André Sels

Potentiation of oxygen toxicity by menadione in Saccharomyces cerevisiae

The cytotoxicity of molecular oxygen can be sharply increased in the yeast Saccharomyces cerevisiae by the use of redox compounds capable of shunting electrons in vivo and of spontaneous reoxidation under aerobic conditions. Among these redox compounds, menadione (Vitamin K3) is particularly able to stimulate the cyanide-resistant respiration of the yeast cells. Under steady-state conditions, the efficiency of menadione is modulated by the physiological state of the yeast cells and also depends on the availability of reducing agents within the cell. Menadione shows lethal effects towards yeast cells in the presence of O2 only, as a result of the production of toxic metabolites like O2-. and H2O2 which are actually detected in the extracellular fluid. Inhibitors of the enzymes scavenging O2-. and H2O2 generally potentiate the lethal effects of this redox compound. On the other hand, superoxide dismutase and/or catalase supplemented into the incubation buffer have been found to protect the cells to various extents from the cytotoxic effects of menadione. Our data support the following conclusions: When the cellular enzymatic defences are functional, the moderate lethality induced by menadione is principally mediated by O2-. ions acting on the outer side of the cell (peripheral region). In the presence of cyanide, but not of azide, the loss of viability also results from additional damage occurring within the inner cell region. In this case, intracellular injury can be caused by H2O2 alone but our data also suggest that during redox cycling more reactive species--O2-. and probably OH.--are generally intracellularly and are involved in the cytotoxic process.

Catalase anabolism in yeast: Loss of regulation by oxygen of catalase apoprotein synthesis after mutation

SummaryA mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media.Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group.Regulation of enzyme synthesis by catabolite repression (glucose effect) persists, unmodified by reference to the wild-type parental strain.Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production.Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.

Sensitivity of yeast cells to reactive oxygen species generated in the extracellular space.

Even when cytoplasmic scavenging activities are plentiful, yeast cells (S. cerevisiae) remain particularly sensitive towards reactive oxygen species generated in the extracellular space (either by the xanthine/xanthine oxidase reaction or by the redox cycling of menadione). A sharp reduction of the extent of cellular alterations when SOD and/or catalase were supplemented in the incubation buffer, points to a contribution of both O-.2 and H2O2 in the toxic process. Although oxygen metabolites as well as t-butylhydroperoxide (tBH), a highly toxic organic peroxide, may be directly responsible for cellular damage, their toxicity is largely reduced in the presence of Desferal. A role of metal ions in potentiating the toxicity points to the involvement of OH. radicals, actually produced in the medium. With tBH, metal cations would be rather active in promoting peroxidative chain reactions. In the case of an extracellular oxidative attack, it may be foreseen that the plasma membrane will form a preferential target. An increased permeability of the plasma membrane towards ionized molecules and uncharged polycarboxylic acids is indeed observed after an oxidative treatment. The loss of selective permeability is, as a rule, correlated with a drop in viability. Early alterations, disrupting the functional organization of the plasma membrane have been sought. The permease involved in the active transport of purine(s) has appeared to be an appropriate marker for checking its functional integrity. This transport function appears to be very sensitive to damage induced by O-.2 generators, particularly under conditions in which the resulting lethality is still kept low and in which the energization of active transport processes remains unimpaired.

Thermosensitive respiratory deficiency in yeast associated with specific effects on particulate cytochrome oxidase.

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37 degrees C, has been selected; at 25 degrees C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a,a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation.