Protective Role of Vitamin D in Attenuating RAI-Induced Liver Injury in Rats

Protective Role of Vitamin D in Attenuating RAI-Induced Liver Injury in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Role of Vitamin D in Attenuating RAI-Induced Liver Injury in Rats Muzaffer ATLI, Aylin AKBULUT, Serdar KURU, Nadide KOCA, Koray DEMİREL, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6700252/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Objective: Radioiodine (RAI) therapy is widely used for thyroid ablation, but I-131 accumulation in non-thyroid tissues may cause adverse effects. Vitamin D, known for its anti-apoptotic and immunomodulatory roles, may help protect against such damage. This study investigates the potential protective effects of vitamin D on RAI-induced liver injury through biochemical, histopathological, and immunohistochemical evaluations. Methods: Thirty male Wistar albino rats were randomly assigned to three groups: Control (Group I, n=10), RAI-treated (Group II, 111 MBq/kg, n=10), and RAI+Vitamin D (Group III, 200 ng/kg/day, n=10). Liver function was evaluated through serum analysis. Liver tissues were examined histopathologically and immunohistochemically. Oxidative stress markers—malondialdehyde (MDA), fluorescent oxidation products (FOP), catalase (CAT), and total sulfhydryl (T-SH)—were measured in liver homogenates. Results: RAI increased apoptotic cell numbers and elevated MDA, FOP, AST, and ALT levels. In contrast, T-SH and CAT levels were highest in the control group. Histopathology showed marked liver damage in the RAI group, which was less severe in the vitamin D group (p<0.001). TUNEL and caspase-3 analyses confirmed increased apoptosis in the RAI group compared to the others (p<0.001). Conclusions: Vitamin D alleviated RAI-induced liver injury, likely through its antioxidant, anti-apoptotic, and anti-inflammatory effects. Iodine-131 Liver injury Radioactive iodine Radioiodine Radioprotection Vitamin D Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Radioactive iodine (Radioiodine: RAI: Iodine-131: I131) treats hyperactive nodules, Graves' disease, and differentiated thyroid carcinoma ablation, preventing relapses and treating metastases [ 1 ]. The oral RAI dose not only affects the thyroid but can also affect other tissues (lower gastrointestinal tract, heart wall, stomach, kidneys, and liver). Side effects are a critical dose-limiting factor. Consequently, repeated RAI doses may be necessary, and the dose administered after each treatment should be increased, which can lead to further risks of side effects [ 1 – 3 ]. The hepatoprotective effects of various agents [ 4 – 7 ] and therapies [ 8 ] on liver injury, fibrosis, radiation toxicity, cellular damage, and radiation-induced DNA damage have been investigated [ 9 , 10 ]. However, there are few studies concerning liver damage following RAI therapy [ 4 , 7 , 11 ]. Common adverse effects of RAI therapy include nausea, vomiting, gastritis, radiation thyroiditis, bone marrow suppression, sialadenitis, hypothermia, xerostomia, ovarian failure, nasolacrimal duct obstruction, dry eyes, leukemia, secondary cancers, and radiation-induced pulmonary fibrosis. In rare instances, liver and kidney dysfunctions may also develop [ 2 , 12 ]. Liver function following RAI therapy has been assessed in a small group of individuals diagnosed with differentiated thyroid cancer [ 13 ]. After RAI therapy, the liver accumulates RAI. Repeated high-dose RAI applications may lead to further RAI accumulation, resulting more in liver damage. Histopathological examination shows that radiation causes inflammation, necrosis, and fibrosis due to microvascular involvement. Damage may be reversible at low radiation doses, but at high doses, significant degeneration of the interstitium with simultaneous inflammation and fibrosis is common [ 10 , 12 , 13 ]. Calcitriol, known as the biologically active variant of Vitamin D, impacts the immune system by interacting with the Vitamin D receptor (VDR), which is found in antigen-presenting cells like dendritic cells, monocytes, and macrophages. Vitamin D enhances the innate immune system by maintaining a balance between inflammatory and anti-inflammatory mechanisms, while it dampens the adaptive immune response, playing a role in the formation of an active immune reaction, and thus depends heavily on the hormonal roles of Vitamin D [ 14 , 15 ]. Vitamin D is essential in managing various inflammatory processes, including gene regulation, activation of signaling pathways, and the production of inflammatory mediators. Its receptors are found in mast cells, macrophages, natural killer (NK) cells, and T and B lymphocytes. Consequently, Vitamin D decreases inflammatory cytokines like IFN-γ, TNF-α, IL-1, and IL-2, while boosting the levels of IL-10, a cytokine with anti-inflammatory properties [ 15 – 17 ]. Vitamin D contributes to cellular homeostasis by facilitating apoptosis and orchestrating cell cycle progression. Its immunoregulatory role involves balancing pro- and anti-inflammatory signals, which supports the functional development of immune cells and maintains immune system equilibrium [ 16 , 17 ]. This experimental study explores the possible liver-protective benefits of Vitamin D during RAI therapy administered at elevated doses. Unlike prior research, it presents the first immunohistochemical analysis using apoptosis markers to evaluate Vitamin D's protective role against RAI-induced liver injury. As a pioneering in vivo study, this research suggests that Vitamin D may mitigate liver damage caused by RAI. MATERIALS AND METHODS All experimental procedures involving animals were in compliance with the European Community Council Directive of 24 November 1986, and ethical approval was granted by the Ethics Committee for Animal Research at Ankara Training and Research Hospital (Approval No: 0086-819; Date: November 1, 2024). Animals In this experimental study, all of the animals were treated humanly and in accordance with standard guidelines. At least seven days in advance of the beginning of the study, the rats were acclimated to the laboratory environment. Next, they were transferred to polypropylene cages under laboratory standard conditions that were maintained at 21±2 °C, using a light-dark cycle regimen of 12 hours and humidity ranging from 65–70%. During the entire experimental duration, the animals had continuous access to a standard diet and drinking water. Experimental Design All experimental procedures were conducted at the Hüsnü Sakal Experimental and Clinical Research Center under the Ankara Training and Research Hospital. Thirty male Wistar albino rats (weighing 200–250 g and aged 3–5 months) were randomly divided into three groups. The control group (n=10) received no treatment, while the RAI group (n=10) was administered 111 MBq/kg of radioactive iodine (MON-IYOT-131; Eczacıbaşı/Monrol, Gebze, Turkey) via oral gavage. The Vitamin D group received active vitamin D in saline suspension [calcitriol, 1,25(OH)2 D3] via intraperitoneal injections at 200 ng/kg daily (Pharmada Ilac et al., Istanbul, Turkey). Vitamin D supplementation began one day before the administration of radioactive iodine (111 MBq/kg) and was maintained throughout the experimental timeline. On the seventh day following RAI treatment, all animals were anesthetized with an intraperitoneal injection of propofol at a dose of 50 mg/kg (Abbott Laboratories, Istanbul, Turkey). Prior to sacrifice, blood and serum samples were collected from all rats and stored at -30 °C for less than 3 Months while awaiting enzymatic activity measurements. Liver tissues obtained after sacrifice were used for biochemical, histopathological, and immunohistochemical evaluations. Biochemical Procedures Measurements of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were performed using a fully automated biochemical analyzer (Roche Cobas 8000, Roche Diagnostics, USA). Oxidative stress parameters, including fluorescent oxidation products (FOP), malondialdehyde (MDA), catalase (CAT) activity, and total sulfhydryl (T-SH) groups, were assessed in liver tissue samples. Liver samples were first weighed and then homogenized using an automated homogenizer (Heidolph DIAX 900, Germany) at a 1:10 (w/v) ratio with ice-cold phosphate-buffered saline (PBS; 50 mM, pH 7.4) serving as the diluent. The homogenization process was carried out under chilled conditions to preserve biochemical stability. Malondialdehyde (MDA) levels were measured using a spectrofluorometric method outlined by Wasowicz et al. [18]. This approach is based on the interaction between MDA and thiobarbituric acid (TBA), resulting in the formation of a fluorescent compound that is subsequently extracted into n-butanol and measured with a fluorometer at excitation and emission wavelengths of 525 nm and 547 nm, respectively. A standard curve was generated using 1,1,3,3-tetraethoxypropane at concentrations between 0 and 5 μmol/L. For the analysis, 50 μL of tissue homogenate was combined with 1 mL of distilled water in 10 mL glass tubes. Subsequently, 1 mL of a 29 mmol/L TBA solution in acetic acid was added, and the mixture was thoroughly vortexed. The tubes were incubated in a water bath at 95–100 °C for 1 hour to facilitate the reaction. After incubation, the samples were left to cool down to room temperature, and 25 μL of 5 mol/L hydrochloric acid (HCl) was introduced. The reaction mixture was then extracted with 3.5 mL of n-butanol by vigorous vortexing for 5 minutes. After centrifuging at 1500 ×g for 10 minutes, the fluorescence intensity of the butanol layer was recorded, and MDA levels were expressed in micromoles per gram of wet tissue. The total sulfhydryl (T-SH) content was determined using a spectrophotometric method based on the procedure outlined by Sedlak and Lindsay [19]. In brief, 250 μL of supernatant from the tissue homogenate was combined with 750 μL of 0.2 M Tris buffer (pH 8.2) and 50 μL of 0.01 M 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). To achieve a final volume of 5 mL, 3950 μL of absolute methanol was incorporated. Alongside the test samples, a reagent blank (lacking homogenate) and a sample blank (omitting DTNB) were prepared using the same procedure. Every tube was sealed with a rubber stopper, kept at ambient temperature for 15 minutes, and then subjected to centrifugation at around 3000×g for the same duration. The absorbance of the supernatant was measured at 412 nm. FOP levels were measured spectrofluorometrically at 360/430 nm after extraction of the homogenates with an ethanol-ether (3:1, v/v) mixture [20]. CAT enzyme activity was assessed using a simple spectrophotometric method as described by Hadwan [21]. Histopathological Procedures For histopathological analysis, tissues were preserved in 10% neutral buffered formalin and then processed using standard paraffin embedding methods. Paraffin-embedded tissue blocks were sectioned into 4 µm slices and subsequently stained using Hematoxylin and Eosin (H&E). Microscopic evaluation and imaging were performed using an Olympus BX-53 microscope (Olympus, Tokyo, Japan) equipped with Olympus Cell B software. The histopathological changes, such as hyperemia, inflammation, fibrosis, vacuolization, multinucleated cells, and granuloma formation, were assessed using a semi-quantitative scoring system (Figure 1 – A, B, C). Scores ranged from 0 (no findings) to 3 (severe). Immunohistochemical Procedures Immunohistochemical analysis was performed in accordance with the protocol outlined by Yumuşak et al. [22], with minor modifications. Tissue sections approximately 4 µm in thickness were obtained from paraffin-embedded samples and placed onto poly-L-lysine-coated slides. After undergoing deparaffinization and rehydration through standard procedures, a streptavidin-biotin complex (ABC) detection system was applied using the Histostain Plus Kit (Zymed, South San Francisco, CA, USA). For immunolabeling, a Caspase-3-specific primary antibody (diluted 1:200; catalog no. PA5-16335, Invitrogen, CA, USA) was used. Chromogenic visualization was achieved with diaminobenzidine (DAB; Dako), which was applied for 10 minutes. Phosphate-buffered saline (PBS) was used for all rinsing steps throughout the staining process. Cytoplasmic immunoreactivity was semi-quantitatively scored on a four-point scale: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong brown staining). All staining steps were carried out in a humid chamber at 37°C (Figure 1 – D, E, F). A TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was utilized to identify apoptotic DNA fragmentation in liver, kidney, and testicular tissues. This analysis was carried out using a commercially available cell death detection kit (In Situ Cell Death Detection Kit, POD; Roche, Mannheim, Germany). The procedure was adapted from the methodology previously described by Yumuşak et al. [22]. The extent of staining was graded based on the proportion of nuclei exhibiting positive labeling: 0 (none, 25–75%), and 3 (strong, >75%) (Figure 1 – G, H, I). Statistical Analysis Statistical analyses were conducted using IBM SPSS Statistics (v27.0; IBM Corporation, Armonk, NY, USA) and MedCalc (v15.8; MedCalc Software, Ostend, Belgium). Descriptive measures, including percentages, means, standard deviations, frequencies, medians, and interquartile ranges (IQRs), were calculated to summarize the dataset. The assumption of normality was evaluated using the Shapiro-Wilk test, along with skewness and kurtosis values, and supported by visual diagnostic tools such as Q-Q plots, histograms, boxplots, and stem-and-leaf graphs. When data conformed to a normal distribution, intergroup comparisons were made using analysis of variance (ANOVA), and Tukey's HSD test was employed for post hoc analyses. For data that did not exhibit normal distribution, the Kruskal-Wallis non-parametric test was applied, followed by Bonferroni-adjusted pairwise comparisons where appropriate. A threshold of p < 0.05 was used to determine statistical significance. Furthermore, a retrospective power calculation was conducted via G*Power software (v3.1.9.7; Franz Faul, University of Kiel, Germany), focusing on malondialdehyde (MDA) values. The sample sizes were n₁ = 10 (100.09 ± 19.24), n₂ = 10 (211.71 ± 52.07), and n₃ = 10 (146.06 ± 45.46), with a pooled standard deviation of 61.38. The analysis was conducted with a significance level of α = 0.05 and an effect size (f) of 0.75, yielding a statistical power of 94%. RESULTS The average measurements for liver function markers and oxidative stress indicators are presented in Table 1 . The biochemical analysis revealed statistically meaningful differences among the control, RAI, and vitamin D groups across all measured variables (p < 0.05). To pinpoint which groups contributed to these differences, further pairwise comparisons were conducted using Tukey’s Honestly Significant Difference (HSD) method: Table 1 Comparison of Biochemical Parameters Between Groups Group I (Control) (n = 10) Group II (RAI) (n = 10) Group III (RAI + VitD) (n = 10) p a Difference b MDA (nmol/g protein) 100,09 ± 19,24 211,71 ± 52,07 146,06 ± 45,46 < 0,001 All FOP (FO/g protein) 99,64 ± 17,41 181,40 ± 32,43 145,42 ± 27,22 < 0,001 All T-SH (umol/g protein) 77,18 ± 14,23 50,48 ± 11,14 62,61 ± 12,10 < 0,001 1 to 2–3 Catalaz (u/g protein) 74,61 ± 7,37 43,26 ± 10,85 58,69 ± 7,76 < 0,001 All AST (U/L) 86,80 ± 19,74 121,60 ± 16,08 95,30 ± 10,22 < 0,001 2 to 1–3 ALT (U/L) 46,30 ± 9,63 64,90 ± 17,43 49,10 ± 5,93 0,004 2 to 1–3 a: One-Way Anova Test (Mean ± SD), b: Post-Hoc Tukey HSD Test, For MDA, FOP, and Catalase variables, all groups differed from each other, For AST and ALT variables, Group II values differed from other groups, For the T-SH variable, Group I values differed from other groups. All assessed parameters, including MDA, FOP, T-SH, catalase, AST, and ALT, displayed statistically significant differences among the experimental groups. Specifically, the animals treated with RAI showed the highest levels of MDA, FOP, AST, and ALT, while the control rats recorded the lowest values. On the other hand, T-SH and catalase levels were highest in the control group, suggesting stronger antioxidant activity. When examining oxidative stress indicators, both the control and vitamin D groups demonstrated substantially reduced levels of MDA and FOP relative to the RAI-treated group (p < 0.001). Similarly, antioxidant markers T-SH and catalase were observed at significantly higher concentrations in the control cohort than in the others (p < 0.001). In terms of hepatic biochemical markers, AST and ALT levels were moderately increased in the vitamin D group relative to the control, yet remained considerably below the elevated levels observed in the RAI group (p < 0.001 and p = 0.004, respectively) (Table 1 and Fig. 2 ). Notable differences were detected between the groups across all histopathological parameters, with statistical results showing p-values less than 0.05. Post hoc analyses indicated that all groups differed significantly in the multiple nucleus parameter (p < 0.001). Group II showed distinct differences from the other groups in terms of hyperemia, inflammation, fibrosis, vacuolization, and granuloma formation. In general, the RAI group exhibited substantially elevated histopathological scores relative to both the control and vitamin D groups, with these differences reaching high statistical significance (p < 0.001 for both) (Table 2 and Fig. 3 ). Table 2 Comparison of Histopathological Parameters Between Groups Group I (Control) (n = 10) Group II (RAI) (n = 10) Group III (RAI + Vitamin D) (n = 10) p a Difference b Hyperemia 0.10 ± 0.32 2.30 ± 0.48 0.30 ± 0.48 < 0,001 2 to 1–3 0* 9 (%90,0) 0 (%0,0) 7 (%70,0) 1* 1 (%10,0) 0 (%0,0) 3 (%30,0) 2* 0 (%0,0) 7 (%70,0) 0 (%0,0) 3* 0 (%0,0) 3 (%30,0) 0 (%0,0) Inflammation 0,10 ± 0,32 2,20 ± 0,42 0,40 ± 0,52 < 0,001 2 to 1–3 0* 9 (%90,0) 0 (%0,0) 6 (%60,0) 1* 1 (%10,0) 0 (%0,0) 4 (%40,0) 2* 0 (%0,0) 8 (%80,0) 0 (%0,0) 3 0 (%0,0) 2 (%20,0) 0 (%0,0) Fibrosis 0,00 ± 0,00 2,20 ± 0,42 0,20 ± 0,42 < 0,001 2 to 1–3 0* 10 (%100,0) 0 (%0,0) 8 (%80,0) 1* 0 (%0,0) 0 (%0,0) 2 (%20,0) 2* 0 (%0,0) 8 (%80,0) 0 (%0,0) 3* 0 (%0,0) 2 (%20,0) 0 (%0,0) Vacuolization 0,20 ± 0,42 2,60 ± 0,52 0,40 ± 0,52 < 0,001 2 to 1–3 0* 8 (%80,0) 0 (%0,0) 6 (%60,0) 1* 2 (%20,0) 0 (%0,0) 4 (%40,0) 2* 0 (%0,0) 4 (%40,0) 0 (%0,0) 3* 0 (%0,0) 6 (%60,0) 0 (%0,0) Multiple Nucleus 0,00 ± 0,00 3,00 ± 0,00 0,60 ± 0,52 < 0,001 All 0* 10 (%100,0) 0 (%0,0) 4 (%40,0) 1* 0 (%0,0) 0 (%0,0) 6 (%60,0) 3* 0 (%0,0) 10 (%100,0) 0 (%0,0) Granuloma 0,00 ± 0,00 2,00 ± 0,00 0,30 ± 0,48 < 0,001 2 to 1–3 0* 10 (%100,0) 0 (%0,0) 7 (%70,0) 1* 0 (%0,0) 0 (%0,0) 3 (%30,0) 2* 0 (%0,0) 10 (%100,0) 0 (%0,0) a: Kruskal-WallisTest (Mean ± SD), b: Post-Hoc Bonferroni Correction, *: n (%), Immunohistochemical evaluation using the TUNEL method indicated notable group-dependent variation, with results demonstrating a high level of statistical significance (p < 0.001). Furthermore, caspase-3 immunoreactivity was markedly elevated in the RAI group relative to both the control and vitamin D groups, and this distinction was also found to be statistically significant (p < 0.001) (Table 3 and Fig. 4 ). Table 3 Comparison of Immunohistochemical Parameters Between Groups Group I (Control) (n = 10) Group II (RAI) (n = 10) Group III (RAI + Vitamin D) (n = 10) p a Difference b Caspase-3 0.00 ± 0.00 2.60 ± 0.52 0.40 ± 0.52 < 0.001 2 to 1–3 0* 10 (%100.0) 0 (%0.0) 6 (%60.0) 1* 0 (%0.0) 0 (%0.0) 4 (%40.0) 2* 0 (%0.0) 4 (%40.0) 0 (%0.0) 3* 0 (%0.0) 6 (%60.0) 0 (%0.0) TUNEL 0.30 ± 0.67 3.00 ± 0.00 1.10 ± 0.88 < 0.001 All 0* 8 (%80.0) 0 (%0.0) 3 (%30.0) 1* 1 (%10.0) 0 (%0.0) 3 (%30.0) 2* 1 (%10.0) 0 (%0.0) 4 (%40.0) 3* 0 (%0.0) 10 (%100.0) 0 (%0.0) a: Kruskal-WallisTest (Mean ± SD), b: Post-Hoc Bonferroni Correction, *: n (%), DISCUSSION Radioactive iodine, a radionuclide emitting beta and gamma rays, has been a primary treatment for thyroid cancer and hyperthyroidism since the 1940s [ 1 , 23 , 24 ]. I-131 accumulation in non-thyroid tissues during RAI treatment can cause adverse effects. Limited research exists on I-131’s toxic and hepatotoxic effects and no universally accepted treatment prevents 131-I tissue damage [ 25 – 28 ]. This study investigates the potential protective role of vitamin D against liver damage induced by radioactive iodine (RAI), using a range of biochemical, histopathological, and immunohistochemical techniques. Our study reveals that RAI treatment causes hepatocellular damage by triggering oxidative stress in the rat liver and that vitamin D shows a protective effect against these harmful processes. RAI administration led to elevated levels of lipid peroxidation markers, MDA and FOP, while reducing the activity of the antioxidant defense system, including T-SH and CAT. Additionally, the significant rise in AST and ALT enzyme levels confirms that RAI causes liver cell damage by disrupting hepatocellular integrity. Histopathological examinations also identified damage findings such as hyperemia, inflammation, fibrosis, and vacuolization. Immunohistochemical analyses confirm that RAI activates apoptosis mechanisms (Caspase-3 expression and TUNEL positive cells). In the vitamin D group, protective effects were observed at various levels, including a reduction in oxidant levels, an increase in antioxidant levels, normalization of liver enzyme levels, alleviation of histopathological damage, and suppression of apoptotic activation. These results experimentally support the idea that vitamin D provides a hepatoprotective effect against RAI-induced liver damage. Iodine is transported within organs or mass lesions via the sodium iodide symporter. The liver itself does not have a sodium iodide symporter. However, hepatic uptake after RAI is a physiological condition resulting from intrahepatic bile ducts containing the sodium iodide symporter. Another reason for hepatic radioactive iodine uptake could be the binding of iodine to thyroxine due to hormonal production in the thyroid glands [ 26 , 27 ]. Hepatic radioactive iodine is positively correlated with the administered I131 dose. As the liver concentrates RAI, serious liver damage can occur due to more RAI accumulation with multiple and increasing high doses. Radiation causes hyperemia, inflammation, necrosis, and fibrosis in the liver, and at lower radiation doses, reversible microvascular hyperemia occurs [ 13 , 28 – 30 ]. Factors like age, gender, nutrition, disease duration, and goiter size may elevate RAI-induced hepatotoxicity [ 30 ]. In a study showing that RAI causes late histopathological damage in rat livers three months after I131 administration, histopathological changes such as hyperemia, steatosis, fibrosis, and capsule thickening were demonstrated [ 4 ]. Hyperemia after RAI-131 administration indicates an increase in blood flow and an inflammatory response. Hyperemia helps transport more oxygen and nutrients to damaged tissues. On the other hand, when the metabolic functions of the liver are affected, especially disruptions in fat metabolism can be observed. Steatosis can be considered an indicator of disruptions in fat metabolism and cellular damage in the liver. Again, damage in the liver triggers a fibrogenic response, leading to increased collagen production and, thus, fibrosis, which is the hardening of the liver. Our histopathological findings also confirm the liver toxicity of RAI in a manner similar to this study. In the analysis of histopathological data in our study, it was found that the RAI group differed from the other groups in the variables of Hyperemia, Inflammation, Fibrosis, Vacuolization, and Granuloma (p < 0.05). Vacuolization, defined as the formation of fluid-filled spaces within the cell, is an adaptive response for cells to store or detoxify harmful substances and wastes. A granuloma is a small lesion formed by the accumulation of macrophages as part of an inflammatory reaction. These histopathological changes observed after RAI-131 administration can be considered indicators of toxic effects and inflammatory responses in the liver. Comparative analysis revealed that the RAI group demonstrated significantly elevated histopathological parameter scores in relation to both the control and vitamin D groups. The reduction of histopathological changes by vitamin D demonstrates its ability to generally protect cellular health, reduce oxidative stress, and control inflammatory responses. Diffuse iodine-131 uptake in the liver has often been documented in scans following RAI treatment in the literature [ 27 , 28 , 31 ]. There are also studies suggesting that liver enzyme elevations in thyroid disease may paradoxically be associated with RAI. The elevation of serum transaminases such as AST and ALT in cases of liver toxicity developing after RAI treatment is an indicator of hepatocyte damage that leads to increased cell membrane permeability, facilitating the leakage of transaminases into the bloodstream [ 28 – 32 ]. Our study found that the values of AST and ALT from liver function tests in the RAI group differed from those in the other two groups. Serum liver enzyme concentrations in the vitamin D group were higher than those in the control group; however, the RAI group exhibited the greatest elevations, with significant intergroup differences observed (p < 0.05). To reduce the effects against liver tissue damage caused by high-dose RAI, the protective effects of different agents such as melatonin [ 7 ], dexmedetomidine [ 11 ], and N-acetyl cysteine [ 33 ] have been demonstrated in the early period. In all three studies, biochemical oxidative stress parameters were evaluated along with histopathological findings. The increase in oxidative stress parameters after RAI-131 treatment is due to a series of factors, such as the direct effects of ionizing radiation, cell damage, weakened antioxidant defense mechanisms, inflammation, and metabolic changes. Similar to the results obtained from these studies, histopathological and biochemical findings parallel our study. Our study's analysis of oxidative stress parameters in liver tissue showed statistically significant differences among the control, RAI, and VitD groups (p < 0.05). Among the groups, oxidant levels were greatest in the RAI group and lowest in the control group. Conversely, antioxidant concentrations reached their highest in the control group. When biological systems are exposed to ionizing radiation, it triggers the production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). These highly active molecules can harm critical macromolecules like DNA, RNA, proteins, and cellular membranes, leading to impaired cell function or even programmed cell death (apoptosis). While ROS are crucial for normal cellular processes, their excessive production can induce cell death through either necrosis or apoptosis. The condition of oxidative stress occurs when there is an imbalance between pro-oxidants and antioxidants, leading to damage such as lipid peroxidation, protein oxidation, and DNA alteration. Oxidative stress is a key contributor to the development of various human conditions, including neurodegenerative diseases, cancer, aging, and chronic inflammatory diseases [ 9 , 34 ]. It is known that ionizing radiation causes damage to cells through free radicals and direct ionization. It is thought that support with antioxidant substances can increase treatment gain by enhancing the tumor response to radiation and reducing its toxic effect on normal cells [ 35 ]. Only a small number of studies have been carried out to evaluate the effect of protective agents against high-dose internal radiation damage in the liver biochemically with oxidative stress parameters [ 4 , 7 , 11 ]. Apoptosis is the process by which the organism kills unwanted cells during homeostasis, development, embryogenesis, immune regulation, or disease [ 36 ]. Morphological, biochemical, and molecular changes can measure this process. During the early and late stages of apoptosis, cysteine proteases called caspases are activated; in particular, caspase-3 mediates both necrotic and apoptotic cell death. The TUNEL assay detects DNA fragmentation occurring in the final stages of apoptosis. In aged rats, increased liver damage caused by increased apoptosis of caspase-3 and TUNEL has been found [ 36 – 39 ]. In our study, we evaluated apoptosis for the first time in the literature regarding the protective effect of vitamin D against the injury caused by 131I in the liver. Our immunohistochemical analysis results showed that RAI significantly activates the apoptosis mechanism in the liver. The notable rise in TUNEL-positive cells and the substantial increase in Caspase-3 expression, a critical effector protein in apoptosis, suggest that RAI induces liver cell death, thereby contributing to tissue damage. The results of this study showed that radiations induced apoptotic pathways (TUNEL- positive cells and increased caspase-3 activity in the RAI group). The significant reduction of these apoptotic markers by vitamin D suggests that it suppresses apoptotic signals through mechanisms such as modulating the Bcl-2/Bax balance or stabilizing calcium homeostasis. This discovery aligns with earlier research emphasizing the dual function of vitamin D, offering anti-apoptotic protection in normal tissues while exhibiting a pro-apoptotic effect in cancer cells [ 40 , 41 ]. Significant differences were identified in the TUNEL evaluation results among the RAI, Control, and Vitamin D groups during the statistical comparison (p < 0.001). In the analysis of Caspase-3 findings, it was found that the values in the RAI group differed from those in the other groups (p < 0.001). The hepatoprotective effects of vitamin D are primarily attributed to its abilities to modulate inflammation and counteract oxidative damage. It reduces oxidative stress by neutralizing free radicals directly or by stimulating the production of endogenous antioxidants, including Superoxide Dismutase and Glutathione Peroxidase [ 15 , 16 ]. Our findings demonstrate that vitamin D is essential in lowering oxidative stress markers such as MDA and FOP, while simultaneously boosting the activity of antioxidant enzymes like Catalase (CAT) and Total Sulfhydryl (T-SH), highlighting its antioxidant properties. Moreover, vitamin D modulates inflammatory responses by lowering pro-inflammatory cytokines like TNF-α and IL-6, while promoting the production of anti-inflammatory cytokines such as IL-10 [ 14 , 42 ]. Histopathological assessments further support these results, showing a reduction in inflammation scores and reinforcing the anti-inflammatory potential of vitamin D. Vitamin D is considered to contribute to liver protection by modulating signaling pathways within hepatic cells particularly non-parenchymal cells through its interaction with the vitamin D receptor (VDR). This receptor, located in various cell types, enables vitamin D to regulate gene transcription. Through this mechanism, vitamin D helps regulate inflammatory activity, supports the synthesis of anti-inflammatory agents, and participates in the regulation of cell death and survival processes. Existing literature suggests that activation of VDR can inhibit liver fibrosis, regulate apoptosis, and promote cell survival [ 43 , 44 ]. Our findings showed decreased levels of fibrosis and apoptotic indicators (TUNEL, Caspase-3), implying a potential role for VDR-dependent mechanisms in the liver-protective effects of vitamin D. Further research is needed to clarify how vitamin D influences intracellular signaling pathways in hepatic cells and to better understand the specific molecular functions of the VDR. In summary, the results of this study indicate that vitamin D may help mitigate liver damage associated with RAI exposure. Its protective influence appears to stem from its anti-inflammatory, antioxidant, and anti-apoptotic functions. These findings may hold particular relevance for individuals with preexisting liver issues or patients receiving RAI-based therapies. In particular, starting vitamin D supplementation before or during RAI treatment in patients undergoing RAI treatment due to thyroid cancer or hyperthyroidism who have concurrent liver disease or are at risk of liver damage may help reduce liver damage and increase treatment tolerance. Abbreviations RAI: I131, Iodine-131, Radioactive iodine, Radioiodine; FOP: Fluorescent oxidation products; MDA: Malondialdehyde; CAT: Catalase; T-SH: Total sulfhydryl; VDR: Vitamin D receptor; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBA: thiobarbituric acid; DTNB: 5,5′-dithiobis-(2-nitrobenzoic acid); H&E: Hematoxylin and Eosin; PBS: Phosphate-buffered saline; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ROS: Reactive oxygen species. Declarations Conflict of interest All authors read and approved the final version of the manuscript. The authors declare that they have no conflicts of interest. They confirm that no financial or non-financial conflicts of interest are relevant to the work conducted or reported in this manuscript. A statement of financial support; This research was conducted independently by the authors and received no specific grant or financial support from any funding agency, commercial entity, or non-profit organization. Acknowledgments; The authors have no acknowledgments to declare. References Luster M, Pfestroff A, Hänscheid H, Verburg FA. Radioiodine Therapy. Semin Nucl Med. 2017 Mar;47(2):126-134 Padma S, Sundaram PS. Radioiodine as an adjuvant therapy and its role in follow-up of differentiated thyroid cancer. J Cancer Res Ther. 2016 Jul-Sep;12(3):1109-1113. Zaletel K, Mihovec A, Gaberscek S. Characteristics of exposure to radioactive iodine during a nuclear incident. Radiol Oncol. 2024 Oct 4;58(4):459-468 Atilgan HI, Yumusak N, Sadic M, Gultekin SS, Koca G, Ozyurt S, Demirel K, Korkmaz M. Radioprotective effect of montelukast sodium against hepatic radioiodine (I131) toxicity: A histopathological investigation in the rat model. Ankara Üniv Vet Fak Derg 2015; 62: 37-43. Hanedan Uslu G, Canyilmaz E, Serdar L, Ersöz Ş. Protective effects of genistein and melatonin on mouse liver injury induced by whole-body ionising radiation. Mol Clin Oncol. 2019 Feb;10(2):261-266. Sadeeshkumar V, Duraikannu A, Aishwarya T, Jayaram P, Ravichandran S, Ganeshamurthy R. Radioprotective efficacy of dieckol against gamma radiation-induced cellular damage in hepatocyte cells. Naunyn Schmiedebergs Arch Pharmacol. 2019; 392(8):1031-1041. Barlas AM, Sadic M, Atilgan HI, Bag YM, Onalan AK, Yumusak N, Senes M, Fidanci V, Pekcici MR, Korkmaz M, Kismet K, Koca G. Melatonin: a hepatoprotective agent against radioiodine toxicity in rats. Bratisl Lek Listy 2017; 118(2): 95-100. Chi CH, Liu IL, Lo WY, Liaw BS, Wang YS, Chi KH. Hepatocyte growth factor gene therapy prevents radiation-induced liver damage. World J Gastroenterol 2005; 11(10): 1496-502. Zhou YJ, Tang Y, Liu SJ, Zeng PH, Qu L, Jing QC, Yin WJ. Radiation-induced liver disease: beyond DNA damage. Cell Cycle. 2023 Mar;22(5):506-526. Kim J, Jung Y. Radiation-induced liver disease: current understanding and future perspectives. Exp Mol Med. 2017 Jul 21;49(7):e359. Kismet K, Sadic M, Bag YM, Atilgan HI, Koca G, Onalan AK, Senes M, Peker SA, Yumusak N, Korkmaz M. Hepatoprotective effect of dexmedetomidine against radioiodine toxicity in rats: evaluation of oxidative status and histopathological changes. Int Surg 2016; 101: 176-84. Fard-Esfahani A, Emami-Ardekani A, Fallahi B, Fard-Esfahani P, Beiki D, Hassanzadeh-Rad A, Eftekhari M. Adverse effects of radioactive iodine-131 treatment for differentiated thyroid carcinoma. Nucl Med Commun 2014; 35(8): 808-17. Wang S, Liang C, Zhao L, Meng Z, Zhang C, Jia Q, Tan J, Yang H, Liu X, Wang X. Influence of radioactive iodine therapy on liver function in patients with differentiated thyroid cancer. Nucl Med Commun 2018; 39(12): 1113-20. Eksioglu U, Atilgan HI, Yakin M, Yazihan N, Altiparmak UE, Yumusak N, Korkmaz M, Demir A, Ornek F, Aribal Ayral P, Koca G. Antioxidant effects of vitamin D on lacrimal glands against high dose radioiodine-associated damage in an animal model. Cutan Ocul Toxicol 2019; 38(1): 18-24. Wimalawansa SJ. Physiology of Vitamin D-Focusing on Disease Prevention. Nutrients. 2024 May 29;16(11):1666. Toniato E, Spinas E, Saggini A, et al. Immunomodulatory effects of vitamin D on skin inflammation. J Biol Regul Homeost Agents 2015; 29: 563-7 Markotić A, Kelava T, Markotić H, Silovski H, Mrzljak A. Vitamin D in liver cancer: novel insights and future perspectives. Croat Med J. 2022 Apr 30;63(2):187-196. Wasowicz W, Nève J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clin Chem 1993; 39(12): 2522–6. Sedlak J, Lindsay RH. Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968; 25(1): 192–205. Wu T, Willett WC, Rifai N, Rimm EB. Plasma fluorescent oxidation products as potential markers of oxidative stress for epidemiologic studies. Am J Epidemiol. 2007; 166(5): 552–60. Hadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC Biochem 2018; 19(1): 7 Yumusak N, Sadic M, Yucel G, Atilgan HI, Koca G, Korkmaz M. Apoptosis and cell proliferation in short-termandlong-termeffects of radioiodine-131-induced kidney damage: an experimental and immunohistochemical study. Nucl Med Commun 2018; 39(2):131-39. Nguyen NC, Anigati EM, Desai NB, Öz OK. Radioactive Iodine Therapy in Differentiated Thyroid Cancer: An Update on Dose Recommendations and Risk of Secondary Primary Malignancies. Semin Nucl Med. 2024 Jul;54(4):488-496. Lee SL. Radioactive iodine therapy. Curr Opin Endocrinol Diabetes Obes 2012; 19(5): 420-8. Hong CM, Ahn BC. Factors Associated with Dose Determination of Radioactive Iodine Therapy for Differentiated Thyroid Cancer. Nucl Med Mol Imaging. 2018 Aug;52(4):247-253. Piantanida E, Ippolito S, Gallo D, Masiello E, Premoli P, Cusini C, Rosetti S, Sabatino J, Segato S, Trimarchi F, Bartalena L, Tanda ML. The interplay between thyroid and liver: implications for clinical practice. J Endocrinol Invest. 2020 Jul;43(7):885-899. Omür O, Akgün A, Ozcan Z, Sen C, OzkiIiç H. Clinical implications of diffuse hepatic uptake observed in postablative and post-therapeutic I-131 scans. Clin Nucl Med 2009; 34(1): 11-4. Jhummon NP, Tohooloo B, Qu S. Iodine-131 induced hepatotoxicity in previously healthy patients with Grave's disease. Thyroid Res. 2013 Mar 16;6:4. Guha C, Kavanagh BD. Hepatic radiation toxicity: avoidance and amelioration. Semin Radiat Oncol 2011; 21(4): 256-63. Lin R, Banafea O, Ye J. I-131 remnant ablation after thyroidectomy induced hepatotoxicity in a case of thyroid cancer. BMC Gastroenterol. 2015 May 7;15:56. Ferris HA, Williams G, Parker JA, Garber JR. Therapeutic implications of diffuse hepatic uptake following I-131 therapy for differentiated thyroid cancer. Endocr Pract. 2013;19(2):263-7. Kim CW, Park JS, Oh SH, Park JH, Shim HI, Yoon JW, Park JS, Hong SB, Kim JM, Le TB, Lee JW. Drug-induced liver injury caused by iodine-131. Clin Mol Hepatol. 2016;22(2):272-5. Pradeep K, Ko KC, Choi MH, Kang JA, Chung YJ, Park SH. Protective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced tissue damage in Sprague-Dawley rats. J Med Food. 2012 May;15(5):419-27. Koc G, Kuskonmaz SM, Demirel K, Koca G, Akbulut A, Yumusak N, Senes M, Kirtil G, Korkmaz M, Culha C. Ameliorating effects of N-acetyl cysteine against early liver damage of radioiodine in rats. Nucl Med Commun. 2021 Nov 1;42(11):1195-1201. Jomova K, Alomar SY, Alwasel SH, Nepovimova E, Kuca K, Valko M. Several lines of antioxidant defense against oxidative stress: antioxidant enzymes, nanomaterials with multiple enzyme-mimicking activities, and low-molecular-weight antioxidants. Arch Toxicol. 2024 May;98(5):1323-1367. Ibáñez B, Melero A, Montoro A, San Onofre N, Soriano JM. Molecular Insights into Radiation Effects and Protective Mechanisms: A Focus on Cellular Damage and Radioprotectors. Curr Issues Mol Biol. 2024 Nov 9;46(11):12718-12732. Moyer A, Tanaka K, Cheng EH. Apoptosis in Cancer Biology and Therapy. Annu Rev Pathol. 2025 Jan;20(1):303-328. Choudhary GS, Al-Harbi S, Almasan A. Caspase-3 activation is a critical determinant of genotoxicstress-inducedapoptosis. Methods Mol Biol 2015; 1219: 1-9. Mustafa M, Ahmad R, Tantry IQ, Ahmad W, Siddiqui S, Alam M, Abbas K, Moinuddin, Hassan MI, Habib S, Islam S. Apoptosis: A Comprehensive Overview of Signaling Pathways, Morphological Changes, and Physiological Significance and Therapeutic Implications. Cells. 2024 Nov 6;13(22):1838. Russo E, Guerra A, Marotta V, Faggiano A, Colao A, Del Vecchio S, Tonacchera M, Vitale M. Radioiodide induces apoptosis in human thyroid tissue in culture. Endocrine. 2013 Dec;44(3):729-34. Şahin S, Gürgen SG, Yazar U, İnce İ, Kamaşak T, Acar Arslan E, Diler Durgut B, Dilber B, Cansu A. Vitamin D protects against hippocampal apoptosis related with seizures induced by kainic acid and pentylenetetrazol in rats. Epilepsy Res. 2019 Jan;149:107-116. Malloy PJ, Feldman D. Inactivation of the human vitamin D receptor by caspase-3. Endocrinology. 2009 Feb;150(2):679-86. Fenercioglu AK. The Anti-Inflammatory Roles of Vitamin D for Improving Human Health. Curr Issues Mol Biol. 2024 Nov 26;46(12):13514-13525. Udomsinprasert W, Jittikoon J. Vitamin D and liver fibrosis: Molecular mechanisms and clinical studies. Biomed Pharmacother 2019;109:1351-60. Barchetta I, Cimini FA, Cavallo MG. Vitamin D and Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD): An Update Nutr 2020; 12(11): 3302 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Jul, 2025 Reviews received at journal 04 Jul, 2025 Reviews received at journal 27 Jun, 2025 Reviews received at journal 17 Jun, 2025 Reviewers agreed at journal 07 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers agreed at journal 01 Jun, 2025 Reviewers invited by journal 01 Jun, 2025 Editor assigned by journal 20 May, 2025 Submission checks completed at journal 20 May, 2025 First submitted to journal 19 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6700252","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465114271,"identity":"d6c102bc-3eb7-4cb8-a8ca-1b0eb24ea8da","order_by":0,"name":"Muzaffer ATLI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFAC5oYDDDxsPPzsDUCOgQUROtgYgVpk+OQkew6AtEgQp4WBwUbO2GBGAohLhBb++Y2Nh27kmCVukHx+dcOPAgkG/vbuBLxaJI4xNhzOOZOWuF06p+xmD9BhEmfObsBvDUhLbs+xxJ2zc9Ju8AC1GEjk4tciD9by73/ihptn0m7+IUaLAdhhPGzGBjfYj90myhbDY4lgLcBAzmG7LWMgwUPQL3KHDx/+nAOOyuPPbr75YyPH395LwPsIwGMAJolVDgLsD0hRPQpGwSgYBSMIAACI4Ut69F3q9gAAAABJRU5ErkJggg==","orcid":"","institution":"Ankara Güven Hospital","correspondingAuthor":true,"prefix":"","firstName":"Muzaffer","middleName":"","lastName":"ATLI","suffix":""},{"id":465114274,"identity":"67ade0c7-bdab-4580-96ec-4d5213c96468","order_by":1,"name":"Aylin AKBULUT","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Aylin","middleName":"","lastName":"AKBULUT","suffix":""},{"id":465114275,"identity":"c15103b8-fe91-4675-930c-3804ddf94fe6","order_by":2,"name":"Serdar KURU","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Serdar","middleName":"","lastName":"KURU","suffix":""},{"id":465114276,"identity":"01211c93-4766-472a-8ed8-ea2f8917a097","order_by":3,"name":"Nadide KOCA","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nadide","middleName":"","lastName":"KOCA","suffix":""},{"id":465114277,"identity":"f8bcaa70-a3bf-4c2c-9a7d-7dbbd28fbfdf","order_by":4,"name":"Koray DEMİREL","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Koray","middleName":"","lastName":"DEMİREL","suffix":""},{"id":465114278,"identity":"a578a2c5-0d6d-47e7-a447-d8854a8587e6","order_by":5,"name":"Gökhan KOCA","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gökhan","middleName":"","lastName":"KOCA","suffix":""},{"id":465114279,"identity":"a4353aaf-2bda-4371-8916-5ccff53c96f7","order_by":6,"name":"Mehmet ŞENEŞ","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"ŞENEŞ","suffix":""},{"id":465114280,"identity":"915be911-94a1-4fd9-8698-f9953f014194","order_by":7,"name":"Nihat YUMUŞAK","email":"","orcid":"","institution":"University of Harran","correspondingAuthor":false,"prefix":"","firstName":"Nihat","middleName":"","lastName":"YUMUŞAK","suffix":""},{"id":465114281,"identity":"0aeb630c-8a88-4248-89a0-e04364a4d5f7","order_by":8,"name":"Meliha KORKMAZ","email":"","orcid":"","institution":"University of Health Sciences, Ankara Training and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Meliha","middleName":"","lastName":"KORKMAZ","suffix":""}],"badges":[],"createdAt":"2025-05-19 15:08:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6700252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6700252/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83855776,"identity":"c8d688bb-2b07-4dc4-8554-e8b789f06967","added_by":"auto","created_at":"2025-06-03 17:29:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":557414,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs showing for Hematoxylin and Eosin (H\u0026amp;E) stained cross-sections of rat livers (-A; showing normal architecture with normal hepatocytes -B; arrow: vacualization, arrowhead: double nuclei, star: perivascular and perilobulary inflammation -C; star: perivascular inflammation) and Immunohistochemical staining of Caspase-3 (-D; arrows: negative expression of Caspase 3, -E; arrows: positive apoptotic cells, -F; arrows: positive apoptotic cells) and TUNEL staining (-G; negative expression of tunel, -H; strong nuclear tunel-positive cells, -I; weak nuclear tunel-positive cells; positive early stage of apoptosis). Micrometric scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"Figure1PathologyVitDRAI.png","url":"https://assets-eu.researchsquare.com/files/rs-6700252/v1/56e29a50840eb9e7d66ff6df.png"},{"id":83855083,"identity":"eb8a04ea-6f8f-4efb-b078-a521448e3559","added_by":"auto","created_at":"2025-06-03 17:21:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288102,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Biochemical Parameters Between Groups\u003c/p\u003e","description":"","filename":"Figure2ComparisonofBiochemicalParameters.png","url":"https://assets-eu.researchsquare.com/files/rs-6700252/v1/4f03bc109dc2b1afdb76fb4a.png"},{"id":83854790,"identity":"04bffca9-e275-45ee-a7a3-18fb9fac03ef","added_by":"auto","created_at":"2025-06-03 17:13:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245326,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Histopathological Parameters Between Groups\u003c/p\u003e","description":"","filename":"Figure3ComparisonofHistopathologicalParameters.png","url":"https://assets-eu.researchsquare.com/files/rs-6700252/v1/f4252cd32d4d9e551b88aa8e.png"},{"id":83855084,"identity":"a54244ef-9333-471b-b7c9-486558f5977e","added_by":"auto","created_at":"2025-06-03 17:21:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135001,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Immunohistochemical Parameters Between Groups\u003c/p\u003e","description":"","filename":"Figure4ComparisonofImmunohistochemicalParameters.png","url":"https://assets-eu.researchsquare.com/files/rs-6700252/v1/4e0b8bb419165329a74a7c12.png"},{"id":83855988,"identity":"3cb3a42f-a704-4be9-b670-4614e0a2c776","added_by":"auto","created_at":"2025-06-03 17:37:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2027816,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6700252/v1/f36ced78-f76e-453c-83e3-34480fb4e63e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Role of Vitamin D in Attenuating RAI-Induced Liver Injury in Rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRadioactive iodine (Radioiodine: RAI: Iodine-131: I131) treats hyperactive nodules, Graves' disease, and differentiated thyroid carcinoma ablation, preventing relapses and treating metastases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The oral RAI dose not only affects the thyroid but can also affect other tissues (lower gastrointestinal tract, heart wall, stomach, kidneys, and liver). Side effects are a critical dose-limiting factor. Consequently, repeated RAI doses may be necessary, and the dose administered after each treatment should be increased, which can lead to further risks of side effects [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The hepatoprotective effects of various agents [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and therapies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] on liver injury, fibrosis, radiation toxicity, cellular damage, and radiation-induced DNA damage have been investigated [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, there are few studies concerning liver damage following RAI therapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCommon adverse effects of RAI therapy include nausea, vomiting, gastritis, radiation thyroiditis, bone marrow suppression, sialadenitis, hypothermia, xerostomia, ovarian failure, nasolacrimal duct obstruction, dry eyes, leukemia, secondary cancers, and radiation-induced pulmonary fibrosis. In rare instances, liver and kidney dysfunctions may also develop [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Liver function following RAI therapy has been assessed in a small group of individuals diagnosed with differentiated thyroid cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. After RAI therapy, the liver accumulates RAI. Repeated high-dose RAI applications may lead to further RAI accumulation, resulting more in liver damage. Histopathological examination shows that radiation causes inflammation, necrosis, and fibrosis due to microvascular involvement. Damage may be reversible at low radiation doses, but at high doses, significant degeneration of the interstitium with simultaneous inflammation and fibrosis is common [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcitriol, known as the biologically active variant of Vitamin D, impacts the immune system by interacting with the Vitamin D receptor (VDR), which is found in antigen-presenting cells like dendritic cells, monocytes, and macrophages. Vitamin D enhances the innate immune system by maintaining a balance between inflammatory and anti-inflammatory mechanisms, while it dampens the adaptive immune response, playing a role in the formation of an active immune reaction, and thus depends heavily on the hormonal roles of Vitamin D [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Vitamin D is essential in managing various inflammatory processes, including gene regulation, activation of signaling pathways, and the production of inflammatory mediators. Its receptors are found in mast cells, macrophages, natural killer (NK) cells, and T and B lymphocytes. Consequently, Vitamin D decreases inflammatory cytokines like IFN-γ, TNF-α, IL-1, and IL-2, while boosting the levels of IL-10, a cytokine with anti-inflammatory properties [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVitamin D contributes to cellular homeostasis by facilitating apoptosis and orchestrating cell cycle progression. Its immunoregulatory role involves balancing pro- and anti-inflammatory signals, which supports the functional development of immune cells and maintains immune system equilibrium [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This experimental study explores the possible liver-protective benefits of Vitamin D during RAI therapy administered at elevated doses. Unlike prior research, it presents the first immunohistochemical analysis using apoptosis markers to evaluate Vitamin D's protective role against RAI-induced liver injury. As a pioneering in vivo study, this research suggests that Vitamin D may mitigate liver damage caused by RAI.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eAll experimental procedures involving animals were in compliance with the European Community Council Directive of 24 November 1986, and ethical approval was granted by the Ethics Committee for Animal Research at Ankara Training and Research Hospital (Approval No: 0086-819; Date: November 1, 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this experimental study, all of the animals were treated humanly and in accordance with standard guidelines. At least seven days in advance of the beginning of the study, the rats were acclimated to the laboratory environment. Next, they were transferred to polypropylene cages under laboratory standard conditions that were maintained at 21±2 °C, using a light-dark cycle regimen of 12 hours and humidity ranging from 65–70%. During the entire experimental duration, the animals had continuous access to a standard diet and drinking water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were conducted at the Hüsnü Sakal Experimental and Clinical Research Center under the Ankara Training and Research Hospital. Thirty male Wistar albino rats (weighing 200–250 g and aged 3–5 months) were randomly divided into three groups. The control group (n=10) received no treatment, while the RAI group (n=10) was administered 111 MBq/kg of radioactive iodine (MON-IYOT-131; Eczacıbaşı/Monrol, Gebze, Turkey) via oral gavage. The Vitamin D group received active vitamin D in saline suspension [calcitriol, 1,25(OH)2 D3] via intraperitoneal injections at 200 ng/kg daily (Pharmada Ilac et al., Istanbul, Turkey). Vitamin D supplementation began one day before the administration of radioactive iodine (111 MBq/kg) and was maintained throughout the experimental timeline. On the seventh day following RAI treatment, all animals were anesthetized with an intraperitoneal injection of propofol at a dose of 50 mg/kg (Abbott Laboratories, Istanbul, Turkey). Prior to sacrifice, blood and serum samples were collected from all rats and stored at -30 °C for less than 3 Months while awaiting enzymatic activity measurements. Liver tissues obtained after sacrifice were used for biochemical, histopathological, and immunohistochemical evaluations. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Procedures \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeasurements of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were performed using a fully automated biochemical analyzer (Roche Cobas 8000, Roche Diagnostics, USA). Oxidative stress parameters, including fluorescent oxidation products (FOP), malondialdehyde (MDA), catalase (CAT) activity, and total sulfhydryl (T-SH) groups, were assessed in liver tissue samples. Liver samples were first weighed and then homogenized using an automated homogenizer (Heidolph DIAX 900, Germany) at a 1:10 (w/v) ratio with ice-cold phosphate-buffered saline (PBS; 50 mM, pH 7.4) serving as the diluent. The homogenization process was carried out under chilled conditions to preserve biochemical stability.\u003c/p\u003e\n\u003cp\u003eMalondialdehyde (MDA) levels were measured using a spectrofluorometric method outlined by Wasowicz et al. [18]. This approach is based on the interaction between MDA and thiobarbituric acid (TBA), resulting in the formation of a fluorescent compound that is subsequently extracted into n-butanol and measured with a fluorometer at excitation and emission wavelengths of 525 nm and 547 nm, respectively. A standard curve was generated using 1,1,3,3-tetraethoxypropane at concentrations between 0 and 5 μmol/L. For the analysis, 50 μL of tissue homogenate was combined with 1 mL of distilled water in 10 mL glass tubes. Subsequently, 1 mL of a 29 mmol/L TBA solution in acetic acid was added, and the mixture was thoroughly vortexed. The tubes were incubated in a water bath at 95–100 °C for 1 hour to facilitate the reaction. After incubation, the samples were left to cool down to room temperature, and 25 μL of 5 mol/L hydrochloric acid (HCl) was introduced. The reaction mixture was then extracted with 3.5 mL of n-butanol by vigorous vortexing for 5 minutes. After centrifuging at 1500 ×g for 10 minutes, the fluorescence intensity of the butanol layer was recorded, and MDA levels were expressed in micromoles per gram of wet tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe total sulfhydryl (T-SH) content was determined using a spectrophotometric method based on the procedure outlined by Sedlak and Lindsay [19]. In brief, 250 μL of supernatant from the tissue homogenate was combined with 750 μL of 0.2 M Tris buffer (pH 8.2) and 50 μL of 0.01 M 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). To achieve a final volume of 5 mL, 3950 μL of absolute methanol was incorporated. Alongside the test samples, a reagent blank (lacking homogenate) and a sample blank (omitting DTNB) were prepared using the same procedure. Every tube was sealed with a rubber stopper, kept at ambient temperature for 15 minutes, and then subjected to centrifugation at around 3000×g for the same duration. The absorbance of the supernatant was measured at 412 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFOP levels were measured spectrofluorometrically at 360/430 nm after extraction of the homogenates with an ethanol-ether (3:1, v/v) mixture [20].\u003c/p\u003e\n\u003cp\u003eCAT enzyme activity was assessed using a simple spectrophotometric method as described by Hadwan [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor histopathological analysis, tissues were preserved in 10% neutral buffered formalin and then processed using standard paraffin embedding methods. Paraffin-embedded tissue blocks were sectioned into 4 µm slices and subsequently stained using Hematoxylin and Eosin (H\u0026amp;E). Microscopic evaluation and imaging were performed using an Olympus BX-53 microscope (Olympus, Tokyo, Japan) equipped with Olympus Cell B software. The histopathological changes, such as hyperemia, inflammation, fibrosis, vacuolization, multinucleated cells, and granuloma formation, were assessed using a semi-quantitative scoring system (Figure 1 – A, B, C). Scores ranged from 0 (no findings) to 3 (severe).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis was performed in accordance with the protocol outlined by Yumuşak et al. [22], with minor modifications. Tissue sections approximately 4 µm in thickness were obtained from paraffin-embedded samples and placed onto poly-L-lysine-coated slides. After undergoing deparaffinization and rehydration through standard procedures, a streptavidin-biotin complex (ABC) detection system was applied using the Histostain Plus Kit (Zymed, South San Francisco, CA, USA). For immunolabeling, a Caspase-3-specific primary antibody (diluted 1:200; catalog no. PA5-16335, Invitrogen, CA, USA) was used. Chromogenic visualization was achieved with diaminobenzidine (DAB; Dako), which was applied for 10 minutes. Phosphate-buffered saline (PBS) was used for all rinsing steps throughout the staining process. Cytoplasmic immunoreactivity was semi-quantitatively scored on a four-point scale: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong brown staining). All staining steps were carried out in a humid chamber at 37°C (Figure 1 – D, E, F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was utilized to identify apoptotic DNA fragmentation in liver, kidney, and testicular tissues. This analysis was carried out using a commercially available cell death detection kit (In Situ Cell Death Detection Kit, POD; Roche, Mannheim, Germany). The procedure was adapted from the methodology previously described by Yumuşak et al. [22]. The extent of staining was graded based on the proportion of nuclei exhibiting positive labeling: 0 (none, \u0026lt;1%), 1 (mild, 1–25%), 2 (moderate, \u0026gt;25–75%), and 3 (strong, \u0026gt;75%) (Figure 1 – G, H, I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using IBM SPSS Statistics (v27.0; IBM Corporation, Armonk, NY, USA) and MedCalc (v15.8; MedCalc Software, Ostend, Belgium). Descriptive measures, including percentages, means, standard deviations, frequencies, medians, and interquartile ranges (IQRs), were calculated to summarize the dataset. The assumption of normality was evaluated using the Shapiro-Wilk test, along with skewness and kurtosis values, and supported by visual diagnostic tools such as Q-Q plots, histograms, boxplots, and stem-and-leaf graphs. When data conformed to a normal distribution, intergroup comparisons were made using analysis of variance (ANOVA), and Tukey's HSD test was employed for post hoc analyses. For data that did not exhibit normal distribution, the Kruskal-Wallis non-parametric test was applied, followed by Bonferroni-adjusted pairwise comparisons where appropriate. A threshold of p \u0026lt; 0.05 was used to determine statistical significance.\u003c/p\u003e\n\u003cp\u003eFurthermore, a retrospective power calculation was conducted via G*Power software (v3.1.9.7; Franz Faul, University of Kiel, Germany), focusing on malondialdehyde (MDA) values. The sample sizes were n₁ = 10 (100.09 ± 19.24), n₂ = 10 (211.71 ± 52.07), and n₃ = 10 (146.06 ± 45.46), with a pooled standard deviation of 61.38. The analysis was conducted with a significance level of α = 0.05 and an effect size (f) of 0.75, yielding a statistical power of 94%.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe average measurements for liver function markers and oxidative stress indicators are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The biochemical analysis revealed statistically meaningful differences among the control, RAI, and vitamin D groups across all measured variables (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To pinpoint which groups contributed to these differences, further pairwise comparisons were conducted using Tukey\u0026rsquo;s Honestly Significant Difference (HSD) method:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Biochemical Parameters Between Groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup I\u003c/p\u003e \u003cp\u003e(Control)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup II\u003c/p\u003e \u003cp\u003e(RAI)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup III\u003c/p\u003e \u003cp\u003e(RAI\u0026thinsp;+\u0026thinsp;VitD)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDifference \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA (nmol/g protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100,09\u0026thinsp;\u0026plusmn;\u0026thinsp;19,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e211,71\u0026thinsp;\u0026plusmn;\u0026thinsp;52,07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e146,06\u0026thinsp;\u0026plusmn;\u0026thinsp;45,46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFOP (FO/g protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e99,64\u0026thinsp;\u0026plusmn;\u0026thinsp;17,41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e181,40\u0026thinsp;\u0026plusmn;\u0026thinsp;32,43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e145,42\u0026thinsp;\u0026plusmn;\u0026thinsp;27,22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-SH (umol/g protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e77,18\u0026thinsp;\u0026plusmn;\u0026thinsp;14,23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e50,48\u0026thinsp;\u0026plusmn;\u0026thinsp;11,14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e62,61\u0026thinsp;\u0026plusmn;\u0026thinsp;12,10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 to 2\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalaz (u/g protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e74,61\u0026thinsp;\u0026plusmn;\u0026thinsp;7,37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e43,26\u0026thinsp;\u0026plusmn;\u0026thinsp;10,85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e58,69\u0026thinsp;\u0026plusmn;\u0026thinsp;7,76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86,80\u0026thinsp;\u0026plusmn;\u0026thinsp;19,74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e121,60\u0026thinsp;\u0026plusmn;\u0026thinsp;16,08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e95,30\u0026thinsp;\u0026plusmn;\u0026thinsp;10,22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e46,30\u0026thinsp;\u0026plusmn;\u0026thinsp;9,63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e64,90\u0026thinsp;\u0026plusmn;\u0026thinsp;17,43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e49,10\u0026thinsp;\u0026plusmn;\u0026thinsp;5,93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0,004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ea: One-Way Anova Test (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), b: Post-Hoc Tukey HSD Test,\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eFor MDA, FOP, and Catalase variables, all groups differed from each other,\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFor AST and ALT variables, Group II values differed from other groups,\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFor the T-SH variable, Group I values differed from other groups.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAll assessed parameters, including MDA, FOP, T-SH, catalase, AST, and ALT, displayed statistically significant differences among the experimental groups. Specifically, the animals treated with RAI showed the highest levels of MDA, FOP, AST, and ALT, while the control rats recorded the lowest values. On the other hand, T-SH and catalase levels were highest in the control group, suggesting stronger antioxidant activity. When examining oxidative stress indicators, both the control and vitamin D groups demonstrated substantially reduced levels of MDA and FOP relative to the RAI-treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, antioxidant markers T-SH and catalase were observed at significantly higher concentrations in the control cohort than in the others (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In terms of hepatic biochemical markers, AST and ALT levels were moderately increased in the vitamin D group relative to the control, yet remained considerably below the elevated levels observed in the RAI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.004, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotable differences were detected between the groups across all histopathological parameters, with statistical results showing p-values less than 0.05. Post hoc analyses indicated that all groups differed significantly in the multiple nucleus parameter (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Group II showed distinct differences from the other groups in terms of hyperemia, inflammation, fibrosis, vacuolization, and granuloma formation. In general, the RAI group exhibited substantially elevated histopathological scores relative to both the control and vitamin D groups, with these differences reaching high statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Histopathological Parameters Between Groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup I\u003c/p\u003e \u003cp\u003e(Control)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup II\u003c/p\u003e \u003cp\u003e(RAI)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup III\u003c/p\u003e \u003cp\u003e(RAI\u0026thinsp;+\u0026thinsp;Vitamin D)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDifference \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e9 (%90,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e7 (%70,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e1 (%10,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e3 (%30,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e7 (%70,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e3 (%30,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflammation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,10\u0026thinsp;\u0026plusmn;\u0026thinsp;0,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,20\u0026thinsp;\u0026plusmn;\u0026thinsp;0,42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,40\u0026thinsp;\u0026plusmn;\u0026thinsp;0,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e9 (%90,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e6 (%60,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e1 (%10,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e4 (%40,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e8 (%80,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e2 (%20,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,20\u0026thinsp;\u0026plusmn;\u0026thinsp;0,42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,20\u0026thinsp;\u0026plusmn;\u0026thinsp;0,42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10 (%100,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e8 (%80,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e2 (%20,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e8 (%80,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e2 (%20,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVacuolization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,20\u0026thinsp;\u0026plusmn;\u0026thinsp;0,42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,60\u0026thinsp;\u0026plusmn;\u0026thinsp;0,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,40\u0026thinsp;\u0026plusmn;\u0026thinsp;0,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e8 (%80,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e6 (%60,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e2 (%20,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e4 (%40,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e4 (%40,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e6 (%60,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple Nucleus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,60\u0026thinsp;\u0026plusmn;\u0026thinsp;0,52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10 (%100,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e4 (%40,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e6 (%60,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e10 (%100,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGranuloma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,30\u0026thinsp;\u0026plusmn;\u0026thinsp;0,48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0,001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10 (%100,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e7 (%70,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e3 (%30,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e10 (%100,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0,0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea: Kruskal-WallisTest (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), b: Post-Hoc Bonferroni Correction, *: n (%),\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical evaluation using the TUNEL method indicated notable group-dependent variation, with results demonstrating a high level of statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, caspase-3 immunoreactivity was markedly elevated in the RAI group relative to both the control and vitamin D groups, and this distinction was also found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Immunohistochemical Parameters Between Groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup I\u003c/p\u003e \u003cp\u003e(Control)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup II\u003c/p\u003e \u003cp\u003e(RAI)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup III\u003c/p\u003e \u003cp\u003e(RAI\u0026thinsp;+\u0026thinsp;Vitamin D)\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDifference \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 to 1\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e10 (%100.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e6 (%60.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e4 (%40.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e4 (%40.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e6 (%60.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTUNEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e0*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e8 (%80.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e3 (%30.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e1*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e1 (%10.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e3 (%30.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e2*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e1 (%10.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e4 (%40.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e3*\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e10 (%100.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e0 (%0.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea: Kruskal-WallisTest (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), b: Post-Hoc Bonferroni Correction, *: n (%),\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eRadioactive iodine, a radionuclide emitting beta and gamma rays, has been a primary treatment for thyroid cancer and hyperthyroidism since the 1940s [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. I-131 accumulation in non-thyroid tissues during RAI treatment can cause adverse effects. Limited research exists on I-131\u0026rsquo;s toxic and hepatotoxic effects and no universally accepted treatment prevents 131-I tissue damage [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This study investigates the potential protective role of vitamin D against liver damage induced by radioactive iodine (RAI), using a range of biochemical, histopathological, and immunohistochemical techniques. Our study reveals that RAI treatment causes hepatocellular damage by triggering oxidative stress in the rat liver and that vitamin D shows a protective effect against these harmful processes. RAI administration led to elevated levels of lipid peroxidation markers, MDA and FOP, while reducing the activity of the antioxidant defense system, including T-SH and CAT. Additionally, the significant rise in AST and ALT enzyme levels confirms that RAI causes liver cell damage by disrupting hepatocellular integrity. Histopathological examinations also identified damage findings such as hyperemia, inflammation, fibrosis, and vacuolization. Immunohistochemical analyses confirm that RAI activates apoptosis mechanisms (Caspase-3 expression and TUNEL positive cells). In the vitamin D group, protective effects were observed at various levels, including a reduction in oxidant levels, an increase in antioxidant levels, normalization of liver enzyme levels, alleviation of histopathological damage, and suppression of apoptotic activation. These results experimentally support the idea that vitamin D provides a hepatoprotective effect against RAI-induced liver damage.\u003c/p\u003e \u003cp\u003eIodine is transported within organs or mass lesions via the sodium iodide symporter. The liver itself does not have a sodium iodide symporter. However, hepatic uptake after RAI is a physiological condition resulting from intrahepatic bile ducts containing the sodium iodide symporter. Another reason for hepatic radioactive iodine uptake could be the binding of iodine to thyroxine due to hormonal production in the thyroid glands [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hepatic radioactive iodine is positively correlated with the administered I131 dose. As the liver concentrates RAI, serious liver damage can occur due to more RAI accumulation with multiple and increasing high doses. Radiation causes hyperemia, inflammation, necrosis, and fibrosis in the liver, and at lower radiation doses, reversible microvascular hyperemia occurs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Factors like age, gender, nutrition, disease duration, and goiter size may elevate RAI-induced hepatotoxicity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In a study showing that RAI causes late histopathological damage in rat livers three months after I131 administration, histopathological changes such as hyperemia, steatosis, fibrosis, and capsule thickening were demonstrated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Hyperemia after RAI-131 administration indicates an increase in blood flow and an inflammatory response. Hyperemia helps transport more oxygen and nutrients to damaged tissues. On the other hand, when the metabolic functions of the liver are affected, especially disruptions in fat metabolism can be observed. Steatosis can be considered an indicator of disruptions in fat metabolism and cellular damage in the liver. Again, damage in the liver triggers a fibrogenic response, leading to increased collagen production and, thus, fibrosis, which is the hardening of the liver. Our histopathological findings also confirm the liver toxicity of RAI in a manner similar to this study. In the analysis of histopathological data in our study, it was found that the RAI group differed from the other groups in the variables of Hyperemia, Inflammation, Fibrosis, Vacuolization, and Granuloma (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Vacuolization, defined as the formation of fluid-filled spaces within the cell, is an adaptive response for cells to store or detoxify harmful substances and wastes. A granuloma is a small lesion formed by the accumulation of macrophages as part of an inflammatory reaction. These histopathological changes observed after RAI-131 administration can be considered indicators of toxic effects and inflammatory responses in the liver. Comparative analysis revealed that the RAI group demonstrated significantly elevated histopathological parameter scores in relation to both the control and vitamin D groups. The reduction of histopathological changes by vitamin D demonstrates its ability to generally protect cellular health, reduce oxidative stress, and control inflammatory responses.\u003c/p\u003e \u003cp\u003eDiffuse iodine-131 uptake in the liver has often been documented in scans following RAI treatment in the literature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. There are also studies suggesting that liver enzyme elevations in thyroid disease may paradoxically be associated with RAI. The elevation of serum transaminases such as AST and ALT in cases of liver toxicity developing after RAI treatment is an indicator of hepatocyte damage that leads to increased cell membrane permeability, facilitating the leakage of transaminases into the bloodstream [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study found that the values of AST and ALT from liver function tests in the RAI group differed from those in the other two groups. Serum liver enzyme concentrations in the vitamin D group were higher than those in the control group; however, the RAI group exhibited the greatest elevations, with significant intergroup differences observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eTo reduce the effects against liver tissue damage caused by high-dose RAI, the protective effects of different agents such as melatonin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], dexmedetomidine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and N-acetyl cysteine [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] have been demonstrated in the early period. In all three studies, biochemical oxidative stress parameters were evaluated along with histopathological findings. The increase in oxidative stress parameters after RAI-131 treatment is due to a series of factors, such as the direct effects of ionizing radiation, cell damage, weakened antioxidant defense mechanisms, inflammation, and metabolic changes. Similar to the results obtained from these studies, histopathological and biochemical findings parallel our study. Our study's analysis of oxidative stress parameters in liver tissue showed statistically significant differences among the control, RAI, and VitD groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among the groups, oxidant levels were greatest in the RAI group and lowest in the control group. Conversely, antioxidant concentrations reached their highest in the control group.\u003c/p\u003e \u003cp\u003eWhen biological systems are exposed to ionizing radiation, it triggers the production of reactive nitrogen species (RNS) and reactive oxygen species (ROS). These highly active molecules can harm critical macromolecules like DNA, RNA, proteins, and cellular membranes, leading to impaired cell function or even programmed cell death (apoptosis). While ROS are crucial for normal cellular processes, their excessive production can induce cell death through either necrosis or apoptosis. The condition of oxidative stress occurs when there is an imbalance between pro-oxidants and antioxidants, leading to damage such as lipid peroxidation, protein oxidation, and DNA alteration. Oxidative stress is a key contributor to the development of various human conditions, including neurodegenerative diseases, cancer, aging, and chronic inflammatory diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is known that ionizing radiation causes damage to cells through free radicals and direct ionization. It is thought that support with antioxidant substances can increase treatment gain by enhancing the tumor response to radiation and reducing its toxic effect on normal cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Only a small number of studies have been carried out to evaluate the effect of protective agents against high-dose internal radiation damage in the liver biochemically with oxidative stress parameters [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApoptosis is the process by which the organism kills unwanted cells during homeostasis, development, embryogenesis, immune regulation, or disease [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Morphological, biochemical, and molecular changes can measure this process. During the early and late stages of apoptosis, cysteine proteases called caspases are activated; in particular, caspase-3 mediates both necrotic and apoptotic cell death. The TUNEL assay detects DNA fragmentation occurring in the final stages of apoptosis. In aged rats, increased liver damage caused by increased apoptosis of caspase-3 and TUNEL has been found [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In our study, we evaluated apoptosis for the first time in the literature regarding the protective effect of vitamin D against the injury caused by 131I in the liver. Our immunohistochemical analysis results showed that RAI significantly activates the apoptosis mechanism in the liver. The notable rise in TUNEL-positive cells and the substantial increase in Caspase-3 expression, a critical effector protein in apoptosis, suggest that RAI induces liver cell death, thereby contributing to tissue damage. The results of this study showed that radiations induced apoptotic pathways (TUNEL- positive cells and increased caspase-3 activity in the RAI group). The significant reduction of these apoptotic markers by vitamin D suggests that it suppresses apoptotic signals through mechanisms such as modulating the Bcl-2/Bax balance or stabilizing calcium homeostasis. This discovery aligns with earlier research emphasizing the dual function of vitamin D, offering anti-apoptotic protection in normal tissues while exhibiting a pro-apoptotic effect in cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Significant differences were identified in the TUNEL evaluation results among the RAI, Control, and Vitamin D groups during the statistical comparison (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the analysis of Caspase-3 findings, it was found that the values in the RAI group differed from those in the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe hepatoprotective effects of vitamin D are primarily attributed to its abilities to modulate inflammation and counteract oxidative damage. It reduces oxidative stress by neutralizing free radicals directly or by stimulating the production of endogenous antioxidants, including Superoxide Dismutase and Glutathione Peroxidase [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our findings demonstrate that vitamin D is essential in lowering oxidative stress markers such as MDA and FOP, while simultaneously boosting the activity of antioxidant enzymes like Catalase (CAT) and Total Sulfhydryl (T-SH), highlighting its antioxidant properties. Moreover, vitamin D modulates inflammatory responses by lowering pro-inflammatory cytokines like TNF-α and IL-6, while promoting the production of anti-inflammatory cytokines such as IL-10 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Histopathological assessments further support these results, showing a reduction in inflammation scores and reinforcing the anti-inflammatory potential of vitamin D. Vitamin D is considered to contribute to liver protection by modulating signaling pathways within hepatic cells particularly non-parenchymal cells through its interaction with the vitamin D receptor (VDR). This receptor, located in various cell types, enables vitamin D to regulate gene transcription. Through this mechanism, vitamin D helps regulate inflammatory activity, supports the synthesis of anti-inflammatory agents, and participates in the regulation of cell death and survival processes. Existing literature suggests that activation of VDR can inhibit liver fibrosis, regulate apoptosis, and promote cell survival [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our findings showed decreased levels of fibrosis and apoptotic indicators (TUNEL, Caspase-3), implying a potential role for VDR-dependent mechanisms in the liver-protective effects of vitamin D. Further research is needed to clarify how vitamin D influences intracellular signaling pathways in hepatic cells and to better understand the specific molecular functions of the VDR.\u003c/p\u003e \u003cp\u003eIn summary, the results of this study indicate that vitamin D may help mitigate liver damage associated with RAI exposure. Its protective influence appears to stem from its anti-inflammatory, antioxidant, and anti-apoptotic functions. These findings may hold particular relevance for individuals with preexisting liver issues or patients receiving RAI-based therapies. In particular, starting vitamin D supplementation before or during RAI treatment in patients undergoing RAI treatment due to thyroid cancer or hyperthyroidism who have concurrent liver disease or are at risk of liver damage may help reduce liver damage and increase treatment tolerance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRAI: I131, Iodine-131, Radioactive iodine, Radioiodine; FOP: Fluorescent oxidation products; MDA: Malondialdehyde; CAT: Catalase; T-SH: Total sulfhydryl; VDR: Vitamin D receptor; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBA: thiobarbituric acid; DTNB: 5,5\u0026prime;-dithiobis-(2-nitrobenzoic acid); H\u0026amp;E: Hematoxylin and Eosin; PBS: Phosphate-buffered saline; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ROS: Reactive oxygen species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript. The authors declare that they have no conflicts of interest. They confirm that no financial or non-financial conflicts of interest are relevant to the work conducted or reported in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA statement of financial support;\u0026nbsp;\u003c/strong\u003eThis research was conducted independently by the authors and received no specific grant or financial support from any funding agency, commercial entity, or non-profit organization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments;\u0026nbsp;\u003c/strong\u003eThe authors have no acknowledgments to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLuster M, Pfestroff A, H\u0026auml;nscheid H, Verburg FA. Radioiodine Therapy. Semin Nucl Med. 2017 Mar;47(2):126-134\u003c/li\u003e\n\u003cli\u003ePadma S, Sundaram PS. Radioiodine as an adjuvant therapy and its role in follow-up of differentiated thyroid cancer. J Cancer Res Ther. 2016 Jul-Sep;12(3):1109-1113.\u003c/li\u003e\n\u003cli\u003eZaletel K, Mihovec A, Gaberscek S. Characteristics of exposure to radioactive iodine during a nuclear incident. Radiol Oncol. 2024 Oct 4;58(4):459-468\u003c/li\u003e\n\u003cli\u003eAtilgan HI, Yumusak N, Sadic M, Gultekin SS, Koca G, Ozyurt S, Demirel K, Korkmaz M. Radioprotective effect of montelukast sodium against hepatic radioiodine (I131) toxicity: A histopathological investigation in the rat model. Ankara \u0026Uuml;niv Vet Fak Derg 2015; 62: 37-43.\u003c/li\u003e\n\u003cli\u003eHanedan Uslu G, Canyilmaz E, Serdar L, Ers\u0026ouml;z Ş. Protective effects of genistein and melatonin on mouse liver injury induced by whole-body ionising radiation. Mol Clin Oncol. 2019 Feb;10(2):261-266.\u003c/li\u003e\n\u003cli\u003eSadeeshkumar V, Duraikannu A, Aishwarya T, Jayaram P, Ravichandran S, Ganeshamurthy R. Radioprotective efficacy of dieckol against gamma radiation-induced cellular damage in hepatocyte cells. Naunyn Schmiedebergs Arch Pharmacol. 2019; 392(8):1031-1041.\u003c/li\u003e\n\u003cli\u003eBarlas AM, Sadic M, Atilgan HI, Bag YM, Onalan AK, Yumusak N, Senes M, Fidanci V, Pekcici MR, Korkmaz M, Kismet K, Koca G. Melatonin: a hepatoprotective agent against radioiodine toxicity in rats. Bratisl Lek Listy 2017; 118(2): 95-100. \u003c/li\u003e\n\u003cli\u003eChi CH, Liu IL, Lo WY, Liaw BS, Wang YS, Chi KH. Hepatocyte growth factor gene therapy prevents radiation-induced liver damage. World J Gastroenterol 2005; 11(10): 1496-502.\u003c/li\u003e\n\u003cli\u003eZhou YJ, Tang Y, Liu SJ, Zeng PH, Qu L, Jing QC, Yin WJ. Radiation-induced liver disease: beyond DNA damage. Cell Cycle. 2023 Mar;22(5):506-526.\u003c/li\u003e\n\u003cli\u003eKim J, Jung Y. Radiation-induced liver disease: current understanding and future perspectives. Exp Mol Med. 2017 Jul 21;49(7):e359.\u003c/li\u003e\n\u003cli\u003eKismet K, Sadic M, Bag YM, Atilgan HI, Koca G, Onalan AK, Senes M, Peker SA, Yumusak N, Korkmaz M. Hepatoprotective effect of dexmedetomidine against radioiodine toxicity in rats: evaluation of oxidative status and histopathological changes. Int Surg 2016; 101: 176-84.\u003c/li\u003e\n\u003cli\u003eFard-Esfahani A, Emami-Ardekani A, Fallahi B, Fard-Esfahani P, Beiki D, Hassanzadeh-Rad A, Eftekhari M. Adverse effects of radioactive iodine-131 treatment for differentiated thyroid carcinoma. Nucl Med Commun 2014; 35(8): 808-17.\u003c/li\u003e\n\u003cli\u003eWang S, Liang C, Zhao L, Meng Z, Zhang C, Jia Q, Tan J, Yang H, Liu X, Wang X. Influence of radioactive iodine therapy on liver function in patients with differentiated thyroid cancer. Nucl Med Commun 2018; 39(12): 1113-20.\u003c/li\u003e\n\u003cli\u003eEksioglu U, Atilgan HI, Yakin M, Yazihan N, Altiparmak UE, Yumusak N, Korkmaz M, Demir A, Ornek F, Aribal Ayral P, Koca G. Antioxidant effects of vitamin D on lacrimal glands against high dose radioiodine-associated damage in an animal model. Cutan Ocul Toxicol 2019; 38(1): 18-24. \u003c/li\u003e\n\u003cli\u003eWimalawansa SJ. Physiology of Vitamin D-Focusing on Disease Prevention. Nutrients. 2024 May 29;16(11):1666.\u003c/li\u003e\n\u003cli\u003eToniato E, Spinas E, Saggini A, et al. Immunomodulatory effects of vitamin D on skin inflammation. J Biol Regul Homeost Agents 2015; 29: 563-7\u003c/li\u003e\n\u003cli\u003eMarkotić A, Kelava T, Markotić H, Silovski H, Mrzljak A. Vitamin D in liver cancer: novel insights and future perspectives. Croat Med J. 2022 Apr 30;63(2):187-196.\u003c/li\u003e\n\u003cli\u003eWasowicz W, N\u0026egrave;ve J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clin Chem 1993; 39(12): 2522\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eSedlak J, Lindsay RH. Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Ellman\u0026rsquo;s reagent. Anal Biochem 1968; 25(1): 192\u0026ndash;205.\u003c/li\u003e\n\u003cli\u003eWu T, Willett WC, Rifai N, Rimm EB. Plasma fluorescent oxidation products as potential markers of oxidative stress for epidemiologic studies. Am J Epidemiol. 2007; 166(5): 552\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eHadwan MH. Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC Biochem 2018; 19(1): 7\u003c/li\u003e\n\u003cli\u003eYumusak N, Sadic M, Yucel G, Atilgan HI, Koca G, Korkmaz M. Apoptosis and cell proliferation in short-termandlong-termeffects of radioiodine-131-induced kidney damage: an experimental and immunohistochemical study. Nucl Med Commun 2018; 39(2):131-39.\u003c/li\u003e\n\u003cli\u003eNguyen NC, Anigati EM, Desai NB, \u0026Ouml;z OK. Radioactive Iodine Therapy in Differentiated Thyroid Cancer: An Update on Dose Recommendations and Risk of Secondary Primary Malignancies. Semin Nucl Med. 2024 Jul;54(4):488-496.\u003c/li\u003e\n\u003cli\u003eLee SL. Radioactive iodine therapy. Curr Opin Endocrinol Diabetes Obes 2012; 19(5): 420-8.\u003c/li\u003e\n\u003cli\u003eHong CM, Ahn BC. Factors Associated with Dose Determination of Radioactive Iodine Therapy for Differentiated Thyroid Cancer. Nucl Med Mol Imaging. 2018 Aug;52(4):247-253.\u003c/li\u003e\n\u003cli\u003ePiantanida E, Ippolito S, Gallo D, Masiello E, Premoli P, Cusini C, Rosetti S, Sabatino J, Segato S, Trimarchi F, Bartalena L, Tanda ML. The interplay between thyroid and liver: implications for clinical practice. J Endocrinol Invest. 2020 Jul;43(7):885-899.\u003c/li\u003e\n\u003cli\u003eOm\u0026uuml;r O, Akg\u0026uuml;n A, Ozcan Z, Sen C, OzkiIi\u0026ccedil; H. Clinical implications of diffuse hepatic uptake observed in postablative and post-therapeutic I-131 scans. Clin Nucl Med 2009; 34(1): 11-4.\u003c/li\u003e\n\u003cli\u003eJhummon NP, Tohooloo B, Qu S. Iodine-131 induced hepatotoxicity in previously healthy patients with Grave\u0026apos;s disease. Thyroid Res. 2013 Mar 16;6:4.\u003c/li\u003e\n\u003cli\u003eGuha C, Kavanagh BD. Hepatic radiation toxicity: avoidance and amelioration. Semin Radiat Oncol 2011; 21(4): 256-63.\u003c/li\u003e\n\u003cli\u003eLin R, Banafea O, Ye J. I-131 remnant ablation after thyroidectomy induced hepatotoxicity in a case of thyroid cancer. BMC Gastroenterol. 2015 May 7;15:56.\u003c/li\u003e\n\u003cli\u003eFerris HA, Williams G, Parker JA, Garber JR. Therapeutic implications of diffuse hepatic uptake following I-131 therapy for differentiated thyroid cancer. Endocr Pract. 2013;19(2):263-7.\u003c/li\u003e\n\u003cli\u003eKim CW, Park JS, Oh SH, Park JH, Shim HI, Yoon JW, Park JS, Hong SB, Kim JM, Le TB, Lee JW. Drug-induced liver injury caused by iodine-131. Clin Mol Hepatol. 2016;22(2):272-5. \u003c/li\u003e\n\u003cli\u003ePradeep K, Ko KC, Choi MH, Kang JA, Chung YJ, Park SH. Protective effect of hesperidin, a citrus flavanoglycone, against \u0026gamma;-radiation-induced tissue damage in Sprague-Dawley rats. J Med Food. 2012 May;15(5):419-27.\u003c/li\u003e\n\u003cli\u003eKoc G, Kuskonmaz SM, Demirel K, Koca G, Akbulut A, Yumusak N, Senes M, Kirtil G, Korkmaz M, Culha C. Ameliorating effects of N-acetyl cysteine against early liver damage of radioiodine in rats. Nucl Med Commun. 2021 Nov 1;42(11):1195-1201.\u003c/li\u003e\n\u003cli\u003eJomova K, Alomar SY, Alwasel SH, Nepovimova E, Kuca K, Valko M. Several lines of antioxidant defense against oxidative stress: antioxidant enzymes, nanomaterials with multiple enzyme-mimicking activities, and low-molecular-weight antioxidants. Arch Toxicol. 2024 May;98(5):1323-1367.\u003c/li\u003e\n\u003cli\u003eIb\u0026aacute;\u0026ntilde;ez B, Melero A, Montoro A, San Onofre N, Soriano JM. Molecular Insights into Radiation Effects and Protective Mechanisms: A Focus on Cellular Damage and Radioprotectors. Curr Issues Mol Biol. 2024 Nov 9;46(11):12718-12732.\u003c/li\u003e\n\u003cli\u003eMoyer A, Tanaka K, Cheng EH. Apoptosis in Cancer Biology and Therapy. Annu Rev Pathol. 2025 Jan;20(1):303-328.\u003c/li\u003e\n\u003cli\u003eChoudhary GS, Al-Harbi S, Almasan A. Caspase-3 activation is a critical determinant of genotoxicstress-inducedapoptosis. Methods Mol Biol 2015; 1219: 1-9.\u003c/li\u003e\n\u003cli\u003eMustafa M, Ahmad R, Tantry IQ, Ahmad W, Siddiqui S, Alam M, Abbas K, Moinuddin, Hassan MI, Habib S, Islam S. Apoptosis: A Comprehensive Overview of Signaling Pathways, Morphological Changes, and Physiological Significance and Therapeutic Implications. Cells. 2024 Nov 6;13(22):1838.\u003c/li\u003e\n\u003cli\u003eRusso E, Guerra A, Marotta V, Faggiano A, Colao A, Del Vecchio S, Tonacchera M, Vitale M. Radioiodide induces apoptosis in human thyroid tissue in culture. Endocrine. 2013 Dec;44(3):729-34.\u003c/li\u003e\n\u003cli\u003eŞahin S, G\u0026uuml;rgen SG, Yazar U, İnce İ, Kamaşak T, Acar Arslan E, Diler Durgut B, Dilber B, Cansu A. Vitamin D protects against hippocampal apoptosis related with seizures induced by kainic acid and pentylenetetrazol in rats. Epilepsy Res. 2019 Jan;149:107-116.\u003c/li\u003e\n\u003cli\u003eMalloy PJ, Feldman D. Inactivation of the human vitamin D receptor by caspase-3. Endocrinology. 2009 Feb;150(2):679-86.\u003c/li\u003e\n\u003cli\u003eFenercioglu AK. The Anti-Inflammatory Roles of Vitamin D for Improving Human Health. Curr Issues Mol Biol. 2024 Nov 26;46(12):13514-13525.\u003c/li\u003e\n\u003cli\u003eUdomsinprasert W, Jittikoon J. Vitamin D and liver fibrosis: Molecular mechanisms and clinical studies. Biomed Pharmacother 2019;109:1351-60.\u003c/li\u003e\n\u003cli\u003eBarchetta I, Cimini FA, Cavallo MG. Vitamin D and Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD): An Update Nutr 2020; 12(11): 3302\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Iodine-131, Liver injury, Radioactive iodine, Radioiodine, Radioprotection, Vitamin D","lastPublishedDoi":"10.21203/rs.3.rs-6700252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6700252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eRadioiodine (RAI) therapy is widely used for thyroid ablation, but I-131 accumulation in non-thyroid tissues may cause adverse effects. Vitamin D, known for its anti-apoptotic and immunomodulatory roles, may help protect against such damage. This study investigates the potential protective effects of vitamin D on RAI-induced liver injury through biochemical, histopathological, and immunohistochemical evaluations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eThirty male Wistar albino rats were randomly assigned to three groups: Control (Group I, n=10), RAI-treated (Group II, 111 MBq/kg, n=10), and RAI+Vitamin D (Group III, 200 ng/kg/day, n=10). Liver function was evaluated through serum analysis. Liver tissues were examined histopathologically and immunohistochemically. Oxidative stress markers—malondialdehyde (MDA), fluorescent oxidation products (FOP), catalase (CAT), and total sulfhydryl (T-SH)—were measured in liver homogenates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eRAI increased apoptotic cell numbers and elevated MDA, FOP, AST, and ALT levels. In contrast, T-SH and CAT levels were highest in the control group. Histopathology showed marked liver damage in the RAI group, which was less severe in the vitamin D group (p\u0026lt;0.001). TUNEL and caspase-3 analyses confirmed increased apoptosis in the RAI group compared to the others (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eVitamin D alleviated RAI-induced liver injury, likely through its antioxidant, anti-apoptotic, and anti-inflammatory effects.\u003c/p\u003e","manuscriptTitle":"Protective Role of Vitamin D in Attenuating RAI-Induced Liver Injury in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 17:13:21","doi":"10.21203/rs.3.rs-6700252/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-06T05:30:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-04T21:46:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T05:27:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-17T18:56:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254821740616175499497411013311459218866","date":"2025-06-08T00:07:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54118148519348292684091417563247618584","date":"2025-06-03T16:13:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275305306258516425097838088908227568533","date":"2025-06-02T01:30:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-01T22:53:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-20T09:02:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-20T08:57:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bratislava Medical Journal","date":"2025-05-19T15:00:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e4cc2e8b-2101-4053-bffe-d29aacffdbec","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-09T13:08:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 17:13:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6700252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6700252","identity":"rs-6700252","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}