Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta)

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1 TOXICOLOGICAL SCIENCES 102(2), (2008) doi: /toxsci/kfn013 Advance Access publication January 21, 2008 Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta) Charlotte E. Hotchkiss,* Michelle E. Bishop, Stephen D. Dertinger, William Slikker Jr, Martha M. Moore, and James T. MacGregor,1,2 *The Bionetics Corporation, Jefferson, Arkansas 72079; National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 72079; Litron Laboratories, Rochester, New York, 14623; and National Center for Toxicological Research, U.S. Food and Drug Administration, Rockville, Maryland, Received November 16, 2007; accepted January 10, 2008 We report evaluation in rhesus monkeys of a flow cytometric procedure (MicroFlow) that has previously been shown to allow assessment of micronucleated reticulocytes (MN-RETs) in the peripheral blood of rats and dogs. Reticulocytes (RETs) were labeled with anti-cd71-fluorescein isothiocyanate, DNA was stained with propidium iodide using RNase treatment, and anti- CD61-phycoerythrin was used to reduce interference from platelets. Flow cytometric data were compared with microscopic scores of peripheral blood and bone marrow using standard acridine orange staining. A single iv administration of cyclophosphamide (CP, 5 mg/kg) induced an approximately 10-fold increase in blood MN-RET frequency, with the peak occurring 2 days after administration. After daily CP treatment to approximate a steadystate condition, the frequency of MN-RETs in peripheral blood was approximately 25% of that in bone marrow, indicating strong selection against MN-RETs. Nonetheless, CP-treated animals exhibited markedly elevated blood MN-RET values ( %, n 5 3; compared to a mean baseline of 0.12%, n 5 6). These measurements closely reflected the increased frequencies observed in the bone marrow compartment (Spearman correlation coefficient , n 5 6). These data suggest that MN-RET measurements in blood are suitable for assessing chemicalinduced chromosomal damage and can be readily integrated into routine toxicity tests, allowing genotoxicity data to be obtained as an integral part of toxicity evaluations. Microscopy-based scoring is challenging due to the low frequency of RETs and MN-RET in monkeys, but sufficient numbers of cells are easily scored with the flow cytometric procedure. Key Words: chromosomal damage; micronucleus; flow cytometry; reticulocytes; blood; bone marrow. The bone marrow erythrocyte micronucleus assay is widely used for regulatory studies conducted to evaluate the potential 1 To whom correspondence should be addressed at Toxicology Consulting Services, 201 Nomini Drive, Arnold, MD Fax: (410) jtmacgregor@earthlink.net. 2 Present address: Toxicology Consulting Services, Arnold, MD 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 conducted in rodents and is based on scoring the incidence of micronuclei in newly formed red blood cells (reticulocytes [RETs]) in the bone marrow, as described in the Food and Drug Administration (FDA) Redbook, Environmental Protection Agency regulations, and International Conferences on Harmonisation and Organization for Economic Cooperation and Development guidelines (D Arcy and Harron, 1998; EPA, 1998; FDA, 2000; OECD, 1997). Conduct of the assay in higher mammalian species has been limited because bone marrow sampling is invasive and peripheral blood has been thought not to be a suitable tissue for analysis because of active splenic selection that removes MN-containing erythrocytes from the peripheral circulation. For instance, it has been demonstrated that for rats (Schlegel and MacGregor, 1984), dogs (Harper et al., 2007; MacGregor et al., 1992), and humans (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. Flow cytometric methods are now available that facilitate the scoring of many times more cells than is possible with microscopic evaluation, thereby making 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), dogs (Harper et al., in press), and humans (Dertinger et al., 2003, 2004, 2007) as well as mice (Dertinger et al., 1996; Torous et al., 2001, 2005). These methods have the potential to be applied to blood samples as a routine part of toxicological investigations in all species commonly used for regulatory studies. 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-ret counts, such as primates, in which manual microscopic scoring is prohibitively labor intensive. The current report describes our work with an important species of toxicological interest, the rhesus monkey. These Published by Oxford University Press 2008.

2 MICRONUCLEI IN BLOOD OF RHESUS MONKEYS 353 experiments were designed with two main objectives: (1) to determine whether analyses of blood samples from clastogenexposed rhesus monkeys reflect the increases in micronucleated reticulocyte (MN-RET) frequency observed in the bone marrow compartment of the same animals and (2) to determine the time course of appearance and disappearance of MN-RETs in peripheral blood following treatment with a wellcharacterized genotoxicant. MATERIALS AND METHODS Chemicals and other reagents. Cyclophosphamide (CP) (CAS No ) was purchased from Mead Johnson Oncology Products (Bristol-Myers Squibb Co., Princeton, NJ) and prepared according to the manufacturer s instructions. Acridine orange (AO) (CAS No ) (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-fluorescein isothiocyanate (FITC), anti-cd61-phycoerythrin (PE), propidium iodide, and fixed malaria-infected rodent blood (malaria biostandard) were from Prototype Nonhuman Primate MicroFlow PLUS Kits and were contributed by Litron Laboratories (Rochester, NY). Animals. Nine female adult captive-bred rhesus monkeys (15 24 years of age) were used. Monkeys were individually housed in an Association for Assessment and Accreditation of Laboratory Animal Care Internationalaccredited facility and allowed monkey chow (Purina High Protein Monkey Diet Jumbo #5047) and water ad libitum. These monkeys had been trained to present limbs for iv injection or blood collection without sedation. Six male adult rhesus monkeys (7 22 years of age) were used for collection of reference blood samples only. All procedures were approved by the Institutional Animal Care and Use Committee. Sample collection. Peripheral blood samples (2 3 ml) were collected by syringe and needle and injected into a lithium heparin Vacutainer tube (Becton, Dickinson and Company, Franklin Lakes, NJ). Within 1 h of collection, an aliquot of whole blood was diluted and fixed according to procedures described in the Prototype Nonhuman Primate MicroFlow Kit and by Harper et al. (Harper et al., 2007.). Samples were stored at 75 Cto 85 C until they were shipped on dry ice via overnight courier to Litron Laboratories for analysis. Upon receipt, the samples were stored at 75 C to 85 C until flow analysis was performed. 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. Bone marrow specimens were obtained from each monkey at necropsy by removing a mid-shaft segment of the femur. The marrow was flushed into a sterile Petri dish containing sterile phosphate-buffered saline and collected into conical tubes for centrifugation at g and 20 C for 5 min. After centrifugation, the supernatant was removed and the cellular pellet 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. MN-RET scoring by flow cytometry. Two-milliliter aliquots of methanolfixed blood samples were washed and labeled for flow cytometric analysis at Litron Laboratories according to procedures that have been described previously (Harper et al., 2007.) and also in the Prototype Nonhuman Primate MicroFlow PLUS Kit. Briefly, cells were washed of fixative and then simultaneously incubated at room temperature with anti-cd71-fitc, anti- CD61-PE, and RNase. Labeled cells were then washed one time with a kitprovided 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 and Company). 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 photomultiplier 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 (v3.3, BD-Immunocytometry Systems, San Jose, CA). The default stop mode was set to score 20,000 CD71-expressing RETs per blood sample for the presence or absence of micronuclei, except in the case of the samples obtained during the approach to steady state (experiment described below). In this case, it was important to obtain the data promptly to determine when a steady state had been achieved; therefore, the nonterminal day specimens (days 0 8) from this experiment were analyzed until either 10,000 RETs or 25 MN-RETs had been scored. The numbers of mature erythrocytes (normochromatic erythrocytes), with and without micronuclei, were also determined. This provided a means for calculating the percentages of RETs and micronucleated normochromatic erythrocytes (MN-NCEs), measures of bone marrow toxicity, and splenic filtration function, respectively. MN-RET scoring by microscopy. Blood and bone marrow smears were scored using the standard scoring technique for AO-stained samples at the FDA National Center for Toxicological Research laboratory as described previously (MacGregor et al., 2006). RET frequencies were determined by scoring 500 or 1000 total erythrocytes per bone marrow or blood sample, respectively. MN- RET incidence was determined by scoring 2000 RETs per sample. Micronuclei were defined by the criteria of Schmid (1975) 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. Spontaneous micronucleus frequency in control animals. Peripheral blood samples were obtained from six untreated female and six untreated male animals under ketamine sedation (10 mg/ml intramuscularly [IM]) to obtain normal reference values. The samples were scored for micronucleus frequency by flow cytometry. Kinetics of appearance and disappearance of micronuclei in peripheral blood following acute treatment. Three female monkeys received a single iv dose of CP (5 mg/kg) on day 0. Each animal was sedated with ketamine (10 mg/kg IM) for physical examination and collection of blood for complete blood count and serum biochemistry to verify health status prior to this treatment. Blood (2 ml heparinized) was collected immediately prior to treatment, and each dayfor7daysfollowing,within1hofthetimeofdosing.themonkeyswere not sedated for blood collection following administration of CP. Flow cytometry was used to assess the time course of micronucleus formation and clearance. Steady-state comparison of peripheral blood and bone marrow micronucleus counts. Six female monkeys were used for the steady-state study. Three monkeys were treated with CP (5 mg/kg iv daily) and three were untreated controls. Blood specimens were analyzed for MN-RET frequency using the flow cytometry approach in order to assess whether a steady-state condition had been reached. For this experiment, the in-life period was terminated 24 h after the last (ninth) injection. At that time, 1.0 ml heparinized blood was collected per animal, and the monkeys were sacrificed with an overdose of pentobarbital for collection of bone marrow. Terminal bone marrow and peripheral blood samples were scored for MN-RET incidence by both microscopy (bone marrow and peripheral blood) and by the flow cytometric method (peripheral blood). Statistical analysis. The incidences of MN-RETs, MN-NCEs, and RETs are expressed as frequency percent. Means and SEM calculations were made with Excel (Office X for Mac; Microsoft Corp., Seattle, Washington). Sexrelated differences were evaluated by two-sided t-tests (Excel) after log transformation to obtain normally distributed data, with significance indicated

3 354 HOTCHKISS ET AL. by p < Spearman coefficients (r s ) were calculated to evaluate the degree of correlation between MN-RET frequencies measured between scoring methods and also between tissues (JMP for Macintosh, v5). RESULTS Assay Optimization Anti-human antibodies that cross-reacted with rhesus monkey blood cells and that provided good fluorescence resolution of RETs (anti-cd71 positive) and also platelets (anti-cd61 positive) were identified, and the optimal labeling incubation conditions were determined. One notable modification to the standard rodent MicroFlow methodology was made: incubation was at room temperature, rather than 4 C. This modification improved anti-cd71 labeling. This is likely attributable to the requirement for this reagent to penetrate fixed cells as the CD71-associated epitope is located on the cytoplasmic membrane s inner leaflet. With this modification in place, good fluorescent resolution between MN-RET and other blood cell populations was achieved (see Fig. 1). Spontaneous MN-RET Frequencies in Peripheral Blood and Bone Marrow The mean incidence of MN-RET measured by flow cytometry in healthy, untreated monkeys was not significantly different between the sexes (six per group): 0.12 ± 0.037% for females and 0.15 ± 0.041% for males ( p ¼ ). These values were similar to those reported previously in rats and mice (MacGregor et al., 2006; Torous et al., 2005). The frequencies of MN-NCE were two orders of magnitude lower than MN-RET, reflecting this species robust splenic selection against circulated MN-containing erythrocytes. Interestingly, MN-NCE values were slightly higher in females compared to males ( ± % vs ± %; p ¼ 0.035). In three female control animals sacrificed as part of the steady-state experiment described below, the mean spontaneous %MN-RET frequency (± SEM) in bone marrow was 0.25 ± % (determined by microscopic scoring). In these same animals, the MN-RET frequency in blood was determined to be 0.12 ± % by flow cytometric analysis and ± % by microscopic scoring. The lower values in peripheral blood reflect splenic removal of micronucleated cells. CP Bolus A single treatment with CP at 5 mg/kg resulted in highly elevated peripheral blood MN-RET frequencies that peaked at 2 days after injection (Fig. 2a). Whereas some variation to absolute MN-RET frequencies was evident, the effect expressed as fold increase were quite consistent across these three female animals, ranging from 9.4- to 9.6-fold over pretreatment values. The elevated frequencies returned baseline values by days 4 5. The frequency of RETs was initially reduced by the treatment, reflecting bone marrow toxicity, and then rebounded during posttreatment days 5 7 (Fig. 2b). It is possible that the particularly large increase in %RETs observed for individual R21 is related to this animal s health status as a diagnosis of endometriosis (which causes blood loss associated with a compensatory reticulocytosis) occurred at the conclusion of this experiment. FIG. 1. Bivariate graphs illustrate the fluorescence signals used to resolve CD71-positive erythrocytes (young RETs, y-axis), and propidium iodide positive events (malaria-infected or micronucleus-containing erythrocytes, x-axis). Note that only gated events are plotted, meaning they had to meet three criteria: light scatter characteristics of single cells, sub-2n DNA content, and negative against an antiplatelet antibody. Panel (a), the prevalence and uniform propidium iodide signal of malaria-infected erythrocytes (arrow) facilitates consistent instrument set up. Panel (b), representative blood specimen from a negative control rhesus monkey; note the rarity of MN-RET events, upper right quadrant. Panel (c), representative blood from a CP-treated monkey; note the elevated incidence of MN-RET.

4 MICRONUCLEI IN BLOOD OF RHESUS MONKEYS 355 FIG. 2. Frequency of peripheral blood MN-RETs (panel a) and RETs (panel b) is graphed as a function of time following a single injection with CP. These measurements were acquired by flow cytometry. CP Repeat Treatment Daily treatment with CP resulted in a sharp increase in MN- RET that increased less rapidly after the third day of treatment (see Fig. 3a). Because %RETs was observed to drop significantly in two of the monkeys by the seventh day (see Fig. 3b), a decision was made to terminate the experiment and collect blood and bone marrow analysis 24 h following the ninth injection rather than risk loss of the experiment due to excessive bone marrow toxicity by continuing treatment in an attempt to achieve a better steady-state condition. Thus, although animals were treated on a daily basis, a true steady state was not obtained. Even so, we judged the observed values to be sufficiently stable to allow comparison of %MN-RET frequencies in blood and bone marrow and thereby assess whether similar increases over control values were observed in these different tissues. For terminal day (approximately steady state) blood specimens, there was very close agreement between the %MN-RET values obtained by the manual and flow cytometric scoring (e.g., the mean MN-RET value for CP-treated monkeys was 2.9% by microscopy and 3.1% for flow cytometry). As seen in Figure 4, bone marrow MN-RET values scored by microscopy were consistently higher than those obtained for peripheral blood specimens at the terminal time point. This is most obvious for the CP-treated animals, whose MN-RET frequencies were on average approximately four times higher than corresponding blood values. Nonetheless, CP-treated FIG. 3. Frequency of peripheral blood MN-RETs (panel a) and RETs (panel b) is graphed as a function of time for each of three monkeys undergoing daily treatment with CP. Data from three untreated monkeys (open symbols) are also graphed. These measurements were acquired by flow cytometry. animals exhibited markedly elevated blood MN-RET values ( %, compared to a mean baseline of 0.12%). Furthermore, these measurements closely reflected those observed in the bone marrow compartment, with a Spearman correlation coefficient of , see Table 1). Individual monkey MN-RET data can be found in Table 2. DISCUSSION Spontaneous MN-NCE frequencies were two orders of magnitude lower than MN-RET frequencies in untreated monkeys, and the absolute frequencies of MN-RET observed in peripheral blood were consistently lower than those found in the bone marrow of the same animal. These observations are consistent with the published report of Zuniga et al. (1996) that splenic removal of micronucleated erythrocytes is robust in rhesus monkeys, as it is in humans (Schlegel et al., 1986). As MN-RET frequencies in peripheral blood of untreated as well as repeat-treated animals were approximately 1/4 of the value found in bone marrow, it appears that the reticuloendothelial system removes spontaneous and CP-induced MN-RET with a similar efficiency, leading to similar fold-increases in both bone marrow and peripheral blood compartments. Splenic selection is greater than that exhibited by Sprague-Dawley rats, in which the MN-RET frequency in peripheral blood was approximately 50% of that in bone marrow during treatment with CP at steady state (MacGregor et al., 2006).

5 356 HOTCHKISS ET AL. TABLE 1 Spearman Correlation Coefficients for MN-RET Measurements Comparison Bone marrow via microscopy vs. blood via microscopy Bone marrow via microscopy vs. blood via flow cytometry Blood via microscopy vs. blood via flow cytometry Correlation coefficient FIG. 4. Frequency of MN-RETs after repeated treatment to achieve an approximate steady-state frequency of MN-RETs in bone marrow and blood. Upper panel, frequency of MN-RETs in bone marrow and blood. Lower panel, expanded scale to allow comparison of relative increases in blood with those observed in bone marrow (upper panel) and to show the greatly reduced micronucleus frequency in the fully matured erythrocytes (normochromatic erythrocytes) due to splenic selection. The response to CP measured by flow cytometry paralleled the response measured manually, both in the peripheral blood and in the bone marrow. The sensitivity of the response of the monkey to CP is similar to that observed with rats and mice (MacGregor et al., 1980, 2006) with increased frequencies MN-RETs approximately 10-fold over the pretreatment values at a dose that causes minimal suppression of the RET frequency. 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 posttreatment. In contrast, in marmosets (a New World primate), treatment with CP resulted in a fivefold increase in MN-RET which peaked at 72 h and remained elevated at 120 h (Zuniga-Gonzalez et al., 2005). Although additional data with other compounds, including aneugenic agents, are obviously desirable, the clearly detectable responses we observed with CP, and the comparability of these values with those observed in the bone marrow and blood of other species, suggest that the sensitivity of detection of responses to other clastogenic agents will be similar. Note that we attempted in several pilot studies to biopsy bone marrow by aspirating marrow from the iliac crest or head of the humerus for MN-RET scoring (needle biopsies), hoping to facilitate microscopic bone marrow analysis without the necessity of sacrificing animals. This was unsuccessful due to variable contamination of the samples with peripheral blood (data not shown). Furthermore, sufficient marrow for analysis could not be obtained from surgical bone biopsies of the iliac crest. Thus, we concluded that nonsacrificial biopsy of bone marrow was not a viable alternative to analysis of peripheral blood in the living animal. Two characteristics of monkey blood specimens make this tissue prohibitively laborious for routine microscopic evaluation: low RET counts and the low frequency of MN-RETs. Automation with flow cytometry makes it feasible to score sufficient RETs for reliable estimation of MN-RET frequency without excessive time/labor requirements. Furthermore, the methodology described herein incorporates an instrument calibration standard, and the flow cytometric measurement eliminates the subjectivity of microscopic inspection. Our data demonstrate the feasibility of monitoring chromosomal damage during the course of toxicology studies in nonhuman TABLE 2 Terminal Day MN-RET Values (%) for Individual Rhesus Monkeys Treated for Eight Consecutive Days with CP Animal ID Bone marrow (microscopy) Blood (microscopy) Blood (flow) Control V R28T R Treated R E

6 MICRONUCLEI IN BLOOD OF RHESUS MONKEYS 357 primates, using the frequency of MN-RETs in peripheral blood as an index of chromosomal breakage in bone marrow. The flow cytometric procedure makes it possible to conduct this analysis relatively noninvasively, using microliter quantities of peripheral blood (up to 3 cc of blood were collected in our studies, but as little as 60 ll is sufficient for the flow cytometric analysis of MN-RET). Measurement in nonhuman primates may be particularly important in assessing the toxicity of compounds that target species-specific receptors, or in cases where metabolism and/or distribution of active metabolites are most appropriately modeled in nonhuman primate species, thereby allowing one to consider the resulting genotoxicity data in the context of appropriate metabolism, pharmacokinetic, and target organ information. FUNDING FDA Office of Science and Health Coordination (Intercenter research award to J.T.M.). 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