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. ,. t,p7,enum Press Publishing In Press International Conference on Male-Mediated Developmental Toxicity September 16-19', 1992 Pittsburgh, Pennsylvani „Ja ~- ~ - ,c r- ANTIOXIDANT PREVENTION OF BIRTH DEFECTS AND CANCER C.G. Fraga, B. N: Ames, P. Motchnih, A MK Shigenaga, and T.M. Hagen Division of Biochemistry and Molecular Biology University of California Berkeley, California 94720 ABSTRACT Metabolism, like other aspects of life, involves trade-offs. Damage to DNA and protein by oxidant by-products of normal metabolism is massive. We argue that this damage (the same as that produced by radiation) is a major contributor to aging, to degenerative diseases of aging, such as cancer, heart disease, cataracts, and brain dysfunetion, and to DNA damage in sperm. Antioxidant defenses against this damage include vitamins C and E and carotenoids, the main sources of which are dietary fruits and vegetables. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake (about four or five portions per day); it also markedly increases the risk of heart disease and cataracts. We will argue also that this deficiency is a major cause of childhood cancer and birth defects. We have shown that oxidative damage to sperm DNA is increased markedly when dietary ascorbate is insufficient to keep seminal fluid ascorbate to an adequate level. A sizeable percentage of the U.S. population, including most stnokers, consumes inadequate levels of dietary ascorbate. The 1000 ppm of NOx in cigarette smoke causes a general depletion in antioxidant levels in smokers. Smokers need to eat two to three times as much ascorbate as non-smokers in order to maintain the same blood levels of ascorbate, but they rarely do. We will review the data showing that smoking fathers increase the risk of birth defects and childhood cancer in their offspring. Since only 9% of the U.S. population eau the recommended five portions of fruits and vegetables per day, widespread changes in diet could have major impact on human health. AGING AND OXIDATION Evolutionary biologists have argued that aging is inevitable because of several tradeoffs (1-4). One tradeoff is that a considerable ptoportion of an animal's resources is devoted to reproduction at a cost to maintenance, which means that the maintenance of somadc tissues is less than that required for indefinite survival Metabolism has costs: oxidant by-products of normal energy metabolism extensively damage DNA, proteins, and other molecules in the cell, and this datttage .atxumulates with age (5-7). A second tradeoff is that nature selects for many genes that have imtnediate survival value, but that may have long teim deleterious consequences. The oxidative burst from phagocytic cells, for example, protects against death from bacterial and viral infections, but contributes to DNA damage, mutation, and cancer (8, 9). Degenerative Diseases of Aging The degenerative diseases associated with aging include cancer, heart disease, brain dysfunction, and cataracts. The degeneration of somatic cells during aging appears, in good part, to contribute to these diseases. The relationship between cancer and age in various mammalian species illustrates this point. Cancer increases with about the fifth power of age in both short-lived species, such as rats, and in long-lived species, such as humans. One important factor in longevity appears to be basal metabolic rate, which is about seven times higher in a rat than a human and which could markedly affect the level of endogneous oxidants and other mutagens produced as by-products of metabolism. The level of oxidative DNA damage appears to be rfiughIy related to metabolic rate in at least four mammalian species (10). 1

:- ~'Jr"sa* Oxidation and Damage to DNA, Protein, and Lipids Oxidadve damage to cells has been postulated to be the most significant endogenous damage leading to aging (11). Superoxide, hydrogen peroxide, and hydroxyl radicals. which are the mutagens produced by radiation, are also by-products of normal metabolism (12, 13). Lipid peroxidation gives rise to mutagenic lipid epoxides, lipid hydroperoxides, lipid alkoxy and hydroperoxy radicals, and enals (a, (3-unsaturated aldehydes) (14). Singlet oxygen, a high energy and mutagenic fonn of oxygen, can be produced by transfer of energy from light, the respintory burst from neutrophils, or lipid peroxidation (15)_ Animals have numerous antioxidant defenses, but since these defenses are not perfect, some DNA is oxidized. Oxidatively damaged DNA is repaired by enzymes that excise the lesions, which are then excreted in the urine. We have developed methods to assay several of these excised damaged bases in the urine of rodents and humans (10, 16). We estimate that the number of oxidative hits per celll per day is about 100,000 in the rat and about 10,000 in the human. DNA repairensywes efficiently remove most, but not all, the lesions formed (5). Oxidative lesions in DNA accumulate with age, so that by the time a rat is old (2 years) it has about two million DNA lesions per cell, which is abourtwice that in a young rat. Mutations also accumulate with age. For example, the somatic mutation frequency in human lymphocytes is about nine times greater in elderly people than in neonates (17). Mitochondrial DNA (mtDNA) from rat liver has more than ten times the level of oxidative DNA damage than does nuclear DNA from the same tissue (18). This increase may be due to a lack of mtDNA repair enzymes, lack of histones protecting mtDNA, and the proximity of mtDNA to oxidants generated during oxidative phosphorylation. The cell defends itself against this high rate of damage by a constant turnover of mitochondria, thus presumably removing those damaged mitochondria that produce increased oxidants. Despiie this turnover, oxidative lesions appear to accumulate with age in mitochondrial DNA at a higher rate than in nuclear DNA (Hagen and Ames, unpublished). Oxidative damage could also account for the higher level of mutations in mtDNA that accumulate with age (19,20) : Endogenous oxidants also damage proteins. Stadunan and his colleagues (7. 21. 22)) have shown that the proteolytic enzymes that hydrolyze oxidized proteins are not sufficient to prevent an age-associated accumulation of oxidized proteins. Imtwo human diseases associated with premature aging, Werner's syndrome and progeria, oxidized proteins accumulate at a much higher ratethan is normal (7). Fluorescent age pigments. which are thought to be due in part to cross-links between protein and lipid peroxidation products, also accumulate with age (23); SOURCES AND EFFECTS OF OXIDANTS Four endogenous sources appear to account for most of the oxidants produced by cells: 1) As a consequence of normal aerobic respiration, mitochondria consume molecular oxygen, reducing it by sequential steps to produce H20. Inevitable by-products of this process are superoxide, hydrogen peroxide, and hydroxyl radical. About 1012 oxygen molecules are processed by each rat cell daily, and the leakage of partially teduced oxygen molecules is about 296, yielding about 2x1010 superoxide and hydrogen peroxide molecules per cell per day (24). 2) Phagocytic cells destroy bacteria or virus-infected cells with an oxidauve burst of NO, O2-, H202, and "OCI. Chronic infection by viruses, bacteria, or parasites, results in a chronic phagocytic activity and consequent chronic inflammation, which is a major risk factor for cancer. Chronic infections are particularly prevalent in third world countries (see below). 3) Cytochrome P450 enzymes in animals constitute one of the primary defense systems against natural toxic chemicals from plants, the majpr source of dietary toxins (25). The induction of these enzymes, prevent acute toxic effects from foreign chemicals, but also results in oxidant by-products that damage DNA [Park, J.-Y. K. and Ames, B.N., unpublished). 4) Peroxisomes, which are organelles responsible for degrading fatty acids and other molecules, produce as a byproduct hydrogen peroxide, which is then degraded by catalase. Evidence suggests that, under certain conditions, some of the peroxide escapes degradation, resulting in its release into other compartments of the cell and in increased oxidative DNA damage (26). Three exogenous sources may significantly increase the large endogenous oxidant load. 1) The NOx in cigarette smoke (about 1000 ppm) causes oxidation of N macromolecules (27-30), and depletes antioxidant levels (31-33), it is therefore likely to (~ contribute significantly to the pathology of smoking. Smoking is a risk factor for heart ~ disease as well as a wide variety of cancers in addition to lung cancer (34-37). 2) Normal ~ diets contain plant food with large amounts of natural phenolic compounds, such as chlorogenic and caffeic acid, that can gtnerale oxidants by redox cycling (25, 38) although w it is not yet clear whether the resulting increment to the oxidant load is significant. 3) Go Iron (and copper) salts promote the generation of oxidizing radicals from peroxides ~ ~ ~ 2

(Fenton chemistry). It has been argued that too much dietaryropper or iron, particularly hetne iron (which is high in tneat), is a risk factor for heart disease and cancer in normal men (3942). Men who absorb significantly more than normal amounts of dietary iron (hemochromatosis disease),are at high risk for both cancer and heart disease (39). Chronic Infection, InAammationand Cancer Leucocytes and other phagocytic cells combat bacteria, parasites, and virus-infected cells by destroying them with powerful oxidants: NO, 02 , H202 and 'OCI. These oxidants protect humans from immediate death from infection, but cause oxidative damage to DNA and also stimulate ceD divisioa; therefore they eontribute to mutation and cancer. Asbestos pathology also appears to be due to chronic inflammation (43. 44). Antioxidants appear to inhibit some of the pathology of chronic iutflamtttation (4349). Cluonic infections contribute to about one-third of the world's cancer, mainly in poorer countria. Hepatitis B and C viruses infect about 500 million people, mainly in Asia and Afiica, and are a major cause of hepatocellular carcinoma (S0-S2). About two million people die prematurely every year from hepatitis. Hepatitis B was ftrs' t established as a cause of liver cancer in Taiwan, which has now vaccinated all children against the virus. Africa and China have not been able to afford widespread vaccination. Since there are no known cures for these viral infections, agents that inhibit inflammation may be the most effective treatments. It remains to be seen whether antioxidant vitamins, such as vitamin C, and anti-inflammatory drugs, such as aspirin, are indeed successful in inhibiting inflammation and cost-effective as well. Helicobacter pylori bacteria, which infect the stomachs of roughly one-third of the world population, appear to be the major cause of stomach cancer, ulcers, and gastritis, (47. 48, S3 SS). In wealthy countries the disease is usually asymptomatic, which indicates that the inflammation is at least partially suppressed, possibly by adequate levels of dietary andoxidants (56). Specific antibiotic treatments usually eliminate the bacteria. Another major chronic infection is schistosomiasis, which is caused by a parasitic worm that is widespread in China and Egypt. The Chinese worm lays its eggs in thetolon, producing inflammation that often leads to colon cancer (57) . The Egyptian worm lays eggs in the bladder, promoting bladder cancer (58). Opisthorchis viverrini; a liver fluke, infects millions of people in Thailgnd and Malaysia. The flukes lodge in bile ducts and increase the risk of cholangiocarcinoma (59). Chlonorchis sinensis infections in millions of Chinese increase their risk for biliary tract cancer (60). OXIDANT STRESS, BIRTH DEFECTS AND CHILDHOOD CANCER Oxidation and Sperm Damage Oxidative lesions in sperm DNA are increased 250% when levels of dietary ascorbate are insufficient to keep seminal fluid ascorbate to an adequate level (61) (Figures 1,2). A sizable percentage of the U.S. population ingest inadequate levels of dietary ascorbate, particularly single males, the poor, and staokers (62). Manied males have about a 12-year longer life ezpectancy than do single males, about eight years of which appear to be due to reduced heart disease and cancer (63). This increase may be due in part to the beneficial influence of wives on promoting good nutrition and mimtmizing their husbands' satokiag and drinking. The oxidants in cigarette smoke deplete the antioxidants itt plasma. Smokers must eattwo to three times mots ascorbate than noon-smokers to achieve the same level of ascorbate in blood (31-33)., but they rarely do. In a comparison of sperm from smokers and nonsmokers Viczian (64) found that the number of sperm and the percent of mobile sperm decrease significantly in smokers, and this decrease is dependent on the dose and duration of smoking. The percentage of pathological spetm increases significantly in smokers and is also dependent on the dose and duration of smoking (64, 6S).paternal smoking, in particular, increases the risk of birth defects and childhood cancer in offspring (see below), and paternal age exacerbates this risk (66) (see below). Due to the high division rate of sperm compared to eggs, the germ line mutation rate in the father greatly exceeds that of the mother. Thus, nutritionally inadequate diets and smoking of fathers result not only in damage to their own DNA but to the DNA of their sperm, an effect that can reverberate down future generations. Parental Smoking and Mutations to the Germline "The much larger number of cell divisions between zygote and sperm than between zygote and egg, the increased age of fathers of children with new dominant mutations, and the greater evolution rate of pseudogenes on the Y chromosome than on autosomes all point to a much higher [germline] mutation rata in males than in females, as first pointed out by Haldane from the study of X-linked hemophilia" (66). Haldat:e (67) estimated the male germline mutation rate to be abouC 10 times that of the female. 3

Dietaty and Semen Ascorbic Acid Content and oxogdG in Sperm DNA 9ue Goe t>rptaioo Mxginal Rzp~ Ascorbau Intake (mg/d) 25 0 5 10 or 20 60 or 250 Diauy Penod (d) 7- 14 32 28 28 Semen 399 t55 203t~72' 115*25' 422t100 Asccrbaa (µ M) 4Qoao8dG 10 0 198 ' 248 ' 159 • a.r..eariy ddrRRN IlYO6SWhC Yl/`C.11 P 4 Om. Figure 1 lteiat= of seminal tluid ascabme to oaidauive DNA dunage in sperm (61). ixi.uus~.r~ao.Wasus.r.ao.r.r~ < 60} o f1.2 LC O? 40 • 2010 oo°g° ° ° qo 0 0 0 200 400 600 800 Seminal Fluid AA ()iM) Figure 2. The data in (61) as reploaed by P. Motchnik. Maternal Smoking and Birth Defects Maternal smoking causes many problems, such as intrauterine growth retardation, leading to spontaneous abortion, premature birth, and low infant birthweight But numerous eptdetnioiogical studies over several tkcades in various countries have failed to consistently find a link between maternal cigarette smoking and genetic (iamage to the fetus. These studies, however, have not examined the following critical factor in determining the link between maternal smoking and birth defects: since a fetus develops from eggs that were fotmed when the tmother.ras in tutro. it is the smoking status of the ;randalother at the time that she waspc+egnant with the mother that is probably the »wst important factor: Khoury eY cl. (68. 69) tiote a dose-response relatioo between the daily amount of maternal smoking and the risk of cleft lip/palate in offspring. They do not control for paternal smoldng, and it is possible that smoking mothers art likely to have smoking mates; thus, the apparenrmatetnal effect may really be a paternal effect Paternal Smoking and Birth Defects We review below those studies that found an effect of paternal smoking and birth defects and childhood tumors. Most studies on smoking and birth defects or childhood tumors looked at maternal smoking. Comstock and Lundin (70) found that neonatal death rate among infants of smoking fathers who smoked was 17.2 per 1000 live births compared to 11.9 (adjusted for sex of child and education of father) among infants of nonsmoking fathers and 26.5 among infants of parents who both smoked. Koo et al. (71) found that wives of smoking fathers have more miscarriages and abortions than those of tton-smoldng fathers. Heary et a1. (72) observed an associadotl' between paternal smoking and increased neural tube defects (4/8 cases vs. 1/17 controls) in offspring. 4

Mau and Netter (73) studied the smoking habits of fathers of 5200 newborns. The rate of major malformations among nonsmoking fathers was 0.8% and among smoking fathers 2.1%. Maternal smoking had no influence on the rate of birth defects. The most striking increase in birth defects concerned major facial clefts. 0.1% of infants of nonsmoking fathers had facial clefts, compared to 0.5% of fathers who smoked 1-10 cigarettes per day and 0.7% of fathers who smoked more than 10 cigarettes per day. There was also an increase in perinaul mortality (unrelated to major birth defects) if the father smoked more than 10 cigarettes per day, even if the mother did not smoke (4.3% compared to 2.896). (By facial cleft the author probably meant cleft lip/palate rather than cleft of the total face, which is a very rare malformation.) Savitz et al. (74) analyzed single live births among 14,685 Kaiser members who pasticipued in ghe child health studies. Father's cigarette smoking was more common among children with cleft lip (with or without cleft palate), hydtvicephalus, ventricular septal defect, and urethral stenosis. However, inverse associations between paternal smoking and birth defects were more common than positive associations. The authors specifically' mention genetically altered sperm as a possible cause of birth defects in infants of smoking fatltetz. They note that several of the anomalies associated with infants of older fathers were also increased among fathers who smoked. The concordance in defects associated with both advanced paternal age and paternal smoking was especially notable for ventricular septal defects and hydrocephalus. Schmid (75) in discussing the Mau and Netter study attributed the effect of paternal smoking on birth defects to passive smoke. However, passive smoking is not a probable explanation for the link between paternal smoking and birth defects because the amount of smoke that reaches the embryo is insignificant compared to the amount that reaches the embryo when the mother herself smokes, and maternal smoking does not appear to significantly inctease the risk of birth defects. Schmidt (76) made an argument similar to ours concerning smoking and genetic damage: "Tobacco smoke contains numerous mutagenic substances. They reach the male gonads via the blood. They show their mutagenic action here openly much more stronger than on egg-cells because the spetzrtatogenesis continues over the whole male reproductive period whereas the formation of eggs is already completed in the fetal phase." Seidman (77) provides a table on the incidence of major and minor congenital malformations by paternal and maternal smoking levels. There was a nonsignificant increase in the incidence of major and minor malformations in the offspring of smoking fathers who mate with nonsmoking mothers. This author does not break down the malformations by specific defect. Smoking and Childhood Tumors Gold et al. (78) looked at maternaL smoking but not paternal smoking. Maternal smoking did not seem to influence the risk of brain tumors in children. Grufferman et al. (79) found that paternal smoking, but not maternal smoking, increased the risk of rhabdomyosarcoma in childhood (relative risk of 3.9). RMS is the sixth most common childhood cancer, with an annual incidence of about 4 cases per million children and a peak incidence at about ages 3-4. The authors suggest that a direct carcinogenic effect of paternal cigarette smoke may be introducedin a prezygotic manner. Grufferman et al. (80)' stated that "In our recent study of childhood rhabdomyosarcoma (RMS) we found an increase in the risk of RMS among children whose fathers smoked cigarettes. However, there was no association between RMS and mothers' smoking. We hypothesize that differential germ cell damage from cigarette smoking underlies our observations and that this risk of germ cell damage from cigarette smoking and from other environmental exposures is gr eater for men than for women. The increased susceptibility for male germ cells may be due to the number and timing of tneiotic and mitotic cell divisions. In males, germ cells undergo large numbers of ineiooic and mitotic divisions throughout the reproductive years. In contrast, in females, generally only one oocyte matures and completes meiosis each month of the reproductive years. Thus, there an very large male-female differences in the number of rapidly dividing germ cells during the reproductive years, and it is rapidly proliferating cells which are most susceptible to genetic damage." John a al. (81) found associations with paternal smoking during the 12 months prior to conception in the absence of maternal smoking during the first trimester were found for all childhood cancers combined (OR=1.2), acute lymphocytic leukemia (OR=1.4), lymphomas (OR=1.6), and brain cancer (OR=1.6): These correladons. however, appear fairly weak, at best equivocal. These figures are similar to those for maternal smoking alone. After adjustment for paternal education, maternal smoking during the first trimester of pregnancy, in the absence of paternal smoking, was associated with an increased risk for all cancers combined (OR=1.3), acute lymphocytic leukemia (OR=1.9), and lymphomas (OR=2.3). Johnston et al. (82) found in their study of children with germ cell tumors, the smoking pattern of fathers was similar for casts and controls. Magnani et al. (83) found tharboth maternal an&paternal smoking up to the child's birth were associated with non-Hodgkin lymphoma in childhood. After adjusting for 5

socioeconomic status, the odds ratio for paternal smoking was 6.7 and for matemal 1.7. The author says that the odds ratio for paternal smoking was not correlated with number of cigarettes. The study showed no correlation between acute lymphocytic leukemia and parental smoking. Neutel and Buck's (84) study only took into consideration the effect of matcrnal smoking on childhood cancer. 'For cancers of all sites, the children of mothers who smoked during pregnancy had a relative risk of 1.3, which doesn i-seem significant. Preston-Martin et al. (85) found that there was a possibly significant increase in risk of childhooa brain tumots (1.5 odds ratio) if during pregnancy the mother lived with a smoker (who was usually the child's father). "Our finding that maternal smoking itself was not related to disease but that living with a smoker (usually the child's father) was may indicate that paternal exposures are im t" Sandier ct al. (86) analyzed cancers all sites, except basal cell cancer of the skin, among people 15-59 years old. Cancer risk was increased 50% among people whose fathers had smoked. (Paternal smoking was defined as the father having smoked before the child reached 10 years of age.) Increased risk associated with paternal smoking was norexplained by demographic factots, social class, or individual smoking habits. There was only a slight increase in overall cancer risk associated with matental smoking (relative risk of 1.1). But maternal and paternal smoking were both associated with risk for hematopoietic cancers, and a dose-response relationship was seen. The relative risk for hematopoieac cancers increased from 1.7 when one parent smoked to 4.6 when both parents smoked. However, the study included a wide range of ages and did not give isolated information on the teenage cases. Therefore, the study does not assess the risk of paternal smoking specifically on childhood cancer. Stewart er al. (87) found the incidence of paternal smoking was similar among children with cancer and without cancer. However, this study did not distinguish between pipe and cigarette smoking. The smoker of pipes generally doesn't inhale much smoke. ANTIOXIDANTS PROTECT AGAINST DISEASE Fruit and Vegetable Consumption Lowers Risk of Degenerative Diseases Block and her colleagues have recently reviewed the 172 studies in the epidemiological literature that relate the amount of fruit and vegetable consumption to cancer incidence (88) (Figure 3). The data are extremely consistent and show a large protective effect: The quarter of the population with low dietary intake of fruits and vegetables compared to the quarter with high intake has double the cancer rate for most types of cancer (lung, larynx, oral cavity, esophagus, stomach, colon and rectum, bladder, pancreas, cervix, and ovary). As Bjelke (89) said 20 years ago: "for colorectal cancer, the large majority of the population may be at increased risk compated to the minority with very high vegetable intakes". Data on the types of cancer known to be associated with hormone levels are not as consistent and show less protection: for breast cancer the protective effect was about 30% (88, 90). There is also literature on the protective effect of fruit and vegetable consumption on hearr disease and stroke (91). Only 9% of Americans eat five servings of fruits and vegetables per day, the intake recommended by the National Cancer Insitute and the National Research Council (88, 92). European countries with low fruit and vegetable intake (e.g., Scotland) are generally in poorer health and have higher rates of heart disease and cancer than countries with high intake (e.g., Greece) (93). The cost of fruits and vegetables is an important factor in discouraging consumption. Poorer people spend a higher percentage of their income on food, eat less fruits and vegetables (94), and have shorter life expectancy than wealthier people. Dietary Antioxidants The effect of the dietary intake of the antioxidants ascorbate, tocopherol, and carotenoids is difficult to disentangle by epidemiological studies from other important vitamins and ingredients in fruits and vegetables (88. 9S), Nevertheless, several arguments suggest that the antioxidant content of fruits and vegetables is a major contributor to their protective effect. 1) Biochemical data, discussed above shows that oxidative damage is massive and is likely to be the major endogenous damage to DNA, proteins, and lipids. 2) Studies showing that oxidative damage to sperm DNA is increased when dietary ascorbate is insufficient. 3) Epidemiological studies and intervention trials on prevention of cancer and heart disease in people taldng antioxidant supplements are suggestive, though larger studies need to be done (91, 96) . Clinical trials using antioxidanu will be the critical test for many of the ideas discussed here. 4) Studies onn oxidative mechanisms or epidemiology on antioxidant protection for individual degenerative diseases, discussed below. ' 6

FRUITS AND VEGETABLES PROTECT AGAINST CANCER (Bloc(. Patterson and Subar.lVutr. Cane..18:1,-29,1992) ReleUve C.noer Sitee FFaction d Sojdes Slwwkg • P+otectln t3teet Ca.0.o51 t8sk 01he5ee) EpKNellal Lung 2425 2.2 Oral' 9/9 2.0 Larynx 4/4 2.3 Esophagus 15/16 2.0 Stomacti 17/19 2.5 Pancreas 9/11 2.8 CeMx 7/8 2.0 Bladder 3/5 2.1 Colorectal' 20135 1.9 Miscellaneous 6/8 - Breast 8/14 1.3 OvanylEndometrium 3/4 1.8 Prostate 4/14 1.3 Total 1291172 F1ewe 3. Review o( epidemioiogical studies showieY protecooo by 6ttius aad vegetables againct wrov (d8).. Small molecule dietary antioxidants such as Vitamin C(ascorbate), Vitamin E (tocophetol), and cat otenoids have genuatedparacuIar interest as anticarcinogens and as defenses against degenerative diseases (97-100). Most carotenoids have antioxidant ac>;vity, particularly against singlet oxygen, though only b-carotene can be metabolizcd to Vitamin A(retinal) (101-103). We have called attention to a number of previously neglected physiological antioxidanu including urttte, bilitubin, carnosine, and ubiquinol (104-107). Ubiquinone (CoQlp), for example, is the critical small molecule for transpotting electrons in mitochondria for the generation of energy. Its reduced form, ubiquinol, is an effective antioxidant in tnembranes and lipoproteins (105,108), Optimal levels of dietary ubiquinoneJubiquinol could be of importance in many of the degenerative diseases. Antioxidants' and Cancer A critical factor in mutagenesis is cell division (9;109,110). When the cell divides, an unrepaired DNA lesion has a certain probability of giving rise to a mutation. Thus an important factor in mutagenesis, and therefore catcinogenesis, is the cell division rate in the precursors of tumor cells. Stem cells are impottant as precursor cells in cancer because they are not on their way to being discarded. Increasing their cell division rate would increase mutation. As expecud, there is liNe cancer in non-dividing cells. Such diverse agents as chronic infection (see below), high levels of particular hormones (Il!) , or chemicals at doses causing cell killing (9, 110, 112, 113) result in increased cell: division and an increased risk for cancer. Oxidants form one important class of agents that stimulate cell division (114, 115). This may be related to the stimulatiott of cell division tbat occurs during the inflammatory process accompanying wound healing (9). Antioxidanu therefore can decrease mutagenesis, and thus canrinogtnesis, ia two ways by dearasittg ouidative DNA datmge and by deaessang cell division Of great iaterest is the mtdetswtding of mechanisms by which tooopherol and cat otenoids aa ptt vent cell division (I16-119). There is an ittct>cuing lice:atute an the ptotective tnle of dietary tocopheroi, ascocbaoe, and b-carotene in lowering the iacidence of a aride vatiety of bumaa cancer (120-122). Antioxidants can couttteract the indttctioo of cancu itt todeaa by a varietx of camirwgens (123-125). Two of the major causes of cancer. cigatrae smoke aad ctuoatc inflammation, both appear to involve oxidanu in their taechanism of action. Almost all of the epidemiological studies that examined the relation between antioxidant levels and ctgarette-induced lung cancer showed a statistically significant protective effect of antioxidants (88, 120, 122). Antioxidants inhibit much of the pathology of cigarette smoke in rodents (126. 127). Inflammatory reactions release large amounts of NO. a radical, nittrnating agent, and mutagenic oxidant (49, 128, 129). Ascorbate inhibits nitrosadon under physiological conditions (130). Antioxidants help to protect against the carcinogenic effects of chronic inflammation, as discussed above. The Optimum Level of Antioxidants The epidemiological evidence and the guidelines of the National Cancer Institute and the National Research Council/National Academy of Sciences suggest that at least two fiuits and three vegetables per day is a desitableptake. Since ascorbate, tocopherol, and b-carotene supplements are inexpensive and high doses are temarkably non-toxic, there is a school that believes that supplements are desirable. Thete is suggestive, but inadequate epidemiological and biochemical evidence bearing on the question (96. 121). What is 7

clear is that fvits and vegetables contain many necessary micronutrients in addition to anrioxidants, some of which also can prevent mutations. Folic acid, for ezample, is required for the synthesis of the nucleottdes in DNA. lnadequa:e intake has been shown to cause chromosome breaks and increased cancer and birth defects (98. 131), Folatc deficiency may'be a risk factor for myocardial infarction as well (132). Niacin is required for making poly (ADP-ribose), a componenrof DNA repair. Other micronutrients are also likely to be part of our defense systems. The U.S. Recommended Daily Allowances (RDAs) for ascorbate and tocopherol intake--there is no guideline for b-catotene-are not adequate for several reasons: 1) The amount recotttmended, e.g., 60 mg/day for ascorbate, is primarily for avoiding an observable deficiency syndnome, e.g., scurvy, and is not necessarily the amount for optimum lifedtne health, which is usually not known. 2) A recommended blood level of each antioxidant, e.g., 60 pM ascotbate, would be a mor'e desirable standard. People vary considerably in the intake required to keep their blood level adequate. A smoker, for example, needs to take in several times as much asoorbate as a non-stnoker to keep the blood level the same. Infections also cause an oxidative stness by activating phagocytic cells. The observation that antioxidant inadequacy is associated with oxidative damage to DNA of the germ line as well as somanc eells, emphasizes the urgency of determining adequate blood levels(61). Since only 9% of Americans, and fewer in most other countries, are eating 5 fruits and vegetables per day, there is a great opportunity to improve health by increasing consumption. ACKNOWLEDGEMENTS This paper has been adapted in part from Ames, B.N!, Shigenaga, M.K. & Hagen. T. "Oxidants, Antioxidants, and the Degenerative Diseases of Aging", submitted. , 8'

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