Sian Rizzo

The haemotoxicity of mitomycin in a repeat dose study in the female CD-1 mouse

Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose‐related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late‐stage or residual bone marrow injury. The present study in female CD‐1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC‐treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6–12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post‐dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post‐dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post‐dosing, many haematological parameters in MMC‐treated mice had returned to control levels; however, there remained evidence of late‐stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte‐macrophage colony‐forming units and erythroid colonies showed a profound decrease immediately post‐dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post‐dosing period, but a return to normal was seen at day 50 post‐dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post‐dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD‐1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post‐dosing period with a return to normal levels at day 50 post‐dosing; however, there was evidence of mild but significant late‐stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow.

A new model of busulphan‐induced chronic bone marrow aplasia in the female BALB/c mouse

Aplastic anaemia (AA) is characterized by hypocellular marrow, pancytopenia, and risk of severe anaemia, haemorrhage and infection. AA is often idiopathic, but frequently occurs after exposure to drugs/chemicals. However, the pathogenesis of AA is not clearly understood, and there are no convenient animal models of drug‐induced AA. We have evaluated regimens of busulphan (BU) administration in the mouse to produce a model of chronic bone marrow aplasia showing features of human AA. Mice were given 8 doses of BU at 0, 5.25 and 10.50 mg/kg over 23 days; marrow and blood samples were examined at 1, 19, 49, 91 and 112 days after dosing. At day 1 post dosing, in mice treated at 10.50 mg/kg, nucleated marrow cells, CFU‐GM and Erythroid‐CFU were reduced. Similarly, peripheral blood erythrocytes, leucocytes, platelets and reticulocytes were reduced. At day 19 and 49 post dosing, there was a trend for parameters to return towards normal. However, at day 91 and 112 post dosing, values remained significantly depressed, with a stabilized chronic bone marrow aplasia. At day 91 and 112 post dosing, marrow cell counts, CFU‐GM and Erythroid‐CFU were decreased; marrow nucleated cell apoptosis and c‐kit+ cell apoptosis were increased; peripheral blood erythrocyte, leucocyte, and platelet counts were reduced. We conclude that this is a model of chronic bone marrow aplasia which has many interesting features of AA. The model is convenient to use and has potential in several areas, particularly for investigations on mechanisms of AA pathogenesis in man.

Further development of a model of chronic bone marrow aplasia in the busulphan-treated mouse

Aplastic anaemia (AA) in man is an often fatal disease characterized by pancytopenia of the peripheral blood and aplasia of the bone marrow. AA is a toxic effect of many drugs and chemicals (e.g. chloramphenicol, azathioprine, phenylbutazone, gold salts, penicillamine and benzene). However, there are no widely used or convenient animal models of drug‐induced AA. Recently, we reported a new model of chronic bone marrow aplasia (CBMA = AA) in the busulphan (BU)‐treated mouse: eight doses of BU (10.50 mg/kg) were administered to female BALB/c mice over a period of 23 days; CBMA was evident at day 91/112 post‐dosing with significantly reduced erythrocytes, platelets, leucocytes and nucleated bone marrow cell counts. However, mortality was high (49.3%). We have now carried out a study to modify the BU‐dosing regime to induce CBMA without high mortality, and investigated the patterns of cellular responses in the blood and marrow in the post‐dosing period. Mice (n = 64/65) were dosed 10 times with BU at 0 (vehicle control), 8.25, 9.0 and 9.75 mg/kg over 21 days and autopsied at day 1, 23, 42, 71, 84, 106 and 127 post‐dosing (n = 7–15); blood and marrow samples were examined. BU induced a predictable bone marrow depression at day 1 post‐dosing; at day 23/42 post‐dosing, parameters were returning towards normal during a period of recovery. At day 71, 84, 106 and 127 post‐dosing, a stabilized, late‐stage, nondose‐related CBMA was evident in BU‐treated mice, with decreased erythrocytes, platelets and marrow cell counts, and increased MCV. At day 127 post‐dosing, five BU‐treated mice showed evidence of lymphoma. In this study, mortality was low, ranging from 3.1% (8.25 mg/kg BU) to 12.3% (9.75 mg/kg BU). It is concluded that BU at 9.0 mg/kg (or 9.25 mg/kg) is an appropriate dose level to administer (10 times over 21 days) to induce CBMA at approximately day 50–120 post‐dosing.

Evaluation of the haemopoietic reservoir in de novo haemolytic paroxysmal nocturnal haemoglobinuria

Summary. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haemopoietic stem cell (HSC). The pathogenetic link with bone marrow failure is well recognized; however, the process of clonal expansion of the glycosylphosphatidylinositol (GPI)‐deficient cells over normal haemopoiesis remains unclear. We have carried out detailed analysis of the stem cell population in 10 patients with de novo haemolytic PNH using the long‐term culture‐initiating cells (LTC‐IC) assay in parallel with measurements of CD34+ cells and mature haemopoietic progenitors, granulocyte–macrophage colony‐forming unit (CFU‐GM) and CFU‐erythroid [burst‐forming units erythroid (BFU‐E) + CFU granulocyte/erythroid/macrophage/megakaryocyte (GEMM)]. All patients had hypercellular bone marrows with erythroid hyperplasia, normal blood counts or mild peripheral blood cytopenias, increased reticulocyte counts and evidence of deficient GPI‐anchored proteins. We found a significant reduction in the LTC‐IC frequency in the CD34+ compartment of PNH patients (mean 2, range 1·3–3·0; n = 6) compared with normal donors (mean 13, range 5·2–45·5; n = 21) (P < 0·0001). Furthermore, there was a significant reduction in the erythroid compartment [CFU‐E/105 bone marrow mononuclear cells (BMMC) and CFU‐E/105 CD34+ cells] of PNH patients, but no significant difference in the granulocyte–monocyte precursors (CFU‐GM/105 BMMC or CFU‐GM/105 CD34+ cells) compared with normal donors, suggesting that there is a defect in the early stem cell pool in PNH patients without clinical or haematological evidence of bone marrow failure.

Near-optimal Conditions for the In Vitro Culture of Hemopoietic Progenitor Cells in Bone Marrow from the Rat

In vitro techniques for the culture of hemopoietic stem cells and committed hemopoietic progenitor cells in rat bone marrow have not been adequately described in the literature. In the present investigations, and using commercially available hemopoietic cytokines and growth factors, the conditions required to perform long-term bone marrow culture (LTBMC) using rat femoral bone marrow were studied, in conjunction with the colony-forming unit cell assay (CFU-C), to quantify the number of progenitor cells. CFU-C production by LTBMCs, set up using Iscove’s modified Dulbecco’s medium supplemented with fetal calf serum and horse serum, ceased after week 2 of culture. However, the duration of CFU-C production was significantly increased by supplementing LTBMCs with the cytokine recombinant mouse stem cell factor or recombinant rat stem cell factor.