Screening of subclinical functional hemoglobin and red blood cell abnormalities among blood donors of Fayoum University Hospital in Egypt: Are RET-He, and IRF useful screening tools?

BACKGROUND: The effectiveness of red cell transfusion in a given blood unit that relied on both quantity and quality of donated cells undoubtedly affects prognostic outcomes. OBJECTIVE: We aimed to determine the frequency of subclinical functional hemoglobin and red cell abnormalities in donated blood of Fayoum University Hospital in Egypt. Additionally, to assess the usefulness of reticulocyte mean hemoglobin content (RET-He) and immature reticulocyte fraction (IRF) as screening measures for such abnormalities. MATERIAL AND METHODS: This cross-sectional study enrolled 200 volunteer blood donors who met the national standard criterion of blood donation. Complete blood count with reticulocyte parameters, serum ferritin, sickling test, G6PD assay, Mentzer index, and naked-eye single tube red cell osmotic fragility test were carried out. RESULTS: Functional red cell abnormalities represented 44 % of this cohort. Out of them, 4.5 % had iron deficiency, 11 % had a positive sickling test, 19 % had G6PD deficiency, and 9.5 % had suspicious thalassemia. The sensitivity and specificity test for RET-He in selective identification of functional hemoglobin abnormalities in donated blood were 83.3 % and 61.2 %, respectively at a cutoff value of 26.9. Though there was no statistically significant effect of RET-He on the selective detection of G6PD deficiency, IRF had a statistically significant high level with a p-value of 0.04. CONCLUSION: Subclinical functional red cell abnormalities seem to be prevalent among blood donors. Reticulocyte/ erythrocyte indices could be useful screening tools for red cell abnormalities. Further studies are required for assessing the impact of transfusing such abnormalities to neonates and other critical recipients.

Red cell membrane disorders: structure meets function.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

Association of abnormal erythrocyte morphology with oxidative stress and inflammation in metabolic syndrome.

In carrying out their role of free radical scavenging, erythrocytes become damaged due to oxidation of membrane lipids and proteins. Such damage may change the morphology of the erythrocytes. The present study aims to demonstrate change in erythrocyte morphology in MetS and associate the changes with increased oxidative stress and inflammation that were shown in our recent study. One hundred participants were recruited from a rural town of Australia. Whole blood viscosity, erythrocyte aggregation, erythrocyte deformability, lipid profile and blood sugar level, oxidative stress markers (erythrocyte reduced glutathione, superoxide dismutase, urinary isoprostanes) and inflammatory markers (high sensitivity C-reactive protein) were measured. Erythrocyte morphological study was performed by scanning electron microscopy. Recruited participants were classified into MetS and non-MetS following the National Cholesterol Education Program Adult Treatment Panel III definition. Data were analyzed by IBM SPSS 20 software. The mean percentages of biconcave cells were decreased whereas acanthocytes, stomatocytes and echinocytes were increased in MetS group compared to healthy controls. Morphologically abnormal erythrocytes were significantly correlated with oxidative stress and chronic inflammation markers. Free radicals generated in increased concentration in MetS seem to damage erythrocyte changing its morphology which possibly could affect other hemorheological parameters.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eß thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study.

Hereditary spherocytosis (HS) is characterised by weakened vertical linkages between the membrane skeleton and the red blood cell's lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. The levels of haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, reticulocytes (Ret), immature reticulocytes fraction (IRF), hypochromic erythrocytes (Hypo-He) and microcytic erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n = 1,488). HS is characterised by a high Ret count without an equally elevated IRF. All 45 HS have Ret >80,000/µl and Ret(10(9)/L)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave predictive positive value and negative predictive value of respectively 75% and 100% (n=1,488), which is much greater than single parameters or existing rules. This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates and ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

Electrical Impedance Characterization of Erythrocyte Response to Cyclic Hypoxia in Sickle Cell Disease.

Cell sickling is the process in which intracellular polymerization of deoxygenated sickle hemoglobin (HbS) leads to distorted, rigid cells, resulting in abnormal blood rheology and painful vaso-occlusion. Current methods for detection of this process mainly rely on optical microscopy of cellular morphology and measurements of cell deformability and blood rheology. As electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics, it can be a promising marker for characterization of abnormal blood rheology and a means more convenient than optics to be integrated into point-of-care devices. In this work, a microfluidics-based electrical impedance sensor has been developed for characterizing the dynamic cell sickling-unsickling processes in sickle blood. The sensor is capable of measuring the continuous variation in the sickle cell suspension due to cyclic hypoxia-induced intracellular HbS polymerization and depolymerization. Simultaneous microscopic imaging of cell morphological change shows the reliability and repeatability of the electrical impedance-based measurements of cell sickling and unsickling processes. Strong correlation is found between the electrical impedance measurement and patients' hematological parameters such as levels of HbS and fetal hemoglobin. The combination of electrical impedance measurement and on-chip hypoxia control provides a promising method for rapid assessment of the dynamic processes of cell sickling and unsickling in patients with sickle cell disease.

Association between myeloid malignancies and acquired deficit in protein 4.1R: a retrospective analysis of six patients.

Constitutional deficit in the erythroid protein 4.1 (4.1R), a structural component of the erythrocyte membrane, is implicated in hereditary elliptocytosis. Acquired deficit in protein 4.1R have been rarely described in myelodysplastic syndromes. Here, we report a series of six patients presenting a myelodysplastic or a myeloproliferative disease in association with an elliptocytosis curve on osmotic gradient ektacytometry and a significant decrease in protein 4.1R level. We confirm that deficit in protein 4.1R is recurrent in myeloid malignancies and should be particularly investigated when deletion del (20 q) is present, since we found this chromosomal abnormality in four out of six patients.

Human erythrocytes are affected in vitro by flavonoids of Aristotelia chilensis (Maqui) leaves.

Aristotelia chilensis (Mol.) Stuntz (A. chilensis), also known as maqui, is a plant of the Elaeocarpaceae family that grows in central and southern Chile as well as southwestern Argentina. Infusions of its leaves have long been used in the traditional native herbal medicine to treat different ailments. Phytochemical studies of the plant's chemical composition of the plant indicate the presence of indolic alkaloids, flavonoids, cianidine glucosides, delfidine, malvidine, petunidine, cumarines and triterpenes. These compounds, particularly the flavonoids, have antioxidant properties. In order to evaluate the mechanisms of its toxicity and their antioxidant properties, the leaves' aqueous extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM), and molecular models of the human erythrocyte membrane. These consisted of multibilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, and large unilamellar vesicles (LUV) of DMPC. The capacity of A. chilensis aqueous extracts to perturb the bilayer structure of DMPC and DMPE was evaluated by X-ray diffraction, DMPC LUV and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). Results of the present study indicate that aqueous extracts of A. chilensis induced an alteration of human erythrocyte morphology from the normal discoid shape to an echinocytic form, changes that are explained in terms of the extract interaction with the membrane's outer phospholipid monolayer.

A nonsense mutation 1669Glu-->Ter within the regulatory domain of human erythroid ankyrin leads to a selective deficiency of the major ankyrin isoform (band 2.1) and a phenotype of autosomal dominant hereditary spherocytosis.

We describe a nonsense mutation in the regulatory domain of erythroid ankyrin associated with autosomal dominant hereditary spherocytosis with a selective deficiency of the ankyrin isoform 2.1 (55% of normal), a deficiency of spectrin (58% of normal) proportional to the decrease in ankyrin 2.1, and a normal content of the other main ankyrin isoform, protein 2.2. PCR amplification of cDNA encoding the regulatory domain of ankyrin revealed a marked decreased in the ratio of ankyrin 2.1 mRNA to the ankyrin 2.2 mRNA. Sequencing of ankyrin gene in the region where the 2.1 and 2.2 mRNA differ detected a nonsense mutation 1669Glu-->Ter (GAA-->TAA) in one ankyrin allele. Only normal ankyrin 2.1 mRNA was detected in the reticulocyte RNA. Since the alternative splicing within the regulatory domain of ankyrin retains codon 1669 in ankyrin 2.1 mRNA and removes it from ankyrin 2.2 mRNA, we propose that the 1669Glu-->Ter mutation decreases the stability of the abnormal ankyrin 2.1 mRNA allele leading to a decreased synthesis of ankyrin 2.1 and a secondary deficiency of spectrin.

Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.

Intracellular polymerization and sickling depend markedly on the cellular concentration of sickle hemoglobin (Hb S). A possible therapeutic strategy for sickle cell disease is based on reducing the cellular concentration of Hb S through prevention of erythrocyte dehydration. The K-Cl cotransporter is a major determinant of sickle cell dehydration and is inhibited by increasing erythrocyte Mg content. We studied 10 patients with sickle cell disease before treatment and after 2 and 4 wk of treatment with oral Mg supplements (0.6 meq/kg/d Mg pidolate). Hematological parameters, erythrocyte Na, K, and Mg content, erythrocyte density, membrane transport of Na and K, and osmotic gradient ektacytometry were measured. We found significant increases in sickle erythrocyte Mg and K content and reduction in the number of dense sickle erythrocytes. Erythrocyte K-Cl cotransport was reduced significantly. We also observed a significant reduction in the absolute reticulocyte count and in the number of immature reticulocytes. Ektacytometric analysis showed changes indicative of improved hydration of the erythrocytes. There were no laboratory or clinical signs of hypermagnesemia. Mild, transient diarrhea was the only reported side effect. We conclude that oral Mg supplementation reduces the number of dense erythrocytes and improves the erythrocyte membrane transport abnormalities of patients with sickle cell disease.

The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus).

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.

Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry.

A proteomic approach using a cleavable ICAT reagent and nano-LC ESI tandem mass spectrometry was used to perform protein profiling of core RBC membrane skeleton proteins between sickle cell patients (SS) and controls (AA), and determine the efficacy of this technology. The data was validated through Peptide/Protein Prophet and protein ratios were calculated through ASAPratio. Through an ANOVA test, it was determined that there is no significant difference in the mean ratios from control populations (AA1/AA2) and sickle cell versus control populations (AA/SS). The mean ratios were not significantly different from 1.0 in either comparison for the core skeleton proteins (alpha spectrin, beta spectrin, band 4.1 and actin). On the natural-log scale, the variation (standard deviation) of the method was determined to be 14.1% and the variation contributed by the samples was 13.8% which together give a total variation of 19.7% in the ratios.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells

Structural organisation of band 3 in Melanesian ovalocytes.

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normocytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.

Hemisodium, a novel selective Na ionophore. Effect on normal human erythrocytes.

Hemisodium is a novel Na ionophore that belongs to the class of compounds called cryptands. These compounds possess an electron-rich cavity for binding of cations and are conformationally organized during synthesis to favor the selective binding of one cation over another. In media containing 145 mM NaCl and 5 mM KCl, hemisodium (10(-5) M) increased erythrocyte Na content from 23 to 345 mmol/kg.dry cell solid (dcs) over 4 h and increased water content from 1.8 to 3.5 liter/kg.dcs over the same period. K content decreased somewhat over the same time period, but this fall in K content was prevented entirely by incubation in either low Na media (to prevent net Na entry) or in Cl free media. Thus, the decrease in K content in high NaCl media was due to cell swelling, which activated KCl cotransport, and not due to a direct action of hemisodium on K permeability. Hemisodium-mediated Na transport was conductive, because erythrocyte membrane potential (Vm), determined by diS-C3-5 fluorescence, changed from -9 to +22 mV in high Na media in the presence of hemisodium and DIDS. In cells equilibrated with sulfamate, an anion with low conductive permeability, Vm changed 54 mV per 10-fold change in external Na concentration with the addition of hemisodium. In contrast, a 10-fold change in the external concentration of K, Rb, Cs, or T1 failed to alter Vm in the presence of hemisodium, suggesting a high Na specificity of the ionophore. Na conductance determined from net fluxes increased from 0.04 to 5.2 microS/cm2 with 10 microM hemisodium, and with that concentration the ratio of Na to K conductance was 45:1. Among the Na ionophores available so far, hemisodium appears to have the greatest specificity. Hemisodium may be a valuable tool in membrane transport studies.

Does higher red blood cell (RBC) lactate transporter activity explain impaired RBC deformability in sickle cell trait?

Lactate and H(+) are suggested to promote the sickling process in red blood cells (RBCs) containing hemoglobin S. We demonstrated greater activity of the RBC monocarboxylate cotransporter MCT-1, lower RBC deformability and impaired hematological indices in sickle cell trait (SCT) carriers compared to control subjects, suggesting an involvement of MCT-1 in hemorheological disturbances in SCT carriers.

Drug-associated "bite cell" hemolytic anemia.

PURPOSE: "Bite cell" hemolytic anemia is a variant of drug-related hemolysis usually associated with methemoglobinemia and Heinz body inclusions in red blood cells secondary to oxidant drug injury. Bite cells are morphologically characterized as poikilocytes with one or more semicircular portions removed from the cell margin. The purpose of this report is to emphasize the importance of peripheral smear examination in patients with possible drug-associated hemolytic anemia. The morphologic characteristics of bite cells by light microscopy and scanning electron microscopy are detailed in this study, and the pathophysiologic mechanism is discussed. PATIENTS AND METHODS: Clinical and laboratory data were retrospectively studied on eight patients (two men and six women, aged 29 to 85 years) who showed evidence of bite cell hemolytic anemia associated with drug exposure. Multiple standard hematologic laboratory evaluations for hemolytic anemia were performed. Five hundred red blood cells were counted from randomly selected peripheral smear fields for the calculation of bite cell percentage. RESULTS: Peripheral smears showed predominantly normochromic normocytic red cells with prominent bite cells and occasional blister cells. Bite cell counts ranged from 5.5% to 13.6% (mean, 8.7 +/- 3.0% SD) associated with a hematocrit reduction of 3.0% to 13.2% (mean, 8.5 +/- 3.8% SD) and concomitant reticulocytosis of varying degree from 2.3% to 15.4% (mean, 7.3 +/- 4.9% SD). Withdrawal of the offending drug(s) and treatment of underlying diseases resulted in improvement of hemolytic anemia and eventual disappearance of bite cells. A close correlation between hematocrit reduction, reticulocyte response, and bite cell percentage increase was seen. The usual biochemical markers of hemolysis were not consistently observed. Scanning electron microscopy confirmed the light microscopic evidence of bite cell morphology and revealed a keratocytic variant. CONCLUSION: This study emphasizes the importance of peripheral smear examination for early diagnosis and management of bite cell hemolytic anemia. Withdrawal of the putative offending drug(s) and treatment of underlying disorders should result in improvement of this form of drug-associated hemolysis.

Hypochromic macrocytes: are they reticulocytes?

Automated full blood counters measuring the size and haemoglobin concentration of individual red cells by high and low angle light scatter can provide estimates of the percentage of cells which are abnormal in either or both of these variables. The hypothesis that an increase in the number of large cells with a reduced haemoglobin content ("hypochromic macrocytes") was indicative of a reticulocytosis was investigated. A correlation was shown between the percentage of hypochromic macrocytes and the reticulocyte count. This correlation was too weak to allow the actual reticulocyte percentage to be predicted from the percentage of hypochromic macrocytes. An increased percentage of hypochromic macrocytes, however, often indicates an increased reticulocyte count and thus serves to chart the haematologist to this possibility.

Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.

Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG-p53 mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time-weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, peripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN-PCE and MN-NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6-month treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose-dependent increase in the frequency of MN-PCE and MN-NCE and in %PCE. Maximal induction of MN-PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration-dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not exclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN-NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans.