Ecotoxicology and Environmental Safety 115 (2015) 75–82

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Evaluation of DNA damage and antioxidant system induced by di-nbutyl phthalates exposure in earthworms (Eisenia fetida) Li Du, Guangde Li n, Mingming Liu, Yanqiang Li, Suzhen Yin, Jie Zhao, Xinyi Zhang College of Resources and Environment, National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer, Key Laboratory of Colleges and Universities in Shandong Province Agricultural Environment, Shandong Agricultural University, 61 Daizong Road, Taian 271018, China

art ic l e i nf o

a b s t r a c t

Article history: Received 14 November 2014 Received in revised form 28 January 2015 Accepted 31 January 2015

Di-n-butyl phthalates (DBP) are recognized as ubiquitous contaminants in soil and adversely impact the health of organisms. The effect of DBP on the activity of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT), malondialdehyde (MDA) content and DNA damage were used as biomarkers to analyze the relationship between DNA damage and oxidative stress and to evaluate the genotoxic effect of DBP on earthworms (Eisenia fetida). DBP was added to artificial soil in the amounts of 0, 5, 10, 50 and 100 mg per kg of soil. Earthworm tissues exposed to each treatment were collected on the 7th, 14th, 21st, and 28th day of the treatment. The results showed that SOD and CAT levels were significantly inhibited in the 100 mg kg
1 treatment group on day 28. MDA content in treatment groups was higher than in the control group throughout the exposure time, suggesting that DBP may lead to oxidative stress in cells. A dose–response relationship existed between DNA damage and total soil DBP levels. The comet assay showed that increasing concentrations of DBP resulted in a gradual increase in the OTM, Comet Tail Length and Tail DNA %. The degree of DNA damage was increased with increasing concentration of DBP. These results suggested that DBP induced serious oxidative damage on earthworms and induced the formation of reactive oxygen species (ROS) in earthworms. The excessive generation of ROS caused damage to vital macromolecules including lipids and DNA. DBP in the soils were responsible for the exerting genotoxic effects on earthworms. & 2015 Elsevier Inc. All rights reserved.

Keywords: Di-n-butyl phthalate Earthworm (Eisenia fetida) DNA damage Oxidative stress Genotoxicity

1. Introduction Phthalic acid esters (PAEs) are organic substances that are used in plastic matter industries because they can increase the plasticity of many materials. The US Environmental Protection Agency (US EPA, 1991) and the China National Environmental Monitoring Center (Wang et al., 1995) have already classified PAEs as priority environmental pollutants. Di-n-butyl phthalate (DBP), one of heavily used PAEs, is widely applied in the production process of plastics and cosmetics. It is ubiquitous environmental contaminant, as indicated by its presence in air, water, and soil worldwide (Huang et al., 1994). Examining urinary phthalate metabolites, Blount et al. (2000) found that the general population appears to be exposed to disproportionately higher amounts of DBP compared with other phthalates. DBP may induce lesions in the reproductive system of rabbits, especially during the intrauterine period (Higuchi et al., 2003). Exposure to low concentrations of DBP impairs spermatogenesis in frogs (Xenopus n

Corresponding author. E-mail address: [email protected] (G. Li).

http://dx.doi.org/10.1016/j.ecoenv.2015.01.031 0147-6513/& 2015 Elsevier Inc. All rights reserved.

laevis) (Lee and Veeramachaneni, 2005) and it is reported that in utero exposure to DBP resulted in reduced testosterone levels, leydig cell aggregates and multinucleated gonocytes in fetal testes (Kleymenova et al., 2005). DBP has been reported to reduce steroidogenesis by fetal-type leydig cells in primates and rodents (Hallmark et al., 2007). In addition, significant changes in the expression of 391 genes were detected during treatments to developmentally toxic phthalates (Liu et al., 2005). DBP can also cause oxidative stress in organisms. Qin et al. (2011a) found that SOD and CAT activities in the mantle were induced significantly and the LPO level was also obviously induced in Perna viridis during chronic DBP exposure. Gill SOD activity in Lutjanus erythropterus increased significantly as the concentration of DBP increased. Gill and liver MDA content increased then decreased after being exposed to DBP (Qin et al., 2011b). Chen et al. (2010) also found that SOD activity in stem and leaves increased first and then decreased with the increase of DBP concentrations and that DBP caused increase in MDA content in Arabidopsis seedlings. DBP has relatively high residual levels in the soils, ranging from 3.18 to 29.37 mg kg
1 in aquic soils of the Handan District (average 14.06 mg kg
1) and 2.75–14.62 mg kg
1 in black soils of the Harbin District (average 7.60 mg kg
1). The total content of four

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kinds of phthalates (DMP, DEP, DBP and DEHP) is 0.89–10.03 mg kg
1 in agricultural soil in China (Hu et al., 2003). Sludges contained DBP of 4.2 to approximately 5.7 mg kg
1 dried solids in Shanghai (Zheng et al., 2008). All non-cultivated soils contain the lowest contents of phthalates, suggesting that the kinds of pollutants are largely derived from human agricultural activities (Xu et al., 2008). Besides the average DBP concentration in the Yangtze River sediment reaches more than 84.3 mg kg
1 dry weight (Wang et al., 2008). The half-life period of DBP is about 20 years. Alkaline single-cell gel electrophoresis (comet assay) offers various advantages over other cytogenetic methods for DNA damage detection, such as chromosome aberrations, sister chromatid exchanges (SCEs) and micronucleus test (MN), because the cells studied need not to be mitotically active (Pavlica et al., 2001). Coelomocytes are the cells that circulate in the coelomic fluid and play an important physiological role in the immune defence of earthworms (Plytycz et al., 2006; Dhainaut and Scaps, 2001). Different biomarkers have been developed on earthworm coelomocytes that are able to detect sensitive physiological responses in animals exposed to toxic chemicals, such as lysosomal membrane stability and phagocytosis (Goven et al., 1993; Hankard et al., 2004). Coelomocytes represent therefore an appropriate target for assessing genotoxic damage in view of the non-invasive method of extraction, easy sample manipulation and short slide-preparation time (Eyambe et al., 1991; Fugère et al., 1996; Tučková and Bilej, 1996) These cells isolated from earthworms maintained in contaminated soils have been shown to exhibit significantly elevated levels of DNA single-strand breaks as measured by the comet assay (Verschaeve and Gilles, 1995; Šalagovič et al., 1996; Martin et al., 2005). Damage to DNA may cause mutations, strand breaks, altered bases (Shugart, 2000), eventually leading to carcinogenesis, teratogenesis and health disorders such as the genotoxic disease syndrome (Kurelec, 1993). Some other lesions of DNA damage such as DNA cross-links (e.g., thymidine dimers) and oxidative DNA damage may also be assessed using lesion-specific antibodies or specific DNA repair enzymes in the comet assay (Dhawan et al., 2009). Because of the high sensitivity and short response time, it has been a useful tool for the determination of genotoxicity. Šalagovič et al. (1996) have applied the comet assay to earthworms for the detection of strand breaks in the DNA. Reactive oxygen species (ROS) has been reported to interfere directly with cell metabolism. Besides cellular damage inflicted by ROS can increase the risk that damaged DNA leads to mutations and increasing the exposure of DNA to mutagens, including ROS (Ames et al., 1993). Superoxide dismutase (SOD) and catalase (CAT) are regarded as antioxidant enzymes that prevent oxidative stress and their levels which reflect change on biological cells may be used to monitor oxidative stress (Yu et al., 2008). Malondialdehyde (MDA), another indicator of oxidative stress, shows the degree of lipid peroxidation (LPO) in the body. It has been reported that antioxidant enzyme activities and LPO levels in fishes and bivalve animals may be used as biomarkers of oxidative stress and reflections of the extent to the organisms (Di Giulio et al., 1993; Solé et al., 1995). Farombi et al. (2006) indicated that the reduction in the activity of CAT observed in rats may reflect the inability of the testes to eliminate hydrogen peroxide possibly produced by activation of DBP and its metabolites or inactivation of the enzymes caused by excess generation of ROS in the testes and that the overwhelming generation of free radicals in the testicular milieu may therefore inactivate SOD as observed in rats. Earthworms play a critical role in soil structure and function (Saint-Denis et al., 1999). Furthermore, they contribute significantly to organic matter decomposition and nutrient cycling (Coleman and Ingham, 1988). Since they are ecologically important and plentiful, earthworms have been one of the most commonly

used organisms to examine biological effects of potentially harmful materials. The earthworm Eisenia fetida has been regarded as a model biomonitor for measuring soil environmental pollution since 1984 (OECD, 1984). Zeng et al. (2010) found that in Esisenia foelide DEHP induced oxidative damage to cells and changed the activity of enzymes. Because DBP, as a kind of PAE, has a similar structural formula and properties to DEHP (Charles et al., 1997), we supposed that E. fetida may be sensitive to DBP and can cause damage in E. fetida. Therefore, E. fetida was chosen for the evaluation of toxicity of DBP in the present study. Our purpose of this paper is to study whether DBP could induce oxidative defense and DNA strand breaks or not, and at the same time, to develop a more comprehensive under standing on the effects of DBP on earthworms, in order to obtain specific information about oxidative DNA damage.

2. Material and methods 2.1. Chemicals DBP (98% pure), was purchased from Shandong Jingbo Agricultural Chemical Co. Ltd. (Beijing). Other chemicals used in this study were also of analytical grade and were purchased from local commercial sources. Glassware was meticulously cleaned to reduce any background contamination of phthalates. All chromic acid washed glassware was placed in a 300 °C oven overnight. After cooling, the glassware was air-dried until use. 2.2. Earthworms E. fetida between 300 and 400 mg each were obtained from laboratory cultures at Shandong Agricultural University. Each culture is maintained with 5 g dry and defaunated cow dung per week on the artificial soil surface. Earthworms were removed from culture, rinsed with tap water and stored in Petri dishes on damp filter paper for 24 h (in the dark at 20 72 °C) to void the gut contents. The earthworms chosen for this assay had all reached adulthood and exhibited well-developed clitellum. 2.3. Acute toxicological tests The acute toxic effects of DBP on E. fetida were investigated by artificial soil tests according to the OECD normal method (OECD, 1984). The composition of artificial soil samples (dry weight) was 70% fine sand, 20% kaolin clay and 10% sphagnum peat. Calcium carbonate (about 1 mg per 100 mg
1 of artificial soil) was added in artificial soil to adjust the pH to a value of 7.070.2. According to the pre-experimental results, five concentrations, i.e., 500, 1000, 2000, 5000 and 10,000 mg DBP kg
1 artificial soil (dry weight), were set for the acute toxicity tests. DBP was dissolved in acetone and thoroughly mixed into the artificial soil at the various concentrations. The soils were placed in a well-ventilated fume hood and turned daily for 7 days in order to evaporate acetone and age the spiked soil. Following acetone evaporation, all soils were rehydrated to 35% moisture and left one day to equilibrate. The control was mixed with the same volume of acetone, distilled water and pH as the treatment groups. Mortality was determined at 14 d. The earthworms were cultivated for 24 h in untreated artificial soil and then they were put in the DBP treated artificial soil. Each 1000 mL container was filled with 500 g of artificial soil (wet weight). Ten worms with uniform body lengths and weights were randomly divided into the five treatment groups with three replicates of each treatment. The containers were sealed with plastic film to reduce evaporation and punched with holes for ventilation.

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Tests were conducted in a climate chamber with a stable temperature of 20 72 °C, moisture of 7572% with a 12:12 h light:dark regimen. 2.4. DBP exposure DBP concentrations of 0, 5, 10, 50 and 100 mg DBP kg
1 artificial soil (dry weight) were used for the toxicity tests. A single earthworm was collected from each replicate bowl on the 7th, 14th, 21st, and 28th day after application of DBP. Toxicity tests were conducted according to the OECD (1984) guidelines in artificial soil and similar to the acute toxicity tests described in detail above. 2.5. Comet assay The comet assay was conducted as described by Eyambe et al. (1991). Singh et al. (1988) with slight modifications. Individual earthworms were rinsed in the extrusion medium (EM) which consisted of 5% ethanol, 95% saline, 2.5 mg mL
1 Na2–EDTA and 10 mg mL
1 guaiacol glyceryl ether (pH 7.3). Coelomocytes were spontaneously secreted in the medium and washed twice with phosphate-buffered saline (PBS) prior to the comet assay. The cells were collected by centrifugation (3000 rpm, 3 min) and kept on ice in 4 °C before the comet assay. Frosted slides were coated with 100 μL of 1% agarose (normal melting point, NMA) in PBS. After agarose was solidified (15 min in refrigerator), coelomocytes suspension mixed with 0.7% agarose (low melting point, LMA) were placed on the first agarose layer. A coverslip was added and agarose was allowed to solidify for 15 min in the refrigerator. After removing the coverslip, the third layer of 0.7% LMA agarose was added and left to solidify as described. The coverslip was removed and the cells were lysed in freshly made lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% TRITON X-100, 1% N-Lauroyl Sarcosine Na), pH 10, for 1 h at 4 °C. After rinsing with distilled water, the slides were placed on a horizontal electrophoresis unit filled with fresh alkaline electrophoresis solution (300 mM NaOH and 1 mM Na2– EDTA, pH 13.0) to a level approximately 0.2 cm above the sides at 20 °C for 30 min to allow DNA unwinding before electrophoresis. Electrophoresis was conducted at 20 °C using 25 V and 300 mA for 30 min. After the electrophoresis, the slides were soaked in cold neutralizing buffer (500 mM Tris buffer, pH 7.5) at 4 °C for 10 min. The DNA were stained with ethidium bromide (EB) (2 μg mL
1), and the slides were examined with a fluorescent microscope. Three slides per treatment were prepared and at least 30 cells were analyzed from each slide. Images were analyzed according to the method of Collins et al. (1995) using the comet assay software project (CASP 1.2.2). 2.6. Enzyme sample preparation Earthworms were placed into a prechilled mortar and pestled under ice-cold conditions homogenized in Tris–HCl buffer (250 mM sucrose, 50 mM Tris (pH 7.5), 1 mM DTT and 1 mM EDTA) in a 1/4 w/v ratio for 1 min using a XHF-D homogenizer. Homogenates were centrifuged at 10,000 rpm for 15 min. After centrifugation, the supernatants were collected and stored at
20 °C until analysis. The samples were carried out at 4 °C. 2.7. SOD activity Superoxide dismutase (SOD) activity was determined according to a modification of the method of Marklund and Marklund (1974). This assay is based on the ability of SOD to inhibit the autoxidation of pyrogallol (50 mM) in a 50 mM Tris–HCl buffer (pH 8.3). The

77

reaction mixture contained 4.5 mL Tris–HCl buffer, 100 mL supernatant and 10 mL pyrogallol. Oxidation of pyrogallol was monitored by measuring absorbance at 325 nm. One unit of SOD activity was defined as 50% inhibition of the oxidation process. Enzymatic activity was expressed as U mg
1 Pr. 2.8. CAT activity Catalase (CAT) activity was determined according to a modified method of Greanwald (1985). A 3 mL solution contained 0.67 M substrate (prepared by adding 0.16 mL 30% H2O2 to 100 mL phosphate buffer, pH 7.0 and 100 mL sample) or blank (digestive gland and gills, respectively). Kinetics was recorded at 240 nm. The concentration of H2O2 was determined at 3–4 min intervals after the initiation of the reaction by addition of supernatant. One unit of CAT activity was defined as 50% H2O2 consumption at 1 min. The results are expressed as U mg
1 Pr. 2.9. MDA content Malondialdehyde (MDA) content was used as an indicator of lipid peroxidation (LPO) level and was quantified by measuring the formation of thiobarbituric acid reactive substances according to the methods described by Ohkawa et al. (1979) with some modifications. The concentration of MDA formed was calculated using the absorbance coefficient 1.56  105 M
1 cm
1 (Flecha et al., 1991) and results are expressed as nmol mg
1 Pr. 2.10. Statistical analysis The data were analyzed using SPSS (Standard Version 13.0, SPSS Inc.). The relationships between DBP concentration and SOD, CAT activity, MDA content, and DNA damage were tested by analysis of variance (ANOVA). When significant differences were considered at level P o0.05 between treatments, multiple comparisons were made by the least significant difference test. All values are presented as mean 7SD.

3. Results Results of the acute toxicity tests showed that the 14 d-LC50 in the artificial soil test was 2364.8 mg kg
1 (2319.5–2509.1 mg kg
1). There was no mortality observed during the following experiment progress. 3.1. Comet assay The amount of DNA that migrates away from the nuclei is used to assess the extent of DNA damage. Data on individual cell response using the OTM, Comet Tail Length and Tail DNA % are presented in Figs. 1–3. The comet assay (Fig. 1) showed that increasing concentrations of DBP resulted in a gradual increase in the OTM. During the entire exposure, the OTM at DBP dose ranging from 5 to 100 mg kg
1 were significantly higher than those of the control. Figs. 2 and 3 exhibit that the majority of cells after exposure to 5–10 mg kg
1 DBP also showed DNA damage. When exposed to 100 mg kg
1 of DBP, the Comet Tail Length and Tail DNA % values reached maximum. The degree of DNA damage was increased with increasing concentration of DBP. The cells in the control group obtained from images showed a nucleoid core with zero or minimal DNA migrating to the tail region (Fig. 4). Slight DNA migration was found under 5–50 mg kg
1 of DBP exposure. Upon treatment with 50 and 100 mg L
1 of DBP, the DNA migration increased significantly, and most of the DNA migrated away from the nuclei (Figs. 5–7).

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0

5

10

50

100 mg/kg

Olive Tail Moment (OTM)

7

* *

6

*

*

5

*

*

4 3

*

2

*

*

*

*

*

*

*

*

*

1 0 7

14

21

28

Treatment times/d Fig. 1. Effect of DBP on earthworm coelomocytes comet olive tail moment.

3.2. SOD activity

4. Discussion

SOD activity changed depending on DBP concentration and exposure duration (Fig. 8). After 28 d of exposure, SOD activity in all treatments obviously decreased. In comparison with the control, SOD activity was significantly inhibited on day 21 when the earthworms were exposed to 100 mg kg
1 of DBP. SOD levels were significantly inhibited in the 10–100 mg kg
1 treatment group on day 28.

As a simple, sensitive, and rapid technique, comet assay has been widely used to detect DNA damage in cells exposed in vivo to a variety of physical or chemical agents in individual cells (Fairbairn et al., 1995a) and therefore can be useful in studies of genetic toxicology, especially ecogenotoxicology (Fairbairn et al., 1995b). Compared to the other parameters of comet assay, the OTM, Comet Tail Length and Tail DNA % are more sensitive to differences in the gel and subsequent variability in DNA migration. The OTM values in the earthworms exposed to 5–100 mg kg
1 of DBP were statistically significant increased (p o0.05) when compared to the control during the entire exposure. In earthworm coelomocytes the OTM increased with increasing DBP concentration. The cells after exposure to 5–10 mg kg
1 DBP also showed DNA damage. Both Comet Tail Length and Tail DNA % increased gradually with an increase in DBP concentration. After exposure at the highest concentration (100 mg kg
1), DBP showed a high level of DNA damage to earthworm coelomocytes. In our study, a positive dose– response relationship was observed between DBP concentration and the corresponding OTM, Comet Tail Length and Tail DNA % values. This might be the reason that the defense mechanism of enhanced ROS scavenging capacity is induced by the stress from increasing doses in earthworms. The possible mechanism could be that highest concentrations of DBP caused severe stress and destroyed the balance of the system in the earthworm. DNA breaks may be directly produced by chemicals such as ROS and H2O2 which result from endogenous metabolism or may be produced in excess from redox cycling or other free radical interactions associated with organic xenobiotics, metabolites, and transition metals. (Mitchelmore and Chipman, 1998). Di Giulio et al. (1989) also found that oxidative stress caused by excessive ROS might lead to

3.3. CAT activity The effect of DBP on the CAT activity in earthworms after different days of exposure is displayed in Fig. 9. The CAT activity of treated earthworms was markedly reduced by a medium level of DBP (5–50 mg kg
1) when compared to the control exposure at 21 days. In addition, CAT activity in all treatment groups showed significant decrease compared with that in the control after 28 d of exposure. 3.4. MDA content The change in MDA content between control and DBP-treated E. fetida is displayed in Fig. 10. No significant perturbations in MDA content were noted between control and DBP-treated earthworms after 7 d of treatment. MDA content increased in the groups that received 50–100 mg kg
1 on day 14. After 21 d of treatment, MDA content in 10–50 mg kg
1 treatment groups increased significantly in comparison with the control. After 28 d of exposure, MDA levels were significantly higher in all treatment groups compared to the control group.

0

5

10

50

100 mg/kg

25

*

Comet Tail Length

* *

20

* *

15 10

*

*

*

*

*

*

* *

*

*

* 5 0 7

14

21 Treatment times/d

Fig. 2. Effect of DBP on earthworm coelomocytes comet tail length.

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L. Du et al. / Ecotoxicology and Environmental Safety 115 (2015) 75–82

0

5

10

50

79

100 mg/ kg

40 *

Tail DNA (%)

35

*

*

30

* *

25 20

*

15 10

*

*

*

*

* *

*

*

*

*

5 0 7

14

21

28

Treatment times/d Fig. 3. Effect of DBP on earthworm coelomocytes tail DNA.

Fig. 4. Typical comet image of earth worm coelomocytes cells in control. Fig. 7. Typical comet image of earth worm coelomocytes cells exposed to DBP (100 mg kg
1).

Fig. 5. Typical comet image of earth worm coelomocytes cells exposed to DBP (5 mg kg
1).

Fig. 6. Typical comet image of earth worm coelomocytes cells exposed to DBP (50 mg kg
1).

DNA, proteins and lipids damage. Environmental pollutants disrupt the normal metabolic processes of the cell, which may leads to a large production of ROS. Antioxidant activities such as SOD and CAT protect cells against adverse effects of ROS and they are able to reduce oxygen to water through their electron transport chains and protect themselves from normal ROS damage (Farber, 1994; Hu et al., 2010). MDA is an

oxidized product of cellular lipid membranes and could be used as a sensitive biomarker of cell injury (Muir et al., 2007; Maity et al., 2009). So SOD and CAT activity and MDA content were chose as biomarkers for the evaluation of the oxidative stress through the formation of ROS caused by DBP. SOD can convert O2
to O2 and H2O2 and protects cells against oxidative stress (Singh et al., 2006). SOD activity reflects changes in oxidative stress in biological cells, which may be induced by xenobiotics. Our study indicated that SOD activity in the lower concentration groups was inhibited during the early period of DBP exposure. After a longer exposure time, SOD activity decreased to levels that were lower than that of the control group. The decrease in SOD activity at 21 and 28 d may result from the elimination of the highly reactive O2- by conversion to H2O2, or may be the reason that the natural antioxidant defenses were saturated (Ma et al., 2012; Wu et al., 2011). The SOD measurements suggest that earthworms suffered more oxidative stress from DBP at higher concentrations and after a longer exposure time. SOD maintaining a dynamic balance can meet the need of the organism to eliminate O2- under normal physiological conditions. However, the balance between the formation and removal of O2- can be easily and quickly destroyed by oxidative stress after longer exposure times. CAT plays a key role in cellular antioxidant mechanisms. CAT catalyzes the conversion of H2O2 to water and oxygen and in this way removes H2O2, providing protection against oxidative damage to the cell. Exposure to chemicals may lead to a decrease in CAT activity and cellular lesions (Wu et al., 2012). Our study indicated that CAT activity was inhibited in earthworms exposed to DBP concentrations of 5–50 mg kg
1 after 21–28 d of exposure. A potential reason for the observed decreased activity may be inactivation of the enzyme by ROS and inhibition of enzyme

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0

5

10

50

100 mg/ kg

SOD activity( U mg-1 Pr)

0. 4 0. 35

*

0. 3 0. 25

* *

*

*

*

*

0. 2

*

*

*

*

0. 15

*

*

0. 1 0. 05 0 7

14

21

28

Treatment times/ d Fig. 8. SOD activity of the earthworm E. fetida exposed to varying concentrations of DBP and measured each week for 28 days.

CAT activity( U mg-1 Pr)

0 20

5

10

50

100 mg/ kg

*

15 10

*

*

*

* *

*

*

* *

5 0 7

14

21

28

Treatment times/ d Fig. 9. CAT activity of the earthworm E. fetida following exposure to various concentrations of DBP measured each week for 28 d.

MDA content ( nmol mg-1 Pr)

0

5

10

50

100 mg/ kg

30 25

*

*

*

*

*

*

*

*

20 15 10 5 0 7

14

21

28

Treatment times/ d Fig. 10. MDA activity of the earthworm E. fetida following exposure to various concentrations of DBP measured each week for 28 d.

synthesis or change in the assembly of enzyme subunits (Sandalio et al., 2001). Changes in the activity of SOD and CAT in earthworms was similar to the results of previous studies in that SOD activity decreased in response to low and high dose exposure (HartleyWhitaker et al., 2001; Lin et al., 2007). Kono and Fridovich (1982) found that O2-inhibited CAT action and the presence of H2O2 inhibited the action of dismutase. It is possible that exposure to DBP generates O2- which then leads to inhibition of CAT activity. As the metabolic product of lipid peroxidation in organisms, MDA indirectly reflects the degree of intracellular injury. Thus, it is a sensitive gauge of cellular oxidative injury (Grundy and Storey, 1998). MDA is a major product of the oxidation reaction between free radicals and unsaturated fatty acids in cellular membranes. Free radicals can react with the free amino groups of proteins to form inter- and intra-molecular protein crosslinks, resulting in cellular injury (Papadimitriou and Loumbourdis, 2002). Shalata and Tal (1998) suggested that one of the most damaging effects of ROS and their products in cells is the peroxidation of membrane lipids, which can be indicated by MDA detection. Many researchers

also considered that stress-dependent peroxidation of membrane lipids of cells was closely related to ROS (Candan and Tarhan, 2003; Fazeli et al., 2007). In the present study, the MDA content in treatment groups was higher than in the control group throughout the exposure time, indicating that more products of peroxidized unsaturated fatty acids had accumulated in tissues. After 28 d of exposure, there was a significant difference in MDA content between each treatment group and the control group. These results suggest the earthworms sustained serious oxidative damage and were not able to efficiently combat oxidative stress. We have investigated and analyzed on the correlation between SOD, CAT activity and DNA damage. The enhancement of DNA damage was due to oxidative stress, indicating that ROS accumulation in tissues caused subsequent DNA damage or due to the activation of DNA repair mechanisms induced by DBP in earthworms. Exposure to higher DBP concentration causes irreversible damage to a series of enzyme processes and physiological behavior in earth worms and eventually decreases the cellulase activity.

L. Du et al. / Ecotoxicology and Environmental Safety 115 (2015) 75–82

5. Conclusion Our results with respect to various antioxidant enzymes and MDA content clearly suggested ROS production in the earthworms exposed to DBP. The results indicated that treatment with DBP resulted in an excessive increase of ROS. ROS, in turn, stimulated the response of antioxidant defenses and resulted in oxidative DNA damage. The study also confirms that comet assay is an appropriate and sensitive method for assessment of genotoxicity resulting from exposure to DBP.

Acknowledgment This project was financially supported by the Shandong Provincial Natural Science Foundation of China.

Appendix A. Suplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.01. 031.

References Ames, B.N., Shigenaga, M.K., Gold, L.S., 1993. DNA lesions, inducible DNA repair, and cell division: three key factors in mutagenesis and carcinogenesis. Environ. Health Perspect. 101 (Suppl. 5), 35–44. Blount, B.C., Silva, M.J., Caudill, S.P., Needham, L.L., Pirkle, J.L., Sampson, E.J., Lucier, G.W., Jackson, R.J., Brock, J.W., 2000. Levels of seven urinary phthalate metabolites in a human reference population. Environ. Health Perspect. 108, 979–982. Candan, N., Tarhan, L., 2003. The correlation between antioxidant enzyme activities and lipid peroxidation levels in Mentha pulegium organs grown in Ca2 þ , Mg2 þ , Cu2 þ , Zn2 þ and Mn2 þ stress conditions. Plant Sci. 165, 769–776. Charles, A.S., Dennis, R.P., Thomas, F.P., 1997. The environmental fate of phthalate esters: a literature review. Chemosphere 35 (4), 667–749. Chen, X.X., Zhang, W., Wu, Y., Yang, X., 2010. Oxidative damage induced by dibutyl phthalate to stem and leaves of Arabidopsis seedlings. Asian J. Ecotoxicol. 5 (3), 402–406 (in Chinese). Coleman, D.C., Ingham, E.R., 1988. Carbon, nitrogen, phosphorus and sulfur cycling in terrestrial ecosystems. Biogeochemistry 5, 3–6. Collins, A.R., Ma, A.G., Duthie, S.J., 1995. The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. 36, 69–77. Dhainaut, A., Scaps, P., 2001. Immune defense and biological responses induced by toxics in Annelida. Can. J. Zool. 79, 233–253. Dhawan, A., Bajpayee, M., Parmar, D., 2009. Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biol. Toxicol. 25, 5–32. Di Giulio, R.T., Habig, C., Gallagher, E.P., 1993. Effects of Black Rock Harbor sediments on indices of biotransformation, oxidative stress, and DNA integrity in channel catfish. Aquat. Toxicol. 26, 15–22. Di Giulio, R.T., Washburn, P.C., Wenning, R.J., Winston, G.W., Jewell, C.S., 1989. Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environ. Toxicol. Chem. 8, 1103–1123. Eyambe, G.S., Goven, A.J., Fitzpatrick, L.C., Venables, B.J., Cooper, E.L., 1991. A noninvasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies. Lab. Anim. 25, 61–67. Fairbairn, D.W., Olive, P.L., O’Neill, K.L., 1995b. The comet assay: a comprehensive review. Mutat. Res. 339, 37–59. Fairbairn, J.J., Khan, M.W., Ward, K.J., Loveridge, B.W., Fairbairn, D.W., O’Neill, K.L., 1995a. Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia. Cancer Lett. 89, 183–188. Farber, J.L., 1994. Mechanisms of cell injury by activated oxygen species. Environ. Health Perspect. 102, 17–24. Farombi, E.O., Abarikwu, S.O., Adedara, I.A., Oyeyemi, M.O., 2006. Curcumin and kolaviron ameliorate di-n-butyl phthalate-induced testicular damage in rats. Basic Clin. Pharmacol. Toxicol. 100, 43–48. Fazeli, F., Ghorbanli, M., Niknam, V., 2007. Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two sesame cultivars. Biol. Plantarum 51, 98–103. Flecha, B.S.G., Repetto, M., Evelson, P., Boveris, A., 1991. Inhibition of microsomal lipid-peroxidation by alpha-tocopherol and alpha-tocopherol acetate. Xenobiotica 21, 1013–1022. Fugère, N., Brousseau, P., Krzystyniak, K., Coderre, D., Fournier, M., 1996. Heavy

81

metal-specific inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes from Lumbricus terrestris (Oligochaeta). Toxicology 109, 157–166. Goven, A.J., Fitzpatrick, L.C., Eyambe, G.S., Venables, B.J., Cooper, E.L., 1993. Cellular biomarkers for measuring toxicity of xenobiotics: effects of polychlorinated biphenyls on earthworm Lumbricus terrestris coelomocytes. Environ. Toxicol. Chem. 12, 863–870. Greanwald, R.A., 1985. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL. Grundy, J.E., Storey, K.B., 1998. Antioxidant defenses and lipid peroxidation damage in estivating toads, Scaphiopus couchii. J. Comp. Physiol. 168, 132–142. Hallmark, N., Walker, M., McKinnell, C., Mahood, I.K., Scott, H., Bayne, R., Coutts, S., Anderson, R.A., Greig, I., Morris, K., Sharpe, R.M., 2007. Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human. Environ. Health Perspect. 115, 390–396. Hankard, P.K., Svendsen, C., Wright, J., Wienberg, C., Fishwick, S.K., Spurgeon, D.J., Weeks, J.M., 2004. Biological assessment of contaminated land using earthworm biomarkers in support of chemical analysis. Sci. Total Environ. 330, 9–20. Hartley-Whitaker, J., Ainsworth, G., Meharg, A., 2001. Copper and arsenateinduced oxidative stress in Holcus lanatus L. clones with differential sensitivity. Plant Cell Environ. 24, 713–722. Higuchi, T.T., Palmer, J.S., Gray, L.E., Veeramachaneni, D.N.R., 2003. Effects of dibutyl phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol. Sci. 72, 301–313. Hu, C.W., Lia, M., Cui, Y.B., Li, D.S., Chen, J., Yang, L.Y., 2010. Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida. Soil Biol. Biochem. 42, 586–591. Hu, X.Y., Wen, B., Shan, X.Q., 2003. Survey of phthalate pollution in arable soils in China. J. Environ. Monit. 5, 649–653. Huang, X., Hang, X.J., Fu, Q., 1994. Assessment for biodegradability of organics in municipal wastewater and controlling measure for its refractory organics. Chin. J. Environ. Sci. 15, 15–19. Kleymenova, E., Swanson, C., Boekelheide, K., Gaido, K.W., 2005. Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat sertoli cells and disrupts sertoli cell-gonocyte contact. Biol. Reprod. 73, 482–490. Kono, Y., Fridovich, I., 1982. Superoxide radical inhibits catalase. J. Biol. Chem. 257, 5751–5754. Kurelec, B., 1993. The genotoxic disease syndrome. Mar. Environ. Res. 35, 341–348. Lee, S.K., Veeramachaneni, D.N.R., 2005. Subchronic exposure to low concentrations of di-n-butyl phthalate disrupts spermatogenesis in Xenopus laevis frogs. Toxicol. Sci. 84, 394–407. Lin, A.J., Zhang, X.H., Chen, M.M., Cao, Q., 2007. Oxidative stress and DNA damages induced by cadmium accumulation. Chin. J. Environ. Sci. 19, 596–602. Liu, K., Lehmann, K.P., Sar, M., Young, S.S., Kevin, W., 2005. Gene expression profiling following in utero exposure to phthalate esters reveals new gene targets in the etiology of testicular dysgenesis. Biol. Reprod. 73, 180–192. Ma, L.L., Ma, C., Shi, Z.M., Li, W.M., Xu, L., Hu, F., Li, H.X., 2012. Effects of fluoranthene on the growth, bioavailability and anti-oxidant system of Eisenia fetida during the ageing process. Eur. J. Soil Biol. 50, 21–27. Maity, S., Bhattacharya, S., Chaudhury, S., 2009. Metallothionein response in earthworms Lampito mauritii (Kinberg) exposed to fly ash. Chemosphere 77, 319–324. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 169–479. Martin, F.L., Piearce, T.G., Hewer, A., Phillips, D.H., Semple, K.T., 2005. A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longain spiked soil. Environ. Pollut. 138, 307–315. Mitchelmore, C.L., Chipman, J.K., 1998. DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutat. Res. 399, 135–147. Muir, M.A., Yunsua, I.A.M., Burchett, M.D., Lawrie, R., Chan, Y.K., Manoharan, V., 2007. Short-term responses of two contrasting species of earthworms in an agricultural soil amended with coal fly ash. Soil Biol. Biochem. 39, 987–992. OECD, 1984. Test 207: Earthworm, Acute Toxicity Tests, Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals. Organization for Economic Cooperation and Development. OECD, Paris, France. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal-tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Papadimitriou, E., Loumbourdis, N.S., 2002. Exposure of the frog Rana nidibunda to copper impact on two biomarkers, lipid peroxidation and glutathione B. Environ. Contam. Tox 69, 885–889. Pavlica, M., Klobučar, G.I.V., Mojaš, N., Erben, R., Papeš, D., 2001. Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res. 490, 209–214. Plytycz, B., Homa, J., Koziol, B., Rózanowska, M., Morgan, A.J., 2006. Riboflavin content in autofluorescent earthworm coelomocytes is species-specific. Folia Histochem. Cytobiol. 44, 275–280. Qin, J.F., Chen, H.G., Cai, W.G., Yang, T., Jia, X.P., 2011a. Effects of di-n-butyl phthalate on the antioxidant enzyme activities and lipid peroxidation level of Perna viridis. Chin. J. Appl. Ecol. 22 (7), 1878–1884 (in Chinese). Qin, J.F., Chen, H.G., Cai, W.G., Yang, T., Jia, X.P., 2011b. Effects of di-n-butyl phthalate (DBP) on activity of enzyms in different tissues of crimson snapper (Lutjanus

82

L. Du et al. / Ecotoxicology and Environmental Safety 115 (2015) 75–82

erythropterus). J. Fish Sci. China 18 (5), 1125–1131 (in Chinese). Saint-Denis, M., Narbonne, J.F., Arnaud, C., Thybaud, E., Ribera, D., 1999. Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil: effects of benzo(a)pyrene. Soil Biol. Biochem. 31, 1837–1846. Šalagovič, J., Gilles, J., Verschaeve, L., Kalina, I., 1996. The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites. Folia Biol. 42, 17–21. Sandalio, L.M., Dalurzo, H.C., Gómez, M., Romero-Puertas, M.C., del Rio, L.A., 2001. Cadmium induces changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115–2126. Shalata, A., Tal, M., 1998. The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt tolerantrelative Lycopersicon pennellii. Physiol. Plant 104, 169–174. Shugart, L.R., 2000. DNA damage as a biomarker of exposure. Ecotoxicology 9, 329–340. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Singh, S., Eapen, S., D’Souza, S.F., 2006. Cadmium accumulation and its influence on lipid peroxidation and antioxidative system in an aquatic plant, Bacopa monnieri L. Chemosphere 62, 233–246. Solé, M., Porte, C., Albaigés, J., 1995. The use of biomarkers for assessing the effects of organic pollution in mussels. Sci. Total Environ. 159, 147–153. Tučková, L., Bilej, M., 1996. Mechanism of antigen processing in invertebrates: are there receptors In: Cooper, E.L. (Ed.), Comparative and Environmental Physiology. Springer–Verlag, Berlin, Heidelberg, New York, pp. 41–72. US EPA, 1991. Risk Assessment Guidance for Superfund (RAGS): Volume I—Human Health Evaluation Manual (HHEM) (Part B, Development of Risk–Based Preliminary Remediation Goals). Office of Emergency and Remedial Response,

Washington, DC (EPA/540/R-92/003, OSWER Directive 9285.7-01B, NTIS PB92– 963333). Verschaeve, L., Gilles, J., 1995. Single cell gel electrophoresis assay in the earthworm for the detection of genotoxic compounds in soils. Bull. Environ. Contamin. Toxicol 54, 112–119. Wang, F., Xia, X.H., Sha, Y.J., 2008. Distribution of phthalic acid esters in Wuhan section of the Yangtze River, China. J. Hazard. Mater. 154, 317–324. Wang, J.L., Liu, P., Qian, Y., 1995. Microbial degradation of di-n-butyl phthalate. Chemosphere 31, 4051–4056. Wu, S.J., Wu, E.M., Qiu, L.Q., Zhong, W.H., Chen, J.M., 2011. Effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (Eisenia fetida) under laboratory conditions. Chemosphere 83, 429–434. Wu, S.J., Zhang, H.X., Zhao, S.L., Wang, J.L., Li, H.L., Chen, J.M., 2012. Biomarker responses of earthworms (Eisenia fetida) exposured to phenanthrene and pyrene both singly and combined in microcosms. Chemosphere 87, 285–293. Xu, G., Li, F.H., Wang, Q.H., 2008. Occurrence and degradation characteristics of dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) in typical agricultural soils of China. Sci. Total Environ. 393 (2–3), 333–340. Yu, M., Li, S.M., Li, X.Y., Zhang, B.J., Wang, J.J., 2008. Acute effects of 1-octyl-3-methylimidazolium bromide ionic liquid on the antioxidant enzyme system of mouse liver. Ecotoxicol. Environ. Saf. 71, 903–908. Zeng, Q., Cai, F.Y., Wang, J., Li, L., Yang, X., 2010. A study on the oxidative damage of Esisenia foelide cells induced by DEHP, In: Proceedings of the 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, Chengdu, China, pp. 1–4. Zheng, Z., He, P.J., Fu, Q., Shao, L.M., Lee, D.J., 2008. Partition of six phthalic acid esters in soluble and solid residual fractions of wasterwater. Environ. Technol. 29, 343–350.