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Antioxidant defense system responses and DNA damage of earthworms exposed to Perfluorooctane sulfonate (PFOS)
Environmental Pollution 174 (2013) 121e127
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Antioxidant defense system responses and DNA damage of earthworms exposed to Perfluorooctane sulfonate (PFOS) Dongmei Xu a, Chandan Li b, Yuezhong Wen b, *, Weiping Liu b a b
College of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2012 Received in revised form 16 October 2012 Accepted 29 October 2012
The use of earthworms as a sublethal endpoint has significantly contributed to the ecological risk assessment of contaminated soils. Few studies have focused on the potential toxicity of PFOS to earthworms in the soil. In this work, artificial soils were tested, and contact filter paper studies were used. The results showed that earthworm growth was generally inhibited. The antioxidant activities of the enzymes superoxide dismutase, peroxidase, catalase and glutathione peroxidase were initially activated and then inhibited. Reduced glutathione content was observed, and malondialdehyde content was elevated over the duration of the exposure. These results suggested that PFOS induced oxidative stress in earthworms. In addition, the values of olive tail moment, tail DNA% and tail length using SCGE showed similar frequency distributions and increased with increases in the PFOS concentration. These results suggest that all concentrations of PFOS cause DNA damage. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: PFOS Soil Ecotoxicity Earthworm Enzyme activity DNA damage
1. Introduction Perfluorooctane sulfonate (PFOS) has been widely used in consumer and industrial products, including food packaging, insecticides, protective coatings for fabrics and carpets, paper coatings, paints, cosmetics and fire-fighting foams (Moody et al., 2002; Noorlander et al., 2011). As a result, a pervasive presence of PFOS in food, drinking water and natural waters has been detected (Noorlander et al., 2011; Kannan et al., 2005; Lechner and Knapp, 2011; Skutlarek et al., 2006; Hansen et al., 2002; Taniyasu et al., 2004; Simcik and Dorweiler, 2005; So et al., 2007). PFOS is not readily biologically degradable and is often released in industrial wastewater directly into the aquatic environment or indirectly via canalization and sewage treatment plants. PFOS is emitted from wastewater treatment plants partially in the effluent and is partially adsorbed on sewage sludge. The use of sewage sludge as a fertilizer in agriculture can result in the contamination of soil with PFOS (Lechner and Knapp, 2011; Sinclair and Kannan, 2006). Due to the persistent and bioaccumulative nature of PFOS, its toxicities to aquatic organisms, animals and cells have attracted much attention. PFOS exposure in aquatic organisms and mammals has been shown to cause adverse effects in many systems, including hepatotoxicity, immunotoxicity, reproductive and developmental * Corresponding author. E-mail address: [email protected] (Y. Wen). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2012.10.030
toxicity, neurotoxicity and increased tumorigenic potential of the liver, pancreas and breast (Harada et al., 2005; Cui et al., 2009). Based on the 72 h oral LD50 of this compound on honeybees, PFOS was classified as “highly toxic” by the International Commission for Bee Botany. However, very few studies have focused on the potential harm of PFOS to terrestrial ecotoxicity (Zhao et al., 2011), despite the high levels of PFOS found in agricultural soils (Renner, 2009). The bioavailability and toxicity of contaminants varies among different soils and knowledge of this variance is essential to develop soil environmental quality guidelines (Yan et al., 2011). Therefore, to develop a comprehensive toxicity profile for PFOS, the toxicity of PFOS-contaminated soils should be assessed with various bioassays. Organisms such as fish, snails and plants have been employed as biomonitors (Dorts et al., 2011; Li, 2009). A study showed that the survival after 21 d EC50 for PFOS was lowest for onions and highest for soybeans in a test of seven species of plants. Although this approach is useful and promising, it is somewhat limited because it is based on a specific combination of living organisms with certain substances (Hirano and Tamae, 2011). Earthworms, the most prevalent animal species in soil, play an irreplaceable role in maintaining the ecological functions of soil. They have been used as important biomonitors to assess the ecological risks of toxic substances in the terrestrial environment (Fent, 2003; Xu et al., 2010). The molecular, cellular and physiological levels of earthworms change significantly when they are
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under contamination stress. These reactions produce a specific biological signal, called a biomarker. The biomarkers can indicate the impact of pollutants on the individuals before the lethal effect becomes apparent and can also be used to monitor soil pollution and provide an eco-toxicological diagnosis as an early warning system (Spurgeon et al., 2003). However, few studies have focused on the potential toxicity of PFOS to earthworms in the soil (Xu et al., 2011). In this paper, Eisenia foetida (widely used in ecotoxicological tests of earthworms) was used as a model organism to study the effects of PFOS on the change in bodyweight, antioxidant defense system and DNA damage. The results revealed the toxic effects of PFOS and provide a scientific basis for the comprehensive evaluation of the effect of PFOS on soil ecosystems. 2. Materials and methods 2.1. Materials and reagents The experimental animals, E. foetida, used in this study were supplied by the active central earthworm breeding farm of Zhejiang University. The experiments were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. Healthy adult earthworms, more than 2 months in age, with a bodyweight of approximately 0.3 g and obvious clitellum, were domesticated for 7 days in an artificial climate incubator before performing the tests. Potassium heptadecafluorooctanesulfonate (PFOS, 98%) was purchased from Tokyo Chemical Industries. N-Lauroyl-sacosine sodium salt, trihydroxymethyl aminomethane (Tris) and guaiacol glyceryl ether were purchased from Amersco. Normal melting agarose (NMA) and Triton X-100 were purchased from Acros. Low melting agarose (LMA) was purchased from Serva. Ethylenediamine tetraacetic acid disodium salt (Na2-EDTA) and dimethyl sulphoxide (DMSO) were purchased from J&K. The other reagents used were AR grade. Artificial soil was prepared according to a standard method from OECD guideline no. 207 (OECD, 1984). Artificial soil was made in the laboratory by mixing a ratio of 69% silica sand, 20% kaolinite clay, 10% peat moss and 1% CaCO3 (on a weight basis). The pH of the artificial soil was 7.0. 2.2. Earthworm exposure protocol and enzyme assays Based on the results of acute toxicity tests and avoidance behavior tests of PFOS used in the field (Xu et al., 2011), six different concentrations (0, 10, 20, 40, 80, 120 mg/kg) were used in the present study. Three replicates were used for each concentration with 500 g of artificial soil in each. The soil was spiked with 175 ml PFOS water solution that the required concentrations of PFOS are 0, 28.57, 57.14, 114.28, 228.57 and 342.86 mg/L, respectively. Final PFOS concentrations in soil were 0, 10, 20, 40, 80 and 120 mg/kg, respectively. The contaminated soils were then rehydrated to give an overall moisture content of approximately 35% of the final weight. They were then mixed thoroughly and left for one day to equilibrate. After voiding their gut contents, 10 healthy earthworms were weighed and introduced into the soil. The containers were then covered with gauze to limit water loss. From days 15 to 42, 0.5 g of finely ground dry horse manure per earthworm was added to the soil surface weekly. All exposures were conducted in an artificial climate incubator at 20 1 C in 80e85% relative humidity with a 16:8 h light:dark regime. The earthworms were collected after 2, 7, 14, 28 and 42 days of exposure. Survival status was recorded, and the earthworms were left for 1 d to void their gut contents. They were then weighed without drying, and the enzyme activities were measured. After toxicant exposure, five live earthworms from three parallel experiments of each treatment were collected. The earthworms were mixed with an amount of Trise HCl buffer (100 mmol/L, pH 7.5) that was four times that of the their bodyweight. They were then homogenized and centrifuged at 9000 g at 4 C for 30 min. The supernatant (post-mitochondrial fraction, S9) was collected and stored at 80 C for use in the enzyme activity assays. The contents of total protein, malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined using kits obtained from the Nanjing Jiancheng Bioengineering Institute.
conducted on ice. The alkaline comet assay was performed as described originally by Singh with slight modifications (Singh et al., 1988). The cell suspension (10 ml) was mixed with 90 ml of 0.7% low melting agar (LMA) in PBS at 37 C and pipette onto fully frosted slides precoated with a layer of 100 ml 0.75% normal (NMA). After solidification, the slides were immerse into a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA (pH 10.0), 1% Na-sarcosinate, supplemented with 10% dimethyl sulfoxide (DMSO) and 1% Triton X-100). Slides were then placed in an electrophoresis tank containing 300 mM NaOH with 1 mM Na2EDTA for 20 min prior to electrophoresis for 25 min at 25 V (300 mA). The slides were then neutralized (0.4 M Tris, PH 7.5) thrice at 5 min intervals and stained with ethidium bromide (2 mg/ml) for fluorescence microscopy analysis. 2.4. Statistical analysis Each treatment was conducted in triplicate. SPSS 17.0 statistical software was used to analyze the experimental data, and the results were expressed in the form of mean SD. An analysis of variance was conducted to analyze the enzyme activities of earthworms in terms of PFOS concentration, time of exposure, and their interaction. Significant differences (P < 0.05) between the treatment groups and the control were determined using the post hoc LSD test. Slides were viewed using a fluorescence microscope (Nikon, Eclipse, Te2000-U) equipped with a CCD camera. Non-overlapping cells were captured at 400 magnification. At least 100 cells per slide were analyzed using the Comet Assay Software Project (CASP). A measurement of olive tail moment (OTM) was used to quantify the extent of DNA damage. Tail length (TL) and tail DNA% (TDNA) were also analyzed. Comet assay data were found to be abnormally distributed. Therefore, non-parametric statistical methods were employed. Significant differences (P < 0.01) between the treatment groups and the control were determined using the ManneWhitney U (Two-tailed) test.
3. Results and discussion 3.1. Effects of PFOS on E. foetida weight Bodyweight development is a somewhat insensitive response, especially when testing copper (Hartmut and Otto, 1997), but it is a macroscopic indicator that shows the effect of pollutants on organisms. The effects of PFOS on earthworm weight are shown in Fig. 1. Earthworm weight in the control group was significantly enhanced relative to incubation duration. The growth of the earthworms were generally inhibited except for earthworms exposed to 10e80 mg/kg PFOS for 2 d. After exposure for 7 d and 28 d, the weight change rates of all earthworm groups treated with PFOS were significantly lower than that of the control groups. When exposed for 14 d and 42 d, only the treatment group exposed to the highest concentration of PFOS (120 mg/kg) was significantly different from the control group. The results suggested that PFOS significantly affected the earthworms’ growth, which may be related to changes at the molecular and physiological level.
30
0 40
10 80
**
18 12 **
*
**
6
2.3. Alkaline comet assay Contact filter paper tests were conducted within 48 h. Seven different concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mg/kg) were used. Ten replicates were used for each concentration, and the controls were exposed to the same volume of deionized water. Earthworm coelomocytes were obtained using the method described by Bu et al. (2006). Individual earthworms were rinsed in 1 ml volume of extrusion medium composed of 5% ethanol, 95% saline, 2.5 mg/ml EDTA, 10 mg/ml guaiacol glyceryl ether (pH 7.3). Coelomocytes were spontaneously secreted in the medium and collected by centrifugation (9000 r/min, 4 C). Phosphate-buffered saline (PBS) was used to wash the cells prior to the comet assay. All the performance was
20 120 mg·kg-1
24 Weight change rate, %
122
*
** *** ***
***
0 -6
2
14
7
28
42
*** Time, day
Fig. 1. Weight change rate of E. foetida exposed to PFOS. Data are expressed as mean sd (n ¼ 3, n: the samples of replicate). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
3.2. Effect of PFOS on E. foetida antioxidant responses-enzymatic system
0.7 0 40
*
0.5
10 80
20 120mg·kg-1 *** * ** * *
***
***
0.4 0.3 0.2 0.1 0.0 14 Time, day
7
2
28
42
Fig. 3. The effect of PFOS on the POD activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
were approximately the same as those observed by Qu et al. (2010). In that study, the toxic effects of different concentrations (0.1e 200 mg/L) of PFOS on wheat were investigated. Their results showed that low concentrations of PFOS (0.1e10 mg/L) induced SOD and POD activities in wheat roots and leaves, a high concentration of PFOS (200 mg/L) inhibited the SOD and POD activities. Pollution induces the expression of antioxidant enzymes that allow organisms to partially or totally overcome stress resulting from exposure to an unsafe environment (Cossu et al., 1997). However, excess toxicity inhibits these enzymes. The decrease of SOD and POD activities may have been due to excessive ROS production. Normally, ROS levels are in a dynamic balance with the antioxidant enzyme level. However, if the free radicals induced directly or indirectly by xenobiotic pollutants cannot be scavenged promptly, the natural antioxidant defenses can be overwhelmed. This can lead to severe sub-cellular injuries, such as ion loss, protein denaturation, and DNA damage (Nel et al., 2006). The CAT and GSH-Px activities are shown in Figs. 4 and 5, respectively. The CAT activity was inhibited in the PFOS-treated
7
50 0 40
6
10 80
20 120mg·kg-1 **
**
5
*
*
*
**
4 ***
3
*** ***
2 1 0 2
0 40
7
14 Time, day
28
42
Fig. 2. The effect of PFOS on the SOD activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01.
10 80
40 CAT activity, U·gprot -1
SOD activity , U·mgprot-1
***
0.6
POD activity, U·mgprot-1
Many studies have reported that PFOS can induce oxidative stress in many organisms (Shi and Zhou, 2010; Liu et al., 2007). The burden of reactive oxygen species (ROS) production is largely counteracted by an intricate antioxidant defense system. Antioxidant defense systems consist of an enzymatic component and a nonenzymatic component. Enzymatic systems include SOD, CAT, POD, GSH-Px and other antioxidant enzymes, while nonenzymatic systems include GSH and other nonenzymatic substances. SOD, CAT, POD and GSH-Px activities are often used as biomarkers to indicate ROS production (Zelikoff et al., 1996). The enzymes SOD, CAT, and POD are involved in the detoxification of O 2 (SOD) and H2O2 (CAT, POD) and prevent the formation of ROS (Wen et al., 2011). In this study, the effects of PFOS on E. foetida antioxidant responses were studied. The activities of SOD, POD, CAT and GSHPx in earthworms exposed to PFOS are presented in Figs. 2e5. As shown in Fig. 2, in general, SOD activity was first activated, then inhibited, except on days 14 and 42. Compared to the controls, SOD activity was significantly higher and extremely higher in the presence of 10, 40, or 120 mg/kg PFOS on day 7, 40 mg/kg PFOS on day 28 and 120 mg/kg PFOS on day 42. The SOD activity declined on day 14 and was induced on day 42 with increasing PFOS concentrations. The same trend was found for POD activity. POD had a rapid toxic response and a short active period (see Fig. 3). After 2 d of treatment, POD activity was significantly greater in the PFOStreated groups compared to the controls, particularly in the presence of 20, 40 or 80 mg/kg PFOS. After 7 d, the POD activity in the PFOS-treated groups was extremely higher than the controls at 10 and 120 mg/kg PFOS. The POD activity of the treatment groups showed few changes compared to the controls until day 42, at which point the POD activity of the PFOS-treated groups was significantly higher than that of the controls. At the initial stage of exposure (2 d, 7 d), the CAT activity, and SOD and POS variation are quite different. Since the 14th day of exposure, the CAT activity has the same trend of variation with the above two ferments. But we have also noticed that, as the extension of exposure time, under the impact of PFOS, CAT activity of earthworm sees more obvious decrease than that of SOD and POD, which might because the accumulation of H2O2, the product of free radical have restrained the activity of ferment. The changes in the SOD and CAT activities
123
20 120mg·kg-1 ** *
**
30 20 *
*
**
10 0 2
7
14
28
42
Time, day Fig. 4. The effect of PFOS on the CAT activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
0 40
16 GSH-Px , U · mg prot-1
*
12
*
*
10 80
*** **
******
8
20 120 mg·kg-1 *** *** **
** *
4
0.6
GSH content, mgGSH·gprot-1
124
0 40
0.5 0.4
*** *** ***
0.3 0.2
10 80
*** ***
20 120mg·kg-1
*
** **
** **
**
*
**
**
0.1 0
2
7
14 Time, day
28
0.0
42
Fig. 5. The effect of PFOS on the GSH-Px activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01.
groups compared to the control group during early exposure (2e 14 d), and it was significantly decreased in the PFOS-treated groups relative to the control at 2 d and 7 d. CAT activity was stimulated at low concentrations of PFOS, while it decreased markedly at high concentrations and exposure times. The GSH-Px activity was stimulated in the PFOS-treated except for in several treatment groups on day 14. Some of the treatment groups on day 2, 7, and 28 showed significant or extremely significant increased GSH-Px activity relative to the control group. The enzyme activity was not markedly different on day 42. Similar results were reported by Shi, after exposure to PFOS (0, 0.2, 0.4, or 1.0 mg/L), CAT and GSH-Px activities in 4 h fertilized zebrafish embryos of the treatment groups were significantly induced (Shi and Zhou, 2010). However, the changes in the CAT and GSH-Px activities in the present study are inconsistent with the results of a previous study by Hu (Hu and Hu, 2009). The results of this previous study showed that high concentrations of PFOS (150 or 200 mmol/L) significantly enhanced CAT activity while strongly reducing GSH-Px activity. The disparity between the results could be attributed to the different organisms studied and different mechanisms of action.
2
Fig. 6. The effect of PFOS on the GSH content of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
damage. The content of MDA can indirectly determine the level of radicals. MDA may cause a variety of cell damage. As depicted in Fig. 7, the MDA content changed slightly during early exposure (2 d, 7 d). With increased exposure duration, the MDA content rose significantly. These results indicate that treatment with PFOS resulted in an increase of ROS. ROS, in turn, stimulated the response of antioxidant defenses and resulted in a decreased earthworm growth rate and impaired its DNA and physiological functions. 3.4. DNA damage induced by PFOS The DNA damage of earthworm coelomocytes was compared with control coelomocytes after being exposed to PFOS for 48 h. Fig. 8a shows a typical comet image of undamaged DNA from earthworm coelomocytes, and Fig. 8b shows typical comet image of damaged DNA of earthworm coelomocytes. Figs. 9e11 show the box charts of the dynamic changes of OTM, TDNA and TL, respectively. As shown in the figures, the frequency distribution of the
3.3. Effect of PFOS on E. foetida antioxidant responsesnonenzymatic system
0 40
1.2
MDA content, nmol·mgprot-1
The enhancement of antioxidant enzyme activities suggested that oxidative stress conditions led to the increased antioxidant capability of earthworms. Reduced glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine (which is attached by normal peptide linkage to a glycine) and the carboxyl group of the glutamate side chain. It exists widely in vivo and plays a central role in coordinating the body’s antioxidant defense process. GSH is an important detoxification substance: its sulphydryl can bind with the carbon or chlorine atom of many pollutants and metabolites. When GSH is depleted, the pollutants and metabolites that are not consumed by GSH act on large biological molecules and cause oxidative stress. Changes in the GSH content in E. foetida are shown in Fig. 6. In general, the GSH content in the treatment groups was lower than that of the control except for the exposure on day 7, suggesting that GSH was markedly consumed in the PFOS-treated groups. Diminished GSH levels elevated cellular vulnerability for oxidative stress, as shown in Fig. 7. MDA is the ultimate lipid peroxidation product of oxidative
42
28
14 Time, day
7
10 80
20 120mg·kg-1 ***
1.0 *
0.8
***
***
***
**
***
0.6 **
** *** **
*
*
0.4 0.2 0.0 2
7
14
28
42
Time, day Fig. 7. The effect of PFOS on the MDA activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
125
300
125
250
100
Tail Length, µm
Olive Tail Moment, %•µm -1
Fig. 8. Typical comet figures. (a) Deionized water control, (b) 2 mg/cm2 PFOS.
75 50 25
200 150 100 50
0
0 0
0.25
0.5
1
2
4
8
0
PFOS Concentration, µg/cm2
0.5
1
2
4
PFOS Concentration, µg/cm
Fig. 9. The changes in olive tail moment caused by different concentrations of PFOS.
TDNA and TL data is similar to that of OTM. Fig. 9 shows that the OTM values of the treatment groups were all higher than the controls and that at 1 mg/cm2 PFOS, these values were low relative to other treatment groups, but not the controls. The dynamic changes of OTM, TDNA and TL values are shown in Table 1. In general, the values of OTM, TDNA, and TL increased
100 80 60
Tail DNA, %
0.25
8
2
Fig. 11. The changes in tail length caused by different concentrations of PFOS.
with the increase in PFOS concentration. The OTM values ranged from 3.60 0.46 to 24.24 2.46, with the highest value observed at 4 mg/cm2 PFOS. The increase of PFOS may cause ROS levels to increase, leading to DNA damage. However, the OTM value decreased at 8 mg/cm2. The data showed a similar trend to those of IR on ovarian carcinoma (David et al., 2010). Our analysis showed that the OTM, TDNA, and TL values of the treatment groups were extremely significantly elevated (P < 0.001) relative to the controls. All of the concentrations of PFOS caused DNA damage. In this study, the enhanced DNA damage was due to oxidative stress, indicating that ROS accumulation in tissues caused subsequent DNA damage. Many studies have shown that ROS is the major source of DNA damage, ROS cause DNA damage by causing strand breaks, removing nucleotides, and modifying the nucleotide bases (Ames, 1983; Cooke et al., 2003). A study of common carp exposed to 0.1,
40 Table 1 DNA damage of earthworm coelomocytes exposed to PFOS.
20 0 -20
0
0.25
0.5
1
2
4
8
PFOS Concentration, µg/cm2 Fig. 10. The changes in tail DNA caused by different concentrations of PFOS.
PFOS concentration (mg/cm2)
OTM (%$mm)
0 0.25 0.5 1 2 4 8
3.60 13.45 18.11 10.90 23.48 24.24 13.56
0.46 1.47*** 2.39*** 1.68*** 2.53*** 2.46*** 1.52***
TL (mm)
TDNA% 9.88 24.59 25.87 19.66 31.65 32.58 25.61
1.12 1.46*** 1.84*** 1.78*** 2.04*** 1.77*** 1.88***
Statostoa; significance versus control group. ***P < 0.001.
25.52 68.32 75.04 54.90 94.64 101.55 56.55
2.42 4.62*** 5.71*** 4.33*** 7.12*** 6.33*** 4.44***
126
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
Table 2 ANOVA results for bodyweight and the anti-oxidation of E. foetida exposed to PFOS. Items
SOD POD CAT GSH-Px GSH MDA Weight change rate
Concentration
Duration
df F
df F
P
df
F
P
4 4 4 4 4 4 4
0*** 0*** 0*** 0*** 0*** 0*** 0***
20 20 20 20 20 20 20
2.39 3.29 4.91 6.35 5.92 3.33 1.26
0.006** 0.001*** 0*** 0*** 0*** 0.001*** 0.24
5 5 5 5 5 5 5
P
1.90 0.111 2.56 0.042* 5.08 0.001*** 8.56 0*** 5.26 0.001*** 13.55 0*** 6.19 0***
8.56 33.12 32.43 33.50 38.11 10.42 18.38
Concentration*duration
df: degree of freedom, *: cases where the concentration or the duration of exposure had a significant effect (*P < 0.05, **P < 0.01, ***P < 0.001).
0.5 and 1 mg/L PFOS in water for 14 d was conducted previously (Hagenaars et al., 2008). The results of this previous study showed that several genes related to energy, reproduction and stress responses were expressed. Furthermore, PFOS may alter the expression levels of genes related to antioxidant enzymes (Nakayama et al., 2008). 3.5. Analysis of variance of bodyweight and anti-oxidation affected by PFOS When E. foetida earthworms were exposed to PFOS, changes in the relative physical index were the result of combined effects of pesticide concentration and exposure duration. Table 2 shows that the PFOS concentration had a very significant effect on the rate of change of weight and other biochemical indicators, except SOD activity. Exposure duration caused extremely significant changes for all indicators. The effects of the interactions between PFOS concentration and exposure duration were significant for all indicators except bodyweight. The fluro structure of the length of 8 carbon chains in the PFOS molecule structures has obvious hydrophobility. Wang et al. observed the toxicity of individual PFCAs and their mixtures to Photobacterium phosphoreum. The results showed that “the maximum tolerance of the cell membrane” led to a tendency of increasing toxicity from C3 to C14 PFCA and a tendency of decreasing toxicity from C14 to C18 PFCA (Wang et al., 2011). Basing on the research outcome and the concentration of PFOS in the practical soil environment, we may deem that the PFOS concentration in current soil environment will not constitute any threat to the earthworm or other animals in the soil, but the bioconcentration and transmission along the food chain (network) may enable the advanced consumers to concentrate such pollutants within their bodies, and suffer from their potential toxicity. And long term low dose exposure is likely to result in negative effect. Acknowledgments The authors acknowledge financial support from the National Natural Science Foundation of China (No. 20907046) and the National Basic Research Program of China (No. 2009CB421603). References Ames, B.N., 1983. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science 221 (4617), 1256e1264. Bu, Y.Q., Luo, Y.M., Teng, Y., 2006. Detection of DNA damage in earthworm (Eisenia foetida) in vivo exposure to copper ion. Asian J. Ecotoxicol. 1 (3), 228e235. Cooke, M.S., Evans, M.D., Dizdaroglu, M., Lunec, J., 2003. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195e1214. Cossu, C., Doyotte, A., Jacquin, M.C., Babut, M., Exinger, A., Vasseur, P., 1997. Glutathione reductase, selenium dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic contamination in field studies. Ecotoxicol. Environ. Saf. 38, 122e131.
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Hansen, K.J., Johnson, H.O., Eldridge, J.S., Butenhoff, J.L., Dick, L.A., 2002. Quantitative characterization of trace levels of PFOS and PFOA in the Tennessee River. Environ. Sci. Technol. 36 (8), 1681e1685. Harada, K., Xu, F., Ono, K., Lijima, T., Koizumi, A., 2005. Effects of PFOS and PFOA on L-type Ca2þ currents in guinea-pig ventricular myocytes. Biochem. Biophys. Res. Commun. 325 (2), 487e494. Hartmut, K., Otto, L., 1997. Development and standardization of test methods for the prediction of sublethal effects of chemicals on earthworms. Soil Biol. Biochem. 29, 635e639. Hirano, T., Tamae, K., 2011. Earthworms and soil pollutants. Sensors 11, 11157e11167. Hu, X.Z., Hu, D.C., 2009. Effects of perfluorooctanoate and perfluorooctane sulfonate exposure on hepatoma Hep G2 cells. Arch. Toxicol. 83, 851e861. Kannan, K., Tao, L., Sinclair, E., Pastva, S.D., Jude, D.J., Giesy, J.P., 2005. Perfluorinated compounds in aquatic organisms at various trophic levels in a great lakes food chain. Arch. Environ. Contam. Toxicol. 48, 559e566. Lechner, M., Knapp, H., 2011. Carryover of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) from soil to plant and distribution to the different plant compartments studied in cultures of carrots (Daucus carota ssp. stativus), potatoes (Solanum tuberosum), and cucumbers (Cucumis sativus). J. Agric. Food Chem. 59, 11011e11018. Li, M.H., 2009. Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to plants and aquatic invertebrates. Environ. Toxicol. 24, 95e101. Liu, C.S., Yu, K., Shi, X.J., Wang, J.X., Lam, P.K.S., Wu, R.S.S., Zhou, B.S., 2007. Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Aquat. Toxicol. 82 (2), 135e143. Moody, C.A., Martin, J.W., Kwan, W.C., Muir, D.C.G., Mabury, S.A., 2002. Monitoring perfluorinated surfactants in biota and surface water samples following an accidental release of fire-fighting foam into etobicoke creek. Environ. Sci. Technol. 36, 545e551. Nakayama, K., Iwata, H., Tao, L., Kannan, K., Imoto, M., Kim, E.Y., Tashiro, K., Tanabe, S., 2008. Potential effects of perfluorinated compounds in common cormorants from Lake Biwa, Japan: an implication from the hepatic gene expression profiles by microarray. Environ. Toxicol. Chem. 27 (11), 2378e2386. Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311, 622e627. Noorlander, C.W., van Leeuwen, S.P.J., Biesebeek, J.D.T., Mengelers, M.J.B., Zeilmaker, M.J., 2011. Levels of perfluorinated compounds in food and dietary intake of PFOS and PFOA in the Netherlands. J. Agric. Food Chem. 59, 7496e 7505. OECD, 1984. TEST No. 207: Earthworm, Acute Toxicity Tests. In: Guideline for Testing of Chemicals, vol. 1(2), pp. 1e9. Qu, B.C., Zhao, H.X., Zhou, J.T., 2010. Toxic effects of perfluorooctane sulfonate (PFOS) on wheat (Triticum aestivum L.) plant. Chemosphere 79, 555e560. Renner, R., 2009. EPA finds record PFOS, PFOA levels in Alabama grazing fields. Environ. Sci. Technol. 43, 1245e1246. Shi, X.J., Zhou, B.S., 2010. The role of Nrf2 and MAPK pathways in PFOS-induced oxidative stress in zebrafish embryos. Toxicol. Sci. 115 (2), 391e400. Simcik, M.F., Dorweiler, K.J., 2005. Ratio of perfluorochemical concentrations as a tracer of atmospheric deposition to surface waters. Environ. Sci. Technol. 39 (22), 8678e8683. Sinclair, E., Kannan, K., 2006. Mass loading and fate of perfluoroalkyl surfactants in wastewater treatment plants. Environ. Sci. Technol. 40 (5), 1408e1414. 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 (1), 184e191. Skutlarek, D., Exner, M., Frber, H., 2006. Perfluorinated surfactants in surface and drinking waters. Environ. Sci. Pollut. Res. Int. 13 (5), 299e307. So, M.K., Miyake, Y., Yeung, W.Y., Ho, Y.M., Taniyasu, S., Rostkowski, P., Yamashita, N., 2007. Perfluorinated compounds in the Pearl River and Yangtze River of China. Chemosphere 68 (11), 2085e2095. Spurgeon, D.J., Weeks, J.M., Van Gestel, C.A.M., 2003. A summary of eleven years progress in earthworm ecotoxicology. Pedobiologia 47 (5e6), 588e606. Taniyasu, S., Yamashita, N., Kannan, K., Horii, Y., Sinclair, E., Petrick, G., Gamo, T., 2004. Perfluorinated carboxylates and sulfonates in open ocean waters of the Pacific and Atlantic oceans. Organohalogen Compd. 66, 4035e4040. Wang, T., lin, Z.F., Yin, D.Q., Tian, D.Y., Zhang, Y.L., Kong, D.Y., 2011. Hydrophobicitydependent QSARs to predict the toxicity of perfluorinated carboxylic acids and their mixtures. Environ. Toxicol. Pharmacol. 32, 259e265. Wen, Y.Z., Chen, H., Shen, C.S., Zhao, M.R., Liu, W.P., 2011. 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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Antioxidant defense system responses and DNA damage of earthworms exposed to Perfluorooctane sulfonate (PFOS) Dongmei Xu a, Chandan Li b, Yuezhong Wen b, *, Weiping Liu b a b
College of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2012 Received in revised form 16 October 2012 Accepted 29 October 2012
The use of earthworms as a sublethal endpoint has significantly contributed to the ecological risk assessment of contaminated soils. Few studies have focused on the potential toxicity of PFOS to earthworms in the soil. In this work, artificial soils were tested, and contact filter paper studies were used. The results showed that earthworm growth was generally inhibited. The antioxidant activities of the enzymes superoxide dismutase, peroxidase, catalase and glutathione peroxidase were initially activated and then inhibited. Reduced glutathione content was observed, and malondialdehyde content was elevated over the duration of the exposure. These results suggested that PFOS induced oxidative stress in earthworms. In addition, the values of olive tail moment, tail DNA% and tail length using SCGE showed similar frequency distributions and increased with increases in the PFOS concentration. These results suggest that all concentrations of PFOS cause DNA damage. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: PFOS Soil Ecotoxicity Earthworm Enzyme activity DNA damage
1. Introduction Perfluorooctane sulfonate (PFOS) has been widely used in consumer and industrial products, including food packaging, insecticides, protective coatings for fabrics and carpets, paper coatings, paints, cosmetics and fire-fighting foams (Moody et al., 2002; Noorlander et al., 2011). As a result, a pervasive presence of PFOS in food, drinking water and natural waters has been detected (Noorlander et al., 2011; Kannan et al., 2005; Lechner and Knapp, 2011; Skutlarek et al., 2006; Hansen et al., 2002; Taniyasu et al., 2004; Simcik and Dorweiler, 2005; So et al., 2007). PFOS is not readily biologically degradable and is often released in industrial wastewater directly into the aquatic environment or indirectly via canalization and sewage treatment plants. PFOS is emitted from wastewater treatment plants partially in the effluent and is partially adsorbed on sewage sludge. The use of sewage sludge as a fertilizer in agriculture can result in the contamination of soil with PFOS (Lechner and Knapp, 2011; Sinclair and Kannan, 2006). Due to the persistent and bioaccumulative nature of PFOS, its toxicities to aquatic organisms, animals and cells have attracted much attention. PFOS exposure in aquatic organisms and mammals has been shown to cause adverse effects in many systems, including hepatotoxicity, immunotoxicity, reproductive and developmental * Corresponding author. E-mail address: [email protected] (Y. Wen). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2012.10.030
toxicity, neurotoxicity and increased tumorigenic potential of the liver, pancreas and breast (Harada et al., 2005; Cui et al., 2009). Based on the 72 h oral LD50 of this compound on honeybees, PFOS was classified as “highly toxic” by the International Commission for Bee Botany. However, very few studies have focused on the potential harm of PFOS to terrestrial ecotoxicity (Zhao et al., 2011), despite the high levels of PFOS found in agricultural soils (Renner, 2009). The bioavailability and toxicity of contaminants varies among different soils and knowledge of this variance is essential to develop soil environmental quality guidelines (Yan et al., 2011). Therefore, to develop a comprehensive toxicity profile for PFOS, the toxicity of PFOS-contaminated soils should be assessed with various bioassays. Organisms such as fish, snails and plants have been employed as biomonitors (Dorts et al., 2011; Li, 2009). A study showed that the survival after 21 d EC50 for PFOS was lowest for onions and highest for soybeans in a test of seven species of plants. Although this approach is useful and promising, it is somewhat limited because it is based on a specific combination of living organisms with certain substances (Hirano and Tamae, 2011). Earthworms, the most prevalent animal species in soil, play an irreplaceable role in maintaining the ecological functions of soil. They have been used as important biomonitors to assess the ecological risks of toxic substances in the terrestrial environment (Fent, 2003; Xu et al., 2010). The molecular, cellular and physiological levels of earthworms change significantly when they are
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under contamination stress. These reactions produce a specific biological signal, called a biomarker. The biomarkers can indicate the impact of pollutants on the individuals before the lethal effect becomes apparent and can also be used to monitor soil pollution and provide an eco-toxicological diagnosis as an early warning system (Spurgeon et al., 2003). However, few studies have focused on the potential toxicity of PFOS to earthworms in the soil (Xu et al., 2011). In this paper, Eisenia foetida (widely used in ecotoxicological tests of earthworms) was used as a model organism to study the effects of PFOS on the change in bodyweight, antioxidant defense system and DNA damage. The results revealed the toxic effects of PFOS and provide a scientific basis for the comprehensive evaluation of the effect of PFOS on soil ecosystems. 2. Materials and methods 2.1. Materials and reagents The experimental animals, E. foetida, used in this study were supplied by the active central earthworm breeding farm of Zhejiang University. The experiments were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. Healthy adult earthworms, more than 2 months in age, with a bodyweight of approximately 0.3 g and obvious clitellum, were domesticated for 7 days in an artificial climate incubator before performing the tests. Potassium heptadecafluorooctanesulfonate (PFOS, 98%) was purchased from Tokyo Chemical Industries. N-Lauroyl-sacosine sodium salt, trihydroxymethyl aminomethane (Tris) and guaiacol glyceryl ether were purchased from Amersco. Normal melting agarose (NMA) and Triton X-100 were purchased from Acros. Low melting agarose (LMA) was purchased from Serva. Ethylenediamine tetraacetic acid disodium salt (Na2-EDTA) and dimethyl sulphoxide (DMSO) were purchased from J&K. The other reagents used were AR grade. Artificial soil was prepared according to a standard method from OECD guideline no. 207 (OECD, 1984). Artificial soil was made in the laboratory by mixing a ratio of 69% silica sand, 20% kaolinite clay, 10% peat moss and 1% CaCO3 (on a weight basis). The pH of the artificial soil was 7.0. 2.2. Earthworm exposure protocol and enzyme assays Based on the results of acute toxicity tests and avoidance behavior tests of PFOS used in the field (Xu et al., 2011), six different concentrations (0, 10, 20, 40, 80, 120 mg/kg) were used in the present study. Three replicates were used for each concentration with 500 g of artificial soil in each. The soil was spiked with 175 ml PFOS water solution that the required concentrations of PFOS are 0, 28.57, 57.14, 114.28, 228.57 and 342.86 mg/L, respectively. Final PFOS concentrations in soil were 0, 10, 20, 40, 80 and 120 mg/kg, respectively. The contaminated soils were then rehydrated to give an overall moisture content of approximately 35% of the final weight. They were then mixed thoroughly and left for one day to equilibrate. After voiding their gut contents, 10 healthy earthworms were weighed and introduced into the soil. The containers were then covered with gauze to limit water loss. From days 15 to 42, 0.5 g of finely ground dry horse manure per earthworm was added to the soil surface weekly. All exposures were conducted in an artificial climate incubator at 20 1 C in 80e85% relative humidity with a 16:8 h light:dark regime. The earthworms were collected after 2, 7, 14, 28 and 42 days of exposure. Survival status was recorded, and the earthworms were left for 1 d to void their gut contents. They were then weighed without drying, and the enzyme activities were measured. After toxicant exposure, five live earthworms from three parallel experiments of each treatment were collected. The earthworms were mixed with an amount of Trise HCl buffer (100 mmol/L, pH 7.5) that was four times that of the their bodyweight. They were then homogenized and centrifuged at 9000 g at 4 C for 30 min. The supernatant (post-mitochondrial fraction, S9) was collected and stored at 80 C for use in the enzyme activity assays. The contents of total protein, malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined using kits obtained from the Nanjing Jiancheng Bioengineering Institute.
conducted on ice. The alkaline comet assay was performed as described originally by Singh with slight modifications (Singh et al., 1988). The cell suspension (10 ml) was mixed with 90 ml of 0.7% low melting agar (LMA) in PBS at 37 C and pipette onto fully frosted slides precoated with a layer of 100 ml 0.75% normal (NMA). After solidification, the slides were immerse into a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA (pH 10.0), 1% Na-sarcosinate, supplemented with 10% dimethyl sulfoxide (DMSO) and 1% Triton X-100). Slides were then placed in an electrophoresis tank containing 300 mM NaOH with 1 mM Na2EDTA for 20 min prior to electrophoresis for 25 min at 25 V (300 mA). The slides were then neutralized (0.4 M Tris, PH 7.5) thrice at 5 min intervals and stained with ethidium bromide (2 mg/ml) for fluorescence microscopy analysis. 2.4. Statistical analysis Each treatment was conducted in triplicate. SPSS 17.0 statistical software was used to analyze the experimental data, and the results were expressed in the form of mean SD. An analysis of variance was conducted to analyze the enzyme activities of earthworms in terms of PFOS concentration, time of exposure, and their interaction. Significant differences (P < 0.05) between the treatment groups and the control were determined using the post hoc LSD test. Slides were viewed using a fluorescence microscope (Nikon, Eclipse, Te2000-U) equipped with a CCD camera. Non-overlapping cells were captured at 400 magnification. At least 100 cells per slide were analyzed using the Comet Assay Software Project (CASP). A measurement of olive tail moment (OTM) was used to quantify the extent of DNA damage. Tail length (TL) and tail DNA% (TDNA) were also analyzed. Comet assay data were found to be abnormally distributed. Therefore, non-parametric statistical methods were employed. Significant differences (P < 0.01) between the treatment groups and the control were determined using the ManneWhitney U (Two-tailed) test.
3. Results and discussion 3.1. Effects of PFOS on E. foetida weight Bodyweight development is a somewhat insensitive response, especially when testing copper (Hartmut and Otto, 1997), but it is a macroscopic indicator that shows the effect of pollutants on organisms. The effects of PFOS on earthworm weight are shown in Fig. 1. Earthworm weight in the control group was significantly enhanced relative to incubation duration. The growth of the earthworms were generally inhibited except for earthworms exposed to 10e80 mg/kg PFOS for 2 d. After exposure for 7 d and 28 d, the weight change rates of all earthworm groups treated with PFOS were significantly lower than that of the control groups. When exposed for 14 d and 42 d, only the treatment group exposed to the highest concentration of PFOS (120 mg/kg) was significantly different from the control group. The results suggested that PFOS significantly affected the earthworms’ growth, which may be related to changes at the molecular and physiological level.
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2.3. Alkaline comet assay Contact filter paper tests were conducted within 48 h. Seven different concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mg/kg) were used. Ten replicates were used for each concentration, and the controls were exposed to the same volume of deionized water. Earthworm coelomocytes were obtained using the method described by Bu et al. (2006). Individual earthworms were rinsed in 1 ml volume of extrusion medium composed of 5% ethanol, 95% saline, 2.5 mg/ml EDTA, 10 mg/ml guaiacol glyceryl ether (pH 7.3). Coelomocytes were spontaneously secreted in the medium and collected by centrifugation (9000 r/min, 4 C). Phosphate-buffered saline (PBS) was used to wash the cells prior to the comet assay. All the performance was
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Fig. 1. Weight change rate of E. foetida exposed to PFOS. Data are expressed as mean sd (n ¼ 3, n: the samples of replicate). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
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3.2. Effect of PFOS on E. foetida antioxidant responses-enzymatic system
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Fig. 3. The effect of PFOS on the POD activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
were approximately the same as those observed by Qu et al. (2010). In that study, the toxic effects of different concentrations (0.1e 200 mg/L) of PFOS on wheat were investigated. Their results showed that low concentrations of PFOS (0.1e10 mg/L) induced SOD and POD activities in wheat roots and leaves, a high concentration of PFOS (200 mg/L) inhibited the SOD and POD activities. Pollution induces the expression of antioxidant enzymes that allow organisms to partially or totally overcome stress resulting from exposure to an unsafe environment (Cossu et al., 1997). However, excess toxicity inhibits these enzymes. The decrease of SOD and POD activities may have been due to excessive ROS production. Normally, ROS levels are in a dynamic balance with the antioxidant enzyme level. However, if the free radicals induced directly or indirectly by xenobiotic pollutants cannot be scavenged promptly, the natural antioxidant defenses can be overwhelmed. This can lead to severe sub-cellular injuries, such as ion loss, protein denaturation, and DNA damage (Nel et al., 2006). The CAT and GSH-Px activities are shown in Figs. 4 and 5, respectively. The CAT activity was inhibited in the PFOS-treated
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Fig. 2. The effect of PFOS on the SOD activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01.
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Many studies have reported that PFOS can induce oxidative stress in many organisms (Shi and Zhou, 2010; Liu et al., 2007). The burden of reactive oxygen species (ROS) production is largely counteracted by an intricate antioxidant defense system. Antioxidant defense systems consist of an enzymatic component and a nonenzymatic component. Enzymatic systems include SOD, CAT, POD, GSH-Px and other antioxidant enzymes, while nonenzymatic systems include GSH and other nonenzymatic substances. SOD, CAT, POD and GSH-Px activities are often used as biomarkers to indicate ROS production (Zelikoff et al., 1996). The enzymes SOD, CAT, and POD are involved in the detoxification of O 2 (SOD) and H2O2 (CAT, POD) and prevent the formation of ROS (Wen et al., 2011). In this study, the effects of PFOS on E. foetida antioxidant responses were studied. The activities of SOD, POD, CAT and GSHPx in earthworms exposed to PFOS are presented in Figs. 2e5. As shown in Fig. 2, in general, SOD activity was first activated, then inhibited, except on days 14 and 42. Compared to the controls, SOD activity was significantly higher and extremely higher in the presence of 10, 40, or 120 mg/kg PFOS on day 7, 40 mg/kg PFOS on day 28 and 120 mg/kg PFOS on day 42. The SOD activity declined on day 14 and was induced on day 42 with increasing PFOS concentrations. The same trend was found for POD activity. POD had a rapid toxic response and a short active period (see Fig. 3). After 2 d of treatment, POD activity was significantly greater in the PFOStreated groups compared to the controls, particularly in the presence of 20, 40 or 80 mg/kg PFOS. After 7 d, the POD activity in the PFOS-treated groups was extremely higher than the controls at 10 and 120 mg/kg PFOS. The POD activity of the treatment groups showed few changes compared to the controls until day 42, at which point the POD activity of the PFOS-treated groups was significantly higher than that of the controls. At the initial stage of exposure (2 d, 7 d), the CAT activity, and SOD and POS variation are quite different. Since the 14th day of exposure, the CAT activity has the same trend of variation with the above two ferments. But we have also noticed that, as the extension of exposure time, under the impact of PFOS, CAT activity of earthworm sees more obvious decrease than that of SOD and POD, which might because the accumulation of H2O2, the product of free radical have restrained the activity of ferment. The changes in the SOD and CAT activities
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Time, day Fig. 4. The effect of PFOS on the CAT activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 5. The effect of PFOS on the GSH-Px activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01.
groups compared to the control group during early exposure (2e 14 d), and it was significantly decreased in the PFOS-treated groups relative to the control at 2 d and 7 d. CAT activity was stimulated at low concentrations of PFOS, while it decreased markedly at high concentrations and exposure times. The GSH-Px activity was stimulated in the PFOS-treated except for in several treatment groups on day 14. Some of the treatment groups on day 2, 7, and 28 showed significant or extremely significant increased GSH-Px activity relative to the control group. The enzyme activity was not markedly different on day 42. Similar results were reported by Shi, after exposure to PFOS (0, 0.2, 0.4, or 1.0 mg/L), CAT and GSH-Px activities in 4 h fertilized zebrafish embryos of the treatment groups were significantly induced (Shi and Zhou, 2010). However, the changes in the CAT and GSH-Px activities in the present study are inconsistent with the results of a previous study by Hu (Hu and Hu, 2009). The results of this previous study showed that high concentrations of PFOS (150 or 200 mmol/L) significantly enhanced CAT activity while strongly reducing GSH-Px activity. The disparity between the results could be attributed to the different organisms studied and different mechanisms of action.
2
Fig. 6. The effect of PFOS on the GSH content of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
damage. The content of MDA can indirectly determine the level of radicals. MDA may cause a variety of cell damage. As depicted in Fig. 7, the MDA content changed slightly during early exposure (2 d, 7 d). With increased exposure duration, the MDA content rose significantly. These results indicate that treatment with PFOS resulted in an increase of ROS. ROS, in turn, stimulated the response of antioxidant defenses and resulted in a decreased earthworm growth rate and impaired its DNA and physiological functions. 3.4. DNA damage induced by PFOS The DNA damage of earthworm coelomocytes was compared with control coelomocytes after being exposed to PFOS for 48 h. Fig. 8a shows a typical comet image of undamaged DNA from earthworm coelomocytes, and Fig. 8b shows typical comet image of damaged DNA of earthworm coelomocytes. Figs. 9e11 show the box charts of the dynamic changes of OTM, TDNA and TL, respectively. As shown in the figures, the frequency distribution of the
3.3. Effect of PFOS on E. foetida antioxidant responsesnonenzymatic system
0 40
1.2
MDA content, nmol·mgprot-1
The enhancement of antioxidant enzyme activities suggested that oxidative stress conditions led to the increased antioxidant capability of earthworms. Reduced glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine (which is attached by normal peptide linkage to a glycine) and the carboxyl group of the glutamate side chain. It exists widely in vivo and plays a central role in coordinating the body’s antioxidant defense process. GSH is an important detoxification substance: its sulphydryl can bind with the carbon or chlorine atom of many pollutants and metabolites. When GSH is depleted, the pollutants and metabolites that are not consumed by GSH act on large biological molecules and cause oxidative stress. Changes in the GSH content in E. foetida are shown in Fig. 6. In general, the GSH content in the treatment groups was lower than that of the control except for the exposure on day 7, suggesting that GSH was markedly consumed in the PFOS-treated groups. Diminished GSH levels elevated cellular vulnerability for oxidative stress, as shown in Fig. 7. MDA is the ultimate lipid peroxidation product of oxidative
42
28
14 Time, day
7
10 80
20 120mg·kg-1 ***
1.0 *
0.8
***
***
***
**
***
0.6 **
** *** **
*
*
0.4 0.2 0.0 2
7
14
28
42
Time, day Fig. 7. The effect of PFOS on the MDA activity of E. foetida. Data are expressed as mean sd (n ¼ 3, n: the number of replicated samples). Statistical significance versus control group: *P < 0.05, **P < 0.01, ***P < 0.001.
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
125
300
125
250
100
Tail Length, µm
Olive Tail Moment, %•µm -1
Fig. 8. Typical comet figures. (a) Deionized water control, (b) 2 mg/cm2 PFOS.
75 50 25
200 150 100 50
0
0 0
0.25
0.5
1
2
4
8
0
PFOS Concentration, µg/cm2
0.5
1
2
4
PFOS Concentration, µg/cm
Fig. 9. The changes in olive tail moment caused by different concentrations of PFOS.
TDNA and TL data is similar to that of OTM. Fig. 9 shows that the OTM values of the treatment groups were all higher than the controls and that at 1 mg/cm2 PFOS, these values were low relative to other treatment groups, but not the controls. The dynamic changes of OTM, TDNA and TL values are shown in Table 1. In general, the values of OTM, TDNA, and TL increased
100 80 60
Tail DNA, %
0.25
8
2
Fig. 11. The changes in tail length caused by different concentrations of PFOS.
with the increase in PFOS concentration. The OTM values ranged from 3.60 0.46 to 24.24 2.46, with the highest value observed at 4 mg/cm2 PFOS. The increase of PFOS may cause ROS levels to increase, leading to DNA damage. However, the OTM value decreased at 8 mg/cm2. The data showed a similar trend to those of IR on ovarian carcinoma (David et al., 2010). Our analysis showed that the OTM, TDNA, and TL values of the treatment groups were extremely significantly elevated (P < 0.001) relative to the controls. All of the concentrations of PFOS caused DNA damage. In this study, the enhanced DNA damage was due to oxidative stress, indicating that ROS accumulation in tissues caused subsequent DNA damage. Many studies have shown that ROS is the major source of DNA damage, ROS cause DNA damage by causing strand breaks, removing nucleotides, and modifying the nucleotide bases (Ames, 1983; Cooke et al., 2003). A study of common carp exposed to 0.1,
40 Table 1 DNA damage of earthworm coelomocytes exposed to PFOS.
20 0 -20
0
0.25
0.5
1
2
4
8
PFOS Concentration, µg/cm2 Fig. 10. The changes in tail DNA caused by different concentrations of PFOS.
PFOS concentration (mg/cm2)
OTM (%$mm)
0 0.25 0.5 1 2 4 8
3.60 13.45 18.11 10.90 23.48 24.24 13.56
0.46 1.47*** 2.39*** 1.68*** 2.53*** 2.46*** 1.52***
TL (mm)
TDNA% 9.88 24.59 25.87 19.66 31.65 32.58 25.61
1.12 1.46*** 1.84*** 1.78*** 2.04*** 1.77*** 1.88***
Statostoa; significance versus control group. ***P < 0.001.
25.52 68.32 75.04 54.90 94.64 101.55 56.55
2.42 4.62*** 5.71*** 4.33*** 7.12*** 6.33*** 4.44***
126
D. Xu et al. / Environmental Pollution 174 (2013) 121e127
Table 2 ANOVA results for bodyweight and the anti-oxidation of E. foetida exposed to PFOS. Items
SOD POD CAT GSH-Px GSH MDA Weight change rate
Concentration
Duration
df F
df F
P
df
F
P
4 4 4 4 4 4 4
0*** 0*** 0*** 0*** 0*** 0*** 0***
20 20 20 20 20 20 20
2.39 3.29 4.91 6.35 5.92 3.33 1.26
0.006** 0.001*** 0*** 0*** 0*** 0.001*** 0.24
5 5 5 5 5 5 5
P
1.90 0.111 2.56 0.042* 5.08 0.001*** 8.56 0*** 5.26 0.001*** 13.55 0*** 6.19 0***
8.56 33.12 32.43 33.50 38.11 10.42 18.38
Concentration*duration
df: degree of freedom, *: cases where the concentration or the duration of exposure had a significant effect (*P < 0.05, **P < 0.01, ***P < 0.001).
0.5 and 1 mg/L PFOS in water for 14 d was conducted previously (Hagenaars et al., 2008). The results of this previous study showed that several genes related to energy, reproduction and stress responses were expressed. Furthermore, PFOS may alter the expression levels of genes related to antioxidant enzymes (Nakayama et al., 2008). 3.5. Analysis of variance of bodyweight and anti-oxidation affected by PFOS When E. foetida earthworms were exposed to PFOS, changes in the relative physical index were the result of combined effects of pesticide concentration and exposure duration. Table 2 shows that the PFOS concentration had a very significant effect on the rate of change of weight and other biochemical indicators, except SOD activity. Exposure duration caused extremely significant changes for all indicators. The effects of the interactions between PFOS concentration and exposure duration were significant for all indicators except bodyweight. The fluro structure of the length of 8 carbon chains in the PFOS molecule structures has obvious hydrophobility. Wang et al. observed the toxicity of individual PFCAs and their mixtures to Photobacterium phosphoreum. The results showed that “the maximum tolerance of the cell membrane” led to a tendency of increasing toxicity from C3 to C14 PFCA and a tendency of decreasing toxicity from C14 to C18 PFCA (Wang et al., 2011). Basing on the research outcome and the concentration of PFOS in the practical soil environment, we may deem that the PFOS concentration in current soil environment will not constitute any threat to the earthworm or other animals in the soil, but the bioconcentration and transmission along the food chain (network) may enable the advanced consumers to concentrate such pollutants within their bodies, and suffer from their potential toxicity. And long term low dose exposure is likely to result in negative effect. Acknowledgments The authors acknowledge financial support from the National Natural Science Foundation of China (No. 20907046) and the National Basic Research Program of China (No. 2009CB421603). References Ames, B.N., 1983. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science 221 (4617), 1256e1264. Bu, Y.Q., Luo, Y.M., Teng, Y., 2006. Detection of DNA damage in earthworm (Eisenia foetida) in vivo exposure to copper ion. Asian J. Ecotoxicol. 1 (3), 228e235. Cooke, M.S., Evans, M.D., Dizdaroglu, M., Lunec, J., 2003. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195e1214. Cossu, C., Doyotte, A., Jacquin, M.C., Babut, M., Exinger, A., Vasseur, P., 1997. Glutathione reductase, selenium dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic contamination in field studies. Ecotoxicol. Environ. Saf. 38, 122e131.
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