Document e7K4mpwZpDO5nJg9anBKL2qEg

PLAINTIFF'S EXHIBIT AL-1209 Vol. 105 No. 23 CHEMICAL CARCINOGENESIS -- FARBER 1379 References 1. Whitienberger JL. Artificial respiration. Physiol Rev. 1955; 35:611*28. 2. LunkeittKjmcr PP, Rafllcnbcul W, Iteller H, et al. Application of iranstrachtfKqressure oscillations as a modification of "diffusion respiration.*' BrS^naesth. 1972; 44:627-8. 3. Sjdstrand U. Revtewbfi^e physiological rationale for and development of high-frequency posmVfe^reuure ventilation -- HFPPV. Acta Anaestbesiol Scand [SupplJ. T9S 64:7-27. 4. Kkain M, Smith RB. High frequehe&Dercutaneous transtracheal jet ventilation. Cril Care Med. 1977; S:2w * 5. Bunnell JB, Karlson KH, Shannon DC. HiglHraquency positive pres sure ventilation in dogs and rabbits. Am Rev Kfespjr Dis. 1978; 117: Supp!:289. abstract. 6. Bohn Di, Miyasaka K, Marchak BE, Thompson WK/'Piqese AB, Bryan AC. Ventilation by high-frequency oscillation. J Appl Phy^ol. 1980; 48:710-6. 7. Butler WJ, Bohn DJ, Bryan AC, Froese AB. Ventilation by high-^, frequency oscillation in humans. Anesih Analg (Cleve). 1980; 59:1 84. 8. Carlon GC, Ray C Jr, Klein M, McCormack PM. Htalrfrequency positive pressure ventilation in management of a patieprtmh broncho pleural fistula. Anesthesiology. 1980; 52:160*2. 9. Slutsky AS, Brown R, Lehr J, RossingTH, Dg*en JM. High frequency ventilation: a promising new approach t^jdechanical ventilation. Med Instrum. 1981; 15:229-33. 10. Froese AB, Bryan AC. High freopcticy ventilation. Am Rev Rcspir Dis. 1981; 123:249-50. 11. Slutsky AS. Drazen JM^kfgram RH Jr, et al. Effective pulmonary ven tilation with smail-vptdme oscillations at high frequency. Science. 1980; 209:609-11. 12. Slutsky AS^4Camm RD, Rossing TH, et al. Effects of frequency, tidal volume-ami lung volume on CO, elimination in dogs by high frequency (2;36^Hz), low tidal volume ventilation. J Clin Invest, (in press). 13. Sjfatrand U. Summary of experimental and clinical features of hu frequency positive-pressure ventilation -- HFPPV. Acta Anaepttesiol Scand (Suppl). 1977; 64:165-78. 14. Frantz ID, Stark AR, Werthammer J. Improvement pulmonary interstitial emphysema with high frequency venUiafSon. Pcdiatr Res. 1981; 15:719. abstract. 15. Goldstein DH, Slutsky AS, Ingram RHirfWesiennan p, Venegas J, Drazen J. CO, elimination by high frequency ventilation (4 to 10 Hz) in normal subjects. Am Rev Respip-Cks. 1981; 123:251-5. 16. Custer JR, Shannon DC. HigkTrequency ventilation enhances oxygen ation and alveolar ventUptfim at reduced airway pressures in an animal model of pulmonagpedan*. Am Rev Respir Dis. 1979; 119: Suppl:266. abstract. 17. Schmid ERtnopp TJ, Rebder K. Intrapulmonary gas transport and perfusipff'auring high frequency oscillation. J Appl Physiol, (in press). 18. Fu)kIwii Y, Roussos CS. Macklem PT, Engel LA. Convection, diffu- (on and cardiogenic mixing of inspired gas in the lung: an experimental approach. Respir Physiol. 1976; 26:77-90. Slutsky AS. Gas mixing by cardiogenic oscillations: a theoretical quaniutive analysis. J Appl Physiol, (in press). 20. fr4berg JJ. Augmented (Effusion in the airways can support pulmonafr^as exchange. J Appl Physiol. 1980; 49:232-8. 21. Haselton FmScberer PW. Bronchial bifurcations and respiratory mass transport. Scienba^lttO; 208:69-71. 22. Taylor G. Disperubvpf soluble matter in solvent Rowing slowly through a tube. Proc RBeqA. 1953; 219:186*203. 23. Idem. The dispersion of mattefrsjurbuient flow through a pipe. Proc R Soc A. 1954; 223:446-68. 24. Chatwin PC. On the longitudinal dispehiqn of passive contaminant in oscillatory flows in tubes. J Fluid Mech. l9S^7l(3):5l3-27. 25. Mead J. Contribution of compliance of airwaystek^frequency-depcndent behavior of lungs. J Appl Physiol. 1969; 26:67> 26. Rossing TH, Slutsky AS, Lehr J, et al. The influence offrd^l volume (VT)on CO, output (VCO,) during high frequency ventilation (^V) in dogs. Air. Rev Respir Dis. 1981: 123(4): Part 2:203. abstract. MEDICAL PROGRESS CHEMICAL CARCINOGENESIS Emmanuel Farber, M.D., Ph.D. ' | 'HE perception of cancer as a disease primarily X related to the environment has been progres sively strengthened since the publication of a Medical Progress article on chemical carcinogenesis in 1971.1 By the mid-1960s, the view of the cause and patho genesis of cancer had already begun to change radi cally from that of previous decades, during which no clear perception of the possible role of the environ ment in the genesis of most of the major forms of can cer was evident. Although the relation of cancer to en vironmental hazards in the work place and to a few unusual cultural patterns around the world was es tablished, the examples were considered exceptional. The general acceptance of cigarette smoking as a major factor in the development of lung cancer and the increasing number of examples of major shifts in the organ or tissue distribution of primary cancers with migration of distinctive ethnic groups from one From the departments of Pathology and Biochemistry, University of To ronto. 100 College St.. Toronto, ON MSG 1L5, Canada, where reprint re quests should be addressed. Supported by research grants from the National Cancer Institute or Can ada. the Medical Research Council or Canada, and the National Cancer insutuie (U.S.A.). geographic location to another were important in changing the whole perception of the genesis of cancer. This radical change has encouraged increasing attention to the nature of the environmental influ ences. The available epidemiologic evidence indicates the likelihood of several (if not many) components in this environmental outlook. Diet, carcinogenic chem icals, radiation, and viruses are among the major fac tors that appear to be involved. Of these, chemicals as carcinogens have been receiving increasing attention as having the greatest role in the genesis of cancer. How justified is this perception of the causation of cancer? About one third of the cancer in North America and Europe is related to the use of cigarettes or other to bacco products. There is incontrovertible evidence that chemicals related to the work place or occupa tion are responsible for a segment of cancer in human beings (Table 1). To these must be added a growing list of naturally occurring chemicals and of drugs used in medicine. All these known agents, together with ul traviolet light as the single most common cause of can cer, can easily account for over 50 per cent of the cases 511967 0305 \ ol. 303 No 23 CHEMICAL CARCINOGENESIS -- FARBER 1381 can be seen during the long so-called latent period. Often, new cell populations appear that probably rep resent stages or steps in the cellular evolution from normal cells through initiated, preneoplastic, and premalignant cells to highly malignant neoplastic cells.15-17 Research in laboratory animals has concen trated heavily on the early events and has suggested the existence of common patterns of early preneo plastic changes in several organ systems and in sever al species.17 Research in human beings has concen trated largely on the later events and has confirmed the occurrence of atypical hyperplasias, dysplasia, and carcinoma in situ as probable precancerous steps in several organs late in the process.,7*19 However, the critical properties of each different cell population that relate to the possible roles of these cells in cancer development and to the manner in which the two aspects of carcinogenesis (the experimental and the human) fit together into some biologically meaning ful pattern remain as challenges.17 The conceptual advances made in the late 1930s and 1940s concerning the first two early steps in chemical carcinogenesis, initiation and promotion, are well known.15'17'70 However, their mechanistic bases are only now unfolding, and these will be topics for review. The basic validity of these concepts for cancer development in human beings has been estab lished in a few instances and as such is reassuring both for the physician and for the scientist. For exam ple, a relatively brief exposure to diethylstilbestrol during pregnancy may be associated with the devel opment of vaginal neoplasia some 15 to 25 years later in the exposed person's daughters.21 Parenthetically, it should be mentioned that it is not known whether diethylstilbestrol acts in this situation as a chemical carcinogen or whether the effect is predominantly hor monal. Again, a limited exposure for only a few months to a known chemical hazard, such as vinyl chloride, may lead many years later to the appear ance of angiosarcoma of the liver.22 Nonneoplastic cel lular and tissue changes are seen during the apparent latent period. some nitrosamides (e.g., alkyl nitrosourea), bis(chloromethyl) ether, and nitrogen mustard, are active by themselves and do not seem to require any metabolic conversion to more reactive metabolites. Activation and Metabolism of Procarcinogens The first major type of metabolic activation discov ered was the conversion of an aromatic amine, 2-acetylaminofluorene, by .V-hydroxylation to an N-OH derivative.20 This type of metabolic product is seen with several aromatic amines, including the human bladder carcinogen 2-naphthylamine (|8-naphthylamine). More recently, much effort has been spent on the clarification of the activation of benzo(a)pyrene and the many other polycyclic aromatic hydrocarbons. Many of them are easily generated by burning (py rolysis), are widely distributed in our environment, and are thought to be partly responsible for the carci nogenicity of coal-tar products, oils, and tars. These compounds undergo epoxidation to form reactive ep oxides, some forms of which are considered to be ulti mate carcinogens.25 In the case of benzo(a)pyrene (and probably other polycyclic aromatic hydrocar bons), the initial site of epoxidation may undergo hy dration to form a dihydrodiol in a reaction catalyzed by epoxide hydrolase. This inactive derivative, in turn, can be converted to another epoxide at a second site to form a dihydrodiol epoxide. These are consid ered to be the most likely ultimate carcinogens for at least some polycyclic aromatic hydrocarbons. This series of reactions for benzo(a)pyrene is illustrated in Figure 1. Another potentially important carcinogen for human beings that is subject to activation through epoxidation is aflatoxin B,.20 This substance under goes oxidation at its 2,3-position, and this derivative, the 2,3-oxide, appears to be one form of ultimate car cinogen of this mycotoxin. Vinyl chloride is also acti vated through epoxidation.20 1? 1 Initiators and Initiation Initiators Until fairly recently, one of the most puzzling and confusing aspects of chemical carcinogenesis was the diversity in the chemical structure of carcinogens. This problem has now been largely resolved by the discovery of metabolic conversion of many carcino gens to highly reactive metabolites.20 The best-known form of reactive moieties, generated from several dif ferent types of carcinogens, is the "electrophilic re actant," which is a positively charged molecule that reacts well with sites of electron densities in many dif ferent cellular components, including DNA, RNA, protein, glutathione, and probably also polysaccha rides.20 A minority of chemical carcinogens, such as Figure 1. Current View of the Activation of Benzo(a)pyrene (BP) to Benzo(a)pyrene 7,8-Dihydrodiol,9,10 Epoxide through the Mixed-Function Oxygenase (MFO) System (Cytochrome P-450 System) and Epoxide Hydrolase. 511961 0307 V'ol 305 No. 23 CHEMICAL CARCINOGENESIS -- FARBER 1383 MNU. BAi.tNNQ ectasia and Fanconi's anemia) also involve deficien cies in DNA repair as well as an increased risk of neo plasia.41 Experimentally, there is a correlation under some conditions between the formation of specific DNA ad ducts (such as 0`-methylguanine in the brain), per sistence of this biochemical lesion, and the ultimate appearance of gliomas.43 Other studies indicate that the genesis and persistence of 0*-methylguanine may be important but is insufficient to account for cancer production with some nitrosamines or nitrosamides44 or 1,2-dimethylhydrazine.43 The major protection against cancer may reside in the efficiency with which we repair DNA that has been damaged by mutagens and carcinogens. As pointed out by German" and by Cairns,11 since fibro blasts from patients with xeroderma pigmentosum show a defect in the repair of DNA damage by some Figure 2. Diagrammatic Representation of Sites of interac tions of Activated Forms of Carcinogens with the Four Bases of DNA. T denotes thymine; A. adenine; C. cytosine; G, guanine; MNU, N-methyl-N-nitrosourea; ENU. N-ethyl-N-nitrosourea; ENNG, N-ethyl-N'-nitro-/V-nitrosoguanidine; 7 Br MBA, 7bromothyl-benzo(a)anthracene; BP epoxide, benzo(a)pyrene 7,8-dihydrodiol,0,10 epoxide; 4NQO, 4-nitroquinoline-Noxide; DMS, dimethylsulfate; DMN, dimethylnitrosamine; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitroAAnitrosoguanidine; 1.2.DMH, 1,2-dimethylhydrazine; DEN, diethylnitrosamine; N-OH-1-naphthylamine, W-hydroxy-1naphthylamine; 2 AAF, 2-acetylaminofluorene; MAM, methylazoxymethanol; EMS, ethyl methane sulfonate; BPL, 0-pro- pioiactone; and MAB, 4-methylaminoazobenzene. chemicals as well as by ultraviolet light, such patients should have an elevated risk for cancer in organs or tissues other than the skin if chemical carcinogens are important in the genesis of many forms of cancer. Yet the available data show no apparent increase in the risk of cancers other than those of the skin in pa tients with xeroderma pigmentosum.12 Patients with Bloom's syndrome do have an elevated risk. Such observations are interpreted as evidence against a role for the many environmental chemical hazards in the causation of human cancers generally, except in wellestablished instances such as respiratory-tract cancer in smokers and in persons with occupational expo sures.12 An alternative hypothesis must be enter studies of susceptibility to nucleases, separation of tained: that the role of repair may be quite different in transcriptionally active from inactive DNA, or isola the dynamics of cancer induction by chemicals, as tion of linker and nucleosome regions in chromatin, compared with induction by ultraviolet light. some of the interactions of carcinogens with DNA The past 10 years have seen a large expansion of have been found to be nonrandom. However, no single studies of DNA repair, largely in vitro. These studies region of high affinity has been found.36 The use of should lead to a much clearer delineation of the types DNA cloning for specific genes in'biologically well- of repair that may occur in carcinogenesis. What does defined preneoplastic and early neoplastic popula happen remains poorly understood. Processes of re tions should be profitable. pair by base excision, by "long-patch versus short- DNA Repair patch" removal, by possible recombination or other forms of post-replication repair, by removal of inter- In view of the essentially irreversible nature of initi strand cross-links or other more complex forms of ation with chemicals and the apparent focal nature of damage, and by other mechanisms are slowly being the initiation process, major emphasis at the molecu studied in many different systems in vitro and to a lar level is given to DNA as a probable target in ini much lesser degree in vivo.36'41 The enzymology, al T tiation. However, the evidence is largely circum though difficult, is also slowly becoming clear. In the stantial. case of methylating carcinogens, an interesting recent The bulk of evidence comes from studies in pa finding is the discovery of an enzyme in bacteria that tients with xeroderma pigmentosum, who have a high removes the methyl group from the O6 position of gua incidence of skin cancers. The etiologic agent is ultra nine in DNA by transmethylation with its transfer to a violet light, and the vast majority of patients have a sulfur-containing moiety in protein.46 This raises the defect in their ability to repair the damage inflicted on question of whether some of the enzymes involved in DNA by this form of radiation. Such patients can be repair of DNA damage perform physiologic functions protected from skin cancer by careful avoidance of ex posure to ultraviolet light.40 Fibroblast cultures from other than repair that subserve normal cellular re quirements. these patients have increased sensitivity not only to ul Obvious failings in many of the attempts to corre traviolet light but also to some chemical carcinogens late patterns of DNA repair with carcinogenicity in and mutagens.41 Other diseases (e.g., ataxia-telangi clude the wide gap between the chemistry and the bi- 511961 0309 Vol. 305 No, 23 CHEMICAL CARCINOGENESIS -- FARBER 1385 crosis induced by viruses, toxic agents, parasites, or dietary deficiencies, followed by cell regeneration, could be a major determinant of cancer initiation in many sites. In addition, the presence of many prolif erating cells is probably one basis for the susceptibili ty of the fetus and neonate to many chemical carcino gens'' and could account for the peak in cancer incidence in the first decade of life. The human fetus, unlike the rodent fetus, acquires the capability of ac tivation of some carcinogens early in development" and thus may be at greater risk than some laboratory animals for cancer development with chemicals. A most important question about initiation con cerns the essential biologic nature of the initiated cells. What properties have they acquired that allows them, as a group, to be precursors for the ultimate de velopment of cancer? The available evidence is against any conclusion that the initiated cells have acquired any autonomy of growth."'" In most systems, the properties critical to initiation remain unknown. In two continuously proliferating tissues, the skin" and the colon,'2 an early property of carcinogen-altered cells may be some disturbance in programming or control, such that the cells do not show the normal progression of differentiated proper ties. In the liver, the cell with acquired resistance to the inhibitory effects of carcinogens on cell prolifera tion has been shown to be one type of initiated cell."'*3 Although a majority of chemical carcinogens fall well within the current paradigm in which initiating effects are related to some form of DNA damage, there are known carcinogens that appear to be exceptions. A growing list of hypolipidemic agents'4 and several pesticides, herbicides, and other xenobiotics" have not been shown to generate mutagenicity or other DNA-damaging effects. Is this merely a reflection of deficiencies in our technology, or are there pathways to cancer that do not involve DNA, damage of exoge nous origin as essential early steps in the process? Promoters and Promotion The term "promotion" is often used for the process whereby neoplastic development, tumor formation, or cancer development is accelerated or encouraged in a tissue that has been exposed to an initiating dose or doses of a carcinogen. Promoters Early in the recent history of chemical carcinogen esis it was found that a noncarcinogen, croton oil, could stimulate tumor formation in the skin after a brief initial exposure to a carcinogen. This naturally encouraged a chemical "attack" on croton oil Hecker" and Van Duuren'7 discovered that esters of the diterpene phorbol, isolated from croton oil, have potent promoting effects on mouse skin. Many bio chemical, metabolic, and biologic effects are induced in many different normal cells by the phorbol esters and related compounds; in the vast majority of cases, the intensity of the particular effect closely parallels the efficacy of the compounds as promoters of skin papillomas in mice after exposure to a single dose of dimethylbenzanthracene or a similar carcinogen."'' These effects include changes in morphology, micro tubule polymerization, cell proliferation, enzyme induction, polyamine synthesis, phospholipid synthe sis, membrane structure and function, ATPase, re lease of prostaglandins, inhibition and sometimes stimulation of differentiation, and many others in a wide variety of normal cells and cell lines. Many of the effects are considered pleiotropic. Recently, with the use of a less active phorbol ester, 3H-phorbol dibutyrate, membranes of fibroblast cul tures and mouse epidermal cells were found to con tain a high-affinity receptor for many phorbol esters, including one of the most active, 12-O-tetradecanoylphorbol-13-acetate."'* The "natural" metabolites for the receptors have not been identified. Given the wide diversity and large number of effects seen in treated cells, it may be that more than one type of receptor is involved. At present it is impossible to relate the findings in vitro to chemical carcinogenesis in vivo. Given the array of phenomena induced by the phorbol esters, how does one select relevance to promotion? A major problem concerns the matching of the tar get cells in vivo and in vitro. In the intact animal, an active promoter does not induce focal proliferations (such as papillomas) in the normal skin, but does so only after initiation. However, many skin promoters do induce general hyperplasia of the epidermis in an imals in which initiation has not taken place. Studies in several laboratories have shown that a general stim ulation of cell proliferation is insufficient for the selec tive or differential stimulation of initiated skin to form papillomas.'1 Promotion A question that requires an early answer is wheth er the differential effect of a promoting environment on initiated tissues is directed primarily to the initiat ed cells or to the surrounding cells.'2 There is consid erable circumstantial evidence to suggest that the first major phenomenon in promotion is the selection of an appropriately altered cell to produce a focal prolifera tion, and that promoting environments create differ ential effects on the initiated cells and on the sur rounding cells." `3''2 Cancer frequently arises in an atrophic tissue or organ, not a hypertrophic one. Is this the consequence of a change in the local environ ment, favoring growth of the rare initiated cell? As observed in vivo in experimental models in com mon use today, cell proliferation is an essential phe nomenon in promotion. It is therefore to be anticipat ed that many biochemical changes that are seen in the different phases of the cell cycle,'3 such as an increase in ornithine decarboxylase activity, will be seen in promotion. Determining whether one or more of such changes in enzymes, the cell membrane, DNA organ ization, or other factors will have a special role in pro- 511961 0311 Vol. 305 No. 23 CHEMICAL CARCINOGENESIS -- FARBER 1387 some patients the risk of second primary tumors varies from 7 to 12 per cent.115 These figures include some risk factors aside from the therapy, that are pos sibly genetic and favor second or multiple primary tumors. However, there is an additional risk from the chemotherapy itself. This risk will increase in volume as current types of chemotherapy for cancer become more successful. However, as mechanisms by which chemicals induce cancer and kill cancer cells are clar ified, ways will be found to prevent the carcinogenic effects without interfering with the drugs' efficacy in - treating cancer. A common problem in cancer chemotherapy is the appearance of resistant cancer cells. Since resistant cells are also produced by carcinogens during carci nogenesis, and since such resistance constitutes one form of initiation,"'1 it is conceivable that the induc tion of resistance to therapy and that of cancer are related. New Bioassays for Carcinogens and Promoters The basic knowledge that has been generated dur ing the past 20 years or so has spawned a large array of short-term tests (mostly in vitro) for potential car cinogens, and more recently for promoters.These tests generally fall into two broad groups: those that use some correlate of DNA damage or chromosomal damage as end points and those that use cell transfor mation as the end point."4'"* The majority require the inclusion of an appropriate activation system. This development is an essential requirement for rapid monitoring of our environment. In addition, these tests are being used increasingly as rapid assays for some more basic aspects of chemical carcinogene sis, such as patterns of activation in tissues and the en dogenous production of carcinogens or mutagens, to name but two of many examples. Conclusions The past decade has produced new insights into the need for metabolic activation, the nature of the active molecules, the enzymology of activation, and the array of products generated, including major ulti mate carcinogens. The interactions of such deriva tives with DNA and other cell constituents and the possible toxicities and other effects on cells are be coming clearer. There is a general belief that DNA damage is involved in cancer initiation in most in stances. The basic studies have generated over 100 short-term assays for possible carcinogens, with many of the assays reflecting damage to DNA or chromo somes. The importance of the modulation of metabolic ac tivation by enzyme induction with hormones, diet, drugs, and chemicals (including many environmental contaminants such as pesticides, herbicides, and poly chlorinated biphenyls) is appreciated. How this ubiq uitous exposure may influence cancer development in human beings is not known. The repair of lesions in DNA is receiving increas ing emphasis. The majority of such studies are in rel atively simple in vitro systems and are generating in sights into what may happen. The study of what does happen remains a major challenge. It may be aided by the development of ultrasensitive enzymatic radioim munoassays for carcinogen-DNA adducts. There is an increasing realization that many post initiation phenomena, including promotion, are of major importance in cancer development. Alterations in diet, hormones, drugs, and xenobiotic agents have major influences through effects on promotion and other later steps. The reversibility of many of the precancerous steps is being studied with vitamin A and its analogues and with other dietary components. Despite the major advances, the study of the later phases of chemical carcinogenesis lags far behind that of the early biochemical events. A nagging uncertain ty in this field in general concerns the role of chemical carcinogens of either exogenous or endogenous origin in the causation and pathogenesis of some of the major forms of cancer. There is a general agreement that cigarette smoking and ultraviolet light are two major factors in the genesis of several important types of cancers. There is also incontrovertible evidence that many chemicals to which persons are exposed in the work place or its environs cause cancer in several organ systems. However, the relative quantitative im portance of these considerations in the majority of pa tients with cancer not related to smoking is unclear. The resolution of this problem, although no doubt dif ficult, will almost certainly have a major impact on our approaches to cancer prevention in coming decades. I am indebted to Miss H6Iine RobitaiUe for assistance in the preparation of this manuscript and to my colleagues, Drs. A. Med line and D. S. R. 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