Litron Laboratories, 1351 Mount Hope Avenue, Rochester, New York 14620
Received March 31, 2003; accepted April 30, 2003
ABSTRACT
A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.
Key Words: micronuclei; genotoxicity; flow cytometry; rats; CD71 antigen.
The micronucleus (MN) assay (Hayashi et al., 2000; Heddle, 1973
; Schmid, 1975
) is the most widely utilized in vivo system for evaluating chemicals potential to induce chromosome breaks or to poison mitotic spindle apparatus. The test is based on the observation that replicating cells with chromatid breaks or dysfunctional mitotic apparatus exhibit disturbances in the anaphase distribution of their chromatin. After telophase, this displaced chromatin can be excluded from the nuclei of the daughter cells and is found in the cytoplasm as a micronucleus. Micronuclei therefore represent chromosome fragments or whole chromosomes resulting from clastogenic or aneugenic events. Erythrocytes are particularly well suited for evaluating MN events since erythroblast precursors are a rapidly dividing population of cells, and their nucleus is expelled a few hours after the last mitosis, making MN-associated chromatin relatively simple to detect.
The MN assay was originally devised to score chromosome damage in mouse bone marrow. MacGregor et al.(1980) demonstrated that micronuclei formed in the bone marrow of mice persist in the peripheral blood. Therefore, assay sensitivity is retained when studying gentoxicant-induced micronucleated erythrocytes in the peripheral blood of mice (CSGMT, 1992
; Hayashi et al., 1990
). However, in the area of product safety assessment, the mouse is not the preferred test species. Rather, the majority of studies are performed with rats (e.g., acute and subchronic toxicology and pharmacokinetic studies). This being the case, a rat peripheral blood-based MN test has tremendous potential for providing investigators with concurrent in vivo endpoints. The peripheral blood compartment is ideal for such integrated studies, since it can be readily sampled at any point during subchronic studies (Asanami et al., 1995
). Thus, if the MN endpoint proves to be an appropriate and sensitive index of genotoxicity in rat peripheral blood, it may be possible to significantly reduce animal usage by eliminating dedicated rodent micronucleus tests.
To date, erythrocyte-based MN studies involving the rat blood compartment have been qualified because it has been assumed that the high efficiency in which the spleen eliminates MN erythrocytes would severely limit assay sensitivity. Even so, data are accumulating which suggest that rat blood may be used effectively to study chemical-induced genotoxicity (Abramsson-Zetterberg et al., 1999; Asanami et al., 1995
; Hamada et al., 2001
; Hayashi et al., 1992
; Hynes et al., 2002
; Torous et al., 2000
; Wakata et al., 1998
). These studies have typically restricted analysis of MN to the youngest fraction of reticulocytes (RETs). The premise is that the impact of spleen function would be reduced by scoring MN in these cells. The flow cytometric (FCM) system for scoring rodent MN-RET frequency developed by this laboratory is based on anti-CD71-FITC staining of RETs (MicroFlow®, Dertinger et al., 1996
; Torous et al., 2000
). By using CD71-based fluorescence as an index of RET age, this system has the potential to focus analysis of MN in the youngest RET population (in analogy to microscopists who rely on RNA content).
Previously, Schlegel and MacGregor (1984) estimated that rat peripheral blood studies would require the analysis of six to eight times the number of RETs in comparison to bone marrow to achieve equivalent statistical power. By analyzing blood samples for the presence of MN at rates up to several thousands of cells/second, a much greater number of cells can be analyzed in comparison to microscopic scoring. Flow cytometry therefore represents a technology that allows MN-RET scoring to be restricted to certain (e.g., youngest) RET cohorts, and also allows for eightfold (or greater) numbers of cells to be scored compared to conventional methodologies. Experiments were initiated to test the hypothesis that rat peripheral blood-based MN tests would benefit from automated scoring which allows for (1) analysis to be restricted to the most immature fraction of RETs, and (2) analysis of 20,000 RETs per animal compared to the standard value of 2000. Dose response data for nine well-characterized genotoxicants is presented herein, both from microscopy- and FCM-derived measurements.
MATERIALS AND METHODS
Animals and treatment regimen.
Six-week-old male Sprague-Dawley rats were purchased from Taconic (Germantown, NY). Animals were group housed and randomly assigned to treatment groups. The animals were acclimated for one week before experiments were initiated. Food and water were available ad libitum throughout the acclimation and experimentation periods. Each treatment group consisted of five rats. The nine genotoxicants studied were evaluated in a total of four separate experiments. Each of these experiments incorporated a concurrent vehicle control. CAS numbers and choice of solvents, as well as administration/bleeding details, can be found in Table 1. The choice of nine chemicals, representing both clastogenic and aneugenic mechanisms of action, was designed to test the robustness of the system for identifying diverse classes of genotoxicants. Note that choice of solvents, dose levels, and blood harvesting times were derived from literature, especially Wakata et al., 1998
, and Abramsson-Zetterberg et al., 1999
.
|
Instrument setup with malaria-infected blood.
All flow cytometric analyses were carried out with a FACSCalibur flow cytometer (Becton Dickinson; 488 nm excitation). The CD71-FITC and PI fluorescence signals were detected in the FL1 and FL3 channels, respectively. To reproducibly calibrate the instrument for the micronucleus scoring application, this laboratory has recommended analyzing rodent erythrocytes infected with the malaria parasite Plasmodium berghei (Tometsko et al., 1993; Torous et al., 2001
). Fixed P. berghei-infected rodent cells were therefore stained in parallel with test samples on each day of analysis. By maximizing the fluorescent resolution of erythrocytes with and without malaria parasites, these MN-erythrocyte mimicking biostandards facilitated optimization of FL1 and FL3 PMT voltages. Blood rich in malaria-infected erythrocytes also guided FL3-%FL2 compensation settings, and the PI-associated fluorescent signal of single parasite-containing cells was used to set the boundary of the quadrant which differentiates erythrocytes with and without MN (see Fig. 1
).
|
|
Statistical analyses were performed with JMP Software (v5, SAS Institute, Cary, NC). For each treatment group, the mean and standard deviation for %RET and %MN-RET was calculated. ANOVA was used to assess whether there were treatment-related changes to RET and MN-RET frequencies (significance indicated by p < 0.05). Positive ANOVA results were followed by Dunnetts pair-wise tests. A trend test was also utilized to evaluate whether a dose-related increase in MN-RET had occurred. This assessment was performed for each chemical using a linear regression model. A regression effect (i.e., a dose-related trend) was indicated when p < 0.05. We considered the MN-RET data sets to exhibit evidence of genotoxicity if either a pair-wise test or the trend test was positive.
To further investigate correspondence between scoring methodologies, the mean %RET value for each treatment group as measured by FCM was plotted against the corresponding mean %RET value obtained through microscopic inspection. A linear correlation coefficient (r value) was calculated for each of the nine chemicals examined. This same analysis was performed for %MN-RET frequencies.
RESULTS
The effect of chemical treatment on RET frequency is presented in Table 2. The frequency of RETs based on AO-coated slides represents all RNA-positive erythrocytes. The frequency of RETs measured by FCM is based on the expression of high levels of CD71 (Fig. 2
). Therefore, while these measurements are closely related, the higher absolute microscopy-based values were expected. Even so, there was good agreement between scoring systems as evidenced by high linear correlation coefficient values. There were two exceptions that correspond to chemicals that produced modest (or no) change in RET levels. In the case of MNNG, it is likely that a reduction in very young RETs detected by FCM reflects an early indication of cytotoxicity. That is, we would expect the youngest cohort of RETs (high CD71 expressing erythrocytes) to be a leading indicator of cytotoxicity that the entire RNA-positive RET cohort (AO staining) will reflect at a somewhat later time point. Acetaldehyde showed no significant effect on RET levels, irrespective of scoring method. The low r value resulted from a best fit line algorithm that was based on very few data points (4), coupled with the fact that the mean %RET values plotted did not differ appreciably over the concentration range tested.
|
In addition to analyses of correlation, it was also informative to consider statistical tests that assess whether significant chemical-dependent MN formation had occurred. When criteria for a genotoxic response was defined as a significant pair-wise test or a significant trend test, then seven of nine chemicals were judged to be positive when based on microscopic inspection. When these same criteria are applied to each FCM-based data set, all nine chemicals were positive. The two discrepancies were with MNNG and benzene. These chemicals were judged positive in the report by Wakata et al.(1998), and indeed the microscopy-based data approached statistical significance in the present study. In both cases, it seems likely that it was the more accurate depiction of the dose-response curve and the lower variability associated with FCM measurements that provided for a more sensitive trend test (Table 2
and Fig. 3
).
|
The induction of DNA damage and the resulting sequelae of mutations and chromosomal rearrangements are primary mechanisms by which cancers arise (Barrett, 1993; Bishop, 1991
). These types of events have also been implicated in diseases such as atherosclerosis, and processes such as aging (Zwijsen et al., 1990
). Therefore, there is an important need for sensitive methods that are capable of identifying chemical or physical agents that can permanently alter DNA. Given the tremendous cost of long-term chronic studies such as two-year bioassays, short- and medium-term systems for predicting DNA reactivity will continue to play a vital role in carcinogen identification, as well as in lead prioritization strategies. In fact, the need for short-term tests that have a high throughput capacity has never been greater (Gollapudi and Krishna, 2000
). Advances in molecular biology and combinatorial chemistry have provided large numbers of potential targets and many novel compounds that may be useful for treating or preventing disease. This situation clearly calls for methods that are able to quickly and reliably determine toxicological profiles of compounds under consideration. In the case of in vivo micronucleus testing, flow cytometry technology may be of great assistance. For the experiments described herein, throughput was as high as 100 blood samples per day. Compared to microscopy, this represents roughly a tenfold higher rate of analysis (also consider that the FCM method evaluated tenfold more cells in this amount of time).
The greatly enhanced efficiency of FCM-based MN measurements may have implications for where in the drug development process the endpoint is first studied. As far as in vivo genotoxicity assessment, the rodent MN erythrocyte test is the most widely utilized system. Even so, it is often applied late in drug development, and often using mice. The rat has traditionally been more vigorously studied for product safety testing. There would be advantages to obtaining genotoxicity data in this species, as this would allow investigators to relate any observed effects to information regarding deposition, metabolism, and elimination. Since the peripheral blood compartment of rats is so amenable to sampling (even repeat sampling), it is conceivable that MN data could be generated in early toxicology investigations, such as during acute rat studies. This strategy could provide important information that might highlight problem chemicals early in the development process, and thereby help redirect resources to more promising leads.
Beyond confirming the appropriateness of the rat peripheral blood compartment for conducting erythrocyte-based MN analyses, the data presented herein are valuable for considering whether this endpoint may also be utilized to study chemical-induced genotoxicity in other species with robust spleen function. For instance, in regard to MN filtering, the human spleen behaves similarly to that of the rat. Therefore, data pertaining to the suitability of an FCM-based rat peripheral blood system may provide clues as to the feasibility of FCM-based human blood measurements (Abramsson-Zetterberg et al., 2000; Dertinger et al., 2002
). Experiments designed to test this scenario are in progress.
ACKNOWLEDGMENTS
This work was supported by a grant from the National Institute of Environmental Health Sciences (NIEHS; grant number R44 ES 09578-03). The contents are the sole responsibility of the authors and do not necessarily represent the official views of NIEHS. The authors would like to thank Drs. James MacGregor and Makoto Hayashi for many valuable discussions.
NOTES
1 To whom correspondence should be addressed. Fax: 585-442-0934. E-mail: sdertinger{at}litronlabs.com.
REFERENCES
Abramsson-Zetterberg, L., Grawe, J., and Zetterberg, G. (1999). The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: The response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry. Mutat. Res. 423, 113124.[ISI][Medline]
Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M., and Grawe, J. (2000). Human cytogenetic biomonitoring using flow-cytometric analysis of micronuclei in transferrin-positive immature peripheral blood reticulocytes. Environ. Mol. Mutagen. 36, 2231.[CrossRef][ISI][Medline]
Asanami, S., Shimono, K., Sawamoto, O., Kurisu, K., and Uejima, M. (1995). The suitability of rat peripheral blood in subchronic studies for the micronucleus assay. Mutat. Res. 347, 7378.[CrossRef][ISI][Medline]
Barrett, J. C. (1993). Mechanisms of multistep carcinogenesis and carcinogen risk assessment. Environ. Health Perspect. 100, 920.[ISI][Medline]
Bishop, J. M. (1991). Molecular themes in oncogenesis. Cell 64, 235248.[ISI][Medline]
CSGMT (The Collaborative Study Group for the Micronucleus Test) (1992). Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS MMS. Mutat. Res. 278, 8398.[CrossRef][ISI][Medline]
Dertinger, S., Torous, D., and Tometsko, K. (1996). Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer. Mutat. Res. 371, 283292.[ISI][Medline]
Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G., Gleason, S. E., Miller, R. K., and Tometsko, C. R. (2002). Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer. Mutat. Res. 515, 314.[ISI][Medline]
Gollapudi, B. B., and Krishna, G. (2000). Practical aspects of mutagenicity testing strategy: An industrial perspective. Mutat. Res. 455, 2128.[ISI][Medline]
Hamada, S., Sutou, S., Morita, T., Wakata, A., Asanami, S., Hosoya, S., Ozawa, S., Kondo, K., Nakajima, M., Shimada, H., et al. (2001). Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th collaborative study by the collaborative study group for the micronucleus test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)Mammalian Mutagenicity Study Group (MMS). Environ. Molec. Mutagen. 37, 93110.[CrossRef][ISI][Medline]
Hayashi, M., Kodama, Y., Awogi, T., Suzuki. T., Asita, A. O., and Sufuni, T. (1992). The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats. Mutat. Res. 278, 209213.[CrossRef][ISI][Medline]
Hayashi, M., MacGregor, J. T., Gatehouse, D. G., Adler, I.-D., Blakey, D. H., Dertinger, S. D., Krishna, G., Morita, T., Russo, A., and Sutou, S. (2000). In vivo rodent erythrocyte micronucleus assay: Aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. A report from the International Workshop on Genotoxicity Test Procedures (IWGTP). Environ. Mol. Mutagen. 35, 234252.[CrossRef][ISI][Medline]
Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, M., Jr. (1990). The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. Mutat. Res. 245, 245249.[CrossRef][ISI][Medline]
Hayashi, M., Sofuni, T., and Ishidate, M., Jr. (1983). An application of acridine orange fluorescent staining to the micronucleus test. Mutat. Res. 120, 241247.[CrossRef][ISI][Medline]
Heddle, J. (1973). A rapid in vivo test for chromosome damage. Mutat. Res. 18, 187190.[ISI][Medline]
Hynes, G. M., Torous, D. K., Tometsko, C. R., Burlinson, B., and Gatehouse, D. G. (2002). The single laser flow cytometric micronucleus test: A time course study using colchicines and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood. Mutagenesis 17, 1523.
MacGregor, J., Wehr, C., and Gould, G. (1980). Clastogen-induced micronuclei in peripheral blood erythrocytes: The basis of an improved micronucleus test. Environ. Mutagen. 2, 509514.[ISI][Medline]
Schlegel, R., and MacGregor, J. (1984). The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: Implications for cytogenetic screening. Mutat. Res. 127, 169174.[CrossRef][ISI][Medline]
Schmid, W. (1975). The micronucleus test. Mutat. Res. 31, 915.[ISI][Medline]
Tometsko, A. M., Torous, D. K., and Dertinger, S. D. (1993). Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. Mutat. Res. 292, 129135.[CrossRef][ISI][Medline]
Torous, D., Dertinger, S., Hall, N., and Tometsko, C. (2000). Enumeration of micronucleated reticulocytes in rat peripheral blood: A flow cytometric study. Mutat. Res. 465, 9199.[ISI][Medline]
Torous, D. K., Hall, N. E., Dertinger, S. D., Diehl, M. S., Illi-Love, A. H., Cederbrant, K., Sandelin, K., Bolcsfoldi, G., Ferguson, L. R., Pearson, A., et al. (2001). Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories. Environ. Mol. Mutagen. 38, 5968.[CrossRef][ISI][Medline]
Wakata, A., Miyamae, Y., Sato, S., Suzuki, T., Morita, T., Asano, N., Awogi, T., Kondo, K., and Hayashi, M. (1998). Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS.MMS. Environ. Mol. Mutagen. 32, 84100.[CrossRef][ISI][Medline]
Zwijsen, R. M. L., van Kleef, E. M., and Alink, G. M. (1990). A comparative study on the metabolic activation of 3,4-benzo(a)pyrene to mutagens by aortic smooth muscle cells of rat and rabbit. Mutat. Res. 230, 111117.[ISI][Medline]
|