J. Carl Barrett,1 Harri Vainio,2 David Peakall,3 and Bernard D. Goldstein4
Definitions
The International Programme on Chemical Safety has defined a biomarker of effect as "A measurable biochemical, physiological, behavioral or other alteration within an organism that, depending upon the magnitude, can be recognized as associated with an established or possible health impairment or disease" (1).
This is a very broad definition. Such biomarkers of effect can be elicited as a result of interaction of the organism with a host of different environmental factors (including chemical, physical, and biologic agents); this definition encompasses biomarkers of effect at the level of the whole organism, at the level of organ function, at the level of tissue and individual cells, and at the subcellular level. For example, the results of neuropsychologic tests may be considered biomarkers of behavioral effects of the organism which may be induced by solvent exposures. Spirometry results may be considered biomarkers of physiological effects on the respiratory system which may be induced by fibrogenic dust exposures. Sperm counts may be considered biomarkers of effect on reproductive cells which may be induced by exposure to synthetic estrogenic compounds. There are many other similar examples of biomarkers at these levels in almost all systems of the body, including biomarkers of hematological toxicity, nephrotoxicity, hepatotoxicity, immunotoxicity, neurotoxicity, pulmonary toxicity, and reproductive and developmental toxicity (1). These will not be considered further here, but rather this review will focus on biomarkers of effect at the subcellular level, particularly at the chromosomal and molecular level, which are particularly useful in assessing susceptibility. These include biomarkers at the genomic level, such as cytogenetic alterations and gene mutations, and biomarkers of gene expression, such as messenger RNA and proteins. Some assays of protein function, such as enzymatic activity, could be included in this category but could also be considered as biomarkers of exposure (e.g., cholinesterase activity in response to OP exposure), and thus they will not be included here. Biomarkers at the genomic level or at the level of gene expression have been most strongly associated with the risk for cancer, although in selected instances they may also be associated with other disease end points. Therefore, in general, we will be concerned here with subcellular biomarkers of effect that are believed to be associated with cancer. We will examine the methodologies employed with reference to the objectives of their use, their potential uses, and their advantages and limitations.
Because in many instances, these biomarkers of effect are believed to represent events in a causal pathway to disease, their occurrence may be viewed as indicative of an acquired susceptibility for that disease. This is in distinction to the biomarkers of susceptibility per se, which in many cases represent indicators of inherited susceptibility to disease due to their influence on steps in the causal pathway. Biological markers of effect, as indicators of acquired susceptibility for disease, have the theoretical potential to exhibit predictive value in identifying individuals who could develop disease at some point in the future or in determining the likely disease progression in terms of severity and prognosis once disease has occurred. In addition, biomarkers of effect, because they occur quite far along the pathway between exposure and disease outcome, essentially provide an effective integration of the influence of biomarkers of susceptibility for all preceding steps in the causal pathway and are thus a summary marker for susceptibility to that point.
Cytogenetic Markers
In humans, cytogenetic alterations are most often analyzed microscopically from peripheral lymphocytes after they have been stimulated to divide in culture by a mitogen (40). Lymphocytes are used as a surrogate for the actual target tissues of genotoxic carcinogens. Some cytogenetic end points (micronuclei, numerical aberrations) can also be studied in interphase cells from target tissues or proximate target tissues, such as exfoliated cells from buccal, nasal, urothelial, bronchial or esophageal epithelium (41,42). Specimens can also be obtained from hair bulbs. In special cases, for instance, in connection with accidental exposures, dividing cells from bone marrow may be available. Furthermore, cytogenetic biomarkers can be studied from samples collected for prenatal diagnosis (amniocytes or chorionic villus cells). Effects on germ cells can be examined from semen specimens (43). Fresh samples are required in most cases, but preservation by freezing or fixation is possible for several of the cytogenetic techniques. The analysis of cytogenetic end points from such samples is used to show exposure to genotoxic carcinogens and may have value in the identification of groups of people at increased risk of cancer (44,45). Cells from different individuals can be challenged with genotoxic agents in vitro, which in some cases may enable the recognition of sensitive individuals (46,47).
Chromosome Aberrations. Chromosome aberrations (CAs) are structural alterations, breaks and rearrangements, in chromosomes, usually observed in metaphase-blocked cells using conventional microscopy (40). Chromosome-type rearrangements, such as translocations and dicentrics inspected in biological dosimetry of radiation, can also be analyzed using chromosome painting based on FISH with chromosome-specific DNA probe libraries (48). This approach has enabled the recognition of new types of complex chromosome rearrangements that have not been detectable with conventional cytogenetic techniques. Recently, a simplified FISH method to detect chromosome breakage and alterations of chromosome number, using tandem DNA probes specific for a region in chromosome number 1, was reported (49). FISH has also been applied to study numerical chromosome aberrations of specific chromosomes (50). Polymerase chain reaction (PCR)-based methods for the analysis of lymphocyte-specific illegitimate chromosome recombination involving human immunoglobulin or immune receptor loci and considered to reflect genetic instability have also been described (51,52). In two recent, independent reports, increased rates of CA in peripheral lymphocytes were shown to be associated with later development of cancer (44,45). Thus, the analysis of CAs is presently regarded as the cytogenetic method of choice in studies of human exposure to genotoxic carcinogens. in vivo inducers of CAs in humans include, among others, ionizing radiation, alkylating cytostatics, tobacco smoking, benzene, and styrene. Besides smoking, factors such as age, gender, and diagnostic and therapeutic X-rays are usually taken into account as possible confounders. In vitro challenging of lymphocytes from healthy individuals with genotoxins has revealed individual differences in CA response to some genotoxic agents (53,54). The reasons for such differences are unknown, except for 1,2:3,4-diepoxybutane, where an association probably exists with the deficiency of glutathione S-transferase T1 (GSTT1) (46,47,55). Among butadiene-exposed chemical workers, GSTT1 null individuals also had an elevated rate of chromosome aberrations in peripheral lymphocytes in vivo (56). On the other hand, the lack of GSTM1 has been associated with elevated CA frequencies in smokers and in bus drivers (57).
Sister Chromatid Exchanges. Sister chromatid exchanges (SCE) represent symmetrical exchanges of DNA segments between the sister chromatids of a duplicated metaphase chromosome (58). In lymphocytes of humans, tobacco smoking, alkylating cytostatics, and ethylene oxide are well documented SCE inducers. Factors to be controlled in the analysis of SCEs include age, gender, and life style. Individuals with enhanced in vitro SCE response to some genotoxins have been described and, in some cases, this has been associated with certain metabolic genotypes or phenotypes (46,47,50,59-66). For example, individuals sensitive to in vitro SCE induction by 1,2:3,4-diepoxybutane were recently found to be deficient of GSTT1 (46,47,62,64,66). Such people also show elevated baseline frequencies of SCEs in their peripheral lymphocytes (47,62,67). SCEs were not, however, elevated in GSTT1 null individuals exposed to butadiene (62). Increased SCE rates have also been indicated in relation to the GSTM1 null genotype and smoking (68) and deficiency of the low Km aldehyde dehydrogenase and alcohol consumption (69).
Micronuclei. Micronuclei (MN) are small additional nuclei observable in interphase cells. Micronucleus induction can be triggered by either clastogens or agents that influence the mitotic apparatus, such as spindle poisons (70,71). Micronuclei can also be scored in exfoliated cells of buccal, nasal, or urothelial mucosa (41). The presence of whole chromosomes in MN can be checked by identifying centromeric DNA sequences (using FISH) or kinetochore proteins in the MN (72,73). in vivo, increased MN frequencies in lymphocytes have been associated with exposure to ionizing radiation, aging, and gender (74). In women, the influence of age on the frequency of MN in lymphocytes has been related to an increased inclusion of the X-chromosome in MN (75,76), while in men the Y-chromosome appears to be responsible for a part of this effect (77). In buccal or nasal mucosa, MN induction has clearly been shown for, e.g., various ethnic chewing habits (3) and exposure to formaldehyde (78). The MN analysis has given negative results in several studies in which another biomarker of genotoxic exposure has been positive. As MN can originate from chromosomal fragments and whole chromosomes, the number of cells presently examined may not be enough to reveal the relatively small effects usually expected in exposed humans. The identification of the centromeric contents of MN and automated analysis may improve the specificity and sensitivity of the MN assay of lymphocytes.
General Considerations. At the population level, structural CA appears to have predictive value for later development of cancer. As similar associations have not been detected for other cytogenetic endpoints, the analysis of CA appears to be, at present, the cytogenetic method of choice in studies of cytogenetic alterations in lymphocytes. However, the sensitivity of the assay does not allow evaluation of cancer risk based on individual values. Analysis of an in vitro cytogenetic response of human cells to genotoxins may, in some cases, be used to identify susceptible individuals, especially when genetic polymorphisms are taken into account. In biological dosimetry of radiation exposure, the analysis of chromosome-type aberrations has been used as the basis for individual dose estimates. Chromosome painting by FISH is becoming the new tool in the biological dosimetry of radiation; the applicability of techniques based on FISH and PCR for the identification of specific chromosome alterations for studies of chemical genotoxins should be evaluated. The MN assay combined with FISH analysis of centromeres in MN may offer an easier technique to score CA, especially in target tissues or proximate target tissue. Because each biomarker depicts a different phenomenon, the use of various exposure and effect biomarkers together is recommended in risk assessment. It is also a useful approach for detecting patterns of human susceptibility. The combination of cytogenetic parameters and information on metabolic genotypes or phenotypes is expected to increase the sensitivity of the cytogenetic assays and allow better understanding of the biological significance of genetic polymorphisms (43). As some polymorphisms have been shown to influence basic or induced levels of cytogenetic damage, information on the genotypes or phenotypes of the individuals studied is becoming important when these end points are used as biomarkers. Automation and analysis techniques utilizing FISH offer faster and easier approaches to detect cytogenetic alterations.
Markers of Gene Mutations
Somatic Gene Mutations in Surrogate Tissues. The detection of mutations in the HPRT gene has been used in experimental mutagenicity studies of mammalian cells, and it is also the most extensively employed assay for human gene somatic mutations in vivo. In humans, HPRT mutations are examined in lymphocytes, and the standard assay involves T-lymphocyte cloning for phenotypic selection of 6-thioguanine-resistant mutant cells. With the development of PCR-based techniques, further molecular characterization of the mutations present in the T-cell clones has also become possible. Such studies have shown, for instance, a clear difference in mutation spectrum between "spontaneous" mutations occurring early in human development and mutations acquired later in life (79). Studies of human lymphocytes in vitro are also considered useful, as they provide information on HPRT mutation spectra obtained after exposure to specific carcinogens in controlled conditions. Several studies have been carried out on HPRT-mutant lymphocytes in human populations exposed to various genotoxic agents, as reviewed by Cole and Skopek (80) and briefly described by Hemminki (11). Only a few studies have thus far tried to correlate HPRT mutations with other measures of exposure and susceptibility. In a study of occupational exposure in a foundry, the frequencies of HPRT-mutant T-lymphocytes correlated with the level of aromatic DNA adducts (81,82). In a recent study on garage workers, a correlation was found between aromatic DNA adducts and mutation frequency at the individual level; genotypes of two xenobiotic-metabolizing genes (GSTM1 and NAT2), alone or combined, did not influence HPRT mutation frequency (83).
The glycophorin A (GPA) assay is another in vivo method for the detection of somatic mutations to study the potential effects of exposure to chemical and physical mutagens (84,85). The assay is based on the autosomal GPA locus that encodes the cell surface sialoglycoprotein expressed in the erythrocytic lineage and responsible for the M,N blood group. It uses immunolabeling and flow cytometry to enumerate, in peripheral blood samples of M/N heterozygotes, erythrocyte variants reflecting mutations in the GPA locus. Most of the variants are considered to derive from mutations that occurred in bone marrow stem cells and are therefore permanent, depicting lifetime accumulation of mutations. The GPA assay has been automated and is thus easy, fast, and cheap to run. The main limitations are that only one half of the human population (M/N heterozygotes) can be studied and an expensive flow cytometer is required. As erythrocytes have no nuclei, the variant cells cannot be clonally expanded and the mutations cannot be characterized at the molecular level.
Both the HPRT and GPA mutation assays show a small number of people with exceptionally high mutant frequencies in healthy subjects (86). While at least part of such high frequencies are due to clonal expansion of a few original mutations, some of them might indicate individuals of enhanced susceptibility. In a study of GPA mutations in reinforced plastics workers, the high frequency individuals were primarily smokers or ex-smokers (87). Recently it was shown that in smokers significantly elevated NN variant frequency was associated with GSTT1 null genotype (88).
Gene Mutations in Target Tissue. Knowledge on gene mutations from other tissue than peripheral blood and lymphocytes is sparse. This relates to problems in availability of samples and difficulties in developing methods suitable for detecting mutations occurring at a low frequency. Growing cells under in vitro conditions circumvents the problem with lymphocytes. Somatic mutations have been detected in genes related to human disease and particularly in cancer-related genes (protooncogenes and tumor suppressor genes). In most cases somatic mutations in disease-related genes do not give rise to any functional change in the cell, which would allow its isolation or expansion in vitro. In the case of cancer tissue, malignant growth involves clonal expansion of cells, which allows detection of mutations by the methods presently available. The methods appear to work best and most reproducibly on fresh frozen tissue, but in many instances only preserved tissue is available.
Mutations in oncogenes and tumor suppressor genes are most common in many types of human cancer. In the context of external exposure to carcinogens, the ras genes have been shown to be mutationally activated in a number of environmental cancers in humans (89), and in a carcinogen-specific manner in animal studies (90). Since the p53 tumor suppressor gene is among the most frequently altered genes in human cancer, molecular analysis of its mutational spectra has been used as a clue to cancer etiology and mechanisms of carcinogenesis (91,92). In the p53 gene, DNA sequences corresponding to the highly conserved domains of the protein have been identified as a "hot spot" region for mutations. A majority of mutations identified in these genes in human cancers are missense mutations, making them suitable targets for such analysis. The exposure-related nature of p53 mutations has been proposed in many studies on various forms of human cancers (91-94). Well-known examples of this are nonmelanoma skin cancer associated with exposure to sunlight (UVB) as well as hepatocellular carcinoma related to dietary exposure to the mycotoxin aflatoxin B1 (AFB1) (92). With the highly sensitive mutation detection assays it has been possible to investigate whether the suggested causative carcinogens (AFB1, UVB) induce similar kinds of mutations in human cells in vitro. In the case of AFB1, the results were in agreement with the etiological role of AFB1, although other types of mutations were also seen (95). In nonmalignant human liver tissue, the frequency of the specific AGG to AGT mutation at codon 249 was found to parallel the level of AFB1 exposure in the geographical areas where the patients lived (96). For UVB exposure, the experimental work on human skin fibroblasts did not find the tandem double CC to TT mutations seen frequently in skin cancer (97). A relationship between certain types of ras mutations or p53 mutations and occupational exposure to agents like solvents, vinyl chloride monomer, some bladder carcinogens, and radon has been suggested (98-100). In addition, smoking has been associated with increased prevalence of p53 mutations in some tumors, and certain types of base substitutions have been linked with exposure to tobacco smoke (101,102). Recently, two studies have reported a higher frequency of p53 mutations in lung cancer patients with the at risk genotype of GSTM1 gene (103-105).
Studies on prognostic significance of p53 protein overexpression (determined immunohistochemically) or mutation have suggested that accumulation of the mutant protein may predict poor survival in many cancer types (92).
Methodological Considerations. An array of methods has been used for detection of point mutations in unknown positions of the target gene. Most of them initially used radioactive labeling but alternative techniques have been developed. One of the first techniques available was the RNase mismatch cleavage analysis method (106). Methods relying on chemical modification of mismatched nucleotides, by either carbodiimide or hydroxylamine and osmium tetroxide (chemical cleavage of mismatch), have been developed; they use mutation detection mainly in genes of inherited diseases (107). Two assays used widely in mutation detection in association with inherited diseases, genetic polymorphisms, and also for somatic mutations in tumor tissue are single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). SSCP assay is based on conformational changes in DNA due to sequence alterations (108,109). SSCP has been successfully used for detection of DNA alterations of many cancer-related genes in human cancer (ras, p53, RB, for example). It is estimated to yield over 90% efficiency in detecting single base substitutions in sequences of 300 bp or less in length (109,110). It is also well suited for detection of polymorphic alleles of various genes (109). DGGE separates DNA molecules based on their sequence-determined ability to melt (separate partially) in longer fragments (low- and high-temperature melting domains) (111). DGGE separates physically wild-type DNA from mutant molecules, and under appropriate conditions all single-base substitutions, frameshifts, and deletions less than about 10 bp can be resolved from the wild-type DNA (112). DGGE, or its application called constant gradient gel electrophoresis (CDGE) (113), has been used to study both germline and somatic mutations in many human genes. These include mutations in cancer-related genes, human ras and p53 genes, as well as chemically induced mutations of HPRT gene in human lymphocytes in vitro (112). More recently, a capillary electrophoresis technique has been applied for CDGE (114).
For detection of known mutations in certain sites of a gene, two much-applied methods include allele-specific oligonucleotide hybridization and genotypic mutation analysis by restriction fragment length polymorphism (RFLP)/PCR method. Allele-specific oligonucleotide hybridization uses labeled probes which are hybridized to PCR-amplified genomic DNA. It has especially been used in studies on ras gene mutations in various human cancers (89,115). More recently, many assays relying on recognition of a certain restriction site which either is present in the wild-type sequence or results from a mutation have been developed. Of these, one of the most sensitive and perhaps most promising for molecular toxicology purposes is the RFLP/PCR assay for detection of codon 12 mutations in the human H-ras gene and codon 247 to 250 mutations in the human p53 tumor suppressor gene (116,117). The method is very sensitive and suitable for detection of very low frequency mutations, such as those in premalignant or in normal cells after chemical treatment in vitro. They are, however, limited to a single base substitution at a certain codon.
DNA sequencing based on dideoxy-mediated chain termination reaction (118) can be performed also after in vitro amplification of the DNA template by PCR. Direct sequencing of PCR amplification products is thus one method of choice for mutation analysis; it is perhaps more labor intensive when performed manually but has been automated (119). Methods using solid phase support in sequencing are being used increasingly (120). Sequencing has of course the advantage of giving precise information on the sequence alterations unlike some other methods (SSCP, DGGE). For targeted use, an application called solid-phase minisequencing has been developed (121). However, direct sequencing of genomic DNA of poor quality may be problematic (fixed and paraffin-embedded tissue samples) and other methods are better suited in such instances.
Markers of Gene Expression
mRNA Expression. Markers of gene expression include assays for detection of mRNA or for detection of proteins. Many of the assays for mRNA employ methodologies similar to those used for DNA analysis noted above. In most cases, the assays have been used for the examination of diseased tissue (e.g., cancer tissue) in the target organ. This poses problems of accessibility. In addition, mRNA has very limited stability, and, therefore, tissue samples must be obtained fresh and processed quite rapidly so that the mRNA is preserved.
With the appropriate oligonucleotide probes, the presence of mRNA in tissue can be detected by in situ hybridization. For example, differences in mRNA for oncogenes may be detectable between cancer tissue and adjacent normal tissue (122). Other disease end points may also be relevant. For example, in situ hybridization can detect increases in mRNA for growth factors, such as platelet-derived growth factor and transforming growth factor-ß, in relation to the stimulation of cell growth and the development of fibrosis resulting from asbestos (123). Alternately, tissue can be analyzed for mRNA expression by isolation of message and amplification by quantitative PCR followed by Northern blot analysis (analogous to the separation techniques of Southern blot analysis for DNA by gel electrophoresis) with appropriate oligonucleotide probes. Quantitative PCR is considerably more difficult to perform reliably compared to qualitative PCR, adding further complexity to this approach.
None of the approaches to mRNA expression have been well developed for routine application. Thus, among other problems, there is generally little standardization of assay techniques, little definition of normal values or values of pathophysiologic significance, and little examination of reproducibility, sensitivity, specificity and predictive value of the tests in terms of the occurrence of or prognosis for disease state (i.e., indication of susceptibility for development or severity of disease). Given these limitations and the other difficulties noted above, it is probably unrealistic to expect that gene expression by mRNA analysis will be very useful as a biomarker of effect. At any rate, the true index of gene expression from the point of view of effect in the cell is at the protein level, not the mRNA level, and it is not even clear that the two always correlate. As noted below, detection of protein expression has the potential to avoid some of the problems inherent in mRNA detection and is thus the more attractive candidate for a biomarker of gene expression for many reasons.
Protein Expression. Methodologies for examining gene expression by the detection of the encoded proteins all depend on the development of antibodies (either polyclonal or monoclonal) that are theoretically specific for the identification of the protein of interest. In many cases, however, the specificity is only theoretical because there is cross reactivity of the antibody with similar epitopes in other, unrelated proteins, raising the possibility of false-positive tests. Conversely, sensitivity of the antibody detection (related to many factors, including avidity and affinity for the epitope, accessibility and stability of the epitope, and the sensitivity of the secondary reaction system used to identify the occurrence of the antigen-antibody interaction) may be insufficient to detect the antigen, leading to false-negative tests. Monoclonal antibodies that are produced from a single cell that is clonally expanded and producing an antibody for only a single epitope are more easily standardized. Polyclonal antibodies can be variable and are thus more difficult to standardize but may have the virtue of reacting with several different epitopes on the protein of interest, increasing the likelihood of detection. Modifications of antibody applications that can improve the results include confirmation of the molecular weight of the identified protein as consistent with what is expected and the use of more than one antibody probe to demonstrate that the protein identified reacts with all expected positive antibodies; it does not react with negative antibodies.
For protein identification of gene expression, antibodies can be used in tissue analysis or in assays of surrogate sites such as extracellular fluids. As with mRNA analysis, tissue analysis for protein expression usually relies on diseased tissue (e.g., cancer tissue) in the target organ, although in some cases the same analysis can be applied to exfoliated cells, for example, from the urothelium or the bronchial epithelium (124). Tissue analysis can be either in situ or in aggregate. in situ analysis relies on the application of the appropriate antibody in immunohistochemistry either to fresh-frozen tissue sections or to fixed, paraffin-embedded tissue sections. The latter approach raises issues of the stability of the antigen with fixation and with storage over time, which can vary with the different proteins. Immunohistochemistry allows for the localization of proteins of interest within cells (e.g., the question of whether the protein is identified at the subcellular site one would expect) as well as within the histology of the specimen (e.g., if the protein is identified in cancer tissue, in precursor lesions, in apparently normal adjacent tissue). Aggregate analysis of tissue can be accomplished with fresh tissue specimens by lysis of the cells and analysis of the appropriate cellular fraction of the lysate (125,126). Analysis of cell lysate can be performed with antibodies applied in Western blotting (separation of the proteins by gel electrophoresis followed by antibody probing), which has the advantage of allowing molecular weight determination as a confirmatory indicator but which can be laborious, difficult, and hard to standardize. Alternately, analysis of cell lysate can be performed with antibodies incorporated into an antigen-competition or an immunosorbent assay, such as radioimmunoasays or enzyme-linked immunosorbent assays (ELISA), which are generally easier, more rapid, and more easily standardized. Stepwise application of assays can be employed, for example, in which an ELISA may be used to screen samples and Western blotting may be used to confirm positive results.
Protein identification in extracellular fluids can be similarly accomplished with antibodies employed in Western blotting or ELISA or related assays. This approach is obviously more accessible and convenient than relying on tissue, but it raises issues such as whether extracellular fluid protein levels accurately reflect cellular protein levels. Identification of certain proteins in extracellular fluids, such as tumor-associated antigens, has been well standardized in convenient assays and widely applied. Prostate-specific antigen (PSA) is a good example (127,128). Even in these cases, however, many important issues remain unresolved. Tumor-associated antigens like PSA may be useful monitoring the course of disease once identified and treated, but the relationship to the pathophysiology of the disease process remains unclear and thus its utility in predicting future occurrence of disease that will be clinically significant in an individual is unproven. Other proteins that represent expression of genes in the presumed causal pathway for disease, such as growth factors and oncoproteins, can also be analyzed with antibody-based techniques in extracellular fluids, as described in depth by Brandt-Rauf (129). Unfortunately, these tests are not as well developed as the others mentioned. In general, they are not standardized; normal values and values of pathophysiologic significance are not well defined; confounders have not been identified; and sensitivity, specificity, and predictive value as indicators of susceptibility for disease occurrence need to be more closely examined. Thus, they are clearly not ready for routine use at this time. As with all biomarkers of gene expression, additional research will be necessary to more clearly define their advantages and limitations and to determine their potential uses.
Conclusions and Recommendations
Considerations for Developing Countries. Little work has been done to monitor the level of exposures and their effects on human populations in developing countries (130). Although trials have been done for cytogenetic monitoring of populations at risk (42,131), more effort should be made to improve the information concerning human exposure possibilities and the identification of exposure-related diseases.
Simple cytogenetic techniques (such as CAs, SCEs and MN) and simple gene expression techniques (ELISA for protein levels) can be applied for monitoring in developing countries. More advanced molecular biomarkers (e.g., based on PCR and gene sequencing) may be considered only if the appropriate resources are available.
General Considerations. Most of the biological markers of effect described here are still experimental tools whose utility remains to be established. For these biomarkers, issues of assay validation, QA/QC, and convenience and cost need to be addressed. As noted above within the context of susceptibility, one objective of their use would be the identification of individuals or groups who, by virtue of the acquired susceptibility to the effect, are likely to develop disease. Some examples, such as the evidence that increased rates of chromosomal aberrations are associated with later development of cancer, suggest that this may be possible, but in most cases of biomarkers of effect, the predictive value remains unproven.
Even if predictive value is ultimately established, problems will remain. The utility of establishing such susceptibility without any way to alter it is of questionable value. In some cases, the effects detected may be irreversible, so effective secondary prevention would be one important option. In other cases, the effects detected may be reversible (as with the reversion of SCE rates following removal of exposure to cytostatic drugs), so that primary prevention may be effective. At any rate, primary prevention through the reduction or elimination of the exposure producing the effect is always good policy, and secondary preventive interventions or the use of biomarkers to eliminate individuals from ongoing exposure situations are not a substitute for primary preventive efforts.
Other important social, legal, and ethical implications also derive from these aspects of biomarkers of effect. For example, the knowledge of truly predictive biomarkers of effect could obviously be misused to detrimentally affect the employability and insurability of the individual concerned, compounding the suffering induced by the psychological burden of the knowledge. Furthermore, such knowledge having a potentially adverse psychological effect raises the issue of whether the detection of a biomarker of effect is itself an impairment and perhaps a disability, even without other demonstrable clinical effects. Could such an impairment/disability be compensable under tort or workers' compensation laws? Would such an individual be entitled to special considerations under laws for protecting the disabled in employment and other societal circumstances? Prior to the general use of biomarkers of effect, such questions will have to be addressed. Some general ethical aspects of these issues are considered in more detail elsewhere in this volume.
Other potential uses of biomarkers of effect are in monitoring of disease progression and prognosis, and as adjuncts to other biomarkers in providing refinements of epidemiology and risk assessments. At the very least, biomarkers of effect, as well as other biomarkers, offer the opportunity to provide scientific confirmation of proposed exposure-disease pathways in vivo in human populations. Biomarkers of effect may be particularly useful for demonstrating the biologic influence of preceding susceptibility factors (e.g., genetic polymorphisms of xenobiotic-metabolizing enzymes). It should be noted once again, however, that at this time these biomarkers of effect should only be employed in the context of carefully designed studies with all due attention to the rights and concerns of the human subjects involved. Nevertheless, we believe that research on biomarkers of effect is worth pursuing because of the potential benefits for disease prevention.
Last Update: June11, 1997