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Bull Environ Contam Toxicol (2016) 96:49–54 DOI 10.1007/s00128-015-1693-3

Evaluation of Biochemical, Genetic and Hematological Biomarkers in a Commercial Catfish Rhamdia quelen Exposed to Diclofenac Ariane Ghelfi1 • Joaõ Luiz Coelho Ribas2 • Izonete Cristina Guiloski2 • Franciele Lima Bettim2 • Lae´rcio Dante Stein Piancini3 • Marta Margarete Cestari3 Aramis Jose´ Pereira4 • Guilherme Lanzi Sassaki4 • Helena Cristina Silva de Assis2

•

Received: 5 February 2015 / Accepted: 11 November 2015 / Published online: 20 November 2015 Ó Springer Science+Business Media New York 2015

Abstract Juveniles Rhamdia quelen fish species were exposed to diclofenac for 96 h at concentrations of 0.2, 2, and 20 lg/L. Biochemical, genetic, and hematological biomarkers were assessed in the liver, kidney, and blood in order to evaluate the toxic effects. No oxidative stress was observed in liver. In kidney the superoxide dismutase activity increased in all concentrations, suggesting an alteration in the hydrogen peroxide production, but DNA damage and lipid peroxidation were not detected. Diclofenac exposure increased the red blood cells number at concentrations of 0.2 and 2 lg/L, and monocytes and neutrophils at 2 and 20 lg/L, respectively. These results suggest that acute exposure to diclofenac, even at low concentrations, caused hematologic and renal enzymatic alterations in R. quelen. Keywords Pharmaceuticals NSAIDs Fish Hematology Biomarkers Pharmaceutical compounds have become the focus of environmental concerns as some of these drugs are not eliminated from environment by conventional wastewater

& Helena Cristina Silva de Assis [email protected] 1

Ecology and Conservation Program of Post-Graduation, Federal University of Parana´, PO Box 19031, Curitiba, Parana´ CEP 81.531-990, Brazil

2

Department of Pharmacology, Federal University of Parana´, PO Box 19031, Curitiba, Parana´ CEP 81.531-990, Brazil

3

Department of Genetics, Federal University of Parana´, PO Box 19031, Curitiba, Parana´ CEP 81.531-990, Brazil

4

Department of Biochemistry, Federal University of Parana´, PO Box 19031, Curitiba, Parana´ CEP 81.531-990, Brazil

treatment processes. In addition, these drugs are also excreted by the organisms, either unchanged or as metabolites (Reis Filho et al. 2007; Sodre´ et al. 2010). Among the pharmaceuticals the most frequently detected in the aquatic environment is the diclofenac. It belongs to the class of NSAIDs (nonsteroidal anti-inflammatory drugs) with analgesic and anti-inflammatory properties and is a widely prescribed drug (Stu¨lten et al. 2008). Diclofenac is found at concentrations ranging from 0.2 to 2.3 lg/L in Brazil and European countries such as Austria, Germany, Sweden, Finland, and Switzerland (Bila and Dezotti 2003; WHO 2012). It has been associated with hepatotoxicity (Van Leeuwen et al. 2011), severe hemorrhagic gastroenteritis, and kidney failure in birds (Oaks et al. 2004). Saravanan et al. (2011) observed that the exposure of carp to diclofenac resulted in hematologic and biochemical alterations. Stepanova et al. (2013) exposed early stages of common carp (Cyprinus carpio) to 3 mg/L of diclofenac for 30 days and observed mortality and oxidative stress. Ajima et al. (2014) investigated the effect of diclofenac in Clarias gariepinus when chronically exposed to 1.57, 3.14, and 6.28 mg/L. Few studies have investigated the effect of environmentally relevant concentrations of diclofenac mainly in catfish with commercial interest such as Rhamdia quelen, commonly known as jundia´. It is a catfish with great potential for aquaculture in South America because of its fast growth (Garcia et al. 2008), and provides own tasty meat and is well accepted for consumption (Baldisserotto 2013). Although its annual production is about 1300 tons, representing \0.5 % of the Brazilian continental aquaculture, it is in clear expansion (Koakoski et al. 2013). In addition, the catfish has been widely studied in various aspects, such as its ecology (Gomiero et al. 2007), physiology (Higuchi et al. 2011), and its response to exposure to pesticides (Mela et al. 2013), metals (Witeck et al. 2011),

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and anti-inflammatory drugs as dipyrone (Pamplona et al. 2011). This catfish is a species susceptible to contaminants effect, as shown in the studies that evaluated several biochemical and morphological changes, neurotoxicity, genetic damage in liver, kidney and gills, and also alterations in hematology, physiological and reproductive parameters. As we used some biomarkers in these studies, the size of the tissues of this fish is important to be compared to other fish species such as Danio rerio. The NSAIDs once entered in the aquatic environment can provide potential risk of causing adverse effects on organisms such as fishes (Bila and Dezotti 2003). These drugs may stimulate reactive oxygen species (ROS) and alteration in oxidative stress parameters (Stepanova et al. 2013). Some biomarkers to assess oxidative stress are the enzymes activities of CAT (catalase), SOD (superoxide dismutase), GPx (glutathione peroxidase), and GST (glutathione S-transferase), responsible for acting in the enzymatic antioxidant system. The reduced glutathione (GSH) is one of the most efficient tools in detoxifying the cells (Pompella et al. 2003). The failure of antioxidant defenses to detoxify the excess of ROS production may lead to significant oxidative damage including DNA damage and lipid peroxidation (Islas-Flores et al. 2013). Other possible observed damages can be of hematological nature, the blood alterations may be associated with environmental alterations and even genetic, because some xenobiotics are able to damage directly or indirectly the DNA structure (Lee and Steinert 2003). Therefore these effects can change with the fish species and is important to focus the study in different fish model with commercial interest. Our hypothesis is that acute exposure to diclofenac even in low concentrations can cause biochemical changes due to oxidative stress, hematological changes, and DNA damage to commercial fish R. quelen. So the aim of this study was to expose the fish to, 0.2, 2.0, and 20 lg/L of diclofenac for 96 h, and analyze some biochemical, hematological, and genetic biomarkers.

Materials and Methods Juvenile specimens of R. quelen (8.80 ± 2.23 g; 10.11 ± 3.17 cm) were obtained from a fish farm (Peixes & Peixes; CEASA/PR, Brazil), transported to the laboratory and maintained in glass aquaria (30 L capacity) under controlled temperature (23 ± 2°C), constant aeration, and a 12-h-light/12-h-dark photoperiod. Water analyses of the degradation of diclofenac were carried out before the exposure. The water was sampled for 96 h by 12 h interval from one aquarium containing only water and another one containing water and fish. The diclofenac

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Bull Environ Contam Toxicol (2016) 96:49–54

concentration was measured by gas chromatography coupled to the mass spectrometry (GC–MS) (Schwaiger et al. 2004). The results of this experiment defined the frequency of the replacement of the pharmaceutical during the experiment. For the exposure, juvenile fish were randomly distributed in four groups (12 fish per group). The bioassay was carried out with 0, 0.2, 2.0, and 20 lg/L of diclofenac sodium (Sigma Aldrich, D6899) in filtered water for a period of 96 h. These concentrations were chosen based on the literature, as a mean of detected concentrations in the aquatic environment of different countries. The protocols of this study were approved by the Animal Experimentation Ethics Committee of Federal University of Parana, under number 646. After 96 h of exposure the animals were anesthetized with benzocaine 1 %. Blood was taken via caudal vein puncture for the evaluation of hematological parameters and comet assay. The fish were euthanized by medullar section and the liver and kidney collected for biochemical and genetic biomarkers. Samples of liver and kidney were homogenized (10 % w/v) in potassium phosphate buffer 0.1 M, pH 7.0. Homogenates were centrifuged at 15,0009g for 30 min at 4°C. In the supernatants were measured SOD, CAT, GPx, and GST activities and GSH concentration and lipoperoxidation (LPO). The CAT activity was measured at 240 nm according to the method of Aebi (1984). SOD activity was measured at 560 nm by the method described by Crouch et al. (1981). GPx activity was measured at 340 nm (Sies et al.1979). GST activity was measured at 340 nm by the method described by Keen et al. (1976). GSH concentration was measured based on the method of Sedlak and Lindsay (1968). The LPO was analyzed at 570 nm (Jiang et al.1992). Protein concentration was determined using Bradford’s method (1976) with bovine serum albumin as the standard. Hematological parameters were performed as described by Tavares-Dias et al. (2008), and the numbers of erythrocytes, leukocytes, thrombocytes as well as hematimetric index were evaluated. The comet assay was performed according to Speit and Hartmann (1999) in blood, liver, and kidney. The data were checked for normality by the Kolmogorov–Smirnov test and one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc tests was used for biochemical and hematological biomarkers. Comet assay data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. All data were statistically analyzed by the GraphPad Prism 5.00 (GraphPad Software, Inc.). All tests were regarded as statistically significant when p \ 0.05.


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Results and Discussion The degradation of diclofenac in water is demonstrated as its half-life is about 12 h, in aquarium containing only water (Fig. 1a), whereas the degradation time of the drug is slightly lower in the aquarium containing fish than only water (Fig. 1b). Based on this result the diclofenac was replaced for every 12 h. The results of the hepatic antioxidant enzymes showed no changes after 96 h of exposure to the diclofenac when

compared to control group (Table 1). We can attribute our results to the short-term exposure and the low concentrations used. Praskova et al. (2013) studied effects of NSAID ketoprofen on early developmental stages of carp exposed for 30 days and not found significant differences in the activity of the antioxidant enzymes GST, GR, and GPx at concentrations 0.003, 2.1, 6.3, and 21 mg/L. Gonzalez-Rey and Bebianno (2014) studied the effects of exposure of mussels (Mytilus galloprovincialis) to 250 ng/L of

Fig. 1 Degradation of diclofenac in water. a Percentage of diclofenac reduction in the aquarium with water. b Percentage of the diclofenac reduction in the aquarium with water and fish

Table 1 Biochemical biomarkers evaluated in liver of R. quelen exposed to diclofenac

Biochemical biomarkers

Concentration of diclofenac (lg/L) Control

0.2

2.0

20 355.4 ± 39.5

CAT

422.3 ± 26.9

392.5 ± 21.2

411.3 ± 36.5

SOD

4.5 ± 0.4

5.1 ± 0.6

5.1 ± 0.5

GPx

142.6 ± 9.0

145.7 ± 11.2

172.1 ± 12.3

171.9 ± 8.6

5.2 ± 0.3

GST

110.3 ± 6.3

114.3 ± 5.3

110.7 ± 4.6

121.9 ± 9.4

GSH

11.3 ± 1.7

11.1 ± 1.5

10.5 ± 1.4

10.1 ± 1.7

LPO

5.2 ± 0.9

3.7 ± 0.3

3.4 ± 0.3

3.4 ± 0.3

-1

The results are expressed as mean ± SE of the mean. n = 12. CAT (lmol min mg of protein-1), SOD (U mg of protein-1) GPx and GST (lmol min-1 mg of protein-1) GSH (lg mg of protein-1) LPO (nmol de hydroperoxide mg of protein-1)

Table 2 Biochemical biomarkers evaluated in kidney of R. quelen exposed to diclofenac

Biochemical biomarkers


0.2

2.0

20

CAT

38.6 ± 4.4

58.5 ± 9.2

40.1 ± 6.8

43.5 ± 4.1

SOD

21.9 ± 3.8

70.4 ± 16.1*

83.7 ± 16.8*

64.6 ± 8.3*

GPx

114.9 ± 11.6

101.1 ± 16.7

106.9 ± 14.9

128.6 ± 9.3

GST

182.5 ± 7.4

199.8 ± 17.5

181.8 ± 18.8

187.7 ± 16.1

GSH

9.9 ± 0.9

11.2 ± 1.7

10.7 ± 2.1

10.3 ± 1.1

LPO

4.5 ± 0.7

3.1 ± 0.6

4.2 ± 0.7

1.0 ± 0.5*

The results are expressed as mean ± SE of the mean. n = 12 * Significant difference (p \ 0.05) compared to the control group. ANOVA, Bonferroni. CAT (lmol min-1 mg of protein-1), SOD (U mg of protein-1) GPx and GST (lmol min-1 mg of protein-1) GSH (lg mg of protein-1) LPO (nmol de hydroperoxide mg of protein-1)

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Table 3 Hematological parameters of R. quelen exposed to diclofenac

Hematological parameters


0.2

2.0

20

Erythrocytes (106 lL-1)

1.0 ± 0.1

0.9 ± 0.03*

0.8 ± 0.02*

Hemoglobin (g dL-1)

3.5 ± 0.2

2.8 ± 0.10

3.5 ± 0.3

3.7 ± 0.1

Hematocrit (%)

1.0 ± 0.04

20.0 ± 1.5

18.5 ± 1.9

17.7 ± 0.9

18.9 ± 1.2

198.5 ± 14.5

213.0 ± 23.5

213.2 ± 12.2

190.0 ± 14.6

MCH (qg cells )

34.3 ± 1.8

32.7 ± 0.8

41.9 ± 3.6

37.2 ± 2.3

MCHC (g dL-1)

17.6 ± 0.8

16.9 ± 1.8

20.1 ± 2.0

20.2 ± 1.3

Leukocytes (103 lL-1)

60.3 ± 3.2

59.4 ± 2.3

62.8 ± 3.4

68.4 ± 4.4

Thrombocytes (103 lL-1)

31.0 ± 2.9

28.0 ± 6.7

23.3 ± 8.0

28.0 ± 6.6

6.1 ± 0.7

7.4 ± 0.7

10.3 ± 0.9*

8.2 ± 0.9

Lymphocytes (103 lL-1)

50.4 ± 3.5

47.6 ± 1.9

47.2 ± 2.9

53.5 ± 3.4

Neutrophills (103 lL-1)

3.8 ± 0.3

4.3 ± 0.5

5.3 ± 0.5

MCV (fL) -1

Monocytes (103 lL-1)

6.7 ± 0.4*

The results are expressed as mean ± SE of the mean. n = 12 * Significant difference (p \ 0.05) compared to the control group. ANOVA, Bonferroni

500

A

200

B

C 300

400

200

100

Control 0,2µg/L 2,0µg/L 20µg/L

200

* 100

50

100 0

Score

Score

Score

150 300

0 Control 0,2µg/L 2,0µg/L 20µg/L

0

Control 0,2µg/L 2,0µg/L 20µg/L

Fig. 2 Scores of genetic damage (Comet assay) of R. quelen exposed for 96 h at 0, 0.2, 2.0 and 20 lg/L of diclofenac sodium. a Liver, b blood, c kidney. The results are presented as median/quartile 1

(25 %) and 3 (75 %). The Kruskal–Wallis test followed by Dunn’s test. *Significant difference (p \ 0.05) compared to the control group

diclofenac for 15 days and observed an increase in SOD, CAT, and LPO, indicating oxidative stress. In the kidney was observed increase in SOD activity in all concentrations (Table 2), suggesting that exposure to diclofenac increased the production of superoxide anion and the enzyme responsible for its metabolism. Cyprinus carpio exposed to industrial effluent containing NSAIDs had an increase in SOD activity in gills, liver, and blood (San Juan-Reyes et al. 2013). Although enzymes responsible for the elimination of hydrogen peroxide (CAT and GPx) were not altered after 96 h. The increase of SOD activity can be the cause of the LPO decreasing at the highest concentration tested, indicating a reduction of the hydroperoxides generated compared to the control group. Other possibility is the protective effects of NSAIDs against oxidative stress (Petersen et al. 2005). Feito et al. (2012) observed a reduction in lipid peroxidation after zebrafish embryos exposure to 90 min to diclofenac at 0.03 lg/L.

Diclofenac altered some hematological parameters as a decreased number of erythrocytes at concentrations of 0.2 and 2.0 lg/L (Table 3). The hemoglobin was also reduced at the low concentration. These results are commonly related to the use of NSAIDs in causing hemolytic effect (Manente et al. 2011) or hemodilution (Delfino et al. 1995). This effect in carp was also observed after ibuprofen exposure (Saravanan et al. 2012) and in R. quelen exposed to dipyrone (Pamplona et al. 2011). The number of leucocytes did not change, but the number of monocytes and neutrophils at 2.0 and 20 lg/L increased, respectively (Table 3). This can indicate an initial organism reaction to the drug. It is possible to expect that, in a long term exposure, this response will be higher. The increased number of neutrophils is considered the most common response for infections and apoptosis in fish (Afonso et al. 1998) (Cohen et al. 1992). This effect can be a defense to the xenobiotic. Monocytes and neutrophils numbers were also increased in C. gariepinus when

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chronically exposed to higher concentrations of diclofenac (Ajima et al. 2014). Genotoxicity was not observed in any of the diclofenac concentrations tested compared to the control group. In the kidney occurred significant decrease of DNA damage in higher concentration (Fig. 2), maybe due to the LPO reduction. However, in others studies diclofenac caused DNA damage. Diclofenac induced nephrotoxicity in rats, by causing oxidative stress and DNA damage (Hickey et al. 2001). There was a DNA fragmentation in hemocytes of the bivalve of the Dreissena polymorpha when exposed to diclofenac for 1 h (Parolini et al. 2009), but these effects were not confirmed when it was exposed to diclofenac for 96 h (Parolini et al. 2011). In the present study, the hematological and biochemical changes were observed in kidney after acute exposure to environmental relevant concentrations demonstrating that, even at low concentrations, the diclofenac may cause negative effects to R. quelen. It is a concern, because this fish species showed sensitivity to diclofenac that is found in freshwater environment. A continuous exposure can result in more drastic effects particularly nephrotoxicity and consequently harming fish farming. Acknowledgments This work was supported in part by CNPq (Brazilian Agency for Science and Technology) and CAPES (National Council for the Improvement of Higher Education).

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