The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7bgene, 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 × 105, 5.3 ± 0.9 × 105, and 5.3 ± 1.0 × 105, 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.

The LEC3mutant 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.

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-β-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 χ2 test using STATVIEW version 4.5 (Abacus Concepts, Inc., Berkeley, CA),respectively.

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 × 105 at 6 weeks of age,and increased with age, culminating at 2.4 ± 1.2 × 105at 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 × 105 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 × 105, 2.4-times the C-B value (2.2 ± 0.7 × 105) 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.

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/mrats 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 κB 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 C→T and G→T(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.

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.

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 lacIgene; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; ROS, reactive oxygen species; GOT, glutamate-oxaroacetate transaminase; GPT,glutamate-pyruvate transaminase.

4

Unpublished data.

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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