Oxygen

by Nick Lane

  In the meantime, one study of an innovative new cancer therapy does suggest that vitamin C can help kill tumours by exacerbating the effect of free radicals. The treatment is called photodynamic therapy, and was touched on in Chapter 6. The procedure uses light to activate a drug.

  Once activated, the drug transfers chemical energy onto oxygen, generating singlet oxygen and various free radicals, which attack the tumour.

  Research teams at the University of Iowa and in China have shown that giving high-dose vitamin C in conjunction with photodynamic therapy enhances the destructive effects of treatment on the tumour. If this turns out to be an important clinical effect (it is still too early to say), the tarnished reputation of the prophet Pauling may yet be restored.

  I have used the example of vitamin C to explore the wider function and behaviour of antioxidants. What have we learnt? The first conclusion is that vitamin C has a repetitive molecular action, constrained by its chemistry. It is not some kind of infinitely flexible superhero, capable of assuming any shape to protect us from evil. All molecular antioxidants are, of necessity, constrained within tight bounds by their chemistry. This does not prevent them from having a wide range of actions. Our second broad conclusion is that a single repetitive action can have many physiological

  Vitamin C and the Many Faces of an Antioxidant • 191

  roles besides an antioxidant effect. We have seen that vitamin C is a cofactor for at least eight enzymes, which affect completely different aspects of bodily function, from regular housekeeping tasks, such as collagen synthesis and fat metabolism, to survival measures, such as our response to stress (noradrenaline synthesis) and our perception of pain (activation of substance P). In fact, of all the actions of vitamin C, its antioxidant properties are probably the least well documented. This is equally true of many other reputed antioxidants.

  The most compelling evidence that vitamin C does behave as an antioxidant in the body comes from its rapid uptake by neutrophils, where it protects them against their own noxious anti-bacterial effusions.

  In this respect, it is worth noting that neutrophils only accumulate vitamin C when they are activated by bacteria. Such a sporadic response may reflect no more than the energetic futility of vacuuming up vitamin C

  when it is not needed, but might equally be a precaution against vitamin C toxicity. This brings us to the third broad conclusion that we can draw from vitamin C. The precise behaviour of an antioxidant depends on its surroundings. Whether vitamin C acts as an antioxidant, or a pro-oxidant, or somewhere in between, depends primarily on its interactions with other molecules. We have seen that vitamin C interacts with some free radicals directly, but also with iron, copper, vitamin E and glutathione. For vitamin C to have a beneficial antioxidant effect, each of these needs to be present in the right amount at the right place, and so each requires its own network of support molecules. If we take a bird’s-eye view, then each of these factors should really be considered antioxidants.

  Where do we draw the line? To appreciate the difficulty of defining an antioxidant, let us draw this chapter to a close with a quick look at the behaviour of activated neutrophils.

  Although neutrophils accumulate vitamin C to 100 times plasma levels, they do not absorb vitamin C itself, but only dehydroascorbate, the oxidized form of vitamin C. Neutrophils have protein pumps in their cell membranes that recognize dehydroascorbate and pump it into the cell.

  Once inside, the dehydroascorbate is useless until it is regenerated into vitamin C. In neutrophils, this step needs an enzyme called glutaredoxin, which takes electrons from glutathione to regenerate vitamin C. If the whole system is not to grind to a halt, the depleted glutathione needs to be regenerated in turn. Glutathione regeneration is achieved by an enzyme called glutathione reductase, using electrons that would otherwise be used to convert oxygen into water in the course of cellular respira-

  192 • PORTRAIT OF A PARADOX

  tion. This amounts to a long-odds gamble on life itself. The physiological balance of the neutrophil is shifted away from normal respiration — from what amounts to breathing — into an emergency holding pattern, which is dedicated to regenerating glutathione and thus vitamin C. In other words, activated neutrophils trade taking a breath for protection, in the hope that they will survive long enough to kill the bacteria.6

  Such heavy betting on the long odds begs the question why vitamin C? As a water-soluble vitamin, it accumulates within the cytosol of the cell. The ‘thin red line’ that holds bacteria at bay is not the inside of the neutrophil, but its surrounding cell membrane, made of fats immiscible with vitamin C. Even when bacteria have been ingested by neutrophils, they are still held in isolation inside the phagocytic vesicles formed from invaginations of the external cell membrane. The neutrophils pour their toxins into these vesicles (as well as the surrounding environment). If they are not to be killed by their own toxins, they must maintain the integrity of their external and internal membranes. If this membrane is damaged in the battle with bacteria, and loses its integrity, the neutrophil will die just as surely as we ourselves die when flayed of our skin. In fact, vitamin C is used to rally and, quite literally, revitalize the front-line forces.

  Lipid-soluble vitamin E is the foremost defender of the cell membrane. Vitamin E donates electrons directly to free radicals that can damage the membrane’s integrity and so neutralizes them, leaving behind its sacrificial corpse, the ␣-tocopheryl radical. Vitamin C breathes life back into this near-inert radical, resurrecting it as vitamin E. The reaction takes place without the aid of an enzyme, but its speed depends on the amount of vitamin C relative to vitamin E. The more vitamin C, the quicker the regeneration of vitamin E, hence the stockpiling of vitamin C by neutrophils. At the same time, however, high levels of vitamin C present a danger, especially in the presence of free radicals such as superoxide, which can release iron from proteins (Chapter 6). Vitamin C might change sides and start acting as a pro-oxidant. To stop this from happening, the main 6 Advocates of mega-dose vitamin C argue that regeneration of vitamin C by glutathione is a liability: it is slow, drains cellular energy and ultimately deepens the crisis. One rationale for taking mega-dose vitamin C when ill is to side-step this dangerous regeneration step. Such an approach might work outside cells, but cannot work inside cells, where the protection is most urgently needed. The problem is that most cells only recognize the oxidized form of vitamin C, dehydroascorbate. For mega-dose vitamin C to protect cells from within, it must first be oxidized in the blood, then taken up by the cell as dehydroascorbate, and finally regenerated to vitamin C using glutathione. No short cuts here.

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  agents provocateurs, iron and copper, must be stowed away. Battening down the hatches demands molecular sensors that detect any free iron or copper within the cell, and the capacity to lock away the excess in protein cages (ferritin and caeruloplasmin, respectively). If the storage capacity is limited, new protein cages may need to be manufactured, which in turn requires the transcription and translation of various genes. In all, some 350 genes are expressed by human neutrophils within 2 hours of activation, including the genes for ferritin and caeruloplasmin.

  Each link of these entwined chains is critical for the system as a whole to work. The fact that neutrophils protect themselves by accumulating vitamin C, but bacteria do not, hangs by a single thread: bacteria cannot detect dehydroascorbate in their surroundings or pump it into themselves. In neutrophils, the entire response is activated by the presence of dehydroascorbate in the surroundings. The more dehydroascorbate there is, the faster the pumps work. Indeed, neutrophils can be activated in the absence of bacteria by the simple expedient of adding a little dehydroascorbate. Bacteria, on the other hand, remain dormant, even when drowning in pools of dehydroascorbate. They have all the cellular machinery they need to regenerate vitamin C, vitamin E and glutathione, or to hide iron and copper, but are blind to the presence of dehydroascorbate. This sin
gle failure may cost them their life. If so, the neutrophils’ gamble on the long odds will have paid off.

  The most striking feature of this scenario is the way in which the whole metabolism of the neutrophil is re-routed in the presence of dehydroascorbate. All of these changes operating together contribute to the overall antioxidant response. We cannot simply define an antioxidant as a molecule with a particular type of action. ‘Seeing’ dehydroascorbate is an antioxidant response. Hiding iron is an antioxidant response. Regenerating glutathione is an antioxidant response. Even lowering the metabolic rate — holding the breath — is an antioxidant response. There is no hard and fast dividing line between factors normally described as antioxidants, such as vitamin C, and physiological adaptations not usually classed as antioxidant responses, such as a reduction in cellular respiration. To have any sense of how such large-scale webs of interactions work in organisms as a whole, we will need to step back from vitamin C. In the next chapter, then, we will take a wider look at how organisms deal with oxidative stress.

  C H A P T E R T E N

  The Antioxidant Machine

  A Hundred and One Ways of Living with Oxygen

  Governments work with tight definitions of words like

  ‘unemployed’, ‘literacy’, or ‘no more taxes!’. Opposition spokes-men and newspaper editors dispute the validity of their definitions. Words fly back and forth, all sound and fury, signifying little.

  Scientists are considered to be above this kind of thing. The terminology of science brooks no opposition: it is clearly defined and quantifiable, albeit unreadable. Scientists strive to render a definition into a mathematical symbol. Only when the term sits comfortably in an equation are we happy. Yet even within the perfect discipline of mathematics, precision is not always possible. The ‘fiddle factor’ is an ever-present bogey, which symbolizes the resistance of the world to petty classification.

  In biology, the trouble with definitions is far worse than in mathematics. Few biologists ever use the word ‘proof’ — it is far too exact. Doctors rarely use the word ‘cure’. Who knows, after all? Remission is a more comfortable word, if only because it means very little: “the illness seems to have gone away for now, for all I can see; I have no idea if it will come back”. Nature deftly sidesteps our clumsy swings at a definition. How does one define ‘life?’ Reproduction and some form of metabolism seem to be musts, but then is a virus alive? It has no metabolism of its own and so falls outside most definitions of life. If you can define life to encompass a virus, then how about the infectious prion, which is simply a protein?

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  How does one define ageing? A relentless decline in faculties ending in death? Or was that just a description? If we can’t define life, what on earth is death? If a prion is not alive, is it dead? If so, does that mean we can’t kill it?

  I’ve no intention of wallowing in this sea of semantics. Of course there are answers, albeit rarely simple. My concern now is to find a wider definition of the term ‘antioxidant’. We saw the potential for confusion in Chapter 9. The problem is one of precision: just how precise can we be in defining such a slippery concept?

  The original definition of ‘antioxidant’ came from chemistry. As befits a science full of symbols, it had a precise meaning. An antioxidant is an electron donor that prevents a substance from being oxidized (or stripped of electrons). The word came of age in food technology in the 1940s. Fatty foods, such as butter, go rancid if left in the air. Technically, they ‘peroxidize’. Peroxidation is a chain reaction started by oxygen free radicals, such as hydroxyl radicals, which attack lipids to get an electron.

  They may seize an electron and run, or become mired in the lipid like a rugby player who got the ball but couldn’t escape the scrum. Either way, the lipid loses an electron: it has become a free radical and so attacks its neighbours to get an electron back. Such chain reactions spread like wildfire through the densely packed lipids in butter. An antioxidant stops all this by ‘scavenging’ free radicals. It gives up electrons to stop the chain reactions from spreading. Antioxidants, such as BHA (butylated hydroxyanisole), are added routinely to manufactured foods.

  Such a precise definition is fine for chemistry or food technology but is not helpful in biology. In the presence of iron, we could just as easily define an electron donor as a pro-oxidant. Everything depends on context. In this chapter, then, I propose to explore the context and drop the precision — to put aside the reductionist approach and see how a broad synthesis works. We’ll therefore look at how whole organisms, whether single cells or multicellular creatures, avoid being oxidized. Rather than thinking only in terms of reactions, I’ll include traits such as morphology and behaviour.

  Antioxidant defences can be divided into five categories: avoidance (sheltering), antioxidant enzymes (prevention), free-radical scavengers (containment), repair mechanisms (first aid) and stress responses (entrench-ment). Some organisms, particularly those that hide from oxygen, rely on just one or two of these mechanisms, while others, including ourselves, draw on the entire armoury. We are, truly, antioxidant machines. To see

  196 • THE ANTIOXIDANT MACHINE

  how these defences work, we will examine a few instances of each mechanism in turn. Rather than an exhaustive analysis, I will highlight some of the insights that these mechanisms offer into our own physical and physiological makeup.

  The simplest form of protection against oxygen toxicity is to hide. Tiny bacteria have a wealth of possible hiding places. Some strictly anaerobic bacteria, which are killed by trace amounts of oxygen, even shelter within larger cells to escape the long reach of oxygen. An extreme example is the methane-producing bacteria that live in the rumen of cattle and sheep. Like a set of Russian dolls, they hide inside the symbiotic microbes that break down cellulose from grass, which in turn hide inside the rumen.

  The hind-guts of animals, from wood-eating termites to elephants, are comfortable hiding places for all manner of anaerobic microbes. Our own guts are inhabited by heaving colonies of so-called commensal bacteria, which are usually harmless or beneficial, but sometimes as noxious as their smell. The total population of gut bacteria is said to have a metabolic capacity equal to that of the liver. Together, the undigested organic matter and bacteria sponge up oxygen, rendering the large intestine virtually anoxic, with an oxygen concentration typically less than 0.1 per cent of atmospheric levels. Under these conditions, anaerobic bacteria such as Bacteroides outnumber their aerobic cousins a hundred-fold.

  On a much larger scale, free-living anaerobic bacteria can hide from oxygen by buffering the outside world. A good example is the sulphate-reducing bacteria, which produce hydrogen sulphide gas as a waste product (see Chapters 3 and 4). Hydrogen sulphide reacts with oxygen to regenerate sulphate, thus simultaneously replenishing the raw material of the sulphate-reducing bacteria, and depleting dissolved oxygen. The effect is to keep the outside world out and sustain an unchanging stagnant environment. Following their evolution, perhaps 2.7 billion years ago, sulphate-reducing bacteria dominated the deep ocean ecosystem for more than 2

  billion years. They are still to be found today in the Black Sea, and indeed in any stagnant, stinking sludge the world over, including our own guts.

  Their success in turning the world to their own advantage is comparable only to the cyanobacteria, which for 3.5 billion years have used sunlight to fill the world with oxygen. The antithesis is almost a biblical pitting of

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  the forces of darkness against the forces of light. Sulphate-reducing bacteria shun the grace of light and air for darkness and slime, the sulphurous, foul-smelling pits of the underworld. Yet their noxious waste helps to maintain ecological diversity. Just as the opposition of light and darkness is central to many religions, with most of us inhabiting the moral middle ground, so too the opposite poles of the living world create a spectrum of conditions that fost
ers the variety of life.

  Many single-celled organisms do not hide from oxygen, but are quick to move away from places where the oxygen levels are too high, exploit-ing the spectrum of conditions maintained by microbes like sulphate-reducing bacteria. We saw in Chapter 3 that free-living ciliates actively swim away from oxygen. This is actually quite a complex response. To do this, they need sensors that can detect the oxygen level in their surroundings. The information gleaned must then be coupled to the beating of their cilia, to drive them away from oxygen. The sensors themselves are proteins containing haem, similar to haemoglobin. Haem proteins are well suited to this purpose, because their physical properties change in the presence of oxygen, as in the colour change of haemoglobin from blue to red. Representatives of all three of the great domains of life use haem proteins as oxygen sensors, implying that LUCA herself (the Last Universal Common Ancestor, see Chapter 8) might have used them for this purpose.

  When used in this way, haem proteins are acting as antioxidants: they play an integral part in maintaining external oxygen levels within congenial limits. Even when no movement is involved, as in the root nodules of leguminous plants (Chapter 8), haem proteins act as antioxidants by binding to excess oxygen and releasing it only slowly, maintaining a constant and low concentration of oxygen in the immediate environment.

  Some microbes tolerate oxygen in their surroundings by physically screening themselves from the worst atrocities. The simplest screen is a pile of dead cells. In the same way, the riddled body of a dead comrade may protect a soldier from the bullets of an execution squad. Anaerobic cells living in stromatolites (Chapter 3) have relied heavily on this device, sheltering behind successive layers of dead cells. They have been doing this for 3.5 billion years.

  A more sophisticated approach to the same problem is to secrete a layer of mucus and lurk within it. All free-living aerobic microbes live inside a mucus capsule as habitually as a crab lives in its shell. Rather