European Journal of Scientific Research (ISSN: 1450 ... - EuroJournals

© European Journal of Scientific Research, Vol 7, No 5, 2005
and other factors that would affect the biological activity of vitamin E would be the
presence of methyl group on the chromanol ring and saturation or branching on the side
chain (18). Studies on human and laboratory animals showed that bioavailability of
naturally occurring vitamin E isomers are three times higher compared to vitamin E
acetate (19). These factors encourage us to use domestically produced vitamin E that
comes from natural source which is the palm oil.
To directly demonstrate the effect of CYP2E1 enzyme on oxidative stress in vitro, Hep
G2 cells which was transfected with CYP2E1 gene was used. Two vitamin E derivatives
α-tocopherol and γ-tocotrienol derived from palm oil were used in this study as
antioxidative agent.
OBJECTIVES
The goal of the current study was to evaluate the effects of vitamin E (α-tocopherol and
γ-tocotrienol) on lipid peroxidation status in transfected Hep G2 cell line expressing
human CYP2E1 enzyme and to compare these effects to control cells that do not express
this enzyme.
MATERIALS AND METHODS
CHEMICALS
Cell culture reagents such as L-glutamine, penicillin, streptomycin, geniticin, pnitrophenol
and NADPH were purchased from Sigma, New York (USA), Fetal Calf
Serum (FCS), Tripsin-EDTA from Flowlab (Australia) and EMEM from PAA Lab
(Austria). Palm oil vitamin E (α-tocopherol and γ-tocotrienol) was obtained from MPOB
(Malaysia). Other chemicals and solvents were supplied by Sigma or Merck
(Darmstadt,Germany).
CELL CULTURE
Transduced Hep G2 subclones that overexpress CYP2E1 enzyme (E47) and the control
cell line (C34) were kindly provided by Prof. A.I. Cederbaum (Mount Sinai School of
Medicine, New York, USA). Both cell lines were established with the use of transfection
and limited dilution techniques (20). The E47 and C34 cell lines were cultured in MEM,
supplemented with 10% fetal calf serum, 100 units of penicillin per milliliter, 100 mg/ml
of streptomycin and 2 mM glutamine in a humidified atmosphere in 5% CO2 at 37°C.
P-NITROPHENOL (PNP) OXIDATION ASSAY
PNP assay was performed as described by Chen & Cederbaum (1998) (20) to determine
the activity of CYP2E1 enzyme in transfected Hep G2 cell line. Cells were washed with
PBS, harvested and sonicated on ice for 45 sec. Microsomes were prepared by
differential centrifugation and resuspended in PBS. Oxidation of PNP was determined
using 100 µg of microsomal protein in a reaction-system containing PBS, 0.4 mM PNP
and 1 mM NADPH. All reactions were carried out in duplicate, initiated with NADPH,
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