Oxidative stress (OS), first described by Helmut Sies in 1985, is a critical factor in the development of numerous diseases (Sies, 2015; Teleanu et al., 2022; Zheng et al., 2022). It arises from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses. ROS, primarily generated in the mitochondrial respiratory chain, are by-products of normal cellular metabolism. However, environmental factors such as pollution, microbial infections, radiation, and smoking can exacerbate ROS production, increasing the risk of cellular damage (Valko et al., 2007; Aseervatham et al., 2013). To counteract ROS-induced harm, cells rely on antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) (Nathan and Cunningham-Bussel, 2013).

While physiological ROS levels support processes like immune defense and wound healing, excessive ROS can overwhelm antioxidant defenses, triggering oxidative stress (Ďuračková, 2010; Bhattacharyya et al., 2014). This leads to cellular damage and inflammation, which contribute to diseases such as cardiovascular disorders (e.g., coronary artery disease, hypertension, and atherosclerosis), vascular diseases (e.g., rheumatoid arthritis and inflammatory bowel disease), neurodegenerative conditions (e.g., Alzheimer’s and Parkinson’s diseases), respiratory disorders (e.g., asthma, acute lung injury, and cystic fibrosis), and cancer (Madamanchi et al., 2005; Matés et al., 2012; Bezerra et al., 2023; Olufunmilayo et al., 2023) (Figure 1).

Hydrogen peroxide (H₂O₂) is a widely used agent to experimentally induce oxidative damage in cell models due to its ability to generate hydroxyl radicals (⋅OH) via the Fenton reaction. It may also directly oxidize thiol groups in redox-sensitive proteins, leading to functional modifications. High H₂O₂ concentrations (>50 µM) are cytotoxic to most cell types, causing DNA strand breaks, nitrogen base alterations, and DNA-protein cross-links (Halliwell, Clement, and Long, 2000; Kołodziej et al., 2018; Ransy et al., 2020). Elevated ROS levels also lead to mitochondrial DNA damage, chromosomal aberrations, and mutations, which promote the development of oxidative stress-related diseases (Singh et al., 2019).

To study the effects of oxidative stress, various assays are employed. For example, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) measures ROS levels, while SOD, CAT, and GPX assays assess antioxidant activity (Weydert and Cullen, 2010). Additionally, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay evaluates cell viability by measuring mitochondrial activity. Metabolomics, a comprehensive tool for analyzing biochemical changes, is particularly effective in investigating oxidative stress-induced metabolic alterations (Liu et al., 2011; Deidda et al., 2018).

Studies on OS are typically conducted using in vitro models, where cell cultures provide a platform to investigate oxidative stress, cellular toxicity, and adaptive responses. These models also help identify compounds with therapeutic potential (Gille and Joenje, 1992; Chung et al., 2022). Cells exposed to pro-oxidant agents like menadione, potassium bromate, or hydrogen peroxide (H₂O₂) represent a gold standard model to examine diverse cellular responses, leading to various cellular reactions and pathways in response to oxidative stress induction (Goffart et al., 2021). This study examines the effects of H₂O₂-induced oxidative stress on human embryonic kidney (HEK-293) and African green monkey kidney (COS-7) cells. The kidney cells, being rich in mitochondria, are highly susceptible to oxidative stress, a key factor in kidney diseases—a growing global health concern (Daenen et al., 2019; Gyurászová et al., 2020). The primary aim is to identify specific metabolic changes linked to ROS-induced mitochondrial dysfunction, particularly those involving the tricarboxylic acid (TCA) cycle. By comparing the resilience of these two cell lines, this work provides novel insights into the biochemical alterations and adaptive responses to oxidative stress at the cellular level.

The biological impact of hydrogen peroxide exposure was first confirmed through MTT and DCF-DA assays, which revealed clear, concentration-dependent responses in both HEK-293 and COS-7 cell lines. A gradual reduction in cell viability was observed starting from 0.1 mM H₂O₂, while ROS production measured by DCF-DA fluorescence, increased significantly at the same threshold, especially in HEK-293 cells. These results indicate that even short-term exposure (30 min) to moderate concentrations of hydrogen peroxide is sufficient to trigger oxidative stress. Notably, COS-7 cells retained higher viability and exhibited a more moderate increase in ROS levels, suggesting greater oxidative resilience. These findings provided a basis for the metabolomic analysis, shedding light on how cells adapt to oxidative stress and which processes are most affected. Concentrations of 100 nM and 1 μM H₂O₂ did not elicit notable changes in the metabolite profiles of HEK-293 and COS-7 cells. However, significant metabolic shifts were detected starting at 0.1 mM hydrogen peroxide. The relatively short incubation time was chosen to capture the early-phase metabolic response to oxidative stress, consistent with previous reports indicating that exposure to a single, concentrated dose of H₂O₂ is sufficient to induce a measurable oxidative stress response (Gille and Joenje, 1992; Wiese et al., 1995; Deferme et al., 2013).

Both cell lines at a higher concentration of H₂O₂ demonstrated a decrease in key intracellular metabolites critical for energy metabolism, including glutathione (GSH), glutamate, NAD⁺, ATP/ADP, and pyruvate, alongside an increase in AMP levels. Under physiological conditions, NAD⁺ is involved in pyruvate conversion to acetyl-CoA and the electron transport chain (ETC.) function via NADH generation. Oxidative stress may disrupt this process by impairing key TCA cycle enzymes, such as isocitrate dehydrogenase and α-ketoglutarate dehydrogenase (α-KGDH), thereby reducing NADH production and ATP turnover (Tretter and Adam-Vizi, 2000; Gray, Tompkins, and Taylor, 2014). The observed decrease in pyruvate suggests disruption of glycolysis and possible oxidative damage to cells (Gray, Tompkins, and Taylor, 2014). Additionally, during oxidative stress, an increase in AMP was observed in both cell lines. Increased intracellular AMP levels indicate an energy-deprived state (Choi et al., 2001; Hawley et al., 2010; Auciello et al., 2014).

Focusing on the HEK-293 cell line, oxidative stress induced profound intracellular metabolic collapse. An increased intracellular GSH at 0.1 mM H₂O₂ indicates an early defense mechanism against OS. However, at higher concentrations, GSH depletion occurred, evidenced by elevated extracellular pyroglutamine levels, a marker of oxidative stress and GSH depletion (Gamarra et al., 2019). The crucial role of glutathione tripeptide (GSH) in protecting cells from oxidative stress is evident, as it acts as a free radical scavenger (Townsend et al., 2003). Glutamate, synthesized from glutamine, plays a vital role in GSH biosynthesis and mitochondrial function (Sappington et al., 2016). Therefore, OS-induced reductions in glutamate levels likely reflect mitochondrial impairment (Straadt et al., 2010).

In COS-7 cells increases in formate, isoleucine, and valine levels were observed, accompanied by a decrease in creatine and aspartate concentration. These changes suggest metabolic reprogramming to support energy production and mitochondrial resilience. Increased levels of branched-chain amino acids (BCAAs), such as isoleucine and valine, which enter the TCA cycle via intermediates like propionyl-CoA, acetyl-CoA, and succinyl-CoA, may reflect adaptations to maintain ATP production under oxidative conditions (Bhagavan and Ha, 2015; Wu et al., 2022). These changes are consistent with activation of mTOR signaling pathways in response to oxidative stress (Bonvini et al., 2018). Reduced aspartate levels may be also explained by mitochondrial impairment and disturbances in the TCA cycle or associated biosynthetic routes (Straadt et al., 2010). A decreasing trend in aspartate was also noted in HEK-293 cells, approaching statistical significance. Elevated formate in COS-7 cells may support nucleotide biosynthesis and oxidative stress mitigation through effects on tetrahydrofolate metabolism, although the exact mechanism remains to be clarified (Oizel et al., 2020; Pietzke et al., 2020).

Significant alterations were also observed in extracellular metabolites, including pyruvate, acetate, glutamine, glutamate, lactate, and unidentified compounds (unknown_1–3). Both cell lines exhibited elevated extracellular acetate levels, which could indicate enhanced de novo synthesis aimed at preserving intracellular acetyl-CoA pool during oxidative stress (Bose, Ramesh, and Locasale, 2019). As a key intermediate in central metabolism, acetate contributes to acetyl-CoA generation and provides an alternative carbon source under nutrient-limited conditions (Liu et al., 2018). A shared characteristic was the elevated extracellular pyruvate concentration observed in both COS-7 and HEK-293 cell lines, with a statistically significant increase detected in COS-7 cells at 37.8 mM H₂O₂ compared to HEK-293 cells at 3.19 mM. Increased extracellular pyruvate is more likely indicative of intracellular efflux due to membrane damage. Additionally, another extracellular metabolite of statistical significance is glutamine, which exhibited a decrease in extracellular medium concentration in both cell lines. It’s worth noting that glutamine serves as a precursor for glutamate, a key component in the biosynthesis of glutathione (Matés et al., 2002). As intracellular glutamine levels decrease, there is a corresponding increase in extracellular glutamate, which might result in a lower concentration of intracellular GSH. Furthermore, extracellular lactate levels showed statistically significant changes in both COS-7 and HEK-293 cell lines compared to the control. In HEK-293 cells, a decrease in lactate was observed at most concentrations, except for the highest H₂O₂ concentration (37.8 mM), where an increase occurred. In COS-7 cells, a statistically significant decrease in extracellular lactate was noted only at 37.8 mM H₂O₂.

HEK-293 cells displayed additional significant changes, including increased extracellular pyroglutamine, hypoxanthine, 3-hydroxybutyrate (BHB), and formate. Formate and hypoxanthine accumulation may reflect mitochondrial dysfunction and chemical damage caused by oxidative stress, potentially resulting in membrane rupture and the release of intracellular contents. Hypoxanthine is routinely evaluated as a marker for OS due to its association with mitochondrial dysfunctions (Makowski, 2021). Elevated BHB suggests an antioxidant response aimed at mitigating oxidative damage and supporting cellular protection (Haces et al., 2008; Rojas-Morales et al., 2020).

In general, the HEK-293 cell line displayed greater vulnerability to oxidative stress. As mentioned earlier, Bian et al. (2015) observed significant oxidative damage in HEK-293 cells, with an IC₅₀ of approximately 0.96 mM following a 2-h exposure to H₂O₂. This study confirms the high sensitivity of HEK-293 cells to oxidative stress. In our study, significant metabolic changes and viability reduction were already evident at similar concentrations following a much shorter 30-min exposure, suggesting an early and rapid onset of oxidative stress responses. GSH depletion, alongside elevated extracellular pyroglutamine, hypoxanthine, and BHB, highlights the sensitivity of HEK-293 cells to oxidative damage. Moreover, HEK-293 cells showed increased extracellular formate and hypoxanthine levels, reflecting mitochondrial dysfunction and disruptions in purine metabolism caused by oxidative stress (Oizel et al., 2020).

Our findings suggest that COS-7 cells, derived from monkey kidney tissue, may exhibit a more stable metabolic profile under oxidative stress conditions. These cells showed smaller reductions in pyruvate, elevated formate levels, and increased BCAAs, reflecting early metabolic adaptations to oxidative challenges. This observation aligns with previous studies, such as Yoon et al. (2016), which reported COS-7 cells maintaining 83.8% viability at 100 μM H₂O₂ and 68% at 400 μM after 24 h. Figure 7 summarizes statistically significant changes in intracellular metabolite levels observed during oxidative stress.

This study demonstrates the utility of metabolomics in uncovering early cellular responses to oxidative stress induced by hydrogen peroxide (H₂O₂). Concentration-dependent metabolic changes were observed, including disruptions in mitochondrial function, the TCA cycle, and amino acid metabolism, accompanied by reductions in GSH and other energy-related metabolites such as ATP/ADP, and AMP – key molecules involved in cellular energy transfer. HEK-293 cells showed changes consistent with a higher susceptibility to oxidative stress, including GSH depletion, elevated oxidative stress markers, and altered purine metabolism. In contrast COS-7 cells exhibited metabolic changes that may reflect an adaptive response, including increases in branched-chain amino acids (BCAAs) and formate, which are associated with mitochondrial support and purine metabolism. These metabolic observations were in line with the results of the MTT and DCF-DA assays, which confirmed reduced viability and increased ROS levels in HEK-293 cells, and a relatively more stable redox status in COS-7 cells.