An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae

Abstract

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

reactive oxygen species, oxidative stress, yeast

Introduction

Oxidants, which include reactive oxygen species (ROS) and reactive nitrogen species, can cause oxidative degradation of biomolecules such as DNA (Salmon, 2004), proteins (Cabiscol, 2000) and lipids (Girotti, 1998). Some ROS are either produced endogenously as part of normal metabolic processes (Halliwell & Gutteridge, 1984) or imposed on organisms by the external environment (Rogers & Clarke, 2007). The superoxide anion radical (O₂^●−) is generated as a byproduct of mitochondrial respiration via reduction of oxygen by electrons that have escaped from the electron transfer chain (Raha & Robinson, 2000). The superoxide anion can then react with nitrogen oxide to form the highly reactive oxidant peroxynitrite (ONOO⁻), which has been implicated in the formation of neurodegenerative diseases (Szabó, 2007). Hydrogen peroxide (H₂O₂) can also be formed via a dismutation reaction of two superoxide radicals (Kohen & Nyska, 2002). The extremely reactive hydroxyl radical (^●OH) could be derived from H₂O₂ through the Fenton reaction in the presence of transition metal ions (Meneghini, 1997). The oxidative stress resulting from the detrimental effects of H₂O₂ and the hydroxyl radical leads to not only numerous medical problems such as Down's syndrome (Busciglio & Yankner, 1995; Cao & Prior, 1998) but also has industrial implications such as affecting yeast cells during the initial stages of beer fermentations (Higgins, 2003a, b).

To counteract the damaging effects of these oxidants, cells have nonenzymatic antioxidant systems, such as the low-molecular-weight antioxidants including glutathione, vitamin E such as α-tocopherol, vitamin C and flavonoids. Cells also have the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, peroxiredoxin, thioredoxin and thioredoxin reductase (Munhoz & Netto, 2004). Under circumstances where these cellular antioxidant systems are not adequate, physiological changes such as growth arrest are used to protect the cell. To assist the cell in preventing oxidative stress, effective antioxidants are in ever-increasing demand in today's society. Efficient screening assays for candidate antioxidant compounds are essential to facilitate the discovery and development of antioxidants with therapeutic or industrial application.

Antioxidant capacity is defined as the capability of a compound to protect an organism from oxidant assault. A living cell system would provide the best platform for determining antioxidant capacity; however, the majority of current assays are chemically based and carried out in vitro. Two widely used methods are the diphenylpicrylhydrazyl (DPPH) assay and the azinobis ethylbenzothiaziline-6-sulphonate (ABTS) assay. The DPPH assay was established by Blois (1958), on the basis of the reduction of the stable free radical DPPH in the presence of hydrogen-donating antioxidants such as phenolics. The same working mechanism applies to the ABTS method that measures the reduction of the radical cation ABTS (Miller, 1993). These assays share the same drawback in that they do not measure the effect of an antioxidant on cell survival. This deficiency could be addressed using living cells and physiologically relevant oxidants. Further, a compound effective against one oxidant may not be active against another; this was demonstrated in previous findings indicating that chemically diverse oxidants had an effect on different molecular mechanisms in yeast (Alic, 2001, 2003, 2004; Thorpe, 2004). This indicates that a useful assay should be able to simultaneously screen antioxidant activity against a range of oxidants.

A key finding in past studies is that Saccharomyces cerevisiae adapts to a nonlethal dose of exogenous oxidant by undergoing a temporary cell cycle arrest. Upon exposure to a low concentration of linoleic acid 13-hydroperoxide (LAH), yeast cells exhibit a G1 arrest or a delay in cell cycle progression (Alic, 2001; Fong, 2008). This oxidant-induced cell cycle arrest extends to H₂O₂, which results in a G2 delay, and menadione, which causes a G1 delay similar to LAH (Flattery-O'Brien & Dawes, 1998). These studies demonstrated that growth arrest is a mechanism whereby yeast adapts to oxidative stress. This characteristic feature of yeast offered a basis for the development of an antioxidant assay. Upon treatment with a compound, an increase in yeast growth above the oxidant-induced arrest, which serves as a base line, indicates positive antioxidant activity. As a result, a S. cerevisiae-based assay was established with known antioxidants against a panel of six oxidants, which mimic or induce naturally occurring forms of oxidative stress.

Materials and methods

Yeast strain

The S. cerevisiae strain BY4743 (MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 met15Δ0/MET15 lys2Δ0/LYS2 ura3Δ0/ura3Δ0) used in the assay was obtained from Euroscarf (Frankfurt, Germany) (Brachmann, 1998).

Yeast growth media and culture conditions

BY4743 was streaked from a frozen glycerol stock to a YEPD agar plate containing 1% w/v yeast extract, 2% w/v peptone, 2% w/v d-glucose and 2% w/v agar, and grown at 30 °C for 48 h. Yeast culture was initiated by the inoculation of a single colony into a liquid synthetic defined minimal (SDM) medium containing 2%d-glucose, 0.17% yeast nitrogen base with neither ammonium sulphate nor amino acids and 0.5% ammonium sulphate, supplemented with 10 mg L⁻¹ adenine, 50 mg L⁻¹l-arginine, 80 mg L⁻¹l-aspartic acid, 20 mg L⁻¹l-histidine HCl, 50 mg L⁻¹l-isoleucine, 100 mg L⁻¹l-leucine, 50 mg L⁻¹l-lysine HCl, 20 mg L⁻¹l-methionine, 50 mg L⁻¹l-phenylalanine, 100 mg L⁻¹l-threonine, 50 mg L⁻¹l-tryptophan, 50 mg L⁻¹l-tyrosine, 140 mg L⁻¹l-valine and 20 mg L⁻¹ uracil. The culture was incubated overnight with shaking at 150 r.p.m. at 30 °C. Yeast growth was monitored by measuring the OD_{600 nm}. Yeast in peroxynitrite and diamide experiments was cultured in phosphate-buffered SDM media.

Antioxidants and oxidants

The following chemicals were purchased from Sigma-Aldrich: antioxidants (ascorbic acid, α-tocopherol, lipoic acid, phenolic compound – gallic acid, trace elements – zinc sulphate and manganese dioxide) and oxidants [H₂O₂, the aromatic hydroperoxide cumene hydroperoxide (CHP), the superoxide-generating agent menadione and the thiol-oxidizing agent diamide].

The lipid peroxide – LAH and peroxynitrite were synthesized as described previously, with some modifications (Evans, 1998). The reaction was started by incubating linoleic acid (1.0 mM) with soybean lipoxygenase (5000 U) in 0.1 M tetra-sodium borate buffer (pH 9.0). After a 30-min incubation at room temperature with vigorous stirring, the reaction mixture was loaded onto an end-capped C₁₈ reverse-phase column (Sepak cartridge, Waters, Australia), and the LAH was eluted in 1.5 mL of methanol. The concentration and purity of LAH were determined spectrophotometrically (λ=234 nm; 1 OD_{234 nm}=25 000 M⁻¹ cm⁻¹).

Peroxynitrite was synthesized using isoamyl nitrite and H₂O₂. Sodium hydroxide (2 M) of 38.5 mL volume was mixed with 11.5 mL 30% H₂O₂ (Sigma-Aldrich) and 0.5 mL 0.5 M EDTA. Isoamyl nitrite (25 mL) was then added to the solution while stirring at 4 °C in a cold room. The bottle was sealed and stirred overnight. The synthesis was manifested by a change in colour from yellow to orange. The excess isoamyl nitrite was separated and the aqueous orange layer containing peroxynitrite was collected and further purified by mixing with an equal volume of chloroform. Manganese oxide (0.25 g) was added to the peroxynitrite fraction for scavenging residual H₂O₂. Peroxynitrite was finally obtained by filtration through 3 mm filter paper to remove the manganese oxide. The concentration and purity were determined by reading the A_{302 nm} (1 OD_{302 nm}=1670 M⁻¹ cm⁻¹). The decomposed peroxynitrite control was prepared by allowing a portion of the synthesized peroxynitrite (10 mL) to undergo complete decomposition at room temperature for 48 h.

Assay for screening scavenging activity in a 96-well microtitre plate

A single colony of BY4743 was inoculated into 30 mL SDM with shaking, at 30 °C overnight. The preculture was then diluted to an OD_{600 nm} of 0.2 in SDM media, which yielded the initial seeding density of yeast in a 96-well plate at an OD_{600 nm} of 0.08. Yeast growth curves were first obtained for each oxidant with a series of concentrations against the untreated control. The oxidant concentration at which yeast growth was arrested for 5 h was determined as the arresting concentration. Yeast cells grew at concentrations lower than the arresting concentration, while any concentrations above it were lethal. Once the appropriate arresting concentrations were selected, the scavenging activities of the following compounds were assayed including ascorbate, tocopherol, gallic acid, lipoic acid, zinc sulphate and manganese oxide. The appropriate nontoxic concentration for each of them was firstly determined using a range of concentrations, and then mixed with individual oxidant at the arresting concentration to a final OD_{600 nm} of 0.2. The mixture (150 μL) was aliquoted into each of six replicate wells. The plates were incubated at 30 °C with shaking at 700 r.p.m. Yeast growth was monitored by reading the OD_{600 nm} at the start of incubation and then at hourly intervals for 5 h using a 96-well plate reader (Multiskan EX, Thermo Electron). Only oxidant controls were included in each experiment.

Assay for screening induction of intracellular antioxidant activity in a 96-well microtitre plate

BY4743 was cultured overnight in 30 mL SDM media containing antioxidants to induce an antioxidant response. The yeast were then centrifuged at 1000 g for 5 min and washed twice with SDM. They were then diluted in the individual oxidant at the arresting concentration to an OD_{600 nm} of 0.2 and a final volume of 150 μL added to microtitre plate wells. The plates were incubated at 30 °C with shaking at 700 r.p.m. Yeast growth was monitored as described above.

Results and discussion

Determination of arresting concentrations of oxidants

The oxidants used in this assay were chemically diverse, covering inorganic peroxide (H₂O₂), organic peroxide (CHP), lipid peroxide (LAH), superoxide-generating menadione, peroxynitrite and the thiol-oxidizing diamide (Fig. 1). To determine the concentration that caused growth arrest in the BY4743 yeast strain, the effect of a range of concentrations on yeast growth was determined for each compound; only two concentrations and a control are representatively shown in Fig. 2. The arresting concentration for each oxidant was determined to be 4 mM for H₂O₂, 150 μM for CHP, 75 μM for LAH, 150 μM for menadione, 10 mM for peroxynitrite and 1.6 mM for diamide. This oxidant-induced yeast growth arrest set the baseline for the antioxidant screening. The data demonstrated that the oxidant-induced growth arrest phenomenon of yeast was replicable in a 96-well plate format, because these results correlated with previous data that confirmed cell cycle arrest (Flattery-O'Brien & Dawes, 1998; Alic, 2001; Fong, 2008). The growth arrest observed with all six oxidants indicated that this was a conserved feature of yeast in response to different forms of oxidative stress. These six oxidants range from water-soluble H₂O₂ to hydrophobic lipid hydroperoxide; therefore, the assay is compatible with different solvents. Many antioxidant capacity assays are limited by the properties of the compound, such as hydrophilicity or hydrophobicity, which govern the compound's solubility in the assay reaction medium. An example of a compound that cannot be measured for oxidant-scavenging activity using the DPPH method is one that is insoluble in 60% or absolute methanol (Prior, 2003; Sharma & Bhat, 2009).

1

Chemical structures of the oxidants used in this study. (a) H₂O₂, (b) menadione, (c) LAH, (d) CHP, (e) peroxynitrite and (f) diamide.

2

Determination of arresting concentrations of oxidants on BY4743 yeast cells. Yeast cells (OD_{600 nm} 0.07) were challenged with two concentrations of (a) H₂O₂ at 2 mM (▪) and 4 mM (▲), (b) CHP at 100 μM (▪) and 150 μM (▲), (c) LAH at 50 μM (▪) and 75 μM (▲), (d) peroxynitrite at 5 mM (▪) and 10 mM (▲), (e) menadione at 100 μM (▪) and 150 μM (▲) and (f) diamide at 0.8 mM (▪) and 1.6 mM (▲). The control for each oxidant treatment is included (◆). Yeast growth (OD_{600 nm}) was monitored every hour for 5 h at 30°C. Data represent the average of six biological replicates with the SD.

Scavenging activity of antioxidants

By adding each oxidant at the arresting concentration to a yeast culture containing an antioxidant candidate compound, its oxidant-scavenging activity was assessed by its capacity to promote yeast growth from the oxidant-induced arrest. The results demonstrated that yeast assaulted with 4 mM H₂O₂ continued to display robust growth when treated with 8 mM ascorbate, indicating that a relatively high concentration of ascorbate is needed to negate the effects of 4 mM H₂O₂. This is in contrast to gallic acid, which had strong scavenging effects at only 2 mM concentration (Fig. 3). This could be due to its three hydroxyl groups, which can donate hydrogen for the reduction of H₂O₂, as it has been demonstrated that the number of hydroxyl groups correlates positively with the antioxidant activity (Sugita, 2004).

3

Screening for scavenging activity of antioxidants. Yeast cells were prepared and mixed with individual antioxidants and oxidants. Out of 36 combinations of six antioxidants and six oxidants performed, representative data are shown. (a) The scavenging activity of 8 mM ascorbate against 4 mM H₂O₂ (▪) the control with 4 mM H₂O₂ only (◆). (b) The scavenging activity of 2 mM gallic acid against 4 mM H₂O₂ (▪) and the control with 4 mM H₂O₂ only (◆). (c) The scavenging activity of 50 μM MnO₂ against 4 mM H₂O₂ (▪) and the control with 4 mM H₂O₂ only (◆). (d) The scavenging activity of 50 μM lipoic acid against 4 mM H₂O₂ (▪) and the control with 4 mM H₂O₂ only (◆). Data represent the average of six biological replicates, with the SD shown.

Ascorbate and gallic acid showed scavenging activity against the majority of oxidants by mitigating the oxidant-induced growth arrest of the BY4743 yeast strain. These results correlate with past studies that have demonstrated that both these antioxidants can scavenge many types of free radicals, including singlet oxygen, superoxide and hydroxyl radicals (Padh, 1991; Cheel, 2007). A major difference in the activity of ascorbate compared with gallic acid was that ascorbate was effective against the thiol-oxidizing chemical, diamide. This was somewhat unexpected, because ascorbate has been reported to be unable to reduce disulphide bonds (Jaffrey, 2001). However, more recent studies have indicated that ascorbate could indeed reduce disulphide bonds (Huang & Chen, 2006; Giustarini, 2008). The oxidized form of ascorbate, dehydroascorbic acid, is actually used to oxidize free thiol groups of proteins in bread dough to generate a more stable dough structure (Nakamura & Kurata, 1998), highlighting the complexity of the mechanisms involved in the activity of ascorbate in thiol chemistry. To further investigate the antidiamide activity of ascorbate, time-course experiments were performed for scavenging activity over an 18-h period (Fig. 4). The scavenging activity of ascorbate reduced the time of cell-cycle delay in yeast from 14 h in diamide-only samples to 4 h for cultures treated with ascorbate in the presence of diamide (Fig. 4a). The scavenging activity of ascorbate against H₂O₂ also resulted in the cells exiting early from the H₂O₂-induced arrest and entering the growth cycle at 8 h, 4 h earlier than the H₂O₂-only cells (Fig. 4b). The mechanism for the ascorbate activity against H₂O₂ is well documented. H₂O₂ oxidizes ascorbate to produce dehydroascorbate and water, with dehydroascorbate being relatively stable and less toxic (Sies, 1992; Deutsch, 1998).

4

The time course of yeast growth in the presence of an oxidant with and without the antioxidant ascorbate. (a) The growth of yeast cells in the media containing diamide (1.6 mM) only (▪) and diamide (1.6 mM) plus ascorbate (3.2 mM) (▲) was measured at the start and at every 2-h interval for 18 h. (b) The same procedure was carried out for yeast cells in the media containing H₂O₂ (4 mM) only (▪) and H₂O₂ (4 mM) plus ascorbate (3.2 mM) (▲). Data represent the average of six biological replicates, with the SD shown.

Manganese dioxide is often used as a catalyst for the decomposition of H₂O₂ (Hasan, 1999). As shown in this study, it is highly active against H₂O₂, with a concentration of 50 μM neutralizing the effect of 4 mM H₂O₂ (Fig. 3). These antioxidant activities exhibited by the manganese ion correlated well with past findings that showed that its homeostasis plays an important role in cell defence against oxidative stress (Lapinskas, 1995). Lipoic acid and zinc sulphate were shown to have no scavenging activity against H₂O₂ because their addition did not negate the growth arrest of 4 mM H₂O₂ (Fig. 3 and Table 1). For lipoic acid, the result is consistent with previous findings that lipoic acid was not an efficient scavenger of free radicals (Kagan, 1992).

1

Scavenging and intracellular induction activities of antioxidants against oxidants

Activity	Antioxidants	H2O2	CHP	LAH	Peroxynitrite	Menadione	Diamide
Scavenging activity	Ascorbate	✓	✓	✓	✓	✓	✓
Tocopherol	✓	×	✓	×	×	×
Gallic acid	✓	✓	✓	✓	✓	×
Lipoic acid	×	×	×	×	×	×
Zinc sulphate	×	×	×	×	×	×
Manganese oxide	✓	×	✓	✓	×	×
Intracellular induction response	Ascorbate	✓	✓	✓	✓	✓	✓
Tocopherol	✓	×	✓	×	×	✓
Gallic acid	✓	✓	✓	✓	✓	×
Lipoic acid	✓	✓	×	✓	✓	✓
Zinc sulphate	✓	✓	×	✓	×	×
Manganese oxide	✓	✓	✓	✓	×	×

✓, Positive activity; ×, no activity.

Induction of intracellular antioxidant activity

In addition to the scavenging activity, the antioxidants were also assayed for their ability to induce an intracellular antioxidant response within yeast cells. Cells treated with the candidate compounds were thoroughly washed, so that no extracellular antioxidant remained, before being exposed to the oxidants.

Similar to the results of the scavenging activity assays, ascorbate and gallic acid displayed activity against the majority of oxidants tested by inducing a growth arrest-resistant phenotype (Table 1). The ability of ascorbate to elicit intracellular antioxidant activity is likely due to its induction of gene expression as well as its intracellular accumulation. Catani (2001) demonstrated that several genes were upregulated by ascorbate, including the fra-1 gene, a member of the Fos family of transcription factors. Such ascorbate-mediated gene expression responses resulted in cellular protection against UV-induced oxidative damage (Catani, 2001). However, the gene expression profile engendered by ascorbate in yeast cells needs to be investigated to pinpoint the exact metabolic pathway/s responsible for its protective role against these oxidants. In addition, during the period of ascorbate treatment, yeast could accumulate the antioxidant, which has been shown to occur in other eukaryotic cells (Welch, 1995). The intracellularly accumulated ascorbate can in turn play a protective role in the ensuing oxidant exposure. It could work in concert with glutathione in the pathway known as the ascorbate–glutathione cycle (del Rio, 1998). Similarly, the wide-ranging induction activity of gallic acid could be a result of its upregulation of antioxidant enzymes such as glutathione synthase, glutathione peroxidase and thioredoxin peroxidase as shown by Abdelwahed (2007).

Zinc sulphate, manganese oxide and lipoic acid also induced an intracellular antioxidant response against a number of oxidants (Table 1). The zinc (Zn)- and manganese (Mn)-treated cultures showed some growth in the media containing 10 mM peroxynitrite, in contrast to the untreated controls. However, their growth did not reach the level of the yeast with decomposed peroxynitrite, which was nontoxic (Fig. 5). The results demonstrated that 50 μM zinc sulphate and 50 μM manganese oxide partially rescued yeast growth from peroxynitrite toxicity. However, neither Zn nor Mn had a positive effect on yeast growth in response to the superoxide-generating oxidant, menadione (Table 1). This finding was unexpected, because Zn and Mn are major components of the SODs. The Zn-SOD (cytosol) and Mn-SOD (mitochondria) are involved in cellular antioxidant defence by catalysing superoxide into H₂O₂ and oxygen (van Loon, 1986). This indicates that the Zn and Mn treatment did not contribute to an increased production of Zn-SOD and Mn-SOD. However, the destination of Zn and Mn might be in the formation of metalloporphyrins or their relative compounds because these Zn- and Mn-porphyrins were previously shown to be effective in neutralizing peroxynitrite oxidation (Szabó, 2007).

5

Screening for induction of intracellular antioxidant activity. Yeast cells were treated with each of the six antioxidants overnight, then washed and each treatment was reacted with six oxidants, respectively. The level of induction of the intracellular antioxidant activity was measured according to the growth of yeast cells. Out of 36 combinations, the representative data of Zn and Mn ions are shown. (a) Growth of Zn-treated yeast cells in the media containing peroxynitrite (▲) and the two controls performed in parallel, i.e. untreated yeast plus peroxynitrite (◆) and Zn-treated yeast plus decomposed peroxynitrite (▪). (b) Growth of Mn-treated yeast cells in the media containing peroxynitrite (▲) and the two controls including untreated yeast plus peroxynitrite (◆) and Mn-treated yeast plus decomposed peroxynitrite (▪). Data represent the average of six biological replicates, with the SD shown.

Lipoic acid was active in antioxidant induction against H₂O₂, cumene peroxide, menadione, peroxynitrite and diamide (Table 1). This is in clear contrast to its lack of scavenging activity. The explanation for this difference could lie in the change in its molecular structure as it moves from the extracellular environment to within the cell. The original lipoic acid, applied in the experiment for scavenging activity, has a cyclic disulphide chemical structure; hence, no free thiol group is available for reducing the oxidant. As a result, no scavenging activity was observed. However, upon entering the cell, the reduced form of lipoic acid, dihydrolipoic acid, is generated. Dihydrolipoic acid is easily oxidized, thus rendering it a strong reductant against superoxide, hydroxyl and peroxyl radicals and singlet oxygen within the cell (Moini, 2002). Another possible mechanism for such an intracellular induction could be the involvement of lipoic acid/dihydrolipoic acid redox coupling in the regeneration of the other antioxidants like ascorbic acid, vitamin E and glutathione (Packer, 1995).

This report has outlined an improved method for evaluating the potency of antioxidant compounds. Previous techniques were based on in vitro assays, and therefore had limitations in assessing the in vivo effects of potentially useful compounds. A more biologically relevant method was required to predict with greater accuracy the industrial or the medical benefits of candidate antioxidants. As yeast is the most characterized eukaryote at the molecular level, it serves as a paradigm for higher eukaryotes like plants and animals in fundamental cellular studies. The high-throughput technique outlined here utilized the important physiological phenomenon of oxidative stress-mediated growth arrest to screen the oxidant-scavenging and intracellular antioxidant activity of a chemically diverse range of compounds. Both of these characteristics are important for proper analyses, and the method was successful in identifying the scavenging and/or the intracellular antioxidant capacity of the compounds tested. Consequently, the assay is superior to previous methods used to determine antioxidant efficacy, and is highly relevant in facilitating antioxidant discovery for industrial or therapeutic applications.

Acknowledgements

This research was supported under Australian Research Council's Linkage Projects funding scheme (project number LP0775238). The authors are grateful for the technical support of Kellie McNamara.

Author notes

Editor: Monique Bolotin-Fukuhara