Low ergosterol content in yeast adh1 mutant enhances chitin maldistribution and sensitivity to paraquat-induced oxidative stress.

Alcohol dehydrogenases catalyse the reversible oxidation of alcohols to aldehydes or ketones, with concomitant reduction of NAD(+) or NADP(+) . Adh1p is responsible for the reduction of acetaldehyde to ethanol, while Adh2p catalyses the reverse reaction, the oxidation of ethanol to acetaldehyde. Lack of Adh1p shifts the cellular redox balance towards excess NADH/NADPH and acetaldehyde, while absence of Adh2p does the opposite. Yeast mutant adh1Δ had a slow growth rate, whereas adh2Δ grew like the isogenic wild-type (WT) during prediauxic shift fermentative metabolism. After 48 h WT and mutants reached the same number of viable cells. When exponentially growing (LOG) cells were exposed to calcofluor white, only mutant adh1Δ displayed an irregular deposition of chitin. Quantitative analyses of both LOG and stationary-phase cells showed that adh1Δ mutant contained significantly less ergosterol than cells of WT and adh2Δ mutant, whereas the erg3Δ mutant contained extremely low ergosterol pools. Both adh1Δ and adh2Δ mutants showed higher-than-WT resistance to heat shock and to H(2) O(2) but had WT resistance when exposed to ultraviolet (UV) light and the DNA cross-linking agent diepoxyoctane, indicating normal DNA repair capacity. Mutant adh1Δ was specifically sensitive to acetaldehyde and to membrane peroxidizing paraquat. Our results link the pleiotropic phenotype of adh1Δ mutants to low pools of ergosterol and to reductive stress, and introduce the two new phenotypes, resistance to heat shock and to H(2) O(2) , for the adh2Δ mutant, most probably related to increased ROS production in mitochondria, which leads to the induction of oxidative stress protection.

DNA repair mutant pso2 of Saccharomyces cerevisiae is sensitive to intracellular acetaldehyde accumulated by disulfiram-mediated inhibition of acetaldehyde dehydrogenase.

Blocking aldehyde dehydrogenase with the drug disulfiram leads to an accumulation of intracellular acetaldehyde, which negatively affects the viability of the yeast Saccharomyces cerevisiae. Mutants of the yeast gene PSO2, which encodes a protein specific for repair of DNA interstrand cross-links, showed higher sensitivity to disulfiram compared to the wild type. This leads us to suggest that accumulated acetaldehyde induces DNA lesions, including highly deleterious interstrand cross-links. Acetaldehyde induced the expression of a PSO2-lacZ reporter construct that is specifically inducible by bi- or poly-functional mutagens, e.g., nitrogen mustard and photo-activated psoralens. Chronic exposure of yeast cells to disulfiram and acute exposure to acetaldehyde induced forward mutagenesis in the yeast CAN1 gene. Disulfiram-induced mutability of a pso2Delta mutant was significantly increased over that of the isogenic wild type; however, this was not found for acetaldehyde-induced mutagenesis. Spontaneous mutability at the CAN1 locus was elevated in pso2Delta, suggesting that growth of glucose-repressed yeast produces DNA lesions that, in the absence of Pso2p-mediated crosslink repair, are partially removed by an error-prone DNA repair mechanism. The use of disulfiram in the control of human alcohol abuse increases cellular acetaldehyde pools, which, based on our observations, enhances the risk of mutagenesis and of other genetic damage.