Investigation of Effects of Silymarin in 5-Fluorouracil Hepatotoxicity and Nephrotoxicity in Mice

Hepatotoxicity and nephrotoxicity are common side effects of 5-Fluorouracil (5-FU). The present study aimed to investigate the effects of Silymarin (SLY) on 5-FU induced hepatotoxicity and nephrotoxicity in mice. In our study, 10 mice in each group were randomly divided into four groups as the control group, 5-FU, SLY50+5-FU, and SLY100+5-FU group. SLY50+5-FU and SLY100+5-FU groups were administered at a dose of 50 and 100 mg/kg for seven days, respectively. 5-FU was administered at a dose of 400 mg/kg intraperitoneally on the fourth day. After the applications, the mice were decapitated under anesthesia. The liver and kidney functions which urea, creatinine, AST, ALT, and total bilirubin levels were analyzed in serum. In liver and renal tissues, MDA and GSH levels, SOD, CAT, and GR activity were determined. Also, histopathological and immunohistochemical changes were examined in liver and kidney sections. Urea, creatinine, ALT, AST, and total bilirubin levels increased 5-FU group according to control and prevented to this increases the especially high dose of SLY. 5-FU also causes histopathological and immunohistochemical changes such as degeneration, necrosis, hyperemia, DNA damage, and IL-6 increase in kidney and liver tissue. High doses of SLY prevented these changes caused by 5-FU. As a result of this study, it was determined that SLY has hepatoprotective and nephroprotective effects on 5-FU-induced liver and kidney damage in mice. tissues obtained from the experimental groups and evaluated them among the groups. The results showed that a particularly high dose of SLM (100mg/kg) prevented 5-FU-induced oxidative damage in kidney and liver tissue and protected these tissues from oxidative damage by increasing SOD, CAT, and GR activity and GSH levels. Besides, SLM (100 mg/kg) can protect kidney and liver tissue by reducing the MDA content. The underlying mechanism may include SLM attenuated ROS formation in renal and hepatic tissue cells. Some studies in recent years show that antioxidants have a certain protective effect on organ toxicity caused by anti-carcinogenic substances. like


Introduction
Cancer is an important public health problem worldwide in recent years (Siegel et al. 2019). In the treatment of cancer are applied to different strategies such as surgery, radiotherapy, and chemotherapy. Chemotherapy is a powerful therapy for cancer and antineoplastic agents. Used for this purpose, 5-Fluorouracil (5-FU) is a commonly used agent in the treatment of various malignancies such as colon, breast cancer, head and neck cancer (Akindele et al. 2018;Grem 2000). Despite a lot of advantages, 5-FU's treatment has been largely limited due to some organ toxicity, inhibition of thymine synthesis, and DNA damage (Akindele et al. 2018;Gelen et al. 2018). Considering the mechanism of action of 5-FU; It affects the S phase of the cell cycle, activates thymidine phosphorylase, thymidylate synthase inhibiting uorodeoxyuridine. Thus, it prevents DNA synthesis, which leads to cell death ). The In previous studies, the therapeutic and protective effects of SLY in nephrotoxicity and hepatotoxicity induced by some pharmacological agents have been determined (Bektur et al. 2016;Kandemir et al. 2017). As a result of the literature review, we determined that the effects of SLY on 5-FU-induced hepatorenal toxicity in mice have not been investigated yet. Therefore, we investigated the protective effects of SLY on 5-FU-induced hepatotoxicity and nephrotoxicity in a mouse model.

Animals
We used 40 male mice in our study. The weight of the mice was chosen to be 30-40 g on average. Mice were obtained from Atatürk University Medical Experimental Research and Application Center. Animals were subjected to standard feeding conditions. The necessary permission for the study was obtained from Atatürk University Rectorate Animal Experiments Local Ethics Committee (Protocol number: 2019/17).

Experimental design
In our study, the nephrotoxicity and hepatotoxicity model was formed by 5-FU (400 mg/kg, intraperitoneal (i.p.), three dose starting from the fourth day) and SLY (50 and 100 mg/kg, 7 days) were administered intraperitoneally. The animals were divided into 4 groups. Separated groups and application methods are as follows.
At the end of the applications, mice in all groups were weighed and then intracardiac blood samples were taken under anesthesia. The mice were decapitated after blood samples were taken. The liver and kidney tissues of all mice in the experimental groups were removed and weighed. The right kidney of mice was placed in 10% formaldehyde for histopathological and immunohistochemical examinations. Left kidneys of mice were washed with cold phosphate buffer and frozen with liquid nitrogen. It was stored at -20 o C until biochemical studies were carried out.

Analysis of liver and renal function parameters
The blood samples taken from the experimental groups were centrifuged at 3500-4000 rpm in a cooled centrifuge at 4 ° C for 12 minutes. Serum samples were taken into tubes. It was stored at 80 ° C until analysis. Serum urea, creatinine, ALT, AST, and total bilirubin levels were determined using the Randox IV Monaco-Auto-Chemistry-Analyzer.

Preparation of liver and renal homogenates
Liver and kidney tissues obtained from experimental groups were homogenized to 5 microns in a Tissue Lyser II (Qiagen) with liquid nitrogen on the day of analysis. Tissues were weighed speci cally, then diluted 1:20 with phosphate buffer (pH 7.4). Subsequently, samples were homogenized in Tissue Lyser II.
After homogenization, the homogenates were centrifuged at 3000 rpm for 20 minutes at 4 o C and the supernatant was used for ELISA analysis.

Statistical analysis
The results of our study were evaluated statistically. The results were given as X ± SD. The quantitative values were statistically analyzed in SPSS 20.00 statistical data program. Then, one-way ANOVA was evaluated by the Tukey test. p <0.05 was considered signi cant.

Results
Effects of SLY on body, liver, and kidney weights in 5-FU-toxicity The live weights of the mice were similar among to groups. Liver weights reduced to 5-FU group so far as control but this decreasing not statistically signi cant. Liver weights of the SLY10+5-FU group were higher than the 5-FU group (p<0.05). Also, were similar among to groups (Table 1). Effects of SLY on liver enzymes and renal function parameters Serum ALT, AST, total bilirubin, urea, and creatinine levels were markedly increased in the 5-FU and SLY50+5-FU groups according to control. In the SLY100+5-FU group lower levels of these parameters by comparison to 5-FU and SLY50+5-FU groups ( Table 2). Effects of SLY on liver MDA and GSH levels MDA levels were signi cantly higher in 5-FU and SLY50 + 5-FU groups compared to control, lower in SLY50 group than 5-FU group, but there was no signi cant difference (p> 0.05). MDA level was higher than control in SLY100 + 5-FU group. However, it is lower than the 5-FU group ( Figure 1A). Also, liver GSH levels decreased signi cantly in the 5-FU and SLY50 + 5-FU groups up to the control and SLY100 + 5-FU groups ( Figure 1C).
Effects of SLY on liver SOD, CAT, and GR activities Liver SOD, CAT, and GR activities were markedly lower in the 5-FU group in comparison with control. These enzyme activities increased in the SLY50+5-FU and SLY100+5-FU groups according to the 5-FU group (p 0.05) and these effects of SLY were dose-dependent and higher doses of SLY more signi cantly prevented the 5-FU-induced reduction in antioxidant enzyme activities ( Figure 1B, 1D, 1E).
Effects of SLY on renal MDA and GSH levels MDA levels in 5-FU and SLY50+5-FU groups had signi cantly higher according to the control and were lower in the low dose group of SLY than 5-FU group (p 0.05). MDA level in SLY100+5-FU group reduced according to 5-FU ( Figure 2A). Renal GSH levels reduced signi cantly in the 5-FU and SLY50+5-FU groups so far as to control and SLY100+5-FU groups ( Figure 2C).
Effects of SLY on renal SOD, CAT, and GR activities Renal SOD, CAT, and GR activities were signi cantly lower in the 5-FU group in comparison with others groups. These enzyme activities increased in the SLY50+5-FU and SLY100+5-FU groups according to the 5-FU group (p>0.05) and these effects of SLY were dose-dependent ( Figure 2B, 2D, 2E).

Histopathological ndings
When the liver and kidney tissues of the control group were examined to histopathologically, it was observed that they were in normal histological structures (Figure 3-4 A). In 5-FU groups, especially in the acinar region of the liver and hepatocytes were observed to severe degeneration, necrosis, and vascular hyperemia ( Figure 3B). In the liver sections of SLY50+5-FU group, moderate degeneration, mild necrosis, and hyperemia in the vessels were detected in hepatocytes ( Figure 3C). When the liver tissues of SLY100+5-FU group were examined histopathologically, mild degeneration in the hepatocytes and mild hyperemia in the vessels were observed ( Figure 3D). The renal tubular epithelium of mice in the 5-FU group were observed severe hydropic degeneration and coagulation necrosis and were detected severe hyperemia in the glomerular and interstitial vessels ( Figure 4B). The renal tubular epithelium of the SLY50+5-FU group was detected moderate degeneration, necrosis, and severe hyperemia in interstitial and glomerular vessels ( Figure 4C). When the kidney tissues of the SLY100+5-FU group were examined histopathologically, mild degeneration in the tubular epithelium and mild hyperemia in the interstitial and glomerular vessels were determined ( Figure 4D). A signi cant difference (p 0.05) was detected when compared with the 5 FU groups (Table 3).

Immunohistochemical ndings
When liver and kidney tissues of the control group were examined immunohistochemically, 8-OHdG and IL-6 expression in liver tissues ( Figure 5-6A) and kidney tissues (Figure 7-8A) were negative. Severe cytoplasmic 8-OHdG expression was detected in hepatocytes in the liver, acinar region, in the 5-FU group ( Figure 5B). Also, severe IL-6 expression was observed in the liver at sinusoidal and portal intervals ( Figure 6B). In the 5-FU group was viewed severe cytoplasmic 8-OHdG expression in kidney tubular epithelium ( Figure 7B), severe IL-6 expression in the glomerulus, intertubular intervals, and vascular circumference ( Figure 8B). In the SLY50+5-FU group were observed moderate cytoplasmic 8-OHdG expression in liver hepatocytes ( Figure 5C) and in this group was detected moderate IL-6 expression in the sinusoidal spaces, vascular environment and portal spaces ( Figure 6C). The SLY50+5-FU group were determined moderate cytoplasmic 8-OHdG expression in the tubular epithelium ( Figure 7C), moderate IL-6 expression in glomeruli, intertubular intervals, and vascular circumference ( Figure 8C). In livers of SLY100+5-FU group were determined mild cytoplasmic 8-OHdG expression in the tubular epithelium ( Figure 5D), sinusoidal intervals and in the portal region was detected to IL-6 expression at mild level ( Figure 6D). In the SLY100+5-FU group, in the kidney tissues were observed 8-OHdG expression in the cytoplasmic mild level in the tubular epithelium ( Figure 7D), and were determined to mild IL-6 expression in the glomerulus, intertubular intervals, and vascular circumference ( Figure 8D). A signi cant difference (p 0.05) was detected when compared with the control group. Immunohistochemical ndings are summarized in table 3.  release has also been reported (Peluso et al.2015). In previous studies, the inhibitory effect of SLY, which is a avonoid, on IL-6 was reported (Shahidi et al.2017). The data we obtained in our study show that 5-FU treatment signi cantly increases IL-6 expression in liver and kidney tissues. However, we found that SLY treatment caused a signi cant decrease in the IL-6 increase caused by 5-FU in liver and kidney tissues. This result is likely due to the anti-in ammatory properties of SLY.

Conclusion
According to our results, the mechanisms of 5-FU-induced hepatorenal toxicity are seen and con rm the potential antioxidant and anti-in ammatory of SLY. Other ndings of this study support the role of oxidative stress, in ammation, DNA damage in the pathogenesis of 5-FU-induced liver and renal toxicity. Availability of data and materials The authors con rm that the data and materials supporting the ndings of this study are available within the article.
Funding information This work was not supported by any institution.  Figure 1 Page 14/20 The MDA (A) and GSH (C) levels and SOD (B), CAT (D), and GR (E) activities in liver tissues, a,b: p <0.001, a,c; b,c: p<0.05).