Association of CYP2C9 genotypes leading to high enzyme activity and colorectal cancer risk

Carmen Martínez, Elena García-Martín1,, José M. Ladero2,, Javier Sastre3,, Francisco Garcia-Gamito4,, Manuel Diaz-Rubio2, and José A. G. Agúndez5,

Department of Pharmacology, Medical School and
1 Department of Biochemistry, School of Biological Sciences, University of Extremadura, Avda. de Elvas s/n, E-06071, Badajoz,
2 Department of Gastroenterology and
3 Department of Medical Oncology, San Carlos University Hospital, Prof. Martin Lagos s/n, E-28040, Madrid and
4 Department of Surgery, Hospital Insalud, Pol. Nueva Ciudad s/n, E-06800, Mérida, Spain


    Abstract
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 Abstract
 Introduction
 References
 
CYP2C9 enzyme activity is involved in the metabolism of substances related to colorectal cancer, and it is functionally linked to a genetic polymorphism. Two allelic variants of the CYP2C9 gene, namely CYP2C9*2 and CYP2C9*3, differ from wild-type CYP2C9*1 by single amino acid substitutions. These mutated alleles encode enzymes with altered properties that are associated with impaired metabolism. The present study evaluates the impact of CYP2C9 genotypes leading to high enzyme activity on colorectal cancer risk. For this, the frequency of allelic variants of the CYP2C9 gene was analysed in genomic DNA from 129 patients with colorectal cancer and in 150 healthy controls. Patients with colorectal cancer showed a statistically significant increase in the frequency of genotypes homozygous for the active CYP2C9*1 gene, as compared with healthy individuals. Such a high frequency is more significant among patients with cancer in proximal segments of the colon (P < 0.025; odds ratio 2.36 95% CI 1.18–4.72), and decreases in more distal tumour locations. We conclude that CYP2C9 polymorphism can be considered as a secondary risk factor for colorectal cancer in the studied population: those individuals with genotypes leading to high enzyme activity were at increased risk. The association of the CYP2C9 polymorphism to colorectal cancer risk could be related to CYP2C9-mediated metabolic activation of polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. In addition, the key role of CYP2C9 in the metabolic inactivation of non-steroidal anti- inflammatory drugs could diminish the protective effect of these drugs against colorectal cancer.

Abbreviations: HCAs, heterocyclic aromatic amines; NSAIDs, non-steroidal anti-inflammatory drugs; PAHs, polycyclic aromatic hydrocarbons.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Colorectal cancer is among the commonest neoplasias worldwide, with incidence rates close to that of lung cancer in Caucasian individuals (1). The pathogenesis of colorectal cancer involves mutations affecting proto-oncogenes, suppressor genes, as well as genes involved in DNA mismatch repair (2,3). In addition, it has been shown that genetic instability affecting mitotic checkpoint genes is especially incident in colorectal cancer (4,5). The prominent role that dietary factors play in the risk of developing colorectal cancer suggests the involvement of dietary mutagens in the initial steps leading to colorectal cancer.

Diverse xenobiotic-metabolizing enzymes that are capable of activating carcinogens and mutagens are expressed in human intestinal epithelium (69). Among them, cytochrome P450 (CYP) enzymes play a key role in the metabolism of xenobiotics. The CYP2C enzyme subfamily accounts for about 20% of the total CYP enzymes in human liver, CYP2C9 being the most abundant (10,11). CYP2C9 is also expressed in intestinal epithelium (6). Since the enzyme is involved in both the activation of dietary carcinogens and mutagens, liver metabolism and local metabolism in intestinal epithelium may occur. Whereas it is widely admitted that colorectal cancer risk is epidemiologically linked to dietary habits, a possible association between high CYP2C9 enzyme activity and colorectal cancer has not been analysed. CYP2C9 enzyme activity in man is modulated by a genetic polymorphism. Two allelic variants of the human CYP2C9 gene have been described as occurring at relatively high frequency, both being related to impaired metabolism. These are classified as CYP2C9*2 and CYP2C9*3, whereas the functional wild-type allele is classified as CYP2C9*1 (12,13). Since the enzyme activity is related to the presence of mutations in the CYP2C9 gene, individuals carrying non-mutated genes are expected to show high CYP2C9 metabolic capacity. Thus, the hypothesis for a role of CYP2C9 activity in colon cancer risk can be studied by analysing the CYP2C9 genotypes. This would indicate whether the CYP2C9 enzyme activity, as inferred from the genotypes, contributes to the risk of developing colorectal cancer.

129 unrelated consecutive patients (68 men and 61 women) suffering from colorectal cancer that attended the collaborating hospitals were included in the study. All patients were evaluated by consultant physicians and the diagnosis was based on histology analyses of endoscopic biopsies and/or surgical resection specimens. All consecutively diagnosed patients were requested to participate in the study, and all of them agreed to participate. The mean age (SD, range) was 67.3 (11.1, 37–89).

Control samples were obtained from healthy volunteers (n = 150), most of them being medical students or university and hospital staff. A medical examination was made to identify subjects in good health. Over 95% of the healthy subjects requested agreed to participate in the study. All participants were white Spanish individuals, living in the same areas as patients (Madrid and surrounding area), and were included in the study after giving informed consent. The protocol was approved by the Ethics Committees of those hospitals involved in the study. The controls were gender matched (80 men and 70 women). The mean age (SD, range) was 43.2 (12.3, 19–95). A possible confounding factor in the present study is that the frequency of a determined CYP2C9 genotype within a population may change with age. If a determined genotype has a `protective effect' against a disease, it may be expected that, in populations composed of older subjects, there is an increased frequency of such a protecting genotype. Therefore we included in the control group a selected subgroup of 41 healthy subjects with ages ranging from 90 to 95 years. The analysis of the CYP2C9 genotype indicated frequencies for each genotype that were identical to that of younger healthy subjects. The frequencies for CYP2C9*2 and CYP2C9*3 were 14.2 and 16.0 for younger controls and 14.6 and 15.8 for older controls, respectively.

Blood samples were immediately frozen after collection and kept at –80°C until analysed. The genomic DNA was prepared from peripheral leucocytes, and dissolved in sterile 10 mM Tris–HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid, at a final concentration of 400–600 µg/ml. The samples were stored at 4°C in sterile plastic vials. Mutations at the CYP2C9 gene locus were analysed by PCR and restriction mapping, as described elsewhere (14).

For the statistical analysis, the intergroup comparison values were calculated by using the {chi}2 test, unless the conditions for the application of this test were not adequate. In such cases, Fisher's Exact test was used to calculate P values. The 95% confidence intervals were calculated according to Bulpitt (15).

After digestion with the appropriate restriction endonucleases, five different genotypes were identified among colorectal cancer patients and healthy controls. The summary of genotypes is shown in Table IGo. The comparison of genotype frequencies among overall colorectal cancer patients and healthy controls indicates a statistically significant increased number of individuals with two active CYP2C9*1 genes. This is also reflected in the allele frequency that is shown in Table IIGo. An increased frequency of the active CYP2C9*1 gene, and a consistent lower frequency of both mutated genes among colorectal cancer patients is observed. Therefore our findings indicate that individuals suffering from colorectal cancer tend to have a higher frequency of genotypes leading to high CYP2C9 enzyme activities, as compared with healthy individuals. The frequency for CYP2C9 mutated genes is higher in the control population analysed, as compared with previously described reports on CYP2C9 genotypes in white individuals (14,16). However, such a frequency is consistent with that observed in a different study carried out in 157 Spanish healthy subjects (17) and with that described in other white populations (18).


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Table I. CYP2C9 genotypes in colorectal cancer patients and healthy controls
 

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Table II. CYP2C9 allele frequencies in colorectal cancer patients and healthy controls
 
In order to elucidate whether the CYP2C9 genotype could be linked to a particular type of colorectal cancer, patients were divided into three subgroups according to the anatomical site of the tumour (rectum, sigmoid colon and non-sigmoid colon). Table IIIGo shows that the excess of individuals carrying two functional genes is more relevant in the subgroup of 47 non-sigmoid colon cancer patients, as compared with the control group. These findings correspond to a significant increase in the frequency of the functional CYP2C9*1 gene in non-sigmoid colon cancer patients when compared with the control group. The increase of patients homozygous for functional genes, as well as the frequency of active CYP2C9*1 genes was not statistically significant in the rest of patients, as compared with the control group.


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Table III. CYP2C9 genotype and allelic distribution in colorectal cancer patients classified according to anatomical tumour site
 
Gender, previous surgical therapy or chemotherapy and Dukes' stage of the tumour did not influence the distribution of the CYP2C9 polymorphism among patients. Age at diagnosis was similar in all subgroups of patients, and this parameter was not related to the CYP2C9 genotype. Both groups, patients and controls, are in Hardy Weinberg's equilibrium, with calculated frequencies for carriers of two mutated genes of 5.5% for patients and 9.2% for controls. These calculated data are in agreement with the actual frequencies, 8.5% for patients and 10.7% for controls, that can be obtained from the data shown in Table IGo.

The involvement of CYP2C9 enzyme in the metabolism of xenobiotics could underlie the mechanism responsible for the association of CYP2C9 genotype and colorectal cancer. A number of xenobiotics that are ingested as pyrolysis products have been related to colorectal cancer risk. These include polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs). Several of these mutagenic substances are CYP2C9 substrates. Regarding HCAs, CYP2C9 has been shown to be involved in the metabolism of the highly carcinogenic 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (19), and in the metabolism of butadiene to the bi-functional alkylating agent diepoxybutane (20). The PAH dibenzo(a,h)anthracene is activated to the trans-3,4-dihydrodiol carcinogenic precursor by CYP2C9 (21). CYP2C9 also plays a role in the metabolic activation of the carcinogenic agent 7,12-dimethylbenz[a]anthracene (22), and in the transformation of benzo[a]pyrene to mutagenic 7,8-diols (23). It is to be expected that individuals with genotypes leading to high CYP2C9 activity, when exposed to these procarcinogens would activate them more efficiently, and therefore have an increased risk of developing colorectal cancer.

Another possible mechanism that may explain the association of CYP2C9 polymorphism and colorectal cancer risk is related to the role of CYP2C9 in drug metabolism. The enzyme plays a key role in the metabolism of non-steroidal anti-inflammatory drugs (NSAIDs) (for a review, see ref. 10). This, together with the protective effect that NSAIDs play in human colorectal cancer (24), raises the possibility that in individuals with high enzyme activity the protective effect of NSAIDs could be diminished. Several NSAIDs decrease cultured cancer cell proliferation and colorectal cancer risk, even when these drugs are not regularly used (for a review, see ref. 25). Langman et al. (24) show statistically significant differences in odds ratios for colorectal cancer in patients with seven or more NSAIDs prescriptions in the 13–36 months before diagnosis. Individuals with high CYP2C9 enzyme activity inactivate NSAIDs with higher efficiency; therefore it is tempting to speculate that the effect of NSAIDs on colorectal cancer risk would be reduced in these individuals.

According to our findings, ~70% of patients with non-sigmoid colorectal cancer may display high metabolism rates of drugs metabolized by CYP2C9. In this regard it should be kept in mind that the CYP2C9 enzyme is involved in the metabolism of the anti-neoplastic prodrugs cyclophosphamide and ifosfamide (26). In addition, the CYP2C9 enzyme plays a key role in the metabolism of NSAIDs that are frequently used in cancer patients (10). Therefore drug management, with special focus on oxazaphosphorines and NSAIDs, should be carefully individualized in these patients.

In summary, we have identified a statistically significant association of CYP2C9 genotype and colorectal cancer risk. However, the risk does not seem to affect all patients as a homogeneous group. Patients with non-sigmoid colon cancer show the strongest association, with a high frequency of genotypes homozygous for the CYP2C9*1 allelic variant. The frequency of patients with such a genotype decreases as the anatomical site of the tumor becomes more distal: patients with rectal cancer have similar frequencies of this genotype as that of healthy controls (Table IIIGo). It should be noted that the statistical significance for the association of the genotype homozygous for CYP2C9*1 and colorectal cancer risk is small, and therefore the CYP2C9 polymorphism should be considered as a secondary factor in the development of colorectal cancer. However, the epidemiological impact on the number of individuals who are protected (those carrying mutated genes) and on the number of individuals that are at increased risk (those carrying two active genes) may be considerable. The findings described in the present study require independent confirmation; since colorectal cancer risk is epidemiologically related to dietary habits, populations with different dietary habits should also be analysed for the association described in the present study.


    Notes
 
5 To whom correspondence should be addressedEmail: jagundez{at}unex.es Back


    Acknowledgments
 
This work was supported by Grants FIS00/0278 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (Madrid, Spain) and IPR00/C022, CSC00/38 and CSC00/51 from Junta de Extremadura (Mérida, Spain).


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Received October 30, 2000; revised February 7, 2001; accepted February 8, 2001.