| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ORIGINAL RESEARCH COMMUNICATION |
1 From the Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland (J-HC); the Departments of Anatomy (GLT and K-ML), Pathology (ECL and HSHS), Pharmacology (W-MT), and Medicine (PCWF) and the Centre for the Study of Liver Disease (GLT and PCWF), Faculty of Medicine, The University of Hong Kong; and the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia (AAN)
2 Supported by the Research Grants Council, Committee on Research and Conference Grants, The University of Hong Kong, and the National Institutes of Health (grant AA12893), Bethesda, MD. 3 Address reprint requests to AA Nanji, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Founders 7-103, 3400 Spruce Street, Philadelphia, PA 19104. E-mail: amin.nanji{at}uphs.upenn.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Objective: We examined the effects of green tea polyphenols in carbon tetrachloride–treated mice, a model of liver injury in which oxidant stress and cytokine production are intimately linked. We tested the effect of a pure form of epigallocatechin gallate (EGCG), the major polyphenol in green tea, in mice treated with carbon tetrachloride.
Design: Eight-week-old ICR mice were administered 20 µL/CCl4 kg dissolved in olive oil. Two different doses of EGCG, 50 and 75 mg/kg, were tested. Control mice were treated with saline and olive oil. We analyzed liver histopathology, lipid peroxidation, and messenger RNA and protein concentrations of inducible nitric oxide synthase. Additionally, nitric oxide–generated radicals were assessed by electron paramagnetic resonance spectroscopy, and protein concentrations were measured by immunohistochemistry and Western blot analysis.
Results: Carbon tetrachloride administration caused an intense degree of liver necrosis associated with increases in lipid peroxidation, inducible nitric oxide synthase messenger RNA and protein, nitrotyrosine, and nitric oxide radicals. EGCG administration led to a dose-dependent decrease in all of the histologic and biochemical variables of liver injury observed in the carbon tetrachloride–treated mice.
Conclusions: Green tea polyphenols reduce the severity of liver injury in association with lower concentrations of lipid peroxidation and proinflammatory nitric oxide–generated mediators. Green tea polyphenols can be a useful supplement in the treatment of liver disease and should be considered for liver conditions in which proinflammatory and oxidant stress responses are dominant.
Key Words: Polyphenols • green tea • nitric oxide • free radicals • lipid peroxidation • carbon tetrachloride
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because oxidative stress plays a major role in several liver diseases, it was of interest to evaluate the role of green tea polyphenols in protecting against liver injury. One study, for example, showed that green tea suppresses D-galactosamine–induced liver injury in rats (7). The mechanism of the protective effect of the crude green tea extract used in the study was thought to be through inhibition of tumor necrosis factor 
–induced apoptosis (7). Other studies have generally used isolated cells to evaluate the effects of green tea polyphenols (8, 9). To examine the in vivo effects of green tea polyphenols, we used the mouse model of carbon tetrachloride–induced liver injury (10). Carbon tetrachloride, a classic hepatotoxidant, causes acute liver injury characterized by centrilobular necrosis (10). The hepatotoxicity involves 2 phases. The initial phase involves metabolism of carbon tetrachloride by cytochrome P450, which leads to the formation of free radicals and lipid peroxides (11, 12). The second step involves activation of Kupffer cells, probably by free radicals. Activation of Kupffer cells is accompanied by production of proinflammatory mediators (10, 11).
As noted above, numerous mediators have been implicated in toxin-induced liver injury, including inflammatory cytokines, eicosanoids, and reactive oxygen species (13, 14). More recent evidence indicates that nitric oxide (NO) plays a significant role in the pathogenesis of toxin-induced liver injury (15–17). NO is a second messenger molecule synthesized by hemoproteins known as NO synthases (NOS). In normal liver, NO is synthesized mainly by the constitutive NOS isoform—the endothelial NOS (18). The inducible isoform of NOS (iNOS) can be induced in response to proinflammatory cytokines and mediators (19).
Inhibition of iNOS in endotoxemia models results in increased liver damage, which suggests a beneficial role for NO (20, 21). In contrast with the documented protective role for NO, its detrimental role in liver injury has also been reported (19). In the current study we evaluated the role of the inhibition of NO production as a possible mechanism for the protective role of green tea polyphenols in toxin-induced liver injury. Instead of using a crude extract of green tea polyphenols, we used the pure form of EGCG.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Eight-week-old male ICR mice weighing 30–40 g were maintained in our animal facilities on a standard laboratory nonpurified diet and received care in compliance with the requirements of the University of Hong Kong and the National Institutes of Health guidelines. The initial experiment (experiment 1) was performed to determine the lowest dose of carbon tetrachloride that produced histologic evidence of liver necrosis. Three groups of mice (n = 3 per group) were administered, intraperitoneally, a single injection of carbon tetrachloride at doses of 10, 20, or 40 µL/kg. The mice were killed 6 h after injection; this time interval was determined to be optimal for the assessment of liver injury on the basis of preliminary observations.
The next experiment (experiment 2) was performed to determine the time of peak expression of iNOS. The lowest dose of carbon tetrachloride (20 µL/kg), which showed consistent histologic liver damage in experiment 1, was administered to 4 groups of mice (n = 3 per group). The mice were killed 1, 3, 6, and 12 h after injection. Additional groups of mice treated with injections of saline and vehicle served as controls.
The third experiment (experiment 3) was designed to test the effect of EGCG on the development of carbon tetrachloride–induced hepatotoxicity. On the basis of previous studies (23) and our preliminary experiments, which showed that green tea polyphenols at a dose of 100 mg EGCG/kg caused considerable morbidity, 2 lower doses of EGCG were chosen: 50 and 75 mg/kg. At these doses of EGCG, there is no increased level of morbidity (PCW Fung, unpublished observations, 2003). We predicted that these lower doses would allow us to determine the lowest effective dose of EGCG that ameliorates liver injury without causing unacceptable morbidity. Mice were randomly divided to receive one of the following 3 treatments (n = 8 per group): olive oil (vehicle; control group), carbon tetrachloride dissolved in olive oil (CCl4 group), and carbon tetrachloride plus 50 or 75 mg EGCG/kg (EGCG-50 and EGCG-75 groups). In the experimental groups, vehicle, carbon tetrachloride, or carbon tetrachloride plus EGCG were administered intraperitoneally for 3 consecutive d before carbon tetrachloride administration. Carbon tetrachloride (20 µL/kg) was then administered on the day the final injections of saline, vehicle, or EGCG were given. All animals received humane care according to the guidelines for care and use of laboratory animals established at The University of Hong Kong and by the National Institutes of Health.
Tissue collection and histopathologic analysis
The whole liver was removed from the mice, and the total liver volume was measured by water displacement. Fresh liver blocks were cut and immediately fixed in 10% phosphate-buffered formalin and then dehydrated in graded alcohols and embedded in paraffin. Paraffin sections of 6 µm thickness were rehydrated and stained with hematoxylin and eosin. Stained sections were observed under light microscopy and later subjected to image analysis (Leica QWIN, Cambridge, United Kingdom).
Determination of alanine aminotransferase activity in serum
Alanine aminotransferase activity in serum was used as a biochemical indicator of hepatic injury. A reaction mixture containing L-alanine (80 mmol/L), NADH (0.2 mmol/L), and lactate dehydrogenase (2 units) in potassium phosphate buffer (0.2 mmol/L, pH 7.4) and serum (0.35 mL) was incubated at 37°C for 3 min to determine the basal rate of NADH consumption at 340 nm. -Ketoglutarate (10 mmol/L) was subsequently added to measure the rate of NADH utilization by alanine aminotransferase present in serum. The activity of the enzyme was expressed in units per liter of serum.
Measurement of lipid peroxidation
The degree of lipid peroxidation in liver tissues was determined by measuring thiobarbituric acid–reactive substances (TBARS). Liver samples were homogenized in ice-cold tris buffer (0.5 mol/L, pH 7.4). An aliquot of 2 mL of the homogenate was added to a reaction mixture containing 1 mL trichloroacetic acid (20%) and 2 mL 2-thiobarbituric acid (0.67%). The amount of TBARS was expressed as nmol/g liver.
RNA extraction and analysis of messenger RNA by reverse transcriptase–polymerase chain reaction
To examine the expression of iNOS in liver tissue, total RNA was extracted by using the RNeasy mini kit (Qiagen Inc, Valencia, CA). The sequences of primer pairs, 5 and 3, and the predicted size of the amplified polymerase chain fragments were previously published (20, 21). Reverse transcription and amplification was performed as described previously (21). After the polymerase chain reaction (PCR) products were subjected to electrophoresis and ethidium bromide staining, the gels were analyzed by densitometry (21). To normalize signals from different RNA samples, 2 µL of the same reverse transcriptase reaction was amplified with GAPDH-specific primers. Varying the number of PCR cycles did not change the relative differences between samples, indicating that the PCR conditions were not within the plateau phase of amplification.
Nonradioactive in situ hybridization for iNOS
The in situ hybridization technique of Massimi et al (24) was modified for use. Three distinct oligonucleotide iNOS probes in an antisense orientation and another 3 probes in a sense orientation were synthesized by Gibco BRL Custom Company (Rockville, MD). All probes were end-labeled with a digoxigenin tailing kit (Roche Molecular Systems Inc, Nutley, NJ). The sections were treated with 15 µg proteinase K/mL in TE buffer (100 mmol tris-HCl/L and 50 mmol EDTA/L, pH 8.0) for 15 min at 37°C, followed by acetylation with acetic anhydride 0.25% (by vol) in 0.1 mol triethanolamine/L (pH 8.0) for 15 min at room temperature. Finally, sections were dehydrated for 3 min in ethanol. After air drying, 20 µL of the hybridization solution containing 2 ng/µL of the oligonucleotide mixtures was applied to each section and hybridized at 37°C for 16–20 h in a humidified chamber. After being washed 3 times for 30 min each at 37°C in 2 x SSC (0.3 mol NaCl/L, 0.03 mol sodium citrate/L, pH 7.0) and then 3 times in 1 x SSC, the sections were incubated for 1 h with 2% normal sheep serum in tris buffer (100 mmol tris-HCl/L, pH 7.5, 150 mmol NaCl/L). The sections were incubated with an alkaline phosphatase–conjugated sheep anti-digoxigenin antiserum (Roche Diagnostics) diluted at 1:250 in 2% normal sheep serum in tris buffer and exposed overnight at 4°C. The alkaline phosphatase was detected by incubating the sections for 3 h at room temperature in a freshly prepared substrate solution containing 20 µL/mL of a mixture of nitroblue tetrazoline and 5-bromo-4-chloro-3-indolyl phosphate in detection buffer (100 mmol tris-HCl/L, pH 9.5; 100 mmol NaCl/L, 50 mmol MgCl2/L) with 1 mmol levamisole/L (Roche Diagnostics). The color development was stopped by incubation in deionized water, and the sections were mounted with Clearmount (ZYMED Laboratories Inc, San Francisco). Controls for probe specificity included hybridization with sense probe or prehybrizidation with RNase-A at a concentration of 30 µg/mL in 2 x SSC for 60 min at 37°C.
Immunohistochemical and Western blot analysis for iNOS
Sections were immunostained with antiserum to iNOS by using the biotin-avidin-peroxidase method. Briefly, endogenous peroxidase activity was blocked by immersing the sections in 3% hydrogen peroxide for 5 min at room temperature. The sections were permeabilized in 0.1% tyrosine and 0.1% CaCl2 in phosphate-buffered saline (0.01 mol/L, pH 7.5). The sections were preincubated with 10% normal goat serum to reduce nonspecific binding of the antiserum, incubated overnight at 4°C, with rabbit polyclonal iNOS antibody (Transduction Laboratories, San Diego), and diluted at 1:100 in phosphate-buffered saline containing 2% normal goat serum or normal rabbit immunoglobulin G (IgG). Sections were washed 3 times in phosphate-buffered saline and then incubated with biotinylated goat anti-rabbit IgG at a dilution of 1:200 for 30 min at 37°C. The sections were further washed and incubated with an avidin and biotinylated peroxidase complex (1:50) for 30 min at 37°C. Finally, the sections were washed and the peroxidase was visualized by immersing in 0.05% diaminobenzidine containing 0.03% hydrogen peroxide in tris-HCl buffer (pH 7.5) for 2 min. The sections were rinsed in water and counterstained with hematoxylin. Positive staining was indicated by a brown color. Control sections were stained with normal rabbit IgG.
The specificity and the relative differences of iNOS protein expression were confirmed by Western blot analysis. Briefly, liver tissue was rapidly homogenized and lysed in 5 volumes of cold lysis buffer (50 mmol tris/L, pH 8.0; 150 mmol NaCl/L, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mmol phenylmethylsulfonyl fluoride/L) on ice for 30 min. The cell debris was removed by centrifugation at 17000 x g (1300 rpm) for 30 min at 4°C. The protein concentration in the supernatant fluid was determined by using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). Polyacrylamide gel electrophoresis with the use of using 20 µg total protein was performed by using the Mighty Small II run gel system (SE 250/260; Hoefer Pharmacia Biotech Inc, San Francisco). The protein was transferred onto a polyvinylidene fluoride blotting membrane by using a TE series transfer electrophoresis unit (Hoefer, Pharmacia Biotech). The membrane was incubated in a blocking buffer for 1 h and then incubated with polyclonal iNOS antibody (Transduction Laboratories, San Diego) overnight at 4°C. The membrane was washed and then incubated with a 1:10000 dilution of secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) at room temperature for 1 h. The iNOS protein was detected by using an ECL Western blotting detection kit (Amersham Pharmacia Biotech).
Immunohistochemical and Western blot analysis of nitrotyrosine
The immunohistochemical staining and Western blot procedures used for nitrotyrosine were similar to those described for iNOS. Instead of using the antibody to iNOS, the sections and liver homogenates were incubated with the anti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) at 1:100 dilution.
Measurement of NO-generated radicals by electron paramagnetic resonance spectroscopy
The NO synthesized was specifically trapped with Fe2+-diethyldithiocarbamate complex (FeDETC2) formed from endogenous iron and diethyldithiocarbamate (DETC), which was administered intraperitoneally 30 min before the animals were killed. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an electron paramagnetic resonance (EPR) signal at g
= 2.035, g|| = 2.02 with triplet hyperfine structure at g. This allowed quantification of the amount of NO formed in the liver (25). Thirty minutes after injection of the spin trap, liver tissue was rapidly removed and cut into small pieces. The tissue was placed in a quartz EPR tube, immediately frozen, and kept in liquid nitrogen temperature until the EPR spectra were recorded. We previously determined that freezing of tissue has no effect on the signal intensity of the EPR spectra and that administration of the spin trap does not increase iron concentrations in liver tissue (PCW Fung, unpublished observations, 2003). The EPR spectra of the mononitrosyl-iron DETC complex were recorded at 77 K with the use of a Brucker EPS 300E spectrometer (Brucker Analytische Messtechnik, Rheinstetten, Germany). As described previously (25), EPR signals from liver preparations in the different experimental groups were compared on the basis of the signal intensity produced from identical volumes of tissue. The concentration of NO-FeDETC2 was estimated by measuring the peak-to-baseline height after normalization of the data. The measurement of the NO-FeDETC2 concentration by EPR spectroscopy provided a measure of the amount of NO available for trapping by Fe0DETC2.
Image analysis
The percentage area of necrosis and the staining for iNOS messenger RNA (mRNA) and protein was determined by dividing the sum area of positive staining by the sum of the reference area of 10 fields. To confirm the validity of our findings, the parameters used to evaluate staining were further expressed as the mean volume in relation to the total volume of the liver.
Statistical analysis
Data from each group were expressed as means ± SEMs. Statistical comparison between groups was done by using analysis of variance (ANOVA) with the STATVIEW 5.0 program (Abacus Concepts, Berkley, CA) or the nonparametric Mann-Whitney U test (two-tailed) followed by Bonferroni's multiple comparisons test for group comparisons. P < 0.05 was considered to be statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
|
) and histology (Figure 2). Mice that received carbon tetrachloride alone showed regions of marked centrilobular necrosis and inflammation, whereas mice pretreated with EGCG had a marked decrease in the concentration of alanine aminotransferase and the degree of necrosis (Figures 1 and 2).
|
). Treatment of carbon tetrachloride–treated rats with EGCG led to a significant decrease in the degree of lipid peroxidation (Figure 3). Although lipid peroxidation in the EGCG-treated rats appeared to be lower than that in the control animals, the differences were not statistically significant.
|
).
|
A). After carbon tetrachloride administration, hepatocytes with mRNA iNOS expression were more abundant (Figure 5B). Pretreatment with EGCG at 50 mg/kg reduced the expression of mRNA iNOS (Figure 5C) compared with carbon tetrachloride treatment alone (Figure 5B). EGCG pretreatment at a higher dose of 75 mg/kg resulted in a further reduction in the expression of mRNA iNOS (Figure 5D). The results from image analysis confirmed that EGCG pretreatment at a dose of 50 mg/kg decreased the mean percentage of cells positive for mRNA iNOS expression, and EGCG pretreatment at a higher dose of 75 mg/kg resulted in further reduction of the percentage of cells expressing mRNA iNOS (Figure 5E).
|
). In the normal mice, occasional hepatocytes were positive for iNOS protein (Figure 6A). After treatment with carbon tetrachloride, iNOS protein expression markedly increased predominantly in the centrilobular regions of the liver (Figure 6B). Mice pretreated with 50 mg EGCG/kg showed a significant reduction in the number of iNOS-positive hepatocytes (Figure 6C) compared with the carbon tetrachloride–treated mice. Pretreatment with EGCG at a higher dose of 75 mg/kg resulted in a further reduction of iNOS protein staining (Figure 6D). The image analysis of the number of cells showing positive staining for iNOS confirmed the dose-dependent effect of EGCG in reducing iNOS protein expression (Figure 6E). The mean volume of cells positive for iNOS protein expression confirmed the results obtained with image analysis (data not shown). To assess the validity of changes in iNOS protein by immunohistochemical analysis, we analyzed iNOS protein expression in the different groups by Western blot analysis. The data obtained by using Western blot analysis confirmed the results obtained by immunohistochemsitry (Figure 6F).
|
). Pretreatment with 50 mg EGCG/kg significantly reduced the level of adduct formation (P < 0.01). Treatment with 75 mg EGCG/kg led to a further reduction in the amount of adduct formation (Figure 7B).
|
). The level of nitrotyrosine in the different treatment groups measured by Western blot analysis corresponded to the levels assessed by immunohistochemistry and image analysis (Figure 8).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effect of EGCG on NO-related oxidants
The notion that NO is involved in acute liver injury is based on several observations that toxin-induced hepatic damage is associated with increased NO production by the liver (15). However, whether the augmented production of NO serves a protective or deleterious role in the liver remains an unresolved issue. The findings that NO inhibits the inflammatory response and reduces liver injury suggests that NO plays a beneficial role in attenuating liver injury (15, 32). On the other hand, there is also growing evidence that excessive NO production by iNOS plays an important role in the induction of toxin-induced liver injury (21). This conclusion is based on several observations. First, the expression of iNOS in the different cell types of the liver coincides with the development of liver injury. Second, administration of iNOS inhibitors attenuates liver injury. Third, extensive nitrotyrosine staining, the footprint of peroxynitrite formation, is detected in sections of liver in animals with liver injury and coincides with liver injury (33, 34).
Thus, several mechanisms exist through which increased production of NO, in the model used in this study, produced by iNOS-mediated hepatic injury. Reaction of NO with superoxide anions produces peroxynitrite, which is a highly oxidative species capable of nitrating tyrosine residues of numerous proteins, which leads to the formation of nitrotyrosine. Nitrotyrosine formation, detected by a specific antibody, was increased in carbon tetrachloride–treated mice and was significantly decreased by EGCG treatment. The fact that iNOS induction and nitrotyrosine formation occurred in the cells exhibiting necrotic changes suggests evidence for a role of NO in liver injury, at least in the model used in the current study. Other pathways through which NO and peroxynitrite mediate tissue injury include inhibition of mitochondrial respiration, inactivation of proteinase inhibitors, and formation of free radicals (34). Of note, the decreased production of NO-derived free radicals in the mice treated with carbon tetrachloride and EGCG provides further evidence for a role of EGCG in down-regulating NO-mediated injury.
Thus, the main finding of the current study was that carbon tetrachloride elicited acute liver injury as indicated by a significant increase in hepatocellular damage, increased alanine aminotransferase activity in serum, increased expression of iNOS, and extensive nitrotyrosine formation. By comparison, the degree of liver injury and expression of iNOS and nitrotyrosine decreased significantly in the EGCG-treated mice. Although our study focused on the role of EGCG in preventing hepatic toxicity, it is important to point out that the overall protective effect of green tea may require the combined actions of several components of tea (35). Relevant to the findings of the current study is the observation by Tedeschi et al (36), which shows that a concentration of green tea extract equivalent to the consumption of 10 cups tea/d exerts inhibitory actions on cytokine-induced tyrosine phosphorylation that blocks the expression of iNOS and reduces NO production. Because green tea can be consumed over long periods of time without any obviously known side effects, its possible role as an adjunct therapeutic agent in human inflammatory liver disease deserves consideration.
|
ACKNOWLEDGMENTS |
|---|
J-HC, GLT, and AAN contributed to the design of the experiment, analysis of data, and writing of the manuscript. ECL, HSHS, K-ML, and W-MT contributed to the collection of data. PCWF contributed to the analysis and interpretation of the EPR data. None of the authors had any financial or other conflicts of interest.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B-kinase activity in macrophages. Biochem Pharmacol 2000;59:357–67.[Medline]
production and lethality in a murine model. J Nutr 1998;128:2334–440.B kinase activity in the intestinal epithelial cell line IEC-6. Mol Pharmacol 2001;60:528–33.
activation. Mol Pharmacol 2004;65:111–20.This article has been cited by other articles:
|
|
 |
|
  M. Roomi, T Kalinovsky, N. Roomi, V Ivanov, M Rath, and A Niedzwiecki A nutrient mixture suppresses carbon tetrachloride-induced acute hepatic toxicity in ICR mice Human and Experimental Toxicology, July 1, 2008; 27(7): 559 - 566. [Abstract] [PDF]
|
|
|
|
|
|
M. Roomi, T Kalinovsky, V Ivanov, M Rath, and A Niedzwiecki A nutrient mixture prevents acetaminophen hepatic and renal toxicity in ICR mice Human and Experimental Toxicology, March 1, 2008; 27(3): 223 - 230. [Abstract] [PDF]
|
|
|
|
 |
|
 J. W. FINLEY Proposed Criteria for Assessing the Efficacy of Cancer Reduction by Plant Foods Enriched in Carotenoids, Glucosinolates, Polyphenols and Selenocompounds Ann. Bot., June 1, 2005; 95(7): 1075 - 1096. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
± SEM; n = 8 per group) of hepatic necrosis induced by carbon tetrachloride and confirmed the findings on histologic analysis (C and D). ANOVA: *Significantly different from the other 3 groups, P < 0.01; 
significantly different from the EGCG-50 group, P < 0.05.
