Evaluation of the effects of acepromazine and xylazine on viability in an equine dermal cell line

Article history Xylazine and acepromazine are drugs used exclusively in veterinary medicine. Xylazine is used as a sedative, analgesic, and tranquilizer while acepromazine is used as a sedative, pre-anesthetic, and anesthetic adjuvant. In vitro drug toxicity experimentation is essential to predict possible damage associated with treatment. This study was carried out to evaluate and compare the in vitro effects of acepromazine and xylazine on cell viability. Equine Dermis cells lines were used to examine different drug concentrations (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL). An MTT assay was carried out to reveal cell viability. Both tested drugs reduced the viability of ED cells at 0.02 and 0.01 mg/mL. At 0.005 mg/mL, only acepromazine presented an effect. These results corroborate previous studies with xylazine. On the other hand, this is the first report about acepromazine and cell viability. Previous studies suggest that the mechanisms involved in reducing cell viability are apoptosis for xylazine and the activation of the autophagic pathway for acepromazine. Both mechanisms have been seen in other drugs of the same classes. These findings reveal that both acepromazine and xylazine cause concentration-dependent cytotoxicity in vitro. Future experiments could further elucidate the mechanisms by which this effect happens and thus circumvent the risk of potential tissue damage in vivo. Received 13 May 2020 Accepted 10 September 2020


INTRODUCTION
Xylazine and acepromazine are drugs used exclusively in the clinical routine of veterinary medicine. Xylazine, an alpha2 adrenergic receptor agonist (RANKIN, 2017), is used because of its sedative and analgesic properties in short-term procedures, in addition to inducing myorelaxation (MASSONE, 2011). It is delivered by subcutaneous (SC), intramuscular (IM) or intravenous (IV) routes (MASSONE, 2011) and goes through hepatic metabolism (TIBÚRCIO et al., 2014) and renal excretion (GROSS, 2003).
Acepromazine is a widely used drug in horses (CASTRO, 2003) and is currently the most widely used phenothiazine derivative in veterinary medicine (CORTOPASSI & FANTONI, 2010). It is used as a tranquilizer, sedative, pre-anesthetic, and anesthetic adjuvant (PAPICH, 2012). Its mechanism of action is mainly mediated by blocking dopaminergic receptors, although alpha1 adrenergic, muscarinic, and histamine receptors are also affected (RANKIN, 2017).  (EDMONDS et al., 2016;MANCINI et al., 2017). As for acepromazine, no studies were found regarding its cytotoxic effect.
Cytotoxic assays such as these can be carried out in vivo but, to decrease the use of animal experimentation, numerous alternative methodologies have been adopted, such as tests in cell cultures, which can produce relevant in vitro results (HENRIQUES & SAMPAIO, 2002;BARROS, 2007). Likewise, assessments of cytotoxicity can be carried out on cells of different lineages and origins, according to each purpose, to achieve an approximation of the real effect of a given drug (ROGERO et al., 2003). Cell viability assays have been widely used to evaluate in vitro cytotoxic effects of different substances -including drugs (SAKAI, 2004;OLIVEIRA, 2015), Among the methods used to measure cytotoxicity on cell viability, the colorimetric 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay is one of the most widely used (ALVES & GUIMARÃES, 2010), due to its efficiency and reliability (BRITO et al., 2001). This method allows researchers to verify changes in mitochondrial metabolic activity in viable cell cultures (ABREU, 2008), making it possible to determine cell viability after a given treatment.
Given this possibility, the present study aims to evaluate the cytotoxic effect of acepromazine and xylazine using an MTT assay. Cell viability results after treatment with acepromazine were also compared with the previously described effects of xylazine (EDMONDS et al., 2016;MANCINI et al., 2017) on cell lines derived from equine dermis.

Evaluation of cell viability
ED cells were plated in 96-well tissue culture dishes in 4×10 4 cells/well. The plate was maintained at 37 °C in a humid environment with 5% CO2. After, the medium was removed and the cells were exposed to four concentrations of xylazine or acepromazine in triplicate (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL) and then incubated for 24 hours at 37°C in 5% CO2. The integrity of the cells was verified with an inverted microscope after which the treatment was removed and 100 µL of MTT 0.5 mg/mL diluted in E-MEM was added, followed by 4 hours of incubation at 37 °C in a humid environment with 5% CO2. Subsequently, the MTT was removed and 100 µL of DMSO (dimethylsulfoxide) was added to dissolve formazan crystals. The optical density (OD) was measured with a spectrophotometer (SpectraMax, Molecular Devices) at 540nm.

Effect of pH on cytotoxicity
Acepromazine and xylazine were diluted in a culture medium and the pHs of the solutions were determined with a pH meter (DM-22, Digimed).

Statistical analysis
Data were obtained from experiments performed in triplicate. The mean and standard deviation for the MTT results were calculated. Data were expressed as mean + standard deviation and analyzed by ANOVA, followed by the Tukey test when necessary, with statistical significance set at p <0.01 (CALLEGARI-JACQUES, 2003). Statistical analysis was performed using VassarStats © .

RESULTS AND DISCUSSION
In this study, xylazine and acepromazine were evaluated for cytotoxic effects on ED cells using an MTT assay. Results indicated that these drugs reduced the viability of ED cells. When compared to control cells (100%), a xylazine dilution of 0.02 mg/mL preserved 30.61% of viable cells, 78.03% at 0.01 mg/mL, 85.11% at 0.005 mg/mL, and 94.68% at 0.0025 mg/mL. Regarding the acepromazine dilutions, 6.11% of viable cells were preserved at 0.02 mg/mL, 29.28% at 0.01mg/mL, 77.37% at 0.005 mg/mL and 93.54% at 0.0025 mg/mL, as shown in Figure 1. Figure 1. Effect of xylazine and acepromazine on ED cell viability. Differences were considered significant when p <0.01. Data were expressed as mean ± standard deviation. * indicates a significant difference in relation to controls; and # indicates a significant difference in relation to xylazine (SILVEIRA et al, 2020).
A decrease in the viability of the ED cell line was observed in a concentration-dependent manner for both drugs (xylazine and acepromazine). Besides what was found concerning drug concentration, the measured pHs of the xylazine (7.69) and acepromazine (7.76) solutions were close to the pH of the E-MEM (7.5) used in the cell cultures. Therefore, the pH values of the solutions used in this study should not have interfered with the results observed.
The results of the experiment corroborate those of other studies which showed that xylazine is toxic to thymocytes (CUPIĆ et al., 2003), splenocytes (CUPIĆ et al., 2001), chondrocytes (MANCINI et al., 2017, mesenchymal cells (EDMONDS et al., 2016) and endothelial cells (SILVA-TORRES et al., 2014). Mancini et al. (2017) assessed the cell morphology of chondrocytes treated with xylazine using cell cytometry and verified cell death due to apoptosis. This finding may be of clinical importance since, according to Edmonds et al. (2016), cell death in the region of application could contribute to a local inflammatory reaction. As such, the authors suggest that sedation with xylazine should be replaced with romifidine -a drug of the same class that did not significantly affect cell viability in that study. Alternatively, they recommend a combination of detomidine with butorphanol, which was also found to cause less cytotoxicity than xylazine.
Regarding acepromazine, so far there have been no reports of its effects on cell viability. Ours is the first study. Some authors have evaluated the role of mitochondrial permeability in cell death induced by phenothiazines in HTC cells (De Faria et al., 2015). Disruptions in mitochondrial function are crucial for regulating cell death, through cytochrome c release and apoptosis. This effect was also shown with promethazine, a drug from the same pharmacologic group as acepromazine (Kemmerich et al., 2017;Medeiros et al., 2018). Previous results showed a 50% concentration-dependent decrease in cell viability from a concentration of 25 µM. This decrease was attributed to the activation of the autophagic pathway (Kemmerichet al., 2017), due to a decrease in mTOR protein expression and an increase in autophagic flux (Medeiros et al., 2018). Recently, Do Carmo et al. (2020) used computational simulations to certify if phenothiazines can compete with pro-apoptotic proteins for their binding-sites, which would explain the cytotoxic potential of phenothiazines. Taking all these data into account, the effects caused by acepromazine in ED cells were possibly triggered by the same mechanisms described for other phenothiazines. However, the relevance of this cytotoxic effect in clinical practice still needs to be described.
Cell death caused by the acidity of the drugs was excluded in this study, corroborating results from previous studies on chondrocyte lines (KARPIE and CHU, 2007;BREU et al., 2013a), mesenchymal cells (BREU et al., 2013b), and cells from human melanoma (KANG et al., 2015) exposed to lidocaine. In those studies, lidocaine was diluted in a buffering solution, a culture medium, or a saline buffered solution, which should have prevented the pH effect. We also avoided this effect in our study. However, it must be remembered that lidocaine belongs to the pharmacological group of local anesthetics (RANKIN et al., 2017). Therefore, the comparison of this drug with xylazine and acepromazine may be a limiting factor in the present study.
Results observed in this experiment may be not identical to those which occur in vivo, since damage may be limited by the abundant extracellular matrix that surrounds tissue (JUNQUEIRA & CARNEIRO, 2013). Similarly, it is not possible to observe an inflammatory response in cell cultures, since this is a complex response of organisms in the face of aggression; one which is impossible to mimic in vitro (MITCHELL et al., 2006

CONCLUSIONS
In this experiment, we observed the cytotoxic activity of xylazine and acepromazine in ED cell lines occurring in a concentration-dependent manner. The pH of the drugs had no impact on the results. For pharmacology, this study is relevant since, until now, there has been no recognized effect of acepromazine on cell viability. In this pilot project, however, the possible cytotoxic activity of this drug in vitro has now been shown. Although this research addressed hypotheses about the effect of the tested drugs on cell viability in vitro, to confirm these findings and elucidate the mechanisms involved, more specific tests are needed.