Antioxidants protect against reactive oxygen species associated with adriamycin-treated cardiomyocytes

doi:10.1016/S0304-3835(98)00306-1

Cancer Letters

Volume 136, Issue 1, 8 February 1999, Pages 41-46

https://doi.org/10.1016/S0304-3835(98)00306-1 Get rights and content

Abstract

Adriamycin (ADM) is a broad-spectrum antineoplastic antibiotic used to treat cancer patients. However, the usefulness of this drug is presently limited by the development of a dose-dependent cardiotoxicity. A current hypothesis for the ADM-induced cardiotoxicity is the production of reactive oxygen radicals by the drug. We utilized the fluorescent indicator 2′,7′-dichlorodihydrofluorescein diacetate (DCFH/DA), in which fluorescence appears if reactive oxygen species (ROS) are present, to investigate the ability of ADM to generate reactive oxygen species and the potential protective effect of antioxidants in a cultured cardiomyocyte model. All three of the antioxidants (alpha-phenyl-tert-butyl nitrone (PBN), trolox, and 5-aminosalicyclic acid (5-ASA)) tested in our ADM-treated myocytes provided protection against the oxidative stress induced by the drug. These findings suggest that antioxidants modulate ADM-induced oxidative stress, and they are discussed in terms of a possible therapeutic strategy in the prevention of cardiotoxicity resulting from ADM administration.

Introduction

Adriamycin (ADM) is an anthracycline antibiotic that is one of the most frequently used antineoplastic agents in the treatment of human malignancies [1]. However, the clinical effectiveness of ADM treatment is diminished due to the dose-limiting side effect of cardiotoxicity [2], [3]. A predominant hypothesis for the mechanism of ADM-induced cardiotoxicity involves the production of free radicals [4], [5], [6], [7]. For example, we showed that ADM treatment of erythrocytes leads to protein oxidation [8], a key marker of oxidative stress [9].

Studies of the ability of antioxidants to protect against the ADM-induced cardiotoxicity in animal models have not shown a consistent pattern. Reports of vitamin E being cardioprotective [10], [11] have been disputed by other investigators who have shown no changes in the mortality of animals during ADM administration [12], [13], [14]. N-Acetylcysteine has also been shown to be ineffective in the prevention of the ADM-induced cardiotoxicity in a rat model [15]. However, other antioxidants have demonstrated a protective effect. Paracchini et al. established that the spin trap agent alpha-phenyl-tert-butyl nitrone (PBN) significantly reduced the myocardial impairment in rats during ADM treatment [16]. Recently, Siveski-Iliskovic et al. showed that the antioxidant probucol was effective in preventing ADM-induced cardiomyopathy in a rat model [17].

A highly sensitive method of assessing oxidative stress in cultured cardiomyocytes involves use of an oxidant-sensitive fluorescence probe combined with high spatial and temporal resolution laser-scanning confocal microscopy. Sarvazyan [18] utilized this method to confirm an oxidative mechanism of ADM toxicity in cardiomyocytes. If ADM were to induce ROS oxidative stress, then one could predict that appropriate antioxidants should modulate the stress. The present study utilizing 2′,7′-dichlororfluorescein fluorescence demonstrates the ability of antioxidants to protect against ADM-generated reactive oxygen radicals in cardiomyocyte cultures.

Section snippets

Chemicals

Adriamycin HCl was purchased from the University of Kentucky Hospital pharmacy. 5-Aminosalicylic acid (5A-SA), alpha-phenyl-tert-butyl nitrone (PBN), and cell culture materials were purchased from Sigma Chemical Co. (St. Louis, MO.). Trolox was purchased from Fisher Scientific (Pittsburgh, PA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH/DA) was obtained from Molecular Probes (Eugene, OR).

Cardiomyocytes

Rat heart myoblasts H9c2 (2-1) were purchased from American Type Culture Collection (Rockville, MD)

Results and discussion

DCFH/DA is a sensitive fluorimetric probe of the production of oxidative stress in living cells [20]. DCFH/DA enters the cell and is deacetylated by esterases to DCFH which, because of the negative charge, remains enclosed within the cell. When the DCFH is oxidized by ROS, it is transformed into the highly fluorescent 2′,7′-dichlororfluorescein (DCF).

Fig. 1 is a representative picture of the DCF fluorescence in myocytes during the various treatments. It is obvious that ADM leads to ROS

Acknowledgements

This work was supported in part by grants from the NIH and from Centaur Pharmaceuticals, Inc.

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Antioxidants protect against reactive oxygen species associated with adriamycin-treated cardiomyocytes

Abstract

Introduction

Section snippets

Chemicals

Cardiomyocytes

Results and discussion

Acknowledgements

Life Sci.

Properties of a clonal muscle cell line from rat heart. Exp. Cell Res.

Free Rad. Biol. Med.

Life Sci.

Exp. Neurol.

Free Rad. Biol. Med.

Free Rad. Biol.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Free Rad. Biol. Med.

Neuroscience

Anthracycline analogs: the past, present, and future

Cancer Chemother. Pharmacol.

Analysis of some probable factors responsible for adriamycin-induced cardiotoxicity

A clinicopathologic analysis of adriamycin cardiotoxicity

Cancer

Role of oxygen radical formation in anthracycline cardiac toxicity

Effect of anthracycline antibiotics on oxygen radical formation in rat heart

Cancer Res.