Antiserum detection of reactive carbonyl species-modified DNA in human colonocytes

Nalini Mistry,Ruth J Bevan,Marcus S Cooke,Mark D Evans,Eugene P Halligan,Damon A Lowes,Karen Nichol,Joseph Lunec

doi:10.1080/10715760802008106

Nalini Mistry, Ruth J Bevan + Show 6 more

Open Access

https://doi.org/10.1080/10715760802008106

Copy DOI

Abstract

Polyunsaturated fats have been linked to occurrences of sporadic colon cancer. One possible cause may be degradation of polyunsaturated fats during cooking, resulting in multiple reactive carbonyl species (RCS) that can damage nuclear DNA and proteins, particularly in rapidly dividing colon crypt cells. This study describes a novel antiserum against RCS-modified DNA, with apparent order of reactivity to DNA modified with 4-hydroxy-trans-2-nonenal > glyoxal > acrolein > crotonaldehyde > malondialdehyde; some reactivity was also observed against conjugated Schiff base-type structures. Anti-(RCS-DNA) antiserum was successfully utilised to demonstrate formation of RCS-DNA in a human colon cell model, exposed to RCS insult derived from endogenous and exogenous lipid peroxidation sources. Further utilisation of the antiserum for immunohistochemical analysis confirmed RCS-modified DNA in crypt areas of ‘normal’ colon tissue. These results fully support a potential role for dietary lipid peroxidation products in the development of sporadic colon cancer.

Highlights

Reactive carbonyl species (RCS) are predominant products of lipid peroxidation, generated primarily as a result of oxidative degradation of lipid hydroperoxides [1, 2]
reactive carbonyl species (RCS)-modified DNA in colonocytes MDA modification of lysine residues. The application of this anti-(RCS-DNA) antiserum is illustrated by the immunocytochemical demonstration of RCS-modified DNA formation in undifferentiated human colonocytes exposed to products of endogenous and exogenous lipid peroxidation
Antigenicity of RCS-modified DNA At regular intervals throughout the immunisation schedule, test bleeds were screened by direct Enzyme-linked immunosorbent assay (ELISA) for reactivity against native DNA, the immunogen and DNA modified with individual RCS

Summary

Introduction

Reactive carbonyl species (RCS) are predominant products of lipid peroxidation, generated primarily as a result of oxidative degradation of lipid hydroperoxides [1, 2]. In an attempt to monitor global carbonyl stress (DNA and protein) we describe the development and characterisation of a novel polyclonal antiserum with apparent reactivity against (a) DNA modified with multiple RCS comprising MDA, HNE, GLY, CROT and ACR, and (b) conjugated Schiff base-type structures formed from

Results

Conclusion