Other Abstract | Reptiles originate in the late Carboniferous period. They are an important part of vertebrates, occupying an important position in the ecosystem. Although the spec ies of reptile records is increasing, the quantity of reptiles is decreasing in China,especially in areas with serious environmental pollution. Therefore, more and more scientists focus on reptile protection tox icology and ecological risk a ssessment
Triazole fungicide s are the sec ond milestone fungicide s and act by inhibiting the synthesis of the demethyl sterols. It has been reported that more than 75% of t hem are chiral pesticides. Although different enantiomers of chiral pesticides have similar phy sicochemical properties, the toxicity of different enantiomers o f triazole pesticides in animals is of great difference . Most current studies reported the toxicity of chiral pesticides based on the form of racemate, wheras the behavior of racemates and ena ntiomers in organisms may vary. The evaluation of chiral pesticides in the form of racemates is not only inaccurate, but may also cause immeasurable losses. Therefore, it is of great significance to explore the enantiomeric differences of chiral pesticides .
Two kinds of chiral triazole fungicides, myclobutanil and fenbuconazole, were selected in this study. The enantiopure enantiomers were prepared by high performance liquid chromatography (HPLC) with chiral OD 3R column. The enantioselective distribution a nd degradation of racemate and enantio pure enantiomers in lizard were measured. After a single exposure, the elimination half lives of myclobutanil were between 0.133 and 14.828 h . T he enantiomer fraction (EF) value s were less than 0.5 i n lizard fat, brain and kidney , suggesting that myclobutanil was more likely to accumulate in these tissues . This phenomenon was pronounced after multiple exposures. After 28 days, ( myclobutanil were detected in all lizard tissues and blood , while myclobutanil was only detected in lizard skin. The elimination rate of fenbuconazole in lizards was significantly lower than that of myclobutanil. Fats couldould enrich fenbuconazoleenrich fenbuconazole withoutwithout enantioselectivity. enantioselectivity. In In lizard blood lizard blood and liver, (+)and liver, (+)--fenbucfenbuconazole onazole waswas enantioselectively degraded. In enantioselectively degraded. In addition, a small amount of chiral conversion was observed in the addition, a small amount of chiral conversion was observed in the eliminationelimination process process of myclobutanil and fenbuconazole in lizards.of myclobutanil and fenbuconazole in lizards.
The results of qualitative and semi--quantitative detection of the metabolites ofquantitative detection of the metabolites of myclobutanil andmyclobutanil and fenbuconazole in lizards showfenbuconazole in lizards showeded that the liver that the liver waswas the main the main metabolic metabolic placeplace for bothfor both two fungicides. The main metabolite of myclobutanil two fungicides. The main metabolite of myclobutanil waswas RHRH--9090, and the main metabolite of fenbuconazole 9090, and the main metabolite of fenbuconazole waswas RHRH--9129&RH9129&RH--9130. The 9130. The chiral configuration ochiral configuration of the metabolites generated from (+)f the metabolites generated from (+)--enantiomer enantiomer werewere completely different from its antipode, indicating that enantioselectivitycompletely different from its antipode, indicating that enantioselectivity existexisteded during the metabolites formation. In addition, the enantiomeric enrichment of the during the metabolites formation. In addition, the enantiomeric enrichment of the metabolites in racemate group metabolites in racemate group werewere difdifferent from that of the enantiomers group, ferent from that of the enantiomers group, especially for fenbuconazole. After exposure to especially for fenbuconazole. After exposure to racrac--fenbuconazole, the highest fenbuconazole, the highest concentration of the metabolite RHconcentration of the metabolite RH--6467 produced by (6467 produced by (--))--enantiomer enantiomer waswas greater than greater than that produced by (+)that produced by (+)--enantiomer, enantiomer, whilewhile the highestthe highest concentration of the metabolite concentration of the metabolite RHRH--9129 & RH9129 & RH--9130 produced by (9130 produced by (--))--enantiomer was less than that produced by enantiomer was less than that produced by (+)(+)--enantiomer. However, the results in the lizard liver and enantiomer. However, the results in the lizard liver and bloodblood were revewere reversed. This rsed. This result indicatedresult indicated a certain interaction between the two enanta certain interaction between the two enantiomers in the metabolism iomers in the metabolism of myclobutanil and fenbuconazole in lizards.of myclobutanil and fenbuconazole in lizards.
Furthermore, the relative expression of metabolic enzyme--related genes in lizard related genes in lizard liver after exposure to myclobutanil enantiomers was studied. It was found that liver after exposure to myclobutanil enantiomers was studied. It was found that (+)(+)--myclobutanil significantly upmyclobutanil significantly up--regulated the regulated the mRNA mRNA expression of cytochrome expression of cytochrome P450 1a1 (P450 1a1 (cyp1a1cyp1a1)), , cyp2d3cyp2d3, , cyp2d6cyp2d6, , cyp3a4cyp3a4 and and cyp3a7cyp3a7 genes, while genes, while ((--))--myclobutanil significantly upmyclobutanil significantly up--regulated the expression levels of regulated the expression levels of cyp1a1cyp1a1, , cyp2d3cyp2d3, , cyp2d6cyp2d6, , cyp2c8cyp2c8 and and cyp3a4cyp3a4 genes. The result indicated that the two enantiomers of genes. The result indicated that the two enantiomers of myclobutanil induced different metabolicmyclobutanil induced different metabolic pathways. The results on the mRNA pathways. The results on the mRNA expression of antioxidant enzyme gene indicateexpression of antioxidant enzyme gene indicatedd that (+)that (+)--myclobutanil mmyclobutanil mightight induce induce more obvious oxidative stress.more obvious oxidative stress. The liver damage was obvious after 28 d exposure to The liver damage was obvious after 28 d exposure to myclobutanil enantiomers.
The racemate of a chiral triahe racemate of a chiral triazole zole fungicide fungicide is like a complex system. The is like a complex system. The eliminationelimination in lizards is not the sum of the in lizards is not the sum of the eliminaitoneliminaiton ofof two enantiomers, but a two enantiomers, but a combined effect. combined effect. The enantioselective distribution and metabolism of myclobutanil in The enantioselective distribution and metabolism of myclobutanil in rats were studied in order to compare the srats were studied in order to compare the similarities and differences of this imilarities and differences of this enantioselectivity between reptiles and other species. Similar to lizards, the enantioselectivity between reptiles and other species. Similar to lizards, the distribution and metabolism of racemate and enantiopure enantiomers were obviously distribution and metabolism of racemate and enantiopure enantiomers were obviously different. The liver was the main metabolism site of myclodifferent. The liver was the main metabolism site of myclobutanil and RHbutanil and RH--9090 was 9090 was the main metabolite. In rat liver, kidney, heart, lunt and testis, (the main metabolite. In rat liver, kidney, heart, lunt and testis, (--))--myclobutanil myclobutanil preferentially enriched. However, myclobutanil eliminated more quickly in rats than preferentially enriched. However, myclobutanil eliminated more quickly in rats than in lizards. This may be explained by the fact that the mRNA exprin lizards. This may be explained by the fact that the mRNA expression of metabolism ession of metabolism enzymes enzymes was higher in rats. In addition, the conversion from (+)was higher in rats. In addition, the conversion from (+)--myclobutanil to myclobutanil to ((--))--myclobutanil was found in rats.myclobutanil was found in rats.
In summary, the interaction interaction betweenbetween enantiomers enantiomers existed not onlyexisted not only in lizards, but in lizards, but also in rats. also in rats. Moreover, the enantioseMoreover, the enantioselectivity of myclobutanil was different between lectivity of myclobutanil was different between lizards and rats. lizards and rats. Therefore, the effects of different enantiomers on organisms Therefore, the effects of different enantiomers on organisms and and different species different species should be considered in assessing the toxic effects of chiral should be considered in assessing the toxic effects of chiral pesticides. pesticides. Reptiles are an indispensable Reptiles are an indispensable part of the ecosystem.part of the ecosystem. The enantioselective The enantioselective metabolic behavior of chiral pesticides in reptiles is unique. Thmetabolic behavior of chiral pesticides in reptiles is unique. Thusus it is imperative to it is imperative to constructconstruct reptile toxicology research systemreptile toxicology research systemss..
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