Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 115
4
Use of Biological Markers
in Assessing Human Exposure
to Airbome Contaminants
INTRODUCTION
As used in this report, biological markers are indicators of changes or
events in human biological systems. For example, a metabolite of some exog-
enous substance found in a person's blood or urine might be considered a
marker of the person's exposure to that substance in the environment. If a
tissue response to the person's exposure to the substance parallels a disease
process, but does not itself constitute a disease process, the tissue response
might be considered a marker of effect. The use of biological indicators of
exposure to toxic substances is not a new concept it has been used in occupa-
tional health monitoring for many decades. However, as the emphasis on
conducting accurate exposure assessments increases, biological markers of
exposure will be used more frequently A dramatic indicator, particularly in
occupational epidemiology studies, is death. Less drastic and more manipula-
ble biological measures of health outcome do exist and continue to be devel-
oped.
Unfortunately, terminology is confusing, the array of technological subtle-
ties is bewildering, and there is a potential for ethical dilemmas. By virtue of
their collaborative and interdisciplinary nature, investigations involvingbiologi-
ca] markers are highly demanding in terms of personnel, cost, and effort to
develop mutual understanding between researchers in different fields. Incor-
poration of biological markers into exposure assessment engenders new and
significant methodological problems in the design of studies that use human
subjects and in analysis of data. It also introduces important ethical questions
into health effects research, particularly when information on biological mark-
ers of uncertain relation to adverse health effects is given to a possibly ex-
posed person. Successful use of biological markers will require an under-
standing of the fate and effects of a contaminant within a person to permit the
115
OCR for page 116
116 ASSESSING HUMAN EXPOSURE
relation of marker data to the exposure and the establishment of relationships
among biological markers of effect and exposure.
This chapter discusses how biological markers might be used as surrogates
for or in combination with other measures of exposure, including comments
on their limitations.
FROM EXPOSURE TO HEALTH EFFECTS:
KINDS OF MARKERS
As defined in Chapter 1' exposure to an airborne contaminant is the prod-
uct of the concentration of the contaminant and the period during which
exposure is at that concentration. "Dose" is defined as the actual amount of a
given contaminant that is absorbed or deposited in the body of an exposed
organism in ~ given period (usually with reference to a single medium or
route of exposure). "Potential dose" assumes total absorption of a contaminant.
The term dose can be subdivided into internal dose and biologically effective
dose~oth are discussed in this chapter with regard to biological markers.
~Effect" is defined as an adverse health outcome or nuisance resulting from
exposure. An exposure might or might not lead to a dose, and a dose to a
health or nuisance effect. Each of these compartments may also be subdivid-
ed into stages representing a progression from exposure to adverse health
effect, as shown in Figure 1.2 in Chapter 1. These stages are not discrete.
The progression from exposure to effect is a result of the physiological chang-
es that can occur within the organism. The progression can be affected at any
point by internal biological factors or by external interventions.
The classes of biological markers are depicted in Figure 4.1, an elaboration
of Figure 1.2 in Chapter 1. Biological markers can be used for each stage in
the progression and can be used either to determine the exposure to the
causative agent or to predict adverse health effects (Committee on Biological
Markers of the National Research Council, 1987~. Markers can be divided
into three broad classes: markers of exposure, markers of health effects, and
markers of susceptibility (NRC, 1989~. The committee focuses in this chapter
on the application of biological markers to assessment of exposure to contami-
nants rather than their use to predict health effects. However, the use of
markers to predict health effects should not be disregarded.
As discussed in Chapter 1, biological markers of exposure integrate all
routes of exposure to a particular contaminant. For example, lead concentra-
tions as measured in a human tissue, such as blood, can reflect exposure via
any or all of such routes, including inhalation of lead in ambient air, ingestion
of vegetables contaminated by deposition of airborne lead and ingestion of
OCR for page 117
BIOLOGICAL MARKERS 117
Exposure
Markers of
exposure
Markers of
health
effects
Internal dose
Biologically
effective dose
Early biological
effect
Altered function
or structure
Clinical disease
~1
13
Markers of
susceptibility
FIGURE 4.1 Kinds of biological markers. Source: Adapted from Committee on
Biological Markers, 1987.
lead in drinking water or dust, especially by children with pica. This integrat-
ing process of biological markers of exposure can be important for risk assess-
ment and risk management. In the case of lead, for example, the amount
inhaled might be assessed with procedures described in Chapter 3. If only a
very small portion of the observed dose of lead could be accounted for via
inhalation, other exposure routes and sources might then be sought.
Biological markers provide information on dose that can be related to
exposure by using pharmacokinetic or pharmacodynamic models (e.g., blood
lead or carboxyhemoglobin). Internal dose is the amount of contaminant that
is absorbed into the body in a given period. Biological markers of internal
OCR for page 118
118 ASSESSING HURON EXPOSURE
dose directly measure the exposure substance or its metabolites in cells, tis-
sues, or body fluids. The biologically effective dose is the amount of contami-
nant or its metabolites that has interacted with a target site over a given peri-
od so as to alter a physiological function. Such an interaction can lead to
disease or be subjected to repair.
Biological markers of health effects (referred to in this chapter as biological
markers of effect) represent a further step away from exposure and a step
closer to the clinical manifestation of disease. A marker of early biological
effect provides information on an event resulting from a toxic interaction,
either at a target or at an analogous site. An early effect is considered an
irreversible step in pathogenesis or a qualitative or quantitative correlate of
a disease process. Biological markers of effect also include preclinical alter-
ations in organs, tissue structure, or function directly associated with disease.
To be useful in disease prevention, these markers of effect should be measur-
able at preclinical stages.
Biological markers of susceptibility span the entire range of markers show n
in Figure 4.1. They indicate an organism's inherent or acquired limitations
that affect its response to an exposure to a particular contaminant. These
markers can be useful in understanding the relationships that exist between
markers of exposure and effect.
Relating a biological marker directly to an exposure becomes more uncer-
ta~n as one obtains information on the stages within the progression shown in
Figure 4.1. Accordingly, the Committee on Biological Markers of the Nation-
al Research Council (1987) has stated that "markers of health effects are often
less readily related to environmental exposures than are the markers of expo-
sure.- Therefore, greater emphasis is given to markers of exposure. Biological
markers of effect can be used for exposure analysis if and only if information
concerning contaminant identity and route of exposure is incorporated into the
exposure assessment.
An attempt to use biological markers as surrogates for measurements of
exposure might raise ethical questions. When humans are directly involved
in biological-marker studies, caution must be exercised in reporting results to
study subjects, to avoid undue psychological stress when the significance of the
marker for future adverse health effects is uncertain (Ashby, 1988~.
Pharmacokinetics is the quantitative description of the rates of absorption,
distribution, metabolism, and elimination of a contaminant taken into a bio-
logical system (Leung and Pastenbach, 1988~. Pharmacodynamics is the de-
scription of the processes that relate biologically effective dose to health ef-
fects. Pharmacokinetic modeling techniques are used to aid in the extrapola-
tion of test results from animals to humans; pharmacodynamic models are just
being developed (Menzel, 1987~. The pha~acokinetic models attempt to
OCR for page 119
BIOLOGICAL ~4RKER5 119
compensate In part for the physiological and biochemical differences between
humans and test animals, which can cause markedly different responses to the
same substance. The models use mechanistic, biochemical, and physiological
information to describe the disposition of a contaminant once taken in.
Mechanistic studies provide information on the target tissues, metabolic path-
ways, and nature of a contaminant's chemically stable metabolites or reactive
intermediates. Pertinent biochemical data include partition coefficients and
permeation coefficients. Pertinent physiological factors include tissue volumes,
blood flow rates, and ventilation rates. Much of this information can be
obtained from published sources or through the use of in vitro techniques.
A physiologically based pharmacokinetic model is constructed as a set of
biologically defined compartments-tissues and target organs are grouped into
well-perfused, moderately perfused, or poorly perfused compartments for
describing the process of metabolic transformation in the lungs, kidneys, or
liver. Each compartment has a defined volume, blood flow, partition coeffi-
cient, and metabolic constants (Travis et al., 1990~. The growing availability
of sophisticated simulation packages that can solve large numbers of mass-
balance differential equations makes the construction and testing of models
ncreasmgly easier.
An example of use of pharmacokinetic techniques to relate various biologi-
cal markers to each other and to an exposure is the study of halothane con-
centrations in the breathing zone and blood of operating-room personnel
(Fiserova-Bergerova, 1987~. Anesthesiologists had the highest breathing-zone
concentrations but lower blood concentrations than nurses, who had substan-
tially lower breathing-zone concentrations. Pharmacokinetics accounted for
the difference: the anesthesiologists had a primarily sedentary job, whereas
the nurses were actively moving about the room and therefore had higher
ventilation rates and cardiac output.
Pharmacokinetics and pharmacodynamics can also be used to indicate
which tissues are to be sampled and when samples should be taken, as dis-
cussed in Chapter 2. For example, the pharmacokinetics of cadmium indicate
that, if one is interested in recent cadmium exposure, one should analyze
blood; if accumulation is of interest, one should analyze urine (ACGIH, 1986~.
Another study involved the effect of ethylene glycol ethers on sperm count.
Pharmacodynamic studies did not show.a relationship between sperm count
and metho~yacetic acid, the primary metabolite of ethylene glycol ethers
(Smith, 1988~. However, because the sperm production cycle takes 80 days,
there is a lag of approximately 80 days between toxic exposure and reduced
sperm count. Therefore, exposure and effect measurements had to be offset
by approximately 80 days.
Art understanding of the pharmacokinetics of a contaminant is important
OCR for page 120
120 ASSESSING HUMAN EXPOSURE
In relating biological markers to the original exposure. As pharmacodynamic
modeling advances, relationships will then be established between biological
markers of exposure and biological markers of effect (Menzel, 1987~.
APPLICATIONS OF HUMAN BIOLOGICAL MARKERS
Markers of Exposure
Sensitive physicochemical methods can detect and measure very low con-
cer~trations of xenobiotic substances in the body (Sheldon et al., 1986~. They
are being used in industrial hygiene with biologic exposure indexes as refer-
ence values for workplace exposure monitoring (ACGIH, 1986, 1988b; Fiser-
ova-Bergerova, 1987~. Concentrations of a contaminant are usually measured
in exhaled air, blood, or urine, but breast milk and semen have also been
used. Each biological medium has a unique relevance to exposure and health
outcome, and interpretation of the resulting data requires an understanding
of the pharmacokinetics of the substance in question. For example, concen-
trations in exhaled air generally reflect only inhalation, whereas concentrations
in blood, other tissues, and other fluids might reflect recent exposures from
several sources and stored body burdens.
Measures of internal dose can be characterized according to chemical
specificity and selectivity. Highly selective markers of exposure typically repre-
sent measures of unchanged contaminants in biological media and thus pro-
vide the most clear-cut evidence of a specific environmental exposure. Studies
of volatile airborne contaminants usually involve the measurement of a specific
substance in exhaled air. The unchanged compound or its metabolite in urine
may also be analyzed where variations in urinary volume can be accounted for
by normalizing the concentration of the analyte to the creatinine levels. If it
is necessary to analyze for a metabolite, rather than its parent substance, a
procedure can lose some specificity in relating the biological marker to expo-
sure. For example, exposure to styrene and exposure to ethyl benzene each
give rise to mandelic acid in urine, so a finding of mandelic acid in urine
would have to be supplemented with another assay (e.g., of breath) to deter-
mine which compound the subject was exposed to.
Measures of biologically effective dose include DNA and hemoglobin ad-
ducts in peripheral blood and other cells and tissues c.g., lung macrophages,
sputum, bronchial washes or bronchoalveolar ravage fluid, buccal mucosa,
bone marrow, placental tissue, and lung tissue. Such measures can be used
as markers of exposure only when the analyzed cells or tissues are the target
of the exposure contaminant or its metabolites. Many carcinogens and repro
OCR for page 121
BIOLOGICAL MARKERS 121
ductive toxicants are metabolically activated to clectrophilic metabolites that
covalently bind to DNA. AdJucts on DNA, if they occur at critical sites and
are not repaired, can cause gene mutation, which has been shown to be an
initiating step in the multistage carcinogenic process. Several methods to
detect DNA-chemical abducts In lymphocytes and target tissues are available,
including radio- or enzyme-linked immunoassays that use polyclonal or mono-
clonal antibodies, 32P-postlabeling, and synchronous fluorescence spectropho-
tometry (Watson, 1987; Santella, 1988~.
The first use of antibodies to detect polycyclic aromatic hydrocarbon
(PAH)-DNA adducts involved lung tissue and peripheral white blood cells
from lung-cancer patients and controls (Perera et al., 1982~. They have since
been used to analyze white blood cells and other tissues from persons exposed
to PAHs in cigarette smoke and in occupational settings (e.g., foundries and
coke ovens) (Harris et al., 1985; Shamsuddin et al., 1985; Haugen et al., 1986;
Perera et al., 1988~. Antibodies are available to assess formation of DNA
abducts in humans with several carcinogenic substances: aflatoxin Be, benzoa-
pyrene (BaP) and other PAHs, cisplatinum, and metho~ypsoralen. Immuno-
assays can detect frequencies as low as one adduct per 108 nucleotides. As-
says that use monoclonal antibodies are highly specific for a given substance,
but those with polyclonal antibodies, such as PAM-DNA antibodies, can react
with multiple structurally related compounds and thus lose specificity. The
value of immunoassays depends on the development of appropriate antibodies,
and the highly specific monoclonal assays can be time-consuming and techni-
cally difficult.
In contrast with the monoclonal assays, 32P-postlabeling can be used to
recognize various adducts without characterizing their chemical compositions.
It can be more sensitive than monoclonal assays; it can detect one abduct per
10~° nucleotides. The method produces an image that constitutes an idiosyn-
cratic ~fingerprint" of the exposure. However, researchers developing this assay
have faced difficulties in identifying the abducts formed. If the adducts can
be isolated and identified, further analyses can be carried out with immunoas-
say techniques, once the appropriate antibody has been developed. The post-
labeling method generally is limited to the measurement of bulky adJucts that
might limit the usefulness of the technique to smaller airborne contaminants,
and the results of the method are only semiquantifiable. Effective use of the
postlabeling method requires the synthesis of an internal standard since the
efficiency of labeling can vary according to the substance.
The technique of 32P-postlabeling has been applied to the identification of
various alkylating and methylating agents (Ready and Randerath, 1987~. It is
possible to characterize methylating agents using HPLC (high performance
liquid chromatography); however, this is not a routine technique. Application
OCR for page 122
122 ASSESSING HUMAN EXPOSURE
to populations exposed to airborne pollutants has been limited to assessment
of BaP and other PAH exposure of foundry workers and roofers (Hemminki
et al., 1988; Phillips et al., 1988~. Some investigators have related placenta
and lung adJuct measurements to cigarette smoking with postlabeling tech-
niques (Emerson et al., 1986~; others have not seen smoker-nonsmoker differ-
ences in bone marrow, white blood cells, or buccal mucosa (Dunn and Stich,
1986; Phillips et al., 1986; Jahnke et al., 1990~.
A third approach uses synchronous fluorescence spectrophotometry, which
has recently been applied to coke-oven workers with a reported sensitivity of
one BaP adduct per lo? nucleotides (Vahakangas et al., 1985~. The method
has been used to confirm the presence of BaP-DNA abducts In placental
tissue from smokers (Weston et al., 1988~. HPLC and fluorescence spectros-
copy have been used to detect excised carcinogen-DNA adJucts in urine
(Autrup et al., 1983~. The technique is limited in that it is useful only for
detecting compounds that fluoresce, such as PAHs.
Assays that measure protein abducts, including adducts of direct binding
agents and metabolites with hemoglobin, can in some cases be a good surro-
gate for DNA-adduct measurements. That use is supported by correlations
in animal studies between protein and DNA binding by BaP, vinyl chloride,
ethylene oxide, methyl methanesulfonate, and transit dimethylaminostilbene
(LARC, 1984; Neumann, 1984; Bartsch, 1988~. Methods available for measur-
ing those adJucts include immunoassays, amino acid analysis by ion-exchange
LC, and the combination of gas chromatography and mass spectrometry
(GC-MS) with both conventional ionization and negative chemical ionization
techniques. GC-MS has been successfully applied to the quantitation of
aminobipheny} (lABP) hemoglobin adJucts in smokers (Bryant et al., 1987;
Perera et al., 1987~. Sensitive GC-MS methods can measure protein adJucts
in persons exposed to ethylene oxide from cigarette smoke and workplace
sources (Osterman-Golkar et al., 1984; Farmer et al., 1986; Tornqvist et al.,
1986~. Because of the 3-month life span of hemoglobin, those assays reflect
relatively recent exposures; DNA adducts in lymphocyte subpopulations reflect
exposure integrated over a longer period. Protein adducts are more abundant
than their DNA counterparts and therefore provide a more sensitive measure
of exposure.
Markers of Effect
Markers of biological effect can be useful for exposure assessment, provid-
ed that they can be related to the exposure responsible for an effect. A num-
ber of markers might signal a preclinical or presymptomatic stage in disease
OCR for page 123
BIOLOGICAL MARKERS 123
development, some of which are specific.to a chemical; others might signal
adaptive changes that are not themselves pathological. For example, the
presence of carboxyhemoglobin in the blood signals that damage related to
carbon monoxide exposure is occurring, but the source could be the inhalation
of carbon monoxide or the metabolism of methylene chloride. Red blood cell
delta-aminolevulinic acid dehydratase (deIta-ALAD) has been used as an
indicator of early toxic effects of lead exposure (Friberg, 1985), but the Ameri-
can Conference of Governmental Industrial Hygienists (ACGIH) has not rec-
ommended its use as a biological exposure index, because of Interpretative
diff~culties~ (ACGIH, 1986~. Reduction in plasma acetylcholinesterase activity
has been specifically linked to organophosphate insecticides, but thiocarba-
mates can induce the same effect (Fiserova-Bergerova, 1987~. Nonspecific
markers of reproductive impairment include plasma ollicle-stimulating hor-
mone, plasma luteinizing hormone, salivary progesterone, and urinary hydro~y-
proline or hydroxylysine (which could reflect tissue remodeling (collagen
turnover) after inhalation exposure to environmental pollutants) (NRC, 1988~.
Cytogenetic techniques provide another direct, although nonspecific, meth-
od of identifying changes that occur on the chromosomal level after exposure
to environmental contaminants. Cytogenetic changes include alterations in
chromosome number, such structural chromosomal changes as breakage and
rearrangement, and exchanges between reciprocal portions of a single chromo-
some referred to as sister-chromatic exchanges (SCE). The mechanism re-
sponsible for inducing SCEs is not well understood. Many classes of carcino-
gens and mutagens are known to increase SCE frequency, and that could limit
the usefulness of SCE frequency for exposure assessment. For example,
increased SCE frequency has been found in workers exposed to ethylene
once, styrene, benzene, arsenic, chloromethyl ether, chloroprene, organophos-
phates, or ionizing radiation (Evans, 1982~.
Chromosomal aberrations have been used successfully as a biological do-
simeter to measure absorbed radiation in humans (LAEA, 1986~; however,
ionizing radiation acts through mechanisms that are different from those of
most other atmospheric contaminants. Micronuclei, fragments of nuclear
material left in the cytoplasm after replication, are considered an indication
of the prior existence of chromosomal aberrations. Cytogenetic markers can
be identified in lymphocytes and sometimes in other tissue with stimulated cell
culture and staining techniques in conjunction with light microscopy (Liv~ng-
ston et al., 1983; Carrano and Natarajan, 1988~. Many types of human cancers
are associated with specific alterations (e.g., adult leukemia) or nonspecific
aberrations, and there is increasing evidence that chromosomal aberrations
might be linked to the carcinogenic process related to exposure to some
chemicals (Yunis, 1983~.
OCR for page 124
124 ASSESSING HUMAN EXPOSURE
New techniques have been based on the fact that a specific mutagen (such
as a chemical agent) induces a specific pattern of gene mutation (Benzer and
Freese, 1958~. Techniques have been developed to screen for patterns In
genes from human peripheral blood lymphocytes that contain mutations in the
non-essential enzyme hypoxanthine guan~ne phosphoribosyl transferase
(HGPRT) (Cariello and Thilly, 1986~. One technique involves the use of
denaturing gradient gel electrophoresis, which can separate short DNA mole-
cules according to their melting (uncoiling) properties. The melting behavior
of the DNA fragments is extremely sequence-dependent; a single base-pair
substitution can change migration on the gel. That type of tool provides a
convenient means of examining HGPRT for genetic alterations (according to
the generation of mutation-related spectra on the gel) and then relating the
alterations to specific causative agents (Cariello et al., 1988~. The technique
depends on the use of a high-fidelity polymerase chain reaction to provide in
vitro amplification of a given region of the genome and thus improve sensitivi-
ty and provide enough material for sequencing. The application of the tech-
nique to exposure assessment holds the potential for being specific for a sub-
stance causing mutation and so would be useful in epidemiological studies,
risk assessment, and risk management. In addition, it leads to a measure of
a biological effect and thus could be more directly related to potential adverse
health outcomes than biological markers of exposure. Extensive validation of
the technique is necessary.
Another new approach to assessing genetic effects involves measurement
of single-strand breaks in lymphocyte DNA. A linear dose-response relation-
ship was found in mice exposed to styrene (Welles and Orsen, 1983), and
increased frequencies of breaks were found in workers exposed to styrene
(Welles et al., 1988~. The assay was also used to demonstrate differential
styrene metabolism and clearance by various organ systems (Welles and Or-
sen, 1983~. Alkylating agents, such as N-nitroso-N-methylnitroguanidine and
ethyl methylsulfonate, which are negative in standard cell transformation
assays, have also been shown to be strong inducers of single-strand breaks
(Lubes et al., 1983~.
DNA hyperploidy measured in e~ol~ated bladder and lung cells has been
shown to be a biological marker of response to exposure to carcinogens. It
has been detected with flow cytometry of stained cells (Melamed et al., 1977),
but this approach requires large samples and cannot be used to evaluate
individual cells. The more recently developed technique of quantitative fluo-
rescence image analysis involves the scanning of the fluorescence of individual
stained cells in a microscopic field (Hemstreet et al., 1986~. This technique
has show n DNA hyperploidy to be highly correlated with magnitude of expo-
sure in workers exposed to aromatic amines (Hemstreet et al., 1988~. Further
OCR for page 125
BIOLOGICAL MARKERS 125
validation is needed to determine the specificity of DNA hyperploidy markers
for causative agents and for actual magnitudes of exposure in the environment.
Activated oncogenes and their protein products can be used as markers of
effect. During oncogenesis, a normal segment of DNA (a proto-oncogene)
is activated to a form that causes cells to become malignant. Activation can
occur through several mechanisms, including gene mutation and chromosomal
breaks and rearrangements. Of particular interest in assessment of ambient
environmental exposures is the ras oncogene, first identified in rat sarcoma,
later in human bladder, colon, and lung cancers (Slamon et al., 1984; Span-
didos and Kerr, 1984~. Activated ras oncogenes containing a point mutation
have been produced in vitro by a number of ambient pollutants, including
BaP, dimethylbenzanthracene, and N-nitroso compounds (Sukumar et al.,
1984~. Activation of the ras oncogene can be measured with complex im-
munoblotting techniques. A more convenient and relatively simple method
involves measurement of the gene's abnormal protein product, designated P21.
P21 can be measured in tissue or sputum with monoclonal antibodies, as well
as in blood and urine with immunoprecipitation techniques. The assay is still
in the early validation stage (Brandt-Rauf, 1988~. It is likely that more than
one oncogene needs to be activated to convert a normal cell to a cancerous
one (Stowers et al., 1987~. More studies will be needed to reveal the sequen-
tial requirements for oncogene activation.
Assays that detect changes in the function of target or analogous tissue,
such as decreased sperm counts after dibromochloropropane exposure, pro-
vide markers that are closely linked to disease end points. An increased
concentration of erythrocyte zinc protoporphyrin (EPP) indicates a later stage
in lead toxicity than does delta-ALAD deficiency (Friberg, 1985) and Is a
better masticator of past chronic exposure to lead then is blood lead concentra-
tion (Blumberg et al., 1977; Franco et al., 1984~. ACGIH recommends the
use of EPP as a measure of lead exposure (ACGIH, 1986), although it points
out that iron deficiency can also increase EPP (Lamola and Yamane, 1974~.
Some markers can indicate the presence of disease at preclinical or early
clinical stages. For example, serum alpha-fetoprotein, although not specific
to liver cancer, has been successfully used in China to indicate preneoplasia
of the liver (Committee on Biological Markers of the National Research
Council, 1987~. However, the lack of specificity of tumor markers (such as
alpha-fetoprotein) severely restricts their usefulness (Hulka and Wilcosky,
1988~.
OCR for page 132
132 ASSESSING HUMAN EXPOSURE
from the same worker population (Hemminki et al., 1988; Phillips et al.,
1988~. However, despite the correlation between DNA adJucts and exposure
at the group level, there was significant variation among individuals within the
exposed groups. Adducts measured with immunoassay ranged from nondetec-
table to 2.8 fmol/~g. On the basis of the work of Lioy et al. (1988), it is
possible that workers were receiving exposures by other routes, mainly food.
Harris et al. (1985) did not find BaP-DNA abducts In 10 of 41 coke-oven
workers evaluated. They suggested that that was probably due to variation in
exposure, ~ individual metabolic balance between activation and deactivation,
and in DNA repair capacity. Because DNA repair is both inducible and
saturable, differences related to dose are likely (Swenberg et al., 1987; Dan-
heiser et al., 1989~.
The manner and timing of sample collection can produce ambiguous or
meaningless results. For example, in a study to determine exposure to envi-
ronmental tobacco smoke with nicotine or its metabolites as markers, differ-
ent results were obtained when different sampling protocols were used. Physi-
ological pharmacokinetic modeling has demonstrated that the single-time point
sampling protocol is of little use, unless a marker has a long half-life, which
is rare for most exposures (Schwartz and Baiter, 1988~. Therefore, a good
understanding of the pharmacokinetics is essential for proper timing of sample
collection.
Difficulty of Establishing Links
Between Exposure and Markers of Effect
Biological markers of effect are usually not exposure-specific. For example,
pathophysiological changes, such as alterations in sperm structure or function
and hormonal changes indicative of early reproductive disease, are not specific
to chemicals and routes of entry. The same is true of most markers of pre-
clinical and clinical immunological, cardiovascular, neurological, and renal
dysfunction (EPA, 1988a). With respect to tumorigenesis, most preclinical
effect markers- such as fetal proteins, oncogene protein products, structural
changes in oncogenes, and chromosomal deletions not reveal the identity
of the environmental exposure responsible. Increased specificity can be
achieved if future studies, such as those suggested by Cariello and Thilly
(1986), show that mutagens are specific with regard to the kind and position
of mutation, and this specificity can be considered as a chemical fingerprint
left on the DNA. The fingerprint would be a powerful tool in establishing the
cause-effect relationship. However, Sukumar et al. (1984) observed the same
OCR for page 133
BIOLOGICAL MARKERS 133
ras oncogene is activated in neoplastic cells derived from exposure to several
different chemicals, so this lesion cannot act as a fingerprint.
The search for effect markers for pulmonary to~ncants is also frustrated by
the lack of specificity of pulmonary responses to etiologic agents (NRC, 1988~.
Those responses include altered breathing patterns and airway constriction,
cell injury leading to inflammation, persistent alteration of lung structure (e.g.,
fibrosis, chronic obstructive pulmonary disease, and granulomatous disease),
and neoplasia.
There is a need for research to identify biochemical correlates of structural
changes, as well as to distinguish effects caused by a chemical from effects
that are responses to cell injury (Smith et al., 1986~. It is therefore desirable
to incorporate chemical-specific markers of exposure and early biological
effect into a study design to complement the nonspecific biological markers.
As discussed later, however, relating even a chemical-specific marker to a
defined cxposurc period is not clear-cut.
Confounding Influences on Biological Markers
Biological markers of exposure reflect diverse behavioral, biological, and
methodological processes. Hence, they can be more subject to confounding
influences than are measurements of ambient airborne contaminants. Droz
(1985) evaluated effects of confounding factors on dose with simulation mod-
els. He identified six confounding factors that could influence the dose of
organic solvents: intraday and interday fluctuation of exposure, repetition of
exposure, physical workload, body build, and metabolism. The factors were
applied to four solvents (benzene, styrene, methyl chloroform, and tetrachloro-
ethylene) that differ in blood solubility and metabolism. Droz suggested that
such models provide a means for assessing the role of confounding factors in
internal dose.
Complexity and Resource Intensiveness
By virtue of their collaborative and interdisciplinary nature, biological
marker investigations are highly demanding in terms of personnel, cost, and
effort to develop understanding among researchers in different fields. Most
also require clinical interaction with subjects. Incorporation of biological
markers engenders new and important methodological problems in study
design and in analysis of data (Schulte, 1987, 1989~. It also introduces impor-
tant ethical questions into health-effects research, particularly if information
OCR for page 134
134 ASSESSING lIUMAN EXPOSURE
is given to a person concerning a biological marker of effect when the rela-
tionship to an adverse health effect is unclear.
Use of Exposure Markers
in Conjunction with Traditional Measures
The most effective method of characterizing exposure is to combine meth-
ods for assessing ambient exposure with various biological markers (Friberg,
1985~. Used together, they enhance the precision and accuracy of exposure
information, as exemplified in a study of hospital sterilizer operators occupa-
tionally exposed to ethylene oxide (Yager et al., 1983~. Ambient and
breathing-zone ethylene oxide concentrations were measured, as well as SCEs.
Workers were classified into two groups according to estimates of amounts
potentially absorbed: low, a mean of 13 mg of ethylene oxide over 6 months;
and high, a mean of 500 mg over 6 months. A nonexposed control group was
also evaluated. The high-exposure group had a mean of 10.7 SCEs/cell, which
was significantly different ~ = 0.002) from both that in the low-exposure
group (7.8 SCEs/cell) and that in the control group (7.6 SCEs/cell). Without
the use of ambient measurements to distinguish the two exposure groups, the
difference in mean SCE frequency between exposed and control groups would
have been only marginally significant (p < 0.05~.
CRITERLt GOVERNING THE VALIDATION
AND USE OF BIOLOGICAL MARKERS
Biological markers can be used to identify discrete components in the
e~osure-health-effects relationship (Figure 4.1~. For a given exposure and
disease, it is possible to identify most of the components of the relationship.
Whether the relationship is linear or follows some other form, such as a
branched network, is uncertain, but the linear concept of a relationship should
suffice for research planning. Use of this model, however, should not obscure
the need for efforts to explore the relationship between markers that might
be represented by more accurate, nonlinear models (see Schulte, 1989, for
review).
The current use of biological markers for exposure assessment is not with-
out its pitfalls, and in some cases it cannot occur without adjustments and a
stipulation of an independent determination of confounding variables. For
example, there will be requirements to account for exposure to background
concentrations of contaminants from the same and other media and to adjust
OCR for page 135
BIOLOGICAL MARKERS 135
for seasonal or regional variation in some markers. These adjustments not-
withstanding, the conventional techniques for assessing e~osure-disease asso-
ciations, screening for exposure of individuals in populations and handing
multiple variables can be practiced for any two components in the relationship
(e.g., exposure and internal dose). The major assumption that permits this
approach is the causal or positive association between components of the
relationship. For investigations involving markers of components from the
central regions of the relationship (e.g., biologically effective dose), there is an
increased need to test assumptions and hypotheses, evaluate misclassification,
and identify and control confounding factors. That is because it is difficult to
define the central markers in terms of either exposure or health effect when
so many confounding factors (e.g., kinetics, medical history, and rates of tran-
sition) can affect the pathognomonic relationships.
Validation and Selection of Biological Markers
The validity of a biological marker for environmental studies should be
determined on the basis of the fundamental criteria reviewed by Perera (1987)
and discussed below.
Exposure Assessment
There must be a clear hypothesis or model of exposure-response relation-
ships defining the role of the specific marker in relation to the possible expo-
sure scenario. The relevance of markers of exposure is generally easier to
establish than that of effect markers when assessing exposure. Relating mark-
ers of early biological effect to an original causative agent and routes of expo-
sure is much more difficult, requiring extensive validation studies with careful
linkage of an exposure to a marker of effect.
Understanding of Pharmacokinetics
and Temporal Relevance
The proper selection and use of biological markers depend on an under-
standing of the underlying pharmacokinetics and pharmacodynamics of sus-
pected contaminants (WHO, 1986; Andersen, 1987; Smith, 1987; Yager, 1988).
Knowledge of pharmacokinetics is important in determining the frequency and
timing of sampling and the tissues or fluids most appropriate for study. It also
OCR for page 136
136 ASSESSING HUMAN EXPOSURE
guides the interpretation of dose and effect data obtained in a target tissue or
a surrogate.
The temporal relationship of markers to external exposure or to disease
end points must be clear. Whether an exposure marker reflects recent or
cumulative exposures, or peaks or averages, depends on the pharmacokinetics
of the contaminant, the persistence of the marker In the biological sample
being assayed (which is in large part a function of the turnover rate or half-life
of the sample), and repair rates.
Understanding of temporal relevance is essential for developing monitoring
strategies and interpreting results. Most measures of internal dose, for exam-
ple, reflect recent exposures. An exception would be a substance that is fat-
soluble and is stored in adipose tissue. Hemoglobin is a good integrating
dosimeter over the Month half-life of erythrocytes, which, unlike white blood
cells, lack repair systems. Human serum albumin has a half-life of 20-25 days.
Albumin is synthesized in the liver, where many carcinogens are metabolically
activated, so it might reveal adducts not detected in hemoglobin.
The period reflected by white blood cells or lymphocytes is considerably
more complex (Perera, 1987; Carrano and Natarajan, 1988~. For example,
adducts on DNA from white cells can reflect both past and current exposures,
because a subset (T cells) is very long-lived. A review of white-cell subpopu-
lations shows that measurements of DNA adducts in these cells will be influ-
enced by the longer-lived T cells and will therefore reflect exposures that
occurred both recently and several decades in the past. T cells make up
approximately 60-90% of lymphocytes, which in turn constitute about 22-28%
of peripheral white cells in circulating blood. Thus, T cells constitute a ma~n-
mum of 25% of white cells. The estimated half-life of T ceils is 3 years In
contrast, B cells and monocytes constitute roughly 1-2% and 1-7%, respective-
ly, of circulating white cells and have lifetimes ranging from days to weeks.
Granulocytes make up the remaining 66-85% of white cells and are short-lived
(hours to days). Thus, one should consider DNA adducts only in cells dam-
aged while in circulation and not white-cell adducts in circulating white cells,
which might result from damage to stem or precursor cells in the bone mar-
row. When all DNA from a sample of peripheral blood is assayed for DNA
adduces, one sees that the long-lived T cells are the major contributor in cases
of past discontinued exposure, largely because of their lifetime, which is 100-
1,000 times longer. In addition, lymphocytes have lower repair activity than
do circling cells, so DNA lesions are likely to be more persistent in lympho-
cytes In cases of current, recent, or long-term uninterrupted exposure to
carcinogens, T cells will contribute less importantly to total abducts. The
preponderance of adducts will be measured in the shorter-lived granulocytes,
B cells, and monocytes.
OCR for page 137
BIOLOGICAL MARKERS 137
In retrospective case-control studies, for example, one would want a perma-
nent marker left decades earlier by an initiating carcinogen. In the case of
discontinued exposures, even the longest-lived markers wiD be diluted by cell
turnover, thereby leading to underestimation of past or cumulative dose. Only
if exposure had been continuous and had not changed substantially over the
decades (and only if the disease had not altered metabolism) would current
measurements of the marker be directly representative of critical prior e~o-
sure. At the very least, however, such markers as adducts reflect a person's
responsiveness to carcinogenic exposures, provided that metabolism was un-
changed by disease. Many other exposure patterns are relevant to case-con-
trol and cohort studies (current but interrupted, continuous but of varying
magnitude, etc.~. Each pattern would lead to a different distribution of ad-
ducts among white-cell populations and hence to a varied pattern of persis-
tence.
Understanding of Background
Variability and Confounding Variables
It is essential to know the range of values of a given biological marker in
a "normal" population. Care must be taken not to be deceived by the extensive
variation in biochemical individuality; what is considered Healthy in some
might indicate a health risk in others (Schulte, 1987~. The range of ~normal"
can be large. For example, it is well known that cholinesterase activity in
people not exposed to organophosphorus insecticides covers a wide range
(WHO, 1975~.
Interindividual variation and intraindividual variation are important contri-
butors to "noise. or background in monitoring or epidemiological studies and
should be characterized before large-scale application of a particular biological
marker. Such data, however, can be generated only by large-scale surveys
with repeated sampling and efforts to control for confounding variables. Thus,
a background study is an important exposure-assessment exercise in itself.
With respect to carcinogen-DNA and carcinogen-protein adducts, substantial
interindividual variation and intraindividual variation have been observed with
PAHs, ABE, and nitrosamines (Harris et al., 1985; Umbenhauer et al., 1985;
Bryant et al., 1987; Perera, 1987) SCEs also vary widely within and between
subjects (Carrano and Natarajan, 1988~.
As discussed earlier, biological markers are subject to greater variability
than conventional exposure-assessment techniques, because the body actively
participates in the collection, distribution, and elimination of absorbed con-
taminants. Confounding variables that must be accounted for in studies that
OCR for page 138
138 ASSESSING HUMAN EXPOSURE
use biological markers include age, sex, race, cigarette smoking, alcohol con-
sumption, diet, drugs or other environmental exposures, genetic factors, and
pre-e~nsting health impairment. In fact, it is known that alcohol intake is the
most common cause of reduced metabolism of industrial chemicals (Fiserova-
Bergerova, 1987~. Thus, in a study of those chemicals in which the chemical
or a metabolite is used as a biological marker of dose, one would have to
account for alcohol consumption if one were to understand and interpret the
data properly.
Most reports of cytogenetic studies have given no information on the ~m-
pact of smoking and other exposures to carcinogens or mutagens on their
results. Life-style factors (e.g., smoking and diet) and other chemical eypo-
sures (e.g., environmental, recreational, and therapeutic) are also potential
confounding factors with respect to SCEs and most other markers. Additional
potential confounders are host factors (eeg., health and immune status) and
exposures that influence the marker of interest. In a study of PAM-DNA
adJucts in workers, for example, it is necessary to account for all other back-
ground exposures to PAHs and, ideally, for factors that could induce or inhibit
metabolism and binding. Those inducers include cigarette smoke, char-broiled
meat, alcohol, sedatives, and PCBs; the inhibitors include methylxanthines in
foods, steroids, solvents, and spray paints. Thus, even pilot studies attempting
to evaluate biological markers can become complex epidemiological exercises
that involve careful interviewing.
Reproducibility, Sensitivity, and Specificity
Chemical specificity is a prime criterion for marker selection. Markers
should be chemical-specific or at least highly correlated with the contaminant
exposure of concern. That is important if effective mitigating steps are to be
taken to prcvcat later exposures.
In addition to being specific, markers should also be "sensitive" to the agents
involved in the exposures, detecting a high percentage of persons in the ex-
posed group. Given interindividual variation, not all exposed persons would
be expected to be positive; but it is important that the method for assaying a
marker be sensitive enough to determine background, so that a true compari-
son of the exposed and unexposed can be made. Another criterion for sensi-
tivity might be biological relevance. Some postlabeling techniques are able to
detect progressively lower numbers of adJucts, so a point might well be
reached where adducts are of no phenotypic significance. That point could be
detennined in animal carcinogenicity studies (Ashby, 1988~.
Assays should be reproducible, with limited variability ascribable to labora
OCR for page 139
BIOLOGICAL MARCHERS 139
tory personnel or the assay method itself (Gann et al., 1985~. Reproducibility
includes interlaboratory and intralaboratory reproducibility. When it is possi-
ble, different methods for assaying a given marker are useful for detecting
artifacts In a method. An example of such a process is the cross-validation of
various DNA-adduct marker techniques with immunoassays, 32P-postlabeling
and GC-MS, such as that carried out on PAH adducts of Finnish foundry
workers.
Feasibility
The biological sample to be assayed must be readily available in humans
so that procedures cause minimal discomfort to the participant. Often, under-
stand~ng of the pharmacokinetics can assist In determining which tissue is
most appropriate for sampling.
Assays must be cost-effective. Most assays are still in the experimental
stage and cost about $300-500/sample. If multiple assays are performed in
tandem on the same biological samples, costs of a study are greatly reduced.
That is also the case if stored samples from a prospective study or biological
specimen banks are available.
Storage of samples must not allow degradation of the relevant marker.
Preparation and assay of samples are generally time-consuming, because
repeat assays are needed to ensure reproducibility, but an assay should not
require unusually complicated processing or storage efforts.
Study Design
Adequate Sample Size
Sample size is determined by the elected differences in marker frequency
or concentration between comparison groups, by the anticipated background
in the exposed group, and by variability in measurement data. Considerable
groundwork is required to establish those characteristics.
Appropriate Control Populations
Ideally, depending on the type of study (e.g., cohort vs. case-control), con-
trols must be selected from persons expected to be nonexposed. However, in
most studies of environmental exposures, there are numerous and often im
OCR for page 140
140 ASSESSING HUMAN EXPOSURE
portent background sources. For example, when one is evaluating inhalation
exposure of PAH, ethylene ounce, or styrene from industrial sources, back-
ground exposures could include cigarette smoke, diet, and indoor-air pollution.
It is necessary to control and analyze for potentially confounding sources of
exposure. The definition of the none~osed group will be the results of analy-
sis of the chemical and biological markers. In many instances, assays have
detected a substantial background of industrial compounds in nonindustrial
persons, owing to the domestic use or misuse of the compounds. Hence, it
may not always be necessary to be marker-free at the beginning of a longitudi-
nal cohort study; however, they should have similar frequencies in each group.
Control of Potential Confounding Variables
This issue has been discussed above. Adequate control necessitates elicit-
ing detailed environmental histories from study subjects. Where it is possible,
an array of biological markers should be used to quantify their relationship to
the exposure of interest, confirm the validity of the markers, and attempt to
detect artifacts. Biological markers of susceptibility are useful to document
the presence of confounding conditions.
Use of Batteries of Biological Markers
As noted previously, combination of markers is necessary to account for
confounding factors. Multiple markers should also be used to distinguish
between recent and earlier exposures. A battery of markers designed to
assess the role of occupational exposure to aromatic amines in bladder cancer,
for example, might include breathing-zone monitoring for the amines, im-
munoassays of specific aromatic amine-DNA adducts in white cells (lifetimes
of days to years), and cytogenetic effects in T cells (half-life, 3 years). Coti-
nine in plasma could be used to learn about cigarette-smoke exposure, a
potential confounding variable for bladder cancer.
Analysis
For a detailed discussion of the analytical considerations in biological-mark-
er studies, the reader is referred to Thompson (1983), Sheldon et al. (1986),
Schulte (1987, 1989), and Margolin (1988~.
OCR for page 141
BIOLOGICAL MARKERS 141
ETHICAL ISSUES
The availability of highly sensitive assays that can identify dose and effects
resulting from the interplay between low-level exposures and genetic or ac-
quired susceptibility raises several thorny ethical and moral questions. The
reader is referred to Ashford (1986), Committee on Biological Markers of the
National Research Council (1987), Schulte (1987), and Weiss (1989) for dis-
cussion of those issues. Primary among them is the use that is to be made of
biological-marker information. Take, for example, the theoretical case of a
biological marker known to reflect susceptibility. Should a worker who tests
positive or has an increased measurement be removed from the workplace?
If so, should he or she be offered an equivalent job in the same industry? Or
should the workplace be cleaned up to protect the most sensitive worker?
In reality, few, if any, biological markers are established as predictors of
individual risk associated with inborn traits, exposure, or a combination of the
two. Therefore, it is important to inform research-study participants in ad-
vance that the results are interpretable only on the group level. Participants
in such studies should be provided test results that are presented and dis-
cussed in context with available information (or lack thereof) on the variability
within and between people in the normal (nonexposed) population, as well as
that observed in the research-study group.
SUMMARY
Biological markers can be divided into three broad classes: markers of
exposure, markers of effects, and markers of susceptibility. The committee
focused on the application of biological markers to assessment of exposure to
contaminants rather than their use to predict health effects. Markers provide
information on dose that can be related to exposure using pharmacokinetic or
pharmacodynamic models. However, these markers integrate all routes of
exposure, and the actual routes cannot be detected without personal or micro-
env~ronmental exposure information. Biological markers, therefore, are best
used in conjunction with conventional measures of exposure.
Biological markers range from measures of the intact original contaminant
to measures of adverse health effects caused by that contaminant. Relating
a marker directly to an exposure becomes more uncertain as measurements
are obtained within the progression toward an effect, because the variability
associated with the human "receptor" increases along that progression.
Properly measured, biological markers can reduce misclassification errors
of degree of exposure and can increase the power of epidemiological studies
OCR for page 142
142 ASSESSING lIUM4N EXPOSURE
to identify associations between exposure and disease. Markers can, in theory,
be useful in developing and validating pharmacokinetic models aimed at relat-
ing exposure to dose. They have the potential to improve risk extrapolation
between species and between populations and to allow better predictions of
human risk. Markers can provide an early warning of hazard or potential risk
of disease. Markers measured in stored specimen banks can allow retrospec-
tive determination of whether a particular contaminant was elevated in the
general population at an earlier time.
Use of biological markers for assessing exposure has several disadvantages
and limitations. A major limitation is that most markers are in the develop-
ment stage and have not been fully validated or field tested. A key question
regarding markers of exposure is what compounds and exposures could have
produced a marker of a measured amount in a specific tissue. The variability
of markers can present problems in establishing quantitative relationships
between exposure and response at low-exposure concentrations. Markers
reflect diverse behavioral, biological, and methodological processes. Hence,
they can be subject to more confounding influences than are measurements
of ambient airborne contaminants. By virtue of their collaborative and inter-
disc~plinary nature, investigations of biological markers are demanding in
terms of personnel, cost, and effort to develop understanding among research
ers.
Analytical techniques with improved chemical specificity and sensitivity for
biologically significant markers are needed to apply to exposure assessments,
especially flexible assays that can analyze a number of markers simultaneously
or be readily adapted to analyze numerous markers sequentially. For such
techniques, validation studies are needed to link conclusively biological mark-
ers to putative causative agents.
Better pharmacokinetic data are needed for an increasing range of chemi-
cals. These data are needed to further the development and validation of
more sophisticated biological-marker models and to further understanding of
how to model multiple metabolism pathways as a function of exposure level.
Representative terms from entire chapter:
biologically effective
