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.
4 comments:
Why did they not use a primary liver cell line? It seems that using an immortal line may skew their viability results. Also, it seems that the high dosage of ethanol may be to counteract any lost from evaporation due to ethanol's volatility and the high temperature during incubation. And I don't know if we're supposed to be using wikipedia or not, but it says that the mechanism for activation of GGT is unclear, so mRNA levels may not be indicative of activation.
I also agree with your worries about their use of high levels of ethanol. I think that using such doses may show viability of cells, but it would skew the levels of oxidative markers because the cells may go into apoptosis immediately, rather than try to protect themselves from the oxidative stress.
On a positive note, I did find that their graphs were easy to read and comprehend. And they clearly show that EGCG is the main source of protection from oxidative stress.
I think the natural viability of HepG2 cells are at least reasonable; they've been pretty hardy the few weeks we've been using them. The paper makes little mention, saying only that HepG2 cells responded similarly to primary liver cells to ethanol.
The alcohol vaporization is a good point, but if that were a concern the researchers should have determined the rate of alcohol loss and accounted for that by adding ethanol as they went.
I think the fact that they're looking for changes in viability offsets that skew of using immortal cells somewhat, but that is still a good point.
The graph in Figure 3 seems to show conclusively that EGCG increases cell viability. But any ideas as to why EGCG appears to significantly increase cell viability more than with the original green tea? Was there a higher concentration of EGCG used in the single compound assay than in the green tea assay, or are there interactions between compounds that need to be considered? This is a cool paper. As I enjoy drinking green tea, I hope that researchers are able to identify all of its health benefits.
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