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9 Biological, Biochemical, and Molecular Markers in Environmental Epidemiology

Downloaded by UNIV LAVAL on November 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0241.ch009

M a r i l y n F. Vine Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, N C 27599-7400

A biological or molecular marker is a cellular, biochemical, or molecular alteration that is measurable in biological media, such as human tissues, cells, or fluids. Biological markers are being explored to determine their ability to help us understand the intermediate steps between exposure and disease occurrence and to improve the precision of exposure and outcome measures in epidemiological studies. The purpose of this chapter is to discuss (1) the potential benefits of using biological markers in epidemiological research, (2) the chief characteristics

of biological markers that make them

particularly

useful in epidemiological studies, (3) the limitations of the use of biological markers in epidemiological research, and (4) the rewards and obstacles involved in collaborative efforts between laboratory scientists, who develop the assays to detect markers, and epidemiologists.

JEPIDEMIOLOGY IS THE STUDY of the d i s t r i b u t i o n a n d d e t e r m i n a n t s of d i s eases i n populations. Traditionally, epidemiologists have taken a " b l a c k b o x " approach to the study of e n v i r o n m e n t a l l y i n d u c e d disease, assessing an e n v i r o n m e n t a l or external exposure b y u s i n g , for e x a m p l e , questionnaires a n d e n v i r o n m e n t a l m o n i t o r i n g a n d t h e n evaluating the association b e t w e e n the exposure measure a n d some h e a l t h outcome. B i o l o g i c a l markers are b e i n g e x p l o r e d to d e t e r m i n e t h e i r ability to h e l p us u n d e r s t a n d the i n t e r m e d i a t e steps b e t w e e n exposure a n d disease occurrence a n d to i m p r o v e the p r e c i sion of exposure a n d o u t c o m e measures. T h i s chapter provides an o v e r v i e w of biological markers u s e d i n e p i d e miological studies. It discusses the properties of markers that m a k e t h e m potentially valuable to epidemiologists, a n d reviews c r i t e r i a for the selection of markers to be u s e d i n e p i d e m i o l o g i c a l investigations. T h e c u r r e n t l i m i 0065-2393/94/0241-0105$08.00/0 © 1994 A m e r i c a n C h e m i c a l Society

In Environmental Epidemiology; Draper, W.; Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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tations of markers i n e p i d e m i o l o g i c a l research are also discussed. Because laboratory assays are i n v o l v e d i n m e a s u r i n g the presence of biological m a r k ers i n h u m a n biological m a t e r i a l , the benefits a n d challenges of collaborative efforts b e t w e e n epidemiologists a n d laboratory scientists are p r e s e n t e d . E x amples of situations i n w h i c h biological markers have b e e n u s e d i n e n v i r o n m e n t a l - o c c u p a t i o n a l investigations are p r o v i d e d .


Types of Biological Markers A biological m a r k e r is a " c e l l u l a r , b i o c h e m i c a l , o r m o l e c u l a r alteration that is measurable i n biological m e d i a such as h u m a n tissues, cells or f l u i d s " (I). B i o l o g i c a l markers can be m e a s u r e d i n a v a r i e t y of target a n d surrogate tissues. Target tissues are tissues that u n d e r g o pathological changes as the r e sult of exposure (2). A n example of a target tissue for cigarette smoke exposure is the l u n g . M a r k e r levels i n surrogate (nontarget) tissues, s u c h as b l o o d or u r i n e , can sometimes be u s e d to estimate m a r k e r levels i n target tissues. B i o l o g i c a l markers can be c o n c e p t u a l i z e d as f o r m i n g a c o n t i n u u m that represents a sequence of events, f r o m exposure to an exogenous agent to disease occurrence (3). F i g u r e 1 (4) presents the categories of biological markers that represent the various stages i n the sequence of events. T h e s e categories i n c l u d e markers of i n t e r n a l dose, biologically effective dose, early response, altered structure a n d f u n c t i o n , a n d disease. T h i s sequence of events begins w h e n an i n d i v i d u a l is exposed to some e n v i r o n m e n t a l or external agent a n d is b e l i e v e d to progress, unless the agent is e l i m i n a t e d from the b o d y a n d the damage that results from the exposure is r e p a i r e d . O u t s i d e of the sequence of events are susceptibility factors that can act at any p o i n t along the c o n t i n u u m to modify the effects of external exposures. T h i s c o n c e p t u a l framework is based o n the w o r k of P e r e r a a n d W e i n s t e i n (5).

i

Exposure-

Internal Dose

1,

Biologically Effective Dose

i

Biological Response

t

p

Altered Structure/ Function

Disease | ^ Prognosis

/ ^ E ^ r o n m e n T N

V^LifeStyleX Figure 1. The relationship of biological markers to exposure and disease. (Modified with permission from reference 4. Copyright 1989 National Academy Press.)


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A m a r k e r o f i n t e r n a l dose indicates that an exogenous c h e m i c a l has e n t e r e d the b o d y of an exposed i n d i v i d u a l . A n exogenous c h e m i c a l m a y e n t e r the b o d y b y inhalation, i n g e s t i o n , or absorption t h r o u g h the s k i n . T h e i n t e r n a l dose m a r k e r may measure the c h e m i c a l itself (unchanged) or a m e t a bolically a l t e r e d state (3). A n example of an i n t e r n a l dose m a r k e r that can be m e a s u r e d u n c h a n g e d is lead i n b l o o d (6). A m a r k e r of a m e t a b o l i c a l l y a l t e r e d c h e m i c a l is cotinine i n the b o d y fluids of smokers. C o t i n i n e , a m e t a b o l i c b y p r o d u c t of nicotine m e t a b o l i s m (7), is a m a r k e r of exposure to tobacco smoke. A m a r k e r of biologically effective dose ( B E D ) indicates " t h e a m o u n t of absorbed c h e m i c a l that has i n t e r a c t e d w i t h c r i t i c a l s u b c e l l u l a r targets, m e a s u r e d i n e i t h e r a target o r a surrogate t i s s u e " (3). W o r k e r s exposed to coke o v e n emissions c o n t a i n i n g p o l y c y c l i c aromatic hydrocarbons are at increased risk o f l u n g cancer (8). D N A adducts (complexes o f chemicals covalently b o n d e d to D N A , d e r i v e d f r o m l u n g tissue, a n d o b t a i n e d at autopsy or t h r o u g h biopsy) c o u l d p o t e n t i a l l y serve as a biologically effective dose m a r k e r for the target tissue. D N A adducts are b e l i e v e d to b e i n v o l v e d i n the initiation of cancer (9). A l t h o u g h it is u n l i k e l y that h e m o g l o b i n adducts (complexes of e l e c t r o p h i l i c chemicals covalently b o n d e d w i t h h e m o g l o b i n i n r e d b l o o d cells) are d i r e c t l y i n v o l v e d i n the carcinogenic process, they may serve as surrogate B E D markers of D N A adduct formation i n target tissues. A n i m a l (10-12) a n d h u m a n studies (13) suggest that for some c h e m i c a l s , an i n crease i n h e m o g l o b i n adduct formation is associated w i t h an increase i n D N A adduct formation i n target tissues. A m a r k e r of biologic response indicates " b i o l o g i c a l or b i o c h e m i c a l changes i n target cells or tissues that result from the action of the c h e m i c a l a n d are thought to be a step i n the pathologic process t o w a r d disease" (I). E x a m p l e s of markers of biological response i n c l u d e c h r o m o s o m a l aberrations and m i c r o n u c l e i . F o r e x a m p l e , b e t e l q u i d chewers w h o have an elevated risk of oral cancer also have elevated levels of m i c r o n u c l e i i n exfoliated b u c cal mucosa cells (14). M i c r o n u c l e i , s m a l l pieces o f D N A that arise i n the cytoplasm w h e n c h r o m a t i d or c h r o m o s o m a l fragments or w h o l e c h r o m o somes are not i n c o r p o r a t e d into daughter n u c l e i d u r i n g mitosis (15), f o r m as a result of exposure to clastogenic agents, such as X - r a y s , a n d agents that cause damage to the c e l l s s p i n d l e apparatus (16). A m a r k e r of disease indicates a " b i o l o g i c a l or c h e m i c a l event that e i t h e r represents a s u b c l i n i c a l stage of disease or is a manifestation of the disease i t s e l f " (I). Disease markers are often the d e p e n d e n t , or o u t c o m e , variables i n e p i d e m i o l o g i c a l research. A n example of a disease m a r k e r is s e r u m otfetoprotein, an indicator of l i v e r cancer (I). A m a r k e r of s u s c e p t i b i l i t y "measures o r is associated w i t h , factors that increase an i n d i v i d u a l s risk o f d e v e l o p i n g a disease after exposure to some exogenous agent" (17). M a r k e r s of s u s c e p t i b i l i t y may be genetically d e t e r m i n e d or a c q u i r e d . X e r o d e r m a p i g m e n t o s u m is an example of a genetically



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d e t e r m i n e d susceptibility. I n d i v i d u a l s w i t h this c o n d i t i o n are at increased risk of d e v e l o p i n g s k i n cancer after exposure to u l t r a v i o l e t radiation (18). O t h e r examples o f genetically d e t e r m i n e d susceptibilities i n c l u d e d e b r i s o quin hydroxylation phenotype and N-acetylation phenotype. Extensive m e tabolizers of d e b r i s o q u i n are thought to be at increased risk of l u n g cancer o n exposure to cigarette smoke (19), a n d slow acetylators are at increased risk o f b l a d d e r cancer o n exposure to N - s u b s t i t u t e d a r y l c o m p o u n d s (20), s u c h as those u s e d i n the d y e industry. E x a m p l e s of a c q u i r e d susceptibility factors i n c l u d e age, diet, life style, a n d previous diseases. F o r example, i n dividuals w h o consume large quantities of alcohol a n d also smoke are at i n creased risk of d y i n g f r o m h e a d a n d neck cancers (21), a n d p e o p l e w h o have b e e n infected w i t h the hepatitis B v i r u s have b e e n f o u n d to have a greater chance of d e v e l o p i n g l i v e r cancer i n association w i t h exposure to aflatoxin (22).

Advantages of Using Biological Markers in Epidemiological Research T h e r e are potentially m a n y reasons for u s i n g biological markers i n e p i d e miological research (1,23): 1. I n t e r n a l dose a n d biologically effective dose markers may p r o v i d e m o r e accurate measures of exposure that are specific to the i n d i v i d u a l than do traditional measures of exposure, such as area air m o n i t o r i n g , i n f o r m a t i o n about j o b title, o r questionnaire data. I n a d d i t i o n , biologically effective dose markers m a y p r o v i d e estimates of the relevant exposure dose to the target tissue. B y p r o v i d i n g exposure i n f o r m a t i o n specific to the i n d i v i d u a l (and to target tissues, i n particular), markers m a y h e l p r e d u c e misclassiflcation i n exposure measures, t h e r e b y e n h a n c i n g o u r ability to detect d o s e - r e s p o n s e r e l a tionships b e t w e e n external exposures a n d h e a l t h o u t c o m e measures. 2. B i o l o g i c a l markers m a y i m p r o v e k n o w l e d g e of participant c o m p l i a n c e w i t h treatment regimens i n i n t e r v e n t i o n trials. I n a typical t r i a l , one g r o u p of participants is r a n d o m i z e d to r e ceive one treatment or i n t e r v e n t i o n , a n d one or m o r e other groups are r a n d o m i z e d to receive another treatment or a p l a cebo. F o r example, i n a study of the effects of (i-earotene s u p p l e m e n t a t i o n o n m i c r o n u c l e i i n d u c t i o n i n b u c c a l cells of b e t e l q u i d chewers over a 3 - m o n t h p e r i o d , one g r o u p of p a r t i c i pants w o u l d be g i v e n (3-carotene capsules, a n d the o t h e r group w o u l d be g i v e n a placebo. A n a l y s i s of b l o o d specimens several times d u r i n g the 3 - m o n t h trial w o u l d indicate w h e t h e r the treatment g r o u p was r e a l l y t a k i n g the (3-carotene capsules


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a n d w h e t h e r the s e r u m (3-earotene levels of treated a n d c o n t r o l groups actually differed. It is possible, for e x a m p l e , that m e m b e r s of the c o n t r o l group w e r e also t a k i n g v i t a m i n s u p p l e m e n t s o r c o n s u m i n g diets h i g h i n (3-earotene. 3. M a r k e r s of disease m a y p r o v i d e e v i d e n c e of p r e c l i n i c a l d i s ease or m a y p r o v i d e a m o r e h o m o g e n e o u s classification of d i s ease. M a r k e r s of p r e c l i n i c a l disease may a l l o w d e t e c t i o n at earlier p r e v e n t a b l e stages. M a r k e r s that classify disease i n t o more homogeneous subclassifications may h e l p identify exposure-disease associations. C e r t a i n l e u k e m i a s , for example, can be d i s t i n g u i s h e d o n the basis o f w h e t h e r oncogene a c t i vation is associated w i t h the disease. T a y l o r et a l . (24) r e p o r t e d that patients occupationally exposed to c e r t a i n c h e m icals are m o r e l i k e l y to d e v e l o p ras-positive t h a n ras-negative acute myelogenous l e u k e m i a ( A M L ) . C o m p a r i s o n s w i t h healthy controls s h o w e d that occupational c h e m i c a l exposure d i d not increase the risk of ras-negative A M L . 4. B i o l o g i c a l markers m a y p r o v i d e clues to m e c h a n i s m s of d i s ease causation b y e l u c i d a t i n g i n t e r m e d i a t e steps i n the p r o cess. M a r k e r s o f response m a y b e u s e d to d e t e r m i n e w h e t h e r exogenous exposures have adverse effects o n genetic m a t e r i a l , an i n t e r m e d i a t e step i n the d e v e l o p m e n t of cancer. F o r exa m p l e , occupational exposure to e t h y l e n e oxide leads to an increased i n c i d e n c e o f sister c h r o m a t i d exchanges a m o n g exposed w o r k e r s (25). 5. M a r k e r s of s u s c e p t i b i l i t y may indicate subpopulations at i n creased risk of disease. I f populations at i n c r e a s e d risk can b e i d e n t i f i e d , t h e n these groups can b e targeted for p r e v e n t i v e measures. 6. M a r k e r s of exposure m a y i m p r o v e k n o w l e d g e of the extent o f p o p u l a t i o n exposures to various exogenous agents b y means of surveillance techniques. Tissue specimens (e.g., b l o o d or fat), o b t a i n e d f r o m a p p r o p r i a t e l y s a m p l e d m e m b e r s of the p o p u l a t i o n , can b e a n a l y z e d to d e t e r m i n e the extent of exposure to specific exogenous e n v i r o n m e n t a l agents i n the p o p ulation. 7. M a r k e r s of exposure m a y be u s e d to validate m o r e traditional measures of exposure. F o r e x a m p l e , i n a study of the c o n s u m p t i o n of p o l y c h l o r i n a t e d b i p h e n y l ( P C B ) - c o n t a m i n a t e d fish a n d adverse r e p r o d u c t i v e effects a m o n g w o m e n i n W i s consin (26), s e r u m P C B levels w e r e u s e d to assess the v a l i d i t y of u s i n g fish c o n s u m p t i o n (obtained v i a questionnaire) as an estimate of P C B intake i n a subgroup of the w o m e n . T h e correlation b e t w e e n the questionnaire i n f o r m a t i o n a n d s e r u m


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P C B levels was 0.666. It was c o n c l u d e d that the questionnaire information, w h i c h was easier to o b t a i n a n d less costly than m e a s u r i n g s e r u m P C B levels, p r o v i d e d a reasonably accurate estimate of P C B intake (26). 8. M a r k e r s may p r o v i d e m o r e accurate measures of p o t e n t i a l confounders i n e p i d e m i o l o g i c a l research. Misclassification of confounder status can result i n inadequate c o n t r o l of c o n f o u n d i n g factors i n the analysis of e p i d e m i o l o g i c a l studies, p o tentially l e a d i n g to a bias i n study results (27). F o r an expanded discussion of the p o t e n t i a l benefits of u s i n g biological m a r k ers i n e p i d e m i o l o g i c a l research m e n t i o n e d , see H u l k a , 1990 (I) a n d 1991 (23).

Criteria for Selecting Biological Markers in Epidemiological Research A n epidemiologist must consider m a n y c r i t e r i a before e m p l o y i n g a b i o l o g i c a l m a r k e r i n an e p i d e m i o l o g i c a l investigation. T h e most i m p o r t a n t c o n s i d e r ation is the study's objective. If it is d e t e r m i n e d that a m a r k e r w i l l benefit the study, the epidemiologist must d e t e r m i n e w h i c h m a r k e r or markers w i l l best meet the study's objective. T h e f o l l o w i n g characteristics of markers should be c o n s i d e r e d before a m a r k e r is u s e d i n an e p i d e m i o l o g i c a l i n v e s tigation.

Availability of Markers.

If o n l y one m a r k e r exists to measure a p a r -

ticular exposure or outcome event, t h e n the c h o i c e of a m a r k e r is s i m p l i f i e d . S o m e t i m e s , however, m o r e than one m a r k e r is available. F o r example, there are many potential markers of tobacco smoke exposure, i n c l u d i n g t h i o c y a nate, c a r b o x y h e m o g l o b i n , n i c o t i n e , c o t i n i n e , D N A a n d h e m o g l o b i n a d ducts, a n d u r i n e mutagenicity. E a c h m a r k e r has different p r o p e r t i e s . T h e choice of m a r k e r d e p e n d s o n h o w w e l l the p r o p e r t i e s of the available m a r k ers coincide w i t h the objectives of the study.

Specificity of the Marker. Specificity refers to the ability of the m a r k e r to indicate the specific exposure or o u t c o m e of interest. F o r e x a m ple, the u r i n e mutagenesis assay is a nonspecific m a r k e r of mutagenic agents i n the u r i n e . It cannot indicate w h i c h exposure l e d to mutagens a p p e a r i n g i n the u r i n e . O n the other h a n d , c o t i n i n e (metabolite of nicotine) i n the u r i n e is a m a r k e r essentially specific to tobacco smoke. T h e r e q u i r e d degree of specificity d e p e n d s o n the research question. Invasiveness of the Technique To Obtain Biological Specimens. M a r k e r selection is often i n f l u e n c e d b y the q u a n t i t y of the s p e c i m e n n e e d e d to measure the m a r k e r a n d the invasiveness (either p h y s i c a l l y or p s y c h o l o g -



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ically) of the s a m p l i n g t e c h n i q u e necessary to o b t a i n the b i o l o g i c a l s p e c i m e n i n w h i c h the m a r k e r is m e a s u r e d . T h e m o r e invasive the p r o c e d u r e a n d the greater the amount of s p e c i m e n n e e d e d , the less l i k e l y it is that p e o p l e w i l l participate i n the study. Assays, for example, that detect m i c r o n u c l e u s form a t i o n can be p e r f o r m e d o n h u m a n b o n e m a r r o w cells, l y m p h o c y t e s , r e d b l o o d cells, a n d exfoliated cells. It is u n l i k e l y that m a n y p e o p l e w o u l d p a r ticipate i n a study i n w h i c h sternal p u n c t u r e to obtain b o n e m a r r o w cells was r e q u i r e d . P a r t i c i p a t i o n w o u l d be greater i n a study that r e q u i r e d the d r a w i n g of b l o o d or the scraping of the oral mucosa to obtain exfoliated cells. T h e invasiveness of the procedures affects not o n l y the n u m b e r s of p a r t i c i pants b u t also t h e i r characteristics. Self-selection for a study o n the basis of the t e c h n i q u e u s e d to obtain specimens c o u l d l i m i t the g e n e r a l i z a b i l i t y of results; it may e v e n lead to a bias i n results because of systematic differences a m o n g o u t c o m e measures of participants, o n the basis of exposure status.

Time to Appearance of the Marker.

T h e " t i m e to appearance of

the m a r k e r " refers to the first p o i n t after the event of interest occurs at w h i c h the m a r k e r can be measured. I n other w o r d s , there may be a delay b e t w e e n the t i m e of the event of interest a n d w h e n the m a r k e r indicates that it has o c c u r r e d . F o r example, i n m i c r o n u c l e u s formation, m i c r o n u c l e i that are sloughed (exfoliated) from the surface of the b u c c a l mucosa indicate damage that has o c c u r r e d i n the basal c e l l layer (14). E x f o l i a t e d cells are d e r i v e d from basal cells. O n l y the basal cells d i v i d e a n d therefore f o r m m i c r o n u c l e i . S t i c h et a l . (28) have estimated that it takes 5 - 7 days for r a d i a t i o n - i n d u c e d m i c r o n u c l e a t e d basal cells to migrate to the surface of the b u c c a l mucosa, w h e r e they are exfoliated. T h u s , evaluating b u c c a l mucosa cells s a m p l e d before the 5 - 7 days w o u l d u n d e r e s t i m a t e the effect of the exposure o n m i cronucleus formation. T h e t i m e to appearance of the m a r k e r is often a greater c o n c e r n i n acute versus c h r o n i c exposure situations a n d w h e n one is t r y i n g to l i n k the t i m e of exposure to a p a r t i c u l a r effect.

Persistence of the Marker.

T h e persistence of the m a r k e r also i n -

fluences the t i m e d u r i n g w h i c h one can evaluate the presence of the m a r k e r i n a biological m e d i u m . T h e persistence of the m a r k e r depends o n the m e tabolism, storage, a n d excretion of the m a r k e r f r o m the b o d y a n d also o n the stability of the biological tissue i n w h i c h the m a r k e r is m e a s u r e d . C o t i n i n e , for example, has a half-life i n s e r u m of about 1 6 - 1 9 h (29), whereas n i c o t i n e , the parent c o m p o u n d , has a half-life of o n l y 2 h (30). N i c o t i n e levels w o u l d indicate s h o r t - t e r m exposures, whereas c o t i n i n e levels w o u l d indicate exposures that took place over the previous 1 or 2 days. H e m o g l o b i n adducts, however, c o u l d be u s e d to indicate exposures o v e r a longer p e r i o d of t i m e , because they are thought to persist for the life of the r e d b l o o d c e l l , about 120 days (31).



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Peak or Integrated Dose. W h e t h e r the l e v e l of the m e a s u r e d m a r k e r represents the peak or integrated dose over t i m e depends o n (1) w h e t h e r the event that p r o d u c e d the m a r k e r was an acute (one time) or c h r o n i c (continuous) event, a n d (2) the tissue i n w h i c h the m a r k e r is m e a sured. M a r k e r levels indicative of acute events, such as exposure to an exogenous c h e m i c a l , rise (peak) a n d fall w i t h t i m e as the b o d y metabolizes, excretes, or stores the c h e m i c a l i n another tissue. T h u s , i n acute events, w h e n one samples the tissue i n relation to the t i m e of the event is i m p o r t a n t . If the tissue i n w h i c h the m a r k e r is m e a s u r e d does not store the marker, c h r o n i c events often lead to a steady state of m a r k e r levels, i n w h i c h s i m i l a r levels of the m a r k e r enter a n d leave the tissue. I f the tissue stores the c h e m ical, one may be able to measure the a c c u m u l a t e d dose of the marker. L e a d , for example, accumulates i n b o n e . B o n e l e a d levels account for about 9 5 % of the total b o d y b u r d e n of l e a d . L e a d has a half-life i n b o n e of about a decade (6). Therefore, analysis of b o n e l e a d levels s h o u l d indicate a c c u m u lated lead exposure. Variability (Intraperson, Interperson).

Variability is a major issue

i n b i o m a r k e r research. L e v e l s of markers can vary w i t h i n a p e r s o n (intraperson variability) from one t i m e to the next a n d e v e n a m o n g tissues w i t h i n the same person. L e v e l s of markers can also v a r y dramatically a m o n g i n d i viduals (interperson variability). Interperson variability i n m a r k e r levels can result from differences i n exogenous exposures as w e l l as genetic v a r i a b i l i t y i n D N A repair processes a n d m e t a b o l i s m of exogenous agents. F o r example, 4 - a m i n o b i p h e n y l ( 4 - A B P ) h e m o g l o b i n adduct levels a m o n g active a n d passive smokers vary w i t h acetylation a n d oxidation p h e n o t y p e (32). It is n e c essary to u n d e r s t a n d the sources of variability i n biological m a r k e r research i n o r d e r to be able to make v a l i d comparisons of m a r k e r levels a m o n g i n d i viduals. F o r a m o r e extensive discussion of the sources of variability i n e p i d e m i o l o g i c a l studies i n v o l v i n g biological m a r k e r s , see H u l k a a n d M a r g o l i n (33).

Stability of the Marker in Storage. Stability of the m a r k e r i n storage influences the feasibility of an e p i d e m i o l o g i c a l investigation. Storage procedures can i n c l u d e freezing or fixing tissue specimens i n a preservative such as formaldehyde. If the m a r k e r is not stable i n storage, analyses often have to be done c o n c u r r e n t l y w i t h sample c o l l e c t i o n . S o m e t i m e s this p r o c e d u r e is not feasible. S o m e t i m e s h a v i n g to analyze specimens at the t i m e of sample collection escalates costs. F o r example, someone has to be e m p l o y e d to do the analyses throughout the study. I f analyses are done over t i m e , it is essential to analyze simultaneously specimens from c o n t r o l a n d exposed or diseased persons i n o r d e r to c o n t r o l for any changes i n laboratory practice over t i m e . I f the m a r k e r is stable i n storage, analyses can be p e r f o r m e d after a l l the samples have b e e n c o l l e c t e d , t h e r e b y increasing study design options.


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Biological,


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Suitability of the Assay for an Epidemiological Study.

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T h e key


issues i n evaluating the s u i t a b i l i t y of an assay for an e p i d e m i o l o g i c a l study i n c l u d e the availability a n d cost of the assay as w e l l as its sensitivity, s p e c i ficity, a n d reliability. S o m e assays are c o m m e r c i a l l y available, whereas o t h ers are e i t h e r v e r y c o m p l i c a t e d or still i n d e v e l o p m e n t , such that o n l y one or two laboratories i n the w o r l d p e r f o r m t h e m . T h e success of e p i d e m i o l o g ical studies i n v o l v i n g assays p e r f o r m e d b y few laboratory investigators d e pends o n the epidemiologist s ability to collaborate w i t h the p a r t i c u l a r l a b oratory investigators w h o p e r f o r m the assay. Costs of assays v a r y w i t h h o w t i m e - c o n s u m i n g a n d labor-intensive t h e i r procedures are. Analyses to d e t e r m i n e s e r u m c o t i n i n e levels, for example, are m u c h less expensive than D N A adduct analyses. H i g h assay costs can e i t h e r r u l e out an assay for a p a r t i c u l a r a p p l i c a t i o n or l i m i t the study's sample size. Sensitivity of the assay must be c o n s i d e r e d . T h e assay must b e able to detect p o p u l a t i o n levels of the marker. I n a d d i t i o n to b e i n g sensitive, it s h o u l d be as specific as possible for the m a r k e r i n q u e s t i o n . F o r example, one c r i t i c i s m of radioimmunoassay techniques is that the antibodies u s e d to detect a p a r t i c u l a r m a r k e r (antigen) can cross-react w i t h similar c o m p o u n d s . A nonspecific assay can lead to misclassification of m a r k e r levels. Similarly, an u n r e l i a b l e assay (one that does not p r o d u c e repeatable results o n the same specimen) can also l e a d to misclassification of m a r k e r levels. F o r a m o r e i n - d e p t h discussion of the p r o p e r t i e s of markers a n d the c r i t e r i a u s e d i n m a r k e r selection, see W i l c o s k y a n d G r i f f i t h (34) a n d W i l cosky (2).

Limitations of Biological Markers in Epidemiological Research T h e r e are several limitations of the use of biological markers i n e p i d e m i o logical research. F o r some situations, suitable markers do not exist or the properties of available markers are inadequate to m e e t the study s objectives. F o r example, analysis o f the m a r k e r may b e too expensive, or available markers may not be specific e n o u g h for a p a r t i c u l a r exposure. I n o t h e r cases, the properties of a potentially useful m a r k e r m a y not be k n o w n . A n s w e r s m a y yet be n e e d e d to such questions as " W h a t is the relationship b e t w e e n levels of the m a r k e r i n the surrogate tissue a n d levels of the m a r k e r i n the target tissue?"; " W h a t are the p h a r m a c o k i n e t i c p r o p e r t i e s of the m a r k e r ? " ; or " H o w stable is the m a r k e r w h e n stored frozen?" I n a d d i t i o n , markers may not have b e e n v a l i d a t e d for the applications i n t e n d e d b y the researcher. V a l i d a t i o n of b i o l o g i c a l m a r k e r s is a c o m p l i c a t e d issue. M e t h o d s of validating a m a r k e r differ i n some respects, d e p e n d i n g o n w h e t h e r the m a r k e r is u s e d to measure an exposure, an i n t e r m e d i a t e e n d p o i n t , or a disease outcome. I n a l l three cases, steps that s h o u l d b e u n d e r taken i n c l u d e p r e l i m i n a r y laboratory studies to establish the l i m i t s of d e t e c -


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t i o n a n d r e l i a b i l i t y of the assay. P r e l i m i n a r y f i e l d studies i n humans s h o u l d be u n d e r t a k e n (1) to establish that the assay can detect p o p u l a t i o n levels of the m a r k e r ; (2) to d e t e r m i n e the range of m a r k e r levels i n h u m a n p o p u l a tions; (3) to identify the factors associated w i t h variation i n m a r k e r levels, such as race, gender, a n d age; a n d (4) to d e t e r m i n e the r e l i a b i l i t y of the assay u n d e r study conditions (35). A l l of this information is k n o w n for o n l y few markers. I n the case of exposure markers a n d markers of i n t e r m e d i a t e e n d points, p r e l i m i n a r y laboratory w o r k s h o u l d also i n c l u d e a n i m a l experiments to d e scribe d o s e - r e s p o n s e relationships b e t w e e n k n o w n doses of an e x t e r n a l exposure a n d i n t e r n a l m a r k e r levels. Information s h o u l d be available for m a r k ers m e a s u r e d after acute a n d c h r o n i c exposure situations (36). H u m a n f i e l d studies c o u l d t h e n be c o n d u c t e d to assess the d o s e - r e s p o n s e relationships b e t w e e n k n o w n levels of an external exposure a n d i n t e r n a l m a r k e r levels i n h i g h - versus low-exposure situations. O n e w o u l d want to see that, o n average, those w i t h the h i g h exposures are m o r e l i k e l y to have h i g h e r levels of the m a r k e r t h a n those w i t h l o w exposures. F i n d i n g a perfect c o r r e l a t i o n b e t w e e n levels of an external exposure a n d m a r k e r levels does not necessari l y invalidate the marker. I n fact, a strength of biological markers is that they integrate exposures f r o m m a n y sources a n d p r o v i d e estimates of exposure to the i n d i v i d u a l that consider the use of protective e q u i p m e n t a n d i n t e r i n d i v i d u a l differences i n , for example, absorption, m e t a b o l i s m , a n d excretion. T h u s , e v e n i f the m a r k e r does not perfectly correlate w i t h external measures of the exposure, it may accurately indicate i n t e r n a l exposure to the i n d i v i d ual. E x t e r n a l exposure i n f o r m a t i o n , such as can be o b t a i n e d b y area air m o n i t o r i n g a n d questionnaires, needs to be assessed to characterize the c o n t r i b u t i o n of other sources of exposure to m a r k e r levels. D a t a c o n c e r n i n g the pharmacokinetic properties of the m a r k e r w o u l d be n e e d e d to i n t e r p r e t m a r k e r levels. M o r e research has concentrated on establishing associations b e t w e e n external levels of exposures a n d i n t e r n a l m a r k e r levels than o n d e t e r m i n i n g the p r e d i c t i v e value of the m a r k e r levels. U l t i m a t e l y , it w o u l d be h e l p f u l to show the l i n k b e t w e e n elevated m a r k e r levels a n d increased risk of some disease o u t c o m e as an indicator of the v a l i d i t y of the marker. M a r k e r s of disease can be v a l i d a t e d against a m e d i c a l l y d e t e r m i n e d " g o l d s t a n d a r d " for diagnosing the disease. A s part of the v a l i d a t i o n process, one w o u l d calculate the sensitivity, specificity, a n d positive p r e d i c t i v e value of the m a r k e r (37). Sensitivity, as defined b y epidemiologists is the p r o b a b i l i t y that the m a r k e r w i l l correctly identify an i n d i v i d u a l as h a v i n g the d i s ease, g i v e n that the i n d i v i d u a l actually has the disease (as d e t e r m i n e d b y the g o l d standard method). T h e specificity is the p r o b a b i l i t y that the m a r k e r w i l l correctly identify the i n d i v i d u a l as not h a v i n g the disease, g i v e n that the i n d i v i d u a l actually does not have the disease. T h e positive p r e d i c t i v e value is the p r o b a b i l i t y that the i n d i v i d u a l has the disease, g i v e n that the


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m a r k e r indicates the presence of the disease. I n each case, one w o u l d want these values to b e as h i g h as possible. F o r example, a recent study (38) evaluated elevated levels of the angiogenic p e p t i d e basic fibroblast g r o w t h factor i n u r i n e as a m a r k e r for b l a d d e r cancer a m o n g b l a d d e r cancer patients diagnosed w i t h cytoscopic analysis a n d radiological studies a n d f o u n d the sensitivity a n d specificity to be modest at 8 1 % a n d 6 4 % , respectively. I f one is t r y i n g to validate a m a r k e r of p r e c l i n i c a l disease, some follow-up of study participants w i l l be necessary to d e t e r m i n e i f the i n d i v i d u a l s w h o test p o s i tive for the m a r k e r actually d e v e l o p the disease.


Quality-Control Issues To p r o d u c e m e a n i n g f u l study results, c e r t a i n q u a l i t y - c o n t r o l procedures s h o u l d be followed. L a b o r a t o r y p e r s o n n e l s h o u l d be b l i n d e d to the exposure and disease characteristics of the participants f r o m w h o m the biological specimens w e r e obtained. Specimens f r o m e x p o s e d - n o n e x p o s e d or d i s e a s e - n o n diseased i n d i v i d u a l s s h o u l d simultaneously be analyzed to a v o i d confusion as to w h e t h e r differences i n the characteristic b e i n g s t u d i e d or differences i n laboratory techniques over t i m e are responsible for any o b s e r v e d differences i n laboratory results b e t w e e n groups. I n a d d i t i o n to a n a l y z i n g s p e c i mens from each of the study participants, it is r e c o m m e n d e d that one anal y z e specimens k n o w n to be positive a n d those k n o w n to be negative for the m a r k e r of interest. A l s o , a n a l y z i n g m o r e than one s p e c i m e n from the same i n d i v i d u a l for a subset of the study participants aids i n assessing the r e l i ability of the assay.

Collaborative Research Involving Biological Markers E p i d e m i o l o g i c a l research, i n general, is b e c o m i n g m o r e a n d m o r e collaborative. P e o p l e i n vastly different fields are w o r k i n g together. T h i s is especially true i n biological m a r k e r research. These collaborations b r i n g w i t h t h e m benefits a n d challenges. T h e major benefit is that questions c a n be addressed b y collaborative efforts that are less w e l l addressed b y the m e m bers of the i n d i v i d u a l fields w o r k i n g separately. T h e interactions b e t w e e n the collaborators f r o m different fields can l e a d to creative solutions to p r o b lems. T h e challenges i n h e r e n t i n collaborative w o r k are many. P r o b l e m s arise because collaborators f r o m different fields t h i n k differently, use the same words to refer to different things, a n d have different agendas (23, 33). T h u s , the v e r y reasons w h y investigators form collaborations present obstacles to the collaborative effort. F o r collaborative efforts to succeed, m e m b e r s of each d i s c i p l i n e must l e a r n the language, strengths, a n d weaknesses of the other discipline. E p i d e m i o l o g i s t s a n d laboratory scientists differ i n t h e i r outlook o n a n u m b e r of issues. W h e r e a s laboratory scientists are often c o n c e r n e d about


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the effects of agents o n i n d i v i d u a l s or small groups of subjects, e p i d e m i o l o gists are c o n c e r n e d w i t h m a k i n g inferences about disease causation i n p o p ulations. E p i d e m i o l o g i s t s can say little about the definite cause of a disease i n a specific i n d i v i d u a l . T h e y can o n l y estimate the p r o b a b i l i t y that a p a r t i c ular agent l e d to the d e v e l o p m e n t of the disease i n that i n d i v i d u a l , o n the basis of p o p u l a t i o n statistics. T h e t i m e frame of laboratory research tends to be short, c o m p a r e d w i t h that of e p i d e m i o l o g i c a l research. L a b o r a t o r y e x p e r i m e n t s can be c o m p l e t e d i n days to months, whereas e p i d e m i o l o g i c a l research often takes years to complete. I n the F r a m i n g h a m H e a r t S t u d y (39), for example, participants have b e e n followed for over 20 years. E v e n i n studies i n w h i c h participants are not followed over t i m e , it can take months to years to identify e l i g i b l e study participants, e n r o l l t h e m i n the study, a n d obtain specimens f r o m them. T h e sample sizes i n laboratory studies t e n d to be s m a l l , c o m p a r e d w i t h those of e p i d e m i o l o g i c a l studies. L a b o r a t o r y research usually involves a h a n d f u l of animals exposed to different doses of a c h e m i c a l . E p i d e m i o l o g i c a l research often must i n v o l v e h u n d r e d s or thousands of participants to p r o d u c e statistically significant results. L a b o r a t o r y scientists may not be e q u i p p e d either to handle the large n u m b e r of specimens r e q u i r e d of m a n y e p i d e m i o l o g i c a l investigations or w a n t to c o m m i t resources for such a l o n g time. T h e n u m b e r of extraneous factors i n laboratory experiments also tends to be s m a l l , c o m p a r e d w i t h those i n e p i d e m i o l o g i c a l research. L a b o r a t o r y researchers often w o r k w i t h genetically similar animals a n d c o n t r o l as m a n y other factors as possible. E p i d e m i o l o g i s t s observe what happens i n the r e a l world. Participants i n e p i d e m i o l o g i c a l studies are often not genetically s i m i l a r a n d have a variety of different exposures a n d life style habits. E p i d e m i o l o gists t r y to c o n t r o l for as m a n y factors as possible i n the statistical analysis of the data. E p i d e m i o l o g i s t s a n d laboratory scientists often have different views of the laboratory assays themselves. L a b o r a t o r y scientists t e n d to want to p e r fect the assay, for example, b y m a k i n g it m o r e sensitive. O n c e the assay is " p e r f e c t e d , " the laboratory scientist may no longer be i n t e r e s t e d i n i t . T h e epidemiologists, o n the other h a n d , want to use the same assay over a n d over again o n a l l the participants i n the study. T h e differences b e t w e e n the two disciplines can be e i t h e r strengths or obstacles. I f laboratory scientists a n d epidemiologists w o r k together, t h e y can l e a r n to m a x i m i z e the strengths a n d m i n i m i z e the obstacles. F o r e x a m p l e , epidemiologists a n d laboratory scientists m a y n e e d to d e v e l o p m o r e c l e v e r a n d specific hypotheses so that large n u m b e r s of subjects are not n e c essary to obtain m e a n i n g f u l study results. Because the operation of l a b o r a tory assays i n p o p u l a t i o n settings is often different from that i n the c o n t r o l l e d


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e x p e r i m e n t a l e n v i r o n m e n t (A. W i l c o x : p e r s o n a l c o m m u n i c a t i o n ) , t e c h n i c a l aspects of the assay often n e e d to be addressed. C o l l a b o r a t i o n s b e t w e e n laboratory scientists a n d epidemiologists have the p o t e n t i a l to p r o v i d e i n teresting avenues of research for investigators f r o m b o t h d i s c i p l i n e s .

Examples of the Use of Biological Markers in Environmental and Occupational Epidemiology Exposure Assessment.

A m o n g the most difficult aspects o f d e t e r -

m i n i n g the h e a l t h consequences of e n v i r o n m e n t a l exposures is i d e n t i f y i n g w h o is exposed a n d q u a n t i f y i n g the levels of exposure o f the affected i n d i -


viduals. B i o l o g i c a l markers of exposure may h e l p alleviate some of these p r o b l e m s . A n example of a situation i n w h i c h b i o l o g i c a l markers m a y b e useful is t r y i n g to assess the possible adverse h e a l t h effects of l i v i n g near a hazardous waste site. O n c e it is d e t e r m i n e d that the agents c o n t a i n e d i n the site have the p o t e n t i a l to cause adverse h u m a n h e a l t h effects (for e x a m p l e , t h r o u g h literature reviews), the next step is to d e t e r m i n e w h e t h e r the r e s i dents near the site are actually exposed to the agents i n the hazardous waste. T h e i d e a l b i o l o g i c a l m a r k e r o f exposure, i n this case, w o u l d b e specific for a particular agent present at the waste site. I f one u s e d a m a r k e r that was not v e r y specific, confusion m i g h t arise as to w h e t h e r persons positive for the m a r k e r w e r e exposed because of agents e m a n a t i n g f r o m the waste site or because of other exposures. W h e n less specific markers are u s e d , sources of other exposures can sometimes be assessed b y questionnaires. T h e A g e n c y for Toxic Substances and Disease R e g i s t r y ( A T S D R ) (40) assessed levels of arsenic i n the u r i n e o f residents l i v i n g near an a b a n d o n e d arsenic p r o d u c t i o n site. U r i n a r y arsenic levels (|xg/g creatinine) w e r e not statistically significantly h i g h e r a m o n g a l l p o t e n t i a l l y exposed residents of the n e a r b y apartm e n t complex, c o m p a r e d w i t h controls w h o w e r e less l i k e l y to have b e e n exposed.

H o w e v e r , three c h i l d r e n i n the n e a r b y a p a r t m e n t c o m p l e x h a d

p a r t i c u l a r l y h i g h u r i n a r y arsenic levels. T h e three c h i l d r e n a n d two adults w i t h arsenic levels greater t h a n 50 |jig/g c r e a t i n i n e w e r e retested after a w a r n i n g to stay away from the c o n t a m i n a t e d area. T h e l o w e r e d arsenic levels a m o n g the retested c h i l d r e n suggest that the site was the source of the a r senic exposure a n d that the w a r n i n g m a y have b e e n effective i n r e d u c i n g exposure.

Intermediate E n d Points.

M o s t of the e n v i r o n m e n t a l research i n -

v o l v i n g biological markers has p r o c e e d e d b y i d e n t i f y i n g an exposure a n d t h e n d e t e r m i n i n g its effect o n the levels o f a p a r t i c u l a r marker. T h i s is exe m p l i f i e d i n occupational settings i n w h i c h the b l o o d cells of w o r k e r s exp o s e d to chemicals, such as e t h y l e n e oxide a n d p e t r o l e u m vapors, have b e e n evaluated for the presence of various m a r k e r s of b i o l o g i c a l response, i n c l u d i n g c h r o m o s o m e aberrations, sister c h r o m a t i d exchanges, a n d m i c r o n u c l e i (25,41). I n m a n y cases, elevations i n these i n t e r m e d i a t e markers have b e e n


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associated w i t h the exposure of interest. A l t h o u g h e v i d e n c e of increased c h r o m o s o m a l breakage is generally not c o n s i d e r e d favorable, no one knows what h a v i n g elevated levels of these markers really means (42). H e r e is an example of a situation i n w h i c h research is n e e d e d to d e t e r m i n e the p r e d i c tive value of markers for adverse health outcomes. K n o w l e d g e of t h e i r p r e d i c t i v e value c o u l d b e beneficial for u n d e r s t a n d i n g disease causation a n d p r e v e n t i n g disease at earlier stages.


Summary E p i d e m i o l o g y is the study of the d i s t r i b u t i o n a n d determinants of diseases i n populations. Traditionally, epidemiologists have taken a " b l a c k b o x " app r o a c h to the study of e n v i r o n m e n t a l l y i n d u c e d disease, assessing an e n v i r o n m e n t a l or external exposure a n d m e a s u r i n g some h e a l t h o u t c o m e . It is h o p e d that, t h r o u g h the use of biological m a r k e r s , often assessed b y m o l e c ular a n d toxicological techniques, w e can b e g i n to u n d e r s t a n d the i n t e r m e diate steps b e t w e e n exposure a n d disease occurrence a n d can i m p r o v e the p r e c i s i o n of exposure a n d o u t c o m e measurements. A biological m a r k e r is a cellular, b i o c h e m i c a l , o r m o l e c u l a r alteration that is measurable i n b i o l o g i c a l m e d i a s u c h as h u m a n tissues cells or f l u i d s . B i o l o g i c a l markers can be classified into categories that represent a sequence of events from exposure to disease, i n c l u d i n g markers of i n t e r n a l dose, biologically effective dose, early response, altered structure a n d f u n c t i o n , a n d disease. O u t s i d e this sequence of events are susceptibility factors that can i n f l u e n c e events at any point along the pathway. Because e p i d e m i o l o g i c a l investigations often i n c l u d e large n u m b e r s of participants, the biological markers that are most desirable for use i n h u m a n studies are those that can be m e a s u r e d w i t h a m i n i m u m a m o u n t of s k i l l or e q u i p m e n t i n a small a m o u n t of b i o l o g i c a l m e d i u m a n d can be o b t a i n e d w i t h m i n i m a l l y invasive techniques. T h e assays u s e d to d e tect the markers s h o u l d be inexpensive, sensitive, specific, r e l i a b l e , a n d able to quantify accurately levels of the markers present i n h u m a n specimens. Unfortunately, the properties of m a n y potentially useful markers are still u n k n o w n , a n d the properties o f available markers m a y not b e appropriate for p a r t i c u l a r applications. F u t u r e research i n this area w i l l r e q u i r e the c o l laborative efforts of epidemiologists a n d laboratory scientists, a m o n g other professionals. C o l l a b o r a t i v e efforts can lead to innovative research. F o r the collaborations to succeed, researchers from each of the various d i s c i p l i n e s must l e a r n the language, strengths, a n d weaknesses of the other d i s c i p l i n e s .

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RECEIVED for r e v i e w S e p t e m b e r 3, 1992. ACCEPTED r e v i s e d m a n u s c r i p t April 8, 1993.


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