The benzene metabolite, hydroquinone, induces dose-dependent hypoploidy in a human cell line.

Chronic exposure to high concentrations of benzene can result in the development of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving high frequency of loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8. The pattern of reoccurring chromosome abnormalities associated with the development of leukemia can be used as a guide in understanding the etiology and pathogenesis of these diseases. Therefore, a research project was designed to determine whether a metabolite of benzene, hydroquinone (HQ), could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7 and 8 probes we demonstrate that 42, 49 and 26 microM HQ induces monosomy 5, 7 and 8, respectively, in the human lymphoblast cell line GM09948. These results demonstrate for the first time that HQ induces a specific chromosome loss found in secondary MDS/AML. The pattern of chromosome 5 and/or 7 loss in benzene-induced MDS/AML is probably due to selective cell survival after HQ exposure rather than specific targeting of HQ for chromosomes 5 or 7.

Characterization and phenotypic analysis of differentiating CD34+ human bone marrow cells in liquid culture.

Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.

Peroxidase activity in murine and human hematopoietic progenitor cells: potential relevance to benzene-induced toxicity.

Peroxidases may be important in the mechanism of toxicity of a number of compounds including benzene, a chemical that has been associated with bone marrow toxicity and leukemia after chronic exposure. The major peroxidase in bone marrow is myeloperoxidase (MPO), which has been previously thought to be expressed at the promyelocytic stage of differentiation. Hematopoietic progenitor cells are important potential cellular targets of bone marrow toxins and leukemogens. We therefore examined peroxidase activity in both murine and human progenitor cells. Murine progenitor populations were purified as lineage-negative cells (> 99% enriched) and human progenitor populations were purified as CD34+ cells (> 95% enriched). Using conventional biochemical assays for peroxidase activity, murine and human progenitor cells were found to have 30% and 11% of the peroxidase activity of murine and human unpurified marrow, respectively. Peroxidase activity was confirmed in purified murine and human progenitor populations by flow cytometry using a 2,7-dichlorofluorescein assay, adapted to measure peroxidase activity. In addition, two-color flow cytometry of murine whole marrow using phycoerythrin-conjugated antibodies to lineage markers confirmed the peroxidase activity of the murine progenitor cell population. A reverse transcription-polymerase chain reaction assay was developed for MPO mRNA, which was detected in murine progenitor cells. These data show that MPO mRNA is expressed in murine progenitor cells and that both murine and human progenitor cells have marked peroxidase activity. These data may have relevance for studies of hematopoietic cell differentiation and for the examination of mechanisms underlying cell-specific toxicity in bone marrow.

Purification of a novel glycosylated ferritin from horse heart.

We have previously shown that mRNA coding for ferritin L subunit is present on both cytosolic ribosomes and endoplasmic reticulum-bound ribosomes in rat heart tissue [Campbell et al. (1989) Arch Biochem Biophys 273:89-98]; from this we infer that heart tissue is capable of making a secreted ferritin. We now report the purification from horse heart, of a ferritin that specifically binds to Concanavalin A-Sepharose and is immunologically cross-reactive with antibodies raised against both horse cellular ferritin and horse serum ferritin. Where cellular ferritin is 10 nm in diameter and contains primarily 21-kDa subunits (as determined by gel exclusion chromatography and electron microscopy), the glycosylated heart ferritin is smaller with diameters of 3-5 nm. Antisera raised against serum ferritin cross-reacted with the glycosylated heart ferritin did but did not show significant cross-reactivity with cellular ferritin thus indicating that serum ferritin and glycosylated heart ferritin have antigenic determinants which may not be present on cellular ferritin. The glycosylated ferritin also differs from cellular ferritin in subunit composition, with subunits of 66, 60.5, 53.5, 43.5, and 29.5 kDa, as shown by SDS-PAGE and Western blot analysis. Interestingly, ferritin purified from horse serum contains subunits of similar size.