Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c, DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening

Micronucleated erythrocytes are selectively removed from the peripheral circulation of normal rats. Splenectomy prevents this selective removal. In normal rats treated daily for 20 days with 0.2 mg/kg triethylenemelamine (TEM), micronucleated normochromatic (mature) erythrocytes did not accumulate in peripheral blood. In these same animals, the frequencies of micronucleated cells among polychromatic (newly formed) erythrocytes increased from 0.21 to 5.25 per thousand in peripheral blood and from 1.75 to 31.5 per thousand in bone marrow. Since both control and induced frequencies in peripheral blood were approximately 15% of those in bone marrow, the removal appears to be equally efficient for cells containing either spontaneously occurring or clastogen-induced micronuclei. In splenectomized rats treated daily for 11 days with 0.2 mg/kg TEM, the frequency of micronucleated normochromatic erythrocytes (NCEs) in the peripheral blood rose rapidly to 9 times the control value and remained elevated for 50-55 days, indicating a life span approximately equivalent to that of normal erythrocytes. Among splenectomized rats exposed to either 0.15 mg/kg triethylenemelamine, 6.5 mg/kg cyclophosphamide, or 300 mg/kg urethane for periods exceeding the erythrocyte life span, the incidences of micronucleated NCEs in the peripheral blood rose steadily from a control value of 1.0 per thousand to maximum values of 15.0, 12.7 and 8.9 per thousand, respectively. During these extended exposures, the mean frequencies of micronucleated polychromatic erythrocytes (PCEs) in peripheral blood increased from a spontaneous value of 0.9 per thousand to 23.0, 13.0 and 6.6 per thousand, respectively, reflecting the frequencies among PCEs in the bone marrow and approximating the maximum values among NCEs in the peripheral blood. Thus, the frequency of micronucleated erythrocytes in the peripheral blood of splenectomized rats can be used as an index of both acute and cumulative chromosomal damage, while in normal rats the use of peripheral blood for cytogenetic monitoring is restricted by the selective removal of these micronucleated cells.     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

The micronucleus test with mouse spleen cells

The results of this study show that the micronucleus test can be carried out with mouse spleen cells as well as with cells from bone marrow. Polychromatic erythrocytes occurred in the spleen at a frequency of about 9% of the whole spleen cells compared with about 13% in the bone marrow. 3 test compounds were used to compare the frequency of micronuclei in cells from the 2 tissues. Mitomycin C and cyclophosphamide induced micronucleated polychromatic erythrocytes in both spleen and bone marrow. Fosfomycin, an antibiotic having a broad spectrum of antimicrobial activities, did not induce micronucleated erythrocytes in either organ.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

An evaluation of the genotoxic properties of some chosen dyes using the micronucleus test in vivo

The frequency of micronucleated polychromatic erythrocytes and the polychromatic to normochromatic erythrocyte ratio was studied in BalbC mice treated with four azo dyes: Direct Blue 74, Direct Blue 296, Direct Blue 297 and Direct Green 98 at two (40and 80% LD₅₀/kg body weight) concentrations. None of the studied compounds revealed a genotoxic activity in the micronucleus test. However, it was found that two dyes, Direct Blue 297 at doses 40% and 80% LD₅₀ and Direct Green 98 at dose 80% LD₅₀, cause a significant decrease in the ratio of polychromatic to normochromatic erythrocytes in bone marrow of mice, which means that at the doses specified above they can affect the proliferation of the blood cells.     

Density-gradient enrichment of newly-formed mouse erythrocytes. Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080-1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60-80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method.

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticulocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticulocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF1 mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF1 mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment. These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.     

Transplacental mutagenesis: The micronucleus test on fetal mouse blood

Summary The induction of cytogenetic damage (micronuclei) in mouse fetal blood was studied with four selected mutagens: cyclophosphamide, procarbazine, trenimon, and mitomycin-C. For comparison the standard micronucleus test on maternal bone marrow was also performed. In contrast to the results obtained from maternal bone marrow the changes in the cellular composition in fetal blood were only slight after treatment with mutagens. A significant and dosepdependent increase in the incidence of micronucleated fetal blood cells was found with all four mutagens. The inducibility of micronuclei by indirect mutagens was particularly interesting. The three mutagens other than mitomycin-C induced a higher frequency of micronucleated polychromatic erythrocytes in fetal blood cells than in maternal bone marrow. The results indicate that this modified micronucleus test is well suited and useful for mutagenicity screening of environmental chemicals and especially for assessment of risks to the fetus when pregnant females are exposed to environmental chemicals.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticuiocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticuiocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF₁ mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF₁ mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment.These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.     

Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.     

Effect of residual splenic function and folate levels on the frequency of micronucleated red blood cells in splenectomized humans.

Frequencies of micronucleated erythrocytes in the peripheral blood of splenectomized individuals can be used as an index of genetic damage to erythrocyte precursor cells in the bone marrow. This is in contrast to non-splenectomized humans, whose micronucleated erythrocytes are removed by the spleen. Many subjects whose spleen has been removed surgically have residual spleen tissue and consequent residual spleen function (RSF), which can be measured by the percentage of 'pitted' peripheral red blood cells. In this study evidence of RSF was associated with decreased frequencies of micronucleated erythrocytes. Analysis of data limited to subjects with minimal spleen function suggested an inverse association between the incidence of micronucleated erythrocytes and serum folate levels that was not apparent in the absence of stringent control for RSF.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane.

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF1 mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF₁ mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Micronucleus test with cyclophosphamide using mouse peripheral blood reticulocytes.

The usefulness of the micronucleus test using supravital staining of peripheral blood reticulocytes with acridine orange was evaluated in two laboratories after administering cyclophosphamide (CYP) as a model chemical by intraperitoneal injection (i.p.) to CD-1 mice. The frequencies of micronucleated peripheral reticulocytes (MNRETs) increased dose-dependently at each sampling time. There were no significant differences in the results obtained with this new method by the two laboratories. Although the induction of MNRETs was delayed by about 24 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optical sampling time, 24 h for MNPCEs and 48 h for MNRETs. Therefore, it is concluded that this method is a suitable alternative to that using femoral marrow cells.     

Statistics of the mouse bone-marrow micronucleus test: counting, distribution and evaluation of results

The distribution of historical control results from male mice tested in the bone-marrow micronucleus test was used to optimize counting procedures and to develop decision rules for evaluating test results. The ratio of normochromatic to polychromatic erythrocytes followed a normal distribution, while the incidence of micronucleated polychromatic and normochromatic erythrocytes followed a binomial distribution. Recommendations for the number of cells to be scored per animal and for the evaluation of results are based on these distributions and the two-hypothesis multiple-decision approach of Selby and Olsen (1981).     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.