Genistein protection against acetaminophen-induced liver injury via its potential impact on the activation of UDP-glucuronosyltransferase and antioxidant enzymes

Food and Chemical Toxicology 55 (2013) 172–181

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Genistein protection against acetaminophen-induced liver injury via its potential impact on the activation of UDP-glucuronosyltransferase and antioxidant enzymes Fan Yuan-jing ⇑, Rong Yu, Li Peng-fei, Dong Wan-ling, Zhang Dong-yin, Zhang Ling, Cui Min-jie School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China

a r t i c l e

i n f o

Article history: Received 15 August 2012 Accepted 3 January 2013 Available online 16 January 2013 Keywords: Genistein APAP-glucuronide UGTs Hepatic detoxification Antioxidant enzymes

a b s t r a c t The purpose of this study was to investigate genistein’s influence on the relationship between the activation of uridine diphosphate glucuronosyltransferase (UGTs) and the protection against acetaminophen-induced liver toxicity. Animal experimental results revealed that genistein (50, 100 or 200 mg/ BWkg) significantly ameliorated the biomarkers alanine aminotransferase, alanine aminotransferase, lactate dehydrogenase and malondialdehyde, as indicators of acute liver damage caused by APAP (200 mg/BWkg). The level of GSH declined sharply after treatment with APAP within 1 h in both the liver and blood with and without genistein. However, after 16 h, the levels approached or returned to the original level. Genistein may accelerate and promote APAP glucuronidation as the results showed that APAP-glucuronide increased by 18.44%, 46.79%, and 66.49% for 4 h of treatment with genistein dosages of 50, 100 or 200 mg/BWkg, respectively, compared with the APAP-only treatment. The activation of UGTs and glutathione peroxidase and the inhibition of CYP2E1 by genistein were observed, and UGTs mRNA expression level with genistein was measured. These findings suggest that genistein can prevent and protect against APAP-induced liver toxicity due to the inhibition of APAP biotransformation and the resistance to oxidative stress via the modulation of the activities of metabolism and the antioxidant enzyme. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Isoflavones (genistein, daidzein and glycitein) can have diverse effects on human and animal health (Dixon, 2004) and may play a role in the prevention of cancer through their capacity to affect antioxidant or protective phase II enzyme activities (Appelt and Reicks, 1999). Early studies have shown that the induction of phase I and phase II enzymes is an important mechanism of cancer chemoprevention and the cancer protective effects of isoflavones have been attributed to a wide variety of mechanisms, including the modulation of enzyme activities that result in the decreased toxicity and carcinogenicity of xenobiotics (Lake, 1999). The detoxification system in body, which includes a wide spectrum of phase I and II drug metabolising enzymes, can compete with Abbreviations: UGTs, uridine diphosphate glucuronosyltransferase; UDPGA, uridine diphosphate-glucuronic acid; APAP, acetaminophen, paracetamol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GSH, glutathione; LDH, lactate dehydrogenase; MDA, malondialdehyde; DTNB, 5,50 -dithio-bis-2-nitrobenzoic acid; 4-MU, 4-methylumbelliferone glucuronide; GSH-Px, glutathione peroxidase; CYP2E1, cytochrome P450(2E1); TAC, the total antioxidant capacity. ⇑ Corresponding author. Tel./fax: +86 551 2902955. E-mail address: [email protected] (Y.-j. Fan). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.01.003

activating enzymes by eliminating reactive electrophiles via conjugation, rendering them more water soluble and more readily excretable from the cell and the body (Jiang et al., 2003). The inhibition of cytochrome P450 (CYP450) enzymes and activation of phase II detoxifying enzymes, such as glutathione S-transferase (GSTs), UDP-glucuronyltransferase (UGTs) and quinone reductase, by flavonoids result in the reduction of oxidant stress and the detoxification of xenobiotics and represent one mechanism of their anticarcinogenic effects (Moon et al., 2006). Acetaminophen (or Paracetamol, APAP) is metabolised, primarily in the liver, into non-toxic products. There are three metabolic pathways: glucuronidation is believed to account for 40–65% of the metabolism of APAP; sulphation (sulphate conjugation) may account for 20–40% (Hendrickson and Kenneth, 2006); and N-hydroxylation and rearrangement, followed by GSH conjugation, accounts for less than 15%. The hepatic cytochrome P450 enzyme system metabolises APAP, particularly CYP2E1, to form reactive, toxic metabolites that in turn cause liver injury in experimental animals and humans (Caro and Cederbaum, 2005) by forming a minor yet significant alkylating metabolite known as N-acetyl-pbenzo-quinoneimine (NAPQI), which is then irreversibly conjugated with the sulfhydryl groups of glutathione (Borne, 1995). In

Y.-j. Fan et al. / Food and Chemical Toxicology 55 (2013) 172–181

the third pathway, the intermediate product NAPQI is toxic and it is primarily responsible for the toxic effects of APAP. High levels of exposure to APAP may induce saturated detoxification pathways, as a consequence of the accumulation of NAPQI, causing acute liver failure (Chun et al., 2009). The process of glucuronidation is a major part of phase II metabolism and is also the major pathway for xenobiotics removal for most drugs, dietary substances, toxins and endogenous substances. UGTs are responsible for glucuronidation and are the most important enzymes in the phase II (conjugative) metabolism. UGTs have been divided into two distinct subfamilies based on their sequence identities, UGT1 and UGT2 (King et al., 2000). Only rare examples show that glucuronides retain their biological activity, therefore this pathway is regarded as a ‘‘detoxification’’ mechanism, as originally proposed by Dutton (Tukey and Strassburg, 2001). It was reported that genistein inhibits cytochrome P450 (CYP450) phase I enzymes and induces phase II xenobiotics metabolism enzymes (Stagos et al., 2012). The mechanism of genistein’s ability to induce phase II xenobiotics metabolism enzymes is not yet understood. The potential impact of the detoxification of xenobiotics on the activations of the UGTs pathways may be an important factor in the multi-target detoxification and anticancer effects of genistein (Bolling et al., 2010). In this study, we carried out experiments to explore the role and mechanism of protection of genistein extracted from soybeans against APAP-induced liver toxicity. The effect of the antioxidation and biological activities of genistein on the activities of CYP2E1, GSH-Px and UGTs were evaluated. Additionally, GSH and APAP-G were analysed quantitatively through animal experiments. 2. Materials and methods 2.1. Chemicals and animals Genistein extracted from soybeans (Gen, 4,5,7-trihydroxyisoflavone, 98% purity, Xian Biological Products Co., Ltd., Xian, China) was dissolved in 1% sodium carboxymethyl cellulose (CMC-Na, Sinopharm Chemical Reagent Co., Ltd.). Acetaminophen (APAP, 99% purity, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was dissolved in a heated physiological salt solution (20% suspension in saline stabilized by 0.2% gum). APAP-glucuronide (4-acetamidophenyl b-D-glucuronide sodium salt, APAP-G) was purchased from Toronto Research Chemicals Inc. SPF KM male mice obtained from Anhui Medical University Centre were 6 weeks old and weighed 25 ± 5 g. The animal rooms were maintained at a temperature of 22 ± 1 °C and a relative humidity of 55 ± 5% throughout a 12-h light/dark cycle. The mice were allowed free access to standard laboratory chow and water. They were weighed and fed for 1 week. Thereafter, they were randomly divided into treatment groups of 10 mice of per group. The research was conducted in accordance with the internationally accepted guidelines for laboratory animal use and care. The experiments reported here were approved by our institutional ethics committee. 2.2. Animal treatments and surgery The mice were divided into three batches. The first batch of male mice were randomly separated into six groups of 10 mice each. The mice in group 1 served as controls and were not treated with APAP or Genistein. They were administered a 1% CMC-Na intragastric treatment of the vehicle solution (saline with 0.2% gum) every day. Group 2 was administered 200 mg/BWkg (i.g.) of Gen as a reagent control (named as Gen3). The mice in group 3 (model group) were treated with APAP to induce hepatic injure; APAP was dissolved in physiological saline then administered by gastric instillation at 200 mg/BWkg, a well-documented dose for inducing hepatic damage (Amimoto et al., 1995). For the genistein treatment groups 4–6, APAP (200 mg/BWkg, i.g.) was first administered followed immediately thereafter by the intragastric (intragastric gavage, i.g.) treatment with genistein at doses of 50 mg/BWkg, 100 mg/BWkg and 200 mg/BWkg for the groups APAP + Gen1, APAP + Gen2 or APAP + Gen3, respectively. The blood of these mice was tested for ALT, AST, LDH and MDA, and the activities of UGTs and CYP450 were detected in their livers. The second batch of mice were divided into six groups: the control group (untreated, control check), the reagent control group (Gen treatment at 200 mg/BWkg i.g., named as Gen3), the APAP group (200 mg/BWkg i.g.), the APAP (200 mg/BWkg i.g.) + Gen1 (50 mg/BWkg i.g.) group, the APAP + Gen2 (100 mg/BWkg i.g.) group and the APAP + Gen3 (200 mg/BWkg) group. After treatment, each group were

173

killed at intervals of 0.5, 1, 2, 4, 8, and 16 h to test for GSH in the serum and liver, and APAP-glucuronide (APAP-G) was tested at intervals of 4 and 16 h in the blood and livers. The time interval between the first and the second or between the second and third groups treatment was half hour (0, 0.5, 1, 2, . . ., 16 h), in order to killed and treated liver and blood of mice within 30 min completely, 6 mice every group was designed. According to the guidelines of appropriate statistical practice in toxicology experiments, it has been recommended that the inclusion of at least 6 animals in each experimental group is a minimum (Festing and Altman, 2002). The third batch of mice was divided into 8 groups of 10 mice each Groups, they were control, APAP (200 mg/BWkg i.g.), Gen1 (50 mg/BWkg i.g.), Gen2 (100 mg/ BWkg i.g.), Gen3 (200 mg/BWkg i.g.), APAP (200 mg/BWkg i.g.) + Gen1 (50 mg/ BWkg i.g.), APAP + Gen2 (100 mg/BWkg i.g.) and APAP + Gen3 (200 mg/BWkg) groups respectively. After treated for 16 h, the mice of every group were killed and their livers were harvested to test for the activities of GSH-Px of control, APAP, Gen1, Gen2 and Gen3 groups, and total antioxidant capacity (TAC) of liver tissue of all groups. The diagram of experiment design and assignment of mice were as shown in Fig. 1, the animals of the first batch were exsanguinated through the eye socket after fasting for 16 h. The collected plasma was centrifuged at 3000 rpm for 10 min and the serum was stored at 4 °C. The livers of the mice were removed and rinsed with 0.05 M Tris–HCl buffer (pH 7.4, containing 0.15 M KCl). The hepatic subcellular and crude microsomes fractions were isolated from the cleaned mice livers to test the activation of the enzymes UGTs and CYP450. Mice liver homogenate was centrifuged at 10,000g for 20 min and the resulting pellet was suspended in a 0.05 M Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose. This homogenate was again centrifuged for an additional 20 min at 10,000g and the resulting supernatant was removed and assessed for GSH and UGTs. Part of the mice livers were used for tissue slices and the RNA test of the mRNA expression of the UGTs by RT-PCR. The second and third batches of mice were also treated according to the described treatment conditions above. 2.3. Tests for ALT, AST, LDH, and MDA Alanine aminotransferase (ALT) and aminotransferase (AST) were measured to determine if the livers were damaged or diseased. Low levels of ALT are normally found in the blood, but when the liver is damaged or diseased, it releases ALT into the bloodstream. The levels of ALT and AST were tested by their assay kits (Jiancheng Bioengineering Institute., Nanjing, China). Lactate dehydrogenase (LDH, Jiancheng Bioengineering Institute, Nanjing, China) and malondialdehyde (MDA, Jiancheng Bioengineering Institute, Nanjing, China) were also determined using kits. 2.4. Measurement of the levels of GSH and TAC GSH was measured in both the blood and liver. A glutathione-SH (GSH) serum assay kit (nmolGSH/ml) and tissue assay kit (nmolGSH/mgprot) were both purchased from Rong sheng Biotech Co., Ltd. (Shanghai, China). Briefly, 2 ml of reagent 1 of the GSH and tissue assay kits was added to the prepared mice serum (0.5 ml) and liver homogenate (10% 0.5 ml), respectively. The mixtures were then centrifuged at 4000 rpm for 10 min. After the colour reaction, the absorbance of the supernatant was measured at 412 nm and the GSH level was calculated according to the reference standard curves. The total antioxidant capacity (TAC) of the liver tissue was measured using the TAC assay kit following the ferric reducing antioxidant power (FRAP) method and the absorbance detection method. The FRAP method was conducted according to the TAC kit’s instructions: 0.15 ml of mice liver homogenate was mixed with 1 ml of reagent 1, 2 ml of reagent 2, 0.5 ml of reagent 3 and 0.1 ml of reagent 4 using a spiral vortex; the mixture was allowed to react at 37 °C for 30 min and the absorbance at 520 nm was measured. 2.5. Tests for APAP and APAP-G To generate standard curves, APAP-G (80, 40, 20, 10, 5, 2.5 lg/ml) and APAP (20, 10, 5, 2.5, 1.25, 0.6 lg/ml) were dissolved in methanol. APAP-G and APAP were detected using HPLC with a Waters RP-18 column (4.6 mm  250 mm, 5 lm) and a mobile phase composed of phosphate buffering solution (pH 4.5) and acetonitrile (97:7) at a flow rate of 1.0 ml/min. The detection wavelength was 250 nm (Girolamo et al., 1998). The APAP-G and APAP concentrations were calculated based on the prepared standard curves. 2.6. Assays for UGTs, GSH-Px and CYP2E1 The UGTs activities of mice liver were measured by observing the drop in fluorescence as the compound converted to 4-methylumbelliferone glucuronide (4-MU, Sigma, St. Louis, MO, USA). The method used is based on the protocol of Aitio (Aitio, 1974). Briefly, 50 ll of microsomal protein (10 mg/ml stock) and 400 ll of 4-MU (1– 1000 lM final concentration) in 0.1 M Tris–HCl buffer, containing 5 mM MgCl2 and 0.05% BSA at pH 7.4, were added to microtubes and preincubated for 2 min. UDPGA (50 ll, 2 mM final concentration) was then added and incubated for 20 min at

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356 mice were divided groups

The first batch separated into 6 groups (60 mice)

control

APAP

The second batch separated into 6 groups (216 mice)

Gen3

APAP+Gen1

APAP+Gen2

The first batch mice divided into 6 groups, 10 mice every group were obtained blood and liver for 16 hours after treatment

The second batch mice divided into 6 groups, 36 mice every group, 6 mice from each group were killed at intervals of 0.5, 1, 2, 4, 8, 16 hours and obtained blood and liver after treatment

. ALT, AST, LDH and MDA in blood; . The activities and expression mRNA of UGTs in liver; . The activity of CYP2E1 in liver

.The level of GSH in blood and liver at intervals of 0, 0.5, 1, 2, 4, 8, 16 hours respective; . APAP-G in blood and liver for 4, 16 hours after treatment; . APAP in blood and liver for 4 or 16 hours after treatment

The third batch separated into 8 groups (80)

APAP+Gen3

Gen1

Gen2

The third batch mice divided into 8 groups, 10 mice every group, were killed and obtained blood and liver for 16 hours after treatment . The activity of GSH-Px acted by APAP and Gen in liver for 16 hours after treatment; . TAC in liver for 16 hours after treatment

Fig. 1. Experimental design and assignment of mice used in this study. Abbreviations: control group, untreated; APAP, acetaminophen 200 mg/BWkg i.g.; Gen1, genistein 50 mg/BWkg i.g.; Gen2, 100 mg/BWkg i.g.; Gen3, 200 mg/BWkg i.g.; APAP + Gen1, 200 mg/BWkg of APAP plus 50 mg/BWkg of Gen; APAP + Gen2, 200 mg/BWkg of APAP plus 100 mg/BWkg of Gen; APAP + Gen3, 200 mg/BWkg of APAP plus 200 mg/BWkg of Gen; APAP-G, APAP-glucuronide; GSH-Px, glutathione peroxidase; UGTs, uridine diphosphate glucuronosyltransferase. 37 °C; the final volume was 0.5 ml. Fluorescence was measured in a fluorescence spectrophotometer (370 nm excitation and 450 nm emission, 930-A, Shanghai Analytical Instruments Co., LTD,). The activities of the UGTs were also tested with using 4-nitrophenol (4-NP, Sigma, St. Louis, MO, USA). The enzyme activity of glutathione peroxidase (GSH-Px) was measured using a cellular glutathione peroxidase assay kit (Jiancheng Bioengineering Institute., Nanjing, China). Liver homogenates were centrifuged at 3000 rpm for 15 min at 4 °C. The oxidation of glutathione (GSH) to oxidised glutathione (GSSG) was catalysed by GSH-Px, coupled to the recycling of GSSG back to GSH, utilising glutathione reductase and NADPH. The decrease in NADPH absorbance measured at 412 nm during the oxidation of NADPH to NADP was indicative of GSH-Px activity; the activity of GSH-Px was assessed from the optical density measurements at 412 nm. The activities of CYP450 (2E1) in the livers were measured as described by the instructions of the mouse CYP2E1 ELISA kit (YanJin BioSci-Tech Co., Ltd., Shanghai, China). Following the kit’s instructions, the weight of the mice liver tissues were determined, then PBS (PH7.2–7.4) was added and the mixture was homogenised by hand or with grinders. The mixture was centrifuged for 20 min at speeds in the range of 3000 rpm and the supernatant was removed. Briefly, the assay procedure is as follows: the sample was diluted and added to the standard then incubated for 30 min at 37 °C; the liquid was then separated and the sample was washed, followed by the addition of the enzyme (HRP-Conjugate reagent 50 ll to each well, except the blank well) and incubation; finally, and the sample was washed and the colour reaction was initiated by the addition of chromogen solutions A and B in the absence of light for 15 min at 37 °C. The absorbances at 450 nm were read within 15 min after the addition of the stop solution. The blank well was taken as zero.

2.7. mRNA expression of the UGTs by RT-PCR The expression levels of the mRNA of UGTs were measured to determine their catalytic effects on the glucuronidation of APAP and genistein. The groups APAP + Gen3, APAP and control were sampled and tested separately. UGTs that were highly expressed in the mouse livers include UGT1a1, UGT1a6, UGT1a10, and UGT2b1 (Zhang et al., 2004; Kiang et al., 2005), which were then tested for their activities. For the mRNA expression of UGTs in the mice hepatic cells by reverse transcription-polymerase chain reaction (RT-PCR) analysis, the total RNA was isolated by the TRIZOL Reagent (Invitrogen., USA) following the manufacturer’s protocol. RT-PCR was performed using a RT-PVR Kit (Takara BIOTECH, Japan). The primers used in this study were based on the GenBank primer sequences. The primer sequences were as follows: UGT1a1(F): 5-CACGCTGGGAGGCTGTTAGT-3, (R): 5-CACAGTGGGCACAGTCAGGTA3; UGT1a6 (F): 5-GTGAACGAGGACATGACATTA-3, (R): 5-CACAGGGGATG TACGTAGAAAG-3; UGT1a10 (F): 5-TTGCCAAGTAT CTGTCACTCCC-3, (R): 5-ACAACGATGCCATGCTCCC-3; UGT2b1(F): 5-TGAAAAATGG GTAGGAAACTGG-3, (R): 5-GCACATAGGAAGGGGGGAG-3. Primers for the mouse were GAPDH (F): 5-CCTTCATTGACCTCAACTACATGG-3, (R): 5-CGTGGTTCACACCCATCACAAAC-3. The mRNA expression of UGTs were quantified by densitometric scanning and the gel was photographed under UV illumination.

2.8. Histological observation of liver tissue slices Hepatic morphology was assessed by light microscopy. The mice liver was sliced and the tissue slices were fixed in 10% buffered-neutral formalin for 6 h. The fixed liver tissue slices were processed and embedded in paraffin wax in an automatic tissue processor. Sections 4 lm in thickness were stained with hematoxylin and eosin (H&E) before histopathological evaluation. 2.9. Statistical analysis The data are expressed as the mean ± SD and were analysed with SPSS, Version 12.0 software. Statistical significance was determined by one-way analysis of variance, followed by Duncan’s posthoc test. The significance level was set at P < 0.05.

3. Results 3.1. Genistein protection against APAP-induced acute liver injury Table 1 showed that ALT, AST and LDH activities rose in the serum of the APAP group of mice quickly; it implied that large doses of APAP produced significant hepatic injury in the mice. Genistein exhibited a dose-dependent inhibitory action against APAP-induced acute hepatotoxicity. MDA is a reactive oxygen species (ROS) and one of the many reactive electrophile species that causes toxic stress in cells; thus it is assayed in vivo as a bio-marker of oxidative stress (Stancliffe et al., 2011). Genistein decreased the production of oxygen free radicals and significantly prevented the increase of MDA in the serum due to APAP hepatic toxins (Table 1). 3.2. Effects of genistein on the hepatic levels of GSH In the case of NAPQI, the reactive cytochrome P450-reactive metabolite formed by APAP, it becomes toxic when GSH was depleted by an overdose of APAP. It was reported that NAPQI rapidly depletes GSH; therefore, the degree of GSH consumption before the manifestation of toxicity is one of the best biomarkers for APAP bioactivation (Bessems et al., 1997; Tan et al., 2008). So the depletion of GSH was measured with respect to time (Fig. 2A and B) and it was observed that the levels of GSH in the livers and blood of the mice were at the lowest levels 1 h after APAP or APAP + Gen was administered. Then, the GSH levels increased gradually after the

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Y.-j. Fan et al. / Food and Chemical Toxicology 55 (2013) 172–181 Table 1 Protective effect of genistein on APAP-induced liver toxicity (values are mean ± SD from ten mice serum in each group). Group

ALT (U/L)

AST (U/L)

LDH (U/L)

MDA (nmol/ml)

Control Gen3 APAP APAP + Gen1 APAP + Gen2 APAP + Gen3

42.09 ± 4.62 41.29 ± 6.05 237.78 ± 20.67a 96.67 ± 3.86a,b 56.38 ± 6.39b 40.77 ± 4.13b

101.39 ± 11.27 100.69 ± 12.88 205.58 ± 22.47a 153.93 ± 23.51a,b 105.37 ± 8.99b 87.3 ± 12.89b

787.16 ± 53.37 789.65 ± 55.24 1876.2 ± 130.61a 1468.1 ± 133.92a,b 1248.15 ± 112.19a,b 777.98 ± 53.79b

8.54 ± 0.31 8.39 ± 0.27 23.56 ± 2.71a 16.65 ± 1.42a,b 14.73 ± 2.00a,b 12.26 ± 1.29a,b

control

Gen 3

APAP

APAP+Gen 1 100

APAP+Gen 2

APAP+Gen 3

80

a

60

a

a

40

a

A

0

a

a

20

0h

0.5 h

a

a

a

1h

2h

a

4h

8h

16 h

GSH in liver (nmol/mgprot)

GSH in blood (nmol/ml)

Values are mean ± SD of 10 mice from each group; a P < 0.05 compared to ck (conteol) or Gen3 (reagent control). b P < 0.05 vs. APAP (model group); aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), malondialdehyde (MDA).

control APAP+Gen 1 40

Gen 3 APAP+Gen 2

APAP APAP+Gen 3

35 30 25 20

a a

a a

15

a

10

a a

5

B

a

a

a

0

0h

0.5 h

1h

2h

4h

8h

16 h

33.352 32.894 32.386 32.486 32.155 32.651 32.744

control

88.94 87.718 86.364 86.463 86.079 86.735 86.316

control

Gen3

88.89 88.87 89.26 89.87 89.98 90.42 90.78

Gen3

33.34 33.87 33.98 34.21 34.59 34.98 35.12

APAP

88.536 44.987 6.9777 14.335 27.623 42.214 64.371

APAP

33.201 16.87 2.6166 5.3757 13.359 16.768 24.139

APAP+Gen 1 89.913 46.522 8.9649 21.839 37.999 45.592 70.309

APAP+Gen1 33.717 17.446 3.3618 8.1896 14.249 17.097 26.366

APAP+Gen 2 91.565 46.3 11.913 23.08 46.123 59.502 78.048

APAP+Gen2 34.337 17.362 4.4672 8.6552 17.296 24.188 29.268

APAP+Gen 3 91.88 50.052 15.935 27.173 55.092 71.299 89.608

APAP+Gen3 34.455 18.77 5.9757 10.19 22.535 28.612 33.603

time (hour)

time (hour)

Fig. 2. The levels of GSH in mice liver (A) and blood (B): -- untreated as control, -h- genistein (200 mg/BWkg i.g.) alone treated as reagent control (Gen3), -j- APAP (200 mg/ BWkg i.g.) alone treated, -- APAP + Gen1 (50 mg/BWkg i.g.) treated, -- APAP + Gen2 (100 mg/BWkg i.g.) treated and -}- APAP + Gen3 (200 mg/BWkg i.g.). After treatment for 0.5, 1, 2, 4, 8 or 16 h, at least 6 mice were killed to measure GSH in the liver and blood. Symbol: ‘‘a’’ shows the significant difference vs. the control or Gen3 groups (P < 0.05).

first hour to the 16th hour. Compared with the APAP group, the APAP + Gen groups showed that genistein may be able to stimulate the restoration of the GSH levels in both the liver and blood for APAP-induced liver injury, but the Gen3 group (Gen treatment without APAP treatment for 16 h) displayed no significant increases in GSH concentration (Fig. 2A and B). 3.3. Effects of genistein on the activities of CYP450, UGTs, GSH-Px and TAC CYP2E1 is the rate-limiting enzyme that initiates the cascade of events leading to APAP hepatotoxicity. The inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation and the role of CYP2E1 in NAPQI formation at higher concentrations has been confirmed in a previous study (Hazai et al., 2002). The activity of CYP2E1 with APAP increased substantially more than the control and Gen3 groups (Fig. 3A); compared with control, there was no significant change for the treatment with genistein at a dose of 200 mg/BWkg (Fig. 3A, Gen3), but there was a significant change in APAP administered with or without genistein. Genistein suppressed the activities induced by APAP, exhibiting dose-dependent effects (Fig. 3A, APAP + Gen groups). Simultaneously, the activities of the UGTs were determined (Fig. 3B), the results showed that genistein enhanced significantly the activities of UGTs during acute liver injury induced by APAP, and the alterative activities of UGTs were in consonance with the rate of APAP glucuronidation (Fig. 4).

The main biological role of glutathione peroxidase (GSH-Px) is to protect the organism from oxidative damage, the total antioxidant capacity (TAC) of tissues and body fluids are equipped with powerful defense systems that help counteract oxidative challenge. The study result showed that the activity of GSH-Px were promoted by genistein and inhibited by APAP (Fig. 3C) and the impact on TAC of liver tissue caused by APAP insult and genistein resistance in hepatic toxin were observably difference (Fig. 3D). 3.4. Effects of genistein on the metabolism of APAP-G APAP and its glucuronide conjugate (APAP-G) were detected in the study. The HPLC chromatograms of the control, sample and standard APAP (Fig. 4A), and APAP-G were shown in Fig. 4A. The results showed that the level of APAP-glucuronide (APAP-G) transformation was the lowest without genistein (Fig. 4B and C, APAP group). Genistein exhibited a promotive action on APAP glucuronidation in the liver and blood (Fig. 4B and C). Genistein may accelerate and promote APAP glucuronidation as the results showed that APAP-glucuronide in liver increased by 18.44%, 46.79%, and 66.49% for 4 h of treatment with genistein dosages of 50, 100 or 200 mg/BWkg, respectively, compared with the APAP-only treatment. Likewise for 16 h APAP-glucuronide in liver increases by 29.60%, 54.64%, and 71.95% corresponding to the same groups respectively. The concentration of APAP-G was evidently lower in

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B control APAP+Gen1

300

a

250

a,b 200

a,b a

150 100 50 0

UGTs activity in liver (% of control)

CYP2E1 activity in liver (% of control)

A

Gen 3 APAP+Gen 3

120

b b

100

a,b

a,b a

80

a

a

a

60 40 20

APAP+Gen 3

APAP+Gen 2

APAP+Gen 1

APAP

Gen3

control

0 detected by 4MU(unit/mgprot)

TAC of liver tissue (% of control)

180

160

a,b

140

a,b

b b

120

a,b

100

120 100 a

80 60 40 20

a,b

80 60

a,b a

40 20 Gen 3+APAP

Gen 2+APAP

Gen 1+APAP

APAP

Gen 3

Gen 2

Gen3

Gen2

Gen1

APAP

control

Gen 1

0

0

a,b

control

GSH-Px activity in liver (% of control)

a,b

140

detected by 4NP(unit/mgprot)

D

C 160

APAP APAP+Gen 3

Fig. 3. The activities of CYP2E1 (A), UGTs (B), GSH-Px (C) and TAC (D) in mice liver: CYP2E1 (Fig. 3A) was detected with treatment of Gen3 alone, APAP alone, APAP + Gen1, APAP + Gen2 or APAP + Gen3 for 16 h after treatment; the UGTs activities (Fig. 3B) in the hepatic microsomes were tested in the groups of Gen3 alone, APAP alone, APAP + Gen1, APAP + Gen2 or APAP + Gen3 treated after 16 h; GSH-Px (Fig. 3C) was detected in the liver of mice treated with genistein alone at 50 mg/BWkg i.g. (Gen1), 100 mg/BWkg i.g. (Gen2) or 200 mg/BWkg i.g. (Gen3) for 16 h, APAP (200 mg/BWkg i.g.) alone for 16 h, and control was the untreated control; the test of TAC (Fig. 3D) in the liver of mice treated with Gen1, Gen2, or Gen3 alone, APAP alone, APAP + Gen1, APAP + Gen2 or APAP + Gen3 for 16 h. Symbol: ‘‘a’’ shows the significant difference vs. the control and regent groups, ‘‘b’’ shows the significant difference vs. the APAP groups (P < 0.05).

the blood at 16 h than at 4 h, it was demonstrated that the part of APAP-G in blood has been eliminated because of its hydrophilic (Fig. 4C). There was a very slight trace of APAP in the liver and it was metabolised in the liver with no free APAP detected in liver homogenates; Fig. 4D indicted the retention of APAP in the blood only.

UGT2b1 in the model (treated with APAP) groups were much lower than in the control and APAP + Gen groups because of partial hepatocyte necrosis or death, but the level of mRNA expression in all the APAP + Gen groups were greatly enhanced. These results demonstrated the effects of genistein on the activation and expression of the UGTs.

3.5. Effect of genistein on the expression of the UGTs

3.6. Histological observation of genistein’s protective effects on APAPinduced hepatic injured tissue slices

The mRNA expressions of the UGT1a1, 1a6, 1a10 and UGT2b1 genes were shown in Fig. 5. Compared with the control, there were obvious expressions differences in the mRNA in the APAP group and APAP + Gen groups (Fig. 5B). UGT1a1, 1a6 and UGT2b1 mRNA were highly expressed in the liver of mice, of which UGT1a1 was at low levels in the duodenum, jejunum, and large intestine. UGT1a6 mRNA was also expressed in large intestine, followed by the lung, stomach, and gonads. UGT2b1 was almost exclusively expressed in liver. UGT1a10 mRNA expression of the UGTs was very low in the liver; it was most notable in the kidney and stomach (David and Curtis, 2007). UGT1a10 showed the lowest level of expression compared with UGT1a1, 1a6 and UGT2b1 in this experiment (Fig. 5B). All the mRNA expressions of UGT1a1, 1a6, 1a10 and

It was reported that the histological examination of APAP-mediated liver damage revealed necrosis, congestion and steatosis. Acetaminophen is a widely prescribed analgesic that causes fulminant hepatic necrosis in overdosed humans; it produced varying degrees of hepatotoxicity in mice, rats, hamsters, guinea pigs and rabbits (Yohe et al., 2006). In our studies, the hepatic slices were dyed with hematoxylin and eosine (H&E) then expediently observed; the liver samples were shown in Fig. 6. The control group was separated from the experimented mice because it was untreated as the control; it clearly showed normal hepatocytes. The cells of the liver from the Gen3 mice treated with 200 mg/BWkg of genistein were scatheless, just as the control group. The liver

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Y.-j. Fan et al. / Food and Chemical Toxicology 55 (2013) 172–181 APAP

B

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1.8 1.5 1.2

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time (hour) Fig. 4. The HPLC derived standard curve for the mice liver from the APAP-only, APAP + Gen and control groups (A); the concentration of APAP glucuronidation in the liver (B) and blood (C) of the mice treated with APAP (200 mg/BWkg i.g.), APAP + Gen1 (50 mg/BWkg), APAP + Gen2 (100 mg/BWkg), and APAP + Gen3(200 mg/BWkg) for 4 and 16 h after treatment; mice survival with APAP in the blood treated for 4 and 16 h (D). Symbol: ‘‘b’’ shows the significant differences vs. the APAP group (P < 0.05).

from the mice treated with APAP (200 mg/BWkg) exhibited markedly massive hepatic cells necrotic injury. The liver slice from the APAP + Gen1 mice treated with 50 mg/BWkg of genistein showed little cell necrosis. The liver slices of the mice treated with APAP + Gen2 (100 mg/BWkg) and APAP + Gen3 (200 mg/BWkg) showed that genistein exhibited the protection against APAP, as observed in the increased dyed nucleoli.

4. Discussion The purpose of this study was to characterise the effects of genistein on the prevention and protection from APAP-induced hepatic

injury via its potential impact on the activation of UGTs and the antioxidant enzyme pathway of the glucuronidation of APAP and antioxidation stress (Raschke et al., 2006). It has been reported that genistein has attracted research attention because of its apparent protective effects against cardiovascular disease, metabolic syndrome, osteoporosis and cancer (Bitto et al., 2009). Advanced biological studies showed that antioxidant phytochemicals, including genistein, promote the induction of phase II enzymes through various signalling pathways, improving metabolism, the detoxification of xenobiotics, antioxidant and free radical scavenging activity capacity (Shon et al., 2007), redox homeostasis and cell viability (Kim and Lee, 1998). Genistein can exert a pleiotropic action in the organism, among various effects; it has engaged the

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M APAP+Gen 3 APAP control

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UGT 2 b1

Fig. 5. The mRNA expression levels of the UGTs impacted by APAP and genistein: (A) UGT1a1,1a6,1a10 and UGT2b1 families expressed in the mice hepatocytes by RT-PCR analysis for the control, APAP (200 mg/kg i.g.) and APAP + Gen3 (200 mg/BWkg i.g) groups treated for 16 h, M was mark; (B) UGT1a and 2b relative mRNA levels expressed by taking the control values obtained from the mouse liver cells. The level of the mRNA expressions of the UGTs in APAP group was much lower than in the control and APAP + Gen groups because of partial hepatocyte necrosis or death; APAP + Gen3 enhanced the level of the UGTs mRNA as it demonstrated genistein’s effects on the activation and expression of the UGTs. Symbol: ‘‘a’’ shows the significant difference vs. control, while ‘‘b’’ shows the significant difference vs. the APAP groups (P < 0.05).

control

APAP+Gen1

Gen3

APAP

APAP+Gen2

APAP+Gen3

Fig. 6. Histological analysis of the liver samples (hepatic tissue slice by H&E) from experimental mice: control was untreated as the control, Gen3 (200 mg/kg i.g.), APAP (200 mg/kg i.g.), APAP + Gen1 (50 mg/kg i.g.), APAP + Gen2 (100 mg/kg i.g.) and APAP + Gen3, all treated for 16 h. The control group clearly showed normal hepatocytes, and the Gen3 hepatic cells were scatheless like the control group. The APAP treated mice liver exhibited markedly massive hepatic cells necrotic injury, while APAP + Gen1 showed few hepatic cell necrosis in the liver slices of the mice in this group. The APAP + Gen2 and APAP + Gen3 groups’ mice liver showed that genistein did its best to protect against APAP, as observed in the increased dyed nucleoli.

research attention of other related field due to their ability to affect metabolic activity and to bring about metabolic antagonism toward exogenous and endogenous compounds. Reports on the activities of isoflavones have been predominantly associated with metabolic and antioxidant enzyme inhibition or induction activity (Lee, 2006; Ha et al., 2012). Isoflavones may also interact with chemotherapeutic drugs used in cancer treatments through the induction or inhibition of their metabolism (Moon et al., 2006). APAP is primarily metabolised via conjugation to form APAP GSH (Ghosh et al., 2010), sulphate and glucuronide (Brunner and Bai, 1999). In the case of APAP-GSH, it was validated that the conjugation to GSH was only a minor route, about 37% glucuronidation

and 3% GSH-conjugate formation, compared with the glucuronidation route (Bessems et al., 1997). GSH is an antioxidant that prevents damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides. Genistein, as an antioxidant, may enhance the activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR) and GST and could be beneficial to cope with oxidative stress in the early stage of injury or oxidative cell damage (Park et al., 2011). It appeared that the protection by genistein depended on its induction of GSH-Px rather than an increase or change in GSH intracellular concentrations by genistein, the utilisation of GSH as a substrate by GSH-Px during peroxide exposure may contribute to the initial

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Fig. 7. Genistein regulated the MAPKs, PI3K or PKC activation of Nrf2 signalling and induction ARE-mediated antioxidative enzymes and phase II enzyme. Genistein activate diverse upstream kinases, which in turn stimulate dissociation of Nrf2 from Keap-1. Nrf2 was released from Keap-1 repression and translocated to nucleus, forms heterodimer with small Maf protein, and binds to ARE/EpRE sequences located in the promoter region of genes encoding antioxidant and detoxifying enzymes. PR, Receptors; PP, Protein phosphatase; PK, Protein kinases; MAPK, Mitogen activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, Extracellular signal-regulated protein kinase; PI3K, Phosphatidylinositol 3-kinase; PKC, Protein kinase C; AER, Antioxidant response element.

depletion of GSH as observed with genistein (Hernandez-Montes et al., 2006). The changes in GSH-Px activities that inhibit peroxide-induced cellular injury may be one of the most important characteristics of the effects of genistein on detoxification and cancer prevention (Suzuki et al., 2002). TAC of liver tissue ranges from the classical enzymes, superoxide dismutases (SOD), catalases (CAT), GSH-Px, to ancillary enzymes quenching or inactivating reactive intermediates, such as quinone oxidoreductases (NQO), and conjugation enzymes, such as GST and UGTs (Sies, 2007), the molecular mechanism of the response to different redox states by the Keap1-Nrf2 system has recently been identified (Tong et al., 2006). The activation and mRNA expression of UGTs enhanced by genistein was very significative, the acceleration of APAP glucuronidation may be an effective route for the prevention of APAP-induced hepatotoxicity because of the reduction and/or inhibition of the APAP biotransformation to NAPQI by cytochrome P450 2E1. On the other hand, the inhibition of APAP glucuronidation by drugs, such as phenobarbital and phenytoin, enhanced APAP toxicity associated with the inhibition of the expression of UGTs and the UGT-mediated metabolism of APAP (Kostrubsky et al., 2005). The present commentary is focused on UGTs which exhibit a central role in the xenobiotic metabolizing enzyme system, acting together with CYPs, and conjugate transporters. Special regulatory mechanisms have been recognised for phase II enzymes such as UGTs and GSTs, AhR and the antioxidant Nrf2/Keap1 pathway are required for induction of UGTs. The commentary emphasises regulation of human hepatic UGTs by Ligand-activated transcription factors and their multilevel crosstalk (Bock, 2011).

The phytochemicals, Such as phenolic flavonoids, regulated the activities of MAPKs, Nrf2, and ARE-mediated phase II enzyme induction and these xenobiotics activate the MAPK pathway via an electrophilic-mediated stress response, leading to the transcription activation of Nrf2/Maf heterodimers on ARE/EpRE enhancers, with the subsequent induction of cellular defense/detoxifying genes including phase II drug metabolizing enzymes, these signalling pathways may yield insights into the fate of cells upon exposure to xenobiotics (Kong et al., 2001). There are several reports indicating that isoflavones can induce the expression of antioxidant and detoxifying genes, phase II detoxifying and antioxidant genes may be modulated by soy isoflavones and involve signalling through transcription factor Nrf2 (Hernandez-Montes et al., 2006; Barve et al., 2008). Genistein-regulated signal transduction networks mainly include 9 receptors, 5 signal adaptors, 13 protein kinases, 2 protein phosphatase regulatory subunits, and 14 transcription regulators (Yan et al., 2011). In the present study, UGT1a1 (homology with Homo sapiens UGT1A1), 1a6 (homology with Homo sapiens UGT1A6), 1a10 (homology with Homo sapiens UGT1A7,8,9 and 10) and UGT2b1 (homology with Homo sapiens UGT2B4) could confer specific expression of distinct UGT isoforms in liver and intestine (Kiang et al., 2005; David and Curtis, 2007), UGT1A6 and UGT1A9 have been shown to undergo APAP biotransformation to APAP-glucuronide; enzyme kinetic studies have demonstrated that UGT1A6 is a relatively high-affinity isoform, whereas UGT1A9 is a low-affinity isoform (Bock et al., 1993). UGT1A1 also contributes to the activity of APAP glucuronidation (de Morais et al., 1992). UGT1A10 showed activities similar to UGT1A6; although UGT1A10 is expressed in low levels in the liver, it may be important in the extrahepatic

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metabolism of APAP, particularly in the gastrointestinal tract (Tukey and Strassburg, 2001). UGT2B4 mRNA was found to be abundant in human liver, suggesting that its role in the liver should not be underestimated in the detoxification of major substrates. Acetaminophen, a widely used analgesic and antipyretic, is known to cause hepatic injury in humans and experimental animals when administered in high doses (Sener et al., 2003), bioactivation of APAP to NAPQI is mediated by P450s 1A2, 2E1, and 3A4 in both mouse and human, P4502E1 has been implicated as the most relevant isozyme in human (Jaeschke and Bajt, 2006). Genistein protection against acetaminophen-induced liver injury may be one of the dominant mechanisms via its potential impact on the activation of UGTs and CYP-2E1. There are many reports on the effects of the isoflavones of daidzein, genistein or glycitein on the activation of mRNA expression of UGTs; despite most endogenous hormones, isoflavones modulate the activities of UGTs to play an important role in detoxification and protection against hepatotoxicity (Galati and O’Brien, 2004; Pfeiffer et al., 2005). Future studies will focus on genistein’s potential impact on the expression level and activation of UGTs through the Nrf2 pathways (Fig. 7). Nevertheless, the data presented in this study shows the mechanism of the pathway for modulating the expression and activation of UGTs by genistein on APAP-induced hepatic injury. We conclude on the basis of the detoxification and hepatoprotection experiments of genistein that the findings presented here provide an important insight into xenobiotics or carcinogen inactivation or detoxification as one primary characteristic of genistein’s multitudinous health protections. Conflict of Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, ‘‘Genistein protects against acetaminophen-induced liver injury via potential impact on activation of UDP-Glucuronosyltransferase and antioxidant enzymes’’. Acknowledgment This study was supported by the National Natural Science Foundation Committee of China (Grant No. 31071535). References Aitio, A., 1974. UDP-glucuronyltransferase activity in various rat tissues. International of Journal Biochemistry 5, 325–330. Amimoto, T., Matsura, T., Yakoyama, 1995. Acetaminophen-induced hepatic injury in mice: the role of lipid peroxidation and effects of pretreatment with coenzyme Q10 and a-tocopherol. Free Radical Biology & Medicine 19 (2), 169– 176. Appelt, L.C., Reicks, M.M., 1999. Soy induces phase II enzymes however, does not inhibit dimethylbenz[a] anthracene-induced carcinogenesis in female rats. Journal of Nutrition 129, 1820–1826. Barve, A., Khor, T.O., Nair, S., Lin, W., Yu, S., Jain, M.R., Chan, J.Y., Kong, A.N., 2008. Pharmacogenomic profile of soy isoflavone concentrate in the prostate of Nrf2 deficient and wild-type mice. Journal of Pharmaceutical Sciences 97, 4528– 4545. Bessems, J.G., Van Stee, L.L., Commandeur, J.N., Groot, E.J., Vermeulen, N.P., 1997. 3,5-Dihalogenated analogues: role of cytochrome P-450 and formation of GSH conjugates and protein adducts. Toxicology in Vitro 11, 9–19. Bitto, A., Altavilla, D., Bonaiuto, A., Polito, F., Minutoli, L., Di Stefano, V., Giuliani, D., Guarini, S., Arcoraci, V., Squadrito, F., 2009. Effects of aglycone genistein in a rat experimental model of postmenopausal metabolic syndrome. Journal of Endocrinology 200, 367–376. Bock, K.W., 2011. From differential induction of UDP-glucuronosyltransferases in rat liver to characterization of responsible ligand-activated transcription factors, and their multilevel crosstalk in humans. Biochemical Pharmacology 82, 9–16.

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