Oncorhynchus mykiss.
Rodrigues, S.1,2, Antunes, S.C.1,2, Correia, A.T.2,3, Nunes, B.4,5
1Departamento de Biologia da Faculdade de Ciências da Universidade do Porto (FCUP),
Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
2Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Terminal
de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4450-208 Matosinhos, Portugal
3Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Rua
Carlos da Maia, 296, 4200-150, Porto, Portugal.
4Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193
Aveiro, Portugal
5Centro de Estudos do Ambiente e do Mar (CESAM), Campus de Santiago,
Universidade de Aveiro, 3810-193 Aveiro, Portugal
Corresponding author
Bruno Nunes: Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
Abstract
Oxytetracycline (OTC) is a tetracycline antibiotic, widely used in human and veterinary medicines, including in aquaculture. Given this use, OTC has been detected in different aquatic environments. Some recent works have demonstrated unintentional biological activity of OTC in non-target aquatic organisms. This study investigated the acute and chronic effects of OTC on the physiology of the fish species Oncorhynchus mykiss (rainbow trout), namely through the quantification of the activity of enzymes involved in different biochemical pathways, such as detoxification (phase II - glutathione S- transferases - GSTs, uridine-diphosphate-glucuronosyltransferases - UGTs), neurotransmission (acetylcholinesterase - AChE) and energy production (lactate dehydrogenase - LDH). The here-obtained data demonstrated the induction of GSTs activity in gills, and inhibition of AChE activity in eyes tissue, in chronically exposed organisms, as well as alterations in LDH activity following both exposures. Considering this set of results, we can infer that OTC exposure may have induced the glutathione pathway of detoxification in gills with the involvement of GSTs, or indirectly due to the metabolites that may have been produced. In turn, these metabolites may have interfered with the mechanism of neurotransmission, also causing physiological and biochemical disturbances in rainbow trout after OTC exposure, namely disturbances in energetic metabolism. In addition, it is important to stress that such occurrences took place at low, environmentally realistic levels of OTC, suggesting that organisms exposed in the wild may be putative targets of toxic effects by commonly used drugs such as antibiotics.
Keywords
Rainbow trout; Antibiotic; Biotransformation enzymes; Acetylcholinesterase; Metabolism
Introduction
Among the vast number of pharmaceuticals commonly used in both human and animal therapeutics, some are antibiotics. According to the statistics, in the European Union, the annual consumption of antibiotics is approximately 5,000 tons, and for tetracycline alone, it is estimated to be around 2,300 tons (nearly 46% of the total amount of the antibiotics class) (Wang and Wei, 2013). These numbers are demonstrative of the actual scenario of widespread consumption of these specific class of drugs, which is also responsible for their presence in the environment. In addition, specific features turn pharmaceutical drugs into particularly troublesome compounds when in the wild, since they are biologically active and may act as potential environmental micropollutants.
Consequently, antibiotics have been considered compounds of high environmental concern (Barros-Becker et al. 2012; Romero et al. 2012; Ambili et al. 2013; Rodrigues et al. 2017a), especially of the aquatic environment.
Tetracyclines are antibiotics, which are widely used in human and veterinary therapeutics (Halling- Sørensen et al. 1998). Among this class, one of the most commonly used is oxytetracycline (OTC) (Nebot et al. 2014), for the treatment of several human diseases (e.g. cholera, anthrax, plague, syphilis, chlamydia, lyme disease, typhus, respiratory infection, relapsing fever, mycoplasma, tularaemia, malaria, rickettsiae, streptococcal infection and acne) (Jayanthi and Subash, 2010; Guerra et al. 2016); the use of OTC is not restricted to human medicine, since it is also used in aquaculture, given its broad-spectrum efficacy in the treatment of several bacterial fish diseases, and as a growth promotor in animal farming (Rodrigues et al. 2017a).
OTC has been detected in ng/L - µg/L range, in wastewater effluents and natural waters (Halling-Sørensen et al. 1998; Fent et al. 2006; Kümmerer, 2009). In surface waters (e.g. rivers), concentrations of OTC were already detected ranging from 0.0003 to 340 ng/L (Kolpin et al. 2002; Calamari et al. 2003; Pailler et al. 2009) and in Chinese rivers its presence was reported in concentrations ranging from 0.235 to 0.712 mg/L (Li et al. 2008). Since OTC is inefficiently eliminated by WWTPs (Jelić et al. 2012), the study of its effects is necessary to prevent the deterioration of ecosystems, and to preserve biodiversity.
Among all potential biological effects caused by pollutants, biochemical changes occur more rapidly and show higher sensitivity. In ecotoxicological analyses, biomarkers have thus been widely used as early warning signs of intoxication because they can anticipate severe alterations in organisms exposed to pollutants that could result in deleterious alterations at the population level (Peakall, 1992). Among all biomarkers available, the assessment of deleterious changes in metabolic, neurological, and respiratory pathways is a successful strategy, considering their physiological importance and the availability of analytic methodologies for their quantification. The activities of phase II conjugating enzymes (including glutathione S-transferases (GSTs) and uridine- diphosphate (UDP)-glucuronosyltransferases UGTs) are particularly important as putative markers of metabolic alterations, since the primary toxicity of drugs is exerted mainly at molecular and biochemical levels. Other frequently assessed endpoints include neuronal toxicity (inhibition of acetylcholinesterase (AChE) activity), and alterations of cellular respiration (lactate dehydrogenase (LDH) activity, which have also been shown to be responsive in a large variety of species; Pari and Gnanasoundari, 2006; Delwing- de Lima et al. 2010; Li et al. 2012; Ambili et al. 2013; Oliveira et al. 2013; Samanta et al. 2014). All mentioned enzymes can be targets for the toxic activity of xenobiotics, but also
by-products of detoxification processes, serving as tools to diagnose the effects of environmental pollutants.
Biotransformation is usually a two-phase process, divided in phase I and phase II. In phase II reactions, a diverse group of enzymes is involved, which only act in the presence of specific cofactors. Glucuronic and glutathione conjugates are main metabolites in fish (Ikenaka et al. 2013). GSTs are a group of biotransformation isoenzymes that catalyze the conjugation of a variety of metabolites or toxic compounds (endogenous or foreign compounds) with reduced glutathione (GSH) to form conjugates that are more easily excretable (Jancova et al. 2010). GSTs have been used as a biomarker in several ecotoxicological studies. Glucuronidation, accomplished by uridine- diphosphate-glucuronosyltransferases (UGTs), represents a major pathway of conjugation biotransformation metabolism in all vertebrates, including fish (Sturm et al. 1999). The synthesis of glucuronides by UGTs is a major pathway for the inactivation and subsequent excretion of both xenobiotics and endogenous compounds (Van der Oost et al. 2003). There are a few studies that claim that OTC affects important biochemical pathways. OTC caused disturbances in phase II detoxification processes, in several aquatic organisms (e.g. fish) (Yonar, 2012; Oliveira et al. 2013; Wang et al. 2016; Ren et al 2017).
AChE plays an important role in the mechanism of neurotransmission; and its inhibition has been used to detect the exposure to anticholinesterase compounds, such as carbamate (CB) and organophosphorus (OP) pesticides (Monteiro et al; 2007; Rodrigues et al. 2012). More recently, several works have demonstrated that AChE is also sensitive to other compounds, including some surfactants, metals and also pharmaceuticals (Nunes et al. 2006; Monteiro et al. 2007; Vieira et al. 2009; Gonçalves et al. 2010; Rodrigues et al. 2011; Rodrigues et al. 2012). Specific AChE inhibitors can induce enzyme inactivation, leading to acetylcholine accumulation, hyperstimulation of post-synaptic receptors and disturbed neurotransmission (Čolović et al. 2013). Additionally, many studies suggest that OTC disrupts redox processes, thus causing oxidative stress (Yonar et al. 2011; Yonar, 2012; Oliveira et al. 2013; Rodrigues et al. 2017a). It is known that tetracyclines such as OTC cause impairment of the nervous system, as reported for the rodent species guinea pig (Said et al. 1995), possibly due to the oxidative stress generated by free radicals (Yonar et al. 2011). Nevertheless, there is little information about the relationship between tetracyclines and AChE inhibition.
LDH is the terminal enzyme of anaerobic glycolysis, being therefore fundamental for muscle physiology and homeostasis, especially in the presence of chemical stress, during which high levels of energy may be necessary for a reduced period of time
(Monteiro et al. 2007; Koenig and Solé, 2014). LDH activity can be used as a biomarker to evaluate the respiratory status of organisms (Antunes et al. 2010), and some studies have considered muscular LDH activity as an indicator of contaminant-induced metabolic disturbances (Nunes et al. 2004; Vieira et al. 2009; Antunes et al. 2010). Furthermore, LDH has been proposed as good indicator of chemical stress induced by antibiotics in fish (Oliveira et al. 2013). Some studies have shown that OTC can modulate LDH activity in aquatic organisms (Ambili et al. 2013; Oliveira et al. 2013), but also in rats (Pari and Gnanasoundari, 2006).
Thus, the main objective of the present study was to evaluate the effects of acute and chronic OTC exposures on several biomarkers (GSTs, UGT, AChE and LDH) of the freshwater fish Oncorhynchus mykiss. By analyzing specific biotransformation enzymes (GSTs and UGTs) activities, we aim to obtain relevant information on the effects of OTC on fish detoxification systems. By analyzing AChE and LDH activities, we aim to better understand the effects of OTC on neurotransmission, and metabolism, respectively. This set of enzymes can thus be used as physiological relevant effect criteria in further ecotoxicological studies, in order to understand the deleterious effects that OTC may exert in fish species.
Material and Methods Chemicals
OTC was acquired from Sigma Aldrich (CAS: 2058-46-0). Two stock solutions of OTC were prepared by dilution of OTC in dechlorinated tap water (6.65 g/L and 67 mg/L for acute exposure, and 1.2 mg/L for chronic exposure), immediately before starting each exposure, and also before the periodic renewal of the exposure media. Test solutions were prepared by dilution of the stock solutions.
Test organisms
Two hundred and twenty O. mykiss juveniles (8.0 - 9.0 cm) were acquired at a mountain range aquaculture facility (Posto Aquícola do Torno - Marão) in northern Portugal. After arrival at the laboratory facilities, individuals were then subjected to an acclimation period of three weeks, in 500 L tanks, under controlled conditions (recirculation of aerated and dechlorinated tap water, controlled photoperiod (12L:12D) and temperature 15.0±0.2°C).
During this period, physical and chemical parameters were monitored: pH 6.9±0.2, dissolved oxygen 8.0±0.4 mg/L, conductivity 274.8±6.1 μS/cm; ammonium (NH3)
0.50±0.04 mg/L and nitrites (NO2) 0.32±0.03 mg/L. Water of the acclimation tanks was
commercial fish food (pellets). Fish were checked every day, and apparently sick or dead fish were immediately discarded.
Exposure conditions Acute exposure
Tests were performed under laboratory-controlled conditions. After the acclimation period, 90 individuals (length of 8.65±0.12 cm and weight of 9.24±0.23 g) were distributed by six groups: one control group (unexposed to OTC - Ctrl) and five concentration groups (exposed to distinct concentrations of OTC - 0.005, 0.05, 0.5, 5, and 50 mg/L. Each group was subdivided into three replicates, of five organisms per replicate. So, trouts were divided into eighteen 50 L aquaria. The selection of the here- used levels of OTC was based on i) already reported levels in the environment (0.0003 ng/L – 0.712 mg/L; Kolpin et al. 2002; Calamari et al. 2003; Li et al. 2008; Wei et al. 2011) and ii) toxicity data for OTC (EC50 > 75 mg/L) for fish species, namely Menticirrhus
saxatilis, Danio rerio and Oreochromis niloticus (Park and Choi, 2008; Oliveira et al.
2013; Botelho et al. 2015). Thus, all selected concentrations were sub-lethal, and the lowest were ecologically relevant. Exposure occurred in agreement with the OECD test guideline 203, during 96h (OECD, 1992) and following the indications of Rodrigues et al. (2017a). Fish were not fed during the acute exposure. 80% of the medium was renewed after 48 h of exposure. According to Doi and Stoskopf (2000), who studied the kinetics of OTC degradation under the influence of various environmental factors (pH, temperature, light conditions, etc), we assume that the levels of OTC were always re- established every two days.
Chronic exposure
Long-term exposures were carried out in agreement with OECD guideline 215 (OECD, 2000). O. mykiss individuals (8.35±0.33 cm, 7.75±0.53 g) were exposed under similar conditions described for the acute exposure (ninety fish, distributed in 18 aquariums, each with five fish; three replicates per concentration). During exposure (28 days), fish were exposed to a range of sub-lethal concentrations of OTC: 0.0 (Ctrl), 0.3125, 0.625, 1.25, 2.5 and 5.0 µg/L. The selected concentrations for the chronic assay were all environmentally relevant, as previously mentioned, and based on the results used by a similar study (Rodrigues et al. 2017a). Feeding was maintained similarly to the acclimation period. Test conditions were similar to those adopted for the acute exposure. Medium renewal (80%) took place every 48 hours.
Water quality control
Water quality was monitored every 48 h during the exposures, as recommended by OECD guidelines (nº 203 and 215). During exposures, no mortality was recorded in the control group [complying with the OECD (1992 and 2000) guidelines requirements: mortality < 10%]. Physical and chemical water parameters (pH, temperature, conductivity and dissolved oxygen) were measured using a hand-held multiparameter probe (YSI, 556 MPS) and for quantification of ammonium and nitrites a photometer (YSI, 9300 Photometer) with water test tablets (Palintest, NH3 and NO2) was used. Physical and
chemical parameters measured during both exposures are presented in Table 1.
Table 1. Physical and chemical parameters measured during the acute and chronic exposures of O. mykiss to oxytetracycline.
OTC concentrati ons pH Temperature (ºC) Conductivity (µS/cm) Dissolv ed oxygen (mg/L) Ammoni um (mg/L) Nitrites (mg/L) Acut e e xpo sure (mg /L) CTRL 7.07± 0.02 15.4±0.2 278.2±0.9 7.27± 0.11 0.29±0.04 0.010± 0.003 0.005 7.09± 0.02 15.4±0.2 278.2±1.1 7.14± 0.17 0.26±0.07 0.012± 0.004 0.050 7.11± 0.02 15.2±0.2 278.7±1.5 7.29± 0.23 0.36±0.03 0.008± 0.002 0.500 7.10± 0.01 15.4±0.2 279.2±1.9 7.28± 0.18 0.37±0.07 0.007± 0.003 5.000 7.07± 0.01 15.5±0.2 279.7±2.0 7.40± 0.40 0.42±0.14 0.008± 0.002 50.00 7.11± 0.03 15.4±0.1 268.5±0.8 7.56± 0.64 0.41±0.01 0.010± 0.001 Ch ro n ic e xpo sur e (µg/L) CTRL 6.74± 0.04 14.5±0.2 274.4±0.3 8.16± 0.11 1.84±0.08 0.020± 0.005 0.3125 6.78± 0.05 14.5±0.2 273.7±0.2 8.08± 0.09 1.71±0.01 0.023± 0.002 0.625 6.77± 0.04 14.5±0.1 274.6±0.6 8.07± 0.07 1.66±0.11 0.038± 0.007 1.250 6.74± 0.04 14.6±0.1 275.5±0.5 8.13± 0.04 1.49±0.11 0.035± 0.006 2.500 6.76± 0.04 14.7±0.1 277.2±1.0 8.08± 0.04 1.51±0.04 0.028± 0.003 5.000 6.80± 0.02 14.6±0.1 272.8±0.8 8.17± 0.08 1.49±0.07 0.051± 0.016
Sacrifice and biological samples collection
After exposures, fish were sacrificed after sedation through immersion in an ice-water (4 °C) bath (Wilson et al. 2009; for more detailed information see Rodrigues et al. 2017a). This method is in agreement with the AVMA Guidelines for the Euthanasia of Animals, and took into consideration the Portuguese animal welfare testing regulations (Decree-
Law 113/2013). The use of these organisms was previously authorized by the Ethical Committee (ORBEA) of the host institution (CIIMAR).
Subsequently, eyes, gills, liver and dorsal muscle were removed, separated into different aliquots (several analyses by organ) and preserved in liquid nitrogen, immediately after tissue/organ collection. Samples were stored at -80ºC until the performance of enzymatic determinations.
Enzymatic determinations Tissue preparation
Homogenization of eyes, dorsal muscle, gills and liver samples was carried out on ice, using a mechanical homogenizer (Yellowline DI 18 basic). Subsequently, samples were
centrifuged in a refrigerated centrifuge (Eppendorf 5810R) to obtain the supernatant fraction. For the quantification of GSTs activity, gills and liver samples were homogenized in 2.5 mL of ice-cold phosphate buffer (50 mM, pH 7.0) with Triton X-100 (0.1%), and centrifuged at 15.000 g for 10 min at 4 °C. UGTs activity was quantified in gills and liver and the samples were homogenized in 500 µL (in gills) and in 750 µL (in liver) of 0.05 M Tris/1.15 % KCl (pH 7.4). Homogenates were then centrifuged at 9.500 g for 20 min at 4°C. For AChE activity determinations, eyes and muscle were homogenized in 1.5 mL of ice-cold phosphate buffer (0.1 M, pH 7.2) and centrifuged at 3300 g for 5 min at 4°C. For LDH activity determinations, dorsal muscle tissues were homogenized in 1.5 mL of ice-cold TRIS buffer (0.1 M, pH=7.2) and centrifuged at 3300 g for 3 min at 4°C. Supernatants were recovered and the enzymatic analysis was performed in this fraction.
Biomarkers assays
Spectrophotometric readings (GSTs, UDPs, AChE, and LDH) were performed in a spectrophotometer microplate reader Thermo Scientific, model Multiskan GO, version 1.00.40, with SkanIt Software 3.2. All enzymatic activities were determined in quadruplicate.
Protein quantification was performed at 595 nm using the Bradford method (Bradford, 1976), adapted to microplate, with standard bovine γ-globulin (1 mg/mL), in order to express enzymatic activities per mg of protein of the analyzed tissues/organs.
GSTs activity was determined by spectrophotometry, according to Habig et al. (1974), and the results were expressed as millimoles of thioeter produced per minute, per milligram of protein. GSTs catalyze the conjugation of the substrate 1-chloro-2,4-
dinitrobenzene (CDNB) with glutathione, forming a thioeter whose formation can be followed by the increment of absorbance at 340 nm.
UGTs activity was determined with p-nitrophenol as the aglycone, in the microsomal fraction, according to Thunberg et al. (1980). Microssomes were prepared by the Ca-aggregation method according to Aitio and Vainio (1976). The results were expressed as the amount of p-nitrophenol consumed per mL, per mg microsomal protein, and was inversely proportional to the absorbance at 400 nm.
AChE activity was determined in eyes and muscle homogenates following the method of Ellman et al. (1961) adapted to microplate. This method follows the formation of a complex by conjugation of thiocholine (resulting from the hydrolytic degradation of acetylthiocholine by acetylcholinesterase) with 5-5’-dithio-bis-2-nitrobenzoate (DTNB). This complex absorbs light at a wavelength of 412 nm, and the increase in absorbance is proportional to the enzyme’s activity.
LDH activity was determined in dorsal muscle homogenates following the method of Vassault (1983) adapted to microplate. Determination of its activity was performed spectrophotometrically by measuring the reduction of absorbance caused by the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) in the presence of pyruvate, at a wavelength of 340 nm, during 5 min. Activity was expressed in millimoles of β-NADH oxidized per minute, per milligram of protein.
Statistical analyses
Nested ANOVA was run for each variable to test for differences among OTC concentrations across experimental units (random nested factor) followed, if needed, by Dunnet multicomparison test to discriminate significantly different chemical treatments relative to the control (using the nested factor variance as the error term). All statistical analyses were performed using SPSS Statistics v23 and MS Excel and a significance level of 0.05 was applied to all analyses.
Results
No significant differences in GSTs activity (Fig. 1) were found in gills after OTC acute exposure (F[5, 12] = 0.344; p = 0.486). However, exposure to OTC caused a significant
increase in gills GSTs activity of organisms chronically exposed to 1.25, 2.5 and 5.0 µg/L (F[5, 12] = 0.137; p < 0.001; Fig. 1), which correspond to an induction of 43, 50 and 84 %,
relative to the control. In liver, no significant differences were found in terms of GSTs activity, after acute (F[5, 12] = 12.781; p= 0.242) and chronic (F[5, 12]=4.371; p = 0.062)
Fig. 1 – Glutathione S-transferase (GSTs) activity on O. mykiss after acute and chronic exposure to oxytetracycline. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates. *Stands for significant differences when compared to control (Dunnett’s test, p < 0.05).
Following acute exposure, the activity of UGTs was not affected in both tissues tested (gills: F[5, 12] = 0.051; p = 0.393; liver: F[5, 12]=1.210; p=0.719 – Fig. 2). Organisms
subjected to chronic exposure (Fig. 2) showed significant changes in this parameter (F[5, 12] = 0.064; p = 0.029; gills) (F[5, 12]= 0.215; p = 0.037; liver), however, in both tissues, the
Dunnett’s test did not detect differences between treatments and respective control (p>0.05).
Fig. 2 – Uridine-diphosphate-glucuronosyltransferase activity (UGT) on O. mykiss after acute and chronic exposure to oxytetracycline. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates. * Stands for significant differences when compared to control (Dunnett’s test, p < 0.05).
Following acute exposure to distinct concentrations of OTC, the AChE activity (Fig. 3) determined in eyes of O. mykiss was not statistically different when compared to the control treatment (Dunnett’s test, p<0.05), despite the occurrence of significant differences among treatments (F[5, 12] = 13.584; p = 0.038). However, chronic exposure
to OTC caused a significant decrease in AChE activity of eyes of exposed organisms, for the two highest concentrations 2.5 and 5.0 µg/L (F[5, 12] = 108.663; p = 0.010; Fig. 3).
These effects correspond to an inhibition of AChE of approximately 27% and 29%, relative to the control. In muscle tissue, no significant differences were found concerning AChE activity (Fig. 3) after acute (F[5, 12] = 219.012; p = 0.112) and chronic (F[5, 12] =
214.662; p = 0.057) exposures.
Fig. 3 – Acetylcholinesterase activity (AChE) on O. mykiss after acute and chronic exposure to oxytetracycline. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates. * Stands for significant differences when compared to control (Dunnett’s test, p < 0.05).
Regarding LDH activity (Fig. 4), significant differences were detected after both exposures: an increase in organisms acutely exposed only to the highest concentration tested – 50.00 mg/L (F[5, 12] = 1088.812; p = 0.018), and a significant decrease of LDH
activity in organisms chronically exposed to the lowest concentrations tested (0.3125 – 1.25 μg/L; F[5, 12] = 244.273; p = 0.012).
expressed as mean ± standard error (SE), five fish per tank, with three replicates. *Stands for significant differences when compared to control (Dunnett’s test, p < 0.05).
Discussion
This study investigated the effects of OTC on specific aspects of fish physiology, evaluating its impact on different biochemical pathways. Ji et al. (2010) already referred the lack of information about the effects of pharmaceutical pollution, mainly of chronic, sublethal exposures and potential underlying mechanisms of toxic effect. Understanding the mechanisms related to the deleterious effects induced by xenobiotics (OTC in our study), upon fish metabolism, is a mandatory step to develop accurate and precise diagnostic methodologies with prognostic capability to assess putative ecotoxicological effects under realistic conditions (Tu et al. 2009). This is important, especially under real scenarios of pollution, such as aquaculture facilities where antibiotics are used, and heavily polluted areas, namely by urban effluents contaminated by large amounts of therapeutic drugs and their residues. Due to its biological activity, intensive use, incorrect