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The role of clinical investigations in biological markers research
ENVIRONMENTAL RESEARCH 50, 1-I0 (1989)
REVIEW The Role of Clinical Investigations in Biological Markers Research MARK R. CULLEN
Yale-New Haven Occupational Medicine Program, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Received October 18, 1988 As the agenda of the occupational and environmental health community proceeds from the study of the effects of high doses of toxins causing grossly altered disease patterns to examination of the impact of lower doses on prevalent disorders, new scientific approaches have become necessary. The emerging concept of molecular epidemiology, substituting biologically determined markers of dose and early effect for traditional measures of exposure and morbidity, offers a heuristically appealing direction for the field. However, the transition to this evolving approach has been slow and its potential has yet to be validated. In this essay, some of the limitations of this approach are discussed and theoretical modifications developed. The central proposition is that existing modalities of clinical research, widely employed in subspecialty medicine research but heretofore unexploited in the study of environmental diseases, offer great promise. By incorporating clinical investigations centrally, some historically rate-limiting problems such as variability of host responses, multiplicity of environmental risks, and differences between human and animal responses could be transformed from major impediments at present to realistic objects for future study. © 1989AcademicPress, Inc.
INTRODUCTION With the emergence of occupational and environmental medicine (OEM) as a relevant and visible clinical discipline in the 1970s and 1980s, increasing examination of the scientific foundation of the field has begun. It is the intention of this paper to summarize and critique existing concepts and, based on this, propose an approach for the future. Ideally, such an approach could more efficiently yield answers to the questions which render current practice so limited for prevention and cure of even the best understood environmentally induced diseases. In the most traditional view, OEM has been conceived as a subdiscipline of preventive medicine, whose scientific tools are chronic disease epidemiology and animal toxicology. The venerated paradigm is that studies in animals would provide evidence of possible human risk for disease which would be confirmed by epidemiologic evaluation of large populations. Alternatively, laboratory investigation could be used to follow-up observations made by clinicians and epidemiologists. Indeed, many of the most notorious and important disease associations, like those linking asbestos with lung cancer and vinyl chloride with hepatic angiosarcoma, were discovered and verified via these pathways. Unfortunately, the great days for these paradigms are rapidly passing. For the epidemiologist, so productive during the 1960s and 1970s at finding the toxins
0013-9351/89 $3.00 Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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which cause rare diseases or drastically increase the risk for common ones, three difficulties have emerged. First, since the strongest associations were quickly uncovered, the outstanding challenge for the future is to elucidate factors which increase risk for diseases modestly (Robins et al., 1987). Though such causes could be extremely important from a clinical or public health point of view (imagine a workplace chemical that increased the risk of cardiovascular disease by a " m e r e " 20 or 30%), these kinds of association are very hard to detect because the relative risk for exposed, compared to nonexposed people, is only slightly elevated; small biases in study design and statistical quirks make interpretation of such findings highly subject to controversy, hence uncertainty for practice. This problem is compounded by the fact that with increasing environmental regulation and control, most exposures of interest are moderately low-dose, at least compared to historic industrial levels. Complicating this central dilemma is the imprecision of modern field methods to accurately quantify human exposures, especially those which occurred years before study commenced. In the earlier search for strong associations, such inaccuracies were relatively unimportant; they could not mask overwhelming excess risk among the exposed. Searching for weaker (but possibly more important) associations, the ability to classify people by the amount of their exposures becomes crucial, if no less difficult. Any misclassification--counting exposed subjects as unexposed or vice versa--would have the effect of reducing the possibility to detect real differences between exposure groups. Finally, the temporal pace of the traditional approach, which must await large populations exposed for long periods, causing widespread effects before analysis is possible, has become a serious drawback. Regulators and clinicians are too impatient to await such uncontrolled "human experimentation" and desperately seek shorter term strategies in which populations need not be "condemned" to be studied. Thus, for example, after years of human exposure and despite impressive levels of study and publication during the past decade, the potential of such ubiquitous dusts as fiberglass and silica to cause human lung cancer remains uncertain, possibly unsoluble by the old model. The problems for toxicology, as traditionally practiced, are quite different though not less critical. Working in virtual isolation from clinicians and field investigators, a substantial portion of ongoing work is disturbingly remote from human application. Such differences as broad genetic variability, coexposure to a multiplicity of "confounding" factors in the environment, and the tendency to idiosyncratic responses all distinguish human from animal responses. These differences need not, of themselves, preclude an extremely valuable role for laboratory-based animal study; they do, however, demand a different focus, or context, if results are to be broadly meaningful to questions of human health. This is especially true in the social context in which environmental concerns must be juxtaposed against other important societal needs; our culture seems, perhaps justifiably, unwilling to defer economic growth to control hypothetical risks based on simple extrapolation from animal models. The importance of genetic and more general host variability has not been lost on the toxicologic community. In the 1970s, with the emergence of technologies for
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effectively studying the human genome, the field of ecogenetics was introduced by Mulvihill and others(Mulvihill, 1977, 1980). This largely experimental, laboratory-based discipline strove to delineate the mechanisms which underlie variations in human responses with the expectation that these findings might illuminate the always peculiar patterns of observations made by clinicians and epidemiologists. However, as recently discussed by Schulte, the products of such work have not yet found major applicability to epidemiologic study (Schulte, 1987). MOLECULAR EPIDEMIOLOGY AND BIOLOGICAL MARKERS RESEARCH
In the early 1980s, the concept of "molecular epidemiology" was coined by investigators at Columbia to describe an evolving approach to research which attempted to synthesize existing scientific tools into a new formulation (Perera and Weinstein, 1982). The theory rests on two biologic tenets: (1) early biologic effects from a toxic exposure are far more prevalent in the population at risk than the late events of direct interest, like cancer or chronic disease, and may sometimes be more specific to the exposure than the outcome itself; (2) given technologic advances, most toxins can be either directly quantified in the body or indirectly measured by identification of some predictable, dose-related biologic response. Combining these two principles, a new approach to human study emerged. Once (prevalent, early) "markers" of effect and (accurate) "markers" of dose could be developed in the laboratory, human epidemiology could proceed without prior constraints--relative risks would be high because the events studied are either very common among the exposed (i.e., sensitive markers) or very rare among the unexposed (i.e., specific markers); exposures could be precisely classified by direct measurement and the lapsed time between first human exposure and an opportunity for study would be foreshortened because endpoints are, by definition, early. In addition to these central theoretical advantages, certain side-benefits may also be anticipated as recently discussed in a critical evaluation by Hulka and Wilcosky (I988). These include (1) a contribution to the understanding of underlying pathogenic mechanisms inherent in study of events at the molecular, cellular or tissue levels; (2) the potential for more accurate and etiologic classifications of environmental diseases; and (3) the possibility that recognition of early effects could prompt strategies for secondary prevention or early disease modification. The conception of research embodied in this new synthesis represents an enormous theoretical advance and has already stimulated reorganization and redirection of some established research agendas. On the other hand, the promise of this nascent movement has not quickly materialized; no single major new association between a toxin and a major health endpoint has been proven, or even suggested, by such an approach. While 5 years may be, perhaps, too short a time for serious judgment, there are certain recognizable limitations to the framework which portend that it will not "revolutionize the field," as proponents have predicted, without significant refinement, both conceptual and practical. Many of these problems have already ben elucidated in the literature (Hulka and Wilcosky, 1988; Hattis, 1987). On the exposure side of the equation, the specificity of markers in relationship to a particular exposure of interest may limit
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the utility of otherwise accessible and mechanistically interesting tools. For example, genetic abberations, including the much studied sister chromatid exchange, may be affected by a broad range of host and environmental factors beyond any agent or agent whose effects are to be investigated; use of such biological markers may introduce confounding which cannot be adequately adjusted for until the operational characteristics of the test itself are conducted and the range of potential confounding factors elucidated. More specific exposure markers, like the level of toxin, or its metabolites or biological reaction products (e.g., toxin-protein or toxin-nucleic acid adducts) in a biologic tissue or fluid may be problematic because of the complexity of the factors which determine such levels. These include host metabolism and toxicokinetics, current physiologic state, length of marker persistence, etc. The underlying problem is that all biological exposure markers, however intimately linked to exposure, entail some "processing" by the host which is likely to be mediated both by intrinsic host factors and by other environmental exposures. In other words, factors which may be associated with the "effect" may also modify the measure of exposure, severely confounding causal reasoning. For example, differences in levels of D N A adducts in urine of workers exposed to polyaromatic hydrocarbons (PAHs) may reflect as much host variability in metabolizing the PAHs as differences in exposure per se; a correlation between this measure of "dose" and a cancer endpoint could falsely suggest a dose response while, in fact, merely proving that those at underlying higher risk have higher risk. The addition to the equation of "susceptibility" markers as proposed by Hulka (Hulka and Wilcosky, 1988) offers a theoretical solution but one which may be very elusive in practice. Establishing a priori a list of such modifiers for a given toxic may represent a formidable challenge. For example, considerable interest has emerged during the past decade in the use of retained asbestos fibers in lung tissue as an exposure marker for asbestos. Unfortunately, enormous interindividual differences have been found even where exposures have ostensibly been comparable (Churg, 1983). Presumably these differences, which do correlate somewhat well with asbestosis grade (Roggli et al., 1986), reflect variability in deposition and/or clearance, in turn likely due to such "susceptibility" factors as breathing pattern, smoking behavior, extent of lung disease (asbestos-related or otherwise), and likely others. Study of these variables is further complicated by methodologic issues, such as timing of exposure relative to collection, the effect of the pathologic process itself on fiber counting, etc. On the effect side of the equation, other serious theoretical and practical problems have been recognized. Most troublesome is the relationship between early effects, e.g., altered tissue morphology or gene expression, and subsequent diseases of interest. Indeally, these could be established experimentally or by longitudinal observations of exposed individuals. In reality, however, both ethical and practical constraints limit this kind of data collection to situations where material obtained from individuals under surveillance or study can be retrospectively evaluated in relation to disease outcomes. An alternative to this approach is juxtaposition of separate, but related, studies. For example, it has been shown that certain host and environmental factors cause alterations in crypt morphology
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of the colonic mucosa (Lipkin and Newmark, 1985). From the separately proven fact that patients with colon cancer demonstrate similar morphologic alterations at sites remote from their tumors, it might be inferred that the host and environmental factors from the former study are causally linked to colon cancer. While this may be true, such reasoning may fall prey to a variation of the ecologic fallacy; "transitivity" of biologic relations cannot be presumed unless or until all of the intervening steps are worked out. Another problem pertains to the practicality of studying early biologic responses of interest. Although technology now exists to obtain virtually any human material for study with minimal health risk, population studies constrain the investigator to examination of only readily obtainable materials or tests, e.g., blood, urine, X-rays, etc. These materials must, somehow, serve as "surrogates" for the end-organ targets of interest. Finding such suitable surrogates becomes, itself, a challenging area of research which heretofore has been largely limited to the use of animals. An elegant example is the recent demonstration that lymphocyte esterase may serve as a surrogate for the more relevant neural esterase in evaluating delayed neurotoxicity of organophosphorous esters (Cherniack, 1988). Unfortunately, as with all of the other relationships discussed above, individual "susceptibility" factors--genetic or acquired--may modify the relationship between the target of interest and surrogates, confounding use of the latter as a reflection of the former. Probably more importantly, a high proportion of endpoints of interest may have no suitable marker easily accessible to the field investigator or only ones which bear too remote a relation to sequelae of interest. THE ROLE FOR CLINICAL INVESTIGATIONS
The concept of molecular epidemiology, or biological markers research as its generalization is coming to be known, is fundamentally sound. The above critique notwithstanding, this new synthesis of the parent scientific disciplines of OEM--toxicology and epidemiology---brings the unique strengths of each to the challenge of studying toxins at doses suspect to cause only low or moderate relative risk for adverse health effects. No other new approach has emerged which offers nearly the same scientific opportunity. It is only with great respect, therefore, that I offer the following ideas about directions future studies may take to obviate some of the problems discussed above and bring some of the promise of molecular epidemiology more quickly to fruition. The most fundamental modification which I propose to the existing paradigms is the addition of a research tool historically underutilized in OEM research center-based clinical investigation. Specifically, I refer to in- or outpatient studies performed under highly controlled conditions on invited subjects which have become the cornerstone of research in clinical subspecialty medicine. Unlike typical epidemiologic studies, study size is necessarily quite small; this limitation is balanced by the breadth and depth of information which can be obtained on each subject, especially in terms of biological parameters, compared to what may be gleaned from larger populations in the field. It is the central tenet of this paper that this type of architecture, so effectively used by colleagues in other disciplines to study disease mechanisms, physiology, and responses to therapeutic interven-
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tions, can be readily transformed to productive use in etiologic research, especially in the context of collaboration with epidemiologists and toxicologists engaged in biologic markers research. In the paragraphs which follow, I shall illustrate some of the potential advantages of this approach in light of problems elucidated above. The first potential role for clinical investigation is in the area of marker identification. Subjects with well-characterized exposures and/or consequences of exposure may be "explored" in search of various biological phenomena which have been hypothesized to be indicative measures of exposure or early effect. Given the controlled setting in a research center environment and truly volunteer subjects, there need be no arbitrary or artificial limit to the source of material (within reason), or, in general, the amount. Conspicuous examples of this type of research are the studies of immunologic lung diseases in which the specific assays have been elucidated for immunoglobulins against sensitizers such as trimellitic anhydride (Patterson et al., 1982) and in vitro lymphocyte proliferative responses to beryllium and other causes of alveolitis (Rossman et al., 1988). Exposure markers, as well, have been explored in this research milieu, such as the determination of mineral fiber burdens in bronchoalveolar lavage fluid of exposed and affected subjects (Dumortier et al., 1988). A close corollary is the opportunity to explore for "surrogate" markers, especially those related to early effect, when primary markers involve inaccessible tissues. For example, our own group has recently completed bone marrow examinations on selected subcohorts of a population previously demonstrated to have hematologic abnormalities on peripheral smears associated with exposure to ethylene glycol ethers (Welch and Cullen, 1988). Simultaneously we examined several peripherally measured indices, including red cell enzymes which have historically been associated with stem cell abnormalities. Although the data are not yet fully analyzed, early analysis suggests a very close correlation between myeloid hypoplasia in the marrow and certain red cell enzymes in peripheral blood, hopefully paving the way for a sensitive early effect marker for use in future field studies. Similarly, investigators in Denver have utilized lavage studies in beryllium workers to develop for the first time a sensitive peripheral lymphocyte assay for hypersensitivity to beryllium (Kreiss et al., in press), an invaluble step in the effort to define the elusive dose-response relationship for this agent. Establishing "linkage" between apparent early effect markers and late health effects of interest may also be facilitated by these kinds of biologically intense clinical investigations. By simultaneously studying diseased as well as nondiseased individuals with varying degrees and lengths of an exposure of interest, one may be able to reconstruct and even sequence temporally disparate events. The Denver beryllium study has illustrated this concept as well. After developing an accessible effect marker, a beryllium-exposed population was screened with center-based follow-up investigation of selected "positives" and "negatives." Surprisingly, almost all with positive tests demonstrated striking alveolitis on bronchoalveolar lavage, despite the absence of clinical symptoms or signs of chronic beryllium disease; alveolitis was not seen among negatives (Kreiss et al., in press). By this clever, cross-sectional design, the investigators have elucidated
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the sequence of most of the events leading to the development of chronic beryllium disease: exposure, followed after variable latency by sensitization, followed after apparently short latency by alveolitis, followed after unknown latency by clinical disease. Clinical investigations may also hold the key to successful evaluation of the "operational characteristics" of new markers, as they have for clinical tests generally. Although ultimately all markers require evaluation in populations sufficiently large to draw strong causal inferences, many facets of marker evaluation may require testing of controls which are too invasive for the field or of subjects with specific unusual diseases. For example, full evaluation of the relationship between red cell enzyme levels and ethylene glycol ethers, alluded to above, will require study of subjects with a range of myeloproliferative and other hematologic disturbances in addition to the more obvious control groups, i.e., healthy exposed and unexposed workers. Similarly, the significance of the beryllium lymphocyte proliferation test was highly uncertain until tested in patients with other granulomatous and inflammatory diseases like sarcoidosis (Rossman et al., 1988). Exposure markers may also need such careful testing given the likelihood that certain disease states may drastically interfere with bioaccumulation and disposition of toxins. This kind of effort is especially important for toxins which are ubiquitous like heavy metals, halogenated hydrocarbons, etc., and the effects of which might include (confounding) damage to excretory organs of relevance. Such has evidently been shown through elegant clinical investigation to be the case for urinary cadmium (Lauwerys et al., 1979), an exposure marker at low level but, at higher values, an index either of high exposure or renal injury due to the metal. A final advantage of this research modality is the opportunity it may afford to study some of the "susceptibility" markers which are crucial both in and of themselves, as well as for correctly interpreting exposure-effect relations. This is an area which heretofore has been least explored, in part because molecular and other tools have only recently become available. Nontheless, the basic structure for such studies is relatively clear: direct comparisons at the appropriate (i.e., hypothesized) biological level between comparably exposed subjects with varying degrees of response. For example, at present our group is in the midst of a study of various exposure and early effect markers in a group of heavily exposed asbestos workers who had been clinically followed for periods up to 6 years prior to study. Among those who had shown evidence of progression of fibrotic disease, markers of alveolitis (e.g. polymorphonuclear cells) are more prominent than in nonprogressors even after controlling for extent of current disease judged radiographically or functionally (Merrill et al., 1989i. In the next phase we plan to explore environmental and host factor differences between the "progressors" and the "nonprogressors" in an attempt to define determinants of ongoing fibrogenesis after exposure has ceased. Given the highly variable disease expression among those comparably exposed, it appears likely that factors other than asbestos per se are important and hopefully more amenable to late modification. Obviously it will ultimately be necessary to test any theories which emerge by establishing accessible surrogate susceptibility markers and applying them on larger, unselected populations. On the other hand, it is hard to envision finding an
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explanation for the differences in progression, which took many years even to discern by noninvasive means, without these intervening clinical investigations. NEW DIRECTIONS FOR TOXICOLOGY AND EPIDEMIOLOGY The insertion of clinical investigation into a prominent position in the collaborative milieu of biological marker research leads, inevitably, to some new challenges for the toxicologist and epidemiologist. I conclude this essay with a brief consideration of these implications. Historically, the toxicologist as independent investigator has served two prime functions in OEM research: first, to predict, on the basis of an experimental design, what effects a toxin might induce in humans (and at what relative doses); second, to explicate effects proven or suspected in humans, and/or revealing underlying mechanisms, by reproducing them in a model free from confounding influences. In the proposed schema, animal experimentation must take some new perspectives defined by the collaborative agenda. Foremost among these is exploration for potential markers of exposure and effect, for accessible surrogates which correlate with these, and for factors, genetic and environmental, which might modify the sequence anywhere between exposure and outcome. While experimental proof of these relations may be appropriate as collaboration proceeds, the fundamental mode is not hypothesis driven but descriptive with its goal to inform parallel human studies. Such efforts might interdigitate in countless ways with coordinated population studies and clinical investigations but would be, in any event, driven by the larger picture, not independently motivated. A particularly crucial area for animal studies in the proposed context would be in "sequencing" effects--establishing the temporal relations between early events, starting with actual exposure, and late ones of medical and public health interest. Given the special problem, elucidated above, of linking early effect markers to relevant adverse health consequences, this portion of the sequence would likely be a major focus, especially since human proof of such connections may take long periods of time and/or create ethical constraints, e.g., if an available intervention is known or suspected to alter the sequence. Although these kinds of fundamentally predictive, experimental studies are not foreign to the independent toxicologist, attention to intermediate, rather than endpoint, events may be. For example, rather than the typical sacrifice of animals at defined time points with comparison of group results at these various intervals, attention to the sequence might prompt strategies to "sample" animals, without destroying them, specifically to observe the temporal relationships of responses in each animal. Such experiments could readily allow the study of the predictive values of findings (i.e., markers) at time q, for findings (i.e., health effects) at a later time t 2. Strong predictive value in a relevant animal model could, in turn, substantially alter the biological significance attached to parallel epidemiologic evidence that the early marker is causally linked to the environmental toxin of interest. The proposed collaborative strategy would also likely modify the traditional role of the epidemiologist in etiologic research. The review of Hulka and Wilcosky admirably addresses many of the opportunities and difficulties in interpretation of data sets which include biological measures of exposure or effect (Hulka and
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Wilcosky, 1988). Not considered, however, are the outstanding interpretative and stochastic problems (and, I would argue, opportunities) created by the necessarily small, but biologically rich, data sets from clinical investigations. While quite obviously these sets, derived on highly selected research volunteers, cannot substitute for observational studies in large, unselected populations of interest, they no less, and probably more, demand attention to inferential strategy. Such strategies no longer entail the standard formula of attempting to adjust observational data for the biases potentially introduced by nonexperimental design but rather call into play extraordinarily diverse bases of belief, e.g., prior hypotheses based on animal findings, biological paradigms, or previous clinical observations, to design and interpret clinical investigations. While certain problems are inevitably difficult to manage, e.g., the effects of selection on volunteer studies, other typical sources of variability, like "background" disease risk, may be easily controlled by virtue of the extent of medical testing. Thus, in the bone marrow study of men with hematologic abnormalities associated with ethylene glycol ether exposure, we could confidently exclude consideration of common causes of anemia and neutropenia in the study population by "ruling them out." Establishing differences between blood counts of exposed and unexposed workers required statistical inference in the original population study (Welch and Cullen, 1988) but could receive quite different treatment once major sources of confounding were excluded biologically. The focus could then be shifted to factors which might explain why some members of the exposed cohort had the specific effect while others did not. To summarize, while the clinical investigations of medical subspecialists, geared toward mechanistic endpoints, have generally entailed limited input from epidemiologists (probably inappropriately so), use of the same modalities in etiologic research will demand not only involvement but supervision of such projects by epidemiologists. At present, to my knowledge, no broad experience with this kind of work or body of theory has yet emerged to guide the design and data interpretation aspects of biological markers research as proposed. SUMMARY
The limitations of current research activities in OEM have fostered conceptualization of a new paradigm for research during the past decade. Unfortunately, efforts at utilizing molecular epidemiology or biological markers research, although adding greatly to our understanding of many toxic effects, have yet to yield substantive new associations. Meanwhile, theoretical consideration has progressed with clear and elegant attention to emerging problems and opportunities. The thrust of this essay has been to propose incorporation of an old research modality into the central schema--research-center-based clinical investigation. Examples of previous and ongoing work of this type illustrate the potential to surmount some o f the recognized problems of biological markers and reap some of their proposed benefits. However, full realization will demand far broader collaboration and modification of the traditional approaches of toxicology and epidemiology if large, presently insoluble, problems are to be successfully attacked.
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REFERENCES Cherniack, M. (1988). Toxicologic screening for organophosphorous-induced delayed neurotoxicity: Complications in toxicity testing. Neurotoxicology 19, 249-272. Churg, A. (1983). Asbestos fiber content of the lungs in patients with and without asbestos airways disease. Amer. Rev. Resp. Dis. 127, 470--473. Dumortier, P., DeVuyst, P., and Yernault, J. C. (1988). Mineralogical analysis of bronchoalveolar lavage fluids. Z. Erkr. Atmungsorgane 17, 50-58. Hattis, D. (1987). The value of molecular epidemiology in quantitative health risk assessment. In (S. Draggan, J. J. Cohrssen, and R. E. Morlison, Eds.), "Environmental Impacts on Human Health" pp. 89-116. Praeger, New York. Hulka, B. S., and Wilcosky, T. (1988). Biological markers in epidemiologic research. Arch. Environ. Health 43, 83-89. Kreiss, K., Newman, L. S., Mroz, M. M., and Campbell, P. A. (in press). Screening blood test identifies subclinical beryllium disease. J. Occup. Med. Lauwerys, R. B., Roels, H. A., Regniers, M., et al. (1979). Significance of cadmium concentration in blood and in urine in workers exposed to cadmium. Environ. Res. 20, 375-391. Lipkin, M., and Newmark, H. (1985). Effect of added dietary calcium on colonic epithelial cell proliferation in subjects at high risk for familial colonic cancer. N. Engl. J. Med. 313, 1381-1384. Merrill, W., Cullen, M. R., and Care, S. B. (1989). Neutrophilic alveolitis and functional deterioration among asbestos workers. CIin. Res. 37, 479A. Mulvihill, J. J. (1977). Genetic repertory of human neoplasia. In "Genetics of Human Cancer" (J. J. Mulvihill, R. W. Miller, J. F. Fraumeni, Eds.). Raven Press, New York. Mulvihill, J. J. (1980). Clinical observations of ecogenetics in human cancer. C. C. Harris, Moderator. Individual differences in cancer susceptibility. Ann. Intern. Med. 92, 809--813. Patterson, R., Zeiss, C. R., and Pruzansky, J. J. (1982). Immunology and immunopathology of trimellitic anhydride pulmonary reactions. J. Allergy Clin. Immunol. 70, 19-23. Perera, F. P., and Weinstein, I. B. (1982). Molecular epidemiology and carcinogen-DNA adduct detection: New approaches to studies of human cancer causation. J. Chron. Dis. 35,581--600. Robins, J. M., Cullen, M. R., and Welch, L. S. (1987). Improved methods for discerning health impacts of current technologies. In "Environmental Impacts on Human Health" (S. Draggan, J. J. Cohrssen, and R. E. Morrison, Eds.), pp. 165-192. Praeger, New York. Roggli, V. L., Pratt, P. C., and Brudy, A. R. (1986). Asbestos content of lung tissue in asbestosassociated diseases. Brit. J Ind. Med. 43, 18-28. Rossman, M. D., Kern, J. A., Elias, J. A., et al. (1988). Proliferative response of bronchoalveolar lymphocytes to beryllium. Ann. Intern. Med. 108, 687-693. Schulte, P. A. (1987). Simultaneous assessment of genetic and occupational risk factors. J. Occup. Med. 29, 884-891. Welch, L. S., and Cullen, M. R. (1988). Effects of exposure to ethylene glycol ethers on shipyard painters. III. Hematologic effects. Amer. J. Ind. Med. 4, 527-536.
REVIEW The Role of Clinical Investigations in Biological Markers Research MARK R. CULLEN
Yale-New Haven Occupational Medicine Program, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Received October 18, 1988 As the agenda of the occupational and environmental health community proceeds from the study of the effects of high doses of toxins causing grossly altered disease patterns to examination of the impact of lower doses on prevalent disorders, new scientific approaches have become necessary. The emerging concept of molecular epidemiology, substituting biologically determined markers of dose and early effect for traditional measures of exposure and morbidity, offers a heuristically appealing direction for the field. However, the transition to this evolving approach has been slow and its potential has yet to be validated. In this essay, some of the limitations of this approach are discussed and theoretical modifications developed. The central proposition is that existing modalities of clinical research, widely employed in subspecialty medicine research but heretofore unexploited in the study of environmental diseases, offer great promise. By incorporating clinical investigations centrally, some historically rate-limiting problems such as variability of host responses, multiplicity of environmental risks, and differences between human and animal responses could be transformed from major impediments at present to realistic objects for future study. © 1989AcademicPress, Inc.
INTRODUCTION With the emergence of occupational and environmental medicine (OEM) as a relevant and visible clinical discipline in the 1970s and 1980s, increasing examination of the scientific foundation of the field has begun. It is the intention of this paper to summarize and critique existing concepts and, based on this, propose an approach for the future. Ideally, such an approach could more efficiently yield answers to the questions which render current practice so limited for prevention and cure of even the best understood environmentally induced diseases. In the most traditional view, OEM has been conceived as a subdiscipline of preventive medicine, whose scientific tools are chronic disease epidemiology and animal toxicology. The venerated paradigm is that studies in animals would provide evidence of possible human risk for disease which would be confirmed by epidemiologic evaluation of large populations. Alternatively, laboratory investigation could be used to follow-up observations made by clinicians and epidemiologists. Indeed, many of the most notorious and important disease associations, like those linking asbestos with lung cancer and vinyl chloride with hepatic angiosarcoma, were discovered and verified via these pathways. Unfortunately, the great days for these paradigms are rapidly passing. For the epidemiologist, so productive during the 1960s and 1970s at finding the toxins
0013-9351/89 $3.00 Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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which cause rare diseases or drastically increase the risk for common ones, three difficulties have emerged. First, since the strongest associations were quickly uncovered, the outstanding challenge for the future is to elucidate factors which increase risk for diseases modestly (Robins et al., 1987). Though such causes could be extremely important from a clinical or public health point of view (imagine a workplace chemical that increased the risk of cardiovascular disease by a " m e r e " 20 or 30%), these kinds of association are very hard to detect because the relative risk for exposed, compared to nonexposed people, is only slightly elevated; small biases in study design and statistical quirks make interpretation of such findings highly subject to controversy, hence uncertainty for practice. This problem is compounded by the fact that with increasing environmental regulation and control, most exposures of interest are moderately low-dose, at least compared to historic industrial levels. Complicating this central dilemma is the imprecision of modern field methods to accurately quantify human exposures, especially those which occurred years before study commenced. In the earlier search for strong associations, such inaccuracies were relatively unimportant; they could not mask overwhelming excess risk among the exposed. Searching for weaker (but possibly more important) associations, the ability to classify people by the amount of their exposures becomes crucial, if no less difficult. Any misclassification--counting exposed subjects as unexposed or vice versa--would have the effect of reducing the possibility to detect real differences between exposure groups. Finally, the temporal pace of the traditional approach, which must await large populations exposed for long periods, causing widespread effects before analysis is possible, has become a serious drawback. Regulators and clinicians are too impatient to await such uncontrolled "human experimentation" and desperately seek shorter term strategies in which populations need not be "condemned" to be studied. Thus, for example, after years of human exposure and despite impressive levels of study and publication during the past decade, the potential of such ubiquitous dusts as fiberglass and silica to cause human lung cancer remains uncertain, possibly unsoluble by the old model. The problems for toxicology, as traditionally practiced, are quite different though not less critical. Working in virtual isolation from clinicians and field investigators, a substantial portion of ongoing work is disturbingly remote from human application. Such differences as broad genetic variability, coexposure to a multiplicity of "confounding" factors in the environment, and the tendency to idiosyncratic responses all distinguish human from animal responses. These differences need not, of themselves, preclude an extremely valuable role for laboratory-based animal study; they do, however, demand a different focus, or context, if results are to be broadly meaningful to questions of human health. This is especially true in the social context in which environmental concerns must be juxtaposed against other important societal needs; our culture seems, perhaps justifiably, unwilling to defer economic growth to control hypothetical risks based on simple extrapolation from animal models. The importance of genetic and more general host variability has not been lost on the toxicologic community. In the 1970s, with the emergence of technologies for
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effectively studying the human genome, the field of ecogenetics was introduced by Mulvihill and others(Mulvihill, 1977, 1980). This largely experimental, laboratory-based discipline strove to delineate the mechanisms which underlie variations in human responses with the expectation that these findings might illuminate the always peculiar patterns of observations made by clinicians and epidemiologists. However, as recently discussed by Schulte, the products of such work have not yet found major applicability to epidemiologic study (Schulte, 1987). MOLECULAR EPIDEMIOLOGY AND BIOLOGICAL MARKERS RESEARCH
In the early 1980s, the concept of "molecular epidemiology" was coined by investigators at Columbia to describe an evolving approach to research which attempted to synthesize existing scientific tools into a new formulation (Perera and Weinstein, 1982). The theory rests on two biologic tenets: (1) early biologic effects from a toxic exposure are far more prevalent in the population at risk than the late events of direct interest, like cancer or chronic disease, and may sometimes be more specific to the exposure than the outcome itself; (2) given technologic advances, most toxins can be either directly quantified in the body or indirectly measured by identification of some predictable, dose-related biologic response. Combining these two principles, a new approach to human study emerged. Once (prevalent, early) "markers" of effect and (accurate) "markers" of dose could be developed in the laboratory, human epidemiology could proceed without prior constraints--relative risks would be high because the events studied are either very common among the exposed (i.e., sensitive markers) or very rare among the unexposed (i.e., specific markers); exposures could be precisely classified by direct measurement and the lapsed time between first human exposure and an opportunity for study would be foreshortened because endpoints are, by definition, early. In addition to these central theoretical advantages, certain side-benefits may also be anticipated as recently discussed in a critical evaluation by Hulka and Wilcosky (I988). These include (1) a contribution to the understanding of underlying pathogenic mechanisms inherent in study of events at the molecular, cellular or tissue levels; (2) the potential for more accurate and etiologic classifications of environmental diseases; and (3) the possibility that recognition of early effects could prompt strategies for secondary prevention or early disease modification. The conception of research embodied in this new synthesis represents an enormous theoretical advance and has already stimulated reorganization and redirection of some established research agendas. On the other hand, the promise of this nascent movement has not quickly materialized; no single major new association between a toxin and a major health endpoint has been proven, or even suggested, by such an approach. While 5 years may be, perhaps, too short a time for serious judgment, there are certain recognizable limitations to the framework which portend that it will not "revolutionize the field," as proponents have predicted, without significant refinement, both conceptual and practical. Many of these problems have already ben elucidated in the literature (Hulka and Wilcosky, 1988; Hattis, 1987). On the exposure side of the equation, the specificity of markers in relationship to a particular exposure of interest may limit
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the utility of otherwise accessible and mechanistically interesting tools. For example, genetic abberations, including the much studied sister chromatid exchange, may be affected by a broad range of host and environmental factors beyond any agent or agent whose effects are to be investigated; use of such biological markers may introduce confounding which cannot be adequately adjusted for until the operational characteristics of the test itself are conducted and the range of potential confounding factors elucidated. More specific exposure markers, like the level of toxin, or its metabolites or biological reaction products (e.g., toxin-protein or toxin-nucleic acid adducts) in a biologic tissue or fluid may be problematic because of the complexity of the factors which determine such levels. These include host metabolism and toxicokinetics, current physiologic state, length of marker persistence, etc. The underlying problem is that all biological exposure markers, however intimately linked to exposure, entail some "processing" by the host which is likely to be mediated both by intrinsic host factors and by other environmental exposures. In other words, factors which may be associated with the "effect" may also modify the measure of exposure, severely confounding causal reasoning. For example, differences in levels of D N A adducts in urine of workers exposed to polyaromatic hydrocarbons (PAHs) may reflect as much host variability in metabolizing the PAHs as differences in exposure per se; a correlation between this measure of "dose" and a cancer endpoint could falsely suggest a dose response while, in fact, merely proving that those at underlying higher risk have higher risk. The addition to the equation of "susceptibility" markers as proposed by Hulka (Hulka and Wilcosky, 1988) offers a theoretical solution but one which may be very elusive in practice. Establishing a priori a list of such modifiers for a given toxic may represent a formidable challenge. For example, considerable interest has emerged during the past decade in the use of retained asbestos fibers in lung tissue as an exposure marker for asbestos. Unfortunately, enormous interindividual differences have been found even where exposures have ostensibly been comparable (Churg, 1983). Presumably these differences, which do correlate somewhat well with asbestosis grade (Roggli et al., 1986), reflect variability in deposition and/or clearance, in turn likely due to such "susceptibility" factors as breathing pattern, smoking behavior, extent of lung disease (asbestos-related or otherwise), and likely others. Study of these variables is further complicated by methodologic issues, such as timing of exposure relative to collection, the effect of the pathologic process itself on fiber counting, etc. On the effect side of the equation, other serious theoretical and practical problems have been recognized. Most troublesome is the relationship between early effects, e.g., altered tissue morphology or gene expression, and subsequent diseases of interest. Indeally, these could be established experimentally or by longitudinal observations of exposed individuals. In reality, however, both ethical and practical constraints limit this kind of data collection to situations where material obtained from individuals under surveillance or study can be retrospectively evaluated in relation to disease outcomes. An alternative to this approach is juxtaposition of separate, but related, studies. For example, it has been shown that certain host and environmental factors cause alterations in crypt morphology
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of the colonic mucosa (Lipkin and Newmark, 1985). From the separately proven fact that patients with colon cancer demonstrate similar morphologic alterations at sites remote from their tumors, it might be inferred that the host and environmental factors from the former study are causally linked to colon cancer. While this may be true, such reasoning may fall prey to a variation of the ecologic fallacy; "transitivity" of biologic relations cannot be presumed unless or until all of the intervening steps are worked out. Another problem pertains to the practicality of studying early biologic responses of interest. Although technology now exists to obtain virtually any human material for study with minimal health risk, population studies constrain the investigator to examination of only readily obtainable materials or tests, e.g., blood, urine, X-rays, etc. These materials must, somehow, serve as "surrogates" for the end-organ targets of interest. Finding such suitable surrogates becomes, itself, a challenging area of research which heretofore has been largely limited to the use of animals. An elegant example is the recent demonstration that lymphocyte esterase may serve as a surrogate for the more relevant neural esterase in evaluating delayed neurotoxicity of organophosphorous esters (Cherniack, 1988). Unfortunately, as with all of the other relationships discussed above, individual "susceptibility" factors--genetic or acquired--may modify the relationship between the target of interest and surrogates, confounding use of the latter as a reflection of the former. Probably more importantly, a high proportion of endpoints of interest may have no suitable marker easily accessible to the field investigator or only ones which bear too remote a relation to sequelae of interest. THE ROLE FOR CLINICAL INVESTIGATIONS
The concept of molecular epidemiology, or biological markers research as its generalization is coming to be known, is fundamentally sound. The above critique notwithstanding, this new synthesis of the parent scientific disciplines of OEM--toxicology and epidemiology---brings the unique strengths of each to the challenge of studying toxins at doses suspect to cause only low or moderate relative risk for adverse health effects. No other new approach has emerged which offers nearly the same scientific opportunity. It is only with great respect, therefore, that I offer the following ideas about directions future studies may take to obviate some of the problems discussed above and bring some of the promise of molecular epidemiology more quickly to fruition. The most fundamental modification which I propose to the existing paradigms is the addition of a research tool historically underutilized in OEM research center-based clinical investigation. Specifically, I refer to in- or outpatient studies performed under highly controlled conditions on invited subjects which have become the cornerstone of research in clinical subspecialty medicine. Unlike typical epidemiologic studies, study size is necessarily quite small; this limitation is balanced by the breadth and depth of information which can be obtained on each subject, especially in terms of biological parameters, compared to what may be gleaned from larger populations in the field. It is the central tenet of this paper that this type of architecture, so effectively used by colleagues in other disciplines to study disease mechanisms, physiology, and responses to therapeutic interven-
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tions, can be readily transformed to productive use in etiologic research, especially in the context of collaboration with epidemiologists and toxicologists engaged in biologic markers research. In the paragraphs which follow, I shall illustrate some of the potential advantages of this approach in light of problems elucidated above. The first potential role for clinical investigation is in the area of marker identification. Subjects with well-characterized exposures and/or consequences of exposure may be "explored" in search of various biological phenomena which have been hypothesized to be indicative measures of exposure or early effect. Given the controlled setting in a research center environment and truly volunteer subjects, there need be no arbitrary or artificial limit to the source of material (within reason), or, in general, the amount. Conspicuous examples of this type of research are the studies of immunologic lung diseases in which the specific assays have been elucidated for immunoglobulins against sensitizers such as trimellitic anhydride (Patterson et al., 1982) and in vitro lymphocyte proliferative responses to beryllium and other causes of alveolitis (Rossman et al., 1988). Exposure markers, as well, have been explored in this research milieu, such as the determination of mineral fiber burdens in bronchoalveolar lavage fluid of exposed and affected subjects (Dumortier et al., 1988). A close corollary is the opportunity to explore for "surrogate" markers, especially those related to early effect, when primary markers involve inaccessible tissues. For example, our own group has recently completed bone marrow examinations on selected subcohorts of a population previously demonstrated to have hematologic abnormalities on peripheral smears associated with exposure to ethylene glycol ethers (Welch and Cullen, 1988). Simultaneously we examined several peripherally measured indices, including red cell enzymes which have historically been associated with stem cell abnormalities. Although the data are not yet fully analyzed, early analysis suggests a very close correlation between myeloid hypoplasia in the marrow and certain red cell enzymes in peripheral blood, hopefully paving the way for a sensitive early effect marker for use in future field studies. Similarly, investigators in Denver have utilized lavage studies in beryllium workers to develop for the first time a sensitive peripheral lymphocyte assay for hypersensitivity to beryllium (Kreiss et al., in press), an invaluble step in the effort to define the elusive dose-response relationship for this agent. Establishing "linkage" between apparent early effect markers and late health effects of interest may also be facilitated by these kinds of biologically intense clinical investigations. By simultaneously studying diseased as well as nondiseased individuals with varying degrees and lengths of an exposure of interest, one may be able to reconstruct and even sequence temporally disparate events. The Denver beryllium study has illustrated this concept as well. After developing an accessible effect marker, a beryllium-exposed population was screened with center-based follow-up investigation of selected "positives" and "negatives." Surprisingly, almost all with positive tests demonstrated striking alveolitis on bronchoalveolar lavage, despite the absence of clinical symptoms or signs of chronic beryllium disease; alveolitis was not seen among negatives (Kreiss et al., in press). By this clever, cross-sectional design, the investigators have elucidated
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the sequence of most of the events leading to the development of chronic beryllium disease: exposure, followed after variable latency by sensitization, followed after apparently short latency by alveolitis, followed after unknown latency by clinical disease. Clinical investigations may also hold the key to successful evaluation of the "operational characteristics" of new markers, as they have for clinical tests generally. Although ultimately all markers require evaluation in populations sufficiently large to draw strong causal inferences, many facets of marker evaluation may require testing of controls which are too invasive for the field or of subjects with specific unusual diseases. For example, full evaluation of the relationship between red cell enzyme levels and ethylene glycol ethers, alluded to above, will require study of subjects with a range of myeloproliferative and other hematologic disturbances in addition to the more obvious control groups, i.e., healthy exposed and unexposed workers. Similarly, the significance of the beryllium lymphocyte proliferation test was highly uncertain until tested in patients with other granulomatous and inflammatory diseases like sarcoidosis (Rossman et al., 1988). Exposure markers may also need such careful testing given the likelihood that certain disease states may drastically interfere with bioaccumulation and disposition of toxins. This kind of effort is especially important for toxins which are ubiquitous like heavy metals, halogenated hydrocarbons, etc., and the effects of which might include (confounding) damage to excretory organs of relevance. Such has evidently been shown through elegant clinical investigation to be the case for urinary cadmium (Lauwerys et al., 1979), an exposure marker at low level but, at higher values, an index either of high exposure or renal injury due to the metal. A final advantage of this research modality is the opportunity it may afford to study some of the "susceptibility" markers which are crucial both in and of themselves, as well as for correctly interpreting exposure-effect relations. This is an area which heretofore has been least explored, in part because molecular and other tools have only recently become available. Nontheless, the basic structure for such studies is relatively clear: direct comparisons at the appropriate (i.e., hypothesized) biological level between comparably exposed subjects with varying degrees of response. For example, at present our group is in the midst of a study of various exposure and early effect markers in a group of heavily exposed asbestos workers who had been clinically followed for periods up to 6 years prior to study. Among those who had shown evidence of progression of fibrotic disease, markers of alveolitis (e.g. polymorphonuclear cells) are more prominent than in nonprogressors even after controlling for extent of current disease judged radiographically or functionally (Merrill et al., 1989i. In the next phase we plan to explore environmental and host factor differences between the "progressors" and the "nonprogressors" in an attempt to define determinants of ongoing fibrogenesis after exposure has ceased. Given the highly variable disease expression among those comparably exposed, it appears likely that factors other than asbestos per se are important and hopefully more amenable to late modification. Obviously it will ultimately be necessary to test any theories which emerge by establishing accessible surrogate susceptibility markers and applying them on larger, unselected populations. On the other hand, it is hard to envision finding an
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explanation for the differences in progression, which took many years even to discern by noninvasive means, without these intervening clinical investigations. NEW DIRECTIONS FOR TOXICOLOGY AND EPIDEMIOLOGY The insertion of clinical investigation into a prominent position in the collaborative milieu of biological marker research leads, inevitably, to some new challenges for the toxicologist and epidemiologist. I conclude this essay with a brief consideration of these implications. Historically, the toxicologist as independent investigator has served two prime functions in OEM research: first, to predict, on the basis of an experimental design, what effects a toxin might induce in humans (and at what relative doses); second, to explicate effects proven or suspected in humans, and/or revealing underlying mechanisms, by reproducing them in a model free from confounding influences. In the proposed schema, animal experimentation must take some new perspectives defined by the collaborative agenda. Foremost among these is exploration for potential markers of exposure and effect, for accessible surrogates which correlate with these, and for factors, genetic and environmental, which might modify the sequence anywhere between exposure and outcome. While experimental proof of these relations may be appropriate as collaboration proceeds, the fundamental mode is not hypothesis driven but descriptive with its goal to inform parallel human studies. Such efforts might interdigitate in countless ways with coordinated population studies and clinical investigations but would be, in any event, driven by the larger picture, not independently motivated. A particularly crucial area for animal studies in the proposed context would be in "sequencing" effects--establishing the temporal relations between early events, starting with actual exposure, and late ones of medical and public health interest. Given the special problem, elucidated above, of linking early effect markers to relevant adverse health consequences, this portion of the sequence would likely be a major focus, especially since human proof of such connections may take long periods of time and/or create ethical constraints, e.g., if an available intervention is known or suspected to alter the sequence. Although these kinds of fundamentally predictive, experimental studies are not foreign to the independent toxicologist, attention to intermediate, rather than endpoint, events may be. For example, rather than the typical sacrifice of animals at defined time points with comparison of group results at these various intervals, attention to the sequence might prompt strategies to "sample" animals, without destroying them, specifically to observe the temporal relationships of responses in each animal. Such experiments could readily allow the study of the predictive values of findings (i.e., markers) at time q, for findings (i.e., health effects) at a later time t 2. Strong predictive value in a relevant animal model could, in turn, substantially alter the biological significance attached to parallel epidemiologic evidence that the early marker is causally linked to the environmental toxin of interest. The proposed collaborative strategy would also likely modify the traditional role of the epidemiologist in etiologic research. The review of Hulka and Wilcosky admirably addresses many of the opportunities and difficulties in interpretation of data sets which include biological measures of exposure or effect (Hulka and
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Wilcosky, 1988). Not considered, however, are the outstanding interpretative and stochastic problems (and, I would argue, opportunities) created by the necessarily small, but biologically rich, data sets from clinical investigations. While quite obviously these sets, derived on highly selected research volunteers, cannot substitute for observational studies in large, unselected populations of interest, they no less, and probably more, demand attention to inferential strategy. Such strategies no longer entail the standard formula of attempting to adjust observational data for the biases potentially introduced by nonexperimental design but rather call into play extraordinarily diverse bases of belief, e.g., prior hypotheses based on animal findings, biological paradigms, or previous clinical observations, to design and interpret clinical investigations. While certain problems are inevitably difficult to manage, e.g., the effects of selection on volunteer studies, other typical sources of variability, like "background" disease risk, may be easily controlled by virtue of the extent of medical testing. Thus, in the bone marrow study of men with hematologic abnormalities associated with ethylene glycol ether exposure, we could confidently exclude consideration of common causes of anemia and neutropenia in the study population by "ruling them out." Establishing differences between blood counts of exposed and unexposed workers required statistical inference in the original population study (Welch and Cullen, 1988) but could receive quite different treatment once major sources of confounding were excluded biologically. The focus could then be shifted to factors which might explain why some members of the exposed cohort had the specific effect while others did not. To summarize, while the clinical investigations of medical subspecialists, geared toward mechanistic endpoints, have generally entailed limited input from epidemiologists (probably inappropriately so), use of the same modalities in etiologic research will demand not only involvement but supervision of such projects by epidemiologists. At present, to my knowledge, no broad experience with this kind of work or body of theory has yet emerged to guide the design and data interpretation aspects of biological markers research as proposed. SUMMARY
The limitations of current research activities in OEM have fostered conceptualization of a new paradigm for research during the past decade. Unfortunately, efforts at utilizing molecular epidemiology or biological markers research, although adding greatly to our understanding of many toxic effects, have yet to yield substantive new associations. Meanwhile, theoretical consideration has progressed with clear and elegant attention to emerging problems and opportunities. The thrust of this essay has been to propose incorporation of an old research modality into the central schema--research-center-based clinical investigation. Examples of previous and ongoing work of this type illustrate the potential to surmount some o f the recognized problems of biological markers and reap some of their proposed benefits. However, full realization will demand far broader collaboration and modification of the traditional approaches of toxicology and epidemiology if large, presently insoluble, problems are to be successfully attacked.
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