Mutagenesis, Vol. 17, No. 4, 353-359,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Comparison of kinetics of induction of DNA adducts and gene mutations by a nitrofuran compound, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in the caecum and small intestine of Big BlueTM mice
Unité de Programmation Moléculaire et de Toxicologie Génétique, CNRS Ura 1444, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris cedex 15, France
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Abstract |
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In previous experiments, i.p. injection of the 5 nitronaphthofuran derivative 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) to lacI transgenic Big BlueTM mice led to an increase in the mutant frequency (MF), especially in the caecum and the small intestine. In the present work, the in vivo genotoxicity of R7000 in these two target organs was further investigated. Big BlueTM mice were treated with a single daily i.p. injection of R7000 of 0.05–0.5 mg/day for five consecutive days and killed 28 days later. These treatments led to significant increases in MF of 1.8-, 3- and 5.4-fold at 0.1, 0.2 and 0.5 mg/day R7000, respectively, in the small intestine. In the caecum, a mutagenic effect, of 4.5-fold, was only observed at the highest dose. DNA adduct formation and MFs resulting from R7000 were also analysed in parallel at various times after the last injection. R7000 led to 14 and seven different nucleotide modifications in the caecum and small intestine, respectively. Three hours after the final injection the level of induced DNA adducts was 10 times higher in the caecum than in the small intestine. From 3 h to 5 days after the final injection, 93 and 58% of DNA adducts disappeared in the caecum and small intestine, respectively. The resulting MF values were similar when comparing the two organs. Analysis of the R7000-induced mutation spectrum in the caecum showed that single G:C and large,
3 bp deletions and GC
CG transversions were the first induced mutations at the end of the treatment. Fifteen days later, the R7000 mutation specificity characteristics already reported in Escherichia coli and in the small intestine of Big BlueTM mice were evident in the caecum, with the two major events being GCTA transversions and deletions of one G:C base pair. In both organs, a relationship between the decrease in R7000–DNA adducts and induction of MF was evident. However, the efficiency of this compound in damaging DNA was not correlated with the capacity of DNA lesions to lead to mutations. Some discrepancies in the R7000 genotoxic effects between the two organs were observed, which may be attributable to differences in the metabolic activation pathway of the compound, as well as to DNA repair proficiency in each tissue. |
Introduction |
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2-Nitrofurans have broad spectrum antimicrobial properties and are used in human and veterinary medicine (Bryan, 1978
; IARC, 1990). Most of them are mutagenic in bacteria and mammalian cells and some are tumorigenic in animal models. These genotoxic properties are due to their metabolic activation, essentially via the reduction of their nitro group (McCalla, 1983). Addition of one or two rings to the nitrofuran molecule results in increased genotoxicity (Arnaise et al., 1986). Thus, the nitro-2-naphthofuran derivative 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) (Royer et al., 1978; Cavier et al., 1981) ranks among the most potent mutagens known in bacteria (Weill-Thévenet et al., 1981, 1982). R7000 is a very efficient inducer of the SOS functions in Escherichia coli (Arnaise et al., 1986). It leads to about 10 different DNA lesions on guanine residues (Touati et al., 1989, 1993), resulting mainly in G:CT:A transversions and deletion of one G:C base pair (Touati et al., 1996). Mutagenic properties have also been detected in cultured mammalian cells (Arnaise et al., 1986). Previous studies reported carcinogenic effects of R7000 in rats and mice, which developed local fibrosarcoma after s.c. injections (Salmon et al., 1985, 1986a). In addition, oral administration of R7000 induced tumours in the forestomach (Salmon et al., 1986b). Due to its extreme mutagenic potency, this compound constitutes an interesting model to study the mutagenic action of nitrofurans.
The development of transgenic mice has opened up new possibilities for mutagenicity testing in small animal models. The lacI transgenic Big BlueTM mouse assay (Kohler et al., 1991) allows spontaneous and induced gene mutations to be studied in any tissue from which DNA can be extracted and mutational events can be also characterized at the nucleotide sequence level. This system responds to a wide range of known carcinogens (Schmezer and Eckert, 1999). In any organ, the time required to express mutations is tissue-specific. Mutagenic response depends on the rate of cell turnover as well as on the time required to convert DNA lesions into mutational events (Heddle et al., 1995; Heddle, 1999a).
Using Big BlueTM mice, we have previously shown that i.p. injection of R7000 significantly increased the mutant frequency (MF), by 2-, 3- and 4-fold the control value, in colon, small intestine and caecum, respectively (Quillardet et al., 2000). In the present study, the in vivo genotoxicity of R7000 was further investigated in the caecum and the small intestine of these transgenic mice. A mutagenic dose–response analysis and a time–course study of DNA adduct formation with relevance to MF were performed. For a better understanding of the implicated mechanism, the sequences of R7000-induced mutations in the caecum were determined, taking into account time after treatment, and compared with results previously obtained in the small intestine (Quillardet et al., 2000). In both organs, a good correlation between a decrease in DNA adducts and MF induction was evident. Differences between the two organs concerning DNA adducts, MF levels and mutation spectra are discussed.
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Materials and methods |
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Animals
The experiments reported here were submitted to and approved by the Central Animal Facility Committee of the Institut Pasteur, in conformity with the French Ministry of Agriculture guidelines for animal care.
Six-week-old pathogen-free male mice, strain C57Bl/6, carrying the 
/lacI transgene (Big BlueTM transgenic mice; Stratagene, La Jolla, CA) were housed in polycarbonate cages and fed a commercial diet with water ad libitum. Animals were acclimatized for 1 week prior to the start of the experiments. All animal experimentation was performed in accordance with institutional guidelines.
Chemicals
R7000 (Royer and Buisson, 1980a,b) was kindly provided by Dr J.P.Buisson. It was dissolved in DMSO/H2O (85:15).
Experimental design
The LD50 was determined by treating C57Bl/6 mice with i.p. injections of R7000 ranging from 0.2 to 1 mg/day in 100 µl of 85% DMSO for four consecutive days. For each condition four mice were treated. Injections of 0.7 and 1 mg/day R7000 killed 50 and 100% of the animals, respectively.
R7000 mutagenic properties were determined on Big BlueTM mice, which received i.p. injections of 100 µl of 0.05–0.5 mg/day R7000 for four consecutive days. Four mice were analysed for each dose. Control mice received i.p. injections of 100 µl of 85% DMSO. Mice were killed 28 days post-treatment by CO2 asphyxiation.
The kinetic study of the induction of DNA adducts and the resulting MF was performed following the same protocol. The experiments consisted of a treated group of eight Big BlueTM mice, which received daily doses of 0.5 mg R7000, and four control mice. At 3 h (D0) and 5 (D5), 15 (D15) and 25 (D25) days after the fifth injection, one mouse from the control and two treated mice were killed. The caecum and small intestine were rapidly collected and stored in liquid nitrogen until DNA isolation.
Mutant frequency (MF) determination
Organs were washed in water and complete tissues quickly disaggregated and digested with proteinase K and RNase. The DNA was extracted using phenol/chloroform and precipitated in 100% ethanol before being resuspended in a suitable volume (200–400 µl) of TE buffer (10 mM Tris–HCl, pH 7.6, 1 mM EDTA). It was then left for solubilization at room temperature for at least 24 h before storage at 4°C.
The lacI-containing shuttle vectors were recovered from genomic DNA preparations using a packaging extract (Transpack; Stratagene), according to the manufacturer's instructions. The phage particules were assayed for lacI mutations by infecting a culture of Escherichia coli SCS8 (recA1, endA1, mrcA, 
[argF-lac]U169, 
80dlacZM15, Tn10 [tetr]) and plating the infected bacteria with prewarmed NZY top agarose (Miller, 1992) containing 1.5 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) (ICN Biochemicals, Aurora, USA), dissolved in N,N-dimethylformamide on square plates (25x25 cm) containing NZY agar. After an overnight incubation at 37°C, the lacI- mutants expressed ß-galactosidase, leading to blue mutant plaques. Colourless plaques corresponded to the LacI+ phenotype. MF was expressed as the number of blue plaques divided by the total number of plaques. Pinpoint plaques (small blue dots peripherally located within a colourless wild-type plaque) and plaques in which less than one-third of the periphery was blue were considered as sectored mutants and were not taken into account (Hill et al., 1999).
DNA sequence analysis
lacI mutants induced in the caecum of R7000-treated and untreated mice were isolated as agar plugs, eluted in 400 µl of SM buffer (20% maltose w/v, 1 M MgSO4) and purified by replating at low density. Agar plugs of purified plaques were re-eluted in the same buffer. The lacI gene was then directly amplified by PCR using forward primer LcI5' (positions –92 to –71 in lacI) and reverse primer LcII3' (positions 1227 to 1249) (Farabaugh, 1978). The resulting PCR products were incubated with exonuclease I and shrimp alkaline phosphatase and sequenced with the BigDye Terminator cycle sequencing kit in an Applied Biosystems 377A DNA sequencer (Applied Biosystems, Foster City, CA). Primers used for DNA sequencing hybridized at the 5'-end of the lacI gene at nt –69 to –56 and at the 3'-end at nt 1191 to 1208 (Farabaugh, 1978). All primers were obtained from Genset (France). Sequence data were analysed with ABI Prism Edit view software. Mutations were detected by comparison with the wild-type lacI nucleotide sequence (Farabaugh, 1978), using DNA Strider software.
32P-post-labelling analysis of DNA adducts
DNA adducts present in the genomic DNA samples (20 µg) isolated from the control and the two treated mice at each time point, D0, D5 and D15, were analysed by the 32P-post-labelling method (Randerath et al., 1981; Gupta et al., 1982), using the nuclease P1 enhancement procedure (Reddy and Randerath, 1986). The DNA samples were extracted from caecum and small intestine, 32P-labelled and analysed by thin layer chromatography. The detection of adducts spots and their measurement were carried out with a phosphorimager 445SI (Molecular Dynamics).
Statistical analysis
For dose–response analysis, differences in MF between groups of treated and untreated mice were determined using a two-tailed t-test. A P 
0.05 was considered significant.
For time–course analysis, correlations between MF and the time until death after the last injection were further determined by calculation of Pearson's coefficient (r). A P 0.05 indicates a significant correlation between the two parameters (Lowry, 1998).
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Results |
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As previously reported, a significant increase in MF was observed with genomic DNA isolated from the caecum and the small intestine of R7000-treated mice (Quillardet et al., 2000
). The main objectives of the present study were to investigate the evolution of R7000–DNA adducts and the resulting mutagenesis in these two different parts of the digestive tract. The purpose of our work was also to determine whether the mutagenic response to R7000 was the same for these two similar tissues.
Dose–response analysis
As described in Materials and methods, Big BlueTM mice were treated i.p. with increasing doses of the compound. Under each tested condition four mice were analysed and killed 28 days later.
The control MFs calculated from DMSO-treated mice were similar in the two organs: mean ± SD = 3.9 ± 1x10-5 (caecum) and 4.8 ± 1.5x10-5 (small intestine) (Table I). These values are in the same range as spontaneous MFs usually found (Morrison and Ashby, 1994). In both small intestine and caecum, the MF increased with dose of R7000 (Table I). In the small intestine MF was significantly increased, by 1.5- (P = 0.05), 1.8- (P < 0.01), 3- (P < 0.01) and 5.4-fold (P < 0.001) at 0.05, 0.1, 0.2 and 0.5 mg/day R7000, respectively, as compared with the control group. In the caecum the MF was significantly induced only at the highest dose, 0.5 mg/day, with a 4.5-fold increase (P < 0.01) above the background level.
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Time–course analysis of DNA adduct levels and lacI MF
To further analyse the mechanism of in vivo genotoxicity of R7000, DNA adducts detected by 32P-post-labelling (Randerath et al., 1981
) and MF were determined on the same DNA samples at various times after R7000 treatment (see Materials and methods). In the so-called `Big Blue' mutagenesis assay it is usually recommended that at least 5 mice/group are analysed (Callahan and Short, 1995). Since the mutagenicity of R7000 after i.p. injection had already been demonstrated for the caecum and the small intestine (Quillardet et al., 2000), a smaller number of mice were treated in this part of the work. Total DNA adduct levels from control mice were similar (0.06 adducts/108 nt) for these two organs but their chromatographic profiles were different (Figure 1). Control MF values were of 8 ± 1.6x10-5 and 10 ± 4x10-5 for the caecum and small intestine, respectively (Table II). They are in agreement with previously reported spontaneous MF levels (Morrison and Ashby, 1994; Quillardet et al., 2000).
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At D0, DNA isolated from the caecum showed at least 14 different adducts (Figure 1A
) corresponding to a total level of 5 adducts/108 nt, which is 45 times the background level (0.11 adducts/108 nt). It decreased rapidly, to reach 0.22 and 0.37 adducts/108 nt at D5 and D15, respectively. Thus, 95% of the induced DNA adducts had disappeared 5 days after the last injection, but one major spot was always observed at D15 (Figure 1A). R7000 led to a progressive increase in MF with time, from 12–19x10-5 at D0 to 32–40x10-5 at D25 (Table II). A significant correlation (P 0.02) was found for the induction of MF and the time of death from D0 to D25. MF varied with time following the equation: MF = [(0.997xt) + 13.43]x10-5, where t is time after last injection. Concomitant with this progressive increase, the level of R7000–DNA adducts decreased drastically.
In the small intestine R7000 induced 0.48 adducts/108 nt, eight times higher than the level in control animals (0.06 adducts/108 nt), at D0. It was 10 times lower than that observed in the caecum, and the chromatographic profiles were different. Eight distinct adducts were detected and most of them remained detectable at D5 and D15 (Figure 1B). Their levels decreased 2.4-fold from that at D0 (0.2 adducts/108 nt). R7000 led to a significant progressive increase in MF from 7–14x10-5 at D0 to 26–27x10-5 at D5 (P = 0.05) (Table II). Due to interindividual variability among the animals tested, it is difficult to conclude whether MF was unchanged from D5 at D15 and D25 or slightly decreased.
Characteristics of mutation spectra induced in the caecum Spontaneous mutations
Thirty-five LacI mutants obtained from the caecum of the four control mice were sequenced. The results are reported in Table III. Two mutants showed double mutations. Seventy per cent of the mutations occurred at G:C base pairs. Substitution base pairs represented 84% of the total events, 48% of which consisted of G:CA:T transitions at 5'-CpG-3' dinucleotides (11 of 15 cases). All type of substitutions were observed except for A:TT:A transversions. Frameshifts represented 16% of the mutants, 67% of which were the addition of one A:T base pair. This spectrum was comparable with those previously published for various tissues (De Boer et al., 1998).
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R7000-induced mutations
Fifty-eight LacI mutants isolated from R7000-treated mice were sequenced. Two of them with GC
AT at position 376 were isolated from the same mouse. In order to avoid a bias due to clonal expansion and not to de novo mutations (Heddle, 1999b), these two mutants were not considered. Eighty-three per cent of the mutations occurred at G:C base pairs. Substitutions represented 77% of all events (Table III) and were essentially found in the first 300 nt of lacI. This part of the gene corresponds to the DNA-binding domain of the lactose repressor in E.coli (Gordon et al., 1988). Forty-nine per cent of base pairs substitutions were G:CT:A transversions and 26% were G:CA:T transitions. Sixty-six per cent of substituted G were preceded by C (on the 5'-side). Nine per cent of the substitution events occurred at A:T base pairs with a G:C on the 5'- and 3'-sides. Single frameshifts represented 18% of all events and consisted essentially of a deletion of one G:C base pair within a run of two or three. Three large deletions were also recovered, between positions –63 to –29, 281 to 369 and 610 to 794 (according to the numbering of the lacI gene sequence; Farabaugh, 1978). It should be noted that the 88 nt segment deleted between nt 281 and 369 is flanked on the 5'- and 3'-sides by the sequence 5'-TCTCGCGC-3'. In the case of the third deletion (nt 610–794), the tetranucleotide 5'-GCGC-3' is found on both sides of the deleted fragment.
The distribution of mutational events was also followed with time after R7000 treatment. Three groups of mutants were compared (Figure 2). Group A mutants were isolated from control mice (35 mutants), group B and group C at D0 and D5 (17 events) and D15 (32 events), respectively. The mutation spectrum corresponding to group B shows that deletions of one G:C base pair (13%) and 3 bp (13%), as well as G:CC:G transversions, (20%) were induced early. Each of them represented only 3% of the mutants in the control sample, group A (Figure 2A). In group B, G:CA:T transitions were still predominant (34%) (Figure 2B). Mutants isolated 15 days later (group C) showed two major R7000-induced events, G:CT:A transversions and single G:C deletions (Figure 2C), already reported in E.coli (Touati et al., 1996) and in the small intestine of mice, 28 days after treatment (Quillardet et al., 2000).
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Several differences in the mutational spectra determined from the caecum and the small intestine samples were observed. In the control samples, AT
TA transversions represented 8% of the events (3/39) in the small intestine, but were not detected in the caecum. The addition of one A.T base pair accounted for 11% of the events in the caecum (4/37), but was not observed in the small intestine. The R7000-induced mutation spectra also showed differences between the two organs. GCCG transversions represented 11% of the events (6/52) in the caecum and only 4% (2/42) in the small intestine. Eight per cent of the mutants (4/42) were addition of one G:C base pair in the small intestine, which was not observed in the caecum. Large deletions (3/52) were observed in the caecum (see above), but were not found in the small intestine. |
Discussion |
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A previous study from our laboratory with Big BlueTM transgenic mice reported that i.p. injection of R7000 led to a significant induction of lacI mutation, especially in the caecum and small intestine, 28 days after treatment (Quillardet et al., 2000
). A schematic view of the mechanisms of R7000-induced mutagenesis is as follows. It is metabolized to genotoxic intermediates, some of which produce DNA lesions of various types (Touati et al., 1989, 1993). These lesions may be processed by error-free or error-prone repair systems before replication occurs. Upon replication, mutations can be generated at the site of the remaining lesion or possibly in its vicinity. In the present study, the induction of DNA adducts and mutagenesis by R7000 was further investigated in Big BlueTM transgenic mice. The analysis was carried out on the caecum and small intestine at different times after treatment. The mutagenic response to increasing doses of the compound showed an increase in MF at the highest dose injected (0.5 mg/day for 5 days), by a factor of 4.5 and 5 compared with the background level, in the caecum and the small intestine, respectively. However, the small intestine was more sensitive than the caecum to R7000 mutagenesis, since doses of 0.1 and 0.2 mg/day led to a significant increase in MF in the former. These results could indicate that these two tissues, albeit spatially close and expected to share similar histological and physiological characteristics, present differences in their susceptibility to R7000 DNA-damaging and mutagenic effects.
Tissue turnover is an important factor in the appearance of somatic mutations and the cells most implicated in this process should be stem cells (Heddle et al., 1996; Heddle, 1999a). Intestinal epithelium is a rapidly dividing tissue, replaced in less than a week. The major histological difference between caecal and small intestine epithelium is the absence of villi in the case of the caecum (Walker and Homberger, 1997). Within 3–5 days, cells divide and migrate from the bottom of the intestinal crypt to the top of the villus, where they are lost (Alberts et al., 1994). This 5 day period is similar to the time required for induction of LacI mutants in the Big BlueTM assay. It is conceivable that in intestinal tissues, where the cell proliferation rate is high, DNA adducts are rapidly eliminated, as shown in the present study.
The DNA adduct level detected by the post-labelling method (Randerath et al., 1981) 3 h after the last injection was 10 times higher in the caecum than in the small intestine, whereas MF values were similar. This suggests that some R7000 lesions in the small intestine could be more mutagenic than those in the caecum. Furthermore, there was no direct correlation between DNA adduct levels and MF. In both organs, the highest levels of adducts were observed 3 h after the last injection (D0). This is consistent with the observation that i.p. administration of [14C]R7000 to rats or mice leads to a high R7000 plasma level 3 h after injection (Maurizis et al., 1985). Qualitative and quantitative variations in R7000–DNA adducts observed between the small intestine and the caecum could be attributed in part to differences in the metabolic pathways or enzymatic activities implicated in activation of the compound. Indeed, analysis of metabolites excreted in urine after oral administration of R7000 to rats indicated three different metabolic pathways, namely demethylation of the methoxy group, hydroxylation of the aromatic ring and cleavage of the furan ring followed by reduction of the nitro group to an amine (Maurizis et al., 1986). The importance of the reduction in metabolic activation of nitrofurans has also been demonstrated in vitro with small intestine wall homogenates from rats (Aufrere et al., 1978). Between D0 and D5, 95 and 58% of the DNA adducts disappeared in the caecum and the small intestine, respectively, concomitant with the increase in MF. At later times, DNA adduct levels remained constant in both cases. Thus, there is a correlation between the disappearance of DNA adducts and the increase in MF. Accordingly, bulky adducts formed by numerous genotoxic compounds have been reported to undergo very slow removal (Tombolan et al., 1999). It is likely that all nucleotide modifications observed are not acting as premutagenic lesions. The decrease in adduct levels could be attributable, in part, to the efficiency of DNA repair systems. However, chemical instability due to the structure of the DNA lesions cannot be ruled out.
The kinetics of DNA adduct formation and mutagenesis is much better documented in Big BlueTM mouse liver. The profile of adduct formation with a peak at early sampling times was reported for other chemicals, including 5,9-dimethyldibenzo[c,g]carbazole (DMDBC), which is carcinogenic specifically in mouse liver (Szafarz et al., 1990; Tombolan et al., 1999). As in our experiments (see Figure 2A), the adduct level decreased rapidly and reached a plateau after 4 days. This decrease in initial DMDBC–DNA adduct level ran parallel to hepatocellular necrosis and was suggested to occur mainly through elimination of seriously damaged cells rather than through repair processes (Tombolan et al., 1999).
Time–course analysis of the R7000-induced mutation spectra in the caecum showed that large (3 bp) and single G:C deletions, as well as G:CC:G transversions, were the first mainly R7000-induced mutations within a few hours after the last injection or during the following days (Figure 2). Two of the large deletions observed were flanked by direct sequence repeats at the deletion sites. This has been frequently reported in bacteria as well as in mammals (Schaaper et al., 1986; Thacker et al., 1992). It is noteworthy that short deletions involving repeated sequences are often implicated in human genetic diseases (Krawczak and Cooper, 1991). The R7000 mutation spectrum reported here at D15, consisting of 48% G:CT:A transversions and 16% single G:C deletions, is very similar to our previous observations in E.coli (Touati et al., 1996) and in the small intestine of mice (Quilllardet et al., 2000). A large number of the nucleotide modifications which disappeared between D0 and D5 could have been repaired by error-free processes. In accordance with this, it was previously reported in E.coli (Quilllardet et al., 1996) that most of the R7000–DNA lesions are removed by the nucleotide excision repair system, which could be as efficient in mouse as in bacteria. Some of the R7000-induced DNA lesions persist for a longer period of time and could account for the specfic R7000 mutation spectra observed at D15.
In spite of the similarities cited above, there are some discrepancies in the distribution of R7000-induced mutation events between the small intestine and the caecum. Most striking are four times more frequent GCCG transversions in the caecum than in the small intestine. This event could result from the induction of abasic sites, since in mammalian cells G and C are more commonly inserted opposite abasic sites, leading to ATCG, GCCG and ATGC. In accordance with this, induction of abasic sites as R7000 premutagenic lesions has already been proposed in E.coli (Touati et al., 1996), where they should result from unstable N7-guanine derivatives through spontaneous cleavage of the N-glycosidic linkage (Loeb and Preston, 1986). Differences in mutation spectra between these two organs are likely to be related to the differences observed in the DNA adduct profiles as evident from the post-labelling analysis.
In conclusion, the genotoxic response of mice to R7000 treatment illustrates a relation between the processing of DNA adducts and mutagenesis. It should be the result of a compromise between metabolic activation of the compound and DNA binding of derivatives, the efficiency of DNA repair systems and cell turnover. This study shows that even with tissues which are histologically and physiologically closely related, specific features may account for their susceptibilities to genotoxic and carcinogenic risks of chemical compounds.
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Acknowledgments |
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We are grateful to Dr Jean-Pierre Buisson for the gift of R7000. We wish to thank Dr Jean-Marie Clément and Dr Sevec Smelczman for their helpful comments and careful reading of the manuscript. This work received financial support from the Direction des Recherches sur l'Environnement et l'Hygiène, Institut Pasteur, Paris.
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Notes |
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1 To whom correspondence should be addressed. Tel: +33 1 45 68 32 88; Fax: +33 1 45 68 88 34; Email: etouati{at}pasteur.fr
This paper is dedicated to the memory of Maurice Hofnung who died during this work
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Received on April 10, 2001; revised on March 21, 2002; accepted on March 25, 2002.
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