Peripheral Blood Reticulocytes Research Articles

Absorption and induction of micronucleated peripheral reticulocytes in mice after oral administration of fragrant hydroxyfuranones generated in the Maillard reaction

Fragrant hydroxyfuranone and dihydroxypyranone derivatives generated in the Maillard reaction of sugars and amino acids are detected in various processed foods and have been shown active to break DNA single-strand in the in vitro studies. In the present study, absorption of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), both found in soy sauce, into plasma after a single intraperitoneal or oral administration at doses of 0.5–1.0 gkg−1 to mice was examined. Both compounds appeared in plasma 15 min after intraperitoneal administration and disappeared 2 h after the administration. They appeared in plasma 5 min after oral administration, reached maximum after 15–45 min, and gradually disappeared after 2 h, indicating that they are absorbed by the digestive tract. Both DMHF and HEMF induced micronucleated reticulocytes (MNRETs) in mouse peripheral blood in a dose-dependent manner after oral administration. The results indicate that DMHF and HEMF can cause genetic damage after oral administration.

Micronucleus induction in somatic cells of mice as evaluated after 1,3-butadiene inhalation

The effect of different 1,3-butadiene (BD) inhalation doses, 130, 250 and 500 ppm, on somatic cells of mice was studied. Two different cell populations with diverse replicative and differentiative activities, namely splenocytes and peripheral blood reticulocytes, were examined and micronucleus (MN) frequencies were estimated. In splenocytes, different postinhalation time intervals were studied with regard to MN induction and characterisation. BD was found to be clastogenic by inducing increased micronucleus frequencies in both cell compartments and also to induce cytotoxicity at the highest level of exposure. In mouse splenocytes, BD has also shown a weak aneugenic effect at a short time interval after the exposure. Postinhalation time influences the induction of chromosome damage in stimulated splenocytes treated in vivo, since MN frequency decreases with time; in addition, BD has shown its aneugenic and cytotoxic potential only at 2 days after exposure.

3-Nitrobenzanthrone, a Powerful Bacterial Mutagen and Suspected Human Carcinogen Found in Diesel Exhaust and Airborne Particulates

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one) was isolated from the organic extracts of both diesel exhaust and airborne particles and was identified as a new class of powerful direct mutagen. Its mutagenicity by Ames Salmonella assay is very high (208 000 revertants/nmol in Salmonella typhimurium TA98 and 6 290 000 revertants/nmol in YG1024) and compares with that of 1,8-dinitropyrene, which is the direct mutagen of strongest activity (257 000 revertants/nmol in TA98 and 4 780 000 revertants/nmol in YG1024) so far reported in the literature. The new mutagen was also shown to induce micronuclei in mouse peripheral blood reticulocytes after intraperitoneal administration (micronucleated reticulocytes, 0.64% against 25 mg/kg dose after 48 h), suggesting its potential genotoxicity to mam malians. 3-Nitrobenzanthrone is most likely to be formed not only during the combustion process of fossil fuels but also from the atmospheric reaction between benzanthrone and lower oxides of nitrogen, since the latter ketone was found to be nitrated quite easily under an artificial atmosphere containing gaseous NO2 (10 ppm) and O3 (5 ppm) to produce the powerfully mutagenic 3-nitro derivative as the major product, along with several other isomeric mononitrobenzanthrones and dinitro descendants as minor products.

P XIV C.9 - P XIV C.9 Molecular characterization of micronuclei in peripheral blood reticulocytes of the mouse by means of an in situ RT-PCR/PRINS approach

P XIV C.9 - P XIV C.9 Molecular characterization of micronuclei in peripheral blood reticulocytes of the mouse by means of an in situ RT-PCR/PRINS approach

An improved method for the mouse liver micronucleus test

We have developed a very practical method for performing the liver micronucleus test in mice. Using this method, we evaluated 11 different types of mutagens, including, 2-acetylaminofluorene, amsacrine, benzene, cyclophosphamide, diethylnitrosamine, 4-dimethylamino-3′-methylazobenzene, N-ethyl- N-nitrosourea, fluorouracil, mitomycin C, potassium chromate (VI) and selenious acid. In order to assess the sensitivity of our method, the peripheral blood reticulocyte micronucleus test was performed in the same mouse. Animals were given test chemicals once and underwent partial hepatectomy (PH) 24 h later in order to induce mitotic stimulation. Peripheral blood was sampled 0, 24, 48 and 72 h after treatment. The incidence of micronucleated hepatocytes was determined 5 days after PH. As a result, diethylnitrosamine and 4-dimethylamino-3′-methylazobenzene, known as liver carcinogens, increased the incidence of micronucleated cells in the liver only. Positive reactions for benzene, on the other hand, were found in the peripheral blood reticulocytes only. The other chemicals showed positive reaction in the liver and peripheral blood reticulocytes with almost the same maximum response of micronucleus induction. Our method was found to have the advantage over Cliets' liver micronucleus test in that it required much less time and was easier to perform procedures and highly sensitive in detecting clastogens. It can be used in combination with the peripheral blood reticulocyte micronucleus test to evaluate test chemicals in two tissues, the liver and the bone marrow, in the same animal. We propose a method of combining this test with the peripheral blood reticulocyte micronucleus test for efficient screening for the clastogenic potential of new chemicals in vivo.

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10 000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.

Micronucleus induction in germ and somatic cells of the mouse after exposure to the butadiene metabolites diepoxybutane and epoxybutene

The genotoxicity of diepoxibutane (DEB) and epoxybutene (EB), two of the main metabolites of 1,3-butadiene, was tested in the germ and somatic cells of the mouse by applying an MN assay in early spermatids, and in peripheral blood reticulocytes of a subgroup of the same animals. DEB (0.17 and 0.35 mmol/kg) and EB (0.35, 0.70 and 1.04 mmol/kg) were administered i.p. In the germ cell assay, significant increases of MN were observed after treatment of premeiotic S-phase cells with both butadiene metabolites, but DEB was shown to be more powerful than EB in the induction of chromosomal damage. A weak effect of the same compounds was also found after treatment of late spermatocytes, approaching the meiotic divisions. From the MN assay in peripheral blood reticulocytes, a statistically significant increase of the frequency of MN was detected at each dose tested for both chemicals. However, the results have again shown that DEB is much more efficient than EB in inducing chromosome damage.

Erythroblastic Inclusions in Dominantly Inherited β Thalassemias

While the precipitation of unstable variant β-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited β thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited β thalassemia due to heterozygosity for the β-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal β-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant β mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated α-globin chains, but also β chains. The data confirm previous suggestions that the cellular pathology underlying this group of β thalassemias is related to the synthesis of highly unstable β-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess α chains, leading to increased ineffective erythropoiesis.

Erythroblastic Inclusions in Dominantly Inherited β Thalassemias

AbstractWhile the precipitation of unstable variant β-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited β thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited β thalassemia due to heterozygosity for the β-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal β-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant β mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated α-globin chains, but also β chains. The data confirm previous suggestions that the cellular pathology underlying this group of β thalassemias is related to the synthesis of highly unstable β-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess α chains, leading to increased ineffective erythropoiesis.

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, an optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment. Environ. Mol. Mutagen. 29:386–393, 1997 © 1997 Wiley-Liss, Inc.

Splenectomy as an adjuvant measure in the treatment of severe aplastic anaemia

The role of splenectomy in aplastic anaemia (AA) is controversial. The hazards of operating on a severely pancytopenic patient, the fear of compromising the patient's immune function, and the improvement of non-surgical treatment have made splenectomy unpopular in this disease. We have evaluated positive and adverse effects of splenectomy in 80 patients with severe aplastic anaemia (SAA) treated with antilymphocyte globulin (ALG) (group A), using 52 nonsplenectomized ALG patients as controls (group B). All patients survived the operation. Nonfatal complications of surgery occurred in 10 (12.5%). Splenectomy induced a significant increase of peripheral blood neutrophils, reticulocytes and platelets within 2 weeks, followed by a continuous increase of all values over the following weeks. 28/132 patients (21%) developed a late clonal disorder of haemopoiesis, paroxysmal nocturnal haemoglobinuria (PNH) or myelodysplastic syndrome (MDS), or both. Their incidence was identical in groups A and B. 13/28 (59%) died, 10/17 (59%) in group A and 3/11 (27%) in group B (not significant (n.s.)). Overall probability of survival at 18 years after ALG was 51+/-6% for group A and 61+/-7% for group B (n.s.). We conclude that splenectomy in AA is safe. It induces an immediate increase of peripheral blood counts and, thereafter, a continuous improvement of haemopoiesis. It does not increase the incidence of late clonal complications but has a borderline effect on mortality from these disorders. Splenectomy should be reconsidered in selective nontransplanted patients who have prolonged transfusion requirements despite otherwise optimal treatment.

Moderate Reduction of β-Globin Gene Transcript by a Novel Mutation in the 5’ Untranslated Region: A Study of Its Interaction With Other Genotypes in Two Families

We have identified two individuals of Greek Cypriot origin with thalassemia intermedia. Molecular analysis has shown that each individual is a compound heterozygote for a previously described beta zero thalassemia allele and a novel mutation, C-->G in position +33, in the 5' untranslated region of the beta globin gene. In both families the beta +33 allele is associated with the same beta haplotype (-++- ) suggesting that it is likely to be of a single origin, beta-cDNAs from normal and mutant beta alleles were isolated from peripheral blood reticulocytes using the technique of reverse transcription-polymerase chain reaction. Because the beta +33 (C-->G) mutation creates a cutting site for the restriction enzyme NlalV, we could demonstrate by differential restriction analysis that the beta gene with +33 mutation showed 25% to 35% residual activity compared with normal. The additive effect of this moderate deficit in beta globin production with the beta zero thalassemia mutation would explain the clinical phenotypes observed in the two probands. In contrast, two siblings of one proband who were also compound heterozygotes for the same beta thalassemia mutations, as well as heterozygotes for a nondeletional alpha thalassemia variant, and two other compound heterozygotes for the beta +33 and a beta+ thalassemia allele were completely asymptomatic. Individuals heterozygous for the beta +33 C-G mutation alone are clinically and hematologically silent, with normal red blood cell indices and normal levels of hemoglobin (Hb) A2. A direct relationship between genotypic and phenotypic severity is clearly demonstrated in these cases with obvious implications for prenatal diagnosis.

Organ variation in the mutagenicity of dimethylnitrosamine in Big Blue mice.

Organ specificity in the lacI mutant frequency (MF) induced by dimethylnitrosamine (DMN) was analyzed in lung, liver, kidney, bone marrow, urinary bladder, and testis of Big Blue mice. Cell proliferative activity was also analyzed in some of these tissues by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Clastogenicity of DMN was concomitantly analyzed by the peripheral blood micronucleus assay with the same animals used for the lacI mutation assay. Five daily intraperitoneal (i.p.) treatments with DMN (1 mg/kg) increased MF in liver (6.2 x control), kidney (2.4 x control), and lung (2.1 x control). These are known target organs for DMN carcinogenesis. No MF increase was observed in nontarget organs studied, i.e., bone marrow, bladder, and testis. Single ip treatment with DMN also increased lacI MF in liver but the increases were smaller than in a 5-daily-treatment regimen. This result suggests that multiple dosing is more effective in the transgenic mutation assay. The enhancement of cell proliferation observed was in bronchial epithelia 7 days after treatment. No micronucleus induction in peripheral blood was observed 24 hours after 2 and 3 daily ip treatments with 1 mg/kg DMN. An increase in the incidence of micronucleated reticulocytes in peripheral blood was observed 48 hours after single ip treatment with 5 or 10 mg/kg DMN. The present study demonstrated organ-specific induction of gene mutations by DMN which suggests a relevance of this assay for the prediction of organ-specific carcinogenesis.

System issues: Organ variation in the mutagenicity of dimethylnitrosamine in Big Blue® mice

Organ specificity in the lacl mutant frequency (MF) induced by dimethylnitrosamine (DMN) was analyzed in lung, liver, kidney, bone marrow, urinary bladder, and testis of Big Blue® mice. Cell proliferative activity was also analyzed in some of these tissues by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Clastogenicity of DMN was concomitantly analyzed by the peripheral blood micronucleus assay with the same animals used for the lacl mutation assay. Five daily intraperitoneal (ip) treatments with DMN (1 mg/kg) increased MF in liver (6.2 × control), kidney (2.4 × control), and lung (2.1 × control). These are known target organs for DMN carcinogenesis. No MF increase was observed in nontarget organs studied, i.e., bone marrow, bladder, and testis. Single ip treatment with DMN also increased lacl MF in liver but the increases were smaller than in a 5-daily-treatment regimen. This result suggests that multiple dosing is more effective in the transgenic mutation assay. The enhancement of cell proliferation observed was in bronchial epithelia 7 days after treatment. No micronucleus induction in peripheral blood was observed 24 hours after 2 and 3 daily ip treatments with 1 mg/kg DMN. An increase in the incidence of micronucleated reticulocytes in peripheral blood was observed 48 hours after single ip treatment with 5 or 10 mg/kg DMN. The present study demonstrated organ-specific induction of gene mutations by DMN, which suggests a relevance of this assay for the prediction of organ-specific carcinogenesis. © 1996 Wiley-Liss, Inc.

Genotoxicity of trophosphamide in mouse germ cells: assessment of micronuclei in spermatids and chromosome aberrations in one-cell zygotes

The genotoxicity of trophosphamide (TP) in mouse germ cells was assessed by the cytogenetic analysis of micronuclei in spermatids and chromosome aberrations in one-cell zygotes and compared with the genotoxicity in somatic cells evaluated by the micronucleus reticulocyte assay. Single acute doses of 50, 75, 100 and 150 mg/kg were studied after i.p. injection. TP was only weakly mutagenic for preleptotene spermatocytes-differentiating spermatogonia, but clear-cut cytotoxic effects were demonstrated after treatment of these cells by a dose-dependent reduction of the ratio between Golgi and cap phase spermatids. Effects induced in post-meiotic stages were estimated, after mating the treated males with untreated superovulated females, by the frequencies of zygotes with chromosome aberrations: a peak of genetic damage was detected in late spermatids, with as many as 55% zygotes with aberrations, but spermatozoa and early spermatids were also clearly affected. When compared with matched solvent-injected controls, the lowest effective dose in spermatozoa and late spermatids was 100 mg/kg, although the 3- to 4-fold increases detected at 50 mg/kg were also statistically significant when compared with a pool of laboratory controls. In peripheral blood reticulocytes, the micronucleus frequencies were increased by 3-20 times the respective baseline values in the individual animals. A marked cytotoxic effect on bone marrow cells was revealed by the reduction of the proportion of early reticulocyte stages, which dropped to 20% of the control value at 150 mg/kg. Both genotoxic and cytotoxic effects were higher in bone marrow than in germ cells of the same animals, pointing to a generalized higher susceptibility of somatic cells to TP, possibly related to chemical distribution and target organ accessibility. The accurate description of stage- and dose-effect relationships in germ cells of experimental models is crucial for genetic risk assessment after chemical exposure. The approaches applied in this study may contribute to this goal.

The micronucleus assay using peripheral blood reticulocytes from X-ray-exposed mice

A new ‘fluorescence’ variant of the micronucleus assay using supravital staining of peripheral blood reticulocytes with an acridine orange coated slide was recently developed. In this study the application of this method to detect a mutagenic response to low-dose exposure of X-irradiation is reported. The mice were exposed to a single dose of 2.5, 5, 10, 15, 25, and 50 cGy. The induction of micronuclei in peripheral blood reticulocytes was recorded with sampling times of 0, 24, 48, 72, and 96 h after exposure without killing of mice. Dose-dependent effects were observed at sampling times of 24–72 h reaching maximum levels at 48 h after X-ray exposure. A highly significant ( p < 0.01) increase of the micronucleated reticulocytes was found for doses of 10, 15, 25, and 50 cGy. Neither the dose of 2.5 nor 5 cGy induced the significant increase in the incidence of micronucleated reticulocytes compared with the respective control group.

The induction of micronuclei in mice hepatocytes and reticulocytes by terrachloroethylene

The clastogenicity of tetrachloroethylene (tetra) was detected by means of the micronucleus assay using hepatocytes and relticulocytes from ddY male mice, to understand its effects in upon hepatocellular carcinomas in mice. The frequency of micronucleated hepatocytes of mice that received a single injection of tetra after partial hepatectomy increased to levels that were significantly higher than those of controls treated with solvent. However, the micronucleus assay using peripheral blood reticulocytes from ddY male mice, revealed that tetra did not induce to a statistically significant increase in micronucleus frequency. These results suggested that tetra metabolites have a clastogenic effect in vivo upon mouse liver but not upon bone marrow cells.

INVESTIGATIONAL AGENTS FOR RHEUMATOID ARTHRITIS

Agents ranging from simple analgesics to antiinflammatory drugs to powerful immunomodulators have been used for the treatment of rheumatoid arthritis with varying success. Despite the availability of agents that are believed to be "second line" or "disease modifying," many patients either do not respond adequately to available agents or must discontinue their use because of intolerable or dangerous adverse reactions. For this reason, researchers continue to search for more efficacious and less toxic agents for patients with rheumatoid arthritis. This article describes pharmaceutical agents currently under investigation for use in rheumatoid arthritis, including the antiinflammatory agents, zileuton and tenidap, and the immunosuppressive agents, leflunomide, mycophenolic acid (RS-61443), tacrolimus (FK-506), sirolimus (rapamycin), amiprilose (therafectin), cladribine (2- chlorodeoxyadenosine), and azaribine.

The suitability of rat peripheral blood in suchronic studies for the micronucleus assay

To examine the suitability of using rat peripheral blood from animals used in subchronic toxicity studies for micronucleus analysis, we orally administered phenacetin or 6-mercaptopurine for 14 days to groups of six rats and compared their micronucleus frequencies to the bone marrow micronucleus frequencies of rats similarly treated for only 2 days. In the 14-day test, phenacetin significantly increased the frequency of micronucleated reticulocytes in peripheral blood at 500 mg/kg starting from day 9, and at 750 and 1500 mg/kg starting from day 6; 6-mercaptopurine topurine gave a positive response at 20 mg/kg starting from day 6. Positive responses in the bone marrow assay were obtained at the same dose levels. In the 2-day test, micronucleated polychromatic erythrocyte frequencies increased significantly at 1000 and 2000 mg/kg for phenacetin, and at 50, 100, and 200 mg/kg for 6-mercaptopurine. These results suggest that micronucleus assays using peripheral blood from rats in subchronic animal studies of phenacetin and 6-mercaptopurine are feasible and at least as sensitive for the assessment of micronuclei as an acute bone marrow micronucleus test.