Molecular Epidemiology of the Human Glutathione S-Transferase Genotypes GSTM1 and GS7T1 in Cancer’

The ,,t (GSTMJ) and 0 (GSTTJ) members of the glutathione S-transferase multigene family are candidate cancer susceptibility genes because of their ability to regulate the conjugation of carcinogenic compounds to excretable hydrophilic metabolites. Deletion variants that are associated with a lack of enzyme function exist at both of these loci. Individuals who are carriers of homozygous deletions in the GSTMJ or GSTTJ genes may have an impaired ability to metabolically eliminate carcinogenic compounds and may therefore be at increased cancer risk. Molecular epidemiological studies have provided three pieces of information about the relationship of GSTMJ and GSTTJ with cancer susceptibility. First, the frequencies of homozygous GSTMJ and GSTTJ deletion carriers is very high (i.e., 20-50%) in most populations studied to date. Second, GSTMI and, possibly, GSTTJ may be involved in the etiology of cancer at more than one site. Third, the risk conferred to individuals who carry homozygous deletions in GSTMJ or GSTTJ appears to be small in magnitude (e.g., odds ratio of <2). However, the magnitude of risk is larger (e.g., odds ratio of 3-5) when interactions of GSTM1 or GSTTJ with other factors (e.g., cigarette smoking) are considered. These findings have implications for studies of GSTMJ and GSTTJ in cancer susceptibility and for future applications of these biomarkers in cancer prevention or control strategies. First, molecular epidemiological studies should consider both the common frequency of deletion genotypes and the relatively low cancer risk these deletion genotypes may impart. For example, the common frequency of deletion variants may improve statistical power in some molecular epidemiological studies, but large samples may still be required to detect relatively small effect sizes or important interaction effects. Second, the fact that deletion genotypes are common implies that the Received 10/23/96; revised 3/24/97: accepted 4/21/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked adsertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 This research was supported by United States Public Health Service Grants CA60798 and ESO8O31 and by a grant from the University of Pennsylvania Cancer Center. 2 To whom requests for reprints should be addressed. at Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, 904 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6021. Phone: (215) 898-1793: Fax: (215) 573-2265: E-mail: [email protected]. proportion of cancer attributable to these variants may be large in the general population. However, these genotypes may be less suited for individual cancer risk assessment because of their relatively small contribution to the absolute risk of cancer. Rationale for GSTMJ and GSTTJ in Cancer Susceptibility The etiology of most commonly occurring cancers cannot be explained by allelic variability at a single locus. Instead, the major burden of cancer in the general population probably results from the complex interactions of multiple genetic and environmental factors over time. An understanding of the interpbay of xenobiotic exposures. endogenous physiology, and genetic variability at multiple loci will facilitate knowledge about cancer etiology and the identification of individuals who are at increased risk for developing cancer. Members ofthe GST3 family (E.C. 2.5.1.18) are important candidates for involvement in susceptibility to commonly occurring forms of cancer because they may regulate an individual’s ability to metabolize environmental carcinogens ( I ). Two genes encode the cytosolic enzymes GST-p. (GSTMI: chromosome lpl3.3) and GST-O (GS77’I; chromosome 22ql 1.2). These enzymes catalyze the addition (conjugation) of aliphatic aromatic heterocyclic radicals, epoxides, or arene oxides to glutathione. conjugation reaction at the electrophilic center of these compounds occurs at the sulfur atom of the glutathione molecule. These molecules act as glutathione peroxidases but do not require a selenium cofactor to accomplish conjugation reactions. A number of recent reviews (2-12) have described the protein structure, enzymology, inducibility, and expression (including tissueand gender-specific expression) of GSTM/ and GS77’l. These reviews summarize the role of GSTMI or GS77’I in the metabolism of and induction by numerous known or suspected carcinogenic compounds. These include benzo(a)pyrene, styrene-7,8-oxide, and trans-stilbene oxide by GSTM/ and epoxybutanes, ethylene oxide, halomethanes, and methyl bromide by GSTTI. As this list implies, there does not appear to be a single class of chemical compounds that is associated with GSTM/ or GS177 metabolism or induction (12). Instead, GSTMI and GS77’J are involved in the metabolism of a wide range of xenobiotics, including environmental carcinogens, chemotherapeutic agents, and reactive oxygen species. Furthermore, GSTMI appears to make a distinct contribution to cancer susceptibility because its substrate specificities may be different from that of the other classes of GSTs ( I 3). Knowledge of GST biochemistry has led to hypotheses about the role of the GSTMJ and GS177 genes in cancer etiology. Normal or increased GST enzyme activity or levels 3 The abbreviations used are: GST, glutathione S-transferase: PAH. polycyclic aromatic hydrocarbon: SCE. sister chramatid exchange: LOH, lass of constitutional heterozygosity: OR. odds ratio: CI, confidence interval. on November 6, 2017. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from 734 Review: GSTMI and GS7.7’l and Cancer Susceptibility may protect susceptible tissues from somatic mutations in DNA by facilitating the conjugation and subsequent elimination of electrophilic carcinogens. Absent or deficient GST enzyme activity may result in poorer elimination of ebectrophilic carcinogens, particularly in the presence of very active electrophilic activation by phase I enzymes (e.g., the cytochromes P-450). GST deficiencies may therefore result in increased risk of somatic mutation, leading to tumor formation (14, 15). If an individual’s inherited genotype at a GST locus does not permit the efficient metabolism of compounds involved in carcinogenesis, then that individual may be at increased cancer risk. This review presents a synopsis of the molecular epidemiobogical evidence that evaluates this hypothesis. Polymorphism in GSTMJ and GSTTJ Carriers of homozygous deletions in the GSTMJ and GS7TI genes have an absence of GSTand GST-O enzyme activity, respectively (I, 16, 17). These deletion variants have been useful for molecular epidemiological studies of cancer because they divide study subjects into two well-defined susceptibility classes: those who are and those who are not able to detoxify potential carcinogens by the metabolic pathways regulated by GSTMJ or GSTfl. Phenotypic assays have been used to identify individuals who lack GSTor GST-O activity (e.g., by induction of protein expression after exposure to trans-stilbene-oxide or methyl chloride, respectively). However, these assays are more time-consuming and susceptible to interassay variability relative to genotyping assays and are therefore not ideally suited for application to molecular epidemiological studies. Fortunately, there is a strong correlation between the phenotypic induction assays and genotype assays in identifying individuals who lack GST( 1 8 -23) or GST-O ( I 7, 24, 25). These studies indicate that the rate of misclassification in GSTand GST-O phenotype by GSTMJ and GS77’l genotype assays is extremely low (i.e. , < 1%). Therefore, genotype assays are appropriate for use in molecular epidemiobogicab studies that require both reliability and inexpensive, high-throughput capability. Three alleles at the GSTMJ locus have been described: GSTMJa (also denoted iii), GSTMJb (also denoted it), and GSTMJ-O (deletion) alleles. The GSTMJa and GSTMJb alleles differ by a C-’s’G substitution at base position 534. This DNA variant results in a Lys-*Asn substitution at amino acid 172 (I). However, there is no evidence to date that the GSTMJa and GSTMJb alleles are functionally different from one another (26). As noted above, comparisons of the homozygous deletion genotype (for simplicity, denoted below as GSTMJ-O) with genotypes containing at least one GSTMJa or GSTMJb allele (collectively denoted here as GSTMI-1) are of primary interest to molecular epidemiologicab studies. GSTMJ-O genotypes can be reliably determined by Southern blotting and PCR-based assays ( 1 8, 19, 22, 27-29), the latter being preferable in epidemiological studies. Because of the high frequency of deletion homozygotes (e.g., 50%), most individuals who are phenotypically GSTMI-1 are expected to be deletion heterozygotes rather than nondeletion homozygotes (42 versus 9%, respectively; Ref. 1). The ability to accurately distinguish deletion heterozygotes from deletion homozygotes by PCR-based methods may be complicated by limitations of consistent PCR product quality and quantity across samples. Scanning densitometry has been used to aid in distinguishing deletion heterozygotes from nondeletion homozygotes (30), but most studies to date have compared GSTMI-O with GSTMJ-1 genotype carriers. Similarly, two functionally different genotypes in Table I Frequencies a f homozygous deletions in GSTMI and GS17’l Locus F requency (range) Population Refs. GSTMJ 0.28-0.35 0.22 0.67 0.38-0.62 0.49-0.54 0.49-0.53