Biochemical responses of freshwater fish Carassius auratus to polycyclic aromatic hydrocarbons and pesticides

Yong JI*, Guang-hua LU Chao WANG Jie ZHANG

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. School of Hydraulic and Ecological Engineering, Nanchang Institute of Technology, Nanchang 330099, P. R. China

Biochemical responses of freshwater fish Carassius auratus to polycyclic aromatic hydrocarbons and pesticides

Yong JI*1,2, Guang-hua LU1, Chao WANG1, Jie ZHANG1,2

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. School of Hydraulic and Ecological Engineering, Nanchang Institute of Technology, Nanchang 330099, P. R. China

The freshwater fishCarassius auratuswas chosen as an experimental subject, and their hepatic biochemical responses to the medium-term exposure of Benzo(k)fluoranthene (BkF) alone and in combination with PCB118 and dichlorodiphenyltrichloroethane (DDT) were investigated by measuring the reduced glutathione (GSH), glutathione S-transferase (GST), and thiobarbituric acid reactive substances (TBARS), to assess sublethal effects. The hepatic GSH content was significantly inhibited by organic pollutants, alone and in mixtures, while the TBARS content was significantly induced after three days of exposure. Bell-shaped concentration-response charts of GST activities were obtained. Significant dose-response relationships were found for hepatic GSH and TBARS contents of all concentrations and for the GST activity, except at the highest concentration. The GSH content, GST activity, and TBARS content inCarassius auratuswere confirmed as useful biomarkers of exposure to organic pollutions.

biochemical response; Carassius auratus; glutathione (GSH); glutathione S-transferase (GST); thiobarbituric acid reactive substances(TBARS)

1 Introduction

Persistent organic pollutants (POPs) are a widespread class of environmental contaminants that may enter the freshwater environment through discharges of industrial and municipal wastewater, shipping activities, offshore oil production, oil spills, fossil fuel combustion, agricultural production, and private and public dwellings (Liu et al. 2009; Xu et al. 2007). In recent years, with the rapid development of the petroleum industry and petrochemical industry, more and more attention has been paid to water environmental safety because of the extensive existence of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorinated pesticide (OCPs) (Hu et al. 2010).

POPs exist extensively in aquatic environments, including lakes, rivers, streams, and other surface waters that support aquatic life (Meyer 2011). PAHs are released into theenvironment as a result of incomplete burning of petrochemical products (Hu et al. 2008). Some toxicological studies on animals have shown that PAHs can cause harmful effects on the body’s defense system that fights disease after both short- and long-term exposures (Xu et al. 2007). PCBs are persistent xenobiotics in aquatic environments, and they elicit diverse toxic effects, such as induction of oxidative stress. Despite numerous previous studies, no detailed information exists on the toxic response of fish (Reyes et al. 2003). DDT is an insecticide used against mosquitoes outdoors and flies in rural areas, as well as fleas and ticks on pets (Monosson 2005).

Under normal physiological conditions, there exists a balance between the generation of reactive oxygen species and their elimination by different antioxidant scavengers. If reactive oxygen species are generated in excess or the cellular antioxidant defense system is impaired, oxidative stress arises. Antioxidative responses of fish exposed to xenobiotics have been extensively explored (Livingstone 2001). The antioxidant enzymes are found in virtually all tissues of vertebrates, but in general at especially high levels in the liver, a major organ for enobiotic uptake and enzymatic transformation of reactive oxygen species. It is known that oxidative damage is an important mechanism of toxicity induced by POPs. Therefore, glutathione (GSH), a ubiquitous non-protein thiol that plays a major role in the maintenance of intracellular redox balance and in the regulation of signaling pathways enhanced by oxidative stress (Peńa-Llopis et al. 2001), was quantified as a nonenzymatic antioxidant defense. The activities of glutathione S-transferase (GST) were selected as biomarkers since they are important enzymatic antioxidant defenses (Livingstone 2001). In addition, the enzymatic activities of GST, a family of multi-functional enzymes involved in phase II of biotransformation that are related to cellular antioxidant defenses due to the conjugation of electrophilic xenobiotics and oxidized components with GSH (Fitzpatrick et al. 1995), were also evaluated. Thiobarbituric acid reactive substances (TBARS), which are a common product of lipid peroxidation and a sensitive diagnostic index of oxidative damage in cells, were used as indicators of oxidative damage in this study.

Most of our current knowledge of the mechanisms of antioxidant biomarkers is acquired from laboratory studies based on single chemicals in clearly defined systems. In contrast, little is known about PAHs in combination with PCBs and DDT. Thus, assessment of the complex toxicity of POPs in mixtures has been an enduring challenge to environmental health research for the past several decades. In the present study,Carassius auratus, one of the main edible fish species in China, was chosen as the experimental subject. The purpose is to study the continual effects of Benzo(k)fluoranthene (BkF) alone and in combination with PCB118 and DDT on the antioxidant defense system ofCarassius auratusduring a medium-term period of 15 days. Hepatic antioxidant defense parameters, such as the GSH content, GST activity, and TBARS content, were assayed to analyze the possible antioxidant defense mechanisms and to study the possibility of taking the antioxidant indices as early biomarkers for the evaluation of POP-polluted aquatic ecosystems.

2 Materials and methods

2.1 Chemicals

BkF, PCB118, and DDT standards were purchased from Sigma-Aldrich Cooperation. Other reagents were obtained from chemical companies in China, at analytical grade or greater.

2.2 Animals

Carassius auratusof both sexes were obtained from an aquatic breeding base (an aquaculture facility in Nanjing, China) with a mean length of 8.5 cm and a mean weight of 25.2 g. The fish were acclimatized to water dechlorinated with activated carbon for two weeks prior to the experiment. They were fed once a day with commercial fish food. Sewage and food residue were removed every other day by suction. During the experiment, the pH value was 7.1 ± 0.2, and the temperature was 18 ± 1 ℃. The dissolved oxygen levels in the water were kept higher than 6 mg/L by continuous aeration. The total mortality of fish was below 1%. The fish were not fed for 24 h prior to the experiment and no food was provided during the test period.

2.3 Experiment

A semi-static method was used to determine the acute median lethal concentrations (LC50 values) of the three pollutants in a pilot study. The 96-h LC50 values were 7.405 mg/kg for BkF, 0.784 mg/kg for PCB118, and 0.648 mg/kg for DDT. During sublethal studies, fish were exposed to LC50 values as well as 1/625, 1/125, 1/25, and 1/5 of LC50 values of the pollutants. Chemical treatments were delivered via intraperitoneal injection. After acclimatization, the fish were randomly classified into six groups (15 fish per group) and kept in glass aquariums with a fish-water ratio of 10.0 g/L. One group receiving corn oil only was designated as the control group and the other groups were designated experimental groups receiving sublethal concentrations of LC50 as well as 1/625, 1/125, 1/25, and 1/5 of LC50 of the BkF dissolved in corn oil, in combination with PCB118 and DDT, respectively, for 15 days on the basis of the results of preliminary tests. After the fish were weighed, a certain amount of drug based on the drug needed for per-kg body weight was injected directly into the bodies of the fish. Mass and dosage data were recorded. Every day, 50% of the water was replaced with fresh water.

2.4 Enzyme assays

Three fish were removed from the tank and killed by cervical transaction by the end of each test period. Their liver tissues were carefully collected, washed in the 0.15 mol/L KCl solution, weighed, and then homogenized in the buffer (19 mL of buffer for 1 g of liver tissue), which was composed of 0.25 mol/L of sucrose, 0.1 mol/L of Tris-HCl, and 1 mmol/L of EDTA and had a pH value of 7.4, and centrifuged at 9 000gfor 25 min (wheregis the gravitational acceleration). The supernatants were collected for determination.

A method described in Habig and Jakoby (1981) and adapted to a microplate reader (Frasco and Guilhermino 2002) was used to determine the GST activity. The total cellularGSH level was measured using the method of Hissin and Hilf (1976). The TBARS content was measured with the thiobarbituric acid assay of Miller and Aust (1989), with some modifications. Protein concentration was determined with the Coomassie protein assay kit (Bradford 1976), with bovine serum albumin as the standard. Measurements were done by a microplate reader at 595 nm.

2.5 Statistical analysis

The data were analyzed with one-way analysis of variance (ANOVA), and the means and standard deviation were presented. Statistical analysis was performed with at-test, and the difference between two samples was considered statistically significant whenp＜ 0.05, wherepis the probability value.

3 Results

In the livers of the fish exposed to corn oil dissolving the organic pollutants, the GSH content, GST activity, and TBARS content did not differ significantly from those in the water. Therefore, the GSH content, GST activity, and TBARS content of the pollutant-exposed fish were compared with those of the corn oil control groups. Fifteen experimental groups of organic pollutants including BkF alone, the binary mixture of BkF and DDT, and ternary mixture of BkF, DDT, and PCB118 were used for the comparison in this study (Table 1).

Table 1 Experimental groups of organic pollutants with different concentrations mg/kg

The in vivo effects and inhibition rates of BkF on the hepatic GSH content, GST activity, and TBARS content are presented in Fig. 1. Compared with the control group, the GSH content decreased continuously in exposure periods at concentrations of 0.01 to 7.41 mg/kg (Fig. 1(a)) in the experimental group. For the 7.41 mg/kg experimental group, GST activities increased first and then decreased from the third day on, while GST activities were significantly induced in the 0.30 and 1.48 mg/kg experimental groups (Fig. 1(b)). The TBARS content was not affected in early periods. Thereafter, the TBARS content increased continuously at concentrations of 1.48 to 7.41 mg/kg (Fig. 1(c)). After three days of exposure, the TBARS content was significantly induced, and it reached a maximal value after 15 days of exposure. The hepatic GSH content showed a trend of initial inhibition, then recovery, and subsequent inhibition for most exposure concentrations (Fig. 1(a)), while the TBARS content showed a gradually increasing trend. Time dependence of GST activities in livers was not apparent, except at 7.41 mg/kg after seven days of exposure.

Fig. 1 Heptic GSH content, GST activity, and TBARS content after 1, 3, 7, and 15 days of exposure to BkF (Bars indicate standard error of the mean, and asterisks indicate values that are significantly higher than control values (p＜ 0.05))

The GSH content was significantly inhibited by all BkF and DDT mixture concentrations after 15 days of exposure (p＜ 0.05; see Fig. 2(a)). The percentage inhibition of the GSH content in livers also increased with mixture concentration. The inhibition of the hepatic GSH content increased significantly within three to seven days, and finally increased slightly on the 15th day for all concentrations and exhibited a good concentration-response relationship. Hepatic GST activities (Fig. 2(b)) and the TBARS content (Fig. 2(c)) did not change significantly after exposure to low-concentration mixtures. However, GST activities and the TBARS content were significantly induced (p＜ 0.05) when the dose of the mixture was increased to 1/5 of the LC50 mixture concentration. The time dependence of hepatic GST activities was not apparent, while the TBARS content increased continuously after one day of exposure.

The hepatic GSH content was also significantly inhibited by the ternary mixture after 15 days of exposure (p＜ 0.05; see Fig. 3(a)). The time response for hepatic GSH content exhibited a trend of initial inhibition, then recovery, and subsequent inhibition for most exposure concentrations (Fig. 3(a)). The GSH content in the fish exposed to ternary mixtures decreased with prolonged exposure and exhibited a good concentration-response relationship for 15 days of exposure. The ternary mixture significantly induced both GST activities and TBARS content after three days of exposure. The concentration-response relationship was similarto that of the BkF and DDT mixture. Changes of GST activities and TBARS content in livers during the periods of exposure (1, 3, 7, and 15 days) are presented in Figs. 3(b) and (c). The percentage induction of the TBARS content increased continuously corresponding to the length of exposure for most concentrations, while GST activities did not exhibit good time-response relationships.

Fig. 2 Heptic GSH content, GST activity, and TBARS content after 1, 3, 7, and 15 days of exposure to binary mixture of BkF and DDT

Fig. 3 Heptic GSH content, GST activity, and TBARS content after 1, 3, 7, and 15 days of exposure to ternary mixture of BkF, DDT, and PCB118

4 Discussion

In this study, a significant decrease (p＜ 0.05) in hepatic GSH levels was found inCarassius auratusexposed to BkF (alone, in a binary mixture with DDT, and in a ternary mixture with DDT and PCB118). The reason may lie in two aspects: (1) The significant reduction in GSH levels was possibly due to the conjugation of pollutants with GSH. During the decline of GSH levels, induction of GST activities was observed, which confirmed the catalytic action of GST in the conjugation of pollutants with GSH. (2) GSH acts as a cellular reducing reagent and protects against the changes of cellular redox cycling induced by toxic substances. From the time-response charts of BkF, a slight reduction in hepatic GSH content inhibition was noted after three days of exposure to all concentrations of pollutants. Subsequently, an obvious increase in inhibition was found. A similar decrease has also been reported inCarassius auratuswhen they were exposed to 0.1 mg/L of 2,4-dichlorophenol (2,4-DCP) for 2, 5, 10, 20 and 40 days (Zhang et al. 2005). GSH is the major cytosolic low-molecular weight sulfhydryl compound that acts as a cellular reducing reagent and a protective reagent against numerous pollutants. Hence, GSH is often the first line of defense against oxidative stress. When the fish cells make contact with some pollutants, such as alachlor, they remove them by conjugation with GSH directly or by means of GST, which decreases the GSH levels.

The concentration-response charts for GSH inhibition did not show obvious characteristics. The inhibitory effects of binary mixtures on the GSH content were greater than those of the corresponding individual pollutant in most cases, suggesting that BkF combined with DDT may induce a partial additive effect of sublethal toxicity on fish. As shown by comparison of Fig. 3(a) with Fig. 2(a), the ternary mixture significantly inhibited the GSH content at all exposure concentrations, while the inhibition level decreased more significantly than that in binary mixtures. The presence of PCB118 seemed to defer the inhibitory effects of the original binary mixture. A change in GSH levels may be a very important indicator of the detoxification ability of an organism. Variations in GSH ofCarassius auratusexposed to a variety of contaminants have been observed, and the responses varied for different species of fish and contaminants (Lu et al. 2010; Luo et al. 2005). A decrease in hepatic GSH content was found inCarassius auratusexposed to 2,4-dichlorophenol for 40 days (Zhang et al. 2004).

In this study, hepatic GST activities in fish exposed to BkF in binary mixtures with DDT and in ternary mixtures with DDT and PCB118 increased obviously in treated groups at higherconcentrations compared with the control group (p＜ 0.05), and GST variation was associated with contaminant levels. Thus, the kinetics of the GSH content and GST activities indicated that the oxidative stress occurred in fish. GST is involved in the biotransformation of several pollutants. Therefore, induction of GST activities has been widely used as an environmental biomarker. Many studies analyzing GST in livers of fish exposed to different insecticides showed enzymatic induction. The increase of GST activities was probably due to a metabolic adaptation ofCarassius auratusexposed to high concentrations of contaminants and as a defense against oxidative damages. The susceptibility of fish to chemical carcinogenesis may be modulated by GST activity (Sun et al. 2006). Hepatic GST activities induced by BkF exhibited bell-shaped concentration-response charts after seven days of exposure and exhibited a good concentration-response relationship in the fish exposed to binary and ternary mixtures. Bell-shaped charts have been reported for various in vitro and in vivo systems after exposure to persistent organic pollutants (Lu et al. 2009; Bosveld et al. 2002). Frasco and Guilhermino (2002) observed a dose-dependent GST effect under exposure ofPoecilia reticulatato dimethoate. In addition, GST detoxifies a number of environmental carcinogens and epoxide intermediates (Wang et al. 2009). However, induction of GST activity was not enhanced by both mixtures in livers.

In this study, the data indicated that hepatic TBARS contents increased in treated groups exposed to BkF (alone, in a binary mixture with DDT, and in a ternary mixture with DDT and PCB118) compared with the control group, which suggested elevation of hepatic lipid peroxidation. In the treated groups exposed to contaminants alone and mixtures, the TBARS content was significantly induced after three days of exposure and showed a gradually increasing trend. The TBARS content reached a maximal value after 15 days of exposure. Time dependence of the TBARS content in livers was apparent except at lower concentrations after seven days of exposure. One of the most damaging effects of these free radicals and their products in cells is the peroxidation of membrane lipids, of which TBARS is an indicator. Detailed studies have provided evidence that many species exhibit increased TBARS following stress produced by some xenobiotics (Shalata and Tal 1998; Heise et al. 2007).

Lipid peroxidation has been suggested as one of the molecular mechanisms involved in pesticide-induced toxicity (Banerjee et al. 1999). A similar change pattern of TBARS in livers was also noted after different doses of 2,4,6-trichlorophenol were injected intraperitoneally into the fish (Carassius auratus) (Li et al. 2007). However, the TBARS content did not show a significant dose-dependent pattern, probably due to the effect of antioxidant system protection. The ternary mixture of higher concentrations significantly induced TBARS contents. However, the inhibition level did not show a significant difference from that in the binary mixture. The induction effects of mixtures on TBARS contents were not shown to be higher than that of the corresponding individual pollutant in most cases, as through comparison of Fig. 3(c) with Fig. 2(c) and Fig. 1(c).

5 Conclusions

In summary, the results of this research indicate that persistent organic pollutants can alter the GSH and TBARS contents and GST activity in livers ofCarassius auratus. The data suggest that BkF (alone, in a binary mixture with DDT, and in a ternary mixture with DDT and PCB118) at sublethal concentrations can induce significant inhibition of hepatic GSH contents inCarassius auratus, while the concentration-response charts for GSH inhibition did not show obvious characteristics. In treated groups exposed to contaminants alone and mixtures, hepatic GST activities and TBARS contents showed significant induction. Hepatic GST activities induced by BkF exhibited bell-shaped concentration-response charts after seven days of exposure and exhibited a good concentration-response relationship in the fish exposed to binary and ternary mixtures. The TBARS content was significantly induced after three days of exposure and showed a gradually increasing trend.

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(Edited by Yun-li YU)

This work was supported by the Special Fund for Public Welfare Industry of Ministry of Water Resources of China (Grant No. 201001056), the Project of Jiangxi Provincial Department of Science and Technology (Grant No. 2010BSA20300), and the Project of Jiangxi Provincial Department of Education (Grants No. GJJ11246 and GJJ11636).

*Corresponding author (e-mail:jiyong@nit.edu.cn)

Received Mar. 3, 2011; accepted Oct. 9, 2011