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Gender differences in the liver micronucleus test in rats with partial hepatectomy
Toxicology Letters 214 (2012) 296–300
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Gender differences in the liver micronucleus test in rats with partial hepatectomy Satoru Itoh ∗ , Chiharu Hattori, Mayumi Nagata, Wataru Takasaki Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Horikoshi 717, Fukuroi-shi, Shizuoka 437-0065, Japan
h i g h l i g h t s
A gender difference in the rat liver micronucleus test was investigated. Responses to structural and numerical chromosome aberration (CA) inducers were examined. Response to CA inducers in males was equal to or stronger than that in females. Use of only male rats in the liver micronucleus test seems to be sufficient.
a r t i c l e
i n f o
Article history: Received 31 July 2012 Received in revised form 4 September 2012 Accepted 5 September 2012 Available online 12 September 2012 Keywords: Gender difference Liver Micronucleus test Partial hepatectomy Structural chromosome aberration Numerical chromosome aberration
a b s t r a c t The liver micronucleus test in rats with partial hepatectomy is a useful method to detect pro-clastogens such as diethylnitrosamine, the active metabolites of which do not reach the bone marrow due to their short lifespan. We have already reported that structural or numerical chromosome aberration inducers should be given before or after partial hepatectomy, respectively, to detect genotoxicity in the liver of rats. In the present study, we found that the percentage of binucleated cells in the liver from naive male rats is approximately 60% of that in female rats, which suggests a gender difference in the response to chromosome aberration inducers. Therefore, we investigated the responses to structural chromosome aberration inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) in male and female rats. The chemicals were given to 8-week-old male and female F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. As the results, diethylnitrosamine and 1,2-dimethylhydrazine produced a significant increase in the frequency of micronucleated hepatocytes in both genders and the responses were comparable. In the case of colchicine and carbendazim, higher frequencies in the micronucleated hepatocytes were obtained in males than in females. Taken together, the response to chromosome aberration inducers in male rats was equal to or stronger than that in female rats. It seems that the use of only male rats in the liver micronucleus test is sufficient, unless existing data indicate a toxicologically meaningful gender difference in rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction According to the new ICH guideline S2(R1), a revised version of the previous ICH S2A and S2B guidelines, an in vivo genotoxicity study using liver is very important to evaluate the genotoxicity of drug candidates (ICH, 2011). The liver micronucleus test in rats can detect the activity of pro-mutagens like diethylnitrosamine, which is not active in the bone marrow micronucleus test because its short-lived active metabolites cannot reach bone marrow (Morita et al., 1997; Tates et al., 1980). Therefore, the liver micronucleus test becomes more important under these circumstances (Itoh
∗ Corresponding author. Tel.: +81 538 42 4356; fax: +81 538 42 4350. E-mail address: [email protected] (S. Itoh). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.09.003
et al., 2012). In the liver micronucleus test, mitotic stimulation is key because mitotic activity in the liver is extremely low (Tates et al., 1980). There are three methods for mitotic stimulation of the liver; one method uses partial hepatectomy (PH) (Tates et al., 1980); another uses post-treatment with a potent liver mitogen (4acetylaminofluorene) (Braithwaite and Ashby, 1998); and the third method uses young (4-week-old) rats (Suzuki et al., 2004) which have proliferating hepatocytes. We have already reported that dosing of the chemical before PH should be used to detect the clastogenicity of structural chromosome aberration inducers and dosing after PH is essential to detect the genotoxicity of numerical chromosome aberration inducers in the liver of rats (Itoh et al., 2012). In that paper, we identified a difference between the percentage of binucleated cells in naive mice and rats, and we speculated that this would cause a species difference in the induction of micronuclei by colchicine.
S. Itoh et al. / Toxicology Letters 214 (2012) 296–300
Gender differences in the induction of micronuclei by chemicals known to induce micronuclei were investigated by the bone marrow micronucleus test in mice (The Collaborative Study Group for the Micronucleus Test, 1986). Of the 20 clastogens included in the study, 4 produced a stronger response in males than in females, 1 produced a stronger response in females, and the remaining compounds produced a similar or slightly stronger response in males than in females. Based on these observations, it was concluded that the use of males is sufficient for general screening. In recent years, reduction of use of animals in genotoxicity testing has been emphasized by the European Centre for the Validation of Alternative Methods (ECVAM). They have published guidance about how to reduce the number of animals in standard genotoxicity tests (Pfuhler et al., 2009). The necessity of using both genders for evaluation of chemicals in the liver micronucleus tests is a major issue for the question of reduction of animal numbers. To the best of our knowledge, there is no publicly available data for gender differences in the liver micronucleus test. Therefore we initiated the present study by investigation of the cell classification in naive male and female rats, and only then investigated the responses of male and female rats to chemicals known to induce structural chromosome aberration [diethylnitrosamine (Natarajan et al., 1976) and 1,2-dimethylhydrazine (Ishidate, 1987)] and numerical chromosome aberration [colchicine (Xu and Adler, 1990) and carbendazim (Vigreux et al., 1998)], which were used in the previous study (Itoh et al., 2012). 2. Materials and methods 2.1. Chemicals Diethylnitrosamine (DEN, CAS no. 55-18-5) and 1,2-dimethylhydrazine dihydrochloride (1,2-DMH, CAS no. 306-37-6) were purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). Colchicine (CAS no. 64-86-8) and carbendazim (CAS no. 10605-21-7) were purchased from Sigma–Aldrich Corporation (MO, USA) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. DEN, 1,2-DMH and colchicine were dissolved in water for injection (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) and carbendazim was suspended in 0.5% methylcellulose (Wako Pure Chemical Industries, Ltd.). 2.2. Animals and treatments Seven-week-old male and female F344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan); housed in cages with bedding; maintained in a room with a 12 h light/dark cycle, temperature of 23 ± 3 ◦ C and relative humidity of 30–70%; and used at 8 weeks of age. Certified pellet food and tap water were provided ad libitum. Two dose levels of each chemical were selected based on the results of the previous study (Itoh et al., 2012). Each chemical was administered once by oral gavage at a dose volume of 10 mL/kg to 4 animals per gender per group. The chemicals were given either before or after PH. In the case of dosing before PH for DEN and 1.2-DMH, the chemical was given on Day 1, PH was performed on Day 2, and the regenerated liver was removed 4 days after PH (i.e., on Day 6). For dosing after PH for colchicine and carbendazim, in contrast, PH was performed on Day −1, the chemical was given on Day 1 and the regenerated liver was removed on Day 4. This study was conducted in compliance with the following law and guidelines; “Law Concerning the Protection and Control of Animals”, Japanese Law No. 105, October 1, 1973, revised on June 22, 2005, “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain”, Notification No. 88 of the Ministry of the Environment, Japan, April 28, 2006 and “Guidelines for Animal Experimentation”, the Japanese Association for Laboratory Animal Science, May 22, 1987. 2.3. Partial hepatectomy (PH) The animal was anesthetized with isoflurane (Mylan Seiyaku Ltd., Tokyo, Japan). Laparotomy of about 2 cm in length was made just under the xiphoid process. The liver was pushed out through the incision; the roots of the medial and lateral left lobes, and of the medial right lobe of the liver were ligated with surgical silk; and then the lobes were removed, the remaining liver was pushed back inside and the incision was sutured. 2.4. Collection of hepatocytes The regenerated liver was removed 4 days after PH. The animal was sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. The liver
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was excised and weighed with an electronic balance. The hepatocytes were isolated by a simplified method for liver perfusion (Igarashi and Shimada, 1997). Briefly, liver perfusion medium (Invitrogen Corporation, CA, USA) prewarmed to 40 ◦ C was perfused through the liver via the vena cava from a stomach tube connected to a 10-mL syringe. Subsequently, a solution of 125 units/mL collagenase (Type IV, Sigma–Aldrich Corporation) was perfused through the liver. The liver was cut into small pieces with scissors, and shaken well in 20 mL of 10% FBS-MEM to separate the hepatocytes. The suspension was filtered through metal meshes and a 70-m mesh strainer (Becton, Dickinson and Company, NJ, USA) and then centrifuged at 50 × g for 2 min. The sediment was resuspended in 10 mL of 10% (v/v) neutral buffered formalin and centrifuged at 50 × g for 2 min, and then this procedure was repeated two more times for each sample. 2.5. Observations The sediment was again resuspended in 10% (v/v) neutral buffered formalin. Equal volumes of liver cell suspension and acridine orange-DAPI solution were mixed well, and one drop was placed on the slide grass just before observation. The specimens were observed under a fluorescence microscope (BX60F5, Olympus, Tokyo, Japan). Two thousand hepatocytes excluding metaphase cells and nuclear fragment cells from each animal were observed for the micronucleated hepatocyte (MNH) count, and classified as mononucleated hepatocytes, binucleated hepatocytes and multinucleated hepatocytes (3 or more nuclei). The number of metaphase cells among 2000 hepatocytes excluding nuclear fragment cells was recorded. Also, the number of nuclear fragment cells (having many nuclei of varying size and no main nucleus) among 2000 hepatocytes excluding metaphase cells was recorded. 2.6. Statistical analyses The following statistical analyses were performed by EXSUS Ver. 7.6 (CAC EXICARE Corporation, Tokyo, Japan) with significance level at 5%. The frequency of MNH and dose dependency including vehicle control group was analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. The relative liver weight to body weight was analyzed by a tree-type algorithm for quantitative data (Hamada et al., 1998).
3. Results 3.1. Naive male and female rats The basal level of MNH frequency and the classification of cells from 4 naive male and female rats are shown in Table 1. The frequencies of MNH in naive male and female rats were comparable to that obtained in the previous study (Itoh et al., 2012). In naive males, the percentage of binucleated cells was 25.1%, which is much lower than that in females (43.9%), and it corresponds to approximately 60% of the value in females. 3.2. Gender difference The frequency of MNH in male and female rats treated with the structural chromosome aberration inducers, DEN or 1,2-DMH, given before PH is shown in Fig. 1, and the relative liver weight and cell classification are summarized in Table 2. Treatment with DEN or 1,2-DMH resulted in a dose-dependent statistically significant increase in MNH regardless of gender, and the magnitude of the induction was comparable in males and females. No statistically significant change by either chemical was observed in the relative liver weight in either gender. The frequency of binucleated cells tended to increase after treatment by either chemical at the higher dose level in both genders, which suggested that liver regeneration was inhibited (Igarashi et al., 2007), and the magnitude of the change in males and females was comparable. No marked change was observed in the frequency of multinucleated hepatocytes, metaphase cells or nuclear fragment cells in any group. The frequency of MNH in male and female rats treated with colchicine or carbendazim, numerical chromosome aberration inducers, given after PH is shown in Fig. 2, and the relative liver weight and cell classification are summarized in Table 3. Treatment with colchicine or carbendazim resulted in a dose-dependent statistically significant increase in MNH, and MNH induction in
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Table 1 MNH and cell classification in naive male and female rats. Gender
Number of animals
MNH (%) Mean ± S.D.
Cell classification (mean)
Nuclear count (%)
Male Female
0.01 ± 0.03 0.00 ± 0.00
4 4
Mononucleated cells
Binucleated cells
Multinucleated cells
74.8 56.1
25.1 43.9
0.2 0.0
Metaphase cells (%)
Nuclear fragmentation cells (%)
0.0 0.0
0.0 0.0
MNH: micronucleated hepatocytes; Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Metaphase cells: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; Nuclear fragmentation cells: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
Fig. 1. Frequency of micronucleated hepatocytes in male and female rats treated with structural chromosome aberration inducers. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed with significance level at 5% on the frequency of MNH by two-tailed Fisher’s exact test (*) and on the dose dependency by two-tailed Cochran-Armitage trend test (#).
males was stronger than that in females. No change in the relative liver weight was observed in either gender. Increased frequency of binucleated cells was observed in the 62.5 and 125 mg/kg carbendazim groups in male rats only. No marked difference between males and females was observed in the frequency of multinucleated cells and nuclear fragment cells in the colchicine and carbendazim groups, but they were higher than those in the DEN and 1,2-DMH groups. The induction in these parameters, including the increased frequency of metaphase cells, is recognized as a typical response to numerical chromosome aberration inducers (Matsuoka et al., 1999).
4. Discussion We have previously reported that micronucleus induction in the liver of rats by structural or numerical chromosome aberration inducers (Itoh et al., 2012). In the present study, we found that the percentage of binucleated cells in naive male rats was approximately 60% of that in naive female rats. A difference in the response to chromosome aberration inducers was suggested, because the percentage of binucleated cells in mice is lower than that in rats and the different temporal response to colchicine between mice and rats was identified in the previous study (Itoh et al., 2012).
Table 2 Relative liver weight and cell classification in male and female rats treated with structural chromosome aberration inducers. Gender
Treatment
Dose (mg/kg)
Number of animals
Relative liver weight (% body weight) Mean ± S.D.
Cell classification (mean)
Nuclear count (%)
Male
Water for injection Diethylnitrosamine
Female
Water for injection Diethylnitrosamine
Male
Water for injection 1,2-Dimethylhydrazine
Female
Water for injection 1,2-Dimethylhydrazine
Meta (%)
NF (%)
Mono
Bi
Multi
0 12.5 25 0 12.5 25
4 4 4 4 4 4
3.07 3.21 2.98 3.07 3.05 2.97
± ± ± ± ± ±
0.07 0.14 0.07 0.12 0.09 0.11
94.3 94.6 90.9 94.4 94.0 87.6
5.7 5.5 9.0 5.6 6.0 12.4
0.0 0.0 0.1 0.0 0.0 0.0
0.2 0.3 0.5 0.8 0.6 0.4
0.0 0.0 0.0 0.0 0.0 0.0
0 6.25 25 0 6.25 25
4 4 4 4 4 4
3.15 3.17 2.74 3.14 3.02 2.97
± ± ± ± ± ±
0.09 0.08 0.28 0.19 0.30 0.11
96.6 95.4 89.1 96.8 95.6 84.8
3.4 4.6 11.0 3.2 4.5 15.2
0.0 0.0 0.0 0.0 0.0 0.0
0.4 0.4 0.4 0.3 0.5 0.3
0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte; Bi: binucleated hepatocyte; Multi: multinucleated hepatocyte; Meta: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; NF: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
S. Itoh et al. / Toxicology Letters 214 (2012) 296–300
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Fig. 2. Frequency of micronucleated hepatocytes in male and female rats treated with numerical chromosome aberration inducers. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed with significance level at 5% on the frequency of MNH by two-tailed Fisher’s exact test (*) and on the dose dependency by two-tailed Cochran-Armitage trend test (#).
Therefore, we investigated the differences between the response of males and females to structural and numerical chromosome aberration inducers in the rat liver micronucleus test with PH. The induction of MNH by administration of chromosome aberration inducers, DEN, 1,2-DMH, colchicine or carbendazim, in male rats in the present study was comparable to that obtained in the previous study (Itoh et al., 2012). Regarding the gender difference, the responses to DEN and 1,2-DMH were similar in male and female rats, but the responses to colchicine and carbendazim were stronger in males. We have reported that the percentage of binucleated cells in naive rats was less than half of that in naive mice, and MNH induction by colchicine occurred earlier in rats than in mice (Itoh et al., 2012). Since the frequency of binucleated cells decreases and that of mononucleated cells increases during proliferation of hepatocytes (Igarashi et al., 2007), the higher frequency of binucleated cells in naive female rats than in male rats may mean that a higher degree of proliferation of the hepatocytes are needed to produce micronuclei at a level similar to that in male rats. Gender difference in the response to the numerical chromosome aberration inducers, colchicine and carbendazim, was identified while no gender difference in response to the structural chromosome aberration inducers, DEN and 1,2-DMH, was obtained. One possible reason for the gender difference in the response to numerical chromosome aberration inducers is the difference in their metabolism of these chemicals. However, there
are no chemicals that are known to induce numerical chromosome aberration after short treatment with metabolic activation (KirschVolders et al., 2003). Therefore, it is unlikely that a difference in the metabolism of these chemicals is the basis for the gender difference in the response to colchicine and carbendazim. Another possible reason is related to the target of numerical chromosome aberration inducers. Their targets are the essential DNA-containing structures (centromeres and telomeres), spindle apparatus (tubulin, microtubule-associated proteins and centrioles) and cell cycle control molecules (cyclins, CDKs and P53), which act during cell proliferation (Kirsch-Volders et al., 2002). In the case of colchicine and carbendazim, exposure in the presence of hepatocyte proliferation is essential for producing MNH in mice and rats (Itoh et al., 2012; Igarashi et al., 2007). Therefore, a lower turnover rate of the hepatocytes may have caused a weaker response to numerical chromosome aberration inducers in female rats. Gender differences in the bone marrow micronucleus test using 20 known clastogens was evaluated in mice, and it was concluded that the use of males is sufficient for general screening purposes (CSGMT, 1986). In the liver micronucleus test, on the other hand, such study has not been performed except for the present study. However, ECVAM reported that data showed no substantial differences in genotoxicity between genders, and thus use of a single gender is an effective way to reduce the number of animals in standard genotoxicity tests (Pfuhler et al., 2009). Therefore, the use
Table 3 Relative liver weight and cell classification in male and female rats treated with numerical chromosome aberration inducers. Gender
Treatment
Dose (mg/kg)
Number of animals
Relative liver weight (% body weight) Mean ± S.D.
Cell classification (mean)
Nuclear count (%) Mono Male
Water for injection Colchicine
Female
Water for injection Colchicine
Male
0.5% Methylcellulose Carbendazim
Female
0.5% Methylcellulose Carbendazim
Bi
Meta (%)
NF (%)
Multi
0 1 2 0 1 2
4 4 4 4 4 4
3.00 2.94 2.92 2.99 3.12 3.05
± ± ± ± ± ±
0.13 0.03 0.20 0.16 0.22 0.28
95.3 92.8 93.8 95.3 95.1 94.2
4.7 7.0 5.7 4.6 4.9 5.4
0.0 0.3 0.6 0.0 0.0 0.4
0.2 0.2 0.2 0.2 0.2 0.1
0.0 0.1 0.5 0.0 0.0 0.1
0 62.5 125 0 62.5 125
4 4 4 4 4 4
3.17 3.10 3.11 3.02 3.11 3.31
± ± ± ± ± ±
0.08 0.13 0.08 0.15 0.20 0.23
95.4 89.2 88.3 96.8 94.3 93.0
4.6 10.2 10.5 3.2 5.5 6.6
0.0 0.6 1.2 0.0 0.2 0.4
0.3 0.3 0.8 0.4 0.4 0.3
0.0 0.3 0.5 0.0 0.0 0.1
Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte; Bi: binucleated hepatocyte; Multi: multinucleated hepatocyte; Meta: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; NF: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
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of only male rats seems to be sufficient in the liver micronucleus test based on the present study, unless there any existing data indicate a toxicologically meaningful gender difference in rats. In summary, DEN and 1,2-DMH, structural chromosome aberration inducers, and colchicine and carbendazim, numerical chromosome aberration inducers, induced MNH in male and female rats. Stronger MNH induction in male rats was observed after treatment with colchicine or carbendazim, and comparable MNH induction in both genders occurred after treatment with DEN or 1,2-DMH. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors appreciate Drs. Takashi Yamoto and Miyuki Igarashi for their useful discussion. References Braithwaite, I., Ashby, J., 1998. A non-invasive micronucleus assay in the rat liver. Mutation Research 203, 23–32. Hamada, C., Yoshino, K., Matsumoto, K., Nomura, M., Yoshimura, I., 1998. Tree-type algorithm for statistical analysis in chronic toxicity studies. Journal of Toxicological Sciences 23, 173–181. ICH Harmonised Tripartite Guideline, 2011. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1). Current Step 4 version dated 9 November 2011. Available at: http://www.ich.org/ products/guidelines/safety/article/safety-guidelines.html Igarashi, M., Shimada, H., 1997. An improved method for the mouse liver micronucleus test. Mutation Research 391, 49–55. Igarashi, M., Setoguchi, M., Takada, S., Itoh, S., Furuhama, K., 2007. Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice. Mutation Research 632, 89–98. Ishidate Jr., M. (Ed.), 1987. Data Book of Chromosomal Aberration Test in vitro. , revised edition. L.I.C., Tokyo, p. 141.
Itoh, S., Hattori, C., Nagata, M., Sanbuissho, A., 2012. Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy. Mutation Research 747, 98–103. Kirsch-Volders, M., Vanhauwaert, A., De Boeck, M., Decordier, I., 2002. Importance of detecting numerical versus structural chromosome aberrations. Mutation Research 504, 137–148. Kirsch-Volders, M., Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., Fenech, M., Ishidate Jr., M., Kirchner, S., Lorge, E., Morita, T., Norppa, H., Surrallés, J., Vanhauwaer, A., Wakata, A., 2003. Report from the in vitro micronucleus assay working group. Mutation Research 540, 153–163. Matsuoka, A., Matsuura, K., Sakamoto, H., Hayashi, M., Sofuni, T., 1999. A proposal for a simple way to distinguish aneugens from clastogens in the in vitro micronucleus test. Mutagenesis 4, 385–389. Morita, T., Asano, N., Awogi, T., Sasaki, Y.F., Sato, S., Shimada, H., Sutou, S., Suzuki, T., Wakata, A., Sofuni, T., Hayashi, M., 1997. Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and 2B). The summary report of the 6th collaborative study by CSGMT/JEMS MMS. Mutation Research 389, 3–122. Natarajan, A.T., Tates, A.D., van Buul, P.P.W., Meijers, M., de Vogel, N., 1976. Cytogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro. I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes. Mutation Research 37, 83–90. Pfuhler, S., Kirkland, D., Kasper, P., Hayashi, M., Vanparys, P., Carmichael, P., Dertinger, S., Eastmond, D., Elhajouji, A., Krul, C., Rothfuss, A., Schoening, G., Smith, A., Speit, G., Thomas, C., Benthem, J.V., Corvi, R., 2009. Reduction of use of animals in regulatory genotoxicity testing: identification and implementation opportunities—report from an ECVAM workshop. Mutation Research 680, 31–42. Suzuki, H., Shirotori, T., Hayashi, M., 2004. A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation. Cytogenetic and Genome Research 104, 299–303. Tates, A.D., Neuteboom, I., Hofker, M., den Engelse, L., 1980. A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo. Mutational Research 74, 11–20. The Collaborative Study Group for the Micronucleus Test, 1986. Sex difference in the micronucleus test. Mutational Research 172, 151–163. Vigreux, C., Poul, J.M., Deslandes, E., Lebailly, P., Godard, T., Sichel, F., Henry-Amar, M., Gauduchon, P., 1998. DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells. Mutation Research 419, 79–90. Xu, W., Adler, I-D., 1990. Clastogenic effects of known and suspect spindle poisons studied by chromosome analysis in mouse bone marrow cells. Mutagenesis 5, 371–374.
Contents lists available at SciVerse ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Gender differences in the liver micronucleus test in rats with partial hepatectomy Satoru Itoh ∗ , Chiharu Hattori, Mayumi Nagata, Wataru Takasaki Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Horikoshi 717, Fukuroi-shi, Shizuoka 437-0065, Japan
h i g h l i g h t s
A gender difference in the rat liver micronucleus test was investigated. Responses to structural and numerical chromosome aberration (CA) inducers were examined. Response to CA inducers in males was equal to or stronger than that in females. Use of only male rats in the liver micronucleus test seems to be sufficient.
a r t i c l e
i n f o
Article history: Received 31 July 2012 Received in revised form 4 September 2012 Accepted 5 September 2012 Available online 12 September 2012 Keywords: Gender difference Liver Micronucleus test Partial hepatectomy Structural chromosome aberration Numerical chromosome aberration
a b s t r a c t The liver micronucleus test in rats with partial hepatectomy is a useful method to detect pro-clastogens such as diethylnitrosamine, the active metabolites of which do not reach the bone marrow due to their short lifespan. We have already reported that structural or numerical chromosome aberration inducers should be given before or after partial hepatectomy, respectively, to detect genotoxicity in the liver of rats. In the present study, we found that the percentage of binucleated cells in the liver from naive male rats is approximately 60% of that in female rats, which suggests a gender difference in the response to chromosome aberration inducers. Therefore, we investigated the responses to structural chromosome aberration inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and numerical chromosome aberration inducers (colchicine and carbendazim) in male and female rats. The chemicals were given to 8-week-old male and female F344 rats a day before or after partial hepatectomy and hepatocytes were isolated 4 days after the partial hepatectomy. As the results, diethylnitrosamine and 1,2-dimethylhydrazine produced a significant increase in the frequency of micronucleated hepatocytes in both genders and the responses were comparable. In the case of colchicine and carbendazim, higher frequencies in the micronucleated hepatocytes were obtained in males than in females. Taken together, the response to chromosome aberration inducers in male rats was equal to or stronger than that in female rats. It seems that the use of only male rats in the liver micronucleus test is sufficient, unless existing data indicate a toxicologically meaningful gender difference in rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction According to the new ICH guideline S2(R1), a revised version of the previous ICH S2A and S2B guidelines, an in vivo genotoxicity study using liver is very important to evaluate the genotoxicity of drug candidates (ICH, 2011). The liver micronucleus test in rats can detect the activity of pro-mutagens like diethylnitrosamine, which is not active in the bone marrow micronucleus test because its short-lived active metabolites cannot reach bone marrow (Morita et al., 1997; Tates et al., 1980). Therefore, the liver micronucleus test becomes more important under these circumstances (Itoh
∗ Corresponding author. Tel.: +81 538 42 4356; fax: +81 538 42 4350. E-mail address: [email protected] (S. Itoh). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.09.003
et al., 2012). In the liver micronucleus test, mitotic stimulation is key because mitotic activity in the liver is extremely low (Tates et al., 1980). There are three methods for mitotic stimulation of the liver; one method uses partial hepatectomy (PH) (Tates et al., 1980); another uses post-treatment with a potent liver mitogen (4acetylaminofluorene) (Braithwaite and Ashby, 1998); and the third method uses young (4-week-old) rats (Suzuki et al., 2004) which have proliferating hepatocytes. We have already reported that dosing of the chemical before PH should be used to detect the clastogenicity of structural chromosome aberration inducers and dosing after PH is essential to detect the genotoxicity of numerical chromosome aberration inducers in the liver of rats (Itoh et al., 2012). In that paper, we identified a difference between the percentage of binucleated cells in naive mice and rats, and we speculated that this would cause a species difference in the induction of micronuclei by colchicine.
S. Itoh et al. / Toxicology Letters 214 (2012) 296–300
Gender differences in the induction of micronuclei by chemicals known to induce micronuclei were investigated by the bone marrow micronucleus test in mice (The Collaborative Study Group for the Micronucleus Test, 1986). Of the 20 clastogens included in the study, 4 produced a stronger response in males than in females, 1 produced a stronger response in females, and the remaining compounds produced a similar or slightly stronger response in males than in females. Based on these observations, it was concluded that the use of males is sufficient for general screening. In recent years, reduction of use of animals in genotoxicity testing has been emphasized by the European Centre for the Validation of Alternative Methods (ECVAM). They have published guidance about how to reduce the number of animals in standard genotoxicity tests (Pfuhler et al., 2009). The necessity of using both genders for evaluation of chemicals in the liver micronucleus tests is a major issue for the question of reduction of animal numbers. To the best of our knowledge, there is no publicly available data for gender differences in the liver micronucleus test. Therefore we initiated the present study by investigation of the cell classification in naive male and female rats, and only then investigated the responses of male and female rats to chemicals known to induce structural chromosome aberration [diethylnitrosamine (Natarajan et al., 1976) and 1,2-dimethylhydrazine (Ishidate, 1987)] and numerical chromosome aberration [colchicine (Xu and Adler, 1990) and carbendazim (Vigreux et al., 1998)], which were used in the previous study (Itoh et al., 2012). 2. Materials and methods 2.1. Chemicals Diethylnitrosamine (DEN, CAS no. 55-18-5) and 1,2-dimethylhydrazine dihydrochloride (1,2-DMH, CAS no. 306-37-6) were purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). Colchicine (CAS no. 64-86-8) and carbendazim (CAS no. 10605-21-7) were purchased from Sigma–Aldrich Corporation (MO, USA) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. DEN, 1,2-DMH and colchicine were dissolved in water for injection (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) and carbendazim was suspended in 0.5% methylcellulose (Wako Pure Chemical Industries, Ltd.). 2.2. Animals and treatments Seven-week-old male and female F344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan); housed in cages with bedding; maintained in a room with a 12 h light/dark cycle, temperature of 23 ± 3 ◦ C and relative humidity of 30–70%; and used at 8 weeks of age. Certified pellet food and tap water were provided ad libitum. Two dose levels of each chemical were selected based on the results of the previous study (Itoh et al., 2012). Each chemical was administered once by oral gavage at a dose volume of 10 mL/kg to 4 animals per gender per group. The chemicals were given either before or after PH. In the case of dosing before PH for DEN and 1.2-DMH, the chemical was given on Day 1, PH was performed on Day 2, and the regenerated liver was removed 4 days after PH (i.e., on Day 6). For dosing after PH for colchicine and carbendazim, in contrast, PH was performed on Day −1, the chemical was given on Day 1 and the regenerated liver was removed on Day 4. This study was conducted in compliance with the following law and guidelines; “Law Concerning the Protection and Control of Animals”, Japanese Law No. 105, October 1, 1973, revised on June 22, 2005, “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain”, Notification No. 88 of the Ministry of the Environment, Japan, April 28, 2006 and “Guidelines for Animal Experimentation”, the Japanese Association for Laboratory Animal Science, May 22, 1987. 2.3. Partial hepatectomy (PH) The animal was anesthetized with isoflurane (Mylan Seiyaku Ltd., Tokyo, Japan). Laparotomy of about 2 cm in length was made just under the xiphoid process. The liver was pushed out through the incision; the roots of the medial and lateral left lobes, and of the medial right lobe of the liver were ligated with surgical silk; and then the lobes were removed, the remaining liver was pushed back inside and the incision was sutured. 2.4. Collection of hepatocytes The regenerated liver was removed 4 days after PH. The animal was sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. The liver
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was excised and weighed with an electronic balance. The hepatocytes were isolated by a simplified method for liver perfusion (Igarashi and Shimada, 1997). Briefly, liver perfusion medium (Invitrogen Corporation, CA, USA) prewarmed to 40 ◦ C was perfused through the liver via the vena cava from a stomach tube connected to a 10-mL syringe. Subsequently, a solution of 125 units/mL collagenase (Type IV, Sigma–Aldrich Corporation) was perfused through the liver. The liver was cut into small pieces with scissors, and shaken well in 20 mL of 10% FBS-MEM to separate the hepatocytes. The suspension was filtered through metal meshes and a 70-m mesh strainer (Becton, Dickinson and Company, NJ, USA) and then centrifuged at 50 × g for 2 min. The sediment was resuspended in 10 mL of 10% (v/v) neutral buffered formalin and centrifuged at 50 × g for 2 min, and then this procedure was repeated two more times for each sample. 2.5. Observations The sediment was again resuspended in 10% (v/v) neutral buffered formalin. Equal volumes of liver cell suspension and acridine orange-DAPI solution were mixed well, and one drop was placed on the slide grass just before observation. The specimens were observed under a fluorescence microscope (BX60F5, Olympus, Tokyo, Japan). Two thousand hepatocytes excluding metaphase cells and nuclear fragment cells from each animal were observed for the micronucleated hepatocyte (MNH) count, and classified as mononucleated hepatocytes, binucleated hepatocytes and multinucleated hepatocytes (3 or more nuclei). The number of metaphase cells among 2000 hepatocytes excluding nuclear fragment cells was recorded. Also, the number of nuclear fragment cells (having many nuclei of varying size and no main nucleus) among 2000 hepatocytes excluding metaphase cells was recorded. 2.6. Statistical analyses The following statistical analyses were performed by EXSUS Ver. 7.6 (CAC EXICARE Corporation, Tokyo, Japan) with significance level at 5%. The frequency of MNH and dose dependency including vehicle control group was analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. The relative liver weight to body weight was analyzed by a tree-type algorithm for quantitative data (Hamada et al., 1998).
3. Results 3.1. Naive male and female rats The basal level of MNH frequency and the classification of cells from 4 naive male and female rats are shown in Table 1. The frequencies of MNH in naive male and female rats were comparable to that obtained in the previous study (Itoh et al., 2012). In naive males, the percentage of binucleated cells was 25.1%, which is much lower than that in females (43.9%), and it corresponds to approximately 60% of the value in females. 3.2. Gender difference The frequency of MNH in male and female rats treated with the structural chromosome aberration inducers, DEN or 1,2-DMH, given before PH is shown in Fig. 1, and the relative liver weight and cell classification are summarized in Table 2. Treatment with DEN or 1,2-DMH resulted in a dose-dependent statistically significant increase in MNH regardless of gender, and the magnitude of the induction was comparable in males and females. No statistically significant change by either chemical was observed in the relative liver weight in either gender. The frequency of binucleated cells tended to increase after treatment by either chemical at the higher dose level in both genders, which suggested that liver regeneration was inhibited (Igarashi et al., 2007), and the magnitude of the change in males and females was comparable. No marked change was observed in the frequency of multinucleated hepatocytes, metaphase cells or nuclear fragment cells in any group. The frequency of MNH in male and female rats treated with colchicine or carbendazim, numerical chromosome aberration inducers, given after PH is shown in Fig. 2, and the relative liver weight and cell classification are summarized in Table 3. Treatment with colchicine or carbendazim resulted in a dose-dependent statistically significant increase in MNH, and MNH induction in
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Table 1 MNH and cell classification in naive male and female rats. Gender
Number of animals
MNH (%) Mean ± S.D.
Cell classification (mean)
Nuclear count (%)
Male Female
0.01 ± 0.03 0.00 ± 0.00
4 4
Mononucleated cells
Binucleated cells
Multinucleated cells
74.8 56.1
25.1 43.9
0.2 0.0
Metaphase cells (%)
Nuclear fragmentation cells (%)
0.0 0.0
0.0 0.0
MNH: micronucleated hepatocytes; Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Metaphase cells: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; Nuclear fragmentation cells: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
Fig. 1. Frequency of micronucleated hepatocytes in male and female rats treated with structural chromosome aberration inducers. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed with significance level at 5% on the frequency of MNH by two-tailed Fisher’s exact test (*) and on the dose dependency by two-tailed Cochran-Armitage trend test (#).
males was stronger than that in females. No change in the relative liver weight was observed in either gender. Increased frequency of binucleated cells was observed in the 62.5 and 125 mg/kg carbendazim groups in male rats only. No marked difference between males and females was observed in the frequency of multinucleated cells and nuclear fragment cells in the colchicine and carbendazim groups, but they were higher than those in the DEN and 1,2-DMH groups. The induction in these parameters, including the increased frequency of metaphase cells, is recognized as a typical response to numerical chromosome aberration inducers (Matsuoka et al., 1999).
4. Discussion We have previously reported that micronucleus induction in the liver of rats by structural or numerical chromosome aberration inducers (Itoh et al., 2012). In the present study, we found that the percentage of binucleated cells in naive male rats was approximately 60% of that in naive female rats. A difference in the response to chromosome aberration inducers was suggested, because the percentage of binucleated cells in mice is lower than that in rats and the different temporal response to colchicine between mice and rats was identified in the previous study (Itoh et al., 2012).
Table 2 Relative liver weight and cell classification in male and female rats treated with structural chromosome aberration inducers. Gender
Treatment
Dose (mg/kg)
Number of animals
Relative liver weight (% body weight) Mean ± S.D.
Cell classification (mean)
Nuclear count (%)
Male
Water for injection Diethylnitrosamine
Female
Water for injection Diethylnitrosamine
Male
Water for injection 1,2-Dimethylhydrazine
Female
Water for injection 1,2-Dimethylhydrazine
Meta (%)
NF (%)
Mono
Bi
Multi
0 12.5 25 0 12.5 25
4 4 4 4 4 4
3.07 3.21 2.98 3.07 3.05 2.97
± ± ± ± ± ±
0.07 0.14 0.07 0.12 0.09 0.11
94.3 94.6 90.9 94.4 94.0 87.6
5.7 5.5 9.0 5.6 6.0 12.4
0.0 0.0 0.1 0.0 0.0 0.0
0.2 0.3 0.5 0.8 0.6 0.4
0.0 0.0 0.0 0.0 0.0 0.0
0 6.25 25 0 6.25 25
4 4 4 4 4 4
3.15 3.17 2.74 3.14 3.02 2.97
± ± ± ± ± ±
0.09 0.08 0.28 0.19 0.30 0.11
96.6 95.4 89.1 96.8 95.6 84.8
3.4 4.6 11.0 3.2 4.5 15.2
0.0 0.0 0.0 0.0 0.0 0.0
0.4 0.4 0.4 0.3 0.5 0.3
0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte; Bi: binucleated hepatocyte; Multi: multinucleated hepatocyte; Meta: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; NF: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
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Fig. 2. Frequency of micronucleated hepatocytes in male and female rats treated with numerical chromosome aberration inducers. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed with significance level at 5% on the frequency of MNH by two-tailed Fisher’s exact test (*) and on the dose dependency by two-tailed Cochran-Armitage trend test (#).
Therefore, we investigated the differences between the response of males and females to structural and numerical chromosome aberration inducers in the rat liver micronucleus test with PH. The induction of MNH by administration of chromosome aberration inducers, DEN, 1,2-DMH, colchicine or carbendazim, in male rats in the present study was comparable to that obtained in the previous study (Itoh et al., 2012). Regarding the gender difference, the responses to DEN and 1,2-DMH were similar in male and female rats, but the responses to colchicine and carbendazim were stronger in males. We have reported that the percentage of binucleated cells in naive rats was less than half of that in naive mice, and MNH induction by colchicine occurred earlier in rats than in mice (Itoh et al., 2012). Since the frequency of binucleated cells decreases and that of mononucleated cells increases during proliferation of hepatocytes (Igarashi et al., 2007), the higher frequency of binucleated cells in naive female rats than in male rats may mean that a higher degree of proliferation of the hepatocytes are needed to produce micronuclei at a level similar to that in male rats. Gender difference in the response to the numerical chromosome aberration inducers, colchicine and carbendazim, was identified while no gender difference in response to the structural chromosome aberration inducers, DEN and 1,2-DMH, was obtained. One possible reason for the gender difference in the response to numerical chromosome aberration inducers is the difference in their metabolism of these chemicals. However, there
are no chemicals that are known to induce numerical chromosome aberration after short treatment with metabolic activation (KirschVolders et al., 2003). Therefore, it is unlikely that a difference in the metabolism of these chemicals is the basis for the gender difference in the response to colchicine and carbendazim. Another possible reason is related to the target of numerical chromosome aberration inducers. Their targets are the essential DNA-containing structures (centromeres and telomeres), spindle apparatus (tubulin, microtubule-associated proteins and centrioles) and cell cycle control molecules (cyclins, CDKs and P53), which act during cell proliferation (Kirsch-Volders et al., 2002). In the case of colchicine and carbendazim, exposure in the presence of hepatocyte proliferation is essential for producing MNH in mice and rats (Itoh et al., 2012; Igarashi et al., 2007). Therefore, a lower turnover rate of the hepatocytes may have caused a weaker response to numerical chromosome aberration inducers in female rats. Gender differences in the bone marrow micronucleus test using 20 known clastogens was evaluated in mice, and it was concluded that the use of males is sufficient for general screening purposes (CSGMT, 1986). In the liver micronucleus test, on the other hand, such study has not been performed except for the present study. However, ECVAM reported that data showed no substantial differences in genotoxicity between genders, and thus use of a single gender is an effective way to reduce the number of animals in standard genotoxicity tests (Pfuhler et al., 2009). Therefore, the use
Table 3 Relative liver weight and cell classification in male and female rats treated with numerical chromosome aberration inducers. Gender
Treatment
Dose (mg/kg)
Number of animals
Relative liver weight (% body weight) Mean ± S.D.
Cell classification (mean)
Nuclear count (%) Mono Male
Water for injection Colchicine
Female
Water for injection Colchicine
Male
0.5% Methylcellulose Carbendazim
Female
0.5% Methylcellulose Carbendazim
Bi
Meta (%)
NF (%)
Multi
0 1 2 0 1 2
4 4 4 4 4 4
3.00 2.94 2.92 2.99 3.12 3.05
± ± ± ± ± ±
0.13 0.03 0.20 0.16 0.22 0.28
95.3 92.8 93.8 95.3 95.1 94.2
4.7 7.0 5.7 4.6 4.9 5.4
0.0 0.3 0.6 0.0 0.0 0.4
0.2 0.2 0.2 0.2 0.2 0.1
0.0 0.1 0.5 0.0 0.0 0.1
0 62.5 125 0 62.5 125
4 4 4 4 4 4
3.17 3.10 3.11 3.02 3.11 3.31
± ± ± ± ± ±
0.08 0.13 0.08 0.15 0.20 0.23
95.4 89.2 88.3 96.8 94.3 93.0
4.6 10.2 10.5 3.2 5.5 6.6
0.0 0.6 1.2 0.0 0.2 0.4
0.3 0.3 0.8 0.4 0.4 0.3
0.0 0.3 0.5 0.0 0.0 0.1
Cell classification: percentage of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte; Bi: binucleated hepatocyte; Multi: multinucleated hepatocyte; Meta: frequency of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells; NF: frequency of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells.
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of only male rats seems to be sufficient in the liver micronucleus test based on the present study, unless there any existing data indicate a toxicologically meaningful gender difference in rats. In summary, DEN and 1,2-DMH, structural chromosome aberration inducers, and colchicine and carbendazim, numerical chromosome aberration inducers, induced MNH in male and female rats. Stronger MNH induction in male rats was observed after treatment with colchicine or carbendazim, and comparable MNH induction in both genders occurred after treatment with DEN or 1,2-DMH. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors appreciate Drs. Takashi Yamoto and Miyuki Igarashi for their useful discussion. References Braithwaite, I., Ashby, J., 1998. A non-invasive micronucleus assay in the rat liver. Mutation Research 203, 23–32. Hamada, C., Yoshino, K., Matsumoto, K., Nomura, M., Yoshimura, I., 1998. Tree-type algorithm for statistical analysis in chronic toxicity studies. Journal of Toxicological Sciences 23, 173–181. ICH Harmonised Tripartite Guideline, 2011. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1). Current Step 4 version dated 9 November 2011. Available at: http://www.ich.org/ products/guidelines/safety/article/safety-guidelines.html Igarashi, M., Shimada, H., 1997. An improved method for the mouse liver micronucleus test. Mutation Research 391, 49–55. Igarashi, M., Setoguchi, M., Takada, S., Itoh, S., Furuhama, K., 2007. Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice. Mutation Research 632, 89–98. Ishidate Jr., M. (Ed.), 1987. Data Book of Chromosomal Aberration Test in vitro. , revised edition. L.I.C., Tokyo, p. 141.
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