Death by apoptosis is an event that characterizes all phylogenetic levels (not only higher organisms), in fact the description of these mechanisms has been made through studies on the nematode (also called Caenorhabditis elegans, worm).  

It is also called "programmed cell death" due to the fact that it is part of the developmental program of all organisms, and is a programmed event. It is therefore a physiological event as part of the program of development and maintenance of homeostasis. Cells actively participate in the program of death (it is also called "suicide").  In necrosis are all situations that the cell undergoes, while in apoptosis the cell participates: it expresses particular genes and produces proteins in order to die. Death by apoptosis is an event that characterizes all phylogenetic levels (not only higher organisms), in fact the description of these mechanisms has been made through studies on nematode (Caenorhabditis elegans).

Starting observation for apoptosis studies: during the development of all C. elegans always 131 cells die by apoptosis and it is therefore a programmed event during its development.  

Apoptosis works to eliminate those cells that are no longer needed within the homeostasis of the organism itself. Cells in tissues that are defined as labile, also known as tissues with high cell turnover, die by apoptosis. It serves to maintain tissue homeostasis and also to eliminate cells that have suffered irreversible damage, such as DNA damage that cannot be repaired or when they have been infected. In order to keep the genetic heritage intact, it is better to eliminate a cell with altered DNA than to keep it with the risk that it may give rise to a clone of altered cells. 

Death by apoptosis does not produce an inflammatory process and can be triggered by extracellular (extrinsic pathway, starts from something exogenous) and intracellular (intrinsic pathway) signals.

 Definitions of apoptosis:

- without repercussion → does not produce an inflammatory process because it does not release any cellular components; in fact, apoptotic bodies are formed, which are then phagocytosed;

- selective → even only two or three cells die by apoptosis, only the cells that need to be eliminated from the tissue;

- regulated → it has a gene regulation, so there are genes that induce or inhibit it.  

Physiological conditions in which apoptosis plays an important role

Apoptosis is part of the embryonic developmental program, even in humans. Here, apoptosis events also occur quite early.

• In humans, cells die by apoptosis before and during implantation of the embryo because they have accomplished their task.  
• During the developmental stage we go through a phase in which we have interdigital membranes that in the next phase are eliminated by apoptosis.
• Apoptosis also occurs during the formation of hollow organs (including the heart), because it is easier to form a full ball and then make it hollow by apoptosis.
• Apoptosis also occurs to CNS cells at the end of its development. In fact, during CNS development more cells are formed than will be needed so that there is a redundancy of cells in case something happens in development and cells are lost (if nothing happens and I don't use the reserve I send them to death by apoptosis at the end of development).

Maintenance of homeostasis of rapidly turnover tissues, e.g., epithelium.

Elimination of cells by cessation of a stimulus:

• involution when a hormone-type stimulus ceases (example of breast and uterus at end of pregnancy and lactation; previously they are hypertrophic and hyperplastic);
• during the inflammatory process the number of granulocytes increases and, when this ends, they are eliminated by apoptosis;
• the same is true for the immune response with involvement of lymphocytes that undergo cloning.

Phases of apoptosis

The event of apoptosis is marked by the succession of phases:   

• Activation of the death program itself 
• Activation of pro-apoptotic proteins.
• During death by apoptosis, cellular components are degraded in a highly regulated manner by the activation of special proteins that are termed pro-apoptotic.  
• Activation of apoptosis executing proteins. 
• They are referred to as caspases and each caspase results in degradation of very specific cellular substrates (each element is degraded by a specific protein). Caspases are pepsidases and degrade specific target proteins.

 Morphological changes

Let us examine what happens at the level of cytoplasmic membranes, nucleus, and mitochondria.  

At the membrane level, the "earliest" events of apoptosis occur. 

• Cells initially isolate themselves from the tissue of which they are a part, they enclose themselves.
• All cell organelles condense, while remaining intact.
• The cell as a whole undergoes a decrease in volume.
• It then emits membrane eversions called blebs.

Thus, it first compacts and then emits membrane eversions. 

In the nucleus, changes are quite early and consist of an initial compaction; this compaction also corresponds to a condensation of chromatin. 

Hematoxylin-eosin staining of the nucleus is diffuse bluish, except in the nucleolus. In apoptosis, chromatin condenses and is arranged in segments below the membrane, so it is no longer uniform. When chromatin is condensed, fragmentation of the nucleus also occurs.

At the mitochondrial level during death by apoptosis there is a change in membrane potential. So their normal permeability or non-permeability also changes. 

This means that they release molecules that are pro-apoptotic, such as cytochrome C.

The cell loses its contact elements, becomes compact, and after the formation of the half-moons in the nucleus it undergoes fragmentation (a number n of fragments are formed); however, all the pieces are always surrounded by the nuclear membrane.  The nuclear fragments will become part of cellular fragments, in the cell will form a number n of fragments with or without fragments of the nucleus inside. These cell fragments are what are called apoptotic bodies (with or without nucleus they are always surrounded by intact plasmamembrane, with no loss of cellular components).  The fate of apoptotic bodies is to be phagocytosed by professional phagocytes (neutrophil granulocytes and macrophages) or by normal cells not affected by the process of apoptosis that are close to dead cells.  

Biochemical changes

A cell that dies by apoptosis synthesizes new proteins in order to go to death. This is a difference from death by necrosis, such as a finger on a hot oven plate. In this case, the cell does not have time to synthesize new proteins. In apoptosis, the cell contributes to its own death program by synthesizing new proteins.

Studies

In the development of a new drug active in cancer cells and able to induce the death of cancer cells. apoptosis avoids the induction of the inflammatory process, hoping to kill them selectively. The different types of fragments that are formed as a result of apoptosis or necrosis can be useful in research for the study of anticancer molecules and to understand if they determine the death of cancer cells by apoptosis (what is expected) or by necrosis (to be avoided because it develops an inflammatory response).

In vitro studies on cultured cells to test the new substance clarify what kind of death occurs. DNA is extracted and electrophoresed by depositing it in wells and analyzing how the DNA fragments arrange and run on the agarose gel. It is important to understand how they are arranged. You deposit the fragments in the wells and then turn on the electric field. Migration depends on the size and charges of the components themselves.

Cellular death by necrosis

Necrosis is a type of cell death, but in fact the term necrosis from a general pathology point of view indicates not only cell death but all the morphological changes that dead cells of a tissue manifest, so yes, it is a type of cell death, which includes all morphological changes that dead cells manifest.

The common characteristics that all dead cells by necrosis exhibit are:

• the plasma membrane discontinuous and cytoplasmic components degraded by enzymes in lysosomes.  
• the cytoplasm corroded because some of the components have been degraded; it will exhibit vacuoles and a decrease in the number of organelles.

With histological staining, you notice the cytoplasm is much more eosinophilic, so it stains much more pink. If you do a histological staining with hemallumen-eosin (or hematoxin-eosin), which is the simplest histological staining, the hemallumen colors the nucleus, while the other dye colors the cytoplasm, and on the slide you will see blue-purple nuclei and pink cytoplasm, in the case of an intact cell. In the cytoplasm of a cell that has gone into necrosis there is a much more pronounced pink staining because this dye, emallume-eosin, binds to degraded proteins and degraded RNA. This shows that there are already degraded proteins present in that cytoplasm.  

Alterations at the level of the nucleus: when a cell dies by necrosis, it undergoes changes that affect mainly the non-nuclear component. In death by necrosis, the nucleus is the part of the cell that undergoes changes later, unlike death by apoptosis. The nucleus of a cell that dies by necrosis undergoes a late random fragmentation of the DNA. Random because nuclease are activated that cut the DNA where it happens, so it will form smaller fragments, larger fragments, of random size. This fragmentation is called nonspecific fragmentation.

In cell death by apoptosis, on the other hand, we have specific fragmentation with the activation of endonucleases that cut the DNA at specific points.

Morphological appearance of the nucleus of a cell dead from necrosis:

• cariolysis: the components of the nucleus are immediately degraded without condensation; nothing remains of the nucleus, only small fragments, all the rest has already been lysed. Depending on the type of damaging agent, different enzyme systems are activated and lysed;
• pycnosis: there is a phenomenon of condensation of the nucleus, which becomes less basophilic and pycnotic (small condensed nucleus), then it shrinks; afterwards, the pycnotic nucleus goes against karyorrhexis, that is it fragments and is digested in its components
• karyorrhexis: a cell dead from necrosis with a pycnotic nucleus, at a somewhat later stage (24-48h), fragments randomly. It is thus the phase following pycnosis.

Necrosis types

• Coagulative. A blood clot is something "stuck"; it is called coagulative necrosis because the cell after two/three days at most, has an appearance of an empty shell. You can still see the outline of the cells, but inside, the cellular components are absent. It is like a blockage: in these situations the damaging agent has also determined the denaturation of enzymatic proteins and blocks autolysis. The cell will be eliminated as part of the inflammatory process. When all tissues (except central nervous tissue or brain tissue within which cells die as a result of ischemia/hypoxia) die as a result of ischemia/hypoxia (=no more blood coming in, =infarction), they take on the appearance of coagulative necrosis. In a slide obtained from a myocardium in which there has been an event of infarction (logically within the first few days) these cardiomyocytes appear as empty shells, the same for a preparation of a columnar infarction, or a preparation of an infarction of the intestine. The speech just made does not apply to a preparation of a cerebral infarction because here the lytic enzymes are present in very high concentration, so there is autolysis even without enzymatic proteins. In coagulative necrosis the basic architecture of the tissue is retained, but not the cellular components, so I see empty shells.
• Colliquative: is characterized by the liquefied appearance that dead cells assume when all cellular components are degraded, lysed immediately. The colliquated appearance occurs when the injurious agent is a bacterial type agent. Bacterial infections attract many lymphocytes that release lytic enzymes. If the leukocytes release so many lytic enzymes, all the components are completely degraded, they are colliquated. Thus, colliquative necrosis is associated with bacterial infections and the exception of death by cerebral infarction.
• Caseous: name indicative of macroscopic appearance; in caseous necrosis the dead cells take on a soft, yellowish-white appearance. It is defined as a subtype of coagulative necrosis and occurs in the type of death we find in tuberculosis granuloma (the bacterium has tropism for the lung, which is the first site of colonization). Tuberculosis, in addition to being an infectious type of pathology, is a chronic type of pathology with granulomatous form, that is, in the course of tuberculosis, these new formations are formed that are called granulomas at the level of the lung. If the bacterium disseminates, they also form in other districts. In this form of chronic inflammatory process, in the center of the tuberculous granuloma, there is an area of caseous necrosis. In tuberculosis, which is localized at the level of the lung, the organism can eliminate the injurious agent, but the system against tuberculosis is not effective. Therefore, the tuberculosis microbacterium cannot be eliminated and attempts are made to circumscribe it. In the central necrosis zone, the cells are dead from the presence of the agent; around it, when the process becomes chronic, cells and collagen fibers are organized to prevent distribution to other areas.
• Gum-like: it occurs at the center of another form of granuloma: that of syphilis. Syphilis is a chronic inflammatory disease in which granulomas form in which the dying cells take on a gum-like appearance. In fact, syphilis used to be called gum disease because in the tertiary phase, subjects develop granulomas in different areas of the body that, when cut, have a white appearance and a gummy consistency. In appearance, gummy and caseous necrosis appear similar. The difference can be felt to the touch: caseous is soft; gummy is like gum or thick glue.
• Hemorrhagic: erythrocytes are also present in the area of dead cells. The cause of necrosis is a stagnation of erythrocytes, so a blockage of flow, a congestion of blood.
• Fibrinoid: occurs in a particular situation, in malignant hypertension. In this case there is an elevation of blood pressure and there is a particularly significant damage that sets in quickly.  This type of necrosis occurs in the vessel walls and is called fibrinoid because, in addition to dead cells, substances that are colored like fibrin accumulate in the vessel wall. The vessel loses its selective permeability and protein components of the blood leak out. If fibrin also comes out, its precursor which is fibrinogen also comes out.
• Steatonecrosis: when cell death is associated with marked lipid degradation leading the cell to take on a chalk-like appearance because saponification of fatty acids liberated by phospholipases occurs. Example: adipocytes due to pancreatic cell death (acute pancreatitis) releasing lipase.
• Gangrene: does not refer to a type of cell death but to a particular situation of death that affects all cells in a tissue, organ or portion of the body. This is a significant cell death phenomenon that can even affect a portion of a limb or an entire limb. When the gangrene episode occurs, we have the superimposition of coagulative necrosis and colliquative necrosis (at first it may have been a phenomenon of ischemia and hypoxia, then coagulative necrosis, followed by colliquative necrosis due to the superimposition of an infection). Usually, gangrene events to portions of the limbs, especially the lower limbs, are the consequence of a perfusion blocking event due to infarct-type events or, in the diabetic, due to a decrease in perfusion in general, to which an infectious-type event is superimposed.

           There are three variants of gangrene (macroscopic reference): 

            - Dry 

            - Wet: the necrotic area has a liquid component; it is the variant observed in diabetic subjects; 

            - Gaseous: superimposition of a Clostridium infection that causes the emission of gaseous type                material; it is accompanied by putrefaction of the tissue.

Markers of cellular damage/necrosis

Necrosis involves disruption of the plasmamembrane and dispersal of cellular components outside the cell, and this event always triggers an inflammatory process. From this unavoidable dispersion, it is possible to diagnose a cell death event in a given tissue.

The knowledge of the molecules that are released from the different tissues are used to analyze the levels of these in the blood and, if these appear in increased amounts, one can hypothesize a massive necrosis event in a given body district. In order to use this as painlessly as possible, a method should be used that allows for a non-invasive blood draw so that the right information can be obtained (not a biopsy of the infarcted tissue because that would be too invasive).

Overview considerations of markers of irreversible damage and cellular necrosis: 

• The markers of cell death are different depending on the type of cell that has died. In a trauma, even a mechanical one, to muscle tissue, the cells of striated tissue die and in the blood there will be proteins typical of that specific tissue (actin and myosin, for example).
• They have different timelines for increasing in the circulation. If there are different types of markers for an injury, the one with the fastest onset should be evaluated to speed up the process.
•  They must be used in association with the evaluation of the history, that is, used to confirm or not a diagnosis made with other tests (for example, if there is a heart attack, in addition to the analysis of markers, also the electrocardiogram).
•  Most markers are proteins with enzyme activity. If there is a suspicion of infarction, an initial sampling on arrival at the hospital is necessary to analyze basal enzyme levels; after a specific time, a second sampling is done. The period between the first and second sampling corresponds to the minimum time for marker elevation. We then exploit the dispersion of cellular components resulting from necrosis to arrive at a diagnosis.

Lactic dehydrogenase (LDH) is an enzyme that catalyzes the transformation of lactate to pyruvate. It is an enzyme expressed in different cell types, but it is expressed as different isoenzymes.

There are 5 different cytoplasmic isoenzymes expressed in different tissues:

- LDH1 mainly in the myocardium;

- LDH2 and LDH3 mainly in the lung;

- LDH4 not majorly produced by a specific tissue;

- LDH5 expressed primarily in the liver.

For a myocardial infarction, the LDH1 assay can be used as a marker, which increases. If, on the other hand, the suspicion is for liver necrosis, LDH5 can be used. Therefore, by measuring the amount of blood of a particular isoenzyme, it is possible to assess whether there has actually been a death by necrosis in that district where it is most expressed.

The increase in LDH, to be significant, requires 10-12h. So, you take a sample at the time of admission and one after 10-12h, and if there is an increase in LDH there is an indication in favor of necrosis.

However, the increase of these enzymes occurs also in other pathological conditions, such as megaloblastic anemia, muscle injury and renal infarction. In fact, the result must be added to the history and other evaluations.

Damage caused by free radicals

We will examine the mechanisms by which an excessive amount of free radicals can damage cells. Excessively produced free radicals damage cells because they take the electrons they need to stabilize themselves and remove them 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 (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.

Let's look in detail at what it consists of: the target is PUFAs, but fatty acids in the cell are essentially the components of the phospholipids that make up the membranes; this means that ultimately, the interaction of free radicals with PUFAs in the membranes will damage all cell membranes.

Let's start with the PUFA, which in this example has three double bonds. R, the free radical, is stabilized by subtracting an electron from the polyunsaturated fatty acid and becoming an RH. The consequence is that this PUFA in turn becomes a radical, thus a fatty acid radical. The fatty acid radical within a lipid bilayer of a membrane: what is its fate? The first thing that a free radical of a fatty acid can do in a membrane is to be stabilized 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, carbonyl-type molecules but much smaller. These products of lipid peroxidation and in particular the aldehydes, are in turn toxic. 

What is the consequence of this process? In the context of what is the structure of a membrane, phospholipids play both a structural function, and precisely because of the presence of polyunsaturated fatty acids, they 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 that has a certain fluidity, at the end you get to a condition where the polyunsaturated fatty acid is no longer there, so it loses the structural function, but it also loses 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. 

The ROS or in general the free radicals, interacting with the membrane phospholipids, determine a structural and functional damage of the membrane itself, so the damage caused by free radicals on 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. 

Free radicals in proteins stabilize 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 an -SH group, are located in the functional center of the protein itself. So if an electron is subtracted from the -SH group of a protein, the functionality is also altered.

If the radical subtracts an electron to a -SH group of a protein and to a -SH group of another protein, disulfide bridges are formed, the functionality is lost and the structure is also lost. This is logically true for both enzymatic and structural proteins.

DNA damage due to interaction with free radicals

Free radical damage to DNA involves tumors, neoplasms. Certainly free radicals damage the DNA, causing the breakage of filaments, cross-links and mutations of bases. 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 check for DNA damage by measuring this marker of oxidative damage.

The free radicals produced in excess create damage to proteins, lipids and DNA; in the context of the discourse of cellular damage, the effects more immediate and more penalizing for the cell, will derive from damage to lipids, proteins or DNA. Mainly to lipids and proteins, but an excess production of free radicals, can also damage DNA. The most immediate repercussions, are those due to membrane phospholipids, and damage to proteins. 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 manifest itself.

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.

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.

METAPLASIA 

Metaplasia is a cellular adaptation that involves the replacement of one differentiated cell type with another that has the same embryonic origin. This replacement occurs because the goal is to change the histotype to better overcome and resist an environmental stress. 

The pulmonary epithelium is a cylindrical ciliated epithelium and if for a long time it is subjected to a stress (for example a chemical stress represented by cigarette smoke) it will undergo a metaplasia: the normal cylindrical ciliated epithelium will be replaced with one more resistant to stress (for example a keratinized epithelium = squamous epithelium). Thus, a more resistant epithelium is obtained but at the same time a loss of function because the ciliated epithelium, in that district, has the function of removing impurities through the cilia.

This adaptation can involve epithelial cells, therefore epithelial metaplasia or mesenchymal cells, mesenchymal metaplasia. In order for this replacement to occur there must be changes in the expression of genes typical of that differentiation. At the base there will be a phenotypic change that has a change at the gene level. 

Metaplasia, as an adaptation, represents a condition that predisposes to the onset of malignancy. Very often on what starts as metaplasia is superimposed a neoplasm. 

Examples of epithelial metaplasia:

• transition from columnar to squamous epithelium in respiratory epithelium that is exposed to chronic irritation
• transition from columnar to squamous epithelium in epithelia of the secretory ducts of the glands as a result of calculosis phenomena. In this case the stressful event is mechanical, not chemical
• "Reverse" metaplasia: a squamous epithelium is replaced by columnar epithelium. This condition occurs in Barret's esophagus as a result of gastric reflux. 
• from transitional epithelium (which we find at the level of the bladder) to squamous epithelium: this transition is caused by the presence of stones at the bladder level and will cause a loss of function as the transitional epithelium is able to modulate its thickness while the squamous one does not.

Mesenchymal metaplasia: there is the formation of a tissue in a district where it is not normally present. We are therefore talking about cartilage, bone and adipose tissue. If a tissue is formed in a site other than normal we will speak of tissue formation in ectopic site: 

• myositis ossificans following intramuscular hemorrhage: formation of bone tissue at the level of a muscle in which a hemorrhage has formed 
• deposition of bone tissue within a scar tissue (scar) in which there is normally connective tissue 
• deposition of bone tissue at the level of atherosclerotic plaques. This deposition will lead to serious consequences in the atherosclerotic phenomenon. 

ORAL LEUKOPLAKIA. In this metaplasia, white plaques form at the level of the oral cavity. (Many oral pathologies will result in white plaques).

Adaptations can be reversible but, in the case of metaplasia, very often this results in a precancerous situation. So potentially, even metaplasia, is reversible unless the transition to a neoplastic condition has already occurred.

DIFFERENCE BETWEEN NEOPLASIA AND METAPLASIA

In neoplasms, cells have alterations at the gene level. These have a different genotype from normal cells that also causes a phenotypic change. 

In metaplasia by replacing the cells there is no mutation at the gene level, only the epithelium varies but remains "normal". Therefore the type of differentiation changes: epithelia tend to renew themselves and if there is an abnormal stimulus, when the cells are replaced, a different epithelium is formed, always consisting of "normal" cells, there are no abnormalities.

CELL DAMAGE 

The capacity of a cell is not unlimited and if it is not able to adapt it will undergo cellular damage. In cellular damage all alterations that affect tissues, organs and our organism in general can be involved. If we adapt the concept of homeostatic condition to each cell we can say that this condition depends on the genetic program of the cell itself, that is it will be conditioned by the function that it performs. A cell of skeletal muscle tissue will need optimal conditions to function that will be different than those of a hepatocyte. Therefore the homeostatic condition of a cell depends on its type of differentiation. It is known that, especially for normal cells, the homeostatic condition of a cell depends on contacts with neighboring cells: normal cells that are part of a tissue do not live alone but in a "condominium" of cells and therefore the homeostatic condition of each depends on contacts with neighboring cells. A cell will be able to live a homeostatic condition even if it has an adequate presence of metabolic substrates and therefore receives a correct amount of nutrients and oxygen. If the cell is in a particularly stressful condition or different from normal it responds adapting itself but if it can't adapt completely it will face a cellular damage that can be of two types:

• REVERSIBLE: if the cell can return to homeostatic conditions 
• IRREVERSIBLE: the cell can no longer return to its original condition and is destined to die

If the exposure to a damaging agent persists over time it causes irreversible damage and the cell can go from a situation of reversible damage to one of irreversible damage that culminates in death. When the cell passes from reversible to irreversible damage, it exceeds the point of no return. This is not a philosophical concept but, in the cell, corresponds to certain changes of biochemical, functional and morphological nature. Therefore, the crossing of the point of no return implies further changes than those that occurred in the reversible damage. If you go along this road from left to right,you will observe an increasing impairment at the level of the cell's functionality, structure and shape.

The effects, which the exposure to the harmful stimulus determines, are correlated with: 

• duration of the harmful stimulus
• intensity of the damaging stimulus
• cell type: a cell in a condition of hypoxia (lack of oxygen) can be damaged, but the severity of this damage will also be related to the cell type. In fact, there are cell types that are much more sensitive to oxygen deficiency (e.g. cardiomyocytes or SN cells). At the same time, this depends on the ability of the cell to adapt, in particular, to chemical injury agents. The effect depends on the ability of the cell to decode chemicals (e.g. cytochromeP 450)
• type of damage: if the damaging agent alters the mitochondrial respiration chain or selectively inhibits a certain enzyme that the cell uses once in a while, different situations occur. The first damaging agent will have a greater effect because it will compromise one of the main functions performed by the cell (ATP production) while the second will act on a "secondary" situation. 

Therefore the exposure to a lesive agent determines a damage that is a function of these aspects.

Atrophy/Hypotrophy

Cells respond when asked to function more, however, they can also find themselves in a situation where they are "forced" to function less. In this case, the cells try to adapt and the condition is called: Atrophy/Hypotrophy.

"Atrophic" means without nourishment, but it should be called hypotrophy since the former term would indicate that the cell is no longer there.

What are the characteristics of atrophy? 

Atrophy is a regressive process in which there is a reduction in the volume of a tissue or organ that has already reached its normal volume. So an organ develops, reaches its normal volume and then regresses by decreasing its volume and therefore also its functionality.

The fact that this organ has already reached its normal volume and then regresses means that atrophy can be described as an acquired defect, that is, one that arises at a later time. 

The decrease in volume of an organ/tissue can occur for two reasons:

• decreases the volume of individual cells
• loss of cells through apoptosis

In atrophy, normally, it is the parenchymal cells that decrease in volume. Instead, in this situation, the stromal component of the cells (the external one) persists more.

The ultimate goal of atrophy is to ensure the survival of the cell even if in conditions of minimal functional activity. A cell to become atrophic on the one hand increases protein catabolism and on the other decreases synthesis. 

Physiologic atrophy: 

• Uterus and mammary gland at termination of pregnancy
• Organs during old age: uterus, ovaries, testes, and even the brain
• Structures during fetal development when they are no longer needed
• Thymus: organ in which T lymphocytes mature, it becomes atrophic at the end of adolescence in all subjects

Pathologic atrophy:

Atrophy is pathological if functional demand decreases and therefore the organ/tissue must function less:

• Skeletal muscle tissue when immobilized (e.g., during a period of cast). In this case, atrophy also depends on the amount of time this remains stationary. 
• Skeletal muscle tissue when there is a lack of innervation which may be due to either gene defects or traumatic events.

Reduced blood perfusion can cause atrophy in any tissue: there has been a decrease in blood flow in a particular district. This can be the result of atherosclerosis phenomena, a pathology that develops at the level of the vessel walls in which the lumen is reduced. As the lumen is reduced, all tissues downstream will receive less blood. An organ can also receive less blood if a neoplasm (tumor mass) compresses the part of the circulatory system that supplies it. 

Tissues become atrophic if there is inadequate nutrition. This may be associated with different geographic contexts (e.g., fasting). Nutrition may be inadequate in both protein and calorie intake. In a diet low in protein and calories, protein synthesis at the tissue level is deficient and tissue will tend to become atrophic. A form of malnutrition involves only protein intake and, again, tissues will become atrophic and the tissue most targeted will be muscle tissue.

The last condition is cachexia. In this situation the subject is subject to a significant loss of weight as a result of a loss of lean mass (muscle) which may or may not be associated with a loss of fat mass. This situation is associated with the most advanced stages of the path of carcinogenesis, in a subject carrying neoplasms, or even in subjects with AIDS in the terminal stages. In these cases the subject is emaciated, no longer has muscle mass and has little fat mass. Therefore cachexia is a condition of pathological atrophy associated with neoplasms, AIDS or chronic inflammatory diseases that can lead to organ failure.