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.

Cholesterol 7␣-hydroxylase (Cyp7a1) and the bile acid pool size are increased 2 to 3-fold in lactating postpartum rats. We investigated the interaction of nuclear receptors with the Cyp7a1 proximal promoter and the expression of regulatory signaling pathways in postpartum rats at day 10 (PPd10) versus female controls to identify the mechanisms of increased expression of Cyp7a1, which is maximal at 16 hours. Liver X receptor (LXR␣) and RNA polymerase II (RNA Pol II) recruitment to Cyp7a1 chromatin were increased 1.5-and 2.5-fold, respectively, at 16 hours on PPd10. Expression of nuclear receptors farnesoid X receptor (FXR), LXR␣, liver receptor homolog (LRH-1), hepatocyte nuclear factor 4␣ (HNF4␣), and short heterodimer partner ( C holesterol 7␣-hydroxylase (Cyp7a1) catalyzes the rate-limiting step of the classical pathway of bile acid synthesis and is the major mechanism for cholesterol catabolism and removal from the body. 1 Bile acids form mixed micelles with phospholipids and cholesterol in bile, which is essential for both the absorption and biliary excretion of cholesterol. Rodent Cyp7a1 expression increases in response to cholesterol-enriched diets, [2][3][4] resulting in increased bile acid synthesis, whereas feedback inhibition prevents the accumulation of cytotoxic hydrophobic bile acids. The regulation of Cyp7a1 expression is thus critical for maintenance of cholesterol and bile acid homeostasis.Several nuclear receptors activate transcription of Cyp7a1. Liver X receptor alpha (LXR␣) binds to LXR response elements in the proximal rat 5,6 and mouse 7

Lactation is associated with increased expression of bile acid transporters and an increased size and hydrophobicity of the bile acid pool in rats. ATP-binding cassette (ABC) transporters multidrug resistance protein 2 (Mdr2), Abcb11 [bile salt export pump (Bsep)], and Abcg5/Abcg8 heterodimers are essential for the biliary secretion of phospholipids, bile acids, and cholesterol, respectively. We investigated the expression of these transporters and secretion of their substrates in female control and lactating Sprague Dawley rats and C57BL/6 mice. Expression of Abcg5/Abcg8 mRNA was decreased by 97 and 60% by midlactation in rats and mice, respectively; protein levels of Abcg8 were below detection limits in lactating rats. Mdr2 mRNA expression was decreased in lactating rats and mice by 47 and 59%, respectively. Despite these changes in transporter expression, basal concentrations of cholesterol and phospholipid in bile were unchanged in rats and mice, whereas increased Bsep mRNA expression in early lactation coincided with an increased basal biliary bile acid concentration in lactating mice. Following taurocholate infusion, coupling of phospholipid and taurocholate secretion in bile of lactating mice was significantly impaired relative to control mice, with no significant changes in maximal secretion of cholesterol or bile acids. In rats, taurocholate infusion revealed a significantly impaired coupling of cholesterol to taurocholate secretion in bile in lactating vs. control animals. These data reveal marked utilization of an Abcg5/Abcg8-independent mechanism for basal biliary cholesterol secretion in rats during lactation, but a dependence on Abcg5/g8 for maximal biliary cholesterol secretion.

Doxorubicin (DOX), an anthracycline antibiotic commonly used as a cancer chemotherapeutic agent, is well known for its ability to induce acute and chronic cardiotoxicity, leading to irreversible cardiomyopathy. In the present study, we investigated the role of multidrug resistance‐associated protein 1 (Mrp1), an ATP‐binding cassette efflux transporter, in DOX induced cardiotoxicity. We cultured cardiomyocytes (CM) and cardiac fibroblasts (CF), which were isolated from C57BL/6J (WT) or Mrp1 knock out (KO) mice (1 – 3 days old). The cells were treated with DOX (0.5 – 3 μM) and examined 24 hrs later. DOX enhanced Mrp1 mRNA (~2 fold) and protein (~2.6 fold) expression similarly in both CM and CF. The MTS assay showed that the CM from KO mice were more sensitive to DOX than WT, with an IC50 in KO of 0.94 μM (95% CI: 0.74 – 1.18) versus 1.60 μM (95% CI: 1.20 – 2.11) in WT. Furthermore, CM and CF from KO mice showed increased caspase 3 and PARP cleavage following DOX treatment, consistent with increased apoptosis in cells from KO. These results imply that Mrp1 protects against DOX induced cardiotoxicity in cultured CM and CF. (CA139844)

Multidrug resistance‐associated protein 1 (Mrp1) is expressed in the sarcolemma of murine heart and effluxes endo‐ and xenobiotics. Doxorubicin (DOX) is a chemotherapeutic drug known to cause cardiactoxicity in cancer patients through oxidative stress. DOX significantly increases Mrp1 in whole heart, submitochondrial fractions, and mitochondria. Mrp1 is adversely affected during oxidative stress by adduction with 4‐hydroxy‐2‐nonenal. We postulate that Mrp1 serves a protective role in the heart by effluxing toxic products of oxidative stress. Wild type C57BL/6J (WT) or Mrp1 knock out (KO) mice were treated with DOX or saline and sacrificed at 24 or 72 hours. Heart tissue slices were used for electron microscopy and glutathione (GSH) and glutathione disulfide (GSSG) measured by HPLC. At 24 and 72h, significantly increased pathologic changes (p<0.05) were seen in cytoplasm and mitochondria of WT and KO mice, with significantly greater damage in mitochondria in both groups. At 72h, Mrp1 KO mice showed evidence of cytoplasmic edema and apoptotic nuclei (13%). GSSG levels were increased in KO mice at 72h (p<0.001). No changes were observed in GSH levels. The GSH to GSSG ratio was decreased in KO mice at 72h, indicating oxidative stress. These results indicate increased DOX induced cardiotoxicity in Mrp1 KO mice. Further studies are needed to elucidate the protective role of Mrp1 in cardiac tissue. NCI(1RO1CA139844‐D2)