Micronucleated Erythrocytes Research Articles

Influence of age and sex on the spontaneous DNA damage detected by Micronucleus test and Comet assay in mice peripheral blood cells

We have investigated the normal variations in basal DNA damage detected by Comet assay in leukocytes and micronucleated erythrocytes (MNE) using the Micronucleus test (MN) in peripheral blood cells from 45 female and male mice from different age groups (newborns, 3.5, 12, and 104 weeks) to clarify age and sex-related changes. Comparison of basal DNA damage detected by Comet assay showed significantly increased values in 104 weeks old mice in relation to the other ages (P < or = 0.01), and newborn mice showed higher values in MNE frequency when compared to all the other groups (P < or = 0.01). A positive correlation was observed between Damage Frequency (r =0.382, P = 0.010) and Damage Index (r = 0.640, P < 0.001) and age. Age was also correlated with the ratio of polychromatic erythrocytes/normachromatic erythrocytes (PCE/NCE) (r = -0.473, P = 0.001), and the MNE frequency was positively correlated with the ratio of PCE/NCE (r = 0.454, P = 0.002). These results suggest an age-related slow down of DNA repair efficiency of DNA damage and/or DNA damage accumulation. Furthermore, data on the spontaneous MNE frequency indicate that the reticuloendothelial system matures with age, and there is a close relationship between erythropoiesis and micronucleus induction in erythrocytes. The influence of sex in the parameters analyzed was less clear. In conclusion, age seems to influence in basal DNA damage and should be considered in genotoxicity studies using mice. Finally, comparisons between assays must be made with care when different cells are compared (e.g. leukocytes and erythrocytes), as found with the Comet assay and MN test.

Blood Cell Morphology and Plasma Biochemistry of Captive Mauremys caspica (Gmelin, 1774) and Mauremys rivulata (Valenciennes, 1833)

Morphological characteristics of peripheral blood cells, micronucleated erythrocyte counts and plasma biochemistry profile were examined in fourteen healthy captive Mauremys caspica and in twenty-three Mauremys rivulata. The size of erythrocyte cells were 19.07 × 11.68 μm and 19.76 × 11.44 μm for M. caspica and M. rivulata, respectively. Nucleus sizes were 6.50 × 5.30 μm for M. caspica and 6.79 × 5.45 μm for M. rivulata. The micronucleated erythrocyte (MNE) values were 0.0008 and 0.0037 for the males and females of M. caspica, respectively. The MNE values were 0.0002 for male and female M. rivulata. We found sex-dependent differences only in the Ca value in the blood biochemistry profile for healthy M. caspica. Sex-dependent differences were found only in albumin and P values in the blood biochemistry profile for healthy M. rivulata. No significant differences were found between males of both species in question with respect to plasma biochemistry values. However, only plasma total protein and Ca content levels differed significantly between the females of the two species.

Aquatic ecotoxicology approaches in Western Mexico

A series of bioindicator organisms for aquatic ecosystems are being maintained under laboratory conditions in order to analyze effects of pollution on aquatic wildlife and potential effects on human health. Growth kinetics of algae Pseudokirchneriella subcapitata was used to evaluate effects of the surfactant nonilphenol (NP). Brachionus calyciflorus was used to set up a model of endocrine disruption using the fungicide vinclozolin (Vc). We exposed salamanders from the genus Ambystoma sp., to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNC) in their shed skin. The presence of spontaneous micronuclei in peripheral blood erythrocytes from 10 fish species in Lake “La Alberca,” Michoacan (Mexico), was evaluated as a possible biological indicator of genotoxic agents. Results confirm the sensivity of Pseudokirchneriella subcapitata to growth kinetics: the range of concentration of NP (20, 200 and 2000 μ g L− 1) shows an inverted U shape in its maximum growth rate; Dimethyl sulfoxide (DMSO) used as a positive control and to solvate NP induced an inverse stimulatory effect on growth rate in the range of concentrations analyzed (0.0023, 0.023 and 0.23% v v− 1). In the use of the rotifer Brachionus calyciflorus, the range of Vc from 0.185 mg L− 1 to 3 mg L− 1 clearly showed an inverted U shape characteristic of endocrine disruptions. We were able to use shed skin from Ambystoma sp., to measure MNC frequencies induced either by an aneugenic or a clastogenic compound. The MNC frequency was increased significantly by all doses of COL and CP, administered either as single or repeated exposures. The presence of MNC in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, as suitable alternative models for detecting genotoxic exposures relevant to aquatic environments. In the survey to determine potential biological fish indicators for genotoxic agents, the frequency of spontaneous micronucleated erythrocytes (MNE) found in the goodeid Xenotoca melanosoma (3.7 ± 1.6 MNC) and the cichlid Oreochromis aureus (2.0 ± 1.0 MNC) suggests that these species can be considered as potential biological indicators.

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.

Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model

Diabetes mellitus (DM) is associated with a high risk of health complications, mainly due to excessive free radical (FRs) production that could result in an increased frequency of micronuclei. The consumption of antioxidants, like folic acid (FA), may mitigate the effects of the FRs. In the present study, micronucleated polychromatic erythrocyte (MNPCE) frequencies were determined in blood sampled weekly from the tails of pregnant female Wistar rats and pregnant Wistar rats with experimental diabetes that were given unsupplemented diets and diets supplemented with FA. At birth, the pups were sampled to analyze micronucleated erythrocyte (MNE) and MNPCE frequencies. Moreover micronucleated cells (MNCs) were evaluated in buccal mucosa samples taken from 81 healthy adult subjects, 48 patients with DM, and 30 DM patients who were sampled before and after FA treatment. Increases in MNPCE frequencies were significant only at the first sampling ( P < 0.01 and P < 0.03) in pregnant rats with experimental diabetes. In addition, the pups from the diabetic group and from diabetic group treated with FA had higher frequencies of MNEs ( P < 0.03 and P < 0.001, respectively) and MNPCEs ( P < 0.009 and P < 0.05, respectively) than the controls. No differences were found in diabetic rats and newborn rats born to diabetic mothers treated with FA compared with untreated animals. Patients with DM had a higher frequency of MNCs compared with healthy subjects ( P < 0.001). Also FA reduced the frequency of MNCs in DM patients ( P < 0.001). The results of this study indicate that diabetes results in elevated frequencies of micronuclei, and that, at least in humans, FA can protect against the elevation.

Los felinos: ¿Una alternativa en estudios de toxicología genética?

The micronuclei (MN) test carry out in peripheral blood is fast, simple, economic and it is used to detect genotoxic environmental agents. MN are fragments of chromosomes or complete chromosomes remaining in the cytoplasm after cell division, which increase when organisms are exposed to genotoxic agents. Therefore, species with the highest values of spontaneous micronucleated erythrocytes (MNE) are the most suitable to be potentials biomonitor of micronucleogenic agents, using a drop of blood. Nine species of Felines that present spontaneous MNE in peripheral blood are shown. From these species, the cat has been previously proven, with positive results and also lion (Panthera leo), yaguaroundi (Felis yagoaroundi), lynx (Lynx ruffus), jaguar (Panthera onca), puma (Puma concolor), tiger (Panthera tigris), ocelote (Felis padalis) and leopard (Panthera pardus) display spontaneous MNE, and with this characteristic this Family can be propose like a potential group to be used in toxicogenetic studies.

Weekly administration of paclitaxel induces long-term aneugenicity in nude mice

We investigated the potential in vivo aneugenic effects associated with paclitaxel treatment. For this purpose, we treated female nude mice with paclitaxel using doses equivalent to those used in weekly schedules at the clinical level (three cycles of 30 mg/kg/week for 3 consecutive weeks followed by 1 resting week). We then evaluated the frequencies of micronucleated erythrocytes (MNE) in peripheral blood using the acridine orange micronucleus assay. The frequency of MNE was evaluated after 24 h and 168 h of administration of the last dose of each paclitaxel cycle (STA mice group) as well as after one year of the first dose of treatment (LTA mice group). We also analyzed the cytology of peripheral blood and bone marrows obtained from these mice at each time period. In the STA mice group, 3 cycles of paclitaxel induced a 2.4-fold increase in MNE frequencies compared to the control group (P

Importance of spontaneous micronucleated erythrocytes in bottlenose dolphin (Tursiops truncatus) to marine toxicology studies

The objective of the work was to characterize the presence of spontaneous micronucleated erythrocytes (MNES) from peripheral blood of bottlenose dolphins (Tursiops truncatus) to evaluate the possibility to use this species as potential bioindicator of genotoxic compounds. Forty-eight blood samples from 12 bottlenose dolphins were obtain from three Mexican dolphinariums, and from 10 dolphins was possible to obtain more than one sample at different sampling times. Smears were processed and observed with an epifluorescence microscope. The average of MNES and polychromatic erythrocytes (PCE) from the 48 samples was 24.3 +/- 6.1 MNES/10,000 total erythrocytes (TE), and 9.1 +/- 5.5 PCE/1,000 TE. MNES and PCE number did not show differences between gender and age. No variations in the MNES values of the bottlenose dolphins that were sampled more than one occasion were found. Comparisons among dolphinariums revealed differences in MNES frequency, with the highest significant frequency observed in dolphins from dolphinarium "A" (26.0 +/- 5.9 MNES/10,000 TE) than dolphinarium "B" (19.5 +/- 3.1 MNES/10,000 TE) (p < 0.05) and dolphinarium "C" (18.6 +/- 3.5 MNES/10,000 TE) (p < 0.007). The presence of MNES and PCE in the bottlenose dolphin may provide a useful marine mammal model to detect DNA damage by means of micronuclei test in peripheral blood erythrocytes to evaluate genotoxicity and cytotoxicity expositions.

Evaluation of Rana snk esculenta blood cell response to chemical stressors in the environment during the larval and adult phases

The assessment of the biological effects on aquatic vertebrate species is frequently employed to monitor water pollution, as it provides significant information on bioavailability and actual concentration levels. In anamniote vertebrates (fish and amphibians), significant correlations have been observed between exposure to contaminants – both natural and experimental – and blood modification. We investigated the changes in some circulating blood cell parameters of green frog ( Rana snk esculenta) tadpoles and adults collected at two sample rice fields, one heavily polluted and the other relatively unpolluted. The frequency of eosinophilic leucocytes, mitotic, anucleated and micronucleated erythrocytes was evaluated also regarding the haemopoietic/haemocatheretic and NOS expression of the liver. Haematological indicators in polluted samples were found to be significantly different from controls as regards both larval and adult exposure, and provided information on long-term background pollution of the habitats under investigation. The population of the polluted area showed evident effects of chronic exposure to contaminants, to a degree which could lead to sub-lethal alterations of their health status. The general nature of responses to this kind of stress emphasizes the role of amphibian peripheral blood as a sensitive indicator regarding contamination in aquatic environments.

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.

Nuclear abnormalities in erythrocytes of parrots (Aratinga canicularis) related to genotoxic damage

Nuclear abnormalities in erythrocytes, as micronuclei and nuclear buds (BE), are considered potential biomarkers of genotoxic exposure. We described previously the frequency of spontaneous micronucleated erythrocytes (MNE) in the species Aratinga canicularis. Here, we have used this species to evaluate the induction of MNE and BE by mitomycin-C. Animals were given a single intracoelomic injection of 0, 2, 3 or 4 mg/kg mitomycin-C on two consecutive days. A drop of blood was obtained after 0, 24, 48 and 72 h, and stained smears were used to count micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes with buds (BPCE)/1000 polychromatic erythrocytes. The number of MNE and BE in 10 000 total erythrocytes was also counted. MNPCE and BPCE frequencies were elevated at 24, 48, and 72 h after the administration of the lower dose (P<0.03). At a 3 mg/kg dose, the frequency of MNPCE increased at 48 and 72 h (P<0.04) whereas the number of BPCE increased, but not significantly. Administration of 4 mg/kg mitomycin-C increased the number of MNE observed at 72 h (P<0.03), the number of MNPCE at 48 h (P<0.01) and 72 h (P<0.006), the BE frequency at 72 h (P<0.05), and the frequency of BPCE at 48 and 72 h (P<0.001). While mitomycin-C appears to produce a parallel increase in MNPCE and BPCE frequencies, the MNE seemed to be a more sensitive indicator of genotoxicity than the BE. This suggests that evaluating BE and MNE in routine haematological analysis should be considered to evaluate environmental genotoxic exposure.

Re: The evaluation of micronucleus frequency by acridine orange fluorescent staining in peripheral blood of rats treated with lead acetate. (Mutagenesis, 20, 411-415, 2005)

The authors thank Dr Raymond J. Proudlock for his comments on their article. First, studies on the genotoxicity of lead salt are inconclusive. Pelclova et al. (1) did not detect a significant increase in structural aberrations (chromosome and chromatid exchanges, and breaks) in 22 workers from a lead battery plant. In contrast, Vaglenov et al. (2) found a significant increase in MN frequency among the lead-exposed staff (millers and assemblers) of a battery plant. The etiology of lead genotoxicity and carcinogenicity is complex. In vitro studies have demonstrated that lead interacts with proteins and nucleic acids, particularly at the sulfhydryl group and the phosphate backbone, respectively (3,4). Yang et al. (5) investigated the effects of lead acetate and found that lead may induce DNA damage through a Fenton reaction by interacting proteins. Lead ion has high affinity for the sulfhydryl groups of proteins. In our study, outbred female Wistar rats were treated by gavage once per week for 10 weeks with cumulative doses of 140, 250 and 500 mg/kg body weight of lead acetate (6). Because this study is a chronic study, peripheral blood is suitable for investigation. The results were compared with negative controls. Results were statistically significant. Studies have shown that mouse peripheral blood cells are an acceptable cell population for detecting micronucleated PCEs (MNPCEs) as long as the sampling schedule accounts for the release of newly-formed micronucleated erythrocytes from bone marrow to the blood (7,8). As indicated by Hayashi (9), cells should be intact and nuclei of nucleated cells and reticulum structure of reticulocytes should fluoresce strongly green and red. In our study, we considered these identifying features and PCEs were identified by their orange–red color, mature erythrocytes by their green color and micronuclei by their yellowish color. Those differences are shown in the paper. Several studies have shown a relationship between lead levels in blood and anemia (10,11). Gurer et al. (12) indicated that lead exposure causes mild anemia along with other haematological disturbances, including considerable variability in the sizes of RBCs (anisocytosis) and irregular shapes in RBCs (poikilocytosis). These data, considered in light of the data suggesting that lead induces oxidative stress in RBCs, support the hypothesis that lead-induced oxidative stress could, in part, be responsible for the lead-induced toxicity to the haematological system (12). There is a strong interaction between the formation of micronuclei and the antioxidant system. These systems vary among living organisms. In our study, we hypothesized that exposure to lead may have the potential for micronucleus formation. In order to understand the mechanisms responsible for the micronucleus formation further cytogenetic studies are needed.

Oral sildenafil citrate lacks genotoxicity and cytotoxicity in a primate model: Callithrix jacchus

To determine sildenafil citrate (SC) genotoxicity and cytotoxicity in the Callithrix jacchus. Fifteen organisms were assigned to one of three groups as follows: experimental (25 mg/kg of SC); negative control (glucose solution 5%); and positive control (3 mg/kg of cytocine arabinoside). Systemic hemodynamic changes were monitored in each animal before and after each treatment. A drop of blood was obtained before and after the treatment at 24-120 h. Smears were made and the frequency of micronucleated erythrocytes (MNE), micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes (PCE) was counted. No significant differences in MNE, MNPCE and PCE were found in the group that received sildenafil and negative control. A significant increase in genotoxicity and cytotoxicity was observed in the positive control group. No changes were observed in systemic hemodynamic changes. The macro-dose of SC lacks genotoxic, cytotoxic or systemic hemodynamic changes effects in this species.

Detection of clastogenic and aneugenic damage in newborn rats

The last 25 years have seen an ever-growing use of the erythrocyte micronucleus test for measuring damage to mammalian chromosomes in vivo. In addition, staining micronuclei with antikinetochore antibodies from CREST serum discriminates aneugenic from clastogenic damage. The use of the micronucleus test in rats, however, has been problematic because the spleen of adult rats efficiently removes micronucleated erythrocytes from the blood. In the present study, we have treated 5-day-old rats with either X-rays (a clastogen) or vinblastine (an aneugen) and measured micronuclei in erythrocytes from the blood and liver. Each treatment increased the frequency of micronuclei in both tissues, with the percentages of CREST-staining micronuclei reflecting the mechanism of micronucleus induction by the two agents. The results indicate that performing the micronucleus assay in the liver and peripheral blood of 5-day-old rats may be a useful approach for detecting the in vivo genotoxicity of chemical and physical agents.

Blood Cell Morphology and Plasma Biochemistry of the Captive European Pond Turtle Emys orbicularis

The morphological characteristics of peripheral blood cells, micronucleated erythrocytes counts and plasma biochemistry profile were examined in ten healthy captive European pond turtles Emys orbicularis. Blood samples were obtained from the caudal vein. The Wright staining method was used for the classification of the blood cells. Mature erythrocytes of captive Emys orbicularis were nucleated ellipsoidal cells (21.7 ±1.27μm × 13.2 ±1.12 μm) with pink cytoplasm. The nucleus (6.9 ± 0.78 μm × 5.4 ± 0.65 μm) was centrally located and stained dark purple. Seven different types of blood cells were determined: erythrocytes, thrombocytes, monocytes, eosinophils, basophils, lymphocytes and heterophils. The micronucleated erythrocyte (MNE) values were 0.0016 and 0.003 for the males and females, respectively. The MNE results were presented as the mean frequency of micronucleated erythrocytes per 1000 cells per animal. The mean plasma concentrations in the total of specimens were as follows: total protein (25 g/L), albumin (7.2 g/L), globulin (17.8 g/L), glucose (2.91 mmol/L), calcium (2.32 mmol/L), phosphorus (1.55 mmol/L), creatinine (46.85 μmol/L), urea (10.93 mmol/L) , triglycerides (0.44 mmol/L), cholesterol (1.48 mmol/L), sodium (125.76 mmol/L), potassium (3.98 mmol/L), chloride (93.94 mmol/L), iron (13.34 μmol/L) and activities of aspartate aminotransferase (2.14 μkat/L), alanine aminotransferase (0.15 μkat/L), gamma glutamyl transpeptidase (2.15 U/L), amylase (8.09 μkat/L), lactate dehydrogenase (19.93 μkat/L). We found sex-dependent differences only in ALT [(0.21 μkat/L and 0.10 μkat/L for the males and females, respectively) (t = 3,107; df = 14; p &lt; 0.05)] value in the blood biochemical profile for healthy Emys orbicularis. We suggest that the biochemical profile described in the present study may be used as a standard profile for healthy Emys orbicularis kept in captivity.

Performance of flow cytometric analysis for the micronucleus assay—a reconstruction model using serial dilutions of malaria-infected cells with normal mouse peripheral blood

To confirm the performance and statistical power of a flow cytometric method for scoring micronucleated erythrocytes, reconstruction experiments were performed. For these investigations, peripheral blood erythrocytes from untreated mice, with a micronucleated erythrocyte frequency of approximately 0.1% were combined with known quantities of Plasmodium berghei (malaria) infected mouse erythrocytes. These cells had an infected erythrocyte frequency of approximately 0.7%, and mimic the DNA content of micronuclei (MN). For an initial experiment, samples with a range of MN/malaria (Mal) content were constructed and analysed in triplicate by flow cytometry until 2000, 20,000 and 200,000 total erythrocytes were acquired. In a second experiment, each specimen was analysed in triplicate until 2000, 20,000, 200,000 and 1,000,000 erythrocytes were acquired. As expected, the sensitivity of the assay to detect small changes in rare erythrocyte sub-population frequencies was directly related to the number of cells analysed. For example, when 2000 cells were scored, increases in MN/Mal frequencies of 3.9- or 2.7-fold were detected as statistically significant. When 200,000 cells were analysed, a 1.2-fold increase was detected. These data have implications for the experimental design and interpretation of micronucleus assays that are based on automated scoring procedures, since previously unattainable numbers of cells can now be readily scored.

Impact of Reperfusion on the Micronucleus Generation Phenomenon in the Bone Marrow Red Blood Cells in Massive Blood Loss

Experiments on non-inbred anesthetized albino male rats weighing 250-350 g have established that blood reperfusion after acute massive blood loss substantially increases the count of micronucleated (MN) erythrocytes in the rat bone marrow by 8.4 times as a whole as compared with the control, which suggests genomic destabilization. The optimum time of elaboration of MN polychromatic erythrocytes is 24 hours after acute blood loss or reinfusion. The intraarterial administration of mexidole (50 mg/kg) just before blood reinfusion drastically decreases the count of MN erythrocytes virtually to the that seen in control animals. The observed effects during micronucleus assay generally correspond to the changes in lipid per-oxidation and overall antioxidative activity, pointing to the free radical mechanism of this phenomenon.

Micronucleated erythrocyte frequencies in old and new world primates: Measurement of micronucleated erythrocyte frequencies in peripheral blood ofCallithrix jacchus as a model for evaluating genotoxicity in primates

Nonhuman primates are of particular relevance in evaluating the potential toxicity of drugs and environmental agents. We have used previously published information and data from the present study to establish a relationship for New World (NW) and Old World (OW) primates on the basis of the frequency of spontaneous micronucleated erythrocytes (MNEs) observed in peripheral blood. Data on spontaneous MNEs in peripheral blood from 15 species of primates, including humans, indicate that NW primates have significantly (P < 0.01) higher MNE frequencies (group mean, 9.5 +/- 7.3 MNEs/10,000 erythrocytes; range, 0.7-20.5/10,000 erythrocytes) than OW primates (group mean, 1.0 +/- 0.9 MNEs/10,000 erythrocytes; range, 0.0-2.6 MNEs/10,000 erythrocytes). Humans are believed to have developed in the OW, and human MNE frequencies were similar to those described for OW primate species. We selected the common marmoset (Callithrix jacchus), a NW primate, to determine whether therapeutic pediatric doses of Metotrexate (MTX; 2.5 mg/kg), Cyclophosphamide (CP; 5 mg/kg), Cytosine-arabinoside (Ara-C; 3 mg/kg), or 5-Fluorouracil (5-FU; 10 mg/kg), administered daily for two consecutive days, increase the frequency of micronuclei. Micronucleated polychromatic erythrocyte frequencies were increased significantly in groups receiving MTX, CP and Ara-C, while MNE frequencies were increased by the Ara-C treatment. The results of this study indicate that NW primates have higher spontaneous MNE frequencies than OW primates, and because of this, NW primates like the common marmoset, may be suitable for evaluating the genotoxicity of chemical agents.

The Mouse Genomic Instability Mutation chaos1 Is an Allele of Polq That Exhibits Genetic Interaction with Atm

chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase theta;. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority ( approximately 90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.

Comparative evaluation of background micronucleus frequencies in domestic mammals

Exposure of human alimentation-destined animals to toxic substances can be an important risk factor for human health. Mutagenicity tests represent a good method for genotoxic effect evaluation of environmental pollutants. The micronucleus frequency in peripheral blood and bone marrow erythrocytes has been evaluated in four domestic Ungulate species (ox, sheep, swine and horse). For each species two or three groups of animals coming from Italy and other European countries were analysed and the results indicate a relatively low mean frequency of micronucleated erythrocytes (ME), both in peripheral blood and bone marrow. The comparison between these two frequencies in the four species studied shows that the ME frequency in sheep and horse is significantly higher in peripheral blood than in bone marrow, whereas in bovines it is higher in the bone marrow, and in swine the difference is not significant. These results could indicate that in ox and swine the spleen is involved in ME removal from the peripheral circulation, as is known for other species including man. Nevertheless, it cannot be excluded that the same occurs in the other two species, since they exhibit a relatively low peripheral blood ME frequency as well. Further studies on domestic mammals are needed to clarify the spleen function and verify the use of the peripheral blood micronucleus test for genotoxic biomonitoring. At present, the application of the micronucleus test to the bone marrow seems a more suitable method, but, being invasive, it can be used only as a control system of animal hygiene and health, in addition to the routine tests, rather than for biomonitoring.