10.1 Challenges Associated with Oxidative Stress
KEY CONCEPTS
By the end of this section, you will be able to do the following:
- Evaluate the importance of reactive oxygen species (ROS) in normal cellular function.
- Describe how ROS are formed and neutralized in normal cellular metabolism, giving specific examples of ROS and antioxidants.
- Compare how oxidative stress impacts proteins, membranes, and DNA.
- Give examples of environments or other factors that result in oxidative stress.
In Chapter 9, we learned that low oxygen levels (hypoxia or anoxia) can cause cellular stress. In this chapter, we’ll explore how oxygen itself can actually be a source of stress. While eukaryotes generally require oxygen to support cellular respiration, normal metabolic processes convert some of that oxygen into reactive oxygen species (ROS). ROS are important for normal cellular function, but in excess they can cause damage to cells in the form of oxidative stress, as we explore below.
Reactive Oxygen Species (ROS) Formation and Neutralization
ROS are produced when oxygen (dioxygen, O2) is incompletely reduced during metabolic reactions (Figure 10.2). ROS can take the form of free radicals (unstable molecules with an unpaired valence electron) such as superoxide (O2●-) or hydroxyl (OH●) radicals, which are the most reactive forms of ROS. Free radical ROS can be further reduced to non-radical ROS (e.g., hydrogen peroxide; H2O2), which are usually less reactive than the radical forms, but still reactive relative to many other molecules in our cells. ROS typically form during cellular metabolism that involves oxygen, including cellular respiration in mitochondria, photosynthesis in chloroplasts, and fatty acid oxidation in peroxisomes. Despite their high reactivity, small concentrations of ROS in cells have an important role in cell functioning. ROS are important in some cell signalling pathways, cell differentiation, proliferation, and programmed cell death.
Since ROS are useful in small concentrations but dangerous at high concentrations, cells have various mechanisms to prevent ROS from accumulating to high levels. Antioxidants are molecules that help complete the reduction of ROS and their byproducts to neutral (non-reactive, non-toxic) substances like water (H2O). For example, glutathione (GSH) is an antioxidant molecule that easily reacts with ROS like hydrogen peroxide (Figure 10.3). This is a redox reaction (see Chapter 6.3 for a redox review), so the ROS is reduced, and the antioxidant is oxidized. Many antioxidants are enzymes that catalyze these transformation of ROS. The example we just described (reaction of GSH with H2O2) is catalyzed by glutathione peroxidase (GPX) enzymes.
Neutralization of ROS can sometimes require multiple enzymes. For example, superoxide dismutase (SOD) catalyzes the conversion of superoxide into hydrogen peroxide, a less-reactive ROS (Figure 10.3). Catalase (CAT) or other enzymes (e.g., GPX) can then catalyze the conversion of hydrogen peroxide into water (Figure 10.3). Superoxide has an electrical charge, so tends to not cross cellular membranes. SOD must therefore be active whether superoxide is produced, e.g., near the ETS of mitochondria. Conversely, hydrogen peroxide is uncharged and can cross cellular membranes. Much of the hydrogen peroxide in cells is neutralized specifically in the peroxisome, which contains abundant CAT and produces hydrogen peroxide during metabolism of fatty acids. There are many other types of antioxidative enzymes (Chapter 10.3), each of which contribute to keeping a safe amount of ROS in cells without causing cellular stress.
Health Connection
It should be noted that antioxidant enzymes require cofactors such as iron, copper zinc, and manganese for optimum functioning. Moreover, there are also non-enzymatic antioxidants such as vitamin C and carotenoids, which the cell can utilize. For example, vitamin C can neutralize hydroxyl radicals, superoxide radicals, and hydrogen peroxide. In humans, these cofactors and micronutrients must be ingested through the diet. Certain foods are rich in these micronutrients. For example, citrus fruits and leafy greens are good sources of vitamin C.
In recent years there has been a dietary trend in which people attempt to eat large amounts of these antioxidant foods in an attempt to prevent disease. The idea is that oxidative stress can cause cancer so, we should eat lots of antioxidants to reduce ROS in our body. However, this idea is not entirely accurate. While it is true that we need these trace metals and micronutrients, we do not need them in large amounts. Moreover, we also use ROS as signalling molecules and need them in small amounts. There is no evidence to suggest that eating an excess of antioxidant rich foods or taking antioxidant supplements reduces our chances of disease.
Oxidative Stress
Oxidative stress occurs when ROS production exceeds antioxidant capacity, leading to an accumulation of ROS. When ROS accumulate, they can react with and damage biological macromolecules such as proteins, lipids, and DNA. Accumulation of ROS can occur in two ways: excess (higher than usual) production of ROS, or insufficient (lower than usual) antioxidant capacity.
ROS can be produced in cells (internally) at an increased rate under some types of environmental stress. For example, if cells experience oxygen limitation (low oxygen), followed by an influx of oxygen, this can lead to a burst of ROS production, especially at the mitochondrial electron transport system. Freezing and thawing are also hypothesized to cause oxidative stress. When ice forms around the cells, it prevents oxygen from passing through the cell membrane (oxygen limitation). When ice thaws, there is an influx of oxygen (reperfusion) that enters the cell. An influx of oxygen can be overwhelming for a cell’s metabolism and can lead to ROS production. Several external factors can also promote ROS formation in cells. For example, smoking cigarettes can cause an overproduction of ROS as tobacco smoke contains many free radicals, promoting ROS formation. When these radicals interact with cells, they are more likely to produce ROS species.
The activity of antioxidant enzymes, like all enzymes, can be affected by various environmental factors. If these environmental factors prevent antioxidant enzymes from functioning, this can result in ROS accumulation. For example, antioxidant enzyme activity is temperature dependent. Therefore, when temperatures are low for prolonged periods of time, the rate at which ROS species are being reduced by antioxidant enzymes is slowed, which allows ROS to accumulate in the cell.
Cellular Effects of Oxidative Stress
Oxidative stress causes damage to various biological macromolecules in the cell, including proteins, lipids and nucleotides (DNA) (Figure 10.4). ROS can cause damage to DNA by oxidizing nucleotides (e.g., guanine) or the sugar-phosphate backbone, ultimately leading to single and double stranded breaks in the DNA double helix (see Chapter 3.5 for a refresher on nucleic acid structure). If unrepaired, this DNA damage can result in cell death. ROS species are also capable of damaging lipids in a process known as lipid peroxidation. Throughout the lipid peroxidation process, ROS react with carbons in double bonds of unsaturated fatty acid chains (see Chapter 3.3 for a refresher on lipid structure). If those fatty acid chains are part of membrane phospholipids, this oxidation can cause damage to cellular membranes, which can also cause cell death. Finally, high concentrations of ROS in cells can cause protein oxidation that leads to protein misfolding. ROS can react with any amino acid but tend to particularly oxidize sulfur-containing amino acids like cysteine and methionine (see Chapter 3.4 for a refresher on protein structure). Additionally, high levels of ROS can cause protein carbonylation, a process in which carbonyl groups (-C=O) is introduced into amino acid R groups. These reactions alter protein structure and can lead to protein denaturation (misfolding). Misfolded proteins have various consequences such as losing their function. However, a more extreme outcome is protein aggregation, which is when proteins clump together causing a disruption in cellular functions, potentially causing cell death.