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Evaluation of the repeated-dose liver micronucleus assay using 2,4-dinitrotoluene: A report of a collaborative study by CSGMT/JEMS.MMS Akihisa Maeda, Hiromi Tsuchiyama, Yoshiji Asaoka, Mikito Hirakata, Tomoya Miyoshi, Keiyu Oshida, Yohei Miyamoto ∗ Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e
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Article history: Received 8 May 2014 Accepted 9 May 2014 Available online xxx Keywords: Liver micronucleus Young adult rat Repeated dose 2,4-Dinitrotoluene (DNT) General toxicity study
a b s t r a c t The liver micronucleus assay using young adult rats has the potential to detect liver carcinogens by repeated dosing, and could be expected to be integrated into repeated-dose toxicity studies using a hepatocyte isolation method without the traditional in situ collagenase perfusion. In this study, to assess the performance of the repeated-dose liver micronucleus assay, 2,4-dinitrotoluene (DNT), which is a rodent liver carcinogen, was administered orally to male rats at doses of 50, 100 and 200 mg/kg/day once daily for 14 or 28 consecutive days, and the frequencies of micronucleated hepatocytes (MNHEPs) and micronucleated immature erythrocytes (MNIMEs) were examined. Significant increases in the MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment, and 50 and 100 mg/kg/day in the 28day treatment. These increases were dependent on both the dose and the number of administrations, which indicates the possibility that the MNHEPs accumulate as a result of repeated dosing. In contrast, no increase in the MNIMEs was observed. In conclusion, the repeated-dose liver micronucleus assay using young adult rats is sufficiently sensitive to detect the genotoxicity of 2,4-DNT at a low dose. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), was undertaken to evaluate the suitability of a repeated-dose liver micronucleus assay using young adult rats. This study using 2,4-dinitrotoluene (DNT) was performed as part of that collaborative study. The micronucleus assay is a useful technique that is widely used to detect genotoxicity for chemicals, including pharmaceuticals. The International Conference on Harmonization (ICH) S2 (R1) guideline includes an in vivo micronucleus assay with rodent hematopoietic cells as part of the standard battery of genotoxicity tests [1]. In recent years, the liver micronucleus assay has been actively investigated because of the high metabolic activity and chemical exposure in the liver. Some carcinogens have showed positive results in the liver micronucleus assay,
∗ Corresponding author. Tel.: +81 467 32 9665; fax: +81 467 32 9768. E-mail address: Youhei [email protected] (Y. Miyamoto).
even when the bone marrow micronucleus assay yielded negative results [2]. However, partial hepatectomy (PH) or mitogen treatment is needed in order to compensate for extremely low mitotic activity of hepatocytes in adult animals [3,4]. These methods not only require much time and labor but may also result in decreased metabolic enzyme activity due to PH or an interaction of mitogen with the test compounds [5,6]. The Collaborative Study Group for the Micronucleus Test (CSGMT)/JEMS.MMS, demonstrated that a liver micronucleus assay using 4-week-old young rats with remarkable hepatocyte proliferation was able to detect the genotoxicity of hepatocarcinogens [2,7]. However, the activities of enzymes, such as the cytochrome P450 2C family, differ between young and adult animals [8]. To overcome these weaknesses, Narumi et al. developed a repeated-dose liver micronucleus assay using young adult rats and showed that the frequency of micronucleated hepatocytes (MNHEPs) increased after the repeated dosing of genotoxic chemicals, even if a low dose was applied [9]. In this study, we evaluated the usefulness of the repeated-dose liver micronucleus assay and assessed whether this assay could be integrated into repeated-dose toxicity studies.
http://dx.doi.org/10.1016/j.mrgentox.2014.05.002 1383-5718/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Maeda, et al., Evaluation of the repeated-dose liver micronucleus assay using 2,4dinitrotoluene: A report of a collaborative study by CSGMT/JEMS.MMS, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.05.002
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2. Materials and methods
3. Results
2.1. Chemicals
3.1. Clinical sign, body weight, necropsy and liver weight
2,4-DNT (CAS No. 121-14-2, Lot No. 18219TA, 97% purity) was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) and was suspended in 0.5% methylcellulose solution (MC, Wako Pure Chemical Industries, Ltd., Osaka, Japan.).
In the 200 mg/kg/day group of the 28-day treatment, 1 of the 5 animals was euthanized because of moribundity on Day 10, and 2 rats died on Day 15. In the 14-day treatment, loss of fur was observed at 200 mg/kg/day. In the 28-day treatment, salivation and loss of fur were observed at 50 mg/kg/day or more and 100 mg/kg/day, respectively. Prone position, lateral position, decrease and loss of locomotor activity, bradypnea, hypothermia, reddish tear, smudge of perinasal area and chromaturia were observed at 200 mg/kg/day. The body weight gains decreased significantly at 100 mg/kg/day or more in the 14- and 28-day treatments (Fig. 1A). In the 14-day treatment, dark red coloration and enlargement of the spleen were observed at 50 mg/kg/day or more and 100 mg/kg/day or more, respectively. In the 28-day treatment, dark red spleen, small testis and reddish spot in the lung were observed at 100 mg/kg/day or more, and reddish spot in the glandular stomach and discoloration of the liver were observed at 200 mg/kg/day. No difference was observed in the absolute liver weight; however, the relative liver weights increased at 100 mg/kg/day or more in the 14-day treatment and 100 mg/kg/day in the 28-day treatment (Fig. 1B and C).
2.2. Animal treatment Crl:CD(SD) male rats (Charles River Japan Inc., Yokohama, Japan) were housed under controlled temperature, lighting and relative humidity conditions (19–25 ◦ C, 12-h light/dark cycle and 40–60%, respectively). The animals were given a standard diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. 2,4-DNT was administered orally (10 mL/kg) to 6-week-old rats by gavage once daily for 14 or 28 consecutive days (Days 1–14 or 1–28) at doses of 50, 100 and 200 mg/kg/day. The three doses were determined on the basis of previous reports [10]. The rats were fasted for approximately 16 h before the necropsy on Day 15 or 29. The 14-day and 28-day repeated-dose tests were conducted separately at different times. During the period from Day 1 to Day 15 or 29, all of the animals were observed for clinical signs. The body weights were measured on Days 1, 4, 8, 11, 15, 18, 22, 25 and 29. At necropsy, all of the animals were euthanized by exsanguination under anesthesia with sodium pentobarbital, and the body surface, body openings, cranium, thoracic cavity, abdominal cavity and contents were examined macroscopically. The whole liver and right femur was removed. The liver weights were measured, and the liver weights relative to the final body weights were calculated. All of the experiments were conducted according to the Guidelines for Animal Experiments, Research & Development Division, Toray Industries, Inc. 2.3. Liver and bone marrow micronucleus assays The micronucleus assays using the liver and bone marrow were performed according to the protocol described in the summary report of this collaborative study [11]. Briefly, sliced livers were treated with 100 units/mL collagenase solution (pH 7.5) at 37 ◦ C for 1 h with shaking at 50 rpm. The tissues were then forcefully pipetted and filtered through a cell strainer. The hepatocyte suspension was fixed with 10 vol% neutral buffered formalin. The hepatocytes were stained with acridine orange (AO) and 4 ,6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope. Two thousands hepatocytes (HEPs) were observed, and the numbers of MNHEPs and metaphase (M-phase) hepatocytes were counted. The bone marrow cells were collected by flushing the cavity of the femur with fetal bovine serum and smeared on a slide glass. The smeared cells were air-dried and fixed with methanol. The bone marrow cells were stained with AO and observed with a fluorescence microscope. Two thousand immature erythrocytes (IMEs) were observed, and the number of micronucleated immature erythrocytes (MNIMEs) was counted. Furthermore, the frequency of IMEs, the number of IMEs per 500 erythrocytes, was determined. 2.4. Statistical analyses The frequencies of MNHEPs and MNIMEs were analyzed statistically using the Kastenbaum–Bowman method [12]. Statistical analyses of the other quantitative data were performed with Dunnett’s multiple comparison using the SAS Ver. 9.1.3 software (SAS institute Japan, Tokyo, Japan).
3.2. Liver micronucleus assay The results of the frequencies of MNHEPs and M-phase HEPs are shown in Table 1. Significant increases in the frequency of MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment and 50 and 100 mg/kg/day in the 28-day treatment. Furthermore, the increases of MNHEPs were dependent on the dose and the number of administrations. The M-phase HEPs in the 100 mg/kg/day group in the 28-day treatment increased significantly compared with those in the vehicle control group. 3.3. Bone marrow micronucleus assay The results of the frequencies of MNIMEs and IMEs are shown in Table 1. The frequencies of MNIMEs in any 2,4-DNT-treatment group were not significantly different from those in the vehicle control groups. No significant difference in the frequency of IMEs was also observed between any 2,4-DNT-treatment group and the vehicle control group. 4. Discussion A liver micronucleus assay with naive young adult animals was reported for the first time in 2012 by Narumi et al. [9]. They found the increase in the frequency of MNHEPs after repeated administration to be the same as that of the micronuclei in the peripheral blood after the repeated administration of a genotoxic agent to mice [13]. Moreover, they established a hepatocyte isolation technique without using in situ collagenase perfusion. We conducted repeated-dose liver micronucleus assays that were integrated into repeated-dose toxicity studies. As a result, a sufficient number of hepatocytes were obtained from only a small part of the liver that MNHEPs could be observed, and it was confirmed that the remaining liver tissue can be used to evaluate other toxicological endpoints. 2,4-DNT is a well-known rodent liver carcinogen. The carcinogenicity of 2,4-DNT would vary between high-grade and technical-grade including approximately 20% 2,6-DNT, which is a complete hepatocarcinogen [10]. In this study, 2,4-DNT with a purity of 97% was used.
Please cite this article in press as: A. Maeda, et al., Evaluation of the repeated-dose liver micronucleus assay using 2,4dinitrotoluene: A report of a collaborative study by CSGMT/JEMS.MMS, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.05.002
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Table 1 Results of 14- and 28-day repeated-dose liver and bone marrow micronucleus assays. No.of dosings
Test article
Dose (mg/kg/day)
No. of animals
Liver
Bone marrow
MNHEPs (%) Individual data
0.5% MC
0
5
50
5
100
5
200
5
0
5
50
5
100
5
200
2a
14 2,4-DNT
0.5% MC
28 2,4-DNT
0.00 0.10 0.10 0.10 0.00 0.20 0.15 0.30 0.10 0.10 0.15 0.45 0.20 0.30 0.20 0.15 0.35 0.40 0.45 0.20 0.05 0.15 0.05 0.05 0.10 0.45 0.40 0.35 0.10 0.20 0.80 0.60 0.35 0.30 0.30 0.55 0.40
M-phase HEPs (%) Mean ± S.D.
0.06 ± 0.05
0.17 ± 0.08*
0.26 ± 0.12**
0.31 ± 0.13**
0.08 ± 0.04
0.30 ± 0.15**
0.47 ± 0.22**
0.48
Individual data 0.10 0.15 0.05 0.00 0.00 0.05 0.10 0.15 0.10 0.00 0.05 0.15 0.05 0.00 0.05 0.05 0.10 0.00 0.10 0.00 0.00 0.00 0.05 0.05 0.05 0.00 0.05 0.10 0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.00 0.15
Mean ± S.D.
0.06 ± 0.07
0.08 ± 0.06
0.06 ± 0.05
0.05 ± 0.05
0.03 ± 0.03
0.07 ± 0.04
0.09 ± 0.02#
0.08
MNIMEs (%) Individual data 0.35 0.30 0.15 0.15 0.15 0.10 0.10 0.10 0.05 0.15 0.50 0.15 0.05 0.15 0.20 0.15 0.00 0.05 0.30 0.30 0.05 0.25 0.30 0.15 0.20 0.15 0.15 0.10 0.30 0.15 0.30 0.10 0.25 0.25 0.15 0.25 0.20
IMEs (%) Mean ± S.D.
0.22 ± 0.10
0.10 ± 0.04
0.21 ± 0.17
0.16 ± 0.14
0.19 ± 0.10
0.17 ± 0.08
0.21 ± 0.08
0.23
Individual data 56.8 51.8 56.8 74.8 48.8 50.4 51.8 42.2 49.8 65.4 65.6 60.0 60.4 51.6 67.0 52.6 48.4 59.8 68.2 52.6 56.6 41.8 43.2 52.2 33.0 54.2 51.2 36.8 28.6 50.4 56.4 52.6 41.8 52.4 42.6 45.0 65.2
Mean ± S.D.
57.8 ± 10.1
51.9 ± 8.4
60.9 ± 6.1
56.3 ± 7.8
45.4 ± 9.3
44.2 ± 11.0
49.2 ± 6.6
55.1
MNHEPs: Micronucleated hepatocytes; M-phase HEPs: Metaphase hepatocytes; MNIMEs: Micronucleated immature erythrocytes; IMEs: Immature erythrocytes MC: Methylcellulose; 2,4-DNT: 2,4-dinitrotoluene. a Two rats died and one rat was moribund because of toxicity during the treatment period. The statistical analysis was not performed in the 200 mg/kg/day group in the 28-day treatment due to 2 animals. * p < 0.05, Kastenbaum and Bowman’s statistical method. ** p < 0.01, Kastenbaum and Bowman’s statistical method. # p < 0.05, Dunnett’s multiple comparison
The clinical signs and the decreased body weight gain observed in this study were consistent with the previous reports [10,14]. The increased relative liver weights were attributed to decreases in the body weight gain in both studies. In the 28-day treatment, death and moribundity in the 200 mg/kg/day group were observed, and the top dose selected was more than the maximum tolerated dose. It is very crucial to set the dose appropriately for the integration of a liver micronucleus assay into a repeated-dose toxicity study. Although other examinations, such as histopathology and blood chemistry, were not conducted, the no observed adverse effect level (NOAEL) in each of these studies was considered to be slightly less than 50 mg/kg/day, given that only slight toxic effects were observed at 50 mg/kg/day. The frequency of MNHEPs in the 2,4-DNT-treated groups increased in a dose-dependent manner at 50 mg/kg/day or more in both the 14- and the 28-day treatments. The frequencies of MNHEPs in the 28-day treatment were higher than those found in the 14-day treatment, which suggests that the increase in MNHEPs also depends on the number of administrations. It was speculated that MNHEPs accumulated with repeated
administration without being selectively eliminated. This tendency was observed with the repeated administrations of diethylnitrosamine, 2,4-diaminotoluene and 1,2-dimethylhydrazine [9,15]. Because the life span of hepatocytes is approximately 200 days in mature rats [16], the MNHEPs might continue to increase for at least a few months after the first administration. The M-phase HEPs in the 100 mg/kg/day group in the 28-day treatment increased significantly compared with the vehicle control group. However, the frequency of M-phase HEPs in the 100 mg/kg/day group was 0.09%, which was the same level observed in the vehicle control groups in our previous study (mean ± S.D.; 0.11 ± 0.08%, unpublished data). In the liver micronucleus assay using young rats, 2,4-DNT induced MNHEPs at a dose of 75 mg/kg/day with the double-dosing method [7], and this repeated-dose micronucleus assay was able to detect the genotoxicity of 2,4-DNT at a lower dose. In contrast, no increase in the MNIMEs was observed, and this result was consistent with previous reports [14,17]. This might be because the concentration of the chemical or its active metabolites did not reach a sufficiently high level in the bone marrow to induce the MNIMEs. Although the ICH S2 (R1) guideline recommends the
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(A)
400 Body weight (g)
b)
14-day
350 300
* *
250
28-day 400
0 mg/kg/day 50 mg/kg/day 100 mg/kg/day 200 mg/kg/day
* *
Body weight (g)
a)
350
0 mg/kg/day 50 mg/kg/day 100 mg/kg/day 200 mg/kg/day
300 *
250
200
200
150
150 0
** **
0
(B)
5
10 Day
15
* *
**
**
10
20
29
Day
Liver weight (g)
15
10
because it is reported that cytotoxicity can lead to a decrease in DNA migration in the comet assay [18]. The UDS test, which detects DNA repair, could detect the genotoxicity of 2,4-DNT in hepatocytes [19]. This study indicates that the repeated-dose liver micronucleus assay using young adult rats has sufficient sensitivity to detect the genotoxicity of 2,4-DNT. The liver micronucleus assay is able to be performed appropriately without disturbing the routine examinations in repeated-dose toxicity studies, achieving a reduction in the animal use, which is strongly desired from the viewpoint of the “3R” principles of animal welfare, but also a decrease in the labor required in the administration of the chemical. The incidence of MNIMEs is affected by hematotoxicity and stresses such as hypothermia [20], and severe hepatotoxicity might increase the frequency of MNHEPs. It has also been reported that histopathological changes related to hepatocyte proliferation correlate with the increase of MNHEPs in liver micronucleus assays [15]. The integration will enable the total toxicity evaluation by the consideration of the toxicological findings such as histopathology, blood chemistry and toxicokinetics in general toxicity studies in general toxicity studies in combination with the results in genotoxicity studies. Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgements 0 0
50
100 200
0
(C)
Relative liver weight (g/100g body weight)
14-day
50
100 200 (mg/kg/day)
28-day
A part of this work was supported by the Health and Labour Sciences Research Grant H24-Chemistry-Designation-008 (Ministry of Health, Labour and Welfare). References
6 **
**
**
4
2
0 0
50
100 200
14-day
0
50
100 200 (mg/kg/day)
28-day
Fig. 1. Body weights (A), liver weights (B), and relative liver weight (C) after the repeated administration of 2,4-DNT at doses of 50, 100 and 200 mg/kg/day to 6week-old rats for 14 or 28 days. The animals were fasted starting on the night of Day 14 or 28 to the necropsy on Day 15 or 29. The data are presented as the mean ± S.D. for 5 animals in each group, with the exception of the 200 mg/kg/day group after Day 18 in the 28-day treatment (2 animals). The statistical analysis was performed using Dunnett’s multiple comparison (*P < 0.05, **P < 0.01), with the exception of the 200 mg/kg/day group after Day 18 in the 28-day treatment because there were just 2 animals.
use of a comet assay as a second in vivo study [1], 2,4-DNT gave negative result in a repeated-dose comet assay using rat liver [14]. The difference in the results obtained from the micronucleus assay and the comet assay using liver might be due to differences in the types of damage detected. In the comet assay, a 2-fold and dose-dependent, but not statistically significant increase in %DNA in tail was observed in the 2,4-DNT-treatment group. The reason why 2,4-DNT induced a weak response might be its cytotoxicity,
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Evaluation of the repeated-dose liver micronucleus assay using 2,4-dinitrotoluene: A report of a collaborative study by CSGMT/JEMS.MMS Akihisa Maeda, Hiromi Tsuchiyama, Yoshiji Asaoka, Mikito Hirakata, Tomoya Miyoshi, Keiyu Oshida, Yohei Miyamoto ∗ Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e
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Article history: Received 8 May 2014 Accepted 9 May 2014 Available online xxx Keywords: Liver micronucleus Young adult rat Repeated dose 2,4-Dinitrotoluene (DNT) General toxicity study
a b s t r a c t The liver micronucleus assay using young adult rats has the potential to detect liver carcinogens by repeated dosing, and could be expected to be integrated into repeated-dose toxicity studies using a hepatocyte isolation method without the traditional in situ collagenase perfusion. In this study, to assess the performance of the repeated-dose liver micronucleus assay, 2,4-dinitrotoluene (DNT), which is a rodent liver carcinogen, was administered orally to male rats at doses of 50, 100 and 200 mg/kg/day once daily for 14 or 28 consecutive days, and the frequencies of micronucleated hepatocytes (MNHEPs) and micronucleated immature erythrocytes (MNIMEs) were examined. Significant increases in the MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment, and 50 and 100 mg/kg/day in the 28day treatment. These increases were dependent on both the dose and the number of administrations, which indicates the possibility that the MNHEPs accumulate as a result of repeated dosing. In contrast, no increase in the MNIMEs was observed. In conclusion, the repeated-dose liver micronucleus assay using young adult rats is sufficiently sensitive to detect the genotoxicity of 2,4-DNT at a low dose. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), was undertaken to evaluate the suitability of a repeated-dose liver micronucleus assay using young adult rats. This study using 2,4-dinitrotoluene (DNT) was performed as part of that collaborative study. The micronucleus assay is a useful technique that is widely used to detect genotoxicity for chemicals, including pharmaceuticals. The International Conference on Harmonization (ICH) S2 (R1) guideline includes an in vivo micronucleus assay with rodent hematopoietic cells as part of the standard battery of genotoxicity tests [1]. In recent years, the liver micronucleus assay has been actively investigated because of the high metabolic activity and chemical exposure in the liver. Some carcinogens have showed positive results in the liver micronucleus assay,
∗ Corresponding author. Tel.: +81 467 32 9665; fax: +81 467 32 9768. E-mail address: Youhei [email protected] (Y. Miyamoto).
even when the bone marrow micronucleus assay yielded negative results [2]. However, partial hepatectomy (PH) or mitogen treatment is needed in order to compensate for extremely low mitotic activity of hepatocytes in adult animals [3,4]. These methods not only require much time and labor but may also result in decreased metabolic enzyme activity due to PH or an interaction of mitogen with the test compounds [5,6]. The Collaborative Study Group for the Micronucleus Test (CSGMT)/JEMS.MMS, demonstrated that a liver micronucleus assay using 4-week-old young rats with remarkable hepatocyte proliferation was able to detect the genotoxicity of hepatocarcinogens [2,7]. However, the activities of enzymes, such as the cytochrome P450 2C family, differ between young and adult animals [8]. To overcome these weaknesses, Narumi et al. developed a repeated-dose liver micronucleus assay using young adult rats and showed that the frequency of micronucleated hepatocytes (MNHEPs) increased after the repeated dosing of genotoxic chemicals, even if a low dose was applied [9]. In this study, we evaluated the usefulness of the repeated-dose liver micronucleus assay and assessed whether this assay could be integrated into repeated-dose toxicity studies.
http://dx.doi.org/10.1016/j.mrgentox.2014.05.002 1383-5718/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Maeda, et al., Evaluation of the repeated-dose liver micronucleus assay using 2,4dinitrotoluene: A report of a collaborative study by CSGMT/JEMS.MMS, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.05.002
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2. Materials and methods
3. Results
2.1. Chemicals
3.1. Clinical sign, body weight, necropsy and liver weight
2,4-DNT (CAS No. 121-14-2, Lot No. 18219TA, 97% purity) was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) and was suspended in 0.5% methylcellulose solution (MC, Wako Pure Chemical Industries, Ltd., Osaka, Japan.).
In the 200 mg/kg/day group of the 28-day treatment, 1 of the 5 animals was euthanized because of moribundity on Day 10, and 2 rats died on Day 15. In the 14-day treatment, loss of fur was observed at 200 mg/kg/day. In the 28-day treatment, salivation and loss of fur were observed at 50 mg/kg/day or more and 100 mg/kg/day, respectively. Prone position, lateral position, decrease and loss of locomotor activity, bradypnea, hypothermia, reddish tear, smudge of perinasal area and chromaturia were observed at 200 mg/kg/day. The body weight gains decreased significantly at 100 mg/kg/day or more in the 14- and 28-day treatments (Fig. 1A). In the 14-day treatment, dark red coloration and enlargement of the spleen were observed at 50 mg/kg/day or more and 100 mg/kg/day or more, respectively. In the 28-day treatment, dark red spleen, small testis and reddish spot in the lung were observed at 100 mg/kg/day or more, and reddish spot in the glandular stomach and discoloration of the liver were observed at 200 mg/kg/day. No difference was observed in the absolute liver weight; however, the relative liver weights increased at 100 mg/kg/day or more in the 14-day treatment and 100 mg/kg/day in the 28-day treatment (Fig. 1B and C).
2.2. Animal treatment Crl:CD(SD) male rats (Charles River Japan Inc., Yokohama, Japan) were housed under controlled temperature, lighting and relative humidity conditions (19–25 ◦ C, 12-h light/dark cycle and 40–60%, respectively). The animals were given a standard diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. 2,4-DNT was administered orally (10 mL/kg) to 6-week-old rats by gavage once daily for 14 or 28 consecutive days (Days 1–14 or 1–28) at doses of 50, 100 and 200 mg/kg/day. The three doses were determined on the basis of previous reports [10]. The rats were fasted for approximately 16 h before the necropsy on Day 15 or 29. The 14-day and 28-day repeated-dose tests were conducted separately at different times. During the period from Day 1 to Day 15 or 29, all of the animals were observed for clinical signs. The body weights were measured on Days 1, 4, 8, 11, 15, 18, 22, 25 and 29. At necropsy, all of the animals were euthanized by exsanguination under anesthesia with sodium pentobarbital, and the body surface, body openings, cranium, thoracic cavity, abdominal cavity and contents were examined macroscopically. The whole liver and right femur was removed. The liver weights were measured, and the liver weights relative to the final body weights were calculated. All of the experiments were conducted according to the Guidelines for Animal Experiments, Research & Development Division, Toray Industries, Inc. 2.3. Liver and bone marrow micronucleus assays The micronucleus assays using the liver and bone marrow were performed according to the protocol described in the summary report of this collaborative study [11]. Briefly, sliced livers were treated with 100 units/mL collagenase solution (pH 7.5) at 37 ◦ C for 1 h with shaking at 50 rpm. The tissues were then forcefully pipetted and filtered through a cell strainer. The hepatocyte suspension was fixed with 10 vol% neutral buffered formalin. The hepatocytes were stained with acridine orange (AO) and 4 ,6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope. Two thousands hepatocytes (HEPs) were observed, and the numbers of MNHEPs and metaphase (M-phase) hepatocytes were counted. The bone marrow cells were collected by flushing the cavity of the femur with fetal bovine serum and smeared on a slide glass. The smeared cells were air-dried and fixed with methanol. The bone marrow cells were stained with AO and observed with a fluorescence microscope. Two thousand immature erythrocytes (IMEs) were observed, and the number of micronucleated immature erythrocytes (MNIMEs) was counted. Furthermore, the frequency of IMEs, the number of IMEs per 500 erythrocytes, was determined. 2.4. Statistical analyses The frequencies of MNHEPs and MNIMEs were analyzed statistically using the Kastenbaum–Bowman method [12]. Statistical analyses of the other quantitative data were performed with Dunnett’s multiple comparison using the SAS Ver. 9.1.3 software (SAS institute Japan, Tokyo, Japan).
3.2. Liver micronucleus assay The results of the frequencies of MNHEPs and M-phase HEPs are shown in Table 1. Significant increases in the frequency of MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment and 50 and 100 mg/kg/day in the 28-day treatment. Furthermore, the increases of MNHEPs were dependent on the dose and the number of administrations. The M-phase HEPs in the 100 mg/kg/day group in the 28-day treatment increased significantly compared with those in the vehicle control group. 3.3. Bone marrow micronucleus assay The results of the frequencies of MNIMEs and IMEs are shown in Table 1. The frequencies of MNIMEs in any 2,4-DNT-treatment group were not significantly different from those in the vehicle control groups. No significant difference in the frequency of IMEs was also observed between any 2,4-DNT-treatment group and the vehicle control group. 4. Discussion A liver micronucleus assay with naive young adult animals was reported for the first time in 2012 by Narumi et al. [9]. They found the increase in the frequency of MNHEPs after repeated administration to be the same as that of the micronuclei in the peripheral blood after the repeated administration of a genotoxic agent to mice [13]. Moreover, they established a hepatocyte isolation technique without using in situ collagenase perfusion. We conducted repeated-dose liver micronucleus assays that were integrated into repeated-dose toxicity studies. As a result, a sufficient number of hepatocytes were obtained from only a small part of the liver that MNHEPs could be observed, and it was confirmed that the remaining liver tissue can be used to evaluate other toxicological endpoints. 2,4-DNT is a well-known rodent liver carcinogen. The carcinogenicity of 2,4-DNT would vary between high-grade and technical-grade including approximately 20% 2,6-DNT, which is a complete hepatocarcinogen [10]. In this study, 2,4-DNT with a purity of 97% was used.
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Table 1 Results of 14- and 28-day repeated-dose liver and bone marrow micronucleus assays. No.of dosings
Test article
Dose (mg/kg/day)
No. of animals
Liver
Bone marrow
MNHEPs (%) Individual data
0.5% MC
0
5
50
5
100
5
200
5
0
5
50
5
100
5
200
2a
14 2,4-DNT
0.5% MC
28 2,4-DNT
0.00 0.10 0.10 0.10 0.00 0.20 0.15 0.30 0.10 0.10 0.15 0.45 0.20 0.30 0.20 0.15 0.35 0.40 0.45 0.20 0.05 0.15 0.05 0.05 0.10 0.45 0.40 0.35 0.10 0.20 0.80 0.60 0.35 0.30 0.30 0.55 0.40
M-phase HEPs (%) Mean ± S.D.
0.06 ± 0.05
0.17 ± 0.08*
0.26 ± 0.12**
0.31 ± 0.13**
0.08 ± 0.04
0.30 ± 0.15**
0.47 ± 0.22**
0.48
Individual data 0.10 0.15 0.05 0.00 0.00 0.05 0.10 0.15 0.10 0.00 0.05 0.15 0.05 0.00 0.05 0.05 0.10 0.00 0.10 0.00 0.00 0.00 0.05 0.05 0.05 0.00 0.05 0.10 0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.00 0.15
Mean ± S.D.
0.06 ± 0.07
0.08 ± 0.06
0.06 ± 0.05
0.05 ± 0.05
0.03 ± 0.03
0.07 ± 0.04
0.09 ± 0.02#
0.08
MNIMEs (%) Individual data 0.35 0.30 0.15 0.15 0.15 0.10 0.10 0.10 0.05 0.15 0.50 0.15 0.05 0.15 0.20 0.15 0.00 0.05 0.30 0.30 0.05 0.25 0.30 0.15 0.20 0.15 0.15 0.10 0.30 0.15 0.30 0.10 0.25 0.25 0.15 0.25 0.20
IMEs (%) Mean ± S.D.
0.22 ± 0.10
0.10 ± 0.04
0.21 ± 0.17
0.16 ± 0.14
0.19 ± 0.10
0.17 ± 0.08
0.21 ± 0.08
0.23
Individual data 56.8 51.8 56.8 74.8 48.8 50.4 51.8 42.2 49.8 65.4 65.6 60.0 60.4 51.6 67.0 52.6 48.4 59.8 68.2 52.6 56.6 41.8 43.2 52.2 33.0 54.2 51.2 36.8 28.6 50.4 56.4 52.6 41.8 52.4 42.6 45.0 65.2
Mean ± S.D.
57.8 ± 10.1
51.9 ± 8.4
60.9 ± 6.1
56.3 ± 7.8
45.4 ± 9.3
44.2 ± 11.0
49.2 ± 6.6
55.1
MNHEPs: Micronucleated hepatocytes; M-phase HEPs: Metaphase hepatocytes; MNIMEs: Micronucleated immature erythrocytes; IMEs: Immature erythrocytes MC: Methylcellulose; 2,4-DNT: 2,4-dinitrotoluene. a Two rats died and one rat was moribund because of toxicity during the treatment period. The statistical analysis was not performed in the 200 mg/kg/day group in the 28-day treatment due to 2 animals. * p < 0.05, Kastenbaum and Bowman’s statistical method. ** p < 0.01, Kastenbaum and Bowman’s statistical method. # p < 0.05, Dunnett’s multiple comparison
The clinical signs and the decreased body weight gain observed in this study were consistent with the previous reports [10,14]. The increased relative liver weights were attributed to decreases in the body weight gain in both studies. In the 28-day treatment, death and moribundity in the 200 mg/kg/day group were observed, and the top dose selected was more than the maximum tolerated dose. It is very crucial to set the dose appropriately for the integration of a liver micronucleus assay into a repeated-dose toxicity study. Although other examinations, such as histopathology and blood chemistry, were not conducted, the no observed adverse effect level (NOAEL) in each of these studies was considered to be slightly less than 50 mg/kg/day, given that only slight toxic effects were observed at 50 mg/kg/day. The frequency of MNHEPs in the 2,4-DNT-treated groups increased in a dose-dependent manner at 50 mg/kg/day or more in both the 14- and the 28-day treatments. The frequencies of MNHEPs in the 28-day treatment were higher than those found in the 14-day treatment, which suggests that the increase in MNHEPs also depends on the number of administrations. It was speculated that MNHEPs accumulated with repeated
administration without being selectively eliminated. This tendency was observed with the repeated administrations of diethylnitrosamine, 2,4-diaminotoluene and 1,2-dimethylhydrazine [9,15]. Because the life span of hepatocytes is approximately 200 days in mature rats [16], the MNHEPs might continue to increase for at least a few months after the first administration. The M-phase HEPs in the 100 mg/kg/day group in the 28-day treatment increased significantly compared with the vehicle control group. However, the frequency of M-phase HEPs in the 100 mg/kg/day group was 0.09%, which was the same level observed in the vehicle control groups in our previous study (mean ± S.D.; 0.11 ± 0.08%, unpublished data). In the liver micronucleus assay using young rats, 2,4-DNT induced MNHEPs at a dose of 75 mg/kg/day with the double-dosing method [7], and this repeated-dose micronucleus assay was able to detect the genotoxicity of 2,4-DNT at a lower dose. In contrast, no increase in the MNIMEs was observed, and this result was consistent with previous reports [14,17]. This might be because the concentration of the chemical or its active metabolites did not reach a sufficiently high level in the bone marrow to induce the MNIMEs. Although the ICH S2 (R1) guideline recommends the
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(A)
400 Body weight (g)
b)
14-day
350 300
* *
250
28-day 400
0 mg/kg/day 50 mg/kg/day 100 mg/kg/day 200 mg/kg/day
* *
Body weight (g)
a)
350
0 mg/kg/day 50 mg/kg/day 100 mg/kg/day 200 mg/kg/day
300 *
250
200
200
150
150 0
** **
0
(B)
5
10 Day
15
* *
**
**
10
20
29
Day
Liver weight (g)
15
10
because it is reported that cytotoxicity can lead to a decrease in DNA migration in the comet assay [18]. The UDS test, which detects DNA repair, could detect the genotoxicity of 2,4-DNT in hepatocytes [19]. This study indicates that the repeated-dose liver micronucleus assay using young adult rats has sufficient sensitivity to detect the genotoxicity of 2,4-DNT. The liver micronucleus assay is able to be performed appropriately without disturbing the routine examinations in repeated-dose toxicity studies, achieving a reduction in the animal use, which is strongly desired from the viewpoint of the “3R” principles of animal welfare, but also a decrease in the labor required in the administration of the chemical. The incidence of MNIMEs is affected by hematotoxicity and stresses such as hypothermia [20], and severe hepatotoxicity might increase the frequency of MNHEPs. It has also been reported that histopathological changes related to hepatocyte proliferation correlate with the increase of MNHEPs in liver micronucleus assays [15]. The integration will enable the total toxicity evaluation by the consideration of the toxicological findings such as histopathology, blood chemistry and toxicokinetics in general toxicity studies in general toxicity studies in combination with the results in genotoxicity studies. Conflicts of interest The authors declare that there are no conflicts of interest.
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Acknowledgements 0 0
50
100 200
0
(C)
Relative liver weight (g/100g body weight)
14-day
50
100 200 (mg/kg/day)
28-day
A part of this work was supported by the Health and Labour Sciences Research Grant H24-Chemistry-Designation-008 (Ministry of Health, Labour and Welfare). References
6 **
**
**
4
2
0 0
50
100 200
14-day
0
50
100 200 (mg/kg/day)
28-day
Fig. 1. Body weights (A), liver weights (B), and relative liver weight (C) after the repeated administration of 2,4-DNT at doses of 50, 100 and 200 mg/kg/day to 6week-old rats for 14 or 28 days. The animals were fasted starting on the night of Day 14 or 28 to the necropsy on Day 15 or 29. The data are presented as the mean ± S.D. for 5 animals in each group, with the exception of the 200 mg/kg/day group after Day 18 in the 28-day treatment (2 animals). The statistical analysis was performed using Dunnett’s multiple comparison (*P < 0.05, **P < 0.01), with the exception of the 200 mg/kg/day group after Day 18 in the 28-day treatment because there were just 2 animals.
use of a comet assay as a second in vivo study [1], 2,4-DNT gave negative result in a repeated-dose comet assay using rat liver [14]. The difference in the results obtained from the micronucleus assay and the comet assay using liver might be due to differences in the types of damage detected. In the comet assay, a 2-fold and dose-dependent, but not statistically significant increase in %DNA in tail was observed in the 2,4-DNT-treatment group. The reason why 2,4-DNT induced a weak response might be its cytotoxicity,
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