Cytotoxic, antioxidative, genotoxic and antigenotoxic effects of Horchata, beverage of South Ecuador

The following reagents and chemicals were purchased from Sigma-Aldrich, St.​ Louis, USA: 2′-7′-dichlorofluorescin diacetate (H₂DCFDA), balanced salt solution (HBSS), sodium hydroxide (NaOH), potassium chloride (KCl), sodium bicarbonate (NaHCO₃), sodium phosphate dibasic anhydrous (NaHPO₄), monobasic potassium phosphate (KH₂PO₄), disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), methanol, ethanol, peroxidemitomycin C, doxurrubicin, (±)-6 hydroxy-2,5,7,8 tetra-methylchromane-2-carboxylic acid (Trolox), 2 diphenyl-1-picrylhydrazyl (DPPH), 2,4,6, tris(2-pyridyl)-s triazine for spectrophometric determination (of Fe) (TPTZ), iron III chloride hexahydrate, gallic acid, Folin–Ciocalteu’s reagent, and cytochalasin B. The Tris and UltraPure Agarose were purchased from Invitrogen, Carlsbad, California, USA. Roswell Park Memorial Institute (RPMI) 1640 medium, HAM-F12, fetal bovine serum (FBS), L–glutamine, antibiotic-antimycotic (100 units·mL⁻¹ penicillin G, 100 μg·mL⁻¹ streptomycin and 0.25 μg·mL⁻¹ amphotericin B), phytohemagglutinin and formaldehyde were purchased from GIBCO, Grovemont Cir, Gaithersburg, MD USA. Sodium acetate trihydrate, fuming hydrochloric acid, acetic acid (glacial) and sodium carbonate were purchased from Merck KGaA, Darmstadt, Germany. Triton X-100, agarose (LMP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTS) for CellTiter 96® Aqueous One Solution Cell Proliferation Assay were purchased from Promega, Madison, Wisconsin, USA. Lymphocyte separation medium was purchased from Lonza, Basilea, Suiza. The Anexin-V/IP kit (sc-4252), PVDF transfer membrane (sc-3723), Bcl-2 (C-21, sc-783), Bax (P-19, sc-526), pS46-TP53 (Ser 46, sc-101,764), p-73 (recognizes all p73 isoforms raised against H-79, sc-7957), pY99-TP73 (Tyr 99, sc-101,769), H2AX (C-20, sc-54,606), pS139-H2A.X (Ser 139, sc-517,348), p21 (187, sc-817), β-tubulin (D-10, sc-5274), goat anti-rabbit (sc-2004), and donkey anti-goat (sc-2020) were purchased from Santa Cruz Biotech, San Jose, California, USA. Goat anti-mouse (AP308P) and a chemiluminescence kit (Luminata Crescendo Western HRP substrate) were purchased from EMD-Millipore, Billerica, Massachusetts, USA.

We used samples from the best-selling HRCHs mixtures [2]. Nine different mixtures were prepared, and the compositions are detailed in Table 1. The specimens were purchased from local markets in Loja, Ecuador and identified by Fani Tinitana, PhD. A voucher specimen was deposited in the Herbarium of Universidad Técnica Particular de Loja, Ecuador.

The HRCHs were prepared according to the traditional methods of Loja, the plants were boiled for 10 min in distilled water and then cooled to an ambient temperature. They were then placed in a laminar flow cabinet and transferred to sterilized glass vials. The samples were freeze-dried in a lyophilizer LABCONCO model 7,754,047 for approximately 48 h. Finally, the samples were stored at 4 °C.

Phytochemical screening showed the presence of secondary metabolites (alkaloids, terpenoids, flavonoids, tannins, saponins, steroids, and quinones), primary metabolites (proteins, carbohydrates, fats), and oils using standard procedures [41].

The total phenolic compounds were measured spectrophotometrically on a microplate reader (EPOCH 2 BioTek, BioTek Instruments Inc., Highland Park, Winooski, USA) according to the Folin-Ciocalteu’s method. This was applied to a 96-well microplate assay using gallic acid as a calibration standard [42]. A 50-μL aliquot of the different concentrations of HRCHs samples and standards were added to 150 μL of freshly prepared Folin–Ciocalteu reagent (1:4 v/v in distilled water) in a 96-well TR5003 cell culture plate (TrueLine, USA). After 10 min at 37 °C, 50 μL of the saturated solution of sodium carbonate was added to each well, and the plate was incubated for 10 min at 37 °C. The absorbance of each solution was measured at 725 nm on a microplate reader (EPOCH 2 BioTek, BioTek Instruments Inc.). The standard curve was linear between 2 and 0.03 mM gallic acid solution. The total amount of phenolics was calculated as mg gallic acid equivalent (GAE)/g of sample.

The DPPH free radical scavenging activity was evaluated on a microplate analytical assay according to the literature [42, 43]. A 30 mL aliquot of the different concentrations of freeze-dried samples and standard were added to 270 μL of DPPH in methanol solution (100 μM) in a 96-well microplate assay. After incubation at 20 °C for 60 min, the absorbance of each solution was determined at 515 nm using a microplate reader (EPOCH 2 BioTek, BioTek Instruments Inc.).

A FRAP assay was performed according to the literature [42, 44]. This was applied to a 96-well microplate assay while monitoring the reduction of Fe³⁺-TPTZ to blue-colored Fe²⁺-TPTZ. Stock solutions of acetate buffer (300 mM) pH 3.6, FeCl₃·6H₂O (20 mM) and 10 mM TPTZ in 40 mM in HCl (10 mM) were prepared. The fresh working solution was prepared by mixing ten volumes of acetate buffer, one volume TPTZ solution, and one volume FeCl₃·6H₂O solution. This was then warmed to 37 °C before use. The FRAP working solution (270 μL) was mixed and incubated with 30 μL HRCHs/standards for 30 min in the dark. The absorbance of each solution was measured at 593 nm using a microplate reader (EPOCH 2 BioTek, BioTek Instruments Inc.). The standard curve was linear between 400 and 25 μM Trolox™ solution.

Cerebral astrocytoma (D384) cells were kindly received from Dr. Mayra Paolillo at the University of Pavia. Colon cancer (RKO) were kindly received from Dr. Patricia Ostrosky at the Universidad Nacional Autonoma de Mexico. Prostate cancer (PC-3), breast cancer (MCF-7), lung cancer (A-549) and Chinese Hamster Ovary (CHO-K1) cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA).

Immortalized Chinese Hamster Ovary (CHO-K1) cells were cultured in HAM-F12 medium. The other cells were cultured in RPMI medium. These media were supplemented with 1% antibiotic-antimycotic, 2 mM L-glutamine, and 10% FBS. The cultures were maintained at 37 °C in a humid atmosphere at 5% CO₂. The doubling times of the PC-3, MCF-7, RKO, and A-549 cells were established as 24 h; the CHO-K1 and D-384 cells were 16 h.

Human peripheral blood lymphocytes (PBL) were isolated by density gradient centrifugation with lymphocyte separation medium from whole peripheral blood from three donors. All donors gave informed written consent to use human blood samples for research purposes (View Ethics approval and consent to participate). The donors had the following characteristics: below age 30, healthy, non-smokers, and with unknown exposure to genotoxic chemicals or radiation. PBL were cultured at 37 °C and 5% CO₂ in RPMI-1640 supplemented with 0.5% phytohemagglutinin; 1% antibiotic antimycotic, 2 mM L-glutamine, and 10% FBS. The cultures were maintained at 37 °C in a humid atmosphere at 5% CO₂.

Cell viability was analyzed with a MTS assay to assess the viability and/or the metabolic state of the cancer cells based on mitochondrial respiratory activity. A total of 5 × 10³ cells were seeded into each well of 96-well plates and allowed to adhere for 24 h. After 24 h, the cells were treated with freeze-dried HRCHs at 50 μg·mL⁻¹. Each concentration/assay was performed in triplicate. Negative control cells were treated with the DMSO vehicle to a final concentration of 0.1% v/v; 0.5 μM doxorubicin was used as a positive control. The cells were then incubated with the treatments for 48 h. After 48 h, MTS (5 mg·mL⁻¹) was added, and the cells were further incubated for 2 h in the dark at 37 °C. Absorbance was measured at 492 nm versus a reference wavelength of 650 nm. In cell lines with a inhibition percentage over 30%, doses of 15, 45, 60, 75 and 100 μg·mL⁻¹ of each HRCHs were applied, and the MTS assay was used to measure the inhibitory concentration 50 (IC₅₀) using non-linear regression.

We next quantified apoptotic cells using an Annexin V-FITC/PI apoptosis detection kit [45]. In brief, the D384 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/well. After treatment at the IC₅₀ for 48 h, both adherent and floating cells were harvested and stained with Annexin V-FITC/PI according to the manufacturer’s procedure. The samples were analyzed with fluorescence microscopy and an excitation wavelength of 475 nm (Zeiss Axioskop 2 plus); 200 cells were counted.

Total protein extraction, quantification, and immunoblots were performed as described [46]. Briefly, 50 μg of total protein were separated by 12–15% SDS-PAGE and transferred to a PVDF membrane (IPVH00010, Immobilon-P, 0.45 μm, Millipore). Rabbit polyclonal antibodies against Bcl-2, Bax, pS46-TP53, p-73, and pY99-TP73 were used with mouse poyclonal antibodies against pS139-H2AX, p21, and β-tubulin and a goat polyclonal antibody against H2AX; all antibodies were used at the dilution factor recommended by the manufacturer with the appropriate goat anti-rabbit, goat anti-mouse, and donkey anti-goat horseradish peroxidase-conjugated immunoglobulins (1:5000). Immunoreactive bands were visualized using a chemiluminescence kit Luminata Crescendo Western HRP substrate. Quantitative analysis of the proteins used a C-Digit Blot Scanner (LICOR).

The detection of ROS used 2′7´-dichlorodihydrofluorescein diacetate (DCHF-DA) [47]. Previously, CHO-K1 cells were cultured in 96-well multi-plates in HAM-F12 culture medium supplemented for 16 h. The cells were incubated with H₂DCFDA (20 μM) and HBSS for 30 min at 37 °C. The cells were exposed to doses of 1000 μg·mL − 1 of the HRCHs and/either H₂O₂ (5 mM) as a positive control; HBSS was applied as a negative control. The multiplate was incubated for 2 h at 37 °C. DCFDA fluoride was measured after exposure on a Fluoroskan Ascent (Thermo Fisher Scientific) fluorometer at a λ_ex of 485 nm and λ_em of 528 nm.

The alkaline comet assay was performed as reported elsewhere with minor modifications [48]. After 16 h of culturing the CHO-K1, two experimental conditions were performed. The first was only HRCHs (1000 μg·mL⁻¹), and the second was concurrent treatment for 16 h of HRCHs (1000 μg·mL⁻¹) and H₂O₂ (5 mM). The CHO-K1 cell cultures were mixed with 150 μL low-melting agarose (0.5%) and added to microscope slides. The slides were immersed in a cooled lysing solution (10% DMSO, 1% Triton X-100, 2.5 M NaCl, 100 mM EDTA, and 10 mM Tris) for 12 h at 4 °C (pH 10). All steps after lysis were performed in the dark to prevent additional DNA damage by another factors. All slides were submerged in electrophoresis buffer solution (300 mM NaOH and 1 mM Na₂EDTA) (pH 13) for 20 min; this allowed the DNA to unwind. Electrophoresis conditions were 25 V and 300 mA for 20 min. A buffer solution was used to neutralize the slides after electrophoresis, (0.4 M Tris, pH 7.5). Each slide received EtBr (60 μL, 30 μg·mL⁻¹) with a cover glass. The length tail of DNA damage was estimated using a ZEISS-Axioskop 2 plus fluorescence microscope, and 100 cells were scored on each slide and confirmed with Comet assay VI software.

The CBMN test was performed as reported elsewhere with minor modifications [49]. After 16 h of culturing CHO-K1, three experimental conditions were performed. The first was only HRCHs and cytochalasin B at a final concentration of 2 μg/ml. The second was 24 h with HRCH, mytomicin C (MMC), and cytochalasin B. The last was MMC for 3 h followed by HRCHs and cytochalasin B. After 16 h of incubation, the cells were fixed in cold (−20 °C) ethanol 96° for 5 min washed with distilled water, re-fixed in acetic acid:methanol (1:3 v/v) for 5 min, and washed. The fixed cells were stained with a drop of DAPI (4′,6′-diamidino-2-phenylindole HCl, Serva), dissolved in Tris-buffer (pH 7.5) at 2 μg/ml. The preparations were evaluated with an Axioskop 2 plus microscope (Zeiss, Germany) equipped with a fluorescence illuminator (HBO50 lamp); the filter set was 365 nm excitation and 420 nm emission (specific for DAPI). Phase contrast was also used, and analysis was performed at high magnification for ~400 Å resolution. To facilitate discrimination of micronuclei (MN) and differentiation between mononucleated, binucleated (which divided once after exposition), and polynucleated cells (which divided twice or more). Scoring was performed blindly. The number of binucleated cells per dish was evaluated after scoring 2000 consecutive cells distributed as a monolayer according to the criteria described previously [50].

All data were reported as the mean ± SEM of independent experiments. The statistical significance was obtained with one-way analysis of variance (ANOVA) followed by a Dunnet post-test; treated cultures were compared to controls. Statistical analyses were carried out in GraphPad Prism 4 (GraphPad Software, San Diego, CA).