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Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast
Biochimica et Biophysica Acta 1781 (2008) 283–287
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p
Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast Ana Cipak a, Morana Jaganjac a, Oksana Tehlivets b, Sepp D. Kohlwein b, Neven Zarkovic a,⁎ a b
Rudjer Boskovic Institute, Zagreb, Croatia Institute of Molecular Biosciences, University of Graz, A8010 Graz, Austria
A R T I C L E
I N F O
Article history: Received 21 December 2007 Received in revised form 7 March 2008 Accepted 28 March 2008 Available online 10 April 2008 Keywords: Fatty acid peroxidation Reactive oxygen species Saccharomyces cerevisiae Fatty acid desaturase Catalase
A B S T R A C T To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Δ12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n − 6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Appearance of oxygen in the atmosphere leads to the evolution of highly efficient cellular energy generation systems, but also introduced the major problem of oxygen toxicity. Molecular oxygen is highly reactive and its partial reduction generates numerous chemically active agents, termed reactive oxygen species (ROS). Although most of the ROS are radical species like hydroxyl and superoxide radical, there are also non-radical species like hydrogen peroxide. ROS are highly damaging to all biological molecules, including DNA, proteins [1–4] and lipids [1,5]. Polyunsaturated fatty acids that are esterified in membrane or storage lipids are subject to ROS-induced peroxidation and may yield cytotoxic aldehydes, like 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA) and acrolein [[1]. The membrane damaging properties of peroxidized fatty acids and their reactive byproducts are believed to be associated with numerous chronic and acute diseases like cancer, cardiovascular diseases, neurodegenerative disorders, Down's syndrome, as well as with ageing [6–9]. Under normal physiological conditions, ROS are produced in low amounts as a result of active aerobic metabolism. Main source of ROS is leakage of electrons from the respiratory chain and by microsomal metabolism, giving rise to superoxide anion [10], which may be catabolized by superoxide dismutase to hydrogen peroxide (H2O2), which can be converted to the highly reactive and cell damaging hydroxyl radical via the metal-ion catalyzed Fenton reaction [11]. ⁎ Corresponding author. Tel.: +385 14560937; fax: +385 14561010. E-mail address: [email protected] (N. Zarkovic). 1388-1981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.03.010
Therefore, cells have evolved efficient enzymatic and non-enzymatic defense mechanisms to deal with potential pro-oxidants and oxidative damage [12–14]. Non-enzymatic mechanisms involve small hydrophobic or hydrophilic molecules that act as radical scavengers, such as glutathione [14–16]. Enzymatic defense systems are more complex and may include cascades of reactions leading to complete detoxification of certain ROS and/or their byproducts. For instance, superoxide dismutase (SOD) disproportionates superoxide radicals to oxygen and hydrogen peroxide, which is further catabolized by catalase to water and molecular oxygen [14]. These mechanisms provide a certain degree of constitutive resistance to oxidative stress, however, it was also demonstrated that ROS may cause distinct adaptive responses. In addition, ROS may induce cross-adaptation, whereby pre-treatment of cells with one ROS increases resistance to another [15]. Saccharomyces cerevisiae is a well-defined unicellular and facultative aerob eukaryotic organism and an excellent model for studying molecular mechanisms of oxidative stress responses. Yeast does not synthesize polyunsaturated fatty acids (PUFAs) due to the lack of enzymes capable of introducing more than a single double bond into its fatty acids [16]. Thus, fatty acids of the yeast cells are more resistant to oxidative attack than those of mammalian cells. However, yeast is well capable of taking up and incorporating exogenous saturated and (poly)unsaturated fatty acids, which normally do not occur in its lipids [17,18], and they are highly sensitive to exogenous long-chain PUFA peroxides [19]. To investigate in a conditional system the effects of PUFA (peroxidation) on cellular physiology, we have constructed a yeast strain that functionally expresses a Δ12 desaturase gene from the tropical
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Fig. 1. Continuous desaturase expression leads to adaptation to oxidative stress. Wild-type (BYctrl; black circles) and Δ12 desaturase-expressing (BYdesa; open circles) strains were grown in standard defined galactose medium by re-inoculation into fresh medium after every 24 h for 11 consecutive days. Aliquots were taken out and cells were incubated with 5 mM hydrogen peroxide for 4 h. After treatment, cells were diluted and plated in triplicate onto YEPD medium to monitor cell viability. Percentage survival is expressed relative to the untreated controls. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). bSignificantly different if compared to the wild type at the same time point (p b 0.05).
rubber tree, Hevea brasiliensis. Only under inducing conditions, this strain produces up to 15% PUFA, mostly 9Z,12Z-C18:2 (linoleic acid) but also 9Z,12Z-C16:2, defining the enzyme as a Δ12 desaturase [20]. In the transgenic strain, the presence of these polyunsaturated fatty acids significantly increases the sensitivity against oxidative stress, induced by the addition of hydrogen peroxide. Oxidative stress and loss of viability of the strain are accompanied by formation of 4-hydroxy-2-nonenal, one of the end products of n−6 polyunsaturated (e.g. linoleic) fatty acid peroxidation [20]. Although the spectrum of lipid peroxidation products generated in this yeast strain does not resemble the major lipid peroxidation products in mammalian cells under similar oxidative stress conditions, the yeast system provides powerful genetic and biochemical means to track the deleterious effects of lipid peroxidation as well as the cellular antioxidant defense mechanisms [19]. While short-term expression of the desaturase rendered the cells more sensitive to H2O2 stress, extended expression and PUFA production resulted in development of increased resistance to this pro-oxidant. This observation indicated some level of adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, induced PUFA production increased levels of endogenous ROS, and was correlated with an increase in catalase activity that is required for hydrogen peroxide detoxification. Deletion mutants defective in peroxisomal catalase lost the ability to adapt to oxidative stress. These data demonstrate the importance of catalase in the process of adaptation to endogenous oxidative stress caused by the presence of PUFA in yeast. 2. Materials and methods 2.1. Yeast strains and cultivation conditions The S. cerevisiae strains used in this study were wild-type BY4742 (MATα his3Δ1 leu2Δ1 lys2Δ0 ura3Δ0; Euroscarf), a catalase A deficient strain, cta1 (MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 YDR256c::KanMX4; Euroscarf) and a catalase T deficient strain, ctt1 (MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 YGR088w::KanMX4; Euroscarf). Cells were grown either in rich YEPD media (2% w/v glucose, 2% w/v Bacto peptone, 1% w/v yeast extract) or in minimal synthetic-defined (SD) media (1% w/v glucose or 1% w/v galactose, 0.17% Difco Yeast Nitrogen Base) supplemented with appropriate amino acids and bases: 0.004% adenine, 0.002% arginine, 0.002% histidine, 0.01% leucine, 0.003% lysine, 0.002% methionine, 0.01% threonine, and 0.002% tryptophan. Media were solidified by the addition of 2% (w/v) agar. 2.2. Transformation of wild-type and deletion mutant strains The cDNA encoding fatty acid Δ12 desaturase from H. brasiliensis was cloned into the yeast episomal expression vector pYES2 (Invitrogen), containing the Gal1/10
promoter and a URA3 selection marker, as described previously [20]. Plasmids pYES2 and pYES2-desa1[21] were transformed into wild-type strain BY4742 and cta1 and ctt1 mutant strains by the lithium acetate method [21], and transformants were selected on media plates lacking uracil. Transformed strains were referred to as BYctrl, Ctactrl and Cttctrl for control strains harbouring the pYES2 plasmid without insert, and BYdesa, Ctadesa and Cttdesa for strains harbouring expression plasmid, pYES2-desa1 encoding the Δ12 fatty acid desaturase [20]. 2.3. Adaptation and stress resistance analysis of PUFA-producing yeast strains Wild-type and cta1 and ctt1 mutants transformed with pYES2 (empty plasmid) or pYES2-desa1 (Δ12 desaturase) were pre-grown overnight in SD medium lacking uracil and containing 1% glucose, washed and inoculated into SD medium without uracil and with 1% galactose, for induction of desaturase expression. To maintain a high level of desaturase expression cells were re-inoculated to an OD600 = 0.1 every 24 h for 11 consecutive days, in the absence of stressor. Cell growth was monitored by OD600 measurement. Sensitivity of cells against hydrogen peroxide was assessed after harvesting cells at the indicated time points and dilution of the cell suspension to OD600 = 1.0. Cells were treated with 5 mM H2O2 or 2 M NaCl for 4 h, aliquots diluted and plated in triplicate on YEPD plates and incubated at 30 °C. Viable cell counts (colony forming units) were scored after 3 days of growth. 2.4. Catalase activity analysis Cells were grown in SD media without uracil, and desaturase expression was induced for 72 h in the presence of 1% galactose. Aliquots of the BYctrl and BYdesa cell cultures were collected and stored at − 80 °C until further analysis. Cells were lysed mechanically with glass beads in phosphate buffer (60 mM K-PO4, pH 7.4) and cell lysates cleared by centrifugation at 13,400 rpm for 15 min. Catalase activity was measured in 25 μl of the supernatant in phosphate buffer (60 mM, pH 7.4), after addition of 100 mM H2O2 as the substrate. The reaction was stopped by addition of 100 mM ammonium molybdate, and color development was measured spectroscopically in a plate reader at 450 nm [22]. Protein concentration was measured according to Lowry et al. [23], using bovine serum albumin as a standard. 2.5. Measurement of ROS production Cellular ROS production was examined by using a nonfluorescent probe for intracellular ROS detection 2',7'-dichlorofluorescin diacetate (DCFH-DA, Fluka). This cell-permeable dye is widely used in both mammalian [24,25] and yeast cells [26,27] for sensitive and rapid quantitation of ROS in response to oxidative stress. Namely, DCFH-DA probe remains nonfluorescent inside the cell until the acetate groups are removed by intracellular esterases and oxidized by intracellular ROS to the fluorescent compound 2',7'-dichlorofluorescein (DCF) which can be detected as a measure for intracellular ROS [28]. After preincubation of yeast cells at an OD600 = 1.0 in 1 ml phosphate-buffered saline (PBS) with 100 μM DCFH-DA at 28 °C for 60 min, the cell suspensions were treated with 5 mM H2O2 for 1 h and then washed and resuspended in 1 ml PBS. Fluorescence intensity was read with a Cary Eclipse Fluorescence Spectrophotometer (Varian) with excitation at 500 nm and emission detection at 530 nm.
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Fig. 2. Desaturase expression causes adaptation to oxidative stress but not to hyperosmotic stress. Wild-type (BYctrl; white bars) and Δ12 desaturase-expressing (BYdesa; gray bars) strains were grown in standard defined galactose medium by re-inoculation into fresh medium after every 24 h for 3 days. Aliquots were taken out and cells were incubated with 5 mM hydrogen peroxide (panel a) or 2 M NaCl (panel b) for 4 h. After treatment, cells were diluted and plated in triplicate onto YEPD medium to monitor cell viability. Percent survival is expressed relative to the untreated controls. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). bSignificantly different if compared to the wild type at the same time point (p b 0.05). 2.6. Statistical analysis All experiments and assays were carried out in triplicates. Mean values were compared using the two tailed Student's t-test, considering values of p b 0.05 as significantly different.
3. Results 3.1. Adaptation analysis of PUFA-producing yeast strains We have previously shown that expression of a heterologous Δ12 desaturase from H. brasiliensis renders yeast wild-type cells more sensitive to oxidative damage by treatment with pro-oxidants, presumably due to the formation of lipid hydroperoxides and reactive aldehydes [20]. To investigate the influence of a long-term expression of the desaturase gene on cellular physiology, both wild-type control and desaturase-expressing strains were cultivated under inducing conditions for up to 11 days. Sensitivity of the strains was tested by treatment with sub-lethal concentrations (5 mM) of hydrogen peroxide for 4 h. Consistent with previous results, acute expression of the desaturase rendered the desaturase-expressing strain more sensitive to H2O2 challenge than wild type (Fig. 1). Interestingly, after 3 days of continued desaturase expression, the BYdesa strain exhibited significantly increased resistance against H2O2 treatment, compared
to the wild-type control, which remained throughout several days. At later stages, after 11 days, both wild-type and desaturase-expressing strains reached the same level of resistance. This time-course of adaptation was analysed in more detail (Fig. 2a) and unveiled that the desaturase-expressing strain adapted within 2 days to a resistance level comparable to wild type and exceeded wildtype resistance by 20% after 3–5 days. In contrast, and consistent with the notion that adaptation to oxidative stress is due to endogenous lipid peroxidation, exposure to 2 M NaCl (hyperosmotic stress) for 4 h did not alter the survival rate of the PUFA-producing strain, compared to wild type, during the first 3 days of desaturase expression (Fig. 2b). 3.2. The role of catalase in adaptation Because adaptation to oxidative stress is linked to the production of PUFA in the transgenic yeast strain, we investigated potential antioxidative defense mechanisms. Catalases are the major enzymes involved in hydrogen peroxide degradation to water and oxygen. Therefore, catalase activity was monitored in both, desaturase-expressing and control strains (Fig. 3a). Whereas wild type did not show any changes in catalase activity during the first 3 days of growth, catalase activity was elevated about 2–2.5-fold, at day two of the experiment, in the desaturase-expressing strain. These data demonstrate that the PUFA
Fig. 3. Role of catalase in adaptation to continuous desaturase expression. Catalase activity assay — Cells were grown in standard defined media without uracil, and desaturase expression was induced for 3 days in the presence of galactose. Aliquots of the BYctrl (white bars) and BYdesa (gray bars) cell cultures were collected and stored each day at −80 °C until further analysis. One catalase unit is the activity decomposing 1.0 μmol of hydrogen peroxide per minute at pH 7.0 at 25 °C. b) H2O2 sensitivity of a cta1 mutant transformed with pYES2 (empty plasmid; white bars) or pYES2-desa1 (Δ12 desaturase; gray bars). Strains were grown in standard defined galactose medium by re-inoculation to fresh medium after every 24 h for 3 days, and treated with 5 mM H2O2 for 4 h prior to plating. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). b Significantly different if compared to the wild type at the same time point (p b 0.05).
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Fig. 4. Impact of PUFA production on reactive oxygen species (ROS) generation. Cellular ROS production was examined by using 2',7'-dichlorofluorescin diacetate (DCFH-DA). a) ROS production in untreated wild-type control cells (BYctrl; white bars) and desaturase-expressing strain (BYdesa; gray bars). b) ROS production in hydrogen peroxide — treated wildtype control cells (BYctrl; white bars) and desaturase-expressing strain (BYdesa; gray bars). Note the different scale, indicating 10 to 20-fold higher ROS levels in cells challenged with H2O2.
production under aerobic conditions leads to increased catalase activity. As an important determinant of the antioxidative defense, total glutathione levels and the activity of glutathione-S-transferase (GST) were also monitored. Neither glutathione levels nor GST activity showed significant differences between wild-type and PUFA-producing strains (data not shown), suggesting that the glutathione system does not play a significant role in adaptation to endogenous PUFA production. The yeast S. cerevisiae expresses two catalase isoenzymes, namely peroxisomal catalase A and cytosolic catalase T. To determine which catalase was responsible for adaptation to PUFA production, the adaptation experiments were repeated making use of specific catalase deletion strains, i.e. cta1 lacking peroxisomal catalase, and ctt1 lacking cytosolic catalase. The ctt1 mutant strain did not survive 5 mM H2O2 treatment for 4 h (results not shown), demonstrating the highly increased sensitivity of this mutant independent of the presence of PUFA. The survival rate of the peroxisomal catalase-deficient mutant Ctadesa expressing the Δ12 desaturase was significantly lower after treatment with H2O2, compared to the control strain Ctactrl (Fig. 3b). The increased H2O2-sensitivity of the Cta1-deficient strain producing PUFAs is consistent with the notion that catalase activity plays an important role in the adaptation and detoxification process resulting from PUFA catabolism. To analyse the impact of PUFA synthesis in yeast on the generation of reactive oxygen species (ROS), the oxidant-sensitive fluorescence probe DCFH-DA was used to monitor ROS production (Fig. 4). ROS production was further increased by 10% (day 1) to 40% (day 3) of Δ12 desaturase expression, compared to the wild-type control. ROS production was increased upon treatment with H2O2, however, no significant differences were observed between strains BYdesa and BYctrl under these conditions, suggesting that both strains are at a maximum capacity under these conditions. Taken together, our data demonstrate that yeast cells conditionally expressing a Δ12 desaturase adapt to PUFA production by increasing catalase activity and that cytosolic catalase T is essential for the cellular survival while peroxisomal catalase A plays an important role in this adaptation process. 4. Discussion Oxidative stress and damage are implicated in many harmful diseases, including cardiovascular and neurodegenerative diseases, autoimmune disorders, and cancer [6–9]. A detailed understanding of the molecular mechanisms underlying the potentially detrimental effects of reactive oxygen species and oxidative damage as well as
cellular defense mechanisms is, therefore, of significant biomedical interest. Lipids are considered potent targets of oxidative damage, and polyunsaturated fatty acids, which are ubiquitously present in mammalian cells, are particularly susceptible. Remarkably, tumor cells have a lower content of polyunsaturated fatty acids compared to nontransformed cells, and it was suggested that this may be one of the reasons for their different responses to oxidative challenge [29,30]. The complexity of potential targets of ROS and their oxidative degradation products in multicellular organisms, however, has obscured the picture as to the molecular mechanisms involved. Recently, we have described a genetically engineered yeast which is able to produce Z9,Z12 polyunsaturated, i.e. hexadecadienoic and octadecadienoic (linoleic) fatty acids, under defined inducing conditions: whereas wild-type yeast cells do not contain PUFAs, this modified yeast strain produces up to 15% PUFAs, thereby providing a novel and intriguing model for lipid peroxidation research. As a consequence of the presence of PUFA in cellular lipids, this strain becomes more sensitive to oxidative challenge by treatment with H2O2 or other prooxidants that induce oxidative stress, such as tert-butylhydroperoxide or paraquat [20]. Remarkably, expression of the heterologous Δ12 desaturase did not increase cellular sensitivity to stress in general, as exposure to other stressors, such as 2 M NaCl or 200 mM acetic acid (weak acid stress) had no or very moderate effects on cellular growth and viability. The initial increased sensitivity of the PUFA-producing yeast strain against hydrogen peroxide was overcome after 2 days of continuous desaturase expression; after 3 days, cells had adapted to this challenge and acquired increased resistance. Notably, also wild type became more resistant to hydrogen peroxide challenge; upon extended reinoculation to inducing media, both wild-type and desaturaseexpressing strain reached similar levels of resistance after 11 days, which may be explained by a genetic selection process, e.g. accumulation of mitochondrial petites, which is currently under investigation. The question raised by this study that will be further evaluated is what triggers the adaptation to H2O2 in the desaturase-expressing strain? The presence of PUFA in aerobically growing yeast cells leads to low but significant levels of the end product of PUFA peroxidation 4-hydroxy-2-nonenal (HNE) even in the absence of exogenous stressors. HNE production might be relevant for the adaptation to the stress because HNE acts as a signaling molecule in regulation of the cellular metabolism, growth and stress response [3,20,30–32]. Growth and survival rates of this strain were, however, identical to wild type and suggested some adaptive mechanisms to deal with the presence of low levels of endogenous PUFA peroxidation and HNE.
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Indeed, cellular catalase activity was increased about 2.5-fold upon PUFA synthesis, leading to increased resistance against H2O2-induced oxidative challenge. Deletion mutants lacking cytosolic catalase T were unable to survive H2O2-induced stress, regardless of PUFA production, demonstrating the essential function of this enzyme in hydrogen peroxide detoxification. The cta1 mutants lacking peroxisomal catalase survived the oxidative challenge, but remained at a constant level of resistance for 3 days, demonstrating the absence of adaptation. They survived the oxidative challenge, but remained more sensitive to hydrogen peroxide if the fatty acid desaturase was expressed, and lacked significant adaptation to PUFA production. These data are consistent with the notion of an important function also of peroxisomal catalase A in the adaptation to oxidative stress caused by PUFA in cellular lipids. Yeast cells are capable of taking up various fatty acids [18], thus in their natural habitat, they may well be exposed to PUFAs derived from other sources, e.g. plants. Even though yeast itself normally does not produce fatty acids that are susceptible to oxidative damage, it has evolved specific defense mechanisms to overcome potentially detrimental problems arising from these compounds. Taken together, our results indicate the crucial importance of cytosolic catalase for the overall survival of the yeast exposed to oxidative stress caused by H2O2, while peroxisomal catalase appears to be important in adaptation to endogenous oxidative stress caused by PUFA. Using an array of yeast deletion mutants expressing the desaturase will aid in understanding the signaling pathways involved in response to PUFA production in the cell and opens new perspectives for understanding cellular processes that cause transformation and abnormalities in cellular function, as a result of lipid peroxidation. Acknowledgements This work was supported by COST B35 Action and Croatian Ministry of Science, Education and Sports to N.Z., as well as from Österreichischer Akademischer Austauschdienst, ÖAD (Project: 9/ 2002) and the Austrian Science Funds, FWF (Project SFP Lipotox, SFB 0030-P05), to S.D.K. References [1] H. Esterbauer, R.J. Schaur, H. Zollner, Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes, Free Radic. Biol. Med. 11 (1991) 81–128. [2] M. Poot, A. Verkerk, J.F. Koster, H. Esterbauer, J.F. Jongkind, Reversible inhibition of DNA and protein synthesis by cumene hydroperoxide and 4-hydroxynonenal, Mech. Age Dev. 43 (1988) 1–9. [3] W. Wonisch, S.D. Kohlwein, R.J. Schaur, F. Tatzber, H. Guttenberger, N. Zarkovic, R. Winkler, H. Esterbauer, Treatment of the budding yeast Saccharomyces cerevisiae with the lipid peroxidation product 4-HNE provokes a temporary cell cycle arrest in G1 phase, Free Radic. Biol. Med. 25 (1998) 682–687. [4] H. Wiseman, B. Halliwell, Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer, Biochem. J. 313 (1996) 17–29. [5] W.A. Pryor, Cancer and free radicals, Basic Life Sci. 39 (1986) 45–59. [6] K. Uchida, Role of reactive aldehyde in cardiovascular diseases, Free Radic. Biol. Med. 28 (2000) 1685–1696. [7] M. Parola, G. Leonarduzzi, G. Robino, E. Albano, G. Poli, M.U. Dianzani, On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis, Free Radic. Biol. Med. 20 (1996) 351–359.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p
Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast Ana Cipak a, Morana Jaganjac a, Oksana Tehlivets b, Sepp D. Kohlwein b, Neven Zarkovic a,⁎ a b
Rudjer Boskovic Institute, Zagreb, Croatia Institute of Molecular Biosciences, University of Graz, A8010 Graz, Austria
A R T I C L E
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Article history: Received 21 December 2007 Received in revised form 7 March 2008 Accepted 28 March 2008 Available online 10 April 2008 Keywords: Fatty acid peroxidation Reactive oxygen species Saccharomyces cerevisiae Fatty acid desaturase Catalase
A B S T R A C T To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Δ12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n − 6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Appearance of oxygen in the atmosphere leads to the evolution of highly efficient cellular energy generation systems, but also introduced the major problem of oxygen toxicity. Molecular oxygen is highly reactive and its partial reduction generates numerous chemically active agents, termed reactive oxygen species (ROS). Although most of the ROS are radical species like hydroxyl and superoxide radical, there are also non-radical species like hydrogen peroxide. ROS are highly damaging to all biological molecules, including DNA, proteins [1–4] and lipids [1,5]. Polyunsaturated fatty acids that are esterified in membrane or storage lipids are subject to ROS-induced peroxidation and may yield cytotoxic aldehydes, like 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA) and acrolein [[1]. The membrane damaging properties of peroxidized fatty acids and their reactive byproducts are believed to be associated with numerous chronic and acute diseases like cancer, cardiovascular diseases, neurodegenerative disorders, Down's syndrome, as well as with ageing [6–9]. Under normal physiological conditions, ROS are produced in low amounts as a result of active aerobic metabolism. Main source of ROS is leakage of electrons from the respiratory chain and by microsomal metabolism, giving rise to superoxide anion [10], which may be catabolized by superoxide dismutase to hydrogen peroxide (H2O2), which can be converted to the highly reactive and cell damaging hydroxyl radical via the metal-ion catalyzed Fenton reaction [11]. ⁎ Corresponding author. Tel.: +385 14560937; fax: +385 14561010. E-mail address: [email protected] (N. Zarkovic). 1388-1981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.03.010
Therefore, cells have evolved efficient enzymatic and non-enzymatic defense mechanisms to deal with potential pro-oxidants and oxidative damage [12–14]. Non-enzymatic mechanisms involve small hydrophobic or hydrophilic molecules that act as radical scavengers, such as glutathione [14–16]. Enzymatic defense systems are more complex and may include cascades of reactions leading to complete detoxification of certain ROS and/or their byproducts. For instance, superoxide dismutase (SOD) disproportionates superoxide radicals to oxygen and hydrogen peroxide, which is further catabolized by catalase to water and molecular oxygen [14]. These mechanisms provide a certain degree of constitutive resistance to oxidative stress, however, it was also demonstrated that ROS may cause distinct adaptive responses. In addition, ROS may induce cross-adaptation, whereby pre-treatment of cells with one ROS increases resistance to another [15]. Saccharomyces cerevisiae is a well-defined unicellular and facultative aerob eukaryotic organism and an excellent model for studying molecular mechanisms of oxidative stress responses. Yeast does not synthesize polyunsaturated fatty acids (PUFAs) due to the lack of enzymes capable of introducing more than a single double bond into its fatty acids [16]. Thus, fatty acids of the yeast cells are more resistant to oxidative attack than those of mammalian cells. However, yeast is well capable of taking up and incorporating exogenous saturated and (poly)unsaturated fatty acids, which normally do not occur in its lipids [17,18], and they are highly sensitive to exogenous long-chain PUFA peroxides [19]. To investigate in a conditional system the effects of PUFA (peroxidation) on cellular physiology, we have constructed a yeast strain that functionally expresses a Δ12 desaturase gene from the tropical
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Fig. 1. Continuous desaturase expression leads to adaptation to oxidative stress. Wild-type (BYctrl; black circles) and Δ12 desaturase-expressing (BYdesa; open circles) strains were grown in standard defined galactose medium by re-inoculation into fresh medium after every 24 h for 11 consecutive days. Aliquots were taken out and cells were incubated with 5 mM hydrogen peroxide for 4 h. After treatment, cells were diluted and plated in triplicate onto YEPD medium to monitor cell viability. Percentage survival is expressed relative to the untreated controls. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). bSignificantly different if compared to the wild type at the same time point (p b 0.05).
rubber tree, Hevea brasiliensis. Only under inducing conditions, this strain produces up to 15% PUFA, mostly 9Z,12Z-C18:2 (linoleic acid) but also 9Z,12Z-C16:2, defining the enzyme as a Δ12 desaturase [20]. In the transgenic strain, the presence of these polyunsaturated fatty acids significantly increases the sensitivity against oxidative stress, induced by the addition of hydrogen peroxide. Oxidative stress and loss of viability of the strain are accompanied by formation of 4-hydroxy-2-nonenal, one of the end products of n−6 polyunsaturated (e.g. linoleic) fatty acid peroxidation [20]. Although the spectrum of lipid peroxidation products generated in this yeast strain does not resemble the major lipid peroxidation products in mammalian cells under similar oxidative stress conditions, the yeast system provides powerful genetic and biochemical means to track the deleterious effects of lipid peroxidation as well as the cellular antioxidant defense mechanisms [19]. While short-term expression of the desaturase rendered the cells more sensitive to H2O2 stress, extended expression and PUFA production resulted in development of increased resistance to this pro-oxidant. This observation indicated some level of adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, induced PUFA production increased levels of endogenous ROS, and was correlated with an increase in catalase activity that is required for hydrogen peroxide detoxification. Deletion mutants defective in peroxisomal catalase lost the ability to adapt to oxidative stress. These data demonstrate the importance of catalase in the process of adaptation to endogenous oxidative stress caused by the presence of PUFA in yeast. 2. Materials and methods 2.1. Yeast strains and cultivation conditions The S. cerevisiae strains used in this study were wild-type BY4742 (MATα his3Δ1 leu2Δ1 lys2Δ0 ura3Δ0; Euroscarf), a catalase A deficient strain, cta1 (MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 YDR256c::KanMX4; Euroscarf) and a catalase T deficient strain, ctt1 (MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 YGR088w::KanMX4; Euroscarf). Cells were grown either in rich YEPD media (2% w/v glucose, 2% w/v Bacto peptone, 1% w/v yeast extract) or in minimal synthetic-defined (SD) media (1% w/v glucose or 1% w/v galactose, 0.17% Difco Yeast Nitrogen Base) supplemented with appropriate amino acids and bases: 0.004% adenine, 0.002% arginine, 0.002% histidine, 0.01% leucine, 0.003% lysine, 0.002% methionine, 0.01% threonine, and 0.002% tryptophan. Media were solidified by the addition of 2% (w/v) agar. 2.2. Transformation of wild-type and deletion mutant strains The cDNA encoding fatty acid Δ12 desaturase from H. brasiliensis was cloned into the yeast episomal expression vector pYES2 (Invitrogen), containing the Gal1/10
promoter and a URA3 selection marker, as described previously [20]. Plasmids pYES2 and pYES2-desa1[21] were transformed into wild-type strain BY4742 and cta1 and ctt1 mutant strains by the lithium acetate method [21], and transformants were selected on media plates lacking uracil. Transformed strains were referred to as BYctrl, Ctactrl and Cttctrl for control strains harbouring the pYES2 plasmid without insert, and BYdesa, Ctadesa and Cttdesa for strains harbouring expression plasmid, pYES2-desa1 encoding the Δ12 fatty acid desaturase [20]. 2.3. Adaptation and stress resistance analysis of PUFA-producing yeast strains Wild-type and cta1 and ctt1 mutants transformed with pYES2 (empty plasmid) or pYES2-desa1 (Δ12 desaturase) were pre-grown overnight in SD medium lacking uracil and containing 1% glucose, washed and inoculated into SD medium without uracil and with 1% galactose, for induction of desaturase expression. To maintain a high level of desaturase expression cells were re-inoculated to an OD600 = 0.1 every 24 h for 11 consecutive days, in the absence of stressor. Cell growth was monitored by OD600 measurement. Sensitivity of cells against hydrogen peroxide was assessed after harvesting cells at the indicated time points and dilution of the cell suspension to OD600 = 1.0. Cells were treated with 5 mM H2O2 or 2 M NaCl for 4 h, aliquots diluted and plated in triplicate on YEPD plates and incubated at 30 °C. Viable cell counts (colony forming units) were scored after 3 days of growth. 2.4. Catalase activity analysis Cells were grown in SD media without uracil, and desaturase expression was induced for 72 h in the presence of 1% galactose. Aliquots of the BYctrl and BYdesa cell cultures were collected and stored at − 80 °C until further analysis. Cells were lysed mechanically with glass beads in phosphate buffer (60 mM K-PO4, pH 7.4) and cell lysates cleared by centrifugation at 13,400 rpm for 15 min. Catalase activity was measured in 25 μl of the supernatant in phosphate buffer (60 mM, pH 7.4), after addition of 100 mM H2O2 as the substrate. The reaction was stopped by addition of 100 mM ammonium molybdate, and color development was measured spectroscopically in a plate reader at 450 nm [22]. Protein concentration was measured according to Lowry et al. [23], using bovine serum albumin as a standard. 2.5. Measurement of ROS production Cellular ROS production was examined by using a nonfluorescent probe for intracellular ROS detection 2',7'-dichlorofluorescin diacetate (DCFH-DA, Fluka). This cell-permeable dye is widely used in both mammalian [24,25] and yeast cells [26,27] for sensitive and rapid quantitation of ROS in response to oxidative stress. Namely, DCFH-DA probe remains nonfluorescent inside the cell until the acetate groups are removed by intracellular esterases and oxidized by intracellular ROS to the fluorescent compound 2',7'-dichlorofluorescein (DCF) which can be detected as a measure for intracellular ROS [28]. After preincubation of yeast cells at an OD600 = 1.0 in 1 ml phosphate-buffered saline (PBS) with 100 μM DCFH-DA at 28 °C for 60 min, the cell suspensions were treated with 5 mM H2O2 for 1 h and then washed and resuspended in 1 ml PBS. Fluorescence intensity was read with a Cary Eclipse Fluorescence Spectrophotometer (Varian) with excitation at 500 nm and emission detection at 530 nm.
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Fig. 2. Desaturase expression causes adaptation to oxidative stress but not to hyperosmotic stress. Wild-type (BYctrl; white bars) and Δ12 desaturase-expressing (BYdesa; gray bars) strains were grown in standard defined galactose medium by re-inoculation into fresh medium after every 24 h for 3 days. Aliquots were taken out and cells were incubated with 5 mM hydrogen peroxide (panel a) or 2 M NaCl (panel b) for 4 h. After treatment, cells were diluted and plated in triplicate onto YEPD medium to monitor cell viability. Percent survival is expressed relative to the untreated controls. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). bSignificantly different if compared to the wild type at the same time point (p b 0.05). 2.6. Statistical analysis All experiments and assays were carried out in triplicates. Mean values were compared using the two tailed Student's t-test, considering values of p b 0.05 as significantly different.
3. Results 3.1. Adaptation analysis of PUFA-producing yeast strains We have previously shown that expression of a heterologous Δ12 desaturase from H. brasiliensis renders yeast wild-type cells more sensitive to oxidative damage by treatment with pro-oxidants, presumably due to the formation of lipid hydroperoxides and reactive aldehydes [20]. To investigate the influence of a long-term expression of the desaturase gene on cellular physiology, both wild-type control and desaturase-expressing strains were cultivated under inducing conditions for up to 11 days. Sensitivity of the strains was tested by treatment with sub-lethal concentrations (5 mM) of hydrogen peroxide for 4 h. Consistent with previous results, acute expression of the desaturase rendered the desaturase-expressing strain more sensitive to H2O2 challenge than wild type (Fig. 1). Interestingly, after 3 days of continued desaturase expression, the BYdesa strain exhibited significantly increased resistance against H2O2 treatment, compared
to the wild-type control, which remained throughout several days. At later stages, after 11 days, both wild-type and desaturase-expressing strains reached the same level of resistance. This time-course of adaptation was analysed in more detail (Fig. 2a) and unveiled that the desaturase-expressing strain adapted within 2 days to a resistance level comparable to wild type and exceeded wildtype resistance by 20% after 3–5 days. In contrast, and consistent with the notion that adaptation to oxidative stress is due to endogenous lipid peroxidation, exposure to 2 M NaCl (hyperosmotic stress) for 4 h did not alter the survival rate of the PUFA-producing strain, compared to wild type, during the first 3 days of desaturase expression (Fig. 2b). 3.2. The role of catalase in adaptation Because adaptation to oxidative stress is linked to the production of PUFA in the transgenic yeast strain, we investigated potential antioxidative defense mechanisms. Catalases are the major enzymes involved in hydrogen peroxide degradation to water and oxygen. Therefore, catalase activity was monitored in both, desaturase-expressing and control strains (Fig. 3a). Whereas wild type did not show any changes in catalase activity during the first 3 days of growth, catalase activity was elevated about 2–2.5-fold, at day two of the experiment, in the desaturase-expressing strain. These data demonstrate that the PUFA
Fig. 3. Role of catalase in adaptation to continuous desaturase expression. Catalase activity assay — Cells were grown in standard defined media without uracil, and desaturase expression was induced for 3 days in the presence of galactose. Aliquots of the BYctrl (white bars) and BYdesa (gray bars) cell cultures were collected and stored each day at −80 °C until further analysis. One catalase unit is the activity decomposing 1.0 μmol of hydrogen peroxide per minute at pH 7.0 at 25 °C. b) H2O2 sensitivity of a cta1 mutant transformed with pYES2 (empty plasmid; white bars) or pYES2-desa1 (Δ12 desaturase; gray bars). Strains were grown in standard defined galactose medium by re-inoculation to fresh medium after every 24 h for 3 days, and treated with 5 mM H2O2 for 4 h prior to plating. aSignificantly different if compared to the growth of the same strain on the previous day (p b 0.05). b Significantly different if compared to the wild type at the same time point (p b 0.05).
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Fig. 4. Impact of PUFA production on reactive oxygen species (ROS) generation. Cellular ROS production was examined by using 2',7'-dichlorofluorescin diacetate (DCFH-DA). a) ROS production in untreated wild-type control cells (BYctrl; white bars) and desaturase-expressing strain (BYdesa; gray bars). b) ROS production in hydrogen peroxide — treated wildtype control cells (BYctrl; white bars) and desaturase-expressing strain (BYdesa; gray bars). Note the different scale, indicating 10 to 20-fold higher ROS levels in cells challenged with H2O2.
production under aerobic conditions leads to increased catalase activity. As an important determinant of the antioxidative defense, total glutathione levels and the activity of glutathione-S-transferase (GST) were also monitored. Neither glutathione levels nor GST activity showed significant differences between wild-type and PUFA-producing strains (data not shown), suggesting that the glutathione system does not play a significant role in adaptation to endogenous PUFA production. The yeast S. cerevisiae expresses two catalase isoenzymes, namely peroxisomal catalase A and cytosolic catalase T. To determine which catalase was responsible for adaptation to PUFA production, the adaptation experiments were repeated making use of specific catalase deletion strains, i.e. cta1 lacking peroxisomal catalase, and ctt1 lacking cytosolic catalase. The ctt1 mutant strain did not survive 5 mM H2O2 treatment for 4 h (results not shown), demonstrating the highly increased sensitivity of this mutant independent of the presence of PUFA. The survival rate of the peroxisomal catalase-deficient mutant Ctadesa expressing the Δ12 desaturase was significantly lower after treatment with H2O2, compared to the control strain Ctactrl (Fig. 3b). The increased H2O2-sensitivity of the Cta1-deficient strain producing PUFAs is consistent with the notion that catalase activity plays an important role in the adaptation and detoxification process resulting from PUFA catabolism. To analyse the impact of PUFA synthesis in yeast on the generation of reactive oxygen species (ROS), the oxidant-sensitive fluorescence probe DCFH-DA was used to monitor ROS production (Fig. 4). ROS production was further increased by 10% (day 1) to 40% (day 3) of Δ12 desaturase expression, compared to the wild-type control. ROS production was increased upon treatment with H2O2, however, no significant differences were observed between strains BYdesa and BYctrl under these conditions, suggesting that both strains are at a maximum capacity under these conditions. Taken together, our data demonstrate that yeast cells conditionally expressing a Δ12 desaturase adapt to PUFA production by increasing catalase activity and that cytosolic catalase T is essential for the cellular survival while peroxisomal catalase A plays an important role in this adaptation process. 4. Discussion Oxidative stress and damage are implicated in many harmful diseases, including cardiovascular and neurodegenerative diseases, autoimmune disorders, and cancer [6–9]. A detailed understanding of the molecular mechanisms underlying the potentially detrimental effects of reactive oxygen species and oxidative damage as well as
cellular defense mechanisms is, therefore, of significant biomedical interest. Lipids are considered potent targets of oxidative damage, and polyunsaturated fatty acids, which are ubiquitously present in mammalian cells, are particularly susceptible. Remarkably, tumor cells have a lower content of polyunsaturated fatty acids compared to nontransformed cells, and it was suggested that this may be one of the reasons for their different responses to oxidative challenge [29,30]. The complexity of potential targets of ROS and their oxidative degradation products in multicellular organisms, however, has obscured the picture as to the molecular mechanisms involved. Recently, we have described a genetically engineered yeast which is able to produce Z9,Z12 polyunsaturated, i.e. hexadecadienoic and octadecadienoic (linoleic) fatty acids, under defined inducing conditions: whereas wild-type yeast cells do not contain PUFAs, this modified yeast strain produces up to 15% PUFAs, thereby providing a novel and intriguing model for lipid peroxidation research. As a consequence of the presence of PUFA in cellular lipids, this strain becomes more sensitive to oxidative challenge by treatment with H2O2 or other prooxidants that induce oxidative stress, such as tert-butylhydroperoxide or paraquat [20]. Remarkably, expression of the heterologous Δ12 desaturase did not increase cellular sensitivity to stress in general, as exposure to other stressors, such as 2 M NaCl or 200 mM acetic acid (weak acid stress) had no or very moderate effects on cellular growth and viability. The initial increased sensitivity of the PUFA-producing yeast strain against hydrogen peroxide was overcome after 2 days of continuous desaturase expression; after 3 days, cells had adapted to this challenge and acquired increased resistance. Notably, also wild type became more resistant to hydrogen peroxide challenge; upon extended reinoculation to inducing media, both wild-type and desaturaseexpressing strain reached similar levels of resistance after 11 days, which may be explained by a genetic selection process, e.g. accumulation of mitochondrial petites, which is currently under investigation. The question raised by this study that will be further evaluated is what triggers the adaptation to H2O2 in the desaturase-expressing strain? The presence of PUFA in aerobically growing yeast cells leads to low but significant levels of the end product of PUFA peroxidation 4-hydroxy-2-nonenal (HNE) even in the absence of exogenous stressors. HNE production might be relevant for the adaptation to the stress because HNE acts as a signaling molecule in regulation of the cellular metabolism, growth and stress response [3,20,30–32]. Growth and survival rates of this strain were, however, identical to wild type and suggested some adaptive mechanisms to deal with the presence of low levels of endogenous PUFA peroxidation and HNE.
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Indeed, cellular catalase activity was increased about 2.5-fold upon PUFA synthesis, leading to increased resistance against H2O2-induced oxidative challenge. Deletion mutants lacking cytosolic catalase T were unable to survive H2O2-induced stress, regardless of PUFA production, demonstrating the essential function of this enzyme in hydrogen peroxide detoxification. The cta1 mutants lacking peroxisomal catalase survived the oxidative challenge, but remained at a constant level of resistance for 3 days, demonstrating the absence of adaptation. They survived the oxidative challenge, but remained more sensitive to hydrogen peroxide if the fatty acid desaturase was expressed, and lacked significant adaptation to PUFA production. These data are consistent with the notion of an important function also of peroxisomal catalase A in the adaptation to oxidative stress caused by PUFA in cellular lipids. Yeast cells are capable of taking up various fatty acids [18], thus in their natural habitat, they may well be exposed to PUFAs derived from other sources, e.g. plants. Even though yeast itself normally does not produce fatty acids that are susceptible to oxidative damage, it has evolved specific defense mechanisms to overcome potentially detrimental problems arising from these compounds. Taken together, our results indicate the crucial importance of cytosolic catalase for the overall survival of the yeast exposed to oxidative stress caused by H2O2, while peroxisomal catalase appears to be important in adaptation to endogenous oxidative stress caused by PUFA. Using an array of yeast deletion mutants expressing the desaturase will aid in understanding the signaling pathways involved in response to PUFA production in the cell and opens new perspectives for understanding cellular processes that cause transformation and abnormalities in cellular function, as a result of lipid peroxidation. Acknowledgements This work was supported by COST B35 Action and Croatian Ministry of Science, Education and Sports to N.Z., as well as from Österreichischer Akademischer Austauschdienst, ÖAD (Project: 9/ 2002) and the Austrian Science Funds, FWF (Project SFP Lipotox, SFB 0030-P05), to S.D.K. References [1] H. Esterbauer, R.J. Schaur, H. Zollner, Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes, Free Radic. Biol. Med. 11 (1991) 81–128. [2] M. Poot, A. Verkerk, J.F. Koster, H. Esterbauer, J.F. Jongkind, Reversible inhibition of DNA and protein synthesis by cumene hydroperoxide and 4-hydroxynonenal, Mech. Age Dev. 43 (1988) 1–9. [3] W. Wonisch, S.D. Kohlwein, R.J. Schaur, F. Tatzber, H. Guttenberger, N. Zarkovic, R. Winkler, H. Esterbauer, Treatment of the budding yeast Saccharomyces cerevisiae with the lipid peroxidation product 4-HNE provokes a temporary cell cycle arrest in G1 phase, Free Radic. Biol. Med. 25 (1998) 682–687. [4] H. Wiseman, B. Halliwell, Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer, Biochem. J. 313 (1996) 17–29. [5] W.A. Pryor, Cancer and free radicals, Basic Life Sci. 39 (1986) 45–59. [6] K. Uchida, Role of reactive aldehyde in cardiovascular diseases, Free Radic. Biol. Med. 28 (2000) 1685–1696. [7] M. Parola, G. Leonarduzzi, G. Robino, E. Albano, G. Poli, M.U. Dianzani, On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis, Free Radic. Biol. Med. 20 (1996) 351–359.
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