Environmental and Molecular Mutagenesis 55:735^740 (2014)
Research Article Assessment of 5-Fluorouracil and 4-Nitroquinoline-1oxide In Vivo Genotoxicity With Pig-a Mutation and Micronucleus Endpoints Changhui Zhou,1 Min Zhang,1 Pengcheng Huang,1 Honggang Tu,1 Zheng Wang,1 Stephen D. Dertinger,2 Dorothea K. Torous,2 and Yan Chang1* 1
National Shanghai Center for New Drug Safety Evaluation and Research, China State Institute of Pharmaceutical Industry, Shanghai, China 2 Litron Laboratories, Rochester, New York
Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5fluorouracil (5-FU) and 4-nitroquinoline-1–oxide (4NQO) as part of an international validation trial of the Pig-a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5-FU for three consecutive days (Days 1–3); blood was sampled on Days 21, 4, 15, 29, and 45. Pig-a mutant phenotype reticulocyte (RETCD592) and mutant phenotype erythrocyte (RBCCD592) frequencies were determined on Days 21, 15, 29, and 45, and percent micronucleated reticulocytes (%MN-RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day.
RBCCD592 and RETCD592 frequencies were determined on Days 21, 15, and 29, and MN-RET were quantified on Day 29. Whereas 5-FU was found to increase %MN-RET, no significant increases were observed for RBCCD592 or RETCD592 at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBCCD592 and RETCD592 frequencies, and this same dose level caused a weak but significantly elevated increase in MN-RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig-a mutation and MN-RET into acute and 28-day repeat-dose studies. Environ. Mol. Mutagen. 55:735–740, C 2014 Wiley Periodicals, Inc. 2014. V
Key words: Pig-a; mutation; flow cytometry; genotoxicity; 5-fluorouracil; 4-nitroquinoline-1-oxide
INTRODUCTION In vivo genotoxicity studies are an important part of the safety assessment of chemical agents and are conducted as part of a standard battery or as a follow-up to positive in vitro findings [Cimino, 2006]. The International Conference on Harmonization S2(R1) guideline recommends the standard in vivo tests to be the bone marrow or peripheral blood micronucleus assay and comet assay, which detect chromosomal damage and primary DNA damage, respectively. Clearly missing is an assessment of gene mutation. Phenotype-based methods for measuring the incidence of in vivo gene mutation at the phosphatidylinositol glycan-class A (Pig-a) locus have been described in recent years [Bryce et al., 2008; Miura et al., 2008a,b; Phonethepswath et al., 2008; Dobrovolsky et al., 2010; Dertinger et al., 2011c]. These assays have several advantageous characteristics that have been detailed in other articles [Miura et al., 2009; Peruzzi et al., 2010; Dertinger C 2014 Wiley Periodicals, Inc. V
et al., 2011a; Schuler et al., 2011]. For example, the Piga gene is highly conserved [Araten et al., 1999], a feature that facilitates studies across species of toxicological interest. Also, mutant cells have been found to accumulate with repeat dosing, a situation that makes the assay attractive for integration into repeat-dose toxicology
Grant sponsor: “The 12th Five-Year Program for Essential Drug Discovery and Development from Ministry of Science and Technology; Grant number: 2012ZX09505001-003. *Correspondence to: Yan Chang, National Shanghai Center for New Drug Safety Evaluation and Research, 199 Guoshoujing Rd., Zhangjiang Hi-tech Park, Pudong, Shanghai 201203, China. E-mail: [email protected] Received 15 May 2014; provisionally accepted 22 July 2014; and in final form 00 Month 2014 DOI 10.1002/em.21893 Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
Environmental and Molecular Mutagenesis. DOI 10.1002/em 736
Zhou et al.
studies. These and other attributes make this endpoint a promising tool for regulatory safety assessments [Dobrovolsky et al., 2010]. A multilaboratory international collaborative trial has been organized to investigate the portability and reproducibility of the Pig-a assay, the sensitivity and specificity of the endpoint, and the incorporation of the Pig-a mutation assay into repeatdose studies. Rat MutaFlowV kits were developed by scientists at Litron Laboratories and provide standardized methods to investigate the merits and limitations of in vivo Pig-a phenotypic mutation assays. Many laboratories across the world are participating in the Pig-a assay validation, and Stage II studies were conducted to investigate interlaboratory transferability of the prototype assay kit using the model compound N-ethyl-N-nitrosourea (ENU), a potent alkylating agent and gene mutagen [Dertinger et al., 2011b]. During Stage III trials, each laboratory chose a prototypical mutagen that had previously been studied at the reference lab (Litron Laboratories). Qualified labs were then supplied with kit-formatted reagents/instructions for experimentation with additional mutagens as well as presumed nonmutagens for the so-called Stage IV studies. Unlike the original Stage III reports, Stage IV work benefits from a more advanced scoring technique that uses immunomagnetic enrichment of mutant phenotype cells before flow cytometric analysis [Dertinger et al., 2011c]. Here, we report our experiences with two Stage IV chemicals. 5-fluorouracil (5-FU) is a thymidylate synthase inhibitor that interferes with DNA synthesis. While it is recognized as an in vivo clastogen, the IWGT Pig-a Expert Workgroup predicted it would be negative for in vivo mutation [IWGT manuscript, submitted for publication]. The second agent is 4-nitroquinoline-1-oxide (4NQO), a genotoxicant that has been one of the weakest inducers of Pig-a mutant phenotype cells evaluated to date [Dertinger et al., 2010; Stankowski et al., 2011]. R
MATERIALS AND METHODS Reagents
Laboratory Animal Company (Shanghai, China). Rats were allowed to acclimate for 1 week. Water and food were available ad libitum throughout the acclimation and experimental periods. A 12 hr light/dark cycle was used and room temperature was maintained between 20 C and 26 C. ENU was freshly prepared each day in PBS (pH 6.0). 5-FU was freshly prepared each day in dH2O. 4-NQO was freshly suspended each day in 0.5% (w/v) methyl cellulose in deionized water. All treatments were via oral gavage in a volume of 10 mL/kg body weight. All animals were weighed daily before dosing. For the 5-FU study, rats were administered 0, 11.5, 23, or 46 mg 5FU/kg/day, or 20 mg ENU/kg/day at 24 hr intervals on days 1, 2, and 3 (n 5 5 per group). Peripheral blood was collected from jugular vein on Days 21 (the day before the first administration), 15, 29, and 45 for Pig-a mutation analysis, and on Day 4 for MN-RET analysis. For the 4NQO study, rats were administered 0, 1.5, 3, or 6 mg/kg/day at 24 hr intervals for 28 consecutive days (n 5 5 per group). Peripheral blood was collected from jugular vein on days 21, 15, and 29 for Pig-a phenotypic mutation and for MN-RET on Day 29. Approximately 200–300 lL of free-flowing blood per rat were collected directly into heparinized capillary tubes. For the Pig-a analysis, 80 lL of each blood sample were transferred to tubes containing 100 lL of kit-supplied heparin solution. For the MN analysis, 100 lL of each blood sample were transferred to tubes containing 350 lL of kit-supplied heparin solution.
Sample Preparation and Data Acquisition Samples for Pig-a Mutation Analysis Determinations of Pig-a mutant cell frequencies were performed on blood samples collected on Days 21, 15, 29, and 45 of the 3-day study, and on Days 21, 15, and 29 of the 28-day study. The sample preparation steps described in the MutaFlowV PLUS-25R kit manual were strictly followed, and used immunomagnetic depletion of wild-type erythrocytes before flow cytometric analysis as has been described previously [Dertinger et al., 2012]. In addition to reducing analysis times to 4 min per sample, immunomagnetic separation made it practical to evaluate many times more cells than is otherwise feasible (e.g., >150 3 106 and >3 3 106 total erythocytes and reticulocytes per sample, respectively). As described previously, an Instrument Calibration Standard (ICS) was generated on each day data acquisition occurred [Dertinger et al., 2011c]. 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 FACSCalibur flow cytometer running CellQuestTM Pro version 5.2.1 software was used for data acquisition and analysis. R
Samples for MN Analysis
ENU (CAS. No. 759-73-9), 5-FU (CAS. No. 51-21-8), and 4-NQO (CAS. No. 56–57-5) were purchased from Sigma-Aldrich (St. Louis, MO). LympholyteV-Mammal cell separation solution was purchased from CedarLane (Ontario, Canada). LS Columns, Anti-PE MicroBeads, and a QuadroMACSTM Separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). CountBrightTM Absolute Count Beads were purchased from Invitrogen (Carlsbad, CA). MutaFlowV PLUS-25R kits (including Anticoagulant Solution, Buffered Salt Solution, Nucleic Acid Dye Solution SYTO13, Stock Anti-CD59-phycoerythrin (PE), and Stock Anti-CD61-PE) were provided by Litron Laboratories (Rochester, NY). MicroFlowV rat blood micronucleus kits were purchased from Litron Laboratories. Fetal bovine serum was purchased from ExCell Biology (Shanghai, China). R
R
Analyses of MN frequency were conducted on blood samples collected on Day 4 (5-FU) or Day 29 (4NQO). Blood was processed, labeled, and analyzed according to the instruction manual received with the MicroFlowV Rat Blood Micronucleus Kit. Approximately 20,000 RET were scored for micronuclei per animal, where available. This method has been described in detail elsewhere [Torous et al., 2003; Dertinger et al., 2004]. A BD FACSCalibur flow cytometer running CellQuestTM Pro version 5.2.1 software was used for data acquisition and analysis. R
R
Animals, Treatment, and Harvests Experiments were conducted with the approval and oversight of the NCDSER Institutional Animal Care and Use Committee. Male SpragueDawley rats, approximately 6–8 week old, were purchased from Slac
Calculations, Statistical Analyses The formulas used to calculate mutant phenotype erythrocyte (RBCCD592) and mutant phenotype reticulocyte (RETCD592) frequencies have been described previously [Dertinger et al., 2012]. The incidence of RETs and MN-RET is expressed as frequency percent, whereas the incidence of mutant phenotype cells is expressed as number per 106. All %RET, mutant phenotype cell frequencies, MN frequencies, averages,
Environmental and Molecular Mutagenesis. DOI 10.1002/em Assessment of 5-FU and 4NQO In Vivo Genotoxicity TABLE I. Body Weight Observations in the 5-Fluorouracil Test ( x 6 s, n 5 5) Dose (mg/kg/day)
0
11.5
23
46
Body weight change 18.2 6 3.9 11.6 6 4.2 21.1 6 8.0a 224.4 6 9.9a (g, days 1-4) a
Significant difference (P < 0.05).
737
point (RBCCD592 and RETCD592) was considered at each individual time point using the nonparametric Kruskal-Wallis test, and pairwise comparisons based on the Wilcoxon rank sum test were performed to evaluate vehicle against each of the other treatment groups. All statistical tests used P < 0.05 to indicate significance. %MN-RET values were transformed using an arcsin transformation, to stabilize group variances (Shapiro-Wilk W-test). Unlike %MN-RET data, all %RET data sets were normally distributed in treatment groups and did not require transformation. Individual %MN-RET and %RET values for animals in each treatment group were evaluated using a oneway analysis of variance (ANOVA) model. If the overall “F” test from the ANOVA model was significant (P < 0.05), comparisons between the drug-treated group means and the concurrent vehicle group mean were made in the context of the ANOVA model, using the Dunnett t-test. Significance was noted for each comparison where P < 0.05.
RESULTS Erythrocyte Pig-a Assay and Micronucleus Assay With 5-FU
Fig. 1. Three Pig-a assay endpoints are graphed for the 3-day 5-fluorouracil (5-FU) study: mean %RET (A), the mean RBCCD592 frequency (B), the mean RETCD592 frequency (C) induced by 0, 11.5, 23, and 46 mg/kg/day 5-FU or 20 mg/kg/day ENU. Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and standard deviation calculations were performed with Excel for Windows. Statistical analyses were based on individual animal data and performed with SAS v9.1 Software. The effect of treatment on each end-
The mean body weight change of groups treated with 5-FU decreased relative to the vehicle control animals, with the magnitude of this change occurring in a doserelated manner on Day 4. Mean body weights treated at 23 mg/kg/day on Day 4 were less than their day 1 body weights, with those treated at 46 mg/kg/day demonstrating even greater body weight loss (Table I). No significant decrease in the %RET was observed for 5-FU treated rats on Days 15, 29, or 45 (Fig. 1A). The mean Pig-a mutant phenotype frequencies in RBCs and RETs of rats treated with 11.5, 23, and 46 mg 5-FU/kg/ day did not yield significant increases above vehicle control at any time points tested, with all groups exhibiting less than 5 3 1026 in both populations of erythrocytes. In contrast, statistically significant increases in both RETCD592 and RBCCD592 frequencies were observed for the concurrent positive control (ENU at 20 mg/kg/day; see Figs. 1B and 1C). Agent-related reductions in %RET were apparent at this early time point, providing an important evidence for bone marrow exposure that was not apparent at Day 15 of the Pig-a analysis. Animals treated with 46 mg/kg/day did not provide sufficient cells for analysis, so percent micronucleated reticulocytes (%MN-RET) data from this dose were not included in the analysis. Statistically significant increases in %MN-RET were observed at doses 11.5 mg 5-FU /kg/day as well as for the concurrent positive control group (Fig. 2). Erythrocyte Pig-a Assay and Micronucleus Assay With 4NQO Weight gain was monitored over the 28-day treatment period. Treatment-related reductions in mean weight gain were evident at 6 mg 4NQO/kg/day from Days 1–8, Days 1–15, and Days 1–29. For example, from Days 1–15, mean weight gain of the rats treated at 3 mg/kg/day
Environmental and Molecular Mutagenesis. DOI 10.1002/em 738
Zhou et al.
Fig. 2. Mean %RET and mean %MN-RET induced by 0, 11.5, 23, and 46 mg/kg/day 5-FU or 20 mg/kg/day ENU. Data presented as mean 6 SD, n 5 5 (high dose group, n 5 2). Statistically significant differences from vehicle controls are indicated at **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was below the vehicle control animals, and this effect was more pronounced for the 6 mg/kg/day group (Table II). RETCD592 and RBCCD592 frequencies were low in all rats at Day 21, as well as in the vehicle control rats at all sampling times, with the vast majority being less than 5 3 1026 in both populations of erythrocytes. Agerelated decreases in %RET were observed in all treatment groups. In addition to the age-related effect, treatment with 6 mg 4NQO/kg/day was observed to reduce mean %RET values compared with vehicle controls on Day 15 (Fig. 3A). Statistically significant increases in RBCCD592 frequencies were noted for the high dose group only on Day 29 (Fig. 3B). On the other hand, the 6 mg/kg/day dose group exhibited elevated RETCD592 frequencies that were significantly higher than those observed in vehicletreated rats on Days 15 and 29, with maximal observed values occurring on day 29 (Fig. 3C). There were no significant changes to %RET compared with the vehicle control at any 4NQO dose tested monitored on Day 29 (Fig. 4). A statistically significant increase in the frequency of %MN-RET, approximately two-fold over the vehicle control mean, was observed only at the high dose group.
DISCUSSION The aim of this study was to contribute to an international validation effort that has been evaluating the in vivo rat Pig-a mutant phenotype assay. Experiments were performed with 5-FU-treated SD rats using a 3-day study design, and 4NQO was studied in the same rat model using the other common treatment schedule, a 28-day repeat dosing design. In addition, the well-established in vivo micronucleus endpoint was integrated into the Pig-a assay. In this study, 5-FU was evaluated at the maximum tolerated dose, as demonstrated by significant decreases in body weight, body weigh gain (Table I), and evident
RET toxicity (Fig. 2) at a dose of 46 mg/kg/day. 5-FU was observed to induce MN-RET in a dose-dependent manner on Day 4. This result is consistent with a report by Zhou et al. [2013]. On the other hand, treatment of rats for 3 days with 5-FU had no statistically significant effect on RETCD592 (Fig. 1C) or RBCCD592 frequencies (Fig. 1B), even at the highly toxic dose level of 46 mg/ kg/day. It would be useful to follow this work up with a 5-FU study that consists of a 28-day exposure period. Regardless, the current data set provides no indication of an in vivo mutagenic potential, despite the fact that the bone marrow was clearly affected. The magnitude and kinetics of the RETCD592 and RBCCD592 mutant phenotype responses in the 4NQO 28day subchronic study were remarkably similar to previous findings which were generated using the intial “Stage III design,” that is, flow cytometry without immunomagnetic separation [Dertinger et al., 2010]. The results were also qualitatively those reported by Stankowski et al. [2011], although the lowest effective dose level was somewhat lower in their case (2.5 mg/kg/day). While the high dose of 4NQO did statistically significantly increase the frequency of MN-RET, the induction was rather weak and inconsistent. The weak positive response for MN-RET on Day 29 in this study is in contrast to the previous negative findings reported for 4NQO on Days 4 and 29 by Dertinger et al. [2010] who studied a top dose level of 5 mg/kg/day as opposed to our 6 mg/kg/day. Our results are closer to the observations of Stankowski et al. [2011], who characterized the MN-RET induction using a 28-day study design as equivocal. Thus, the relatively weak indication of clastogenic activity reported herein is not surprising. Significant in-house and inter-laboratory testing has made it clear that spontaneous Pig-a mutant phenotype RET and RBC values are typically quite low, probably on the order of about 1 3 1026 [Dertinger et al., 2010; Phonethepswath et al., 2010]. Our data support this assessment, and indicate that the immunomagnetic separation method used herein has the advantages of achieving higher rates of data acquisition in order to make it practical to interrogate many times more RBCs and RETs for Pig-a mutant response than is otherwise possible. Overall, our results confirm the interlaboratory reproducibility of the Pig-a assay endpoint previously observed in short-term studies with ENU (the positive control) in the Stage II phase [Dertinger et al., 2011b], and in repeatdose toxicity studies with 4NQO in the Stage III phase [Stankowski et al., 2011]. This report also contributes data regarding a presumed nonmutagen, 5-FU, an agent that was applied at a very toxic dose level. Moreover, the current study supports the premise that Pig-a and micronucleus endpoints can be readily integrated into acute and repeat-dose studies, thereby providing coverage of both gene mutation and chromosomal damage in the same
Environmental and Molecular Mutagenesis. DOI 10.1002/em Assessment of 5-FU and 4NQO In Vivo Genotoxicity
739
TABLE II. Body Weight Observations in the 4NQO Test ( x 6 s, n 5 5) Dose (mg/kg/day) Body weight change (g)
a
Days 1-8 Days 1-15 Days 1-29
0
1.5
3
6
53.9 6 8.1 84.6 6 13.0 127.7 6 24.9
64.7 6 8.5 100.8 6 13.8 162.6 6 22.3
57.6 6 10.2 34.5 6 10.7a 146.1 6 32.7
14.2 6 22.0a 5.8 6 11.9a 60.1 6 34.8a
Significant difference (P < 0.05).
Fig. 4. Mean %RET and mean %MN-RET induced by 0, 1.5, 3, or 6 mg/kg/day 4NQO Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
AUTHOR CONTRIBUTIONS Changhui Zhou, Min Zhang, Stephen D. Dertinger, Dorothea K. Torous, and Yan Chang designed the study. Changhui Zhou, Min Zhang, Pengcheng Huang, Honggang Tu, and Zheng Wang collected the data. Changhui Zhou and Min Zhang analyzed the data and prepared draft figures. Changhui Zhou prepared the manuscript draft with important intellectual input from Stephen D. Dertinger and Yan Chang. All authors approved the final manuscript. Changhui Zhou and Yan Chang had completed access to the study data. REFERENCES
Fig. 3. Three Pig-a assay endpoints are graphed for the 28-day 4-nitroquinoline-1-oxide (4NQO) study: mean %RET (A), the mean RBCCD592 frequency (B), and the mean RETCD592 frequency (C) induced by 0, 1.5, 3, or 6 mg/kg/day. Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at *P < 0.05 and **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
experiment. This has the potential to significantly reduce the number of dedicated in vivo genotoxicity studies required [Kirkland et al., 2011], while at the same time improving the extent and value of available genotoxicity information.
Araten DJ, Nafa K, Pakdeesuwan K, Luzzatto L. 1999. Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc Natl Acad Sci USA 96:5209–5214. Bryce SM, Bemis JC, Dertinger SD. 2008. In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen 49:256– 264. Cimino MC. 2006. Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes. Environ Mol Mutagen 47:362–390. Dertinger SD, Camphausen K, MacGregor JT, Bishop ME, Torous DT, Avlasevich S, Cairns S, Tometsko CR, Menard C, Muanza T, Chen Y, Miller RK, Cederbrant K, Sandelin K, Ponten I, Bolcsfoldi G. 2004. Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood. Environ Mol Mutagen 44:427–435.
Environmental and Molecular Mutagenesis. DOI 10.1002/em 740
Zhou et al.
Dertinger SD, Phonethepswath S, Franklin D, Weller P, Torous DK, Bryce SM, Avlasevich S, Bemis JC, Hyrien O, Palis J, MacGregor JT. 2010. Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies. Toxicol Sci 115:401–411. Dertinger SD, Phonethepswath S, Weller P, Avlasevich S, Torous DK, Mereness JA, Bryce SM, Bemis JC, Bell S, Portugal S, Aylott M, MacGregor JT. 2011a. Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes. Environ Mol Mutagen 52: 748–755. Dertinger SD, Phonethepswath S, Weller P, Nicolette J, Murray J, Sonders P, Vohr H-W, Shi J, Krsmanovic L, Gleason C, Custer L, Henwood A, Sweder K, Stankowski LF Jr, Roberts DJ, Giddings A, Kenny J, Lynch AM, Defrain C, Nesslany F, van der Leede B-JM, Van Doninck T, Schuermans A, Tanaka K, Hiwata Y, Tajima O, Wilde E, Elajouji A, Gunther WC, Thiffeault CJ, Shutsky TJ, Fiedler RD, Kimoto T, Bhalli JA, Heflich RH, Mac-Gregor JT. 2011b. International Pig-a gene mutation assay trial: Evaluation of transferability across 14 laboratories. Environ Mol Mutagen 52:690–698. Dertinger SD, Bryce SM, Phonethepswath S, Avlasevich SL. 2011c. When pigs fly: Immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis. Mutat Res 721:163–170. Dertinger SD, Phonethepswath S, Avlasevich SL, Torous DK, Mereness J, Bryce SM, Bemis JC, Bell S, Weller P, MacGregor JT. 2012. Efficient monitoring of in vivo Pig-a gene mutation and chromosomal damage: Summary of 7 published studies and results from 11 new reference compounds. Toxicol Sci 130:328–348. Dobrovolsky VN, Miura D, Heflich RH, Dertinger SD. 2010. The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ Mol Mutagen 51:825–835. Kirkland DJ, Hayashi M, Jacobson-Kram D, Kasper P, Gollapudi B, M€uller L, Uno Y. 2011. Summary of major conclusions from the 5th IWGT, Basel, Switzerland, 17–19 August 2009. Mutat Res 723:73–76. Miura D, Dobrovolsky VN, Y Kasahara, Heflich RH. 2008a. Development of an in vivo gene mutation assay using the endogenous Pig-a gene: I. Flow cytometric detection of CD59-negative
peripheral red blood cells and CD48-negative spleen T cells from the rat. Environ Mol Mutagen 49:614–621. Miura D, Dobrovolsky VN, Mittelstaedt RA, Kasahara Y, Heflich RH. 2008b. Development of an in vivo gene mutation assay using the endogenous Pig-a gene: II. Selection of Pig-a mutant rat spleen T-cells with proaerolysin and sequencing Pig-a cDNA from the mutants. Environ Mol Mutagen 49:622–630. Miura D, Dobrovolsky VN, Kimoto T, Kasahara Y, Heflich RH. 2009. Accumulation and persistence of Pig-a mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea. Mutat Res 677:86–92. Peruzzi B, Araten DJ, Notaro R, Luzzatto L. 2010. The use of Pig-a as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo. Mutat Res 705:3–10. Phonethepswath S, Bryce SM, Bemis JC, Dertinger SD. 2008. Erythrocyte-based Pig-a gene mutation assay: Demonstration of crossspecies potential. Mutat Res 657:122–126. Phonethepswath S, Franklin D, Torous DK, Bryce SM, Bemis JC, Raja S, Avlasevich S, Weller P, Hyrien O, Palis J, MacGregor JT, Dertinger SD. 2010. Pig-a mutation: Kinetics in rat erythrocytes following exposure to five prototypical mutagens. Toxicol Sci 114:59–70. Schuler M, Gollapudi BB, Thybaud V, Kim JH. 2011. Need and potential value of the Pig-a in vivo mutation assay, a HESI perspective. Environ Mol Mutagen 52:685–689. Stankowski LF Jr, Roberts DJ, Chen H, Lawlor T, McKeon M, Murli H, Thakur A, Xu Y. 2011. Integration of Pig-a, micronucleus, chromosome aberration, and comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide. Environ Mol Mutagen 52:738–747. Torous DK, Hall NE, Murante FG, Gleason SE, Tometsko CR, Dertinger SD. 2003. Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol Sci 74:309–314. Zhou C, Wang Q, Wang Z, Chang Y. 2013. Comparison of three-colour flow cytometry and slide-based microscopy for the scoring of micronucleated reticulocytes in rat bone-marrow and peripheral blood. Mutat Res 758:12–17.
Accepted by— M. Honma
Research Article Assessment of 5-Fluorouracil and 4-Nitroquinoline-1oxide In Vivo Genotoxicity With Pig-a Mutation and Micronucleus Endpoints Changhui Zhou,1 Min Zhang,1 Pengcheng Huang,1 Honggang Tu,1 Zheng Wang,1 Stephen D. Dertinger,2 Dorothea K. Torous,2 and Yan Chang1* 1
National Shanghai Center for New Drug Safety Evaluation and Research, China State Institute of Pharmaceutical Industry, Shanghai, China 2 Litron Laboratories, Rochester, New York
Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5fluorouracil (5-FU) and 4-nitroquinoline-1–oxide (4NQO) as part of an international validation trial of the Pig-a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5-FU for three consecutive days (Days 1–3); blood was sampled on Days 21, 4, 15, 29, and 45. Pig-a mutant phenotype reticulocyte (RETCD592) and mutant phenotype erythrocyte (RBCCD592) frequencies were determined on Days 21, 15, 29, and 45, and percent micronucleated reticulocytes (%MN-RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day.
RBCCD592 and RETCD592 frequencies were determined on Days 21, 15, and 29, and MN-RET were quantified on Day 29. Whereas 5-FU was found to increase %MN-RET, no significant increases were observed for RBCCD592 or RETCD592 at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBCCD592 and RETCD592 frequencies, and this same dose level caused a weak but significantly elevated increase in MN-RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig-a mutation and MN-RET into acute and 28-day repeat-dose studies. Environ. Mol. Mutagen. 55:735–740, C 2014 Wiley Periodicals, Inc. 2014. V
Key words: Pig-a; mutation; flow cytometry; genotoxicity; 5-fluorouracil; 4-nitroquinoline-1-oxide
INTRODUCTION In vivo genotoxicity studies are an important part of the safety assessment of chemical agents and are conducted as part of a standard battery or as a follow-up to positive in vitro findings [Cimino, 2006]. The International Conference on Harmonization S2(R1) guideline recommends the standard in vivo tests to be the bone marrow or peripheral blood micronucleus assay and comet assay, which detect chromosomal damage and primary DNA damage, respectively. Clearly missing is an assessment of gene mutation. Phenotype-based methods for measuring the incidence of in vivo gene mutation at the phosphatidylinositol glycan-class A (Pig-a) locus have been described in recent years [Bryce et al., 2008; Miura et al., 2008a,b; Phonethepswath et al., 2008; Dobrovolsky et al., 2010; Dertinger et al., 2011c]. These assays have several advantageous characteristics that have been detailed in other articles [Miura et al., 2009; Peruzzi et al., 2010; Dertinger C 2014 Wiley Periodicals, Inc. V
et al., 2011a; Schuler et al., 2011]. For example, the Piga gene is highly conserved [Araten et al., 1999], a feature that facilitates studies across species of toxicological interest. Also, mutant cells have been found to accumulate with repeat dosing, a situation that makes the assay attractive for integration into repeat-dose toxicology
Grant sponsor: “The 12th Five-Year Program for Essential Drug Discovery and Development from Ministry of Science and Technology; Grant number: 2012ZX09505001-003. *Correspondence to: Yan Chang, National Shanghai Center for New Drug Safety Evaluation and Research, 199 Guoshoujing Rd., Zhangjiang Hi-tech Park, Pudong, Shanghai 201203, China. E-mail: [email protected] Received 15 May 2014; provisionally accepted 22 July 2014; and in final form 00 Month 2014 DOI 10.1002/em.21893 Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
Environmental and Molecular Mutagenesis. DOI 10.1002/em 736
Zhou et al.
studies. These and other attributes make this endpoint a promising tool for regulatory safety assessments [Dobrovolsky et al., 2010]. A multilaboratory international collaborative trial has been organized to investigate the portability and reproducibility of the Pig-a assay, the sensitivity and specificity of the endpoint, and the incorporation of the Pig-a mutation assay into repeatdose studies. Rat MutaFlowV kits were developed by scientists at Litron Laboratories and provide standardized methods to investigate the merits and limitations of in vivo Pig-a phenotypic mutation assays. Many laboratories across the world are participating in the Pig-a assay validation, and Stage II studies were conducted to investigate interlaboratory transferability of the prototype assay kit using the model compound N-ethyl-N-nitrosourea (ENU), a potent alkylating agent and gene mutagen [Dertinger et al., 2011b]. During Stage III trials, each laboratory chose a prototypical mutagen that had previously been studied at the reference lab (Litron Laboratories). Qualified labs were then supplied with kit-formatted reagents/instructions for experimentation with additional mutagens as well as presumed nonmutagens for the so-called Stage IV studies. Unlike the original Stage III reports, Stage IV work benefits from a more advanced scoring technique that uses immunomagnetic enrichment of mutant phenotype cells before flow cytometric analysis [Dertinger et al., 2011c]. Here, we report our experiences with two Stage IV chemicals. 5-fluorouracil (5-FU) is a thymidylate synthase inhibitor that interferes with DNA synthesis. While it is recognized as an in vivo clastogen, the IWGT Pig-a Expert Workgroup predicted it would be negative for in vivo mutation [IWGT manuscript, submitted for publication]. The second agent is 4-nitroquinoline-1-oxide (4NQO), a genotoxicant that has been one of the weakest inducers of Pig-a mutant phenotype cells evaluated to date [Dertinger et al., 2010; Stankowski et al., 2011]. R
MATERIALS AND METHODS Reagents
Laboratory Animal Company (Shanghai, China). Rats were allowed to acclimate for 1 week. Water and food were available ad libitum throughout the acclimation and experimental periods. A 12 hr light/dark cycle was used and room temperature was maintained between 20 C and 26 C. ENU was freshly prepared each day in PBS (pH 6.0). 5-FU was freshly prepared each day in dH2O. 4-NQO was freshly suspended each day in 0.5% (w/v) methyl cellulose in deionized water. All treatments were via oral gavage in a volume of 10 mL/kg body weight. All animals were weighed daily before dosing. For the 5-FU study, rats were administered 0, 11.5, 23, or 46 mg 5FU/kg/day, or 20 mg ENU/kg/day at 24 hr intervals on days 1, 2, and 3 (n 5 5 per group). Peripheral blood was collected from jugular vein on Days 21 (the day before the first administration), 15, 29, and 45 for Pig-a mutation analysis, and on Day 4 for MN-RET analysis. For the 4NQO study, rats were administered 0, 1.5, 3, or 6 mg/kg/day at 24 hr intervals for 28 consecutive days (n 5 5 per group). Peripheral blood was collected from jugular vein on days 21, 15, and 29 for Pig-a phenotypic mutation and for MN-RET on Day 29. Approximately 200–300 lL of free-flowing blood per rat were collected directly into heparinized capillary tubes. For the Pig-a analysis, 80 lL of each blood sample were transferred to tubes containing 100 lL of kit-supplied heparin solution. For the MN analysis, 100 lL of each blood sample were transferred to tubes containing 350 lL of kit-supplied heparin solution.
Sample Preparation and Data Acquisition Samples for Pig-a Mutation Analysis Determinations of Pig-a mutant cell frequencies were performed on blood samples collected on Days 21, 15, 29, and 45 of the 3-day study, and on Days 21, 15, and 29 of the 28-day study. The sample preparation steps described in the MutaFlowV PLUS-25R kit manual were strictly followed, and used immunomagnetic depletion of wild-type erythrocytes before flow cytometric analysis as has been described previously [Dertinger et al., 2012]. In addition to reducing analysis times to 4 min per sample, immunomagnetic separation made it practical to evaluate many times more cells than is otherwise feasible (e.g., >150 3 106 and >3 3 106 total erythocytes and reticulocytes per sample, respectively). As described previously, an Instrument Calibration Standard (ICS) was generated on each day data acquisition occurred [Dertinger et al., 2011c]. 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 FACSCalibur flow cytometer running CellQuestTM Pro version 5.2.1 software was used for data acquisition and analysis. R
Samples for MN Analysis
ENU (CAS. No. 759-73-9), 5-FU (CAS. No. 51-21-8), and 4-NQO (CAS. No. 56–57-5) were purchased from Sigma-Aldrich (St. Louis, MO). LympholyteV-Mammal cell separation solution was purchased from CedarLane (Ontario, Canada). LS Columns, Anti-PE MicroBeads, and a QuadroMACSTM Separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). CountBrightTM Absolute Count Beads were purchased from Invitrogen (Carlsbad, CA). MutaFlowV PLUS-25R kits (including Anticoagulant Solution, Buffered Salt Solution, Nucleic Acid Dye Solution SYTO13, Stock Anti-CD59-phycoerythrin (PE), and Stock Anti-CD61-PE) were provided by Litron Laboratories (Rochester, NY). MicroFlowV rat blood micronucleus kits were purchased from Litron Laboratories. Fetal bovine serum was purchased from ExCell Biology (Shanghai, China). R
R
Analyses of MN frequency were conducted on blood samples collected on Day 4 (5-FU) or Day 29 (4NQO). Blood was processed, labeled, and analyzed according to the instruction manual received with the MicroFlowV Rat Blood Micronucleus Kit. Approximately 20,000 RET were scored for micronuclei per animal, where available. This method has been described in detail elsewhere [Torous et al., 2003; Dertinger et al., 2004]. A BD FACSCalibur flow cytometer running CellQuestTM Pro version 5.2.1 software was used for data acquisition and analysis. R
R
Animals, Treatment, and Harvests Experiments were conducted with the approval and oversight of the NCDSER Institutional Animal Care and Use Committee. Male SpragueDawley rats, approximately 6–8 week old, were purchased from Slac
Calculations, Statistical Analyses The formulas used to calculate mutant phenotype erythrocyte (RBCCD592) and mutant phenotype reticulocyte (RETCD592) frequencies have been described previously [Dertinger et al., 2012]. The incidence of RETs and MN-RET is expressed as frequency percent, whereas the incidence of mutant phenotype cells is expressed as number per 106. All %RET, mutant phenotype cell frequencies, MN frequencies, averages,
Environmental and Molecular Mutagenesis. DOI 10.1002/em Assessment of 5-FU and 4NQO In Vivo Genotoxicity TABLE I. Body Weight Observations in the 5-Fluorouracil Test ( x 6 s, n 5 5) Dose (mg/kg/day)
0
11.5
23
46
Body weight change 18.2 6 3.9 11.6 6 4.2 21.1 6 8.0a 224.4 6 9.9a (g, days 1-4) a
Significant difference (P < 0.05).
737
point (RBCCD592 and RETCD592) was considered at each individual time point using the nonparametric Kruskal-Wallis test, and pairwise comparisons based on the Wilcoxon rank sum test were performed to evaluate vehicle against each of the other treatment groups. All statistical tests used P < 0.05 to indicate significance. %MN-RET values were transformed using an arcsin transformation, to stabilize group variances (Shapiro-Wilk W-test). Unlike %MN-RET data, all %RET data sets were normally distributed in treatment groups and did not require transformation. Individual %MN-RET and %RET values for animals in each treatment group were evaluated using a oneway analysis of variance (ANOVA) model. If the overall “F” test from the ANOVA model was significant (P < 0.05), comparisons between the drug-treated group means and the concurrent vehicle group mean were made in the context of the ANOVA model, using the Dunnett t-test. Significance was noted for each comparison where P < 0.05.
RESULTS Erythrocyte Pig-a Assay and Micronucleus Assay With 5-FU
Fig. 1. Three Pig-a assay endpoints are graphed for the 3-day 5-fluorouracil (5-FU) study: mean %RET (A), the mean RBCCD592 frequency (B), the mean RETCD592 frequency (C) induced by 0, 11.5, 23, and 46 mg/kg/day 5-FU or 20 mg/kg/day ENU. Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and standard deviation calculations were performed with Excel for Windows. Statistical analyses were based on individual animal data and performed with SAS v9.1 Software. The effect of treatment on each end-
The mean body weight change of groups treated with 5-FU decreased relative to the vehicle control animals, with the magnitude of this change occurring in a doserelated manner on Day 4. Mean body weights treated at 23 mg/kg/day on Day 4 were less than their day 1 body weights, with those treated at 46 mg/kg/day demonstrating even greater body weight loss (Table I). No significant decrease in the %RET was observed for 5-FU treated rats on Days 15, 29, or 45 (Fig. 1A). The mean Pig-a mutant phenotype frequencies in RBCs and RETs of rats treated with 11.5, 23, and 46 mg 5-FU/kg/ day did not yield significant increases above vehicle control at any time points tested, with all groups exhibiting less than 5 3 1026 in both populations of erythrocytes. In contrast, statistically significant increases in both RETCD592 and RBCCD592 frequencies were observed for the concurrent positive control (ENU at 20 mg/kg/day; see Figs. 1B and 1C). Agent-related reductions in %RET were apparent at this early time point, providing an important evidence for bone marrow exposure that was not apparent at Day 15 of the Pig-a analysis. Animals treated with 46 mg/kg/day did not provide sufficient cells for analysis, so percent micronucleated reticulocytes (%MN-RET) data from this dose were not included in the analysis. Statistically significant increases in %MN-RET were observed at doses 11.5 mg 5-FU /kg/day as well as for the concurrent positive control group (Fig. 2). Erythrocyte Pig-a Assay and Micronucleus Assay With 4NQO Weight gain was monitored over the 28-day treatment period. Treatment-related reductions in mean weight gain were evident at 6 mg 4NQO/kg/day from Days 1–8, Days 1–15, and Days 1–29. For example, from Days 1–15, mean weight gain of the rats treated at 3 mg/kg/day
Environmental and Molecular Mutagenesis. DOI 10.1002/em 738
Zhou et al.
Fig. 2. Mean %RET and mean %MN-RET induced by 0, 11.5, 23, and 46 mg/kg/day 5-FU or 20 mg/kg/day ENU. Data presented as mean 6 SD, n 5 5 (high dose group, n 5 2). Statistically significant differences from vehicle controls are indicated at **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was below the vehicle control animals, and this effect was more pronounced for the 6 mg/kg/day group (Table II). RETCD592 and RBCCD592 frequencies were low in all rats at Day 21, as well as in the vehicle control rats at all sampling times, with the vast majority being less than 5 3 1026 in both populations of erythrocytes. Agerelated decreases in %RET were observed in all treatment groups. In addition to the age-related effect, treatment with 6 mg 4NQO/kg/day was observed to reduce mean %RET values compared with vehicle controls on Day 15 (Fig. 3A). Statistically significant increases in RBCCD592 frequencies were noted for the high dose group only on Day 29 (Fig. 3B). On the other hand, the 6 mg/kg/day dose group exhibited elevated RETCD592 frequencies that were significantly higher than those observed in vehicletreated rats on Days 15 and 29, with maximal observed values occurring on day 29 (Fig. 3C). There were no significant changes to %RET compared with the vehicle control at any 4NQO dose tested monitored on Day 29 (Fig. 4). A statistically significant increase in the frequency of %MN-RET, approximately two-fold over the vehicle control mean, was observed only at the high dose group.
DISCUSSION The aim of this study was to contribute to an international validation effort that has been evaluating the in vivo rat Pig-a mutant phenotype assay. Experiments were performed with 5-FU-treated SD rats using a 3-day study design, and 4NQO was studied in the same rat model using the other common treatment schedule, a 28-day repeat dosing design. In addition, the well-established in vivo micronucleus endpoint was integrated into the Pig-a assay. In this study, 5-FU was evaluated at the maximum tolerated dose, as demonstrated by significant decreases in body weight, body weigh gain (Table I), and evident
RET toxicity (Fig. 2) at a dose of 46 mg/kg/day. 5-FU was observed to induce MN-RET in a dose-dependent manner on Day 4. This result is consistent with a report by Zhou et al. [2013]. On the other hand, treatment of rats for 3 days with 5-FU had no statistically significant effect on RETCD592 (Fig. 1C) or RBCCD592 frequencies (Fig. 1B), even at the highly toxic dose level of 46 mg/ kg/day. It would be useful to follow this work up with a 5-FU study that consists of a 28-day exposure period. Regardless, the current data set provides no indication of an in vivo mutagenic potential, despite the fact that the bone marrow was clearly affected. The magnitude and kinetics of the RETCD592 and RBCCD592 mutant phenotype responses in the 4NQO 28day subchronic study were remarkably similar to previous findings which were generated using the intial “Stage III design,” that is, flow cytometry without immunomagnetic separation [Dertinger et al., 2010]. The results were also qualitatively those reported by Stankowski et al. [2011], although the lowest effective dose level was somewhat lower in their case (2.5 mg/kg/day). While the high dose of 4NQO did statistically significantly increase the frequency of MN-RET, the induction was rather weak and inconsistent. The weak positive response for MN-RET on Day 29 in this study is in contrast to the previous negative findings reported for 4NQO on Days 4 and 29 by Dertinger et al. [2010] who studied a top dose level of 5 mg/kg/day as opposed to our 6 mg/kg/day. Our results are closer to the observations of Stankowski et al. [2011], who characterized the MN-RET induction using a 28-day study design as equivocal. Thus, the relatively weak indication of clastogenic activity reported herein is not surprising. Significant in-house and inter-laboratory testing has made it clear that spontaneous Pig-a mutant phenotype RET and RBC values are typically quite low, probably on the order of about 1 3 1026 [Dertinger et al., 2010; Phonethepswath et al., 2010]. Our data support this assessment, and indicate that the immunomagnetic separation method used herein has the advantages of achieving higher rates of data acquisition in order to make it practical to interrogate many times more RBCs and RETs for Pig-a mutant response than is otherwise possible. Overall, our results confirm the interlaboratory reproducibility of the Pig-a assay endpoint previously observed in short-term studies with ENU (the positive control) in the Stage II phase [Dertinger et al., 2011b], and in repeatdose toxicity studies with 4NQO in the Stage III phase [Stankowski et al., 2011]. This report also contributes data regarding a presumed nonmutagen, 5-FU, an agent that was applied at a very toxic dose level. Moreover, the current study supports the premise that Pig-a and micronucleus endpoints can be readily integrated into acute and repeat-dose studies, thereby providing coverage of both gene mutation and chromosomal damage in the same
Environmental and Molecular Mutagenesis. DOI 10.1002/em Assessment of 5-FU and 4NQO In Vivo Genotoxicity
739
TABLE II. Body Weight Observations in the 4NQO Test ( x 6 s, n 5 5) Dose (mg/kg/day) Body weight change (g)
a
Days 1-8 Days 1-15 Days 1-29
0
1.5
3
6
53.9 6 8.1 84.6 6 13.0 127.7 6 24.9
64.7 6 8.5 100.8 6 13.8 162.6 6 22.3
57.6 6 10.2 34.5 6 10.7a 146.1 6 32.7
14.2 6 22.0a 5.8 6 11.9a 60.1 6 34.8a
Significant difference (P < 0.05).
Fig. 4. Mean %RET and mean %MN-RET induced by 0, 1.5, 3, or 6 mg/kg/day 4NQO Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
AUTHOR CONTRIBUTIONS Changhui Zhou, Min Zhang, Stephen D. Dertinger, Dorothea K. Torous, and Yan Chang designed the study. Changhui Zhou, Min Zhang, Pengcheng Huang, Honggang Tu, and Zheng Wang collected the data. Changhui Zhou and Min Zhang analyzed the data and prepared draft figures. Changhui Zhou prepared the manuscript draft with important intellectual input from Stephen D. Dertinger and Yan Chang. All authors approved the final manuscript. Changhui Zhou and Yan Chang had completed access to the study data. REFERENCES
Fig. 3. Three Pig-a assay endpoints are graphed for the 28-day 4-nitroquinoline-1-oxide (4NQO) study: mean %RET (A), the mean RBCCD592 frequency (B), and the mean RETCD592 frequency (C) induced by 0, 1.5, 3, or 6 mg/kg/day. Data presented as mean 6 SD, n 5 5. Statistically significant differences from vehicle controls are indicated at *P < 0.05 and **P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
experiment. This has the potential to significantly reduce the number of dedicated in vivo genotoxicity studies required [Kirkland et al., 2011], while at the same time improving the extent and value of available genotoxicity information.
Araten DJ, Nafa K, Pakdeesuwan K, Luzzatto L. 1999. Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc Natl Acad Sci USA 96:5209–5214. Bryce SM, Bemis JC, Dertinger SD. 2008. In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen 49:256– 264. Cimino MC. 2006. Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes. Environ Mol Mutagen 47:362–390. Dertinger SD, Camphausen K, MacGregor JT, Bishop ME, Torous DT, Avlasevich S, Cairns S, Tometsko CR, Menard C, Muanza T, Chen Y, Miller RK, Cederbrant K, Sandelin K, Ponten I, Bolcsfoldi G. 2004. Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood. Environ Mol Mutagen 44:427–435.
Environmental and Molecular Mutagenesis. DOI 10.1002/em 740
Zhou et al.
Dertinger SD, Phonethepswath S, Franklin D, Weller P, Torous DK, Bryce SM, Avlasevich S, Bemis JC, Hyrien O, Palis J, MacGregor JT. 2010. Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies. Toxicol Sci 115:401–411. Dertinger SD, Phonethepswath S, Weller P, Avlasevich S, Torous DK, Mereness JA, Bryce SM, Bemis JC, Bell S, Portugal S, Aylott M, MacGregor JT. 2011a. Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes. Environ Mol Mutagen 52: 748–755. Dertinger SD, Phonethepswath S, Weller P, Nicolette J, Murray J, Sonders P, Vohr H-W, Shi J, Krsmanovic L, Gleason C, Custer L, Henwood A, Sweder K, Stankowski LF Jr, Roberts DJ, Giddings A, Kenny J, Lynch AM, Defrain C, Nesslany F, van der Leede B-JM, Van Doninck T, Schuermans A, Tanaka K, Hiwata Y, Tajima O, Wilde E, Elajouji A, Gunther WC, Thiffeault CJ, Shutsky TJ, Fiedler RD, Kimoto T, Bhalli JA, Heflich RH, Mac-Gregor JT. 2011b. International Pig-a gene mutation assay trial: Evaluation of transferability across 14 laboratories. Environ Mol Mutagen 52:690–698. Dertinger SD, Bryce SM, Phonethepswath S, Avlasevich SL. 2011c. When pigs fly: Immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis. Mutat Res 721:163–170. Dertinger SD, Phonethepswath S, Avlasevich SL, Torous DK, Mereness J, Bryce SM, Bemis JC, Bell S, Weller P, MacGregor JT. 2012. Efficient monitoring of in vivo Pig-a gene mutation and chromosomal damage: Summary of 7 published studies and results from 11 new reference compounds. Toxicol Sci 130:328–348. Dobrovolsky VN, Miura D, Heflich RH, Dertinger SD. 2010. The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ Mol Mutagen 51:825–835. Kirkland DJ, Hayashi M, Jacobson-Kram D, Kasper P, Gollapudi B, M€uller L, Uno Y. 2011. Summary of major conclusions from the 5th IWGT, Basel, Switzerland, 17–19 August 2009. Mutat Res 723:73–76. Miura D, Dobrovolsky VN, Y Kasahara, Heflich RH. 2008a. Development of an in vivo gene mutation assay using the endogenous Pig-a gene: I. Flow cytometric detection of CD59-negative
peripheral red blood cells and CD48-negative spleen T cells from the rat. Environ Mol Mutagen 49:614–621. Miura D, Dobrovolsky VN, Mittelstaedt RA, Kasahara Y, Heflich RH. 2008b. Development of an in vivo gene mutation assay using the endogenous Pig-a gene: II. Selection of Pig-a mutant rat spleen T-cells with proaerolysin and sequencing Pig-a cDNA from the mutants. Environ Mol Mutagen 49:622–630. Miura D, Dobrovolsky VN, Kimoto T, Kasahara Y, Heflich RH. 2009. Accumulation and persistence of Pig-a mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea. Mutat Res 677:86–92. Peruzzi B, Araten DJ, Notaro R, Luzzatto L. 2010. The use of Pig-a as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo. Mutat Res 705:3–10. Phonethepswath S, Bryce SM, Bemis JC, Dertinger SD. 2008. Erythrocyte-based Pig-a gene mutation assay: Demonstration of crossspecies potential. Mutat Res 657:122–126. Phonethepswath S, Franklin D, Torous DK, Bryce SM, Bemis JC, Raja S, Avlasevich S, Weller P, Hyrien O, Palis J, MacGregor JT, Dertinger SD. 2010. Pig-a mutation: Kinetics in rat erythrocytes following exposure to five prototypical mutagens. Toxicol Sci 114:59–70. Schuler M, Gollapudi BB, Thybaud V, Kim JH. 2011. Need and potential value of the Pig-a in vivo mutation assay, a HESI perspective. Environ Mol Mutagen 52:685–689. Stankowski LF Jr, Roberts DJ, Chen H, Lawlor T, McKeon M, Murli H, Thakur A, Xu Y. 2011. Integration of Pig-a, micronucleus, chromosome aberration, and comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide. Environ Mol Mutagen 52:738–747. Torous DK, Hall NE, Murante FG, Gleason SE, Tometsko CR, Dertinger SD. 2003. Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol Sci 74:309–314. Zhou C, Wang Q, Wang Z, Chang Y. 2013. Comparison of three-colour flow cytometry and slide-based microscopy for the scoring of micronucleated reticulocytes in rat bone-marrow and peripheral blood. Mutat Res 758:12–17.
Accepted by— M. Honma
