Document Z4RxORQvQOekDo6w5owMmVL6L

Vol. 305 No. 23 CHEMICAL CARCINOGENESIS - FARBER 1379 References 1. WhiucnbcrR&r JL. Artificial respiration. Physiol Rev. 1955: 35:611-28. 2. Lunkenheimer PP, Rafflcnbeul W, Keller H, et al. Application of transtracheal pressure oscillations as a modification of "diffusion respiration." Br J Anaesth. 1972; 44:627-8. 3. SjOstrand U. Review of the physiological rationale for and development of high-frequency positive-pressure ventilation -- HFPPV. Acta Anaesthesiol Scand {Suppl]. 1977; $4:7-17. 4. Klain M, Smith RB. High frequency percutaneous transtracheal jet ventilation. Crit Care Med. 1977; 5:280-7. 5. Bunnell IB, Karlson KH, Shannon DC. High frequency positive pres sure ventilation in dogs and rabbits. Am Rev Respir Dis. 1978; U7: Suppl:289. abstract. 6. Bohn DJ, Miyasaka K, Marchak BE, Thompson WK, Frocse AB, Bryan AC. Ventilation by high-frequency oscillation. J Appl Physiol. 1980; 48:710-6. 7. Butler WJ, Bohn DJ, Bryan AC, Froese AB. Ventilation by highfrequency oscillation in humans. Ancsth Analg (Cleve). 1980; 59:57784. 8. Carlon GC, Ray C Jr, Klain M, McCormack PM. High-frequency positive pressure ventilation in management of a patient with broncho pleural fistula. Anesthesiology. 1980; 52:160-2. 9. Slutsky AS, Brown R, Lehr J, RossingTH, Druen JM. High frequency ventilation: a promising new approach to mechanical ventilation. Med Instrum. 1981; 15:229-33. 10. Froese AB, Bryan AC. High frequency ventilation. Am Rev Respir Dis. 1981; 123:249-50. 11. Slutsky AS, Drazen JM, Ingram RH Jr, et al. Effective pulmonary ven tilation with small-volume oscillations at high frequency. Science. 1980; 209:609-11. 12. Slutsky AS, Kamm RD, Rossing TH, el al. Effects of frequency, tidal volume and lung volume on CO, elimination in dogs by high frequency (2-30 Hz), low tidal volume ventilation. J Clin Invest, (in press). 13. SjSstrand U. Summary of experimental and clinical features of high- frequenev positive-pressure ventilation -- HFPPV. Acta Anaesthesiol Scand [Suppl]. 1977; 64:165-78. 14. Frantz ID. Stark AR, Werthammer J. Improvement in pulmonary interstitial emphysema with high frequency ventilation. Pediatr Res. 1981; 15:719. abstract 15. Goldstein DH, Slutsky AS, Ingram RH Jr, Westerman P, Venegas j, Drazen J. CO, elimination by high frequency ventilation (4 to 10 Hz) in normal subjects. Am Rev Respir Dis. 1981; 123:251*5. 16. Custer JR, Shannon DC. High frequency ventilation enhances oxygen ation and alveolar ventilation at reduced airway pressures in an animal model of pulmonary edema. Am Rev Respir Dis. 1979; 119: Suppl:266. abstract. 17. Schmid ER, Knopp TJ, Rehder K. Intrapulmonary gas transport and perfusion during high frequency osculation. J Appl Physiol, (in press). 18. Fukuchi Y, Roussos CS, Macklem PT, Engel LA. Convection, diffusion and cardiogenic mixing of inspired gas in the lung: an experimental approach. Respir Physiol. 1976; 26:77-90. 19. Slutsky AS. Gas mixing by cardiogenic oscillations: a theoretical quan titative analysis. J Appl Physiol, (in press). 20. Fredberg JJ. Augmented diffusion in the airways can support pul monary gas exchange. J Appl Physiol. 1980; 49:232-8. 21. Haselton FR, Scherer PW. Bronchial bifurcations and respiratory mass transport. Science. 1980; 208:69-71. 22. Taylor G. Dispersion of soluble matter in solvent flowing slowly through a tube. Proc R Soc A. 1953; 219:186-203. 23. Idem. The dispersion of matter in turbulent flow through a pipe. Proc R Soc A. 1954; 223:44W8. 24. Chatwin PC. On the longitudinal dispersion of passive contaminant in oscillatory flows in tubes. J Fluid Mech. 197$; 7l(3):5l3-27. 25. Mead J. Contribution of compliance of airways to frequency-depend ent behavior of lungs. J Appl Physiol. 1969; 26:670*3. 26. Rossing TH, Slutsky AS, Lehr J, et al. The influence of tidal volume (VT) on CO, output (VCO,) during high frequency ventilation (H FV) in dogs. Am Rev Respir Dis. 1981; 123(4): Part 2:203. abstract. MEDICAL PROGRESS CHEMICAL CARCINOGENESIS Emmanuel Farber, M.D., Ph.D. THE perception of cancer as a disease primarily related to the environment has been progres geographic location to another were important in changing the whole perception of the genesis of sively strengthened since the publication of a Medicalcancer.2,5 Progress article on chemical carcinogenesis in 1971.' This radical change has encouraged increasing By the mid-1960s, the view of the cause and patho attention to the nature of the environmental influ genesis of cancer had already begun to change radi ences. The available epidemiologic evidence indicates cally from that of previous decades, during which no the likelihood of several (if not many) components in dear perception of the possible roie of the environ this environmental outlook. Diet, carcinogenic chem ment in the genesis of most of the major forms of can icals, radiation, and viruses are among the major fac cer was evident. Although the relation of cancer to en tors that appear to be involved. Or these, chemicals as vironmental hazards in the work place and to a few carcinogens have been receiving increasing attention unusual cultural patterns around the world was es as having the greatest role in the genesis of cancer. tablished, the examples were considered exceptional. How justified is this perception of the causation of The general acceptance of cigarette smoking as a cancer? major factor in the development of lung cancer and About one third of the cancer in North America and the increasing number of examples of major shifts in Europe is related to the use of cigarettes or other to the organ or tissue distribution of primary cancers bacco products. There is incontrovertible evidence with migration of distinctive ethnic groups from one that chemicals related to the work place or occupa tion are responsible for a segment of cancer in human From the depmmenu of Pathology and Biochemistry, University of To ronto, 100 College St., Toronto, ON M5G 1L5, Canada, where reprint requests should be addressed. Supported by research grants from the National Cancer Institute of Can ada, the Medical Research Council of Canada, and the National Cancer In stitute (U.SA.). 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 AP00024019 1380 THE NEW ENGLAND JOURNAL OF MEDICINE Dec. 3, 1981 v Table 1. Chemicals Known or Strongly Suspected to be Car cinogenic for Human Beings. Chemical Occupational Contacts Aromatic amines Arsenic Asbestos Benzene Bis(chloromethyl) ether Bis(chlorocthyl)sulfide Cadmium Chrome ores Coke ovens Nickel ores Soots, tan, and oils Wood dust Vinyl chloride Srre of Cancer Bladder Skin tad bronchus Bronchus, pleura, and peritoneum Marrow Bronchus Respiratory tract Prostate Bronchus Bronchus Bronchus and nasal sinuses Skin and lungs Nasal sinuses Liver Medical Agents Alkylating agents (c.g., melphaian and cyclophosphamide) Anabolic steroids Arsenic Chlornaphazine Diethylstilbestrol Immunosuppressive drugs Phcnaoetin (acctophenetidin) Bladder Hemstopoi stic tissue Liver Skin Bladder Vagina Lymphoid tissue Renal pelvis Others Aflatoxins Tobacco smoke Oral contraceptives Betel nut and lime Liver Bronchus, mouth, pharynx, larynx, esophagus, bladder, and pancreas Liver Mouth seen in the United States, Canada, and several other Western countries. What about the remaining cancers -- those in the colon, breast, uterus, hematopoietic-lymphoid sys tem, prostate, and other sites, including a substantial proportion of those in the urinary bladder and pan creas? Can these be closely related to the chemical contamination of the air, water, and food that has been occurring in the industrialized countries? 4'5 No definitive answer is available. Evidence can be offered in favor of such a view or against it. On the positive side are the following considera tions: the known relation, mentioned above, between many different types of cancer and exposure to chem icals in tobacco and in the work place; the wide spread presence of chemical mutagens and potential carcinogens in the environment in industrialized countries, as monitored by short-term in vitro tests in prokaryotes or eukaryotes; the observations that some religious groups in Utah and elsewhere, who have dif ferent patterns of tobacco and alcohol use and differ ent codes of behavior, appear to have a lower inci dence of some forms of cancer in the respiratory, gastrointestinal, and genitourinary systems4; the in creasing evidence that cancers of many systems, re sembling cancers in human beings, can be induced by chemical carcinogens in one or more laboratory ani mals; and the observations that cancers of the esoph agus or liver have an unusually high incidence in some geographic locations in China, and that domestic ani mals in close contact with human beings in these lo cations frequently have similar cancers in the same anatomic sites.1'10 On the negative side, no clear-cut clinical experi ence or epidemiologic evidence has been presented to implicate environmental chemicals as causative agents for the wide variety of cancers seen clinically. According to German" and Cairns,11 patients with xeroderma pigmentosum, who have a defect in their fibroblasts in the repair of DNA damage by some chemicals as well as by ultraviolet light, have no ele vated risk of cancer in orgarts or tissues other than the skin. If the general environmental exposure to chemi cals were effective in the causation of cancer, such pa tients could be expected to have elevated incidences of cancer not related to ultraviolet light. In some can cers, such as leukemia and cancers of the stomach and cervix, there were decreases in age-adjusted inci dence and in mortality from 1969 to 1971 and from 1973 to 1976 among white persons in the United States, while there were only small increases in most of the other tumors, as compared with cancers of the lung and with melanoma." No increase comparable to that of lung cancer has been seen in the past two decades in the patterns of cancer incidence in the United States. None of the above considerations are by any means conclusive. Most of the arguments on either side have enough contingencies to leave the question in major doubt. The close monitoring of cancer occurrence over the next two decades may allow a more definitive po sition to be taken. In the meantime, a greater under standing of how chemicals are involved in cancer de velopment would seem important in helping us to arrive at' a conclusive position. In addition, the large increase in the use of chemi cal carcinogens in the development of new experi mental models for the study of the possible cause, de velopment (pathogenesis), or treatment of cancers in specific organ or tissue sites in human beings further emphasizes the importance of the study of chemical carcinogenesis. Such studies should generate a much more critical understanding of the relative roles of exogenous and endogenous factors, including diet and hormones, in cancer development and behavior. The orientation in this presentation will be mecha nistic, not descriptive, and will attempt to relate fun damental concepts and understanding derived from experimentation in animals and from other sources to the causation, prevention, and (to some degree) ther apy of cancer in human beings. For this reason, little coverage will be given to the many different types of chemical carcinogens themselves or to their distri bution.14 Cancer as a Chronic Multistage Process It is now appreciated that the development of cancer in human beings in various sites takes many years and that progressive tissue and cellular changes AP00024020 Vol. 305 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 prcmalignant cells to highly malignant neoplastic cells.'5'17 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.17-1* 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.I5"17'20 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 development 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-naphfhylamine 05-naphthyl- amine). 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.23 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 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 I Cytochrom> P450 system 1 (mixed (unction 7 9 * oxygenm (MFOJ) 8*nzo(4)pyrene (BP) 6 oio - (MFO) HO-' BP 7.8-diol*9.lO-poxiti0 BP 7.8-dihydrodioi Figure 1. Current View of the Activation of Benzo(a)pyrene (BP) to Benzo(a)pyrene 7,8-Dlhydrodlol,9,10 Epoxide through the Mixed-Function Oxygenase (MFO) System (Cytochrome P-450 System) and Epoxide Hydrolase. I AP0002402I 1382 THE NEW ENGLAND JOURNAL OF MEDICINE Dec. 3, 1981 Other types of metabolic conversions are also con sidered important in carcinogenesis. Nitrosamines arc converted to highly reactive alkylating moieties, prob ably through oxidation. Aromatic or heterocyclic nitro-compounds, such as nitrofurans and 4-nitroquinoline-A'-oxide, probably undergo initial reduction to hydroxy-amino-derivatives. In the case of 4-nitro- quinoline-^'-oxidc, this form is considered to be an ul timate carcinogen. For the majority of known conversions, the system most active in the cell is the "mixed-function oxygen ase" system, consisting of several cytochromes P-450, reduced nicotinamide-adenine dinucleotide phos phate cytochrome reductase, and lipid.24 This induci ble system is located predominantly in the microso mal fraction of the cell (endoplasmic reticulum), although recent work increasingly points to a second comparable system in the nucleus.15 In addition to these oxidative systems, reducing systems also exist for some procarcinogens, such as the aromatic or het erocyclic nitro-compounds. Diaphorase or other re duced nicotinamide-adenine dinucleotide phosphate or reduced nicotinamide-adenine dinucleotide reduc tases are widely distributed among different cells and are effective in reducing the nitro-group to the hy droxy-amino-derivative. The liver is by far the most active and most versa tile organ in the metabolism of procarcinogens and of xenobiotic agents generally. It and other organs have an ability to detoxify potential carcinogens as well as activate them, and the ultimate fate of a chemical de pends largely on the balance between activation and inactivation -- a balance that is easily modulated in major ways by drugs and other chemicals, age, nutri tion, and hormones, as well as genetics.:*'31 For ex ample, the carcinogenicity of several aromatic amines for the liver can be completely prevented by simulta neous exposure to phcnobarbital or 3-methyicholan- threne -- agents that induce many liver enzymes.32-13 This phenomenon of resistance of the induced liver to some carcinogens may be of great practical impor tance to human beings. Virtually all people in the Western world have levels of several xenobiotic agents, such as chlorophenothane, dieldrin, aldrin, polychlorinated biphenyls, and dioxins, in their adi pose and other tissues. These agents as a group are ef fective enzyme inducers in the liver. In addition, many drugs induce various microsomal or other enzymes in the liver or other tissues. Are these inducers making tissues more or less susceptible to the acute toxic or carcinogenic effects of other environmental agents? The organ or tissue distribution of the carcinogenmetabolizing enzymes may be involved in the organ otropism of carcinogens. Since there is an almost certain requirement for activation, and since the met abolic patterns affecting carcinogens and other xeno biotic agents are so variable from cell to cell or from organ to organ, the absolute activities of the various enzymes and especially their relative balance may have key roles in determining which organ will be a target for a particular carcinogen. An interesting ex ample of modulation at this level is the liver-kidney axis with dimethylnitrosamine. This potent carcino gen normally induces liver cancer when taken through the gastrointestinal tract. However, if the animal is placed on a low-protein diet, the hepatic activation and metabolism fall off. This pattern allows more of the carcinogen to be available for action on the kid ney and changes the dominant cancer pattern from the liver to the kidney.34 Clearly, the pathologic consequences of exposure to any potentially toxic xenobiotic are related not only to the pathways available for its metabolism but also to the physiologic state at the time of exposure. Thus, the presence of a known mutagen or carcinogen in the environment is by no means synonymous with a mutagenic or carcinogenic response by the exposed person. Possible Molecular Targets (or Ultimate Carcinogens The molecular targets for the electrophilic react ants are many. To date, the bulk of the effort has been concentrated on DNA, with some interest in RNA and protein. In principle, many other molecular tar gets exist at the macromolecular and "micromolecular" levels. Because of the ease of relating changes in DNA to a permanent change in the biologic behavior of cells, DNA has received the lion's share of atten tion. Since the first discovery of the chemical nature of an adduct of a carcinogen with nucleic acids in a tar get organ in vivo,35 there has been an increasing inter est in establishing the chemical nature and site of in teraction with several different carcinogens.54 As shown in Figure 2, virtually every potential site for in teraction. with DNA bases (as well as with phos phates) reacts with one or more carcinogens. In at tempts to narrow down the possibilities for relevance to cancer, sites that are more likely to be involved in hydrogen bonding between bases (purines and py rimidines) in DNA, and thus to be involved in possi ble miscoding, are often considered most relevant. This presupposes that the major or only biochemical lesion in DNA that is relevant to cancer initiation is a miscoding lesion. This judgment may not be justi fied, since the evidence is circumstantial and incon clusive. Conceivably, other types of alterations in DNA besides miscoding, such as recombinations and gaps, may be important to the early events in cancer development. I23*3, In fact, Cairns has suggested that mutagenesis may be of only minor importance in the initial events in chemical carcinogenesis, and that genetic transposition including relatively large re gions of the genome may be more relevant to this process.12 The application of this newer technolo gy to appropriate "clean" cell populations during the development of cancer is to be awaited with interest. Another important aspect is the site of interaction of the ultimate carcinogen within the long DNA mol ecules. With several different approaches, such as AP00024022 (< Vol. 305 No. 23 CHEMICAL CARCINOGENESIS - FARBER 1383 UW.BtU.IN**a 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-nltrosourea; ENU, N-ethyl-N-nitrosourea; ENNG, N-ethyl-N '-nitro-N-nitrosoguanidine; 7 Br MBA, 7bromothyl-benzo(a)anthracene; BP epoxide, benzo(a)pyrene 7,8-dihydrodlol,0,10 epoxide; 4NQO, 4-nitroquinoline-Noxide; DMS, dimethylsulfate; DMN, dimethylnitrosamine; MMS, methy! methanesulfonate; MNNG, N-methyl-N'-nitroN-nitrosoguanidine; 1.2.DMH, 1,2-dimethylhydrazine; DEN, dlethylnltrosamlne; N-OH-1-naphthylamine, N-hydroxy-1naphthylamine; 2 AAF, 2-acetylaminolluorene; MAM, methylazoxymethanol; EMS, ethyl methane sulfonate; BPL. 0-pro- piolactone; and MAB, 4-methylaminoazobenzene. studies of susceptibility to nucleases, separation of transcriptionally active from inactive DNA, or isola- ., tion of linker and nucleosome regions in chromatin, some of the interactions of carcinogens with DNA have been found to be nonrandom. However, no single region of high affinity has been found." The use of DNA cloning for specific genes in biologically welldefined prcneoplastic and early neoplastic popula tions should be profitable. DNA Repair In view of the essentially irreversible nature of initi ation with chemicals and the apparent focal nature of the initiation process, major emphasis at the molecu lar level is given to DNA as a probable target in ini tiation. However, the evidence is largely circum stantial. The bulk of evidence comes from studies in pa tients with xeroderma pigmentosum, who have a high incidence of skin cancers. The etioiogic agent is ultra violet light, and the vast majority of patients have a defect in their ability to repair the damage inflicted on DNA by this form of radiation. Such patients can be protected from skin cancer by careful avoidance of ex posure to ultraviolet light.'"' Fibroblast cultures from these patients have increased sensitivity not only to ul traviolet light but also to some chemical carcinogens and mutagens.41 Other diseases (e.g., ataxia-telangi ectasia and Fanconi's anemia) also involve deficien cies in DNA repair as well as an increased risk of neo plasia.42 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.45 ' 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,12 since fibro blasts from patients with xeroderma pigmentosum show a defect in the repair of DNA damage by some 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.13 An alternative hypothesis must be enter tained: that the role of repair may be quite different in the dynamics of cancer induction by chemicals, as compared with induction by ultraviolet light. The past 10 years have seen a large expansion of studies of DNA repair, largely in vitro. These studies should lead to a much clearer delineation of the types of repair that may occur in carcinogenesis. What does happen remains poorly understood. Processes of re pair by base excision, by "long-patch versus shortpatch" removal, by possible recombination or other forms of post-replication repair, by removal of interstrand cross-links or other more complex forms of damage, and by other mechanisms are slowly being studied in many different systems in vitro and to a much lesser degree in vivo.34-41 The enzymology, al though difficult, is also slowly becoming clear. In the case of methylating carcinogens, an interesting recent finding is the discovery of an enzyme in bacteria that removes the methyl group from the 0` position of gua nine in DNA by transmethylation with its transfer to a sulfur-containing moiety in protein.46 This raises the question of whether some of the enzymes involved in repair of DNA damage perform physiologic functions other than repair that subserve normal cellular re quirements. Obvious failings in many of the attempts to corre late patterns of DNA repair with carcinogenicity in clude the wide gap between the chemistry and the bi- i AP00024023 1384 THE NEW ENGLAND JOURNAL OF MEDICINE Dec. 3, 1981 ology and the primitive state of the art of identifying and quantitating discrete biologic steps in the carci nogenic process. Carcinogen Metabolism In Human Tissues A gratifying aspect of recent studies of the early chemical and biochemical events in carcinogenesis is the overall similarity in patterns of activation and types of adducts seen in tissues from human beings and in those from laboratory animals. Studies with some nitrosamines, benzo(a)pyrene, aflatoxin B,, and other carcinogens47-53 have shown that human tissues in general resemble one or more animal tissues in their patterns of activation and in the type of carcinogen-DNA adducts formed. An interesting development that may help to clarify some aspects of this important phase of activation is the use of radioimmunoassays for specific adducts in DNA, especially with highly sensitive new ap proaches to radioimmunoassay.S4"54 Such detection of minute quantities of carcinogen-induced adducts in DNA opens up the possibility of studying exposure patterns to carcinogens. This technique, coupled with adequate medical examination and appropriate epi demiologic studies, might well offer new approaches to the ultimate problem of risk assessment. Although it is far too early to discuss such possibilities except in general terms, adduct formation is an index of expo sure, not initiation, and persistence of a chemical le sion in DNA is by no means synonymous with cancer development. As discussed below, initiation of the car cinogenic process is not an inevitable consequence of adduct formation. Human cells resisted in vitro transformation with chemicals for several years. However, the past three years have seen positive results in some human-cell systems.57-41 Initiation In the classical system of skin carcinogenesis in mice and rabbits, a single or brief exposure to a car cinogen induces some change in the tissues, called ini tiation, such that focal proliferations called papillo mas can be made to appear by application of croton oil or other agents called promoting agents, which by themselves are noncarcinogenic or only weakly car cinogenic. More recently, this phenomenon was also found in several other organs, such as the liver, brain, mammary gland, colon, and urinary bladder.'4'" Such focal proliferations (papillomas, polyps, or hyperplastic nodules), in turn, are sites for further evolution to neoplasia. The term neoplastic refers to a cell that can proliferate without the need for an added or known stimulus for growth -- i.e., a cell that has acquired some degree of autonomy. Initiation at the skin, liver, and mammary gland, and probably at most sites, is generally permanent. This suggests that the very early carcinogen-induced altered cells are not recognized by the host in a manner that leads to their destruction, and it raises serious doubts about the va lidity of the concept of immune surveillance at an early stage in carcinogenesis,42 as has been proposed for host control of cancer development.41 These observations are most easily interpreted as reflecting some permanent change in the DNA in the rare cell-affected during initiation in vivo. Whether the change is a mutation in a structural or regulatory seg ment of DNA44'44 or whether it involves more complex rearrangements of larger segments of DNA, as in transpositions, remains unknown. Pertinent are the observations on transformation initiated with x-rays or with polycyclic aromatic hydrocarbons, in which some change or changes other than a mutation in a rare cell appear to be involved as an early step in ini tiation in vitro with a highly selected cell type.47'4* One cannot equate activation of carcinogens leading to DNA-adduct formation with initiation. For example, many carcinogens, such as benzo(a)pyrene and 7,12-dimethylbenz(c)anthracene, can be activat ed by the liver to form adducts without initiating or inducing liver cancer in adult animals. However, if coupled with one round of cell proliferation, many can initiate54-72 and induce liver cancer.73 In vitro, with dif ferent cell systems, cell proliferation early in the car cinogenic process is required for transformation with x-rays,74 some viruses,75 and some chemicals.74 Thus, as shown in Figure 3, initiation is at least a two-step process: a biochemical step that is reparable, fol lowed by a round of cell proliferation that "fixes" some change so as to make it essentially permanent. The cell proliferation can be generated by some pri mary mitogenic stimulus, such as partial hepatectomy, by a chemical mitogen,64 or as a response to cell death.77-The mechanistic role of cell proliferation in initiation is not understood. A popular hypothesis in volves DNA synthesis or replication as somehow fix ing damage in a daughter strand. The probable requirement for cell proliferation for initiation has important implications for cancer de velopment in many sites. In the nonproliferating tissues, such as the pancreas, salivary gland, liver, kid ney, urinary bladder, and brain, an important ratelimiting step for beginning the carcinogenic process may be cell proliferation. Thus, concomitant cell ne- IM - AN** CNm PPtMoU^ltWokNfoA Figure 3. Steps In the Initiation of Carcinogenesis with Chemicals. The listings under "Initiation" refer to some properties either used in the next phase, promotion (liver), or associated with the initiated tissue (skin or colon). AP00024024 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 gens7' 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 development7''*0 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.15'17 In most systems, the properties critical to initiation remain unknown. In two continuously proliferating tissues, the skin*1 and the colon,*8 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.17'*1 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" and several pesticides, herbicides, and other xenobiotics*5 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 pleiotrqpic. Recently, with the use of a less active phorbol ester, 5H-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, 12-0-tetradecanoylphorbol-13-acetate.**''4 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 Promotlon 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.'8 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.17''3'" 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- AP00024025 1386 THE NEW ENGLAND JOURNAL OF MEDICINE Dec. 3, 1981 motion over and above that related to the cell cycle itself remains an interesting challenge. There is a growing belief that one of the major fac tors in determining cancer development is the pres ence and intensity of promoting environments.'4 In addition to some specific agents such as phenobarbital, butylated hydroxytoluene, and polychlorinated biphenyls for the liver," prolactin for the mammary gland," and bile acids for the colon,'7" diet has been found to influence carcinogenesis in several sys tems.1'3 However, the specific roles of individual die tary components and of their balances remain to be established in most instances. There is also a suspicion on the part of some inves tigators that tissues or organs may create a physio logic promoting or selecting environment. The most provocative evidence relates to carcinogenesis in the colon"'100 and the urinary tract.'01'101 In each of these locations, diversion of the normal movement of con tents has been found to lead to a decreased incidence of neoplasia with known carcinogens. There are several examples in which synergism or promotion may be operating in human beings. For ex ample, exposure to asbestos, nickel, or uranium may be associated with only a relatively low risk of lung cancer. However, when coupled with cigarette smok ing, the risk becomes extremely large -- much larger than that associated with smoking alone. Subsequent Steps Progression Focal proliferative lesions resulting from a promot ing environment, such as papillomas, nodules, or pol yps, undergo a number of further changes before ma lignant behavior is expressed. In many organs, including the bronchial tree in the smoker, one consistently sees several types of putative precancerous lesions, such as atypical hyperplasias, dysplasias, and carcinoma in situ.1*1' How these le sions relate to each other and to altered biologic be havior of cells transformed in vitro (e.g., atypical growth, growth in soft agar, or tumorigenicity) is not clear and remains a potentially fruitful area for study. An important property of many of these changes is reversibility. There is considerable evidence in exper imental models and in human beings that some focal proliferative lesions, such as hyperplastic nodules in the liver (and their probable human equivalent, liver "adenomas," which are seen rarely in women using oral contraceptives), polyps in the colon, and papillo mas in the skin, can undergo regression or remodel ing with normal differentiation.17'103 This area is be coming of particular interest in view of the reported efficacy of vitamin A analogues, such as cw-rctinoic acid, in preventing or delaying cancer development initiated with chemical carcinogens104 in several sites, such as the bronchus and the urinary bladder. Transfection An interesting recent development is the induction of transformation by the transfer of DNA or chroma tin to susceptible cell lines. DNA from several types of cells transformed in vitro or in vivo with different car cinogens was shown to induce transformation.I0s.i< Of great interest is the observation that DNA from nor mal cells was also effective.104 These new approaches to chemical carcinogenesis open up many areas of ex ploration, including the question of whether the in formation contributions of the DNA are positive (i.e., they code for an identifiable component that is im portant in cell transformation) or negative (i.e., they disorganize the host geijiome in an appropriate manner). Diet and Carcinogenesis The composition of the diet may influence the inci dence of cancer in some sites, such as the colon, breast, and endometrium.107-10' The mechanism or mechanisms through which diets exert their influ ences are not well understood. In some instances, car cinogens occur in the food as natural substances, as contaminants (e.g., aflatoxins), or as products of foodpreparation methods (e.g., pyrolysis). Dietary altera tions also modulate the activation of carcinogens. In other instances, micronutrients have a role in the endogenous formation of carcinogens. The past 10 years or so have seen a revival of the concept of the en dogenous formation of carcinogens. This hypothesis was proposed in the 1930s in relation to cholesterol or other sterols, but it was progressively abandoned as the evidence failed to materialize. The resurrec tion of this idea assumes a new form in the genesis of nitroso-compounds from nitrite and secondary or tertiary amines.110 Dietary amines or drugs such as oxytetracycline of chlorpromazine can react with ni trous acid, generated in the stomach from nitrite, to form nitroso-compounds. Some of these substances are carcinogenic in laboratory animals. The forma tion of such nitroso-compounds can be effectively in hibited by dietary ascorbic acid111 and by vitamin E. This general model is being explored in colon cancer in human beings."1 The hypothesis states that carcinogens can be generated in the feces and that the levels of the carcinogens, as measured by mutagenic action, are influenced by the dietary composition, in cluding vitamins C and E and fiber content. Chemotherapy and Carcinogenesis Some of the most effective chemotherapeutic agents for cancer are carcinogenic. For the treatment of can cer in patients above 50 or 60 years old, the impor tance of the risk of carcinogenicity is clearly minimal, given the long latent period for cancer development. However, in children and young adults, the devel opment of second cancers years after the effective treatment of the primary tumor is now becoming a recognizable problem."13'114 Over 25 drugs used in chemotherapy have been found to be carcinogenic in animals, and some have also been implicated in human beings. The magnitude of the risk associated with the many treatment regimens is not fully known. However, for AP00024026 t 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,n',s 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.11* 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.l,,", 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 arc 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 postinitiation 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. 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Promoting ef fect of bile acids on colon carcinogenesis after intrarecta! installation of V-methyl-A^'-nitro-AZ-nitrosoguanidine in rats. J Natl Cancer Inst. 1974;53:1093-7. 98. Reddy BS. Watanabe K, Weisburger JH, Wynder EL. Promoting ef fect of bile acids in colon carcinogenesis in germ-free and convention al F344 rats. Cancer Res. 1977; 37:3238-42. 99. Navarrette A, Spjut HJ. Effect of colostomy on experimentally pro duced neoplasms of the colon of the rat. Cancer. 1967; 20:1466-72. 100. Gennaro AR, Villanueva R, Sukonthamon Y, Vathanophas V, Rosemond GP. Chemical carcinogenesis in transposed intestinal segments. Cancer Res. 1973; 33:53641. 101. Oyasu R, Hirao Y, Izumi K. Promqtion by urine of urinary bladder carcinogenesis. Proc Am Assoc Cancer Res Am SocClin Oncol. 1980; 21:70. abstract. 102. Rowland RG, Henneberry MO, Oyasu R, Grtybtck JT. Effects of urine and continued exposure to carcinogen on progression of early neoplastic urinary bladder lesions. 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New York: Cold Spring Harbor Laboratory, 1977:557-65. 108. Wynder EL, Reddy BS. Diet and cancer of the colon. In: Winick M, ed. Current concqpti in nutrition. New York: John Wiley, 1977; 6:5571. 109. Carroll KK. Lipids and carcinogenesis. J Environ Pathol Toxicol. 1980; 3:353-71. 110. Lijinsky W. Significance of In vivo formation of N-nitroso com pounds. Oncology. 1980; 37:223-6. 111. Mirvish SS. Formation of W-nitroso compounds: chemistry, kinetics and iit vivo occurrence. Toxicol Appl Pharmacol. 1975; 31:325-51. 112. Bruce WR, Ey&scn GM, Ciampi A, Dion PW, Boyd N. Strategies for dietary intervention studies in colon cancer. Cancer. 1981; 47:11215. 113. Harris CC. A delayed complication of cancer therapy -- cancer. J Natl Cancer Inst. 1979; 63:275-7. 114. Hoover R, Fraumeni JF Jr. Drugs.* 185-98. 115. Meadows AT, Krejmas NL, Belasco JB. The medical cost of cure: se quelae in survivors of childhood cancer. In: van Eys J, Sullivan MP, eds. Status of the curability of childhood cancers. New York: Raven Press, 1980:263-76. 116. Stich HF. San RHC, eds. Short-term testa for chemical carcinogens. New York: Springer-Verlag, 1981. 117. Hollstein MJ, McCann J. Angelosanto FA, Nichols WW. Short-term test for carcinogens and mutagens. Mutat Res. 1979; 65:133-226. 118. Bartsch H, Malaveille C, Camus A-M, et al. Validation and compara tive studies on 180 chemicals with S. typhimurium strains and V79 Chi nese hamster cells in the presence of various metabolizing systems. Mutat Res. 1980; 76:1-50. AP00024029 1390 THE NEW ENGLAND JOURNAL OF MEDICINE Dec. 3, 1981 PULMONARY ALVEOLAR PROTEINOSIS IN FOUR SIBLINGS Kuldeep Teja, M.D., Phiup H. Cooper, M.D., Jerry E. Squires, M.D., and Patricia T. Schnatterly, M.S. "pULMONARY alveolar proteinosis (PAP) is a distinctive pathologic lesion in which alveoli are filled with periodic acid-Schifl-positive proteinaceous material. It is usually idiopathic, but it has been re ported in association with a variety of other conditions.1'1 PAP is usually seen in adults, although the first description of cases included a young child.' Since then, there have been several reports of PAP in infants and children, and the condition has been found at birth.' This report describes four siblings in whom PAP developed during infancy. Our review of the litera ture disclosed several other affected sibships, yet the familial aspect of PAP has been only briefly consid ered.10 Analysis of the foregoing observations sug gests that some cases of PAP may have a genetic basis, with a possible autosomal-recessive inheritance. Case Reports The patients were the first four children of a healthy black cou ple. The parents are second cousins (Fig. 1). The mother was in her teens during all four pregnancies and had preeclampsia in the third trimester of each. Despite normal gestational periods as deter mined by history, the birth weights of the infants ranged from 1540 to 2320 g. The parents subsequently had two additional healthy off spring who had normal birth weights. These pregnancies were not accompanied by preeclampsia. We identified no environmental ex posure affecting the parents or their children. The parents were not aware of illnesses in other close family members. Patient 1 This girl seemed well until she was seven months old, when she was admitted to the University of Virginia Hospital because of vom iting and fever. Respiratory distress developed, and chest radio graphs showed pneumonitis. The white-cell count was 29,100 per cubic millimeter. Cultures of blood, urine, stool, and cerebrospinal Quid were negative. The patient was treated with penicillin and dis charged. She was readmitted three weeks later because of cough, vomiting, and diarrhea. A skin test with purified protein derivative of tuberculin (PPD) and a throat culture were negative. Respirato ry distress worsened, and the patient died on the seventh hospital day. An autopsy was not performed. Patient 2 This six-month-old girl was admitted to the University of Virgin ia Hospital with anemia and cough. The chest was normal by phys ical examination, but the respiration rate was 60 per minute. Radiographs showed diffuse, stringy pulmonary densities. The white-cell count was 24,300 per cubic millimeter. Blood transfu sions, iron, and antibiotics led to clinical improvement, but the ra diographic appearance did not change. Cultures of blood, urine, stool, and cerebrospinal fluid were negative, as were skin tests with PPD and fungal antigens. Serum protein electrophoresis gave nor mal results. The patient was discharged, but respiratory distress necessitated readmission. Her respiratory status worsened, and she died on the fifth hospital day. Ah autopsy was performed. Patient 3 This 16-month-old boy was admitted to the University of Vir ginia Hospital for failure to thrive. He appeared cachectic, and there was clubbing of the fingers. Chest radiographs showed bilat eral pulmonary infiltrates with hilar prominence. Negative labora tory studies included a sweat test, skin tests with PPD and fungal antigens, and serum protein electrophoresis. The hematocrit was 36 per cent, and the white-cell count 14,500 per cubic millimeter. An open-lung biopsy was performed. During the next 14 Vi years, the radiographic findings showed progression, with fibrosis and honey combing. When the patient was 16 years old, he was again admitted to the University of Virginia Hospital because of acute pneumonitis. An open-lung biopsy was performed. The patient was treated with antibiotics and discharged. During the next 26 months he had mod erate intolerance to exercise and required treatment for recurrent respiratory-tract infections. The patient was readmitted when he was 19 years old because of extreme dyspnea. Radiographs showed a large area of consolida- 6J From the departments of Pathology and Pediatrics, University of Vir ginia School of Medicine. Addtess reprint requests to Dr. Teja at the De partment of Pathology, Boa 214, University of Virginia School of Medicine, Charlottesville, VA 22908. Squares denote males, circles females, and shaded symbols affected children. The year of birth of each sibling is indicated. AP00024030 NOTICE oT * material may b* MHHBi Use of Serum Bile Acids in the Identification of Vinyl Chloride Hepatotoxicity GARY M. USS, M.D., M.S.RICHARD A. GREENBERG, Ph.D. CARLO H. TAMBURRO, M.D. Louisville. Kentucky From the Uver Research Center, Division of Gastroenterology/Hepatology, Department of Medicine, and the Divisions of Occupational Health and Epidemiology and Blostatlstlcs, Department Ol Community Health, University of Louisville School of Medicine, Louisville, Kentucky. This work was supported in part by National Cancer Institute contract N01-CN-5512 and was pre sented in part at the American Public Health As sociation Annual Meeting. November 1982. Montreal, Canada. Dr. Uss was a Resident in Oc cupational Medicine, Department of Environmental Health, University of Cincinnati Medical Center at the time of this study. Requests for reprints should be addressed to Dr. Carlo H. Tamburo, University of Louisville School of Medicine, Uver Research Center, HSC 55A Room 119, Louisville, Kentucky 40292. Manuscript accepted July 19,1984. * Current address: Special Studies and Services Branch. Mirtstry of taboir. 400 LWversity Avenue, Toronto, Ontario, Canada. Most previous studies proposing serum bile acids as Indicators of hepatic function have been performed in hospitalized patients in whom overt symptomatic liver disease was present. The ability of fasting levels of serum bile acids to identify mild, clinically inapparent chemical liver injury in an occupational setting was compared with that of indocyanine green clearance and routine biochemical liver tests in 67 asymptomatic chemical workers in whom liver biopsies had been performed for medical indications. Histologically, 15 were found to have chemical liver injury, 27 had nonchemical liver dis ease, and 25 were normal. Two serum bile acids, cholylglyclne and conjugates of cholic acid, were determined by radioimmunoassay, using 466 "normal" males from the same worker cohort as a ref erence range. The geometric mean concentrations of cholylglyclne in patients with chemical liver injury, patients with nonchemical liver disease, and normal subjects were 47.9,19.1, and 20.0 fig/dl, re spectively (p = 0.036 by analysis of variance). Conjugates of cholic acfd showed similar differences (p = 0.027), as did indocyanine green clearance with mean halt-life oi 4.2, 3.2, and 3.3 minutes in the three biopsy subgroups, respectively (p = 0.043). Such differ ences were not observed for biochemical liver tests. The fasting level of serum bile acids provided high specificity but lower sensitivity in ihe detection of ail types of liver disease. However, serum bile acids and indocyanine green clearance provided a higher specificity and sensillvity for chemical liver injury than for nonchemical liver disease. An index of average exposure to vinyl chloride was sig nificantly greater in the subgroup with chemical liver injury than In the other two groups, further supporting the association of chemical type injury with impaired anion uptake. These data identify the fasting level of serum bile acids as a clinically usable indicator of early chemical injury in chemically exposed asymptomatic worker populations with liver dysfunction. Further investigation is needed in other occupational hepatotoxlc environments to determine if this association is limited to vinyl monomer type injury. Occupational and nonoccupationaf exposure to hepatotoxic agents can lead to acute, subacute, or chronic liver Injury. The decrease in acute occupational hepatic injury in recent decades has focused concern that chronic hepatic disease, including malignant neoplasms, may develop as a result of prolonged low-level industrial exposure [ 1,2]. Currently, there are no specific clinical means to identify hepatic injury from chronic low-level exposure to chemical hepatotoxins. The inability of standard biochemical enzyme studies to identify the early phases or progression of liver injury has been shown [3-10]. M January 198S The American Journal e< Medicine Volume 78 l AP00024031 ble acio identification of chemical hepatotoxicity--uss et al Serum enzyme studies are of limited value because they primarily reflect acute disruption of cell membrane integrity (liver cell "leaking") rather than the uptake, metabolism, storage, or excretion functions of the liver cell [8]. In subacute, chronic, and end-stage liver dis ease, the enzymes often return to normal levels after initial elevations [9], and fail to reflect the decreased functional capacity of the remaining parenchyma. En zyme levels can also be increased in nonhepatic dis eases. In severe hepatic injury (fulminant hepatitis or severe toxic necrosis), decreasing enzyme levels may reflect worsening disease [8]. At present, measuring clearance rates of substances primarily or solely removed from circulation by the liver provides the most sensitive and specific indicator of liver function [5]. Such substances include exogenous anionic dyes (bromosulphthalein or indocyanine green) and endogenous metabolites (serum bile acids). How ever, few studies have examined the ability of these tests to identify early subclinical liver disease In asymptomatic populations. The discovery of vinyl chloride-related hepatic an giosarcoma and nonmalignant hepatocellular injury in 1974 [11] provided an opportunity to study chronic occupational chemical liver injury. Recent studies [4,10] have shown that indocyanine green clearance provides the best combined sensitivity and specificity for detecting persons with subclinical hepatic disease. However, this test has certain limitations. It is invasive, involves intravenous injection of a synthetic dye, and requires multiple blood samplings. Further, blood samples are best analyzed the same day and not stored for prolonged periods of time due to indocyanine green degradation. Serum bile acids, which have been proposed as a clinical test of hepatic function, are natural substances, cleared only by the liver. Bile acid studies have been conducted in the fasting [12-16] and postprandial [12,13,17,18] states and following exogenous loading, either by the intravenous [19-22] or, more recently, the oral [23,24] route. Most of these studies, however, have been conducted in persons with overt disease with liver damage of mixed origins. The objectives of this study were to determine if serum bile acid levels could identify chemical workers who had had chemical liver injury after exposure to vinyl chloride and other vinyl mono mers, and to compare the capability of bile acid levels with those of indocyanine green dye clearance and conventional biochemical tests. PATIENTS AND METHODS The study population consisted of 1,200 employees of a vinyl chloride/synthetic rubber manufacturing plant. The occu pational surveillance program that monitored these vinyl monomer workers for a seven-year period provided medical data including annual history,- physical examination, 45 bio chemical and hematologic parameters, urine analysis, chest radiography, liver/spleen scanning, indocyanine green dye clearance tests, and serologic studies for viral infection. Fasting serum samples were obtained from all employees who participated in the screening program. Criteria for par ticipation in this bile acid study included male employees who had worked for one or more years, had undergonejthe con ventional biochemical screening tests, had indocyanine green clearance test performed at the low-dose (0.5 mg/kg) level, and had fasting serum samples available for bile acid deter mination. The latter two tests had to have been performed on the same day. A total of 589 employees met these inclusion criteria. At Ihe beginning of the study, prior to any serum bile acid de terminations, these employees were divided into three groups on the basis of the results of medical examination, liver/ 6pleen scanning, and enzyme studies. Employees who had undergone cholecystectomy were excluded. None had had bowel resection. Group I included 466 employees who had no clinical, radiologic, serologic, or biochemical evidence of liver disease; Group II included 56 workers who had re peated biochemical test abnormalities consistent with liver injury but in whom no liver biopsies had been performed; and Group III consisted of 67 employees who had liver biopsies performed, 30 because of biochemical abnormalities, 24 because of radiologic abnormalities, and 13 Incidental biopsies performed during norv-hepatobiliary abdominal surgery. Group III forms the basis for this report. Serum bile acids, cholylglycine and conjugates of cholic acid, were determined by radioimmunoassay. Radioimmu noassay method used had the same selectivity for cholyl glycine as the present-day radioimmunoassay (Diagnostic Kits, Abbott Laboratories, Chicago, Illinois). All assays were performed at one time. In the healthy workers (Group I), Ihe normal range [25] for cholylglycine was 0 to 48 ftg/dl, and 0 to 74 /tg/dl for conjugates of cholic acid. The indocyanine green clearance, expressed as the half-time, was'calculated following intravenous injection of 0.5 mg/kg indocyanine green as previously described [26,27]. The normal range in the same standard population is 1.8 to 3.6 minutes [28]. The following biochemical tests were performed in serum using standard methods: alkaline phosphatase, alanine amino transferase. aspartate aminotransferase, gamma-glutamyl transpeptidase. The normal ranges used were also those previously described in this population [28], All biochemical data from Group I did not significantly differ from generally published norms of similar occupational and general popu lations [5,6,8], Biopsy results were Interpreted in a double-blind duplica tive fashion as previously described [29]. All were classified, a priori into one of three categories; (1) chemical liver injury, (2) nonchemical liver disease, or (3) normal biopsy results, on the basis of known histologic features of vinyl chlorideassociated chemical liver injury [3,29-31]. These Include focal hepatocellular hyperplasia, mixed focal hyperplasia with focal Increased reticulum and/or collagen, and focal sinus oidal dilatation and vascular changes. All Indocyanine green clearance and serum bile acid de- * January 1985 The American Journal of Medicine Volume 78 S9 AP00024032 BILE ACID IDENTIFICATION OF CHEMICAL H6PATOTOXICITY--USS ET AL Figure 1. Diagnostic scheme of subdivision of chemical workers into study groups. terminations in a person were from blood samples drawn on the same day. The biochemical blood studies were not per formed at the same time as indocyanine green clearance and serum bile acid tests in all cases. In 44 (66.7 percent) of 66 patients, serum samples for Indocyanine green clearance, bile acid determination, and biochemical studies were ob tained from blood samples drawn on the same day. The re mainder were obtained from blood samples drawn within six weeks. The date of biochemical tests chosen for analysis was that Closest to the serum bile acids/indocyanine green clearance test date. To assess If this had any effect, the re sults for thosa persons with versus those without simulta neous data ware compared for each biopsy subgroup. The time interval between liver biopsy and serum bile acid de terminations varied, but the median interval was similar in the three biopsy subgroups: six months In patients with chemical liver injury, nine months in patients with nonchemical liver disease, and nine months in normal subjects. Chemical exposure was assessed through an exposure ranking system [32], which consists of a work and job classification based on an ordered, six-level exposure rating regarding the intensity of exposure to 22 potentially hepatotoxic chemicals, including vinyl chloride. Cumulative ex posure rank months and average exposure indexes were determined for each employee. For analysis, logarithmic transformation was performed when skewed distributions were encountered (serum bile acids, biochemical data). Differences among the three biopsy groups were assessed by analysis of variance on the logtransformed data. The differences In exposure indexes be tween biopsy groups were assessed as described by Mantel and Haenszel [33] and confidence limits for the odds ratio were calculated with the pro^am of Rothman and Boiee [34]. A tost for linear trend in proportions of liver function abnor malities with cumulative exposure categories was assessed by the method of Fleiss [35]. The Kruskal-Wallis test was used to compare ages between biopsy subgroups. Hie WIIcoxon rank-sum test was used to compare results between simultaneously and nonslmuttaneously obtained data RESULTS The derivation of the study group from cohort is sum marized in Figure 1. Group I laboratory values were used as the reference group for "normal values range" since all tests were performed in an Identical manner in the same laboratory. The 67 workers who underwent biopsy (Group 111) are further subdivided into 25 with no liver disease (normal biopsy results) and 42 with liver disease on biopsy. This latter subgroup was further subdivided into 15 wiih chemical liver injury and 27 with nonchemical liver disease, usually of viral origin or al cohol-induced. Biopsy findings in the subgroup with nonchemical liver disease consisted of mild or mod erate steatosis and/or portal fibrosis or portal triaditis. Only two patients were described as having "beginning cirrhosis" with steatosis or portal fibrosis. The ages of the workers in the three biopsy subgroups were similar 45.6 2.7 (mean SEM) in patients with chemical liver injury; 46.9 1.9 In patients with nonchemical liver disease; 45.9 1.9 in normal subjects (p >0.8 by Kruskal-Wailis test). An analysis of the levels of the four enzymes, indo cyanine green clearance, and both serum bile acid values revealed no significant differences in results between persons in whom the determinations had been made simultaneously and those in whom it had not This was observed for all three biopsy subgroups (p >0.1 for alt comparisons). The data were thus pooled in further analyses. Serum bile acid levels differed among the three bi opsy subgroups of Group Ilf. Analysis of variance on the log-transformed data revealed significant differences in bile acid levels (p <0.05) among groups. Moreover, considering a priori contrasts, patients with chemical liver injury had the highest mean value, which was significantly greater than that In normal subjects, and therefore in patients with nonchemical liver disease (p <0.05). However, the mean cholylglycine level in pa tients with liver disease as a whole (chemical liver injury and nonchemical liver disease pooled) was not signif icantly different from that in normal subjects. Geometric mean level of cholylglycine was 47.9 pg/dl in patients with chemical liver injury, 19.1 in patients with nonchemical liver disease, and 20.0 in normal subjects. There were simitar findings for conjugates of cholic acid (Table I). The sensitivity and specificity were determined by examining individual serum bile acid values in relation to the normal ranges described earlier. For cholyiglycine, the sensitivity in chemical liver injury was 40 percent and in nonchemical liver disease was 14.8 percent; the specificity was 80 percent based on the normal group. For conjugates of cholic acid, the sen sitivity in chemical liver injury was 26.7 percent and in 70 January 1S The American Journal of Medicine Volume 7* ' .. AP00024033 Ill bile acid identification of chemical HEPATOTOXtCfTY--USS ET AL TABLE I Mean Values lor Serum Bile Acids and Indocyanine Green Dye Clearance, by Biopsy Group Test (normil rang*) Cholyglycine (0-48 ftg/dl) Geometric mean Log,o. mean SEM Conjugates of cholic acid (0-74 fig/HI) Geometric mean Log ,o, mean SEM Indocyanine green clearance (1.8 -3.6 minutes) Mean SEM iopsy Subgroup Honehemieil Uvr Chemical Uvsr Injury (IS) Disease (27) Normal Subjects (25) 47.9 1.68 0.14 19.1 1.28 0.09 20.0 1.30 0.17 45.7 1.66 0.13 17.4 1.24 0.08 19.1 1.2 0.11 4.2 0.6 3.2 0.2 3.3 * 0.2 F 3.52 3.83 3.35 P ^0.038 0.027 0.043 nonchemical liver disease, 7.4 percent; specificity was 92 percent. The indocyanine green clearance half-times revealed similar significant differences among biopsy groups (p <0.05). A priori contrasts again showed that mean in docyanine green clearance was highest in the subgroup with chemical liver injury, significantly greater (p <0.05) than in the normal subgroups or the subgroup with nonchemical liver disease whereas the mean value in patients with liver disease (chemical liver injury and nonchemical liver disease pooled] was not significantly different from that in normal sub|ects (Table I). The sensitivity of indocyanine green clearance was found to be 77.8 percent in chemical liver injury and 26.1 percent in nonchemical liver disease (40.6 percent for all liver disease). Specificity was determined to be 90.5 percent. Analysis of the biochemical data (Table II) revealed significant differences among the three biopsy subgroups (p <0.05 by analysis of variance) for only one enzyme, aspartate aminotransferase. In this case, the mean value was greatest in the subgroup with non chemical liver disease, the opposite of the situation found with serum bile acids and indocyanine green clearance. The relative sensitivity and specificity of serum bile acids (cholylglycine and conjugates of cholic acid) versus standard biochemical tests and indocyanine green clearance in detecting chemical liver injury, nonchemical liver disease, and all liver disease are shown in Figure 2. The top panel shows the sensitivity for identifying all types of liver disease; cholylglycine and conjugates of cholic acid provided relatively lower sensitivity whereas gamma-glutamyl transpeptidase had TABLE II Mean Values for Biochemical Studies, by Biopsy Subgroup / Biopsy Subgroup Test (normal range) Alkaline phosphatase (42-109 units/liter) Geometric mean Log io, mean SEM Aspartate aminotransferase (11-32 units/liter) Geometric mean Log io. mean SEM Alanine aminotransferase (1-36 units/liter) Geometric mean Log io, mean SEM Gamma*glutamyl transpeptidase (10-41 mU/litor) Geometric mean i Logic, mean SEM Nonchemical Liver Chemical Liter Injiay (15) Disease (27) Normal Subjects (25) 87.1 1.94 0.04 24.0 1.38 0.11 17.0 1.23 0.11 44.7 1.65 0.16 77.6 1.89 0.04 31.6 1.50 0.06 25.7 1.41 0.09 43.7 1.64 0.08 66.1 1.82 0.03 20.9 1.32 0.04 13.8 1.14 0.07 25.1 1.40 0.07 F 2.45 3.42 2.89 2.5$ P 0.094 0.039 0.083 0.087 January IMS The American Journal of Medicine Volume 7S 71* AP00024034 B1E ACC DENTIFICATION OF CHEMICAL tEPATOTOXlOTY--USS ET AL << Ut H | Figure 2. Sensitivity and specificity of serum bile acids (cholylglycine and conjugates of cholic acid) versus standard biochemical tests and Indocyanine green clearances in de tecting chemical liver injury, nonchemicai liver injury, and all liverdisease. fiP - alkaline phosphatase: AST" aspartate aminotransferase; ALT= alanine aminotransferase; GGT gamma-glutamyl transpeptidase; CG = cholylglycine; CCA m conjugates of cholic acid; ICG -- indocyanine green. the highest, followed by Indocyanine green clearance. The middle panel shows the sensitivity for chemical liver injury and nonchemicai liver disease; indocyanine green clearance was by far the most sensitive in detecting chemical liver injury, followed by gamma-glutamyl transpeptidase and cholylglycine. The bottom panel illustrates the specificity of the tests; specificity in creased, in tum, with gamma-glutamyl transpeptidase, cholylglycine, alanine aminotransferase, aspartate aminotransferase, indocyanine green, conjugates of cholic acid, and alkaline phosphatase, the latter showing 100 percent specificity. The ratio of frequency of positivity of these test re sults in chemical liver injury relative to nonchemicai liver disease was determined (Figure 3). Levels of the parenchymal enzymes (aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpep tidase) were more frequently elevated in nonchemicai liver disease, whereas results of tests that reflected overall hepatic clearance (serum bile acids, indocyanine green) were far more often positive in chemical liver injury. Examining average exposure indexes in the different biopsy subgroups revealed that the highest exposure rankings (4 or greater) were found in seven (50 percent) of 14 patients with chemical liver injury but in only 11 (21.6 percent) of 51 without chemical liver injury, five (19.2 percent) of 26 with nonchemicai liver disease, and six (24 percent) of 25 normal subjects, supporting the relationship between average and cumulative exposure indexes and the specificity of the histologic findings on liver biopsy. The association of chemical liver injury histologic characteristics with high exposure ranking was statistically significant (chi-square 4,4; odds ratio 3.64; 95 percent confidence interval 1.08, 12.2). 72 January 1985 Ttia American Journal of Medicine Volume 78 Figure 3. The ratio of frequency of positivity in chemical liver injury versus nonchemicai liverdisease. Abbreviations as In Figure 2. AP00024035 in a ' ' ~ *'`n\ - * \f- v- <*'? BIE ACID IDENTIFICATION OF CFEMICAL HEPATOTOXICtTY--LISS ET AL TABLE III Frequency of Abnormality of Liver Function Test Results in Entire Population by Cumulative Vinyl Chloride Exposure Category Vinyl Chloride Exposure Category 20-500 500-900 900-1,200 1.200-1,600 >1,600 Cholytglyclhe 22/238 (9.2%) 8/123(4.9%) 5/62(8.1%) 5/47(10.6%) 4/25(16%) Test Conjugates of Cholic Acid 15/238(6.3%) 2/123(1.6%) 5/62(8.1%) 4/47(8.5%) 3/25(12%) Indocyanine Green Clearance 4/190 (2.1%) 4/115(3.5%) 3/50(6%) , 1/57(1.8%) 2/18(11.1%) Finally, the frequency of abnormalities of liver function in the entire population determined for the various cumulative exposure categories is shown in Table III. A trend is suggested by the increasing fre quency of abnormality with Increasing exposure cate gory (dose-response relationship) for both serum bile acids and indocyanine green clearance. The trend was statistically significant (0.01 <p <0.025) for indocya nine green clearance, but did not quite reach statistical significance for cholylglycine and conjugates of cholic acid. COMMENTS This study has illustrated that mean serum bile acid levels are significantly greater in asymptomatic exposed chemical workers with histologically proved chemical liver injury than in those workers with nonchemical liver disease or with normal findings on biopsy. A similar relationship is seen with indocyanine green clearance values but not with the conventional biochemical tests. In severe or massive hepatic injury, all these tests have high sensitivity. This capability, however, is clinically irrelevant when a test's suitability for the identification of early chemical injury among asymptomatic workers Is concerned. Therefore, the clinical significance of these results is related to the increasing heed for means of identifying the causal agent in chemically exposed persons who are discovered to have persistent liver enzyme abnormalities. Determinations of Indocyanine green clearance and bile acids provide a better means of differentiating the underlying causative agent(s) for Such abnormalities. Liver enzyme abnormalities in the presence of normal results of bile acid studies are more characteristic of asymptomatic persons with histolog ically acute-type cellular injury. In contrast, persistent enzyme abnormalities with elevated levels of serum bile acids are shown to be more indicative of persistent subclinical injury associated with increased collagen deposition and vascular changes due to low-grade chemical exposure. The predictive values of such tests are greatly de pendent upon the prevalence of the disease they are to detect We can provide an approximation of their predictive value only by using a hypothetic model since the actual prevalence of chemical liver injury cannot be determined without obtaining a liver biopsy specimen in ail exposed workers with biochemical abnormalities. If the assumption is made that our biopsy group has Identified all cases of chemical liver injury (the lowest prevalence possible), then the prevalence rate would be 13 per 1,000 exposed workers. If our biopsy group reflects the distribution of disease throughout the cohort and the biochemical screening tests have identified all cases (reasonable assumption since we have three to seven-year follow-up on 90 percent of all workers), then the highest prevalence rate for chemical liver injury would be 30 per 1,000 exposed workers. The positive predictive values for alanine aminotransferase, gamma-glutamyl transpeptidase, conjugates of cholic acid, cholylglycine, and indocyanine green clearance were 5, 7, 9, 6, and 21 percent, respectively, in a working population with the aforementioned assumption regarding prevalence of chemical liver injury. It can be seen that conjugates of cholic acid bile acid test and indocyanine green clearance have better predictive value than the most sensitive (gamma-glutamyl trans peptidase) and most specific (alanine aminotransferase) of enzyme tests. The positive predictive values of these tests are lower than is usually accepted for clinical use. It must be kept in mind, however, that we were screening for latent subclinical liver injury in an asymptomatic pop ulation at high risk of exposure. These persons were not the customary symptomatic patients with clinically overt liver disease seen in the hospital. This is an Important difference. Physicians are often cailed upon to rule out occupationally related hepatic Injury in an asymptomatic person incidentally discov ered to have persistent enzyme abnormalities. A pos itive screening result will either direct the patient to further medical workup for nonoccupational disease or indicate a need to remove the patient from a toxic en vironment until the exact nature of the hepatic dys function is determined. Serum bile acids, especially If January 19*5 Tha American journal of Medicine Volume 7S 73 AP00024036 B1L ACID IDENTIFICATION OF CHEMICAL tePATOTOXIOTY--LISS ET AL conjugates of cholic acid, provide a suitable substitute for indocyanine green clearance as a means of differ* entiating these types of causes. Essentially all previous studies examining serum bile acids considered hospitalized or ill patients or at least persons being assessed for overt clinical liver disease. Moreover, comparisons between these studies are limited by the heterogeneous types of hepatic disease considered and the methods of serum bile acid deter* mination (enzymatic and gas-liquid chromatography) that preceded radioimmunoassay. Although it Is not certain that vinyl chloride monomer exposure alone is accountable for ail the differences observed, several pieces of evidence suggest it. First the subgroups were formed on the basis of "a priori" identification of pathologic features shown to be char acteristic of vinyl chloride-type chemical liver injury. Second, greater mean cumulative and average expo sure ratings for vinyl chloride (but not for any other of 22 chemicals assessed) were observed in the group with chemical liver injury. Third, in this analysis, tissue documentation was used as a criterion. The alcoholrelated type Injury was diagnosed in over half the pa tients with nonchemical liver disease and in none of the subgroups with chemical liver injury. Thus, alcohol is not a likely explanation for the observed differences. Fourth, there is a strong suggestion of a dose-response relationship between the frequency of abnormality of serum bile acid and indocyanine green clearance test results and increasing cumulative exposure category. However, we have observed but do not yet have a de finitive explanation for the relatively high proportion of abnormalities in the low exposure category in the overall worker population. Future sequential studies of these abnormalities may provide an explanation. The differences in the frequency with which results of clearance tests versus enzyme tests were positive in the two groups with liver disease (chemical liver injury/nonchemical liver disease ratio less than 1) prob ably reflect the more cytotoxic nature of the cellular injury seen in nonchemical liver disease, due to alcohol, drugs, and/or viral hepatitis. On the other hand, long term, low-level chemical injury to the liver tends to have less observable cytotoxicity and more evidence of chronic injury with fibrotic repair. The less frequent enzyme elevations and more frequently abnormal serum bile acid levels and indocyanine green clearance (chemical liver injury/nonchemical liver disease ratio greater than 1) better reflect the type of overall func tional impairment of the hepatocytes. Although there were no significant differences in the biochemical studies between the subgroup with non chemical liver disease and the normal subgroup, the mean value for all tests' was greater in the subgroup with nonchemical liver disease than in the normal subgroup. The absence of significance is not unusual considering the mild nature of the liver injury in the subgroup with nonchemica! liver disease and the variability in enzyme test values. It is not unexpected that the sensitivity of serum bile acids found in our present study is below that found in various studies in the literature [12]. The subjects in those studies wjere, for the most part, symptomatic hospitalized patients with more cytotoxically severe and acute disease. A few studies have attempted to look primarily at anicteric subjects or those with minor hepatic dysfunction. In 16 anicteric subjects with chronic liver disease (chronic active hepatitis, al coholic cirrhosis, and primary biliary cirrhosis) studied by Gilmore and Thompson [24], fasting concentrations of bile acids by the enzymatic method were abnormal In 10 (63 percent). Kobayashi et al [36] Investigated 70 patients with histologic evidence of liver disease but with normal results of routine liver function tests. They found a sensitivity of 24 percent for fasting serum bile acid concentrations (enzymatic method), which com pares closely with that in our study. Their patients in cluded 10 with minimal methotrexate-induced liver in jury, 13 with hepatic sarcoidosis, seven with resolving viral hepatitis with bridging necrosis, 12 with fatty in filtration of unknown cause, and 28 with miscellaneous hepatic lesions. Douglas et al [20] examined fasting and postprandial levels of serum bile acids by radioimmu noassay in 20 patients with anicteric liver disease who had only minor abnormalities on conventional liver function tests. The diagnoses included alcoholic hep atitis in six, alcoholic cirrhosis in eight, and one case each of nonspecific reactive hepatitis, alcoholic hem osiderosis, cirrhosis with hemosiderosis, alpha1-antitrypsin deficiency cirrhosis, and mild alcoholic liver disease. Bilirubin level was abnormal in nine of 20, al anine aminotransferase level abnormal in four of 20, aspartate aminotransferase level abnormal in seven of 20, alkaline phosphatase level abnormal in four of 20, and gamma-glutamyl transpeptidase level abnormal in 10 of 20. The authors found abnormal fasting cholato levels in seven of 20 and abnormal postprandial cholate levels in 10 of 20. These studies illustrate the higher frequency of liver disease detection by functional type tests (bilirubin and bile acids) when the disease process is mild In activity and/or chronic and cumulative in its severity. Comparisons between studies, however, are limited because of the heterogeneity in the types of liver injury and the various pathologic stages. Consistent with our findings Is Berk et al's [37] observation of abnormal choiylgiycine clearance as the sole liver dysfunction present in a case of persistent vinyl chloride-induced liver injury. A potential limitation to this prospective study might 1 1A January 198S The American Journal ot Medicine Volume 78 AP00024037 - rv>v- BLE ACID CEKTinCATIQN OF CHEMICAL HEPATOTOXICCTY-USS ET AL be the time relationship between the performance of the liver biopsy, biochemical studies, and the serum bile add determinations and the indocyanine green clear ances. Three factors mitigate against the time differ ences influencing the results. First, there is the nonprogressive nature of these early histologic lesions associated with vinyl monomer chemical injury. Second, the microcirculatory rather than the hepatocellular nature of the injury, which has already been described [8,9], is highly correlated with increased collagen de position and appears to be persistent. Third, the per manent nature of the histologic lesion has been docu mented by serial biopsy in the 3ame person [28,29]. Finally, the results for each test were similar in each biopsy subgroup between simultaneous and nonsimultaneous determinations as shown before. In conclusion, fasting serum bile acid levels, espe cially conjugates of cholic acid, provided a high degree of specificity (i.e., very few false-positive results) in persons with normal biopsy findings--a highly desirable characteristic for a diagnostic test to be used in asymptomatic populations. Fasting serum bile acid levels, because of their lower sensitivity in detecting acute but mild cytotoxic disease, are not appropriate for use as a screening test by themselves. They do, however, when used In combination and in the proper sequence, identify those among the exposed with a very high probability of having low-grade, chronic, or pro gressive chemical liver injury and needing more ex tensive clinical investigation. The low sensitivity may be improved by use of an exogenous load, preferably by the oral route [23,24], but this remains to be dem onstrated. Finally, the serum bile acid determinations provide a clearance-type liver test with greater acceptability with regard to time, cost storage of blood samples, and ease of performance than does indocyanine green clearance. This diagnostic use of bile acids should continue to undergo further validation in other distinct types of hepatotoxie liver injury. ACKNOWLEDGMENT We wish to thank Abbott Laboratories, Chicago, Illinois, and Mr. Philip Miller, Director of New Technology Re search, for supplying the bile acid radioimmunoassay materials. REFERENCES 1. Zimmerman HJ: Hepatotoxlcity. New York: Appleton-Cantury-Crofts, 1978. 2. Tomatia L The IARC program on the evaluation of the carci nogenic risk of chemicals to man. Ann NY Acad Sci 1976; 271: 396-409. 3. Popper H, Gerber MA, Schaffner F, et al: Environmental he patic injury in man. In: Popper H, Schaffner F, eds. Progress In liverdiseases, vol VI. New York: Grune A Stratton, 1979; $05-638. 4. Tamburro CH: Chemical hepatitis: pathogenesis, detection and management Med Clin North Am 1979; 63: 545566. 5. Brody DH, Leichter L Clearance tests of liver function. Med Clin North Am 1979; 63: 621-630. 6. Goldman MA. Schwartz CC, Swell Let al: Bile acid metabo lism In health and disease. In: Popper H, Schaffner F, eds. Progress in liver diseases, vol VI. New York: Grune & Stratton, 1979; 225-241. 7. Korman MG, Hofmann AF, Summerskill WHJ: Assessment of activity In chronic acute liverdisease. Serum bile adds compared with conventional tests and histology. N Engl J Med 1974; 290: 1399-1402. 8. Zimmerman HJ, Seeff LB: Enzymes in hepatic disease. In: Coodiey EL, ed. Diagnostic enzymology. vol. 1. Philadelphia: Lea AFebiger, 1970:1-18. 9. Cornish HFi: Problems posed by observation of serum enzyme 'f changes in toxicology. CRC Celt Rev Toxicol 1971; 1:1- 32. 10. Tamburro CH. Greenberg RA: Effectiveness ot federally re quired medical laboratory screening in the detection of chemical liver injury. Environ Health Perspact 1981: 41: 117-122. 11. Creech JL, Johnson MN: Angiosarcoma of liver in the manu facture of polyvinyl chloride. J Occup Med 1974; 16: 150-151. 12. Kaplowitz N, Kok E, Javitt NB: Postprandial serum bile acids for the detection of hepatobiliary disease. JAMA 1973; 225: 292-293. 13. Pennington CR, Ross PE, Bouchler IAD: Serum bile acids in the diagnosis of hepatobiliary disease. Gut 1977; IS: 903-908. 14. James O, Lesna M, Robert SH, et al: Liver damage after paracetamol overdose. Comparison of liver function tests, fasting serum bile acids, and liver histology. Lancet 1975; II: 579-581. 15. Osuga T, Mitamura K, Mashige F, et al: Evaluation of fluo- rometrically estimated serum bile acid in Fiver disease. Cfn Chim Acta 1977; 75:81-90. 16. Pennington CR. Ross PE. Bouchier IAD: Serum bile acids in patients with viral hepatitis. Scand J Gastroenterol 1978; 13:77-80. 17. Barnes S. Gallo GA, Trash D6, etal: Diagnostic value of serun bile acid extractions in liver disease. J Clin Pathol 1975; 28:506-509. 18. Fausa O. GJone E: Serum bile acid concentrations in patients with liver disease. Scand J Gastroenterol 1976: 11: 637-543. 19. Gilmore IT, Thompson RPH: Kinetics of ,4C-glycochol!c add clearance In normal man and in patients with liver disease. Gut 1978; 19:1110-1115. 20. Douglas JG, BeckettGj,NlmmolA. etal: Clinical value of bits salt tests in anicteric liver disease. Gut 1981; 22: 141148. 21. LaRusso NF. Hoffman NF, Hofmann AF, et al: Validity ana l k January 1985 The American Journal of Medicine Volume 79 75 AP00024038 BCE AOD IDENTIFICATION OF CHEMICAL HEPATOTOXIdTY--US3 ET AL sensitivity of an intravenous bile acid tolerance test In paBert* with liver disease. N Engl J Med 1975; 292:12091214. 22. ThjodieHsson B. Barnes S. Chitranukroh A, et al: Assessment of the plasma disappearance of chalyl-1,4C-glyctne as a last of hepatocellular disease. Gut 1977; 18: 697-702. 23. Matern S, Haag M, Hans C, et al: Oral choiate tolerance test. An application of specific radioimmunoassays for deter, mfciation of serum conjugated cholic and deoxychofic adds. Ik Paumgartner Q, StleN A, eds. Bile acids in health and dtesase. Lancaster M.T.P. Press. 1977; 2S3-261. 24. Gilmore IT, Thompson RPH: Plasma clearance or oral and "ravenous cholic ackj in subjects with and without ctronic Bver disease. Gut 1980; 21: 123-127. 25. Use GM, Tamburro CH: Normal ranges for SBA in a healthy working population (unpublished data). 26. Laevy CM: Dye extraction by the liver. In: Popper H, Schaffner F. eds. Progress In liver diseases. New York: Grune 4 Stratton, 1961; 174-186. 27. Leevy CM, Smith F, Longuevilie J, et al: Indocyanine green clearance as a test for hepatic function. Evaluation by di chromatic ear densitometry. JAMA 1967; 200: 236-240. 28. Tamburro CH, Greenberg RA, Newby LG, etat Implementation and assessment of a demonstration cancer control, de tection and prevention program in a cohort of industrial workers. Propam contract N01-CN-55212, Final Report. Division of Cancer Control and Rehabilitation, National Cancer Institute, Bethesda. Maryland, 1978. 29. Tamburro CH, Makk L, Popper H: Early hepatic alterations among chemical (vinyl monomer) workers. Hepatology 1984; 4: 413-418. 30. Popper H. Thomas LB. Telles NC, et at Development of he patic angiosarcoma in man Induced by vinyl chloride, tho rotrast and arsenic: comparison with eases of unknown etiology. Am J Pathol 1978:92:349-378. 31. Popper H, Maltoni C. Solikoff U: Vinyl chloride-induced hepatic lesions in man and rodents. A comparison. Liver 1981; 1: 7-20. 32. Greenberg RA, Tamburro CH: Exposure indices for epidemi- ologica! surveillance of carcinogenic agents in an Industrial chemical environment J Occup Med 1981; 23; 353- 358. 33. Mantel N, Haenszel W: Statistical aspects on the analysis of data from retrospective studies of disease. JNC11959; 22: 719-748. 34. Rothman KJ, Bolce JO: Epidemiological analysis with a pro grammable calculator. National Institutes of Health publi cation no. 79-1649, program 2. Bethesda. Maryland: United States Department of Health and Human Services, 1979: 8. 35. Fleiss JL: Statistical methods ror rates and proportions. New York; John Wiley & Sons, 1973; 98-98. 36. Kobayashi K, Allen RM, Bloomer RJ, et al: Enzymatic fluorometry lor estimating serum totai bile add concentration. JAMA 1979; 241; 2043-2045. 3T. Berk PC, Martin JF, Waggoner JG: Persistence ol vinyl chlo ride-induced liver injury after cessation of exposure. Ann NY Acad Sci 1975; 246: 70-77. I 76 January 1985 The American Journal of Medicine Volume 76 ... CSi'. AP00024039 Vol. 294 Wo. 12 PUBLIC-HEALTH ROUNDS AT THE HARVARD SCHOOL OF PUBLIC HEALTH 653 PUBLIC-HEALTH ROUNDS AT THE disease most commonly results from an unusual group of HARVARD SCHOOL OF PUBLIC clinical manifestations or disease occurring in an industrial HEALTH Edited by Peter Braun, M.D., and Eleanor Druckman, M.S. cluster, as was true of vinyl chloride. Vinyl chloride is a gas used to make polyvinyl chloride, the basis of widely used plastic products such as phono Vinyl Chloride: Can the Worker Be Protected? graph records, meat wrappers, upholstery covering, plastic containers, toys and pipe for water supplies. The produc tion of vinyl chloride and polyvinyl chloride is a post World Introduction War II industry that has been growing for the last 20 to 25 David H. Wegman, M.D* years. In January, 1974, in Louisville, Kentucky, three cases of a OVER 80 million American men and women spend one quarter of their lives in a workplace outside the home. rare neoplasm, angiosarcoma of the liver, w^re reported in workers exposed to vinyl chloride.' If these had not been The deterioration in health quality that results from exposuch unusual tumors, they would have gone unnoticed. If sure to hazards in their workplace is not widely known. The cancer of the lung, bladder or bowel had developed in these annual toll of job-related injuries is estimated at 2.2 mil workers, we would probably not know about the connection lion, and these injuries result in 14,000 deaths.1 The Na between vinyl chloride and cancer. These tumors occurred tional Health Survey has estimated that the average worker in workers who had been exposed for roughly 20 years. It is experiences six days of absence and more than 16 days of re probable that we are just beginning to see a larger epidemic. stricted activity because of some type ofjob-related disabil It is instructive to examine the accumulation, over time, ity per year and that one out of eight workers will receive a of information about the biologic effects of vinyl chloride. In job-related injury each year. The incidence of occupational 1938 there was a report describing acute toxicity from vinyl disease was recently estimated at 390,000 new cases and as chloride to guinea pigs, mice and dogs.' In 1949 a report of many as 100,000 deaths per year. Job-related disabilities ac cases of liver damage from vinyl chloride in human beings count for 10 times as many lost man days as from strikes.' was published.* In 1961, studies of chronic toxicity on rats, This human cost can, in large part, be assigned to well rabbits, guinea pigs and dogs revealed that liver damage oc known hazards of the workplace or to sporadic human er curred at levels above 50 parts per million.' In 1966 another ror. The first, at least, is subject to control if we are very unusual disease associated with vinyl chloride, acro- prepared to accept an increment in price or inconvenience. osteolysis, was described.* This condition, characterized by In a technologically innovative society, the worker is sub the dissolution of the bones of the fingers and toes, was ject to an additional risk. Morbidity or death may also clearly related to vinyl chloride exposure. In 1970 a study result from exposure to new processes or agents with unan ticipated potential For injury. showing the development of cancer in animals appeared.' Since announcement in January, 1974. of the first three pa The quality of a society should be judged in part on its tients with angiosarcoma of the liver an additional 26 such awareness of the potential for injury in innovative technolo cases have been identified.' Several epidemiologic stud gy. There must be procedures for evaluating the level of ies''" have consistently found an association of vinyl chlo danger to which workers are exposed, and for responding to ride exposure with tumors in sites other than the liver. One demonstrated dangers. The following account illustrates study indicates a time trend with increasing rates of all can how we have dealt with one specific problem. cers in the exposed population.' Perspectives John M. Peters, M.D.' It is hard to escape the conclusion that, well before 1974, there was enough evidence to support the presumption of a serious occupational hazard. What was, and is, lacking is a Most of the known occupational diseases are chronic, un- national policy of screening new materials for potential haz Ireatable and fatal. Despite the fact that the prevention of ard before their dissemination in the workplace, a policy occupational diseases is highly desirable, they are not sub that might have prevented widespread exposure to vinyl ject to systematic surveillance in the United States today by chloride. Should we have a policy for screening chemicals? industry, unions, the government or universities. In addi Could we implement such a policy if we had one? tion, physicians frequently fail to recognize occupational Dr. Rudolph j. Jaeger will tell us what we might have disease. In this context the discovery of a new occupational learned from animal studies. From the Occupational Health Program. Kresge Center for Environmen tal Health, the Center For the Analysis or Health Practices of the Harvard School of Public Health, and the Division of Occupational Hygiene. Mas sachusetts Department of Labor and Industries (address reprint requests to Dr. David Wegman at 665 Huntington Ave., Eoslon. MA 02115). These Rounds are supported in part by grants From the Robert Wood Johnson Foundation and the Commonwealth Fund through the Center for the Analysis of Health Practices. 'Massachusetts Division of Occupational Hygiene and Occupational Health Program. 'Occupational Health Program. Toxicology Rudolph J. Jaeger, Ph.D.* The central problem in toxicology is extrapolation to man of the toxic responses observed in the laboratory animal. In dividual species vary widely in their susceptibility to injury from a given compound, and none meet the requirements of equivalency to man in biologic response, short latency, low cost and convenience across the spectrum of agents to be fToxieology Program. Kresge Center for Environmental Health. v .y tFS'/.VtW't- AP00024040 t>S>4 THE NEW ENGLAND JOURNAL OF MEDICINE March 18, 1976 tested. Despite these shortcomings inherent in toxicologic studies as now conducted, experiments in traditional labo ratory animals could have supported a stronger and earlier presumption of the dangers of vinyl chloride, Under normal environmental conditions vinyl chloride is a colorless, nearly odorless gas that is highly explosive at modest concentrations in the air -- i.e., 4 per cent and above. At 8 per cent and above, it is an anesthetic -- a use that was suggested in the 1930's" but abandoned owing, in part, to flammability and myocardial sensitization." What tests of toxicity might be performed on an agent like vinyl chloride? As a first step, experiments might be conducted in which the gas is inhaled by a test species at a given concentration -- e.g., 100; 1000; 10,000; 100,000 parts per million (ppm) -- for a single acute period com parable to a normal work day of eight hours or less. Except for death due to central-nervous-system depression, vinyl chloride is without immediate lethal action.'* After a single subacute exposure, the animals will recover without residual damage and live without apparent ill effects for a normal life-span. This lack of acute toxic effect in rats is reflected in the experience of workers who have been rendered unconscious by high concentrations of vinyl chloride and then moved to other jobs with little or no sub sequent exposure. They have had no apparent sequelae. A second test of toxicity in animals involves repeated in halation exposure for days, weeks, or months. Such experi ments are comparable to short-term work situations. In ex periments such as these, vinyl chloride produced no dra matic effect on repeated exposure at concentrations up to 20,000 ppm for 92 days." In a study done in 1961 by the Toxicology Laboratory of the Dow Chemical Company, rats, rabbits, guinea pigs, and dogs were exposed to vinyl chloride for seven hours a day, five days a week for periods up to six months al concentrations of 500, 200, 100 and 50 ppm of vinyl chloride. In this study the animals were killed at the end of the exposure period and autopsied.' Growth rates and hematologic indexes were within normal limits. Slight liver enlargement was noted after exposures at con centrations of 100 ppm or more. Minor pathologic changes were also detected in other species at doses higher than 100 ppm. Fifty ppm, a concentration without effect on liver weight, was suggested by Dow as a maximum time-weight ed concentration for eight-hour exposure of workers, on the grounds that these conditions of exposure represented lev els that did not affect rats exposed for six months. A somewhat different approach to the study of toxicity is represented by the studies of Maltoni'1 and Viola,' which showed that exposure of rats to 30,000 ppm of vinyl chloride four hours a day, five days a week, for a year, caused malig nant neoplasms in a variety of sites, including the liv er, brain, kidneys and vascular tissue. In these studies the pathologic findings were sought after the animals' lifetime rather than at an earlier experimental sacrifice point. This was a crucial change of experimental design for the iden tification of potent carcinogenicity of vinyl chloride. Maltoni found cancers at exposure concentrations as low as 250 ppm in rats,'* Furthermore, his data on tumor rate with decreasing dose suggest that there is risk of cancer at concentrations of vinyl chloride below 50 ppm. Thus, a compound with little acute lethal action and negligible short-term toxicity is a potent carcinogen in rodents under worklike exposure conditions of 50 ppm and less. What can be learned from this series of events? It re quired two decades for the development of malignant tumors in human beings, when 12 months of experimenta tion in lower vertebrates could have established the car cinogenicity of vinyl chloride at exposures comparable to those in the working environment. Although a negative ex perimental result in animals cannot exclude potential car cinogenicity in man, a positive result under these circum stances is an unambiguous warning. The perns described here suggest that useful toxicologic screening can be car ried out in locations other than the workplace, and in spe cies other than man. Engineering William A. Burgess, S.M-* The manner in which workers are exposed to vinyl chlo ride varies greatly, depending on their specific roles in the process of production. The industry can be divided into three manufacturing stages -- the production of vinyl chlo ride monomer (VCM), the reaction of this material to form the solid polyvinyl chloride (PVC), and the fabrication of plastic products, from polyvinyl chloride. Over 350,000 workers are exposed to potential hazard at various stages of this industrial sequence. Vinyl chloride monomer is manufactured by a number of different pro cesses in the United States in 15 plants with a total work force of about 1000. In a common process ethylene is com bined with chlorine to form ethylene dichloride, which is pyrolyzed to form vinyl chloride. Although this continuous operation is enclosed, leaks from various valves, pumps, and fittings result in exposure of the worker to airborne vinyl chloride gas. Maintenance of process equipment is a major problem in this industry. Limited data on air concentration are available from the monomer production plants. In normal operations the con centration may be at the level of 1 ppm. Occasional spills, however, will result in peak exposures of hundreds of parts per million. Fortunately, effective methods of control are available in some parts of the industry. Maintenance obviously must be improved to reduce leaks into the workplace. New proce dures must be developed to permit transfer of the monomer and sampling of the product so that the worker need not be exposed during these operations. In the second industrial stage, vinyl chloride monomer is reacted to form the solid polymer, polyvinyl chloride (PVC). Polymer production is carried out in this country in 36 plants with about 5000 employees. The major exposures to vinyl chloride monomer occur in this part of the industry, and most of the cases of angiosarcoma of the liver have come out of these plants. Polyvinyl chloride is produced in a batch operation in a glass-lined reactor or pressure vessel. Vinyl chloride mono mer is introduced into the reactor with an initiator, a cata lyst, and water. The temperature is raised to 60C, the pres- Krcsge Center for Environments! Heslth. -iVV iW*?* \ i' } t AP00024041 Vol. 294 No. 12 PUBLIC-HEALTH ROUNDS AT THE HARVARD SCHOOL OF PUBLIC HEALTH 655 sure is increased, and the material polymerizes over a peri od of 10 to 16 hours. At the end of this interval, the solid granular polyvinyl chloride is formed, drops to the bottom of the reactor, and is then dried. Some unreacted vinyl chloride monomer remains in the polymer and presents an exposure problem to fabricators of end products. Still, fabrication offers Fewer hazards than polymer production. The most common exposure occurs when workers periodi cally crawl into the reactor to clean the inside surface, a sit uation in which exposures may exceed 1000 ppm. How can this exposure be reduced? The industry would like to auto mate the cleaning, but this step has not yet been imple mented widely. Is control of vinyl chloride feasible in this part of the in dustry? This question sets the stage for the major debate. In testimony before the Occupational Safety and Health Ad ministration, the spokesman for the Society of the Plastics Industry had stated that it is not economically feasible to reduce the levels below 25 ppm. A consulting firm retained by the Department of Labor had stated that it is feasible to bring the concentration down to 2 to 5 ppm in this part of the industry. Other authorities said that air concentrations of vinyl chloride monomer of 1 ppm can be achieved.* In the third stage, the fabrication sector of the indus try, the granular polyvinyl chloride is made into useful com mercial and consumer products. The bulk polymer is com pounded with various plasticizers, stabilizers, and other ad ditives depending on the ultimate application. The prod ucts made from the polyvinyl chloride include intermediate industrial materials such as tubing, rod, bar and sheet, and a host of consumer products. There are 7500 fabrication plants with nearly 550,000 workers. The exposure to vinyl chloride monomer in these plants is due to the release of the residual monomer from the solid material. The residual monomer in many polymers is in the range of 1000 to 5000 ppm by weight. The hazard in the fabrication industry could be eliminated if the polymer manufacturers reduced the residual amount to 1 ppm. In summary, although there should be no disagreement that controls must be implemented promptly, there has been much dispute over the levels of control for vinyl chlo ride that can be achieved, and over their costs. Economics Leslie I, Bodtn, B.S.{ On the list of the 50 most common industrial chemicals, vinyl chloride ranks 23d. Annual United States production or this gas is over 2.3 billion kg. By far the most important use of vinyl chloride is in the production of polyvinyl chlo ride, of which we also produce about 2.3 billion kg a year at a cost of over tl billion. United States production of this im portant plastic has been growing steadily since 1939 at an average annual rate of 12 per cent, if the polyvinyl chloride Official Report of Proceedings before the Occupational Safely and Health Administration of the U.S Department of Labor in the matter of proposed permanent standards for occupational exposure to vinyi chloride. Washington. D.C., June - July, 1974 (Available from H R Company. Of ficial Reporters, 320 Massachusetts Avenue, N.E.. Washinilon. DC 20002). 'Occupational Health Program. products industry were to be shut down before substitutes could be found and their manufacture begun, perhaps two million jobs and 75 billion dollars worth of production would be lost.1' The basic institutions of the economy. Firms competing for profits, have been well suited to increase rapidly the availability of goods and services. Through the struggle for profits, business has played a central part in producing the innovations that have raised the standard of living in this country to levels that most people formerly believed impos sible. On the other hand, there are several things 'for which these industrial organizations are poorly suited. They have few incentives to educate the population, lower unemploy ment, promote income equality, protect the environment, or help improve the health of the public. Often, these goals come into direct conflict with the profit motive. Concern for workers' health, for example, might require the individual company to conduct expensive research, and cause it to de lay the introduction of new products and processes. Both re sponses would place it at a competitive disadvaniage. This situation has led to government intervention in the private market to diminish the impact of occupational dis ease. There are at present two types of laws designed to make business more concerned about the health and safety of workers. Worker's compensation laws have created stateregulated programs ensuring that a company pays for med ical expenses and for part of lost income if an occupational disease develops in a worker. Unfortunately, these laws do not provide the companies with adequate incentives to pro tect the health of their workers. For example, the worker's compensation costs for medical expenses and support pay ments to the families of the men who died in Louisville. Kentucky, average about 164,000. If 1000 workers a year died, the cost of paying this level of compensation would amount to approximately S0.0I per pound of polyvinyl chlo ride production, or less than 3 per cent of the cost of its manufacture. This proportion is substantially below the costs of rigid manufacturing controls. In 1970, Congress passed the Federal Occupational Safe ty and Health Act." This law attempts to prevent disease by compelling companies to conform to standards that are de signed to prevent illness due to toxic exposure in the work place. The enforcement of this act has, however, been weak, and its effectiveness questionable. How, then, might the government stimulate adequate testing of the potential toxicity of chemicals that enter the industrial environment? One way to achieve this goal would be the implementation of an occupational health act similar to the Pure Food and Drug Act -- that is, new industrial chemicals with definite potential for human exposure would have to be demonstrated to be nontoxic before being ap proved for use. This step would compel industry to test these substances before they were widely used. Laws like those just mentioned can help to protect the health of workers, but at a cost. This cost might be ex pressed in a slower rate of technologic innovation,, and thus lower production of some goods and services. It might also be felt in increased prices for goods. These increases result from the added costs of experimentation and changes in the iv: ) i.. r b. K ' u4<. "t- k 1; ' i AP00024042 <556 THE NEW ENGLAND JOURNAL OF MEDICINE March 18, 1976 production process that help to protect workers' health. For example, a spokesman for Firestone Plastics has stated that the costs of lowering vinyl chloride exposure from the 1973 standard to 1 ppm would increase the cost of polyvinyl chlo ride by 50 per cent.' The problem is further complicated by the fact that the United States produces less than 30 per cent of the world supply of polyvinyl chloride. If other countries do not im plement strict standards, what is to prevent the multina tional corporations that produce polyvinyl chloride from building new factories overseas, "exporting" both jobs and disease? Regulation David H. Wtgman, M.D.'1 In my position with the Massachusetts Division of Oc cupational Hygiene, my first contact with vinyl chloride came in early 1973 with an attempt to evaluate the problem of acro-osteolysis induced by vinyl chloride in Massachu setts workers. The attempt was short lived because other oc cupational health problems had higher priority. The issue of vinyl chloride, however, was brought back to me with the reports of the cases of angiosarcoma of the liver in January, 1974. As happened elsewhere, we immediately gave the vi nyl chloride problem a high priority and began to investi gate the four local firms manufacturing polyvinyl chloride from vinyl chloride. Although we were easily able to evalu ate current exposure levels in these plants, our efforts to evaluate possible chronic disease foundered because of the small numbers of employees and our inability to locate those who had left employment. At the same time lhat we were attempting to determine the magnitude of the problem in Massachusetts, the Federal Occupational Safety and Health Administration (OSHA) was faced with the more difficult problem of establishing national standards. In July, 1971, OSHA established its first standard on asbestos as a result of heavy pressure from labor." The car cinogenic effect of asbestos was ignored, and the standard was set solely with asbestosis in mind. The next standard promulgated referred to 14 carcinogens but set no "safe" exposure level." Then the vinyl chloride story broke public ly, and the resulting widespread interest forced OSHA to deal directly with a single cancer-causing agent. The agen cy knew that vinyl chloride was carcinogenic at exposure levels yet to be determined, and its use was widespread, and that control of exposure was difficult. The first step was to set an emergency standard at 50 ppm, the level that the Dow Chemical Company had recommended as an upper limit in 1961.* The agency then had six months, by law, to promulgate the final standard. This period included devel opment of both economic and environmental impact state ments and the announcement of three days of public hear ings. The response to the announcement was so great that hearings continued for three weeks. As a medical consultant to OSHA, I was asked to address Proceeding! before the Occupational Safely and Health Administration. tMass. Diviiinn of Occupational Hygiene and Occupational Health Pro gram. the problem of the human health effects, and how well sub stantiated they were. The federal government, through the Food and Drug Administration (FDA), had indicated be fore the hearings had begun that exposure to vinyl chloride was a serious human health problem that needed immedi ate attention. On February 21,1974. the FDA received a pe tition to withdraw vinyl chloride as a propellent in aerosol products (including hair sprays, aerosolized household products and pesticides), and in only six weeks such an or der was issued. Information on how widespread the use of these products had been was unavailable. It was clear that more than the identification of cases of a rare tumor would be required if a recommendation of any exposure level below 50 ppm were to be made. General ly, standards are set on the basis of acute effects of a tox ic material. Vinyl chloride was not known to cause acute problems below the lower explosive limit, and since provi sions had been made to prevent explosions, concentrations that could cause acute disease were unlikely. The chronic effects, however, were difficult to evaluate. "Slap-in-the-face epidemiology" had succeeded in bringing the problem to our attention, it was "quick-and-dirty" epidemiology that gave an initial idea of the size of the problem. Although several attempts to conduct mortality surveys had be?n made, only a few were sufficiently well designed and carried out to provide useful data. Even they fell short of allowing specific estimates of risk. They did tell the unfortunate story of cancer of the liver as well as lung, brain, skin, and lymphatic system in vinyl chloride work ers. Three other studies of current workers showed that vi nyl chloride and polyvinyl chloride were also associated with chronic disease of liver and spleen, bleeding condi tions, skin and bone disease, and chronic lung disease.11'" On the basis of these findings, my first recommendation was for a standard of 1 ppm as the maximum exposure ac ceptable on medical grounds. The response of the director of OSHA to this recommendation was most interesting. Al though initially he had asked for a medical evaluation of the potential health effects, his final request was for a legal de fense of the levels. He believed lhat regardless of what level he set, he would be taken to court for setting too lenient (by labor) or too stringent (by industry) a standard. This raised the core question: How well-supported do health effects need to be to be acted upon? For vinyl chloride it seemed reasonable to assume that human beings arc at least as sensitive as laboratory animals and that the standard would have to be below 50 ppm. But how' far be low 50 ppm can a number be selected and defended? The most convincing argument for the figure of 1 ppm was the unusual behavior of vinyl chloride. It would be difficult to imagine a material with more potential for human damage, for it is associated not only with cancer at multiple sites but also with non-neoplastic disease of at least six different or gans or organ systems. Epilogue On October 4, 1974, the Federal Occupational standard for vinyl chloride was issued.11 Although it accepted in prin ciple I ppm as the maximum possible exposure, work prac tices temporarily permitted exposures of up to 25 ppm with- } ' * ,'S .> * \ '3* t w* > o AP00024043 Vol. 294 No. 12 PUBLIC-HEALTH ROUNDS AT THE HARVARD SCHOOL OF PUBLIC HEALTH 657 out respiratory protection. This standard was immediately challenged by the Society of the Plastics Industry and vari ous companies when they petitioned the federal courts for a stay in the implementation of the standards." The petition was denied, and the United States Supreme Court refused to review the decision." Since the promulgation of the stan dard, apparently only one small plant has closed, and four new plants are nearing completion." Polyvinyl chloride was briefly used to make containers for alcoholic beverages. The discovery that migration of detec table amounts of vinyl chloride from the containers to the beverage led to its removal from this use. Definite evidence on the migration of vinyl chloride from food wraps or pipes for drinking water does not exist. Inadequate study has been performed to date to determine whether there is a hazard ol vinyl chloride exposure to consumers using nonfood-related polyvinyl chloride products. References 1. United States Department of Labor. United States Department of Health. Education, and Welfare. The President's Report on Occupa tional Safetv and Health. Washington. DC, Government Priming Of fice. May, 1972, pill 2. Creech JL Jr. Johnson MN: Angiosarcoma of the liver in manufacture of polyvinyl chloride. J Occup Med 16:150-151, 1974 3. Toxicology and Hygiene of Industrial Solvents. Edited by KB Lehmann, F Flury. Baltimore, The Williams and Wilkins Company, 1943, p 143* 144 4. Tribukh SL. Tikhomirova NP, Levina SV, et al: Working conditions and measures for their improvement in the production and usd of vinyl chlo ride plastics. Gig Sanit 14(10):38-44, 1949 5.Torkelson TR, Oyen F, Rowe VR-. The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Am Ind Hyg Assoc J 22:354-361. 1961 6. Cordier JM. Fievez C. LeFevre MJ, et al: Acroosteolyse ct lesides cutanies issociie* chez deux ouvriers, affecte* au nettoyages d'tutoclavw. Cah Med Travail 4:14-19. 1966 7. Viola PL, Bigotti A, Caputo A: Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Res 31:516*522, 1971 8. Lloyd J'V: Angiosarcoma of the liver in vinyl chloride/polyvinyl chlo ride workers. J Occup Med 17:333*334, 1975 9. Monson RR, Peters JM, Johnson MN: Proportional mortality among vinyl-chloride workers. Lancet 2:397-398, 1974 10. Tabershaw IR. GalTey WR: Mortality study of workers in the manufac ture of vinyl chloride and its polymers. J Occup Med 16:509-518, 1974 11. Nicholson WJ, Hammond EC,5cidman H, el al: Mortality experience of a cohort of vinyl chloride-polyvinyl chloride workers. Ann NY Acad Sci 246:225-230. 1975 12. von Oettingen WF: The Halogenated Aliphatic. Olefinic, Cyclic, Aro- 'matic and Aliphatic-Aromatic Hydrocarbons Including the Halogenat ed Insecticides, Their Toxicity and Potential Dangers. (PHS Publication No. 414). Washington, DC, Government Printing Office. 1955 13. Irish DD: Aliphatic halogenated hydrocarbons, Industrial Hygiene and Toxicology. Second edition. Edited by FA Patty. Vol 2. Toxicology. Ed ited by DW Fisset, DD Irish. New York, Interscience Publishers, 1963, pp 1241-1332 ) 14. Mastromatteo E, Fisher AM. Christie H, et al: Acute inhalation toxicity of vinyl chloride to laboratory animals. Am Ind Hyg Assoc J 21:394-398, I960 15. Lesier D. Greenberg LA. Adams WR: Effects of single and repeated ex posures of humans and rats to vinyl chtoride. Am Ind Hyg Assoc J 24:265-275. 1963 16. Maltoni C. Lefemine G: Carcinogenicity bioassays of vinyl chloride. I. Research plan and early results. Environ Res 7:387-405, 1974 (7. U.S. chemical industry: the products it makes. Chem Eng News 53(22):31-34, 1975 18. Occupational Safety and Health Act. 29 US Code $ 651 (December 29, 1970) 19. 29 Code of Federal Regulations 1910.1001 20. 29 Code of Federal Regulations \ 1910.1003-16 21. Veltman G. Lange CE, JOhe S, et al: Clinical manifestations and course of vinyl chloride disease. Ann NY Acad Sci 246:6-17, 1975 22. Lilis R. Anderson H, Nicholson WJ, et al: Prevalence of disease among vinyl chloride and polyvinyl chloride workers. Ann NY Acad Sci 246:22- 41. 1975 23. Miller A, Teirstein AS. Chuang M, et al: Changes in pulmonary function in workers exposed to vinyl chloride and polyvinyl chloride. Ann NY Acad Sci 246:42-52, 1975 24.29 Code of Federal Regulations $ 1910.1017 25. Industry lines up to fight standard for worker exposure to vinyl chloride. Occup Safety Health Rep 4:523-524, 1974 26. Supreme Court declines review of second circuit ruling in VC care. Oc cup Safety Health Rep 4:1719. 1975 27. Rauner S: Did industry cry wolf? Polyvinyl chloride health rules can be met. A'rw York Times, December 28, 1975, Sec 3, p 1 J53JS7T AP00024044