Resistance of HEK-293 and COS-7 cell lines to oxidative stress as a model of metabolic response

Abstract

Oxidative stress (OS), arising from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, plays a pivotal role in cellular dysfunction and the pathogenesis of numerous diseases. This study evaluates the impact of oxidative stress induced by hydrogen peroxide on the metabolomic profiles of the human embryonic kidney (HEK-293) and African green monkey kidney (COS-7) cell lines. Viability (MTT) and free radical accumulation (DCF-DA) assays confirmed a dose-dependent cytotoxic effect of hydrogen peroxide, with COS-7 cells exhibiting greater resistance and producing lower levels of intracellular ROS compared to HEK-293. Metabolomic profiling was conducted using nuclear magnetic resonance spectroscopy (¹H NMR) to identify and quantify metabolic changes. Exposure to a free radical inducer significantly altered both intracellular and extracellular metabolites compared to control H₂O₂-free samples. The analysis revealed common changes in intracellular metabolites between the two lines, including glutamate, NAD⁺, glutathione, ATP/ADP, AMP, and pyruvate - key molecule for mitochondrial function, as well as extracellular metabolites such as glutamate, glutamine, acetate, lactate, and pyruvate. Metabolomic differences observed in COS-7 cells suggest a potentially greater capacity for metabolic adaptation to oxidative stress. These included elevated levels of branched-chain amino acids (BCAA), supporting energy production, and increased formate production, which may aid purine synthesis and cellular resilience. These findings highlight the distinct metabolic adaptations of COS-7 cells to oxidative stress in comparison to the HEK-293 cell line. They also provide insights into the direct cellular responses to altered redox potential, offering possible therapeutic strategies aimed at targeting metabolic pathways to mitigate oxidative stress.

Keywords: TCA cycle; cell culture; metabolomics; oxidative stress; reactive oxygen species (ROS).

FIGURE 1

Mitochondria in oxidative stress and diseases Created using BioRender.com.

FIGURE 2

Effect of hydrogen peroxide (H₂O₂) concentration on HEK-293 and COS-7 cell viability and intracellular ROS levels. Orange bars indicate the H₂O₂ concentrations selected for metabolomic profiling. The black solid line represents the control, normalized to 100% viability or fluorescence. Statistically significant differences (p ≤ 0.05) are marked with an asterisk (*). (A) Viability of HEK-293 cells (left) measured by the MTT assay and intracellular ROS levels (right) measured by the DCF-DA assay after 30-min incubation with increasing concentrations of H₂O₂. (B) Viability of COS-7 cells (left) and ROS levels (right) assessed under the same experimental conditions.

FIGURE 3

PCA score plot of ¹H NMR data obtained from comparison of groups controls with: 100 nM, 1 μM, 0.1 mM, 3.19 mM, and 37.8 mM for cell lines. (A) for HEK-293 cell line. (B) for COS-7 cell line.

FIGURE 4

Relative concentrations of intracellular metabolites showing statistically significant differences between the control and treatment groups exposed to increasing concentrations of hydrogen peroxide (0.1 mM, 3.19 mM, and 37.8 mM). (A) Statistically significant changes in metabolite levels in the HEK-293 cell line. (B) Statistically significant changes in metabolite levels in the COS-7 cell line. Significant differences are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Note: “ATP/ADP” represents a partially overlapping NMR signal region that includes both ATP and ADP resonances, which cannot be unambiguously separated due to limited spectral resolution.

FIGURE 5

PCA score plot of ¹H NMR data obtained from comparison of groups controls with various concentrations of H₂O₂ for different cell lines. (A) for HEK-293 cell line. (B) for COS-7 cell line.

FIGURE 6

Relative concentrations of extracellular metabolites showing statistically significant differences between the control and treatment groups exposed to increasing concentrations of hydrogen peroxide (0.1 mM, 3.19 mM, and 37.8 mM). (A) Statistically significant changes in metabolite levels in the HEK-293 cell line. (B) Statistically significant changes in metabolite levels in the COS-7 cell line. Significant differences are indicated as follows: *p ≤ 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Metabolomic changes during oxidative stress Created using BioRender.com.