Jun-Yan Hong and Chung S. Yang
College of Pharmacy, Rutgers University, Piscataway, New Jersey
Key words: cytochrome P450, metabolism, genetic polymorphisms, biomarker, susceptibility, cancer risk, environmental toxicity
This paper was prepared as background for the Workshop on Susceptibility to Environmental Hazards convened by the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC) held 17-22 March 1996 in Espoo, Finland. Manuscript received at EHP 5 November 1996; accepted 18 November 1996.
We thank T.J. Smith for helpful discussion and Y. Wang and J. Hong for assistance in the manuscript preparation.
Address correspondence to Dr. J-Y. Hong, Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08854. Telephone: (908) 445-5358. Fax: (908) 445-0687. E-mail: jyhong@rci.rutgers.edu
Abbreviations used: cDNA, complementary DNA; CYP, cytochrome P450; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcription coupled with PCR.
It is well established that most environmental toxic chemicals and carcinogens need to be metabolically activated to exert their toxic or carcinogenic effects. As a major enzyme system in xenobiotic metabolism, CYPs play a critical role in the metabolic activation of many environmental chemicals. In some cases, CYP-catalyzed metabolism leads to the detoxification of toxic chemicals.
Of 15 human CYP enzymes so far characterized, 8 forms of CYP (CYP1A1, 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) have been shown to be polymorphic at the phenotypic or genotypic level, or both (3-5). In addition to metabolizing various drugs, these polymorphic CYP enzymes are involved in metabolizing a large number of environmental carcinogens and toxic compounds (1). Because an individual's capability to metabolize these toxicants can be altered by carrying the variant alleles, genetic polymorphisms of CYP enzymes have been proposed as a biomarker of susceptibility to environmental carcinogenesis and toxicity. This paper briefly reviews the methodologies for CYP polymorphism studies and emphasizes the comparison between the genotyping and phenotyping approaches. The use of genetic polymorphisms of human CYP enzymes as a susceptibility biomarker and the direction of future research will be discussed.
Besides the metabolism in vivo, the polymorphisms of CYP enzymes can be determined at other phenotypic levels. These include enzyme activity determinations for metabolism in vitro with microsomes, CYP enzyme protein levels detected by immunological methods such as immunoblot and immunohistochemical analyses, and CYP mRNA levels measured by different nucleic acid hybridization techniques (Northern and slot blotting, RNase protection, and in situ hybridization). In recent years researchers have developed reverse transcription coupled with polymerase chain reaction (RT-PCR) to detect CYP mRNA in a small amount of tissue sample. If an appropriate internal standard is included in the cDNA synthesis reaction, the RT-PCR can be quantitative in assessing the amount of CYP mRNA in the samples. Expression of CYP1A1 mRNA in human lymphocytes and its regulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin have been successfully determined by this method (8).
The phenotyping approach, especially with activity-related assays such as metabolism in vivo and in vitro, directly links the expression of a given CYP form to the metabolism of its probe substrate. However, a critical issue is the difficulty in finding the right probe drugs that are specific for diagnosing a particular CYP form and safe enough for in vivo metabolism studies. Another major concern with the phenotyping approach is the confounding effects of dietary factors or coadministration of drugs that could affect expression of the metabolizing CYP enzymes (9). This could, in turn, alter the metabolism of the probe drug. Induction or suppression of CYP enzyme activities by xenobiotics, including dietary compounds, has been well documented in animals (9). In humans, induction of CYP2E1 by alcohol drinking (10) and of CYP1A2 by ingestion of charbroiled meats or cruciferous vegetables have been observed (11). In addition, human CYP1A2 is induced by cigarette smoking (12,13). Finally, the availability of substantial amounts of quality-assured human tissues (except peripheral blood cells) could be a limiting factor for determination of CYP enzyme activity in vitro, as well as for determination of CYP protein and mRNA levels.
Once the metabolic polymorphism involving a particular CYP enzyme is demonstrated, various molecular biology approaches and techniques can be used to look for possible genetic changes. A successful example of this approach is the discovery of CYP2D6 genetic polymorphism by Gonzales et al. (14). After cDNA cloning and DNA sequencing, they 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 (15). Prior to labor-intensive DNA sequencing work, the possible DNA sequence alterations can now be screened by single-strand conformation polymorphism analysis that detects the mutation-caused mobility shift of the DNA fragments on gel electrophoresis (16) or by other methods such as denaturing gradient gel electrophoresis (17). For functional analysis, the catalytic activity of CYP enzymes can be studied by expressing different variant CYP proteins with various cDNA expression systems if the polymorphic changes are localized in the coding region. 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.
Recent advances in PCR technology have greatly increased our capability to detect genetic polymorphisms of CYP enzymes. With small amounts of human tissue or cell samples, DNA amplification can be carried out with proper PCR primers for any particular sequence of a polymorphic CYP gene. The DNA source can be from blood leukocytes, buccal epithelial cells, hair roots, or normally exfoliated cells, such as bladder epithelium in the urine. DNA can also be obtained from stored pathological tissue sections, which provide great advantage for retrospective studies. It is now feasible to determine several CYP genetic polymorphisms with less than 10 µl of blood collected from the finger tip. The PCR-amplified DNA sequence containing the polymorphic sites can then be analyzed by RFLP with restriction digestion and visualized on a stained gel after electrophoresis. Comparing the wild-type samples, 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. If the PCR primers are designed to be within the missing sequence of a deletion polymorphism, there will be no PCR product formed.
Obviously, the RFLP method cannot be used to screen the CYP genetic polymorphisms in which the DNA sequence alterations cause no changes at suitable restriction sites. In this case, genotyping can be carried out by allele-specific PCR with a set of mutation-specific primers for amplification. Several polymorphisms of CYP1A1, 2A6, and 2D6 have been identified by the allele-specific PCR method (18-20). If necessary, the results from PCR-RFLP and allele-specific PCR can be confirmed by PCR-direct sequencing.
In contrast to the phenotyping approach, genotyping is not affected by drugs or dietary factors that might modulate the metabolic activity of CYP enzymes. As mentioned previously, the PCR-based genotyping techniques require only a small amount of DNA, which can be obtained by less invasive or noninvasive means or from longtime stored pathological samples. The genotyping approach also allows accurate prediction of the homozygous or heterozygous status of an individual who carries the variant allele. All of these are particularly useful for large population studies in which genetic polymorphisms of CYP enzymes may be susceptibility markers.
a) With current PCR-based techniques and rapid development of molecular biology approaches, identification of CYP genetic polymorphisms and genotypic screening of a subpopulation are not difficult. More new CYP genetic polymorphisms are expected to be discovered. The most critical challenge, however, is to establish the functional importance of different polymorphic variants, especially for those polymorphic sites located in the noncoding regions of the CYP genes. Using an in vitro transfection system with CAT as a reporter gene, it has been observed that the RsaI polymorphic site in the 5´-flanking sequence of the CYP2E1 gene caused a 10-fold increase in transcriptional activity in comparison with the wild-type sequence (31). It is important to demonstrate whether such extent of regulation occurs in vivo. Similarly, the results from the in vitro activity assays with the expressed variant CYP proteins need to be verified in phenotypic studies with human populations.
b) Our current efforts have been focused on the association of CYP genetic polymorphisms with cancer risks and less on the occupational toxicity in which the biological end points are not cancer occurrence. Human carcinogenesis is a long-term, multistep process. Although metabolic activation by CYP enzymes is known to constitute the first and critical step in environmental carcinogens, there are many other important steps involved, such as phase II enzyme detoxification, DNA repair, and immunosurveillance, as well as many modulating factors such as dietary components. In addition, it is difficult or impossible to know the number and identity of the carcinogens involved or the exposure levels. Assessing the role of genetic polymorphism of a particular CYP form by using cancer occurrence as a biological end point is therefore a very difficult task. We believe that at this stage it is more feasible to determine the role of CYP genetic polymorphisms in the susceptibility of workers to chemical toxicity in occupational exposures in which cancer occurrence is not an end point. An advantage in using worker populations is that the identity and the exposure levels of the toxic chemicals are in general clearly known. In addition, the workers are probably more homogeneous than the general population and more easily accessible for follow-up studies.
c) More research is needed for characterization of the substrate specificity and the enzyme kinetics of human CYP enzymes. Knowledge of which environmental toxicants and carcinogens are the substrates of the polymorphic CYP enzymes is important, but that knowledge is not enough in designing a population study. Although it is known that CYP enzymes have overlapping substrate specificities, the CYP form with a high Vmax value and the lowest Km value is usually the principal one involved in the metabolism and is therefore believed to be the one most relevant to the situation in vivo. Obviously, if a particular CYP form is found to be able to activate a toxic chemical but the Km value is much higher than the physiological concentration of that chemical after exposure, that CYP form may be of little relevance to the real in vivo situation. Studies in this direction will also help in developing more selective "probe" drugs for different CYP forms, which is inevitably required in establishing the relationship between CYP genetic polymophisms and metabolic activity in vivo.
d) More information is needed to understand the regulation of CYP enzymes in humans. The studies should include the effect of dietary compounds on the expression of CYP enzymes and the expression profile of CYP enzymes in the tissues that are targets for chemical toxicity or carcinogenesis. Without this information, the role of CYP genetic polymorphism in the biological consequences may not be accurately assessed.
e) Humans are exposed to numerous environmental toxicants. For example, more than 40 carcinogens have been found in tobacco products and tobacco smoke (32). Multiple polymorphic CYP enzymes are involved in metabolizing these carcinogens. Even for a given subpopulation mainly exposed to a single toxic chemical, it is possible that more than one form of CYP enzyme is involved in the activation or detoxification. Therefore, it is recommended that whenever possible a combinational polymorphism analysis on all the involved CYP forms be carried out to obtain a complete picture of the role of CYP enzymes in susceptibility to cancer and toxicity. The availability of current PCR technology allows us to use DNA samples stored for extensive periods (e.g., blood dotted on filter paper or pathological tissue sections) for the analysis of different CYP genetic polymorphisms. In addition, these DNA samples can be used for combinational genetic polymorphism analysis with the phase II metabolizing enzymes such as glutathione S-transferase and N-acetyltransferase, or DNA repair enzymes.
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Last Update: June 13, 1997