Effects of Erdosteine on Acetaminophen-induced Hepatotoxicity in Rats

Abstract

We investigated the effects of erdosteine on acetaminophen (APAP)-induced hepatotoxicity in rats. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), AST (aspartate aminotransferase), and ALT (alanine transaminase) activities, and malonyldialdehyde (MDA) and nitric oxide levels as oxidant/antioxidant biochemical parameters were investigated with light microscopic evaluation in adult female Wistar Albino rats. APAP administration produced a decrease in hepatic SOD, CAT, and GSH-Px activities, and coadministration of erdosteine (150 and 300 mg/kg) resulted in increases in the activities. MDA and NO levels increased in the APAP group, and erdosteine treatments prevented these increases. Significant elevations in serum AST and ALT levels were observed in the APAP group, and when erdosteine and APAP were coadministered, their serum levels were close to those in the control group. Light microscopic evaluation of livers showed that there were remarkable centrilobular (zone III) hepatic necrosis and mild to moderate sinusoidal congestion in the APAP group, whereas in the erdosteine group, cellular necrosis was minimal and the hepatocytes maintained a better morphology when compared to the APAP group. Erdosteine prevented APAP-induced liver injury and toxic side effects probably through the antioxidant and radical scavenging effects of erdosteine.

Introduction

Paracetamol or acetaminophen (APAP) is one of the most commonly used medications worldwide (Kaufman et al. 2002). However, the risk of hepatotoxicity becomes substantial even at low dosages, such as 7.5 g in adults or 150 mg/kg in children. Additionally, chronic APAP usage may cause hepatotoxicity even in low dosages such as 4 g in adults or 90 mg/kg in children (Geiger and Howard 2007). Damaged hepatocytes release characteristic liver enzymes such as aspartate amino-transferase (AST) and alanine aminotransferase (ALT) into serum. Measurement of the levels of these enzymes in the blood provides a reliable clinical measure of hepatotoxicity (Singer et al. 1995).

About 4% of the drug is metabolized to N-acetyl-p-benzo-quinoneimine (NAPQI) via the cytochrome P-450 isoenzyme mixed-function oxidase system (Corcoran et al. 1980). This potentially toxic intermediate conjugates with glutathione, forming the nontoxic metabolites cysteine and mercaptouric acid conjugates. An alternative view is that oxidative stress has a role in hepatotoxicity. There are many characteristic features of oxidative stress in APAP hepatotoxicity, including lipid peroxidation, mitochondrial damage, ATP depletion, and formation of nitrotyrosine adducts in proteins, presumably owing to formation of superoxide-derived peroxynitrite (Jaeschke et al. 2003). However, these processes may be consequences of the damage mediated by protein adduction rather than the direct effect of hepatotoxicity (Josephy 2005).

Many antioxidant agents have been studied in experimental and clinical studies to reduce or prevent APAP-induced hepatotoxicity. The most popular antioxidant for APAP hepatotoxicity is N-acetyl-L-cysteine (NAC) (Buckley et al. 1999; Rumack 2002; Yagmurca et al. 2007). Clinically, the cysteine prodrug NAC (Yagmurca et al. 2007) is the longstanding standard therapy for APAP overdose (Prescott et al. 1977). Protection by NAC is believed to be attributable to its ability to regenerate glutathione (GSH) stores because of its capacity to provide cysteine residues (Harrison et al. 1991). Furthermore, because NAC is known as an antioxidant, it might be useful in the setting of liver injuries with different etiologies (Zwingmann and Bilodeau 2006).

Erdosteine, a widely and orally used mucolytic and expectorant in clinics, has two blocked thiol groups. The active metabolites of erdosteine exhibit free radical scavenging activity via its sulphydryl groups released following the catabolism of erdosteine in the liver (Dechant and Noble 1996; Fadillioglu et al. 2003; Gazzani et al. 1989). There are various clues about the antioxidant effects of erdosteine in animals, for example, an oral erdosteine treatment prevents lipid peroxidation in rats with doxorubicin-induced heart toxicity (Fadillioglu et al. 2003; Sogut et al. 2004), bleomycin-induced pulmonary fibrosis (Sogut et al. 2004), and rotenone-induced hepatotoxicity (Terzi et al. 2004). The aim of this study was to investigate the protective effects of erdosteine on APAP-induced hepatotoxicity in rats.

Material and Methods

Animals and Experimental Procedures

Adult female Wistar Albino rats with body weights between 220 and 280 g were used in the experiments. The animals were housed in silent rooms with a twelve-hour light-dark cycle (7:00 am–7:00 pm), and the animals were treated according to the guidelines of the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996), and the Ethics Committee of the Veterinary School of the Mustafa Kemal University approved the study protocols.

The rats were randomly divided into five groups, with an equal number of rats in each group: the control group; the erdosteine (150 mg/kg)-alone group; the APAP (1 g/kg)-alone group; the erdosteine (150 mg/kg)-plus-APAP (1 g/kg) group, in which rats were treated orally with a single dose of erdosteine forty minutes after oral administration of APAP; and the erdosteine (300 mg/kg)-plus-APAP (1 g/kg) group. The erdosteine (Ilsan-Iltas Pharmaceuticals, Istanbul, Turkey) was prepared in distilled water and administered orally via gavage. Twenty-four hours after APAP treatment, anesthesia was administered intramuscularly by using ketamine hydrochloride (75 mg/kg) and xylazine hydrochloride (8 mg/kg), a venous blood sample was taken, and a serum sample was separated. Rats were decapitated, and the liver was rapidly excised and pieced into two parts for microscopic examination and biochemical analysis. The liver tissue and serum were stored in a freezer at −30°C until measurement of enzymatic activities.

Oxidant and Antioxidant Parameters of Liver

Hepatic tissue samples were homogenized (for two minutes at 5000 rpm) in four volumes of ice-cold Tris-HCl buffer (50 mMol, pH 7.4) by using a glass teflon homogenizer (Ultra Turrax IKA T25 Basic, Jonke & Kunkel GmbH & Co. KG, Staufen, Germany). Malonyldialdehyde (MDA), nitric oxide (NO), and protein levels were measured in the homogenate, and the homogenate was centrifuged at 5000 rpm for sixty minutes to remove debris. Supernatant fluids were collected, and analyses of catalase (CAT) and glutathione peroxidase (GSH-Px) activities as well as measurement of protein concentration were performed. The supernatant solutions were mixed with an equal volume of an ethanol/chloroform mixture (5/3, volume per volume). After centrifugation at 5000 rpm for thirty minutes, the clear upper layer (the ethanol phase) was collected and used for the analysis of superoxide dismutase (SOD) activity and protein assays. All preparation procedures were carried out at +4°C.

The MDA level was determined by a method based on its reaction with thiobarbituric acid at 90°C–100°C (Esterbauer and Cheeseman 1990). Since NO measurement is very difficult in biological specimens, tissue nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) were estimated as an index of NO production, and the colorimetric assay based on the Griess reaction for assessment of NO activity was used (Cortas and Wakid 1990). The total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to a method adapted from Sun et al. (1988). The principle of the method is based on the inhibition of nitroblue tetrazolium reduction by the xanthine–xanthine oxidase system as a super-oxide generator. CAT (EC 1.11.1.6) activity was determined according to the method of Aebi (1984). GSH-Px (EC 1.6.4.2) activity was measured according to a method that was defined by Paglia and Valentine (1967). Protein assays were conducted using the method of Lowry et al. (1951).

Serum AST and ALT levels

Hepatic injury was assessed using the serum levels of AST and ALT and by histologic studies. The serum AST and ALT levels were determined using automated enzyme analyzers (Syncron LX 20, Ireland) and commercial Beckman Coulter diagnostic kits.

Histological Evaluation

For the light microscopic examinations, hepatic samples from the left lobe were fixed in 10% neutral buffered formalin and embedded in paraffin. The paraffin blocks were cut into pieces with a thickness of 4 μm. The sections were stained with hematoxylin and eosin (H&E) for evaluation of hepatocyte injury.

Statistical Analysis

Data were analyzed using a commercially available statistics software package (SPSS for Windows, version 11.5, Chicago, IL, USA). The distributions of each group were analyzed with a one-sample Kolmogrov-Smirnov test. All groups showed normal distribution, so parametric statistical methods were used to analyze the data. One-way analysis of variance (ANOVA) test was used, and post hoc multiple comparisons were performed with least significant difference (LSD). Results are presented as means ± standard deviation. P values less than .05 were accepted as statistically significant.

Results

Biochemical Results

All biochemical analyses were based on single measurements.

Oxidant and Antioxidants

The hepatic MDA and NO levels with SOD, CAT, and GSH-Px activities are presented in Table 1. The tissue MDA levels of the APAP-alone group were significantly higher in comparison to the control and erdosteine groups (p < .001). Erdosteine therapy prevented this increase with both the 150 and 300 mg/kg doses (p <.001). The activities of SOD, CAT, and GSH-Px were decreased in the APAP-alone group in comparison to the control groups (p <.05). The activities of SOD, CAT, and GSH-Px were higher in the erdosteine-treated APAP groups than in the APAP-alone group (p <.05). There was a statistically significant difference between the levels of NO in the APAP-treated groups in comparison with the control and erdosteine-alone groups (p <.05). Erdosteine therapy of the APAP groups caused a decrease in the NO levels in comparison with the APAP-alone group (p <.05).

Serum AST and ALT Levels

A statistically significant increase was detected in the serum AST and ALT levels of the APAP-alone rats in comparison with the control and erdosteine-alone groups (p <.05) (Table 2). Erdosteine therapy of the APAP-treated rats caused a decrease in the serum AST and ALT levels (p <.05). Although the serum AST levels were significantly lower in the erdosteine group treated with 150 mg/kg compared with the APAP-alone group, it was still higher than that of the control group (p <.05).

Light Microscopic Examination Results

Light microscopic examination of liver samples was assessed semiquantitatively by visual inspection of the sections, following the procedures of Valentovic et al. (2004) and Nishida et al. (2006). Sections of erdosteine groups showed no abnormality in liver histology. Hepatocyte plates were normal, and sinusoidal narrowing or congestion was not observed. In addition, hepatocellular vacuolization was not seen, and the lobuli were regular in shape (Figure 1).

APAP administration to the rats revealed a remarkable centrilobular (zone III) hepatic necrosis and mild to moderate sinusoidal congestion. Cytoplasmic changes and sinusoidal narrowings around the central vein were slightly more visible in the APAP-alone than the erdosteine-alone and APAP-plus-erdosteine groups. Necrosis of the centrilobular cells was advanced, with nuclear disintegration and cytoplasmic eosinophilia. The centrilobular zones were infiltrated with mononuclear and occasionally polymorphonuclear cells in the APAP group, in which there was a loss of basophilic granules in the cytoplasm of centrilobular hepatocytes with vacuolation (Figure 1b).

In the erdosteine (150 and 300 mg/kg)-treated groups, cellular necrosis was minimal and the hepatocytes maintained a better morphology when compared with the APAP-alone group. In these groups, marked decreases in cytoplasmic changes of the hepatocytes, sinusoidal narrowings, and congestions around the central vein were noticed when compared with the APAP-alone group. The inflammatory cell infiltration in the central vein was less in the APAP-plus-erdosteine groups than the APAP-alone group (Figures 1c and 1d).

Discussion

The study demonstrated that APAP causes liver injury in rats. There was various evidence of hepatotoxicity, including ALT and AST activity as well as histopathological findings. We found that the serum levels of both ALT and AST were elevated almost fourfold in the APAP-treated group in comparison with the control group. Similarly, different studies have reported that hepatocytes are targeted by APAP (Gazzani et al. 1989; Prescott et al. 1977; Slattery et al. 1987). Another finding in the present study is that the APAP-plus-erdosteine groups showed reduced microscopic changes of the liver compared with the APAP-alone group. There were infiltrations of mononuclear and occasionally polymorphonuclear cells of the centrilobular zones in the APAP groups. Bauer et al. (2000) have shown that when rats were treated with APAP at a dose of 1 mg/kg, there was an APAP-induced inflammatory response in the liver. This inflammatory reaction was, at least in part, owing to the release of chemotactic factors from the hepatocytes. Our findings were consistently similar, and treatment with erdosteine considerably reduced the inflammatory cells induced by the APAP injection. Additionally, histological findings showed that APAP administration to rats revealed a remarkable centrilobular (zone III) necrosis, cytoplasmic changes, and sinusoidal narrowings around the central vein, and it has also been reported in some other studies that APAP intoxication can result in severe hepatic damage characterized by hemorrhagic centrilobular (CL) necrosis in both humans and animals (Thomas 1993; Valentovic et al. 2004). On the other hand, the severity of vacuolization in hepatocytes also decreased after injection of erdosteine.

Tissue MDA level is an important indicator of lipid peroxidation. The present study showed that there was a higher liver MDA level in the APAP group in comparison with the control group. Moreover, our findings indicated that APAP therapy causes depletion of the antioxidant enzymes, including SOD, CAT, and GSH-Px. Approximately 90% to 95% of APAP is metabolized by conjugation with glucuronides and sulfates, which increases urinary excretion. The remaining 5% is normally converted into NAPQI through the action of the cytochrome P450 system, primarily the CYP2E1, in the liver. NAPQI, as one of the metabolites of APAP, is probably responsible for the primary hepatotoxicity of the drug (Kaplowitz 2004). Glutathione is an important protective agent against tissue injury, and the sulfhydryl part of glutathione conjugates with electrophilic and highly reactive NAPQI to detoxify it. During overdosing of the toxic agent, detoxification is limited because of insufficient glutathione resulting from depletion (Geiger and Howard 2007; Josephy 2005). Our study also showed a depletion of GSH-Px activity in APAP toxicity. GSH-Px is an important enzyme for protection from GSH depletion in tissues. High doses of APAP cause formation of NAPQI, so sulfate stores are consumed (Josephy 2005). The accumulating NAPQI reacts with other sulfhydryls in hepatocytes. Hepatotoxicity may directly result from either protein damage or by the accumulation of reactive oxygen species and proinflammatory cytokines after the initial insult (James et al. 2003). One of the other results is NO level in liver tissue. NO is an important indication of the inflammatory process. Recent data have shown that nitrated tyrosine residues as well as APAP adducts occur in the necrotic cells following toxic doses of APAP (James et al. 2003). Formation of peroxynitrite by rapid reaction of superoxide anion and NO mediates nitrotyrosination. During detoxification of peroxynitrite, GSH has an important role. In our study, tissue NO level was dramatically increased following APAP administration. Moreover, the significance of cytokines and chemokines in the development of toxicity and repair processes has been demonstrated by several recent studies. Kupffer cells may also release a number of inflammatory cytokines, and multiple cytokines are released in APAP toxicity (Bourdi et al. 2002; Hogaboam et al. 2000; Laskin et al. 1995).

As also confirmed by the microscopic and biochemical findings, the present study demonstrated that treatment with erdosteine markedly improves hepatotoxicity induced by APAP. Moreover, all of the parameters indicating the presence of oxidative injury were markedly reversed by erdosteine treatment, suggesting that erdosteine has a potent anti-inflammatory effect on APAP hepatotoxity. GSH is an important constituent of intracellular protective mechanisms against various noxious stimuli, including oxidative stress. Erdosteine prevented the depletion of antioxidant enzymes including GSH-Px in the present study. We used two different dosages for erdosteine treatment; however, according to our results, there was no difference between them. Yagmurca et al. (2007) demonstrated that erdosteine treatment against doxorubicin-induced hepatotoxicity improved increased lipid peroxidation, protein oxidation, and NO level. They found that erdosteine treatment with a dose of 50 mg/kg/day recovered the hepatotoxicity histopathologically. Our results confirm the results of Yagmurca et al., that erdosteine treatment prevents hepatotoxicity via both prevention of lipid peroxidation and production of NO, since erdosteine has two sulfhydryl compounds, and its reactive metabolites may have role in this protection.

As a conclusion, erdosteine probably protects from APAP-induced hepatotoxicity by preventing free radical damaging cascades, oxidant radical release, and through its prevention from proinflammatory processes, but further experimental and clinical studies are required to confirm these findings.