Administration of Chromium(III) and Manganese(II) as a PotentialProtective Approach Against Daunorubicin-Induced Cardiotoxicity:in vitro and in vivo Experimental Evidence

Yang Liu & Debin Wang

Received: 7 September 2013 /Accepted: 22 October 2013 /Published online: 5 November 2013# Springer Science+Business Media New York 2013

Abstract Daunorubicin (DNR) is a widely used antitumordrug, but its application is limited because of its cardiotoxicside effects. The present study was designed to investigate theinteraction between DNR and cardiac myosin (CM) in thepresence of chromium(III) (Cr3+) and manganese(II) (Mn2+)using fluorescence spectrometry under simulative physiolog-ical conditions with the aim of exploring the influence ofmetal ion onDNR-CM complex and finding out an aggressiveapproach to abrogate of DNR-induced cardiotoxicity. In de-tail, the quenching and binding constant of ternary system,including metal ion, DNR, and CM, were measured andcompared with the DNR-CM. The data from in vitro experi-ments indicate that the presence of Cr3+ or Mn2+ distinctlydecreased the binding force between DNR and CM, andalleviated the cardiac toxicity caused by DNR. In addition,the variations in mice body weight and myocardial enzymelevel were examined by in vivo experiments. Animals receiv-ing Cr3+ or Mn2+ supplementation of DNR showed preserva-tion of the normal pattern of the heart, especially 2.0 mg Cr3+/kg body wt or 50.0 mg Mn2+/kg body wt exhibited an obvi-ously protective effect accompanied with body weight raisewhen compared with the mice treated with DNR alone, de-creased the ratio of heart to body weight (BW) and the ratio ofleft ventricular mass to BW to the normal levels, and inhibitedthe leak of myocardial enzyme caused by DNR. As a result,this study suggests that pretreatment of lower dose of Cr3+

(2 mg/kg wt) and moderate dose ofMn2+ (50 mg/kg wt) mightbe useful and play an important role in ameliorating thecardiotoxicity of DNR treatment in cancer patients.

Keywords Chromium(III) . Manganese(II) . Daunorubicin .

Cardiac myosin . Cardiotoxicity

Introduction

Daunorubicin (DNR; Fig. 1) is an important part of manytreatment protocols in paediatric oncology [1]. It is a cytostaticanticancer drug showing activity both in leukaemia and insolid tumours. For the treatment of many forms of leukaemia,anthracyclines, including DNR, doxorubicin (DOX) andidarubicin, are necessary to achieve induction of remission[2]. Unfortunately, the applicability of DNR, the firstanthracycline antibiotic to be used, is limited due to its signif-icant and irreversible cardiotoxicity with the cumulative doseas the main risk factor leading to congestive heart failure [3].So far, the precise cellular mechanisms responsible for thischronic cardiotoxicity of DNR remain enigmatic, making itsomewhat difficult to enable the development of therapies forpreventing and/or treating DNR cardiomyopathy. However,De Beer et al. studied the inotropic effect of DOX on skinnedcardiac preparations after both acute drug incubation and afterchronic treatment of rats with DOX, and found a strong anddirect positive inotropic action of DOX on the actin-myosincontractile system after acute administration in both skeletal[4] and cardiac muscle [5]. Additionally, compared withthe control group, significant composition changes areobserved in the myosin heavy chains in left and rightventricular of DOX-treated rats [6]. Based on the previousfindings, we suggested that DNR binds in vivo to myosin,affects the contractile function of myocardium and causes

Y. LiuSchool of Life Science, Wuchang University of Technology, Wuhan,Hubei Province 430223, People’s Republic of Chinae-mail: znmdly@sina.com

D. Wang (*)Medicinal Chemistry and Pharmacology Third-Level Laboratory ofthe State Administration of Traditional ChineseMedicine, College ofPharmacy, South-Central University for Nationalities, Wuhan, HubeiProvince 430074, People’s Republic of Chinae-mail: 15827027744@139.com

Biol Trace Elem Res (2013) 156:253–261DOI 10.1007/s12011-013-9851-0

cardiac dysfunction by a direct interaction of DNR withthe cardiac myosin.

Cardiac myosin (CM), the molecular motor of the heart,has two heavy chains of ∼220 kDa and two light chainsubunits (MLC1, 27 kDa and MLC2, 20 kDa, respectively)[7]. It is the major constituent and contracting protein of thecardiac muscle, which possesses ATPase activity. Accumulat-ing evidences indicate that taking DNR can cause significantimpairment of cardiac contractile function [8, 9].

Although the biochemical and nutritional properties ofchromium(III) (Cr3+) have been well investigated for overfour decades, little consensus has been achieved on its essen-tiality, function and mode of action. Recent evaluations of therole of Cr3+ have come to significant conclusions and hypoth-eses [10]. These advances document the nutritional role of Cr,but the response to Cr is dependent upon chemical species andamount of supplemental Cr [11]. Hexavalent Cr has long beenmarked as an environmental pollutant and a potent inorganiccarcinogen, which is several folds more toxic than the trivalentnutritional Cr and adversely affects the human health [12]. Crin the trivalent form appears to be required for proper carbo-hydrate, lipid, protein and nucleic acid metabolism in mam-mals [13–16]. In addition, it is known that the normal dietaryintake of Cr for most humans is not sufficient, which leads tosigns and symptoms that are similar to those observed for type2 diabetes and cardiovascular and related diseases [17]. Epi-demiological studies suggest a link between Cr status andcardiovascular diseases and subjects who had died from cor-onary artery disease had significantly lower Cr levels in theiraortic tissue compared to those who died from accidents [18,19]. The association of Cr intake with cardiovascular end-points is largely unknown [20].

On the other hand, manganese(II) (Mn2+) is an essentialtrace metal and cofactor for the mitochondrial antioxidantenzyme including pyruvate carboxylase and arginine syn-thase, which has been found to be elevated in diabetic ratsand mice [21, 22]. It suggests that Mn is physiologicallyindispensable for the normal insulin synthesis and secretion

[23, 24]. Moreover, many investigations indicate that Mn-superoxide dismutase (MnSOD), a Mn-assisted enzymaticantioxidant, may play roles to protect against atherosclerosisand plaque formation [25, 26]. It was reported that myocardi-um is the most sensitive target tissue for MnSOD activitycompared to other tissues, and the MnSOD activity and itsmessenger RNA expression in the heart are positively relatedto dietary Mn concentrations in chicks [27]. In cultured cells,Mn supplementation (15 mg daily) significantly increasedlymphocyte MnSOD expression [28], Mn also inducedMnSOD expression in a dose- and time-dependent mannerin human breast cancer Hs578T [29]. A Mn-deficient diet hasbeen reported to decline the activities of MnSOD in mice andrats [30, 31]. Additionally, research suggests that Mn may actas enzyme inhibitor if its concentration is different from theactual physiological requirement, which may lead to either atoxic effect or to inhibition of growth [32]. Conversely, Mnaccumulation has also been suggested to reduce cardiac mito-chondrial integrity and energy production through competi-tive binding to and inhibition of Mg2+- or Ca2+-dependentmitochondrial enzymes [33–35], although the implications ofexcess Mn on heart function have not been studied[36].

In view of above facts, it is important to study the interac-tion between DNR and CM in the presence of Cr3+ and Mn2+,in order to explore different approaches to rescuing DNR-triggered cardiotoxicity. Furthermore, little information isavailable on the effect of Cr3+ and Mn2+ on alleviation ofcardiac toxicity, while some essential trace elemental concen-trations in biological samples of humans have been reported tohave physiological disorders, such as diabetes and myocardialinfarction [37, 38].

In the present study, effects of metal ions, Cr3+ and Mn2+,on CM and the binding of DNR to CM were investigated byfluorescence spectrometry, and the results of different ionswere compared, which attempt to obtain a detailed under-standing of molecular mechanism and protective effect ofCr3+ and Mn2+ on cardiotoxicity caused by DNR. Fluores-cence technique is preferred to study the interaction of drugand protein because of its high sensitivity, rapidity and ease ofimplementation [39–43]. Furthermore, studies on metal ionschanging the body weight and myocardial enzyme level inmice co-administered with DNR were performed, in order todemonstrate the potential therapeutic value of Cr3+ and Mn2+

with appropriate dose to inhibit myocardial damage in DNR-induced cardiac toxicity.

Materials and Methods

Reagents

Daunorubicin hydrochloride for injection was supplied byPfizer Italia S.r.1. Cardiac myosin, prepared and determined

Fig. 1 Structure of daunorubicin (DNR)

254 Liu and Wang

according to the protocols of our previous study [44], wasdiluted to 0.4μMwith 0.01MTris–HCl buffer saline (pH 7.4)containing 0.6 M KCl. CrCl3·6H2O and MnCl2·4H2O werepurchased from Chinese Medicine (Group) Shanghai Chem-ical Reagent Company. Other chemical reagents were made inChina and were of analytical grades, and ultrapure water wasused throughout the studies.

Fluorescence Measurements

Three millilitres of 0.4 μMCM solution was added to a quartzcuvette, and then a given volume of 80 μM DNR or CrCl3(0.2 mM), MnCl2 (1.2 mM) was added to the cuvette using amicropipet, whose total volume was up to 75 μl. Fluorescenceemission spectra of DNR-CM or Cr-CM or Mn-CM wasobtained by spectrofluorimeter (LS-55, Perkin Elmer) using10 nm/5 nm slit widths at excitation and emission wave-lengths of 295 and 320–440 nm, respectively. Additionally,the mixed solution of CM with various amounts of metal ionswas shaken and equilibrated at 4 °C for 2 h and was addedDNR according to the above method, then other fluorescencespectra of ternary system Cr-DNR-CM or Mn-DNR-CMcould be obtained. All operations were performed at roomtemperature.

Animal Care and Diets

Healthy adult male Kunming mice, 4 weeks old and weighingbetween 18 and 20 g, provided by Wuhan Institute of Biolog-ical Products (Reg. No. SCXK(Hubei)2008-0003). They werehoused in a room at a mean constant temperature of 24±1 °C,humidity of 45±5%with a 12 h light–dark cycle, and allowedfree access to standard mouse diet (Wuhan Institute ofBiological Products, China) and distilled water. The exper-imental protocol for this study was approved by the AnimalCare and Use Committee of South Central University forNationalities, China.

Experimental Groups and Treatment

Appropriate concentration of CrCl3 and MnCl2 solution wasprepared with physiological saline. DNR was intraperitoneal-ly injected, and CrCl3 or MnCl2 solution was oral adminis-trated. To study the changes in body weight and myocardialenzyme levels, 80 male mice were randomly divided intoeight groups with ten mice each as following: normal controlgroup (group 1), normal mice only treated with 0.9 % saline.Mice in group 2 received DNR, 3.0 mg/kg body wt alone ondays 15, 17, 19, 21 and 23. Mice in groups 3–5 have the sameadministration of DNR with group 2, but meanwhile received0.1 ml CrCl3 solution (2.0, 4.0, and 8.0 mg Cr3+/kg body wt,respectively) from day 1 until killing on day 24. Similarly,mice in groups 6–8 were performed as the same way of group

3–5 but using 0.1 mlMnCl2 solution (25.0, 50.0 and 100.0 mgMn2+/kg body wt, respectively) instead of CrCl3 solution. Allanimals were killed by cervical dislocation after drawing theblood from eyeball. The mice blood in the same group wasamalgamated together, and separated sera were divided intothree for assaying the level of myocardial enzyme, and heartsamples were collected as well.

Determination of the BodyWeight and Ratio of Heart to BodyWeight

The body weights (BW) of mice were weighed on day 1before the experiment and 24 h after the last administration,respectively. When the animals were killed, the hearts (H)were washed with pre-cooling saline, dried and then weighed.After that, atrial, right ventricular portions and blood vesselswere removed. Then, left ventricular mass (LVM) was accu-rately weighed. The ratio of heart to body weight (H/BW) andthe ratio of LVM/BW were calculated.

Biochemical Analysis

The serum myocardial enzyme level of aspartate amino-transferase (AST), lactate dehydrogenase (LDH), creatinekinase (CK), creatine kinase MB isoenzyme (CK-MB)and α-hydroxybutyrate dehydrogenase (α-HBDH) weremeasured by Hubei Province Academy of TraditionalChinese Medicine, using automatic biochemistry analyser( Dimension Xpand, America).

Statistical Analysis

The data presented are expressed as means±standard error ofthe mean (SEM). To test whether differences between groupswere statistically significant, Student’s t test was used. Resultswere considered significant at p <0.05.

Results

Effect of Cr3+ or Mn2+ on Fluorescence Spectra of CM

The effect of Cr3+ or Mn2+ on CM was investigated byfluorescence spectrometry at room temperature. As shown inFig. 2, the results revealed that the fluorescence emissionintensity of CM had an obvious decrease with the gradualincrease of Cr3+ concentrations. In particular, 24.6 % reduc-tion was observed when concentration of Cr3+ ranged from0.0 to 7.0 μM. On the other hand, the effect of Mn2+ onfluorescence intensity of CM was quite different from that ofCr3+, and the results are shown in Fig. 3. It was found that thefluorescence intensity was enhanced, while the levels of Mn2+

were <1.0 μM (Fig. 3a). However, the system performed

Administration of Cr3+ and Mn2+ Against Cardiotoxicity 255

fluorescence quenching when Mn2+ concentration rangedfrom 1.2 to 3.2 μM (Fig. 3b).

In addition, according to Eqs. (1) and (2) [45], the Stern–Volmer quenching constant KSV value, binding constant KLB

value, the number of binding sites n and correlation coeffi-cient R of Stern–Volmer and Lineweaver–Burk curves werecalculated and the data for binary Cr3+–CM and Mn2+–CMsystem are listed in Table 1. The results showed that KSV

values were much >100, indicating the static quenching inter-action between metal ion, either Cr3+ or Mn2+, and CMoccurred [46]. Moreover, the quenching constant of Cr3+ andCM was three times stronger than that of Mn2+, resulting in aclear decrease in fluorescence intensity of CM and evenreaching 24.6 % decline as exhibited in Fig. 2. However, thebinding force betweenMn2+ and CMwas roughly 1,000 timesstronger than that of Cr3+. Overall, the findings indicated that,compared with Cr3+, it was easier for Mn2+ to combine withCM, whereas it was not easier to make CM fluorescencequenching and then to impact the physiological function ofthe protein. It also could be seen that the number of bindingsite, n was near 1.0, suggesting that Cr3+ or Mn2+ could bindto CM with the molar ratio of 1:1.

Effect of Cr3+ or Mn2+ on Binding of DNR and CM

The interaction between DNR and CM was performed in ourprevious work [46], and their quenching constant KSVand thebinding constant KLB were calculated to be 2.649×102 M−1

and 1.337×105 M−1, respectively. In this study, effects ofdifferent concentrations of Cr3+ or Mn2+ on fluorescencespectra of DNR and CM were monitored at room temperatureto gainmore information on the binding of metal ion, drug andprotein. The Stern–Volmer and Lineweaver–Burk curves areshown in Fig. 4. It was found that the Stern–Volmer plots(Fig. 4a) had fine linear relationship, and the slope, that is,quenching constant KSV, decreased in the order of Cr3+ andMn2+. Figure 4b exhibited the Lineweaver–Burk curves andthe binding constant KLB could be calculated from theintercept.

The data of KSV and KLB of DNR–CM complex in thepresence of Cr3+ or Mn2+ under same conditions are listed inTable 2. It was shown that the levels ofKSVandKLB of DNR–CM system have been changed with different ion concentra-tions, either Cr3+ or Mn2+. Moreover, all of the KSVof ternarysystem, including metal ion, drug and protein, was greater

Fig. 2 Fluorescence quenching spectra of CM in the presence of Cr(III)(λex=295 nm) ([Cr3+] (1→7): 0.0, 0.7, 1.4, 2.8, 4.2, 5.6 and 7.0 μM;[CM]=0.4 μM; pH 7.4; 20 °C)

Fig. 3 Effect of Mn(II) concentration on the fluorescence intensity ofCM (a) and the fluorescence quenching spectra (b) ([Mn2+] (1→7): 0.0,1.2, 1.6, 2.0, 2.4, 2.8 and 3.2 μM ; other conditions as in Fig. 2)

Table 1 The quenching and binding constants between metal ionand CM a

Ions KSV (×104, M−1) RSV KLB (M−1) n RLB

Cr3+ 5.129 0.9924 3.978×102 0.9993 0.9920

Mn2+ 1.269 0.9922 2.710×105 1.246 0.9982

a [Cr3+ ]: 0.7–7.0 μM; [Mn2+ ]: 1.2–3.2 μM; [CM]=0.4 μM

256 Liu and Wang

than the DNR–CM binary system, which illustrated that thepresence of metal ion directly affected the interaction betweenDNR and CM and promoted the fluorescence quenching ofCM induced by DNR.

Variation in BW, H/BW and LVM/BW

The effects of DNR alone or metal ion co-administration onBW, H/BWand LVM/BWare listed in Table 3. The adminis-tration of DNR without metal ion caused a considerableincrease (p <0.001) of H/BW and LVM/BW (10.2 and13.3 %, respectively) in mice (group 2) as compared to thosein the controls of group 1. The data indicated that the DNR-induced cardiac toxicity was associated with cardiac hyper-trophy and ventricular remodeling. The result was consistentwith the previous literature [47].

As for Cr3+-treated groups 3–5, the extremely significantelevation (p <0.001) of LVM/BW and normal H/BW were

apparent when 4.0–8.0 mg Cr3+/kg were co-administered withDNR (groups 4 and 5). Dramatically, the data of H/BW andLVM/BW were no abnormal when 2.0 mg Cr3+/kg were co-administered with DNR (group 3). The results inferred thatlower dose of Cr3+ might have a protective effect on theattenuation of DNR-induced cardiotoxicity, but could lead toventricular remodeling at higher concentrations.

As for Mn2+-treated groups 6–8, the extremely significantelevation (p <0.001) of H/BW was observed when 100.0 mgMn2+/kg were co-administered with DNR (group 8), and thehighly significant elevation (p <0.01) of LVM/BW was alsofound when 25.0 mg and 100.0 mg Mn2+/kg were co-administered with DNR (groups 6 and 8). Furthermore, thedata of H/BW and LVM/BW were not abnormal when50.0 mg Mn2+/kg were co-administered with DNR (group7). The results indicated that moderate dose of Mn2+

(50.0 mg/kg wt) might be weakened the myocardial injurycaused by DNR, and higher dose of Mn2+(100.0 mg/kg wt)would not inhibit the DNR-induced cardiotoxicity and evenhave aggravated the heart damage.

Additionally, a conspicuous change was noted in the bodyweight of all co-administered groups 3–8. The mean bodyweight level of DNR-treated mice (group 2) was lower thanthat of normal mice (group 1), although that decrease was notstatistically significant. The similar result was reported in pre-vious work [47]. In the fed state, Cr3+ orMn2+ co-administratedwith DNR mice showed sign of body mass gaining whencompared to the mice in group 2. Collectively, the findingsdemonstrated that pretreatment with appropriate dosage of Cr3+

(2.0 mg/kg wt) or Mn2+ (50.0 mg/kg wt) might contribute toameliorate the drug-associated weight loss and toxicity.

Fig. 4 Stern–Volmer curves (a) and Lineweaver–Burk curves (b) of CMquenched by DNR, in the presence of metal ion [Cr3+ (square), Mn2+

(star)] ([Cr3+]=0.7 μM; [Mn2+]=0.4 μM; other conditions as in Fig. 2)

Table 2 Effect of Cr3+ and Mn2+on DNR–CM complex

metal ion Concentration(μM)

KSV (M−1) RSV KLB (M−1) RLB

Cr3+ 0.0 2.649×102 0.9946 1.337×105 0.9963

0.7 4.948×105 0.9914 1.103×102 0.9989

1.4 3.486×105 0.9967 1.062×102 0.9928

2.8 2.429×105 0.9968 1.441×102 0.9922

4.2 1.896×105 0.9958 2.058×103 0.9980

5.6 3.356×105 0.9863 1.047×102 0.9923

7.0 4.043×105 0.9946 1.679×102 0.9912

Mn2+ 0.0 2.649×102 0.9946 1.337×105 0.9963

0.2 1.702×105 0.9924 2.346×102 0.9943

0.4 1.564×105 0.9940 9.086×104 0.9953

0.8 1.997×105 0.9986 1.731×104 0.9990

1.2 2.416×105 0.9928 6.506×102 0.9972

1.6 1.228×105 0.9896 3.528×102 0.9922

2.0 1.148×105 0.9982 2.471×102 0.9986

[DNR]: 0–2.0 μM;[CM]=0.4 μM

Administration of Cr3+ and Mn2+ Against Cardiotoxicity 257

Effect of Cr3+ or Mn2+ on Level of Myocardial Enzyme

The myocardial enzyme levels of mice in the study were deter-mined to understand the pathogenesis of cardiac toxicity andeffect of metal ion, and the results are listed in Table 4. In DNRgroup (group 2), the levels of AST, LDH, CK, CK-MB, and α-HBDHwere higher than the corresponding values of the controlgroup (group 1). In group 3 (2.0mg/kgCr+DNR), the inhibitionrate of AST, CK, and α-HBDH increase induced by DNR wereup to 66.9, 50.6 and 13.1 %, respectively. Moreover, althoughgroup 4 (4.0 mg/kg Cr+DNR) and group 5 (8.0 mg/kg Cr+DNR) were observed inhibition AST increase induced by DNR,and other enzyme levels, like CK, CK-MB, andα-HBDH, werehigher than the corresponding values of group 2. The findingsrevealed that lower dose of Cr3+ just were conducive to theprotection against DNR-induced myocardial cells injury.

Interestingly, the five kinds of serum myocardial enzymeindexes in all of Mn2+ experimental groups (group 6–8) werelower than correspondence of levels in group 2 treated alonewith DNR and approached to normal control group (group 1).In addition, group 7 (50.0 mg/kg Mn+DNR) was more effec-tive than group 8, in which the inhibition rate of AST, LDH,CK, CK-MB andα-HBDH increase induced byDNRwere up

to 73.2, 34.3, 59.6, 22.2 and 40.6 %, respectively. The datahinted that the protective effect of 50.0 mg Mn2+/kg body wtwas better than other dosages.

Discussion

Effect of co-ions on binding of DNR to CM

Anthracycline drugs are significant treatment for most malig-nant tumour, but the serious, irreversible cardiac toxicity limitstheir further application, seriously affecting the patient qualityof life. Therefore, prevention of cardiac toxicity is necessary.According to previous works [6, 45], NDRmight bind to CM,change the conformation of the protein, effect the contractilityand lead to the impaired heart function. The aim of the presentwork was to investigate whether metal ions, Cr3+ and Mn2+,change the conformation of CM and influence DNR–CMcomplex fluorescence spectra. The conformation changes inCM or DNR–CM were evaluated by measuring the intrinsicfluorescence intensity of protein tryptophan residues in theabsence and presence of metal ion [48].

Table 3 Summary of body weight (BW), ratio of heart to body weight (H/BW) and ratio of left ventricular mass to body weight (LVM/BW) (n =10)

Experimental group Initial body weight(g) Final body weight (g) H/BW (mg/g) LVM/BW (mg/g)

Group 1 (control group) 19.93±0.20 35.33±0.37 4.30±0.02 1.13±0.09

Group 2 (DNR group) 20.24±0.24 30.28±0.23 4.74±0.11** 1.28±0.09**

Group 3 (2.0 mg/kg Cr+DNR) 20.18±0.20 34.42±0.35 4.33±0.16 1.18±0.09

Group 4 (4.0 mg/kg Cr+DNR) 19.90±0.16 32.59±0.42 4.24±0.10 1.27±0.06**

Group 5 (8.0 mg/kg Cr+DNR) 20.38±0.18 32.87±0.32 4.11±0.14 1.28±0.07**

Group6 (25.0 mg/kg Mn+DNR) 20.22±0.22 31.46±0.38 4.42±0.05 1.22±0.04*

Group 7 (50.0 mg/kg Mn+DNR) 20.32±0.25 33.78±0.29 4.29±0.17 1.17±0.03

Group 8 (100.0 mg/kg Mn+DNR) 20.05±0.21 30.56±0.34 4.97±0.22** 1.22±0.05*

Data are presented as means±standard error of the mean (SEM)

Cr chromium (III),Mn manganese(II)

*p <0.01, **p<0.001, indicate values significantly different from the control group (Student’s t test)

Table 4 Effect of Cr(III) andMn(II) on serum myocardialenzyme of levels (U/L) inDNR-administrated mice

Data are represented themeans of 10 mice

Cr chromium(III),Mn manganese(II)

Experimental group AST LDH CK CK-MB α-HBDH

Group 1 (control group) 133 1346 643 241 520

Group 2 (DNR group) 946 2,683 3,450 491 1,172

Group 3 (2.0 mg/kg Cr+DNR) 313 2,761 1,705 551 1,019

Group 4 (4.0 mg/kg Cr+DNR) 676 – 2,697 777 1,477

Group 5 (8.0 mg/kg Cr+DNR) 587 3,797 2,251 1,111 1,342

Group6 (25.0 mg/kg Mn+DNR) 358 2,300 2,046 522 919

Group 7 (50.0 mg/kg Mn+DNR) 254 1,763 1,394 382 696

Group 8 (100.0 mg/kg Mn+DNR) 373 1,993 1,309 395 788

258 Liu and Wang

Due to the presence of tryptophan, tyrosine and phenylal-anine residues, proteins usually possess intrinsic fluorescence.However, the fluorescence emission peak is only derived fromtryptophan residues while fixing the excitation wavelength at295 nm [49]. In this work, CM has a strong fluorescenceemissionwith a peak at 345 nm on excitation at 295 nm (ratherthan 280 nm), and the addition of Cr3+ or Mn2+ caused aconspicuous decrease in the fluorescence emission intensity ofCM with no shift in the maximum emission wavelength(Figs. 2 and 3). It can be seen that a higher level of Cr3+ orMn2+ led to more effective quenching of the chromophoremolecules fluorescence, which indicated that the binding ofthe metal ion to CM changed the microenvironment of tryp-tophan residue and the tertiary structure of CM. Moreover,according to Stern–Volmer and Lineweaver–Burk equations,the quenching constant, binding constant and the number ofbinding sites in the interaction processing of metal–CM sys-tem are listed in Table 1. It was shown that the quenchingmechanism of CM initiated by Cr3+ or Mn2+ was suggested tobe static quenching, and the molar ratio of metal ion combinedto CM was 1:1 according to the fluorescence measurement.

Further experiment was carried out to examine theeffect of inorganic cations on the solution system ofDNR–CM, which can be used as a model for investigatingthe interaction of DNR to CM [48]. It was reported thatLineweaver–Burk equation, namely Scatchara equation[50], was also applied to calculate the binding constantsof ternary complexes, including ion, pharmaceutical andprotein [48, 51]. Because of susceptible to other optical, amodified Stern–Volmer equation can be used to reduceinterference and calculate binding constants in fluores-cence experiments [52]. In addition, the present studiesindicate that the calculated results are basically same.

Figure 4 shows the Stern–Volmer and Lineweaver–Burk curves of CM quenched by DNR in the presenceof Cr3+ and Mn2+; the result exhibited a good linearrelationship (R >0.9900). In addition, Table 2 shows thatthe binding constants between DNR and CM waschanged in the presence of common ions, implying thatthere was a binding between metal ions and CM and thepresence of metal ions directly affected the binding be-tween DNR and CM. Meanwhile, the binding constantsKLB for both metal ions exhibited non-proportional var-iations to the applied concentration, which might berelated to the different ionic strength. However, the spe-cific reasons are not incompletely clear, and further stud-ies are needed. It was also seen that the binding forcebetween protein and pharmaceutical dramatic decreased,because of the competition between metal ion and DNR.Therefore, animal experiment was then carried out inorder to prove the theoretical results and then to providepractical protection against DNR-induced cardiac toxicityto clinical patients.

Effect of co-ions on H/BW, LVM/BW and MyocardialEnzyme of DNR-treated Mice

Heart damage after anthracycline chemotherapy can be divid-ed into acute and chronic cardiotoxicity. By definition, chroniccardiotoxicity occurs at least 1 year after completion of ther-apy [53], of which electron microscopic examination ofendomyocardial biopsy samples from patients treated withanthracyclines shows myofibril loss, swelling of thesarcoplasmic reticulum and mitochondria, cytoplasmicvacuolisation and widespread damage with necrosis ofmyocardial cells in the left ventricular [54]. Therefore,changes of H/BW and LVM/BW were applied to eval-uate the effect of different concentration metal ions ondamage to the heart of DNR-treated mice in this paper.Table 3 suggested that 2.0 mg Cr3+/kg wt might have aprotective effect on the attenuation of DNR-inducedcardiotoxicity. On the other hand, 50.0 mg Mn2+/kg wtmight be reduced the myocardial injury caused by DNR.

Under normal conditions, AST, LDH,CK, CK-MB and α-HBDH were mainly distributed in the heart, bone, skeletalmuscle and other tissues, especially in cardiac myocytes wasthe most. When the heart damage occurs, myocardial enzymeactivity was significantly increased. Therefore, the myocardialenzyme spectrum is an important index of myocardial func-tion monitoring [55].

In the present study, myocardial enzyme levels in DNR-administrated mice (group 2) showed an increase; the factmight be explained by the production of oxygen free radicalswhenDNR combinedwith myocardial cells [56]. Oxygen freeradicals induced myocardial cell membrane and mitochondriamembrane lipid peroxidation reaction [57], changed the struc-ture of cell membrane, destroyed the cell membrane integrity,decreased fluidity, increased permeability and finally led tomyocardial cell injury or death. The heart is thought to beparticularly susceptible to this damage because it has a largenumber of mitochondria, which are a site of free radicalgeneration and because it has low levels of antioxidant en-zymes [58]. The damaged myocardial cell membrane caused avariety of intracellular enzyme leakage and seriously affectedcell physiological function [59]. The results of serum myocar-dial enzyme levels in mice co-administrated ion and DNRsuggested that the protective effect of 2.0 mg Cr3+/kg body wtor 50.0 mg Mn2+/kg body wt was better than other dosages.

Conclusions

Effects of metal ions on the interaction between CM and DNRwere performed by fluorescence spectroscopy under simula-tive physiological conditions. The results proved that theintrinsic fluorescence of CM was quenched through staticquenching mechanism, and the binding force between DNR

Administration of Cr3+ and Mn2+ Against Cardiotoxicity 259

and CM greatly decreased in the presence of Cr3+ or Mn2+. Inaddition, at specific concentrations, pretreatment 2.0 mg Cr3+/kg body wt or 50.0 mg Mn2+/kg body wt in mice couldeffectively inhibit the increase in serum myocardial enzymelevels induced by DNR and reduce myocardial cell injury.Moreover, they could protect against the decrease in bodyweight, prevent myocardial hypertrophy and ventricular re-modeling of DNR-treated mice. Therefore, the results indicat-ed that appropriate dose of 2.0 mgCr3+/kg bodywt or 50.0mgMn2+/kg body wt might ameliorate the influence of DNR onCM and alleviate the cardiac toxicity induced by DNR.

Acknowledgments This work was financially supported by the NaturalScience Foundation of Hubei Province (2012FFB07409).

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