Free radical-mediated transgene inactivation of macrophages by endotoxin

Citation
S. Dokka et al., Free radical-mediated transgene inactivation of macrophages by endotoxin, AM J P-LUNG, 279(5), 2000, pp. L878-L883
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
da verificare
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
ISSN journal
1040-0605 → ACNP
Volume
279
Issue
5
Year of publication
2000
Pages
L878 - L883
Database
ISI
SICI code
1040-0605(200011)279:5<L878:FRTIOM>2.0.ZU;2-M
Abstract
Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investig ated the effect of endotoxin on gene transfection efficiency and the role o f reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a r eporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expres sion in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicit y induced by endotoxin. Neutralizing the endotoxin by the addition of polym yxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin -treated cells and their inhibition by free radical scavengers. The ROS sca venger N-t-butyl-alpha -phenylnitrone, the H2O2 scavenger catalase, and the . OH scavenger sodium formate effectively inhibited endotoxin-induced effe cts, whereas the O-2(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that . OH formed by H2O2-dependent, m etal-catalyzed Fenton reaction play a major role in this process.