Cellular damage induced by a sequential oxidative treatment on Penicillium digitatum.

AIM: To investigate the cellular damage on Penicillium digitatum produced by a sequential oxidative treatment (SOT), previously standardized in our laboratory, to prevent the conidia growth. Lethal SOT consists of 2-min preincubation with 10 ppm NaClO followed by 2-min incubation with 6 mmol l(-1) CuSO(4) and 100 mmol l(-1) H(2)O(2) at 25°C. METHODS AND RESULTS: After the application of lethal SOT or sublethal SOT (decreasing only the H(2)O(2) concentration), we analysed several conidia features such as germination, oxygen consumption, ultrastructure and integrity of the cellular wall and membrane. Also, we measured the production of reactive oxygen species (ROS) and the content of thiobarbituric acid-reactive species (TBARS). With the increase of H(2)O(2) concentration in the SOT, germination and oxygen consumption of conidia became inhibited, while the membrane permeability, ROS production and TBARS content of conidia increased. Several studies revealed ultrastructural disorganization in P. digitatum conidia after lethal SOT, showing severe cellular damage without apparent damage to the cell wall. In addition, mycelium of P. digitatum was more sensitive than conidia to the oxidative treatment, because growth ceased and permeability of the membranes increased after exposure of the mycelium to a SOT with only 50 mmol l(-1) H(2)O(2) compared to a SOT of 100 mmol l(-1) for these effects to occur on conidia. CONCLUSION: Our insights into cellular changes produced by the lethal SOT are consistent with the mode of action of the oxidant compounds, by producing both alteration of membrane integrity and intracellular damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results allow the understanding of SOT effects on P. digitatum, which will be useful to develop a reliable treatment to control postharvest diseases in view of its future application in packing houses.

Sites of electron transfer to membrane-bound copper and hydroperoxide-induced damage in the respiratory chain of Escherichia coli.

Previous studies in Escherichia coli as a model system for peroxide toxicity (L. Rodríguez-Montelongo, L. C. De la Cruz-Rodríguez, R. N. Farías, and E. M. Massa, 1993, Biochim. Biophys. Acta 1144, 77-84) have shown that electron flow through the respiratory chain supports a membrane-associated Cu(II)/Cu(I) redox cycle involved in irreversible impairment of the respiratory system by tert-butyl hydroperoxide (t-BOOH). In this paper, E. coli mutants deficient in specific respiratory chain components have been used to determine the sites of copper reduction and the targets inactivated by t-BOOH. Two sites of electron transfer to membrane-bound copper were identified: one in the region between NADH and ubiquinone supported by NADH as electron donor and another localized between ubiquinone and the cytochromes supported by electrons coming from NADH, succinate, or D-lactate. Electron flow through the former site in the presence of t-BOOH led to inactivation of NADH dehydrogenase II, whereas electron flow through the latter site in the presence of the hydroperoxide led to damage of ubiquinone. In agreement with the above in vitro results with isolated membranes, copper-dependent inactivation of NADH dehydrogenase and ubiquinone was demonstrated in E. coli cells exposed to t-BOOH. It is proposed that the t-BOOH-induced damage is a consequence of t-butylalkoxy radical generation through a Fenton-type reaction mediated by redox cycling of membrane-bound copper at those two loci of the respiratory chain.

Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli.

We are studying the action of tert-butylhydroperoxide (t-BOOH) on Escherichia coli as a model system for peroxide toxicity. In our previous report (De la Cruz-Rodriguez, L.C., Farías, R.N. and Massa, E.M. (1990) Biochim. Biophys. Acta 1015, 510-516), the respiratory chain was identified as a major target of t-BOOH. In the present paper, we study further the effect of t-BOOH on the NADH oxidase of the E. coli respiratory chain to clarify the mechanism of damage, especially regarding the identity and role of the metal ion involved. The results are: (a) t-BOOH toxicity is mediated by membrane-bound copper ions; (b) a small pool of the membrane-bound copper is reduced from Cu(II) to Cu(I) in the presence of NADH and other respiratory substrates (succinate, D-lactate); (c) this reduction of copper occurs at 37 degrees C but not at 0 degrees C or when the membranes are inactivated by previous heating; (d) the Cu(I) generated by reduction of Cu(II) during membrane preincubation with NADH, is oxidized by t-BOOH with simultaneous inactivation of the NADH oxidase, whereas treatment with only t-BOOH (without NADH) has no effect on the oxidase. It is concluded that the effect of t-BOOH on the respiratory chain is mediated by redox cycling of copper. It is proposed that the damage results from activation of the hydroperoxide through its interaction with Cu(I) in a site-specific Fenton-type reaction.