or most of their evolution, humans have been hungry, assailed by both large predators and tiny microorganisms and eating a largely plant-based diet. These plants were not the selectively bred toxin-free varieties we consume today. Instead, they probably contained a range of noxious agents which they developed over time to protect themselves from consumption by animals. Human lifespan was cut short by accidents, predation, infectious diseases, and malnutrition caused by calorie, protein, vitamin, or mineral deficiencies.

Evolution selects genes that promote survival and fertility even if the traits might prove deleterious in post-reproductive years. Nature will not favour a mutation that increases lifespan but decreases fecundity. Consider the opposite - a mutation that makes a male highly sexed and attractive to women with the downside of a life expectancy no longer than 45 years. This mutation will be passed on, provided of course that this male locates fertile females who find him irresistible.

Because of natural selection, humans evolved a preference for fat and an aggressive immune system to defend against infection. The body easily stores fat, a rich source of calories, to keep it going in times of famine. Abundant fat stores in women allow pregnancy to reach term, even when the quality of the diet is poor. Fat also transports fat-soluble vitamins.

Humans evolved a huge range of detoxifying enzymes to nullify xenobiotics (substances foreign to the body) in dietary plants. Some humans developed a mutation in a gene regulating intestinal iron intake. This mutation may have enabled prolonged survival on iron-poor diets, especially because many plants contain agents that impair iron absorption. Females who absorbed iron more efficiently from such diets might again be more likely to carry pregnancies to term, ensuring propagation of offspring carrying the mutant gene.

In the "advanced" contemporary societies of North America, Europe, and parts of Asia such as Singapore, people live long past their reproductive years, and age-related diseases have become burdensome medical and social problems. Death comes most often as the result of cancer and cardiovascular disease, and the incidence of age-related neurodegenerative diseases (such as Alzheimer's and Parkinson's) is rising fast. In a society of free-flowing food, the old inherent craving for fat and calories, if satisfied, contributes to cardiovascular disease and type 2 diabetes, especially with a decline in energy expenditure that accompanies an increasingly sedentary lifestyle. Mutations affecting the regulation of iron absorption give rise to iron overload later in life, which consequently damages almost all body tissues in the form of a silent killer known as idiopathic haemochromatosis.

At the moment caloric restriction serves as the only intervention reproducibly shown to increase lifespan in laboratory rats and mice. Similar studies are under way in monkeys. Caloric restriction means maintaining an adequate intake of protein, vitamins, and minerals while halving the total calorie content of the diet. Laboratory animals sit in their cages with little to do; they exercise minimally, and they eat the food supplied. Such laboratory conditions resemble sedentary lifestyles in wealthy societies. Singapore, like many such societies, has converted from the malnutrition of deficiency to the malnutrition of excess.

Because nobody really knows the precise causes of ageing, theories proliferate. In fact, people confuse ageing with age-related disease whereas the latter constitutes the true problem. As people age, the efficiency of all organs declines but at a variable rate, and sufficient redundancy remains in most systems for reasonable functionality until very late in life. However, ageing people are increasingly likely to develop age-related diseases: cancer, cardiovascular disease, osteoporosis, and dementia, among others.

One popular theory for why this happens is the "free-radical theory." This theory of ageing, first proposed by Denham Harman in 1956, states that ageing happens as a result of accumulated free-radical damage over the human lifespan. In an attempt to illustrate this theory, he fed synthetic antioxidants to laboratory animals, but did not achieve a convincing outcome. More recently, scientists have induced animals to live longer by manipulating genes causing over-expression of antioxidant enzymes. Limiting food intake in animals strikingly decreases levels of free-radical damage to DNA, lipids, and proteins, a result consistent with its lengthening of lifespan and its effect in decreasing cancer development.

Link to Evolution

In the attempt to explain free radicals and antioxidants, we see a link with evolution, but this time occurring much earlier. Humans breathe oxygen (O₂) to drive respiration, which produces the energy needed to grow, keep warm, move around, and survive, but the irony is that O₂ is poisonous. The first organisms to evolve on earth lived under an anaerobic atmosphere - with almost zero O₂.

The gas began to appear in the atmosphere in greater quantities about 2.5 billion years ago, when some blue-green algae evolved a mechanism to harness the energy of sunlight to split water, yielding hydrogen, so as to drive metabolism. Like many new chemical reactions, splitting water also produced pollution; the unwanted by-product, O₂, was dumped into the atmosphere and gradually altered it into a powerfully oxidising environment.

Anaerobes cannot survive in the current atmospheric concentration of 21% O₂. Although most anaerobes must have died, a few survived by restricting themselves to environments that O₂ did not reach - in deep layers of soil, faecal matter, dental plaque, gangrenous wounds, and other insalubrious places.

This natural selection forced another group of organisms to do something very clever - they evolved antioxidant defences to protect themselves against O₂ toxicity. They could survive and spread into new areas in which competition with the aerointolerants did not exist. Another change occurred: some organisms evolved to use O₂ in respiration, a process highly efficient in delivering more energy from less food. Indeed, over 80% of O₂ that living organisms breathe is used in the mitochondria to generate adenosine triphosphate (ATP), the universal cellular energy currency.

The essential poison paradox is not unique to oxygen. Glucose serves as a vital metabolic fuel for the brain, nervous system, and red blood cells, but prolonged hyperglycaemia causes widespread tissue damage, as in diabetes. Interestingly, high glucose causes harm by promoting excessive production of free radicals. Prolonged exposure to high levels of O₂ also causes damage in humans and animals; a textbook example is retinopathy of premature infancy - retinal injury caused by exposing premature babies to too much O₂.

Sources of Antioxidants

Organisms that have to cope with a lot of O₂ are logical sources of antioxidants. During photosynthesis, chloroplasts in green plants have to tolerate pure O₂, and repairing the cellular damage caused by this high O₂ load consumes much of the ATP synthesised from sunlight's energy. Plants are rich in the antioxidants vitamins C and E, carotenoids such as beta-carotene and lycopene, and the flavonoids. Others antioxidants remain to be discovered.

Plants have evolved metabolic pathways to make all the above antioxidants - humans can make none, perhaps because they missed the evolutionary driving force of pure O₂ and because their early plant-rich diet precluded a need to retain this capacity. This inability to make the above antioxidants, with the exception of vitamin C, holds true for other animals. Rats, mice, cats, dogs, and many other domesticated animals readily make vitamin C, but because of a mutation in the gene encoding the last enzyme in the vitamin C biosynthetic pathway, guinea pigs and primates cannot. This mutation is the root of a universal inborn error of human metabolism that remained silent when humans lived on a plant-rich diet. The mutation becomes severely deleterious when plants are removed from the human diet. So the question becomes: can we prevent ageing and age-related diseases by taking antioxidants?

Antioxidant Paradox

Oxygen toxicity derives from the fact that a small portion of the O₂ humans inhale converts into O₂-free radicals and the other reactive O₂ species, including superoxide radical (O₂·-) hydrogen peroxide (H₂O₂) and hydroxyl radical (OH·) (the superscript dot denotes a free radical). Antioxidants made in the human body dispose of these species. Thus superoxide dismutase (SOD) enzymes convert O₂·- to H₂O₂ and O₂, and the catalase, peroxiredoxins, and glutathione peroxidase enzymes remove H₂O₂. Iron overload (as in idiopathic haemochromatosis) harms body tissues because it converts H₂O₂ into the highly damaging hydroxyl radical. Like glucose and O₂, iron, although essential for life, becomes toxic if allowed to accumulate.

Human antioxidant defences do not remove all reactive O₂ species generated in vivo. Several remain to cause damage. Highfat diets, hypertension, hyperglycaemia, high-plasma low-density lipoprotein (LDL) levels, low high-density lipoprotein (HDL) levels, and cigarette smoking predispose the body to atherosclerosis, in which a key event is free-radical damage causing oxidation (lipid peroxidation) of LDL lipids in blood vessel walls.

Reactive O₂ species also damage DNA, creating mutagenic lesions. A battery of repair systems struggle to keep up with the DNA damage caused by reactive O₂ species and other genotoxic agents, but over the human lifespan sufficient damage eludes these repair systems to cause mutations that initiate and promote cancer. Thus, towards the end of life about onethird of the population develops cancer; the same is true for the shorter lifespan of rats, mice, and other smaller mammals. The major cancers are thus age-related diseases.

Why have better antioxidant defences to prevent LDL oxidation and DNA damage failed to evolve? Evidence increasingly indicates that some free-radical production is useful. At sites of inflammation, white cells produce bursts of reactive O₂ species that aid in the killing of bacteria and fungi and in the inactivation of viruses. Natural selection would therefore pick this essential defence as a desirable one during evolution, as it would help prevent early death from infection. Many bacteria have evolved to respond to the defence-cell challenge with a rapid up-regulation of antioxidant enzymes, enabling them to resist host immune defences. The excessive damage caused by free radicals leading to cancer in the post-reproductive years is immaterial because few people live that long.

The immune system's free-radical production remains a powerful protective tool, but if unchecked, it becomes dangerous. The risks of vascular damage and cancer increase if production of reactive species accelerates, for example in chronic inflammation triggered by autoimmunity or by infection with microorganisms resistant to killing by bodily defence systems.

Important Antioxidants in Diet

Experts associate lower risk of cancer, cardiovascular disease, stroke, type 2 diabetes, and perhaps dementia with diets rich in fruits, grains, and vegetables. These diets have a high antioxidant content. Important dietary antioxidants include vitamin C. Essential for preventing scurvy by acting as a cofactor for several enzymes, it also has antioxidant properties. Vitamin E protects lipids against free-radical damage and constitutes the most important antioxidant contained within LDL lipids. Carotenoids, powerful antioxidants in plants, offer humans a source of vitamin A.

Despite all the hype on commercial supplement labels, no evidence exists that beta-carotene is an important antioxidant or anticancer agent in the human body, and indeed high doses may harm smokers. Several research groups, including my own, have conducted studies on healthy individuals to measure the effects of diet on free-radical damage to DNA. The studies concluded that eating more plants decreases damage levels, but giving the well-fed volunteers more vitamin C, vitamin E, or beta-carotene as supplements does not. Perhaps these data explain the disappointing results obtained in recent intervention trials using such supplements.

My group feels that the antioxidant effects of plant-rich diets do not derive from those three agents acting alone. The identity of the most important dietary antioxidants remains uncertain. Currently, many researchers are focusing on flavonoids, polyphenolic compounds that are powerful in vitro inhibitors of lipid peroxidation. Suppliers of red wine, green tea, and chocolate tout the flavonoid content of their products, yet the evidence that flavonoids constitute important antioxidants in vivo remains inconclusive. Indeed, they may be mild toxins that benefit by upregulation of defence systems, perhaps an example of hormesis (see "Slowing down Ageing from Within" on page 20). Antioxidant supplements have been marketed on the basis of limited scientific evidence and an overly simplistic view of how a plantrich diet works to prevent oxidative damage in the body.

Nevertheless, antioxidant therapies may have use. Increased free-radical damage in the brain accompanies neurodegenerative disease, and agents that can cross the blood-brain barrier to decrease such harm may be neuroprotective. Since increased oxidative damage accompanies diabetes, I suspect that antioxidants (in diet and perhaps as supplements) would be beneficial in delaying disease onset and modulating the severity of type 2 diabetes. Rigorous clinical trials are essential.

Reactive O₂ species are a ubiquitous part of human life. Like O₂, glucose, and iron, they offer both benefit and harm. Researchers are actively investigating the role of reactive O₂ species in the process of ageing and human disease, especially in the area of neurodegenerative disease where increased neuronal free-radical damage is so apparent.

In terms of disease prevention, high doses of single antioxidants are unlikely to be useful, and at the moment, all that we can conclusively say is that some populations would benefit from eating less fat and more fresh fruits and vegetables and taking a multivitamin/mineral supplement with modest amounts of the ingredients. Even Hippocrates, who knew nothing about antioxidants, came to the same conclusion using simple clinical observations and relying on his own common sense.

For more information contact Barry Halliwell at bchbh@nus.edu.sg

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