The high-throughput metabolic viability-based colorimetric MTT test is commonly employed to screen the cytotoxicity of different chemotherapeutic drugs. The assay assumes a cell density-dependent linear correlation with the MTT spectral absorbance. Therefore, the present study aimed to compare the cytotoxicity assessment between the MTT assay and gold standard cell number enumeration. The cytotoxicity was induced by Cisplatin, Etoposide, and Doxorubicin in human lung epithelial adenocarcinoma cells (A549) and cervix carcinoma (HeLa) cell lines. The mitochondrial mass was estimated, and immunoblotting of succinate dehydrogenase (SDH-A) was performed following drug treatment in both cell lines. Student’s t-test paired analysis was employed to calculate the significance of the results, where the value p < 0.05 was considered statistically significant. The drug-induced cytotoxic response estimated by MTT absorbance did not show any significant difference with respect to control, and no correlation was observed with the enumerated cell number in both A549 and HeLa cells. Interestingly, per-cell metabolic viability was found to be increased by 1.18 to 3.26-fold (p < 0.05) following drug treatment. Further, mechanistic investigation revealed a drug concentration-dependent significant increase in mitochondrial mass (1.21 to 4.2-fold) and upregulation of SDH protein (50–70%) as well as enzymatic activity with respect to control in both A549 and Hela cells. The limitation of the MTT assay for drug-induced cytotoxicity assessment is due to increased mitochondrial mass and SDH upregulation in surviving cells, leading to enhanced formazan formation. This leads to a lack of correlation between cell number and MTT spectral absorbance, suggesting that the MTT assay may provide an erroneous conclusion for cytotoxicity assessment.

基于高通量代谢活力的比色MTT测试通常用于筛选不同化疗药物的细胞毒性。该测定假设细胞密度与 MTT 光谱吸光度呈线性相关。因此，本研究旨在比较MTT测定法和金标准细胞计数法之间的细胞毒性评估。顺铂、依托泊苷和阿霉素在人肺上皮腺癌细胞 (A549) 和宫颈癌 (HeLa) 细胞系中诱导细胞毒性。估计线粒体质量，并在两种细胞系中进行药物处理后进行琥珀酸脱氢酶（SDH-A）的免疫印迹。采用学生 t 检验配对分析来计算结果的显着性，其中p  < 0.05 的值被认为具有统计显着性。通过 MTT 吸光度估计的药物诱导的细胞毒性反应与对照相比没有显示任何显着差异，并且在 A549 和 HeLa 细胞中未观察到与计数细胞数的相关性。有趣的是， 药物治疗后，每个细胞的代谢活力增加了 1.18 至 3.26 倍 ( p < 0.05)。此外，机制研究揭示了 A549 和 Hela 细胞中线粒体质量（1.21 至 4.2 倍）和 SDH 蛋白上调（50-70%）以及酶活性的药物浓度依赖性显着增加。 MTT 测定法用于药物诱导的细胞毒性评估的局限性是由于存活细胞中线粒体质量增加和 SDH 上调，导致甲臜形成增强。这导致细胞数量和MTT光谱吸光度之间缺乏相关性，表明MTT测定可能为细胞毒性评估提供错误的结论。