Abstract
Epidemiological as well as experimental evidence exists in support of chemopreventive and anticancer properties of green tea and its constituents. The gallocatechin, epicatechin-3-gallate is a major polyphenol present in green tea, shown responsible for these effects. Plant-derived polyphenolic compounds are established natural antioxidants which are capable of catalyzing oxidative DNA degradation of cellular DNA, alone as well as in the presence of transition metal ions, such as copper. Here we present evidence to support that, similar to various other polyphenoic compounds, epicatechin-3-gallate also causes oxidative degradation of cellular DNA. Single cell alkaline gel electrophoresis (Comet assay) was used to assess DNA breakage in lymphocytes that were exposed to various concentrations of epicatechin-3-gallate. Inhibition of DNA breakage in the presence of scavengers of reactive oxygen species (ROS) suggested involvement of ROS generation. Addition of neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation, dose-dependently, in intact lymphocytes. In contrast, bathocuproine, which does not permeate cell membrane, was observed to be ineffective. We further show that epicatechin-3-gallate degrades DNA in cell nuclei, which can also be inhibited by neocuproine, suggesting mobilization of nuclear copper in this reaction as well. Our results are indicative of ROS generation, possibly through mobilization of endogenous copper ions, and support our long-standing hypothesis of a prooxidant activity of plant-derived polyphenols as a mechanism for their documented anticancer properties.
Highlights
Plant-derived polyphenols possess several pharmacological properties, mechanisms of which are not clearly understood
Bathocuproine, impermeable to cell membrane, could still traverse through permeabilized cells, and directly interact with cell nuclei. These results suggest that epicatechin-3-gallate mobilizes chromatin-bound copper, leading to an oxidative DNA breakage
(−)-Epicatechin-3-gallate, tannic acid, cupric chloride, bathocuproine disulphonate, neocuproine, superoxide dismutase (SOD), agarose, Histopaque 1077, Triton X-100 and Trypan blue were purchased from Sigma
Summary
Plant-derived polyphenols possess several pharmacological properties, mechanisms of which are not clearly understood. Cellular DNA Breakage by Epicatechin-3--GGaallllaattee iinn IInnttaacctt CCeellllss aanndd PPeerrmmeeaabbiilliizzeedd CCeellllssaassMMeeaassuurreeddbbyy CCoommeett AAssssaayy. AA ccoommppaarriissoonn ooff cceelllluullaarr DDNNAA bbrreeaakkaaggee iinndduucceedd bbyy eeppiiccaatteecchhiinn--33--ggaallllaattee iinn iinnttaacctt cceellllss ((fifilllleedd cciirrcclleess)) aanndd ppeerrmmeeaabbiilliizzeedd cceellllss ((fifilllleedd ssqquuaarree)) aass aa ffuunnccttiioonn ooff ccoommeett tatailillelennggththss. CCeelllluullaarr DDNNAA bbrreeaakkaaggee bbyy eeppiiccaatteecchhiinn--33--ggaallllaattee iinn hhuummaann ppeerriipphheerraall llyymmpphhooccyytteess iinn tthhee aabbsseennccee aannddprpesreensecnecoef Couf(ICI)u. EEffffeecctt ooff RReeaaccttiivvee OOxxyyggeenn SSccaavveennggeerrss oonn tthhee EEppiiccaatteecchhiinn--33--GGaallllaattee IInndduucceedd CCeelllluullaarr DDNNAA BBrreeaakkaaggee iinn PPeerrmmeeaabbiilliizzeedd CCeellllss. TTaabbllee 11 pprroovviiddeess rreessuullttss ooff tthhee eexxppeerriimmeenntt wwhheerree aann eeffffeecctt ooff ccaattaallaassee,, SSOODD,, aanndd tthhiioouurreeaa wweerree tteesstteedd oonn eeppiiccaatteecchhiinn--33--ggaallllaattee--iinndduucceedd DDNNAA ddeeggrraaddaattiioonn iinn ppeerrmmeeaabbiilliizzeedd llyymmpphhooccyytteess. EEffffeecctt ooff rreeaaccttiivvee ooxxyyggeenn ssppeecciieess ssccaavveennggeerrss oonn eeppiiccaatteecchhiinn--33--ggaallllaattee iinndduucceedd cceelllluullaarr DDNNAA bbrreeaakkaaggee iinn ppeerrmmeeaabbiilliizzeedd cceellllss. Bathocuproine, impermeable to cell membrane, could still traverse through permeabilized cells, and directly interact with cell nuclei These results suggest that epicatechin-3-gallate mobilizes chromatin-bound copper, leading to an oxidative DNA breakage
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