Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats

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Abstract

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague–Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP ω-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.

Introduction

Doxorubicin (DOX, adriamycin) is a potent anthracycline chemotherapeutic agent used to treat a wide variety of human malignancies. However, the clinical use of this highly effective drug is limited by a significant DOX-induced cardiotoxicity which can progress to end-stage heart failure (Christiansen and Autschbach, 2006, Outomuro et al., 2007). The exact mechanism of DOX-induced cardiotoxicity and its progression to heart failure has not been fully elucidated yet; however, several mechanisms have been proposed. These mechanisms include increased oxidative stress, alteration of myocardial energy metabolism, altered molecular signaling, and apoptotic cell death (Nakamura et al., 2000, Ueno et al., 2006, Takemura and Fujiwara, 2007).

We have previously shown that DOX induces the expression of several cytochrome P450 (CYP) genes in the cardiac derived H9c2 cells (Zordoky and El-Kadi, 2008a). The importance of CYP enzymes in the cardiovascular physiology emerges from their ability to metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) (Roman, 2002). The cardioprotective effect of EETs has been demonstrated in ischemia–reperfusion injury (Seubert et al., 2007), cardiac hypertrophy (Xu et al., 2006), and recently in DOX-induced cardiotoxicity (Zhang et al., 2009). On the other hand, 20-HETE is known to have a detrimental effect in many cardiovascular diseases (Chabova et al., 2007, Lv et al., 2008, Minuz et al., 2008). Therefore, intricate homeostatic mechanisms are needed to keep the balance between these metabolites. Isoproterenol-induced cardiac hypertrophy has been shown to disturb this balance with increased formation of the cardiotoxic 20-HETE and decreased formation of the cardioprotective EETs (Zordoky et al., 2008). Therefore, we hypothesize that DOX-induced cardiotoxicity will cause a similar disturbance to the CYP-mediated arachidonic acid metabolism.

In addition to CYP enzymes, soluble epoxide hydrolase (sEH) is another major player in determining the level of EETs. The cardioprotective EETs are hydrolyzed by sEH to the less biologically active dihydroxyeicosatrienoic acids (DHETs) (Imig et al., 2002). EPHX2, the gene encoding sEH, has been found to be a susceptibility factor for heart failure (Monti et al., 2008). In addition, EPHX2 gene expression has been reported to increase in animal models of angiotensin II- and isoproterenol-induced cardiac hypertrophy (Zordoky et al., 2008, Ai et al., 2009). Moreover, sEH inhibitors have been shown to prevent and/or reverse the development of cardiac hypertrophy in several models (Xu et al., 2006, Loch et al., 2007, Ai et al., 2009).

Therefore, in the current study, we have investigated the effect of acute DOX cardiotoxicity on the expression of several CYP and sEH enzymes in the heart of male Sprague–Dawley (SD) rats. In addition, we investigated the effect of acute DOX cardiotoxicity on the formation of arachidonic acid metabolites to determine whether the changes in CYP and sEH expression have lead to changes in CYP-mediated arachidonic acid metabolites. Our findings show that DOX-induced cardiotoxicity causes induction of several CYP and EPHX2 genes in vivo as well as in vitro. In addition, our results provide the first evidence that DOX-induced cardiotoxicity is associated with alteration in cardiac CYP-mediated arachidonic acid metabolism.

Section snippets

Materials

High-Capacity cDNA Reverse Transcription Kit, SYBR Green SuperMix, and 96-well optical reaction plates with optical adhesive films were purchased from Applied Biosystems (Foster City, CA). Real-time PCR primers were synthesized by Integrated DNA Technologies Inc. (San Diego, CA) according to previously published sequences. Dulbecco's modified Eagle's medium (DMEM) base, arachidonic acid, 4-hydroxybenzophenone, and DOX were purchased from Sigma-Aldrich (St. Louis, MO). Amphotericin B was

Effect of DOX treatment on LDH and on the hypertrophic markers

In order to confirm the occurrence of acute cardiotoxicity by DOX treatment, serum LDH was determined. LDH was significantly increased in the serum of DOX-treated rats to about 180% of its control value at 24 h after DOX administration; however, LDH level was not changed at 6 and 12 h after DOX administration (Fig. 1A). In order to investigate the effect of acute DOX cardiotoxicity on the hypertrophic markers, we measured the cardiac gene expression of atrial natriuretic peptide (ANP) and brain

Discussion

We have previously demonstrated that DOX causes a concentration-dependent induction of several CYP genes in the cardiac derived H9c2 cells (Zordoky and El-Kadi, 2008a). The physiological importance of CYP enzymes depends on their ability to produce various metabolites that confer cardioprotective or cardiotoxic effects (Zordoky and El-Kadi, 2008b). With regard to their role in arachidonic acid metabolism, CYP enzymes are considered one of the major metabolic pathways for arachidonic acid in

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

This study was supported by a grant from the Heart and Stroke Foundation of Alberta, NWT, and Nunavut (to A.O.S.E.-K.). B.N.M.Z. and M.E.A. are the recipients of Egyptian Government Scholarships. A.A-M is the recipient of Mike Wolowyk graduate scholarship award. We thank Dr.Vishwa Somayaji for excellent technical assistance with the LC-ESI-MS. We are grateful to Dr. Bruce Hammock for providing us with tAUCB.

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