- Email: [email protected]
Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O
Mutat Res Gen Tox En 846 (2019) 403072
Contents lists available at ScienceDirect
Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox
Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O Wen Tong1, Changhui Zhou1, Pengcheng Huang, Jing Ma, Yan Chang
T
⁎
Shanghai Innostar Bio-tech Co. Ltd./National Shanghai Center for New Drug Safety Evaluation and Research, China State Institute of Pharmaceutical Industry, Shanghai, China
ARTICLE INFO
ABSTRACT
Keywords: Micronucleus assay Comet assay Pig-a assay Auramine O
A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59−) or mutant reticulocyte frequencies (RETCD59−) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.
1. Introduction The micronucleus (MN) assay is an important and the most widely used assay system for the detection of chromosomal damage in vivo, mainly in hematopoietic cells [1]. 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 [2]. Recently, due to the high metabolic activity and chemical exposure of genotoxic material in the liver, the liver MN assay has been actively investigated. The Collaborative Study Group for the Micronucleus Test (CSGMT) of the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group (JEMS/MMS) has completed a collaborative study of the repeated-dose liver MN assay in 22 chemicals including 16 hepatocarcinogens. Some hepatocarcinogens have showed positive results in the liver MN assays, while the bone marrow (BM)/peripheral blood (PB) MN assay yielded negative results [3]. The collaborative study has proved the sensitivity and specificity of the liver MN assay, especially in predicting hepatocarcinogenicity and evaluating genotoxic substances that target the liver.
The ICH guideline S2(R1) recommends the integration of in vivo genotoxicity endpoints into routine general toxicity studies with the aim of reducing the number of experimental animals used [2]. Now, there are more discussions about how the liver MN assay should be the second in vivo experiment apart from bone marrow or peripheral blood as the target tissue. Single-cell gel electrophoresis - more frequently referred to as the Comet assay - can detect DNA damages caused at the single-cell level. Single- and double-strand DNA breaks, alkali-labile sites, and singlestrand breaks associated with incomplete repair can be detected through the comet assay. These types of DNA lesion are short-lived and may undergo rapid repair [4]. Therefore sampling for comet assays is recommended within the interval of 3-6 h of the final dose [5]. The phosphatidylinositol glycan-class A (Pig-a) gene mutation assay is a new approach to detect gene mutation. It can be the alternative for the transgenic rodent gene mutation assay when addressing bone marrow mutation. This assay has several advantages, e.g., the Pig-a gene is highly conserved, making this assay applicable to many other species of toxicological interest. Mutant cells can also be accumulated with repeat dosing, enabling the assay to be integrated into repeat-dose
Corresponding author. E-mail address: [email protected] (Y. Chang). 1 These authors contributed to the work equally. ⁎
https://doi.org/10.1016/j.mrgentox.2019.07.002 Received 7 November 2018; Received in revised form 30 June 2019; Accepted 2 July 2019 Available online 03 July 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
Mutat Res Gen Tox En 846 (2019) 403072
W. Tong, et al.
toxicology studies. In this study, we evaluated the genotoxicity of Auramine O (AO) with the liver MN assay, liver comet assay, peripheral blood MN assay, and erythrocyte Pig-a assay in one experiment. We used the 15-day treatment protocol that was used here as determined by Hamada et al. [3] as adequate for conducting the rat liver MN assay. AO is a liver carcinogen and in vitro genetox positive that was negative in the bloodbased MN assay. The integrated approach evaluated here will more comprehensibly test such liver-specific compounds for in vivo genetoxicity. With the multiple-endpoint genotoxicity assay, the evaluation of tested chemical can be more precise and reduce false positive rate.
Peripheral blood was collected from the jugular vein on Days -1 (the day before the first administration) for Pig-a mutation analysis. On Day 15, the sampling process was started 3 h after final administration. First, peripheral blood was collected from jugular vein for Pig-a mutation and MN-RET analysis. Then, two parts of the liver was cut from the left lobe of liver for %MNHEP and liver DNA-tail% analysis. 2.4. Sample preparation and data acquisition 2.4.1. Sampling for liver MN assay To prepare the cell suspension, a piece of the left lateral lobe was sliced into many pieces. These pieces were then rinsed with HBSS and treated with HBSS containing 0.05% of collagenase in a centrifuge tube shaken for 1 h at 37 °C. The flask was shaken vigorously 50 times every 30 min. The resulting material was filtered through a 100-μm cell strainer. The cell suspension was then added with 10% neutral buffered formalin and then centrifuged at 500 rpm for 5 min. and washed with 10% neutral buffered formalin. Centrifugation and the washing steps were repeated two times or more. The HEP suspension was fixed with 10% neutral buffered formalin. Immediately before microscope observation, the cell suspension was stained with SYGO-Gold. The mixtures were dropped onto clean glass slides and spread with coverslips. The slide specimens were observed at 100× magnification under a fluorescence microscope with a B excitation filter and emission filter for SYGO, and the numbers of micronucleated hepatocytes in 2000 hepatocytes per rat was recorded.
2. Materials and method 2.1. Animals Male Crl:CD(SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China) and used at 5–6 weeks of age. The animals were housed in an air-conditioned room with a 12-h light/dark cycle with free access to food and drinking water. Prior to beginning these studies, the experimental protocol was reviewed and approved by the Innostar Institutional Animal Care and Use Committee. 2.2. Reagents AO (CAS 2465-27-2, ＞97% purity; Lot No.: MKBX1556 V), ethylnitrosourea (ENU) (CAS. No. 759-73-9; Lot No.: MCKB9275), ethylmethane sulfonate (EMS) (CAS.NO.62-50-0; Lot No.: BCB55100 V), and cyclophosphamide (CP) (CAS.No. 50-18-0; Lot No.: LRAB3626), were purchased from Sigma-Aldrich (St. Louis, MO). AO was suspended in olive oil each day before dosing. For the liver MN liver MN assay, collagenase IV (Sigma-Aldrich, St. Louis, MO) was dissolved to 0.05% (w/v) in Hank’s balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA). SYGO-Gold was purchased from Invitrogen (Oregon, USA). Lympholyte Mammal cell separation solution was purchased from Cedar-Lane (Ontario, Canada). LS Columns, Anti-PE MicroBeads, and a Quadro-MACS™Separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Count Bright™ Absolute Count Beads were purchased from Invitrogen (Carlsbad, CA). MutaFlow® PLUS-25R kits (including Anticoagulant Solution, Buffered Salt Solution, Nucleic Acid Dye Solution SYTO13, Stock Anti-CD59phycoerythrin (PE), and Stock Anti-CD61-PE) were provided by Litron Laboratories (Rochester, NY). MicroFlow® rat blood micronucleus kits were purchased from Litron Laboratories. Fetal bovine serum was purchased from ExCell Biology (Shanghai, China).
2.4.2. Sampling for peripheral blood MN assay Analyses of the MN frequency were conducted on blood samples collected on Day 15. The blood was processed, labeled, and analyzed according to the instruction manual included in the Micro-Flow® Rat Blood Micronucleus Kit. Approximately 20,000 RET were scored for micronuclei per animal (where available). This method has been described in detail elsewhere [6,7]. A BD FACS Calibur flow cytometer running Cell Quest ™ Pro version 5.2.1 software was used for data acquisition and analysis. 2.4.3. Sampling for liver comet assay A 50- to 100-mg piece of liver was excised from the left lateral lobe within minutes of death of the animal (previous experiments in our lab with liver showed that delays greater than 10 min result in increased baseline % tail intensity) and was briefly rinsed in ice-cold mincing buffer (calcium- and magnesium-free Hank’s Balanced Salt Solution, HBSS [Gibco, Grand Island, NY], with 20 mM disodium EDTA (Sigma–Aldrich, St. Louis, MO, USA], pH 7.5) containing 10% dimethylsulfoxide (DMSO; Sigma–Aldrich). The tissue was then finely minced in a fresh cold buffer using small dissection scissors. Comet slides: 30 μl of suspension mixed with 300 μl of 0.05% molten agarose (approximately 37 °C). The cell/agarose mixture was spread on pre-coated microscope slides. Cover slips were added and the slides were then placed on a metal tray on ice for solidification for 310 min. The cover slips were then removed and the slides were immediately placed in an ice-cold lysing solution (100 mM disodium EDTA, 2.5 M sodium chloride, 10 mM Tris hydroxymethyl aminomethane, pH 10.0 with 1% Triton-X 100, by volume, and 10% DMSO, by volume, added on the day of use) and held at 4–10 °C overnight protected from light. Lysed slides were rinsed briefly in water before submerging in alkaline unwinding solution (300 mM sodium hydroxide, pH > 13) on an electrophoresis box platform connected to a circulating refrigerated water bath. Slides were distributed across the platform and electrophoresis ran in a balanced manner with respect to dose group and animal. After 20 min of unwinding, slides were electrophoresed for 20 min. at 14 V (approximately 0.7 V/cm, 300 mA). Temperature was maintained at 6–8 ℃ for unwinding and electrophoresis. Slides were then neutralized, dehydrated in ethanol, air-dried, and coded for scoring. SYBR Gold (Invitrogen, Grand Island, NY, USA)
2.3. Dose levels and treatments Based on the dose-finding study, body weight decrease, dark red eye discharge, and activity reduction was noted at 400 mg/kg/day. Thus, three dose levels of 100, 200, and 400 mg AO/kg/day (maximum tolerance dose (MTD)) were determined for the 15-day repeated dose study. Animals that received the vehicle and positive control served as the negative control group and the positive control group respectively. Five animals were used in each group. Based on the different genotoxicity assays previously established in our lab, rats were divided into 8 groups, including three groups of AO, negative control (saline, 0 mg/kg/day) group, DEN (12.5 mg/kg/day) as the positive control group for the liver MN assay, EMS (100 mg/kg/ day) as the positive control group for the comet assay, CP (10 mg/kg/ day) as the positive control group for the PBMN assay, and ENU (20 mg/kg/day) as the positive control group for the Pig-a assay. From Day 1, rats were orally administrated at approximately 24-hr interval for 15 consecutive days, except for the rats orally treated with ENU for the first three days and the rats treated with CP for the last four days. 2
Mutat Res Gen Tox En 846 (2019) 403072
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diluted 1:10000 in water was used to stain slides just prior to reading. Comet Assay IV Image Analysis Software (Perceptive Instruments, Haverhill, UK) together with a monochrome video camera and a fluorescence microscope (Olympus model BH-2) was used to score slides. 150 comets from different areas of each of two slides per tissue sample were scored. Comets that were misshapen, overlapping, or debris-covered were not scored. Comets exhibiting very high levels of DNA damage (“hedgehogs”, small or non-existent heads and large diffused tails) were not scored but the percentage of hedgehogs among the scorable comets were determined. 2.4.4. Sampling for Pig-a gene mutation assay Determinations of Pig-a mutant cell frequencies were performed on blood samples collected on Days -1 and 15. The sample preparation steps described in the MutaFlow® PLUS-25R kit manual were strictly followed and immunomagnetic depletion of wild-type erythrocytes before flow cytometric analysis as described previously [5]. In addition to reducing analysis duration to 4 min per sample, immunomagnetic separation made it practical to evaluate many times more cells than is otherwise feasible. As described previously, an Instrument Calibration Standard (ICS) was generated on each day data was acquired [8]. The ICS was used to optimize photo multiplier tube voltages and set fluorescence compensation. It was also used to rationally and consistently define the position of mutant phenotype cells. A BD FACS Calibur flow cytometer running Cell Quest TM Pro version 5.2.1 software was used for data acquisition and analysis.
Fig. 1. Mean %MNHEP induced by 0, 100, 200, and 400 mg/kg/day AO or 12.5 mg/kg/day DEN. Data presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at **P＜0.01.
2.5. Statistical analyses All data are expressed as the mean ± standard deviation (n = 5). For the liver MN assay, we counted the micronuclei in 2000 hepatocytes from each animal in each dose group. ANOVA test was used for comparisons of %MNHEP across different dose groups. When significant differences were observed in the ANOVA test (P＜0.05), the Dunnett’s two-tailed test was performed as a post hoc analysis (a Pvalue of ＜0.05 was considered significant). For PBMN and comet assay, after verifying normal distribution, the data obtained from the PBMN and comet assay were submitted to analysis of variance (ANOVA) and the Dunnett’s two-tailed test (a P-value of ＜0.05 was considered significant). For the Pig-a gene mutation assay, the effect of the treatment on each individual time point using the nonparametric Kruskal-Wallis test and pairwise comparisons based on the Wilcoxon rank sum test were evaluated and compared against each of the other treatment groups (a P-value of ＜0.05 was considered significant).
Fig. 2. Mean %RET and mean %MN-RET induced by 0, 100, 200, and 400 mg/ kg/day AO or 10 mg/kg/day CP. Data presented as mean ± SD, N = 5. Statistically significant differences from vehicle controls are indicated at **P < 0.01.
On the peripheral blood MN assay, no significant decrease in the % RET was observed for AO-treated rats on Day 15. In comparison with negative control group, no significant increase in the %MN-RET was observed in three test groups of AO (Fig. 2). 3.3. Liver comet assay The results of the liver comet assay can be found in Fig. 3. The administration of AO produced statistically significant increases in the group’s mean % tail DNA in the liver in the mid- and high-dose group, with a dose-dependent manner. There were no effects on the frequency of “hedgehog” comets.
3. Results 3.1. Liver MN assay The results of the liver MN assay are graphically summarized in Fig. 1. The mean MNHEP frequencies in the vehicle-treated control group ranged from 0.1% to 0.2%. The mean MNHEP frequencies in the positive control group ranged from 1.35% to 1.60%. The MNHEP frequencies increased significantly after repeated administration of AO at 200 and 400 mg/kg/day for 15 days. The frequency of MNHEP increase was directly proportional to the dose. There were no deaths during the scheduled treatment period at any dose level. 3.2. Peripheral blood MN assay After the establishment of the FCM-MN template, kit-supplied negative and positive standards were used as quality control samples. % RET and %MN-RET of negative standard were 2.89 and 0.08 respectively. The corresponding numbers of positive standards were 1.53 and 1.01. The results of the standards were within the required range in the kit.
Fig. 3. Mean DNA-Tail Intensity induced by 0, 100, 200 and 400 mg/kg/day AO or 100 mg/kg/day EMS. Date presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at *P＜0.05, P＜0.01. 3
Mutat Res Gen Tox En 846 (2019) 403072
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mouse peripheral blood [13]. Conversely, a significant increase in MNHEP frequencies was detected in the 15-day mid- and high-dose study groups. Although the requirement for metabolic activation regarding AO is unresolved, AO is considered to be a procarcinogen. It possesses methylated amino groups. It will undergo resonance conjugation rendering the amino N atom positively charged, thus more prone to hydroxylation. The conflicting results of the liver MN assay and PBMN assay of auramine may be due to weak electrophilicity at the unsubstituted positively charged central N atom in the other extreme resonance form [14]. Apart from that, because it is a procarcinogen, the main target organ of AO is the liver. It may affect the metabolic activation in the liver and not have enough exposure in peripheral blood. This is the first report on the liver MN induction by AO; there has been no report on liver MN assays with conventional methods using hepatectomy or juvenile rats. One of the disadvantages of the genotoxicity assay integrated into repeat-dose toxicity assays is that, because the possible dose levels for administration or the maximum tolerated doses are usually lower than those in short-term assays, there might be failure in detection of a genotoxic response. However, in this study, a positive response of liver MN and comet assay was detected at the doses of 200 and 400 mg/kg. Apart from the comet assay, the repeat-dose liver MN assay can be considered to be very suitable for integration into the repeat-dose toxicity assay. A new and simplified methodology for preparing MNHEP specimens will also make it feasible for the repeated dose live micronucleus (RDLMN) assay to be integrated, as this method requires only a small portion of the liver, does not require collagenase perfusion or hepatectomy, and does not hinder either the preparation of histopathological specimens or liver weight measurement [15]. For the erythrocyte Pig-a assay, there is no significant increase of mutant RBC or RET in any dose group, suggesting that AO has no mutagenic effects in the erythrocyte. The blood MN and Pig-a assays of AO showed little to no significant effect of treatment on percentage of reticulocytes, indicating minimal bone marrow exposure. The Ames assay of AO exhibited positive results [16], the negative results of the Pig-a assay may be because the target organ of AO does not include bone marrow or erythrocyte. For certain kinds of compounds, genotoxicity may not be detected by a bone marrow micronucleus assay because those substances do not reach the bone marrow because of instability. Also, there is a possibility that non-genotoxic substances are converted into genotoxic substances by intestinal bacteria. A good characteristic of the gastrointestinal micronucleus assay is its ability to evaluate micronucleus inducibility in the stomach (directly exposed to high concentrations of orally administered test substances) and large intestine (high incidence of cancer). Thus, addition of the gastrointestinal micronucleus assay of AO may elevate the value of the multiple-endpoint genotoxicity assay. In conclusion, the repeat-dose liver MN assay integrated into a multiple-endpoint genotoxicity assay is able to help detect MN induction by AO, a genotoxic hepatocarcinogen, which cannot be detected by a peripheral blood MN assay. The concurrent assay of AO also confirmed the target organ and the potential genotoxic mechanism. This new method of using a multiple-endpoint genotoxicity assay makes it possible to perform a comprehensive risk assessment of the carcinogenicity of chemicals.
Fig. 4. Three Pig-a assay endpoints are graphed for AO study: mean %RET (A), the mean RBCCD59- frequency (B), the mean RETCD59- frequency (C) induced by 0, 100, 200, and 400 mg/kg/day AO or 20 mg/kg/day ENU. Data presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at **P＜0.01.
3.4. Pig-a gene mutation assay The results of the erythrocyte Pig-a assay are graphed in Fig. 4. No significant decrease in the %RET was observed for AO-treated rats on Day 15 (Fig. 4A). The mean Pig-a mutant phenotype frequencies in RBCs and RETs of rats treated with 100, 200, and 400 mg/kg/day AO did not yield significant increases above the vehicle control at any time point tested, with all groups exhibiting less than 5 × 10−6 in both populations of erythrocytes. In comparison with AO treated groups, statistically significant increases in both RETCD59- and RBCCD59- frequencies were observed for concurrent positive control (ENU at 20 mg/ kg/day, see Fig. 4B and C).
Acknowledgment
4. Discussion
This work was funded by The Major Projects Foundation of the National Health Commission of the People’s Republic of China, Grant/ Award Number: 2018ZX09201017-008.
Treatment with Auramine reportedly causes carcinoma in the liver of rats [9]. The carcinogenicity is thought to be caused by its genotoxicity, which is supported by the positive results of the Ames test, in vitro micronucleus assay of primary rat and human hepatocytes, in vitro chromosome aberrations assay, and in vivo rat liver comet assay [10–12]. However, AO was reported to have no effect on %MN in
References [1] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, D.H. Blakey, S.D. Dertinger, L. Abramsson-Zetterberg, G. Krishna, T. Morita, A. Russo, N. Asano, H. Suzuki,
4
Mutat Res Gen Tox En 846 (2019) 403072
W. Tong, et al.
[2] [3]
[4] [5]
[6] [7]
W. Ohyama, D. Gibson, In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single doselevel limit test, Mutat. Res. 627 (2007) 10–30. ICH, Harmonized Tripartite Guideline, Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceutical Intended for Human Use S2 (2012) R1. 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. 2-17 (March) (2015) 780–781. A. Hartman, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A.R. Collins, A. Smith, G. Speit, V. Thybaud, R.R. Tice, Recommendations for conducting the in vivo alkaline Comet assay, Mutagenesis 18 (2003) 45–51. B. Burlinson, R.R. Tice, G. Speit, E. Agurell, S.Y. Brendler-Schwaab, A.R. Collins, P. Escobar, M. Honma, T.S. Kumaravel, M. Nakajima, Y.F. Sasaki, V. Thybaud, Y. Uno, M. Vasquez, A. Hartmann, Fourth international workshop on genotoxicity testing: results of the in vivo comet assay workgroup, Mutat. Res. 627 (2007) 31–35. D.K. Torous, N.E. Hall, F.G. Murante, S.E. Gleason, C.R. Tometsko, S.D. Dertinger, Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy, Toxicol. Sci. 74 (2003) 309–314. S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.T. Torous, S. Avlasevich, S. Cairns, C.R. Tometsko, C. Menard, T. Muanza, Y. Chen, R.K. Miller, K. Cederbrant, K. Sandelin, I. Ponten, G. Bolcsfoldi, Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood,
Environ. Mol. Mutagen. 4 (4) (2004) 427–435. [8] S.D. Dertinger, S.M. Bryce, S. Phonethepswath, S.L. Avlasevich, When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis, Mutat. Res. 721 (2011) 163–170. [9] Carcinogenic Potency Database (CPDB), U.S. National Library of Medicine, 2019. [10] A. Martelli, G.B. Campart, R. Canonero, F. Mattioli, L. Robbiano, M. Cavanna, Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat, Mutat. Res. 414 (1998) 37–47. [11] S.M. Galloway, M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson, E. Zeiger, Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals, Environ. Mol. Mutagen. 10 (1987) 1–175. [12] D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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. [13] T. Morita, N. Asano, T. Awogi, Y.F. Sasaki, S. Sato, H. Shimada, S. Sutou, T. Suzuki, A. Wakata, T. Sofuni, M. Hayashi, Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens _ Groups 1, 2A and 2B/ the summary report of the 6th collaborative study by CSGMT/JEMS.MMS, Mutat. Res. 389 (1997) 3–122. [14] R.D. Combes, R.B. Haveland-Smith, A review of the genotoxicity of food, drug and cosmetic colours and other azo, triphenylmethane and xanthene dyes, Mutat. Res. 98 (1982) 101–248. [15] K. Narumi, K. Ashizawa, R. Takashima, H. Takasawa, S. Katayama, Y. Tsuzuki, H. Tatemoto, T. Morita, M. Hayashi, S. Hamada, Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene, Mutat. Res. 747 (2012) 234–239. [16] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests. V. Results from the testing of 311 chemicals, Environ. Mol. Mutagen. 19 (Suppl. 21) (1992) 2-141.
5
Contents lists available at ScienceDirect
Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox
Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O Wen Tong1, Changhui Zhou1, Pengcheng Huang, Jing Ma, Yan Chang
T
⁎
Shanghai Innostar Bio-tech Co. Ltd./National Shanghai Center for New Drug Safety Evaluation and Research, China State Institute of Pharmaceutical Industry, Shanghai, China
ARTICLE INFO
ABSTRACT
Keywords: Micronucleus assay Comet assay Pig-a assay Auramine O
A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59−) or mutant reticulocyte frequencies (RETCD59−) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.
1. Introduction The micronucleus (MN) assay is an important and the most widely used assay system for the detection of chromosomal damage in vivo, mainly in hematopoietic cells [1]. 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 [2]. Recently, due to the high metabolic activity and chemical exposure of genotoxic material in the liver, the liver MN assay has been actively investigated. The Collaborative Study Group for the Micronucleus Test (CSGMT) of the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group (JEMS/MMS) has completed a collaborative study of the repeated-dose liver MN assay in 22 chemicals including 16 hepatocarcinogens. Some hepatocarcinogens have showed positive results in the liver MN assays, while the bone marrow (BM)/peripheral blood (PB) MN assay yielded negative results [3]. The collaborative study has proved the sensitivity and specificity of the liver MN assay, especially in predicting hepatocarcinogenicity and evaluating genotoxic substances that target the liver.
The ICH guideline S2(R1) recommends the integration of in vivo genotoxicity endpoints into routine general toxicity studies with the aim of reducing the number of experimental animals used [2]. Now, there are more discussions about how the liver MN assay should be the second in vivo experiment apart from bone marrow or peripheral blood as the target tissue. Single-cell gel electrophoresis - more frequently referred to as the Comet assay - can detect DNA damages caused at the single-cell level. Single- and double-strand DNA breaks, alkali-labile sites, and singlestrand breaks associated with incomplete repair can be detected through the comet assay. These types of DNA lesion are short-lived and may undergo rapid repair [4]. Therefore sampling for comet assays is recommended within the interval of 3-6 h of the final dose [5]. The phosphatidylinositol glycan-class A (Pig-a) gene mutation assay is a new approach to detect gene mutation. It can be the alternative for the transgenic rodent gene mutation assay when addressing bone marrow mutation. This assay has several advantages, e.g., the Pig-a gene is highly conserved, making this assay applicable to many other species of toxicological interest. Mutant cells can also be accumulated with repeat dosing, enabling the assay to be integrated into repeat-dose
Corresponding author. E-mail address: [email protected] (Y. Chang). 1 These authors contributed to the work equally. ⁎
https://doi.org/10.1016/j.mrgentox.2019.07.002 Received 7 November 2018; Received in revised form 30 June 2019; Accepted 2 July 2019 Available online 03 July 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
Mutat Res Gen Tox En 846 (2019) 403072
W. Tong, et al.
toxicology studies. In this study, we evaluated the genotoxicity of Auramine O (AO) with the liver MN assay, liver comet assay, peripheral blood MN assay, and erythrocyte Pig-a assay in one experiment. We used the 15-day treatment protocol that was used here as determined by Hamada et al. [3] as adequate for conducting the rat liver MN assay. AO is a liver carcinogen and in vitro genetox positive that was negative in the bloodbased MN assay. The integrated approach evaluated here will more comprehensibly test such liver-specific compounds for in vivo genetoxicity. With the multiple-endpoint genotoxicity assay, the evaluation of tested chemical can be more precise and reduce false positive rate.
Peripheral blood was collected from the jugular vein on Days -1 (the day before the first administration) for Pig-a mutation analysis. On Day 15, the sampling process was started 3 h after final administration. First, peripheral blood was collected from jugular vein for Pig-a mutation and MN-RET analysis. Then, two parts of the liver was cut from the left lobe of liver for %MNHEP and liver DNA-tail% analysis. 2.4. Sample preparation and data acquisition 2.4.1. Sampling for liver MN assay To prepare the cell suspension, a piece of the left lateral lobe was sliced into many pieces. These pieces were then rinsed with HBSS and treated with HBSS containing 0.05% of collagenase in a centrifuge tube shaken for 1 h at 37 °C. The flask was shaken vigorously 50 times every 30 min. The resulting material was filtered through a 100-μm cell strainer. The cell suspension was then added with 10% neutral buffered formalin and then centrifuged at 500 rpm for 5 min. and washed with 10% neutral buffered formalin. Centrifugation and the washing steps were repeated two times or more. The HEP suspension was fixed with 10% neutral buffered formalin. Immediately before microscope observation, the cell suspension was stained with SYGO-Gold. The mixtures were dropped onto clean glass slides and spread with coverslips. The slide specimens were observed at 100× magnification under a fluorescence microscope with a B excitation filter and emission filter for SYGO, and the numbers of micronucleated hepatocytes in 2000 hepatocytes per rat was recorded.
2. Materials and method 2.1. Animals Male Crl:CD(SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China) and used at 5–6 weeks of age. The animals were housed in an air-conditioned room with a 12-h light/dark cycle with free access to food and drinking water. Prior to beginning these studies, the experimental protocol was reviewed and approved by the Innostar Institutional Animal Care and Use Committee. 2.2. Reagents AO (CAS 2465-27-2, ＞97% purity; Lot No.: MKBX1556 V), ethylnitrosourea (ENU) (CAS. No. 759-73-9; Lot No.: MCKB9275), ethylmethane sulfonate (EMS) (CAS.NO.62-50-0; Lot No.: BCB55100 V), and cyclophosphamide (CP) (CAS.No. 50-18-0; Lot No.: LRAB3626), were purchased from Sigma-Aldrich (St. Louis, MO). AO was suspended in olive oil each day before dosing. For the liver MN liver MN assay, collagenase IV (Sigma-Aldrich, St. Louis, MO) was dissolved to 0.05% (w/v) in Hank’s balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA). SYGO-Gold was purchased from Invitrogen (Oregon, USA). Lympholyte Mammal cell separation solution was purchased from Cedar-Lane (Ontario, Canada). LS Columns, Anti-PE MicroBeads, and a Quadro-MACS™Separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Count Bright™ Absolute Count Beads were purchased from Invitrogen (Carlsbad, CA). MutaFlow® PLUS-25R kits (including Anticoagulant Solution, Buffered Salt Solution, Nucleic Acid Dye Solution SYTO13, Stock Anti-CD59phycoerythrin (PE), and Stock Anti-CD61-PE) were provided by Litron Laboratories (Rochester, NY). MicroFlow® rat blood micronucleus kits were purchased from Litron Laboratories. Fetal bovine serum was purchased from ExCell Biology (Shanghai, China).
2.4.2. Sampling for peripheral blood MN assay Analyses of the MN frequency were conducted on blood samples collected on Day 15. The blood was processed, labeled, and analyzed according to the instruction manual included in the Micro-Flow® Rat Blood Micronucleus Kit. Approximately 20,000 RET were scored for micronuclei per animal (where available). This method has been described in detail elsewhere [6,7]. A BD FACS Calibur flow cytometer running Cell Quest ™ Pro version 5.2.1 software was used for data acquisition and analysis. 2.4.3. Sampling for liver comet assay A 50- to 100-mg piece of liver was excised from the left lateral lobe within minutes of death of the animal (previous experiments in our lab with liver showed that delays greater than 10 min result in increased baseline % tail intensity) and was briefly rinsed in ice-cold mincing buffer (calcium- and magnesium-free Hank’s Balanced Salt Solution, HBSS [Gibco, Grand Island, NY], with 20 mM disodium EDTA (Sigma–Aldrich, St. Louis, MO, USA], pH 7.5) containing 10% dimethylsulfoxide (DMSO; Sigma–Aldrich). The tissue was then finely minced in a fresh cold buffer using small dissection scissors. Comet slides: 30 μl of suspension mixed with 300 μl of 0.05% molten agarose (approximately 37 °C). The cell/agarose mixture was spread on pre-coated microscope slides. Cover slips were added and the slides were then placed on a metal tray on ice for solidification for 310 min. The cover slips were then removed and the slides were immediately placed in an ice-cold lysing solution (100 mM disodium EDTA, 2.5 M sodium chloride, 10 mM Tris hydroxymethyl aminomethane, pH 10.0 with 1% Triton-X 100, by volume, and 10% DMSO, by volume, added on the day of use) and held at 4–10 °C overnight protected from light. Lysed slides were rinsed briefly in water before submerging in alkaline unwinding solution (300 mM sodium hydroxide, pH > 13) on an electrophoresis box platform connected to a circulating refrigerated water bath. Slides were distributed across the platform and electrophoresis ran in a balanced manner with respect to dose group and animal. After 20 min of unwinding, slides were electrophoresed for 20 min. at 14 V (approximately 0.7 V/cm, 300 mA). Temperature was maintained at 6–8 ℃ for unwinding and electrophoresis. Slides were then neutralized, dehydrated in ethanol, air-dried, and coded for scoring. SYBR Gold (Invitrogen, Grand Island, NY, USA)
2.3. Dose levels and treatments Based on the dose-finding study, body weight decrease, dark red eye discharge, and activity reduction was noted at 400 mg/kg/day. Thus, three dose levels of 100, 200, and 400 mg AO/kg/day (maximum tolerance dose (MTD)) were determined for the 15-day repeated dose study. Animals that received the vehicle and positive control served as the negative control group and the positive control group respectively. Five animals were used in each group. Based on the different genotoxicity assays previously established in our lab, rats were divided into 8 groups, including three groups of AO, negative control (saline, 0 mg/kg/day) group, DEN (12.5 mg/kg/day) as the positive control group for the liver MN assay, EMS (100 mg/kg/ day) as the positive control group for the comet assay, CP (10 mg/kg/ day) as the positive control group for the PBMN assay, and ENU (20 mg/kg/day) as the positive control group for the Pig-a assay. From Day 1, rats were orally administrated at approximately 24-hr interval for 15 consecutive days, except for the rats orally treated with ENU for the first three days and the rats treated with CP for the last four days. 2
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diluted 1:10000 in water was used to stain slides just prior to reading. Comet Assay IV Image Analysis Software (Perceptive Instruments, Haverhill, UK) together with a monochrome video camera and a fluorescence microscope (Olympus model BH-2) was used to score slides. 150 comets from different areas of each of two slides per tissue sample were scored. Comets that were misshapen, overlapping, or debris-covered were not scored. Comets exhibiting very high levels of DNA damage (“hedgehogs”, small or non-existent heads and large diffused tails) were not scored but the percentage of hedgehogs among the scorable comets were determined. 2.4.4. Sampling for Pig-a gene mutation assay Determinations of Pig-a mutant cell frequencies were performed on blood samples collected on Days -1 and 15. The sample preparation steps described in the MutaFlow® PLUS-25R kit manual were strictly followed and immunomagnetic depletion of wild-type erythrocytes before flow cytometric analysis as described previously [5]. In addition to reducing analysis duration to 4 min per sample, immunomagnetic separation made it practical to evaluate many times more cells than is otherwise feasible. As described previously, an Instrument Calibration Standard (ICS) was generated on each day data was acquired [8]. The ICS was used to optimize photo multiplier tube voltages and set fluorescence compensation. It was also used to rationally and consistently define the position of mutant phenotype cells. A BD FACS Calibur flow cytometer running Cell Quest TM Pro version 5.2.1 software was used for data acquisition and analysis.
Fig. 1. Mean %MNHEP induced by 0, 100, 200, and 400 mg/kg/day AO or 12.5 mg/kg/day DEN. Data presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at **P＜0.01.
2.5. Statistical analyses All data are expressed as the mean ± standard deviation (n = 5). For the liver MN assay, we counted the micronuclei in 2000 hepatocytes from each animal in each dose group. ANOVA test was used for comparisons of %MNHEP across different dose groups. When significant differences were observed in the ANOVA test (P＜0.05), the Dunnett’s two-tailed test was performed as a post hoc analysis (a Pvalue of ＜0.05 was considered significant). For PBMN and comet assay, after verifying normal distribution, the data obtained from the PBMN and comet assay were submitted to analysis of variance (ANOVA) and the Dunnett’s two-tailed test (a P-value of ＜0.05 was considered significant). For the Pig-a gene mutation assay, the effect of the treatment on each individual time point using the nonparametric Kruskal-Wallis test and pairwise comparisons based on the Wilcoxon rank sum test were evaluated and compared against each of the other treatment groups (a P-value of ＜0.05 was considered significant).
Fig. 2. Mean %RET and mean %MN-RET induced by 0, 100, 200, and 400 mg/ kg/day AO or 10 mg/kg/day CP. Data presented as mean ± SD, N = 5. Statistically significant differences from vehicle controls are indicated at **P < 0.01.
On the peripheral blood MN assay, no significant decrease in the % RET was observed for AO-treated rats on Day 15. In comparison with negative control group, no significant increase in the %MN-RET was observed in three test groups of AO (Fig. 2). 3.3. Liver comet assay The results of the liver comet assay can be found in Fig. 3. The administration of AO produced statistically significant increases in the group’s mean % tail DNA in the liver in the mid- and high-dose group, with a dose-dependent manner. There were no effects on the frequency of “hedgehog” comets.
3. Results 3.1. Liver MN assay The results of the liver MN assay are graphically summarized in Fig. 1. The mean MNHEP frequencies in the vehicle-treated control group ranged from 0.1% to 0.2%. The mean MNHEP frequencies in the positive control group ranged from 1.35% to 1.60%. The MNHEP frequencies increased significantly after repeated administration of AO at 200 and 400 mg/kg/day for 15 days. The frequency of MNHEP increase was directly proportional to the dose. There were no deaths during the scheduled treatment period at any dose level. 3.2. Peripheral blood MN assay After the establishment of the FCM-MN template, kit-supplied negative and positive standards were used as quality control samples. % RET and %MN-RET of negative standard were 2.89 and 0.08 respectively. The corresponding numbers of positive standards were 1.53 and 1.01. The results of the standards were within the required range in the kit.
Fig. 3. Mean DNA-Tail Intensity induced by 0, 100, 200 and 400 mg/kg/day AO or 100 mg/kg/day EMS. Date presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at *P＜0.05, P＜0.01. 3
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mouse peripheral blood [13]. Conversely, a significant increase in MNHEP frequencies was detected in the 15-day mid- and high-dose study groups. Although the requirement for metabolic activation regarding AO is unresolved, AO is considered to be a procarcinogen. It possesses methylated amino groups. It will undergo resonance conjugation rendering the amino N atom positively charged, thus more prone to hydroxylation. The conflicting results of the liver MN assay and PBMN assay of auramine may be due to weak electrophilicity at the unsubstituted positively charged central N atom in the other extreme resonance form [14]. Apart from that, because it is a procarcinogen, the main target organ of AO is the liver. It may affect the metabolic activation in the liver and not have enough exposure in peripheral blood. This is the first report on the liver MN induction by AO; there has been no report on liver MN assays with conventional methods using hepatectomy or juvenile rats. One of the disadvantages of the genotoxicity assay integrated into repeat-dose toxicity assays is that, because the possible dose levels for administration or the maximum tolerated doses are usually lower than those in short-term assays, there might be failure in detection of a genotoxic response. However, in this study, a positive response of liver MN and comet assay was detected at the doses of 200 and 400 mg/kg. Apart from the comet assay, the repeat-dose liver MN assay can be considered to be very suitable for integration into the repeat-dose toxicity assay. A new and simplified methodology for preparing MNHEP specimens will also make it feasible for the repeated dose live micronucleus (RDLMN) assay to be integrated, as this method requires only a small portion of the liver, does not require collagenase perfusion or hepatectomy, and does not hinder either the preparation of histopathological specimens or liver weight measurement [15]. For the erythrocyte Pig-a assay, there is no significant increase of mutant RBC or RET in any dose group, suggesting that AO has no mutagenic effects in the erythrocyte. The blood MN and Pig-a assays of AO showed little to no significant effect of treatment on percentage of reticulocytes, indicating minimal bone marrow exposure. The Ames assay of AO exhibited positive results [16], the negative results of the Pig-a assay may be because the target organ of AO does not include bone marrow or erythrocyte. For certain kinds of compounds, genotoxicity may not be detected by a bone marrow micronucleus assay because those substances do not reach the bone marrow because of instability. Also, there is a possibility that non-genotoxic substances are converted into genotoxic substances by intestinal bacteria. A good characteristic of the gastrointestinal micronucleus assay is its ability to evaluate micronucleus inducibility in the stomach (directly exposed to high concentrations of orally administered test substances) and large intestine (high incidence of cancer). Thus, addition of the gastrointestinal micronucleus assay of AO may elevate the value of the multiple-endpoint genotoxicity assay. In conclusion, the repeat-dose liver MN assay integrated into a multiple-endpoint genotoxicity assay is able to help detect MN induction by AO, a genotoxic hepatocarcinogen, which cannot be detected by a peripheral blood MN assay. The concurrent assay of AO also confirmed the target organ and the potential genotoxic mechanism. This new method of using a multiple-endpoint genotoxicity assay makes it possible to perform a comprehensive risk assessment of the carcinogenicity of chemicals.
Fig. 4. Three Pig-a assay endpoints are graphed for AO study: mean %RET (A), the mean RBCCD59- frequency (B), the mean RETCD59- frequency (C) induced by 0, 100, 200, and 400 mg/kg/day AO or 20 mg/kg/day ENU. Data presented as mean ± SD, n = 5. Statistically significant differences from vehicle controls are indicated at **P＜0.01.
3.4. Pig-a gene mutation assay The results of the erythrocyte Pig-a assay are graphed in Fig. 4. No significant decrease in the %RET was observed for AO-treated rats on Day 15 (Fig. 4A). The mean Pig-a mutant phenotype frequencies in RBCs and RETs of rats treated with 100, 200, and 400 mg/kg/day AO did not yield significant increases above the vehicle control at any time point tested, with all groups exhibiting less than 5 × 10−6 in both populations of erythrocytes. In comparison with AO treated groups, statistically significant increases in both RETCD59- and RBCCD59- frequencies were observed for concurrent positive control (ENU at 20 mg/ kg/day, see Fig. 4B and C).
Acknowledgment
4. Discussion
This work was funded by The Major Projects Foundation of the National Health Commission of the People’s Republic of China, Grant/ Award Number: 2018ZX09201017-008.
Treatment with Auramine reportedly causes carcinoma in the liver of rats [9]. The carcinogenicity is thought to be caused by its genotoxicity, which is supported by the positive results of the Ames test, in vitro micronucleus assay of primary rat and human hepatocytes, in vitro chromosome aberrations assay, and in vivo rat liver comet assay [10–12]. However, AO was reported to have no effect on %MN in
References [1] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, D.H. Blakey, S.D. Dertinger, L. Abramsson-Zetterberg, G. Krishna, T. Morita, A. Russo, N. Asano, H. Suzuki,
4
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W. Tong, et al.
[2] [3]
[4] [5]
[6] [7]
W. Ohyama, D. Gibson, In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single doselevel limit test, Mutat. Res. 627 (2007) 10–30. ICH, Harmonized Tripartite Guideline, Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceutical Intended for Human Use S2 (2012) R1. 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. 2-17 (March) (2015) 780–781. A. Hartman, E. Agurell, C. Beevers, S. Brendler-Schwaab, B. Burlinson, P. Clay, A.R. Collins, A. Smith, G. Speit, V. Thybaud, R.R. Tice, Recommendations for conducting the in vivo alkaline Comet assay, Mutagenesis 18 (2003) 45–51. B. Burlinson, R.R. Tice, G. Speit, E. Agurell, S.Y. Brendler-Schwaab, A.R. Collins, P. Escobar, M. Honma, T.S. Kumaravel, M. Nakajima, Y.F. Sasaki, V. Thybaud, Y. Uno, M. Vasquez, A. Hartmann, Fourth international workshop on genotoxicity testing: results of the in vivo comet assay workgroup, Mutat. Res. 627 (2007) 31–35. D.K. Torous, N.E. Hall, F.G. Murante, S.E. Gleason, C.R. Tometsko, S.D. Dertinger, Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy, Toxicol. Sci. 74 (2003) 309–314. S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.T. Torous, S. Avlasevich, S. Cairns, C.R. Tometsko, C. Menard, T. Muanza, Y. Chen, R.K. Miller, K. Cederbrant, K. Sandelin, I. Ponten, G. Bolcsfoldi, Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood,
Environ. Mol. Mutagen. 4 (4) (2004) 427–435. [8] S.D. Dertinger, S.M. Bryce, S. Phonethepswath, S.L. Avlasevich, When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis, Mutat. Res. 721 (2011) 163–170. [9] Carcinogenic Potency Database (CPDB), U.S. National Library of Medicine, 2019. [10] A. Martelli, G.B. Campart, R. Canonero, F. Mattioli, L. Robbiano, M. Cavanna, Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat, Mutat. Res. 414 (1998) 37–47. [11] S.M. Galloway, M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson, E. Zeiger, Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals, Environ. Mol. Mutagen. 10 (1987) 1–175. [12] D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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. [13] T. Morita, N. Asano, T. Awogi, Y.F. Sasaki, S. Sato, H. Shimada, S. Sutou, T. Suzuki, A. Wakata, T. Sofuni, M. Hayashi, Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens _ Groups 1, 2A and 2B/ the summary report of the 6th collaborative study by CSGMT/JEMS.MMS, Mutat. Res. 389 (1997) 3–122. [14] R.D. Combes, R.B. Haveland-Smith, A review of the genotoxicity of food, drug and cosmetic colours and other azo, triphenylmethane and xanthene dyes, Mutat. Res. 98 (1982) 101–248. [15] K. Narumi, K. Ashizawa, R. Takashima, H. Takasawa, S. Katayama, Y. Tsuzuki, H. Tatemoto, T. Morita, M. Hayashi, S. Hamada, Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene, Mutat. Res. 747 (2012) 234–239. [16] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests. V. Results from the testing of 311 chemicals, Environ. Mol. Mutagen. 19 (Suppl. 21) (1992) 2-141.
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