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Pyrene-stimulated reactive oxygen species generation and oxidative damage in Carassius auratus a

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Ying Yin , Jun Jia , Hong Y. Guo , Liu Y. Yang , Xiao R. Wang & Yuan Y. Sun

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State Key Laboratory of Pollution Control and Resources Reuse, School of Environment, Nanjing University , Nanjing , China b

Department of Hydrosciences , Nanjing University , Nanjing , China Published online: 30 Oct 2013.

To cite this article: Ying Yin , Jun Jia , Hong Y. Guo , Liu Y. Yang , Xiao R. Wang & Yuan Y. Sun (2014) Pyrene-stimulated reactive oxygen species generation and oxidative damage in Carassius auratus , Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:2, 162-170, DOI: 10.1080/10934529.2013.838846 To link to this article: http://dx.doi.org/10.1080/10934529.2013.838846

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Journal of Environmental Science and Health, Part A (2014) 49, 162–170 C Taylor & Francis Group, LLC Copyright  ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2013.838846

Pyrene-stimulated reactive oxygen species generation and oxidative damage in Carassius auratus YING YIN1, JUN JIA1, HONG Y. GUO1, LIU Y. YANG1, XIAO R. WANG1 and YUAN Y. SUN2 1

State Key Laboratory of Pollution Control and Resources Reuse, School of Environment, Nanjing University, Nanjing, China Department of Hydrosciences, Nanjing University, Nanjing, China

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Laboratory experiments were carried out to understand the toxicology of pyrene in the goldfish Carassius auratus and investigate the potential oxidative stress induced by reactive oxygen species (ROS) in vivo in a time-dependent manner. Pyrene bioaccumulation, induction of reactive oxygen species and the consequent biochemical responses in the liver of the fish were examined. Fish were exposed to 0.05 mg/L pyrene for different periods. The pyrene concentration in fish liver was analyzed by high performance liquid chromatography (HPLC). Free radicals were detected by electron paramagnetic resonance (EPR). The activities of antioxidant enzymes, contents of nonenzymatic antioxidants and malondialdehyde (MDA) in fish liver were also determined. Results indicated that the pyrene concentrations in fish liver reached a maximum level on day 1, and then declined to a low steady state level over 7 days. The free radical significantly increased at 6 h and reached a maximum on day 2, while the superoxide dismutase (SOD) activity and MDA content were induced, and the reduced glutathione (GSH) content was inhibited by day 2. The catalase (CAT) and glutathione-S-transferase (GST) activities were significantly induced at 12 h. These results indicated that pyrene was rapidly bioaccumulated in fish resulting in redox cycling, and the production of free radical is an important mechanism of pyrene toxicity in C. auratus. The indicators of antioxidant system are sensitive and useful for the study of early biomarkers of pyrene exposure in fish. Keywords: Time-dependent, pyrene, free radical, early biomarker, oxidative damage.

Introduction Polycyclic aromatic hydrocarbons (PAHs) have received considerable attention for their mutagenesis and carcinogenesis activities. As a typical class of persistent organic pollutants, PAHs has been extensively studied with regard to their distribution and biological effects in the environment.[1,2] PAHs are widely distributed in aquatic systems, which consequently results in harmful effects toward aquatic animals such as fish that are directly exposed to the contaminated environments.[3] Therefore, studies concerning the fate and effects of PAHs in aquatic organisms are critical for assessing the ecological risk of these chemicals. High-molecular weight (HMW) PAHs (≥ four benzene rings) are less bioavailable and appear to be more resistant to biological degradation. Pyrene, a tetracyclic PAH, has been studied extensively over the last two decades. Because of its wide distribution in aquatic environments and as a major component in PAH mixtures, Address correspondence to Hong Yan Guo, State Key Laboratory of Pollution Control and Resources Reuse, School of Environment, Nanjing University, Nanjing 210023, China; E-mail: [email protected] or [email protected] Received April 1, 2013.

pyrene was chosen as a model HMW PAH to serve as an indicator of PAH pollution.[4] Free radical production and subsequent oxidative damage may be an important mechanism of toxicity in organisms exposed to xenobiotics.[5] Xenobiotics, sometimes termed “protertogens,” which are relatively nontoxic, can be enzymatically bioactivated to highly toxic, electrophilic, or free radical reactive intermediates.[6] If not detoxified, electrophilic reactive intermediates can bind covalently to embryonic cellular macromolecules, while free radical reactive intermediates can react directly or indirectly with molecular oxygen to form reactive oxygen species (ROS), such as superoxide anion (O•− 2 ), hydrogen peroxide • (H2 O2 ), and hydroxyl radicals ( OH).[7] Among the ROS, •OH exhibits the strongest oxidative activity. It is the most toxic radical known as it can nonspecifically oxidize all classes of biological macromolecules including lipids, proteins, and nucleic acids at virtually diffusion-limited rates.[8] •OH radicals produced in vivo have extremely short half-lives and are present at low concentrations, thus making detection difficult. The electron paramagnetic resonance (EPR) spin-trapping technique has proved to be the most direct method for the detection of short-lived reactive free radicals at low concentrations in biological systems. The highly reactive oxygen species

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Reactive oxygen species generation and oxidative damage in C. auratus can be trapped by spin trap agents to form highly stable adducts that can be detected by EPR spectroscopy.[9] The adverse effects of many chemicals on animals are related to their capacity for undergoing reactions to produce ROS and lipid peroxidation.[10] Antioxidant defense systems neutralize chemical reactive intermediates produced by endogenous pathways and/or xenobiotic metabolism. Antioxidant parameters, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione and ascorbate play a crucial role in scavenging ROS generated during aerobic metabolism. Their induction reflects a specific response to pollutants, and they have been proposed as biomarkers of contaminant-mediated oxidative stress in a variety of aquatic organisms.[11,12] Increasing evidence suggests that in vivo free radical formation has been cited as a contributing factor to the deleterious effects of many chemical pollutants.[13] Contaminant-stimulated ROS production and subsequent oxidative damage could be a mechanism of toxicity in aquatic organisms. Among the various possible biochemical responses, induction of the antioxidative defense system comprises one of the most widely accepted methods for detecting organic pollution in an aquatic environment.[14] PAHs enter into an organism, interact with the aromatic hydrocarbon receptor (AhR) and induce cytochrome P4501A, leading to production of ROS and oxidative stress.[15] The toxic effects of PAHs such as phenanthrene on aquatic organisms have been documented. Fish and submerged macrophytes are known to accumulate phenanthrene in their tissues, providing a concentration- dependent measurement of environmental contamination, with observable cellular and physiological responses.[16,17] However in order to fully evaluate the toxic mechanisms of PAHs, both the dose-effect relationship and the timeintegrated exposure should be taken into account. With an aim to better understand the toxicology of pyrene, the objective of this study was to investigate the potential oxidative stress induced by ROS in vivo in a timedependent manner. A secondary radical spin trapping technique was used to test the hypothesis that pyrene induced ROS production, which in turn could mediate oxidative stress and subsequent oxidative damage in the freshwater fish Carassius auratus. Lipid peroxidation was used as the indicator of oxidative damage. Analysis of antioxidant defense mechanisms, including SOD, CAT, GST, GSH and oxidized (GSSG) forms, were used to study their interactions with ROS. And study was conducted to measure the ROS and antioxidant levels as early biomarkers to access the stress.

Materials and methods Fish collection and treatment Fish (C. auratus) were purchased from a local aquatic breeding base, with a mean body length and weight of

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about 16.0 cm and 28.2 g. All fish were acclimated to water dechlorinated with activated carbon for 10 days before the experiment. Artificial dry food was provided once a day. During the experiment, the water pH was 7.0 ± 0.3, the temperature was 25 ± 2◦ C and the dissolved oxygen levels were kept higher than 5 mg/L by continuous aeration. Fish (n = 8 for each group) were randomly exposed to 0.05 mg/L pyrene in a 35 L glass aquaria for 3 h, 6 h, 12 h, 1 d, 2 d, 4 d, 7 d, 14 d, and 21 d, while one control group was designated for each exposure group. During the experiment, 50% of the water was replaced daily by fresh pyrene solution to minimize contamination from metabolic waste. Fish were taken out for parallel sampling after every period of exposure. Fish samples were dissected and fresh livers were obtained for EPR. The rest of the livers were homogenized at 4◦ C for other experiments. Determination of pyrene Homogenized with anhydrous Na2 SO4 , the liver samples were extracted three times (20 min each) by an ultrasonic wave method with acetone/hexane (3/2; v/v). All the extracted solutions were combined. The combined supernatants were concentrated to about 1 mL by rotary evaporator, and then cleaned up with dichloromethane using an anhydrous Na2 SO4 /Florisil column. The elution was evaporated gently and the obtained dry residue was dissolved in methanol for HPLC analysis. The level of pyrene in methanol was analyzed by a Hewlett Packard (HP) 1100 HPLC with a HP DAD detector (Agilent, Waldbronn, Germany) by monitoring the absorption wavelength at 238 nm, using a mobile phase of methanol/ water (85:15; v/v). The column was a Zorbax Eclipse XDB-C8 (4.6 mm i.d × 150 mm length, Palo Alto, CA, USA). Procedure blanks, reagent blanks and spiked blanks were carried out during the analyses. The recovery obtained by spiking liver samples with pyrene was 88.2 ± 2.01% (n = 4) for the entire procedure. Pyrene was not detected in the control samples. PBN adduct extraction and EPR analysis PBN adduct extraction was performed according to Luo et al.[13] The entire operation was conducted in an incubation system with continuous purging of N2 . After rinsing with ice-cold physiological salt water, the fish livers were immediately weighed and a fraction (0.1 g) was removed and homogenized quickly in 1.0 mL 50 mM PBN (dissolved in DMSO) using a Teflon pestle in a Potter homogenizer (Nanjing Sunshine Biotechnology, Ltd., Nanjing, China). Then 0.1 mL supernatants was transferred to a capillary tube with a diameter of 0.9 mm, and frozen in liquid nitrogen for EPR analysis. The EPR spectra were recorded with a Bruker EMX 10/12 X-band spectrometer (Bruker, Karlsruhe, Germany) at room temperature. The operation conditions were: center field, 3470 G; scan range, 200 G;

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modulation frequency, 100 kHz; modulation amplitude, 0.5 G; receiver gain, 5 × 104 scans, 5 times; microwave power, 20 mW. The central peak of the EPR signals was used to calculate the intensity of the ROS.

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Determination of glutathione levels and enzymatic activities About 0.10 g of liver tissue was homogenized at a 1/10 (w/v) ratio in 10.0 mM ice-cold Tris–HCl buffer (pH 7.5) for the enzyme assays. About 0.10 g of liver tissue was homogenized after the addition of 1.0 mL of 1.0 mM EDTA and 10 µL diluted HClO4 (4000-fold dilution) for measurement of GSH and GSSG levels. The homogenates were centrifuged at 12,000 × g for 10 min at 4◦ C. Aliquots of the supernatants obtained after centrifugation were used for the enzyme assays. All the above operations were carried out at a temperature below 4◦ C. SOD activity was assayed according to Marklund and Marklund.[18] One unit of enzyme activity was defined as the amount of the enzyme required to cause 50% inhibition of the auto-oxidation rate of 0.1 mM pyrogallol in 1 mL of solution at 25◦ C. CAT activity was assayed according to Vasylkiv et al.[19] One unit of enzyme activity was defined as the amount of the enzyme that decreased the concentration of H2 O2 by half after 100 s at 25◦ C. GST activity was assayed according to Habig et al.[20] The enzyme activity was calculated as the amount (µmol) of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate formed/min/mg of protein using a molar extinction coefficient of 9.6 mM−1cm−1. GSH and GSSG levels were determined fluorometrically according to the method of Hissin and Hilf,[21] and the fluorescence intensity was recorded at 420 nm after excitation at 350 nm on a Hitachi (Tokyo, Japan) fluorescence spectrophotometer. Protein levels were measured by the method of Bradford [22] using bovine serum albumin (BSA) as the standard. Determination of MDA MDA content was determined according to Ohkawa et al.[23] The amount of MDA formed was calculated by measuring the absorbance at 532 nm using a molar extinction coefficient of 1.56 × 105 M−1 cm−1. Statistical analysis Data were expressed as the means ± standard deviation (SD). Significant differences (P < 0.05) were determined by the Student’s t-test using Microsoft Excel 2003 (Microsoft, Redmond, WA, USA). Graphs of free radicals were prepared using Origin 7.5 (OriginLab, North Hampton, MA, USA).

Results Bioaccumulation of pyrene in the liver of fish The accumulation of pyrene in fish livers during the uptake period is shown in Figure 1. Typically, the concentrations

Fig. 1. Concentrations of pyrene in the liver of C. auratus during the exposure period.

of pyrene in fish liver increased rapidly shortly after the start of the exposure, and reached a maximum level after approximately 1 day. The levels then decreased until they stabilized after 7 days. Hydroxyl radical production during exposure to pyrene The EPR spectrums of fish hepatic hydroxyl radical trapped by PBN in nitrogen after pyrene exposure are shown in Figure 2a. The EPR spectrum exhibited 3 × 2 lines after PBN spin trapping. The six-line EPR spectra were composed of three groups, with two hyperfine splitting peaks in each. The hyperfine splitting constants for the PBN-radical adduct were aN = 14.9 G, aH = 3.4 G and g = 2.0057. This is consistent with the characteristics of PBN/•OH from the literature.[13] Thus, the EPR investigation showed that the free radical generated after pyrene exposure in freshwater fish was the hydroxyl radical (•OH). • OH production was indicated by the difference between the signal intensity of the second hyperfine splitting peaks in the EPR spectra of PBN radical adducts. The signal intensity of the •OH increased rapidly as the exposure to pyrene increased, and was significantly different at 6 h, and reached a maximum level after 2 days (Fig. 2b). After that, it decreased slowly until the end of the measured exposure period. Activities of antioxidant defense enzymes and glutathione content The hepatic antioxidant enzyme responses of fish to pyrene exposure are reported in Table 1. The activity of SOD at the early exposure periods did not reveal a significant difference from that in the control group. After 2 days of exposure, the activity of SOD was significantly induced and reached a maximum level, and then began to decrease until day 21, the last day of measurement. Compared with the control group, CAT activity was significantly increased at 12 h after

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Reactive oxygen species generation and oxidative damage in C. auratus

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Fig. 3. MDA content in the liver of C. auratus exposed to pyrene (n = 6). (Significantly different from the control, P < 0.05.)

MDA content Changes in MDA content are shown in Figure 3. Compared with the control group, the MDA content was slightly enhanced shortly after the start of the pyrene exposure, then became significantly different at day 2 and day 14, and maintained at constant level from 4 days onward.

Discussion Fig. 2. (a) EPR spectra of the hydroxyl radical detected in the liver of C. auratus. (b) Hydroxyl radical signal intensity in the liver of C. auratus during the exposure period (n = 3). ∗ Significantly different from the control, P < 0.05.

pyrene treatment. After 1 day of exposure, CAT activity remained depressed for the measured period. GST activity exhibited a pattern similar to that of CAT, with a maximum activity at 12 h. From day 1 onwards, GST activity began to decrease and reached the lowest value on day 21 (P < 0.05). Pyrene exposure significantly inhibited the GSH content (P < 0.05) throughout the experiment period (Table 2). GSH content was significantly different from that in the control group at 6 h. After 2 days of exposure, the GSH content reached its minimum level, and then it increased for the remainder of the measurement period. GSSG content at early exposure periods was elevated slightly, but revealed a significant difference from that in the control group 1 day after exposure, and reached a maximum level at 2 days, and then declined quickly. The trend of the GSH/GSSG ratio was similar to the GSH content; when the exposure time of pyrene was 6 h, the GSH/GSSG ratio decreased significantly and reached the minimum at day 2 (P < 0.05).

Because of their low water solubility and strong lipophilic character, PAHs accumulate in the environment and are preferably concentrated in aquatic organisms.[24] The accumulation is a result of an overall combination of uptake, metabolism and excretory processes. The liver is a major site of PAH metabolism, and exposure experiments with PAHs have shown that their concentrations in liver are approximately 100 times higher than that in muscle.[25] The fish liver is a main target organ for toxic mechanism and biomarker studies because of its central role in detoxification processes.[26] In the present study, results showed that pyrene could be rapidly accumulated in fish liver shortly after the start of the exposure and reached the maximum level at 1 day of exposure after which a decrease of pyrene concentrations in fish liver occurred (Fig. 1). These findings were consistent with previous studies that had measured PAHs bioaccumulation in aquatic organisms. Baussant et al.[27] reported that concentrations of PAHs in juvenile turbot exposed to dispersed oil increased rapidly at the beginning, reached the maximum level after approximately 3 days, and then declined quickly to low steady state levels. Sun et al.[16] showed that crucian carp rapidly accumulated phenanthrene into its tissue after 2 days. In this study, pyrene was accumulated in fish tissue faster than phenanthrene because of its higher lipophilicity (logKow = 4.88). After 1 day of exposure, the

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166 decrease of pyrene concentration in the liver might be the result of metabolism and excretion. Alteration in the accumulation of toxic compounds in the liver of fish has long been of interest as an indicator of contaminant stress. PAHs are known to enter into an organism, interact with the aromatic hydrocarbon receptor (AhR) and induce cytochrome P4501A, leading to enhanced free radical production.[5] The EPR method can be used for estimating the type and content of most stable free radicals which accumulate in tissue during periods of oxidative stress. EPR spectroscopy allows both quantification and characterization of free radicals. O•− 2 , produced by xanthine oxidase, tryptophan dioxygenase, diamine oxidase and activated neutrophils, can be a source of additional harmful ROS. O•− 2 is a toxic by-product of oxidative metabolism and can interact with H2 O2 to form the highly reactive •OH radical, which is thought to be primarily responsible for oxygen toxicity in the cell.[28] Previous work suggested that PAHs could induce production of ROS such • as O•− 2 and OH in the liver of mice and fish. This is supported by data which indicate that the formation of •OH follows similar trends as the bioaccumulation of pollutants with changes in the exposure concentration.[29] In this study, using spin trapping by EPR spectroscopy, we have clearly demonstrated the ability of pyrene to stimulate the production of •OH in the liver of C. auratus, as •OH increased significantly after 6 h of exposure, increasing sharply by 1 day and reaching a maximum by 2 days (Fig. 2b). In contrast to the peak pyrene accumulation time, the maximum •OH concentrations experienced a lag. The control group showed a weaker •OH signal in the EPR spectra, which could be explained by •OH formation during normal cellular functions, a process that may be increased by the presence of pyrene. Luo et al.[13] indicated that •OH was significantly induced by 0.001 mg/L of pentachlorophenol exposure, and reached a maximum by day 7. A strong positive correlation was observed between the pentachlorophenol liver concentration and •OH intensities within 7 days. Some studies show that ROS has been employed as a potential biomarker to indicate the risk associated with pollutants in aquatic ecosystems.[30] Our current study demonstrated that the production of •OH under conditions of pyrene stress happened very quickly, thus •OH is a sensitive indicator of pyrene. Evidence of •OH formation in vivo by EPR analysis suggested possible mechanisms responsible for the generated oxidative stress induced by pyrene in freshwater fish. The deleterious effects associated with an increasing pyrene level in the liver have been confirmed by other parameters known to be markers of the antioxidant defense system. Under normal physiological conditions, the production of ROS and other oxygen reactive species are thought to be held in check by antioxidant defense systems, i.e., a balance exists between pro-oxidant and antioxidant processes. Antioxidant defenses include α-tocopherol, GSH and enzymes such as SOD, CAT, and GPx. An important fea-

Yin et al. ture of these enzymes and nonenzymatic antioxidants is their inducibility under oxidative stress. However, severe oxidative stress may suppress the activities of these enzymes, leading to a loss in compensatory mechanisms with a consequence of oxidative damage. As reported herein, the levels or activities of antioxidants are potential biomarkers of a contaminant-mediated biological effect on the organism.[12,31] The present results showed that the activities of SOD, CAT, GST and the content of GSH were affected during the 21-day exposure period compared to the control group. These changes also indicated a possible mechanism of oxidative stress induced by pyrene on fresh water fish. SOD and CAT protect organisms from oxidative damage by the removal of partially reduced oxygen species, while GST is involved in the detoxification of many xenobiotics and plays an important role in the protection of tissues from oxidative stress. SOD catalyzes the dismutation of O•− 2 to H2 O2 and O2 , and H2 O2 is converted to H2 O and O2 by CAT. Thus, the induction of SOD and CAT activities [29] suggests the involvement of O•− 2 and H2 O2 formation. •− O2 may invoke two pathways, formation of H2 O2 via the action of SOD and formation of •OH via the Haber-Weiss reaction in biological systems in the presence of H2 O2 .[32] The increased •OH production in this study may suggest the involvement of O•− 2 and H2 O2 . Of all the possible ROS generated in biological systems, • OH is the most reactive oxygen radical. In this study, when fish were exposed to 0.05 mg/L pyrene, it accumulated in tissue, resulting in an increase of O•− 2 and H2 O2 , and presumably the induction of SOD and CAT may follow. In the • presence of O•− 2 , H2 O2 could form the OH radical, resulting in an increase in ROS production, such that it ultimately exceeds the capabilities of SOD, and CAT, or GST elimination, so that eventually, the ROS can inhibit the enzyme resulting in its inactivation. SOD, CAT and GST activities were increased at first, and then decreased during the 21day exposure period. Induction of SOD activity reached a maximum at 2 days, when •OH production also reached a peak, while CAT and GST activities reached a maximum at 12 h. CAT and GST activities, unlike SOD, exhibited a significant decrease from day 1 onwards. It can be assumed that SOD was more tolerant than CAT and GST to pyrene concentrations in the liver of C. auratus. GST is known to play an important role in the biotransformation of pyrene by microalgal species.[33] Yin et al.[34] reported that GST was clearly increased in treated groups and sensitive to pyrene exposure in C. demersum. Similarly, it is likely that GST was sensitive to the pyrene exposure in the liver of C. auratus. Compared with SOD, GST activity was induced earlier and decreased faster. It has been proven that GSH is one of the most efficient scavengers of ROS arising as by-products of cellular metabolism or during oxidative stress.[35] When fish cells come into contact with pollutants such as pyrene, they remove them by conjugation with GSH directly or by the action of GST, which decreases GSH levels. The most

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5.79 ± 1.07 13.7 ± 3.72 394 ± 67.3

4.95 ± 0.94 11.0 ± 2.01 404 ± 55.9

SOD CAT GST

5.60 ± 1.57 12.6 ± 3.21 462 ± 105

6h

Data are expressed as mean ± sd, n = 6. a Significantly different from the control, P < 0.05.

3h

0

Group 6.39 ± 1.47 18.3 ± 4.09a 496 ± 26.8a

12 h 5.92 ± 2.06 11.5 ± 3.44 275 ± 97.1a

1d 7.68 ± 1.56a 8.22 ± 1.54a 322 ± 108

2d

Exposure times of pyrene

6.92 ± 1.68a 9.07 ± 2.87 230 ± 69.0a

4d

Table 1. Antioxidant defense enzyme activities in the livers of C. auratus after exposure to pyrene (U/mg Pr).

7d 5.81 ± 1.53 5.61 ± 0.95a 259 ± 61.0a

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4.83 ± 1.17 8.75 ± 3.17 179 ± 32.0a

14 d

4.73 ± 0.28 8.36 ± 1.77 83.7 ± 22.0a

21 d

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436 ± 40.2 386 ± 38.8 1.14 ± 0.21

0

475 ± 4.11 345 ± 6.78 1.38 ± 0.04

Data are expressed as mean ± sd, n = 4. a Significantly different from the control, P < 0.05.

GSH GSSG GSH/GSSG

Group 423 ± 35.9a 361 ± 23.0 1.18 ± 0.11a

6h 442 ± 31.9 370 ± 26.7 1.20 ± 0.17

12 h 400 ± 6.78a 376 ± 9.69a 1.06 ± 0.03a

1d 285 ± 27.8a 446 ± 8.90a 0.63 ± 0.07a

2d

Exposure times of pyrene

354 ± 43.9a 373 ± 16.4a 0.95 ± 0.08a

4d

Table 2. Changes in GSH and GSSG content in the livers of C. auratus after exposure to pyrene (µg g−1 fresh weight).

7d 352 ± 35.7a 348 ± 23.3 1.02 ± 0.15a

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483 ± 26.6 366 ± 14.0a 1.32 ± 0.09

14 d

460 ± 6.10a 371 ± 13.2a 1.24 ± 0.05a

21d

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Reactive oxygen species generation and oxidative damage in C. auratus obvious direct effect of certain pollutants is a decrease in thiol status, i.e., the ratio of reduced to oxidized glutathione (GSH/GSSG), due to either direct radical scavenging or increased peroxidase activity. In the present study, pyrene exposure resulted in a significant decrease in the GSH level compared with the control. GSSG levels changed along with GSH levels, which indicated there was a transformation from GSH to GSSG under conditions of oxidative stress. The generation of GSSG is generally higher than the reduction back to GSH, as GSSG is accumulated and translocated outside of the cell by specific transporters to avoid NADPH exhaustion.[36] Sun et al.[29] noted that a decrease in hepatic GSH levels was found in fish after exposure to different concentration of pyrene, and the reduction of GSH was due to the conjugation of pyrene or metabolite with GSH. Van der Oost et al.[37] demonstrated that the GSH and GSSG ratio may be a potential biomarker for oxidative stress in a number of laboratory and field studies. Previous work reported that the GSH/GSSG ratio in the cell correlated well with the corresponding exposure concentration of pollutants and is a good indicator of the level of oxidative stress.[17,38] Our results indicated that both the GSH and GSH/GSSG ratio were sensitive to pyrene exposure and suitable as indicators of pyrene exposure. Lipid peroxidation is considered to be one of the key events in the process of oxidative damage. MDA is the final product of lipid peroxidation and a diagnostic index of oxidative injury in cells. Detailed studies have provided evidence that many species exhibit an increased MDA following stress produced by some xenobiotics.[10,39] A strong positive correlation between •OH and MDA content suggested hydroxyl-triggered lipid peroxidation takes place in fish.[13] In the present study, the increase of MDA content proved that lipid peroxidation in fish liver was promoted (Fig. 3). MDA content significantly increased at 2 days when the • OH production and SOD activity also reached a peak, while the GSH content reached a minimum. The MDA content indicated the prevalence of free radical reactions in tissues suggesting that pyrene-induced membrane lipid peroxidation could be attributed to the decreases in CAT, GST activities and GSH content. These decreased enzyme activities and antioxidants favored accumulation of ROS, which could result in lipid peroxidation. The longer the pyrene exposure, the more likely it is that the toxic effects on the organism are enhanced. Our earlier studies showed that pyrene exposure induced the production of a large number of free radicals and led to oxidative damage of tissues of plants and fish, and the parameters of antioxidant defense mechanisms and lipid peroxidation correlated with the pyrene concentration.[29,34] In the present study, we found that the exposure time to pyrene had a significant influence on the response characteristics of oxidative damage. It was clearly demonstrated that pyrene could accumulate and be metabolized in fish

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and induce hydroxyl radical production, leading to liver oxidative stress. The SOD, CAT and GST activities and GSH and GSSG content were affected during the 21-day exposure period compared to the control group. The second day of exposure time was an important time point, when the • OH production, SOD activity, and GSH and GSSG content both had maximal values, after which the SOD, GSH and GSSG levels returned to those of the control. This suggested that the antioxidant defense mechanisms can be induced by oxidative stress because of a compensatory response; however, severe oxidative stress suppresses the activities of these enzymes because of the oxidative damage and a loss in compensatory pathways (such as CAT and GST).

Conclusions The mechanisms of the toxic effects of pyrene originated from its activation of redox cycling and as a result of its promotion of ROS formation and oxidative stress in vivo, the assumption of free radical damage by pyrene has been confirmed. Further studies are needed to determine the mechanisms of accumulation, toxicity, stress resistance and the production of other free radicals in aquatic organisms upon exposure to pyrene. In addition, many factors have not been considered in this experiment, such as those that fish face in a nonlaboratory environment that will play a part in the response of the antioxidant defense system. However, further study is performed to use these sensitive parameters for the study of early warning of pyrene exposure.

Acknowledgments This work was supported by the National Natural Science Foundation of China (20907020) and the Public Welfare Project of the Yellow River (201001010).

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Pyrene-stimulated reactive oxygen species generation and oxidative damage in Carassius auratus.

Laboratory experiments were carried out to understand the toxicology of pyrene in the goldfish Carassius auratus and investigate the potential oxidati...
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