Chemosphere 132 (2015) 32–39
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Inhibitory effects and oxidative target site of dibutyl phthalate on Karenia brevis Feng-min Li a,⇑, Miao Wu a, Yuan Yao a, Xiang Zheng b, Jian Zhao a, Zhen-yu Wang a, Bao-shan Xing a,c a
Key Lab of Marine Environmental Science and Ecology, Ministry of Education, College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China School of Environment and Natural Resources, Renmin University of China, Beijing 100872, China c Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA b
h i g h l i g h t s Dibutyl phthalate triggered an oxidative response in the algae Karenia brevis. Different SOD isoforms showed different activity under DBP exposure. Mitochondria could be the main target sites of DBP in Karenia brevis.
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Article history: Received 14 October 2014 Received in revised form 17 January 2015 Accepted 23 January 2015
Handling Editor: Frederic Leusch Keywords: DBP Karenia brevis Oxidative stress Toxicity mechanism
a b s t r a c t The inhibitory action and possible damage mechanism of dibutyl phthalate (DBP) on the red tide algae Karenia brevis were investigated. The results showed that the algae experienced oxidative stress after exposure to 5 mg L 1 DBP. Malondialdehyde (MDA) peaked after 72 h, with a value approximately 2.3 times higher than that observed for untreated cells. The superoxide dismutase (SOD) and catalase (CAT) activities significantly increased as an adaptive reaction after 48 h. DBP induced the overproduction of reactive oxygen species (ROS), the OH concentration showed a peak of 33 U mL 1 at 48 h, and the highest H2O2 content was approximately 250 nmol/107 cells at 72 h; these latter two values were 2.5 and 4.4 times higher than observed for the control, respectively. TEM images showed that a number of small vacuoles or apical tubers were commonly found around the cell membrane, and the membrane structure was ultimately disintegrated. Further experiments were carried out to locate the original ROS production sites following DBP exposure. The activity of CuZn-SOD (a mainly cytosolic isoform, with some also found in chloroplasts) under DBP exposure was approximately 2.5 times higher than the control, whereas the Mn-SOD (mitochondrial isoform) activity was significantly inhibited. No significant difference was observed in the activity of Fe-SOD (chloroplastic isoform). In addition, dicumarol (an inhibitor of the electron transport chain in the plasma membrane) stimulated DBP-induced ROS production, whereas rotenone (an inhibitor of the mitochondria electron transport chain complex I) decreased DBP-induced ROS production. These results suggested that mitochondria could be the main target sites for DBP attack. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the use of phthalate esters (PAEs) as food additives and plasticizers has caused food safety problems, thereby raising concerns from both the scientific community and governments. In late May 2011, the Yu-shen Chemical Company (Taiwan, China) was found to be producing an emulsifier known as a clouding agent that had been adulterated with a high ⇑ Corresponding author. Tel.: +86 053266782780. E-mail addresses: [email protected], [email protected] (F.-m. Li), [email protected] (X. Zheng), [email protected] (B.-s. Xing). http://dx.doi.org/10.1016/j.chemosphere.2015.01.051 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
concentration (600 ppm) of di(2-ethylhexyl) phthalate (DEHP) to reduce the cost of production. In November 2012, Jiugui Liquor, known for producing the best white wine in the world, was reported to have exceeded plasticizer content and dibutyl phthalate (DBP) in particular exceeded national regulation by 260%. The breakout of serious food safety issues related to PAEs at this time shocked all of China. Since then, PAEs have received considerable attention from the media, legislative bodies, and environmental organizations. PAEs have become widely distributed environmental pollutants. The global annual production of PAEs was more than 5 million tons in 2010 (Zhang et al., 2012), mostly used for packaging
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and construction materials. The production of PAEs in China increased from 1.25 million tons in 2006 to 2.6 million tons in 2007 (Xu et al., 2008). Moreover, PAEs are not covalently bound to the polymer molecules; therefore, they can migrate into the surrounding environment (Benson, 2009). Although some studies have indicated that PAEs can be biodegraded by microorganisms (Chi et al., 2006; Babu and Wu, 2010), PAEs can still be detected at measurable concentrations in marine and terrestrial ecosystems because of their high consumption and continuous release into the environment (Kang et al., 2012). Some PAEs have been proven to possess reproductive and developmental toxicities to animals and are suspected of causing endocrine-disrupting effects to humans (Howdeshell et al., 2008; Weiss, 2011; Liu et al., 2014). DBP is a low-molecular-weight PAE and has the second-highest consumption rate among all PAEs (Huang et al., 1999). DBP is a pollutant that can now be detected in most aquatic ecosystems in China. DBP was also found to be one of main pollutants in the Wuhan section of the Yangtze River in both high and low water periods, exceeding China’s Surface Water Quality Criteria in 82.4% of the studied water samples (Wang et al., 2008a). Given its mutagenic, teratogenic, and carcinogenic effects, both the US Environmental Protection Agency (EPA) and the Chinese State Environmental Protection Administration (SEPA) have listed DBP as an environmental priority pollutant (Huang et al., 1999; Xu et al., 2008). Although most organisms can partially excrete or degrade PAEs, DBP has aquatic toxic effects and has become a threat to the aquatic ecosystem in the past twenty years (Thurén and Woin, 1991; Kuang et al., 2003b). Toxicity and eco-toxicological studies investigating the effect of DBP on algae, which are known to be the primary producers of the aquatic ecosystem, have also been reported. The toxic effects of DBP on Isochrysis galbana, Phaeodactylum tricornutum, Scenedesmus obliquus, and Anabaena sp. PCC7120 have been studied under experimental conditions (Kuang et al., 2003a; Wang et al., 2006). The results showed that DBP inhibits cell growth, decreases cell concentration and damages chlorophyll a content. The inhibitory effects on I. galbana and P. tricornutum were significantly enhanced when the DBP concentration was 5.0 mg L 1. The 96-h EC50 of DBP on cell densities and chlorophyll a content of S. obliquus were 30.2 and 44.7 mg L 1, respectively. The algal density became lower with increasing DBP content for Anabaena sp. PCC7120. It is not common to detect DBP concentrations as high as 5.0 mg L 1 in the environment. In a national source water survey of China, DBP was the most frequently detected with high concentrations ranging from nd– 1.52 lg L 1, whereas in surface water, concentrations ranged from 10 to 100 lg L 1 (Huang et al., 1999; Liu et al., 2014). However, the bioaccumulation of DBP in the aquatic and terrestrial food chain progressively increases with trophic level (Stales et al., 1997). Taking it into consideration is important. Studies investigating the toxicity of DBP typically use freshwater algae (Huang et al., 1999; Jonsson and Baun, 2003). However, the inhibitory mechanism has rarely been reported in the literature, especially for ocean algae. Karenia brevis (formerly Gymnodinium breve, Ptychodiscus brevis) is one of the ocean species responsible for harmful algal bloom (HAB) formation. This organism was first identified and described in 1948 (Davis, 1948). In our previous study, we found that DBP was an effective allelochemical, and the growth inhibitory effect of DBP on K. brevis exhibited a 72-h EC50 value of 1.1 mg L 1 (Wang et al., 2008b). However, it is unclear whether inhibitory effects are mediated through the production of ROS. If so, the way in which DBP affects ROS production and the target sites of ROS induced by DBP remain unknown. More mechanisms explaining how cellular structure is damaged by ROS and how oxidative stress can lead to cell apoptosis need to be explored. The present study investigated the
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oxidative response induced by DBP on K. brevis. The algal ultrastructure, lipid peroxidation (malondialdehyde, MDA) and reactive oxygen species (ROS) level in K. brevis cells exposed to DBP were analyzed. We also performed further experiments to elucidate the potential modes of action of DBP on K. brevis from the perspective of electron transport chain inhibitors and to identify possible sites of ROS generation. 2. Materials and methods 2.1. Culture conditions K. brevis samples were provided by the Institute of Oceanology, Chinese Academy of Sciences (IOCAS). Sterile cultures were maintained in f/2 medium (–Si) (Guillard, 1975) prepared with natural seawater filtered through a GF/C glass fiber filter, subjected to a 14:10 h light/dark cycle with a light intensity of 4000 lux at 23 ± 1 °C, and shaken once daily by hand to prevent adherent cell growth. 2.2. Exposure of K. brevis to DBP DBP (analytical purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Nanjing, China). A stock solution of DBP was prepared in acetone. For the tests, an additional solvent control was employed, which contained the maximal acetone concentration (0.05% v/v). Statistical t-test analysis revealed no significant difference (p > 0.05) between the growth in controls and acetone-treated samples. Stock solution was diluted with acetone, and 100-lL aliquots were added to 200-mL culture samples, yielding final concentrations of 0, 0.5, 1.0, and 5.0 mg L 1, respectively. The actual DBP content in the culture were reduced but within 5% (measured by HPLC, Agilent Technologies, USA). Each concentration was analyzed in triplicate to determine the effect of DBP on the MDA content, the activity of superoxide dismutase (SOD; E.C 1.15.1.1) and catalase (CAT; E.C 1.11.1.6), and the hydroxyl radical (OH), hydrogen peroxide (H2O2) and superoxide anion (O2 ) content. Samples were monitored after 0, 24, 48, and 72 h. To determine the ultrastructure, SOD isoforms, and site of ROS production, groups were exposed to DBP concentrations of 0 and 5 mg L 1 for 24 h. 2.3. Lipid peroxidation determination and enzyme activity assays To evaluate lipid peroxidation in algal cells, MDA content was quantified as thiobarbituric acid reacting substances (TBARS) using a spectrophotometer (UV-2550, SHIMADZU, Japan) (Wang et al., 2008a). After 24 h of exposure to 5 mg L 1 DBP, the algal cultures were collected and centrifuged, and the pellet was then resuspended in 3 mL of 0.05 M sodium phosphate buffer (pH 7.8) and ultrasonicated at 4 °C for 5 min. Then, the homogenate were centrifuged at 6000 rpm for 5 min at 4 °C, and the supernatant was collected for SOD (E.C 1.15.1.1) and CAT (E.C 1.11.1.6) assays. The activity of SOD was assayed by nitroblue tetrazolium (NBT) photoreduction, according to the method of Beauchamp and Fridovich (Beauchamp and Fridovich, 1971). The activity of CAT was measured by using an assay kit (Jiancheng, Nanjing, China), following the manufacturer’s instructions. 2.4. Measurement of cellular ROS content Cellular ROS production activity was determined by using a DCFH-DA fluorescence probe as described in the assay kit instructions (Beyotime Institute of Biotechnology, China). A fluorescence
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spectrophotometer (F-4600, HITACHI, Japan) was used to detect ROS content. O2 was determined according to Ke et al. (2001) as follows. Approximately 1 mL of 10 mM oxammonium hydrochloride solution was added into 2 mL of the sample supernatant prepared using the procedure for H2O2 determination. The resulting solution was then mixed and incubated for 20 min at 25 °C. Up to 1 mL of 58-mM 4-aminobenzene sulfonic acid solution and 1 mL 7-mMa-naphthylamine solution were then quickly added and mixed, and the mixture was incubated at 30 °C for 30 min. After incubation, 4 mL chloroform was added to remove pigments by liquid–liquid extraction. The upper pink aqueous layer was collected, and its absorbance was measured at 530 nm. The O2 production rate was then calculated and expressed as nmol min cell 1. OH was analyzed by an assay kit (Jiancheng, Nanjing, China), following the manufacturer’s instructions. The OH content was given in units of U per milliliter (U mL 1), where U is the ability of 106 cells to increase the H2O2 concentration by 1 mM. H2O2 was determined according to Patterson (Patterson et al., 1984) with modifications and calculated using a standard curve that was prepared with known concentrations of H2O2. The algal cells were collected by centrifugation (3381 g, 4 min), then homogenized in 5 mL 20 °C acetone using an ultrasonic cell pulverizer for 5 min in an ice bath. The homogenate was then centrifuged (4428 g, 4 °C, 10 min) to obtain exact volumes of supernatant for hydrogen peroxide assays. A solution containing 1 mL 20 °C acetone, 0.5 mL 10% (V:V) titanium tetrachloride dissolved in hydrochloric acid, and 1 mL ammonia was quickly added into 4 mL of the sample supernatant. The resulting mixture was mixed and allowed to react for 5 min. The mixture was centrifuged (4428 g, 4 °C, 10 min) to obtain the precipitate and then rinsed (4428 g, 10 min) three times with 20 °C acetone to remove pigments. The final precipitate was dissolved in 3 mL of 2 M sulfuric acid for 6 h, and the absorbance of the solution was detected at 412 nm. The data are reported as ‘‘nmol cell 1’’. 2.5. Transmission electron microscopy analysis After K. brevis cultures were exposed to DBP (0, 5 mg L 1) for 24 h in the abovementioned culture parameters, and the algal cells were collected by centrifugation (3381 g, 3 min). Samples of the control and DBP-treated algae were pre-fixed in 2.5% glutaraldehyde solution dissolved in 0.1 M phosphate buffer (pH 7.8) at 4 °C for 8 h. The samples were then washed in 0.1 M phosphate buffer (pH 7.8) (4428 g, 10 min) three times, re-fixed for 1 h in 1% osmium tetroxide at 4 °C, and then washed again (4428 g, 10 min) with 0.1 M phosphate buffer (pH 7.8) three times. After this, the samples were dehydrated using a series of alcohol solutions at concentrations of 30%, 50%, 70%, 90%, 100%, and 100%, allowing 10 min contact time with each solution. The samples were then embedded in Epon 812 resin and fixed using a temperature program of 37, 45, and 65 °C, for 24 h at each temperature. Samples were then prepared using a microtome (Ultracut E, Cambridge Instrument, UK) and stained with uranyl acetate and lead citrate. A JEOL-JEM-1200EX (Japan) transmission electron microscope (TEM) was utilized to observe the ultrastructure of the algal samples.
ROS and SOD activity of the eight groups were measured according to the methods mentioned in Section 2.4 above. 2.7. Statistical analysis The experiments were performed in triplicate for all treatments. The results are presented as the means of three replicates (mean ± SD, n = 3) and tested by two-way and one-way analysis of variance (ANOVA) and Tukey’s test using the software spss 19.0. p-values < 0.05 were considered significant.
3. Results 3.1. MDA content under DBP exposure MDA, as an indicator of lipid peroxidation, was measured after DBP exposure for 3 d. The results do not show significant differences in cultures treated with lower DBP concentrations (0 and 0.5 mg L 1) during 72 h of treatment (Fig. 1). Additionally, in the 0, 0.5, and 1 mg L 1 DBP groups, MDA content varied following a similar trend, in which concentrations peaked after 24 h and then returned to a base level at 72 h. However, the MDA content in samples exposed to 5 mg L 1 DBP at 72 h was approximately 2.3 times higher than in the control group, with values as high as 0.34 lmol/ (109 cells). 3.2. Influence of DBP exposure on SOD and CAT activity The cellular enzymatic activities of SOD and CAT were investigated to study the response of K. brevis cells when exposed to different DBP concentrations. The SOD and CAT activities are shown in Fig. 2. In high-dose groups (1, 5 mg L 1), DBP stimulated the activity of SOD and CAT during 72 h of exposure. The SOD activity in algal cells treated with 5 mg L 1 DBP was 2–4 times higher than the control after 48 h of exposure. However, SOD activity increased slightly but was still lower than the control in the 0.5 mg L 1 treatment. The CAT activity shows a different trend: values increased continuously with increasing DBP level. The cells treated with 5 mg L 1 DBP showed the peak value, 78 U/(107 cells) at 72 h exposure, which was 7–9 times higher than the control.
2.6. Test of electron transport chain inhibitors Diuron, rotenone, and dicumarol were purchased from Sigma– Aldrich Corporation (analytical purity). The test concentrations of these chemicals were pre-tested to ensure that they were harmless to algal growth. Three groups of cells were treated with DBP for 12 h after being pretreated with 5 lM rotenone, 1.25 lM Diuron, and 5 lM dicumarol for 30 min. Algae treated with DBP and inhibitors independently were conducted as comparison groups. The
Fig. 1. MDA content of K. brevis exposed to different DBP concentrations at different culture times. Different letters represent significant differences between the treatment groups. Bars represent the SD (p < 0.05, Tukey, n = 3).
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Fig. 2. SOD and CAT activity of K. brevis exposed to different DBP concentrations at different culture times. Different letters represent significant differences between the treatments. Bars represent the SD (p < 0.05, Tukey, n = 3).
Fig. 3. (a) ROS content of K. brevis in 5 mg L 1 DBP treatment. Rosup is a positive control indicating high ROS content. ⁄⁄(p < 0.01) indicates a statistically significant difference compared to the control. Effects of different DBP concentrations on (b) the production rate of O2 , (c) the OH concentration and (d) the H2O2 concentration in K. brevis cells. Different letters above the columns represent significant differences in the treatment groups (p < 0.05, Tukey, n = 3).
3.3. Effect of DBP on ROS production We investigated the ROS content to determine the level of the damage caused by xenobiotics on K. brevis cells. Fig. 3a shows that the total ROS content of the cells treated with 5 mg L 1 DBP was
significantly higher than that of the control. The influence of DBP exposure on the content of the three ROS species O2 , OH and H2O2 in algae is shown in Fig. 3. There is no obvious effect of DBP on the production rate of O2 . During the experiment, the production rate of O2 in K. brevis cells showed an increase after 24 h but did not
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increase with higher DBP concentrations. Furthermore, the O2 production rates of the algal cells after exposure to 0.5 and 1 mg L 1 DBP were lower than those in the control group (Fig. 3b). The OH content for each DBP treatment is plotted against time in Fig. 3c. The OH content in algal cells showed an initial increase over the first 48 h, with an ultimate decrease at 72 h. In addition, the results show an increasing trend of OH with increased DBP concentration, regardless of exposure time. The highest OH content of 33 U mL 1 occurred after 48 h in algae treated with 5 mg L 1 DBP, a value 2.5 times higher than in the control (untreated algal cells). The final OH contents under different DBP concentrations, however, were close to or lower than the initial values (5 U mL 1). DBP had an obvious effect on H2O2 production in algae (Fig. 3d). The H2O2 content in algae treated with 1 mg L 1 DBP increased from 110 nmol/ (107 cells) to 160 nmol/(107 cells) at 24 h, followed by a downward trend from 48 h to 72 h. In addition, the groups treated with 0 and 0.5 mg L 1 DBP shared a similar trend, with an obvious decline from 48 h to 72 h. However, a peak value of 250 nmol/(107 cells), which is approximately 4.4 times the control value, was observed at 72 h at 5 mg L 1 DBP. 3.4. Influences of DBP on K. brevis subcellular structure TEM was used to investigate the changes in the ultrastructure of K. brevis resulting from DBP treatment, and the results are shown in Fig. 4. Fig. 4a shows the intact ultrastructure of the control group with chloroplasts, exhibiting a clear and complicated architecture and containing numerous thylakoids. Fig. 4b and c are zoom images of Fig. 4a. The cell membrane is easily distinguishable in Fig. 4c and was smooth, intact, and close to the cell wall. Algal cells treated with 5 mg L 1 DBP after 24 h are shown in Fig. 5. The structural damage is distinct. The cells were disorganized after DBP exposure, and almost all normal cell organelles were indistinguishable (Fig. 5); these changes are particularly severe in Fig. 5b and c. Additionally, the plasma membrane was detached from the cell wall (Fig. 5c). Over-accumulation of small vacuoles near the cell membrane was observed in cells treated with DBP (Fig. 5a and b). Small tubules or apical parts were commonly found around the cell surface (black arrow). 3.5. Possible site of ROS generation induced by DBP in K. brevis The above experiments have proven that ROS accumulated after exposure to DBP. However, the generation site of ROS in algal cells is not yet clear. Thus, to investigate the possible site of generation of ROS radicals, SOD isoforms were measured, and the results are
shown in Fig. 6. The activity of CuZn-SOD under DBP exposure is approximately 2.5 times higher than that in the control, and no change in Fe-SOD activity is evident. By contrast, a significant inhibition was observed in the activity of Mn-SOD. To explore the ROS production site, we performed an additional experiment to analyze the effects of electron transport chain inhibitors on the ROS content induced by DBP. The effects of diuron, rotenone and dicumarol on ROS production are shown in Fig. 6. Diuron did not affect ROS production, whereas rotenone and dicumarol decreased ROS production. The effects of DBP on ROS production in K. brevis cells pretreated in advance with diuron, rotenone, and dicumarol were also measured. Compared with the algal cells only treated with DBP, ROS production in cells pretreated with diuron showed no notable difference. Rotenone decreased and dicumarol stimulated the ROS production induced by DBP. 4. Discussion In spite of the known growth inhibition caused by DBP, its role in inducing an oxidative stress response in K. brevis remains largely unknown. This study demonstrates that DBP induces oxidative responses. The MDA content dramatically increased under high DBP concentration (5 mg L 1), suggesting that excess ROS in the algal cells attacked the cell membrane and resulted in lipid peroxidation. ROS (OH, H2O2, and O2 ) content varied by species throughout the experiment. The production of ROS is important in several cellular processes (e.g., cellular signaling and defense against infection). It has been suggested that the algae Selenastrum capricornutum was stimulated by ROS to increase its metabolic activity (Hong et al., 2008), but another report showed that the presence of ROS was more often associated with damage to cellular components such as proteins, lipids, and nucleic acids (Pospíšil, 2009). The reported increase in the activities of the antioxidant enzymes SOD and CAT in the study is an adaptive strategy. SOD catalyzes the dismutation of O2 to H2O2 and O2, while CAT directly eliminates H2O2 predominantly in the peroxisome and transforms H2O2 into H2O. In plants, CAT scavenges H2O2 generated during photorespiration and the b-oxidation of fatty acids and plays a significant role in the protection of aerobic organisms from the toxic effects of H2O2 (Apel and Hirt, 2004). The increased CAT activity and increased MDA content in algal cells suggests that the release of hydrogen peroxide is involved in the response of K. brevis cells to DBP. The antioxidant SOD activities of cells in the 1 mg L 1 DBP group initially increased rapidly and then decreased at 72 h, indicating that SOD in K. brevis respond with a short-term adaption to protect the algae against
Fig. 4. The ultrastructure of K. brevis in the control group after 24 h. CHL, chloroplast; M, mitochondrion; N, nucleus; S, starch grain; PM, plasma membrane; CW, cell wall. Scale bars are 1 lm in Fig. 4a and 200 nm in b and c.
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Fig. 5. The ultrastructure of K. brevis in the 5 mg L are 1 lm in Fig. 5 a and c, and 200 nm in b.
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DBP treatment group after 24 h. Fig. 5b is a magnified image of Fig. 5a. N, nucleus; V, vacuole; L, lipid globule. Scale bars
Fig. 6. Activities of SOD isoforms under DBP exposure.
peroxidation induced by DBP. Similar results were found when algae were exposed to enhanced UV-radiation (Tian and Yu, 2009). TEM images of the ultrastructure of K. brevis treated with DBP show altered cell organelles. The less-dense appearance of chloroplasts and mitochondria suggests that the cells were strongly affected by DBP (Fig. 5a and c). There is a high possibility that the structural disruptions to the chloroplasts and mitochondria induced by DBP were accompanied by reductions in photosynthetic and respiratory activities (Morlon et al., 2005). Moreover, the dense appearance of vacuoles around the inner cell surface was observed, indicating that the function of the vacuole, an organelle involved in detoxification, was strengthened. The compromised integrity of cell membrane caused by lipid peroxidation may result in ion leakage (Buchmann and Becker, 2009) and therefore leads to osmotic regulation of vacuoles. The distinct morphological phenomenon in which apical parts or small tubules are commonly found around the cell outer surface when exposed to DBP has seldom been discussed. To the best of our knowledge, the accumulation of vacuoles around the inner cell surface and changes on the outer cell surface may be related to algal cell secretions associated with metabolism. Another explanation may be that the apical changes resulted from a combination of the secretion process and the weak permeability of the cell membrane caused by lipid peroxidation. The transport of metabolites through algal membranes from/to seawater is an important and efficient
life-sustaining factor of algae in aquatic environments. These excretion points, however, obviously need further investigation. In photosynthetic algal species, ROS are continuously produced as byproducts of various metabolic pathways, which mainly derive from the electron transport chains in chloroplasts, mitochondria and the plasma membrane (Apel and Hirt, 2004). The production source of ROS in K. brevis exposed to DBP was investigated. The first step was to measure the SOD, a type of enzyme that can be highly induced under stress. Based on the metal cofactor, SOD isoforms in plants are classified into three groups: CuZn-SOD, Mn-SOD and Fe-SOD (Scandalios, 1993). More importantly, the different SOD isoforms are located in different cell compartments. Mn-SOD is known to be mitochondrial; Fe-SOD is located in chloroplasts in most plants examined to date; and CuZn-SOD is mainly found in cytosol, as well as a small amount in chloroplasts (Alscher et al., 2002). These SOD isoforms react differently under different forms of environmental stress (Khanna-Chopra and Sabarinath, 2004). Under DBP exposure, there was almost no change in the activity of Fe-SOD, whereas the increase in CuZnSOD activity indicated that the cytoplasm may have participated in ROS production. In contrast, the activity of Mn-SOD was significantly inhibited, which may have been caused by excessive ROS, because ROS can damage protein structure. It has been reported that antioxidant systems are prone to protein carbonylation if ROS exceeds the tolerance threshold (Gonçalves et al., 2007). One group of scientists investigated the effects of anthracene, cadmium and chloridazone on three Scenedesmus species by analyzing changes in SOD isoforms; they found that the applied substances disturbed metabolic processes in chloroplasts and indicated that chloroplasts may be the main target site of interaction for ROS overproduction in cells (Zbigniew and Wojciech, 2006). Thus, the ROS observed in K. brevis cells exposed to DBP could derive from cytosolic or mitochondrial sources, according to our results. The ROS production site was further investigated by testing the effects of inhibitors that affect different electron transfer chains. Diuron is an inhibitor of photosynthetic electron transport (McKim and Durnford, 2006). In the group exposed to diuron and then DBP, the ROS level did not differ notably compared with the group exposed only to DBP. This result suggests that chloroplasts were not the main ROS accumulation site in our experiment. However, in Lemna gibba, chloroplasts were found to be the main organ in which O2 induced by anthraquinone accumulated (Babu et al., 2001). Different test organisms and xenobiotics have been found to lead to various results. Rotenone, an inhibitor of the mitochondria electron transport chain complex (Møller, 2001), decreased DBP-induced ROS production, indicating that the mitochondria electron transport chain is related to the target site
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and Technology Program for Water Pollution Control and Treatment (2012ZX07203004), the Public Science and Technology Research Funds Projects of Ocean (No. 201305003), the NSFCShandong Joint Fund for Marine Science Research Centers (No. U1406403) and the Qingdao Science and Technology Project (No. 12-4-1-58-HY).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.01.051.
References
Fig. 7. Effects of inhibitors on DBP-induced ROS production. Control (A), DBP (B), Diuron (C), Rotenone (D), Dicumarol (E), DBP + Diuron (F), DBP + Rotenone (G), DBP + Dicumarol (H). ⁄⁄(p < 0.01) indicates a statistically significant difference compared to the control. Bars represent SD, n = 3.
of DBP. In contrast, dicumarol, an inhibitor of the redox enzyme system in the plasma membrane (Liu et al., 2007), stimulated DBP-induced ROS production. The reason for this, however, is not clear. We suggested that DBP may interact with dicumarol and result in joint toxicity. Liu et al. (2007) also made an attempt to identify the site of ROS in the algae Chattonella marina, using 3-(3,4-dichlorophenyl)-1,1-dimethylurea, KCN, Vitamin K3 and dicumarol, and suggested that ROS production in C. marina was related to a plasma membrane enzyme system. Considering the activity of SOD isoforms and the ROS content under exposure to different inhibitors, we concluded that DBP affected the plasma membrane and mitochondria, particularly inhibiting the mitochondria, because the protein Mn-SOD had been inactivated and ROS accumulated, resulting in the beginning of cell apoptosis and eventually death. Comparing the ROS values after adding electron transfer inhibitors (Fig. 7), only rotenone blocked the pathway of ROS production, indicating that ROS production induced by DBP in K. brevis in our culture conditions was related to the mitochondria. 5. Conclusion Our work showed that oxidative stress is the main toxicity mechanism for the observed growth inhibition of the algae K. brevis by DBP. Based on our results, we assumed that DBP caused an increase in ROS in the algae K. brevis and that it stimulated an increase of antioxidant enzymes as an adaptive response. Under our culture conditions, ROS could be generated both in the mitochondria and cytosol; furthermore, mitochondria could be the main target sites of interaction with DBP. When antioxidant enzymes in the mitochondria are damaged and can no longer scavenge the increased ROS, imbalance and the accumulation of ROS could occur, causing cell apoptosis. Disruption of the cell’s subcellular structure, including the plasma membrane and organelles, was observed. Because the inner mitochondrial membrane is impermeable to DBP, DBP may act on mitochondrial membrane enzymes and the electron transport respiratory chain in the form of metabolites. The interaction mechanism affecting the cell membrane still needs further research. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant No. 51378480), the Major Science
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Inhibitory effects and oxidative target site of dibutyl phthalate on Karenia brevis Feng-min Li a,⇑, Miao Wu a, Yuan Yao a, Xiang Zheng b, Jian Zhao a, Zhen-yu Wang a, Bao-shan Xing a,c a
Key Lab of Marine Environmental Science and Ecology, Ministry of Education, College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China School of Environment and Natural Resources, Renmin University of China, Beijing 100872, China c Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA b
h i g h l i g h t s Dibutyl phthalate triggered an oxidative response in the algae Karenia brevis. Different SOD isoforms showed different activity under DBP exposure. Mitochondria could be the main target sites of DBP in Karenia brevis.
a r t i c l e
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Article history: Received 14 October 2014 Received in revised form 17 January 2015 Accepted 23 January 2015
Handling Editor: Frederic Leusch Keywords: DBP Karenia brevis Oxidative stress Toxicity mechanism
a b s t r a c t The inhibitory action and possible damage mechanism of dibutyl phthalate (DBP) on the red tide algae Karenia brevis were investigated. The results showed that the algae experienced oxidative stress after exposure to 5 mg L 1 DBP. Malondialdehyde (MDA) peaked after 72 h, with a value approximately 2.3 times higher than that observed for untreated cells. The superoxide dismutase (SOD) and catalase (CAT) activities significantly increased as an adaptive reaction after 48 h. DBP induced the overproduction of reactive oxygen species (ROS), the OH concentration showed a peak of 33 U mL 1 at 48 h, and the highest H2O2 content was approximately 250 nmol/107 cells at 72 h; these latter two values were 2.5 and 4.4 times higher than observed for the control, respectively. TEM images showed that a number of small vacuoles or apical tubers were commonly found around the cell membrane, and the membrane structure was ultimately disintegrated. Further experiments were carried out to locate the original ROS production sites following DBP exposure. The activity of CuZn-SOD (a mainly cytosolic isoform, with some also found in chloroplasts) under DBP exposure was approximately 2.5 times higher than the control, whereas the Mn-SOD (mitochondrial isoform) activity was significantly inhibited. No significant difference was observed in the activity of Fe-SOD (chloroplastic isoform). In addition, dicumarol (an inhibitor of the electron transport chain in the plasma membrane) stimulated DBP-induced ROS production, whereas rotenone (an inhibitor of the mitochondria electron transport chain complex I) decreased DBP-induced ROS production. These results suggested that mitochondria could be the main target sites for DBP attack. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the use of phthalate esters (PAEs) as food additives and plasticizers has caused food safety problems, thereby raising concerns from both the scientific community and governments. In late May 2011, the Yu-shen Chemical Company (Taiwan, China) was found to be producing an emulsifier known as a clouding agent that had been adulterated with a high ⇑ Corresponding author. Tel.: +86 053266782780. E-mail addresses: [email protected], [email protected] (F.-m. Li), [email protected] (X. Zheng), [email protected] (B.-s. Xing). http://dx.doi.org/10.1016/j.chemosphere.2015.01.051 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
concentration (600 ppm) of di(2-ethylhexyl) phthalate (DEHP) to reduce the cost of production. In November 2012, Jiugui Liquor, known for producing the best white wine in the world, was reported to have exceeded plasticizer content and dibutyl phthalate (DBP) in particular exceeded national regulation by 260%. The breakout of serious food safety issues related to PAEs at this time shocked all of China. Since then, PAEs have received considerable attention from the media, legislative bodies, and environmental organizations. PAEs have become widely distributed environmental pollutants. The global annual production of PAEs was more than 5 million tons in 2010 (Zhang et al., 2012), mostly used for packaging
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and construction materials. The production of PAEs in China increased from 1.25 million tons in 2006 to 2.6 million tons in 2007 (Xu et al., 2008). Moreover, PAEs are not covalently bound to the polymer molecules; therefore, they can migrate into the surrounding environment (Benson, 2009). Although some studies have indicated that PAEs can be biodegraded by microorganisms (Chi et al., 2006; Babu and Wu, 2010), PAEs can still be detected at measurable concentrations in marine and terrestrial ecosystems because of their high consumption and continuous release into the environment (Kang et al., 2012). Some PAEs have been proven to possess reproductive and developmental toxicities to animals and are suspected of causing endocrine-disrupting effects to humans (Howdeshell et al., 2008; Weiss, 2011; Liu et al., 2014). DBP is a low-molecular-weight PAE and has the second-highest consumption rate among all PAEs (Huang et al., 1999). DBP is a pollutant that can now be detected in most aquatic ecosystems in China. DBP was also found to be one of main pollutants in the Wuhan section of the Yangtze River in both high and low water periods, exceeding China’s Surface Water Quality Criteria in 82.4% of the studied water samples (Wang et al., 2008a). Given its mutagenic, teratogenic, and carcinogenic effects, both the US Environmental Protection Agency (EPA) and the Chinese State Environmental Protection Administration (SEPA) have listed DBP as an environmental priority pollutant (Huang et al., 1999; Xu et al., 2008). Although most organisms can partially excrete or degrade PAEs, DBP has aquatic toxic effects and has become a threat to the aquatic ecosystem in the past twenty years (Thurén and Woin, 1991; Kuang et al., 2003b). Toxicity and eco-toxicological studies investigating the effect of DBP on algae, which are known to be the primary producers of the aquatic ecosystem, have also been reported. The toxic effects of DBP on Isochrysis galbana, Phaeodactylum tricornutum, Scenedesmus obliquus, and Anabaena sp. PCC7120 have been studied under experimental conditions (Kuang et al., 2003a; Wang et al., 2006). The results showed that DBP inhibits cell growth, decreases cell concentration and damages chlorophyll a content. The inhibitory effects on I. galbana and P. tricornutum were significantly enhanced when the DBP concentration was 5.0 mg L 1. The 96-h EC50 of DBP on cell densities and chlorophyll a content of S. obliquus were 30.2 and 44.7 mg L 1, respectively. The algal density became lower with increasing DBP content for Anabaena sp. PCC7120. It is not common to detect DBP concentrations as high as 5.0 mg L 1 in the environment. In a national source water survey of China, DBP was the most frequently detected with high concentrations ranging from nd– 1.52 lg L 1, whereas in surface water, concentrations ranged from 10 to 100 lg L 1 (Huang et al., 1999; Liu et al., 2014). However, the bioaccumulation of DBP in the aquatic and terrestrial food chain progressively increases with trophic level (Stales et al., 1997). Taking it into consideration is important. Studies investigating the toxicity of DBP typically use freshwater algae (Huang et al., 1999; Jonsson and Baun, 2003). However, the inhibitory mechanism has rarely been reported in the literature, especially for ocean algae. Karenia brevis (formerly Gymnodinium breve, Ptychodiscus brevis) is one of the ocean species responsible for harmful algal bloom (HAB) formation. This organism was first identified and described in 1948 (Davis, 1948). In our previous study, we found that DBP was an effective allelochemical, and the growth inhibitory effect of DBP on K. brevis exhibited a 72-h EC50 value of 1.1 mg L 1 (Wang et al., 2008b). However, it is unclear whether inhibitory effects are mediated through the production of ROS. If so, the way in which DBP affects ROS production and the target sites of ROS induced by DBP remain unknown. More mechanisms explaining how cellular structure is damaged by ROS and how oxidative stress can lead to cell apoptosis need to be explored. The present study investigated the
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oxidative response induced by DBP on K. brevis. The algal ultrastructure, lipid peroxidation (malondialdehyde, MDA) and reactive oxygen species (ROS) level in K. brevis cells exposed to DBP were analyzed. We also performed further experiments to elucidate the potential modes of action of DBP on K. brevis from the perspective of electron transport chain inhibitors and to identify possible sites of ROS generation. 2. Materials and methods 2.1. Culture conditions K. brevis samples were provided by the Institute of Oceanology, Chinese Academy of Sciences (IOCAS). Sterile cultures were maintained in f/2 medium (–Si) (Guillard, 1975) prepared with natural seawater filtered through a GF/C glass fiber filter, subjected to a 14:10 h light/dark cycle with a light intensity of 4000 lux at 23 ± 1 °C, and shaken once daily by hand to prevent adherent cell growth. 2.2. Exposure of K. brevis to DBP DBP (analytical purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Nanjing, China). A stock solution of DBP was prepared in acetone. For the tests, an additional solvent control was employed, which contained the maximal acetone concentration (0.05% v/v). Statistical t-test analysis revealed no significant difference (p > 0.05) between the growth in controls and acetone-treated samples. Stock solution was diluted with acetone, and 100-lL aliquots were added to 200-mL culture samples, yielding final concentrations of 0, 0.5, 1.0, and 5.0 mg L 1, respectively. The actual DBP content in the culture were reduced but within 5% (measured by HPLC, Agilent Technologies, USA). Each concentration was analyzed in triplicate to determine the effect of DBP on the MDA content, the activity of superoxide dismutase (SOD; E.C 1.15.1.1) and catalase (CAT; E.C 1.11.1.6), and the hydroxyl radical (OH), hydrogen peroxide (H2O2) and superoxide anion (O2 ) content. Samples were monitored after 0, 24, 48, and 72 h. To determine the ultrastructure, SOD isoforms, and site of ROS production, groups were exposed to DBP concentrations of 0 and 5 mg L 1 for 24 h. 2.3. Lipid peroxidation determination and enzyme activity assays To evaluate lipid peroxidation in algal cells, MDA content was quantified as thiobarbituric acid reacting substances (TBARS) using a spectrophotometer (UV-2550, SHIMADZU, Japan) (Wang et al., 2008a). After 24 h of exposure to 5 mg L 1 DBP, the algal cultures were collected and centrifuged, and the pellet was then resuspended in 3 mL of 0.05 M sodium phosphate buffer (pH 7.8) and ultrasonicated at 4 °C for 5 min. Then, the homogenate were centrifuged at 6000 rpm for 5 min at 4 °C, and the supernatant was collected for SOD (E.C 1.15.1.1) and CAT (E.C 1.11.1.6) assays. The activity of SOD was assayed by nitroblue tetrazolium (NBT) photoreduction, according to the method of Beauchamp and Fridovich (Beauchamp and Fridovich, 1971). The activity of CAT was measured by using an assay kit (Jiancheng, Nanjing, China), following the manufacturer’s instructions. 2.4. Measurement of cellular ROS content Cellular ROS production activity was determined by using a DCFH-DA fluorescence probe as described in the assay kit instructions (Beyotime Institute of Biotechnology, China). A fluorescence
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spectrophotometer (F-4600, HITACHI, Japan) was used to detect ROS content. O2 was determined according to Ke et al. (2001) as follows. Approximately 1 mL of 10 mM oxammonium hydrochloride solution was added into 2 mL of the sample supernatant prepared using the procedure for H2O2 determination. The resulting solution was then mixed and incubated for 20 min at 25 °C. Up to 1 mL of 58-mM 4-aminobenzene sulfonic acid solution and 1 mL 7-mMa-naphthylamine solution were then quickly added and mixed, and the mixture was incubated at 30 °C for 30 min. After incubation, 4 mL chloroform was added to remove pigments by liquid–liquid extraction. The upper pink aqueous layer was collected, and its absorbance was measured at 530 nm. The O2 production rate was then calculated and expressed as nmol min cell 1. OH was analyzed by an assay kit (Jiancheng, Nanjing, China), following the manufacturer’s instructions. The OH content was given in units of U per milliliter (U mL 1), where U is the ability of 106 cells to increase the H2O2 concentration by 1 mM. H2O2 was determined according to Patterson (Patterson et al., 1984) with modifications and calculated using a standard curve that was prepared with known concentrations of H2O2. The algal cells were collected by centrifugation (3381 g, 4 min), then homogenized in 5 mL 20 °C acetone using an ultrasonic cell pulverizer for 5 min in an ice bath. The homogenate was then centrifuged (4428 g, 4 °C, 10 min) to obtain exact volumes of supernatant for hydrogen peroxide assays. A solution containing 1 mL 20 °C acetone, 0.5 mL 10% (V:V) titanium tetrachloride dissolved in hydrochloric acid, and 1 mL ammonia was quickly added into 4 mL of the sample supernatant. The resulting mixture was mixed and allowed to react for 5 min. The mixture was centrifuged (4428 g, 4 °C, 10 min) to obtain the precipitate and then rinsed (4428 g, 10 min) three times with 20 °C acetone to remove pigments. The final precipitate was dissolved in 3 mL of 2 M sulfuric acid for 6 h, and the absorbance of the solution was detected at 412 nm. The data are reported as ‘‘nmol cell 1’’. 2.5. Transmission electron microscopy analysis After K. brevis cultures were exposed to DBP (0, 5 mg L 1) for 24 h in the abovementioned culture parameters, and the algal cells were collected by centrifugation (3381 g, 3 min). Samples of the control and DBP-treated algae were pre-fixed in 2.5% glutaraldehyde solution dissolved in 0.1 M phosphate buffer (pH 7.8) at 4 °C for 8 h. The samples were then washed in 0.1 M phosphate buffer (pH 7.8) (4428 g, 10 min) three times, re-fixed for 1 h in 1% osmium tetroxide at 4 °C, and then washed again (4428 g, 10 min) with 0.1 M phosphate buffer (pH 7.8) three times. After this, the samples were dehydrated using a series of alcohol solutions at concentrations of 30%, 50%, 70%, 90%, 100%, and 100%, allowing 10 min contact time with each solution. The samples were then embedded in Epon 812 resin and fixed using a temperature program of 37, 45, and 65 °C, for 24 h at each temperature. Samples were then prepared using a microtome (Ultracut E, Cambridge Instrument, UK) and stained with uranyl acetate and lead citrate. A JEOL-JEM-1200EX (Japan) transmission electron microscope (TEM) was utilized to observe the ultrastructure of the algal samples.
ROS and SOD activity of the eight groups were measured according to the methods mentioned in Section 2.4 above. 2.7. Statistical analysis The experiments were performed in triplicate for all treatments. The results are presented as the means of three replicates (mean ± SD, n = 3) and tested by two-way and one-way analysis of variance (ANOVA) and Tukey’s test using the software spss 19.0. p-values < 0.05 were considered significant.
3. Results 3.1. MDA content under DBP exposure MDA, as an indicator of lipid peroxidation, was measured after DBP exposure for 3 d. The results do not show significant differences in cultures treated with lower DBP concentrations (0 and 0.5 mg L 1) during 72 h of treatment (Fig. 1). Additionally, in the 0, 0.5, and 1 mg L 1 DBP groups, MDA content varied following a similar trend, in which concentrations peaked after 24 h and then returned to a base level at 72 h. However, the MDA content in samples exposed to 5 mg L 1 DBP at 72 h was approximately 2.3 times higher than in the control group, with values as high as 0.34 lmol/ (109 cells). 3.2. Influence of DBP exposure on SOD and CAT activity The cellular enzymatic activities of SOD and CAT were investigated to study the response of K. brevis cells when exposed to different DBP concentrations. The SOD and CAT activities are shown in Fig. 2. In high-dose groups (1, 5 mg L 1), DBP stimulated the activity of SOD and CAT during 72 h of exposure. The SOD activity in algal cells treated with 5 mg L 1 DBP was 2–4 times higher than the control after 48 h of exposure. However, SOD activity increased slightly but was still lower than the control in the 0.5 mg L 1 treatment. The CAT activity shows a different trend: values increased continuously with increasing DBP level. The cells treated with 5 mg L 1 DBP showed the peak value, 78 U/(107 cells) at 72 h exposure, which was 7–9 times higher than the control.
2.6. Test of electron transport chain inhibitors Diuron, rotenone, and dicumarol were purchased from Sigma– Aldrich Corporation (analytical purity). The test concentrations of these chemicals were pre-tested to ensure that they were harmless to algal growth. Three groups of cells were treated with DBP for 12 h after being pretreated with 5 lM rotenone, 1.25 lM Diuron, and 5 lM dicumarol for 30 min. Algae treated with DBP and inhibitors independently were conducted as comparison groups. The
Fig. 1. MDA content of K. brevis exposed to different DBP concentrations at different culture times. Different letters represent significant differences between the treatment groups. Bars represent the SD (p < 0.05, Tukey, n = 3).
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Fig. 2. SOD and CAT activity of K. brevis exposed to different DBP concentrations at different culture times. Different letters represent significant differences between the treatments. Bars represent the SD (p < 0.05, Tukey, n = 3).
Fig. 3. (a) ROS content of K. brevis in 5 mg L 1 DBP treatment. Rosup is a positive control indicating high ROS content. ⁄⁄(p < 0.01) indicates a statistically significant difference compared to the control. Effects of different DBP concentrations on (b) the production rate of O2 , (c) the OH concentration and (d) the H2O2 concentration in K. brevis cells. Different letters above the columns represent significant differences in the treatment groups (p < 0.05, Tukey, n = 3).
3.3. Effect of DBP on ROS production We investigated the ROS content to determine the level of the damage caused by xenobiotics on K. brevis cells. Fig. 3a shows that the total ROS content of the cells treated with 5 mg L 1 DBP was
significantly higher than that of the control. The influence of DBP exposure on the content of the three ROS species O2 , OH and H2O2 in algae is shown in Fig. 3. There is no obvious effect of DBP on the production rate of O2 . During the experiment, the production rate of O2 in K. brevis cells showed an increase after 24 h but did not
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increase with higher DBP concentrations. Furthermore, the O2 production rates of the algal cells after exposure to 0.5 and 1 mg L 1 DBP were lower than those in the control group (Fig. 3b). The OH content for each DBP treatment is plotted against time in Fig. 3c. The OH content in algal cells showed an initial increase over the first 48 h, with an ultimate decrease at 72 h. In addition, the results show an increasing trend of OH with increased DBP concentration, regardless of exposure time. The highest OH content of 33 U mL 1 occurred after 48 h in algae treated with 5 mg L 1 DBP, a value 2.5 times higher than in the control (untreated algal cells). The final OH contents under different DBP concentrations, however, were close to or lower than the initial values (5 U mL 1). DBP had an obvious effect on H2O2 production in algae (Fig. 3d). The H2O2 content in algae treated with 1 mg L 1 DBP increased from 110 nmol/ (107 cells) to 160 nmol/(107 cells) at 24 h, followed by a downward trend from 48 h to 72 h. In addition, the groups treated with 0 and 0.5 mg L 1 DBP shared a similar trend, with an obvious decline from 48 h to 72 h. However, a peak value of 250 nmol/(107 cells), which is approximately 4.4 times the control value, was observed at 72 h at 5 mg L 1 DBP. 3.4. Influences of DBP on K. brevis subcellular structure TEM was used to investigate the changes in the ultrastructure of K. brevis resulting from DBP treatment, and the results are shown in Fig. 4. Fig. 4a shows the intact ultrastructure of the control group with chloroplasts, exhibiting a clear and complicated architecture and containing numerous thylakoids. Fig. 4b and c are zoom images of Fig. 4a. The cell membrane is easily distinguishable in Fig. 4c and was smooth, intact, and close to the cell wall. Algal cells treated with 5 mg L 1 DBP after 24 h are shown in Fig. 5. The structural damage is distinct. The cells were disorganized after DBP exposure, and almost all normal cell organelles were indistinguishable (Fig. 5); these changes are particularly severe in Fig. 5b and c. Additionally, the plasma membrane was detached from the cell wall (Fig. 5c). Over-accumulation of small vacuoles near the cell membrane was observed in cells treated with DBP (Fig. 5a and b). Small tubules or apical parts were commonly found around the cell surface (black arrow). 3.5. Possible site of ROS generation induced by DBP in K. brevis The above experiments have proven that ROS accumulated after exposure to DBP. However, the generation site of ROS in algal cells is not yet clear. Thus, to investigate the possible site of generation of ROS radicals, SOD isoforms were measured, and the results are
shown in Fig. 6. The activity of CuZn-SOD under DBP exposure is approximately 2.5 times higher than that in the control, and no change in Fe-SOD activity is evident. By contrast, a significant inhibition was observed in the activity of Mn-SOD. To explore the ROS production site, we performed an additional experiment to analyze the effects of electron transport chain inhibitors on the ROS content induced by DBP. The effects of diuron, rotenone and dicumarol on ROS production are shown in Fig. 6. Diuron did not affect ROS production, whereas rotenone and dicumarol decreased ROS production. The effects of DBP on ROS production in K. brevis cells pretreated in advance with diuron, rotenone, and dicumarol were also measured. Compared with the algal cells only treated with DBP, ROS production in cells pretreated with diuron showed no notable difference. Rotenone decreased and dicumarol stimulated the ROS production induced by DBP. 4. Discussion In spite of the known growth inhibition caused by DBP, its role in inducing an oxidative stress response in K. brevis remains largely unknown. This study demonstrates that DBP induces oxidative responses. The MDA content dramatically increased under high DBP concentration (5 mg L 1), suggesting that excess ROS in the algal cells attacked the cell membrane and resulted in lipid peroxidation. ROS (OH, H2O2, and O2 ) content varied by species throughout the experiment. The production of ROS is important in several cellular processes (e.g., cellular signaling and defense against infection). It has been suggested that the algae Selenastrum capricornutum was stimulated by ROS to increase its metabolic activity (Hong et al., 2008), but another report showed that the presence of ROS was more often associated with damage to cellular components such as proteins, lipids, and nucleic acids (Pospíšil, 2009). The reported increase in the activities of the antioxidant enzymes SOD and CAT in the study is an adaptive strategy. SOD catalyzes the dismutation of O2 to H2O2 and O2, while CAT directly eliminates H2O2 predominantly in the peroxisome and transforms H2O2 into H2O. In plants, CAT scavenges H2O2 generated during photorespiration and the b-oxidation of fatty acids and plays a significant role in the protection of aerobic organisms from the toxic effects of H2O2 (Apel and Hirt, 2004). The increased CAT activity and increased MDA content in algal cells suggests that the release of hydrogen peroxide is involved in the response of K. brevis cells to DBP. The antioxidant SOD activities of cells in the 1 mg L 1 DBP group initially increased rapidly and then decreased at 72 h, indicating that SOD in K. brevis respond with a short-term adaption to protect the algae against
Fig. 4. The ultrastructure of K. brevis in the control group after 24 h. CHL, chloroplast; M, mitochondrion; N, nucleus; S, starch grain; PM, plasma membrane; CW, cell wall. Scale bars are 1 lm in Fig. 4a and 200 nm in b and c.
F.-m. Li et al. / Chemosphere 132 (2015) 32–39
Fig. 5. The ultrastructure of K. brevis in the 5 mg L are 1 lm in Fig. 5 a and c, and 200 nm in b.
1
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DBP treatment group after 24 h. Fig. 5b is a magnified image of Fig. 5a. N, nucleus; V, vacuole; L, lipid globule. Scale bars
Fig. 6. Activities of SOD isoforms under DBP exposure.
peroxidation induced by DBP. Similar results were found when algae were exposed to enhanced UV-radiation (Tian and Yu, 2009). TEM images of the ultrastructure of K. brevis treated with DBP show altered cell organelles. The less-dense appearance of chloroplasts and mitochondria suggests that the cells were strongly affected by DBP (Fig. 5a and c). There is a high possibility that the structural disruptions to the chloroplasts and mitochondria induced by DBP were accompanied by reductions in photosynthetic and respiratory activities (Morlon et al., 2005). Moreover, the dense appearance of vacuoles around the inner cell surface was observed, indicating that the function of the vacuole, an organelle involved in detoxification, was strengthened. The compromised integrity of cell membrane caused by lipid peroxidation may result in ion leakage (Buchmann and Becker, 2009) and therefore leads to osmotic regulation of vacuoles. The distinct morphological phenomenon in which apical parts or small tubules are commonly found around the cell outer surface when exposed to DBP has seldom been discussed. To the best of our knowledge, the accumulation of vacuoles around the inner cell surface and changes on the outer cell surface may be related to algal cell secretions associated with metabolism. Another explanation may be that the apical changes resulted from a combination of the secretion process and the weak permeability of the cell membrane caused by lipid peroxidation. The transport of metabolites through algal membranes from/to seawater is an important and efficient
life-sustaining factor of algae in aquatic environments. These excretion points, however, obviously need further investigation. In photosynthetic algal species, ROS are continuously produced as byproducts of various metabolic pathways, which mainly derive from the electron transport chains in chloroplasts, mitochondria and the plasma membrane (Apel and Hirt, 2004). The production source of ROS in K. brevis exposed to DBP was investigated. The first step was to measure the SOD, a type of enzyme that can be highly induced under stress. Based on the metal cofactor, SOD isoforms in plants are classified into three groups: CuZn-SOD, Mn-SOD and Fe-SOD (Scandalios, 1993). More importantly, the different SOD isoforms are located in different cell compartments. Mn-SOD is known to be mitochondrial; Fe-SOD is located in chloroplasts in most plants examined to date; and CuZn-SOD is mainly found in cytosol, as well as a small amount in chloroplasts (Alscher et al., 2002). These SOD isoforms react differently under different forms of environmental stress (Khanna-Chopra and Sabarinath, 2004). Under DBP exposure, there was almost no change in the activity of Fe-SOD, whereas the increase in CuZnSOD activity indicated that the cytoplasm may have participated in ROS production. In contrast, the activity of Mn-SOD was significantly inhibited, which may have been caused by excessive ROS, because ROS can damage protein structure. It has been reported that antioxidant systems are prone to protein carbonylation if ROS exceeds the tolerance threshold (Gonçalves et al., 2007). One group of scientists investigated the effects of anthracene, cadmium and chloridazone on three Scenedesmus species by analyzing changes in SOD isoforms; they found that the applied substances disturbed metabolic processes in chloroplasts and indicated that chloroplasts may be the main target site of interaction for ROS overproduction in cells (Zbigniew and Wojciech, 2006). Thus, the ROS observed in K. brevis cells exposed to DBP could derive from cytosolic or mitochondrial sources, according to our results. The ROS production site was further investigated by testing the effects of inhibitors that affect different electron transfer chains. Diuron is an inhibitor of photosynthetic electron transport (McKim and Durnford, 2006). In the group exposed to diuron and then DBP, the ROS level did not differ notably compared with the group exposed only to DBP. This result suggests that chloroplasts were not the main ROS accumulation site in our experiment. However, in Lemna gibba, chloroplasts were found to be the main organ in which O2 induced by anthraquinone accumulated (Babu et al., 2001). Different test organisms and xenobiotics have been found to lead to various results. Rotenone, an inhibitor of the mitochondria electron transport chain complex (Møller, 2001), decreased DBP-induced ROS production, indicating that the mitochondria electron transport chain is related to the target site
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and Technology Program for Water Pollution Control and Treatment (2012ZX07203004), the Public Science and Technology Research Funds Projects of Ocean (No. 201305003), the NSFCShandong Joint Fund for Marine Science Research Centers (No. U1406403) and the Qingdao Science and Technology Project (No. 12-4-1-58-HY).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.01.051.
References
Fig. 7. Effects of inhibitors on DBP-induced ROS production. Control (A), DBP (B), Diuron (C), Rotenone (D), Dicumarol (E), DBP + Diuron (F), DBP + Rotenone (G), DBP + Dicumarol (H). ⁄⁄(p < 0.01) indicates a statistically significant difference compared to the control. Bars represent SD, n = 3.
of DBP. In contrast, dicumarol, an inhibitor of the redox enzyme system in the plasma membrane (Liu et al., 2007), stimulated DBP-induced ROS production. The reason for this, however, is not clear. We suggested that DBP may interact with dicumarol and result in joint toxicity. Liu et al. (2007) also made an attempt to identify the site of ROS in the algae Chattonella marina, using 3-(3,4-dichlorophenyl)-1,1-dimethylurea, KCN, Vitamin K3 and dicumarol, and suggested that ROS production in C. marina was related to a plasma membrane enzyme system. Considering the activity of SOD isoforms and the ROS content under exposure to different inhibitors, we concluded that DBP affected the plasma membrane and mitochondria, particularly inhibiting the mitochondria, because the protein Mn-SOD had been inactivated and ROS accumulated, resulting in the beginning of cell apoptosis and eventually death. Comparing the ROS values after adding electron transfer inhibitors (Fig. 7), only rotenone blocked the pathway of ROS production, indicating that ROS production induced by DBP in K. brevis in our culture conditions was related to the mitochondria. 5. Conclusion Our work showed that oxidative stress is the main toxicity mechanism for the observed growth inhibition of the algae K. brevis by DBP. Based on our results, we assumed that DBP caused an increase in ROS in the algae K. brevis and that it stimulated an increase of antioxidant enzymes as an adaptive response. Under our culture conditions, ROS could be generated both in the mitochondria and cytosol; furthermore, mitochondria could be the main target sites of interaction with DBP. When antioxidant enzymes in the mitochondria are damaged and can no longer scavenge the increased ROS, imbalance and the accumulation of ROS could occur, causing cell apoptosis. Disruption of the cell’s subcellular structure, including the plasma membrane and organelles, was observed. Because the inner mitochondrial membrane is impermeable to DBP, DBP may act on mitochondrial membrane enzymes and the electron transport respiratory chain in the form of metabolites. The interaction mechanism affecting the cell membrane still needs further research. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant No. 51378480), the Major Science
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