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Interactions of Free Radical Initiators and Antioxidants in Erythrocytes: An Ex Vivo Study

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Mammalian erythrocyte is an ideal cell model to study free radical-induced injury since it is enucleated and has a short life span. Various factors can lead to the generation of reactive oxygen species (ROS) in erythrocytes. Evidence indicates that many physiological and pathological conditions develop due to ROS. Erythrocytes when exposed to free radical initiators (FRI) such as H2O2 and azobis (2-amidinopropane) dihydrochloride (AAPH) can result in oxidative stress (OS). Several antioxidants have been employed in various OS conditions to prevent cell damage. However, the alterations due to FRI and antioxidants in erythrocytes are still unclear. Therefore, an in vitro study was conducted using blood samples from male Wistar rats to investigate the influence of FRI and antioxidants such as caffeic acid and p-coumaric acid on erythrocytes. The samples were divided into controls (without FRI; n = 8) and experimentals (with FRI and antioxidants; n = 8). Erythrocyte suspension were assessed for hemoglobin (Hb) and hemolysis, and the hemolysates were analyzed for the following OS markers: SOD, Catalase, TBARS, SH, and AOPP. There were variations in certain OS markers in the experimental groups with respect to controls. Hb significantly decreased in CH group and AOPP significantly increased in CCA, CCO, CCOA, and CCAH groups. SOD and catalase elevated in CCAH and CCOH groups, respectively. TBARS, SH, and hemolysis were maintained in all the groups. The endogenous antioxidant system could scavenge the ROS and protect the erythrocytes from oxidative damage. Azo compounds (AAPH) generate more free radicals when compared to H2O2. OS was minimal in experimental groups as exogenous antioxidants augmented the endogenous antioxidants, however, p-coumaric acid was more efficient than caffeic acid. Hence, these antioxidants can be further employed in similar OS situations.

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ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Leela Iyengar, Dr. Soumya Ravikumar, Dr. Manasa Mithun, Magdaline Christina Rajanand, and JAIN (Deemed-to-be University) for their support.

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The authors received no specific funding for this study.

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Authors and Affiliations

  1. Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be University), 560027, Karnataka, Bengaluru, India

    Anusha Berikai Ananthakrishna, Carl Hsieh, Masannagari Pallavi, Anagha Papinassery, Anu Sunny, Haripriya Gopinath, Prasad Varshith, Shreya Shriyan,  Smita, Sneha Mathew, Tania Arora & Vani Rajashekaraiah

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  1. Anusha Berikai Ananthakrishna
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Contributions

Vani Rajashekaraiah: conceptualization, methodology, supervision, writing- reviewing and editing; Carl Hsieh, Masannagari Pallavi, Anagha Papinassery, Anu Sunny, Haripriya Gopinath, Prasad Varshith, Shreya Shriyan, Smita, Sneha Mathew, Tania Arora: investigation, data curation, formal analysis; Anusha Berikai Ananthakrishna: writing- original draft preparation, formal analysis.

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Correspondence to Vani Rajashekaraiah.

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Conflict of interest. The authors report there are no conflicting interests to declare.

Statement on the welfare of animals. This study was performed in accordance with the guidelines of Institutional Ethics Committee.

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Abbreviations: AAPH—2,2-Azobis(2-amidinopropane) dihydrochloride; AOPP—advanced oxidation protein product; ARE—AU-rich elements; CA—controls with AAPH; CCA— controls with caffeic acid; CCAH—controls with caffeic acid and H2O2; CCO—controls with coumaric acid; CCOA—controls with coumaric acid and AAPH; CCOH—controls with coumaric acid and H2O2; CON—controls without any treatment; FRI—free radical initiators; Keap1—Kelch-like ECH-associated protein 1; Nrf2—nuclear factor erythroid 2–related factor 2; OS—oxidative stress; SOD—superoxide dismutase; TBARS—thiobarbituric acid reactive substances.

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Anusha Berikai Ananthakrishna, Hsieh, C., Pallavi, M. et al. Interactions of Free Radical Initiators and Antioxidants in Erythrocytes: An Ex Vivo Study. Cell Tiss. Biol. 17, 256–264 (2023). https://doi.org/10.1134/S1990519X23030033

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