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A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats
Mutation Research 742 (2012) 26–30
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats Kunio Wada ∗ , Aya Ohnuma, Sayuri Kojima, Toshinori Yoshida, Kyomu Matsumoto Toxicology Division, The Institute of Environmental Toxicology, 4321, Uchimoriya-machi, Joso-shi, Ibaraki 303-0043, Japan
a r t i c l e
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Article history: Received 3 February 2011 Received in revised form 4 November 2011 Accepted 16 November 2011 Available online 28 November 2011 Keywords: Comet assay Urinary bladder N-Methyl-N-nitrosourea Ethyl methanesulfonate o-Anisidine
a b s t r a c t Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague–Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Numerous classes of genotoxic chemicals have been identified as bladder carcinogens in rodents. Some of these have also been identified as carcinogenic in humans, including, most notably, aromatic amines, nitrosamines, and cyclophosphamide [1]. Several tests have been used for identifying agents with genotoxic activity in urinary bladders, including the single-cell gel electrophoresis (Comet) assay, the unscheduled DNA synthesis (UDS) assay, and 32 P-post-labeling; the Comet assay is a sensitive, rapid, and inexpensive method. The Comet assay was introduced by Singh et al. [2] as a simple method for detecting DNA damage at the individual cell level. It can detect double-strand breaks, single-strand breaks, alkali labile sites, oxidative base damage, incomplete excision repair sites, and DNA cross-linking with DNA or protein. The in vivo rodent Comet assay has two advantages. First, it can be used as a tool for the evaluation of local genotoxicity, especially for organs/cell types (e.g., urinary bladder) that cannot easily be evaluated with other assays. Second, the assay is particularly useful as a second in vivo test (e.g., as an alternative to the in vivo UDS test) [3]. In mechanistic studies,
∗ Corresponding author. Tel.: +81 297 27 4539; fax: +81 297 27 4518. E-mail address: [email protected] (K. Wada). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.11.008
the in vivo Comet assay has been applied to distinguish between genotoxic and non-genotoxic modes of action, causing the neoplastic changes observed in long-term rodent studies. Negative results obtained through an in vivo Comet assay can exclude a mechanism that acts directly on DNA, or can thus suggest the existence of a threshold. The Comet assay thus enables us to evaluate genotoxicity in the urinary bladder for the sake of investigation of local DNA damage in the tissue per se or a follow-up analysis on findings from carcinogenicity studies; however, there is no general agreement on the best method for collecting cells. The Comet assay has been conducted on urinary bladder cells in some laboratories, but using different cell-collecting methods. These methods can be categorized roughly into three types: those in which the bladder is digested by solutions that contain enzymes to harvest the transitional cells [4,5] or cells from the biopsied bladder [6] (the digestion method); those in which the bladder is scraped physically to obtain mucosal cells [7–11] (the scraping method); and those using whole cells obtained by mincing an intact urinary bladder with a pair of fine scissors [12,13] (the mincing method). Most malignancies of the urinary bladder arise from the epithelium and from transitional cell types [1]. The digestion method, which can selectively collect epithelial cells, is thought to be ideal for the in vivo Comet assays, but enzymatic digestion of the epithelium prevents further histopathological examination of the same bladder. Histopathology
K. Wada et al. / Mutation Research 742 (2012) 26–30
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is considered the “gold standard” for assessing levels of necrosis and apoptosis following positive results in an in vivo Comet assay [14]. Therefore, the scraping or mincing methods are appropriate for Comet assays accompanied by histopathological examination. However, the two methods have not been compared in urinary bladder tissues. Hence, we administered three known carcinogens (N-methyl-N-nitrosourea, ethyl methanesulfonate, and o-anisidine) to rats to compare the results of the Comet assay conducted on cells obtained from a bladder using the mincing and scraping methods. Furthermore, we morphologically analyzed urinary bladder cell types in homogenates prepared by the mincing method because this method may produce a mixture of different cell types. Factors such as the simplicity of the process with histopathological examination and the capability to detect DNA damaging agents are discussed in this paper.
coded, and the agarose was allowed to solidify at 4 ◦ C for 10 min. Then, the slides were immersed in lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, 1% Triton X-100, and 10% DMSO) at 4 ◦ C for one or two days. Next, the slides were rinsed with chilled pure water to remove residual detergent and salts and placed in electrophoresis solution (0.3 M NaOH and 1 mM Na2 EDTA; pH 13) for 20 min at 4 ◦ C to allow the DNA to unwind. Electrophoresis was conducted in a horizontal electrophoresis platform in fresh, chilled electrophoresis solution for 20 min. The amperage was set to 300 mA and the voltage was 25 V (0.7 V/cm). The slides were neutralized with Tris–HCl buffer (pH 7.5) for 5 min, dehydrated by immersion into absolute ethanol for 5 min, and stained with SYBR gold. Fifty cells per slide (100 cells per rat) were analyzed with an Olympus fluorescence microscope equipped with an automatic digital analysis system (Komet 5.5; Andor Technology, Belfast, UK), and the percentage of DNA in the tail (% tail DNA), which was the most suitable measurement parameter of DNA-break frequency [17], was calculated. Heavily damaged cells that showed large diffuse tails containing 90% or more of the DNA were excluded from data collection; however, the frequency of such comets (commonly referred to as hedgehogs) was measured as a reference for severe DNA damage per sample based on the visual scoring of 100 cells per slide (200 cells per rat). Cytotoxic damage in the urinary bladder was assessed by gross pathology.
2. Materials and methods
2.6. Morphological analysis
2.1. Chemicals
To examine a proportion of the epithelial cells obtained by the mincing method, the samples were cytocentrifuged and stained with May–Grünwald and Giemsa. A total of 400 cells in the control groups in each sex were counted under light microscopy and subclassified into five types by their shape of nucleus, as follows: (1) an epithelial cell with multiple round nuclei (umbrella cell); (2) an epithelial cell with a large elliptical nucleus; (3) an epithelial cell with a small round nucleus; (4) a mesenchymal cell with a spindle-shape nucleus; and (5) a mesenchymal cell with a nucleus of the other shapes. Selected slides were stained with anti-cytokeratin antibody by using the peroxidase-labeled polymer method (Envision kit) to confirm epithelial cells. Immunohistochemical products were visualized with the substrate 3,3 -diaminobenzidine and counterstained with hematoxylin. The morphological analysis in the scraping method could not be conducted because only a very small amount of cell pellets were obtained by the method.
N-Methyl-N-nitrosourea (MNU, CAS no. 684-93-5) and 2-methoxyaniline (oanisidine; OA, CAS no. 90-04-0) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethyl methanesulfonate (EMS, CAS no. 62-50-0) was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). SYBR gold was from Invitrogen Corp. (Carlsbad, CA, USA). Normal melting point (NMP) agarose (GP-42) was from Nacalai Tesque (Kyoto, Japan). Low melting point (LMP) agarose (NuSieve GTG) was from Lonza (Basel, Switzerland). Anti-cytokeratin antibody and Envision kit were from Dako (Glostrup, Denmark). 2.2. Rats The current study was conducted in accordance with the code of Ethics for Animal Experimentation in the Institute of Environmental Toxicology. Animals used in this study were female and male Sprague–Dawley [Crl: CD (SD)] rats purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan) at seven weeks old. The rats were randomly assigned to groups of four animals and acclimated for seven days. Pellet diet MF (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water in plastic water bottles were available ad libitum. The animal room was maintained at 22 ± 2 ◦ C and 50 ± 20% humidity on a 12-h light cycle. The ranges of body weight at treatment were 185–232 g and 277–320 g in females and males, respectively. 2.3. Treatment to rats MNU and EMS were dissolved in physiological saline (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan). OA was dissolved in corn oil (Wako). Physiological saline and corn oil were used as the solvent controls. These chemicals or solvent controls were administered to animals using oral gavages twice at 21-h intervals. MNU and EMS were given at doses of 41 mg/kg × 2 and 200 mg/kg × 2, respectively. The doses used for MNU and EMS were expected to give detectable DNA damage without clinical signs of toxicity. OA was given up to the maximum tolerated dose of 700 mg/kg × 2. All animals were sacrificed 3 h after the second administration. 2.4. Tissue sampling and cell collecting A urinary bladder was removed from a rat immediately after sacrifice and processed for the Comet assay and the morphological analysis. The urine of the rats treated with OA was examined for hematuria using urine test papers (Uriace® -Kc, Terumo Co., Tokyo, Japan). The urinary bladder sampled was incubated for 20–40 min in cold mincing buffer (20 mM Na2 EDTA and 10% DMSO in Hank’s balanced salt solution [Ca2+ , Mg2+ free], pH 7.5), and then turned inside out on a flat stainless steel bar to expose mucosa. The bar was positioned almost vertically to scrape one side of the mucosa up and down several times with a small spatula for isolating epithelial cells. Isolated cells were put into cold mincing buffer, and then provided for the Comet assay (the scraping method). The scraped part was cut off and discarded. The remaining part was rinsed with cold mincing buffer, put into a microtube containing the buffer, and minced with a pair of scissors for 30 s; after large clumps settled out, the supernatant of the tissue homogenate was used for the Comet assay and morphological analysis. 2.5. Comet assay The Comet assay was conducted under alkaline conditions as described in Tice et al. [15] and Hartmann et al. [16]. The cell suspension was mixed with 0.5% LMP agarose. The mixture containing cells and agarose was dropped onto a bottom layer of 1.0% NMP agarose on glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan) and spread using a cover slip. Two slides were prepared for each sample; they were
2.7. Statistics Comet data were expressed as the mean ± standard deviation. The mean % tail DNA in each treatment was statistically compared with the concurrent control group to evaluate the potential of each chemical to induce DNA damage. The frequency of heavily damaged cells in each treatment was statistically compared with the concurrent control group. The data were first analyzed for homogeneity of variance using the F-test. When the variances were homogeneous, results were evaluated using Student’s t-test. When the variances were not homogeneous, results were evaluated using the Aspin–Welch test. A p value of <0.05 was considered to be statistically significant.
3. Results 3.1. Comet assay results in female rats Table 1 shows the results of the Comet assay in female rats. When each test substance treatment group was compared with the concurrent solvent control group, statistically significant increases in % tail DNA were found in the MNU and EMS treatment groups, whereas no statistically significant increase was found in the OA treatment group, regardless of the cell-collecting methods. The frequency of heavily damaged cells in each treatment group was lower with the mincing method than with the scraping method. Statistically significant increases in the frequency of heavily damaged cells were found in the MNU and EMS treatment groups in the mincing method and in the MNU treatment group in the scraping method. Abnormalities in clinical signs and gross pathological changes were not observed in any animals in the MNU and EMS treatment group. In the OA treatment group, on the other hand, clinical abnormalities such as a decrease in spontaneous motor activity, loss of spontaneous motor activity, and lachrymation were detected in a few animals. In addition, reddish-brown urine was observed in animals treated with OA, but it was confirmed not to be hematuria by a urine test. This abnormally colored urine was considered to be attributed to excreting OA or its metabolites because the urine was similar to OA in color. No gross pathological changes were observed in this group.
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Table 1 DNA damage in the urinary bladders of female rats by the Comet assay with the mincing and scraping methods. Treatment
Dose (mg/kg)
No. of animals
Mincing
Scraping
% Tail DNA PS Corn oil MNU (in PS) EMS (in PS) OA (in corn oil)
0×2 0×2 41 × 2 200 × 2 700 × 2
4 4 4 4 4
6.88 8.29 48.08 33.94 9.88
± ± ± ± ±
1.67 2.83 3.31*** 4.06*** 2.59
Heavily damaged cells (%)a 1.0 2.6 3.9 4.1 2.6
± ± ± ± ±
0.9 1.6 1.4* 1.7* 2.8
% Tail DNA 5.70 9.09 48.38 28.47 8.91
± ± ± ± ±
2.39 2.70 2.10*** 4.04*** 2.18
Heavily damaged cells (%)a 2.5 6.3 13.0 10.4 5.5
± ± ± ± ±
1.1 4.6 5.8* 6.0 3.7
PS, physiological saline; MNU, N-methyl-N-nitrosourea; EMS, ethyl methanesulfonate; OA, o-anisidine. Mean of four animals per group ± S.D. a Frequency of cells with 90% or more of DNA in the tail. * Significantly different from the concurrent control group (p < 0.05). *** Significantly different from the concurrent control group (p < 0.001).
3.2. Comet assay results in male rats Table 2 shows the results of the Comet assay in male rats. Although the same protocol was used, the background level of DNA damage in the control groups was obviously higher in male than in female rats. When the MNU treatment groups were compared with the concurrent control groups, statistically significant increases were found regardless of the cell-collecting methods. When the EMS treatment groups were compared with the concurrent control groups, a statistically significant increase was found with the mincing method, whereas no statistically significant increase was found with the scraping method. When the OA treatment groups were compared with the concurrent control groups, no statistically significant increase was found regardless of the cell-collecting method. The frequency of heavily damaged cells in each treatment group was lower with the mincing method than with the scraping method. No statistically significant increase in the frequency of heavily damaged cells was found in any treatment group in either method. Abnormalities in clinical signs and gross pathological changes were not observed in any of the animals in the MNU and EMS treatment groups, whereas a decrease in spontaneous motor activity was detected in animals of the OA treatment group. Reddish brown urine and no gross pathological changes were also observed in the OA treatment group in the same manner as females. Although heavily damaged cells of 33.9% were observed in the OA treatment group in the scraping method, they did not result in a significant positive response in the Comet assay 3.3. Cells collected by the mincing method The suspension of cells and nuclei obtained by the mincing method is shown in Fig. 1. Epithelial cells with a large ellipsoid nucleus were confirmed by immunostaining for cytokeratin (Fig. 2),
Fig. 1. Bladder cells (nuclei) from female rat treated with corn oil, obtained by the mincing method. Almost all of the cells in this figure are epithelial cells. May–Grünwald stain (×400).
Fig. 2. Epithelial cells (arrow) from a male rat treated with physiological saline, obtained by the mincing method. Cytokeratin positive (×400).
whereas mesenchymal cells with a small spindle-shape nucleus were not stained (Fig. 3). The frequencies of epithelial cells, as determined by their nuclear morphology, were 82.6% in female and 87.4% in male rats (Table 3). These results indicated that epithelial cells were preferentially harvested by the mincing method. 4. Discussion The present study demonstrated that the mincing method detected DNA damage in bladders of MNU- and EMS-treated rats as well as the scraping method, indicating the mincing method had a sufficient capability to detect DNA damaging agents. The mincing method is superior to the scraping method in its simplicity. It only requires mincing for 30 s with a pair of scissors. In a standard in vivo Comet assay for evaluating its genotoxicity of a substance, we usually remove target tissues from at least 20 animals per sex
Fig. 3. A clump of mesenchymal cells from a male rat treated with physiological saline, obtained by the mincing method. Cytokeratin negative (×400).
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Table 2 DNA damage in the urinary bladders of male rats by the Comet assay with the mincing and scraping methods. Treatment
Dose (mg/kg)
No.of animals
Mincing
Scraping Heavily damaged cells (%)a
% Tail DNA PS Corn oil MNU (in PS) EMS (in PS) OA (in corn oil)
0×2 0×2 41 × 2 200 × 2 700 × 2
4 4 4 4 4
24.68 17.49 44.48 38.67 37.61
± ± ± ± ±
6.24 4.89 3.00** 7.40* 17.51
5.8 3.1 4.3 4.5 11.0
± ± ± ± ±
2.7 2.0 2.2 3.0 8.0
% Tail DNA 31.68 20.94 47.18 37.75 42.60
± ± ± ± ±
7.32 9.68 2.20* 12.22 26.71
Heavily damaged cells (%)a 18.1 5.9 12.5 9.0 33.9
± ± ± ± ±
11.9 3.0 8.0 6.1 25.4
PS, physiological saline; MNU, N-methyl-N-nitrosourea; EMS, ethyl methanesulfonate; OA, o-anisidine. Mean of four animals per group ± S.D. a Frequency of cells with 90% or more of DNA in the tail. * Significantly different from the concurrent control group (p < 0.05). ** Significantly different from the concurrent control group (p < 0.01).
(five dose levels including negative and positive controls, four animals per dose) in a day, and cells need to be collected as soon as possible after sacrifice. Technical simplicity is one of critical points for such a large scale in vivo investigation. Another advantage of the mincing method is that it is suitable for conducting a concurrent histopathological examination. In this study, a urinary bladder was turned inside out to expose mucosa for scraping; however, when a concurrent histopathological examination is conducted, this step should be omitted to avoid disruptions of the tissue structure. When a urinary bladder is cut in half at first for both the Comet assay and histopathological examination, the halved tissue is thin and small, and curls readily. Anyone would be able to mince it; however, only a skillful technician would be able to scrape mucosa from it. Therefore, we recommend the mincing method, without eversion, as follows: a bladder is taken from an animal and cut longitudinally into two halves. One half is fixed for histopathological examination and the other is subjected to the Comet assay as described in Section 2. In each sex, MNU gave positive results regardless of the cellcollecting methods. MNU is an alkylating agent and a carcinogen for many organs. Bladder cancer was caused by the intravesical administration of MNU to rats [18]. Our study indicated DNA damage induced by MNU in the urinary bladder as a cause for a bladder cancer. EMS gave positive results in both the mincing and scraping methods in female rats and in the mincing method in male rats. Although a negative response was obtained by the scraping method in male rats, we cannot exclude the possibility that the high background level (31.68%) in the control group of the scraping method decreased the sensitivity of DNA damage detection. The mean % tail DNA in the EMS treatment group of the scraping method was similar to that of the mincing method. Therefore, we concluded that EMS induced DNA damage in the urinary bladder. EMS induced DNA damage in other organs such as the liver, kidney, and lung of mice after oral administration [19]. It is a potent clastogen [20] and induced adenocarcinoma in the mammary glands, mesenchymal tumor in the kidney, and leiomyosarcoma in the uterus of rats [21]. Although EMS induced urothelial hyperplasia in rats after two-year exposure, no neoplasms were seen in urinary bladder [22]. Sasaki et al. [23] reported that the mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. OA gave negative results regardless of the cell-collecting methods in each sex when it was given up to the maximum tolerated
dose. OA is possibly carcinogenic to humans (Group 2B) [24], and produced transitional cell carcinomas of the urinary bladder in mice and rats of each sex [25]. Despite its carcinogenicity, almost all rodent genotoxicity assays have failed to detect OA as a genotoxin in vivo. Only two assays were successful in detecting the genotoxicity of OA in the urinary bladder: (1) Ashby et al. [26] reported a small increase in mutation frequency in the absence of OA–DNA adducts in the urinary bladder; (2) Sasaki et al. [27] showed a positive response in the Comet assay by the scraping method in the urinary bladder of male CD-1 mice. Our result contradicts Sasaki et al.’s study, and this discrepancy might be explained by differences in metabolic activation systems between animal species used. Previous studies have suggested a possible role for one or more peroxidation enzymes in the metabolic activation of OA to a genotoxic species [28,29]. Further experiments should be conducted to clarify a genotoxic mechanism of OA. Heavily damaged cells were difficult to score by image analysis automatically, and thus they are listed separately in Tables 1 and 2. The frequency of such cells seems to depend on preparation methods; scraping was associated with higher levels of these cells compared with mincing, especially in males. A sample containing approximately 80% epithelial cells was collected by the mincing method. Epithelial cells had a tendency to become isolated single cells while mesenchymal cells did not. Fig. 3 demonstrates the clumps of mesenchymal cells obtained after mincing. These clumps precipitated quickly at the bottom of the buffer and were not used for the Comet assay. As a result, a high frequency of epithelial cells was obtained by the mincing method. Assuming that the scraping method was able to collect the only epithelial cells, the sensitivity to detect DNA damage in the minced samples might be lower than that in the scraped samples. Nevertheless, the detecting capabilities of both the methods appeared comparable. This fact suggests that the scraped samples contain mesenchymal cells with almost the same proportion as the minced samples, or that not only epithelial but also mesenchymal cells are suffered DNA damage induced by direct or indirect effects of the test substance and/or its metabolites. We should emphasize an additional interesting finding noted in this study. Although the identical protocol was employed for both sexes, the mean % tail DNA in each concurrent control group was higher in male than in female rats, regardless of the cell-collecting method. The same tendency occurred in a pre-experiment of the Comet assay (data not shown). These data strongly indicate the existence of a sex difference in the background level of DNA
Table 3 Profiles of the urinary bladder cells obtained by the mincing method in female and male rats. Transitional epithelial cell (%)
Female Male
Mesenchymal cell (%)
Umbrella cell
Large ellipsoid
Small round
Total
Spindle
Others
Total
3.5 4.4
43.1 47.2
36.0 35.8
82.6 87.4
12.9 10.1
4.5 2.5
17.4 12.6
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damage in the urinary bladder of SD rats, but there is no report on the sex difference in urinary bladder carcinogenicity in SD rats. We are presently investigating to confirm the universality of such a sex difference on the background level in the Comet assay in urinary bladders using other strains of rats. In conclusion, the mincing method can be used as a standard procedure for the isolation of urinary bladder cells, though the contaminating non-epithelial cells with over 10% are inevitable. The use of the Comet assay with the mincing method for evaluating genotoxicity in urinary bladders is expected to increase in the future. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We thank Mr. Yuzo Takezawa and Ms. Misaki Abe for their technical assistance and Mrs. Hitomi Wada for critical reading of the manuscript. References [1] S.M. Cohen, Urinary bladder carcinogenesis, Toxicol. Pathol. 26 (1998) 121–127. [2] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [3] S. Brendler-Schwaab, A. Hartmann, S. Pfuhler, G. Speit, The in vivo Comet assay: use and status in genotoxicity testing, Mutagenesis 20 (2005) 245–254. [4] L. Robbiano, R. Carrozzino, M. Bacigalupo, C. Corbu, G. Brambilla, Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity, Toxicology 179 (2002) 115–128. [5] A. Wang, J.L. Robertson, S.D. Holladay, A.H. Tennant, A.J. Lengi, S.A. Ahmed, W.R. Huckle, A.D. Kligerman, Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols, Mutat. Res. 634 (2007) 51–59. [6] D.D. Miranda, D.P. Arc¸ari, J. Pedrazzoli Jr., P.O. Carvalho, S.M. Cerutti, D.H. Bastos, M.L. Ribeiro, Protective effects of mate tea (Ilex paraguariensis) on H2 O2 -induced DNA damage and DNA repair in mice, Mutagenesis 23 (2008) 261–265. [7] Y.F. Sasaki, E. Nishidate, F. Izumiyama, M. Watanabe-Akanuma, N. Kinae, N. Matsusaka, S. Tsuda, Detection of in vivo genotoxicity of 3-chloro-4(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs, Mutat. Res. 393 (1997) 47–53. [8] A. Martelli, L. Robbiano, R. Carrozzino, C.P. Puglia, F. Mattioli, M. Angiola, G. Brambilla, DNA damage induced by 3,3 -dimethoxybenzidine in liver and urinary bladder cells of rats and humans, Toxicol. Sci. 53 (2000) 71–76. [9] S. Tsuda, M. Murakami, N. Matsusaka, K. Kano, K. Taniguchi, Y.F. Sasaki, DNA damage induced by red food dyes orally administered to pregnant and male mice, Toxicol. Sci. 61 (2001) 92–99. [10] K. Sekihashi, A. Yamamoto, Y. Matsumura, S. Ueno, M. Watanabe-Akanuma, F. Kassie, S. Knasmüller, S. Tsuda, Y.F. Sasaki, Comparative investigation of multiple organs of mice and rats in the comet assay, Mutat. Res. 517 (2002) 53–75. [11] M.G. Nascimento, M.L. de Oliveira, A.S. Lima, J.L. de Camargo, Effects of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the urinary bladder of male Wistar rats, Toxicology 224 (2006) 66–73.
[12] R.R. Tice, J.W. Yager, P. Andrews, E. Crecelius, Effect of hepatic methyl donor status on urinary excretion and DNA damage in B6C3F1 mice treated with sodium arsenite, Mutat. Res. 386 (1997) 315–334. [13] R.M. Rosa, N.C. Hoch, G.V. Furtado, J. Saffi, J.A. Henriques, DNA damage in tissues and organs of mice treated with diphenyl diselenide, Mutat. Res. 633 (2007) 35–45. [14] B. Burlinson, R.R. Tice, G. Speit, E. Agurell, S.Y. Brendler-Schwaab, A.R. Collins, P. Escobar, M. Honma, T.S. Kumaravel, M. Nakajima, Y.F. Sasaki, V. Thybaud, Y. Uno, M. Vasquez, A. Hartmann, Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup, Mutat. Res. 627 (2007) 31–35. [15] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (2000) 206–221. [16] A. Hartmann, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A. Collins, A. Smith, G. Speit, V. Thybaud, R.R. Tice, Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop, Mutagenesis 18 (2003) 45–51. [17] T.S. Kumaravel, A.N. Jha, Reliable, Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals, Mutat. Res. 605 (2006) 7–16. [18] G.D. Steinberg, C.B. Brendler, T. Ichikawa, R.A. Squire, J.T. Isaacs, Characterization of an N-methyl-N-nitrosourea-induced autochthonous rat bladder cancer model, Cancer Res. 50 (1990) 6668–6674. [19] Y.F. Sasaki, F. Izumiyama, E. Nishidate, N. Matsusaka, S. Tsuda, Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow), Mutat. Res. 391 (1997) 201–214. [20] L. den Engelse, B.G. Floot, R.J. de Brij, A.D. Tates, The induction of chromosomal damage in rat hepatocytes and lymphocytes. II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine, dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects, Mutat. Res. 107 (1983) 153–166. [21] H. Ueo, R. Takaki, H. Yamagami, K. Sugimachi, Mammary carcinoma induced by oral administration of ethyl methanesulphonate. Determination of some of the parameters affecting tumor induction, Carcinogenesis 2 (1981) 1223–1228. [22] R.J. Tudor, N.J. Severs, R.M. Hicks, The induction of urothelial hyperplasia by methyl methanesulphonate and ethyl methanesulphonate, Br. J. Cancer 48 (1983) 289–300. [23] Y.F. Sasaki, K. Sekihashi, F. Izumiyama, E. Nishidate, A. Saga, K. Ishida, S. Tsuda, The Comet assay with multiple mouse organs: comparison of Comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database, Crit. Rev. Toxicol. 30 (2000) 629–799. [24] IARC Monographs on Evaluation of Carcinogenetic Risks of Chemicals to Humans, Ortho-Anisidine, vol. 73, International Agency for Research on Cancer, 1999, pp. 49–58. [25] IARC Monographs on Evaluation of Carcinogenetic Risks of Chemicals to Humans, Some Aromatic Amines, Anthraquinones and Nitroso Compounds, and Inorganic Fluorides Used in Drinking Water and Dental Preparations, vol. 27, International Agency for Research on Cancer, 1982, pp. 63–80. [26] J. Ashby, J.M. Short, N.J. Jones, P.A. Lefevre, G.S. Provost, B.J. Rogers, E.A. Martin, J.M. Parry, K. Burnette, B.W. Glickman, H. Tinwell, Mutagenicity of o-anisidine to the bladder of lacI− transgenic B6C3F1 mice: absence of 14 C or 32 P bladder DNA adduction, Carcinogenesis 15 (1994) 2291–2296. [27] Y.F. Sasaki, E. Nishidate, Y.Q. Su, N. Matsusaka, S. Tsuda, N. Susa, Y. Furukawa, S. Ueno, Organ-specific genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine, Mutat. Res. 412 (1998) 155–160. [28] D.C. Thompson, P.D. Josephy, J.W. Chu, T.E. Eling, Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity, Mutat. Res. 279 (1992) 83–89. [29] M. Stiborova, M. Miksanova, V. Havlicek, H.H. Schmeiser, E. Frei, Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA, Mutat. Res. 500 (2002) 49–66.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats Kunio Wada ∗ , Aya Ohnuma, Sayuri Kojima, Toshinori Yoshida, Kyomu Matsumoto Toxicology Division, The Institute of Environmental Toxicology, 4321, Uchimoriya-machi, Joso-shi, Ibaraki 303-0043, Japan
a r t i c l e
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Article history: Received 3 February 2011 Received in revised form 4 November 2011 Accepted 16 November 2011 Available online 28 November 2011 Keywords: Comet assay Urinary bladder N-Methyl-N-nitrosourea Ethyl methanesulfonate o-Anisidine
a b s t r a c t Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague–Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Numerous classes of genotoxic chemicals have been identified as bladder carcinogens in rodents. Some of these have also been identified as carcinogenic in humans, including, most notably, aromatic amines, nitrosamines, and cyclophosphamide [1]. Several tests have been used for identifying agents with genotoxic activity in urinary bladders, including the single-cell gel electrophoresis (Comet) assay, the unscheduled DNA synthesis (UDS) assay, and 32 P-post-labeling; the Comet assay is a sensitive, rapid, and inexpensive method. The Comet assay was introduced by Singh et al. [2] as a simple method for detecting DNA damage at the individual cell level. It can detect double-strand breaks, single-strand breaks, alkali labile sites, oxidative base damage, incomplete excision repair sites, and DNA cross-linking with DNA or protein. The in vivo rodent Comet assay has two advantages. First, it can be used as a tool for the evaluation of local genotoxicity, especially for organs/cell types (e.g., urinary bladder) that cannot easily be evaluated with other assays. Second, the assay is particularly useful as a second in vivo test (e.g., as an alternative to the in vivo UDS test) [3]. In mechanistic studies,
∗ Corresponding author. Tel.: +81 297 27 4539; fax: +81 297 27 4518. E-mail address: [email protected] (K. Wada). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.11.008
the in vivo Comet assay has been applied to distinguish between genotoxic and non-genotoxic modes of action, causing the neoplastic changes observed in long-term rodent studies. Negative results obtained through an in vivo Comet assay can exclude a mechanism that acts directly on DNA, or can thus suggest the existence of a threshold. The Comet assay thus enables us to evaluate genotoxicity in the urinary bladder for the sake of investigation of local DNA damage in the tissue per se or a follow-up analysis on findings from carcinogenicity studies; however, there is no general agreement on the best method for collecting cells. The Comet assay has been conducted on urinary bladder cells in some laboratories, but using different cell-collecting methods. These methods can be categorized roughly into three types: those in which the bladder is digested by solutions that contain enzymes to harvest the transitional cells [4,5] or cells from the biopsied bladder [6] (the digestion method); those in which the bladder is scraped physically to obtain mucosal cells [7–11] (the scraping method); and those using whole cells obtained by mincing an intact urinary bladder with a pair of fine scissors [12,13] (the mincing method). Most malignancies of the urinary bladder arise from the epithelium and from transitional cell types [1]. The digestion method, which can selectively collect epithelial cells, is thought to be ideal for the in vivo Comet assays, but enzymatic digestion of the epithelium prevents further histopathological examination of the same bladder. Histopathology
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is considered the “gold standard” for assessing levels of necrosis and apoptosis following positive results in an in vivo Comet assay [14]. Therefore, the scraping or mincing methods are appropriate for Comet assays accompanied by histopathological examination. However, the two methods have not been compared in urinary bladder tissues. Hence, we administered three known carcinogens (N-methyl-N-nitrosourea, ethyl methanesulfonate, and o-anisidine) to rats to compare the results of the Comet assay conducted on cells obtained from a bladder using the mincing and scraping methods. Furthermore, we morphologically analyzed urinary bladder cell types in homogenates prepared by the mincing method because this method may produce a mixture of different cell types. Factors such as the simplicity of the process with histopathological examination and the capability to detect DNA damaging agents are discussed in this paper.
coded, and the agarose was allowed to solidify at 4 ◦ C for 10 min. Then, the slides were immersed in lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, 1% Triton X-100, and 10% DMSO) at 4 ◦ C for one or two days. Next, the slides were rinsed with chilled pure water to remove residual detergent and salts and placed in electrophoresis solution (0.3 M NaOH and 1 mM Na2 EDTA; pH 13) for 20 min at 4 ◦ C to allow the DNA to unwind. Electrophoresis was conducted in a horizontal electrophoresis platform in fresh, chilled electrophoresis solution for 20 min. The amperage was set to 300 mA and the voltage was 25 V (0.7 V/cm). The slides were neutralized with Tris–HCl buffer (pH 7.5) for 5 min, dehydrated by immersion into absolute ethanol for 5 min, and stained with SYBR gold. Fifty cells per slide (100 cells per rat) were analyzed with an Olympus fluorescence microscope equipped with an automatic digital analysis system (Komet 5.5; Andor Technology, Belfast, UK), and the percentage of DNA in the tail (% tail DNA), which was the most suitable measurement parameter of DNA-break frequency [17], was calculated. Heavily damaged cells that showed large diffuse tails containing 90% or more of the DNA were excluded from data collection; however, the frequency of such comets (commonly referred to as hedgehogs) was measured as a reference for severe DNA damage per sample based on the visual scoring of 100 cells per slide (200 cells per rat). Cytotoxic damage in the urinary bladder was assessed by gross pathology.
2. Materials and methods
2.6. Morphological analysis
2.1. Chemicals
To examine a proportion of the epithelial cells obtained by the mincing method, the samples were cytocentrifuged and stained with May–Grünwald and Giemsa. A total of 400 cells in the control groups in each sex were counted under light microscopy and subclassified into five types by their shape of nucleus, as follows: (1) an epithelial cell with multiple round nuclei (umbrella cell); (2) an epithelial cell with a large elliptical nucleus; (3) an epithelial cell with a small round nucleus; (4) a mesenchymal cell with a spindle-shape nucleus; and (5) a mesenchymal cell with a nucleus of the other shapes. Selected slides were stained with anti-cytokeratin antibody by using the peroxidase-labeled polymer method (Envision kit) to confirm epithelial cells. Immunohistochemical products were visualized with the substrate 3,3 -diaminobenzidine and counterstained with hematoxylin. The morphological analysis in the scraping method could not be conducted because only a very small amount of cell pellets were obtained by the method.
N-Methyl-N-nitrosourea (MNU, CAS no. 684-93-5) and 2-methoxyaniline (oanisidine; OA, CAS no. 90-04-0) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethyl methanesulfonate (EMS, CAS no. 62-50-0) was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). SYBR gold was from Invitrogen Corp. (Carlsbad, CA, USA). Normal melting point (NMP) agarose (GP-42) was from Nacalai Tesque (Kyoto, Japan). Low melting point (LMP) agarose (NuSieve GTG) was from Lonza (Basel, Switzerland). Anti-cytokeratin antibody and Envision kit were from Dako (Glostrup, Denmark). 2.2. Rats The current study was conducted in accordance with the code of Ethics for Animal Experimentation in the Institute of Environmental Toxicology. Animals used in this study were female and male Sprague–Dawley [Crl: CD (SD)] rats purchased from Charles River Laboratories Japan Inc. (Kanagawa, Japan) at seven weeks old. The rats were randomly assigned to groups of four animals and acclimated for seven days. Pellet diet MF (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water in plastic water bottles were available ad libitum. The animal room was maintained at 22 ± 2 ◦ C and 50 ± 20% humidity on a 12-h light cycle. The ranges of body weight at treatment were 185–232 g and 277–320 g in females and males, respectively. 2.3. Treatment to rats MNU and EMS were dissolved in physiological saline (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan). OA was dissolved in corn oil (Wako). Physiological saline and corn oil were used as the solvent controls. These chemicals or solvent controls were administered to animals using oral gavages twice at 21-h intervals. MNU and EMS were given at doses of 41 mg/kg × 2 and 200 mg/kg × 2, respectively. The doses used for MNU and EMS were expected to give detectable DNA damage without clinical signs of toxicity. OA was given up to the maximum tolerated dose of 700 mg/kg × 2. All animals were sacrificed 3 h after the second administration. 2.4. Tissue sampling and cell collecting A urinary bladder was removed from a rat immediately after sacrifice and processed for the Comet assay and the morphological analysis. The urine of the rats treated with OA was examined for hematuria using urine test papers (Uriace® -Kc, Terumo Co., Tokyo, Japan). The urinary bladder sampled was incubated for 20–40 min in cold mincing buffer (20 mM Na2 EDTA and 10% DMSO in Hank’s balanced salt solution [Ca2+ , Mg2+ free], pH 7.5), and then turned inside out on a flat stainless steel bar to expose mucosa. The bar was positioned almost vertically to scrape one side of the mucosa up and down several times with a small spatula for isolating epithelial cells. Isolated cells were put into cold mincing buffer, and then provided for the Comet assay (the scraping method). The scraped part was cut off and discarded. The remaining part was rinsed with cold mincing buffer, put into a microtube containing the buffer, and minced with a pair of scissors for 30 s; after large clumps settled out, the supernatant of the tissue homogenate was used for the Comet assay and morphological analysis. 2.5. Comet assay The Comet assay was conducted under alkaline conditions as described in Tice et al. [15] and Hartmann et al. [16]. The cell suspension was mixed with 0.5% LMP agarose. The mixture containing cells and agarose was dropped onto a bottom layer of 1.0% NMP agarose on glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan) and spread using a cover slip. Two slides were prepared for each sample; they were
2.7. Statistics Comet data were expressed as the mean ± standard deviation. The mean % tail DNA in each treatment was statistically compared with the concurrent control group to evaluate the potential of each chemical to induce DNA damage. The frequency of heavily damaged cells in each treatment was statistically compared with the concurrent control group. The data were first analyzed for homogeneity of variance using the F-test. When the variances were homogeneous, results were evaluated using Student’s t-test. When the variances were not homogeneous, results were evaluated using the Aspin–Welch test. A p value of <0.05 was considered to be statistically significant.
3. Results 3.1. Comet assay results in female rats Table 1 shows the results of the Comet assay in female rats. When each test substance treatment group was compared with the concurrent solvent control group, statistically significant increases in % tail DNA were found in the MNU and EMS treatment groups, whereas no statistically significant increase was found in the OA treatment group, regardless of the cell-collecting methods. The frequency of heavily damaged cells in each treatment group was lower with the mincing method than with the scraping method. Statistically significant increases in the frequency of heavily damaged cells were found in the MNU and EMS treatment groups in the mincing method and in the MNU treatment group in the scraping method. Abnormalities in clinical signs and gross pathological changes were not observed in any animals in the MNU and EMS treatment group. In the OA treatment group, on the other hand, clinical abnormalities such as a decrease in spontaneous motor activity, loss of spontaneous motor activity, and lachrymation were detected in a few animals. In addition, reddish-brown urine was observed in animals treated with OA, but it was confirmed not to be hematuria by a urine test. This abnormally colored urine was considered to be attributed to excreting OA or its metabolites because the urine was similar to OA in color. No gross pathological changes were observed in this group.
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K. Wada et al. / Mutation Research 742 (2012) 26–30
Table 1 DNA damage in the urinary bladders of female rats by the Comet assay with the mincing and scraping methods. Treatment
Dose (mg/kg)
No. of animals
Mincing
Scraping
% Tail DNA PS Corn oil MNU (in PS) EMS (in PS) OA (in corn oil)
0×2 0×2 41 × 2 200 × 2 700 × 2
4 4 4 4 4
6.88 8.29 48.08 33.94 9.88
± ± ± ± ±
1.67 2.83 3.31*** 4.06*** 2.59
Heavily damaged cells (%)a 1.0 2.6 3.9 4.1 2.6
± ± ± ± ±
0.9 1.6 1.4* 1.7* 2.8
% Tail DNA 5.70 9.09 48.38 28.47 8.91
± ± ± ± ±
2.39 2.70 2.10*** 4.04*** 2.18
Heavily damaged cells (%)a 2.5 6.3 13.0 10.4 5.5
± ± ± ± ±
1.1 4.6 5.8* 6.0 3.7
PS, physiological saline; MNU, N-methyl-N-nitrosourea; EMS, ethyl methanesulfonate; OA, o-anisidine. Mean of four animals per group ± S.D. a Frequency of cells with 90% or more of DNA in the tail. * Significantly different from the concurrent control group (p < 0.05). *** Significantly different from the concurrent control group (p < 0.001).
3.2. Comet assay results in male rats Table 2 shows the results of the Comet assay in male rats. Although the same protocol was used, the background level of DNA damage in the control groups was obviously higher in male than in female rats. When the MNU treatment groups were compared with the concurrent control groups, statistically significant increases were found regardless of the cell-collecting methods. When the EMS treatment groups were compared with the concurrent control groups, a statistically significant increase was found with the mincing method, whereas no statistically significant increase was found with the scraping method. When the OA treatment groups were compared with the concurrent control groups, no statistically significant increase was found regardless of the cell-collecting method. The frequency of heavily damaged cells in each treatment group was lower with the mincing method than with the scraping method. No statistically significant increase in the frequency of heavily damaged cells was found in any treatment group in either method. Abnormalities in clinical signs and gross pathological changes were not observed in any of the animals in the MNU and EMS treatment groups, whereas a decrease in spontaneous motor activity was detected in animals of the OA treatment group. Reddish brown urine and no gross pathological changes were also observed in the OA treatment group in the same manner as females. Although heavily damaged cells of 33.9% were observed in the OA treatment group in the scraping method, they did not result in a significant positive response in the Comet assay 3.3. Cells collected by the mincing method The suspension of cells and nuclei obtained by the mincing method is shown in Fig. 1. Epithelial cells with a large ellipsoid nucleus were confirmed by immunostaining for cytokeratin (Fig. 2),
Fig. 1. Bladder cells (nuclei) from female rat treated with corn oil, obtained by the mincing method. Almost all of the cells in this figure are epithelial cells. May–Grünwald stain (×400).
Fig. 2. Epithelial cells (arrow) from a male rat treated with physiological saline, obtained by the mincing method. Cytokeratin positive (×400).
whereas mesenchymal cells with a small spindle-shape nucleus were not stained (Fig. 3). The frequencies of epithelial cells, as determined by their nuclear morphology, were 82.6% in female and 87.4% in male rats (Table 3). These results indicated that epithelial cells were preferentially harvested by the mincing method. 4. Discussion The present study demonstrated that the mincing method detected DNA damage in bladders of MNU- and EMS-treated rats as well as the scraping method, indicating the mincing method had a sufficient capability to detect DNA damaging agents. The mincing method is superior to the scraping method in its simplicity. It only requires mincing for 30 s with a pair of scissors. In a standard in vivo Comet assay for evaluating its genotoxicity of a substance, we usually remove target tissues from at least 20 animals per sex
Fig. 3. A clump of mesenchymal cells from a male rat treated with physiological saline, obtained by the mincing method. Cytokeratin negative (×400).
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Table 2 DNA damage in the urinary bladders of male rats by the Comet assay with the mincing and scraping methods. Treatment
Dose (mg/kg)
No.of animals
Mincing
Scraping Heavily damaged cells (%)a
% Tail DNA PS Corn oil MNU (in PS) EMS (in PS) OA (in corn oil)
0×2 0×2 41 × 2 200 × 2 700 × 2
4 4 4 4 4
24.68 17.49 44.48 38.67 37.61
± ± ± ± ±
6.24 4.89 3.00** 7.40* 17.51
5.8 3.1 4.3 4.5 11.0
± ± ± ± ±
2.7 2.0 2.2 3.0 8.0
% Tail DNA 31.68 20.94 47.18 37.75 42.60
± ± ± ± ±
7.32 9.68 2.20* 12.22 26.71
Heavily damaged cells (%)a 18.1 5.9 12.5 9.0 33.9
± ± ± ± ±
11.9 3.0 8.0 6.1 25.4
PS, physiological saline; MNU, N-methyl-N-nitrosourea; EMS, ethyl methanesulfonate; OA, o-anisidine. Mean of four animals per group ± S.D. a Frequency of cells with 90% or more of DNA in the tail. * Significantly different from the concurrent control group (p < 0.05). ** Significantly different from the concurrent control group (p < 0.01).
(five dose levels including negative and positive controls, four animals per dose) in a day, and cells need to be collected as soon as possible after sacrifice. Technical simplicity is one of critical points for such a large scale in vivo investigation. Another advantage of the mincing method is that it is suitable for conducting a concurrent histopathological examination. In this study, a urinary bladder was turned inside out to expose mucosa for scraping; however, when a concurrent histopathological examination is conducted, this step should be omitted to avoid disruptions of the tissue structure. When a urinary bladder is cut in half at first for both the Comet assay and histopathological examination, the halved tissue is thin and small, and curls readily. Anyone would be able to mince it; however, only a skillful technician would be able to scrape mucosa from it. Therefore, we recommend the mincing method, without eversion, as follows: a bladder is taken from an animal and cut longitudinally into two halves. One half is fixed for histopathological examination and the other is subjected to the Comet assay as described in Section 2. In each sex, MNU gave positive results regardless of the cellcollecting methods. MNU is an alkylating agent and a carcinogen for many organs. Bladder cancer was caused by the intravesical administration of MNU to rats [18]. Our study indicated DNA damage induced by MNU in the urinary bladder as a cause for a bladder cancer. EMS gave positive results in both the mincing and scraping methods in female rats and in the mincing method in male rats. Although a negative response was obtained by the scraping method in male rats, we cannot exclude the possibility that the high background level (31.68%) in the control group of the scraping method decreased the sensitivity of DNA damage detection. The mean % tail DNA in the EMS treatment group of the scraping method was similar to that of the mincing method. Therefore, we concluded that EMS induced DNA damage in the urinary bladder. EMS induced DNA damage in other organs such as the liver, kidney, and lung of mice after oral administration [19]. It is a potent clastogen [20] and induced adenocarcinoma in the mammary glands, mesenchymal tumor in the kidney, and leiomyosarcoma in the uterus of rats [21]. Although EMS induced urothelial hyperplasia in rats after two-year exposure, no neoplasms were seen in urinary bladder [22]. Sasaki et al. [23] reported that the mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. OA gave negative results regardless of the cell-collecting methods in each sex when it was given up to the maximum tolerated
dose. OA is possibly carcinogenic to humans (Group 2B) [24], and produced transitional cell carcinomas of the urinary bladder in mice and rats of each sex [25]. Despite its carcinogenicity, almost all rodent genotoxicity assays have failed to detect OA as a genotoxin in vivo. Only two assays were successful in detecting the genotoxicity of OA in the urinary bladder: (1) Ashby et al. [26] reported a small increase in mutation frequency in the absence of OA–DNA adducts in the urinary bladder; (2) Sasaki et al. [27] showed a positive response in the Comet assay by the scraping method in the urinary bladder of male CD-1 mice. Our result contradicts Sasaki et al.’s study, and this discrepancy might be explained by differences in metabolic activation systems between animal species used. Previous studies have suggested a possible role for one or more peroxidation enzymes in the metabolic activation of OA to a genotoxic species [28,29]. Further experiments should be conducted to clarify a genotoxic mechanism of OA. Heavily damaged cells were difficult to score by image analysis automatically, and thus they are listed separately in Tables 1 and 2. The frequency of such cells seems to depend on preparation methods; scraping was associated with higher levels of these cells compared with mincing, especially in males. A sample containing approximately 80% epithelial cells was collected by the mincing method. Epithelial cells had a tendency to become isolated single cells while mesenchymal cells did not. Fig. 3 demonstrates the clumps of mesenchymal cells obtained after mincing. These clumps precipitated quickly at the bottom of the buffer and were not used for the Comet assay. As a result, a high frequency of epithelial cells was obtained by the mincing method. Assuming that the scraping method was able to collect the only epithelial cells, the sensitivity to detect DNA damage in the minced samples might be lower than that in the scraped samples. Nevertheless, the detecting capabilities of both the methods appeared comparable. This fact suggests that the scraped samples contain mesenchymal cells with almost the same proportion as the minced samples, or that not only epithelial but also mesenchymal cells are suffered DNA damage induced by direct or indirect effects of the test substance and/or its metabolites. We should emphasize an additional interesting finding noted in this study. Although the identical protocol was employed for both sexes, the mean % tail DNA in each concurrent control group was higher in male than in female rats, regardless of the cell-collecting method. The same tendency occurred in a pre-experiment of the Comet assay (data not shown). These data strongly indicate the existence of a sex difference in the background level of DNA
Table 3 Profiles of the urinary bladder cells obtained by the mincing method in female and male rats. Transitional epithelial cell (%)
Female Male
Mesenchymal cell (%)
Umbrella cell
Large ellipsoid
Small round
Total
Spindle
Others
Total
3.5 4.4
43.1 47.2
36.0 35.8
82.6 87.4
12.9 10.1
4.5 2.5
17.4 12.6
30
K. Wada et al. / Mutation Research 742 (2012) 26–30
damage in the urinary bladder of SD rats, but there is no report on the sex difference in urinary bladder carcinogenicity in SD rats. We are presently investigating to confirm the universality of such a sex difference on the background level in the Comet assay in urinary bladders using other strains of rats. In conclusion, the mincing method can be used as a standard procedure for the isolation of urinary bladder cells, though the contaminating non-epithelial cells with over 10% are inevitable. The use of the Comet assay with the mincing method for evaluating genotoxicity in urinary bladders is expected to increase in the future. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We thank Mr. Yuzo Takezawa and Ms. Misaki Abe for their technical assistance and Mrs. Hitomi Wada for critical reading of the manuscript. References [1] S.M. Cohen, Urinary bladder carcinogenesis, Toxicol. Pathol. 26 (1998) 121–127. [2] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [3] S. Brendler-Schwaab, A. Hartmann, S. Pfuhler, G. Speit, The in vivo Comet assay: use and status in genotoxicity testing, Mutagenesis 20 (2005) 245–254. [4] L. Robbiano, R. Carrozzino, M. Bacigalupo, C. Corbu, G. Brambilla, Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity, Toxicology 179 (2002) 115–128. [5] A. Wang, J.L. Robertson, S.D. Holladay, A.H. Tennant, A.J. Lengi, S.A. Ahmed, W.R. Huckle, A.D. Kligerman, Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols, Mutat. Res. 634 (2007) 51–59. [6] D.D. Miranda, D.P. Arc¸ari, J. Pedrazzoli Jr., P.O. Carvalho, S.M. Cerutti, D.H. Bastos, M.L. Ribeiro, Protective effects of mate tea (Ilex paraguariensis) on H2 O2 -induced DNA damage and DNA repair in mice, Mutagenesis 23 (2008) 261–265. [7] Y.F. Sasaki, E. Nishidate, F. Izumiyama, M. Watanabe-Akanuma, N. Kinae, N. Matsusaka, S. Tsuda, Detection of in vivo genotoxicity of 3-chloro-4(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs, Mutat. Res. 393 (1997) 47–53. [8] A. Martelli, L. Robbiano, R. Carrozzino, C.P. Puglia, F. Mattioli, M. Angiola, G. Brambilla, DNA damage induced by 3,3 -dimethoxybenzidine in liver and urinary bladder cells of rats and humans, Toxicol. Sci. 53 (2000) 71–76. [9] S. Tsuda, M. Murakami, N. Matsusaka, K. Kano, K. Taniguchi, Y.F. Sasaki, DNA damage induced by red food dyes orally administered to pregnant and male mice, Toxicol. Sci. 61 (2001) 92–99. [10] K. Sekihashi, A. Yamamoto, Y. Matsumura, S. Ueno, M. Watanabe-Akanuma, F. Kassie, S. Knasmüller, S. Tsuda, Y.F. Sasaki, Comparative investigation of multiple organs of mice and rats in the comet assay, Mutat. Res. 517 (2002) 53–75. [11] M.G. Nascimento, M.L. de Oliveira, A.S. Lima, J.L. de Camargo, Effects of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the urinary bladder of male Wistar rats, Toxicology 224 (2006) 66–73.
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