Vol.3, No.5, 288-291 (2011)
doi:10.4236/health.2011.35050
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
A new risk assessment method for evaluation of
oxidative chemicals using catalase mutant mouse
primary hepatocytes
Da-Hong Wang1*, Yasuo Ishikawa1, Masahiro Miyazaki 2, Hirofumi Fujita3, Ken Tsutsui4,
Kuniaki Sano5, Noriyo shi Masuoka6, Keiki Ogino1
1Department of Public Health, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama,
Japan; *Corresponding Author:
2Department Okayama Gakuin University, Kurashiki, Japan;
3Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,
Okayama, Japan;
4Department of Genome Dynamics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,
Okayama, Japan;
5Department of Neurogenomics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Oka-
yama, Japan;
6Department of Life Science, Okayama University of Science, Okayama, Japan.
Received 8 March 2011; revised 20 April 2011; accepted 28 April 2011.
ABSTRACT
We examined the possibility of developing a
new risk assessment method for potentially
oxidative chemicals by using mouse primary
hepatocytes from acatalasemic mice (Csb) and
the wild-type (Csa) as predictive model. Chemi-
cal-induced cytotoxicities, such as hydrogen
peroxide and lawsone, a main hair dye ingredi-
ent of henna, were examined. We observed the
differences in cell survival betw een the Csa and
Csb in a dose-dependant manner after treatment
with either hydrogen peroxide or lawsone, sup-
porting the usefulness of this new ly established
method for hazard identification of oxidative
chemicals in a risk assessment process. More
chemicals will be tested to confirm the useful-
ness of this method for the preliminary screen-
ing of oxidative chemicals before animal ex-
perimentation.
Keywords: Catalase-Mutant Mouse Hepatocytes;
Reactive Oxygen Species; Hydrogen Peroxide;
Hazard Assessment; Lawsone Cytotoxicity
1. INTRODUCTION
More and more chemicals are synthesized for indus-
trial and consumer use, it is impossible to finish
long-term rodent bioassay for detection of carcinogens
and identification of hazards in all chemicals because it
involves large numbers of animals and is extremely ex-
pensive. Therefore, simple and efficient pre-screening
alternatives to animal experimentation are desirable [1].
Nowadays, there has been an increasing awareness that
the cellular formation of highly reactive oxygen species
(ROS) such as hydrogen peroxide (H2O2) or hydroxyl
radicals likely cause the DNA lesions [2]. Catalase and
glutathione peroxidase are the most important enzymes
capable of removing intracellular H2O2 in biological
systems [3-5], and catalase particularly plays a critical
role when H2O2 is overproduced. Catalase-mutant acata-
lasemic mouse was established by Feinstein et al.
through a large scale screening of the progeny of irradi-
ated C3H mice [6]. A point mutation at amino acid 11
(from glutamine to histidine) of Csb mouse catalase is
responsible for its catalase deficiency [7,8]. In the pre-
sent study, we examined the possibility of developing a
new risk assessment method for evaluation of oxidative
chemicals using mouse primary hepatocytes from acata-
lasemic mice (Csb) and the wild-type (Csa) as predictive
model.
2. MATERIALS AND METHODS
2.1. Chemicals
Hydrogen peroxide, lawsone (2-hydroxy-1,4-naph-
thoquinone), kanamycin sulfate, insulin, and dexa-
methasone sodium phosphate, were purchased from
Wako Pure Chemical Industries (Osaka, Japan). Cell
counting kit (WST-8) from Dojindo Company (Osaka,

D.-H. Wang et al. / Health 3 (2011) 288-291
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
289289
Japan). All other chemicals were obtained from Sigma
Chemical Co (St. Louis, USA) unless otherwise men-
tioned.
2.2. Hepatocyte Isolation
Adult male wild-type (C3H/AnLCsaCsa) and acata-
lasemic (C3H/AnLCsbCsb) mice were used in this study.
They were anaesthetized intraperitoneally with 9μl/g
body weight of pentobarbital sodium (10% in PBS) and
then the liver was first perfused at 37˚C with EGTA at a
flow rate of 5.5 ml/minute for 10 minutes and thereafter
with collagenase at the same flow rate for 15 minutes as
described previously [9,10]. The yield of isolated hepa-
tocytes was determined with a hemocytometer, and their
viability was evaluated with the standard trypan blue
exclusion method. The viability of the isolated hepato-
cytes was around 90%. The isolated hepatocytes (5 × 106)
were seeded into 100-mm-diameter plates or 96-well
plates containing serum free hepatocyte growth medium
(HGM) [11,12] supplemented with 1 μM insulin (Sigma
Chemical Co, St. Louis, MO), 1 μM dexamethasone so-
dium phosphate, 0.1 mg/ml kanamycin sulfate, and al-
lowed to attach for 24 h before used in the experiments.
2.3. Confirmation of the Genotype of
Catalase Mutant Mouse Hepatocytes
Genomic DNA was purified from primary cultured
hepatocytes of Csa and Csb using Wizard Genomic DNA
Purification Kit (Promega Corporation, WI) and was
used as a template for PCR amplification of the catalase
gene segment encompassing the Csb mutation site. The
primers, 5’-CGGGTGGAGACCAGACCGCT-3’ and
5’-TGCGAGGCCCGCTGCTCCTT-3’, were used to
target a 141 bp fragment of the mouse catalase gene [13].
PCR amplification was performed for 35 cycles (30 s at
98˚C for denaturation, 20 s at 58˚C for annealing, and 40
s at 68˚C for extension) in a final volume of 20 μl with
50 ng of genomic DNA, 50 nM primers, and 0.05 U/ml
LA Taq DNA polymerase under the conditions recom-
mended by the manufacturer (TaKaRa Biochemicals,
Japan). Amplified products were purified by ethanol
precipitation, digested with restriction enzyme NdeI, and
subjected to agarose gel electrophoresis (4% NuSieve
GTG agarose). DNA bands were visualized by staining
with ethidium bromide.
2.4. Cell Viability Assay
Primary hepatocytes seeded into 96-well plates at a
density of 1.2 × 104 per well were used to evaluate the
cytotoxic effect of chemicals. Various concentrations of
test compounds (H2O2 and lawsone were added to the
cells and incubated at 37˚C for 24 h. Cell viability was
measured using the WST-8 assay, based on the reduction
of the tetrazolium salt to a water-soluble formazan
product by the cellular dehydrogenase [14]. Absorbance
was measured at 450 nm by microplate reader. The sur-
vival of cells exposed to chemicals was expressed as a
percentage of cell survival in the negative control group
based upon the following formula: Survival (%) = (Ab-
sorbance of sample – Absorbance of blank)/(Absorbance
of negative control – Absorbance of blank). At least
three tests were performed in each experiment.
2.5. Statistic Analysis
Significant differences (p < 0.05) among groups were
determined by two-way analyses of variance (two-way
ANOVA) by SPSS 15.0 statistical program package
(SPSS Inc., Illinois, USA).
3. RESULTS AND DIS CUSSION
3.1. The Csb Catalase Mutant Gene Is
Susceptible to NdeI Digestion
Since a point mutation at amino acid 11 (from gluta-
mine to histidine) of Csb mouse catalase gene is respon-
sible for the deficient catalase activity [7,8], there is a
recognition site for NdeI that can cut the targeted 141 bp
fragment of Csb band into 2 fragments of 108 and 33 bp.
Figure 1 showed the genomic PCR products with or
without the enzymatic digestion of NdeI. The 141-bp
target region in the wild-type mouse Csa catalase gene
was not cleaved by the enzyme, however, all the 141-bp
products of the Csb catalase mutant gene were cleaved to
2 fragments of 108 and 33 bp (the 33 bp fragment is not
visible here) by NdeI. The results agreed with that of
previous reported [13].
3.2. Difference in Cyto toxic Effects Induced
by Oxidant H2O2 on Mouse Primary
Hepatocytes
In Table 1, catalase-mutant mouse Csb hepatocytes
showed a significantly higher susceptibility to H2O2 in
comparison with the wild-type Csa, and the cytotoxic
effects of H2O2 on both cell groups were dose dependent.
Since a point mutation at amino acid 11 of Csb mouse
catalase gene is responsible for catalase deficiency, H2O2
exposed to Csb hepatocytes could not be fully decom-
posed, which markedly affected the cell survival com-
pared to that of the wild-type Csa. Therefore, based on
the cell viability, we can use the Csa and Csb as predic-
tive models to assume whether H2O2 involves in the
chemical-induced cytotoxicity, which would be helpful
for the preliminary screening of the potential oxidative
chemicals.

D.-H. Wang et al. / Health 3 (2011) 288-291
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
290
141 bp
108 bp
Cs
a
Cs
b
N
deI
N
deI
(-) (+) (-) (+)
Figure 1. The genomic PCR products with or without the en-
zymatic digestion of NdeI.
Ta bl e 1 . Comparison of H2O2-induced cytotoxic effect on pri-
mary hepatocytes#.
Treatment H2O2## Csa Csb
None 100 100
3.75 mM 74.2 ± 2.5 18.3 ± 0.4
7.5 mM 54.0 ± 3.8 15.6 ± 1.6
15 mM 36.0 ± 4.1 12.8 ± 1.1
#Data are expressed as percentage of mean ± SD. At least three tests were
performed in each experiment. Differences within and among groups were
evaluated by two-way ANOVA; ##Concentrations of H2O2 were expressed
as final concentrations in the reaction cultures. P1 < 0.01(comparison be-
tween groups of Csa and Csb). P2 < 0.001 (comparison among groups of
H2O2 concentrations).
3.3. Comparison of Lawsone-Induced
Cytotoxicity on Mouse Primary
Hepatocytes
Then, we further tested the cytotoxic effect of lawsone,
a main color ingredient of hair dye henna, on mouse
primary hepatocytes. Lawsone has been reported causing
mutagenicity/genotoxicity both in vitro and in vivo [15]
by the Scientific Committee on Cosmetic Products and
Non-Food Products (SCCNFP), a scientific advisory
body to the European Commission in matters of con-
sumer protection. Figure 2 demonstrated that in com-
parison with the wild-type Csa hepatocytes, the survival
of catalase-mutant mouse Csb hepatocytes was signifi-
cantly reduced by lawsone treatment, showing 86.1% in
Csa, 38.0% in Csb even at 100 µM lawsone exposure.
Both cell groups dose-dependently detected cytotoxic
effects of lawsone.
Catalase is an important component of the cellular
defenses against the toxicity induced by H2O2 and its
products [4]. Wang et al. reported that the catalase activ-
ity in liver of Csb mice were 39.6% of that in wild-type
Csa [16]. Lawsone was reported to generate H2O2 in
phosphate buffer system [17]. The present study found
lawsone treatment markedly reduced cell viabilities in
Figure 2. Comparison of lawsone-induced cytotoxic effect on
primary hepatocytes. Data are expressed as percentage of mean
± SD. At least three tests were performed in each experiment.
There were significant differences between Csa and Csb (P <
0.001) and among exposure concentrations of lawsone (P <
0.001) evaluated by two-way ANOVA.
catalase-mutant mouse Csb hepatocytes in comparison
with in wild-type Csa, suggesting that H2O2 was increas-
ingly produced in the process of lawsone cytotoxicity.
4. CONCLUSIONS
The present study has attempted to develop a new as-
sessment method for evaluation of oxidative chemicals
using mouse primary hepatocytes from catalase-mutant
mouse Csb and the wild-type Csa as predictive model.
The differences in cell survival between the Csa and Csb
in a dose-dependant manner support the usefulness of
this newly established method for hazard identification
of oxidative chemicals in a risk assessment process.
More chemicals will be tested to confirm the usefulness
of this method for the preliminary screening of oxidative
chemicals before animal experimentation.
It is known that most primary cell cultures have lim-
ited lifespan. The primary cultured hepatocytes em-
ployed in our study can be used for 5 - 6 days, during
which the hepatocytes are supposed to maintain most of
their in vivo functional characteristics in culture [18,19],
and they are expected to be possible predictive models
for large scale screening of oxidative chemicals if the
issue of long-term storage of primary hepatocytes can be
solved.
5. ACKNOWLEDGEMENTS
This work was supported by Grant-in-Aid for Scientific Research
from the Ministry of Education, Science and Culture of Japan
(21310022).
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