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1 International Agency for Research on Cancer (IARC), Lyon, France,
2 Cancer Research United Kingdom Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A) being an initiating lesion in the development of these tumors. As alkylpurine-DNA-N-glycosylase (APNG) releases A from DNA in vitro, wild-type and APNG-/- C57Bl/6J mice were treated with Vcar and levels of A and 3,N4-ethenocytosine (C), which is not a substrate of APNG, were analyzed in liver and lung DNA. At 6 h after the last dose, levels of A were 1.6-fold higher in DNA from APNG-/- mice and subsequently persisted at higher levels for longer than in DNA from wild-type animals, confirming that A is released by APNG in vivo. In contrast, 
14-fold lower levels of C were induced by Vcar, and the kinetics of formation and persistence of C was similar in the two mouse strains. The carcinogenicity of Vcar was compared in APNG-/- and wild-type suckling mice given a single dose of Vcar (30 or 150 nmol/g). After 1 year, only mice in the high-dose group developed hepatocellular carcinoma; however, the incidence was not higher in APNG-/- mice. Although higher levels and increased persistence of A was observed in hepatic DNA from APNG-/- mice at 150 nmol/g Vcar, apoptosis and cell proliferation levels were similar in both strains of mice. This may explain why differences in A formation/persistence observed here did not result in higher susceptibility of APNG-/- mice to hepatocarcinogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, C, and N2,3-ethenoguanine. These four DNA adducts have been detected in vivo after exposure of rodents to urethane, Vcar or Vcar epoxide (reviewed in Ref. 8
). In addition, background levels of these three etheno adducts have been detected in DNA from unexposed humans and rodents (9)
, and are thought to be generated by the lipid peroxidation product, 4-hydroxy-2,3-epoxynonanal (10)
.
Ethenobases lead to point mutations in site-directed mutagenesis assays, or in kinetic or primer extension assays and can induce bp substitution mutations in vitro, in Escherichia coli, and in mammalian cells (reviewed in Ref. 8
). The mutations induced by urethane or Vcar in the ras genes of mouse tumors are compatible with the promutagenic properties of ethenobases. In particular, a high proportion of skin, liver, and lung tumors induced by urethane or Vcar in mice contain a Ha-ras or Ki-ras gene activated through an AT
TA transversion at the 2nd base of codon 61 (11, 12, 13)
. Furthermore, this mutation has been detected in preneoplastic lesions (14, 15, 16)
and in target tissues very early after treatment with Vcar (17
, 18)
, suggesting the formation of an initiating DNA lesion; A could be this initiating lesion, because it has been shown to induce AT TA transversions in mammalian cells (19)
.
Tumor initiation ultimately depends on the relative rates of formation and repair of the promutagenic lesions, and of cell proliferation and apoptosis. Ethenobases are repaired through the base excision repair pathway, involving different DNA N-glycosylases. In vitro, A is excised by the human or rodent APNG, whereas C is cleaved by the human mismatch-specific thymine DNA-N-glycosylase (20
, 21)
. However, the repair pathways and repair kinetics of etheno adducts have not yet been investigated in vivo. Knockout mice deficient in the APNG protein have been generated previously (22)
, and cell-free tissue extracts from APNG-/- mice lack any activity toward oligonucleotides containing A, but retain activity for C and 8-oxoguanine (23)
. In this work, we used APNG null mutant mice to investigate the role of base excision repair in the clearance of A from DNA in vivo and to obtain additional insight into the role of this ethenobase in Vcar-induced carcinogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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32 P]ATP (specific activity >5000 Ci/mmol) and T4 polynucleotide kinase were obtained from Amersham Biosciences (Orsay, France). Micrococcal nuclease and calf spleen phosphodiesterase were obtained from Worthington (Lakewood, NJ) Polyethyleneimine-cellulose plates (Polygram CEL 300 PEI) for TLC were obtained from Macherey-Nagel (Duren, Germany). Kits for TUNEL and BrdUrd labeling were from Roche Molecular Biochemicals (Mannheim, Germany). Vectashield D mounting medium was obtained from Vector Laboratories (Burlingame, CA). All of the other chemicals were from Sigma-Aldrich.
Animals.
APNG-/- mice were generated as described previously (22)
and backcrossed onto a C57BL/6J background for nine generations. The APNG-/- and APNG+/+ mice used in these experiments were obtained by intercrossing APNG heterozygotes. Mice were genotyped by PCR as described previously (22)
. All of the animal procedures were performed in accordance with the Animals (Scientific Procedures) Act 1986, in the United Kingdom.
For the study on the formation and persistence of etheno adducts, 6-week-old male and female mice received five daily i.p. injections of 250 nmol/g of Vcar dissolved in 0.9% saline. Controls were treated with saline. Mice were sacrificed at different time intervals (from 6 h to 96 h) after the last dose. Liver and lung were collected, snap frozen in liquid nitrogen, and stored at -80°C until analysis.
For the carcinogenesis experiment, 14–15-day-old male mice were treated with a single i.p. dose of 30 or 150 nmol/g Vcar dissolved in 0.9% saline. Control animals received a single i.p. injection of saline. As the mice were genotyped after weaning, the numbers of mice in each group was not identical (Table 1)
. Mice were sacrificed after 1 year and examined for tumor development.
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A and C.
DNA Isolation.
DNA was purified from tissues using a phenol-chloroform extraction method. Tissues were disintegrated in a Potter-Elvejehm homogenizer in 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, containing 0.5% (w/v) SDS (10–15 ml/g of tissue). After addition of a solution of RNases (RNase A, 15 units/ml of homogenate; RNase T1, 95 units/ml), the homogenate was incubated at 37°C for 1 h. Proteinase K was added (0.3 mg/ml homogenate) and the mixture incubated overnight at 37°C. After removal of undigested debris, proteins were extracted sequentially with buffer-saturated phenol (pH 7.5–7.8), buffered phenol-chloroform-isoamyl alcohol (25:24:1, v/v) and chloroform-isoamyl alcohol (24:1 v/v). DNA was precipitated by the addition of 1/10 volume of 5 M NaCl and 1 volume cold, absolute ethanol, and then collected on the (closed) tip of a Pasteur pipette. The precipitate was rinsed twice in cold, 70% ethanol, and after gentle air drying, the DNA was redissolved in water. A second digestion of RNA with RNases A and T1 was then performed, and the DNA precipitated and washed as above. After drying, the DNA was redissolved in 500 µl of water. For liver DNA, glycogen was removed by ultracentrifugation at 100,000 x g for 1 h at 4°C. DNA samples were stored at -80°C until analysis.
Analysis of A and C in DNA.
A and C were analyzed by immunoaffinity purification and 32P-postlabeling (9)
. In brief, 25 µg of DNA was hydrolyzed to nucleoside 3'-monophosphates using micrococcal nuclease and calf spleen phosphodiesterase. Normal nucleotides were quantified by high-performance liquid chromatography of part of the digest, and the ethenonucleotides were enriched on immunoaffinity columns prepared using the monoclonal antibodies EM-A-1 (anti-dA) and EM-C-1 (anti-dC; Ref. 25
) coupled to Protein A-Sepharose CL-4B with dimethyl pimelimidate (26)
. The adducts and the internal standard, deoxyuridine 3'-monophosphate, were labeled with [32P]ATP and T4 polynucleotide kinase before resolution by two-dimensional TLC on polyethyleneimine-cellulose plates. Elution was achieved in 1 M acetic acid (pH 3.5) in the first dimension and in saturated ammonium sulfate (pH 3.5) in the second dimension. The adduct spots and the internal standard were measured with a PhosphorImager (Molecular Dynamics, Paris, France). Etheno adduct levels were calculated as molar ratios of ethenobase per 108 parent bases in DNA. At least three samples were analyzed per experimental group, and each sample was analyzed at least twice.
Apoptosis.
Apoptotic hepatocytes were detected in liver sections using the TUNEL assay. Formalin-fixed liver sections embedded in paraffin were dewaxed, rehydrated, and submitted to a protease treatment [Proteinase K; 10 µg/ml in 10 mM Tris-HCl (pH 7.6); 30 min at 37°C]. Slides were rinsed twice in PBS, and after addition of 50 µl TUNEL reaction mixture (containing terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP), slides were incubated in the dark in a humidified atmosphere for 60 min at 37°C. Negative controls were incubated with 50 µl of label solution (without terminal transferase) instead of TUNEL reaction mixture. Positive controls consisted of liver tissue sections from mice treated with tumor necrosis factor 
. Slides were then rinsed three times in PBS and covered with a drop of Vectashield D mounting medium, containing 4',6-diamidino-2-phenylindole for nuclear staining. Double-stained sections were analyzed by fluorescence microscopy (Axioplan 2; Zeiss, Le Pecq, France). Observations were made at excitation wavelengths of 360 nm and 488 nm, with an emission wavelength in the range of 515–565 nm. For each sample, 100 cells were examined (at a magnification of x400) per field, four fields per tissue section, and three sections per sample.
Cell Proliferation.
Cell proliferation was measured by incorporation of BrdUrd in DNA and detection using a monoclonal antibody against BrdUrd. Mice were sacrificed 1 h after an i.p. injection of 1 µmol/g of BrdUrd (dissolved in PBS). Liver was fixed in formalin and embedded in paraffin. After dewaxing, specimens were rehydrated in PBS, treated with 4 M HCl for 20 min, dipped in a 50 mM borax-boric acid buffer (pH 7.6) for 10 min, and rinsed twice in PBS. They were then incubated with proteinase K [15 µg/ml in 10 mM Tris-HCl (pH 7.6)] for 30 min at 37°C and dipped into a 2.5% solution of BSA in PBS for 1 h. After careful drying of the zone around the area to be stained, sections were covered with a solution of anti-BrdUrd mouse monoclonal antibody (containing nucleases for DNA denaturation) and incubated for 30 min at 37°C in a humid atmosphere. After washing in PBS, sections were covered with a solution of antimouse-immunoglobulin-fluorescein and slides incubated for an additional 30 min at 37°C. Slides were then washed three times in PBS and examined by fluorescence microscopy with the same settings as above. The percentage of BrdUrd-incorporating hepatocytes was scored for each liver specimen, after the same procedure adopted for counting apoptotic cells.
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RESULTS |
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A and C in Juvenile Mice Treated with Vcar.A and C were measured in liver and lung DNA from 6-week-old mice after 5 daily i.p. injections of Vcar. Control animals consisted of mice injected with saline and sacrificed after 24 h. Data obtained from male and female mice were pooled, as there was no sex difference in either adduct formation or persistence. Background levels of A in control mice, expressed as molar ratios of A to A (x10-8), were: for liver DNA, 1.83 ± 1.16 (n = 6) in APNG+/+ mice and 2.45 ± 1.26 (n = 6) in APNG-/- mice; and for lung DNA, 1.23 ± 0.45 (n = 3) in APNG+/+ mice and 2.06 ± 0.71 (n = 3) in APNG-/- mice. Six h after the last dose, high levels of A were formed in liver DNA (Fig. 1)
. At this time point, APNG-/- mice exhibited 1.6-fold more A than wild-type animals (P < 0.05, unpaired t test): the molar ratio A:A was 42 ± 15 x 10-8 (n = 8) in wild-type and 66 ± 26 x 10-8 (n = 9) in knockout mice. At longer time intervals, the level of A decreased in both strains although at a slower rate in the APNG-/- mice. At 96 h, the molar ratio A:A reached background values in APNG+/+ mice (1.77 ± 0.52 x 10-8; n = 3) but was still relatively high (15.8 ± 4.8 x 10-8; n = 3) in APNG-/- mice. The estimated half-life of A in hepatic DNA was 21 ± 3 h in wild-type mice and 44 ± 3 h in knockout animals. Similar data were obtained for lung DNA (Fig. 2)
. At 6 h, the molar ratio A:A x 10-8 was 43 ± 12 (n = 6) in APNG+/+ mice and 77 ± 20 (n = 5) in APNG-/- mice (significantly different, P < 0.01, unpaired t test); at 96 h, this ratio was 1.43 ± 0.50 (n = 3) and 29.70 ± 3.50 (n = 3), respectively. The half-life of A in lung DNA was 20 ± 3 h in APNG+/+ mice and 65 ± 7 h in APNG-/- mice. Compared with A, Vcar formed very low levels of C. In liver DNA (Fig. 3)
, the molar ratio C:C at 6 h after treatment was 5.86 ± 2.43 x 10-8 (n = 7) in wild-type mice and 5.73 ± 2.82 x 10-8 (n = 7) in knockout mice; background values were 3.10 ± 1.39 x 10-8 (n = 6) and 3.43 ± 2.03 x 10-8 (n = 6), respectively. The molar ratio C:C measured at the other time points was close to the background values in both strains (Fig. 3)
. Therefore, there was no significant difference between the wild-type and the APNG-deficient mice with regard to the formation and persistence of C in liver DNA. Similar observations were made in lung DNA (data not shown).
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. At the lower dose, no Vcar-induced tumors were observed in either tissue after 1 year (data not shown). This confirms the results of Stanley et al. (27)
, who found that liver tumors were not observed until after 1 year of age in C57Bl/6J mice injected neonatally with 30 mol/g Vcar. Therefore, at this dose of Vcar, our results indicate that the lack of APNG did not increase the induction of liver tumors in this strain of mice over the time scale studied.
A similar outcome was observed at the higher dose of Vcar (Table 1)
. Although the numbers of animals are too few to obtain significance, APNG-deficient mice tended to be more resistant to tumor induction by Vcar than their normal littermates. Thus, whereas the livers from all of the APNG+/+ mice exhibited some pathological lesion, 5 of 21 (24%) of the APNG-/- mice exhibited no abnormalities (Table 1)
. However, Vcar tumor induction between wild-type and APNG-/- was not significantly different when either the number of hepatocellular carcinomas alone (P = 0.11, Fisher’s exact test) or in conjunction with basophilic adenomas and basophilic foci (P = 0.086, Fisher’s exact test), both precarcinoma lesions (28)
, are taken into account. No abnormalities were observed in the lungs of any of the mice treated with the higher dose of Vcar.
Effects of Vcar on Cell Cycle and DNA Damage in Hepatocytes from Suckling Mice.
Fourteen-day-old mice received a single dose of Vcar (150 nmol/g), and were sacrificed after 6, 24, and 48 h.
Apoptosis.
Apoptosis was measured in liver tissue sections using the TUNEL assay. Vcar treatment induced apoptosis in 15–18% of the hepatocytes in both APNG+/+ and APNG-/- mice (Fig. 4)
.
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, when 17% of hepatocytes were proliferating in both the wild-type and knockout mice. In liver from control mice, 3% of hepatocytes were in proliferation.
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A.A and C were measured in liver DNA from APNG+/+, APNG+/-, and APNG-/- mice after a single dose of 150 nmol/g of Vcar. After 6 h, the mean molar ratio of A:A x 10-8 was 52 ± 8 (n = 4) in APNG+/+ mice, 48 ± 6 (n = 4) in APNG+/- mice, and 76 ± 9 (n = 3) in APNG-/- mice (Fig. 6)
. After 24 h, this ratio decreased to 16 ± 5 (n = 4) in APNG+/+ mice, 21 ± 7 (n = 3) in APNG+/- mice, and 54 ± 17 (n = 4) in APNG-/- mice. Therefore, at both time points the level of A was significantly higher in APNG knockout mice compared with APNG+/+ and APNG+/- mice (P < 0.01, Tukey-Kramer Multiple Comparisons Test). In contrast, C levels in Vcar-treated mice did not differ between genotypes and were only marginally above background values (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A in vivo and to determine whether defective repair of A could enhance tumor induction by Vcar.
Firstly, we measured the formation and persistence of A (repaired by the APNG protein) and C (not repaired by APNG) in two known target organs for Vcar-induced carcinogenesis in juvenile mice, of wild-type and APNG-/- mice after repeated administration of Vcar (2)
. Fernando et al. (29)
were the first to demonstrate the formation of A and C in liver and lung DNA from mice exposed to Vcar. Our data show that A is a major promutagenic lesion of Vcar, being formed at 14-fold higher levels than C in liver DNA in vivo (Figs. 1
and 3
). It is not known whether this difference results from the intrinsic reactivity of Vcar epoxide toward DNA bases and/or from a very efficient repair of C. Park et al. (7)
analyzed the adducts formed in reactions of Vcar epoxide with double-stranded DNA and found that N2,3-ethenoguanine and A were formed at 4.4% and 0.6%, respectively, of the level of 7-(2-oxoethyl)guanine, the major DNA adduct. In rodents treated with Vcar or Vcar epoxide, the level of N2,3-ethenoguanine was between 12% and 24% that of 7-(2-oxoethyl)guanine (7)
. The formation of high levels of A (4 A residues per 107 adenine bases in DNA) in two target organs for carcinogenesis is additional evidence that A could be involved in the generation of AT TA transversions in the ras genes in mouse tumors associated with exposure to Vcar or to urethane (11, 12, 13)
.
Greater than 1.5-fold higher levels of A were formed in liver and lung DNA from APNG-/- mice, compared with APNG+/+ animals (Figs. 1
and 2
; 6-h time point), and these decreased with time more slowly in the knockout mice. At 96 h, the A:A ratio was similar to the background value in wild-type mice, but was still significantly higher in both liver (9-fold) and lung (20 fold) DNA from APNG-/- mice. In contrast, the kinetics of formation and persistence of C in DNA was similar in both strains of mice (Fig. 3)
. These results show that APNG and the base excision repair pathway are involved in the repair of A in mice, thus confirming observations obtained in vitro (20
, 21
, 23)
. The residual clearance of A from the DNA of APNG-/- mice may be due to an alternative repair system and/or to dilution of the adduct through cell apoptosis or proliferation. Analysis of DNA from young and aged APNG-/- and APNG+/+ mice failed to reveal an accumulation of A with age in both strains (i.e., arising from the continuous formation from endogenous lipid peroxidation products), suggesting the existence of an alternative system for the repair of this lesion (data not shown).
To examine whether A could be involved in the initiation of carcinogenesis by Vcar, we compared tumor development in wild-type and APNG-/- male mice, after a single dose of Vcar at 14–15 days of age. Using the treatment schedule for C57BL/6J mice reported by Stanley et al. (27)
, we treated the mice with either a low (30 nmol/g) or a high (150 nmol/g) dose of Vcar and sacrificed the mice 1 year after treatment. At the low dose no liver tumors were observed in any of the treated mice. The comparatively short duration of our experiment, which was used to avoid an increased yield in spontaneous tumors, may in part explain why we did not observe liver tumors in the low-dose group. It is clear, however, that the lack of APNG did not increase the sensitivity of the C57Bl/6J mice to Vcar-induced hepatocarcinogenesis over the time scale studied. This was somewhat surprising, because nearly 50% of the liver tumors in the earlier low-dose study had either AT or AG mutations at Ha-ras codon 61, base changes that could be attributed to A (27)
.
At the higher dose, Vcar induced many hepatic lesions, including hepatocellular carcinoma (Table 1)
. Again, APNG-/- mice were not more susceptible to tumor induction by Vcar than wild-type mice under these experimental conditions. They even yielded less liver tumors than the repair-competent animals, but the difference was not statistically significant due to the relatively low number of mice examined. No tumors were observed in other tissues, except a few lymphoma in liver, which were not treatment-related. The C57BL/6 genetic background of the mice used in this work is known to be relatively resistant to spontaneous and chemical carcinogenesis, especially in the liver and lung (6
, 27
, 30)
. More recently, Takahashi et al. (28)
compared Vcar-induced carcinogenesis in five strains of mice, including C57BL/6 mice, using the same protocol over a 2-year experimental period. They also observed essentially liver lesions. The prevalence of hepatocellular carcinoma after 2 years was 0.6% in control C57BL/6 mice, 18.7% in mice treated with the low dose, and 22.5% in mice treated with the high dose of Vcar. The latter value is very close to the incidence we measured in APNG+/+ mice exposed to 150 nmol/g of Vcar (Table 1)
.
We investigated factors other than the formation of etheno DNA adducts, which could be involved in Vcar-induced hepatocarcinogenesis. We examined the effects of Vcar on cell cycle in liver under the same conditions as used for the carcinogenesis experiment. A single dose of 150 nmol/g of Vcar induced both apoptosis and proliferation of hepatocytes. Fifteen to 18% of the hepatocytes underwent apoptosis between 6 and 24 h after treatment (Fig. 4)
. This was followed by a wave of cell regeneration, with 15–20% of the hepatocytes replicating at 24 h after Vcar administration (Fig. 5)
. For both biological end points, the response of APNG-/- mice was similar to that of wild-type mice. Etheno adducts were also measured in liver DNA from APNG+/+, APNG+/-, and APNG-/- mice under the same experimental conditions, i.e., after a single dose of 150 nmol/g of Vcar at 14 days of age (Fig. 6)
. Etheno adducts were found to reach their highest levels 6 h after treatment (data not shown). At this point, the molar ratio A:A was similar in APNG+/+ and APNG+/- mice, amounting to 5 x 10-7, but 1.5-fold higher in APNG knockout mice. After 6 h, A levels again decreased more rapidly in wild-type and heterozygote mice compared with APNG-/- mice. At 24 h, the level of A in DNA was 3-fold higher in the knockout animals compared with the two other strains. These analyses thus confirm the results obtained on juvenile mice after repeated treatment with Vcar. In addition, they show that APNG heterozygotes have the same capacity to repair A as wild-type mice.
The two adducts measured in this study represent only a small part of the spectrum of DNA damage generated by Vcar. Using the data from Park et al. (7)
and a level of 5 x 10-7 A per adenine, we can estimate that a dose of 150 nmol/g of Vcar should also induce 4 x 10-6 N2,3-ethenoguanine and 8 x 10-5 7-(2-oxoethyl)guanine residues per guanine. Because Vcar is oxidatively metabolized by cytochrome P450 2E1 (31)
, this can result in oxidative damage to DNA, especially in the liver, which has the highest cellular level of P450 2E1 and is, thus, the major tissue for metabolic activation of Vcar.
The stimulation of cell proliferation associated with a high level of hepatic DNA damage is probably a major factor in the development of liver tumors after exposure to Vcar. However, the formation of A may play a critical role in this process, as suggested by the mutation spectra observed in the Ha-ras gene in adenoma and hepatocellular carcinoma induced by Vcar in mice (11
, 14)
. Under the conditions of extensive liver damage as seen in this study, however, the elevated level and the longer persistence of promutagenic A in liver DNA from APNG knockout mice were apparently not directly associated with a higher susceptibility to hepatocarcinogenesis.
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ACKNOWLEDGMENTS |
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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.
Request for reprints: Rhoderick H. Elder, Cancer Research United Kingdom Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, United Kingdom. Phone: 44-161-446-3124; Fax: 44-161-446-3109; E-mail: relder{at}picr.man.ac.uk
3 The abbreviations used are: Vcar, vinyl carbamate; APNG, alkylpurine DNA-N-glycosylase; A, 1,N6-ethenoadenine; C, 3,N4-ethenocytosine; TUNEL, terminal deoxynucleotidyl transferase mediated UTP nick end-labeling; BrdUrd, bromodeoxyuridine. 
Received 5/19/03. Revised 8/28/03. Accepted 9/ 4/03.
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