Free Radicals and Human Disease

CRC Handbook of Free Radicals and Antioxidants ,vol 1 (1989), p209-221.

Peter H. Proctor, PhD, MD

Oxygen? "I rarely use it myself, sir. It promotes rust." Robby the Robot, Forbidden Planet(1956)

( Earlier versions: Radical Disease, 1972and Radical Disease, 1984)

Free radical ( "Redox:" ) signaling: Conference on Active Oxygen and Medicine, Honolulu, March,(1979). Abstract .

Indirect evidence suggests that free radicals and excited-state species play a key role in both normal biological function and in the pathogenesis of certain human diseases. For example, generation of activated species by inflammatory cells is a major microbiocidal mechanism and may also mediate important components of the inflammatory response. Activated processes may also be key components in the toxicity of many drugs, in aging, and in carcinogenesis. They may also figure in the etiology of certain ocular, neurological, and psychiatric diseases.

The evidence for a role for electronically activated species in human disease has long been prevalent. For example, Darwin repeats the well-known observation that white, blue-eyed cats are usually deaf. Similarly, the relationship between pigmentary abnormalities and human deafness (for example, in Waardenberg's or Usher's syndromes) is commonplace in audiology(4). Likewise, physicians have long recognized the association between radical-generating metals such as copper or iron and fibrotic changes, e.g., interocular fibrosis in vitreous chalcosis and liver cirrhosis in Wilson's disease and Hemochromatosis.

Further, free radicals and other activated species are so difficult to measure under biological conditions that the evidence for their role in any biological process - much less a human disease state - is normally indirect and circumstantial. This flawed scientific basis often results in heated controversy over methodology, results, and conclusions. Even less should be expected of the clinical evidence. Nonetheless, there is significant circumstantial evidence that active oxygen (Figures 1 and 2) is involved in some of the most fundamental mechanisms in pathogenesis and in the etiology of many human diseases.

Figure 1 The Active Oxygen System

FIGURE 1. The active oxygen system. Molecular oxygen is reduced to water in four single-electron steps. Reduction of nonradical forms of oxygen is a " forbidden " process and thus usually involves spin-orbit coupling by a heavy metal or a halide or excitation to singlet state. An example is Fenton's reaction, the reduction of peroxide to water and hydroxyl radical by ferrous iron. Hydroxyl radical is one of the most powerful oxidizing agents known. Simply put, reducing agents act as prooxidants by reducing nonradical forms of oxygen to radical forms, usually with heavy atom involvement. Similarly, they can act as antioxidants by reducing radical forms of oxygen, by terminating radical chain reactions, or by, for example, reducing hydroperoxides. This dual property can be of great significance. For example, in humans uric acid is probably the primary extracellular antioxidant. On the other hand, a Fenton-type reaction of phagocytized urate with granulocyte-produced peroxide may contribute to the etiology of gout.

Figure 2: Neuromelanins

FIGURE 2. Neuromelanin. A: Dopaminergic pigmented neurons in pars compactaof substantia nigraand B: Noradrenergic pigmented neurons from locus ceruleus. ( Autopsies by the author).

Most, if not all, central catecholaminergic neurons contain a stable free radical, melanin. Specific dying-off of pigmented neurons in the substantia nigrais the apparent cause of Parkinson's disease. Dopaminergic neurons may also be concerned in schizophrenia and in various movement disorders ( e.g., choreoathetosis in the Lesch-Nyhan syndrome ). Noradrenergic neurons may figure in endogenous depression and Altzheimer dementia. The function, if any, of melanin in such neurons is unknown but it may be related to its antioxidant and semiconductor properties. G. C. Cotzias on neuromelanins: " The neuromelanin granule may be the secret key to the understanding of Parkinsonism. I don't believe God put the melanin granule in the central nervous system for nothing. It must be doing something. Something big... "

Later note: Go here and here for examples of the drop-out of melanin-containing neurons in Parkinson's disease. Also, melanin-bound iron, increases in Parkinsonism-- vis, the parkinsonian-like symptomology occasionally found in hemochromatosis. Go here for an example of the antioxidant properties of melanin.

The evidence for a role in disease is of several types. For example, many human diseases present with increased production of activated species or with increased levels of radical-generating substances. Examples include granulocyte activation in inflammation or copper in Wilson's disease. Additionally, the progression of many diseases may be modulated pharmacologically by ectopically administered superoxide dismutase (SOD), catalase, or free radical scavengers. Finally, many such diseases are also associated with one or more characteristic symptoms (Table 1).

The Oxygen-Dependent Microbiocidal System
As summarized in Figure 3, granulocytes and other phagocytic cells possess a membrane NADPH oxidase, which-takes reducing equivalents from the hexose monophosphate shunt and transfers these to molecular oxygen to produce superoxide and other active oxygen species. A further myeloperoxidase converts peroxide produced in this system to microbiocidal products, probably including hypochlorite (2). Production of activated products by this system probably plays a key role in cell-mediated immunity and microbiocidal activity. There is evidence for similar systems in T-lymphocytes,(15) platelets,(6) and mucus.(17) An NADPH oxidase of noninflammatory cells may have a role in mediating cyclic nucleotide metabolism ( 18-20 ).

Figure 3: The Role of Active Oxygen Species in Inflammation

FIGURE 3. Role of active oxygen species in inflammation.
Inflammatory cells ( granulocytes, macrophages, some T-lymphocytes, etc. ) produce active species of oxygen as part of the microbiocida1/citocidal system. In turn, active oxygen species can modulate specific elements of the inflammatory response in vitro. Examples include protein immunomodulator substances such as granulocyte migratory factors, prostaglandins, cyclic nucleotides, as well as formed elements such as platelets. Which, if any, of these are relevant to the in vivosituation is unknown.

Antioxidant Defenses and Solid State Defenses
Biological systems protect themselves against the damaging effects of activated species by several means (21-22). These include free radical scavengers and chain reaction terminators: enzymes such as SOD, catalase, and the glutathione peroxidase system; and " solid-state " defenses such as the melanins.

Chemical antioxidants act by donating an electron to a free radical and converting it to a nonradical form. Likewise, such reducing compounds can terminate radical chain reactions and reduce hydroperoxides and epoxides to less reactive derivatives. However, chemical antioxidant defense is a double-edged sword. When an antioxidant scavenges a free radical, its own free radical is formed. Many antioxidants can act as pro-oxidants by, for example, reducing nonradical forms of oxygen to their radical derivatives, particularly if redox cycling occurs. The exact mix of pro- and antioxidant properties of a reducing compound is a complex interaction involving pH, relative reactivities of radical derivatives, availability of metal catalysts, and so forth. For example, the anti- or pro-oxidant properties of sulfhydryl compounds depend upon pH (29-31), those of beta carotene upon oxygen concentration (69). Likewise, uric acid, probably a significant antioxidantin higher primates (32-36) participates in a Fenton-type reaction with peroxide(35, a property which may be important in the etiology of gouty inflammatory disease.

Similarly, stable radical formers, such as the melanins or the nitroxide spin labels, scavenge odd electrons to form stable radical species, thus terminating radical chain reactions. Interestingly, minoxidil, noteworthy because of its ability to stimulate hair growth and reverse pattern balding, is a nitroxide closely analogous to commonly used spin labeling compounds.

Later Note : Go here for a discussion of how free radicals may modulate hair growth. This is another manifestation of free radical ( " redox " ) signaling. Also, nitroxide spin labels such as TEMPOL are potent SOD-mimetics. Both spin traps and spin labels have significant potential drug applications for most of the diseases considered in this review. Go here for more on this.

Enzymatic defenses against active species include SODases, catalases, and the glutathione reductase/peroxidase system. While there have been some thoughtful questions raised, SOD appears to be one of the most specific enzymes known. Inhibition of a biological process by SOD is often taken as putative evidence for a role for superoxide in that process. However, some of the actions of SOD appear to be due to peroxide production rather than superoxide destruction (4).

Most recent work in free radical defense centers on chemical antioxidants and enzymatic defenses. However, a third class of mechanism, solid-state defense, may be at least as important, particularly with respect to human disease. In solid-state defense, a macromolecule binds a radical-generating compound, deexcites an excited-state species, or quenches a free radical. In many ways the internal action of SOD matches this definition. However, the most important solid-state defense is probably the black pigment melanin. Melanin is also important because it is the only biological polymer which is a stable free radical and was the first free radical established in biological systems.

In the same manner, a visible pigmentary response often occurs in the presence of a radical-dependent process, be it a dermal inflammatory process, UV light, or the chronic presence of a pro-oxidant such as iron in hemochromatosis. This dermal pigmentary response is the only part of the defensive reaction to active species which is a clinically apparent symptom. Thus, it forms a part of the radical-dependent symptom complex (Table 1) and represents a visible outward sign of an otherwise invisible active process.

While chemically inert, melanin is a very active " amorphous semiconductor ". In amorphous semiconductors, photon or electronic energy in the form of motions of electrons is very strongly coupled to molecular vibrations or " phonons ". Heat is one manifestation of phonon energy, while sound vibrations are another. That is, in the melanins electronic or excited-state energy readily exchanges with vibrational energy in the form of heat or sound.
Such seemingly esoteric considerations may explain much of the physical properties and biological functions of the melanins (35-41). For example, the melanins are black, photoprotective, and nonfluorescent because most photon energy ( e.g., from light or chemically produced excited-state species ) absorbed by them readily converts into heat ( 36~37 ). This likely explains the presence of melanins in such energy-transducing areas as the skin, retina, and inner ear. Conversely, rate limitations for such conversions mean that the melanins may themselves be toxic to the cells which contain them by electronically-activated mechanisms (4-9). This may be important in the etiology of such disorders as Parkinsonism, senile macular degeneration, and senile deafness (4,9-27,41 ).
Likewise, the ability of melanins to readily convert vibrational energy in the form of sound into electronic energy means that they are by far the best sound-absorbing materials known (38). This may account for their presence ( as protective devices ? ) in the inner ear (4,9-27,41). It is also relevant to the well-known association between pigmentary abnormalities or the presence of melanin-binding drugs in deafness ( e.g., Waardenburg's syndrome and aminoglucoside ototoxicity ), as well as the association of deafness with pigmentary retinopathies in Usher's syndrome and chloroquine toxicity.
The melanins also have some rather exotic electronic properties. For example, they can act as threshold switches and can store electrical energy like batteries( 39 ). Such properties may explain the presence of melanins in electrically active tissues such as the substantia nigra, where it may play a role, for example, in Parkinson's disease ( Figure 2 ).

Later note : By nearly a decade, melanin was the first organic semiconductor used in an " active " electronic device ( a bistable switch), where an electric field controls current flow. Bistable switches are the basic unit of your computer. Since the "on" state of this switch has almost metallic electrical conductivity, melanin is also the first organic compound shown to have a high-conductivity state.
Melanins are polyacetylenes and vice versa. Much to our surprise, the later discovery of high conductivity produced by chemical means in another polyacetylene ( i.e., another "melanin" ) won the 2000 Nobel Prize in Chemistry. This is like recognizing simple semiconductivity in silicon, while ignoring its previous use in a transistor. Interestingly, nearly all organic semiconductor devices since have technically used "melanin" as their active element.

There is no evidence the Nobel Committee was aware of our previous work, though it was published in a major journal, Science. See organicsemiconductors.com for the details. But enough complaints-- back to the paper....

Melanin also forms stable free radicals, quenches excited states, and binds radical-forming agents such as transition-series metals. All likely contribute to its putative role in antioxidant defense. On the other hand, the ability of melanin to bind toxic radical-generating agents may sometimes be detrimental, as in chloroquine retinopathy and aminoglycoside ototoxicity (41). Finally, melanin can function as an efficient S0Dase and may retain this function in pigmented organs. Thus, the melanins ( which can form abiologically ) may be the oldest evolved system for defense against oxygen radicals, rather than SOD/catalase.
Free radicals are produced by environmental causes such as light or ionizing radiation. However, three physiological processes can result in extraordinarily high levels of radical species. These include the mixed-function oxidase system of endoplasmic reticulum, the NADPH oxidase system of inflammatory cells, and the presence of high levels of autoxidation-mediating charge-transfer agents. Production of activated species by such mechanisms can exceed the capacity of local protective mechanisms and produce tissue injury.
Inflammatory cells produce active species of oxygen in antimicrobial defense (1,2). While such species may directly damage surrounding tissues, their major secondary role may be to mediate important components of the inflammatory response. For example, Figure 3 lists some of the inflammatory immunomodulators reported to be affected in vitro by one or more components of the active oxygen system.