This manuscript was prepared as part of the Environmental
Epidemiology Planning Project of the Health Effects Institute, September
1990 - September 1992.
Introduction
Most studies of health effects in humans exposed to complex air-pollutant
mixtures have used such outcome measures as hospital admission rates during
air pollution episodes, symptom reports from questionnaires or diaries,
and disease prevalence or mortality rates in communities with different
levels of air pollutants. A few studies in controlled exposure settings
have attempted to assess the combined as well as separate effects of mixture
components. Such approaches provide useful information; but studies of overt
disease often are insensitive to low-dose exposure effects, and they focus
on the extreme end of the disease spectrum, where only a small proportion
of the exposure-related disease burden occurs.
Biological markers of intermediate health outcomes (i.e., early pathologic
changes or events predictive of disease) could provide a superior alternative
to traditional measures of pollutant-related disease. Markers can have greater
sensitivity to exposure effects, they may appear sooner after exposure and
at younger ages, and they may detect a greater proportion of the exposure-related
disease burden compared to measures used in the past. Because some early
pathologic changes detected by markers do not progress to symptomatic conditions,
more people will show positive marker responses than overt disease; so studies
using markers can potentially have increased statistical power due to the
more numerous outcomes. However, the markers are useful only to the extent
that they have a known relationship to clinical diseases of interest.
In general, biological markers are indicators of events occurring in
the body that are difficult to measure directly. Markers can indicate that
an exposure, a response to exposure, or an early pathologic change has occurred;
other markers, often enzyme phenotypes, indicate an individual's increased
susceptibility to disease from a particular exposure. Such markers differ
from genetic markers, which are usually defined in the current genetic literature
as discrete phenotypes controlled by genes that occur in close proximity
on chromosomes to other genes of interest. A good genetic marker will be
correlated highly with the presence of the gene of interest, which may be
undetectable.
The following presentation discusses some methodologic issues associated
with the use of biological markers of intermediate health outcomes arising
from exposures to complex pollutant mixtures. In most instances, this article
focuses on outcomes related to four examples of complex mixtures: a)
environmental tobacco smoke (ETS) and nitrogen dioxide (NO2),
b) acid aerosols and oxidant outdoor pollution, c) ETS and
radon, and d) volatile organic compounds (VOCs). No standard definition
exists for the term biological marker. For example, the National Research
Council's (NRC) 1989 book Biologic Markers in Pulmonary Toxicology
(1) defines biological markers as "indicators of events in biologic
systems or samples," while others [e.g., Hulka et al. (2)] limit
the definition to indicators measured in biological specimens obtained from
a person. The NRC definition includes, for example, spirometry, which involves
no assays of biological samples. This discussion uses the more restrictive
definition of Hulka et al. (2). These two references provide additional
information about markers in general and about the specific markers discussed
here.
Methodologic Issues
Link with Disease
The most important property of any biological marker of effect is its
link with the health outcome of interest. Although markers in a target tissue
(i.e., tissues that give rise to the disease of interest) usually provide
the best early indicator of an adverse event, markers outside of the pathogenic
pathway can be superior for a variety of reasons. For example, target tissues
such as the lung are relatively inaccessible, markers in the target tissue
may require higher exposures to show a response than do markers in a nontarget
tissue, and markers in a target tissue may have less persistence due to
rapid cell turnover or other mechanisms of marker loss.
A study of micronuclei measured in exfoliated epithelial cells in sputum
of uranium workers exposed to radon and tobacco smoke illustrates how markers
outside of the pathogenic pathway can show a stronger statistical association
with the disease under consideration. Micronuclei, which are caused by agents
that damage DNA, are small secondary nuclei formed during mitosis when whole
chromosomes or chromosome fragments fail to become incorporated into daughter
nuclei. As summarized by Loomis et al. (3), the micronucleus assay
in exfoliated buccal cells is sensitive to ionizing radiation as well as
tobacco smoke; uranium miners show a clear radon-related excess of lung
cancer, but neither radon exposure nor cigarette smoking was associated
with a higher prevalence of micronuclei in Loomis's study of sputum cells
from the miners. In contrast, other studies of uranium miners (4)
and persons with residential radon exposure (5,6) show increased
levels of structural chromosome aberrations in blood lymphocytes. Although
chromosome aberrations in lymphocytes have no direct role in lung cancer,
their association with radon exposure suggests that this marker, compared
to micronuclei in sputum cells, has a stronger statistical link with lung
cancer arising from radon exposure.
Other markers of premalignant changes with potential interest for studies
of carcinogenic exposures (e.g., VOCs or radon and ETS) include sputum cytology,
the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay for in
vivo mutations, and assays of oncogene activation. To date, human studies
using the HGPRT assay primarily have examined peripheral lymphocytes, but
the assay could be adapted for pulmonary macrophages obtained through bronchoalveolar
lavage (BAL) (1). By using macrophages, the marker would detect mutations
occurring in the lung, although lung tumors do not arise from macrophages
themselves. Theoretically, activated oncogenes could be detected in exfoliated
cells in lavage fluid to characterize developing lung tumors (1).
The use of almost any marker of intermediate outcomes would increase the
number of exposure-related outcomes (compared to the number of cancers)
in the study while reducing the necessary time interval between exposure
and measurement of the outcome. At this point, however, most markers of
intermediate outcomes have an unknown relationship to clinical disease;
so their value is limited.
The rationale for using nontarget tissues and cells for assessing evidence
of early disease is that pathologic changes observed in nontarget tissues
often occur in the target tissues. For example, exposure to acid aerosols
and oxidants can exacerbate airway hyperreactivity in asthmatics (1),
with a resulting increase in pulmonary and blood eosinophils. Chronic deposition
of eosinophils in the lung may cause airway inflammation, but eosinophils
are much easier to measure in blood than in the lung. Even if markers in
target tissues compared to nontarget tissues do show a stronger association
with the ultimate outcome of interest, their inaccessibility may preclude
their use in observational studies.
In general, the selection of markers involves a tradeoff between a marker's
positive predictive value for the disease and such practical issues as specimen
availability, marker sensitivity, and assay cost. Unfortunately, many potentially
useful markers have an unknown relationship to lung disease. For example,
the relationship between changes in constituents of BAL fluid (a potential
source of myriad markers of intermediate outcomes) and pollutant-induced
injury requires further study (1). Markers of events that occur further
down the disease pathway, such as fibrosis, moderate airspace enlargement,
or mutations, have clearer biological links with clinically apparent disease.
In the absence of important advantages associated with other markers, biologically
plausible markers having obvious biological links with disease are preferable
to markers without such links. The validity of markers that occur in nontarget
tissues or early in the pathogenic process would be assessed ideally in
prospective studies, where their positive predictive value for subsequent
overt disease can be estimated. Such studies would be difficult, especially
when cancer is the outcome of interest, because of the need for large study
populations and long follow-up periods.
Studies of associations that can be measured cross-sectionally are more
feasible. One could, for example, ascertain whether a marker measured in
blood has a high positive predictive value for inflammation in the lung.
A marker with a high predictive value for inflammation could be used as
an outcome variable in a study to determine whether exposure to a complex
mixture causes inflammation.
Specificity and Confounding
Some markers respond to specific environmental exposures, while others
respond to a wide variety of agents. For studies of complex mixtures, nonspecificity
can be an asset, because the marker response will reflect the combined effects
of multiple, sometimes unidentified, agents. Furthermore, the airways can
react to inhaled toxic materials in a limited number of ways, so a wide
variety of exposures lead to a small number of health effects. For example,
many inhaled toxicants, such as acid aerosols and oxidant gases like NO2
and ozone, cause inflammation in the respiratory tract. Exposure to ETS
and NO2 in children increases the risk of respiratory infections,
which also cause inflammation. Chronic or repeated inflammation may in turn
lead to irreversible lung injury and, eventually, clinically apparent diseases
such as emphysema. Therefore, indicators of inflammation or early loss of
elasticity can serve as markers of intermediate outcomes from numerous exposures,
the effects of which converge on a common pathologic pathway.
The convenience of using a nonspecific marker that responds to a variety
of complex mixtures is offset by the possibility of dilution and confounding
from exposures other than those of interest. As discussed by Weiss and Liff
(7), the problem of dilution, where an exposure-response association
is obscured by other associations, arises when different causal pathways
lead to the same end point, as is the case with nonspecific markers of intermediate
outcomes. If two different exposures (or sets of exposures), E1
and E2, cause the same marker response through independent pathways,
they increase the overall marker response rate in an additive manner; but
relative measures of association (e.g., relative risk, odds ratio, etc.)
are based on the assumption of a multiplicative model of association. As
a result, the relative risk of the response due to E1 will be
influenced by the background incidence of the response due to E2.
In this situation, use of the risk difference rather than relative risk
to compare marker responses in persons exposed and unexposed to E1
helps avoid the problem of dilution from a high background incidence from
E2.
Another strategy for mitigating the problem of dilution is to stratify
an overall group of end points into its more homogeneous components (7).
Inflammation from different exposures, for example, may have slightly different
manifestations detectable by different markers. Each marker would have greater
specificity for a given exposure than would a marker that detected overall
inflammation. The feasibility of this approach for studies of complex mixtures
is unclear until additional basic information on properties of markers of
intermediate outcomes becomes available.
Weiss and Liff (7) point out that studies of intermediate outcomes
sometimes facilitate the identification of a particular causal pathway.
For example, if exposure to one complex mixture leads to pulmonary disease
through inflammation, while another exposure causes the same disease through
a noninflammatory process, the complex mixture would show a stronger association
with inflammation than with the pulmonary disease. However, this approach
is feasible only when the intermediate outcome has a known relationship
to the clinical outcome of interest--a rare situation.
Confounding could arise in studies of inflammation due to exposure to
acid aerosols and oxidants, for example, if exposed persons tend to be heavy
smokers or have occupational exposures that also cause inflammation. Problems
with confounding are essentially the same whether one uses nonspecific markers
of intermediate outcomes or actual diseases as study end points. The usual
epidemiologic approaches for controlling confounders (i.e., stratified analysis,
matching, or restriction) can remove the effects of extraneous variables.
Exposure-specific markers would be less prone to confounding than would
nonspecific markers, but outcome markers that arise only from single agents
would have limited value for studies of complex mixtures.
Sensitivity
In many instances, different markers can be used to detect the same outcome.
Inflammation, for example, involves numerous physiological changes that
can be used as markers of the inflammatory response. For a given degree
of inflammation, however, some markers will be easier to detect than will
others. Markers that detect the mildest inflammation (i.e., those that are
positive with the lowest exposures) would have the greatest sensitivity.
An animal study (8) illustrates several issues associated with
marker sensitivity for intermediate outcomes. The investigators evaluated
different markers of connective tissue metabolism (a response to injury
in the lung) in urine or BAL fluid. In one exposure protocol using 0.5-ppm
NO2 exposure for 4 weeks, hydroxylysine urinary excretion increased
significantly, but levels of hydroxylysine and angiotensin-converting enzyme
activity in lavage fluid remained normal and lung histology showed no damage.
Compared to other markers of effects on connective tissue, urinary hydroxylysine
apparently has greater sensitivity.
Although this controlled study of NO2 exposure in rats only
has indirect relevance to free-living human populations exposed to complex
mixtures, it does illustrate that different markers vary in sensitivity,
and that the same marker measured in different biological materials also
can have different sensitivities. For reasonably benign exposures, such
as many commonly occurring complex mixtures, exposure chamber studies can
characterize a promising marker's properties (e.g., sensitivity, dose-response,
and interindividual and intraindividual variability) in humans under controlled
conditions. These studies can evaluate markers of acute outcomes but precise
estimates of such properties will rarely, if ever, be available for marker
responses from chronic exposures.
In general, a marker's sensitivity and positive predictive value can
be increased by studying susceptible populations. For example, exposure
to acid aerosols-oxidants can exacerbate symptoms of asthma. Sensitized
asthmatics compared to nonsensitized asthmatics and nonasthmatics are likely
to show an inflammatory response at lower exposure levels, so markers of
inflammation will have greater sensitivity in studies of sensitized asthmatics.
Similarly, ETS-NO2 exposure may increase the risk of respiratory
infections in children more than in adults, possibly through alterations
in immune function; theoretically, markers of such alterations may have
a greater sensitivity (i.e., occur at relatively low exposure levels) in
children given their apparent increased susceptibility to infections compared
to adults. Given the known susceptibility of such groups as asthmatics and
children to some mixtures, they also may be susceptible to other pollutant
mixtures, so that adverse health outcomes and associated markers could be
detected at relatively low exposure levels.
Sensitivity also can be increased for a given ambient concentration by
studying people with a relatively high internal dose of a pollutant mixture,
such as those having a high rate of ventilation due to physical activity.
Persons who spend a large proportion of time outdoors also will have relatively
high doses of ambient outdoor air pollutants. Thus, for a given exposure
level, marker responses would probably be more pronounced in persons with
biological susceptibility and in those with behaviors that increase either
their internal dose or their contact with ambient pollutants.
Temporal Aspects
Markers can appear hours, days, or years after exposure. For example,
nasal irritation is commonly associated with indoor air pollution (e.g.,
VOCs and other complex mixtures). Markers of cell and mediator changes in
nasal lavage fluid could be useful for studies of such pollutants (1),
and the markers would probably appear within hours of exposure. In contrast,
several months or years of exposure to acid aerosols and oxidants may be
necessary to detect airspace enlargement using morphometry, while changes
in alveolar cell populations may appear after days or weeks of exposure.
For transient markers, the timing of measurements is especially crucial.
The influx of neutrophils and eosinophils into the respiratory tract, for
example, usually occurs during the first 3 to 7 days of an inflammatory
response (1). Measurements of these markers of inflammation in BAL
fluid immediately after exposure would underestimate the inflammatory response,
as would measurements taken after the response subsided. Protein influx
reflecting pulmonary epithelial damage, however, should be measured relatively
soon after exposure. For sustained ongoing exposures, such as occurs with
VOCs or residential radon exposure and ETS, transient markers will be replenished,
and measurements can be made any time during seasons when buildings are
likely to be poorly ventilated.
The timing of measurements is less important for markers of chronic exposure-related
changes. Irreversible airspace enlargement, for example, can be measured
long after exposure ends, and it will reflect cumulative exposure effects.
Altered populations of alveolar epithelial cells due to oxidant air pollution
exposure eventually revert to normal proportions, but these markers can
probably be detected for at least several weeks after the end of exposure.
Timing is still important in the sense that the exposure must be sufficiently
long for the marker response to occur. Note that for some markers of chronic
pathogenic processes, such as the markers of connective tissue degradation
in the study by Evans et al. (8), the marker response diminishes
after the exposure stops ,even though the associated damage may be irreversible.
Approaches for Using
Markers
The effective use of a marker in epidemiologic studies of complex mixtures
depends not only on the marker's properties but also on the availability
of suitable biological materials and moderately priced assays. Numerous
markers of intermediate outcomes are inappropriate for field studies because
of their invasive nature. The following section mentions some noninvasive
markers with potential usefulness for studying complex mixtures, and it
discusses strategies for using invasive markers.
Noninvasive Markers
In general, markers measured in such materials as urine, sputum, blood,
and nasal lavage fluid are well suited for field studies because specimen
collection involves relatively little inconvenience or risk for study participants.
Urine could be valuable especially for studies of ETS and NO2.
ETS exposure can be estimated from urine samples, as can some markers of
connective tissue metabolism associated with NO2 exposure. Sputum
cytology, a marker of disease that is nonspecific with regard to exposure,
may reveal early evidence of carcinogenic changes from such exposure as
VOCs or ETS and radon gas. Standardization of sputum collection and preparation
might alleviate the problems encountered by Loomis et al. (3) and
allow detection of increased micronuclei from these exposures. Loomis et
al., who used archived specimens, could not control the source of sputum
and its cellular content; and laboratory manipulation of the old samples
may have caused a loss of some cell structures.
Blood is a source of numerous and diverse markers. As noted earlier,
radon exposure at levels that increase the risk of lung cancer are associated
with chromosome abnormalities in blood lymphocytes. Markers of altered immune
function, which increases the risk of respiratory infections, also can be
measured in blood. Some studies suggest that markers of pulmonary hypertension,
which apparently is caused by several toxic chemicals, also may be present
in blood (1); possible markers include elevated plasma copper levels
and ristocetin cofactor activity relative to plasma von Willebrand factor.
Many changes in constituents of blood and nasal lavage fluid reflect
the changes that occur in less accessible BAL fluid. For example, the distribution
of lymphocyte subpopulations, a marker of air pollution effects, is similar
in blood and BAL fluid (1). Nasal lavage fluid, which contains several
markers that respond to a variety of constituents found in complex mixtures,
may be especially useful in studies of indoor air pollutants that cause
nasal irritation (1). Comparisons between markers in nasal and BAL
fluid are necessary to ascertain the usefulness of nasal lavage markers
as indicators of events in the lower respiratory tract.
Invasive Markers
Some of the most informative markers of intermediate outcomes occur in
relatively inaccessible biological materials such as the lung. One approach
to obtaining lung specimens is BAL, which uses a modified bronchoscope for
collecting pulmonary cells and fluids. Lavage fluid contains a variety of
biological materials in which to measure markers of intermediate outcomes.
The method is used primarily for diagnostic purposes and in controlled-exposure
chamber studies. Although its invasive nature precludes the routine use
of BAL in field studies, small studies of individuals with exposures to
naturally occurring complex mixtures could detect numerous potential exposure-related
effects. The use of personal monitors in conjunction with lavage measurements
would provide a high degree of precision in estimates of exposure-outcome
associations. Retrospective collaborative studies using archived lavage
specimens also may be feasible if unexposed controls from chamber studies
in cities having different levels of air pollution could be identified.
Differences in specimen collection and storage among research centers, however,
may preclude such retrospective studies.
Much of the recent animal research and some human studies of air pollution
have used morphometric measurements for assessing pulmonary damage. The
morphometric approach uses an overlay of points or lines that is placed
over an electron micrograph or other two-dimensional image of a lung section.
By estimating the proportion of points or numbers of lines that fall on
cell or airway structures, one can use a set of formulas to estimate various
cell and tissue parameters, such as cell size and structure, proportions
of cell types, or airway diameters (9).
Morphometric studies of lung tissue are limited to specimens obtained
from surgery or autopsy. This severe constraint raises a host of methodologic
problems, but the potential value of morphometric measurements argues in
favor of further exploring this promising technique. For example, morphometry
has been used to study postnatal lung growth (1), an outcome relevant
to ETS-NO2 exposur; and numerous morphometric studies have found
pulmonary changes in animals exposed to single pollutants and pollutant
mixtures (10). Limited studies of air pollution using morphometry
with human autopsy specimens point to the feasibility of moving from animal
to human tissues. The application of morphometric techniques to human lung
specimens may eventually provide the most direct evidence of pulmonary damage
from chronic low exposures to complex mixtures. However, basic work remains
to be done. Studies are necessary to describe lung lesions in persons of
different ages exposed to ubiquitous background pollutants (ETS, automobile
exhaust, etc.) and to investigate the effects on lesion measurement of different
protocols for collecting, handling, and storing lung specimens in autopsy
settings.
Conclusion
Indoor and outdoor air pollutants potentially can affect the health of
virtually everyone in the United States. Animal studies using controlled
chronic exposures are important for identifying the pollutants responsible
for adverse health effects, but epidemiologic studies address the effects
of complex pollutant mixtures that actually occur in exposed humans. Biological
markers of intermediate outcomes offer new opportunities for advancing the
study of these pollutants.
Unfortunately, many research opportunities associated with markers remain
theoretical. Much basic information critical for valid application of markers
is lacking. For example, few markers have been characterized regarding their
statistical properties, such as assay variability and variability in samples
collected from the same individual at different times. Furthermore, protocols
for collecting, storing, and analyzing biological specimens have not been
standardized. More important, the relationship between markers of intermediate
outcome and clinical disease remains a matter of speculation. Investigators
studying exposures to complex mixtures therefore cannot interpret the health
implications of a positive marker response, nor can they confidently attribute
a lack of an exposure-response association to a true absence of a biological
effect when a marker's sensitivity and variability are unknown. Also, the
nonspecific nature of currently used markers of intermediate outcomes leads
to potential problems with dilution and confounding of exposure-outcome
associations. A combination of controlled exposure studies and systematically
conducted epidemiologic studies could readily address the validity issues,
but the high cost of many marker assays discourages their use in large-scale
epidemiologic studies.
Progress in applying markers to studies of human exposures and diseases
requires considerable effort from both toxicologists and epidemiologists.
However, neither group has great incentive to undertake the mundane systematic
studies necessary to characterize marker properties in a statistically valid
manner: bench scientists usually prefer to investigate promising new techniques
while epidemiologists are primarily interested in etiologic associations.
Progress in the use of markers is likely to occur slowly until answers to
basic questions become available.
