No abstract

Biological models have long been used to establish the cytotoxicity and cytostaticity of natural and/or synthetic chemical compounds. Current assay techniques, however, typically require the use of expensive technological equipment or chemical reagents, or they lack adequate testing sensitivity. The poissoner quantitative drop test (PQDT) assay is a sensitive, inexpensive and accurate method for evaluation of cytotoxicity and/or cytostatic effects of multiple chemical compounds in a single experiment. In this study, the sensitivity of the PQDT assay was evaluated in a wild-type Saccharomyces cerevisiae strain using 4-nitroquinoline-N -oxide (4-NQO) and methyl methanesulfonate (MMS), both cytotoxic and genotoxic standard compounds, and cytostatic 5-fluorouracil, an antitumoral drug. Yeast cell colony growth was measured in culture media containing increasing concentrations of the three chemical agents. The results showed that the PQDT assay was able to clearly differentiate the cytotoxic effect of 4-NQO and MMS from the cytostatic effect of 5-fluorouracil. Interestingly, the cytostatic effect of 5-fluorouracil followed an exponential decay curve with increasing concentrations, a phenomenon not previously described for this drug. The PQDT assay, in this sense, can be applied not only for cytotoxic/ cytostatic assays, but also for pharmacodynamic studies using Saccharomyces cerevisiae as a model. Cellular viability following DNA damage has been a topic of intense research in fields, such as genotoxicity, toxicology, carcinogenesis and cancer therapy [1]. Microorganism models can be used to evaluate cellular viability, because they can be easily and inexpensively cultured in the laboratory. Additionally, their small size, simple morphology and large surface area in relation to their size give microorganisms greater sensitivity than more complex organisms [2]. The yeast Saccharomyces cerevisiae is a model eukaryotic microorganism with a short generation time and very simple growth requirements. The molecular biology and genetics of this organism are well established. Mutants can be easily constructed and are commercially available, and plasmids and promoters for gene expression are well developed [3]. As such, Saccharomyces cerevisiae has been used extensively as a model organism for the study of mammalian diseases and pathways and for evaluation of toxic compounds [4], such as genotoxic and cytotoxic agents.Yeast cell colony growth can be monitored on agar plates containing dilutions of toxic agents to establish cytotoxicity/ cytostaticity of a compound [5]. Many toxic compounds introduce DNA damage and cause formation of bulky lesions that block DNA replication and/or RNA transcription, leading to cell cycle arrest and the inability of the cells to form colonies [6]. Current colony counting techniques and methods used to measure the size of yeast colonies have drawbacks that include: (i) inadequate sensitivity to determine the effect of low doses of cytotoxic/cytostatic compounds; (ii) high expense due to...

Calcium (Ca(2+)) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca(2+) homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca(2+) homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca(2+) transporter of Golgi complex, interacts with Cod1p, which regulates Ca(2+) levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Delta, cod1Deltarad4Delta, and cod1Deltapmr1Delta strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Deltapmr1Delta and cod1Deltarad4Delta strains have a strong arrest at G(2)/M phases of cell cycle after 4-NQO treatment, while pmr1Deltarad4Delta have a similar sensitivity and cell cycle arrest profile when compared to rad4Delta after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca(2+) transporters affect the repair of 4-NQO-induced DNA damage.

No abstract