2015 Volume 21 Issue 1 Pages 137-143
Yeast lacking Cu,Zn-superoxide dismutase (sod1Δ) exhibits methionine (Met) auxotrophy, primarily due to the depletion of NADPH required in the sulfur assimilation pathway by oxidative stress. In this study, we measured the ability of natural compounds, including ingredients in foods and dietary supplements, to restore the cell growth of sod1Δ in liquid and solid media without Met. Homocysteine at around 0.1 mM fully suppressed auxotrophy but in excess (> 0.4 mM) had a harmful effect. Methionine sulfoxide at concentrations from 0.05 to 0.8 mM and S-adenosylmethione (0.05 to 6.2 mM) completely relieved the growth defects caused by sod1Δ. Ascorbic acid (1 to 50 mM) restored sod1Δ growth, indicating that the action of this antioxidant could improve Met biosynthesis. However, ascorbic acid derivatives, ascorbic acid 2-glucoside and dehydroascorbic acid, did not show any activity. The suppressive effects of cysteine, N-acetylcysteine, and glutathione peaked at 0.1 mM, 0.5 mM, and 0.05 mM, respectively, but an excess of these agents was less effective. Suppression by dithiothreitol confirms that the thiol group is responsible for the amelioration of Met biosynthesis. No growth was restored for other categories of antioxidants including polyphenols, Trolox, melatonin, astaxanthin, and others.
Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radical, are continuously generated during respiration and cause oxidative deterioration of cellular macromolecules. ROS damage is linked to aging and many different human diseases, including cancer and neurodegenerative diseases (Halliwell and Gutteridge, 2007). Furthermore, ROS generation is elevated by environmental oxidative stressors such as exposure to ultraviolet or ionizing radiation and diverse chemical pollutants. Enzymes involved in the elimination of ROS include superoxide dismutases (SODs), catalase, and glutathione peroxidase. Low molecular weight antioxidants in cells (e.g., reduced glutathione) and obtained from foods (e.g., vitamins C, E, carotenoids and phenolic compounds) non-enzymatically alleviate the harmful effects of ROS. While dietary supplements with antioxidant activity are commonly consumed, the scientific evidence for their beneficial health effects is inconclusive (Bouayed and Bohn, 2010; Bergström et al., 2012; Sadowska-Bartosz and Bartosz, 2014). Therefore, biological tests to determine the antioxidant activity of natural compounds are important for the nutritional evaluation of ingredients in foods and dietary supplements.
Cu,Zn-SOD is one of the principal defenses against superoxide stress in the cytoplasm and mitochondrial intermembrane space. The yeast Saccharomyces cerevisiae lacking Cu,Zn-SOD (sod1Δ) has a number of oxygen-dependent defects, including sensitivity to oxygen, redox cycling drugs, and hyperosmotic stress (Chang et al., 1991; Krasowska et al., 2003; Kozioł et al., 2005; Saffi et al., 2006; Tamura et al., 2010), a moderate mutator phenotype (Gralla and Valentine, 1991; Nagira et al., 2013), metabolic alterations including auxotrophy for the amino acids Lys and Met (Chang and Kosman, 1990; Wallace et al., 2004; Sehati et al., 2011), and shortening of the replicative life span (Krzepiłko et al., 2004; Owsiak et al., 2010). These detrimental phenotypes of sod1Δ could be alleviated by the addition of vitamin C (ascorbic acid, AsA), suggesting the possibility that the yeast mutant can be used as a biosensor for low molecular weight antioxidants (Zyracka et al., 2005b; Tamura et al., 2010; Nagira et al., 2013).
The disruption of Met biosynthesis in sod1Δ appears to be caused by the depletion of NADPH by oxidative stress, because sulfur assimilation requires a large amount of NADPH (Slekar et al., 1996; Jensen et al., 2004). In the present study, a simple test to biologically evaluate natural compounds such as ingredients of foods and dietary supplements was developed on the basis that sod1Δ growth could be restored in liquid and solid media without Met. We also applied this assay to measure the biological activity of several categories of natural antioxidants and an actual food extract.
Strains and media The sod1Δ strain (clone ID, 16913) of S. cerevisiae and the wild-type (WT) strain (BY4742; Matα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The strains were routinely grown aerobically in complete SD medium, which contains a 0.67% Difco yeast nitrogen base w/o amino acids (Becton, Dickinson and Co., Sparks, MD, USA), 2% glucose, and 1.5% of an amino acid/nucleotide base mixture (containing 3.3 mg/mL of each amino acid His, Leu, Lys, and Met, and 1.3 mg/mL uracil).
Assay for suppression of Met auxotrophy in liquid culture The sod1Δ cells were precultured in complete SD medium at 28°C for 24 h. The cells were inoculated into 2 mL of fresh Met-minus SD medium in a test tube (16.5 mm diameter, 165 mm length) to a final absorbance of 0.01 at 600 nm. Test compounds were supplemented to the medium at various concentrations using a two-fold serial dilution series. The aerobic culture was performed on a reciprocal shaker (250 rpm) at 28°C for 24 h, and the absorbance of each culture was measured at 600 nm.
Assay for suppression of Met auxotrophy on agar plate The sod1Δ and parental cells were grown overnight in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose), and washed with sterilized water. Cell density was adjusted to a final absorbance of 0.3 at 660 nm. The cells were diluted 10-fold serially from 10−1 to 10−4 in sterilized water. Dilutions (8 µL) were spotted onto Met-minus SD agar plates supplemented with test compounds. Test compounds at various concentrations were added to the agar medium cooled to 50°C just before pouring of the plate. After 3 days of growth at 28°C, photographs of colonies were taken.
Reagents AsA, L-methionine sulfoxide (Met-O), S-adenosyl-L-methione (AdoMet), N-acetylcysteine (NAC), and the reduced form of glutathione (GSH) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Other compounds were from Wako Pure Chemical Industries (Osaka, Japan). Lipophilic antioxidants were dissolved with 100% dimethyl sulfoxide (DMSO), and added to culture media to keep the final concentration of DMSO below 1%.
Suppression of sod1Δ Met auxotrophy by organic sulfur compounds The effects of organic sulfur compounds on sod1Δ strain growth in Met-minus medium were quantitatively measured using a liquid culture assay (Fig. 1). Met between 0.025 mM and 0.4 mM restored the growth of sod1Δ in a dose-dependent manner, and at higher concentrations cell growth gradually decreased. The growth curve of sod1Δ in the presence of 0.4 mM Met was almost the same as that of wild-type cells (data not shown). Homocysteine (Hcy) also suppressed Met auxotrophy and peaked at near 0.1 mM, suppressing growth by 90% in the presence of Met at 0.4 mM, but almost all activity was lost at high concentrations (> 0.4 mM). Met-O and AdoMet between 0.00625 mM and 0.05 mM quantitatively restored the growth of sod1Δ, and at higher concentrations the suppression activities of these compounds were saturated (insets in Fig. 1C and 1D). Similarly to Met, a high dosage of Met-O (> 1.6 mM) gradually decreased cell growth. On the other hand, AdoMet sustained considerable activity without any inhibition at the higher concentrations tested. These results indicate that Met and its metabolites could efficiently alleviate the growth defect of sod1Δ in Met-minus medium, but adverse effects were different among those compounds.
Suppression of Met auxotrophy of sod1Δ cells by Met and its derivatives
sod1Δ cells were inoculated into Met-minus SD liquid medium supplemented with Met metabolites at various concentrations using a two-fold serial dilution series. Met (A), Hcy (B), Met-O (C), and AdoMet (D). The cells were cultured aerobically at 28°C for 24 h, and the absorbance at 600 nm of each culture was measured. Dotted line in (B) to (D) shows the growth level of sod1Δ cells in SD medium supplemented with 0.4 mM Met. Results are the mean ± SD for 3 experiments. Inset in C and D shows the same graph with an expanded horizontal axis (lower than 0.25 mM of Met-O and AdoMet).
Suppression of sod1Δ Met auxotrophy by AsA The ability of antioxidants to restore cell growth in Met-minus medium was quantitatively assayed. A potent antioxidant, AsA exhibited a dose-dependent protective effect at concentrations ranging from 1 to 10 mM (Fig. 2). At around 10 mM AsA, the growth of sod1Δ cells was restored to near the level of the Met-plus culture. The activity of AsA at 10 mM to 50 mM was saturated. Although culture on agar plates achieved a more effective aerobic environment than in liquid medium, the growth of sod1Δ cells on plates was also restored by adding 5 mM to 50 mM of AsA (Fig. 3). These results indicate that AsA can suppress sod1Δ Met auxotrophy and that the defect of Met biosynthesis is certainly due to the accumulation of endogenous ROS in the cells. Ascorbic acid 2-glucoside, which is a stable AsA derivative used as a nutritional supplement and in cosmetic products, did not show any suppressive effect of sod1Δ Met auxotrophy (maximum concentration examined, 50 mM), probably due to its inability to be absorbed into yeast cells. Dehydroascorbic acid, an oxidized form of AsA, also showed no protective effect, even at 5 mM.
Suppression of Met auxotrophy of sod1Δ cells by AsA
Assays were performed as described in the Materials and Methods. Dotted line shows the growth level of sod1Δ cells in SD medium supplemented with 0.4 mM Met. Inset shows the same graph with an expanded horizontal axis (lower than 4 mM of AsA). Results are the mean ± SD for 3 experiments.
Suppression of Met auxotrophy of sod1Δ cells by AsA and thiols
Plate assays were performed using Met-minus SD agar plates supplemented with Met (0.4 mM) or antioxidants AsA (A), Cys (B), GSH (C), and NAC (D) at concentrations indicated in the figure.
Suppression of sod1Δ Met auxotrophy by thiol compounds The effects of thiol compounds on sod1Δ strain growth in Met-minus medium were assayed in liquid culture (Fig. 4). Cys, GSH, and NAC were able to restore the growth of sod1Δ cells in Met-minus medium. As little as 0.1 mM of Cys and GSH in the medium was detected, and at the optimal concentration they restored cell growth to nearly 70% of the level of Met-plus medium. NAC had a slightly higher optimal concentration at around 0.5 mM. High doses of these thiol antioxidants proved to be less effective in restoring the growth of sod1Δ cells, probably due to adverse effects of an excess of these compounds. In the agar plate assay, Cys at 0.1 and 0.5 mM restored the growth of sod1Δ, but at higher concentrations (> 5 mM), it exhibited severe toxicity, even to wild-type cells (Fig. 3). GSH efficiently restored the growth of sod1Δ with a wide optimal range (0.05 mM to 5 mM). NAC showed activity at 1 and 5 mM. These compounds possess potent antioxidant activity and simultaneously serve as metabolites that are converted to Met in yeast cells (Thomas and Surdin-Kerjan, 1997). The reducing agent dithiothreitol (DTT) at around 0.5 mM restored growth to nearly 50% of the level of Met-plus medium, indicating that the reducing activity of the thiol group is certainly responsible for restoring the growth of sod1Δ in Met-minus medium (Fig. 4D). On the other hand, DTT is known to cause endoplasmic reticulum stress in yeast cells by interfering with disulfide bond formation (Jämsä et al., 1994). In this study, DTT at higher concentrations (> 1 mM) severely inhibited the growth of sod1Δ (Fig. 4D).
Suppression of Met auxotrophy of sod1Δ cells by thiol compounds
Assays were performed as described in the Materials and Methods. Cys (A), GSH (B), NAC (C), and DTT (D). Dotted line shows the growth level of sod1Δ cells in SD medium supplemented with 0.4 mM Met. Results are the mean ± SD for 3 experiments.
Effects of natural antioxidants on sod1Δ Met auxotrophy In the liquid culture assay, no apparent suppression of Met auxotrophy was observed for other categories of natural antioxidants: resveratrol (maximum concentration examined, 0.2 mM), quercetin (0.2 mM), catechin (1 mM), Trolox (1 mM), vitamin A acetate (1.6 µM), α-lipoic acid (5 mM), carnosine (5 mM), L-carnitine (5 mM), ureic acid (1 mM), melatonin (5 mM), and astaxanthin (0.2 mM). Similarly, in our previous studies, polyphenol antioxidants and a vitamin E analogue did not ameliorate the hypertonic stress-sensitivity and mutator phenotype of sod1Δ (Tamura et al., 2010; Nagira et al., 2013).
Suppression of Met auxotrophy of sod1Δ cells by lemon juice The suppression test using sod1Δ cells was applied to measure the activity of an actual food extract (Fig. 5). Fresh-squeezed lemon juice quantitatively suppressed sod1Δ Met auxotrophy. The addition of 100 µL of fresh juice into 2 mL of culture medium restored the growth of sod1Δ to 65% of the level in Met-plus medium. This suppression efficiency was likely evaluated as the overall activity of various components in lemon juice. Heat treatment (100°C, 30 min) of the juice decreased the suppression activity by about 70%, indicating that the lost activity in fresh juice was heat-labile. At present, it is not clear which compounds in lemon juice were responsible for the effective suppression of sod1Δ Met auxotrophy.
Suppression of Met auxotrophy of sod1Δ cells by lemon juice
Fresh-squeezed lemon juice was neutralized with NaOH to pH 6, and passed through a 0.22-µm filter. The sod1Δ cells were grown in 2 mL of Met-minus SD liquid medium supplemented with various volumes of the fresh lemon juice (closed circle). Heat-treated (100°C, 30 min) juice was also tested for suppression activity (open circle). The cells were cultured aerobically at 28°C for 24 h, and absorbance at 600 nm of each culture was measured. Dotted line shows the growth level of sod1Δ cells in the medium supplemented with 0.4 mM Met. Results are the mean ± SD for 3 experiments.
A simple test for the biological evaluation of food and dietary supplement ingredients was developed on the basis of the suppression of sod1Δ Met auxotrophy. The sod1Δ strain used here was obtained from the collection of the Saccharomyces Genome Deletion Project, and exhibited Met auxotrophy under atmospheric oxygen levels, as well as sensitivity to oxygen and hypertonic stress, and a mutator phenotype (Tamura et al., 2010; Nagira et al., 2013). In the present study, we modified the method of Zyracka et al. (2005a; 2005b) in which the effects of antioxidants on Met and Lys auxotrophy of another sod1Δ strain were measured on agar plates under different oxygen concentrations. They quantified cell growth on solid medium by suspending the obtained colonies in water and reading the optical density of the suspension. Our method, which uses a commercially available strain, is quite simple and suitable for quantitative evaluation of a large number of test compounds for biological activity to rescue sod1Δ auxotrophy.
Met and its metabolites could efficiently rescue the growth defect of sod1Δ in Met-minus medium with different optimum concentrations and side effects. Hcy is converted by remethylation to Met or by transsulphuration to Cys thorough the intermediate cystathione (Thomas and Surdin-Kerjan, 1997). Although Hcy suppressed auxotrophy close to the growth level in the presence of Met, high doses of Hcy caused severe toxicity. Hyperhomocysteinemia in humans is a risk factor for cardiovascular disease and thrombosis (Wierzbicki, 2007; Cattaneo, 1999). Several lines of evidence show that Hcy exerts an adverse effect on endothelial cells by increasing oxidative stress (Kolling et al., 2011; Leung et al., 2013). In yeast cells, high levels of Hcy can lead to mitochondrial dysfunction, which could potentially initiate pro- apoptotic pathways (Kumar et al., 2011). Met-O naturally occurs in protein as the oxidized form of Met residue as well as a free form amino acid, and free Met-O can be reversed by the methionine sulfoxide reductase family enzymes MsrA and fRMsr (Moskovitz et al., 2000; Le et al., 2009). AdoMet is generated from ATP and Met by the methionine adenosyltransferases SAM1 and SAM2, and serves as a methyl donor (Thomas and Surdin-Kerjan, 1997). AdoMet has been available as a nutritional supplement in the USA (i). We found that Met-O and AdoMet are good supplements to alleviate sod1Δ growth defects (Fig. 1C and 1D). AdoMet exhibits efficacy over a broad dose range without any adverse effects.
Exogenous AsA could suppress sod1Δ Met auxotrophy in both liquid and solid media, indicating that antioxidant action could prevent the depletion of NADPH required for Met biosynthesis in yeast cells. Zyracka et al. (2005b) showed the same protective effect of AsA using a different sod1Δ strain. Although AsA acts as a strong ROS scavenger in vitro, it also has a pro-oxidant effect in the presence of free transition metal ions (Bergström et al., 2012; Du et al., 2012). Therefore, the role of AsA in protecting against oxidative damage in living cells is controversial. Healthy human plasma contains AsA at a level between 0.04 and 0.08 mM (Du et al., 2012). Our result showed that AsA exhibits a protective effect in vivo even at a very high concentration (50 mM). In agreement with the result of Zyracka et al. (2005a), thiol compounds Cys, GSH, and NAC restored the growth of sod1Δ in Met-minus medium (Fig. 4). Unlike AsA, an excess of these compounds exhibited adverse effects. The optimal concentration needed to restore sod1Δ growth on agar plates seems to be higher than that measured in liquid culture, probably due to differences in aerobic conditions (Fig. 3). In the plate assay, GSH at a wide range of concentrations (0.05 mM to 5 mM) efficiently protected sod1Δ cells with less toxicity. The result may indicate that GSH plays a pivotal role in the intracellular antioxidant system of yeast.
In this study, we measured the effect of other categories of natural antioxidants, including polyphenols, a vitamin E analogue, melatonin, and astaxanthin; however, no apparent activity to ameliorate sod1Δ Met auxotrophy was observed. These antioxidants have been demonstrated to have effective antioxidant activities in various in vitro assays, and most compounds are ingredients in commercially available dietary supplements (Bouayed and Bohn, 2010; Bergström et al., 2012; Sadowska-Bartosz and Bartosz, 2014). At present, a limited number of antioxidants (AsA and thiol antioxidants) can suppress Met auxotrophy of the sod1Δ strain by exogenous supplementation. The same pattern of antioxidant action was found when studying the restoration of sod1Δ from oxidative stress-sensitive phenotypes (Lewinska et al., 2004; Kozioł et al., 2005; Tamura et al., 2010; Kwolek-Mirek et al., 2012) and a mutator phenotype (Nagira et al., 2013).
The effect of exogenous antioxidants on cells and organisms may be complicated by their permeability into the cells, induction of endogenous signal transduction pathways and adverse effects of the reaction products with biological molecules (Bednarska et al., 2008; Bouayed and Bohn, 2010; Bergström et al., 2012; Sadowska-Bartosz and Bartosz, 2014). With respect to yeast, the ability to lower the redox potential of the extracellular medium and to maintain critical protein thiols in a reduced state are important aspects of the antioxidant action (Bednarska et al., 2008). Supplementation of the growth medium with the lipophilic antioxidant α-tocopherol increased oxidative stress and decreased the lifespan of yeast cells, and antioxidant activity and the membrane retention ability of α-tocopherol were involved in the lifespan-reducing effect (Lam et al., 2010). Therefore, mutant yeast cells could provide a good model system to study the difference in protective effects of antioxidants in vivo and in vitro, especially when conventional and biological evaluations of antioxidants are apparently different (e.g., polyphenols, Trolox, melatonin, astaxanthin, etc. used in this study).
Acknowledgment This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Financial Assistance Program of the Social Cooperation Study (2006–2010).
