Cisplatin-Induced Hepatotoxicity Is Enhanced by Elevated
Expression of Cytochrome P450 2E1
Yongke Lu and Arthur I. Cederbaum1
The Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029
Received October 17, 2005; accepted October 21, 2005
In this study, the possible potentiation of cisplatin-induced
hepatotoxicity by cytochrome P450 2E1 (CYP2E1) was examined
both in vitro and in vivo. Transfected HepG2 cells expressing
CYP2E1 (E47 cells) and not expressing CYP2E1 (C34 cells) were
used as an in vitro model, and mice drinking 2% acetone for 7
days to induce CYP2E1 were used as an in vivo model. Exposure
of E47 cells to cisplatin caused a much greater loss of cell viability,
more striking depletion of reduced glutathione (GSH), and higher
reactive oxygen species (ROS) production as compared with C34
cells. The prooxidant L-buthionine-[R,S]-sulfoximine (BSO),
which depletes GSH, enhanced cisplatin-induced loss of cell via-
bility, whereas the antioxidant glutathione ethyl ester, or the iron
chelator deferoxamine mesylate (DFO) protected against the
cisplatin-induced loss of E47 cell viability. Diallyl sulfide (DAS),
an inhibitor of CYP2E1, also protected against the cisplatin tox-
icity in the E47 cells. After being injected with cisplatin (ip, 45mg/
kg), mice drinking 2% acetone with increased CYP2E1 levels
exhibited elevated levels of serum ALT and AST, liver caspase-3
activity and positive staining of TUNEL increased, and histopa-
thology indicated the presence of necrotic foci in livers of acetone
plus cisplatin-treated mice. Lipid peroxidation and protein
oxidation as indicated by carbonyl formation, staining of 3-nitro-
tyrosine (3-NT) and iron were higher in the cisplatin plus acetone
group, compared with cisplatin alone group. Both in vitro and
in vivo results indicate that elevated CYP2E1 enhances cisplatin-
induced hepatotoxicity, and the mechanism may involve increased
production of ROS and oxidative stress.
hepatotoxicity; oxidative stress; HepG2 cell.
Cisplatin is one of the most potent anticancer drugs used in
chemotherapy (Thigpen et al., 1994; Van Basten et al., 1997).
In spite of its significant anticancer activity, the clinical use of
cisplatin is often limited by its undesirable side effects such as
nephrotoxicity (Madias and Harrington, 1978). Hepatotoxicity
can also occur when cisplatin is administered at high doses
(Cavalli et al., 1978; Cersosimo, 1993; Pollera et al., 1987).
Oxidative stress appears to play an important role in cisplatin-
induced hepatotoxicity. For example, metallothionein protects
against liver injury induced by high doses of cisplatin in mice
(Liu et al., 1998); selenium and high dose of vitamin E
administration protect against cisplatin-induced oxidativedam-
age to liver (Naziroglu et al., 2004); heme oxygenase (HO) and
catalase are important protective responses against cisplatin
toxicity in the livers of tumor-bearing mice (Christova et al.,
2003). L-Buthionine-[R,S]-sulfoximine (BSO), which lowers
cellular reduced glutathione (GSH) levels, enhances cisplatin-
induced cytotoxicity to primary cultured rat hepatocytes, while
L-cysteine, the precursor of GSH, protects against it com-
pletely (Lu et al., 2004).
Many cellular pathways have been suggested to contribute to
induction of a state of oxidative stress. Cytochrome P450 2E1
(CYP2E1) is mainly expressed in liver and in small amounts
in brain, kidney, lung, gastrointestinal tract, and lymphocytes
(Lieber, 1997). Due to being poorly coupled with NADPH-
cytochrome P450 reductase, CYP2E1 exhibits enhanced
NADPH oxidase activity and elevated rates of production of
superoxide anion radical (O2??) and hydrogen peroxide (H2O2)
and, in the presence of iron catalysts, produces powerful oxi-
dants such as the hydroxyl radical (Boveris et al., 1983;
Ekstrom and Ingelman-Sundberg, 1989; Rashba-Step et al.,
1993). It has been shown previously that CYP2E1 induction is
involved in hepatotoxicity of iron and polyunstaturated fatty
acids subsequent to reactive oxygen species (ROS) production
(Castillo et al., 1992; Dai et al., 1993; Sakurai and Cederbaum,
1998), and elevated expression of CYP2E1 enhances liver
injury induced by Fas agonist Jo2 (Wang et al., 2005) and
endotoxin (Lu et al., 2005). It is possible that cisplatin-induced
oxidative stress and CYP2E1-mediated oxidative stress syner-
gize to produce hepatotoxicity. In the present study, the effect
of increased CYP2E1 expression on cisplatin-induced hepato-
toxicity was investigated. The results showed that HepG2 cells
transfected with a CYP2E1 vector were more sensitive to cis-
platin than the cells transfected with empty vector. Moreover,
compared with normal mice, mice with increased CYP2E1
levels revealed enhanced hepatotoxicity after injection of cis-
platin. Thus, CYP2E1 enhances cisplatin hepatotoxicity.
1To whom correspondence should be addressed at The Department of
Pharmacology and Biological Chemistry, Box 1603, Mount Sinai School of
Medicine, One Gustave L. Levy Place, New York, NY 10029. Fax: (212)
996-7214. E-mail: Arthur.email@example.com.
? The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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TOXICOLOGICAL SCIENCES 89(2), 515–523 (2006)
Advance Access publication October 26, 2005
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MATERIALS AND METHODS
Cell cultures and cytotoxicity determination. E47 cells are HepG2 cells
that overexpress human CYP2E1, whereas C34 cells are HepG2 cells that were
transfected with the pCi vector only and do not express CYP2E1 (Chen and
Cederbaum, 1998). Cells were cultured in minimal essential medium (MEM)
supplemented with 10% fetal bovine serum plus 100 units/ml penicillin and
100 lg/ml streptomycin in 5% CO2at 37?C. Before the experiment, cells were
trypsinized and inoculated into 24-well plates, at an amount of 1 3 105cells/
well. Cells were cultured in the plates for 2 days, and then varying concen-
trations of cisplatin were added. In some experiments, cisplatin was added in
the presence of 40 lM BSO (Sigma), or 5 mM of the iron chelator deferoxa-
mine mesylate (DFO, Sigma), or 5 mM glutathione ethyl ester (GSHE,
Calbiochem, Temecula, CA), or 2 mM of the CYP2E1 inhibitor diallyl sulfide
(DAS, Sigma). Cisplatin-induced cytotoxicity was determined by reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as de-
scribed previously (Chen and Cederbaum, 1998).
Measurement of intracellular GSH. Reduced glutathione (GSH) was
assayed using a modified method of Hissin and Hilf (1976). Cells were
inoculated into 6-well plates; the numbers of cells were 2 3 105cells/well. The
cells were treated with cisplatin for 12 and 24 h, the medium was discarded, the
cells were washed with phosphate buffered saline (PBS), and then 0.4 ml of 5%
trichloroacetic acid (TCA) was added. After 30 min of incubation at 4?C to
extract GSH, 10 ll of the TCA extract was added to tubes containing 1 mg/ml
o-phthalaldehyde (200 ll) in 50 mM phosphate/5 lM EDTA buffer (pH 8). The
tubes were incubated at 37?C in the dark (15 min). Fluorescence was measured
(excitation 350 nm, emission 420 nm) using a Perkin-Elmer LC-50B spectro-
fluorometer. The concentration of GSH was determined from a GSH standard.
Intracellular ROS production. Fluorescence spectrophotometry was used
to measurethe levels ofintracellular reactiveoxygenspecies(ROS), with2#,7#-
dichlorofluorescein diacetate (DCF-DA, Sigma) as the probe (Bai et al., 1999).
DCF-DA readily diffuses through the cell membrane and is enzymatically hy-
drolyzed by intracellular esterases to the nonfluorescent DCFH, which can then
be rapidly oxidized to highly fluorescent DCF in the presence of ROS. Cells
incubated in medium alone or cells under different treatment conditions were
incubated with 5 lM DCF-DA in MEM for 30 min at 37?C in the dark. The
cells werewashed in PBS, trypsinized, and resuspended in 3 ml of PBS, and the
intensity of fluorescence was immediately read in a fluorescence spectropho-
tometer (Perkin-Elmer 650-10S, Hitachi, Ltd.) at 503 nm for excitation and at
529 nm for emission. Results are expressed as arbitrary fluorescence units.
Animals and treatments. Male C57BL-6 mice, were purchased from
Charles River Breeding Laboratories (Boston, MA), housed in temperature-
and light-controlled animal facilities, and permitted consumption of tap water
and standard food ad libitum. Mice used in this study received humane care,
and experiments were carried out according to the criteria outlined in the Guide
for the Care and Use of Laboratory Animals and with approval of the Mount
Sinai Animal Care and Use Committee. Mice drank water containing 2%
acetone (FisherChemicals, Fair Lawn, NJ) to induceliverCYP2E1,or tap water
as control. The water containing 2% acetone was changed everyday. After
1 week, cisplatin (Sigma) was injected intraperitoneally at a concentration
of 45 mg/kg body weight; this concentration has been reported to induce
hepatotoxicity (Liu et al., 1998). Control mice were injected with 0.9% saline.
Twenty-four h after cisplatin or saline injection, blood was collected from the
retro-orbital venous sinus under anesthesia, and then the mice were sacrificed.
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
were assayed using diagnostic kits (Thermo Electron, Louisville, CO). Livers
were removed, washed with cold saline, and excised into fragments. Two
aliquots of tissue were fixed in 10% formalin solution or methanol/chloroform/
glacial acetic acid (6:3:1 in volume) for paraffin blocking, while the other
aliquots were stored at –20?C for subsequent assays.
Liver pathology and immunohistochemistry. Liver samples were fixed in
10% formalin solution and embedded in paraffin. Sections (5 lm thick) were
stained with H&E for pathological evaluation. Immunohistochemical staining
for 3-nitrotyrosine (3-NT) was performed by using polyclonal rabbit antisera
against 3-NT (Upstate, Lake Placid, NY) and a rabbit ABC staining system
(Santa Cruz Biotechnology, Santa Cruz, CA). Samples were counterstained
In situ detection of iron. Liver samples were fixed in methanol/
chloroform/glacial acetic acid (6:3:1 in volume), and iron was detected ac-
cording to the method of Sayre et al. (2004). Briefly, liver section slides were
covered with freshly made 7% potassium ferrocyanide and incubated for 2 h at
37?C, followed by freshly prepared DAB/H2O2solution for 5 min, and finally,
the slides were dehydrated and covered with coverslips.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphos-
phate nick-end labeling assay. DNA fragmentation was assessed by in situ
terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate
nick-end labeling assay using an ApopTag in situ apoptosis detection kit
(Chemicon, Temecula, CA). Slides with liver tissue sections were pretreated
with Proteinase K and H2O2, and then incubated with the reaction mixture
containing terminal deoxynucleotidyl transferase and digoxigenin-conjugated
deoxyuridine triphosphate for 1 h. The labeled DNA was visualized with
horseradish peroxidase-conjugated anti-digoxigenin antibody with diamino-
benzidine as the chromogen. Samples were counterstained with 0.5% methyl
Caspase-3 activity. Hepatic tissues were placed in 0.15 M KCl and
homogenized in a polytron homogenizer for 10 strokes. The resulting homog-
enate was centrifuged at 3000 rpm for 12 min, and the supernatant fraction was
used for caspase-3 activity measurement. Caspase-3 activity was determined by
measuring enzymatic cleavage of the substrate Ac-DEVD-AMC (Sigma). Ac-
DEVD-AMC (final concentration 0.2 mM) was dissolved in assay buffer
containing20 mMHEPES(pH7.5),10%glycerol,and2mMdithiothreitol. 5ll
of homogenate was added to this assay buffer (0.5 ml) and incubated at 30?C
overnight. The fluorescence associated with the released AMC (excitation at
380 nm, emission at 460 nm) was assayed in a PerkinElmer spectrofluorometer
(Wellesley, MA). The data are expressed as arbitrary fluorescence units per
milligram of protein.
Lipid peroxidation. Malondialdehyde (MDA) and 4-hydroxyalkenals
(HAE) were measured in liver homogenate as an index of lipid peroxidation
by using the Lipid Peroxidation Assay Kit (Calbiochem, Temecula, CA). The
chromogenic reagent N-methyl-2-phenylindole (MPI) reacts with MDA and
HAE at 45?C, and condensation of one molecule of either MDA or HAE with
two molecules of MPI yields a stable chromophorewith maximal absorbance at
586 nm.MDAwasused asthe standard,and resultsareexpressed asnmolMDA
Protein carbonyl formation. Liver protein oxidation was determined by
measuring protein carbonyls using the OxyBlot protein oxidation detection kit
(Calbiochem, Temecula, CA). Briefly, 5 ll of homogenate obtained as above
was denatured by adding 5 ll of 12% SDS. The carbonyl groups were
derivatized by adding 10 ll of 13 2,4-dinitrophenyl hydrazine (DNPH). The
negative control involved addition of derivatization-control solution instead of
the DNPH solution. The resulting samples were then subjected to Western blot
analysis. Proteins which have undergone oxidative modification were identified
by appearing as a band only in the lane containing the derivatized sample, but
not in the lane containing the negative control.
Preparation of microsomes. Microsomes were prepared as described by
Funae and Imaoka (1985). Tissues were placed in 0.15 M KCl and homog-
enized in a polytron homogenizer for 10 strokes. The homogenate was cen-
trifuged at 9,000 3 g for 20 min, and the resulting supernatant fraction was
centrifuged at 105,000 3 g for 60 min. The resulting pellets (microsomes) were
resuspended in 50 mM sodium phosphate buffer (pH 7.4). All procedures were
carried out under cold conditions.
Western blots. Microsomal proteins (40 lg) were resolved by electropho-
resis using 10% SDS–PAGE and then transferred to nitrocellulose membranes
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CISPLATIN TOXICITYAND CYTOCHROME P450 2E1
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