Mutagenesis Advance Access originally published online on December 19, 2005
Mutagenesis 2006 21(1):15-20; doi:10.1093/mutage/gei068
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Practical threshold for micronucleated reticulocyte induction observed for low doses of mitomycin C, Ara-C and colchicine
Toxicological Research Center, Nitto Denko Corporation, 1-1-2, Shimohozumi, Ibaraki Osaka 567-8680, Japan, 1Litron Laboratories, 200 Canal View Boulevard, Rochester, NY 14623, USA, 2Division of Safety Information on Drug, Food and Chemicals and 3Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
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Abstract |
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Micronucleus induction was studied for the DNA target clastogens mitomycin C (MMC) and 1-ß-D-arabinofuranosylcytosine (Ara-C), and also the non-DNA target aneugen colchicine (COL) in order to evaluate the dose–response relationship at very low dose levels. The acridine orange (AO) supravital staining method was used for microscopy and the anti-CD71-FITC based method was used for flow cytometric analysis. In the AO method, 2000 reticulocytes were analysed as commonly advised, but in the flow cytometric method, 2000, 20 000, 200 000 and 1 000 000 reticulocytes were analysed for each sample to increase the detecting power (i.e. sensitivity) of the assay. The present data show that increasing the number of cells scored increases the statistical power of the assay when the cell was considered as a statistical unit. Even so, statistically significant differences from respective vehicle controls were not observed at the lowest dose level for MMC and Ara-C, or the lower four dose levels for COL, even after one million cells were analysed. When the animal was considered as a statistical unit, only the top dose group for each chemical showed significant increase of micronucleated reticulocytes frequency. As non-linear dose–response curves were obtained for each of the three chemicals studied, these observations provide evidence for the existence of a practical threshold for the DNA target clastogens as well as the non-DNA target aneugen studied.
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Introduction |
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As chemical safety evaluations are performed, the existence of a threshold is an important issue when considering genotoxic carcinogens. There are two important threshold concepts, i.e. ‘absolute’ threshold and ‘practical or biological’ threshold in genotoxicity. The ‘absolute’ threshold is defined as a concentration below which a cell would not ‘notice’ the presence of the chemical, and the ‘practical or biological’ one is considered as a concentration below which any effect is biologically unimportant (1
–3). Some chemicals clearly exhibit a threshold, and non-DNA target mechanisms of action (e.g. spindle apparatus disturbance, topoisomerase II inhibition, DNA synthesis inhibition, overloading homeostatic defence, and physiological perturbation) provide rationale for the non-linear responses that are observed (4). For instance, the spindle poison, colchicine (COL), damages spindle fibres, but the effect on chromosome movement should be detected only at the concentration that damages enough microtubules to impair the anchorage of the chromosome. This mechanism is thought to explain the threshold that is observed for this particular non-DNA target chemical (5). Up to the present, a widely held view is that genotoxic carcinogens do not have a threshold, and thus it has been difficult to determine the acceptable daily intake (ADI) safety exposure level. For this reason, such chemicals have been banned for use in daily life, most notably in food and food-related chemicals. Recently, however, discussion on the strategy for evaluating genotoxicity for risk assessment has been initiated (6–8). Moreover, in many cases and especially in the European Union, the principle of reducing exposure to unavoidable toxic compounds to levels that are as-low-as-reasonably-achievable (ALARA) has been advocated (9–11).
Adding to this complex discussion are reports by Fukushima and his group (12–14) who have demonstrated the existence of practical thresholds for the genotoxic hepatocarcinogens 2-amino-3, 8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) and N-nitrosodiethylamine, and even hormesis for phenobarbital. These investigators studied carcinogenicity, glutathione S-transferase placental form (GSTP) positive focus, gene mutation, DNA oxidative damage and DNA adduct formation at very low dose levels. They showed GSTP positive focus induction at the dose level at which carcinogenicity could not be detected; gene mutation could be observed at the dose level at which GSTP positive foci could not be detected, and so on. Therefore, they concluded that at least practically, a threshold for carcinogenicity existed. It has been claimed that one of the shortcomings of their proposal is the lack of discussion of the sensitivity of the assays they performed, because, from a statistical view point, the power of the assay (i.e. sensitivity) largely depends on the number of cells analysed.
The micronucleus assay has been widely used for evaluating chemical genotoxicity in vivo. One of the characteristics of the rodent peripheral blood micronucleus assay is its simple endpoint, i.e. a small DNA containing cell inclusion in the cytoplasm of enucleated erythrocytes. Because of this simplicity, automation of analysis has been achieved by image analysis (15–17) and flow cytometry (18–23). We have developed a high performance manual method using acridine orange (AO) supravital staining (24,25). We have also developed a flow cytometric method utilizing an erythrocyte surface antigen for CD71 to identify young erythrocytes and the use of malaria infected erythrocytes as an instrument calibration standard for accurate measurement (21–23,26). In the present study, we aimed to show the dose–effect relationship of micronucleus inducers with different modes of action at extremely low dose levels. We applied two methods, i.e. the manual AO supravital staining method and flow cytometry, on three model chemicals: mitomycin C (MMC), which is a cross-linking agent and typical micronucleus inducer (27) frequently used as a positive control in the micronucleus assay; 1-ß-D-arabinofuranosylcytosine (Ara-C), which is a long-patch repair inhibitor and known inducer of small micronuclei (28); and COL, a spindle poison that induces large micronuclei (29). Another aim was to evaluate the degree to which the statistical power of the assay depends on the number of target cells interrogated.
Further impetus to study low dose effects came from reports by Grawé et al. (30) and Abramsson-Zetterberg (31), who measured genotoxicant-induced micronuclei using an alternate, dual laser flow cytometric technique. We discuss the interpretation of very weak micronucleus induction, if any, at low dose levels and also the existence of a threshold effect.
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Materials and methods |
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Chemical substances
Three micronucleus inducers with different modes of action, MMC (CAS number: 50-07-7), Ara-C (CAS number: 147-94-4), and COL (CAS number: 64-86-8) were obtained from Kyowa Hakko (Tokyo, Japan), Merck (PA, USA) and Sigma (Mo, USA), respectively. MMC was dissolved with sterile distilled water and COL and Ara-C were dissolved with physiological saline. Solvents were used as negative controls.
AO pre-coated slides for supravital staining were obtained from Toyobo Co., Osaka. Fixative, anticoagulant and other all materials necessary for diluting, fixing and shipping specimens for flow cytometric analysis were from Mouse MicroFlow® Basic Kits (transported from Litron Laboratories, NY).
Animals
Healthy seven-week-old male CD-1 mice (ICR, Charles River Japan Inc., Hino, Japan) with a mean body weight of 34.6 g were used after a week's acclimation. The mice were housed at 21 ± 1°C and 55 ± 10% relative humidity and exposed to 12-h light-dark cycle. Five mice per group were assigned randomly and were given commercial food pellets (CE-2, CLEA Japan Inc., Tokyo Japan) and tap water ad libitum throughout the acclimation and the experimental period.
Micronucleus assay
The highest dose level of each chemical was determined experimentally or by referring to the published data (27,32–34). The objective was to choose a high dose level that induced micronuclei slightly, but statistically significantly. We selected 0.3 mg/kg for MMC, 6 mg/kg for Ara-C, and 0.8 mg/kg for COL as the high dose levels. The six dose levels for MMC and five dose levels for Ara-C and COL were spaced by the square root of 10. A solvent control was assigned to each experiment as reference. Each chemical was delivered intraperitoneally (10 ml/kg) once and blood was collected 48 h after treatment based on data from a previous paper (25). Treatment, sample preparation and AO scoring were performed at Nitto Denko, Osaka, Japan and fixed blood samples were sent by air on dry ice to Litron Laboratories, NY for flow cytometric analysis.
All AO supravital staining slides were coded and analysed without knowledge of treatment information. All tubes containing fixed blood cells for flow cytometric analysis were also coded. All codes were broken only after analysis was completed.
AO supravital staining micronucleus assay
The AO supravital staining micronucleus assay was performed according to the method of Hayashi et al. (24,25). Aliquots of 5 µl of peripheral blood, obtained from the tail blood vessel of each mouse, was put on an AO coated glass slide, and immediately covered with a glass coverslip. Two thousand reticulocytes were analysed by fluorescence microscopy (Model:AHBT3-RFC, Olympus, Tokyo Japan) with blue-excitation filter set, and the number of micronucleated reticulocytes (MNRET) was scored.
Flow cytometric analysis
At the same time of AO supravital sampling, another 100 µl blood was collected via orbital sinus into the Mouse MicroFlow® basic kit-supplied anticoagulant solution using a cleaned glass capillary. Each sample was fixed in duplicate with ultracold (–80°C) methanol, agitated vigorously and immediately placed at –80°C until shipment on dry ice from Nitto Denko to Litron Laboratories for flow cytometric analysis.
On the day of analysis, samples were washed out of fixative with 
12 ml Hank's Balanced Salt Solution (HBSS). A high-density/CD71-associated fluorescence thresholding technique was used (35,36). Briefly, with this method, 80 µl of each washed cell pellet was added to polypropylene tubes containing 80 µl RNase/antibodies (1.0 ml HBSS, 10 µl anti-CD71-FITC, 5 µl anti-CD61-PE and RNase A at 5 mg/ml). Antibodies, and all other flow cytometry reagents, including fixed malaria-infected rodent blood (malaria biostandard), were from Mouse MicroFlowPLUS® Kits (available from Litron Laboratories, Rochester, NY, and BD Biosciences-Pharmingen).
Following successive 30-min incubations at 4°C and 37°C, the cells were placed at 4°C until analysis (same day). For analysis, each sample was resuspended in approximately 1.5 ml propidium iodide (PI) staining solution. Of the stained blood sample 100 µl was transferred to a separate tube containing 400 µl PI. This diluted sample was used to determine the percentages of reticulocytes and micronucleated normochromatic erythrocyte (MN-NCE) of each blood sample by the analysis of 1 000 000 (1 M) total erythrocytes.
The corresponding undiluted sample was then analysed using the CD71-thresholding technique whereby CD71-negative erythrocytes (the majority of the cells) were omitted from acquisition (35,36). The frequency of micronuclei was then measured for each sample using each of the following stop modes: 2000 (2 K), 18 000 (18 K), 180 000 (180 K) and 800 000 (800 K) reticulocytes. By adding the successive values, percentage of MNRET frequencies could be calculated based on the analysis of 2 K, 20 000 (20 K), 200 000 (200 K) and 1 M reticulocytes.
Statistical analysis
P-values for each comparison with respective controls were calculated by Fisher's exact method. For the flow cytometry data based on 20 K or more cells analysed, a Student's t-test was used after normality of the data was confirmed. When determining statistical significance, a Bonferroni correction was used to adjust for the multiple (i.e. 5) comparisons made.
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Results |
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The group means of five mice/group are summarized in Table I for the AO supravital staining method based on the observation of 2 K reticulocytes and for the flow cytometric method based on the observation of 2, 20 and 200 K, and 1 M reticulocytes. The P-values for all three chemicals were <0.01 at the highest dose group when the Fisher's exact test was applied. However, when considering individual differences, this was not the case for all COL high dose datasets when evaluated using the Student's t-test. Dose-response relationship curves of MMC, Ara-C and COL are shown in Figure 1. Dose-response curves of each chemical were similar between AO supravital analysis and flow cytometric analysis, although there was a tendency for the absolute values of induced MNRET to be higher by the AO supravital method than by the flow cytometric method, especially at higher dose levels.
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It is evident that the variation among mice in each dose group decreased depending on the number of cells analysed. Even so, individual differences among animals in each group were observed even when 1 M reticulocytes were scored per sample. Likewise, the smoothness of the dose–response curves tended to increase as the number of cells analysed increased. As a representative example, Figure 2 shows individual scattergrams of MMC at 2, 20 and 200 K, and 1 M reticulocytes analysed. There are not, however, essential differences among results based on the number of cells analysed.
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Discussion |
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According to the present data, MNRET frequencies obtained using the AO supravital staining method tended to be higher than those by flow cytometric analysis. This phenomenon may be explained by modest differences in the age cohort of reticulocytes analysed by each method, i.e. AO supravital staining, where the analysis is restricted to Types I, II and III reticulocytes, and the flow cytometric method, where the analysis was restricted to reticulocytes with the CD71-positive phenotype. In both analysis procedures, the method of defining reticulocytes was kept consistent for all samples.
For flow cytometry, our data show that 20 and 200 K were sufficient to obtain reliable data for the evaluation of micronucleus induction. While flow cytometric data associated with analysis of 2 K reticulocytes were slightly more variable than corresponding microscopy-based measurements, automated acquisition of 20 K reticulocytes yielded essentially equivalent power of detection compared to the AO method. When 200 K and 1 M reticulocytes were analysed per specimen, assay sensitivity was observed to improve significantly, as evidenced by the lower doses at which statistical significance was noted when evaluated by Fisher's exact test. This was true when cells were the statistical unit evaluated, but not when individual mice were considered to be the unit. This issue is discussed in a companion paper wherein data from a reconstruction experiment are presented (37). In that study it was observed that when 200 K or 1 M cells per sample were analysed, the scoring error decreased and converged to a respective value. However, in the present study, the differences among the individual animals became apparent and there was more data variability within each dose group.
Grawé et al. (30) reported low dose effects on MMC, diepoxybutane, cyclophosphamide and COL using flow cytometry (stained with Hoechst 33342 for DNA and thiazole orange for RNA). Generally, one animal per dose level was used and 200 K polychromatic erythrocytes were analysed. Our present data agree with the COL data showing a non-linear dose–response, but in contrast to our present results, they showed linear dose–response relationships for extremely low dose levels of MMC, 0.007 mg/kg; diepoxybutane, 0.44 mg/kg; and cyclophosphamide, 0.3 mg/kg. Abramsson-Zetterberg et al. (31) also showed linear dose–response curves for acrylamide down to very low dose levels (1 mg/kg body wt) and observed no threshold. Although we did not evaluate acrylamide as a model chemical in this study, we did not observe linear dose–response curves, even for MMC and Ara-C, which are DNA-reactive clastogens. We could not find any rationale to explain such differences at the present time, and believe it is necessary to continue studying chemicals that interact directly with DNA to better understand their effect at extremely low dose levels.
To confirm biological and statistical relevance of the present study data, we performed a reconstruction model experiment using serial dilutions of malaria-infected blood with non-treated mouse blood (37). The samples were analysed by flow cytometry based on 2, 20 and 200 K (Experiment 1) and up to 1 M (Experiment 2) cells. These data show extremely high performance of the flow cytometric assay in terms of accuracy, especially when 200 K or more reticulocytes are evaluated per specimen. This result shows that the statistical power of the assay depended on the number of analysed cells. This dilution experiment supports our conclusion that thresholds were present for micronucleus induction in reticulocytes for the three model chemicals analysed at very low dose levels.
It might be difficult to prove the existence of thresholds in toxicology or biology in general using statistical methods (38), and it is not easy to discuss the threshold concept from the biological viewpoint. However, when we only consider mean values, for example in the case of Ara-C, the MNRET frequencies appeared to increase linearly, but when the individual values (Table II) were evaluated, the differences among animals became clearer. Two individuals at the lowest dose group (mouse ID 3 and 5) demonstrated the lowest MNRET frequency (0.16%) even when including the vehicle control group. The control data variation in the Ara-C experiment was less than that in the MMC or COL experiments. When the Student's t-test was applied to the data obtained by flow cytometry based on the analysis of 
20 K cells, only the highest dose groups were significantly different from the concurrent control for MMC and Ara-C. Therefore, we should consider the individual animal differences to determine the micronucleus induction ability of the chemical being studied. It is likely that at very low doses of genotoxicant, as were studied here, individual differences in DNA repair activity, metabolism related cytochrome P450 activity, or anti-oxidant concentration etc. play a larger role in dictating the micronucleus incidence of each individual of an exposure group.
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In addition, Figure 3 shows the dose–response curves of these three model chemicals in a wider range of dose levels superimposed on the results of published data (27
,32,34) using the same strain of mouse and same manner of experiments by AO supravital staining. Closed circles represent the data from the present study and open circles are published data for higher dose levels. The dose–response curves became clearer by adding the data from higher dose levels and the practical threshold or the threshold were shown. Moreover, COL even shows the tendency of inhibition in induction of micronuclei at extremely low dose levels (U- or J-shape response). Hormesis usually implies increased repair capability or some other protective, adaptive response in the field of radiobiology (1). The COL data presented herein is suggestive of a hormesis-like effect, and further work aimed at elucidating the mechanisms and significance of this observation is warranted.
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Considering the data detailed above, an important conclusion is the existence of a biological or practical threshold in the genotoxicity assay on DNA target chemicals as well as non-target chemicals. Although we used only three model chemicals in the present study, we could draw the following conclusions: (i) the flow cytometric micronucleus assay method is a powerful tool when
20 K cells were analysed; (ii) the AO supravital staining micronucleus assay method and the flow cytometric method gave qualitatively similar results; (iii) when the cell is considered the statistical unit and more cells are analysed, both power and assay sensitivity at lower dose levels is significantly enhanced as evidenced by the significant differences observed when compared to vehicle control; and (iv) non-linear dose–response curves were obtained for the model chemicals studied here when evaluating the individual animal as a unit, suggesting the existence of a practical threshold for the DNA target micronucleus inducers (MMC and Ara-C) as well as the non-DNA target chemical (COL). |
Acknowledgments |
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This work supported by the Health and Labor Sciences Research Grants (H15-Food-008).
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Notes |
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* To whom correspondence should be addressed. Tel: +81 (0) 72 621 0492; Fax: +81 (0) 72 621 0315; Email: asanonri{at}nitto.co.jp
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Received on May 24, 2005; revised on October 27, 2005; accepted on November 15, 2005.
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