Enantioselective Bioaccumulation, Tissue Distribution, and Toxic

Mar 14, 2017 - Effects of Myclobutanil Enantiomers in Pelophylax nigromaculatus. Tadpole. Cheng Cheng, Shanshan Di, Li Chen, Wenjun Zhang, Jinling Dia...
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Enantioselective Bioaccumulation, Tissue Distribution, and Toxic Effects of Myclobutanil Enantiomers in Pelophylax nigromaculatus Tadpole Cheng Cheng, Shanshan Di, Li Chen, Wenjun Zhang, Jinling Diao, and Zhiqiang Zhou* Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China ABSTRACT: Research on the enantioselective behavior of chiral pesticides on amphibians has received growing attention, because amphibians are experiencing a population decline and amphibian metamorphosis shares many similarities with human fetal development. In this study, the enantioselective behavior of myclobutanil on Pelophylax nigromaculatus tadpole was studied. The antioxidant enzyme (SOD, GST) activities and malondialdehyde (MDA) content were investigated to assess the different toxic effects when tadpoles were exposed to myclobutanil enantiomers for 96 h. In the chronic exposure experiment, the bioaccumulation concentration of (−)-myclobutanil in tadpoles is significantly higher than that of (+)-myclobutanil, and the concentration of myclobutanil in tadpole intestine and liver was higher compared with other tissues. During the elimination experiment, about 95% of myclobutanil in tadpoles was eliminated within only 24 h. On the basis of these data, the enantiomeric differences should be taken into consideration in the risk assessment of myclobutanil. KEYWORDS: myclobutanil, tadpole, enantioselective, bioaccumulation, toxic effect

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INTRODUCTION Environmental contamination by pesticides and their residues has become an issue of general concern, because the ecosystem and human health can be adversely affected by these compounds. Previous studies indicate that most applied pesticides do not reach their targets but are lost into the environment via surface runoff and infiltration.1 As a result, the massive use of pesticides is causing severe accumulation of the pesticides in the aquatic environment, including underground streams, rivers, lakes, or even coastal sea waters.2 In China, >40% of currently used pesticides are chiral pesticides.2 They have the same physicochemical properties and consist of two or more enantiomers.3 However, the enantiomers may vary in environmental behaviors, excretion, metabolism, toxicity, and bioactivity.4−6 In the practical application, many chiral pesticides are often released as racemates into the environment, although only one enantiomer has active effects.7 Actually, the existing knowledge of racemate pesticides cannot sufficiently describe their ecological risks and actual environmental fate,7 so study of the potentially detrimental effects of chiral pesticide enantiomers on aquatic organisms such as tadpoles is required. Amphibians have experienced population declines and even extirpations globally from such pervasive stressors as overexploitation, contaminants, and habitat loss.8 Around the world, the present data of amphibian population show significant declines.9 Nearly 600 amphibian populations studied in western Europe show 53% declines beginning in the 1950s.8 In New Zealand and Australia, 70% of the populations have shown declines. In South and North America, as much as 60 and 54% of the amphibian populations studied have declined, respectively. During the years 2000 and 2004, the number of extinctions of amphibians has increased further, and there are 34 species that are considered to be extinct.8 Pesticides in particular appear to be a major threat to amphibians,10,11 but © XXXX American Chemical Society

the mechanism of their role remains enigmatic. The amphibians are sensitive to pesticides because of their unshelled eggs, highly permeable skin, and exposure to terrestrial and aquatic environments at different life stages.12 In addition, the effects of sublethal concentrations are more relevant to amphibian communities because they may directly affect size and time of metamorphosis or indirectly affect survival.13 Moreover, after amphibians have been exposed to sublethal concentrations of pesticides, the amphibian species have exhibited a variety of physiological, histological, or biochemical alterations, leading to morphological abnormalities, delayed development, retardation in growth, and reduction in numbers.14−16 Therefore, they have been regarded as bioindicators, and to evaluate the effects of chemicals on both agricultural and aquatic ecosystems, they are broadly used as test animals.17 The black-spotted frog (Pelophylax nigromaculatus) is a very widespread and abundant species in China, and it lives in a variety of habitats including agricultural sites such as paddy fields. The lifespan of the P. nigromaculatus tadpole is short, but this time is generally the time of pesticide application in spring. Thus, the affects of the pesticides on P. nigromaculatus individuals start from the beginning of their lives. In this study, we take P. nigromaculatus tadpoles as a target for the investigation. Moreover, amphibian metamorphosis shares many similarities with vertebrate development, such as brain development, intestinal remodeling, and bone differentiation.18,19 In addition, because the difficulty of manipulating uterus-enclosed embryos to investigate interferences from maternal hormones can be eliminated, the free-living tadpoles are a favorable alternative Received: Revised: Accepted: Published: A

January 7, 2017 March 1, 2017 March 14, 2017 March 14, 2017 DOI: 10.1021/acs.jafc.7b00086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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over mammalian models.20 Therefore, amphibian metamorphosis serves as an ideal model to simulate the process of human fetal development. Additionally, environmental contamination by pesticides in aquatic ecosystems may cause bioaccumulation of the pesticides in the aquatic organisms, and via the food web those aquatic organisms may likely affect higher trophic levels such as pregnant women.21 Therefore, investigating the effect of pesticides on tadpoles may reflect the effects of pesticides on the human fetus to a certain extent. Myclobutanil, (RS)-2-(4-chlorophenyl)-2-(1H-1,2,4-triazol1-ylmethyl) hexanenitrile (Figure 1), consists of a pair of

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MATERIALS AND METHODS

Chemicals and Reagents. All analytical grade reagents were from Yili Fine Chemicals (Beijing, China). Water was purified by a Millipore Purification Systems (Milli-Q system). 3-Aminobenzoic acid ethyl ester (MS-222) was provided by Sigma-Aldrich (USA). racMyclobutanil (purity ≥ 99.0%) was obtained from the China Ministry of Agriculture’s Institute for Control of Agrochemicals. The enantiomers of myclobutanil were prepared on an Agilent highperformance liquid chromatograph (HPLC) with a chiral column (ADMPC-CSP) that was provided by the College of Science, China Agricultural University, Beijing. Care and Breeding of Animals. P. nigromaculatus tadpoles were obtained from State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (Beijing, China). Tadpoles were reared under controlled laboratory conditions of water temperature of 22 ± 1 °C and 12/12 h light/dark cycles. The dechlorinated tap water quality was as follows: dissolved oxygen concentration, >5 mg L−1, pH 7.3− 7.8, and water hardness (CaCO3), approximately 150 mg L−1. Tadpoles were fed commercially available Labdiet Frog Diet (Labdiet, America) twice daily throughout the chronic exposure and elimination test, whereas tadpoles were not fed in the acute exposure test. Healthy tadpoles (Gonser stage 26)26 were randomly selected for the exposure experiment. Acute Exposure Experiment. The tadpoles were exposed to various sublethal concentrations of rac-myclobutanil, (+)-myclobutanil, and (−)-myclobutanil (0, 7, 8, 9, and 10 mg/L). Each treatment was performed in triplicate. Test solutions were prepared by diluting the stock solutions with dechlorinated tap water containing 0.01% (v/ v) dimethyl sulfoxide. The control solvent group received the same amount of dimethyl sulfoxide. Ten tadpoles for each group were treated in a glass beaker containing 500 mL of solution. The medium was changed daily using freshly prepared solutions. Dead tadpoles were recorded and removed. The assay was performed for 96 h. The design of the experiment refers to the standard practice for conducting acute tests with fishes, macroinverbrates, and amphibians (ASTM 1993). The tadpoles were anesthetized with 100 mg/L MS-222 and then stored at −20 °C prior to biochemical analyses (SOD, GST, MDA). Chronic Exposure and Elimination Experiment. This experiment comprised bioaccumulation, tissue distribution, and removal assay of myclobutail. The bioaccumulation assay is to determine the uptake kinetics; the distribution assay is to examine the bioaccumulation in different tissues of tadpoles; and the removal assay in clean water is to study the elimination kinetics. In the chronic exposure experiment, 150 tadpoles were placed in 5 L of dechlorinated water spiked with 3 mg/L of rac-myclobutanil for 117 days of exposure. Each treatment was performed in triplicate. All solutions were renewed every 2 days. The tadpole samples were anesthetized and collected at time points of 4, 10, 26, 46, 58, 69, 80, 97, and 117 days during the exposure. At the end of the exposure, tadpoles were anesthetized, and whole-body, liver, intestine, lung, gill, tail, and residual carcass were collected prior to the tissue distribution assay. After exposure for 26 days, 35 tadpoles were cleaned and removed to a clean beaker containing 5 L of dechlorinated water for the elimination experiment. Each treatment was performed in triplicate. The solutions were renewed every 2 days. The tadpole samples were anesthetized and collected at time points of 1, 2, 3, 5, 7, 10, and 16 days during the elimination experiment. Preparation of Protein Extracts. The extraction and measurment of proteins were carried out according to the method of Bradford.27 The tadpole samples were put into a prechilled mortar and homogenized in 4 mL of 50 mM phosphate buffer (pH 7.8). The disrupted samples were then centrifuged at 10000 rpm for 10 min at 4 °C, and the supernatant protein extract was stored at −80 °C. Bovine serum albumin was used as the standard to determine the total soluble protein concentration. Determination of Antioxidant Enzyme Activity. The determination of superoxide dismutase (SOD) activity was performed

Figure 1. Chemical structures of myclobutanil stereoisomers.

enantiomers and has an asymmetrically substituted C atom. It is a kind of broad-spectrum systemic triazole fungicide and a sterol demethylation inhibitor with protective and curative action.22 Although it has a low acute toxicity, myclobutanil has been found to disrupt steroid hormone homeostasis in rodents, cause varying degrees of hepatic toxicity, and affect the reproductive abilities of test animal in vivo models.5 At present, myclobutanil is widely used as a fungicide in many countries. It is used to control plant diseases, such as rice sheath blight, rice false smut,23 and Rhizoctonia solani Kuhn24 in paddy fields. Moreover, P. nigromaculatus often inhabits the paddy field, which increases the likelihood of contact with myclobutanil. Therefore, we designed a high-concentration acute exposure experiment to simulate the situation of paddy water just after spraying pesticide and a low-concentration chronic exposure experiment like that of a long-term situation of paddy water after pesticide application. After some time, because of digestion, there will be little or no pesticide in the paddy water, so we also designed an elimination experiment in clean water. Furthermore, the extensive use of myclobutanil may result in its direct (water) or indirect (food web) exposure to humans. Amphibian metamorphosis is seen as an ideal model to simulate the process of human fetal development. Therefore, investigating the potential effects of myclobutanil on tadpoles will give some valuable reference data for the studies on human fetal exposure to myclobutanil. In this study, we assessed the sublethal effects of myclobutanil on P. nigromaculatus tadpoles. Different biomarkers such as antioxidant enzyme (SOD, GST) activities and malondialdehyde (MDA) content in P. nigromaculatus tadpoles were investigated when the tadpoles were treated with myclobutanil for 96 h. This information is important to provide clues on the organisms’ ability to cope with oxidative stress and may further serve as an early-warning indicator of contamination.25 We also investigated the enantioselective bioaccumulation, distribution, and elimination of myclobutanil enantiomers in tadpoles in a chronic exposure experiment. B

DOI: 10.1021/acs.jafc.7b00086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry according to the method of Giannopolitis and Ries,28 which was based on its ability to inhibit nitroblue tetrazolium (NBT) reducing. Three milliliters of reaction mixture contained 0.1 mM EDTA-Na2, 50 mM phosphate buffer (pH 7.8), 130 mM methionine (Met), 0.75 mol/L tetrazolium (NBT), 20 mM riboflavin, and an appropriate aliquot of enzyme extract. The reaction mixtures were illuminated at a light intensity of 4000 lx for 20 min, and then the optical density at 560 nm light wavelength was analyzed. Glutathione-S-transferase (GST) activity was measured spectrophotometrically using 1 mmol/L 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate and 2 mmol/L GSH in phosphate buffer saline (PBS, pH 7.2), according to the method of Habig et al.29 Determination of Malondialdehyde. MDA content was assayed according to the report by Heath and Packer.30 In the ice bath, the tadpole sample was homogenized with 1 mL of 0.1% TCA and then centrifuged at 10000 rpm for 10 min. The reaction mixtures containing 5% TCA, which included 0.5% TBA and the supernatant, were kept in boiling water for 15 min and then centrifuged at 4500 rpm for 10 min. The MDA content was calculated based on A532 − A600 using the extinction coefficient of 155 mmol/cm. Chemical Analysis. All of the tadpole samples were anesthetized with MS-222 and rinsed with distilled water, blotted dry gently with a tissue, and weighed. Then, they were homogenized and vortex-mixed with 3 mL of ethyl acetate for 3 min, exposed to ultrasonic vibration for 15 min, and then centrifuged at 4000 rpm for 3 min. The sample was re-extracted in the same way twice, and the supernatants were combined and evaporated to dryness under a gentle nitrogen stream. All of the samples were redissolved in acetonitrile and purified with nhexane. The acetonitrile layer was passed through a 0.22 μm filter into a sample vial for HPLC-MS/MS analysis. HPLC-MS/MS analyses were performed using an instrument from Thermo Fisher Scientific (Waltham, MA, USA). Myclobutanil enantiomers were separated on a LuxCellulose-1 column (4.6 × 250 mm i.d.; Phenomenex, China). The mobile phase was a mixture of 70% acetonitrile and 30% water at a flow rate of 0.5 mL/min. Chromatographic separation was conducted at 40 °C. The HESI source and mass spectrometer were used with the selected reaction monitoring (SRM) mode in a positive-ion mode to determine the concentrations of myclobutanil. Transition m/z 289 → 70 was used for quantification, m/z 289 → 125 was used for confirmation, and collision energies were 18 and 31 eV, respectively. The detection limit of myclobutanil was 0.1 μg/L, and the mean recoveries were 84− 107%. Results of the quality control showed that the method was successfully used for the analyses of myclobutanil in tadpoles. Date Analysis. Data were presented as the mean ± SD of the mean and were tested for statistical significance using analysis of variance (ANOVA) followed by SPSS 17.0. The means were considered significantly different when the probability (P) was liver > whole body > lung > residual carcass > tail > gill. Similarly, Mann et al.49 reported that the content of Cd in European lacertid lizard gut and liver was higher after chronic exposure to Cd-contaminated food. Liu et al.48 also found a higher concentration of erythromycin in the liver than in the muscle

Figure 4. Malondialdehyde (MDA) content in tadpoles exposed to different concentrations of the racemate and enantiomers of myclobutanil (96 h). Different letters (a−c) represent statistically significant differences among enantiomers and racemate (SNK) at P < 0.05; ∗ denotes significant difference between control and treatments (SNK) at P < 0.05. D

DOI: 10.1021/acs.jafc.7b00086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

bioaccumulation concentration increased gradually and eventually remained stable. At the end of the experiment, the concentrations of (−)-myclobutanil and (+)-myclobutanil were 0.338 ± 0.048 and 0.228 ± 0.029 mg/L, respectively. After 46 days of exposure, there was a significant trend of enantioselective bioaccumulation in the tadpoles with a preferential accumulation of (−)-myclobutanil. It was consistent with that in bioaccumulation of myclobutanil to Tenebrio molitor larvae,50 in which the concentration of (−)-myclobutanil in T. molitor larvae was higher than that of (+)-myclobutanil for the 20 mg/kg dietary exposure experiment. This may be due to the type and/or content of enzymes in tadpoles changing during development. After exposure for 26 days, some tadpoles were withdrawn from the exposed aquaria and transferred to the myclobutanilfree water for the elimination experiment. The depuration of myclobutanil enantiomers in tadpole samples is shown in Figure 8. Both enantiomers were rapidly eliminated by 95%

after exposure for 28 days. The higher concentration of myclobutanil in the liver than in the whole body suggests that the liver detoxification mechanism may be an important clearance mechanism for this chemical. The concentration of myclobutanil in the intestine is the highest. The possible reason is that it could be exposed to myclobutanil via the feeding system, and it has a better absorption function. Moreover, the enterohepatic circulation may also be one of the reasons.45 Furthermore, only in the liver, intestine, and whole body is the bioaccumulation concentration of (−)-myclobutanil significantly higher than that of (+)-myclobutanil. The reason may be that in the liver and intestine myclobutanil is metabolized and/ or absorbed enantioselectively. Bioaccumulation and Elimination of Myclobutanil in Tadpoles. As seen from Figure 6, P. nigromaculatus tadpoles

Figure 6. Bioaccumulation concentrations of myclobutanil enantiomers in tadpole samples. ∗ denotes significant differences between two enantiomers (SNK) at P < 0.05.

readily accumulated myclobutanil from the surrounding water. At the first sample time point (4 days), the detected concentrations of (−)-myclobutanil and (+)-myclobutanil in tadpoles were 0.175 ± 0.002 and 0.184 ± 0.012 mg/L, respectively. After that, the changes of (−)-myclobutanil and (+)-myclobutanil concentration in tadpoles showed a fluctuation trend until the 97th day of exposure. The reason for this phenomenon may be the growth dilution. The fresh weight− time curve of tadpoles exposed to myclobutanil for 117 days is presented in Figure 7. It was observed that when the mass growth rate increased, the bioaccumulation concentration decreased. It could be explained that the bioaccumulation rate is lower than the mass growth rate, resulting in dilution of the bioaccumulation concentration. After exposure for 80 days, the weight of tadpoles was almost steady, and the

Figure 8. Elimination curves for myclobutanil enantiomers in tadpole samples.

within only 24 h and finally reached their noneliminated residue levels after 10 days of depuration, that is, 0.770 ± 0.310 μg/kg (+)-myclobutanil and 0.623 ± 0.440 μg/kg (−)-myclobutanil. Although myclobutanil was eliminated rapidly, it still leaves noneliminated residues in tadpoles that may influence the future growth and development of tadpoles.

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AUTHOR INFORMATION

Corresponding Author

*(Z.Z.) Phone: +8610-62733547. Fax: +8610-62733547. Email: [email protected]. ORCID

Zhiqiang Zhou: 0000-0002-0816-6203 Funding

This work was supported by funds from the National Natural Science Foundation of China (Contract Grant 21577171). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED SOD, superoxide dismutase; GST, glutathione-S-transferase; MDA, malondialdehyde; ROS, reactive oxygen species Figure 7. Fresh weight−time curve of tadpoles in the chronic exposure experiment.

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DOI: 10.1021/acs.jafc.7b00086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.7b00086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX