Studies have shown that intake of green tea or green tea polyphenols (GTPs) can reduce the development and progression of various diseases such as cancer, cardiovascular disease, and neurodegenerative diseases [1
]. The principal hypothesis associated with the benefits of green tea is related to the strong free radical scavenging, antioxidant, and anti-inflammatory properties of these polyphenol compounds [3
]. In addition, GTPs can change drug metabolism by modulating drug-metabolizing enzymes and transporters [6
]. These actions may change the fate of drug metabolism and toxicity. Because of the many polyphenolic components in GTPs, discrepancies exist concerning their effects on drug metabolism and toxicity [6
Among the various tea polyphenols, epigallocatechin-3-gallate (EGCG) is the most abundant and active polyphenol in green tea. Recently, research into the beneficial effects of green tea on health promotion has focused on EGCG [2
]. Studies have shown that EGCG can protect the liver from thioacetamide and triptolide-induced hepatotoxicity [7
]. However, some studies have also shown that high-dose EGCG administration to animals (through an intragastric tube or intraperitoneal injection) can cause oxidative damage to the liver [9
]. To our knowledge, the dose and route by which EGCG is given to animals can be important factors in determining whether oxidative damage will occur. To date, various commercial EGCG products are on the market worldwide. However, little is known about the effect of dietary EGCG on oxidative stress and drug-metabolizing systems, especially its effect on the metabolism and toxicity of prescribed drugs such as acetaminophen (N
-aminophenol, APAP). Therefore, it is of considerable importance to evaluate the interactions between EGCG and APAP and their effects on hepatotoxicity.
APAP is widely used as an over-the-counter analgesic and antipyretic agent. APAP overdose is now the most common cause of acute hepatic failure [12
]. APAP is metabolized primarily by glucuronidation and sulfation reactions to produce the nontoxic metabolites APAP–glucuronate and APAP–sulfate [13
]. APAP overdose can increase the cytochrome P-450 (CYP)-mediated bioactivation of APAP to form a highly reactive metabolite, N
-benzoquinone imine (NAPQI), which exerts its toxicity by covalent binding to cellular macromolecules [14
]. In addition, NAPQI reacts with glutathione (GSH), leading to cellular GSH exhaustion, mitochondrial damage, and cell apoptosis in the liver [15
]. The removal of damaged organelles, including mitochondria, by autophagy can protect hepatocytes against APAP-induced mitochondrial damage and subsequent necrosis [17
]. The other way to lower APAP toxicity is to facilitate the excretion of glucuronate, sulfate, GSH conjugates, and oxidative stress products from the liver by increasing the expression of membrane transporters such as multidrug resistance-associated protein (Mrp)2/3 or reduced uptake transporters such as organic anion-transporting polypeptide (OATP) 1a1 and OATP 1b2 [18
Administration of GTPs has been shown to provide protection against APAP-induced liver injury [21
]. In our pilot study, supplementation with EGCG (0.54%, w
) in the diet for one week had an inhibitory effect against APAP-induced liver injury in rats [22
]. However, the reactive oxygen species (ROS) level in the liver might also have been increased by EGCG treatment (Table S1
), suggesting that the oxidative stress was mildly increased by short-term exposure of EGCG. In addition, the mechanism by which EGCG lowers APAP-induced liver damage is still not clear. In the present study, rats were fed a diet containing EGCG for a longer time (four weeks) to investigate the effects of EGCG on oxidative stress, drug-metabolizing enzymes, and membrane transporters in the liver. Then, the effects of EGCG on the metabolism and toxicity of APAP in the liver were investigated.
In the present study, the results showed that EGCG supplementation for four weeks significantly reduced oxidative stress and the activities of several drug-metabolizing enzymes in the rat liver. After challenge with APAP, EGCG reduced CYP-mediated APAP bioactivation and apoptosis and mildly increased autophagy in the liver. In addition, EGCG increased the activities of antioxidant enzymes, including GSH peroxidase and NQO-1, and decreased the expression of the uptake membrane transporter, OATP 1a1, after APAP treatment. These results indicate that dietary EGCG may reduce APAP-induced hepatotoxicity by lowering CYP-mediated APAP bioactivation, increasing antioxidant enzyme activity, and reducing the accumulation of toxic products in the liver.
EGCG shows both antioxidant and pro-oxidant effects in biological systems. EGCG acts as a pro-oxidant compound when it undergoes metabolic processes that produce ROS [9
]. At high doses, the oxidized form of EGCG is EGCG o-quinone, which reacts with glutathione to form thiol conjugates, resulting in the accumulation of EGCG o-quinone in hepatocytes and causing liver damage [40
]. In our preliminary study, EGCG supplementation in the diet (0.54%, w
) caused a transient increase in ROS level in the liver during the first week of EGCG treatment, but the production of ROS may act as a signal to upregulate GSH synthesis and GSH peroxidase activity in the liver. Therefore, EGCG did not cause any hepatotoxicity (Table S1
). In this study, hepatic ROS, GSSG, and TBARS contents were significantly decreased after four weeks of treatment with the same dose of EGCG, indicating that the oxidative stress in the liver was diminished. This phenomenon could be partly explained by the rats having adapted to the high dose of EGCG (0.6% in the diet) and maintained a high antioxidant capacity in the liver after four weeks of EGCG treatment. The other possibility is that EGCG supplementation in the diet may have lowered the intestinal absorption rate of EGCG compared with EGCG administered to the animal by intragastric [10
] or intraperitoneal injection [11
], resulting in a lower concentration of plasma EGCG. Therefore, in this study, EGCG administration for four weeks reduced oxidative stress in the liver and caused no hepatotoxicity.
After 12 h of administration of a single dose of APAP to rats, APAP caused liver damage, which was characterized by an increase in the plasma ALT concentration, a change in hepatocyte morphology, a dramatic decrease in GSH content, and a decrease in liver GSH peroxidase activity (Figure 2
). In addition, APAP increased the Bax/Bcl2 ratio with little or no change in the LC3B-II/LC3B-I ratio in the liver, indicating that APAP induced hepatocyte apoptosis without affecting autophagy. Notably, treatment with EGCG caused a lower Bax/Bcl2 ratio and a higher LC3B II/LC3B I ratio, indicating that EGCG could reduce apoptosis and induce autophagy (Figure 3
B,C). Activation of autophagy by EGCG has been demonstrated to protect against APAP-induced hepatotoxicity [41
]. These results suggest that inhibition of apoptosis and induction of autophagy after EGCG challenge may protect the liver against APAP-induced hepatotoxicity.
In this study, the increased GSH peroxidase activity due to EGCG may have reduced ROS production during CYP-mediated APAP metabolism. EGCG has been shown to be proficient at scavenging free radicals [5
]. Therefore, the reduced oxidative stress by EGCG after APAP challenge may be partially attributed to its direct and/or indirect increase in antioxidant activity or decrease in ROS production in the liver, even though the oral bioavailability of EGCG is low (<1%) [42
Regarding the drug-metabolizing enzyme activity, a previous study showed that CYP3A, sulfotransferase, and GST enzyme activities in the liver were significantly reduced after one week of EGCG feeding (0.54%, w
]. In this study, hepatic CYP3A, CYP2E1, CYP1A2, UGT, and GST activity was suppressed after four weeks of EGCG feeding. This observation is consistent with previous results showing that orally administered EGCG reduces the activities of hepatic drug-metabolizing enzymes [6
]. These results suggest that dietary EGCG may lower the metabolism of drugs or toxic compounds in the liver.
Consistent with previous findings, in this study, APAP treatment for 12 h reduced the activity of drug-metabolizing enzymes and antioxidant enzymes. It is known that APAP-induced liver toxicity is mediated by covalent binding to critical proteins or enzymes with NAPQI [44
]. A toxic dose of APAP to animals decreases the catalytic activity of the hepatic enzymes, including CYP enzymes, UGT, GST, and glutathione peroxidase, probably due to covalent binding to these cellular proteins [22
]. In addition to CYP2E1 and CYP1A2, CYP3A is an important enzyme responsible for the CYP-mediated bioactivation of APAP to generate the electrophile NAPQI in both human and rats, especially when an overdose of APAP is administered [35
]. In this study, EGCG administration caused lower hepatic CYP3A, CYP2E1, and CYP1A2 activity prior to APAP injection (Table 1
). These changes caused by EGCG may result in a lower CYP-mediated NAPQI production after APAP treatment. Indeed, a lower formation of APAP–GSH and APAP–protein adducts was observed in the liver (Table 3
). In addition to APAP–protein adducts, APAP–GSH is toxic to the liver because the conjugate can induce mitochondrial impairment, which can lead to enhanced ROS production [49
]. In this study, EGCG increased NQO1 activity after APAP treatment, which may enhance the conversion of NAPQI back to the parent APAP. Therefore, EGCG suppressed CYP enzyme activity prior to APAP injection and had higher NQO1 activity after APAP challenge resulted in lower NAPQI production, which might lead to lower formation of APAP–protein adducts and APAP–GSH in the liver (Table 3
). Therefore, EGCG lowering APAP-induced hepatotoxicity is likely due to its ability to reduce CYP enzyme activity and enhance NQO1 activity and, thus, lower CYP-mediated APAP bioactivation.
Several studies have indicated that the expression of efflux membrane transporters such as Mrp2/3 and p-glycoprotein is increased after APAP intoxication [18
]. In general, APAP–glucuronide and APAP–GSH are mainly excreted into bile, while APAP–sulfate is mainly excreted into urine [51
]. These membrane proteins can remove toxic metabolites and oxidative products from the liver via the urine or bile. Uptake transporters of OATPs in the liver, such as Oatp1a1 and Oatp1b2, which mediate the uptake of numerous drugs and xenobiotics into cells, were reduced after APAP treatment [18
]. Roth et al. [52
] showed that the expression of Oatp1a1 is inhibited by EGCG. In this study, the protein expression of Mrp2 and Mrp3 was slightly increased and that of OATP1a1 was decreased after 12 h of APAP treatment. EGCG had no effect on Mrp2/3 and p-glycoprotein protein expressions in the liver; however, EGCG significantly reduced (−37.5%) OATP1a1 expression. These results suggest that EGCG may reduce the hepatic uptake of APAP and its metabolites from the circulation into the liver. On the other hand, in this study, EGCG supplementation also lowered fecal β-glucuronidase activity, which might diminish deconjugation of APAP–glucuronide and thus increase fecal APAP–glucuronide excretion. This observation is similar to the results of a previous study [53
]. Therefore, the reduced hepatic OATP1a1 protein expression and microbial β-glucuronidase activity due to EGCG may lead to lower reabsorption of APAP into the liver from the circulation and intestine, respectively. These actions may lower repeated CYP-mediated APAP bioactivation in the liver.
In summary, the results of this study show that EGCG supplementation for four weeks reduced APAP-induced liver damage in rats. The mechanisms contributing to the detoxification of APAP by EGCG may include reduced CYP-mediated APAP bioactivation, oxidative stress, and apoptosis; and increased autophagy and lower accumulation of toxic products in the liver.