| Bees are exposed to pesticide residues during the growth process,especially when collecting nectar and pollen,leading to pesticide residue contamination in honey and causing product quality and safety problems in bee products.Bioavailability is a critical factor in assessing the risk of dietary exposure to pesticide residues on human health.Investigating the bioavailability of pesticide residues in honey is now a pressing issue that demands attention for the scientific evaluation of pesticide residue safety in food.In this thesis,honey was chosen as the research subject,and pyrethroids with high pesticide residues were selected as representative pesticides in actual samples.We established an in vitro gastrointestinal simulation method of bioavailability based on Tenax adsorbent,and investigated the bioavailability of pyrethroids based on animal experiments.Furthermore,we established an in vitro-in vivo correlation（IVIVC）model to evaluate the bioavailability of the pyrethroids in honey from different honey plants.We used the Caco-2 cell model to investigate the absorption and transport patterns of pyrethroids,and we combined metabolomics and molecular docking technology to analyze the transporter-mediated regulatory mechanism of the lambda-cyhalothrin in the Caco-2 cell model.These results provide a theoretical basis for enriching and expanding the study of pesticides in environmental toxicology and health risk assessment.The main findings are as follows:1.Optimisation of in vitro bioavailability assay based on Tenax adsorption kineticsQu ECh ERS-UPLC-MS/MS method was developed for the residue analysis of sevenpyrethroids,including fenpropathrin,beta-cypermethrin,deltamethrin,fenvalerate,lambda-cyhalothrin,bifenthrin,and beta-cyfluthrin,in six simulated intestinal fluids:SBRC,IVG,PBET,DIN,RIVM,and SHIME.The method demonstrated good linearity within a concentration range of 1-500μg/L,with correlation coefficients（R2）exceeding 0.99.The limits of detection（LODs）and the lowest limits of quantification（LOQs）for the method were 2μg/L and 10μg/L,respectively.The average recoveries of the seven pyrethroids in the six simulated intestinal fluids,at spiked concentrations of 10,100,and 1000μg/L,ranged from 74.90%to 116.80%,with relative standard deviations（RSDs）between 0.62%and12.60%.The method exhibited accuracy,precision,and sensitivity that meet the requirements for analyzing multi-residues of pesticides in six simulated intestinal fluids.Tenax was chosen as the adsorbent to enhance the bioavailability of pyrethroids in honeywithin an in vitro gastrointestinal simulation method.The results of the Tenax adsorption kinetics experiments demonstrated a high adsorption rate of pyrethroids in the simulated intestinal fluid of SHIME,with an adsorption equilibrium reached in 38.06-50.73 minutes.Moreover,as the quantity of Tenax added increased,the adsorption efficiency for the test pesticide residues reached 95.61-97.03%when the mass of Tenax reached 0.2 g.The utilization of Tenax-based adsorbents in the intestinal fluid significantly improved adsorption efficiency.The bioavailability(BAin vitro)values of the tested pesticides were41.93%,48.83%,63.35%,48.92%,47.73%,40.59%,and 43.02%,respectively.These values showed a significant increase compared to the SHIME method without Tenax,with enhancements ranging from 12.39%to 43.81%.Therefore,0.2 g Tenax was selected as the additive amount for the in vitro assay of bioavailability of SHIME method.2.Analysis of accumulation and dose-response relationship of pyrethroids in miceA method was developed for analyzing pesticide residues in seven biological samples（the heart,liver,spleen,lung,kidney,stomach,and intestine of mice）.The method exhibited good linearity in the concentration range of 1-500μg/L,with R2 all exceeding 0.99.The limits of detection（LODs）and the lowest limits of quantification（LOQs）for the method were 0.4μg/L and 2μg/L,respectively.The average spiked recoveries of the seven pyrethroid in the seven biological samples from mice,at spiked concentrations of 2,50,and500μg/L,ranged from 73.13%to 107.08%,with relative standard deviations（RSDs）between 1.82%and 12.05%.The accuracy,precision,and sensitivity of the method meet the requirements for analyzing multi-residues of pesticides in seven biological samples.The results of the dose-effect analysis of pyrethroids in various organ tissues of miceshowed that the cumulative distribution of residues for the tested pesticides in different organ tissues of mice followed this order:stomach>kidney>liver>intestine>lung>spleen>heart.Notably,there was a strong linear relationship between the dose of exposure and the cumulative amounts in the liver,kidney,and the combined target organs of the liver and kidney in mice,with correlation coefficients（R2）ranging from 0.9569 to 0.969,0.969 to0.969,and 0.969 to 0.969,respectively.As a result,the liver,kidney,and the combination of the liver and kidney were selected as target organs for bioavailability evaluation.3.Establishment and application of the IVIVC model for the bioavailability of pyrethroids in honeyThe relative bioavailability(RBAin vivo)of pyrethroids,based on different bioassay targets such as the liver,kidney,and combined liver and kidney,was evaluated.The results indicated that the RBAin vivo of pyrethroids ranged from 9.20%to 83.03%,16.58%to 92.01%,and 16.36%to 85.05%when the assay targets were the liver,kidney,and combined liver and kidney,respectively.In vivo-in vitro correlation（IVIVC）analyses were conducted for RBAin vivo using mice model and BAin vitro employing Tenax adsorbent-based SHIME.The study explored significant IVIVC correlations,focusing on the liver and kidneys as the assay targets,revealing correlation coefficients（R2）ranging from 0.7815 to 0.9746.The application of the IVIVC model to evaluate the RBAin vivo results of pyrethroids inhoney from different honey plants showed that the bioavailability of fenpropathrin,beta-cypermethrin,deltamethrin,fenvalerate,lambda-cyhalothrin,bifenthrin,and beta-cyfluthrin ranged from 18.60%to 22.43%,17.54%to 22.76%,70.94%to 85.71%,45.24%to 88.07%,57.19%to 79.55%,47.25%to 56.79%,and 61.83%to 85.92%,respectively.4.Caco-2 cell model to investigate the uptake and transport of pyrethroidsCaco-2 cell model was established to investigate the uptake and transport patterns related to the bioavailability of pyrethroids.The results demonstrated a significant positive correlation between the amount of transport and time in the Caco-2 cell model for seven pyrethroids.The apparent permeability coefficients(Papp AB→BL)and efflux ratio（ER）ranged from 4.32×10-4 to 6.64×10-3 and from 1.27×10-3 to 5.52×10-2,respectively,indicating that the pyrethroids are readily absorbed,with a passive diffusion uptake and transport mechanism.Efflux-type transporters（P-gp,MRP,BCRP）and uptake-type transporters（OATs,OATPs,OCTs）were found to mediate the transport of pyrethroids.As a result,the Papp AB→BL values decreased to the range of 3.01×10-5–7.36×10-4,1.46×10-4–3.19×10-3,3.96×10-5–3.38×10-4,2.55×10-5–2.91×10-4,2.01×10-4–8.75×10-4,and 2.05×10-5–5.16×10-4.5.Mechanisms of transporter-mediated regulation of lambda-cyhalothrin in the Caco-2 cell modelUsing UPLC-Q-TOF-MS-based non-targeted metabolomics and molecular docking techniques,the regulatory mechanism of transporter-mediated uptake and transport of the test pesticide residues in the Caco-2 cell model was investigated,with lambda-cyhalothrin as the representative compound.The results revealed the identification of a total of 390metabolites,and the differential metabolic pathways primarily included the ABC transporter pathway,purine metabolism,alanine,aspartate,and glutamate metabolism,protein digestion and absorption,glutathione metabolism,cholesterol metabolism,and the TCA cycle.P-gp,BCRP,and MRP were the transporter proteins that mediated the action of lambda-cyhalothrin on the ABC transporter protein pathway in Caco-2 cells. |
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