Protective effects of morin against acrylamide-induced hepatotoxicity and nephrotoxicity: A multi-biomarker approach

Journal Pre-proof Protective effects of morin against acrylamide-induced hepatotoxicity and nephrotoxicity: A multi-biomarker approach Fatih Mehmet Kandemir, Serkan Yıldırım, Sefa Kucukler, Cuneyt Caglayan, Ekrem Darendelioğlu, Muhammet Bahaeddin Dortbudak PII:

S0278-6915(20)30078-8

DOI:

https://doi.org/10.1016/j.fct.2020.111190

Reference:

FCT 111190

To appear in:

Food and Chemical Toxicology

Received Date: 1 December 2019 Revised Date:

7 February 2020

Accepted Date: 11 February 2020

Please cite this article as: Kandemir, F.M., Yıldırım, S., Kucukler, S., Caglayan, C., Darendelioğlu, E., Dortbudak, M.B., Protective effects of morin against acrylamide-induced hepatotoxicity and nephrotoxicity: A multi-biomarker approach, Food and Chemical Toxicology (2020), doi: https:// doi.org/10.1016/j.fct.2020.111190. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author contribution The Author contribution as follows was accepted by all authors: Fatih Mehmet Kandemir: investigation, data curation, software, formal analysis, supervision, project administration. Serkan Yıldırım: methodology, investigation, conceptualization, formal analysis. Sefa Kucukler: investigation, methodology, validation. Cuneyt Caglayan: conceptualization, investigation, visualization, writing - review & editing, supervision. Ekrem Darendelioğlu: methodology, investigation, data curation, software, formal analysis. Muhammet Bahaeddin Dortbudak: methodology, investigation.

Protective effects of morin against acrylamide-induced hepatotoxicity and nephrotoxicity: A Multi-Biomarker Approach

Fatih Mehmet Kandemira, Serkan Yıldırımb, Sefa Kucuklera, Cuneyt Caglayanc*, Ekrem Darendelioğlud, Muhammet Bahaeddin Dortbudakb

a

Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey b

Department of Pathology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey

c

Department of Biochemistry, Faculty of Veterinary Medicine, Bingol University, Bingol, Turkey

d

Department of Molecular Biology and Genetics, Faculty of Science and Literature, Bingol University, Bingol, Turkey

*Corresponding author: Assist Prof. Cuneyt Caglayan Address: Department of Biochemistry, Faculty of Veterinary Medicine, Bingol University, 12000-Bingol, TURKEY Phone : +90 4262160027 Fax : +90 4262160036 E-mail: [email protected]

Abstract Acrylamide (ACR) is a heat-induced carcinogen substance that is found in some foods due to cooking or other thermal processes. The aim of present study was to assess the probable protective effects of morin against ACR-induced hepatorenal toxicity in rats. The rats were treated with ACR (38.27 mg/kg b.w., p.o.) alone or with morin (50 and 100 mg/kg b.w., p.o.) for 10 consecutive days. Morin treatment attenuated the ACR-induced hepatorenal tissue injury by diminishing the serum AST, ALP, ALT, urea and creatinine levels. Morin increased activities of SOD, CAT and GPx and levels of GSH, and suppressed lipid peroxidation in ACR induced tissues. Histopathological changes and immunohistochemical expressions of p53, EGFR, nephrin and AQP2 in the ACR-induced liver and kidney tissues were decreased after administration of morin. In addition, morin reversed the changes in levels of apoptotic, autophagic and inflammatory parameters such as caspase-3, bax, bcl-2, cytochrome c, beclin1, LC3A, LC3B, p38α MAPK, NF-κB, IL-1β, IL-6, TNF-α and COX-2 in the ACR-induced toxicity. Morin also affected the protein levels by regulating the PI3K/Akt/mTOR signaling pathway and thus alleviated ACR-induced apoptosis and autophagy. Overall, these findings may shed some lights on new approaches for the treatment of ACR-induced hepatotoxicity and nephrotoxicity. Keywords: Acrylamide, Apoptosis, Hepatotoxicity, Morin, Nephrotoxicity, Oxidative stress

Graphical abstract

1. INTRODUCTION Acrylamide (ACR) is a water-soluble compound that is widely used in many fields, such as dye synthesis, soil coagulation, treatment of wastewater, paper packaging, and laboratory purposes (Elblehi et al., 2020; Mehri et al., 2012). ACR does not occur naturally. It was reported that carbohydrate-rich foods heated or fried at high temperatures contain relatively high levels of ACR (Zhang et al., 2012). ACR is formed during frying, grilling, roasting or baking carbohydrate-rich foods such as crackers, potato crisps, cereals, bread, and french fries at temperatures above 120 °C through interactions of amino acids (e.g. asparagine) with reducing sugar (e.g. glucose) (Uthra et al., 2017; Zamani et al., 2018). ACR has been reported to cause genotoxic, neurotoxic, hepatotoxic, nephrotoxic effects along with carcinogenic, developmental and reproductive toxicities in rodents (Chen et al., 2016; Ghorbel et al., 2017; Sun et al., 2018; Zhang et al., 2013b). The International Agency for Research on Cancer (IARC) classified ACR as 2A, a probable human carcinogen (Cao et al., 2008). Glycidamide, a metabolite of ACR, attaches to DNA and leads to damage in genes. Effect of ACR on deterioration of oxidative status and enzyme activities and production of inflammatory cytokines were also reported (Alturfan et al., 2012; Pan et al., 2018) revealing detrimental effect of ACR to human health that cannot be neglected. Nowadays, the use of phytochemicals is common for overcoming hepatotoxicity and nephrotoxicity. Flavonoids are natural polyphenolic phytochemicals, with their universal presence in almost all fruits, vegetables, dietary plants, nuts and seeds (Ahmad et al., 2012; Benzer et al., 2018; Mangwani et al., 2019). Among them, morin (2′,3,4′,5,7pentahydroxyflavone), a flavonoid isolated from Maclura tinctoria, Maclura pomifera and from leaves of Psidium guajava, has a wide range of pharmacological properties including, anti-inflammatory, antioxidant, anti-autophagy and anti-apoptosis (Kuzu et al., 2018; Singh et al., 2018). The most important advantage of morin as antioxidant is its minimal toxicity even

1

at higher dose usage (KV et al., 2016). The anti-oxidant, anti-inflammatory and anti-apoptotic role of morin on doxorubicin-induced hepatotoxicity and nephrotoxicity in rats were revealed in our previous study (Kuzu et al., 2019). The main objective of this study was to assess the protective effects of morin against ACR-induced oxidative stress, inflammatory, apoptotic and autophagic responses in the rat liver and kidney tissues. 2. MATERIAL AND METHODS 2.1. Chemicals and reagents Acrylamide (≥99%), morin hydrate (CAS Number: 654055-01-3) and all other reagents were supplied by Sigma Chemical Co. (St. Louis, MO). Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) assay kits were obtained from TML, Diagnostic Medical Products, (Ankara, Turkey). Serum urea and creatinine assay kits were purchased from Diasis Diagnostic Systems, (İstanbul, Turkey). Antibodies for immunohistochemical staining and western blot analysis were supplied by Abcam (United Kingdom) and Santa Cruz Biotechnology (USA), respectively. Tumor necrosis factor-alpha (TNF-α), p38α mitogen-activated protein kinase (p38α MAPK), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), cyclooxygenase-2 (COX-2), bcl-2 associated X protein (Bax), b-cell lymphoma-2 (Bcl-2), p53, beclin-1, light chain 3B (LC3A), light chain 3B (LC3B), protein kinase B (Akt), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) enzymelinked immunosorbent assay (ELISA) kits were purchased from YL Biont, (Shangai, China). Cysteine aspartate specific protease-3 (caspase-3) assay kit was obtained from Sunred Biological Technology (Shanghai, China). 2.2. Animals and experimental design

2

Thirty five adult male Sprague Dawley rats (approximately 2-month old and weighing 250 ± 20 g) were obtained from the Experimental Research and Application Center, Ataturk University (Erzurum, Turkey). The rats were housed in a clean well-ventilated room at an adjusted temperature (24 ± 1 °C), humidity (45 ± 5%) and 12 h light/dark cycle. During the study, commercial pellet diet and water were given ad libitum. The experimental protocol was approved by the Ataturk University Ethical Committee for Animal Experiments. (Approval no: 2017-13/161). The animals were randomly divided into 5 groups consisting of 7 rats each and all chemicals were applied with oral gavage for 10 days. The toxic dose of ACR (1/3rd of LD50) has been determined by Uthra et al. (2017). And it has sufficient to elicit mild or moderate oxidative stress in rats. The doses of morin to be administered to rats was determined according to the study by Kuzu et al. (2018). Group I (Control): Rats received oral saline for 10 consecutive days, to serve as the control. Group II (ACR): Rats received ACR (38.27 mg/kg b.w./day orally) for 10 days. Group III (Morin): Rats received morin (100 mg/kg b.w./day orally) for 10 days. Group IV (ACR + Morin 50): ACR (38.27 mg/kg b.w.) and morin (50 mg/kg b.w.) administered orally for 10 days. Group V (ACR + Morin 100): ACR (38.27 mg/kg b.w.) and morin (100 mg/kg b.w.) administered orally for 10 days. At the end of the study period, the rats were anesthetized with mild sevoflurane and blood samples were withdrawn. The blood samples allowed to coagulate and then centrifuged at 3000 rpm for 10 min. Serum was maintained at -20 °C until biochemical parameters such as AST, ALP, ALT, urea, and creatinine were evaluated. The liver and kidney tissues were dissected and used for biochemical, molecular, histopathological and immunohistochemical examinations.

3

2.3. Liver and kidney function tests Serum activities of ALT, AST and ALP were measured as indicators of hepatic injury using standard diagnostic kits. Also, the levels of serum creatinine and urea were determined in an ELISA reader (Bio-Tek, Winooski, VT, USA) with commercial kits according to the manufacturer’s instructions. 2.4. Oxidative stress markers in the liver and kidney tissues Liver and kidney tissues for biochemical analyses were homogenized in the appropriate buffer on ice by homogenizer (Tissue Lyser II, Qiagen, Netherlands) after grinding in liquid nitrogen for each method separately. For malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) assays, homogenates were centrifuged at 3500 rpm for 15 min at 4°C. To assay glutathione (GSH) level and glutathione peroxidase (GPx) activity, homogenates were centrifuged for 20 min at 10000 rpm at +4 °C and the supernatant was used for assaying of the biochemical analyses. The activities of SOD, CAT, and GPx enzymes using the methods of Sun et al. (1988), Aebi (1984) and Lawrence and Burk (1976), respectively. The MDA levels (indicator of lipid peroxidation) were assayed colorimetrically according to the methods of Placer et al. (1966). GSH level was assessed by method of Sedlak and Lindsay (1968). The protein concentration in liver and kidney homogenates were determined according to the method of Lowry et al. (1951). 2.5. Inflammation markers in the liver and kidney tissues In this part, commercial rat ELISA kits have been used to measure inflammatory cytokines in the kidney and liver tissues. The IL-1β, IL-6, TNF-α, and NF-κB levels and p38α MAPK and COX-2 activities have been measured by using ELISA kit. Tissue homogenization for all rat ELISA kits was performed according to our previous study (Caglayan et al., 2019b).

4

2.6. Apoptotic and autophagic markers in the liver and kidney tissues Caspase-3, p53, Bax, Bcl-2, beclin-1, LC3A and LC3B levels in the liver and kidney homogenates were measured by a rat ELISA kit following the manufacturer’s instructions. Analyses have been carried out by ELISA Plate Reader (Bio-Tek, Winooski, VT, USA) according to the standard manufacturer’s instructions. Absorbance was read at 450 nm. 2.7. Determination of mTOR, PI3K and Akt in the liver and kidney tissues The mTOR, PI3K and Akt activities in the liver and kidney homogenates were measured by a rat ELISA kit following the manufacturer's recommendations. 2.8. Western blotting analysis for apoptosis The experimentally treated rat tissues were homogenized and western blotting analysis was performed as given in our previous study (Caglayan et al., 2019a). Protein samples were prepared in Laemmli buffer (Tris-HCl pH 6.8, glycerol, bromophenol blue, sodium dodecyl sulfate, 2-mercaptoethanol). Following that, the same volumes of protein samples were run onto 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to nitrocellulose membranes. The membranes were washed 2 times in TBS-0.05% Tween-20 (TBS-T) for 5 min and blocked for 1 h before use of a primary antibody in 5% Bovine Serum Albumin. β-Actin (sc-47778), cytochrome c (sc-13156), procaspase-3 (sc-271759), Bcl-2 (sc-7382) and Bax (sc-20067) was used as primary antibodies. Following that, nitrocellulose membranes were left in the presence of primary antibodies at 4 °C for overnight. The blots were then washed in TBS-T for 5 times for 5 min and incubated in the presence of anti-mouse secondary antibody, at 37 °C for 90 min. Quantification of density of protein band in X-ray films from ECL (Advansta, CA) was developed to identify specific binding and were analyzed densitometrically with a photo analysis system (GelDoc, Bio-Rad, USA).

5

2.9. Histopathological Examination Tissues obtained from necropsied rats were placed into 10% neutral buffered formalin solution for the histopathological application. A routine tissue follow-up procedure was applied to the fixed tissues for 48 hours. After the follow-up, the tissues were formed into paraffin blocks. The paraffin blocks were cut into 4 µm thick sections and placed on slides. Hematoxylin-Eosin (H&E) stain was applied, and the tissue sections were examined under a light microscope (LM). Histological findings of these slides were graded according to their severity as none (-), mild (+), moderate (++), and severe (+++). 2.10. Immunohistochemical Examination Tissue sections were cut from paraffin blocks and placed on positively charged slides for immunohistochemical staining. These tissues were kept in the oven for 1 h and then immersed in xylol-alcohol series for deparaffinization and dehydration. After these processes, the tissue sections were washed with phosphate buffer solution (PBS) and treated in a 3% hydrogen peroxide (H2O2) solution for 10 min. Subsequently, to clarify the antigens in tissues with endogenous peroxidase inactivation, microwave treatment was carried out twice for 5 min at 500 watts in antigen retrieval solution. After cooling the tissues at room temperature and washing with PBS, the protein block was applied to prevent nonspecific antibody binding. After removal of protein block from tissues, tissue was incubated with anti-nephrin (ab136894), anti-aquaporin-2 (ab110418), anti-p53 (ab131442), and anti-EGFR (ab52894) antibodies. This was followed by washing with PBS and secondary antibody treatment. After washing again with PBS, 3-3’ Diaminobenzidine (DAB) chromogen was applied to the tissues. After a sufficient amount of time, the tissues were washed for the last time with PBS and were passed through tap water, then counterstained with Mayer's hematoxylin. Tissues, subsequently, were graded according to their immune responses as none (-), mild (+), moderate (++), and severe (+++). 6

2.11. Statistical Analysis The biochemical analysis was compared by one-way analysis of variance (ANOVA). Tukey’s multiple comparison test were performed. P < 0.05 were considered statistically significant. The results are represented as mean ± standard error. Western Blot Analysis was repeated at least three times. Statistical analysis and comparable data groups were assessed using GraphPad Prism 5 by one-way ANOVA Newman-Keuls Post-Hoc Test; p < 0.05 was considered as significant. In the histopathological examination, for the analysis of the differences between the groups in the semiquantitatively obtained data, the Kruskal-Wallis test was used for nonparametric tests and the Mann Whitney U test was used for comparison of the two groups. 3. RESULTS 3.1. Effect of morin on ACR-induced serum hepatic markers As presented in Table 1, administration of ACR caused a remarkable (p < 0.05) rise in serum activities of ALT, ALP and AST in comparison to control group. However, co-administration of morin with ACR significantly (p < 0.05) reduced the above-mentioned parameters compared to ACR group. Only morin treated group showed no significant difference compared to the control group. 3.2. Effect of morin on ACR-induced serum renal markers Oral administration of ACR for 10 days resulted in a significant increase (p < 0.05) in serum urea and creatinine levels as compared to the control group. Morin effectively (p < 0.05) reduced the serum levels of urea and creatinine in ACR + M 100 group comparison to the only ACR group (Table 2). 3.3. Effect of morin on ACR-induced liver and kidney oxidative stress and antioxidant status

7

The present study revealed that ACR-induced oxidative stress in the liver and kidney as indicated by a significant (p < 0.05) increase in MDA levels. In the meantime, activities of SOD, GPx, and CAT and level of GSH were found to decrease as compared to the control group. In contrast, co-administration of morin with ACR significantly decreased MDA levels and increased the activities of SOD, CAT and GPx, and levels of GSH compared to only ACR-treated group (p < 0.05) (Table 1 and 2). 3.4. Anti-inflammatory effect of morin on ACR-induced inflammation In the present study, ACR induced a series of inflammatory changes that mediated liver and kidney injury. The levels of NF-κB, TNF-α, IL-1β, IL-6, and activities of COX-2 were significantly higher in ACR group compared to control group. However, morin treatment (50 and 100 mg/kg) significantly (p < 0.05) inhibited the overproduction of these inflammatory biomarkers as compared to ACR-induced group (Fig 1A-F). The p38α MAPK activities were significantly (p < 0.05) increased in the ACR-treated group when compared to the control group. However, there was a significant decrease in the activities of p38α MAPK in the morin treated group in dose-dependent manner as compared to ACR group. 3.5. Anti-apoptotic effect of morin on ACR-induced apoptosis To study the detailed biomolecular mechanisms of the anti-apoptotic effects of the morin on ACR-induced apoptosis in kidney and liver tissues (Fig. 3 and 4), the protein expressions of pro-apoptotic Bax, cytochrome c and anti-apoptotic Bcl-2 and procaspase-3 were investigated. The ratio Bax/Bcl-2 was also analysed as it is more important in defining apoptosis (Reed, 1997). It was determined that the Bax/Bcl-2 ratio was considerably increased by ACR and decreased by morin in both tissues (Fig. 3B-III and 4B-III). The level of cytochrome c remarkably increased in the ACR treated groups and considerable reduction was seen in expression level of cytochrome c in the ACR + M 50 and ACR + M 100 groups

8

(Fig. 2B-IV and 3B-IV). Moreover, the levels of procaspase-3 were meaningfully decreased in ACR treated group and increased in the ACR + M 100 group (Fig. 3B-V and 4B-V). An increase in procaspase-3 level proposes a reduction in level of active caspase-3. These results further suggest that morin could have an anti-apoptotic role in rats treated with ACR. Consistent with the results obtained by the Western blotting method, the ELISA results revealed that the levels of p53, caspase-3, Bax, and Bcl-2 were markedly increased in the liver and kidney of the ACR-treated rats. However, treatment with morin decreased in the levels of these apoptotic markers (Fig. 2A-D). 3.6. Morin regulated the PI3K/Akt/mTOR signaling pathways in the liver and kidney As summarized in Fig. 5A-C, levels of PI3K, Akt and mTOR were decreased after ACR administration. Administration of both of morin with ACR significantly (p < 0.05) increased in the these parameters compared to only ACR group. 3.7. Anti-autophagic effect of morin on ACR-induced autophagy Liver and kidney autophagic protein levels measured using the ELISA method is shown in Fig 6A-C. There were significant (p < 0.05) increases in the beclin-1, LC3A and LC3B levels in the rats that received ACR alone. On the other hand, administration of morin (50 and 100 mg/kg) significantly decreased the levels of these autophagic parameters as compared to ACR alone treated rats. 3.8. Histopathological findings of liver and kidney tissues Histopathological examination of the liver tissues in the control and morin groups revealed that the parenchymal tissue and serosa were of normal histological structure. Hepatocytes in the ACR group liver tissue showed severe hydropic degeneration and coagulation necrosis; vessels showed severe hyperemia. There was a statistically significant difference when compared with the control group (p<0.05). Hepatocytes in the ACR+M 50 group, especially in the central region of the liver, showed moderate hydropic degeneration

9

and necrosis; vessels showed hyperemia. Hepatocytes in the acinar region of ACR+M 100 group liver tissues showed mild degeneration. There was a statistically significant difference when compared with the ACR group (p<0.05) (Fig. 7 and Table 3). The kidney tissues in the control and morin groups revealed showed normal histological appearance. Renal tubular epithelium of ACR kidney group showed severe hydropic degeneration and coagulation necrosis; interstitial vessels showed severe hyperemia. There was a statistically significant difference when compared with the control group (p<0.05). Renal tubular epithelium of ACR+M 50 kidney group showed moderate hydropic degeneration and coagulation necrosis; interstitial vessels showed severe hyperemia. Renal tubular epithelium of ACR+M 100 kidney group showed mild degeneration but no necrosis; the interstitial and glomerular vessels were of normal histological appearance. There was a statistically significant difference when compared with the ACR group (p<0.05) (Fig. 8 and Table 4). 3.9. Immunohistochemical findings of the liver and kidney tissues It was detected that p53 and EGFR expression was negative in control and morin group liver hepatocytes. Severe levels of cytoplasmic p53 and EGFR expressions were observed in hepatocytes in the ACR group, especially in the liver acinar region. There was a statistically significant difference when compared with the control group (p<0.05). Moderate cytoplasmic p53 and EGFR expressions were detected in the ACR+M 50 group liver acinar hepatocytes. Mild cytoplasmic p53 and EGFR expressions were detected in the ACR+M 100 group liver hepatocytes. There was a statistically significant difference when compared with the ACR group (p<0.05) (Fig. 7 and Table 3). Severe levels of aquaporin-2 and nephrin expressions were detected in the control and morin group renal tubular epithelium. Aquaporin-2 and nephrin expressions were detected in in tubular epithelium of ACR group kidney renal cortex and medulla. There was a statistically

10

significant difference when compared with the control group (p<0.05). Moderate levels of aquaporin-2 and nephrin expressions were detected in ACR+M 50 group renal tubular epithelium. Severe levels of aquaporin-2 and nephrin expressions were detected in ACR+M 100 group renal tubular epithelium. There was a statistically significant difference when compared with the ACR group (p<0.05) (Fig. 8 and Table 4).

4. DISCUSSION ACR forms when protein and carbohydrate rich foods are cooked above 120 °C. ACR is an immensely toxic molecule that rapidly dissolves in water and can easily spread to tissues after the intake (Besaratinia and Pfeifer, 2007). Once ACR is absorbed in the digestive system, 1 mol of hemoglobin is transferred to the liver for each 4 mol of ACR, which is then metabolized and disrupted in the body in two different pathways (Gedik et al., 2017). In the first pathway ACR is converted into a mutagenic and genotoxic active substance, glycidamide, in the liver by the CYP2E1 enzyme system (Watzek et al., 2012). The second pathway is conjugation of ACR with reduced GSH by GST, an enzyme that catalyzes the conversion of ACR into non-toxic N-Acetyl-S- (2-carbamoylethyl) cysteine (Gedik et al., 2017; Sumner et al., 1999). ACR has been reported to form free radicals, disrupt antioxidant status and ultimately lead to carcinogenesis and oxidative stress (Uthra et al., 2017). Also, it reacts with the biomolecules (mainly hemoglobin) via interaction between its vinyl, NH2 and SH moieties (Abdel-Daim et al., 2014; Adams et al., 2010). An imbalance between antioxidant capacity and generation of reactive oxygen species (ROS) rises oxidative injury that has a key role in ACR-induced toxicity (Ghorbel et al., 2017). The harmful effects of ACR on liver and kidney tissues have been studied in rodent models (Alturfan et al., 2012; Ghorbel et al., 2017; Rizk et al., 2018; Uthra et al., 2017). As shown in Table 1 and Table 2, ACR significantly increased the lipid peroxidation, reduced the antioxidant enzyme activities (GPx, SOD, and CAT) and level of GSH in both tissues. Morin is a pentahydroxyflavone, 11

hydroxyl group at the C‐3°‐5 and at C‐4 of which assists to quench free radicals produced and is thought to contribute to antioxidant activity (Bachewal et al., 2018; Çelik et al., 2020). In the present study, morin provided protection against ACR-induced toxicities by significantly decreasing lipid peroxidation product (MDA) level and significantly increasing the antioxidant enzyme activities. Serum ALT, AST and ALP activities are used in the diagnosis of specific liver diseases, while serum urea and creatinine levels are considered as markers of kidney function and renal structural integrity (Eldutar et al., 2017; Turk et al., 2019). ACR orally administered to rats caused a significant increase in serum ALP, AST, ALT activities along with urea, and creatinine levels. The finding of this study is correlated with the findings of previous studies (Gedik et al., 2017; Ghorbel et al., 2017). In addition, our histopathological examinations supported the biochemical effects of ACR on both tissues. ACR administration caused degeneration, necrosis, and hyperemia in interstitial vessels in the tissues. The key histopathological finding of our study was that morin treatment contributed to the recovery of liver and kidney architecture induced by ACR. The transfer of water through the cell plasma membrane is mediated by aquaporins (AQPs) (Jaffuel et al., 2013). To date, 13 AQP isoforms, named AQP0 to AQP12, were identified in mammals (Kandemir et al., 2018). Among them, AQP2 is situated in the kidney assembling duct, spread in the cell plasma membrane and cellular vesicles (Chang et al., 2017). Dysregulation of AQP2 is considerably linked with several clinical circumstances including nephrotic syndrome, acute and chronic renal failure, hereditary nephrogenic diabetes insipidus, electrolytes disturbance, ureteral obstruction, and congestive heart failure (Kwon et al., 2013). On the other hand, nephrin is an immunoglobulin‐like adhesion molecule and is responsible for ultrafiltration in kidney tissue (Li et al., 2011; Saito et al., 2010). Previous studies have shown that oxidative damage is closely associated with a decrease in

12

glomerular nephrine expression (Arozal et al., 2010; Shibata et al., 2007). In our study, AQP2 and nephrin expressions were markedly reduced in ACR group with significant changes in kidney immunohistochemical staining. In contrast, morin increased AQP-2 and nephrin expressions reduced by ACR in a dose-dependent manner. Inflammation is caused by an imbalance between anti-inflammatory and proinflammatory cytokines and can be mediated by related proteins (He et al., 2017). Several transcription factors that include NF-κB and p53, have been reported to induce inflammation (Acaroz et al., 2018). These transcription factors could be upregulated by p38 MAPK as p38 MAPK affect NF-κB levels by promoting phosphorylation of IκB, resulting in the degradation and dissociation of NF-κB and IκB complexes (Yang et al., 2016). In this regard, activation of NF-κB stimulates inflammation and immune response in response to proinflammatory cytokine genes such as TNF-α, IL-1β, IL-6, and COX-2 (Caglayan et al., 2018; Kandemir et al., 2017; Temel et al., 2019). Administration of ACR to experimental animals at different doses, such as 40 mg/kg (Alturfan et al., 2012) and 20 mg/kg (Abdel-Daim et al., 2014) has been reported to significantly increase levels of inflammatory cytokine including TNF-α IL1β and IL-6. To investigate the effects of morin on ACR-induced hepatorenal inflammation, we evaluated COX-2, IL-1β, IL-6 TNF-α NF-κB, and p38α MAPK levels by rat ELISA kits. In the present study, morin treatment inhibited the liver and kidney inflammation induced by ACR by decreasing the levels of inflammation-related parameters. Therefore, we speculate that the protective effects of morin on liver and kidney injury may be partially attributable to the inhibition of inflammation. Reactive oxygen species is primarily produced in the mitochondrion and has a crucial role in apoptosis (Chen et al., 2013; Zhao et al., 2015). Apoptotic pathways include Bcl-2 and the p53 protein family. Antitumor protein p53 impacts the apoptotic pathway and could change the levels of the Bcl-2 protein family (Yang et al., 2016). One of the vital apoptotic

13

pathways is the mitochondrial (intrinsic) pathway that is characterized by a rise in cytochrome c levels in the cytosol owing to the diminished Bax/Bcl-2 ratio. The elevated level of cytochrome c stimulates apoptosis, leading to activation of caspase-3 protein. The expression of active caspase-3 increases and consequently induces apoptosis through the intrinsic pathway (Darendelioglu et al., 2016). ACR-induced toxicity is linked with oxidative stress and long-time exposure to ACR induced mitochondrial decline and lastly resulted in apoptosis (Zhao et al., 2015). ACR was also reported to induce apoptotic pathways by increased the expression ratio of Bax/Bcl-2, the release of cytochrome c, and the activation of caspase 9, while it decreased the mitochondrial membrane potential MMP ratio in human astrocytoma cells (Chen et al., 2013). In this study ELISA and immunoblotting results confirmed that ACR-induced kidney and liver cells death via the increase activation of p53, Bax, cytochrome c and caspase-3, and decreased the expression of Bcl-2 protein level. However, morin exposured with ACR defends the tissues by keeping the p53, cytochrome c, caspase-3, Bax and Bcl-2 protein expressions like the control group. To learn more about the likelihood machineries participated in the hepatotoxicity and nephrotoxicity of ACR and the protective effects of morin, we focused on PI3K/Akt/mTOR signaling pathways. PI3K induces a signaling cascade generating phosphatidylinositol triphosphate and provoking the activation of Akt (Sun et al., 2011). Phosphorylated Akt mediates the activation of target genes, thereby regulating cell survival and proliferation (Wu et al., 2016). The mTOR is an atypical serine/threonine-protein kinase and is positively regulated by Akt activation (Mohamed et al., 2018). Inhibition of PI3K/Akt/mTOR signaling pathway causes cell autophagy and/or apoptosis (Zhang et al., 2013a). Several studies have shown that PI3K/Akt/mTOR pathways are closely related to various liver diseases (Matsuda et al., 2013; Wang et al., 2013). On the other hand, the role of the PI3K/Akt pathway in kidney diseases is controversial. Some researchers have reported that PI3K/Akt activation is

14

harmful to the kidney tissue (Das et al., 2008), whereas some of the other groups reported protective role of PI3K/Akt activation (Ma et al., 2017). Relationship between ACR administration and PI3K/Akt/mTOR signaling pathway has not been studied until date. In our study, ACR decreased the levels of PI3K, Akt, and mTOR. However, morin treatment (50 and 100 mg/kg) significantly increased the activities of these enzymes compared to only ACR group. Autophagy is the main intracellular degradative process that provides the lysosomal degradation of unnecessary or damaged organelles and misfolded proteins (Choi, 2012). Beclin 1, LC3A and LC3B are three key proteins that are commonly used to study autophagy (Giatromanolaki et al., 2014). Beclin-1 is a scaffold protein for the formation of the PI3K complex (Cao and Klionsky, 2007). This protein complex is among the first components recruited by the developing autophagosome and is essential for autophagy (Giatromanolaki et al., 2018). LC3A and LC3B have been reported to be abundantly expressed in the brain, liver, heart, testis and skeletal muscle (Huang et al., 2010; Zois et al., 2011). LC3B is the most widely used autophagic marker. Recent reports have also demonstrated that LC3B contributes to one type of autophagosome induced under stress, which is different from LC3A positive autophagosome (Nassar et al., 2017). However, the effect of ACR on autophagic cell death in both tissues remains unknown. In a previous study, lipoic acid treatment has been shown to reduce the expression of autophagy-related proteins such as beclin-1 and LC3 in ACRinduced neurotoxicity (Song et al., 2017). Our results demonstrated that morin played an important anti-autophagic role on ACR-induced liver and kidney toxicities. 5. CONCLUSION In conclusion, this study demonstrated that morin attenuates ACR-induced liver and kidney damage. In this context, the protective effect of morin against ACR-induced liver and kidney toxicity could be partially mediated through its antioxidant, anti-inflammatory, anti-apoptosis 15

and anti-autophagic action. However, further studies are required to clarify the exact mechanism at the molecular level and to propose clinical practice. Funding This study was supported by Ataturk University, Foundation of Scientific Researches Projects (Project number: TSA-2018-6490). Conflict of interest No potential conflict of interest was reported by the authors.

REFERENCES Abdel-Daim, M.M., Abd Eldaim, M.A., Hassan, A.G., 2014. Trigonella foenum-graecum ameliorates acrylamide-induced toxicity in rats: Roles of oxidative stress, proinflammatory cytokines, and DNA damage. BCB 93, 192-198. Acaroz, U., Ince, S., Arslan-Acaroz, D., Gurler, Z., Kucukkurt, I., Demirel, H.H., Arslan, H.O., Varol, N., Zhu, K., 2018. The ameliorative effects of boron against acrylamideinduced oxidative stress, inflammatory response, and metabolic changes in rats. Food Chem. Toxicol. 118, 745-752. Adams, A., Hamdani, S., Van Lancker, F., Méjri, S., De Kimpe, N., 2010. Stability of acrylamide in model systems and its reactivity with selected nucleophiles. Food Res. Int. 43, 1517-1522. Aebi, H., 1984. [13] Catalase in vitro, Methods Enzymol. Elsevier, pp. 121-126. Ahmad, S.T., Arjumand, W., Nafees, S., Seth, A., Ali, N., Rashid, S., Sultana, S., 2012. Hesperidin alleviates acetaminophen induced toxicity in Wistar rats by abrogation of oxidative stress, apoptosis and inflammation. Toxicol. Lett. 208, 149-161. Alturfan, A.A., Tozan-Beceren, A., Şehirli, A.Ö., Demiralp, E., Şener, G., Omurtag, G.Z., 2012. Resveratrol ameliorates oxidative DNA damage and protects against acrylamideinduced oxidative stress in rats. Mol. Biol. Rep. 39, 4589-4596. Arozal, W., Watanabe, K., Veeraveedu, P.T., Ma, M., Thandavarayan, R.A., Sukumaran, V., Suzuki, K., Kodama, M., Aizawa, Y., 2010. Protective effect of carvedilol on daunorubicin-induced cardiotoxicity and nephrotoxicity in rats. Toxicology 274, 18-26. Bachewal, P., Gundu, C., Yerra, V.G., Kalvala, A.K., Areti, A., Kumar, A., 2018. Morin exerts neuroprotection via attenuation of ROS induced oxidative damage and neuroinflammation in experimental diabetic neuropathy. BioFactors 44, 109-122. Benzer, F., Kandemir, F.M., Ozkaraca, M., Kucukler, S., Caglayan, C., 2018. Curcumin ameliorates doxorubicin‐induced cardiotoxicity by abrogation of inflammation, apoptosis, oxidative DNA damage, and protein oxidation in rats. J. Biochem. Mol. Toxicol. 32, e22030. 16

Besaratinia, A., Pfeifer, G.P., 2007. A review of mechanisms of acrylamide carcinogenicity. Carcinogenesis 28, 519-528. Caglayan, C., Kandemir, F.M., Darendelioğlu, E., Yıldırım, S., Kucukler, S., Dortbudak, M.B., 2019a. Rutin ameliorates mercuric chloride-induced hepatotoxicity in rats via interfering with oxidative stress, inflammation and apoptosis. J. Trace Elem. Med. Biol. 56, 60-68. Caglayan, C., Kandemir, F.M., Yildirim, S., Kucukler, S., Eser, G., 2019b. Rutin protects mercuric chloride‐induced nephrotoxicity via targeting of aquaporin 1 level, oxidative stress, apoptosis and inflammation in rats. J. Trace Elem. Med. Biol. 54, 69-78. Caglayan, C., Temel, Y., Kandemir, F.M., Yildirim, S., Kucukler, S., 2018. Naringin protects against cyclophosphamide-induced hepatotoxicity and nephrotoxicity through modulation of oxidative stress, inflammation, apoptosis, autophagy, and DNA damage. Environmental Science and Pollution Research 25, 20968-20984. Cao, J., Liu, Y., Jia, L., Jiang, L.-P., Geng, C.-Y., Yao, X.-F., Kong, Y., Jiang, B.-N., Zhong, L.-F., 2008. Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. J. Agric. Food Chem. 56, 12059-12063. Cao, Y., Klionsky, D.J., 2007. Physiological functions of Atg6/Beclin 1: a unique autophagyrelated protein. Cell Res. 17, 839. Chang, Z., Zhang, H., Wu, X., Nabi, F., Rehman, M., Yuan, X., Mehmood, K., Zhou, D., 2017. Renal dose dopamine mediates the level of aquaporin-2 water channel (Aqp2) in broiler chickens. Brazilian Journal of Poultry Science 19, 387-392. Chen, J.-H., Yang, C.-H., Wang, Y.-S., Lee, J.-G., Cheng, C.-H., Chou, C.-C., 2013. Acrylamide-induced mitochondria collapse and apoptosis in human astrocytoma cells. Food Chem. Toxicol. 51, 446-452. Chen, W., Su, H., Xu, Y., Bao, T., Zheng, X., 2016. Protective effect of wild raspberry (Rubus hirsutus Thunb.) extract against acrylamide-induced oxidative damage is potentiated after simulated gastrointestinal digestion. Food Chem. 196, 943-952. Choi, K.S., 2012. Autophagy and cancer. Exp. Mol. Med. 44, 109. Çelik, H., Kucukler, S., Çomaklı, S., Özdemir, S., Caglayan, C., Yardım, A., Kandemir, F.M., 2020. Morin attenuates ifosfamide-induced neurotoxicity in rats via suppression of oxidative stress, neuroinflammation and neuronal apoptosis. Neurotoxicology 76, 126137. Darendelioglu, E., Aykutoglu, G., Tartik, M., Baydas, G., 2016. Turkish propolis protects human endothelial cells in vitro from homocysteine-induced apoptosis. Acta Histochem. 118, 369-376. Das, F., Ghosh‐Choudhury, N., Venkatesan, B., Li, X., Mahimainathan, L., Choudhury, G.G., 2008. Akt kinase targets association of CBP with SMAD 3 to regulate TGFβ‐induced expression of plasminogen activator inhibitor‐1. J. Cell. Physiol. 214, 513-527. Elblehi, S.S., El Euony, O.I., El-Sayed, Y.S., 2020. Apoptosis and astrogliosis perturbations and expression of regulatory inflammatory factors and neurotransmitters in acrylamideinduced neurotoxicity under ω3 fatty acids protection in rats. Neurotoxicology 76, 4457. Eldutar, E., Kandemir, F.M., Kucukler, S., Caglayan, C., 2017. Restorative effects of Chrysin pretreatment on oxidant–antioxidant status, inflammatory cytokine production, and 17

apoptotic and autophagic markers in acute paracetamol‐induced hepatotoxicity in rats: An experimental and biochemical study. J. Biochem. Mol. Toxicol. 31, e21960. Gedik, S., Erdemli, M.E., Gul, M., Yigitcan, B., Bag, H.G., Aksungur, Z., Altinoz, E., 2017. Hepatoprotective effects of crocin on biochemical and histopathological alterations following acrylamide-induced liver injury in Wistar rats. Biomed. Pharmacother. 95, 764-770. Ghorbel, I., Elwej, A., Fendri, N., Mnif, H., Jamoussi, K., Boudawara, T., Grati Kamoun, N., Zeghal, N., 2017. Olive oil abrogates acrylamide induced nephrotoxicity by modulating biochemical and histological changes in rats. Ren. Fail. 39, 236-245. Giatromanolaki, A., Koukourakis, M.I., Georgiou, I., Kouroupi, M., Sivridis, E., 2018. LC3A, LC3B and Beclin-1 Expression in Gastric Cancer. Anticancer Res. 38, 6827-6833. Giatromanolaki, A., Sivridis, E., Mitrakas, A., Kalamida, D., Zois, C.E., Haider, S., Piperidou, C., Pappa, A., Gatter, K.C., Harris, A.L., 2014. Autophagy and lysosomal related protein expression patterns in human glioblastoma. Cancer Biol. Ther. 15, 14681478. He, Y., Tan, D., Mi, Y., Zhou, Q., Ji, S., 2017. Epigallocatechin-3-gallate attenuates cerebral cortex damage and promotes brain regeneration in acrylamide-treated rats. Food Funct. 8, 2275-2282. Huang, X., Bai, H.-M., Chen, L., Li, B., Lu, Y.-C., 2010. Reduced expression of LC3B-II and Beclin 1 in glioblastoma multiforme indicates a down-regulated autophagic capacity that relates to the progression of astrocytic tumors. J. Clin. Neurosci. 17, 1515-1519. Jaffuel, A., Lemoine, J., Aubert, C., Simon, R., Léonard, J.-F., Gautier, J.-C., Pasquier, O., Salvador, A., 2013. Optimization of liquid chromatography–multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine. J. Chromatogr. 1301, 122-130. Kandemir, F.M., Kucukler, S., Caglayan, C., Gur, C., Batil, A.A., Gülçin, İ., 2017. Therapeutic effects of silymarin and naringin on methotrexate‐induced nephrotoxicity in rats: Biochemical evaluation of anti‐inflammatory, antiapoptotic, and antiautophagic properties. J. Food Biochem. 41, e12398. Kandemir, F.M., Yildirim, S., Kucukler, S., Caglayan, C., Mahamadu, A., Dortbudak, M.B., 2018. Therapeutic efficacy of zingerone against vancomycin-induced oxidative stress, inflammation, apoptosis and aquaporin 1 permeability in rat kidney. Biomed. Pharmacother. 105, 981-991. Kuzu, M., Kandemir, F.M., Yildirim, S., Kucukler, S., Caglayan, C., Turk, E., 2018. Morin attenuates doxorubicin-induced heart and brain damage by reducing oxidative stress, inflammation and apoptosis. Biomed. Pharmacother. 106, 443-453. Kuzu, M., Yıldırım, S., Kandemir, F.M., Küçükler, S., Çağlayan, C., Türk, E., Dörtbudak, M.B., 2019. Protective effect of morin on doxorubicin-induced hepatorenal toxicity in rats. Chem.-Biol. Interact. 308, 89-100. KV, A., Madhana, R.M., Kasala, E.R., Samudrala, P.K., Lahkar, M., Gogoi, R., 2016. Morin Hydrate Mitigates Cisplatin‐Induced Renal and Hepatic Injury by Impeding Oxidative/Nitrosative Stress and Inflammation in Mice. J. Biochem. Mol. Toxicol. 30, 571-579.

18

Kwon, T.-H., Frøkiær, J., Nielsen, S., 2013. Regulation of aquaporin-2 in the kidney: a molecular mechanism of body-water homeostasis. Kidney research and clinical practice 32, 96-102. Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in selenium-deficient rat liver. BBRC 71, 952-958. Li, M., Armelloni, S., Ikehata, M., Corbelli, A., Pesaresi, M., Calvaresi, N., Giardino, L., Mattinzoli, D., Nisticò, F., Andreoni, S., 2011. Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors. The Journal of pathology 225, 118-128. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Ma, Y., Chen, F., Yang, S., Duan, Y., Sun, Z., Shi, J., 2017. Silencing of TRB3 ameliorates diabetic tubule interstitial nephropathy via PI3K/AKT signaling in rats. Medical science monitor: international medical journal of experimental and clinical research 23, 2816. Mangwani, N., Singh, P.K., Kumar, V., 2019. Medicinal plants: Adjunct treatment to tuberculosis chemotherapy to prevent hepatic damage. J. Ayurveda Integr. Med. Matsuda, S., Kobayashi, M., Kitagishi, Y., 2013. Roles for PI3K/AKT/PTEN pathway in cell signaling of nonalcoholic fatty liver disease. ISRN Endocrinol. 2013. Mehri, S., Abnous, K., Mousavi, S.H., Shariaty, V.M., Hosseinzadeh, H., 2012. Neuroprotective effect of crocin on acrylamide-induced cytotoxicity in PC12 cells. Cell. Mol. Neurobiol. 32, 227-235. Mohamed, E.A., Ahmed, H.I., Zaky, H.S., 2018. Protective effect of irbesartan against doxorubicin-induced nephrotoxicity in rats: implication of AMPK, PI3K/Akt, and mTOR signaling pathways. Can. J. Physiol. Pharmacol. 96, 1209-1217. Nassar, M., Samaha, H., Ghabriel, M., Yehia, M., Taha, H., Salem, S., Shaaban, K., Omar, M., Ahmed, N., El-Naggar, S., 2017. LC3A silencing hinders aggresome vimentin cage clearance in primary choroid plexus carcinoma. Sci. Rep. 7, 8022. Pan, X., Wu, X., Yan, D., Peng, C., Rao, C., Yan, H., 2018. Acrylamide-induced oxidative stress and inflammatory response are alleviated by N-acetylcysteine in PC12 cells: Involvement of the crosstalk between Nrf2 and NF-κB pathways regulated by MAPKs. Toxicol. Lett. 288, 55-64. Placer, Z.A., Cushman, L.L., Johnson, B.C., 1966. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. AnBio 16, 359-364. Reed, J.C., 1997. Double identity for proteins of the Bcl-2 family. Nature 387, 773. Rizk, M., Abo-El-matty, D., Aly, H., Abd-Alla, H., Saleh, S., Younis, E., Elnahrawy, A., Haroun, A., 2018. Therapeutic activity of sour orange albedo extract and abundant flavanones loaded silica nanoparticles against acrylamide-induced hepatotoxicity. Toxicology reports 5, 929-942. Saito, Y., Okamura, M., Nakajima, S., Hayakawa, K., Huang, T., Yao, J., Kitamura, M., 2010. Suppression of nephrin expression by TNF-α via interfering with the cAMP-retinoic acid receptor pathway. American Journal of Physiology-Renal Physiology 298, F1436F1444. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. AnBio 25, 192-205. 19

Shibata, S., Nagase, M., Yoshida, S., Kawachi, H., Fujita, T., 2007. Podocyte as the target for aldosterone: roles of oxidative stress and Sgk1. Hypertension 49, 355-364. Singh, M.P., Chauhan, A.K., Kang, S.C., 2018. Morin hydrate ameliorates cisplatin-induced ER stress, inflammation and autophagy in HEK-293 cells and mice kidney via PARP-1 regulation. Int. Immunopharmacol. 56, 156-167. Song, G., Liu, Z., Wang, L., Shi, R., Chu, C., Xiang, M., Tian, Q., Liu, X., 2017. Protective effects of lipoic acid against acrylamide-induced neurotoxicity: involvement of mitochondrial energy metabolism and autophagy. Food Funct. 8, 4657-4667. Sumner, S.C., Fennell, T.R., Moore, T.A., Chanas, B., Gonzalez, F., Ghanayem, B.I., 1999. Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice. Chem. Res. Toxicol. 12, 1110-1116. Sun, C.H., Chang, Y.H., Pan, C.C., 2011. Activation of the PI3K/Akt/mTOR pathway correlates with tumour progression and reduced survival in patients with urothelial carcinoma of the urinary bladder. Histopathology 58, 1054-1063. Sun, J., Li, M., Zou, F., Bai, S., Jiang, X., Tian, L., Ou, S., Jiao, R., Bai, W., 2018. Protection of cyanidin-3-O-glucoside against acrylamide-and glycidamide-induced reproductive toxicity in leydig cells. Food Chem. Toxicol. 119, 268-274. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34, 497-500. Temel, Y., Kucukler, S., Yıldırım, S., Caglayan, C., Kandemir, F.M., 2019. Protective effect of chrysin on cyclophosphamide-induced hepatotoxicity and nephrotoxicity via the inhibition of oxidative stress, inflammation, and apoptosis. Naunyn-Schmiedeberg's Archives of Pharmacology, 1-13. Turk, E., Kandemir, F.M., Yildirim, S., Caglayan, C., Kucukler, S., Kuzu, M., 2019. Protective effect of hesperidin on sodium arsenite-induced nephrotoxicity and hepatotoxicity in rats. Biol. Trace Elem. Res. 189, 95-108. Uthra, C., Shrivastava, S., Jaswal, A., Sinha, N., Reshi, M.S., Shukla, S., 2017. Therapeutic potential of quercetin against acrylamide induced toxicity in rats. Biomed. Pharmacother. 86, 705-714. Wang, P., Guo, Q.-s., Wang, Z.-w., Qian, H.-x., 2013. HBx induces HepG-2 cells autophagy through PI3K/Akt–mTOR pathway. Mol. Cell. Biochem. 372, 161-168. Watzek, N., Scherbl, D., Feld, J., Berger, F., Doroshyenko, O., Fuhr, U., Tomalik‐Scharte, D., Baum, M., Eisenbrand, G., Richling, E., 2012. Profiling of mercapturic acids of acrolein and acrylamide in human urine after consumption of potato crisps. Mol. Nutr. Food Res. 56, 1825-1837. Wu, J., Xue, X., Zhang, B., Jiang, W., Cao, H., Wang, R., Sun, D., Guo, R., 2016. The protective effects of paeonol against epirubicin-induced hepatotoxicity in 4T1-tumor bearing mice via inhibition of the PI3K/Akt/NF-kB pathway. Chem.-Biol. Interact. 244, 1-8. Yang, D., Tan, X., Lv, Z., Liu, B., Baiyun, R., Lu, J., Zhang, Z., 2016. Regulation of Sirt1/Nrf2/TNF-α signaling pathway by luteolin is critical to attenuate acute mercuric chloride exposure induced hepatotoxicity. Sci. Rep. 6, 37157.

20

Zamani, E., Shokrzadeh, M., Ziar, A., Abedian-Kenari, S., Shaki, F., 2018. Acrylamide attenuated immune tissues’ function via induction of apoptosis and oxidative stress: Protection by l-carnitine. Hum. Exp. Toxicol. 37, 859-869. Zhang, D.-M., Liu, J.-S., Deng, L.-J., Chen, M.-F., Yiu, A., Cao, H.-H., Tian, H.-Y., Fung, K.-P., Kurihara, H., Pan, J.-X., 2013a. Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway. Carcinogenesis 34, 1331-1342. Zhang, L., Wang, E., Chen, F., Yan, H., Yuan, Y., 2013b. Potential protective effects of oral administration of allicin on acrylamide-induced toxicity in male mice. Food Funct. 4, 1229-1236. Zhang, L., Zhang, H., Miao, Y., Wu, S., Ye, H., Yuan, Y., 2012. Protective effect of allicin against acrylamide-induced hepatocyte damage in vitro and in vivo. Food Chem. Toxicol. 50, 3306-3312. Zhao, M., Wang, P., Zhu, Y., Liu, X., Hu, X., Chen, F., 2015. The chemoprotection of a blueberry anthocyanin extract against the acrylamide-induced oxidative stress in mitochondria: unequivocal evidence in mice liver. Food Funct. 6, 3006-3012. Zois, C.E., Giatromanolaki, A., Sivridis, E., Papaiakovou, M., Kainulainen, H., Koukourakis, M.I., 2011. " Autophagic flux" in normal mouse tissues: focus on endogenous LC3A processing. Autophagy 7, 1371-1378.

21

Table 1. Effect of morin on hepatic serum markers and oxidative stress biomarkers in ACRinduced hepatotoxicity Parameters

Control

ACR

Morin

ACR+M 50

ACR+M 100

ALP (U/L)

60.37±0.89a

120.23±3.19d

61.39±1.56a

96.07±2.07c

72.52±1.23b

ALT(U/L)

39.18±0.90a

78.71±1.54d

40.53±0.91a

66.21±1.64c

49.34±0.73b

AST (U/L)

45.70±0.67a

101.39±2.44d

46.37±0.91a

85.14±1.16c

55.28±1.45b

MDA(nmol/g tissue)

14.27±0.47a

26.14±0.57d

13.08±0.33a

21.05±0.79c

18.13±0.48b

GSH (nmol/g tissue)

7.38±0.15d

4.31±0.09a

7.50±0.11d

5.57±0.11b

6.49±0.15c

CAT(katal/g protein)

45.03±1.06d

31.02±0.64a

46.01±0.67d

35.33±0.49b

39.07±1.20c

SOD (U/g tissue)

35.91±0.67d

24.67±0.47a

35.37±0.66d

27.74±0.61b

30.35±0.57c

GPx (U/g tissue)

38.37±0.64d

25.18±0.47a

38.36±0.86d

30.86±0.61b

34.48±0.59c

Different superscripts (a–d) in the same row indicate significant difference (p<0.05) among groups.

Table 2. Effect of morin on renal serum markers and oxidative stress biomarkers in ACRinduced nephrotoxicity Parameters

Control

ACR

Morin

ACR+M 50

ACR+M 100

Urea (mg/dL)

2.08±0.04a

5.38±0.56c

2.10±0.04a

4.19±0.11b

3.36±0.10b

Creatinine (mg/dL)

0.44±0.01a

1.86±0.05d

0.42±0.01a

1.07±0.03c

0.63±0.01b

MDA(nmol/g tissue)

16.74±0.55a

31.24±0.61d

15.39±0.37a

24.74±0.47c

19.77±0.52b

GSH (nmol/g tissue)

6.17±0.12d

3.67±0.07a

6.38±0.14d

4.69±0.07b

5.41±0.11c

CAT(katal/g protein)

35.40±0.75d

23.27±0.54a

37.05±0.64d

27.26±0.57b

30.46±0.69c

SOD(U/g tissue)

30.05±0.66d

15.82±0.38a

30.65±0.60d

22.47±0.74b

26.09±0.61c

GPx(U/g tissue)

30.36±0.44d

16.79±0.56a

31.03±0.61d

22.12±0.56b

25.82±0.76c

Different superscripts (a–d) in the same row indicate significant difference (p<0.05) among groups.

Table 3. Histopathological and immunohistochemical finding and their scores in liver tissue in rats. Parameters

Control

ACR

Morin

ACR+M 50

ACR+M 100

Degeneration of hepatocytes Necrosis in hepatocytes Hyperemia in interstitial vessels and sinusoids P53

-

+++

-

++

+

-

+++

-

+

-

-

+++

-

+++

++

-

+++

-

++

+

EGFR

-

+++

-

++

+

(–) No change, (+) Mild change, (++) Moderate change, (+++) Severe change

Table 4. Histopathological and immunohistochemical finding and their scores in kidney tissue in rats. Parameters Degeneration of tubular epithelium Necrosis of tubular epithelium Hyperemia in interstitial vessels

Control

ACR

Morin

ACR+M 50

ACR+M 100

-

+++

-

++

+

-

+++

-

++

-

-

+++

-

+++

++

Nephrin

+++

+

+++

++

+++

Aquaporin-2

+++

+

+++

++

+++

(–) No change, (+) Mild change, (++) Moderate change, (+++) Severe change

Figures Fig. 1. (A) Effect of morin on ACR-induced liver and kidney NF-κB levels. (B) Effect of morin on ACR-induced liver and kidney TNF-α levels. (C) Effect of morin on ACR-induced liver and kidney IL-1β levels. (D) Effect of morin on ACR-induced liver and kidney IL-6 levels. (E) Effect of morin on ACR-induced liver and kidney COX-2 activities. (F) Effect of morin on ACR-induced liver and kidney p38α MAPK activities. Values are expressed as mean ± SEM. Different letters (a-d) on the columns show a statistical difference (p < 0.05).

Fig. 2. (A) Effect of morin on ACR-induced liver and kidney p53 levels. (B) Effect of morin on ACR-induced liver and kidney caspase-3 activities. (C) Effect of morin on ACR-induced liver and kidney Bax levels. (D) Effect of morin on ACR-induced liver and kidney Bcl-2 levels. Values are expressed as mean ± SEM. Different letters (a-d) on the columns show a statistical difference (p < 0.05).

Fig. 3. Anti-apoptotic effects of morin on ACR-induced apoptosis in liver tissue. Protein levels (A) Bax (23 kDa), Bcl-2 (26 kDa), Sitokrom c (15 kDa) ve procaspase-3 (34 kDa) were measured by Western blotting analysis. β-Actin was used as reference. (B) Data were presented as mean ± SEM. ***p < 0.001 Control vs Others, **p < 0.01 Control vs Others, *p < 0.05 Control vs Others; ###p < 0.001 ACR vs Others, ##p < 0.01 ACR vs Others, #p < 0.05 ACR vs Others; ns: not significant.

Fig. 4. Anti-apoptotic effects of morin on ACR-induced apoptosis in kidney tissue. Protein levels (A) Bax (23 kDa), Bcl-2 (26 kDa), Sitokrom c (15 kDa) ve procaspase-3 (34 kDa) were measured by Western blotting analysis. β-Actin was used as reference. (B) Data were presented as mean ± SEM. ***p < 0.001 Control vs Others, **p < 0.01 Control vs Others, *p < 0.05 Control vs Others; ###p < 0.001 ACR vs Others, ##p < 0.01 ACR vs Others, #p < 0.05 ACR vs Others; ns: not significant.

Fig. 5. (A) Effect of morin on ACR-induced liver and kidney mTOR levels. (B) Effect of morin on ACR-induced liver and kidney PI3K activities. (C) Effect of morin on ACR-induced liver and kidney PKB activities. Values are expressed as mean ± SEM. Different letters (a-d) on the columns show a statistical difference (p < 0.05).

Fig. 6. (A) Effect of morin on ACR-induced liver and kidney Beclin-1 levels. (B) Effect of morin on ACR-induced liver and kidney LC3A levels. (C) Effect of morin on ACR-induced liver and kidney LC3B levels. Values are expressed as mean ± SEM. Different letters (a-d) on the columns show a statistical difference (p < 0.05).

Fig. 7. Histopathological examination of rat liver tissue. (A and C) Normal histological appearance of liver tissue. (B) Severe hydropic degeneration and coagulation necrosis in hepatocytes, and severe hyperemia. (D) Moderate hydropic degeneration, necrosis and hyperemia in hepatocytes. (E) Mild degeneration in hepatocytes. (F and K) Negative p53 and EGFR expression in liver hepatocytes. (G and L) Severe p53 and EGFR expression in hepatocytes. (H and M) Negative p53 and EGFR expression in liver hepatocytes. (I and N) Moderate expression of p53 and EGFR in hepatocytes. (J and O) Mild p53 and EGFR expression in hepatocytes.

Fig. 8. Histopathological examination of rat kidney tissue. (A and C) Normal histological appearance of kidney tissue. (B) Severe hydropic degeneration and coagulation necrosis in tubular epithelium, and severe hyperemia in interstitial tissue. (D) Moderate degeneration and coagulation necrosis of tubular epithelium, and severe hyperemia. (E) Mild degeneration of tubular epithelium, mild hyperemia in interstitial and glomerular tissue. (F,K,H and M) Severe expression of aquaporin-2 and nephrin in tubular epithelium. (G and L) Mild aquaporin-2 and nephrin expression in tubular epithelium of cortex and medulla. (I and N) Moderate aquaporin-2 and nephrin expression in tubular epithelium. (J and O) Severe aquaporin-2 and nephrin expression in tubular epithelium.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Highlights •

Morin protects rats against ACR-induced hepatotoxicity and nephrotoxicity.

•

Morin reduces apoptosis and autophagy in the ACR-induced toxicity.

•

Morin depressed levels of oxidative stress and pro-inflammatory cytokines.

•

Morin regulated the PI3K/Akt/mTOR signaling pathway in the ACR-induced toxicity.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The authors declare that there are no conflicts of interest.