Chromosomal aberrations (CA) contribute to cancer development in humans and experimental animals [25
], and elevated lymphocyte CA and MN frequencies have been shown to be biomarkers of cancer risk within a population of healthy subjects [26
]. The use of MN as a surrogate for CA is supported by a number of validation studies showing a strong correlation between MN and CA frequencies within the same cell population [30
]. In experimental animals, induction of CA or MN in appropriate target cells following defined exposures is considered a biomarker of genotoxic exposure and predictive of an agent’s potential to induce cancer [31
]. Because scoring for micronucleated erythrocytes is much easier than scoring for CA in bone marrow cells, the in vivo
rodent erythrocyte MN assay is a standard component of safety assessment protocols for environmental agents, industrial and commercial chemicals, and pharmaceuticals (i.e., it is part of the genotoxicity test battery required by regulatory agencies world-wide [34
]). The NTP includes the in vivo
rodent erythrocyte MN assay as part of its standard genetic toxicology test battery used in the comprehensive toxicological evaluation of substances of public health concern.
The peripheral blood erythrocyte MN assay has advantages over the bone marrow assay because it permits serial sampling of animals over an extended exposure period, permits assessment of the effects of chronic dosing in animals without splenic scavenging, and allows the assay to be combined with other toxicity evaluations. The mouse model has been extensively validated by international collaborative studies [6
]. FCM methods have recently emerged as powerful tools for assaying MN in peripheral blood erythrocytes of mice. Studies comparing the traditional slide-based assay to the FCM-based assay have shown the FCM-based assay to be a robust method characterized by high reproducibility and rapid, objective scoring [9
]. FCM methods can be used to score over a million cells per animal, if needed, and FCM methods can accurately distinguish subpopulations of erythrocytes so that appropriate target populations can be interrogated for MN based on the specific exposure protocol employed. Distinguishing RET from mature erythrocytes by microscopy relies on the staining of residual RNA, which can be somewhat subjective, while FCM objectively identifies RET by the presence of the CD71 cell surface marker [9
In rats, MN have traditionally been evaluated in bone marrow RET due to the rapid and efficient removal of MN-RET from the peripheral blood by the rat spleen [35
]. However, results of recent studies combined with accumulated published data have suggested that the FCM-based peripheral blood MN assay can be used in place of the slide-based bone marrow assay in rats if analysis is restricted to the most highly expressing CD71 RET (very young RET), and if an adequate number of cells is sampled per animal [12
]. Data from the tests conducted with rats in this study revealed generally higher frequencies of MN-RET detected in bone marrow slide preparations than by FCM analysis of peripheral blood (–). This observation is consistent with highly efficient splenic scavenging, even though FCM analysis was restricted to the most highly expressing CD71 subpopulation of erythrocytes. Although frequencies of MN-RET in rats were lower in FCM-analyzed blood samples, the magnitude of the increases induced by the genotoxic chemicals was generally similar in bone marrow and blood (–). The one exception was the CP study in rats, where analysis of BM slides showed a 50-fold increase in MN-RET while FCM analysis of peripheral blood samples showed only a 6-fold increase over baseline at the 10 mg/kg dose level ().
In general, good agreement was seen in both rats and mice between peripheral blood MN-RET frequencies obtained by microscopy-based and FCM-based methods for both the nongenotoxic and genotoxic compounds (). In addition, for the genotoxic compounds, the dose response curves in both rats and mice showed a high degree of correspondence between microscopy- and FCM-based MN-RET determinations (–). Thus, the results obtained from peripheral blood samples analyzed either by FCM or microscopy are comparable.
While microscopy-based and FCM-based assessments of MN-RET frequencies in peripheral blood were in close agreement, some significant differences were observed between MN-RET frequencies in bone marrow slide preparations and in blood samples analyzed by FCM (). In general, the actual counts obtained by scoring bone marrow slides were higher than those obtained through FCM analysis of peripheral blood. Because FCM and microscopic analysis of blood provided similar actual counts, the difference in slide-based counts of bone marrow and FCM-based counts of blood appears to reflect a true biological difference between bone marrow and peripheral blood rather than a difference related to scoring procedure. Furthermore, the difference is not unexpected, particularly in the rats, where the differences between the two scoring methods were most pronounced (). Despite these differences in absolute counts, the magnitude of the responses (fold-increases) observed with the genotoxic compounds between bone marrow slide evaluation and FCM-based blood evaluation were similar and significant increases were detected at the same dose levels across all three methods (bone marrow slides, blood slides, blood FCM). In fact, for vincristine sulfate in rats, FCM analysis of blood detected a significant rise in MN-RET frequency at a lower dose than did manual scoring of bone marrow or peripheral blood slides (). Thus, FCM scoring did not miss any of the genotoxic compounds in rats or mice, and in fact, it correctly identified the negative result with acrylamide in rats (comparison with the “gold standard” bone marrow evaluation method of MN frequency in rats). These data indicate that the NTP would not compromise its hazard identification capability by using FCM methods to evaluate MN frequency.
To gain insight into mechanism of MN formation, 100 MN-RET from each animal in each treatment group for each of the four genotoxic agents were analyzed for PI-associated fluorescence intensity, providing a quantitative description of DNA content within the MN-RET [16
]. Only MN induced by vincristine sulfate showed an upward shift in distribution of PI-associated fluorescence intensity in rats and in mice, indicating an increased fraction of MN containing larger amounts of DNA, and therefore, whole chromosomes rather than fragments (). These results are consistent with vincristine’s aneugenic mechanism of action. The three other genotoxic chemicals are known clastogens. Because these data are consistent with the primary modes of genotoxicity for each of these four agents, we recommend evaluating this endpoint for all compounds that show a positive response in the MN assay.
Some investigators have expressed concern about the ability to detect MN induced by aneugenic compounds in rat blood samples because the rat spleen preferentially removes RETs with larger sized MN [39
]. In the studies reported here, the induction of MN by the aneugenic compound vincristine sulfate was clearly detected in blood of both rats and mice, and median channel fluorescence data showed a clear “signature” of aneugenic potential. Thus, our protocol appears to be compatible with detection of both aneugens and clastogens.
Due to the large number of cells that can be routinely analyzed, the FCM-based assay has a higher power of detection relative to traditional microscopy-based methods. The relationship between sample size (ranging from 2000 to 1 × 106
cells per animal) and statistical power of the FCM-based peripheral blood MN assay was recently examined [23
]. Based on their analysis, the authors recommended an ideal sample size of 20,000 RET per animal. Tests reported here with the nongenotoxic compounds were conducted prior to this analysis, and thus, for these five compounds, only 10,000 RET were analyzed per mouse by FCM (still a reasonable number of cells with good statistical sensitivity). For the MN tests with the four genotoxic compounds, conducted at a later date, 20,000 RET were analyzed per blood sample. Overall, the sensitivity of the FCM-based MN assay used with the compounds tested here (clearly genotoxic or nongenotoxic compounds) was not enhanced compared to microscopy-based enumeration of MN-RET. However, because the FCM MN assay has the ability to interrogate very large numbers of cells, even stringent and conservative statistical analyses will at times be expected to detect very small increases in MN-RET (<0.1%) as significant. Thus, it will be important to include experimental reproducibility among the criteria used to evaluate biological relevance.
In conclusion, this study extends previous validation studies of the FCM-based MN assay in mice and rats. FCM-based enumeration of MN-RET has several distinct advantages over microscopy-based scoring and these are supported by the present results as well as by the published data from studies conducted by other laboratories. Based on these results, the NTP has begun to use the in vivo FCM-based rodent peripheral blood MN assay as a standard part of its efforts to evaluate the genetic toxicity of substances of public health concern.