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Enzymatic biomarkers of earthworms Eisenia fetida in response to individual and combined cadmium and pyrene
Ecotoxicology and Environmental Safety 86 (2012) 162–167
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Enzymatic biomarkers of earthworms Eisenia fetida in response to individual and combined cadmium and pyrene Xiaoxia Yang a,b, Yufang Song a,n, Jianrong Kai a,b, Xiufeng Cao a,b a b
Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Shenyang 110016, People’s Republic of China Graduate School, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2012 Received in revised form 7 September 2012 Accepted 8 September 2012 Available online 12 October 2012
The responses of enzymatic biomarkers of earthworms Eisenia fetida to low-level exposures of cadmium (Cd) (2.50 mg kg 1), pyrene (0.96 mg kg 1) or their combination were investigated in this study. A set of enzymatic biomarkers, namely, cytochrome P450 (CYP) as a family of phase I enzymes, glutathiones-transferase (GST) as one of phase II enzymes and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT) ), was selected to evaluate the responses of the earthworms in a period upto eight weeks. The earthworms exposed to the mixture of Cd and pyrene demonstrated different responses of the enzymatic biomarkers from those exposed to Cd or pyrene alone. The responses of enzymatic biomarkers to the combined exposure were time-dependent, with initial antagonistic effects on CYP content and activities of GST and SOD, but with additive effects at the end of experiment causing the reductions of CYP content and GST activity and the enhancement of activities of SOD and CAT. Our results indicated the toxicity of low-level pyrene may be prolonged by the co-presence of Cd. & 2012 Elsevier Inc. All rights reserved.
Keywords: Cadmium Earthworm Enzymatic biomarkers Pyrene
1. Introduction Soils contamination due to discharge of various organic and inorganic pollutants has been a worldwide issue for human health and agriculture. Hence, terrestrial organisms living on contaminated soils are typically exposed to a mixture of toxicants of both related and distinct classes. Combined effects of toxicants may be stronger (synergism) or weaker (antagonism) than expected from the observed effects of individual exposures of toxicants. The responses to exposure to multiple pollutants are dependent upon the components of the mixture and may vary significantly, such as additive effects for narcotic compounds (Jensen and Sverdrup, 2002), synergistic lethal effects among metals and between metals and organic compounds (Fleeger et al., 2007; Maria and Bebianno, 2011). The toxicity of mixed toxicants has been well studied in aquatic environment (Banni et al., 2009; Benedetti et al., 2007; Bouraoui et al., 2009; Dondero et al., 2011), but not in soil environment. Soil environment is so complex that different types of interactions may occur, including chemical and physico– chemical interactions affecting sorption and bioavailability, physiological interactions in the organisms affecting uptake from soil, or mechanistic processes at the target receptors. Individual toxicants may interfere with each other in these steps, leading to changes in toxicity and responses.
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Corresponding author. Fax: þ86 2483970426. E-mail address: [email protected] (Y. Song).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.09.022
Heavy metals and polycyclic aromatic hydrocarbons (PAHs) usually co-occur in soils. Cadmium (Cd) as a non-essential heavy metal is well known to cause adverse effects on organisms (Friberg et al., 1985). PAHs, all made up of 2–7 benzene rings, are ubiquitous in the terrestrial environment. Of the PAHs, pyrene, a tetracyclic PAH, is considered to be non-carcinogenic by the International Agency for Research on Cancer and the US Environmental Protection Agency (Brown et al., 2004; Jensen and Sverdrup, 2003). Toxicities of this PAH are thought to result principally from non-polar narcosis. Benzo[a]pyrene (B[a]P) has been extensively studied in invertebrates due to its known mammalian carcinogenicity (Banni et al., 2009; Saint-Denis et al., 1999), however, the toxicities of pyrene in terrestrial invertebrates are not well investigated. Earthworms may represent 60–80 percent of the total soil biomass and have favorable effects on soil structure and function (Saint-Denis et al., 1999). These traits make them one of the most suitable organisms to examine biological effects of chemicals under laboratory conditions. A set of standard test guidelines has been established (ISO, 1998; OECD, 1984) focusing upon acute toxicity and chronic bioassay endpoints of reproduction and growth of earthworms Eisenia fetida as the standard species. Biomarkers that apply molecular endpoints may also be valuable tools for toxicity assessment. A range of biomarkers have been developed in recent years to evaluate organisms’ stress response to individual toxicants (Banni et al., 2009; Bouraoui et al., 2009; Saint-Denis et al., 2001). Cytochrome P450 (CYP) is a huge family of phase I enzymes of detoxification process,
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catalyzing the oxidative conversion of lipophilic xenobiotics into entities which are more water-soluble and can be readily excreted and detoxified. Glutathione-s-transferase (GST) as one of phase II enzymes can facilitate conjugation of electrophilic substances or tripipetide glutathione and exert the function of detoxification. This enzyme also plays a role in cellular protection against oxidative stress. Previous studies have documented that both heavy metals (such as Cd, Copper (Cu)) and PAHs (such as B[a]P, pyrene) may induce the responses of CYP and GST in earthworms (Brown et al., 2004; Ribera et al., 2001; Saint-Denis et al., 1999). A by-product of the metabolism of xenobiotics by CYP is the production of free radicals. To counter these effects, earthworms possess a suite of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). SOD dismutates superoxide to hydrogen peroxide (H2O2), acting as the first line of defense against reactive oxygen species (ROS). H2O2 is subsequently detoxified by CAT and other enzymes. Many previous studies focused on the individual effects of Cd and pyrene on earthworms’ survival, reproduction and even the responses of detoxification system as well. The number of earthworms did not change significantly when exposed to 1–10 mg/kg Cd in soil upto 4 months (Lapinski and Rosciszewska, 2008). Reinecke et al. (1999) concluded that earthworm E. fetida developed the resistance to Cd during an exposure even for 3 years to low-level Cd. It has been reported that the survival, cocoon production rate and weight change did not vary significantly for earthworms Lubricus rubellus exposed to soil with pyrene ranging from 0 to 40 mg/kg for 42 days (Brown et al., 2004). The CYP content and antioxidant enzymes varied subtly when earthworms E. fetida were exposed for 14 days to pyrene less than 0.96 mg/kg (Zhang et al., 2007). Nevertheless, significant increases of CAT and SOD activities were observed when earthworms E. fetida were exposed to pyrene of 50 or 100 mg /kg soil (Wu et al., 2012). While the effects of individual exposure of Cd and pyrene on enzymatic biomarkers were documented on earthworms, the combined effects of low-level Cd and pyrene on earthworms E. fetida are rarely reported. Moreover, exposure duration is generally less than 30 days in most previous studies paying attention to the response of enzymatic biomarkers of earthworms. Therefore, the study on the response of earthworms E. fetida to environmentally relevant low-level exposure of the mixture of Cd and pyrene for a longer period is needed for better evaluating ecological risk of Cd and pyrene in laboratory and field conditions. This study aims to investigate individual and combined effects of cadmium (2.5 mg/kg) and pyrene (0.96 mg/kg) on a set of enzymatic biomarkers (CYP, SOD, CAT and GST) of earthworms E. fetida upto eight weeks, and to provide valuable information for ecological risk assessment of the interacted effects of Cd and pyrene at environmentally relevant levels.
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The environmentally relevant concentrations of Cd (2.50 mg kg 1) and pyrene (0.96 mg kg 1) were selected in the experiment based on previous studies (Brown et al., 2004; Lapinski and Rosciszewska, 2008; Reinecke et al., 1999; Zhang et al., 2007) and the average level of Cd (2.50 mg kg 1) in agricultural soil of Shenyang, China. The worms were randomly divided into four groups with four replicates: (1) control, (2) pyrene: 0.96 mg kg 1, (3) Cd: 2.50 mg kg 1 and (4) the mixture of pyrene and Cd (pyrene: 0.96 mg kg 1 þCd: 2.50 mg kg 1). Pyrene and CdCl2 were dissolved in acetone and water, respectively, and then transferred to the soil. 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 40 percent of water holding capacity and left one day to equilibrate. 5000 g treated-soil for each replicate was placed in an aerated container (45 cm 30 cm 20 cm) and matured earthworms (130) were transferred to each container. Containers were subsequently covered with a perforated lid to limit water loss and kept for eight weeks at (207 2)1C in a 12:12-h lightdark regime. Worms were fed with dry and defaunated cow dung twice a month during the whole incubation period, and a few milliliters of distilled water were added daily into each container to maintain suitable humidity for earthworm activity. Worms were collected for the biochemical analysis after one, two, three, four, six and eight weeks of exposure. Five samples (Four worms per sample) at each container were collected every time. Each sample was measured three times. Both unhatched earthworm cocoons and new-born earthworms were removed throughout the experiment.
2.2. Biochemical assays Worms were first rinsed with distilled water, and placed on the petri dish with moistened filter paper to purge their gut contents for 3 days. Filter paper was changed every day during this period. Samples were subsequently immobilized in ice-cold 20 percent (V/V) glycerol solution for 3 min. The guts were then separated, washed afterwards with cold 0.15 M KCl solution and homogenized manually in a vitreous tissue homogenizer with 5 ml homogenization buffer (250 mM sucrose, 50 mM Tris pH 7.5, 1 mM DTT and 1 mM EDTA). Homogenates were centrifuged at 4 1C for 20 min at 15,000 g to produce the postmitochondrial fraction. One part of the supernatant was collected for the determination of activities of SOD, CAT and GST. The other part was further centrifuged at 150,000 g for 90 min to obtain microsomal pellets for CYP content determination. Cytochrome P450 content was determined by the method of Omura and Sato (1964) by means of sodium dithionite reduced carbon monoxide. Microsomal protein concentrations were evaluated by the method of Bradford (1976) using bovine serum albumin (BSA) as standard. GST activity was assayed by the method of Habig et al. (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The assay was carried out by monitoring the appearance of the conjugated complex of CDNB and glutathione (GSH) at 340 nm. The mixture contained 190 ml of 0.1 M Tris buffer pH 7.0, 0.5 ml of 1 mM GSH, 1 ml of 1 mM CDNB and 10 ml enzyme extract. The reaction was initiated by the addition of GSH. SOD activity was assayed by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp and Fridovich, 1971). The assay mixture contained 1.5 ml of 50 mM phosphate buffer pH 7.8, 0.3 ml of 130 mM methionine, 0.3 ml of 750 mM NBT, 0.3 ml of 0.1 mM EDTA, 0.3 ml of 20 mM riboflavin, 0.05 ml of deionized water and 0.05 ml enzyme extract in a total volume of 3 ml. Riboflavin was added finally, and the tubes were shaken and then illuminated for 15 min. The absorbance was recorded at 560 nm and the absorbance of the nonirradiated reaction mixture served as the control. CAT activity was assayed according to the method of Aebi (1984). The assay mixture contained 0.2 ml supernatant, 1.5 ml of 50 mM phosphate buffer pH 7.8, 0.3 ml of 0.1 M H2O2 and 1 ml deionized water. The decrease of the absorbance of the mixture was recorded at 240 nm for 4 min.
2.3. Statistical analysis 2. Materials and methods 2.1. Animals and treatment Earthworms, E. fetida, with well-developed clitella 300–400 mg were purchased from Shenyang Agricultural University, China and kept in control soil in the dark at (207 2)1C prior to the start of toxicant exposure. No sexual differences were considered since earthworms are hermaphroditic. The test soil (0–20 cm) was collected from the Ecological Experiment Station of Chinese Academy of Science in Shenyang, China. The soil was screened through a 5 mm sieve, kept at 4 1C and normal field moisture until use. The soil had the following characteristics: pH 6.20, Kjeldahl nitrogen 0.09 percent, total phosphorus 0.04 percent, total potassium 0.18 percent, organic matter content 1.65 percent, cation exchange capacity 12.30 cmol kg 1, water holding capacity 32.00 percent. The particle distribution of this soil was as follows: sand (450 mm) 22 percent, silt (1–50 mm) 64 percent, clay (o1 mm) 14 percent. The background level of Cd in the soil is 1.80 mg kg 1, and pyrene was not detectable in this soil.
All data were represented as the means7 standard deviation (SD). Statistical analysis for all measurements was performed by SPSS 16.0 software (SPSS Inc, Chicago, USA). Normality and variance homogeneity were first tested using Kolmogornov–Smirnov and Levene’s tests, respectively. However, even though mathematical transformation was applied to dependent variables, the variations after data transformation were still heteroscedastic or non-normally distributed in this study. Therefore the non-parametric Kruskal–Wallis test for independent samples (K 42) was adopted to test the difference between the control and treated samples at the same exposure time. A statistical difference at P o 0.05 was considered to be significant, and that at P o0.01 very significant.
3. Results The concentrations of Cd and pyrene in the soil treated with Cd and pyrene were verified as 4.2270.05 and 0.8070.03 mg kg 1,
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Fig. 1. CYP content in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at P o0.01.
respectively, which were close to the nominal concentraions after deducting the corresponding background values. Exposures to Cd and/or pyrene did not cause significant mortality of worms during the eight-week experiment. The response of each biomarker in the control group did not change significantly during the eightweek experiment as shown in Figs.1–4. The individual and combined effects of Cd and pyrene on CYP content were shown in Fig. 1. In the worms exposed to Cd alone, a significant elevation of CYP content was observed at week three (29.72 percent) and four (104.84 percent), and a declining trend occurred since week six. For the worms exposed to pyrene alone, a similar bell-shape change of CYP content was also observed, except that the enhancement of CYP content appeared earilier, between week two (155.17 percent) and three (76.03 percent), and the decrease of CYP content also appeared earilier. For the mixture-exposed worms, a pronounced increase of CYP content was observed from week two to four (93.32 percent, 42.02 and
Fig. 2. GST activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at Po 0.01.
33.42 percent, respectively), while a significant decrease (20.48 percent) was recorded at week eight. Fig. 2 presented the responses of GST activity in the tested worms. In the worms exposed to Cd alone, GST activity showed a tendency of increase, but only became significant (Po0.05) at week six (33.93 percent) compared to the control. In the worms exposed to pyrene alone, GST activity was elevated very significantly (P o0.01) between week two and three (147.20 percent and 115.07 percent, respectively). A flat bell shape was observed for GST activity in the mixture-exposed worms. At week two, the extent of increase of GST activity for the worms exposed to the mixture (20.36 percent) was much lower than that for those exposed to pyrene alone (147.20 percent). At week three, the mixture inhibited the increase of GST activity induced by pyrene since GST activities induced by pyrene and mixture exposure were 115.07 percent and 33.40 percent higher than the control, respectively. At week eight, GST activity levels induced by individual and combined exposure of Cd and pyrene
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Fig. 3. SOD activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at P o0.05, and **indicated a significant difference at P o0.01.
were recorded as 111.96 percent, 70.59 percent and 79.26 percent of the control, respectively. The responses of SOD activity of the earthworms were shown in Fig. 3. In the Cd alone-exposed worms, the SOD activity varied insignificantly between week one and four, but show pronounced increases at week six (53.45 percent) and eight (33.50 percent). SOD activity of the worms exposed to pyrene alone was significantly elevated between week one and two (100.86 percent and 36.23 percent, respectively); it decreased subtly with the exposure time increased from four weeks. For the mixture-exposed worms, no significant variation was observed on SOD activity except an increase of 66.16 percent at week one. The results on CAT activity were shown in Fig. 4. The kinetic shape of CAT activity of the Cd alone-exposed worms was similar to that of SOD activity. CAT activity levels after six and eight weeks of exposure were higher than their corresponding controls (79.16 percent and 53.26 percent, respectively), and no significant variation was exhibited at other exposure time. CAT activity levels of the mixture-exposed worms increased by 33.13 percent and
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Fig. 4. CAT activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at Po 0.01.
38.93 percent, respectively, after six and eight weeks of exposure. However, CAT activity in the pyrene alone-exposed worms demonstrated insignificant variation during the whole experimental period.
4. Discussion The application of multiple biomarkers to evaluate the effect of complex pollutants is prevalent in recent years. The study of biological responses of organisms exposed to different classes of contaminants is being considered as a helpful method for the evaluation of environmental quality. While the acute effects of exposure of high concentrations of cadmium or pyrene alone on terrestrial organisms were well documented (Brown et al., 2004; Scaps et al., 1997), few studies explored the chronic response of earthworms E. fetida exposed to the mixture of Cd and pyrene at environmental levels. The present study investigated the chronic
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effects of environmentally relevant low-level cadmium, pyrene and their mixture, since both accumulation and the appearance of toxicity of a low-level chemical within an organism need time. CYP family is particularly important in the metabolism of xenobiotics (Benedetti et al., 2007; Henczova´ et al., 2006), and has been widely used as a biomarker of organic and inorganic pollution in fish and sea worms as well as earthworms (Banni et al., 2009; Benedetti et al., 2007; Saint-Denis et al., 1999). In this study, compared to the effects of Cd or pyrene alone, the significant increase of CYP content in the mixture-exposed worms lasted three weeks from week two, indicating higher metabolism processed longer. The obvious additive decrease of CYP content induced by the co-exposure of Cd and pyrene was observed at week eight, suggesting that Cd may cause the enhancement of pyrene toxicity. This is in accordance with the results of Wang et al. (2011), in which a significant inhibition of the activity of aryl hydrocarbon hydroxylase (AHH) in calm Ruditapes philippinarum was induced by co-exposure of B[a]P and Cd compared to that by B[a]P exposure. A similar kinetic pattern was presented in GST activity of the worms exposed to the mixture of Cd and pyrene (Fig. 2). GST activity in the mixture-exposed earthworms was higher than that in the pyrene alone-exposed worms at the end of experiment, implying that the jointed effects generated higher oxidative stress. Broerse et al. (2012) demonstrated that the coexposure of Cd and pyrene decreased significantly pyrene uptake and elimination rates and resulted in a prolonged half life of pyrene. Many studies investigated the combined effects of heavy metals (Cd, Cu) and B[a]P on aquatic organisms such as sea worms, fish and so on. Similar to our results, Banni et al. (2009) showed an obvious additive increase of NADPH CYP C red activity and GST activity by the co-exposure of Cd and B[a]P equimolar (each 0.2 mM) to the sea worm Hediste diversicolor. Differently, exposure to Cu with the presence of B[a]P did not affected the level of NADPH CYP C in H. diversicolor within 48-h exposure (Bouraoui et al., 2009). Benedetti et al. (2007) reported the co-exposure of Cd suppressed completely the induction of ethoxyresorufin-o-deethylase (EROD) caused by B[a]P in the Antarctic fish Trematomus bernacchii. The possible inhibitory effect of Cd on B[a]P was also found by Jensen and Krøkje (2008) in a ternary mixture (0.7 mM Cd/B[a]P/Tetrachlorobiphenyl) in rat hepatoma cells. These results range from additive, independent, or antagonistic to synergistic mixture effects, depending on the combination of metals and PAHs, the exposure medium, test duration, test organism and the studied endpoint. The interactive effects of Cd and pyrene demonstrated in this study were exposure time-dependent, with the appearance of antagonism at week two to three and of additive effect at week eight for CYP content and GST activity. It is well established that both Cd and pyrene are able to induce ROS in earthworms (Brown et al., 2004; Scaps et al., 1997). Fortunately, the SOD-CAT system acts as the key part to fight against oxygen damage and free radicals generated in phase I. SOD activity was considered as an important biomarker for the assessment of pollutants on ecosystems. In this study, an enhancement of SOD activity was recorded for more than six weeks in the Cd alone-exposed worms and for less than three weeks in the pyrene alone-exposed worms, confirming that the ROS induction occurred in the metabolism of either Cd or pyrene. However, at the end of experiment, the increase of SOD activity was 15.64 percent with the mixture exposure, compared to the reduction by 18.65 percent with pyrene alone-exposure and the enhancement by 33.50 percent with the Cd alone-exposure, showing an additive effect again. Therefore, we deduced that the toxicity of pyrene is higher at week eight when it is coexposed with Cd due to the enhancement of pyrene accumulation by the co-presence of Cd. However, the co-exposure of Cd and
pyrene inhibited the enhancement of SOD activity caused by pyrene at week one and two, and similar interactive effects were also observed on CYP content and GST activity at week two. The fact that Cd decreases the uptake and elimination of pyrene and increases its hydroxylation in short time (Broerse et al., 2012) may be accountable for this kind of interaction. Moreover, our results confirmed the results of Broerse et al. (2012) from the aspect of biochemical responses that the interaction of Cd and pyrene is time dependent. Similarly to our findings, Wang et al. (2011) observed a decrease in SOD activity of mussel induced by exposure of B[a]P (500 mg/l) alone compared to that induced by co-exposure of B[a]P (500 mg/l) and Cd (80 mg/l). However, three binary mixtures of B[a]P and Cu (5, 10 and 25 mg L 1 for each chemical) did not cause significant differences compared to controls in the digestive gland of mussels (Maria and Bebianno, 2011). Similarly, a strong synergistic interaction of phenanthrene and pyrene on SOD activities of earthworm E. fetida was observed by Wu et al. (2012). Insignificant change in CAT activity of earthworms exposed to pyrene with respect to the control was exhibited in this study. Lei et al. (2006) reported that CAT activity in pyrene-treated cells (0.1 mg/ml) remained almost the same as the control for all microalgae species. Due to the low concentration of pyrene, the hydrogen peroxide can thus be eliminated by themselves or by other antioxidant enzymes. However, CAT activity increased significantly in the mixture-exposed worms in this work in six to eight weeks, similarly to the trend found in the Cd alone-exposed worms, so we concluded that Cd may influence the toxicity of pyrene on earthworms. Banni et al. (2009) demonstrated that an additive effect on the activation of CAT activity in worms co-exposed to 0.2 mM Cd/B[a]P, while CAT activity kept at the control level when co-exposed to 1.0 mM Cd/ B[a]P in the sea worm H. diversicolor. An additive effect on the activation of CAT activity in worms with exposure of the mixture of Cd and pyrene at week eight was also observed in our study, confirming the interacted toxicity of Cd and pyrene. Though the modeling computation (Dondero et al., 2011; Jonker et al., 2005) is prevalent to predict the interaction of mixed pollutants, simple mathematical calculation combined with statistical analysis (Banni et al., 2009, 2011; Bouraoui et al., 2009; Maria and Bebianno, 2011; Wu et al., 2012) was still applied widely. In this work, the modeling calculation is inapplicable since only a single concentration was adopted for each chemical in the mixture herein, resulting in the absence of relative parameters for modeling calculation such as the toxic unit. Moreover, many researchers who highlight the biochemical responses also utilize the simple mathematical method instead of modeling for the judgment of interaction types (Banni et al., 2011; Maria and Bebianno, 2011; Wang et al., 2011; Wu et al., 2012). The interacted effects of Cd and pyrene on enzymatic biomarkers may be elucidated based on chemical toxicokinetics. Pyrene uptake and elimination can be significantly decreased with the presence of Cd, resulting in more pyrene accumulation in body and a prolonged elimination half life. Meanwhile, pyrene hydroxylation can also be increased but its further metabolism is postponed due to the co-presence of Cd, causing the half life prolonged as well (Broerse et al., 2012). The interacted effects might also be interpreted based on toxicodynamics by the fact that both Cd and pyrence can cause oxidative stress (Broerse et al., 2012). Cd can be sequestered by GSH to prevent its adverse interaction with biomolecules. GSH and other thiols play a crucial role in cleaning ROS (Broerse et al., 2012). When exposed to the mixture of pyrene and Cd, GSH and other thiols may be consumed fast due to the presence of Cd, and ROS levels may be elevated indirectly (Broerse et al., 2012). Cd may also postpone further metabolism of the phase I products of pyrene by a depletion of
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possible conjugates. In the second phase, reactive phase I metabolites are conjugated with chemicals like GSH, which may be also involved in scavenging and detoxifying Cd (Baird et al., 2005). It may also account for the prolonged toxicity of pyrene by the copresence of Cd.
5. Conclusions The binary mixtures of Cd and pyrene can account for the different responses in earthworms E. fetida from those observed with single exposures. Antagonistic effects were observed on CYP content, GST activity and SOD activity initially, but additive effects were responsible for the reduction of CYP content and GST activity and the enhancement of activity of SOD and CAT at the end of experiment. The interaction of pyrene and Cd varied over time, indicating that the exposure time may be more important than the exposure dose for the combined effect. Moreover, the presence of cadmium prolonged the toxic effect of pyrene. Short-term toxicity may overlook the interactive effects as the accumulation of toxicants is time dependent. A suite of biomarkers is helpful in the evaluation of the interactive toxicity of PAHs and heavy metals on earthworms and could be instrumental in monitoring soils contaminated with multiple pollutants.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Enzymatic biomarkers of earthworms Eisenia fetida in response to individual and combined cadmium and pyrene Xiaoxia Yang a,b, Yufang Song a,n, Jianrong Kai a,b, Xiufeng Cao a,b a b
Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Shenyang 110016, People’s Republic of China Graduate School, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2012 Received in revised form 7 September 2012 Accepted 8 September 2012 Available online 12 October 2012
The responses of enzymatic biomarkers of earthworms Eisenia fetida to low-level exposures of cadmium (Cd) (2.50 mg kg 1), pyrene (0.96 mg kg 1) or their combination were investigated in this study. A set of enzymatic biomarkers, namely, cytochrome P450 (CYP) as a family of phase I enzymes, glutathiones-transferase (GST) as one of phase II enzymes and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT) ), was selected to evaluate the responses of the earthworms in a period upto eight weeks. The earthworms exposed to the mixture of Cd and pyrene demonstrated different responses of the enzymatic biomarkers from those exposed to Cd or pyrene alone. The responses of enzymatic biomarkers to the combined exposure were time-dependent, with initial antagonistic effects on CYP content and activities of GST and SOD, but with additive effects at the end of experiment causing the reductions of CYP content and GST activity and the enhancement of activities of SOD and CAT. Our results indicated the toxicity of low-level pyrene may be prolonged by the co-presence of Cd. & 2012 Elsevier Inc. All rights reserved.
Keywords: Cadmium Earthworm Enzymatic biomarkers Pyrene
1. Introduction Soils contamination due to discharge of various organic and inorganic pollutants has been a worldwide issue for human health and agriculture. Hence, terrestrial organisms living on contaminated soils are typically exposed to a mixture of toxicants of both related and distinct classes. Combined effects of toxicants may be stronger (synergism) or weaker (antagonism) than expected from the observed effects of individual exposures of toxicants. The responses to exposure to multiple pollutants are dependent upon the components of the mixture and may vary significantly, such as additive effects for narcotic compounds (Jensen and Sverdrup, 2002), synergistic lethal effects among metals and between metals and organic compounds (Fleeger et al., 2007; Maria and Bebianno, 2011). The toxicity of mixed toxicants has been well studied in aquatic environment (Banni et al., 2009; Benedetti et al., 2007; Bouraoui et al., 2009; Dondero et al., 2011), but not in soil environment. Soil environment is so complex that different types of interactions may occur, including chemical and physico– chemical interactions affecting sorption and bioavailability, physiological interactions in the organisms affecting uptake from soil, or mechanistic processes at the target receptors. Individual toxicants may interfere with each other in these steps, leading to changes in toxicity and responses.
n
Corresponding author. Fax: þ86 2483970426. E-mail address: [email protected] (Y. Song).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.09.022
Heavy metals and polycyclic aromatic hydrocarbons (PAHs) usually co-occur in soils. Cadmium (Cd) as a non-essential heavy metal is well known to cause adverse effects on organisms (Friberg et al., 1985). PAHs, all made up of 2–7 benzene rings, are ubiquitous in the terrestrial environment. Of the PAHs, pyrene, a tetracyclic PAH, is considered to be non-carcinogenic by the International Agency for Research on Cancer and the US Environmental Protection Agency (Brown et al., 2004; Jensen and Sverdrup, 2003). Toxicities of this PAH are thought to result principally from non-polar narcosis. Benzo[a]pyrene (B[a]P) has been extensively studied in invertebrates due to its known mammalian carcinogenicity (Banni et al., 2009; Saint-Denis et al., 1999), however, the toxicities of pyrene in terrestrial invertebrates are not well investigated. Earthworms may represent 60–80 percent of the total soil biomass and have favorable effects on soil structure and function (Saint-Denis et al., 1999). These traits make them one of the most suitable organisms to examine biological effects of chemicals under laboratory conditions. A set of standard test guidelines has been established (ISO, 1998; OECD, 1984) focusing upon acute toxicity and chronic bioassay endpoints of reproduction and growth of earthworms Eisenia fetida as the standard species. Biomarkers that apply molecular endpoints may also be valuable tools for toxicity assessment. A range of biomarkers have been developed in recent years to evaluate organisms’ stress response to individual toxicants (Banni et al., 2009; Bouraoui et al., 2009; Saint-Denis et al., 2001). Cytochrome P450 (CYP) is a huge family of phase I enzymes of detoxification process,
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catalyzing the oxidative conversion of lipophilic xenobiotics into entities which are more water-soluble and can be readily excreted and detoxified. Glutathione-s-transferase (GST) as one of phase II enzymes can facilitate conjugation of electrophilic substances or tripipetide glutathione and exert the function of detoxification. This enzyme also plays a role in cellular protection against oxidative stress. Previous studies have documented that both heavy metals (such as Cd, Copper (Cu)) and PAHs (such as B[a]P, pyrene) may induce the responses of CYP and GST in earthworms (Brown et al., 2004; Ribera et al., 2001; Saint-Denis et al., 1999). A by-product of the metabolism of xenobiotics by CYP is the production of free radicals. To counter these effects, earthworms possess a suite of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). SOD dismutates superoxide to hydrogen peroxide (H2O2), acting as the first line of defense against reactive oxygen species (ROS). H2O2 is subsequently detoxified by CAT and other enzymes. Many previous studies focused on the individual effects of Cd and pyrene on earthworms’ survival, reproduction and even the responses of detoxification system as well. The number of earthworms did not change significantly when exposed to 1–10 mg/kg Cd in soil upto 4 months (Lapinski and Rosciszewska, 2008). Reinecke et al. (1999) concluded that earthworm E. fetida developed the resistance to Cd during an exposure even for 3 years to low-level Cd. It has been reported that the survival, cocoon production rate and weight change did not vary significantly for earthworms Lubricus rubellus exposed to soil with pyrene ranging from 0 to 40 mg/kg for 42 days (Brown et al., 2004). The CYP content and antioxidant enzymes varied subtly when earthworms E. fetida were exposed for 14 days to pyrene less than 0.96 mg/kg (Zhang et al., 2007). Nevertheless, significant increases of CAT and SOD activities were observed when earthworms E. fetida were exposed to pyrene of 50 or 100 mg /kg soil (Wu et al., 2012). While the effects of individual exposure of Cd and pyrene on enzymatic biomarkers were documented on earthworms, the combined effects of low-level Cd and pyrene on earthworms E. fetida are rarely reported. Moreover, exposure duration is generally less than 30 days in most previous studies paying attention to the response of enzymatic biomarkers of earthworms. Therefore, the study on the response of earthworms E. fetida to environmentally relevant low-level exposure of the mixture of Cd and pyrene for a longer period is needed for better evaluating ecological risk of Cd and pyrene in laboratory and field conditions. This study aims to investigate individual and combined effects of cadmium (2.5 mg/kg) and pyrene (0.96 mg/kg) on a set of enzymatic biomarkers (CYP, SOD, CAT and GST) of earthworms E. fetida upto eight weeks, and to provide valuable information for ecological risk assessment of the interacted effects of Cd and pyrene at environmentally relevant levels.
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The environmentally relevant concentrations of Cd (2.50 mg kg 1) and pyrene (0.96 mg kg 1) were selected in the experiment based on previous studies (Brown et al., 2004; Lapinski and Rosciszewska, 2008; Reinecke et al., 1999; Zhang et al., 2007) and the average level of Cd (2.50 mg kg 1) in agricultural soil of Shenyang, China. The worms were randomly divided into four groups with four replicates: (1) control, (2) pyrene: 0.96 mg kg 1, (3) Cd: 2.50 mg kg 1 and (4) the mixture of pyrene and Cd (pyrene: 0.96 mg kg 1 þCd: 2.50 mg kg 1). Pyrene and CdCl2 were dissolved in acetone and water, respectively, and then transferred to the soil. 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 40 percent of water holding capacity and left one day to equilibrate. 5000 g treated-soil for each replicate was placed in an aerated container (45 cm 30 cm 20 cm) and matured earthworms (130) were transferred to each container. Containers were subsequently covered with a perforated lid to limit water loss and kept for eight weeks at (207 2)1C in a 12:12-h lightdark regime. Worms were fed with dry and defaunated cow dung twice a month during the whole incubation period, and a few milliliters of distilled water were added daily into each container to maintain suitable humidity for earthworm activity. Worms were collected for the biochemical analysis after one, two, three, four, six and eight weeks of exposure. Five samples (Four worms per sample) at each container were collected every time. Each sample was measured three times. Both unhatched earthworm cocoons and new-born earthworms were removed throughout the experiment.
2.2. Biochemical assays Worms were first rinsed with distilled water, and placed on the petri dish with moistened filter paper to purge their gut contents for 3 days. Filter paper was changed every day during this period. Samples were subsequently immobilized in ice-cold 20 percent (V/V) glycerol solution for 3 min. The guts were then separated, washed afterwards with cold 0.15 M KCl solution and homogenized manually in a vitreous tissue homogenizer with 5 ml homogenization buffer (250 mM sucrose, 50 mM Tris pH 7.5, 1 mM DTT and 1 mM EDTA). Homogenates were centrifuged at 4 1C for 20 min at 15,000 g to produce the postmitochondrial fraction. One part of the supernatant was collected for the determination of activities of SOD, CAT and GST. The other part was further centrifuged at 150,000 g for 90 min to obtain microsomal pellets for CYP content determination. Cytochrome P450 content was determined by the method of Omura and Sato (1964) by means of sodium dithionite reduced carbon monoxide. Microsomal protein concentrations were evaluated by the method of Bradford (1976) using bovine serum albumin (BSA) as standard. GST activity was assayed by the method of Habig et al. (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The assay was carried out by monitoring the appearance of the conjugated complex of CDNB and glutathione (GSH) at 340 nm. The mixture contained 190 ml of 0.1 M Tris buffer pH 7.0, 0.5 ml of 1 mM GSH, 1 ml of 1 mM CDNB and 10 ml enzyme extract. The reaction was initiated by the addition of GSH. SOD activity was assayed by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp and Fridovich, 1971). The assay mixture contained 1.5 ml of 50 mM phosphate buffer pH 7.8, 0.3 ml of 130 mM methionine, 0.3 ml of 750 mM NBT, 0.3 ml of 0.1 mM EDTA, 0.3 ml of 20 mM riboflavin, 0.05 ml of deionized water and 0.05 ml enzyme extract in a total volume of 3 ml. Riboflavin was added finally, and the tubes were shaken and then illuminated for 15 min. The absorbance was recorded at 560 nm and the absorbance of the nonirradiated reaction mixture served as the control. CAT activity was assayed according to the method of Aebi (1984). The assay mixture contained 0.2 ml supernatant, 1.5 ml of 50 mM phosphate buffer pH 7.8, 0.3 ml of 0.1 M H2O2 and 1 ml deionized water. The decrease of the absorbance of the mixture was recorded at 240 nm for 4 min.
2.3. Statistical analysis 2. Materials and methods 2.1. Animals and treatment Earthworms, E. fetida, with well-developed clitella 300–400 mg were purchased from Shenyang Agricultural University, China and kept in control soil in the dark at (207 2)1C prior to the start of toxicant exposure. No sexual differences were considered since earthworms are hermaphroditic. The test soil (0–20 cm) was collected from the Ecological Experiment Station of Chinese Academy of Science in Shenyang, China. The soil was screened through a 5 mm sieve, kept at 4 1C and normal field moisture until use. The soil had the following characteristics: pH 6.20, Kjeldahl nitrogen 0.09 percent, total phosphorus 0.04 percent, total potassium 0.18 percent, organic matter content 1.65 percent, cation exchange capacity 12.30 cmol kg 1, water holding capacity 32.00 percent. The particle distribution of this soil was as follows: sand (450 mm) 22 percent, silt (1–50 mm) 64 percent, clay (o1 mm) 14 percent. The background level of Cd in the soil is 1.80 mg kg 1, and pyrene was not detectable in this soil.
All data were represented as the means7 standard deviation (SD). Statistical analysis for all measurements was performed by SPSS 16.0 software (SPSS Inc, Chicago, USA). Normality and variance homogeneity were first tested using Kolmogornov–Smirnov and Levene’s tests, respectively. However, even though mathematical transformation was applied to dependent variables, the variations after data transformation were still heteroscedastic or non-normally distributed in this study. Therefore the non-parametric Kruskal–Wallis test for independent samples (K 42) was adopted to test the difference between the control and treated samples at the same exposure time. A statistical difference at P o 0.05 was considered to be significant, and that at P o0.01 very significant.
3. Results The concentrations of Cd and pyrene in the soil treated with Cd and pyrene were verified as 4.2270.05 and 0.8070.03 mg kg 1,
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Fig. 1. CYP content in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at P o0.01.
respectively, which were close to the nominal concentraions after deducting the corresponding background values. Exposures to Cd and/or pyrene did not cause significant mortality of worms during the eight-week experiment. The response of each biomarker in the control group did not change significantly during the eightweek experiment as shown in Figs.1–4. The individual and combined effects of Cd and pyrene on CYP content were shown in Fig. 1. In the worms exposed to Cd alone, a significant elevation of CYP content was observed at week three (29.72 percent) and four (104.84 percent), and a declining trend occurred since week six. For the worms exposed to pyrene alone, a similar bell-shape change of CYP content was also observed, except that the enhancement of CYP content appeared earilier, between week two (155.17 percent) and three (76.03 percent), and the decrease of CYP content also appeared earilier. For the mixture-exposed worms, a pronounced increase of CYP content was observed from week two to four (93.32 percent, 42.02 and
Fig. 2. GST activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at Po 0.01.
33.42 percent, respectively), while a significant decrease (20.48 percent) was recorded at week eight. Fig. 2 presented the responses of GST activity in the tested worms. In the worms exposed to Cd alone, GST activity showed a tendency of increase, but only became significant (Po0.05) at week six (33.93 percent) compared to the control. In the worms exposed to pyrene alone, GST activity was elevated very significantly (P o0.01) between week two and three (147.20 percent and 115.07 percent, respectively). A flat bell shape was observed for GST activity in the mixture-exposed worms. At week two, the extent of increase of GST activity for the worms exposed to the mixture (20.36 percent) was much lower than that for those exposed to pyrene alone (147.20 percent). At week three, the mixture inhibited the increase of GST activity induced by pyrene since GST activities induced by pyrene and mixture exposure were 115.07 percent and 33.40 percent higher than the control, respectively. At week eight, GST activity levels induced by individual and combined exposure of Cd and pyrene
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Fig. 3. SOD activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at P o0.05, and **indicated a significant difference at P o0.01.
were recorded as 111.96 percent, 70.59 percent and 79.26 percent of the control, respectively. The responses of SOD activity of the earthworms were shown in Fig. 3. In the Cd alone-exposed worms, the SOD activity varied insignificantly between week one and four, but show pronounced increases at week six (53.45 percent) and eight (33.50 percent). SOD activity of the worms exposed to pyrene alone was significantly elevated between week one and two (100.86 percent and 36.23 percent, respectively); it decreased subtly with the exposure time increased from four weeks. For the mixture-exposed worms, no significant variation was observed on SOD activity except an increase of 66.16 percent at week one. The results on CAT activity were shown in Fig. 4. The kinetic shape of CAT activity of the Cd alone-exposed worms was similar to that of SOD activity. CAT activity levels after six and eight weeks of exposure were higher than their corresponding controls (79.16 percent and 53.26 percent, respectively), and no significant variation was exhibited at other exposure time. CAT activity levels of the mixture-exposed worms increased by 33.13 percent and
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Fig. 4. CAT activity in worms exposed to 2.50 mg/kg of Cd (A), 0.96 mg/kg of pyrene (B) and their mixture (C) for eight weeks. Data are expressed as the means7 SD of quadruplicate analyses. *indicated a significant difference at Po 0.05, and **indicated a significant difference at Po 0.01.
38.93 percent, respectively, after six and eight weeks of exposure. However, CAT activity in the pyrene alone-exposed worms demonstrated insignificant variation during the whole experimental period.
4. Discussion The application of multiple biomarkers to evaluate the effect of complex pollutants is prevalent in recent years. The study of biological responses of organisms exposed to different classes of contaminants is being considered as a helpful method for the evaluation of environmental quality. While the acute effects of exposure of high concentrations of cadmium or pyrene alone on terrestrial organisms were well documented (Brown et al., 2004; Scaps et al., 1997), few studies explored the chronic response of earthworms E. fetida exposed to the mixture of Cd and pyrene at environmental levels. The present study investigated the chronic
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effects of environmentally relevant low-level cadmium, pyrene and their mixture, since both accumulation and the appearance of toxicity of a low-level chemical within an organism need time. CYP family is particularly important in the metabolism of xenobiotics (Benedetti et al., 2007; Henczova´ et al., 2006), and has been widely used as a biomarker of organic and inorganic pollution in fish and sea worms as well as earthworms (Banni et al., 2009; Benedetti et al., 2007; Saint-Denis et al., 1999). In this study, compared to the effects of Cd or pyrene alone, the significant increase of CYP content in the mixture-exposed worms lasted three weeks from week two, indicating higher metabolism processed longer. The obvious additive decrease of CYP content induced by the co-exposure of Cd and pyrene was observed at week eight, suggesting that Cd may cause the enhancement of pyrene toxicity. This is in accordance with the results of Wang et al. (2011), in which a significant inhibition of the activity of aryl hydrocarbon hydroxylase (AHH) in calm Ruditapes philippinarum was induced by co-exposure of B[a]P and Cd compared to that by B[a]P exposure. A similar kinetic pattern was presented in GST activity of the worms exposed to the mixture of Cd and pyrene (Fig. 2). GST activity in the mixture-exposed earthworms was higher than that in the pyrene alone-exposed worms at the end of experiment, implying that the jointed effects generated higher oxidative stress. Broerse et al. (2012) demonstrated that the coexposure of Cd and pyrene decreased significantly pyrene uptake and elimination rates and resulted in a prolonged half life of pyrene. Many studies investigated the combined effects of heavy metals (Cd, Cu) and B[a]P on aquatic organisms such as sea worms, fish and so on. Similar to our results, Banni et al. (2009) showed an obvious additive increase of NADPH CYP C red activity and GST activity by the co-exposure of Cd and B[a]P equimolar (each 0.2 mM) to the sea worm Hediste diversicolor. Differently, exposure to Cu with the presence of B[a]P did not affected the level of NADPH CYP C in H. diversicolor within 48-h exposure (Bouraoui et al., 2009). Benedetti et al. (2007) reported the co-exposure of Cd suppressed completely the induction of ethoxyresorufin-o-deethylase (EROD) caused by B[a]P in the Antarctic fish Trematomus bernacchii. The possible inhibitory effect of Cd on B[a]P was also found by Jensen and Krøkje (2008) in a ternary mixture (0.7 mM Cd/B[a]P/Tetrachlorobiphenyl) in rat hepatoma cells. These results range from additive, independent, or antagonistic to synergistic mixture effects, depending on the combination of metals and PAHs, the exposure medium, test duration, test organism and the studied endpoint. The interactive effects of Cd and pyrene demonstrated in this study were exposure time-dependent, with the appearance of antagonism at week two to three and of additive effect at week eight for CYP content and GST activity. It is well established that both Cd and pyrene are able to induce ROS in earthworms (Brown et al., 2004; Scaps et al., 1997). Fortunately, the SOD-CAT system acts as the key part to fight against oxygen damage and free radicals generated in phase I. SOD activity was considered as an important biomarker for the assessment of pollutants on ecosystems. In this study, an enhancement of SOD activity was recorded for more than six weeks in the Cd alone-exposed worms and for less than three weeks in the pyrene alone-exposed worms, confirming that the ROS induction occurred in the metabolism of either Cd or pyrene. However, at the end of experiment, the increase of SOD activity was 15.64 percent with the mixture exposure, compared to the reduction by 18.65 percent with pyrene alone-exposure and the enhancement by 33.50 percent with the Cd alone-exposure, showing an additive effect again. Therefore, we deduced that the toxicity of pyrene is higher at week eight when it is coexposed with Cd due to the enhancement of pyrene accumulation by the co-presence of Cd. However, the co-exposure of Cd and
pyrene inhibited the enhancement of SOD activity caused by pyrene at week one and two, and similar interactive effects were also observed on CYP content and GST activity at week two. The fact that Cd decreases the uptake and elimination of pyrene and increases its hydroxylation in short time (Broerse et al., 2012) may be accountable for this kind of interaction. Moreover, our results confirmed the results of Broerse et al. (2012) from the aspect of biochemical responses that the interaction of Cd and pyrene is time dependent. Similarly to our findings, Wang et al. (2011) observed a decrease in SOD activity of mussel induced by exposure of B[a]P (500 mg/l) alone compared to that induced by co-exposure of B[a]P (500 mg/l) and Cd (80 mg/l). However, three binary mixtures of B[a]P and Cu (5, 10 and 25 mg L 1 for each chemical) did not cause significant differences compared to controls in the digestive gland of mussels (Maria and Bebianno, 2011). Similarly, a strong synergistic interaction of phenanthrene and pyrene on SOD activities of earthworm E. fetida was observed by Wu et al. (2012). Insignificant change in CAT activity of earthworms exposed to pyrene with respect to the control was exhibited in this study. Lei et al. (2006) reported that CAT activity in pyrene-treated cells (0.1 mg/ml) remained almost the same as the control for all microalgae species. Due to the low concentration of pyrene, the hydrogen peroxide can thus be eliminated by themselves or by other antioxidant enzymes. However, CAT activity increased significantly in the mixture-exposed worms in this work in six to eight weeks, similarly to the trend found in the Cd alone-exposed worms, so we concluded that Cd may influence the toxicity of pyrene on earthworms. Banni et al. (2009) demonstrated that an additive effect on the activation of CAT activity in worms co-exposed to 0.2 mM Cd/B[a]P, while CAT activity kept at the control level when co-exposed to 1.0 mM Cd/ B[a]P in the sea worm H. diversicolor. An additive effect on the activation of CAT activity in worms with exposure of the mixture of Cd and pyrene at week eight was also observed in our study, confirming the interacted toxicity of Cd and pyrene. Though the modeling computation (Dondero et al., 2011; Jonker et al., 2005) is prevalent to predict the interaction of mixed pollutants, simple mathematical calculation combined with statistical analysis (Banni et al., 2009, 2011; Bouraoui et al., 2009; Maria and Bebianno, 2011; Wu et al., 2012) was still applied widely. In this work, the modeling calculation is inapplicable since only a single concentration was adopted for each chemical in the mixture herein, resulting in the absence of relative parameters for modeling calculation such as the toxic unit. Moreover, many researchers who highlight the biochemical responses also utilize the simple mathematical method instead of modeling for the judgment of interaction types (Banni et al., 2011; Maria and Bebianno, 2011; Wang et al., 2011; Wu et al., 2012). The interacted effects of Cd and pyrene on enzymatic biomarkers may be elucidated based on chemical toxicokinetics. Pyrene uptake and elimination can be significantly decreased with the presence of Cd, resulting in more pyrene accumulation in body and a prolonged elimination half life. Meanwhile, pyrene hydroxylation can also be increased but its further metabolism is postponed due to the co-presence of Cd, causing the half life prolonged as well (Broerse et al., 2012). The interacted effects might also be interpreted based on toxicodynamics by the fact that both Cd and pyrence can cause oxidative stress (Broerse et al., 2012). Cd can be sequestered by GSH to prevent its adverse interaction with biomolecules. GSH and other thiols play a crucial role in cleaning ROS (Broerse et al., 2012). When exposed to the mixture of pyrene and Cd, GSH and other thiols may be consumed fast due to the presence of Cd, and ROS levels may be elevated indirectly (Broerse et al., 2012). Cd may also postpone further metabolism of the phase I products of pyrene by a depletion of
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possible conjugates. In the second phase, reactive phase I metabolites are conjugated with chemicals like GSH, which may be also involved in scavenging and detoxifying Cd (Baird et al., 2005). It may also account for the prolonged toxicity of pyrene by the copresence of Cd.
5. Conclusions The binary mixtures of Cd and pyrene can account for the different responses in earthworms E. fetida from those observed with single exposures. Antagonistic effects were observed on CYP content, GST activity and SOD activity initially, but additive effects were responsible for the reduction of CYP content and GST activity and the enhancement of activity of SOD and CAT at the end of experiment. The interaction of pyrene and Cd varied over time, indicating that the exposure time may be more important than the exposure dose for the combined effect. Moreover, the presence of cadmium prolonged the toxic effect of pyrene. Short-term toxicity may overlook the interactive effects as the accumulation of toxicants is time dependent. A suite of biomarkers is helpful in the evaluation of the interactive toxicity of PAHs and heavy metals on earthworms and could be instrumental in monitoring soils contaminated with multiple pollutants.
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