Assessment of the genotoxicity and cytotoxicity of etazolate in cultured human lymphocytes

Etazolate is a pyrazolopyridine compound that exhibits a range of pharmacological mechanisms, including inhibition of phosphodiesterase-4 activity and modulation of the γ-aminobutyric acid (GABA) signaling pathway. Etazolate has shown promising results in treating depression, traumatic brain injury, and Alzheimer’s disease in animal models and clinical trials. However, limited information is available regarding its genotoxicity and cytotoxicity to human cells. Therefore, the current study aims at testing the effect of etazolate on the genetic material using two genotoxicity assays. In addition, the study aims to evaluate the effect of etazolate on the mitotic index (MI) of cultured human lymphocytes. Different concentrations of etazolate (0.01, 1, 10, and 50 µM) were used. Genotoxicity was assessed using the chromosomal aberrations (CAs) assay, and oxidative DNA damage was assessed using the 8-hydroxy-2-deoxyguanosine (8-OHdG) assay, while cytotoxicity was assessed using the MI assay. Treatment of cultured human lymphocytes with etazolate did not induce CAs at all concentrations examined ( p > 0.05). In addition, the normal level of 8-OHdG detected in the control group was not affected by etazolate treatment ( p > 0.05). Furthermore, no effect was observed for etazolate on the MI ( p > 0.05). In conclusion, current results indicate that etazolate is neither genotoxic nor cytotoxic to cultured human lymphocytes using the indicated assays at the examined concentrations.


INTRODUCTION
Etazolate is a pyrazolopyridine-derived compound that exhibits a wide range of pharmacological properties Megat et al., 2021;Siopi et al., 2013). Etazolate enhances GABAergic signaling through positive allosteric modifications of GABA(A) receptors, which could explain its anxiolytic activity (Marcade et al., 2008;Thompson et al., 2002). Etazolate is also a selective phosphodiesterase-4 inhibitor, and thus it can be used to manage mood and cognitive disorders (Ankur et al., 2013;Jindal et al., 2015;Wang et al., 2014). For example, using animal models, etazolate showed positive results in the treatment of neuropsychiatric disorders such as depression and anxiety associated with traumatic brain injury via normalization of oxidative stress and restoring the Cyclic adenosine monophosphate (cAMP) signaling pathway (Jindal et al., 2017). In addition, etazolate was shown to stimulate soluble N-terminal amyloid precursor protein fragment (sAPPalpha) secretion and subsequent elevations in the soluble amyloid precursor protein that can protect neurons from the development of beta-amyloid and Alzheimer's disease (Marcade et al., 2008). Moreover, etazolate has been shown to improve spatial learning and memory performance related to the homing task in aged rats (Drott et al., 2010). A pilot phase IIA, randomized, double-blind, multicenter clinical trial that included 159 patients with Alzheimer's disease demonstrated the safety and tolerability of etazolate and its ability to alter disease progression (Vellas et al., 2011). Similarly, data generated from phase 1 clinical trials of Alzheimer's disease showed that the etazolate has an acceptable safety profile with fewer side effects than other phosphodiesterase-4 inhibitors (Desire et al., 2008). This made etazolate an attractive agent to be implicated in Alzheimer's disease treatment (Drott et al., 2010;Vellas et al., 2011). Additionally, etazolate was shown to protect against memory impairment anxiety-and depression-like behaviors induced by posttraumatic stress disorder (Alzoubi et al., 2017(Alzoubi et al., , 2019 and Parkinson's disease . The majority of studies with etazolate have focused on the clinical uses of this drug Jindal et al., 2017;Marcade et al., 2008;Megat et al., 2021). However, studies that examine the genotoxicity and cytotoxicity of etazolate to human cells are needed. Previous studies showed that some pyrazolopyridine derivatives might have DNA-binding affinity (El-Gohary and Shaaban, 2018;El-Gohary et al., 2019;Suntharalingam et al., 2010). In addition, some pyrazolopyridine derivatives showed antitumor and antiproliferative activity and could interfere with cell cycle progression (Baviskar et al., 2013;Butu et al., 2014Butu et al., , 2015Gavriil et al., 2016;Rodino et al., 2014). Therefore, the current investigation aims to evaluate the genotoxicity and cytotoxicity of different concentrations of etazolate in cultured human lymphocytes using the chromosomal aberrations (CAs) assay, the oxidative DNA damage 8-hydroxy-2-deoxyguanosine (8-OHdG) assay, and the mitotic index (MI) assay.

Blood sampling
Five healthy young men (aged 20-27 years) donated blood for laboratory experiments. Blood donors were nonsmokers and nonalcoholics and had not taken any medications or supplements for at least 3 months before donating blood (Rababa et al., 2021). Blood samples were collected in lithium heparin tubes and cultured fresh within 1 hour of donation (Al-Eitan et al., 2020).

Ethical approval
The study was approved by the Institutional Review Board of King Abdullah University Hospital (Approval No. 23/93/2016). Informed consent was taken from the donors before blood sampling.

Etazolate preparation
Etazolate hydrochloride (Cas No. 35838-58-5) was obtained from Bio-Apex (Houston, TX, Catalog No. B6304) as a powder. The drug was freshly prepared before each experiment by dissolving it in sterile distilled water to obtain a 10 mM stock solution. The stock solution was sterilized using a Millipore filter (0.2 μM) before being used to treat cultured blood lymphocytes. Cultures were treated with different doses of etazolate (final concentrations: 0.01, 1, 10, and 50 µM). These doses were within the range that was previously used with cultured cells (Barnes et al., 1983;Marcade et al., 2008;Mehta and Ticku, 1987).

CAs assay
Cultured cells were treated with different concentrations of etazolate and the positive control (cisplatin, Sigma-Aldrich, USA; final concentration 0.4 μg/ml) in the last 24 hours of the 72hour incubation period (Azab et al., 2019). After cell incubation, harvesting was initiated by arresting the cultured cells for 2 hours in the mitotic phase with 0.1 µg/ml colcemid (Sigma-Aldrich) (Mhaidat et al., 2016). Cultures were then transferred to 15 ml conical tubes and centrifuged at 500 × g for 5 minutes. The supernatant was removed, and cells were then resuspended in 9 ml of prewarmed hypotonic solution (0.56% KCL) and then incubated at 37°C for 20 minutes. Swelled cells were collected by centrifugation at 500 × g for 5 minutes, the supernatant was removed, and cells were then fixed in methanol: acetic acid (3:1) for 10 minutes at room temperature . The cells were then washed with the above fixative 3×, and finally, cells were resuspended in about 1.5 ml of the fixative. The suspended cells were then dropped onto prechilled glass slides to obtain metaphase chromosomal spreads. Chromosomes were then stained by immersing the slides in slide jars containing 5% Giemsa stain (Sigma-Aldridge, USA) in a phosphate buffer for 7 minutes. Slides were then air-dried, and metaphases were visualized using a Nikon medical microscope at 1,000 × magnification. CAs were examined in 500 cells per treatment (100 cells from each donor). CAs were categorized into breaks/exchanges and gaps (Khabour et al., 2013).

MI assay
The MI is a measure of the degree of cellular proliferation and is used to measure cell division (Romansik et al., 2007). The MI was assessed by random analysis >5,000 cells per drug concentration (at least 1,000 cells per concentration per donor) along with scoring of cells that were in the metaphase stage. The MI was expressed as the ratio of the number of metaphases seen over the total number of intact cells seen (Al-Sweedan et al., 2012).

The 8-OHdG assay
8-Hydroxyl-2-deoxy guanosine is a naturally occurring hydroxyl radical-damaged guanosine that is generated during cellular processes (Esmadi et al., 2016). The 8-OHdG test is widely used to assess oxidative DNA damage induced by exposure to chemical agents and disease conditions (Urbaniak and Boguszewska, 2020;Valavanidis et al., 2009). Lymphocytes were cultured and incubated as described for the CAs assay (see above). After 72 hours of incubation, cultured cells were centrifuged at 500 × g for 5 minutes, and the supernatant was removed. The cell pellets were then washed 5× with 9 ml of RPMI 1,640 medium each, and then the cells were resuspended in 1 ml of RPMI 1,640 medium . Thereafter, the cell suspension was treated with etazolate or the positive control (cisplatin) for 6 hours at 37°C. Tubes were then centrifuged at 500 × g, and 8-OHdG levels were quantified in the supernatant using an enzyme-linked immunoassay (ELISA) kit from Abcam (Cambridge, UK; Catalog No. ab201734) according to the protocol supplied with the kit. Plates were read at 450 nm using an automated ELISA reader (Bio-Tek, Winooski, VT). A standard for known concentrations of 8-OHdG was provided by the kit and was used to calculate the 8-OHdG biomarker in samples.

Statistical analysis
Statistical analysis was conducted using the GraphPad Prism software (version 5). All parameters were compared using analysis of variance and Tukey's post hoc test. A p < 0.05 indicates a statistical difference. Table 1 shows the frequency of gap aberrations after lymphocyte cultures were treated for 24 hours under different concentrations (0.1, 1, 10, and 50 μM) of etazolate. None of the examined etazolate concentrations increased the frequency of gap aberrations observed in the control group (p > 0.05). On the other hand, the positive control cisplatin at 0.4 μg/ml caused significant increases in the frequency of gap aberrations by several folds.

RESULTS
When considering CAs (breaks/exchanges), results similar to those of gap aberrations were obtained ( Table 2). Thus, etazolate did not induce genotoxicity in cultured human lymphocytes at concentrations up to 50 μM as examined using the CA assay. Figure 1 shows the level of 8-OHdG in cultured lymphocytes treated with different concentrations of etazolate. 8-OHdG levels were not significantly different between the etazolate groups and the control group (p > 0.05). The treatment of cultures with cisplatin, the positive control, caused significant (p < 0.01) increases in 8-OHdG at a concentration of 0.4 μg/ml. Thus, etazolate did not induce oxidative DNA damage as assessed by the 8-OHdG biomarker.
The MI assay was used to evaluate the antiproliferative potential of etazolate in cultured human lymphocytes. Cultures treated with different concentrations of etazolate showed similar mitotic indices to those of the control group (Fig. 2, p > 0.05). On the other hand, the positive control, cisplatin, at 0.4 µg/ml significantly reduced the MI (p < 0.05). Thus, etazolate did not have antiproliferative effects on cultured human lymphocytes at all examined concentrations.

DISCUSSION
In the current study, the genotoxicity and antiproliferative effects of etazolate were evaluated in cultured human lymphocytes. The results showed that etazolate at concentrations up to 50 μM did not cause CAs or oxidative DNA damage as measured in cultured human lymphocytes by the CAs and 8-OHdG assay, respectively. In addition, etazolate did not affect the MI of cultured cells.
Etazolate is a neuroprotective agent that showed promising results in the treatment of Alzheimer's disease, depression, anxiety, and memory impairments Ankur et al., 2013;Drott et al., 2010;Jindal et al., 2015). Etazolate is a pyrazolopyridine derivative, and previous studies showed that some pyrazolopyridine-derived compounds could interact with DNA (El-Gohary and Shaaban, 2018;El-Gohary et al., 2019;Suntharalingam et al., 2010). In addition, some pyrazolopyridine derivatives showed antitumor and antiproliferative activity and could block the progression of the cell cycle (Baviskar et al., 2013;Gavriil et al., 2016). Furthermore, some studies have reported the ability of phosphodiesterase inhibitors to modulate DNA repair systems via alterations of the cellular cAMP-Protein Kinase A pathway (Ghorbani et al., 2015;Parkkonen et al., 2008;Sabisz and Skladanowski, 2008). These reports prompted us to evaluate the potential genotoxic effect of etazolate in cultured human cells using the CAs assay and the 8-OHdG assay. These assays are widely used, sensitive, and robust indicators of genotoxicity (Mateuca et al., 2006;Urbaniak Alzoubi et al., 2017;Jindal et al., 2015Jindal et al., , 2017. Thus, etazolate appears to be a promising and safe neuroprotective agent. Further studies using other cell types and/or other in vivo genotoxicity models are needed to confirm the present findings. In the current study, etazolate at the concentrations examined did not impact the proliferative capacity of cultured human lymphocytes. In a study on Leishmania donovani promastigotes, etazolate was shown to have an antiproliferative effect at a dose of 24 µM (Saha et al., 2020). In addition, in an animal study on mice epidermal mitosis, etazolate has been shown to inhibit mitosis at concentrations between 10 and 100 µM (Birnbaum et al., 1976). On the other hand, etazolate has been shown to delay the apoptosis and cell death of intestinal epithelial cells induced by chemical agents (Joseph et al., 2005). Thus, the potential antiproliferative effect of etazolate can vary according to the cell type and/or animal model used.
In the current study, an in vitro culture system was used. Previous studies have shown that metabolic activation within the body may significantly impact the genotoxicity and the cytotoxicity of chemical compounds (Ku et al., 2007;Oliveira et al., 2021;Zhou et al., 2007). Therefore, in vivo studies are needed to confirm the present in vitro findings.

CONCLUSION
In the current study, using the CAs, 8-OHdG, and MI assays, etazolate was found to be neither genotoxic nor cytotoxic to cultured human lymphocytes. More in vivo studies are needed to confirm these results.