Full text of DOI:10.1007/s11745-002-0931-1

Dietary Fish Oil and Vitamin E Enhance Doxorubicin
Effects in P388 Tumor-Bearing Mice
Qi-Yuan Liu and Benny K.H. Tan*
Department of Pharmacology, Faculty of Medicine, National University of Singapore, Singapore 117597,
Republic of Singapore
ABSTRACT: In this study, four kinds of rodent diets, CO, FO, green parts of higher plants. It is generally agreed that the rela-
CVe, and FVe, were used by addition of canola oil, oil mixture
(fish oil + canola oil), canola oil plus vitamin E, and oil mixture
plus vitamin E, respectively, to a basic diet, AIN-93G, to inves-
tigate the influence of dietary fish oil and vitamin E on doxoru-
bicin (DOX) treatment in P388 ascitic mice. Animal life span
(LS) and heart damage were recorded in mice fed the four dif-
ferent diets and treated with different doses of DOX. The opti-
mal doses of DOX for antitumor effect as manifested by in-
creased LS were 6.0 and 9.0 mg/kg. Both fish oil and vitamin E
significantly enhanced this effect. On the other hand, DOX at
12.0 mg/kg induced severe heart damage, which was also sig-
tive antioxidant activity of the tocopherols in vivo is in the
order of α > β > γ > δ (6). In addition to their antioxidant prop-
erty, tocopherols and their derivatives have been reported to
have pro-oxidant properties in some biological systems (7,8).
Doxorubicin (DOX) has been widely used in the treatment
of a variety of solid tumors and hematological malignancies.
Several of its biological effects are supposedly derived from its
quinone moiety acting as an alkylating/arylating electrophile
or a pro-oxidant. The quinone structure permits DOX to act as
an electron acceptor in reactions mediated by oxoreductases,
nificantly aggravated by both fish oil and vitamin E, as shown such as cytochrome P450 reductase (9), NADH dehydrogenase
by both decreased LS and increased serum creatine phosphoki-
nase activity. Fish oil and vitamin E appeared to enhance the
antitumor effect of optimal doses of DOX but to aggravate car-
diotoxicity owing to DOX overdose.
(10), and xanthine oxidase (11), to convert the quinone to a
semiquinone free radical. Under anaerobic conditions, the
semiquinone undergoes further reduction accompanied by re-
ductive cleavage of the sugar residue to generate a quinone me-
thide, which binds covalently to nucleophiles and is known to
form adducts with DNA (12). In the presence of O2, however,
this semiquinone radical is spontaneously and rapidly reoxi-
Paper no. L8891 in Lipids 37, 549–556 (June 2002).
The effects of PUFA on tumor development and tumor growth dized in a process that generates a superoxide radical (13). The
have been intensively investigated in both animal models and superoxide radical and its dismutation product, hydrogen per-
humans in the past two decades. Although rigid scientific oxide, undergo Haber–Weiss and Fenton reactions, respec-
proof has yet to be obtained, it is generally believed that n-6 tively, to form the hydroxyl radical, which is able to extract a
PUFA in terrestrial plant oils enhance tumor growth, but n-3 doubly allylic hydrogen atom from PUFA and thus initiate lipid
PUFA in fish oil have a protective effect (1). Furthermore, two peroxidation (14). The nonenzymatic formation of reactive
long-chain n-3 PUFA that are found in significant amounts in oxygen species is mediated by the formation of complexes be-
fish oil, viz., EPA (20:5n-3) and DHA (22:6n-3), may exert tween DOX and iron, which can either react with O2 to yield
multiple activities to retard the growth of tumor cells and superoxide radical and hydrogen peroxide or bind O2 in a su-
xenografts (2,3). On the other hand, it is known that certain peroxo- or peroxo-like form (15). Despite its antitumor effect,
physical and functional properties of cell membranes can be clinical effectiveness of DOX is hindered by its unique dose-
modified when PUFA content is changed, and lipid nutrition limiting cardiotoxicity (16), possibly because of the sensitivity
directed at producing this modification has been suggested in of the heart to reactive free radicals, due to a low activity of an-
cancer therapy (4). By altering the properties of membrane tioxidant enzymes (17). Separates studies, however, have found
lipids, FA-based diets may provide a new approach for en- both effectiveness (18,19) and ineffectiveness (20) of vit E in
hancing the effectiveness of certain antineoplastic therapies.
Vitamin E (vit E) includes eight naturally occurring com-
cardioprotection in DOX-treated animals.
In this study, we prepared four experimental diets by
pounds of two classes, tocopherols and tocotrienols, with dif- adding natural oils to the basic components of AIN-93G, a
ferent biological activities. Tocopherols are the most important purified diet for laboratory rodent growth (21). These diets
chain-breaking antioxidants within cellular membranes, mainly differed in their contents of n-3 PUFA and tocopherols. We
owing to their ability to donate phenolic hydrogens to lipid free tested their effects on the therapeutic efficacy of various doses
radicals (5). They are present in oily seeds, leaves, and other of DOX in mice induced with P388 ascitic tumor [which is
the standard transplantable murine tumor most sensitive to
DOX (18)], recording life span (LS) of the tumor-bearing
mice, and examining animal heart damage by measurement
of serum creatine phosphokinase (CPK) level (a biochemical
marker of animal myocardial damage).
*To whom correspondence should be addressed at Dept. of Pharmacology,
Faculty of Medicine, National University of Singapore, MD2, 18 Medical
Dr., Singapore 117597, Republic of Singapore.
E-mail: phctankh@nus.edu.sg
Abbreviations: CPK, creatine phosphokinase; DOX, doxorubicin; HNE, 4-
hydroxynonenal; LS, life span; MDA, malondialdehyde, vit E, vitamin E.
Copyright © 2002 by AOCS Press
549
Lipids, Vol. 37, no. 6 (2002)

550
Q.-Y. LIU AND B.K.H. TAN
AIN-93G basic diet contains 75 mg/kg d-α-tocopherol acetate
EXPERIMENTAL PROCEDURES
(21). The natural vit E used in our experiments is a mixed to-
®
Cell lines and chemicals. The P388 cell line was obtained
from ATCC (American Type Culture Collection, Manassas,
VA), and the cells were grown in DBA/2 mice for one gener-
ation before use in the following animal experiments. All
chemicals were purchased from Sigma-Aldrich (St. Louis,
MO) unless otherwise stated.
Diets. Four experimental diets with AIN-93G as a base
food were prepared by Specialty Feed Service (Glen Forest,
Australia) as follows: (i) CO: AIN-93G + 10% canola oil;
(ii) FO: AIN-93G + 8.5% fish oil + 1.5% canola oil; (iii) CVe:
AIN-93G + 500 mg/kg natural vit E + 10% canola oil;
(iv) FVe: AIN-93G + 500 mg/kg natural vit E + 8.5% fish oil
+ 1.5% canola oil.
Canola oil, a favorable dietary oil, is an appropriate control
to use to study the effect of long-chain n-3 PUFA in fish oil be-
cause it contains a relatively high proportion of linoleic acid
(18:2n-6) and α-linolenic acid (18:3n-6), and a low proportion
of EPA and DHA. Canola oil (1.5%) was added to fish oil
(8.5%) in FO and FVe diets in order to increase linoleic acid
content to above 1%, which is the requirement for optimal ro-
dent growth. The FA composition of the four diets is shown in
Table 1. Total n-6 PUFA content was similar in the four diets,
whereas n-3 PUFA contents, particularly EPA and DHA, were
greater in FO and FVe than in CO and CVe. On the other hand,
copherol concentrate, Covi-ox T-70 (Cognis Nutrition &
Health, Broadmeadows, Victoria, Australia), with relative
amounts of α- (14%), β- (1%), γ- (62%), and δ-tocopherols
®
(23%). Five hundred milligrams of Covi-ox T-70 contains
350 mg of total tocopherols. According to the manufacturer
(Specialty Feed Service, Glen Forest, Australia), canola oil
contained 330, 16, 846 and 56 mg/kg of α-, β-, γ-, and δ-to-
copherols, respectively, whereas fish oil contained 340 mg/kg
of α-tocopherol only. The calculated contents of individual vit
E species in four diets are shown in Table 2, indicating that CVe
and FVe contained a significantly higher content of all tocoph-
erol species compared to CO and FO, respectively.
Comparisons between CO and FO and between CVe and
FVe were made to evaluate the effect of fish oil, whereas
comparisons between CO and CVe and between FO and FVe
were made to examine the effect of vit E on tumor-bearing or
normal mice with or without DOX treatment.
Measurements of LS. All experiments with animals were
conducted according to guidelines in “International Guiding
Principles for Animal Research,” WHO Chronicles, Vol. 39,
1985. Male BDF1 mice purchased from Animal Resources
Center (Canning Vale, Western Australia) were used at 6–8 wk
of age when their body weights ranged between 19.0 and 21.0
g. The animals were kept in filter-covered plastic cages,
housed in a temperature-controlled room with a diurnal 12 h
light cycle, and provided with tap water and the assigned ex-
perimental diets ad libitum for 10 d prior to experimentation
and continuously thereafter. This period of time was found to
be sufficient to modify the PUFA composition of phospho-
lipids in most organs to stable levels in mice (22). The animals
were inoculated intraperitoneally on day 0 with P388 cells
(106 cells/mouse), followed by injection of DOX or saline also
intraperitoneally on days 1 and 6. Deaths among the experi-
mental animals were recorded at least twice a day.
Determination of DOX cardiotoxicity. In the second experi-
ment, the BDF1 mice were maintained, fed, and administered
DOX or saline as described above without the inoculation of
tumor cells. The animals were killed on day 7 by cervical dislo-
cation, blood was collected by cardiac puncture, and serum was
obtained after the clotting of blood at room temperature for 1 h.
Serum was kept frozen (−76°C) until analysis. Cardiotoxicity
was determined by measuring serum CPK activity, calculated
according to the Sigma Diagnostics Procedure No. 47-UV. A
TABLE 1
FA Composition in Four Experimental Dietsa
FA (%)
CO
FO
CVe
FVe
4.5
19.2
5.0
4.1
18.4
ND
13.5
ND
1.7
Individual
14:0
16:0
ND
5.6
4.3
19.8
2.2
ND
4.6
2.0
ND
60.3
1.5
20.1
10.5
0.6
18:0
1.3
16:1
18:1
20:1
ND
60.1
1.5
5.1
19.3
ND
16.6
ND
1.3
1.9
8.7
20.9
100
18:2n-6
18:3n-3
18:4n-3
20:4n-6
20:5n-3
22:6n-3
Total
20.3
10.2
0.6
ND
0.5
ND
0.5
ND
1.9
9.1
22.5
100
ND
100
100
Classb
∑SFA
6.9
61.6
31.6
26.3
24.4
49.4
6.6
61.8
31.7
28.7
22.5
48.7
∑MUFA
∑PUFA
TABLE 2
∑n-3 PUFA
∑n-6 PUFA
11.3
20.3
30.9
19.8
11.6
20.7
33.3
17.1
Vitamin E Content (mg/kg) in Four Experimental Dietsa
Species of vitamin E
CO
FO
CVe
FVe
∑n-3 PUFA/∑n-6 PUFA
Average double bondc
0.56
1.4
1.56
2.4
0.56
1.4
1.95
2.4
α-Tocopherol acetate
α-Tocopherol
β-Tocopherol
γ-Tocopherol
δ-Tocopherol
75
33
1.6
75
33.9
0.2
75
82
5.1
75
82.9
3.7
229.7
81.3
aDiet CO, AIN-93G + 10% canola oil; FO, AIN-93G + 8.5% fish oil + 1.5%
canola oil; CVe, AIN-93G + 500 mg/kg natural vitamin E + 10% canola oil;
FVe, AIN-93G + 500 mg/kg natural vitamin E + 8.5% fish oil + 1.5% canola
oil; ND, not detected.
84.6
5.6
12.7
0.8
301.6
86.1
bSFA, saturated FA; MUFA, monounsaturated FA.
Total
200
122.2
549.8
472.6
c”Average double bond” means average number of double bonds per mole-
cule of FA.
aFor description of diets see Table 1.
Lipids, Vol. 37, no. 6 (2002)

FISH OIL, VITAMIN E, AND DOXORUBICIN ANTITUMOR
551
TABLE 3
CPK Test kit (CK-20; Sigma Chemical Co.) based on the
ANOVA on Life Span (LS) of P388 Ascitic Micea
method described by Szasz et al. (23) was used in the study.
Lipid analysis of P388 ascitic tumor cells and cardiac tis-
sue. In the third experiment, BDF1 mice were fed experimen-
tal diets 10 d prior to the experimentation and thereafter. P388
cells (107 cells/mouse) were intraperitoneally inoculated into
animals on day 0. On day 7, the mice were killed by cervical
dislocation. The hearts were collected and ascitic tumor cells
obtained by laving the peritoneum with ice-cold saline. The
tumor cells were pelleted at 700 × g for 5 min and washed twice
with the same buffer solution. Total lipid was extracted using a
method described by Folch et al. (24). The phospholipid frac-
tion was separated by silicic acid chromatography (25), saponi-
fied in alkali (26), and methylated with BF3/methanol (27). The
fractions of FAME with different chain lengths and unsatura-
tions were separated in a Hewlett-Packard 5890 gas chromato-
graph using a DB-225 column (J&W Scientific, Folsom, CA).
Peaks were identified by comparison with the retention times
of known standards and peak areas quantified.
Two-way ANOVA
Source
P0 (n = 28)
P12 (n = 24)
Fish oil
Vit E
Fish oil × vit E
0.052
0.010
0.854
0.018
0.023
1.000
Multiway ANOVA
Source
P6,9 (n = 48)
DOX
Fish oil
Vit E
DOX × fish oil
DOX × vit E
Fish oil × vit E
0.262
0.025
<0.001
0.567
0.844
0.601
aCanola oil served as the control for fish oil. Column P0 considers data on
the LS of animals without doxorubicin (DOX) treatment; P12, with 12.0
mg/kg DOX treatment; and P6,9, with 6.0 and 9.0 mg/kg DOX treatment; n,
number of mice; vit E, vitamin E.
Statistical analysis. Data were analyzed by Student’s t-test
or ANOVA as shown in the text and the legends for the tables
and figures. Experimental data are shown as mean SD. P <
0.05 was set as the level of significance throughout the study.
RESULTS
LS of P388 ascitic mice. The individual LS of P388 ascitic
mice fed the four experimental diets and administered vari-
FIG. 1. Effects of different doses of doxorubicin (DOX) and experimental diets on life span (LS)
of P388 ascitic mice. Each column represents the LS of one animal, whereas the value above
the cluster of columns represents the mean LS in each group. *P < 0.05 represents the differ-
ence between the saline and DOX-treated groups on the same diet (Student’s t-test). Diets:
CO, AIN-93G + 10% canola oil; FO, AIN-93G + 8.5% fish oil + 1.5% canola oil; CVe, AIN-
93G + 500 mg/kg natural vitamin E + 10% canola oil; FVe, AIN-93G + 500 mg/kg natural
vitamin E + 8.5% fish oil + 1.5% canola oil.
Lipids, Vol. 37, no. 6 (2002)

552
Q.-Y. LIU AND B.K.H. TAN
FIG. 2. Effects of fish oil and vitamin E (vit E) on LS of P388 ascitic mice treated with various
doses of DOX. The values outside and inside the parentheses at each point represent the mean
LS and number of animals, respectively, in the group. (A) Effect of fish oil; (B) effect of vit E.
Canola oil served as the control for fish oil. For abbreviations see Figure 1.
ous doses of DOX are shown in Figure 1. The results of sta- of 12.0 mg/kg DOX (increase of 0.7 unit/mL serum; P = 0.002,
tistical analyses of the data are shown in Figures 1 and 2 and two-way ANOVA). On the other hand, fish oil increased DOX-
Table 3. In the absence of DOX treatment, vit E supplement induced CPK activity to an extent proportional to DOX dose
increased the mean LS from 17.7 to 18.8 d (P = 0.010, two- (increases of 0, 0.3, 0.6, and 1.5 unit/mL serum in 0, 6.0, 9.0,
way ANOVA) (Fig. 2B, Table 3). Both 6.0 and 9.0 mg/kg and 12.0 mg/kg DOX-treated mice with P = 0.935, 0.005,
DOX significantly increased animal LS in all diet groups (P <0.001, and <0.001, respectively, two-way ANOVA) (Fig. 3).
< 0.05, Student’s t-test) (Fig. 1), but there was no significant
FA composition of ascitic tumors and animal hearts. Lipid
difference (P = 0.262, multiway ANOVA) in therapeutic effi- analysis of the FA composition in phospholipids indicated that
cacy between these two doses (Table 3). Both fish oil and vit n-6 PUFA content was comparable in both P388 ascitic tumor
E, however, were found to significantly enhance the effect of and mouse heart of all four groups, whereas n-3 PUFA as well
DOX at these doses (P = 0.025 and <0.001, respectively, mul- as total PUFA content was significantly larger in both tissues of
tiway ANOVA) (Table 3). Fish oil increased the mean LS FO- and FVe-fed mice than those in CO- and CVe-fed mice
from 40.8 to 49.6 d in mice treated with 6.0 mg/kg DOX and
from 43.6 to 58.3 d in mice treated with 9.0 mg/kg DOX (Fig.
2A). Vit E increased the mean LS from 33.8 to 56.6 d in mice
treated with 6.0 mg/kg DOX and from 40.5 to 61.4 d in mice
treated with 9.0 mg/kg DOX (Fig. 2B). When DOX dose was
increased to 12.0 from 9.0 mg/kg, animal LS was signifi-
cantly decreased (P < 0.001, multiway ANOVA followed by
Tukey’s test for DOX only), suggesting the negative effect of
DOX at this high dose. Both fish oil and vit E significantly
aggravated this negative effect (P = 0.018 and 0.023, respec-
tively, two-way ANOVA) (Table 3), with fish oil decreasing
the mean LS from 28.2 to 20.8 d (Fig. 2A), and vit E decreas-
ing the mean LS from 28.0 to 21.0 d (Fig. 2B).
TABLE 4
Effects of Fish Oil and Vit E on CPK Activity in Mice
Treated with Various Doses of DOXa
A: CPK activity (unit/mL)b
DOX (mg/kg)
Diet
0
6.0
9.0
12.0
CO
FO
CVe
FVe
0.6 0.1
0.7 0.1
0.4 0.1
0.3 0.1
0.8 0.1
1.1 0.3
0.6 0.2
0.8 0.2
1.1 0.2
1.7 0.3
0.9 0.2
1.6 0.2
1.9 0.2
3.3 0.1
2.5 0.3
4.1 0.7
B: Results of two-way ANOVAb
Source
P0 (n = 24) P6 (n = 24) P9 (n = 24) P12 (n = 24)
DOX-induced CPK activity. The experimental data of
plasma CPK levels in mice fed the four experimental diets and
treated with various doses of DOX are shown in Table 4, part
A. The results of statistical analysis on the data are shown in
Table 4, part B, and Figure 3. DOX increased plasma CPK ac-
tivity dose-dependently (Table 4). Two-way ANOVA (Table 4)
showed that vit E inhibited plasma CPK activity in mice with-
out DOX (decrease of 0.3 unit/mL serum; P < 0.001) or with
6.0 mg/kg DOX (decrease of 0.3 unit/mL serum; P = 0.007)
but did not significantly modify CPK activity with 9.0 mg/kg
DOX. In contrast, vit E enhanced CPK activity in the presence
Fish oil
Vit E
Fish oil × vit E
0.935
<0.001
0.142
0.005
0.007
0.456
<0.001
0.095
0.965
<0.001
0.002
0.687
aThe experimental data are shown as mean SD of six determinations on
the samples obtained from four mice in each diet group. Canola oil served
as the control of fish oil. Column P0 considers the data on groups given
saline treatment; P6, with 6.0 mg/kg DOX treatment; P9, with 9.0 mg/kg
DOX treatment; and P12, with 12.0 mg/kg DOX treatment. n, number of de-
terminations.
bMultiway ANOVA followed by Tukey’s test for DOX only, P < 0.001 for 0
vs. 6.0 mg/kg, P < 0.001 for 6.0 vs. 9.0 mg/kg, and P < 0.001 for 9.0 vs. 12.0
mg/kg. For description of diets see Table 1. For abbreviations see Table 3.
Lipids, Vol. 37, no. 6 (2002)

FISH OIL, VITAMIN E, AND DOXORUBICIN ANTITUMOR
553
FIG. 3. Effects of fish oil/vit E interactions with DOX on CPK activity in normal mice. The value
at each point represents the mean CPK activity from 12 determinations on the samples from
eight mice in each group. (A) Effect of fish oil; (B) effect of vit E. Canola oil served as the con-
trol for fish oil. CPK, creatine phosphokinase; for other abbreviations see Figures 1 and 2.
FIG. 4. Effects of experimental diets on PUFA in phospholipids of P388 ascitic tumor and
mouse heart. (A) P388 ascitic tumor; (B) animal heart. The columns represent mean SD from
five separate determinations on samples from three mice in each group. For diets see Figure 1.
Key: n-3 PUFA, bars with stippling; n-6 PUFA, bars with vertical and horizontal lines.
(Fig. 4). The increased content of n-3 PUFA in FO and FVe (28–30). Vit E may inhibit tumor growth via the cytotoxic ef-
groups can be attributed largely to EPA and DHA (data not fects of tocopherols and their derivatives (31–33), induction
shown), both of which were present in large amounts in FO and of apoptosis through activation of P21WAF1/CIP1 (34), reduc-
FVe diets.
tion of prostaglandin E2 production (35), and abatement of
muscle wasting in cancer cachexia (36).
In the present study, the optimal DOX antitumor effect was
observed at 6.0 and 9.0 mg/kg doses. When DOX dose was
DISCUSSION
Four AIN-93G-based rodent diets, CO, FO, CVe, and FVe, increased to 12.0 mg/kg, the animal LS significantly de-
were designed for this study. CO and CVe contained 10% creased, indicating that DOX at this high dose produced a se-
canola oil, whereas FO and FVe contained 8.5% fish oil plus vere negative effect. The major negative effect of DOX might
1.5% canola oil. CVe and FVe additionally included 500 be its cardiotoxicity, as it was shown that serum CPK activity
mg/kg natural vit E. FO and FVe with CO and CVe as their increased dose-dependently. Transmission electron micros-
respective controls were used to investigate the effect of fish copy also demonstrated that DOX at 12.0 mg/kg induced
oil, and CVe and FVe with CO and FO as their respective con- myofibrillar fragmentation and damaged mitochondria in the
trols were applied to assess the effect of vit E.
heart (data not shown).
This study demonstrated the therapeutic effect of vit E in
The effects of DOX at various doses were modified by
P388 mice (6.2% increase in mean LS; P = 0.010, two-way both fish oil and vit E. Fish oil not only significantly enhanced
ANOVA). This was consistent with the previous reports of 6.0 and 9.0 mg/kg DOX therapeutic efficacy in P388 ascitic
tumor inhibitory and chemopreventive effects of tocopherols mice (21.6 and 33.7% increases in mean LS, respectively;
Lipids, Vol. 37, no. 6 (2002)

554
Q.-Y. LIU AND B.K.H. TAN
P = 0.025, multiway ANOVA) but also elevated mouse overdose DOX, vit E supplement aggravated 12.0 mg/kg DOX
plasma CPK level to an extent proportional to the DOX dose cardiotoxicity and thus decreased animal LS (25%; P = 0.023
administered, implying that fish oil might enhance DOX cy- in two-way ANOVA). Tocopherols transform to chromanoxyl
totoxicity in both tumor cells and heart. The chemistry of radicals in the scavenging of lipid radicals. In the absence of
DOX lends itself to enzymatic and nonenzymatic generation adequate reductants, such as ascorbic acid, the chromanoxyl
of superoxide radical and the secondary reactive oxygen radicals will be irreversibly degraded to tocopheryl quinones
species derived from superoxide, including hydrogen perox- (44), which are able to initiate and/or enhance lipid peroxida-
ide and hydroxyl radical (10,14). The hydroxyl radical is able tion (6). Owing to the potential pro-oxidant activity of vit E, it
to extract a doubly allylic hydrogen atom from the unsatu- may interact synergistically with DOX to produce reactive free
rated lipids to form lipid radicals (37), which may react radicals and induce lipid peroxidation. Overdose DOX treat-
quickly with O2 to form lipid peroxyl radicals that in turn pro- ment produces oxidative stress, which may overwhelm and/or
duce lipid endoperoxides (37), or else initiate the lipid perox- reduce the antioxidant capability in cells. In the absence of an
idation cycle, converting other unsaturated lipids into lipid efficient antioxidant apparatus, such as in the heart, the reac-
hydroperoxides (38). A number of lipid endoperoxides and tive oxygen metabolites of tocopherols, together with DOX,
hydroperoxides and their aldehyde derivatives have been im- engender the production of vast amounts of oxygen free radi-
plicated as causative agents for cytotoxic processes in cells cals, thereby causing extreme cytotoxicity to cells through
(39). Lipid peroxidation has been suggested to be the cause drastically increased lipid peroxidation.
of DOX cytotoxicity in both tumor (40) and heart tissue (41).
It thus seems that, at least in animals, vit E may express
The increased membrane lipid unsaturation would conse- antioxidant or other activities to protect against heart damage
quently provide more targets for peroxidative events gener- when combined with DOX at optimal doses, but produce a
ated by the metabolism of DOX, thus increasing the cytotoxi- pro-oxidant effect to enhance DOX-induced cardiotoxicity
city to tissues. On the other hand, the interaction between fish when combined with overdose DOX. This may explain why,
oil and DOX cytotoxicity might also occur through the en- in some studies, vit E was reported to be ineffective in car-
hancement of DOX accumulation owing to the increased dioprotection (20) or even to be deleterious to the heart of
membrane lipid unsaturation and thus fluidity (4,42). The DOX-treated animals (45). Another point to note is that, al-
lipid analysis in the present study showed that PUFA content though α-tocopherol content was similar between fish oil and
(an index of cell membrane unsaturation) was increased in canola oil, the latter had a greater γ-tocopherol content than
both ascitic tumor and animal heart in mice fed FO and FVe, fish oil. However, fish oil but not canola oil enhanced the ther-
suggesting the possibility that dietary fish oil could directly apeutic efficacy of DOX, implying that α-tocopherol, but not
enhance DOX-induced lipid peroxidation as well as elevate γ-tocopherol, may be the dominant species of vit E with re-
intracellular DOX accumulation in both tumors and heart. Al- spect to its activity. This is consistent with the fact that α-to-
though fish oil markedly exacerbated 12.0 mg/kg DOX car- copherol has both a higher (absolute value) tissue distribution
diotoxicity and abolished the DOX therapeutic effect, the in- (46) and a higher antioxidant activity (6) than γ-tocopherol.
creased LS was achieved when DOX was administered at op-
In addition, the function of both PUFA and tocopherols as
timal doses, i.e., tumor inhibition could be achieved with alkylating/arylating agents is known to account for some of
DOX at optimal doses while deleterious effects on the heart their biological effects. The electrophiles generated from lipid
occurred only with DOX overdose. This suggests that heart peroxidation, such as malondialdehyde (MDA) and 4-hydroxy-
and tumor may have differential responses to the interaction nonenal (HNE), may react with DNA bases. MDA modifies
of fish oil and DOX and/or differential sensitivities to the cy- purine bases to generate a tricyclic adduct with guanine (47)
totoxicity induced by DOX combined with fish oil.
and an acyclic adduct with adenine (48). HNE reacts directly
Vit E seemed to have an inhibitory effect on 6.0 mg/kg with 2′-deoxyguanosine to produce a tricyclic substituted
DOX-induced CPK release. Many studies found that vit E in- propane adduct and is readily oxidized to the epoxide deriva-
jection protected against DOX cardiotoxicity (18,19). The tive, which can covalently modify both 2′-deoxyguanosine and
cardioprotective effect was achieved in the present study by 2′-deoxyadenosine to form etheno adducts (49). Failure to re-
dietary vit E. The scavenging activity of vit E on lipid peroxi- pair these DNA lesions can lead to apoptosis (50). On the other
dation has been proposed to account for the cardioprotective hand, γ- and δ-tocopherol quinones generated from γ- and δ-to-
effect of DOX in DOX-treated animals (18,19), although copherols, respectively, are cytotoxic arylating electrophiles
mechanism(s) other than its antioxidant activity might also because they contain an α,β unsaturated carbonyl structure that
contribute to the cardioprotection (43). On the other hand, vit forms Michael adducts with compounds containing a thiol nu-
E has been reported to directly enhance the inhibitory effect cleophilic group, such as glutathione and proteins (51).
of DOX on tumor growth (34), and this may partly explain Whether treatment with PUFA, tocopherols, and DOX leads to
the enhanced therapeutic effectiveness of 9.0 mg/kg DOX. It the interactions through their alkylating/arylating activities and
is thus possible that vit E might enhance the therapeutic effi- thereby enhances therapeutic efficacy remains to be studied.
cacy of DOX at optimal doses via cardioprotection and/or en-
hancement of tumor growth inhibition.
Animal xenograft studies found that human lung cancer
(52) and mammary carcinoma (53) in nude mice showed a
Through a synergistic interaction between tocopherols and greater sensitivity to DOX when the mice were fed fish oil.
Lipids, Vol. 37, no. 6 (2002)

FISH OIL, VITAMIN E, AND DOXORUBICIN ANTITUMOR
555
14. Gutteridge, J.M. (1984) Lipid Peroxidation and Possible Hy-
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A Clinicopathologic Analysis of Adriamycin Cardiotoxicity,
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Furthermore, the present work comprehensively investigated
the influence of dietary fish oil and vit E, individually and in
combination, on the therapeutic efficacy of DOX and has
shown their additively enhancing effects on optimal doses of
DOX in P388 ascitic mice. Increasing DOX dose, however,
led to severe heart damage, which was exacerbated by fish oil
and vit E. Thus overall, it appears that both fish oil and vit E
modulate the effects of DOX in the laboratory mouse like a
double-edged sword, on the one hand enhancing its antitumor
effect and on the other, aggravating its cardiotoxicity.
ACKNOWLEDGMENTS
This work was supported by a Research Grant from the National
University of Singapore (No. R-184-000-018-112). The authors
thank Annie Hsu for technical assistance in animal experiments and
Frances Lim for her help in the use of the gas chromatograph for the
estimation of FAME.
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