A new risk assessment method for evaluation of oxidative chemicals using catalase mutant mouse primary hepatocytes

doi:10.4236/health.2011.35050

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Vol.3, No.5, 288-291 (2011)

doi:10.4236/health.2011.35050

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: [email protected]

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

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

290

141 bp

108 bp

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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