ToxSci Advance Access originally published online on September 13, 2007
Toxicological Sciences 2007 100(2):406-414; doi:10.1093/toxsci/kfm241
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Published by Oxford University Press 2007.
Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes III. An Efficient Method of Monitoring Chromosomal Damage in the Beagle Dog
* Department of Veterans Affairs, Washington, DC 20422
Litron Laboratories, Rochester, NY 14623
National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079
GlaxoSmithKline Research & Development, Herts, SG12 0DP, UK
¶ U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Laurel, MD 20708
|| Toxicology Consulting Services, Arnold, MD 21012
4 To whom correspondence should be addressed at Toxicology Consulting Services, Arnold, MD 21012. Fax: (410) 975-0481. E-mail: jtmacgregor{at}earthlink.net.
Received June 11, 2007; accepted September 5, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Erythrocyte-based micronucleus tests have traditionally analyzed bone marrow because splenic filtration in most species removes micronucleated cells from peripheral blood. We have evaluated a flow cytometric method for monitoring micronucleated reticulocyte frequencies (%MN-RET) in the peripheral blood of beagle dogs treated with cyclophosphamide (CP) and have found that analysis of micronucleated reticulocytes (MN-RETs) in peripheral blood is a suitable surrogate for bone marrow analysis. The three-color flow cytometric method uses anti-CD71 labeling to identify reticulocytes and Plasmodium berghei–containing erythrocytes as a calibration standard. The spontaneous %MN-RET determined by flow cytometry was 0.31 ± 0.09% (n = 22) for peripheral blood, compared with 0.38 ± 0.13% (SD, n = 12) for bone marrow, and 0.27 ± 0.08% (n = 12) for peripheral blood by microscopic scoring with acridine orange staining. The kinetics of appearance and disappearance of MN-RETs in blood were determined by collecting daily samples after iv treatment with CP. The maximum frequency occurred
48 h after dosing. Frequencies of MN-RETs in peripheral blood at steady state following daily CP treatment were 55–68% of corresponding bone marrow values assessed by microscopy and 55–112% as assessed by flow cytometry. This difference is presumably due to splenic removal, which appears slightly less stringent than that previously reported for CP-treated Sprague-Dawley rats. Responses in bone marrow and peripheral blood were highly correlated and similar to or greater than those reported in mice and rats at equitoxic doses. Key Words: chromosomal damage; micronucleus; flow cytometry; reticulocytes; blood; bone marrow.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The bone marrow erythrocyte micronucleus assay is widely used for regulatory studies conducted to evaluate the potential for induction of chromosomal damage or chromosome loss due to exposure to drugs, food additives, pesticides, industrial chemicals, and other products. The assay has traditionally been based on scoring the incidence of micronuclei, which result from chromosome breakage or chromosome loss during cell division, in newly formed (immature) red blood cells in bone marrow, as described in the FDA "Redbook", EPA regulations, and ICH and OECD Guidelines (D'Arcy and Harron, 1998
; U.S. EPA, 1998; FDA, 2000; OECD, 1997; ICH, 1998a; ICH, 1995b). Mice or rats have most commonly been used for this assay. Although micronucleated cells formed in bone marrow enter the peripheral circulation, bone marrow has been the traditional target tissue sampled because, with the exception of the mouse (MacGregor et al., 1980), most species employed routinely for laboratory toxicology evaluations selectively remove micronucleated erythrocytes from the peripheral circulation. In species with active splenic removal of micronucleated cells, including the rat (Schlegel and MacGregor, 1984), dog (MacGregor et al., 1992; this publication), and the human (Everson et al., 1988; Schlegel et al., 1986), the frequency of micronuclei in bone marrow cells is greater than that in the peripheral blood due to the removal of micronucleated cells from the circulation by the spleen. The combination of labor intensiveness and the perceived necessity of invasive sampling of bone marrow has limited the application of this assay mainly to mice and rats. It would be highly advantageous to be able to conduct the assay using small volumes of peripheral blood obtained during key safety assessment studies, especially when those studies are conducted in higher mammals such as dogs or non–human primates. This would not only eliminate the need for a separate animal study to assess genotoxicity but would also allow direct comparison of genotoxicity data with pharmacokinetic and pharmacodynamic information in the species most relevant to the safety assessment of a particular agent.
Flow cytometric methods have recently become available that have made possible the use of peripheral blood samples in place of bone marrow in rats (Dertinger et al., 2006; MacGregor et al., 2006; Torous et al., 2003). These methods have the potential to be applied to blood samples of other species including the human—in which the splenic effect is particularly strong (Abramsson-Zetterberg et al., 2000; Dertinger et al., 2003, 2004, 2007; Grawé et al., 2005; Offer et al., 2005; Schlegel et al., 1986; Stopper et al., 2005; Witt et al., 2007). Analysis of peripheral blood using flow cytometry makes it practical to use species in which bone marrow sampling is not easily accomplished and species with low reticulocyte counts, such as primates, in which manual microscopic scoring is prohibitively labor intensive.
Using cyclophosphamide (CP) as a model clastogenic agent, we demonstrate here that the flow cytometric analysis of micronucleated reticulocytes (MN-RETs) in the peripheral blood of beagle dogs provides an accurate index of chromosomal damage in bone marrow. We also define the kinetics of appearance and disappearance of MN-RETs after induction of chromosomal breakage and show that the sensitivity of this assay is similar to that previously demonstrated in mice and rats. The results confirm that the flow cytometric method correlates with manual scoring of micronuclei from bone marrow samples or peripheral blood but greatly reduces the labor of scoring and provides greater reproducibility than microscopic scoring.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Chemicals and Other Reagents
CP (CAS No. 6055-19-2) was purchased from Mead Johnson Oncology Products (Bristol-Myers Squibb Co., Princeton, NJ) and was prepared according to the manufacturer's instructions. Acridine orange (AO) (CAS No. 65-61-2) (Sigma Chemical Co., St Louis, MO) was prepared as a staining solution according to Hayashi et al. (1983)
. Flow cytometry reagents, including anticoagulant and fixative solutions, anti-CD71–FITC, anti-CD61–PE, propidium iodide, and fixed malaria–infected rodent blood (malaria biostandard), were from Prototype Canine MicroFlowPLUS Kits and were contributed by Litron Laboratories, Rochester, NY.
Animal Source and Care
All experiments involving CP treatment were conduced at the U.S. Food and Drug Administration laboratory at the Center for veterinary Medicine, Laurel, Maryland (FDA Laboratory). In addition to samples from the FDA laboratory, control blood and bone marrow samples were obtained from SRI International, Menlo Park, CA (SRI Laboratory) and the GlaxoSmithKline Research and Development Laboratory, Herts, UK (GSK Laboratory). All animal studies were conducted according to the recommendations of the National Research Council (1996) Guide for the Care and Use of Laboratory Animals and were approved by the appropriate Institutional Animal Care and Use Committees.
FDA Laboratory.
Healthy male and female beagle dogs, ranging in age from 4 to 6 months, were used for all studies (Harlan, Indianapolis, IN). The seven males ranged in body weight from 6.5 to 10.2 kg and the six females ranged in body weight from 5.2 to 7.75 kg at the start of the study. Each dog was individually identified by the vendor by a permanent tattoo located on the inside earflap and an accompanying USDA tag. Throughout the study, dogs were housed in inside runs, individually or in compatible pairs, and received a commercially available laboratory diet for growing dogs (Harlan Teklad 2021) with ad libitum access to drinking water. As environmental enrichment, dogs were provided with toys in their runs, two separate group play sessions each day (14 sessions per week), and a variety of commercial food treats.
SRI Laboratory.
Two separate studies were conducted with eight dogs in 1992 (previously presented and published in abstract form) and six dogs in 2004. Healthy male and female beagle dogs, ranging in age from 6 to 8 months, were used for all studies (Marshall Farms, North Rose, NY). Each dog was individually identified by the vendor by a permanent tattoo located on the inside earflap and an accompanying USDA tag. Throughout the study, dogs were housed in group runs inside and received a commercially available laboratory diet for growing dogs (Vendor) with ad libitum access to drinking water. As environmental enrichment, dogs were provided with daily exercise sessions (seven per week).
GSK Laboratory.
All animal treatment and husbandry were in accordance with approved procedures of the Animals (Scientific Procedures) Act, UK, 1986. Healthy male and female beagle dogs (Harlan, Oxon, UK), ranging in age from 11 to 16 months, were individually identified by microchip and housed in groups of three of the same sex in concrete-floored/solid-floored pens with sawdust litter (Datesand, Manchester, UK). The three males ranged in body weight from 9.98 to 13.13 kg, and the three females ranged in body weight from 7.97 to 10.55 kg at the start of the study. They received a commercially available laboratory diet (Harlan Teklad 2021) with ad libitum access to drinking water. Environmental controls were set at 21°C ± 2°C, 55 ± 15% relative humidity, and fluorescent lighting between 06.00 h and 18.00 h GMT. As part of environmental enrichment, all dogs had access to toys in their kennels and received some form of enrichment every weekday, that is, playtimes or kennel socialization periods (generally five playtimes and two socialization periods per week).
Comparison of MN-RETs in Peripheral Blood and Bone Marrow of Normal Canines
SRI Laboratory.
Bone marrow specimens were obtained at necropsy as described below for terminal marrow sample collection from the humerus and ilium at the FDA laboratory.
GSK Laboratory.
Six dogs were dosed orally with a control vehicle consisting of 75% (vol/vol) polyethylene glycol 400/25% (vol/vol) vitamin E D-alpha tocopherol polyethylene glycol succinate at a constant dose volume of 5.0 ml/kg for 21 days. On day 22, 3.0 ml peripheral blood sample was collected from the jugular vein of each dog for microscopy and flow cytometric scoring of circulating micronuclei. Within 1 h of blood collection, each dog was euthanized by exsanguination under deep barbiturate (pentobarbital) anesthesia prior to terminal bone marrow collection. The femur was removed and sectioned lengthways to expose the marrow cavity. Marrow samples were obtained from the proximal and midshaft regions by flushing with fetal calf serum. Samples were filtered through bolting cloth and collected into conical tubes for centrifugation at 1000 rpm at room temperature for 5 min. After centrifugation, most of the supernatant was discarded, leaving sufficient volume to resuspend the cellular pellet. The resulting suspension was used to prepare bone marrow smears on clean glass slides. After air drying, the samples were fixed in absolute methanol for 10 min prior to staining.
SRI and GSK Laboratories—Peripheral blood sample preparation and storage.
Blood was collected by jugular venipuncture into lithium heparin tubes. Within 1 h of collection, 1 ml aliquot of whole blood was transferred into 5 ml diluent solution. The diluent was phosphate-buffered saline (PBS) (Invitrogen, Paisley, UK) with 500 USP/ml sodium heparin (Molekula, Dorset, UK). The diluted sample was inverted to ensure a homogeneous suspension and then 1.0 ml was removed and forcefully injected into 11 ml ultracold methanol (– 75°C to – 85°C) and vortexed for 3–5 s. Immediately after fixation, the samples were stored at – 75°C to – 85°C until flow analysis was performed. Five fixed samples were prepared for each blood sample.
Fifteen whole-blood smears were prepared on glass slides for each sample, allowed to air-dry, and then fixed in absolute methanol for 10 min for storage prior to staining.
Comparison of MN-RETs in Peripheral Blood and Bone Marrow of Treated Canines
FDA Laboratory—CP dose-range finding study.
One male 4-month-old beagle dog was used to identify an appropriate dose of CP that would produce an optimal level of circulating micronuclei with minimal adverse events. Prior to dosing, two 3.0-ml samples of peripheral blood were obtained approximately 2 weeks apart. In addition, percutaneous bone marrow aspirates were obtained under anesthesia from the ilium, humerus, and femur after each blood sample for microscopy scoring of micronuclei.
One week after preliminary samples were acquired, 3.0 ml peripheral blood sample was obtained at baseline. Within 1 h of sampling, the dog was dosed with 50 mg/m2 CP by iv bolus. This dose represents the therapeutic dose recommended for the treatment of neoplastic disease (Plumb, 2002). Daily 3.0 ml peripheral blood samples were obtained approximately every 24 h over the next 7 days for microscopy and flow cytometric scoring of circulating micronuclei.
After a 7-day recovery period to provide for a 14-day washout interval from the initial CP administration, a physical examination and complete blood count were performed to verify satisfactory health status and to ensure that leukocyte and platelet counts were within the normal range.
A baseline peripheral blood sample was obtained and 25 mg/m2 CP was administered by iv bolus, followed by the same sampling schedule above.
Results (not shown) of the two dosing regimens were used to select the doses employed in subsequent experiments.
FDA Laboratory—kinetics study.
Three male and three female beagle dogs, ranging in age from 4 to 6 months, were used to evaluate the kinetics of micronucleus appearance and disappearance in the peripheral circulation and to assess the sensitivity of the proposed flow cytometric assay. Peripheral blood sample (3 ml) was obtained from each dog at baseline. Within 1 h of blood collection, the dog was manually restrained and administered 25 mg/m2 CP by iv bolus. Daily 3.0 ml peripheral blood samples was obtained approximately every 24 h over the next 7 days for microscopy and flow cytometric scoring of circulating micronuclei.
FDA Laboratory—steady-state studies.
Six male and six female beagle dogs, ranging in age from 4 to 6 months, were used to compare the frequency of micronuclei in circulating reticulocytes to those in bone marrow reticulocytes at steady state. Peripheral blood sample (3 ml) was obtained from each dog on one or more days at baseline. Within 1 h of blood collection, the dog was manually restrained and administered 25 mg/m2 CP by iv bolus. Daily 3.0 ml peripheral blood samples was obtained, followed by CP administration repeated approximately every 24 h over the next 7–8 days for microscopy and flow cytometric scoring of circulating micronuclei. Circulating micronuclei were determined to be at a constant steady state of production by flow cytometric scoring. Conditions for "steady state" were satisfied when there was no significant change in micronuclei production over a 48-h period. Approximately 24 h after the last administration of CP, a final blood sample was obtained and each dog was euthanized by iv administration of an overdose of pentobarbital prior to terminal bone marrow collection.
FDA Laboratory—peripheral blood sample collection, preparation, and storage.
To obtain peripheral blood, each dog was manually restrained and a 3 ml blood sample was collected from the cephalic vein of the front leg into a lithium heparin Vacutainer tube (Becton, Dickson and Company, Franklin Lakes, NJ).
Within 1 h of collection, 0.5 ml aliquot of whole blood was transferred into 2.5 ml heparinized PBS (Litron Laboratories, Rochester, NY) at 4°C and gently inverted to ensure a homogeneous suspension. Using a P-1000 pipette, 450 µl of the diluted sample was forcefully injected into 5 ml ultracold methanol (– 75°C to – 85°C) and vortexed for 3–5 s. Immediately after fixation, the samples were stored at – 75°C to – 85°C until flow analysis was performed. A minimum of three fixed samples were prepared for each blood sample.
A minimum of five whole-blood smears were prepared on glass slides for each sample, allowed to air-dry, and then fixed in absolute methanol for 10 min for storage prior to staining.
FDA Laboratory—percutaneous bone marrow sample collection.
The dog was sedated by iv administration of diazepam (0.28 mg/kg body weight) and ketamine (5.5 mg/kg body weight) and maintained on 0–2% isoflurane inhalation anesthesia for the remainder of the procedure. The biopsy site was surgically prepped and a sterile hollow core biopsy needle with stylet (Allegiance Healthcare Corp., McGraw Park, IL) was manually introduced through the skin into the marrow cavity. Approximately 0.5–1.0 ml of marrow was aspirated into a clean syringe and immediately transferred to a sterile dipotassium EDTA vacutainer with sterile PBS on ice. The suspended marrow was transferred into conical tubes for centrifugation at 1000 x g and 20°C for 5 min. After centrifugation, the supernatant was discarded and the cellular pellet was resuspended in an approximately equal volume of sterile PBS. The resulting suspension was used to prepare bone marrow smears on clean glass slides. After air drying, the samples were fixed in absolute methanol for 10 min prior to staining.
A high percentage of bone marrow samples obtained by percutaneous aspiration were heavily contaminated with peripheral blood during sampling, resulting in a cytology sample of low cellularity with regard to marrow elements. As a result of the significant variation between samples, percutaneous sampling was discontinued for subsequent studies.
FDA Laboratory—terminal bone marrow sample collection, preparation, and storage.
Bone marrow specimens were obtained from each dog at necropsy by removing a segment of the femur (proximal and midshaft), ilium (dorsal crest of the wing), and humerus (proximal). The marrow was flushed into a sterile Petri dish containing sterile PBS and collected into conical tubes for centrifugation at 1000 x g and 20°C for 5 min. After centrifugation, the supernatant was discarded and the cellular pellet was resuspended in an approximately equal volume of sterile PBS. The resulting suspension was used to prepare bone marrow smears on clean glass slides. After air drying, the samples were fixed in absolute methanol for 10 min prior to staining.
Microscopy-Based Scoring (FDA)
Blood and bone marrow smears were scored using the standard scoring technique for AO-stained samples at the FDA-NCTR laboratory as described previously (MacGregor et al., 2006). Methanol fixation leads to a diffuse distribution of RNA, and erythrocytes are classified as normochromatic or as RETs based on the presence or absence of RNA-associated orange fluorescence. RET frequencies were determined by inspecting 500 or 1000 total erythrocytes per bone marrow or blood sample, respectively. MN-RET incidence was determined by inspecting 2000 RETs per sample. Micronuclei were defined by the criteria of Schmid (1976) with the added requirements that they exhibit the characteristic yellow to yellow-green fluorescence characteristic of AO staining and that they exhibit the smooth boundary expected from a membrane-bound body.
Flow Cytometry-Based Scoring (Litron Laboratories)
Methanol-fixed blood samples were washed and labeled for flow cytometric analysis by Litron Laboratories according to procedures described in the Prototype Canine MicroFlowPLUS Kit. Briefly, cells were washed out fixative and then simultaneously incubated with anti-CD71–FITC, anti-CD61–PE, and RNase. Labeled cells were then washed one time with a kit-provided balanced salt solution supplemented with 1% fetal bovine serum. Thereafter, samples were resuspended in working propidium iodide solution and maintained on ice until flow cytometric analysis (same day).
Samples were analyzed with a FACSCalibur instrument (Becton Dickinson). Anti-CD71–FITC, anti-CD61–PE, and propidium iodide fluorescence signals were detected in the FL1, FL2, and FL3 channels, respectively (stock filter sets). Calibration of the flow cytometer for the MN scoring application was accomplished by staining kit–supplied Plasmodium berghei–infected rodent blood (malaria biostandards) in parallel with test samples on each day of analysis (Dertinger et al., 2000; Tometsko et al., 1993; Torous et al., 2001). By adjusting voltages applied to the photo-multiplier tube, it was possible to standardize the FL3 fluorescence channel into which erythrocytes with single (micronucleus-like) parasites fell. In this manner, analysis regions were consistent between experiments. Data were acquired with CellQuest software (version 3.3, BD-Immunocytometry Systems, San Jose, CA), with the stop mode set so that 20,000 CD71-expressing RETs were analyzed per blood sample. The number of mature (CD71 negative) erythrocytes was determined concurrently, providing an index of cytotoxicity (%RETs).
Calculations.
All calculations were performed with Excel (Office X for Mac, Microsoft Corp., Seattle, WA). The incidence of MN-RETs among total RETs is expressed as frequency percent (i.e., %MN-RET). The incidence of RETs among total erythrocytes is also expressed as frequency percent (i.e., %RET).
Statistics.
Correlations between flow cytometric and microscopic scoring of blood samples and between microscopic bone marrow scores and flow cytometric peripheral blood scores were evaluated using JMP v5 for Macintosh to calculate Spearman's (nonparametric) and Pearson's correlation coefficients.
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RESULTS AND DISCUSSION |
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Control Values in Bone Marrow and Peripheral Blood
Control values were determined in bone marrow and/or peripheral blood of purpose-bred beagle dogs from three different sites: the FDA, GSK, and SRI Laboratories identified above. These samples were used to compare baseline values in blood and bone marrow scored by the microscopic method and also to compare these with blood measurements obtained by flow cytometry. These values are reported in the online appendix (see supplementary material), and mean values (± SEM) obtained in the present studies are presented in Table 1.
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Control bone marrow and peripheral blood samples obtained at the GSK and SRI sites were scored at the FDA-NCTR site using microscopic scoring with AO staining in order to directly compare frequencies in bone marrow and blood of control animals (Supplementary Appendix 1). No control animals were sacrificed at the FDA site, so control bone marrow samples are not available from that site. The spontaneous frequencies of MN-RETs in blood and bone marrow were significantly higher than those previously reported for mouse and rat, averaging 0.38% in bone marrow and 0.27–0.31% in peripheral blood (Table 1). Values reported for mice and rats are generally in the range of 0.1–0.2%, with rats tending to exhibit somewhat lower frequencies than mice (e.g., Hamada et al., 2001
; MacGregor et al., 1990, 2006; Torous et al., 2005).
Values in bone marrow and blood determined by microscopic scoring were similar: values in blood from the GSK animals averaged 78% of bone marrow values and those in blood from the SRI animals averaged 67% of bone marrow values. The spontaneous frequencies in female bone marrow samples were slightly higher than in those of males but were not statistically significantly different in the small groups examined (Kastenbaum and Bowman, 1970). These values indicate somewhat less splenic selection against micronucleated cells than the values reported previously by SRI International (MacGregor et al., 1992), which had indicated that values of MN-RETs in peripheral blood averaged only 31% of those in bone marrow (values given in Supplementary Appendix 1).
The results indicate relatively uniform MN-RET frequency values obtained from three distinct bone marrow sites, femur, humerus, and ilium (Supplementary Appendix 1). Thus, any of these three sampling sites should be satisfactory when bone marrow samples are required.
Frequencies of MN-RETs in peripheral blood determined by the flow cytometric method were comparable to those determined by microscopic scoring. As shown by the summary Table 1, the average value obtained by microscopy was 0.27% compared to 0.31% for flow cytometry. It should be noted that the mean microscopy value is based on 12 dogs from two laboratory sites (GSK and SRI), whereas the flow cytometry value is based on 22 dogs from three laboratories (GSK and SRI, as well as pretreatment specimens from FDA animals). In any event, these methodologies are clearly generating comparable values.
Kinetics of Appearance and Loss of MN-RET in Peripheral Blood
The time course of appearance and disappearance of MN-RET in peripheral blood following a single dose of CP is shown in Figure 1. Microscopic scoring of AO-stained smears and flow cytometric scoring gave similar results, with a Pearson correlation coefficient of 0.96 and a nonparametric Spearman's correlation coefficient of 0.91 (36 matched samples, Fig. 2). The sensitivity of the response of the dog to CP is similar to that observed with rats and mice (MacGregor et al., 1980, 2006) with increased frequencies of MN-RETs greater than 10- to 20-fold over the pretreatment values at a dose that causes minimal suppression of the bone marrow reticulocyte frequency.
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The kinetics of appearance and disappearance of MN-RETs are similar to those reported for the Sprague-Dawley rat (MacGregor et al., 2006
). The maximum frequency was observed at 48 h after treatment and had returned nearly to the pretreatment values by 96 h after treatment. Because samples were obtained at 24-h intervals, the exact time of the peak frequency cannot be determined, but the slight asymmetry of the obtained curve suggests that the peak was just slightly later than 48 h (i.e., in all animals the frequency had barely risen at 24 h, was highest at 48 h, and was clearly still elevated at 72 h). Thus, the optimum sampling time following a single treatment is likely to be approximately 50 h when there is no significant bone marrow suppression and possibly slightly longer for doses that cause significant bone marrow toxicity (i.e., that delay the progression of cell division).
Comparison of Blood and Bone Marrow Values at Steady State
Two experiments were conducted in which CP was administered on a daily basis in order to establish a steady state at which the frequency of MN-RETs in bone marrow and blood would be expected to be approximately equal in the absence of selective removal of the micronucleated cells from the peripheral circulation. The frequency of MN-RETs in peripheral blood was monitored each day to verify establishment of the steady state and animals were sacrificed at the end of the experiment to obtain bone marrow samples free of peripheral blood. This proved necessary because repeated attempts at percutaneous needle biopsies of the proximal femur, humerus, and iliac crest did not consistently yield samples sufficiently free of contamination with peripheral blood to allow the reliable determination of the relative MN-RET scores in bone marrow versus peripheral blood. Fortunately, the data demonstrated that the observed increases in MN-RET frequency in peripheral blood accurately reflected those in bone marrow and so routine sampling of bone marrow should not be necessary to monitor the genotoxic response in the future.
Figure 3 shows the daily values of %MN-RET and %RET. In experiment 1, the %MN-RET was not as constant as had been hoped on the two days prior to sacrifice. In experiment 2, treatment was continued for a longer period and a better steady state was achieved. In both cases, microscopic scoring of the MN-RET frequency of the terminal steady-state samples using methanol-fixed, AO-stained, bone marrow and peripheral blood smears showed that the frequencies in peripheral blood were approximately 55–68% of those in bone marrow (Fig. 4). Relative increases in the MN-RET frequency in peripheral blood and in bone marrow were approximately the same in both males and females. The results show that splenic selection against MN-RETs in the peripheral circulation of the beagle dog is similar to that in Sprague-Dawley rats, in which we found the MN-RET frequency in peripheral blood to be approximately 50% of that in bone marrow during treatment with CP at steady state and 59 and 39% with cis-platin and vinblastine, respectively (MacGregor et al., 2006). Schlegel and MacGregor (1984) report a greater difference between blood and bone marrow frequencies of MN-RETs in Fisher 344 rats treated with TEM under steady-state conditions, suggesting a stronger effect of splenic selection in that strain of rat.
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The relative increases due to the CP treatment were similar in bone marrow and peripheral blood and are similar in magnitude to increases at steady state observed in rats and mice treated with CP or other clastogenic agents (Hamada et al., 2001
; MacGregor et al., 1990, 2006; Witt et al., 2000). The flow cytometric scores of %MN-RET frequencies in peripheral blood were highly correlated with corresponding bone marrow scores obtained by microscopic scoring (Pearson correlation coefficient of 0.93 and Spearman nonparametric correlation coefficient of 0.88; 22 matched samples of blood vs. bone marrow). |
CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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These data show that in beagle dogs, as in mice and rats, cytogenetic damage can be easily monitored by using the frequency of MN-RETs in peripheral blood as an index of damage in bone marrow erythropoietic cells. Although data from a single clastogen cannot be considered to fully validate the regulatory application of the model, the biology of the micronucleated cell response and the dependence of the response on cell kinetics and splenic selection are well understood, and there is no reason to expect significant differences with other clastogenic agents. With additional data, including information on the response to aneugenic agents, it is expected that it will prove possible to monitor micronucleus induction as a routine part of other clinical pathology measurements conducted during toxicological assessments in rats, dogs, mice, and perhaps other species. This practice would also allow the micronucleus response to be related directly to other endpoints of toxicity and to pharmacokinetic and metabolic information that is obtained during toxicology studies.
With daily or continuous treatments, an approximate steady-state frequency would be expected after approximately 3 or 4 days of treatment and a single sample obtained within 24 h of the last treatment (or any treatment after the third) would then suffice. A practical approach would be to obtain blood samples for flow cytometric analysis near the end of 1- or 2-week repeat-dose toxicity studies in each species. Routine assessment during toxicology studies in rodents and dogs would eliminate the necessity of using any additional animals for the genetic toxicology assessment and would be more cost-effective than the present practice of conducting purpose-specific experiments because the only additional cost to the studies already being conducted for toxicity evaluations would be the minor cost of the flow cytometric analysis. For experiments that are based on a single treatment with test article, it would be appropriate to obtain two specimens per animal within a time window of approximately 48–54 h after treatment. Pretreatment blood samples could be used in lieu of a concurrent vehicle control group when possible vehicle-related effects are not a concern. Thus, at less than the cost of a traditional mouse micronucleus assay, data in two species using a relevant route and duration of exposure could be obtained under conditions that allow direct comparison with pharmacokinetic, metabolism, and toxicity endpoints being evaluated in the course of safety testing. As data with more agents are accumulated, it is hoped that separate animal studies are no longer required for in vivo bone marrow clastogenicity screening but that this information can be obtained from analysis of peripheral blood samples during the course of toxicology studies.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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U.S. FDA Office of Science Coordination and Communication.
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NOTES |
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1 Present address: Johns-Hopkins University, Baltimore, MD 21218.
2 Present address: U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, MD 21702.
3 Previous address: U.S. Food and Drug Administration, National Center for Toxicological Research, Rockville, MD 20857.
Disclaimer: The contents of this article are the sole responsibility of the authors and do not necessarily reflect the views or policies of the institutions or companies with which they are affiliated.
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ACKNOWLEDGMENTS |
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The authors would like to thank Russell Frobish, Ph.D., and James Weaver, Ph.D., of the FDA for their support of these studies. We also express thanks for the invaluable technical assistance of Steven Bryce, Svetlana Avlasevich, Joanne Collins, Ph.D., Jonathan Howe, B.Sc., and Gregory Black. We also appreciate the assistance of Sandra Phillips, Linda Rausch, and Dr Jon Mirsalis of SRI International who provided control bone marrow and blood samples from the SRI laboratory. Conflict of interest statement: S.D.D. is employed by a company that sells flow cytometry kits and conducts contract studies using the flow cytometric assay described in this article.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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