Effect of green tea and (-)-epigallocatechin gallate on ethanol-induced toxicity in HepG2 cells.

Effect of green tea and (-)-epigallocatechin gallate on ethanol-induced toxicity in HepG2 cells.

Sang Il Lee, Hyo Jung Kim and Yong Chool Boo

Phytother Res. 2008 May; 22(5):669-74.

http://www3.interscience.wiley.com/cgi-bin/fulltext/117935658/PDFSTART

Summary:

The research described in this paper seeks to identify the source of the antitoxic and antioxidative properties of green tea. It is widely known that excessive alcohol can lead to liver damage and a variety of liver ailments. Although there are a variety of different mechanisms involve in liver toxicity, there is evidence that ethanol leads to oxidative stress and can therefore be prevented by antioxidants such as those found in green tea, which contains a variety of different compounds such as (-)-epigallocatechin gallate (EGCG), L-theanine, and caffeine, the three of which will be investigated by this study, by studying a marker of general oxidative stress, gamma-glutamyltransferase, a membrane enzyme that is responsible for catalyzing glutathione (GSH) breakdown and appears to be associated particularly with ethanol consumption.

The compounds in green tea were first extracted and identified using high pressure liquid chromatography, which found green tea extract to be composed of 5.6% caffeine and 13.6% ECCG. HepG2 cells, used to simulate human liver cells, were used for this study and were seeded at densities of 10^4 cells/cm^2, which reached 50-60% confluency in 24 hours. Standard cell culture procedures were followed to raise the cells, which were then treated with the various test compounds (L-Theanine, Caffeine, and EGCG) for one hour, before being incubated with 1.0 M ethanol (corresponding to 5.8% by volume) for 48 hours. A GGT assay using gamma-glutamyl-p-nitroanilide was used to determine the amount of GGT content of the media, cell viability was determined using the MTT assay and Alamar Blue Assays, and intracellular GSH content was assayed using DTNB after collecting cell lysate.

Figure 2: Charts showing the cell viability results of alcohol exposure and the GGT activity of the control cells that were not exposed to any of the components of green tea. There is a marked decrease of cell viability at ethanol concentrations above 1M and a substantial increase in GGT activity with ethanol concentrations of greater than 0.5M. * indicates significant (p<0.05)>

Figure 3: Charts showing the effects of varying concentrations of different components of green tea on the viability and GGT activity of HepG2 cells relative to controls (vehicle). As described, cells were exposed to 1.0 M concentration of ethanol after being pretreated with concentrations of the hypothesized antioxidants. # indicates a significant (p<0.05)>

The data indicates that GGT activity in the serum is closely associated with cell death, which is understandable considering that cell death releases GGT into the serum through membrane breakdown. When the green tea compounds were tested, it appears that only EGCG made a significant difference in the viability and GGT activity of cells. EGCG appears to substantially increase cell viability, which was reflected in the GGT assays. The researchers then decided to examine the mechanism of EGCG protection, by comparing it against other modulators of “ethanol metabolism, reactive nitrogen/oxygen species production and GSH homeostasis.” These results revealed that acivicin, “an irreversible GGT inhibitor”, rescued the cells very effectively.

This lead the researchers to examine the effects of EGCG and Acivicin directly on extracellular GGT, and it was found that both EGCG and Acivicin strongly inhibited GGT. Interestingly, further studies on intracellular GSH revealed that EGCG depleted the intracellular GSH levels. However, this suggests that mechanism of action of EGCG is very much like that of acivicin, protecting cells by inhibiting GGT and slowing ethanol induced oxidative damage.

Critique:

This paper is very interesting in that it tries to essentially tell a narrative about a series of experiments instead of merely summarizing the results of a single one. By following what is effectively a train of thought, there is a sort of natural organization to the experiments and results. However, I felt that the procedure for this paper was lacking in two respects. First, the only thing they measured was effectively cell viability, as it was shown that GGT activity was reasonably related to the number of cells alive. Secondly, to more thoroughly understand the mechanism behind which EGCG or green tea prevents ethanol-induced oxidative damage, they should have run a RT-PCR for GGT expression, along with other known markers of oxidative stress. An RT-PCR would have been much more diagnostic for changes induced by ethanol.

Finally, what struck me is that 1M of ethanol is an extremely high concentration to be exposing liver cells to, corresponding to approximately 5% by volume of ethanol. The lethal dosage for ethanol is a blood concentration of less than 1%, meaning that the conditions used in the experiment are by no means reflective of actual physiological states. Alcohol damage also occurs over long periods of time, not the 48 hour incubation period used in this paper. With such a high dosage and such a short exposure time, it may be the case that the cells are effectively “shocked” by the sudden influx of the ethanol, revealing short-term instead of long-term responses to ethanol and oxidative stress.