J. Carl Barrett,1 Harri Vainio,2 David Peakall,3 and Bernard D. Goldstein4
Definition of Susceptibility Markers
A biomarker of susceptibility is defined as an indicator or a measure of an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance (1). Biomarkers of susceptibility are concerned with factors in kinetics and dynamics of uptake and metabolism of exogenous chemicals. Thus the concept encompasses enzymes of activation and detoxication, repair enzymes, and changes in target molecules for toxic chemicals. Many of the latter conditions include factors that confer highly increased risks to predisposed individuals (i.e., repair gene defects, "fragile" DNA conditions, etc.). Susceptibility factors occur along a continuum from "near-obligatory" determinants of high risk to contributory low risk factors (such as metabolic polymorphisms). In this document, we focus on those factors that involve a major contribution of gene-environment interaction to become manifest.
Monogenic traits are inherited in a Mendelian fashion. A genetic polymorphism refers to a monogenic variation that occurs with at least two phenotypes with sufficient frequency (>1%) to cause population differences. Genetic variations occur as either germline inheritance or somatic cell mutations. Rare inherited metabolic diseases such as Crigler-Najjar syndrome occur as a germline mutation (132); a well-known example of a somatic cell mutation is hepatocellular carcinoma caused by dietary exposure to AFB1 (91).
Nongenetic factors such as age and sex are obviously important but need further refinement in terms of molecular mechanisms and their interplay with environmental factors before being useful for any intervention. Acquired factors (for example physiological changes, disease-induced changes, induction and inhibition of enzymes by dietary factors, etc.) are also critical but difficult to study because of their individual nature. Such changes may not be permanent and thus may be impractical to take into consideration in any long-term or retrospective study.
Interindividual variability is observed for practically all diseases and toxic responses. It is certainly related in part to different qualitative and quantitative environmental exposures, but differences in susceptibility between individuals in response to the same level of exposure are also common. In most chronic diseases, a long sequence of events leads to a final response, which is seen as a result of an interplay of multiple genetic and other host factors and environmental factors, each contributing to an overall risk of having a manifest response.
Although biomarkers of susceptibility identify those individuals in a population who have a difference in susceptibility to the effects of chemical exposure, only in some circumstances can they predict an individual's risk with any confidence. It is important to not generalize inappropriately from IN VITRO evidence of susceptibility, as has occurred in inferring that individuals with red blood cell glucose 6-phosphate dehydrogenase deficiency are particularly sensitive to inhaled ozone (133).
Objectives of Use of Susceptibility Markers
Interindividual variation occurs as a result of different genetically inherited background modified by dietary and environmental exposure and revealed by genotypic and phenotypic variation. Susceptibility markers are useful because they can partially explain interindividual variation inherent in the general population and thus provide a biological rationale for investigation of inherent vulnerability prior to exposure to environmental hazards.
The objectives of the use of susceptibility markers are the following:
Methodology in Studying Susceptibility-related Genes
The approaches and methods described in this section will focus on studies of polymorphisms in xenobiotic-metabolizing enzymes. However, most of these methods, in particular the molecular techniques, can be used for studying polymorphisms of other susceptibility-related genes, such as DNA repair enzyme genes or disease genes.
In addition to the polymorphisms of susceptibility-related genes, other genetic alterations such as "mini- and microsatellites" are also believed to be important in human carcinogenesis and are involved in individual susceptibility (134).
There are two important parts in studies of genetic polymorphisms. The first is how to identify a new polymorphism and its possible functional significance. The second is how to define the role of a known polymorphism in human susceptibility to environmental toxicity in which the frequency distribution of the polymorphic genotypes or phenotypes should be determined. In both cases the PCR technology has greatly increased our ability to study genetic polymorphisms. In the PCR reaction specific stretches of the DNA can be amplified exponentially by thermostable DNA polymerases with a pair of specific primers. Several methods are widely used in studies of genetic polymorphisms and interindividual variations in chemical metabolism.
Methods for Studying Phenotypic Expression. USE OF PROBE DRUGS IN VIVO. When an appropriate probe drug (with specificity and safety) is available, metabolic polymorphisms can be identified by determining the metabolic ratio, i.e., the ratio of the blood or urinary concentration of the parent drug over its metabolite in different individuals administered the probe drug. Metabolic polymorphism is usually indicated in a population if the frequency distribution of the metabolic ratio is shown to be bimodal or trimodal. For example, a bimodal distribution of debrisoquine 4-hydroxylation (catalyzed by CYP2D6) is observed due to the existence of "poor" and "extensive" metabolizers (135,136). The role of gender-based CYP1A2 variability in susceptibility to bladder cancer has been explored using the caffeine breath test (137).
USE OF PROBE DRUGS IN VITRO. The probe drugs can be used for IN VITRO metabolism studies to look for possible polymorphisms of the metabolizing enzymes, which are usually indicated if large interindividual variations in the activities are observed. Diagnostic probe drugs for individual CYP enzymes have been developed such as coumarin for CYP2A6 and caffeine for CYP1A2 (138).
OTHER METHODS. Polymorphisms of xenobiotic-metabolizing enzymes can be determined at other phenotypic levels. Enzyme protein levels can be measured by immunological methods such as immunoblotting and immunohistochemical analyses. mRNA levels are quantified by different nucleic acid hybridization techniques (Northern and slot blotting, RNase protection, and in situ hybridization). Quantitative RT-PCR (reverse transcription of the mRNA to cDNA followed by PCR amplification) has been developed for detection of mRNA in a small amount of tissue samples. Expression of CYP1A1 mRNA in human lymphocytes and its regulation by tetrachloro-dibenzo-p-dioxin (TCDD) have been successfully determined by this method (139).
Most genotoxic chemicals need to be metabolically activated to exert their effects. Therefore variations in the activities of the xenobiotic metabolizing enzymes by polymorphic changes can influence the genotoxic effects. Biomarkers of effect, such as cytogenetic damage caused by a genotoxic compound, can be used as an indirect measurement to evaluate the metabolic activity of cells from donors with different polymorphic genotypes or phenotypes. The commonly used cytogenetic parameters include CAs, SCEs, and MN (46).
Methods for Studying Genetic Polymorphisms. Identification of novel genetic polymorphisms. Once a metabolic polymorphism (or a polymorphism at other phenotypic expression levels) is demonstrated, various molecular biology techniques including cloning and DNA sequencing can be used to look for possible genetic changes of the enzyme. A successful example is the discovery of CYP2D6 genetic polymorphism (140). After cDNA cloning and DNA sequencing, it was demonstrated that a mutant 2D6 allele is responsible for the majority of "poor metabolizers." Further work established that a mutation at a splicing site caused the production of defective 2D6 mRNA and a total absence of 2D6 protein (141).
The DNA sequence alterations can be screened by SSCP analysis, which detects the mutation-caused mobility shift of the DNA fragments on gel electrophoresis (109), or by DGGE, in which DNA molecules are separated based on differences in their melting temperatures due to the sequence alterations (113). In some cases, the variant alleles are detected by sequence comparison with no knowledge of the related phenotypic polymorphism. Most of the polymorphisms will not have a functional significance. Functional analysis is then critically important in establishing the phenotypic significance of these variant alleles. Catalytic activity of enzymes can be studied by expressing different variant proteins with the cDNA expression systems if the polymorphic changes are localized in the coding region, such as in CYP2A6 polymorphism (142). If the polymorphic loci are in the noncoding region, their effects on the transcriptional regulation can be studied by linking the mutated sequence with a reporter gene, such as in CYP2E1 RsaI polymorphism (143).
Genotyping of populations. It is rather simple to determine an individual's genotype with respect to a specific locus by current molecular biology techniques, when the polymorphic sites of a xenobiotic-metabolizing enzyme gene are clearly identified. If a polymorphic site changes the recognition sequence of a restriction enzyme, or the polymorphic alteration involves a large deletion, the genetic polymorphism can be identified by RFLP analysis in which DNA is subjected to Southern blotting after digestion with appropriate restriction enzymes and hybridized with specific probes.
PCR technology has greatly increased our ability to detect genetic polymorphisms. DNA amplification can be carried out with specific PCR primers for any particular sequence of a polymorphic gene, with small amounts of human tissue or cell samples. The DNA source can be from blood leukocytes, buccal epithelial cells, hair roots, and exfoliated cells such as bladder epithelial cells in the urine. The PCR-amplified DNA sequence containing the polymorphic sites can be analyzed by RFLP with restriction digestion and visualized on a stained gel after electrophoresis. If the genetic polymorphism results in a loss, or in some cases a gain, of a restriction site, the band pattern on the gel will be different from the wild-type samples, such as in CYP2E1 polymorphisms (143). If the PCR primers are designed to be within the missing sequence of a deletion polymorphism, no PCR product will be formed.
Obviously, the RFLP method cannot be used to screen the genetic polymorphisms in which the sequence alterations cause no changes at suitable restriction sites. In this case, genotyping can be carried out by creating a restriction site by mismatch primers (144,145) or by allele-specific PCR (146). If necessary, the results from PCR-RFLP and allele-specific PCR can be confirmed by PCR-direct sequencing (147).
Advantages and Limitations of Genetic Susceptibility Markers
Determinants of the Quality/Appropriateness of the Approaches. ASSESSMENT OF AN EXPOSURE. The reliable characterization and assessment of the level of exposure(s) in the population studied is an important task. However, there are difficulties in measuring exposure because assessment methodologies frequently depend on personal recall (148,149). In many studies published so far on metabolic polymorphisms and susceptibility to environmentally induced diseases, the exposure data are either very scarce or not available. This may contribute in part to the divergent findings reported for potential host factors in individual responses to toxicants. Knowledge of the substrate specificity of the polymorphic enzyme studied is needed before any interpretations concerning its potential effect on individual response to a given exposure can be made.
SELECTION OF THE STUDY GROUP. The cases and controls should have comparable exposures in studies on individual responses to environmental agents. Age and gender matching should also be performed. If these potential confounding factors are not controlled, the effect may be nullified (150). Where matching is not possible, logistic regression can be used to evaluate the individual contributions by multiple factors and to identify confounding factors (151).
SAMPLE COLLECTION. The sample collection is one important aspect of the appropriate assays. The proper treatment and storage of the samples is a prerequisite for obtaining good results. For instance, blood samples for DNA analyses can be stored in a refrigerator for several days and subsequently may be transported on ice or frozen for transportation or for further storage. In contrast, the samples collected for RNA analyses have to be frozen immediately, preferably in liquid nitrogen, and stored at -80°C before use. Blood, which has been the most used source of DNA samples, should not be collected in heparin tubes for DNA extraction. Interference with the PCR assay occurs when compared to the DNA extracted from blood collected in EDTA tubes. Although the DNA-bound heparin can be removed using heparinases, they are far too expensive to be used in studies involving large sample sizes. The inhibition can be decreased by extensive dilution of the samples (152).
In future studies, increased use of alternative sources of the DNA for genotyping studies, such as cells from buccal mucosa (153), is expected. These samples are easily collected by scraping or mouth washing and will thus overcome some of the problems associated with blood sampling. Aside from the samples collected for "prospective" studies, the DNA can also be from stored pathological tissue sections, which provide great advantage for retrospective studies but have additional problems due to the small amount of DNA obtained and due to possible DNA degradation.
NUMBER OF MARKERS STUDIED SIMULTANEOUSLY. Given the number and variability in expression of the xenobiotic metabolizing enzymes, assessment of a single polymorphic phenotype or genotype cannot be expected to be sufficient for evaluating individual susceptibility to environmental agents. The ultimate goal should thus be to concurrently assess individual phenotypes or genotypes for all the metabolic genes relevant for a given exposure (154). This may raise an urgent need for software to aid in the interpretation of the overall risk contributed by several different host factors.
Phenotyping versus Genotyping. Phenotyping has been widely offered as a more appropriate method compared to the new DNA-based techniques in studies on individual metabolic capacity. Determination of the actual phenotype of an individual, although more time consuming, is justified when no genotyping methods are available or when the correlation between the genotype and phenotype is very poor. However, in most cases the correlation is fairly good. Moreover, phenotyping is easily affected by confounders such as food or drug intake prior to testing, which do not affect genotyping analyses.
Protein and mRNA levels, although they give some indication of the expression level of a given metabolic enzyme, do not necessarily reflect the metabolic activity (155). On the other hand, the power of PCR to detect only a few copies of the target sequence makes it particularly vulnerable to errors caused by contaminating DNA. Both approaches will be discussed in more detail below.
PHENOTYPING ASSAYS. Use of probe drugs in vivo. The identification of polymorphic traits has repeatedly been by in vivo probe and involves the collection of blood or urine usually over a specific period. The use of in vivo probes gives a picture of the whole body capacity for a specific enzyme to metabolize a given substance. Probes are not yet available for all known polymorphically expressed enzymes. In many cases the probe is metabolized by other enzymes as well, which may complicate interpretation. It is also important to have a probe that is both specific and safe for use in humans.
Use of probe drugs in vitro. Because of drug/chemical toxicity, the use of an in vitro probe may be the only safe alternative to perform phenotyping. A simple in vitro assay can be developed, if the polymorphic enzyme is expressed in blood cells. The use of other tissues, such as liver biopsies, may provide an opportunity to measure the expression level of the enzyme as well. Concerns for patient safety limit the availability of these tissues for study.
GENOTYPING ASSAYS. DGGE and SSCP. DGGE and SSCP methods are widely used for detection of alterations in oncogenes and tumor suppressor genes, but they could also be used to track down sequence variations in the genes coding for xenobiotic-metabolizing enzymes. Recently they have also been used as a genotyping methodology (156), but at present the RFLP- and PCR-based analyses may be more useful in these studies.
RFLP assays. The early RFLP-based genotyping studies by Southern blotting were time consuming and labor intensive and usually incorporated the less desirable use of radioactive probes. Southern blotting has been mainly replaced by PCR-based analyses, which have greatly facilitated studies of individual susceptibility. This is best exemplified by the rapid expansion of the studies on GSTM1 polymorphism after elucidation of PCR-based methods (157) for detection of the gene deficiency (158). Although several PCR-based methods are available for detection of homozygous gene deletions (157,159), amplifications (160) or heterozygous deletions (158) of the genes are still detectable only by the Southern blotting analyses.
PCR-based methods. As mentioned above, the PCR methods have mainly replaced the genomic DNA methods in genotyping studies. The RT-PCR is also used to quantitate tissue-specific expression of the metabolic genes (161). While rare mRNA transcripts can be detected by the RT-PCR approach, determination of relative or absolute copy number may be more problematic (162). Consequently, there is debate about whether RT-PCR is suitable for these studies.
One advantage of PCR methods is the requirement of only nanogram quantities of the DNA or RNA template. The need for starting material can be further diminished by employing a multiplex PCR that gives the genotype of several metabolic genes from the same sample. However, optimization of the multiplex PCRs may be time consuming and the number of assays one can add to one reaction is relatively limited.
The evident disadvantage in PCR methods is their extreme sensitivity to contaminating DNA. Consequently, special emphasis has to be put on controlling the contamination (163). This can be achieved by minimizing the possibilities for contamination to occur, by UV radiating the reaction mixture before adding template DNA, and by using carefully selected positive and negative internal controls in all amplifications. An additional limitation, specific for RT-PCR, is that measurement of mRNA levels does not necessarily reflect metabolic activity.
Sequencing. Sequencing is the only method that provides the actual DNA sequence of interest. Consequently, it is still used mainly to identify novel allelic variants and to confirm the applicability of new genotyping methods. As the methodology improves, sequencing may be more commonly used to determine genetic markers (164).
Ethical considerations. Ethical review of all studies regarding human risk is appropriate. Specific issues arise with the use of archival samples for which donor permission may not have been obtained for the current research investigation. Current practice is variable regarding the need for consent. A second issue is the type and quantity of information derived from the project that should be provided to the subject [see below and Soskolne (15)].
Conclusions and Recommendations
There are a large number of different methods to measure susceptibility factors, ranging from the activity of an enzyme to the detection of mutant alleles of a gene associated with a modified phenotypic trait. In an era of molecular biology, exceedingly sensitive and specific methods to detect alleles and expression of genes of interest have taken center stage; but one has to keep in mind that it is actually the phenotype that is of importance for the final response to the hazardous insult.
One serious problem in phenotyping studies is lack of knowledge of the levels of enzymes in the target cells and organs relevant to the whole body metabolism and the correlation of genotype with in vivo phenotype. In humans, practical considerations dictate to a large extent that one has to resort to surrogate tissues.
Genotyping has some advantages, such as unequivocal identification and lack of interference with confounding factors; but a modified sequence within a gene (polymorphism) must be demonstrated to generate a different phenotype.
in vivo phenotyping has the advantage in some cases of assigning a value with one assay that might require determination of multiple genotyping assays when several alleles are present in the population. in vivo methods to measure phenotype may lead to misclassification of different genotypes, which in the case of rare alleles can have a large effect on the frequency.
To fully exploit the genetics of xenobiotic metabolizing enzymes as risk assessment tools, much more information on the structure/function relationships and regulation of these enzymes correlated to the in vivo phenotype must be elucidated.
On the basis of the current knowledge of the field and of the above conclusions, the following recommendations are made:
a) Phenotyping should be continued along with genotyping, until we gain experience with large numbers to enable interpretation of more complex relationships.
b) The combined impact of all relevant genes for a given exposure (as far as they are known) needs to be assessed through population-based studies using multiple markers. New insights into the physiologic function of genes encoding xenobiotic metabolizing enzymes will be revealed by studies of transgenic "knockout" or "insert" models.
c) Better kinetic characterization of enzyme substrate and inhibitor specificity must be determined through studies of human tissues, as well as through detailed metabolic studies of whole organisms. The future availability of crystal structure of drug metabolizing enzymes in combination with computational chemistry will provide better information on structure/function predictions.
d) A greater understanding of the regulation of enzyme expression by environmental agents is needed.
e) The value of intermediate markers of exposure such as DNA adducts, should be studied and related to other end points of disease, e.g., cancer, by cohort studies of individuals who have had unintentional exposure.
f) Widely available software for interpretation of the overall risk contributed by several different host factors is needed.
g) Better methods of DNA sample collection should be devised to facilitate storage and transport and to reduce costs because of the need to perform complex DNA investigations at remote laboratories.
h) A future approach will be to use information obtained from analysis of populations of interethnic groups, in an attempt to modify risk by reducing exposure to potentially hazardous environmental and dietary factors linked to disease. The effects of these actions must be evaluated to validate proposed interventions.
i) All of these areas need further exploration before we will be able to assign individual specific prevention strategies.
Last Update: June11, 1997