We have postulated that the p-NO2 group of chloramphenicol (CAP) is the structural feature underlying aplastic anemia from this drug. In a series of studies to examine this hypothesis we have demonstrated the toxic nature of the CAP-reduction intermediate nitroso CAP (NO-CAP) and its damaging effect on isolated DNA in vitro. The present study was designed to examine the comparative effects of CAP, NO-CAP, and thiamphenicol (TAP) on the integrity of DNA in intact cells. By using the alkaline elution technique of Kohn, DNA damage in the form of single strand breaks could be readily demonstrated in cultured Raji cells and in PHA-stimulated normal human lymphocytes by small concentrations of NO-CAP (0.05-0.1 mM). A small but reproducible effect was observed from large concentrations of CAP (2 mM). In contrast, TAP, lacking the p-NO2 group, was without effect.

To explore the potential role of some bacterial metabolites of chloramphenicol (CAP) in CAP-induced hematotoxicity, we examined their cytotoxic effects on bone marrow cells in vitro using a number of cytotoxicity parameters. Among the metabolites tested, dehydro-CAP (DHCAP) and p-nitrophenyl-2-amino-3 hydroxypropanone-HCI (NPAP) were more toxic than CAP. DHCAP was at least as toxic as nitroso-CAP. At concentrations of less than or equal to 10(-4) mol/L, DHCAP caused total irreversible inhibition of myeloid colony (CFU-GM) growth and 80% inhibition of DNA synthesis in human bone marrow. Incubation of human bone marrow cells with 10(-4) mol/L nitroso-CAP or DHCAP for 24 hours resulted in 75% and 65% cell death respectively. Although DHCAP was 10- to 20-fold more cytotoxic than CAP, it was only one third as effective in inhibiting mitochondrial protein synthesis, indicating that DHCAP exerts its toxic effect by alternate mechanisms. The cytotoxicity of DHCAP and its relative stability, compared to the unstable nitroso CAP, suggest that this bacterial metabolite of CAP, and possibly others, may play a significant role in CAP-induced hematotoxicity.

Four chloramphenicol (CAP) metabolites known to be produced by intestinal bacteria were examined with respect to their capacity to induce DNA damage in intact cells. The induction of DNA single-strand breaks in Raji cells, activated human lymphocytes, and human marrow cells was assayed by the alkaline elution technique. One of the four compounds tested, dehydro-CAP, was capable of inducing DNA single-strand breaks in all three cell systems at concentrations of 10(-4) M. This effect is comparable to that observed previously with nitroso-CAP, the nitroreduction intermediate of CAP. The nitroreduction of dehydro-CAP by human bone marrow cell homogenate was detected by the production of the corresponding amino derivative amounting to 5.6 X 10(-5) M from 2 X 10(-3) M substrate under aerobic conditions. In sharp contrast, nitroreduction of CAP by bone marrow could not be demonstrated. The genotoxicity of dehydro-CAP, its relative stability compared to the nitroso-CAP, and its nitroreducibility by bone marrow suggest that this bacterial metabolite of CAP may play a key role as a mediator of aplastic anemia in the predisposed host.

Mutation and killing induced by X radiation and 60CO gamma radiation were studied in six different histidine-requiring auxotrophs of Salmonella typhimurium. Strain TA100, which is sensitive to base-pair substitutions, and strains TA2637 and TA98, which are sensitive to frameshifts, carry the pKM101 plasmid and exhibit significantly higher radiation-induced mutations compared to their plasmidless parent strains TA1535, TA1537, and TA1538, respectively. Among the plasmid-containing strains, TA98 and TA2637 are much more sensitive to the mutagenic action of radiation than is TA100 based on a comparison with their respective spontaneous mutation rates; however, no uniformity was observed in the responses of the strains to the lethal action of ionizing radiation. The pKM101 plasmid provides partial protection against lethality in TA100 and TA2637, whereas the same plasmid enhances the lethal action of ionizing radiation in TA98. The following conclusions are consistent with these observations: (1) the standard Ames Salmonella assay correctly identifies ionizing radiation as a mutagenic agent; (2) frameshift-sensitive parent strains are more sensitive to the mutagenic effects of ionizing radiation than is the only strain studied that is sensitive to base-pair substitutions; and (3) enhancement of mutagenesis and survival is related to plasmid-mediated repair of DNA damage induced by ionizing radiation and does not involve damage induced by Cerenkov-generated uv radiation which is negligible for our irradiation conditions.

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