Role of oxygen and antioxidants in the response of yeast to heat and oxidative stresses

Abstract

A study was conducted on the physiological effects of heat and oxidative stress on the yeast 'Saccharomyces cerevisiae'. The effects of oxygenation and synthetic and naturally-occurring antioxidants on cell survival were examined. Two cell types were used as experimental systems at 25°C. In one case, cells were grown aerobically in an orbital shaker and, in the other, cells were grown anaerobically in water-jacketted fermenters under a constant stream of high purity nitrogen. Cells were subjected to a mild heat shock from 25°C to 37°C for 45 min before exposure to a heat stress at 48°C over a time course. The ability of cells to survive, this heat protocol, as determined by plate counts, was termed induced thermotolerance. On the other hand, the ability of cells to survive a direct heat stress from 25°C to 48°C over a time course was termed intrinsic thermotolerance. Oxidative stress was measured by incubation of cells at 25°C with H₂0₂ (5-10 mM). In general, aerobically grown cells were more stress tolerant than anaerobically grown cells. This observation was correlated, to some extent, with the membrane fatty acyl composition of the cells. The addition of synthetic antioxidants (butylated hydroxytoluene, propyl gallate) was detrimental to cell survival against heat. By contrast, naturally-occurring antioxidants (ascorbate, α-tocopherol,β-carotene) in general appeared to act as growth promotants. Oxygenation, either during or after the heat treatments, was highly beneficial (3 to 15-fold) to cell survival. However, oxygenation prior to thermal treatment was detrimental as was oxygenation in the presence of cycloheximide which inhibited protein synthesis. Heat shock induced (37°C/45 min) protein synthesis was monitored by ³⁵S-methionine labelling and SDS-PAGE analysis. The classical heat shock proteins (hsp) hsp 100, hsp 90, hsp 70, and hsp 60 were identified in aerobic and anaerobic cells. Additional bands at 36 and 40 kDa were observed in cells oxygenated (45 min) at 25°C but no additional bands were observed in cells subjected simultaneously to heat treatment and oxygenation. Trehalose concentration in anaerobic cells (8-12% w/w) was relatively high compared to aerobic cells (< O.5% w/w). Heat shock induced a modest increase in trehalose levels. These results indicated that heat shock proteins but not trehalose may play a role in stress tolerance. The major conclusions from the present studies were that exogenous antioxidants, synthetic or naturally-occurring, had no significant protective effect on yeast thermotolerance or oxytolerance. The marked beneficial effects on cell survival by oxygenation were proposed to be partly due to protection of key mitochondrial functions.