Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2010/06/18/dmd.110.033597.DC1
0090-9556/10/3809-1545–1552$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:1545–1552, 2010
Vol. 38, No. 9
33597/3618712
Printed in U.S.A.
Methemoglobinemia Induced by 1,2-Dichloro-4-nitrobenzene in
Mice with a Disrupted Glutathione S-Transferase Mu 1 Gene□S
Shingo Arakawa, Takanori Maejima, Naoki Kiyosawa, Takashi Yamaguchi, Yukari Shibaya,
Yoshie Aida, Ryota Kawai, Kazunori Fujimoto, Sunao Manabe, and Wataru Takasaki
Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Shizuoka, Japan (S.A., T.M., N.K., T.Y., Y.S., Y.A., R.K.,
K.F.); Global Project Management Department, Daiichi Sankyo Co., Ltd., Tokyo, Japan (S.M.); and Daiichi Sankyo Inc., Edison,
New Jersey (W.T.)
Received March 28, 2010; accepted June 18, 2010
ABSTRACT:
compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses
in the spleen showed exclusive up-regulation of hematopoiesisrelated genes in Gstm1-null mice. These changes were considered
to be adaptive responses to methemoglobinemia and attenuated
the higher predisposition to methemoglobinemia observed in
Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were
higher in Gstm1-null mice than those in wild-type mice, resulting
from the Gstm1 disruption. In conclusion, it is suggested that the
higher exposure to DCNB due to Gstm1 disruption was reflected in
methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.
Introduction
classified into seven classes (Alpha, Mu, Pi, Theta, Sigma, Zeta, and
Omega) (Hayes et al., 2005). Furthermore, each class of GSTs is
composed of several isoforms in both experimental animals and
humans. When normal animal models are used, it is difficult to reveal
the contribution of the isoforms that are responsible for metabolism
and toxicity, especially in vivo (Gonzalez, 2003). Among the seven
isoforms (GSTM1–GSTM7) of Mu class GST, GSTM1 is highly
expressed in the liver and is considered to be a crucial GST isoform
in mice, rats, and humans (Mannervik et al., 1985; Mitchell et al.,
1997; Eaton and Bammler, 1999), and species differences in substrate
specificity have been reported (Hansson et al., 1999).
In general, drug metabolism is widely recognized as an important
factor that determines the incidence of toxicity by xenobiotics. In the
last decade, genetically modified animals have been produced and
used to examine the role of drug-metabolizing enzymes in vivo. In
phase I enzymes, Cyp1a1, Cyp1a2, Cyp1b1, and Cyp2e1 knockout
(null) mice were produced, and many studies were performed to
examine in vivo metabolism, toxicity, and carcinogenesis (Gonzalez
and Kimura, 2003). In addition, knockout mice of the Cyp3a family
were recently produced and applied to the generation of CYP3A4
humanized mice to predict in vivo human metabolism (van Herwaarden et al., 2007). In phase II enzymes, toxicological approaches using
Glutathione S-transferases (GSTs) are major phase II conjugation
enzymes that catalyze the conjugation of electrophilic compounds to
GSH. Electrophilic compounds, which are produced in the metabolic
process of several chemicals, such as acetaminophen (Larson, 2007),
bromobenzene (Lau et al., 1980), and aflatoxin B1 (Guengerich et al.,
1998), covalently bind to cellular macromolecules and induce severe
toxicity. Accordingly, GSTs are generally recognized as important
enzymes that detoxify electrophilic compounds produced by environmental carcinogens/toxicants, pesticides, and drugs (Eaton and
Bammler, 1999).
However, the contribution of each GST isoform to xenobioticinduced toxicity has not been well elucidated. Various isoforms of
GST have been identified and classified into three families in terms of
cellular localization, namely, cytosolic, mitochondrial, and microsomal GSTs. Cytosolic GSTs comprise the largest family and are further
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033597.
□
S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: GST, glutathione S-transferase; GSTM1, glutathione S-transferase Mu 1; DCNB, 1,2-dichloro-4-nitrobenzene; TK, toxicokinetic; RT, reverse transcriptase; PCR, polymerase chain reaction; RBC, red blood cell count; RET, reticulocyte count; MetHB, methemoglobin; M0,
methylsulfon-N-acetyl form of 1,2-dichloro-4-nitrobenzene.
1545
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A specific substrate to Mu class glutathione S-transferase (GST),
1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice
with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate
the in vivo role of murine Gstm1 in toxicological responses to
DCNB. A single oral administration of DCNB at doses of 500 and
1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to
methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600
mg/kg demonstrated a marked increase in blood MetHB in both
wild-type and Gstm1-null mice. However, marked increases in the
blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice
1546
ARAKAWA ET AL.
Materials and Methods
Generation and Maintenance of Gstm1-Null Mice. Gstm1-null mice were
generated by homologous recombination in embryonic stem cells as described
previously (Fujimoto et al., 2006). Wild-type and Gstm1-null mice were
maintained in a C57BL/6J and 129S1 mixed background.
Animal Care. All mice described in these studies were kept in a controlled
environment at a room temperature of 23 ⫾ 2°C and humidity of 55 ⫾ 10%
with an illumination period of 12 h (7:00 AM to 7:00 PM) per day. Each mouse
was housed individually in a bracket cage and fed ad libitum with solid feed
(Certified Rodent Diet 5002; PMI Nutrition International, Inc., Tokyo, Japan)
sterilized by radiation (irradiated with a 60Co-␥ ray of 30 kGy), and tap water
was supplied by an automatic watering system. The studies were approved by
the Ethics Review Committee for Animal Experimentation of Sankyo Co.,
Ltd., and were conducted in compliance with the “Law Concerning the
Protection and Control of Animals” (Japanese Law 105, October 1, 1973;
revised on June 22, 2005).
Study Designs. Wild-type and Gstm1-null mice (males and females) at 14
to 16 weeks of age were orally administered DCNB (Wako Pure Chemical
Industries, Ltd., Osaka, Japan), which was suspended in 0.5% methylcellulose
solution. The dose levels of DCNB were 0, 500, and 1000 mg/kg in the
single-dose study and 0, 150, 300, and 600 mg/kg in the 14-day repeated-dose
study. An additional 14-day repeated-dose study was conducted to perform a
pathological examination. The dose levels for this pathological study were set
at 0, 150 (only females), and 300 mg/kg to avoid mortality. The first day of
administration was defined as day 1, and autopsies were performed 24 h after
the last dosing in each study.
Study Designs of Toxicokinetics Monitoring and Gene Expression Analysis. The wild-type and Gstm1-null mice in the satellite groups of the 14-day
repeated-dose study were orally treated with DCNB for 14 days at dose levels
of 0 and 300 mg/kg. Blood was sampled 2 h after dosing on days 1 and 14,
based on the previous information in which maximal concentrations of plasma
DCNB were exhibited approximately 2 h after the DCNB treatment (Fujimoto
et al., 2006). Then, the blood samples were centrifuged at 10,000 rpm for 5 min
at 4°C to obtain samples of the plasma and blood cells. The TK samples were
stored in a freezer set at ⫺80°C. The spleens of female mice were also stored
in a freezer set at ⫺80°C and used for microarray and quantitative RT-PCR
analyses.
Hematological Examination. On the day of autopsy, the animals were
anesthetized with ether, and blood samples were collected from the inferior
vena cava. These samples were placed in blood sampling tubes (Microtainer;
Nippon Becton Dickinson Company, Ltd., Tokyo, Japan) coated with EDTA2K. The samples for analysis, along with an automated blood cell counter,
were mixed with equal volumes of physiological saline (Otsuka Pharmaceutical Industries, Tokyo, Japan). The red blood cell count (RBC) and reticulocyte count (RET) were determined using an ADVIA120 automated blood cell
counter (Bayer Medical Ltd., Tokyo, Japan). Blood methemoglobin (MetHB)
was determined by the spectrophotometric method with a 7011 clinical spectrophotometer (Hitachi, Ltd., Tokyo, Japan).
Pathological Examination. Macroscopic observations of organs and tissues were performed in each study during the autopsy. For the microscopic
examinations, the organs and tissues were removed and then fixed in 10%
neutral buffered formalin, embedded in paraffin, sectioned at a thickness of 2
to 3 ␮m and stained with hematoxylin and eosin. The grading score of
increased extramedullary hematopoiesis in the spleen was determined based on
the area percentage of the hematopoietic cells within spleen sections. The
criteria for the grading score were defined as follows. No change (⬍10%
increase) in area percentage of hematopoietic cells within spleen sections
compared with the control was defined as grade 0. A slight increase (10 – 40%
increase), moderate increase (41– 60% increase), and marked increase (⬎60%
increase) were defined as grade 1, grade 2, and grade 3, respectively.
Microarray Analysis. Microarray analysis was performed according to the
Affymetrix standard protocol. In brief, total RNA was isolated from an
individual female mouse spleen, and the pooled RNA samples were prepared
for both wild-type and Gstm1-null mice by mixing an equal amount of RNA
samples within the groups. Five micrograms of the pooled RNA was used for
cDNA synthesis using a T7-(dT)24 primer (GE Healthcare, Little Chalfont,
Buckinghamshire, UK). A biotin-labeled cRNA mix was transcribed using a
BioArray High Yield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). Every biotin-labeled cRNA target sample (approximately 15 ␮g)
was fragmented and hybridized to a Mouse 430 2.0 GeneChip array (Affymetrix, Santa Clara, CA) at 45°C for 18 h, followed by washing and staining
with streptavidin-phycoerythrin using a Fluidics Station 400 (Affymetrix).
Microarray image data were analyzed with the Comparison Analysis algorithm
(Affymetrix).
Microarray Data Analysis. Probe sets whose Change Call showed “NC”
(Not Changed) and/or whose 兩Signal Log Ratio兩 values were lower than 0.6
were excluded from the analysis. In addition, probe sets whose sequences are
not annotated with an Entrez Gene identification number were excluded from
the data analysis as well. In the present study, we hypothesized that Gstm1-null
mice-specific gene expression regulation might be associated with the marked
increase in extramedullary hematopoiesis in the spleens of Gstm1-null mice.
Accordingly, genes whose expression levels increased in the Gstm1-null mice
(兩expression ratio to control兩 ⬎1.4) but not in the wild-type mice (兩expression
ratio to control兩 ⬍1.4) after DCNB treatment were extracted in Table 3. Genes
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
knockout mice are very limited, although six lines of knockout mice
for cytosolic GSTs have been established so far. Gstp1/p2 (Henderson
et al., 1998), Gsta4 (Engle et al., 2004), Gstm1 (Fujimoto et al., 2006),
Gstt1 (Fujimoto et al., 2007), Gstz1 (Fernández-Cañón et al., 2002;
Lim et al., 2004), and Gsto1 (Chowdhury et al., 2006) knockout mice
have already been established, and their phenotypes have been characterized. A few studies, such as acetaminophen administration to
GSTp1/p2 knockout mice (Elsby et al., 2003), paraquat administration
to Gsta4 knockout mice (Engle et al., 2004), and carbon tetrachloride
administration to Gsta4 knockout mice (Dwivedi et al., 2006), have
been performed to investigate toxicological responses to xenobiotics
using knockout mice for GSTs. Although the compounds in these
investigations, such as acetaminophen, paraquat, and carbon tetrachloride, were typical toxicants and provided significant information, they
were not necessarily specific substrates for GST isoforms that were
disrupted in each study. Accordingly, in vivo administration of specific substrates for disrupted GST isoforms would also be a useful
approach to investigate the relationship between the drug metabolism
mediated by GSTs and the subsequent incidence of toxicity.
Based on these backgrounds, we generated mice with a disrupted
Gstm1 gene (Gstm1-null mice) by gene targeting and characterized its
phenotypes by in vitro and in vivo metabolic studies using 1,2dichloro-4-nitrobenzene (DCNB) (Fujimoto et al., 2006). As notable
phenotypes, Gstm1-null mice showed markedly low GST activity to
DCNB in the in vitro studies using cytosol prepared from the liver and
kidney. In addition, in vivo administration of DCNB showed a high
plasma concentration of DCNB in Gstm1-null mice compared with
that in the wild-type control mice. From these results, DCNB was
considered to be a specific substrate for murine GSTM1, and the in
vivo administration of DCNB to Gstm1-null mice would provide
useful information on the use of genetically modified animals. In
practice, we are curious as to whether higher exposure to DCNB in
Gstm1-null mice causes severe toxicity. DCNB is a basic chemical for
the synthesis of intermediates, which are further processed to herbicides, bactericides, and dyestuffs. In addition, it has been registered in
the Hazardous Substances Data Bank (http://toxnet.nlm.nih.gov/cgi-bin/
sis/htmlgen?HSDB), which is a database of potentially hazardous
chemicals. In addition, the biological threshold limit value in human
urine has been established for nitro and amino aromatics, including
DCNB because it has been reported to have a potential risk of
methemoglobinemia (Linch, 1974). p-Nitrochlorobenzene and nitrobenzene, chemical analogs of DCNB, have been reported to cause
methemoglobinemia in experimental animals and humans (Watanabe
et al., 1976; Schimelman et al., 1978). In this study, we investigated
in vivo toxicological responses to DCNB in Gstm1-null mice and
focused on methemoglobinemia and its related changes.
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METHEMOGLOBINEMIA INDUCED BY DCNB IN Gstm1-NULL MICE
TABLE 1
Primers for quantitative RT-PCR
Gene Symbol
Entrez Gene
Forward (5⬘ 3 3⬘)
Reverse (3⬘ 3 5⬘)
Product Size
Mmp9
Chi3l3
Trem3
Ms4a3
Lcn2
Gapdh
17395
12655
58218
170813
16819
24383
AACACCACCGAGCTATCCAC
GAAGGAGCCACTGAGGTCTG
GGAGGATTATCCCACCGAAT
TTCAAGGGTTGCCAATCTTC
CTGAATGGGTGGTGAGTGTG
GTGGACCTCATGGCCTACAT
AGGAGTCTGGGGTCTGGTTT
CACGGCACCTCCTAAATTGT
CCACACGTCAGAACGATCAC
AAACATTTCCCAAGCACCAC
GCTCTCTGGCAACAGGAAAG
TGTGAGGGAGATGCTCAGTG
163
141
180
151
101
148
bp
bp, base pair.
software (Microsoft Office Excel 2003; Microsoft, Redmond, WA). A parametric Dunnett’s test (for comparison among three groups or more) or an F-t
test (for comparison between two groups) was performed to analyze the
statistical differences in the mean values. The statistical analysis was performed with statistical software (SAS version 6.1.2; SAS Institute Inc., Cary,
NC). A 5% level of probability was considered to be statistically significant.
For the F-t test, the homogeneity of variance was estimated by an F test and
significant differences in the mean values were evaluated by a Student’s t test
(for homogeneous data) or an Aspin-Welch t test (for heterogeneous data). The
mean values that consisted of one or two data points were excluded from the
statistical analysis.
Results
Single-Dose Study of DCNB. The toxicological responses to
DCNB in wild-type and Gstm1-null mice were examined in the
single-dose study. In the hematological examination, a marked increase in MetHB was observed in Gstm1-null mice, which was similar
in both sexes (Fig. 1). Although a statistically significant increase in
MetHB was observed in the wild-type females given 1000 mg/kg, the
magnitude of the increase was slight compared with that in Gstm1null mice.
14-Day Repeated-Dose Study of DCNB. Toxicological responses
to DCNB in wild-type and Gstm1-null mice were examined in the
14-day repeated-dose study. Three wild-type males given 600 mg/kg
died on days 8 and 10. One wild-type female given 600 mg/kg died on
day 5. Three Gstm1-null females given 600 mg/kg died on days 4, 5
and 6. Although decreases in activity and fecal volume were observed
in the dead animals, no pathological changes were observed in the
results of the unscheduled autopsies (data not shown), and the cause
of death could not be confirmed. In the hematological examination, a
marked increase in MetHB was observed in both wild-type and
Gstm1-null mice, which was similar in both sexes (Fig. 2A). On the
other hand, a marked increase in RET was observed in the Gstm1-null
mice (Fig. 2B). Although a statistically significant increase in RET
was observed in the wild-type mice, the magnitude of the increase was
slight compared with that in the Gstm1-null mice. A slight decrease in
RBC was observed in wild-type males and Gstm1-null females (Fig.
2C). In the relative organ weight, an increase in spleen weight was
observed in the Gstm1-null mice (Fig. 3). In the pathological examination conducted as an additional study, increased extramedullary
MetHB
Male
Female
20
20
10
㪁
10
5
5
0
0
Wild-type
Gstm1-null
㪁
15
㪁㪁㪁
%
%
15
0 mg/kg
500 mg/kg
/
㪁㪁㪁
1000 mg/kg
Wild-type
Gstm1-null
FIG. 1. Blood concentrations of MetHB in the single-dose study of
DCNB. Blood concentrations of MetHB were measured 24 h after
a single-dose oral administration of DCNB to wild-type control or
Gstm1-null mice. Open, light gray, and dark gray bars indicate dose
levels of 0, 500, and 1000 mg/kg DCNB , respectively. The values
are depicted as the mean ⫾ S.D. of four or five mice per group.
Significant differences from the control (0 mg/kg) group by Dunnett’s test: ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
whose increasing ratio between Gstm1-null and wild-type mice (Gstm1-null/
wild-type) exhibited values greater than 2.5 are listed in the table.
Quantitative RT-PCR. Expression levels of mouse matrix metallopeptidase 9 (Mmp9), chitinase 3-like 1 (Chi3l3), triggering receptor expressed on
myeloid cells 3 (Trem3), membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3), lipocalin 2 (Lcn2), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) genes were measured by quantitative RT-PCR. For each
sample, 1 ␮g of total RNA was reverse-transcribed by SuperScript II using an
anchored oligo(dT) primer as described by the manufacturer (Invitrogen,
Carlsbad, CA). The resultant cDNA (1.0 ␮l) was used as the template in a
30-␮l PCR reaction containing 0.1 ␮M concentrations each of forward and
reverse gene-specific primers designed using Primer3 (Rozen and Skaletsky,
2000), 3 mM MgCl2, 1.0 mM dNTPs, 0.025 IU of AmpliTaq Gold, and 1⫻
SYBR Green PCR buffer (Applied Biosystems, Foster City, CA). PCR amplification was conducted in MicroAmp Optical 96-well reaction plates (Applied Biosystems) on an Applied Biosystems Prism 7000 Sequence Detection
System using the following conditions: initial denaturation and enzyme activation for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C
for 1 min. A dissociation protocol was performed to assess the specificity of
the primers and the uniformity of the PCR-generated products. Each plate
contained duplicate standards of purified PCR products of known template
concentrations covering 8 orders of magnitude to interpolate relative template
concentrations of the samples from the standard curves of log copy number
versus the threshold cycle. The copy number of each unknown sample per gene
was standardized to that of the Gapdh gene to control for differences in RNA
loading, quality, and cDNA synthesis. Primer sequences and amplicon sizes
are shown in Table 1.
TK Analysis for DCNB and Its Metabolite, M0. Fifty microliters of blood
cells were hemolyzed with 200 ␮l of distilled water and the hemolysate
obtained was used for the TK analysis. Fifty microliters of each plasma or
hemolysate sample was mixed with 150 ␮l of ethanol and centrifuged at 3000
rpm for 5 min at 4°C. To determine the concentration of DCNB, the supernatant was subjected to a high-performance liquid chromatography system
(Shimadzu Corporation, Kyoto, Japan) consisting of a system controller (SCL10A), a pump (LC-10AD), an autosampler (SIL-10AXL), a column oven
(CTO-10AC), and a UV detector (SPD-10A). The HPLC conditions for this
analysis were as follows: the column was an L-column ODS (150 ⫻ 4.6 mm
i.d.; Chemicals Evaluation and Research Institute, Tokyo, Japan); the column
temperature was 40°C; the mobile phase was water-acetonitrile-1 M ammonium acetate (55:45:1, v/v/v); the flow rate was 1.0 ml/min; the injection
volume was 10 ␮l; and the UV detector wavelength was 270 nm.
Statistical Analyses. The results are expressed as the mean ⫾ S.D. The
values of the mean and S.D. in each group were calculated with calculation
1548
ARAKAWA ET AL.
A
MetHB
Male
10
㪁
㪁
%
%
㪁
5
0
㪁㪁
5
Wild-type
Gstm1-null
Female
15
15
㪁㪁
10
㪁㪁
10
㪁
㪁㪁
0 mg/kg
㪁㪁
㪁㪁
%
%
150 mg/kg
300 mg/kg
RET
Male
150 mg/kg
300 mg/kg
5
600 mg/kg
0
Wild-type
C
Gstm1-null
Wild-type
RBC
Male
Female
15
Millions/µL
L
15
Gstm1-null
㪁㪁
5
0
㪁
10
0 mg/kg
㪁㪁
150 mg/kg
300 mg/kg
5
600 mg/kg
0
Wild t
Wild-type
G t 1 ll
Gstm1-null
Wild t
Wild-type
G t 1 ll
Gstm1-null
hematopoiesis was observed in both strains with a higher grade score
in the Gstm1-null mice (Table 2). Microarray analysis in the spleen
demonstrated genes whose expression levels increased in the Gstm1null mice (兩expression ratio to control兩 ⬎1.4) but not in the wild-type
mice (兩expression ratio to control兩 ⬍1.4) after DCNB treatment. All of
the microarray data are presented as Supplemental Table S1. Genes
whose increasing ratio between Gstm1-null and wild-type mice
(Gstm1-null/wild-type) exhibited a greater value than 2.5 are listed in
Table 3. From these genes, the representative five genes that are
related to hematopoiesis (i.e., Mmp9, Chi3l3, Trem3, Ms4a3, and
Relative spleen weight
Male
Female
1.0
1.0
㪁㪁
㪁㪁
㪁㪁
%
%
FIG. 2. Hematological examination in the 14-day repeated-dose
study of DCNB. Blood concentrations of MetHB (A), RET (B), and
RBC (C) were measured after a repeated-dose oral administration of
DCNB to wild-type control or Gstm1-null mice. Open, light gray,
dark gray, and filled bars indicate dose levels of 0, 150, 300, and
600 DCNB mg/kg, respectively. The values are depicted as the
mean ⫾ S.D. of four or five mice per group. The values of wild-type
male given 600 mg/kg and Gstm1-null female mice given 600
mg/kg are depicted as the mean of two animals and are excluded
from statistical analysis. Significant differences from the control (0
mg/kg) group by Dunnett’s test: ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ,
P ⬍ 0.001.
0.5
0.5
0.0
150 mg/kg
300 mg/kg
600 mg/kg
0.0
Wild-type
0 mg/kg
Gstm1-null
Wild-type
FIG. 3. Relative spleen weight in the 14-day repeated-dose study of
DCNB. Relative spleen weight was measured after a repeated-dose
oral administration of DCNB to wild-type control or Gstm1-null
mice. Open, light gray, dark gray, and filled bars indicate dose
levels of 0, 150, 300, and 600 DCNB mg/kg, respectively. The
values are depicted as the mean ⫾ S.D. of four of five mice per
group. The values of wild-type male mice given 600 mg/kg and
Gstm1-null female mice given 600 mg/kg are depicted as the mean
of two animals and are excluded from statistical analysis. Significant differences from the control (0 mg/kg) group by Dunnett’s test:
ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.
Gstm1-null
TABLE 2
Pathological grade of increased extramedullary hematopoiesis in the spleen after DCNB treatment for 14 days
Grade 0, no change compared with the control (0 mg/kg); grade 1, slight change; grade 2, moderate change; grade 3, marked change.
Dose Levels of DCNB
Grade
Male
Wild-type
Gstm1-null
Female
Wild-type
Gstm1-null
150 mg/kg
0
2
1
4
2
2
4
300 mg/kg
3
No. Examined
6
6
0
1
2
5
1
5
3
3
3
3
No. Examined
6
5
3
6
6
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
0
L
Millions/µL
0 mg/kg
㪁㪁
600 mg/kg
Gstm1-null
B
10
㪁㪁
0
Wild-type
5
Female
10
1549
METHEMOGLOBINEMIA INDUCED BY DCNB IN Gstm1-NULL MICE
TABLE 3
List of genes that were exclusively up-regulated in the spleen of female Gstm1-null mice after DCNB treatment for 14 days
Genes whose expression levels increased in the Gstm1-null mice (兩expression ratio to control兩 ⬎1.4) but not in the wild-type mice (兩expression ratio to control兩 ⬍ 1.4) after DCNB treatment
are extracted. In the table, genes whose increasing ratio between Gstm1-null and wild-type mice (Gstm1-null/wild-type) exhibited a value greater than 2.5 are listed. Expression levels of
underlined genes were also measured by quantitative RT-PCR.
DCNB/Control
Mmp8
Tm7sf3
1500009C09Rik
AA467197
Chi3l3 (Ym1)
Lcn2
Spag5
1100001G20Rik
Taf7
S100a9
Cenpa
Chi3l1
Mmp9
Ccdc74a
Trem3
2310061C15Rik
Hipk2
Cd177
Stfa2l1
Epx
S100a8
Ms4a3
Entrez Gene
17394
67623
76505
433470
12655
16819
54141
66107
24074
20202
12615
12654
17395
72315
58218
66531
15258
68891
268885
13861
20201
170813
Wild-Type
Gstm1-Null
0.93
1.23
1.00
0.93
0.93
1.07
1.07
1.07
1.07
1.00
0.87
1.15
0.87
0.87
0.93
0.87
1.32
1.23
0.93
0.76
1.15
1.15
7.46
9.19
6.50
5.66
4.92
4.92
4.59
4.29
4.00
3.73
3.03
3.48
2.64
2.46
2.64
2.30
3.48
3.25
2.46
2.00
3.03
3.03
Lcn2) were evaluated by quantitative RT-PCR. Quantitative RTPCR showed exclusive up-regulation in Gstm1-null mice after
DCNB treatment (Fig. 4), consistent with the results of microarray
analysis.
TK Monitoring. TK monitoring in plasma and blood cells was
performed in the wild-type and Gstm1-null mice in the satellite groups
of the 14-day repeated-dose study. DCNB concentrations in the
Gstm1-null mice on days 1 and 14 were higher than those in the
wild-type mice in both plasma and blood cells (Fig. 5, A and B). There
were no apparent changes in DCNB concentrations after repeated
dosing for 14 days. DCNB concentrations in blood cells were higher
than those in plasma, and the ratio of blood cells/plasma was similar
in both strains (Fig. 5C). Although a statistically significant increase
0
Re
elative expression (Arbitrary unit)
Re
elative expression (Arbitrary unit)
㩻
5
㩻㩺
10
5
0
Wild-type
Matrix metallopeptidase 8
Transmembrane 7 superfamily member 3
RIKEN cDNA 1500009C09 gene
Expressed sequence AA467197
Chitinase 3-like 3
Lipocalin 2
Sperm associated antigen 5
RIKEN cDNA 1100001G20 gene
TAF7 RNA polymerase II, TBP-associated factor
S100 calcium binding protein A9 (calgranulin B)
Centromere protein A
Chitinase 3-like 1
Matrix metallopeptidase 9
Coiled-coil domain containing 74A
Triggering receptor expressed on myeloid cells 3
RIKEN cDNA 2310061C15 gene
Homeodomain interacting protein kinase 2
CD177 antigen
Stefin A2 like 1
Eosinophil peroxidase
S100 calcium binding protein A8 (calgranulin A)
Membrane-spanning 4-domains, subfamily A, member 3
was observed in the wild-type females, the magnitude of the increase
was slight. Plasma M0 concentrations on days 1 and 14 in the
Gstm1-null mice were lower than those in the wild-type mice (Fig.
5D). M0 concentrations in blood cells were below the lower limit of
quantitation in most of the samples and could not be evaluated
appropriately.
Discussion
We investigated the effect of murine Gstm1 disruption on the
toxicological response to DCNB, a specific substrate to Mu class
GST, using Gstm1-null mice. In the single-dose study, a marked
increase in blood MetHB was observed in the Gstm1-null mice, and
the magnitude of the increase was greater than that in wild-type mice.
Gstm1-null
5
㪁
4
3
2
1
㩺
0
Wild-type
Gstm1-null
Lcn2
㩻㩺
10
5
0
Gstm1 null
Gstm1-null
Relative express
sion (Arbitrary unit)
Ms4a3
Relative express
sion (Arbitrary unit)
8.0
7.5
6.5
6.1
5.3
4.6
4.3
4.0
3.7
3.7
3.5
3.0
3.0
2.8
2.8
2.6
2.6
2.6
2.6
2.6
2.6
2.6
Trem3
15
Gstm1-null
15
Wild type
Wild-type
Gene Title
Chi3l3
M
Mmp9
9
10
Wild-type
Gstm1-Null/Wild-Type
20
㪁
15
0 mg/kg
10
300 mg/kg
5
0
Wild type
Wild-type
Gstm1 null
Gstm1-null
FIG. 4. Quantitative RT-PCR analysis
in the spleens of female mice in the
14-day repeated-dose study of DCNB.
Quantitative RT-PCR was performed
after a repeated-dose oral administration of DCNB to wild-type control or
Gstm1-null mice. Open and dark gray
bars indicate dose levels of 0 and 300
DCNB mg/kg, respectively. The values
are depicted as the mean ⫾ S.D. of four
or five mice per group and are expressed as an arbitrary unit, whose values of wild-type control groups are set
at 1 unit. Significant differences from
the control (0 mg/kg) group: $, P ⬍
0.05 (F test); ⴱ, P ⬍ 0.05 (Student’s t
test); #, P ⬍ 0.05 (Aspin-Welch t test).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
1449366_at
1442801_x_at
1452827_at
1434046_at
1419764_at
1427747_a_at
1427498_a_at
1434484_at
1441982_at
1448756_at
1441864_x_at
1451537_at
1416298_at
1445437_at
1460271_at
1427922_at
1429566_a_at
1424509_at
1442339_at
1449136_at
1419394_s_at
1420572_at
Gene Symbol
Re
elative expression (Arbitrary unit)
Affymetrix No.
1550
ARAKAWA ET AL.
㪁㪁
0
Day1
Female
㪁㪁㪁
㪁㪁
5
Wild-type
Gstm1-null
0
Day1
Day14
㪁㪁
30
㪁㪁
20
10
0
Day1
Day14
DCNB concentration (µg/mL)
D
Male
40
Female
㪁㪁㪁
30
㪁㪁㪁
20
Wild-type
10
Gstm1-null
0
Day1
Day14
Blood cells / Plasma ratio: DCNB
Male
Blood cells / Plassma ratio
10
5
0
Day1
y
10
㪁
5
Wild-type
Gstm1-null
0
Day14
y
D
Female
FIG. 5. DCNB concentrations in plasma (A), blood cells (B), and
the ratio of blood cells/plasma (C) after 2 h from the first (day 1) and
last (day 14) dosing in a 14-day oral administration of DCNB (300
mg/kg) to wild-type control or Gstm1-null mice. D, M0 concentrations in plasma after 2 h from the first (day 1) and last (day 14)
dosing in a 14-day oral administration of DCNB (300 mg/kg) to
wild-type control or Gstm1-null mice. Open and dark gray bars
indicate wild-type and Gstm1-null mice, respectively. The values
are depicted as the mean ⫾ S.D. of five mice per group. Significant
differences from the wild-type control by F-t test: ⴱ, P ⬍ 0.05; ⴱⴱ,
P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.
Day1
y
Day14
y
Plasma: M0
Male
2
1
㪁
㪁
0
Day1
Day14
2
Female
1
㪁
Wild-type
㪁㪁
Gstm1-null
0
Day1
Day14
Therefore, Gstm1-null mice were considered to be more predisposed
to methemoglobinemia induced by a single dosing of DCNB, and it is
suggested that the effect of Gstm1 disruption was reflected in methemoglobinemia in the single-dose study of DCNB. Because Gstm1-null
mice showed markedly low GST activity to DCNB (Fujimoto et al.,
2006) and higher exposure to DCNB in Gstm1-null mice was confirmed in TK monitoring, the predisposition to methemoglobinemia
observed in Gstm1-null mice in the single-dose study is considered to
be attributed to the higher exposure to DCNB.
In the 14-day repeated-dose study, in contrast, a marked increase in
blood MetHB was observed in both wild-type and Gstm1-null mice,
and the magnitude of the increase was similar in both strains. However, marked increases in blood RET, relative spleen weight, and
extramedullary hematopoiesis in the spleen were observed in Gstm1null mice. These changes were considered to be adaptive responses to
methemoglobinemia, because it has been reported that repeated-dose
studies of typical compounds that cause methemoglobinemia, such as
nitrobenzene (Cattley et al., 1994), p-nitrochlorobenzene (Nair et al.,
1986), and aniline (Hejtmancik et al., 2002), showed similar responses
in experimental animals. The increase in blood RET is a compensatory hematopoiesis response to elevation of damaged erythrocytes,
and the increase in relative spleen weight is attributed to sequestration
of damaged erythrocytes and extramedullary hematopoiesis. In addition, these adaptive responses were not observed in the single-dose
study of DCNB, and it has been reported that an increase in spleen
weight was not observed 24 h after dosing with aniline to rats (Khan
et al., 1997). Therefore, it is suggested that the adaptive responses
enhanced by the repeated dosing for 14 days in Gstm1-null mice
attenuated the higher predisposition to methemoglobinemia in the
single-dose study. In addition, a single dosing seems to be not enough
to show sufficient adaptive responses to methemoglobinemia. To get
an insight into the molecular mechanism of adaptive responses, microarray analysis was conducted in the spleen of female mice and
revealed that a number of genes were up-regulated exclusively in
Gstm1-null mice after DCNB treatment. Five hematopoiesis-related
genes, namely, Mmp9, Chi3l3, Trem3, Ms4a3, and Lcn2, were subjected to quantitative RT-PCR analysis to confirm their up-regulation,
which was observed exclusively in the Gstm1-null mice. It has been
reported that Mmp9 activation enhanced stem and progenitor cell
recruitment and was found to be an essential factor for hematopoiesis
(Heissig et al., 2002). The activation of the Mmp9-mediated response
is also suggested by the up-regulation of Chi3l3, which is a chitinase
family protein reported to be a substrate of Mmp9 and expressed in
macrophages (Greenlee et al., 2006). Another macrophage-expressed
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
DCNB concentration (µg/mL)
D
Day14
10
Blood cells: DCNB
40
C
Blood cells / Plassma ratio
㪁
5
B
M0 concentra
ation (µg/mL)
Plasma: DCNB
DC
CNB concentration (µg
g/mL)
Male
10
M0 concentra
ation (µg/mL)
DC
CNB concentration (µg
g/mL)
A
METHEMOGLOBINEMIA INDUCED BY DCNB IN Gstm1-NULL MICE
showed lower values than those of wild-type mice and the results of
the previous study were reproduced.
In conclusion, Gstm1-null mice showed marked methemoglobinemia and adaptive responses in single- and repeated-dose studies of
DCNB, respectively. These findings were considered to be based on
the higher exposure to DCNB due to Gstm1 disruption. It is suggested
that the effect of Gstm1 disruption was reflected in toxicological
responses related to methemoglobinemia. This study showed an example of a new approach to use genetically modified animals for the
evaluation of toxicities that depend on drug metabolism.
Acknowledgments. We thank Toshio Matsuura, Kiyomi Terada,
Yasushi Yamazaki, Noriyo Niino, and Makoto Tomida for their
technical assistance.
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mask the toxicities and careful interpretation might be necessary if
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toxicities.
In TK monitoring conducted in the satellite groups of the 14-day
repeated-dose study, DCNB concentrations on days 1 and 14 in
plasma and blood cells were higher in Gstm1-null mice than in
wild-type mice. In addition, it seems that the movement of DCNB
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mercapturic acid pathway and is eliminated into the bile or urine. In
the present study, plasma M0 concentrations in Gstm1-null mice
1551
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Address correspondence to: Dr. Shingo Arakawa, Medicinal Safety Research
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