Are Females More Susceptible to EGCG Induced ...

International Journal of Pharmacology and Clinical Trials, ISSN: 2051-8293, Vol.26, Issue.1 dx.doi.org/10.12817/20518293.26.1.27703420

1183

Are Females More Susceptible to EGCG Induced Hepatotoxicity? Ibrahim G. Saleh National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Zulfiqar Ali National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA

Floyd D. Wilson Mississippi Veterinary Research and Diagnostic Laboratory, College of Veterinary Medicine, Mississippi State University, Pearl, MS. 39208, USA

Farid M. Hamada Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Mohamed F. Abd-Ellah Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Larry A. Walker National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677, USA

Ikhlas A. Khan National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11557, Saudi Arabia

Mohammad K. Ashfaq National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Corresponding Author E-mail: [email protected]

ABSTRACT Many recent studies and case reports have implicated Epigallocatechin-3-gallate (EGCG) to be hepatotoxic. As females consume weight control medications more than males, they are relatively more exposed to the harmful effects of EGCG on their livers. This study was conducted to assess the effect of EGCG in female mice either under normal or inflammatory conditions induced by lipopolysaccharide (LPS). Some biochemical parameters associated with liver functions were assessed. Also, survival of mice was studied and histopathological examination of liver samples was performed. Administration of lower IG doses of EGCG in normal female mice did not show any harmful effect on liver, however, administration of higher IG doses of EGCG in normal conditions was associated with an early stage of hepatotoxicity. Under inflammatory conditions, the effect of administration of EGCG on liver was severe. The results presented in this study suggest that females were susceptible to risk of liver injury, especially under an inflammatory insult as histopathology showed severe acute liver injury. In our previous study with male mice (published earlier), histopathology was relatively less severe. This may suggest that females are relatively more susceptible to EGCG induced liver injury than males.

Keywords-Green Tea (GT), Epigallocatechin-3-gallate (EGCG),Lipopolysaccharide Hepatotoxicity.

(LPS),

Inflammation,

1. INTRODUCTION There are a number of reports on hepatotoxicity of plantderived drugs [1]. The reports on severe hepatitis or necrosis resulting in irreversible liver damage are sporadic. In many cases, it is difficult to decide if the plant-derived drug was in fact the cause of the liver symptoms or if another factor led to or aggravated these symptoms [2]. Constituents of plant-derived drugs may damage the hepatocyte directly or after metabolic activation [3]. Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydroalcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU [4, 5]. Experimental studies and clinical case reports have documented the hepatotoxic effect of GT and its extracts [6-8]. In experimental animals and mammalian cells, EGCG undergoes oxidative metabolism [9] and methylation by

© RECENT SCIENCE PUBLICATIONS ARCHIVES |April 2014|$25.00 | 27703420| *This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*

International Journal of Pharmacology and Clinical Trials, ISSN: 2051-8293, Vol.26, Issue.1

catechol-O-methyltransferase, and glucuronidation at various positions[10]. Absorption from the intestine was suggested to be limited by Multidrug resistance proteins (Mrps) such as Mrp 2 since Mrp inhibitors enhanced cellular accumulation in HT-29 colon carcinoma cells in vitro. Epigallocatechin-gallate (EGCG) is a major contributor to the cytotoxic effect of GT extracts in cultured rat hepatocytes. The mechanism of the cytotoxic action of EGCG is unclear [5]. A number of studies were aimed at explaining the inhibitory effects of GT on the proliferation of tumor cells. In vitro, EGCG inhibited cell cycle progression in Hep G2 hepatoma cells and led to apoptosis [11]. Inhibition of fatty acid synthase was also suggested to play a role in growth inhibition of tumor cells [12]. Recent studies showed that EGCG can induce apoptosis in lymphocytic leukemia cells via different pathways [13]. It is also reported, however, to be cytotoxic in normal cells. Likewise, cytotoxicity of EGCG was shown in cultured human hepatic stellate cells (reported in [5]). This effect was linked to a possible prevention of liver fibrosis by GT, mediated via suppression of the production of pro-fibrotic factors by hepatic stellate cells [14]. Since the mechanism of cytotoxicity of EGCG is not known in detail, however, it is proposed to cause cytotoxicity via induction of apoptosis. Other factors (infections, drugs) and/or alternative mechanisms of liver damage are more likely to explain the effects seen. According to statistical data, obesity is more prevalent in females compared to males [15] Also females being more concerned about weight control, makes them a larger population that consumes weight reducing supplements. Green tea extract is sold as one of the weight reducing supplements and is commercially available in mega doses in capsular form. Thus consumption of such supplements with high EGCG content by the female population may put them at higher risk for liver toxicity. Our previous results showed hepatotoxicity associated with EGCG in male mice [16]. This study was undertaken to assess the hepatotoxic potential of EGCG in female mice under normal and under inflammatory stress conditions (induced by LPS)

2. MATERIALS AND METHODS 2.1 Animals Female ND-4 mice were obtained from Harlan Lab (Indianapolis, IN, USA) at 5 weeks of age and 23 - 28 g body weight, housed in micro isolator cages with corn cob bedding, on 12 h light/dark cycle, at 72°F (22 °C)and 3550% relative humidity. Mice were fed on (TekLad 57001) laboratory chow and water ad libitum. All animals were fasted for at least 8 hours before any treatment. All animal study protocols were approved by the Institutional Animal Care and Use Committee (IACUC), University of Mississippi, USA.

2.2 Chemicals 1- Epigallocatechin - 3- gallate (EGCG) 99%, obtained from Sigma-Aldrich (St Louis, MO, USA).

1184

2-Lipopolysaccharide (from E. coli O55:B5), obtained from Sigma-Aldrich (St Louis, MO, USA).

2.3 Experimental design (Table.1) Group n=10 G1

Treatment

Dose

10% DMSO (vehicle) LPS

6 mg/kg single dose IP

LPS 10% DMSO EGCG

6 mg/kg single dose IP (5 days) IG 750 mg/kg (5 days IG)

G5

LPS EGCG

6 mg/kg single dose IP 750 mg/kg (5 days IG)

G6

EGCG

1500 mg/kg (5 days IG)

G7

LPS EGCG

6 mg/kg single dose IP 1500 mg/kg (5 days) IG

G2 G3 G4

(5 days) IG

2.4 Sample collection Blood samples were collected in heparinized micro tubes from the mice by venipuncture in the mandibular region. After euthanization by CO2 asphyxiation, tissue samples of liver (left median lobe) were taken and processed by standard histological techniques.

2.5 Relative reduction of body weight of mice Mice were weighed before the start of the experiment (initial body weight), during the course of the study and after the end of experiment just before euthanasia (final body weight).

2.6 Relative weight of liver After euthanization of mice, livers were dissected out and placed in phosphate buffered saline (pH 7.4) and washed out, then blotted dry on filter paper. Each liver was weighed.

2.7 Clinical chemistry The levels of alanine amino transferase (ALT), alkaline phospatase (ALP), albumin to globulin ratio (A/G ratio), amylase, blood urea nitrogen (BUN) and total bilirubin were measured immediately after blood sampling using an automated VetScan dry chemistry analyzer with Comprehensive Diagnostic Profiles (Abaxis, Union City, CA, USA).

2.8 Animal survival Death in each group was monitored and recorded during the course of the experiment. Mortality was calculated using Kaplan-Meier test of survival using Graph Pad Prism software (La Jolla, CA).

2.9 Liver histopathological examination Liver samples were kept in formalin solution (10%) for 24 hours then washed in tap water for 12 hours followed by absolute ethyl alcohol for dehydration of tissues. Tissues were cleared in xylene and embedded in paraffin blocks. Three



2.10 Statistical analysis Numerical data were analyzed by one way ANOVA test followed by Tukey Cramer multiple comparisons using Graph Pad Prism software (La Jolla, CA). A P-value of less than 0.05 was considered to show a significant difference between vehicle and other groups.

3. RESULTS 3.1 Change in body weight (Fig. 3.1)

Plasma ALT U/L

150

* 100

*

50

0

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

G3 Vh/LPS

G2 LPS

G1Vh(10%DMSO)

0

G7 EGCG 1500/LPS

G6 EGCG 1500

G4 EGCG 750

G3 Vh/LPS

G2 LPS

G1 10% DMSO

G5 EGCG 750/LPS

Relative change in body weight (%)

*

200

Except for the group of mice that was administered EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (G7), all groups did not show any significant change in body weight percentage compared to vehicle control (G1). This group (G7) showed a significant reduction of body weight (about 26 folds) compared to the vehicle control (G1). 5

did not show any significant change in plasma level of ALT compared to the vehicle control (G1). While, a combination of LPS (6 mg/kg IP) and EGCG (750 mg/kg IG) (G5) significantly increased the plasma level of ALT (about 2.5 folds) compared to the vehicle control (G1). Higher dose of EGCG (1500 mg/kg IG) (G6) caused about 3.5 folds elevation of plasma level of ALT compared to the vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (1500 mg/kg IG) (G7) showed further elevation of plasma level of ALT (about 5 folds) compared to the vehicle control (G1).

G7 EGCG 1500/LPS

micron-thick sections were stained by hematoxylin (RICCA chemical Co., Arlington, TX) and eosin (EMD Chemicals, Gibbstown, NJ). The slides were observed for lesions and analyzed using routine light microscopy.

1185

Fig. 3.3: Plasma level of Alanine transaminase (ALT) of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

-5

-10

-15

*

-20

Fig. 3.1: Relative change in body weight percentage of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

3.2 Effect on liver weight (Fig.3.2) Except for the group of mice that was administered EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (G7), all groups did not show any significant change of relative weight of liver compared to vehicle control (G1). This group (G7) showed a significant increase in the relative weight of liver (about 1.5 folds) compared to the vehicle control (G1).

3.4 Effect on plasma ALP (Fig. 3.4) Administration of LPS (G2), the combination of LPS with the vehicle control (G3) or EGCG (750 mg/kg IG) (G4) did not show any significant change in plasma level of ALP compared to the vehicle control (G1). While, a combination of LPS (6 mg/kg IP) and EGCG (750 mg/kg IG) (G5) significantly increased the plasma level of ALP (about 2.5 folds) compared to the vehicle control (G1). Higher dose of EGCG (1500 mg/kg IG) (G6) caused about 2.5 folds elevation of plasma level of ALT compared to vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (1500 mg/kg IG) (G7) showed further elevation of plasma level of ALT (about 3 folds) compared to the vehicle control (G1).

*

3.3- Effect on plasma ALT (Fig. 3.3) Administration of LPS (G2), the combination of LPS with the vehicle control (G3) or EGCG (750 mg/kg IG) (G4)

G6 EGCG 1500

100 50 0

G7 EGCG 1500/LPS

Fig. 3.2: Relative weight of liver of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

*

G5 EGCG 750/LPS

G7 EGCG 1500/LPS

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

G3 Vh/LPS

G2 LPS

G1 10% DMSO

0

*

150

G4 EGCG 750

2

*

200

G3 Vh/LPS

4

250

G2 LPS

6

G1Vh(10%DMSO)

8

Plasma ALP U/L

Relative wight of liver

10

Fig. 3.4: Plasma level of alkaline phosphatase (ALP) of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001


*

80

*

*

1

* G7 EGCG 1500/LPS

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

G3 Vh/LPS

0

Fig. 3.6: Plasma albumin/globulin (A/G) ratio of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

3.7 Effect on total bilirubin (Fig. 3.7) Only the group of mice that was administered EGCG (1500 mg/kg IG) and LPS (6 mg/kg IP) (G7 showed 10 folds increase in Plasma level of total bilirubin compared to the vehicle control (G1). All other groups did not show any significant change of plasma level of total bilirubin compared to the vehicle control (G1).

60

4

*

Fig. 3.5: Blood urea nitrogen of female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

3.6 Effect on plasma albumin to globulin (A/G) ratio (Fig. 3.6) Administration of LPS (G2), the combination of LPS with the vehicle control (G3) or EGCG (750 mg/kg IG) (G4) did not show any significant change in plasma A/G ratio compared to the vehicle control (G1). While, a combination of LPS (6 mg/kg IP) and EGCG (750 mg/kg IG) (G5) significantly decreased the plasma A/G ratio (about 2 folds reduction) compared to the vehicle control (G1). Higher dose of EGCG (1500 mg/kg IG) (G6) caused a significant reduction of plasma A/G ratio to one half compared to vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (1500 mg/kg IG) (G7) showed a further reduction of A/G ratio (about 3 folds reduction) compared to the vehicle control (G1).

1

G7 EGCG 1500/LPS

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

0

G3 Vh/LPS

G7 EGCG 1500/LPS

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

G3 Vh/LPS

G2 LPS

G1Vh(10%DMSO)

0

2

G2 LPS

* 20

3

G1Vh(10%DMSO)

*

40

* Total bilirubin mg/dL

BUN level mg/dL

100

2

G2 LPS

Administration of LPS (G2) or the combination of LPS with the vehicle control (G3) did not show any significant change BUN level compared to the vehicle control (G1). While, administration of EGCG 750 mg/kg (IG) (G4) caused a significant increase of BUN level (about 1.5 folds) compared to the vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (750 mg/kg IG) (G5) significantly increased the level of BUN (about 2 folds) compared to the vehicle control (G1). Higher dose of EGCG (1500 mg/kg IG) (G6) caused about 2.5 folds elevation of BUN level compared to the vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (1500 mg/kg IG) (G7) showed a further elevation of the level of BUN (about 4.5 folds) compared to the vehicle control (G1).

1186

3

G1Vh(10%DMSO)

3.5 Effect on BUN concentration (Fig. 3.5)

Plasma albumin/globulin ratio


Fig. 3.7: Total bilirubin concentration in female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1). P< 0.001

3.8 Effect on plasma amylase (Fig. 3.8) Administration of LPS (G2) or the combination of LPS with the vehicle control (G3) did not show any significant change in the plasma level of amylase compared to the vehicle control (G1). While, administration of EGCG (750 mg/kg IG) (G4) caused a significant increase in the plasma level of amylase (about 1.5 folds) compared to the vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (750 mg/kg IG) (G5) significantly increased the plasma level of amylase (about 1.75 folds) compared to the vehicle control (G1). Higher dose of EGCG (1500 mg/kg IG) (G6) caused about 2.15 folds elevation of the level of amylase compared to the vehicle control (G1). Also, a combination of LPS (6 mg/kg IP) and EGCG (1500 mg/kg IG) (G7) showed a further elevation of the plasma level of amylase (about 2.8 folds) compared to the vehicle control (G1).



Plasma amylase U/L

2000

*

1500

* 1000

*

*

1187

vacuolar degeneration evidenced by clear cytoplasm and shrunken nuclei, accompanied with apparent mononuclear granulomatous inflammatory cell infiltration (yellow arrows). Apparent sinusoidal congestion was also present (circles).

500

G7 EGCG 1500/LPS

G6 EGCG 1500

G5 EGCG 750/LPS

G4 EGCG 750

G3 Vh/LPS

G2 LPS

G1Vh(10%DMSO)

0

Fig. 3.8: Amylase concentration in female mice after treatment with EGCG IG and/or LPS IP for 5 days. * Significantly different from vehicle control (G1).

3.9 Survival of mice (Fig. 3.9) No mortality was observed in the mice treated with the vehicle control (G1) or other treatments except in the group of mice that were administered EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (G7). In this later group mortality started on day 3 of treatment when 20% of animals died. On day 4 the mortality percentage reached 100%, taking in consideration that the mice that showed signs of severe illness were sacrificed within hours of the 4th dose, after taking blood samples for chemical analysis, and were considered dead.

Percent survival

150

G1 Vh (10% DMSO) G2 LPS G3 Vh/LPS

100

G4 EGCG 750 G5 EGCG 750/LPS G6 EGCG 1500

50

G7 EGCG 1500/LPS

0 0

2

4

6

Days Exposed

Fig. 3.9: Percent survival of female mice after treatment with EGCG IG and/or LPS IP for 5 days.

3.10 Histopathology of liver (Fig. 3.10) Mice in groups (G1, G2, G3 and G4) did not show any marks of liver injury, while mice in groups (G5, G6 and G7) showed some lesions as follows: A- Vehicle control (10% DMSO oral): Normal liver structure with no signs of injury. B- EGCG 750 mg/kg Oral + LPS 6 mg/kg IP (G5): The dark nuclei within the sinusoids appear to be Kupffer cells (yellow arrows). Mild congestion is present (circles). Possible minimal hepatocellular vacuolar changes present immediately around the central vein. C- EGCG 1500 mg/kg Oral (G6): Hepatocytes exhibit some minimal vacuolar or fatty changes (yellow arrows), with mild congestion in the sinuses and portal veins (circles). D- EGCG 1500 mg/kg Oral + LPS 6 mg/kg IP (G7): Hepatocytes showed a multifocal hepatic necrosis (green arrows) that is surrounded by a zone of hepatocellular

Fig. 3.10: Photomicrographs of representative liver samples of mice after treatment with EGCG and /or LPS (H & E stain), magnification power = 200X. CV= Central vein, PV= Portal vein.

4. DISCUSSION Previously, we had shown the hepatotoxic effect of EGCG on male mice [16, 17]. As females consume weight control medications more than male, they may be more liable to EGCG consumption. This study was conducted to assess the effect of EGCG on female mice either in normal conditions or under the influence of LPS as an inflammagen. The results of the current study showed that treatment of female mice with EGCG 1500 mg/kg IG for 5 days under the influence of 6 mg/kg LPS IP, markedly reduced the body weight and it also increased the relative weight of liver. These results may be indicative of a metabolic disorder associated with liver and/or liver cirrhosis. Neither LPS alone nor EGCG (750 or 1500 mg/kg IG) showed any significant change in body weight or relative weight of liver. Also, a combination of EGCG 750 mg/kg IG and LPS 6 mg/kg IP did not show any effects on body weight or relative weight of liver. Plasma level of ALT was raised upon treatment of mice with a combination of EGCG 750 mg/kg IG and LPS 6 mg/kg IP. The rise was more in the mice treated with EGCG 1500 mg/kg IG and was much pronounced in mice that received a combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP. Our results coincide with those reported earlier where marked elevation of plasma levels of ALT and high rates of mortality were documented upon treatment of female Swiss-Webster mice with EGCG [18]. The hepatotoxic effects in that mentioned study were more severe than of ours; this may be due to the strain difference. Compared to our previous observations on male mice [16], the rise in ALT level was about 1.3 fold more in males than in the females. Similar trend was observed in plasma level of ALP, where the elevation of the enzyme was almost the same in mice



treated with a combination of EGCG 750 mg/kg IG and LPS 6 mg/kg IP and those which received EGCG 1500 mg/kg IG alone. The elevation was much marked in the mice received a combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP. Plasma level of total bilirubin (TB) showed a marked rise in the mice that received a combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP. The effect of EGCG 750 mg/kg was not remarkable even with a combination with LPS. Also EGCG 1500 mg/kg alone did not affect the level of TB. Taken together, the observations of ALP and TB suggest occurrence of obstructive hepatic disorder as it is well known that obstructive liver diseases can cause alkaline phosphatase to rise. This is suggestive of intrahepatic obstructive disease that could be secondary to biliary cirrhosis [19] or induced by drug administration [20]. Besides, impaired ability of liver to conjugate bilirubin, leading to elevation of TB, is a sign of hepatic failure or biliary cirrhosis [21]. Compared to our previous observations on male mice, elevation of ALP was about 1.5 fold higher in males than in the females that received the same combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP. It also shows similar elevation of TB in both males and females [17]. The plasma A/G ratio showed a marked decrease upon treatment of mice with a combination of EGCG 750 mg/kg IG and LPS 6 mg/kg IP. The decrease was more in the mice treated with EGCG 1500 mg/kg IG and was markedly noticeable in the mice that received a combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP. Albumin levels decrease in case of hepatic cirrhosis [22]. These observations in addition to the ones of ALT, ALP and TB in this study, confirm the occurrence of liver injury. Although plasma amylase level could be non-specific and may point primarily to pancreatic disorder, it may indicate a liver injury when combined with high ALT, ALP, TB and low A/G ratio. It was documented earlier that serum amylase tends to rise in viral hepatitis [23] and in hepatic cirrhosis [24]. Usually, BUN tends to decrease in marked liver injury due to loss of ability of liver to detoxify ammonia to urea that results in a decrease in BUN level. However, in the current study the levels BUN increased upon treatment with EGCG (750 and 1500 mg/kg IG), either alone or combined with 6 mg/kg LPS IP. This may reflect an impairment of renal excretion of urea. The effect of a combination of EGCG and LPS on kidney needs to be evaluated. The results of A/G ratio, amylase and BUN reported here in female mice are similar to our previous reported observations in male mice. Data of survival of mice indicate the severity of the combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP on female mice. On day 3, 20% of mice died and the rest of 100% of animals died on day 4 post treatment. Comparing with our previous observations on male mice, where mortality started on the day 3 of treatment when 10% of animals died. On the day 4, the mortality was 50% that increased to 60% on day 5 [17]; it can be concluded

1188

that under the influence of LPS, female mice showed faster onset and higher percentage of mortality than males. The results of histopathological examination of female liver samples confirm the mortality data. Mice treated with the combination of EGCG 1500 mg/kg IG and LPS 6 mg/kg IP showed signs of severe multifocal hepatocellular necrosis. While, the mice that were treated with EGCG 1500 mg/kg IG alone showed signs of minimal vacuolar or fatty changes accompanied with mild congestion. Comparing these results with our observations in male mice, the severity of hepatocellular injury is more in females than in males. Comparing with our previous study, the effect of EGCG/LPS on the biochemical parameters was more prominent in male than in female mice. While, histopathology and survival data indicated that the effect of EGCG/LPS on female was more prominent than on male mice. To explain this paradox it should be borne in mind that the hormonal changes in females may influence or alter liver enzymes profile and that is the reason that males are preferably used in preclinical studies rather than females. However, our results of histopathology and mortality point towards severe toxicity in females.

5. CONCLUSIONS The results of the current study confirm our previous results on male mice. Administration of EGCG in high doses can become hepatotoxic especially under inflammatory conditions. The hepatotoxic effect of EGCG under the influence of LPS occurred in male and in female mice. Compared together, it appears that the hepatotoxic effect of EGCG is more pronounced in female than in male mice. In other words, EGCG may be more tolerable in males than in females. In view of possible higher consumption of supplements containing mega doses of EGCG for weight control by females, caution need to be exercised on its use under febrile or other inflammatory conditions.

ACKNOWLEDGMENTS This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01FD004246. Authors would like to thank Ms. Penny Bolton and her vivarium staff for animal caring during the experiment.

CONFLICT OF INTEREST All authors declare no conflict of interest with the work done in this study.

REFERENCES [1]

R. Teschke, A. Genthner, and A. Wolff, “Kava hepatotoxicity: comparison of aqueous, ethanolic, acetonic kava extracts and kava–herbs mixtures,” Journal of ethnopharmacology, vol. 123, no. 3, pp. 378-384, 2009.



[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

M. K. Ashfaq, “Impact of High EGCG Consumption on Mouse Liver,” Journal of Biomedical and Pharmaceutical Research, vol. 1, no. 02, 2012. M. Pittler, and E. Ernst, “Systematic review: hepatotoxic events associated with herbal medicinal products,” Alimentary pharmacology & therapeutics, vol. 18, no. 5, pp. 451-471, 2003. G. Mazzanti, F. Menniti-Ippolito, P. A. Moro, F. Cassetti, R. Raschetti, C. Santuccio, and S. Mastrangelo, “Hepatotoxicity from green tea: a review of the literature and two unpublished cases,” European journal of clinical pharmacology, vol. 65, no. 4, pp. 331-341, 2009. M. Schmidt, H.-J. Schmitz, A. Baumgart, D. Guedon, M. Netsch, M.-H. Kreuter, C. Schmidlin, and D. Schrenk, “Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture,” Food and Chemical Toxicology, vol. 43, no. 2, pp. 307-314, 2005. G. Galati, A. Lin, A. M. Sultan, and P. J. O'Brien, “Cellular and in vivo hepatotoxicity caused by green tea phenolic acids and catechins,” Free Radical Biology and Medicine, vol. 40, no. 4, pp. 570-580, 2006. M. Molinari, K. D. Watt, T. Kruszyna, R. Nelson, M. Walsh, W. Y. Huang, B. Nashan, and K. Peltekian, “Acute liver failure induced by green tea extracts: case report and review of the literature,” Liver transplantation, vol. 12, no. 12, pp. 18921895, 2006. R. Gloro, I. Hourmand-Ollivier, B. Mosquet, L. Mosquet, P. Rousselot, E. Salamé, M.-A. Piquet, and T. Dao, “Fulminant hepatitis during selfmedication with hydroalcoholic extract of green tea,” European journal of gastroenterology & hepatology, vol. 17, no. 10, pp. 1135-1137, 2005. H.-K. Na, and Y.-J. Surh, “Modulation of Nrf2mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG,” Food and Chemical Toxicology, vol. 46, no. 4, pp. 1271-1278, 2008. H. Lu, X. Meng, and C. S. Yang, “Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (−)epigallocatechin gallate,” Drug metabolism and disposition, vol. 31, no. 5, pp. 572-579, 2003.

[11]

P.-L. Kuo, and C.-C. Lin, “Green tea constituent (−)-epigallocatechin-3-gallate inhibits Hep G2 cell proliferation and induces apoptosis through p53dependent and Fas-mediated pathways,” Journal of biomedical science, vol.10, no.2, pp.219-227, 2003.

[12]

X. Wang, K.-S. Song, Q.-X. Guo, and W.-X. Tian, “The galloyl moiety of green tea catechins is the critical structural feature to inhibit fatty-acid

1189

synthase,” Biochemical pharmacology, vol. 66, no. 10, pp. 2039-2047, 2003. [13]

E. C. Stuart, M. J. Scandlyn, and R. J. Rosengren, “Role of epigallocatechin gallate (EGCG) in the treatment of breast and prostate cancer,” Life sciences, vol. 79, no. 25, pp. 2329-2336, 2006.

[14]

Y. C. Hsu, Y. T. Chiu, C. C. Cheng, C. F. Wu, Y. L. Lin, and Y. T. Huang, “Antifibrotic effects of tetrandrine on hepatic stellate cells and rats with liver fibrosis,” Journal of gastroenterology and hepatology, vol. 22, no. 1, pp. 99-111, 2007.

[15]

C. L. Ogden, M. D. Carroll, B. K. Kit, and K. M. Flegal, "Prevalence of obesity in the United States, 2009-2010," US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics, 2012.

[16]

I. G. Saleh, Z. Ali, N. Abe, F. D. Wilson, F. M. Hamada, M. F. Abd-Ellah, L. A. Walker, I. A. Khan, and M. K. Ashfaq, “Effect of Green Tea and its Polyphenols on Mouse Liver (GT and Mouse Liver),” Fitoterapia, 2013.

[17]

I. Saleh, Z. Ali, F. Hamada, M. Abd-Ellah, I. Khan, L. Walker, and M. Ashfaq, “Consumption of EGCG can Become Hepatotoxic Under Inflammatory Condition,” Planta Medica, vol. 79, no. 05, pp. P95.

[18]

M. G. Goodin, and R. J. Rosengren, “Epigallocatechin gallate modulates CYP450 isoforms in the female Swiss-Webster mouse,” Toxicol Sci, vol. 76, no. 2, pp. 262-70, 2003.

[19]

E. G. Giannini, R. Testa, and V. Savarino, “Liver enzyme alteration: a guide for clinicians,” Cmaj, vol. 172, no. 3, pp. 367-79, 2005.

[20]

L.S. Velayudham, and G. C. Farrell, “Drug-induced cholestasis,” Expert Opin Drug Saf, vol. 2, no. 3, pp. 287-304, 2003.

[21]

J. Heathcote, “Update on primary biliary cirrhosis,” Can J Gastroenterol, vol. 14, no. 1, pp. 43-8, 2000.

[22]

B. T. Doumas, and T. Peters, Jr., “Serum and urine albumin: a progress report on their measurement and clinical significance,” Clin Chim Acta, vol. 258, no. 1, pp. 3-20, 1997.

[23]

S. G. Tewari, S. P. Tyagi, A. K. Vaid, and Sibghatullah, “Serum amylase and its isoenzymes in cases of viral hepatitis,” Gastroenterol Jpn, vol. 16, no. 5, pp. 441-6, 1981.

[24]

I. H. Bhutta, and M. A. Rahman, “Serum amylase activity in liver disease,” Clinical chemistry, vol. 17, no. 12, pp. 1147-1149, 1971.
