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Abstract

Introduction: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and a major cause of morbidity and mortality throughout the world. Today we know that exposure to exogenous chemicals (xenobiotics) combined with a modified ability to detoxify carcinogens increases the risk of developing cancer. Xenobiotic metabolizing enzymes (XMEs) play a major role in the activation and detoxification of several carcinogens but their expression and activity in human colorectal tissue as well as their role in the development of CRC has not been sufficiently explored. In order to shed light on this question, this study aimed to link XME metabolism, lifestyle and risk factors with the etiology of CRC investigating two different aims. Therefore, the thesis is split into two parts:

1) Evaluation of mRNA, protein and enzyme activities of relevant phase I and phase II xenobiotic metabolizing enzymes in normal colorectal tissue of colorectal cancer patients: In this study, three different layers of XME abundance (mRNA, protein, and enzyme activity) were evaluated in cytochromes P450 (CYPs), glutathione S-transferases (GSTs), and UDP-glucuronosyltransferases (UGTs). Gene expression was assessed by quantitative real-time PCR (qRT-PCR), protein expression evaluated by immunoassay detection, and enzymatic activities measured by biochemical assays, in the normal tissue of 97 patients with CRC. The mean relative expression levels in normal colorectal tissue were highest for GSTP1 [mean (± standard deviation): 7.70 (0.60)] and lowest for GSTM1 [mean: 4.08 (1.80)]. Associations of xenobiotic metabolism-related gene expression, protein level and enzyme activities with clinical parameters in patients with CRC, were evaluated by the Mann-Whitney U-test and the Kruskal-Wallis test. Results of the univariate analysis revealed a 1.2-fold lower UGT1A8 expression and 1.7-fold lower UGT activity in normal tissue of rectal compared to colon cancer patients (p=0.008; pFDR=0.34 and p=0.002; pFDR=0.17, respectively). Furthermore, lower GSTP1 expression levels among recent nonsteroidal anti-inflammatory drug (NSAIDs) users compared to non-users (p=0.04; pFDR=0.58) were detected. Associations with lifestyle and dietary factors were evaluated using linear regression models. Results of the multivariable models adjusted for relevant covariables, showed that regular consumption (>1x/week) of cooked vegetables was associated with higher CYP3A4 protein levels (β=5.62, pn=0.009; pFDR=0.65) and regular consumption of raw vegetables was associated with lower UGT activities (β=-0.56; pn=0.03; pFDR=0.72) compared to non-regular consumption of raw and cooked vegetables (≤ 1x/week) in the normal mucosa of CRC patients.

Relation of mRNA expression, protein levels and enzyme activities were assessed using Spearman correlation coefficients. No statistically significant associations were found between mRNA expression and neither protein levels, nor enzymatic activities for the CYPs and GSTs. For the UGTs, statistically significant, albeit weak, positive associations between UGT1A8 protein and mRNA abundance (r=0.20, p≤ 0.05) and UGT1A10 mRNA levels and UGT activity (r=0.28, p ≤0.01) were observed. However, between UGT1A10 protein level and UGT activity, a statistically significant negative correlation (r= -0.27, p ≤0.01) was detected.

2) Evaluation of differentially expressed XMEs in normal and tumor colorectal tissue: The expression of eight selected XMEs (GSTP1, GSTA1, GSTM1, UGT1A10, UGT1A8, CYP2W1, CYP2C9, and CYP3A4) in colorectal carcinomas and adjacent normal mucosa (n=71) was compared and associations of sociodemographic, lifestyle and dietary factors with the expression of these genes were investigated. Differences between XMEs’ gene expression (IlluminaHT-12 Expression BeadChips) in tumor and normal mucosa were tested by the paired Wilcoxon-Rank-sum test. Among the genes analyzed, GSTM1, GSTA1, UGT1A8, UGT1A10, CYP3A4, and CYP2C9 were down-regulated in tumor tissue as compared to normal tissue, while GSTP1 and CYP2W1 were up-regulated. Linear regression models were used to evaluate potential associations between sociodemographic, lifestyle and dietary factors and the relative gene expression in tumor and normal mucosa tissue. Although none of these relationships remained statistically significant after false discovery rate (FDR)-adjustment for multiple testing, a trend toward significance (β=-0.21; pn=0.0005; pFDR=0.05) with lower CYP2C9 expression in normal tissue of rectal compared to colon cancer patients was detected. CYP2C9 plays a key role in the metabolic activation of many environmental and dietary mutagens and interactions of its expression with these factors should be considered in larger studies.

Overall conclusions: In summary, it can be concluded that in normal tissue of CRC patients: - Correlations between XMEs’ mRNA, protein and enzyme activities are moderate to poor - Colon and rectum showed considerable differences regarding expression and activities of several XMEs - Regular consumption of cooked vegetables was associated with CYP3A4 gene expression and protein levels Taken together, the results of this thesis suggest some interesting differences, which should be considered in larger studies to elucidate its potential contribution to CRC etiology.