Correlation of frameshift mutagenicity with DNA intercalation by CGS 20928A using an in vitro DNA unwinding assay.

A compound's mutagenicity in different Salmonella tester strains can suggest its mechanism of reaction with DNA. Clear confirmation of such a mechanism, however, requires a direct test of the compound's reaction with DNA, often relying on specific in vitro studies. We report the use of a rapid in vitro test designed to measure DNA unwinding, a characteristic of DNA intercalators and many frameshift mutagens. CGS 20928A, an adenosine antagonist, produced a significant (> 2-fold) increase in revertants only for Salmonella tester strain TA1537, and only without metabolic activation. These data indicated that the compound was a direct acting frameshift mutagen and possibly intercalated into DNA. Our DNA unwinding assay indicated that at concentrations of > 0.1 mM CGS 20928A behaved like known intercalating compounds in that it unwound DNA. These concentrations of compound are comparable to those found mutagenic to TA1537. By comparison, the frameshift mutagen and known intercalating compound 9-aminoacridine unwound DNA in this assay in a concentration dependent fashion between 6-12 microM. ICR-191, another acridine frameshift mutagen, also unwound DNA. A compound structurally related to CGS 20928A, which was not mutagenic in Salmonella tester strains, did not produce any DNA unwinding even at 10 mM. Because the assay uses microgram quantities of material, it should be ideal for screening small amounts of congeneric series suspected of frameshift mutagenicity.

Inter-laboratory ring trial of in vitro DNA repair tests using rat hepatocytes: further testing and conclusive remarks.

The results of the extension of a collaborative study for the detection of chemical-induced DNA damage in rat hepatocytes in vitro are presented in this report. Three coded compounds, i.e. 1,4-butanediol dimethanesulphonate, hydrazine sulphate and sodium dichromate, were tested for DNA repair synthesis by seven different laboratories, either using autoradiographic procedures or the liquid scintillation counting technique. Inter-laboratory standardization was intentionally not requested in order to investigate the validity of each study design under routine conditions. 1,4-Butanediol dimethanesulphonate was clearly positive in most laboratories; sodium dichromate was generally positive, while the results on hydrazine sulphate were contradictory.

Testing of hydralazine in in vivo-in vitro hepatocyte assays for UDS and stimulation of replicative DNA synthesis.

The vasodilator hydralazine was tested for induction of DNA-repair synthesis and stimulation of replicative DNA synthesis in rat hepatocytes after administration in vivo, either once or repetitively. No increase in unscheduled or replicative DNA synthesis was observed. By contrast, positive controls clearly induced DNA-repair synthesis, either after a single treatment (4-aminobiphenyl, dimethylnitrosamine and methyl methanesulphonate) or after repetitive treatment (benzo[a]pyrene), or stimulated replicative DNA synthesis (carbon tetrachloride and dimethylnitrosamine). Thus, hydralazine displayed no genotoxic and no tumour-promoting activity in these in vivo-in vitro test systems.

Anchorage-independent muscle cell differentiation.

Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures. Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not only were the same protein species synthesized, but also the relative intensities of the spots corresponding to known muscle-specific proteins as well as the patterns of the many unidentified spots were similar. Synthesis of creatine kinase subunits B and M at different times was determined. In both suspended and reattached cells there was, as expected for differentiating myogenic cells, a marked increase by day 5 in the ratio of M to B subunits synthesized. Immunofluorescent staining with antibodies against an M-line protein with M(r) 165,000 revealed myofibrils partially wound about the nuclei of suspended cells; these became strung out in the axis of the cell as reattached cells elongated. The synthesis of muscle proteins, their assembly into myofilaments, and formation of well-organized myofibrils is evidently not anchorage dependent. However, proper alignment of parallel arrays of myofibrils in register does appear to require cell attachment to substrate.