Enantioselective Distribution, Degradation, and ... - ACS Publications

Jun 29, 2018 - Real-Time Nowcasting of Microbiological Water Quality at Recreational Beaches: A Wavelet and Artificial Neural Network Based Hybrid ...
0 downloads 0 Views 1009KB Size
Subscriber access provided by FORDHAM UNIVERSITY

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Environmental Science & Technology

1

Enantioselective Distribution, Degradation, and Metabolite

2

Formation of Myclobutanil and Transcriptional Responses of

3

Metabolic-related Genes in Rats

4

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

5

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

6

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

7

Shuangqing RD 18, Beijing 100085, China

8

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

9

China

10

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

11

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

12 13

Abstract

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

ACS Paragon Plus Environment

Environmental Science & Technology

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

findings of this study provide more specific insights into the enantioselective

36

metabolic mechanism of chiral triazole fungicides.

37

Introduction

38

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

39

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

40

activity1. 75% of the triazole fungicides are chiral2.

41

compound have similar physicochemical properties, but they may behave differently

42

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

43

enzymes and biological receptors, are chiral compounds, enantioselective metabolism

44

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

45

different ecotoxicological effects to the environment. Therefore, studying the

46

enantioselective metabolism of triazoles is valuable for risk assessment and regulatory

47

decisions.

Different enantiomers of a chiral

2

ACS Paragon Plus Environment

Page 2 of 26

Page 3 of 26

Environmental Science & Technology

48

Myclobutanil

(MT),

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

pure enantiomers14. These results suggest that the metabolic mechanisms and

63

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

64

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

65

differences between the enantiopure enantiomers and the racemate have not been

66

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

67

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

68

such as glutathione transferase (gst)

69

has rarely been reported.

70

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

71

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

72

investigated the stereospecific distribution, degradation and main chiral metabolites of

16-18

and UDP-glucuronosyltransferase (ugt) 19, 20

3

ACS Paragon Plus Environment

Environmental Science & Technology

73

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

74

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

75

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

76

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

77

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

78

fungicides.

79

Materials and Methods

80

Reagents

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

Extraction of MT and metabolites

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

4

ACS Paragon Plus Environment

Page 4 of 26

Page 5 of 26

Environmental Science & Technology

98

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

99

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

100

0.22 µm filter.

101

Enantioselective LC-MS/MS Analysis

102

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

103

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

104

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

105

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

106

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

107

Gene expression analysis

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

Exposure experiments

119

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

120

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

121

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

122

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

5

ACS Paragon Plus Environment

Environmental Science & Technology

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

analysis of gene expression.

134

Data analysis

135

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

136

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

137

2.0 software (DAS 2.0, Chinese Pharmacological Society of Mathematical

138

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

139

‫ܥ = ܥ‬଴ ݁ ି௞௧ − ‫ି ݁ܭ‬௞ೌ ௧

140

‫ݐ‬ଵ/ଶ =

௟௡ଶ ௞

=

଴.଺ଽଷ ௞

(1) (2)

141

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

142

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

143

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

144

equal to C0.

145

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

146

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

147

EF =

஼ሺశሻషಾ೅ ஼ሺశሻషಾ೅ ା஼ሺషሻషಾ೅

(3)

6

ACS Paragon Plus Environment

Page 6 of 26

Page 7 of 26

Environmental Science & Technology

148

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

149

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

150

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

151

and tukey’s multiple range tests.

152

Results and Discussion

153

Enantioselective distribution and enrichment of MT in rats

154

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

155

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

156

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

157

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

158

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

159

27

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

7

ACS Paragon Plus Environment

Environmental Science & Technology

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

a study31 investigated the metabolism of

193

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

194

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

195

for the two enantiomers were different.

196

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

197

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

14

C-myclobutanil in rats and found the

8

ACS Paragon Plus Environment

Page 8 of 26

Page 9 of 26

Environmental Science & Technology

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

Chiral conversion in tissues

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

because very low concentration of (+)-MT (