DISTRIBUTION OF CYTOCHROME

Distribution of cytochrome P450 2C, 2E1, 3A4, 3A5 in human colon mucosa

Abstract

Inspite of the reality that the alimentary tract is part of the body's first line of defense contrary to orally ingested xenobiotica, slight is known about the distribution and expression of cytochrome P450 (CYP) enzymes in human colon. Therefore, expression and protein grades of four representative CYPs (CYP2C(8), CYP2E1, CYP3A4, and CYP3A5) were determined in human colon mucosa biopsies got from ascending, descending and sigmoid colon. Expression of CYP2C, CYP2E1, CYP3A4, and CYP3A5 mRNA in colon mucosa was very resolute by RT-PCR. Protein concentration of CYPs was determined using Western blot methods. In this paper, the current data suggest that the expression of CYP2C, CYP2E1, and CYP3A5 varies in distinct parts of the colon.

Table of Contents

Chapter 1: Introduction4

Chapter 2: Literature Review6

Chapter 3: Research Methodology8

Research Design8

Literature Research8

Tissues and isolation of total RNA and protein9

Electrophoresis and immunoblotting9

Reverse transcription and PCR10

Statistical analysis11

Chapter 4: Conclusion12

Reference13

Appendix14

Chapter 1: Introduction

Throughout the last decades drug metabolism in the alimentary tract has received growing attention as part of the body's first line of defense contrary to orally ingested harmful xenobiotica. Most xenobiotic compounds need an enzymatic activation to form a carcinogen or toxicant. The reactive intermediates resulting from enzymatic drug metabolism are often unstable and thus are unlikely to be transported from the liver to other tissues to exert toxicity. Therefore, the chemical toxicity discovered in extrahepatic tissue often outcomes from cellular metabolic activities in the organ. However, knowledge on the variability and guideline of expression of drug metabolizing enzymes in the human gastrointestinal tract and particularly the large intestine is poor in comparison with the "classical" drug metabolizing organs (e.g. liver) (McKay JA, 2002).

Cytochrome P450 (CYP) is a multi-gene superfamily of heme-containing enzymes catalyzing the oxidative metabolism of numerous compounds. CYP families 1, 2, and 3, which are the major CYP families participating in the metabolism of xenobiotics, are highly conveyed inside the liver, but are furthermore conveyed in extrahepatic tissues (for reconsider see). Members of the CYP families 2 and 3, and herein particularly CYP2C and CYP3A4, are present in relation high concentrations in small intestinal epithelium, and it has been proposed that they facilitate a barrier function to defends the small intestine from toxic xenobiotics. Furthermore, it has been shown that total CYP content increases somewhat in the progression from duodenum to jejunum and subsequently declines considerably in the ileum. Despite the fact that it has been proposed that the nonattendance of some of these microsomal enzymes in the colon may be engaged in the comparably high incidence of carcinoma in this organ, information accessible on the expression of CYPs in the large intestine of humans is restricted and some of the accessible facts and numbers are contradictory. Therefore, the major reason of the present study was to evaluate the expression pattern, protein concentration, and distribution of four representative CYPs in ascending, descending and sigmoid human colon mucosa of virtually healthy subjects. Furthermore, protein levels were associated to mRNA expression pattern (Mercurio MG, ...