key: cord-0789066-yfudp5ko authors: da Luz, Thiarlen Marinho; da Costa Araújo, Amanda Pereira; Estrela, Fernanda Neves; Braz, Helyson Lucas Bezerra; Jorge, Roberta Jeane Bezerra; Charlie-Silva, Ives; Malafaia, Guilherme title: CAN USE OF HYDROXYCHLOROQUINE AND AZITHROMYCIN AS A TREATMENT OF COVID-19 AFFECT AQUATIC WILDLIFE? A STUDY CONDUCTED WITH NEOTROPICAL TADPOLE date: 2021-03-18 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2021.146553 sha: 01d79c65e08e23b1feb419ab4601cec4e1d9ed45 doc_id: 789066 cord_uid: yfudp5ko The impacts on human health and the economic and social disruption caused by the pandemic COVID-19 have been devastating. However, its environmental consequences are poorly understood. Thus, to assess whether COVID-19 therapy based on the use of azithromycin (AZT) and hydroxychloroquine (HCQ) during the pandemic affects wild aquatic life, we exposed (for 72 hours) neotropical tadpoles of the species Physalaemus cuvieri to the water containing these drugs to 12.5 μg/L. We observed that the increase in superoxide dismutase and catalase in tadpoles exposed to AZT (alone or in combination with HCQ) was predominant to keep the production of NO, ROS, TBARS and H2O2 equitable between the experimental groups. In addition, the uptake of AZT and the strong interaction of AZT with acetylcholinesterase (AChE), predicted by the molecular docking analysis, were associated with the anticholinesterase effect observed in the groups exposed to the antibiotic. However, the unexpected increase in butyrylcholinesterase (BChE) in these same groups suggests its constitutive role in maintaining cholinergic homeostasis. Therefore, taken together, our data provide a pioneering evidence that the exposure of P. cuvieri tadpoles to AZT (alone or in combination with HCQ) in a predictably increased environmental concentration (12.5 μg/L) elicits a compensatory adaptive response that can have, in the short period of exposure, guaranteed the survival of the animals. However, the high energy cost for maintaining physiological homeostasis, can compromise the growth and development of animals and, therefore, in the medium-long term, have a general negative effect on the health of animals. Thus, it is possible that COVID-19 therapy, based on the use of AZT, affects wild aquatic life, which requires greater attention to the impacts that this drug may represent. It is known that amphibians to comprise one of the most endangered groups (Beebee & Griffiths, 2005; Green et al., 2020) , a fact that has been discussed for some time (Wake, 1991 , Wake, 1998 ; but that, more recently, discussions have been more urgent (Green et al., 2020; Bolochio et al., 2020) . This is because the increasing loss of natural habitats (Mayani-Parás et al., 2020; Semper-Pascual et al., 2021) , increased UV-B irradiation (Lundsgaard et al., 2020; Morison et al., 2020) , emergence of emerging diseases (Blaustein et al., 2018; Fisher & Garner, 2020; Brannelly et al., 2021) , introduction of non-native species (Nunes et al., 2019) , climate change (Bucciarelli et al., 2020 ) and the increase in pollution of freshwater ecosystems (Wesner et al., 2020; Meindl et al., 2020; Lent et al., 2020) has greatly intensified the reduction and distribution of various species of amphibians. Regarding the impacts of pollutants on these animals, most studies have directed their designs to assess the effects of classic chemical compounds, such as pesticides and their degradation products, heavy metals, nitrogen-based fertilizer, among others [see review by Blaustein et al. (2003) ]. However, fewer ecotoxicological studies addressing the impacts of emerging pollutants on amphibians are less [see reviews by McConnell et al. (2010) and Egea -Serrano et al. (2012) ]. Such pollutants include synthetic or natural chemicals that are not part of the list of those included in routine (inter) national monitoring programs, but that have the potential to enter different environmental compartments and cause ecological and/or human health effects (Geissen et al., 2015; Calvo-Flores et al., 2018; NORMA, 2021) . Two chemical compounds considered as emerging pollutants, whose impacts on amphibians have never been studied, refer to azithromycin (AZT) and hydroxychloroquine (HCQ) (Mendez et al., 2017; Dabić et al., 2019; Gomes et al., 2020) . While AZT is a macrolide antibiotic which inhibits bacterial protein synthesis (Parnham et al., 2014) also used in the treatment of cancer and autoimmune and inflammatory diseases (Patel & Hashimi, 2020) ; HCQ is used in the prevention and treatment of malaria (Shippey et al., 2018) and as a therapeutic option in the treatment of rheumatoid arthritis (Lane et al., 2020) , lupus erythematosus (Jakhar & Kaur, 2020), porphyria cutanea tarda ( Malkinson & Levitt, 1980) , Q fever (Hartzell et al., 2008; Cherry & Kersh, 2020) and photosensitive diseases (Millan & Quijano, 1957) . Due to the COVID-19 pandemic (started in late 2019), the use of these drugs has increased considerably (Yazdany & Kim, 2020; Agarwal et al., streams or via leaching from landfills, which in many countries do not receive adequate treatment (Ansari et al., 2019; Urban & Nakada, 2021) or the processes used are insufficient to remove these pollutants or are financially inaccessible (Ali et al., 2017; Khan et al., 2019) . In cities with a high incidence of COVID-19, for example, the dramatic increase in the production of hospital waste in health facilities has been an additional administrative challenge (Sarkodie & Owusu, 2020) , in addition to amplifying the presumed concentrations of AZT and HCQ in the aquatic environment. In India, for example, after the approval of the Indian Council of Medical Research for the empirical use of HCQ for prophylaxis of COVID-19, the stocks available in pharmacies have been reduced dramatically, especially when hospitals and health professionals began to prescribe the drug to their patients . The King Abdullah University Hospital in Jordan produced, at the height of the pandemic, ten times more medical waste compared to average production during the days before the spread of SARs-Cov-2 (Abu-Qdais et al., 2020). In Spain, an increase of more than 300% was observed (Klemeš et al., 2020) and in Asia, it is estimated that the total of hospital waste generated exceeds 16.5 thousand tons/day, with India, followed by Iran, Pakistan, Saudi Arabia, Bangladesh and Turkey are the largest producers of this waste in the context of the COVID-19 pandemic (Sangkham, 2020) . In this sense, as discussed by Farias et al. (2020) , questions about the impact that therapy against COVID-19 has on aquatic wildlife. Particularly in amphibians, how can this increase affect the health of these animals and the decline of their natural populations? Thus, to assume the ecotoxicological effects of these drugs on the natural populations of anurans, we exposed tadpoles of Physalaemus cuvieri (Anura, Leptodactylidae) to AZT and HCQ (alone or in combination). This species, in particular, occurs in several countries in South America (Mijares et al., 2011; De-Oliveira-Azithromycin (AZT) and hydroxychloroquine (HCQ) used in our study, [similar to the study by Amaral et al. (2019) ] were intentionally acquired in common commercial facilities in order to bring our experimental design as close to the most realistic condition as possible. For the preparation of the AZT stock solution, we used AZT dihydrate dragees (500 mg) (Brainfarma Indústria Química e Farmacêutica S.A., Anápolis, GO, Brazil) and for the HCQ solution, HCQ sulfate dragees (400 mg), manufactured by Apsen Farmacêutica SA (São Paulo, SP, Brazil) were used. Both solutions were prepared by diluting the pills in acetonitrile solution (0.01 M), according to Shen et al. (2010) . From these solutions, the aliquots added to the exposure waters were removed. Table 1 presents general information about the drugs used in our study. To assess the aquatic toxicity of AZT and HCQ, we used tadpoles of the species Physalaemus cuvieri (Leptodactylidae) as a model system. Its wide geographical distribution in South America (Miranda et al., 2019) , stability and population abundance in the areas that occur (Frost, 2017) , in addition to good adaptability in the laboratory and early biological response to changes in its environment justify the choice of species in our study, as well as in other recent (eco) toxicological studies (Herek et al., 2020; Araújo et al., 2020ab; Rutkoski et al., 2020 ether extract, 5% crude fiber, 14% mineral matter and 87% dry matter). After the eggs hatched, the tadpoles remained in these conditions until they reached stage 26G, according to Gosner (1960) (body biomass: 70 mg ± 4.1 mg and total length: 20.1 mm ± 0.7 mm -mean ± SEM). Then, 800 healthy tadpoles (i.e., with normal swimming movements and without morphological deformities or apparent lesions) were distributed into four experimental groups (n = All experimental groups were kept in glass containers containing 2 L of naturally dechlorinated water, in which the drugs were diluted, with an exposure period of 72 h, simulating an ephemeral exposure. During the exposure, the animals were fed once a day with commercial fish feed and the waters were not renewed (i.e., static system). The drug concentrations were based on previous studies that identified them in surface waters. Fernandes et al. (2020) reported that AZT concentration of up to 2.8 µg/L was detected in a river in northern Portugal and, in Olaitan et al. (2014) , the median concentration of chloroquine (chemically similar to HQC, its derivative) identified in different water samples from Nigeria was 2.12 µg/L. Therefore, the concentration tested in our study (ie: 12.5 µg/L) simulates a potential increase (approximately 6 times) in AZT and HCQ concentrations in aquatic environments (associated with the COVID-19 pandemic), which can be considered a predictive environmentally relevant concentration. Prior to biochemical assessments, the samples to be analyzed were prepared, similarly to . In this case, we used 96 tadpoles/group, distributed in eight samples composed of a pool of 12 animals/each. These animals were weighed (12.5 g ± 0.0004 -mean ± standard error) and subsequently macerated in 1 mL of phosphate buffered saline (PBS), centrifuged at 13,000 rpm for 5 min (at 4°C). The supernatant was separated into aliquots to be used in different biochemical evaluations. Entire bodies were used in the experiment due to the hard time isolating certain organs from small animals. Unlike adult anurans, organ-specific biochemical assessment in tadpole requires highly accurate dissection due to their small size, which makes it difficult processing The effects of exposure à AZT e HCQ (alone or in combination) on oxidative stress reactions Sies et al., 2020) . The Griess colorimetric reaction [as described in Bryan et al., (2007) ] was used to measure nitrite and the TBARS levels were determined based on procedures described by Ohkawa et al. (1979) and modified by Sachett et al. (2020) . The production of H2O2 and ROS was evaluated according to Elnemma et al. (2004) and Maharajan et al. (2018) , respectively. The activation or suppression of antioxidant activity by treatments was evaluated by determining the activity of catalase and superoxide dismutase (SOD), which are considered first-line antioxidants important for defense strategies against oxidative stress (Ighodaro & Akinloye, 2018) . While catalase activity was assessed according to Sinha et al. (1972) [see details in Montalvão et al. (2021) ]; SOD levels were determined according to the method originally described by Del-Maestro & McDonald (1985) and adapted by Estrela et al. (2021) . The possible neurotoxic effects induced by AZT and HCQ (alone and in combination) were evaluated by determining the activity of acetylcholinesterase (AChE) (Trott & Olseon, 2010) . The binding affinity and interactions between residues were used to determine the best molecular interactions. The results were visualized using ADT, Biovia Discovery Studio v4.5 and UCSF Chimera X (Pettersen et al., 2021) . Interestingly, we also observed that the treatments induced a differentiated effect on the animals' cholinesterase system. While AChE activity was reduced in the -AZT‖ and -AZT + HCQ‖ groups ( Figure 3A ); BChE concentrations increased in these same groups ( Figure 3B ). In addition, we observed that the animals' exposure period was sufficient to induce uptake of AZT and HCQ in the animals, which suggests that drugs dispersed in water were absorbed by the tadpoles. While the subtracted from the samples from the tadpoles exposed to the treatments. As for the analysis of molecular docking, our data predicted the affinity between the drugs and the enzymes AChE and BChE, as well as the existence of interactions with residues from all tested moorings. Figure 5 shows that the binding energies required for AZT to bind to AChE (-8.8 ± 1.12 kcal/mol) and BChE (-9.1 ± 0.65 kcal/mol) were comparable, similar to that observed for HCQ and its molecules target [HCQ / AChE (-6.9 ± 0.97 kcal/mol) and HCQ/BChE (-6.2 ± 0.69 kcal/mol)] ( Figure 5B ). However, the activation energies required for the connection between AZT and the evaluated enzymes were lower than those required for HCQ ( Figure 5 ), which suggests greater stability of the AZT-AChE and AZT-BChE complex and, consequently, the most likely to be formed in the evaluated biological system. Initially, we evaluated whether the tadpoles' exposure to AZT and HCQ (alone or in combination) could induce an increase in oxidative processes, from different biomarkers. However, no difference was observed between the experimental groups, regarding the concentrations of nitrite, TBARS, ROS and H2O2 (Figure 1 ). These results are interesting, as they disagree with some previous reports and corroborate the findings of others. In the study by Li et al. (2020) , for example, the increased production of ROS in Daphnia magna exposed to AZT (after 96h of feeding Chlorella pyrenoidosa exposed to AZT) was related to changes in feeding behavior, nutritional status, and digestive physiology of these animals. Similarly, Mhadhbi et al. (2020) reported that exposure to AZT similarly to our findings, Shiogiri et al. (2017) found no evidence of increased oxidative stress in Oreochromis niloticus (juveniles) exposed for 14 days to different concentrations of AZT (1, 50 and 100 mg/L). In relation to HCQ, studies involving aquatic organisms have not evaluated biomarkers of oxidative stress, despite having already observed effects of chloroquine on the enzymatic and histopathological physiology of Cyprinus carpio fish , immobilization of D. magna ( Zurita et al., 2005; Rendal et al., 2010) , reduction of lysosomal function in Poeciliopsis lucida fish cells, inhibition of luminescence in Vibrio fischer bacteria and inhibition of the growth of Chlorella vulgaris algae (Zurita et al., 2005) , as well as transpiration inhibition in Salix viminalis plants (Rendal et al., 2010) . Therefore, this scenario denounces not only the lack of studies focusing on the ecotoxicological effects of AZT and HCQ, but also shows that the biological response to drugs is dependent on the species, period and concentrations used in the exposures. In our study, it is possible to attribute the absence of oxidative effect induced by AZT and HCQ to the action of the enzymes SOD and catalase. Although only the group co-exposed to the drugs showed a significant increase in SOD activity (Figure 2A ), in the groups exposed to AZT and HCQ (alone) the enzyme activity increased by 34.9% and 30.6% (respectively) compared to the control group, which biologically may have been preponderant to inhibit the increase of cellular oxidative processes. Similar reasoning can be used to increase catalase in the -AZT‖ and -AZT + HCQ‖ groups ( Figure 2B ), since both enzymes are important for antioxidant defense against free radicals. While SOD converts the superoxide anion radical to H2O2, catalase converts H2O2 into H2O and O2 molecules (Lee et al., 2018; Ransy et al., 2020; Damiano et al., 2020) . As for the reduction of catalase activity in animals exposed to HCQ (alone), it is possible that it has been compensated for by the performance of other peroxisomal enzymes (which also aid in the decomposition of H2O2 and other reactive oxygen and nitrogen species) to maintain the oxidative homeostasis in this group. Such enzymes include, for example, peroxiredoxin 5, glutathione S-transferase kappa, 'microsomal' glutathione S-transferase, and epoxide hydrolase 2 (Fransen et al., 2012) . On the other hand, our data show AZT's anticholinesterase effect on the studied tadpoles, marked by a significant reduction in AChE activity in the -AZT‖ and -AZT + HCQ‖ groups ( Figure 3A ), like the reports by Mhadhbi et al. (2020) , in which juveniles D. labrax exposed to AZT (0.05 and 0.08 mg/L, during 4 and 14 days) also showed a reduction of this enzyme in the gills and liver. As discussed by Massoulié et al. (1993) , AChE is one of the most prominent constituents of central cholinergic pathways. It ends the synaptic action of ACh through its hydrolysis and produces the choline portion necessary for recycling the neurotransmitter. Therefore, any changes in the activity of this enzyme can lead to important neurological consequences. In our study, it is plausible to assume that the uptake of AZT in animals ( Figure 4 ) and, especially, its greater affinity with AChE (in relation to HCQ, Figure 5) were preponderant for the occurrence of the observed anticholinesterase effect. J o u r n a l P r e -p r o o f As suggested by molecular docking, this effect may have been due to the probable -AZT-AChE‖ interaction, via connections involving different amino acid residues (Tyr430, Asn254, Pro256, Pro427 and Trp549 - Figure 6 ). Although these residues are not part of any AChE active or catalytic site [see details in Harel et al. (1993) , Silman & Sussman, (2005) , Johnson & Moore (2006) and ], it is possible that AZT acted as an allosteric modulator, changing the conformation of the enzyme and decreasing its activity, which would not have occurred in the -HCQ-AChE‖ interaction. In this case, in addition to the binding energy for this interaction being higher than that required for the -AZT-AChE‖ interaction ( Figure 5) , it is possible that such connections did not cause sufficient conformational changes to alter the activity/functionality of the enzyme or that the uptake concentration of HCQ (Figure 4 ) was insufficient to induce changes in the enzyme or even if biologically (ie: in vivo) such connections did not occur. As discussed by Kitchen (2004), the greater the free binding energy predicted in molecular docking, the less favored is the interaction between the ligand and the target biomolecule and, therefore, less likely to occur. On the other hand, interestingly, we found an increase in BChE concentrations in the same groups in which AChE activity was reduced (ie: -AZT‖ and -AZT + HCQ‖ groups; Figure 3 ), which suggests an adaptive (compensatory) response to break down excess ACh in synaptic clefts caused by reduced AChE. Despite being encoded by different genes, the enzymes AChE and BChE have high structural homology, differing in their affinity for substrates and sensitivity to inhibitors. While AChE is an esterase that hydrolyzes predominantly ACh (Soreq & Seidman, 2001) , BChE hydrolyzes different types of choline esters, including butyrylcholine (BCh), succinylcholine (SCh) and ACh (Darvesh et al., 2003; Nurulain et al., 2020) . However, considering that these enzymes have different Km values (Michaelis-Menten constant), they are expected to have different kinetic responses to ACh concentrations in synaptic clefts. According to Silver (1974) , at low concentrations of ACh, AChE is highly efficient but BuChE is much less efficient. However, at higher ACh concentrations BuChE's efficiency in the hydrolysis of ACh is significantly increased. Thus, this evidence, associated with other studies that have already reported the compensatory support role of BChE in response to the absence or decrease of AChE, reinforces the hypothesis about the occurrence of a physiological adaptation to maintain cholinergic homeostasis in tadpoles exposed to AZT (alone or in combination with HCQ) (Norel et al., 1993; Li et al., 2000; Xie et al., 2000; Mesulam et al., 2002ab) . Alternatively, we cannot rule out the hypothesis that the interaction between AZT and BChE (molecular docking; Figure 6 -7) has also caused changes in the normal activity of the enzyme. However, contrary to the effects of the -AZT-AChE‖ interaction, such changes would have favored the enzyme's activity, with AZT acting as a positive allosteric modulator. Anyway, regardless of the mechanisms that explain our findings, it is important that new studies expand the understanding of the intrinsic factors involved in the physiological response of J o u r n a l P r e -p r o o f Journal Pre-proof tadpoles exposed to different treatments. Although our data strongly suggest that BChE could potentially replace AChE in the context of tadpole's exposure to AZT and HCQ, as well as playing a constitutive role (rather than just back-up) in the hydrolysis of ACh, there is no way to guarantee (in our study) that this compensatory action has, in fact, regulated the concentrations of this neurotransmitter in the synaptic clefts. As is well known, both the increase and the decrease in the amounts of ACh in the synaptic clefts can induce effects on different physiological functions in the organisms, which include a wide spectrum of clinical manifestations (eg: dysfunctional gland disorders, respiratory processes, and disorders in the functioning of the central nervous system). In this case, in vivo evaluations to determine the concentrations of ACh in the tadpoles exposed to the treatments (AZT and HCQ), constitute interesting future investigative perspectives. Equally important will be the conduct of in vivo and in vitro studies to confirm the mechanisms of action predicted by molecular docking and, once confirmed, whether the interactions of drugs with the target molecules are reversible or irreversible. Finally, taken together, our data point to an unexpected response from P. cuvieri tadpoles to exposure to drugs, in which the REDOX and cholinergic imbalance induced by AZT would have been counterbalanced by the compensatory increase in enzyme activity that neutralized production excessive free radicals and apparently reestablished the central cholinergic pathways affected by the reduction in AChE. As discussed by Biagianti-Risbourg et al. (2013) , this type of response constitutes an individual-level physiological adaptation and, therefore, can (in the short term) increase animal survival and maintain the highest possible fitness under stressful conditions (Hoffman, 1995; Collier et al., 2019) . However, this physiological adaptation is energetically expensive for the organism and, depending on the nature and intensity of environmental stress, can trigger a physiological trade-off might, including the reduction of life expectancy or the reproductive success of individuals (Wilson & Franklin, 2002; Wood & Harrison, 2002; Farwell et al., 2012; Loria et al., 2019) . Therefore, when transposing these considerations to the context of our study, we cannot guarantee that the prolonged exposure of tadpoles to drugs will have its harmlessness sustained by the physiological tolerance observed in the short exposure. Considering that the metamorphosis of amphibians, in itself, consists of a high energy cost phase (Pfab et al., 2020) , the reallocation of energy from other processes (eg: growth and development) to maintain physiological homeostasis, will have a general negative effect on animal health. In this sense, it will be essential to assess how much the biological responses observed in our study will be able to guarantee the survival of the tadpoles until their complete metamorphosis, without affecting their reproductive performance. J o u r n a l P r e -p r o o f Journal Pre-proof In conclusion, our study demonstrated that the short exposure of P. cuvieri tadpoles to AZT and HCQ (alone or in combination) unexpectedly induced an adaptive physiological response marked by increased activity of the enzymes SOD and catalase (for the maintenance of homeostasis oxidative) and by increasing BChE (to -possibly -counteract the anticholinesterase effect induced by AZT). In addition, the uptake of AZT in tadpoles and the strong link between this drug and AChE, suggested by molecular docking, were preponderant in triggering the animals' physiological response. When considering the pioneering nature of the present study, our results constitute only the -tip of the iceberg‖ that can represent the physiological effects of COVID-19 therapy based on AZT/HCQ in animal physiology. Therefore, we strongly suggest that studies of this nature be continued. Conflict of interest: The authors declare no conflict of interest. Ethical approval: All experimental procedures were carried out in compliance with ethical guidelines on animal experimentation. Meticulous efforts were made to assure that animals suffered the least possible and to reduce external sources of stress, pain and discomfort. The current study did not exceed the number of animals necessary to produce trustworthy scientific data. This article does not refer to any study with human participants performed by any of the authors. 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