Toxicology in Vitro 15 (2001) 1324

www.elsevier.com/locate/toxinvit

Antioxidant and prooxidant roles for b-carotene,

a-tocopherol and ascorbic acid in human lung cells
P. Zhang, S.T. Omaye *
Environmental Sciences and Health Graduate Program and the Department of Nutrition, Mail Stop 142,
University of Nevada, Reno, NV 89557, USA
Accepted 1 September 2000

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

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14

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

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 torr oxygen tension

0
2.310.52
31.262.4
AAPHc
0.1
30.593.28
0.2
27.121.06
0.4
22.503.59*f
0.8
21.585.02*
1.6
16.782.85**

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***

143 torr oxygen tension

0
3.781.2
AAPH
34.253.27
0.1
31.892.1
0.2
26.593.65
0.4
22.592.9**
0.8
22.395.3*
1.6
20.221.58**

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***

722 torr oxygen tension

0
8.633.4
AAPH
44.121.9
0.1
42.015.23
0.2
41.272.09
0.4
45.384.12
0.8
44.567.2
1.6
49.252.26*

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).

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

2.3. Treatment regimen

Exponentially growing cells were exposed to varying
concentrations of b-carotene, a-tocopherol, ascorbic
acid, and mixtures of the three compounds for 1 h and
providing sucient time for lipid peroxidation and p53
protein to be expressed. Fresh solutions of each compound were prepared on the day of each experiment.
Solutions of b-carotene and a-tocopherol were prepared
in tetrahydrouran (THF), 99.9% with 0.025% (w/v)
butylated hydroxytoluene (BHT) (Aldrich Chemical,
Milwaukee, WI, USA). THF was added by a hypodermic needle purged with nitrogen to prevent oxidation
of the solvent. Stock THF was stored in a bottle sealed
with a Sure/Seal lid (Aldrich Chemical) designed to
prevent peroxide formation. Ascorbic acid was dissolved in deionized and distilled water with 0.025% (w/v)
BHT. At the end of 1 h, 30 mm AAPH was introduced
to cells to initiate free radical reaction for 4 h.
2.4. Analysis of -carotene, -tocopherol and ascorbic acid
The concentrations of b-carotene in the cell medium
were assayed by HPLC analysis (Ringer et al., 1991),

15

with detection at 450 nm (Perkin-Elmer LC-95 UV-VIS

detector; Perkin-Elmer Corp., Norwalk, CT, USA). The
mobile phase consisted of acetonitrile/methanol/methylene chloride 50:30:20 (by vol.) and ran isocratically
with a ow rate of 1.0 ml/min (Perkin-Elmer LC-250
pumping system). Calibration was by extracted external
standards of synthetic all-trans b-carotene (Sigma Chemical Company).
a-Tocopherol was extracted from cell medium by the
following method of Kahlon et al. (1986). The mobile
phase remained at 100% methanol for 15 min, followed
by a gradient from 100% methanol to 93% methanol/
water (v/v) in 2 min at 1.0 ml/min (Perkin-Elmer LC250). a-Tocopherol was detected at 292 nm using a
Perkin-Elmer LC-95 UV/VIS detector. Extracted external standards were used for calibration.
Ascorbic acid concentrations were determined using
the method of Wei et al. (1996).
2.5. Oxygen tension
Flasks containing growing cells in complete medium
were tted with sterile rubber septa and exposed to a
continuously owing humidied atmosphere comprised

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 torr oxygen tension

0
0.0840.023
AAPH
0.2100.014
0.1
0.1840.052
0.2
0.1620.021*
0.4
0.1580.033
0.8
0.1520.011**
1.6
0.1270.028*

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***

143 torr oxygen tension

0
0.1020.037
AAPH
0.2630.062
0.1
0.2130.024
0.2
0.1950.016
0.4
0.1870.038
0.8
0.1520.044
1.6
0.1490.028*

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**

722 torr oxygen tension

0
0.1330.056
AAPH
0.2540.044
0.1
0.2460.032
0.2
0.2270.021
0.4
0.260.078
0.8
0.2580.041
1.6
0.2310.055

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

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

of varying amounts of N2, air and oxygen. Three oxygen

tensions were created: 0 torr (95% N2 and 5% CO2); 143
torr (95% air and 5% CO2); and 722 torr (95% O2 and
5% CO2) (Zhang and Omaye, 2000). At 0 torr the calculated initial and nal oxygen (0 and 1 h) concentrations
were 44 and 30 mm, respectively. At the end of 1 h, AAPH
was added to the asks by injection through the rubber
septum without disturbing the oxygen tension.
2.6. Lipid peroxidation
Lipid peroxidation of the cells was determined by measuring isoprostanes (8-epi-PGF2a, 15-F21-isoprostane or
8-iso-F2a) (Liu et al., 1998), prostaglandin-like compounds that are products of free radical-induced peroxidation of arachidonic acid (Isoprostane ELISA Kit,
Oxford Biomedical Research, Inc., Oxford, MI, USA).
2.7. Protein oxidation
The cultured cells were lysed in 150 mm NaCl, 50 mm
TrisHCl, pH 8.0, 5 mm EDTA, 1% NP40, 1 mm PMSF
for 30 min on ice and the extracts were centrifuged at
30,000 g for 30 min. Cell extracts were mixed with 500
ml 2,4-dinitrophenylhydrozine (DNP) (Sigma). Sample
tubes were incubated in the dark at room temperature
with vortexing every 15 min for 1 h. The DNP-derivated
protein was dialysed against 2 l of phosphate buered
saline (PBS) overnight to eliminate vitamins, unbound
DNP and AAPH. The nal protein content of each
sample was determined by BCA Protein assay (Pierce,
Rockford, IL, USA) adapted for microtitre plates using
bovine serum albumin (BSA) as the standard.
Carbonyl standards were performed according to
Levine (1993), with minor modication. After dissolving
in 6 m guanidine HCl, the protein was dialysed against 2
l of PBS overnight to remove the guanidine HCl. The
nal carbonyl content was determined by spectrophotometer, calculated from its peak absorbency at 370
nm using an absorbency coecient  of 22,000 m 1
cm 1. Working standards were prepared by serial dilution of the stock solution to nal carbonyl concentrations between 0.01 and 0.5 nmol/ml.
Samples and standards were diluted with PBS to 5 mg
protein/ml. Each sample (200 ml containing 1 mg protein) was added to wells (triplicate) of a microtitre plate.
The plate was incubated overnight at 4 C, followed by
three washings with PBS. To each well the following
was added: 250 ml of 3% BSA in PBS with 0.02%
sodium azide and incubated for 2 h at room temperature. Following three washings with PBS, 200 ml of antiDNP antibody (1:15,000 in PBS) was added to the
samples and incubated for 1 h at 37 C. To remove the
unbound antibody, samples were washed three times
with PBS. 200 ml of anti-IgG alkaline phosphatase diluted 1:12,000 in PBS were added to the samples and

incubated at room temperature for 1 h followed by

three washings with PBS. This was followed by adding
200 ml of 1 mg/ml p-nitrophenylphosphate, disodium
(pNPP) in ethanolamine buer (Sigma) to each well and
incubating the plate at 37 C for 1 h. The endpoint color
change for each sample on the microtitre plate was read
at 405 nm and carbonyl content was calculated from the
standard curve using the KCJr, Bio-Tek's PC-based
curve tting and statistical software.
2.8. DNA damage
We measured the level of p53 expression as an indication of DNA damage. p53 is a nuclear phosphoprotein with a short half-life. Its normal concentration in
the nucleus is low (Levine, 1993); however, p53 protein
accumulates in the nuclei of cells exposed to UV irradiation, ionizing radiation, and DNA damage-inducing
agents. The cultured cells (5107) were lysed in 90 C
Laemmli sample buer to make whole-cell lysates. The
whole-cell lysates were separated by SDSpolyacrylamide gel electrophoresis (SDSPAGE) on a 10%
gel and transferred onto nitrocellulose membrane in a
Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell
for 4 h, at 4 C and 150 mA in transfer buer (25 mm
Tris, 190 mm glycine and 20% methanol). Pre-stained
molecular mass markers (Bio-Rad, Hercules, CA, USA)
were run in parallel. The blots were blocked in 0.1%
Tween-20 in PBS for 1 hr, probed overnight at 4 C
with mouse monoclonal anti-p53 IgG2a (DO-1) (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and
washed in 0.1% Tween-20 in PBS. The blots were then
incubated for 1 hr at room temperature in horseradish
peroxidase (HRP)conjugated goat anti-mouse IgG
(Santa Cruz Biotechnology, Inc.), diluted 1:1000, and
washed again in 0.1% Tween-20 in PBS. The density of
p53 protein bands was analyzed by Gel-Doc densitometer (Bio-Rad).
3. Results
THF concentrations of 0.5% used to deliver b-carotene and a-tocopherol had no toxic (obvious morphological) eects on cells. We measured the concentrations
of various compounds in cell media analogous to making measurements in plasma for in vivo studies. The
quantity of all three compounds within cells during the
time frame of our studies was found to be negligible
relative to media concentrations (data not shown).
3.1. Lipid peroxidation
3.1.1. Hypoxia
There were signicant dierences in the formation of
isoprostane between the positive control group (treated

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

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

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

concentrations tested, although the extent of protection

was less than that found for the lower oxygen tension
(Table 2). The mixtures of these three compounds protected protein oxidation by 39% at mixture group 2,
47% at mixture group 4, and 53% at mixture group 5,
less eective than at lower oxygen tension (Table 2).
3.3. DNA damage
A wide variety of compounds are able to produce
damage in DNA, inducing the accumulation of p53
protein. The accumulation of p53 in the human lung cell
in response to AAPH treatment in the presence of bcarotene, a-tocopherol, ascorbic acid and a mixture of
these three compounds at dierent oxygen tensions was
examined by immunoblotting.
3.3.1. Hypoxia
Plate 1(a) illustrates a concentration-dependent antioxidant eect of b-carotene (lanes 16) and a-tocopherol (lanes 712). The level of p53 decreased in
response to increasing concentrations of both b-carotene and a-tocopherol. b-Carotene attenuated the
DNA damage by 91% at the highest concentration (1.6
mm), while a-tocopherol decreased DNA damage by
94% at the highest concentration (80 mm). Plate 1(b)
illustrates the antioxidant eect of vitamin C and

mixtures of three compounds. Ascorbic acid eectively

attenuated DNA damage by blocking 95% of the
p53 accumulation at the concentration of 160 mm.
Mixtures of these three compounds exhibited better
protection against AAPH-induced DNA damage. At
the highest concentration group, almost 100% protection was achieved (lane 12). All these responses were
dose dependent. Oxygen tension had no impact on the
antioxidant eects of these compounds or mixtures of
antioxidants.
3.3.2. 143 torr oxygen tension
b-Carotene decreased p53 accumulation by 53, 80, 83,
91 and 97% at concentrations of 0.1, 0.2, 0.4, 0.8 and
1.6 mm, respectively (Plate 2a, lanes 16). Incubation of
CCD-8lu cells with a-tocopherol resulted in a dosedependent inhibition of AAPH-induced DNA damage
by 63, 85, 96, 96 and 98% at the doses of 5, 10, 20, 40
and 80 mm, respectively (Plate 2a, lanes 712). Adding
ascorbic acid resulted in a dose-dependent antioxidant
eect by decreasing 48, 63, 73, 86 and 89% of p53
accumulation at 10, 20, 40, 80 and 160 mm vitamin C,
respectively (Plate 2b, lanes 16). The mixtures of three
compounds provided a more eective protection against
AAPH-induced DNA damage. At the highest dose
group, the mixture provided 99.3% protection (Plate
2b, lanes 712). Oxygen tension below 143 torr had no

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.

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

signicant eect on antioxidant functions of the three

compounds.
3.3.3. 722 torr oxygen tension
Plate 3(a) illustrates that b-carotene lost its antioxidant eect in response to higher b-carotene concentration and higher oxygen tension. b-Carotene
exhibited antioxidant eects at or below 0.20 mm (Plate
3a, lanes 1 and 2, not lane 3). b-Carotene addition
resulted in decreased p53 accumulation by 67% at 0.20
mm. However, the antioxidant eect was attenuated 0.4
mm with 66, 53 and 25% reduction of p53 accumulation
at 0.4, 0.8 and 1.6 mm, respectively (Plate 3a, lanes 46).
The p53 accumulation at 1.49 mm was less than that of
the AAPH control (75% of control). Thus, b-carotene
demonstrated an antioxidant eect, not a prooxidant
eect. The most eective concentration was 0.2 mm with
67% protection. At 0 torr oxygen tension, b-carotene
achieved the most protection at 1.5 mm with 91% protection. At 143 torr, b-carotene produced maximum
protection 97% at 1.5 mm. At 722 torr, maximum protection (67%) was found at 0.2 mm. Thus, oxygen tension and concentration of b-carotene played important
roles in determining the antioxidant eect of b-carotene.
a-Tocopherol exhibited an antioxidant eect with 96%
protection at 80 mm. The protection aorded by a-tocopherol was dose dependent. In comparison with the
results of lower oxygen tension, no impact of higher oxy-

19

gen tension was found to reduce the antioxidant eect of

a-tocopherol (Plate 3a, lanes 712). Like b-carotene,
ascorbic acid demonstrated a protective eect at lower
concentrations (Plate 3b, lanes 16). Adding ascorbic
acid decreased p53 accumulation by 34, 62, 76 and 85%
at doses of 10, 20, 40 and 80 mm, respectively (Plate 3b,
lanes 15). However, at the highest concentration, 160
mm, ascorbic acid only provided 72% protection or 13%
less than that of the previous concentration (80 mm).
This value is also 23 and 17% less than that of the same
concentration levels at 0 and 143 torr of oxygen tension,
respectively. The protective eect of ascorbic acid was
compromised by the higher oxygen tension at the higher
concentration of ascorbic acid. The mixtures of these
three compounds exhibited a concentration-dependent
protective eect. The higher oxygen tension did not show
signicant eect on the protective eect of the mixture of
these three compounds (Plate 3b, lanes 712).
4. Discussion
Carotenoids are a class of pigments widely distributed
in nature and responsible for the bright colors of various
fruits and vegetables. People who ingest more dietary
carotenoids have a reduced risk for several chronic diseases, including cardiovascular and photosensitivity
diseases, cataracts, age-related macular degeneration

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

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

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.

and some cancers (Mayne, 1996). Recent ndings from

intervention trials, however, indicate that supplemental
b-carotene is of little or no value in preventing cardiovascular diseases and the major cancers that occur in
well-nourished populations (Greenberg et al., 1996;
Hennekens et al., 1996), and may actually increase rather
than reduce lung cancer incidence in smokers (ATBC
Study Group, 1994; Omenn et al., 1996a,b). As a consequence of these ndings, there has been interest in elucidating the mechanism(s) by which carotenoids might
function in human tissues. Thus, lung cells were used as
our model because of their relevance to tissues for which
epidemiological studies have shown an inverse association between cancer and high intakes of b-carotene.
However, there are limitations with extrapolation using
such cells because CCD-8Lu are brobast-like and cancer is regarded as epithelial in origin (Chen et al., 1998).
Both antioxidant and/or prooxidant eects have been
suggested as possible mechanisms by which b-carotene
may exert physiological action. The chemical structure
of conjugated double bonds associated with b-carotene,
which can trap a free radical (Terao, 1989), is the proposed mode of antioxidant action for b-carotene. However, carotenoids do not have the structural features
commonly associated with chain-breaking antioxidants.
The extensive system of conjugated double bonds in

their molecules imparts a prooxidant character and

makes them very susceptible to attack by free radical
species. Others (Stocker et al., 1987; Burton, 1989;
Palozza and Krinsky, 1991) have indicated that whether
b-carotene exhibits an antioxidant or prooxidant eect
is dependent on oxygen tension and b-carotene concentrations, rather than structure. In agreement, we
have found that isoprostane production was signicantly reduced at or above 143 torr of oxygen tension and this reduction is concentration dependent.
However, the antioxidant eects of b-carotene were
decreased and replaced with prooxidant eects at 722
torr of oxygen tension. Although the prooxidant properties of b-carotene at high oxygen tension have been
extensively demonstrated in in vitro studies (Burton and
Ingold, 1984; Vile and Winterbourn, 1988; Kennedy
and Liebler, 1992; Palozza et al., 1995, 1997, 1998), it is
still unclear how the oxygen tension might change the
action of b-carotene from anti- to prooxidant. Burton
and Ingold (1984) showed that b-carotene could inhibit
peroxyl radical to form either a carotenoid radical species or a resonance-stabilized carbon-centered radical.
The antioxidant-prooxidant activity of this carotenoid
radical would be dependent on the oxygen tension in the
system. In the presence of oxygen, both carotenoid
radical and resonance-stabilized carbon-centered radical

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

could react with oxygen to form equilibrium reactions.

If the oxygen tension is high, the equilibrium of the
reaction would shift to form either carotenoid radical
species or a resonance-stabilized carbon-centred radical,
and b-carotene forms a peroxyl radical capable of acting
as a prooxidant and undergoes autoxidation. Although
none of the potential intermediate forms proposed by
Burton and Ingold have been isolated, a variety of products arising from the reactions of radicals with b-carotene have been described (Handelman et al., 1991;
Kennedy and Liebler, 1991; Liebler and McClure,
1996). Based on an experiment using CCl3OO as a
model of peroxyl radical, Hill et al. (1995) suggested
that products of b-carotene with CCl3OO depend on
oxygen concentration. At low oxygen concentration the
only reaction observed was electron transfer producing
a radical cation of b-carotene (bC +). In air and in
oxygen-saturated solutions, two species were produced,
the radical cation and a second species (CCl3OO-b-C) ,
which decayed to produce the radical cation itself.
Truscott (1996) suggested that oxygenated products of
b-carotene may have prooxidant activity and may lead
to further oxidation of b-carotene.
In contrast, Kennedy and Liebler (1992) suggested
that the loss in antioxidant eciency of b-carotene at
high oxygen tension may be due to b-carotene autoxidation rather than to the formation of a b-caroteneoxygenated radical and our ndings would not exclude
this posibility. The increased autoxidation would consume b-carotene without scavenging peroxyl radicals
and would attenuate b-carotene antioxidant eciency.
Prooxidant eects have also been reported in vitro
and in vivo at high concentrations of b-carotene (Alam
and Alam, 1983; Lomnitsky et al., 1991; Anderson and
Anderson, 1993; Palozza, 1998; Zhang and Omaye, 2000).
Also, we cannot exclude a provitamin A eect of bcarotene (Dartigues et al., 1990; Quick and Ong, 1990;
Scita et al., 1992; Wamer et al., 1993) resulting in the
antioxidant eects observed in 0 and 143 torr of oxygen
tension because we did not measure retinol content of
the cells and media. The extraction procedure does not
permit recovery of retinol from cell extracts and media.
Possible additive and synergistic eects of the antioxidant and provitamin A activities of of b-carotene
have not been fully explored in in vitro assays. Additional studies are necessary for evaluating the importance of retinol production associated with in vivo
exposure to b-carotene.
In the present study, the combination of the three
compounds exhibited more antioxidant protection than
any single compound, but such protection was not
additive. Non-additive eects of the three compounds
may be explained by: (1) length of incubation with the
compound, that is, incubation time may impact on maximum uptake and breakdown of the compounds, and (2)
optimum level and balance of the three compounds that

21

would provide maximum ecacy, that is, additive

response may be a function of concentrations that do
not promote prooxidant eects. The concentrations of
each compound and compound mixtures were selected
because they were representive of the normal range of
these compounds in human plasma. Average serum atocopherol, b-carotene and ascorbic acid concentrations
have been found to be 20, 80 and from 0.1 to 1 mm,
respectively (Combs, 1998).
Thus, the resulting eects could be the co-operative
interaction among these compounds. Several studies
demonstrate that interactions can occur between carotenoids and tocopherols, carotenoids and ascorbic
acid. Tocopherols may protect carotenoids from autoxidation (Handelman et al., 1991; Kennedy and Liebler,
1992). a-Tocopherol protects b-carotene from oxidation
in toluene under 100% oxygen at 60 C (Handelman et
al., 1991). The protection was concentration dependent
and only evident at high concentrations of a-tocopherol.
Conversely, d-tocopherol enhances the protective eects
of b-carotene from 1O2-initiated photooxidation of
methyl linoleate (Terao et al., 1980). Similarly, g-tocopherol can prevent against the prooxidant eects of bcarotene in puried triacylglycerol fraction of rapeseed
oil exposed to light and the prooxidant eect of lutein
and lycopene in autoxidized triglycerides (Haila and
Heinonen, 1994; Haila et al., 1996). Both of these studies show that even minor concentrations of g-tocopherol
may inhibit the prooxidant eect of the carotenoids. In
addition, a combination of b-carotene and g-tocopherol,
or a combination of lutein and g-tocopherol were more
ecient than g-tocopherol alone in inhibiting hydroperoxide formation (Haila and Heinonen, 1994; Haila et
al., 1996); thus, tocopherols may protect b-carotene
during free radical-induced lipoperoxidation. Also, carotenoidtocopherol interaction has been demonstrated
in a membrane model (Palozza and Krinsky, 1992) and
a combination of b-carotene and a-tocopherol inhibits
free radical-induced lipid peroxidation signicantly and
greater than the sum of the individual inhibitions. Because
of a-tocopherol degradation, these ndings suggest that
a-tocopherol might be consumed to retard the formation of carotenoid radical adducts and their further
degradation to autoxidation products. These ndings
also suggest that tocopherols may limit the prooxidant
eects of carotenoids in biological systems and have
potential benets because of an association between
tocopherols and carotenoids in biologic membranes.
The possibility of interactions in vitro between b-carotene and ascorbic acid (Packer, 1993) was demonstrated when ascorbic acid added to a preparation of
low-density lipoproteins (LDL) acted synergistically
with b-carotene in protecting LDL from oxidation and
b-carotene from its consumption. Similar results were
obtained by Jialal and Grundy (1991), who found that
added ascorbate protected the endogenous tocopherols

22

P. Zhang, S.T. Omaye / Toxicology in Vitro 15 (2001) 1324

and b-carotene in human LDL oxidized with Cu2+.

Moreover, dietary antioxidant (ascorbic acid, a-tocopherol and b-carotene) resulted in the protection of
LDL (Reaven et al., 1994) and imply that b-carotene
and ascorbate may be sparing each other in vivo.
Ascorbic acid and a-tocopherol are well known antioxidants (Burton and Ingold, 1986; Frei et al., 1989,
1990). When the two antioxidants are present in an
aqueous environment, ascorbic acid eciently inhibits
in vitro lipid peroxidation (Frei et al., 1988, 1989) due to
a combination of direct radical interception by ascorbic
acid (where aqueous radicals are involved) and interaction with a-tocopherol as a co-antioxidant. The regeneration of a-tocopherol from a-tocopheroxyl radical by
ascorbate, with concomitant generation of the ascorbyl
radical is well established (Packer et al., 1979; Barclay et
al., 1985; Doba et al., 1985; Kagan et al., 1992; Sharma
and Buettner, 1993; Witting et al., 1996). Addition of
ascorbate to LDL undergoing oxidation induced by
aqueous ROO. results in immediate cessation of atocopherol consumption and lipid oxidation. Upon
removal of ascorbate, consumption of a-tocopherol and
oxidation of LDL resumes, attaining the same rates as
those prior to the addition of ascorbate (Bowry and
Stocker, 1993).
We have found that there may be dierences in the
extent of protection when these three compounds are
used against AAPH-induced cell damage. All compounds and AAPH were added to the cell medium and
b-carotene and a-tocopherol were dissolved in THF, a
moderate organic solvent, allowing b-carotene and atocopherol to be delivered into the aqueous solution.
Cellular uptake of these compounds was negligible;
therefore, the dierent protective eects observed for
each compound may be attributable to the concentrations of each compound in the media. However, in vivo,
cellular location of each compound may be an important factor in protecting the cell from oxidative damage.
In vivo, b-carotene is more lipophilic and located in the
interior of membranes or lipoproteins, and a-tocopherol
is also lipophilic but conned to the interface between
membranes and water. Ascorbic acid is located in the
extracellular and hydrophilic regions of the cell (Niki et
al., 1995). Ascorbic acid, in the extracellular matrix, is
the antioxidant that rst defends cells. Free radicals
must pass across the cell membrane to come into contact with the intracellular components. We found that at
high oxygen tension and at high b-carotene concentrations there were no prooxidant eects, for example,
protein and DNA damage. However, the antioxidant
eect of b-carotene at the lower oxygen tension was
signicantly reduced (Plate 9a, lanes 16). This result
could be explained by the structure of the cell and the
location of the nucleus. Located in the heart of the cell,
the nucleus is protected by many antioxidant defenses.
When the cell is under free radical stress, free radicals

have to pass the rst defense of the cell, the membrane

where lipid soluble antioxidants reside. Next, they have
to conquer the aqueous phase of the cell in which water
soluble antioxidants and antioxidant enzymes reside.
After passing these defenses, free radicals nally reach
the DNA. If concentrations of free radicals are not sufcient enough and/or antioxidant concentration is high,
the nucleus may survive free radical damage.
Acknowledgements
This project was funded by the Nevada Experimental
Station, University of Nevada, Reno. We would like to
thank Sher-Janel T. Todd for her critical review of this
manuscript.

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