Use of PMA1 as a housekeeping biomarker for assessment of toxicant-induced stress in Saccharomyces cerevisiae.
The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence. The reporter assay deployed a simple and robust protocol for the rapid detection of toxic effects within a 96-well microplate format. Fluorescence emissions were normalized to cell growth determined by absorption and were correlated to internal reference standards. The results were expressed as effective concentrations (EC20). Dose-response experiments were conducted in which yeast cells were exposed in minimal medium and in the presence of 20% fetal calf serum to sublethal concentrations of an array of heavy metals, salt, and a number of stress-inducing compounds (Diclofenac, Lindane, methyl-N-nitro-N-nitrosoguanidine [MNNG], hydroxyurea, and caffeine). Long-term exposure (7 h) played a considerable role in the adaptive response to intoxication compared to early responses at 4 h exposure. The data obtained after 4 h of exposure and expressed as EC20 were compared to 50% inhibitory concentration values derived from cell line and ecotoxicological tests. This study demonstrates the versatility of the novel biomarker to complement existing test batteries to assess contaminant exposure and effects.
Schmitt
Marcel
M
Institut für Zelluläre und Molekulare Botanik, AG Molekulare Bioenergetik, Universität Bonn, Kirschallee 1, 53115 Bonn, Germany.
Schwanewilm
Petra
P
Ludwig
Jost
J
Lichtenberg-Fraté
Hella
H
D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D002417:Cattle; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D066298:In Vitro Techniques; D019216:Metals, Heavy / Q000633:toxicity; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D011401:Promoter Regions, Genetic; D006180:Proton-Translocating ATPases / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
D015415:Biomarkers; D004271:DNA, Fungal; D019216:Metals, Heavy; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008769:Methylnitronitrosoguanidine; D049452:Green Fluorescent Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases