Slide preparation and sampling as a major source of variability in the mouse micronucleus assay

This paper describes the results of a study in which mouse bone marrow micronucleus assay slides were assessed for homogeneity of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE). The slides were prepared by 3 distinct methods and several methods of slide reading were assessed. Observations made using our slides were confirmed by re-analysis of slides from 3 independent laboratories. It is concluded that the method of slide preparation and assessment can significantly influence the variability of data obtained from a study. The extent of this variability casts doubt upon the validity of certain assumptions concerning this assay--such as sex differences in MPE incidence, responder variability, etc. Results are discussed within the context of the very recent literature for this assay. Some laboratories appear to have adequate methods of slide preparation and data accumulation, while others do not. Methods to improve the sensitivity of this assay are suggested within the context of the recommendations made by the Gene-Tox review group. In particular, it is suggested that individual investigators present evidence of the adequacy of their data accumulation techniques in order to enhance the value of future studies.     

The micronucleus test in pigs: induction of micronuclei in polychromatic erythrocytes by various doses of X-rays

This paper describes the results of a study in which pigs were used in the bone marrow micronucleus assay. In a first experiment the spontaneous frequency of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE) was investigated in 78 animals. It was found that it is low with individual values of 0-4 MPE/1000 PE and a group average of 1.76 +/- 1.06% (mean +/- SD). In a second set of investigations animals were exposed to 0.25, 0.5, 1.0, 1.5, 2.25 and 2.75 Gy of 9-MeV X-irradiation performed as a single whole-body exposure. Time- and dose-dependent changes in micronucleus incidence were observed. Maximal group averages appeared nearly uniform 36 h post irradiation (p.i.). Considering the 36-h values in the dose range of 0-2.25 Gy there is a marked dose-effect relationship (r = 0.971). The data yield best to a regression curve of a third-grade polynomial indicating a complex interaction between dose and micronucleus formation. In conclusion, the results demonstrate that it appears feasible to use swine as target organisms in the micronucleus test to estimate the cytogenetic damage caused by ionizing radiations or, potentially, chemical compounds.     

The assessment of micronucleated polychromatic erythrocytes in rat bone marrow. Technical and statistical considerations

Traditionally, the mouse is the species of choice for the rodent bone marrow micronucleus assay (MN). However, the rat is used for most other toxicological studies. The suitability of the rat as a test species for the MN was therefore investigated. In this paper, the methodological aspects of the assay have been considered. The distribution and incidence of micronucleated polychromatic erythrocytes (MPEs) on bone marrow slides prepared by two techniques, the conventional smear and the paint-brush technique, were assessed in control and cyclophosphamide-dosed male and females rats. MPEs were shown to be homogeneously distributed when assessed over a large number of PEs on slides prepared by both techniques, but when viewed over a few hundred PEs (less than 500 PEs), the incidence of MPEs on the same slides was seen to vary considerably (0-10 MPEs/500 PEs). Variability was within acceptable limits when at least 1000 PEs/animal were analysed. The spontaneous incidence of MPEs in the AP rat is low (0-2 MPEs/1000 PEs). Cyclophosphamide increased the incidence markedly and there was a wide inter-animal variability in the response (10-40 MPEs/1000 PEs). The paint-brush technique is considered technically simpler and recommended over the smear technique. This study shows that MPEs can be accurately scored in the bone-marrow of the rate provided due consideration is given to staining and sample size of PEs analysed per animal.     

Expression Profile of Three Splicing Factors in Pleural Cells Based on the Underlying Etiology and Its Clinical Values in Patients with Pleural Effusion

Splicing factors (SFs) are involved in oncogenesis or immune modulation, the common underlying processes giving rise to pleural effusion (PE). The expression profiles of three SFs (HNRNPA1, SRSF1, and SRSF3) and their clinical values have never been assessed in PE. The three SFs (in pellets of PE) and conventional tumor markers were analyzed using PE samples in patients with PE (N = 336). The sum of higher–molecular weight (Mw) forms of HNRNPA1 (Sum-HMws-HNRNPA1) and SRSF1 (Sum-HMws-SRSF1) and SRSF3 levels were upregulated in malignant PE (MPE) compared to benign PE (BPE); they were highest in cytology-positive MPE, followed by tuberculous PE and parapneumonic PE. Meanwhile, the lowest-Mw HNRNPA1 (LMw-HNRNPA1) and SRSF1 (LMw-SRSF1) levels were not upregulated in MPE. Sum-HMws-HNRNPA1, Sum-HMws-SRSF1, and SRSF3, but neither LMw-HNRNPA1 nor LMw-SRSF1, showed positive correlations with cancer cell percentages in MPE. The detection accuracy for MPE was high in the order of carcinoembryonic antigen (CEA, 85%), Sum-HMws-HNRNPA1 (76%), Sum-HMws-SRSF1 (68%), SRSF3, cytokeratin-19 fragments (CYFRA 21-1), LMw-HNRNPA1, and LMw-SRSF1. Sum-HMws-HNRNPA1 detected more than half of the MPE cases that were undetected by cytology and CEA. Sum-HMws-HNRNPA1, but not other SFs or conventional tumor markers, showed an association with longer overall survival among patients with MPE receiving chemotherapy. Our results demonstrated different levels of the three SFs with their Mw-specific profiles depending on the etiology of PE. We suggest that Sum-HMws-HNRNPA1 is a supplementary diagnostic marker for MPE and a favorable prognostic indicator for patients with MPE receiving chemotherapy.     

Membrane phospholipid organization in calcium-loaded human erythrocytes

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.     

Flow cytometric detection of erythropoietic cytotoxicity in mouse bone marrow.

Erythroblast proliferation and maturation in bone marrow are the processes leading to the formation of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE), respectively. PE contain RNA but no DNA, and can therefore be distinguished both from NE (which lack both RNA and DNA) and from nucleated cells (which contain both DNA and RNA). Cytotoxic agents that induce impairment of the maturation process change the PE:NE ratio. We have developed a simple and rapid method of determining the PE:NE ratio, based on flow cytometric analysis of formaldehyde-fixed, acridine orange (AO)-stained cells. The effects of cyclophosphamide (CP), mitomycin C (MMC), and vincristine (VC) were tested and the PE:NE ratio was evaluated over 7 days of treatment. In this study we monitored the kinetics of these compounds and were able to demonstrate both a time- and a dose-dependent effect. We detected a difference between the effects of the alkylating agents tested and those induced by the spindle inhibitor tested. Flow cytometry of fixed bone marrow samples stained with AO provides more information, better and more rapid statistical analysis, than conventional microscopic methods for counting the PE:NE ratio.     

Some observations on the primitive and definitive erythrocytes of the developing chick

Summary The cytological changes in the primitive and definitive erythrocytes of the incubating chick have been followed. Observations have been made on the nucleoli, vital granules, mitochondria,Golgi apparatus, reticulum ofSinigaglia and the reticulation patterns of the basophilic substance. The cells of the primitive and definitive lines are ordinarily readily distinguished from one another. Data are included on the rate of disappearance of the primitive cells from the circulation. They may persist as long as two weeks after hatching. Giant primitive erythrocytes are common during the first week of incubation. The cells have one, two three or four nuclei. The nuclearplasma relationship is maintained somewhere near a constant. These atypical cells are due to aberrations in mitosis. Data on the percentage of mitosis in both types of erythrocytes are also included. The initial activity of the spleen and bone-marrow is reflected in the blood stream. There is a distinct rise in the proportion of young definitive erythrocytes. An attempt is made to correlate the findings ofHall (1934) on the changing affinity of the hemoglobin for oxygen with the changing blood picture. The primitive line does not persist long enough to account for the phenomenon. It is suggested, however, that the hemoglobin of the erythrocytes produced by the yolk sac may differ from that of the cells produced by the spleen and bone-marrow. With Plates I–III.     

Visualization of erythrocytes in the zebrafish brain

We found that erythrocytes of zebrafish have cytoplasmic peroxidase activity. Blood in the zebrafish brain was visualized using a standard peroxidase staining method after formaldehyde fixation. The erythrocytes in the brain were heavily stained, but neurons and glias were not stained at all. This easy method enables the distribution of erythrocytes in the whole brain to be determined, and enables the actual number of erythrocytes in each area in the brain to be calculated. The paths of major, thick blood vessels in zebrafish brain are similar to those in higher vertebrates, however, the distribution of thin blood vessels is different. We also found that the erythrocytes were unevenly distributed in the brain. For example, the density of erythrocytes in the surface layer of the tectum was more than 30-fold higher than in the deeper granular layer. Very few erythrocytes were found in bundles of axons like cranial nerves and the medial longitudinal fascicle. In general, fewer erythrocytes were found in areas near the ventricle, whereas many more were found closer to the surface of the brain. The distribution of erythrocytes in the brains of sleeping, awake and actively moving fish were compared. In the brains of sleeping fish, most of the erythrocytes were present in large vessels. This was not observed in brains of awake or actively moving fish. We found that the blood supply to motor neurons in the ventral horn of the spinal cord increased during active movement compared to that in awake or sleeping fish.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

Relevant assay to study the adhesion of Plasmodium falciparum-infected erythrocytes to the placental epithelium

In placental malaria, Plasmodium falciparum-infected erythrocytes adhere to the apical plasma membrane of the placental epithelium, triggering an impairment of placental function detrimental to the fetus. The design of anti-adhesion intervention strategies requires a detailed understanding of the mechanisms involved. However, most adhesion assays lack in vivo relevance and are hardly quantitative. Here, we describe a flow cytometry-based adhesion assay that is fully relevant by using apical epithelial plasma membrane vesicles as the adhesion matrix, and being applicable to infected erythrocytes directly isolated from patients. Adhesion is measured both as the percentage of pathogens bound to epithelial membrane vesicles as well as the mean number of vesicles bound per infected erythrocytes. We show that adhesins alternative to those currently identified could be involved. This demonstrates the power of this assay to advance our understanding of epithelial adhesion of infected erythrocytes and in the design of intervention strategies.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

The effect of reducing the number of cells scored on the performance of the in vivo rat liver UDS assay

The most labour-intensive feature of the in vivo rat liver UDS assay is the scoring of hepatocyte autoradiograms by microscope. Even with image analyser and computer equipment the scoring phase of a full study might require half of the technical effort applied. Practice recommended by guidelines has been to score 50 cells/slide and two slides per animal. Now sufficient data have been accumulated, an evaluation was made to observe whether this procedure was necessary. An analysis of the accumulated UDS database in our laboratory was made to determine the sources of variability of mean net nuclear grain count, [N - C]. It was observed that the two largest components of variation in negative control animal mean [N - C]. were between-day and interanimal variability. The contribution from sampling error during slide scoring was relatively small. Theoretical calculations showed that the greater sampling error derived from scoring 30 rather than 50 cells/slide would result in only a marginal increase in total assay variation. To test this, 30 cells/slide were randomly selected from the 50 cells scored originally in negative control animals in each of 18 studies over an 18-month period. It was confirmed that reducing the number of cells had a negligible effect on the variation of negative control animal mean [N - C] values. Furthermore, analysis of data from 10 more studies confirmed that within-study variation would be essentially unaffected by scoring 30 cells/slide. The use of 30 rather than 50 cells per slide (a total of 60 cells per animal) has therefore been adopted for all current studies and scoring procedures modified to avoid operator bias during the selection of a smaller number of cells.     

Cellular changes in the bone marrow of Plasmodium berghei-infected mice: II. Blast transformation and phagocytosis

The present work is concerned with early cellular changes occurring during a malaria infection. Blast transformation by lymphoid cells and phagocytosis by adherent cells from the bone marrow was performed, using immune and nonimmune Balb/c mice. Nonadherent bone marrow cells from immune mice show an increased specific lymphoblast transformation. This increase was not observed during a lethal infection (PI). Adherent bone marrow cells were assayed for phagocytosis of parasitized (PE) or normal erythrocytes (NE). Cells from immune mice show an increase in phagocytosis of PE and NE. Cells from PI mice showed a decreased phagocytosis throughout the infection, beginning at Day 1 after challenge.     

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes.

In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost.     

ATP-dependent transport of phosphatidylserine analogues in human erythrocytes

The plasma membrane of most cells contains a number of lipid transporters that catalyze the ATP-dependent movement of phospholipids across the membrane and assist in the maintenance of lipid asymmetry. The most well-characterized of these transporters is the erythrocyte aminophospholipid flippase, which selectively transports phosphatidylserine (PS) from the outer to the inner monolayer. Previous work has demonstrated that PS and to a lesser extent phosphatidylethanolamine (PE) are substrates for the flippase and that other phospholipids move across the membrane only by passive flip-flop. The present study re-evaluates these results. The incorporation and transbilayer movement of a number of short-chain (dilauroyl) phospholipid analogues in human erythrocytes was measured by observing lipid-induced changes in cell morphology, and the effect of an ATPase inhibitor (vanadate) and a sulfyhdryl reagent (N-ethylmaleimide) was determined. Incubation of cells with these lipids causes the rapid formation of echinocytes, because of the accumulation of the lipid in the outer monolayer. While dilauroylphosphatidylcholine-treated cells retained this shape, cells treated with sn-1,2-DLP-l-S, sn-1,2-DLP-d-S, or N-methyl-DLPS rapidly changed morphology to stomatocytes, which is consistent with the transport and accumulation of the lipid in the inner monolayer. A similar, although slower, stomatocytic shape change was induced by sn-2,3-DLP-l-S. Other lipids that were tested (dilauroylphosphatidylhydroxypropionate, dilauroylphosphatidylhomoserine, DLPS-methyl ester, or sn-2,3-DLP-d-S) reverted to discocytes only. In all cases, pretreatment with vanadate or N-ethylmaleimide inhibited the conversion of echinocytes to discocytes or stomatocytes. This is the first report of a protein- and energy-dependent pathway for the inwardly directed transbilayer movement of lipids other than PS and PE in the erythrocyte membrane and suggests that the flippase has broader specificity for substrates or that other lipid transporters are present.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.     

Role of monocyte chemoattractant protein-1, tumor necrosis factor-alpha and interleukin-6 in the control of malignant pleural effusion and survival in patients with primary lung adenocarcinoma

This study aimed at assessing the role of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the control of pleural effusion (PE) and survival in patients with primary lung adenocarcinoma. The concentrations of the 3 cytokines were measured in PE from 79 lung adenocarcinoma patients with malignant pleural effusion (MPE) and 23 patients with tuberculosis. Data were correlated with the efficacy of MPE control and patient survival. The level of MCP-1 in PE was significantly higher in patients with lung adenocarcinoma than those with tuberculosis. By contrast, the levels of TNF-alpha and IL-6 were significantly lower in patients with lung adenocarcinoma than those with tuberculosis. An MCP-1 level greater than 3,187 pg/mL (which was used as a cutoff point) indicated failure to control MPE (odds ratio [OR]=2.82, 95% confidence interval [CI]=1.02-7.82, p=0.04). In multivariate analysis, MCP-1 was confirmed as an independent prognostic factor for progression-free survival (hazard ratio [HR]=2.02, 95% CI=1.24-3.30, p=0.01). The level of MCP-1 in PE appears to be a reliable surrogate marker for evaluating the therapeutic efficacy in the control of MPE and predicting survival in lung adenocarcinoma patients with MPE.     

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.     

Identification of bartonella trw host-specific receptor on erythrocytes

Each Bartonella species appears to be highly adapted to one or a limited number of reservoir hosts, in which it establishes long-lasting intraerythrocytic bacteremia as the hallmark of infection. Recently, we identified Trw as the bacterial system involved in recognition of erythrocytes according to their animal origin. The T4SS Trw is characterized by a multiprotein complex that spans the inner and outer bacterial membranes, and possesses a hypothetical pilus structure. TrwJ, I, H and trwL are present in variable copy numbers in different species and the multiple copies of trwL and trwJ in the Bartonella trw locus are considered to encode variant forms of surface-exposed pilus components. We therefore aimed to identify which of the candidate Trw pilus components were located on the bacterial surface and involved in adhesion to erythrocytes, together with their erythrocytic receptor. Using different technologies (electron microscopy, phage display, invasion inhibition assay, far western blot), we found that only TrwJ1 and TrwJ2 were expressed and localized at the cell surface of B. birtlesii and had the ability to bind to mouse erythrocytes, and that their receptor was band3, one of the major outer-membrane glycoproteins of erythrocytes, (anion exchanger). According to these results, we propose that the interaction between TrwJ1, TrwJ2 and band 3 leads to the critical host-specific adherence of Bartonella to its host cells, erythrocytes.