Effect of Green tea and EGCG on Liver : in vivo and in vitro Study

1. Effect of Green tea and EGCG on Liver : in vivo and in vitro Study Ibrahim Ghalib Saleh 1, 2, Zulfiqar Ali 1, Farid M.A. Hamada 2 , M.F. Abd-Ellah 2 , Ikhlas A. Khan 1, 3 Larry A. Walker 1 and Mohammad K. Ashfaq 1 1 National Center for Natural Products Research, University of Mississippi, University, MS 38677, USA 2 Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt 3 Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA A: Effect of different doses of EGCG (IG) on plasma ALT level with/without LPS (IP) administration Abstract Epigallocatechin 3-gallate (EGCG), the principal component of green tea (GT), is well known for its beneficial effects. However, high doses of EGCG may cause liver toxicity. We studied the effect of GT and EGCG in vivo and in vitro. In the in vivo study, we examined the potential hepatotoxicity of high doses of EGCG under febrile conditions induced by lipopolysaccharide (LPS). EGCG was given intra gastric (IG) or intraperitoneal (IP), while LPS was given IP to mice (ND4). Plasma ALT levels were determined and liver histopathology was performed. Results suggested that administration of high doses of EGCG can lead to mild liver toxicity. However, under febrile conditions (induced by LPS), this liver toxicity could become severe. In the in vitro study, HepG2 cells were treated with different concentrations of GT and EGCG with and without pre-sensitization with LPS. At lower concentrations of GT or EGCG, cell viability was increased regardless of pre-sensitization with LPS. However, at higher concentrations, both GT and EGCG decreased cell viability, especially, in cells that were presensitized with LPS. Furthermore, TGFß1 and RXRα were also over-expressed in HepG2 cells that were pre-sensitized with LPS and treated with high concentration of EGCG. These in vitro results lend support to the in vivo results indicating that EGCG probably acts as an anti-oxidant at lower doses but at higher doses it may cause liver toxicity possibly due to its pro-oxidant activity. C: Effect of different doses of EGCG (IG) on plasma ALP level with/without LPS (IP) administration D: Effect of different doses of EGCG (IP) on plasma ALP level with/without LPS (IP) administration In vivo Experimental Design In vivo Results B: Effect of different doses of EGCG (IP) on plasma ALT level with/without LPS (IP) administration Fig. 3: Histopathology micrograph of liver sections after treatment with different doses of EGCG IG or IP and/or LPS intraperitoneal (IP) 400X, H and E stain. Congestion (arrows), Vacuoles (arrow heads), Degenerative hepatocytes (stars) and Prominence of Kupffer cells and other inflammatory cells (rectangles). [A= E1, B= E2, C= E3, D= E4, E= E5, F= E6]. Figure 2. Histopathology micrograph of liver sections after treatment with different doses of EGCG IG and/or LPS intraperitoneal (IP) 400X, H and E stain. Congestion (arrows), Vacuoles (arrow heads), Degenerative hepatocytes (stars) and Prominence of Kupffer cells and other inflammatory cells (rectangles). [A= E2, B= E9, C= E10, D= E11, E= E12, F= E6]. Figure 1. Effect of different doses of EGCG on plasma ALT and ALP levels with/without LPS administration. In vitro Results Fig. 7: Expression of RXRα after treatment of HepG2 cells with different concentrations of GT water extract, with /without LPS. Blue = Nucleus, Red = RXRα. Fig. 6: Expression of TGFß1 after treatment of HepG2 cells with different concentrations of a water extract of GT with /without LPS (Green = F-Actin, Blue = Nucleus, Red = TGFß1). Fig. 5: Cell viability of HepG2 after treatment with a water extract of GT with/ without LPS using Acridin orange stain (Green = Viable, Yellow and Red = Dead). Fig. 4: Cell viability of HepG2 after treatment with a water extract of GT with/ without LPS using MTT assay. Acknowledgments Conclusions Under febrile conditions, the use of low doses of polyphenols does not pose risk of liver damage. Continuous high doses of EGCG itself can lead to mild liver injury and under febrile condition it can cause severe liver injury. This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01FD004246. The authors thank Ms. Penni Bolton and her vivarium staff for animals care services. Fig. 8: Cell viability of HepG2 after treatment with EGCG with/ without LPS using MTT assay. Fig.8: Expression of RXRα after treatment of HepG2 cells with different concentrations of EGCG with /without LPS (Blue = Nucleus, Red = RXRα). Fig. 10: Expression of TGFß1 after treatment of HepG2 cells with different concentrations of EGCG with /without LPS (Green = Cell structure, Blue = Nucleus, Red and orange = TGFß1. Fig. 9: Cell viability of HepG2 after treatment with EGCG with/ without LPS using Acridin orange stain (Green = Viable, Yellow and Red = Dead).