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Diclofenac-Induced Cytotoxicity in Cultured Carp Leukocytes

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Diclofenac-Induced Cytotoxicity in Cultured Carp Leukocytes Physiol. Res. 69 (Suppl. 4): S607-S618, 2020 https://doi.org/10.33549/physiolres.934609 Diclofenac-Induced Cytotoxicity in Cultured Carp Leukocytes Monika NEMCOVA1,2, Jiri PIKULA1,2, Jan ZUKAL3,4, Veronika SEIDLOVA1,2 1Department of Ecology and Diseases of Zoo Animals, Game, Fish and Bees, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic, 2Department of Zoology, Fisheries, Hydrobiology and Apiculture, Mendel University in Brno, Czech Republic, 3Institute of Vertebrate Biology of the Czech Academy of Sciences v.v.i., Brno, Czech Republic, 4Institute of Botany and Zoology, Masaryk University, Brno, Czech Republic Received October 10, 2020 Accepted October 26, 2020 Summary Game, Fish and Bees, University of Veterinary and Diclofenac is a drug commonly used in human and veterinary Pharmaceutical Sciences Brno, Palackého tř. 1946/1, 612 42 medicine for the treatment of diseases associated with Brno, Czech Republic. E-mail: [email protected] inflammation and pain. Medicinal products enter waste and surface waters on an everyday basis and contaminate the aquatic Introduction environment. Fish are therefore permanently exposed to these chemicals dissolved in their aquatic environment. To simulate Human and wildlife health and disease are variable environmental conditions, the aim of our study was to intricately interlinked (Forget and Lebel 2001, Zinsstag examine adverse effects of diclofenac under different et al. 2011). Development and use of efficient drugs temperatures of cell incubation (18, 21, 24, 27 and 30 °C). Cyto- improves the quality of human and animal life. On the toxic and -static effects of diclofenac in concentrations of other hand, industrially produced medicinal products are 0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml and mostly non-natural, hence, adverse effects from 100 μg/ml for the carp (Cyprinus carpio) cultured leukocytes environmental drug residues may be a serious cause for were quantified using detection of lactate dehydrogenase concern (Arnold et al. 2013, Hruba et al. 2019, Mezzelani released from damaged cells. Overall DCF cytotoxicity was et al. 2016). Pharmaceuticals can influence aquatic relatively low and its impact was pronounced at higher organisms as well as contaminate ground and drinking temperature and DCF concentration. Cells growth inhibition is water sources and other parts of ecosystems. changing more rapidly but it is high mainly at the highest Diclofenac, a non-steroidal anti-inflammatory concentration from low temperature. DNA fragmentation was not drug (NSAID), is amongst the most widely prescribed detected in tested leukocyte cell line. CYP450 increased medicines worldwide and, consequently, one of the most diclofenac cytotoxicity only at the highest concentration but at frequently detected compounds in waste and surface incubation temperatures 18 and 27 °C. Leukocyte viability is waters (McGettigan and Henry 2013, Schwaiger et al. essential for immune functions and any change can lead to 2004). It is used in the treatment of pain, physical reduction of resistance against pathogens, mainly in cold year disability in rheumatic diseases as well as for its anti- seasons, when the immune system is naturally suppressed. inflammatory and antipyretic effects (Kołodziejska and Kołodziejczyk 2018). In general, diclofenac, as a non- Key words selective cyclooxygenase (COX) inhibitor, acts on both Fish • Immune system • Non-steroidal anti-inflammatory drug • types of COX, namely COX-2 synthesized during Toxicity • Temperature variation • Environmental pollution inflammatory processes (therapeutic effect) and COX-1 with physiological function in synthesis of Corresponding author prostaglandins, which are important for protection of V. Seidlova, Department of Ecology and Diseases of Zoo Animals, gastrointestinal epithelial cells (Hawkey 2000, Laine PHYSIOLOGICAL RESEARCH • ISSN 1802-9973 (online) 2020 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: [email protected], www.biomed.cas.cz/physiolres S608 Nemcova et al. Vol. 69 1996), for blood pressure balance, correct thrombocyte the liver, kidney and gills suggestive of inflammatory and aggregation and some other cardiovascular system necrotic processes associated with exposure to diclofenac characteristics (Al-Saeed 2011, Grosser et al. 2006). (Ajima et al. 2015, Hoeger et al. 2005, Schwaiger et al. Therefore, adverse effects are commonly associated with 2004, Stepanova et al. 2013), resulting in general health the above-mentioned systems (Al-Saeed 2011, Laine impairment (Schwaiger et al. 2004). 1996). Importantly, however, detoxification and The majority of ecotoxicology experiments were elimination pathways of xenobiotics including diclofenac designed to examine effects of diclofenac at the level of are located in the liver and kidney, where diclofenac can whole fish organisms (Ajima et al. 2015, Hoeger et al. be concentrated and/or metabolized by the enzyme 2005, Schwaiger et al. 2004, Stepanova et al. 2013), complex of cytochrome P450 (CYP450) to form toxic maintaining a narrow range of aquarium water parameters metabolites (Boelsterli 2002, Shen et al. 1999, Tang et al. including the temperature. Immune defences of non- 1999). Nephro- (Hickey et al. 2001, Murray and Brater, homeothermic vertebrates are temperature-dependent 1993) and hepato-toxicity (Aithal and Day 2007, Helfgott (Heger et al. 2020, Morvan et al. 1998). While et al. 1990) can be observed consequently. immunotoxicity of diclofenac in fish is still understudied The primary source of drugs and their (Schwaiger et al. 2004), fish cell cultures provide useful metabolites in the environment are patients. The active tools for experimental approaches with temperature substances and/or their metabolites are excreted in faeces variation. Here, we performed diclofenac toxicity testing and urine. First, pollutants contaminate wastewater using cultured carp leukocytes and predicted both higher treatment plants where they should ideally be eliminated. cytotoxicity rates and proliferation inhibition at lower However, imperfectly captured micropollutants pose temperatures. We also aimed to obtain data applicable for a risk because “treated” wastewater is released into the environmental pollution risk evaluation of diclofenac in environment. Second, drugs used to treat animals can the common carp (Cyprinus carpio), an important directly get into the soil with manure and later they may aquaculture species. be flushed with rainwater. Expired drugs entering the cycle in the form of seepage from landfills is another Materials and Methods source of pollution (Lonappan et al. 2016, Zuccato et al. 2000). Cell line and chemicals The dose, chemical structure and route of We used common carp (Cyprinus carpio) administration are important factors influencing the leukocytes cell culture number 95070628 (Merck Sigma- environmental burden of each biologically active Aldrich, Germany) for diclofenac toxicity testing. substance (Daughton and Ruhoy 2013). Environmental Following chemicals were used during cultivation and studies revealed concentrations amounting up to a few testing: culture medium Dulbecco's Modified Eagle hundred nanograms of diclofenac per liter of water. In the Medium/Ham's F-12 (1:1), assay medium Dulbecco's Czech Republic, the highest observed value of 1080 ng/l Modified Eagle Medium (DMEM) w/o sodium pyruvate, was detected in the Elbe river with relatively high flow bovine calf serum (BCS) 10 %, antibiotics (ATB) rate of more than 60 m3/s. At the same time, significantly (penicillin 100 IU/ml and streptomycin 100 μg/ml, higher levels of diclofenac residues can be detected in Biosera, Boussens, France), diclofenac sodium salt smaller watercourses with low flow rate, rural localities (Sigma-Aldrich, Germany), 1A2 Cytochrome P450 with intensive agriculture, in rivers below wastewater human expressed in Pichia pastoris (CYP450) (Merck, treatment plants and in the lower parts of streams (Buser Sigma-Aldrich, Germany), lactate-dehydrogenase (LDH) et al. 1998, Lacina et al. 2013, Marsik et al. 2017, Tixier activity assay kit (Roche Sigma-Aldrich, Germany), et al. 2003, Ternes, 1998). dimethyl sulfoxide (DMSO), phosphate saline buffer, and While diclofenac concentrations detected in trypsin solution (Sigma-Aldrich, Germany). water usually do not induce acute toxicity with lethal effects (Lonappan et al. 2016), it accumulates in fish Solutions for treatment of cultures organs in a concentration-dependent manner (Schwaiger After reduction of light intensity in the et al. 2004). It was found that fish show abnormal laboratory, diclofenac was solubilized in DMSO and behavior, respiratory distress, changes in biochemical and diluted with DMEM w/o sodium pyruvate to obtain final hematological parameters and pathological alterations in testing concentrations of diclofenac. Two experiments 2020 Immunotoxicity of Diclofenac in Fish S609 with different concentrations of diclofenac were (the medium was removed and cells were detached from performed (see Table 1). The tested concentration range the cultivation surface using trypsin solution). DNA for started with 1 ng/ml based on the value detected in the detection of DNA fragmentation was isolated using Elbe river (Marsik et al. 2017). It can also be assumed NucleoSpin® Tissue (Macherey-Nagel GmbH & Co. KG, that diclofenac concentrations in the aquatic environment Dueren, Germany). Isolated DNA was then analyzed by can be higher than the observed
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