J. Carl Barrett,1 Harri Vainio,2 David Peakall,3 and Bernard D. Goldstein4
Many of the biomarkers of effect are used also to assess exposure, and some of these will be discussed in this section. Not all biomarkers can be used in all settings, particularly in developing nations (5).
Objectives for Using Biomarkers of Exposure
Exposure can be viewed as both a population and an individual phenomenon. Exposure markers can function in several ways. a) They may lead to early detection of exposure at a point where significant health effects have not occurred. This must focus attention on external means of reducing exposure, i.e., on primary prevention. b) They may provide validation of exposure for use in epidemiologic studies. c) They may facilitate comparison of exposure levels in different compartments (external, blood, cellular) to identify susceptibility differences.
Criteria for Screening, Using Biomarkers. The application of any biomarker approach must be conducted within the context of a viable screening and prevention program. The World Health Organization (WHO) has established a set of criteria to be met before instituting a screening program. These will be discussed in relation to exposure markers.
CRITERION 1. The screening must be conducted for a condition of public health significance. Overall the exposure, health status, or susceptibility of populations or subpopulations is clearly of public health significance. Thus, criterion 1 is usually met (but see the section on ethical considerations).
CRITERION 2. The natural history of the exposure marker must be well understood, and there must be a distinguishable subclinical phase. There is tremendous variability in our knowledge regarding the natural history of various exposure markers and their relation to subsequent effects.
CRITERION 3. There must be a scientifically defensible and socially and personally acceptable intervention. The usual intervention when an exposure is detected is to remove the hazard and reduce the exposure.
CRITERION 4. The tests used must have appropriate sensitivity and specificity. Usually the sensitivity limitation is imposed by the analytic method available. Increasingly sophisticated instrumentation has allowed the measurement of various analytes at infinitesimally small levels, although in most cases this sensitivity is not required. However, it is essential to choose the appropriate tissue or fluid and methodology to assure adequate test sensitivity. Sensitivity also depends on the proper timing of analysis with respect to exposure.
Measuring the agent of concern is usually highly specific. For example, a blood lead determination reflects lead exposure. However, it may not be specific to the source of lead being investigated since there are many sources of lead in our environment. Specificity in part depends on the question being asked. For example, one might be interested in recent exposure or historic exposure and would design a different testing approach for each. In some cases, to address specific questions, chemical speciation of the agent or analyte is necessary (e.g., chromium VI vs chromium III, methyl vs inorganic mercury, organic vs inorganic arsenic) and speciation, in turn, may reduce sensitivity.
The metabolites one measures may be specific or nonspecific. Thus phenol is commonly used as a marker of benzene exposure but is not specific (other compounds are metabolized to phenol and some consumer products, such as cough medicines, contain phenol). Nonetheless it may still be useful in quantifying exposure in individuals known to have relatively high levels of exposure to benzene. Other metabolites are more highly specific (e.g., muconic acid for benzene), and others are very specific (DDE for DDT). Some metabolites are also active agents.
CRITERION 5. The condition or exposure being sought must be sufficiently common that the tests have acceptable predictive value. Exposure is a phenomenon common to both a population and an individual. On the individual level, the predictive value of a screening test depends not only on its sensitivity and specificity, but on the underlying prevalence of the exposure. One can estimate population exposure by randomly sampling a subgroup of the population before extending screening for exposure biomarkers to a larger population.
CRITERION 6. The testing must be acceptable to the target population. Biomarkers of exposure are most often detected with noninvasive (breath sampling, urine testing) or minimally invasive (milk collection, blood testing) approaches. At the other end of the spectrum are fat biopsies which are limited to clinical diagnostic settings or to specialized research protocols.
CRITERION 7. The testing program must be cost effective (see below, on analysis and quality assurance).
Use of Biomarkers
Applications of Biomarkers of Exposure. Monitoring biomarkers of exposure is usually part of a preventive activity. It can contribute to identifying and reducing exposure or to identifying at-risk or susceptible populations that need to be protected in special ways. In occupational health, the major goal of detecting biomarkers is the prevention of health impairment by the recognition of excessive exposure and the elimination of hazards (6). Ideally, exposure is controlled and measured at the source so that no excessive exposure occurs (7), but often there is supplemental reliance on biological monitoring as part of a medical surveillance program, to assure that the primary preventive strategies have been effective.
A marker can be applied on an individual basis to estimate the amount of pollutant absorbed or retained, usually through the measurement of the agent or its specific metabolites (8). A marker can reflect individual differences in the rate of absorption and in toxicokinetics (metabolism, distribution, and excretion).
The traditional definition of an exposure biomarker involves measurement of a xenobiotic or its metabolite in a body tissue or fluid, whereas markers of effect include any measurable alteration attributable to a xenobiotic that can be recognized as a health impairment. Between the two is a continuum of subtle effects caused by chemicals that can be measured but which are not indicative of a disease. Some of these are early stages in a significant pathogenic process (e.g., DNA adducts of alkylating agents), others are early, often trivial, stages in a pathogenic process (zinc protoporphyrin [ZPP] elevation in lead exposure), and others may be the early signs of damage that are reversible and not of clinical significance (for example, depression of cholinesterase activity or elevation of some tubular proteins in persons exposed to heavy metals). In the latter case, it is recognized that kidney function may show no clinically detectable decrement until about 90% of the nephrons are damaged; hence substantial changes can occur with no immediate overt clinical significance except a reduction in reserve capacity.
Blood lead is a marker of exposure and a surrogate marker of effect. Elevations of ZPP or erythrocyte protoporphyrin and urinary 
-aminolevulinic acid (-ALA) or the activity of -ALA dehydratase are all effects caused by lead; the inhibition of enzymes in the heme synthesis pathway results in anemia, yet anemia is rarely the end point of concern in lead poisoning, especially in adults. Thus, they are useful as exposure as well as effect markers.
A major criterion for distinguishing markers of exposure from markers of effect is the purpose to which they are put. In many cases enzymatic or cellular damage markers are used for dose reconstruction or for the classification of exposure status for epidemiologic studies. Van Schooten et al (9), for example, treat DNA adducts in smokers as a means of estimating their polycyclic aromatic hydrocarbons (PAH) exposure.
For the purposes of this discussion we recognize the following as biomarkers of exposure: a) measurement of the xenobiotic itself (i.e., lead in blood, DDT in milk); b) measurement of specific metabolites; c) measurement of specific subclinical effects that are reversible. Many effect markers that are used to estimate exposure or tissue dose are described in more detail below.
Choice of Tissue or Fluid and Analyte. Factors that influence the choice of tissue or fluid and analyte include the toxicodynamics of the agent (where it is distributed, stored, excreted), speciation (its chemical form), and chronology (acute vs chronic exposures and temporal aspects of its kinetics in the body).
An exposure marker should be able to distinguish long-term from short-term exposures (10). Thus blood lead may reflect recent exposure, while bone lead reflects cumulative exposure.
An example of the importance of appropriate quality control (QC) is reflected in the experience with routine blood lead determinations by clinical laboratories. As recently as the early 1990s, when laboratory proficiency testing programs were concerned with accuracy in the presumed toxic range of 40 µg/dl, it was found that most laboratories were unable to provide reliable results below 15 µg/dl, even though the Centers for Disease Control had designated 10 µg/dl as a "level of concern." Two years later, when proficiency testing began to include unknowns in the 10 µg/dl range, most laboratories rapidly improved to the point where they provided acceptable results. It is essential that a quality assurance (QA) program be designed for the range of values that will be encountered in a population.
DNA and Protein Adducts. Many compounds form covalent adducts with nucleic acids and other macromolecules including hemoglobin and other proteins. In the case of DNA adducts, the changes in the DNA molecule may relate directly to mutational or repair events and the possibility of subsequently developing cancer. Elevated levels of DNA adducts are seen in a number of populations with known elevated cancer risks, including smokers and coke-oven workers.
DNA adducts can be measured by the 32P-postlabeling technique or by immunoassays. For certain adducts, mass spectrometry, fluorescence spectrometry, and liquid chromatography/electrochemical detection have been used. For quantitative analysis a standard compound is necessary for the calculation of recovery as discussed by Hemminki (11). In the case of complex mixtures, identification of individual adducts may not be possible. In such cases recoveries cannot be calculated and quantification is not possible. Data on half-lives of adducts in humans are very limited. Biomonitoring of protein adducts applies to either hemoglobin or albumin. Adducts can be released hydrolytically from the protein and assayed by gas or liquid chromatography, or by mass spectrometry. N-terminal valine of hemoglobin can be specifically released in the Edman degradation process and assayed by mass spectrometry. Adducts are generally not thought to alter the half-lives of the proteins.
DNA-protein cross-links are another end point that has been used to estimate exposure to mutagens such as hexavalent chromium (12). A standardized DNA extraction procedure is used that fails to extract cross-linked DNA that can be quantified as a percentage of the total DNA.
Assessing Population Exposures. Examples of exposure markers used to document the effectiveness of regulatory interventions are the dramatic decline in blood leads in countries that have eliminated lead from gasoline (13), and the decline in the levels of polychlorinated dioxins and dibenzofurans in breast milk in Sweden (14), attributable partly to the ban of chlorinated phenol herbicides.
Overall Ethical Considerations. The estimation of exposure is performed to benefit the individual subject or population, usually by subsequently reducing their exposure to hazards. It is unethical to perform a testing program merely for its own sake, without having the goal of reducing harmful exposures that may be detected. Therefore, in a workplace, a biological monitoring program using markers of exposure should be performed as part of a comprehensive medical surveillance program linked to an industrial hygiene program capable of discovering and eliminating hazards.
Zielhuis (10) cautioned that the results of a screening examination must be examined in the light of many individual factors. In turn the physician must impart objective information to the participant. "In the perception of the examined subject, biological sampling for assessment of internal exposure, and henceforth of health risk, is not distinguished from assessment of their health" (10). Individuals should be informed of their health risks, along with the degree of uncertainty, regardless of the technologic and economic consequences.
Some question the ethics of biological monitoring for exposure, arguing that humans should not be the guinea pigs or the detectors of their own exposure. However, biological monitoring offers the advantage of taking into account absorption by all routes (whereas air monitoring would not detect potential exposure by other routes). We caution that biological monitoring can be an adjunct, but it cannot substitute for environmental monitoring and controls. [See below and Soskolne (15) elsewhere in this issue for a further discussion of ethical issues.]
Examples of Biological Monitoring for Selected Agents (Table 1)
Heavy Metals. Most heavy metals are of toxicologic concern, although some are essential trace elements in humans as well. Since these elements are not metabolized, they can be tracked in the body, and can be measured in various body compartments and fluids. A variety of standardized analytic procedures are described in readily available references (16). Most of these are analyzed with atomic absorption spectrophotometry (AAS), usually using a graphite furnace as the source. Flame photometry works with higher concentrations of metals. Mercury is usually measured with a cold vapor technique.
Lead. A variety of tests of lead exposure and early effects have been used, including: -ALA in urine, -ALA dehydratase activity in red blood cells, free erythrocyte protophorporin or ZPP in blood (still widely used), urinary coproporphyrin (very limited use), and blood lead (the standard measure).
The measurement of blood lead is considered the best sampling approach for both adults and children (17). Analysis for populations with negligible lead exposure (no occupational exposure in countries without leaded gasoline) requires the sensitivity of graphite furnace AAS; this is currently the most widely used instrument for any population, although anodic stripping voltammetry is also used. In the absence of this capability, excessive lead exposure can be detected by measuring ZPP in a finger-stick blood sample with a portable fluorometer. Alternative methods still available in some laboratories include measuring the urinary excretion of -ALA.
These measurements reflect relatively recent and ongoing exposure. They do not provide information regarding the body burden or effects of long-term accumulation. The challenge test, which uses a dose of a chelating agent (usually EDTA) and monitors urinary lead excretion in the following 24 hr, provides an estimate of the amount of lead that can be mobilized and is used to judge whether chelation will be effective. The chelation test carries a level of risk, including the mobilization of large amounts of lead which can then reach the brain and kidneys.
The past decade has seen the development of in vivo X-ray fluorescence (XRF) of bone as a way of measuring the relative concentration of lead stored in the skeleton. Although increasingly available, the technique is still under development, and there is not adequate concordance among laboratories (18). Two types of machines are used, the K-wave and L-wave sources, which differ in their penetration and apparently in the precision of measurements. Almost all machines in use today employ K-wave XRF.
An example of using blood lead as a marker of exposure and susceptibility is a recent study in Mexico. Romieu et al. (19) found a significant correlation among the lead content of ceramics used to prepare food, the soil lead on children's hands, and the children's blood lead; yet all of the environmental variables explained only 19% of the variance in blood lead. Thus even assuming that there are uncertainties in measurement of the independent variables, this leaves tremendous room for individual variability in the uptake and distribution of inorganic lead in these children. Thus blood lead identifies variation in susceptibility as well as exposure among these children.
Analysis of lead in urine is of relatively little value for quantifying exposure, even in those with organolead exposure.
Many studies have shown that there is a low correlation between lead in blood and lead in air, due in part to the sampling duration; concurrent exposures through water, diet or other jobs; alternative routes of exposure (ingestion); variations in the use of protective equipment; variations in circumstances of exposure, including respiratory rate, exercise, and other microniche characteristics; and individual variation of either a genetic or epigenetic nature (17).
MERCURY. The uptake, toxicokinetics, and end points associated with organic mercurials (particularly methyl mercury) differ greatly from those associated with inorganic mercurials. Inorganic mercury is excreted mainly in urine, organic mercury mainly in feces. Both mercurials can be deposited in hair. Accordingly, inorganic exposure is usually monitored with urinary mercury, although blood mercury testing is also useful and helps distinguish exposure within the past week from that occurring in the past month. Blood mercury is used to assess organic mercury exposure. When dietary exposure (especially fish consumption) is the source of mercury, it is usually not necessary to speciate the mercury, since almost all of the mercury is methyl mercury. There is a strong correlation between either blood or urine mercury and air mercury (20).
Hair mercury is useful for screening populations. The digestion of hair, however, causes difficulties in some laboratories (21). Urinary mercury testing is mainly used for occupational exposure or for residential exposure to metallic (elemental) mercury.
CADMIUM. Cadmium is usually measured in blood or urine by graphite furnace AAS. Lauwerys et al. (7) documented the relationship between exposure and blood levels of cadmium in humans. Many sources recommend the concomitant measurement of ß2-microglobulin, but that marker of renal tubular damage is insensitive and should not be relied on. By the time it becomes elevated, significant kidney damage has occurred. However, a variety of new tubular markers such as retinal binding protein (RBP) and N-acetyl-ß-d-glucosaminidase (NAG) are more sensitive but not more specific (22).
The body burden of cadmium has been measured by neutron activation in vivo (23), but this is a highly specialized technique of only research application.
Pesticides. Pesticides include any substance or mixture that destroys or controls plant or animal pests or vectors of disease. "Pesticide" is a broad term including, in addition to pesticides, a variety of biocides (fungicides, herbicides, acaricides, molluscicides, rodenticides, etc.) Pesticides remain a global occupational health problem (24).
Some pesticides, such as the organophosphates, are short-lived both in the environment and in the body, and it is possible to detect only acute exposure (by measuring a metabolite) or recent exposure (past 3 months) by measuring cholinesterase levels. Chlorinated hydrocarbon pesticides are persistent both in the environment and in the body and can be measured for many months or years after exposure has terminated (25,26).
PESTICIDE EXPOSURE. Absorption resulting from dermal exposure is the most important route of uptake for pesticide-exposed workers, while ingestion is the most important nonoccupational route. Within the body, the pesticide may be eliminated or transferred to a target, unchanged or after metabolism. Organophosphates are typically broken down rapidly, while many organochlorine pesticides are stored in the fat. The actual pesticide exposure (uptake) can be measured by biological monitoring of human tissues and body fluids. Insecticides and their metabolites can be measured after occupational exposures (26-29).
WHERE TO MEASURE VARIOUS PESTICIDES. Different sampling approaches are required for different classes of pesticide. Examples include the measurement of dialkylphosphates in urine after exposure to organophosphorus insecticides (27), of p-nitrophenol after exposure to parathion and methylparathion (28), and of 1-naphthol after exposure to carbaryl (29). Altered liver enzyme activities have been reported among pesticide workers exposed to organophosphorus pesticides alone or in combination with organochlorine or other pesticides (30). Monitoring changes in vitamin K levels is useful for identifying exposure to anticoagulant rodenticides (31).
Adducts to hemoglobin have been detected with several pesticides (32). The advantages of such measurements include the possibility of assessing dose closer to the target, of assessing individual capacity to form electrophiles, and of extrapolating data on toxicity more easily across species. When the mechanism of action of a pesticide is understood, more specific markers can be used (30).
ORGANOCHLORINES. Certain organochlorine (OC) pesticides are highly persistent. Although DDT is metabolized to DDE and DDD, these metabolites persist in the body fat for many years after exposure. They can be detected at much lower levels in serum. The reported levels in fat vary among countries, the highest levels of DDT being found in countries where the compound is still used. Fat biopsies are not suitable for routine screening of pesticide exposure.
Some studies of the concentration of pesticides in human milk are summarized by Anwar (26). The contaminants found most frequently in human milk have been DDT, its main metabolite DDE, hexachlorobenzene, hexachlorocyclohexane, dieldrin, heptachlor epoxide, and the nonpesticide polychlorinated biphenyls (33).
ORGANOPHOSPHATES. Whereas persistent organochlorine pesticides tend to have relatively low human toxicity, the organophosphates (OP) are highly toxic to humans and are responsible for many deaths around the world. These compounds bind various cholinesterase enzymes, thereby interfering with transmission in the nervous system. A widely used biochemical biomarker is cholinesterase depression. It is frequently used in occupational health as a marker of OP exposure and is regularly used clinically as a marker of OP effect. With a specific antibody it is possible to measure the concentration of a particular esterase in plasma or serum. Diagnostic kits to measure specific activities of blood esterases are being developed for use in the field (30). However, it is essential that the workers with potential exposure have two baseline cholinesterase determinations, since cholinesterase activity varies greatly between individuals and within the same individual over time (21,34).
Further markers in pesticide application workers are described by Anwar (26).
Polycyclic Aromatic Hydrocarbons. PAH are usually present in complex mixtures of 3- to 6-ring compounds, of which hundreds of congeners may be present in various proportions. Analysis is usually by gas or liquid chromatography, where selected compounds, such as benzo[a]pyrene (B[a]P) are quantified.
Biological methods have been developed to measure 1-hydroxypyrene (1-HP), a metabolite of pyrene, in urine. 1-Hydroxypyrene is a prevalent species in PAH mixtures. It is excreted in the urine and is a good biomarker of exposure to PAH (35). An advantage is that urine can be stored for at least a year frozen at -20°C without preservative. After hydrolysis and cleanup, the 1-HP is extracted and analyzed on high performance liquid chromatography (HPLC). The result is expressed as µmols/mol of creatinine (35).
Øvrebø et al. (36) found levels up to 13.5 µmol/mol creatinine in coke oven workers compared to 1.54 µmol/mol in the surrounding community and up to only 0.2 µmol/mol in a control area.
Adducts of hemoglobin or albumin are measured after hydrolytic cleavage of PAH from the protein, followed by quantification by mass spectrometry. DNA adducts are measured by the 32P-postlabeling technique and by immunoassay. Neither of these techniques quantifies individual PAHs.
Jongeneelen (37) studied the S9 supernatant fraction of liver samples from 15 kidney transplant donors for variation in PAH metabolism in vitro. 1-Hydroxypyrene was the major metabolite of pyrene and 3-OH-B[a]P was the major metabolite of B[a]P. The Vmax for B[a]P and for purine were highly correlated. They found individual variation in the Vmax and the apparent affinity for the substrates. The Vmax for B[a]P ranged from 0.002 to 0.710, with a geometric mean of 0.012.
Volatile Organics. Ethylene oxide is discussed here as an example of a volatile organic compound. Ethylene oxide is used as sterilant in hospitals. It is also the principle metabolite of ethene, a precursor to polyethylene plastics and other synthetic chemicals. Ethylene oxide can be measured by gas chromatography in air or biological specimens. Ethylene oxide reacts in the body with hemoglobin; N-terminal valine may be released by a modified Edman degradation process and measured by gas chromatography-mass spectrometry. DNA adducts can be measured by a number of techniques [above; Hemminki (11)], including 32P-postlabeling mass spectrometry and liquid chromatography/electrochemical detection.
Ionizing Radiation. Various cytogenetic techniques have been used extensively to study populations exposed to ionizing radiation, most notably the survivors of the bombing of Hiroshima and Nagasaki. Giemsa-banding techniques are labor intensive and provide a resolution of about 1 million base pairs. They give information on the frequency of chromosomal aberrations: breaks, dislocations, deletions, and translocations. The fluorescent in situ hybridization (FISH) technique (38) correlates well with traditional chromosomal aberration analysis and allows a more rapid screening of individual samples for dose reconstruction. This is also an effect marker. Reciprocal translocations are apparently permanent, hence are potentially useful for dose reconstruction even years after the exposure event. The use of hypoxanthine phosphoribosyltransferase (HPRT) mutants and other molecular biologic approaches to detect gene alteration, including radiation specific lesions in DNA (39) is dealt with below.
Sampling Considerations
Toxicokinetics: The Importance of Timing. Many endogenous chemicals and probably also xenobiotics do not maintain a constant level in the body but undergo diurnal variation, sometimes in a deterministic fashion. Therefore it is important to standardize techniques by sampling at one time of day. However, it is also important to understand the time course of excretion, which is not always a uniphasic half-life (negative exponential curve) but may be biphasic or triphasic with an initial rapid elimination followed by much slower elimination. If a substance is mainly excreted within 24 to 48 hr, it is possible only to monitor for acute exposure. Even for substances with a half-life of 6 to 12 months, it may be impossible to reconstruct the exposure that may have occurred in years past.
Conclusions and Recommendations
Biological monitoring of exposure can play an important role in the detection of exposed individuals or groups at an early stage before significant or irreversible adverse effects have occurred. Biological markers of exposure also play a role in quantifying or classifying exposure for epidemiologic studies. Biological monitoring is also useful in identifying variations in susceptibility among members of a population. It is important that any biological monitoring for exposure markers be conducted as part of a comprehensive framework that includes steps for intervening and reducing any hazards that are identified. A screening program should meet the criteria proposed by WHO. The information from such programs should be shared with the stakeholders.
Last Update: June11, 1997