Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation

Is the hydroxyl radical really the most damaging radical species?

M. A. Khalid, Muhammad Ashraf

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (·OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of ·OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of ·OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83±0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of ·OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094±0.021 units/mg protein) and increased fivefold after 120 minutes (0.428±0.069 units/mg protein). Similarly, MDA increased from 0.78±0.14 nmol/mg protein at 15 minutes to 1.65±0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the ·OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 μM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate ·OH endogenously; 2) maximum ·OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in ·OH generation; 4) there was no significant production of ·OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional ·OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than ·OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.

Original languageEnglish (US)
Pages (from-to)725-736
Number of pages12
JournalCirculation research
Volume72
Issue number4
StatePublished - Jan 1 1993
Externally publishedYes

Fingerprint

Cardiac Myocytes
Hydroxyl Radical
Muscle Cells
Malondialdehyde
L-Lactate Dehydrogenase
Wounds and Injuries
Deferoxamine
Proteins
Cell Membrane Permeability
Trypan Blue
Salicylates
Enzymes
Horseradish Peroxidase
Blister
Cell Extracts
Fluorescent Dyes
Free Radicals
Permeability
Cell Survival
Coloring Agents

Keywords

  • anoxia
  • deferoxamine
  • diphenylphenylenediamine
  • high-performance liquid chromatography
  • hydroxyl radical
  • myocytes
  • reoxygenation
  • salicylate

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

@article{3f2e4c9d1aec4116afab29f51c33449a,
title = "Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation: Is the hydroxyl radical really the most damaging radical species?",
abstract = "Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (·OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of ·OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of ·OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83±0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of ·OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094±0.021 units/mg protein) and increased fivefold after 120 minutes (0.428±0.069 units/mg protein). Similarly, MDA increased from 0.78±0.14 nmol/mg protein at 15 minutes to 1.65±0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the ·OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 μM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate ·OH endogenously; 2) maximum ·OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in ·OH generation; 4) there was no significant production of ·OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional ·OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than ·OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.",
keywords = "anoxia, deferoxamine, diphenylphenylenediamine, high-performance liquid chromatography, hydroxyl radical, myocytes, reoxygenation, salicylate",
author = "Khalid, {M. A.} and Muhammad Ashraf",
year = "1993",
month = "1",
day = "1",
language = "English (US)",
volume = "72",
pages = "725--736",
journal = "Circulation Research",
issn = "0009-7330",
publisher = "Lippincott Williams and Wilkins",
number = "4",

}

TY - JOUR

T1 - Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation

T2 - Is the hydroxyl radical really the most damaging radical species?

AU - Khalid, M. A.

AU - Ashraf, Muhammad

PY - 1993/1/1

Y1 - 1993/1/1

N2 - Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (·OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of ·OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of ·OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83±0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of ·OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094±0.021 units/mg protein) and increased fivefold after 120 minutes (0.428±0.069 units/mg protein). Similarly, MDA increased from 0.78±0.14 nmol/mg protein at 15 minutes to 1.65±0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the ·OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 μM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate ·OH endogenously; 2) maximum ·OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in ·OH generation; 4) there was no significant production of ·OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional ·OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than ·OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.

AB - Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (·OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of ·OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of ·OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83±0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of ·OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094±0.021 units/mg protein) and increased fivefold after 120 minutes (0.428±0.069 units/mg protein). Similarly, MDA increased from 0.78±0.14 nmol/mg protein at 15 minutes to 1.65±0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the ·OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 μM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate ·OH endogenously; 2) maximum ·OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in ·OH generation; 4) there was no significant production of ·OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional ·OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than ·OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.

KW - anoxia

KW - deferoxamine

KW - diphenylphenylenediamine

KW - high-performance liquid chromatography

KW - hydroxyl radical

KW - myocytes

KW - reoxygenation

KW - salicylate

UR - http://www.scopus.com/inward/record.url?scp=0027461348&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027461348&partnerID=8YFLogxK

M3 - Article

VL - 72

SP - 725

EP - 736

JO - Circulation Research

JF - Circulation Research

SN - 0009-7330

IS - 4

ER -