Differential cardiotoxicity in response to chronic doxorubicin treatment in male spontaneous hypertension-heart failure (SHHF), spontaneously hypertensive (SHR), and Wistar Kyoto (WKY) rats

Toxicology and Applied Pharmacology 273 (2013) 47–57

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

Differential cardiotoxicity in response to chronic doxorubicin treatment in male spontaneous hypertension-heart failure (SHHF), spontaneously hypertensive (SHR), and Wistar Kyoto (WKY) rats Leslie C. Sharkey a,⁎, M. Judith Radin b, Lois Heller c, Lynette K. Rogers d, Anthony Tobias a, Ilze Matise e, Qi Wang f, Fred S. Apple g, Sylvia A. McCune h a

Veterinary Clinical Sciences Department, University of Minnesota, 1352 Boyd Ave, St. Paul, MN 55108 USA Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210 USA c Department of Biomedical Sciences, University of Minnesota Medical School—Duluth, 1035 University Drive, Duluth, MN 55812-3031, USA d Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive, Columbus, OH 43205 USA e Veterinary Population Medicine Department, University of Minnesota, 1365 Gortner Ave, St Paul, MN, USA f Clinical and Translational Science Institute (CTSI), University of Minnesota, 717 Delaware St SE, Minneapolis, MN, USA g Department of Laboratory Medicine and Pathology, Hennepin County Medical Center and University of Minnesota, 701 Park Ave S, Minneapolis, MN USA h Department of Integrative Physiology, University of Colorado at Boulder, 354 UCB, Clare Small 114, Boulder, CO 80309, USA b

a r t i c l e

i n f o

Article history: Received 20 March 2013 Revised 28 July 2013 Accepted 10 August 2013 Available online 28 August 2013 Keywords: Doxorubicin Cardiotoxicity Hypertension Soluble epoxide hydrolase Epoxyeicosatrienoic acid Arachidonic acid

a b s t r a c t Life threatening complications from chemotherapy occur frequently in cancer survivors, however little is known about genetic risk factors. We treated male normotensive rats (WKY) and strains with hypertension (SHR) and hypertension with cardiomyopathy (SHHF) with 8 weekly doses of doxorubicin (DOX) followed by 12 weeks of observation to test the hypothesis that genetic cardiovascular disease would worsen delayed cardiotoxicity. Compared with WKY, SHR demonstrated weight loss, decreased systolic blood pressure, increased kidney weights, greater cardiac and renal histopathologic lesions and greater mortality. SHHF showed growth restriction, increased kidney weights and renal histopathology but no effect on systolic blood pressure or mortality. SHHF had less severe cardiac lesions than SHR. We evaluated cardiac soluble epoxide hydrolase (sEH) content and arachidonic acid metabolites after acute DOX exposure as potential mediators of genetic risk. Before DOX, SHHF and SHR had significantly greater cardiac sEH and decreased epoxyeicosatrienoic acid (EET) (4 of 4 isomers in SHHF and 2 of 4 isomers in SHR) than WKY. After DOX, sEH was unchanged in all strains, but SHHF and SHR rats increased EETs to a level similar to WKY. Leukotriene D4 increased after treatment in SHR. Genetic predisposition to heart failure superimposed on genetic hypertension failed to generate greater toxicity compared with hypertension alone. The relative resistance of DOX-treated SHHF males to the cardiotoxic effects of DOX in the delayed phase despite progression of genetic disease was unexpected and a key finding. Strain differences in arachidonic acid metabolism may contribute to variation in response to DOX toxicity. © 2013 Elsevier Inc. All rights reserved.

Introduction Increased success of cancer treatment has resulted in a growing population of cancer survivors, due in part to chemotherapeutics (Ganz and Abbreviations: BW, body weight; cTnT, cardiac troponin T; CYP, cytochrome P450; DHET, dihydroxyeicosatrienoic acid; DOX, doxorubicin; EET, epoxyeicosatrienoic acid; HE, hematoxylin and eosin; HETE, hydroxyeicosatetraenoic acid; LTD4, leukotriene D4; LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internal diameter in systole; %FS, left ventricular fractional shortening; SAL, saline; SBP, systolic blood pressure; sEH, soluble epoxide hydrolase; SHHF, spontaneous hypertension heart failure rat; SHR, spontaneously hypertensive rat; WKY, Wistar Kyoto rat. ⁎ Corresponding author. Fax: +1 612 624 0751. E-mail addresses: [email protected] (L.C. Sharkey), [email protected] (M.J. Radin), [email protected] (L. Heller), [email protected] (L.K. Rogers), [email protected] (I. Matise), [email protected] (Q. Wang), [email protected] (F.S. Apple), [email protected] (S.A. McCune). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.08.012

Hahn, 2008). Some of the most commonly used chemotherapeutic drugs, however, result in delayed toxicities, causing disease years to decades after treatment. Of childhood cancer survivors, 2/3 will experience at least one late-onset complication, and 1/3 will experience a severe or life threatening effect from treatment with chemotherapy, radiation, or a combination of the two (Landier and Bhatia, 2008). Anthracyclines are often successful in treating hematopoietic and solid tumors, but result in delayed development of subclinical to severe complications in some patients (Longhi et al., 2012; Octavia et al., 2012; Smith et al., 2010). Multisystemic late effects after doxorubicin treatment include cardiomyopathy, congestive heart failure and nephropathy (Fumoleau et al., 2006; Ganz and Hahn, 2008; Hudson et al., 2007; Jones et al., 2008; Santin et al., 2007; Steinherz et al., 1991). Congestive heart failure is a common disabling health risk in long-term cancer survivors, with exposure to high doses (250 mg/m2 or higher) of

48

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

anthracyclines increasing the relative risk of congestive heart failure compared with untreated siblings by a factor of five (Mulrooney et al., 2009; Volkova and Russell, 2011). Therefore, there is significant interest in understanding the role of predisposing factors, especially those of cardiovascular origin. Spontaneously hypertensive rats (SHR) have been used to evaluate effects of doxorubicin (DOX) in hypertension (Hazari et al., 2009; Herman et al., 1985; Herman et al., 1988; Herman et al., 1998; Zhang et al., 1996). Hypertension exacerbates cardiac and renal toxicity in studies in which measurements are collected during the treatment period. Progressive cardiac damage after exposure to DOX or its derivatives is observed in normotensive rats (Chugun et al., 2000; Cirillo et al., 2000; Lebrecht et al., 2003; Lebrecht et al., 2006); however, studies of delayed toxicity in strains with genetic cardiovascular disease such as the SHR and the spontaneously hypertensive heart failure rat (SHHF) are lacking. SHHF exhibit progressive systolic and diastolic dysfunction characterized by initial hypertension and compensated left ventricular hypertrophy followed by deleterious cardiac remodeling culminating in congestive heart failure (Heyen et al., 2002). While precise mechanisms for cardiomyopathy are not entirely known, SHHF rats develop characteristic histopathology (myocardial degeneration/ necrosis, fibrosis, mononuclear inflammatory infiltrates) and increased systemic and cardiac expression of inflammatory mediators. Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites that are products of cytochrome P-450 (CYP) epoxygenase enzymes (See Supplemental Fig. 1). EETs promote vasodilation, angiogenesis, and cardiac myocyte contraction, and inhibit inflammation, platelet aggregation, and cardiac hypertrophy (Imig, 2012). Soluble epoxide hydrolase (sEH) is the primary catabolic enzyme that degrades EETs into less cardioprotective dihydroxyeicosatrienoic acid isoforms (DHETs) (Imig, 2012). Acute DOX exposure modifies the cardiac expression of CYP and sEH enzymes in male Sprague–Dawley rats, thus reducing EETs and increasing DHETs, which may be a novel mechanism of DOX cardiotoxicity (Zordoky et al., 2010). Alterations in EETs and sEH activity in the central nervous system of SHR are implicated in the development of hypertension (Sellers et al., 2005). Linkage analysis and genomewide expression profiling demonstrated a mutation in the sEH gene of SHHF, causing up-regulation of transcription suggested to be associated with progression to heart failure (Monti et al., 2008). The purpose of our study was to examine the role of genetic predisposition to cardiovascular disease on late-onset DOX toxicity. Our hypothesis was that genetic hypertension would worsen late-onset DOX toxicity, and that hypertension and propensity to heart failure together would further accelerate progression. We compared the delayed toxic effect 12 weeks after the cessation of DOX treatment in normotensive rats (Wistar Kyoto, WKY), genetically predisposed hypertensive rats (SHR) and genetically predisposed hypertension and cardiomyopathy rats (SHHF). Although our data showed enhanced delayed toxic effects as compared to WKY, there was clearly an attenuated delayed toxic response in SHHF as compared to the SHR. Given the suggested role of EETs and sEH in DOX cardiotoxicity and in the development of hypertension and heart failure in the SHHF rat, we suspected that alterations in arachidonic acid metabolism may contribute to the observed strain differences in response to DOX. As proof of principle for the potential role of this mechanism in strain differences in response to DOX, we subsequently examined cardiac sEH content and arachidonic acid metabolite production by electrospray mass spectroscopy in response to acute DOX in the 3 strains of rats. As expected, cardiac sEH activity was higher and cardiac EETs were lower in untreated SHR and SHHF than WKY, however, paradoxically, SHHF and SHR rats significantly increased cardiac EETs in response to DOX with no significant change in cardiac sEH activity or DHETs. This suggests alternative mechanisms for EET metabolism and subsequent response to DOX in genetic disease but does not explain the observed differences in sensitivity between SHHF and SHR. DOX significantly increased cardiac levels of leukotriene D4 (LTD4) in SHR but not in WKY and SHHF, suggesting a potential role

for this leukotriene in DOX sensitivity in SHR. These data demonstrate the potential for a significant and complex role for arachidonic acid metabolism in influencing the interaction between the effects of DOX and genetic predisposition to cardiovascular disease that should be explored further in longer-term studies. Methods Assessment of delayed post-DOX toxicity Thirteen 8 to 10-week old SHR and WKY male rats and fourteen 8 to 10-week old phenotypically lean SHHF male rats were obtained from Charles River Laboratories. Male rats were chosen for this investigation due to the existing data documenting the response to DOX in shortterm studies and because of earlier expression of hypertension. All animals were housed in an AAALAC accredited facility according to NIH guidelines for the care and use of laboratory animals; protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. After a one week acclimation period, DOX rats received 8 weekly doses of pharmacologic grade doxorubicin (Bedford Laboratories, Bedford, OH 44146) at a dose of 2 mg/kg by subcutaneous injection (n = 7 for each strain). Saline treated control rats (SAL) received an equivalent volume of sterile saline (n = 7 for SHHF, n = 6 for SHR and WKY). Injection sites were rotated to avoid repeated use of a single site. An 8 dose protocol was determined based on preliminary studies to optimize the protocol. During the 12 weeks following the final DOX injection, rats were monitored for general health as well as signs of heart failure (cyanosis, tachypnea and increased respiratory effort, edema, or body cavity effusions). Body weights (BWs), systolic blood pressures (SBPs), and echocardiography studies were performed at 1 and 12 weeks after the last dose of DOX or saline (designated week 1 and week 12) to evaluate the progression of delayed toxicity. Blood samples for the determination of serum cardiac troponin T (cTnT) were collected at 1 and 12 weeks after the last dose of DOX or saline and frozen at − 80 °C until analysis. Unless euthanasia was indicated earlier for humane reasons, rats were humanely euthanized 12 weeks after the final DOX injection by isoflurane anesthesia followed by CO2. Necropsies were performed to document gross lesions, obtain organ weights for heart and kidneys, and to collect heart and renal tissue for histopathologic studies. Systolic blood pressure measurement (SBP) Rats were acclimated to the tail cuff blood pressure method. Briefly, rats were gently restrained in a warmed environment. Tail cuff measurements were taken using a BP-2000 Blood Pressure Analysis System™ for mice and rats (Visitech Systems, Inc., Apex, NC). The average of 3 stable readings was recorded. Echocardiographic studies Echocardiography was performed by a board certified veterinary cardiologist (AT) who was blinded to strain and treatment group. Anesthesia was induced with 5% isoflurane in a chamber until movement ceased. Anesthesia was then maintained with isoflurane administered via face mask with concentration reduced to 1–2% titrated to the lightest anesthetic plane that eliminated movement and retraction of limbs during restraint for echocardiography. Echocardiography was performed using an ATL 5000CV ultrasound system (Philips Medical Systems, Maplewood, MN) and a 12 to 5 MHz multifrequency transducer. All images were captured digitally for off-line analysis. Right parasternal echocardiography was performed to obtain both long- and short axis two-dimensional imaging planes, followed by routine M-mode echocardiography. M-mode measurements (to the nearest 0.1 mm) included left ventricular internal diameter in diastole (LVIDd), left ventricular internal diameter in systole (LVIDs), aortic root diameter, and left atrial

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

dimension at end-systole. Fractional shortening (%FS) was calculated as (LVIDd − LVIDs) / LVIDd × 100. The possibility of fluid retention and volume loading was assessed by normalizing LVIDd to body weight and making comparisons between groups. Assuming no differences in cardiac or vascular compliance, an increase in the LVIDd/BW ratio over the control group could be attributed to fluid retention and a compensatory increase in cardiac preload, however the potential for dilation of the left ventricle secondary to apoptosis or autophagy cannot be fully excluded. Serum cardiac troponin T measurement (cTnT) Serum cTnT was measured using a commercially available electrochemiluminescence immunoassay kit validated for rats (Roche Diagnostics, Indianapolis, IN, Apple et al., 2008) Because of skewness of the data, the change from week 1 to week 12 was calculated as LN(week 12 cTnT) − LN(week 1 cTnT). Histopathologic evaluation Tissue sections collected from the left ventricular free wall and the left kidney were fixed in 10% neutral formalin, processed and embedded in paraffin using standard methods. Four-micron tissue sections were cut and stained with hematoxylin and eosin (HE). Histopathologic evaluation was done in blinded fashion by a single board certified veterinary pathologist (IL). The effect of DOX on myocardium of WKY, SHR, and SHHF rats was assessed by light-microscopic examination of one HE section of the left ventricular myocardium from each rat for the following lesions: (1) myocyte vacuolization and loss of myofibrils (according to scoring scheme described by Herman et al., 1985), (2) myocyte necrosis characterized by coagulation necrosis and coagulative myocytolysis (Zenker's necrosis), and (3) interstitial proliferation and inflammation. Each lesion was scored separately on 0–4+ scale. Score 0 was given if the lesion was not present; 1+ if the lesion was minimal (rare and/or minimal myocyte vacuolization/necrosis and inflammation); 2+ if the lesion was mild (a few myocytes with several large cytoplasmic vacuoles and myofibril loss/necrosis and inflammation); 3+ if the lesion was moderate (moderate numbers of myocytes with several large cytoplasmic vacuoles/necrosis and moderate numbers of foci with interstitial proliferation and inflammation); 4+ if the lesion was severe (extensive, marked myocyte vacuolization involving more than 50% of myocytes/frequent myocyte necrosis/extensive interstitial proliferation and inflammation). The total score of myocardial damage was the sum of individual scores (range of possible score of 0–12). The effect of DOX on kidneys of WKY, SHR, and SHHF rats was assessed by light-microscopic examination of one HE section of left kidney from each rat. A 1–4+ scale was used as described above. The following lesions were assessed in each kidney: (1) the presence of glomerulonephritis with increased mesangial matrix, mesangial or epithelial cell vacuolization, distention of capillary loops and adhesions between parietal and visceral podocytes, (2) tubular lesions included dilatation, protein casts, epithelial cell degeneration, atrophy and regeneration, and basement membrane thickening, (3) interstitial fibrosis and inflammation. The total score of renal damage was sum of individual scores (range of possible score of 0–12). EETs and DHETs in response to acute doxorubicin treatment 8–10 week old rats of each strain (WKY, SHR, SHHF, n = 14/strain) were obtained from Charles River Laboratories and housed under conditions described above. After a one week accommodation period, each DOX rat (n = 7/strain) received a single intraperitoneal injection of DOX at a dose of 15 mg/kg as described by Zordoky et al., 2010. Nontreatment rats received a similar volume of sterile saline (SAL, n = 7/ strain). Rats were euthanized 24 h after DOX treatment by intraperitoneal

49

injection of 100 mg/kg sodium pentobarbital. Tissue was collected from the left ventricle, snap frozen in liquid nitrogen and stored at − 80 °C until analysis for sEH protein and arachidonic acid metabolite concentrations. Western blot analysis for sEH protein Tissue from the left ventricles of the acutely treated rats was homogenized and protein was extracted for Western blot analysis using an SDS-PAGE gel (Barakat et al., 2001; Zordoky et al., 2010). 15 μg of protein were loaded into each well. Proteins were transferred to a nitrocellulose membrane, which was washed and incubated with blocking buffer (5% BSA in 1× TBS-Tween) for 1 h at room temperature. Blots were washed and then incubated overnight at 4 °C with primary antibody for either sEH (polyclonal rabbit anti-mouse diluted 1:1500 in 1× TBS-Tween/5% BSA, courtesy of Christophe Morisseau, UC Davis, CA; Yamada et al., 2000) or GAPDH (diluted 1:2000, sc-25778, Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a loading control. Membranes were then rinsed and washed prior to incubation for 1 h at room temperature with the secondary antibody, goat anti-rabbit ECL Plex Cy5 (GE Healthcare Life Sciences, Pittsburgh, PA) diluted 1:1000 in blocking buffer (5% BSA in 1× TBS-Tween). Blots were again washed and rinsed (5% BSA in 1× TBS without Tween), then scanned on a Typhoon 9410 Variable Mode Imager (Amersham Biosciences, Piscataway, NJ.) at 650 V using the red laser (633) and 670 BP 30 filter setting. Bands were quantified using ImageQuant TL 1D gel analysis (GE Healthcare Life Sciences, Pittsburgh, PA). Arachidonic acid metabolite determination by high pressure liquid chromatography (HPLC)/electrospray mass spectrometry Cardiac tissue from the acutely treated rats was pulverized over liquid nitrogen then homogenized on ice using a Power Gen Homogenizer equipped with a sawtooth generator (Fisher, Fair Lawn, NJ) in a 1 M potassium hydroxide (KOH) solution containing 0.1% butylated hydroxytoluene. The samples were spiked with a mixed deuterated internal standard (Cayman Chemical, Ann Arbor, MI) and placed on ice for 1 h allowing for tissue hydrolysis to occur, then neutralized to a pH of 5 using 1 M hydrochloric acid (HCl) containing 5% NaCl. The samples were extracted with chloroform:methanol (2:1) using 4× volume the first extraction and 2× volume the second extraction and then combining. The extracts were dried under a stream of nitrogen, and reconstituted in 200 μL ethanol followed by 1.8 mL water, pH 3 (using HCl). This was applied to certified C-18 Sep-Pak columns (3 cm3, 500 mg) (Waters, Milford, MA) preconditioned with one bed volume each of methanol, water, and water/HCl, pH 3. After the samples were loaded, the columns were washed with 5 mL of water/HCl, pH 3 and 5 mL 10% methanol, then 1 mL hexane. The samples were eluted with 3 mL methanol, dried under nitrogen, and finally reconstituted in 100 μL ethanol. Samples were analyzed by an AB Sciex 4000 QTrap LC/MS/MS (AB Sciex, Framingham, MA, USA) equipped with a Shimadzu HPLC (Cambridge, MA). A binary system set at a flow rate of 0.3 mL/min executed a gradient elution with 8.3 mM acetic acid, pH 5.7 with ammonium hydroxide (mobile phase A) and acetonitrile:2-propanol (50:50) (mobile phase B) as follows: 3 min hold at 15% B, 10 min linear to 55% B, 15 min linear to 80% B, 5 min wash at 100% B, 7 min re-equilibration at 15% B on a Zorbax SB-C18 Narrow Bore column, 2.1 × 100 mm, 5 μm, with a corresponding guard column at 40 °C. The injection volume was 20 μL. The samples were analyzed by Multiple Reaction Monitoring using electro-spray ionization in negative mode. Individual calibration curves were generated for each metabolite, and sample concentrations were calculated as ng of metabolite/g of tissue (Harkewicz, 2007; Hevko et al., 2001). Metabolites measured included epoxyeicosatrienoic acids (5,6EET, 8,9EET, 11,12EET and 14,15EET) and their respective DHET isomers, leukotrienes (B4, C4, D4, and E4), lipoxin A4, prostaglandins (F2-

50

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

α, E2, and D2), thromboxane B2, 8-iso-prostaglandin F2-α, 9-Shydroxyoctadecadienoic acid, 13-S-hydroxyoctadecadienoic acid, and hydroxyeicosatetraenoic acids (5-S-HETE, 8-S-HETE, 9-S-HETE, 11-SHETE, 12-S-HETE, 15-S-HETE, and 20-S-HETE). Statistical analysis To focus on the changes occurring as a result of the delayed phase of toxicity that are novel to this investigation, and to normalize for expected strain variations and the effects of DOX during the initial 8 week dosing period, data were analyzed as change over time from one week to 12 weeks after the last dose of DOX for data. The changes from week 1 to week 12 for SBP, BW, FS, LVIDd/BW, and cTnT were calculated by subtracting the measures at week 1 from the corresponding measures at week 12. Continuous terminal measures included (heart weight, atrial weight, right ventricular weight, left ventricle + septum weight, combined kidney weight and Western blot analysis of sEH). Two-way analysis of variance (ANOVA) F-tests were performed to evaluate the effect of treatment, strain, and the interaction between treatment and strain. This was followed by F tests with a multiple comparisons analysis of the least square (LS) means. Tukey–Kramer adjustment for the p-values was used to account for multiple testing. Arachidonic acid metabolites were assessed for outliers using the Grubbs test and for normality using the Shapiro–Wilk test. Strain differences within a treatment were compared by 2-way ANOVA with Bonferroni multiple comparison test for mean separation. For the rest of the measures which were all ordinal including histopathologic grading of lesions, a non-parametric method (the Kruskal– Wallis test) was used (the normality assumption of ANOVA was not met) to evaluate whether the distribution functions were equal across the three strains for each treatment or across the two treatments for each strain. Overall effects of treatment and strain were also assessed using the same methods. SAS or GraphPad statistical packages were used for performing the statistical analysis. Results Systemic indicators of delayed DOX toxicity: mortality, body weight, and SBP There were no deaths in any of the experimental groups during the eight week treatment period and all rats appeared healthy at the end of the treatment. In contrast to all other groups, the DOX-SHR group had 3 rats die spontaneously during the last week of the post-treatment observation period. Gross lesions at necropsy included pallor, cachexia, and marked thoracic and abdominal effusions characterized by a thin clear transudative fluid. Kidneys were enlarged, yellow, and pitted. Over the 12 week post-treatment period, DOX-WKY exhibited similar weight gain to the SAL-WKY. So while they had a lower starting weight at week 1, they resumed growth once the DOX was withdrawn. In contrast, DOX-SHHF exhibited growth restriction compared to SAL-

Fig. 1. A) Body weights at 1 week and 12 weeks after 8 weekly doses of 2 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). Weight gain was determined as the difference in body weight between week 12 and week 1. The interaction between treatment and strain was significant (p b 0.0001). B) Tail cuff systolic blood pressure (SBP) in mm Hg at 1 week and 12 weeks after 8 weekly doses of 2 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). The change in SBP was determined as the difference in SBP between weeks 12 and 1. The interaction between treatment and strain was significant (p = 0.0014). C) Cardiac troponin T (cTnT) in μg/L at 1 week and 12 weeks after 8 weekly doses of 2 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). The increase in the cTnT was determined as the difference between week 12 and week 1. The interaction between treatment and strain was significant (p b 0.0001). Data expressed as mean ± SE. Statistical analysis was done on the change from week 1 to week 12 and indicated as follows: *p b 0.05, ***p b 0.001 indicates significant difference between DOX and SAL within a strain; †p b 0.001 indicates DOX-SHR is significantly different from DOX-WKY and DOX-SHHF; ††p b 0.001 indicates SAL-SHR and SAL-SHHF are significantly different from SAL-WKY.

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

SHHF and DOX-SHR exhibited severe weight loss compared to SAL-SHR (Fig. 1A). Similar to body weight, there were no differences between the strains in the change in SBP over the 12-week period for SAL. SHR again demonstrated a more severe effect of DOX in the delayed phase, with a significant fall in SBP in DOX-SHR rats despite no effect in DOX-WKY or DOX-SHHF (Fig. 1B).

Indicators of cardiotoxicity: LVIDd/body weight ratio, % FS, serum cTnT, heart weight, and cardiac histopathology Changes in the ratio of LVIDd to BW may suggest changes in fluid retention. In the WKY and SHHF strains, changes in the ratio of LVIDd/BW during the delayed phase were minimal and not statistically significant between treatment groups or strains (SAL-WKY = −0.08 ± 0.27, DOXWKY = − 0.29 ± 0.26, SAL-SHHF = − 0.31 ± 0.24, DOX-SHHF = 0.13 ± 0.32 mm/g). SAL-SHR rats demonstrated a similar change in the LVIDd/BW ratio (−0.27 ± 0.12 mm/g); however, in the DOX-SHR group, the LVIDd increased considerably at the same time that body weight decreased, significantly increasing the LVIDd/BW ratio between weeks 1 and 12 (1.0 ± 0.37 mm/g; p b 0.0001). This finding indicates left ventricular dilatation and may suggest fluid loading and increased cardiac preload in DOX-SHR that parallels mortality, weight loss, and falling SBP in the post-treatment period. LV fractional shortening data did not reveal any significant strain, treatment or interaction effects during the 12 week post-treatment period. The %FS for week 1 and week 12 were as follows for each group (± SEM): SAL-WKY 42% ± 2 and 36% ± 4, SAL-SHR 29% ± 3 and 18% ± 2, SAL-SHHF 29% ± 2 and 25% ± 2, DOX-WKY 37 ± 3 and 29.5 ± 3, DOX-SHR 24% ± 2 and 17% ± 2, DOX-SHHF 25% ± 3 and 14% ± 2. The SHR and SHHF tended to have lower %FS than the WKY at both week 1 and week 12. All groups tended to show a decline %FS but the change over time was not significantly different between groups (SAL-WKY = − 6.1 ± 3.1%, SAL-SHR = − 11.5 ± 3.1%, SAL-SHHF = − 3.9 ± 2.9%, DOX-WKY = − 7.7 ± 2.9%, DOXSHR = − 9.0 ± 3.4%, DOX-SHHF = − 10.7 ± 2.9%). Within SAL groups, the increase in cTnT was greater in SAL-SHR (p b .0001) and SAL-SHHF (p b .0001) than in SAL-WKY. DOX caused a significantly greater increase in serum cTnT compared with SAL in WKY (Fig. 1C). This is in contrast to the absence of a treatment effect in DOX-SHR or DOX-SHHF rats with no measurable exacerbation of cTnT elevation beyond that associated with genetic disease (p b .0001 for a strain effect). Thus the sensitivity to DOX in SHR was not specifically reflected in significantly worse left ventricular function or cardiac biomarker indicators. Expected strain effects were observed with SHR and SHHF having greater overall total heart (p = 0.0008) and left ventricle + septum weights (p = 0.0002) than WKY due to the hypertrophy associated

Table 1 Total heart, right ventricle (RV), left ventricle and septum (LVS), and total kidney weights at week 12 following the last dose of 2 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). Group

Strain

Total heart (g)

RV (g)

LVS (g)

Kidney weight (g)

SAL

WKY SHR SHHF

1.193 ± 0.087 1.413 ± 0.105 1.491 ± 0.035

0.197 ± 0.022 0.223 ± 0.011 0.237 ± 0.015

0.883 ± 0.061 1.028 ± 0.084 1.087 ± 0.037

2.12 ± 0.13 2.37 ± 0.17 2.64 ± 0.16

DOX

WKY SHR SHHF

1.161 ± 0.043 1.559 ± 0.121a 1.403 ± 0.052

0.189 ± 0.013 0.213 ± 0.032 0.206 ± 0.017

0.809 ± 0.019 1.037 ± 0.056b 1.029 ± 0.027b

2.73 ± 0.09 3.66 ± 0.29a,c 3.48 ± 0.10a,c

Data expressed as mean ± SE, n = 6–7/group. a p b 0.01 significantly different from WKY within a treatment. b p b 0.05 significantly different from WKY within a treatment. c p b 0.01 significantly different from SAL within the same strain.

51

with hypertension (Table 1). However, when analyzed as individual treatment groups, these differences only achieved significance in the DOX groups. The distribution of scores for all cardiac lesion subtypes comprising the cumulative score is described in Table 2, while cumulative scores were used for statistical analysis. SAL-SHHF rats demonstrated more significant baseline pathology, including myocyte vacuolization and fibril loss, necrosis, and non-cardiomyocyte proliferation compared with minimal lesions in the other SAL-treated strains, consistent with their genetic predisposition to heart failure. DOX exacerbated cardiac pathology most significantly in SHR and less so in WKY, whereas paradoxically, SHHF appear protected from further drug-induced damage (Table 2 and Fig. 2). Cardiac pathology in DOX-WKY was primarily driven by balanced increases in myocyte vacuolization/loss and interstitial cell proliferation, while in DOX-SHR, globally increased lesions were dominated by interstitial cell proliferation and inflammation. Kidney weights and histopathology While no strain differences in kidney weights were observed in SAL, DOX increased kidney weight in all strains, but only significantly so in DOX-SHR and DOX-SHHF (Table 1). While both strain and treatment significantly impacted kidney weight (both p b .0001), these two factors appeared to act independently since their interaction factor was not significant (p = 0.15). The distribution of scores for all renal lesion subtypes comprising the cumulative score is described in Table 3, while cumulative scores were used for statistical analysis. SAL-SHR had mild renal lesions, predominantly due to mild interstitial inflammation and fibrosis. Renal lesions were not observed in SAL-WKY or SAL-SHHF. DOX caused moderate renal lesions with a slightly greater glomerular component in DOX-WKY. In DOX-SHR and DOX-SHHF, the increase was balanced between lesion types and more substantial. Histopathologic findings are demonstrated in Fig. 3. Cardiac sEH protein content and arachidonic acid metabolism following acute DOX treatment Determination of left ventricular sEH content showed the previously described strain differences, with significantly higher sEH in the hearts of SAL-SHR and SAL-SHHF rats compared with SAL-WKY (Fig. 4). While the sEH content of the SHR hearts appeared intermediate between WKY and SHHF, the differences between SHR and SHHF were not statistically significant. Acute DOX treatment failed to increase cardiac sEH in any of the strains. As anticipated, the concentrations of all EET isomers (5,6-EET, 8,9EET, 11,12-EET, 14,15-EET, and total EET), were greater in SAL-WKY than SAL-SHHF (Fig. 5A–E). EETs in SAL-SHR were either similar to SAL-SHHF (5,6-EET, 14,15-EET, and total EET), or were intermediate between SAL-WKY and SAL-SHHF (8,9-EET and 11,12-EET). This supports increased catabolism of EETs as a result of increased expression of cardiac sEH. DOX failed to significantly alter cardiac EETs compared with SAL in WKY rats (Fig. 5). In contrast, DOX tended to increase expression of all EET isomers and total EETs in SHHF rats to a level similar to the WKY, with p-values for specific isomers ranging from 0.055 to 0.099. DOXSHR showed a similar trend for increased cardiac EET. There were no statistically significant strain or treatment differences in the DHET metabolites (Table 4) or for EET/DHET ratios (data not shown). There were no differences in production of HETEs either by CYP ω-hydrolase (20-S-HETE) or by lipoxygenase pathways (Table 5). Cyclooxygenasemediated prostaglandin production was unchanged by DOX treatment. Of the additional arachidonic acid metabolites that were measured only LTD4 showed any strain or treatment effect (Fig. 5). Cardiac LTD4 content was lower in SAL-SHR compared to SAL-WKY while SAL-SHHF was intermediate. Cardiac LTD4 concentration significantly increased in DOX-SHR compared with SAL-SHR (Fig. 5F).

52

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

Table 2 Distribution of left ventricular histopathology scores with summary statistics of mean cumulative scores based on evaluation of hematoxylin and eosin (HE) stained sections. Each cell contains the number of animals with the corresponding score to reflect the frequency of distribution of each score. Mean, median, and 95% confidence interval (CI) are reported for the cumulative heart score. Group

Strain

Myocyte vacuolization; loss of myofibrils score 0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

Mean

Median

95% CI

SAL

WKY SHR SHHF

2 4 0

4 2 6

0 0 1

0 0 0

0 0 0

5 3 0

1 3 7

0 0 0

0 0 0

0 0 0

3 1 0

2 5 2

1 0 5

0 0 0

0 0 0

1.50 1.67 3.86

2 1.5 4a

0.2–2.8 0.8–2.5 3.2–4.5

DOX

WKY SHR SHHF

0 0 0

0 0 2

6 3 5

1 4 0

0 0 0

1 0 3

5 3 3

1 4 1

0 0 0

0 0 0

0 0 0

1 0 1

4 0 2

2 5 4

0 2 0

5.29 7.43 4.86

5b 7c 5

4.1–6.4 6.9–7.9 3.5–6.2

a b c

Coagulation necrosis; coagulative myocytolysis score

Interstitial cell proliferation; inflammation score

Cumulative heart score/12

p = 0.01 indicates SHHF is significantly different from WKY and SHR within SAL. p b 0.01 for DOX is significantly different from SAL within a strain. p b 0.05 indicates SHR is significantly different from WKY and SHHF within DOX.

Discussion Our results support the hypothesis that a genetic predisposition to cardiovascular disease is a risk factor for delayed-onset complications

of cancer chemotherapy. In people, incomplete genetic characterization of disease, variation in treatment protocols, and long time frames make it difficult to establish these links. Rodent models support an association between hypertension and susceptibility to the effects of DOX during

Fig. 2. Cardiac lesions in Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), and spontaneously hypertensive and heart failure (SHHF) male rats 12 weeks after the last dose of 8 weekly doses of saline (SAL) or 2 mg/kg doxorubicin (DOX). No microscopic lesions are present in the hearts of SAL-WKY (A) or SAL-SHR (C). There is mild interstitial proliferation in SAL-SHHF (E) rats. (B) Moderate, multifocal myocyte vacuolization (arrows) in DOX-WKY rat. (D) Mild myocyte vacuolization (arrow) in DOX-SHR rat. (F), Mild interstitial proliferation and minimal inflammation (arrows) in DOX-SHHF rat. Hematoxylin and eosin stain.

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

53

Table 3 Distribution of kidney histopathology scores with summary statistics of mean cumulative scores based on evaluation of hematoxylin and eosin (HE) stained sections. Each cell contains the number of animals with the corresponding score to reflect the frequency of distribution of each score. Mean, median, and 95% confidence interval (CI) are reported for the cumulative kidney score. Group

Strain

Glomerular lesions score 0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

Mean

Median

SAL

WKY SHR SHHF

6 5 7

0 0 0

0 1 0

0 0 0

0 0 0

6 4 7

0 1 0

0 1 0

0 0 0

0 0 0

6 3 7

0 1 0

0 2 0

0 0 0

0 0 0

0 1.67 0

0 1 0

DOX

WKY SHR SHHF

0 0 0

0 0 0

1 0 0

6 0 3

0 7 4

0 0 0

1 0 0

5 0 0

1 0 2

0 7 5

0 0 0

2 0 0

5 0 0

0 2 7

0 5 0

5.57 11.71 10.43

6a 12a,b 11a,b

a b

Tubular lesions score

Interstitial inflammation and fibrosis score

Cumulative kidney score/12 95% CI 0–0 0–4.1 0–0 4.5–6.7 11.3–12.2 9.4–11.5

p b 0.0001 DOX is significantly greater than SAL within a strain. p b 0.001 significantly greater than WKY within DOX treatment.

treatment. Our study suggests that genetic hypertension in SHR exacerbated delayed-onset effects of DOX, demonstrated by weight loss, falling systolic blood pressure, significantly greater cardiac and renal

pathology, and increased mortality. In contrast, genetic predisposition to heart failure superimposed on hypertension (SHHF) failed to increase toxicity compared with hypertension alone (SHR). The relative resistance

Fig. 3. Renal lesions Wistar-Kyoto strain (WKY), spontaneously hypertensive (SHR), and spontaneously hypertensive and heart failure (SHHF) male rats 12 weeks after the last dose of 8 weekly doses of saline (SAL) or 2 mg/kg doxorubicin (DOX). Areas of normal histology in the kidneys of SAL-WKY (A), SAL-SHR (C) or SAL-SHHF (E) rats. (B) Moderate focal interstitial mononuclear inflammation (arrow) and tubular dilatation (D) in DOX-WKY. (D) Marked mesangial matrix deposition in the glomerulus with podocyte adhesions (arrow), moderate tubular dilation with protein casts (P) and moderate interstitial fibrosis with mild mononuclear inflammation in DOX-SHR. (F) Marked mesangial matrix deposition in the glomerulus with podocyte adhesions and mesangial cell vacuolization (arrows), marked tubular dilation (T) with protein casts (P) and minimal mononuclear interstitial inflammation in DOX-SHHF. Hematoxylin and eosin stain.

54

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

Fig. 4. Soluble epoxide hydrolase (sEH) protein content in the left ventricle by Western blot analysis 24 h after a single intraperitoneal dose of 15 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). Data are normalized to GAPDH and are expressed as mean ± SE. SHR and SHHF are never significantly different from each other. There are no significant differences between SAL and DOX treatment groups within a strain. *p b 0.001 difference from WKY within a treatment group. Representative Western blots are shown above their respective groups. n = 7/group. The positive control (recombinant mouse sEH, courtesy of C. Morisseau) is in the left most lane.

of the male SHHF to the cardiotoxic effects of DOX in the delayed phase despite progression of genetic disease is a key finding of this study. Other studies using normotensive rats have demonstrated delayed progressive cardiotoxicity following cessation of DOX treatment similar to ours, including weight loss, decreases in blood pressure, increased heart weight, and comparable histologic lesions in the heart and kidney (Cirillo et al., 2000; Lebrecht et al., 2003, 2006). DOX treated male Wistar rats experience progressive depression of heart muscle contraction in vitro (Chugun et al., 2000) and in vivo (Teraoka et al., 2000) 8 to 18 weeks after a multi-dose DOX protocol. Our study did not detect significant decrements in left ventricular function between the different strains despite other evidence of progressive delayed onset effects. The reason is unclear, however the 12 week delayed phase may have been insufficient for fulminant expression of pathology in the SHHF and WKY, so survival studies may be informative. Direct comparisons between studies are hampered by divergent dosing protocols and study duration, strain variations, and different methods for assessing organ damage. Perhaps enhanced sensitivity to the isoflurane anesthesia used in our study contributed to the unexpectedly low %FS in SHR and SHHF. Shorter-term multi-week DOX protocols using SHR rats corroborate our delayed phase study, reporting similar growth restriction, cardiac and renal lesions, and death during treatment (Hazari et al., 2009; Herman et al., 1985, 1988, 1998; Zhang et al., 1996). There are no other comparable studies with the SHHF rat; however, the failure of genetically programmed hypertension and heart failure to accelerate progression of toxicity above that of the SHR is intriguing and counterintuitive. It is possible that our study ended too soon to detect significant decrements in cardiac function and progressive lesions. In the absence of intervention, SHHF males exhibit myocardial hypertrophy by 2–3 months and have functional decompensation from 15 to 18 months of age (McCune et al., 1995), several months later than the termination of our study. While increased cTnT and histopathologic lesions in the DOX-SHHF suggest that additional time may have revealed cardiac functional changes, the cardiac histopathologic changes in SHHF were still less severe than in the DOXSHR. Unlike DOX-SHR, no DOX-SHHF died during the 12 week post treatment period. Serum cTnT is a biomarker for DOX-induced myocardial damage, and increases in cTnT can precede deterioration of ventricular function in treated male Wistar rats (Herman et al., 1999; Koh et al., 2004). DOX-WKY exhibited a greater increase in cTnT compared to SAL-WKY that paralleled an increase in histologic lesions in the myocardium.

Both SAL-SHR and SAL-SHHF had increased cTnT compared to SALWKY, and this may reflect cardiac damage due to the genetic hypertension in the SHR and hypertension with cardiomyopathy in the SHHF rat strains. Interestingly, the exacerbation of cardiac lesions demonstrated by histopathology in the DOX-SHR was not reflected by greater increases in serum cTnT over the 12 week post treatment period. The higher week 1 cTnT in DOX-SHR may indicate that cardiac injury developed earlier in the course of treatment. In people, elevations in serum cardiac troponin T have been described in association with renal disease, however this is interpreted as a reflection of the high prevalence of cardiovascular morbidity in these patients and is used as an indicator of cardiac pathologies frequently observed in patients with chronic kidney disease (Keddis et al., 2013). DOX exposure in rodents is an experimental model of a self-perpetuating renal syndrome beginning with glomerular sclerosis followed by interstitial and tubular involvement (Lebrecht et al., 2004; Zheng et al., 2005) and is used as a model of chronic nephropathy in humans. Therefore, it is possible that renal pathology may have caused secondary cardiac effects that contributed to serum cTnT elevations in SHR and SHHF. Because data suggest that upregulation in the EPHX gene that encodes for sEH in SHHF rats is associated with the development of heart failure (Monti et al., 2008) and that the epoxide pathway is involved in mediating DOX cardiotoxicity (Zhang et al., 2009; Zordoky et al., 2010), we evaluated cardiac sEH and epoxide content as a potential contributor to the relative resistance of SHHF rats to DOX. To explore the role of this mechanism in the better characterized acute phase of toxicity, we used a single higher dose of DOX for this experiment, which also offered the advantage of facilitating comparison of our results with data from other investigators using other strains. A significant limitation to this approach is that direct extrapolation of these findings to delayed toxicity is premature until they can be confirmed in more chronic studies. In this context, consistent with previous reports, we found increased sEH protein in the left ventricle of SAL-SHHF and SALSHR compared with SAL-WKY. (Monti et al., 2008). As expected based on the sEH results, both SAL-SHHF and SAL-SHR had decreased cardiac EETs. In contrast to a previous study (Monti et al., 2008), we did not observe the expected increase in cardiac DHETs in our strains, possibly because of utilization of other EET degradation pathways or further catabolism of the DHETs (Imig, 2012). Unlike previous reports in male Sprague–Dawley rats, DOX did not increase cardiac sEH in any of the strains we examined. Regardless, DOX increased all EETs in the hearts of SHHF rats, and some isomers in SHR, equalizing the post-treatment EET values among all strains without any corresponding change in DHETs. There were no demonstrable differences between SHHF and SHR that might have accounted for enhanced sensitivity of DOX-SHR and relative sparing of DOXSHHF to delayed toxicity. Our data indicate that there are significant strain differences in the cardiac epoxide pathway and that upregulation of cardiac EETs in the face of DOX treatment could be cardioprotective adaptive mechanism for SHHF. However, the differences between SHR and SHHF in the epoxide pathway components that we evaluated appear insufficient to fully explain the disparity in response to DOX. Alterations in other branches of the arachidonic acid pathway also might contribute to the strain differences. Our finding that DOX significantly increased leukotriene D4 exclusively in the SHR could be consistent with that possibility. The global pro-inflammatory effects of this class of arachidonic acid metabolites are consistent with the interstitial cell proliferation and inflammation observed in DOX-SHR hearts. The CystLT2 receptor for the cysteinyl leukotriene D4 is expressed throughout the heart and vasculature where it mediates vasoconstriction and increased capillary permeability (Singh et al., 2010) and worsens ischemia/reperfusion injury (Ni et al., 2011). Further studies on alternative pathways for arachidonic acid metabolism in response to DOX are warranted. The epoxide pathway is gaining attention as an important mediator of cardiovascular disease, modulating atherosclerosis, heart failure, and

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

55

Fig. 5. Epoxyeicosatrienoic acids (EETs) and leukotriene D4 in the left ventricle of male WKY, SHR, and SHHF rats 24-h after a single intraperitoneal dose of 15 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). Data expressed as the mean ± SE.*p b 0.05, **p b 0.01, ***p b 0.001 significantly different from WKY within a treatment group. ‡p b 0.10, ‡‡p b 0.05 significant difference between DOX and SAL treatment groups within a strain. n = 6–7/group.

chemotherapy-induced damage through effects on blood pressure, inflammation, and cell survival (Imig, 2012; Qiu et al., 2011; Wang et al., 2010). Therefore, this pathway is a high-value target for therapeutic manipulation. The major proposed mechanisms for amelioration of disease focus on cardioprotective EETs, which can be increased by up-

regulating CYP-mediated production (especially CYP2J2) or downregulating degradation by sEH. Soluble epoxide hydrolase inhibitors have shown promising results in laboratory animal studies (Qiu et al., 2011; Wang et al., 2010). The study of this pathway using rodent genetic models of hypertension has been complicated by strain polymorphisms

Table 4 Heart muscle content of dihydroxyeicosatrienoic acids (DHETs) in ng/g of ventricle 24 h after a single intraperitoneal dose of 15 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL). Treatment

Strain

5,6-DHET

8,9-DHET

11,12-DHET

14,15-DHET

Total DHET

SAL

WKY SHR SHHF

0.0980 ± 0.0219 0.0854 ± 0.0131 0.0690 ± 0.0084

0.1294 ± 0.0120 0.1243 ± 0.0120 0.1047 ± 0.0148

0.7434 ± 0.1628 0.7294 ± 0.1498 0.7554 ± 0.1691

0.4166 ± 0.0739 0.3524 ± 0.0549 0.3153 ± 0.0485

1.387 ± 0.268 1.291 ± 0.232 1.231 ± 0.241

DOX

WKY SHR SHHF

0.0989 ± 0.0058 0.1006 ± 0.0160 0.0824 ± 0.0052

0.1949 ± 0.0373 0.1639 ± 0.0321 0.1317 ± 0.0215

0.7478 ± 0.1052 0.8900 ± 0.2379 0.7990 ± 0.1342

0.5010 ± 0.0812 0.5006 ± 0.1041 0.3820 ± 0.0543

1.767 ± 0.357 1.655 ± 0.385 1.395 ± 0.209

Data expressed as mean ± SE. There were no significant strain or treatment effects.

56

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57

Table 5 Heart muscle content of arachidonic acid metabolites in ng/g of ventricle 24 h after a single intraperitoneal dose of 15 mg/kg doxorubicin (DOX) or an equivalent volume of saline (SAL).

5(S)HETE 8(S)HETE 9(S)HETE 11(S)HETE 12(S)HETE 15(S)HETE 20(S)HETE 9(S)HODE 13(S)HODE PGD2 PGE2 PGF2α TXB2 8-iso-PGF2α LTB4 LTC4 LTE4 LXA4

SAL-WKY

SAL-SHR

36.12 6.45 4.77 4.58 47.89 460.8 2.23 28.50 680.1 0.232 0.191 0.197 3.55 0.054 0.084 0.119 0.019 7.07

33.34 ± 6.50 ± 4.43 ± 4.33 ± 47.05 ± 475.2 ± 2.02 ± 33.19 ± 798.5 ± 0.175 ± 0.164 ± 0.246 ± 4.57 ± 0.087 ± 0.079 ± 0.059 ± 0.004 ± 4.72 ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.19 1.70 1.21 1.14 12.56 113.1 0.30 6.13 175.6 0.041 0.031 0.043 1.04 0.017 0.014 0.030 0.010 1.33

7.50 1.66 0.97 1.03 11.87 113.4 0.47 7.13 207.1 0.031 0.021 0.037 0.77 0.017 0.017 0.012 0.001 0.48

SAL-SHHF

DOX-WKY

DOX-SHR

DOX-SHHF

30.12 ± 5.71 ± 3.89 ± 3.95 ± 40.62 ± 416.3 ± 1.55 ± 29.77 ± 711.6 ± 0.155 ± 0.138 ± 0.193 ± 3.89 ± 0.075 ± 0.082 ± 0.063 ± 0.007 ± 4.93 ±

35.48 ± 6.92 ± 4.96 ± 4.55 ± 49.68 ± 594.6 ± 2.54 ± 22.75 ± 485.9 ± 0.197 ± 0.157 ± 0.200 ± 4.29 ± 0.085 ± 0.163 ± 0.092 ± 0.009 ± 6.81 ±

38.70 ± 5.03 ± 4.91 ± 4.86 ± 47.05 ± 529.6 ± 2.06 ± 34.98 ± 807.7 ± 0.268 ± 0.201 ± 0.249 ± 5.76 ± 0.127 ± 0.096 ± 0.058 ± 0.011 ± 7.62 ±

30.77 5.72 4.31 3.99 42.89 417.1 1.99 26.74 461.6 0.178 0.168 0.179 3.73 0.067 0.070 0.084 0.005 5.88

6.77 1.46 0.90 0.99 10.37 101.6 0.27 6.08 175.6 0.022 0.022 0.044 0.87 0.021 0.013 0.009 0.003 0.79

5.07 1.34 0.78 0.64 9.18 139.3 0.18 2.19 58.5 0.025 0.013 0.029 0.69 0.023 0.044 0.014 0.003 1.05

10.29 0.92 1.22 1.31 11.87 147.1 0.46 11.14 280.8 0.091 0.051 0.075 2.01 0.055 0.022 0.006 0.005 2.64

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.12 1.32 0.92 0.84 9.84 95.58 .34 6.28 89.48 0.037 0.030 0.045 0.89 0.021 0.015 0.017 0.0003 1.11

Data expressed as mean ± SE. There were no significant strain or treatment effects. HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; PG, prostaglandin; TXB2, thromboxane B2; LT, leukotriene; LX, lipoxin.

in the EPHX gene in which even the animal source can be important. This has been extensively documented in SHR lines (Corenblum et al., 2008; Fornage et al., 2002). Furthermore, a direct correlation between the alleles and hypertension in SHR could not be established in one study (Fornage et al., 2002), while another study using Ephx2 knockout mice revealed no alteration in basal blood pressure because compensatory changes elsewhere in the pathway maintained homeostasis (Luria et al., 2007). Regardless, there is substantial evidence that pharmacologic or genetic modification of this pathway improves cardiovascular indicators. Strain differences in sEH activity have been observed in rats and in mice. Fornage et al., 2002 reported that the increased sEH activity observed in the kidneys of SHR rats obtained from Charles River Laboratories was also present in heart tissue, although EET and DHET data were not reported. Our data from SHHF rats corroborate the findings of Monti et al. (2008) that sEH activity was higher in SHHF than WKY, although direct comparison with their study is not possible for SHR since they used the stroke-prone strain. The cause for an increase in some cardiac EET isomers in SHHF and SHR in response to DOX is not obvious, but may involve compensatory mechanisms in other pathways as has been described for genetically modified mice (Luria et al., 2007). Upregulation of CYP epoxygenases is a consideration. CYP may also protect from DOX cardiotoxicity by altering DOX metabolism (Zhang et al., 2009). The relative resistance of SHHF rats to delayed-onset effects of DOX compared to the related SHR may be multifactorial and influenced by mechanisms not directly addressed in this study. Proposed mechanisms for DOX induced cardiotoxicity include induction of apoptosis, oxidative damage, alterations in gene expression, mitochondrial dysfunction, proteolysis via MMP activation of the ubiquitin-proteasome system, and changes in calcium homeostasis (Boucek et al., 1999; Minotti et al., 2004); most of which could be influenced directly or indirectly by epoxides. There likely are numerous important mechanisms, although it is not clear which may be most significant in the delayed phase of toxicity or how different mechanisms contributing to heart failure may interact (see Carll et al., 2011 for an excellent review of noninvasive rat models of left ventricular heart failure). Another potential factor influencing the response of SHHF rats to DOX is the presence of a leptin receptor defect in this strain. Homozygous wild type SHHF are unaffected, however obese SHHF homozygous for the mutation develop heart failure at a significantly earlier age than wild type animals, with heterozygous rats being intermediate (McCune et al., 1995). Therefore, the zygosity status of the SHHF rats could potentially influence the response to DOX. Due to limitations on the availability of genotyped animals, we used phenotypically lean SHHF for this study, which likely

consisted of a mix of animals homozygous for the wild type leptin gene and some heterozygous for the leptin receptor defect. The response to DOX in the phenotypically lean animals appeared identical to preliminary dose optimization studies performed using a limited number of available known homozygous wild type rats, although we did not systematically evaluate the impact of zygosity on the response to DOX, which is a potential limitation of our results. Based on accelerated progression of heart failure, theoretically the inclusion of heterozygous SHHF would have increased the likelihood of negative cardiac outcomes rather than ameliorated the effects as we observed. There is limited data evaluating leptin and epoxide biology; we located a report that pharmacologic inhibition of sEH reduced weight gain in diet-induced obesity in mice, which subsequently reduced plasma leptin (do Campo et al., 2010). Additional studies investigating the effect of DOX in obese SHHF could be informative in exploring the complex relationships between DOX, obesity and leptin, and genetic predisposition to heart failure. Our data are consistent with the potential for alterations in the epoxide pathway to ameliorate DOX toxicity in SHHF. Additional mechanisms are needed to explain the disparity in susceptibility between the SHHF and the SHR to DOX toxicity, including routing of arachidonic acid metabolites to alternative pathways such as leukotriene production. Future experiments utilizing a range of receptor blockers in these complex pathways will likely be informative and may bring us closer to pharmacologic strategies for modulating doxorubicin cardiotoxicity. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2013.08.012. Acknowledgments Research is supported by an internal Grant-in-Aid of Research, Artistry, and Scholarship from the University of Minnesota, Office of the Vice President for Research. We would like to thank Toni Hoepf, Tess DeBlieck and Pamela Fettig for technical assistance and Dr. Jaime Modiano for critical review of the manuscript. Conflict of Interest The authors declare that there are no conflicts of interest. References Apple, F.S., Murakami, M.M., Ler, R., Walker, D., York, M., HESI Technical Committee of Biomarkers Working Group on Cardiac Troponins, 2008. Analytical characteristics of commercial cardiac troponin I and T immunoassays in serum from rats, dogs, and monkeys with induced acute myocardial injury. Clin. Chem. 54, 1982–1989.

L.C. Sharkey et al. / Toxicology and Applied Pharmacology 273 (2013) 47–57 Barakat, M.M., El-Kadi, A.O., du Souich, P., 2001. L-NAME prevents in vivo the inactivation but not the downregulation of hepatic cytochrome P450 caused by an acute inflammatory reaction. Life Sci. 69, 1559–1571. Boucek, R.J., Miracle, A., Anderson, M., Engelman, R., Atkinson, J., Dodd, D.A., 1999. Persistent effects of doxorubicin on cardiac gene expression. J. Mol. Cell. Cardiol. 31, 1435–1446. Carll, A.P., Willis, M.S., Lust, R.M., Costa, D.L., Farraj, A.K., 2011. Merits of non-invasive rat models of left ventricular heart failure. Cardiovasc. Toxicol. 11, 91–112. Chugun, A., Temma, K., Oyamada, T., Suzuki, N., Kamiya, Y., Hara, Y., Sasaki, T., Kondo, H., Akera, T., 2000. Doxorubicin-induced late cardiotoxicity: delayed impairment of Ca2+-handling mechanisms in the sarcoplasmic reticulum in the rat. Can. J. Physiol. Pharmacol. 78, 329–338. Cirillo, R., Sacco, G., Venturella, S., Brightwell, J., Giachetti, A., Manzini, S., 2000. Comparison of doxorubicin- and MEN 10755-induced long-term progressive cardiotoxicity in the rat. J. Cardiovasc. Pharmacol. 35, 100–108. Corenblum, M.J., Wise, V.E., Georgi, K., Hammock, B.D., Doris, P.A., Fornage, M., 2008. Altered soluble epoxide hydrolase gene expression and function and vascular disease risk in the stroke-prone spontaneously hypertensive rat. Hypertension 51, 567–573. do Campo, J.M., da Silva, A.A.K., Morgan, J., Wang, Y.X., Munusamy, S., Hall, J.E., 2010. Inhibition of soluble epoxide hydrolase reduces food intake and increases metabolic rate in obese mice. Nutr. Metab. Cardiovasc. Dis. 22, 598–604. Fornage, M., Hinojos, C.A., Nurowska, B.W., Boerwinkle, E., Hammock, B.D., Morisseau, C.H.P., Doris, P.A., 2002. Polymorphisms in soluble epoxide hydrolase and blood pressure in spontaneously hypertensive rats. Hypertension 40, 485–490. Fumoleau, P., Roché, H., Kerbrat, P., Bonneterre, J., Romestaing, P., Fargeot, P., Namer, M., Monnier, A., Montcuquet, P., Goudier, M.J., Luporsi, E., 2006. Long-term cardiac toxicity after adjuvant epirubicin-based chemotherapy in early breast cancer: French adjuvant study group results. Ann. Oncol. 17, 85–92. Ganz, P.A., Hahn, E.E., 2008. Implementing a survivorship care plan for patients with breast cancer. J. Clin. Oncol. 26, 759–767. Harkewicz, R., 2007. Eicosanoids. Presented at LIPID MAPS Lipidomics Workshop, April 28, 2007 (http://www.lipidmaps.org). Hazari, M.S., Haykal-Coates, N., Winsett, D.W., Costa, D.L., Farraj, A.K., 2009. Continuous electrocardiogram reveals differences in the short-term cardiotoxic response of WistarKyoto and spontaneously hypertensive rats to doxorubicin. Toxicol. Sci. 110, 224–234. Herman, E.H., El-Hage, A., Ferrans, V.J., Ardalan, B., 1985. Comparison of the severity of the chronic cardiotoxicity produced by doxorubicin in normotensive and hypertensive rats. Toxicol. Appl. Physiol. 78, 202–214. Herman, E.H., El-Hage, A., Ferrans, V., 1988. Protective effect of ICRF-187 on doxorubicininduced cardiac and renal toxicity in spontaneously hypertensive (SHR) and normotensive (WKY) rats. Toxicol. Appl. Pharmacol. 92, 42–53. Herman, E.H., Zhang, J., Hasinoff, B.B., Tran, K.T., Chadwick, D.P., Clark, J.R., Ferrans, V.J., 1998. Comparison of the chronic toxicity of piroxantrone, losoxantrone and doxorubicin in spontaneously hypertensive rats. Toxicology 128, 35–52. Herman, E.H., Zhang, J., Lipshultz, S.E., Rifai, N., Chadwick, D., Takeda, K., Yu, Z.X., Ferrans, V.J., 1999. Correlation between serum levels of cardiac troponin-T and the severity of the chronic cardiomyopathy induced by doxorubicin. J. Clin. Oncol. 17, 2237–2243. Hevko, J., Bowers, R., Murphy, R., 2001. Synthesis of 5-oxo-6,8,11,14-eicosatetaenoic acid and identification of novel omega-oxidized metabolites in the mouse macrophage. J. Pharmacol. Exp. Ther. 296, 293–305. Heyen, J.R., Blasi, E.R., Nikula, K., Rocha, R., Daust, H.A., Friderdich, G., Van Veleet, J.F., De Ciechi, P., McMahon, E.G., Rudolph, A.E., 2002. Structural, functional, and molecular characterization of the SHHF model of heart failure. Am. J. Physiol. Heart Circ. Physiol. 283, H1775–H1784. Hudson, M.M., Rai, S.N., Nunez, C., Merchant, T.E., Marina, N.M., Zalamea, N., Cox, C., Phipps, S., Pompeu, R., Rosenthal, D., 2007. Noninvasive evaluation of late anthracycline cardiac toxicity in childhood cancer survivors. J. Clin. Oncol. 25, 3635–3643. Imig, J.D., 2012. Epoxides and soluble epoxide hydrolase in cardiovascular physiology. Physiol. Rev. 92, 101–130. Jones, D.P., Spunt, S.L., Green, D., Springate, J.E., 2008. Renal late effects in patients treated for cancer in childhood: a report from the children's oncology group. Pediatr. Blood Cancer 51, 724–731. Keddis, M.T., El-Zoghby, Z.M., El Ters, M., Rodrigo, E., Pellikka, P.A., Jaffe, A.S., Cosio, F.G., 2013. Cardiac troponin T before and after kidney transplantation: determinants and implications for posttransplant survival. Am. J. Transplant. 13, 406–414. Koh, E., Nakamura, T., Takahashi, H., 2004. Troponin-T and brain natriuretic peptide as predictors for adriamycin-induced cardiomyopathy in rats. Circ. J. 68, 163–167. Landier, W.L., Bhatia, S., 2008. Cancer survivorship: a pediatric perspective. Oncologist 13, 1181–1192. Lebrecht, D., Setzer, B., Ketelsen, U.P., Haberstroh, J., Walker, U.A., 2003. Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation 108, 2423–2429. Lebrecht, D., Setzer, B., Rohrbach, R., Walker, U.A., 2004. Mitochondrial DNA and its respiratory chain products are defective in doxorubicin nephrosis. Nephrol. Dial. Transplant. 19, 329–336.

57

Lebrecht, D., Geist, A., Ketelsen, U.P., Haberstroh, J., Setzer, B., Kratz, F., Walker, U.A., 2006. The 6-maleimidocaproyl hydrazone derivative of doxorubicin is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage. Int. J. Cancer 120, 927–934. Longhi, A., Ferrari, S., Tamburini, A., Luksch, R., Fagioli, F., Bacci, G., Ferrari, C., 2012. Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients. Cancer 18, 5050–5059. Luria, A., Weldon, S.M., Kabcenell, A.K., Ingraham, R.H., Matera, D., Jiang, H., Gill, R., Morisseau, C., Newman, J.W., Hammock, B.D., 2007. Compensatory mechanism for homeostatic blood pressure regulation in Ephx2 gene-disrupted mice. J. Biol. Chem. 282, 2891–2898. McCune, S., Park, S., Radin, M.J., Jurin, R., 1995. SHHF/Mcc-facp rat model: a genetic model of congestive heart failure. In: Singal, P., Dixon, I., Beamish, R., Dhalla, R. (Eds.), Mechanisms of Heart Failure. Kluwar Academic Publishers, Norwell, MA, pp. 91–106. Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L., 2004. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 56, 185–229. Monti, J., Fischer, J., Paskas, S., Heinig, M., Schulz, H., Gosele, C., Heuser, A., Fischer, R., Schmidt, C., Schirdewan, A., Gross, V., Hummel, O., Maatz, H., Patone, G., Saar, K., Vingron, M., Weldon, S.M., Lindpaintner, K., Hammock, B.D., Rohde, K., Dietz, R., Cook, S.A., Schunck, W.H., Luft, F.C., Hubner, N., 2008. Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease. Nat. Genet. 40, 529–537. Mulrooney, D.A., Yeazel, M.W., Kawashima, T., Mertens, A.C., Mitby, P., Stovall, M., Donaldson, S.S., Green, D.M., Sklar, C.A., Robison, L.L., Leisenring, W.M., 2009. Cardiac outcomes in a cohort of adult survivors of childhood and adolescent cancer: retrospective analysis of the Childhood Cancer Survivor Study cohort. BMJ 339, b4606. http://dx.doi.org/10.1136/bmj.b4606. Ni, N.C., Yan, D., Ballantyne, L.L., Barajas-Espinosa, A., St Amand, T., Pratt, D.A., Funk, C.D., 2011. A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice. J. Pharmacol. Exp. Ther. 339, 768–778. Octavia, Y., Tocchetti, C.G., Gabrielson, K.L., Janssens, S., Crijns, H.J., Moens, A.L., 2012. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J. Mol. Cell Biol. 52, 1213–1225. Qiu, H., Liu, J.Y., Harris, T.R., Hammock, B.D., Chiamvimonvat, N., 2011. Soluble epoxide hydrolase inhibitors and heart failure. Cardiovasc. Ther. 29, 99–111. Santin, J.C., Deheinzelin, D., Junior, S.P.C., Lopoes, L.F., de Camargo, B., 2007. Late echocardiography assessment of systolic and diastolic function of the left ventricle in pediatric cancer survivors after anthracycline therapy. J. Pediatr. Hematol. Oncol. 29, 761–765. Sellers, K.W., Sun, C., Diez-Freire, C., Waki, H., Morisseau, C., Falck, J.R., Hammock, B.D., Paton, J.F., Raizada, M.K., 2005. Novel mechanism of brain soluble epoxide hydrolase-mediated blood pressure regulation in the spontaneously hypertensive rat. FASEB 6, 626–628. Singh, R.K., Gupta, S., Dastidar, S., Ray, A., 2010. Cysteinyl leukotrienes and their receptors: molecular and functional characteristics. Pharmacology 85, 336–349. Smith, L.A., Cornelius, V.R., Plummer, C.J., Levitt, G., Verrill, M., Canney, P., Jones, A., 2010. Cardiotoxicity of anthracycline agents for the treatment of cancer: systematic review and meta-analysis of randomized controlled trials. BMC Cancer 10, 337. Steinherz, L.J., Steinherz, P.G., Tan, C.T.C., Heller, G., Murphy, M.L., 1991. Cardiac toxicity 4– 20 years after completing anthracycline therapy. J. Am. Med. Assoc. 266, 1672–1677. Teraoka, K., Hirano, M., Yamaguchi, K., Yamashina, A., 2000. Progressive cardiac dysfunction in adriamycin-induced cardiomyopathy rats. Eur. J. Heart Fail. 2, 373–378. Volkova, M., Russell, R., 2011. Anthracycline cardiotoxicity: prevalence, pathogenesis and treatment. Curr. Cardiol. Rev. 7, 214–220. Wang, Y.J., Ulu, A., Zhang, L.N., Hammock, B., 2010. Soluble epoxide hydrolase in atherosclerosis. Curr. Atheroscler. Rep. 12, 174–183. Yamada, T., Morisseau, C., Maxwell, J.E., Argiriadi, M.A., Christianson, D.W., Hammock, B.D., 2000. Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase. J. Biol. Chem. 275, 23082–23088. Zhang, J., Clark, J.R., Herman, E.H., Ferrans, V.J., 1996. Doxorubicin-induced apoptosis in spontaneously hypertensive rats: differential effects in heart, kidney, and intestine, and inhibition by ICRF-187. J. Mol. Cell. Cardiol. 28, 1931–1943. Zhang, Y., El-Sikhry, H., Chaudhary, K.R., Batchu, S.N., Shayeganpour, A., Jukar, T.O., Bradbury, J.A., Graves, J.P., DeGraff, L.M., Myers, P., Rouse, D.C., Foley, J., Nyska, A., Zeldin, D.C., Seubert, J.M., 2009. Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity. Am. J. Physiol. Heart Circ. Physiol. 297, H37–H46. Zheng, Z., Schmidt-Ott, K.M., Chua, S., Foster, K.A., Frankel, R.Z., Pavlidis, P., Barasch, J., D'Agati, V.D., Gharavi, A.G., 2005. A Mendelian locus on chromosome 16 determines susceptibility to doxorubicin nephropathy in the mouse. PNAS 102, 2502–2507. Zordoky, B.N.M., Anwar-Mohamed, A., Aboutabl, M.E., El-Kadi, A.O.S., 2010. Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats. Toxicol. Appl. Pharmacol. 24, 38–46.