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Abstract
Experiments were conducted to determine the antioxidant and prooxidant eects of b-carotene, a-tocopherol and ascorbic acid
on human lung cells at dierent oxygen (O2) tensions. Free radical initiator, 2,20 -azobis (2-amidinopropane) dihydrochloride
(AAPH), was used to induce the cellular damage associated with lipid peroxidation, protein oxidation and DNA breaks. Under
hypoxic conditions (0 torr O2 tension) all compounds produced a concentration-dependent antioxidant eect. Mixtures of the three
compounds exhibited greater protective aects than any individual compound. At 143 torr O2 tension, all compounds exhibited
concentration-dependent protective eects against AAPH-induced cellular lipid, protein and DNA damage. At 722 torr O2 tension,
cells exhibited a consistent increase in lipid peroxidation (isoprostane formation), protein oxidation (carbonyl formation) and DNA
damage (p53 protein accumulation). b-Carotene (1.5 mm) produced a prooxidant eect by promoting 12% isoprostane formation.
Protein oxidation and DNA damage at 722 torr O2 tension was not increased by b-carotene; however, the antioxidant eect of bcarotene was attenuated. The antioxidant eects of a-tocopherol, ascorbic acid, and mixtures of the three antioxidant compounds
also were reduced by the high O2 conditions. These results partially substantiate the hypothesis that the antioxidant and prooxidant
eects of b-carotene are dependent on O2 tension and concentration of b-carotene. Such ndings may partially explain why selected
populations, such as smokers, respond adversely when supplemented with b-carotene. # 2001 Elsevier Science Ltd. All rights
reserved.
Keywords: b-Carotene; a-Tocopherol; Ascorbic acid; Human lung cell line CCD-8Lu; Antioxidant; Prooxidant
1. Introduction
Intervention trials and animal studies exploring the
relationship between b-carotene and disease suggest that
b-carotene is of little or no value in preventing cardiovascular disease and the major cancers that occur in
well-nourished populations (ATBC Study Group, 1994;
Greenberg et al., 1996; Hennekens et al., 1996; Omenn
et al., 1996a; Wang et al., 1999). The mechanism
involved, so far understood, is the prooxidant eect of
b-carotene at high oxygen concentration. The relationship between the prooxidant eect of b-carotene and
Abbreviations: AAPH, 2,20 -azobis (2-amidinopropane) dihydrochloride; BHT, butylated hydroxytoluene; BSA, bovine serum
albumin; DNP, 2,4-dinitrophenylhydrozine; HRP, disodium horseradish peroxidase; PBS, phosphate buered saline; pNPP, p-nitrophenylphosphate; THF, tetrahydrouran;
* Corresponding author. Tel.: +1-775-784-6447; fax: +1-775-7846449.
E-mail address: omaye@unr.edu (S.T. Omaye).
oxygen concentration and consequences of the prooxidant eect of b-carotene have been studied in in vitro
(Vile and Winterbourn, 1988; Palozza et al., 1995,
1997). In vitro, whether b-carotene is antioxidant or
prooxidant is dependent on oxygen tension and the
concentration of b-carotene (Zhang and Omaye, 2000).
The purpose of this study was to test the anti- or
prooxidant eects of b-carotene on human lung cells
with respect to dierent oxygen tensions, thus allowing
us to examine the eects of intact cells on the interactions between b-carotene, oxidation and the subsequent
impact of antioxidant mixtures. Several epidemiological
studies provided data showing the negative eects of bcarotene on lung cancer incidence (ATBC Study Group,
1994; Omenn et al., 1996a,b; Omenn, 1998; Albanes,
1999; Wang et al., 1999). However, the mechanism of
such negative eects is unclear. Wang et al. (1999) suggested that diminished retinoid signalling, resulting from
the suppression of RAR b gene expression and overexpression of activator protein-1, could be a mechanism
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0887-2333(00)00054-0
14
to enhance lung tumorigenesis after high-dose b-carotene supplementation and exposure to tobacco smoke.
To mimic dierent partial pressure of oxygen to
which the lung is exposed (Guyton, 1991), we studied
oxygen tensions of 0, 143 and 722 torr to represent the
hypoxic, normal and extreme conditions respectively of
oxygen tension in the human body.
2. Materials and methods
The concentration of compounds used in the present
in vitro study was within the range of levels which may
be achieved in sera of human subjects (Combs, 1998).
Media concentrations were within the range found in
human subjects and between 0.5 and 7% of the targeted
concentrations (Tables 1 and 2). The composition of
antioxidant mixtures was: (1) 0.1 mm b-carotene, 5 mm atocopherol and 10 mm ascorbic acid; (2) 0.2 mm b-carotene, 10 mm a-tocopherol and 20 mm ascorbic acid; (3)
0.4 mm b-carotene, 20 mm a-tocopherol and 40 mm
ascorbic acid; (4) 0.8 mm b-carotene, 40 mm a-tocopherol
and 80 mm ascorbic acid; (5) 1.6 mm b-carotene, 80 mm
a-tocopherol and 160 mm ascorbic acid. Actual antioxidant concentrations found in the media were
between 0.5 and 7% of the added concentrations.
2.1. Chemicals
b-Carotene, a-tocopherol (vitamin E), ascorbic acid
(vitamin C) were purchased from Sigma Chemical
Company (St Louis, MO, USA). 2,20 -Azobis (2-amidinopropane) dihydrochloride (AAPH) was purchased
from Wako Chemicals (Richmond, VA, USA). Fresh
stock solutions of b-carotene, a-tocopherol and ascorbic
acid were prepared as needed. Concentrations of each
compound are listed in Table 1.
2.2. Cell line
Human lung cells (CCD-8Lu) were obtained from the
American Type Culture Collection (Manassas, VA,
USA) and grown in Eagle's minimum essential medium
supplemented with 10% fetal bovine serum (Atlanta
Biologicals, Norcross, GA, USA) and maintained at
37 C with a 5% CO2/95% atmosphere.
Table 1
ELISA analysis of isoprostane from human lung cell line CCD-8Lu cultured with b-carotene, a-tocopherol, ascorbic acid and a mixture of these
three compounds at 0, 143 and 722 torr oxygen tension, respectively
a-Tocopherol
(mm)
Isoprostane
(ng/ml)
Ascorbic
aci(mm)
Isoprostane
(ng/ml)
Mixture no.b
(mm)
Isoprostane
(ng/ml)
0
AAPH
5
10
20
40
80
3.020.61
29.563.57
27.451.39
25.123.58
24.892.36
19.474.12*
13.643.11**
0
AAPH
10
20
40
80
160
4.560.78
32.334.19
29.43.28
23.82.13*
22.564.75
21.312.69*
12.63.28**
0
AAPH
1
2
3
4
5
3.581.21
33.584.26
18.652.36**
18.213.45**
14.582.57**
11.851.38**
8.692.38***
0
AAPH
5
10
20
40
80
0
AAPH
10
20
30
80
160
2.890.41
29.692.35
32.15.01
30.272.14
25.14.21
16.53.27**
12.82.47***
0
AAPH
1
2
3
4
5
4.220.95
32.162.36
20.583.65**
17.221.25***
16.233.56***
12.542.77***
7.593.2***
0
AAPH
5
10
20
40
80
0
AAPH
10
20
40
80
160
7.140.59
43.212.36
36.12.1*
40.583.88
33.542.31**
24.35.09**
28.554.17**
0
AAPH
1
2
3
4
5
9.661.25
42.181.08
39.75.07
31.776.7
30.84.15*
18.351.05***
15.224.11***
b-Carotene
(mm)
Isoprostane
(ng/ml)a
2.360.58
31.22.78
30.53.11
31.52.03
21.64.19*
19.72.01**
14.21.51***
8.912.03
45.266.2
42.112.77
37.283.28
36.56.5
26.363.87*
26.81.66**
Data represent mean S.D. (triplicate). Values signicantly dierent from AAPH-treated are labeled with * (P < 0.05), ** (P < 0.01) and ***
(P < 0.001).
b
Concentration of the mixture can be found in Table 1.
c
AAPH 2,20 -azobis (2-amidinopropane) dihydrochloride (positive control).
15
Table 2
ELISA analysis of carbonyl from human lung cell line CCD-8Lu cultured with b-carotene, a-tocopherol, ascorbic acid and a mixture of these three
compounds: 0, 143 and 722 torr oxygen tension, respectively
a-Tocopherol
(mm)
Carbonyl
(nmol/mg protein)
Ascorbic acid
(mm)
Carbonyl
(nmol/mg protein)
Mixture no.b
(mm)
Carbonyl
(nmol/mg protein)b
0
AAPHc
5
10
20
40
80
0.0950.014
0.2300.024
0.1680.018*
0.1390.008**
0.1380.044*
0.0950.021**
0.0810.024**
0
AAPH
10
20
40
80
160
0.0880.041
0.1970.005
0.1820.051
0.1580.033
0.1240.018**
0.0810.029**
0.0760.038**
0
AAPH
1
2
3
4
5
0.1010.051
0.2510.024
0.1920.042
0.1680.011**
0.1090.038**
0.1060.022**
0.0630.024***
0
AAPH
5
10
20
40
80
0.1130.025
0.2570.045
0.2010.009
0.1850.013*
0.1770.025*
0.1250.033*
0.1110.053*
0
AAPH
10
20
40
80
160
0.0980.022
0.2410.033
0.1940.016
0.1920.041
0.1660.028*
0.1230.037*
0.0920.018**
0
AAPH
1
2
3
4
5
0.1220.038
0.2190.028
0.1660.044
0.1610.009*
0.0980.022**
0.1060.034*
0.0740.027**
0
AAPH
5
10
20
40
80
0.1420.026
0.2320.029
0.2190.048
0.2250.039
0.1850.027
0.1770.056
0.1670.018*
0
AAPH
10
20
40
80
160
0.1260.022
0.2280.016
0.180.021*
0.1870.009*
0.1620.054
0.1320.044*
0.1310.033*
0
AAPH
1
2
3
4
5
0.130.033
0.2480.044
0.1970.036
0.1520.028*
0.1540.061
0.1320.015*
0.1770.037*
b-Carotene
(mm)
Carbonyl
(nmol/mg protein)a
Data represent mean S.D. (triplicate). Values signicantly dierent from AAPH-treated are labeled with * (P < 0.05), ** (P < 0.01) and ***
(P < 0.001).
b
Concentration of the mixture can be found in Table 1.
c
AAPH, 2,20 -azobis (2-amidinopropane) dihydrochloride (positive control).
16
with AAPH only) and the b-carotene groups of CCD8Lu supplemented with 0.4, 0.8 and 1.6 mm b-carotene
(Table 1). Supplementation of b-carotene reduced isoprostane formation by 28, 31 and 46%, respectively. aTocopherol protected cells from lipid peroxidation signicantly at 40 and 80 mm by reducing isoprostane formation by 34 and 54%, respectively (Table 1). Also,
ascorbic acid provided a dose-dependent protection
against lipid peroxidation, at 80 and 160 mm with 34 and
61% protection, respectively (Table 1). The mixtures of
these compounds produced better protection than any
single compound. Mixture #1 containing 0.1 mm b-carotene, 5 mm a-tocopherol and 10 mm ascorbic acid
decreased isoprostane formation by 44%. All mixtures
provided 44, 46, 57, 66 and 74% protection with increasing concentrations of each participating compound
(Table 1).
3.1.2. 143 torr oxygen tension
At 143 torr oxygen tension, all compounds demonstrated protection patterns that were similar to those
found at 0 torr oxygen tension (Table 2). The addition
of b-carotene resulted in 34, 35 and 41% protection at
0.38, 0.77 and 1.56 mm, respectively. The addition of atocopherol resulted in 31, 37 and 54% protection at
18.9, 38.2 and 78.2 mm, respectively. The addition of
ascorbic acid decreased isoprostane production by 44
and 57% at 78.3 and 156.3 mm, respectively. Mixtures of
these compounds provided CCD-8Lu cells protection
against AAPH-induced lipid peroxidation by decreasing
isoprostane formation by 36, 46, 50, 61 and 76% for
mixture groups 1, 2, 3, 4 and 5, respectively.
3.1.3. 722 torr oxygen tension
No protection against AAPH-induced lipid peroxidation was found between b-carotene concentrations of
0.10.8 mm. However, b-carotene produced 12% more
isoprostane at concentration of 1.5 mm (Table 1). aTocopherol and ascorbic acid still exhibited an antioxidant eect, but the antioxidant eect of a-tocopherol
was not dose dependent. a-Tocopherol reduced isoprostane formation by 42% at 40 mm, while only 41%
reduction at 80 mm (Table 1). Ascorbic acid reduced
isoprostane formation by 44% at 80 mm, while only
34% at 160 mm (Table 1). The mixtures of these compounds demonstrated a dose-dependent antioxidant
eect. Unlike our ndings with 0 and 143 torr oxygen
tensions, the mixtures provided signicant protection
only at the highest three concentrations of combinations
(Table 1).
3.2. Protein oxidation
3.2.1. Hypoxia
AAPH (30 mm) treated CCD-8Lu cell resulted in the
production of 2.5 times more protein oxidation product,
17
carbonyls, than background (untreated samples). Addition of b-carotene at 0.20 mm to AAPH-treated CCD8Lu cells leads to a signicant protective of protein oxidation, decreasing carbonyl formation by 23% (Table 2).
The protection eect of b-carotene was even more signicant with the increasing concentrations of b-carotene. Carbonyl production decreased by 28 and 48%
at 0.80 mm and 1.60 mm, respectively. Adding a-tocopherol resulted in more protection of protein oxidation.
A signicant protection was observed at the lowest
concentration of 4.6 mm b-carotene, producing 27% less
carbonyl than AAPH treated samples. Adding b-carotene provided 40, 40, 59 and 65% protection at 10,
120, 40 and 80 mm, respectively (Table 2).
The protective eect of ascorbic acid was dose
dependent. Adding ascorbic acid resulted in 37, 59 and
61% decrease of carbonyl formation at 40, 80 and 160
mm, respectively (Table 2). In the presence of a mixtures
of the three compounds, carbonyl was markedly
decreased in a dose-dependent manner, 33, 57, 58 and
75% reduction at mixture groups 2, 3, 4 and 5, respectively (Table 2).
3.2.2. 143 torr oxygen tension
In the presence of 143 torr oxygen tension, the protective eect of b-carotene in CCD-8Lu cells was
decreased. The only signicant suppression of carbonyl
formation (43%) was observed at the highest concentration of b-carotene (1.6 mm). The protective eect
of lower concentrations was insignicant, although the
reduction of carbonyl was in a concentration-dependent
manner (Table 2).
The antioxidant function of a-tocopherol was not
aected by the oxygen tension. A dose-dependent protection against AAPH-induced protein oxidation was
found (Table 2). Ascorbic acid produced 31, 49 and
62% reduction of carbonyl formation at 40, 80 and 160
mm, respectively (Table 2). A mixture of these three
compounds provided a weaker protection than that of 0
torr oxygen tension condition, that is, 26, 55, 52 and
66% reduction of carbonyl at mixture groups 2, 3, 4 and
5, respectively (Table 2).
3.2.3. 722 torr oxygen tension
Table 2 illustrates the induction of protein oxidation
in CCD-8Lu cells by the addition of 30 mm AAPH, as
measured by increased carbonyl formation, in the presence of b-carotene, a- tocopherol, ascorbic acid, and a
mixture of these three compounds. As with adding bcarotene, no signicant antioxidant or prooxidant eect
was observed at all the concentrations tested (Table 2).
High oxygen tension diminished the antioxidant eect
of b-carotene but did not induce prooxidant eects.
Only a 28% reduction of carbonyl was observed at the
highest concentration of a-tocopherol, 80 mm (Table 2).
Ascorbic acid exhibited an antioxidant eect at all
18
Plate 1. Immunoblot analysis of p53 protein from human lung cell line CCD-8Lu cultured with b-carotene, a-tocopherol, ascorbic acid, and a
mixture of these three compounds at 0 torr of oxygen. 2,20 -azobis (2-amidinopropane) dihydrochloride (AAPH) was used to stimulate p53 accumulation. The protective eects of each compound were quantied by calculating the relative volume of each band to the AAPH-treated control and
shown on the top of each band. (a) Lane 1: 30 mm AAPH; lane 2: 30 mm AAPH and 0.1 mm b-carotene; lane 3: 30 mm AAPH and 0.2 mm b-carotene;
lane 4: 30 mm AAPH and 0.4 mm b-carotene; lane 5: 30 mm AAPH and 0.8 mm b-carotene; lane 6: 30 mm AAPH and 1.6 mm b-carotene; lane 7: 30
mm AAPH; lane 8: 30 mm AAPH and 5 mm a-tocopherol; lane 9: 30 mm AAPH and 10 mm a-tocopherol; lane 10: 30 mm AAPH and 20 mm atocopherol; lane 11: 30 mm AAPH and 40 mm a-tocopherol; lane 12: 30 mm AAPH and 80 mm a-tocopherol; (b) lane 1: 30 mm AAPH only (30 mm);
lane 2: 30 mm AAPH and 10 mm ascorbic acid; lane 3: 30 mm AAPH and 20 mm ascorbic acid; lane 4: 30 mm AAPH and 40 mm ascorbic acid; lane 5:
30 mm AAPH and 80 mm ascorbic acid; lane 6: 30 mm AAPH and 160 mm ascorbic acid; lane 7: 30 mm AAPH; lane 8: 30 mm AAPH and mixture 1;
lane 9: 30 mm AAPH and mixture 2; lane 10: 30 mm AAPH and mixture 3; lane 11: 30 mm AAPH and mixture 4; lane 12: 30 mm AAPH and mixture 5.
19
Plate 2. Immunoblot analysis of p53 protein from human lung cell line CCD-8Lu cultured with b-carotene, a-tocopherol, ascorbic acid, and mixture
of these three compounds at 143 torr of oxygen. 2,20 -azobis (2-amidinopropane) dihydrochloride (AAPH) was used to stimulate p53 accumulation.
The protection eects of each compound were quantied by calculating the relative volume of each band to the AAPH-treated control and shown on
the top of each band. (a) Lane 1: 30 mM AAPH; lane 2: 30 mm AAPH and 0.1 mm b-carotene; lane 3: 30 mm AAPH and 0.2 mm b-carotene; lane 4:
30 mm AAPH and 0.4 mm b-carotene; lane 5: 30 mm AAPH and 0.8 mm b-carotene; lane 6: 30 mm AAPH and 1.6 mm b-carotene; lane 7: 30 mm
AAPH; lane 8: 30 mm AAPH and 5 mm a-tocopherol; lane 9: 30 mm AAPH and 10 mm a-tocopherol; lane 10: 30 mm AAPH and 20 mm a-tocopherol;
lane 11: 30 mm AAPH and 40 mm a-tocopherol; lane 12: 30 mm AAPH and 80 mm a-tocopherol; (b) lane 1: 30 mm AAPH only (30 mm); lane 2: 30
mm AAPH and 10 mm ascorbic acid; lane 3: 30 mm AAPH and 20 mm ascorbic acid; lane 4: 30 mm AAPH and 40 mm ascorbic acid; lane 5: 30 mm
AAPH and 80 mm ascorbic acid; lane 6: 30 mm AAPH and 160 mm ascorbic acid; lane 7: 30 mm AAPH; lane 8: 30 mm AAPH and mixture 1; lane 9:
30 mm AAPH and mixture 2;lane 10: 30 mm AAPH and mixture 3; lane 11: 30 mm AAPH and mixture 4; lane 12: 30 mm AAPH and mixture 5.
20
Plate 3. Immunoblot analysis of p53 protein from human lung cell line CCD-8Lu cultured with b-carotene, a-tocopherol, ascorbic acid, and mixture
of these three compounds at 722 torr of oxygen. 2,20 -azobis (2-amidinopropane) dihydrochloride (AAPH) was used to stimulate p53 accumulation.
The protective eects of each compound were quantied by calculating the relative volume of each band to the AAPH-treated control and shown on
the top of each band. (a) Lane 1: 30 mm AAPH; lane 2: 30 mm AAPH and 0.1 mm b-carotene; lane 3: 30 mm AAPH and 0.2 mm b-carotene; lane 4: 30
mm AAPH and 0.4 mm b-carotene; lane 5: 30 mm AAPH and 0.8 mm b-carotene; lane 6: 30 mm AAPH and 1.6 mm b-carotene; lane 7: 30 mm AAPH;
lane 8: 30 mm AAPH and 5 mm a-tocopherol; lane 9: 30 mm AAPH and 10 mm a-tocopherol; lane 10: 30 mm AAPH and 20 mm a-tocopherol; lane 11:
30 mm AAPH and 40 mm a -tocopherol; lane 12: 30 mm AAPH and 80 mm a-tocopherol; (b) lane 1: 30 mm AAPH only (30 mm); lane 2: 30 mm
AAPH and 10 mm ascorbic acid; lane 3: 30 mm AAPH and 20 mm ascorbic acid; lane 4: 30 mm AAPH and 40 mm ascorbic acid; lane 5: 30 mm AAPH
and 80 mm ascorbic acid; lane 6: 30 mm AAPH and 160 mm ascorbic acid; lane 7: 30 mm AAPH; lane 8: 30 mm AAPH and mixture 1; lane 9: 30 mm
AAPH and mixture 2; lane 10: 30 mm AAPH and mixture 3; lane 11: 30 mm AAPH and mixture 4; lane 12: 30 mm AAPH and mixture 5.
21
22
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