ARTICLE pubs.acs.org/est
Enantioselectivity Tuning of Chiral Herbicide Dichlorprop by Copper: Roles of Reactive Oxygen Species Yuezhong Wen,^ † Hui Chen,^ † Chensi Shen,† Meirong Zhao,§ and Weiping Liu‡,* ,
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†
Institute of Environmental Sciences and ‡Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China § Research Center of Green Chirality, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China
bS Supporting Information ABSTRACT: Reactive oxygen species (ROS) are considered to be the key players in cell toxicity. However, cross talk between the enantioselective toxicity of pesticides, heavy metals, and ROS is poorly understood. To decipher the puzzle, the effects of copper (Cu) on the enantioselective ecotoxicity of the chiral pesticide dichlorprop (DCPP) to Scenedesmus obliquus were investigated. The results showed that the presence of DCPP and Cu, both individually and in combination, caused a sudden increase of ROS. This in turn stimulated the response of antioxidant defenses, impaired subcellular structure and physiological function, and finally resulted in cell growth inhibition. In the absence of Cu, ROS production after exposure to the herbicidally active (R)-enantiomer was higher than that of the (S)-enantiomer, suggesting a preference for an (R)-enantiomerinduced production of ROS. When DCPP and Cu were both added to algae simultaneously, (R)-DCPP preferentially induced production of ROS was observed. However, the enantioselective induced production of ROS was reversed when DCPP was mixed with Cu for 24 h prior to addition to the algae solution. It was also found that the generation of ROS, antioxidant response, and growth inhibition rate in Scenedesmus obliquus were all (R)-enantiomer preferentially induced. These findings implied that ROS play a primary role in chemical contaminant toxicity, and interactions between contaminants can tune the enantioselectivity of chiral herbicides, which should be considered in future risk assessment.
’ INTRODUCTION Because of the long-term discharge of untreated domestic and industrial wastewater runoff, accidental spills, and direct soil waste dumping, a variety of heavy metals have recently detected in different environmental compartments, including soil, water, and air.13 However, heavy metals are toxic to most organisms.4,5 Because of bioaccumulation and the negative effects of heavy metals, global heavy metal pollution is a growing threat to the environment. Generally, both pesticides and heavy metals exist in the environment as anthropogenic chemical pollutants due to runoff and the leaching of these compounds through the soil, resulting in the contamination of both surface and groundwater.1,5 Exposure to these environmental chemicals is involuntary, and often the exposure is to mixtures of chemicals, either simultaneously or sequentially. Therefore, the joint toxicity of pesticides and heavy metals has previously been studied.6 As many as 25% of all pesticide active ingredients are chiral, and this percent is increasing as compounds with more complex structures are introduced into use.7,8 Enantiomers of a chiral chemical generally undergo identical physical and chemical r 2011 American Chemical Society
processes and reactions in the environment; however, enantiomers may behave differently in biologically mediated environmental processes 7,9 and their biological effects (e.g., toxicity, mutagenicity, carcinogenicity, and endocrine-disrupting activity) are typically enantioselective.10 Some studies have also found that environmental factors can affect the enantioselectivity of pesticides.11 Our recent work showed that the enantioselective behaviors of chiral compounds in the environment can be shifted when interactions with other chiral receptors coexist.12 Therefore, it is necessary to reveal how heavy metals affect the bioavailability and ecotoxicity of chiral pesticides. However, to the best of our knowledge, not much work about this has been published in the literature. Microalgae are important tools in monitoring water quality and aquatic toxicity because algae are not only the primary producers in the food chain but also are often more sensitive to Received: February 1, 2011 Accepted: April 21, 2011 Revised: April 8, 2011 Published: May 05, 2011 4778
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contaminants than are fish and invertebrates. When algae are exposed to abnormal conditions, they may respond by a burst of reactive oxygen species (ROS).13,14 Under oxidative conditions, algae respond by increasing their antioxidant defenses, notably enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). In the present study, we used dichlorprop as a representative chiral herbicide and copper (Cu) as a representative heavy metal. The growth inhibition rate and chlorophyll content of the aquatic unicellular alga Scenedesmus obliquus were studied and the ultrastructural morphology of cells was observed by transmission electron microscopy. Taking into account that in algae ROS production is dependent on Cu and dichlorprop, their effects on the production of ROS, malondialdehyde (MDA), and the activities of the antioxidant enzymes SOD, POD, and CAT were also examined to evaluate oxidative damage in algae. Results from this study will provide insights on the roles of reactive oxygen species in the enantioselectivity tuning of chiral herbicide dichlorprop by copper.
’ MATERIALS AND METHODS Chemicals. (R)-dichlorprop (DCPP), (S)-dichlorprop, and
(Rac)-dichlorprop with 99% purity were synthesized as described previously.15 20 ,70 -Dichlorodihydrofluorescein diacetate (H2DCFDA), FITC-conjugated lectin (FITC-ConA), and fluorescein acetate (FDA) were purchased from Sigma Aldrich. Details on the preparation of reagents and related methods to determine total soluble protein (TSP), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), chlorophyll contents, and cell permeability are given in Text S1 of the Supporting Information. All other reagents were analysis purity. All glassware was sterilized in an autoclave. Algal Growth Inhibition Assay. The freshwater microalgae, Scenedesmus obliquus, were used as a test organism. Initial stock organisms were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Before the assay, the algae were cultivated as described previously12 and the sensitivity was tested using K2Cr2O7. First, the toxicities of dichlorprop and copper chloride were assessed independently in growth inhibition test (Figures S1 and S2 and Table S1 of the Supporting Information). To keep the pH of the culture solution for Scenedesmus obliquus, both dichlorprop and copper chloride were dissolved in shuisheng-4 (HB-4) medium. Then DCPP (430 μM) and CuCl2 (10.0 μM) were selected in this study, which was discussed in Text S2 of the Supporting Information. To resemble common pollution scenarios, we investigated the effects of Cu on the toxicology of DCPP in two ways. The first involved adding separate solutions of dichlorprop and CuCl2 into the culture solution simultaneously (DCPP-Cu-1). The second involved adding a premixed solution of dichlorprop and CuCl2 (DCPP-Cu-2) that had been stirred at ambient temperature for 24 h into the solution containing Scenedesmus obliquus. During the entire experiment, the pH of the culture solution varied by less than 1 pH unit, which met the requirements of OECD 201 (Organization for Economic Co-operation and Development 201). Scenedesmus obliquus cell density was monitored at 680 nm with a Shimadzu UV-2401 spectrophotometer after 72 h. Detection of ROS Production. ROS production was measured by using the cell permeable indicator H2DCFDA.16 For the distribution and quantitative determination of ROS, 1.0 mL algal cells grown for 72 h were centrifuged at 10 000 rpm for 10 min,
Figure 1. Imaging of ROS production in Scenedesmus obliquus by CLSM and the chlorophyll autofluorescence (red (al)) and fluorescence due to FITC-ConA (green, (a)) and DCF (green, (cl)). (ab) control, (c) Cu, (d) (R)-DCPP, (e) (S)-DCPP, (f) (Rac)-DCPP, (g) (R)-DCPPCu-1, (h) (S)-DCPP-Cu-1, (i) (Rac)-DCPP-Cu-1, (j) (R)-DCPP-Cu-2, (k) (S)-DCPP-Cu-2, (l) (Rac)-DCPP-Cu-2. For scale, the bar in (a) = 5.0 μm, and in (bl) the bar = 7.5 μm.
after which the supernatant was discarded, followed immediately by the addition of 10 μM H2DCFDA to the cell pellet. Next they were incubated in a water bath at 37 C for 2 h in the dark. The algal cells were recentrifuged at 10 000 rpm for 10 min. The resulting pellet was washed twice with HB-4 medium. Subsequently, the cell pellet was resuspended in HB-4 medium. The distributions of ROS in algal cells were examined with the Leica TCS SP5 confocal laser scanning microscopy (CLSM) by excitation at 485 nm and emission at 530 nm. Chlorophyll autofluorescence was detected by excitation at 633 nm and emission at 680 nm. For the quantitative determination of ROS, 200 μL resuspended algae solution was transferred to a 96 well microplate. Fluorescence was measured with a SpectraMax M5 multilabel microplate reader, with excitation and emission filters of 485 and 530 nm, respectively. The production of ROS was expressed as absolute fluorescent units (FU) of dichlorodihydrofluorescein (DCF) by 105 algae cells. Statistical Analysis. The data were analyzed using the Origin 8.0 software (OriginLab, Northampton, MA, USA) according to the methods provided by the manufacturer of the test kit. Comparisons were made using one-way analyses of variance (ANOVA) followed by a multiple comparison test of means (Tukey test). The differences were considered statistically significant when p was less than 0.05.
’ RESULTS AND DISCUSSION ROS Distribution and Quantitative Analysis in Algae. ROS can be triggered by a number of external contaminants and their hazards have long been recognized. Therefore, the production of ROS in algae was investigated. In part a of Figure 1, we can see clearly the shape of the algae cell (the green fluorescence from 4779
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Figure 2. Production of ROS in Scenedesmus obliquus after 72 h. Different letters above adjacent bars indicate a significant difference (p < 0.05) between each treatment, whereas the same letter indicates no significant difference.
FITC-ConA was used to recognize the algae cell wall) and almost all of the cell volume was taken up by the chloroplast (red fluorescence). The ROS dependent on fluorescence of DCF was almost not visible in cells from the control treatment (part b of Figure 1), but bright green fluorescence was observed in all part of the Cu-treated cells (part c of Figure 1). Some fluorescence spots apart from the chloroplasts were also observed in the cytoplasm, which may have been peroxisomes or mitochondria. These results showed that copper ions can stimulate the formation of free radicals. In DCPP-treated algal cells (parts df of Figure 1), the green fluorescence was localized in parts of the chloroplasts and the area of the fluorescence was smaller. Additionally, a round bright spot was found that may have been related to peroxisomes or mitochondria. Green fluorescence was also found in the DCPP-Cu-1 treated algal cells (parts gi of Figure 1) that, to a lesser extent, overlapped with the chlorophyll autofluorescence. In addition, green fluorescence was also observed in regions other than the chloroplasts, which may have been from the cavum between the cell wall and protoplast (part g of Figure 1). The release of ROS can be an important process to protect the cells against enhanced cellular ROS concentrations.17 Some of the fluorescence was also detected within the inner side of cell wall or plasma membrane (part h of Figure 1). Both combined treatments of DCPP and CuCl2 shared a similar pattern of ROS release (parts jl of Figure 1). Figure 2 showed that all of the treatments stimulated the production of ROS. For (R)-DCPP, (S)-DCPP, (Rac)-DCPP and Cu, the productions of ROS measured 202.36, 146.93, 145.6, and 227.51 units, respectively, whereas the control was only 133.02 units. Also, the production of ROS in Scenedesmus obliquus treated by DCPP-Cu was significantly higher than the individual treatments. For example, the (R)-DCPP-Cu-1 treatment stimulated the production of ROS to levels 1.69-fold higher that of the (R)-DCPP treatment and 1.51-fold that of the Cu treatment. It should be noticed that the production of ROS of the herbicidally active (R)-enantiomer was higher than that of the (S)-enantiomer in the absence of Cu, suggesting a (R)-enantiomer preferentially induced production of ROS. When DCPP and Cu were added into the solution simultaneously (DCPP-Cu-1), (R)-DCPP preferentially induced production of ROS was also
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observed. However, the (R)-enantiomer preferentially induced production of ROS was reversed when DCPP was mixed with Cu 24 h before being added to the algae solution (DCPP-Cu-2). The carboxyl group of DCPP can provide complex sites for copper ions, but in this experiment, the quantity of Cu ions was far lower than that of DCPP molecule (DCPP/Cu = 43:1, molar ratio). Therefore, Cu should quantitatively form the DCPP-Cu complex, which may have induced this chiral inversion. The inversion of molecular chirality has been reported when N,N-Dialkylmethionines form complexes with Cu. N,N-Dialkylmethionines is optically active and can act as ligand when form complexes with Cu.18 Response of Antioxidant Defenses. The burden of ROS production is largely counteracted by an intricate antioxidant defense system. The enzymes SOD, CAT, and POD are involved in the detoxification of O2 (SOD) and H2O2 (CAT, POD), thereby preventing the formation of ROS. As shown in part a of Figure 3, the SOD activity of Scenedesmus obliquus treated by DCPP was higher than that of the control. The SOD activity of (R)-DCPP-treated algae increased significantly by 2.92-fold and (S)-DCPP-treated algae showed a 1.75-fold increase compared to the control. It was obvious that the (R)-DCPP caused a higher SOD activity in the algae compared to the (S)-DCPP and the same trends were found with CAT activity and POD activity. When added with Cu, SOD activity showed a significant increase compared to any individual contaminant treatment. The measured SOD activities were 11.25, 8.60, 7.01, and 10.29 U/(103 mg protein) when treated with (R)-DCPP-Cu-1, (S)-DCPPCu-1, (R)-DCPP-Cu-2, and (S)-DCPP-Cu-2, respectively. We noticed that (R)-DCPP-Cu-1 also caused a higher SOD activity than the (S)-DCPP-Cu-1; however, this trend was reversed when treated with a premixed solution (DCPP-Cu-2). These results were consistent with the observed trend found with the production of ROS reported above. This phenomenon was also discovered with CAT and POD activity. The enhancement of antioxidant enzyme activities suggested that the oxidative stress conditions led to an increased antioxidant capability of algal cells. However, this increase might not match the production of ROS and if accumulation of ROS exceeds the capacity of antioxidant systems, the cell will be damaged in various ways including peroxidation. The extent of lipid peroxidation is considered an important parameter for the identification of oxidative stress. Decomposition of lipid peroxides generates many products, including MDA.19 In our experiment, all treatments provoked a boom in the MDA level in Scenedesmus obliquus. It can be seen from part d of Figure 3 that the production of MDA in Scenedesmus obliquus treated by DCPP-Cu was significantly higher than any of the individual treatments. Hence, these results provided evidence of oxidative stress and the inability to maintain cellular homeostasis, which could have resulted from either diminished antioxidants or the increased production of ROS.19 Change of Chlorophyll Contents. Imaging of ROS production in Scenedesmus obliquus by CLSM showed that the ROS production may have been associated with the chloroplasts. Therefore, we examined the concentration of chlorophylls in the cell as a parameter to follow the growth of the algae culture. The inhibitory effects of DCPP and Cu, both individually and in combination, on chlorophyll a (Chla), chlorophyll b (Chlb), and the Chla/Chlb ratio in algal cells after 72 h of exposure are shown in Table 1. Compared to the control, the Chla and Chlb concentrations decreased after exposure to (R)-DCPP and (Rac)-DCPP, whereas for (S)-DCPP there was a small increase 4780
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Figure 3. Change of SOD (a), CAT (b), POD (c), and MDA (d) in Scenedesmus obliquus after 72 h. Different letters above adjacent bars indicate a significant difference (p < 0.05) between each treatment, whereas the same letter indicates no significant difference.
Table 1. Chlorophyll A and Chlorophyll B Concentration of Scenedesmus obliquusa Chla (mg/L)
Chlb (mg/L)
Chla/chlb
control
2.53 ( 0.0237a
1.52 ( 0.0061b
1.69
(R)-DCPP
1.42 ( 0.0122c
0.79 ( 0.0046d
1.79
(S)-DCPP
1.64 ( 0.0094b
1.63 ( 0.0124a
0.99
(Rac)-DCPP
1.41 ( 0.0151c
0.70 ( 0.0068e
2.04
(R)-Cu-1
0.67 ( 0.0083 g
0.47 ( 0.0099 h
1.37
(S)-Cu-1
0.85 ( 0.0084f
0.51 ( 0.0064 h
1.66
(Rac)-Cu-1 (R)-Cu-2
0.98 ( 0.0047e 0.85 ( 0.0052f
0.63 ( 0.007f 0.59 ( 0.009 g
1.54 1.43
(S)-Cu-2
0.55 ( 0.007 g
0.37 ( 0.0064i
1.47
(Rac)-Cu-2
1.12 ( 0.0634d
0.69 ( 0.0059 e
1.58
Cu
1.24 ( 0.0359d
0.87 ( 0.008c
1.39
a
Note: a. Chla = chlorophyll a; b. Chlb = chlorophyll b; c. Values represent the mean ( S.D. Data with significant differences (p
