Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cerevisiae.

BACKGROUND: Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. METHODOLOGY/PRINCIPAL FINDINGS: Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Delta and gtt2Delta) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. CONCLUSIONS/SIGNIFICANCE: These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification

Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae

Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 ºC, in 0.04% sulphite, 1 mol L-1 NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors of substrates into ethanol (Yp/s), glycerol (Yg/s) and acetic acid (Yac/s), were calculated. The highest values of Yp/s in sugar cane juice fermentation were obtained by four strains, one isolated from fruit (0.46) and the others from sugar cane (0.45, 0.44 and 0.43). These values were higher than the value obtained using traditional yeast (0.38) currently employed in the Brazilian bioethanol industry. The parameters Yg/s and Yac/s were low for all strains. The UFLA FW221 presented the higher values for parameter related to bioethanol production. Thus, it was tested in co-culture with Lactobacillus fermentum. Besides this, a 20-L vessel for five consecutive batches of fermentation was performed. This strain was genetically stable and remained viable during all batches, producing high amounts of ethanol. The UFLA FW221 isolated from fruit was suitable to produce bioethanol in sugar cane juice. Therefore, the study of the biodiversity of yeasts from different environmental can reveal strains with desired characteristics to industrial applications

<i>S. cerevisiae</i> tolerance to plumbagin.

<p>Yeast cells harvested in mid log phase, were directly stressed with 7.0 μM plumbagin (white bars) or were previously treated with 0.5 μM plumbagin/1 h and then submitted to severe stress conditions (black bars). Tolerance was expressed as percentage of survival.</p

Enhancement of lipid peroxidation in wild-type and Gtt deficient cells caused by plumbagin.

<p>The increase in lipid peroxidation was expressed as a ratio between the levels of lipid peroxidation of plumbagin stressed and non-stressed cells. Yeast cells, harvested in mid log phase, were directly stressed with 7.0 μM plumbagin (white bars) or were previously treated with 0.5 μM plumbagin before been submitted to severe plumbagin stress (black bars).</p

Determination of cellular response to naphthoquinones after cross-protection treatment.

<p>First exponential cells of the wild-type strain were submitted to a lethal stress either with 20 mM menadione or with 7.0 μM plumbagin (white bars), or were previously adapted with 0.5 mM menadione (gray bars) or 0.5 μM plumbagin (black bars).</p

Effect of menadione and plumbagin on glutathione.

<p>The GSH and GSSG levels were expressed in nmols/mg of cell. Normal Growth refers to a non-stress condition while menadione or plumbagin stressed refers to cells stressed with 20 mM menadione or 7.0 μM plumbagin for 1 h, respectively. Menadione and Plumbagin treated and stressed refers to cells pre-treated with 0.5 mM menadione or 0.5 μM plumbagin for 1 h before being stressed with the same drugs. The means obtained for drug treated cells were compared with the normal growth condition of each strain using a t-student test and ^* represents statistically different results at <i>P</i><0.05.</p><p>ND–not determined.</p