Thiocyanates

Frozen fecal samples were thawed on ice, 100 mg of each sample was suspended in 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA, and the samples were then beaten with zirconia beads using a FastPrep FP100A instrument (MP Biomedicals, USA). DNA was extracted from the bead-treated suspensions using a Magtration System 12GC and GC series MagDEA DNA 200 (Precision System Science, Japan). DNA concentrations were estimated by spectrophotometry using an ND-1000 instrument (NanDrop Technologies, USA), and the final concentration of the DNA sample was adjusted to 10 ng/µL.

All blood analyses were performed using a free radical analyzer system (FREE Carpe Diem, Wimerll Company Ltd., Tokyo, Japan) that included a spectrophotometric device reader and a thermostatically regulated mini-centrifuge, and the measurement kits were optimized to the FREE Carpe Diem System, according to the manufacturer's instructions. Based on the recommendation from the manufacturer, all analyses were performed within 48 hours of venous blood collection to avoid falsely high or low results. To analyze the plasma levels of reactive oxygen metabolites, antioxidant capacity, and thiol-antioxidant capacity, diacron reactive oxygen metabolite (dROM), biological antioxidant potential (BAP), and sulfhydryl (SH) tests were performed, respectively.
The dROM test reflects the amount of organic hydroperoxides that is related to the free radicals from which they are formed. When the samples are dissolved in an acidic buffer, the hydroperoxides react with the transition metal (mainly iron) ions liberated from the proteins in the acidic medium and are converted to alkoxy and peroxy radicals. These newly formed radicals oxidize an additive aromatic amine (N,N-diethyl-para-phenylen-diamine) and cause formation of a relatively stable colored cation radical that is spectrophotometrically detectable at 505 nm [33] (link), [36] (link). The results are expressed in arbitrary units (U. Carr), one unit of which corresponds to 0.8 mg/L of hydrogen peroxide [33] (link), [36] (link).
The BAP test provides an estimate of the global antioxidant capacity of blood plasma, measured as its reducing potential against ferric ions. When the sample is added to the colored solution obtained by mixing a ferric chloride solution with a thiocyanate derivative solution, decoloration results. The intensity of the decoloration is spectrophotometrically detectable at 505 nm and is proportional to the ability of plasma to reduce ferric ions [34] (link), [37] (link). The results are expressed in µmol/L of the reduced ferric ions.
The SH test provides an estimate of the total thiol groups in the biologic samples, using a modified Ellman method [38] (link), [39] (link). When the sample is added to the solution, sulfhydryl groups in the sample react with 5,5-dithiobus-2-nitrobenzoic acid, which is followed by development of a stained complex that is spectrophotometrically detectable at 405 nm and is proportional to their concentration according to the Beer-Lambert law [34] (link), [36] (link). The results are expressed as µmol/L of the sulfhydryl groups.

The level of intracellular iron was measured as previously described [42 (link)]. After treatment with NPs, cells were trypsinized, washed with phosphate-buffered saline (PBS), centrifuged and counted. The cell pellet was digested in 5 N HCl for 24 h at 37 °C and then centrifuged at 2000× g rpm for 10 min. A volume of 50 μL of supernatant was mixed with 50 μL 1% ammonium persulfate solution (to convert the Fe²⁺ to Fe³⁺ ions) and 100 μL of 0.5 M potassium thiocyanate and shaken for 5 min in order to develop the red colored iron-thiocyanate. The formed complexes were spectrophotometrically detected at 450 nm. A standard curve with FeCl₃•6H₂O ranging from 0–250 μg Fe³⁺/mL was used to quantify the intracellular iron. The total amount of iron was related to the cell number.

Total RNA was isolated from tissue samples using the acid guanidium thiocyanate/phenol/chloroform method and was purified with lithium chloride, as previously reported [11 (link),22 (link)]. cDNAs were synthesized using a ImProm Ⅱ Reverse-Transcription system (Promega Corporation, Madison, WI, USA), and real-time polymerase chain reactions were performed on a StepOne Plus Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) [11 (link)]. Primer sequences for mouse Cdn15 were 5′-TCT TTC TAG GCA TGG TGG GA-3′ and 5′-TCA GTA GTG ATG TTG ACG GC-3′, and those for mouse Il6, Il17a, Tnf, Il1b, and Rn18s (the gene encoding 18S ribosomal RNA) were previously reported [22 (link),54 (link)]. The mRNA values were normalized to the Rn18s levels.

Air-dried leaf samples (leaf blades) were ground and dry-ashed in concentrated HNO₃ vapours, and the ash was dissolved in a 3% HCl solution. Wet digestion in H₂SO₄ and HNO₃ was used for N and S detection, respectively. Atomic absorption spectroscopy, using an acetylene-air flame atomizer (Perkin Elmer AAnalyst 700) and microwave plasma atomic emission spectrometry (4200 MP-AES, Agilent), was used for the measurement of K, Ca, Mg, Fe, Mn, Zn, and Cu according to the manufacturer’s instructions. Levels of P, Mo, N, and B were determined by colorimetry: P by ammonium molybdate in an acid-reduced medium, Mo by thiocyanate in a reduced acid medium, B by hinalizarine in a sulphuric acid medium, N by modified Kjeldal method using Nessler’s reagent in an alkaline medium, and S through the turbidimetric method by adding BaCl₂, using a spectrophotometer Jenway 6300 as described previously [60 (link)]. All the values were expressed as mass % and mg kg⁻¹ on a dry matter basis for each macronutrient and micronutrient evaluated, respectively.