Trying To Unlock The Mysteries Of Free Radicals And Antioxidants

SIDEBAR : Resources For Free Radical And Antioxidant Research Professional Societies: Oxygen Society 74 New Montgomery, Suite 230 San Francisco, Calif. 94105 (415) 546-3124 - Fax: (415) 764-4915 E-mail: 76125.3071@compuserve.com 750 members President: Bruce Freeman Executive Director: Carol Holland Parlette Journal: Free Radicals in Biology and Medicine Internet home page: http://www.biophysics.mcw.edu/oxsoc/ International Society for Free Radical Research c/o Lester Packer, president-elect Department of Molecular and Cell Biology 251 Life Science Addition University of California, Berkeley Berkeley, Calif. 94720-3200 (510) 642-1873 - Fax: (510) 642-8313 E-mail: packer@garnet.berkeley.edu 3,000 members Internet home page: http://http1.brunel.ac.uk:8080/~bcstrlw/sfrr.html Internet Site For additional information on free radical and antioxidant research, contact the National Institutes of Health's home page at http://www.nih.gov. Aerobic organisms exist in a perpetual catch-22. Oxygen sustains them, but it also poisons them via reactive intermediates produced during respiration. The powerful oxidants produced in this process -- including the superoxide anion, hydroxyl radicals, and hydrogen peroxide -- are known as free radicals. These highly reactive molecules have been fingered as agents not merely of disease, but also of the aging process itself. But evolution has not left aerobes defenseless against reactive oxygen species; their cells also produce antioxidants to keep free radicals in check. Thus, scientists are trying to understand how free radicals cause destruction as well as how antioxidants protect cells from damage, which could provide clues to treat or prevent disease and perhaps even aging. Reports on free radicals in living systems fill many specialized journals and are featured prominently throughout the biochemical literature. Yet as recently as 30 years ago, reactive oxygen species were not thought to occur in living cells, recalls longtime researcher William Pryor, director of Louisiana State University's Biodynamics Institute in Baton Rouge. "The cant in the field [at that time] was that radicals were so reactive and unselective that they could not occur in biological systems," Pryor explains. That view changed in 1967 when biochemist Irwin Fridovitch of Duke University and Joe McCord (then a graduate student at Duke; now a professor at the University of Colorado School of Medicine in Denver) discovered the antioxidant enzyme superoxide dismutase (SOD), an important means of cellular defense against free radical damage. Today, researchers agree that aerobically respiring cells are veritable radical factories, producing "good radicals and bad radicals both: those that are under control and perform desirable biochemical transformations, and those that cause pathology-as well as toxins that work through radical reactions," Pryor says. RADICAL THINKING: William Pryor recalls that 30 years ago, scientists thought radicals couldn't occur in biological systems. Research concerning the cellular origins and physiological consequences of free radicals now occupies thousands of investigators worldwide. Some of these scientists are examining the potential role of reactive oxygen species in a long list of maladies, including atherosclerosis, cancer, inflammatory disease, and cataracts. Free radicals are also thought to cause reperfusion injury, which occurs when tissues are temporarily deprived of blood flow, such as after heart attack, stroke, and organ transplantation. Recent explorations of possible links between oxidative damage and neurodegenerative disease have proved especially fruitful. Yet, as Pryor mentions, free radicals aren't all bad. That's particularly true of nitric oxide (NO), a molecule that's lately achieved celebrity status. Ubiquitous NO apparently regulates all manner of neurological, vascular, and immunological functions; it even seems to act as an antioxidant under certain conditions. But NO also has its "dark side," according to biochemist Bruce Freeman, a professor in the department of anesthesiology at the University of Alabama in Birmingham (Hot Papers, The Scientist, Sept. 16, 1996, page 13). Scientists are only beginning to explore that unknown territory. HOPING FOR A PAYOFF: Pamela Starke-Reed notes NIA's free radical funding. Theories of aging come and go, but one of the most enduring suggests that free radicals play a significant role in biological senescence, explains Pamela Starke-Reed, director of the Office of Nutrition at the National Institute on Aging (NIA). "It's far from being proven," she says, "but it hasn't been disproved, either." That's mainly because there's no direct way to measure free radicals in vivo, she elaborates, "but we're getting close." Thus, NIA generously funds research in free radical biology. Starke-Reed estimates that 15 percent of the institute's 1995-96 budget of $71 million supported research on oxidative damage related to aging; that figure does not include studies of related topics such as genetic mutation. As Starke-Reed indicates, the key challenge in this field is to identify the mechanism by which oxidative damage induces age-related physiological phenomena. Denham Harman, an emeritus professor of medicine at the University of Nebraska Medical Center in Omaha and the originator of the free radical theory of aging, thinks that humans may decline along with their mitochondria. There is, Harman says, a growing consensus among biogerontologists that in mammals, aging -- defined as an increase in the risk of death -- results from deleterious cellular changes produced by free-radical reactions. These cell-damaging processes are largely initiated in the course of mitochondrial respiration, he says, while life span is determined by the rate of damage to the mitochondria. PIONEER: Free radical theory originator Denham Harman. Numerous studies supporting that consensus were recently described by Rajindar Sohal, a professor in the department of biological sciences at Southern Methodist University in Dallas, and Richard Weindruch, a professor of medicine at the University of Wisconsin Medical School in Madison (Science, 273:59-63, 1996). Four conditions need to be met to validate the free radical theory of aging, the authors wrote: first, oxidative damage must increase as aging progresses; second, longer-lived species must sustain lower rates of damage; third, known life span-lengthening regimes such as caloric restriction must be shown to reduce oxidative damage to cells; and finally, experimental increases in antioxidant defenses should lengthen life span. On all of these counts, says Sohal, "there is enough evidence to give good credence to the free radical theory of aging." MULTIFACTORIAL: Biogerontologist Leonard Hayflick believes aging has multiple causes, including free radical damage. Other scientist take abroader view of aging. For example biogerontologist Leonard Hayflick, a professor at the University of California, San Francisco, believes aging probably results from multiple causes, one of which is free radical damage. "The most intriguing aspect of studies done with free radicals," Hayflick writes in his recent book, How and Why We Age (2d ed., New York, Ballantine Books, 1996), "is, perhaps, not what they might be telling us about aging, but what they are telling us about disease." In particular, free radical studies are speaking volumes about the link between oxidative damage and neurodegenerative disorders such as Huntington's (HD), Parkinson's (PD), and Alzheimer's diseases, as well as amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease. "The more people have looked for free radical effects in neurodegenerative diseases, the more they've found," observes Dale Bredesen, director of the program on aging at the Burnham Institute in La Jolla, Calif. Neurologists suspect that glutamate, a neurotransmitter, sparks free radical production if it accumulates in the brain (J.T. Coyle, P. Puttfarcken, Science, 262:689-94, 1993). The resulting reactive oxygen and nitrogen species may directly damage cell membranes and proteins, Bredesen explains; they may also act as signaling molecules to initiate programmed cell death, or apoptosis, a process suspected to be involved in several neurodegenerative disorders. Free radicals may damage compromised brain cells that might otherwise resist such an attack. For example, mutations in the copper-zinc form of SOD are associated with a rare subtype of familial ALS, a degenerative disease of motor neurons. To further study the role of this enzyme in protecting motor neurons, a team led by scientists from Cephalon Inc., a pharmaceutical company based in West Chester, Pa., and from the Washington University School of Medicine in St. Louis created a Cu/Zn knockout mouse (A.G. Reaume et al., Nature Genetics, 13:43-7, 1996). Although "overtly normal" through young adulthood, these knockout mice showed "increased vulnerability to neuronal death following injury," states author Richard Scott of Cephalon. Cu/Zn SOD, he concludes, while unnecessary for normal development, "seems to protect neurons against unexpected bursts of oxidative damage," such as those known to occur following injury. Besides mutation, another way that antioxidant proteins may be weakened is through the action of nitric oxide. In this and other instances in which it plays the cellular villain, NO is thought to react with superoxide radicals to produce an even more reactive species, peroxynitrite. A combination oxidant and nitrating agent, peroxynitrate subsequently reacts with the amino acid tyrosine in cellular proteins, converting it to nitrosotyrosine. Nitration may in turn affect protein function, according to Joseph Beckman, a professor in the departments of anesthesiology and biochemistry at the University of Alabama in Birmingham. Beckman and colleagues recently raised antibodies that recognize nitrated proteins and have used them to visualize nitrated proteins in tissues affected by ALS, HD, and atherosclerosis (J.S. Beckman et al., Biological Chemistry Hoppe-Seyler, 375:81-8, 1994), among others. Subsequently, Lee Ann MacMillan-Crow, a postdoctoral fellow in the Alabama laboratory of Anthony Thompson in the department of surgery, optimized an immunoprecipitation procedure that enabled her and coworkers to isolate nitrated proteins from rejected kidney transplant tissue. Most interestingly, she reports, they recovered nitrated Mn SOD, the mitochondrial form of the enzyme. (Cu/Zn SOD is found in the cytosol, the fluid portion of the cytoplasm.) They also found decreased Mn SOD activity in rejected transplant extracts, as compared with healthy kidney tissue controls. In follow-up experiments, the researchers found that the degree of nitration in the extracts paralleled enzyme inactivation, according to MacMillan-Crow. Once Mn SOD is inactivated, she points out, superoxide-and thus peroxynitrite-probably runs rampant in the cell. "We think this positive-feedback mechanism is a realistic model which may account for end-stage kidney disease, and which could be operating in other diseases as well," she concludes. SNO-BALLING EVIDENCE: Jonathan Stamler found that S-nitrosothiols (SNOs) regulate protein activity. Nitric oxide also reacts with thiol groups of proteins to form powerful compounds called S-nitrosothiols (SNOs), which have been shown to regulate protein activity and even confer novel function on some proteins. Researchers led by Jonathan Stamler, an associate professor of medicine at Duke University, recently demonstrated that SNOs are a major regulator of gas exchange and blood pressure (L. Jia et al., Nature, 380:221-6, 1996). And this month, Stamler and coworkers reported that SNOs up-regulate genes encoding proteins that, in turn, destroy excessive SNOs in the cell (A. Hausladen et al., Cell, 86:719-29, 1996). Previously, only oxidative stress was known to activate such a genetic feedback loop. This discovery not only strengthens NO's reputation as a universal molecular signal, but also indicates that it may rival oxygen as a cellular toxin. Thus, Stamler has coined the term "nitrosative stress" to describe the cellular consequences of excess NO. Nitrosative stress, Stamler asserts, "parallels oxidative stress in that it may lead to a host of diseases, as well as the cumulative damage of aging." While oxidative and nitrosative stresses can be synergistic, he says, the latter represents "a new type of stress that can occur in the absence of oxygen." Although many studies focus on damage wrought by free radicals, a significant body of research describes the many cellular defenses deployed against this assault. A small number of pharmaceutical researchers are also engaged in discovering and designing chemical antioxidants to treat disorders associated with oxidative stress. Scientists continue to identify new natural antioxidants. Recent recruits to the ranks of SOD and vitamin E include the so-called thiol-specific antioxidant enzyme discovered by researchers at the National Heart, Lung, and Blood Institute (M.B. Yim et al., Journal of Biochemistry, 269:1621-6, 1994) and, perhaps, the hormone melatonin (R. Reiter, European Journal of Endocrinology, 134:412-20, 1996). Chemical mimics of the active site of Mn SOD, developed by Eukarion Inc., a Bedford, Mass.-based biotech company, have also been shown to mediate beta-amyloid toxicity, thought to cause neuronal degeneration in Alzheimer's disease (A.J. Bruce et al., Proceedings of the National Academy of Sciences, 93:2312-6, 1996). 'GOOD CREDENCE': Rajindar Sohal says the free radical theory is promising. Based on observations that the steroid hormone methylprednisolone reduced central nervous system injury in animal models, researchers at Pharmacia and Upjohn Inc. of Kalamazoo, Mich., have developed a series of chemical variants of this molecule, which they dubbed "lazaroids," after the biblical character Lazarus, whom Jesus raised from the dead, according to senior scientist Edward Hall. One of these compounds, tirilazad, is being tested in clinical trials for the treatment of brain and spinal cord trauma and stroke-induced injury. Hall says his company is also studying another class of antioxidant molecules, the pyrrolopyrimidines, for the treatment of PD and related diseases. Similarly, Centaur Pharmaceuticals of Sunnydale, Calif., is developing a family of novel antioxidants based on phenylbutylnitrone-a compound initially used as an indicator of free radical activity in biological systems-primarily as therapeutics for neurodegenerative disorders, reports founder and chief technical officer John Carney. Evaluating antioxidants' ability to prevent disease is also a topic of intense research. Although mounting evidence testifies to the benefits of eating antioxidant-rich foods, more studies are required to confirm whether supplementing a healthy diet with the antioxidant vitamins E, C, and beta-carotene actually reduces free radical levels in vivo, notes NIA's Starke-Reed. Recently, however, researchers described a way to measure free radical byproducts in urine (M. Reilly et al., Circulation, 94:19-25, 1996) and therefore gauge the effect of various treatments on free radical generation. "The antioxidant field is extremely exciting because both animal and in vitro data have long suggested that antioxidants would be potent protectors against chronic diseases-particularly cancer and heart disease," reflects Pryor. Recent epidemiological and clinical studies have been almost uniformly supportive of this hypothesis, he says. Alison Mack is a freelance science writer based in Wilmington, Del.