Pharmacological dose of α-tocopherol induces cardiotoxicity in Wistar rats determined by echocardiography and histology

Abstract

The effect of pharmacological dose of α-tocopherol on heart health was determined in Wistar rats. Animals were randomly assigned to either C (control, n = 11) or E (α-tocopherol, n = 11) group. Animals received corn oil (C) or α-tocopherol dissolved in corn oil (250 mg α-tocopherol/[kg body wt/day]) (E) by gavage for a 7-week period. Rats underwent echocardiogram and were analyzed for cardiomyocyte histology and cardiac α-tocopherol absorption at the end of the study period. As compared to the C group, α-tocopherol-supplemented group showed significantly (p < 0.05) lower body weight (E, 412.8 g vs C, 480.3 g) and total cardiac weight (E, 0.94 g vs C, 1.08 g); cardiomyocyte histological impairment; smaller left ventricle (LV) (LV end-diastolic diameter (E, 7.22 mm vs C, 7.37 mm), lower LV systolic [left ventricle fractional shortening (E, 47.6% vs C, 53.6%) and ejection fraction ratio (E, 85.4 vs C, 89.9)] and diastolic [early peak velocities of diastolic transmitral flow (E, 64.6 cm/sec vs C, 75.1 cm/sec)] function. The α-tocopherol uptake in target tissue was confirmed by determination of α-tocopherol concentration medians in cardiac tissue (E, 109.91 nmol/kg vs C, 52.09 nmol/kg). The current study indicates that pharmacological dose of α-tocopherol supplementation can induce cardiotoxicity in healthy rats.

Keywords

α-tocopherol cardiotoxicity rat Doppler-echocardiogram

Introduction

Based on antioxidant function of vitamin E, several studies^1–9 using vitamin E have been conducted to prevent or to attenuate various chronic diseases. In particular, studies on the effect of dietary or supplemental vitamin E against cardiovascular diseases are still controversial. Several observational studies that examined mortality^1,2 and coronary artery event risks³ in humans showed inverse association with high intake of diet rich in vitamin E. In addition, beneficial effect of vitamin E and ascorbic acid on rabbit myocardium infarction-induced-left ventricle dilatation has been identified.⁴On the other hand, there was no beneficial effect of vitamin E supplementation on coronary disease in humans⁵ and on cardiotoxicity in experimental animal models such as rabbits⁶ and dogs.⁷ A meta-analysis of clinical trials studying vitamin E suggests that high doses of vitamin E (greater than 400 IU/day) may actually increase mortality in patients with coronary disease.⁸ In addition, recent GISSI-Prevenzione trial showed a 50% increased risk of developing congestive heart failure in vitamin E-supplemented patients with ventricular dysfunction.⁹ These studies indicate that vitamin E can exert either anti-oxidant or pro-oxidant action.

It is plausible that the factors determining the switch from anti-oxidant to pro-oxidant performance of vitamin E may associate, in part, with the vulnerability of organs already compromised by a disease-induced heavy oxidative stress.¹⁰ However, to the best of our knowledge, the effect of vitamin E supplementation on healthy heart has not been studied.

The present study was undertaken to evaluate the effect of pharmacological dose of α-tocopherol on heart from animals without previous cardiac disease. In addition, α-tocopherol uptake in the target tissue, heart, has been determined in the current study.

Methods

Chemical products

α-tocopherol (dl-α-tocopherol-acetate) was provided by Relthy Labs (Indaiatuba, SP, Brazil). HPLC-grade methanol and water were purchased from J. T. Baker Chemical (Philipsburg, New Jersey, USA). Methyl-tert-butyl ether was purchased from Aldrich Chemical (Milwaukee, Wisconsin, USA). All HPLC solvents were passed through a 0.45-mm membrane filter and degassed before use. The standard (dl-α-tocopherol acetate) was stored at −70°C until use. Pentobarbital sodium (Cristalia, Paulinia, SP, Brazil), ketamine chlorhydrate (Vetbrands, Jacarei, SP, Brazil) and xylazine (Bayer, São Paulo, SP, Brazil) were used for anesthesia.

Animals

Male Wistar rats (age, 14−16 weeks; weight, 350−400 g) from Centro Multidisciplinar de Investigação Biológica (CEMIB) UNICAMP, Campinas, SP, Brazil, were housed for 7 weeks (wks) and kept three per cage in plastic cages in the animal facility at the Experimental Laboratory of Internal Medicine Department, Botucatu School of Medicine, UNESP, where they consumed water and commercial diet (Nuvilab CR-1, Nuvital, Colombo, PR, Brazil) ad libitum. Rats were randomly assigned either to C (control, n = 11) or E (α-tocopherol, n = 11). α-Tocopherol dissolved in corn oil or corn oil (Mazola ®, São Paulo, SP, Brazil) was administered daily by gavage every morning for the entire 7-wk period. The α-tocopherol supplement mixed with corn oil (250 mg α-tocopherol/[kg body wt/day]) was given to E group, while the C group received only corn oil. To avoid differences in the energy provided, all groups received the same amount of corn oil (~0.5 mL/kg body wt/day). One day, before euthanasia, all animals underwent a transthoracic Doppler echocardiogram under light anesthesia (ketamine 50 mg/kg body wt and xylazine 1 mg/kg body wt). Rats were fasted overnight before euthanasia under pentobarbital sodium IP (50 mg/kg body wt) anesthesia and necropsied immediately after death. Hearts were harvested and weighted after animal euthanasia. Left ventricles (LV) with the interventricular septum were carefully separated from other heart chambers by dissection for morphological and α-tocopherol analyses. Fresh coronal sections of the LV were fixed with 10% buffered formalin for 48 hours for morphological evaluation, whereas other LV fragments were stored at −80°C until α-tocopherol analysis. The protocol used was in accordance with the Ethical Principles for Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and was approved by Animal Research Ethics Committee at UNESP (Document # 445/2004). Chow contained 30 mg vitamin E/kg diet, and corn oil contained 0.15 mg vitamin E/mL oil, according to the respective manufactures.

α-Tocopherol preparation given by gavage

The α-tocopherol-corn oil mixture was stirred for 20 minutes in a water bath at 54°C before being fed to the Wistar rats. Each milliliter of solution contained 250 mg of α-tocopherol. The α-tocopherol was monitored at 294 nm by an HPLC system and its stability was confirmed by diode-array spectra, as previously described.¹¹ To dissolve α-tocopherol, the solution was sonicated for three times at 4°C (1 minute each) and also vortexed for 30 seconds.

α-Tocopherol analyses in myocardium

Myocardium was analyzed as previously described.¹² Saponified cardiac tissue (left ventricle) was extracted using CHCl₃/CH₃OH and hexane. A 50-μL aliquot was used for HPLC analysis. All sample analyses were done in duplicate. All sample processing was carried out under red light. The recovery of the added internal standard was consistently >90%. The HPLC system was a Waters Alliance 2695 (Waters, Wilmington, Massachusetts, USA) that consisted of a pump and chromatography bound to a 2996 programmable photodiode array detector and a 2475 fluorescence detector, a C30 column (3.0 μm, 150 × 4.6 mm, YMC, Wilmington, North Carolina, USA), and Empower software. The Waters 2996 programmable photodiode array detector was set at 292 nm for α-tocopherol analysis. The HPLC mobile phase was methanol/methyl-tert-butyl ether/water (83:15:2, v/v/v, 15 g/L ammonium acetate in water, solvent A) and methanol/methyl-tert-butyl ether/water (8:90:2, v/v/v, 10 g/L ammonium acetate in water, solvent B). The gradient procedure, at a flow rate of 1 mL/min (16°C), was as follows: (1) 100% solvent A was used for 2 min followed by a 6-minute linear gradient to 70% solvent A; (2) a 3-minute hold followed by a 10-minute linear gradient to 45% solvent A; (3) a 2-min hold, then a 10-minute linear gradient to 5% solvent A; (4) a 4-minute hold, then a 2-minute linear gradient back to 100%. An interval of 4 minutes between each run was provided to stabilize the column and allow new α-tocopherol determination. α-Tocopherol was quantified by determining peaks in the HPLC chromatograms calibrated against known amounts of standard. The amounts were corrected for extraction and handling losses by monitoring the recovery of the internal standards. The lower limit of detection was 2.7 pmol for tocopherol.

Echocardiographic evaluation

Six days after final dose of α-tocopherol supplementation, all animals were evaluated in vivo by transthoracic Doppler-echocardiogram using a SONOS 2000 (Hewlett- Packard Medical Systems, Andover, Massachusetts, USA), equipped with a 7.5 MHz phased array transducer as previously reported¹³ with slight modification. Briefly, rats were lightly anesthetized and subjected to the echocardiographic evaluation. Aorta diameter was measured at the end of diastole. LV end-diastolic dimension (LVD) and posterior wall thickness (PWT) were obtained at maximal diastolic dimension, and the end-systolic dimension (LVS) was taken at maximal anterior motion of the posterior wall. Free wall relative thickness was calculated by the PWT/LVD ratio. Global ventricular systolic function was evaluated by the following indexes: fractional shortening (FS, %) = [(LVD – LVS) / LVD] × 100, ejection fraction (EF) (LVD³ – LVS³) / LVD³ and aortic flux velocity (VAo). All examination was performed by the same experienced examiner, who was blinded to the animal treatments, and followed the leading-edge method recommended by the American Society of Echocardiography.¹⁴ Measurements represented the mean of at least five consecutive cardiac cycles. In these conditions, the intra-observer variability is less than 6%.

Histological evaluation

Coronal sections of the left ventricle were fixed in 10% buffered formalin and embedded in paraffin. Five-micron-thick sections were cut from the blocked tissue and stained with haematoxylin-eosin (H&E). The severity of morphological features identified by H&E was graded blindly by two pathologists on a scale from zero (no alterations) to three (marked abnormality) using semi-quantitative analysis according to the modified Billingham method¹⁵ as follows: zero, normal morphology; 1, few affected cells with early myofibrilar changes (loss) and cytoplasmic vacuolation; 2, group of affected cells with loss of contractile elements (marked myofibril loss) and cytoplasmic vacuolation; 3, extensively affected tissue characterized by severe changes. The following items were evaluated: (a) cytoplasmic vacuoles; (b) myocytic necrosis; (c) myocytic degeneration; (d) inflammatory infiltrate and; (e) cell organization. Myocyte necrosis was characterized by nuclear (pyknosis, karyorrhexis, and karyolysis) and by cytoplasmic (acidophilic appearance and myofibrillar loss) changes. Myocytic degeneration was recognized by identification of cytoplasmic vacuolation, smaller cells with pale and homogenous cytoplasm. These investigations were performed using light microscope (Axio Imager A1; magnification ×400) attached to a digital video camera (Zeiss Vision) and connected to a personal computer equipped with image analyzer software (Axio Vision software rel., version 4.3; Carl Zeiss, Germany).

Statistical analysis

Data for transthoracic Doppler echocardiogram, body and cardiac weights were assessed by ANOVA complemented with the Tukey test for multiple comparisons. Results are expressed as means ± SD. Histological evaluation and concentrations of lipid protein and cardiac α-tocopherol were assessed by a nonparametric variance analysis complemented with the Dunn test for multiple comparisons, and results are expressed as median ± semi-range. All calculations were performed with SigmaStat version 2.0 for Windows 95, NT & 3.1. A p level of 0.05 was used to determine significance (Jandel Scientific Software, San Rafael, California, USA).

Results

General characteristics

Animals from C and E groups were healthy throughout the study period. However, aggressive behaviour and bleeding between the mouth and nostrils were observed among rats in E group. No death or in vivo congestive failure symptom was detected in any animal of both groups.

Body and cardiac weights

Both groups had similar weight gain profiles up to the 3rd week. However, rats supplemented with pharmacological dose of α-tocopherol showed reduced weight gain (p < 0.05) after 3 wks as compared to those of controls. At the end of the study (7th wk), body weight was significantly lower in E (412.82 ± 23.40 g) than in C (480.27 ± 27.82 g) group (Figure 1 ). In addition, administration of α-tocopherol led to a lower cardiac weight in E (0.94 ± 0.07 g) as compared to that of C (1.08 ± 0.09 g) group at the end of the study (p < 0.05). Both groups were similar when cardiac weight was adjusted for body weight (C: 0.0022 ± 0.0016; E: 0.0023 ± 0.0017).

Figure 1.

Effect of α-tocopherol supplementation on animal body weights. Values represent percentage; groups: Control (corn oil orally for 7 wks); α-tocopherol (250 mg α-tocopherol/kg body wt/day orally for 7 wks). ANOVA with Tukey test for multiple comparisons was used to compare groups: *, different from control.

α-Tocopherol uptake and absorption

After 7 weeks of α-tocopherol supplementation (250 mg α-tocopherol/kg body wt/day), cardiac α-tocopherol concentrations were higher in E (109.91 ± 18.43 nmol/kg) than C (52.09 ± 17.62 nmol/kg) group (p < 0.05). There was detectable α-tocopherol in cardiac tissue of C group due to its presence in the corn oil.

Histological analysis

Rat myocardium from C group presented no pathological changes (Figure 2 , Panel A). Supplementation with pharmacological dose of α-tocopherol led to the damage of the ventricular myocytes. When compared with the C group, the blinded semi-quantitative analysis revealed marked nuclear necrosis (p < 0.05), myocyte degeneration (p < 0.05), and disorganization of myofibrillar morphology (p < 0.05) in animals in E group (Table 1 ). The main myocardial histological alterations are shown in Figure 2.

Figure 2.

Effect of α-tocopherol supplementation (250 mg α-tocopherol/kg body wt/day) for 7 wks on rat myocardium histology. No alteration was found in the control group (Panel A), while myofibrillar disorganization (Panel B), nuclear necrosis, lymphomononuclear inflammatory infiltration, interstitial edema (Panel C), and cytoplasmic vacuolation of myocyte (Panel D) were identified in E group (α-tocopherol supplemented; HE, scale bar: 20 microns).

Table 1.

Effect of α-tocopherol supplementation on myocardium histology in Wistar rats^a

	Control (n = 11)	E (n = 11)
Nuclear necrosis	ND	2.00 ± 0.50^b
Myocyte degeneration	ND	2.00 ± 1.00^b
Myofibrillar disorganization	ND	1.00 ± 0.50^b

Abbreviation: ND: not detected.

^a Values are medians ± total semi-range; groups: Control (corn oil orally for 7 wks); E (250 mg α-tocopherol/kg body wt/day orally for 7 wks). Nonparametric variance analysis complemented with Dunn test for multiple comparisons was used to compare groups.

^b Different from Control.

Echocardiographic evaluation

Echocardiographic examination allowed the evaluation of cardiac remodelling using morphological and functional variables, which are presented in Tables 2 and 3 . Pharmacological dose of α-tocopherol was associated with significant decrease in left ventricular end-diastolic diameter (LVD) and aortic diameter (Ao), left ventricle fractional shortening (FS), ejection fraction (EF) and transmitral flow early peak velocity (E). There were no echocardiographic changes in the control group.

Table 2.

Echocardiographic morphology findings in Wistar rats after supplementing α-tocopherol (250 mg α-tocopherol/kg body wt/day/7 wks)^a

	Control (n = 11)	E (n = 11)
LA (mm)	3.5 ± 0.4	3.5 ± 0.5
LVD (mm)	7.37 ± 0.62	7.22 ± 0.41^b
LVS (mm)	3.43 ± 0.43	3.78 ± 0.41
PWT (mm)	1.64 ± 0.26	1.42 ± 0.14
PWT / LVD (ratio)	0.23 ± 0.05	0.20 ± 0.03
Ao (mm)	3.8 ± 0.2	3.4 ± 0.4^b
LA / Ao (ratio)	0.93 ± 0.10	1.05 ± 0.13

Abbreviations: LA: left atrium, LVD: left ventricular end-diastolic diameter, LVS: left ventricular end-systolic diameter, PWT: posterior wall thickness, PWT/LVD: posterior wall thickness normalized to the LVD, Ao: aorta diameter, LA/Ao: left atrium diameter normalized to the aortic diameter.

^a Values are mean ± SD; groups: Control (corn oil orally for 7 wks); E (250 mg α-tocopherol/kg body wt/day orally for 7 wks). ANOVA with the Tukey test for multiple comparisons was used to compare groups:

^b Different from Control.

Table 3.

Haemodynamic and left ventricular function in Wistar rats after supplementing α-tocopherol (250 mg α-tocopherol/kg body wt/day/7 wks)^a

	Control (n = 11)	E (n = 11)
A (cm/sec)	57.3 ± 14.3	50.1 ± 13.8
E (cm/sec)	75.1 ± 7.9	64.6 ± 10.2*
E/A (ratio)	1.4 ± 0.3	1.3 ± 0.3
FS (%)	53.6 ± 2.67	47.6 ± 4.47*
EF (ratio)	89.9 ± 1.85	85.4 ± 3.92*
VAo (cm/sec)	90.45 ± 7.01	79.27 ± 13.73
HR (bpm)	331 ± 36	329 ± 29

Abbreviations: A: late peak velocities of diastolic transmitral flow, E: early peak velocities of diastolic transmitral flow, E/A: left ventricle diastolic function index, FS: left ventricle fractional shortening, EF: ejection fraction, VAo: aortic flux velocity, HR: heart rate.

^b Different from Control.

Discussion

The current study indicates for the first time that pharmacological dose of α-tocopherol induces a cardiotoxicity in healthy Wistar rats. The dosage of α-tocopherol (250 mg α-tocopherol/kg body wt/day) was selected based on the previous studies showing protective effect of α-tocopherol against cardiac diseases.^16,17 It has been also found that pharmacological dose of α-tocopherol resulted in the reduction of body and cardiac weights. In contrast, previous study¹⁸ evaluating cardiac weight upon vitamin E supplementation (10,000 IU and 25,000 IU of vitamin E/kg of diet) for 8 months reported a significant heart enlargement in Wistar rats.

Oral administration of α-tocopherol (250 mg α-tocopherol/kg body wt/day) for 7 weeks led to appearance of α-tocopherol (median, 109.91 nmol/kg tissue) in the cardiac tissue confirming the tissue uptake although this level was lower than that in cardiac tissue of Sprague-Dawley rats, which were not supplemented with α-tocopherol, as previously¹⁹ reported. However, it is difficult to compare the previous report with the current study since the α-tocopherol content in the diet of the previous study was not provided. In addition, the bioavailibity of α-tocopherol in Wistar rat may be lower than that of Sprague-Dawley rat.

The marked myofibril loss and cell disorganization in rats supplemented with α-tocopherol suggest a destructive effect of α-tocopherol supplementation on cardiomyocytes. Interestingly, previous study²⁰ showed that α-tocopherol deficiency led to similar changes, including myofilament loss, cytoplasmic vacuolation, cell necrosis, and myocyte loss. To the best of our knowledge, there is no report available indicating the cardiomyocyte damage by α-tocopherol supplementation.

We observed that α-tocopherol administration was associated with significant decrease in ventricular size and systolic (FS and EF indexes) and diastolic dysfunction (E velocity). These alterations were accompanied by necrosis of cardiomyocyte. The decrease of left ventricle size may have resulted from at least two factors: myocyte necrosis and delayed growth of myocytes. However, the systolic function damage suggests that the first factor is more important. The effect of pharmacological dose of α-tocopherol either on systolic and diastolic dysfunction or on remodelling damage has not previously been reported in rats in vivo. Only a non-significant impairment of contractile function in isolated heart of α-tocopherol-supplemented rabbits (300 mg α-tocopherol/kg body wt/day) for 12 wks was previously described by others.⁶ In contrast to the current study, Wahab et al. did not identify cardiotoxicity in mice even using the same dose of vitamin E as the current study.¹⁶ This result can be attributed in part to the higher oral α-tocopherol tolerance of mice, as compared to the rat.²¹

The adverse myocardial effects of α-tocopherol supplementation found in the current study may be due to the non-physiological high dose. The antioxidant function of vitamin E depends on several factors, such as the dose and the presence of co-antioxidants. It is plausible that augmented concentrations of α-tocopherol resulted in increase of α-tocopheroxyl radicals, which can initiate the lipid peroxidation cascade.²² When the antioxidant system is balanced, this pro-oxidant action of vitamin E radicals is inhibited by co-antioxidants which can reduce the radical back to vitamin E. However, elevated concentrations of α-tocopherol only may over-produce α-tocopheroxyl radicals, which can no longer be efficiently detoxified by the co-antioxidants.²³ This mechanism may lead to the cardiotoxicity found in the current study.

The α-tocopherol-mediated peroxidation process has been demonstrated by experimental studies using lipoprotein^24–27 in which its oxidation was initiated and promoted by a-tocopheroxyl radicals. Rabbits receiving similar dose of vitamin E (10,000 UI/kg chow ~ 417 UI/kg body wt/day) for 28 days exhibited endothelial vasodilator dysfunction²⁸ and sclerotic thickening of the aorta’s intima (300 mg α-tocopherol/kg body wt/day for 12 wks).⁶ Low doses of vitamin E supplementation (~47mg /kg body wt/day for 2 wks and ~94 mg/kg body wt/day for 4 wks) significantly reduced genes coding for superoxide dismutase, an oxygen detoxifing enzyme, and intracellular adhesion molecule (ICAM)-1 in cardiac tissue of Lewis rats.²⁹ In humans, the α-tocopherol pro-oxidative action has been suggested by high susceptibility to hydrogen peroxide-induced peroxidation of erythrocytes found in non-smoking men supplemented with d-α-tocopherol acetate (1050 mg/day) for 20 wks.³⁰ A meta-analysis of clinical trials studying vitamin E suggests that the use of vitamin E greater than 400 IU/day may increase mortality in patients with coronary disease.⁸ Mortality increase due to the hemorrhagic stroke was also identified in smoking male volunteers exposed to daily vitamin E supplementation (50 mg/day) for 6.1 years.³¹

The current study indicates a cardiac harmful effect of pharmacological dose of α-tocopherol even under healthy conditions. Dietary vitamin E, however, provided with various other antioxidants showed no toxic effects.^32,33 Thus, an appropriate dietary supplementation with antioxidants, including vitamin E, and other co-antioxidants could constitute part of a protective strategy to minimize chronic oxidative damage. Although our experimental study provided information for the harmful effect of pharmacological dose of α-tocopherol supplementation on healthy cardiac conditions, it remains to be determined whether these effects may be generalized to humans. Clinical studies focusing the role of dietary and supplemented co-antioxidants both alone and in combination with α-tocopherol are warranted.

In conclusion, administration of pharmacological dose of α-tocopherol (250 mg α-tocopherol/kg body wt/day) for 7 wks to Wistar rats, which did not have any prior cardiac disease, induced body weight loss and cardiotoxicity confirmed by echocardiography and histology.

Footnotes

Acknowledgements

We thank Alexandre L Loureiro, Corina Correa, Jose A Souza, Jose C Georgete, Mario A Dallaqua, Mario B Bruno, Rogerio A Monteiro, Sandra A Fábio, Sueli Clara, Vitor M Souza for their help in this study.

This research was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo grant, FAPESP # 05/52571-5 and by Conselho Nacional de Pesquisa grant, CNPq PQ-II # 301943/2007-9. M.C.M.O. Nascimento, a PhD student, was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Abbreviations

References

Stampfer

Hennekens

Manson

Colditz

Rosner

Willet

WCV

. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 1993; 328: 1444–1449.

Kushi

Folsom

Prineas

Mink

Bostick

. Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women. N Engl J Med 1996; 334: 1156–1162.

Rimm

Stampfer

Ascherio

Giovannucci

Colditz

Willett

. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993; 328: 1450–1456.

Qin

Yan

Liu

Dong

Vitamins C and E attenuate apoptosis, ß-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction. Free Radic Biol Med 2006; 40: 1827–1842.

The

HOPE

Trial Investigators

HOPE-TOO

. Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. JAMA 2005; 293: 1338–1347.

Breed

Zimmerman

Dormans

Pinedo

. Failure of the antioxidant vitamin E to protect against adriamycin-induced cardiotoxicity in the rabbit. Cancer Res 1980; 40: 2033–2038.

Van Vleet

Ferrans

Weirich

. Cardiac disease induced by chronic adriamycin administration in dogs and an evaluation of vitamin E and selenium as cardioprotectants. Am J Pathol 1980; 99: 13–42.

Miller

ERr

Pastor-Barriuso

Dalal

Riemersma

Appel

Guallar

. Meta-analysis: Highdosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005; 142: 142: 137–146.

Marchioli

Levantesi

Macchia

Marfisi

Nicolosi

Tavazzi

Vitamin E increases the risk of developing heart failure after myocardial infarction: results from de GISSI-Prevenzione trial. J Cardiovasc Med 2006; 7: 347–350.

10.

Rietjens

IMCM

Boersma

de Haan

Spenkelink

Awad

Cnubben

NHP

The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environ Toxicol Phar 2002; 11: 321–333.

11.

Yeum

K-J

Ferland

Patry

Russell

. Relationship of plasma carotenoids, retinol and tocopherols in mothers and newborn infants. J Am Coll Nutr 1998; 17: 442–447.

12.

Ferreira

ALA

Yeum

K-J

Matsubara

Correa

Pereira

Doxorubicin as an antioxidant: maintenance of myocardial levels of lycopene under doxorubicin treatment. Free Radic Biol Med 2007; 43: 740–751.

13.

Ferreira

ALA

Russell

Rocha

Ladeira

MSP

Salvadori

DMS

Nascimento

MCMO

Effect of lycopene on doxorubicin-induced cardiotoxicity: an echocardiographic, histological, and morphometrical assessment. Basic Clin Pharmacol Toxicol 2007; 101: 16–24.

14.

. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978; 58: 1072–1083.

15.

Billingham

Mason

Bristow

Daniels

. Anthracycline cardiomyopathy monitored by morphologic changes. Cancer Treat Rep 1978; 62: 865–872.

16.

Wahab

MHA

Akoul

EEMS

Abdelaziz

. Modulatory effects of melatonin and vitamin-E on doxorubicin-induced cardiotoxicity in Ehrlich ascites carcinoma bearing mice. Tumori 2000; 86: 157–162.

17.

Giakoustidis

Papageorgiou

Iliadis

Kontos

Kostopoulou

Papachrestou

Intramuscular administration of very high dose of alpha-tocopherol protects liver from severe ischemia-reperfusion injury. World J Surg 2002; 26: 872–877.

18.

Yang

NYJ

Desai

. Effect of high levels of dietary vitamin E on hematological indices and biochemical parameters in rats. J Nutr 1977; 107: 1410–1417.

19.

Danelisen

Palace

Lou

Singal

. Maintenance of myocardial levels of vitamin A in heart failure due to adriamycin. J Mol Cell Cardiol 2002; 34: 789–795.

20.

Legha

Wang

Mackay

Ewer

Hortobagyi

Benjamin

Ali

. Clinical and pharmacologic investigation of the effects of alpha-tocopherol on adriamycin cardiotoxicity. Ann N Y Acad Sci 1982; 393: 411–418.

21.

Demole

. Pharmakologisches uber vitamin E. Int Z Vitaminforsch 1939; 8: 338–341.

22.

Upston

Terentis

Stocker

. Tocopherol-mediated peroxidation (TMP) of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J 1999; 13: 977–994.

23.

Stocker

. The ambivalence of vitamin E in atherogenesis. TIBS 1999; 24: 219.

24.

Neuzil

Thomas

Stocker

. Requirement for, promotion, or inhibition by alpha-tocopherol of radical-induced initiation of plasma lipoprotein lipid peroxidation. Free Radic Biol Med 1997; 22: 57–71.

25.

Bowry

Stocker

Tocopherol-mediated peroxidation. The prooxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J Am Chem Soc Society 1993; 115: 6029–6044.

26.

Witting

Westerlund

Stocker

. A rapid and simple screening test for potential inhibitors of tocopherol-mediated peroxidation of LDL lipids. J Lipid Res 1996; 37: 853–867.

27.

Upston

Terentis

Morris

Keaney

JFJ

Stocker

. Oxidized lipid accumulates in the presence of alpha-tocopherol in atherosclerosis. Biochem J 2002; 363: 753–760.

28.

Keaney

Gaziano

Frei

Curran-Celentano

Shwaery

Low-dose alpha-tocopherol improves and high-dose alpha-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest 1994; 93: 844–851.

29.

Schulte

Bektas

Klempnauer

Borlak

. Vitamin E in heart transplantation: effects on cardiac gene expression. Transplantation 2006; 81: 736–745.

30.

Brown

Morrice

Duthie

. Erythrocyte vitamine E and plasma ascorbate concentrations in relation to erythrocyte peroxidation in smokers and nonsmokers: dose response to vitamin E supplementation. Am J Clin Nutr 1997; 65: 496–502.

31.

Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med 1994; 330: 1029–1035.

32.

Bendich

Machlin

. Safety of oral intake of vitamin E. Am J Clin Nutr 1988; 48: 612–619.

33.

Meydani

Barklund

Liu

Meydani

Miller

Cannon

Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am J Clin Nutr 1990; 52: 557–563.