* Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 358 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; and
Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 161, Hachinohe, Aomori 039-1192, Japan
Received April 8, 2002; accepted June 14, 2002
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ABSTRACT |
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Key Words: flumequine; carcinogenicity study; DNA damage; comet assay; DNA gyrase; topoisomerase II.
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INTRODUCTION |
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MATERIALS AND METHODS |
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The study period consisted of a 2-week initiation phase and a 13-week promotion phase. Mice in Groups 13 were fed basal diet (BD, Oriental Yeast, Tokyo, Japan) throughout the study period. Groups 4 and 5 received a diet containing FL at 4000 ppm for 2 weeks, followed by BD for 13 weeks, and Group 6 was fed a diet containing the same dose of FL throughout the study period. Groups 26 were intraperitoneally injected with Gal at 300 mg/kg during weeks 2 and 5. In addition, Groups 3 and 5 were exposed to 500 ppm of PB in their drinking water. The treatments with both Gal and PB were employed to provide tumor promotion effects within a relatively short experimental period. The animals were observed daily and weighed once a week throughout the study.
At the end of the experiment, all survivors were killed by exsanguination under ether anesthesia, and were necropsied. The livers were weighed and fixed in 10% phosphate-buffered formalin. The left lobe was embedded in paraffin, sectioned for staining with hematoxylin and eosin (H&E), and examined under a light microscope.
Quantitative data were analyzed using the F-test followed by a t-test to detect any significant differences between treatment and corresponding control groups. Incidences of hepatocellular foci were analyzed with Fishers exact test. A p-value less than 0.05 was considered statistically significant.
Comet assay in CHL/IU cells.
The Chinese hamster lung cell line CHL/IU was routinely maintained in monolayer culture in Dulbeccos modified MEM medium supplemented with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Exponentially growing cells were treated with FL dissolved in DMSO (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 1 h. The dose range was chosen in order to obtain both damaged and highly damaged cells as observed in a dose range-finding study. Following FL treatment, cells were embedded in GP42 agarose (Nakalai Tesque, Kyoto, Japan) dissolved in saline at 1% (Miyamae et al., 1997). Cell number and cell viability (Trypan blue exclusion method) were determined for each dose. Slides were placed in a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM TrisHCl, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) and kept at 0°C in the dark for >60 min, then in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 20 min in the dark at 0°C. Electrophoresis was conducted at 0°C in the dark for 20 min at 25 V (0.96 V/cm) and approximately 250 mA. The slides were then neutralized and stained with 50 µl of 20 µg/ml ethidium bromide.
The length of the whole comet was measured for 50 nuclei for each dose, and differences between the means in treated and control plates were compared with the Dunnett test after 1-way ANOVA. A p-value less than 0.05 was considered statistically significant.
Comet assay in mice.
Infant and young-adult male ddY mice were obtained from Japan SLC, Inc. at 4 and 7 weeks of age, respectively, and were used after 1 week of acclimatization. Groups were treated once orally with FL at <500 mg/kg. Adult mice were sacrificed at 3 and 24 h after treatment, and 8 organs, the stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow, were removed. Infant mice were sacrificed 3 and 24 h after treatment, and the livers were excised. In another study, the genotoxicity of FL was studied in the regenerating liver of adult mice. For this purpose, male mice at 8 weeks-of-age were anesthetized with ether and 3 major lobes of the liver, left lateral lobe, left medial lobe, and right lateral lobe, were removed. Four days after the hepatectomy, mice were subjected to oral administration of FL once. They were sacrificed 3 h after FL-treatment and regenerated livers were sampled. Slides for the comet assay were prepared at each set time, according to our methods established for multiple mouse organs (Sasaki et al., 2000). Slides prepared from nuclei isolated by homogenization were placed in a chilled lysing solution as described above, then in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 10 min in the dark at 0°C. Electrophoresis was conducted at 0°C in the dark for 15 min at 25 V (0.96 V/cm) and approximately 250 mA. The slides were neutralized and thereafter stained with 50 µl of 20 µg/ml ethidium bromide.
We calculated migration as the difference between the whole comet length and the diameter of the head for each. Mean migration of the 50 nuclei from each organ was calculated for each individual animal and differences between the averages for 4 treated animals and untreated controls were compared with the Dunnett test after 1-way ANOVA. A p-value less than 0.05 was considered statistically significant.
In vitro DNA gyrase/topoisomerase II inhibitory assay.
As control compounds, 3 quinolones, enrofloxacin (Bayer AG, Leverkusen, Germany), nalidixic acid (Sigma, St. Louis, MO) and levofloxacin (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan) were employed. Purified subunits A and B of DNA gyrase and relaxed pBR322 plasmid DNA were kindly supplied by Daiichi Pharmaceutical Co., Ltd. Human topoisomerase II and Topo II Assay Kit were purchased from TopoGEN, Inc. (Columbus, OH) and kinetoplast DNA (kDNA) was obtained from Nippon Gene (Toyama, Japan). Inhibitory activities of quinolones against DNA gyrase and topoisomerase II were assayed electrophoretically as described previously (Akasaka et al., 1998). For the supercoiling assay of DNA gyrase, 20 µl of reaction mixture containing subunits A and B (1 U of each), 0.2 µg of relaxed pBR322 plasmid DNA, and various concentrations of quinolones were incubated for 1h in buffer containing 20 mM TrisHCl (pH 7.5), 20 mM KCl, 4 mM MgCl2, 1 mM spermidine-HCl, 1 mM ATP, 1 mM dithiothreitol and 20 µg/ml of bovine serum albumin at 37°C. After stopping the reaction by adding sarcosyl with gel-loading buffer, supercoiled and relaxed pBR322 were separated by agarose gel electrophoresis. For the decatenation assay of topoisomerase II, 20 µl of reaction mixture containing 0.4 µg of kinetoplast DNA (kDNA, Nippon Gene, Toyama, Japan) with 1 U of topoisomerase II and serial dilutions of each antibacterial agent were incubated for 5 min at 37°C in buffer containing 20 mM TrisHCl (pH 7.5), 120 mM KCl, 10 mM MgCl2, 1 mM ATP, 0.5 mM dithiothreitol, and 30 µg/ml of bovine serum albumin. After stopping the reaction by adding sarcosyl with gel-loading buffer, catenated and decatenated kDNAs were separated by agarose gel electrophoresis. The gels were stained with ethidium bromide and photographed using UV light (302 nm) with a Gel Print 200i/VGA (Bio Image Co., Ann Arbor, MI), and the brightness of bands was traced with an Image Analyzer (Bio Image Co.). Each band was quantified and the amount of DNA treated with each concentration of quinolone was measured to determine the 50% inhibitory concentration against DNA gyrase and topoisomerase II.
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RESULTS |
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DISCUSSION |
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It is generally considered that initiation is closely related to adverse effects on DNA. The main reason that FL has been considered a promoter but not an initiator is its lack of genotoxicity in mutagenicity studies, including assays for gene mutation in bacteria and mammalian cells in vitro and for chromosomal aberrations in mammalian cells in vivo (WHO, 1997). In the present study, we revaluated its genotoxicity using the comet assay to measure DNA breakage in CHL/IU cells and showed FL to induce dose-dependent damage. In the liver of adult mice, only sporadic genotoxic effects were observed although FL targets the liver with regard to its carcinogenicity. In the regenerating liver of adult mice and the liver of infant mice, FL induced dose-dependent DNA damage; the most prominent effects were exerted in the regenerating liver. In B6C3F1 male mice, the frequency of hepatocytes in growth-phase is high (about 1%) at 5 weeks of age and constantly low (below 0.25%) after 6 weeks of age (Miyagawa et al., 1995
). From our results, FL would be expected to induce dose-dependent DNA damage in the liver in growth-phase, in line with the dose-dependent present findings for 3 organs with relatively high mitotic activities in adult mice: the stomach, colon, and urinary bladder.
Concerning the mechanism of DNA breakage induced by FL, Yoshida et al. (1999) demonstrated that staining of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was increased in the mouse liver treated with FL. They therefore concluded that oxidative stress might be a crucial factor for FL carcinogenicity. As another possible mechanism, inhibitory effects on DNA topoisomerases may be important. Though quinolone antibacterial agents work by inhibiting DNA gyrase (bacterial topoisomerase II) or topoisomerase IV, they are also known to have slight inhibitory effects on eukaryotic topoisomerase II, which is responsible for the double-strand DNA breakage/reunion reaction (Wang et al., 2001), due to similarities in the biochemical mechanisms and amino acid sequences between these enzymes (Lynn et al., 1986
). Accordingly, information on the relative selectivity toward bacterial target enzymes versus eukaryotic topoisomerase II is of significance, and a great deal of effort has been made to improve the specificity for gyrase or topoisomerase IV versus topoisomerase II (Lawrence, 2001). Both FL and nalidixic acid belong to the first generation of quinolones and their selectivity regarding bacterial gyrase is extremely low when compared to those of new quinolones such as enrofloxacin and levofloxacin, as confirmed in the present study. Actually, it has been demonstrated that topoisomerase-II inhibitors impair the DNA strand rejoining function of the enzyme, resulting in DNA single- and double-strand breaks (Snyder, 2000
). The comet assay can detect DNA damage caused directly by faulty repair or through alkali-labile lesions (Fairbairn et al., 1995
), and it has been already reported that it can detect the induction of DNA strand breaks by topoisomerase II inhibitors (Godard et al., 1999
). Thus etoposide, a typical example, has been shown to induce DNA strand breaks in dividing cells by the comet assay in vitro and in vivo (Godard, 1999). In the in vitro study, DNA damage was observed in CHO cells exposed for 1 h. In rats at 4 to 10 weeks of age, etoposide intraperitoneally injected at 5 or 50 mg/kg yielded DNA damage in the liver, intestine, thymus, and bone marrow but not in the kidney. Since the ages of the rats whose livers were damaged was not cited in their report, any growth-dependence is not clear. In the mucosa of gastrointestinal organs, however, positive results with etoposide in comet assay coincide well with those for FL. Therefore, the positive results of comet assay in the liver in the growth-phase and in different mucosa are consistent.
If flumequines genotoxicity is essentially limited to dividing cells, the observed lack of effects in the bone marrow, a tissue with significant mitotic activity, is puzzling. In fact, the highest incidence of DNA fragmentation with etoposide was observed in the bone marrow 1 h after its injection (Godard et al., 1999). One possible explanation for the lack of DNA-damage induction by FL might be that FL and/or its metabolites do not reach to the bone marrow. Since there are no reliable data on kinetics of this compound in mice, an answer to this question remains for further studies.
How do the genotoxic effects shown by the in vivo comet assay relate to rodent hepatic carcinogenicity? In carcinogenicity studies, including that reported here, adult animals are generally employed. As shown by Miyagawa et al. (1995), the frequency of hepatocytes in growth-phase is lower than 0.25% in mice after 6 weeks of age. One technical disadvantage of the comet assay is that it cannot evaluate large numbers of cells and fails to detect damaged cells if present at only very low frequency, in spite of its generally accepted high sensitivity (Tice et al., 1999). Therefore, the failure to detect clear dose-dependent induction of DNA damage in the livers of adult mice might reflect the small number of growth-phase hepatocytes. In addition, since FL has been shown to induce cell proliferation in hepatocytes of heterozygous p53-deficient mice initiated with DMN (Takizawa et al., 2002), it cannot be ruled out that FL enhances fixation of spontaneous and FL-induced DNA damage of these cells due to increased cell proliferation, and might result in its hepatocarcinogenicity.
FL has generally been considered to be a non-genotoxic carcinogen with only promoting activity (WHO, 1997). However, recent data in the literature and the results of the present study provide evidence that FL was not only a hepatic tumor promoter but also a hepatic tumor initiator. In addition, our results suggest that the initiating activity is due to its induction of DNA strand breaks. Accordingly, more extensive safety assessment of this compound is warranted.
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ACKNOWLEDGMENTS |
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NOTES |
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