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High dietary level of synthetic vitamin E on lipid peroxidation, membrane fatty acid composition and cytotoxicity in breast cancer xenograft and in mouse host tissue
© Cameron et al; licensee BioMed Central Ltd. 2003
Received: 27 January 2003
Accepted: 12 March 2003
Published: 12 March 2003
d-α-tocopherol is a naturally occurring form of vitamin E not previously known to have antitumor activity. Synthetic vitamin E (sE) is a commonly used dietary supplement consisting of a mixture of d-α-tocopherol and 7 equimolar stereoisomers. To test for antilipid peroxidation and for antitumor activity of sE supplementation, two groups of nude mice bearing a MDA-MB 231 human breast cancer tumor were fed an AIN-76 diet, one with and one without an additional 2000 IU/kg dry food (equivalent to 900 mg of all-rac-α-tocopherol or sE). This provided an intake of about 200 mg/kg body weight per day. The mice were killed at either 2 or 6 weeks after the start of dietary intervention. During necropsy, tumor and host tissues were excised for histology and for biochemical analyses.
Tumor growth was significantly reduced by 6 weeks of sE supplementation. Thiobarbituric acid reactive substances, an indicator of lipid peroxidation, were suppressed in tumor and in host tissues in sE supplemented mice. In the sE treated mice, the fatty acid composition of microsomal and mitochondrial membranes of tumor and host tissues had proportionately less linoleic acid (n-6 C 18-2), similar levels of arachidonic acid (n-6 C 20-4), but more docosahexanoic acid (n-3 C 22-6). The sE supplementation had no significant effect on blood counts or on intestinal histology but gave some evidence of cardiac toxicity as judged by myocyte vacuoles and by an indicator of oxidative stress (increased ratio of Mn SOD mRNA over GPX1 mRNA).
At least one of the stereoisomers in sE has antitumor activity. Synthetic vitamin E appears to preferentially stabilize membrane fatty acids with more double bonds in the acyl chain. Although sE suppressed tumor growth and lipid peroxidation, it may have side-effects in the heart.
Vitamin E is an essential fat-soluble vitamin. The general term vitamin E refers to eight naturally occurring and synthetic tocopherols and tocotrienols and their acetate and succinate derivatives. Although the naturally occurring forms of vitamin E have lipid-soluble antioxidant properties that protect cell membranes against damage by free radicals, the acetate and succinate derivatives that are esterified at the C-6 position of the chromanol ring do not have antioxidant properties unless the esterification is hydrolyzed and free tocopherol is regenerated [1–3].
Synthetic vitamin E, a form of vitamin E commonly used as a dietary supplement, is the form used in the in vivo studies reported here. It is a mixture of eight stereoisomers in equal amounts designated "dl" or all-rac tocopherol and does have antioxidant properties . Thus, different forms of vitamin E can have different antioxidant properties as well as differences in their absorption, distribution, and metabolism . There are also reports that some forms of vitamin E have activity against cancer cells in vitro [3, 5]. For example, tocotrienol and vitamin E succinate are reported to have antiproliferative activity in human cancer cells whereas some forms of vitamin E are reported to induce apoptosis in cancer cell types but not in normal cell types . Specifically, these apoptogenic forms include RRR-α-tocopheryl succinate and RRR-δ-tocopherol while the α, β, and γ tocopherol and acetate derivatives of tocopherol were not apoptogenic . There are few reports on the use of vitamin E for treating tumorous cancers in vivo but the results are inconsistent [4–10]. It is therefore essential that the form of vitamin E used in any research study as well as the dosage and the mode of administration be defined.
The study reported on here was originally designed to test the ability of a high dietary dose of sE to prevent lipid peroxidation in nude mice bearing a human breast cancer xenograft MDA-MB-231 [11, 12]. To assure suppression of lipid peroxidation, sE was added to the diet at a dose level. For example, the vitamin E requirement for mice when all-rac-α-tocopherol acetate is used as the dietary source is 32 mg/kg for diets in which lipids comprise less than 10 percent of the diet . Thus, the 900 mg/kg diet used in this study is 28 fold higher than this estimated requirement. This is in contrast to human recommendations which are given in mg of α-tocopherol equivalents with the adult RDA set at 15 mg/day and the upper limit set at 1000 mg/day. The observed antitumor activity of this sE dietary supplementation was an unexpected experimental result as sE was not previously reported to have antitumor activity although vitamin E may give modest protection against the risk of breast cancer . Based on the observed antitumor activity of high dose sE in the diet, it became important to investigate if the sE supplement, when fed at such a high dose, was associated with the modification of the fatty acid composition of mitochondrial and microsomal membranes and if the sE was acting as an antioxidant or possibly a prooxidant in tumor and in host tissues. Given that such a high dose of sE has antitumor activity, it also became important to look for possible side-effects in host tissues prior to further preclinical trials.
Fatty acid composition of microsomal and of mitochondrial membranes
The percent of total fatty acid composition of linoleic acid (LA, n6 18-2), arachidonic acid (AA, n6 20-4), and docosahexaenoic acid (DHA, n3 22-6) in microsomal and in mitochondrial fractions of tumor, of liver and of colon following a two week dietary intervention of AIN·76 food formula without (-) and with (+) supplementation with synthetic vitamin E (2000 IU/kg dry food). Mean ± SD and number of mice (n) are listed.
Vitamin E supplement
8.7 ± 5.0
7.4 ± 12
0.3 ± 0.3
1.9 ± 2.1
7.9 ± 4.2
1.3 ± 1.3
7.0 ± 5.9
9.6 ± 2.8
0.2 ± 0.5
4.4 ± 5.9
5.8 ± 3.7
2.3 ± 4.9
12.9 ± 5.1
4.9 ± 1.8
1.0 ± 0.9
11.3 ± 1.7
5.4 ± 0.9
1.8 ± 0.3
14.2 ± 1.5
8.0 ± 0.9
0.5 ± 0.1
12.9 ± 1.2
6.1 ± 1.8
1.4 ± 0.5
5.3 ± 2.1
6.3 ± 2.2
0.3 ± 0.3
4.6 ± 1.2
4.1 ± 1.3
2.4 ± 1.1
9.4 ± 2.1
2.9 ± 0.2
0.8 ± 0.5
6.3 ± 1.3
3.7 ± 1.6
1.8 ± 1.0
Change in body weight (g) at two weeks and at six weeks from the start of dietary intervention with AIN·76 formula ± supplementation with 2000 IU/kg of dry food with synthetic vitamin E (mean ± SE, number of mice in parenthesis).
Vitamin E supplement
0.38 ± 0.30 (15)
-0.25 ± 0.53 (8)
0.22 ± 0.22 (17)
-0.16 ± 0.29 (5)
Hematological measurements in nude mice bearing MDA-MB-231 breast carcinoma xenografts after supplementation with (+) or without (-) synthetic Vitamin E (2000 IU/kg food) (mean ± SE, numbers of mice in parentheses) for two weeks.
Vitamin E supplement
0.62 ± 0.14
8.77 ± 0.04
471 ± 173
1.04 ± 0.43
8.50 ± 0.15
461 ± 130
Colon crypt height expressed in number of cells from the crypt base to the crypt mouth was scored as an indicator of damage attributable to sE supplementation. The crypt column heights, (number of cells, mean ± SE) was 21.24 ± 0.54 (15) for mice without the sE supplementation and 21.63 ± 0.51 (11) for mice with the sE supplementation. These means are not significantly different. No histological differences attributable to the dietary intervention with sE were observed.
Heart histopathology and antioxidant enzyme gene expression
Results of quantitative RT-PCR for antioxidant enzyme gene expression in the heart of mice fed the AIN·76 food formula with (+) or without (-) a synthetic vitamin E supplement (2000 IU/kg dry food) for six weeks.
Vitamin E supplement
Mn SODb GPX-1
.325 ± .019
.495 ± .048
.915 ± .038
2.106 ± .166
.064 ± .005
.020 ± .002
6.55 ± 0.56
.252 ± .028
.522 ± .029
.915 ± .054
2.538 ± .175
.059 ± .005
.013 ± .002
10.49 ± 1.16
Assay for chromosomal breakage or loss
Results of peripheral erythrocyte (RBC) assay for chromosomal breakage or loss. Frequency of micronuclei (MN) per 2000 erythrocytes counted in blood smear from each mouse (mean ± SE, n = number of mice).
Vitamin E supplementa
7.6 ± 1.4d
15.5 ± 2.5e
5.6 ± 1.1d
19.3 ± 1.9e
Vitamin E was established as an essential micronutrient for proper fetal development  and the amount of vitamin E required to maintain proper fetal development has been defined in International Units. In addition to its role in reproduction, vitamin E is known to be a lipid-soluble antioxidant that blocks peroxidation of polyunsaturated fatty acids in cellular membranes and is known to stabilize biological membranes [14–17]. Because vitamin E has multiple biological activities, it is best to report dosage in weight units. The recommended dietary allowance and tolerable upper intake limit for sE, the commercially available form, has been calculated to be about 22 and 1470 mg per day respectively for 70 kg adult humans .
In animal species, oral intake of up to 200 mg/kg body weight per day was reported not to result in noticeable side effects . Thus, we selected to use a dietary dose of sE at the level of 200 mg/kg body weight per day in tumor bearing mice in an attempt to maximize inhibition of lipid peroxidation in the cellular membranes of the tumor without an expectation of finding harmful side-effects. Specifically, this experiment was originally designed to test the hypothesis that DOX, a prooxidative cancer chemotherapeutic drug, inhibits tumor growth by increasing lipid peroxidation products in the tumor to cytostatic or cytotoxic levels and that suppression of lipid peroxidation by sE would suppress the antitumor effects of DOX [11, 12]. The results demonstrated that sE supplementation did markedly suppress DOX-induced lipid peroxidation, yet tumor growth was still suppressed in mice treated with DOX and sE. It was concluded that increased levels of lipid peroxidation products were not the sole cause of tumor growth inhibition by DOX. As reported here (Fig. 1), the dietary supplementation with this high dose of sE, by itself, retarded tumor growth to a significant extent.
It is not known how such a high intake of sE works to suppress tumor growth. What is known from the current study is that the sE supplement reduced lipid peroxidation (Fig. 1) and preferentially stabilized membrane polyunsaturated fatty acids with more double bonds (Fig. 3). It has been proposed that vitamin E may incorporate into cellular membranes by association of the tocopherol side chain with the polyenoic fatty acid residues in the membrane fatty acids [15, 19]. This interaction may stabilize cell membranes by making highly unsaturated fatty acids less liable to peroxidation as well as by making highly unsaturated fatty acids less available for phospholipid hydrolysis by phospholipase [15, 16]. The latter action may reduce mobilization of arachidonic acid (AA) from the membranes and in this way reduce the amount of AA available as a substrate to cyclooxygenase and/or lipoxygenase to produce eicosanoids with mitogenic and with inflammatory properties [17, 20]. Other possible antitumor actions include: induction of apoptosis, interference with hormone production, modulation of cellular signaling and gene transcription, and induction of differentiation [4, 21–24].
In comparison to the inhibitory effect of vitamin E on mammary tumor growth (this report), Cognault et al. report a stimulatory effect of vitamin E on mammary tumor growth. Specifically, Cognault et al. report that when the diet was high in omega-3 PUFAs, tumor growth was significantly increased by vitamin E supplementation. However, when the diet was relatively low in omega-3 PUFAs, the promotional effect of vitamin E was not observed . It is noteworthy that the diet used in the presently reported study was very low in omega-3 PUFAs and that this diet, combined with the high dietary level of sE, resulted in suppression of tumor growth. Thus, the different results may be attributable to the type and the amount of omega-3 polyunsaturated fatty acids (PUFAs) in the diet.
Evidence for cardiotoxicity, as observed in this study, was not expected as supplementation with vitamin E at extremely high doses gave no significant indications of harmful side-effects in animals or in humans [18, 25]. Perhaps vitamin E supplementation acts to inhibit lipid peroxidation but at the same time acts to down regulate other protective antioxidant systems as suggested by the antioxidant enzyme gene expression data in Table 4. If gene expression levels in Table 4 reflect antioxidant enzyme activity, then an increase in SOD and decrease in GPX without an increase in CAT would result in accumulation of inorganic and organic peroxides and hydroperoxides that can react with Fe2+ to yield highly reactive free radical products that can damage cellular proteins and DNA as well as lipids. Likewise, the sE supplementation used caused low expression of the stress-induced isoform of heme oxygenase, (HO-1). HO-1 catalyzes the breakdown of prooxidant heme to biliverdin with release of carbon monoxide and iron ion. It should be realized that these observations are suggestive of cardiotoxicity but are not definitive.
Early studies suggested that vitamin E lessened the cardiotoxic effects of DOX chemotherapy  whereas other studies indicate potentiation of DOX induced cardiotoxicity [27, 28]. Shinoyawa et al.  suggest that vitamin E, when combined with DOX, may increase levels of toxic products in the heart and in the tumor while Liu and Tan  suggest that vitamin E may have prooxidant properties at high dosage levels.
The micronuclei results in Table 5 suggest that while sE protects against lipid peroxidation, it did not protect the genome of erythroblasts against the prooxidant effects of DOX in this study. Thus, while the toxic effect of DOX was not significantly enhanced by vitamin E supplementation, neither was the toxic effect of DOX lessened by vitamin E supplementation.
The adage that more is better may not hold true for vitamin E as there may be beneficial effects at certain levels but not at high levels. Clearly, more research is needed on the antitumor effects of sE but caution is advised that sE may also have side-effects in the heart.
Methods and Materials
Methods involving: preparation of cells, animals, tumor and body weight measurements, necropsy and tissue processing, products of lipid peroxidation, gas chromatography, and statistical analyses have been described in a prior issue of this journal . The source of dl-α-tocopherol (synthetic vitamin E) was ICN Biochemical (Costa Mesa, CA). This experiment was conducted at the University of Texas Health Science Center at San Antonio. All animal use and handling was approved by UTHSCSA Institutional Animal Care and Use Committee.
Diet components, chemicals and composition
Purified high nitrogen casein, pure cornstarch, Alphacel (non-nutritive bulk cellulose), AIN-76 vitamin mixture, AIN-76 mineral mixture and choline bitartrate (99% pure) were obtained from ICN Nutritional Biochemicals, Cleveland, OH. Imperial brand (Sugarland, TX) extra fine pure cane sugar and 100% pure corn oil (Wesson) were purchased locally. D.L. methionine was purchased from Sigma, St. Louis, MO. Corn oil contains about 50% linoleic acid, 23% oleic acid, 10% C16 fatty acids and <1% n-3 PUFAs. The composition of the diet was as follows: 5% corn oil, 50% sugar, 20% casein, 15% cornstarch, 1% AIN-76 vitamin mix (the mix contains 20 g/kg of dl-α-tocopherol acetate), 3.5% AIN-76 mineral mix, 0.2% choline bitartrate, 0.3% D.L. methionine, and 5% Alphacel fiber .
Histological analyses of heart and colon
At sacrifice, the heart and colon were removed. The heart was crosscut through the ventricles 1/3 of the distance between the apex and the atrium, and then the apex section was fixed in OmniFix II (Mt. Vernon, NY) and then embedded, cut surface down, in molten paraffin. This provided a consistent histological section for estimates of myocyte vacuolization and toxicity. Four μm thick sections were stained with hematoxylin and eosin. The number of myocytes with vacuoles in the subendocardial region of the left ventricle was counted. Cross sections of the descending colon were scores for number of cells in a crypt column from crypt base to mouth.
Blood was obtained by cardiac puncture at sacrifice and assayed within 3 hours of collection. A Coulter STKS hematology analyzer was used for counts of red blood cells, white blood cells, and platelets in EDTA anticoagulated blood of the mice.
At the time of necropsy, blood was collected by heart puncture using a 1 ml syringe fitted with a 22G needle. Immediately, small drops of blood were placed on clean microscopic slides. The blood was pushed behind another slide held at a 45° angle to form a thin smear over an area of 3–4 cm. All smears were air-dried for 30–45 minutes. The cells on the slides were fixed in absolute methanol for 30 minutes. All slides were air-dried and stored in a box kept in a dark place at room temperature or at 4°C. The slides were stained with acridine orange before microscopic evaluation.
All slides were examined under 1000× magnification using a fluorescent microscope fitted with appropriate filters for acridine orange stain. For each animal, the percentage of 1000 erythrocytes with micronuclei was scored.
RNA extraction and quantitative reverse transcription-polymerase chain reaction
All heart samples were immediately frozen in liquid nitrogen for RNA extraction. RNA was extracted by standard methods  and analyzed by reverse transcription followed by real-time quantitative polymerase chain reaction (PCR) for the transcripts of interest. The methodology of quantitative PCR has been described in detail previously . Specific quantitative assays were designed from mouse sequences available in GenBank™. Primers and probes were designed from nonconserved sequences of the genes (allowing for isoform specificity), spanning sites where two exons join (splice sites) when such sites are known (preventing recognition of the assay to any potential contaminating genomic DNA). Standard RNA was made for all assays by the T7 polymerase method (Ambion, Austin, TX), using total RNA isolated from the mouse heart. The correlation between the Ct (the number of polymerase chain reaction cycles required for the fluorescent signal to reach a detection threshold) and the amount of standard was linear over at least a 5-log range of RNA for all assays (data not shown). The level of transcripts for the constitutive gene product β-actin was quantitatively measured in each sample to control for sample-to-sample differences in RNA concentration. Polymerase chain reaction data are reported as the number of transcripts/number of β-actin molecules.
Supported by the Susan G. Komen Breast Cancer Foundation and by American Institute for Cancer Research. Mr. N. Short is thanked for help with graphs and typing.
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