[Cancer Research 60, 5080-5086, September 15, 2000]
© 2000 American Association for Cancer Research
Increased Mutant Frequency and Altered Mutation Spectrum of the lacI Transgene in Wilson Disease Rats with Hepatitis1
Hideko Sone2,
Ying Jie Li,
Mayumi Ishizuka,
Yasunobu Aoki and
Minako Nagao
Regional Environment Division [H. S., Y. J. L., M. I.] and Environmental Health Division [Y. A.], National Institute for Environmental Studies, Ibaraki 305-0053, and Tokyo University of Agriculture, Tokyo 156-8502 [M. N.], Japan
 |
ABSTRACT
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The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates
copper in the liver because of a mutation in the Atp7b
gene, encoding a copper-ATPase, is a model of Wilson disease. It
spontaneously develops hepatitis, and subsequently hepatocellular
carcinoma and cholangiofibrosis. Excess intracellular copper has been
thought to induce DNA damage through reactive oxygen species produced
by Cu (II)/Cu (I) redox cycling, and also by direct interaction with
DNA. We have developed lacI transgenic Wilson disease
(WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda
shuttle vector harboring the lacI gene.
lacI mutations of the livers of C-B heterozygous
(Atp7b w/m, lacI) and WND-B homozygous
(Atp7b m/m, lacI) rats at 6, 24, and 40
weeks of ages were analyzed. Mutant frequencies in the WND-B rats were
2.0 ± 0.7 x 10-5, 5.3 ± 0.9 x 10-5, and 5.3 ± 1.0 x 10-5, respectively,
significantly higher than those of C-B rats. Nucleotide sequence
analysis revealed that the frequency of deletion mutations of more than
two nucleotides were much higher, 15% in WND-B rats, but only 2% in
C-B rats. In addition, the average size of deletion was larger in the
former. Loss of oligonucleotide-repeat units was specific and
relatively frequent in WND-B rats. This type of mutation might be
implicated in the induction of DNA strand scissions by reactive oxygen
species. These findings suggest that the increase in mutant frequencies
and/or the specific type of mutation according to copper accumulation
play a crucial role in hepatocarcinogenesis in LEC rats.
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INTRODUCTION
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The LEC3
mutant rat developed at Hokkaido University accumulates copper in the
liver because of a mutation in the Atp7b gene encoding a
copper-ATPase (1, 2, 3)
. This genetic defect is the same as
that which exists in Wilson disease patients (4)
. Under
standard breeding conditions, the LEC rat develops hepatitis at around
20 weeks of age, and HCCs at around 18 months of age. Hepatitis
development has also been linked to copper accumulation in the studies
using F1 backcross rats (5
, 6)
. Further, administration of
copper chelating agents has been seen to prevent hepatitis development
and HCC development (7
, 8)
. It has been reported by us and
others that 8-OHdG (9)
,
1,N6-ethenodeoxyadenosine (varepsilon
dA), and 3,N4
-ethenodeoxycytidine
(varepsilon dC) DNA adducts in the livers are increased
(10)
, whereas levels of antioxidant, such as ascorbate and
ubiquinol in plasma, are decreased in LEC rats (11)
.
Etheno-adducts produced from other bases are also known to be produced
under oxidative conditions (12
, 13)
. Thus, it is
considered that the pro-oxidant status associated with copper
accumulation causes cellular damage through ROS produced by Cu (II)/Cu
(I) redox cycling. It has been reported that copper itself directly
interacts with DNA and results in DNA alterations (14
, 15)
. There is no evidence of infiltration of inflammatory cells
in LEC rat livers during hepatitis development, and no induction of
nitric oxide synthase (iNOS) has been
observed.4
In previous studies, levels of oxidative DNA damage, including 8-OHdG
and etheno-adducts, were shown to be higher in the acute phase of
hepatitis than before onset or during the chronic phase (9
, 10)
. Because the hepatocyte turnover rate also peaks with acute
hepatitis (16)
, DNA modifications caused by oxidative DNA
damage could be efficiently fixed as mutations.
Although 8-OHdG and etheno-adducts are known to produce mutations
in vitro (17, 18, 19)
, their roles in
vivo with regard to cancer development have not been well
elucidated. The LEC rat model has distinct advantages for clarifying
the role of oxidative DNA damage in hepatitis-hepatoma development. In
particular, analysis of the spectra of mutants induced during hepatitis
development might provide information on the types of mutation induced
in vivo by oxidative DNA damage.
In this study, we analyzed the MF in the livers of WND-B rats harboring
homozygous Atp7b mutations (Atp7b m/m) and the
lacI gene, with reference to hepatitis development. Further,
the mutational spectrum of the lacI mutants was analyzed.
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MATERIALS AND METHODS
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Animals.
Female and male Big Blue rats (F344 Tac[LIZd]; homozygous) at 6 weeks
of age were purchased from Stratagene (La Jolla, CA). The rats were
maintained at 24 ± 1°C with a 12-h light and dark
cycle and fed a diet (MF, Oriental Yeast, Japan) and tap water ad
libitum. Male and female LEC rats purchased from Charles River
Japan Inc. were mated with Big Blue rats. The F1 generation was then
mated with LEC rats again. A subset of rats that are homozygous for the
Atp7b mutation (Atp7b m/m) and harbor the
lacI gene, named WND-B rats, were used as the experimental
group. Another subset of rats that are heterozygous for the
Atp7b mutation (Atp7b w/m) and harbor the
lacI gene, named C-B rats, were used as a control group. For
detection of the lacI gene, which should be heterozygous,
dot blot analysis of tail DNA at 4–6 weeks of age was performed
according to the method previously reported (20)
.
Genotyping of each rat for Atp7b was performed by Southern
blot analysis of the tail or liver DNA with a cDNA probe of rWDF41R30.
In this analysis, the wild-type allele of the Atp7b gene
appeared as a single band, and the mutant allele showed no signals.
All animals were cared for and maintained in accordance with the
National Institute for Environmental Studies animal care guidelines.
Determination of lacI Gene MF.
Liver DNA extraction and transgenic lambda phage rescue were carried
out according to the manufacturer’s instructions (Stratagene, La
Jolla, CA). Briefly, liver DNA was packaged by mixing with a phage
packaging extracts, Transpack. Rescued phages were then plated on an
SCS-8 bacterial cell lawn in the presence of
5-bromo-4-chloro-3-indolyl-ß-D-galactoside, and
blue-colored plaques were counted as lacI mutants. MF was
obtained as the number of blue-colored plaques over the total number of
plaques. Blue plaques were isolated and subjected to mutation analysis.
Analysis and Classification of Mutations.
DNA was extracted by SM buffer from the blue plaques subcloned.
The lacI gene covering the coding and promoter regions was
amplified by PCR in a thermal cycler using a primer pair of
5'-GACACCATCGAATGGTGCAA-3' and 5'-TTCCACACAACATACGAGCC-3'. The PCR
products were subjected to restriction-single-stranded conformational
polymorphism analysis according to the method described by
Ushijima et al. (21)
. After locating the
mutation in either of the A-I fragments, the PCR products of the
fragment containing the mutation were then directly sequenced using ABI
388 or ABI 310 sequencers (Applied Biosystems, Japan). The jackpot
mutants were excluded to avoid the influence of clonal growth. When bp
deletion or insertion mutations were detected in the repeats of a
sequence, the first position in the 5' upstream site was assigned as
the mutation site.
Determination of Plasma GOT and GPT Levels.
GOT and GPT were determined using a Hitachi 736 autoanalyzer (Hitachi
Tokyo, Japan).
Statistical Analysis of Data.
Statistical analyses of MF data and mutation spectra were carried out
by the t test and 
2 test using
STATVIEW version 4.5 (Abacus Concepts, Inc., Berkeley, CA),
respectively.
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RESULTS
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MF in the lacI Gene of the Liver.
The results for MF in the lacI gene of the livers of C-B
(Atp7b w/m, lacI) and WND-B (Atp7b
m/m, lacI) rats at 6, 24, and 40 weeks of age are
summarized in Table 1
. MFs in the C-B were 1.3 ± 0.3 x 10-5 at 6 weeks of age,
and increased with age, culminating at 2.4 ± 1.2 x 10-5
at 40 weeks, in line with values reported for F344 Big Blue rats
(22
, 23)
. The MF in the WND-B rats was 2.0 ± 0.7 x 10-5 at 6 weeks, slightly
but significantly higher than that of C-B rats. Plasma levels of GOT
and GPT as markers of hepatitis onset, however, were not elevated. At
24 weeks of age, MF in the WND-B rats was 5.3 ± 0.9 x 10-5, 2.4-times the C-B
value (2.2 ± 0.7 x 10-5) and plasma levels of
GOT (417 IU/liter) and GPT (317 IU/liter) were much higher. At 40 weeks
of age, when the WND-B rats were in the chronic phase of hepatitis, MFs
in the livers were almost the same as that at 24 weeks.
Mutational Spectra of the lacI DNA Sequence.
The DNA sequences of a total of 200 lacI mutants isolated
from the livers of C-B rats (47 at 24 weeks and 52 at 40 weeks) and
WND-B rats (49 at 24 weeks and 52 at 40 weeks) were analyzed, and 186
independent mutations (95 of C-B and 91 of WND) were detected. The
mutational types and locations of all mutants are listed in Tables 2
3
4
5
, and a summary of mutational types is given in Tables 6
and 7
. The majority of the recovered mutations in both genotypes were base
substitutions (C-B, 81%; WND-B, 78%), giving rise to stop codons or
amino acid substitutions (Tables 2
, 3
, 6
, and 7)
. The others were all
simple deletions or insertions of 1–358 bp (Tables 4
and 5)
. Because
mutational types did not principally differ between 24 and 40 weeks in
either C-B or WND-B rats (Tables 6
and 7)
, a comparison between C-B and
WND-B was made for the total mutations at 24 and 40 weeks.
The most frequent mutations were G:C to A:T transitions in both strains
with frequencies of 41% and 49% in C-B and WND-B, respectively. They
were mostly present at CpG sites with frequencies of 62% and 87% of
the total G:C to A:T mutations in C-B and WND-B, respectively (Tables 6
and 7)
, the difference being significant (P = 0.0164).
A:T to G:C transitions were observed with a significantly higher
frequency in C-B (P = 0.0121). Further, it is
worthy to note that mutations at the A:T site, including transitions
and transversions, were significantly more prevalent in C-B than in
WND-B, with frequencies of 24% versus 10%, respectively
(P = 0.0184).
Total frequencies for the frameshifts (one or two bp), deletion (more
than two bp), and insertions (more than two bp) were almost the same in
C-B and WND-B rats, being 19% (18 of 95) and 22% (20 of 91) of the
total, respectively, as shown in Tables 6
and 7
. Frameshift mutations
were more frequent in C-B (13 of 18; 72%) than in WND-B (6 of 20;
30%). In contrast, only 5 (5%) deletion and insertion mutations
ranging from 4 to 358 bp were found in C-B, but 14 (16%) were found in
WND-B rats. Of these, one C-B but nine WND-B mutants involved >10 bp
deletions. Thus, a tendency toward large deletion mutations was seen in
WND-B rats, with the average size of 71 bp in WND-B, in contrast to 16
bp in C-B rats.
Mutational hot spots, defined as more than three mutations, were
detected at nucleotide positions 92, 329, and 791 in C-B rats and 92,
95, 131, 180, and 329 in WND-B rats, with totals of 10 and 18
mutations, respectively. All these sites were CpG, and 8 of 10 and 17
of 18 mutations in C-B rats and WND-B rats, respectively, were G:C to
A:T transitions.
Insertion of 5'-CTGG-3' was observed twice in C-B rats and once in a
WND-B rat, at nucleotide positions 620–623 where a three-consecutive
repeat of 5'-(CTGG)3-3' exists. In contrast,
three deletion mutations, with loss of one of the three repeats, were
detected in WND-B rats but none in C-B. In the two strains of rats, 5
of 19 insertion and deletion mutations were at nucleotide number 620,
indicating the 5'-CGT(CTGG)3 CAT-3' to be a
target in both C-B and WND rats.
Other characteristic mutations were also found: A mutation in C-B rats
(plaque no. 665) had an ATGCG insertion resulting in a repeat of this
sequence. Another mutation in WND-B rats (plaque no. 99113) was
implicated with a palindrome structure composed of an inverted 6 bp
separated by 45 bp, while 46 bp were deleted.
|
DISCUSSION
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The present study demonstrated the MF in the liver of WND-B rats
to be 1.5 times higher than that in the C-B rat, even before the onset
of hepatitis at 6 weeks of age. At this age, the copper level in the
WND-B rat liver was much higher, 65.3 µg/g wet tissue
(n = 8; range, 27–95), than in the C-B case
(15.8 µg/g wet tissue; n = 5; range,
5.4–26.4
Thus, the higher MF in the WND-B
rat could have been attributable to accumulation of copper, which is
known to induce mutations by direct interaction with DNA or through
production of ROS. The BrdUrd labeling index of LEC rats at 6
weeks of age is the same as that of the wild-type rat
(24)
, suggesting that cell proliferation itself played no
major role in the difference in MF. The MF ratio of WND-B:C-B was
increased to 2.4 at 24 weeks, when hepatitis had developed in the WND-B
rats, with high plasma GOT and GPT levels. Some Atp7b m/m
rats in fact died of fulminant jaundice at around 21 weeks. At 24 weeks
of age, the levels of copper in WND-B rats were highest, with an
average of 200 µg/g (n = 7; range,
113–275), in line with data for oxidative DNA damage (9
, 10)
and cell proliferation rate (2)
. Thus, the DNA
lesions would be expected to be efficiently fixed as mutations. Among
WND-B rats at 24 weeks of age, there was a positive correlation between
the copper levels and MF (r = 0.398);
however, no correlation between plasma GOT/GPT levels and MF
(r = -0.231) was observed. At 40 weeks of
age, the MF values in C-B and WND-B were the same as those at 24 weeks,
and this lack of increase might be partly explained by the lower levels
of copper [178 µg/g (n = 7; range,
31–301)], DNA adducts (9
, 10)
, and cell proliferation
rate (2)
at 24 weeks. All LEC rats surviving the acute
phase of hepatitis develop HCCs. Thus, the increased MF occurring in
the acute phase could play important roles in hepatocarcinogenesis,
along with signal transduction induced by ROS, such as through the
nuclear factor 
B pathway (25)
.
Ratios of base substitution mutations did not basically differ between
C-B and WND-B rats. However, some differences were observed in
mutational types: mutations at A:T sites were significantly decreased,
and G
A transitions at the CpG site were significantly increased in
WND-B rats. Additionally, large deletions were observed at a high
frequency in WND-B rats. Recently it was found that 2-OHdA is produced
70–80 times more efficiently in the nucleotide pools than on DNA.
2-OHdA is misinserted opposite dC to give G:C to A:T transitions in
subsequent DNA replications (26)
. Our experimental
results, showing a higher tendency for mutation of G:C to A:T in WND-B
rats than in C-B rats, suggest 2-OHdA as a possible cause. The
predominant base substitutions produced by incubation of
single-stranded M13mp2DNA with Cu were CT and GT
(27)
. Thus, involvement of direct interaction of copper
with DNA in base substitution mutation cannot be ruled out. ROS
produced by H2O2 or metals,
including Fe2+, Cu2+, and
Ni2+, in contrast, is reported to induce CC to
TT mutations (27
, 28) ; ROS produced by bleomycin is
associated with single bp deletions at hot spots of 5'-GTC-3' or
5'-GCC-3' in CHO cells (29)
. However, no such bp
substitutions or preferential sites for frameshift were observed
here in either C-B or WND-B rats. The oxidative stress in this model
system might be attributable to another type of ROS, which resembles
spontaneously accumulated mutations during aging.
It has been reported that singlet oxygen contributes to strand breakage
by lead acetate
(Pb(CH3COO)2; Ref.
30
), and that cobaltous chloride
(CoCl2) induces deletion mutations, specifically
at direct repeat sequences in Escherichia coli
(31)
. There were only two deletions at direct repeat
sequences in C-B but four in WND-B rats, and one at an inverted repeat
sequence in WND-B rats. Further, some mutation spectra of mutant
plaques were not determined because their lacI gene fragment
could not be amplified by PCR, suggesting that these plaques might
include some large deletions.
In conclusion, the present results suggest that the increase of MF and
changes in the mutational spectrum in WND-B rats are attributable to
DNA damage induced by copper accumulation itself and/or associated
oxidative stress. In addition, we hypothesize that the remarkable
increase in MF in the LEC rat liver may play a crucial role in its
hepatocarcinogenesis.
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ACKNOWLEDGMENTS
|
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We thank Dr. Michihiro C. Yoshida of Hokkaido University for
providing rWDF41R30; Drs. Hitoshi Nakagama and Toshikazu Ushijima
(National Cancer Center Research Institute) for helpful discussions;
and Hiromi Takanaga and Naoko Tetsura (National Institute for
Environmental Studies) for support in performing the experiment.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by a Grant-in-Aid for Cancer Research
from the Ministry of Health and Welfare of Japan, and by a grant from
the Ministry of Education, Sciences, Sports and Culture, Japan. 
2 To whom requests for reprints should be
addressed, at Regional Environment Division, National Institute for
Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan.
Fax: 81-298-50-2571; E-mail: hsone{at}nies.go.jp
3 The abbreviations used are: LEC,
Long-Evans Cinnamon (rat); HCC, hepatocellular carcinoma; MF, mutant
frequency; C-B rat, rat heterozygous for the WND gene (Atp7b
w/m) with lacI gene; WND-B rat, rat homozygous
for the WND gene (Atp7b m/m) with lacI
gene; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; ROS, reactive oxygen
species; GOT, glutamate-oxaroacetate transaminase; GPT,
glutamate-pyruvate transaminase.
4 Unpublished data.
Received 2/28/00.
Accepted 7/20/00.
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ABSTRACT
INTRODUCTION
