Petite mutagenesis in Saccharomyces cerevisiae by a series of bis-cationic trypanocidal drugs

Abstract

A group of bis-cationic imidazo[1,2-a]pyridinium salts and related compounds, some of which exhibit in vivo trypanocidal activity, have been investigated for induction of petite mutagenesis in Saccharomyces cerevisiae. All of the compounds which are active trypanocides induce mutagenesis. There appears to be a correlation between trypanocidal activity and mutagenic activity which may have its structural origin in the spatial separation of the cationic centers.A series of imidazo[1,2-a]pyridinium and related heteroaromatic salts was recently reported to have good activity against the bloodstream form of Trypanosoma rhodesiense in mice (22). The structures of these compounds resemble those of various bis-cationic antitumor compounds such as diamidines, phthalanilides, and bis-quaternary ammonium heterocycles (1), which are generally believed to interact with DNA by binding in the minor groove (6, 11-13 For all experiments, the 1-ml sample was thawed, added to 10 ml of fresh medium, and grown for exactly 2 h before use.Microtiter assay for petite mutagenesis. The microtiter assay has been described in detail (7). Briefly, a log-phase culture was washed and then diluted into fresh growth medium. A 96-well microtiter tray (A/S Nunc, Roskilde, Denmark) was inoculated with (usually) 100-pd aliquots of the diluted yeast culture, and drugs were added at various dilutions to the wells to a maximum of 200 ,ug/ml. Drugs were dissolved in 50% ethanol, and dilutions were made so that there was no more than 1% ethanol in each well. Trays were incubated for 24 h at 30°C, an appropriate dilution was * Corresponding author.made from each well into saline, and 100 ,ul was plated onto each of three YC plates. Cell numbers were calculated so that the dilutions at this point were at least 1:104, thereby effectively washing drugs from cells by dilution (7).In some experiments, cells were tested in nongrowing conditions by diluting the cultures into 0.87% saline rather than complete medium. For a limited number of the compounds, samples were taken at various intervals in order to establish the rate of petite formation.Plates were incubated at 30°C for 3 days, and then all colonies were counted with an Artek model 880 counter. Scores of some plates were checked by manual counting. Colonies were then overlaid with tetrazolium in order to score petite colonies (16). Following a preliminary experiment to establish the correct concentration range, dose response of all drugs was tested at least twice and the data were compared for reproducibility. Maximum frequencies of petites were recorded. The dose to reduce numbers of yeast colonies to 50% of the value for the untreated control and the dose to convert 50% of the colonies to petite were both estimated by linear regression analysis. The presented data have been averaged. Table 1 gives the 50% lethal dose and 50% petite mutant conversion dose measured for the test compounds. Comparative data for berenil, stilbamidine, and the phthalanilides structures shown in Fig. 2 have been reported prev...

A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii agents

“…Other respiratory inhibitors, i.e., antimycin A and 2,4-dinitrophenol, were also effective at low concentrations. Ethidium bromide has also been reported to target yeast mitochondria by induction of petite mutants, which are deficient in respiration (27). Ethidium bromide had a marked ability to reduce the ATP pools of organism populations but was quite toxic in the animal model of infection, with little apparent reduction of organism burden at the concentrations used (45).…”

Microsomal Catalyzed N-Hydroxylation of Guanabenz and Reduction of the N-Hydroxylated Metabolite: Characterization of the Two Reactions and Genotoxic Potential of Guanoxabenz1

“…This result is in harmony with other investigations in which guanabenz was found to be nonmutagenic in various in vitro tests () and where other N -hydroxyamidinohydrazones were found to be mutagenic (). However, it does not hold in every case that amidinohydrazones are not mutagenic while N -hydroxyamidinohydrazones are because mutagenic activities have also been detected for some amidinohydrazones ( , ). The decrease in the mutagenicity of guanoxabenz in the presence of a metabolizing system could be attributable to the retroreduction to guanabenz even when the conditions of the Ames test (NADPH, pH 7.4) are not optimal for the N -reduction.…”

Guanylhydrazones in therapy of Pneumocystis carinii pneumonia in immunosuppressed rats

“…Current investigative interest in the mechanism of action of these drugs has focused on binding to the minor groove of DNA and inhibition of topoisomerase (8,10,17,20,22), although other pathways have also been studied (2,3,25). Studies of these drugs with trypanosomes have shown good correlation in the ability of these agents to bind to DNA, induce petite mutants in Saccharomyces cerevisiae, and exert trypanocidal activity in the mouse model (8,12,24). This approach has also been applied to P. carinii and other microbes, but further studies are needed to determine its value for prediction of in vivo activity (9,11,25,27).…”