Chinese Journal of Oceanology and Limnology Vol. 27 No. 4, P. 816-824, 2009 DOI: 10.1007/s00343-009-9222-z

Effects of tributyltin (TBT) on enzyme activity and oxidative stress in hepatopancreas and hemolymph of small abalone, Haliotis diversicolor supertexta*

JIA Xiwei (贾锡伟)†,††, ZHANG Ziping (张子平)††, WANG Shuhong (王淑红)††, LIN Peng (林鹏)††, ZOU Zhihua (邹志华)††, HUANG Bangqin (黄邦钦)†, WANG Yilei (王艺磊)††,** † State Key Laboratory of Marine Environmental Science, Ministry of Education, Environmental Science Research Center, Xiamen University, Xiamen 361005, China †† The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen 361021, China

Received Oct. 15, 2008; revision accepted Feb. 3, 2009

Abstract We investigated the effect of tributyltin (TBT) exposure on the concentration of malondialdehyde (MDA) and the activity levels of the superoxide dismutase (SOD), catalase (CAT), and acid and alkaline phosphatase (ACP and AKP) enzymes in the small abalone, Haliotis diversicolor

supertexta. We collected samples of the hepatopancreas and hemolymph 2, 6, 24, 48, 96, and 192 h after exposure to 0.35 μg (Sn)/L TBT. In the hepatopancreas, ACP activity was significantly higher in animals exposed to TBT 2, 24, and 96 h post-exposure compared with the control animals. AKP activity was also higher after 2 h, but SOD and CAT activity was unchanged. The concentration of MDA in the hemolymph was significantly higher than the control animals 2 and 6 h post-exposure. In the hemolymph of animals exposed to TBT, ACP activity was significantly lower than in the control animals 192 h post-exposure, whereas AKP activity was significantly lower 2 and 192 h post-exposure. Hemolymph SOD activity and levels of MDA were significantly lower than in the control animals 24 h after exposure but significantly higher after 96 h. Our results demonstrate that exposure to TBT cause rapid changes in ACP and AKP activity as well as altering the concentration of MDA in the hepatopancreas and hemolymph. SOD and CAT do not appear to be involved in the detoxification of TBT in the hepatopancreas of small abalone.

Keyword: tributyltin (TBT); superoxide dismutase (SOD); catalase (CAT); acid and alkaline phosphatase (ACP and AKP); malondialdehyde (MDA); Haliotis diversicolor supertexta

1 INTRODUCTION Tributyltin (TBT) is a powerful anti-fouling

biocide that has been used widely on vessels in both seawater and freshwater environments since the mid 1960s. TBT has adverse effects on many target and non-target marine organisms. Therefore, many countries have regulated its application as an antifoulant (Champ, 2000). In 2003, the International Marine Organization (IMO) imposed a worldwide ban on TBT which the maritime industry scheduled to come into effect by 2008 (Bekri et al., 2006). However, TBT is still used in antifouling paints applied to larger ships (>25 m in length) in most developed countries. Furthermore, restrictions on its

use have not been implemented in most developing countries, including China (Yang et al., 2006). High concentrations of TBT are still found in seawater and sediment throughout the world (Axiak et al., 2000; Shim et al., 2005; Buggy et al., 2006). In China, Jiang et al. (2001) measured TBT concentrations of 977 ng (Sn)/L and 425.3 ng (Sn)/L in the Qingdao North Sea Shipyard and Shanghai Huangpu River, respectively. Furthermore, the concentrations of

∗ Supported by the Committee of Xiamen Science and Technology, Xiamen, China (No. 502Z20055024), the National Natural Science Foundation of China (No. 20877034), and the Innovation Team Foundation of Jimei University (No. 2008A001) ∗∗ Corresponding author: ylwang@jmu.edu.cn

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TBT in the coastal environment of Xiamen, China ranged from nd (not detected) to 60 ng (Sn)/L in the water, nd to 20 ng (Sn)/L in suspended particulate matter, and nd to 26 ng (Sn)/g (dry weight) in the sediment (Wang et al., 2008). It is well documented that TBT is responsible for a number of adverse effects, including: imposex (Horiguchi et al., 2000, 2002, 2005; Svavarsson, 2000), endocrine disruption (Siah et al., 2003; Santos et al., 2006), immunological changes and cell death (Gronczewska et al., 2004), inhibition of embryo development, and inhibition of development and survival in larvae and sperm (Rurangwa et al., 2002; Grzyb et al., 2003; Inoue et al., 2006, 2007; Shimasaki et al., 2006). Consequently research on the fate, toxicity, and ecological risk of organotin compounds remains a necessity.

A biomarker is defined as a change in a biological response (ranging from molecular through cellular and physiological responses to behavioral changes) which can be related to exposure to environmental chemicals (Peakall, 1994). The most compelling reason for using biomarkers is that they can provide information on the biological effects of pollutants rather than a mere quantification of their environmental levels. Biomarkers may also provide insight into the potential mechanisms of contaminant effects (Ron et al., 2003). We measured several biochemical parameters in small abalone to evaluate environmental stress resulting from TBT exposure and determine their utility as biomarkers.

Abalone is marine gastropods that are found throughout the world (Geiger, 1999). They are very sensitive to environmental stress so they are an ideal bio-indicator of the health of marine environments (Hunt, 1989). Small abalone, Haliotis diversicolor supertexta, is a commercially important species for fisheries and aquaculture in southern China (Liao et al., 2004). The wild population of small abalone has been in decline for a number of years. Furthermore, cultured stocks have been subject to an increasing number of infectious disease outbreaks (Wang, 2004). Many factors, such as environmental pollution or other stressors, may contribute to these problems. Given these factors, reliable biomarkers for diagnosing the health of small abalone populations are urgently needed. However, there is a lack of both acute and chronic toxicity data for this species. Many pollutants (or their metabolites) may exert toxicity via oxidative stress. Because of this, antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), have been widely studied as

potential biomarkers in environmental risk assessment programs. In addition, the products of lipid peroxidation (e.g. malondialdehyde (MDA)) are often evaluated concurrently (Ron et al., 2003). We have shown that acid and alkaline phosphatase (ACP and AKP) are important for innate immune defense in small abalone (Wang, 2004). Our current objective is to evaluate the effect of TBT exposure on a range of biochemical parameters, including SOD, CAT, ACP, and AKP activity, and MDA concentrations in small abalone. Our results may be used to evaluate the utility of these parameters as biomarkers for TBT exposure.

2 MATERIALS AND METHODS 2.1 Animals

We collected abalones (shell width: 4.65±0.27 cm, weight: 10.07±2.03 g) from a commercial farm (Futian, Dongshan, Fujian Province). The animals were transferred into polyethylene tanks and acclimated for up to 2 weeks. Each tank contained 60 animals in 180 L of aerated and sand filtered seawater at 23–25°C. Fresh seawater was changed everyday. The animals were fed with fresh kelp (Laminaria japonica Aresch) during each water change.

2.2 Chemicals

We purchased tributyltin chloride (TBTCl) from Sigma (St. Louis, USA). We made a stock solution of TBTCI by dissolving in 100% ethanol (3.5 g (Sn)/L).

2.3 Acute Toxicity Assay

We exposed 12 small abalones to either 0.0 (control), 0.5, 1.0, 2.0, 5.0, 7.5, or 10.0 μg (Sn)/L TBT in series for 96 h. Each concentration was replicated. The lethal concentrations (LCx) were calculated using a maximum likelihood regression with a probit model. Significant differences between LCx values were defined by the criterion of nonoverlapping 95% confidence intervals (C.I.).

2.4 Exposure challenge

The 96 h lethal concentration (LC50) value was 3.5 μg (Sn)/L. The stock solution was then diluted in fresh seawater to a final concentration of 0.35 μg (Sn)/L for exposure. The control animals were exposed to an. equal volume of fresh seawater containing 0.1% of 100% ethanol. We changed the holding water with fresh seawater containing the appropriate concentration of TBT once a day. The animals were fed with fresh kelp during each water

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change. Because the individual response of small abalone to the environment is highly variable, we incorporated a control group in each phase to eliminate sampling error. We collected the hepatopancreas and hemolymph from at least fifteen abalones in both the treatment and control groups 2, 6, 24, 48, 96, and 192 h after the challenge. The hepatopancreas tissue was then frozen at -80°C until used. The volume of fresh seawater was reduced following the removal of animals for sample collection to maintain the appropriate density of small abalones.

2.5 Homogenate and hemolymph preparation

The hepatopancreas was homogenized in physiological saline (1 ml per 50–100 mg of tissue). The homogenate was centrifuged at 1 000 g for 5 min (4°C), and the supernatant was collected for evaluation of enzymatic activity. We collected the hemolymph (approximately 1.0 ml/animal) using a capillary to withdraw fluid from a central incision in the pleopod muscle. The samples were transferred into a 1.5 ml centrifuge tube and immediately centrifuged at 150 g for 5 min (4°C) to remove the hemocytes. The cell-free hemolymph was frozen at -80°C until used.

2.6 Biochemical assays

Acid and alkaline phosphatase (ACP and AKP) activity assays were carried out according to Barrett’s (1972) method using a commercial kit (Nanjing Jiancheng Biotech Company, China). Analysis was performed in 96-well flat-bottomed microtiter plates. The concentration of phenol was measured spectrophotometrically at 520 nm after incubation at 37°C for 30 min (for ACP), or for 20 min at 37°C (for AKP). ACP and AKP activity was defined as the amount of phenol (mol) produced per milligram protein.

Superoxide dismutase (SOD) activity was determined using a modification of the xanthine-xanthine oxidase assay (McCord et al., 1969), adapted using a kit (Nanjing Jiancheng Biotech Company, China). The assay was carried out in a 96-well flat-bottomed microtiter plate. One nitrite unit (NU) of SOD was defined as the quantity of enzyme (per mg protein) that inhibits the reduction of cytochrome c by 50%.

Catalase (CAT) activity was determined following the method of Aebi (1984) by measuring the decrease in absorbance at 240 nm due to H2O2 consumption. Results were expressed as units per milligram of protein.

Malondialdehyde (MDA) concentration was determined spectrophotometrically using the thiobarbituric acid method (Esterbauer et al., 1990). We used a commercial kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). Briefly, 5% cold trichloroacetic acid was added to the homogenate to precipitate the protein. The precipitate was centrifuged (5 000 g for 10 min) and the supernatant was added to an equal volume of 1% thiobarbituric acid in a boiling water bath for 10 min. After cooling, TBAR levels were estimated at 532 nm against a blank consisting of 5% cold trichloroacetic acid mixed with 1% thiobarbituric acid. We used malondialdehyde bis acetal as a standard. Concentrations of lipid peroxidation compounds were expressed as nmol mg-1 protein.

Total protein levels were determined according to Bradford (1976).

2.7 Statistics

All data are presented as the mean and standard error of at least 15 specimens. We used an independent-sample T test to compare among groups. P <0.05 were considered to be significantly different.

3 RESULTS 3.1 Lethal concentration evaluation

The mortality of small abalone after 96 h exposure to TBT is shown in Table 1. The TBT concentrations were log-transformed prior to fitting data to the regression models. Following this the acute toxicity results were log-transformed. The 96 h LC50 value was 3.5 (95% confidence interval: 2.02–4.75) µg (Sn)/L.

3.2 Changes in ACP and AKP activity in the hepatopancreas of small abalone during exposure to TBT

ACP activity was significantly higher in the hepatopancreas of abalone that were exposed to 0.35 μg (Sn)/L TBT than in the controls after 2 h (P<0.05), 24 h and 96 h (P<0.01) (Fig.1).

Table 1 TBT concentrations in test solutions and the acute toxicity to small abalone during a 96 h exposure

Concentration of TBT μg (Sn)/L LC50 95% C.I. *R 0.5 1.0 2.0 5.0 7.5 10.0 μg (Sn)/L μg (Sn)/L

A 4.2 8.3 29.2 79.2 87.5 83.3 Mortality (%) B 4.2 16.7 20.8 62.5 70.8 91.7

3.5 2.02–4.75

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AKP activity was significantly higher in the hepatopancreas of abalone exposed to 0.35 μg (Sn)/L TBT than in the control animals after 2 h (P<0.01) (Fig.2).

Fig.1 Changes in ACP activity in the hepatopancreas during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. One asterisk denotes a significant (P<0.05) difference from the control group, two asterisks denote a highly significant difference (P<0.01) from the controls

Fig.2 Changes in AKP activity in the hepatopancreas during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. The two asterisks denote a highly significant difference (P<0.01) from the controls. 3.3 Changes in ACP and AKP activity in the hemolymph of small abalone during exposure to TBT

ACP activity was significantly lower in the hemolymph of abalone exposed to 0.35 μg (Sn)/L TBT compared with the controls after 192 h (P<0.05) (Fig.3).

AKP activity was significantly lower in the hemolymph of abalone exposed to 0.35 μg (Sn)/L TBT after 2 and 192 h (P<0.05) compared with the control animals (Fig.4).

Fig.3 Changes in ACP activity in the hemolymph during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. The asterisk denotes a significant (P<0.05) difference from the control group.

Fig.4 Changes in AKP activity in the hemolymph during exposure to TBT

All data are presented as the mean and standard errors of a minimum of 15 abalones. The asterisk denotes a significant (P<0.05) difference from the

control group. 3.4 Changes in SOD activity in the hepatopancreas of small abalone during exposure to TBT

There was no statistical difference between the two groups at any time (Fig.5).

3.5 Changes in SOD activity in the hemolymph of small abalone during exposure to TBT

SOD activity was significantly lower in the hemolymph of abalone exposed to 0.35 μg (Sn)/L TBT than in the controls 24 h (P<0.01) post-exposure. In contrast, activity levels were significantly higher compared with the controls after 96 h (P<0.05) (Fig.6).

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Fig.5 Changes in SOD activity in the hepatopancreas during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones.

Fig.6 Changes in SOD activity in the hemolymph during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. One asterisk denotes a significant (P<0.05) difference from the control group. Two asterisks denotes a highly significant (P<0.01) difference from the control group.

3.6 Changes in CAT activity in the hepatopancreas of small abalone during exposure to TBT

There was no difference in CAT activity in the hepatopancreas between the two groups at any time (Fig.7).

We were not able to measure CAT activity in the hemolymph.

3.7 Changes in MDA concentrations in the hepa-topancreas of small abalone during exposure to TBT

The concentration of MDA in abalone exposed to 0.35 μg (Sn)/L TBT was significantly higher than that of control group 2 h (P<0.05) and 6 h (P<0.01) after exposure (Fig.8).

Fig.7 Changes in CAT activity in the hepatopancreas during

exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones.

Fig.8 Changes in the concentration of MDA in the

hepatopancreas during exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. One asterisk denotes a significant (P<0.05) difference from the control group. Two asterisks denote a highly significant (P<0.01) difference from the control group.

3.8 Changes in MDA concentrations in the hemo-lymph of small abalone during exposure to TBT

The concentration of MDA from abalone exposed to 0.35 μg (Sn)/L TBT was significantly lower than that of control group after 24 h (P<0.05), and significantly higher than that of the control group 96 h(P<0.05) after exposure (Fig.9).

4 DISCUSSION ACP and AKP are involved in a variety of

metabolic processes such as detoxification, metabolism, and the biosynthesis of energetic macromolecules for various essential functions (Rahman et al., 2004). Interference with either of these two enzymes can lead to biochemical impairment of cellular functions and

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Fig.9 Changes in the concentration of MDA in the

hemolymph during exposure to TBT All data are presented as the mean and standard errors of a minimum of 15 abalones. One asterisk denotes a significant (P<0.05) difference from the control group.

tissue lesions (Enan et al., 1982). For example, ACP acts as a marker enzyme for the detection of lysosomes in cell fractions and can be altered by the presence of xenobiotics (Cajaraville et al., 2000; Rajalakshmi et al., 2005). It can also be used as a biomarker for a number of diseases (Samman et al., 1996) and for xenobiotic-induced lysosomal membrane damage in earthworms (Hønsi et al., 2000). In mussels exposed to 400 ng/g copper, gill ACP activity remained high throughout the period of exposure, indicating derangement of defense mechanisms or a delay in revival (Rajalakshmi et al., 2005). AKP is a phosphomonoesterase that detoxifies contaminants during normal living conditions (Zhang, 2004).

AKP and ACP are sensitive to metals. Mazorra et al. (2002) demonstrated that the effect of metals on ACP and AKP activity could be modeled using the Michaelis–Menten model. Furthermore, Rao (2006) measured an increase in plasma activity of ACP and AKP following exposure to organophosphorus insecticides in a euryhaline fish (Oreochromis mossambicus). Imposex has been reported in marine gastropods following exposure to TBT (McClellan- Green et al., 2000; Svavarsson, 2000; Meng et al., 2005). A similar phenomenon has also been reported in abalone, Haliotis gigantean (Horiguchi et al., 2000, 2002).

In present study, we found that ACP activity was significantly higher in the hepatopancreas of animals exposed to TBT 2, 24, and 96 h post- exposure when compared with the control group. Similarly, AKP activity was significantly higher in the hepatopancreas after 2 h, relative to the control

animals. The elevation in ACP and AKP activity suggests an increase in lysosomal mobilization and cell necrosis caused by TBT. Feng (1988) suggested that an elevation in lysosome enzyme (including lysozyme, ACP, and AKP) activity is only one indicator of an immune response. Equally important is that a decrease in lysosome enzyme activity might also be an indication of disease. Our results indicated that TBT induced ACP and AKP activity in the hepatopancreas and activated the immune defense response in abalone. In contrast, ACP activity declined significantly in the hemolymph 192 h after exposure to TBT. AKP activity also declined significantly in the hemolymph 2 and 192 h after exposure to TBT. We hypothesize that the decrease of ACP and AKP activity in the hemolymph induced an increase in activity in the hepatopancreas to counter the effect of TBT. AKP activity was not significantly affected by oil-derived products in the eleven-armed asteroid Coscinasterias muricata (Georgiades et al., 2003). In the present study hemolymph AKP activity in the animals exposed to TBT was only different from the control animals at 2 time points. Together, these results suggest that hydrolases function primarily in the hepatopancreas. Hemolymph ACP and AKP activity in small abalone was induced by injection with Vibrio parahaemolyticus (Wang et al., 2004). This contrasts with our current findings that showed a difference in the response of ACP and AKP to TBT exposure. Thus, we speculate that ACP and AKP may be used as biomarkers to distinguish particular pollution sources.

It is well known that both organic and inorganic contaminants can induce oxidative stress in animals by producing reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide anion (O2̄). ROS may initiate a sequence of reactions which produce free lipid radicals and hydroperoxides that are extremely toxic to cells (Livingstone, 2001). Superoxide dismutase (SOD) and catalase (CAT) are the primary enzymes for the defense against ROS-mediated toxicity (Matozzo et al., 2004).

SOD activity was much lower in the hemolymph of the small abalone than in the hepatopancreas, suggesting that SOD is not widely distributed in the hemolymph in this species. Liu (2006) reported that SOD activity increased in the liver of rats after 3 d of TBT exposure. Similarly, Wang (2005) noted that hepatic SOD and CAT activity increased significantly in Sebastiscus marmoratus following exposure to 9.6, 19.3, or 193 μg TBT/kg for 4 d. In

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contrast, we did not detect any change in SOD or CAT activity in the hepatopancreas of the small abalone. A number of reports have shown that SOD and CAT activity is induced by a range of contaminants (Prieto et al., 2006; Oruc et al., 2007; Vega -López et al., 2007). However, our results suggest that SOD and CAT are not involved in the detoxification of TBT in the hepatopancreas. In hemolymph however, SOD may play a role in minimizing the effects of TBT. The interaction between TBT and anti-oxidant enzymes requires further study.

Lipid peroxidation (LPO) or the oxidation of polyunsaturated fatty acids is an important consequence of oxidative stress and has been investigated extensively (Ron et al., 2003). Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation (Pampanin et al., 2005). Therefore, measurement of MDA is widely used as an indicator of lipid peroxidation. It is particularly interesting to associate the measurement of antioxidant molecules (such as CAT) with an oxidative damage assay, such as the measurement of MDA concentrations.

MDA concentrations increased significantly in the hepatopancreas 2 and 6 h after exposure to TBT. However, levels tended to rise more slowly in the hemolymph and were not significantly different from the controls until after 96 h in the animals exposed to 0.35 μg (Sn)/L TBT. Thus, it appears that the animals were under oxidative stress during these time periods. Geret et al. (2002) reported a similar effect in the gill of the clam Ruditapes decussates after 3 d of exposure to 25 mg Cu/L. A number of studies have shown that MDA concentrations are negatively correlated with CAT or SOD activity (Funes et al., 2006; Liu et al., 2006; Pampanin et al., 2005). In contrast, other studies have noted a positive correlation between these indicators. For example, MDA concentrations and SOD activity were both higher in an ischemia-reperfusion group in the ileum tissue of rats (Topcu et al., 2007). Similarly, MDA levels increased slightly and CAT and SOD increased significantly in the liver and kidney of rats treated with trichloroacetic acid (TCA) (Celik, 2007). We also found that the changes in MDA concentration after 24 and 96 h mirrored the change in SOD activity in the hemolymph. We hypothesize that the increase in MDA induced an increase in SOD activity to reduce the oxidative stress.

5 CONCLUSION Exposure to TBT caused rapid changes in ACP

and AKP activity as well as altering the concentration of MDA in the hepatopancreas and hemolymph. Our results suggest that ACP and AKP are involved in the detoxification of TBT. In contrast, SOD and CAT did not play a key role in detoxification of TBT in the hepatopancreas.

6 ACKNOWLEDGMENTS We thank Mr. Scot V. Libants (Department of

Fisheries & Wildlife, Michigan State University, USA) for critical reading of the manuscript.

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