Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response

Abstract

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho0] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho0] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 microg ml-1). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37 degrees C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.

FIG. 1

Sensitivity of yeast cells to LoaOOH. (A) Wild-type cells grown to an OD₆₀₀ of 1 in YEPD medium were treated with LoaOOH for 1 h at 30°C. Cells were diluted and plated in triplicate onto YEPD medium to monitor cell viability. Percent survival is expressed relative to the untreated control culture (100%). Symbols: □, LoaOOH-treated cells; ◊, methanol-treated control; ○, linoleic acid control; ▵, lipoxygenase control. (B) The kinetics of cell death was determined by sampling untreated and treated cultures at 20-min intervals for 1 h, using different concentrations of LoaOOH as indicated. Data are means of triplicates from a representative experiment.

FIG. 2

Adaptation of wild-type cells to a lethal dose of LoaOOH. Cells can mount an inducible adaptive response to LoaOOH which is dependent on protein synthesis. Cells were pretreated with a sublethal dose of LoaOOH (0.05 mM; 1 h) prior to challenge with a lethal dose (0.2 mM; 1 h). The cytosolic protein synthesis inhibitor CHX was used at a concentration of 25 μg ml⁻¹. Data are means of triplicates from a representative experiment.

FIG. 3

The presence of oxygen is required for maximal sensitivity to LoaOOH. Pure oxygen or argon was bubbled through culture aliquots for 15 min prior to addition of different concentrations of LoaOOH for 1 h. Cell viability was determined as described for Fig. 1 and compared to that of a control which received LoaOOH treatment only. Data are means of triplicates from a representative experiment.

FIG. 4

Cross-adaptation to other stresses indicates overlapping response systems. (A) Adaptation of wild-type cells to a lethal dose of LoaOOH (0.2 mM; 1 h) following pretreatment with a sublethal dose of MDA (1 mM; 1 h). (B) Adaptation of wild-type cells to a lethal dose of LoaOOH (0.2 mM; 1 h) following pretreatment with a mild heat shock (37°C; 1 h). (C) Fold adaptation, following pretreatment with a sublethal dose of LoaOOH (0.05 mM; 1 h), to the organic hydroperoxides cumene hydroperoxide (COOH; 4 mM) and tert-butyl hydroperoxide (tBOOH; 12 mM), the superoxide-generating agents paraquat (PQ; 15 mM) and menadione (MD; 6 mM), MDA (5 mM), and H₂O₂ (5 mM). An asterisk indicates that no adaptive response was detected. Data are means of triplicates from a representative experiment.

FIG. 5

Sensitivities of the wild type and oxidative-stress mutants to LoaOOH. Yeast strains CY4 (wild type), CY4p ([rho⁰] petite mutant), CY9 (gsh1 petite mutant), CY97 (gsh2), CY7 (glr1), and CY29 (yap1) were grown to exponential phase and treated with various concentrations of LoaOOH for 1 h. Samples were diluted and plated on YEPD to monitor cell viability. Data are means of triplicates from a representative experiment.

FIG. 6

Inhibition of respiration or ATP synthesis can increase cellular resistance to LoaOOH. (A) Wild-type cells were treated with a range of concentrations of one of the respiratory inhibitors flavone (0.05 to 0.2 mM), antimycin (0.1 to 0.5 mM), KCN (0.1 to 0.5 mM), and sodium azide (0.1 to 0.5 mM) or with an inhibitor of ATP synthesis, oligomycin (0.2 mM), for 1 h prior to treatment with a lethal dose of LoaOOH (0.2 mM). (B) The wild-type strain CY4 (wt grande) and its isogenic coq3 and cox6 mutants (lacking ubiquinone and cytochrome c oxidase subunit 6, respectively) were tested for their sensitivities to LoaOOH as described for Fig. 5. Data are means of triplicates from a representative experiment.