Elevated Glutathione Is Not Sufficient to Protect against Doxorubicin-Induced Nuclear Damage in Heart in Multidrug Resistance–Associated Protein 1 (Mrp1/Abcc1) Null Mice

Abstract

Cardiotoxicity is a major dose-limiting adverse effect of doxorubicin (DOX), mediated in part by overproduction of reactive oxygen species and oxidative stress. Abcc1 (Mrp1) mediates the efflux of reduced and oxidized glutathione (GSH, GSSG) and is also a major transporter that effluxes the GSH conjugate of 4-hydroxy-2-nonenal (HNE; GS-HNE), a toxic product of lipid peroxidation formed during oxidative stress. To assess the role of Mrp1 in protecting the heart from DOX-induced cardiac injury, wild-type (WT) and Mrp1 null (Mrp1 2/2 ) C57BL/6 littermate mice were administered DOX (15 mg/kg) or saline (7.5 ml/kg) i.v., and heart ventricles were examined at 72 hours. Morphometric analysis by electron microscopy revealed extensive injuries in cytosol, mitochondria, and nuclei of DOX-treated mice in both genotypes. Significantly more severely injured nuclei were observed in Mrp1 2/2 versus WT mice (P 5 0.031). GSH and the GSH/GSSG ratio were significantly increased in treatmentnaïve Mrp1 2/2 versus WT mice; GSH remained significantly higher in Mrp1 2/2 versus WT mice after saline and DOX treatment, with no changes in GSSG or GSH/GSSG. GS-HNE, measured by mass spectrometry, was lower in the hearts of treatment-naïve Mrp1 2/2 versus WT mice (P , 0.05). DOX treatment decreased GS-HNE in WT but not Mrp1 2/2 mice, so that GS-HNE was modestly but significantly higher in Mrp1 2/2 versus WT hearts after DOX. Expression of enzymes mediating GSH synthesis and antioxidant proteins did not differ between genotypes. Thus, despite elevated GSH levels in Mrp1 2/2 hearts, DOX induced significantly more injury in the nuclei of Mrp1 2/2 versus WT hearts.

Loss of Multidrug Resistance–Associated Protein 1 Potentiates Chronic Doxorubicin-Induced Cardiac Dysfunction in Mice

“…We recently found that 72 hours after a single dose of DOX (15 mg/kg body weight, i.v. ), there is significantly more nuclear injury in the Mrp1 2/2 heart compared with the WT heart (Deng et al;2015). On the basis of these findings, we hypothesized that Mrp1 protects against DOX-induced cardiac dysfunction.…”

“…GS-HNE was somewhat elevated at 48 hours after DOX and significantly increased 2 weeks after the last dose of DOX, but this increase was similar in WT and Mrp1 2/2 mice. By contrast, after a single dose of DOX, GS-HNE was significantly decreased in WT mice relative to saline treatment, whereas there was no change in GS-HNE in Mrp1 2/2 mice (Deng et al, 2015). We have shown that in WT mice, a single dose of DOX (20 mg/kg i.p.)…”

“…higher GSH levels present in hearts of Mrp1 2/2 mice due to loss of GSH efflux (Deng et al, 2015), loss of Mrp1 potentiated DOX-induced cardiac dysfunction and greater nuclear damage.…”

“…The protein concentrations of heart ventricle homogenates were determined with the bicinchoninic acid protein assay. The measurement of GSH and GSSG by high-performance liquid chromatography was performed exactly as described (Deng et al, 2015) and used by others (Yamazaki et al, 2008(Yamazaki et al, , 2012Yamazaki and Villarreal, 2011;Kennedy et al, 2013;Barajas-Espinosa et al, 2014). The use of authentic GSH and GSSG to develop the standard curves ensured quantitation of the correct entities by high-performance liquid chromatography, thus validating the assay.…”

“…The heart ventricle was prepared by removal of the atrium and attached fat tissue and vessels. GS-HNE measurement by liquid chromatography-tandem mass spectrometry was performed exactly as described (Deng et al, 2015).…”