Bioscience Methods 2024, Vol.15, No.4, 173-183 http://bioscipublisher.com/index.php/bm 179 5.8 Lipid peroxidation assay The levels of lipid peroxidation in the tissue homogenates were determined following the procedures reported by (Devasagayam et al. 2003). 1 ml of the tissue homogenate was mixed with 1ml of 10% trichloroacetic acid (TCA) solution. Two to three drops of butylated hydroxytoluene (BHT) solution were added to the mixture, vortexed, carefully positioned on ice for 10 min, and centrifuged at 10,000 ×g for 15 min at 4 °C. To the supernatant was added same volume of thiobarbituric acid (TBA), and the mixture was incubated in a boiling water bath for 45 min to allow for the color to develop. After incubation, the sample was cooled in an ice bath and then centrifuged at 2000 rpm for 10 min at 4 °C to eliminate any precipitates. The absorbance of the supernatant was then measured at 532 nm wavelength using a UV-spectrophotometer. Level of lipid peroxidation was expressed as μM TBARS/mg protein using a molar extinction coefficient of 156,000 M-1/cm-1. 5.9 Protein estimation Using bovine serum albumin as a reference, the Bradford test (Bradford, 1976) was used to measure the protein content of the tissue homogenate. 5.10 Data analysis One-way analysis of variance was used to examine the data obtained (since the group are independent) in order to find the differences among the means of the various values obtained from the treatment groups. The means were then separated by Tukey’s multiple comparison tests to check significant difference among the means. IBM SPSS version 21 was used for all statistical calculations. For reporting purposes, data were expressed as mean ± standard deviation, and statistical significance was assumed at p = 0.05. Acknowledgments The authors appreciate Mrs. Alade Toyin of the Department of Biology, Federal University of Technology, Akure for assistance with some assay procedures. Conflict of Interest Disclosure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Adeyemi J.A., and Klerks P.L., 2013, Occurrence of copper acclimation in the least killifish heterandria formosa, and associated biochemical and physiological mechanisms, Aquat. Toxicol., 130: 51-57. https://doi.org/10.1016/j.aquatox.2013.01.001 Adeyemi J.A., Atere T.G., Oyedara O.O., Olabiyi K.O., and Olaniyan O.O., 2013, Hematological assessment of health status of african catfish clarias gariepinus (Burchell 1822) experimentally challenged with escherichia coli and vibrio fischeri, Comp. Clin. Path, 23: 1309-1313. https://doi.org/10.1007/s00580-013-1780-y Adeyemi J.A., 2014, Oxidative stress and antioxidant enzymes activities in the african catfish, clarias gariepinus, experimentally challenged with escherichia coli and vibrio fischeri, Fish Physiol. Biochem., 40: 347-354. https://doi.org/10.1007/s10695-013-9847-x Aliu C., Ajayi O.O., Olawuyi T.S., Gbadamosi O.K., Barbosa F., Adedire C.O., and Adeyemi J.A., 2023, Tissue accumulation, cytotoxicity, oxidative stress, and immunotoxicity in african catfish, clarias gariepinus exposed to sublethal concentrations of hexavalent chromium, Biol. Trace Elem. Res., 11: 1-14. https://doi.org/10.1007/s12011-023-03812-y Arunachalam K.D., Annamalai S.K., and Kuruva J.K., 2013, In vivo evaluation of hexavalent chromium induced DNA damage by alkaline comet assay and oxidative stress in catla, Am. J. Environ. Sci., 9(6): 470-482. https://doi.org/10.3844/ajessp.2013.470.482 Aydın A., Orhan H., Sayal A., Özata M., Şahin G., and Işımer A., 2001, Oxidative stress and nitric oxide related parameters in type II diabetes mellitus: effects of glycemic control, Clin. Biochem., 34: 65-70. https://doi.org/10.1016/S0009-9120(00)00199-5 Baki M.A., Hossain M.M., Akter J., Quraishi S.B., Shojib M.F.H., Ullah A.A., and Khan M.F., 2018, Concentration of heavy metals in seafood (fishes, shrimp, lobster and crabs) and human health assessment in saint martin island, Bangladesh. Ecotoxicol. Environ. Saf., 159: 153-163. https://doi.org/10.1016/j.ecoenv.2018.04.035 Barata C., Varo I., Navarro J.C., Arun S., and Porte C., 2005, Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran daphnia magna exposed to redox cycling compounds, Comp. Biochem. Physiol. Part-C: Toxicol. Pharm., 140(2): 175-186. https://doi.org/10.1016/j.cca.2005.01.013
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