Dose-Dependent Protective and Inductive Effects of Xanthohumol on Oxidative DNA Damage in Saccharomyces cerevisiae

The prenylated chalcone xanthohumol ((E)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]-3-(4-hydroxyphenyl)prop-2-en-1-one) is the principal prenylfl avonoid of the female infl orescences (hops) of the hop plant Humulus lupulus L. (1,2). Hops are used in beer production to add bitt erness and fl avour, whereby beer is the main human dietary source of xanthohumol (3,4). This phenolic compound has received considerable att ention in recent years, since it has shown interesting biological properties with potential for disease prevention and therapeutic applications: anti-infl ammatory, antioxidant, antilipoperoxidative activities as well as antiangiogenic and antiproliferative eff ects (3–5).

Xanthohumol has been shown to exhibit potent antioxidant activity through the direct scavenging of reactive oxygen species (ROS) generated by xanthine oxidase, while its isomer isoxanthohumol is inactive (6).The radicals formed by oxidative stress can modify polyunsaturated lipids, proteins and nucleic acids, and are therefore associated with the early stages of carcinogenesis and apoptosis (4).Xanthohumol can also inhibit Cu 2+ -mediated oxida tion of low-density lipoproteins, but also promote oxidation by accelerating the formation of hydroxyl radicals in mixtures with H 2 O 2 (7,8).The capacity of xanthohumol to induce intracellular ROS was shown to activate the mitochondrial apoptotic pathway in human malignant glioblastoma cells (9).Other studies support this fi nding, showing that it is able to inhibit the growth and to induce apoptosis in several cancer cell lines: prostate (10), ovarian (11), glioblastoma and malignant astrocytes (9,12), lymphocytic leukaemia (13) and colon (14).
The brewing industry has been particularly att entive to the health-promoting properties of xanthohumol aiming at the production of xanthohumol-enriched beer.The fermentation process is responsible for a strong reduction of xanthohumol content, since almost 60-90 % of it is lost by adsorption to Saccharomyces cerevisiae cells (15,16), which explains the relatively low xanthohumol content found in commercial beer.Therefore, diff erent studies have focused on the evaluation of the parameters responsible for the decrease of xanthohumol in beer and how to overcome this problem to obtain its higher levels in the fi nal product.However, in order to maintain the overall organoleptic properties and quality of the fi nal beer, it is important to understand how the increased xanthohumol content might aff ect S. cerevisiae physiology.Yeast cells produce ROS as a by-product of oxidative phosphorylation, which can aff ect fermentation performance as well as the oxidative stability of the fi nal beer (17).Xanthohumol has recently been shown to exhibit a dose-dependent eff ect on mitochondria of mammalian cells (18).Thus, the potential problems or benefi ts that it could bring to yeast physiology, due to its antioxidant properties, is an important issue of brewing research.A recent study, developed at pilot-scale, showed that xanthohumol has a positive dose-dependent eff ect on the physiological conditions of brewer's yeast during fermentation (19).
The aim of the present work is to reveal how xanthohumol may aff ect the yeast cell viability in order to allow improvements of the brewing process such as effi ciency of fermentation and production of xanthohumol-enriched beer.We have investigated the mechanism of action of xanthohumol on the yeast cells by assessing its intracellular antioxidant activity using fl ow cytometry with dichlorofl uorescein diacetate (H 2 DCFDA).The in vitro antioxidant capacity of xanthohumol was also evaluated using the metmyoglobin antioxidant assay, based on the capacity to inhibit the ABTS ((2,2'-azino-di-[3-ethylbenzthiazoline sulphonate]) radical formation by metmyoglobin.Finally, the antigenotoxic/genotoxic eff ects of xanthohumol on yeast cells were investigated using the yeast comet assay to assess oxidative DNA damage.

Reagents and standards
All the reagents used, except when otherwise stated, were purchased from GIBCO (Paisley, Scotland) and were of analytical grade.High-purity water (resistivity not lower than 18.2 MΩ•cm) from a Direct-Q ® 3UV water purifi cation system (Millipore Iberica, S.A., Madrid, Spain) was used for all analyses.Xanthohumol (>98 % purity) was kindly donated by Martin Biendl from Hopsteiner (S. S. Steiner, New York, NY, USA, and Steiner Hopfen GmbH, Mainburg, Germany).Dichlorofl uorescein diacetate (H 2 DCFDA) was purchased from Life Technologies (Carlsbad, CA, USA), H 2 O 2 (30 %) was purchased from Merck (Darmstadt, Germany), quercetin (>98 %) and anhydrous ethanol, used as solvent, were purchased from Sigma-Aldrich (St. Louis, MO, USA).Acetonitrile (high--performance liquid chromatography grade) and formic acid (for mass spectrometry) were obtained from Sigma--Aldrich.Xanthohumol and quercetin stock solutions of 1 g/L were prepared by diluting the appropriate amount of reagent in ethanol and were stored at -20 °C.These solutions were used to prepare diluted solutions in all experiments.

Yeast strains, media and culture conditions
Two Saccharomyces cerevisiae strains were used in this study, S. cerevisiae BY4741 (MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and yap1, which is the same strain also carrying the yap1(YKL114c)::kanMX4 replacement allele.Both were obtained from the European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF, Institute for Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany).Stock cultures were maintained on solid YPD medium (containing in % by mass per volume: yeast extract 1, peptone 2, glucose 2 and agar 2) at 30 °C for 2 days and then maintained at 4 °C for one week.Cells were cultivated in liquid YPD medium (without agar), in an orbital shaker at 30 °C and 200 rpm.Growth was monitored by measuring the absorbance at 600 nm (A 600 nm ).

Yeast culture preparation
Saccharomyces cerevisiae cells were removed with an inoculation loop from a solid stock culture, then suspended in 5 mL of YPD medium (pre-inoculum) and incubated overnight at 30 °C and 200 rpm.An appropriate volume of the pre-inoculum was diluted in fresh YPD medium to obtain A 600 nm =0.1 and the culture was incubated under the same conditions for two generations to reach A 600 nm =0.4.Subsequently, cells were harvested by centrifugation at 5000×g and 4 °C for 2 min, washed once with the same volume of sterile deionised water and suspended in sterile deionised water, an appropriate buff er or liquid YPD medium depending on the experiment.This procedure ensures that cells are in an early exponential phase of growth.

Viability assays
A volume of 100 μL of cell suspension was collected, serially diluted to 10 -4 in sterile deionised water and 100 μL were spread on solid YPD medium in order to obtain the viability count before treatments.Next, diff erent dilutions of xanthohumol were added to aliquots of the initial culture in YPD medium for fi nal concentrations of 1, 5, 10, 20 and 50 mg/L (fi nal volume of 1 mL was maintained in all samples).Alternatively, the solvent (ethanol) was added to similar cell suspensions for control.In all samples the maximum fi nal ethanol volume fraction was 5 %.The suspensions were incubated at 30 °C and 200 rpm, and 100 μL were taken from each aliquot at 30, 60 and 150 min, diluted and spread on solid YPD.Cells were incubated at 30 °C for 48 h and the colonies were counted.Survival rates were calculated as percentage of colony--forming units (CFU), assuming 100 % survival of cells before any treatment (ethanol or xanthohumol).In order to exclude the eff ect of cell proliferation in culture media in viability assays, the procedure of the viability assays was the same, except that the cells were suspended in phosphate-buff ered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 and 1.47 mM KH 2 PO 4 , pH=7.4) instead of sterile deionised water, aft er harvesting the culture, and all treatments were performed in PBS instead of YPD.

Evaluation of the growth of yeast cultures
An appropriate volume of the pre-inoculum was diluted in liquid YPD medium to achieve A 600 nm =0.1 as stated above and the culture was incubated with xanthohumol (1, 5, 10, 20 or 50 mg/L) and 10 mM H 2 O 2 .Ethanol (similar volume as xanthohumol solution) was added to a similar culture to be used as control.Cultures were incubated at 30 °C and 200 rpm, and growth was monitored by measuring the absorbance at 600 nm for 300 min.Specifi c growth rate was calculated from periodical measures of A 600 nm .

Comet assay
The yeast comet assay was performed to quantitatively determine DNA damage as described by Oliveira and Johansson (20).The yeast culture was prepared as mentioned above, cells were harvested by centrifugation at 5000×g and 4 °C for 2 min and washed twice with the same volume of ice-cold deionised water.Cells were resuspended in 2 mg/mL of zymolyase 20T (20 000 U/g; Im-munO™, MP Biomedicals, LLC, Santa Ana, CA, USA) in S buff er (1 M sorbitol and 25 mM KH 2 PO 4 , pH=6.5) containing 50 mM β-mercaptoethanol and incubated at 30 °C and 200 rpm for 30 min, in order to obtain spheroplasts.Spheroplasts were collected by centrifugation at 5000×g and 4 °C for 2 min, washed twice with the same volume of ice--cold S buff er, resuspended in the same volume of S buffer and distributed in aliquots of 100 μL.For treatments, aliquots of suspended cells were incubated for 20 min at 30 °C in the presence of xanthohumol (1, 2, 5, 7.5, 10 or 20 mg/L), ethanol and H 2 O 2 (positive control; 10 mM H 2 O 2 and 5 %, by volume, ethanol), mixtures of xanthohumol and H 2 O 2 (1, 2, 5 or 10 mg/L of xanthohumol and 10 mM H 2 O 2 ) or ethanol (negative control; same volume as xanthohumol solution).For the assessment of genotoxicity, spheroplasts were incubated only with xanthohumol, and of antigenotoxicity with xanthohumol and H 2 O 2 .Aft er incubation, spheroplasts were collected by centrifugation at 5000×g and 4 °C for 2 min, washed twice with S buff er and then suspended in 50 μL of low melting agarose (1.5 % by mass per volume in S buff er) at 35 °C.Subsequently, samples were analysed by the yeast comet assay as described elsewhere (20).At least 20 comets from representative images were acquired from each sample.The tail length (μm) was measured using the CometScore v. 1.5 soft ware (21) and used as a parameter for DNA damage quantifi cation.

Flow cytometry analysis
The yeast culture was prepared as described in Yeast culture preparation section.Cells were harvested by centrifugation at 5000×g and 4 °C for 2 min and washed twice with the same volume of PBS.The suspension was diluted to reach A 600 nm =0.02 with PBS and 500 μL were collected for autofl uorescence measurement.The antioxidant activity was measured as described by Marques et al. (22) with minor modifi cations.Dichlorofl uorescein diacetate (H 2 DCFDA) was added to the cell suspension (fi nal concentration 50 μM) and cells were further incubated at 30 °C and 200 rpm for 1 h in the dark.Cells were washed twice with the same volume of PBS and aliquots of 1 mL were incubated with xanthohumol (1, 2 or 5 mg/L) and 5 mM H 2 O 2 for 20 min at 30 °C.Each sample containing 20 000 cells was analysed by fl ow cytometry in an Epics ® XLTM cytometer (Beckman Coulter, Brea, CA, USA) equipped with a 15 mW argon-ion laser emitt ing at 488 nm.Green fl uorescence was collected through a 488-nm blocking fi lter, a 550-nm long-pass dichroic and a 225-nm band-pass fi lter.Data were analysed and histograms were made with the FlowJo v. 10.0.7 soft ware (23).

HPLC-DAD-ESI-MS/MS analysis of xanthohumol oxidation products
The identifi cation of the oxidation products of xanthohumol was confi rmed online by high-performance liquid chromatography with diode array detector (HPLC--DAD) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS).The samples were prepared by adding 5 mM H 2 O 2 to 10 mg/L of xanthohumol solution prepared in 10 mM ammonium bicarbonate buff er at pH=7.The mixture was incubated for 20 min at 30 °C in the dark.Control assays without H 2 O 2 and/or xanthohumol were also prepared.The formation of oxidation products was investigated by HPLC-DAD-ESI-MS/MS.The HPLC Accela system was equiped with Accela PDA detector, Accela Autosampler and Accela 600 Pump (Thermo Fischer Scientifi c, Bremen, Germany).Separations were carried out on a Phenomenex Gemini 3 μm C18 100 Å LC column, 150 mm×4.6 mm (Phenomenex Inc., Torrance, CA, USA) with a pre-column (4 mm×3.0 mm) using a binary solvent gradient (solvent A: 0.1 % formic acid in water and solvent B: acetonitrile), starting the injection with 40 % B, followed by a linear increase to 100 % B in 20 min and maintained for 5 min under these conditions.The fl ow rate was 0.4 mL/min and a total volume of 20 μL of a sample was injected into the column, which was kept at 20 °C.
An LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientifi c) controlled by LTQ Tune Plus v. 2.5.5 soft ware and equipped with an electrospray ionization (ESI) source was used.Simultaneous acquisition of mass spectral data and photodiode array (PDA) data was processed by using Xcalibur v. 2.2 soft ware (24).The capillary voltage of the ESI was set to 3000 V.The capillary temperature was 275 °C.The sheath gas and auxiliary gas fl ow rate (nitrogen) were set to 40 and 10, respectively (arbitrary units as provided by the soft ware sett ings).The capillary voltage was 9 V and the tube lens voltage 60 V. Acquisition of the mass data was performed between m/z=80 and 2000 and the Orbitrap resolution was set to 30 000.All experiments were done using MS E (Waters, Milford, MA, USA) for data recording without discrimination of ions or their preselection (30.0 energy collision).The positive ion polarity mode was selected in this work due to a bett er signal-to-noise ratio in comparison with negative ion mode.

Metmyoglobin antioxidant assay
The Antioxidant Assay Kit was purchased from Cayman Chemical Company (Ann Harbor, MI, USA), and the assay was developed according to the manufacturer's protocol.The assay was performed in 96-well plates by adding to each well 10 μL of xanthohumol or quercetin (0-200 mg/L), 10 μL of metmyoglobin and 150 μL of ABTS •+ .At the end, 50 μL of H 2 O 2 (441 μM) were added to the mixture and incubated in a shaker for 5 min at room temperature.Antioxidant capacity was quantifi ed by reading the absorbance at 750 nm using a BioTek ® Power-Wave XS (Winooski, VT, USA) microplate reader and evaluating the half maximal inhibitory concentration (IC 50 ) responsible for inhibition of 50 % of ABTS oxidation.

Statistical analysis
Comet assay results are presented as the mean value±standard deviation (S.D.) obtained from at least 20 comets of each of three independent experiments.All other results are presented as the mean value±S.D. from at least three independent experiments.One-way analysis of variance (ANOVA) was used for comparison of more than two mean values and Tukey's test was used for multiple comparisons.Spearman's rank correlation coeffi cient (r s ) was applied to measure the strength of association between two ranked variables.

Eff ect of xanthohumol on yeast viability
Yeast cells were incubated with xanthohumol (1, 5, 10, 20 or 50 mg/L) for diff erent time periods, and then plated on solid YPD medium to evaluate the eff ect of xanthohumol on viability.This concentration range was chosen according to the suggested fi nal xanthohumol concentration in enriched beer (up to 30 mg/L) (25).A control without xanthohumol, but containing the same volume of the solvent (ethanol), was included in order to evaluate the viability of untreated and non-stressed cells.When yeast cells were exposed to xanthohumol concentrations equal to or higher than 10 mg/L, a signifi cant increase (p>0.05) in the rate of loss of viability was observed (Fig. 1a), while concentrations equal to or lower than 5 mg/L did not have signifi cant impact on the yeast viability.Incubation in the presence of PBS for 150 min led to substantially higher viabilities (Fig. 1b) compared to cultures in YPD (Fig. 1a), particularly in the presence of 20 mg/L of xanthohumol.This result strongly suggests that the toxic activity of xanthohumol is mediated by active yeast metabolism, which is att enuated in the nutrient-free PBS solution.

Xanthohumol has in vitro antioxidant activity
The antioxidant capacity of xanthohumol was tested by the metmyoglobin assay, a rapid method for the assessment of antioxidant protection of a substance (26,27).This assay has been used to measure the total antioxidant capacity of various biological samples related to the production of ROS as a consequence of normal aerobic metabolism.The assay relies on the ability of compounds to inhibit the oxidation of ABTS to ABTS •+ by metmyoglobin.The amount of produced radical is suppressed to a given level, which is proportional to the antioxidant capacity of the tested compound.In this case, diff erent xanthohumol concentrations were tested based on its antioxidant capacity and compared with quercetin, a naturally occurring polyphenol well-known for its antioxidant properties.Xanthohumol displayed a weak antioxidant capacity aft er the addition of H 2 O 2 (IC 50 =31.47mg/L) when com- pared to quercetin (IC 50 =39.97mg/L), which correlates with the decreased toxicity in cells suspended in PBS, presented above, where cellular metabolism is att enuated.

Xanthohumol protects yeast cells under stress conditions
The low antioxidant capacity of xanthohumol observed in the metmyoglobin assay could potentially protect yeast cells from oxidative stress.To test this hypothesis, we measured yeast growth in the presence of 10 mM H 2 O 2 with and without xanthohumol.As the xanthohumol is dissolved in 5 % ethanol, the same volume fraction of ethanol without xanthohumol was included in the control.Yeast cultures did not grow in the presence of 10 mM H 2 O 2 (with 5 % ethanol) or in combination with 1 or 2 mg/L of xanthohumol (Fig. 2).The presence of 5 mg/L of xanthohumol had a protective eff ect against H 2 O 2 as yeast growth could be observed aft er 120 min of incubation when compared with cultures containing 10 mM H 2 O 2 and 5 % ethanol.

Xanthohumol decreases intracellular oxidation
The protective eff ect observed in Fig. 2 could be due to a decrease in the levels of ROS as a consequence of a scavenging activity of xanthohumol.We tested this hypothesis by incubating cells loaded with the redox-sensitive fl uorochrome H 2 DCFDA with xanthohumol and H 2 O 2 .This probe is able to diff use freely into the cells through the plasma membrane where it is deacetylated to reduced dichlorofl uorescein (H 2 DCF).This form is hydrophilic and becomes trapped inside cells due to impermeability of plasma membranes.In the presence of oxidants, or depending on the intracellular redox environment, H 2 DCF is converted to oxidized fl uorescent form, dichlorofl uorescein (DCF), which can be measured in a fl ow cytometer upon excitation at 530 nm and emission at 485 nm.As can be seen in Fig. 3, 5 mM H 2 O 2 induced an increase of intracellular oxidation and, hence, higher fl uo-rescence in H 2 DCFDA-loaded cells (272 AU) than in non--treated cells (120 AU).Increasing concentrations of xanthohumol from 1 to 2 and 5 mg/L decreased the intracellular oxidation of cells induced by H 2 O 2 in a dose-dependent manner (Fig. 3), which resulted in a lower fl uorescence (250, 222 and 145 AU, respectively).Therefore, the antioxidant activity of xanthohumol observed in vitro is also present intracellularly when yeast cells are incubated with xanthohumol and H 2 O 2 .

Xanthohumol oxidation products determined by HPLC-DAD-ESI-MS/MS
The oxidation reaction of the prenylated fl avonoid xanthohumol was investigated in order to infer an oxidation induced by H 2 O 2 .The generated compounds were subsequently detected by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS).A 40-minute chromatographic run was performed and three major peaks were observed in HPLC-DAD and HPLC-MS/MS chromatograms (Fig. 4).The peak with retention time of 17.36 min showed a protonated molecule at m/z=355.15 in the ESI-Orbitrap mass spectrum and a UV spectrum with a maximum at 368 nm.This is the most abundant peak in the chromatogram and corresponds to the xanthohumol fraction that was not oxidized (fragment ions at m/z=179.03 and 299.09 were observed).Two new peaks were detected as the oxidation products of xanthohumol, since they are not present in the blank/control assays.The new compound corresponding to the retention time of 12.64 min showed a protonated molecule at m/z=369.13 (Fig. 4).This oxidation product resulted from the net addition of one oxygen atom to xanthohumol and the loss of two hydrogen atoms.Collision-induced fragmentation of the selected ion yielded an abundance of prenyl cleavage fragments with m/z=313.07 [MH-C 4 H 8 ] + .Accordingly, the compound was identifi ed as the endoperoxyxanthohumol (EPOX, Fig. 5a), which The observed m/z=387.14 for the peak with retention time of 11.95 min (Fig. 4) represents a gain in molecular mass of 32 Da, corresponding to an introduction of two oxygen atoms at the xanthohumol structure.The presence of the fragment ion at m/z=301.11 (Fig. 5b) suggested that the prenyl group could be att acked by the hydroperoxide.In eff ect, this fragment corresponds to the loss of the prenyl group, as shown in Fig. 5b.The double bond in the prenyl group has a higher electron density than the conjugated chalcone skeleton and is therefore more prone to an att ack by the peroxyl radical.Consequently, addition of the peroxyl radical to the prenyl group gives rise to the cyclic peroxide with m/z=387.14 (C 21 H 23 O 7 + ).The presence of this oxidation product has not been reported for xanthohumol.However, several natural cyclic peroxides from marine sources have been tested for a broad range of activities including antiviral, antimalarial, antimicrobial activity and cytotoxicity (29).

Xanthohumol protects yeast cells from DNA damage induced by H 2 O 2
The integrity of DNA is crucial to cells, but it is susceptible to damage induced by ROS (26).If a compound is able to prevent DNA damage it may have a potential benefi t to cells and possibly to health in humans.The antioxidant activity of xanthohumol could contribute to genome protection in yeast cells, which, to our knowledge, has not been investigated before.The genome-protective eff ect of xanthohumol against oxidative DNA damage induced by H 2 O 2 was evaluated using the yeast comet assay.The yeast comet assay is a well-established, simple, versatile, sensitive and inexpensive technique used to assess DNA oxidative damage quantitatively and qualitatively in eukaryotic cell populations (30)(31)(32)(33).Yeast spheroplasts were incubated with diff erent xanthohumol concentrations and 10 mM H 2 O 2 in S buff er (containing 1 M sorbitol) in order to maintain osmotic protection.Ethanol (5 % by volume) was present in all samples since xanthohumol was dissolved in ethanol.The DNA of the cells in the presence of ethanol alone was not signifi cantly damaged (measured as comet tail length; Fig. 6), whereas H 2 O 2 dramatically increased (p<0.01)comet tail length.When xanthohumol was added, a decrease in comet tail length was observed at concentrations below 10 mg/L (Fig. 6).However, at the highest concentration of xanthohumol (10 mg/L), DNA damage was similar to that of cells incubated only with 10 mM H 2 O 2 .Therefore, dose-dependent antigenotoxic activity of xanthohumol is only observed at concentrations below 10 mg/L.

Xanthohumol induces DNA damage
Considering that xanthohumol was shown to be toxic to yeast cells only at concentrations higher than 10 mg/L (Figs.1a and b) and at the same time protective at lower concentrations (Figs. 2, 3 and 6), it was hypothesized that xanthohumol could be genotoxic instead of protective at high concentrations.Cells were incubated with xanthohumol alone, without H 2 O 2 , and the DNA damage was assessed by the comet assay.As shown in Fig. 7, cells treated with xanthohumol at concentrations above 5 mg/L exhibited a statistically signifi cant (p<0.001) increase in comet tail length when compared with cells treated only with ethanol (5 % by volume).This strongly suggests that xanthohumol also has a genotoxic activity at higher concentrations.The genotoxic eff ect did not promote longer comet tails at concentrations above 10 mg/L (Fig. 7), possibly due to the limited capacity of the genomic DNA to unwind and migrate during electrophoresis in the comet assay (30).

Xanthohumol genotoxicity and prooxidant activity
In an att empt to investigate the mechanism involved in xanthohumol genotoxicity observed at higher concentrations, a mutant yeast strain, yap1, sensitive to oxidative stress (34), was incubated with xanthohumol.The yap1 mutant strain is sensitive to oxidative stress because YAP1 encodes a transcription factor involved in the oxidative stress response triggering the transcription of genes encoding proteins required for scavenging and degrading ROS (35,36).Strains yap1 and the corresponding parental strain (BY4741) were cultured in rich medium in the presence of diff erent concentrations of xanthohumol for 300 min and growth was monitored periodically by absorbance.Growth rate inhibition was calculated taking as reference the growth rate of the same strain without xanthohumol (Table 1).The growth of both strains was aff ected by xanthohumol, the yap1 being more sensitive.Growth of the yap1 was signifi cantly aff ected at all tested concentrations, while strain BY4741 was aff ected at 7.5 and 10 mg/L of xanthohumol.Higher concentrations of xanthohumol aff ected both strains.The increased sensitivity of the yap1 strain adds genetic evidence to the previous results, indicating that xanthohumol is prooxidant at high concentrations.

Discussion
Anticarcinogenic and antioxidant properties of xanthohumol present in Humulus lupulus L. have been reported before (3,5,(9)(10)(11)(12)(13)(14)(37)(38)(39).However, scarce information is available on the eff ect of xanthohumol on yeast cells.This work presents a fi rst approach to the study of antigenotoxic and genotoxic eff ects of xanthohumol on Saccharomyces cerevisiae cells focusing on the brewer's yeast physiology.The potential benefi cial use of xanthohumol in human health has led beer manufacturers to The results presented in Figs.1a and b demonstrate that yeast cells are aff ected by xanthohumol and hence, fermentation can be highly infl uenced by the addition of this compound in the previous industrial steps.For this reason, forthcoming investigation on the production of xanthohumol-enriched beer should include monitoring of xanthohumol concentration during fermentation in order to improve fermentation performance.These results are in agreement with those reported by Magalhães et al. (19) where viability slightly decreased aft er the addition of 10 mg/L of xanthohumol at the beginning of fermentation.The diff erences with our study, namely in the fermentation conditions at 4 °C for one week and the yeast species (Saccharomyces pastorianus), might explain the less intense response in viability.Moreover, lower temperatures can decrease xanthohumol solubility and consequent bioavailability since it is only slightly soluble in aqueous medium.
In the same study, Magalhães et al. (19) report that the increase of yeast vitality (fermentative capacity) could be ascribed to the protective antioxidant activity of xanthohumol.Accordingly, xanthohumol has been reported to be an eff ective antioxidant (8) and free radical scavenger (5,37).Results of our study remain in line with these reports for concentrations below 5 mg/L, assessed by both, viability assays (Fig. 2) and fl ow cytometry (Fig. 3).In this range of concentrations we report that xanthohumol has antigenotoxic activity, which correlates with the antioxidant activity reported here (Fig. 3, r s =0.90) and elsewhere (5,8,37), and with the prevention of amino-3-methylimidazo[4,5-f]quinoline-induced DNA damage in liver cells (41).The antioxidant activity of xanthohumol was evidenced by the identifi cation of two major oxidation products of xanthohumol molecule induced by H 2 O 2 (Figs. 4 and 5).Concentrations of xanthohumol above 5 mg/L provoked the opposite eff ect, leading to a slower growth rate (Table 1) and genotoxicity (Fig. 6).When a yeast mutant sensitive to oxidative stress (yap1) was used, the decrease in growth rate in the presence of xanthohumol was even higher (Table 1).This is also in accordance with previous studies showing that xanthohumol was able to induce intracellular ROS, leading to a decrease of cell viability and induction of apoptosis in several cancer cell lines (9,42).Flavonoids with a phenol B ring are able to form phenoxyl radicals, which catalyze glutathione (GSH) or nicotinamide adenine dinucleotide (NADH) oxidation and consequent generation of ROS (42).Similarly, xanthohumol has an open C-ring with a hydroxyl group on the B--ring that may support the production of phenoxyl radical and ROS production.Prooxidant activity also appears to be related to the presence of transition metals (such as iron and copper), peroxidases and high fl avonoid concentrations, increasing the formation of Fe 2+ and Cu + that react with H 2 O 2 in the Fenton reaction (7,43).
Taken together, our results strongly suggest that xanthohumol has a dose-dependent dual eff ect on yeast cells: at low concentrations it is antigenotoxic, whereas at high concentrations it becomes genotoxic.In vitro antioxidant activity also revealed a mild antioxidant activity that correlates with this property.For fl avonoids, this eff ect can be ascribed to the prooxidant activity at elevated doses, combined with the agents that induce oxidative damage (42).These results are in accordance with the denominated Janus eff ect proposed for substances with dual eff ect (44).These results agree qualitatively with a recent fi nding that xanthohumol protects mammalian cancer cells at low concentrations, while doses higher than 1.8 mg/L had a detrimental eff ect (18).

Conclusions
This work presents the fi rst approach to the study of antigenotoxic and genotoxic eff ects of xanthohumol on Saccharomyces cerevisiae, contributing to an optimized utilization of xanthohumol in the production of xanthohumol-enriched beer and reinforcing the benefi cial and harmful eff ects of this compound on yeast cells.Xanthohumol protects yeast cells from oxidative stress and oxidative DNA damage induced by H 2 O 2, in a dose-dependent manner at concentrations up to 5 mg/L.The protective eff ect of xanthohumol involves a ROS scavenging mechanism with a consequent decrease of intracellular oxidation of cells.However, once a concentration threshold (5 mg/L) has been reached, xanthohumol triggers the opposite eff ect leading to a slower growth rate and genotoxicity.The evaluation of the impact of xanthohumol on yeast mutants aff ected by oxidative stress (yap1) proved that xanthohumol toxicity is mediated by oxidative stress, indicating its prooxidant eff ect at concentrations higher than 5 mg/L.

Fig. 1 .
Fig. 1.Eff ect of xanthohumol (XN) on the viability of Saccharomyces cerevisiae BY4741 cells in: a) YPD cultures and in b) PBS suspensions.Yeast cells were incubated with XN or 5 % (by volume) ethanol at each time point (150 min in Fig. b) aliquots were collected, diluted to 10 -4 and spread on YPDA plates.Colonies were counted aft er 48 h of incubation at 30 °C and the percentage of viability was calculated taking 0 min as reference.Data are the mean value±S.D. of three independent experiments (*p<0.05,**p<0.01)

Fig. 2 .Fig. 3 .
Fig. 2. Xanthohumol (XN) protective eff ect on S. cerevisiae BY4741 cells under oxidative stress.Yeast cells in YPD cultures were coincubated at 30 °C with XN (1, 2 or 5 mg/L) and 10 mM H 2 O 2 for 5 h.Culture growth was monitored by reading A 600 nm at each time-point and the percentage of growth was calculated taking 0 min as reference.Data are the mean value±S.D. of three independent experiments

Table 1 .
Growth rates of S. cerevisiae BY4741 and yap1 strains in the presence of xanthohumol Inhibition of growth was calculated as percentage of growth rate decrease of each strain taking as reference the control culture without xanthohumol (XN).Growth rates are the mean value of three independent experiments (*p<0.05 and **p<0.01)