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The ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and several cultured human malignant blood cell lines to analogues of "activated" cyclophosphamide, namely, 4-hydroperoxycyclophosphamide and mafosfamide, and to phosphoramide mustard was quantified with and without concurrent exposure to an inhibitor of aldehyde dehydrogenase activity, namely, disulfiram, cyanamide, diethyldithiocarbamate, or ethylphenyl(2-formylethyl)phosphinate. Inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of 4-hydroperoxycyclophosphamide and mafosfamide toward all of the hematopoietic progenitors; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Potentiation of the cytotoxic action of mafosfamide toward cultured human malignant blood cells was minimal. Spectrophotometric assay revealed little NAD-linked aldehyde dehydrogenase activity present in the cultured human tumor cell lines as compared to that found in normal mouse liver or oxazaphosphorine-resistant L1210 cells. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major intermediate in cyclophosphamide bioactivation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that human multipotent hematopoietic progenitor cells contain the relevant aldehyde dehydrogenase activity, the relevant activity is retained upon differentiation to progenitors committed to the megakaryocytoid, granulocytoid/monocytoid, and erythroid lineages, and the relevant activity may be lost or diminished upon transformation of hematopoietic progenitors to malignant cells.

A spectrophotometric assay was developed and utilized to directly characterize aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide to carboxyphosphamide by soluble and solubilized particulate fractions prepared from mouse liver homogenates. Vmax values of 3310 and 1170 nmol/min/g liver were obtained for the soluble and solubilized particulate fractions respectively. Km values were 22 and 84 microM respectively. Alkaline pH optimums were observed in each case. Aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide by the soluble fraction was markedly more temperature responsive. Catalysis of aldophosphamide and acetaldehyde or benzaldehyde oxidation was apparently by the same isozyme(s) in the soluble fraction. Similarly, low Km (acetaldehyde/benzaldehyde) and high Km (acetaldehyde/benzaldehyde) isozymes each apparently catalyzed the oxidation of aldophosphamide in the solubilized particulate fraction. Our findings suggest that (1) oxidation of aldophosphamide to carboxyphosphamide by mouse liver is catalyzed largely by the predominant aldehyde dehydrogenase isozyme present in the soluble fraction (cytosol) of this tissue, and (2) isozymes that catalyze aldophosphamide oxidation are not different from those that catalyze the oxidation of acetaldehyde and benzaldehyde, though the relative contribution of each isozyme within the solubilized particulate fraction to the catalysis of aldophosphamide oxidation remains to be determined.