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In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether
Mutation Research 816 (2017) 7–11
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
In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether Shinji Takasu a , Yuji Ishii a , Yuh Yokoo a , Takuma Tsuchiya a , Aki Kijima a , Yukio Kodama b , Kumiko Ogawa a , Takashi Umemura a,∗ a b
Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Division of Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e
i n f o
Article history: Received 7 November 2016 Received in revised form 8 February 2017 Accepted 21 February 2017 Available online 22 February 2017 Keywords: in vivo genotoxicity Decabromodiphenyl ether gpt Delta mouse
a b s t r a c t Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, have been widely used as additive flame retardants. Recently, the use of brominated flame retardants has been restricted or prohibited under various legislative acts because of the persistence, bioaccumulation potential, and toxicity of these compounds. However, there are also additional concerns regarding environmental contamination and human exposure to PBDEs resulting from informal recycling technology. Decabromodiphenyl ether (decaBDE), one type of PBDE, has carcinogenic potential in the livers of rodents. Although one study has shown that decaBDE exerts genotoxic effects, the other in vitro and in vivo studies were negative for such effects. Thus, it remains unknown whether genotoxic mechanisms are involved in decaBDE-induced hepatocarcinogenesis in rodents. In this study, to explore the genotoxicity of decaBDE in mice, particularly in the context of carcinogenesis, we performed micronucleus assays in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice treated with decaBDE at carcinogenic doses for 28 days. Our results demonstrated negative results in micronucleus tests and reporter gene mutation assays. Thus, decaBDE did not exert genotoxic effects at carcinogenic target sites and did not show positive results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Overall, comprehensive evaluation using in vivo genotoxicity data in rats and our data indicated that nongenotoxic mechanisms may be responsible for decaBDE-induced hepatocarcinogenesis. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, consist of 209 theoretical congeners depending on the numbers and positions of bromine atoms on the phenyl rings [1]. PBDEs have been widely used as additive flame retardants in plastics used in televisions, computers, and electronic equipment. During the lifetime of a product, however, PBDEs can migrate from product and become distributed throughout various environments, such as indoor air and dust [2]. In addition, some brominated flame retardants, including PBDEs, show persistence, bioaccumulation potential, and toxicity. Thus, the use of brominated flame retardants has been restricted or prohibited under various legislative acts. With regard to PBDEs, commercially available octabrominated diphenyl ether (octaBDE), which is mainly composed of hexa- and heptaBDE, and commercially available pentaBDE, which is mainly
∗ Corresponding author. E-mail address: [email protected] (T. Umemura). http://dx.doi.org/10.1016/j.mrgentox.2017.02.003 1383-5718/© 2017 Elsevier B.V. All rights reserved.
composed of tetra- and pentaBDE, are listed at the Stockholm Convention on persistent organic pollutants [3,4]. Although commercial PBDE products have been restricted worldwide, the increasing environment contamination of PBDEs resulting from informal recycling technology and activities for electronic waste has been reported [5]. Indeed, soils and plants at major destinations for e-waste disposal, such as China and Nigeria, are highly contaminated with PBDEs [6]. Decabromodiphenyl ether (decaBDE), a type of PBDE, has carcinogenic potential in the livers of rodents. In long-term bioassays, decaBDE has been reported to be a hepatocarcinogen in rats [7]. Moreover, in mice, the incidences of hepatocellular tumors in dosed male mice were marginally increased, although the difference was not statically significant [7]. Regarding its genotoxicity, one in vitro comet assay showed that decaBDE exerted genotoxic effects [8]; however, other in vitro studies, including Ames tests, mouse lymphoma TK+/− assays, sister-chromatid exchanges, or chromosomal aberration tests, were negative for such effects [8–10]. For in vivo tests, only one chromosomal aberration test has been performed in rat bone marrow, and the results were negative [8,9]. Thus, it
8
S. Takasu et al. / Mutation Research 816 (2017) 7–11
remains unclear whether decaBDE exerts genotoxic effects in mice or at its carcinogenic target site. Considering the growing concerns regarding environmental contamination and human exposure to PBDEs [11,12], it is important to clarify whether the genotoxic mechanisms of decaBDE include hepatocarcinogenesis in rodents. Comprehensive evaluation using in vivo genotoxicity data in rats and mice could be helpful to understand decaBDE-induced hepatocarcinogenesis. Thus, we examined the in vivo genotoxicity of decaBDE using micronucleus assays [13] in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice to explore the genotoxicity of decaBDE in mice and at the carcinogenic target site.
B6C3F1 gpt delta mice carrying 80 tandem copies of the transgene lambda EG10 per haploid genome were raised by mating C57BL/6 gpt delta and nontransgenic C3H/He mice (Japan SLC, Inc., Shizuoka, Japan). Twenty male B6C3F1 gpt delta mice were randomized by body weight into 4 groups. Mice were housed in polycarbonate cages with hardwood chips for bedding in a conventional animal facility, air-conditioned to 23 ± 2 ◦ C and 55% ± 5% humidity, on a 12-h light-dark cycle. The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences (Tokyo, Japan).
by in vitro packaging. For 6-TG selection, the packaged phages were incubated with Escherichia coli YG6020, which expressed Cre recombinase, and converted to a plasmid carrying gpt and chloramphenicol acetyltransferase. Infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol and 6-TG. To determine the total number of rescued plasmids, infected cells were also poured onto plates containing chloramphenicol without 6-TG. The plates were then incubated at 37 ◦ C. Positively selected colonies were counted on day 3 and collected on day 4. The gpt mutant frequencies (MFs) were calculated by dividing the number of gpt mutants by the number of rescued phages. gpt mutations were characterized by amplifying a 739-bp DNA fragment containing the 456-bp coding region of the gpt gene and sequencing the polymerase chain reaction (PCR) products with an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). For Spi- selection, packaged phages were incubated with E. coli XL1-Blue MRA for survival titration and E. coli XL1-Blue MRA P2 for mutant selection. Infected cells were mixed with molten lambdatrypticase soft agar and poured onto lambda-trypticase agar plates. The next day, plaques (Spi- candidates) were punched out with sterilized glass pipettes, and the agar plugs were suspended in SM buffer. The Spi- phenotype was confirmed by spotting the suspensions on three types of plates on which XL1-Blue MRA, XL1-Blue MRA P2, or WL95 P2 strains were spread with soft agar. Spi- mutants, which produced clear plaques on every plate, were counted.
2.2. Animal treatments
2.5. Statistical analysis
After 1 week of acclimatization, 6-week-old male B6C3F1 gpt delta mice were given decaDBE (CAS No. 1163-19-5; SigmaAldrich Co. LLC., St. Louis, MO, USA) at concentrations of 25000 or 50000 ppm in the basal diet or administered ethyl methanesulfonate (EMS) daily at a dose of 100 mg/kg body weight by gavage for 4 weeks for micronucleus and in vivo mutation assays. Dose levels of decaDBE were selected based on an NTP technical report of a 2-year carcinogenicity study [7]. That of EMS was determined as positive control for micronucleus assays in the bone marrow and in vivo mutation assays in the livers of B6C3F1 gpt delta mice [14]. At the end of the experiments, all animals were euthanized under deep anesthesia. The bone marrow was excised for micronucleus assays. Livers were collected, frozen immediately in liquid nitrogen, and stored at −80 ◦ C for in vivo mutation assays. A portion of each of the harvested livers was fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination.
The data for body and liver weights, proportion of MNPCE, ratios of polychromatic erythrocytes, gpt MFs, specific mutation frequencies of gpt mutants, and Spi- MFs in control and decaBDE-treated groups were analyzed by Dunnett’s tests. The significance of differences between control and EMS-treated groups was evaluated with Student’s t-tests.
2. Materials and methods 2.1. Animals
3. Results
Cells were collected from the bone marrow using fetal bovine serum and were smeared on slide glass. After drying, the cells were fixed in methanol, and the slides were stored at room temperature. The cells were stained with acridine orange solution and immediately observed by fluorescence microscopy. Micronucleated polychromatic erythrocytes (MNPCEs) were recorded based on the observation of 2000 polychromatic erythrocytes (PCEs), and MNPCE/PCE ratios were calculated. In addition, 200 total erythrocytes (PCEs plus normochromatic erythrocytes) were scored for PCE frequency.
Data for final body weights and absolute and relative liver weights are summarized in Table 1. No significant changes were observed in the final body weights of decaDBE-treated mice. Absolute and relative liver weights of mice treated with decaDBE were significantly increased compared with those of the control group. Histopathological examination revealed centrilobular hepatocellular hypertrophy in the livers of mice treated with decaBDE. The results of bone marrow micronucleus assays are shown in Fig. 1. There were no significant differences in the proportions of MNPCE in any decaBDE-treated or control mice, although that in the EMS-treated group was significantly increased compared with that in the control group. Data for gpt MFs and mutation spectra of gpt mutants in the livers of mice treated with decaBDE or EMS are summarized in Tables 2 and 3, respectively. Data for Spi- MFs are summarized in Table 4. There were no significant differences in gpt MFs among decaBDE-treated groups, although that in the EMS-treated group was significantly increased. No significant differences in specific mutation frequencies of gpt mutants and Spi- MFs were observed among groups.
2.4. In vivo mutation assays
4. Discussion
6-Thioguanine (6-TG) and Spi- selection were performed as previously reported [15]. Briefly, genomic DNA was extracted from the livers, and lambda EG10 DNA was rescued as lambda phages
In the present study, we examined the in vivo genotoxicity of decaBDE using gpt delta mice to clarify whether genotoxic mechanisms were involved in decaBDE-induced hepatocarcinogenesis in
2.3. Micronucleus assays in the bone marrow
S. Takasu et al. / Mutation Research 816 (2017) 7–11
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Table 1 Final body and liver weights in B6C3F1 gpt delta mice treated with decaBDE for 4 weeks. Group
No. of animals
Control 25000 ppm decaBDE 50000 ppm decaBDE
Final body weight (g)
27.3 ± 0.8a 27.6 ± 1.0 27.9 ± 0.8
5 5 5
Liver weight Absolute (g)
Relative (g/100 g BW)
1.21 ± 0.05 1.33 ± 0.06* 1.34 ± 0.05**
4.44 ± 0.15 4.81 ± 0.09** 4.80 ± 0.12 **
*, **Significantly different from the control group at p < 0.05 and 0.01, respectively. a Mean ± SD.
Fig. 1. Bone marrow micronucleus assay in B6C3F1 mice treated with decaBDE or EMS. (A) Proportion of micronucleated polychromatic erythrocytes (MNPCEs). (B) Ratios of polychromatic erythrocytes. NCE, normochromatic erythrocyte. Data represent the mean ± SD. *, **Significantly different from the control at p < 0.05 and 0.01, respectively. Table 2 gpt MFs in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Group
Animal No.
CmR colonies (x105 )
6-TGR and CmR colonies
Mutant frequency (x10-5 )
Mean ± SD
Control
1 2 3 4 5
8.8 10.6 7.3 20.9 11.1
5 6 9 7 9
0.57 0.57 1.23 0.33 0.81
0.70 ± 0.34
25000 ppm decaBDE
11 12 13 14 15
11.0 21.5 7.4 5.7 16.1
8 9 3 6 7
0.73 0.42 0.41 1.05 0.43
0.61 ± 0.28
50000 ppm decaBDE
21 22 23 24 25
24.1 21.9 15.3 22.6 17.7
19 11 12 10 3
0.79 0.50 0.78 0.44 0.17
0.54 ± 0.26
100 mg/kg EMS
31 32 33 34 35
20.2 9.8 14.6 14.4 14.8
23 14 17 16 12
1.14 1.43 1.16 1.11 0.81
1.13 ± 0.22*
*
Significantly different from the control at p < 0.05.
rodents. Therefore, the present study was carried out under conditions in which the doses of the chemicals and the background strain of gpt delta mice were the same as those in the NTP carcinogenesis study [7]. The present results showed that treatment with decaBDE increased liver weights. In association with these increases in liver weights, centrilobular hepatocellular hypertrophy was histopathologically observed in groups treated with decaDBE. DecaDBE has been previously reported to increase liver weights and cause hepatocyte hypertrophy due to Cyp2b induction in mice at a carcinogenic dose [16]. The present results were consistent with these previous findings.
Under these conditions, there were no significant differences in the proportions of MNPCE and gpt/Spi- MFs in the decaBDE-treated group, although those in the EMS-treated group were significantly increased. EMS is a well-known positive control compound for in vivo micronucleus assays in the bone marrow. Moreover, daily treatment with 100 mg/kg EMS for 28 days increases gpt MFs in the liver [14]. According to OECD guidelines, exposure for 4 weeks is thought to be sufficient for producing accumulation of mutations by weak mutagens and for providing adequate exposure for detecting mutations in organs with low proliferating activity [17]. Thus, the present results strongly indicated that decaBDE did not exert genotoxicity at the carcinogenic target site in addition to the negative
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Table 3 Mutation spectra of gpt mutants in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Control
25000 ppm decaBDE
50000 ppm decaBDE
100 mg/kg EMS
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
Transversion G:C-T:A G:C-C:G A:T-T:A A:T-C:G
6a (16.7) 0 (0.0) 0 (0.0) 0 (0.0)
0.12 ± 0.10b 0 0 0
9 (27.3) 3 (9.1) 1 (3.0) 0 (0.0)
0.14 ± 0.10 0.05 ± 0.08 0.01 ± 0.03 0
9 (16.4) 1 (1.8) 0 (0.0) 1 (1.8)
0.09 ± 0.07 0.01 ± 0.02 0 0.01 ± 0.02
16 (19.5) 0 (0.0) 4 (4.9) 2 (2.4)
0.23 ± 0.10 0 0.05 ± 0.09 0.03 ± 0.04
Transition G:C-A:T A:T-G:C
16 (44.4) 0 (0.0)
0.32 ± 0.21 0
12 (36.4) 3 (9.1)
0.21 ± 0.10 0.08 ± 0.15
23 (41.8) 2 (3.6)
0.23 ± 0.08 0.02 ± 0.04
31 (37.8) 3 (3.7)
0.44 ± 0.17 0.05 ± 0.09
Deletion Single bp Over 2 bp Insertion Complex Total
8 (22.2) 3 (8.3) 0 (0.0) 3 (8.3) 36
0.17 ± 0.14 0.05 ± 0.05 0 0.05 ± 0.08 0.70 ± 0.34
4 (12.1) 1 (3.0) 0 (0.0) 0 (0.0) 33
0.08 ± 0.07 0.03 ± 0.06 0 0 0.61 ± 0.28
8 (14.5) 5 (9.1) 3 (5.5) 3 (5.5) 55
0.09 ± 0.14 0.04 ± 0.04 0.03 ± 0.04 0.03 ± 0.02 0.54 ± 0.26
16 (19.5) 6 (7.3) 0 (0.0) 4 (4.9) 82
0.20 ± 0.14 0.07 ± 0.07 0 0.06 ± 0.04 1.13 ± 0.22
a b
Number of colonies with independent mutations. Mean ± SD.
Table 4 Spi- MFs in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Group
Animal No.
Plaques within XL-1 Blue MRA (x105 )
Plaques within WL95 (P2)
Mutant frequency (x10-5 )
Mean ± SD
Control
1 2 3 4 5
12.7 21.1 8.0 20.3 11.4
4 3 0 13 5
0.32 0.14 0.00a 0.64 0.44
0.38 ± 0.25
25000 ppm decaBDE
11 12 13 14 15
21.6 27.0 14.5 6.2 27.2
2 7 5 1 4
0.09 0.26 0.35 0.16 0.15
0.20 ± 0.11
50000 ppm decaBDE
21 22 23 24 25
9.1 16.0 25.7 20.5 20.1
5 3 4 3 2
0.55 0.19 0.16 0.15 0.10
0.23 ± 0.20
100 mg/kg EMS
31 32 33 34 35
35.8 14.3 14.3 19.7 16.5
3 5 5 8 5
0.08 0.35 0.35 0.41 0.30
0.30 ± 0.13
a
No mutant colonies were detected on the plate and this data was excluded from the calculation of mutant frequency.
results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Thus, based on our findings and previous studies, which almost all showed negative results for the genotoxicity of decaBDE, genotoxic mechanisms may not be involved in decaBDE-induced hepatocarcinogenesis in rodents. Cell proliferation is thought to play an important role in rodent hepatocarcinogenesis involving nongenotoxic mechanisms. Moreover, decaBDE does not increase hepatocyte proliferation after 1 week of treatment. However, decaBDE treatment for 27 weeks following diethylnitrosamine treatment increases the multiplicity of hepatocellular adenomas, suggesting that decaBDE has tumorpromoting potential [16]. Further studies are required to elucidate the nongenotoxic mechanisms involved in rodent hepatocarcinogenesis. In conclusion, although some genotoxic carcinogens require longer treatment duration to significantly increase MF [18], the present results showed that decaBDE did not exert genotoxic effects in mice when administered at carcinogenic doses under OECD guideline condition. Comprehensive evaluation using the previ-
ous rat data and the present results suggested that genotoxic mechanisms might not be involved in decaBDE-induced hepatocarcinogenesis. These findings contribute to the human risk assessment of environmental decaBDE contamination.
Acknowledgment This work was supported by a grant-in-aid from the Ministry of Health, Labour, and Welfare of Japan, Japan (H26-Kagaku-ippan005).
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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
In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether Shinji Takasu a , Yuji Ishii a , Yuh Yokoo a , Takuma Tsuchiya a , Aki Kijima a , Yukio Kodama b , Kumiko Ogawa a , Takashi Umemura a,∗ a b
Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Division of Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e
i n f o
Article history: Received 7 November 2016 Received in revised form 8 February 2017 Accepted 21 February 2017 Available online 22 February 2017 Keywords: in vivo genotoxicity Decabromodiphenyl ether gpt Delta mouse
a b s t r a c t Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, have been widely used as additive flame retardants. Recently, the use of brominated flame retardants has been restricted or prohibited under various legislative acts because of the persistence, bioaccumulation potential, and toxicity of these compounds. However, there are also additional concerns regarding environmental contamination and human exposure to PBDEs resulting from informal recycling technology. Decabromodiphenyl ether (decaBDE), one type of PBDE, has carcinogenic potential in the livers of rodents. Although one study has shown that decaBDE exerts genotoxic effects, the other in vitro and in vivo studies were negative for such effects. Thus, it remains unknown whether genotoxic mechanisms are involved in decaBDE-induced hepatocarcinogenesis in rodents. In this study, to explore the genotoxicity of decaBDE in mice, particularly in the context of carcinogenesis, we performed micronucleus assays in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice treated with decaBDE at carcinogenic doses for 28 days. Our results demonstrated negative results in micronucleus tests and reporter gene mutation assays. Thus, decaBDE did not exert genotoxic effects at carcinogenic target sites and did not show positive results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Overall, comprehensive evaluation using in vivo genotoxicity data in rats and our data indicated that nongenotoxic mechanisms may be responsible for decaBDE-induced hepatocarcinogenesis. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, consist of 209 theoretical congeners depending on the numbers and positions of bromine atoms on the phenyl rings [1]. PBDEs have been widely used as additive flame retardants in plastics used in televisions, computers, and electronic equipment. During the lifetime of a product, however, PBDEs can migrate from product and become distributed throughout various environments, such as indoor air and dust [2]. In addition, some brominated flame retardants, including PBDEs, show persistence, bioaccumulation potential, and toxicity. Thus, the use of brominated flame retardants has been restricted or prohibited under various legislative acts. With regard to PBDEs, commercially available octabrominated diphenyl ether (octaBDE), which is mainly composed of hexa- and heptaBDE, and commercially available pentaBDE, which is mainly
∗ Corresponding author. E-mail address: [email protected] (T. Umemura). http://dx.doi.org/10.1016/j.mrgentox.2017.02.003 1383-5718/© 2017 Elsevier B.V. All rights reserved.
composed of tetra- and pentaBDE, are listed at the Stockholm Convention on persistent organic pollutants [3,4]. Although commercial PBDE products have been restricted worldwide, the increasing environment contamination of PBDEs resulting from informal recycling technology and activities for electronic waste has been reported [5]. Indeed, soils and plants at major destinations for e-waste disposal, such as China and Nigeria, are highly contaminated with PBDEs [6]. Decabromodiphenyl ether (decaBDE), a type of PBDE, has carcinogenic potential in the livers of rodents. In long-term bioassays, decaBDE has been reported to be a hepatocarcinogen in rats [7]. Moreover, in mice, the incidences of hepatocellular tumors in dosed male mice were marginally increased, although the difference was not statically significant [7]. Regarding its genotoxicity, one in vitro comet assay showed that decaBDE exerted genotoxic effects [8]; however, other in vitro studies, including Ames tests, mouse lymphoma TK+/− assays, sister-chromatid exchanges, or chromosomal aberration tests, were negative for such effects [8–10]. For in vivo tests, only one chromosomal aberration test has been performed in rat bone marrow, and the results were negative [8,9]. Thus, it
8
S. Takasu et al. / Mutation Research 816 (2017) 7–11
remains unclear whether decaBDE exerts genotoxic effects in mice or at its carcinogenic target site. Considering the growing concerns regarding environmental contamination and human exposure to PBDEs [11,12], it is important to clarify whether the genotoxic mechanisms of decaBDE include hepatocarcinogenesis in rodents. Comprehensive evaluation using in vivo genotoxicity data in rats and mice could be helpful to understand decaBDE-induced hepatocarcinogenesis. Thus, we examined the in vivo genotoxicity of decaBDE using micronucleus assays [13] in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice to explore the genotoxicity of decaBDE in mice and at the carcinogenic target site.
B6C3F1 gpt delta mice carrying 80 tandem copies of the transgene lambda EG10 per haploid genome were raised by mating C57BL/6 gpt delta and nontransgenic C3H/He mice (Japan SLC, Inc., Shizuoka, Japan). Twenty male B6C3F1 gpt delta mice were randomized by body weight into 4 groups. Mice were housed in polycarbonate cages with hardwood chips for bedding in a conventional animal facility, air-conditioned to 23 ± 2 ◦ C and 55% ± 5% humidity, on a 12-h light-dark cycle. The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences (Tokyo, Japan).
by in vitro packaging. For 6-TG selection, the packaged phages were incubated with Escherichia coli YG6020, which expressed Cre recombinase, and converted to a plasmid carrying gpt and chloramphenicol acetyltransferase. Infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol and 6-TG. To determine the total number of rescued plasmids, infected cells were also poured onto plates containing chloramphenicol without 6-TG. The plates were then incubated at 37 ◦ C. Positively selected colonies were counted on day 3 and collected on day 4. The gpt mutant frequencies (MFs) were calculated by dividing the number of gpt mutants by the number of rescued phages. gpt mutations were characterized by amplifying a 739-bp DNA fragment containing the 456-bp coding region of the gpt gene and sequencing the polymerase chain reaction (PCR) products with an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). For Spi- selection, packaged phages were incubated with E. coli XL1-Blue MRA for survival titration and E. coli XL1-Blue MRA P2 for mutant selection. Infected cells were mixed with molten lambdatrypticase soft agar and poured onto lambda-trypticase agar plates. The next day, plaques (Spi- candidates) were punched out with sterilized glass pipettes, and the agar plugs were suspended in SM buffer. The Spi- phenotype was confirmed by spotting the suspensions on three types of plates on which XL1-Blue MRA, XL1-Blue MRA P2, or WL95 P2 strains were spread with soft agar. Spi- mutants, which produced clear plaques on every plate, were counted.
2.2. Animal treatments
2.5. Statistical analysis
After 1 week of acclimatization, 6-week-old male B6C3F1 gpt delta mice were given decaDBE (CAS No. 1163-19-5; SigmaAldrich Co. LLC., St. Louis, MO, USA) at concentrations of 25000 or 50000 ppm in the basal diet or administered ethyl methanesulfonate (EMS) daily at a dose of 100 mg/kg body weight by gavage for 4 weeks for micronucleus and in vivo mutation assays. Dose levels of decaDBE were selected based on an NTP technical report of a 2-year carcinogenicity study [7]. That of EMS was determined as positive control for micronucleus assays in the bone marrow and in vivo mutation assays in the livers of B6C3F1 gpt delta mice [14]. At the end of the experiments, all animals were euthanized under deep anesthesia. The bone marrow was excised for micronucleus assays. Livers were collected, frozen immediately in liquid nitrogen, and stored at −80 ◦ C for in vivo mutation assays. A portion of each of the harvested livers was fixed in 10% neutral-buffered formalin. Fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination.
The data for body and liver weights, proportion of MNPCE, ratios of polychromatic erythrocytes, gpt MFs, specific mutation frequencies of gpt mutants, and Spi- MFs in control and decaBDE-treated groups were analyzed by Dunnett’s tests. The significance of differences between control and EMS-treated groups was evaluated with Student’s t-tests.
2. Materials and methods 2.1. Animals
3. Results
Cells were collected from the bone marrow using fetal bovine serum and were smeared on slide glass. After drying, the cells were fixed in methanol, and the slides were stored at room temperature. The cells were stained with acridine orange solution and immediately observed by fluorescence microscopy. Micronucleated polychromatic erythrocytes (MNPCEs) were recorded based on the observation of 2000 polychromatic erythrocytes (PCEs), and MNPCE/PCE ratios were calculated. In addition, 200 total erythrocytes (PCEs plus normochromatic erythrocytes) were scored for PCE frequency.
Data for final body weights and absolute and relative liver weights are summarized in Table 1. No significant changes were observed in the final body weights of decaDBE-treated mice. Absolute and relative liver weights of mice treated with decaDBE were significantly increased compared with those of the control group. Histopathological examination revealed centrilobular hepatocellular hypertrophy in the livers of mice treated with decaBDE. The results of bone marrow micronucleus assays are shown in Fig. 1. There were no significant differences in the proportions of MNPCE in any decaBDE-treated or control mice, although that in the EMS-treated group was significantly increased compared with that in the control group. Data for gpt MFs and mutation spectra of gpt mutants in the livers of mice treated with decaBDE or EMS are summarized in Tables 2 and 3, respectively. Data for Spi- MFs are summarized in Table 4. There were no significant differences in gpt MFs among decaBDE-treated groups, although that in the EMS-treated group was significantly increased. No significant differences in specific mutation frequencies of gpt mutants and Spi- MFs were observed among groups.
2.4. In vivo mutation assays
4. Discussion
6-Thioguanine (6-TG) and Spi- selection were performed as previously reported [15]. Briefly, genomic DNA was extracted from the livers, and lambda EG10 DNA was rescued as lambda phages
In the present study, we examined the in vivo genotoxicity of decaBDE using gpt delta mice to clarify whether genotoxic mechanisms were involved in decaBDE-induced hepatocarcinogenesis in
2.3. Micronucleus assays in the bone marrow
S. Takasu et al. / Mutation Research 816 (2017) 7–11
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Table 1 Final body and liver weights in B6C3F1 gpt delta mice treated with decaBDE for 4 weeks. Group
No. of animals
Control 25000 ppm decaBDE 50000 ppm decaBDE
Final body weight (g)
27.3 ± 0.8a 27.6 ± 1.0 27.9 ± 0.8
5 5 5
Liver weight Absolute (g)
Relative (g/100 g BW)
1.21 ± 0.05 1.33 ± 0.06* 1.34 ± 0.05**
4.44 ± 0.15 4.81 ± 0.09** 4.80 ± 0.12 **
*, **Significantly different from the control group at p < 0.05 and 0.01, respectively. a Mean ± SD.
Fig. 1. Bone marrow micronucleus assay in B6C3F1 mice treated with decaBDE or EMS. (A) Proportion of micronucleated polychromatic erythrocytes (MNPCEs). (B) Ratios of polychromatic erythrocytes. NCE, normochromatic erythrocyte. Data represent the mean ± SD. *, **Significantly different from the control at p < 0.05 and 0.01, respectively. Table 2 gpt MFs in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Group
Animal No.
CmR colonies (x105 )
6-TGR and CmR colonies
Mutant frequency (x10-5 )
Mean ± SD
Control
1 2 3 4 5
8.8 10.6 7.3 20.9 11.1
5 6 9 7 9
0.57 0.57 1.23 0.33 0.81
0.70 ± 0.34
25000 ppm decaBDE
11 12 13 14 15
11.0 21.5 7.4 5.7 16.1
8 9 3 6 7
0.73 0.42 0.41 1.05 0.43
0.61 ± 0.28
50000 ppm decaBDE
21 22 23 24 25
24.1 21.9 15.3 22.6 17.7
19 11 12 10 3
0.79 0.50 0.78 0.44 0.17
0.54 ± 0.26
100 mg/kg EMS
31 32 33 34 35
20.2 9.8 14.6 14.4 14.8
23 14 17 16 12
1.14 1.43 1.16 1.11 0.81
1.13 ± 0.22*
*
Significantly different from the control at p < 0.05.
rodents. Therefore, the present study was carried out under conditions in which the doses of the chemicals and the background strain of gpt delta mice were the same as those in the NTP carcinogenesis study [7]. The present results showed that treatment with decaBDE increased liver weights. In association with these increases in liver weights, centrilobular hepatocellular hypertrophy was histopathologically observed in groups treated with decaDBE. DecaDBE has been previously reported to increase liver weights and cause hepatocyte hypertrophy due to Cyp2b induction in mice at a carcinogenic dose [16]. The present results were consistent with these previous findings.
Under these conditions, there were no significant differences in the proportions of MNPCE and gpt/Spi- MFs in the decaBDE-treated group, although those in the EMS-treated group were significantly increased. EMS is a well-known positive control compound for in vivo micronucleus assays in the bone marrow. Moreover, daily treatment with 100 mg/kg EMS for 28 days increases gpt MFs in the liver [14]. According to OECD guidelines, exposure for 4 weeks is thought to be sufficient for producing accumulation of mutations by weak mutagens and for providing adequate exposure for detecting mutations in organs with low proliferating activity [17]. Thus, the present results strongly indicated that decaBDE did not exert genotoxicity at the carcinogenic target site in addition to the negative
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S. Takasu et al. / Mutation Research 816 (2017) 7–11
Table 3 Mutation spectra of gpt mutants in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Control
25000 ppm decaBDE
50000 ppm decaBDE
100 mg/kg EMS
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
No. (%)
Specific mutation frequency (x10-5 )
Transversion G:C-T:A G:C-C:G A:T-T:A A:T-C:G
6a (16.7) 0 (0.0) 0 (0.0) 0 (0.0)
0.12 ± 0.10b 0 0 0
9 (27.3) 3 (9.1) 1 (3.0) 0 (0.0)
0.14 ± 0.10 0.05 ± 0.08 0.01 ± 0.03 0
9 (16.4) 1 (1.8) 0 (0.0) 1 (1.8)
0.09 ± 0.07 0.01 ± 0.02 0 0.01 ± 0.02
16 (19.5) 0 (0.0) 4 (4.9) 2 (2.4)
0.23 ± 0.10 0 0.05 ± 0.09 0.03 ± 0.04
Transition G:C-A:T A:T-G:C
16 (44.4) 0 (0.0)
0.32 ± 0.21 0
12 (36.4) 3 (9.1)
0.21 ± 0.10 0.08 ± 0.15
23 (41.8) 2 (3.6)
0.23 ± 0.08 0.02 ± 0.04
31 (37.8) 3 (3.7)
0.44 ± 0.17 0.05 ± 0.09
Deletion Single bp Over 2 bp Insertion Complex Total
8 (22.2) 3 (8.3) 0 (0.0) 3 (8.3) 36
0.17 ± 0.14 0.05 ± 0.05 0 0.05 ± 0.08 0.70 ± 0.34
4 (12.1) 1 (3.0) 0 (0.0) 0 (0.0) 33
0.08 ± 0.07 0.03 ± 0.06 0 0 0.61 ± 0.28
8 (14.5) 5 (9.1) 3 (5.5) 3 (5.5) 55
0.09 ± 0.14 0.04 ± 0.04 0.03 ± 0.04 0.03 ± 0.02 0.54 ± 0.26
16 (19.5) 6 (7.3) 0 (0.0) 4 (4.9) 82
0.20 ± 0.14 0.07 ± 0.07 0 0.06 ± 0.04 1.13 ± 0.22
a b
Number of colonies with independent mutations. Mean ± SD.
Table 4 Spi- MFs in the liver of B6C3F1 gpt delta mice treated with decaBDE or EMS. Group
Animal No.
Plaques within XL-1 Blue MRA (x105 )
Plaques within WL95 (P2)
Mutant frequency (x10-5 )
Mean ± SD
Control
1 2 3 4 5
12.7 21.1 8.0 20.3 11.4
4 3 0 13 5
0.32 0.14 0.00a 0.64 0.44
0.38 ± 0.25
25000 ppm decaBDE
11 12 13 14 15
21.6 27.0 14.5 6.2 27.2
2 7 5 1 4
0.09 0.26 0.35 0.16 0.15
0.20 ± 0.11
50000 ppm decaBDE
21 22 23 24 25
9.1 16.0 25.7 20.5 20.1
5 3 4 3 2
0.55 0.19 0.16 0.15 0.10
0.23 ± 0.20
100 mg/kg EMS
31 32 33 34 35
35.8 14.3 14.3 19.7 16.5
3 5 5 8 5
0.08 0.35 0.35 0.41 0.30
0.30 ± 0.13
a
No mutant colonies were detected on the plate and this data was excluded from the calculation of mutant frequency.
results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Thus, based on our findings and previous studies, which almost all showed negative results for the genotoxicity of decaBDE, genotoxic mechanisms may not be involved in decaBDE-induced hepatocarcinogenesis in rodents. Cell proliferation is thought to play an important role in rodent hepatocarcinogenesis involving nongenotoxic mechanisms. Moreover, decaBDE does not increase hepatocyte proliferation after 1 week of treatment. However, decaBDE treatment for 27 weeks following diethylnitrosamine treatment increases the multiplicity of hepatocellular adenomas, suggesting that decaBDE has tumorpromoting potential [16]. Further studies are required to elucidate the nongenotoxic mechanisms involved in rodent hepatocarcinogenesis. In conclusion, although some genotoxic carcinogens require longer treatment duration to significantly increase MF [18], the present results showed that decaBDE did not exert genotoxic effects in mice when administered at carcinogenic doses under OECD guideline condition. Comprehensive evaluation using the previ-
ous rat data and the present results suggested that genotoxic mechanisms might not be involved in decaBDE-induced hepatocarcinogenesis. These findings contribute to the human risk assessment of environmental decaBDE contamination.
Acknowledgment This work was supported by a grant-in-aid from the Ministry of Health, Labour, and Welfare of Japan, Japan (H26-Kagaku-ippan005).
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