Since anthracycline-induced cardiotoxicity (AIC), a complication of anthracycline-based chemotherapies, is thought to involve iron, concerns exist about using iron for anaemia treatment in anthracycline-receiving cancer patients. This study evaluated how intravenous ferric carboxymaltose (FCM) modulates the influence of iron deficiency anaemia (IDA) and doxorubicin (3–5 mg per kg body weight [BW]) on oxidative/nitrosative stress, inflammation, and cardiorenal function in spontaneously hypertensive stroke-prone (SHR-SP) rats. FCM was given as repeated small or single total dose (15 mg iron per kg BW), either concurrent with or three days after doxorubicin. IDA (after dietary iron restriction) induced cardiac and renal oxidative stress (markers included malondialdehyde, catalase, Cu,Zn-superoxide dismutase, and glutathione peroxidase), nitrosative stress (inducible nitric oxide synthase and nitrotyrosine), inflammation (tumour necrosis factor-alpha and interleukin-6), and functional/morphological abnormalities (left ventricle end-diastolic and end-systolic diameter, fractional shortening, density of cardiomyocytes and capillaries, caveolin-1 expression, creatinine clearance, and urine neutrophil gelatinase-associated lipocalin) that were aggravated by doxorubicin. Notably, iron treatment with FCM did not exacerbate but attenuated the cardiorenal effects of IDA and doxorubicin independent of the iron dosing regimen. The results of this model suggest that intravenous FCM can be used concomitantly with an anthracycline-based chemotherapy without increasing signs of AIC.
Cytotoxic cancer treatments frequently include anthracyclines [
Considering that anaemia and iron deficiency (ID) are frequent complications in cancer patients [
The primary objective of this study was to evaluate whether i.v. iron treatment (ferric carboxymaltose, FCM, Vifor Pharma) modulates doxorubicin- (DOX-) induced AIC. For this purpose, we established a model of DOX-induced cardiotoxicity in spontaneously hypertensive stroke-prone (SHR-SP) rats with iron deficiency anaemia (IDA). Since DOX is also nephrotoxic [
All experiments were approved by the Hospital Aleman Animal Care and the Teaching and Research Committee and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Three-week-old male SHR-SP rats (Charles River Laboratories, USA) were acclimatized in a temperature-controlled room (
Haematological, functional, and oxidative/nitrosative stress and inflammatory markers according to iron diet and DOX treatment.
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Iron diet | LID | LID | LID | LID | ND | ND |
DOX dose (mg/kg BW) | 3 | 4 | 5 | 0 | 4 | 0 |
Hb (g/dL) |
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TSAT (%) |
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LVDD (mm) |
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LVSD (mm) |
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FS (%) |
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Caveolin-1 (%/area) |
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Cu,Zn-SOD (U/mg protein) |
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GPx (U/mg protein) |
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MDA (nmol/mg protein) |
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GSH : GSSG ratio |
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Nitrotyrosine (%/area) |
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TNF- |
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Data presented as mean
After 4 weeks of acclimatization, 48 SHR-SP rats were randomized into six groups (
After 3 weeks of acclimatization, 64 SHR-SP rats were randomized into eight groups (
Two weeks after the last DOX or saline injection (end of study), anaesthetized rats were euthanized by subtotal exsanguination, and tissue samples were taken for histological assessments.
At study end, systolic and diastolic blood pressure (SBP and DBP) were measured in nonanaesthetized rats as described [
Blood hemoglobin (Hb) and serum iron were measured using routine blood analyzers (Hb: HemoCue, Sweden; SYSMEX XT 1800i, Japan; iron: Roche Diagnostics Autoanalyser Modular-P800, Germany). Serum transferrin was determined by radial immunodiffusion (Biocientifica, Argentina) and transferrin saturation (TSAT) was calculated as described previously [
Thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and glutathione, as well as the activities of catalase, Cu,Zn superoxide dismutase (Cu,Zn-SOD), and glutathione peroxidase (GPx) were quantified in tissue homogenates of the saline-perfused heart (left ventricular free wall [LVFW], left ventricular septum [LVS], and right ventricle [RV]) as previously described [
Three-micron sections of LVFW, LVS, and RV were fixed in phosphate-buffered, 10% formaldehyde (
Immunohistochemistry was performed with antibodies against tumour necrosis factor-alpha (TNF-
All histological stainings are expressed as percentage of positive staining per area from 20 random images that were viewed and evaluated independently by two investigators who were blinded to sample identity.
Data are shown as mean ± standard deviation (SD). Statistical analyses used absolute values and GraphPad Prism 5.01 for Windows (GraphPad Software, USA). Normally distributed parameters were compared among groups using ANOVA. Parameters with non-Gaussian distribution (e.g., histological data) were compared using Kruskal-Wallis test (nonparametric ANOVA) and Dunn’s multiple comparison test.
LID-fed rats developed IDA as indicated by reduced Hb and TSAT (Groups D versus F;
As previously described in SHR-SP rats [
In the heart of LID-fed rats (Group D), markers of oxidative stress (Cu,Zn-SOD, GPx, and MDA), nitrosative stress (nitrotyrosine), and inflammation (TNF-
In contrast to LID alone, the combination of LID and DOX resulted in a dose-dependent increase of mortality in SHR-SP rats; 13%, 25%, and 50% for DOX doses of 3, 4, and 5 mg/kg, respectively.
Hb, serum iron, and TSAT as well as L-ferritin in the heart were significantly reduced in LID-fed rats compared to ND-fed rats (Groups 6 versus 7;
Anaemia and iron status according to iron diet, DOX treatment, and supplementation with FCM.
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1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
Treatment | ||||||||
Iron diet | LID | LID | LID | LID | LID | LID | ND | ND |
DOX (4 mg/kg BW) | + | + | + | + | + | − | − | + |
FCM (mg iron/kg BW) | 3 | 3 | 15 | 15 | − | − | − | 15 |
Concurrent with DOX | Three days after DOX | Concurrent with DOX | Three days after DOX | Concurrent with DOX | ||||
Saline | − | − | − | − | + | + | + | − |
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Hb (g/dL) |
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Serum iron ( |
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TSAT (%) |
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L-Ferritin (%/area) |
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Data presented as mean
The increase in mean blood pressure from baseline (SBP 160–167 mm Hg and DBP 93-94 mm Hg) to end of study (SBP 194–205 mm Hg and DBP 120–123 mm Hg) was similar in all treatment groups.
Echocardiography indicated that LID-fed rats had increased LVDD, LVSD, and LV mass (LVM) and decreased FS compared to ND-fed (Groups 6 versus 7;
Echocardiographic outcomes according to iron diet, DOX treatment, and supplementation with FCM.
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1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
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LVDD (mm) |
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LVSD (mm) |
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LVPW (mm) |
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IVS (mm) |
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FS (%) |
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LVM (g) |
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Data presented as mean ± SD. A brief tabular overview of treatments in Groups 1–8 is included in Table
Markers of oxidative stress (TBARS, Cu,Zn-SOD, GPx, and GSH : GSSG ratio), nitrosative stress (nitrotyrosine and iNOS), inflammation (TNF-
Cardiac oxidative/nitrosative stress and inflammation according to iron diet, DOX treatment, and supplementation with FCM.
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1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
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MDA (nmol/mg protein) |
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Cu,Zn-SOD (U/mg protein) |
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GPx (U/mg protein) |
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GSH : GSSG ratio |
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Nitrotyrosine (%/area) |
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iNOS (%/area) |
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TNF- |
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IL-6 (%/area) |
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Collagen III (%/area) |
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Data presented as mean ± SD. A brief tabular overview of treatments in Groups 1–8 is included in Table
Immunohistochemical detection of nitrosative stress in the heart. Light micrographs of heart sections from rats treated as outlined in Materials and Methods Section and Table
Immunohistochemical detection of inflammation in the heart. Light micrographs of heart sections from rats treated as outlined in Materials and Methods Section and Table
Increased extracellular matrix expansion and interstitial fibrosis was seen in LID-fed compared to ND-fed rats (Groups 6 versus 7; Table
Histomorphometric assessment of the heart according to iron diet, DOX treatment, and supplementation with FCM.
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1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
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Masson’s trichrome (% area) |
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Sirius Red (% area) |
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Cardiomyocyte width (µm) |
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Cardiomyocyte density ( |
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Capillary density ( |
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Cardiomyocyte/capillary ratio |
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Data presented as mean ± SD. A brief tabular overview of treatments in Groups 1–8 is included in Table
ND-fed rats treated with DOX and a single dose of FCM on the same day as the second weekly DOX treatment (Group 8) had higher levels of serum iron and TSAT compared to LID-fed rats given the same DOX and FCM treatments (Group 3; Table
No deaths occurred in saline-treated, ND-fed rats (Group 7) or any LID-fed group regardless of treatment (Groups 1–6). Deaths were reported for two of eight ND-fed rats treated with concomitant DOX and FCM (15 mg iron/kg) (Group 8).
The data presented herein show that FCM at a total dose of 15 mg iron/kg BW resolves IDA and attenuates IDA- and DOX-associated cardiorenal toxicity in SHR-SP rats. In line with prior clinical and nonclinical studies, IDA alone increased cardiac and renal oxidative/nitrosative stress and inflammation as well as abnormalities in cardiac morphology and function in the models described here [
The development of hypertension in SHR-SP rats [
Beneficial effects of FCM were observed whether administered as a single dose or as repeated 3 mg/kg doses; yet single dose administration was associated with a trend towards higher Hb as well as less oxidative stress, LVSD, and urinary NGAL (an early marker of acute kidney damage) [
Anthracycline-mediated production of ROS is considered an important mechanism of AIC, and iron loading has been suggested to potentiate this mechanism [
In rodents, DOX-induced cardiotoxicity is increased by dietary and genetic iron overload [
Notably, AIC can occur as “acute” cardiotoxicity within hours or days of treatment as well as early-onset or late-onset AIC within one year or after one year of treatment, respectively [
Since this study focused on the characterisation of DOX-induced cardiotoxicity and its modulation with i.v. iron, rats were nontumour bearing. However, comorbid hypertension was addressed by using SHR-SP rats. Dietary iron restriction resulted in IDA as well as significant oxidative stress and inflammation, which were aggravated by DOX. The decrease in Hb associated with DOX alone suggests that iron-independent effects on erythropoiesis, possibly mediated by oxidative stress and inflammation, are also addressed in this model. Hence, the model combines features of IDA and chemotherapy-induced anaemia. Nevertheless, the results of this study are based on a model of absolute iron deficiency and should not be extrapolated to patients with functional iron deficiency since the biodistribution and erythropoietic efficacy of i.v. iron may be different in the clinical setting of cancer-associated anaemia.
FCM did not aggravate DOX-induced cardiotoxicity in iron-deficient hypertensive rats receiving chronic DOX treatment. Moreover, administration of FCM attenuated cardiorenal oxidative/nitrosative stress, inflammation, and fibrosis as well as effects on cardiac and renal morphology and function compared to saline controls.
This work was previously presented at 17th Congress of the European Hematology Association, Amsterdam, The Netherlands, June 14–17, 2012 (Jorge Toblli, Gabriel Cao, Jorge Giani, Fernando Dominici, and M. Angerosa, “Evaluation of ferric carboxymaltose for reduction of doxorubicin-induced cardiotoxicity in iron deficient spontaneously hypertensive stroke-prone rats,” Haematologica, vol. 97(s1), pp. 392, abstract 949, 2012).
The authors declare that there is no conflict of interests regarding the publication of this paper.
The study was sponsored by Vifor (International) AG, Switzerland, and Beate Rzychon (Vifor Pharma) reviewed and commented on the paper. Editorial support from SFL Regulatory Affairs & Scientific Communication was funded by Vifor Pharma. Jorge Eduardo Toblli has received Research Grants and consultancy fees from Vifor Pharma. Felix Funk and Lee Mizzen are employees of Vifor Pharma.