Oxidative stress, free radicals and antioxidants

The biological concept of oxidative stress describes a state where the equilibrium between pro-oxidant molecules and antioxidant defence mechanisms is shifted in favour of the former^[1]. First defined in 1985, oxidative stress is formally described as an imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage^[1]. This phenomenon is a common denominator across many chronic diseases, which, despite their multifactorial origins, share this underlying biochemical disturbance^[1].

It is essential to distinguish between harmful oxidative stress and physiological redox regulation. A physiological level of oxygen/nitrogen free radicals and non-radical reactive species is termed oxidative eustress or “good stress”^[1]. This state is characterized by low to mild levels of oxidants involved in the regulation of various biochemical transformations such as carboxylation, hydroxylation, peroxidation, or modulation of signal transduction pathways^[1]. These pathways include the Nuclear factor-κB (NF-κB) cascade, Mitogen-activated protein kinase (MAPK) pathways, and the nuclear factor erythroid 2–related factor 2 (Nrf2) system^[1].

When the normal steady state is disturbed, the resulting imbalance may provoke pathological reactions causing a range of respiratory and non-respiratory diseases, such as chronic obstructive pulmonary disease. The term oxidative stress is therefore a double-edged sword; while essential for host defence and physiological signaling, it becomes harmful when reactive species overwhelm the availability of antioxidants^[1]. In clinical practice, this balance is critical; for example, in older adults or patients with chronic inflammatory conditions, the capacity to maintain this steady state is often compromised, leading to the persistence of inflammation. ^[2]

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are chemical entities that often contain at least one unpaired electron in their outer shell, a feature that typically confers high reactivity^[1]. These species are formed during electron transfer reactions, either by losing or accepting electrons^[1]. The collective term "reactive species" encompasses both free radicals, such as the superoxide radical and hydroxyl radicals, and non-radical reactive species, such as hydrogen peroxide^[1].

The chemistry of these species is central to their biological impact. For instance, the superoxide radical (O2•-) is a primary reactive species that is managed by the enzyme superoxide dismutase (SOD), which facilitates its removal through the reaction: 2O2•– + 2H+ → H2O2 + O2. Hydrogen peroxide, while not a free radical itself, is a critical component of the ROS family that participates in various signaling cascades^[1]. Other significant species include nitric oxide (·NO), peroxyl radicals, and peroxynitrite (ONOO−), which contribute to the overall oxidative and nitrosative burden within the cell^[1].

The study of these molecules has evolved significantly since oxygen was first recognized as a component of combustion processes in the 18th century. Since the 1960s, it has been understood that oxygen is necessary for energy generation in aerobic life, yet it simultaneously possesses the potential to threaten life through the generation of these reactive molecules. In medical contexts, distinguishing between these species is vital. For example, hydroxyl radicals (·OH) are particularly damaging due to their extreme reactivity, while nitrogen-based species like peroxynitrite (ONOO−) are central to the nitrosative stress observed in chronic kidney disease^[1]. Illustrative example: A patient with chronic kidney disease may exhibit elevated levels of both ROS and RNS, necessitating a multi-faceted approach to understanding their specific oxidative profile.

Oxidative stress arises from both endogenous and exogenous sources^[1]. Endogenous production occurs primarily within the mitochondria and through the activity of NADPH oxidases^[1]. Exogenous sources that contribute to the total oxidant load include exposure to radiation, certain drugs, specific food components, cigarette smoking, and environmental pollution^[1]. When these sources increase the concentration of ROS beyond the threshold of 10 to the power of minus 6 M, the system shifts from eustress to oxidative distress, leading to significant damage to biomolecules^[1].

The damage caused by oxidative stress is broad, affecting the fundamental building blocks of the cell: DNA, proteins, and membrane lipids^[1]. In the context of chronic kidney disease, for example, damage is mediated through distinct pathways, including ROS-mediated oxidative stress, RNS-mediated nitrosative stress, HOCl-mediated chlorinated stress, and carbonyl stress resulting from advanced glycation end products^[1]. This damage is not limited to a single organ; it is a systemic issue that can affect any tissue. In Alzheimer’s disease, the extent of oxidative damage observed in brain tissues provides clear evidence that antioxidants may be helpful in ameliorating such damage^[1].

The body has evolved sophisticated systems to scavenge these radicals and maintain homeostasis. However, when these systems are overwhelmed, the resulting oxidative damage contributes to the pathogenesis of various chronic states^[1]. This is particularly relevant in vulnerable populations, such as those in low-resource settings where environmental pollutants may exacerbate the baseline oxidant load, or in pregnancy where metabolic demands increase the production of ROS. Understanding these pathways is critical for medical students, as the modulation of oxidative stress remains a significant area of research for therapeutic intervention in inflammatory and degenerative diseases^[1].

The human body maintains cellular integrity through a sophisticated antioxidant defence mechanism designed to neutralize reactive species immediately upon their formation^[1]. This system is essential because approximately 4–5% of oxygen consumed in the human body is converted into reactive oxygen species (ROS) by biological reductants. Under normal physiological conditions, this conversion rate is lower, typically around 1–2% of total consumed oxygen being converted into superoxide radical anions and hydrogen peroxide. However, under hyperglycemic conditions, this conversion can increase up to 10%. To counter this, cells employ a concerted action of both enzymatic and low molecular weight antioxidants.

The primary enzymatic defences include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Superoxide dismutases are critical for the initial dismutation of superoxide radicals. Catalase and glutathione peroxidases subsequently manage the resulting hydrogen peroxide. The glutathione system is particularly robust, involving glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and glutathione itself^[1]. Glutathione peroxidase is a selenium-dependent enzyme, containing four units of selenium as cofactors, which facilitates the breakdown of both organic hydroperoxides and hydrogen peroxides^[1]. Specifically, glutathione peroxidase 1 is an efficient scavenger of hydrogen peroxide, whereas glutathione peroxidase 4 demonstrates higher efficacy against lipid hydroperoxides^[1]. These enzymes are found in high concentrations in the liver, where they play a vital role in detoxification metabolism^[1]. ^[2]

Beyond enzymes, low molecular weight antioxidants—such as vitamins C and E, flavonoids, carotenoids, and melatonin—provide essential support. Vitamin E is recognized as the first line of defence against the peroxidation of lipids. The overall balance of oxidative stress is maintained by either restricting the activity of radical-producing enzymes, such as xanthine oxidase and NAD(P)H oxidase, or by enhancing the expression of the aforementioned antioxidant enzymes^[1]. When these defences are overwhelmed, oxidative stress contributes to the pathology of numerous conditions, including cardiovascular disease, diabetes, and various neurodegenerative disorders.

The respiratory burst represents a specialized mechanism where cells, particularly those involved in inflammation and immune response, produce reactive oxygen species to neutralize pathogens. This process is primarily mediated by NADPH oxidases, which generate superoxide radicals and hydrogen peroxide as a second line of attack following the onset of initial pathology or injury-mediated inflammation. While this burst is a necessary physiological response to infection, it highlights the dual nature of ROS; they are essential for signalling and host defence, yet their overproduction leads to oxidative stress.

The regulation of this burst is critical. For instance, in pulmonary endothelial cells, the herbicide paraquat triggers a redox cycling mechanism that results in the formation of superoxide radical anions via exogenous NADPH. This illustrates how exogenous factors can hijack cellular pathways to induce oxidative stress. Conversely, therapeutic interventions can modulate these pathways. Curcumin has been shown to exert a protective effect against gentamicin-induced oxidative stress by increasing the levels of superoxide dismutase and glutathione, while simultaneously suppressing proapoptotic proteins like Bax and the executioner caspase, caspase-3.

The respiratory burst is also implicated in the progression of chronic diseases. For example, cancer cells often produce high amounts of ROS, forcing them to adapt to enhanced oxidative stress to survive. Similarly, in type 2 diabetes, the mitochondria serve as a major source of ROS-induced oxidative stress. The interplay between the respiratory burst and the antioxidant system is a dynamic process. When the production of ROS via NADPH oxidases exceeds the scavenging capacity of the antioxidant enzymes, the resulting oxidative stress leads to disturbed signalling pathways, impaired mitochondrial function, and protein modification. Understanding this balance is fundamental to clinical medicine, as oxidative stress is observed as a primary cause in atherosclerosis and radiation-induced lung injury, and as a secondary factor in the progression of diseases like Alzheimer’s and chronic pulmonary disease. ^[1]

Oxidative stress represents a fundamental mechanism of cellular injury across diverse clinical scenarios, ranging from acute drug toxicity to chronic systemic conditions. A classic example of drug-induced oxidative stress is paracetamol (acetaminophen) hepatotoxicity. At therapeutic doses, paracetamol is primarily conjugated in the liver. However, when taken in excess, the saturation of these pathways leads to the increased production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). This metabolite depletes intracellular glutathione (GSH) stores. Glutathione is a tripeptide occurring in high millimolar concentrations in the intracellular environment, and it is essential for maintaining cellular redox homeostasis. When GSH is depleted, the cell loses its primary defence against reactive oxygen species (ROS), leading to covalent binding of NAPQI to hepatic proteins, mitochondrial dysfunction, and subsequent centrilobular necrosis. ^[1]

Hyperoxia provides another critical model of oxidative stress. The lung is the only organ that is under more exposure to ambient air than the skin, making it uniquely susceptible to inhaled oxidants and high partial pressures of oxygen. When the equilibrium between the antioxidants and oxidants in the lung lining fluid is compromised, it leads to oxidative stress. This triggers an inflammatory response, resulting in the release of cytokines and pro-inflammatory chemokines, which facilitates the invasion of leukocytes and monocytes into the inflammatory environment. ^[2]

Ischaemia-reperfusion (I/R) injury represents a paradoxical phenomenon where the restoration of blood flow to an ischaemic tissue exacerbates cellular damage. During the ischaemic phase, the lack of oxygen disrupts the electron transport chain. Upon reperfusion, a sudden influx of oxygen leads to a burst of ROS production. This oxidative surge overwhelms the endogenous antioxidant defence systems, such as glutathione reductase (GRd), which is responsible for maintaining the balance between oxidised and reduced glutathione. In the cells, the GSH/GSSG ratio is greater than 30 (Hwang et al. ). ^[1][2]

A significant pitfall in the study of oxidative stress is the assumption that lower levels of a specific antioxidant, such as glutathione, are always synonymous with high oxidative stress. In reality, lower GSH levels do not necessarily have to be related to increased oxidative stress, but rather to suppressed cysteine pools as a result of a specific diet. Furthermore, while many studies evaluate GSH concentration in erythrocytes because it is high, whether it is a good measure of systemic oxidative stress remains under question. ^[1]

Another common error is the misinterpretation of the causal relationship between oxidative stress and disease states. While oxidative stress is a common denominator of a wide range of diseases, including cancer, Alzheimer’s disease, and cardiovascular disorders, it is often difficult to determine if oxidative stress is a cause or a consequence of the disease state. For instance, in mental and psychiatric disorders like schizophrenia or bipolar disorder, the association is well-documented, but the causal direction is problematic to reliably determine.

Researchers and clinicians must also be cautious when interpreting antioxidant interventions. While it is intuitive to assume that supplementing antioxidants will always mitigate damage, recent evidence suggests complexity. In lung cancer, for example, ROS-induced oxidative stress is maintained by the enhanced transcription of antioxidant enzymes. In response to this stress, Nrf2 accumulates and stabilizes the pro-metastatic transcription factor BACH1 by inducing heme oxygenase-1 (HO-1). Consequently, oxidative stress suppressed by antioxidants can, in some cases, increase the stability of BACH1 and promote its accumulation, thereby facilitating metastasis. This highlights that antioxidant therapy is not universally beneficial and may inadvertently support pathological signalling pathways. ^[1]

Finally, relying on a single biomarker is rarely sufficient. While 8-OH-Gua is one of the best-studied oxidative DNA markers, it represents only one facet of damage. To date, more than 100 DNA adducts with ROS have been detected. Relying on a single marker may overlook the complexity of the redox environment. A comprehensive understanding requires evaluating multiple markers, such as thioredoxin (TRX) levels, which are elevated in patients with cancer and AIDS, or peroxiredoxin (PRX) levels, which are used as biomarkers in diabetes and rheumatoid arthritis. ^[1]

In clinical practice, the assessment of oxidative stress is increasingly relevant for monitoring chronic disorders and evaluating therapeutic efficacy. Biomarkers are used to track the progression of conditions where redox signalling is disturbed, such as diabetes mellitus, where direct oxidation of biomolecules and impaired signalling occur simultaneously.

Clinical research is currently focused on identifying key biomarkers to establish a direct relationship between the level of oxidative damage and the progression of chronic disorders. For example, PRX4 has been proposed as a suitable biomarker of oxidative stress specifically in diabetes. Similarly, oxidized PRX6 in cerebrospinal fluid has been identified as a correlate of oxidative stress following traumatic brain injury. These specific enzymes are part of a family that detoxifies peroxides and peroxynitrites with high turnover, making them sensitive indicators of the cellular redox state. ^[1]

In the context of oncology, the measurement of oxidative stress markers is being explored to predict metastatic potential. Given that lung cancer cells are characterized by increased ROS production, the inhibition of HO-1 is currently being investigated as a promising therapy to prevent metastasis. Furthermore, the quantification of oxidized nucleosides in urine, such as 8-OH-Gua, provides a non-invasive method to assess the total oxidative stress of an organism. The most sensitive method for the detection of such oxidative DNA damage remains the combination of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) electrochemical detection. ^[1]

Ultimately, the goal of clinical practice is to move beyond descriptive associations. By understanding the specific mechanisms—whether it is the depletion of GSH in paracetamol toxicity or the upregulation of antioxidant enzymes in cancer—clinicians can better target the underlying redox imbalance. As research progresses, it is expected that these biomarkers will play a larger role in the management of aging and chronic inflammatory conditions, where the equilibrium between antioxidants and oxidants is frequently compromised.