## Changes in conditions cause oxidative stress in cells

In addition to temperature changes, other factors can cause stress in the cell too. Oxidative stress, which is defined as a disruption of the balance between the concentration of oxidants and antioxidants in the cell, is very common and dangerous for the cell. Oxidative stress is caused not only by changes in external but also internal conditions, for example by damage to various cellular structures. Oxidants or reactive particles are molecules that, if they are presented in excess in the cell and at the same time this excess is not removed quickly enough by antioxidants, will cause serious damage to macromolecules, especially proteins, lipids and DNA. The main source of reactive particles in the cell are mitochondria, which are created during cellular respiration when adenosine triphosphate (ATP) molecules are formed on the inner mitochondria membrane (Figure 8.1). ATP is the main source and storehouse of energy for cells and can be released at any time by splitting the so-called high-energy bonds.

During the ATP synthesis, a large number of electrons are released, which must be stored somewhere, and most often the location for storage is the oxygen molecule. Since oxygen has a high electronegativity (in the periodic table of elements, it is located in sixth group, so it lacks only two electrons in the second valence layer to reach the full complement of eight electrons). Oxygen can bind electrons relatively strongly to form unstable particles known as reactive oxygen species (ROS) (Table 8.1). The most dangerous ROS for cells are the these with extremely short half-life, namely superoxide (O2•-) and hydroxyl (OH•) radicals. The superoxide radical is produced in cells naturally during cellular respiration and the hydroxyl radical by the Fenton reaction, when in the presence of metal ions (Fe2+ or Cu+) hydrogen peroxide is degraded into a hydroxyl radical and a hydroxide anion.

Reactive species | Average time of occurence
Radicals | Superoxide (O2·-) | 10-6 seconds
Hydroxyl (OH·) | 10-10 seconds
Peroxyl radical (ROO·) | 17 seconds
Nitric oxide (NO·) | a few seconds
Non-radical compounds | Hydrogen peroxide (H2O2) | Stable
Singlet oxygen (1O2) | 10-6 seconds
Ozone (O3) | a few seconds
Nitrogen trioxide (N2O3) | a few seconds
Nitrous acid (NHO2) | a few seconds

Table 8.1 Examples of some reactive oxygen species (ROS) with the average time of occurance in the cell.

Since the processes that cause the formation of reactive particles occur naturally in cells, there are of course mechanisms for their regulation. These are referred to as antioxidant mechanisms and are provided by molecules called antioxidants. Depending on how antioxidants perform their function, they are classified as enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants are protein complexes that ensure the breakdown of reactive particles into stable, safe molecules. The best-known enzymatic antioxidants are superoxide dismutase, catalase, and glutathione peroxidase.

Superoxide dismutase is an enzyme that ensures the decomposition (dismutation) of the superoxide radical into hydrogen peroxide and molecular oxygen. Since the superoxide radicals are produced primarily in mitochondria, superoxide dismutase also is primarily located there in order to eliminate superoxide as soon as possible. Activity of superoxide dismutase produces hydrogen peroxide, which is also a reactive particle. While hydrogen peroxide is not as dangerous for the cell as the superoxide radical, if it is not eliminated, it can enter the Fenton reaction and creates an extremely reactive and dangerous hydroxyl radical. To prevent this, the cell eliminates hydrogen peroxide with the action from another antioxidant catalase, to form a water molecule and molecular oxygen. In addition to catalase, the cell can also activate glutathione peroxidase, which also eliminates hydrogen peroxide with the help of the non-enzymatic antioxidant glutathione. Which enzyme the cell chooses depends on the conditions inside the cell. If the concentration of peroxide is low, the cell will primarily use glutathione peroxidase because it eliminates hydrogen peroxide relatively slowly, even at low concentrations. However, as soon as hydrogen peroxide concentration rises sharply, glutathione peroxidase cannot effectively degrade it, thus catalase is activated, which eliminates the toxic hydrogen peroxide more quickly.

The second group of antioxidants that the cell uses to eliminate reactive particles are non-enzymatic antioxidants. They are small molecules that can remove or give an electron to reactive particles and thus return them to their original stable state. Such "used" antioxidants can either be degraded in the cell or recycled and used to eliminate other reactive particles. The advantage of non-enzymic antioxidants compared to enzymes is their size - they are much smaller than enzymatic antioxidants, so they can move relatively quickly to places that are inaccessible to the enzyme. The best-known non-enzymatic antioxidant is glutathione. It is a short peptide that contains the amino acid cysteine with a thiol functional group (-SH) in its structure. Under standard conditions, a hydrogen cation is bound to sulphur. However, in the presence of reactive particles, oxidation of the thiol group occurs, i.e. the transfer of an electron and formation of a disulphide bond (S-S) between two glutathiones (Figure 8.2). Such cysteines can no longer donate more electrons, so they are returned to their original state by the enzyme glutathione reductase.

To cause oxidative stress in the cell, the concentration of reactive particles must be so high that antioxidants cannot remove them effectively. This condition can occur either due to the influence of the external environment, i.e. reactive particles can enter the cells by passing through the cytoplasmic membrane, or they are created in excessive amounts directly in the cell due to the influence of foreign substances, or by processes inherent in the cell (for example, during cellular respiration). If the reactive particles are not eliminated quickly enough, damage of various macromolecules can occur. Oxidation of macromolecules is random, taking place based on which are located near reactive particles.

One of the most frequently oxidized macromolecules in cell are proteins. Their oxidation can cause a change in conformation, overall charge, fragmentation of the amino acid chain or aggregation. The most common oxidative damage of proteins is carbonylation. In this process, the carbonyl group (-C=O) binds to amino acids in the protein. Carbonylation can occur in several places of the protein, which can significantly change its conformation (Figure 8.3A). The cell is unable to repair such damage, so the protein have to be degraded. The second relatively frequent damage is the formation of disulfide bonds, like in glutathione. Disulfide bonds may not only occur between two proteins, but also within one protein (Figure 8.3B). With such damage, the active site may be covered, or another part of the protein may be exposed, which may react with other proteins in the cell, thus causing protein aggregation. However, this modification can be easily repaired so protein does not have to be degraded.

The second often target of reactive particles are lipids. Lipids are the main component of membranes, placed right next to each other to form a phospholipid bilayer. They are composed of a hydrophilic head, which is oriented to the environment (toward the intracellular and extracellular space) and a hydrophobic tail composed of higher fatty acid tails (Figure 8.4A). The main task of lipids are to form a partially permeable membrane between the environment and the inside of the cell. However, reactive particles presented in the cell can oxidize fatty acids chains, causing them to bend. The oxidized chains change conformation leading to the movement of lipids in the bilayer and therefore creating a hole in the membrane (Figure 8.4B).

A third macromolecule that can be targeted by reactive particles is DNA. Although DNA is protected in the nucleus, covered by nuclear proteins and nucleus membrane, reactive particles still manage to reach and cause damage to DNA. Oxidative damage of DNA is relatively frequent, the cell must repair 10,000-100,000 instances of them per day. The most common are oxidized DNA bases, of which guanine is the most susceptible to this type of damage. Oxidation of guanine by reactive particles produces a modified 8-oxoguanine. It differs from standard guanine only by the presence of oxygen on the eighth carbon. Although this change is not noticeable at first glance, it causes 8-oxoguanine to pair with thymine instead of cytosine. If the cell does not repair the damage, a mutation will occur after DNA replication.

Another type of DNA damage that can result from oxidative stress are strand breaks, where the sugar backbone of DNA is oxidised and thus is broken, or interrupted. Primarily, single-strand breaks are formed (Figure 8.5A), because reactive particles can disrupt only one strand at a time, but if co-oxidation of both strands in close proximity occurs, the hydrogen bonds between the bases are not able to hold both strands together and a double-strand break occurs (Figure 8.5B). For cells, double-strand breaks are a serious damage, because they cause chromosome fragmentation, and the cell cannot replicate or transcribe such damaged DNA (more in chapter 5 - Mutations: how they arise and what to do with them).