ANTIOXIDANT EFFECTS OF LYCOPENE AND
UBIQUINOL-10 ON THE OXIDATIVE STRESS IN RAT HEPATOCYTES INDUCED BY
Laboratory Medicine Department, Paramedicine Faculty
Hamedan University of Medical Sciences & Health Services,
Fax: +98 811 8281442
Free radicals especially reactive oxygen metabolites can
damage DNA, protein, enzymes, and membrane lipids. Lipid
peroxidation in hepatocyte membrane may be involved in hepatic
diseases. Antioxidants may inhibit this reaction. Due to
oxidant-antioxidant imbalance, free radicals may cause destructive
effects. For several years, scientists tried to find antioxidant
compounds. In this study, the effects of lycopene and ubiquinol-10
on the oxidative stress in rat hepatocytes induced by t-buthyl
hydroperoxide was determined.
First, rat hepatocytes were isolated and then incubated in
the presence of tert-buthyl hydroperoxide and the amount of
malondialdehyde, as a marker of lipid peroxidation, was determined.
Then, this reaction was performed in the presence of various
concentrations of each lycopene and ubiquinol-10, and the
malondialdehyde level was determined.
The results of this study showed that in the presence of various
concentrations of lycopene and ubiquinol-10 the levels of
lipid peroxidation products significantly decreased
Thus, lycopene and ubiquinol-10 have inhibitory effects on
lipid peroxidation reaction. This study showed the potential
utility of lycopene and ubiquinol-10 in prevention of hepatic
Hepatocytes, which make up the majority of the liver structure,
are very active in the metabolism of exogenous chemicals, and this
is one of the major reasons why the liver is a target for toxic
substances (1). In recent studies, aging and related diseases, such
as cancer and coronary heart disease and neurodegenerative
disorders such as Alzheimerۥs disease were found to be related to
oxidative and free radical mediated reactions (2). One of the most
important of free radicals, reactive oxygen species (ROS) are
generated which cause oxidative stress (3). The tipping of the
balance towards prooxidant status in oxidative stress induced
damage to cellular and extra-cellular macromolecules, such as
proteins, lipids and nucleic acids, and affects the immune function
(4). Endogenous or dietary factors play a major role in the
antioxidative defense of the organism against the ROS generated
during normal cellular aerobic respiration (5). Therefore, it is
generally assumed that increased intake of dietary antioxidants may
help to shift the balance towards an adequate antioxidant status.
The epidemiological data support this reputed benefit of dietary
It was postulated that incubation of lipophylic agent with
rat hepatocytes, may prevent against oxidative damage. To
test this hypothesis, lycopene and ubiquinol-10 were used. These
compounds are highly lipophylic and operate in membranes or
lipoproteins (6). Lycopene is a carotenoid found in high quantities
in tomatoes and tomato-rich products and a potent donor antioxidant
(reductant), which increases the lipoprotein resistance against the
oxidative modification. In lipid solutions and dispersions, it
inhibits formation of radicals linearly with time until consumed in
the process (7). Ubiquinol-10, the reduced form of ubiquinone-10
(coenzyme Q10), is a well-known proton-electron carrier in inner
mitochondria membrane and a potent lipophylic antioxidant in
different cell membranes. It is well established that both
ubiquinols and ubiquinones are active against lipid peroxidation in
mitochondria and liposomes but that the quinols are much more
powerful antioxidants than the corresponding quinones (8).
Recently, we reported that both compounds can protect human
lipoprotein more efficiently against lipid peroxidation than
antioxidant vitamins (9, 10). The aim of the present study was to
investigate the effects of lycopene and ubiquinol-10 on liver
injury induced by tert-butyl hydroperoxide (t-BHP), a short-chain
analogue of lipid peroxide in rat livers
Materials and Methods
Chemicals: tert-Butyl hydroperoxide (t-BHP),
thiobarbituric acid (TBA), lycopene, ubiquinol-10 and other
chemical substances were prepared from Merck, Germany.
Animals: In this study, male Wister rats (with a body weight of
250±20 g) were purchased from Pasteur Institute Tehran, Iran. These
animals were housed in laboratory cages and maintained on a 12 hr
light-dark cycle with free access to food and water throughout the
Isolation and culture of rat hepatocytes: Rat hepatocytes were
prepared by collagenase perfusion, as described previously (11).
First, rats were anesthetized with ether. The liver were then
removed, minced and homogenized at 4oC in 3ml per g liver, of a
buffer made of 20mM Tris-chloride, 0.15m NaCl, 1mM CaCl2 and 1m
PMSF (phenyl methyl sulfonyl fluride) with pH=7.4. The samples were
centrifuged at 800g for 10min. The supernatant was filtered through
Miracloth (Calbiochem-Behring). The resulting filtrate was quickly
frozen in liquid nitrogen and can be stored at -70 oC for
performing the other experiments.
In vitro hepatocytes treatment: First, to the primary cultured rat
hepatocytes, various doses of each : lycopene or ubiquinol-10 (0,
10, 20 and 50 µmol/L) were added to the rat hepatocytes for 4 hr at
37oC. The media were replaced by phosphate-buffered saline. Then,
hepatocytes were incubated with t-BHP (1.5Mm) for 30 min at 37oC
Lipid peroxidation assay: Hepatocytes were pretreated with lycopene
and ubiquinol-10, and then with t-BHP as described above. The lipid
peroxidation product was assayed according to an improved TBA
method by spectrophotometer at 532 nm wavelenght absorption,
using 1,1,3,3-tetra-methoxypropane as a standard (13). The results
were expressed as nmol of malondialdehyde (MDA) equivalents per mg
Statistical analysis: The results obtained were expressed as a mean
±SD. The Studentۥs t-test was used to perform the statistical
comparison between the group by one-way analysis of variance.
Significant differences were taken as p<0.05.
Cytotoxicity of t-BHP: The TBA method showed that t-BHP
expressed toxicity effects to the primary cultures of rat
hepatocytes in 0.5, 1 and 1.5 mM concentrations, and in 1.5 mM
concentration the highest toxicity effect was observed (Table
Table 1. Cytotoxicity effects of tert-butyl hydroperoxide
(t-BHP) assessed by the MDA assay in primary cultured rat
hepatocytes. Hepatocyte cultures were treated with various doses of
t-BHP for 30 min at 37oC. Data represent mean ± SD (n=3).
* p<0.05, ** p<0.01, compared with the group non-treated with
Effects of lycopene on t-BHP-induced hepatotoxicity: The effects
of lycopene on the cytotoxicity induced by t-BHP in the primary
cultured hepatocytes were expressed by the MDA formation. As shown
in Table 2, the MDA formation caused by t-BHP (1.5 mM for 30 min)
was significantly suppressed by lycopene pretreatment ( 51% for 50
µmol/L , p<0.001, 46% for 20 µmol/L, p<0.001, and 20%
for 10 µmol/L lycopene, p<0.01).
Table 2. Effect of lycopene on the lipid peroxidation induced by
tert-butyl hydroperoxide (t-BHP) in 1.5 mM
concentration for 30 min in primary cultured rat hepatocytes. Lipid
peroxidation was evaluated by malondialdehyde formation. Data
represent mean ± SD (n=3).
* p<0.01, ** p<0.001, compared with the group treated with
Effects of ubiquinol-10 on t-BHP-induced hepatotoxicity:
The effects of ubiquinol-10 on the cytotoxicity induced by t-BHP in
the primary cultured hepatocytes were expressed by the MDA
formation. As shown in Table 3, the MDA formation caused by t-BHP
(1.5 mM for 30 min) was significantly suppressed by ubiquinol-10
pretreatment ( 40% for 50 µmol/L , p<0.001, 26% for 20
µmol/L, p<0.01, and 10% for 10 µmol/L ubiquinol-10,
Table 3. Effect of ubiquinol-10 on the lipid peroxidation
induced by tert-butyl hydroperoxide in 1.5 mM concentration for 30
min in primary cultured rat hepatocytes. Lipid peroxidation was
evaluated by MDA formation. Data represent mean ± SD (n=3).
* p<0.01, ** p<0.001, compared with the group treated with
We employed t-BHP, a short-chain analog of lipid peroxides to
induce acute oxidative damage in cultured rat hepatocytes. The
model of t-BHP induced acute hepatic damage in animals has been
used in several studies, and the capacity of a test compound to
reduce the injurious effects or to preserve the normal hepatic
physiological mechanisms which have been disturbed by t-BHP is the
index of its hepato-protective effect (1, 14).
Reactive oxygen species (ROS; .OH, O2-, RO, ROO, NO) can originate
from a number of internal and external sources, such as metabolic
reactions, infections, dietary intake and cigarette smoking. The
body, however, possesses defense mechanisms to reduce the oxidative
damage, and such mechanisms use both enzymes and antioxidant
nutrients or medicine to arrest the damaging properties of excited
oxygen species. Only when the normal protective mechanism breaks
down, or when the effectiveness of antioxidant sources is reduced,
which leads to the excessive amount of free radicals in the body,
the irreversible oxidative damage may occur. A great
number of studies have suggested that antioxidant nutrients and/or
medicines play a protective role for the human health (3,
Two distinctive pathways are involved in the metabolism of t-BHP in
hepatocytes. The first employs the microsomal cytochrome P-450
system leading to the production of ROS that initiate lipid
peroxidation (16); while the second concerns a reaction involving
glutathione peroxidase and its substrate glutathione (GSH).
Decreased GSH is considered to be a major event in t-BHP-
induced toxicity (17).
In the hepatotoxicity and lipid peroxidation experiments, lycopene
and ubiquinol-10 were shown to possess a high antioxidative and
protective capacity against the injury induced by t-BHP, as
reflected in the decreased formation of MDA. Lycopene and
ubiquinol-10 are known to be a highly efficient lipid-soluble
antioxidants in the protection of lipids in a variety of biological
and model systems including lipoproteins and liposomes (18, 19). It
is generally assumed that they may exhibit their protective effects
by preventing the formation of lipid free radicals and/or by
eliminating them. On the other hand, the possibility that the
inhibition of lipid peroxidation is related to their interactions
on the lipid structure of the hepatocytes membranes needs further
investigation. Lycopene and ubiquinol-10 contain shielding methyl
(-CH3) groups and are optimally positioned in the membranes by
their phytyl side-chain. Also, the inhibition of photosensitized
oxidants of lipids and lipoproteins by both compounds mediated by
singlet oxygen have been demonstrated (20). Incubation of lycopene
and ubiquinol-10 with hepatocytes over a long period of time may
enrich their membranes sufficiently to make them less susceptible
to oxidative reactions.
In the present study, lycopene and ubiquinol-10 were observed to
inhibit the oxidative damage in in vitro cultured intact cells.
This provides biological evidence supporting the use of both
compounds for managing liver disorders.
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