Alleviation of cisplatin‐induced hepatotoxicity by gliclazide: Involvement of oxidative stress and caspase‐3 activity

Abstract Aims Cisplatin (CP), as an effective alkylating agent, is widely used in cancer treatment, while hepatotoxicity is one of its side effects. Gliclazide (GLZ), as an oral hypoglycemic drug, has antioxidant and anti‐inflammatory properties. This study was designed to investigate the protective effect of GLZ against CP‐induced hepatotoxicity in mice. Methods In this experimental study, 64 adult male mice randomly were allocated into eight groups (8 mice/group). Control, GLZ (5, 10, and 25 mg/kg, orally), CP (10 mg/kg, single dose, intraperitoneally), and CP+GLZ (in three doses). GLZ was administrated for 10 consecutive days. CP was injected on the 7th day of the study. At the end of the experiment, hepatotoxicity was evaluated by serum and tissue biochemical, histopathological, and immunohistochemical assessments. Results The data were revealed that CP increased oxidative stress (increased MDA and reduced GSH), liver damage enzymes (ALT, AST, and ALP), and immunoreactivity of caspase‐3 in liver tissue of CP‐injected mice. Also, CP induced histopathological changes such as eosinophilic of hepatocytes, dilatation of sinusoids, congestion, and proliferation of Kupffer cells. GLZ administration significantly ameliorated serum functional enzyme and hepatic oxidative stress markers in CP‐injected mice. In addition, the histological and immunohistochemical alterations were ameliorated in GLZ‐treated mice. Of the three doses, 10 and 25 mg/kg were more effective. Conclusions In conclusion, GLZ with its antioxidant, anti‐inflammatory, and anti‐apoptotic activities, can be suggested as a promising drug in the treatment of CP‐induced hepatotoxicity.

involved in hepatotoxicity induced by CP. 3 Furthermore, CP affects the tumor-suppressor protein p53 through the generation of reactive oxygen species (ROS) and induces apoptosis via intrinsic caspases. 4 Loss of liver histoarchitecture, positive caspase-3 reactions, decrease in GSH, and an increase in MDA levels were reported in CP treatment. 2 Since oxidative stress is the most important mechanism of the induction of hepatotoxicity, the use of antioxidants can play a major role in reducing CP-induced toxicity. 5 Gliclazide (GLZ) as an oral hypoglycemic drug and a secondgeneration sulfonylurea, is used in diabetic patients. 6 GLZ is able to improve the histopathological changes and DNA damage underlying both the process of aging and type 2 diabetes by reducing free radical generation and an increase in free radical scavenging. 6 In addition to its antioxidant property, GLZ has potentially prevented tissue damage by acting anti-apoptotic and anti-inflammatory effects. 7,8 In diabetic experimental models, it has been demonstrated that GLZ preserves brain and sciatic injury 9 and DNA damage in peripheral blood lymphocytes. 10 Only one study reported the synergistic effect of metformin and GLZ on liver injury in diabetic patients. 11 And until now, no study reported the protective effect of GLZ on CP-induced hepatotoxicity.
Two case studies reported that GLZ at high doses causes acute hepatitis. 12,13 However, several studies have shown that low-dose GLZ with having antioxidant properties mitigates DNA damage induced by Type 2 diabetes mellitus (T2DM) patients. 14 With this background, in this study, we have investigated the effects of GLZ on oxidative stress, apoptosis, and histopathological changes induced by CP on liver tissue.
Primary antibodies of caspase-3 and a secondary antibody conjugated with horseradish peroxidase were purchased from Abcam.
Gliclazide was from a Pharmaceutical Co. (Tehran Daru).

| Animals and experimental design
In this experimental study, 64 male BALB/c mice (25- In this study, animals were randomly divided into eight groups (8 mice/group). Groups were defined as follows: Group I; as control animals received distilled water (vehicle of GLZ). Group II, III, and IV; animals were administered with GLZ at doses of 5, 10, and 25 mg/ kg/day for 10 consecutive days orally. Group V; hepatotoxicity was induced in mice by a single-dose intraperitoneal injection of CP (10 mg/kg/day) on the 7 th day. Groups VI, VII, and VIII were administered GLZ at three doses of 5, 10, and 25 mg/kg via oral administration for 10 consecutive days, and CP injected on the seventh-day study. The doses of CP chosen were based on our previous study 16 and GLZ based on the basis of the literature studies. 9 A study design scheme is represented in Figure 1.

| Sample collections
One week after receiving the last drug, the animals were euthanized with ketamine (50 mg/kg) and xylazine (5 mg/kg).
Immediately blood samples were collected from the heart using a heparin syringe, then poured into the gel clot tube. After 15 min of centrifugation at 3000 × g for 15 min, the serum samples were separated and stored at −20°C for serum biochemical evaluation.
Then, immediately the liver was carried out. One part of the liver was fixed in a 10% buffer formalin solution for histological and immunohistochemical examination and another piece of liver was washed in PBS, weighed, and then freshly frozen and stored at −70°C for biochemical analysis.

| Oxidative stress assay
The liver samples were homogenized in cold phosphate buffer, centrifuged, and the supernatants were used for oxidative stress markers. The concentration of GSH as an antioxidant parameter was determined in the homogenate samples and measured with spectrophotometric. Data were expressed as μmol/gr tissue. 17 Malondialdehyde (MDA) as a lipid peroxidation marker was assessed by thiobarbituric acid (TBA) and the colorimetric absorbance was measured at 535 and 520 nm in a spectrophotometer. Results were expressed as nmol/gr tissue. 18

| Histopathological assay
The liver samples were fixed in 10% neutral-buffered formalin solution, dehydrated in alcohol series, embedded in paraffin, and then sections with 5 mm thickness were stained with H&E (hematoxylin and eosin). The liver slides were evaluated blindly by histologist by light microscopy at 400X magnification (Olympus light microscope, Japan). For semi-quantitative evaluation of liver damage, histological photomicrographs were examined by the liver scoring system.
In this study, the extent of sinusoidal dilatation, inflammatory cell infiltration, congestion, degeneration, the proliferation of Kupffer cells, and cytoplasmic vacuolization were studied. For each mouse, five sections, and in each section, 10 fields were randomly assessed.

| Immunohistochemical assay
An immunohistochemical examination was done according to the guidance kit company (Abcam Company). Slides were deparaffinized with xylene and rehydrated in alcohol series, endogenous peroxidase activities were blocked by 0.3% H 2 O 2 in methanol (30 min). Then, tissue sections were incubated with primary antibodies (anti-caspase-3 rabbit polyclonal antibody, 1:100 in PBS, v/v, Abcam, lat: GR224831-2) at 4°C overnight. After incubation with secondary antibody conjugated with horseradish peroxidase (Mouse and Rabbit Specific HRP/DAB, Abcam, Lat: GR2623314-4) for 2 h, immunohistochemical staining was carried out by incubation with diaminobenzidine tetrahydrochloride for 5 min. Then, the slides were dehydrated and mounted. 20 The primary antibody was omitted for negative controls. For the quantitative analysis, immunohistochemical photomicrographs were estimated by densitometry using MacBiophotonics ImageJ 1.41asoftware in 5 fields/each section in all groups. The positive staining severity was assessed as the ratio of the stained area to the entire field assessment.

| Statistical analysis
The results were expressed as mean ± SD. The Kolmogorov-Smirnov (K-S) normality test was used in order to evaluate the normality of the data. All statistical comparisons were performed by one-way ANOVA followed by Tukey test. Scoring data analyzed by Kruskal-Wallis H Test between groups. p-value less than .05 (p < .05) was considered statistically significant (Prism software).

| Effect of GLZ on serum liver function enzymes in CP-treated mice
The effects of CP, GLZ, and their combination of liver function enzymes (AST, ALT, and ALP levels) are shown in Table 1. In this study, CP-treated mice showed a significant increase in serum AST, ALT, and ALP levels compared with the control group (p < .0001).
However, administration of GLZ with 5, 10, and 25 mg/kg decreased significantly the serum liver enzymes in the CP-treated mice. This decrease was dose-dependent. For ALT and AST markers, a dose of 10 and 25 mg/kg was more effective than the other dose.

| Effect of GLZ on histopathology of liver tissue in CP-treated mice
Histopathological micrographs in all groups are shown in Furthermore, semi-quantitative evaluation of liver damage with the scoring system showed that CP administration caused a significant increase in liver injury score (0.29 ± 0.49) compared with control and GLZ alone groups (0.14 ± 0.38). This change with GLZ administration was reduced in CP-treated mice ( Figure 3F).

| DISCUSS ION
Cisplatin is an anti-neoplastic agent for solid organ tumors has side effects on most organs including hepatotoxicity. 21,22 In this study, we showed that CP can significantly induce hepatotoxicity in mice confirmed by elevations in serum liver enzyme activities and histopathological changes. GLZ improved these parameters. The protective effects of GLZ are attributed to a decrease in oxidative stress, suppressing caspase-3 immunoreactivity.
CP is a small molecule that easily passes through the cell membrane and reaches the cell nucleus and changes the structure of DNA. Although the pathophysiological mechanism of CP-induced hepatotoxicity is not yet well understood, oxidative stress and apoptosis are important inducers of liver injury. 5 Researchers have shown that CP increases MDA as a lipid peroxidation marker and reduces GSH as an antioxidant marker in liver tissues. 23 In oxidative stress, deficiencies occur in the overproduction of reactive oxygen species or the antioxidant system. 24 GLZ at a low dose has antioxidant activity and can scavenge free radicals. 25 Our data suggested that GLZ with this potential, protects hepatotoxicity via mitigating oxidative stress and apoptosis in the CP-treated mice. These findings were consistent with the study of others. 26  showed that GLZ had a protective effect on liver tissue and reduced the amount of these enzymes by reducing oxidative stress. Patients with type 2 diabetes are at risk for liver damage and subsequently elevated liver enzymes. Belcher has shown that GLZ intake in diabetic patients is able to reduce liver enzymes. 29 Alsharidah et al.
also showed that, the combined metformin/GLZ therapy was able to reduce liver enzymes in diabetic patients. 11 This study showed that CP led to histopathological changes in liver structure such as sinusoidal dilatation, congestion, central reported that the use of GLZ for a long time leads to hepatitis (acute necro-inflammation of the liver). 12,13 In this study, the protective effect of GLZ on liver tissue structure was demonstrated.
Our data showed that GLZ at all three selected doses improved CP-induced liver damage. However, this effect was more effective at doses of 10 and 25 mg/kg as compared to the dose of 5 mg/kg. In the scoring system, the highest score was also for the CP group.
And of the three GLZ+CP groups, the dose of 25 mg/kg had the lowest score. it also recommended that GLZ be used at the usual dose in cancer patients before starting cisplatin administration.
In conclusion, cisplatin-induced liver injury is associated with oxidative stress (increased MDA and decreased GSH), disruption of tissue structure, and apoptosis. GLZ treatment improved liver function and histological damage by suppressing oxidative stress and apoptosis activity.

ACK N OWLED G M ENT
The authors thank the Vice-Chancellor of Research, Mazandaran University of Medical Sciences, Sari, Iran, for their financial support.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest in this study and publication.

AUTH O R S ' CO NTR I B UTI O N S
Taghizadeh and Mirzaei conducted the experiments. Talebpour, Zargari, and Karimpour performed the data analysis. Talebpour and Hosseinimehr participated in research design and wrote or contributed to the writing of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated in this study are included in this manuscript.