Metabolism of hexamethylene bisacetamide and its metabolites in leukemic cells

We investigated whether leukemic cell lines could convert hexamethylene bisacetamide (HMBA) to any of the metabolites previously identified and quantified in the urine and plasma of patients treated with HMBA. After 5-7 days of incubation with 1-2 mM HMBA, HL60 human promyelocytic leukemic cells, L1210 and P388 murine lymphoblastic leukemic cells, and Friend murine erythroleukemia cells contained 4 of the previously identified metabolites of HMBA. Gas chromatography/mass spectrometry confirmed the presence of N-acetyl-1,6-diaminohexane (NADAH), 1,6-diaminohexane (DAH), 6-acetamidohexanoic acid (AcHA), and 6-aminohexanoic acid (AmHA). Gas chromatography with nitrogen-phosphorus selective detection was used to quantify cellular concentrations of each metabolite. Cellular concentrations of AmHA and DAH were greater than those of NADAH and AcHA but no concentration of a metabolite exceeded that of HMBA. Metabolites were not detected in media from cells incubated with HMBA. Friend murine erythroleukemia cells that were resistant to HMBA contained only HMBA and NADAH. Moreover, the concentrations of NADAH in Friend murine erythroleukemia cells that were resistant to HMBA were less than those in the other cell lines studied. HL60 cells accumulated HMBA rapidly. NADAH, DAH, AcHA, and AmHA appeared sequentially in HL60 cells that were incubated with HMBA. NADAH appeared very rapidly, but concentrations of DAH were greater than or equal to those of NADAH by 8 h. AcHA and AmHA were not detected in cells before 24-48 h of incubation with HMBA. HL60 cells incubated with individual HMBA metabolites were able to accumulate each compound and to interconvert some: cells incubated with NADAH also contained DAH, AcHA, and AmHA; cells incubated with AcHA also contained low concentrations of AmHA; cells incubated with DAH also contained AmHA; and cells incubated with AmHA contained no other HMBA metabolites. HMBA was not present in cells incubated with any of its known metabolites. These results document the ability of various leukemic cells to metabolize HMBA, indicate the unidirectional catabolism of that compound, and may have implications as to its mechanism of action.