Appl Biochem Biotechnol DOI 10.1007/s12010-013-0662-8

DNA Damage and Effects on Antioxidative Enzymes in Earthworm (Eisenia fetida) Induced by Flumorph Xiangyu Cao & Chao Yang & Jianli Liu & Xiujuan Hui & Wei Yang & Shuangshuang Li & Yanan Tian & Leiming Cai

Received: 6 September 2013 / Accepted: 28 November 2013 # Springer Science+Business Media New York 2013

Abstract Flumorph is an Oomycete fungicide, which is used extensively as an effective fungicide in vegetables and fruits, but little is known about its effect on nontarget soil organisms. In the present study, biochemical responses including changes in the activity of antioxidative enzymes catalase (CAT), superoxide dismutase (SOD), glutathione-Stransferase (GST), malondialdehyde (MDA), and DNA damage induced by flumorph were investigated in earthworms (Eisenis fetida). The CAT concentrations were stimulated at 5.0 mg kg−1 over 28 days and inhibited at 10 and 20 mg kg−1, except 10 mg kg−1 on days 21 and 28 compared with the controls. The overall SOD activities were inhibited except 5 mg kg−1 on day 28 and 10 mg kg−1 on days 7 and 14. Meanwhile, the GST activities were stimulated on day 7 and decreased on the other days in summary. The MDA activities were increased notably at 5, 10, and 20 mg kg−1 after 14 days. Clear dose-dependent DNA damage to Eisenia fetida was observed by olive tail moments in comet assay compared with controls. The results demonstrate that flumorph induces oxidative stress and DNA damage to earthworms, and the effects may be the important mechanisms of its toxicity. Keywords Flumorph . Earthworm . Oxidative stress . Genotoxicity . Comet assay

X. Cao : J. Liu : W. Yang School of Life Science, Liaoning University, Shenyang 110036, China C. Yang Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China X. Hui (*) : S. Li : Y. Tian School of Environment Science, Liaoning University, Shenyang 110036, China e-mail: [email protected] L. Cai National Shenyang Center for New Drug Safety Evaluation Research Center, Shenyang 110021 Liaoning, China

Appl Biochem Biotechnol

Introduction In worldwide agriculture, there is an increasing concern about soil contamination and other contaminations due to the widespread use of agricultural fungicide [1]. Although they were designed for specific targets, when introduced to the environment, they affect nontarget organisms [2]. It has been shown that soil contamination with fungicides can be detrimental to earthworm populations, which are important biological monitor in determining the ecological hazards that resulted from pesticidecontaminated soil in ecotoxicological studies [3, 4]. Therefore, understanding the adverse effects of fungicides on earthworms is essential to predict the potential food chain effects on soil contamination [1]. Flumorph,(4-[3-(3,4-dimethoxyphenyl)-3-(4-fluorophenyl)-1-oxo-2-propenyl]morpholine), is introduced as an Oomycete carboxylic acid amide fungicide. It is structurally analogous to dimethomorph and has been classified into morpholine group with dimethomorph based on its chemical structure. Both of its isomers (50 % Z-isomer, 50 % E-isomer) have considerable fungicide activities against Peronospora and Phytophthora diseases at a dose rate of 100–200 g ai/ha. It was developed by Shenyang Research Institute of Chemical Industry (Shenyang, China) in 1994 and has been granted patents in China (ZL.96115551.5), the USA (US6020332), and Europe (0860438B1) [5–7]. Although flumorph is a novel fungicide, its toxicity is barely studied. In Hu et al., no degradations in buffers of various pH values or water with different physical and chemical properties were observed. Meanwhile, the assessment of flumorph photodegradation in various aqueous solutions under normal and controlled conditions indicated that photoreaction was an important dissipation pathway for flumorph in natural water system [8]. Hu et al. suggested that flumorph may be involved in the impairment of cell polar growth by directly or indirectly disrupting the organization of F-actin [9]. Flumorph was also concluded to have thyroid disruption effects and likely a thyroid disrupter [10]. However, data on the biochemical response and genotoxicity to earthworms is very limited as flumorph is becoming widely used. Therefore, additional studies must be performed on flumorph toxicity. Biochemical responses in organisms against environmental stress are regarded as early warning indices of pollution in the environment. Many enzymatic activities have been considered as biomarkers of environmental pollution [1]. It has been reported that reactive oxygen species (ROS) can be generated in living organisms exposed to environmental contaminants. The increased level of ROS can produce oxidative damage to macromolecules such as proteins, nucleic acids, and lipids eventually leading to the damage of cell machineries [11]. Some of these damages have been attributed to changes in the activities of ROS scavenging enzymes, such as catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST). Therefore, changes of these enzymatic activities are good indicators of the toxic effects of contaminants on living organisms. Comet assay, developed by Singh et al. [12] on the basis of a neutral electrophoresis system, is a widely used technique to detect DNA damage caused by environmental stress. It has been acknowledged by different scientific communities that the comet assay is a simple, reliable, and straightforward method for the detection of genotoxins in a wide variety of eukaryotic cells [1, 13, 14]. Whether flumorph has potential biochemical response and genotoxicity to earthworms or it is nontoxic to no-target soil organisms has not been investigated. Therefore, the aim of this study was to evaluate the toxicity of flumorph, particularly on the oxidative stress and DNA damage in earthworms.

Appl Biochem Biotechnol

Materials and Methods Material and Chemicals Flumorph of 95 % purity was obtained from Shenyang Research Institute of Chemical Industry. All other chemicals were of reagent grade and purchased from Sigma (St. Louis, MO, USA). Toxicological Tests The earthworm (Eisenia fetida) species was selected as the test organism. After being purchased from ChengGong earthworm cultivation farm in Dongli District, Tianjin, China, the earthworms were acclimated for 2 weeks in laboratory. Before test, the earthworms were rinsed in distilled water to allow voiding of gut contents. Adults weighing about 350 mg (live weight) were selected for all experiments. The Organization for Economic Co-operation and Development (OECD) artificial soil was included in this study, and sample composition strictly followed the OECD guideline: 10 % sphagnum peat moss, 20 % kaolin clay, and 70 % sand. The soil moisture was adjusted to 30 % using distilled water [15]. Flumorph was dissolved in acetone and mixed into OECD artificial soil at rates of 5, 10, and 20 mg kg−1. The control group was mixed with the same volume of acetone. Three replicates were set for each treatment group and the control group. Each pot was filled with 750 g of OECD artificial soil (wet weight), and 25 earthworms had been incubated in the OECD artificial soil. Five earthworm specimens were assigned for enzyme and comet assay, respectively, and then collected on days 7, 14, 21, and 28 following the application of flumorph. The tests were all conducted in a chamber with manual climatic control with the temperature of 22 ± 1 °C, moisture of 75 ± 2 %, and a photoperiod of 12 h light/12 h dark. For the duration of the experiment, the moist content was kept by the regular spraying of distilled water. Mortality had not yet been observed during the process. Preparation of Earthworm Extracts All procedures were carried out at 4 °C. Earthworms were placed into a prechilled mortar and pestled under ice-cold conditions in 50 mM phosphate buffer (1:9, w/v), pH 7.0. The homogenate was centrifuged at 9,000 rpm for 30 min. The supernatant was used for the assay of enzyme activity and protein examination. Biochemical Assays The activity of CAT was determined as described by Song et al. [1]. A solution of H2O2 was used as substrate for the enzyme. The enzyme activity was calculated from the decrease in ultraviolet absorption at 250 nm, following the degradation of H2O2 by CAT present in the sample. One unit of CAT activity was defined as the enzyme quantity required consuming half of H2O2 in 100 s at 25 °C. The activity of SOD was measured to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) as described by Giannopolitis and Ries with slight modification [16]. One unit of the SOD activity was considered to be the amount of the enzyme required to inhibit NBT reduction by 50 %, and the result was expressed as units per milligram of fresh mass (FM). Absorbance of the reaction mixture was read at 560 nm.

Appl Biochem Biotechnol

The activity of GST was assayed according to the method of Saint-Denis et al. [17]. It was measured using 1-chloro-2, 4-dinitro-benzene as its substrate. One unit of the GST activity is defined as the amount of the enzyme catalyzing the formation of 1 μmol of the product per min under the assay conditions. Estimation of MDA content was based on the formation of thiobarbituric acid reactive substances according to the method described by Livingstone et al. [18] and expressed as nanomoles per gram. The supernatant was mixed with 0.75 % thiobarbituric acid and heated at 100 °C for 15 min. Following further addition of 70 % trichloroacetic acid, the malonaldehyde-like lipid peroxide products were quantified by reference to malonaldehyde bis-(dimethylacetal) standards. Comet Assay After being exposed to the pollutant at various levels, earthworm coelomocytes were obtained using the noninvasive extrusion method as described by Eyambe et al. [19]. Individual earthworms were rinsed in the extrusion medium (5 % ethanol, 95 % saline, 2.5 mg mL−1 EDTA, and 10 mg mL−1 guaiacol glyceryl ether, pH 7.3). Coelomocytes were spontaneously secreted in the medium and washed with phosphate-buffered saline (PBS) for 3 min, and the final cell density was adjusted to about 1×105–1×106 cells mL−1 with PBS. Viability of the cells obtained by the tryphan blue exclusion method was in the range of ≥90 % for all groups. The cells were collected by centrifugation 9,000 rpm, 10 min at 4 °C, and placed on ice prior to the comet assay. An alkaline comet assay was performed according to Singh et al. [12] with slight modifications. All steps were conducted under dim yellow light and performed at 4 °C to prevent additional DNA damage. In our study, the basal layer of 35 μL of NMA was added to increase the adhesion of the colloidal before the first layer, and then the cell suspension was mixed with 65 μL of 0.7 % (w/v in PBS) low melting agar (LMA) in PBS at 37 °C and pipetted onto fully frosted slides precoated with a layer of 75 μL 0.8 (w/v in PBS) normal melting agar (NMA). After solidification on ice for 15 min, another layer of 60 μL LMA was added, and the slides were immersed into a lysis solution for 1.5 h (4 °C, 2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10.0), 10 % dimethyl sulfoxide and 1 % Triton X-100 just before use. Slides were then incubated in an electrophoresis tank containing 300 mM NaOH with 0.2 mM Na2EDTA (pH 10.0) for 40 min at 4 °C prior to electrophoresis in the same buffer for 20 min at 25 V (300 mA). The slides were then neutralized (0.4 M Tris, pH 7.5) thrice at 5-min intervals and stained with 40 μL ethidium bromide (20 μL mL−1) for fluorescence microscopy analysis (BX50/BX-FLA fluorescence microscope) using a digital imaging system. For each parallel set of slides, the randomly select and nonoverlapping cells were captured at ×400 magnification, and a total of 200 cells were scored for each sample. The images of the comet assay were analyzed lastly using CASP [20]. The parameter used to quantify the extent of DNA damage was the olive tail moment (OTM). OTM is the product of the distance between the center of gravity of the head and the center of gravity of the tail and the percent tail DNA [1, 25]. Statistical Analysis Each treatment was performed in triplicate and the values are presented as the means±standard deviation (SD). Independent-sample t tests were performed to evaluate the statistical significance of the results using the SPSS software (SPSS 15.0).

Appl Biochem Biotechnol

Results Biochemical Assays The possible biochemical effects of flumorph on the activity of various enzymes in E. fetida were evaluated, and the relevant results are shown in Figs. 1, 2, 3, and 4, respectively. As shown in Fig. 1, the CAT activity changed depending on flumorph concentration and exposure duration. The CAT activity at 5 mg kg−1 flumorph was remarkably higher than the control over the course of the experiment. The CAT activity at 10 mg kg−1 flumorph was lowered on days 7 and 14, and increased on days 21 and 28. The CAT activity of the 20 mg kg−1 was lower than the control group, and statistically significant differences (p