nitrosochloramphenicol and Anemia--Aplastic

Reduction of the nitro group of chloramphenicol (CAP) gives rise to more highly reactive intermediates which may in involved in the aplastic anemia associated with CAP use. One such intermediate, nitroso-chloramphenicol (NO-CAP), has been found to be a potent agent for mediating degradation of isolated DNA. In a reaction mixture containing 100 microM NO-CAP, 100 microM CuCl2, and 5 mM NADH, 7 micrograms of Escherichia coli [3H]DNA was completely degraded to acid-soluble fragments in 30 min. Damage to DNA was in the form of single-stranded scissions. The requirement for copper was specific, and copper chelating reagents blocked the degradation. The need for a reducing agent could be met equally well by NADH or NADPH, but not by sulfhydryl reagents such as glutathione, dithiothreitol and 2-mercaptoethanol. Oxygen was also necessary for the NO-CAP mediated DNA damage, with reduced forms of oxygen participating in the reaction. A role for H2O2 was indicated by the inhibition of the degradation seen when catalase was included in the mixture. Hydroxyl radicals are known to be produced in the reaction of H2O2 with certain transition metals. Scavangers of hydroxyl radicals also inhibited strand-scission, suggesting that the radicals may be the primary agents in DNA degradation. The importance of the nitroso moiety of NO-CAP was evidenced by the lack of DNA damage seen when NO-CAP was replaced by CAP under the conditions tested.

Topics: Anemia, Aplastic; Chloramphenicol; DNA; Free Radicals; Humans; Oxidation-Reduction

A comparative study of the cellular transport of CAP and its nitroso derivative (NO-CAP) was carried out in Raji cells, a transformed human lymphoblastoid cell line. Both agents were concentrated by the cells by a factor of 3 (cellular/extracellular concentration ratio). The cellular uptake of NO-CAP, like that of CAP, was found to be rapid and temperature-independent. Thus the greater cytotoxicity of NO-CAP is apparently not due to an enhanced uptake of the nitroso derivative relative to CAP. In contrast to the similarity of uptake, NO-CAP becomes covalently bound to both Raji cells and freshly isolated human bone marrow cells to a much higher extent (15-fold). Also, cells previously loaded with CAP or NO-CAP retain three times as much of the nitroso compound during a 24 hr dialysis against a drug-free isotonic solution. The increased binding of NO-CAP to human hematopoietic cells attests to the greater reactivity of the p-substituted aromatic nitroso group and is consistent with the postulate that reduction products of the nitro group of CAP may be responsible for CAP-induced aplastic anemia.

Topics: Anemia, Aplastic; Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Line; Chloramphenicol; Dialysis; Humans; Tritium

We compared the effects of CAP and its nitroso derivative on DNA synthesis, CFU-C growth, and cell viability in human bone marrow and on mouse CFU-S viability in vitro. As previously reported from this laboratory, CAP inhibited DNA synthesis only when used in high concentrations (greater than 3 x 10(-4)M), and the inhibition was largely reversible. It also caused a reversible concentration-dependent inhibition of CFU-C growth but did not affect marrow cell viability. In sharp contrast, nitroso-CAP inhibited DNA synthesis in much lower concentrations and caused irreversible inhibition of CFU-C growth and cell death at 5 x 10(-5)M as well as irreversible mouse CFU-S damage. In a rapidly growing human lymphoid cell line, nitroso-CAP caused accumulation of cells in the G2M phase and increasing cell death within the arrested population. We have postulated from these and other observations that CAP-induced aplastic anemia occurs in the predisposed host who provides the milieu for the transformation of the p-NO2 group of the drug to toxic intermediates.

Topics: Anemia, Aplastic; Animals; Bone Marrow Cells; Chloramphenicol; Colony-Forming Units Assay; DNA; Hematopoietic Stem Cells; Hydroxylamines; Male; Mice; Structure-Activity Relationship; Thiamphenicol