In chemistry, a radical (or free radical) is defined as a highly reactive molecular entity with a very short half-life, consisting of an atom or a molecule formed by several atoms, which has an unpaired electron: this electron makes the radical extremely reactive, able to bind to other radicals or to subtract an electron from other nearby molecules.
The term radical and free radical are often used with the same meaning. The first stable free radical, triphenylmethyl, was identified by Moses Gomberg in 1900 at the University of Michigan. In spite of their reactivity, most of them have sufficiently long lifetimes that they can be observed by spectroscopic methods. They are formed spontaneously in nature or in the laboratory, by the action of light or heat following the homolytic cleavage of a covalent bond.
Radicals play an important role in phenomena such as combustion, polymerization and in photochemistry, and many other chemical processes, including those involving human physiology. In the latter case, superoxide and nitric oxide have a very important function in regulating many biological processes, such as the control of vascular tone.
Free radical is any chemical species, atom or molecule, of organic or inorganic nature, which, having unpaired electrons in its orbitals, tends to couple them, in reactions with other species, giving up or acquiring them to eliminate the situation of decoupling. For this reason it is generally said that free radicals are extremely reactive chemical species. This is always true from the thermodynamic point of view, which expresses the tendency of the species to gain or lose electrons. From the kinetic point of view, which expresses the rate at which this tendency occurs, this may not be true, so that there are extremely stable free radicals, due to their molecular structure. One of these stable radicals is oxygen, which reacts only under conditions of proper catalysis.
Oxygen constitutes about 20% of the Earth’s atmosphere and is used by aerobic life forms (i.e. that need this element to live) to produce energy at the level of specialized membrane structures that are located, in eukaryotic organisms, in the mitochondria. In these structures, oxygen is activated by specific enzymes, which make it rapidly accept the electrons needed to be transformed into its most stable reducing molecule, water. In this process, oxygen passes through intermediate reduction steps which, under physiological conditions, are only minimally released outside the respiratory chain. These intermediate states are called ‘reactive forms of oxygen’ because of their mostly radical nature and, if their quantity increases under pathological conditions, they can be very harmful for molecular structures essential to the life of the cell (oxidative damage).
In recent years, the meaning of free radical has expanded to become one of the key concepts for understanding all the functions of organisms that use molecular oxygen for energy production. While oxidative free radical damage involves irreversible chemical modifications of the molecular target affected and indiscriminate with respect to the type of molecule and chemical group involved, other effects of a physiological nature have been discovered that are expressed through reversible and selective chemical alterations and, therefore, potentially subject to metabolic regulation.
Initially the free radicals of biological importance were considered only the intermediate products of oxygen reduction in the various districts of the cell (superoxide, hydrogen peroxide and hydroxyl radical) and the peroxides resulting from the addition of oxygen to the radicals of the fatty acids of the membranes. These species are currently indicated by the acronym ROS (Reactive oxygen species). A new family of radical species has been added to them, characterized by the presence, in addition to oxygen, of a nitrogen atom, and therefore indicated as RNS (Reactive nitrogen species). The progenitor of these radicals is nitric oxide or nitric oxide, NO, which in biological systems is generated by oxidation of one of the azides of the guanidine group of the amino acid arginine, catalyzed by the enzyme nitric oxide synthetase or NO-synthase (NOS). ROS and RNS interact with each other in various ways: for example, NOS is activated by physiological doses of hydrogen peroxide, and the direct interaction of superoxide and NO is at the origin of an oxidative damage-generating species, peroxynitrite.
More recent studies, however, have shown that most of the effects of RNS are at the level of physiological regulation of body functions. In 1998, only eleven years after the discovery of the cellular production of nitric oxide, until then considered a gas of inorganic chemistry, the Nobel Prize for medicine or physiology was awarded to U.S. scientists Robert F. Furchgott, Louis J. Ignarro and Ferid Murad for their research on the role of nitric oxide as a mediator of physiological signals within the cardiovascular system, in particular those that regulate vasodilation due to smooth muscle relaxation. These properties of nitric oxide are at the basis of the vasodilating activity of nitroglycerin, discovered by the Italian Ascanio Sobrero in 1847, together with its explosive action that will be exploited a few years later by Alfred Nobel for the preparation of dynamite. The most recent consequences of the studies on the biological properties of this free radical have been the discovery of the molecular mechanisms of penile erection and the subsequent diffusion of today’s popular drugs for the treatment of male impotence.
Another species of which no biological effects were known so far is the carbonate radical, CO32-. It has been seen recently that it is very important in the mechanism of the additional activity of the enzyme copper-zinc superoxide dismutase (SOD 1), which occurs in some pathological situations. This enzyme is the cell’s main defense against ROS, because it eliminates the primary oxygen radical, O2– or superoxide anion, by a dismutation reaction that transforms it into hydrogen peroxide, H2O2. This reaction product, if not decomposed by the other enzymes catalase or glutathione peroxidase, generates hydroxyl radicals at the active site of the enzyme, inactivating it.
In the presence of bicarbonate, carbonate radicals are formed, and this reaction spares the active center of the enzyme, but produces oxidative damage on other biological targets. These reactions configure a pro-oxidant activity of an antioxidant enzyme and are relevant, for example, when there are genetic variants of SOD 1 that, for the same superoxide-dismutase activity, have a conformation of the active site that favors the reaction with hydrogen peroxide. The case of nitric oxide with its duality of effects, oxidative damage and physiological regulation, has stimulated new studies, which have shown that ROS can also intervene as physiological mediators of the initiation of many functional processes.
Role of free radicals in biological signal transduction
Membrane receptors transfer inside the cell the chemical signal that comes from outside carried by hormones, growth factors and other bioactive molecules, such as drugs and food components, which are not able to cross the cell membrane (signal transduction). A typical case concerns the hormone essential to cellular glucose metabolism, insulin. The binding of insulin to its receptor produces the activation of a membrane enzyme (NADPH oxidase), similar to that which is activated on the membrane of phagocytes in the inflammatory response, capable of producing hydrogen peroxide from the oxidation of a reduced coenzyme (NADPH), which is generated exclusively by direct oxidation of glucose and therefore acts as a detector of its concentration in tissues. It is this ROS flux that reversibly oxidizes specific protein cysteines embedded in the intracellular part of the receptor.
In such a mildly oxidized state, the receptor increases its affinity for phosphoric groups and, in its phosphorylated form, can activate enzymes in the cell that are crucial for glucose metabolism and that it would not have ‘encountered’ and ‘recognized’ in its phosphorus-free form. This ROS-phosphorylation tandem, mediated by mild oxidation of cysteines of specialized proteins, operates in many other cases of receptor activation and is a special case of the more general phenomenon of regulation of kinases by ROS. Kinases are precisely the enzymes that catalyze the transport of phosphoric groups from the universal donor adenosine triphosphate (ATP) to certain proteins, whose activity is thus increased or reduced.
Many kinases are involved in cell cycle regulation, for example those regulated by mitogens or MAPK (Mytogen activated protein kinase). We now know that very narrow intervals in the flux of ROS and RNS can direct the cell cycle toward its activation or arrest, through oxidative modification of cysteines of proteins that intercalate between the primary source of radicals and MAPKs. These reactions thus play a very important role in determining the acceleration of cell reproduction (proliferation), as in the development of organisms and in carcinogenesis, or conversely its slowdown, as in aging and programmed cell death or apoptosis. Other kinases are also subject to oxidoreductive regulation, which can occur not only by oxidation of their specific cysteines by ROS, but also, conversely, by the binding of antiradical molecules, such as vitamins A and E, which prevent their oxidation.
If the oxidative stimulus from the environment were limited to the formation of ROS and RNS, it would be ineffective as a regulator of physiological processes of organisms, because these species have a very short life in the cell, due to their instability and their indiscriminate reactivity with surrounding molecules. Instead, the oxidative potential of ROS, when produced in physiological concentrations, is fixed on molecules that in the oxidized state are relatively stable and restore their reduced state only by reactions endowed with high specificity. For these processes, hydrogen peroxide and nitric oxide are more suitable than their hydroxyl radical and peroxynitrite derivatives, which are responsible for more powerful, indiscriminate, and irreversible oxidizing reactions. The chemical system that molecular evolution has selected as the ideal target of these mild oxidations consists of compounds containing, in their reduced state, the sulfhydryl or thiol group (-SH) of the amino acid cysteine. By the action of hydrogen peroxide it becomes sulphenic group (-SOH) or disulfide group (-S-S-), while by the action of nitric oxide it is transformed into nitrosotiolic group (SNO).
These reactions occur as postgenetic modifications of cysteines of proteins specifically deputed to the transport of the signal triggered by free radicals or, in a more ubiquitous and quantitatively relevant way, at the level of the cysteine of the tripeptide glutathione (glutamic acid, cysteine and glycine: abbreviated as GSH in the thiolic state and as GSSG in the most common state of mild oxidation, the disulfuric one). By virtue of its abundance, glutathione is the true buffer for radical reactions in the cell, because excess free radicals react with GSH, transforming it into its radical form, and two GSH radicals then react with each other to generate GSSG. The latter can propagate the radical signal by a succession of thiol-disulfide reactions at selected proteins within a given regulatory process, such as in signal transduction and gene activation.
Free radicals and genes
The defense of organisms against oxidative damage is essentially based on the expression, constitutive or inductive, of genes that, with their products, regulate the synthesis of antiradical molecules or mediate the physiological action of the radicals themselves. This is equivalent to saying that radical reactions intervene both downstream, on signal terminator proteins, and upstream, on molecular devices of signal initiation at the genetic level. These devices are also proteins, called ‘transcription factors’, because, by binding to specific areas of the DNA of genes (promoters), they trigger the synthesis of complementary RNA (transcription) that will dictate the synthesis of the corresponding protein (translation).
In prokaryotes, free radicals have direct effects on the gene because transcription factors themselves contain an ROS-sensitive element, such as reduced cysteine groups or iron-sulfur centers, consisting of an iron atom placed at the center of a sulfhydryl complex. In bacteria, an oxidative stress response system has been identified that includes a regulatory protein, OxyR, which, when its cysteines are oxidized by low levels of ROS to sulfenic acid and disulfide, promotes gene expression of numerous antiradical molecules.
In eukaryotes, gene expression is more often controlled by ROS in an indirect and complex manner because of the different cellular compartments that contribute to the successive steps of induction and execution of the gene activation process, and which may have different oxidoreductive homeostases.
For example, many gene transcription regulatory factors are activated in the cytoplasm by phosphorylation by kinases that are in turn activated by ROS, but the event that terminates the process, i.e., binding of the factor to DNA to initiate transcription, requires, unlike in prokaryotes, reducing conditions, which are provided in the nucleus by high concentrations of GSH and a thiol-rich protein, thioredoxin.
The need to accurately regulate the flow of free radicals, in order to ensure this dual requirement of pro-oxidant and antioxidant conditions, is the basis of the exposure of higher organisms to risks of oxidoreductive imbalance that result in various pathological situations.
From free radical pathology to a molecular medicine of oxidoreductive regulation
The knowledge gained on the role of radicals in all stages of generation and transfer of biological regulatory signals have allowed, in recent years, to revisit the pathology of free radicals, looking for their involvement in the most intimate and subtle pathogenetic mechanisms, beyond the mere observation of a terminal damage hypothetically of oxidative nature. This has also opened new horizons to therapeutic perspectives. All medicine, in its molecular aspects, has been involved in this transition, but some fields have witnessed more significant advances.
Free radical aging, i.e., the simplistic hypothesis that senescence results from the accumulation of cellular damage caused by ROS produced during oxygen metabolism, has become almost commonplace since D. Hartman first proposed it in 1956, pointing to it, in the context of the knowledge of the time, as a theory based on the chemistry of free radicals and radiation. Now, however, we have less descriptive and phenomenological data based on genetics.
In animals or transgenic cells, alteration of gene expression of an antioxidant enzyme alters the lifespan of the transformed biological system. Regarding the mechanism underlying these effects, the most recent data have focused on the mitochondrion as the main site of the connection between free radicals and aging, not only as the main site of ROS production in the cell, but also as a target of choice, by free radicals, in the processes of senescence.
It has been shown that programmed cell death or apoptosis, a very important process in tissue senescence, is largely related to the release in the cytoplasm of mitochondrial factors, which in turn is made possible by the loss of integrity of the organelle membrane due to the action of free radicals. ROS can be produced in excess by the mitochondrion itself not only in relation to pathological situations, but also as a result of the action of molecules that signal the expiration, genetically programmed, of the time assigned to the life of a particular biological system.
A gene locus implicated in signals of this type has been identified through targeted mutations of the shc gene: the suppression of the synthesis of one of its protein products, called p66 from its molecular weight, has given to treated mice a lower incidence of diseases related to aging, such as atherosclerosis, and a significant extension of life. It was then discovered that p66 is an oxidoreductive enzyme that, when activated by pro-apoptotic oxidative signals that modulate its degree of phosphorylation, is capable of diverting electrons from their normal flow in the mitochondrial membrane toward the production of hydrogen peroxide, resulting in oxidative stress, senescence and death of the affected system.
Alzheimer’s disease and Parkinson’s disease occur late in life in specific areas of the brain that, to autopsy findings, show signs of oxidative damage and appear as local cases of aging in a tissue, such as the nerve, particularly susceptible to oxidative damage due to the intense aerobic metabolism and the relative lack of antioxidant defenses. In Alzheimer’s disease, the improper binding of some metal ions to a certain neuronal protein could give rise to monovalent reactions with oxygen (auto-oxidation), which trigger the radical chain of ROS. In Parkinson’s disease, the auto-oxidation involves the metabolites of dopamine, a neurotransmitter that is released in high concentrations in the brain areas affected by the disease.
But it is in the study of amyotrophic lateral sclerosis (ALS) that the molecular medicine of oxidative-reductive phenomena has provided the most interesting insights, and once again progress has been made thanks to the contribution of genetics. ALS is a progressive paralysis that usually arises in mature age and leads inexorably to death, in a few years, for specific degeneration of nerve cells that send the impulses for muscle contraction (motor neurons or motoneurons). It has been seen that in a very small fraction of ALS cases, with well-defined familial incidence, there are mutations in the gene of SOD 1, which alter the function of the enzyme by decreasing it or adding a new activity that, instead of being antioxidant, is peroxidase-like and therefore pro-oxidant.
In each case there is an increase in ROS that leads to the death of motor neurons. The cases, much more numerous, of ALS without this mutation (‘sporadic form’) are indistinguishable from the familial forms for symptoms, course and autopsy findings. It can be hypothesized that in these latter cases another factor intervenes that however increases the flow of ROS in motor neurons. The appearance of this disease, typical of the age around 50 years, in young people who practice competitive sports, often accompanied by drug abuse, is in line with this hypothesis. In Italy, a significant number of cases have been diagnosed in football players at the end of their career.
In the states treated above it is clear that the oxidoreductive balance is shifted towards pro-oxidant conditions that promote cell degeneration and death. The growth of a tumor is instead marked by an uncontrolled tendency for proliferation of certain cells. In reality in cancer free radicals have a different role depending on the different phases of the tumor process: carcinogenesis (transformation phase), tumor development (progression phase) or therapy.
While in the first phase dominates the mutagenic role of ROS (it is well established the relationship between cancer and the presence in the environment of conditions known for their ability to produce radicals, such as chemical or radioactive pollution), for the development of cancer are more favorable local conditions that promote cell proliferation, that is, generally, a reduced state of tissue antioxidants.
Instead, at the time of therapy we try to produce massive radical fluxes at the level of the neoplasm, with radio- or chemotherapeutic treatments, taking advantage of the higher sensitivity of cancer cells to free radicals due to the higher reproductive rate and the lower presence of antioxidants typical of poorly differentiated cells.
Nutrition and physical exercise
A large part of the calories introduced with food go to build up the potential for oxygen reduction in the mitochondrion in order to produce energy in the form of ATP. In physiological conditions, 2-5% of the oxygen consumed in this process gives rise to superoxide and other ROS. Thus, the higher the caloric content of a diet, the higher the flux of ROS from the mitochondria. Because mitochondrial production of ROS has been seen to correlate with aging processes, the question has been raised whether reducing the calories consumed during an individual’s lifetime (calorie restriction) could lead to life extension.
Research on experimental animals has shown that this relationship exists and is associated with reduced oxidative damage of mitochondrial enzymes involved in the reduction of oxygen by metabolic derivatives of energy-rich food components.
In addition to this relevance of ROS in the overall amount of dietary intake, the specific pro- or anti-ROS activities of various dietary components, both natural and added (additives and dietary supplements), are the subject of extensive studies. Many vitamins (especially A, C and E) directly neutralize ROS, while some mineral ions (copper and iron in particular) are essential coenzymes of enzymes that neutralize ROS, such as superoxide dismutase and catalase. In addition to these essential nutrients, the vegetables are rich in substances that, although not necessary to our metabolism, also have a positive action on the prevention of diseases related to free radicals, precisely because of their high antioxidant capacity: these are polyphenols, compounds that give vegetables very intense colors and are very abundant especially in drinks such as tea and red wine.
The need to preserve the antioxidant properties of food, in the conditions of dilated distribution in time and space typical of modern society, has spread the use of chemical additives with antiradical action. It is also increasingly common to substitute the antioxidant capacity of a varied and balanced natural diet with the intake of vitamins and minerals in pills or concentrated extracts. It should be borne in mind, however, that an antioxidant is such as it has a reducing capacity, and therefore its assumption outside the synergies and compensations present in a food or diet can lead to interference with the physiological actions of free radicals, or pro-oxidant effects only apparently paradoxical, because a reducing agent in excessive concentrations tends to self-oxidize in the presence of oxygen, triggering in turn the production of ROS.
Exercise is related to nutrition, as it consumes in the muscle the ATP derived from food calories. In this sense it has a protective action similar to caloric restriction. Excessive exertion, however, leads to increased production of ROS, both because of increased oxygen consumption and because of the repeated cycles of hypoxia/reoxygenation that occur especially in anaerobic sports and that reproduce a situation, at the level of the whole body, comparable to that which is created locally in heart attack. These complications are prevented by training that has, among its many effects, to induce the expression of genes of antioxidant enzymes.
A particular type of exercise is that which is carried out in high mountains, such as on the summit of Mount Everest where the partial pressure of oxygen decreases to one third of that present at low altitude. It has been shown that, in extreme conditions, oxygen consumption produces 50% free radicals (against the physiological 25%), due to the drastic reduction and inefficiency of mitochondria metabolism and the alternation of hypoxic phases during the effort and reoxygenation during rest.
Molecular adaptations of altitude-acclimatized populations include increased gene expression of antioxidant enzymes, and this is also seen in individuals living at lower altitudes but who have undergone a congruent period of altitude training.
- Dröge 2002: Dröge, Wulf, Free radicals in the physiological control of cell functions, “Physiological reviews”, 82, 2002, pp. 47-95.
- Filomeni 2002: Filomeni, Giuseppe – Rotilio, Giuseppe – Ciriolo, Maria Rosa, Cell signalling and the glutathione redox system, “Biochemical pharmacology”, 64, 2002, pp. 1057-1064.
- Filomeni 2005: Filomeni, Giuseppe – Rotilio, Giuseppe – Ciriolo, Maria Rosa, Disulfide relays and phosphorylative cascades: partners in redox-mediated signaling pathways, “Cell death and differentiation”, 12, 2005, pp. 1555-1563.