Protective effects of green tea polyphenols against subacute hepatotoxicity induced by microcystin-LR in mice

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Abstract

Green tea polyphenols (GTP) have been shown to possess anti-oxidative, anti-mutagenic and anti-carcinogenic activities. The present study aimed to evaluate the chemopreventive efficacy of GTP against subacute hepatotoxicity induced by microcystin-LR (MC-LR) in mice and also elucidates the underlying mechanisms. In this study, healthy Kunming male mice (24–26 g bw) were randomly assigned to five groups. Group I was fed on normal diet and water ad libitum as control. Group II was maintained on normal diet and received MC-LR intraperitoneal injection (10 μg/kg/day) from day 6 till sacrifice. Mice in groups III, IV and V were daily pre-treated with GTP through intragastric administration at doses of 50, 100 and 200 mg/kg/day from day 0 prior to MC-LR intoxication, consecutively 18 days. The results showed MC-LR alone led to oxidative stress and to damage antioxidant defense system, as evidenced by elevation of serum and liver lipid peroxidation. Additionally, hepatocellular apoptosis and injury were significantly observed. GTP pre-treatment caused a significant elevation in serum antioxidant enzymes GSH and SOD activities as well as a decrease in hepatic lipid peroxidation MDA level and serum ALT, AST, ALP activities. GTP pre-treatment obviously inhibited hepatocellular apoptosis and up-regulated Bcl-2 protein expression. The damages in liver were less severe in GTP pre-treated mice in correlation with the biochemical parameters. In summary, this study confirmed that repeated exposure to MC-LR could induce hepatotoxicity. Our study demonstrated that GTP can reduce MC-LR-induced oxidant stress and prevent biochemical parameters and pathological changes caused by MC-LR in a dose-dependent manner. The results indicated that tea polyphenols have a potential to be developed as a preventive agent against MC-LR-induced toxicity and the mechanism involved in the protection could be due to their antioxidant activities.

Introduction

The monocyclic heptapeptides microcystins (MCs) are the most common and abundant cyanotoxins and pose a world health threat to humans and animals (de Figuereido et al., 2004). MCs are characterized as monocyclic heptapeptide with over 80 different MCs isoforms identified (WHO, 2004), but the most frequent and commonly studied isoform is microcystin-LR (MC-LR), followed by MC-RR, MC-YR and MC-LA.

MCs can strongly and irreversibly inhibit the activities of several serine/threonine protein phosphatases in hepatocytes, including PP1 and PP2A. This subsequently induces various responses leading to the disruption of normal cell metabolism and functions, such as protein hyper-phosphorylation, morphological changes of hepatocytes and tumour promotion (Nishiwaki-Matsushima et al., 1992). It is well known that MCs are responsible for illness and death not only in domestic and wildlife animals but also in humans (Guzman and Solter, 1999, Jochimsen et al., 1998). At present, the exact mechanisms by which MCs induce hepatotoxicity and tumour promotion have not been fully elucidated. Several pieces of evidences strongly suggest that oxidative stress may play a significant role in the pathogenesis of MCs-induced toxicity in mammals (Ding and Ong, 2003). Previous studies have shown that intraperitoneal injection of MC-LR resulted in a decrease in the antioxidant enzymes and induced elevated lipid peroxidation (LPO) in serum and relative organs in vivo (Moreno et al., 2005, Guzman and Solter, 1999), which may be related to changes in the antioxidant defense system.

Because of the rapid, irreversible and severe damage to the liver caused by MCs, therapy is likely to have little or no value, and effective prophylaxis is critical. In spite of the potential human hazards associated with MCs, very little work has been done on development of effective chemoprotectants or antidotes against these toxins. GTP, the main components of green tea used worldwide as a popular beverage that exert a wide range of biochemical and pharmacological effects, were numerously reported in the past decades that have significant anti-oxidative, anti-mutagenic and anti-carcinogenic activities. These results suggest that GTP might be a useful cancer chemopreventive agent in the human population (Lung et al., 2002, Yang et al., 1999). The main components of GTP are catechins, which have a polyphenol structure, including [(−)-epigallocatechin-3-gallate (EGCG)], [(−)-epigallocatechin (EGC)], [(−)-epicatechin-3-gallate (ECG)] and [(−)-epicatechin (EC)], all these catechins have strong antioxidant activity (Rice-Evans et al., 1996, Dufresne and Farnworth, 2001, Higdon and Frei, 2003) and are considered as potent scavengers of ROS, such as superoxide, hydrogen peroxide, hydroxyl radicals and nitric oxide produced by various chemicals (Guo et al., 1996, Jovanovic et al., 1995, Khan et al., 1992, Schroeder et al., 2003). The antioxidant activity increased in the following order: EC < ECG < EGC < EGCG. It has been demonstrated that the chemically induced lipid peroxidation in animal's liver and kidney could be inhibited by the intake of tea catechins (Sano et al., 1995).

Considering the diverse functional properties of GTP and the lack of studies on chemoprotectants against MCs-induced toxicity, we therefore undertook the present study to demonstrate whether subacute exposure to MC-LR in mice could modify the antioxidant defense system and induce oxidant damages. In addition, we evaluated the chemopreventive potential benefits of GTP pre-treatment prior to MC-LR and investigated the mechanisms of GTP protection effects.

Section snippets

Chemicals

The cyanobacterial toxin MC-LR (95%) was obtained from Alexis Biochemicals (Switzerland). GTP chemical was obtained from Tianbao biochemicals Co. Ltd., China (Lot# 20041030), among which the main components were 55% of EGCG, 17% of EGC, 14% of ECG, 10% of EC and 0.5% of caffeine, the other ingredients included amino acids (1%), sugars (0.5%), ash (0.5%), water (0.5%), minor elements, etc. GSH, SOD and MDA detection kits, in situ cell apoptosis detection kit (TUNEL) and Bcl-2 SABC detection kit

General observations

No mortality associated with MC-LR and GTP administration was observed throughout the experimental period and the behavior of the MC-LR-treated mice could not be distinguished from that of the controls and GTP pre-treated mice in any of the groups. However, The setting dose of MC-LR exposure has caused a subtle decrease in food consumption, subsequently sufficient to cause detectable weight loss.

Data for initial and final body weights, absolute and relative liver weights are summarized in Table

Discussion

Increasing evidences support the hypothesis that MCs-induced tissue damage may be a consequence of oxidative stress (Ding and Ong, 2003). In the present study, hepatotoxicity was observed either histologically or by clinical chemistry after MC-LR intoxication and the antioxidant defense system is modified by MC-LR treatment, suggesting the role of ROS in the pathogenesis of MC-LR-induced toxicity. We demonstrated that GTP remarkably protected mice from MC-LR-induced oxidative stress and liver

Acknowledgements

The authors would like to thank Dr. Jun-Yan Hong (School of Public Health/Environmental and Occupational Health Sciences Institute University of Medicine and Dentistry of New Jersey, USA) for critically reviewing this manuscript and kindly giving some precious advice. This work was supported by grants from National Natural Science Foundation of China (Grant No. 30630056), Chinese National Program for Key S&T Project (Grant No. 2003BA869C) and Program of S&T (Chongqing) Project (Grant No.

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