Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo and Universidade de Franca, Av. do Café S/N, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil1
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil2
Instituto de Fisiologia Celular-UNAM, Mexico City, Mexico3
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID, USA4
Author for correspondence: Gustavo H. Goldman. Tel: +55 16 6024280/81. Fax: +55 16 6331092/6024280. e-mail: ggoldman{at}usp.br
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ABSTRACT |
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Keywords: catalase, heat shock, stress tolerance, Aspergillus nidulans
Abbreviations: ROS, reactive oxygen species
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INTRODUCTION |
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Oxidative stress is the result of an imbalance between pro-oxidant species and the levels of the defences resulting from the generation of reactive oxygen species (ROS) (Santoro & Thiele, 1997 ). ROS, including the superoxide anion (
), hydrogen peroxide (H2O2) and the hydroxyl radical (OH·), are noted for their ability to alter or inactivate proteins, lipid membranes and DNA by reacting chemically with them. Biological systems have evolved several defence mechanisms that enable cells to cope with lethal oxidative environments (Moradas-Ferreira et al., 1996
). These antioxidant defence systems include enzymic activities such as superoxide dismutase and catalase, which detoxify
and H2O2, respectively, and non-enzymic protective molecules including glutathione, vitamins C and E, and uric acid (Scandalios, 1990
). In addition to the induction of proteins directly involved in detoxifying and repairing damage by oxidants and free radical species, some of the inducible proteins overlap with heat-shock-inducible polypeptides. For example, a Salmonella typhimurium oxyR1 mutant selected under oxidative conditions and resistant to H2O2 constitutively overexpresses five heat-shock proteins (Morgan et al., 1986
). The oxidative stress response and its relationship to the heat-shock phenomenon have also been intensively investigated in the eukaryotic organism S. cerevisiae (Jamieson et al., 1994
; Davidson et al., 1996
). Experiments with promoter fusions have demonstrated that one of the heat-inducible forms of HSP70 from S. cerevisiae is inducible with H2O2 (Jamieson et al., 1994
). Recently, Godon et al. (1998)
analysed by comparative two-dimensional gel electrophoresis of total cell proteins the changes in gene expression underlying the yeast adaptative stress response to H2O2. The hydrogen-peroxide-responsive targets include a number of heat-shock proteins and proteins with reactive oxygen intermediate scavenging activities.
Here, we have examined the relationship between the heat-shock response and the response to peroxide treatment in Aspergillus nidulans germlings in order to investigate whether oxidative stress is involved in the lethal effect of heat exposure. We show that hydrogen peroxide treatment actually increases A. nidulans germling viability when cells are exposed to a heat shock. In addition, mutants deficient in the key antioxidant enzyme catalase are more sensitive to a 50 °C heat exposure.
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METHODS |
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Gas chromatography analysis of A. nidulans cell extracts.
Approximately 0·1 g dried mycelium was boiled in 10 ml 80% ethanol with 100 µg -D-phenyl glucopyranoside ml-1 (added as an internal standard) in a tube, then centrifuged at 7900 g for 10 min to pellet the insoluble debris. The debris was washed twice with 80% ethanol. The washes were combined with the initial supernatant and passed through an activated Sep-Pak Plus C-18 cartridge (Waters; WAT020515) and evaporated to dryness in a vacuum oven at 60 °C.
The dried extracts were derivatized using a modification of standard protocols (Sweeley et al., 1963 ; Brobst & Lott, 1966
). Briefly, 15 µl 2-dimethylaminoethanol and 400 µl pyridine containing 30 mg methoxyamine ml-1 were added to each dried extract. The vials were then capped and heated to 7580 °C for 1 h, then allowed to cool to room temperature. After cooling, 20 µl trifluoroacetic acid and 400 µl hexamethyldisilazane were added. The contents were mixed, then incubated for 1 h at room temperature. The insoluble material was removed by centrifuging at 15000 g for 5 min and the supernatant was transferred to a clean vial.
Derivatized samples were separated on a Hewlett Packard HP 5890 series II Gas Chromatograph with a 30 cm CP-SIL 8 C8-MS column (Chrompack 5860) and detected with an HP 5989A mass spectrometer. Retention times and derivatization efficiencies were established using solutions of known composition for requested compounds. Total peak areas were corrected for derivatization efficiency and weight of material processed to arrive at µg (g dry weight)-1 values.
Heat and oxidative stress treatments.
Six-hour-old germlings were heat-shocked by incubating them at 50 °C for 30, 45 and 60 min. Oxidative stress treatments were performed by incubating 6-h-old germlings (heat-shocked or non-heat-shocked) in 50 mM hydrogen peroxide for 20 min at 37 °C. In all cases appropriate dilutions were made and 100 µl aliquots spread on plates of YAG medium containing 0·01% Triton X-100 to restrict colony growth (Donnely et al., 1994 ).
DNA and RNA manipulations.
Restriction enzyme digests and DNA ligations were performed in accordance with the suppliers (Boehringer and Amersham Pharmacia) recommendations. Plasmid DNA isolation from Escherichia coli was performed using standard procedures (Sambrook et al., 1989 ). DNA probes were constructed using a random primer system according to the manufacturer (Boehringer).
For Northern analysis, A. nidulans mycelia from the heat and oxidative stress treatments were harvested by filtration through a Whatman no. 1 filter, washed thoroughly with sterile water, disrupted and total RNA extracted (Sambrook et al., 1989 ). Twenty micrograms of RNA from each treatment were then fractionated in a 2·2 M formaldehyde, 1·5% agarose gel and then transferred to Hybond-N+ filters (Amersham) in the presence of 20x SSPE (3·5 M NaCl, 0·2 M sodium phosphate pH 7·7, 0·02 M disodium EDTA). Prehybridization and hybridization were performed according to Sambrook et al. (1989)
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RESULTS AND DISCUSSION |
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In order to understand the mechanisms underlying such a process, we performed a similar set of experiments using catalase-deficient A. nidulans mutants. It is known that catalase genes have an important role in protecting cells against damage caused by oxidative and heat-shock stresses (Piper, 1997 ; Moradas-Ferreira et al., 1996
; Davidson et al., 1996
). Two catalase genes, catA and catB, have been characterized in A. nidulans. The catA gene expresses a developmentally regulated catalase that accumulates to high levels in asexual and sexual spores (conidia and ascospores, respectively; Navarro et al., 1996
; Navarro & Aguirre, 1998
), whilst catB expresses a catalase induced during vegetative mycelial growth and stationary phase (Kawasaki et al., 1997
). The effect of mutations in catA, catB and the double mutant catA catB on cell survival was determined after heat shock at 50 °C for 30, 45 and 60 min, and 75 min for the wild-type strain TWA22 (Fig. 2
). The catA mutant and the double mutant showed much less ability to recover from heat shock than the catB deletion strain, whilst this strain showed lower survival than the wild-type.
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Since both catalase genes are thought to encode enzymically similar proteins, the treatment-dependent differences between catA and catB phenotypes might be explained in one of two ways. Either each gene is induced by only one of the two inducers, or each is induced by both inducers but to very different levels. In order to distinguish between these two possibilities, we assayed steady-state levels of catA and catB mRNA after heat shock and after hydrogen peroxide treatment. We found that both messengers accumulated to low basal levels in unstressed cultures. On the other hand, catA mRNA did accumulate significantly upon heat shock and heat shock plus hydrogen peroxide treatments but not after hydrogen peroxide treatment alone. By contrast, catB mRNA accumulation was only induced upon hydrogen peroxide treatment (Fig. 4 and Kawasaki et al., 1997
). Taken together, our results indicate that germling survival after either heat shock or oxidative stress depends primarily on the catA-encoded catalase. Based on the phenotypic analyses, we nevertheless conclude that catB contributes somewhat to cell survival in both conditions, despite the fact that it is not induced significantly after heat stress. Our studies demonstrate the complexity of stress defence processes. Aspergillus germlings may accumulate small soluble molecules like trehalose to stabilize enzyme structure at high temperatures, but our results indicate that heat-shock-induced oxidative stress remains one of the major factors contributing to death of heat-treated cells. In order to protect themselves, germlings must accumulate agents such as mannitol and enzymes like catalase in order to scavenge some of the ROS that are produced by the interruption of normal electron transfer. The lack of catalase activity leads to an increase in formation of hydroxyl radicals as peroxide is not converted to water and oxygen. The increasing amount of hydroxyl radicals probably has the most damaging effect, although the diffusion of hydroxyl radicals is more limited than that of peroxide. Presumably, survival is also dependent on the production of additional antioxidant defences that are induced by H2O2 treatments although we have not yet identified these components. Other results support this interpretation. Godon et al. (1998)
have shown that during the yeast adaptive stress response to H2O2, a number of heat-shock proteins accumulate. Davidson et al. (1996)
have shown that mutants deficient in the key antioxidant enzymes catalase, superoxide dismutase and cytochrome-c peroxidase are sensitized to a 50 °C heat exposure, whilst overexpression of catalase and superoxide dismutase provides protection from lethal heat shock. The yeast cytoplasmic catalase T gene is under stress-response element (STRE) control with the result that catalase T is induced by heat shock except in cells with high protein kinase A activity. Lack of catalase T causes a small reduction in thermotolerance in both proliferating and stationary-phase cells except when kinase A activity levels are high (Piper, 1997
; Wieser et al., 1991
). These influences of the levels of different antioxidant activities on thermotolerance are thought to reflect the severity of ROS-induced damage to cellular proteins, nucleic acids and lipids that results from respiration at higher temperatures.
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ACKNOWLEDGEMENTS |
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Received 23 February 1999;
revised 15 June 1999;
accepted 8 July 1999.