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Yerspect I SCIENCE, Voi.'249, No. 4972, pages 97C - 971 (31 August 1990) TOO Many y Rodent C. ~C1,:1®g~~.S ® 1VIit®grenesis Increases . Muta'.p,enesis BRUCE N. .AAES* AND LOIS SWIRSRY C'sOLD CLARIFIC A~'IOBI OF THE MECHANISM OF CARCINOGENESIS is devclctping at a rapid rate. This new understanding undermines many assumptions of current regulatory policy toward todent caranogrias and necessitates rethinking the utility and meaning of routine animal cancer tests. At a recent watershed meeting on cariiogeclesis, much evidence was presented suggesting that mitogenesis (induced cell division) plays a dominant role in carcinogenesis (1). The work of Cohen and Ellwein in this issue (2) is illustrative. Our own rethi_nlring of mechanism was prompted by our findings that: (i) spontaneous DNA damage caused by endoge- nous oxidants is remarkably frequent (3) and (ii) in chronic testing at the maximimg tolerated dose (MTD), more than half of all chemicals tested (both natural and synthetic) are carcinogens in rodents, and a high percentage of these carcinogens are not muta- gens (4). Mitogenesis incnea.ses vnutagenesis. Many "promoters" of eareinogene- sis have been described and have been thought to increase mitogene- . sis or selective lpn~wth of preneoplastic cells, or both. The concept of promotion, however, has been fuzzy compared to the clearer understanding of the role of mutagenesis in carcinogenesis. The idea i that mitogenesis increases mutagenesis helps to explain promotion and other aspeca; of carcinogenesis (2, 5). A dividing adl is much more at risk of mutating than a quiescent cell (4). Mutagens arc often thought to be only exogenous agents, but endogenaus mutagens cause massive DNA damage (by forma- tion of oxidative and other adducts) that can be converted to stable mutations during cell division. We estimate that the DNA hits per cell per day from endogenous oxidants are normally --105 in the rat and ^-1W in the human (3). This promutagenic damage is effectively but not perfectly repaired; for example, the normal steady-state level of 8-hydroxydwxTguanosine (1 of about 20 known oxidative DNA adducts) in rat DNA has been measured as 1 per 130,000 bases, or ' about 47,000 per cell (3). We have argued that this oxidative DNA damage is a major contributor to aging and to the degenerative diseases associated with aging, such as cancer. Thus, any agent causing chronic mitogenesis can be indirectly mutagenic (and consequently carcinogenic) because it increases the probability of converting endogenous DNA damage into mutations. Nongeno- toxic agents [for example, saccharin (2)] can be carcinogens at high Ihe authors are in the Division of Biochcmistry and Molecular Biology, Barker Hall, University of Califoaiia, and the Lawrcnce Berkcley Laboratory, Berkeley, CA 94720. *To whom corresporidcace should be addres.ud. doses just by causing chronic mitogenesis and inflammation, and the dose response would be expected to show a threshold. Genotoxic chemicals [for example, N-2-fluorenylacetamide (2-AAF) (2)] are even more effective than nongenotoxic chemicals at causing mito- genesis at high doses (as a result of cell killing and cell replacement). Since genotoxic chemicals also act as mutagens, they can produce a multiplicative interaction not found at low doses, leading to an upward curving dose response for carcinogenicity. Furthermore, endogenous rates of DNA damage' are so high that it may be difficult for exogenous mutagens to increase them significantly at low doses that do not increase mitogenesis. Therefore, mitogenesis, which can be increased by high doses of chemicals, is indirectly mutagenic, and seems to explain much of carcinogenesis (1, 4, 5). Nevertheless, the potent mutagen 2-AAF (3) induces liver tumors at moderate doses in the presence of only background rates of mito- genesis. Detailed studies of mechanism, particularly in the case of apparent exceptions, are critically important Causes of human cancer. Henderson and co-workers (6), and others (4), have discussed the importance of chronic mitogenesis for many, if not most, of the known causes of human cancer, for example, the importance of hormones in breast cancer, hepatitis B (7) or C viruses or alcohol in liver cancer, high salt or.Helicvbacter (Campyla " bacter) infection in stomach cancer, papilloma virus in cervical cancer, asbestos or tobacco smoke in lung cancer, and excess animal fat and low calaum in colon cancer. For chemical carcinogens associated with occupational' cancer, worker exposure has been primarily at high, near-toxic doses that might be expected to induce mitogenesis. Epidemiologists are frequently discovering dues about the causes of human cancer, and their hypotheses are then refined by animal and metabolic studies. During the next decade, it appears likely that this approach will lead to an understanding of the causes of the major human cancers (8). Cancer dusters in small areas are expected to be common by chance alone, and epidemiology lacks the power to esrablish causality in these cases (9). It is important to show that pollution exposure that purportedly causes a cancer cluster is significantly higher than the- background of exposures to naturally occurring rodent carcinogens (4). Causes of cancer in animal tests. Animal cancer tests are conducted at near toxic doses (the maximum tolerated dose, MTD) of the test chemical, for long periods of time, which can cause chronic mito- genesis (1). Chronic dosing at the MTD can be thought of as a chronic wounding, which is known to be both a promoter of carcinogenesis in animals and a risk factor fbr cancer in humans. Thus, a high percentage of all chemicals might be expected to be carcinogenic at chronic, near toxic doses and this is exactly what is found. About half of all chemicals tested chronically at the MTD are carcinogens (4). - Synthetic chemicals account for 82% (350/427) of the chemicals adequately tested in both rats and mice (4). Despite the fact that humans eat vastly more natural than synthetic chemicals, the world of natural chemicals has never been tested systematically. Of the natural chemicals tested, approximately half (37/77) are carcino- gens, which is approximately the same as has been found for synthetc chemicals (212/350). It is unlikely that the high propor- tion of carcinogens in rodent studies is due simply to selection of suspicious chemical structures; most chemicals were selected because of their use as industrial compounds, pesticides, drugs, or food additives. The human diet consists of thousands of natural pesticides (chemicals that plants produce to defend themselves) (4); we calculate that 99.99% (by weight) of the pesticides in our diet are natural. Of the natural pesticides that have been tested in at least one rodent species, about half (27/52) are rodent carcinogens. These 27 SCIINCE, VOL. 249 2025546369

occur commonly in plant foods (10). We estimate that the average intake of the; e pesticides is about 1500 mg per person per day (4). By comparison, the average intake per day of residues of 100 synthetic pesiicides is 0.09 mg per person per day (4). In addition, of the mold toxins tested at the MTD (including aflatoxin), 11 out of 16 are rodent carcinogens. The cooking of food produces thousands of pyrolysis products, and we estimate that dietary intake of these products is roughly 2000 mg peI person per day. Few of these have been tested; for example, of 826 volatile chemicals that have been identified in roasted cofh'ee; only 21 have been tested chronically, and 16 are rodent carcinogens; caffeic aid, a non-volatile carcinogen, is also present. A cup of coffee contains at least 10 mg (40 ppm) of rodent carcinogens (mostly caffeic acid, catechol, furfural, hydrogen perox- ide, and hydroquinone) (4). '1'hus, very low exposures to pesticide residues or other synthetic chemicals should be compared to the enormous bac*ground of natural substances. In the evolutionary war between plants and animals, animals have developed layers of general defenses, almost all inducible, against toxic chemf,cils (4). This means that humans are well buffered against toxidty at low doses from both man-made and natural chemicals. Given the high proportion of carcinogens among those natural chemicals tested, human exposure to rodent carcinogens is far more colrunon than generally thought; however, at the low doscs of most human exposures (where cell-killing and mitogenesis do not occur), the hazards may be much lower than is commonly assumed and often will bezero (4). Thus, without studies of the mechanism of carcinogmesis, the fact that a chemical is a carcinogen at the MTD in rodc:nts provides no information about low-dose risk to humans. Trade-offs. Pesticide residues (or water pollution) must be put in the context,of the enormous background of natural substances, and there is no convincing evidence from either epidemiology or toxicology that they are of interest as causes of human cancer (4, 9). Minimizing pollution is a separate issue, and is clearly desirable for reasons other than effects on public health. Efforts to regulate synthetic pesticides or other synthetic chemicals at the parts per billion level because these chelnicals are rodent carcinogens must include an uniersranding of the economic and health-related trade- of£s. For example, synthetic pesticides have markedly lowered the cost of foocl fiom plant sources, thus encouraging increased con- sumption. Incxeased consumption of fiuits and vegetables, along with deaea:oi consumption of fat, may be the best way to lower risks of can~:er and heart disease, other than giving up smoking. Also, some of the vitamins, antioAdants, and fiber found in many plant foods are anticarcinogenic. The control of the,major cancer risks that have been reliably identified should be a major focus, and attention should not be diverted from these major causes by a succession of highly publi- cized scares about low levels of synthetic chemicals that may be of little or no importance as causes of human disease. Moreover, we must increase research to identify more major cancer risks, and to better understand the hormonal determulants of breast cancer, the viral determinants of cervical cancer, and the dietary determinants of stomach and colon cancer. In this cmltext, the most important contribution that animal studies can offer is insight into carcinogen- esis mechanisms and into the complex natural world in which we Gve. REFERENCES AND NOTES 1. B. E. Bumaworth and T. Slaga, Eds. Chenri.ify Lduce3 Cdl Pmlfua6an: Impli=ions for Risk Aasessmeru (Wilcy-Liss, New York, in precc). 2. S. M. Cohen and L B. Eltwdn, Sciaa 249, 1007 (1990). 3. B. N. Ames, Frce Rad. Res. Caemnm. 7,121(1989); G G. Fraga, M. K. Shigauga, J: W. Park, P. Degan, B. N. Ames, Proc. Nal1. Ared. Sd. U.S.A. 87,4533 (1990). 4. B. N. Ames, M. Profet, L S. Gold, Proc. Nad. Amd. Sd. U.S.A., in peess; B. N. Ames and L S. Gold, iW., in press; Med.Onml. TianarPkmnarahn. 7,69 (1990); B. N. Ames, 6wiron. Mol. Mutagen.14, 66 (1989); R Mag7w, L S. Gold, Sciaae 236,271 (1987); L S. Gold et al., 6:virm. Hrald: Perpea. 81, 211 (1989). 5. J. E. Tcosko, J. Am. CoII. Toximl. 8, 1121 (1989); - G C Chang, B. V. Msdhui2r, S. Y. Oh, !n Yum Toximl. 3, 9, 1990; Trosko has proposed that &PprCSSM °f S'P NncTional innarodhilar oommuniaiion in ooatxt-inlubited cells muld tnd to cal pcc'ifmrion by cell deth, odl xanovaL, p:omoang dhaninls, spcc:&c O°t~ Prod~ growth fxtacs, and 6oc-. 6. B. E. Henderson, R Ross, L Bernstein, Cmvn Rea. 48, 246 (1988); S. Presaon- Mutin a d., in (7ianirdly Irducei Cd1 Proltferation: Lrcpfirau~xes fi'ir Risk Assavruxt, B. E. Buttesw+orth and T. Slaga, Eds. (Liss, New York, in press). 7. H. A. Dunsford, S. Shc, F. V. C7usui, CmKO Rrs. 50, 3400 (1990). 8. Current ep;aemiobgic aata point to these risk fictocs for h-~ cancer agarcar snokang (which is responsible for 30% of cancer dczdhs), dienry imbalanccs, hotmones, vinUses, and oca>patioa "1TJhc agc-aditrtted moctatity rate for all caccers combined acept hmg cancer has bom dediuing snce 1950 for all individual age grotips ecapt 85 and above° (Natioml Cmncer Institate, 1987 AmrJa! ('.muu Sleristics Revieu,lxdudiag Cmvn Trends: 1950-1985, NIH Publiearion 88-2789 (National Imtinuu of Health, Bethesda, MD, 1988), p.1L31. Although inddrnx rates for some cancers have bem rising, tcmds in recorded 'uldderre rates may be biascd by improved regimaoon and diagnosis. Even if particular cancers can be shown to be increasing (far aampk, non-Hodgiass lymphoma and mdanocua) or decreasing (for ea@ple, stomach, cervical, and rectal rirke), estiMislumg calses remains di&cu}t because of the mury cbmgmg aspects of our E'c-styk.Lff~ mpomnicyoonti- m~everyy=. 9. J. Higginson, Canar Rtv. 48, 1381 (1988). 10. A search in foods for the pnaenoe of just tbae 27 nanual pesticide rodent carcinogens indicates that they ocaa naturally in the fol6ow'vig (those at levels over 10 ppm of a singic aednogen am listed in italics): mdse, apple, banana, basg, bcocaodi, Bn4sseir sp-- , -U-ge , -tttalo~ ---y, -, -d.t- , cefery, ilse+y, danamon, doves, aoma, coffa (Leu-), canjq w, lill, Wrm¢, ad;vc, frmr(, grapefiuit.iuice, grape, Irauy, honeydew mdoa, borsem&sk kak, terrke, r+aa, ximega. -lnomn, -#ad (6row:) ra,rn-g, amgC juia, r-sky, 1-srP, Pe-h+ P-, pePaer (bla&), PincaPPk, plu-. P-, radid, , rupbarry, rose-y, sW, sesaw seeds (keated), strawberry, rmregan, dsy:-e, and turnip (4). Pattieuhr nuutal pesticides that arr carcISograic in rodents can be bnod out of cxops if sndies of inedlu»un indieme that they may be signifiaint hazards to humans. 11. This aak was supported by National Cancer Institute Outstanding Investigator g,rant CA39910, by Nataooal Institute of Eaviconmmnl Health Sciences Center grant ES01896 and by DOE Contract D£-AC03-76SF00098. We thank M. Profet, S. Luin, B. Bumsvwcdt, and R Peto for critidmms. 31 AUGUST Iq90 ; PERSPECTIVE 971