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Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats
Mutation Research 780-781 (2015) 81–84
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats Hajime Sui a,∗ , Hirotaka Matsumoto a,1 , Yumi Wako b , Kazufumi Kawasako b a b
Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257-8523, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2014 Accepted 1 October 2014 Available online 18 October 2014 Keywords: Repeated-dose liver micronucleus assay Integrated into general toxicological studies Thioacetamide
a b s t r a c t The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14and 28-day RDLMN assays to assess the performance of the assay. The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20 mg/kg/day. Hepatocytes were collected approximately 24 h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1–4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150 mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7]. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Environmental Mutagen Society of Japan (JEMS), was conducted to evaluate the suitability of the repeated dose liver and gastrointestinal tract micronucleus assays using young adult rats. Using thioacetamide (TAA), this study was performed as a part of that collaborative study. Micronucleus assays using rodents typically target bone marrow cells. However, unstable active metabolites may not reach their target organ, i.e., the bone marrow. Therefore, evaluation of the genotoxic potential of these chemicals cannot reasonably be performed when only bone marrow cells are used. The liver is generally known to be the major site of metabolism of absorbed chemicals. Therefore, the liver is considered to be an important target organ for evaluation of the genotoxicity. In this study, the performance of the repeated-dose liver micronucleus (RDLMN) assay was assessed.
2.1. Animals Five-week-old male Sprague-Dawley rats [Crl:CD (SD), SPF] were purchased from the Atsugi Breeding Center at Charles River Laboratories Japan, Inc. (Yokohama, Japan). The rats were used for testing at 6 weeks of age. The animals were housed individually during the experimental period in metal cages (220 mm × 270 mm × 190 mm) with a wire mesh floor in a room with a temperature of 21–25 ◦ C, relative humidity of 40–75%, a ventilation exchange rate of approximately 15 times per hour and a 12-h lighting cycle. The animals were given free access to pellet feed and drinking water. The animal experiments in this study were conducted according to the Guidelines for Animal Experimentation in our laboratory.
2.2. Chemicals Thioacetamide (TAA, CAS No. 62-55-5, >98.0% purity; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in distilled water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan).
2.3. Dosage and administration ∗ Corresponding author at: Laboratory of Genetics, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257-8523, Japan. Tel.: +81 463 82 4751; fax: +81 463 82 9627. E-mail address: [email protected] (H. Sui). 1 Present address: 2-35-21 Shinmachi, Setagaya-ku, Tokyo 154-0014, Japan. http://dx.doi.org/10.1016/j.mrgentox.2014.10.001 1383-5718/© 2014 Elsevier B.V. All rights reserved.
TAA was administered to rats orally at dosages of 5, 10 and 20 mg/kg/day once daily for 14 or 28 days. The highest dose was determined in reference to the rat TD50 of TAA (11.5 mg/kg/day), which was previously calculated [8] using the concentration of TAA in the diet at which hepatic carcinoma was significantly induced [9]. The dose was given at a volume of 10 mL/kg. The general condition was observed once a
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H. Sui et al. / Mutation Research 780-781 (2015) 81–84
day during the administration period. The rats were weighed twice per week during the administration period.
2.7. Statistical analysis The differences in the incidence of MNHEPs and MNIMEs were analyzed using Kastenbaum and Bowman tables at 5% and 1% significance levels. For analysis of the MI, the body weight (BW), the liver weight (LW) and the relative liver weight (LW per BW), one-way ANOVA was performed between the TAA-treated group and negative control group at the 5% and 1% significance levels because a homogeneous variance was demonstrated. Furthermore, the logarithmic dose dependency of the frequencies of MI, MNHEPs and MNIMEs was analyzed by the trend test of Cochran–Armitage (one-tailed) at the 5% significance level.
2.4. Liver micronucleus assay Micronucleus assays using the liver were performed according to the protocol described in the summary report of this collaborative study [10]. Briefly, the liver was weighed (LW) and hepatocyte specimens were prepared approximately 24 h after the last administration. A part of the left lateral lobe was sliced, and then incubated in a digestion solution containing collagenase (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) with reciprocal shaking at 37 ◦ C for 1 h. The supernatant was removed, and hepatocyte pellet was suspended in Hank’s balanced salt solution (GIBCO-Invitrogen, Carlsbad, CA, USA) warmed at 37 ◦ C by pipetting. The hepatocyte suspension was filtered through a nylon gauze, and then through 70 m nylon Cell Strainer. The hepatocyte suspension filtered was mixed with 10% neutralbuffered formalin, and then centrifuged at 50 × g for 2 min. The hepatocyte pellet was resuspended with 10% neutral-buffered formalin, and then centrifuged at 50 × g for 1 min. The hepatocyte pellet was resuspended with 10% neutral-buffered formalin, and kept in a refrigerator until analysis. The hepatocyte suspension was mixed with an equal volume of an acridine orange (AO, 500 g/mL) and 4 ,6-diamidino-2phenylindole dihydrochloride (DAPI, 10 g/mL) staining solution, dropped onto a glass slide, and covered with a coverslip. The slide specimens were observed using a fluorescence microscope (600× magnification with UV excitation). Two thousand parenchymal hepatocytes (HEPs) per rat were observed by two individuals, and the number of micronucleated hepatocytes (MNHEPs) was recorded. Additionally, the number of cells at mitotic phase for every 2000 HEPs in each rat was counted to determine the mitotic index (MI).
3. Results and discussion In this study, no change in the general condition was observed in any rat. No statistically significant difference in the BW and the LW was observed, but the relative liver weights were significantly increased in 20 mg/kg/day TAA-treated group with 14- and 28-day repeat dosing (p < 0.01, negative control groups: 3.44 ± 0.12 and 3.08 ± 0.26; TAA-treated groups: 4.26 ± 0.42 and 4.07 ± 0.38). The histopathological examination showed centrilobular hypertrophy of hepatocytes, single cell necrosis of centrilobular hepatocytes and a prominent nucleolus in the centrilobular hepatocytes in the group that received 20 mg/kg/day TAA (Table 1). These findings were observed in the TAA-treated group, but were not observed in the negative control group. Thus, it was confirmed that the liver was exposed to TAA at a high dose. The frequency of MI was investigated for hepatocyte proliferation in this study, and consequently no significant difference was observed in any TAA-treated groups with 14- and 28-day repeat dosing regardless of whether the histopathological findings were observed. It was previously reported that single oral gavage of TAA gave positive results both in the rat hepatocyte replicative DNA synthesis (RDS) test [11] and the BrdU immunohistochemistry evaluation [12]. It was also reported that the increase in hepatocyte proliferation was due to a regenerative liver response following cytotoxicity by TAA treatment [12]. Unfortunately, the regenerative liver response following cytotoxicity was not clearly observed in this study. When the frequency of MI is considered alone as in this study, it may be difficult to detect the alteration of hepatocyte proliferation because the frequency of hepatocyte proliferation is normally very low and available information about cell-cycle progression is limited. Thus, the integration of the immunohistochemistry evaluation into the RDLMN may be useful to investigate cell proliferation induced by hepatotoxic hepatocarcinogens such as TAA.
2.5. Bone marrow micronucleus assay Micronucleus assays using bone marrow were performed according to the protocol described in the summary report of this collaborative study [10]. Briefly, bone marrow cell specimens were prepared approximately 24 h after the last administration. After the liver removal, left femur was excised from the same animals. Both ends of each femur were cut off, and bone marrow cells were flushed out with fetal bovine serum (GIBCO-Invitrogen, Carlsbad, CA, USA) into a centrifuge tube followed by centrifugation at 200 × g for 5 min to remove the supernatant. The bone marrow cells suspension was smeared on glass slides. The slides were dried at room temperature, and then fixed with methanol for 5 min to prepare specimens. The specimens were stained with 40 g/mL AO solution, and covered with a coverslip. The slide specimens were observed using a fluorescence microscope (1000× magnification with blue excitation). Two thousand immature erythrocytes per rat were observed by two individuals, and the number of micronucleated immature erythrocytes (MNIMEs) was recorded.
2.6. Histopathological examination The histopathological examination was performed according to the protocol described in the summary report of this collaborative study [10]. Before the liver removal for liver micronucleus assay, a part of the left lateral lobe was excised from the same animals. The left lateral lobe was fixed with 10% neutral-buffered formalin, and then used for the histopathological examination.
Table 1 Results of histopathological examination of the liver with TAA for 14- and 28-day repeat dosings in male young adult rats. Findings (14-day repeat dosing)
Dose (mg/kg/day) 0
5
10
20
Animal no.
Hypertrophy, hepatocytes, centrilobular Single cell necrosis, hepatocytes, centrilobular Prominent nucleolus, hepatocytes, centrilobular Findings (28-day repeat dosing)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− + +
− − −
− ++ +
− ++ +
− ++ +
Dose (mg/kg/day) 0
5
10
20
Animal no.
Hypertrophy, hepatocytes, centrilobular Single cell necrosis, hepatocytes, centrilobular Prominent nucleolus, hepatocytes, centrilobular −, none; + minimal; ++, mild.
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − +
− + +
− + +
− + +
+ + +
H. Sui et al. / Mutation Research 780-781 (2015) 81–84
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Table 2 Results of liver and bone marrow micronucleus assays with TAA for 14-day repeat dosing in male young adult rats. Group
Animal No.
No. of cells scored
Micronucleated hepatocytesa
Mitotic index of hepatocytesb
Micronucleated immature erythrocytesc
No. of MN cells
% of frequency (mean ± S.D.)
No. of cells
% of frequency (mean ± S.D.)
No. of MN cells
% of frequency (mean ± S.D.)
Negative control Water for injection JP (10 mL/kg/day)
1 2 3 4 5 Total
2000 2000 2000 2000 2000 10,000
1 1 1 1 1 5
0.05 0.05 0.05 0.05 0.05 (0.05 ± 0.00)
3 1 3 2 3 12
0.15 0.05 0.15 0.10 0.15 (0.12 ± 0.04)
6 6 4 7 8 31
0.30 0.30 0.20 0.35 0.40 (0.31 ± 0.07)
TAA (5 mg/kg/day)
6 7 8 9 10 Total
2000 2000 2000 2000 2000 10,000
0 0 0 2 2 4
0.00 0.00 0.00 0.10 0.10 (0.04 ± 0.05)
3 2 1 2 2 10
0.15 0.10 0.05 0.10 0.10 (0.10 ± 0.04)
7 8 6 9 3 33
0.35 0.40 0.30 0.45 0.15 (0.33 ± 0.12)
TAA (10 mg/kg/day)
11 12 13 14 15 Total
2000 2000 2000 2000 2000 10,000
2 0 1 1 3 7
0.10 0.00 0.05 0.05 0.15 (0.07 ± 0.06)
0 1 6 1 2 10
0.00 0.05 0.30 0.05 0.10 (0.10 ± 0.12)
7 6 6 7 6 32
0.35 0.30 0.30 0.35 0.30 (0.32 ± 0.03)
TAA (20 mg/kg/day)
16 17 18 19 20 Total
2000 2000 2000 2000 2000 10,000
1 2 0 4 3 10
0.05 0.10 0.00 0.20 0.15 (0.10 ± 0.08)
4 5 5 4 2 20
0.20 0.25 0.25 0.20 0.10 (0.20 ± 0.06)
4 3 8 9 4 28
0.20 0.15 0.40 0.45 0.20 (0.28 ± 0.14)
a b c
Two thousand parenchymal hepatocytes per rat were observed. The number of cells at mitotic phase for every 2000 hepatocytes in each rat was counted. Two thousand immature erythrocytes per rat were observed.
The results of the micronucleus assay with 14- and 28-day repeat dosing are summarized in Tables 2 and 3. In both experiments, the frequencies of MNHEPs in any TAA-treated group were not significantly higher than those in the negative control group. However, the trend test indicated that there was a significant dose-dependent increase only in the frequency of MNHEPs for TAA with repeat dosing for 28 days (p < 0.05). The mean frequency of MNHEPs in the 20 mg/kg/day TAA-treated group (0.17%) for 28 days seemed to slightly increase compared with the concurrent negative control group (0.09%). One animal (0.20%) in the 10 mg/kg/day TAA-treated group and one (0.30%) in the 20 mg/kg/day TAA-treated group (animal no. 34 and 36, respectively) showed minor increases in the frequency of MNHEPs compared with the range of the individual frequencies of MNHEPs (0.00–0.15%) in the concurrent negative control group for 28 days. In addition, all mean frequencies of MNHEPs in TAA-treated groups after both 14- and 28-day repeat dosing (0.04–0.10% and 0.09–0.17%, respectively) were within the fluctuation range for the negative control group (i.e., mean ± 2 × S.D.; 0.06 ± 0.12 for both repeat dosing) described in the summary report of this collaborative study [10]. Thus, it was concluded that no clear increase in the frequency of MNHEPs was observed in any of the TAAtreated groups after 14 and 28 days of repeat dosing in this study. TAA is known as a liver carcinogen in rats [13]. However, TAA gave negative results in the short-term genotoxic studies, such as the Ames test [1–3], the chromosomal aberration test using CHL/IU cells [4] and the liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150 mg/kg) [5]. Only bone marrow micronucleus test using mice, conducted up to near the maximum tolerated dose, showed positive results [6,7]. On the other hand, the intermediate metabolite of TAA, thioacetamide-S-oxide, gave positive results at a high dose only without S9 mix in the Ames test [14], and the bone marrow micronucleus test using mice orally
dosed twice [6]. The ultimate metabolite of TAA, acetamide, gave negative result in the Ames test [2,3], but gave positive results in the bone marrow micronucleus test using mice orally dosed twice [6]. It was also reported that thioacetamide-S-oxide induced the highest frequency of MNIMEs, and acetamide induced the lowest frequency on a molar basis [6]. Thus, it is considered that the amount of thioacetamide-S-oxide generated from metabolism of TAA (rather than TAA itself) is strongly related to the genotoxic activity of TAA. In this study, microscopic observation revealed no significant increase in the frequency of MNIMEs in any TAA-treated groups compared with the negative control group (Tables 2 and 3). However, TAA gave strong positive results in the bone marrow micronucleus test using male C57BL/6 mice [7], and weak positive responses were observed in female C57BL/6 mice [6,7] and both sexes of BALB/c mice [7]. Also, these micronucleus tests were performed up to near the maximum tolerated dose. Thus, the species-dependent difference of clastogenic effect by TAA treatment and dose selection is thought to be responsible for the failure of the increased frequency of MNIMEs in the present study. TAA is also known as a hepatotoxic hepatocarcinogen because rat hepatocyte proliferation was increased due to a regenerative liver response following cytotoxicity by TAA treatment [10]. This evidence suggests that the exposure to its metabolite at a high dose (i.e., initiation effect) and the mitogenic activation of hepatocytes following cytotoxicity by TAA and its metabolites (i.e., promotion effect) are necessary conditions for hepatocarcinogenesis. In the present study, we concluded that there was no clear increase in the frequency of MNHEPs. However, the significant dose-dependent increase was observed in the frequency of MNHEPs for 28-day repeat dosing of TAA, but not for 14-day repeat dosing. This result could imply that there is accumulation of initiation and promotion effects by TAA treatment. In the RDLMN assay, achieving doses close
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Table 3 Results of liver and bone marrow micronucleus assays with TAA for 28-day repeat dosing in male young adult rats. Group
Animal No.
No. of cells scored
Micronucleated hepatocytesa , *
Mitotic index of hepatocytesb
Micronucleated immature erythrocytesc
No. of MN cells
No. of cells
% of frequency (mean ± S.D.)
No. of MN cells
% of frequency (mean ± S.D.)
% of frequency (mean ± S.D.)
Negative control Water for injection JP (10 mL/kg/day)
21 22 23 24 25 Total
2000 2000 2000 2000 2000 10,000
3 0 2 2 2 9
0.15 0.00 0.10 0.10 0.10 (0.09 ± 0.05)
1 2 1 1 1 6
0.05 0.10 0.05 0.05 0.05 (0.06 ± 0.02)
3 5 7 5 6 26
0.15 0.25 0.35 0.25 0.30 (0.26 ± 0.07)
TAA (5 mg/kg/day)
26 27 28 29 30 Total
2000 2000 2000 2000 2000 10,000
1 2 3 1 2 9
0.05 0.10 0.15 0.05 0.10 (0.09 ± 0.04)
2 2 1 0 0 5
0.10 0.10 0.05 0.00 0.00 (0.05 ± 0.05)
6 6 2 4 4 22
0.30 0.30 0.10 0.20 0.20 (0.22 ± 0.08)
TAA (10 mg/kg/day)
31 32 33 34 35 Total
2000 2000 2000 2000 2000 10,000
3 1 0 4 3 11
0.15 0.05 0.00 0.20 0.15 (0.11 ± 0.08)
3 3 1 2 2 11
0.15 0.15 0.05 0.10 0.10 (0.11 ± 0.04)
6 4 4 7 8 29
0.30 0.20 0.20 0.35 0.40 (0.29 ± 0.09)
TAA (20 mg/kg/day)
36 37 38 39 40 Total
2000 2000 2000 2000 2000 10,000
6 3 2 3 3 17
0.30 0.15 0.10 0.15 0.15 (0.17 ± 0.08)
2 2 4 1 2 11
0.10 0.10 0.20 0.05 0.10 (0.11 ± 0.05)
4 6 7 6 11 34
0.20 0.30 0.35 0.30 0.55 (0.34 ± 0.13)
a b c *
Two thousand parenchymal hepatocytes per rat were observed. The number of cells at mitotic phase for every 2000 hepatocytes in each rat was counted. Two thousand immature erythrocytes per rat were observed. Significant dose-dependent increase was observed by the trend test of Cochran–Armitage (p < 0.05).
to the maximum tolerated, and careful consideration of selection of test species, are thought to be very important factors to detect clastogenic effects with hepatotoxic hepatocarcinogen such as TAA. The number of doses and the sampling time used in the present study were the same as those used in a repeated-dose 28-day oral toxicity study in rodents. Thus, the RDLMN assay can be integrated into the general repeated-dose 28-day oral toxicity study and is useful for the evaluation of genotoxicity in the liver. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment 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 [1] D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1–256. [2] J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Nat. Acad. Sci. U. S. A. 72 (1975) 5135–5139. [3] L.E. Kier, D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Prival, T.K. Rao, V. Ray, The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutat. Res. 168 (1986) 69–240. [4] M. Ishidate Jr., M.C. Harnois, T. Sofuni, A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures, Mutat. Res. 195 (1988) 151–213.
[5] 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. [6] E. Chieli, F. Aliboni, M. Saviozzi, G. Malvaldi, Induction of micronucleated erythrocytes by primary thioamides and their metabolites in the mouse, Mutat. Res. 192 (1987) 141–143. [7] E.T. Mirkova, Activities of the rodent carcinogens thioacetamide and acetamide in the mouse bone marrow micronucleus assay, Mutat. Res. 352 (1996) 23–30. [8] L.S. Gold, T.H. Slone, N.B. Manley, G.B. Garfinkel, L. Rohrbach, B.N. Ames, Carcinogenic potency database, in: L.S. Gold, E. Zeiger (Eds.), Handbook of Carcinogenic Potency and Genotoxicity Databases, CRC Press, Inc., Florida, 1997, pp. 488–489. [9] K. Kuroda, K. Terao, M. Akao, Inhibitory effect of fumaric acid on hepatocarcinogenesis by thioacetamide in rats, J. Natl. Cancer Inst. 79 (1987) 1047– 1051. [10] S. Hamada, W. Ohyama, R. Takashima, K. Shimada, K. Matsumoto, S. Kawakami, F. Uno, H. Sui, Y. Shimada, T. Imamura, S. Matsumura, H. Sanada, K. Inoue, S. Muto, I. Ogawa, A. Hayashi, T. Takayanagi, Y. Ogiwara, A. Maeda, E. Okada, Y. Terashima, H. Takasawa, K. Narumi, Y. Wako, K. Kawasako, M. Sano, N. Ohashi, T. Morita, H. Kojima, M. Honma, M. Hayashi, Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the collaborative study group for the micronucleus test (CSGMT)/the Japanese Environmental Mutagen Society (JEMS) – Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 780–781 (2015) 2–17. [11] Y. Uno, H. Takasawa, M. Miyagawa, Y. Inoue, T. Murata, K. Yoshikawa, An in vivo–in vitro replicative DNA synthesis (RDS) test using rat hepatocytes as an early prediction assay for nongenotoxic hepatocarcinogens screening of 22 known positives and 25 noncarcinogens, Mutat. Res. 320 (1994) 189–205. [12] M. Ohtsuka, K. Fukuda, H. Yano, M. Kojiro, Immunohistochemical measurement of cell proliferation as replicative DNA synthesis in the liver of male Fischer 344 rats following a single exposure to nongenotoxic hepatocarcinogens and noncarcinogens, Exp. Toxicol. Pathol. 50 (1998) 13–17. [13] F.F. Becker, Thioacetamide hepatocarcinogenesis, J. Natl. Cancer Inst. 71 (1983) 553–558. [14] A.P. Breau, W.M. Mitchell, D.W. Karkhanis, L. Field, Thiono compounds, 3. Chemical oxidation of thioacetamide to a mutagenic S-oxide, Mutat. Res. 139 (1984) 1–4.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Evaluation of in vivo genotoxicity by thioacetamide in a 28-day repeated-dose liver micronucleus assay using male young adult rats Hajime Sui a,∗ , Hirotaka Matsumoto a,1 , Yumi Wako b , Kazufumi Kawasako b a b
Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257-8523, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 27 September 2014 Accepted 1 October 2014 Available online 18 October 2014 Keywords: Repeated-dose liver micronucleus assay Integrated into general toxicological studies Thioacetamide
a b s t r a c t The repeated-dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens and can be integrated into general toxicological studies. In this study, thioacetamide (TAA) was tested in 14and 28-day RDLMN assays to assess the performance of the assay. The test substance, TAA, was administered orally to 6-week-old male Crl:CD (SD) rats once daily for 14 or 28 days at a dosage of 5, 10 or 20 mg/kg/day. Hepatocytes were collected approximately 24 h after the last TAA administration, and the incidence of micronuclei was assessed. In this study, bone marrow micronucleus assays were also conducted in the same animals. The 14- and 28-day RDLMN assays indicated that none of the TAA dosages significantly increased the proportion of micronucleated hepatocytes. Bone marrow micronucleus assays with TAA also provided negative results. It is known that TAA is a liver carcinogen in mice and rats. In the previous genotoxic studies, the Ames test and the chromosomal aberration test using CHL/IU cells have yielded negative results [1–4]. The liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150 mg/kg) also produced negative results [5]. TAA gave positive results only in the mouse bone marrow micronucleus assays [6,7]. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Environmental Mutagen Society of Japan (JEMS), was conducted to evaluate the suitability of the repeated dose liver and gastrointestinal tract micronucleus assays using young adult rats. Using thioacetamide (TAA), this study was performed as a part of that collaborative study. Micronucleus assays using rodents typically target bone marrow cells. However, unstable active metabolites may not reach their target organ, i.e., the bone marrow. Therefore, evaluation of the genotoxic potential of these chemicals cannot reasonably be performed when only bone marrow cells are used. The liver is generally known to be the major site of metabolism of absorbed chemicals. Therefore, the liver is considered to be an important target organ for evaluation of the genotoxicity. In this study, the performance of the repeated-dose liver micronucleus (RDLMN) assay was assessed.
2.1. Animals Five-week-old male Sprague-Dawley rats [Crl:CD (SD), SPF] were purchased from the Atsugi Breeding Center at Charles River Laboratories Japan, Inc. (Yokohama, Japan). The rats were used for testing at 6 weeks of age. The animals were housed individually during the experimental period in metal cages (220 mm × 270 mm × 190 mm) with a wire mesh floor in a room with a temperature of 21–25 ◦ C, relative humidity of 40–75%, a ventilation exchange rate of approximately 15 times per hour and a 12-h lighting cycle. The animals were given free access to pellet feed and drinking water. The animal experiments in this study were conducted according to the Guidelines for Animal Experimentation in our laboratory.
2.2. Chemicals Thioacetamide (TAA, CAS No. 62-55-5, >98.0% purity; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in distilled water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan).
2.3. Dosage and administration ∗ Corresponding author at: Laboratory of Genetics, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257-8523, Japan. Tel.: +81 463 82 4751; fax: +81 463 82 9627. E-mail address: [email protected] (H. Sui). 1 Present address: 2-35-21 Shinmachi, Setagaya-ku, Tokyo 154-0014, Japan. http://dx.doi.org/10.1016/j.mrgentox.2014.10.001 1383-5718/© 2014 Elsevier B.V. All rights reserved.
TAA was administered to rats orally at dosages of 5, 10 and 20 mg/kg/day once daily for 14 or 28 days. The highest dose was determined in reference to the rat TD50 of TAA (11.5 mg/kg/day), which was previously calculated [8] using the concentration of TAA in the diet at which hepatic carcinoma was significantly induced [9]. The dose was given at a volume of 10 mL/kg. The general condition was observed once a
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day during the administration period. The rats were weighed twice per week during the administration period.
2.7. Statistical analysis The differences in the incidence of MNHEPs and MNIMEs were analyzed using Kastenbaum and Bowman tables at 5% and 1% significance levels. For analysis of the MI, the body weight (BW), the liver weight (LW) and the relative liver weight (LW per BW), one-way ANOVA was performed between the TAA-treated group and negative control group at the 5% and 1% significance levels because a homogeneous variance was demonstrated. Furthermore, the logarithmic dose dependency of the frequencies of MI, MNHEPs and MNIMEs was analyzed by the trend test of Cochran–Armitage (one-tailed) at the 5% significance level.
2.4. Liver micronucleus assay Micronucleus assays using the liver were performed according to the protocol described in the summary report of this collaborative study [10]. Briefly, the liver was weighed (LW) and hepatocyte specimens were prepared approximately 24 h after the last administration. A part of the left lateral lobe was sliced, and then incubated in a digestion solution containing collagenase (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) with reciprocal shaking at 37 ◦ C for 1 h. The supernatant was removed, and hepatocyte pellet was suspended in Hank’s balanced salt solution (GIBCO-Invitrogen, Carlsbad, CA, USA) warmed at 37 ◦ C by pipetting. The hepatocyte suspension was filtered through a nylon gauze, and then through 70 m nylon Cell Strainer. The hepatocyte suspension filtered was mixed with 10% neutralbuffered formalin, and then centrifuged at 50 × g for 2 min. The hepatocyte pellet was resuspended with 10% neutral-buffered formalin, and then centrifuged at 50 × g for 1 min. The hepatocyte pellet was resuspended with 10% neutral-buffered formalin, and kept in a refrigerator until analysis. The hepatocyte suspension was mixed with an equal volume of an acridine orange (AO, 500 g/mL) and 4 ,6-diamidino-2phenylindole dihydrochloride (DAPI, 10 g/mL) staining solution, dropped onto a glass slide, and covered with a coverslip. The slide specimens were observed using a fluorescence microscope (600× magnification with UV excitation). Two thousand parenchymal hepatocytes (HEPs) per rat were observed by two individuals, and the number of micronucleated hepatocytes (MNHEPs) was recorded. Additionally, the number of cells at mitotic phase for every 2000 HEPs in each rat was counted to determine the mitotic index (MI).
3. Results and discussion In this study, no change in the general condition was observed in any rat. No statistically significant difference in the BW and the LW was observed, but the relative liver weights were significantly increased in 20 mg/kg/day TAA-treated group with 14- and 28-day repeat dosing (p < 0.01, negative control groups: 3.44 ± 0.12 and 3.08 ± 0.26; TAA-treated groups: 4.26 ± 0.42 and 4.07 ± 0.38). The histopathological examination showed centrilobular hypertrophy of hepatocytes, single cell necrosis of centrilobular hepatocytes and a prominent nucleolus in the centrilobular hepatocytes in the group that received 20 mg/kg/day TAA (Table 1). These findings were observed in the TAA-treated group, but were not observed in the negative control group. Thus, it was confirmed that the liver was exposed to TAA at a high dose. The frequency of MI was investigated for hepatocyte proliferation in this study, and consequently no significant difference was observed in any TAA-treated groups with 14- and 28-day repeat dosing regardless of whether the histopathological findings were observed. It was previously reported that single oral gavage of TAA gave positive results both in the rat hepatocyte replicative DNA synthesis (RDS) test [11] and the BrdU immunohistochemistry evaluation [12]. It was also reported that the increase in hepatocyte proliferation was due to a regenerative liver response following cytotoxicity by TAA treatment [12]. Unfortunately, the regenerative liver response following cytotoxicity was not clearly observed in this study. When the frequency of MI is considered alone as in this study, it may be difficult to detect the alteration of hepatocyte proliferation because the frequency of hepatocyte proliferation is normally very low and available information about cell-cycle progression is limited. Thus, the integration of the immunohistochemistry evaluation into the RDLMN may be useful to investigate cell proliferation induced by hepatotoxic hepatocarcinogens such as TAA.
2.5. Bone marrow micronucleus assay Micronucleus assays using bone marrow were performed according to the protocol described in the summary report of this collaborative study [10]. Briefly, bone marrow cell specimens were prepared approximately 24 h after the last administration. After the liver removal, left femur was excised from the same animals. Both ends of each femur were cut off, and bone marrow cells were flushed out with fetal bovine serum (GIBCO-Invitrogen, Carlsbad, CA, USA) into a centrifuge tube followed by centrifugation at 200 × g for 5 min to remove the supernatant. The bone marrow cells suspension was smeared on glass slides. The slides were dried at room temperature, and then fixed with methanol for 5 min to prepare specimens. The specimens were stained with 40 g/mL AO solution, and covered with a coverslip. The slide specimens were observed using a fluorescence microscope (1000× magnification with blue excitation). Two thousand immature erythrocytes per rat were observed by two individuals, and the number of micronucleated immature erythrocytes (MNIMEs) was recorded.
2.6. Histopathological examination The histopathological examination was performed according to the protocol described in the summary report of this collaborative study [10]. Before the liver removal for liver micronucleus assay, a part of the left lateral lobe was excised from the same animals. The left lateral lobe was fixed with 10% neutral-buffered formalin, and then used for the histopathological examination.
Table 1 Results of histopathological examination of the liver with TAA for 14- and 28-day repeat dosings in male young adult rats. Findings (14-day repeat dosing)
Dose (mg/kg/day) 0
5
10
20
Animal no.
Hypertrophy, hepatocytes, centrilobular Single cell necrosis, hepatocytes, centrilobular Prominent nucleolus, hepatocytes, centrilobular Findings (28-day repeat dosing)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− + +
− − −
− ++ +
− ++ +
− ++ +
Dose (mg/kg/day) 0
5
10
20
Animal no.
Hypertrophy, hepatocytes, centrilobular Single cell necrosis, hepatocytes, centrilobular Prominent nucleolus, hepatocytes, centrilobular −, none; + minimal; ++, mild.
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − −
− − +
− + +
− + +
− + +
+ + +
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Table 2 Results of liver and bone marrow micronucleus assays with TAA for 14-day repeat dosing in male young adult rats. Group
Animal No.
No. of cells scored
Micronucleated hepatocytesa
Mitotic index of hepatocytesb
Micronucleated immature erythrocytesc
No. of MN cells
% of frequency (mean ± S.D.)
No. of cells
% of frequency (mean ± S.D.)
No. of MN cells
% of frequency (mean ± S.D.)
Negative control Water for injection JP (10 mL/kg/day)
1 2 3 4 5 Total
2000 2000 2000 2000 2000 10,000
1 1 1 1 1 5
0.05 0.05 0.05 0.05 0.05 (0.05 ± 0.00)
3 1 3 2 3 12
0.15 0.05 0.15 0.10 0.15 (0.12 ± 0.04)
6 6 4 7 8 31
0.30 0.30 0.20 0.35 0.40 (0.31 ± 0.07)
TAA (5 mg/kg/day)
6 7 8 9 10 Total
2000 2000 2000 2000 2000 10,000
0 0 0 2 2 4
0.00 0.00 0.00 0.10 0.10 (0.04 ± 0.05)
3 2 1 2 2 10
0.15 0.10 0.05 0.10 0.10 (0.10 ± 0.04)
7 8 6 9 3 33
0.35 0.40 0.30 0.45 0.15 (0.33 ± 0.12)
TAA (10 mg/kg/day)
11 12 13 14 15 Total
2000 2000 2000 2000 2000 10,000
2 0 1 1 3 7
0.10 0.00 0.05 0.05 0.15 (0.07 ± 0.06)
0 1 6 1 2 10
0.00 0.05 0.30 0.05 0.10 (0.10 ± 0.12)
7 6 6 7 6 32
0.35 0.30 0.30 0.35 0.30 (0.32 ± 0.03)
TAA (20 mg/kg/day)
16 17 18 19 20 Total
2000 2000 2000 2000 2000 10,000
1 2 0 4 3 10
0.05 0.10 0.00 0.20 0.15 (0.10 ± 0.08)
4 5 5 4 2 20
0.20 0.25 0.25 0.20 0.10 (0.20 ± 0.06)
4 3 8 9 4 28
0.20 0.15 0.40 0.45 0.20 (0.28 ± 0.14)
a b c
Two thousand parenchymal hepatocytes per rat were observed. The number of cells at mitotic phase for every 2000 hepatocytes in each rat was counted. Two thousand immature erythrocytes per rat were observed.
The results of the micronucleus assay with 14- and 28-day repeat dosing are summarized in Tables 2 and 3. In both experiments, the frequencies of MNHEPs in any TAA-treated group were not significantly higher than those in the negative control group. However, the trend test indicated that there was a significant dose-dependent increase only in the frequency of MNHEPs for TAA with repeat dosing for 28 days (p < 0.05). The mean frequency of MNHEPs in the 20 mg/kg/day TAA-treated group (0.17%) for 28 days seemed to slightly increase compared with the concurrent negative control group (0.09%). One animal (0.20%) in the 10 mg/kg/day TAA-treated group and one (0.30%) in the 20 mg/kg/day TAA-treated group (animal no. 34 and 36, respectively) showed minor increases in the frequency of MNHEPs compared with the range of the individual frequencies of MNHEPs (0.00–0.15%) in the concurrent negative control group for 28 days. In addition, all mean frequencies of MNHEPs in TAA-treated groups after both 14- and 28-day repeat dosing (0.04–0.10% and 0.09–0.17%, respectively) were within the fluctuation range for the negative control group (i.e., mean ± 2 × S.D.; 0.06 ± 0.12 for both repeat dosing) described in the summary report of this collaborative study [10]. Thus, it was concluded that no clear increase in the frequency of MNHEPs was observed in any of the TAAtreated groups after 14 and 28 days of repeat dosing in this study. TAA is known as a liver carcinogen in rats [13]. However, TAA gave negative results in the short-term genotoxic studies, such as the Ames test [1–3], the chromosomal aberration test using CHL/IU cells [4] and the liver micronucleus assay using young adult rats singly dosed with TAA (75 and 150 mg/kg) [5]. Only bone marrow micronucleus test using mice, conducted up to near the maximum tolerated dose, showed positive results [6,7]. On the other hand, the intermediate metabolite of TAA, thioacetamide-S-oxide, gave positive results at a high dose only without S9 mix in the Ames test [14], and the bone marrow micronucleus test using mice orally
dosed twice [6]. The ultimate metabolite of TAA, acetamide, gave negative result in the Ames test [2,3], but gave positive results in the bone marrow micronucleus test using mice orally dosed twice [6]. It was also reported that thioacetamide-S-oxide induced the highest frequency of MNIMEs, and acetamide induced the lowest frequency on a molar basis [6]. Thus, it is considered that the amount of thioacetamide-S-oxide generated from metabolism of TAA (rather than TAA itself) is strongly related to the genotoxic activity of TAA. In this study, microscopic observation revealed no significant increase in the frequency of MNIMEs in any TAA-treated groups compared with the negative control group (Tables 2 and 3). However, TAA gave strong positive results in the bone marrow micronucleus test using male C57BL/6 mice [7], and weak positive responses were observed in female C57BL/6 mice [6,7] and both sexes of BALB/c mice [7]. Also, these micronucleus tests were performed up to near the maximum tolerated dose. Thus, the species-dependent difference of clastogenic effect by TAA treatment and dose selection is thought to be responsible for the failure of the increased frequency of MNIMEs in the present study. TAA is also known as a hepatotoxic hepatocarcinogen because rat hepatocyte proliferation was increased due to a regenerative liver response following cytotoxicity by TAA treatment [10]. This evidence suggests that the exposure to its metabolite at a high dose (i.e., initiation effect) and the mitogenic activation of hepatocytes following cytotoxicity by TAA and its metabolites (i.e., promotion effect) are necessary conditions for hepatocarcinogenesis. In the present study, we concluded that there was no clear increase in the frequency of MNHEPs. However, the significant dose-dependent increase was observed in the frequency of MNHEPs for 28-day repeat dosing of TAA, but not for 14-day repeat dosing. This result could imply that there is accumulation of initiation and promotion effects by TAA treatment. In the RDLMN assay, achieving doses close
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Table 3 Results of liver and bone marrow micronucleus assays with TAA for 28-day repeat dosing in male young adult rats. Group
Animal No.
No. of cells scored
Micronucleated hepatocytesa , *
Mitotic index of hepatocytesb
Micronucleated immature erythrocytesc
No. of MN cells
No. of cells
% of frequency (mean ± S.D.)
No. of MN cells
% of frequency (mean ± S.D.)
% of frequency (mean ± S.D.)
Negative control Water for injection JP (10 mL/kg/day)
21 22 23 24 25 Total
2000 2000 2000 2000 2000 10,000
3 0 2 2 2 9
0.15 0.00 0.10 0.10 0.10 (0.09 ± 0.05)
1 2 1 1 1 6
0.05 0.10 0.05 0.05 0.05 (0.06 ± 0.02)
3 5 7 5 6 26
0.15 0.25 0.35 0.25 0.30 (0.26 ± 0.07)
TAA (5 mg/kg/day)
26 27 28 29 30 Total
2000 2000 2000 2000 2000 10,000
1 2 3 1 2 9
0.05 0.10 0.15 0.05 0.10 (0.09 ± 0.04)
2 2 1 0 0 5
0.10 0.10 0.05 0.00 0.00 (0.05 ± 0.05)
6 6 2 4 4 22
0.30 0.30 0.10 0.20 0.20 (0.22 ± 0.08)
TAA (10 mg/kg/day)
31 32 33 34 35 Total
2000 2000 2000 2000 2000 10,000
3 1 0 4 3 11
0.15 0.05 0.00 0.20 0.15 (0.11 ± 0.08)
3 3 1 2 2 11
0.15 0.15 0.05 0.10 0.10 (0.11 ± 0.04)
6 4 4 7 8 29
0.30 0.20 0.20 0.35 0.40 (0.29 ± 0.09)
TAA (20 mg/kg/day)
36 37 38 39 40 Total
2000 2000 2000 2000 2000 10,000
6 3 2 3 3 17
0.30 0.15 0.10 0.15 0.15 (0.17 ± 0.08)
2 2 4 1 2 11
0.10 0.10 0.20 0.05 0.10 (0.11 ± 0.05)
4 6 7 6 11 34
0.20 0.30 0.35 0.30 0.55 (0.34 ± 0.13)
a b c *
Two thousand parenchymal hepatocytes per rat were observed. The number of cells at mitotic phase for every 2000 hepatocytes in each rat was counted. Two thousand immature erythrocytes per rat were observed. Significant dose-dependent increase was observed by the trend test of Cochran–Armitage (p < 0.05).
to the maximum tolerated, and careful consideration of selection of test species, are thought to be very important factors to detect clastogenic effects with hepatotoxic hepatocarcinogen such as TAA. The number of doses and the sampling time used in the present study were the same as those used in a repeated-dose 28-day oral toxicity study in rodents. Thus, the RDLMN assay can be integrated into the general repeated-dose 28-day oral toxicity study and is useful for the evaluation of genotoxicity in the liver. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment 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 [1] D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1–256. [2] J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Nat. Acad. Sci. U. S. A. 72 (1975) 5135–5139. [3] L.E. Kier, D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Prival, T.K. Rao, V. Ray, The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutat. Res. 168 (1986) 69–240. [4] M. Ishidate Jr., M.C. Harnois, T. Sofuni, A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures, Mutat. Res. 195 (1988) 151–213.
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