Menthol exposure induces reversible cardiac depression and reduces lipid peroxidation in the heart tissue of tambaqui Colossoma macropomum
Introduction
Different operations such as sampling, biometry, labeling, handling, sorting and transportation induce stress in farmed fish (Parodi et al., 2014; Hoseini et al., 2018). Anesthetics have been recommended to mitigate stress (Benovit et al., 2015) and decrease physical injuries (Johny and Inasu, 2016) in intensive farming systems. Moreover, there is an ever growing concern over ethical and fish welfare issues. In recent years many studies have shown the activity and suitability of herbal products to replace synthetic fish anesthetics, as some of them may cause undesirable side effects (Hoseini et al., 2018; Aydın and Barbas, 2020; Barbas et al., 2021). Some natural anesthetics can increase antioxidant defenses and mitigate physiological stress in different fish species (Yousefi et al., 2018; Boaventura et al., 2020); however, the precise mechanisms of action of natural anesthetics or their impacts at the physiological and biochemical levels in fish still need further clarification.
Exposure to natural and synthetic anesthetics leads to full body immobilization and loss of sensation, followed by presumable unconsciousness (Ross and Ross, 2008). In addition to describing behavioral patterns upon exposure (agitation, loss of equilibrium, immobilization, etc.) and the monitoring of anesthesia through electrophysiological (Barbas et al., 2017; Cantanhêde et al., 2020; Barbas et al., 2021) and biochemical responses (Boaventura et al., 2020; Hoseini et al., 2020), the monitoring of vital organs such as the heart should always be considered as anesthesia may directly affect cardiac function in fish (Barbas et al., 2017; De Souza et al., 2019).
The electrocardiogram allows to assess the impact of anesthetics on the cardiac function, as they can elicit arrhythmias depending on the concentration used. Moreover, bradycardia and cardiac arrest during induction, maintenance or recovery could also be observed (Ross and Ross, 2008). Quali-quantitative analyses of the electrocardiographic patterns and tracings in experiments dealing with novel products or evaluating the safety of existing fish anesthetics can be made through characterization of the heart rate, P and T waves, amplitude and duration of the QRS complex and duration of the QT and RR intervals (Choi et al., 2010).
Biochemical markers of oxidative stress are widely used to assess biological responses of fish exposed to various types of chemical substances and compounds (Regoli and Giuliani, 2014). Although anesthetics are used to minimize fish stress during and after handling, they can also contradictorily induce undesirable responses in fish, such as biochemical alterations or stress (Parodi et al., 2014; Souza et al., 2018). Depending on the concentration and type of anesthetic they can cause oxidative and tissue damage in fish (Velisek et al., 2011; Souza et al., 2018; Yousefi et al., 2019; Hoseini et al., 2020). Since the cardiac tissue has a high oxidative metabolic rate and the rates of the main antioxidants are relatively low, it becomes rather prone to oxidative damage (Ascensão et al., 2003). Therefore, the assessment of cardiac and biochemical parameters of oxidative stress is a valuable tool to reinforce the use or raise awareness on potential risks to fish before a broad use of an anesthetic is recommended.
Among many of the plant derived essential oils or compounds that have been suggested for use as fish anesthetics (Aydın and Barbas, 2020), menthol has been evaluated as a potential alternative anesthetic to replace some traditionally used synthetic drugs (Façanha and Gomes, 2005; Gonçalves et al., 2008; Simões and Gomes, 2009; Pádua et al., 2010) or even eugenol, which is a widely used natural compound but has been recently reported to cause epileptiform alterations in fish (Barbas et al., 2021). Menthol is a compound obtained from the essential oil of mint (Mentha spp), or by laboratory synthesis, through the hydrogenation of mentone or isometone (Manuale et al., 2010). Mentha spp., belongs to the Lamiaceae family, and is popularly known as mint, peppermint or English mint. According to some reports it has important analgesic and anesthetic actions, with the advantages of being a low cost and widely available product (Gonçalves et al., 2009), in addition to its important biological actions as an antibacterial and antifungal product (Mimica-Dukic et al., 2003).
Colossoma macropomum, popularly known as tambaqui, is the main farmed native fish species in Brazil and other countries in South and Central America (FAO, 2020). Tambaqui is an Amazonian fish species of great economic importance. After the pirarucu (Arapaima gigas), it is the second largest freshwater scalefish in the Amazon (Sousa et al., 2016). Its good adaptation to captivity, resistance to diseases and high market acceptance have made it one of the most farmed and marketed species in the Amazon region (Inoue and Boujink, 2011). Tambaqui has been used as a promising model to design studies with new anesthetic-like products for tropical freshwater fish species, including electrocardiographic responses (Barbas et al., 2017; De Souza et al., 2019; Barbas et al., 2021). Therefore, the objective of this study was to analyze the effects of different concentrations of menthol on the cardiac function and oxidative stress responses of Colossoma macropomum exposed to short-term anesthetic baths.
Section snippets
Experimental fish and acclimation period
The animals used in this study were juveniles of tambaqui fish C. macropomum which were purchased from a commercial farm and obtained through controlled spawning process. The fish (12.9 ± 2.5 g and 6.61 ± 0.79 cm) were acclimated in 250 L aerated fish tanks (5.0 g fish L−1; 10 fish per tank) for 15 days at the Experimentation vivarium of the Laboratory of Pharmacology and Toxicology of Natural Products at the Universidade Federal do Pará, with controlled room temperature (25 to 28 °C) and a
Normal electrocardiographic activity of Colossoma macropomum
The control fish presented a regular sinus rhythm and the tracing clearly showed the P (atrial depolarization) and T (ventricular repolarization) waves, the QRS complex (ventricular depolarization time) and the RR and QT intervals. All cardiac parameters were considered normal and regular (Fig. 1 A and B).
Electrocardiographic activity of Colossoma macropomum during exposure to menthol
Upon exposure, fish presented concentration-dependent alterations in the ECG tracings. Distinct ECG wave and complex patterns of fish exposed to menthol can be observed in fragment
Discussion
The assessment of additional information such as cardiac responses and biochemical parameters of oxidative stress could allow for a better understanding of the safety levels and effectiveness of a given fish anesthetic, as well as the most appropriate concentrations to be used.
The baseline ECG records of the C. macropomum cardiac muscle clearly showed the patterns in the cardiac rhythm. While the T wave represents ventricular repolarization, the P wave reflects atrial excitation and the QRS
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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