- Email: [email protected]
Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats
G Model ETP 50979 No. of Pages 8
Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats Toshinori Yoshidaa,* , Hirotada Murayamaa , Masahi Kawashimaa , Rei Nagaharaa , Yumi Kangawaa , Sayaka Mizukamia,b , Masayoshi Kimuraa,b , Hajime Abea,b , Shim-mo Hayashic, Makoto Shibutania a
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan c Global Scientific and Regulatory Affairs, San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan b
A R T I C L E I N F O
Article history: Received 11 February 2016 Received in revised form 5 September 2016 Accepted 15 October 2016 Keywords: NADPH oxidase Preneoplastic liver cell lesions Two-stage hepatocarcinogenesis model High fat diet Apocynin Enzymatically modified isoquercetin
A B S T R A C T
We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilinnegative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver pathology in the United States, and is estimated to affect approximately 20–30% of the population (Younossi, 2008). NAFLD is a progressive disease characterized by intrahepatic accumulation, i.e. liver steatosis and hepatic inflammation in the early stage, driving the transition from steatosis toward more advanced non-alcoholic steatohepatitis (NASH) (Sheedfar et al., 2013). Ultimately, NASH may progress to an irreversible form of cirrhosis that can lead to liver cancer (Sheedfar et al., 2013). The pathogenesis of NAFLD is multifactorial, including factors such as the impairment of lipid metabolism, redox imbalance, and insulin
* Corresponding author. E-mail address: [email protected] (T. Yoshida).
resistance in the liver. However, the details of the progression to hepatic steatosis and hepatitis in NAFLD and NASH, respectively, followed by liver cancer, are not fully understood. The intracellular redox status is established by several redox pairs, such as reduced glutathione/oxidized glutathione, NADH/ NAD+, and NADPH/NADP+ (Kalyanaraman, 2013). NADPH oxidase (NOX) is a dedicated reactive oxygen species (ROS)-generating enzyme that acts through the transfer of electrons from NADPH across biological membranes to release molecular oxygen, and broadly and specifically regulates redox-sensitive signalling pathways. The mammalian NOX family comprises seven isoforms: NOX1-5, and Duox 1 and 2, which are distributed in a cell specific manner (Montezano and Touyz, 2014). Each NOX and DUOX consists of several subunits; for instance, NOX2, comprise the cytosolic regulatory subunits p47phox, p67phox, p40phox, the membrane regulatory subunit p22phox, and the catalytic subunit gp91phox; NOX4 comprises p22phox and Polidip2. NOX activation
http://dx.doi.org/10.1016/j.etp.2016.10.003 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
2
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
is involved in killing pathogens in phagocytes (Kalyanaraman, 2013), vascular diseases such as hypertension and stroke (Montezano and Touyz, 2014), and cancer development and progression (Block and Gorin, 2012). NOX-mediated ROS generation is also associated with high-fat diet (HFD)-fed mice and rats, which develop NAFLD, NASH, or liver cirrhosis (Gupte et al., 2009; Gabele et al., 2011; Chatterjee et al., 2013); however, the significant role of NOX, especially p22phox, in the liver, including in steatosis and preneoplastic lesions, is not yet clearly understood. Current pharmaceutical drugs such as insulin-sensitizers, lipid lowering agents, anti-inflammatory drugs, and antioxidants that target the mechanisms involved in NAFLD have been evaluated in animal and clinical studies (Ibrahim et al., 2013). Much attention has been paid to the identification of potential dietary substances from fruits, vegetables, and edible plants to provide a new strategy for NAFLD treatment (Pan et al., 2014). Quercetin is a natural flavonol that is typically present in onions, broccoli, and leafy green vegetables. Many in vivo studies have supported the preventive and therapeutic efficacy of quercetin against NAFLD. In diet-mediated obese animals, feeding quercetin reduces hepatic lipid accumulation, inflammatory markers, and portal fibrosis, and improves lipid peroxidation (Marcolin et al., 2012; Pisonero-Vaquero et al., 2015). Enzymatically-modified quercetin (EMIQ) prepared by enzymatic deglycosylation and subsequent a-oligoglucosylation of quercetin 3-O-b-rutinoid (rutin) significantly increased plasma levels of quercetin metabolites as compared with quercetin 3-O-b-glucoside (isoquercitrin) and rutin (Murota et al., 2010; Makino et al., 2009). Our research groups demonstrated that EMIQ supplementation prevents chemical-induced liver promotion in a mediumterm liver carcinogenesis bioassay (initiation and promotion model) (Kuwata et al., 2011; Nishimura et al., 2010; Morita et al., 2011; Shimada et al., 2010). We hypothesized that the antioxidant EMIQ prevents steatosisrelated liver promotion in rats fed a HFD through the NOXmediated mechanism, and compared the results with those by the NOX inhibitor APO. To investigate this hypothesis, we examined the effects of EMIQ or APO on hyperlipidemia and liver histopathology including the activities of cell proliferation and apoptosis, and the expression of NOX subunits in GST-P-positive liver foci in rats red a HFD that were administered both DEN and malachite green (MG). MG is not only an enhancer of liver promotion but also a lipogenic substance in rats (Sundarrajan et al., 2000; Gupta et al., 2003). We found that MG was an antihyperlipidemic substance, and that this effect was enhanced by both EMIQ and APO. In the liver, p22phox-expressed GST-Ppositive foci are prevented by EMIQ and APO, suggesting that NOXmediated liver promotion might be involved in rats under the conditions studied. 2. Materials and methods 2.1. Chemicals DEN (CAS No. 55-18-5, purity >99%) and APO (CAS No. 498-02-2, purity >98%) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan) and Sigma-Aldrich Co., LLC., Mo. USA, respectively. EMIQ (purity >97%) was provided by San-Ei Gen F.F.I., Inc. (Osaka, Japan). 2.2. Animals and treatment A total of 48 of 5-week-old male F344/N rats were purchased from Japan SLC, Inc. (Shizuoka, Japan), maintained in an airconditioned room (room temperature, 22 3 C; relative humidity, 56 11%; 12-h light/dark cycle), and given free access to a powdered diet (Oriental MF; Oriental Yeast, Tokyo, Japan) and tap water. After a 1-week acclimatization period, a medium-term
liver carcinogenesis bioassay (Ito et al., 2003) was conducted by the following procedure. All animals received an intraperitoneal injection of DEN at a dose of 200 mg/kg body weight to initiate liver carcinogenesis, and were fed a HFD (D12451; Research Diets, Lane, NJ, USA) containing 0% or 0.01% MG for 6 weeks starting 2 weeks after DEN initiation. Animals who received MG were also supplied with 0.2% APO or 0.5% EMIQ in their drinking water. The dose selection of each substance was based on previously-reported results: 0.01% MG increased eosinophilic liver cell foci in rats initiated with DEN (Sundarrajan et al., 2000); 0.2% Apo ameliorated cisplatin-induced renal injuries in rats (Chirino et al., 2008); 0.5% EMIQ prevented a liver promoter piperonyl butoxide-induced preneoplastic liver cell foci in rats subjected to a medium-term liver carcinogenesis bioassay (Hara et al., 2014). All the animals were subjected to two-thirds partial hepatectomy 1 week after MG treatment to enhance cell proliferation. At the end of the experiment, we euthanized the rats by exsanguination under isoflurane anaesthesia, and collected blood from the vena cava. The livers were excised and weighed, and the sliced liver samples were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered formalin (pH 7.4; Wako Pure Chemicals Industries, Ltd.) for histopathology and immunohistochemistry. The liver pieces were frozen in dry ice and stored at 80 C until needed for further analysis. All procedures in this study were conducted in compliance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006) and according to the protocol approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology. Three of 48 animals were excluded from the evaluations because one animal died during the study, and two animals had proliferation of bile duct in the liver, caused by partial hepatectomy. 2.3. Blood biochemistry We obtained serum samples from the blood samples described above. We sent the samples for analysis of the following parameters at the LSI Medicine Corporation (Tokyo, Japan): alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transferase, T.CHOL, triglycerides (TGs), and glucose (GLU). 2.4. Histopathology and immunohistochemistry The fixed liver slices were routinely dehydrated in graded ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination. The pathological changes including steatotic cells, ballooning cells, and inflammatory foci were graded by the NAFLD activity score (NAS) (Kleiner et al., 2005). Immunohistochemical staining was performed using primary antibodies of adipophilin (1:390; Abcam, Cambridge, MA, USA), GST-P (1:1000; Medical & Biological Laboratories, Nagoya, Japan), Ki-67 (1:50; Dako, Glostrup, Denmark), active caspase-3 (1:200; Novus Biologicals, Ltd., Cambridge, UK), p22phox (1:200; Bioss Inc., Woburn, MA, USA), p47phox (1:1000; Bioworld Technology, Inc.), NOX4 (1:1000; Bioworld Technology, Inc.), and LC3a (1:900; Abcam). The deparaffinised liver sections were treated with 0.3% H2O2 in methanol for 30 min, and then antigen retrieval by autoclaving at 121 C for 10 min in citrate buffer, pH6.0 (for Ki-67) or antigen retrieval solution (Dako), pH9.0 (for active caspase-3), or microwaving at 90 C for 10 min in citrate buffer, pH6.0 (for adipophilin and LC3a). No antigen retrieval was conducted for the other primary antibodies. The sections were incubated with the primary antibodies overnight at 4 C, and reacted with an avidin–biotin– peroxidase complex method with VECTASTAIN1 Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA), followed by
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
victualing with 3,30 -diaminobenzidine as a chromogen and staining with light hematoxylin. For negative control, phosphate-buffered saline replaced the primary antibodies. For positive control, liver tissues from rats treated with lipopolysaccharide (Shimada et al., 2010) were stained to confirm expression of p22phox, p47phox, and NOX4 in hepatocytes with inflammation. The numbers and areas of GST-P-positive foci (>0.2-mm diameter) and the total areas of the liver sections were quantified using Scion Image (Scion Corp., Frederick, MD, USA) (Hara et al., 2014). Ki-67, active caspase-3, p22phox, p47phox, or NOX4-positive cells were examined in a total of 1000 cells in GST-P-positive foci or non-GCTP-positive foci per animal. LC3a-positive foci were counted in GSTP-positive foci. 2.5. Statistical analysis All data were expressed as means with standard deviations. The statistical significance of the differences between the control and the treated group(s) was determined the Tukey or Steel-Dwass multiple comparison test. A p-value <0.05 was considered statistically significant. 3. Results 3.1. MG increases liver weight with reduction of plasma total cholesterol and alkaline phosphatase, while EMIQ reduced the plasma parameters There were no-treatment-related clinical signs or changes to body weight, food intake, and water intake in any group (Table 1). Absolute and relative liver weight tended to increase in the MGtreated groups when compared with the control group, with a significant increase in relative weight in the MG + EMIQ co-treated group (p = 0.038). ALP (p = 0.002) and T.CHOL (p = 0.005) were significantly lower in the MG-treated group than in the control group, even though no treatment-related changes in AST, ALT, and GLU levels were detected (Table 2). Both ALP (p = 0.012) and T.CHOL (p = 0.003) were significantly lower in the MG + EMIQ co-treated group when compared with the MG-treated group. TGs tended to decrease in the MG + EMIQ co-treated group when compared with the MG-treated group. 3.2. HDF induces hepatocellular steatosis with adipophilin expression, except in preneoplastic liver cell foci Slight or moderate hepatocellular steatosis, ballooning, and inflammatory foci were observed in the liver in all groups. There were some individual differences in the levels of steatosis, with marked heterogeneity in some parts of liver sections. Scoring of the sections by a pathologist revealed that MG tended to increase the NAS score, with a significant increase in the MG + APO co-treated groups when compared with the control group (Table 3). Altered
3
Table 2 Blood biochemistry in rats treated with MG with or without APO or EMIQ after DEN initiation.d Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
AST (U/L) ALT (U/L) ALP (U/L) GLU (mg/dL) T.CHOL (mg/dL) TG (mg/dL)
82 5 48 7 1673 124 160 36 90 10 316 128
81 4 48 4 1517 71a 136 18 80 6a 327 71
80 4 47 5 1582 102 150 29 78 7 293 102
80 6 52 6 1389 95a,b,c 151 19 69 5a,b,c 265 92
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; DEN, N-diethylnitrosamine; AST, aspartate aminotransaminase; ALT, alanine aminotransaminase; ALP, alkaline phosphatase; GLU, glucoase; T.CHOL, total cholesterol; TG, triglyceride. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b p < 0.05 versus MG (Tukey’s or the Steel-Dwass test). c p < 0.05 versus MG + APO (Tukey’s or the Steel-Dwass test). d All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
Table 3 Quantitative analysis of NAFLD steatosis, and p47phox+ cells, NOX4+ cells, and LC3a+ cells in GST-P+ foci in rats treated with MG with or without APO or EMIQ after DEN initiation.b Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
NAS (Score) p47phox (%) NOX4 (%) LC3a (%)
3.5 0.8 7.0 6.1 2.9 2.1 1.4 2.2
4.5 1.2 5.8 3.9 3.2 3.4 3.5 1.9a
5.0 1.1a 4.1 2.6 1.8 2.1 2.6 2.5
4.3 1.1 8.3 5.5 1.2 1.7 4.2 3.1
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; DEN, N-diethylnitrosamine; NAS, NAFLD acticity score. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
liver cell foci (eosinophilic, clear cell, or basophilic) were observed in all groups (Fig. 1); eosinophilic and clear cell foci corresponded to GST-P-positive foci. Adipophilin was observed in the margin of variable-sized vacuoles in hepatocytes and sinusoidal cells in nonGST-P-positive foci outside GST-P-positive foci, not inside), while its expression was restricted in sinusoidal cells in GST-P-positive foci (inside GST-P-positive foci). 3.3. MG increases the number of preneoplastic liver cell foci, while coadministration of apocynin prevents MG-induced preneoplastic liver cell foci The number of GST-P-positive foci was significantly increased in the MG, MG + APO, and MG + EMIQ groups when compared with the control group (Fig. 2A). This number was significantly reduced in the MG + APO co-treated group when compared with the MG-
Table 1 Final body weight, liver weight, food intake, and water intake in rats treated with MG with or without APO or EMIQ after DEN initiation.b Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
Final body weight (g) Absolute liver weight (g) Relative liver weight (%BW) Food intake (g/rat/day) Water intake (g/rat/day)
276.7 14.1 8.26 0.73 2.98 0.17 9.5 3.4 17.3 2.2
277.6 9.7 8.70 0.44 3.13 0.08 9.3 3.4 18.6 2.7
282.1 16.3 8.73 0.72 3.10 0.20 9.4 3.2 17.5 1.9
280.7 13.7 8.88 0.62 3.16 0.15a 9.6 3.1 16.4 1.7
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; BW, body weight; DEN, N-diethylnitrosamine. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
4
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Fig. 1. Representative images of altered liver foci in rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by malachite green treatment. Adipophilin is not expressed in phenotypically altered hepatocytes in eosinophilic (A and B), clear cell (C and D), and basophilic cell foci (E and F). Adipophilin is expressed in sinusoidal cells in- or out-side foci and surrounding hepatocytes outside foci. (A) Control group; (B and C) MG group. Bar = 100 um.
treated group. The ratios in the MG, MG + APO, and MG + EMIQ groups were 1.7, 1.4, and 1.5-fold higher than that in the control group, respectively. The area of GST-P-positive foci in the MGtreated group was 1.4-fold higher than the control values, but this was not statistically significant (Fig. 2B). The area in the MG + APOcotreated group was comparable with that in the control group. 3.4. MG increases cell proliferation and apoptosis in preneoplastic liver cell foci, while coadministration of apocynin prevents MG-induced cell proliferation and apoptosis in the foci, and coadministration of EMIQ increases apoptosis in liver parenchymal cells Ki-67 labelling index in GST-P-positive foci was 1.6-fold higher in the MG-treated group than in the control group, but this was not statistically significant (Fig. 2C). The index in the MG + APOcotreated group was comparable with that in the control group. No treatment-related effects on the index in non-GST-P-positive foci were detected in any group (Fig. 2D). The labelling index of active caspase-3 in GST-P-positive foci was increased 3-fold in the MGtreated group when compared with the control group, and this was not statistically significant (Fig. 2E). APO tended to decrease the
index, and the value was 1.7-fold higher than the control value. The index in the MG + EMIQ-cotreated group was significantly (3.7fold) higher than that in the control group. A similar treatmentrelated change was detected in non-GST-P-positive foci, the values in the MG, MG + APO, and MG + EMIQ groups being 2.5-, 1.7-, or 6.8fold higher than those in the control group, respectively (Fig. 2F). The value in the MG + EMIQ-cotreated group was significantly different. 3.5. Coadministration of apocynin or EMIQ prevents MG-induced expression of a NOX subunit p22phox in preneoplastic liver cell foci NOX-related molecules, p22phox, p47phox, and NOX4 were detected in the cytoplasm or cell membrane of hepatocytes. Expression of p22phox in GST-P-positive foci was slightly increased in the MG treated group when compared with that in the control group but this was not statistically significant; the value was significantly lower in the MG + APO and MG + EMIQ cotreated group than the MG-treated group (Figs. 3 and 4). No treatment-related changes were detected with respect to the expression of p22phox in non-GST-P-positive foci in any group. No
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
5
Fig. 2. Quantitative analysis of glutathione S-transferase placental (GST-P)-positive foci and Ki-67-positive cells and active caspase-3-positive cells in GST-P-positive foci and non-GST-P-positive foci in the liver of rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by malachite green (MG) treatment with or without cotreatment with apocynin (APO) or enzymatically modified isoquercetin (EMIQ). The number (A) and area (B) of GST-P-positive foci, and quantitative data (%) of Ki-67-positive cells (C and D) and active caspase-3-positive cells (E and F) in GST-P-positive foci (C and E) and non-GST-P-positive foci (D and F). Columns represent means with standard deviations. *p < 0.05 (Tukey or Steel-Dwass multiple comparison test).
treatment-related changes were detected with respect to the expression of p47phox and NOX4 in GST-P-positive foci in any group (Table 3). A marker of autophagy, the LC3a-positive hepatocyte, was highly expressed in GST-P-positive foci in the MG-treated group, and this was statistically significant. No clear treatment-related effect by APO or EMIQ was detected with respect to LC3a expression in the liver foci. 4. Discussion There are clear parallels between aging and the pathological progression from NAFLD to HCC, as older people generally experience these liver diseases more commonly than people in younger age groups (Sheedfar et al., 2013). Health tends to decline particularly dramatically with aging in the modern world due to
lifestyle factors such as low levels of physical activity and poor nutritional habits, decreases in hormone regulation, and imbalances in the immune system. The steatosis-related pathological processes in humans are similar to those in animal models, where it takes a long time to develop NASH and HCC when fed a HFD (Gauthier et al., 2006; Duval et al., 2010), even with initiation with DEN (de Lima et al., 2008). To effectively observe hyperlipidaemia and liver steatosis followed by precancerous lesions, i.e. GST-Ppositive liver foci preceding HCC in rats, we fed the rats a HFD in a well-established initiation and promotion hepatocarcinogenesis model (Ito et al., 2003). This model can be useful in determining both lipid metabolism and liver promotion activity in rats over a short time, e.g. 8 weeks, assuming that hypothetical acceleration of ageing induces preneoplastic liver lesions through liver steatosis. We recently confirmed that rats fed a HFD had significantly
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
6
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Fig. 3. Representative images of p22phox-positive cells in GST-P-positive foci in rats treated with MG and MG + EMIQ. p22phox-positive cells (B and D) are observed in GST-Ppositive foci (A and C) in MG group (A and B), but not MG + EMIQ group (C and D). Bar = 50 um.
increased GST-P-positive liver foci together with hyperlipidaemia and liver steatosis compared with control rats fed a basal diet (Murayama et al., unpublished data). As demonstrated by blood biochemistry, we confirmed hyperlipidaemia in rats fed a HFD: the TG level (316 128 mg/dL) was 7.5 times the control value (42 8 mg/dL) (Kojima et al., 2009), and T. CHOL level (90 10 mg/dL) was 2 times the control value (44 2 mg/dL) (Kojima et al., 2009). We also selected a lipogenic substance (MG) to determine the effect of EMIQ and APO on hyperlipidaemia and fatty change in the livers of rats fed a HFD. Feeding rats a diet with MG for 28 days induces hepatocellular fatty change in the liver (Culp et al., 1999). Interestingly, MG reduced the levels of blood lipids (T.CHOL), which were further reduced by EMIQ. With no effects of MG on TG in blood, EMIQ tended to also
reduce TG. Additionally, ALP level (1673 124 U/L) in rats fed a HFD was 2.2 times the control value (770 36 U/L) (Kojima et al., 2009). ALP is known to be increased by a HFD. Serum ALP level is correlated with jejunal ALP activity, which is crucial to intestinal function and controlling inflammation (Lalles et al., 2012). ALP coordinated the treatment-related effects on T.CHOL and TG. Accordingly, the present model is useful for determining the effects on blood lipid levels by the test substances. As we showed in our present study, MG appears to have a weak liver promotion effect with a statistical significant difference detected only in the number of GST-P-positive foci. The effects seemed to be associated with relatively higher activity of cell proliferation and apoptosis as shown in Ki-67 and active caspase3-labelling indices. The data were almost consistent with
Fig. 4. Quantitative analysis of p22phox-positive cells in glutathione S-transferase placental (GST-P)-positive foci in the liver of rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by MG treatment with or without co-treatment with APO or EMIQ. Quantitative data (%) of p22phox-positive cells in GST-P-positive foci (A) and non-GST-P-positive foci (B). Columns represent means with standard deviations. *p < 0.05 (Tukey or Steel-Dwass multiple comparison test).
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
previously reported findings in vivo (Fernandes and Rao, 1991; Sundarrajan et al., 2000, 2001; Gupta et al., 2003). The NOX inhibitor APO reduced these effects against GST-P-positive foci, and this effect might be correlated with reduced expression of p22phox in the hepatocytes of GST-P-positive foci. EMIQ also reduced the expression of p22phox in GST-P-positive liver foci, but not significantly. p22phox is a membrane regulatory subunit of NOX1, 2, or 4 (Montezano and Touyz, 2014). NOX1, 2 and 4 served as a prominent source of ROS in hepatocytes, while NOX2 serves the same role in phagocytes (Levin et al., 2012; Choi et al., 2014). NOX4 depends on only p22phox in order to be active, is constitutively activated, and its ROS production is regulated by Poldip2 (Montezano and Touyz, 2014). Although NOX4 expression was not altered, reduced p22phox expression in the precancerous lesion might be related to lower cell proliferation activity or a greater apoptosis response to APO or EMIQ supplementation, respectively. This finding could be in part consistent with evidence that upregulation of NOX4 by TGF-b is required for its proapoptotic activity in hepatocytes, while impairment of the TGFb-induced response confers apoptosis resistance to HCCs (Carmona-Cuenca et al., 2008). GST-P-positive liver foci reduced the expression of adipophilin in phenotypically altered hepatocytes. Adipophilin is a well-known lipid droplet protein involved in lipid droplet homeostasis, and works primarily by stimulating fat packing inside lipid droplets in order to avoid catabolic pathways, which can induce oxidative stress (Grasselli et al., 2010). The results of these studies suggest that a nutritional niche within the precancerous lesion could be regulated separately from the surrounding hepatocytes, which highly express adipophilin together with lipid droplets. Contrary to our expectations, LC3a, a marker of autophagy, was highly expressed in the hepatocytes of precancerous lesion. This expression was enhanced by MG treatment compared with the control group. Therefore, we speculated that a precancerous liver lesion might be resistant to lipid accumulation, resulting in starvation in comparison with the surrounding intact hepatocytes and activating autophagy to control intracellular nutritional levels. This interpretation is supported by the possible bifunctional role of autophagy in cancer development: autophagy protects cells by removing ROS-damaged organelles to maintain normal cellular condition before tumour development, while autophagy promotes tumour cell survival even in low nutrient and oxygen conditions at a later phase (Czaja et al., 2013). In conclusion, APO or EMIQ ameliorated hyperlipidaemia under the current study conditions when combined with the lipogenic substance MG in HFD-fed rats. The effects of both agents on liver steatosis remain unclear. Further studies are required to determine the lipid metabolism pathways taking place in the liver, and in particular time-course changes. These results emphasize that the antioxidant EMIQ and NOX inhibitor APO reduce the expression of the NOX subunit p22phox in preneoplastic liver foci. By taking advantage of animal models, our data suggest that the NOX subunit p22phox is a possible candidate for the chemoprevention of steatosis-related liver cancer. Conflict of interest statement All the authors declare no conflicts of interest. Acknowledgements The authors thank Mrs Shigeko Suzuki for her technical assistance in preparing the histological specimens. This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and
7
Welfare of Japan (H25-Shokuhin-Ippan-005) and the Japan Food Chemical Research Foundation. References Block K, Gorin Y. Aiding and abetting roles of NOX oxidases in cellular transformation. Nat. Rev. Cancer 2012;12:627–37. Carmona-Cuenca I, Roncero C, Sancho P, Caja L, Fausto N, Fernandez M, et al. Upregulation of the NADPH oxidase NOX4 by TGF-beta in hepatocytes is required for its pro-apoptotic activity. J. Hepatol. 2008;49:965–76. Chatterjee S, Ganini D, Tokar EJ, Kumar A, Das S, Corbett J, et al. Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis. J. Hepatol. 2013;58:778–84. Chirino YI, Sanchez-Gonzalez DJ, Martinez-Martinez CM, Cruz C, Pedraza-Chaverri J. Protective effects of apocynin against cisplatin-induced oxidative stress and nephrotoxicity. Toxicology 2008;245:18–23. Choi J, Corder NL, Koduru B, Wang Y. Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma. Free Radic. Biol. Med. 2014;72:267–84. Culp SJ, Blankenship LR, Kusewitt DF, Doerge DR, Mulligan LT, Beland FA. Toxicity and metabolism of malachite green and leucomalachite green during shortterm feeding to Fischer 344 rats and B6C3F1 mice. Chem. Biol. Interact. 1999;122:153–70. Czaja MJ, Ding WX, Donohue Jr. TM, Friedman SL, Kim JS, Komatsu M, et al. Functions of autophagy in normal and diseased liver. Autophagy 2013;9:1131–58. de Lima VM, Oliveira CP, Alves VA, Chammas MC, Oliveira EP, Stefano JT, et al. A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma. J. Hepatol. 2008;49:1055–61. Duval C, Thissen U, Keshtkar S, Accart B, Stienstra R, Boekschoten MV, et al. Adipose tissue dysfunction signals progression of hepatic steatosis towards nonalcoholic steatohepatitis in C57BL/6 mice. Diabetes 2010;59:3181–91. Fernandes C, Rao KV. Enhanced expression of low molecular weight keratins during progressive diethylnitrosamine-induced hepatocarcinogenesis in rats is associated with the presence of high levels of gamma-glutamyl transpeptidase and glycogen-deficient islands. Cancer Lett. 1991;59:211–9. Gabele E, Dostert K, Hofmann C, Wiest R, Scholmerich J, Hellerbrand C, et al. DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH. J. Hepatol. 2011;55:1391–9. Gauthier MS, Favier R, Lavoie JM. Time course of the development of non-alcoholic hepatic steatosis in response to high-fat diet-induced obesity in rats. Br. J. Nutr. 2006;95:273–81. Grasselli E, Voci A, Pesce C, Canesi L, Fugassa E, Gallo G, et al. PAT protein mRNA expression in primary rat hepatocytes: effects of exposure to fatty acids. Int. J. Mol. Med. 2010;25:505–12. Gupta S, Sundarrajan M, Rao KV. Tumor promotion by metanil yellow and malachite green during rat hepatocarcinogenesis is associated with dysregulated expression of cell cycle regulatory proteins. Teratog. Carcinog. Mutagen. 2003;301–12. Gupte RS, Floyd BC, Kozicky M, George S, Ungvari ZI, Neito V, et al. Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver. Free Radic. Biol. Med. 2009;47:219–28. Hara S, Morita R, Ogawa T, Segawa R, Takimoto N, Suzuki K, et al. Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model. Exp. Toxicol. Pathol. 2014;66:225– 34. Ibrahim MA, Kelleni M, Geddawy A. Nonalcoholic fatty liver disease: current and potential therapies. Life Sci. 2013;92:114–8. Ito N, Tamano S, Shirai T. A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci. 2003;94:3–8. Kalyanaraman B. Teaching the basics of redox biology to medical and graduate students: oxidants, antioxidants and disease mechanisms. Redox Biol. 2013;1:244–57. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Nonalcoholic Steatohepatitis Clinical Research Network: design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21. Kojima S, Sasaki J, Tomita M, Saka M, Ishizuka K, Kawakatsu H, et al. Multiple organ toxicity, including hypochromic anemia, following repeated dose oral administration of phenobarbital (PB) in rats. J. Toxicol. Sci. 2009;34:527–39. Kuwata K, Shibutani M, Hayashi H, Shimamoto K, Hayashi SM, Suzuki K, et al. Concomitant apoptosis and regeneration of liver cells as a mechanism of livertumor promotion by beta-naphthoflavone involving TNFalpha-signaling due to oxidative cellular stress in rats. Toxicology 2011;283:8–17. Lalles JP, Orozco-Solis R, Bolanos-Jimenez F, de Coppet P, Le Drean G, Segain JP. Perinatal undernutrition alters intestinal alkaline phosphatase and its main transcription factors KLF4 and Cdx1 in adult offspring fed a high-fat diet. J. Nutr. Biochem. 2012;23:1490–7. Levin I, Petrasek J, Szabo G. The presence of p47phox in liver parenchymal cells is a key mediator in the pathogenesis of alcoholic liver steatosis. Alcohol. Clin. Exp. Res. 2012;36:1397–406. Makino T, Shimizu R, Kanemaru M, Suzuki Y, Moriwaki M, Mizukami H. Enzymatically modified isoquercitrin, alpha-oligoglucosyl quercetin 3-O-
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
glucoside, is absorbed more easily than other quercetin glycosides or aglycone after oral administration in rats. Biol. Pharm. Bull. 2009;32:2034–40. Marcolin E, San-Miguel B, Vallejo D, Tieppo J, Marroni N, Gonzalez-Gallego J, et al. Quercetin treatment ameliorates inflammation and fibrosis in mice with nonalcoholic steatohepatitis. J. Nutr. 2012;142:1821–8. Montezano AC, Touyz RM. Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid. Redox Signal. 2014;20:164–82. Morita R, Shimamoto K, Ishii Y, Kuwata K, Ogawa B, Imaoka M, et al. Suppressive effect of enzymatically modified isoquercitrin on phenobarbital-induced liver tumor promotion in rats. Arch. Toxicol. 2011;85:1475–84. Murota K, Matsuda N, Kashino Y, Fujikura Y, Nakamura T, Kato Y, et al. alphaOligoglucosylation of a sugar moiety enhances the bioavailability of quercetin glucosides in humans. Arch. Biochem. Biophys. 2010;501:91–7. Nishimura J, Saegusa Y, Dewa Y, Jin M, Kawai M, Kemmochi S, et al. Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats. Arch. Toxicol. 2010;84:143–53. Pan MH, Lai CS, Tsai ML, Ho CT. Chemoprevention of nonalcoholic fatty liver disease by dietary natural compounds. Mol. Nutr. Food Res. 2014;58:147–71.
Pisonero-Vaquero S, Martinez-Ferreras A, Garcia-Mediavilla MV, Martinez-Florez S, Fernandez A, Benet M, et al. Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT pathway in a diet-induced mouse model of nonalcoholic fatty liver disease. Mol. Nutr. Food Res. 2015;59:879–93. Sheedfar F, Di Biase S, Koonen D, Vinciguerra M. Liver diseases and aging: friends or foes?. Aging Cell 2013;12:950–4. Shimada Y, Dewa Y, Ichimura R, Suzuki T, Mizukami S, Hayashi SM, et al. Antioxidant enzymatically modified isoquercitrin suppresses the development of liver preneoplastic lesions in rats induced by beta-naphthoflavone. Toxicology 2010;268:213–8. Sundarrajan M, Fernandis AZ, Subrahmanyam G, Prabhudesai S, Krishnamurthy SC, Rao KV. Overexpression of G1/S cyclins and PCNA and their relationship to tyrosine phosphorylation and dephosphorylation during tumor promotion by metanil yellow and malachite green. Toxicol. Lett. 2000;116:119–30. Sundarrajan M, Prabhudesai S, Krishnamurthy SC, Rao KV. Effect of metanil yellow and malachite green on DNA synthesis in N-nitrosodiethylamine induced preneoplastic rat livers. Indian J. Exp. Biol. 2001;39:845–52. Younossi ZM. Review article: current management of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Aliment. Pharmacol. Ther. 2008;28:2–12.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats Toshinori Yoshidaa,* , Hirotada Murayamaa , Masahi Kawashimaa , Rei Nagaharaa , Yumi Kangawaa , Sayaka Mizukamia,b , Masayoshi Kimuraa,b , Hajime Abea,b , Shim-mo Hayashic, Makoto Shibutania a
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan c Global Scientific and Regulatory Affairs, San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan b
A R T I C L E I N F O
Article history: Received 11 February 2016 Received in revised form 5 September 2016 Accepted 15 October 2016 Keywords: NADPH oxidase Preneoplastic liver cell lesions Two-stage hepatocarcinogenesis model High fat diet Apocynin Enzymatically modified isoquercetin
A B S T R A C T
We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilinnegative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver pathology in the United States, and is estimated to affect approximately 20–30% of the population (Younossi, 2008). NAFLD is a progressive disease characterized by intrahepatic accumulation, i.e. liver steatosis and hepatic inflammation in the early stage, driving the transition from steatosis toward more advanced non-alcoholic steatohepatitis (NASH) (Sheedfar et al., 2013). Ultimately, NASH may progress to an irreversible form of cirrhosis that can lead to liver cancer (Sheedfar et al., 2013). The pathogenesis of NAFLD is multifactorial, including factors such as the impairment of lipid metabolism, redox imbalance, and insulin
* Corresponding author. E-mail address: [email protected] (T. Yoshida).
resistance in the liver. However, the details of the progression to hepatic steatosis and hepatitis in NAFLD and NASH, respectively, followed by liver cancer, are not fully understood. The intracellular redox status is established by several redox pairs, such as reduced glutathione/oxidized glutathione, NADH/ NAD+, and NADPH/NADP+ (Kalyanaraman, 2013). NADPH oxidase (NOX) is a dedicated reactive oxygen species (ROS)-generating enzyme that acts through the transfer of electrons from NADPH across biological membranes to release molecular oxygen, and broadly and specifically regulates redox-sensitive signalling pathways. The mammalian NOX family comprises seven isoforms: NOX1-5, and Duox 1 and 2, which are distributed in a cell specific manner (Montezano and Touyz, 2014). Each NOX and DUOX consists of several subunits; for instance, NOX2, comprise the cytosolic regulatory subunits p47phox, p67phox, p40phox, the membrane regulatory subunit p22phox, and the catalytic subunit gp91phox; NOX4 comprises p22phox and Polidip2. NOX activation
http://dx.doi.org/10.1016/j.etp.2016.10.003 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
2
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
is involved in killing pathogens in phagocytes (Kalyanaraman, 2013), vascular diseases such as hypertension and stroke (Montezano and Touyz, 2014), and cancer development and progression (Block and Gorin, 2012). NOX-mediated ROS generation is also associated with high-fat diet (HFD)-fed mice and rats, which develop NAFLD, NASH, or liver cirrhosis (Gupte et al., 2009; Gabele et al., 2011; Chatterjee et al., 2013); however, the significant role of NOX, especially p22phox, in the liver, including in steatosis and preneoplastic lesions, is not yet clearly understood. Current pharmaceutical drugs such as insulin-sensitizers, lipid lowering agents, anti-inflammatory drugs, and antioxidants that target the mechanisms involved in NAFLD have been evaluated in animal and clinical studies (Ibrahim et al., 2013). Much attention has been paid to the identification of potential dietary substances from fruits, vegetables, and edible plants to provide a new strategy for NAFLD treatment (Pan et al., 2014). Quercetin is a natural flavonol that is typically present in onions, broccoli, and leafy green vegetables. Many in vivo studies have supported the preventive and therapeutic efficacy of quercetin against NAFLD. In diet-mediated obese animals, feeding quercetin reduces hepatic lipid accumulation, inflammatory markers, and portal fibrosis, and improves lipid peroxidation (Marcolin et al., 2012; Pisonero-Vaquero et al., 2015). Enzymatically-modified quercetin (EMIQ) prepared by enzymatic deglycosylation and subsequent a-oligoglucosylation of quercetin 3-O-b-rutinoid (rutin) significantly increased plasma levels of quercetin metabolites as compared with quercetin 3-O-b-glucoside (isoquercitrin) and rutin (Murota et al., 2010; Makino et al., 2009). Our research groups demonstrated that EMIQ supplementation prevents chemical-induced liver promotion in a mediumterm liver carcinogenesis bioassay (initiation and promotion model) (Kuwata et al., 2011; Nishimura et al., 2010; Morita et al., 2011; Shimada et al., 2010). We hypothesized that the antioxidant EMIQ prevents steatosisrelated liver promotion in rats fed a HFD through the NOXmediated mechanism, and compared the results with those by the NOX inhibitor APO. To investigate this hypothesis, we examined the effects of EMIQ or APO on hyperlipidemia and liver histopathology including the activities of cell proliferation and apoptosis, and the expression of NOX subunits in GST-P-positive liver foci in rats red a HFD that were administered both DEN and malachite green (MG). MG is not only an enhancer of liver promotion but also a lipogenic substance in rats (Sundarrajan et al., 2000; Gupta et al., 2003). We found that MG was an antihyperlipidemic substance, and that this effect was enhanced by both EMIQ and APO. In the liver, p22phox-expressed GST-Ppositive foci are prevented by EMIQ and APO, suggesting that NOXmediated liver promotion might be involved in rats under the conditions studied. 2. Materials and methods 2.1. Chemicals DEN (CAS No. 55-18-5, purity >99%) and APO (CAS No. 498-02-2, purity >98%) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan) and Sigma-Aldrich Co., LLC., Mo. USA, respectively. EMIQ (purity >97%) was provided by San-Ei Gen F.F.I., Inc. (Osaka, Japan). 2.2. Animals and treatment A total of 48 of 5-week-old male F344/N rats were purchased from Japan SLC, Inc. (Shizuoka, Japan), maintained in an airconditioned room (room temperature, 22 3 C; relative humidity, 56 11%; 12-h light/dark cycle), and given free access to a powdered diet (Oriental MF; Oriental Yeast, Tokyo, Japan) and tap water. After a 1-week acclimatization period, a medium-term
liver carcinogenesis bioassay (Ito et al., 2003) was conducted by the following procedure. All animals received an intraperitoneal injection of DEN at a dose of 200 mg/kg body weight to initiate liver carcinogenesis, and were fed a HFD (D12451; Research Diets, Lane, NJ, USA) containing 0% or 0.01% MG for 6 weeks starting 2 weeks after DEN initiation. Animals who received MG were also supplied with 0.2% APO or 0.5% EMIQ in their drinking water. The dose selection of each substance was based on previously-reported results: 0.01% MG increased eosinophilic liver cell foci in rats initiated with DEN (Sundarrajan et al., 2000); 0.2% Apo ameliorated cisplatin-induced renal injuries in rats (Chirino et al., 2008); 0.5% EMIQ prevented a liver promoter piperonyl butoxide-induced preneoplastic liver cell foci in rats subjected to a medium-term liver carcinogenesis bioassay (Hara et al., 2014). All the animals were subjected to two-thirds partial hepatectomy 1 week after MG treatment to enhance cell proliferation. At the end of the experiment, we euthanized the rats by exsanguination under isoflurane anaesthesia, and collected blood from the vena cava. The livers were excised and weighed, and the sliced liver samples were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered formalin (pH 7.4; Wako Pure Chemicals Industries, Ltd.) for histopathology and immunohistochemistry. The liver pieces were frozen in dry ice and stored at 80 C until needed for further analysis. All procedures in this study were conducted in compliance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006) and according to the protocol approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology. Three of 48 animals were excluded from the evaluations because one animal died during the study, and two animals had proliferation of bile duct in the liver, caused by partial hepatectomy. 2.3. Blood biochemistry We obtained serum samples from the blood samples described above. We sent the samples for analysis of the following parameters at the LSI Medicine Corporation (Tokyo, Japan): alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transferase, T.CHOL, triglycerides (TGs), and glucose (GLU). 2.4. Histopathology and immunohistochemistry The fixed liver slices were routinely dehydrated in graded ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination. The pathological changes including steatotic cells, ballooning cells, and inflammatory foci were graded by the NAFLD activity score (NAS) (Kleiner et al., 2005). Immunohistochemical staining was performed using primary antibodies of adipophilin (1:390; Abcam, Cambridge, MA, USA), GST-P (1:1000; Medical & Biological Laboratories, Nagoya, Japan), Ki-67 (1:50; Dako, Glostrup, Denmark), active caspase-3 (1:200; Novus Biologicals, Ltd., Cambridge, UK), p22phox (1:200; Bioss Inc., Woburn, MA, USA), p47phox (1:1000; Bioworld Technology, Inc.), NOX4 (1:1000; Bioworld Technology, Inc.), and LC3a (1:900; Abcam). The deparaffinised liver sections were treated with 0.3% H2O2 in methanol for 30 min, and then antigen retrieval by autoclaving at 121 C for 10 min in citrate buffer, pH6.0 (for Ki-67) or antigen retrieval solution (Dako), pH9.0 (for active caspase-3), or microwaving at 90 C for 10 min in citrate buffer, pH6.0 (for adipophilin and LC3a). No antigen retrieval was conducted for the other primary antibodies. The sections were incubated with the primary antibodies overnight at 4 C, and reacted with an avidin–biotin– peroxidase complex method with VECTASTAIN1 Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA), followed by
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
victualing with 3,30 -diaminobenzidine as a chromogen and staining with light hematoxylin. For negative control, phosphate-buffered saline replaced the primary antibodies. For positive control, liver tissues from rats treated with lipopolysaccharide (Shimada et al., 2010) were stained to confirm expression of p22phox, p47phox, and NOX4 in hepatocytes with inflammation. The numbers and areas of GST-P-positive foci (>0.2-mm diameter) and the total areas of the liver sections were quantified using Scion Image (Scion Corp., Frederick, MD, USA) (Hara et al., 2014). Ki-67, active caspase-3, p22phox, p47phox, or NOX4-positive cells were examined in a total of 1000 cells in GST-P-positive foci or non-GCTP-positive foci per animal. LC3a-positive foci were counted in GSTP-positive foci. 2.5. Statistical analysis All data were expressed as means with standard deviations. The statistical significance of the differences between the control and the treated group(s) was determined the Tukey or Steel-Dwass multiple comparison test. A p-value <0.05 was considered statistically significant. 3. Results 3.1. MG increases liver weight with reduction of plasma total cholesterol and alkaline phosphatase, while EMIQ reduced the plasma parameters There were no-treatment-related clinical signs or changes to body weight, food intake, and water intake in any group (Table 1). Absolute and relative liver weight tended to increase in the MGtreated groups when compared with the control group, with a significant increase in relative weight in the MG + EMIQ co-treated group (p = 0.038). ALP (p = 0.002) and T.CHOL (p = 0.005) were significantly lower in the MG-treated group than in the control group, even though no treatment-related changes in AST, ALT, and GLU levels were detected (Table 2). Both ALP (p = 0.012) and T.CHOL (p = 0.003) were significantly lower in the MG + EMIQ co-treated group when compared with the MG-treated group. TGs tended to decrease in the MG + EMIQ co-treated group when compared with the MG-treated group. 3.2. HDF induces hepatocellular steatosis with adipophilin expression, except in preneoplastic liver cell foci Slight or moderate hepatocellular steatosis, ballooning, and inflammatory foci were observed in the liver in all groups. There were some individual differences in the levels of steatosis, with marked heterogeneity in some parts of liver sections. Scoring of the sections by a pathologist revealed that MG tended to increase the NAS score, with a significant increase in the MG + APO co-treated groups when compared with the control group (Table 3). Altered
3
Table 2 Blood biochemistry in rats treated with MG with or without APO or EMIQ after DEN initiation.d Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
AST (U/L) ALT (U/L) ALP (U/L) GLU (mg/dL) T.CHOL (mg/dL) TG (mg/dL)
82 5 48 7 1673 124 160 36 90 10 316 128
81 4 48 4 1517 71a 136 18 80 6a 327 71
80 4 47 5 1582 102 150 29 78 7 293 102
80 6 52 6 1389 95a,b,c 151 19 69 5a,b,c 265 92
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; DEN, N-diethylnitrosamine; AST, aspartate aminotransaminase; ALT, alanine aminotransaminase; ALP, alkaline phosphatase; GLU, glucoase; T.CHOL, total cholesterol; TG, triglyceride. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b p < 0.05 versus MG (Tukey’s or the Steel-Dwass test). c p < 0.05 versus MG + APO (Tukey’s or the Steel-Dwass test). d All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
Table 3 Quantitative analysis of NAFLD steatosis, and p47phox+ cells, NOX4+ cells, and LC3a+ cells in GST-P+ foci in rats treated with MG with or without APO or EMIQ after DEN initiation.b Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
NAS (Score) p47phox (%) NOX4 (%) LC3a (%)
3.5 0.8 7.0 6.1 2.9 2.1 1.4 2.2
4.5 1.2 5.8 3.9 3.2 3.4 3.5 1.9a
5.0 1.1a 4.1 2.6 1.8 2.1 2.6 2.5
4.3 1.1 8.3 5.5 1.2 1.7 4.2 3.1
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; DEN, N-diethylnitrosamine; NAS, NAFLD acticity score. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
liver cell foci (eosinophilic, clear cell, or basophilic) were observed in all groups (Fig. 1); eosinophilic and clear cell foci corresponded to GST-P-positive foci. Adipophilin was observed in the margin of variable-sized vacuoles in hepatocytes and sinusoidal cells in nonGST-P-positive foci outside GST-P-positive foci, not inside), while its expression was restricted in sinusoidal cells in GST-P-positive foci (inside GST-P-positive foci). 3.3. MG increases the number of preneoplastic liver cell foci, while coadministration of apocynin prevents MG-induced preneoplastic liver cell foci The number of GST-P-positive foci was significantly increased in the MG, MG + APO, and MG + EMIQ groups when compared with the control group (Fig. 2A). This number was significantly reduced in the MG + APO co-treated group when compared with the MG-
Table 1 Final body weight, liver weight, food intake, and water intake in rats treated with MG with or without APO or EMIQ after DEN initiation.b Group No. of animals
CTL 11
MG 13
MG + APO 9
MG + EMIQ 12
Final body weight (g) Absolute liver weight (g) Relative liver weight (%BW) Food intake (g/rat/day) Water intake (g/rat/day)
276.7 14.1 8.26 0.73 2.98 0.17 9.5 3.4 17.3 2.2
277.6 9.7 8.70 0.44 3.13 0.08 9.3 3.4 18.6 2.7
282.1 16.3 8.73 0.72 3.10 0.20 9.4 3.2 17.5 1.9
280.7 13.7 8.88 0.62 3.16 0.15a 9.6 3.1 16.4 1.7
Abbreviations: CTL, control; MG, malachite green; APO, apocynin; EMIQ, enzymatically modified isoquercitrin; BW, body weight; DEN, N-diethylnitrosamine. Data are shown as the mean standard deviation. a p < 0.05 versus CTL (Tukey’s or the Steel-Dwass test). b All animals were subjected to two-thirds partial hepatectomy at week 1 after starting MG promotion.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
4
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Fig. 1. Representative images of altered liver foci in rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by malachite green treatment. Adipophilin is not expressed in phenotypically altered hepatocytes in eosinophilic (A and B), clear cell (C and D), and basophilic cell foci (E and F). Adipophilin is expressed in sinusoidal cells in- or out-side foci and surrounding hepatocytes outside foci. (A) Control group; (B and C) MG group. Bar = 100 um.
treated group. The ratios in the MG, MG + APO, and MG + EMIQ groups were 1.7, 1.4, and 1.5-fold higher than that in the control group, respectively. The area of GST-P-positive foci in the MGtreated group was 1.4-fold higher than the control values, but this was not statistically significant (Fig. 2B). The area in the MG + APOcotreated group was comparable with that in the control group. 3.4. MG increases cell proliferation and apoptosis in preneoplastic liver cell foci, while coadministration of apocynin prevents MG-induced cell proliferation and apoptosis in the foci, and coadministration of EMIQ increases apoptosis in liver parenchymal cells Ki-67 labelling index in GST-P-positive foci was 1.6-fold higher in the MG-treated group than in the control group, but this was not statistically significant (Fig. 2C). The index in the MG + APOcotreated group was comparable with that in the control group. No treatment-related effects on the index in non-GST-P-positive foci were detected in any group (Fig. 2D). The labelling index of active caspase-3 in GST-P-positive foci was increased 3-fold in the MGtreated group when compared with the control group, and this was not statistically significant (Fig. 2E). APO tended to decrease the
index, and the value was 1.7-fold higher than the control value. The index in the MG + EMIQ-cotreated group was significantly (3.7fold) higher than that in the control group. A similar treatmentrelated change was detected in non-GST-P-positive foci, the values in the MG, MG + APO, and MG + EMIQ groups being 2.5-, 1.7-, or 6.8fold higher than those in the control group, respectively (Fig. 2F). The value in the MG + EMIQ-cotreated group was significantly different. 3.5. Coadministration of apocynin or EMIQ prevents MG-induced expression of a NOX subunit p22phox in preneoplastic liver cell foci NOX-related molecules, p22phox, p47phox, and NOX4 were detected in the cytoplasm or cell membrane of hepatocytes. Expression of p22phox in GST-P-positive foci was slightly increased in the MG treated group when compared with that in the control group but this was not statistically significant; the value was significantly lower in the MG + APO and MG + EMIQ cotreated group than the MG-treated group (Figs. 3 and 4). No treatment-related changes were detected with respect to the expression of p22phox in non-GST-P-positive foci in any group. No
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
5
Fig. 2. Quantitative analysis of glutathione S-transferase placental (GST-P)-positive foci and Ki-67-positive cells and active caspase-3-positive cells in GST-P-positive foci and non-GST-P-positive foci in the liver of rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by malachite green (MG) treatment with or without cotreatment with apocynin (APO) or enzymatically modified isoquercetin (EMIQ). The number (A) and area (B) of GST-P-positive foci, and quantitative data (%) of Ki-67-positive cells (C and D) and active caspase-3-positive cells (E and F) in GST-P-positive foci (C and E) and non-GST-P-positive foci (D and F). Columns represent means with standard deviations. *p < 0.05 (Tukey or Steel-Dwass multiple comparison test).
treatment-related changes were detected with respect to the expression of p47phox and NOX4 in GST-P-positive foci in any group (Table 3). A marker of autophagy, the LC3a-positive hepatocyte, was highly expressed in GST-P-positive foci in the MG-treated group, and this was statistically significant. No clear treatment-related effect by APO or EMIQ was detected with respect to LC3a expression in the liver foci. 4. Discussion There are clear parallels between aging and the pathological progression from NAFLD to HCC, as older people generally experience these liver diseases more commonly than people in younger age groups (Sheedfar et al., 2013). Health tends to decline particularly dramatically with aging in the modern world due to
lifestyle factors such as low levels of physical activity and poor nutritional habits, decreases in hormone regulation, and imbalances in the immune system. The steatosis-related pathological processes in humans are similar to those in animal models, where it takes a long time to develop NASH and HCC when fed a HFD (Gauthier et al., 2006; Duval et al., 2010), even with initiation with DEN (de Lima et al., 2008). To effectively observe hyperlipidaemia and liver steatosis followed by precancerous lesions, i.e. GST-Ppositive liver foci preceding HCC in rats, we fed the rats a HFD in a well-established initiation and promotion hepatocarcinogenesis model (Ito et al., 2003). This model can be useful in determining both lipid metabolism and liver promotion activity in rats over a short time, e.g. 8 weeks, assuming that hypothetical acceleration of ageing induces preneoplastic liver lesions through liver steatosis. We recently confirmed that rats fed a HFD had significantly
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
6
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
Fig. 3. Representative images of p22phox-positive cells in GST-P-positive foci in rats treated with MG and MG + EMIQ. p22phox-positive cells (B and D) are observed in GST-Ppositive foci (A and C) in MG group (A and B), but not MG + EMIQ group (C and D). Bar = 50 um.
increased GST-P-positive liver foci together with hyperlipidaemia and liver steatosis compared with control rats fed a basal diet (Murayama et al., unpublished data). As demonstrated by blood biochemistry, we confirmed hyperlipidaemia in rats fed a HFD: the TG level (316 128 mg/dL) was 7.5 times the control value (42 8 mg/dL) (Kojima et al., 2009), and T. CHOL level (90 10 mg/dL) was 2 times the control value (44 2 mg/dL) (Kojima et al., 2009). We also selected a lipogenic substance (MG) to determine the effect of EMIQ and APO on hyperlipidaemia and fatty change in the livers of rats fed a HFD. Feeding rats a diet with MG for 28 days induces hepatocellular fatty change in the liver (Culp et al., 1999). Interestingly, MG reduced the levels of blood lipids (T.CHOL), which were further reduced by EMIQ. With no effects of MG on TG in blood, EMIQ tended to also
reduce TG. Additionally, ALP level (1673 124 U/L) in rats fed a HFD was 2.2 times the control value (770 36 U/L) (Kojima et al., 2009). ALP is known to be increased by a HFD. Serum ALP level is correlated with jejunal ALP activity, which is crucial to intestinal function and controlling inflammation (Lalles et al., 2012). ALP coordinated the treatment-related effects on T.CHOL and TG. Accordingly, the present model is useful for determining the effects on blood lipid levels by the test substances. As we showed in our present study, MG appears to have a weak liver promotion effect with a statistical significant difference detected only in the number of GST-P-positive foci. The effects seemed to be associated with relatively higher activity of cell proliferation and apoptosis as shown in Ki-67 and active caspase3-labelling indices. The data were almost consistent with
Fig. 4. Quantitative analysis of p22phox-positive cells in glutathione S-transferase placental (GST-P)-positive foci in the liver of rats after N-diethylnitrosamine (DEN) initiation or DEN initiation followed by MG treatment with or without co-treatment with APO or EMIQ. Quantitative data (%) of p22phox-positive cells in GST-P-positive foci (A) and non-GST-P-positive foci (B). Columns represent means with standard deviations. *p < 0.05 (Tukey or Steel-Dwass multiple comparison test).
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
previously reported findings in vivo (Fernandes and Rao, 1991; Sundarrajan et al., 2000, 2001; Gupta et al., 2003). The NOX inhibitor APO reduced these effects against GST-P-positive foci, and this effect might be correlated with reduced expression of p22phox in the hepatocytes of GST-P-positive foci. EMIQ also reduced the expression of p22phox in GST-P-positive liver foci, but not significantly. p22phox is a membrane regulatory subunit of NOX1, 2, or 4 (Montezano and Touyz, 2014). NOX1, 2 and 4 served as a prominent source of ROS in hepatocytes, while NOX2 serves the same role in phagocytes (Levin et al., 2012; Choi et al., 2014). NOX4 depends on only p22phox in order to be active, is constitutively activated, and its ROS production is regulated by Poldip2 (Montezano and Touyz, 2014). Although NOX4 expression was not altered, reduced p22phox expression in the precancerous lesion might be related to lower cell proliferation activity or a greater apoptosis response to APO or EMIQ supplementation, respectively. This finding could be in part consistent with evidence that upregulation of NOX4 by TGF-b is required for its proapoptotic activity in hepatocytes, while impairment of the TGFb-induced response confers apoptosis resistance to HCCs (Carmona-Cuenca et al., 2008). GST-P-positive liver foci reduced the expression of adipophilin in phenotypically altered hepatocytes. Adipophilin is a well-known lipid droplet protein involved in lipid droplet homeostasis, and works primarily by stimulating fat packing inside lipid droplets in order to avoid catabolic pathways, which can induce oxidative stress (Grasselli et al., 2010). The results of these studies suggest that a nutritional niche within the precancerous lesion could be regulated separately from the surrounding hepatocytes, which highly express adipophilin together with lipid droplets. Contrary to our expectations, LC3a, a marker of autophagy, was highly expressed in the hepatocytes of precancerous lesion. This expression was enhanced by MG treatment compared with the control group. Therefore, we speculated that a precancerous liver lesion might be resistant to lipid accumulation, resulting in starvation in comparison with the surrounding intact hepatocytes and activating autophagy to control intracellular nutritional levels. This interpretation is supported by the possible bifunctional role of autophagy in cancer development: autophagy protects cells by removing ROS-damaged organelles to maintain normal cellular condition before tumour development, while autophagy promotes tumour cell survival even in low nutrient and oxygen conditions at a later phase (Czaja et al., 2013). In conclusion, APO or EMIQ ameliorated hyperlipidaemia under the current study conditions when combined with the lipogenic substance MG in HFD-fed rats. The effects of both agents on liver steatosis remain unclear. Further studies are required to determine the lipid metabolism pathways taking place in the liver, and in particular time-course changes. These results emphasize that the antioxidant EMIQ and NOX inhibitor APO reduce the expression of the NOX subunit p22phox in preneoplastic liver foci. By taking advantage of animal models, our data suggest that the NOX subunit p22phox is a possible candidate for the chemoprevention of steatosis-related liver cancer. Conflict of interest statement All the authors declare no conflicts of interest. Acknowledgements The authors thank Mrs Shigeko Suzuki for her technical assistance in preparing the histological specimens. This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and
7
Welfare of Japan (H25-Shokuhin-Ippan-005) and the Japan Food Chemical Research Foundation. References Block K, Gorin Y. Aiding and abetting roles of NOX oxidases in cellular transformation. Nat. Rev. Cancer 2012;12:627–37. Carmona-Cuenca I, Roncero C, Sancho P, Caja L, Fausto N, Fernandez M, et al. Upregulation of the NADPH oxidase NOX4 by TGF-beta in hepatocytes is required for its pro-apoptotic activity. J. Hepatol. 2008;49:965–76. Chatterjee S, Ganini D, Tokar EJ, Kumar A, Das S, Corbett J, et al. Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis. J. Hepatol. 2013;58:778–84. Chirino YI, Sanchez-Gonzalez DJ, Martinez-Martinez CM, Cruz C, Pedraza-Chaverri J. Protective effects of apocynin against cisplatin-induced oxidative stress and nephrotoxicity. Toxicology 2008;245:18–23. Choi J, Corder NL, Koduru B, Wang Y. Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma. Free Radic. Biol. Med. 2014;72:267–84. Culp SJ, Blankenship LR, Kusewitt DF, Doerge DR, Mulligan LT, Beland FA. Toxicity and metabolism of malachite green and leucomalachite green during shortterm feeding to Fischer 344 rats and B6C3F1 mice. Chem. Biol. Interact. 1999;122:153–70. Czaja MJ, Ding WX, Donohue Jr. TM, Friedman SL, Kim JS, Komatsu M, et al. Functions of autophagy in normal and diseased liver. Autophagy 2013;9:1131–58. de Lima VM, Oliveira CP, Alves VA, Chammas MC, Oliveira EP, Stefano JT, et al. A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma. J. Hepatol. 2008;49:1055–61. Duval C, Thissen U, Keshtkar S, Accart B, Stienstra R, Boekschoten MV, et al. Adipose tissue dysfunction signals progression of hepatic steatosis towards nonalcoholic steatohepatitis in C57BL/6 mice. Diabetes 2010;59:3181–91. Fernandes C, Rao KV. Enhanced expression of low molecular weight keratins during progressive diethylnitrosamine-induced hepatocarcinogenesis in rats is associated with the presence of high levels of gamma-glutamyl transpeptidase and glycogen-deficient islands. Cancer Lett. 1991;59:211–9. Gabele E, Dostert K, Hofmann C, Wiest R, Scholmerich J, Hellerbrand C, et al. DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH. J. Hepatol. 2011;55:1391–9. Gauthier MS, Favier R, Lavoie JM. Time course of the development of non-alcoholic hepatic steatosis in response to high-fat diet-induced obesity in rats. Br. J. Nutr. 2006;95:273–81. Grasselli E, Voci A, Pesce C, Canesi L, Fugassa E, Gallo G, et al. PAT protein mRNA expression in primary rat hepatocytes: effects of exposure to fatty acids. Int. J. Mol. Med. 2010;25:505–12. Gupta S, Sundarrajan M, Rao KV. Tumor promotion by metanil yellow and malachite green during rat hepatocarcinogenesis is associated with dysregulated expression of cell cycle regulatory proteins. Teratog. Carcinog. Mutagen. 2003;301–12. Gupte RS, Floyd BC, Kozicky M, George S, Ungvari ZI, Neito V, et al. Synergistic activation of glucose-6-phosphate dehydrogenase and NAD(P)H oxidase by Src kinase elevates superoxide in type 2 diabetic, Zucker fa/fa, rat liver. Free Radic. Biol. Med. 2009;47:219–28. Hara S, Morita R, Ogawa T, Segawa R, Takimoto N, Suzuki K, et al. Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model. Exp. Toxicol. Pathol. 2014;66:225– 34. Ibrahim MA, Kelleni M, Geddawy A. Nonalcoholic fatty liver disease: current and potential therapies. Life Sci. 2013;92:114–8. Ito N, Tamano S, Shirai T. A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci. 2003;94:3–8. Kalyanaraman B. Teaching the basics of redox biology to medical and graduate students: oxidants, antioxidants and disease mechanisms. Redox Biol. 2013;1:244–57. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Nonalcoholic Steatohepatitis Clinical Research Network: design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21. Kojima S, Sasaki J, Tomita M, Saka M, Ishizuka K, Kawakatsu H, et al. Multiple organ toxicity, including hypochromic anemia, following repeated dose oral administration of phenobarbital (PB) in rats. J. Toxicol. Sci. 2009;34:527–39. Kuwata K, Shibutani M, Hayashi H, Shimamoto K, Hayashi SM, Suzuki K, et al. Concomitant apoptosis and regeneration of liver cells as a mechanism of livertumor promotion by beta-naphthoflavone involving TNFalpha-signaling due to oxidative cellular stress in rats. Toxicology 2011;283:8–17. Lalles JP, Orozco-Solis R, Bolanos-Jimenez F, de Coppet P, Le Drean G, Segain JP. Perinatal undernutrition alters intestinal alkaline phosphatase and its main transcription factors KLF4 and Cdx1 in adult offspring fed a high-fat diet. J. Nutr. Biochem. 2012;23:1490–7. Levin I, Petrasek J, Szabo G. The presence of p47phox in liver parenchymal cells is a key mediator in the pathogenesis of alcoholic liver steatosis. Alcohol. Clin. Exp. Res. 2012;36:1397–406. Makino T, Shimizu R, Kanemaru M, Suzuki Y, Moriwaki M, Mizukami H. Enzymatically modified isoquercitrin, alpha-oligoglucosyl quercetin 3-O-
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003
G Model ETP 50979 No. of Pages 8
8
T. Yoshida et al. / Experimental and Toxicologic Pathology xxx (2016) xxx–xxx
glucoside, is absorbed more easily than other quercetin glycosides or aglycone after oral administration in rats. Biol. Pharm. Bull. 2009;32:2034–40. Marcolin E, San-Miguel B, Vallejo D, Tieppo J, Marroni N, Gonzalez-Gallego J, et al. Quercetin treatment ameliorates inflammation and fibrosis in mice with nonalcoholic steatohepatitis. J. Nutr. 2012;142:1821–8. Montezano AC, Touyz RM. Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid. Redox Signal. 2014;20:164–82. Morita R, Shimamoto K, Ishii Y, Kuwata K, Ogawa B, Imaoka M, et al. Suppressive effect of enzymatically modified isoquercitrin on phenobarbital-induced liver tumor promotion in rats. Arch. Toxicol. 2011;85:1475–84. Murota K, Matsuda N, Kashino Y, Fujikura Y, Nakamura T, Kato Y, et al. alphaOligoglucosylation of a sugar moiety enhances the bioavailability of quercetin glucosides in humans. Arch. Biochem. Biophys. 2010;501:91–7. Nishimura J, Saegusa Y, Dewa Y, Jin M, Kawai M, Kemmochi S, et al. Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats. Arch. Toxicol. 2010;84:143–53. Pan MH, Lai CS, Tsai ML, Ho CT. Chemoprevention of nonalcoholic fatty liver disease by dietary natural compounds. Mol. Nutr. Food Res. 2014;58:147–71.
Pisonero-Vaquero S, Martinez-Ferreras A, Garcia-Mediavilla MV, Martinez-Florez S, Fernandez A, Benet M, et al. Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT pathway in a diet-induced mouse model of nonalcoholic fatty liver disease. Mol. Nutr. Food Res. 2015;59:879–93. Sheedfar F, Di Biase S, Koonen D, Vinciguerra M. Liver diseases and aging: friends or foes?. Aging Cell 2013;12:950–4. Shimada Y, Dewa Y, Ichimura R, Suzuki T, Mizukami S, Hayashi SM, et al. Antioxidant enzymatically modified isoquercitrin suppresses the development of liver preneoplastic lesions in rats induced by beta-naphthoflavone. Toxicology 2010;268:213–8. Sundarrajan M, Fernandis AZ, Subrahmanyam G, Prabhudesai S, Krishnamurthy SC, Rao KV. Overexpression of G1/S cyclins and PCNA and their relationship to tyrosine phosphorylation and dephosphorylation during tumor promotion by metanil yellow and malachite green. Toxicol. Lett. 2000;116:119–30. Sundarrajan M, Prabhudesai S, Krishnamurthy SC, Rao KV. Effect of metanil yellow and malachite green on DNA synthesis in N-nitrosodiethylamine induced preneoplastic rat livers. Indian J. Exp. Biol. 2001;39:845–52. Younossi ZM. Review article: current management of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Aliment. Pharmacol. Ther. 2008;28:2–12.
Please cite this article in press as: T. Yoshida, et al., Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j. etp.2016.10.003