Genetic polymorphisms in UDP-glucuronosyltransferases and glutathione S-transferases and colorectal cancer risk

E.M.J. van der Logt1, S.M. Bergevoet1, H.M.J. Roelofs1, Z. van Hooijdonk1, R.H.M. te Morsche1, T. Wobbes2, J.B. de Kok3, F.M. Nagengast1 and W.H.M. Peters1,4

1 Department of Gastroenterology, 2 Department of Surgery and 3 Department of Clinical Chemistry, University Medical Centre St Radboud, Nijmegen, The Netherlands

4 To whom correspondence should be addressed Email: w.peters{at}mdl.umcn.nl


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Colorectal cancer (CRC) is one of the most common malignancies in the Western world showing an increasing incidence, and has been associated with genetic and lifestyle factors. Individual susceptibility to CRC may be due partly to variations in detoxification capacity in the gastrointestinal tract. Genetic polymorphisms in detoxification enzymes may result in variations in detoxification activities, which subsequently might influence the levels of toxic/carcinogenic compounds, and this may influence the risk for CRC. To determine whether genetic polymorphisms in detoxification enzymes predispose to the development of CRC, 371 patients with sporadic CRC and 415 healthy controls were genotyped for polymorphisms in the important detoxification enzymes UDP-glucuronosyltransferase UGT1A1, UGT1A6, UGT1A7 and UGT1A8, and glutathione S-transferase GSTA1, GSTM1, GSTP1 and GSTT1. Patients and controls were all of Caucasian origin. DNA was isolated from either blood or tissue and tested by polymerase chain reaction followed by restriction fragment length polymorphism analyses. Logistic regression analyses showed significant age- and gender-adjusted risks for CRC associated with variant genotypes of UGT1A6 [OR 1.5, 95% (confidence interval) CI 1.03–2.3] and UGT1A7 (OR 2.4, 95% CI 1.3–4.6), whereas no associations were found between CRC and the other polymorphic genes as mentioned above. In conclusion, the data suggest that the presence of variant UGT1A6 and UGT1A7 genotypes with expected reduced enzyme activities, might enhance susceptibility to CRC.

Abbreviations: CI, confidence interval; CRC, colorectal cancer; HCA, heterocyclic amines; ITC, isothiocyanates; RFLP, fragment length polymorphism; UGT, UDP-glucuronosyltransferase


    Introduction
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 Abstract
 Introduction
 Materials and methods
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Colorectal cancer (CRC) is an important cause of death in Western countries. In The Netherlands, it is the second cause of death from malignant disease in women, and the third cause of death in men (1). It is estimated that up to 10% of CRC cases can be attributed to hereditary factors of high penetrance (2) leaving ~90% so called sporadic CRC cases, which may be attributed to diet, lifestyle factors and genetic factors of low penetrance. Epidemiological studies have shown that diets low in fruit and vegetables, and high in red meat and fat are associated with an increased risk of CRC (3,4). Humans may be exposed daily to a large variety of toxic or even carcinogenic compounds, present in food (5) or as a result of lifestyle habits such as smoking of cigarettes (6,7). However, humans also possess a highly efficient system of defence against such harmful compounds, and the detoxification enzymes are a main part of this. Detoxification enzymes such as UDP-glucuronosyltransferases (UGTs) (8) and glutathione S-transferases (GSTs) (9) are responsible for the efficient modification of harmful molecules, making them less biologically active and facilitating their excretion. These enzymes are present predominantly in the gastrointestinal tract, especially in the liver, where detoxification enzymes have been identified at very high levels. However, these enzymes have also been distinguished in extra-hepatic tissues, including skin, kidney, intestine and many other organs (811). Since the gastrointestinal tract is in direct contact with potentially carcinogenic agents, ingested by food, medication, drugs, etc., the intestinal mucosa acts as a first-line barrier. Tissue-specific expression of the various isoforms of detoxification enzymes in colon and liver was shown to contribute to the differences in enzyme activities observed in these tissues (1013). In addition, variations due to the presence of genetic polymorphisms may contribute to the inter-individual differences in expression levels and enzyme activities of these enzymes (9,14). These genetic polymorphisms may result in variations in detoxification activities, which also might influence the levels of toxic/carcinogenic compounds in the colon. Therefore, polymorphisms in the detoxification enzymes could contribute to individual susceptibility to CRC.

UGT and GST are important families of detoxification enzymes. UGTs catalyze the conjugation of a wide variety of exogenous (e.g. drugs, pesticides, components of tobacco smoke) and endogenous (e.g. bilirubin, bile acids, steroid hormones) compounds to glucuronic acid (8), while GSTs catalyze the reaction of glutathione with exogenous electrophiles (e.g. polycyclic aromatic hydrocarbons, heterocyclic amines) and endogenous products of oxidative stress (9,10). The metabolites formed by these reactions are generally less toxic and more water-soluble, which facilitates their biliary and renal excretion. Polymorphic variations in these detoxification enzymes may influence the rates of conversion of toxic or carcinogenic compounds. Many genetic polymorphisms in UGTs or GSTs have been described and some have been associated with increased CRC susceptibility (1518).

In humans, two UGT families have been classified: UGT1A and UGT2 (19). So far, nine functional UGT1A isoenzymes (UGT1A1, UGT1A3–UGT1A10) have been characterized, all derived from a single gene locus on chromosome 2 (8,20). UGT1A enzymes are involved mainly in the metabolism of exogenous compounds; this is not strictly the case however as bilirubin and steroid hormones are important endogenous substrates. UGT2 isoenzymes 2A1, 2B4, 2B7, 2B10, 2B11, 2B15 and 2B17 are involved mainly in the glucuronidation of endogenous compounds and therefore were not studied further here. Most UGT1A family members are expressed at low levels in the colon. UGT1A7, UGT1A8 and UGT1A10 are expressed only extra-hepatically and may be highly relevant for colonic detoxification (21). Several functional polymorphisms in UGT1A family isoenzymes have been identified (8,14,22,23). In Caucasians homozygosity for the UGT1A1*28 polymorphism in the UGT1A1 promoter results in significantly reduced hepatic bilirubin UGT enzyme activity (24,25), leading to a mild form of hyperbilirubinemia, known as Gilbert's syndrome (26,27). Two missense mutations in exon 1 of UGT1A6 have been described, which results in T181A and R184S amino acid changes (28). These polymorphisms are usually linked on one allele (UGT1A6*2), although alleles carrying only the R184S polymorphism (UGT1A6*3) are found occasionally. Metabolism rates of phenols by recombinant UGT1A6*2 were lower than those of the most common enzyme. For UGT1A7, eight allelic variants of the most common UGT1A7*1 allele have been described (29,30); however, only UGT1A7*1 to *4 have been identified in Caucasians. Complete loss, or a very strong reduction, of activity was reported for UGT1A7*3 (29), whereas substantial reduction of activity was demonstrated for UGT1A7*2 and UGT1A7*4. In UGT1A8, two missense mutations in exon 1 were identified, resulting in an A173G substitution with little impact on catalytic activity, in contrast to the substitution of C277Y yielding an inactive enzyme (22).

In humans, the GST family comprises four main classes (alpha, mu, pi, theta), which genes are mapped on different chromosomes (9). Coles et al. described a polymorphism at nucleotide 69 in the 5'-regulatory region of the GSTA1 gene (31). Homozygotes for this polymorphism (GSTA1*B) have reduced enzyme activity compared with GSTA1*A homozygotes. Five GST Mu class genes (M1 to M5) have been identified clustered on chromosome 1 (32). For intestine GSTM1 seems most important, however 40–60% of Caucasians do not express GSTM1 due to the GSTM1 null genotype (16,33). GSTP1, the only member of the GST Pi class, appears to be the most widely distributed GST isoenzyme (10). A functional polymorphism has been described for GSTP1 resulting in an I105V substitution (34) and leading to a lower enzyme activity (35,36). Two GST Theta class genes, GSTT1 and GSTT2, have been characterized and in humans a GSTT1 null genotype may be present at a frequency of ~10–20% in Caucasians (16,37).

Aim
To determine whether genetic polymorphisms in several important isoenzymes of the UGT and GST family predispose to the development of CRC, 371 Caucasian patients with sporadic CRC and 415 Caucasian healthy controls were genotyped for polymorphisms in UGT1A1, UGT1A6, UGT1A7, UGT1A8, GSTA1, GSTM1, GSTP1 and GSTT1.


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Patients and control subjects
A total of 371 patients (212 males, 159 females; mean age 64 ± 11 years) with sporadic CRC were recruited at the Departments of Gastroenterology and General Surgery, University Medical Centre St Radboud, Nijmegen, The Netherlands. 415 subjects (168 males, 247 females; mean age 42 ± 12 years) served as controls and were recruited by advertisement in a local paper. All subjects were of Caucasian origin.

DNA isolation
Whole blood from 280 CRC patients and 415 healthy controls was obtained by venapuncture in sterile vacutainer tubes anti-coagulated with EDTA and stored at –20°C until use. Most blood samples from CRC patients were collected at the Department of Clinical Chemistry. DNA was isolated from whole blood using the Pure Gene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the instructions of the manufacturer and stored at 4°C. For 91 patients with CRC, no blood samples were available and DNA was isolated from resected normal colorectal tissue obtained at the Department of Surgery. After surgical resection, tissue specimens were immediately frozen in liquid nitrogen and stored at –80°C until use. DNA was extracted from normal colorectal mucosa using phenol–chloroform–isoamylalcohol extraction according to Maniatis et al. (38). Cancer diagnosis was confirmed by histopathological investigation of tissue specimens by a pathologist. Cases were classified according to Dukes' stages (A, B, C, D) and according to location of the tumour in the large intestine as either proximal (cecum, ascending or transverse) or distal (descending, sigmoid, rectosigmoid junction or rectum).

Genotyping
UGT1A1. The number of TA repeats in the promoter region of the UGT1A1 gene was analyzed using polymerase chain reaction (PCR) conditions and primers (Table I) as described by Monaghan et al. (24). Amplification was confirmed by agarose electrophoresis before fragments were resolved on 12% polyacrylamide gels (19:1 acrylamide/bisacrylamide; Bio-Rad) in Tris–borate–EDTA buffer. Gels (20 x 20 x 0.075 cm) were run at 400 V for 3 h and stained with ethidium bromide for 30 min (25). Fragments of 98 bp indicate the UGT1A1*1 allele, containing six TA repeats, and fragments of 100 bp indicate the UGT1A1*28 allele, containing seven TA repeats.


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Table I. Primers and restriction enzymes used for genotyping analyses

 
GSTM1 and GSTT1. The GSTM1 genetic polymorphism was determined by PCR according to the method of Brockmöller et al. (33). As an internal positive control, the ß-globin gene was co-amplified, whereas sterile H2O was substituted for genomic DNA and served as a negative control. PCR primers are given in Table I. PCR products were subjected to electrophoresis on 1% agarose gels and analyzed for the presence of a 650-bp product, which is indicative of at least one functional GSTM1 allele. The lack of an amplification product is consistent with the null genotype. The procedure followed to detect the GSTT1 null polymorphism is similar to that of GSTM1 and it is based on the method of Pemble et al. (37). PCR reactions and electrophoretic analyses were performed under the same conditions as described for GSTM1. Amplifications were performed in duplicate. The visualization of a 480-bp product indicates the presence of a least one functional GSTT1 allele.

Other investigated genes. The polymorphisms in all other investigated genes were studied using PCRs followed by restriction fragment length polymorphism (RFLP) analyses. The primers and restriction enzymes used for PCR–RFLP are shown in Table I. PCR–RFLP assays were adapted from methods described earlier and were used to identify the polymorphic variants of the following genes: UGT1A6 (28,39), GSTA1 (31) and GSTP1 (35).

Polymorphic variants in the UGT1A7 and UGT1A8 genes were identified by PCR–RFLP methods developed in our laboratory. In short, to detect the variations at UGT1A7 codons 129/131, we used the forward primers F1 and F2 (the ‘a’ in the primer sequence denotes site-directed mutagenesis for introduction of a VspI restriction site in the wild-type allele) and the reverse primer R1 (Table I). F1 only detects the N129K/R131K polymorphism; F2 detects both the N129K/R131K and N129R/R131K polymorphisms (see Figure 1A and B). To detect the W208R alteration, we used the forward primer F3 and the reverse primer R2 (Table I and Figure 1C). To determine whether the N129K/R131K or N129R/R131K and W208R occur cis or trans, we use the primers F4 and R1 (Table I and Figure 1D). PCR conditions were 5 min at 95°C, then 37 cycles of 30 s at 95°C, 30 s at 55°C (codons 129/131)/65°C (codon 208)/56°C (allele specific), and 30 s at 72°C, and finally an elongation step at 72°C for 5 min. Aliquots of 10 µl of the PCR product were digested with the restriction enzyme VspI (codons 129/131) or RsaI (codon 208/allele specific) for at least 1 h at 37°C (see Figure 1). The polymorphisms in the UGT1A8 gene corresponding with amino acid substitutions at position 173 and 277 were analyzed with two separate PCRs followed by RFLP analyses. The primers used for the PCR to detect the A173G substitution are shown in Table I. The PCR conditions were 4 min at 95°C, then 35 cycles of 30 s at 95°C, 1 min at 58°C, and 1 min at 72°C, and finally an elongation step at 72°C for 7 min. A 750-bp product was amplified and aliquots of 5 µl of the PCR mixture were digested for 1 h at 37°C with the restriction enzyme AluI, followed by electrophoresis on 3% agarose gel, containing ethidium bromide. The UGT1A8*2 allele (A173G) contains only one restriction site for AluI, instead of two restriction sites for the UGT1A8*1 and UGT1A8*3 alleles (see Figure 2A). The primers used to detect the C277Y substitution by PCR are shown in Table I (the last G of the forward primer creates a restriction site for PvuII in the wild-type allele). Except for the annealing temperature, which was 1 min at 49°C, similar PCR conditions were used as described above for detection of the A173G substitution. The digestion of the 215 bp PCR product with PvuII was carried out under similar conditions as described above. The UGT1A8*3 allele (C277Y) contains no restriction site for PvuII, distinct from the UGT1A8*1 and UGT1A8*2 alleles, which have one PvuII restriction site (Figure 2B). During each PCR analysis, sterile H2O was added instead of genomic DNA in several wells of the 96-well PCR plate, which served as negative control for amplification. All genotypes analyzed are summarized in Table II.



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Fig. 1. Genotyping of UGT1A7 129/131 and 208 polymorphisms with PCR–RFLP. (A) N129K/R131K PCR fragment digested with VspI: most common: 315 + 18 bp; heterozygous: 333 + 315 + 18 bp; homozygous: 333 bp. (B) N129K/R131K or N129R/R131K PCR fragment digested with VspI: most common: 315 + 18 bp; heterozygous: 333 + 315 + 18 bp; homozygous: 333 bp. (C) W208R PCR fragment digested with RsaI: most common: 440 bp; heterozygous: 440 + 337 + 103 bp; homozygous: 337 + 103 bp. (D) Allele-specific PCR for the detection of N129K/R131K or N129R/R131K variations, digested with RsaI: no product: the 129/131 mutations are not present; 332 bp product: only the 129/131 mutations are present or the 129/131 mutations are not in the same allele as the 208 mutation; 253 + 79 bp products: the 129/131 mutations are in the same allele as the 208 mutation; 332 + 253 + 79 bp products: the 129/131 mutations are present in one allele and in the other allele the 129/131 mutations and the 208 mutation are present. Lane M, 100 bp marker; lane 1, UGT1A7*1*1; lane 2, UGT1A7*1*2; lane 3, UGT1A7*1*3; lane 4, UGT1A7*2*2; lane 5, UGT1A7*2*3; lane 6, UGT1A7*3*3; lane 7, UGT1A7*1*10 (genotype UGT1A7*2*4 was not identified; alleles carrying only the W208R polymorphism were not found). The sizes of the PCR fragments are indicated.

 


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Fig. 2. Genotyping of UGT1A8 173 and 277 polymorphisms with PCR–RFLP. (A) Electrophoresis patterns of PCR fragments after digestion with AluI for detection of the A173G polymorphism and (B) after digestion with PvuII for the C277Y polymorphism. Lane M, 100 bp marker; lane 1, homozygosity for the common allele; lane 2, heterozygosity; lane 3, homozygosity for the variant allele (homozygosity for the C277Y polymorphism was not found). The sizes of the PCR fragments are indicated.

 

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Table II. Polymorphic variants of the detoxification enzymes investigated

 
Statistical analyses
Differences between characteristics of patients and controls were analyzed with {chi}2 test and t-test. All genotypes investigated among controls and patients were tested whether they were distributed according to the Hardy–Weinberg equilibrium. Furthermore, the {chi}2 statistic was used to test for differences in the distribution of the genotypes between the two study groups, or to estimate differences in allele frequencies. In total, four genetic polymorphisms of the UGT1A family were analyzed. Because the different UGT1A isoforms are derived from one single gene locus, we corrected for multiple testing with Bonferroni, meaning that a P-value of <0.013 instead of 0.05 was considered to represent statistical significance. Odds ratios (OR) with 95% confidence interval (95% CI) were calculated by logistic regression analyses for genotypes associated with normal enzyme activity versus genotypes associated with expected reduced enzyme activity (variant genotypes). All statistical analyses were performed with SAS (version 8.0; SAS Institute, Cary, NC).


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Characteristics of patients and controls
Descriptive statistics of CRC patients and controls are given in Table III. The mean age of CRC patients (64 ± 11 years) is significantly higher compared with that of the control group (42 ± 12 years; P < 0.0001). There is also a statistically significant difference in gender between CRC patients and healthy controls, with more female subjects in the control group (P < 0.0001).


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Table III. Clinical characteristics of patients with sporadic CRC and controls

 
Polymorphisms in genes of biotransformation enzymes
Genotype distributions of the UGT and GST biotransformation enzymes investigated are summarized in Table IV, and corresponding allele frequencies are shown in Table V. The genotype distributions and allele frequencies in patients are based on PCR–RFLP, using DNA extracted from either whole blood or normal colorectal mucosa. All genotype distributions tested here fulfilled the Hardy–Weinberg criteria. {chi}2 analyses revealed no significant differences for the investigated polymorphisms between CRC patients and controls. Calculation of odds ratios of genotypes associated with normal enzyme activity versus genotypes associated with expected reduced enzyme activity (variant genotypes) with logistic regression analyses showed significant age- and gender-adjusted risks for CRC associated with variant genotypes of UGT1A6 (OR 1.5, 95% CI 1.03–2.3) and UGT1A7 (OR 2.4, 95% CI 1.3–4.6). There were no statistically significant differences between cases and controls for the other genotype distributions investigated in this study (Table IV).


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Table IV. Distribution of genotypes of UGT and GST detoxification enzymes in patients with CRC and controls

 

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Table V. Allele frequencies of UGT and GST genes investigated in patients with CRC and controls

 
{chi}2 analyses revealed no significant differences in the presence of variant alleles between CRC patients and controls, for all enzymes investigated. When combining alleles that provide normal catalytic properties and alleles that may yield lower enzyme activity we calculated the odds ratios for CRC risk (Table V). Logistic regression analyses showed significant age- and gender-adjusted risks for CRC associated with variant UGT1A7 alleles (OR 1.5, 95% CI 1.1–2.0).

Possible associations of genotype distributions of the UGT and GST biotransformation enzymes and clinical characteristics, such as tumour location and tumour stage were also investigated and the results are summarized in Table VI. These analyses revealed an association of UGT1A6 variant genotypes with proximal CRC (adjusted OR 2.1, 95% CI 1.1–4.1), whereas UGT1A7 variant genotypes were associated with distal CRC (adjusted OR 3.0, 95% CI 1.5–6.2). In addition, logistic regression analyses showed significant age- and gender-adjusted risks for Dukes C/D CRC associated with variant genotypes of UGT1A6 (OR 2.0, 95% CI 1.2–3.2), whereas presence of GSTM1 was associated with a reduced risk for Dukes A/B CRC (OR 0.47, 95% CI 0.25–0.83). Risk for CRC associated with variant UGT1A7 genotypes was independent of tumour stage [Dukes A/B: OR 2.5 (1.1–6.3); Dukes C/D: OR 2.8 (1.4–6.0)].


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Table VI. Distribution of genotypes of UGT and GST detoxification enzymes with respect to tumour location and tumour stage in patients with CRC

 

    Discussion
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The risk of sporadic CRC is associated mainly with lifestyle factors and may be further modulated by several genetic factors of low penetrance (16). Since the gastrointestinal tract is in direct contact with potentially toxic or (pre-)carcinogenic agents, the intestinal mucosa acts as a first-line barrier. In humans, detoxification enzymes are prominently present in the liver. However, these enzymes have also been distinguished in extra-hepatic tissues of the gastrointestinal tract (811). Polymorphic variations in the detoxification enzymes may modulate the rate of conversion of toxic or carcinogenic compounds in the epithelium lining the lumen of the gastrointestinal tract. Several polymorphisms of genes encoding for the detoxification enzymes have been described and have sometimes been associated with increased CRC susceptibility (1517). In the present study we investigated the relationship between sporadic CRC and polymorphisms in UGT and GST genes, which are associated with functional changes in enzyme activity.

In this study, a significant age- and gender-adjusted risk for CRC associated with variant genotypes of UGT1A6 was revealed. In particular, variant genotypes of UGT1A6, with an expected reduction of the corresponding enzyme activities, were associated with tumours of the proximal colon and with Dukes C/D tumour stages. The frequency of the UGT1A6*2 allele in healthy subjects was estimated at 35%, which is higher than the 16.8% reported by Ciotti et al. (28), but comparable with 30.7 and 32.5% found by Lampe et al. (40) and Köhle et al. (41), respectively. Earlier, Bigler et al. (42) described an inverse association with intake of aspirin and colon adenoma risk, for individuals who carried the variant UGT1A6 alleles. The low activity alleles were protected against adenomas by aspirin intake whereas the individuals bearing only high activity alleles were not. UGT1A6 primarily metabolizes simple phenols (43) and planar heterocyclic amines (HCAs) (44). HCAs are formed in protein-rich foods, such as meat, as a result of pyrolysis during cooking. Meat intake, specifically red meat, has been associated with higher CRC risk (45). Recently, Butler et al. (46) examined the association between CRC and meat intake categorized by the stage of the cooking process, cooking method and estimated levels of HCAs. They reported moderate, dose-dependent associations between CRC and red meat intake, in particular for pan-fried and well-done red meat, which showed the strongest correlations with the investigated HCAs. Individuals bearing variant UGT1A6 genotypes, associated with reduced enzyme activity to detoxify these meat-derived HCAs sufficiently, may therefore be at an increased risk for CRC.

Strassburg et al. (18) showed a significant association of CRC and the presence of the variant UGT1A7*3 allele (OR 2.8 95% CI 1.6–4.7). We found only a weak association; presence of the UGT1A7*3 allele yielded an OR of 1.2 (95% CI 0.91–1.6), which was not statistically significant. However, the combination of all variant alleles revealed a significant association with CRC (OR 1.5 95% CI 1.1–2.0). Furthermore, an increased risk for CRC was specifically observed among patients with distal CRC who had the variant UGT1A7 genotypes compared with subjects who had the most common genotype (OR 3.0). The differences between our results and those published by Strassburg et al. could be explained by the relatively low frequency of the UGT1A7*3 allele in their control subjects (18). In our study, the frequency of the UGT1A7*3 allele averaged 40% in healthy controls. This is comparable with frequencies of 32, 36 or 37% in Caucasian control subjects reported by Villeneuve et al., Guillemette et al. and Köhle et al., respectively (29,30,41), but is much higher than values of 16–21%, as reported by Strassburg et al. (18,4749). UGT1A7 has been demonstrated to catalyze the glucuronidation of tobacco smoke-derived polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, as well as dietary-derived heterocyclic amines (21,50), all of which are known carcinogens. The risk factors for CRC identified here, the polymorphisms UGT1A7*2 and UGT1A7*3, have been shown to result in a significant reduction of enzyme activity towards carcinogenic metabolites of benzo[a]pyrene in catalytic studies (18). Because these polymorphisms can result in reduced detoxification activities, accumulation of carcinogenic compounds, such as benzo[a]pyrene, may occur in individuals bearing variant UGT1A7 genotypes, which eventually may result in an increased risk for developing CRC. In addition, epidemiological studies showed that smoking of cigarettes is a risk factor for CRC with a long lag time of up to 35–40 years (51). This association between smoking and CRC risk may be due partly to insufficient detoxification of carcinogenic components of cigarette smoke by genetic polymorphisms in UGT1A7.

For UGT1A1 and UGT1A8 we found no difference in the distribution of the polymorphic variants in healthy controls compared with CRC patients. This suggests that individuals carrying the UGT alleles, encoding for less active enzymes do not have a higher risk for developing CRC. The frequency of the UGT1A1*28 allele was 33% in healthy controls. This is comparable with the frequencies (29–39%) reported in Caucasian control subjects by several other groups (40,41,5254). The detected frequency of 2.5% for the UGT1A8*3 allele in healthy controls was similar to 2.2% as reported by Huang et al. (22).

The different UGT1A isoforms investigated here are derived from one single gene locus and recently, frequent co-occurrence of variant alleles of UGT1A1, UGT1A6 or UGT1A7 has been reported (39,41). Both UGT1A6 and UGT1A7 variant genotypes were associated with an increased CRC risk after adjustment for age and gender, as described above. Therefore, we also performed logistic regression analyses with both UGT1A6 and UGT1A7 in the regression model. This revealed an age- and gender-adjusted OR of 1.3 (95% CI 0.85–2.1) for UGT1A6 and an OR of 2.0 (95% CI 0.98–3.9) for UGT1A7. This means that the UGT1A7 polymorphism is the most important risk factor for development of CRC in comparison with the other UGT1A polymorphisms investigated here.

In this study, similar frequencies for the homozygous GSTA1*B genotype were observed in controls (14%) and cases (14%), which is not consistent with the data of Coles et al. (55) that 14% of the control subjects and 24% of the CRC patients bore this genotype. This discrepancy could possibly be explained by the much larger population of controls (415) and CRC patients (371) we examined, as compared with the 226 control and 100 case subjects investigated by Coles et al.

The relationship between CRC risk and GSTM1 polymorphism has been most extensively studied and recently, two meta-analyses have been published by de Jong et al. (16) and Houlston and Tomlinson (17). Both pooled analyses revealed no association of the GSTM1 polymorphism with CRC. Our results are in accordance with these observations. In contrast, a significant association for GSTM1 null genotype carriers and an increased CRC risk was described in a recent study by Sachse et al. (15). Earlier, Zhong et al. (56) reported that the risk of CRC associated with GSTM1-deletion was restricted to proximal disease, but this association was not seen in our study or in the meta-analyses, mentioned above (16,17). A striking observation was the association between GSTM1 null genotype carriers and a reduced risk of Dukes A/B CRC. The primary hypothesis is that individuals with the GSTM1 null genotypes eventually are at higher cancer risk because of reduced capacity to dispose of carcinogens. However, GSTM1 also plays an important role in the disposition of isothiocyanates (ITC), breakdown products of glucosinolates, which are abundant in cruciferous vegetables, and which are strong inducers of the GSTs and other detoxification enzymes. The GSTM1 null polymorphism, associated with reduced enzyme activity, may result in longer circulating half-lives of ITC and potentially greater chemopreventive effects of cruciferous vegetables (57). Some case-control studies provide evidence that the presence of GSTM1 in conjunction with low intake of cruciferous vegetable is an important risk factor for CRC or pre-cancerous lesions (58,59). Using ITC as a biomarker of cruciferous vegetable exposure, Seow et al. and London et al. (60,61) further strengthened the understanding of this gene–diet interaction. Seow et al. reported that high dietary ITC is associated with a significantly lower risk of CRC among individuals who are both GSTM1 null and GSTT1 null and London et al. observed that men with null genotypes of GSTM1 or GSTT1, who had consumed cruciferous vegetables were at a lower risk for lung cancer. A similar explanation as given above may be valid here, and it may be hypothesized that: in the early stages of CRC, ITC may be involved in prevention of the development of CRC in GSTM1 null or GSTT1 null genotype carriers. However, we do not have any data on consumption of cruciferous vegetables by the CRC patients or controls investigated here to test this hypothesis.

Several studies reported on the genetic polymorphism in codon 105 of the GSTP1 gene as a possible risk factor for CRC (15,6264). However, pooled analyses by de Jong et al. (16) and Houlston and Tomlinson (17) did not show an increased risk of this polymorphism for CRC, which is in accordance with our results.

Conflicting findings have also been reported in the relationship between GSTT1 status and CRC (1517). We found no association between the GSTT1 null genotype and CRC risk, in agreement with the results found in the meta-analysis by Houlston and Tomlinson. (17). However, the meta-analysis by de Jong et al. (16) reported the opposite. The overall odds ratio for GSTT1 calculated by de Jong et al. was based on the crude data of the different studies, without corrections for age and gender. This may be crucial, as outlined recently by Butler et al. (65); crude data revealed a statistically significant association between GSTT1 null genotype and CRC risk, whereas after adjustment for age the increased risk was not observed anymore. Subgroup analysis for tumour location revealed no association between GSTT1 null and CRC in our study, which is in accordance with the results of the meta-analysis by de Jong et al. (16).

Our findings have to be viewed from the perspective of potential limitations. Odds ratios can only be calculated correctly when confounding factors, such as age, gender, diet and lifestyle factors, are taken into consideration. In this study we observed a statistically significant difference in age and gender between CRC patients and controls. By including both age and gender in the logistic regression analyses we corrected the calculated odds ratios for differences in these factors. This is necessary because younger control subjects in comparison with CRC patients, have a shorter time of exposure to carcinogens and thus at the moment they reach the age of the CRC patients, some of them may also have developed CRC. It would be preferable to better match control and patient populations implicating that no afterward correction is needed, but in practice it appeared very difficult to realize. In this study no information was available on the dietary habits, alcohol use and smoking patterns of both patients and controls, which may also be confounding factors. Possibly some of the low-penetrance genes investigated here only contribute to CRC in combination with (some of) these dietary or lifestyle factors.

Polymorphic variations in detoxification enzymes may determine in part the rates of conversion of toxic or carcinogenic compounds, and thus may influence their levels in the gastrointestinal tract. We conclude that individuals carrying the low enzyme activity associated genotypes of UGT1A6 and UGT1A7 might be more prone to develop CRC. It is hard to estimate the actual impact of a respective 1.5- and 2.4-fold increased risk, however these findings may guide research towards the search of relevant substrates of these enzymes, as important risk factors for CRC. Such factors may be components of cigarette smoke and heterocyclic amines, present in protein-rich food such as meat. However, other UGT family members may compensate reduced conversion rates because detoxification enzymes possess overlapping substrate-specificity. Furthermore, dietary anticarcinogens are able to enhance the activity of detoxification enzymes. Therefore, it is also a challenge to investigate whether such anticarcinogens can compensate for the reduced enzyme activities caused by the genetic polymorphisms studied here.


    Acknowledgments
 
The authors wish to thank Dr R.J.F.Laheij for his contribution to the statistical analyses and Numico Research Netherlands for their interest and financial support.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 10, 2004; revised June 30, 2004; accepted July 25, 2004.





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