Mutagenesis, Vol. 15, No. 3, 239-244,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
DNA breakage in asbestos-treated normal and transformed (TSV40) rat pleural mesothelial cells
1 INSERM E99.09, IM3, EA 2345, Faculté de Médecine, 8 rue du Général Sarrail, Créteil, 94010, France and 2 Lung Biology Center, UCSF Box 0854, San Francisco, CA, USA
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Abstract |
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Asbestos has been shown to induce cell cycle arrest, DNA repair and some abnormalities consistent with DNA damage but not DNA breakage. The purpose of the study was to investigate DNA breakage in asbestos-exposed rat pleural mesothelial cells (RPMC). RPMC were compared with their transformed counterparts, RPMC-TSV40 (i.e. p53-inactivated by infection with a retroviral recombinant encoding the SV40 large T antigen), as in the latter cells the cell cycle does not arrest and DNA repair is deficient due to ineffective p53-dependent cell cycle control. RPMC and RPMC-TSV40 were exposed to chrysotile and crocidolite asbestos and also to camptothecin for comparison. The presence of DNA breakage was determined using the single cell gel (Comet) assay with alkaline electrophoresis and quantified by measuring comet tail length (TL) and the percentage of total DNA in the tail and calculating tail moment (TM). We found that comets were generated by both types of asbestos in RPMC and in RPMC-TSV40 as well as by camptothecin in RPMC. On a per weight basis, chrysotile induced more abnormalities in comet parameters than did crocidolite. The comet TL and TM increased with fibre concentration, although less so with crocidolite than with chrysotile. When exposed to chrysotile at similar concentrations, RPMC consistently showed more abnormal comet parameters than did RPMC-TSV40. We concluded that asbestos causes DNA breakage and suggest that some of the DNA breakage measured was due to repair mechanisms in the normal RPMC.
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Introduction |
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Knowledge of the mechanisms by which asbestos alters cellular DNA is of great importance for understanding the mechanisms of fibre-induced neoplastic cell transformation. From the results published in the literature it can be suggested that the action of proliferative signals on cells with chromosomal aberrations could induce neoplastic transformation. In asbestos-exposed mesothelial cells, DNA and chromosomal damage have been observed in vitro (Pelin et al., 1992
; Yegles et al., 1993) and in asbestos-exposed rodents cell proliferation has been demonstrated (Brody and Overby, 1989; Jaurand, 1996). Recently, a lack of control of cell proliferation by binding of SV40 large T antigen (TAg) to the tumour suppressor genes p53 and pRb has been suggested as an important factor in the pathogenesis of mesothelioma (Wiman and Klein, 1997). Therefore, DNA alterations may be of major importance in the mechanisms of mesothelial cell transformation for two reasons: direct potential damage to critical genes by asbestos and weakening of repair processes resulting from the abrogation of p53 function in cell cycle control by SV40 or other similar viruses.
Although DNA damage has been demonstrated in rat pleural mesothelial cells (RPMC) exposed to asbestos fibres, neither DNA strand breaks specifically nor DNA breakage in general have been directly demonstrated. DNA strand breaks have been suggested by activation of poly(ADP)ribose polymerase (PARP) (Dong et al., 1995) and deoxyguanosine hydroxylation (Chen et al., 1996; Fung et al., 1997). DNA breakage in asbestos-exposed RPMC has been also suggested by triggering of DNA repair processes, such as a stimulation of unscheduled DNA synthesis (UDS) (Renier et al., 1990), an increase in AP endonuclease gene expression (Fung et al., 1998) and an arrest or delay of cell cycle progression through the G1/S transition in cultures of mesothelial cells (Levresse et al., 1997). Cell cycle arrest was associated with induction of p53 and p21Cip1/WAF1 (Levresse et al., 1997), in agreement with activation of a cell cycle checkpoint allowing repair of damage by a p53-dependent process, as seen with other DNA-damaging agents (Sanchez and Elledge, 1995; Smith et al., 1995; McDonald et al., 1996). More interestingly, we found that p53-inactivated RPMC did not arrest in G1 and continued to proliferate, suggesting that these cells would not have the opportunity to repair their DNA or that the repair process would be limited (Levresse et al., 1998). DNA breakage is an important signal for arrest or apoptosis and failure to detect that signal may lead to inadequate repair of genetic abnormalities.
The aim of the present work was to investigate DNA breakage in asbestos-exposed RPMC. To carry out this work, we studied DNA breakage by the single cell gel electrophoresis (SCGE) assay in normal and in p53-inactivated RPMC. The Comet assay permits, under alkaline conditions, the detection of a wide range of DNA damage, including single- and double-strand breaks, lesions transformed into single-strand breaks (i.e. alkali-labile sites) and excision repair events in progress (Collins et al., 1997). Our results are the first to demonstrate that DNA breakage occurs in asbestos-exposed normal RPMC and suggest that some of the breakage is due to repair processes. If so, the lower DNA breakage in RPMC-TSV40 may be due to an inability of p53-inactivated mesothelial cells to repair DNA damage, a finding of interest regarding the possible involvement of p53 inactivation in some human mesothelioma cells.
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Materials and methods |
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Fibres and chemicals
Two samples of asbestos fibres were used. Rhodesian chrysotile and crocidolite were obtained from the Union Internationale Contre le Cancer. Mean length and diameter were 1.7 ± 2.2 and 0.05 ± 0.04 µm, respectively, for chrysotile and 2.1 ± 3.6 and 0.19 ± 0.12 µm for crocidolite as determined by transmission electron microscopy (Yegles et al., 1995
). In some experiments, National Institute of Health and Environmental Sciences (NIEHS) crocidolite (mean length 11.4 ± 8.0 µm) was used (Broaddus et al., 1996). Fibres (1 mg/ml) were dispersed by sonication in complete medium (50 W, vibra cell; Sonic Materials) for 5 min to obtain a homogeneous suspension. Camptothecin was purchased from Sigma (La Verpillière, France).
Cell culture
Normal RPMC from Sprague–Dawley rats were cultured according to a method described elsewhere (Jaurand et al., 1981). RPMC were cultured in Ham's F10 medium (ATGC, Noisy le Grand, France) supplemented with 10% foetal bovine serum (FBS) (ATGC, Noisy le Grand, France), 10 mM HEPES (Eurobio, les Ulis, France), 100 IU/ml penicillin and 50 µg/ml streptomycin (Eurobio, les Ulis, France). We also used RPMC expressing the large T antigen (TAg) from SV40 virus. These cells (RPMC-TSV40) were generated by infecting RPMC with a retroviral recombinant encoding the SV40 TAg as described elsewhere (Levresse et al., 1998). It was verified that RPMC-TSV40 expressed TAg and that p53 was inactivated (Levresse et al., 1998).
Treatments
The experimental conditions, doses and duration of incubation were chosen according to previous investigations on cell cycle control and apoptosis in asbestos-treated RPMC (Levresse et al., 1997). Therefore, RPMC were exposed to 0.5, 2, 5 or 10 µg/cm2 chrysotile or 2 or 10 µg/cm2 crocidolite for 24 h during exponential growth. Briefly, cells were plated in 75 cm2 tissue culture flasks (Costar, Dutscher, Brumath, France). Twenty-four hours later the medium was changed and replaced with complete medium containing the desired fibre concentrations. After 24 h incubation, cells were detached and collected as described in the following. In addition, RPMC exposed to camptothecin served as reference treatments for the Comet assay (Lebailly et al., 1997).
SCGE (Comet) assay
At the end of the treatment, RPMC were washed with Ca2+, Mg2+-free phosphate-buffered saline (PBS), trypsinized, centrifuged at 350 g for 10 min and washed several times in PBS with Ca2+ and Mg2+. Cells were resuspended at a final concentration of 5x105 cells/ml in 0.5% low melting point agarose (LMA) (Sigma, La Verpillère, France). Sixty microlitres of the suspension were sandwiched between a lower layer of 0.5% normal melting point agarose (Life Technologies, Cergy Pontoise, France) and an upper layer of 0.5% LMA on fully frosted microscope slides. The slides were immersed in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, 1% v/v Triton X-100) for 1 h at 4°C. Then, the slides were placed in a horizontal gel electrophoresis tray filled with electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 40 min at 4°C in darkness, allowing DNA unwinding and formation of alkali-labile damage before electrophoresis. Electrophoresis was carried out in the same buffer at 4°C for 25 min at 300 mA, 25 V. After the electrophoresis run, the slides were neutralized three times with 0.4 M Tris, pH 7.5, for 5 min at room temperature and cells were stained with a 75 µg/ml propidium iodide solution. Slides were examined at 250x magnification using a fluorescence microscope (Zeiss, Oberkocken, Germany) equipped with a dichroic mirror (excitation filter 515–560 nm, barrier filter 590 nm) coupled to a CDD camera. Image analysis was performed with the software Fenestra Komet v.3.0 (Kinetic Imaging, Liverpool, UK) on 150 randomly selected cells (50 cells each of three replicate slides).
Quantitative analysis was performed by the measurement of several parameters: (i) tail length, i.e. distance between the trailing edge of the nucleus and end of the tail; (ii) head and tail DNA as percentages of total DNA; (iii) tail moment calculated as the product of tail length and tail DNA. The arithmetic mean of tail moment is abbreviated as MTM. Histograms report number of cells according to the tail moment within classes of two tail moment values (e.g. 0–
2, >2–4). Since the distribution of tail moments is not Gaussian (Bauer et al., 1998), the MTM was depicted by two parameters: the median and the 75% percentile (the MTM value at which 75% of cells had a lower MTM). Comets with a tail moment lower than 2 are usually considered as arising from cells with unbroken DNA. Graphical output was performed with Microsoft Excel. Statistical analyses were performed using the non-parametric Mann–Whitney test using GraphPad PRISM v.2.0 software for the Macintosh.
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Results |
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DNA breakage induced in RPMC exposed to asbestos fibres
In untreated cells, comets exhibited a high percentage of DNA in the head and the mean tail length was 10.6 µm (mean of all untreated series). The MTM was lower than 2, demonstrating that the majority of cells had unbroken DNA, as confirmed by the typical picture observed in untreated cultures (Figure 1
). The distribution of tail moments demonstrates that the percentage of cells with a tail moment greater than 2 is very low (Figure 2). In general at least, 85% of the untreated cells had unbroken DNA (tail moment <2). The 75% percentile of the tail moment distribution (i.e. 75% of the cells had a lower tail moment) ranged between 0.53 and 0.91 for the five experiments.
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Camptothecin was used as a reference agent for comet generation since DNA topoisomerase I inhibitors are known to induce DNA breakage in other cell types (Lebailly et al., 1997
) and camptothecin produces damage in RPMC (Levresse et al., 1997). In RPMC treated with camptothecin (5 µg/ml for 24 h) compared with untreated cells, the mean tail length of the comets was greater (Table I) and moment distribution shifted towards the higher tail moments (P = 0.004 and P < 0.0001, n = 2). The MTM reached 5.2 ± 2.1 (mean ± SD); similarly, the 75% percentile of tail moment was 2.5 and 5.9 in two experiments, versus 0.9 and 0.5 in the respective untreated cells. Atypical comets, likely corresponding to apoptotic cells (Marsteinstredet et al., 1997) were present in this population (Figure 1
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Both types of asbestos fibres formed comets (Figure 1
). Compared with no treatment, chrysotile even at 0.5 µg/cm2 produced an enhancement in the tail moment (Table I and Figure 2A) of approximately the same level as with 5 µg/ml camptothecin. This enhancement was associated with an increase in tail length (Table I). Although tail length was increased in a dose-dependent manner, the increase in MTM reached a plateau at a fibre concentration of 2 µg/cm2(Table I) because of a decrease in tail DNA at fibre concentrations >2 µg/cm2. Therefore the plateau may be related to a limitation of the method since comets exhibiting high DNA damage, as may be found after treatment with high doses of chrysotile, may be difficult to analyse. There was a significant difference in tail moment distribution between untreated and chrysotile-treated cultures at all concentrations (P < 0.0001) (Figure 2A). The 75% percentile for tail moment was strongly enhanced in chrysotile-treated cells in comparison with untreated cells: The range was 0.53–0.56 for untreated cells and 3.9–5.0 and 12.9–18.6 in cultures treated with 0.5 and 2 µg/cm2 chrysotile, respectively.
UICC crocidolite also produced a dose-dependent increase in tail length and moment, but much less effectively than did chrysotile (Table I). By analysis of MTM and of tail moment distribution, only at high concentrations (10 µg/cm2) did crocidolite induce greater comets than in untreated cells (P < 0.0001). Similarly, the median of the tail moment distribution was enhanced at this concentration (untreated, 0.18 ± 0.03; 2µg/cm2, 0.18 ± 0.06; 10µg/cm2, 0.75 ± 0.44) and the 75% percentile reached 9.6 with 10 µg/cm2 (controls, 0.53). In a comparative study, RPMC treated with NIEHS crocidolite also presented comets but the values found were lower than with UICC crocidolite (Figure 3). The distribution was statistically different from untreated cells in all exposed groups (P = 0.0016, 0.003 and <0.0001 in cultures treated with 2 and 10 µg/cm2 NIEHS crocidolite and 10 µg/cm2 UICC crocidolite, respectively). In these series exposed to 2 and 10 µg/cm2 NIEHS crocidolite and 10 µg/cm2 UICC crocidolite the 75% percentile of tail moment distribution (1.9, 1.6 and 5.2) was enhanced in comparison with untreated cells (0.8). The lower values obtained with NIEHS crocidolite in comparison with UICC crocidolite may be related to the different fibre parameters.
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DNA breakage in RPMC-TSV40 exposed to asbestos fibres
Untreated RPMC-TSV40 had baseline comet parameters very close to those of the untreated RPMC, i.e. a high percentage of DNA in the head and a short tail length (Table I
and Figure 4). In addition, the MTM was lower than 2, showing a low rate of spontaneous DNA breakage. The median of tail moment distribution was 0.24 ± 0.01 and the 75% percentile ranged between 0.62 and 0.75 (n = 3).
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After exposure of RPMC-TSV40 cells to chrysotile fibres (Table I
and Figure 4) tail length and MTM increased in a dose-dependent manner. The changes, however, were smaller than that of chrysotile-exposed RPMC at all experimental doses. Tail length never exceeded 20 µm and tail moment never exceeded 6. Tail moment distribution was different from that of untreated cells after treatment with 5 and 10 µg/cm2 chrysotile (P < 0.0001). The 75% percentiles found in two experiments were 1.15—1.55, 3.2—4.5 and 3.6—7.8 in cultures treated with 2, 5 and 10µg/cm2 chrysotile, respectively. With UICC crocidolite fibres (10 µg/cm2) an enhancement of the comet parameters was also found (Table I) and the distribution was statistically different from that of untreated cells (P < 0.0001). The comet parameters were not significantly different from those of crocidolite-exposed RPMC. |
Discussion |
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Although the capacity of asbestos fibres to induce DNA breaks in cell-free systems has been established (Chao and Aust, 1994
; Hardy and Aust 1995; Leanderson et al., 1988), the clastogenic potential of asbestos in cultured cells is unclear. Some authors have failed to detect DNA breaks or point mutations in various cell types exposed to asbestos fibres (Fornace et al., 1982; Kodama et al., 1993). In contrast, Turver and Brown (1987) demonstrated by a S1 nuclease digestion method that crocidolite fibres (25–200 µg/cm2) induced single-strand breaks in C3H10T1/2 cells. Libbus et al. (1989), using a nick translation method, demonstrated that low concentrations of crocidolite (0.5–2 µg/cm2) increased the rate of tritiated thymidine incorporation in rat embryo cells. Recently DNA single-strand breaks have been reported in TSV40-transformed (MeT-5A) human pleural mesothelial cells (Ollikinen et al., 1999). However, in that study the effect of asbestos on normal cells was not studied; moreover, only crocidolite fibres were investigated. The present work compares DNA breakage in both normal and TSV40-transformed rat pleural mesothelial cells, a model of great interest to study the specific mechanisms of action of asbestos. In this study, we applied a new method, the SCGE assay, to the study of the genotoxicity of both types of asbestos fibres.
Numerous studies have validated the Comet assay as a sensitive method to quantify DNA breakage and to evaluate the genotoxic potential of a xenobiotic (Lee et al., 1996; Leroy et al., 1996). The assay has been shown to be insensitive to low concentrations of hydrogen peroxide (Ollikainen et al., 1999) and inert particles such as carbon black (Zhong et al., 1997). The different parameters measured give complementary information about the degree of DNA breakage. Comet tail length provides information about the average size of DNA fragments, tail DNA percentage provides information about the total percentage of DNA fragmented and tail moment, as the product of the two, provides information about both the quantity and average length of DNA fragments. In contrast to other methods more likely performed on growth-arrested cell cultures, such as nick translation or UDS, the SCGE assay can be used with growing cells and offers the opportunity to study individual cells. While the Comet assay was first used to detect the ability of some agents to generate single-strand and/or double-strand breaks in DNA (Fairbairn et al., 1995), some authors have suggested that the DNA breaks leading to comet generation could also result from repair of some DNA lesions. For example, it has been demonstrated that repair of oxidized bases or ligation of bases can lead to the generation of DNA breaks (Fortini et al., 1996; Alapetite et al., 1997). Thus we chose to study comet generation in both normal and SV40-transformed mesothelial cells, in which repair is abnormal, to learn more about the contribution of repair in our system.
In our study, we found that both types of asbestos fibres, chrysotile and crocidolite, generate comets in RPMC, in agreement with a genotoxic potential of these fibres. Comet generation could be related to both the fibres' ability to induce DNA breaks directly (Turver and Brown, 1987; Libbus et al., 1989) and to repair of DNA damage. The first series of breaks could be due to production of oxygen species by the fibres (Kamp et al., 1995) or by the cells during phagocytosis. Several sorts of breaks can result from the generation of hydroxylated bases [8-oxoguanosine and 8-hydroxydeoxyguanosine (8-OHdG)], as demonstrated in mesothelial cells (Chen et al., 1996) or in HL-60 cells (Takeuchi and Moromoto, 1994). This type of lesion can be converted into apurinic or apyrimidic sites by DNA repair processes and form DNA breaks under alkaline conditions (Collins et al., 1997).
A greater cellular toxic effect of chrysotile compared with crocidolite asbestos has been noted by others in different cell systems and assays. One possibility is that chrysotile, despite its lower iron content, induces more reactive oxygen or reactive nitrogen stress in cells than does crocidolite, as has been suggested more or less directly in certain studies (Marsh and Mossman, 1991; Perkins et al., 1991; Thomas et al., 1994; Tanaka et al., 1998). In the present study when the two UICC samples were compared, chrysotile induced DNA breaks at a lower mass concentration than did crocidolite (0.5 versus 10 µg/cm2) and induced higher MTM and mean tail length values than did crocidolite. The chrysotile was more potent than crocidolite even when compared at concentrations where the number of fibres was equivalent: chrysotile 0.5 µg/cm2, MTM 5.3; UICC crocidolite 2 µg/cm2, MTM 1.1. The high values of comet parameters in cells exposed to chrysotile fibres is unlikely to be related to cytotoxicity: for instance, comets appeared in 66.6% of the cells exposed to 0.5 µg/cm2 chrysotile although ~90% of the cells were viable as determined by MTT assay (Levresse et al., 1997). Moreover, NIEHS crocidolite, which appears to be more toxic than UICC crocidolite on the basis of apoptotic yield, produced lower comet values than did UICC crocidolite. Therefore, our results may demonstrate a higher genotoxic potential of UICC chrysotile fibres versus crocidolite and/or a higher repair proficiency of cells exposed to them. These results are interesting since crocidolite fibres, because of their higher iron content, are thought to produce more reactive oxygen species than chrysotile (Vallyathan et al., 1992; Ghio et al., 1994; Janssen et al., 1994). Our results may indicate that, under these experimental conditions, chrysotile was the more effective producer of reactive oxygen species or that DNA damage occurred by other mechanisms. However, other factors may influence the results. For instance, in the study performed by Howden and Faux (1996a) H2O2 known to potentiate the generation of 8-OHdG by crocidolite was added to the culture medium. Furthermore, reactive oxygen species are not the only factors able to produce DNA damage. Products of oxido-reduction reactions may consist of more stable factors, i.e. lipid peroxidation products may form DNA adducts (Howden and Faux, 1996b). Although not identified, Emerit et al. (1991) demonstrated the ability of chrysotile-exposed RPMC to produce stable clastogenic factors.
Since repair processes may account for the formation of comets, the results we obtained could also be related to higher repair proficiency in cells exposed to chrysotile in comparison with crocidolite. However, this explanation seems unlikely since Renier et al. (1990) reported similar levels of repair in RPMC exposed to both types of fibres measured using an UDS assay. To investigate the contribution of DNA repair in the Comet assay, it would be necessary to remove fibres from the cells in order to permit repair of fibre-induced DNA damage and a return to baseline levels. Such experiments carried out with HeLa cells and lymphocytes treated with H2O2 or UVC have demonstrated a reversibility of comet formation with time (Collins et al., 1997). Similar experiments cannot be made with fibres because of their internalization. In order to answer this question, we studied RPMC-TSV40, a cell line expressing the SV40 TAg and deficient in control of the G1/S transition (Levresse et al., 1998). These cells may have an inactivation of some DNA repair processes, as found in fibroblasts expressing TAg (Zhan et al., 1994), where an impairment of DNA repair is characterized by a lack of recruitment of GADD45 and GADD153 proteins after irradiation. We showed that these cells had less comet development than normal cells when exposed to chrysotile. Although a dose-dependent increase in the number of comets in RPMC-TSV40 cultures was observed after treatment with asbestos, the percentage was lower than in similarly exposed RPMC cultures. Several hypotheses could be drawn to explain these results. First, RPMC-TSV40 could be less sensitive to chrysotile fibres than RPMC; second, their repair mechanism could be inactivated. Such a difference in comet production was not found in RPMC-TSV40 versus RPMC after exposure to UICC crocidolite. We may assume that DNA repair is impaired in RPMC-TSV40 and that chrysotile induces both greater DNA breakage and repair than UICC crocidolite. These results are in agreement with our previous findings showing a stronger cell cycle arrest in chrysotile-exposed RPMC in comparison with crocidolite-exposed RPMC (Levresse et al., 1998). These hypotheses suggest therefore that the DNA breaks induced by asbestos fibres in normal RPMC would reflect both direct breaks induced by asbestos and breaks resulting from activation of excision resynthesis processes. The inactivation of DNA repair processes as well as the inactivation of cell cycle control could lead to the generation of highly damaged cells accounting for the higher rate of apoptosis observed in RPMC-TSV40, as described elsewhere (Levresse et al., 1998).
Given previous results on DNA repair synthesis, activation of PARP, cell cycle arrest, activation of cell cycle checkpoints and, more recently of AP endonuclease induction, these results are important in leading to the hypothesis that certain types of DNA damage by asbestos can be repaired, especially at the G1/S transition. Other types of DNA damage, such as those resulting in nuclear aberration and mitotic abnormalities, may be of more concern as evidence of asbestos toxicity if they are not subject to repair. Finally, DNA damage, repair of damage and the quality of repair are very important questions for understanding mesothelial cell malignancy (Broaddus, 1997).
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Acknowledgments |
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The authors thank Dominique Renault and Dominique Brault (Drug Safety Department, Rhône-Poulenc Rorer, France) for the Comet analyses and Sylviane Moritz (INSERM U.402 Paris) for technical assistance.
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Notes |
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3 Present address: Department of Medicine, University of Colorado Health Science Center, Renal Division C281, 4200 East 9th Avenue, Denver, CO 80262, USA
* To whom correspondence should be sddressed. Tel: +33 1 49 81 36 66; Fax: +33 1 49 81 35 33; Email: jaurand{at}im3.inserm.fr
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Received on October 11, 1999; accepted on December 22, 1999.
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