Sister chromatid exchange induced by etheno-ATP derivatives in vitro.

Genotoxic activities of a series of commercially purchased 1,N6-ethenoadenosine (epsilon-Ado) and epsilon-deoxyadenosine (epsilon-dAdo) derivatives were assessed using the sister chromatid exchange (SCE) assay in murine spleen lymphocytes in vitro. Of the epsilon-Ado adducts evaluated for SCE induction epsilon-ATP and epsilon-dATP were highly active (5x baseline) SCE inducers over a concentration range of 50-150 microM. Moderate SCE-inducing activities were seen with epsilon-dAdo, epsilon-A, and epsilon-AMP. epsilon-A was of particular interest in that spleen lymphocytes from a single mouse were highly sensitive to SCE (greater than 50 SCE/cell at 75 microM). epsilon-Ado was weakly effective and epsilon-ADP and epsilon-dAMP did not produce significantly elevated SCEs. Cocanavalin A-stimulated T-lymphocytes and lipopolysaccharide-stimulated B-lymphocytes exhibited comparable SCE responses to epsilon-A, epsilon-AMP, and epsilon-dATP. However, B-lymphocytes were considerably less sensitive than T-lymphocytes to epsilon-dAdo and epsilon-ATP. Evaluation of the purities of specific epsilon-Ado derivatives, as performed by high-performance liquid chromatography and thin layer chromatography, failed to detect potential contaminants as cytogenetically active agents. However, a difference (about threefold) in cytogenetic activities of two lot numbers of epsilon-ATP paralleled the difference in UV absorbance of quivalent concentrations (mg/ml), prepared according to the manufacturers stated purity. Any impurities likely to be present were consistent with inactive nonchromophoric compounds such as buffer salts. Because of the direct genotoxic activity of epsilon-A in intact mammalian cells, we suggest that intracellular adenylate pools, including the prominent ubiquitous nucleotide ATP, are non-DNA targets for epsilon-modification by active metabolites and the resulting epsilon-adducts are likely to be active moieties in SCE induction and in neoplastic transformation produced by ethyl carbamate.

Identity of differentiation inducing factor and tumour necrosis factor.

Human myelogenous leukaemic cells can be induced to differentiate into the monocyte/macrophage pathway by protein inducers called differentiation inducing factors (DIF) in conditioned media of mitogen-stimulated human peripheral blood leukocytes. However, human DIF has not yet been well characterized. DIF is known to be a T-cell lymphokine, as it can be obtained from the T-cell line HUT-102 and can be partially purified from medium conditioned by phytohaemagglutinin (PHA)-stimulated lymphocytes. We found that monocytes also produce factor(s) that induce differentiation of human myelogenous leukaemia cell lines to cells with macrophage-like characteristics. This factor(s) has activity different from that of colony-stimulating factor(s) or interferons. We have now purified a DIF to homogeneity from medium conditioned by PHA-stimulated leukocytes using a human myeloblastic leukemia cell line, ML-1, as target cells. The purified DIF has a relative molecular mass (Mr) of approximately 17,000, with an NH2-terminal sequence the same as that of human tumour necrosis factor (TNF). Recombinant human TNF (rHuTNF) induces differentiation of ML-1 cells and an anti-pDIF monoclonal antibody can neutralize both differentiation inducing activity and cytotoxic activity of DIF and rHuTNF. The findings indicate that one of the DIF(s) produced by leukocytes is probably TNF.