Synergistic toxicity of epigallocatechin-3-gallate and diethyldithiocarbamate, a lethal encounter involving redox-active copper
Graphical abstract
Introduction
Green tea (Camellia sinensis) is a widely consumed beverage. Epigallocatechin-3-gallate (EGCG) is the major catechin form of green tea and is believed to be the most active constituent. Many reports indicate that tea catechins and EGCG have preventive effects against many types of chronic diseases, including cancer, obesity, type-2 diabetes mellitus and cardiovascular diseases [1]. Some of the biological effects of EGCG are associated with its antioxidant or pro-oxidant properties, depending upon the dose levels and the biological environments [1], [2], [3]. Green tea extracts, with EGCG being the most abundant catechin, are readily available as dietary supplements, mainly for the purpose of body weight reduction. The benefit-to-risk ratio of high levels of EGCG supplementation has not been well defined. Hepatotoxicity, nausea, abdominal pain and diarrhea owing to copious consumption of green tea extracts have been identified in some individuals [4]. The adverse effect of excessive EGCG has been ascribed to its pro-oxidant actions [3], [5]. Upon EGCG auto-oxidation, reactive oxygen species (ROS) including hydrogen peroxide and superoxide anion are formed [6], [7]; in the meantime, EGCG is transformed to quinones, which can covalently bind to the free thiol group of cysteine residues of proteins, leading to the formation of quinoproteins [8]. Moreover, transition metals, such as copper and iron ions, promote EGCG oxidation and amplify EGCG-triggered ROS production [6], [9].
Versatile dithiocarbamates (DTC), a class of thiono-sulfur containing compounds, have been used in industry as vulcanization accelerators for rubber manufacture, in agriculture as insecticides, herbicides and fungicides, and in medicinal domain as an antidote against metal poisoning or for treatment of viral, fungal and bacterial infections [10]. Disulfiram (DSF), which is metabolized to diethyldithiocarbamate (DEDTC) in vivo, has been used for more than 60 years as a drug for alcohol aversion therapy of alcohol abuses [11]. A wide range of new medical applications of DTC have been explored. DEDTC and DSF are receiving considerable attention as promising anticancer agents, mainly owning to their binding to copper [12], [13], [14]. Copper is the third most abundant trace element in the human body, with the highest concentration in the liver [15]. Copper plays a wide and pivotal role in biological systems because it serves as a co-factor of many enzymes, such as Cu/Zn-superoxide dismutase (SOD), cytochrome oxidase, ascorbate oxidase and tyrosinase. Due to its exceptionally high redox activity, excessive copper causes DNA damage, lipid peroxidation and protein dysfunction [16]. Therefore, intracellular free copper concentrations need to be well regulated. Biological systems have evolutionarily developed a sophisticated network of proteins to modulate redox activity of copper ions and to keep their quantities in line with the cellular needs [16], [17]. The upper limit of pools of free copper has been estimated to be far less than a single free copper ion per cell [18]. DTC have a strong capacity of chelating copper through forming Cu(DEDTC)2 complex. Such copper complex is lipophilic and can act as a copper ionophore to transport copper into the cells, causing intracellular copper overload [19], [20]. Animal studies have found that DSF causes redistribution of copper and significantly increases hepatic copper levels [21], [22] and DEDTC can efficiently inhibit SOD activity [23].
As the most active catechin in green tea, the interactions between EGCG and various drugs or nutrients have been explored. Dietary quercetin can enhance bioavailability of EGCG via inhibiting catechol-O-methyltransferase that promotes EGCG methylation and reduces bioactivity of EGCG. Consequently, the combination of green tea polyphenols and quercetin, like the co-treatment of EGCG and entacapone or tolcapone, two catechol-O-methyltransferase inhibitors, generates enhanced anti-cancer effects [24], [25]. Vardenafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, has been found to significantly potentiate therapeutic efficacy of EGCG on tumors in animal models [26]. In this experimental system, EGCG binds to 67-kDa laminin receptor and initiates cGMP-dependent apoptosis, and vardenafil inhibits the hydrolysis of cGMP by phosphodiesterase 5 to enhance EGCG-induced apoptosis. The efficacy of clinically-used bortezomib and other boronic acid-based proteasome inhibitors are dramatically attenuated by green tea polyphenols, mainly due to covalent binding of EGCG to boron that is essential for this class of anti-cancer drugs to disrupt proteasome [27].
DEDTC not only modulates copper distribution but also inactivates SOD. Since SOD participates in attenuating EGCG auto-oxidation [28] and copper ions promote EGCG oxidation, we thus hypothesize that DEDTC would promote pro-oxidant actions and enhance toxicity of EGCG. The present study tested this hypothesis by investigating the interactions between EGCG and DEDTC in mice. We found that co-administration of EGCG and DEDTC, both at tolerable doses, caused a lethal consequence mainly due to EGCG auto-oxidation catalyzed by redox-active copper.
Section snippets
Chemicals and drugs
EGCG (99%) purified from green tea was obtained from Ebeikar Tea & Extracts Co., Ltd. (Hangzhou, China). DSF, DEDTC, 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), thiobarbituric acid, glyceraldehyde phosphate dehydrogenase (GAPDH), nitroblue tetrazolium (NBT) and bovine serum albumin was purchased from Sigma (St. Louis, Missouri, USA). ECL Plus reagent and PVDF membrane were products of Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The primary antibodies against histone H2AX (γH2AX) and
Co-administration of EGCG and DSF/DEDTC causes toxicity and lethality in mice
Mice were i.p. injected with saline as control, 45 mg/kg EGCG, 300 mg/kg DSF or the combination, once daily for 3 days. Mice were tolerant to treatment with EGCG or DSF alone; surprisingly, 83% of mice in the combination group died within the two-week observation period (Fig. 1a). In a second experiment, mice were i.p. injected with saline as control, 30 mg/kg EGCG, 500 mg/kg DEDTC or the combination, once daily for 3 days; while the individual treatment did not trigger toxic reactions, the
Discussion
EGCG has been suggested to have many beneficial health effects. However, consumption of large quantities of green tea extracts, in which EGCG is a major functional ingredient, causes hepatotoxicity in some individuals. The current study demonstrates for the first time that DEDTC substantially reduces the safety threshold of EGCG, or copious consumption of EGCG enhances the toxicity of DEDTC, as outlined in Fig. 7.
The toxicological mechanism of EGCG is related to its pro-oxidant action [3], [5],
Acknowledgements
This work was supported by National Natural Science Foundation of China (31771971 and 31170648). We thank Guangshan Zhao for the performing on animal experiment in Fig. 1a, and Pingping Chen on animal experiment in Fig. 6a, b.
Competing interests
The authors declare no competing financial interests.
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Contributed equally.