Essential cellular systems and targets of injurious agents

In order to cause cellular damage, damaging agents must act on cellular systems that are essential for cell survival and function. Once these systems are identified, it will be easy to understand that these same systems represent the main target of the different types of damaging agents.

Systems essential to the survival of a cell: 

• the maintenance of the integrity of the plasma membrane. This system is essentially represented by all the proteins that act as pumps and function at the plasma membrane, ensuring ionic and osmotic homeostasis.
• protein synthesis (both structural and enzymatic proteins). In order to survive, the cell needs to implement an adequate protein synthesis
• energy production, mainly for ATP synthesis: the various systems that allow aerobic respiration
• the defense of the genetic heritage from any kind of damage 

The systems for the defense of the genetic patrimony are essential, in fact our cells are equipped with various systems for DNA repair. It is essential that all these systems work properly because a normal cell, if it undergoes an alteration of its genetic heritage, goes to meet two phenomena:

• if it is a proliferating cell, then it goes into lockdown and stops proliferating so that these enzyme systems can repair the DNA;
• if the enzyme systems fail to repair the DNA properly, then normal cells would rather send the cell itself to death by apoptosis than have a cell with an altered genetic makeup that is potentially a cancer cell.

These 4 systems are essential for the survival of a cell, and are also the primary targets of injurious agents.

Some damaging agents have very specific cellular targets. This is the case of cyanide, a poison that alters mitochondrial respiration by blocking the functioning of the enzyme cytochrome oxidase located at the level of the respiratory chain.

 Another example is the bacterium 'Clostridium perfringens' which produces a molecule that goes to inhibit a specific target: phospholipase, which degrades phospholipids.

For some injurious agents specific targets have not yet been identified but potentially we know they are inherent to the four points listed above.

Biochemical changes involved in cellular damage

Regardless of the type of injurious agent, in a cell that suffers even reversible damage, the following biochemical changes occur: 

• ATP depletion, which is a decrease, a lack of the amount of ATP produced and available. The depletion may be due to an alteration at the mitochondrial level which may be:

- a direct consequence of the injurious agent (such as cyanide described above)

- an indirect consequence, in the sense that the agent in question has not directly altered mitochondria but indirectly has decreased their functionality and therefore the ability to produce ATP.  A decrease in the production of ATP certainly involves an alteration of all ATP-dependent functions, such as the sodium-potassium pump or the calcium pump calcium pump, but it can also occur a decrease in protein synthesis because, in order for the proteins are synthesized, the presence of ATP is necessary.

• Alteration of intracellular calcium homeostasis, as the amount of calcium within the cell changes and in particular there is an increase in calcium levels. The increase in calcium may be due to alterations in the functioning of the calcium pump, or it may be due to increased release from stores. In the cell, calcium is found in free form in the cytoplasm or it is stored in mitochondria and endoplasmic reticulum.

If the amount of calcium inside the cell increases, the most serious consequence is the activation of all calcium-dependent enzymes, such as proteases, nucleases and calcium-dependent lipases. 

If there is the activation of all these enzymes, the cell will undergo a greater degradation at the level of substrates, mainly due to the action of proteases and nucleases. Therefore inside the cell there will be an increase in catabolic activity.

In addition, an increase in intracellular calcium can result in an increase in mitochondrial membrane permeability and subsequent induction of death by apoptosis. Apoptosis can be induced by two pathways: intrinsic and extrinsic. In this case, the increase in intracellular calcium leads to an alteration of membrane permeability, which is the initial event of the intrinsic pathway of apoptosis. The intrinsic pathway is the one that begins with an alteration in the ratio of pro-apoptotic to anti-apoptotic proteins.

• Alteration of plasma membrane permeability. Damage to the plasma membrane can be:

- direct, in case of substances such as bacterial toxins, chemical agents or the activation of the immune response (antibodies), which have as a direct consequence a damage to the plasma membrane through the activation of the Complement System

- indirect, usually due to the formation of ROS (reactive oxygen species), which if produced in excessive quantities damage the cell with a whole series of mechanisms. Free radicals damage the membranes because they alter the fatty acids of the membrane phospholipids. Moreover, when free radicals interact with the fatty acids of membrane phospholipids, they determine the peroxidation of these fatty acids and the products of peroxidation are in turn toxic, they have a detergent function and therefore an amplification of the damage. Indirect damage can also result from the activation of calcium-dependent enzymes such as lipase and protease.

If there is a decrease in the metabolism of the cell there will also be a decrease in phospholipid synthesis and thus an alteration in the structure of the membrane itself.

Cellular Damage and Death: Free Radical Damage

It is essential to dwell on the characteristic aspects of the molecular species of free radicals, especially the mechanisms by which free radicals in general, induce damage in the cell. In particular on the pathological aspects of these molecules, compared to the physical and biochemical aspects.

Free radicals are chemical species characterized by an unpaired electron in the outermost orbital, this electronic arrangement makes them in general highly reactive species because they try to stabilize themselves. This search for stability makes the molecule itself highly reactive, a free radical tries to stabilize itself by subtracting an electron from another molecule. A chain reaction starts, the free radical stabilizes at the expense of another molecule which loses an electron and becomes reactive.

The best known free radicals are oxygen free radicals, but in reality there can be nitrogen radicals (RNS). Let us focus our attention on those that are reactive oxygen species, i.e. the so-called ROS. There are different species of ROS: those that are more abundant for production and those that are more reactive towards other biological molecules.  

• Superoxide anion
• Hydroxyl radical
• Hydroperoxyl radical (forms quite frequently and is also highly reactive)
• Peroxide ion. It is one of the "components" of hydrogen peroxide. Although it is an ion, it falls into the category of ROS because it has a behavior and a reactivity, towards other biological molecules, very similar to that of radicals, so it is assimilated to them even if from a purely chemical point of view it is not.

Free radicals, are formed in our body but the big problem is the fact that in case of damage the amount of ROS increases. Now let's see what are the sources that normally produce free radicals:

• Exogenous sources. Free radicals can be formed within our body, whenever a phenomenon of absorption of radiant energy occurs. Or free radicals are formed from certain carcinogenic substances present in tobacco (exposure to cigarette smoke). Finally, radicals are formed due to environmental pollutants. They can be exogenous sources of radical production, all those foods that we consume and that contain iron and copper.
• Endogenous sources: production of free radicals due to normal metabolic processes within the cells of our body. In an electron transport chain, free radicals are always formed. Endogenous sources leading to free radical production include the normal enzymatic metabolism of xenobiotics (xeno = foreign ----> xenobiotics. These are exogenous molecules/compounds foreign to the body), especially at the level of hepatocytes. The metabolization system of exogenous substances, located at the level of the smooth endoplasmic reticulum: the cytochrome p450 system also called "drug metabolism system". The cytochrome p450 system is a chain of electron transport; the passage of the exogenous substance along this chain of electron transport, can detoxify or activate this chain that transforms the substance, but also leads to the production of free radicals.

Why are free radicals produced? Because there is a chain of electron transport and in fact free radicals are molecular species that, based on their arrangement of electrons, are unstable and therefore "hunting for electrons". So whenever there is a chain of electron transport, there is also the possibility that free radicals are formed.

Free radicals are formed physiologically in our cells thanks to the activity of peroxisomes. At the level of peroxisomes take place many metabolisms including the β-oxidation of fatty acids, a reaction of oxidation-reduction, ie a reaction in which there is an exchange of electrons. Free radicals are also formed "voluntarily" during the inflammatory process.

 Role of physiologically produced free radicals

The cell normally produces, mainly through metabolisms, radical oxygen species. The free radicals of oxygen, when produced in "physiological" quantities, not only do not damage the cells, but can also play a function of second messengers (a molecule is formed as a result of a reaction and gives rise to a biological effect), then still play a role in the regulation of many pathways of signal transduction. Through the role played on signal transduction pathways, free radicals, always produced in physiological quantities, can also regulate the expression of certain genes. They can also give rise to reversible modifications of proteins (e.g. phosphorylation/dephosphorylation).

If the cell produces free radicals in physiological quantities and in a controlled environment, as in phage-lysosomes, it can use this weapon to damage the molecular components of an agent that it must eliminate. So certainly products in excess damage the cell and tissues, but there are situations in which the cell produces them in an almost physiological way.

Free radical scavenger molecules and enzymes

If the cells produce free radicals in quantities higher than physiological ones, it is necessary that the same cells have defense mechanisms: they must make sure not to suffer damage resulting from the increased production of free radicals. This means that both our cells and the organism in toto, possess molecules and enzymatic systems that have the function to block the action of free radicals: these systems are called free radical scavenger.

 Here's how these antioxidant molecules and systems work, starting with the molecules:

Cysteine: Cysteine is the simplest of these molecules, it has antioxidant properties because it contains the sulfhydryl group -SH. 

Glutathione: from a biochemical point of view, it is defined as a false tripeptide, because although it consists of three amino acids (cysteine, glycine and glutamic acid), there is a canonical peptide bond and an abnormal peptide bond. Glutathione owes its antioxidant properties, again to the SH group of cysteine it contains. From a quantitative point of view, glutathione is the most abundant antioxidant present in our cells and logically if it has an antioxidant function, it means that it can be present both in reduced form (GSH) and in oxidized form (GSSG). GSH means is present the group -SH of cysteine, able to give up an electron. GSSG means that glutathione is oxidized because the -SH group has been lost and a disulfide bond (SS) has been formed between two glutathione molecules that have given up two electrons to two free radicals stabilizing them. The transition from the reduced to the oxidized form represents the glutathione cycle. Crucial to the antioxidant activity of glutathione is the enzyme glutathione reductase, which uses NADPH acting as a coenzyme to convert GSSG back to GSH so that it can function as an antioxidant. Glutathione reductase has NADPH as its coenzyme. The cofactor generally is inorganic and does not participate in the reaction, the coenzyme does.

Vitamin C: used to maintain the cell's antioxidant defenses, it is a free radical scavenger. It also performs the important function of contributing to the regeneration of vitamin E. Vitamin C also cycles between an oxidized form (L-dehydroascorbic acid) and a reduced form (L-ascorbic acid). In addition, vitamin C is important for iron absorption and metabolism.

Vitamin E: also called α-tocopherol. The term α-tocopherol does not refer to a single substance, but to eight different isomers, which however perform the same function. Vitamin E is the most important fat-soluble antioxidant.

Free radical scavenger enzyme systems

Glutathione peroxidase: is an enzyme belonging to the class of oxidoreductases, which catalyzes the following reaction:

2 glutathione + H₂O₂ ⇄ glutathione disulfide + 2 H₂O

Superoxide dismutase SOD: catalyzes the reaction of transformation of the superoxide anion radical into hydrogen peroxide. There are 3 different isoforms of SOD, with different localization: mitochondrial, extracellular and cytoplasmic.

Catalase: catalyzes the degradation of hydrogen peroxide, which is formed as a result of the action of superoxide dismutase. Catalase is localized in peroxisomes.

Other molecules also play an antioxidant function or are involved in the regulation of redox balance in the cells of our body: some of these molecules are produced by our body, others we can take from outside, exogenous nature.

Transferrin and ferritin are involved in iron metabolism, ceruloplasmin is involved in copper metabolism. These molecules are responsible for the mobilization or otherwise accumulation of elements such as Fe and Cu, so these molecules also function as electron acceptors and donors.

Retinol or Vitamin A and the function of coenzyme Q10 located within the mitochondria, in the electron transport system are related to the antioxidant function related to the transport of electrons in the mitochondrial chain.

These substances are produced by our body, but there are other antioxidants that you can take through food (fruits and vegetables).

Among the molecules with antioxidant function: polyphenols, flavonoids, catechins, terpenes, lipoic acid, resveratrol (a phenol produced and contained in red grapes, is transferred in red wine); lycopene, a molecule that is abundant in tomatoes and recommended especially to males to avoid prostate problems.  These are all molecules found in natural foods that fight excess free radicals.

Damage caused by free radicals

Excessively produced free radicals damage the cell because they take the electrons they need to stabilize themselves, taking them away from the main cellular biomolecules: lipids, proteins and nucleic acids.

Damage to lipids by interaction with free radicals

The main target of the action of free radicals is represented by the so-called polyunsaturated fatty acids or PUFA (acronym of PolyUnsaturated Fatty Acids) that form the tails of membrane phospholipids. The radicals go to get the electron they need to stabilize, going to subtract it at the level of the double bond of polyunsaturated fatty acids. Lipid peroxidation is the phenomenon of damage to polyunsaturated fatty acids, due to interaction with free radicals.

The target are PUFAs. It is known that the fatty acids in the cell are essentially the components of phospholipids that make up the membranes: ultimately, the interaction of free radicals with the PUFA of the membranes, will damage all cell membranes.

Suppose PUFA, has three double bonds, R is the free radical, which is stabilized by subtracting an electron from the polyunsaturated fatty acid and becoming a RH. The consequence is that this PUFA in turn becomes a radical, so you will have a radical of a fatty acid. The first thing that a free radical of a fatty acid can do in a membrane is to stabilize itself by another fatty acid: a chain reaction occurs, in which one fatty acid stabilizes at the expense of another. In the presence of oxygen and then iron (both must be present, because the first phase is triggered by the presence of oxygen and then the subsequent phases by the presence of iron) the fatty acid radical can also meet another fate: a break down, that is a break down of oxidative type, a progressive degradation of oxidative type. The polyunsaturated fatty acid radical breaks down progressively, until it reaches the level of the products of lipid peroxidation, which are always carbonyl type molecules but much smaller. These products of lipid peroxidation and in particular the aldehydes, are in turn toxic.

So, starting from the PUFA, which becomes a lipid radical when an electron is subtracted, there are intermediate reactions or lipid peroxidation and finally the phenomenon of fragmentation, we arrive at carbonyl products: aldehyde and ethane. Ultimately: loss of structure, loss of functionality, even get degradation products that are toxic. This concerns the damage to lipids, due to free radicals.  Cause of this phenomenon: within what is the structure of a membrane, phospholipids play both a structural function, and precisely because of the presence of polyunsaturated fatty acids, also play a role as modulators of membrane fluidity. Starting from a plasma-membrane that has all the polyunsaturated fatty acids in the right place and has a certain fluidity, at the end you get to a condition where the polyunsaturated fatty acid is no longer there, so you lose the structural function, but you also lose the function that ensures membrane fluidity.

The lipid peroxidation triggered by free radicals at the expense of PUFA of membrane phospholipids, ultimately determines a structural alteration (there is no more fatty acid) and a consequent functional alteration. 

ROS or in general free radicals, interacting with membrane phospholipids, cause a structural and functional damage of the membrane itself, so the damage caused by free radicals to lipids is due to lipid peroxidation.

Damage to proteins by interaction with free radicals

Free radicals can also stabilize at the expense of proteins: the target of free radical interaction in a protein is the sulfhydryl group of the amino acid cysteine, which is present in proteins. The radicals will subtract there an electron to stabilize themselves. It is the same principle as the antioxidant function of cysteine and glutathione, but in this case it is in an offensive sense, while previously it was in a defensive sense.

Free radicals in proteins, are stabilized by subtracting an electron always at the level of -SH groups. In proteins, the presence of -SH groups is important, because usually amino acids that have a -SH group, are located in the functional center of the protein itself. So if you subtract an electron from the -SH group of a protein, the functionality is altered.

DNA damage due to interaction with free radicals

Free radicals damage DNA, leading to strand breaks, cross-links, and base mutations. When these damages occur, we speak of an oxidative type of damage and there is a marker of oxidative damage of free radicals to DNA: 8-hydroxy-deoxyguanosine (8-OHDG). If you expose cells to a source of free radicals, you can test for DNA damage by assaying this oxidative damage marker.

The free radicals produced in excess create a damage to proteins, lipids and DNA; regarding the cellular damage, the most immediate repercussions are those due to the membrane phospholipids, and the damage to proteins. In this context, the cell dies first because it has an altered membrane, not because it has altered DNA. The DNA damage will become evident when the altered gene has to be transcribed and translated, if the damage was to a portion of DNA that is not transcribed and translated, that damage would not occur.

 Morphological alterations of the cell in case of damage

Biochemical changes accompany the cellular damage, now let's see what are the morphological alterations. It is necessary to distinguish between morphological alterations typical of reversible damage and those typical of irreversible damage (damage in which the point of no return is exceeded).

Morphological alterations in reversible damage

• Cellular swelling: The first morphological alteration visible in a cell that suffers reversible damage is cellular swelling as there is an alteration in electrolyte balance, due to a decrease in ATP, resulting in an alteration in pump function (e.g. Sodium/Potassium). They increase the electrolytes, the solutes, within the cell. If the solutes inside the cell increase, it is as if this cell is more concentrated and tries to overcome this situation by introducing water from outside, so by introducing water from outside, the cell swells.  This accumulation of water inside the cell will also cause a swelling of organelles surrounded by membrane: we will have inside the cell, swollen mitochondria (but without other morphological alterations), mitochondrial ridges will be intact as reversible damage. In reversible damage condition, mitochondria are more swollen, but they do not undergo an alteration at the level of the ridges, because if they undergo an alteration at the level of the ridges, the damage would become irreversible. We will also have an endoplasmic reticulum both smooth and wrinkled, swollen. Let's analyze the wrinkled one: if a wrinkled, bulging endoplasmic reticulum is present, ribosomes can be detached. In the wrinkled endoplasmic reticulum if there is the detachment of some ribosome, the consequence will be a reduction of protein synthesis, without rupture.
• Chromatin thickening. Another morphological change already present in reversible damage is chromatin thickening. Chromatin will not be distributed more or less uniformly within the whole nucleus but will be condensed, darker from a histological point of view because concentrated in some areas.
•  Accumulations of Lipids: depending on the cell type, accumulations of lipids in the cytoplasm.
• Vacuoles: within the cytoplasm there are generally vacuoles.
• Blebs: In some cases the formation of blebs on the plasma membrane can be observed. Blebs are extroversions of the cell membrane. They occur when there is a detachment of the plasma membrane from the underlying cytoskeleton.  In the formation of blebs in reversible damage, the cell membrane is still intact.