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Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy
Mutation Research 747 (2012) 98–103
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Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy Satoru Itoh ∗ , Chiharu Hattori, Mayumi Nagata, Atsushi Sanbuissho Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Horikoshi 717, Fukuroi-shi, Shizuoka-ken 437-0065 Japan
a r t i c l e
i n f o
Article history: Received 23 February 2012 Received in revised form 27 March 2012 Accepted 17 April 2012 Available online 23 April 2012 Keywords: Liver Micronucleus test Structural chromosome aberration Numerical chromosome aberration Partial hepatectomy
a b s t r a c t The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89–98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8–10 days after PH. © 2012 Elsevier B.V. All rights reserved.
1. Introduction According to the ICH S2A and S2B guidelines [1,2], genotoxicity tests for drug candidates should comprise two in vitro tests, a bacterial reverse mutation test and an in vitro cytogenetic test; one in vivo test, a rodent micronucleus test in bone marrow or blood, should be conducted when the two in vitro tests show no genotoxic potential. The reality is, however, many compounds indicate positive responses in cytogenetic tests using mammalian cells and a second in vivo test is performed frequently as a follow-up. Recently, a revised version of the ICH guideline, [S2(R1) a revised version of ICH S2A and S2B], has reached step 4 [3], and it will move immediately to the final step of the process, which is regulatory implementation in the European Union, the US and Japan. According to that new guideline, there are two options for the standard battery for genotoxicity evaluation and it recommends selection of the target organ in the second in vivo test. Namely, when the
∗ Corresponding author. Tel.: +81 538 42 4356; fax: +81 538 42 4350. E-mail address: [email protected] (S. Itoh). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2012.04.007
drug candidate indicates a positive response under the presence of S9 in the in vitro mammalian cell test in option 1, the follow-up strategy will generally focus on in vivo studies of the liver. In the case of option 2, a second in vivo test, using a tissue that is well exposed to the drug or its metabolites, should be included in the battery to assure of lack of genotoxicity. Therefore, an in vivo genotoxicity study using liver is very important to evaluate whether a compound is a suitable candidate for drug development. The liver micronucleus test can detect the activity of promutagens, such as active metabolites that do not reach bone marrow due to their short lifespan, and this is one method of choice for a second in vivo genotoxicity test of a drug candidate [3]. In the liver micronucleus test, partial hepatectomy (PH) is used to stimulate mitosis of liver because mitotic activity of the liver is extremely low, and PH facilitates detection of chromosome damage by the test compound [4]. In rat studies, the liver micronucleus test is more sensitive to a structural chromosome aberration inducer when the compound is given before PH than when given after PH [4,5]. We have already reported that dosing after PH is essential to detect genotoxicity of a numerical chromosome aberration inducer in mice [6]. In recent years, rats have been used for in vivo
S. Itoh et al. / Mutation Research 747 (2012) 98–103
genotoxicity studies instead of mice in many cases in consideration of the toxicokinetics. In the present study, therefore, we first investigated the cell classification in naive rats and compared it with that in naive mice. Since a big difference was recognized in the proportion of binucleated cells between rats and mice, the responses to compounds known to induce structural or numerical chromosome aberrations – diethylnitrosamine [7] and 1,2-dimethylhydrazine [8], or cholchicine [9] and carbendazim [10], respectively – in rats were investigated; the compounds were given before or after PH. 2. Materials and methods 2.1. Test compounds Diethylnitrosamine (DEN, CAS no. 55-18-5) and 1,2-dimethylhydrazine dihydrochloride (1,2-DMH, CAS no. 306-37-6), structural chromosome aberration inducers, were purchased from Sigma–Aldrich Corporation (MO, USA) and Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan), respectively. Colchicine (CAS no. 6486-8) and carbendazim (CAS no. 10605-21-7), numerical chromosome aberration inducers, were purchased from Sigma–Aldrich Corporation 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., Osaka, Japan). 2.2. Animals Seven-week-old male 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 (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water were provided ad libitum. 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. Dose levels and treatment schedules Treatment schedules are illustrated in Fig. 1. Dose levels of 1,2-DMH or colchicine were determined based on the results of preliminary toxicity tests. Namely, the test compound was administered to 2–3 animals per group one day before or after PH and the general toxicity was observed at least for 4 days after PH. The maximum tolerable dose was selected for the highest dose level in the main study. In the case of DEN and carbendazim, the highest dose levels were determined by reference to previous studies [6,11]. The middle and low dose levels were set by dividing the highest dose by a common ratio of 2. Each test compound was administered once by oral gavage at a dose volume of 10 mL/kg to 4 animals per group on Day 1. 2.3.1. Partial hepatectomy (PH) Animals were anesthetized with isoflurane (Abbott Japan Co., 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 medial right lobe of the liver were ligated with surgical silk; and then the lobes were removed. The incision was sutured. The regenerated liver was removed 4 days after PH. 2.3.2. PH schedule The test substances were given either before or after PH. In the case of dosing before PH, the test compound 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). In contrast, PH was performed one day before the dosing of test compound (i.e., on Day -1), the test
Dosing before PH Day 1
Dosing
Day 2
PH
Day 3
99
compound was given on Day 1 and the regenerated liver was removed on Day 4 in the dosing after PH. 2.4. Measurement of regenerated liver weight and collection of hepatocytes Animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. The liver was excised and weighed with an electronic balance. The hepatocytes were isolated by a simplified method for liver perfusion [12]. 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, Inc.) was perfused through the liver. The liver was cut into small pieces with scissors, and they were 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 vol% neutral buffered formalin and centrifuged at 50 × g for 2 min, and then this procedure was repeated two times more for each sample. 2.5. Observations The sediment was resuspended in 10 vol% neutral buffered formalin at a volume ratio of 1:3–4. Equal volumes of liver cell suspension and acridine orange-DAPI solution were mixed well, one drop was placed on a slide glass, and then the slide was sealed with a cover glass. A fluorescence microscope (BX60F5, Olympus, Tokyo, Japan) with a total magnification of at least 400× was used for observation of the specimens. Two thousand hepatocytes excluding metaphase cells and nuclear fragment cells from each animal were observed for 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 EXSAS Ver. 7.6 (Arm Corporation, Osaka, Japan) with significance level at 5%. The incidence of MNH and dose dependency across all groups including vehicle control group was analyzed by twotailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. The relative liver weight was analyzed by a tree-type algorithm for quantitative data [13].
3. Results 3.1. MNH and cell classification in naive rats The basal level of MNH and the classification of cells from 5 naive rats and the respective data in mice from a previous study [6] in both species at 8 weeks of age are shown in Table 1. In naive rats, the percentage of binucleated cells was 27.3%, which is much lower than that in mice (83.6%). The incidence of MNH in rats was very low and comparable to that in mice. 3.2. Structural chromosome aberration inducers (DEN and 1,2-DMH) The incidence of MNH after treatment by DEN or 1,2-DMH, structural chromosome aberration inducers, given before and after PH is shown in Fig. 2, and the relative liver weight and cell classification are summarized in Table 2.
Dosing after PH Day 4
Day 5
Day 6
Hepatocyte isolation
Day -1
PH
Day 1
Dosing
Day 2
Day 3
Day 4
Hepatocyte isolation
Fig. 1. Treatment schedules. The test compound was administered to rats one day before or after PH and the hepatocytes were isolated 4 days after PH. The day of dosing is defined as Day 1.
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Table 1 MNH and cell classification in rats and mice. Species
Number of animals
Age (weeks old)
MNH (%)
Cell classifications (Mean) Nuclear count (%)
Rats Mice [6]
8 8
5 3
0.03 0.10
Mononucleated cells
Binucleated cells
Multinucleated cells
72.7 16.0
27.3 83.6
0.0 0.4
Metaphase cells (%)
Nuclear fragmentation cells (%)
0.0 0.0
0.0 0.0
MNH: micronucleated hepatocytes.
DEN Dosing before PH
6
1,2-DMH Dosing after PH
#
#
5
*
4
*
*
*
*
*
3 2 1 0
0
12.5 25
50
0 12.5
25
50
Incidence of MNH (%)
Incidence of MNH (%)
7
Dosing before PH
10
Dosing after PH
#
8
*
6
* *
4
* *
2 0
0
25
Dose level (mg/kg)
50 100
0
37.5
*
75 150
Dose level (mg/kg)
Fig. 2. Incidence of micronucleated hepatocytes in rats treated with structural chromosome aberration inducers before and after PH. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%. Table 2 Relative liver weight and cell classification in rats treated with structural chromosome aberration inducers before and after PH. Timing of PH
Test compound
Dosing before PH
Water for injection Diethylnitrosamine
Dosing after PH
Water for injection Diethlnitrosamine
Dosing before PH
Water for injection 1,2-Dimethylhydrazine
Dosing after PH
Water for injection 1,2-Dimethylhydrazine
Dose (mg/kg)
0 12.5 25 50 0 12.5 25 50 0 25 50 100 0 37.5 75 150
Number of animals
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
3.11 3.14 2.95 2.66 2.96 2.85 2.62 2.42 3.04 2.96 2.66 1.93 2.89 2.63 2.25 2.18
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.06 0.07* 0.06* 0.20 0.12 0.12* 0.20* 0.24 0.08 0.27 0.18* 0.12 0.22 0.05* 0.11*
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
94.3 92.0 89.7 84.9 92.9 89.5 85.3 87.7 95.4 88.0 81.7 76.9 93.9 88.0 90.8 91.4
5.7 8.0 10.3 15.1 7.1 10.5 14.6 12.3 4.6 11.9 18.3 23.1 6.1 12.0 9.2 8.6
0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0
Meta (%)
NF (%)
0.5 0.4 0.4 0.3 0.2 0.3 0.2 0.2 0.6 0.9 0.3 0.2 0.1 0.1 0.2 0.1
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. * Statistically significant decrease in relative liver weight (P < 0.05, a tree-type algorithm for quantitative data).
Treatment with DEN resulted in a dose-dependent statistically significant increase in MNH at 12.5, 25 and 50 mg/kg regardless of whether the test drug was given before or after PH, and the magnitude of the change was comparable in both methods. Meanwhile, 1,2-DMH exhibited statistically significant increase in MNH with dose-dependent reduction in both methods, and MNH induction was stronger when the test drug was given before PH than when it was given after PH. The relative liver weight in the groups treated with DEN at 25 and 50 mg/kg decreased significantly in both methods, and the incidence of binucleated cells was increased, which suggested that liver regeneration was inhibited [6]. In the case of 1,2-DMH, a dose-dependent decrease in relative liver weight and an increase in the incidence of binucleated cells were observed,
similar to the results with DEN. No marked change was observed in the incidence of multinucleated hepatocytes, metaphase cells or nuclear fragment cells in either DEN or 1,2-DMH treatment groups. An additional experiment with lower dose levels of 1,2-DMH in both methods was conducted because the peak effect of MNH induction by 1,2-DMH was not obtained in the first experiment due to inhibition of liver regeneration. The incidence of MNH by lower dose levels of 1,2-DMH given before and after PH is shown in Fig. 4, and the relative liver weight and cell classification are listed in Table 4. In the groups given 1,2-DMH before PH, a dose-dependent statistically significant increase in the incidence of MNH was observed
S. Itoh et al. / Mutation Research 747 (2012) 98–103
Colchicine
101
Carbendazim
5
Incidence of MNH (%)
# *
3 #
2 1
*
*
*
3
6
0 0
12
0
0.5
1
2
Incidence of MNH (%)
Dosing after PH
Dosing before PH
4
12
Dosing after PH
Dosing before PH
#
10
*
8
*
6 4
*
2 0 0
Dose level (mg/kg)
62.5 125 250
0
62.5 125 250
Dose level (mg/kg)
Fig. 3. Incidence of micronucleated hepatocytes in rats treated with numerical chromosome aberration inducers before and after PH. Mean of 4 animals. The vertical bars represent S.D. All animals died between PH and hepatocyte isolation in the group given12 mg/kg of colchicine before PH. One animal died just after PH in the group given 62.5 mg/kg of carbendazim before PH. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%.
Table 3 Relative liver weight and cell classification in rats treated with numerical chromosome aberration inducers before and after PH. Timing of PH
Test compound
Dose(mg/kg)
Dosing before PH
Water for injection Colchocine
Dosing after PH
Water for injection Colchicine
Dosing before PH
0.5% methylcellulose Carbendazim
Dosing after PH
0.5% methylcellulose Carbendazim
0 3 6 12 0 0.5 1 2 0 62.5 125 250 0 62.5 125 250
Number of animals
4 4 4 0 4 4 4 4 4 3a 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
3.06 ± 0.10 2.90 ± 0.16 3.01 ± 0.25 ND 2.98 ± 0.29 2.98 ± 0.25 2.92 ± 0.24 2.95 ± 0.10 3.18 ± 0.10 3.24 ± 0.12 3.16 ± 0.25 2.99 ± 0.09 3.03 ± 0.16 3.11 ± 0.17 3.23 ± 0.09b 3.03 ± 0.32
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
94.7 94.9 95.3 ND 93.8 93.5 94.5 93.9 92.9 93.7 93.8 93.1 93.7 90.2 86.4 85.8
5.3 5.1 4.7 ND 6.2 6.5 5.4 5.8 7.1 6.3 6.2 6.9 6.3 9.7 12.8 13.7
0.0 0.0 0.0 ND 0.0 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.1 0.9 0.6
Meta (%)
NF (%)
0.1 0.2 0.5 ND 0.2 0.1 0.1 0.1 0.1 0.4 0.4 0.6 0.1 0.1 0.0 0.1
0.0 0.0 0.0 ND 0.0 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.8
ND: no data because all animals died between PH and hepatocyte isolation; Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. a One animal died just after PH. b No data in one animal because the caudate lobe was inadvertently left in the animal.
1,2-DMH
Incidence of MNH (%)
10
Dosing before PH
at doses from 3.13 to 25 mg/kg without inhibition of liver regeneration, based on the relative liver weight and the cell classification. In dosing after PH, a dose-dependent gradual increase in the incidence of MNH was obtained although slight inhibition of liver regeneration was observed at higher dose levels.
Dosing after PH
#
8
* 6
3.3. Numerical chromosome aberration inducer (colchicine and carbendazim)
#
4
* *
2
*
*
*
*
* 0 0
3.13 6.25 12.5 25
0
4.69 9.38 18.8 37.5
Dose level (mg/kg) Fig. 4. Incidence of micronucleated hepatocytes in rats treated with 1,2-DMH before and after PH (additional experiment). Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%.
The incidence of MNH after treatment by colchicine or carbendazim, numerical chromosome aberration inducers, given before and after PH is shown in Fig. 3, and the relative liver weight and cell classification are summarized in Table 3. In dosing before PH, all animals in the 12 mg/kg of colchicine treatment group died between the time of PH and hepatocyte isolation. Treatment with colchicine resulted in a dose-dependent statistically significant increase in MNH whether given before or after PH, however, the incidences of MNH in the groups given 3 and 6 mg/kg colchicine before PH were within the historical control range of our laboratory (mean ± 3 S.D.: 0–0.74). Therefore, those increases were judged to have no toxicological relevance. In dosing before PH, 1 animal in the 62.5 mg/kg of carbendazim treatment
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Table 4 Relative liver weight and cell classification in rats treated with 1,2-DMH before and after PH (additional experiment). Timing of PH
Test compound
Dosing before PH
Water for injection 1,2-Dimethylhydrazine
Dosing after PH
Water for injection 1,2-Dimethylhydrazine
Dose (mg/kg)
0 3.13 6.25 12.5 25 0 4.69 9.38 18.75 37.5
Number of animals
4 4 4 4 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
2.98 3.00 3.06 3.03 2.86 3.25 3.06 2.94 2.46 2.44
± ± ± ± ± ± ± ± ± ±
0.20 0.11 0.08 0.16 0.10 0.27 0.15 0.08 0.16* 0.18*
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
95.1 94.6 95.0 93.9 89.3 96.2 93.2 91.1 86.6 90.7
4.9 5.4 5.0 6.1 10.7 3.8 6.8 8.9 13.4 9.3
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Meta (%)
NF (%)
0.5 0.3 0.5 0.7 0.5 0.4 0.2 0.6 0.5 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. * Statistically significant decrease in relative liver weight (P < 0.05, a tree-type algorithm for quantitative data).
group died just after PH. Treatment with carbendazim resulted in a dose-dependent statistically significant increase in MNH at 62.5, 125 and 250 mg/kg given after PH, but no MNH induction was obtained when dosing occurred before PH. No change in the relative liver weight was observed in colchicine or carbendazim treatment groups in either method. The incidence of binucleated cells in the groups given 125 and 250 mg/kg carbendazim after PH was higher than that in the vehicle control group, suggesting that inhibition of liver regeneration had occurred [6]. For metaphase cells, a relatively higher incidence was obtained when higher dose levels of colchicine and carbendazim were given before PH, and high incidences of multinucleated and nuclear fragment cells were also observed at higher dose levels given after PH. Such induction is recognized as a typical response of numerical chromosome aberration inducers [14]. 4. Discussion We have already reported on micronucleus induction in the liver of mice by numerical chromosome aberration inducers, and that dosing after PH is essential to detect their genotoxicity [6]. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the liver micronucleus test using rats. DEN and 1,2-DMH, structural chromosome aberration inducers, exhibited markedly and significantly increased incidence of MNH when given before PH and also induced MNH when given after PH. The magnitude of induction when the drug was given after PH was comparable to, or weaker than, that when the drug was given before PH for DEN or 1,2-DMH, respectively. The in vivo clastogenicity of mitomycin C and cyclophosphamide have been investigated by dosing before and after PH in mice [12]. For both compounds, the incidence of MNH was higher when the compound was given before PH than when it was given after PH. The results with 1,2-DMH in the present study are consistent with these findings. The cytotoxicity of those compounds may be stronger when given after PH, when cell proliferation is activated, than when given before PH, when the cells are not dividing. This is supported by the fact that a slight increase in MNH was observed when a range of dose levels of 1,2-DMH (4.69–150 mg/kg) were given after PH. However, more research on the structural chromosome aberration inducers given both before and after PH is needed because DEN showed comparable potency for MNH induction in both methods in the present study. Recently, the relationship between DNA damage and micronucleus induction in mice has been reported [15] in a study of structural chromosome aberration inducers. DNA damage and micronucleus induction in
the liver were examined by Comet assay and micronucleus test with PH, respectively; it was concluded that the optimal timing of PH to detect clastogenicity of these compounds is 24 h after dosing. Furthermore, Tates et al. reported that DEN and dimethylnitrosamine induced hepatocytes with micronuclei when they were administered before and after PH, and concluded that the administration of the test compound before PH has advantages such as exposure to the compound in physiologically normal animals with intact liver, and examination under the influence of persistent DNA lesions [4]. From the above findings, the potency for induction of structural chromosome aberrations in the liver should be examined by dosing of the test compound before PH. Colchicine and carbendazim, numerical chromosome aberration inducers, significantly increased MNH frequency only when given after PH, not before PH, and the results were similar to those in mice [6]. Taken together, it was confirmed that exposure to a numerical chromosome aberration inducer under hepatocyte proliferation is essential for detecting genotoxic activity. The reason for this is that the inducer of numerical chromosome aberrations targets the essential DNA-containing structures (centromeres and telomeres), spindle apparatus (tubulin, MAP and centrioles) and cell cycle control molecules (cyclins, cdk’s and P53), which act during cell proliferation [16]. It seems that exposure to the test compound before PH is insufficient to induce micronucleus in the liver because most of the test compound is metabolized by the time that activation of hepatocyte proliferation begins 24 h after PH. DEN and 1,2-DMH have also been reported to increase the incidence of MNH in young rats at 4 weeks of age [17–19]. In young rats, the percentage of S-phase cells is 40 times higher than that in naive adult rats [20], therefore, hepatocytes are proliferating, which is similar to the condition in adult rats when the test compound is administered after PH. Induction of MNH by administration of DEN to adult rats after PH in the present study was stronger that in young rats in the published study, while MNH induction by 1,2-DMH administered after PH was comparable to that in young rats [19]. The reason for the different results of DEN between young intact rats and adult rats with PH is not clear, however, a difference in proliferative activity of the liver may contribute to this discrepancy. Actually, the proliferation rate in young rats is lower than that in adult rats after PH, since the percentage of S-phase cells in young rats is less than 29% of that in regenerating liver [20]. Unfortunately, since no numerical chromosome aberration inducer was used in the experiments with young rats [11,17–19,21], no comparison with the present results is possible. Interestingly, from our preliminary study using young rats,
S. Itoh et al. / Mutation Research 747 (2012) 98–103
colchicine did not induce MNH even at a sublethal dose (data not shown). Regarding the species differences in the liver micronucleus test, the percentage of binucleated cells in naive rats was less than half that in naive mice and a difference in temporal response to structural and numerical chromosome aberration inducers was suggested. Induction of MNH by administration of DEN or carbendazim to adult rats before or after PH, respectively, in the present study was similar to that in young rats in the published study [6,12], but a difference in the time course of the response to colchicine was identified. Colchicine increased the MNH frequency at 4 days after PH in rats, although the induction in mice was observed later, at 8–10 days after PH [6]. The incidence of binucleated cells decreased and mononucleated cells increased during proliferation of hepatocytes [6], and thus the high incidence of binucleated cells in naive mice may mean that more time will be required to enhance turnover of the hepatocytes enough to produce micronuclei at a level similar to that with rats. In summary, DEN and 1,2-DMH, structural chromosome aberration inducers, induced MNH when administered either before or after PH. Stronger MNH induction by 1,2-DMH was observed in the dosing before PH, and comparable MNH induction was obtained in both methods for DEN. Dosing before PH should be examined to detect the clastogenicity of structural chromosome aberration inducers in the liver of rats. Colchicine and carbendazim, numerical chromosome aberration inducers, increased the MNH frequency only when dosing was after PH in rats, the same as in mice [6]. Dosing after PH is essential to detect the genotoxicity of numerical chromosome aberration inducers in the liver of rats. Similar results in rats and mice were obtained with DEN and carbendazim in the liver micronucleus test with PH, but the responses to colchicine were different. The MNH induction by colchicine in rats clearly occurred earlier than that in mice. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors appreciated Drs. Takashi Yamoto and Miyuki Igarashi for their useful discussion. References [1] ICH Harmonised Tripartite Guideline, Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Available at: http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm065007.htm. [2] ICH Harmonised Tripartite Guideline, Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Available at: http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm065007.htm. [3] ICH Harmonised Tripartite Guideline, 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.
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[4] A.D. Tates, I. Neuteboom, M. Hofker, L. den Engelse, A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo, Mutat. Res. 74 (1980) 11–20. [5] A.D. Tates, I. Neuteboom, Micronucleus test with rat hepatocytes to detect induction of clastogenic damage, in: J. Ashby (Ed.), Evaluation of ShortTerm Tests for Carcinogens, Cambridge University Press, Cambridge, 1987, pp. 1400–1404. [6] M. Igarashi, M. Setoguchi, S. Takada, S. Itoh, K. Furuhama, Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice, Mutat. Res. 632 (2007) 89–98. [7] A.T. Natarajan, A.D. Tates, P.P.W. van Buul, M. Meijers, N. de Vogel, 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, Mutat. Res. 37 (1976) 83–90. [8] M. Ishidate Jr. (Ed.), Data Book of Chromosomal Aberration Test in vitro, revised ed., L.I.C., Tokyo, 1987, p. 141. [9] W. Xu, I.-D. Adler, Clastogenic effects of known and suspect spindle poisons studied by chromosome analysis in bone marrow cells, Mutagenesis 5 (1990) 371–374. [10] C. Vigreux, J.M. Poul, E. Deslandes, P. Lebailly, T. Godard, F. Sichel, M. HenryAmar, P. Gauduchon, DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells, Mutat. Res. 419 (1998) 79–90. [11] H. Suzuki, T. Shirotori, M. Hayashi, A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation, Cytogenet. Genome Res. 104 (2004) 299–303. [12] M. Igarashi, H. Shimada, An improved method for the mouse liver micronucleus test, Mutat. Res. 391 (1997) 49–55. [13] C. Hamada, K. Yoshino, K. Matsumoto, M. Nomura, I. Yoshimura, Tree-type algorithm for statistical analysis in chronic toxicity studies, J. Toxicol. Sci. 23 (1998) 173–181. [14] A. Matsuoka, K. Matsuura, H. Sakamoto, M. Hayashi, T. Sofuni, A proposal for a simple way to distinguish aneugens from clastogens in the in vitro micronucleus test, Mutagenesis 4 (1999) 385–389. [15] M. Igarashi, M. Nagata, S. Itoh, T. Yamoto, S. Tsuda, Relationship between DNA damage and micronucleus in mouse liver, J. Toxicol. Sci. 35 (2010) 881–889. [16] M. Kirsch-Volders, A. Vanhauwaert, M. De Boeck, I. Decordier, Importance of detecting numerical versus structural chromosome aberrations, Mutat. Res. 504 (2002) 137–148. [17] H. Takasawa, H. Suzuki, I. Ogawa, Y. Shimada, K. Kobayashi, Y. Terashima, H. Matsumoto, K. Oshida, R. Ohta, T. Imamura, A. Miyazaki, M. Kawabata, S. Minowa, A. Maeda, M. Hayashi, Evaluation of a liver micronucleus assay in young rats (IV): a study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 698 (2010) 24–29. [18] H. Suzuki, N. Ikeda, K. Kobayashi, Y. Terashima, Y. Shimada, T. Suzuki, T. Hagiwara, S. Hatakeyama, K. Nagaoka, J. Yoshida, Y. Saito, J. Tanaka, M. Hayashi, Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats: a study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 583 (2005) 133–145. [19] H. Suzuki, H. Takasawa, K. Kobayashi, Y. Terashima, Y. Shimada, I. Ogawa, J. Tanaka, T. Imamura, A. Miyazaki, M. Hayashi, Evaluation of a liver micronucleus assay with 12 chemicals using young rats (II): a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society–Mammalian Mutagenicity Study Group, Mutagenesis 24 (2009) 9–16. [20] J.W. Parton, M.L. Garriott, An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four week-old rats treated with known clastogens, Environ. Mol. Mutagen. 29 (1997) 379–385. [21] H. Takasawa, H. Suzuki, I. Ogawa, Y. Shimada, K. Kobayashi, Y. Terashima, H. Matsumoto, C. Aruga, K. Oshida, R. Ohta, T. Imamura, A. Miyazaki, M. Kawabata, S. Minowa, M. Hayashi, Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 698 (2010) 30–37.
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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
Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy Satoru Itoh ∗ , Chiharu Hattori, Mayumi Nagata, Atsushi Sanbuissho Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Horikoshi 717, Fukuroi-shi, Shizuoka-ken 437-0065 Japan
a r t i c l e
i n f o
Article history: Received 23 February 2012 Received in revised form 27 March 2012 Accepted 17 April 2012 Available online 23 April 2012 Keywords: Liver Micronucleus test Structural chromosome aberration Numerical chromosome aberration Partial hepatectomy
a b s t r a c t The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89–98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8–10 days after PH. © 2012 Elsevier B.V. All rights reserved.
1. Introduction According to the ICH S2A and S2B guidelines [1,2], genotoxicity tests for drug candidates should comprise two in vitro tests, a bacterial reverse mutation test and an in vitro cytogenetic test; one in vivo test, a rodent micronucleus test in bone marrow or blood, should be conducted when the two in vitro tests show no genotoxic potential. The reality is, however, many compounds indicate positive responses in cytogenetic tests using mammalian cells and a second in vivo test is performed frequently as a follow-up. Recently, a revised version of the ICH guideline, [S2(R1) a revised version of ICH S2A and S2B], has reached step 4 [3], and it will move immediately to the final step of the process, which is regulatory implementation in the European Union, the US and Japan. According to that new guideline, there are two options for the standard battery for genotoxicity evaluation and it recommends selection of the target organ in the second in vivo test. Namely, when the
∗ Corresponding author. Tel.: +81 538 42 4356; fax: +81 538 42 4350. E-mail address: [email protected] (S. Itoh). 1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2012.04.007
drug candidate indicates a positive response under the presence of S9 in the in vitro mammalian cell test in option 1, the follow-up strategy will generally focus on in vivo studies of the liver. In the case of option 2, a second in vivo test, using a tissue that is well exposed to the drug or its metabolites, should be included in the battery to assure of lack of genotoxicity. Therefore, an in vivo genotoxicity study using liver is very important to evaluate whether a compound is a suitable candidate for drug development. The liver micronucleus test can detect the activity of promutagens, such as active metabolites that do not reach bone marrow due to their short lifespan, and this is one method of choice for a second in vivo genotoxicity test of a drug candidate [3]. In the liver micronucleus test, partial hepatectomy (PH) is used to stimulate mitosis of liver because mitotic activity of the liver is extremely low, and PH facilitates detection of chromosome damage by the test compound [4]. In rat studies, the liver micronucleus test is more sensitive to a structural chromosome aberration inducer when the compound is given before PH than when given after PH [4,5]. We have already reported that dosing after PH is essential to detect genotoxicity of a numerical chromosome aberration inducer in mice [6]. In recent years, rats have been used for in vivo
S. Itoh et al. / Mutation Research 747 (2012) 98–103
genotoxicity studies instead of mice in many cases in consideration of the toxicokinetics. In the present study, therefore, we first investigated the cell classification in naive rats and compared it with that in naive mice. Since a big difference was recognized in the proportion of binucleated cells between rats and mice, the responses to compounds known to induce structural or numerical chromosome aberrations – diethylnitrosamine [7] and 1,2-dimethylhydrazine [8], or cholchicine [9] and carbendazim [10], respectively – in rats were investigated; the compounds were given before or after PH. 2. Materials and methods 2.1. Test compounds Diethylnitrosamine (DEN, CAS no. 55-18-5) and 1,2-dimethylhydrazine dihydrochloride (1,2-DMH, CAS no. 306-37-6), structural chromosome aberration inducers, were purchased from Sigma–Aldrich Corporation (MO, USA) and Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan), respectively. Colchicine (CAS no. 6486-8) and carbendazim (CAS no. 10605-21-7), numerical chromosome aberration inducers, were purchased from Sigma–Aldrich Corporation 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., Osaka, Japan). 2.2. Animals Seven-week-old male 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 (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water were provided ad libitum. 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. Dose levels and treatment schedules Treatment schedules are illustrated in Fig. 1. Dose levels of 1,2-DMH or colchicine were determined based on the results of preliminary toxicity tests. Namely, the test compound was administered to 2–3 animals per group one day before or after PH and the general toxicity was observed at least for 4 days after PH. The maximum tolerable dose was selected for the highest dose level in the main study. In the case of DEN and carbendazim, the highest dose levels were determined by reference to previous studies [6,11]. The middle and low dose levels were set by dividing the highest dose by a common ratio of 2. Each test compound was administered once by oral gavage at a dose volume of 10 mL/kg to 4 animals per group on Day 1. 2.3.1. Partial hepatectomy (PH) Animals were anesthetized with isoflurane (Abbott Japan Co., 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 medial right lobe of the liver were ligated with surgical silk; and then the lobes were removed. The incision was sutured. The regenerated liver was removed 4 days after PH. 2.3.2. PH schedule The test substances were given either before or after PH. In the case of dosing before PH, the test compound 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). In contrast, PH was performed one day before the dosing of test compound (i.e., on Day -1), the test
Dosing before PH Day 1
Dosing
Day 2
PH
Day 3
99
compound was given on Day 1 and the regenerated liver was removed on Day 4 in the dosing after PH. 2.4. Measurement of regenerated liver weight and collection of hepatocytes Animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. The liver was excised and weighed with an electronic balance. The hepatocytes were isolated by a simplified method for liver perfusion [12]. 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, Inc.) was perfused through the liver. The liver was cut into small pieces with scissors, and they were 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 vol% neutral buffered formalin and centrifuged at 50 × g for 2 min, and then this procedure was repeated two times more for each sample. 2.5. Observations The sediment was resuspended in 10 vol% neutral buffered formalin at a volume ratio of 1:3–4. Equal volumes of liver cell suspension and acridine orange-DAPI solution were mixed well, one drop was placed on a slide glass, and then the slide was sealed with a cover glass. A fluorescence microscope (BX60F5, Olympus, Tokyo, Japan) with a total magnification of at least 400× was used for observation of the specimens. Two thousand hepatocytes excluding metaphase cells and nuclear fragment cells from each animal were observed for 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 EXSAS Ver. 7.6 (Arm Corporation, Osaka, Japan) with significance level at 5%. The incidence of MNH and dose dependency across all groups including vehicle control group was analyzed by twotailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. The relative liver weight was analyzed by a tree-type algorithm for quantitative data [13].
3. Results 3.1. MNH and cell classification in naive rats The basal level of MNH and the classification of cells from 5 naive rats and the respective data in mice from a previous study [6] in both species at 8 weeks of age are shown in Table 1. In naive rats, the percentage of binucleated cells was 27.3%, which is much lower than that in mice (83.6%). The incidence of MNH in rats was very low and comparable to that in mice. 3.2. Structural chromosome aberration inducers (DEN and 1,2-DMH) The incidence of MNH after treatment by DEN or 1,2-DMH, structural chromosome aberration inducers, given before and after PH is shown in Fig. 2, and the relative liver weight and cell classification are summarized in Table 2.
Dosing after PH Day 4
Day 5
Day 6
Hepatocyte isolation
Day -1
PH
Day 1
Dosing
Day 2
Day 3
Day 4
Hepatocyte isolation
Fig. 1. Treatment schedules. The test compound was administered to rats one day before or after PH and the hepatocytes were isolated 4 days after PH. The day of dosing is defined as Day 1.
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S. Itoh et al. / Mutation Research 747 (2012) 98–103
Table 1 MNH and cell classification in rats and mice. Species
Number of animals
Age (weeks old)
MNH (%)
Cell classifications (Mean) Nuclear count (%)
Rats Mice [6]
8 8
5 3
0.03 0.10
Mononucleated cells
Binucleated cells
Multinucleated cells
72.7 16.0
27.3 83.6
0.0 0.4
Metaphase cells (%)
Nuclear fragmentation cells (%)
0.0 0.0
0.0 0.0
MNH: micronucleated hepatocytes.
DEN Dosing before PH
6
1,2-DMH Dosing after PH
#
#
5
*
4
*
*
*
*
*
3 2 1 0
0
12.5 25
50
0 12.5
25
50
Incidence of MNH (%)
Incidence of MNH (%)
7
Dosing before PH
10
Dosing after PH
#
8
*
6
* *
4
* *
2 0
0
25
Dose level (mg/kg)
50 100
0
37.5
*
75 150
Dose level (mg/kg)
Fig. 2. Incidence of micronucleated hepatocytes in rats treated with structural chromosome aberration inducers before and after PH. Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%. Table 2 Relative liver weight and cell classification in rats treated with structural chromosome aberration inducers before and after PH. Timing of PH
Test compound
Dosing before PH
Water for injection Diethylnitrosamine
Dosing after PH
Water for injection Diethlnitrosamine
Dosing before PH
Water for injection 1,2-Dimethylhydrazine
Dosing after PH
Water for injection 1,2-Dimethylhydrazine
Dose (mg/kg)
0 12.5 25 50 0 12.5 25 50 0 25 50 100 0 37.5 75 150
Number of animals
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
3.11 3.14 2.95 2.66 2.96 2.85 2.62 2.42 3.04 2.96 2.66 1.93 2.89 2.63 2.25 2.18
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.06 0.07* 0.06* 0.20 0.12 0.12* 0.20* 0.24 0.08 0.27 0.18* 0.12 0.22 0.05* 0.11*
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
94.3 92.0 89.7 84.9 92.9 89.5 85.3 87.7 95.4 88.0 81.7 76.9 93.9 88.0 90.8 91.4
5.7 8.0 10.3 15.1 7.1 10.5 14.6 12.3 4.6 11.9 18.3 23.1 6.1 12.0 9.2 8.6
0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0
Meta (%)
NF (%)
0.5 0.4 0.4 0.3 0.2 0.3 0.2 0.2 0.6 0.9 0.3 0.2 0.1 0.1 0.2 0.1
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. * Statistically significant decrease in relative liver weight (P < 0.05, a tree-type algorithm for quantitative data).
Treatment with DEN resulted in a dose-dependent statistically significant increase in MNH at 12.5, 25 and 50 mg/kg regardless of whether the test drug was given before or after PH, and the magnitude of the change was comparable in both methods. Meanwhile, 1,2-DMH exhibited statistically significant increase in MNH with dose-dependent reduction in both methods, and MNH induction was stronger when the test drug was given before PH than when it was given after PH. The relative liver weight in the groups treated with DEN at 25 and 50 mg/kg decreased significantly in both methods, and the incidence of binucleated cells was increased, which suggested that liver regeneration was inhibited [6]. In the case of 1,2-DMH, a dose-dependent decrease in relative liver weight and an increase in the incidence of binucleated cells were observed,
similar to the results with DEN. No marked change was observed in the incidence of multinucleated hepatocytes, metaphase cells or nuclear fragment cells in either DEN or 1,2-DMH treatment groups. An additional experiment with lower dose levels of 1,2-DMH in both methods was conducted because the peak effect of MNH induction by 1,2-DMH was not obtained in the first experiment due to inhibition of liver regeneration. The incidence of MNH by lower dose levels of 1,2-DMH given before and after PH is shown in Fig. 4, and the relative liver weight and cell classification are listed in Table 4. In the groups given 1,2-DMH before PH, a dose-dependent statistically significant increase in the incidence of MNH was observed
S. Itoh et al. / Mutation Research 747 (2012) 98–103
Colchicine
101
Carbendazim
5
Incidence of MNH (%)
# *
3 #
2 1
*
*
*
3
6
0 0
12
0
0.5
1
2
Incidence of MNH (%)
Dosing after PH
Dosing before PH
4
12
Dosing after PH
Dosing before PH
#
10
*
8
*
6 4
*
2 0 0
Dose level (mg/kg)
62.5 125 250
0
62.5 125 250
Dose level (mg/kg)
Fig. 3. Incidence of micronucleated hepatocytes in rats treated with numerical chromosome aberration inducers before and after PH. Mean of 4 animals. The vertical bars represent S.D. All animals died between PH and hepatocyte isolation in the group given12 mg/kg of colchicine before PH. One animal died just after PH in the group given 62.5 mg/kg of carbendazim before PH. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%.
Table 3 Relative liver weight and cell classification in rats treated with numerical chromosome aberration inducers before and after PH. Timing of PH
Test compound
Dose(mg/kg)
Dosing before PH
Water for injection Colchocine
Dosing after PH
Water for injection Colchicine
Dosing before PH
0.5% methylcellulose Carbendazim
Dosing after PH
0.5% methylcellulose Carbendazim
0 3 6 12 0 0.5 1 2 0 62.5 125 250 0 62.5 125 250
Number of animals
4 4 4 0 4 4 4 4 4 3a 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
3.06 ± 0.10 2.90 ± 0.16 3.01 ± 0.25 ND 2.98 ± 0.29 2.98 ± 0.25 2.92 ± 0.24 2.95 ± 0.10 3.18 ± 0.10 3.24 ± 0.12 3.16 ± 0.25 2.99 ± 0.09 3.03 ± 0.16 3.11 ± 0.17 3.23 ± 0.09b 3.03 ± 0.32
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
94.7 94.9 95.3 ND 93.8 93.5 94.5 93.9 92.9 93.7 93.8 93.1 93.7 90.2 86.4 85.8
5.3 5.1 4.7 ND 6.2 6.5 5.4 5.8 7.1 6.3 6.2 6.9 6.3 9.7 12.8 13.7
0.0 0.0 0.0 ND 0.0 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.1 0.9 0.6
Meta (%)
NF (%)
0.1 0.2 0.5 ND 0.2 0.1 0.1 0.1 0.1 0.4 0.4 0.6 0.1 0.1 0.0 0.1
0.0 0.0 0.0 ND 0.0 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.8
ND: no data because all animals died between PH and hepatocyte isolation; Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. a One animal died just after PH. b No data in one animal because the caudate lobe was inadvertently left in the animal.
1,2-DMH
Incidence of MNH (%)
10
Dosing before PH
at doses from 3.13 to 25 mg/kg without inhibition of liver regeneration, based on the relative liver weight and the cell classification. In dosing after PH, a dose-dependent gradual increase in the incidence of MNH was obtained although slight inhibition of liver regeneration was observed at higher dose levels.
Dosing after PH
#
8
* 6
3.3. Numerical chromosome aberration inducer (colchicine and carbendazim)
#
4
* *
2
*
*
*
*
* 0 0
3.13 6.25 12.5 25
0
4.69 9.38 18.8 37.5
Dose level (mg/kg) Fig. 4. Incidence of micronucleated hepatocytes in rats treated with 1,2-DMH before and after PH (additional experiment). Mean of 4 animals. The vertical bars represent S.D. Statistical analyses were performed on the incidence of MNH and dose dependency by two-tailed Fisher’s exact test (*) and two-tailed Cochran-Armitage trend test (#), respectively, with significant level at 5%.
The incidence of MNH after treatment by colchicine or carbendazim, numerical chromosome aberration inducers, given before and after PH is shown in Fig. 3, and the relative liver weight and cell classification are summarized in Table 3. In dosing before PH, all animals in the 12 mg/kg of colchicine treatment group died between the time of PH and hepatocyte isolation. Treatment with colchicine resulted in a dose-dependent statistically significant increase in MNH whether given before or after PH, however, the incidences of MNH in the groups given 3 and 6 mg/kg colchicine before PH were within the historical control range of our laboratory (mean ± 3 S.D.: 0–0.74). Therefore, those increases were judged to have no toxicological relevance. In dosing before PH, 1 animal in the 62.5 mg/kg of carbendazim treatment
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Table 4 Relative liver weight and cell classification in rats treated with 1,2-DMH before and after PH (additional experiment). Timing of PH
Test compound
Dosing before PH
Water for injection 1,2-Dimethylhydrazine
Dosing after PH
Water for injection 1,2-Dimethylhydrazine
Dose (mg/kg)
0 3.13 6.25 12.5 25 0 4.69 9.38 18.75 37.5
Number of animals
4 4 4 4 4 4 4 4 4 4
Relative liver weight (%BW) Mean ± S.D.
2.98 3.00 3.06 3.03 2.86 3.25 3.06 2.94 2.46 2.44
± ± ± ± ± ± ± ± ± ±
0.20 0.11 0.08 0.16 0.10 0.27 0.15 0.08 0.16* 0.18*
Cell classification (Mean) Nuclear count (%) Mono
Bi
Multi
95.1 94.6 95.0 93.9 89.3 96.2 93.2 91.1 86.6 90.7
4.9 5.4 5.0 6.1 10.7 3.8 6.8 8.9 13.4 9.3
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0
Meta (%)
NF (%)
0.5 0.3 0.5 0.7 0.5 0.4 0.2 0.6 0.5 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cell classification: number of each type of cell among 2000 hepatocytes excluding metaphase and nuclear fragment cells; Mono: mononucleated hepatocyte, Bi: binucleated hepatocyte, Multi: multinucleated hepatocyte, Meta: number of metaphase cell among 2000 hepatocytes excluding nuclear fragment cells, NF: number of nuclear fragment cell among 2000 hepatocytes excluding metaphase cells. * Statistically significant decrease in relative liver weight (P < 0.05, a tree-type algorithm for quantitative data).
group died just after PH. Treatment with carbendazim resulted in a dose-dependent statistically significant increase in MNH at 62.5, 125 and 250 mg/kg given after PH, but no MNH induction was obtained when dosing occurred before PH. No change in the relative liver weight was observed in colchicine or carbendazim treatment groups in either method. The incidence of binucleated cells in the groups given 125 and 250 mg/kg carbendazim after PH was higher than that in the vehicle control group, suggesting that inhibition of liver regeneration had occurred [6]. For metaphase cells, a relatively higher incidence was obtained when higher dose levels of colchicine and carbendazim were given before PH, and high incidences of multinucleated and nuclear fragment cells were also observed at higher dose levels given after PH. Such induction is recognized as a typical response of numerical chromosome aberration inducers [14]. 4. Discussion We have already reported on micronucleus induction in the liver of mice by numerical chromosome aberration inducers, and that dosing after PH is essential to detect their genotoxicity [6]. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the liver micronucleus test using rats. DEN and 1,2-DMH, structural chromosome aberration inducers, exhibited markedly and significantly increased incidence of MNH when given before PH and also induced MNH when given after PH. The magnitude of induction when the drug was given after PH was comparable to, or weaker than, that when the drug was given before PH for DEN or 1,2-DMH, respectively. The in vivo clastogenicity of mitomycin C and cyclophosphamide have been investigated by dosing before and after PH in mice [12]. For both compounds, the incidence of MNH was higher when the compound was given before PH than when it was given after PH. The results with 1,2-DMH in the present study are consistent with these findings. The cytotoxicity of those compounds may be stronger when given after PH, when cell proliferation is activated, than when given before PH, when the cells are not dividing. This is supported by the fact that a slight increase in MNH was observed when a range of dose levels of 1,2-DMH (4.69–150 mg/kg) were given after PH. However, more research on the structural chromosome aberration inducers given both before and after PH is needed because DEN showed comparable potency for MNH induction in both methods in the present study. Recently, the relationship between DNA damage and micronucleus induction in mice has been reported [15] in a study of structural chromosome aberration inducers. DNA damage and micronucleus induction in
the liver were examined by Comet assay and micronucleus test with PH, respectively; it was concluded that the optimal timing of PH to detect clastogenicity of these compounds is 24 h after dosing. Furthermore, Tates et al. reported that DEN and dimethylnitrosamine induced hepatocytes with micronuclei when they were administered before and after PH, and concluded that the administration of the test compound before PH has advantages such as exposure to the compound in physiologically normal animals with intact liver, and examination under the influence of persistent DNA lesions [4]. From the above findings, the potency for induction of structural chromosome aberrations in the liver should be examined by dosing of the test compound before PH. Colchicine and carbendazim, numerical chromosome aberration inducers, significantly increased MNH frequency only when given after PH, not before PH, and the results were similar to those in mice [6]. Taken together, it was confirmed that exposure to a numerical chromosome aberration inducer under hepatocyte proliferation is essential for detecting genotoxic activity. The reason for this is that the inducer of numerical chromosome aberrations targets the essential DNA-containing structures (centromeres and telomeres), spindle apparatus (tubulin, MAP and centrioles) and cell cycle control molecules (cyclins, cdk’s and P53), which act during cell proliferation [16]. It seems that exposure to the test compound before PH is insufficient to induce micronucleus in the liver because most of the test compound is metabolized by the time that activation of hepatocyte proliferation begins 24 h after PH. DEN and 1,2-DMH have also been reported to increase the incidence of MNH in young rats at 4 weeks of age [17–19]. In young rats, the percentage of S-phase cells is 40 times higher than that in naive adult rats [20], therefore, hepatocytes are proliferating, which is similar to the condition in adult rats when the test compound is administered after PH. Induction of MNH by administration of DEN to adult rats after PH in the present study was stronger that in young rats in the published study, while MNH induction by 1,2-DMH administered after PH was comparable to that in young rats [19]. The reason for the different results of DEN between young intact rats and adult rats with PH is not clear, however, a difference in proliferative activity of the liver may contribute to this discrepancy. Actually, the proliferation rate in young rats is lower than that in adult rats after PH, since the percentage of S-phase cells in young rats is less than 29% of that in regenerating liver [20]. Unfortunately, since no numerical chromosome aberration inducer was used in the experiments with young rats [11,17–19,21], no comparison with the present results is possible. Interestingly, from our preliminary study using young rats,
S. Itoh et al. / Mutation Research 747 (2012) 98–103
colchicine did not induce MNH even at a sublethal dose (data not shown). Regarding the species differences in the liver micronucleus test, the percentage of binucleated cells in naive rats was less than half that in naive mice and a difference in temporal response to structural and numerical chromosome aberration inducers was suggested. Induction of MNH by administration of DEN or carbendazim to adult rats before or after PH, respectively, in the present study was similar to that in young rats in the published study [6,12], but a difference in the time course of the response to colchicine was identified. Colchicine increased the MNH frequency at 4 days after PH in rats, although the induction in mice was observed later, at 8–10 days after PH [6]. The incidence of binucleated cells decreased and mononucleated cells increased during proliferation of hepatocytes [6], and thus the high incidence of binucleated cells in naive mice may mean that more time will be required to enhance turnover of the hepatocytes enough to produce micronuclei at a level similar to that with rats. In summary, DEN and 1,2-DMH, structural chromosome aberration inducers, induced MNH when administered either before or after PH. Stronger MNH induction by 1,2-DMH was observed in the dosing before PH, and comparable MNH induction was obtained in both methods for DEN. Dosing before PH should be examined to detect the clastogenicity of structural chromosome aberration inducers in the liver of rats. Colchicine and carbendazim, numerical chromosome aberration inducers, increased the MNH frequency only when dosing was after PH in rats, the same as in mice [6]. Dosing after PH is essential to detect the genotoxicity of numerical chromosome aberration inducers in the liver of rats. Similar results in rats and mice were obtained with DEN and carbendazim in the liver micronucleus test with PH, but the responses to colchicine were different. The MNH induction by colchicine in rats clearly occurred earlier than that in mice. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors appreciated Drs. Takashi Yamoto and Miyuki Igarashi for their useful discussion. References [1] ICH Harmonised Tripartite Guideline, Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Available at: http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm065007.htm. [2] ICH Harmonised Tripartite Guideline, Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Available at: http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm065007.htm. [3] ICH Harmonised Tripartite Guideline, 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.
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