Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout, Oncorhynchus mykiss

Chemico-Biological Interactions 185 (2010) 241–246

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Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout, Oncorhynchus mykiss Zhi-Hua Li a,b,∗ , Vladimir Zlabek a , Roman Grabic a,c , Ping Li a,b , Jana Machova a , Josef Velisek a , Tomas Randak a a University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic b Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou 434000, China c Department of Chemistry, Umea University, SE-90187 Umea, Sweden

a r t i c l e

i n f o

Article history: Received 15 January 2010 Received in revised form 19 February 2010 Accepted 22 February 2010 Available online 1 March 2010 Keywords: Fish intestine Residual pesticide Digestive enzyme Oxidative stress RNA/DNA ratio Na+ -K+ -ATPase

a b s t r a c t The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 ␮g l−1 ) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na+ -K+ -ATPase). Exposure to 500 ␮g l−1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 ␮g l−1 ). At the highest concentration (500 ␮g l−1 ), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na+ -K+ -ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 ␮g l−1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Toxicological and environmental problems resulting from the widespread use of pesticides in agriculture have raised concerns, particularly with respect to the potential toxic effects in man and animals [1]. Propiconazole (PCZ) is a triazole fungicide used to slow or stop the growth of fungus infecting agricultural fields. Although the triazole fungicides have shorter half-lives and lower bioaccumulation than the organochlorine pesticides, possible detrimental effects on the aquatic ecosystem may arise from spray drift or surface run-off after rainfall [2].

Abbreviations: PCZ, propiconazole; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; LPO, lipid peroxidation; TBARS, thiobarbituric acid-reactive substances; CP, carbonyl protein; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; PCA, principal component analysis. ∗ Corresponding author at: University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic. Tel.: +420 387 774 622; fax: +420 387 774 634. E-mail address: [email protected] (Z.-H. Li).

The physiological and biochemical changes in fish exposed to contaminants are complex, and although liver, gill and brain are generally the targets in aquatic toxicological studies [3–5], it is important to identify responses in other organs. Intestine is an important organ for digestion and absorption, although it is not mainly responsible for detoxification [6]. Nonetheless, information on the effects of residual fungicides on intestinal function is largely lacking. The enzymatic systems, including digestive enzymes, energy metabolic enzymes, and antioxidant enzymes, are sensitive to environmental stress [7,8]. Consequently, changes in their activity are used as biomarkers for monitoring environmental pollutants. Furthermore, pollutants that interfere with energy-yielding reactions indirectly inhibit the synthesis of RNA, DNA, and protein [9]. Hence, the RNA to DNA ratio not only provides a measure of the synthetic capacity of the cell, but also could be a tool for revealing environmental stress [10,11]. The objectives of this study were to investigate the effects of the fungicide PCZ on intestinal enzyme activity and the RNA/DNA ratio in rainbow trout, Oncorhynchus mykiss, by analyzing digestive enzymes (proteolytic enzymes and amylase), energy metabolic enzymes (Na+ -K+ -ATPase), and antioxidant enzymes (SOD, CAT, GPx, GR), as well as the oxidative stress indices (TBARS and CP).

0009-2797/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.02.040

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2. Materials and methods 2.1. Chemicals Propiconazole and other chemicals were purchased from SigmaAldrich Corporation (USA). The PCZ was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at a concentration of 100 mg ml−1 . 2.2. Fish Rainbow trout (n = 110), weighing 292.83 ± 45.70 g (mean ± S.D.), were obtained from a local commercial hatchery (Husinec, Czech Republic). They were held in aquaria containing 250 l of freshwater continuously aerated to maintain dissolved oxygen values at 7.5–8.0 mg l−1 . Temperature was 15 ± 1 ◦ C, and pH was 7.4 ± 0.2. The photoperiod was 12:12 h light–dark. Fish were acclimatized for 14 days before the beginning of the experiment and fed daily at a fixed time with commercial fish pellets at 1% total body weight; uneaten food was removed. Fish were starved for 24 h prior to experimentation to avoid prandial effects during the assay. 2.3. Exposure to PCZ A 200 l semi-static system was used in which 11 rainbow trout were randomly placed in each of ten aquaria. The nominal concentrations of PCZ used were 0.2 ␮g l−1 (group E1, approximating to environmental concentration), 50 ␮g l−1 (group E2), and 500 ␮g l−1 (group E3). Propiconazole was dissolved in DMSO with a final concentration less than 0.01%. Two additional groups were used as controls: a group exposed to plain freshwater and a DMSO group exposed to the volume of DMSO (v/v, 0.01%) used for the highest PCZ concentration. Fish were exposed to PCZ for 30 days. Each experiment was duplicated. Eighty percent of the PCZ solution was replaced each day after 2 h of feeding, to maintain the appropriate concentration of PCZ and DMSO and to maintain water quality. The test equipment was cleaned every 7 days. To ensure agreement between nominal and actual compound concentrations in the aquaria, water samples were analyzed during the experimental period by LC–MS/MS. Water samples were collected from the test aquaria 1 h and 24 h after renewing the solutions. The mean concentration of PCZ in the water samples was always within 20% of the intended concentration.

Amylase (EC 3.2.1.1) activity was assayed with 2% (w/v) starch solution as substrate [13]. Activity was determined from the maltose standard curve and expressed as moles of maltose released from starch/min/mg protein at 37 ◦ C. 2.6. Antioxidant responses The TBARS method described by Lushchak et al. [14] was used to evaluate branchial LPO. The concentration of TBARS was calculated from the absorption at 535 nm and a molar extinction coefficient of 156 mM cm−1 . The value was expressed as nM of TBARS per g wet weight tissue. Carbonyl derivatives of proteins were detected by reaction with DNPH according to the method of Lenz et al. [15]. The amount of CP was measured spectrophotometrically at 370 nm using a molar extinction coefficient of 22 mM cm−1 . The values were expressed as nM of CP per g of wet weight tissue. Total superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by the method of Marklund and Marklund [16]. This assay depends on the autoxidation of pyrogallol. Superoxide dismutase activity was assessed spectrophotometrically at 420 nm and expressed as the amount of enzyme per mg of protein. The catalase (CAT; EC 1.11.1.6) activity assay, using the spectrophotometric measurement of H2 O2 breakdown, measured at 240 nm, was performed following the method of Beers and Sizer [17]. Glutathione peroxidase (GPx; EC 1.11.1.9) activity was assayed following the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase. The specific activity was determined using the extinction coefficient of 6.22 mM cm−1 [18]. Glutathione reductase (GR; EC 1.6.4.2) activity was determined spectrophotometrically, measuring NADPH oxidation at 340 nm [19]. 2.7. RNA/DNA ratio and Na+ -K+ -ATPase Free nucleotides were removed using a series of washes with cold perchloric acid (HClO4 ). RNA was then hydrolyzed with potassium hydroxide and the hydrolysate was acidified with cold HClO4 to remove the RNA from the DNA and protein. Intestine RNA/DNA ratio was measured using the method of Kuropat et al. [20] as modified by Mercaldo-Allen et al. [21]. Na+ -K+ -ATPase activity was measured by liberating PO4 from a hydrolysis reaction with ATPase, as described previously [22]. The ATPase activity was expressed as ␮mol Pi liberated/mg protein/h. 2.8. Protein estimation

2.4. Tissue samples At the end of the exposure period, all the fish were killed and the intestine of each fish was quickly removed, immediately frozen and stored at −80 ◦ C until analysis. Frozen tissue samples were weighed and divided into three sets for all the assays performed: the first set for measuring digestive enzymes and RNA/DNA ratio, the second set for Na+ -K+ -ATPase study, and the third set for antioxidant response assay. 2.5. Digestive enzymes Proteolytic enzyme activity was determined by the casein digestion method of Kunitz [12] as modified by Debnath et al. [13]. The reaction mixture consisted of 1% casein as substrate, phosphate buffer (pH 7.5), and homogenate which was incubated at 37 ◦ C for 20 min. A reagent blank was prepared by adding tissue homogenate just before stopping the reaction without incubation. Tyrosine was used as standard; one unit of enzyme activity was expressed as moles of tyrosine released/min/mg protein at 37 ◦ C.

Protein levels were estimated spectrophotometrically by the method of Bradford [23] using bovine serum albumin as a standard. 2.9. Statistical analysis All values were expressed as mean ± S.D. and analyzed by SPSS for Win 13.0 software. One-way ANOVA with Tukey’s test was used to determine whether results of treatments were significantly different from the control group (p < 0.05). To get a general picture of the trends and groupings in the material, the data from individual fish were subjected to multivariate data analyses (PCA method) using the software Statistic 6.0. 3. Results 3.1. Digestive enzymes Changes in activity of two digestive enzymes in the intestine are shown in Fig. 1. A slight but non-significant decrease in amylase

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Fig. 1. Effect of 30 days exposure to PCZ on amylase and proteolytic enzyme activity in rainbow trout intestine. Data are means ± S.D., n = 6. Significant differences compared with control value * p < 0.05, ** p < 0.01.

Fig. 2. Effect of 30 days exposure to PCZ on Na+ -K+ -ATPase enzyme activity and RNA/DNA ratio in rainbow trout intestine. Data are means ± S.D., n = 6. Significant differences compared with control value * p < 0.05, ** p < 0.01.

activity was observed in PCZ treated groups at the lower concentrations (0.95-fold in E1 and 0.90-fold in E2) (p > 0.05). However, amylase activity was significantly inhibited (p < 0.01) at the highest PCZ concentration (0.62-fold in E3). Proteolytic enzyme activity was also significantly inhibited in E2 (0.70-fold, p < 0.05) and E3 (0.51-fold, p < 0.01).

3.3. RNA/DNA ratio and Na+ -K+ -ATPase Alterations in intestine RNA/DNA ratio and Na+ -K+ -ATPase activity are shown in Fig. 2. Compared to controls, RNA/DNA ratio was significantly depressed (p < 0.05) in E2 (0.71-fold, p < 0.05) and E3 (0.60-fold, p < 0.01). Changes of Na+ -K+ -ATPase for PCZ treated groups with lower concentration (0.93-fold in E1 and 0.87-fold in E2) were inhibited slightly (p > 0.05). Compared with the controls, Na+ -K+ -ATPase in E3 was significantly inhibited (0.63-fold, p < 0.01) and exhibited the lowest value.

3.2. Antioxidant enzymes The oxidative stress and antioxidant responses in intestine are shown in Table 1. Although there was no significant (p > 0.05) induction of LPO formation at lower concentrations of PCZ (0.98-fold in E1 and 1.12-fold in E2), a significantly higher (1.35-fold, p < 0.01) LPO level was observed in the E3 after 30 days exposure, compared to both controls. There was no significant change (0.97-fold, p > 0.05) in CP level in E1 following exposure. However, compared with the control groups, CP level was significantly induced in E2 (1.15-fold, p < 0.05) and E3 (0.31-fold, p < 0.01). Superoxide dismutase activity was significantly induced (p < 0.05 or p < 0.01) with low PCZ concentrations (1.24-fold in E1 and 1.37-fold in E2). With increasing PCZ concentration, SOD activity was inhibited slightly in E3 (0.93-fold, p > 0.05) compared with the control. The CAT activity at low concentration was slightly increased (1.07-fold in E1, p > 0.05) relative to the control groups. At higher concentration (E2), CAT activity was significantly induced (1.27-fold, p < 0.05), but significantly inhibited (0.81-fold, p < 0.05) in E3 compared with the controls. The activity of GR was slightly inhibited (0.85-fold, p > 0.05) in E1, but not significantly. With exposure concentration increasing, GR activities returned to control level (1.02-fold in E2 and 0.92-fold in E3). The GPx activity was induced in E2 compared to controls, but not significantly (1.14fold, p > 0.05); however, a significant inhibition of GPx activity was observed in E3 (0.81-fold, p < 0.05).

3.4. Chemometrics Based on the bilinear decomposition of the original data, PCA was used to transform a multivariate data array into a new data set, in which the new variables are orthonormal and explain the maximum variance. In the present study, a data matrix including 10 analyzed biomarkers was constructed as independent variables and 30 sampled individuals as grouping variables. All the parameters measured were distinguished on the ordination plots corresponding to the first (59.70%) and second (26.69%) principle components (Fig. 3). This showed the relationship of all the biomarkers: (a) LPO (expressed as TBARS level) was positively correlated with CP; (b) digestive enzymes (proteolytic enzymes and amylase) and energy metabolic parameters (RNA/DNA ratio, Na+ -K+ -ATPase) were positively correlated; (c) the correlation among all antioxidant enzymes (SOD, CAT, GR and GPx) was positive; (d) oxidative stress indices were negatively correlated with digestive enzymes and metabolic parameters. Based on the parameters analyzed, three groups with 86.36% of the total accumulated variance were distinguished (Fig. 4). Individuals in the same area had similar biochemical responses. Group A comprised the control and DMSO groups and E1. Groups with

Table 1 Effect of PCZ on antioxidant responses in rainbow trout intestine. Indices

Units

TBARS CP SOD CAT GR GPx

nM/gww nM/gww U/mg protein U/mg protein mU/mg protein mU/mg protein

Test groups Control 4.71 60.29 11.26 1.62 4.18 35.74

± ± ± ± ± ±

DMSO 0.52 3.78 1.14 0.24 0.47 3.76

4.28 63.81 10.67 1.58 3.71 39.27

Data are means ± S.D., n = 6. Significant differences compared with control value. * p < 0.05. ** p < 0.01.

E1 ± ± ± ± ± ±

0.29 5.84 1.95 0.40 0.56 3.52

4.62 58.33 13.93 1.73 3.46 37.14

E2 ± ± ± ± ± ±

0.33 4.29 1.04* 0.21 0.44 4.27

5.29 69.14 14.41 2.05 4.27 41.48

E3 ± ± ± ± ± ±

0.61 4.01* 1.88* 0.37* 0.61 4.85

6.37 71.26 10.47 1.36 3.85 29.38

± ± ± ± ± ±

0.57** 5.93* 1.07 0.13* 0.52 5.92*

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silis exposed to lead nitrate [6], and activity of amylase, trypsin, and pepsin were induced in intestine of Channa punctaus after 20 days exposure to mercuric chloride [27]. Therefore, the influence of pollutants on digestive enzymes in fish is unclear, and requires further study. 4.2. Antioxidant responses

Fig. 3. Ordination diagram of PCA of biochemical parameters in rainbow trout intestine after 30 days exposure to PCZ.

higher concentrations of PCZ, E2 and E3, were separated into independent groups B and C, respectively. 4. Discussion 4.1. Digestive enzymes Digestive enzyme patterns can reflect the feeding rate and digestive capacity of fish; hence, digestive enzyme activity can be used as bio-indicators of growth and health status of fish [13,24]. In the present study, the activities of proteolytic enzymes and amylase in the intestine were significantly inhibited with higher levels of PCZ exposure. This indicated that long-term exposure to PCZ could have negative effects on digestive enzyme activity in intestine. This is in agreement with Quistad and Casida [25], who found three digestive enzymes (␣-chymotrypsin, elastase and trypsin) to be highly sensitive to organophosphorus pesticides. In addition, studies have found that the activity of various digestive enzymes is inhibited in fish exposed to heavy metals [8,26]. However, activity of maltase and lactase remained unchanged in intestine of Heteropneustes fos-

Fig. 4. Individual variations in biochemical parameters in rainbow trout intestine after 30 days exposure to PCZ using PCA.

Lipid peroxidation and protein carbonylation are widely used as oxidative stress indicators in aquatic animals, and they have been reported to be major contributors to the loss of cell function under oxidative stress conditions [5,28]. Considering that the typical reaction during ROS-induced damage involved the peroxidation of unsaturated fatty acids, the present results clearly showed that exposure to PCZ for 30 days led to oxidative stress, with higher levels of LPO and CP in the intestine, compared to controls. These results suggested that ROS-induced oxidative damage can be one of the main toxic effects of PCZ. Observed alterations in the activity of antioxidant enzymes upon exposure to sublethal concentrations of PCZ suggest that changes could be in response to ROS. Superoxide dismutase made a defense response under oxidative stress at lower concentrations of PCZ (E1 and E2), demonstrating that SOD is the first enzyme to break down oxyradicals [29]. Catalase is chiefly located in the peroxisomes and facilitates the removal the hydrogen peroxide, which is metabolized to molecular oxygen and water [30]. In this study, CAT activity was significantly induced in E2, but significantly inhibited in E3. This indicated that a low concentration of PCZ can induce CAT in intestine but the accumulation of ROS could make it inhibited strongly. In the present study, GR activity decreased first but then slightly elevated, probably due to the change in the availability of NADPH in vivo [31]. Glutathione peroxidase catalyzes the reduction of both hydrogen peroxide and lipid peroxide [32,33]. The activity of GPx was induced slightly at the lower PCZ concentration which indicated the generation of ROS and the adaptive response to oxidative stress. 4.3. Energy metabolic parameters Measurement of RNA/DNA ratio in fish has been used widely as an indicator of protein syntheses and specific growth [34,35], but the use of RNA/DNA ratio as a biomarker of environmental pollutant exposure in fish has not been confirmed. In this study, a significant reduction in the RNA/DNA ratio was found in the higher concentration PCZ treatment groups, E2 and E3. These results are in agreement with the previous studies showing a depressed RNA/DNA ratio in fish chronically exposed to metal and organic contaminants. For instance, a significant reduction in the RNA/DNA ratio was found in fingerlings of gilthead sea bream exposed to dichlorvos (0.05 and 0.1 mg/l) [10] and juvenile tilapia exposed to Cd2+ (6 mg/l) for 45 days [36]. Barron and Adelman [37] observed that exposure to various toxicants (Cr6+ , CN− , ethyl acetate, pcresol, benzophenone) decreased the RNA/DNA ratio in fathead minnow larvae. In contrast, some studies have not revealed a relationship between the RNA/DNA ratio and environmental contaminants [9]. Benton et al. [38] found that the RNA/DNA ratio was not significantly affected in juvenile Sailfin mollies after 21 days exposure to various concentrations of DDT. Similar results were observed in Atlantic salmon fry exposed to Al3+ concentrations of 124 or 264 ␮g/l for 60 days [39] and in juvenile common carp exposed to Cu2+ concentration of 125 and 250 ␮g/l for 28 days [40]. These results might be explained by species differences, stage of fish life cycles, types of toxicants and experimental conditions. Miliou et al. [41] pointed out that in early life stages of fish, when RNA synthesis is high, the RNA/DNA ratio is a useful indicator of chronic toxicant

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effects, but more studies should be carried out to investigate the variation in this index during the life of different fish species and relative to genetic mechanisms. Adenosine triphosphatase is a group of enzymes that play an important role in intracellular functions and are considered to be sensitive indicators of toxicity [42]. These enzymes, especially Na+ K+ -ATPase, play a central role in whole body osmoregulation [43], in that they provide energy for the active transport of Na+ and K+ across the cell membrane and also affect the trans-epithelial movements of cations [31]. In this study, the inhibition of Na+ -K+ -ATPase in intestine probably represented disturbance to the Na+ -K+ pump, resulting in a erratic entry of Na+ into the cell along the concentration gradient with the water molecule following the osmotic gradient [44]. 5. Conclusion In summary, long-term exposure of PCZ was associated with both enzymatic changes and differences in RNA/DNA ratio in the intestines of rainbow trout. Multiple enzymatic systems were affected, including digestive enzymes, antioxidant responses, and energy metabolic indices. According to the multivariate data analysis (PCA), fish exposed to the environmental concentration of PCZ (E1) had similar biochemical responses to controls, indicating that O. mykiss tolerates the levels of PCZ in the natural environment. However, the results indicated that long-term exposure to PCZ at higher concentrations had a distinct effect on fish. Compared the present results with our previous studies focused on biochemical responses in different tissues of rainbow trout after 42 days exposure to sublethal Carbamazepine (another pharmaceutical widely present in aquatic environment) [3,5,31], antioxidant defense system in fish showed tissue-specific differences under pharmaceuticals-induced oxidative stress: (1) the liver, as the central organ in detoxifying xenobiotics and processing metabolic products for degradation, has a typically developed antioxidant defense system under pharmaceuticals stress; (2) the gill, as the first system affected by pollutants, becomes the prime target to toxic chemicals because of not only its large surface area facilitates greater toxicant interaction but also its detoxification system not as robust as that of liver; (3) the brain, as an organ in which homeostasis must be strictly maintained, is particularly vulnerable to attacks of free radical induced by residual pharmaceuticals in aquatic environment, because it contains large amounts of polyunsaturated fatty acids and a weak antioxidant defense system; (4) the intestine, as the major digestive organ, has a relative low content of mitochondria and low intensity of oxidative metabolism, which results in the activities of all antioxidant enzymes in fish intestine are much lower than in liver. Additional, fish intestine showed tissue-specific responses under PCZ-induced stress: digestive enzymes and energy metabolic indices had more negative correlations with oxidative stress than did antioxidant enzymes. In short, the alterations of intestine-related biochemical markers measured in this study may be attributed to different mechanisms. Therefore, these sensitive parameters could potentially be new indicators for monitoring the effects of residual PCZ on fish. However, body weight changes of fish exposed to environmental contaminants could give some hint about potential toxic actions, which should be given more attention in the future. Furthermore, more detailed study is needed before these findings could be applied in a field investigation. Moreover, precise molecular mechanisms for these biochemical responses in the fish intestine are unclear and also need further investigation. Conflict of interest The authors declare that there are no conflicts of interest.

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