Effect And Mechanism Of Bile Acid Perturbation Induced By Triazole Fungicides On β-receptor Blockers Oral Absorption

Triazole fungicides are highly efficient fungicides that hinder fungal cell membrane synthesis by inhibiting fungal CYP 51,and are used to control various fungal diseases on crops.However,the inhibitory effect of triazole fungicides on CYP enzymes is nonspecific and can cause health hazards to humans.Although many studies have shown that pesticide exposure at no adverse reaction levels(No observed adverse effect level,NOAEL)does not harm health,CYP enzymes are involved in various physiological processes,including the synthesis of physiological surfactant(bile acids)that promote lipid absorption.Whether exposure to triazole fungicides may disrupt bile acid homeostasis by affecting the activity of CYP enzymes and in turn affects the absorption of oral drugs remains to be explored.Objective: To construct a low-dose tebuconazole exposure model in rats with tebuconazole as a representative triazole fungicides for short-and long-term oral exposure.Taking β-receptor blockers with different lipophilicity as model drugs,the effect of tebuconazole exposure on its absorption was evaluated,and the mechanism of tebuconazole exposure on drug absorption was studied based on bile acid perturbation.Methods:(1)Short-term(7 d)and long-term(28 d)oral transoral exposure models of1/10 and 1/2 NOAEL doses of tebuconazole were established by gavage.Low-dose tebuconazole exposure’s effects on rats survival status were evaluated by recording body weight and morphological changes;the tissue distribution of tebuconazole was analyzed by enzyme-linked immunoassay and LC-MS/MS.The tissue distribution of tebuconazole and the efficiency,content,specific activity and cholesterol and bile acid content of bile acid synthesis-related enzymes(CYP7/27A1)catalyzing cholesterol in liver tissues were examined;(2)Propranolol hydrochloride(log P =3.77),bisoprolol fumarate(log P =2.47)and atenolol(log P = 1.54)were given by gavage to normal rats and tebuconazole-exposed rats,and the blood concentrations of the drugs at different time points were measured by liquid-quality coupling and blood concentration-time curves were plotted to analyze the effects of tebuconazole exposure on the oral absorption behavior of drugs with different hydrophilicβ-receptor blockers;(3)Simulated intestinal fluids were prepared in vitro according to the content and proportion of bile acids in normal and exposed model rats.The effect of dissolving propranolol hydrochloride in simulated intestinal fluid on the dissolution status was evaluated.The intestinal mucus was incubated with simulated intestinal fluid to evaluate the effect of bile acid disturbance on the physicochemical properties of the mucus;an in vitro intestinal mucus permeability model was constructed to evaluate the effect of bile acid disturbance on the infiltration behavior of β-receptor blockers in mucus.An in situ intestinal perfusion model was developed to evaluate the effect of bile acid perturbations on β-receptor blockers intestinal absorption and intestinal epithelial barrier function.Results: Tebuconazole exposure had no significant effects on survival status and major organ indices of rats,tebuconazole accumulated mainly in the liver and kidney after exposure,and the degree of accumulation depended on the exposure time and dose;tebuconazole exposure reduced the activity of bile acid synthase CYP7/27A1 ratio but increased its expression level,and short-term exposure increased the ability of CYP7/27A1 to catalyze cholesterol;meanwhile,short-term exposure increased the content of bile acids and decreased the content of cholesterol in rat liver tissues.(2)Tebuconazole exposure significantly affected the absorption of β-receptor blockers:short-term tebuconazole exposure significantly increased the oral absorption of propranolol hydrochloride,the most lipophilic drug,but high-dose tebuconazole exposure significantly increased the oral absorption of bisoprolol fumarate and atenolol,the moderately lipophilic drug.(3)Short-term tebuconazole exposure significantly increased the bile acid content and decreased the size of propranolol hydrochloride/bile acid micelles;disrupted the intestinal mucus barrier and epithelial barrier and increased the ability of the drug to penetrate the mucus barrier and epithelial barrier.Conclusion: Low-dose tebuconazole exposure did not significantly affect the survival status of rats,but altered the dissolution status of drugs in the intestine,reduced the intestinal mucus barrier and epithelial barrier function by affecting the hepatic bile acid composition,and thus affected the in vivo absorption behaviour of β-receptor blockers.The results of this study can provide a research basis and reference for the safety assessment of triazole fungicides.