Evaluating the Protective Effects of L-Arginine against Cisplatin-Induced Cytotoxicity, Genotoxicity, and Oxidative Stress in Normal Kidney Cells and Human Blood Lymphocytes

Abstract

Background and purpose: L-arginine is an essential amino acid used for glutathione synthesis and as a precursor to nitric oxide, it can act as a free radical scavenger and inhibitor of lipid peroxidation. Therefore, this study aimed to investigate the protective effect of L-Arginine on cisplatin-induced cytotoxicity, genotoxicity, and oxidative stress in normal kidney cells and human blood lymphocytes. Materials and methods: In this experimental study, normal kidney cells (Vero cell line) and human blood lymphocytes were used. Vero cells were pre-treated with various concentrations of L-Arginine (9.35, 18.75, 37.5, 75, 150, and 300 µg/mL) and a damaging dose of cisplatin (1.7 µg/mL). Cell viability and IC50 (inhibitory concentration) were evaluated using the MTT assay. For genotoxicity assessment, 5 mL of venous blood sample was collected from a healthy, non-smoking, non-alcoholic volunteer using a heparin syringe and after isolating lymphocytes, different concentrations of L-Arginine were pre-treated with cisplatin at an optimal genotoxic dose. To evaluate micronucleus formation in cytokinesis-blocked binucleated lymphocytes, the slide was prepared and was evaluated by light microscopy. Oxidative stress tests, including ROS and MDA levels, were conducted. ROS levels were measured using a fluorimeter device and DA-DCFH reagent. To measure the amount of malondialdehyde (MDA) produced during the lipid peroxidation process Thiobarbituric acid (TBA) was used as a reagent. Data analysis was performed using one-way ANOVA with GraphPad Prism.8 software. Results: Cisplatin exhibited dose-dependent cytotoxicity at concentrations (0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μg/ml) after 48 hours incubation, with an IC50 of 1.7 μg/mL. Based on the results of this study, Pre-treatment with L-arginine at concentrations of 18.75, 37.5, 75, 150, and 300 μg/ml with 1.7 μg/ml cisplatin significantly reduced cytotoxic effects, so that with the increase of L-arginine concentration, increasing the viability of normal lung cells compared to cisplatin alone as a positive control group. On the other hand, micronucleus test results showed that L-arginine significantly inhibited the genotoxicity of cisplatin in human blood lymphocytes. So in the concentration of 18.75 µg/mL, there was a significant difference with the positive control group (P<0.05), and in concentrations 37.5 to 300 µg/ml this significant difference was evident (P<0.001). Also, L-arginine in different concentrations, reduced oxidative stress caused by cisplatin by decreasing reactive oxygen species (ROS) and malondialdehyde (MDA) production. Statistically, L-arginine had a significant difference with the positive control group in all concentrations (P<0.001). Conclusion: The study demonstrated that L-Arginine, as an antioxidant compound, moderated cisplatin-induced cytotoxicity, genotoxicity, and oxidative stress in normal kidney (Vero) cells and human blood lymphocytes, showing significant protective effects. Therefore, it can be hoped for potential use as a preventive agent.

Keywords

• l-arginine
• cisplatin
• micronucleus assay
• cytotoxicity
• oxidative stress