Protective effects of 2,4-dihydroxybenzophenone against acetaminophen-induced hepatotoxicity in mice

Abstract

AIM: To examine the effects of 2,4-dihydroxybenzophenone (BP-1), a benzophenone derivative used as an ultraviolet light absorbent, on acetaminophen (APAP)-induced hepatotoxicity in C57BL/6J mice.

METHODS: Mice were administered orally with BP-1 at doses of 200, 400 and 800 mg/kg body weight respectively every morning for 4 d before a hepatotoxic dose of APAP (350 mg/kg body weight) was given subcutaneously. Twenty four hours after APAP intoxication, the serum enzyme including serum alaine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) were measured and liver histopathologic changes were examined.

RESULTS: BP-1 administration dramatically reduced serum ALT, AST and LDH levels. Liver histopathological examination showed that BP-1 administration antagonized APAP-induced liver pathological damage in a dose-dependent manner. Further tests showed that APAP-induced hepatic lipid peroxidation was reduced significantly by BP-1 pretreatment, and glutathione depletion was ameliorated obviously.

CONCLUSION: BP-1 can effectively protect C57BL/6J mice from APAP-induced hepatotoxicity, and reduction of oxidative stress might be part of the protection mechanism.

Key Words: 2,4-dihydroxybenzophenone; Acetaminophen; Hepatotoxicity

INTRODUCTION

Acetaminophen (APAP), which is also named paracetamol, is a widely used antipyretic and analgesic drug. However, it can cause hepatic necrosis and even death in human and experimental animals when taken in overdose[1]. Overdose of APAP can lead to acute liver injury and histopathological changes characterized by centrilobular necrosis[2,3]. Most of APAP is cleared by glucuronic acid or sulphate combination while a part of APAP is metabolized by cytochrome P450 system and generates reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI can be conjugated by glutathione and hence excreted from the body as sulfhydryl adducts. If the formation of NAPQI is beyond the clearing capacity of reduced glutathione (GSH), NAPQI combined with liver cellular protein will cause liver cell injury[4,5]. APAP has been widely used to establish acute liver injury models for liver protective agent research. Since C57BL/6J mice were more susceptible to APAP, C57BL/6J mice were used in the present study.

2,4-dihydroxybenzophenone (BP-1), a benzophenone derivative, is often used as an ultraviolet light absorbent. BP-1 has also been used in some products such as nail polish, polish remover, shaving cream, body cleanser and so on. Previous researches about BP-1 mainly focused on its estrogenic activities. Matsumoto et al[6] demonstrated that BP-1 exhibited estrogenic activities by estrogen receptor using MCF-7 (a human breast cancer cell line) cell proliferation assay. In this study, we examined the effects of BP-1 in APAP-induced hepatotoxicity in mice.

MATERIALS AND METHODS

Chemicals

BP-1 was purchased from Huangshi Meifeng Chemical Company (Huangshi, China) and APAP from Jiaozuo Xin’an Science and Technology Company (Jiaozuo, China). BP-1 suspension was prepared with 1% Tween80 from Amresco Company (Solon, USA). TBARS assay kit was purchased from Cell Biolabs (San Diego, USA). Glutathione assay kit was purchased from Calbiochem (Darmstadt, Germany).

Animals and treatment

Healthy and clean male C57BL/6J mice weighing 18-22 g were used in all experiments. They were bought from Peking University Laboratory Animal Department. Mice were housed in a well-ventilated room and fed adaptively for 3 d before experiment. Room temperature was controlled at 21-25°C with a 12 h/12 h light-dark cycle and humidity at 65%-70%. All mice were allowed free access to water and fed with forage supplied by Laboratory Animal Center of Academy of Military Medical Sciences.

Thirty mice were randomly divided into 5 groups. Group 1 was vehicle control group and Group 2 was APAP alone group, both of which administered intragastrically with 1% Tween80 for 4 d. Groups 3, 4 and 5, administered intragastrically with BP-1 at doses of 200, 400 and 800 mg/kg body weight respectively for 4 d. On the 4th day, all mice except those in vehicle control group were injected with APAP (350 mg/kg body weight) subcutaneously 30 min after the final administration. Twenty-four hours after APAP injection, blood samples were collected from orbital venous plexus. Then all mice were sacrificed, and livers were moved out immediately and washed with saline, dried on a filter paper and weighed. Liver samples were prepared for further tests. The animal experiments and surgical procedures were all performed in compliance with the Guidelines for Animal Care and Use issued by Peking University.

Biochemical test

The serum enzyme levels including serum alaine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were determined by HITACHI-7170A automatic analyzer. Liver homogenization was carried out in ice-cold KCl solution (0.15 mol/L) or phosphate buffered solution to yield a 5% (w/v) tissue homogenates for malonaldehyde (MDA) and GSH tests. The MDA, GSH and oxidized glutathione (GSSG) levels in liver tissues were determined according to the manufacturer’s protocols. GSH/GSSG ratio was calculated.

Histological examination

The left liver lobes were cut out and fixed in 10% formalin solution. After pathological sectioning and HE staining, liver histopathologic changes were examined under inverted phase contrast microscope.

Data treatment and statistical analysis

Data were expressed as mean ± SE. Statistical comparison between groups was performed by one-way analysis of variance (ANOVA) with SPSS 13.0 software. Significance was accepted at P < 0.05.

RESULTS

BP-1 administration reduced ALT, AST and LDH

Twenty-four hours after a single dose of APAP (350 mg/kg body weight), serum levels of ALT, AST and LDH increased significantly (P < 0.05), showing characteristic acute liver injury induced by APAP. Compared with APAP group, BP-1 groups lowered serum levels of ALT, AST and LDH dramatically. At a dose of 200 mg/kg body weight, the difference was not significant; at doses of 400 and 800 mg/kg body weight, there were significant differences between BP-1 groups and APAP group (P < 0.05). The effect of BP-1 was dose-dependent and serum levels of ALT, AST and LDH in the highest dose groups were comparable to that of vehicle control group (P > 0.05) (Figure 1).

Figure 1 Effects of 2,4-dihydroxybenzophenone administration on serum levels of alaine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in mice. Acetaminophen (APAP) was administered at 350 mg/kg sc and 2,4-dihydroxybenzophenone (BP-1) was administered ig at doses of 200, 400 and 800 mg/kg respectively 4 d before acetaminophen treatment. Alaine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase(LDH) levels were measured 24 h after acetaminophen treatment. Results were expressed as mean ± SE and data was analyzed by one-way analysis of variance using SPSS 13.0 software. ^aP < 0.05 vs acetaminophen group; ^cP < 0.05 vs control group.

Effect of BP-1 administration in liver tissue MDA and GSH/GSSG ratio

Compared with vehicle control group, MDA content in liver tissue of APAP alone group increased while the ratio of GSH/GSSG decreased dramatically (P < 0.05), indicating an oxidation stress and depletion of GSH. With increasing dose of BP-1, MDA level decreased and GSH/GSSG ratio increased. In the highest dose group (800 mg/kg body weight), the MDA and ratio of GSH/GSSG were close to the normal level as shown in vehicle control group (Table 1).

Table 1 Effects of 2,4-dihydroxybenzophenone administration on malonaldehyde level and GSH/GSSG ratio in mice.

Groups	MDA (μmol/g)	GSH/GSSH ratio
Control	0.30 ± 0.27	10.5 ± 2.7
Acetaminophen	0.90 ± 0.70c	7.1 ± 2.1c
BP-1 (200 mg/kg)	1.08 ± 0.68	5.1 ± 1.4a
BP-1 (400 mg/kg)	0.58 ± 0.33	6.8 ± 1.5
BP-1 (800 mg/kg)	0.50 ± 0.10	9.6 ± 1.9a

^aP < 0.05 vs acetaminophen alone group;

^cP < 0.05 vs control group. BP-1: 2,4-dihydroxybenzophenone; MDA: Malonaldehyde.

BP-1 alleviated pathological injury induced by APAP

Observed by naked eyes, the livers of vehicle control group were deep red, moist, glossy and resilient. In APAP group, the livers lost luster and yellow necrosis foci were often found on the surface. Liver injury of BP-1 pretreated mice was attenuated dramatically in a dose-dependent manner.

Under light microscope, liver lobular structures in vehicle control group were clear and regular, and single layer of hepatocytes arranged around the central vein in a radical pattern. There were abundant basophilic granular cytoplasms in the hepatocytes (Figure 2A). In APAP-intoxicated mice, normal liver lobular structures were damaged and collapsed. The hepatocytes showed vacuolization, sinusoidal dilation and congestion. Infiltration of inflammatory cells and loss of cell boundaries were also observed (Figure 2B). Pre-administration of BP-1 showed mild injuries in a dose-related manner. BP-1 at 200 mg/kg could not effectively prevent the damage (Figure 2C). There was a moderate injury in the 400 mg/kg group (Figure 2D) while in the 800 mg/kg group liver lobular structure was well comparable to that in the vehicle control group (Figure 2E).

Figure 2 Liver histology of all groups 24 h after acetaminophen treatment (350 mg/kg). Hematoxylin-eosin-stained liver sections displayed representative hepatocellular morphological changes. Original magnification × 200. A: Liver section in the vehicle control group showed a normal lobular structure; B: Liver section in acetaminophen alone group showed large areas of centrilobular necrosis, vacuolar degeneration and inflammatory cell infiltration; C: Liver section in the 200 mg/kg group showed necrosis with the same degree as in acetaminophen group; D: Liver section in the 400 mg/kg group showed a significant alleviation of liver injury; E: Liver section in the 800 mg/kg group showed absence of necrosis and almost normal lobular structure.

DISCUSSION

APAP is often used in animal experiments to establish acute liver injury models. Our previous researches found that C57BL/6J mice were more susceptible to APAP[7], so C57BL/6J mice were chosen in the present study as experimental animals. The serum levels of ALT, AST and LDH are the main indexes which reflect liver injury[8]. Our study demonstrated that BP-1 protected mice from APAP-induced acute liver injury as shown by the significant decrease in serum ALT, AST and LDH. BP-1 could also antagonize tetrachloromethane-, cocaine- and thioacetamide-induced acute liver injuries, which was evidenced by reduced serum ALT, AST and LDH levels, as well as ameliorated pathological changes in the liver (data not shown).

BP-1 showed a broad protective effect in hepatotoxic chemicals-induced acute liver injury, implying that it may target on the oxidative stress, which was the key event in liver injuries caused by these hepatotoxic chemicals. To confirm the hypothesis, we examined the content of MDA in liver tissues which was often used as a biomarker to measure the level of oxidative stress in organisms[9], as well as the GSH/GSSG ratio which indicated the capability to antagonize the oxidative injury[10]. The results demonstrated that, MDA levels in APAP-intoxicated mice increased obviously and GSH/GSSG ratio decreased, implying the existence of oxidative damage and depletion of GSH. However, pre-administration of BP-1 decreased the MDA level and increased GSH/GSSG ratio in a dose-dependent manner. The results indicated that the hepatoprotective effect of BP-1 was associated with its antioxidant activity.

COMMENTS

Background

Acetaminophen (APAP)-induced acute liver injury model has been widely used as hepatoprotective agent screening. The authors have conducted the screening for hepatoprotective agents for a long time. To the best of their knowledge, there has been no report about the hepatoprotective effects of 2,4-dihydroxybenzophenone (BP-1) up to date.

Innovations and breakthroughs

This study shed a light on the hepatoprotective effects of BP-1. The results indicated that the hepatoprotective effect of BP-1 was associated with its antioxidant activity.

Applications

This study provided evidence for the protective effect of BP-1 against APAP-induced liver injury, which implied that BP-1 might be a promising agent for acute liver injury in human. Of course, before possible clinical use, more researches are needed to confirm that BP-1 is both effective and safe for humans.

Peer review

The authors investigated the effect of BP-1 pretreatment on APAP-induced hepatotoxicity in mice. The results demonstrated that: BP-1 pretreatment antagonized APAP-induced liver pathological damage; BP-1 pretreatment reduced APAP-induced hepatic lipid peroxidation; and BP-1 pretreatment ameliorated glutathione depletion.