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Ecotoxicology and Human Environmental Health

Enantioselective Distribution, Degradation, and Metabolite Formation of Myclobutanil and Transcriptional Responses of Metabolic-related Genes in Rats Weiyu Hao, Xiao Hu, Feilong Zhu, Jing Chang, Jitong Li, Wei Li, Huili Wang, Baoyuan Guo, Jianzhong Li, Peng Xu, and Yanfeng Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01721 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Enantioselective Distribution, Degradation, and Metabolite

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Formation of Myclobutanil and Transcriptional Responses of

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Metabolic-related Genes in Rats

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Weiyu Hao1, 2, Xiao Hu1, 2, Feilong Zhu1, 2, Jing Chang1, 2, Jitong Li1, Wei Li1, Huili

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Wang1, Baoyuan Guo1, Jianzhong Li1, Peng Xu1, Yanfeng Zhang1 *

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1. Research Center for Eco-Environmental Science, Chinese Academy of Sciences,

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Shuangqing RD 18, Beijing 100085, China

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2. University of the Chinese Academy of Sciences, Yuquan RD 19 a, Beijing 100049,

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China

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*Corresponding author: Yanfeng Zhang, address: Shuangqing RD 18, Beijing,

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China; E-mail: [email protected]; phone: +010-6284-9385; fax: +010-6284-9790

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Abstract

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Myclobutanil (MT), a chiral fungicide, can be metabolized enantioselectively in

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organisms. In this work, the associated absorption, distribution, metabolism and

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transcriptional responses of MT in rats were determined following a single-dose (10

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mg·kg-1 body weight) exposure to rac-, (+)- or (-)-MT. The enantiomer fractions (EFs)

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were less than 0.5 with time in the liver, kidney, heart, lung and testis, suggesting

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preferential enrichment of (-)-MT in these tissues. Furthermore, there was conversion

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of (+)-form to (-)-form in the liver and kidney after 6 h exposure to enantiopure

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(+)-MT. Enrichment and degradation of the two enantiomers differed between

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rac-MT and MT-enantiomers groups, suggesting that MT bioaccumulation is 1

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enantiomer-specific. Interestingly, the degradation half-life of MT in the liver with

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rac-MT treatment was shorter than that with both MT-enantiomer treatments. One

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reason may be that the gene expression levels of cytochrome P450 1a2 (cyp1a2) and

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cyp3a2 genes in livers treated with rac-MT were the highest among the three

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exposure groups. In addition, a positive correlation between the expression of cyp2e1

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and cyp3a2 genes and rac-MT concentration was found in livers exposed to rac-MT.

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Simultaneously, five chiral metabolites were detected, and the enantiomers of three

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metabolites, RH-9090, RH-9089 and M2, were separated. The detected enantiomers

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of (+)-MT metabolites were in complete contrast with those of (-)-MT metabolites.

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According to the results, a metabolic pathway of MT in male rats was proposed,

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which included the following five metabolites: RH-9089, RH-9090, RH-9090 Sulfate,

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M1 and M2. The possible metabolic enzymes were marked in the pathway. The

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findings of this study provide more specific insights into the enantioselective

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metabolic mechanism of chiral triazole fungicides.

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Introduction

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Triazole fungicides, containing 1,2,4-triazole groups in the main chain, are used

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worldwide to protect fruits, vegetables and crops because of their excellent antifungal

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activity1. 75% of the triazole fungicides are chiral2.

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compound have similar physicochemical properties, but they may behave differently

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in reactions with chiral molecules3. As most biomolecules, especially metabolic

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enzymes and biological receptors, are chiral compounds, enantioselective metabolism

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frequently occurs in organisms2,4. Enantioselectivity in the process may result in

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different ecotoxicological effects to the environment. Therefore, studying the

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enantioselective metabolism of triazoles is valuable for risk assessment and regulatory

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decisions.

Different enantiomers of a chiral

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Myclobutanil

(MT),

(RS)-2-(4-chlorophenyl)-2-(1,2,4-triazol-1-ylmethyl)

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hexanenitrile, is a typical triazole fungicide used for the control of powdery mildew

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and scabbing of plants5. Fungicidal action occurs through inhibition of the sterol

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14-demethylase enzyme6. It has a single chiral center and consists of two enantiomers.

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Though the acute toxicity of MT is very low, it can cause an increase in serum

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testosterone levels7 and cytochrome P450 (CYP) activities8, 9, which may further lead

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to reproductive inhibition and liver toxicity. Currently, MT is applied and released

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into the environment in the form of the racemate. However, the enantiomeric effects

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of MT have been demonstrated on bioactivity and metabolism. The fungicidal activity

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of (+)-MT is more efficient than that of (-)-MT10-12. Enantioselective enrichment and

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degradation of MT have been previously reported in tadpoles13 and rabbits11.

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Enantioselectivity in these processes can result in differences in toxicological effects.

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Tadpoles treated with (+)-MT exhibited higher oxidative stress13. A cytotoxicity

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research proved that the EC50 for the racemate was lower than those of the optically

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pure enantiomers14. These results suggest that the metabolic mechanisms and

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toxicities of rac-MT and MT-enantiomers are different15. Nevertheless, existing

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metabolic studies were carried out under the condition of rac-MT. The metabolic

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differences between the enantiopure enantiomers and the racemate have not been

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studied. In addition, studies of the effects of MT on metabolic enzymes have been

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limited to CYPs. The impact of MT on the regulation of other metabolic enzymes,

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such as glutathione transferase (gst)

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has rarely been reported.

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Therefore, the current study evaluated the enantioselective metabolic pathways of MT

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in male rats after one oral administration of rac-MT or enantiopure enantiomers. We

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investigated the stereospecific distribution, degradation and main chiral metabolites of

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and UDP-glucuronosyltransferase (ugt) 19, 20

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MT in rats. In addition, the time-dependent mRNA expression of metabolic enzymes

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genes (cyp1a2, cyp2d6, cyp2c8, cyp3a2, gst, ugt) in the liver was measured by

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real-time quantitative polymerase chain reaction (qPCR). Based on the results, we

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developed a metabolic pathway of MT in male rats. The findings of this study may

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contribute to a better understanding of the metabolic mechanism of chiral triazole

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fungicides.

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Materials and Methods

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Reagents

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MT (racemate, >98% purity, CAS# 88671-89-0) was provided by the Institute for

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Control of Agrochemicals, Ministry of Agriculture (Beijing, China). Both enantiopure

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enantiomers of MT (purity > 99%) were collected in our laboratory. Detailed

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descriptions of separated methods are available in the supporting information (SI).

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Acetonitrile, n-hexane, acetone and formic acid (HPLC-grade) were obtained from

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Dikma (Beijing, China). TRNzol A+, reverse transcription (RT) kit and qPCR SYBR

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green kit were purchased from TIANGEN Biotech (Beijing, China).

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Extraction of MT and metabolites

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The extraction of MT and its metabolites was carried out by the QuEChERS

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(acronym for quick, easy, cheap, effective, rugged, and safe) method. In brief, 1 g

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homogenized samples were mixed with 15 mL of acetonitrile in a 50 mL

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polypropylene centrifuge tube, and ultrasonically extracted for 20 min. After

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centrifugation at a relative centrifugal force (RCF) of 6000 g for 5 min, the

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acetonitrile (upper) layer was transferred into a 250 mL separatory funnel, and the

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precipitate was extracted repeatedly with another 15 mL of acetonitrile. The combined

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extracts were partitioned in a separatory funnel with 3×10 mL of n-hexane. The

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acetonitrile layer was filtered through a column containing 4 g of anhydrous sodium

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sulfate into a round bottom flask. The sample was then evaporated to dryness on a

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vacuum rotary at 38 °C, redissolved in 1.0 mL of acetonitrile and filtered through a

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0.22 µm filter.

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Enantioselective LC-MS/MS Analysis

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High-performance liquid chromatography (HPLC) tandem Q Exactive mass

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spectrometry (MS) (Thermo Fisher Scientific, San Jose, CA) equipped with a heated

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electrospray ion (HESI) source was used to measure quantitation of MT enantiomers

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and identification of metabolites. Chiral separation was achieved using a CHIRAL

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OD-3R column. Additional methodological details are provided in SI.

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Gene expression analysis

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As livers are the crucial place to xenobiotic metabolism and rich in metabolic

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enzymes21, liver samples were used for qPCR. In this study, the transcriptional

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changes of cyp1a2, cyp2d6, cyp2e1, cyp3a2, gst and ugt genes were investigated due

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to relatively high expression in livers. Specific primers (Table S1) and methods are

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detailed in SI. Briefly, total RNA was extracted using TRNzol-A+ reagent following

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the manufacturer’s instructions. RNA samples were used for cDNA synthesis using

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RT kit. Relative abundance of the mRNA level was assessed using a MX3005P

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real-time qPCR system (Stratagene, USA) by SYBR green PCR kit. Relative

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quantification of each mRNA level was normalized according to the gapdh mRNA

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level. The relative expression of the target genes were calculated using 2-∆∆Ct.

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Exposure experiments

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Male Sprague-Dawley rats (200 ± 25 g) were purchased from Vital River Laboratory

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Animal Technology (Beijing, China). All rats were housed under a 12-h light/dark

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cycle at 25 °C. 24 h before experiments, the rats were fasted but had free access to

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water. 10 mg·kg-1 myclobutanil is considered to be the no observed effect level

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(NOEL) for any number of traditional toxicity endpoints22-24. After single exposure,

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MT is well absorbed and degraded in rats, and mRNA may be regulated to metabolize

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MT. Therefore, 10 mg·kg-1 body weight (bw) was chosen to study the chiral

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metabolism and gene expression. MT was dissolved in ethanol and then diluted to 2

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mg·mL-1 with corn oil. The proportion of ethanol in the whole system was less than

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10%. Thirty-six rats were randomly divided into three groups. The rats were dosed

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orally with 10 mg·kg-1 bw of rac- /(+)- /(-)-MT. Two rats in each group were

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euthanized at time points of 1, 3, 6, 12, 24 and 48 h after oral exposure. Hearts, lungs,

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kidneys, livers, intestines and testis were excised and frozen at -20 °C for later

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analysis. A portion of the liver was minced and stored in RNA storage solution for

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analysis of gene expression.

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Data analysis

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The degradation of MT enantiomers in rats followed pseudo-first-order kinetics with

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absorption (equation 1). The kinetics was fitted using Drug and Statistic for Windows

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2.0 software (DAS 2.0, Chinese Pharmacological Society of Mathematical

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Pharmacology Committee). Half-life (‫ݐ‬ଵ/ଶ , h) was calculated according to equation 2.

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‫ܥ = ܥ‬଴ ݁ ି௞௧ − ‫ି ݁ܭ‬௞ೌ ௧

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‫ݐ‬ଵ/ଶ =

௟௡ଶ ௞

=

଴.଺ଽଷ ௞

(1) (2)

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where k and ka are the degradation rate constant and the absorption rate constant,

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respectively. C represents the concentration of MT enantiomers at t hours, and C0 is

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the highest concentration in the tissue. K is the fitting parameter and is approximately

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equal to C0.

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The enantiomer fraction (EF) was used to measure the enantioselectivity of MT in rats

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(equation 3). EF values range from 0 to 1 and equal 0.5 for the racemate.

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EF =

஼ሺశሻషಾ೅ ஼ሺశሻషಾ೅ ା஼ሺషሻషಾ೅

(3)

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where C(+)/(-)-MT represents the concentration of (+)/(-)-MT in rats.

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All data are expressed as the mean ± SD. Statistically significant differences (p < 0.05)

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among three exposure groups were evaluated using analysis of variance (ANOVA)

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and tukey’s multiple range tests.

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Results and Discussion

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Enantioselective distribution and enrichment of MT in rats

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The distribution of MT into different tissues can provide valuable information for the

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underlying mechanism of bioaccumulation in vivo25. Therefore, the concentrations of

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Myclobutanil in heart, liver, lung, kidney, testis and intestine were measured. After

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oral exposure, MT flowed into the stomach and was absorbed by the intestinal villus.

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Then, it entered the blood through the gastrointestinal tract and reached other tissues26,

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Figure 1. The concentration of MT increased initially and reached maximum levels at

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6 h, then decreased rapidly with time in the liver, intestine, kidney and testis of rats

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exposed to rac-MT and (+)-MT. Both enantiomers peaked at 12 h in the lung and

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heart of rac-MT-exposed rats. Interestingly, the concentration of (+)-MT in the lung

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and heart of rats exposed to (+)-MT were lower than those in rats exposed to rac-MT.

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As conversion of (+)-MT to (-)-MT was not found in the lung and heart, this

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phenomenon may be due to the faster absorption and slower degradation in rac-MT

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samples. In the (-)-MT treatment group, the maximum (-)-MT concentration (Cmax)

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was reached at 3 h in the liver, intestine, testis and heart, while the concentration of

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MT decreased with time in the kidney and lung. The result indicated that optically

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pure (-)-MT can be absorbed more easily than (+)- and rac-MT. After 24 h exposure,

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the concentrations of both enantiomers in tissues reduced to less than 5 µg·kg-1,

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indicating that rats can rapidly metabolize both (+)- and (-)-MT. Rapid metabolism of

. The concentration-time curves of MT in different tissue samples are shown in

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MT was also found in other organisms, such as rabbits exposed by intravenous

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injection11, Tenebrio molitor exposed through food28, and earthworms29 treated with

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an environment containing MT. A previous study reported that a chemical with log

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KOW > 1.75 and log KOA ≥ 5.25 has bioaccumulation potential, unless it is

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metabolized at a sufficiently rapid rate30. The log KOW and log KOA values of MT are

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2.94 and 9.70, respectively. Therefore, rapid metabolism of both MT enantiomers

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could be the main reason for non-bioaccumulation of MT.

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In rac-MT treatment, the sum of the Cmax in six tissues was 3711 µg·kg-1 for (+)-MT

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and 4738 µg·kg-1 for (-)-MT. However, the sum of the Cmax in six tissues was 659

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µg·kg-1 for the (+)-form and 1817 µg·kg-1 for the (-)-form in enantiopure enantiomer

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exposure groups (Figure 2). (-)-MT was enantio-enriched in rats regardless of

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racemate or optically pure enantiomers exposure. Surprisingly, rac-MT was easily

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enriched in rats than MT-enantiomers, suggesting a more steady state of rac-form.

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The highest and second-highest (+)-MT concentrations were found in the intestine

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and kidney of both rac-MT and (+)-MT treatment, while the Cmax values of (-)-MT in

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the intestine and liver were higher than those in other tissues. Interestingly, the Cmax

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of (-)-MT in the kidney of rac-MT treatment was significantly higher than that in the

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kidney of (-)-MT treatment. It was speculated that certain renal enzymes that can be

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induced by (+)-MT may play an important role in the enrichment of MT. Consistently,

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a study31 investigated the metabolism of

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highest concentrations of MT in the intestine, liver and kidney. Furthermore, the

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different distribution of (+)-MT and (-)-MT implied that the clearance mechanisms

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for the two enantiomers were different.

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EFs were calculated for the rac-MT treatment to further assess the enantiomeric

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enrichment (Figure 1C). The EF value in the intestine was approximately 0.5,

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C-myclobutanil in rats and found the

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suggesting that there was no enantioselectivity. This could be explained by the

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intestine absorbing drugs by passive diffusion, in which lipid solubility is the main

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influence32, and this property of (+)- and (-)-enantiomer are same. The EF values were

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less than 0.5 with time in the liver, kidney, heart, lung and testis, indicating

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enantioselective enrichment of (-)-MT in these tissues. Similarly, preferential

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enrichment of (-)-MT was also found in the kidney, heart and lung of lizards exposed

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to 20 mg·kg-1 bw rac-MT33. In contrast, a study reported that there was an enrichment

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of (+)-MT in Scenedesmus obliquus34. These data demonstrated that enantioselective

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enrichment of MT in non-target organisms is species-specific.

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Chiral conversion in tissues

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The chromatographic peak corresponding to (-)-MT clearly appeared in the liver and

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kidney after exposure to enantiopure (+)-MT (Figure S1). The conversion rate was

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used to express the inversion degree of one enantiomer into the other. It was defined

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as the concentration of the enantiomer from conversion divided by the concentration

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of total MT in this study. Approximately 5.9% conversion in the liver and 5.7%

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conversion in the kidney were observed at 6 h after (+)-MT exposure. This

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observation suggested that conversion of (+)-MT to (-)-MT took place along with the

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degradation process. However, the conversion of (-)-MT to (+)-MT was uncertain

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because very low concentration of (+)-MT (