Phase II Metabolism

27 May

Of the phase II genes, those studies most extensively  are the N-acetyltransferases  and glu- tathione-S-transferases  in relation to the pharmacogenetics  of neoplasia. It is the former gene family that have been studied in relation to human cancer for the longest period. In the human, at least three N-acetyltransferase  (NAT) gene loci exist. One of these is probably a pseudogene, while the two expressed genes, NAT 1 and NAT 2, are both located on chromosome 8 but are separated by at least 25 kb (Grant, 1993). It is the NAT 2 gene locus that is involved in human acetylation polymorphism. A schematic diagram of most of the variant alleles is shown in Figure 5.15. In this diagram the wildtype alleles are designated  as R, the mutant alleles as S. These

Figure 5.12 Structure of the polymorphic  site in exon 7 of the P-450IA1 gene. The cysteine residue, a heme-binding thiolate ligand, is noted as enclosed with a box. The mutant codon is indicated by the G over the A reflecting  the transition  giving  rise to the valine  (Val)  as noted  below  the normal  amino  acid sequence. (Adapted from Hayashi et al., 1992, with permission of authors and publisher.)

Figure 5.13 Relative distribution  of cumulative  cigarette consumption  of patients in the Ile-Val geno- types, where the total number of patients with each of the genotypes is considered as 100%. Vertical arrow- heads indicate mean cigarette consumption  of patients with each genotype. (Adapted from Nakachi et al.,

1993, with permission of authors and publisher.)

designations also indicate the rate at which the transacetylation is catalyzed, R indicating rapid and S slow acetylator phenotypes.

Although the mechanism of the ineffective acetylation by the mutant slow acetylator phe- notypes is not generally known, the M1 mutant appears to cause a decrease in the amount of NAT 2 protein in the liver because of defective translation, whereas M2 translation results in an unstable enzyme (Blum et al., 1991).

Since N-acetyltransferases  are involved in the formation of the ultimate forms of aromatic amine carcinogens (Chapter 3), studies of the frequency of the rapid and slow acetylator pheno- types in workers exposed to such agents became an important project. Table 5.13 shows the rela- tionship of bladder cancer incidence in industrial chemical workers classified as to whether each individual was a slow or rapid acetylator. From these data, workers with the slow acetylator phe- notype were at a far greater risk of developing bladder cancer than rapid acetylators (cf. Nebert,

1991). Fettman et al. (1991) have suggested that such slow acetylators appear to be predisposed to bladder cancer as a result of the shift in metabolism of arylamines to oxidation and glucu- ronidation with concentration of the metabolites in the urine, thus increasing the risk of bladder cancer. In contrast, according to these investigators, the rapid acetylator phenotype may be iden- tified in a disproportionately  large number of colorectal cancer patients presumably through the enhanced production of mutagenic arylamides and acetoxyarylamines  initiated by the N-acetyl- transferase pathway. However, not all investigations have supported this latter concept (Probst- Hensch et al., 1995; Oda et al., 1994). A scheme of the metabolic  pathway of an arylamine showing the two alternative paths indicated above may be seen in Figure 5.16.

Figure 5.14 Diagram  of the wild-type  (wt) and variant alleles of the human CYP2D6  8P and 7P are pseudogenes, while 6 is the functional CYP2D6 gene. The box with a question mark may be an additional CYP2D gene or pseudogene. The mutations that have been characterized are denoted by asterisks. X1, X2, X3, and X4 are restriction sites (XbaI). In the 11.5 allele the functional gene is completely missing, while in the 44 allele a large insertion (heavy bar) is present in the 5′ half of the functional CYP2D6 gene. Other alleles are also known (cf. Shields, 1994), and a particularly  interesting allele is that exhibiting a 12-fold amplification  of the functional  gene resulting  in an ultrarapid  metabolism  of the enzyme’s  normal  sub- strates (Johansson et al., 1993). The major association with human cancer is the strong association of the EM phenotype with lung cancer as shown in several studies (Caporaso et al., 1992; Hirvonen et al., 1993). CYP2D6 polymorphism  is not related to human colorectal cancer (Ladero et al., 1991a), but these individ- uals have presented some evidence suggesting  an increased risk of breast cancer among women with the PM phenotype (Ladero et al., 1991b). (Adapted from Nebert, 1991, with permission of the author and pub- lisher.)

Another metabolic phase II pathway is the inactivation of various chemical carcinogens by their conjugation  with the tripeptide  glutathione,  catalyzed  by glutathione  S-transferase.  One form of this enzyme, termed GST1, is dominant in the population with approximately  50% of individuals exhibiting the recessive characteristic resulting in little or no measurable GST1 ac- tivity (Seidegård and Pero, 1985). The distribution of GST1 in lung cancer patients and appro- priate controls expressed as a fraction or percentage of patients expressing the gene (dominant expression) in relation to the total patients studied is seen in Table 5.14. Again, patients with lung cancer exhibit lower proportions of the dominant gene activity than do controls.

When the expression of both genes is studied in the same population, the relative risks of the patients bearing the mutant form of P450IA1 and the recessive expression of GST1 exhibit extremely high relative risks, demonstrating the complexity of the genetic background of indi- viduals and their risk of developing cancer on exposure to chemical carcinogens (Table 5.15). As noted in Figure 5.13, the relative risks can increase to 40 or 50 when one takes into consideration the age and total cigarette consumption.

Figure 5.15 Schematic diagram of variant alleles at the NAT 2 gene locus. Identities of the nucleotides at the six variable regions of the 870-bp NAT 2 coding sequence are indicated for the allele, R1. Deviations from this are indicated as base changes (G, C, A, T) on each of the other alleles are as any gains and losses of recognition  sites for the various restriction  endonucleases  indicated.  (Adapted from Grant, 1993, with permission of author and publisher.)

Figure 5.16 Potential pathways for the metabolism of arylamines. Abbreviations used: UDPGA, uridine diphosphoglucuronic acid; UDP-GT, UDP-glucuronosyltransferase; PAPS, phosphoadenosine  phosphosul- fate; ST, sulfotransferase.  (From Fettman et al., 1991, with permission of the author and publisher.)

In addition to the above noted relationship of alterations in glutathione S-transferase I ex- pression and lung cancer, the null or decreased expression of this gene has also been associated with increased risk for hepatocellular carcinoma (McGlynn et al., 1995), adenocarcinoma of the stomach and colon (Strange et al., 1991), bladder cancer (Bell et al., 1993), and mesothelioma (Hirvonen et al., 1995). In addition, the null phenotype of the glutathione-S-transferase I gene is associated with larger amounts of polycyclic aromatic hydrocarbon-dGMP  adducts in the lungs of smokers.

Table 5.14 Distribution of Glutathione S-Transferase I in Smokers with Various Types of Lung Cancer and in Matched Controls

Table 5.15 Relative Risk Estimate of the Combined Genotypes of the P450IA1 and GST1 Genes for Lung Cancer

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