Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans

The frequency of micronuclei (also known as Howell-Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen's effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71-positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space.     

Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans

The frequency of micronuclei (also known as Howell–Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen’s effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71-positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space.     

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.     

Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes

Transgenic mice that differed in their p53 genetic status were exposed to an acute dose of highly charged and energetic (HZE) iron particle radiation. Micronuclei (MN) in two distinct populations of circulating peripheral blood erythrocytes, the immature reticulocytes (RETs) and the mature normochromatic erythrocytes (NCEs), were measured using a simple and efficient flow cytometric procedure. Our results show significant elevation in the frequency of micronucleated RETs (%MN-RETs) at 2 and 3 days post-radiation. At 3 days post-irradiation, the magnitude of the radiation-induced MN-RET was 2.3-fold higher in the irradiated p53 wild-type animals compared to the unirradiated controls, 2.5-fold higher in the p53 hemizygotes and 4.3-fold higher in the p53 nullizygotes. The persistence of this radiation-induced elevation of MN-RETs is dependent on the p53 genetic background of the animal. In the p53 wild-type and p53 hemizygotes, %MN-RETs returned to control levels by 9 days post-radiation. However, elevated levels of %MN-RETs in p53 nullizygous mice persisted beyond 56 days post-radiation. We also observed elevated MN-NCEs in the peripheral circulation after radiation, but the changes in radiation-induced levels of MN-NCEs appear dampened compared to those of the MN-RETs for all three strains of animals. These results suggest that the lack of p53 gene function may play a role in the iron particle radiation-induced genomic instability in stem cell populations in the hematopoietic system.     

Genetics of leukocyte telomere length and its role in atherosclerosis

Malnutrition is a serious public health problem that affects approximately one third of all children. Developing countries have the highest incidence of malnourished children, and approximately 60% of deaths that occur in children under five are directly related to malnutrition and associated diseases. The relationship between malnutrition and genetic damage has been widely studied in humans and animal models. The micronucleus (MN) assay is useful in detecting chromosome damage induced by several factors. The aim of this study was to evaluate the effects of infection and malnutrition on the frequency of MN in erythrocytes from the peripheral blood of well-nourished, uninfected (WN) and well-nourished, infected (WNI) children, and moderately malnourished (UNM) and severely malnourished (UNS) children, both with infection, using a flow cytometric analysis technique. The percentage of reticulocytes (RETs) was significantly higher (1.5-fold) in WNI children than well-nourished controls. In addition, the UNS group had a 2.2-fold increase in the percentage of RETs compared to the WNI group. The frequency of micronucleated reticulocytes (MN-RETs) was 2.5 times greater, in WNI group compared to the WN group. These frequencies were significantly higher (1.7- and 2.1-fold) in UNM and UNS, respectively, compared to the WNI group. The results suggest that infection and malnutrition induce DNA damage in children.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.     

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     

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     

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., ≤0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

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.     

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose-response and time-course profiles measured by flow cytometry

A flow cytometric, anti-CD71-based method was used to measure peripheral blood reticulocyte and micronucleated reticulocyte frequencies in response to (137)Cs total body irradiation (TBI). In three independent experiments, groups of five female C57BL/6N mice were irradiated at graded doses up to 3 Gy, and peripheral blood specimens were collected at 43 h post-irradiation. Whereas the frequency of reticulocytes declined over the range of doses studied, micronucleated reticulocyte incidence was observed to increase in a dose-dependent manner up to 1 Gy. At doses greater than approximately 1 Gy, micronucleated reticulocyte frequencies declined with increasing exposure. These responses were highly reproducible, with significant effects on reticulocyte and micronucleated reticulocyte frequencies observed for the lowest dose studied (0.125 Gy). A time-course experiment was performed to test whether radiation-induced cell cycle delay may explain saturation of the micronucleated reticulocyte endpoint at doses >1 Gy. For this experiment, groups of four female C57BL/6N mice were exposed to 1, 1.5, or 2 Gy TBI, and blood collection occurred at 12h intervals from 43 to 115 h post-exposure. Reduced reticulocyte frequencies were observed for each dose studied, and the recovery of reticulocytes was increasingly delayed with higher radiation doses. Maximal micronucleated reticulocyte frequencies were observed at 43 or 55 h, with progressively lower values at later time points. At no time did micronucleated reticulocyte frequencies induced by 1.5 or 2 Gy significantly exceed that observed for 1 Gy at 43 h. These time-course data suggest that radiation-induced cell cycle delay cannot account for the micronucleated reticulocyte downturn phenomenon observed at doses greater than 1 Gy. An alternate hypothesis is discussed whereby apoptotic elimination of severely damaged bone marrow erythroid precursors plays a dominant role in saturating the radiation-induced micronucleated reticulocyte response observed for C57BL/6N mice.     

The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-beta-D-arabinofuranosylcytosine.

Dose-dependent induction of micronuclei with 1-beta-D-arabinofuranosylcytosine (ara-C) was clearly shown in CD-1 mouse peripheral blood reticulocytes (RETs) using an acridine orange (AO) supravital staining method, as well as in the conventional bone marrow assay. The maximum frequencies of micronucleated RETs (MNRETs) in peripheral blood and of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow were comparable, as shown in two laboratories independently. The maximum frequencies of MNRETs in peripheral blood lagged about 24 and 12 h behind those of MNPCEs in bone marrow in experiments with 24- and 12-h sampling intervals, respectively. The proportion of each type of RET was examined periodically after treatment with ara-C at doses ranging from 6.25 to 50.0 mg/kg. The proportion of type I RETs among total RETs decreased 24 or 48 h after treatment according to the dose level. This suggest that this ratio could be a good indicator of the bone marrow cell toxicity of test chemicals.     

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.     

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose–response and time-course profiles measured by flow cytometry

A flow cytometric, anti-CD71-based method was used to measure peripheral blood reticulocyte and micronucleated reticulocyte frequencies in response to ¹³⁷Cs total body irradiation (TBI). In three independent experiments, groups of five female C57BL/6N mice were irradiated at graded doses up to 3 Gy, and peripheral blood specimens were collected at 43 h post-irradiation. Whereas the frequency of reticulocytes declined over the range of doses studied, micronucleated reticulocyte incidence was observed to increase in a dose-dependent manner up to 1 Gy. At doses greater than approximately 1 Gy, micronucleated reticulocyte frequencies declined with increasing exposure. These responses were highly reproducible, with significant effects on reticulocyte and micronucleated reticulocyte frequencies observed for the lowest dose studied (0.125 Gy). A time-course experiment was performed to test whether radiation-induced cell cycle delay may explain saturation of the micronucleated reticulocyte endpoint at doses >1 Gy. For this experiment, groups of four female C57BL/6N mice were exposed to 1, 1.5, or 2 Gy TBI, and blood collection occurred at 12 h intervals from 43 to 115 h post-exposure. Reduced reticulocyte frequencies were observed for each dose studied, and the recovery of reticulocytes was increasingly delayed with higher radiation doses. Maximal micronucleated reticulocyte frequencies were observed at 43 or 55 h, with progressively lower values at later time points. At no time did micronucleated reticulocyte frequencies induced by 1.5 or 2 Gy significantly exceed that observed for 1 Gy at 43 h. These time-course data suggest that radiation-induced cell cycle delay cannot account for the micronucleated reticulocyte downturn phenomenon observed at doses greater than 1 Gy. An alternate hypothesis is discussed whereby apoptotic elimination of severely damaged bone marrow erythroid precursors plays a dominant role in saturating the radiation-induced micronucleated reticulocyte response observed for C57BL/6N mice.     

Increase in micronucleated erythrocytes associated with babesiosis in Syrian golden hamsters

The effect of infection by Babesia microti, a tick-borne piroplasm endemic to the northeastern United States, on the temporal pattern of micronucleated erythrocyte frequencies in peripheral blood was investigated in male Syrian golden hamsters. Significantly greater frequencies of micronucleated erythrocytes occurred in the blood of infected hamsters from 26 to 46 days after injection with B. microti, the magnitude of which within individual hamsters correlated highly with the percentage of polychromatic erythrocytes and the extent of parasitization. These data suggest that parasitic infection and other factors which alter the rate of erythropoiesis should be considered when the micronucleus assay is used in environmental or laboratory studies of genetic toxicity.     

Flow cytometric analysis of blood cells stained with the cyanine dye DiOC1[3]: reticulocyte quantification

The fluorescent dye 3,3'-dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes less than immature erythrocytes congruent to platelets less than leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, new methylene blue, brilliant cresyl blue (BCB), and acridine orange (AO). However, the population of imRBC as quantified by DiOC1[3] fluorescence is a subset of reticulocytes (reticulocytes as determined by BCB assay). The advantages and disadvantages of using DiOC1[3], AO, or pyronine Y as reticulocyte stains are discussed.     

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.     

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.     

Assessment of the genotoxicity of propineb in mice bone marrow cells using micronucleus assay

Propineb, a dithiocarbamate fungicide, is commonly used for the control of disease in a wide range of crops in agriculture. The genotoxic effects of commercial formulation of propineb in bone marrow cells of mice was investigated in vivo by micronucleus (MN) assay. The three different concentrations of propineb (12.5, 25 and 50 μg/mL; 0.01 mL per gram) were injected intraperitoneally (i.p.) to mice for 24 and 48 h. The results of the MN assay indicated that propineb induced a significant increase in frequency of micronucleated polychromatic erythrocytes (MNPCE) at 25 and 50 μg/mL concentrations for 24 h and at the highest (50 μg/mL) concentration for 48 h when compared with negative control. Also significant reduction for the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio which is indicative for bone marrow cytotoxicity was observed at the same concentrations for 24 and 48 h. These results lead us to the conclusion that propineb may have genotoxic and cytotoxic potential due to induction in the frequency of MN and a reduction in PCE/NCE ratio in the bone marrow cells of mice.