Formation of Acrolein-derived 2′-Deoxyadenosine Adduct in an Iron-induced Carcinogenesis Model

Yoshichika Kawai,Atsunori Furuhata,Shinya Toyokuni,Yasuaki Aratani,Koji Uchida

doi:10.1074/jbc.m309057200

Yoshichika Kawai, Atsunori Furuhata + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m309057200

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Abstract

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2' -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2' -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2' -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2' -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.

Highlights

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation
The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress
Based on these and the in vitro observations [6] that FDP-lysine was detected in the oxidatively modified low density lipoprotein with Cu2ϩ and that a metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, we proposed that polyunsaturated fatty acids might represent the potential sources of acrolein under oxidative stress

Summary

EXPERIMENTAL PROCEDURES

Calf thymus DNA, 2Ј-deoxyribonucleosides, acrolein, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. The reaction mixture (100 ␮l) of 2Ј-deoxyadenosine (2 mM) and acrolein (10 mM) was injected and fractionated (every 1 min) with a reversephase HPLC using a Develosil ODS-HG-5 column (4.6 ϫ 250 mm). The major product in the immunoreactive fraction was isolated with a reversephase HPLC using a Develosil ODS-HG-5 column (20.0 ϫ 250 mm) equilibrated in 1% acetonitrile containing 0.05% acetic acid at a flow rate of 6.0 ml/min. Calf thymus DNA (1 mg/ml) was mixed with aldehydes (10 mM) and incubated in phosphate buffer (pH 7.4) at 37 °C for 24 h. Cells postconfluency on glass coverslips was exposed to acrolein in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum

General Procedures

RESULTS

DISCUSSION