Methods for monitoring oxidative stress response in ...

J BIOCHEM MOLECULAR TOXICOLOGY Volume 19, Number 4, 2005

Methods for Monitoring Oxidative Stress Response in Yeasts Polona Jamnik and Peter Raspor Food Science and Technology Department, Chair of Biotechnology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; E-mail: [email protected] Received 21 January 2005

ABSTRACT: Changes in the chemical or physical conditions of the cell that impose a negative effect on growth demand rapid cellular responses, which are essential for survival. Molecular mechanisms induced upon exposure of cells to such adverse conditions are commonly designated as stress responses. Herein, different methods which can be used to monitor oxidative stress response in yeasts are presented including monitoring of oxygen partial pressure during yeast cultivation, cell viability determination, measuring activity of enzymatic and level of nonenzymatic primary antioxidant defense systems, and examination of transcriptome and proteome changes. Additionally, some studies are given as examples of particular method’s application for studying oxidative stress response in C 2005 Wiley Periodicals, Inc. J Biochem Mol yeasts. Toxicol 19:195–203, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI /jbt.20091 KEYWORDS: Yeasts;

Yeast Physiology; Oxidative Stress Response; Oxygen Partial Pressure; Yeast Viability; Antioxidant Defense Systems; Transcriptome; Proteome

INTRODUCTION Stress Response

detrimental effects of stress factors and repair damages that are already done. They include changes at the level of enzyme activities and gene expression and lead to acquisition of stress tolerance [1]. We can distinguish between early and late responses. Early responses (e.g., the activation of some protein kinases or of trehalose metabolism) appear to have two functions: they provide a minimal protection against sudden stress, and they initiate delayed or late responses, e.g. the synthesis of heat shock proteins or enzymes scavenging toxic oxygen radicals. These delayed responses will protect cells permanently and more effectively by allowing adaptation to persistent stress [2].

Oxidative Stress Response Yeasts like other organisms living in aerobic conditions are continuously exposed to reactive oxygen species (ROS) formed as by-products during normal cellular metabolism. Under physiological conditions, cell defense systems are able to avoid molecular damages. Different stress conditions (oxidants, heat shock, ethanol, metal ions) increase levels of ROS leading to induction of antioxidant defense systems—the oxidative stress response [4].

Definition Stress responses are designated as molecular mechanisms induced in the cells upon exposure to stress conditions [1]. In the case of unicellular organisms like yeasts, stress conditions are broadly defined as those environmental factors that cause a reduction in growth rate [2,3]. Stress responses aim to protect cells against Correspondence to: Peter Raspor. Contract Grant Sponsor: Ministry of Science and Technology, Republic of Slovenia. Contract Grant Number: J4-7454-490. Contract Grant Sponsor: Ministry of Education and Sport, Republic of Slovenia. Contract Grant Number: 35/8. c 2005 Wiley Periodicals, Inc.

MONITORING OF OXIDATIVE STRESS RESPONSE IN YEASTS Monitoring of Oxygen Partial Pressure (pO2 ) During Yeast Cultivation Monitoring of pO2 during yeast cultivation can present an important indicator concerning determination of stress factor concentration induced oxidative or other stress response. Jamnik and Raspor [5] demonstrated that pO2 measurement during yeast cultivation is reasonable to determine concentration of Cr(VI) as a stress factor that induces oxidative stress response. Yeast cells were exposed to Cr(VI) in different concentrations 50, 100, 300, and 500 M, and the growth 195

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was monitored by some bioprocess parameters pO2 , pH, and optical density. The most pronounced changes after Cr(VI) addition were evident from the curve indicating pO2 , specifically at 100 M Cr(VI), where increased oxygen consumption was observed. Increased oxygen consumption indicates increased metabolism intensity, and authors connected this observation to stress response induction.

Determination of Cell Viability Since stress conditions in the case of unicellular organisms are related to reduction of growth rate, determination of cell viability as CFU [6] is another simple assay to estimate condition of cell population and can be considered to determine concentration at which stress response is activated. Therefore, decrease in cell viability indicates growth inhibition and consequently the stress response induction. Cell viability determination is usually carried out at the beginning of studies where the effect of different environmental factors on cells is investigated, see [7–11].

Measuring Activity of Enzymatic and Levels of Nonenzymatic Primary Antioxidant Defense Systems Primary antioxidant defense systems are induced in the cell upon increased ROS production; and there are those enzymes and compounds that prevent the initiation or propagation of radical/oxidant damage, or that act as radical chain terminating agents. Therefore, they neutralize the reactive oxygen species and hence are

classical (or true) antioxidants [4,12]. As it is shown in Table 1, they can be divided into enzymatic and nonenzymatic primary antioxidant defense systems. Furthermore, study of primary antioxidant defense systems also indirectly gives information about toxicity mechanism of particular stress factor. Namely, to establish the mechanism of toxicity as ROS mediated, there are many direct and indirect methods. Direct methods are related to the ROS measurement, but these species are very reactive and their quantification can be difficult. Therefore, indirect methods, such as measurement of changes in endogenous antioxidant enzyme activity, are often used [13]. A number of methods are available and include spectrophotometric and electrophoretic methods for measurement of activity of particular enzymatic systems and spectrophotometric and chromatographic methods for measurement of levels of nonenzymatic systems. There are given some studies for measuring activity of particular enzymes such as catalase [13,18– 22], superoxide dismutase [13,23–27], glutathione peroxidase [13,28–30], glutahione reductase [13,31–34], glucose-6-phosphate dehydrogenase [35–37], thioredoxin reductase [38–41], and cytochrome c peroxidase [42]. From nonenzymatic systems, several methods for measuring levels of glutathione [5,43–47], thioredoxin [38–41], and metallothioneins [48,49] were developed. However, it has to be mentioned that results obtained by different methods analyzing particular characteristics may differ due to differences in analytical procedure (for instance, different buffers with different concentrations, monitoring time etc.)

TABLE 1. Yeast Primary Antioxidant Defense Systems [14–17] Antioxidant defense system

Function Enzymatic defense systems

Catalase A Catalase T Cu/Zn superoxide dismutase Mn superoxide dismutase Glutathione reductase Thioredoksin reductase Glutathione peroxidase Cytochrome c peroxidase Glucose-6-phosphate dehydrogenase

Decomposition of hydrogen peroxide (cytoplasm) Decomposition of hydrogen peroxide (peroxisome) Dismutation of superoxide anion (cytoplasm) Dismutation of superoxide anion (mitochondria) Reduction of oxidized glutathione Reduction of oxidized thioredoxin Reduction of hydrogen peroxide Reduction of hydrogen peroxide Generation of NADPH via the pentose phosphate cycle Nonenzymatic defense systems

Glutathione Metallothioneins Phytochelatins Polyamines Thioredoxin Glutharedoxin Lipofylic antioxidants (red yeasts)

Scavenging of oxygen free radicals Metals binding, scavenging superoxide, and hydroxyl radicals Scavenging of oxygen free radicals Protection of lipids from oxidation Reduction of protein disulfides Reduction of protein disulfides Scavenging of oxygen free radicals


Several studies have shown the utility of measuring activity of antioxidant enzymes and levels of nonenzymatic systems to monitor oxidative stress response in yeasts [5,7,8,11,50–58].

Monitoring of Transcriptome Change Discovery of methods that are able to assess transcriptome gave us new possibility and new dimension on the what is happening in the cell. The transcriptom comprises mRNAs that are present in a cell at a particular time. Transcriptomes can have highly complex profile, with hundreds or thousands of different mRNAs present; each makes up a different fraction of the overall population [59]. In the past, the most common technique for mRNA analysis was the Northern blotting [60], where RNA is separated on a gel, blotted on a membrane, and detected by the hybridization of labeled sequence specific probes. This hybridization step followed by autoradiography allows the detection of a specific mRNA [61]. An alternative to the Northern blotting is the RNAase protection assay (RPA) [62], where a labeled antisense cRNA is transcribed from a DNA cloned in an appropriate vector, hybridized with an mRNA sample, and single-stranded RNA digested with RNAase, then run out on a gel. The amount of labeled surviving RNA is directly proportional to the amount of target mRNA present in the sample. In situ hybridization (ISH) [63] is an effective approach for the localization of gene expression at the cytological level. It includes specific annealing of a labeled probe to complementary sequences of desired mRNA of interest in fixed specimen, followed by detection and visualization of nucleic acid hybrids with cytological methods. All these traditional methods work well, but they generally require a large amount of RNA. The development of reverse transcriptase polymerase chain reaction (RT-PCR) [64] has provided some advantages; only a small amount of RNA is required, and a large number of samples can be analyzed in one experiment. Furthermore, real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) quantitates the initial amount of the template most specifically, sensitively, and reproducibly and is a preferable alternative to RTPCR that detects the amount of final amplified product at the endpoint by agarose gels [65]. Real-time PCR [66–68] provides a sensitive, reproducible, and accurate method for determining mRNA levels in the cells. It monitors the fluorescence emitted during exponential phase of the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection. The

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method combines amplification and detection in one step. Differential display PCR (DD-PCR) [69] is another approach that facilitates the identification of many genes expressed in a variety of physiological conditions. The method utilizes a set of oligonucleotide primers: one being anchored to the polyadenylate tail of a subset of mRNAs, and the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA defined by these primer pairs is amplified after reverse transcription and is resolved on a DNA sequencing gel. The major advantages of this method are its high sensitivity, the ability to carry out rapid mRNA analyses using total RNA, and to test multiple tissues in parallel. Its limitations are the need for many primer combinations for adequate representation of mRNAs and the large number of bands displayed [70]. The most direct way to characterize a transcriptome is to convert its mRNA into cDNA and then to sequence every clone in the resulting cDNA library [59]. Comparisons between the cDNA sequences and the genome sequence allow identification of particular genes that are activated as part of stress response. Serial analysis of gene expression (SAGE) [71] is another method, which rather than studying complete cDNA yields short sequences as little as 12 bp in length, each of which presents an mRNA that is present in the transcriptome. These short sequences enable to identify the gene that is coded for the mRNA. In the past few years, DNA microarrays (sometimes called DNA chips), which allow the rapid and simultaneous screening of many thousands of genes, have been widely used. DNA segments from known genes are amplified by PCR and spotted on a solid surface using robotic devices [72]. An alternative strategy is to synthesize DNA directly on the solid surface, using photolithography [73]. The total complement of mRNA is isolated from cells, converted to cDNA, and the cDNA are labeled with nucleotides that fluoresce. The fluorescent cDNA are used as probes, each hybridizing to complementary sequence on the microarray. The intensity of the hybridization signal enables an estimation of the distinct mRNA level in the investigated cell [74]. The yeast Saccharomyces cerevsiae is an excellent model organism for the study of stress factor action. Its genome has been fully sequenced and well characterized, and with the development of whole-genome microarrays, it is now possible to monitor globally gene expression changes in response to various experimental conditions [75]. Several of the genes that participate in cellular defense against oxidative stress are known to display increased expression under oxidative stress conditions

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as part of an oxidative stress response pathway [14]. Northern blotting was followed as an appropriate method for study of the oxidative stress response in yeasts, which is confirmed by several studies [11,76– 82]. As an alternative method for study, the expression of selected genes appears through RT-PCR [77,80,83]. In the recent time, most of the DNA microarrays were described as a powerful tool to provide information about the changes in gene expression in yeast cells after stress factor exposure. This is expected because this technique allows the analysis of the mRNA levels of all genes of a microbial genome at one time [84]. Many studies have revealed their application in studying oxidative stress response in yeasts [85–89]. Afshai et al. [90] and Nuwaysir et al. [91] suggest the use of microarrays in toxicology as bioassays for risk assessment. Similarly, Momose and Iwahashi [92] conclude that DNA microarrays are very useful instruments for creating new bioassay systems and finding genetic promoters of stress indicators.

Monitoring of Proteome Change An examination of the transcriptome gives an accurate indication of which genes are active in particular cell, but gives a less accurate indication of the proteins that are present [59]. Therefore, proteome analysis is conceptually attractive because it fits within the concept that the determination of protein rather than RNA levels has a major advantage as it is the proteins that carry out functions [93]. Proteome analysis means an analysis of the entire protein complement expressed by a genome [94], but generally proteome analysis, which cannot cover the total number of proteins existing in a biological system at a given moment, involves the expression profile of many proteins at a time [95]. Proteome analysis mainly includes two steps: (1) separation of proteins and (2) protein analysis and identification [93]. The standard methods for separation of proteins include isoelectric focusing (IEF) [96], where proteins are separated according to their isoelectric points; sodium dodecyl sulfate gel electrophoresis (SDS GE) [97], which separates proteins on the basis of mass (molecular weight); and the most commonly used electrophoretic method is two-dimensional gel electrophoresis (2-D GE) [98,99], which combines IEF and SDS GE sequentially. It is a more sensitive method than either electrophoretic method alone. It separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but with different molecular weights [74]. The result of two-dimensional gel electrophoresis is a series of spots, each one representing a differ-


ent protein. Not all the components of the proteome will be visible because the staining methods used to reveal the spots have a detection limit, but a clear picture of the most abundant proteins is obtained. Differences between two proteomes are seen as changes in the position and/or intensity of one or more spots on the gel [59]. Two-dimensional electrophoresis has been seen as an ideal tool for proteome analyses. Although it has shortcomings, for example, poor ability to separate hydrophobic proteins and trace-quantity expressed ones, immobilized pH-gradient (IPG) strips used in the first dimension provide a basis for reproducible separation according to proteins’ isoelectric points [95]. Proteome analysis utilizing 2-D GE protein separation is frequently criticized as being low throughput, in part due to the time-consuming process of image analysis that is necessary to determine differential protein expression. This process can be laborious because of gel-to-gel variations that confound the analysis process. Fluorescence 2-D difference gel electrophoresis (2-D DIGE) [100,101] enables comparative proteome analysis of multiple samples on the same two-dimensional gel. Up to three protein extracts, for example one control and two treated, are labeled with different fluorescent dyes (Cy2, Cy3, and/or Cy5), then combined and separated by 2D GE on the same gel. Up to three images of the gel are captured—using the Cy2, Cy3, and/or Cy5 excitation wavelengths. The images are then merged, and differences between them can be determined using image analysis software. A main disadvantage to this technology is the high cost involved in acquiring equipment as well as expendable supplies, such as the fluorescent dyes. Rather than relying on 2-D GE separation and subsequent image analysis of proteins immobilized within gels, isotope-coded affinity tagging (ICATTM ) [102] utilizes stable isotope labeling to perform quantitative analysis of paired protein samples, followed by separation and identification of proteins within these complex mixtures by liquid chromatography and mass spectrometry. The strength of this technique lies in its ability to allow quantification and identification within a single analysis. It can also be applied to samples from any source as it does not require metabolic labeling. Since the procedure targets cysteine residues, certain proteins and peptides will be missed—in particular those that undergo post-translational modifications. Another proteomic technology involved in quantitative analysis of protein mixtures is known as surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) [103]. This technique utilizes varied chemical and biochemical bait surfaces that allow differential capture of proteins based on their intrinsic properties. Energy-absorbing molecules are added to


retained proteins. Following laser desorption and ionization of proteins, time-of-flight (TOF) mass spectrometry accurately determines their masses. Patterns of masses rather than actual protein identifications are produced by SELDI-TOF analysis. Protein arrays [104] represent a proteomic tool that closely emulates the DNA microarray technology and are becoming widely available to be used as tools to easily and quickly monitor the expression of specific proteins. They accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. The most popular ones currently rely on antibody–antigen interactions. The potential of antibody arrays is limited by the availability of antibodies that have both high specificity (to eliminate cross reactions with nonspecific proteins within the sample) and high affinity for the target of interest (to allow detection of small quantities within a sample). Another challenge of protein array technology is the ability to preserve proteins in their biologically active shape and form. Protein identification is mainly carried out by mass spectrometry, specifically matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [105,106] on the basis of peptide mass matching following in-gel digestion with trypsin. Proteins that cannot be identified by MALDI-TOF MS, for example the low-molecular mass proteins that yield only a few peptides [107], are analyzed by tandem mass spectrometry, using the Qq-TOF approach. Peptide mass fingerprinting identifies a protein based on molecular weights of its peptides obtained by mass spectrometry after digestion by a specific protease, classically trypsin [108]. With the help of specific software, the list of experimental molecular weights is compared with the theoretical peptide fingerprinting of protein sequences existing in the current databases. The developed bioinformatics [109] allows the identification of most of the proteins. Of course, other protein identification methods, such as amino acid composition analysis and Nterminal sequencing or immunochemistry, as well as column chromatography can be used [110], or other biochemical techniques can be applied for protein enrichment [111]. From all above-mentioned proteomic techniques, 2-D GE [77,112–114] and also 2-D DIGE [115] were found to be applied to studying oxidative stress response in yeasts. Other new emerging techniques such as ICATTM , SELDI-TOF, and protein arrays, which are extensively used in medical research to find diagnostic markers for diseases [116–120] and in toxicology to find markers of pollution exposure [121], also have huge potential for study of the oxidative and other stress responses in yeasts.


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CONCLUDING REMARKS Monitoring of enzyme activity and levels of particular primary antioxidant defense systems as well as analysis of gene expression on the transcriptome and proteome level reveal the ways by which a given stress factor affects the cell and provides important clues about the mechanisms involved in the adaptation and survival of organisms under adverse environmental conditions. This review gives an overview of selected methods that can be used and also have already been applied to monitor oxidative stress response in yeasts, and new emerging methods that are now widely used in the medical research also have potential to monitor oxidative or other stress response in yeasts. The selection of methods presented in this paper is based on the principle of most common methods applied in the field and therefore not all published studies are cited. Furthermore, methods for assessing oxidative damage of proteins, lipids, and DNA are not included, because according to definition of stress response [1] oxidative damage is not part of stress response, but it is consequence of oxidative stress when ROS levels exceed cell’s antioxidant capacity.

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