Effects of mulberry extract on the liver pathology and serum biochemical parameters in carmustine administrated rats

Abstract

BACKGROUND:

Carmustine is a chemotherapeutic agent that is mainly used in the treatment of glioblastoma and can cause toxic effects on various organs, including the liver. The white mulberry extract has anti-apoptotic and anti-oxidant effects.

OBJECTIVE:

The study aimed at investigating the effects of the dried white mulberry extract on the pathology, apoptosis, and oxidative stress in the liver, as well as the levels of serum adenosine deaminase, glutathione peroxidase, superoxide dismutase, ceruloplasmin, paraoxonase, and malondialdehyde in carmustine-administrated rats.

METHODS:

Forty-two rats divided into six groups were used in this study. BCNU was administrated intraperitoneally (IP) (5 mg/kg body weight (BW)/week) for 10 weeks to the BCNU and BCNU-DWME groups. DWME was administered (600 mg/kg-BW by oral gavage) daily for 10 weeks to the DWME and BCNU-DWME groups. After the experimental procedure, histopathological, immunohistochemical, and biochemical analyses were performed.

RESULTS:

Carmustine caused biliary hyperplasia at a dose of 5 mg/kg. However, the mulberry extract was not effective in alleviating this pathology. Furthermore, the administration of carmustine induced apoptosis in hepatocytes, and the mulberry extract had an anti-apoptotic effect. Carmustine increased the 8-OHdG activity in the liver, and dried mulberry extract ameliorated this activity. Although there was no significant difference in the serum oxidative stress parameters between the groups, carmustine significantly increased the adenosine deaminase activity during the recovery period, while mulberry extracts partially ameliorated these effects in the recovery period.

CONCLUSIONS:

Dried white mulberry extract has anti-apoptotic and anti-oxidative effects against carmustine-induced toxicity.

Keywords

Apoptosis biochemistry carmustine liver white mulberry extract

1 Introduction

Chemotherapeutic drugs of the nitrosourea group have negative effects on the liver [1]. Carmustine (1,3-bis (2-chloroethyl)-1-nitrosourea or BCNU) is an alkylating and carbamoylating agent, which causes the degradation and alkylation of DNA and RNA [2]. BCNU is used as an alkylating agent in the treatment of glioblastoma and less commonly in the treatment of melanoma, gastrointestinal and pancreatic cancer, and Hodgkin and non-Hodgkin lymphoma. This mutagenic and chemotherapeutic drug reacts with the DNA through the SN1 mechanism, resulting in the formation of a 12-base adduct and phosphotriesters [3].

BCNU has long been known to cause delayed hepatoxicity in rats and humans [4, 5]. Hepatitis is also a common side effect of BCNU [1]. Mulberry fruit has previously been reported to possess hepatoprotective [6], antioxidant [7, 8], and anti-inflammatory effects [9]. These effects have been attributed to phenolics, flavonoids [10, 11], and anthocyanins [12]. To the best of the authors’ knowledge, there is a lack of information regarding the effects of dried white mulberry extract (DWME) on BCNU-induced liver injury. Hence, this study aimed to investigate the effects of DWME on liver pathology and some serum biochemical parameters after the administration of BCNU in rats.

2 Methods

2.1 DWME extraction

The methods for the extraction of DWME were adopted from previous studies [13, 14] with some modifications. Briefly, white mulberry fruits were obtained from Erzincan, Turkey. They were dried and powdered, following which, 500 g of the powder was macerated in a dark glass tube with 1.5 L methanol. This mixture was shaken for 1 h every 12 h for 15 days. Then, the mulberry-methanol mixture was filtered into a sterile container using a Whatman filter paper, and the solvent was evaporated using a vacuum evaporator at 50°C. The concentrated DWME was stored at –20°C until further use.

2.2 Total phenolic content of DWME

The total phenolic content of DWME was determined according to a previous study [15]. Briefly, 1 g of dried mulberry was extracted in 10 mL methanol and centrifuged at 6000 rpm. An aliquot of 500μL of the extract was taken from the supernatant, mixed with 500μL of Folin reagent in centrifuge tubes, and kept for 3 min. Then, 3 mL of 2% Na₂CO₃ was added to the tube and kept standing for 2 h in the dark. At the end of this period, the absorbance against the blank prepared under the same conditions was read at 760 nm using a spectrophotometer (Shimadzu®, Japan). A calibration curve was prepared using a gallic acid solution under the same conditions, and the concentration was calculated. The extract was tested five times, and the results were expressed in mg/g. Eventually, the phenolic level of the mulberry extract was determined as 4.77 mg/g.

2.3 Total flavonoid content of DWME

The total flavonoid content of DWME was calculated according to a previous study [15]. Briefly, 200μL of the methanolic extract of DWME was taken, to which 0.3 mL of 5% NaNO₂ was added, and the mixture was kept for 5 min. Then, 0.3 mL of 10% AlCl₃, 2 mL of 1 M NaOH, and 2.4 mL of distilled water were added and mixed. The absorbance of the sample was read at 510 nm, against a blank prepared immediately under the same conditions, using a spectrophotometer (Shimadzu®, Japan). The concentration of flavonoids was determined by plotting a calibration curve from the catechin concentration and absorbance recorded under the same conditions. The extract was analyzed five times, and the mean was calculated. The concentration of flavonoids was determined as 9.95 mg/g.

2.4 Gas chromatography-mass spectrometry (GC-MS) analysis of DWME

The GC-MS analysis was performed using gas chromatography-mass spectrometry (QP 2010, Shimadzu) in electron ionization mode at 0.70 kW and with a split/split-less injector system. Helium was used as the carrier gas in a capillary column-RTX-5MS (30 mx 0.25 mm; 0.25μm). The interface and ion source were heated at 250°C and 200°C, respectively. The oven temperature was programmed initially at 40°C for 3 min, followed by an increase of 4°C/min until it reached 240°C, and then maintained at 240°C for 10 min. A volume of 1μL/mL of the derivatized mixture was injected into the inlet heated at 250°C, with a 1 : 10 split ratio. The acquisition mass range was at 45–450 m/z. The compounds were determined based on a direct comparison of their retention times with Wiley libraries and the reference literature [16].

2.5 Animals and treatment design

Forty-two healthy adult male (8-weeks-old, mean weight 369.6±38.65 g) rats were used in this study. The animals were obtained from Mehmet Akif Ersoy University’s Experimental Research Center, Burdur, Turkey. They were maintained at a temperature of 20±3°C with a 12-hour light/dark cycle and fed a commercial pellet diet with fresh drinking water ad libitum. The experimental protocol was approved by Mehmet Akif Ersoy University’s Local Ethics Committee on Animal Research (04.12.2020/707). BCNU (Tokyo Chemical Industry, C2634) was diluted with physiological saline according to the manufacturer’s instructions. BCNU was administrated intraperitoneally (IP) (5 mg/kg body weight (BW)/week) according to a previous study [17]. Concentrated DWME was administered to animals by gavage after dilution with fresh drinking water. Since there was no previous study with DWME, the dose (600 mg/kg-BW by oral gavage) was chosen from a previous study on mulberry leaves extract [18]. The doses of BCNU and DWME were calculated according to the weekly body weights of the rats.

The study duration was 20 weeks and included two periods: experiment (first 10 weeks) and recovery (final 10 weeks when the drugs were withdrawn). The rats were divided randomly into the following four groups: Group 1-Control group (Control, n = 7), who received physiological saline (5 mg/kg-BW/week) IP (placebo) and fresh drinking water daily (0.5 mL tap water by oral gavage) for 10 weeks; Group 2-Carmustine (BCNU) group (C, n = 14) rats, who received carmustine (5 mg/kg-BW/week) IP and fresh drinking water daily (0.5 mL tap water by oral gavage) for 10 weeks; Group 3-BCNU + DWME group (n = 14) rats, who received carmustine (5 mg/kg-BW/week) IP and DWME (600 mg/kg-BW/daily by oral gavage) for 10 weeks; Group 4-DWME group (DWME; n = 7) rats, who received physiological saline (5 mg/kg-BW/week) IP and DWME (600 mg/kg-BW/daily by oral gavage) for 10 weeks. Seven rats from each of the BCNU and BCNU + DWME groups and all rats from the other groups (control and DWME) were euthanized after the collection of blood samples under ketamine/xylazine anesthesia at the end of the experiment period (10 weeks), while the remaining rats from the BCNU and BCNU + DWME groups were euthanized after blood sample collection at the end of the recovery period (20 weeks).

2.6 Calculation of the human-equivalent dose

To determine the human-equivalent dose (HED) of DWME, we used a previously reported reference method [19]. The formula used was: HED (mg/kg) = Animal DWME (mg/kg) x (Weight_animal [kg])^(1 –0.67) (The body surface correction factor was considered as 0.67).

2.7 Sample collection

The blood samples were collected from the heart under anesthesia, and the sera were used for the biochemical analysis. Liver samples were placed in 10% neutral buffered formaldehyde for histopathological and immunohistochemical examinations.

2.8 Histopathological examination

Liver samples were fixed with 10% neutral buffered formalin for 1 day. After routine follow-up procedures, 4μm sections were taken, and the slides were stained with hematoxylin-eosin solution. Histopathologically, the lesions were scored using a semi-quantitative method by examining 10 different fields under the 40X magnification of a microscope (Nikon Eclipse E600, Japan). Scoring was done as: 0 (none), 1 (mild), 2 (moderate), and 3 (severe).

Histopathologically, vascular congestion, inflammation (portal and parenchymal), degeneration, necrosis, hemorrhage, bile duct hyperplasia, and fibrosis were evaluated under light microscopy (Nikon Eclipse E600) and compared between the groups.

2.9 Determination of apoptosis and oxidative stress by immunohistochemistry

Caspase-3 (GTX110543, caspase-3 antibody, GeneTex) protein was examined immunohistochemically to determine the level of apoptosis. The avidin-biotin complex method was used, with DAB as the chromogen. Ten high-power areas were assessed for each animal, and the mean positive cell values were used for the statistical analysis.

For the evaluation of oxidative stress, 8-hydroxydeoxyguanosine (bs-1278R, 8-OHdG polyclonal antibody, Bioss) and secondary kit (MP-7601, ImmPRESS Excel Amplified Polymer Staining Kit, VectorLab) was used for the immunostaining. Relative levels of positive cells (0 = 0–5%, 1 = 0, 5–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%) and staining intensity (0 = negative, 1 = weak, 2 = moderate, and 3 = strong) were calculated in five high-power areas (x400) for each animal. The IHC scores were calculated as the score obtained from % positive cells (0–4) x score of intensity (0–3). Then, the total score obtained (between 0 and 12) were evaluated statistically. The evaluations were performed according to a previous report, with some modifications [20].

2.10 Biochemical analysis

The activity levels of adenosine deaminase (ADA), glutathione peroxidase (GPx), superoxide dismutase (SOD), ceruloplasmin (CP), paraoxonase (PON), and malondialdehyde (MDA) were evaluated from the blood sera. ADA was measured by spectrophotometric analysis according to the procedure described by Guisti [21]. GPx activity was assessed according to the method of Paglia and Valentine [22]. SOD activity was determined by the method of Sun et al. [23]. Protein concentration in the serum samples was determined using the Bradford assay [24]. CP values were determined according to the method of Cerón and Martínez-Subiela [25], and PON activity was assessed using the method of Armstrong [26]. MDA levels were assessed according to the method of Yoshioka et al. [27].

2.11 Statistical analysis

The statistical program Minitab® version 16.1.1 was used for the statistical analysis. One-Way ANOVA with Tukey post hoc analysis was performed to compare the results of serum biochemical analysis, as well as the histopathological and immunohistochemical analysis of the liver between groups. The data in the tables are expressed as X±standard error, along with the level of significance (determined at P < 0.05).

3 Results

3.1 Bioactive compounds of DWME

Based on the GC-MS analysis, the ten major components of DWME were 5-hydroxy-methyl-furfural (5-HMF, 31.67%), 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP, 19.54%), Propanoic acid, 2-methyl-, methyl ester (5.71%), 1,2,3-Propanetriol, monoacetate (5.47%), cis-Vaccenic acid (4.06%), 2-Furanmethanol (3.82%), 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl- (2.51%), 5-Acetoxymethyl-2-furaldehyde (2.09%), Palmitic acid (1.94%) and (S)-5-Hydroxymethyl-2[5H]-furanone (1,73%).

3.2 Pathological results

Histopathologically, there was no significant difference between the groups in terms of vascular congestion, inflammation, degeneration, necrosis, bleeding, and fibrosis, while a significant increase in the occurrence of bile duct hyperplasia was observed in the BCNU (Recovery) and BCNU + DWME (Recovery) groups (P < 0.01) (Table 1) (Fig. 1). Immunohistochemically, a significant increase in the levels of caspase-3 was observed in the BCNU group compared to the control and DWME groups (P < 0.01). A significant decrease in the caspase-3 levels was observed in the BCNU + DWME group compared to the BCNU group (P < 0.01). The positivity of caspase-3 was significantly lesser in the BCNU (Recovery) and BCNU + DWME (Recovery) groups compared to the BCNU group. Moreover, the decrease in caspase-3 levels in the BCNU + DWME (Recovery) group was significantly higher than that in the BCNU (Recovery) group (P < 0.01) (Fig. 2). The 8-OHdG scores were significantly higher in the BCNU group than in the control and DWME groups (P < 0.05). In the BCNU + DWME group, the 8-OHdG scores were significantly lower than the BCNU group (P < 0.05). The 8-OHdG scores in treatment groups (BCNU and BCNU + DWME) were not significantly different from those in the recovery groups; only a slight decrease was observed in the recovery period groups (P > 0.05).

Table 1
Mean±SEM values of histopathological and immunohistochemical parameters

Groups Caspase-3 8-OHdG Biliary Hyperplasia Portal Inflammation

X±SE X±SE X±SE X±SE

Control 3.014±0.188^bc 1.800±1.110^b 0.000±0.000^a 1.200±0.200^a

DWME 2.071±0.605^c 1.833±0.910^b 0.286±0.184^a 1.286±0.184^a

BCNU 7.100±0.299^a 7.800±1.800^a 0.000±0.000^a 1.400±0.245^a

BCNU + DWME 1.857±0.199^c 3.000±1.520^b 0.500±0.500^ab 1.333±0.422^a

BCNU (Recovery) 3.871±0.524^b 2.400±0.678^b 1.857±0.459^b 1.571±0.202^a

BCNU + DWME (Recovery) 1.486±0.182^c 2.670±1.410^b 2.000±0.516^b 1.833±0.401^a

P value 0.000^*** 0.029^* 0.005^** 0.388

Groups	Caspase-3	8-OHdG	Biliary Hyperplasia	Portal Inflammation
Control	3.014±0.188^bc	1.800±1.110^b	0.000±0.000^a	1.200±0.200^a
DWME	2.071±0.605^c	1.833±0.910^b	0.286±0.184^a	1.286±0.184^a
BCNU	7.100±0.299^a	7.800±1.800^a	0.000±0.000^a	1.400±0.245^a
BCNU + DWME	1.857±0.199^c	3.000±1.520^b	0.500±0.500^ab	1.333±0.422^a
BCNU (Recovery)	3.871±0.524^b	2.400±0.678^b	1.857±0.459^b	1.571±0.202^a
BCNU + DWME (Recovery)	1.486±0.182^c	2.670±1.410^b	2.000±0.516^b	1.833±0.401^a
P value	0.000^***	0.029^*	0.005^**	0.388

a, b, c: The difference between the means with different letters is statistically significant. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 1

Histopathology of the portal area. Normal appearance of the portal areas of the control (A), DWME (B), BCNU (C), and BCNU-DWME (D) groups. Biliary hyperplasia is characterized by numerous biliary tracts in the BCNU (Recovery) (E) and BCNU-DWME (Recovery) (F) groups (arrows). H&E. Bars: 50μm.

Fig. 2

Caspase-3 immunostaining of hepatocytes (arrows). Mild immunostaining in the Control (A) and DWME (B). Numerous and moderate positivity in BCNU (C) and BCNU (Recovery) (E) groups, respectively. Decreased positivity in BCNU-DWME (D) and BCNU-DWME (Recovery) (F) groups. Bars: 50μm.

Fig. 3

8-OHdG immunostaining of hepatocytes (arrows). Mild positivity in the Control (A) and DWME (B), severe positivity in the BCNU (C), decreased positivity in the BCNU-DWME (D), and mild to moderate positivity in the BCNU (Recovery) (E) and BCNU-DWME (Recovery) (F) groups. Bars: 50μm.

3.3 Biochemical results

Biochemically, ADA, one of the indicators of oxidative stress, increased significantly in the BCNU (Recovery) group compared to the control and treatment groups (P < 0.01). However, there was no increase in the BCNU group. Interestingly, a decrease in ADA was observed in the BCNU + DWME (Recovery) group compared to the BCNU (Recovery) group (P > 0.05). No statistically significant difference was observed between the groups in terms of CP, PON, SOD, GPx activities, and MDA levels. However, a slight increase in the CP activity was observed compared to the control and DWME groups (P > 0.05). In the PON evaluation, only a slight decrease was observed in the BCNU (Recovery) group compared to the other groups (P > 0.05) (Table 2).

Table 2
Mean±SEM values of biochemical parameters

Groups ADA CP PON SOD GPX MDA

X±SE X±SE X±SE X±SE X±SE X±SE

Control 2.253±0.532^b 0.1444±0.0274^a 49.10±4.73^a 9.650±1.350^a 0.03687±0.00124^a 10.329±0.685^a

DWME 2.309±0.860^b 0.1543±0.0388^a 43.51±9.23^a 7.119±0.753^a 0.03000±0.00180^a 9.7600±1.150^a

BCNU 2.016±0.250^b 0.2153±0.0161^a 41.81±5.19^a 7.589±0.678^a 0.03272±0.00260^a 10.222±0.656^a

BCNU + DWME 2.889±0.556^b 0.2173±0.0204^a 40.05±4.41^a 9.880±1.200^a 0.03035±0.00181^a 13.700±1.130^a

BCNU (Recovery) 6.371±0.675^a 0.2021±0.0199^a 34.69±5.71^a 9.454±0.881^a 0.03073±0.00405^a 10.030±1.250^a

BCNU + DWME (Recovery) 3.660±1.180^ab 0.2162±0.0409^a 40.56±3.88^a 8.525±0.785^a 0.03158±0.00149^a 10.594±0.831^a

P value 0.001^* 0.228 0.526 0.255 0.364 0.066

Groups	ADA	CP	PON	SOD	GPX	MDA
Control	2.253±0.532^b	0.1444±0.0274^a	49.10±4.73^a	9.650±1.350^a	0.03687±0.00124^a	10.329±0.685^a
DWME	2.309±0.860^b	0.1543±0.0388^a	43.51±9.23^a	7.119±0.753^a	0.03000±0.00180^a	9.7600±1.150^a
BCNU	2.016±0.250^b	0.2153±0.0161^a	41.81±5.19^a	7.589±0.678^a	0.03272±0.00260^a	10.222±0.656^a
BCNU + DWME	2.889±0.556^b	0.2173±0.0204^a	40.05±4.41^a	9.880±1.200^a	0.03035±0.00181^a	13.700±1.130^a
BCNU (Recovery)	6.371±0.675^a	0.2021±0.0199^a	34.69±5.71^a	9.454±0.881^a	0.03073±0.00405^a	10.030±1.250^a
BCNU + DWME (Recovery)	3.660±1.180^ab	0.2162±0.0409^a	40.56±3.88^a	8.525±0.785^a	0.03158±0.00149^a	10.594±0.831^a
P value	0.001^*	0.228	0.526	0.255	0.364	0.066

a, b: The difference between the means with different letters is statistically significant. ^*P < 0.01.

3.4 HED Calculation for DWME

The DWME value in a rat weighing approximately 370 g is 600 mg/kg. So, the HED was calculated as HED (mg/kg) = 600×(0.37/60)^(0.33) = 110 mg/kg. Thus, for a human weighing 60 kg, the dose could be approximately 6.6 g. This dose is further divided by a factor value of 10 for initiation in human studies; so, the dose is 0.66 g.

4 Discussion/conclusion

White mulberry contains phenolic and flavonoid components, as well as various vitamins, oils (mainly linoleic acid, palmitic acid, oleic acid), and minerals [28]. The fruits of this plant have been reported to have anti-diabetic, anti-oxidative [28], anti-inflammatory, anti-apoptotic [29], and anti-hyperlipidemic [30], anti-microbial [31] functions. Moreover, the fruit, root, bark, and leaves are used for the treatment of various ailments such as inflammation, jaundice, hepatitis, cancer, diarrhea, cough, edema, fever, anemia, atherosclerosis, and hypertension [32 –35]. In our study, the effects of dried mulberry fruit on carmustine-induced liver injury were investigated due to the above-mentioned properties, especially the anti-oxidative, anti-apoptotic, and hepatoprotective effects.

BCNU has been reported to cause an increase in liver enzymes that could not be explained by a primary disease in 26% of the patients [5]. In another study, hepatitis due to BCNU was noted as a common finding [1]. Krell et al. [36] reported that BCNU could contribute to the development of hepatotoxic lesions by causing an increase in the paracellular permeability in hepatocytes. Phillips et al. [37] reported that in patients with autologous bone marrow transplantation, BCNU at doses higher than 1200 mg/m² was associated with hepatic necrosis in 3% of the patients, while in 25% of the cases, the increase in levels of bilirubin and alkaline phosphatase continued for up to 4 months after treatment. In another study, abnormal liver function (increased transaminase and alkaline phosphatase) and hyperbilirubinemia were reported in 90% of the patients during the first week following BCNU administration [38]. In our study, no statistically significant difference was observed between the groups in terms of vascular congestion, degeneration, necrosis, inflammation, and fibrosis in the histopathological evaluation of the liver. However, a slight increase in portal inflammation was observed in other groups compared to the control and DWME groups, which was slightly higher in the recovery period (P > 0.05).

Laquerriere et al. [39] observed severe lesions in the liver of six animals on day-30 after a single dose of 20 mg/kg BCNU administration. The lesions included dystrophy in the biliary epithelium, dense peri-canalicular fibrosis, and cholangiolysis in two animals. On the 60th day, irregularities in the connective tissue network of the periportal areas were observed, along with biliary changes. On the 90th day, it was observed that the lesions regressed, and five animals had minor hepatocytic lesions. For the 50 mg/kg dose, bile duct changes characterized by minor lesions in three animals, sclerosed cholangitis in four animals, and polymorphic granulomatous infiltrates in four animals were observed on day-30. On day-60, minor lesions were seen in five animals and changes like those in the 20 mg/kg group were observed in four animals. Biliary ducts were destroyed in one animal. On day-90, reversible lesions were present in two animals, while six animals had split or unchanged scar lesions. Thompson and Larson [4] reported the cholestatic effect of BCNU in rats. Demir et al. [40] observed that the biliary flow decreased and serum-conjugated bilirubin levels increased in the BCNU group in the intrahepatic cholestasis model, which was formed due to the administration of BCNU in rats. In our study, 5 mg/kg BCNU administered weekly for 10 weeks did not cause any damage to the biliary epithelium. Regarding the prevalence of biliary hyperplasia, no difference was observed in the BCNU group compared to the control and DWME groups, while a significant increase was formed in the BCNU (Recovery) and BCNU + DWME (Recovery) groups compared to the other groups (P < 0.01). This finding indicates that the repeated administration of BCNU at low doses causes biliary hyperplasia in the long term, but mulberry extract does not have a positive effect on this pathology.

The effect of BCNU mainly involves the alkylation of DNA and RNA and the stimulation of apoptosis [41 –46]. BCNU also induces apoptosis through oxidative stress [17 , 48]. BCNU induces oxidative stress by inhibiting chymotrypsin, alcohol dehydrogenase, and glutathione reductase as a result of severe carbamoylation in proteins, mainly cysteine, as well as by significantly increasing the leakage of lactate dehydrogenase [6 , 49–51]. BCNU-induced oxidative stress formation is partially associated with an increased uptake of calcium from the extracellular spaces [52]. In our study, caspase-3 levels were investigated immunohistochemically in terms of apoptosis in the liver, and the caspase-3 activity was observed to be significantly higher in the BCNU group compared to the control and DWME groups (P < 0.001). Also, 8-OHdG, a marker of DNA damage resulting from oxidative stress in cells, was examined in the liver tissue using immunohistochemistry. The 8-OHdG is commonly studied and is considered to be a biomarker for the oxidative damage of DNA [53, 54]. Interaction of hydroxyl radicals with the bases of DNA leads to the formation of 8-OHdG [55]. Numerous studies have shown that the formation of 8-OHdG is increased in various liver diseases, including viral [55], toxic [56 –58], alcoholic [59], and non-alcoholic fatty liver disease [60, 61], chronic liver diseases, and cancer [62]. Some studies have also stated that the expression of 8-OHdG contributes to the development of hepatocellular carcinoma [20]. Moreover, an age-related increase in the expression of 8-OHdG was observed in various organs of rats, including the liver [63]. In our study, BCNU increased the expression of 8-OHdG in hepatocytes compared to the control and DWME groups (P < 0.05). Furthermore, DWME ameliorates the expression of 8-OHdG (P < 0.05). Furthermore, some slight effects were observed in the recovery period of BCNU and DWME-BCNU groups compared to the treatment groups (P > 0.05). The serum oxidative stress parameters were also evaluated. Despite the observation that BCNU caused oxidative stress-mediated apoptosis as mentioned above, we did not observe any statistically significant differences between the groups in terms of serum CP, PON, SOD, and GPX activities (P > 0.05). This may be related to the use of BCNU at lower doses in our study. Nevertheless, there was a numerical increase in CP activity in all other groups compared to the control and DWME groups (P > 0.05). This suggests that a slight increase in CP was caused by the application of BCNU, but DWME did not have any positive counter effect on this increase. Moreover, there was a decrease in PON levels in the BCNU (Recovery) group compared to the control and DWME groups, while a slight increase was observed in the BCNU + DWME (Recovery) group (P > 0.05). This situation may indicate that there was a slight decrease in the PON level in the long term following the application of BCNU and that DWME alleviated this effect.

Khan et al. [64] reported that the glutathione levels decreased rapidly after BCNU administration in isolated rat hepatocytes, followed by lipid peroxidation and cytotoxicity. It was also observed that this cytotoxicity was delayed by the use of antioxidants [butylated hydroxyanisole (BHA), alpha-tocopherol, the ferric iron-chelator deferoxamine, or the radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl]. In our study, it was observed that the DWME extract, which has antioxidant properties, significantly reduced the activation of caspase-3. It was also observed that a waiting period of just two weeks following BCNU administration resulted in a decrease in caspase activation in the liver tissue. This suggests that the apoptotic effect of BCNU on the liver is transient at a dose of 5 mg/kg and can be significantly reduced with the use of DWME.

ADA activity increases significantly in serum with liver diseases, hematological malignancies, and infectious diseases [65]. Raczynska et al. [66] reported an increase in the ADA activity in plasma with liver diseases, including cirrhosis, cancer, and hemochromatosis. Kobayashi et al. [67] demonstrated that high ADA isoenzyme activities (ADA1 and ADA2) in patients with liver diseases included acute hepatitis, alcoholic hepatic fibrosis, chronic active hepatitis, liver cirrhosis, and hepatoma, and suggested that ADA may be a marker for liver disease. Although no increase in the serum ADA levels was observed in the BCNU group compared to the control and DWME groups in our study, a significant increase was observed in the BCNU (Recovery) group (P < 0.01). Moreover, a slight decrease was noticeable in the BCNU + DWME (Recovery) group, although it was not significant (P > 0.05).

Numerous studies have demonstrated that the mulberry fruit has antioxidant properties [14 , 68–70]. According to the GC-MS analysis in our study, the major components of DWME were 5-HMF (31.69%) and DDMP (19.54%). 5-HMF is the end product of carbohydrate metabolism [71, 72]. It has been reported that 5-HMF has antioxidant [73], anti-hypoxia [74], inhibitor of erythrocyte sickling [75], and hepatoprotective [76 –78] effects. DDMP has also been reported to have antioxidant activities [79 –81]. In our study, it was observed that DWME has an anti-apoptotic effect on the liver. Although DWME did not make a significant difference in terms of systemic biochemical oxidative stress parameters, it possibly achieved this anti-apoptotic and anti-oxidative effect through substances such as 5-HMF and DDMP in its content.

The main limitations of our study are the inability to analyze the blood parameters from liver samples and the inability to evaluate different cumulative BCNU doses or DWME doses. Furthermore, the human equivalent dose (HED) was calculated based on the dose of DWME in our study (600 mg/kg), and this dose was assumed as the NOAEL (no observed adverse effect levels) of the DWME, which is another limitation of our study. Thus, a higher dose of DWME may be a candidate for NOAEL. Nevertheless, our findings indicate that while BCNU significantly increased the activity of caspase-3 and 8-OHdG in the short term, DWME ameliorated these effects. In the longer term, it was observed that BCNU was associated with bile hyperplasia in the liver and an increase in serum ADA activity. Although no positive effect of DWME on biliary hyperplasia was observed, it did cause a slight decrease in ADA activity. These findings may be related to the doses of BCNU used in our study. Further studies involving different doses, durations, and parameters will make it possible to reveal the effects of DWME on BCNU-induced damage more thoroughly.

Footnotes

Acknowledgments

The authors thank Gaffari Turk and Okkes Yilmaz for the analysis of total phenol and flavonoid levels and Serpil Dag, Emin Karakurt, and Ayfer Yildiz for providing the oxidative stress antibody.

Funding

This study was financed under the project supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 218O162).

Conflict of interest

The authors have no conflict of interest to report.

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Groups	ADA	CP	PON	SOD	GPX	MDA
	X±SE	X±SE	X±SE	X±SE	X±SE	X±SE
Control	2.253±0.532^b	0.1444±0.0274^a	49.10±4.73^a	9.650±1.350^a	0.03687±0.00124^a	10.329±0.685^a
DWME	2.309±0.860^b	0.1543±0.0388^a	43.51±9.23^a	7.119±0.753^a	0.03000±0.00180^a	9.7600±1.150^a
BCNU	2.016±0.250^b	0.2153±0.0161^a	41.81±5.19^a	7.589±0.678^a	0.03272±0.00260^a	10.222±0.656^a
BCNU + DWME	2.889±0.556^b	0.2173±0.0204^a	40.05±4.41^a	9.880±1.200^a	0.03035±0.00181^a	13.700±1.130^a
BCNU (Recovery)	6.371±0.675^a	0.2021±0.0199^a	34.69±5.71^a	9.454±0.881^a	0.03073±0.00405^a	10.030±1.250^a
BCNU + DWME (Recovery)	3.660±1.180^ab	0.2162±0.0409^a	40.56±3.88^a	8.525±0.785^a	0.03158±0.00149^a	10.594±0.831^a
P value	0.001^*	0.228	0.526	0.255	0.364	0.066