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Oxygen

Page 29

by Nick Lane


  of bilirubin — help to protect against the oxidative stress of traumatic injury, when blood-borne antioxidants may not be able to get through.

  In many cases, the balance of risks and benefits with chain-breaking antioxidants is far from clear. Take uric acid, for example — a potent antioxidant, certainly, but one that at high levels causes the painful inflammation of gout as it crystallizes in the joints. Uric acid is sometimes considered to be a risk factor for cardiovascular disease as well, because the people with the highest levels of uric acid in their blood stream are also at the greatest risk of a heart attack. In reality, this simple association may be quite misleading. Those at greatest risk of cardiovascular disease tend to have the lowest intake of dietary antioxidants. What could be more sensible for the body than to step up levels of endogenous antioxidants? The worse the disease, the more uric acid is needed to combat it; hence the association between the severity of disease and plasma uric acid levels. This line of argument is speculative, but highlights the danger of bare associations. In this case, treatments aimed at lowering plasma uric acid might easily prove detrimental, unless coupled with a better diet; and of course, if we change diet, we can hardly come to any sensible conclusions about the true role of uric acid. There are few straight relationships between antioxidants and health.

  Two small sulphur-containing compounds, glutathione and thioredoxin, have now cropped up several times in this chapter and the last. Both donate electrons, either to regenerate antioxidants such as vitamin C, or to detoxify hydrogen peroxide and organic peroxides directly. Far from being bit players, these sulphur compounds are the gatekeepers between genes and diet, between health and disease. It is time they took centre stage, for they are responsible for orchestrating the last two of the five antioxidant mechanisms that I outlined at the start of this chapter —

  repair mechanisms and stress responses. Sulphur is now seen as the most important counterbalance to oxygen inside individual cells, as well as in the wider ecosystem.

  Here I must beg indulgence for a single paragraph of unmitigated biochemistry, a diffident request after the geneticist Steve Jones’s dismissal of biochemistry as a subject unfit for popularization. However, the concept is not difficult, and is critical to understanding how sulphur can act as a molecular switch that tells the cell when it is ill. I’m picking out a single

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  example of how such processes work; a baffling array of other mechanisms are superimposed over this one, eroding or strengthening the signal.

  Nonetheless, the behaviour of sulphur is as exemplary as can be, and is without doubt one of the most important areas in the hot science of biological signalling.

  Sulphur, bound to hydrogen (–SH), is a constituent of just one of the 20 amino acids — a nondescript little molecule with 14 atoms called cysteine. The unique sulphur group in cysteine, the –SH group, is called a thiol group. Thiols are delicate structures, easily susceptible to oxidation. I imagine them waving their yellow sulphur heads gently, delicate as seed-ing dandelions. When thiol groups are oxidized, one of two things can happen. First, if the hydrogen atom (proton plus electron) is extracted, the sulphur stubs of adjacent thiols may bind together to form sulphur-to-sulphur bonds, known as disulphide bridges. In the presence of oxygen, disulphide bridges are more robust than unoxidized thiols and have long been recognized as important structural features of extracellular proteins, helping to stabilize their three-dimensional shapes. The second possible fate for delicate thiols, formidably named S-nitrosylation, began to emerge in the late 1990s under the searching intellect of Jonathan Stamler, latest in a distinguished line of free-radical biochemists at Duke University.

  Stamler and his colleagues argue that oxidative stress increases the production of another free radical, nitric oxide (NO•). Nitric oxide itself is sluggishly reactive with most substances, but it acts synergistically with other free radicals to oxidize thiols. In this case, the product is not a disulphide bridge, but an S-nitrosothiol (–SNO), which is almost as stable.

  The formation of either a disulphide bridge or an S-nitrosothiol modifies the structure of the protein in a reversible way. It is possible to regenerate the delicate thiol group using hydrogen atoms from glutathione or thioredoxin.

  For proteins, structure corresponds to activity. If thiols influence the structure of proteins, then they also affect their activity. In other words, the oxidation state of thiols can act as a molecular switch for thiol-containing proteins, turning their activity on and off. The list of proteins with sensitive thiol groups is growing all the time, and is now known to include some of the most important transcription factors (proteins that bind to DNA and stimulate the transcription of genes to produce new proteins). Whether these factors bind to DNA or not, even whether they journey into the nucleus at all, depends on the status of their thiol groups.

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  The inside of a healthy cell is full of delicately waving thiols. They are kept in the unoxidized state by the continuous policing of glutathione and thioredoxin. Any thiols oxidized by ‘mistake’ are reconverted back into the pristine state. Glutathione and thioredoxin are regenerated by tapping the energy of cellular respiration, as we saw in Chapter 9 while discussing vitamin C. Under normal conditions, this is not much of a drain on the resources of the cell. The situation is reversed, however, when the cell is under oxidative stress.

  Picture a cell under oxidative stress. The problem might be too much oxygen, or perhaps an infection or a disease. The end result is the same: there are free radicals everywhere. Chain-breaking antioxidants such as vitamin C are used up quickly. They are regenerated by glutathione, but not without some loss. As antioxidants are depleted, the same number of free radicals causes more damage. Thiol groups in proteins are now being oxidized. Some are patched up by glutathione and thioredoxin, but the balance is shifting. This is a war zone. The defenders cannot rebuild bombed bridges every night for ever. Now half the proteins in the cell have oxidized thiols. Some of the proteins are switched off by the oxidized thiols, shut down to await the end of the war. Others are switched on. A guerrilla militia is taking charge of the nucleus, the last stronghold.

  The guerrillas are mostly transcription factors. Inside the nucleus they bind to DNA and stimulate the production of new proteins. But which new proteins? This is no random selection. The cell has a serious decision to make: entrench for battle or commit suicide (apoptosis) for the good of the body as a whole. The decision depends on the odds of success, in particular on the number and state of the transcription factors that made it safely to the nucleus. At the molecular level, Nietzsche’s aphorism holds sway — what doesn’t kill you makes you stronger.

  If a cell opts to slug it out rather than take an honourable exit, the kinds of defences it draws on are qualitatively similar in all living things, from E. coli to humans. At present, the system in E. coli is better understood, in part because bacterial genes are organized into operating units called operons. This makes it easier to identify exactly which genes are involved.

  In E. coli, two main transcription factors are activated by the oxidation of sulphur groups. One is a thiol-containing protein known, in the opaque shorthand of molecular biology, as OxyR, and the other is the protein SoxRS, which contains sulphur in the form of an iron–sulphur cluster, (this acts in much the same way for our purposes here). When oxidized,

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  each of these transcription factors controls the transcription of a dozen or more genes, the products of which strengthen the antioxidant defences of the cell.

  In people, the list of transcription factors whose activity is controlled by the oxidation of thiol groups is growing all the time, and includes NF␬B (pronounced NF-kappa B), Nrf-2, AP-1 and P53. For our purposes, the two most important are NF␬B and Nrf-2. NF␬B marshals the ‘stress’

  response by activating a mixture of offensive (inflammatory) a
nd defensive (antioxidant) genes. Nrf-2 has a purely defensive (antioxidant) role, actually switching off the offensive inflammatory genes. Both NF␬B and Nrf-2 therefore strengthen the cell, but otherwise their effects are opposing. They act like two generals in a council of war, one of whom advocates all-out war, the other appeasement. The outcome depends on how successful they are at persuading the rest of the council to accept their view.

  For transcription factors, this comes down to numbers on the ground. If 1000 activated NF␬B proteins reach the nucleus but only 100 activated Nrf-2 proteins, the cell will go to war, launching an inflammatory attack on the invader and bolstering its own defences. If Nrf-2 holds sway in the nucleus, then the cell effectively barricades itself in bunker and waits out the war. In either case, the extra defences provide immediate benefits, but they also confer resistance to future attacks, regardless of their exact nature. Forewarned is forearmed, whatever the threat may turn out to be.

  What are the products of these defensive genes? Some have not yet been identified, others are familiar to us already. As we might expect by now, the levels of SOD, catalase and other antioxidant enzymes are stepped up; new metabolic proteins divert cellular respiration towards the regeneration of glutathione and thioredoxin; and extra iron-sensors and iron-binding proteins are made that detect and store free iron. A number of so-called ‘stress’ proteins are also produced, which busy themselves with clearing up the mess and dusting down the salvageable, like the emergency services picking over rubble in the aftermath of a bombing raid. Irredeemably broken proteins are earmarked for destruction and recycling. Less seriously damaged proteins, bruised but unbroken, are refolded and repackaged with the aid of proteins known as molecular chaperones. Other proteins set to work on DNA, cutting out oxidized fragments, inserting replacements and re-joining breaks in the DNA strands.

  Taken together, these actions restore the cell to health, but (with the exception of antioxidant enzymes) have little effect on its longer-term capacity to withstand a similar assault. There are some proteins that can

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  strengthen a cell’s resistance to future assault, however. Two of the most powerful are metallothionein and one of the versions of haem oxygenase

  — the stress protein HO-1. These proteins bolster the resistance of cells to a whole range of insults, from heavy-metal contamination to radiation poisoning and infection — conditions linked by the common thread of oxidative stress. They are more powerful protectors than any dietary antioxidant, although they achieve this by imposing what amounts to a curfew, in which the normal daily life of the cell is held in check.

  The firmness of their rearguard action unveils a final irony: antioxidant supplements, which behave more like a scattered vanguard on a looting trip, have the potential to exacerbate some diseases. The reason is as follows. The signal to produce metallothionein and haem oxygenase is the oxidation of thiols. Thiols become oxidized when the supply of antioxidants is depleted. Raising our intake of antioxidants can therefore suppress the signal by sparing thiols, thereby robbing the cell of its most powerful allies. This is not just an idle fancy. Roberto Motterlini and Roberta Foresti, at the Northwick Park Institute for Medical Research, London, have shown that adding antioxidants to cells under oxidative stress (especially thiol-regenerating antioxidants, like N-acetylcysteine) does indeed prevent the cell from making new haem oxygenase. This renders the cells more vulnerable to injury. In the body, suppression or loss of haem oxygenase activity can have some dire consequences.

  Just how important such a loss might be is illustrated by an exceptional case study reported in 1999 by a team at Kanazawa University in Japan: the first known case of haem oxygenase deficiency in humans. An unfortunate six-year-old boy suffered from severe growth retardation, abnormal blood coagulation, haemolytic anaemia and serious renal injury.

  All this misery stemmed from the loss of a single enzyme, a stress protein supposedly produced only sporadically in times of oxidative stress. Similar problems have been reported in animals. Kenneth Poss and Susumu Tonegawa, at the Massachusetts Institute of Technology, have shown that

  ‘knock-out’ mice deficient in haem oxygenase succumb to a serious inflammatory condition, which resembles haemochromatosis in people (the iron-overload syndrome discussed in Chapter 9). The mice lacking haem oxygenase suffer from serious iron overloading in tissues and organs, which leads to pathological enlargement of the spleen, liver injury and fibrosis, various immune disorders, weight loss, decreased mobility and premature mortality. Mice without haem oxygenase also have shrunken testes, 25 per cent smaller than similarly sized littermates, and a lack of

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  libido, both of which are characteristic of hereditary haemochromatosis in people.

  For the effects of haem oxygenase deficiency to be so profound we may well wonder whether some degree of oxidative stress is in fact the norm. If so, then haem oxygenase levels presumably change continuously as part of a flexible and self-regulating feedback mechanism. We shall see in Chapter 15 that a continuously high haem oxygenase activity probably does have beneficial effects on our health in old age.

  We have come a long way from vitamin C. If nothing else, I hope that this chapter has exploded the notion of antioxidants as dietary elements that will help us live forever. Our whole body is an antioxidant machine, from the physical structure of individual cells to the physique of a human being. This perspective is obscured by our deeply seated, almost childlike, yearning to allocate purpose, which is seldom echoed in biology. We want to say a car is ‘for’ driving, a business is ‘for’ making money, life ‘for’

  living. Similarly, mitochondria are ‘for’ producing energy, haemoglobin is ‘for’ delivering oxygen and vitamin C ‘for’ protecting against free radicals. I compared the skin to a layer of dead cells in a stromatolite.

  Certainly it is. We cannot breathe through our skin like amphibians. But the skin is equally a barrier that prevents evaporation and infection, and an important criterion of beauty. The point is that few things serve a single purpose, least of all in biology, and we must fight our instinct to impose one. Apart from anything else, this means we must resist the scientific urge to apply tight — and therefore often singular — definitions.

  In Chapter 9, we saw that vitamin C is used in diverse ways, united only by the same molecular action. This also applies to SOD, catalase or haem oxygenase. At the molecular level, their actions are always identical.

  The effects, however, are diverse and may serve quite different purposes.

  Take SOD. Its action is simple: to remove superoxide radicals. But is this purely and simply an antioxidant action, or is it also a signal? If the formation of superoxide radicals outstrips their removal by SOD, some of the extra free radicals will oxidize the thiol groups in proteins, sending transcription factors scurrying to the nucleus. In the nucleus, these transcription factors bind to DNA and stimulate the production of new proteins, which help restore the cell to health. In other words, the cell adapts to a small change in circumstances, such as a slight increase in oxidative

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  stress, by a subtle change in its repertoire of proteins. Are we really helping if we block this pathway by swamping it in an excess of SOD or other antioxidants? Who knows. We have noted one reason why dabbling in this way might be detrimental: haem oxygenase is produced in response to thiol oxidation. If it is true that the levels of haem oxygenase flux continuously in response to small changes in oxidative stress, then smother-ing this subtle interplay may dull our response to sudden challenges such as infection. Perhaps this is why it has been so hard to confirm any protective effects of vitamin C against the common cold, as claimed by Pauling. The benefits of vitamin C may be offset by the disadvantages of suppressing the synthesis of stress proteins like haem oxygenase.

  Despite these uncertainties, we cannot dispute that eati
ng fruit and vegetables is good for us. If this has anything to do with antioxidants, we might conclude that our sophisticated antioxidant machine falls short of perfection. Presumably, the extra antioxidants mop up free radicals that would otherwise damage DNA and proteins, undermining the vitality of cells and ultimately the whole body. On the other hand, it is quite possible that the benefits of fruit also relate to other factors, such as mild doses of toxins that stimulate the production of stress proteins like haem oxygenase. These toxins are designed to prevent fruit from being eaten before it is properly ripe, or by the wrong sort of vegetarian. Perhaps it is the balance of antioxidants and mild toxins that confers the benefits of fruit. Certainly the benefits of fruit and vegetables have never been reproduced simply by taking antioxidant supplements.

  Half a century ago, the pioneer of free-radical biology Rebeca Gerschman questioned whether a continuous small ‘slipping’ in antioxidant defences might contribute to aging and death. This idea is still the basis of the free-radical theory of ageing. I hope this chapter has shown that our antioxidant defences are far more complex than Gerschman, or for that matter Pauling, could ever have imagined. These are the fruits of molecular biology in the past few years. But we are left on the horns of a dilemma. Can ageing be slowed down by blocking free-radical slippage, or is some degree of slippage necessary for marshalling our resistance to stress? If chain-breaking antioxidants are relatively unimportant compared with the stress response, might we be able to slow down ageing by manipulating the stress response directly? Armed with our new understanding of antioxidants, we will look at the balance of evidence in the next four chapters.

  C H A P T E R E L E V E N

  Sex and the Art of Bodily

  Maintenance

  Trade-offs in the Evolution of Ageing

  Gilgamesh, King of Uruk,sets out on a quest for immortality, the pain of loss in his heart. Passing through twelve leagues of darkness, where there is no light from the rising of the sun to the setting of the sun, he seeks Utnapishtim the Faraway, who knows the secret of everlasting life. Utnapishtim tells Gilgamesh that his quest is like a search for the wind, but then reveals the mystery of the gods: a plant that grows under the water, which restores lost youth to a man. By its virtue, a man may win back all his former strength. Its name is “The Old Men Are Young Again”. Gilgamesh finds the plant, only to have it snatched away by a serpent before he can eat of it. The serpent sloughs away its skin and disappears into a spring of water. Gilgamesh weeps and returns to Uruk empty-handed. He has his story engraved on clay tablets.

 

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