Study The Effects of 5 Selenocyantouracil Compound on The liver and Kidney of Male Rats After Acetaminophen-Induced Liver Injury

Selenium compounds have long been assumed to protect organs against inflammation and cancer development by reducing oxidative stress and organ injury. Drug-induced liver injury is a common side effect of many medications such as acetaminophen, with main symptoms ranging from an increase in asymptomatic liver enzymes to liver failure. The main objective of the present work is to evaluate the possible protective effect of 5 selenocyantouracil against liver and kidney damage induced by acetaminophen. Fourty male rats were divided into 4 groups : Group A : rats were freely excess to food and water (negative control). Group B : rats were treated with acetaminophen (500mg/kg) (positive control). Group C : rats were given acetaminophen in the preceding dose, and 5 selenocyantouracil (50 mg/kg). Group D : rats were treated with acetaminophen orally at the prior dose, and 5 selenocyantouracil (100mg/kg) orally once daily for 21 days. According to biochemical data on alanine aminotransferase, aspartate aminotransferase, and creatinine serum level of 5 selenocyantouracil-treated groups significantly decreased when compared to AAP-treated groups, but no significant differences were observed when compared to the negative control group. Histopathological examinations confirm such results. As a conclusion, 5 selenocyantouracil compound has a protective activity against acetaminophen -induced tissue damage.


Introduction
Selenium (Se) has a vital physiological role as a potent antioxidant (1) .Se is required by living organisms because it is a part of the compound selenocysteine (Sec), which can be found in a number of selenoproteins (2) .Se is found in at least 25 different selenoproteins (3) .These proteins play several roles, the most important of which is redox homeostasis.
Thirteen selenoproteins are oxidoreductases, with selenocysteine as the catalytic redox-active residue.The remaining twelve selenoproteins either have no known function or have just a limited understanding of their functions (4) .However, because of dietary and environmental factors as well as Se's limited safety range, diseases brought on by Se excess or deficiency have become much more prevalent in recent years (5) .In humans, Se deficiency is a very common condition all around the world.It occurs when dietary intake is less than 40µg per day.Se deficiency has been linked to higher exposure to viral RNA infections (6) .
severe Se deficiency causes Keshan disease (cardiomyopathy) and Kashin-Beck disease (osteochondropathy) (2) .Antioxidants are compounds that have the ability to scavenge free radicals and decrease damage to biological tissues by lowering cellular oxidative stress (7) .Due to the antioxidant characteristics of most selenoproteins, selenium has long been assumed to protect organs against inflammation and cancer development by reducing oxidative stress (8).Artificial selenium-containing compounds can be used to treat a variety of diseases, including cancer and infections due to their antioxidant properties (9) .When the balance between reactive oxygen species (ROS) generation and elimination favors an elevation in ROS levels, oxidative stress occurs, causing cellular function to be disrupted (10) .ROS are a group of radicals or ions created when oxygen is added, they are characterized by their high capacity for oxidation and instability (11) .Druginduced liver injury is a common side effect of many medications, with main symptoms ranging from an increase in asymptomatic liver enzymes to liver failure (12) .For many years, paracetamol has been widely used as an analgesic and antipyretic throughout the world (13) .In our study, acetaminophen (AAP) was used as an oxidative stress inducer to cause liver injury.Many studies have found that oxidative stress is associated with various toxicities with AAP, and numerous antioxidants have been studied to see if they can protect against AAP-induced liver toxicity (14) .Laboratory testing is essential for identifying and monitoring liver disease and injury.Plasma markers of injury the aspartate and alanine aminotransferases (AST and ALT, respectively) are the most commonly used injury markers (15) .So It is commonly accepted that the serum level of ALT/AST ratio is a better indicator of liver damage (16)   .The ALT enzyme is found in serum and organ tissues, especially liver, although significant concentrations are also found in kidney, skeletal muscle, and myocardium, whereas AST is produced in the liver, heart and muscles (17) .AST concentrations in the human plasma range from 5 to 40 U/L, while ALT concentrations range from 5 to 35 U/L.In cases of heart, liver or muscle disease or injury, the levels of AST or ALT increase (18) .Another enzyme found in the liver, kidneys, intestines, and bone is called ALP. primarily used for cholestasis diagnosis (19) .Renal function test evaluation is now used as an indicator in the longterm treatment of a variety of disorders because it is quick, easy -to -understand, accurate, relatively inexpensive, and precise (20) .Two crucial parameters, serum creatinine and urea, are used to diagnose and treat kidney diseases and modify drug doses.Since creatinine is thought to be a more accurate indicator of kidney function than urea, the requirement for a perfect filtration indicator is justified (21) .Urea is a metabolic waste product excreted in urine by the kidneys (22) .while creatinine is derived from the breakdown of creatine and phosphocreatine in muscle (23) .

Materials
Drugs and chemicals that are used in this study are: Acetaminophen powder from (SAMA AL FAYHAA for Pharmaceutical Industries), 10% formalin solution (SDFCL, India) 5 Selenocyanatouracil (5SeU ) (a novel synthesized compound newly prepared from the reaction of KSeCN with 5-Iodouracil in an aqueous medium, in the central lab/College of Pharmacy/University of Basrah), 0.9% normal saline (pioneer, Iraq)

Animals
Forty male albino rats were purchased from a pet vet clinic in Erbil, in the north of Iraq.The rats (150-250g) in weight were housed in polypropylene cages (5 rats per cage).Rats were fed commercial pellets with distilled water.The rats were first housed under the natural lighting and temperature of the laboratory (21±4°C).

Experimental design
Forty male albino rats were randomly allocated into (4) groups, each group with (10) rats: Group A: rats were freely excess to food and water (negative control).Group B: rats were given acetaminophen (500mg/kg dissolved in 2 ml of DW once per day for three weeks) (24) (positive control).Group C: rats were given acetaminophen in the preceding dose, and 30 minutes later, they were given 5SeU (50 mg/kg dissolved in 2 ml of DW) orally once daily for 21 days.Group D: rats were given acetaminophen orally at the prior dose, and 30 minutes later, they were given 5SeU (100mg/kg dissolved in 2ml DW) orally once daily for 21 days.After a 21-day of treatment, the rats were anesthetized for blood drawing (about 3ml) from the heart, blood samples were collected in gel tubes and centrifuged for 10 minutes at 4000 rpm and stored at -20 °C until biochemical parameters were assessed (liver enzymes (ALT, AST), urea, and creatinine).The liver and kidneys were removed and washed with 0.9% saline solution.After being preserved in 10% formalin then embedded in paraffin wax, the samples were cut into 5 μm slices, and Hematoxylin and eosin staining were applied to the slices (H and E) in Al BASMA Lab in Baghdad.The result was discussed by a specialist histopathologist at the College of Veterinary Medicine/Basrah University.

Statistical analysis
Results are expressed as the mean± S.D. Statistical analysis was performed using the SPSS statistical package.One-way ANOVA comparisons were used to examine differences between test groups.Differences with p<0.05 were regarded as statistically significant.

Effect of 5 selenocyantouracil on liver enzymes Alanine aminotransferase (ALT)
Alanine aminotransferase serum level increased significantly (p<0.05) in B group when compared with A group after AAP treatment.While ALT serum levels decreased significantly (p<0.05) in C group in comparison with B group.Serum ALT levels were significantly (p<0.05)lower in D group when compared with B group as shown in the Figure (1) and Table (1) below.

Aspartate aminotransferase (AST)
Aspartate aminotransferase serum level increased significantly (p<0.05) in B group when compared with A group.While AST serum levels in C group decreased significantly (p<0.05) when compared with B group.D group reported significantly (p<0.05)lower serum AST levels when compared to B group as shown in Figure (2) and Table (1).

Effect of 5 selenocyantouracil on Renal Functions Serum creatinine
The difference between the serum creatinine levels of C, and D groups were significantly decreased than in B group , only B group increased significantly than A group as shown in Figure (3) and Table (2).

Serum Urea
The difference between the serum urea levels of C, and D groups were not statistically significant eithor with A group (negative control) or with B group (positive control), as shown in Figure (4) and Table (2).a -refer to significant differences with control group, b-refer to mean significant differences with positive group.p<0.05.

Effect of 5 Selenocyantouracil on liver enzymes
In general, liver enzymes are used in both humans and animals to diagnose liver diseases and monitor how the liver reacts to toxic drugs (26) .As a result of AAP overdose, variations in serum AST, ALT levels regarded as vital signs of hepatocyte physical injury.Liver enzymes play a role in energy metabolism involving the transamination of amino acids proceeding at many tissues, such as liver, cardiac muscle, and brain.AST, ALT can overflow into the circulation in cases of cellular damage, resulting in elevated activity (27) .According to the results of our study, selenium has been shown to enhance liver function by reducing serum AST and ALT levels.The study's findings agree with those of Reja et al. 2020 and Ozardali et al. 2004.Therefore, selenium supplementation may be useful in the treatment of severe liver disease.So because of their higher antioxidant properties, selenium may have hepatoprotective activity (28),(29) , The National Academy of Sciences (NAS) suggests dietary intake of about 55 μg of Se/day for adults for maximum glutathione activity (30) .

Effect of 5 Selenocyantouracil on renal parameters
The kidneys are crucial for the removal of wastes and toxins like urea, creatinine, and uric acid in addition to performing other functions.Renal function tests are essential for determining the occurrence of renal disease and for monitoring the progression of the kidneys' recovery by observing how they react to treatment (31) .In clinical findings, urea and creatinine are key indicators of renal dysfunction.Thus, serum urea and creatinine levels were measured to determine kidney damage (32) .The liver produces urea, a nitrogenous waste product that is dispersed throughout both intracellular and extracellular fluid as a result of the catabolism of protein and amino acids.The kidneys' glomeruli eliminate urea from the blood, and some of it is then partially reabsorbed.Based on muscle mass, the body creates creatinine at a consistent rate as a byproduct of the breakdown of creatine phosphate in muscle (33) .
In the present study, urea levels of postive control group (B group) and negative control (A group), showed no significant altered by Se treatment, while creatinine levels increased significantly in positive group in comparison with negative control group, but 5SeU treated group revealed no significant changes compared to control, so our findings are in agreement with Omrani et al.2016, who found that in, selenium supplementation seemed to have no effect on blood urea nitrogen (BUN) or creatinine levels (34) .El-Shenawy and Hassan's 2008 findings were also in agreement with our results that rats given selenium orally did not show any significant changes in serum levels of BUN or creatinine (35) .Another study (Musik, Kieczykowska, and Donica 2013) found that cyclic selenoorganic compounds didn't result in many significant changes in the studied parameters, so the results of selenium on biochemistry parameters of blood revealed that urea remained unchanged, as in our research (36) .Our findings contradict those of Hasanvand et al. 2017, who discovered that in the ischemia-reperfusion injury group (which resulted in renal dysfunction) the levels of urea and serum creatinine (Cr) increased significantly more than in the control group, and that Se treatment significantly reduced these indicators.Finally, selenium protects against ischemiareperfusion injury.This effect could be believed to be due to selenium's antioxidant properties (37) .Another study, Nadheerah et al. 2019, which presupposed that synthetically produced novel selenadiazole derivatives have minor effects on the kidney, contradicts our findings (9) .

Histopathological examination Effect on liver
The liver is the primary organ of metabolism and detoxification of many drug and toxin.AAP administration has caused parenchymal cell necrosis as well as cholestatic injury, as demonstrated by increased serum liver injury biomarkers and decreased protein synthesis from the liver (38) .Hepatocytes are quiescent, nonproductive for the cell cycle (G0 phase).Following AAP induced liver injury induction, cells begin to arrive the cell cycle and restart other phases, G1 mitosis process and S phase, when replication of DNA arises, followed by means of G2 in which the cell is divided (39) .When paracetamol is consumed in large amounts, the P-450-dependent metabolism creates the reactive product N-acetyl-pbenzoquinone imine (NAPQI), this excess NAPQI causes liver necrosis by depleting glutathione resulting in release of hepatocellular content into the blood (40) .The inflammatory response, which may be a cellular factor in healing, is primarily cause the death of necrotic cells.Prior to the onset of cellular mitosis, the two main inflammatory cells involved in AAP-induced liver damage are neutrophils and macrophages.By removing injured tissues that will be replaced with new cells, macrophage activation has been shown to aid hepatocyte proliferation (39) .
As shown in fig. 5 above, a control rats liver, it consists of outer hepatic triplets or tetrads embedded in the connective tissue and hexagonal or pentagonal lobules with central veins, hepatocytes are organized into bands that radiate from the central vein, and Kupffer cells-containing sinusoids serve as the dividers between the bands.They have a regular shape, a sizable spheroidal nucleus, a well-defined nucleolus, and a dispersion of chromatin along the periphery.Some cells each contain two nuclei.Liver slices photographed under a microscope after taking paracetamol concurrently, the trabecular structure is blurred in the majority of hepatic lobules, sinusoid congestion and dilated central veins.Empty regions resembling vacuoles of cytoplasmic fatty changes can be observed in the hepatocytes and pyknotic nuclei.In the sinusoid walls, a sizeable number of Kupffer cells, sinusoid congestion, and dilated central veins are seen.Also, rat's liver in positive control group exhibits a focal area of a microabscess made up of inflammatory cells and necrotic debris as shown in fig.6 above.Rat's liver following a three-week exposure to AAP (500 mg/kg) and 5SeU (50 mg/kg) was examined.There are usually no evidence of blood vessel dilation and mostly intact epithelium in most hepatic lobules, trabecular structures are slightly distorted and divided by sinusoids containing Kupffer cells.Occasionally, cells with empty areas resembling vacuoles can be seen in the cytoplasm.In the sinusoid walls, there are slightly more Kupffer cells than elsewhere.Hepatocytes are regular and have a big spheroidal nucleus as shown in fig.7 above.The section in the liver from 5SeU (100mg/kg)&AAP group, showed hepatocytes are arranged into stripes that spread beginning from central vein, and the bars separated via sinusoids that have Kupffer-cells.In most hepatic lobules, they have a regular shape, a large spheroidal nucleus, an obviously identified nucleolus, no signs of blood vessel dilation, and largely intact epithelium as shown in fig.8 above.Similar to our study, studies of Nadheerah et al. 2019 and Sabah Ansar et al. 2017 discovered minor differences in liver physiology between control and Se treated groups.Histopathological investigation revealed that selenium has a protective effect against AAP-induced liver damage (41)(42) .

Effects on kidney
As shown in the fig.9 above, a control rat's kidney (cortical portion).The structure of the renal glomeruli is normal.The usual thick cubic epithelium that lines the renal tubules is present.The cubic epithelium density, and the lumen of the tubules are quite uniform and distinct.It is possible to see the glomerule's lobular arrangement, the flat epithelium liner the glomerular capsule.Regarding the kidney section, Typical histological features of the kidney followed AAP alone or in combination with 5SeU (50mg or 100)mg/kg BW (H&E staining).Renal tissue slices photographed under a microscopic examination showed normal glomerular structure, and renal tubules.No signs of congestion or necrosis or glomerular atrophy as shown in the fig.(10, 11 and 12) above.The kidney is involved in the elimination of many drugs and their metabolites (38) .The nephrotoxic effects of AAP overdose are caused by complex events.In most cases, AAP-induced nephrotoxicity appears after hepatotoxicity, Some of the possible mechanisms of nephrotoxicity identified in clinical and animal studies include the cytochrome P-450 pathway, N-deacetylase enzymes , and prostaglandin synthesis (40) .Selenium works as a cofactor for an antioxidant enzyme, but it is not a pure antioxidant.The effects of selenium on chronic kidney disease may be related to the following factors.The first effect of selenium is an increase in Glutathione peroxidase 3 activity, which may protect kidney function by preventing mitochondrial damage caused by high oxidative stress (43) .Second, selenium influences phagocytes, immunoglobulin, and T cells to enhance human immunity.Third, selenium is crucial for thyroid function, which affects renal hemodynamics (44) .In a study with 1,871 participants, Hiragi et al. (2018) provided epidemiological data on the relationship between AAP overdose and the frequency of acute renal damage.The outcomes revealed no association between AAP and acute renal damage, confirming general physicians' assumptions that AAP is less harmful to the kidneys and agree with our study (45) .A study by Tsekhmistrenko et al. 2021 found that giving albino rats selenium for 30 days did not significantly alter their weights in the kidneys.In agreement with the findings, there were no detectable variations among the selenium groups and control group (46) .

Conclusion
In a dose-dependent manner, 5 SeU exhibits good antioxidant and anti-inflammatory effects.(5SeU) has a protective effect against paracetamol damage effect on the liver, and kidney.Histopathological study confirms such results can be concluded that 5SeU has a protective effect against acetaminophen -induced liver damage.

Figure3.
Figure3.Creatinine serum level in A, B, C and D groups.

Figure 4 .
Figure 4 .Urea level in serum of the rats in A, B, and D groups

Table 2 . Renal functions parameters of experiment groups.
Data C. Association between Selenium Status and Chronic Kidney Disease in Middle-Aged and Older Chinese Based on.2022; 44.Neamah NF, Al-Jadaan SAN, Al-Ali AM.Study of some of novel selena-diazole derivative activities on hematological parameters, differentiate lymphocytes cells in addition to thyroid hormones levels in female rats (One of the series of studies on the impact of the new compound).Systematic Reviews in Pharmacy.2020;11(11):975-80.45.Hiragi S, Yamada H, Tsukamoto T, Yoshida K, Kondo N, Matsubara T, et al.Acetaminophen administration and the risk of acute kidney injury: A self-controlled case series study.This work is licensed under a Creative Commons Attribution 4.0 International License.