Protective effects of 5-methoxypsoralen against acetaminophen-induced hepatotoxicity in mice

Abstract

AIM: To investigate the hepatic protective effects of 5-methoxypsoralen (5-MOP) and to learn if 5-MOP causes hepatotoxicity at protective doses.

METHODS: C57BL/6J mice were administrated orally with 5-MOP at doses of 12.5, 25 and 50 mg/kg body weight respectively every morning for 4 d before given acetaminophen (APAP) subcutaneously at a dose of 500 mg/kg. The 5-MOP alone group was treated with 5-MOP orally at a dose of 50 mg/kg body weight for 4 d without APAP. Twenty-four hours after APAP administration, blood samples of mice were analyzed for serum enzyme alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) levels, and malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) of liver tissues were measured and histopathologic changes of the liver were observed.

RESULTS: Compared with the vehicle control group, the serum levels (IU/L) of ALT, AST and LDH were all increased significantly in APAP group (8355 ± 3940 vs 30 ± 21, P < 0.05; 6482 ± 4018 vs 146 ± 58, P < 0.05; 24627 ± 10975 vs 1504 ± 410, P < 0.05). Compared with APAP group, the serum ALT levels (IU/L) (1674 ± 1810 vs 8355 ± 3940, P < 0.05; 54 ± 39 vs 8355 ± 3940, P < 0.05; 19 ± 9 vs 8355 ± 3940, P < 0.05), AST levels (IU/L) (729 ± 685 vs 6482 ± 4108, P < 0.05; 187 ± 149 vs 6482 ± 4108, P < 0.05; 141 ± 12 vs 6482 ± 4108, P < 0.05) and LDH levels (IU/L) (7220 ± 6317 vs 24 627 ± 10 975, P < 0.05; 1618 ± 719 vs 24 627 ± 10 975, P < 0.05; 1394 ± 469 vs 24 627 ± 10 975, P < 0.05) were all decreased drastically in the three-dosage 5-MOP pretreatment groups. Pretreatment of 5-MOP could attenuate histopathologic changes induced by APAP, including hepatocellular necrosis and infiltration of inflammatory cells, and the effect was dose-dependent. MDA levels (nmol/mg) were decreased by 5-MOP in a dose-dependent manner (0.98 ± 0.45 vs 2.15 ± 1.07, P > 0.05; 0.59 ± 0.07 vs 2.15 ± 1.07, P < 0.05; 0.47 ± 0.06 vs 2.15 ± 1.07, P < 0.05). The pretreatment of 5-MOP could also increase the GSH/GSSG ratio (3.834 ± 0.340 vs 3.306 ± 0.282, P > 0.05; 5.330 ± 0.421 vs 3.306 ± 0.282, P < 0.05; 6.180 ± 0.212 vs 3.306 ± 0.282, P < 0.05). In the group treated with 5-MOP but without APAP, the serum enzyme levels, the liver histopathologic manifestation, and the values of MDA and GSH/GSSG ratio were all normal.

CONCLUSION: 5-MOP can effectively protect C57BL/6J mice from APAP-induced hepatotoxicity and possesses an antioxidative activity, and does not cause liver injury at the protective doses.

Key Words: 5-Methoxypsoralen; Protection; Acetaminophen; Hepatotoxicity; Antioxidation

INTRODUCTION

Acetaminophen (APAP), a widely used antipyretic and analgesic drug, could induce hepatotoxicity and even acute liver failure (ALF) when taken at overdose[1]. APAP overdose is a common cause of adult and children ALF in the United States and other countries[2-5]. APAP can be metabolized by cytochrome P450 enzymes (CYPs) to N-acetyl-P-benzoquinoneimine (NAPQI)[6]. At overdoses of APAP, a large number of NAPQIs is generated, which can deplete reduced glutathione (GSH) and then bind to mitochondrial proteins to cause mitochondrial dysfunction and oxidant stress[7,8], leading to hepatocellular damage and centrilobular hepatic necrosis. In this process, APAP can increase the level of malondialdehyde (MDA) both in the liver and plasma[9] and NAPQI is capable of lowering GSH/oxidized glutathione (GSSG) ratio by oxidizing the thiol group of GSH[10]. Oxidant stress plays a central role in the hepatic damage induced by APAP[11].

5-methoxypsoralen (5-MOP), a furocoumarin found in many medicinal plants, possesses slight antioxidative activity evidenced from researches in vitro[12,13]. 5-MOP has been used in combination with UV radiation in skin photochemotherapy for decades[14], and some studies also found that it has anticancer[15-19], antidepressant[20-24], anticonvulsion[25] and anti-inflammatory effects[26,27], but none of previous studies have shown that 5-MOP could prevent hepatotoxicity.

In addition, some patients suffered from toxic hepatitis induced by 5-MOP when it was used as photochemotherapeutic agent[28,29], and one animal experiment demonstrated that high doses of 5-MOP can induce hepatotoxicity in mice[30]. So it is essential to examine if 5-MOP can cause liver injury at therapeutic doses.

This study was designed to determine the protective effects of 5-MOP in APAP-induced hepatotoxicity using mouse hepatotoxic models, and to investigate if 5-MOP can cause hepatotoxicity in mice at effective doses.

MATERIALS AND METHODS

Chemicals

5-MOP was purchased from Tokyo Chemical Industry (Tokyo, Japan), and APAP from Jiaozuo Xin’An Science and Technology Company (Henan, China). Tween 80, which was used to prepare 5-MOP suspension, was bought from Biodee Biotechnology Company (Beijing, China). APAP was dissolved in normal saline before use. GSH and N-ethylmathione gained from Lizhudongfeng Biotechnology Company (Shanghai, China), GSSG from Hongxing Biotechnology Company (Beijing, China), o- phthalaldehyde (OPT) from Jinlong Chemical Company (Beijing, China), and thiobarbituric acid (TBA) from Acros (United States).

Animals and treatment

Male C57BL/6J mice, 18-22 g in weight, were purchased from Peking University Laboratory Animal Department, Beijing, China. They were housed in a well-ventilated room and the room temperature was controlled at 21 °C-23 °C and humidity at 65%-70% with a 12 h light-12 h dark cycle. All the mice were fed adaptively for three d before experiment, and they had free access to water and were fed with forage supplied by Laboratory Animal Center of Military Medical Science Academy.

5-MOP was suspended in 1% Tween 80 at different concentrations of 1.25 mg/mL, 2.5 mg/mL, and 5 mg/mL, and all of these suspensions were administrated to mice at 10 mL/kg body weight; that is, mice were administrated with 5-MOP at doses of 12.5 mg/kg, 25 mg/kg and 50 mg/kg, respectively. A 5-d experiment was performed with 36 mice which were randomly divided into 6 groups by weight. Group 1 was the vehicle control group and group 2 was APAP alone group, both groups were orally treated with 1% Tween 80 (10 mL/kg body weight) every morning for 4 d. Groups 3, 4 and 5 were 5-MOP multiple-dose groups administered with oral 5-MOP at doses of 12.5, 25 and 50 mg/kg body weight respectively every morning for 4 d. Group 6 was 5-MOP alone group treated with oral 5-MOP at a dose of 50 mg/kg body weight also for 4 d. Thirty minutes after the administrations, all mice except those in the vehicle control group and 5-MOP alone group were subcutaneously administrated with APAP (500 mg/kg body weight). Twenty hours after APAP administration, blood samples were collected from orbital venous plexus of the mice. After the mice were sacrificed, their livers were dissected out immediately and washed with normal saline, dried on a filter paper and weighted. Then the livers were prepared immediately for further examinations.

The animal care and surgical procedures were performed in compliance with the Guidelines for Animal Care and Use of Peking University.

Biochemical test

The blood samples were collected to determine serum enzyme [alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH)] levels by HITACHI-7170A automatic analyzer. The liver tissues were homogenized with potassium chloride (KCl) solution (0.15 mol/L) on ice to yield a 5% (w/v) homogenates for MDA test. The hepatic MDA levels were determined as thiobarbituric acid reactive substances levels using a published colorimetric method[31].

The liver tissues were homogenized with phosphate buffered solution on ice to yield a 5% (w/v) homogenates for glutathione test. The GSH and GSSG levels in liver tissues were measured by the improved Hission method[32], a fluorometric method that uses OPT as a fluorescent reagent. Then GSH/GSSG ratio was calculated.

Histopathologic examination

The left liver lobes were scissored out and fixed in 10% formalin solution for 48 h. The liver samples were then cut into thin transverse sections with the help of microtome and permanent slides were prepared with HE staining. Liver histopathologic changes were examined under an optical microscope with the original magnification × 200.

Data treatment and statistical analysis

The experimental results were expressed as mean ± SE (standard error). Statistical comparison between groups was performed by one-way analysis of variance with SPSS 13.0 statistical software. A P value < 0.05 was indicated as a statistically significant difference.

RESULTS

Effects of APAP alone, 5-MOP multiple-dose and 5-MOP alone on serum enzyme levels

The hepatocellular damage induced by a toxic dose (500 mg/kg) of APAP and the effects of pretreatment with 5-MOP were investigated by measuring the serum levels of ALT, AST and LDH. As shown in Table 1, APAP significantly increased the serum ALT, AST and LDH levels compared with the control group, and the multiple-dose 5-MOP pretreatment significantly prevented the increases of serum enzyme levels. The effect of 5-MOP was dose-dependent, and in the highest dose group, serum levels of ALT, AST and LDH were close to the normal levels as compared with the vehicle control group (P > 0.05).

Table 1 Effects of acetaminophen alone, 5-methoxypsoralen multi-dose and 5-methoxypsoralen alone on alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase activities, hepatic malondialdehyde and reduced glutathione/oxidized glutathione ratio in mice (mean ± SE).

Treatments groups	ALT (IU/L)	AST (IU/L)	LDH (IU/L)	MDA (nmol/mg)	GSH/GSSH
Vehicle control	30 ± 21	146 ± 58	1504 ± 410	0.18 ± 0.11	6.045 ± 0.629
APAP alone at 500 mg/kg	8355 ± 3940a	6482 ± 4018a	24 627 ± 10975a	2.15 ± 1.07a	3.306 ± 0.282a
5-MOP at 12.5 mg/kg	1674 ± 1810c	729 ± 685c	7220 ± 6317c	0.98 ± 0.45	3.834 ± 0.340
5-MOP at 25 mg/kg	54 ± 39c	187 ± 149c	1618 ± 719c	0.59 ± 0.07c	5.330 ± 0.421c
5-MOP at 50 mg/kg	19 ± 9c	141 ± 12c	1394 ± 469c	0.47 ± 0.06c	6.180 ± 0.212c
5-MOP alone at 50 mg/kg	37 ± 20	138 ± 22	1471 ± 191	0.15 ± 0.09	6.858 ± 0.678

^aP < 0.05 vs vehicle control group;

^cP < 0.05 vs acetaminophen (APAP) alone group. 5-MOP: 5-methoxypsoralen; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; LDH: Lactate dehydrogenase; MDA: Malondialdehyde; GSH: Reduced glutathione; GSSG: Oxidized glutathione.

The influences of 5-MOP alone on serum enzyme levels were also observed. There were no statistically significant differences in the serum levels of ALT, AST and LDH between 5-MOP alone group (50 mg/kg) and the vehicle control group (P > 0.05).

Effects of APAP alone, 5-MOP multiple-dose and 5-MOP alone on liver tissue MDA and GSH/GSSG ratio

As seen in Table 1, compared with the vehicle control group, a toxic dose of APAP elevated liver MDA and lowered the hepatic GSH/GSSG ratio. With the escalating dose of 5-MOP (12.5, 25 and 50 mg/kg), the content of MDA decreased and ratio of GSH/GSSG increased.

In the 5-MOP alone group, the MDA level in liver was as low as that in the vehicle control group (0.15 ± 0.09 vs 0.18 ± 0.11, P > 0.05). In addition, the hepatic GSH/GSSG ratio in the 5-MOP alone group was not significantly changed as compared with that in the vehicle control group (6.858 ± 0.678 vs 6.045 ± 0.629, P > 0.05).

Effects of APAP alone, 5-MOP multi-dose and 5-MOP alone on histopathologic changes

The liver histopathologic changes of mice in the six groups are shown in Figure 1. The liver sections displayed the representative hepatocellular morphological changes of each group.

Figure 1 Representative pathological changes of liver section of the six groups (original magnification × 200). A: Section of liver from the vehicle control group showing a normal lobular structure; B: Section of liver from acetaminophen alone group showing large areas of centrilobular necrosis with inflammatory cell infiltration; C: Section of liver from the 12.5 mg/kg 5-methoxypsoralen (5-MOP) dose group showing absence of hepatocellular necrosis and infiltration of inflammatory cells but presence of hepatocellular hydropic degeneration and sinusoidal dilation; D: Section of liver from the 25 mg/kg 5-MOP dose group showing normal hepatocellular morphology but liver lobule structure damage; E: Section of liver from the 50 mg/kg 5-MOP dose group showing a significant alleviation of liver pathological injury with an almost normal lobular structure; F: Section of liver from 5-MOP alone group (50 mg/kg) showing presence of normal lobular structure.

In the vehicle control group, hepatocytes, presenting normal morphology, arranged around the central vein in a radical pattern, and liver lobule structures were clear and regular (Figure 1A). Normal liver lobule structures were damaged and collapsed in the APAP alone group. Large areas of hepatocellular necrosis and infiltration of inflammatory cells were also observed (Figure 1B). 5-MOP administration could alleviate the pathological injury induced by APAP in a dose-dependent manner. 5-MOP at a dose of 12.5 mg/kg could slightly relieve the pathological injury. In this group, no hepatocellular necrosis and infiltration of inflammatory cells were observed, but hepatocellular hydropic degeneration and sinusoidal dilation occurred (Figure 1C). There was no necrosis and hydropic degeneration of hepatocytes, no sinusoidal dilation and infiltration of inflammatory cells in the 25 mg/kg 5-MOP dose group. However, liver lobule structures were still not clear in this group (Figure 1D). 5-MOP at a dose of 50 mg/kg could significantly prevent APAP-induced hepatotoxicity with an almost normal lobular structure comparable to the vehicle control group (Figure 1E). There were no significant liver histopathologic changes in the 5-MOP alone group (Figure 1F).

DISCUSSION

The protective effect of 5-MOP against hepatocellular injury and oxidative stress, and the potential toxic effect of 5-MOP on the liver were investigated in this study. C57BL/6J mice were used because our previous research found that C57BL/6J mice were more susceptible to APAP[33]. The serum levels of ALT, AST and LDH are main indices of liver injury[34] and the levels of MDA, GSH and GSSG can be used as indices of oxidative stress[9]. We evaluated the hepatic protective effect of 5-MOP based on these indices. It is well known that any chemical can be toxic if its dose is high enough, so a 5-MOP alone group was designed to see if the highest therapeutic dose of 5-MOP could cause hepatotoxicity.

APAP used alone can significantly increase the serum levels of ALT, AST and LDH and cause pathological changes as compared with the vehicle control group. Oxidative stress also took place as shown by the increase of MDA level and decrease of GSH/GSSG ratio. The model of APAP-induced hepatotoxicity was successfully established in this experiment.

5-MOP can protect mice from APAP-induced acute liver injury based on the fact that it can decrease the serum ALT, AST and LDH levels in a dose-dependent manner and alleviate the liver histopathologic alterations. Moreover, 5-MOP decreased the MDA level and increased the GSH/GSSG ratio in a dose-related manner, which reflected that 5-MOP could significantly attenuate the oxidative stress induced by APAP and suggested that the hepatoprotective effect of 5-MOP may be associated with its antioxidative activity.

However, besides antioxidant activity, 5-MOP also possesses biological activities to inhibit the mouse and human CYPs both in vivo and in vitro[14]. And CYPs-catalyzed formation of NAPQI is the key mechanism in APAP-induced hepatotoxicity[35]. So we presume that inhibition of CYPs of 5-MOP may also account for the protective mechanism against APAP-induced hepatotoxicity, which should be further investigated.

In the 5-MOP alone group, the serum enzyme (ALT, AST and LDH) levels and histopathologic changes were as normal as in the vehicle control group, which indicated that 5-MOP could not cause liver injury at a dose of 50 mg/kg (the highest therapeutic dose used in this study). The MDA level and the GSH/GSSG ratio were not significantly changed as compared with the vehicle control group, which showed that 5-MOP did not influence the normal oxido-reduction levels.

In conclusion, 5-MOP could protect against APAP-induced hepatotoxicity in mice and had an antioxidative activity, and caused no hepatotoxicity at protective doses.

COMMENTS

Background

Overdose of acetaminophen (APAP) can induce hepatotoxicity and oxidative stress plays a central role in the hepatic damage. Though 5-methoxypsoralen (5-MOP) possesses antioxidative activity suggested by researches in vitro, none of previous studies has found that 5-MOP could prevent APAP-induced hepatotoxicity.

Research frontiers

It is important to search for effective methods to protect human from APAP-induced hepatotoxicity. Despite the various applications of 5-MOP, no research has been conducted to determine if 5-MOP could prevent APAP-induced hepatotoxicity. In addition, although the antioxidative activity of 5-MOP has been evidenced from researches in vitro, this activity was not manifested in vivo. Besides, 5-MOP may also cause hepatotoxicity when taken at high doses.

Innovations and breakthroughs

This study manifested that 5-MOP could protect mice from APAP-induced hepatotoxicity in vivo, and this hepatoprotective effect was associated with its antioxidation activities. In addition, 5-MOP caused no hepatotoxicity at protective doses.

Applications

This study has suggested that 5-MOP can be used at appropriate doses as a drug against APAP-induced hepatotoxicity in human. However, before clinical use, more researches are needed to confirm the safety of APAP administration at protective doses.

Peer review

The authors investigated the protective effects of 5-MOP against APAP-induced hepatotoxicity and whether 5-MOP could cause hepatotoxicity in mice. The results suggested that 5-MOP resisted APAP-induced hepatotoxicity, reduced APAP-induced oxidative stress, and did not cause liver injury at protective doses.