Article pubs.acs.org/JAFC
Chemical Modification and Degradation of Atrazine in Medicago sativa through Multiple Pathways Jing Jing Zhang,†,‡ Yi Chen Lu,†,‡ and Hong Yang*,† †
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China
‡
S Supporting Information *
ABSTRACT: Atrazine is a member of the triazine herbicide family intensively used to control weeds for crop production. In this study, atrazine residues and its degraded products in alfalfa (Medicago sativa) were characterized using UPLC-TOF-MS/MS. Most of atrazine absorbed in plants was found as chemically modified derivatives like deisopropylated atrazine (DIA), dehydrogenated atrazine (DHA), or methylated atrazine (MEA), and some atrazine derivatives were conjugated through different functional groups such as sugar, glutathione, and amino acids. Interestingly, the specific conjugates DHA+hGSH (homoglutathione) and MEA-HCl+hGSH in alfalfa were detected. These results suggest that atrazine in alfalfa can be degraded through different pathways. The increased activities of glycosyltransferase and glutathione S-transferase were determined to support the atrazine degradation models. The outcome of the work uncovered the detailed mechanism for the residual atrazine accumulation and degradation in alfalfa and will help to evaluate whether the crop is suitable to be cultivated in the atrazinepolluted soil. KEYWORDS: atrazine, Medicago sativa, toxicity, accumulation, degradation
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INTRODUCTION Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5triazine] is a member of the triazine herbicide family. It is predominantly used for controlling broadleaf weeds in the areas with cultivated graminaceous crops such as corn, sorghum, and sugar cane. Because of its low cost and effective nature in application,1 it is widely used in agronomic practice. It is estimated that atrazine has been used for 40 years in more than 80 countries including the United States and China.2 Atrazine is well dispersed in ground, surface waters, and atmosphere.3 According to the study of Jablonowski et al., atrazine is still detectable twenty-two years after the last application, indicating that atrazine is unexpectedly persistent in soil.4 The massive atrazine residue in ecological systems has resulted in a negative effect not only on crop production5,6 but also on environmental quality.7−10 Alfalfa (Medicago sativa L.) is one of the most important legume crops in temperate areas worldwide; it has a great biological value owing to its high nutritional quality (e.g., high protein storage) and adaptability to different habitats.11 Alfalfa is also commonly used for forage or in crop rotation practices to contribute organic nitrogen to soils via its symbiosis with nitrogen fixing bacteria.12 The wide use and massive residues of atrazine in soil have a great potential to impact alfalfa growth.13 According to previous studies, atrazine, when applied to corn field a year before, could still affect the current rotation atrazine-sensitive crops.14 In this case, farmers using corn− alfalfa rotations have to face the problem that atrazine residues most likely contaminate the alfalfa crops, which may further affect the dairy and livestock production. Thus, investigation on the accumulation and degradation of atrazine in alfalfa is of © 2014 American Chemical Society
great importance from the environmental and agricultural points of view.15 Despite the fact that a certain genotype of crops is sensitive to organic xenobiotics,16 there are genetic differences of the crops in the uptake of herbicides.17 Such differences occur not only among plant species but also in cultivars (or varieties) within the same species.18 Most ecotypes of alfalfa grow under adverse environmental conditions. These diverse ecotypes are adaptive to various environmental conditions such as cold and dry climates and even organic and inorganic polluted soils. For example, recent studies have highlighted alfalfa as a tolerant crop to toxic heavy metals because some of the cultivars are able to remove metal ions from contaminated soil and aqueous solution.19,20 In fact, some plants have developed tolerant mechanisms responsible for removal of organic xenobiotics by taking them up and allowing them to degrade in plants via a certain metabolic pathway.21 This makes it possible that some genotypes of crop even growing in the atrazine-contaminated soils may accumulate the herbicide at a very low level. Herbicide residues in plants can be detoxified by means of conjugation with polar donor metabolic molecules.22 SGlutathionylation and glycosylation with herbicides are the common processes that allow plant attenuation at the toxic levels of toxicants.23−25 However, the mechanism underlining herbicide detoxification and degradation in plants is largely unknown. To date, no report is available on how atrazine is degraded in alfalfa plants. Hence, the aims of this study were Received: Revised: Accepted: Published: 9657
July 6, 2014 September 12, 2014 September 16, 2014 September 16, 2014 dx.doi.org/10.1021/jf503221c | J. Agric. Food Chem. 2014, 62, 9657−9668
Journal of Agricultural and Food Chemistry
Article
Figure 1. Effect of atrazine on the elongation (A), biomass (B), and the content of chlorophyll (C) of Medicago sativa. Medicago sativa seedlings were cultured in the 1/2 strength Hoagland nutrient solution containing atrazine at 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg L−1 for 6 d. Values are the means ± SD (n = 3). Asterisks indicate significant differences between the treatments and the control (p < 0.05). DW: dry weight. FW: fresh weight. spectrophotometrically, and the content of chlorophyll was expressed as mg g−1 fresh weight (FW). Analysis of Atrazine. Alfalfa plants were cultured in 1/2 strength Hoagland nutrient solutions containing 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg L−1 atrazine, respectively. Shoots and roots of plants were separately harvested after atrazine treatment. Fresh shoots or roots (3.0 g) were ground and dissolved in 15 mL of acetone−water (3:1, v:v) and ultrasonicated for 30 min. The homogenate was centrifuged at 4000g for 7 min and filtrated. The above steps were repeated in triplicate. The filtrate was vaporized to remove acetone at 40 °C using a rotary vacuum evaporator. The residual water was partitioned in petroleum ether three times, each time with 10 mL of the solvent. The petroleum ether was concentrated to dryness at 40 °C. The residue was dissolved in 0.5 mL of methanol and diluted with 20 mL of water. The mixture was passed through an LC-18 solid-phase extraction (SPE) column. Eluates were discarded, and the column was washed with 2 mL of methanol−water (80:20, v:v). The washing solution was collected for analysis of high performance liquid chromatography (HPLC, Waters 515; Waters Technologies Co. Ltd., USA) with ultraviolet (UV) detector. The operating conditions of HPLC were as follows: wavelength, 225 nm; room temperature; Hypersil reversedphase C18 column (Thermo, 250 mm × 4.6 mm i.d.); mobile phase, methanol:water (70:30, v:v); flow rate, 0.6 mL min−1; injection volume, 20 μL. Atrazine in the plant-culture solution was also measured with the LC-C18 SPE column as described above. The spiked recovery and determination limit of the above method are shown in Table S1 in the Suppporting Information. Assay of Enzyme Activities. Fresh shoot (1 g) or root tissues (1.5 g) were homogenized in 6 mL of ice-cold sodium phosphate buffer (50 mM, pH 7.8) containing 1 mM EDTA and 1% (w/w) polyvinylpyrrolidone. The homogenate was centrifuged at 12000g at 4 °C for 20 min. Glutathione S-transferase (GST, EC 2.5.1.18) activity
(1) to assess the ability of alfalfa to accumulate and degrade atrazine and (2) to investigate the possible atrazine catabolic pathway with detailed characterization of atrazine derivatives or atrazine-conjugated products in alfalfa.
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MATERIALS AND METHODS
Materials and Treatments. The herbicide atrazine was obtained from the Institute of Pesticide Science, Academy of Agricultural Sciences in Nanjing, China, with 99% purity. Alfalfa seeds (Medicago sativa, cv. Golden Empress) were surface-sterilized with 5% solution of sodium hypochlorite for 15 min, rinsed with distilled water, and placed on moist filter paper for germination. Germinating seeds were placed on a plastic net floating on distilled water in the dark at 25 °C for 2 d. Then, uniform seedlings were transferred to black polyvinyl chloride pots (1 L) that contained 1/2 strength Hoagland nutrient solution.26 Culture solutions were renewed every 2 days. When the third true leaf was well developed, seedlings were treated with atrazine at 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg L−1 for 6 d, respectively, in 1/2 strength nutrient solution. Treatment solutions were renewed every 2 d. Each pot contained 30 seedlings, which were grown in a growth chamber under the conditions of 14 h photoperiod, 200 μmol of photons m−2 s−1, and 25/20 °C (day/night). Each treatment was repeated thrice. Determination of Growth and Chlorophyll Content. The elongation of shoots and roots of fresh individual plants was measured with a normal ruler. A forced-air oven was used for determination of biomass. Fresh shoots and roots were dried at 105 °C for 20 min, then oven-dried at 80 °C for 48 h, and weighed afterward.26 Chlorophyll was assayed according to the method of extraction with 80% acetone27 Fresh leaves (0.1 g) were extracted with 8 mL of 80% acetone (pH 7.8). After extracting solution was centrifuged at 5000g for 10 min, the supernatant was collected, the chlorophyll content was assayed 9658
dx.doi.org/10.1021/jf503221c | J. Agric. Food Chem. 2014, 62, 9657−9668
Journal of Agricultural and Food Chemistry
Article
Figure 2. Accumulation of atrazine in shoots (A) and roots (B) of Medicago sativa. Alfalfa seedlings were cultured in the 1/2 strength Hoagland nutrient solution containing atrazine at 0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg L−1 for 2, 4, and 6 d, respectively. Values are the means ± SD (n = 3). was determined by the method of Song et al.28 The reaction mixture consisted of 3 mL of 100 mM sodium phosphate buffer (pH 6.5) containing 1 mM GSH, 75 μL of 40 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 100 μL of enzyme extract. The change in absorbance was recorded at 340 nm during 3 min. Fresh shoots or roots (1 g) were ground and extracted with 1 mM EDTA, 50 mM NaCl, 1% (w/v) polyvinylpolypyrrolidone, and 50 mM Tris−HCl (pH 8.0). The homogenate was centrifuged at 10000g at 4 °C for 30 min. The supernatant was collected as crude extract for measurement of UDP-glycosyltransferases (GTs, EC 2.4.x.y).9,29 The 250 μL of reaction mixture contained 100 μL of the enzymatic extraction, 0.04 mM p-nitrophenol, and 2 mM UDP-glucose. The reaction was carried out at 30 °C for 2 h, terminated by addition of 250 μL of methanol, and chilled to −20 °C for 0.5 h. The reduced rate of the p-nitrophenol concentration was assayed by HPLC. One unit of the GT activity was defined as the amount of enzyme activity needed to consume 1 mol of p-nitrophenol per minute. The protein concentration in enzyme extract was assayed by the method of dyebinding according to Bradford.30 Analysis of Atrazine Metabolites and Conjugates in Alfalfa. Fresh shoots or roots (7.0 g) were ground with liquid nitrogen. The process of extraction and purification was run with the same analytical method indicated above except petroleum ether extraction. The washing solution for the LC-18 column was collected for analysis. Metabolites and conjugates of atrazine in alfalfa were analyzed using ultrahigh performance liquid chromatography (UHPLC) (Thermo, USA) coupled to a linear ion trap−Orbitrap hybrid mass spectrometer (LTQ Orbitrap XL) equipped with a heated-electrospray ionization probe. Instrument control was through Tune 2.6.0 and Chromeleon programs. Separations were performed on a Hypersil gold C18 (100 mm × 2.10 mm, 3 μm particle size, Thermo Fisher Scientific). Mobile phase was composed of (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. A linear gradient program was performed in 36 min at a flow rate of 0.20 mL/min under the following conditions: 5% B for 1 min, 1−15 min from 5% to 35% B, 15−25 min from 35% to 95% B, 95% B for 5 min, 30−31 min from 95% to 5% B, and 5% B for 5 min. Column oven and autosampler temperature were set at 35 and 10 °C, respectively. The injection volume was 10 μL. The mass spectrometer was operated in negative mode. HESIsource parameters were as follows: capillary temperature 300 °C, the source voltage 4 kV, and auxiliary gas 25. Accurate mass spectra were recorded from 100 to 800 m/z. For fragmentation study, a data dependent scan was performed by deploying collision-induced dissociation (CID). The product ions were generated by the LTQ ion trap at normalized collision energy of 35% and q-activation of 0.25 using an isolation width of 2 Da. The external mass calibration of the Orbitrap was performed once a week to ensure a working mass accuracy
