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Cíntia de David, Graziella Rodrigues, Silvia Bona, Luise Meurer, Javier González-Gallego, María Jesús Tuñón and NormaRole of Quercetin in Preventing Thioacetamide-Induced Liver Injury in Rats

  

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Role of Quercetin in Preventing Thioacetamide-InducedLiver Injury in Rats

CINTIA DE DAVID1, GRAZIELLA RODRIGUES

1, SILVIA BONA1, LUISE MEURER

1, JAVIER GONZALEZ-GALLEGO2,

MARIA JESUS TUNON2, AND NORMA POSSA MARRONI

1

1Laboratory of Experimental Hepatology and Physiology, Porto Alegre Clinical Hospital, Federal University of

Rio Grande do Sul, Porto Alegre, RS, Brazil2Institute of Biomedicine, University of Leon; and Centro de Investigacion Biomedica en Red de Enfermedades

Hepaticas y Digestivas (CIBERehd), Campus of Vegazana s/n, PC 24071 Leon, Spain

ABSTRACT

In hepatic toxicity induced in rats by two injections of thioacetamide (TAA, 350 mg/kg with an interval of 8 hr), the action of quercetin was

investigated. After 96 hr, TAA administration resulted in hepatic necrosis, significant increases in serum transaminase activity, and increases in hepatic

lipoperoxidation. Thioacetamide-induced hepatotoxicity also showed changes in antioxidant enzymes in the liver of rats, with alterations in p-ERK 1/2

(phosphorylated extracellular-signal related kinase 1/2) as well as an imbalance between proapototic protein Bax and anti-apoptotic protein Bcl-2

expression. With administration of the flavonoid quercetin (50 mg/Kg i.p.) for four consecutive days following TAA, serum aspartate

aminotransferase (AST) and alanine aminotransferase (ALT) activity were close to normal values in rats. Histological findings suggested that quercetin

had a preventive effect on TAA-induced hepatic necrosis. Quercetin treatment caused significant decreases in lipid peroxide levels in the TAA-treated

rats, with some changes in antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Quercetin also

inhibited the change of the p-ERK1/2 by TAA and significantly prevented the increase in Bax/Bcl-2 ratio, thus preventing apoptosis. Findings indicate

that quercetin may have a preventive effect on TAA-induced hepatotoxicity by modulating the oxidative stress parameters and apoptosis pathway.

Keywords: quercetin; thioacetamide; hepatotoxicity; oxidative stress

INTRODUCTION

Appropriate animal models have contributed to our under-

standing of the mechanisms responsible for hepatotoxic injury.

The pathological lesions caused by hepatotoxins may be simi-

lar to many forms of liver disease, contributing to the evalua-

tion of novel potential hepatoprotectants (Abul et al. 2002;

Bruck, Aeed, Avni, et al. 2004).

Hepatotoxins initially damage the centrilobular regions of

liver where there are high levels of cytochrome P450 oxidases

that mediate their conversion to toxic intermediates, followed

by reactive oxygen species (ROS) production, lipid peroxida-

tion, and release of pro-inflammatory cytokines (Luster et al.

2000). Thus, thioacetamide (TAA) is a hepatotoxin causing

centrilobular necrosis (Zimmerman 1978; Diez-Fernandez

et al. 1993; Diez-Fernandez, Sanz, and Cascales 1996; Mangi-

pudy et al. 1998; Chu et al. 2000), which has been shown to

also induce apoptosis and periportal inflammatory cell infiltra-

tion in rat liver (Ledda-Columbano et al. 1991). Thioacetamide

causes an elevation of oxidative stress, enhancing free radical–

mediated damage to proteins, lipids and deoxyribonucleic acid

(DNA) (Lu et al. 1999; Bruck, Aeed, Shirin, et al. 2004; Tunez

et al. 2005; Uskokovic-Markovic et al. 2007). Thioacetamide

induces hepatotoxicity via its S-oxide metabolite (thioaceta-

mide-S-dioxide), an unstable, reactive metabolite, that initiates

necrosis and the generation of reactive oxygen species (ROS)

by binding covalently to liver macromolecules (Porter and Neal

1978). Several studies have demonstrated the beneficial effect

of antioxidants in protecting the liver against TAA-induced

injury (Balkan et al. 2001; Uskokovic-Markovic et al. 2007;

Baskaran, Periyasam, and Venkatraman 2010). TAA induces

hepatocyte damage following its metabolism to thioacetamide

sulphene and sulphone, via a critical pathway that involves

CYP4502E1-mediated biotransformation (T. Wang et al.

2000; Ramaiah, Apte, and Mehendale 2001).

Address correspondence to: Dr. Norma Possa Marroni, Laboratorio de

Hepatologia Experimental—Fisiologia do HCPA, Rua Ramiro Barcelos

2350, Centro de Pesquisas—90035-003—Porto Alegre, RS, Brazil; phone:

55 (51) 3359 8937; e-mail: nmarroni@terra.com.br.

FIPE: Fundo de Incentivo a Pesquisa e Eventos (Fund to Encourage

Research and Events) sponsored the experiments. Cıntia de David received a

PhD scholarship from CNPq: Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (National Council for Scientific and Technological

Development and from CAPES (Coordenacao de Aperfeicoamento de

Pessoal de Nıvel Superior)).

Abbreviations: AST, aspartate aminotransferase; ALT, alanine

aminotransferase; CAT, catalase; CO, control group; DNA, deoxyribonucleic

acid; ERK, extracellular signal-related protein kinase; GPx, glutathione perox-

idase; HCPA, Hospital de Clınicas of Porto Alegre; H2O2, hydrogen peroxide;

HRP, horseradish peroxidase; ip., intraperitoneal; KCl, potassium chloride;

MAPK, mitogen-activated protein kinase; NADPH, nicotinamide adenine

dinucleotide phosphate; NO, nitric oxide; PARP, poly (ADP-ribose) polymer-

ase; p-ERK1/2, phosphorylated extracellular-signal related kinase 1 and 2; Q,

quercetin; ROS, reactive oxygen species; SOD, superoxide dismutase; S.D.,

standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel

electrophoresis; TAA, thioacetamide; TBARS, thiobarbituric acid reactive

substances; TNF-a, tumor necrosis factor-alpha.

949

Toxicologic Pathology, 39: 949-957, 2011

Copyright # 2011 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623311418680

Flavonoids are a large group of natural polyphenolic

substances widely distributed in the plant kingdom that can act

as antioxidants in biological systems. Quercetin (3, 3’, 4’, 5, 7-

pentahydroxyflavone), one of the most abundant flavonoids, is

present in large amounts in vegetables, fruits, tea, and olive oil.

It contains a number of phenolic hydroxyl groups and is a potent

oxygen free radical scavenger and a metal chelator (Middleton

1998). It has been demonstrated that quercetin exhibits its thera-

peutic potential against many diseases, including ischemic heart

diseases, atherosclerosis, liver fibrosis, renal injury, and chronic

biliary obstruction (Peres et al. 2000; Singh, Chander, and Chopra

2004; Tieppo et al. 2007). In this context, the present study was

designed to highlight the mechanisms involved in an experimen-

tal model of TAA-induced hepatotoxicity and to determine if the

treatment with quercetin exerts a beneficial effect.

MATERIALS AND METHODS

Animals: All procedures related to the rats were carried

out according to the guidelines of the Ethical Research Com-

mission in Health of the Research and Graduate Group of the

Hospital de Clınicas of Porto Alegre (HCPA) (Goldin and Ray-

mundo 1997). Two-month-old male Wistar rats with a mean

weight of 250 g were used. The animals were obtained from the

State Foundation in Health Production and Research, Porto

Alegre. They were kept in the Animal Experimentation Unit

at the Research Center of the HCPA in plastic cages measuring

47 � 34 � 18 cm lined with wood chips, under a 12 hr light/

dark cycle and temperature between 20–25�C. They received

water and food ad libitum.

Experimental Procedure: Twenty-eight male Wistar rats

were randomized in four groups: CO ¼ Control Group (n ¼7), Q ¼ Control Group undergoing treatment with quercetin

(n ¼ 7), TAA ¼ Thioacetamide Group undergoing treatment

with thioacetamide (n ¼ 7), and TAA þ Q ¼ Thioacetamide

Group undergoing treatment with quercetin (n ¼ 7). The

TAA groups were injected with two doses of TAA (350

mg/kg i.p.) with an interval of 8 hr. Treatment with quercetin

(50 mg/kg) was initiated 2 hr after the second dose of TAA,

and the animals received four doses of quercetin (Sigma

Chemical, St. Louis, MO, USA) i.p., at intervals of 24 hr.

At day 5, rats were anesthetized using ketamine (100 mg/

kg) and xylazine (10 mg/kg) and killed by exsanguination.

Tissues were collected and stored at –80�C.

Blood Analysis: Serum aspartate aminotransferase (AST)

and alanine aminotransferase (ALT) were determined by com-

mercial kits (Boehringer Mannheim, Mannheim, Germany).

Histological Analysis: Thin slices of the left lobes of the

livers from each group were collected and kept for 48 hr in a

10% formaldehyde solution. After paraffin embedding, tissues

were sectioned and stained with hematoxylin and eosin (H&E)

for histological studies.

Oxidative Stress and Antioxidant Assay: Frozen tissue

from each rat was homogenized in ice-cold phosphate buffer

(140 mM KCl, 20 mM phosphate, pH 7.4) and centrifuged at

1,500 g for 10 minutes. Oxidative stress was determined by

measuring the concentration of aldehydic products by thiobar-

bituric acid reactive substances (TBARS) (Buege and Aust

1978). Spectrophotometric absorbance of the supernatant at

535 nm was determined. Cytosolic superoxide dismutase

(SOD) (EC 1.5.1.1) was assayed at 30�C according to Misra

and Fridovich (Misra and Fridovich 1972). The autoxidation

rate of epinephrine, which is progressively inhibited by

increasing amounts of SOD in the homogenate, was monitored

spectrophotometrically at 560 nm. The amount of enzyme that

inhibited 50% of epinephrine autoxidation was defined as 1 U

of SOD activity. Catalase (CAT) activity was determined by

measuring the decrease in absorption at 240 nm in a reaction

medium containing 50 mM phosphate buffer (pH 7.2) and

0.3 M hydrogen peroxide (Boveris and Chance 1973). The

enzyme activity was assayed spectrophotometrically at 240

nm. The glutathione peroxidase (GPx) activity was determined

by the oxidation rate of nicotinamide adenine dinucleotide

phosphate (NADPH) in the presence of reduced glutathione

and glutathione reductase (Flohe and Gunzler 1984).

Nitric Oxide Metabolites: Nitric oxide production in liver

tissue was measured indirectly using a quantitative colori-

metric assay based on the Griess reaction as previously

described by Granger et al. (1999). It measures one of the stable

end products of nitric oxide, that is, nitrite. Aliquots of 50 mL

were incubated with enzyme cofactors and nitrate reductase for

30 minutes at room temperature for the conversion of nitrate to

nitrite. The nitrite formed was then analysed by reaction with

the Griess reagent, forming a coloured compound that was

measured by spectrophotometer at a wavelength of 540 nm

(Granger et al. 1999).

Western Blot Analysis: For protein isolation, liver tissues

in different treatment groups were prepared in the presence of

proteases and phosphatase inhibitors. Lysate proteins were

fractionated by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE), and Western blotting was per-

formed using the corresponding primary antibodies. Bound

antibody was detected by enhanced chemiluminescence. Mem-

brane rehybridization with b-actin antibody was performed for

loading accuracy (Tunon et al. 2003). Antibodies anti-p-ERK1/

2, anti-Bcl-2, and anti-Bax were obtained from Santa Cruz Bio-

technology (Santa Cruz, CA, USA). Polyclonal anti-b-actin

TABLE 1.—Effect of quercetin on serum hepatic enzymes in TAA-

induced liver toxicity.

Experimental Groups

Parameters Control Q TAA TAAþQ

AST (U/L) 177.9 + 29.2 189.8 + 66.5 497.8 + 123.5a 198.9 + 41.8

ALT (U/L) 42 + 4.4 36.8 + 9.8 271.4 + 87.3a 37.3 + 7.9

Results represent mean + SD.aSignificant difference between TAA group and groups Control, Q, and TAAþQ,

p � .05.

950 DE DAVID ET AL. TOXICOLOGIC PATHOLOGY

antibody was obtained from Sigma (St. Louis, MO, USA). Sec-

ondary HRP (horseradish peroxidase) conjugated antibody was

obtained from Dako (Glostrup, Denmark). The ECL detection

kit was purchased from Amersham Pharmacia (Uppsala,

Sweden).

Statistical Analysis: The results were expressed as mean

+ SD. The data were compared by analysis of variance

(ANOVA); when the analysis indicated the presence of a sig-

nificant difference, the means were compared with the Duncan

test. Significance was accepted at p � .05. All calculations

were performed using the SPSS 14.0 statistical software.

RESULTS

Blood Analysis: TAA administration resulted in a

marked increase in the levels of serum aminotransferase, com-

pared with untreated rats (Table 1). This increase was

significantly decreased in rats that received quercetin for four

days (p � .05).

Histopathology: Examination was performed on liver

specimens obtained four days after TAA injection to confirm

the biochemical findings. The histological analysis of liver tis-

sue from animals in the control group (Figure 1a) or from the

rats receiving quercetin alone (Figure 1b) exhibited normal

hepatocytes with well-preserved cytoplasm and nucleus. The

livers of rats treated with TAA alone exhibited residual perive-

nular necrosis with infiltration of lymphocytes and macro-

phages (Figure 1c). The changes were minimal in rats treated

with quercetin after TAA-induced hepatic injury. This group

exhibited less inflammatory cell infiltration (Figure 1d).

Oxidative Stress and Antioxidant: The TBARS levels

were significantly increased in rats that received TAA alone,

compared with control rats (Figure 2). Quercetin treatment

FIGURE 1.—Micrographs of hepatic tissue (original magnification: 200x). Livers of control rats (a); control treated with quercetin rats (b);

tioacetamide-injected rats (c); tioacetamide-injected rats treated with quercetin (d). Tissue samples were stained with hematoxylin-eosin (HE).

CV, centrilobular vein. Control liver shows normal structures (a, b), whereas residual necrosis and infiltration of lymphocytes and macrophages

were seen in perivenular area of TAA-injected rats (c). Quercetin treatment shows reduced amount of periportal cell infiltration (d).

Vol. 39, No. 6, 2011 HEPATOPROTECTIVE QUERCETIN 951

significantly reduced hepatic TBARS levels when compared

with TAA alone, but did not restore the TBARS to control lev-

els (p � .05; Figure 2). Our results showed that the hepatic

SOD activity increased significantly in the group that

received TAA alone (p � .05; Table 2). However, in the

group that received TAA and quercetin, the hepatic SOD

activity was significantly decreased in comparison with the

TAA group, returning to levels near to controls. In contrast,

the hepatic CAT activity was significantly decreased in the

TAA group (p � .05; Table 2), while treatment with querce-

tin after TAA significantly increased the CAT activity com-

pared to the control group. A significant increase in GPx

activity from the liver homogenates was observed in rats

treated with TAA (p � .05; Table 2). However, in the group

treated with quercetin after TAA administration, GPx activity

was significantly decreased in comparison to the TAA group

(p � .05), but higher than in the control group.

Nitric Oxide Metabolite: Figure 3 shows that nitrite/

nitrate levels in liver homogenates were significantly elevated

in the TAA group (p � .05). The treatment with quercetin

showed markedly decreased nitrite/nitrate levels compared to

the TAA-induced group (p � .05), returning to the levels sim-

ilar to the control.

Western Blot: Phosphorylated ERK1/2 was signifi-

cantly decreased in rats treated with TAA compared with

the control group. There were no changes observed in

ERK1/2 phosphorylation by quercetin treatment (Figure

4). Figure 5 demonstrates representative Western blots of

Bcl-2 and Bax expression in animals without and with quer-

cetin treatment. Densitometric analysis revealed that Bax

expression was significantly increased and Bcl-2 expression

significantly reduced in TAA group. Treatment with querce-

tin significantly prevented these changes, resulting in a sig-

nificantly lower Bax/Bcl-2 ratio when compared to TAA

rats (Figure 5).

DISCUSSION

The present study evaluated the effects of quercetin treat-

ment on liver injury resulting from TAA administration. TAA

treatment caused a significant increase in the activity levels of

serum ALT and AST, and quercetin reduced those levels. His-

topathological data also point toward a protective effect of

quercetin against TAA-induced liver injury. Histopathological

analysis showed that lesions of periportal hepatic cells with

periportal necrosis and macrophage infiltration in the TAA

group were ameliorated in rats receiving quercetin following

the induction of liver damage. Nitric oxide (NO) is a potent

biological mediator produced by hepatocytes after exposure

to cytokines (Geller et al. 1994; Kang, Berthiaume, and Yar-

mush 2002) that influences physiological processes in every

organ and tissue (X. Yao, Chen, and Li 2009). It is rapidly oxi-

dized in blood and tissues to form nitrate and nitrite (Shaker et

al. 2010). Production of NO can be determined by measuring

liver nitrite and nitrate levels (X. Yao, Chen, and Li 2009), and

its activity is regulated at the transcriptional level by cytokines

and the exposure of cells to other inflammatory stimuli such as

FIGURE 2.—TBARS levels in TAA-induced toxicity of rats treated

with quercetin or not. Results represent mean + SD. aSignificant dif-

ference between TAA group and groups Control, Q and TAAþQ; p�.05. bSignificant difference between TAAþQ group and group TAA,

Control and Q; p � .05.

TABLE 2.—Effect of quercetin on antioxidant enzymes in TAA-induced

liver toxicity.

Experimental Groups

Parameters Control Q TAA TAAþQ

SOD(U/L) 6.32 + 1.89 6.19 + 2.21 21.70 + 7.31a 10.67 + 5.00

CAT (U/L) 10.86 + 2.20 9.11 + 3.06 4.41 + 1.74a 18.40 + 3.71b

GPx (U/L) 0.011 + 0.004 0.013 + 0.003 0.144 + 0.039a 0.093 + 0.020b

Results represent mean + SD.aSignificant difference between TAA group and groups CO, Q, and TAAþQ, p � .05.bSignificant difference between the TAAþQ group and group TAA, CO, and Q,

p � .05.

FIGURE 3.—Levels of liver nitrites/nitrates in rats with TAA, treated

with quercetin or not. Results represent mean + SD. aSignificant dif-

ference between TAA group and groups Control, Q and TAAþ Q, p�.05.

952 DE DAVID ET AL. TOXICOLOGIC PATHOLOGY

endotoxin or ROS (Manjeet and Ghosh 1999). The indiscri-

minate destruction of cells and tissues by NO and its reactive

nitrogen intermediates may be involved in the pathology of

many inflammatory disorders, such as sepsis and severe gas-

troenteritis, where the levels of nitrate and nitrite are greatly

increased (Moncada and Higgs 1993; Herulf et al. 1999;

Crawford et al. 2004). Quercetin and its glycoside quercitrin

have been found previously to have anti-inflammatory effects

both in in vitro and in experimental models of inflammatory

diseases (Camuesco et al. 2004; Comalada et al. 2006). Quer-

cetin is a strong oxygen radical scavenger and also a good

metal chelator (Jovanovic et al. 1998). In vitro studies have

suggested that quercetin has a potent inhibitory activity

against the production of NO and tumor necrosis factor in

lipopolysaccharide-stimulated Kupffer cells (Kawada et al.

1998). Quercetin was also shown to reduce the serum levels

of NO in streptozotocin-treated rats (Coskun et al. 2005) and

to scavenge superoxide in ischemia-reperfusion injury (Huk

et al. 1998). Thus, the elevated liver nitrite and nitrate levels

observed in our study are in agreement with previous data in

different animal models of liver injury (Isobe et al. 2000;

Shaker et al. 2010). This indicates that the hepatic injury pro-

duced by TAA treatment could be a consequence of oxidative

stress. This oxidative stress results from the increased gener-

ation of ROS, which have been reported to attack various

biological molecules and causing lipid peroxidation.

In the current study, the increased activity of SOD reflects

an activation of the compensatory mechanism through the

effects of TAA on progenitor cells, and its extent depends on

the magnitude of the oxidative stress and, hence, on the dose

of the stressors (Prakasam, Sethupathy, and Lalitha 2001).

Increased activity of GPx could be due to the significant pro-

duction of H2O2 in TAA-induced toxicity (Chen, Hsu, and

Weng 2006).

Quercetin maintained the activities of SOD and GPx similar

to control levels. However, TAA reduced the levels of CAT

activity, which may cause the accumulation of O2–, H2O2, or

their products of decomposition. Loss of CAT activity results

in oxygen intolerance and triggers a number of deleterious

reactions such as protein and DNA oxidation and cell death

(Bruck et al. 1999). Treatment with quercetin may have pre-

vented large increases in activity of antioxidant enzymes SOD

and GPx, as observed for TAA group. Animals treated with the

antioxidant quercetin also showed an increased catalase activ-

ity. Flavonoids, and quercetin in particular, are potent antioxi-

dants and are known to modulate the activities of different

enzymes due to their interactions with various biomolecules.

The cytoprotective effect of quercetin may also be due to its

ability to interact with and penetrate the lipid bilayer (Behling

et al. 2006).

There are two overlapping signaling pathways leading to

apoptosis, termed intrinsic and extrinsic pathways. The Bcl-2

protein family plays a role in the regulation of the intrinsic

pathway. Two key representative members of this family are

the anti-apoptotic Bcl-2 and the pro-apoptotic Bax (Narita

et al. 1998). The two apoptotic pathways are not mutually

FIGURE 4.—Western blot analysis of the effects of quercetin treatment on ERK1/2 phosphorylation in rat livers with TAA-toxicity. Upper panel:

representative Western blots. Lower panel: densitometric analysis. Values, normalized against b-actin, are presented as percentage change from

the Control group and expressed as mean + SD. ap � .05 significantly different from control animals.

Vol. 39, No. 6, 2011 HEPATOPROTECTIVE QUERCETIN 953

exclusive in hepatocytes but are closely interrelated, as the

mitochondrial pathway is often required to amplify the rela-

tively weak death receptor-induced apoptotic signal (Ghavami

et al. 2005). The Bcl-2 functions to prevent cell death, whereas

Bax, which forms hetero-dimers with Bcl-2, appears to accel-

erate the cell death signal (Oltvai, Milliman, and Korsmeyer

1993). Increases in ROS production and mitogen-activated

protein kinase (MAPK) activation result in an imbalance

between anti- and pro-apoptotic Bcl-2 family proteins, mem-

brane permeability transition, release of cytochrome c, and fur-

ther production of ROS, which contributes to the susceptibility

of a given cell to apoptosis (Oltvai, Milliman, and Korsmeyer

1993). In a nonalcoholic steatohepatitis model, it was observed

that significant increases of Bax protein concentration in the

livers of rats fed the high-fat diet occurred. This increase of

Bax pro-apoptotic protein without changes of Bcl-2/Bcl-xl

anti-apoptotic proteins, associated with oxidative stress, may

lead to an imbalance within the Bcl-2 family, which then med-

iates mitochondrial dysfunction and cell apoptosis (Y. Wang

et al. 2008). Overexpression of Bcl-2 and Bcl-xl, reducing the

Bax/Bcl-2 and Bax/Bcl-xl ratio, have recently been reported to

inhibit ROS production, cytosolic cytochrome c release and

poly (ADP-ribose) polymerase (PARP) degradation-mediated

apoptosis and mediated autophagy in a renal ischemia/

reperfusion study (Wu et al. 2009). Cytosolic protein content

for both molecules was studied by Western blot. Our results

confirm that Bax expression was significantly increased and

Bcl-2 expression significantly reduced at four days after

TAA-induced liver toxicity in rats. Treatment with quercetin

significantly prevented those changes, resulting in a signifi-

cantly lower Bax/Bcl-2 ratio when compared with untreated

rats. These results are in agreement with findings in a study

of apoptosis mediated by H2O2. Quercetin markedly inhibited

the apoptotic characteristics via reduction of intracellular

reactive oxygen species generation. Also, it prevented the

H2O2-mediated mitochondrial dysfunction, including disrup-

tion of mitochondria membrane permeability transition as well

as an increase in expression of apoptogenic Bcl-2 proteins,

Bcl-2 and Bcl-xl (Park et al. 2003).

MAPKs are proposed to play a key role in intracellular sig-

naling cascades in normal and pathogenic conditions (Raman,

Chen, and Cobb 2007; Y. Wang 2007). The activation of

MAPK is linked with cell death in the rat liver via oxidative

stress (Iida et al. 2009). Extracellular-signal related kinase

(ERK) functions cytoprotectively against apoptosis that is trig-

gered by oxidative stress (Czaja, Liu, and Wang 2003;

Y. Wang et al. 2004; Tommasini et al. 2005), tumor necrosis

factor-alpha (TNF-a) (Xia et al. 1995; Gardner and Johnson

1996), NO (Bhat et al. 1998; Mi Jeong, Davaatseren and Kim

2009), and proapoptotic drugs (Stadheim and Kucera 1998).

Phosphorylated ERK1/2 significantly increased soon after liver

injuries (Kishioka et al. 2007; Garcia-Lastra et al. 2010;

Kitamura et al. 2010). In a rat model of liver ischemia for

45 min and reperfusion, phosphorylated ERK2 increased

FIGURE 5.—Western blot analysis of the effects of quercetin treatment on liver expression of Bax and Bcl-2. Bax/Bcl-2 ¼ ratio. Upper panel:

representative Western blots. Lower panel: densitometric analysis. Values, normalized against b-actin, are presented as percentage change from

the Control group and expressed as mean + SD. ap � .05 significantly different from control animals.

954 DE DAVID ET AL. TOXICOLOGIC PATHOLOGY

significantly 1.5 hr after reperfusion in both the ischemic and

non-ischemic regions (Kitamura et al. 2010). A model of rabbit

hemorrhagic disease showed that expression of phosphorylated

p-ERK1/2 was significantly elevated at 12 hr pi, and at 48 hr pi

there was no p-ERK1/2 expression (Garcia-Lastra et al. 2010).

In the present study, p-ERK1/2 was decreased in TAA-induced

rats four days after liver toxicity. These findings imply that the

activation of p-ERK1/2 could take place earlier and that the

strong apoptotic and necrotic signals induced by a high dose

of TAA could block cell responses to growth and survival fac-

tors acting through the ERK pathway.

Currently, the most effective therapy for acute or chronic

hepatic failure is orthotopic transplantation (Ahmed and Keeffe

2007). A number of beneficial effects of quercetin on human

health have been shown (P. Yao et al. 2007; Boots, Haenen,

and Bast 2008), and some studies have indicated an important

role for quercetin in protecting against the deleterious effects of

reactive oxygen species and in the inhibition of redox-sensitive

signaling pathways in several diseases (Moreira et al. 2004;

Dias et al. 2005; Amic et al. 2007; Tieppo et al. 2009).

In summary, we confirmed literature findings that hepato-

cyte apoptosis was significantly increased in liver injury

induced by TAA. Our findings indicate that the increased oxi-

dative stress and its associated pERK1/2 activation as well as

an imbalance of pro- and anti-apoptotic proteins in the Bcl-2

family all contributed to high hepatocyte apoptosis that may

play a significant role in the pathogenesis of TAA-induced

liver injury. Quercetin, inhibiting the oxidative stress and/or

directly intervening in apoptotic pathways, as demonstrated

in our study, could represent a potential valid therapeutic drug

for liver toxicity.

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