2,4,6-Trihydroxyphenanthrene, a trans-resveratrol photoreaction byproduct: First evidences of genotoxic risk

Phytochemistry Letters 30 (2019) 362–366

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2,4,6-Trihydroxyphenanthrene, a trans-resveratrol photoreaction byproduct: First evidences of genotoxic risk

T

Antonio Franciosoa,b, ,1, Luciana Moscaa, Ivette María Menéndez-Perdomob,2, Sergio Fanellia, Mario Fontanaa, Maria D’Ermea, Fabiana Fuentes-Leonb, Angel Sanchez-Lamarb ⁎

a b

Department of Biochemical Sciences, “Sapienza” University of Rome, Italy Plants Biology Department, Faculty of Biology, University of Havana, Cuba

ARTICLE INFO

ABSTRACT

Keywords: Resveratrol 2,4,6-Trihydroxyphenanthrene (THP) Photochemical reaction product DNA damage Genotoxicity Cytotoxicity

Resveratrol, a natural product with well-known multiple beneficial effects, has been widely used as a bioactive principle for the development of anti-aging topical phytoproducts. These products are usually applied to skin that is often exposed to solar light for a long time, leading to potentially harmful resveratrol derivatives. In this study we investigated the cytotoxicity and genotoxicity of 2,4,6-trihydroxyphenanthrene (THP), derived from trans-resveratrol photochemical isomerization and electrocyclization. To achieve this goal, we used Caulobacter crescentus as the experimental model for evaluating the colony-forming ability and the SOS response induction of cells exposed to THP. DNA-strand breaks and oxidative damage were assessed by the cell-free pCMut plasmid. The obtained data demonstrated that THP induces a cytotoxic and genotoxic effect of THP even at submicromolar concentrations, through a pro-oxidant mechanism leading to DNA damage.

1. Introduction Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a bioactive polyphenol occurring in a variety of vegetable sources like grapes, peanuts, blueberry and Japanese knotweed (Polygonum cuspidatum) and is one of the most important natural polyphenols for its well documented beneficial effects on human health (Francioso et al., 2014b). The 2,4,6trihydroxyphenanthrene (THP), derived from resveratrol photochemical isomerization and ring closure electrocyclization (Fig. 1), has been isolated and characterized in our previous studies (Francioso et al., 2014a). The unequivocal demonstration of the production of phenantrenoid species arising from trans-resveratrol photo-oxidation raises some concerns regarding the increasing use of this compound in cosmetic products and medical devices. Nowadays, trans-resveratrol is widely used in a variety of products as an anti-aging and antioxidant agent, many of which are exposed to solar UV irradiation for a long time in direct contact with human skin. Resveratrol derivatives, formed during exposure to sunlight, might lead to undesirable effects on the skin. THP, produced following resveratrol photocyclization, is a

polycyclic aromatic compound. The polycyclic aromatic hydrocarbons (PAHs) are widely dispersed contaminants in the environment and seriously affect human health. Cancer is a primary risk of exposure to PAHs, which have been linked to skin, lung, bladder, liver, and stomach cancers in well-established animal model studies (Alves et al., 2017; Brinkmann et al., 2013). PAHs exposure is also associated with cardiovascular diseases and poor fetal development (Bostrom et al., 2002; Poursafa et al., 2017). Some carcinogenic PAHs are genotoxic and induce mutations that initiate cancer, while others affect cancer promotion or progression. Mutagenic metabolites of PAHs include diol epoxides, quinones, and radical PAH cations (Androutsopoulos et al., 2009). These metabolites can bind to DNA at specific sites, forming bulky complexes called DNA adducts that can be stable or unstable. Stable adducts may lead to DNA replication errors, while unstable adducts react with the DNA strand, removing a purine base (either adenine or guanine) (Henkler et al., 2012). Such damage, if not properly repaired, can transform gene-encoding for normal cell signaling proteins into cancer-causing oncogenes (Agudo et al., 2017). Quinones can also generate reactive oxygen species that may independently damage DNA (Xue and Warshawsky,

Corresponding author at: Department of Biochemical Sciences, “Sapienza” University of Rome, Italy. E-mail address: [email protected] (A. Francioso). 1 Current address: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany. 2 Current address: Department of Biological Sciences, University of Calgary, Calgary, Canada. ⁎

https://doi.org/10.1016/j.phytol.2019.02.025 Received 13 October 2018; Received in revised form 30 January 2019; Accepted 19 February 2019 Available online 26 February 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. THP (right) produced after trans-resveratrol (left) UV-light irradiation.

2005). The present study investigates the cytotoxicity and genotoxicity of THP photoproduct. To this end, the Caulobacter crescentus experimental model was used to evaluate the colony-forming ability and the SOS induction of cells exposed to THP. Additionally, the cell-free pCMut plasmid was used to evaluate DNA-strand breaks and oxidative damage induced by this compound. The obtained data demonstrate a cytotoxic and genotoxic effect of this compound also at sub-micromolar concentration.

Fig. 3. Levels of β-galactosidase activity displayed by Caulobacter crescentus cells exposed to THP at different concentrations, evaluated by SOS Chromotest assay. Data are mean ± SD of 5 experiments with 4 replicates each. (*) Significant in Dunnett test, p < 0.05.

2. Results To evaluate the possible toxic effect of THP, two different models were selected. Caulobacter crescentus was used to assay cyto- and genotoxic effects caused by THP. The NA 1000 pP3213 LacZ strain of Caulobacter crescentus possesses a transcriptional fusion of the imuA promoter with lacZ gene, as reporter. The imu (inducible mutagenesis) operon expression levels are increased in response to UVC damage, thereby UVC irradiation was used as an agent capable to induce large amounts of DNA damage (Alves et al., 2017; Lopes-Kulishev et al., 2015; Menéndez-Perdomo et al., 2017) On the other hand DNA plasmid assay is an excellent tool to investigate the direct effect of a substance on the DNA molecule. The use of pCmut allows to demonstrate whether THP may induce DNA strand breaks or produce oxidative damages. In this context, UVA irradiation was used as control because it causes large DNA oxidative damage.

0.01–100 μM. Data obtained under our experimental conditions indicate that 100 μM THP induced a 100% cell death, and that the estimated IC50 value is 10.5 ± 0.7 μM. 2.2. Genotoxicity 2.2.1. SOS chromotest in Caulobacter crescentus To further investigate whether THP induced primary DNA damage, we determined the activation of SOS genes in Caulobacter crescentus. As shown in Fig. 3, the THP treatment increased β-galactosidase activity as a function of its concentration. In particular, a significant enhancement of enzyme activity was reached at THP concentrations as low as 0.1 μM, and increased proportionally with THP amount.

2.1. Cytotoxicity

2.2.2. Cell-free plasmid DNA assay in pCMut To investigate whether THP had a direct interaction with DNA molecule, pCMUT plasmid was utilized. Two DNA-damage endpoints were investigated: DNA-strand breaks (SSB or DSB) and bases-oxidative damage. Fig. 4 shows that THP treatment did not generate DNA breaks. To evaluate the oxidative damage exerted by THP treatment, Fpg activity was assayed. Fpg nicks DNA when it encounters 8-Oxoguanine nucleic bases. As shown in Fig. 4, oxidative DNA damage increased with THP concentration and was markedly high at all concentrations tested.

The effect of THP was ascertained by Caulobacter crescentus colonyforming ability. This assay is an established method for assessing the cytotoxic effect of natural products by determining the IC50. Results show that THP caused a decrease in colony-formation with respect to a control in a concentration dependent manner, with a cytotoxicity of about 20–40% observed in the concentration range 0.1–10 μM (Fig. 2). IC50 of THP was determined with the dose-response curves in the range

3. Discussion Because of the potential toxic effects of polycyclic aromatic hydrocarbons (PAHs), this work aimed to evaluate whether the resveratrol derivative THP might have a potential toxic effect on health. This research focused on the evaluation of genetic damage at the level of the primary structure of DNA. The use of plasmid DNA molecules (pCMUT) and Caulobacter crescentus bacteria in the experimental procedure allowed analysis of the effect of THP on the genetic material, either in the cellular context (SOS Chromotest) or by direct chemical action (ex vivo interaction). In particular, the results obtained in the experiments with Caulobacter crescentus suggest a high genotoxic potential of THP. When equal concentrations of THP were applied in order to evaluate the cytotoxic and genotoxic effects, significant genotoxic responses were observed at THP concentrations (0.1 μM) in which cell survival was greater than 50%. Specifically, the concentration of 1 μM THP reduces bacteria survival by only 25% while increasing two fold the induction of SOS genes compared to basal induction in these cells. These results raise concerns not only about DNA damage generated by THP, but also

Fig. 2. Cytotoxicity of Caulobacter crescentus cells exposed to different concentrations of THP. The graph bars represent the number of colonies expressed as percent vs CTRL. Data are mean ± SD of 5 experiments with 4 replicates each. (*) Significant in Dunnett test, p < 0.05. 363

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Fig. 4. Quantification of induced DNA-damage (strand breaks and oxidized bases) on pCMut plasmid after treatment with increasing THP concentrations.

about genotoxic potentiality to affect the next generations of cells. In this study, ex vivo assays with plasmid DNA have been used to determine the effects of THP on DNA. The evidence of oxidative damage in the primary structure of the genetic material presented here constitutes a new finding, and represents an important initial qualitative approach to understand the kind of DNA-damage generated by THP. It has been reported that PAHs derivatives have the capacity to enter redox cycles and induce the production of reactive oxygen species (ROS), thereby causing oxidative stress (An et al., 2011a, b; An et al., 2011c). Auto-oxidation of polycyclic hydroxylated derivatives may result in the formation of quinones (Briede et al., 2004a, b). These metabolites can undergo redox-cycling and produce superoxide anions which are then converted to hydroxyl radicals by the Haber-Weiss reaction (Lesko and Lorentzen, 1985). Free radicals react with guanine and cause DNA damage, including the production of 7-hydro-8-oxo-2´deoxyguanosine (8-oxodG) (Chatgilialoglu and O’Neill, 2001). The OGG1 gene codes for a DNA glycosylase involved in base excision repair of 8-oxo-dG that arises from ROS. When this system fails, there is an increase in mutation rate (Bonner et al., 2005). Balance between generation of ROS species and scavenging of these molecules is fundamental in repairing DNA damage. If the rate of ROS generation is greater than their removal it is likely that more DNA damage will result. PAHs may absorb light energy in the UV region (280–400 nm) and may induce DNA damage by production of ROS. For example, chrysene, induces apoptosis and DNA damage in human keratinocytes by generating ROS in response to UVB irradiation (Ali et al., 2011). Given that THP is a hydroxylated phenanthrene that can spontaneously undergo radical reactions, we also demonstrate a cytotoxic and genotoxic effect of this molecule in the sub-micromolar range and, in particular, that DNA damage is primary exerted by an oxidative mechanism. Further experiments will be performed to test the possible mutagenic and/or cancerogenic effects of this photoreaction byproduct.

carcinogenic effects. Taking into account the use of resveratrol in cosmetics and the potential skin absorption of resveratrol, human exposure to UV-light is a key aspect related to the THP production. Our data demonstrate the cytotoxic and genotoxic effects of THP even in the submicromolar range, and notably we demonstrated that the DNA damage is primarily exerted by a pro-oxidant mechanism. This new finding represents an important starting point for in vivo investigation of THP as a contaminant in view of its possible secondary effects on human health. Our future challenge will be to develop an analytical method to determine the presence of resveratrol photoproducts in complex matrices, which can represent a good tool to detect THP in biological, pharmaceutical, environmental and agricultural samples after UV-light exposure. Furthermore, it should be necessary to determine whether resveratrol and its derivatives can induce mutagenic effects in other biological systems (human cell cultures, animal models, etc.) and to study in more detail the photochemistry and the photobiology of this molecule.

4. Conclusions

5.2.1. Bacterial strain and culture Two Caulobacter crescentus strains were used in this study: NA1000 strain (wild type) used in Survival assays, and NA 1000 pP3213 LacZ strain used in SOS Chromotest, both kindly provided by the Department of Microbiology (Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil). The latter was previously obtained by the transformation

5. Materials and methods 5.1. Chemicals and reagents Trans-resveratrol was purchased from Shangai Novanat Co., Ltd. (Shanghai, China). THP was obtained from resveratrol photochemical reaction and purified as described in our previous work (Francioso et al., 2014a). The concentration of THP was checked spectrophotometrically by using the molar extinction coefficient (ε1M 257,5nm = 31,456) calculated on freshly purified product (Fig. 5). All other reagents were from analytical grade. 5.2. Evaluation of THP cytotoxicity in Caulobacter crescentus

Like a double edged sword, resveratrol may exert positive and negative effects on health. A deeper investigation of the photochemical and metabolic byproducts of this compound may provide clues to explain why some researchers have evidenced pro-oxidant and pro364

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U = (DO420nm x 1000)/(DO600nm x Vol x t) Where: U: β-galactosidase enzymatic activity; OD420nm: optical density value registered at 420 nm after the enzymatic reaction has occurred; OD600nm: optical density value registered at 600 nm after incubation; Vol: cell culture volume = 0.05 mL; t: enzymatic reaction time = 5 min. The statistically significant increase of the β-galactosidase enzymatic activity compared to the negative control was taken as genotoxicity parameter. All measurements were done in triplicate, and the experiments were repeated five times. Fig. 5. THP UV–vis spectrum and molar extinction coefficient determination (λmax at 2575 nm).

5.3. Evaluation of THP genotoxicity in Cell-free plasmid DNA assay 5.3.1. Plasmid Plasmid pCMUT (1762 bp) (C – chloramphenicol resistance, and MUT – supF, mutation target gene) derived from pAC189 plasmid was used (Schuch et al., 2009). Purification of DNA samples was performed by using Qiagen Plasmid Maxi Kit (Valencia, CA, USA) with freshly transformed E. coli strain DH10b and stored in TE buffer (10 mM Tris−HCl [pH 8.0], 1 mM ethylenediaminetetraacetic acid) at -20 °C before starting the experiments. For all procedures, the plasmid DNA samples were dissolved in TE solution (10 mM Tris HCl, 1 mM EDTA, pH 7.5) at an initial concentration of 100 μg/μL. The negative control was 1 μL DNA, 1 μL TE solution and 18 μL NET solution (100 mM NaCl, 10 mM EDTA, 10 mM Tris HCl, pH 8). The positive control was 1 μL of UVA (100 kJ/m²) irradiated DNA in 18 μL of NET solution, 1 μL of Fpgenzyme. The UVA irradiated plasmid was kindly donated by the Department of Microbiology (Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil).

of wild NA 1000 strain with pP3213 plasmid, containing the imuA SOS response gene promoter in transcriptional fusion with the lacZ gene, coding for β-galactosidase enzyme (Galhardo et al., 2005).The use of this bacteria was conducted as reported. Briefly, cells were grown overnight at 30 °C with constant shaking (100 rpm) in PYE medium (Ely, 1991). The culture was then diluted ten-fold in fresh medium and grown under similar conditions until the optical density at 600 nm (OD600nm) was 0.4 (6 × 107 cells/mL), corresponding to the phase of mid-log growth. Then, cells were collected by centrifugation (ColeParmer, Eppendorf, US) in 1.5 mL microcentrifuge tubes. 5.2.2. Toxicity The cells were resuspended with PYE medium and THP at different concentrations (range 0.01–100 μM). Afterwards, tubes were incubated for 30 min at 4 °C in a refrigerator and then 2 h at 30 °C under constant shaking (100 rpm). In both assays, cells harvested in the medium were used as a negative control and UVC-irradiated as a positive control. Subsequently for each strain, aliquots of each treatment were taken to perform the Survival Assay, and SOS Chromotest as described below.

5.3.2. THP exposition of plasmid DNA In order to evaluate the ability of the THP to produce DNA singlestrand (SSB) or double strands (DSB), 1 μl of THP, prepared at different concentrations (0.1, 1, and 10 μM),was added to 1 μL of plasmid DNA samples and 18 μL of NET solution. To evaluate whether oxidative DNA damages is produced, the formamidopyrimidine-DNA glycosylase (Fpg protein) was used in a parallel set of plasmid DNA samples (Schuch et al., 2017). The samples were incubated at 37 °C for 30 min.

5.2.3. UVC irradiation Cells were irradiated in 3 cm diameter Petri dishes. UVC irradiation (λ = 254 nm, E = 45 J/m2) was carried out using a Vilber Lourmat Lamp T15 M 15 W (Vilber Lourmat, Suebia, Germany) at room temperature. All steps were carried out in the dark to avoid photo-reactivation.

5.3.3. Electrophoretic conditions - DNA band quantification All plasmid DNA treatments were submitted to a 0.8% agarose gel electrophoresis in 0.5X TBE (44.6 mM Tris, 44.5 mM boric acid, 50 mM EDTA, pH 8, containing ethidium bromide 1 μg/μL). The electrophoretic run was developed in 0.5X TBE solution at 80 V and 60 mA for 60 min. After electrophoresis, DNA bands corresponding to supercoiled (FI) and/or circular relaxed (FII) forms of plasmid were separated and visualized by fluorescence. The genotoxicity criterion used was the appearance of the FII and reduction of FI. The intensity of electrophoretic bands was quantified according to the image analysis program, Image J (ImageJ Basics, version 1.44, National Institutes of Health, USA. gov / ij /). The number of break sites per kbp of plasmid DNA was calculated, assuming a Poisson distribution adapted to this technique, by the following equation:

5.2.4. Survival assay The influence of the THP on cell survival was evaluated by colonyforming ability. A 10 μL aliquot was removed after each treatment for serial dilutions and plated on solid PYE medium for cell viability determination after 48 h incubation at 30 °C, and the number of colonies was assessed. Survival was expressed as a percentage of the control values. Experiments were performed five times with four replicates each. The evaluation of the IC50 values was performed by the GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA). 5.2.5. SOS chromotest assay After cell treatments OD600nm for each sample was determined and the SOS Chromotest modified protocol was performed (Galhardo et al., 2005). Then 50 μL aliquots were dispensed in tubes containing 800 μL of a permeabilization solution for cells disruption (Buffer Z: Na2HPO4 8.5 g/L; NaH2PO4 7.18 g/L; KCl 0.75 g/L; MgSO4.7H20 0.51 g/L); 50 μL of chloroform and 2.88 μL of β mercaptoethanol; then mixed, and incubated for 5 min at room temperature. Afterwards, 200 μL of 4 mg/mL o-nitrophenyl-β-D-galactopyranoside (ONPG) substrate in phosphate buffer (Na2HPO4 16.1 g/L; NaH2PO4 5.5 g/L) was added, and after 5 min of incubation the reaction was stopped using 400 μL of 1 M Na2CO3. Finally, the OD420nm was measured and β-galactosidase activity was calculated as describe previously (Zhang and Bremer, 1995), by the following relationship:

X = -ln (1.4*FI/1.4*FI + FII) / 1.8 where FI represents the intensity measured in the supercoiled DNA bands, FII the intensity in the relaxed DNA bands, 1.4 is a factor employed for correcting the increased fluorescence of ethidium bromide when bound to the relaxed form compared to the supercoiled form, and 1.8 is pCMUT vector size in kbp (Schuch et al., 2009). 5.3.4. Statistical analysis The data are expressed as mean ± standard deviation (SD) determined for each treatment. Controls and treatments were compared using the Kolmogorov-Smirnov test for Normality, Brown-Forsythe test 365

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for variance homogeneity, and the single classification ANOVA, the Dunnett test for all the assays. Statistical significance was defined as p < 0.05. All the tests were performed by the software Statistica v.6.

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Conflict of interest The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Acknowledgments This work was supported by grants from Sapienza University of Roma (Italy), “Avvio alla Ricerca Ateneo 2014-2015” to AF and “Visiting Professorship 2016” to ASL. Authors wish to thank Ms. Jane Reynolds for revising the English language. References Agudo, A., Peluso, M., Munnia, A., Lujan-Barroso, L., Barricarte, A., Amiano, P., Navarro, C., Sanchez, M.J., Quiros, J.R., Ardanaz, E., Larranaga, N., Tormo, M.J., Chirlaque, M.D., Rodriguez-Barranco, M., Sanchez-Cantalejo, E., Cellai, F., Bonet, C., Sala, N., Gonzalez, C.A., 2017. Aromatic DNA adducts and breast cancer risk: a case-cohort study within the EPIC-Spain. Carcinogenesis 38, 691–698. Ali, D., Verma, A., Mujtaba, F., Dwivedi, A., Hans, R.K., Ray, R.S., 2011. UVB-induced apoptosis and DNA damaging potential of chrysene via reactive oxygen species in human keratinocytes. Toxicol. Lett. 204, 199–207. Alves, I.R., Lima-Noronha, M.A., Silva, L.G., Fernandez-Silva, F.S., Freitas, A.L.D., Marques, M.V., Galhardo, R.S., 2017. Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus. DNA Repair (Amst) 59, 20–26. An, B.C., Lee, S.S., Lee, E.M., Lee, J.T., Wi, S.G., Jung, H.S., Park, W., Lee, S.Y., Chung, B.Y., 2011a. Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress. Cell Stress Chaperones 16, 317–328. An, B.C., Lee, S.S., Wi, S.G., Bai, H.W., Lee, S.Y., Chung, B.Y., 2011b. Improvement of chaperone activity of 2-Cys peroxiredoxin using gamma ray. J. Radiat. Res. 52, 694–700. An, H., Zhai, Z., Yin, S., Luo, Y., Han, B., Hao, Y., 2011c. Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus. J. Agric. Food Chem. 59, 3851–3856. Androutsopoulos, V.P., Tsatsakis, A.M., Spandidos, D.A., 2009. Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention. BMC Cancer 9, 187. Bonner, M.R., Rothman, N., Mumford, J.L., He, X., Shen, M., Welch, R., Yeager, M., Chanock, S., Caporaso, N., Lan, Q., 2005. Green tea consumption, genetic susceptibility, PAH-rich smoky coal, and the risk of lung cancer. Mutat. Res. 582, 53–60. Bostrom, C.E., Gerde, P., Hanberg, A., Jernstrom, B., Johansson, C., Kyrklund, T., Rannug,

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