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Institute of General Pathology, Catholic University, 00168 Rome, Italy * Department of Biology, Tor Vergata University, 00133 Rome, Italy
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • canthaxanthin • carotenoids • antioxidant enzymes • iron status • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Numerous epidemiologic studies have shown
that a high consumption of fruit and vegetables, the main dietary
sources of carotenoids, is associated with a low risk for cancer
(Van Poppel and Goldbohm 1995
). Although some
intervention trials have indicated that supplemental ß-carotene is of
little or no value in preventing cancer incidence (Hennekens et al. 1996
) or mortality (Greenberg et al. 1996
)
and may actually increase lung cancer in smokers
(Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group
1994
, Omenn et al. 1996
), several other studies
have shown that carotenoids, alone or in combination with other
nutrients, may function as potent chemopreventive agents in humans
(Blot et al. 1995
) and animals (Gerster 1995
, Krinsky 1989
). One of the mechanism(s) by
which carotenoids function as antitumor agents is their ability to
modulate cell redox status. Carotenoids may act as antioxidant
(Krinsky 1993
, Palozza and Krinsky 1992
,
Sies and Stahl 1995
) or prooxidant (Palozza 1998
, Schwartz 1996
) agents in vitro. However,
it is still unclear whether such a modulation occurs in vivo and
whether it is due to intrinsic properties of carotenoid molecules per
se or to carotenoid-induced changes in cell antioxidant defenses.
The hypothesis of a modulation of antioxidants by carotenoids is
supported by experiments in vitro. ß-Carotene treatment significantly
decreased the levels of superoxide dismutase
(SOD)3
and glutathione S-transferase in human oral carcinoma cells
(Schwartz et al. 1992
). Moreover, carotenoids induce
dose-dependent changes in the activities of antioxidant enzymes in
chicken embryo fibroblasts (Lawlor and O’Brien 1995
and 1997
). Finally, a loss of endogenous 
-tocopherol was
observed in murine thymoma cells after ß-carotene treatment
(Palozza et al. 1997b
).
Although oral carotenoid supplementation can reduce or increase plasma
and tissue levels of tocopherols in animals (Bendich and Shapiro 1986
, Blakely et al. 1991
, Lambert et al. 1994
, Mayne and Parker 1989
, Tang et al. 1995
, Woodall et al. 1996
, Xu et al. 1992
) and humans (Mobarhan et al. 1994
,
Xu et al. 1992
), few studies exist on the effect of
carotenoids on cell antioxidant status in vivo.
Mice fed carotenoid-fortified supplements accumulate them in serum
and tissues, although this accumulation is not as efficient as that in
humans (van Vliet 1996
). We previously reported that Balb/c mice
incorporated canthaxanthin into liver within 15 d (Palozza et al. 1997a
). We also found that supplementation with this
carotenoid had an antitumor effect (Palozza et al. 1997a
) and altered tocopherol status (Palozza et al. 1998
) in Balb/c mice. These effects were more pronounced in
female than in male mice. In view of the hypothesis that carotenoids
may modify the course of chronic diseases by affecting the protective
ability of cells against oxidative stress, we investigated whether a
15-d canthaxanthin treatment alters lipid peroxidation and/or changes
antioxidant enzymes in liver of Balb/c mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Canthaxanthin (kindly provided by Hoffmann La Roche, Basel,
Switzerland) was administrated to the mice as stock solutions in olive
oil (3.0 g/L in the oil) as indicated (Palozza et al. 1997a
). Canthaxanthin solution was prepared every day. The
level of all-trans-canthaxanthin in the total material
was 97% and its purity was verified as indicated (Palozza et al. 1998
). Ammonium acetate and BHT were obtained from Sigma
Chemical (St. Louis, MO); tetrahydrofuran (99%) was purchased from
Aldrich Chemical (St. Louis, MO) and always used under a nitrogen
atmosphere; hexane, methanol, acetonitrile and ethanol were obtained
from Fluka Chemika-Biochemika (Buchs, Switzerland). All of the
solvents used were HPLC grade.
Animals.
Female Balb/c mice (Catholic University laboratories, Rome, Italy), aged 6 wk, with a mean body weight of 22.5 ± 1.0 g, were allocated to cages housing 5 mice per cage in a room with a 12-h day:night cycle, at a temperature of 25°C and at a constant humidity. Throughout the experiment, mice were fed a nonpurified commercial diet (Altromin-Rieper diet, Rieper, Bz, Italy).4 The mice had free access to water. Mice were examined and weighed at the start of the experiment and twice a week during the study period. All animal procedures were reviewed and approved by the Ministry of Health, Veterinary Service, Rome, Italy.
Experimental design.
The mice were assigned to one of two groups of 10. The control group
(C) received olive oil alone (vehicle) for 15 d; the
canthaxanthin-treated group (Cx) received canthaxanthin at a dose
of 14 µg/(g body wt·d) for the same period of time.
Canthaxanthin and olive oil were administered daily by gavage. Fourteen
µg/(g body wt·d) corresponds to a carotenoid dose of ~68 mg/d for
a 60 kg-man by the following formula: (dose in mg/m2)
= Km·(dose in mg/kg), where
Km is the appropriate factor for converting
doses from mg/kg to mg/m2 surface area for each species
(Km = 3 for mice and 37 for humans)
(Freireich et al. 1966
). Therefore, the dose of
carotenoid administered in this study was similar to that given in
several human studies (Delmas-Beauvieux et al. 1996
,
Johnson et al. 1997
, Xu et al. 1992
), in
which the amount of carotenoid supplemented reached 60 mg/d. In
addition, this dose represented the maximum dose of the carotenoid that
can be dissolved in an amount of olive oil tolerated by the mice.
At the end of the treatment, mice were killed by cervical dislocation and liver was excised, frozen in liquid nitrogen and stored at -80°C.
Canthaxanthin assay.
Canthaxanthin was extracted from liver samples (0.16–0.20 g) and
analyzed by HPLC according to Palozza et al. (1997a)
.
Chromatography was carried out with a LC-18-DB Supelcosil column, 15 cm
x 0.46 cm, 3-µm particle size (Supelco,
Bellefonte, PA). A C18-DB Supelcosil precolumn, 2 cm x 0.46 cm,
5-µm packing, was used. The mobile phase was 85%
acetonitrile/15% methanol, containing 0.1 g/L ammonium acetate. The
flow rate was 1 mL/min and the detection was 460 nm. Canthaxanthin
concentration in samples was calculated as
all-trans-canthaxanthin from a calibration curve
generated from a peak height of canthaxanthin in calibration samples.
Malondialdehyde (MDA) assay.
MDA was extracted and analyzed as indicated (Tatum et al. 1990
). The isobutanol extract was mixed with methanol (2:1)
before injecting into the HPLC. The column was packed with Supelcosil
LC-18 material, 3-µm particle size, in a 15 cm x 4.6 mm cartridge format (Supelco). A 2-cm cartridge precolumn
containing 5 µm LC-18 Supelcosil packing was used. The
mobile phase was a 1:1 (v/v) mixture of methanol and
double-distilled water, containing tetrabutyl ammonium dihydrogen
phosphate (0.5 g/L). The thiobarbituric acid (TBA)-MDA adduct was
detected by a fluorimeter set at an excitation wavelength of 515 nm and
an emission wavelength of 550 nm. At a flow rate of 1 mL/min, the
retention time of the TBA-MDA adduct was 5 min. MDA concentration
was calculated from a calibration curve generated from a peak height of
the MDA standard, prepared by acid hydrolysis of
1,1,3,3-tetramethoxypropane (Tatum et al. 1990
).
Assay of glutathione peroxidase (GSH-Px).
Liver samples (0.20 g) were homogenized in 0.25 mol/L sucrose and
centrifuged at 100,000 x g for 60 min. The
reaction mixture (1 mL) containing 0.05 mol/L potassium phosphate (pH
7.0), 0.001 mol/L sodium azide, 0.002 mol/L GSH, 0.0002 mol/L NADPH,
0.001 mol/L EDTA, 1 kU/L glutathione reductase and 20–100
µL of sample (0.5–1 g/L protein) was incubated at
25°C for 5 min. Addition of 0.01 mol/L
t-butylhydroperoxide started the reaction, which was
followed spectrophotometrically at 340 nm (25°C) for 3 min
(Wendel 1981
).
Glutathione assay.
Total and oxidized GSH were extracted and analyzed by HPLC as indicated
(Neuschwander-Tetri and Roll 1989
). Chromatography was
carried out with a LC-18 Supelcosil column, 25 cm x 0.46 cm,
5-µm particle size (Supelco). A C18 Supelcosil
precolumn, 2 cm x 0.46 cm, 5-µm packing, was
used. The mobile phase was 7.5% methanol/92.5% acetate buffer (0.15
mol/L, pH 7.00). The flow rate was 1.5 mL/min. Peaks were detected by
fluorescence measurement at 420 nm after excitation at 340 nm. GSH and
GSSG concentrations in samples were derived by comparing the derivative
peak area to a standard curve generated by derivatizing known amounts
of GSH.
Assay of catalase activity.
Liver samples (0.20 g) were homogenized in isotonic buffer and
centrifuged at 700 x g for 5–10 min. Ethanol
(0.01 mL EtOH/mL) was added to an aliquot of the supernatant; samples
were incubated for 30 min in an ice-water bath and Triton X-100 was
then added to a final concentration of 10 g/L. The enzyme activity was
measured spectrophotometrically, measuring the ultraviolet absorption
of H2O2 at 240 nm (Beers and Sizer 1952
).
Assay of CuZnSOD and MnSOD activities.
Liver samples (0.15 g) were excised and homogenized on ice in 0.05
mol/L potassium phosphate, 0.0001 mol/L EDTA, pH 7.4, for 2 min at full
speed with a Polytron homogenizer. Total SOD activity was assayed on 48
h-dialyzed homogenate by the method of inhibition of hematoxylin
autoxidation to hematin (Martin et al. 1987
), monitored
at 560 nm (pH 7.5, 25°C) using a standard curve obtained by purified
bovine blood enzyme. MnSOD was measured in the presence of 0.003 mol/L
Na cyanide. CuZnSOD resulted from the difference between total and
MnSOD.
Northern blot analysis.
Because it has been reported that MnSOD is an inducible enzyme and that
oxidative stress is one of the major factors regulating its gene
expression (Borrello et al. 1992
), we evaluated the mRNA
of MnSOD in liver of mice fed olive oil (control) or canthaxanthin for
15 d. Total RNA was isolated according to the method of
Chirgwin et al. (1979)
. RNA concentrations were
determined spectrophotometrically by absorbance at 260 nm. Samples of
denatured RNA (12 µg) were size-fractionated by
formaldehyde-agarose (15 g/L) gel electrophoresis and capillary
transferred to nylon membranes. Hybridization was performed at 42°C
with rat MnSOD cDNA radiolabeled by the Multiprime DNA labeling system
(Amersham). Autoradiograms were obtained at -70°C using Kodak XAR
films (Sigma Chemical, St. Louis, MO).
Hepatic Fe, Cu and Mn concentrations.
Evidence suggests that iron (De Leo et al. 1997
), copper
and manganese (Borrello et al. 1992
) are implicated in
pre- and post-transcriptional regulation of SOD; catalytic metal
ions (Halliwell and Gutteridge 1990
) are reported to
play a role in oxidative processes through Fenton reactions. For this
purpose, we measured the levels of these ions in murine liver after a
15-d treatment with olive oil (control) or canthaxanthin. Metal
concentrations were determined by atomic absorption using a Perkin
Elmer 272 Spectrophotometer (Norwalk, CT) utilizing thin slices of
fresh tissues (0.50 g), dried overnight at 100°C and digested with
nitric acid (Calviello et al. 1994
).
Statistical analysis.
Unpaired t tests were used to determine differences between the two groups. Differences, analyzed using Minitab Software (Minitab, State College, PA), were considered significant at P < 0.05. Values are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In agreement with previous results (Palozza et al. 1997a
), canthaxanthin treatment for 15 d at the dose of 14
µg/(g body wt·d) resulted in hepatic incorporation of
the carotenoid, which was maximum in liver (data not shown) and reached
0.52 ± 0.05 nmol/g liver. On the other hand, no traces of
canthaxanthin were found in liver from the control group. Nevertheless,
the presence of canthaxanthin had no discernible effect on lipid
peroxidation, measured as endogenous production of MDA. After 15 d
of canthaxanthin supplementation, liver MDA concentration was 29.8
± 0.5 and 28.7 ± 0.6 nmol/g in control and
canthaxanthin-treated mice, respectively.
Canthaxanthin treatment significantly (P < 0.01)
reduced GSH-Px activity by 35% (Fig. 1
, upper
panel). In contrast, the carotenoid treatment did not significantly modify
the hepatic GSH and GSSG concentrations. Hepatic GSH was 3.88 ± 0.40 and 3.50 ± 0.40 µmol/g wet tissue in control and
canthaxanthin-treated mice, respectively. Hepatic GSSG was 0.50
± 0.06 and 0.38 ± 0.04 µmol/g wet liver in control and
canthaxanthin-treated mice, respectively. In contrast,
canthaxanthin treatment significantly (P < 0.005)
enhanced catalase by 59% (Fig. 1
, middle panel). Although
canthaxanthin supplementation did not significantly modify CuZnSOD
activity, it significantly (P < 0.05) increased MnSOD
activity by 28% relative to controls (Fig. 1
, lower panel).
|
shows the Northern blot analysis of MnSOD mRNA and Figure 2B
shows the respective densitometric analysis of autoradiographic bands.
MnSOD mRNA levels were greater (P < 0.05) in
canthaxanthin-treated mice than in controls.
|
), but copper and manganese concentrations did not differ. Copper
concentrations were 0.12 ± 0.01 nmol/mg dry liver in control
group and 0.13 ± 0.01 nmol/mg dry liver in the
canthaxanthin-treated group. Manganese concentrations were 0.07
± 0.01 and 0.06 ± 0.01 nmol/mg dry liver in the control and
canthaxanthin groups, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), but also the activity of enzymatic antioxidants. The
carotenoid reduced the activity of GSH-Px and increased those of
catalase and MnSOD. Moreover, we have shown that it is possible to
induce the expression of the MnSOD gene in vivo by carotenoid
treatment. Changes in the activities of antioxidant enzymes by other
carotenoids, such as ß-carotene, have been reported previously in
vitro in cultured cells. It has been demonstrated that ß-carotene
treatment of human oral carcinoma cells significantly reduces the
activities of SOD and glutathione S-transferase (Schwartz et al. 1992
). Evidence that carotenoids may modify the activities
of antioxidant enzymes has been also reported in vivo. A significant
decrease in erythrocyte SOD activity was observed in adult women
consuming a ß-carotene–deficient diet (Dixon et al. 1994
). In the same study, dietary ß-carotene repletion (15
mg/d for 28 d) increased SOD activity. In addition,
carotenoid-rich food extracts [250 µg/(kg body
wt·d) for 14 wk] lowered plasma glutathione, blood SOD and blood
catalase in rats treated with aflatoxin B1 (He et al. 1997
) Finally, dietary supplementation of rats with
ß-carotene (80000 IU/kg diet for 6 wk) modulated the
increase of SOD induced by peroxyl radicals produced by a high fat diet
(Blakely et al. 1988
).
Although these results are rather controversial, they point out that
carotenoids may alter the activities of oxygen-protective enzymes;
consequently, they may induce cellular oxidative stress by acting as
prooxidants. According to this hypothesis, powerful oxidants, such as
paraquat, similarly modulate antioxidant enzymes in cultured cells,
increasing SOD and catalase activities and decreasing GSH-Px
activity (Lawlor and O’Brien 1997
). Moreover, it has
been demonstrated that MnSOD activity is increased by generators of
reactive oxygen species, such as oxidants, ionizing radiations,
cytokines, tumor necrosis factor- and lipopolysaccharide in
different experimental models (De Leo et al. 1997
).
Thus, the different responses of the three antioxidant enzymes to
canthaxanthin treatment may be due to their different roles in
oxidative processes as well as their different locations in the cells.
MnSOD is located in the mitochondria, catalase in peroxisomes and
GSH-Px in cytoplasm. On the other hand, the reduced activity of
GSH-Px may be responsible for the lack of increased GSSG levels. It
is also possible that the dose as well as the concomitant presence of
an oxidative stress may influence the modulating effect of carotenoids
on the antioxidant enzymes in vivo. Such a hypothesis is supported by
experiments in vitro. In chicken embryo fibroblasts, ß-carotene
modulated the activity of SOD, catalase and GSH-Px in a
concentration-dependent manner, i.e., at a high concentration (10
µmol/L) it increased SOD and catalase activities and
decreased GSH-Px activity, whereas at a low concentration (0.1
µmol/L), it induced the opposite effects (Lawlor and O’Brien 1995
). In addition, when a prooxidant agent such
as paraquat was used in the same model, ß-carotene prevented the
paraquat-induced elevation of catalase and SOD activities and the
reduction of GSH-Px activity at low but not at high concentrations
(Lawlor and O’Brien 1997
). It is interesting to note
that such a modulation also varied under different partial pressures of
oxygen (Lawlor and O’Brien 1997
).
It has been hypothesized that carotenoids may act as prooxidant agents
by forming carotenoid radical species in the presence of oxygen
(Burton and Ingold 1984
). This study suggests that
carotenoid molecules could also act as prooxidants by modulating the
iron content in tissues. A significantly greater iron concentration was
observed in liver of mice treated with canthaxanthin than in controls.
This is inconsistent with the observation of Blakely et al. (1991)
, who reported that canthaxanthin feeding lowered liver
nonheme iron concentrations in rats. This difference may be due to the
different doses of canthaxanthin as well as to the different animal
model. Blakely et al. (1991)
supplemented canthaxanthin
to the diet of rats for 8 wk, but at a much higher canthaxanthin
concentration [2 g/kg diet, which corresponds to an oral dose of
~100 µg/(g body wt·d) estimated from food intakes and body
weights] than that administered by gavage to mice in this study. In
contrast, it has been shown recently that vitamin A (from 0.37 to 2.78
µmol/100 g cereal) and ß-carotene (from 0.58 to 2.06
µmol/100 g cereal) can improve iron absorption from foods
in humans (Garcia-Casal et al. 1998
). The mechanism may
involve the formation of a complex between carotenoids and iron,
keeping them soluble in the intestinal lumen and preventing the
inhibitory effects of phytates and polyphenols on iron absorption
(Garcia-Casal et al. 1998
). Several studies have shown
that iron increased the levels of endogenous free oxy-radicals
through Fenton reactions (Halliwell and Gutteridge 1990
). On the other hand, it has been suggested that this ion
is also involved in the transcriptional regulation of antioxidant
enzymes (De Leo et al. 1997
).
It is interesting to note, however, that canthaxanthin did not modify in vivo lipid peroxidation as measured by MDA production, suggesting that the modulation of the endogenous antioxidant defenses is still able to limit possible prooxidant effects of the carotenoid on cell membranes.
Further investigations are required to clarify the prooxidant role of
carotenoids and the mechanisms of their interactions with cell
antioxidants in vivo by varying carotenoid dose as well as cell
antioxidant status. However, our study suggests that canthaxanthin may
alter protection against oxidative stress in vivo. This observation
could justify the dual role of carotenoids as antitumor or
tumor-promoting agents. When the prooxidant activity of carotenoids
occurs in normal cells, this could generate oxidative damage, which may
depress cell integrity and/or induce neoplastic transformation. In
contrast, when carotenoids act as prooxidants in already transformed
cells, which exhibit low antioxidant defenses (Masotti et al. 1988
), they could have beneficial effects, inhibiting tumor
growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: C, control group; Cx,
canthaxanthin-treated group; GSH, glutathione; GSH-Px,
glutathione peroxidase; GSSG, oxidized glutathione; MDA,
malondialdehyde; SOD, superoxide dismutase; TBA, thiobarbituric acid.
4 The diet had the following composition (g/100
g): crude protein, 23; fat, 5.5; fiber, 5; minerals, 8; carbohydrates,
58.5; and water, 12. The diet provided (g/kg): (n-6) polyunsaturated
fatty acids (18:2), 7.5 and (n-3) polyunsaturated fatty acids (18:3,
18:4, 20:5 and 22:6), 1.02. The vitamin mixture added to the diet was
2.5 g/kg and contained: all-rac--tocopherol
acetate, 0.1; retinyl palmitate, 0.06; cholecalciferol, 0.00005;
vitamin C, 0.1; choline chloride, 1; biotin, 0.2; folic acid, 0.01;
DL-methionine, 3.5; vitamin B-12, 0.00003; calcium
pantothenate, 0.00005; and thiamin·HCl, 0.00015. The mineral mixture
added to the diet was 0.52 g/kg and contained: Fe, 0.12; Mg, 0.06; Zn,
0.02; Cu, 0.005; Co, 0.0004; and Se, 0.0005.
Manuscript received September 13, 1999. Initial review completed October 20, 1999. Revision accepted January 26, 2000.
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