The Protective Effect Of Melatonin On High-dose (-)-Epigallocatechin-3-gallate (EGCG)-triggered Hepatotoxicity And Potential Molecular Mechanism In Mice

(-)-Epigallocatechin-3-gallate(EGCG), a naturally occurring constituent in green tea,has been considered to have numerous health promotion effects. It is commonly believed that the biological activity of EGCG is highly related to its pro-oxidant property and doses level, high-dose levels of EGCG is commonly used to investigate biological effects in animal experiments in recently years. Clinical studies also frequently observed adverse effects of oral administration of high-dose green tea extracts, such as hepatotoxicity,nausea, abdominal pain, and diarrhea, and which has been listed by the US Food and Drug Administration as a major reason for regulating supplements of green tea extracts. Further animal studies confirmed that high-dose levels of EGCG cause toxicity, especially hepatotoxicity, and the toxic effects of EGCG are attributed to its pro-oxidant property.However, the molecular targets and toxicological mechanism of high-dose levels of EGCG not been reported, hence, it is important to systemic explore the mechanism and drug targets of EGCG by different treatment methods and dose levels in vivo.Major antioxidant enzyme, mianly including superoxide dismutase(SOD), catalase(CAT) and glutathione peroxidase(Gpx), converts peroxide to low toxic and innoxious substance via redox cycle. Moreover, the highly stable expressions of antioxidant enzymes maintain redox environment balance regarding as the first line of defense against oxidative damage. Nuclear factor E2-related factor 2(Nrf2) controls antioxidant response element(ARE)-mediated signaling pathway, Nrf2-target antioxidant and phase 2 enzymes consist of the second defense line against oxidative stress. Therefore, antioxidant enzymes and Nrf2 signaling pathway play an important role in protecting xenobiotic compounds against oxidative damage. In this paper, the experimental purpose is conducted to determine the hepatotoxicity of EGCG, and to investigate the toxicological mechanism of high-dose levels of EGCG involve in inhibiting antioxidant enzymes biosynthesis and regulating Nrf2-mediated signaling pathway.Mice were repeated intaperitioneally(i.p.) injected with 55 and 75 mg/kg EGCG(subacute toxicity model) or a signle dose of 200 mg/kg EGCG(acute toxicity model)casued hepatotoxicity and oxidative stress. Chemical analysis showed that EGCG-treatment(55 and 75 mg/kg, i.p.) significantly inhibited hepatic antioxidant enzymes biosynthesis. Furthermore, EGCG(75 mg/kg, i.p.) induced hepatic Nrf2-target genes expression and initiated adaptive recuse pathway via up-regulation of transcriptional level and nuclear distribution of Nrf2. In acute toxicity model, EGCG treatment(200mg/kg, i.p.) not only significantly inhibited hepatic antioxidant enzymes biosynthesis, but also inhibited hepatic Nrf2 transcription, nucleus protein and target genes expression.Simultaneously inhibition of first line antioxidant enzymes and second line Nrf2 pathway by high-dose EGCG treatment aggravated the toxic outcome. These changes were also verified by the oral administration of EGCG in mice. In general, toxic dose of EGCG inhibited antioxidant enzymes biosynthesis, and induced Nrf2 pathway, but lethal dose of EGCG simultaneously inhibited antioxidant enzymes biosynthesis and Nrf2 pathway. In addition, EGCG at non-toxic pharmacological dose(45 mg/kg, i.p.) did not affect antioxidant enzymes biosynthesis, and induced some of Nrf2-target genes expression,whereas, the induction of Nrf2 target-genes expression can be eliminated by co-treatment with antioxidant melatonin.Mice were repeated intaperitioneally(i.p.) injected with 55 mg/kg EGCG(subacute and acute toxicity model) or a signle dose of 125 mg/kg EGCG(acute toxicity model)casued hepatotoxicity and oxidative stress, and the toxic outcome can be alleviated by co-treamtent of melatonin. Melatonin can protect toxic doses of EGCG-triggered hepatotoxicity, hepatic pathological damage, inflammation and oxidative stress. Potential molecular mechanism involves directly attenuating oxidative stress and positive regulating Nrf2 response. Pharmacological effects of EGCG in regulating the metabolism of glucose and lipid genes and activating adenosine monophosphate-activated protein kinase(AMPK)did not affect by melatonin treatment.In conclusion, high-dose EGCG is hepatotoxic to mice, its mechanism of EGCG toxicity involves suppression of hepatic major antioxidant enzymes, and Nrf2 rescue pathway plays a vital role for counteracting the toxicity of EGCG. Nrf2 signaling pathway is biphasic regulated by dosage levels of EGCG, the induction is occurred in toxic dose levels and the suppression is occurred in lethal dose levels. Melatonin can reduce EGCG toxicity thought directly attenuating oxidative stress and positive regulating Nrf2 response,but without compromising its pharmacological action in mice.