Amitraz induced cytotoxic effect on bovine cumulus cells and impaired oocyte maturation

Abstract

The aim of this study was to evaluate the genotoxic and cytotoxic effects of amitraz (AMZ) on the primary culture of bovine cumulus cells (CC) and oocyte nuclear maturation. Cytotoxicity was evaluated by assessing mitochondrial activity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Genotoxicity was estimated using the alkaline single cell gel electrophoresis (SCGE) assay. Apoptosis was detected with the Annexin V-affinity assay. The in vitro maturation test was performed in bovine oocytes. To understand AMZ action, glutathione content, superoxide dismutase enzyme activity, and lipid peroxidation were evaluated in CC. Results showed that AMZ lethal concentration (LC 50 24h ) for bovine CC was 32.55 μg/mL (MTT assay). A 25 μg/mL induced late apoptosis and necrotic cells (p < 0.05); however, DNA damage was decreased at the same concentration (SCGE assay; p < 0.05). A decrease in metaphase II was observed at 25 μg/mL, and degenerate oocytes were observed at 15 and 25 μg/mL (p < 0.05). None of the oxidative stress parameters evaluated showed significant differences. This study contributes to a better understanding of AMZ in this model, suggesting its potential cytotoxicity and impact on bovine reproduction.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

“…The location of formazan formation and its intracellular transportation has remained controversial. While the role of mitochondria in MTT reduction [ 2 , 4 , 5 ] has been a justification for the common application of the assay to measure mitochondrial activity [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ], biochemical and microscopic studies have located formazan in various intracellular organelles. Intracellular formazan granules have been observed in the endoplasmic reticulum, cytosolic lipid droplets [ 13 , 14 ], plasma membranes [ 1 , 15 ], nucleus, and microsomes [ 5 ].…”

“…Several studies have revealed limitations of the MTT assay [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] and various confounding factors that are needed to be considered in designing, performing, analyzing, and interpreting the assay results [ 5 , 9 , 10 , 11 , 12 , 13 , 14 ]. However, the MTT assay is still commonly used and interpreted, overlooking these limitations and escaping the necessary optimization steps.…”

Effects of an indole derivative on cell proliferation, transfection, and alternative splicing in production of lentiviral vectors by transient co-transfection

“…Effects of indole on cell proliferation and morphology were evaluated by label-free cell count and brightfield microscopy at 24 h post-transfection. This method was preferable to e.g., MTT assay due to fewer limitations to assay validation [53][54][55][56][57][58][59][60][61] such as interferences from potentially confounding factors, e.g., cell culture variations, DMSO, optical effects, cell manipulation, and transfection effects on metabolic activity [62][63][64][65][66][67][68][69][70][71][72][73]. Cells were imaged at 40X magnification using the high contrast brightfield detection channel of the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek CYT1FAV).…”