Animals
The study protocol was designed in accordance to the International Medical Board of Animal Experiments guidelines accepted by the Inonu University Ethics Committee on Experimental Animal Research. It was approved by the Inonu University Ethics Committee on Experimental Animal Research (reference number: 2022/5 − 1). The study was conducted at Inonu University Experimental Animal Research Center (IUEARC). Forty female Wistar Albino rats (weighing 221.7–245.1 g; mean, 233,4 g) were used in the study. Rats were supplied by IUEARC. The rats were randomly divided into four groups (ten rats each). All animals were fed standard commercial pellet diet and water ad libitum. Rats were fasted for 12 hours before the experiment. All rats were housed under standard conditions, at 21 ± 2°C and 60 ± 5% humidity, with a 12-hour dark-light cycle.
Preparation of the HA and cupper
Humic acid and copper sulfate were dissolved in isotonic saline used as a vehicle solvent and administered orally to each rat at a volume of 5 mL/kg[10, 18].
Experimental Design
The experimental study will consist of 4 groups.
Group I (control, n = 10): normal diet, 14 days
Group II (HA, n = 10): normal diet + HA 536 mg/kg/day (Humic acid sodium salt technical grade, 100G Sigma Aldrich Fine Chemicals Biosciences) po, 14 days
Group III (Cu, n = 10): Normal diet + Copper sulfate solution (Copper(II) sulfate, 98%, pure, anhydrous, Thermo Scientific Chemicals) 75mg/kg/day po, in the morning, 14 days
Group IV(Cu + HA, n = 10):Normal diet + HA(536 mg/kg/day, po, evening) + Copper sulfate solution, 75 mg/kg/day, po, morning, 14 days.
At the end of the study (day 15), euthanasia was performed under xylazine + ketamine anesthesia, and 4–5 cc of intracardiac blood was taken by puncture for biochemical parameters. ALT, AST, ceruloplasmin and copper were measured from blood samples. For histopathological examination, samples were taken from the liver and brain and placed in 10% formaldehyde. For biochemical evaluation, some of the tissue samples were stored in liquid nitrogen solution. After tissue samples were thawed at room temperature, oxidative stress (malondialdehyde, total oxidative stress, oxidative stress index) and antioxidant system (superoxide dismutase, catalase and markers of glutathione peroxidase, total antioxidant system and reduced glutathione, which is a non-enzymatic antioxidant system, were analyzed. Additionally, caspase 3 and 9 antibodies and dry copper were investigated in the tissues to show apoptosis in the tissues. It was stored in a deep freezer at -80 C until analysis.
Biochemical Analysis
The weights of the tissues taken were weighed and noted. It was homogenized with 7.4 Tris-HCl buffer solution using an IKA-WERKE T 25 B brand device. The homogenates were centrifuged and the supernatant was separated. Relevant spectrophotometric readings were made with SHIMADZU UV-160A and BİOTEK SYNERGY LX multi-mode reader brand devices.
Determination of Malondialdehyde
The method of Uchiyama and Mihara was used to measure the malondialdehyde(MDA). MDA, a derivative of lipid peroxidation, is combined with thiobarbituric acid at 95°C to form the method. After the procedures applied to the standards and homogenats, the n-butanol phase in the tubes of the samples was read with a spectrophotometer at 535 and 520 nm wavelengths and the difference was recorded. The results obtained were calculated from the standard chart and expressed as nmol/g tissue[19].
Determination of Protein Content
The analysis of the amount of protein, which was the basis for the calculation of the data for the markers under investigation, was carried out according to the modified Lowry method[20]. The alkaline copper-protein solution containing the samples was mixed with Folin reagent. The standards and the samples were read in the spectrophotometer at a wavelength of 750 nm. The results were calculated according to the standard curve obtained and expressed as µg/ml.
Determination of Superoxide Dismutase Activity
Superoxide Dismutase (SOD) activity was determined by the inhibition of the reduction of nitroblue tetrazolium chloride (NBT) according to the method described by Sun et al[21]. SOD activity is inversely proportional to the absorbance of formazon at a wavelength of 560 nm. The results were calculated as U/mg protein.
Determination of Catalase Activity
Catalase(CAT) activity was determined by the Aebi method[22] based on the determination of the rate constant (k; s− 1) of H2O2 dissociation at a wavelength of 240 nm. The supernatant was added to the H2O2 solution. The decomposition was monitored over a period of time in a spectrophotometer. The supernatant was added to the H2O2 solution, the decomposition was monitored for a while on a spectrophotometer, and the rate constant was calculated from the absorbance change. Results are presented as K/g protein.
Determination of Glutathione Peroxidase Activity
Glutathione Peroxidase (GPx) activity was measured in accordance with the Paglia and Valentine method[23]. An enzymatic reaction was initiated by the addition of supernatant to a tube containing NADPH, reduced glutathione, sodium azide and glutathione reductase. H2O2 was then added to the incubated tubes and the reaction was monitored with a spectrophotometer at a wavelength of 340 nm for a period of time. The activity, expressed as U/mg protein, was calculated from the observed change in absorbance.
Determination of Glutathione Content
Glutathione (GSH) levels were determined by the Ellman method[24] using spectrophotometric assay. Each homogenate sample was mixed with 10 mM 5,5-dithiobis 2-nitrobenzoic acid (DTNB) in 100 mM potassium phosphate buffer (pH 7.5) and 17.5 M ethylene diamine tetraacetic acid (EDTA). The reaction was initiated by the addition of 0.5 units of glutathione reductase and 0.4 mM NADPH. After a 5-minute interval, the samples' absorbance was measured at a wavelength of 410 nm, and the concentration of GSH was calculated using a standard curve. Results expressed as µmol/g tissue.
Determination of Total Anti-oxidant Capacity
Total antioxidant status (TAS) was measured in the resulting supernatant using the Erel method[25]. This method uses the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) residue which changes colour from blue to green depending on the amount of antioxidants and antioxidant capacity, and the colour change is evaluated by measuring at a wavelength of 660 nm and the concentration of antioxidants. unit of measurement is Trolox equivalent/L.
Determination of Total Oxidant Status
Total oxidant status (TOS) was measured using a fully automated colorimetric method developed by Erel[25]. The TOS is expressed in terms of µmol H2O2 equivalent/L.
Measurement of Oxidative Stress Index
The oxidative stress index (OSI) was calculated by the division of the TOS by the total antioxidant level (TAS). OSI was expressed in arbitrary units (AU) [25].
Histopathological analysis
Fixed liver, kidney, and brain tissues were fixed in 10% neutral formaldehyde. After fixation, specimens were dehydrated in 70%, 80%, 90%, 96%, and 99% alcohol series. In the next step, the tissue specimens were cleared in xylene and embedded in paraffin. Paraffin-embedded specimens were cut into 4 µm thick sections, and mounted on slides. For histopathological analyses, the tissue sections were stained with hematoxylin and eosin (H&E).
The liver slides were assessed in terms of hepatocyte degeneration (hypereosinophilic and concentrated cytoplasm, pyknotic nuclei, hydropic degeneration) in 10 random fields per slide depending on the presence and severity of the damage. The following scale was used for semiquantitative scoring: absence = 0, slight = 1, moderate = 2, and severe = 3 [26].
The kidney slides were evaluated in terms of tubular damage (dilatation, desquamation, swelling/vacuolization, and necrosis of tubular epithelial cells) and glomerular damage (dilatation of glomerular capillaries). Ten randomly selected areas for tubular damage and randomly selected at least 30 glomeruli for glomerular damage were examined. The damage was semiquantitatively graded as follows according to the degree of histopathological changes; 0: no change, 1: mild, 2: moderate, 3: severe change [27].
The brain slides were evaluated in terms of neuronal degeneration in the cerebral cortex. The severity of neuronal degeneration was obtained by determining the number of degenerated neurons (neurons with shrunken hypereosinophilic cytoplasm and pycnotic nuclei) in 10 randomly selected areas for each slide under x400 magnification [28].
Immunohistochemical analysis
For immunohistochemical staining, 4 µm thickness tissue sections were deparaffinized, rehydrated, placed in antigen retrieval solution (citrate buffer), boiled in a pressure cooker for 20 minutes, and cooled to room temperature for 20 min. Then the sections were washed with phosphate-buffered saline (PBS), then for block endogenous peroxidase activity the slides were incubated in hydrogen peroxide solution for 15 min at room temperature and were washed in PBS. After blocking non-specific antigen-binding sites with protein block, the caspase-3 antibody (Santa Cruz Biotechnology, Inc) was applied for 60 minutes at room temperature. After being rinsed with PBS, sections were incubated with biotinylated secondary antibody and streptavidin peroxidase for 20 minutes at room temperature. Samples were visualized with the chromogenic substrates AEC, counterstained with hematoxylin, and mounted in the glass slide.
The cytoplasm of caspase-3 positive cells stained brown color. For immunohistochemical evaluation, ten randomly selected areas in each slide were examined. The immunostaining was semi-quantitatively scored based on the density of immunoreactive cells (0: 0–25%, 1:26–50, 2:51–75%, 3:76–100%) and the severity of immunoreactivity (0: none, 1: mild, 2: moderate, 3: severe). The total score was calculated as follows [29].
The total score = (density of immunoreactive cell) X (immunostaining severity)
Statistical Analysis
Data were analyzed using the IBM SPSS software program for Windows, version 26.0 (SPSS Inc., Chicago, IL). The normal distribution of quantitative data was evaluated using the Kolmogorov-Smirnov and Shapiro-wilk test. Differences in quantitative variables between groups were analysed using one-way analysis of variance (One-way ANOVA) for markers that comply with normal distribution. Multiple comparisons were conducted using either Tukey HSD or Tamhane test, depending on the homogeneity of variances. When the assumption of normal distribution was not met, we used the Kruskal Wallis-H test to determine differences between groups in terms of quantitative variables. Pairwise comparisons were made with the Mann-Whitney-U test with Bonferroni correction. Quantitative data within the scope of the research were presented as mean ± standard deviation in parametric tests and median (minimum-maximum) (Med (Min.-Max.)) in non-parametric tests. Results were considered statistically significant at p < 0.05.