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Applied and Environmental Microbiology, January 2007, p. 572-580, Vol. 73, No. 2
0099-2240/07/$08.00+0     doi:10.1128/AEM.01963-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Green Tea Polyphenols Function as Prooxidants To Activate Oxidative-Stress-Responsive Transcription Factors in Yeasts

Kazuhiro Maeta, Wataru Nomura, Yoshifumi Takatsume, Shingo Izawa, and Yoshiharu Inoue^*

Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received 17 August 2006/ Accepted 12 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epigallocatechin gallate (EGCG) is the most abundant polyphenolic flavonoid in green tea. Catechin and its derivatives, including EGCG, are widely believed to function as antioxidants. Here we demonstrate that both EGCG and green tea extract (GTE) cause oxidative stress-related responses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe under weak alkaline conditions in terms of the activation of oxidative-stress-responsive transcription factors. GTE as well as EGCG induced the nuclear localization of Yap1 in S. cerevisiae, which was repressed by the addition of catalase but not by the addition of superoxide dismutase. The same phenomena were observed for the nucleocytoplasmic localization of Msn2 in S. cerevisiae and Pap1, a Yap1 homologue, in S. pombe. The formation of intramolecular disulfide bonds has been proposed to be crucial for the H₂O₂-induced nuclear localization of Yap1, and we verified the importance of cysteine residues of Yap1 in response to EGCG and GTE. Additionally, we show that EGCG and GTE produce H₂O₂ in a weak alkaline medium. Finally, we conclude that tea polyphenols are able to act as prooxidants to cause a response to oxidative stress in yeasts under certain conditions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Green tea is one of the beverages consumed in the highest quantity in the world. Epidemiologic research has revealed that individuals who drink a lot of green are less likely to develop cancer (28, 30, 51, 64). Very recently, a relationship between the consumption of green tea and a reduced risk of type 2 diabetes was reported (23). Green tea contains many ingredients considered to promote health such as polyphenolic flavonoids, of which epigallocatechin gallate (EGCG) is the major constituent. Evidence is mounting that EGCG has anticarcinogenic activity in vitro (3, 8, 27, 61), which may support the results of the epidemiologic research on the correlation between drinking green tea and the risk of morbidity from cancer.

Many studies have been done on the biological activity of green tea extract (GTE) and individual catechins in vitro. EGCG is widely accepted as an antioxidant. For example, EGCG scavenges superoxide anion radicals (O₂^·–), hydrogen peroxide (H₂O₂), hydroxy radicals (HO^·), peroxyl radicals, singlet oxygen, and peroxynitrite (5, 16, 17, 45, 47, 49, 56). The one-electron reduction potential of EGCG under standard conditions is 550 mV, a value lower than that of glutathione (920 mV) and comparable to that of -tocopherol (480 mV) (13, 24, 25). Besides directly scavenging reactive oxygen/nitrogen species, EGCG chelates redox-active metal ions, such as iron and copper, leading to a reduction in the production of reactive oxygen species. Accordingly, many food supplements or beverages containing a high concentration of EGCG (>1 mM) have been developed, and therefore, the physiological function of EGCG in vivo with a high-dose ingestion remains to be elucidated.

In contrast to its antioxidative activity, recent experiments in vitro indicate that EGCG produces reactive oxygen species. For example, EGCG promotes apoptosis and has bactericidal activity, which is attributed to its ability to reduce O₂ to yield H₂O₂ (2, 43). We have previously found that EGCG has an adverse effect on the protection of Escherichia coli cells against oxidative damage in the presence of H₂O₂ and copper ions (21). In addition, we have recently revealed that mutant strains of the budding yeast Saccharomyces cerevisiae that are defective in Yap1 and/or Skn7 show increased sensitivity to GTE (52). Both Yap1 and Skn7 are transcription factors critical for the response to oxidative stress. We have also reported that the expression of TRX2, which codes for the antioxidant enzyme thioredoxin and is one of the target genes for both Yap1 and Skn7 (32, 41, 55), is induced following treatment with GTE (52). Consequently, mutants defective in Yap1 and/or Skn7 showed increased susceptibility to GTE (52). These results imply that GTE induces a response to oxidative stress in yeast.

In spite of numerous reports about the effects of polyphenolic flavonoids on the mammalian system, intriguingly, little attention has been paid to the use of yeast to analyze the biological activity of tea polyphenols, though this organism has provided an excellent model for the study of many biological events. In the present study, to assess whether GTE actually causes oxidative stress-related responses in yeast, we determined the activity levels of oxidative-stress-responsive transcription factors, i.e., Yap1 and Msn2 in S. cerevisiae and Pap1 in the fission yeast Schizosaccharomyces pombe. The activities of these transcription factors are regulated through their nucleocytoplasmic localization (14, 31, 34). We show that these transcription factors are concentrated in the nuclei of cells treated with GTE or EGCG. Additionally, we demonstrate that the nuclear localization of Yap1 and Msn2 is repressed by catalase but not by superoxide dismutase. In regard to Yap1, the formation of intramolecular disulfide bonds has been proposed as crucial for its nuclear localization (10, 11, 33, 34, 58). We verify the correlation between the GTE- and EGCG-induced nuclear localization of Yap1 and the transcriptional activation of TRX2 using various Cys-replaced Yap1 mutants. Finally, we show that both GTE and EGCG produce H₂O₂ under weak alkaline conditions, which induce a response to oxidative stress in yeast cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
Green tea extract (sunphenon) was obtained from Taiyo Kagaku Co., Ltd., Japan. EGCG, bovine catalase, and Cu,Zn-superoxide dismutase were purchased from Wakenyaku Co., Ltd., Japan.

Strains.
S. cerevisiae strains used in this study have the YPH250 background (MATa trp1-1 his3-200 leu2-1 lys2-801 ade2-101 ura3-52). Construction of mutants yap1 (yap1::HIS3) and msn2 (msn2::URA3) was described previously (22). The YAP2/CAD1 locus of YPH250 was disrupted with the yap2::kanMX4 cassette (Invitrogen). S. cerevisiae W303-1A, carrying the temperature-sensitive xpo1-1 allele [MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 xpo1::LEU2, pKW466 (xpo1-1)] (50), which was donated by K. Weis, was also used. S. pombe TP108-3c (h^– leu1-32 ura4-D18 pap1::ura4⁺) (53) was provided by K. Shiozaki.

Media.
The yeasts were cultured in SD medium (2% glucose, 0.67% yeast nitrogen base without amino acids [pH 5.5]) supplemented with appropriate amino acids and nucleobases. YPD medium (2% glucose, 1% yeast extract, 2% peptone [pH 5.5]), H medium (1% glucose, 0.2% yeast extract, 0.5% peptone, 0.03% K₂HPO₄, 0.03% KH₂PO₄, 0.01% MgCl₂) (20, 52), and EMM medium (40) were also used as necessary.

GFP-tagged proteins.
A series of green fluorescent protein (GFP)-tagged Yap1 proteins, described previously, were used (34, 38). The plasmids for Msn2-GFP (14), Yap2-GFP (4), and GFP-Pap1 (31) were obtained from C. Shüller, P. Sunnerhagen, and M. Yoshida, respectively.

Treatment with chemicals.
Cells were cultured at 28°C until a log phase of growth was reached, collected aseptically by centrifugation, and suspended in fresh H medium at different pH levels as described previously (52). One-tenth percent GTE or 0.05% (1.1 mM) EGCG was added to the cell suspension, and cells were cultured for the prescribed time.

ß-Galactosidase assay.
The TRX2-lacZ reporter plasmid was donated by S. Kuge (32). The preparation of cell extracts and the measurements of ß-galactosidase activity were carried out as described previously (22). One unit of activity was defined as the amount of enzyme that increases the A₄₂₀ by 1,000 per hour at 30°C. Protein concentrations were determined by the method of Bradford (6).

Fluorescence microscopy.
Cells carrying each GFP-tagged protein were cultured in SD medium (for S. cerevisiae) or EMM medium (for S. pombe) until log phase was reached and treated with chemicals after being transferred to H medium as described above. The subcellular distribution of each GFP-tagged protein was observed using a fluorescence microscope (Olympus BX51).

Measurement of H₂O₂.
The production of H₂O₂ in H medium without cells (cell-free medium) after the addition of 0.1% GTE or 0.05% EGCG for 10 min at 28°C was measured by the ferrous ion oxidation-xylenol orange method as described previously (36, 43, 48).

Survival test.
Cells were cultured in SD medium until the A₆₁₀ reached 0.5. A portion of the cells was transferred to H medium (pH 5.5 or 7.6) containing 0.1% GTE and/or 1 mM H₂O₂. After incubation at 28°C for 60 min, cells were taken, diluted appropriately with the sterilized 0.85% NaCl solution, and spread on YPD agar plates. Viable cell numbers were determined by counting the colonies after 3 days of incubation at 28°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of pH on the nuclear localization of Yap1 following GTE treatment.
We have recently reported that GTE induces the expression of TRX2 in S. cerevisiae (52). The efficiency of TRX2 expression following GTE treatment varied with the pH of the medium, for which the optimal pH for the production of thioredoxin (Trx2) was pH 7.6. Yap1 is one of the transcription factors critical for the expression of TRX2 under oxidative stress conditions. To explore whether Yap1 is activated with GTE treatment, we monitored the distribution of GFP-tagged Yap1 in cells in GTE-containing media at different pH levels. As shown in Fig. 1, Yap1 was concentrated in the nucleus following treatment with 0.1% GTE at pH 7.6 for 10 min. Hence, we treated yeast cells with GTE at pH 7.6 thereafter. Adjustment of the pH of the medium per se did not cause the nuclear accumulation of Yap1.

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FIG. 1. Effect of pH on the nuclear localization of Yap1 without (w/o) and with (w/) GTE. S. cerevisiae yap1 mutant cells expressing GFP-Yap1 were cultured in SD medium until the A610 reached 0.5. Cells were transferred to H medium preparations of different pH levels as indicated in the figure, and 0.1% GTE was added. The subcellular distribution of GFP-Yap1 was monitored after 10 min.

Reversibility of GTE-induced nuclear localization of Yap1.
In mammals, orally ingested catechins are degraded through methylation, glucuronidation, and sulfonation in the intestines, liver, and kidneys (61); however, intracellular concentrations of catechins and their metabolized derivatives are fairly low even when a high dose of catechins has been ingested (13). To our knowledge, there is no report examining the uptake and bioconversion of catechins by yeast cells. Rather, Lopez-Toledano et al. (37) have reported that yeast inhibits the degradation of catechins under certain conditions. To gain a clue as to whether the nuclear localization of Yap1 is attributable to catechins and/or other ingredients in GTE, either taken up by the cell or present outside of the cell, we examined the reversibility of the activation of Yap1. As shown in Fig. 2, Yap1 was rapidly returned to the cytoplasm when cells were washed to remove GTE from the medium. Additionally, the nuclear Yap1 was redistributed in the cytoplasm after 60 min even though ample GTE was present in the medium. These results indicate that the activation of Yap1 by GTE is reversible and that yeast cells adapt to GTE in terms of the nuclear localization of Yap1.

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FIG. 2. Reversibility of GTE-induced nuclear localization of Yap1. S. cerevisiae yap1 mutant cells expressing GFP-Yap1 were treated with 0.1% GTE at pH 7.6, and the subcellular distribution of GFP-Yap1 was monitored periodically. After 10 min, GTE was removed by washing the cells with fresh H medium (pH 7.6) without GTE, and the cells were resuspended in the same medium.

Nuclear localization of Pap1.
Pap1 is a homologue of Yap1 in the fission yeast S. pombe. The activity of Pap1 is also regulated through its subcellular distribution (31). We determined whether GTE affects the nucleocytoplasmic localization of Pap1. As shown in Fig. 3A, Pap1 was concentrated in the nucleus 10 min after 0.1% GTE at pH 7.6 was added and was redistributed to the cytoplasm after GTE was removed.

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FIG. 3. Nuclear localization of bZIP transcription factors in yeasts with GTE. (A) S. pombe pap1 mutant cells expressing GFP-Pap1 were cultured in EMM medium until the A595 reached 0.5. For H2O2 treatment, 0.3 mM H2O2 was added directly. For GTE treatment, cells were transferred to H medium (pH 7.6) and 0.1% GTE was added. After 10 min, cells were collected by centrifugation and washed with fresh EMM medium or fresh H medium to remove chemicals. (B) The Yap2-GFP plasmid was introduced into S. cerevisiae carrying the temperature-sensitive xpo1-1 allele, and the transformant was cultured in SD medium at 28°C. When the A610 reached 0.5, cells were transferred to an incubator preheated at 37°C, and the subcellular localization of Yap2-GFP was examined after 15 min. (C) S. cerevisiae yap1 mutant cells expressing GFP-Yap1 or yap2 mutant cells expressing Yap2-GFP were cultured in SD medium until the A610 reached 0.5. For H2O2 treatment, 0.4 mM H2O2 was added directly. For GTE treatment, cells were transferred to H medium (pH 7.6) and 0.1% GTE was added. After 10 min, cells were collected by centrifugation and washed with fresh SD medium or fresh H medium to remove chemicals.

Yap2/Cad1, a paralogue of Yap1, is not concentrated in the nucleus with GTE treatment.
We have demonstrated that two oxidative-stress-responsive, basic-leucine zipper (bZIP) transcription factors in yeasts, i.e., Yap1 in S. cerevisiae and Pap1 in S. pombe, are concentrated in the nucleus following treatment with GTE. The regulatory mechanisms of the nucleocytoplasmic localization of these transcription factors are well conserved, i.e., these factors are concentrated in the nucleus under conditions of oxidative stress, and the nuclear export of these bZIP proteins is performed by Crm1/Xpo1 (31, 35, 54, 60). Both Yap1 and Pap1 have a conserved Cys cluster at their C terminus, which is referred to as the C-terminal Cys-rich domain (c-CRD), where nuclear export signals for Crm1/Xpo1 overlap (7, 31, 34). Since Yap2, also referred to as Cad1 (59), is a paralogue of Yap1 in S. cerevisiae, it has a c-CRD in its C terminus; however, there is no direct evidence that the nuclear export of Yap2/Cad1 is performed by Crm1/Xpo1. We verified whether Crm1/Xpo1 exports Yap2/Cad1 from the nucleus by using a temperature-sensitive xpo1-1 mutant. As shown in Fig. 3B, Yap2-GFP was accumulated in the nucleus upon exposure of xpo1-1 mutant cells to a nonpermissive temperature, indicating that Crm1/Xpo1 is the nuclear export factor for Yap2/Cad1.

Next, we examined whether Yap2/Cad1 is also concentrated in the nucleus following GTE treatment. As shown in Fig. 3C, however, Yap2/Cad1 was not concentrated in the nucleus under conditions where the nuclear accumulation of both Yap1 and Pap1 is achieved. Additionally, Yap2-GFP was not concentrated in the nucleus following treatment with 0.4 mM H₂O₂, conditions under which Yap1 is concentrated in the nucleus (Fig. 3C). We confirmed that Yap2/Cad1 was virtually restricted to the cytoplasm after 60 min of treatment with 0.4 mM H₂O₂ or with 0.1% GTE (Fig. 3C).

Nuclear localization of Msn2.
Besides Yap1, Msn2 is also one of the transcription factors in S. cerevisiae whose function is crucial for an oxidative stress-evoked response, and its nucleocytoplasmic localization changes under oxidative stress conditions. We examined whether Msn2 is also concentrated in the nucleus following treatment with GTE. As shown in Fig. 4, Msn2 accumulated in the nucleus for 5 min when 0.1% GTE was added at pH 7.6 and then returned to the cytoplasm after 60 min. In addition, as was observed for Yap1, Msn2 was rapidly redistributed to the cytoplasm when GTE was removed from the medium.

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FIG. 4. Effect of GTE on the nuclear localization of Msn2. S. cerevisiae msn2 mutant cells expressing Msn2-GFP were treated with 0.1% GTE at pH 7.6, and the subcellular distribution of Msn2-GFP was monitored periodically. After 10 min, GTE was removed by washing the cells with fresh H medium (pH 7.6) without GTE, and the cells were resuspended in the same medium.

Catalase represses the nuclear localization of Yap1 following GTE treatment.
Considering the results obtained above, GTE seems to cause a response to oxidative stress in yeast cells. To assess whether reactive oxygen species are formed during GTE treatment, we determined the effect of antioxidant enzymes on the GTE-induced nuclear localization of Yap1. As shown in Fig. 5A, catalase repressed the nuclear accumulation of Yap1 with GTE treatment, whereas Cu,Zn-superoxide dismutase did not. Essentially the same results were obtained in the case of Msn2 (Fig. 5B). These results suggest that H₂O₂ is produced when GTE is present in the medium.

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FIG. 5. Effects of antioxidant enzymes on GTE-induced nuclear localization of Yap1 and Msn2. S. cerevisiae yap1 mutant cells expressing GFP-Yap1 (A) or msn2 mutant cells expressing Msn2-GFP (B) were cultured in SD medium until the A610 reached 0.5. Cells were transferred to H medium (pH 7.6), and 0.1% GTE was added with or without catalase or Cu,Zn-superoxide dismutase (Cu,Zn-SOD). The subcellular distributions of GFP-Yap1 and Msn2-GFP were monitored after 10 min.

Production of H₂O₂ by GTE.
To obtain direct evidence that H₂O₂ is actually generated under conditions in which Yap1, Msn2, and Pap1 are concentrated in the nucleus, we measured the H₂O₂ level in the cell-free medium. As shown in Fig. 6, the amount of H₂O₂ produced increased in accordance with the pH, and approximately 0.37 mM H₂O₂ was generated by 0.1% GTE at pH 7.6 for 10 min. This tendency coincided well with the pH dependency of the nuclear localization of Yap1 following treatment with GTE (Fig. 1).

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FIG. 6. Effect of pH on the production of H2O2 by GTE. GTE (0.1%) was dissolved in H medium at different pH levels, and the production of H2O2 for 10 min was measured by the ferrous ion oxidation-xylenol orange method as described in the text. Results indicate the averages ± standard deviations of three independent experiments.

Several catechins are present in green tea, and EGCG is a major component (50%). We also determined the amount of H₂O₂ produced in cell-free medium containing 0.05% (approximately 1.1 mM) EGCG. As a result, 0.24 mM H₂O₂ was generated at pH 7.6. Therefore, the GTE-induced nuclear localization of oxidative-stress-responsive transcription factors is attributed to H₂O₂ produced in the presence of GTE.

Effect of H₂O₂ generated by GTE or EGCG on Yap1 activity.
We have demonstrated here that EGCG as well as GTE produces H₂O₂ in yeast medium under weak alkaline conditions (pH 7.6). To address whether the H₂O₂ generated by GTE or EGCG has the physiological effect of an oxidative stressor on yeast cells, we determined the nuclear localization of several Cys-replaced Yap1 mutants and their transcriptional activities by using the TRX2-lacZ reporter gene.

Figure 7A illustrates the configurations of Yap1 mutants tagged with GFP. Besides the c-CRD, Yap1 has another cluster of Cys residues in the N terminus (n-CRD). All the Cys residues in the Yap1 sequence are in these two CRDs. As shown in Fig. 7B, the replacement of all Cys residues (Yap1^6Cys mutant) abolished the nuclear accumulation of Yap1 following treatment with 0.1% GTE or 0.05% EGCG. The replacement of Cys residues in the n-CRD (Yap1^3Cys mutant) or c-CRD (Yap1^cm46A5 mutant) also prevented Yap1 from accumulating in the nucleus following treatment with GTE or with EGCG. Accordingly, these Yap1 mutants were not able to activate the expression of TRX2 (Fig. 7C).

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FIG. 7. Effect of Cys substitution on the nuclear localization of Yap1 with GTE and EGCG. (A) Schematic configuration of each Yap1 mutant. The formation of two disulfide bonds (Cys303-Cys598 and Cys310-Cys629) has been reported following H2O2 treatment (11, 15, 58). (B) yap1 mutant cells carrying each Yap1 mutant and the TRX2-lacZ reporter gene were cultured in SD medium until the A610 reached 0.5. Cells were transferred to H medium (pH 7.6), and 0.1% GTE or 0.05% EGCG was added. The subcellular distribution of each GFP-tagged Yap1 was observed after 10 min. (C) Cells were treated with 0.1% GTE or 0.05% EGCG as described above, and cell extracts were prepared after 60 min of incubation. ß-Galactosidase activity derived from TRX2-lacZ was measured as described in the text. It should be noted that the n-fold increase in the expression of TRX2-lacZ in the presence of 0.05% EGCG was lower because the amount of H2O2 produced by 0.05% EGCG was less than that attained with 0.1% GTE. Results indicate the averages ± standard deviations of three independent experiments.

Recent publications indicate that intramolecular disulfide bonds between n-CRD and c-CRD are formed in Yap1 upon H₂O₂ treatment (11, 14, 57, 58). To assess the importance of Cys residues for the GTE- and the EGCG-induced activation of Yap1, we determined the nuclear localization of Yap1 mutants, in which each of three Cys residues in the c-CRD was replaced. As shown in Fig. 7B, GFP-tagged mutants Yap1^C598T and Yap1^C629T failed to accumulate in the nucleus following treatment with GTE or EGCG, and consequently, these mutants did not induce TRX2 expression. As far as we could find, Cys⁶²⁰ was dispensable for the nuclear localization and the transcriptional activation of Yap1.

Previously, we have reported that Yap1 is activated by methylglyoxal (38), a natural metabolite of glycolysis (18, 39). The methylglyoxal-induced activation occurs without the formation of an intramolecular or intermolecular disulfide bond, i.e., any one of three Cys residues in the c-CRD is sufficient for the activation of Yap1 (38). To examine such a possibility, we determined the responsiveness of Yap1 mutants containing a single Cys residue in the c-CRD (mutants Yap1^3Ccm56, Yap1^3Ccm46, and Yap1^3Ccm45) following treatment with GTE or EGCG. As shown in Fig. 7B, these mutants resided in the cytoplasm, and therefore, they did not activate TRX2 expression (Fig. 7C).

Effect of GTE on the viability of yeasts.
We have demonstrated that GTE seems to function as a prooxidant to activate yeast transcription factors under weak alkaline conditions. Conversely, numerous reports have proven that GTE as well as EGCG has antioxidative activity. To verify that GTE does not function as an antioxidant for yeast cells under our experimental conditions, we determined the effect of GTE on the viability of yeast cells in the presence of H₂O₂ at pH 5.5 or 7.6. As shown in Fig. 8, 0.1% GTE did not repress the growth of yeast at pH 5.5, whereas viability was slightly reduced after 60 min of incubation at pH 7.6. When they were treated with 1 mM H₂O₂ for 60 min, approximately 45% of cells died at both pH levels. The cell death induced by 1 mM H₂O₂ was suppressed if 0.1% GTE was present at pH 5.5, indicating that GTE alleviates the toxic effect of H₂O₂; however, no such effect was observed at pH 7.6.

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FIG. 8. Evaluation of antioxidative activity of GTE. S. cerevisiae cells (wild type) were cultured in SD medium until the A610 reached 0.5. Cells were transferred to H medium at pH 5.5 or 7.6, and 0.1% GTE and/or 1 mM H2O2 was added. After 60 min, cell viability was determined as described in the text. The viability of cells before chemical treatment was taken as 100%. Results indicate the average ± standard deviations of three independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
H₂O₂ production by tea polyphenols.
In the present study, we have presented evidence that GTE as well as EGCG causes oxidative-stress-related responses in yeast cells under weak alkaline conditions. We have previously reported the adverse effect of EGCG on the viability of E. coli cells in the presence of H₂O₂ and copper ions (21), despite many studies demonstrating that EGCG scavenges reactive oxygen species directly and chelates redox active metals in vitro (1, 16, 26, 29, 42, 46, 56). Hence, we propose that the antioxidative activity of catechins should be reevaluated under certain conditions (21). Previously, it had been established that catechins as well as GTE kill Streptococcus mutans via unknown mechanisms. Later, it was reported that H₂O₂ is produced by EGCG (9, 62, 63). Recently, Arakawa et al. (2) pointed out the importance of the pH of the solution in which EGCG as well as GTE is dissolved for the production of H₂O₂, which is attributed to the bactericidal activity of GTE and EGCG. Independently, Nakagawa et al. (43) also reported that EGCG produces a considerable amount of H₂O₂ in a weak alkaline buffer. In our previous report (52), we found that the GTE-induced expression of TRX2 in S. cerevisiae varied with the pH of the medium. In the present study, we showed that a marked nuclear localization of oxidative-stress-responsive transcription factors in yeasts (Yap1, Msn2, and Pap1) following treatment with GTE (Fig. 1, 3, and 4) and EGCG (Fig. 7, data not shown) was attained at pH 7.6, conditions under which the maximal expression of TRX2 was achieved (52). We also revealed that concentrations of 0.37 mM and 0.24 mM H₂O₂ are produced by 0.1% GTE and 0.05% (1.1 mM) EGCG in yeast medium at pH 7.6, respectively. However, in a weakly acidic medium, GTE protects yeast cells from oxidative damage caused by H₂O₂ (Fig. 8), suggesting that the antioxidative activity of GTE is exerted, whereas at pH 7.6, the viability of yeast cells was slightly decreased in the presence of GTE per se. These results indicate that the antioxidative activity of GTE, which is due to polyphenolic flavonoids such as EGCG, depends upon the pH of the milieu.

Arakawa et al. (2) proposed the following model of H₂O₂ production in a solution at high pH: HO^– ions dissociate two protons from the hydroxy moieties of EGCG, and remaining electrons on the resultant phenoxyl radicals are accepted by O₂ to form H₂O₂. Presumably, each of two electrons is transferred sequentially to O₂ to form O₂^2– (O₂ + e^– O₂^·– + e^– O₂^2–), and consequently, protonation of O₂^2– will occur to yield H₂O₂ (O₂^2– + 2H⁺ H₂O₂). Recently, Elbling et al. (12) have reported that GTE and EGCG exhibit cytotoxic and genotoxic effects but no antioxidative activity at all in HL60 cells, and such adverse effects are suppressed only when catalase and superoxide dismutase are added simultaneously. Therefore, they argue that an additional effect of O₂^·– generated from EGCG should be considered. Nakagawa et al. (43) have also reported that the EGCG-mediated cytotoxic effect in Jurkat cells is partially suppressed by the addition of superoxide dismutase. However, under the conditions we used with yeast medium, catalase alone was able to suppress the nuclear localization of Yap1 and Msn2 following treatment with GTE, whereas superoxide dismutase was not (Fig. 5). Since the half-life of O₂^·– is shorter than that of H₂O₂, superoxide dismutase used in our system might not be enough to trap O₂^·–.

Activation of AP-1-like bZIP transcription factors by GTE.
Kuge et al. (33) have reported that an intramolecular disulfide bond is formed between Cys⁵⁹⁸ and Cys⁶²⁰ within the c-CRD of Yap1 following H₂O₂ treatment in vitro. A fusion construct consisting of the nuclear localization signal of Gal4 protein followed by GFP and Yap1-c-CRD (Gal4-GFP-c-CRD) is sensitive to H₂O₂ in terms of its nuclear localization (32), suggesting that the disulfide bond between Cys⁵⁹⁸ and Cys⁶²⁰ masks the nuclear export signal within the c-CRD to inhibit the interaction with Crm1/Xpo1, thereby resulting in the nuclear localization. However, even though the Yap1^3Cys mutant concentrated in the nucleus following treatment with 0.4 mM H₂O₂, it resides there for a shorter period than the wild-type Yap1 and, consequently, cannot induce the expression of the Yap1 target gene (38). Here we confirmed that Yap1^3Cys failed to induce TRX2 expression in response to GTE and EGCG (Fig. 7C).

Delaunay et al. (11) have reported that an intramolecular disulfide bond between Cys³⁰³ and Cys⁵⁹⁸ is formed to induce TRX2 expression. This suggests that the disulfide bond between n-CRD and c-CRD is necessary for the nuclear accumulation and transcriptional activation of Yap1. Wood et al. (57, 58) have shown that two intramolecular disulfide bonds, one between Cys³¹⁰ and Cys⁶²⁹ and the other between Cys³⁰³ and Cys⁵⁹⁸, in Yap1 formed in vitro during oxidation experiments with H₂O₂ and that such an oxidized Yap1 can bind the TRX2 promoter. More recently, Gulshan et al. (15) have reported that the folding of Yap1 to form an intramolecular disulfide bond between n-CRD and c-CRD is necessary for the recruitment of a mediator protein, Rox3, to the TRX2 promoter. We have demonstrated in this study that Cys⁵⁹⁸ and Cys⁶²⁹ are essential but Cys⁶²⁰ is dispensable for the GTE- and EGCG-induced nuclear localization and transcriptional activation of Yap1 (Fig. 7). Considering that the Yap1^3Cys mutant was incompetent in inducing the expression of TRX2 in response to GTE, EGCG, and H₂O₂, a conformational change of Yap1 caused by folding through the formation of a disulfide bond between the domains of n-CRD and c-CRD may determine the activity of Yap1 as a transcription factor. The behavior of a Yap1 protein mutated at Cys⁶²⁹ may provide a clue to this issue. Delaunay et al. (10) have reported that the Yap1^C629A mutant was impaired in the induction of TRX2 expression by H₂O₂, comparable to that of Yap1^C598A. Kuge et al. (33) have also reported that the Yap1^C629T mutant was incompetent in its response to H₂O₂ in terms of TRX2 expression. On the other hand, Wood et al. (57) have reported that the disulfide bond of Cys³⁰³-Cys⁵⁹⁸ is less stable in Yap1^C629A. It is feasible that the disulfide bond between Cys³⁰³ and Cys⁵⁹⁸ is formed with the aid of Gpx3 in the presence of H₂O₂ (11), and subsequently, the second disulfide bond between Cys³¹⁰ and Cys⁶²⁹ is formed presumably as the distance between n-CRD and c-CRD shortens and, consequently, the dual disulfide bond stabilizes the folding of Yap1. Hence, both Cys³⁰³-Cys⁵⁹⁸ and Cys³¹⁰-Cys⁶²⁹ may be necessary to maintain a stable folding of Yap1 under our experimental conditions, in which a relatively small amount of H₂O₂ is supplied and Cys³¹⁵ may not affect the localization of Yap1 following treatment with GTE as well as EGCG. This also suggests that the response of Yap1 to GTE/EGCG has requirements similar to those for H₂O₂.

The responsiveness of Yap2/Cad1 may provide another aspect to the reactive oxygen species generated by GTE and EGCG. Even though Cys residues in the c-CRD are well conserved in Yap2/Cad1 and Crm1/Xpo1 is the nuclear export factor for this Yap1 paralogue, Yap2-GFP was not concentrated in the nucleus following treatment with either GTE or H₂O₂ (Fig. 3). On the other hand, Bilsland et al. (4) have reported that Yap2/Cad1 is accumulated in the nucleus following treatment with 1.5 mM tert-butyl hydroperoxide, a model compound of lipid hydroperoxide. We have demonstrated that approximately 0.4 mM H₂O₂ was generated from 0.1% GTE. The oxidation potential of H₂O₂ is not strong enough to cause lipid peroxidation; however, if redox-active metals such as iron and copper are present, a so-called Fenton reaction (H₂O₂ + Fe²⁺ HO^· + HO^– + Fe³⁺) occurs to generate HO^·, which can extract bis-allylic hydrogen atoms from unsaturated fatty acids to initiate the radical chain reaction to yield lipid hydroperoxide (19). Additionally, Bilsland et al. (4) have reported that Yap2/Cad1 accumulated in the nucleus following treatment with 10 mM Zn²⁺ (65.4 ppm) or 30 µM Cd²⁺ (3.32 ppm). Although the GTE used in the present study contains a trace amount of heavy metals (<1 ppm dry weight; manufacturer's specifications), the final concentration of heavy metals with 0.1% GTE is 0.001 ppm, and such low levels of metals were not likely to contribute to the accumulation of Yap2/Cad1 in the nucleus. In addition, the reduction of oxidized metal ions (e.g., Fe³⁺ + e^– Fe²⁺) is necessary to trigger the Fenton reaction to produce HO^· constantly. Nakagawa et al. (44) have reported that HO^· is produced in a cell-free system containing 100 µM (5.85 ppm) Fe³⁺ and 100 µM EGCG. However, HO^·-mediated lipid peroxidation allowing Yap2/Cad1 to accumulate in the nucleus would not be feasible under the conditions we employed, where only trace amounts of heavy metals are involved. Collectively, the reactive oxygen species believed to cause the oxidative stress response of yeast cells to GTE as well as EGCG is H₂O₂.

Here we have presented the first evidence that a polyphenolic flavonoid in green tea provokes oxidative stress-related responses in yeasts. Our data will provide new insights into the cellular response of yeast to polyphenolic compounds.

 
We thank K. Shiozaki, C. Shüller, P. Sunnerhagen, M. Yoshida, and S. Kuge for generously providing yeast strains and plasmids.

This work was supported in part by grants from the Bio-oriented Technology Research Advancement Institution.

 
* Corresponding author. Mailing address: Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. Phone: 81 774-38-3773. Fax: 81 774-33-3004. E-mail: y_inoue{at}kais.kyoto-u.ac.jp.

Published ahead of print on 22 November 2006.

Present address: Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, January 2007, p. 572-580, Vol. 73, No. 2
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