ISIS Report 30/05/12
Green tea polyphenol antioxidant protects against bystander effects of low dose ionizing radiation that damage cells and cause numerous diseases including cancer Dr. Mae-Wan Ho
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The recent discovery of bystander effects from low levels of ionizing radiation has thrown risk assessment and radioprotection into disarray  (Bystander Effects Multiply Dose and Harm from Ionizing Radiation, SiS 55). However, it has also led to the discovery of potential mitigating measures against exposure to radioactivity, especially from nuclear accidents like Chernobyl (and Fukushima), the devastation health impacts of which are still surfacing 25 years later  (Chernobyl Deaths Top a Million Based on Real Evidence. SiS 55).
Ionizing radiation has been known to produce free radicals and reactive oxygen species (ROS), predominantly by ionizing water, the most abundant molecules in tissues and cells (see  for an explanation of ROS). ROS are responsible for oxidative damage to DNA, proteins, and lipids, initiating cell death, genomic instability and other consequences of radiation, both in cells that have been directly targeted, and in bystander cells that have not been irradiated . There is evidence that various antioxidants can protect cells against bystander radiation damages, and new findings published online in Mutation Research appear particularly promising.
Ashu Tiku and Benila Richi at Jawaharlal Nehru University, New Delhi and Roasaheb Kale at Central University of Gujarat in India may have found the ideal antioxidant for radioprotectopm .
Non-toxic compound needed for radioprotection
One main problem in radioprotection is to find compounds that are non-toxic or minimally so, and natural compounds fit the bill in being both non-toxic and easily available. Green tea is a rich source of polyphenols with strong antioxidant activities. Green tea extracts and its polyphenols have been shown to possess many health benefits attributed to their antioxidant and anti-inflammatory properties (see [4, 5] Green Tea, The Elixir of Life? and Green Tea Against Cancers, SiS 33). Most of the health benefits of green tea have been credited to the major polyphenol EGCG (epigallocatechin-3-gallate) (Figure 1), which constitutes 55 – 70 % of total polyphenols in green tea extract. Its antioxidant potential is believed to be far greater than vitamin E and vitamin C, the two main antioxidants among vitamins .
Figure 1 EGCG (epigallocatechin-3-gallate) from green tea
The team exposed both pBR322 plasmid DNA as well as spleen cells from mice to g-radiation at different concentrations of EGCG. Preliminary experiments found that EGCG concentrations above 125 mM were toxic to the cells, so the highest concentration used was restricted to 100 mM. The effects of quercetin – another polyphenol found in fruits, vegetables, leaves and grains – and vitamin C were also investigated. The plasmid DNA and cells were incubated for 2 hours with EGCG at different concentrations or quercetin and vitamin C, both at 100 mM, before being irradiated. Afterwards, the plasmid and cells were assessed for DNA damage, and the cells for viability, lipid peroxidation, membrane fluidity, and for activities of enzymes and cofactors involved in detoxification and scavenging of ROS.
Tea compound protects against DNA breaks and cell death
The intact plasmid is supercoiled in a compact form, while the cut plasmid is circular, and the two forms can be clearly distinguished and quantified by electrophoresis. The control (unexposed) sample is about 85% supercoiled. EGCG was found to protect plasmid DNA against breaks at high (50 Gy) or low (3 Gy) dose radiation: >82.5 % protection even at the lowest concentration of EGCG tested (10 mM) and complete 100 % protection at 50 mM. EGCG was better at protection against DNA breaks than quercetin or vitamin C at the same concentration of 100 mM.
The viability of cells was determined with a vital dye that depends on active mitochondria. At 3 to 7 Gy of g-irradiation, cell viability was significantly decreased, and at the highest dose, to 53 % of unexposed controls; but pre-incubation with EGCG protected the cells and restored viability in a concentration dependent manner, at 100 mM, viability was restored to >96 % of control.
Single cell comet assay was used to determine the extent of DNA degradation in the cells. In this assay, cells are trapped in agar gel on a microscope slide, lysed to expose their DNA for electrophoresis, and stained with a fluorescent dye. Cells with intact DNA will appear as a small compact bright spot, while cells with degraded DNA will appear as a diffuse spot with a tail, like a comet, hence the name of the assay. The bigger the tail, the greater is the extent of degradation, which can be quantified with computer software under a fluorescent microscope. Exposing the cells to 3 Gy led to substantial DNA degradation, which was reduced in a concentration dependent manner by EGCG. Quercetin and vitamin C also protected the cells against DNA damage, though not as effectively as EGCG.
Protection against lipid peroxidation
Peroxidation of membrane lipids by ROS destroys membrane structure and function. The results showed that lipid peroxidation increased with radiation dose from 0 to 7 Gy; and membrane fluidity also increased but more slowly. Pre-incubation with EGCG prevented lipid peroxidation and increase in membrane fluidity in a concentration dependent manner. Quercetin and vitamin C similarly protected against peroxidation and increase in membrane fluidity, but again, less efficiently than EGCG.
Key enzyme activities for antioxidant defence restored
Glutathione-S-transferase (GST) is a family of enzymes catalyzing the conjugation of reduced glutathione (GSH) to peroxidized lipids to detoxify them. Reduced glutathione GSH is a tripeptide antioxidant that takes part in reduction-oxidation reactions; in the process, it is oxidized into glutathione disulphide (GSSG). The ratio of reduced to oxidized glutathione is important in the cell’s antioxidant defence. Superoxide dismutase (SOD) catalyses the conversion of superoxide (a reactive oxygen species) into oxygen and hydrogen peroxide and is an important ROS scavenger in cells. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactic acid with simultaneous interconversion of NADH and NAD (reduced and oxidized nicotinamde adenine dinucleotide), which is important in maintaining the cell’s electronic balance and antioxidant defence.
g-irradiation reduced the activities of both GST and SOD. The reduction was countered by EGCG, and also by quercetin and vitamin C. The level of LDH, an indicator of damage was increased in g-irradiated cells, while glutathione was decreased, as indicative of oxidative stress. EGCG was able to counteract those effects, and almost completely at 100 mM. Quercetin was just as effective in reducing LDH and restoring GSH levels to those of controls, but vitamin C less so.
EGCG intercalates in DNA double helix
The authors suggest that EGCG can intercalate in the DNA double helix and protect it from free radical attack. EGCG binding to both DNA and RNA was documented for the first time by researchers at the Tokushima Bunri University and the Saitama Cancer Centre in Japan . They found that EGCG binds to both single-stranded DNA and RNA, as well as double-stranded DNA. Moreover, EGCG binding appears to stabilize double-stranded DNA.
Previous work has also demonstrated that due to the presence of abundant phenolic hydroxyl groups on aromatic rings (see Figure 1), EGCG is a highly efficient free radical scavenger, effectively disarming the free radicals and rendering harmless .
Most importantly, in the absence of g-radiation, EGCG did not have any significant effect. Thus the innocuous habit of drinking two cups of green tea a day may indeed have surprisingly beneficial effects [4, 5] that include protecting against ionizing radiation.