The study protocol was approved by the Ethics Committee of the medical association Stuttgart, Germany, and followed the ethical guidelines of the 1975 Declaration of Helsinki. Informed consent was obtained from all subjects. Colonic adenoma and surrounding normal colon mucosa biopsies (each 5–10 mg) were obtained during routinely performed coloscopies from 25 patients, aged 50 – 70 years (Table 1). In addition, biopsy specimens of normal colon mucosa were obtained from 27 disease-free controls during routine endoscopy (age 50 – 70 years) displaying no signs of colonic neoplasia or other diseases of the colon. Immediately after removal of biopsies, colon mucosa specimens were frozen in liquid nitrogen and stored at -80°C. All patients completed a questionnaire concerning factors that may influence the expression of cytochrome P450 such as medication, smoking, and alcohol consumption (Table 2). Neither anthropometrics nor medication differed significantly between the patient group and that of disease-free controls. Patients and disease-free controls did not consume drugs known to interfere with/ or to induce cytochrome P450 enzymes investigated in this study.

All colon mucosa specimens were immediately frozen in liquid nitrogen after excision and stored at -80°C. Both total RNA and protein were isolated using Trizol reagent (Invitrogene, Gaithersburg, MD, USA).

Antibodies used for the detection of CYP2E1 and CYP3A4 were a generous gift of Dr. M. Ingelman-Sundberg, Karolinska Institute, Stockholm, Sweden. Primary antibodies for the measurements of CYP2C8 and CYP3A5 were purchased from Chemicon, Inc. (Frankfurt, Germany). Microsomes and Supersomes were from cell lines/clones over-expressing human CYP2C8, CYP2E1, CYP3A4, and CYP3A5, respectively. They were used as internal standards for quantification and to test for cross reactivity of antibodies (Gentest Corporation, Woburn, MA, USA). Protein concentration was determined by the method of Bradford [15], using a commercial preparation (BioRad, Munich, Germany).

Twenty to 30 μg of total protein were separated by SDS-polyacrylamid gel electrophoresis and transferred to nitrocellulose membranes. To enable quantification of CYP protein, a serial dilution of the appropriate standard was proceeded identically. To ensure equal loading of samples, membranes were pre-stained with Ponceau red before blocking and incubation with antibodies. Membranes were blocked in 5% non-fat milk in Tris-buffered saline-Tween 20 (TBST, 0.01% v/v Tween 20) and probed with dilutions of primary antibodies in TBS, followed by an incubation with the secondary antibody. The protein/antibody complex was visualized by enhanced chemiluminescence (SuperSignal^® West Dura, Pierce, KTM, Bad Godesberg, Germany). Blots were evaluated (Camera LAS 1000, Fuji, USA) and densitometric analysis was performed using the software AIDA (Raytest, Isotopenmessgeraete, Straubenhardt, Germany). Signal intensities of the samples were adjusted to the intensities of the serially diluted standards. To ensure equal loading some blots were probed for β-actin (Sigma, St Louis, USA).

The integrity and concentration of RNA was analyzed in a 1.2% agarose gel. First-strand complementary DNA was synthesized from 200 ng of total RNA using a First-Strand cDNA Synthesis Kit (Invitrogen, Gaithersburg, MD, USA). Sequences of primers are summarized Table 3. The PCR reaction consisted of 0.6 μl of cDNA, 10 x PCR buffer, 200 μM dNTPs (Boehringer, Mannheim, Germany), BSA (0.25 mg/ml), DMSO (2% v/v), 0.5 μM of specific primer and 0.5 U Taq-polymerase (Promega, Madison, WI, USA), and water to a final volume of 10 μl. For amplifications of the four cytochrome P450 cDNAs, PCR-conditions were as follows: 3 s at 94°C, 3 s at 45°C, 30 s at 72°C, for 32 cycles. Amplification of histone 3.3 was performed applying the following conditions: 3 s at 94°C, 3 s at 45°C, and 30 s at 72°C for 30 cycles. All PCR amplifications were carried out in triplicate in a Rapid Cycler (Idaho Tec., USA) within the linear range of the reaction. PCR products were separated in a 1.5% agarose gel, stained with ethidium bromide and photographed using a digital camera from Biometra (Goettingen, Germany). To ensure the success of PCR, human liver cDNA was used as a positive control.

Results are presented as means ± standard error of the mean (SEM) unless otherwise indicated. Fisher's exact test was used to compare lifestyle data. The Mann-Whitney U-test was used for the comparison of relative mRNA concentration and protein levels measured in normal colon mucosa obtained from patients with adenoma and disease-free controls. Wilcoxon's t-test was used for the comparison of relative mRNA expression and protein concentration measured in normal colon mucosa and colonic adenoma of the same subjects. Differences accepted as significant had a significance level (P) of less than 0.05.

High quality, non-degraded mRNA was obtained from normal tissue of 20 disease-free controls and unaffected tissue as well as adenomas of 18 patients with colonic adenoma. Expression of histone 3.3, which was used as housekeeping gene, was detected in all samples. Figure 1 depicts representative agarose gels of RNA integrity and results of RT-PCR measurements. Results of CYP2C, CYP2E1, CYP3A4, and CYP3A5 mRNA expression are summarized in Figures 1C and 1D. When comparing the relative mRNA concentration of CYP2C, CP2E1, CYP3A4, and CYP3A5 between adenomatous tissue and normal tissue of patients with adenoma, significant differences were only found for the expression of CYP3A5. Specifically, CYP3A5 mRNA expression was significantly higher by ~48% in adenomatous tissue in comparison to normal colonic tissue. No differences were found when comparing CYP expression in normal tissue of patients with adenoma and disease-free controls.

Western blot analyses of CYP2E1, CYP3A4, and CYP3A5 were performed with specimens obtained from 25 patients with colonic adenoma and 27 disease-free controls. Since higher protein concentrations were needed for the detection of CYP2C8, protein concentration of CYP2C8 was only determined in biopsies obtained form 19 cases and 12 disease-free controls. No differences were found when comparing mean protein levels of CYP2C8, CYP2E1, CYP3A4, and CYP3A5 measured in normal and neoplastic tissue of patients with adenoma. Representative Western blots and quantitative analysis of blots are depicted in Figure 2.

In addition, protein levels of CYPs were also determined in colon mucosa biopsies of disease-free controls and compared with those determined in macroscopically normal tissue of patients with colonic adenoma. Figure 3 depicts representative Western blots and quantitative analysis of protein.

Protein levels of CYP2E1 in mucosal biopsies obtained from normal colon mucosa of patients with adenoma did not differ significantly from those measured in disease-free controls. In contrast, mean protein level of CYP2C8 was significantly lower in unaffected, macroscopically normal colon mucosa obtained from patients with adenoma than in samples obtained from disease-free controls. Specifically, protein concentration of CYP2C8 in colon mucosa obtained from patients with adenoma was ~85% lower than in tissue obtained from disease-free controls. Similar results were found when comparing protein levels of CYP3A4 and CYP3A5 between patients with adenoma and disease-free controls. CYP3A4 protein concentration was ~69% lower in colon mucosa of patients with adenoma in comparison to disease-free controls. Protein level of CYP3A5 was ~54% lower in biopsies obtained from patients with adenoma when compared to disease-free controls.

To further investigate whether differences found in protein levels of CYPs between normal tissue of patients with adenoma and disease-free controls are related to the mRNA expression of CYPs, protein levels of CYP2C8, CYP3A4, and CYP3A5 of subjects with detectable mRNA expression were compared to those with undetectable mRNA expression of the respective CYP. CYP2E1 was excluded from this comparison, since CYP2E1 expression was only detected in three disease-free controls and two patients with adenoma. No comparison was performed with adenomatous tissue since the sample sizes were too small (n < 5) for most of the CYPs investigated. Results are summarized in Figure 4.

Despite the fact that expression of CYP2C was detected in ~50 % of patients with adenoma and disease-free controls, no differences were found when comparing protein levels of patients with adenoma and disease-free controls with detectable and undetectable mRNA expression. Similar, protein levels of CYP3A4 in disease-free controls with undetectable mRNA expression did not differ from those with detectable mRNA expression of CYP3A4. In contrast, CYP3A4 protein levels of patients with adenoma with undetectable mRNA expression of CYP3A4 were significantly higher when compared to those with detectable mRNA expression. Specifically, protein levels were ~68 % higher in patients with adenoma with undetectable mRNA expression. In disease-free controls, protein concentration of CYP3A5 did not differ between disease-free controls with detectable und undetectable CP3A5 mRNA expression. Similar to CYP3A4, protein levels of CYP3A5 were ~46 % higher in patients with undetectable mRNA expression in comparison to those with detectable mRNA expression of CP3A5. However, due to a large inter-individual variability differences were not statistically significant (p = 0.098).

Carcinogens and pro-carcinogens present in the diet are critical environmental factors influencing the development of carcinoma in the large intestine. Cytochrome P450-mediated metabolism of (pro-)carcinogens has been shown – depending upon the compound – to either result in detoxification or toxification (for review see [1]) and might therefore have potential impact on the development of neoplasia. Furthermore, most human CYPs have been found to be genetically polymorphic and these polymorphisms may affect the enzyme expression and activity subsequently leading to an increased risk to develop several forms of cancer but also effecting treatment (for review see [16]). Only a few extensive studies on the mRNA and protein expression of cytochrome P450 in colonic tissue and colonic adenoma have been performed thus far. The presence of CYP2C-, CYP2E- and CYP3A- in normal and in neoplastic colonic mucosa along with substantial inter-individual variability has been reported by others before [12,14,17,18]. However, some of the available data are contradictory and most studies either determined mRNA expression or protein levels [14,18]. For example, Yokose et al. [14] showed the presence of CYP2C (CYP2C8-CYP2C19) protein in both unaffected and neoplastic human colon mucosa using immunohistochemical methods. In contrast, Western blot analyses of de Waziers et al. [18] and Massaad et al. [12], who both used conventional immunoperoxidase staining procedures, showed neither cytochrome P450 2C8-10 nor 2E1 to be present in normal colon mucosa and in colon carcinoma. McKay et al. [17], who used immunohistochemical methods, detected CYP3A protein more frequently in neoplastic tissue in colon mucosa specimens of patients with neoplasia than in the morphologically normal mucosa of these patients. Expression of CYP3A4 and CYP3A5 has been reported for human colon mucosa [19,20]. In the present study, the expression and protein levels of four representative CYPs were determined in macroscopically normal colon mucosa and adenoma of patients with adenoma and disease-free controls. At the level of mRNA expression, only the expression of CYP3A5 was found to be significantly altered between neoplastic tissue and normal, unaffected mucosa of patients with adenoma. However, as reported by others before, mRNA expression of all four CYPs varied extensively between individuals even though the expression of the housekeeping gene histone 3.3 was detected in all samples and was used for normalization. At the level of protein, CYP2C8, CYP3A4, and CYP3A5 protein concentration was found to be significantly lower in normal tissue of patients with adenoma than in colon mucosa of disease-free controls. Even though previous studies have indicated that the abundance of CYP protein is altered between neoplasia and normal tissue [17], in the present study protein levels of CYPs were found to be similar in neoplasia and normal tissue of patients with adenoma. Taken together, these data suggest that CYP expression not only varies extensively between individuals but that protein levels of some CYPs (e.g. CP2C8, CYP3A4, and CYP3A5) are considerably lower in normal tissue of patients with adenoma in comparison to those of disease-free controls.

It has been suggested that expression of CYPs is not solely regulated at the level of gene transcription [11]. This is supported by the results of animals studies, reporting a dissociation of mRNA expression and protein levels of CYP2C7 (corresponding to human CYP2C8) and CYP2E1 in rat colon mucosa as well as CYP3A4 and CYP3A5 in duodenum and kidney. Furthermore, in vitro studies performed in rat hepatocytes indicate that CYP2E1 is regulated by posttranscriptional ligand-dependent stabilization of the enzyme [21]. Similar mechanisms have been described for CYP3A in rats and humans [22,23]. Using cultured hepatocytes it also has been showed that only ~60–70% of mRNA encoding for CYP2E1 is translated [24]. Indeed, in the present study, no relation with respect to protein levels of subjects with detectable and undetectable mRNA expression of the CYPs was found. Instead, protein levels of CYP2C8, CYP3A4, and CYP3A5 did not differ in disease-free controls. In contrast, in normal tissue of patients with adenomas protein levels of CYP3A4 and CYP3A5 were contrary to mRNA expression pattern. Taken together, these data suggest that in colon mucosa the expression of CYP2C8, CYP3A4, and CYP3A5 is not solely regulated at the level of transcription and that the mechanism of regulation, at least for some CYPs (e.g. CYP3A4 and CYP3A5), might differ between patients with colonic adenoma and healthy subjects.