide concentration in hair, urine, and plasma.

　　Materials and methods: Animal experiment Animal reproduction: 68 female LE rats (180-200g), 2 in a cage, light-dark cycle experiment in a controlled environment (temperature 22±3°C, humidity 55±10%) Before, the admission conditions of food, water and animals were domesticated for 2 weeks. Change the special bedding twice a week to reduce the external pollution of the hair caused by the pesticide released by the urine. In order to evaluate possible external contamination caused by urine excretion, the litter of the highest exposure group was placed in the sentinel cage of four untreated rats and analyzed at the end of the experiment. The sentinel rat was placed on a dirty bed and changed twice a week during the entire experiment. Analysis of the hair of sentinel rats showed that the waste does not contaminate the hair. Animal management: There are 8 animals in each experimental group. These rats were given three doses of calorie-containing low-calorie hydrogel daily by gavage for 90 days. The doses of the pesticide mixture exposed were 4, 10, 20, 40, 100, 200 and 400 ug/kg body weight. The minimum dose is the lowest level of pesticide allowed to contact hair after 90 days. Low-level testing does not matter because certain compounds are no longer detected. In addition, for some other compounds, there was no difference between the minimum exposure level of the control group and the background exposure. The maximum dose is set according to the toxicity of the compound. This is 1/20 of the minimum lethal dose. Before each administration, the animals should be weighed to adapt to the effect of the pesticide mixture on the animal's body weight.

　　Pesticide compulsory feeding mixture: Prepare a pesticide mixture stock solution made of ethanol every two weeks. The gel mixture was poured into an aluminum mold and allowed to cool at room temperature. Add an appropriate amount of pesticide mix stock solution to the gel. The ethanol is dried at room temperature (25°C) until completely evaporated. Then add a second layer of gel to trap pesticides. The control group received the same pesticide-free gel. Determine the best evaporation time according to the ethanol gel study. Headspace sampling-gas chromatography-mass spectrometry (GC-MS) was used to evaluate the ethanol content at various time points. Sample collection: Before the start of the experiment, the hair of these animals was shaved to ensure that the hair collected at the end of the study accurately reflected the 90-day exposure time. Collect white hair and black hair separately, put them in aluminum foil, and store at -20°C until analysis. Three hours after oral administration on the 90th day. Collect blood from the tail vein and place it in an EDTA tube. Centrifuge each sample (500-750uL) at 5000G for 3 minutes at room temperature. In order to collect urine, the rats were placed in another metabolic cage 24 hours (88-89 days) after gavage.

　　Pesticide analysis: Tandem mass spectrometry lacks background noise in chromatographic analysis, so the limit of detection (LOD) method based on background noise is not applicable. Result: For all chemicals found in hair and urine (the drug itself and its metabolites), the matrix concentration is closely related to the exposure level. In plasma, only diethyl phosphate (DEP) and DETP are significantly associated with different exposure levels because their metabolites are always present. The slope of the analyte concentration in the hair (linear fit) depends on the slope of the exposure level. The "slope" of urine and plasma is also completely different from the "exposure level" of various pesticides.

　　Organochlorine pesticides: Organochlorine compounds can be detected at most exposure levels in hair and plasma. P,P'-dichlorodiphenyldichloroethylene (P,P'-DDE) was observed in the hair, but not in the control group. Only P, P'-DDE and P, P'-DDD were detected at concentrations of 20 and 40 ug/kg. In the control group, P, P'DDT and its metabolites were not detected in plasma. P, P'DDD and its metabolites were only detected in the 40ug/kg dose group. In contrast, urine samples were not frequently detected, and only γ-hexachlorocyclohexane and pentachlorophenol were detected in all groups. In addition, p,p'-DDT and p,p'-DDE were only detected in urine samples of the 200ug/kg dose group. B-endosulfan and P,P'-DDD were not detected in urine samples of all groups. Large differences in concentration were found in hair samples. Except for β-endosulfan, it is only found in highly exposed people. P, P'DDT and P, P'DDD only appeared in the 20 and 40 ug/kg dose groups. Organochlorines in plasma are similar to those in hair. However, the differences between the groups are not as important as the hair samples. The differences in pesticides and their metabolites between the urine sample groups were not as obvious as the hair and plasma groups. Under the same exposure level, β-endosulfan is the lowest of all organochlorine compounds in hair and plasma, and it is not detectable in urine. The highest concentration of beta-hexachlorocyclohexane was observed in hair, and the highest concentration of pentachlorophenol was observed in plasma and urine samples.

　　Organophosphorus pesticides: quantify organophosphorus in plasma and urine samples, but only in hair. The pesticide itself cannot be detected in the urine sample. Only one toxic rif was detected in the blood of animals treated with two pesticides, and the average concentration of the control group was in the range of 0.15±0.01g/ml to 0.41±0.09g/ml of the highest concentration group. Regardless of the exposure concentration, DEP, DETP and TCPy can be detected, including the control group. Except for DEP and DETP in plasma, there is always a significant correlation between the concentration of metabolites in the test sample and the exposure level. For hair and urine samples, DETP showed the highest exposure dose-concentration correlation. For DEP, the best difference between the two groups is urine, followed by hair, and the worst is plasma. Synthetic pyrethrin: Cyfluthrin was detected when the hair exposure was 20ug/kg and the blood exposure was 4ug/kg. The exposure dose of cypermethrin in hair is 200ug/kg, and the content in plasma is 10ug/kg. Conclusion: The results of this study demonstrated for the first time that there is a significant correlation between pesticide exposure levels and the concentrations produced in the hair. Although the number of pesticides used in our research on rats is limited, it seems reasonable to assume that other chemicals and other species (such as humans) have similar behaviors. For human migration, although the concentration of pesticides in the hair is high, the intake of pesticides cannot be determined due to differences between species. Current research still shows that hair analysis can reliably classify individuals based on different exposure levels. Therefore, proof of concept is a step in epidemiological research using hair analysis as a reliable tool to investigate the adverse health effects associated with exposure.