I want to determine the toxicity and genotoxicity of organic substances with a yeast-bioassay. Which test is the best one? Maybe the Green Screen Assay?
Is it also possible to identify the protein toxicity of organic substances?
i don't think there are difference in protocol for toxicity assay or minimum inhibitory concentration (MIC) assay. although i have screened the library of small molecules. in that many of them are toxicants and disinfectant like compounds and i follow the MIC determination protocol. for the determination of genotoxicity of compound comet assay or similar kind of protocol can be followed at sub inhibitory concentration of compound after treatment..
Dear Stephanie,
For checking general toxicity, as mentioned above it's better to check its effect on growth of yeast. Subsequently, you can focus on cell viability (arrest or death) and effect on cell cycle (FACS) will help in getting an idea about the toxicity.
In case of genotoxicity, I suggest you to do comet assay, tunnel assay, and other genotoxicity markers.
For studying toxicity of any molecule, yeast is a most easier, feasible and acceptable model now a days.
Good Luck...!!!
you could try dropping the yeast cultive (treated/untreated) as do in these papers, is easy and fast (but not quantitative)
-Identification and Dissection of a Complex DNA Repair Sensitivity Phenotype in Baker's Yeast. Ann Demogines, Erin Smith, Leonid Kruglyak, Eric Alani
-Shp1, a regulator of protein phosphatase 1 Glc7, has important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans. Kangdi Hua, Wanjie Lia, Haitao Wanga, Kun Chena, Yue Wangb,
Hi Stephanie,
Did you also think of a genotoxicity assay using the well described diploid yeast strain D7, with which you can test several agents for mitotic crossing-over (via ade2 and red/pink colonies), convertants (trp5) or revertants (ilv1). This strain is often used for toxicity screenings and was first described 1975 by Zimmermann, Kern & Rasenberger.
Thanks for your answers.
I have already thought about the Comet assay and also the yeast strain D7. I will have a look for the other tests.
I don't want to test mycotoxins, but different organic substances.
I want to test substances like alcohols, aldehydes, esters, ethers, aromates, carboxylic acids, amines, amides, haloalkanes, ...
Dear Stephanie,
First, results of your toxicity or genotoxicity evaluation depend of organism and strain you choose. IC50, MIC or CL 50 for a substance are different between two strains of a same organism. in other terms, toxicity and genotoxicity aren't absolute phenomenon. Because of it none bioassay is better than other. If you want to determine toxicity and genotoxicty of organic coumpounds, it's better to use several bioassays ( yeast, bacteria, human cells,...).
Second, toxicity is not a linear phenomenon. It can be acute, sub-letal, chronicle, long term, with or without a windows range. Even if noe alone bioassay can be report all of these situation, classicals bioassays, like MIC determination or growth inhibition in liquid or solid plate, give a global information about level impact. This level including "postive" impact (growth stimulation for example) and "negative" impact ( inhibition).
Third, for genotoxicity, as for toxicity, none bioassay can report all type of DNA attack ( clastogen or aneugen for example; with or without metabolic activation).
Difference between bioassays concern sensibility (50 cut for Comet assay, 1 for H2ax,...), level of bad answer (for example, menthol is genotoxic in some bio-assay), and type of DNA attack take account.
About yeast you have Comet assay, Radarscreen assay, Greenscreen assay, or ways suggested by Daniela, Fernando, Upendar or Anubhav. To have a good idea of genotoxic impact is better to use a series of tests including at least a dimension of metabolic activation ( by S9 for example) .
Predictive Models for carcinogenicity and Mutagenicity : frameworks, state-of-the-art, and Perpectives E. benfenati Jornal of Environmental Science and Health PartC, 27: 57-90, 2009
For MIC use a standard dose-response, we do these in liquid (quantified on a plate reader at 600nm) and on solid (which is semi quantitative).
For toxicity/cell death use a colony-forming-unit assay. Treat cells with MIC and remove, wash with water and plate onto YPD agar plates, count the number of colonies relative to control. While "toxicity" can be due to several reasons this assay will identify compounds that are toxic, or at least irreversible growth inhibition.
For genotoxicity, we tend to use rad mutant sensitivity. Alternatively you can assay for resistance to canavanine as a test for mutagenesis.
Protein toxicity: Think about what this actually means. The only thing I can think of is that a compound might be a protein alkylating agent. You should be able to guess how likely this is by looking for electrophilic/reactive centres on the molecules.
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