Pharmacognosy Magazine

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 15  |  Issue : 61  |  Page : 342--347

Protective effects of genistein alleviate alcohol-induced liver injury in rats


Wanlapa Leelananthakul1, Duangporn Werawatganon1, Naruemon Klaikeaw2, Maneerat Chayanupatkul1, Prasong Siriviriyakul1,  
1 Department of Physiology, Alternative and Complementary Medicine for GI and Liver Diseases Research Unit, Bangkok, Thailand
2 Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Correspondence Address:
Duangporn Werawatganon
Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330
Thailand

Abstract

Background: Alcohol is a major contributor of chronic liver disease worldwide. Medical treatment for alcoholic liver disease (ALD) is limited. Due to the roles of oxidative stress in the development of ALD, genistein, a natural antioxidant, might be beneficial in alleviating alcohol-induced liver injury. Materials and Methods: Eighteen male Sprague–Dawley® rats were divided into three groups (n = 6 each). Control group received distilled water, while alcohol group received 50% alcohol (8 g/kg body weight [BW] per day), and genistein group received genistein (16 mg/kg BW per day) dissolved in 50% alcohol (8 g/kg BW per day) for 4 weeks. At the end of the study, liver tissue was obtained for histopathology and immunohistochemistry for interleukin-18 (IL-18), hepatic malondialdehyde (MDA), and glutathione (GSH) measurement. Serum samples were analyzed for alanine transaminase (ALT) and tumor necrosis factor-α (TNF-α). Results: Alcohol-fed rats gained significantly less weight than control and genistein ones (48.83 ± 14.59, 142.83 ± 10.06 vs. 69.17 ± 7.33 g, respectively, P < 0.01). Serum ALT levels were also significantly lower in genistein group than in alcohol group (32.43 ± 12.90 vs. 120.30 ± 75.30; P < 0.05). Hepatic MDA levels were higher in alcohol group (0.13 ± 0.02 nmol/mg protein), while the levels were comparable between genistein (0.09 ± 0.02 nmol/mg protein) and control groups (0.1 ± 0.01 nmol/mg protein). There was a trend toward a decrease in GSH levels in alcohol-fed rats as compared to control ones. On the contrary, GSH levels were significantly increased in GSH-treated rats. Markers of inflammatory responses, such as IL-18 and TNF-α, were higher in alcohol group and declined toward the control group with genistein administration. Conclusion: Alcohol-induced hepatic cell damages through oxidative stress and inflammatory responses. Genistein could alleviate alcohol-induced liver injury through its antioxidant and anti-inflammatory properties. Abbreviations used: ALD: Alcoholic liver disease; NAFLD: Nonalcoholic fatty liver disease; ALT: Alanine transaminase; TNF-α: Tumor necrosis factor-alpha; IL-18: Interleukin-18; LPS: Lipopolysaccharide; MDA: Malondialdehyde; GSH: Glutathione; TBARS: Thiobarbituric acid-reactive substances; ELISA: Enzyme-linked immunosorbent assay; DMSO: Dimethyl sulfoxide; DAB: Diaminobenzidine.



How to cite this article:
Leelananthakul W, Werawatganon D, Klaikeaw N, Chayanupatkul M, Siriviriyakul P. Protective effects of genistein alleviate alcohol-induced liver injury in rats.Phcog Mag 2019;15:342-347


How to cite this URL:
Leelananthakul W, Werawatganon D, Klaikeaw N, Chayanupatkul M, Siriviriyakul P. Protective effects of genistein alleviate alcohol-induced liver injury in rats. Phcog Mag [serial online] 2019 [cited 2021 Jun 24 ];15:342-347
Available from: http://www.phcog.com/text.asp?2019/15/61/342/253493


Full Text



[INLINE:1]

SUMMARY

Genistein alleviated alcohol-induced liver injury through its antioxidant, anti-inflammatory, and glutathione restoration properties.

 Introduction



Alcoholic liver diseases (ALDs) cause significant health problems worldwide. Globally, in 2010, alcohol-attributable liver cirrhosis was responsible for 493,300 deaths and 14,544,000 disability-adjusted life years.[1] The spectrum of ALD ranges from simple steatosis, alcoholic steatohepatitis, progressive fibrosis to cirrhosis and hepatocellular carcinoma.[2] The current medical therapies for ALD are limited and with disappointing efficacy. Prednisolone, a standard treatment for severe alcoholic hepatitis, has been shown in a recent randomized controlled trial to have no benefits in long-term survivals and increased risk of infectious complications.[3] Therefore, the quest for safe and effective treatment for alcoholic hepatitis has been an area of active research.

Liver plays a pivotal role in alcohol metabolism. Once absorbed, alcohol is metabolized to acetaldehyde by alcohol dehydrogenase enzyme and subsequently to acetic acid by aldehyde dehydrogenase. Moreover, alcohol can be metabolized through microsomal ethanol-oxidizing system using cytochrome P450 2E1. This process creates reactive oxygen species (ROS).[4],[5] Both acetaldehyde and ROS create lipid peroxidation leading to the production of end products such as malondialdehyde (MDA).[5],[6],[7] In addition, acetaldehyde can deplete the natural antioxidants such as mitochondrial glutathione (GSH) and S-adenosylmethionine, further intensifying the oxidative stress.[5] These chain reactions lead to hepatocyte injury, inflammatory responses, hepatic stellate cell activation, and extracellular matrix production.

Other than the direct effects from its metabolites, alcohol can increase intestinal permeability and liver exposure to lipopolysaccharide (LPS).[8] LPS then binds with Toll-like receptor-4 (TLR-4) on Kupffer cells leading to inflammatory cytokine releases, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, and IL-18.[8],[9] These cytokines are major players in the hepatocyte injury and apoptosis. Given the roles of oxidative stress in the development of ALD, the natural products with antioxidant properties appear to be the attractive options for the treatment of ALD. One of the leading candidates would be genistein.

Genistein is one of the major isoflavones found in soy and soy products. Genistein exerts several functions such as reducing lipid peroxidation,[10],[11],[12] enhancing fatty acid catabolism,[11] and promoting extracellular matrix degradation.[12] Despite being studied extensively in nonalcoholic fatty liver disease (NAFLD), little is known about the effects of genistein in ALD. In the present study, we aimed to evaluate the effects of genistein on alcohol-induced liver injury in rats determined by the changes in histopathology, antioxidants, lipid peroxidation, and inflammatory markers. The protocol for this study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Approval No. 08/2556).

 Materials and Methods



Animal preparation

Male Sprague–Dawley® rats weighing 180–220 g were purchased from the National Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand. Animals were housed in a standard room with controlled temperature of 25°C ± 1°C and a 12-h light-dark cycle. The research was conducted according to the policies and procedures proposed by the Chulalongkorn University Animal Care and Ethics Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Approval No. 08/2556).

Experimental protocol

Eighteen rats were randomly divided into three groups (n = 6 in each group). All animals were fed ad libitum with a custom diet containing 35% of total calories from fat (corn oil), 18% from protein, and 47% from carbohydrates.[13],[14] Genistein powder was purchased from Cayman Chemical Company (Michigan, USA). All animals were fed with either alcohol or distilled water twice a day through an intragastric tube for 4 weeks. Group 1 or control group received distilled water (16 mL/kg body weight [BW] per day); Group 2 or alcohol group received 50% alcohol (8 g/kg BW per day); and Group 3 or genistein group received daily genistein (16 mg/kg BW per day) dissolved in 50% alcohol (8 g/kg BW per day) for the entire 4 weeks of the experiment. All rats were weighed on the 1st day of the study before drug administration and the end of the study before euthanasia to calculate the changes in BW.

At the end of 4 weeks, all rats were euthanized with sodium thiopental overdose after a 12-h fast. Total hepatectomy was performed. Liver was immediately washed with 25°C saline and then cut into several pieces. Three small pieces of the liver were frozen in liquid nitrogen and stored at −80°C until MDA and GSH analyses. The remaining liver specimens were fixed in 40 g/L formaldehyde for IL-18 expression analysis. Blood samples were obtained by cardiac puncture and kept at room temperature for 2 h. Specimens were then centrifuged at 2000 × g for 20 min, and serum samples were collected for alanine transaminase (ALT) and TNF-α analysis.

Hepatic malondialdehyde determination

MDA level was measured using a commercial assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). The test involves the measurement of thiobarbituric acid reactive substances production rate under high temperature and acidic conditions. The methodology is as follows: 1 g of liver tissue was homogenized in radioimmunoprecipitation assay buffer containing protease inhibitor and sonicated on ice for 15 s. Supernatants were obtained after centrifugation at 1600 × g for 10 min at 4°C. The absorbance of supernatant fraction was read at a wavelength of 532 nm. MDA levels were calculated from a standard curve and expressed as nmol/mg protein.

Hepatic glutathione determination

GSH level was quantified using a commercial assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Liver tissues were washed with phosphate-buffered saline (PBS) solution. Tissues were then homogenized with cold (morpholino) ethanesulfonic acid buffer before being centrifuged at 10,000 × g for 15 min at 4°C. Supernatants were collected and deproteinated. The absorbance of supernatant fraction was read at a wavelength of 405 nm, and GSH values were calculated from a standard curve and expressed as nmol/mg protein.

Immunohistochemistry for hepatic IL-18 expression

After being fixed in formaldehyde, liver samples were embedded in paraffin and sliced at a thickness of 3 μm. Tissue sections were then deparaffinized with xylene and ethanol for 10 min. The antigen retrieval was achieved by treating the slides with citrate buffer pH 6.0 and heating in a microwave for 13 min. Slides were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity for 5 min and with 3% normal horse serum to block nonspecific binding for 20 min. Tissues were then washed with PBS solution. Subsequently, sections were incubated with mouse monoclonal antibody against IL-18 (GeneTex, Inc., Irvine, CA, USA) at a dilution of 1:200 for 1 h at a room temperature and washed again with PBS solution. Slides were then incubated with secondary antibody for IL-18 (Dako Denmark AS, Glostrup, Denmark) for 30 min at a room temperature. When the color development with diaminobenzidine was detected, sections were counterstained with hematoxylin. Under light microscopy, IL-18-positive cells were defined as Kupffer cells with dark brown-stained nuclei. Images of each sample were taken at high magnifications (×40). Five hundred Kupffer cells were manually counted for each rat, and the proportions of IL-18-positive cells were calculated.

Serum tumor necrosis factor alpha determination

Serum levels of TNF-α were determined by a solid-phase enzyme immunoassay technique using commercially available kits (R and D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. TNF-α levels were expressed as pg/mL.

Pathological examination

After fixation in 10% formalin, liver tissues were processed by routine histology procedures and embedded in paraffin. The 5-μm thick tissue sections were stained with hematoxylin and eosin (H and E) and then put on glass slides for light microscopy. An experienced pathologist blinded to the experiment evaluated all samples. The entire sections were examined for histologic grading of steatosis (0–3), hepatocyte ballooning (0–3), and lobular inflammation (0–3) according to the criteria described by Brunt et al.[15]

Statistical analysis

Continuous data were presented as mean ± standard deviation. One-way ANOVA and post hoc Tukey's honestly significant difference tests were used for comparisons among groups. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics for Windows version 17 (SPSS, Inc., Chicago, IL, USA).

 Results



Effect of genistein on body weight changes

As depicted in [Figure 1]a, rats in the alcohol group gained significantly less weight than those in genistein and control groups with the BW changes of 48.83 ± 14.59, 69.17 ± 7.33, and 142.83 ± 10.06 g in alcohol, genistein, and control groups, respectively (P < 0.01). Comparing with alcohol group, rats in the genistein group gained more weight at 4 weeks (48.83 ± 14.59 g in alcohol group and 69.17 ± 7.33 g in genistein group, P < 0.05).{Figure 1}

Serum alanine aminotransferase level

The level of serum ALT in alcohol group was higher than control group (120.30 ± 75.30 vs. 59.6 ± 9.78 U/L). However, serum ALT level significantly decreased in rats treated with genistein when compared with alcohol group (32.43 ± 12.90 vs. 120.30 ± 75.30 U/L, P < 0.05) [Figure 1]b.

Effect of genistein on liver oxidative stress marker and hepatic glutathione levels

Hepatic MDA levels in alcohol group were significantly higher than in control group (0.13 ± 0.02 vs. 0.1 ± 0.01 nmol/mg protein, respectively; P < 0.05). With genistein coadministration, hepatic MDA levels in genistein group decreased significantly and closely resembled those in control group (0.09 ± 0.02 vs. 0.13 ± 0.02 vs. 0.1 ± 0.01 nmol/mg protein in genistein, alcohol, and control groups, respectively; P < 0.01) [Figure 1]c.

Hepatic GSH levels in the control group were higher than in alcohol group, albeit not statistically significant (12.66 ± 1.08 vs. 11.55 ± 1.15 nmol/mg protein, respectively). However, hepatic GSH levels were significantly higher in genistein group than in alcohol and control ones (14.77 ± 2.80 vs. 11.55 ± 1.15 vs. 12.66 ± 1.08 nmol/mg protein, respectively; P < 0.05) [Figure 1]d.

Effect of genistein on inflammatory responses

Serum TNF-α levels in alcohol group were significantly higher than in control group (13.17 ± 2.34 vs. 10.64 ± 0.8 pg/mL, respectively; P < 0.05), while a fall in serum TNF-α levels was noted in rats receiving both genistein and alcohol as opposed to those receiving alcohol alone (11.43 ± 1.31 vs. 13.17 ± 2.34 pg/mL; P < 0.05, respectively) [Figure 1]e. As presented in [Figure 2], the percentage of IL-18 positive cells in alcohol group was significantly higher than in control group (57%. 4 ± 2.54% vs. 16.87% ± 5.01%, respectively; P < 0.01). The receipt of genistein led to a decline in IL-18 positive cells in genistein group comparing with alcohol group (19.52% ± 10.9% vs. 57.4% ± 2.54%, respectively; P < 0.01).{Figure 2}

Effect of genistein on liver pathology

As shown in [Figure 3] and [Table 1], liver histopathology from alcohol group exhibited liver injury features, including steatosis, lobular inflammation, and moderate ballooning degeneration of hepatocytes. On the other hand, liver histopathology in genistein group improved compared with alcohol group.{Figure 3}{Table 1}

 Discussion



Malnutrition is common among patients with chronic alcohol consumption due to ingestion of calories devoid of protein, vitamins, and minerals and possibly the direct effect of alcohol on nutrient absorption.[16] In the present study, we found that rats in alcohol group gained significantly less weight than those in control and genistein group. These findings were in line with the study by Zhuo et al., which showed that alcohol-fed rats had less weight gain than control rats and this could partially be restored by receiving taurine, epigallocatechin gallate, and genistein.[17] Interestingly, weight changes were more prominent in control group than genistein one. We hypothesized that less weight gain in genistein group happened as a result of genistein effects on the reduction of total body fat and leptin production.[18],[19]

Oxidative stress and lipid peroxidation happened as a result of ROS production from alcohol metabolism through microsomal ethanol-oxidizing system and from acetaldehyde effect.[20] In this study, we used hepatic MDA level as a marker for oxidative stress and found that hepatic MDA levels were significantly higher in alcohol group than in control group, and this detrimental effect of alcohol was attenuated by genistein administration as evidenced by the comparable MDA levels between genistein and alcohol groups. Our results were similar to a previous study by Teare et al.,[21] which showed a significant increase in hepatic MDA levels in alcohol-fed rats compared with control rats, suggesting that oxidative stress is a mechanism of alcohol-induced liver damage. Studies in rat models of alcoholic and NAFLD demonstrated that genistein could alleviate oxidative stress and lipid peroxidation as shown by the decline in MDA levels in genistein-treated rats.[22],[23]

In addition to increased ROS production, alcohol could also cause depletion in hepatic GSH, further increasing liver susceptibility to oxidative stress.[22],[23],[24],[25] In the current study, we found a trend toward a decline in GSH levels in alcohol group. Studies have shown that acute alcohol exposure could reduce GSH levels through the binding of its metabolites with GSH and the inhibition of GSH synthesis.[26] On the contrary, chronic alcohol exposure could lead to increased GSH efflux into hepatic sinusoids, GSH synthesis, and GSH levels.[27],[28] The relatively unchanged GSH levels in alcohol-fed rats in our study could happen as a result of the balance between the direct effects of alcohol metabolites on GSH and the consequences of chronic alcohol exposure on GSH synthesis and efflux.

Unequivocally, GSH levels significantly increased in genistein-treated group as compared with alcohol and control groups. There are several proposed mechanisms of how genistein promotes GSH synthesis. The rate of GSH synthesis is mainly controlled by the activity of γ-glutamylcysteine ligase, which is regulated by redox-sensitive transcription factor, nuclear factor erythroid-2-related factor 2 (Nrf2).[29] An in vitro study showed that genistein administration could induce the expression of Nrf2 gene and increase superoxide dismutase, catalase, and GSH levels.[30] Other antioxidant effects of genistein include the induction of GSH peroxidase 1[31] and (nicotinamide adenine dinucleotide-phosphate quinone oxidoreductase 1,[32] the enzymes responsible for the prevention of ROS formation.

Alcohol can also cause liver injury through its effects on gut dysbiosis and LPS exposure to the liver.[8] LPS then binds to TLR-4 on Kupffer cells, which in turn release inflammatory cytokines such as TNF-α and IL-18.[33] IL-18 plays a pivotal role in LPS-activated Propionibacterium acnes-induced liver injury through the augmentation of TNF-α release and the induction of interferon- γ and Fas ligand.[9],[34],[35] In this study, we found the rise in serum TNF-α levels and IL-18 positive cells in alcohol-fed rats, supporting the role of LPS in alcohol-induced liver injury. These effects could be attenuated by genistein use. Our results were similar to a study by Zhao et al., which demonstrated that genistein treatment could reduce TNF-α levels and partially protect against hepatocyte apoptosis from alcohol.[22] In vitro studies also suggested that genistein could reduce LPS-induced inflammation through the inhibition of TNF-α, IL-6, and nuclear factor kappa B production.[36] Despite not being studied directly in ALD, genistein has been shown to reduce IL-18 levels in other conditions such as metabolic syndrome.[37]

 Conclusion



Genistein exerted the antioxidant and anti-inflammatory properties in rat models of alcohol-induced liver injury and could prove beneficial in the prevention or treatment of ALD. Human studies are warranted to confirm its usefulness in clinical practice.

Acknowledgements

We would like to thank for the Ratchadapisek Sompotch Fund by Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Financial support and sponsorship

This study was financially supported by Ratchadapisek Sompotch Fund, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Conflicts of interest

There are no conflicts of interest.

References

1Rehm J, Samokhvalov AV, Shield KD. Global burden of alcoholic liver diseases. J Hepatol 2013;59:160-8.
2Mathurin P, Bataller R. Trends in the management and burden of alcoholic liver disease. J Hepatol 2015;62:S38-46.
3Thursz MR, Richardson P, Allison M, Austin A, Bowers M, Day CP, et al. Prednisolone or pentoxifylline for alcoholic hepatitis. N Engl J Med 2015;372:1619-28.
4Mello T, Ceni E, Surrenti C, Galli A. Alcohol induced hepatic fibrosis: Role of acetaldehyde. Mol Aspects Med 2008;29:17-21.
5Dunn W, Shah VH. Pathogenesis of alcoholic liver disease. Clin Liver Dis 2016;20:445-56.
6Mylonas C, Kouretas D. Lipid peroxidation and tissue damage.In Vivo 1999;13:295-309.
7Tilg H, Day CP. Management strategies in alcoholic liver disease. Nat Clin Pract Gastroenterol Hepatol 2007;4:24-34.
8Vassallo G, Mirijello A, Ferrulli A, Antonelli M, Landolfi R, Gasbarrini A, et al. Review article: Alcohol and gut microbiota – The possible role of gut microbiota modulation in the treatment of alcoholic liver disease. Aliment Pharmacol Ther 2015;41:917-27.
9Tsutsui H, Matsui K, Okamura H, Nakanishi K. Pathophysiological roles of interleukin-18 in inflammatory liver diseases. Immunol Rev 2000;174:192-209.
10Fang YC, Chen BH, Huang RF, Lu YF. Effect of genistein supplementation on tissue genistein and lipid peroxidation of serum, liver and low-density lipoprotein in hamsters. J Nutr Biochem 2004;15:142-8.
11Kim MH, Kang KS, Lee YS. The inhibitory effect of genistein on hepatic steatosis is linked to visceral adipocyte metabolism in mice with diet-induced non-alcoholic fatty liver disease. Br J Nutr 2010;104:1333-42.
12Huang Q, Huang R, Zhang S, Lin J, Wei L, He M, et al. Protective effect of genistein isolated from hydrocotyle sibthorpioides on hepatic injury and fibrosis induced by chronic alcohol in rats. Toxicol Lett 2013;217:102-10.
13Enomoto N, Yamashina S, Kono H, Schemmer P, Rivera CA, Enomoto A, et al. Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells. Hepatology 1999;29:1680-9.
14Lieber CS, DeCarli LM. The feeding of ethanol in liquid diets. Alcohol Clin Exp Res 1986;10:550-3.
15Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467-74.
16Lieber CS. Alcohol and malnutrition in the pathogenesis of liver disease. JAMA 1975;233:1077-80.
17Zhuo L, Liao M, Zheng L, He M, Huang Q, Wei L, et al. Combination therapy with taurine, epigallocatechin gallate and genistein for protection against hepatic fibrosis induced by alcohol in rats. Biol Pharm Bull 2012;35:1802-10.
18Kim HK, Nelson-Dooley C, Della-Fera MA, Yang JY, Zhang W, Duan J, et al. Genistein decreases food intake, body weight, and fat pad weight and causes adipose tissue apoptosis in ovariectomized female mice. J Nutr 2006;136:409-14.
19Szkudelski T, Nogowski L, Pruszyńska-Oszmałek E, Kaczmarek P, Szkudelska K. Genistein restricts leptin secretion from rat adipocytes. J Steroid Biochem Mol Biol 2005;96:301-7.
20Caballería J. Current concepts in alcohol metabolism. Ann Hepatol 2003;2:60-8.
21Teare JP, Greenfield SM, Watson D, Punchard NA, Miller N, Rice-Evans CA, et al. Lipid peroxidation in rats chronically fed ethanol. Gut 1994;35:1644-7.
22Zhao L, Wang Y, Liu J, Wang K, Guo X, Ji B, et al. Protective effects of genistein and puerarin against chronic alcohol-induced liver injury in mice via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. J Agric Food Chem 2016;64:7291-7.
23Yalniz M, Bahcecioglu IH, Kuzu N, Poyrazoglu OK, Bulmus O, Celebi S, et al. Preventive role of genistein in an experimental non-alcoholic steatohepatitis model. J Gastroenterol Hepatol 2007;22:2009-14.
24Fernández-Checa JC, Hirano T, Tsukamoto H, Kaplowitz N. Mitochondrial glutathione depletion in alcoholic liver disease. Alcohol 1993;10:469-75.
25Vogt BL, Richie JP Jr. Glutathione depletion and recovery after acute ethanol administration in the aging mouse. Biochem Pharmacol 2007;73:1613-21.
26Mitchell MC, Raiford DS, Mallat A. Effects of ethanol on glutathione metabolism. In: Watson RR, editor. Liver Pathology and Alcohol. Totowa, NJ: Humana Press; 1991. p. 169-94.
27Callans DJ, Wacker LS, Mitchell MC. Effects of ethanol feeding and withdrawal on plasma glutathione elimination in the rat. Hepatology 1987;7:496-501.
28Morton S, Mitchell MC. Effects of chronic ethanol feeding on glutathione turnover in the rat. Biochem Pharmacol 1985;34:1559-63.
29Steele ML, Fuller S, Patel M, Kersaitis C, Ooi L, Münch G, et al. Effect of nrf2 activators on release of glutathione, cysteinylglycine and homocysteine by human U373 astroglial cells. Redox Biol 2013;1:441-5.
30Zhang T, Wang F, Xu HX, Yi L, Qin Y, Chang H, et al. Activation of nuclear factor erythroid 2-related factor 2 and PPARγ plays a role in the genistein-mediated attenuation of oxidative stress-induced endothelial cell injury. Br J Nutr 2013;109:223-35.
31Suzuki K, Koike H, Matsui H, Ono Y, Hasumi M, Nakazato H, et al. Genistein, a soy isoflavone, induces glutathione peroxidase in the human prostate cancer cell lines LNCaP and PC-3. Int J Cancer 2002;99:846-52.
32Wiegand H, Wagner AE, Boesch-Saadatmandi C, Kruse HP, Kulling S, Rimbach G, et al. Effect of dietary genistein on phase II and antioxidant enzymes in rat liver. Cancer Genomics Proteomics 2009;6:85-92.
33Neuman MG, Maor Y, Nanau RM, Melzer E, Mell H, Opris M, et al. Alcoholic liver disease: Role of cytokines. Biomolecules 2015;5:2023-34.
34Tsutsui H, Matsui K, Kawada N, Hyodo Y, Hayashi N, Okamura H, et al. IL-18 accounts for both TNF-alpha – And Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice. J Immunol 1997;159:3961-7.
35Sakao Y, Takeda K, Tsutsui H, Kaisho T, Nomura F, Okamura H, et al. IL-18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock. Int Immunol 1999;11:471-80.
36Ji G, Zhang Y, Yang Q, Cheng S, Hao J, Zhao X, et al. Genistein suppresses LPS-induced inflammatory response through inhibiting NF-κB following AMP kinase activation in RAW 264.7 macrophages. PLoS One 2012;7:e53101.
37Azadbakht L, Kimiagar M, Mehrabi Y, Esmaillzadeh A, Hu FB, Willett WC, et al. Soy consumption, markers of inflammation, and endothelial function: A cross-over study in postmenopausal women with the metabolic syndrome. Diabetes Care 2007;30:967-73.