The ameliorative effects of boron against acrylamide-induced oxidative stress, inflammatory response, and metabolic changes in rats

Accepted Manuscript The ameliorative effects of boron against acrylamide-induced oxidative stress, inflammatory response, and metabolic changes in rats Ulas Acaroz, Sinan Ince, Damla Arslan-Acaroz, Zeki Gurler, Ismail Kucukkurt, Hasan Hüseyin Demirel, Halil Ozancan Arslan, Nuray Varol, Kui Zhu PII:

S0278-6915(18)30403-4

DOI:

10.1016/j.fct.2018.06.029

Reference:

FCT 9850

To appear in:

Food and Chemical Toxicology

Received Date: 17 May 2018 Revised Date:

12 June 2018

Accepted Date: 15 June 2018

Please cite this article as: Acaroz, U., Ince, S., Arslan-Acaroz, D., Gurler, Z., Kucukkurt, I., Demirel, Hasan.Hü., Arslan, H.O., Varol, N., Zhu, K., The ameliorative effects of boron against acrylamideinduced oxidative stress, inflammatory response, and metabolic changes in rats, Food and Chemical Toxicology (2018), doi: 10.1016/j.fct.2018.06.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT The ameliorative effects of boron against acrylamide-induced oxidative stress, inflammatory response, and metabolic changes in rats Ulas Acaroz1*, Sinan Ince2, Damla Arslan-Acaroz3, Zeki Gurler1, Ismail Kucukkurt4,

Afyon Kocatepe University, Veterinary Faculty, Department of Food Hygiene and

Technology, Afyonkarahisar, Turkey 2

SC

1

RI PT

Hasan Hüseyin Demirel3, Halil Ozancan Arslan5 , Nuray Varol6, Kui Zhu7

Afyon Kocatepe University, Veterinary Faculty, Department of Pharmacology and

M AN U

Toxicology, Afyonkarahisar, Turkey 3

Afyon Kocatepe University, Bayat Vocational School, Afyonkarahisar, Turkey

4

Afyon Kocatepe University, Veterinary Faculty, Department of Biochemistry,

Afyonkarahisar, Turkey

University of Zurich, Vetsuisse Faculty, Clinic of Reproductive Medicine,

Switzerland 6

TE D

5

Afyon Kocatepe University, Faculty of Medicine, Department of Medical Genetics,

7

EP

03200 Afyonkarahisar, Turkey

China Agricultural University, College of Veterinary Medicine, Beijing, National

AC C

Center for Veterinary Drug Safety Evaluation, China 100193

* Corresponding author: Ulaş ACARÖZ E-mail: [email protected] Tel: +90272281312-16162 Fax: +90272281349

ACCEPTED MANUSCRIPT The ameliorative effects of boron against acrylamide-induced oxidative stress, inflammatory response, and metabolic changes in rats Abstract

RI PT

Acrylamide (ACR) is a hazardous substance associated with the accumulation of excessive reactive oxygen species and causes oxidative stress. Presence of ACR in foods leads to public health concerns due to its known neurotoxic, genotoxic, and carcinogenic effects. The present

SC

study investigated the ameliorative effects of boron (B) against ACR exposed rats. Forty Wistar albino male rats, fed with low-boron diet, were randomly and equally allocated into 5

M AN U

groups. The control group was orally treated with physiological saline as placebo, the second group was orally given 15 mg/kg ACR. The other groups were orally treated with 15 mg/kg ACR and B at the levels of 5, 10, and 20 mg/kg/day for 60 days, respectively. ACR-treatment significantly increased malondialdehyde levels whereas decreased glutathione levels in rat

TE D

tissues. Also, ACR-treatment increased the activities of superoxide dismutase and catalase in erythrocytes and tissues. Meanwhile, mRNA expression levels of NFĸB, IFN-γ, IL-1β, and TNF-α in liver and brain of rats were increased under ACR treatment. Additionally, ACR

EP

caused a significant decrease in the level of high-density lipoprotein, with increase in the levels of low-density lipoprotein, triglyceride, cholesterol, glucose, urea nitrogen, and

AC C

creatinine. Lastly, B alleviated histopathological alterations induced by ACR in rat tissues. Keywords: Acrylamide, Boron, Oxidative stress, Gene expression, Histopathology, Rat 1. Introduction

Acrylamide (ACR) is also known as 2-propenamide an odorless and colorless crystalline monomer with significantly high chemical activity (Zhang et al., 2009; Abdel-Daim et al., 2014). ACR does not exist naturally occurring via Maillard reaction between amino acids, particularly asparagine, and reducing sugars when carbohydrate-rich foods are prepared at 1

ACCEPTED MANUSCRIPT high temperature (Hamdy et al., 2012; Zhang et al., 2012; Alturfan et al., 2012). ACR is recognized as a by-product of deep-frying and described as cooking carcinogen due to its spontaneous formation during the cooking process of food (Alturfan et al., 2012). It is widely found in many foodstuffs including fried, deep-fried, oven-baked foods, chips, biscuits, bread,

RI PT

crackers, coffee, and breakfast cereals (Zhang et al., 2010; Hamdy et al., 2012; Mehri et al., 2015; Zhang et al., 2012; Alturfan et al., 2012). Due to its high water solubility and low molecular weight, ACR can pass biological membranes such as blood-milk and blood-

SC

placenta barriers (Abdel-Daim et al., 2014). By this way, it could be found in placenta and breast milk of humans (Zhang et al., 2010). FAO, FDA, and WHO stated that foodborne ACR

M AN U

is hazardous to human health (Rosen 2002; Gedik et al., 2017). Also, Agency for Research on Cancer has classified ACR as Group 2A carcinogen (IARC, 1994; Zhang et al., 2017). Moreover, it has diverse well-known toxic effects including neurotoxicity, genotoxicity, reproductive toxicity, and immunotoxicity (Zamani et al., 2017), in cell line and animal

TE D

models (Zhang et al., 2012; Liu et al., 2015; Song et al., 2013; Zamani et al., 2017). Only it is known that in the body ACR is partially metabolised to glycidamide by cytochrome P450-2E1 which is more toxic than ACR itself (Song et al., 2013). Consequently, oxidative stress plays

EP

crucial roles in ACR-induced toxicity (Abdel-Daim et al., 2014) due to over-production of

AC C

reactive oxygen species (Song et al., 2013). Boron (B) is an essential element for plants. Various foodstuffs such as raw avocado, salted dry roasted peanuts, creamy peanut butter, canned grape juice, bottled prune juice, sweetened chocolate powder, table wine, and several fruits, beans, peas, and nuts are rich boron sources. The amount of boron in a single diet for adults are between 0.15 and 40 mg. Approximately, 20% of this amount is provided by vegetable, fruit, and fruit drink products (Hunt, 2005). Also, its compounds which are boric acid, borax, ulexite, kernite, colemanite and other borates are used for various applications such as water softeners, food preservatives, 2

ACCEPTED MANUSCRIPT stabilizers, emulsifiers, pH adjusters, buffers, and neutralizers in food industry and in health care systems (Beyer et al., 1983; Scorei 2012). Besides, B plays an important role in the health of humans and animals. In the metabolism, mineral substance B has effects on vitamin D, enzymes, hormones, mineral metabolisms, biochemical parameters and reactive oxygen

RI PT

species (Nielsen et al., 1987; Meacham et al., 1994; Hunt, 1996; Armstrong et al., 2001; Devirian and Volpe, 2003; Turkez et al., 2007; Ince et al., 2014a). Beside these, B shows hepatoprotective, and antigenotoxic effects, and antioxidant activity by inhibiting production

SC

of reactive oxygen species (Coban et al., 2015). Due to the above mentioned health effects of B, it can be considered that the consumption of boron-rich food and foodstuffs could attenuate

M AN U

the adverse effects of hazardous substances such as ACR.

This empirical study evaluated the effect of B against ACR induced biochemical parameters [glucose, cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), urea nitrogen (BUN), and creatinine in plasma], lipid peroxidation (LPO), antioxidant

TE D

status in blood and in kidney, liver, heart, brain, lung, and testis tissues of Wistar albino rats. Moreover, histopathological changes were examined in tissues and the expression levels of

EP

the inflammation-related genes were determined in liver and brain.

AC C

2. Material and methods

1.1. Materials

ACR (C3H5NO) was obtained from Merck (Darmstadt, Germany) and boric acid (H3BO3) as a B source and boron diet components were purchased from Sigma-Aldrich Company (St. Louis, Missouri, USA). Also, ACR and B were dissolved in physiological saline before administration to rats. All the other chemicals and reagents, employed in this work were of analytical and molecular grade, which were acquired from commercial sources. 3

ACCEPTED MANUSCRIPT

2.2. Experimental protocol 2.2.1. Animals and experimental groups

RI PT

The local committee of Afyon Kocatepe University approved all experimental protocols (Approval No: 49533702-33). Healthy male Wistar albino rats weighing approximately 250300 g were procured by Afyon Kocatepe University, Experimental Animal Application and

SC

Research Center, Afyonkarahisar, Turkey. Prior to experiment for seven days, rats were objected to the adaptation to the animal facility circumstances. Rats were housed in a well-

M AN U

ventilated room in a 12 h light/dark cycle (25 oC, 50-55% relative humidity) with free access to low B diet (Bourgeois et al., 2007) and fresh deionized water. The B content of rat diet was determined by employing ICP-OES (Optima 8300 DV, Perkin Elmer, Waltham-MA, USA). Rats in the Group I (control group) were treated orally with physiological saline and Group II

TE D

was administered ACR by gastric gavage at the dose of 15 mg/kg/day (Patel et al., 2015). Group III, IV and V were also administered the same dose of ACR followed by oral treatment of B at the doses of 5, 10, 20 mg/kg/day (Ince et al., 2014a), respectively for 60 days. Each

EP

group consists of eight animals.

AC C

2.2.3. Collection of blood and tissue samples Twenty-four hours after the final treatment, blood samples of rats were taken via cardiac puncture under anaesthesia (ketamine/xylazine). Then, animals were sacrificed and their tissues (kidney, liver, heart, brain, lung, and testis) were collected. Erythrocytes, prepared from blood samples, were used for the measurement of antioxidant enzyme activities. Tissue samples of kidney, liver, heart, brain, lung, and testis were taken for biochemical and histopathological analyses. Also, liver and brain samples were taken for the molecular analyses. 4

ACCEPTED MANUSCRIPT 2.3. Preparation of erythrocytes and tissue homogenates For the preparation of erythrocytes, method described by Winterbourn et al. (1975) was employed. Briefly, erythrocytes were obtained by centrifugation (3500 rpm, 15 min, 4 oC) of blood samples followed by 3 times washing with isotonic saline buffer solution (pH 7.4).

RI PT

Then, erythrocytes were transferred into Eppendorf tubes with the equal volume of isotonic saline buffer solution and stored in vials at -20 oC. Prior to the analysis, destruction of

within 3 days and it was stored at 4 oC during this time.

SC

erythrocyte suspension was mediated by cold deionised water. Measurement was performed

The preparation of tissue samples was carried out according to Ince et al., (2014a). Briefly,

M AN U

the animals were sacrificed and their kidney, liver, heart, brain, lung, and testis tissues were directly washed using an ice-cold isotonic saline buffer. Tissue samples were cleaned free of extraneous tissue and rinsed in cooled Tris-HCl buffer (0.15 M, pH 7.4) and then were blotted dry. Homogenates of samples (10%, w/v) were prepared using a Tris-HCl buffer. Thereafter,

TE D

centrifugation of homogenized tissues was performed at 3500 rpm for 10 min at 4oC and stored in -20oC until the measurement. Erythrocytes and tissue homogenates were used for the

EP

measurement of lipid peroxidation parameters including MDA, GSH, SOD, and CAT. 2.4. Determination of lipid peroxidation (MDA) and reduced glutathione (GSH)

AC C

The level of malondialdehyde (MDA) was served as a reliable biomarker of lipid peroxidation (LPO). MDA was determined for whole blood samples and tissue homogenates according to Draper and Hardley (1990) and Ohkawa et al., (1979), respectively. These methods spectrophotometrically measure the yielded colour of the thiobarbituric acid and MDA reaction. MDA concentration was determined by the absorbance coefficient of thiobarbituric acid-MDA complex (nmol/ml in blood and nmol/g in wet tissue) and determined at 532 nm using a double beam UV–Visible spectrophotometer (Shimadzu 1601, Tokyo, Japan). GSH is the major component of a non-enzymatic defence system against reactive oxygen species and 5

ACCEPTED MANUSCRIPT its consumption can be induced by oxidative stress (Guerin et al., 2001). The concentration of GSH in blood and tissue samples was determined as previously described (Beutler et al., 1993). Briefly, sample (0.2 ml) and distilled water (1.8 ml) mixed together, followed by addition of 3 ml precipitating solution (1.67 g HPO3, 30 g NaCl, 0.2 g EDTA in 100 ml

RI PT

distilled water) to the sample. This mixture was filtered (Whatman No. 42) after standing for about 5 min. Then, filtrate (2 ml) was mixed with 0.3 M Na2HPO4 (8 ml) and 5,5′-Dithiobis (2-nitrobenzoic acid) (1 ml) in another tube. The spectrophotometrically determination of

SC

optical density was performed at 412 nm (Shimadzu 1601 UV–VIS spectrophotometer,

M AN U

Tokyo, Japan). Results were expressed as nmol/ml blood and nmol/ g wet tissue. 2.5. Determination of SOD and CAT activity

SOD and CAT are involved in the protection of cells against oxidative damage (Guerin et al., 2001). The SOD activity in tissue homogenate and erythrocyte lysate was determined as

TE D

previously described (Sun et al., 1988). The principle of this method is based on the reaction between xanthine oxidase and xanthine which are generating a superoxide flux and reduce nitroblue tetrazolium (NBT) to blue formazon as an indicator of superoxide. The

EP

spectrophotometric measurement was performed at 560 nm and the activity of SOD was expressed as U/gHb in erythrocyte and U per g protein in tissue.

AC C

The determination of CAT activity was performed in tissue homogenate and erythrocyte lysate according to the methods described by Aebi (1974) and Luck (1955), respectively. These methods work through the decomposition of H2O2 to water and oxygen via catalase. H2O2 provides maximal absorption at 240 nm in the ultraviolet spectrum. The reduction rate was measured at 240 nm for 45 s. at room temperature and expressed as k/gHb in erythrocyte and k/µg protein in tissue (k; nmol/min).

6

ACCEPTED MANUSCRIPT 2.6. Determination of protein and hemoglobin concentrations The content of protein in tissues and hemoglobin in erythrocytes were spectrophotometrically determined according to Lowry et al. (1951) and Drabkin and Austin (1935), respectively.

RI PT

2.7. Histopathological analysis Brain, lung, heart, kidney, liver, heart tissues of dissected rats were collected and fixed in 10% formaldehyde solution. Then, these tissues analysed based on histological tissue follow-

SC

up procedures and embedded in paraffin blocks. After the cutting of 5-6 µm thick paraffin sections, haematoxylin-eosin staining was performed and they were analysed with Olympus

M AN U

Bx51 light microscope equipped with Olympus DP20 camera. Histopathological alterations in tissues and scoring of findings were shown as follows; normal histology (–), mild (+), moderate (++), and severe (+++), and the results are shown in Table 6. 2.8. Measurement of biochemical parameters

TE D

To determine the concentrations of plasma glucose, cholesterol, triglyceride, HDL, LDL, BUN, and creatinine diagnostic commercial kits were used as described by the manufacturer

EP

(Biolabo Laboratories, Maizy, Picardy, France),

AC C

2.9. Expression levels of IF-γ, IL-1β, TNF-α, and NFĸB The expression levels of IF-γ, IL-1β, TNF-α, and NFĸB were determined by Real time-PCR. Total RNA extraction of brain and liver tissues was performed by Tri-Pure Reagent (Roche, Germany). Nanodrop ND-1000 (Thermo, USA) was employed for the quantification of isolated RNA and its purity was detected at OD 260/280. The reverse transcription of total RNA was performed by RT2 HT First Strand kit (Qiagen, Germany) according to the manufacturer’s instructions. Specific primers were purchased from Ella Biotech GmbH (Martinsried, Germany) GAPDH (GAPDH forward: 5′-ACCACAGTCCATGCCATCAC-3′,

7

ACCEPTED MANUSCRIPT reverse: TCCACCACCCTGTTGCTGTA). IFN-γ (forward: 5′-CACGCCGCGTCTTGGT-3′, reverse:TCTAGGCTTTCAAT-GAGTGTGCC),

IL-1β

(forward:

5′-

CACCTCTCAAGCAGAGCACAG-3′, reverse: GGGTTCCATGGTGAAGTCAAC), TNF-α (forward:

5′-CTTCTGTCTACTGAACTTCGG-3′,

reverse:

GTGCTTGATCTGT-

RI PT

TGTTTCC), and NFĸB (forward: 5′-GGGACTATGACTTGAATGCGGTCC −3′, reverse: CAGGTCCCGTGAAATACACCTCA-A) . A Rotor-Gene Q using RT2 SYBR-Green ROX master mix (Qiagen, Germany) was employed for qRT-PCR analysis. The analysis of gene

SC

expression levels of each sample was performed in triplicate. The results were normalized to the expression level of GAPDH. The obtained data are expressed as relative gene expression

M AN U

via the 2−∆∆Ct method. 2.16. Statistical analyses

Statistical analyses of the obtained data were carried out by SPSS (20.0) software by one-way

TE D

analysis of variance (followed by Duncan post hoc test) and expressed as means and standard

3. Results

AC C

EP

deviation (±SD). The statistically significance was considered as p< 0.05.

3.1. Boron content analysis of animal feed Before starting the experiment boron diet was evaluated in terms of its boron compound and found to be at <0.018 mg/kg. When this value was examined, due to the fact that the boron proportion in the prepared diet was significantly low, this diet found suitable for the study and it did not have an additional effect on boron doses.

8

ACCEPTED MANUSCRIPT 3.2. Effect on LPO and GSH MDA is the end product of polyunsaturated fatty acid and usually served as a marker of lipid peroxidation. Statistically significant increase was determined in blood, kidney, liver, brain, lung, testis (p < 0.001), and heart (p < 0.05) MDA levels of ACR group compared to control.

RI PT

However, B treatment ameliorated these deteriorations in whole blood and tissues. B treatment caused a significant decrease in MDA levels comparing with ACR group and MDA levels of blood and tissues are presented in Table 1. GSH, non-enzymatic antioxidant

SC

substance, has a fundamental detoxification role against toxic metabolites. GSH levels of whole blood, kidney, liver, brain, testis (p < 0.001), lung (p < 0.05), and heart (p > 0.05) of

M AN U

ACR group were found to be decreased compared to the control group. Contrarily, B treatment showed higher GSH levels of blood and tissues of rat than the ACR group which is illustrated in Table 2.

TE D

3.3. Effect on antioxidant enzymes

SOD and CAT, endogenous and enzymatic antioxidants, take place as a part of the enzyme defense system against oxidative stress. Their activities were assessed in erythrocyte, kidney,

EP

liver, heart, brain, lung, and testis tissues of rats and given in Table 3 and 4, respectively. In ACR treated group, the activities of SOD were higher in liver, heart, brain, testis (p < 0.001),

AC C

erythrocyte, kidney (p < 0.05), and lung (p < 0.01) than control group (Table 3). Similarly, CAT activities of ACR treated group were also increased in erythrocyte, kidney, liver, heart, brain, lung, and testis (p < 0.001) tissues compared to control group (Table 4). Yet, B administration reversed the alterations in the SOD and CAT activities.

9

ACCEPTED MANUSCRIPT 3.4. Effect on the level of gene expression In the current study, expression levels of inflammation-associated genes IF-γ, IL-1β, TNF-α, and NFĸB determined based on PCR method (Fig. 1A-D). Expression levels of these genes were found to be higher in the brain and liver of ACR group than control group (p < 0.05).

RI PT

However, B administration (especially, 10 and 20 mg/kg of B) reversed ACR-treated expression levels of related genes and inhibited inflammation in rat tissues (p < 0.05).

SC

3.5. Effects on biochemical parameters

The administration of ACR (15 mg/kg, via gastric gavage) significantly decreased HDL while

M AN U

increased glucose, LDL, cholesterol, triglyceride, creatinine, and BUN levels (p<0.05). Boron treatment ameliorated ACR-induced alterations of biochemical parameters in male rats (Table 5). 3.6. Effects on histopathology

TE D

The histopathological alterations in the tissues of the rats were demonstrated detailed in Table 6 and shown in supplementary material (Fig. S1-6). In ACR group, neuronal degeneration and focal gliosis were determined in the brain (Fig. S1-B), focal gliosis (Fig. S1-C), and

EP

thickening of interalveolar septal tissue were also seen in the lung (Fig. S2-B) of rats. A mild

AC C

hyaline degeneration in the myocardium was observed (Fig. S3-B). In the kidney, enlargement of Bowman’s space and tubular epithelial cells degenerations (Fig. S4-B), and hyperemia in glomerulus (Fig. S4-C) were sighted. In the liver, sinusoidal dilatation, hyperemia, and Kupffer cell activation (Fig. S5-B) and a slight decrease in Kupffer cells (Fig. S5-C) were found. In testis, reduced spermatogenic density and giant cells formation in tubulus seminiferus contortus (Fig. S6-B) were observed. In B treated groups (particularly B20), moderate histopathological alterations were observed in the rat tissues (brain, lung, heart, kidney, liver, and testis) compared to ACR group (Fig. S1-6 C, D and E, respectively). 10

ACCEPTED MANUSCRIPT No significant histopathological alterations were detected in brain, lung, heart, kidney, liver, and testis tissues of rats in the control group (Fig. S1-6 A, respectively).

RI PT

4. Discussion

ACR causes a potentially important risk to human health due to its mutagenic, genotoxic, and carcinogenic features. Also, it is well known that ACR leads to free radicals

SC

generation by disturbing the oxidant-antioxidant balance and finally causing oxidative stress (Alturfan et al., 2012; Gey, 1993). Due to enhanced oxidative stress, the level of TBARS or

M AN U

MDA are increased which are the most essential oxidative stress biomarkers (Ince et al., 2014a; Gasparrini et al., 2017; Giampieri et al., 2017). In the present study, the administration of ACR at the dose of 15 mg/kg markedly increased the lipid peroxidation in the brain, lung, heart, kidney, liver, and testis tissues of rats. Also, the augmented production of lipid

TE D

peroxides in blood and tissues was consistent with other studies (Alturfan et al., 2012; AbdelDaim et al., 2014; Catalgol et al., 2009). In addition to MDA level, ACR-induced intense effects of the oxidative stress represented also by the decreased level of GSH, as well as

EP

increased activities of antioxidant enzymes (SOD and CAT). In accordance with our results, previous studies have also determined that GSH plays a major role in the detoxification of the

AC C

reactive oxygen species generated by ACR (Chen et al., 2016; Catalgol et al., 2009). In this study, the augmented lipid peroxidation with depletion of GSH levels in ACR group shows that the increased peroxidation could result from depleted GSH stores. B-treatment of rats yielded normal levels of GSH, which could be arised from the B-mediated decline of peroxidation activity by its role of as a scavenger of singlet molecular oxygen, superoxide and hydroxyl radical (Ince et al., 2010; Ince et al., 2012; 2014a; Ince et al., 2014b). Antioxidant enzymes are able to repress free radical formation. Both CAT and SOD can degrade free radicals into H2O2, decreasing oxidative stress and preventing cells from deleterious effects of 11

ACCEPTED MANUSCRIPT free radicals (Ince et al., 2014a). Some in vivo and in vitro studies suggested that ACR increased the activities of SOD (Catalgol et al., 2009; Yousef and El-Demerdash 2006) and CAT (Gedik et al., 2017). In the present study, the increase in the levels of SOD and CAT activities in erythrocytes and tissues could be attributed to the increase of oxidative stress in

RI PT

ACR group. The activities of these enzymes in the B groups were lower than the ACR group. This points out that B treatment reduced oxidative stress and prevented consuming of enzymes activities due to its potent antioxidant effect (Ince et al., 2017).

SC

The effects of ACR in different doses and duration on biochemical parameters were investigated by several studies (Uthra et al., 2017; Mahmood et al., 2015; Atef et al., 2017;

M AN U

Alturfan et al. 2012; Totani et al., 2007). Uthra et al., (2017) determined that the oral administration of ACR (38.27 mg/kg bw) for 10 days markedly increased levels of serum metabolites such as triglycerides, cholesterol, bilirubin, creatinine, uric acid and urea. However, quercetin-treatment ameliorated ACR induced alterations. Comparably, Atef et al.

TE D

(2017) investigated the renal effects of ACR at the dose of 20 mg/kg for 4 weeks in rats observed that ACR augmented the serum concentration of Creatinine and blood urea nitrogen whereas Vitamin E treatment closed these data to control group. Also, Alturfan et al. (2012)

EP

reported that creatinine and blood urea nitrogen levels significantly increased by ACR

AC C

treatment (40 mg/kg, i.p.) whereas resveratrol showed ameliorative effects on these parameters. The results of current study are in line with the above mentioned studies. ACR treatment induced an increase of plasma glucose, LDL, triglyceride, cholesterol, blood urea nitrogen and creatinine levels. However, a decrease in HDL level was observed. Coadministration of ACR with B closed these biochemical parameters were to the control group. This showed that ACR-induced biochemical alterations were ameliorated by antioxidant effects of B on rat tissues (Turkez ve ark., 2007; Ince ve ark., 2014a).

12

ACCEPTED MANUSCRIPT Inflammation could be induced by oxidative stress that causes many chronic diseases including cancer, diabetes, cardiovascular and neurological diseases. On the other hand, various transcription factors such as AP-1, NFĸB, p53 could be activated by this situation (İnce et al. 2017). The application of ACR to experimental animals in different doses such as

et al., 2013)

RI PT

20 mg/kg (Abdel-daim et al., 2014), 40 mg/kg (Alturfan et al., 2012), and 50 mg/kg (Zhang increased

significantly the serum and/or plasma levels of

inflammatory

cytokines, IL-1β, IL-6, and TNF-α. However, co-administration of antioxidant substances

SC

(fenugreek oil, resveratrol, and allicin, respectively) reversed ACR induced alterations in their studies. The results of the present study are in line with above-mentioned studies and

M AN U

exhibited that ACR caused the activation of inflammatory cells and afterwards augmented the inflammatory answer via releasing several cytokines. ACR administration increased the expression levels of inflammation-related genes. When the rats were given B, the expression level of IF-γ, TNF-α, IL-1β, and NFĸB markedly decreased. These results showed that B

TE D

could ameliorate tissue damage induced by ACR by suppressing inflammatory answer. The current study revealed that orally treatment of 15 mg/kg ACR induced apparent histopathological changes in the brain, lung, heart, kidney, and testis tissues of Wistar albino

EP

rats. The observed pathological changes were as follows; focal gliosis in the brain;

AC C

interalveolar septal thickening in the lungs and hyperemia in the interalveolar capillary vessels; hyaline degeneration in myocardial cells and hyperemia in vessels; enlargement in Bowman space of glomerulus in kidneys and degenerative and necrobiotic changes in tubulus epithelial cells; sinusoidal dilatation, degeneration and Kupffer cell activation in the liver; decrease in spermatogenic density, giant cell formation, cell debris in the tubulus seminiferous contortus lumen of the testis. Hammad et al. (2013) reported that dietary supplementation of ACR for 6 weeks caused histopathological changes of rat tissues in a dose-dependent manner. 10 mg/kg ACR induced hepatocyte degeneration. In addition to this,

13

ACCEPTED MANUSCRIPT 30 mg/kg ACR caused congestion of the blood vessels in the heart. Also, 60 mg/kg ACR resulted in generalized necrosis of the hepatic cells. Besides, 90 mg/kg ACR induced degeneration and necrosis of the glomeruli. Uthra et al., (2017) suggested that peroral administration of ACR at the dose of 38.27 mg/kg for 10 days caused alterations in the liver

RI PT

such as inflammatory cell infiltration, cytoplasmic vacuolization, and disruption in sinusoidal spaces and hepatic cords. Also, degeneration in glomeruli was observed in kidney. Besides, vacuolization, degenerated pyramidal cells in cerebral cortex and cerebral atrophy was seen.

SC

Ahmed et al. (2010) observed that the administration of ACR (50 mg/kg, bw, i.p.) caused neurotoxicity in adult female rats showing the dark neurons possessing corkscrew dendrites,

M AN U

granular or amorphous gray cytoplasm, coarse clumping of hyperchromatic nuclear chromatin. Similarly, Lakshmi et al. (2012) revealed that intraperitoneal administration of ACR (30 mg/kg) for 30 days resulted in condensed nuclei along with damaged cells in the cerebral cortex of rats. Khan et al., (2011) reported in their work that animals treated with

inflammation,

TE D

ACR (6 mg/kg bw; i.p.) for 15 days showed severe changes in their livers including lymphocytes

infiltration,

disruption

of

hepatic

cords,

centrilobular

vacuolization of hepatocytes, necrosis and congestion of the central canal wall. Yang et al.,

EP

(2005) reported that 60 mg/kg of ACR-treatment caused the formation of many multinucleated giant cells in seminiferous tubules and thickening and multiple layering of the

AC C

tubular endothelium in testis tissues of rats. Similarly, Camacho et al. (2012) observed that 50 mg/kg ACR for 14 d caused various testicular lesions such as depletion of germ cells, exfoliated germ cells, spermatid retention in male F344 rats. The present study determined that B, based on its antioxidant properties (İnce et al. 2014a, 2014b), ameliorated ACR induced alterations of rat tissues.

14

ACCEPTED MANUSCRIPT Conclusion The present study, to the best of author’s knowledge, is the first work investigating the effects of B on ACR-induced toxicity on blood and brain, lung, heart, kidney, liver and testis tissues of Wistar albino rats by evaluating oxidant-antioxidant, inflammatory, biochemical, and

RI PT

histopathological parameters. Our results showed that B effectively ameliorated ACR-caused oxidative stress, inflammation, altered biochemical parameters and alleviated tissue damage

M AN U

Acknowledgement

SC

by preventing the consumption of antioxidant enzymes and inhibiting lipid peroxidation.

This study was financially supported by the Afyon Kocatepe University Scientific Research Council, Afyonkarahisar, Turkey (Project no: 16.VF.02).

TE D

The summary of this study was partly presented as an oral presentation at the 4th International VETIstanbul Group Congress 11-13 May 2017 Almaty KAZAKHSTAN

AC C

EP

5. 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. Biochem. Cell Biol. 93(3), 192-198. Aebi, H., 1974. Catalase in vitro, in: U. Bergmeyer (Ed.), Methods of enzymatic analysis. Academic Press, New York and London. pp. 673-677.

15

ACCEPTED MANUSCRIPT Ahmed, H.H., Elmegeed, G.A., El-Sayed, E.S. M., Abd-Elhalim, M.M., Shousha, W.G., Shafic, R.W. 2010. Potent neuroprotective role of novel melatonin derivatives for management of central neuropathy induced by acrylamide in rats. Eur. J. Med. Chem.

RI PT

45(11), 5452–5459. Almoeiz, A.Y., Hammad, Osman, M.E., Abdelgadir, W.S. 2013. Histopathological Assessment and hematotoxicity of Dietary acrylamide on Wistar rats. IJLSCI, 7(1),

SC

21-25.

Alturfan, A.A., Tozan-Beceren, A., Şehirli, A.Ö., Demiralp, E., Şener, G., Omurtag, G.Z.

M AN U

2012. Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats. Mol. Biol. Rep. 39(4), 4589-4596. Armstrong, T.A., Spears, J.W., Lloyd, K.E., 2001. Inflammatory response, growth, and thyroid hormone concentrations are affected by long-term boron supplementation in

TE D

gilts. J. Anim. Sci. 79, 1549–1556.

Atef, H., Attia, G.M., Rezk, H. M., El-Shafey, M. 2017. Effect of vitamin E on biochemical

EP

and ultrastructural changes in acrylamide-induced renal toxicity in rats. Int. J. Sci. Rep. 3(5), 134-143.

AC C

Beutler, E., Duron, O., Kelly, B.M., 1993. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882-888.

Beyer, K.H., Bergfeld, W.F., Berndt, W.O., Boutwell, R.K., Carlton, W.W., Hoffmann, D.K., Schroeter, A.L. 1983. Final report on the safety assessment of sodium borate and boric acid. J. Am. Coll. Toxicol. 2(7), 87-125.

16

ACCEPTED MANUSCRIPT Bourgeois, A.C., Scott, M.E., Sabally, K., Koski, K.G., 2007. Low dietary boron reduces parasite (Nematoda) survival and alters cytokine profiles but the infection modifies liver minerals in mice. J. Nutr. 137(9), 2080-2086.

RI PT

Camacho, L., Latendresse, J.R., Muskhelishvili, L., Patton, R., Bowyer, J.F., Thomas, M., Doerge, D.R. 2012. Effects of acrylamide exposure on serum hormones, gene expression, cell proliferation, and histopathology in male reproductive tissues of

SC

Fischer 344 rats. Toxicol Lett. 211(2), 135–143.

Catalgol, B., Özhan, G., Alpertunga, B. 2009. Acrylamide-induced oxidative stress in human

M AN U

erythrocytes. Hum. Exp. Toxicol. 28(10), 611-617.

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.

TE D

Coban, F.K., Liman, R., Cigerci, İ.H., Ince, S., Hazman, O., Bozkurt, M.F., 2015. The antioxidant effect of boron on oxidative stress and DNA damage in diabetic

EP

rats. Fresen. Environ. Bull. 24(11 B), 4059-4066. Devirian, T.A., Volpe, S.L., 2003. The physiological effects of dietary boron. Crit. Rev. Food

AC C

Sci. Nutr. 43, 219–231.

Drabkin, D.L., Austin, J.H., 1935. Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J. Biol. Chem. 112, 51-65.

Draper, H.H., Hardley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421-431. Gasparrini, M., Forbes-Hernandez, T.Y., Giampieri, F., Afrin, S., Alvarez-Suarez, J.M., Mazzoni, L., Mezzetti, B., Quiles, J.L., Battino, M., 2017. Anti-inflammatory effect of

17

ACCEPTED MANUSCRIPT strawberry extract against LPS-induced stress in RAW 264.7 macrophages. Food Chem. Toxicol. 102, 1–10. https://doi.org/10.1016/j.fct.2017.01.018

Gedik, S., Erdemli, M.E., Gul, M., Yigitcan, B., Bag, H.G., Aksungur, Z., Altinoz, E. 2017.

RI PT

Hepatoprotective effects of crocin on biochemical and histopathological alterations following acrylamide-induced liver injury in Wistar rats. Biomed. Pharmacother. 95, 764-770.

M AN U

cardiovascular disease. Br. Med. Bull., 49: 679-99

SC

Gey, K.F., 1993. Prospects for the prevention of free radical disease, regarding cancer and

Giampieri, F., Alvarez-Suarez, J.M., Cordero, M.D., Gasparrini, M., Forbes-Hernandez, T.Y., Afrin, S., Santos-Buelga, C., González-Paramás, A.M., Astolfi, P., Rubini, C., Zizzi, A., Tulipani, S., Quiles, J.L., Mezzetti, B., Battino, M., 2017. Strawberry consumption

TE D

improves aging-associated impairments, mitochondrial biogenesis and functionality through the AMP-activated protein kinase signaling cascade. Food Chem. 234, 464– 471. https://doi.org/10.1016/j.foodchem.2017.05.017

EP

Guerin, P., El Mouatassim, S., Menezo, Y., 2001. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum.

AC C

Reprod. Update 7(2), 175-189.

Hamdy, S.M., Bakeer, H.M., Eskander, E.F., Sayed, O.N., 2012. Effect of acrylamide on some hormones and endocrine tissues in male rats. Human Exp. Toxicol. 31(5), 483491. Hunt, C.D., 1996. Biochemical effects of physiological amounts of dietary boron. J. Trace Elem. Exp. Med. 9, 185–213.

18

ACCEPTED MANUSCRIPT Hunt, C.D., 2005. Boron, in: Coates, P.M., Blackman, M., Cragg, G.M., Levine, M.A., Moss, J., White, J.D. (Eds.), Encyclopedia of Dietary Supplements, Marcel Dekker, New York, pp. 55-63.

RI PT

IARC, 1994. Some Industrial Chemicals. Lyon, France: International Agency for Research on Cancer

Ince, S., Arslan-Acaroz, D., Demirel, H. H., Varol, N., Ozyurek, H. A., Zemheri, F.,

SC

Kucukkurt, I. 2017. Taurine alleviates malathion induced lipid peroxidation, oxidative stress, and proinflammatory cytokine gene expressions in rats. Biomed. Pharmacother.

M AN U

96, 263-268.

Ince, S., Keles, H., Erdogan, M., Hazman, O., Kucukkurt, I. 2012. Protective effect of boric acid against carbon tetrachloride–induced hepatotoxicity in mice. Drug Chem. Toxicol. 35(3), 285-292.

TE D

Ince, S., Kucukkurt, I., Cigerci, I.H., Fidan, A.F., Eryavuz, A., 2010. The effects of dietary boric acid and borax supplementation on lipid peroxidation, antioxidant activity, and

EP

DNA damage in rats. J. Trace Elem. Med. Biol. 24, 161–164. Ince, S., Kucukkurt, I., Demirel, H.H., Acaroz, D.A., Akbel, E., Cigerci, I.H. 2014a.

AC C

Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats. Chemosphere 108, 197-204.

Ince, S., Acaroz, D. A., Neuwirth, O., Demirel, H. H., Denk, B., Kucukkurt, I., Turkmen, R. 2014b. Protective effect of polydatin, a natural precursor of resveratrol, against cisplatin-induced toxicity in rats. Food Chem. Toxicol. 72, 147-153. Rosen, J.D. 2002. Acrylamide in food: is it a real threat to public health? Am. Counc. Sci. Health. 19

ACCEPTED MANUSCRIPT Khan, M.R., Afzaal, M., Saeed, N., Shabbir, M. 2011. Protective potential of methanol extract of Digera muricata on acrylamide induced hepatotoxicity in rats. Afr. J. Biotechnol. 10(42), 8456–8464.

RI PT

Lakshmi, D., Gopinath, K., Jayanthy, G., Anjum, S., Prakash, D., Sudhandiran, G. 2012. Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex. Neurochem. Res. 37(9), 1859–1867.

SC

Liu, Z., Song, G., Zou, C., Liu, G., Wu, W., Yuan, T., Liu, X., 2015. Acrylamide induces mitochondrial dysfunction and apoptosis in BV-2 microglial cells. Free Radic. Biol.

M AN U

Med. 84, 42-53.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-75.

Luck, H., 1955. Catalase. in: Bergmeyer, H.U. (Ed.), Methods in Analysis. Academy Press,

TE D

London.

Mahmood, S.A., Amin, K.A., Salih, S. F. 2015. Effect of acrylamide on liver and kidneys in

EP

albino wistar rats. Int. J. Curr. Microbiol. App. Sci. 4(5), 434-44. Meacham, S.L., Taper, L.J., Volpe, S.L., 1994. Effects of boron supplementation on bone

AC C

mineral density and dietary, blood, and urinary calcium, phosphorus, magnesium, and boron in female athletes. Environ. Health Perspect. 102, 79–82.

Mehri, S., Abnous, K., Khooei, A., Mousavi, S.H., Shariaty, V.M., Hosseinzadeh, H., 2015. Crocin reduced acrylamide-induced neurotoxicity in Wistar rat through inhibition of oxidative stress. Iran J. Basic Med. Sci. 18(9), 902.

20

ACCEPTED MANUSCRIPT Nielsen, F.H., Hunt, C.D., Mullen, L.M., Hunt, J.R., 1987. Effect of dietary boron on mineral, estrogen, and testosterone metabolism in postmenopausal women. FASEB J. 1, 394– 397.

thiobarbituric acid reaction. Anal. Biochem. 95, 351-358.

RI PT

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by

Patel, P.G., Kapadiya, K.B., Patel, B.J., 2015. Protective effect of vitamin E on biochemistry,

M AN U

(Rattus norvegicus). Vet. Sci. Res. J. 6(1), 16-22.

SC

oxidative stress and histopathological alterations induced by acrylamide in wistar rats

Scorei, R., 2012. Is Boron a Prebiotic Element? A Mini-review of the Essentiality of Boron for the Appearance of Life on Earth. Orig. Life Evol. Biosph. 42, 3–17. Song, J., Zhao, M., Liu, X., Zhu, Y., Hu, X., Chen, F., 2013. Protection of cyanidin-3glucoside against oxidative stress induced by acrylamide in human MDA-MB-231

TE D

cells. Food Chem. Toxicol. 58, 306-310. Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide

EP

dismutase. Clin. Chem. 34, 497-500.

Totani, N., Yawata, M., Ojiri, Y., Fujioka, Y. 2007. Effects of trace acrylamide intake in

AC C

Wistar rats. J. Oleo Sci. 56(9), 501-506. Turkez, H., Geyikoglu, F., Tatar, A., Keles, S., Ozkanc, A., 2007. Effects of some boron compounds on peripheral human blood. Z. Naturforsch. 62, 889–896.

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.

21

ACCEPTED MANUSCRIPT Winterbourn, C.C., Hawkins, R.E., Brain, M., Carrell, R.W., 1975. The estimation of red cell superoxide activity. J. Lab. Clin. Med. 55, 337-341. Yang, H.J., Lee, S.H., Jin, Y., Choi, J.H., Han, C.H., Lee, M.H., 2005. Genotoxicity and toxicological effects of acrylamide on reproductive system in male rats. J. Vet. Sci.

RI PT

6(2), 103–109.

Yousef, M.I., El-Demerdash, F.M., 2006. Acrylamide-induced oxidative stress and

SC

biochemical perturbations in rats. Toxicology 219(1-3), 133-141.

Zamani, E., Shaki, F., AbedianKenari, S., Shokrzadeh, M., 2017. Acrylamide induces

apoptosis

in

mice

M AN U

immunotoxicity through reactive oxygen species production and caspase-dependent splenocytes

via

the

mitochondria-dependent

signaling

pathways. Biomed. Pharmacother. 94, 523-530.

Zhang, Y., Ren, Y., Zhang, Y., 2009. New Research Developments on Acrylamide:

TE D

Analytical Chemistry, Formation Mechanism, and Mitigation Recipes. Chem. Rev. 109, 4375–4397. https://doi.org/10.1021/cr800318s

EP

Zhang, J.X., Yue, W.B., Ren, Y.S., Zhang, C.X., 2010. Enhanced fat consumption potentiates acrylamide-induced oxidative stress in epididymis and epididymal sperm and effect

AC C

spermatogenesis in mice. Toxicol. Mech. Methods 20(2), 75-81. Zhang, L., Xu, Y., Li, Y., Bao, T., Gowd, V., Chen, W., 2017. Protective property of mulberry digest against oxidative stress–A potential approach to ameliorate dietary acrylamide-induced cytotoxicity. Food Chem. 230, 306-315. 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(9), 3306-3312.

22

ACCEPTED MANUSCRIPT

Figure Legends Fig 1. Effects of ACR and ACR plus B5, B10, and B20 on the liver and brain of IF-γ (A), IL-

RI PT

1β (B), NFĸB (C), and TNF-α (D) gene expressions by RT-PCR in male rats. ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at

AC C

EP

TE D

M AN U

SC

20 mg/kg.

23

ACCEPTED MANUSCRIPT

rats. Kidney

Liver

Heart

Brain

Lung

Testis

(nmol/ml)

(nmol/g tissue)

(nmol/g tissue)

(nmol/g tissue)

(nmol/g tissue)

(nmol/g tissue)

(nmol/g tissue)

Control

1,84±0,18c

2,02±0,48c

1,55±0,41c

1,41±0,16b

5,46±0,62d

3,42±0,85c

2,87±0,68b

ACR

3,41±0,83a

4,38±0,72a

3,22±0,27a

2,02±0,5a

14,78±1,43a

5,68±0,63a

5,63±0,80a

B5 + ACR

2,57±0,63b

3,11±0,71b

2,22±0,65b

1,97±0,29a

11,38±0,92b

5,25±0,73a

5,52±0,84a

B10 + ACR

2,02±0,36bc

3,50±0,49b

1,77±0,30c

1,83±0,45a

11,41±1,18b

4,50±0,53b

5,25±0,62a

B20 + ACR

2,07±0,49bc

3,27±0,83b

1,70±0,47c

1,66±0,37ab

7,83±1,13c

3,59±0,74c

3,45±0,64b

M AN U

SC

Blood

TE D

Treatment Design

RI PT

Table 1. Effects of ACR and ACR plus B5, B10, and B20 on malondialdehyde levels in blood, kidney, liver, heart, brain, lung, and testis of

ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

In the same column values with different letters show statistically significant differences in blood, kidney, liver, brain, lung, testis (p <

0.001), and heart (p < 0.05).

AC C

a,b,c

EP

Values are mean ± Standard deviations; n = 8.

1

ACCEPTED MANUSCRIPT

Blood

Kidney

Liver

Heart

Lung

Testis

(nmol/ml)

(nmol/g tissue)

(nmol/g tissue)

Control

42,92±5,83a

3,97±0,69a

4,80±1,02a

6,86±1,51

ACR

30,83±4,63c

2,05±0,65d

2,06±0,64c

B5 + ACR

34,77±4,99bc

2,21±0,52cd

B10 + ACR

35,90±3,81b

B20 + ACR

38,25±3,35ab

Brain

(nmol/g tissue)

(nmol/g tissue)

9,98±1,74a

6,44±1,41a

11,01±1,41a

5,05±0,99

5,07±0,89c

4,73±1,09b

5,17±1,31b

2,41±0,67c

5,89±1,23

5,40±1,04c

6,37±1,08a

5,50±1,52b

2,83±0,71bc

2,73±0,62bc

5,54±1,10

5,37±1,14c

6,27±1,17a

5,69±1,40b

3,18±0,60b

3,24±0,67b

5,62±0,95

8,29±2,00b

5,68±0,79ab

5,75±1,60b

M AN U

SC

(nmol/g tissue) (nmol/g tissue)

TE D

Treatment Design

RI PT

Table 2. Effects of ACR and ACR plus B5, B10, and B20 on glutathione levels in blood, kidney, liver, heart, brain, lung, and testis of rats.

ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

In the same column values with different letters show statistically significant differences in whole blood, kidney, liver, brain, testis (p <

0.001), and lung (p < 0.05).

AC C

a,b,c,d

EP

Values are mean ± Standard deviations; n = 8.

2

ACCEPTED MANUSCRIPT

testis of rats. Liver

Heart

Brain

Lung

Testis

(U/gHb)

(U/µg protein)

(U/µg protein)

(U/µg protein)

(U/µg protein)

(U/µg protein)

(U/µg protein)

Control

10,02±1,30b

7,09±1,48c

3,76±1,40d

10,09±1,89c

8,41±2,04c

5,44±1,50c

7,19±1,34b

ACR

16,67±6,06a

10,01±2,3a

7,15±0,77a

20,51±2,72a

23,12±4,42a

7,76±1,27a

10,95±2,13a

B5 + ACR

13,89±5,19ab

9,71±1,92ab

6,61±1,20ab

14,83±1,44b

18,66±3,15b

7,56±1,46ab

10,14±1,48a

B10 + ACR

13,76±3,57ab

8,56±1,90abc

5,46±1,31bc

14,37±2,01b

16,40±3,45b

6,19±1,24bc

8,50±1,40b

B20 + ACR

10,75±1,60b

7,91±1,78bc

5,17±1,15c

13,89±1,63b

17,53±3,55b

5,79±1,29c

8,18±1,18b

M AN U

SC

Kidney

TE D

Treatment Design Erythrocyte

RI PT

Table 3. Effects of ACR and ACR plus B5, B10, and B20 on superoxide dismutase activities in erythrocyte, kidney, liver, heart, brain, lung, and

ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

In the same column values with different letters show statistically significant differences in liver, heart, brain, testis (p < 0.001),

AC C

a,b,c

EP

Values are mean ± Standard deviations; n = 8.

erythrocyte, kidney (p < 0.05), and lung (p < 0.01).

3

ACCEPTED MANUSCRIPT

Liver

Heart

Brain

Lung

Testis

(k/gHb)

(k/µg protein)

(k/µg protein)

(k/µg protein)

(k/µg protein)

(k/µg protein)

(k/µg protein)

Control

13,18±2,32d

0,30±0,07c

26,12±5,96d

11,72±2,23c

4,59±1,31c

5,15±0,95c

4,28±1,35c

ACR

27,82±3,98a

0,76±0,11a

125,55±17,21a

54,75±9,06a

21,64±3,71a

10,15±1,29a

9,19±2,42a

B5 + ACR

21,12±4,07b

0,54±0,14b

71,89±14,68b

22,67±4,55b

5,17±1,55bc

10,14±1,13a

6,77±2,06b

B10 + ACR

18,46±4,35bc

0,51±0,11b

61,81±6,82bc

20,91±5,49b

5,21±1,56bc

10,11±1,07a

6,34±1,45b

B20 + ACR

17,02±3,94cd

0,43±0,11b

53,81±9,91c

12,12±3,13c

7,19±1,87b

6,76±1,43b

6,15±1,76bc

M AN U

SC

Kidney

TE D

Treatment Design Erythrocyte

RI PT

Table 4. Effects of ACR and ACR plus B5, B10, and B20 on catalase activities in erythrocyte, kidney, liver, heart, brain, lung, and testis of rats.

a,b,c,d

AC C

Values are mean ± Standard deviations; n = 8.

EP

ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

In the same column values with different letters show statistically significant differences in erythrocyte, kidney, liver, heart,

brain, lung, and testis (p < 0.001).

4

ACCEPTED MANUSCRIPT

Cholesterol

Triglyceride

HDL

LDL

BUN

Creatinine

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

84.54±7.15c

83.9±3.86c

78.58±5.34b

47.21±4.74a

27.19±2.88c

35.48±3.61c

0.91±0.15c

ACR

105.92±14.73a

94.59±5.34a

102.42±11.24a

38.03±4.27b

37.46±3.52a

43.77±3.51a

1.53±0.3a

B5 + ACR

99.97±10.85ab

89.8±5.12ab

84.12±6.71b

42.41±5.66ab

34.79±3.83ab

40.24±4.86ab

1.27±0.31ab

B10 + ACR

94.11±11.77bc

89.51±4.77ab

80.5±5.21b

43.42±6.32ab

34.19±2.07b

37.32±3.11bc

1.09±0.32bc

B20 + ACR

91.6±6.27bc

86.93±5.84bc

82.89±7.1b

45.56±7.95a

30.2±2.7c

37.21±5.33bc

1.02±0.25bc

M AN U

Control

SC

Glucose

TE D

Treatment Design

RI PT

Table 5. Effects of ACR and ACR plus B5, B10, and B20 on plasma glucose, cholesterol, triglyceride, HDL, LDL, BUN, and creatinine of rats.

a,b,c

AC C

Values are mean ± Standard deviations; n = 8.

EP

ACR: Acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

In the same column values with different letters show statistically significant differences (p < 0.05).

5

ACCEPTED MANUSCRIPT

Table 6 Effects of ACR and B5, B10, and B20 plus ACR on histopathological alterations in the brain, lung, heart, kidney, liver, and testis

Tissue

Histopathological

Control

ACR

-(8/8)

+(3/8)

RI PT

tissues of rats

B5+ACR

Degenerative changes of

+(6/8)

-(7/8)

-(4/8)

-(2/8)

+(1/8)

+(3/8)

-(8/8)

-(8/8)

+(4/8)

M AN U

Brain

B20+ACR

SC

alterations

B10+ACR

++(3/8)

neurons and focal gliosis

+++(2/8) Thickening in the

-(8/8)

interalveolar septal tissue

Heart

Hyaline degeneration

++(2/8)

-(5/8)

+(8/8)

-(6/8)

-(6/8)

-(7/8)

+(2/8)

+(2/8)

+(1/8)

-(1/8)

-(3/8)

-(7/8)

-(8/8)

+(4/8)

+(3/8)

+(1/8)

++(2/8)

++(2/8)

EP

alveolar capillaries

-(8/8)

AC C

Hyperemia in intra-

+(6/8)

TE D

Lung

-(8/8)

6

ACCEPTED MANUSCRIPT

Dilatation of sinusoids

-(8/8)

and hyperemia

Kidney

-(4/8)

-(4/8)

++(1/8)

++(4/8)

+(4/8)

+(8/8)

-(5/8)

-(4/8)

-(7/8)

+(3/8)

+(4/8)

+(1/8) -(8/8)

-(8/8)

Enlargement of

M AN U

Kupffer cell activation

+(7/8)

-(8/8)

Bowman’s space and

-(6/8)

-(6/8)

++(7/8)

+(2/8)

+(2/8)

-(1/8)

-(6/8)

-(7/8)

+(6/8)

+(2/8)

+(1/8)

tubulus epithelial cells

-(7/8)

-(6/8)

-(7/8)

Reduced spermatogenic density in tubulus

+(1/8)

+(2/8)

+(1/8)

seminiferus contortus

-(5/8)

-(4/8)

-(7/8)

-(8/8)

++(2/8)

-(8/8)

AC C

Testis

EP

necrobiotic changes in

TE D

-(8/8)

-(8/8)

+(1/8)

hyperemia Degenerative and

SC

Liver

RI PT

+++(1/8)

Giant cells formation in

-(8/8)

+(8/8)

+(8/8)

7

ACCEPTED MANUSCRIPT

tubulus seminiferus

+(3/8)

RI PT

contortus Normal histology (–), mild (+), moderate (++), and severe (+++)

+(4/8)

AC C

EP

TE D

M AN U

SC

ACR: acrylamide at dose 15 mg/kg; B5: boron at 5 mg/kg; B10: boron at 10 mg/kg; B20: boron at 20 mg/kg.

8

+(1/8)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT • Boron (B) reduces acrylamide (ACR) induced toxicity.

• B inhibits oxidative stress and restores LPO, GSH, SOD, and CAT in rats.

• B regenerates ACR-induced histopathological alterations in rat tissues.

AC C

EP

TE D

M AN U

SC

RI PT

•B attenuates inflammatory response and ameliorates biochemical alterations.