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Dietary Trans Fats Enhance Doxorubicin-InducedCardiotoxicity in MiceMei-chin Mong, Te-chun Hsia, and Mei-chin Yin

Abstract: This study investigated the combined effects of trans fat diet (TFD) and doxorubicin upon cardiac oxidative,inflammatory, and coagulatory stress. TFD increased trans fatty acid deposit in heart (P < 0.05), and decreased proteinC and antithrombin-III activities in circulation (P < 0.05). TFD plus doxorubicin treatment elevated activities ofplasminogen activator inhibitor-1, lactate dehydrogenase, and creatine phosphokinase (P < 0.05). This combination alsoraised xanthine oxidase activity, and enhanced cardiac levels of reactive oxygen species, interleukin (IL)-6, IL-10, tumornecrosis factor-alpha, and monocyte chemoattractant protein-1 than TFD or doxorubicin treatment alone (P < 0.05).TFD alone increased cardiac nuclear factor kappa B (NF-κB) activity (P < 0.05), but failed to affect expression of NF-κBand mitogen-activated protein kinase (MAPK) (P > 0.05). Doxorubicin treatment alone augmented cardiac activity,mRNA expression, and protein production of NF-κB and MAPK (P < 0.05). TFD plus doxorubicin treatment furtherupregulated cardiac expression of NF-κB p65, p-p38, and p-ERK1/2 (P < 0.05). These findings suggest that TFDexacerbates doxorubicin-induced cardiotoxicity.

Keywords: cardiac injury, coagulation, doxorubicin, MAPK, NF-κB, trans fat

IntroductionIt has been documented that the intake of trans fat (TF) rich

diet increased the risk of coronary insufficiency, myocardial infarc-tion, atherosclerosis, and metabolic disorders (Micha and Mozaf-farian 2009; Mozaffarian and others 2009; Chen and others 2011).Harvey and others (2008) reported that TF activated inflammatoryresponses and led to endothelial dysfunctions. Iwata and others(2011) indicated that elaidic acid (C18:1, trans) and linoelaidicacid (C18:2, trans) induced superoxide production in endothelialcells. Those studies suggest that TF could promote oxidative andinflammatory progression, and cause cardiovascular injury. Ourprevious study found that TF diet changed hepatic fatty acid pro-file, and increased hepatic inflammatory cytokines including in-terleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1in mice (Liu and others 2010). Thus, it is possible that dietaryTF alters cardiac fatty acid composition, and enhanced cardiacoxidative and inflammatory stress.

Doxorubicin (also called adriamycin) is a widely usedchemotherapeutic agent for a variety of cancers. Its practical ther-apeutic use is limited because of its acute and chronic cardiotox-icities (Richard and others 2011; Octavia and others 2012). It isreported that doxorubicin evokes oxidative reactions in NADPHmicrosomal systems, and stimulates superoxide formation in heart,especially submitochondrial particles (Kalivendi and others 2005;Li and Yu 2008), which leads to overproduction of reactive oxy-gen species (ROS) and causes cardiomyocyte apoptosis. Further-more, it has been indicated that nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) are involvedin doxorubicin-induced cardiotoxicity (Kang and others 2000;

MS 20130456 Submitted 4/3/2013, Accepted 8/2/2013. Authors Mong andYin are with Dept. of Health and Nutrition Biotechnology, Asia Univ., TaichungCity, Taiwan. Author Hsia is with Dept. of Respiratory Therapy, China Medi-cal Univ., Taichung City, Taiwan. Author Yin is also with Dept. of Nutrition,China Medical Univ., Taichung City, Taiwan. Direct inquiries to author Yin (E-mail:mcyin@mail.cmu.edu.tw).

Kalivendi and others 2005), and the activation of these path-ways facilitates the production of inflammatory cytokines suchas tumor necrosis (TNF)-alpha. Since TF and doxorubicin haveadverse effects upon cardiac health, it is hypothesized that their in-teraction may exacerbate cardiac injury. In addition, Swystun andothers (2011) reported that doxorubicin augmented thrombin–antithrombin complex formation, and favored coagulation orthrombosis. It seems hemostatic disorder also warrants attentionwhen doxorubicin is used. Thus, our present study was designedto investigate the effects of TF and/or doxorubicin upon the vari-ation of coagulation factors such as fibrinogen and anticoagulationfactors such as protein C in order to understand their impact uponhemostatic balance.

The major purpose of this present study was to examine thecombined effects of TF-rich diet plus doxorubicin upon car-diac oxidative, inflammatory, and coagulatory stress. The influenceof TF and/or doxorubicin on cardiac expression of NF-κB andMAPK was also evaluated.

Materials and Methods

MaterialsShortening was purchased from Tai-Yi Co. (Taichung City,

Taiwan). Doxorubicin hydrochloride was purchased from Sigma-Aldrich Co. (St. Louis, Mo., U.S.A.). All chemicals used in thesemeasurements were the highest purity commercially available.

Animals and dietsThree-week-old male C57BL/6 mice were obtained from Natl.

Laboratory Animal Center (Natl. Science Council, Taipei City,Taiwan). Use of the mice was reviewed and approved by ChinaMedical Univ. animal care committee. Mice were housed on a12-h light-12-h dark schedule, and fed with mouse standard dietfor 1-wk acclimation. Mice were then divided into 2 groups,one continuously consumed normal diet (ND), and the other wasswitched to a diet prepared by a commercial shortening, whichwas rich in trans fatty acids, and defined as TF diet (TFD). Dietcomposition is presented in Table 1.

C© 2013 Institute of Food Technologists R©

doi: 10.1111/1750-3841.12257 Vol. 78, Nr. 10, 2013 � Journal of Food Science H1621Further reproduction without permission is prohibited

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Table 1–Composition (g/kg) of normal diet (ND) and trans fatdiet (TFD).

NDMean TFD

Cornstarch 410 410Sucrose 203 203Casein 200 200fiber 20 20Vitamin mix 10 10Mineral mix 35 35Olive oil 97 0a

Soybean oil 25 25Shortening 0 97a

Protein (%) 20.0 20.0Carbohydrate (%) 62.0 62.0Total fat (%) 12.5 12.5

aSignificantly lower or higher than ND (P < 0.05).

Table 2–Fatty acid composition (mg/g) in shortening.

ShorteningMean ± SD, n = 10

C14:0 15.41 ± 1.70C16:0 196.64 ± 6.18C16:1, cis 5.44 ± 0.66C16:1, trans 82.80 ± 3.21C18:0 128.56 ± 5.32C18:1, cis 90.72 ± 2.48C18:1, trans 362.10 ± 7.60C18:2, cis 30.48 ± 2.89C18:2, trans 84.88 ± 4.13Total SFA 340.61 ± 2.38Total MFA 541.06 ± 9.36Total PUFA 115.36 ± 5.89Total trans 529.78 ± 7.42

Experimental designAfter 8-wk feeding, ND or TFD groups were further divided

into 2 subgroups, in which doxorubicin (25 mg/kg) or saline wasgiven via one single i.p. injection. Each group had 10 mice (n =10). After 48 h, mice were killed with carbon dioxide. Blood wascollected, and plasma was separated from erythrocytes immedi-ately. Heart from each mouse was collected and weighted. Then,0.1 g cardiac tissue was homogenized in 2 mL phosphate bufferedsaline (PBS, pH 7.2) on ice, and the homogenate was collected.The protein concentration of homogenate was determined by themethod of Lowry and others (1951) using bovine serum albuminas a standard. Sample was diluted to 1 mg protein/mL, and usedfor measurements.

Triglyceride and fatty acid composition analysesTotal lipids were extracted from cardiac tissue; triglyceride

concentration (mg/g wet tissue) was quantified by a triglyceridekit (Boehringer Mannheim, Germany). Fatty acid compositionof shortening, diet, or heart was analyzed by a HP5890 gaschromatography equipped with FID and 30-m Omegawaxcapillary column (Supelco Chromatography Products, Bellefonte,Pa., U.S.A.). Fatty acids were quantified by measuring areas underidentified peaks, and heptadecanoic acid was used as an internalcontrol. Results are reported as mg/g diet or tissue. The fatty acidcompositions of shortening, ND, and TFD are shown in Table 2and 3.

Table 3–Fatty acid composition (mg/g) in normal diet (ND) andtrans fat diet (TFD).

NDMean ± SD, n = 10 TFD

C12:0 0.63 ± 0.10 0.59 ± 0.17C14:0 0.48 ± 0.08 0.84 ± 0.15C16:0 16.34 ± 1.28 18.96 ± 2.03b

C16:1, cis 1.36 ± 0.15 0.84 ± 0.19b

C16:1, trans –a 4.06 ± 0.47b

C18:0 34.84 ± 3.02 20.65 ± 2.34b

C18:1, cis 35.70 ± 2.77 24.31 ± 2.24b

C18:1, trans 0.58 ± 0.10 32.59 ± 3.06b

C18:2, cis 31.88 ± 2.42 16.87 ± 1.57b

C18:2, trans – 5.07 ± 0.85b

C18:3 1.51 ± 0.29 –b

C20:4 0.92 ± 0.15 –b

Total SFA 52.29 ± 3.31 41.04 ± 3.16b

Total MFA 37.64 ± 2.44 61.80 ± 3.03b

Total PUFA 34.31 ± 2.71 21.94 ± 1.25b

Total trans 0.58 ± 0.10 41.72 ± 3.19b

aMeans too low to be detected.bSignificantly lower or higher than ND (P < 0.05).

Blood analysesPlasma lactate dehydrogenase (LDH) and creatine phosphoki-

nase (CPK) activities were assayed using commercial kits (Randox,U.K.). C-reactive protein (CRP) level (μg/mL) was determinedby a commercial ELISA kit (Anogen, ON, Canada). For hemo-static analyses, blood samples were anticoagulated using sodiumcitrate. Plasma fibrinogen level (g/L) was measured using a com-mercial kit (Iatroset Fbg, Iatron Laboratory, Tokyo, Japan). Plas-minogen activator inhibitor (PAI)-1 activity (kU/L) was assayed bya commercial kit (Trinity Biotech plc, Co. Wicklow, Ireland). Theactivities of antithrombin (AT)-III and protein C in plasma weredetermined by chromogenic AT-III and protein C kits (SigmaChemical Co., St. Louis, Mo., U.S.A.), and were shown as per-centage of those in normal human plasma.

Determination of oxidative and antioxidative statusMalondialdehyde (MDA) and glutathione (GSH) levels in car-

diac tissue were measured by using commercial kits (OxisResearch,Portland, Oreg., U.S.A.). The method described in Privratsky andothers (2003) was used to analyze the production of ROS in cardiactissue. Briefly, 10 mg tissue was homogenized in 1 mL of ice cold40 mM Tris-HCl buffer (pH 7.4). After filtrating through a what-man Nr. 1 filter paper to remove tissue debris, the homogenatewas further diluted to 0.25% with the same buffer. Then, sam-ples were loaded with 40 μL 1.25 mM nonfluorescent dye 2′,7′-dichlorofluorescin in methanol, and incubated at 37 ◦C for 30min in the dark. The fluorescence intensity of sample was mea-sured using a fluorescent microplate reader (Perkin-Elmer Inc.,Waltham, Mass., U.S.A.) with excitation wavelength at 488 nmand emission wavelength at 535 nm. Results are expressed as rela-tive fluorescence unit (RFU) per mg protein. Cardiac activities ofglutathione peroxidase (GPX) and superoxide dismutase (SOD)were determined by commercial assay kits (Calbiochem Inc.,San Diego, Calif., U.S.A.). Xanthine oxidase (XO) activity wasmeasured spectrophotometrically by monitoring the formation ofuric acid from xanthine through the increase in absorbance at293 nm.

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Cardiac inflammatory factors analysesCardiac tissue was homogenized in 10 mM Tris-HCl buffered

solution (pH 7.2) containing 2 M NaCl, 1 mM ethylenediaminete-traacetic acid, 0.01% Tween 80, 1 mM phenylmethylsulfonyl flu-oride, and centrifuged at 9000 × g for 30 min at 4 ◦C. Theresultant supernatant was used for cytokine determination. Thelevels of IL-6, IL-10, TNF-alpha, and MCP-1 were assayed bycytoscreen immunoassay kits (BioSource Intl., Camarillo, Calif.,U.S.A.).

NF-κB p50/65 assayNF-κB p50/65 DNA binding activity in nuclear extract of

heart was determined by a commercial kit (Chemicon Intl. Co.,Temecula, Calif., U.S.A.). The binding of activated NF-κB wasexamined by adding a primary polyclonal anti-NF-κB p50/p65antibody, and a secondary antibody conjugated with horseradishperoxidase, and the 3,3′,5,5′-tetramethylbenzidine substrate. Theabsorbance at 450 nm was read. Values are expressed as relativeoptical density (OD) per mg protein.

Real-time polymerase chain reaction for mRNA expressionTotal RNA was isolated using Trizol reagent (Invitrogen, Life

Technologies, Carlsbad, Calif., U.S.A.). One μg RNA was used togenerate cDNA, which was amplified using Taq DNA polymerase.PCR was carried out in 50 μL reaction mixture containing TaqDNA polymerase buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl,200 mM dNTP, 2.5 mM MgCl2, 0.5 mM of each primer) and 2.5U Taq DNA polymerase. The specific oligonucleotide primers oftargets are as follow: NF-κB p50, forward, 5’-GGA GGC ATGTTC GGT AGT GG-3’, reverse, 5’-CCC TGC GTT GGA TTTCGT G-3’; NF-κB p65, 5’-GCG TAC ACA TTC TGG GGAGT-3’, reverse, 5’-CCG AAG CAG GAG CTA TCA AC-3’;glyceraldehyde-3-phosphate dehydrogenase (GAPDH, the house-keeping gene), forward, 5’-TGA TGA CAT CAA GAA GGTGGT GAA G-3’, revere, 5’-CCT TGG AGG CCA TGT AGGCCA T-3’. The cDNA was amplified under the following re-action conditions: 95 ˚C for 1 min, 55 ˚C for 1 min, and 72˚C for 1 min. Twenty-eight cycles were performed for GAPDHand 35 cycles were performed for others. Generated fluorescencefrom each cycle was quantitatively analyzed by using the Taqmansystem based on real-time sequence detection system (ABI Prism7700, Perkin-Elmer Inc., Foster City, Calif., U.S.A.). In this study,mRNA level was calculated as percentage of the ND group withsaline treatment.

Western blot analysisCardiac tissue was homogenized in buffer containing 0.5%

Triton X-100, and protease-inhibitor cocktail (1:1000, Sigma-Aldrich Chemical Co., St. Louis, Mo., U.S.A.). This homogenatewas further mixed with buffer (60 mM Tris-HCl, 2% SDS, and2% β-mercaptoethanol, pH 7.2), and boiled for 5 min. Sampleat 40 μg protein was applied to 10% SDS-polyacrylamide gelelectrophoresis, and transferred to a nitrocellulose membrane(Millipore, Bedford, Mass., U.S.A.) for 1 h. After blocking with asolution containing 5% nonfat milk for 1 h to prevent nonspecificbinding of antibody, membrane was incubated with mouse anti-NF-κB p50 (1:1000), anti-NF-κB p65 (1:1000), anti-p38,anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-ERK1/2, anti-phospho-ERK1/2 (1:2000) monoclonal antibody(Boehringer-Mannheim, Indianapolis, Ind., U.S.A.) at 4 ˚Covernight, and followed by reacting with horseradish peroxidase-conjugated antibody 3.5 h at room temperature. The detectedbands were quantified by an image analyzer (ATTO, Tokyo,

Japan) and GAPDH was used as a loading control. The blot wasquantified by densitometric analysis. Results were normalized toGAPDH, and given as arbitrary units.

Statistical analysisThe effect of each treatment was analyzed from 10 mice (n =

10) in each group. All data were expressed as mean ± standard de-viation (SD). Statistical analysis was done using one-way analysisof variance, and post hoc comparisons were carried out using Dun-net’s t-test. Differences with P < 0.05 were considered statisticallysignificant.

ResultsIn current study, shortening rich in trans fatty acids (529 mg/g,

Table 2) was used to prepare TFD, which led to 71.93-fold increasein trans fatty acids when compared with ND (Table 3, P < 0.05).After 8-wk TFD intake, cardiac triglyceride content increased40%; and cardiac levels of saturated and trans fatty acids also raised1.52-fold and 32.5-fold, respectively (Table 4, P < 0.05). As shownin Table 5, doxorubicin treatment alone caused 6.05- and 4.11-foldelevation in LDH and CPK activities; and TFD treatment alonelowered 15% protein C and 31% AT-III activities (P < 0.05).However, TFD plus doxorubicin further raised 1.32-fold LDH,1.26-fold CPK, and 1.16-fold PAI-1 activities when comparedwith doxorubicin treatment alone (P < 0.05).

Doxorubicin treatment alone decreased GSH content, increasedROS and MDA levels, declined GPX and SOD activities, and en-hanced XO activity in heart (Table 6, P < 0.05). TFD intakealone decreased GSH level and raised XO activity (P < 0.05).Compared with doxorubicin alone, the combination of TFD anddoxorubicin enhanced 33% ROS production and 42% XO ac-tivity, and reduced 16% GSH content (P < 0.05). As shown inTable 7, doxorubicin treatment alone increased circulating CRPlevel, and cardiac IL-10 and MCP-1 levels (P < 0.05), but TFDdid not affect test inflammatory factors. Compared with doxoru-bicin alone, TFD plus doxorubicin led to 2.61-, 2.04-, 2.92-,and 1.63-fold elevation in cardiac IL-6, IL-10, TNF-alpha, andMCP-1 levels (P < 0.05).

Doxorubicin treatment alone augmented cardiac activity,mRNA expression, and protein production of NF-κB (Figure 1A-1C, P < 0.05). TFD elevated NF-κB activity (P < 0.05), butfailed to affect mRNA expression and protein production (P >

0.05). Compared with doxourbicin alone, TFD plus doxorubicinled to 53%, 55%, and 81% increase in NF-κB activity, NF-κBmRNA expression, and NF-κB p65 protein production. Dox-orubicin treatment alone upregulated p-p38, p-ERK 1/2, andp-JNK (Figure 2, P < 0.05), but TFD did not affect MAPK (P >

0.05). Compared with doxourbicin alone, TFD plus doxorubicinled to 52% and 43% elevation in p-p38 and p-ERK1/2 production(P < 0.05).

DiscussionSanders and others (2003) reported that 2-wk trans fatty acids

intake did not affect procoagulant and fibrinolytic markers suchas fibrinogen and PAI-1 in young healthy men. Our data revealedthat 8-wk TFD intake did not change these factors in circulationof mice. However, we found this diet diminished the activities ofAT-III and protein C, 2 anticoagulatory factors. AT-III inhibitsthe activities of several proteases in the coagulation cascade, andprotein C inactivates coagulation factors such as factors Va andVIIIa (Asakawa and others 2000). Since dietary TF decreased an-ticoagulatory protection, it is highly possible that long-term TF

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Table 4–Cardiac weight (g), triglyceride content (mg/g tissue) and fatty acid composition (mg/g tissue) in mice fed by normal diet(ND), or trans fat diet (TFD) for 8 wk and followed with or without one single i.p. injection of doxorubicin (25 mg/kg).

ND ND + doxorubicin TFD TFD + doxorubicin

Heart, g 1.37 ± 0.12a 1.41 ± 0.09a 1.33 ± 0.16a 1.29 ± 0.13a

Triglyceride, mg/g 25.66 ± 2.07a 25.10 ± 1.85a 35.74 ± 2.88b 35.69 ± 2.56b

C14:0, mg/g 0.23 ± 0.06 0.27 ± 0.08 0.36 ± 0.05 0.33 ± 0.04C16:0 4.63 ± 0.21 4.30 ± 0.14 6.64 ± 0.17b 6.17 ± 0.20b

C16:1, cis 0.38 ± 0.03 0.43 ± 0.06 0.75 ± 0.11 0.62 ± 0.05C16:1, trans –a – 1.09 ± 0.08b 1.13 ± 0.06b

C18:0 6.28 ± 0.27 6.55 ± 0.19 9.91 ± 0.35b 10.10 ± 0.36b

C18:1, cis 6.57 ± 0.30 6.64 ± 0.25 6.75 ± 0.47 7.55 ± 0.53C18:1, trans 0.12 ± 0.02 0.09 ± 0.04 1.69 ± 0.13b 1.53 ± 0.16b

C18:2, cis 5.66 ± 0.18 5.45 ± 0.22 4.64 ± 0.23b 4.34 ± 0.29b

C18:2, trans – – 1.12 ± 0.09b 1.15 ± 0.07b

C18:3 0.49 ± 0.05 0.40 ± 0.09 0.57 ± 0.06 0.53 ± 0.08C20:4 0.90 ± 0.10 0.77 ± 0.07 1.75 ± 0.04b 1.69 ± 0.06b

Total SFA 11.14 ± 0.89 11.12 ± 1.02 16.91 ± 1.14b 16.60 ± 0.85b

Total MFA 7.07 ± 0.21 7.16 ± 0.31 10.28 ± 0.97b 10.83 ± 0.72b

Total PUFA 7.05 ± 0.44 6.62 ± 0.28 8.08 ± 0.56 7.71 ± 0.59Total trans 0.12 ± 0.02 0.09 ± 0.04 3.90 ± 0.35b 3.81 ± 0.40b

Values are mean ± SD, n = 10.aMeans too low to be detected.bSignificantly different from ND (P < 0.05).

Table 5–Blood activities or levels of LDH, CPK, PAI-1, fibrinogen, protein C, and AT-III in mice fed by normal diet (ND) or transfat diet (TFD) for 8 wk and followed with or without one single i.p. injection of doxorubicin (25 mg/kg).

ND ND + doxorubicin TFD TFD + doxorubicin

LDH, IU/L 34.5 ± 2.1 214.3 ± 9.4a 36.6 ± 1.3 285.1 ± 11.2a,b

CPK, IU/L 60.8 ± 3.0 268.4 ± 10.2a 68.1 ± 4.1 337.5 ± 14.7a,b

PAI-1, kU/L 7.3 ± 0.5 7.9 ± 0.4 7.4 ± 0.8 9.2 ± 0.6Fibrinogen, g/L 2.41 ± 0.17 2.60 ± 0.14 2.52 ± 0.16 2.65 ± 0.20Protein C, % 97 ± 6 95 ± 7 82 ± 4a 79 ± 5a

AT-III, % 128 ± 12 119 ± 13 90 ± 15a 86 ± 9a

Values are mean ± SD, n = 10.aSignificantly different from ND (P < 0.05).bSignificantly different from ND + doxorubicin (P < 0.05).

Table 6–Cardiac levels of GSH, ROS, MDA, and activities of GPX, SOD, and XO in mice fed by normal diet (ND) or trans fat diet(TFD) for 8 wk and followed with or without one single i.p. injection of doxorubicin (25 mg/kg).

ND ND + doxorubicin TFD TFD + doxorubicin

GSH, nmol/mg protein 19.2 ± 1.0 15.3 ± 0.7a 17.4 ± 0.8a 12.9 ± 1.2a,b

ROS, RFU/mg protein 0.21 ± 0.03 1.19 ± 0.11a 0.26 ± 0.06 1.57 ± 0.14a,b

MDA, μmol/mg protein 0.53 ± 0.07 1.26 ± 0.15a 0.67 ± 0.09 1.33 ± 0.17a

GPX, U/mg protein 35.3 ± 0.8 31.2 ± 1.3a 33.7 ± 1.0 26.3 ± 1.5a,b

SOD, U/mg protein 30.4 ± 0.6 25.5 ± 1.0a 29.8 ± 0.9 22.1 ± 1.4a,b

XO, U/mg protein 0.22 ± 0.04 0.84 ± 0.10a 0.53 ± 0.07a 1.17 ± 0.12a,b

Values are mean ± SD, n = 10.aSignificantly different from ND (P < 0.05).bSignificantly different from ND + doxorubicin (P < 0.05).

intake promotes coagulatory progression and favors the develop-ment of thrombosis.

In our present study, TF intake increased cardiac deposit oftrans fatty acids; and markedly lowered GSH level, raised XOactivity, and enhanced NF-κB activity in heart, which in turnenhanced cardiac oxidative responses. Moreover, we notified thatTF intake increased cardiac levels of arachidonic acid (C20:4)and saturated fatty acids such as stearic acid (C18:0). It is knownthat these fatty acids promote lipotoxicity to cardiovascular system(Estadella and others 2013), in which arachidonic acid is aprecursor for several key inflammatory mediators includingprostaglandins (Calder 2009) and stearic acid could induce Toll-like receptor 4/2-independent inflammation (Anderson and oth-ers 2012). Although TFD alone did not affect cardiac levels of

test inflammatory factors, the increased C18:0 and C20:4 in heartcontributed to raise inflammatory potential. These findings sup-port that TF intake alters cardiac fatty acid profile and impairscardiovascular functions.

As reported by others (Yamamoto and others 2008; Trivedi andothers 2011), our results agreed that doxorubicin activated bothNF-κB and MAPK pathways, and caused severe cardiac oxidativestress via declining enzymatic antioxidant defense and increasingROS and MDA formation. Morris and others (2011) reportedthat elevated CRP was an early biomarker of doxorubicin inducedcardiotoxicity. Besides CRP, our present study further found thatthis drug augmented cardiac formation of MCP-1 and IL-10, 2inflammatory biomarkers. MCP-1, a chemotactic factor for acti-vating monocytes and macrophages, could recruit monocytes to

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Table 7–Circulating CRP level, and cardiac IL-6, IL-10, TNF-alpha, and MCP-1 levels in mice fed by normal diet (ND) or trans fatdiet (TFD) for 8 wk and followed with or without one single i.p. injection of doxorubicin (25 mg/kg).

ND ND + doxorubicin TFD TFD + doxorubicin

CRP, μg/mL 15 ± 3 75 ± 7a 19 ± 2 88 ± 9a

IL-6, pg/mg protein 20 ± 6 35 ± 9 27 ± 8 91 ± 11a

IL-10, pg/mg protein 17 ± 2 94 ± 6a 24 ± 5 187 ± 12a,b

TNF-alpha, pg/mg protein 22 ± 4 37 ± 12 26 ± 5 108 ± 10a

MCP-1, pg/mg protein 16 ± 5 98 ± 7a 25 ± 9 161 ± 13a,b

Values are mean ± SD, n = 10.aSignificantly different from ND (P < 0.05).bSignificantly different from ND + doxorubicin (P < 0.05).

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Figure 1–NF-κB p50/65 DNA binding activity, expressed as relative OD per mg protein (A); NF-κB p50 and NF-κB p65 mRNA expression determinedby RT-PCR (B); NF-κB p50 and NF-κB p65 protein production determined by western blot analysis (C) in heart from mice fed by normal diet (ND) or transfat diet (TFD) for 8 wk and followed with or without one single i.p. injection of doxorubicin (25 mg/kg). Values are mean ± SD, n = 10. ∗Significantlydifferent from ND (P < 0.05). #Significantly different from ND + doxorubicin (P < 0.05). (Figure 1 continued on next page)

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the sites of injury (Martinovic and others 2005). The increasedMCP-1 release from doxorubicin treatment indicated that car-diac tissue was injured and local immune response was activated.Teng and others (2010) reported that doxorubicin raised serum IL-10 level, and contributed to myocardial inflammation. However,IL-10 is an anti-inflammatory cytokine. Thus, the overproducedcardiac IL-10 in our present study not only revealed cardiac on-going inflammatory status, but also highly implied that this tissuemight intend to suppress inflammatory response via self-protectiveaction.

TFD plus doxorubicin substantially raised the activity of PAI-1,the primary physiologic inhibitor of fibrinolysis (Vaughan 2005),which subsequently suppressed fibrinolysis and favored thrombo-sis and atherogenesis (Urano and others 26; Coffey and others2011). Thus, our findings suggest that TFD plus doxorubicindisturbs hemostatic balance, promoted coagulation, and deterio-rated cardiovascular functions. In addition, this combined treat-ment led to less GSH content, lower GPX and SOD activities,greater ROS, IL-6, TNF-alpha and MCP-1 production in heartthan either TFD or doxorubicin treatment alone. These findingsindicated that this combination synergistically enhanced cardiac

oxidative and inflammatory injury. It is interesting to find thatthis combination effectively raised XO activity. XO is a powerfuloxygen radical-generating source, and inhibition of XO activitycould improve some biomarkers in patients with cardiovasculardiseases (Szasz and others 2008; Doehner and Landmesser 2011).The raised cardiac XO activity from this combined treatmentdefinitely facilitated ROS generation and diminished cardiac an-tioxidative defense. Our data regarding increased LDH and CPKactivities, 2 cardiac injury markers, also agreed that this combina-tion caused severe cardiotoxicity. Furthermore, our data indicatedthat this combination aggravated the activation of NF-κB p65,p-p38, and p-ERK1/2. Apparently, TFD plus doxorubicin moreefficiently activated NF-κB and MAPK, upstream regulatory path-ways responsible for the production of oxidative and inflammatoryfactors such as ROS and TNF-alpha. These findings suggest thatcardio-toxicity from TFD plus doxorubicin could be ascribed tothe upregulation of both NF-κB and MAPK pathways.

After calculated and justified, the daily TF consumed by micein our present study was equal to 4 g/d for a 70-kg human male.Our results revealed that TFD alone at this dose did not causesevere cardiovascular disorder, and was not an effective activator for

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Figure 1–Continued.

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*,#

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

p-p38 p-ERK1/2 p-JNK

AU

ND ND+doxorubicin TFD TFD+doxorubicin

Figure 2–MAPK expression determined by western blot analysis in heart from mice fed by normal diet (ND) or trans fat diet (TFD) for 8 wk andfollowed with or without one single i.p. injection of doxorubicin (25 mg/kg). Values are mean ± SD, n = 10. ∗Significantly different from ND (P < 0.05).#Significantly different from ND + doxorubicin (P < 0.05).

NF-κB and MAPK pathways. It is highly possible that TF intakealtered cardiac fatty acid composition, and abated antioxidative andanti-inflammatory defense in this tissue. Thus, the following use ofdoxorubicin easily destroyed cardiovascular protective system andcaused damage. These results suggest that TF should be eliminatedfor patients with doxorubicin therapy in order to avoid additivelyor synergistically oxidative and/or inflammatory injury in heart.

ConclusionsIn conclusion, the combination of diet rich in trans fats and

doxorubicin enhanced cardiac oxidative and inflammatory stressvia activating NF-κB and MAPK pathways, as well as increasing

cardiac generation of ROS, TNF-alpha, and MCP-1. This com-bination also caused hemostatic imbalance. These findings supportthat dietary trans fat promotes doxorubicin-induced cardiotoxicity.

Conflicts of interest statementThe authors declare that there are no conflicts of interest.

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