Chemicals and Biochemicals
The low melting point (LMP) and normal melting point (NMP) agarose were purchased from Sigma-Aldrich (USA) and Bioline (UK), respectively. The 96% ethyl alcohol, anhydrous ethyl alcohol, boric acid, and xylene were purchased from POCH (Poland). The sodium chloride, sodium hydroxide, chloroform, ethylenediaminetetraacetic acid (EDTA), Trizma-base, Tris-HCl, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) were from Sigma-Aldrich (USA). In this study, the proteinase K from Tritirachium album (Blirt S.A. DNA-Gdańsk, Poland) and ribonuclease A (RNase A, EC 220.127.116.11), 100 μg mL−1 in 10-mM sodium acetate buffer pH 5.2 from Sigma-Aldrich (USA) were used. For nucleic acid and tissue staining on microscope slides, Hoechst 33342 (1 μL mL−1 working solution in water, ThermoScientific, USA), methyl green–pyronin Y (MGP, ready to use solution, Sigma-Aldrich, USA), eosine (Serva), Delafields hematoxyline, and GelRed (10,000 × concentrated, ImmuniQ, Poland) were used. In comet assays, nucleic acids were stained with SYBR Green (Sigma-Aldrich, USA) 10,000 × diluted in tris-EDTA (TE) buffer (10-mM Tris-HCl, 1-mM EDTA, pH 7.5). During nucleic acid agarose gel separation, the DNA size marker M10 kbp with DNA fragments from 200 bp to 10 kbp and 6 × concentrated loading buffer (DNA Ladder, DNA-Gdańsk, Poland) were applied. For isolation of nucleic acids, the DNeasy Blood & Tissue Kit (Qiagen GmbH, Germany) and the phenol/chloroform/isoamyl alcohol reagent (25:24:1, v/v/v, Sigma-Aldrich, USA) were used. Additionally, for preparation of paraffin-embedded food samples, histological paraffin (Histosec pastilles, Merck, Germany), 4% formaldehyde solution in phosphate buffer (Mega Herba, Poland), 36–38% formaldehyde (POCH, Poland), glacial acetic acid (Merck, Germany) and Neo-Mount anhydrous resin for mounting of stained histological samples (Merck, Germany) were used. Water was purified with MilliQ (Millipore) system (Merck, Germany).
The sirloin meat portions were derived from the single cut of a pork meat, and after slaughter and 24-h maturation period, was bought at a local meat-processing plant that owns a slaughterhouse in the Pomerania region, Poland. The meat came from a domestic pig (Sus scrofa f. domestica), females 6 months old, fed with bio fit concentrate, grain, triticale, and their mixtures. Each pork loin was divided into samples for culinary processing, and processed on the same day. The chicken meat portions, derived from single broiler, after slaughter and 48-h cold storage (4 °C) were obtained from the local village in the Pomerania region, Poland. Green bean (Phaseolus vulgaris) (0.5 kg), kohlrabi (Brassica oleracea var. gongylodes) (one piece), potato (Solanum tuberosum) (0.5 kg), and celery (Apium graveolens var. dulce) (one bunch of stalks) were bought at the local supermarket in the Pomerania region, Poland. All samples were stored at −80°C until used. Industrial meat products included steamed white sausage (one piece) and baked meat pate (0.5 kg) from the regular portfolio of Meat Company Nowak, Jankowo, Northern Poland.
Meat Sample Preparation
In this study, raw chicken meat and raw or home-processed pork (sirloin) or industrial pork meat products were used. Typical culinary home treatments or those applied by the food industry were used to obtain samples of pork processed meat. The home-processed pork was basically suitable for human consumption, but without addition of salt or spices, unless indicated otherwise. Three types of thermal treatments were applied to receive a product suitable for human consumption. A pork slice 1.5 cm thick was either boiled for 60 min in 1 L of distilled water without salt addition or grilled in a microwave oven (Bartscher GmbH, Germany) using the full power of the heater (1000 W) for 20 min on each side of a slice.
Preparation of Paraffin-Embedded Meat Samples
The nuclei and nucleic acids in meat tissue were visualized by fluorescent staining of paraffin-embedded meat sections. Directly after culinary treatment, from each meat sample, the fragments with dimensions of 0.5 × 1.0 × 2.0 cm (H × W × L) (at least 5–10 portions per sample) were cut out. Raw meat was fixed in 4% (v/v) formaldehyde in phosphate buffer for 24 h, then rinsed under running water for 6 h and transferred to 50% ethanol for 1 h. From the sausage slices and from meat pate, fragments with dimensions of 0.5 × 1.0 × 2.0 cm (H × W × L) were cut out. All meat samples were dehydrated initially with 60% (v/v) ethanol aqueous solution for about 1 h. Thereafter, the meat samples were transferred to a series of staining chambers containing increasing concentrations of ethanol (70%, 80%, 96% v/v of ethanol) and finally absolute ethanol. In each solution, the meat samples were kept for about 1 h. In the case of meat samples for which two washing steps in absolute alcohol were not enough to enable their saturation with xylene in whole volume, one or two additional washing steps (in total 3 or 4 times) in absolute ethanol were performed before the saturation with xylene. After dehydration, the samples were submerged for 10 min in a mixture of xylene:absolute ethanol (1:1 v/v) to pre-saturate the tissues with xylene, then placed in pure xylene to complete the saturation of the tissue. The xylene-saturated samples were characterized by a glassy structure within the whole volume. Finally, the meat samples were placed in a mixture of xylene and paraffin 9:1 (v/v) for 1 h and transferred to a liquid histological paraffin (Histosec pastilles, Merck, Germany) thermostated at 58 °C in a thermostat (Binder GmbH, Germany), where they remained for 24 h. This step allowed the saturation of tissue with paraffin. The paraffin-saturated samples were placed in histological metal molds previously filled with deaerated liquid paraffin. The paraffin was deaerated by immersing red-hot tweezers in it until the air bubbles were not visible. The blocks were allowed to solidify at room temperature for 24 h. The solid paraffin blocks were removed from the histological molds, placed in the arm of manual rotary microtome (SM 2000 M, Jung Heidelberg, Germany), and cut into 7-μm-thick slices. The obtained paraffin-embedded tissue sections were placed on a water surface prewarmed to 37 °C, where the cuts were straightened and transferred on the surface of clean, defatted microscope glass slides. The slices were left on the heated surface (37 °C) until fully dried and attached to glass.
Preparation of Paraffin-Embedded Plant Samples
Paraffin-embedded plant samples were prepared according to the procedure proposed by Weigel and Glazebrook (2008) with modifications. Small pieces of plant samples were submerged in formalin–acetic acid–alcohol fixative (48% ethyl alcohol, 3.7% formaldehyde, 5% glacial acetic acid, FAA) in beakers then placed in a desiccator. The vacuum was turned on and the reduced pressure was maintained for 15 min. The procedure was repeated twice. Samples were placed in a new portion of FAA and incubated for 4 h. After fixation, all plant samples were dehydrated initially with 60% (v/v) ethanol aqueous solution for about 30 min. Thereafter, the plant samples were transferred to a series of staining chambers containing increasing concentrations of ethanol (70%, 80%, 96% v/v) and finally absolute ethanol (twice). In each solution, the plant samples were kept for about 30 min (except for the first portion of absolute ethanol in which the samples were kept for 1 h). After dehydration, the samples were submerged in solutions with increasing xylene concentration relative to absolute ethyl alcohol (1:3, 1:1, 3:1 v/v). In each solution, the tissues were kept for 30 min. The tissues were then rinsed twice in pure xylene, each time for 1 h. Further preparation stages were proceeded as described for meat samples.
Basic Tissue Staining
The raw and culinary treated meat tissue sections as well as fragments of plant were stained with hematoxylin and eosin to visualize the overall tissue structures. These dyes are water-soluble; therefore, to enable the staining of meat and plant tissue, it was necessary to dewax and rehydrate paraffin-embedded meat and plant slides by two washes in xylene (2 min per slide) and in a series of solutions with decreasing ethanol concentration (absolute alcohol – twice, 96%, 80%, 70%, 60%, 50% v/v, water – 2 min in each). Staining in hematoxylin lasted up to 45 s, or until the nuclei became visible as purple or dark blue spots. The excess dye was removed by washing the microscope slides under a gentle stream of tap water for about 20 min. After brief immersion in distilled water, the slides were transferred to 70% ethanolic eosin solution for about 10 s. The time of exposure of the sample to the dye solution was dependent on the concentration of the dye, and lasted until the cytoplasm turned red-orange colored. Subsequently, a brief immersion of stained microscope slides in a series of increasing ethanol concentration solutions for dehydration (from 70% up to absolute alcohol) was performed. The ethanol present in tissue sections was removed by washing in two portions of xylene, for 2 and 5 min, respectively. Dehydrated tissue sections were coated with a drop of anhydrous Neo-Mount resin for closing and covered with a cover slip. After molting, the microscope slides were analyzed using a light Olympus BX 60 microscope, under ×10 and ×20 magnification.
Fluorescent Staining of Nucleic Acids
The meat and plant sections were stained with DNA and RNA selective fluorescent dyes: Hoechst 33342 and MGP mixture, which should enable the staining of deoxyribonucleic acid in nuclei in purple-bluish (methyl green) and the ribonucleic acid and cell plasma in red (pyronin Y). Hoechst 33342 and the MGP mix were applied on separate microscope slides. Due to the aqueous nature of these dyes, it was necessary to dewax and rehydrate the meat and plant tissue sections as described before. After a short rinse in distilled water, 1–2 drops of dye working solution was applied on the surface of the tissue for 10 and 30 min for Hoechst 33342 and MGP, respectively. From that point, the microscope slides were protected from light. After staining, the slides were washed in deionized water for 1 min, mounted with a drop of anhydrous resin Neo-Mount, covered with a cover slip, and immediately analyzed under fluorescent light using Olympus BX 60 microscope, applying ×10 and ×20 magnifications. Staining with a methyl green and pyronin Y dye mixture was done by green, red, and blue fluorescent channel overlay, according to Li et al. (2002).
DNA Integrity in Meat Samples by Comet Assay
The comet assay protocol used to assess DNA fragmentation in cells of raw and culinary treated meat samples was adapted from European Norm EN-13784, with modifications. Briefly, a suspension of nuclei from deep frozen meat samples was prepared as follows: about 1 g of frozen material was cut or scraped off with a sharp scalpel, cut into thin slices, and transferred to a beaker containing 5 mL of cold phosphate-buffered saline solution (PBS). The suspended meat flakes were stirred using a magnetic stirrer (500 rpm) for 5 min on an ice bath. Then, the suspension was filtered through gauze with a pore size of about 500 μm, to remove the tissue particulates. The filtrate was allowed to settle on ice for about 5 min and was used as a cell extract. The cell extract (20 μL) was mixed with 200 μL of melted 0.8% (w/v) LMP agarose in PBS thermostated at 45 °C. The suspension (35 μL) was applied dropwise on a microscope slides pre-coated with NMP agarose 1% (w/v) in distilled water, immediately covered with a cover slip, and allowed to solidify on ice for 5 min. The cover slips were carefully removed and agarose embedded nuclei were lysed for 10 min at room temperature in Tris-borate-EDTA buffer (TBE; 1× concentrated) containing 2.5% (w/v) SDS. The slides were transferred to electrophoresis apparatus (Bio-Rad, USA) filled with 1× TBE buffer and topped with 2–4 mm of the buffer layer. Under these conditions, the chromatin unwinding stage was carried out for 5 min. Electrophoresis was carried out in the same 1× TBE buffer for 2 min at 2 V cm−1, at room temperature. Then, the microscope slides were rinsed with water for 5 min and air-dried. The nuclei present on microscope slides were stained with 10,000× diluted SybrGreen dye solution for 30 min, rinsed for 10–15 min in water, and covered with cover slips. The DNA fragmentation (DNA comets) was observed under a fluorescence microscope coupled with a camera for recording of images. Comets were analyzed at ×50 magnification using METAFER 4 (Metasystems, Germany) software. The assay was performed in four replicates for each meat sample, and on each slide 100 (or all when there was less than 100 objects), consecutive comets were counted.
DNA Integrity in Plant Samples by Comet Assay
The comet assay protocol used to assess DNA fragmentation in plant cells was adapted from Pourrut et al. (2014) with modifications. Briefly, a suspension of nuclei from deep frozen plant samples was prepared as follows: about 450 mg of frozen material was placed in Petri dish (kept on ice) containing 1.5 mL of cold 400-mM Tris-HCl buffer (pH = 7.5). The tissues were vigorously chopped with a razor for 30 s. The Petri dish was then kept on ice for 5 min. Then, the suspension was filtered through gauze with a pore size of about 500 μm, to remove the tissue particulates, and the obtained cell extract was stored on ice. The cell extract (90 μL) was mixed with 150 μL of melted 1% (w/v) LMP agarose in PBS thermostated at 45 °C. The suspension (80 μL) was applied dropwise on a microscope slides pre-coated with NMP agarose 1% (w/v) in distilled water, immediately covered with a cover slip, and allowed to solidify on ice for 5 min. The cover slips were carefully removed and the slides were transferred to electrophoresis apparatus (Bio-Rad, USA) filled with cold electrophoretic buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) and topped with 2–4 mm of the buffer layer. Under these conditions, the chromatin unwinding stage was carried out for 15 min. Electrophoresis was carried out in the same buffer for 5 min (300 mA, 0.72 V/ cm, 4 °C). Then, the microscope slides were rinsed three times with 400-mM Tris-HCl buffer for 5 min and air-dried. The nucleic stained and analysis stages were performed analogously to meat samples, but at ×100 magnification.
DNA Isolation from Meat Samples
Raw and processed meat samples were used for the isolation of nucleic acids by phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) solvent extraction method. For all meat samples, the DNA isolation with and without the RNase A (100 μg mL−1) addition was performed. The tissue was scraped with a scalpel from frozen meat sample. About 25 mg of meat sample was weighed, placed in a 1.5-mL centrifuge tube, and combined with 200 μL of lysis buffer (20-mM Tris pH 8.0, 20-mM NaCl, 20-mM EDTA pH 8.0, 1% (w/v) SDS, 600 μg mL−1 proteinase K). The sample was homogenized with a hand homogenizer (Pellet Mixer, VWR, USA) and incubated in a dry heating bath (QBD4 Grant, UK) for 1.5 h at 37 °C, then 1.5 h at 56 °C or until completely digested (no floating particles visible), but not longer than for 6 h. After incubation, the lysate was diluted 1:1 (v/v) with dilution buffer (150-mM NaCl, 5-mM EDTA). Phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) in an aliquot of 1:1 (v/v) was added to the sample and it was thoroughly shaken for about 30 s on a laboratory shaker (IKA Vortex, China). The phases were separated by centrifugation for 2 min at 13,000 rpm with Eppendorf centrifuge (model 5415 R, Germany) at room temperature. The aqueous phase (top) was transferred to a new 1.5-mL centrifuge tube, extracted with chloroform 1:1 (v/v) and thoroughly vortexed for a few seconds. It was again centrifuged for 2 min at 13,000 rpm with Eppendorf centrifuge (model 5415 R, Germany) at room temperature. The separated aqueous phase was collected into a new 1.5-mL centrifuge tube, combined with 5-M NaCl, to reach the final concentration of 250-mM NaCl. The resultant solution was mixed thoroughly. Two volumes of 100% ethanol were added and the sample was kept overnight at 4 °C. The precipitated DNA was collected by centrifugation for 30 min at 13,000 rpm at 4 °C (Eppendorf centrifuge, model 5415 R, Germany). The obtained supernatant was discarded; the precipitate was washed with cold 70% (v/v) ethanol and centrifuged for 10 min at 13,000 rpm at 4 °C. The supernatant was carefully aspirated and discarded. The precipitate was air-dried and dissolved in TE buffer (10-mM Tris, pH 8.0, 1-mM EDTA) and kept at 4 °C for further analysis or at −20 °C for long-term storage.
Profiling of Isolated Nucleic Acids
Qualitative analysis of the isolated nucleic acids was performed using spectrophotometric and electrophoretic techniques. Determination of the presence of RNA in the samples was performed by comparing the fragmentation profile of isolated nucleic acid isolated with and without of RNase A addition. The concentration and purity of isolated nucleic acids were determined using NanoDrop 2000 C spectrophotometer (Thermo Scientific, USA). The isolated nucleic acids were separated either by agarose gel (OmniPAGE Electrophoresis System VS20 DGGE, Bio-Rad Cleaver Scientific Ltd) or by capillary electrophoresis (QIAxcel Advanced, Qiagen GmbH, Germany). Agarose electrophoresis was performed in a 1% (w/v) agarose gel prepared in 1× TBE buffer (89-mM Trizma-Base, 89-mM boric acid, 0.5-mM EDTA, pH 8.3) with 2 μL of ready to use DNA GelRed dye for 6.5 × 11.5 × 1.0 cm agarose gel. A M10 kbp DNA fragment marker was used. The separation was performed at room temperature, at a voltage of 100 V for 1.5 h. The QIAxcel High Resolution DNA cartridge, the QX Alignment Marker 15 bp size marker and QX DNA Size Marker 250 bp - 8 kbp v 2.0 (Qiagen GmbH, Germany) supplemented with 10 kbp and 20 kbp DNA fragments (NoLimits DNA Fragments, ThermoFischer Scientific, USA) were used for capillary electrophoresis. The separation was performed by the OM1200 method, available on the QIAxcel Advanced device. The samples of isolated nucleic acids were analyzed without additional dilutions. The nucleic acid profiles were analyzed with QIAxcel ScreenGel software.