Oxidative Stress and Genotoxicity Induced by Ketorolac on the Common Carp Cyprinus carpio mez-Olivan,3 I. Pe rez-Coyotl,1 M. Galar-Martınez,1 S. Garcıa-Medina,2 L. M. Go 1 D. J. Mendoza-Monroy, R. E. Arrazola-Morgain 1
tica, Escuela Nacional de Departamento de Farmacia, Laboratorio de Toxicologıa Acua gicas, Instituto Polite cnico Nacional, Ciudad de Me xico, Me xico Ciencias Biolo
2
Unidad Analıtica de la Unidad de Farmacologıa Clınica, Facultad de Medicina - UNAM, yotl, Edo de Me xico Nezahualco 3
Departamento de Farmacia, Facultad de Quımica, Laboratorio de Toxicologıa Ambiental, noma del Estado de Me xico, Toluca, Me xico Universidad Auto
Received 11 August 2014; revised 23 December 2014; accepted 29 December 2014 ABSTRACT: The nonsteroidal anti-inflammatory drug ketorolac is extensively used in the treatment of acute postoperative pain. This pharmaceutical has been found at concentrations of 0.2–60 mg/L in diverse water bodies around the world; however, its effects on aquatic organisms remain unknown. The present study, evaluated the oxidative stress and genotoxicity induced by sublethal concentrations of ketorolac (1 and 60 mg/L) on liver, brain, and blood of the common carp Cyprinus carpio. This toxicant induced oxidative damage (increased lipid peroxidation, hydroperoxide content, and protein carbonyl content) as well as changes in antioxidant status (superoxide dismutase, catalase, and glutathione peroxidase activity) in liver and brain of carp. In blood, ketorolac increased the frequency of micronuclei and is therefore genotoxic for the test species. The effects observed were time and concentration dependent. C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. V
Keywords: ketorolac; oxidative stress; lipid peroxidation; hydroperoxides; oxidized proteins; antioxidant enzymes; genotoxicity; micronucleus; DNA; Cyprinus carpio
INTRODUCTION Pharmaceutical products are biologically active and persistent substances that have been recognized as a constant threat to the stability of the environment. These toxicants form part of the group termed “emerging contaminants” and many tons of them are produced and consumed annually
Correspondence to: M. Galar-Martınez; e-mail: [email protected]; [email protected] or S. Garcıa-Medina, e-mail: [email protected]; [email protected] Contract grant sponsor: Secretarıa de Investigaci on y Posgrado of the Instituto Politecnico Nacional SIP-IPN. Contract grant numbers: 20131086; 20141042 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22113
around the world. This explains their occurrence in the environment, particularly in aquatic ecosystems which they enter through municipal and hospital wastewater discharges (Christen et al., 2010; Ginebreda et al., 2010). These compounds have been designed to elicit a specific biological action in the body and often resist inactivation prior to inducement of their intended therapeutic effect, these properties being paradoxically responsible for both their bioaccumulation and toxic effect on hydrobionts (Santos et al., 2010). One of the most frequently used pharmaceutical groups is that of the nonsteroidal anti-inflammatory drugs (NSAIDs), which includes ketorolac (KTC). Many of these remedies are sold without prescription, favoring selfmedication, and overuse, and increasing the concentrations at which they are found in the environment (Parolini et al., 2010; Islas-Flores et al., 2013). In the case of KTC, it has
C 2015 Wiley Periodicals, Inc. V
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been detected in hospital effluents at concentrations ranging from 0.2 to 59.5 mg/L (Gomez et al., 2006), and in effluents of treatment plants around 1.2 mg/L, since it is difficult to remove (Martınez-Bueno et al., 2012). KTC is an NSAID with a potent analgesic and antiinflammatory activity induced through interference with prostaglandin synthesis via the nonselective inhibition of cyclooxygenase. It is extensively used for clinical management of acute postoperative and cancer-related pain (Jalkut, 2014). Since the compound in the form of free acid is scarcely soluble in water, it is marketed as tromethamine salt, which increases its solubility in water for oral, intramuscular, intravenous, and ophthalmic administration (Radwan et al., 2010). This pharmaceutical is readily and completely absorbed in humans, reaching 100% bioavailability and maximum plasma concentrations about 1 h after administration. KTC has a low apparent volume of distribution: it is more than 99% bound to plasma proteins and has a half-life of 3.5–9.2 h in young adults and 4.7–8.6 h in the elderly (Prathibha et al., 2014). In mammals this compound is metabolized in the liver via Phase I reactions involving cytochrome P450 (CYP) 2C8/9-mediated oxidation to form phydroxy ketorolac as well as Phase II reactions through which it is conjugated with glucuronic acid, the unaltered pharmaceutical and its metabolites being eliminated mostly through the urine (91%) and feces (Kulo et al., 2013). Like many other NSAIDs, KTC induces diverse adverse effects in mammals including reduced cytoprotection of the gastric mucosa, alterations in renal and hepatic function, and inhibition of platelet aggregation (Vitale et al., 2003). Also, since other NSAIDs have been shown to induce oxidative stress in some aquatic species—e.g., diclofenac and ibuprofen, which increase lipid peroxidation (LPX) and protein carbonyl content (PCC) and modify the antioxidant status of carp exposed to sublethal concentrations of the compound (Islas-Flores et al., 2013, 2014); and ibuprofen, diclofenac, and naproxen in the case of Daphnia magna (Gomez-Olivan et al., 2014b), KTC may elicit similar effects on C. carpio; however, there are no reports regarding its toxicity on aquatic species, even though this drug has been found at environmentally relevant concentrations (above 1 mg/L) at diverse kinds of effluents, and is of difficult removal by water treatment plants. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses in the body, and many xenobiotics including some NSAIDs share this mechanism of action. Organisms are able to adapt to oxidative stress through the up-regulation of antioxidant defenses—both enzymatic, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and nonenzymatic such as glutathione—in order to promote ROS detoxification. Failure in this up-regulation results in significant oxidative damage including enzyme inactivation, protein degradation, LPX, and damage to genetic material (Valavanidis et al., 2006; Islas-Flores et al., 2013).
Environmental Toxicology DOI 10.1002/tox
The common carp Cyprinus carpio has a cosmopolitan distribution. In Mexico it has been introduced in 80% of freshwater bodies and is an ecologically and economically important species. In 2012 alone, some 7 tons of this species were grown, harvested and consumed, with an approximate commercial value of 6 million US dollars (Base de Datos del Registro Nacional de Pesca y Acuacultura. 2013). Furthermore, C. carpio is used as a bioindicator in toxicity assays due to its sensitivity and easy maintenance under laboratory conditions. The present study, aimed to evaluate KTC-induced oxidative stress and genotoxicity in liver, brain, and blood of the common carp Cyprinus carpio.
MATERIALS AND METHODS Specimen Procurement and Maintenance Common carp (Cyprinus carpio) with an average weight of 2.3 g, obtained from the carp culturing facility in Tiacaque, (Estado de Mexico), were transported to the laboratory and acclimated for two weeks. During acclimation, specimens were maintained in 80 L glass tanks equipped with filtration systems under 12:12 h light:dark conditions and constant R every third aeration, and were fed Pedregal Silver CorpV day. Tank water had the following physicochemical properties: dissolved oxygen 6.4 6 0.5 mg/L, ammonia concentration 0.32 6 0.2 mg/L, nitrates 0.26 6 0.001 mg/L, temperature 20 6 2 C, oxygen saturation 90–100% and pH 7.5–8.0.
Toxicity Assays Prior to the present study, an acute toxicity assay was performed in order to verify that the test concentrations to be used were not lethal to C. carpio. Exposure systems were set up with six 80 L tanks (one per each exposure time) and six carp were placed in each. The test concentrations used were 1 and 60 mg KTC/L (Sigma, CAS 74103-07-4). The former is the threshold safety value in water established by the European Medicines Agency (EMEA) (Ginebreda et al., 2010) and the latter is a previously reported concentration found in the environment (Gomez et al., 2006; Santos et al., 2010). The exposure periods used were 12, 24, 48, 72, and 96 h. Conditions during the study were similar to those used for specimen maintenance. At the end of the exposure period, carp were anesthetized by immersion in 2% lidocaine for 30 s. Anesthetized specimens were placed in a lateral position and a blood sample was taken by puncture of the caudal vessel using a heparinized 1 mL hypodermic syringe and performed laterally near the base of the caudal peduncle, at mid-height of the anal fin and ventral to the lateral line. After which, specimens were placed in an ice bath, sacrificed and the liver and brain were removed. Tissue samples were supplemented with 2 mL phosphate buffer solution pH 7.4,
KETOROLAC INDUCES TOXICITY IN COMMON CARP
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then homogenized and centrifuged at 12,500 rpm and 24 C for 15 min. Whole blood samples were used to evaluate genotoxicity by micronucleus test, while oxidative stress was assessed in liver and brain homogenates using the following biomarkers: LPX, hydroperoxide content (HPC), and PCC, as well as SOD, CAT, and GPx activity. Total protein content was used to express biomarker results. Three replicate tanks were set up per each test concentration and exposure time, and solutions were not replaced. This protocol was reviewed and approved by the Bioethics Committee of the Escuela Nacional de Ciencias Biolgicas (ENCB-IPN) to ensure it was carried out in accordance with institutional standards for the care of animal test subjects. The provisions set out in the official Mexican norm on the production, care and use of laboratory animals (NOM-062-ZOO-1999) were also taken into account.
guanidine (Sigma) pH 2.3, and incubated at 37 C for 30 min. Absorbance was read at 366 nm and results were expressed as nmol reactive carbonyls formed/mg protein based on their MEC of 21,000 M/cm.
Determination of LPX
CAT activity was determined as proposed by Radi et al. (1991). To 20 lL of supernatant was added 1 mL isolation buffer solution (0.3M saccharose, 1 mM EDTA, 5 mM HEPES, and 5 mM KH2PO4) and 0.2 mL hydrogen peroxide (20 mM). Absorbance was read at 240 nm, at 0 and 60 s. Results were obtained by substituting the absorbance value obtained for each time in the formula: catalase activity 5 (A60 2 A0)/MEC, where the MEC of H2O2 equals 0.043 mM/ cm. Results were expressed as mM H2O2/mg protein.
LPX was determined as in B€uege and Aust (1978). The cell pellet was reconstituted with Tris–HCl buffer (pH 7.4) until a 5 mL volume was attained, and 1 mL of this solution was incubated at 37 C for 30 min. Next, 2 mL TBA-TCA reagent (0.375% thiobarbituric acid in 15% trichloroacetic acid) was added; the sample was shaken in a vortex, placed in a bath of boiling water for 45 min, allowed to cool, and the precipitate removed by centrifugation at 3,000 rpm for 10 min. Absorbance was read at 535 nm using a reaction blank. Results were expressed as mM malondialdehyde (MDA)/mg protein, using the molar extinction coefficient (MEC) of 1.56 3 105 M cm.
Determination of HPC HPC was evaluated by the Jiang et al. (1991) method. To 100 mL of supernatant (previously deproteinized with 10% TCA) was added 900 mL of the reaction mixture [0.25 mM FeSO4, 25 mM H2SO4, 0.1 mM xylenol orange, and 4 mM butylated hydroxytoluene in 90% (v/v) methanol]. The mixture was incubated for 60 min at ambient temperature and absorbance was determined at 560 nm against a blank containing only reaction mixture. Results were interpolated on a type curve and were expressed as nM cumene hydroperoxide (CHP)/mg protein.
Determination of PCC PCC was determined by the method of Levine et al. (1994). Soluble proteins were obtained by centrifugation of samples at 10,500 rpm for 30 min. To 100 lL of this supernatant was added 150 lL of 10 mM dinitrophenylhydrazine in 2M HCl (Sigma) prior to incubation at room temperature for 1 h in the dark. Next, 500 lL of 20% TCA was added and the sample allowed to rest for 15 min at 4 C, then centrifuged at 16,000 rpm for 5 min. The bud was rinsed thrice in 1:1 ethanol:ethyl acetate (Baker), dissolved in 150 lL of 6M
Determination of SOD Activity SOD activity was determined according to the Misra and Fridovich (1972) method. To 20 lL of supernatant in a 1 cm cuvette was added 150 lL carbonate buffer solution (50 mM sodium carbonate and 0.1 mM EDTA) pH 10.2, plus 100 lL adrenaline (30 mM). Absorbance was read at 480 nm, at 30 s and 5 min. SOD activity was determined by interpolating the data on a type curve. Results were expressed as IU SOD/mg protein.
Determination of CAT Activity
Determination of GPx Activity GPx activity was determined using Paglia and Valentine’s (1967) methodology. To 100 lL of supernatant was added 900 lL buffer reagent solution (5M K2HPO4, 5M KH2PO4, 3.5 mM reduced glutathione, 1 mM sodium azide, 2 U glutathione reductase, and 0.12 mM NADPH, pH 7.0; Sigma) plus 200 lL H2O2 (20M). Absorbance was read at 340 nm, at 0 and 60 s. Activity was estimated using the MEC of NADPH (6.2 mM/cm). Results were expressed as mM NADPH/mg protein.
Determination of Total Protein Content To 25 lL of supernatant was added 75 lL deionized water and 2.5 mL Bradford’s reagent (0.05 g Coomassie Blue dye, 25 mL of 96% ethanol, and 50 mL H3PO4, in 500 mL deionized water). The test tubes were shaken and allowed to rest for 5 min prior to reading of absorbance at 595 nm and interpolation on a bovine albumin curve (Bradford, 1976).
Micronucleus Test A smear of the cell suspension obtained from each specimen was fixed with pure ethanol for 5 min, then treated with 10% Giemsa stain for 9 min. A total of 1000 erythrocytes from each sample were examined under a light microscope, and frequency of micronuclei (MNi) was expressed as the number of micronucleated cells per 1000 cells (Cavas and
Environmental Toxicology DOI 10.1002/tox
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Fig. 1. Lipid peroxidation (LPX) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
centration, but decreased drastically thereafter and attained values comparable to those of the control group from 48 h on. Meanwhile in fish exposed to the higher test concentration, LPX increased in liver beginning at 24 h, reached its maximum value at 72 h (376%) (p < 0.05) and maintained increased values up to 96 h. In brain of carp exposed to either concentration, there was a significant increase in LPX as compared to the control group (p < 0.05) which began at 12 h and was maintained up to the end of the experiment, this damage being greater with the higher concentration. HPC results (Fig. 2) show that KTC induced a major increase in this parameter with respect to the control group from 24 h on in liver of common carp exposed to either concentration. However, in specimens exposed to 1 mg/L, HPC peaked at 48 h (a 165% increase as compared to the control group, p < 0.05) and thereafter decreased until values similar to those of the control group were attained, while in carp exposed to 60 mg/L the damage induced was not reversed. On the other hand, in brain of carp exposed to either test concentration, HPC increased with respect to the control group at 12 and 24 h (p < 0.05), decreasing thereafter and reaching values comparable to those of the control group from 48 h on. Figure 3 shows PCC results. In liver of carp exposed to either concentration this biomarker increased significantly
Ergene-G€ oz€ ukara, 2005). The criteria used to determine the presence of MNi were nonlinkage of small ovoid or round nuclei with the main nucleus, color, and staining intensity similar to the main nucleus (II-Yong and Chang-Kee, 2006), and diameter 1/5–1/20 of the main nucleus (Bolognesi et al., 2006).
Statistical Analysis Results of oxidative stress determination were subjected to a one-way analysis of variance (ANOVA) (p < 0.05), while differences between means were analyzed using the StudentNewman-Keuls method. Mni frequency was analyzed with a Kruskal–Wallis nonparametric ANOVA (p < 0.05), followed by Tukey’s post hoc test for multiple comparison of means. Sigma Stat v3.5 was used throughout.
RESULTS Oxidative Stress Figure 1 shows LPX results in common carp exposed to sublethal concentrations of KTC. This biomarker increased markedly with respect to the control group during early exposure periods (12 and 24 h, 70 and 246%, respectively) (p < 0.05) in liver of specimens exposed to the lower con-
Environmental Toxicology DOI 10.1002/tox
Fig. 2. Hydroperoxide content (HPC) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
KETOROLAC INDUCES TOXICITY IN COMMON CARP
with respect to the control group (p < 0.05) from the earliest exposure period (12 h) on. This trend was maintained during the course of the experiment in the group exposed to the higher concentration and reached its peak at 72 h with a 1,388% increase with respect to the control group. However, in liver of specimens exposed to the lower concentration, PCC values were similar to those of the control group from 72 h on. In brain of carp exposed to either concentration, PCC increased as compared to the control group (p < 0.05) at 12 h only and thereafter decreased until values similar to those of the control group were reached. As regards the antioxidant status of carp exposed to KTC, SOD activity (Fig. 4) was seen to increase with respect to the control group from 12 h on in liver of fish exposed to either test concentration (p < 0.05). This trend was maintained throughout the experiment in the group exposed to 60 mg/L and reached its peak at 72 h with a 264% increase with respect to the control group. However, in specimens exposed to 1 mg/L, SOD activity obtained values as comparable to those of the control group from 72 h on. In brain, this activity increased in specimens exposed to either concentration during early exposure periods (12 and 24 h) and decreased significantly thereafter compared to the control group in both cases (72% at 72 h with 1 mg/L, and 51% at 96 h with 60 mg/L, p < 0.05). CAT activity (Fig. 5) increased from 24 h on with respect to the control group (p < 0.05) in liver of carp exposed to
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Fig. 4. Superoxide dismutase (SOD) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA, and SNK (p < 0.05).
either concentration, peaking in both cases at 72 h (659 and 1335% with 1 and 60 mg/L, respectively). A similar behavior was found to occur in brain, although the maximum value was reached at 24 h in carp exposed to the higher concentration (239%) and at 72 h in those exposed to the lower concentration (56%). Figure 6 shows GPx activity results. In liver of carp exposed to the lower concentration, activity increased with respect to the control group (p < 0.05) during early exposure periods (85 and 380% at 12 and 24 h, respectively), but this effect was reversed from 48 h on. At the higher concentration, this enzyme activity decreased markedly compared to the control group from 12 h on and this tendency was maintained throughout the experiment. A similar behavior was observed in brain: increased activity in fish exposed to the lower test concentration and reduced activity in those exposed to the higher concentration.
Genotoxic Response
Fig. 3. Protein carbonyl content (PCC) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
Micronucleus test results are shown in Figure 7. MNi frequency increased remarkably from 12 h on with exposure to either concentration and reached peak values at 96 h (1600 and 2400% with 1 and 60 mg/L respectively), damage being much more marked in specimens exposed to the higher concentration (p < 0.05).
Environmental Toxicology DOI 10.1002/tox
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Fig. 5. Catalase (CAT) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
DISCUSSION Oxidative stress is one of the most important mechanisms of toxicity, given the impact that an imbalance between reactive species and antioxidant defenses has on vital biomolecules such as lipids, proteins, and genetic material, as well as the countless toxicants capable of inducing it (metals, hydrocarbons, pesticides, and NSAIDs such as ibuprofen, diclofenac, paracetamol, etc.) (Hernandez et al., 2013; Sureda et al., 2013; Espın et al., 2014; Islas-Flores et al., 2014; Nava Alvarez et al., 2014). NSAIDs are extensively used globally and various studies indicate that several members of this group of pharmaceuticals can induce oxidative stress on aquatic species, e.g. ibuprofen, naproxen, paracetamol, and acetylsalicylic acid in D. magna (G omez-Olivan et al., 2014a,b) or diclofenac in Myti lus spp. (Schmidt et al., 2011) and C. carpio (Nava-Alvarez et al., 2014). However, no reports are available on the effects of KTC on aquatic species. Results obtained in the present study show that KTC at sublethal and environmentally relevant concentrations (1 and 60 mg/L) induces this type of damage, since LPX, HPC, and PCC increases are elicited in both liver and brain of C. carpio exposed to this pharmaceutical, such damage being concentration and organ dependent.
Environmental Toxicology DOI 10.1002/tox
Fig. 6. Glutathione peroxidase (GPx) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA, and SNK (p < 0.05).
As regards LPX, the increase found in brain is both larger and sustained, while in liver this damage is smaller and shows a tendency towards recovery of normal values near the end of exposure with the lower concentration. A similar effect was observed in the latter organ in relation to HPC and PCC, even though in brain these parameters were less strongly affected and also evidenced a tendency towards recovery of normal values, damage in both organs being greater in specimens exposed to the higher concentration.
Fig. 7. Frequency of micronuclei (MNi) in blood of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and Tukey’s (p < 0.05).
KETOROLAC INDUCES TOXICITY IN COMMON CARP
In mammalian cells, NSAID toxicity is related to mitochondrial dysfunction, transporter efflux, and CYP-mediated metabolism (Van Leeuwen et al., 2012) since metabolites and reactive species may be produced during biotransformation. It is worth noting that CYP can produce an oxygenated intermediate—the oxy-cytochrome P450 complex [P450 (Fe31) O22]—during the metabolic cycle and release the superoxide anion (Doi et al., 2002), promoting oxidative stress and subsequent damage to biomolecules such as lipids, proteins, and genetic material. In this sense, it is worth noting that diverse CYP gene families have been characterized in C. carpio including CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, and CYP19 (Stegeman and Livingstone, 1998); the CYP2 gene family, particularly CYP2C8/9, being responsible for the biotransformation of KTC to p-hydroxy-ketorolac via phase I oxidation reactions (Kulo et al., 2013). It is thus probable that this metabolite as well as potentially reactive species including the superoxide anion are produced also in the common carp, which may explain the increases found in our study in biomarkers of oxidative damage (LPX, HP, and PCC). As regards the differences observed between the organs evaluated, it is worth noting that the liver is the primary biotransforming organ, so that formation of reactive metabolites is enhanced. However, the brain is particularly vulnerable to ROS due to the large amounts of oxygen required to carry out its activities as well as the fact that its membranes are rich in polyunsaturated fatty acids (Halliwell, 2006), which may favor LPX and hence increased HPC. On the other hand, the reactive metabolites and ROS produced during CYP-mediated biotransformation can also affect proteins, inducing increased PCC (as in our study), which leads to loss of sulfhydryl groups and changes in the resonance structures of amino acids, altering their function and therefore the integrity of the body (Parvez and Raisuddin, 2000). The antioxidant defense system is essential in the neutralization of ROS and related damage (Regoli et al., 2002). This system is mediated by a cascade of antioxidant enzymes that sequester ROS and convert them to less toxic and reactive species. This group of enzymes includes SOD, CAT, and GPx. In our study, SOD activity in liver of specimens exposed to 60 mg KTC/L increased from 12 h on and this trend was maintained up to the end of the experiment, while in specimens exposed to 1 mg KTC/L this increase occurred only at 48 h and later values were similar to those of the control group. A comparable effect was seen in brain of carp exposed to the lower concentration, while exposure to the higher one induced an activity reduction. SOD is the main enzyme responsible for offsetting the effects induced by the superoxide ion (Van der Oost et al., 2003), which it transforms to hydrogen peroxide. As stated previously, since CYP is present in C. carpio, formation of the oxycytochrome P450 complex [P450 (Fe31) O22] and release of the superoxide ion are possible, activating SOD in order to
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offset the oxidative effect of this free radical. This may explain the results in liver and brain obtained in our study during early exposure periods with either concentration. Increased SOD activity may act as a signal of oxidative stress, leading to activation and/or induction of antioxidant enzymes associated with a system of H2O2-sequestration such as CAT or GPx (Vlahogianni et al., 2007). In our study, CAT activity in brain of specimens exposed to either concentration increased at the start of exposure and decreased thereafter, while in liver there was a steady increase over the course of the experiment. In both organs, the increase observed in CAT activity may be due to higher concentrations of H2O2 as a result of SOD activity. Furthermore, Bagnyukova et al. (2006) state that LPX products may be involved in the regulation of some antioxidant enzymes, so that the LPX increase found in our study may likewise explain the increased antioxidant enzyme (SOD, CAT, and GPx) activity observed. Finally, GPx activity increased in our study at the beginning of exposure in liver and brain of specimens exposed to the lower concentration of KTC and thereafter the damage was reversed, while exposure to the higher concentration induced a reduction in both organs. One consequence of oxidative stress is protein damage (Parvez and Raisuddin, 2000). In our study, the higher concentration of KTC may induce ROS in larger numbers, exceeding the neutralizing capacity of the antioxidant system and damaging the enzymes that compose it—in this case GPx. A similar effect was observed by Orhan and Sahin (2001) in human erythrocytes exposed to therapeutic concentrations of KTC. Chandran et al. (2005) state that ROS, particularly the superoxide ion, directly affect the activity of antioxidant enzymes modifying their structure, which may explain the reduction found in our study. Many environmental contaminants are not only hazardous to the physiology and survival of organisms, but can also induce genetic changes leading to mutations which may put at risk species survival (Russo et al., 2004). Mutagenic chemicals may induce DNA lesions such as chain breaks, base modifications and crosslinks in aquatic organisms, producing adverse effects on ecosystem stability. Diverse techniques enable their evaluation, including MNi frequency determination, which detects MNi resulting from chromosomal breakage during cell division, loss of chromosomes due to damage in anaphase, and short-term cytogenetic damage (Russo et al., 2004; Kim and Hyun, 2006). In fish, the micronucleus technique has proved useful in vivo for evaluating genotoxicity and in situ for monitoring water quality (Kim and Hyun, 2006). In fish, this technique is usually performed on erythrocytes, which in these organisms are nucleated and very sensitive to different contaminants (Ragugnetti et al., 2011). In our study, MNi frequency evidenced a time- and concentration-dependent increase from 48 h on. Although KTC has not been studied in terms of its potential genotoxicity, various authors have demonstrated that NSAIDs can
Environmental Toxicology DOI 10.1002/tox
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induce damage to DNA, e.g., Ragugnetti et al. (2011), who found that MNi frequency increases in Oreochromis niloticus subchronically exposed to ibuprofen; or Parolini et al. (2010), who demonstrated that paracetamol induces DNA translocations, inversions, and fragmentation in Dreissena polymorpha in addition to increased apoptotic cell frequency and reduced lysosomal membrane stability, probably due to reactive metabolites and ROS formed during its biotransformation. This may also be the case of KTC which, as stated above, is metabolized by CYP. It is important to mention that in addition to the fact that phase I of the biotransformation process can produce reactive metabolites, glucuronide complexes of paracetamol induce an increase in MNi frequency and also inhibit cell division and nondisjunction of chromosomes. KTC may also undergo phase II reactions during which it is conjugated with glucuronic acid to form ketorolac-glucuronide by means of the enzyme uridine diphosphate glucuronosyltransferase (UGT) 2B7, analogs of which have been found in fish (UDP-glucuronosyltransferase, UDPGT; George and Taylor, 2002), it being probable that this metabolite is also produced in C. carpio. On the other hand, Asensio et al. (2007) state that NSAIDs inhibit glucose-6-phosphate dehydrogenase due to formation of NSAID-acyl-CoA or NSAID-CoA complexes, and as a result ribose-5-phosphate synthesis is halted and consequently also nucleic acid synthesis, it being possible therefore that KTC elicits a similar effect. Furthermore, this kind of damage may be indirectly induced if reactive metabolites or ROS formed in the biotransformation of KTC damage the proteins responsible for DNA synthesis and repair (G omez-Olivan et al., 2014a). n conclusion, results of the present study show that KTC at sublethal and environmentally relevant concentrations (1 and 60 mg/L) induces oxidative stress and genotoxicity on C. carpio.
REFERENCES Asencio C, Levoin N, Guillaume C, Guerquin MJ, Rouguieg K, Chretien F, Chapleur Y, Netter P, Minn A, Lapicque F. 2007. Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: A key to oxidative stress induced by non-steroidal anti-inflammatory drugs? Biochem Pharmacol 73:405–416. Bagnyukova T, Chahrak O, Lushchak V. 2006. Coordinated response of goldfish antioxidant defenses to environmental stress. Aquat Toxicol 78:325–331.
Bradford M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. B€uege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods Enzymol 52:302–310. Cavas T, Ergene-G€oz€ukara S. 2005. Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquat Toxicol 74:264–271. Chandran R, Sivakumar AA, Monadas S, Aruchami M. 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica. Comp Biochem Physiol C: Toxicol Pharmacol 140:422–426. Christen V, Hickmann S, Rechenberg B, Fent K. 2010. Highly active human pharmaceuticals in aquatic systems: A concept for their identification based on their mode of action. Aquat Toxicol 96:167–181. Doi H, Iwasaki H, Masubushi Y, Nishigaki R, Horie T. 2002. Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes. Chem–Biol Interact 140:109–119. Espın S, Martınez-Lopez E, Leon-Ortega M, Martınez JE, GarcıaFernandez AJ. 2014. Oxidative stress biomarkers in Eurasian eagle owls (Bubo bubo) in three different scenarios of heavy metal exposure. Environ Res 131:134–144. George SG, Taylor B. 2002. Molecular evidence for multiple UDP-glucuronosyltransferase gene families in fish. Mar Environ Res 54:253–257. Ginebreda A, Mu~noz I, Lopez de Alda M, Brix R, Lopez-Doval J, Barcelo D. 2010. Environmental risk assessment of pharmaceuticals in rivers: Relationships between hazard indexes and aquatic macroinvertebrate diversity indexes in the Llobregat River (NE Spain). Environ Int 36:153–162. Gomez MJ, Petrovic´ M, Fernandez-Alba AR, Barcelo D. 2006. Determination of pharmaceuticals of various therapeutic classes by solid-phase extraction and liquid chromatography-tandem mass spectrometry analysis in hospital effluent wastewaters. J Chromatogr A 1114:224–233. Gomez-Olivan L, Galar-Martınez M, Garcıa-Medina S, ValdesAlanıs A, Islas-Flores H, Neri-Cruz N. 2014a. Genotoxic response and oxidative stress induced by diclofenac, ibuprofen and naproxen in Daphnia magna. Drug Chem Toxicol 37:391–399. Gomez-Olivan L, Galar-Martınez M, Islas-Flores H, GarcıaMedina S, SanJuan-Reyes N. 2014b. DNA damage and oxidative stress induced by acetylsalicylic acid in Daphnia magna. Drug Chem Toxicol 164:21–26. Halliwell B. 2006. Oxidative stress and neurodegeneration: Where are we now? J Neurochem 97:1634–1658.
Base de Datos del Registro Nacional de Pesca y Acuacultura. 2013. (RNPA), Consejo Nacional de Pesca de la Secretarıa de Agricultura, Ganaderıa, Desarrollo Rural, Pesca y Alimentacion (SAGARPA), Mexico.
Hernandez AF, Lacasa~na M, Gil F, Rodrıguez-Barranco M, Pla A, Lopez-Guarnido O. 2013. Evaluation of pesticide-induced oxidative stress from a gene-environment interaction perspective. Toxicology 307:95–102.
Bolognesi C, Perrone E, Roggieri P, Pampanin DM, Sciutto A. 2006. Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions. Aquat Toxicol 78:93–98.
II-Yong K, Chang-Kee H. 2006. Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicol Environ Saf 64:288–297.
Environmental Toxicology DOI 10.1002/tox
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Islas-Flores H, Gomez-Olivan LM, Galar-Martınez M, Colın-Cruz A, Neri-Cruz N, Garcıa-Medina S. 2013. Diclofenac-induced oxidative stress in brain, liver, gill and blood of common carp (Cyprinus carpio). Ecotoxicol Environ Safe 92:32–38. Islas-Flores H, Gomez-Olivan LM, Galar-Martınez M, GarcıaMedina S, Neri-Cruz N, Dublan-Garcıa O. 2014. Effect of ibuprofen exposure on blood, gill, liver and brain of common carp (Cyprinus carpio) using oxidative stress biomarkers. Environ Sci Pollut Res 21:5157–5166. Jalkut MK. 2014. Ketorolac as an analgesic agent for infants and children after cardiac surgery: Safety profile and appropriate patient selection. AACN Adv Crit Care 25:23–30. Jiang ZY, Wollard ACS, Wolff SP. 1991. Lipid hydroperoxide measurement by oxidation of Fe21 in the presence of xylenol orange-comparison with the TBA assay and an iodometric method. Lipids 26:853–856. Kim IY, Hyun CK. 2006. Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicol Environ Safe 64:288–297. Kulo A, Hendrickx S, de Hoon J, Mulabegovic N, van Calsteren K, Verbesselt R, Allegaert K. 2013. The impact of pregnancy on urinary ketorolac metabolites after single intravenous bolus. Eur J Drug Metabol Pharmacokinet 38:1–4. Levine R, Williams J, Stadtman E, Shacter E. 1994. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357. Martınez-Bueno MJ, Gomez MJ, Herrera S, Hernando MD, Ag€uera A, Fernandez-Alba AR. 2012. Occurrence and persistence of organic emerging contaminants and priority pollutants in five sewage treatment plants of Spain: Two years pilot survey monitoring. Environ Pollut 164:267–273. Misra P, Fridovich I. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175. Nava-Alvarez R, Razo-Estrada AC, Garcıa-Medina S, GomezOlivan LM, Galar-Martınez M. 2014. Oxidative stress induced by mixture of diclofenac and acetaminophen on common carp (Cyprinus carpio). Water Air Soil Pollut 225:1. NORMA Oficial Mexicana (NOM-062-ZOO-1999). Especificaciones tecnicas para la produccion, cuidado y uso de los animales de laboratorio. Secretarıa de Agricultura, Ganaderıa, Desarrollo Rural, Pesca y Alimentacion (SAGARPA).
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postoperative pain after third molar surgery—A prospective randomized study. J Craniomaxillofac Surg 42:629–633. Radi R, Turrens J, Chang Y, Bush M, Capro D, Freeman A. 1991. Detection of catalase in rat heart mitochondria. J Biol Chem 22: 20028–22034. Radwan MA, AlQuadeib BT, Aloudah NM, Aboul Enein HY. 2010. Pharmacokinetics of ketorolac loaded to polyethylcyanoacrylate nanoparticles using UPLC MS/MS for its determination in rats. Int J Pharm 397:173–178. Ragugnetti M, Adams ML, Guimar~aes ATB, Sponchiato G, Carvalho de Vasconcelos E, Ribas de Oliveira CM. 2011. Ibuprofen genotoxicity in aquatic environment: An experimental model using Oreochromis niloticus. Water Air Soil Pollut 218:361–364. Regoli F, Gorbi S, Frenzilli G, Nigro M, Corsi I, Focardi S, Winston GW. 2002. Oxidative stress in ecotoxicology: From the analysis of individual antioxidants to a more integrated approach. Mar Environ Res 54:419–423. Russo C, Rocco L, Morescalchi MA, Stingo V. 2004. Assessment of environmental stress by the micronucleus test and the comet assay on the genome of teleost populations from two natural environments. Ecotox Environ Safe 57:168–174. Santos LHMLM, Araujo AN, Fachini A, Pena A, Delerue-Matos C, Montenegro MCBSM. 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J Hazard Mater 175:45–95. Schmidt W, O’Rourke K, Hernan R, Brian Q. 2011. Effects of the pharmaceuticals gemfibrozil and diclofenac on the marine mussel (Mytilus spp) and their comparison with standardized toxicity tests. Mar Pollut Bull 62:1389–1395. Stegeman JJ, Livingstone DR. 1998. Forms and functions of cytochrome P450. Comp Biochem Physiol C: Toxicol Pharmacol 121:1–3. Sureda A, Tejada S, Boxsa A, Deudero S. 2013. Polycyclic aromatic hydrocarbon levels and measures of oxidative stress in the Mediterranean endemic bivalve Pinna nobilis exposed to the Don Pedro oil spill. Mar Pollut Bull 71:69–73. Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M. 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Safe 64:178–189.
Paglia DE, Valentine WN. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158–169.
Van der Oost R, Beyer J, Vermeulen N. 2003. Fish bioaccumulation and biomarkers in environment risk assessment a review. Environ Toxicol Pharmacol 13:57–149. € u B, Vermeulen NPE, Vos JC. 2012. DifferVan Leeuwen JS, Unl€ ential involvement of mitochondrial dysfunction, cytochrome P450 activity, and active transport in the toxicity of structurally related NSAIDs. Toxicol Vitro 26:197–205.
Parolini M, Binelli A, Cogni D, Provini A. 2010. Multi-biomarker approach for the evaluation of the cyto-genotoxicity of paracetamol on the zebra mussel (Dreissena polymorpha). Chemosphere 79:489–498.
Vitale MG, Choe CJ, Hwang WM, Bauer MR, Hyman EJ, Lee YF, Roye PD. 2003. Use of ketorolac tromethamine in children undergoing scoliosis surgery: an analysis of complications. Spine 3:55–62.
Parvez S, Raisuddin S. 2000. Protein carbonyls: Novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch). Environ Toxicol Pharmacol 20:112–117.
Vlahogianni T, Dassenakis M, Scoullos M, Valavanidis A. 2007. Integrated use of biomarkers (superoxide dismutase, catalase and lipid peroxidation) in mussels Mytilus galloprovincialis for assessing heavy metals’ pollution in coastal areas from the Saronikos Gulf of Greece. Mar Pollut Bull 54:1361.
Orhan H, Sahin G. 2001. In vitro effects of NSAIDS and paracetamol on oxidative stress-related parameters of human erythrocytes. Exp Toxicol Pathol 53:133–140.
Prathibha G, Ramanujapuram ML, Kavitha P. 2014. Comparative study of intravenous Tramadol versus Ketorolac for preventing
Environmental Toxicology DOI 10.1002/tox
tica, Escuela Nacional de Departamento de Farmacia, Laboratorio de Toxicologıa Acua gicas, Instituto Polite cnico Nacional, Ciudad de Me xico, Me xico Ciencias Biolo
2
Unidad Analıtica de la Unidad de Farmacologıa Clınica, Facultad de Medicina - UNAM, yotl, Edo de Me xico Nezahualco 3
Departamento de Farmacia, Facultad de Quımica, Laboratorio de Toxicologıa Ambiental, noma del Estado de Me xico, Toluca, Me xico Universidad Auto
Received 11 August 2014; revised 23 December 2014; accepted 29 December 2014 ABSTRACT: The nonsteroidal anti-inflammatory drug ketorolac is extensively used in the treatment of acute postoperative pain. This pharmaceutical has been found at concentrations of 0.2–60 mg/L in diverse water bodies around the world; however, its effects on aquatic organisms remain unknown. The present study, evaluated the oxidative stress and genotoxicity induced by sublethal concentrations of ketorolac (1 and 60 mg/L) on liver, brain, and blood of the common carp Cyprinus carpio. This toxicant induced oxidative damage (increased lipid peroxidation, hydroperoxide content, and protein carbonyl content) as well as changes in antioxidant status (superoxide dismutase, catalase, and glutathione peroxidase activity) in liver and brain of carp. In blood, ketorolac increased the frequency of micronuclei and is therefore genotoxic for the test species. The effects observed were time and concentration dependent. C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. V
Keywords: ketorolac; oxidative stress; lipid peroxidation; hydroperoxides; oxidized proteins; antioxidant enzymes; genotoxicity; micronucleus; DNA; Cyprinus carpio
INTRODUCTION Pharmaceutical products are biologically active and persistent substances that have been recognized as a constant threat to the stability of the environment. These toxicants form part of the group termed “emerging contaminants” and many tons of them are produced and consumed annually
Correspondence to: M. Galar-Martınez; e-mail: [email protected]; [email protected] or S. Garcıa-Medina, e-mail: [email protected]; [email protected] Contract grant sponsor: Secretarıa de Investigaci on y Posgrado of the Instituto Politecnico Nacional SIP-IPN. Contract grant numbers: 20131086; 20141042 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22113
around the world. This explains their occurrence in the environment, particularly in aquatic ecosystems which they enter through municipal and hospital wastewater discharges (Christen et al., 2010; Ginebreda et al., 2010). These compounds have been designed to elicit a specific biological action in the body and often resist inactivation prior to inducement of their intended therapeutic effect, these properties being paradoxically responsible for both their bioaccumulation and toxic effect on hydrobionts (Santos et al., 2010). One of the most frequently used pharmaceutical groups is that of the nonsteroidal anti-inflammatory drugs (NSAIDs), which includes ketorolac (KTC). Many of these remedies are sold without prescription, favoring selfmedication, and overuse, and increasing the concentrations at which they are found in the environment (Parolini et al., 2010; Islas-Flores et al., 2013). In the case of KTC, it has
C 2015 Wiley Periodicals, Inc. V
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GALAR-MARTINEZ ET AL.
been detected in hospital effluents at concentrations ranging from 0.2 to 59.5 mg/L (Gomez et al., 2006), and in effluents of treatment plants around 1.2 mg/L, since it is difficult to remove (Martınez-Bueno et al., 2012). KTC is an NSAID with a potent analgesic and antiinflammatory activity induced through interference with prostaglandin synthesis via the nonselective inhibition of cyclooxygenase. It is extensively used for clinical management of acute postoperative and cancer-related pain (Jalkut, 2014). Since the compound in the form of free acid is scarcely soluble in water, it is marketed as tromethamine salt, which increases its solubility in water for oral, intramuscular, intravenous, and ophthalmic administration (Radwan et al., 2010). This pharmaceutical is readily and completely absorbed in humans, reaching 100% bioavailability and maximum plasma concentrations about 1 h after administration. KTC has a low apparent volume of distribution: it is more than 99% bound to plasma proteins and has a half-life of 3.5–9.2 h in young adults and 4.7–8.6 h in the elderly (Prathibha et al., 2014). In mammals this compound is metabolized in the liver via Phase I reactions involving cytochrome P450 (CYP) 2C8/9-mediated oxidation to form phydroxy ketorolac as well as Phase II reactions through which it is conjugated with glucuronic acid, the unaltered pharmaceutical and its metabolites being eliminated mostly through the urine (91%) and feces (Kulo et al., 2013). Like many other NSAIDs, KTC induces diverse adverse effects in mammals including reduced cytoprotection of the gastric mucosa, alterations in renal and hepatic function, and inhibition of platelet aggregation (Vitale et al., 2003). Also, since other NSAIDs have been shown to induce oxidative stress in some aquatic species—e.g., diclofenac and ibuprofen, which increase lipid peroxidation (LPX) and protein carbonyl content (PCC) and modify the antioxidant status of carp exposed to sublethal concentrations of the compound (Islas-Flores et al., 2013, 2014); and ibuprofen, diclofenac, and naproxen in the case of Daphnia magna (Gomez-Olivan et al., 2014b), KTC may elicit similar effects on C. carpio; however, there are no reports regarding its toxicity on aquatic species, even though this drug has been found at environmentally relevant concentrations (above 1 mg/L) at diverse kinds of effluents, and is of difficult removal by water treatment plants. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses in the body, and many xenobiotics including some NSAIDs share this mechanism of action. Organisms are able to adapt to oxidative stress through the up-regulation of antioxidant defenses—both enzymatic, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and nonenzymatic such as glutathione—in order to promote ROS detoxification. Failure in this up-regulation results in significant oxidative damage including enzyme inactivation, protein degradation, LPX, and damage to genetic material (Valavanidis et al., 2006; Islas-Flores et al., 2013).
Environmental Toxicology DOI 10.1002/tox
The common carp Cyprinus carpio has a cosmopolitan distribution. In Mexico it has been introduced in 80% of freshwater bodies and is an ecologically and economically important species. In 2012 alone, some 7 tons of this species were grown, harvested and consumed, with an approximate commercial value of 6 million US dollars (Base de Datos del Registro Nacional de Pesca y Acuacultura. 2013). Furthermore, C. carpio is used as a bioindicator in toxicity assays due to its sensitivity and easy maintenance under laboratory conditions. The present study, aimed to evaluate KTC-induced oxidative stress and genotoxicity in liver, brain, and blood of the common carp Cyprinus carpio.
MATERIALS AND METHODS Specimen Procurement and Maintenance Common carp (Cyprinus carpio) with an average weight of 2.3 g, obtained from the carp culturing facility in Tiacaque, (Estado de Mexico), were transported to the laboratory and acclimated for two weeks. During acclimation, specimens were maintained in 80 L glass tanks equipped with filtration systems under 12:12 h light:dark conditions and constant R every third aeration, and were fed Pedregal Silver CorpV day. Tank water had the following physicochemical properties: dissolved oxygen 6.4 6 0.5 mg/L, ammonia concentration 0.32 6 0.2 mg/L, nitrates 0.26 6 0.001 mg/L, temperature 20 6 2 C, oxygen saturation 90–100% and pH 7.5–8.0.
Toxicity Assays Prior to the present study, an acute toxicity assay was performed in order to verify that the test concentrations to be used were not lethal to C. carpio. Exposure systems were set up with six 80 L tanks (one per each exposure time) and six carp were placed in each. The test concentrations used were 1 and 60 mg KTC/L (Sigma, CAS 74103-07-4). The former is the threshold safety value in water established by the European Medicines Agency (EMEA) (Ginebreda et al., 2010) and the latter is a previously reported concentration found in the environment (Gomez et al., 2006; Santos et al., 2010). The exposure periods used were 12, 24, 48, 72, and 96 h. Conditions during the study were similar to those used for specimen maintenance. At the end of the exposure period, carp were anesthetized by immersion in 2% lidocaine for 30 s. Anesthetized specimens were placed in a lateral position and a blood sample was taken by puncture of the caudal vessel using a heparinized 1 mL hypodermic syringe and performed laterally near the base of the caudal peduncle, at mid-height of the anal fin and ventral to the lateral line. After which, specimens were placed in an ice bath, sacrificed and the liver and brain were removed. Tissue samples were supplemented with 2 mL phosphate buffer solution pH 7.4,
KETOROLAC INDUCES TOXICITY IN COMMON CARP
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then homogenized and centrifuged at 12,500 rpm and 24 C for 15 min. Whole blood samples were used to evaluate genotoxicity by micronucleus test, while oxidative stress was assessed in liver and brain homogenates using the following biomarkers: LPX, hydroperoxide content (HPC), and PCC, as well as SOD, CAT, and GPx activity. Total protein content was used to express biomarker results. Three replicate tanks were set up per each test concentration and exposure time, and solutions were not replaced. This protocol was reviewed and approved by the Bioethics Committee of the Escuela Nacional de Ciencias Biolgicas (ENCB-IPN) to ensure it was carried out in accordance with institutional standards for the care of animal test subjects. The provisions set out in the official Mexican norm on the production, care and use of laboratory animals (NOM-062-ZOO-1999) were also taken into account.
guanidine (Sigma) pH 2.3, and incubated at 37 C for 30 min. Absorbance was read at 366 nm and results were expressed as nmol reactive carbonyls formed/mg protein based on their MEC of 21,000 M/cm.
Determination of LPX
CAT activity was determined as proposed by Radi et al. (1991). To 20 lL of supernatant was added 1 mL isolation buffer solution (0.3M saccharose, 1 mM EDTA, 5 mM HEPES, and 5 mM KH2PO4) and 0.2 mL hydrogen peroxide (20 mM). Absorbance was read at 240 nm, at 0 and 60 s. Results were obtained by substituting the absorbance value obtained for each time in the formula: catalase activity 5 (A60 2 A0)/MEC, where the MEC of H2O2 equals 0.043 mM/ cm. Results were expressed as mM H2O2/mg protein.
LPX was determined as in B€uege and Aust (1978). The cell pellet was reconstituted with Tris–HCl buffer (pH 7.4) until a 5 mL volume was attained, and 1 mL of this solution was incubated at 37 C for 30 min. Next, 2 mL TBA-TCA reagent (0.375% thiobarbituric acid in 15% trichloroacetic acid) was added; the sample was shaken in a vortex, placed in a bath of boiling water for 45 min, allowed to cool, and the precipitate removed by centrifugation at 3,000 rpm for 10 min. Absorbance was read at 535 nm using a reaction blank. Results were expressed as mM malondialdehyde (MDA)/mg protein, using the molar extinction coefficient (MEC) of 1.56 3 105 M cm.
Determination of HPC HPC was evaluated by the Jiang et al. (1991) method. To 100 mL of supernatant (previously deproteinized with 10% TCA) was added 900 mL of the reaction mixture [0.25 mM FeSO4, 25 mM H2SO4, 0.1 mM xylenol orange, and 4 mM butylated hydroxytoluene in 90% (v/v) methanol]. The mixture was incubated for 60 min at ambient temperature and absorbance was determined at 560 nm against a blank containing only reaction mixture. Results were interpolated on a type curve and were expressed as nM cumene hydroperoxide (CHP)/mg protein.
Determination of PCC PCC was determined by the method of Levine et al. (1994). Soluble proteins were obtained by centrifugation of samples at 10,500 rpm for 30 min. To 100 lL of this supernatant was added 150 lL of 10 mM dinitrophenylhydrazine in 2M HCl (Sigma) prior to incubation at room temperature for 1 h in the dark. Next, 500 lL of 20% TCA was added and the sample allowed to rest for 15 min at 4 C, then centrifuged at 16,000 rpm for 5 min. The bud was rinsed thrice in 1:1 ethanol:ethyl acetate (Baker), dissolved in 150 lL of 6M
Determination of SOD Activity SOD activity was determined according to the Misra and Fridovich (1972) method. To 20 lL of supernatant in a 1 cm cuvette was added 150 lL carbonate buffer solution (50 mM sodium carbonate and 0.1 mM EDTA) pH 10.2, plus 100 lL adrenaline (30 mM). Absorbance was read at 480 nm, at 30 s and 5 min. SOD activity was determined by interpolating the data on a type curve. Results were expressed as IU SOD/mg protein.
Determination of CAT Activity
Determination of GPx Activity GPx activity was determined using Paglia and Valentine’s (1967) methodology. To 100 lL of supernatant was added 900 lL buffer reagent solution (5M K2HPO4, 5M KH2PO4, 3.5 mM reduced glutathione, 1 mM sodium azide, 2 U glutathione reductase, and 0.12 mM NADPH, pH 7.0; Sigma) plus 200 lL H2O2 (20M). Absorbance was read at 340 nm, at 0 and 60 s. Activity was estimated using the MEC of NADPH (6.2 mM/cm). Results were expressed as mM NADPH/mg protein.
Determination of Total Protein Content To 25 lL of supernatant was added 75 lL deionized water and 2.5 mL Bradford’s reagent (0.05 g Coomassie Blue dye, 25 mL of 96% ethanol, and 50 mL H3PO4, in 500 mL deionized water). The test tubes were shaken and allowed to rest for 5 min prior to reading of absorbance at 595 nm and interpolation on a bovine albumin curve (Bradford, 1976).
Micronucleus Test A smear of the cell suspension obtained from each specimen was fixed with pure ethanol for 5 min, then treated with 10% Giemsa stain for 9 min. A total of 1000 erythrocytes from each sample were examined under a light microscope, and frequency of micronuclei (MNi) was expressed as the number of micronucleated cells per 1000 cells (Cavas and
Environmental Toxicology DOI 10.1002/tox
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GALAR-MARTINEZ ET AL.
Fig. 1. Lipid peroxidation (LPX) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
centration, but decreased drastically thereafter and attained values comparable to those of the control group from 48 h on. Meanwhile in fish exposed to the higher test concentration, LPX increased in liver beginning at 24 h, reached its maximum value at 72 h (376%) (p < 0.05) and maintained increased values up to 96 h. In brain of carp exposed to either concentration, there was a significant increase in LPX as compared to the control group (p < 0.05) which began at 12 h and was maintained up to the end of the experiment, this damage being greater with the higher concentration. HPC results (Fig. 2) show that KTC induced a major increase in this parameter with respect to the control group from 24 h on in liver of common carp exposed to either concentration. However, in specimens exposed to 1 mg/L, HPC peaked at 48 h (a 165% increase as compared to the control group, p < 0.05) and thereafter decreased until values similar to those of the control group were attained, while in carp exposed to 60 mg/L the damage induced was not reversed. On the other hand, in brain of carp exposed to either test concentration, HPC increased with respect to the control group at 12 and 24 h (p < 0.05), decreasing thereafter and reaching values comparable to those of the control group from 48 h on. Figure 3 shows PCC results. In liver of carp exposed to either concentration this biomarker increased significantly
Ergene-G€ oz€ ukara, 2005). The criteria used to determine the presence of MNi were nonlinkage of small ovoid or round nuclei with the main nucleus, color, and staining intensity similar to the main nucleus (II-Yong and Chang-Kee, 2006), and diameter 1/5–1/20 of the main nucleus (Bolognesi et al., 2006).
Statistical Analysis Results of oxidative stress determination were subjected to a one-way analysis of variance (ANOVA) (p < 0.05), while differences between means were analyzed using the StudentNewman-Keuls method. Mni frequency was analyzed with a Kruskal–Wallis nonparametric ANOVA (p < 0.05), followed by Tukey’s post hoc test for multiple comparison of means. Sigma Stat v3.5 was used throughout.
RESULTS Oxidative Stress Figure 1 shows LPX results in common carp exposed to sublethal concentrations of KTC. This biomarker increased markedly with respect to the control group during early exposure periods (12 and 24 h, 70 and 246%, respectively) (p < 0.05) in liver of specimens exposed to the lower con-
Environmental Toxicology DOI 10.1002/tox
Fig. 2. Hydroperoxide content (HPC) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
KETOROLAC INDUCES TOXICITY IN COMMON CARP
with respect to the control group (p < 0.05) from the earliest exposure period (12 h) on. This trend was maintained during the course of the experiment in the group exposed to the higher concentration and reached its peak at 72 h with a 1,388% increase with respect to the control group. However, in liver of specimens exposed to the lower concentration, PCC values were similar to those of the control group from 72 h on. In brain of carp exposed to either concentration, PCC increased as compared to the control group (p < 0.05) at 12 h only and thereafter decreased until values similar to those of the control group were reached. As regards the antioxidant status of carp exposed to KTC, SOD activity (Fig. 4) was seen to increase with respect to the control group from 12 h on in liver of fish exposed to either test concentration (p < 0.05). This trend was maintained throughout the experiment in the group exposed to 60 mg/L and reached its peak at 72 h with a 264% increase with respect to the control group. However, in specimens exposed to 1 mg/L, SOD activity obtained values as comparable to those of the control group from 72 h on. In brain, this activity increased in specimens exposed to either concentration during early exposure periods (12 and 24 h) and decreased significantly thereafter compared to the control group in both cases (72% at 72 h with 1 mg/L, and 51% at 96 h with 60 mg/L, p < 0.05). CAT activity (Fig. 5) increased from 24 h on with respect to the control group (p < 0.05) in liver of carp exposed to
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Fig. 4. Superoxide dismutase (SOD) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA, and SNK (p < 0.05).
either concentration, peaking in both cases at 72 h (659 and 1335% with 1 and 60 mg/L, respectively). A similar behavior was found to occur in brain, although the maximum value was reached at 24 h in carp exposed to the higher concentration (239%) and at 72 h in those exposed to the lower concentration (56%). Figure 6 shows GPx activity results. In liver of carp exposed to the lower concentration, activity increased with respect to the control group (p < 0.05) during early exposure periods (85 and 380% at 12 and 24 h, respectively), but this effect was reversed from 48 h on. At the higher concentration, this enzyme activity decreased markedly compared to the control group from 12 h on and this tendency was maintained throughout the experiment. A similar behavior was observed in brain: increased activity in fish exposed to the lower test concentration and reduced activity in those exposed to the higher concentration.
Genotoxic Response
Fig. 3. Protein carbonyl content (PCC) in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
Micronucleus test results are shown in Figure 7. MNi frequency increased remarkably from 12 h on with exposure to either concentration and reached peak values at 96 h (1600 and 2400% with 1 and 60 mg/L respectively), damage being much more marked in specimens exposed to the higher concentration (p < 0.05).
Environmental Toxicology DOI 10.1002/tox
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Fig. 5. Catalase (CAT) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and SNK (p < 0.05).
DISCUSSION Oxidative stress is one of the most important mechanisms of toxicity, given the impact that an imbalance between reactive species and antioxidant defenses has on vital biomolecules such as lipids, proteins, and genetic material, as well as the countless toxicants capable of inducing it (metals, hydrocarbons, pesticides, and NSAIDs such as ibuprofen, diclofenac, paracetamol, etc.) (Hernandez et al., 2013; Sureda et al., 2013; Espın et al., 2014; Islas-Flores et al., 2014; Nava Alvarez et al., 2014). NSAIDs are extensively used globally and various studies indicate that several members of this group of pharmaceuticals can induce oxidative stress on aquatic species, e.g. ibuprofen, naproxen, paracetamol, and acetylsalicylic acid in D. magna (G omez-Olivan et al., 2014a,b) or diclofenac in Myti lus spp. (Schmidt et al., 2011) and C. carpio (Nava-Alvarez et al., 2014). However, no reports are available on the effects of KTC on aquatic species. Results obtained in the present study show that KTC at sublethal and environmentally relevant concentrations (1 and 60 mg/L) induces this type of damage, since LPX, HPC, and PCC increases are elicited in both liver and brain of C. carpio exposed to this pharmaceutical, such damage being concentration and organ dependent.
Environmental Toxicology DOI 10.1002/tox
Fig. 6. Glutathione peroxidase (GPx) activity in liver and brain of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA, and SNK (p < 0.05).
As regards LPX, the increase found in brain is both larger and sustained, while in liver this damage is smaller and shows a tendency towards recovery of normal values near the end of exposure with the lower concentration. A similar effect was observed in the latter organ in relation to HPC and PCC, even though in brain these parameters were less strongly affected and also evidenced a tendency towards recovery of normal values, damage in both organs being greater in specimens exposed to the higher concentration.
Fig. 7. Frequency of micronuclei (MNi) in blood of Cyprinus carpio exposed to ketorolac. Values are the mean 6 SE. *Significantly different from control group, ANOVA and Tukey’s (p < 0.05).
KETOROLAC INDUCES TOXICITY IN COMMON CARP
In mammalian cells, NSAID toxicity is related to mitochondrial dysfunction, transporter efflux, and CYP-mediated metabolism (Van Leeuwen et al., 2012) since metabolites and reactive species may be produced during biotransformation. It is worth noting that CYP can produce an oxygenated intermediate—the oxy-cytochrome P450 complex [P450 (Fe31) O22]—during the metabolic cycle and release the superoxide anion (Doi et al., 2002), promoting oxidative stress and subsequent damage to biomolecules such as lipids, proteins, and genetic material. In this sense, it is worth noting that diverse CYP gene families have been characterized in C. carpio including CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, and CYP19 (Stegeman and Livingstone, 1998); the CYP2 gene family, particularly CYP2C8/9, being responsible for the biotransformation of KTC to p-hydroxy-ketorolac via phase I oxidation reactions (Kulo et al., 2013). It is thus probable that this metabolite as well as potentially reactive species including the superoxide anion are produced also in the common carp, which may explain the increases found in our study in biomarkers of oxidative damage (LPX, HP, and PCC). As regards the differences observed between the organs evaluated, it is worth noting that the liver is the primary biotransforming organ, so that formation of reactive metabolites is enhanced. However, the brain is particularly vulnerable to ROS due to the large amounts of oxygen required to carry out its activities as well as the fact that its membranes are rich in polyunsaturated fatty acids (Halliwell, 2006), which may favor LPX and hence increased HPC. On the other hand, the reactive metabolites and ROS produced during CYP-mediated biotransformation can also affect proteins, inducing increased PCC (as in our study), which leads to loss of sulfhydryl groups and changes in the resonance structures of amino acids, altering their function and therefore the integrity of the body (Parvez and Raisuddin, 2000). The antioxidant defense system is essential in the neutralization of ROS and related damage (Regoli et al., 2002). This system is mediated by a cascade of antioxidant enzymes that sequester ROS and convert them to less toxic and reactive species. This group of enzymes includes SOD, CAT, and GPx. In our study, SOD activity in liver of specimens exposed to 60 mg KTC/L increased from 12 h on and this trend was maintained up to the end of the experiment, while in specimens exposed to 1 mg KTC/L this increase occurred only at 48 h and later values were similar to those of the control group. A comparable effect was seen in brain of carp exposed to the lower concentration, while exposure to the higher one induced an activity reduction. SOD is the main enzyme responsible for offsetting the effects induced by the superoxide ion (Van der Oost et al., 2003), which it transforms to hydrogen peroxide. As stated previously, since CYP is present in C. carpio, formation of the oxycytochrome P450 complex [P450 (Fe31) O22] and release of the superoxide ion are possible, activating SOD in order to
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offset the oxidative effect of this free radical. This may explain the results in liver and brain obtained in our study during early exposure periods with either concentration. Increased SOD activity may act as a signal of oxidative stress, leading to activation and/or induction of antioxidant enzymes associated with a system of H2O2-sequestration such as CAT or GPx (Vlahogianni et al., 2007). In our study, CAT activity in brain of specimens exposed to either concentration increased at the start of exposure and decreased thereafter, while in liver there was a steady increase over the course of the experiment. In both organs, the increase observed in CAT activity may be due to higher concentrations of H2O2 as a result of SOD activity. Furthermore, Bagnyukova et al. (2006) state that LPX products may be involved in the regulation of some antioxidant enzymes, so that the LPX increase found in our study may likewise explain the increased antioxidant enzyme (SOD, CAT, and GPx) activity observed. Finally, GPx activity increased in our study at the beginning of exposure in liver and brain of specimens exposed to the lower concentration of KTC and thereafter the damage was reversed, while exposure to the higher concentration induced a reduction in both organs. One consequence of oxidative stress is protein damage (Parvez and Raisuddin, 2000). In our study, the higher concentration of KTC may induce ROS in larger numbers, exceeding the neutralizing capacity of the antioxidant system and damaging the enzymes that compose it—in this case GPx. A similar effect was observed by Orhan and Sahin (2001) in human erythrocytes exposed to therapeutic concentrations of KTC. Chandran et al. (2005) state that ROS, particularly the superoxide ion, directly affect the activity of antioxidant enzymes modifying their structure, which may explain the reduction found in our study. Many environmental contaminants are not only hazardous to the physiology and survival of organisms, but can also induce genetic changes leading to mutations which may put at risk species survival (Russo et al., 2004). Mutagenic chemicals may induce DNA lesions such as chain breaks, base modifications and crosslinks in aquatic organisms, producing adverse effects on ecosystem stability. Diverse techniques enable their evaluation, including MNi frequency determination, which detects MNi resulting from chromosomal breakage during cell division, loss of chromosomes due to damage in anaphase, and short-term cytogenetic damage (Russo et al., 2004; Kim and Hyun, 2006). In fish, the micronucleus technique has proved useful in vivo for evaluating genotoxicity and in situ for monitoring water quality (Kim and Hyun, 2006). In fish, this technique is usually performed on erythrocytes, which in these organisms are nucleated and very sensitive to different contaminants (Ragugnetti et al., 2011). In our study, MNi frequency evidenced a time- and concentration-dependent increase from 48 h on. Although KTC has not been studied in terms of its potential genotoxicity, various authors have demonstrated that NSAIDs can
Environmental Toxicology DOI 10.1002/tox
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induce damage to DNA, e.g., Ragugnetti et al. (2011), who found that MNi frequency increases in Oreochromis niloticus subchronically exposed to ibuprofen; or Parolini et al. (2010), who demonstrated that paracetamol induces DNA translocations, inversions, and fragmentation in Dreissena polymorpha in addition to increased apoptotic cell frequency and reduced lysosomal membrane stability, probably due to reactive metabolites and ROS formed during its biotransformation. This may also be the case of KTC which, as stated above, is metabolized by CYP. It is important to mention that in addition to the fact that phase I of the biotransformation process can produce reactive metabolites, glucuronide complexes of paracetamol induce an increase in MNi frequency and also inhibit cell division and nondisjunction of chromosomes. KTC may also undergo phase II reactions during which it is conjugated with glucuronic acid to form ketorolac-glucuronide by means of the enzyme uridine diphosphate glucuronosyltransferase (UGT) 2B7, analogs of which have been found in fish (UDP-glucuronosyltransferase, UDPGT; George and Taylor, 2002), it being probable that this metabolite is also produced in C. carpio. On the other hand, Asensio et al. (2007) state that NSAIDs inhibit glucose-6-phosphate dehydrogenase due to formation of NSAID-acyl-CoA or NSAID-CoA complexes, and as a result ribose-5-phosphate synthesis is halted and consequently also nucleic acid synthesis, it being possible therefore that KTC elicits a similar effect. Furthermore, this kind of damage may be indirectly induced if reactive metabolites or ROS formed in the biotransformation of KTC damage the proteins responsible for DNA synthesis and repair (G omez-Olivan et al., 2014a). n conclusion, results of the present study show that KTC at sublethal and environmentally relevant concentrations (1 and 60 mg/L) induces oxidative stress and genotoxicity on C. carpio.
REFERENCES Asencio C, Levoin N, Guillaume C, Guerquin MJ, Rouguieg K, Chretien F, Chapleur Y, Netter P, Minn A, Lapicque F. 2007. Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: A key to oxidative stress induced by non-steroidal anti-inflammatory drugs? Biochem Pharmacol 73:405–416. Bagnyukova T, Chahrak O, Lushchak V. 2006. Coordinated response of goldfish antioxidant defenses to environmental stress. Aquat Toxicol 78:325–331.
Bradford M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. B€uege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods Enzymol 52:302–310. Cavas T, Ergene-G€oz€ukara S. 2005. Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquat Toxicol 74:264–271. Chandran R, Sivakumar AA, Monadas S, Aruchami M. 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica. Comp Biochem Physiol C: Toxicol Pharmacol 140:422–426. Christen V, Hickmann S, Rechenberg B, Fent K. 2010. Highly active human pharmaceuticals in aquatic systems: A concept for their identification based on their mode of action. Aquat Toxicol 96:167–181. Doi H, Iwasaki H, Masubushi Y, Nishigaki R, Horie T. 2002. Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes. Chem–Biol Interact 140:109–119. Espın S, Martınez-Lopez E, Leon-Ortega M, Martınez JE, GarcıaFernandez AJ. 2014. Oxidative stress biomarkers in Eurasian eagle owls (Bubo bubo) in three different scenarios of heavy metal exposure. Environ Res 131:134–144. George SG, Taylor B. 2002. Molecular evidence for multiple UDP-glucuronosyltransferase gene families in fish. Mar Environ Res 54:253–257. Ginebreda A, Mu~noz I, Lopez de Alda M, Brix R, Lopez-Doval J, Barcelo D. 2010. Environmental risk assessment of pharmaceuticals in rivers: Relationships between hazard indexes and aquatic macroinvertebrate diversity indexes in the Llobregat River (NE Spain). Environ Int 36:153–162. Gomez MJ, Petrovic´ M, Fernandez-Alba AR, Barcelo D. 2006. Determination of pharmaceuticals of various therapeutic classes by solid-phase extraction and liquid chromatography-tandem mass spectrometry analysis in hospital effluent wastewaters. J Chromatogr A 1114:224–233. Gomez-Olivan L, Galar-Martınez M, Garcıa-Medina S, ValdesAlanıs A, Islas-Flores H, Neri-Cruz N. 2014a. Genotoxic response and oxidative stress induced by diclofenac, ibuprofen and naproxen in Daphnia magna. Drug Chem Toxicol 37:391–399. Gomez-Olivan L, Galar-Martınez M, Islas-Flores H, GarcıaMedina S, SanJuan-Reyes N. 2014b. DNA damage and oxidative stress induced by acetylsalicylic acid in Daphnia magna. Drug Chem Toxicol 164:21–26. Halliwell B. 2006. Oxidative stress and neurodegeneration: Where are we now? J Neurochem 97:1634–1658.
Base de Datos del Registro Nacional de Pesca y Acuacultura. 2013. (RNPA), Consejo Nacional de Pesca de la Secretarıa de Agricultura, Ganaderıa, Desarrollo Rural, Pesca y Alimentacion (SAGARPA), Mexico.
Hernandez AF, Lacasa~na M, Gil F, Rodrıguez-Barranco M, Pla A, Lopez-Guarnido O. 2013. Evaluation of pesticide-induced oxidative stress from a gene-environment interaction perspective. Toxicology 307:95–102.
Bolognesi C, Perrone E, Roggieri P, Pampanin DM, Sciutto A. 2006. Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions. Aquat Toxicol 78:93–98.
II-Yong K, Chang-Kee H. 2006. Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicol Environ Saf 64:288–297.
Environmental Toxicology DOI 10.1002/tox
KETOROLAC INDUCES TOXICITY IN COMMON CARP
Islas-Flores H, Gomez-Olivan LM, Galar-Martınez M, Colın-Cruz A, Neri-Cruz N, Garcıa-Medina S. 2013. Diclofenac-induced oxidative stress in brain, liver, gill and blood of common carp (Cyprinus carpio). Ecotoxicol Environ Safe 92:32–38. Islas-Flores H, Gomez-Olivan LM, Galar-Martınez M, GarcıaMedina S, Neri-Cruz N, Dublan-Garcıa O. 2014. Effect of ibuprofen exposure on blood, gill, liver and brain of common carp (Cyprinus carpio) using oxidative stress biomarkers. Environ Sci Pollut Res 21:5157–5166. Jalkut MK. 2014. Ketorolac as an analgesic agent for infants and children after cardiac surgery: Safety profile and appropriate patient selection. AACN Adv Crit Care 25:23–30. Jiang ZY, Wollard ACS, Wolff SP. 1991. Lipid hydroperoxide measurement by oxidation of Fe21 in the presence of xylenol orange-comparison with the TBA assay and an iodometric method. Lipids 26:853–856. Kim IY, Hyun CK. 2006. Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicol Environ Safe 64:288–297. Kulo A, Hendrickx S, de Hoon J, Mulabegovic N, van Calsteren K, Verbesselt R, Allegaert K. 2013. The impact of pregnancy on urinary ketorolac metabolites after single intravenous bolus. Eur J Drug Metabol Pharmacokinet 38:1–4. Levine R, Williams J, Stadtman E, Shacter E. 1994. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357. Martınez-Bueno MJ, Gomez MJ, Herrera S, Hernando MD, Ag€uera A, Fernandez-Alba AR. 2012. Occurrence and persistence of organic emerging contaminants and priority pollutants in five sewage treatment plants of Spain: Two years pilot survey monitoring. Environ Pollut 164:267–273. Misra P, Fridovich I. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175. Nava-Alvarez R, Razo-Estrada AC, Garcıa-Medina S, GomezOlivan LM, Galar-Martınez M. 2014. Oxidative stress induced by mixture of diclofenac and acetaminophen on common carp (Cyprinus carpio). Water Air Soil Pollut 225:1. NORMA Oficial Mexicana (NOM-062-ZOO-1999). Especificaciones tecnicas para la produccion, cuidado y uso de los animales de laboratorio. Secretarıa de Agricultura, Ganaderıa, Desarrollo Rural, Pesca y Alimentacion (SAGARPA).
9
postoperative pain after third molar surgery—A prospective randomized study. J Craniomaxillofac Surg 42:629–633. Radi R, Turrens J, Chang Y, Bush M, Capro D, Freeman A. 1991. Detection of catalase in rat heart mitochondria. J Biol Chem 22: 20028–22034. Radwan MA, AlQuadeib BT, Aloudah NM, Aboul Enein HY. 2010. Pharmacokinetics of ketorolac loaded to polyethylcyanoacrylate nanoparticles using UPLC MS/MS for its determination in rats. Int J Pharm 397:173–178. Ragugnetti M, Adams ML, Guimar~aes ATB, Sponchiato G, Carvalho de Vasconcelos E, Ribas de Oliveira CM. 2011. Ibuprofen genotoxicity in aquatic environment: An experimental model using Oreochromis niloticus. Water Air Soil Pollut 218:361–364. Regoli F, Gorbi S, Frenzilli G, Nigro M, Corsi I, Focardi S, Winston GW. 2002. Oxidative stress in ecotoxicology: From the analysis of individual antioxidants to a more integrated approach. Mar Environ Res 54:419–423. Russo C, Rocco L, Morescalchi MA, Stingo V. 2004. Assessment of environmental stress by the micronucleus test and the comet assay on the genome of teleost populations from two natural environments. Ecotox Environ Safe 57:168–174. Santos LHMLM, Araujo AN, Fachini A, Pena A, Delerue-Matos C, Montenegro MCBSM. 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J Hazard Mater 175:45–95. Schmidt W, O’Rourke K, Hernan R, Brian Q. 2011. Effects of the pharmaceuticals gemfibrozil and diclofenac on the marine mussel (Mytilus spp) and their comparison with standardized toxicity tests. Mar Pollut Bull 62:1389–1395. Stegeman JJ, Livingstone DR. 1998. Forms and functions of cytochrome P450. Comp Biochem Physiol C: Toxicol Pharmacol 121:1–3. Sureda A, Tejada S, Boxsa A, Deudero S. 2013. Polycyclic aromatic hydrocarbon levels and measures of oxidative stress in the Mediterranean endemic bivalve Pinna nobilis exposed to the Don Pedro oil spill. Mar Pollut Bull 71:69–73. Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M. 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Safe 64:178–189.
Paglia DE, Valentine WN. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158–169.
Van der Oost R, Beyer J, Vermeulen N. 2003. Fish bioaccumulation and biomarkers in environment risk assessment a review. Environ Toxicol Pharmacol 13:57–149. € u B, Vermeulen NPE, Vos JC. 2012. DifferVan Leeuwen JS, Unl€ ential involvement of mitochondrial dysfunction, cytochrome P450 activity, and active transport in the toxicity of structurally related NSAIDs. Toxicol Vitro 26:197–205.
Parolini M, Binelli A, Cogni D, Provini A. 2010. Multi-biomarker approach for the evaluation of the cyto-genotoxicity of paracetamol on the zebra mussel (Dreissena polymorpha). Chemosphere 79:489–498.
Vitale MG, Choe CJ, Hwang WM, Bauer MR, Hyman EJ, Lee YF, Roye PD. 2003. Use of ketorolac tromethamine in children undergoing scoliosis surgery: an analysis of complications. Spine 3:55–62.
Parvez S, Raisuddin S. 2000. Protein carbonyls: Novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channa punctata (Bloch). Environ Toxicol Pharmacol 20:112–117.
Vlahogianni T, Dassenakis M, Scoullos M, Valavanidis A. 2007. Integrated use of biomarkers (superoxide dismutase, catalase and lipid peroxidation) in mussels Mytilus galloprovincialis for assessing heavy metals’ pollution in coastal areas from the Saronikos Gulf of Greece. Mar Pollut Bull 54:1361.
Orhan H, Sahin G. 2001. In vitro effects of NSAIDS and paracetamol on oxidative stress-related parameters of human erythrocytes. Exp Toxicol Pathol 53:133–140.
Prathibha G, Ramanujapuram ML, Kavitha P. 2014. Comparative study of intravenous Tramadol versus Ketorolac for preventing
Environmental Toxicology DOI 10.1002/tox
