open access

Vol 68, No 4 (2009)
ORIGINAL ARTICLES
Published online: 2009-11-26
Submitted: 2012-02-06
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NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells

J. Antosiewicz, J.H. Spodnik, M. Teranishi, A. Herman-Antosiewicz, Ch. Kurono, T. Soji, M. Woźniak, A. Borkowska, T. Wakabayashi
Folia Morphol 2009;68(4):247-255.

open access

Vol 68, No 4 (2009)
ORIGINAL ARTICLES
Published online: 2009-11-26
Submitted: 2012-02-06

Abstract

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2’-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Abstract

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2’-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.
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Keywords

AAPH; ethanol; apoptosis

About this article
Title

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells

Journal

Folia Morphologica

Issue

Vol 68, No 4 (2009)

Pages

247-255

Published online

2009-11-26

Bibliographic record

Folia Morphol 2009;68(4):247-255.

Keywords

AAPH
ethanol
apoptosis

Authors

J. Antosiewicz
J.H. Spodnik
M. Teranishi
A. Herman-Antosiewicz
Ch. Kurono
T. Soji
M. Woźniak
A. Borkowska
T. Wakabayashi

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