Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 04/08/2025 Aceptado: 19/12/2025 Publicado: 14/01/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 9 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico ABSTRACT Acrylamide is a known neurotoxic and potenally carcinogenic compound. It remains as a major public health concern due to its widespread presence in heat-processed foods. Despite extensive research on acrylamide-induced toxicity, eﬀecve dietary strategies to migate its harmful impact remain limited. Intermient fasng has recently emerged as a promising metabolic intervenon shown to enhance cellular stress resistance and improve anoxidant capacity. This study was designed to invesgate the eﬀects of intermient fasng on acrylamide-induced toxicity in rats. Wistar rats were randomly divided into four groups: control, acrylamide, intermient fasng, and acrylamide + intermient fasng. Intermient fasng was applied every other day, while acrylamide was administered intraperitoneally at a dose of 30 mg/kg/day, three mes per week. On day 28, behavioral assessments were performed using the Elevated Plus Maze, Open Field Test, hotplate, and rotarod tests. Biochemical analyses were conducted on blood samples, and oxidave stress parameters Catalase, Glutathione peroxidase, Superoxide dismutase were measured in liver, kidney, and brain ssues. Histopathological evaluaons were also carried out. Histopathological ﬁndings indicated ssue damage in the acrylamide group and paral improvement in the acrylamide + intermient fasng group. In the rotarod test, performance of the acrylamide + intermient fasng group was similar to the control group, suggesng a protecve eﬀect. Catalase, Glutathione peroxidase, and Superoxide dismutase levels showed paral amelioraon in kidney and brain ssues due to intermient fasng. The results suggest that intermient fasng may exert a protecve eﬀect against acrylamide-induced oxidave stress and behavioral impairments in rats. These ﬁndings highlight the potenal of intermient fasng as a non-pharmacological strategy to migate acrylamide toxicity. Key words: Acrylamide, intermient fasng, behavior, oxidave stress RESUMEN La acrilamida es un compuesto neurotóxico y potencialmente cancerígeno conocido. Sigue siendo un importante problema de salud pública debido a su amplia presencia en alimentos procesados térmicamente. A pesar de la extensa invesgación sobre la toxicidad inducida por acrilamida, las estrategias dietécas eﬁcaces para migar su impacto nocivo siguen siendo limitadas. El ayuno intermitente se ha converdo recientemente en una prometedora intervención metabólica que ha demostrado mejorar la resistencia celular al estrés y la capacidad anoxidante. Este estudio se diseñó para invesgar los efectos del ayuno intermitente sobre la toxicidad inducida por acrilamida en ratas. Las ratas Wistar se dividieron aleatoriamente en cuatro grupos: control, acrilamida, ayuno intermitente y acrilamida + ayuno intermitente. El ayuno intermitente se aplicó día por medio, mientras que, la acrilamida se administró por vía intraperitoneal a una dosis de 30 mg/kg/día, tres veces por semana. En el día 28 se realizaron evaluaciones conductuales mediante el Laberinto en Cruz Elevado, la Prueba de Campo Abierto, la prueba de placa caliente y la prueba de rotarod. Se efectuaron análisis bioquímicos en muestras de sangre, y se midieron parámetros de estrés oxidavo —Catalasa, Glutaón peroxidasa, Superóxido dismutasa— en hígado, riñón y tejido cerebral. También se llevaron a cabo evaluaciones histopatológicas. Los hallazgos histopatológicos indicaron daño sular en el grupo acrilamida y una mejora parcial en el grupo acrilamida + ayuno intermitente. En la prueba de rotarod, el rendimiento del grupo acrilamida + ayuno intermitente fue similar al del grupo control, lo que sugiere un efecto protector. Los niveles de Catalasa, Glutaón peroxidasa y Superóxido dismutasa mostraron una mejora parcial en riñón y cerebro debido al ayuno intermitente. Los resultados sugieren que el ayuno intermitente podría ejercer un efecto protector contra el estrés oxidavo y las alteraciones conductuales inducidas por acrilamida en ratas. Estos hallazgos resaltan el potencial del ayuno intermitente como una estrategia no farmacológica para migar la toxicidad de la acrilamida. Palabras clave: Acrilamida, ayuno intermitente, conducta, estrés oxidavo Eﬀect of intermient fasng on behavioral, biochemical and histopathological parameters in rats under Acrylamide exposure Efecto del ayuno intermitente sobre los parámetros conductuales, bioquímicos e histopatológicos en ratas expuestas a acrilamida Okan Arihan 1* , Asli San Dagli Gul 1 , Hurrem Turan Akkoyun 2 , Mahire Akkoyun 3 , Ahmet Uyar 4 , Tuncer Kutlu 4 , Ahmet Onur Dastan 1 , Aysen Erdem 1 , Sezgi Firat Ozgur 1 ¹Haceepe University, Faculty of Medicine, Department of Physiology, Ankara, Türkiye ²Siirt University, Veterinary Faculty, Department of Physiology, Siirt, Türkiye ³Siirt University, Veterinary Faculty, Department of Biochemisty, Siirt, Türkiye ⁴Hatay Mustafa Kemal University, Veterinary Faculty, Department of Pathology, Hatay, Türkiye Correspondence author: okanarihan@gmail.com https://doi.org//10.52973/rcfcv-e361765

Protecve eﬀect of intermient fasng against Acrylamide / Arihan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Acrylamide is a chemical formed in certain condions and unintenonally consumed with food. There are increasing number of publicaons which study eﬀect of acrylamide in nervous system, blood viscosity, cancer or nutrion [1]. In order to model diﬀerent exposure schemes of acrylamide on laboratory animals diﬀerent dosages and protocols are chosen [2],3,4]. Intermient fasng (IF), which involves restricng or completely abstaining food for certain periods, has recently become a widely studied subject due to its posive eﬀects on health [5]. Among the most well-known posive eﬀects of IF are weight loss and reducon of adipose ssue [6], increased insulin sensivity, control of blood glucose levels, dyslipidemia, altered blood pressure as well as a reducon in metabolic syndrome [7 , 8]. Addionally, its contribuon to cellular repair processes posively aﬀects the health of many ssues, including the brain ssue. It has been shown to aenuate neuroinﬂammaon and increase certain molecules such as brain-derived neurotrophic factor (BDNF) in brain ssue [9]. Various human studies have demonstrated the slowing of disease progression in Alzheimer’s disease, epilepsy as well as mulple sclerosis through IF. Its eﬀects on Parkinson’s disease, ausm spectrum disorder, and anxiety are mostly studied through animal research, with promising ﬁndings [10 , 11]. Intermient fasng can help the body detoxify and eliminate metabolic waste. It is known to reduce the eﬀects of exposure to harmful chemicals on the body. Although some animal studies have shown promising results in reducing the side eﬀects of chemotherapeucs, its eﬀecveness in cancer treatment is controversial due to the potenal adverse eﬀects of fasng in cancer paents [12]. Evaluang the eﬀects of IF on stress and anxiety through human studies can be challenging when considering individuals’ beliefs, especially with religious fasts. Nonetheless, a meta- analysis conducted in 2021 involving 1,436 paents indicated that symptoms of stress, anxiety, and depression decreased in individuals aﬅer Ramadan fasng compared to control groups [13]. Conversely, a 2023 meta-analysis that excluded the inﬂuence of religious beliefs found no signiﬁcant diﬀerences in anxiety and mood changes, although there was a decrease in depression scale scores [14]. Considering the diverse outcomes in the literature aim of this study is to invesgate the potenal protecve eﬀects of IF on behavioral, biochemical, histological, and oxidave stress parameters during acrylamide exposure in male rats (Raus norvegicus). MATERIALS AND METHODS Twenty-eight male Wistar-albino rats, aged 4-8 weeks and weighing between 200-250 grams (g) (Meler PM600), were used. During the acclimizaon, the rats were provided with water and food ad libitum without restricon. To ensure standard condions prior to the study, all rats were given the same standard pellet food, water, and care condions (temperature: 20 ± 2 ºC, 12-hour light/dark cycle). Then the animals were randomly assigned to four groups (n = 7 in each groups). The experimental procedures was carried out over 4 weeks. All administraons were conducted aﬅer an ethical approval from Haceepe university Animal Experiments Ethics Commiee (decision number= 2020/11-09). Control Group (C): Intraperitoneal (i.p.) saline injecons were given three mes a week. Acrylamide Group (AC): Rats received AC at a dose of 30 mg/kg/day (d) i.p. on three non-consecuve d per week (e.g. Monday, Wednesday and Friday), for a total duraon of 4 weeks (12 injecons; cumulave dose: 360 mg/kg). The IF Group: Was applied to the rats every other d (24 h fasng / 24 h feeding). Acrylamide+Intermient Fasng Group (ACIF): 30mg/kg/d i.p. acrylamide was given three mes a week+intermient fasng was applied every other d. Aﬅer the experimental procedures, Open Field Test and Elevated Plus Maze anxiety tests as well as rotarod and hot plate tests were conducted in the given order. In the literature diﬀerent exposures are given to animals [2]. Over the 28-d experimental period, this protocol resulted in a cumulave acrylamide dose of 360 mg/kg (3 injecons/week × 30 mg/kg × 4 weeks). Behavioral tests All behavioral tests were performed on d 28 of the experimental protocol. The IF schedule was arranged to ensure that all animals—whether in the IF or non-IF groups—were tested on a morning following a non-fasted night, minimizing potenal confounding eﬀects of acute fasng. Food was available ad libitum on d 1, removed on d 2 morning, and reintroduced on d 3 morning, connuing in a 24-h fasng / 24-h feeding cycle throughout the 28-d protocol. Thus, behavioral tests were conducted on the morning of d 28, when both IF and C groups had food available overnight. On d 28, all animals underwent behavioral tesng in the following ﬁxed order: Open Field Test → Elevated Plus Maze → Rotarod → Hot Plate Test. All tests were performed during 09:00–12:00. Open ﬁeld test Locomotor acvity and anxiety-related behavior were assessed with open ﬁeld test. Time spent in the center and perimeter area, the number of rearing events, and the number of grooming behaviors was recorded (5 min) in an open ﬁeld square box with a deﬁned perimeter and center. The apparatus was wiped with 70 % ethanol aﬅer each animal trial. Elevated plus maze test Following Open Field Test, rats were placed in the center of the plus-shaped maze facing an open arm. The number of entries into the open and closed arms, the me spent in these arms, and the percentages were evaluated in an elevated plus maze (5 min). An increase in the me and percentage spent in the closed arm was considered an indicator of increased anxiety. The apparatus was wiped with 70 % ethanol aﬅer each animal trial. 2 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Rotarod test Following elevated plus maze test, Rotarod test was conducted to assess locomotor acvity in the rats in which, the duraon of the rats on a rod rotang at 15 rpm was recorded. Following a habituaon period three consecuve rotarod tesng sessions were recorded and those recordings were included in the stascal analyses. Hotplate test The rats were placed on a plaorm heated to 52 °C, and the time to withdraw, lick or shake their paws due to the heat was recorded (Rotarod and hotplate devices were manufactured by the Haceepe University researchers according to relevant literature). Blood biochemistry Immediately aﬅer compleon of behavioral tesng, animals were sacriﬁed by high-volume blood collecon under anesthesia, and measurements were made from the blood withdrawn into tubes. Relevant parameters; alkaline phosphatase (ALT), aspartate aminotransferase (AST), triglycerides, high density lipoproteins (HDL), low density lipoproteins (LDL), total cholesterol, creanine, total protein, blood glucose, insulin were evaluated (Beckman coulter AU5800 and Unicel DXL800, USA) [15]. Oxidave stress analyses Catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) were examined (Ransod SD-125, Ransel RS- 504, Randox, UK) in the liver, kidney, and brain ssues of the rats. Relevant ssues were washed with 0.9 % NaCl and stored at -80 °C until analysis. Tissue samples (0.5 g) were homogenized in 5 mL of ice-cold homogenization buffer (1 mmol/L) using an Ultra Turrax T25 homogenizer (IKA, Staufen, Germany) and an ultrasonic homogenizer (20 KHz frequency, Bandelin Sonupuls). The buffer contained EDTA, 0.32 mol/L sucrose, and 10 nmol/L Tris-HCl, pH 7.4. The homogenization process lasted 8 min, followed by centrifugation at 10118 g (Hech EBA 20 UK) for 30 min [16]. Procedures were carried out at 4 °C. The supernatants were used to determine the activity of antioxidant enzymes. The supernatants from liver, kidney, and brain tissue homogenates were used to evaluate the antioxidant enzymes SOD, glutathione peroxidase (GSH-Px), and CAT. CAT Determinaon: CAT (EC 1.11.1.6) acvity was measured at 240 nm wavelength using the method described by Aebi [17]. GPX Determinaon: Tissue GSH-Px (EC1.11.1.9) acvity was measured at 340 nm wavelength according to the method of Paglia and Valenne [18]. SOD Determinaon: Tissue SOD (EC 1.15.1.1) acvity was determined at 505 nm wavelength using the method of Sun et al. [19]. CAT, GPX and SOD was determined with Shimadzu UV- 1800 UV-VIS Spectrophotometer, Kyoto, Japan. Histopathological examinaon Liver and kidney ssues were ﬁxed in 10 % buﬀered formaldehyde soluon for 72 h. The ssue samples were placed in an automac ssue processor (Leica TP 1020, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and underwent roune processing: dehydraon through a series of alcohols (70, 80, 90 and 100 %), clearing through a series of Xylene, and embedding in paraﬃn. From these blocks, serial secons of 5 microns thick were taken using a microtome (Leica RM 2135, Leica TP 1020, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and stained with roune Hematoxylin-Eosin (HE) staining technique. All stained slides were examined and photographed using a light microscope with a digital imaging system (Olympus DP12BSW, Tokyo, Japan) Histopathological alteraons in the liver and kidney ssues of each group (congeson, degeneraon in hepatocytes, bile duct proliferaon, and degeneraon in tubular epithelium) were evaluated qualitavely by two pathologists according to relevant scoring systems [20 ,[21]. The scoring system was as follows: (-) score (negave score); no structural changes, (+) score (1 posive score); mild degree, (++) score (2 posive scores); moderate degree, (+++) score (3 posive scores); severe structural changes. Stascal analysis SPSS 20 soﬅware was used for stascal analysis. Data are presented as mean ± standard deviaon and P < 0.05 was considered signiﬁcant. The analyzes were determined according to whether the data was parametric or not. The Shapiro−Wilk test was used to determine the normality of data distribuons, while the variance homogeneity of groups was determined by the Levene test. Data were evaluated using the Kruskal-Wallis test. For comparisons, the Mann-Whitney-U test and Bonferroni correcon was used. RESULTS AND DISCUSSION Protecve eﬀect of IF against acrylamide exposure was invesgated through behavioral, oxidave stress, blood biochemical and histopathological analyses. Results from the examined parameters indicate that intermient fasng exhibits a migang eﬀect on the negave eﬀects caused by acrylamide. Weight monitoring of groups The average weights on the ﬁrst and last days of the experiment were as follows: • C group: 229 g-266 g • AC group: 230 g-238 g • IF group: 224 g-214 g • ACIF group: 225 g-195 g 3 of 9

Protecve eﬀect of intermient fasng against Acrylamide / Arihan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Behavioral ﬁndings Open ﬁeld test The number of unsupported rearing events was signiﬁcantly lower in all groups (AC 3.43 ± 5.65, IF 2.43 ± 2.70, ACIF 2.0 ± 1.53) compared to the C group (12.29 ± 7.32). Elevated plus maze A signiﬁcant diﬀerence was observed in the percentage of me spent in the closed/open areas between the AC (68.43 ± 41.58) and IF (7.86 ± 11.25) groups. The percentage of me spent in the closed/open arms was signiﬁcantly higher in the AC group but not in ACIF group compared to the IF group. This indicates an increase in anxiety due to AC exposure and a paral protecve eﬀect induced by IF against AC. Acrylamide is known to cause toxicity at both macro and cellular levels in the nervous system [22]. It causes oxidave damage, neuroinﬂammaon as well as disrupng metabolism [23]. Its causes disturbance to the secreon of neurotransmiers, altering acetylcholinesterase acvity [24]. On the other hand, evidence from animal studies suggests that IF reduces beta-amyloid accumulaon, increases synapc adaptaons in the hippocampus, improves cognive funcons, reduce neurodegeneraon, neuronal and cognive damage in mice following traumac brain injury or epilepsy [25 , 26 , 27]. It also augments BDNF, Neurotrophin 3 levels in rats thereby alleviang behavioral outcomes in some condions such as diabetes [28]. Another protecve eﬀect of IF comes from decreasing oxidave stress and balancing authophagy mechanisms in certain condions [29]. It is also suggested that IF also causes alleviaon in the brain energy metabolism via ketone bodies [30]. Rotarod The lowest value was observed in the AC group (16.1 ± 14.1s), and the highest value was observed in the IF group (50.7 ± 14.9s). In the C group, this value was found to be 47 ± 16.9s, while in the ACIF group, it was 23 ± 19.1s. The AC group walked for a signiﬁcantly shorter me compared to the C and IF groups (P < 0.05). Consistent with expectaons [31], AC administraon reduced walking me in the rotarod test in this current study. Fasng in the ACIF group parally reversed the decrease caused by AC, although not stascally signiﬁcant. AC and its derivaves causes peripheral nerve damage and myelin loss which causes morphological damage to such nerves [32]. It also causes mitochondrial dysfuncon and triggers necroptosis in Purkinje cells [33]. IF is known to have posive changes in mitochondrial funcon [34]. During IF some molecular markers are suggested such as BDNF, Gamma-Aminobutyric Acid, Growth Hormone/ Insulin-like Growth Factor-1, Fibroblast Growth Factor 2 and ketone bodies which end up with augmented resilience against cellular stress resistance and enhanced neurogenesis and synapc plascity [35]. The results observed in the present study may be linked to the ﬁndings reported in scienﬁc literature. Hot plate The longest withdrawal me was observed in the AC group (11.7 ± 3.5s), while the shortest me was observed in the C (7 ± 1.7s). IF group had a me close to that of the C group (7.6 ± 3s). Although ACIF group (8.3 ± 3.2s) was lower than the AC group, there was no signiﬁcant diﬀerence among the groups (P > 0.05). Experimental procedure was set to 28 d in accordance with relevant literature [36]. Studies showed signiﬁcant alteraons in hotplate test due to acrylamide administraon [37]. This unparallel results may be due to diﬀerent experimental protocols. Blood biochemistry Blood cholesterol (HDL, LDL, total cholesterol (TCHOL)) and TG levels were reduced in IF and ACIF groups compared to C and AC which are signiﬁcant in TG (C versus ACIF), TCHOL (C and AC versus ACIF), HDL (C and AC versus ACIF) and LDL (C and AC versus ACIF). A similar paern is also present in total protein (TPROT) where AC have signiﬁcantly higher value compared to IF and ACIF (TABLE I). TABLE I Blood biochemistry results Groups C AC IF ACIF HDL 41.00±6.87 a 41.57±8.60 a 30.71±6.47 ab 28.14±1.21 b LDL 21.33±4.32 a 20.71±4.72 a 14.43±3.41 ab 13.00±1.15 b CRE 0.18±0.02 ab 0.21±0.04 a 0.14±0.01 b 0.16±0.04 ab TPROT 6.24±0.31 ab 6.33±0.41 a 5.73±0.35 b 5.78±0.27 b TG 64.83±15.38 a 48.29±18.38 ab 43.43±11.47 ab 40.86±8.05 b TCHOL 61.50±9.35 a 60.43±12.82 a 44.57±9.55 ab 41.57±1.62 b C: Saline, AC: Acrylamide, IF: intermient fasng, ACIF: acrylamide + intermient fasng (n = 7 in each group). Diﬀerent leers (a, b, c) indicate stascally signiﬁcant diﬀerence. P < 0.05. HDL: High Density Lipoprotein, LDL: Low Density Lipoprotein. CRE: Creanine. TPROT: Total protein. TG: Triglyceride. TCHOL: Total cholesterol These observed aenuaons can be linked with every other day fasng. However increased creanine (CRE) in AC can be due to eﬀects of AC administraon on kidney ssue. Since CRE value is not increased in ACIF as in AC it can be suggested that fasng may have protected kidneys agains harmful eﬀects of AC exposure which is in parallel with histopathology. On the other hand no signiﬁcant change in ALT and AST values needs to clariﬁed since liver histopathology showed prominent impact of AC on liver. Oxidave stress Oxidave stress ﬁndings indicated a similar paern in the brain, kidneys, and liver, showing comparable levels of CAT, GPX, and SOD enzymes. It was observed that the results, which were similar between the control and IF, decreased in the AC group, indicang that intermient fasng either parally reversed or maintained this decrease at the same level, as seen in the ACIF group (TABLE II). 4 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE II Results of the biochemical analyses of diﬀerent oxidave parameters found in the liver, kidney, and brain ssues Biochemical Parameters C AC IF ACIF Liver CAT (EU/mg protein) 9.95 ± 1.54 a 5.25 ± 0.90 b 9.27 ± 1.69 a 5.49 ± 1.90 b SOD (EU/mg protein) 6448.57 ± 44.78 a 2746.31 ± 964.59 b 6516.00 ± 156.85 a 2733.05 ± 1615.77 b GPX (EU/mg protein) 0.21 ± 0.01 a 0.15 ± 0.02 ab 0.21 ± 0.04 a 0.14 ± 0.02 b Kidney CAT (EU/mg protein) 18.37 ± 3.12 a 11.04 ± 0.98 b 17.63 ± 2.36 a 14.10 ± 2.92 ab SOD (EU/mg protein) 1493.26 ± 131.46 a 1040.52 ± 70.92 b 1485.59 ± 274.88 a 1213.59 ± 144.06 ab GPX (EU/mg protein) 14.35 ± 1.40 a 9.83 ± 1.89 b 14.34 ± 2.09 a 10.63 ± 1.12 b Brain CAT (EU/mg protein) 65.56 ± 4.55 a 34.97 ± 4.78 b 58.83 ± 3.97 a 38.95 ± 5.39 ab SOD (EU/mg protein) 7159.33 ± 691.14 a 2740.39 ± 928.63 b 6736.57 ± 1083.74 a 2863.79 ± 499.14 b GPX (EU/mg protein) 0.24 ± 0.06 a 0.12 ± 0.02 c 0.22 ± 0.03 ab 0.13 ± 0.02 bc C: Saline, AC: Acrylamide, IF: Intermient fasng, ACIF: Acrylamide + intermient fasng (n = 7 in each group). Diﬀerent leers (a, b, c) indicate stascally signiﬁcant diﬀerence. P < 0.05. Catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) Studies indicate that acrylamide induces oxidave stress in the kidneys. Rats administered AC at 38.27 mg/kg showed decreased levels of SOD and GSH and increased levels of malondialdehyde (MDA) in their kidneys [22]. AC also increases interleukin-1β which is an inﬂammatory interleukin [38]. In another study the gastric gavage administraon of 50 mg/ kg acrylamide to rats for 11 d leads to kidney damage. Diﬀerent substances such as plant based ingredients [39] or endogenous molecules such as melatonin are tested to alleviate negave eﬀects of AC [40]. However this current study presents another choice for migang adverse eﬀects of AC via IF. IF is known to exhibit protecve eﬀects against various condions, including oxidave stress. IF is a process that supports the degradaon of damaged organelles through autophagy and maintains cellular homeostasis. Important signaling molecules such as mechanisc Target of Rapamycin, Adenosine monophosphate-Acvated Protein Kinase, and Sirtuin 1 are involved in this process. There is increasing evidence suggesng that IF plays a role in reducing damage in condions of oxidave stress by restoring the oxidant-anoxidant balance and supporng autophagy [41]. IF enhances intracellular levels of the protecve anoxidant GSH while reducing MDA, a terminal product of lipid peroxidaon [42]. In diabec rats, it has been shown that 4 weeks of IF reduces resisn, Sterol Regulatory Element-Binding Protein-1c (SREBP- 1c) and inﬂammatory cytokines/enzymes (TNF-α, IL-6, IL-1ß, Myeloperoxidase (MPO)), and decreases the elevated levels of SOD, CAT, and GSH associated with diabetes [43]. This current study data demonstrate that IF reduces increased oxidave stress induced by acrylamide administraon, aligning with literature ﬁndings. Notably, the scienﬁc literature reports that alternang IF enhances anoxidant capacity and aenuates oxidave stress to a greater extent than restricted feeding [44], aligning well with our ﬁndings. Histopathological ﬁndings The livers of rats in the C and IF groups exhibited normal histological structure (FIG. 1a). In the AC group, moderate congeson and hepatocellular hydropic degeneraon were present. Addionally, mild fay degeneraon, Kupﬀer cell proliferaon, and bile duct proliferaon were observed (FIG.1b and 1c). In the ACIF group, degenerave changes were mild (FIG.1d). Intermient fasng was found to reduce the hydropic degeneraon and congeson induced by AC in the liver (TABLE III). FIGURE 1. Histopathological ﬁndings observed in the liver, H.E. a) Normal histological struc- ture in the liver, C group. b) Degenerave changes (arrows) in the liver, AC group. c) Bile duct proliferaon (arrows) and Kupﬀer cell proliferaon (arrowheads) in the liver, AC group. d) Mild degenerave changes in the liver, ACIF group. C: Saline, AC: Acrylamide, IF: intermit- tent fasng, ACIF: acrylamide+intermient fasng (n = 7 in each group) Signiﬁcant hepatocellular hydropic degeneraon was observed in the AC group. In the ACIF group, however, the presence of less damage and a similar paern observed in the hyperemia parameter demonstrates a persistent detrimental eﬀect of AC exposure. Bile duct proliferaon was observed only in the AC group. Unlike these three parameters, fay degeneraon was observed in both the AC and ACIF groups. 5 of 9

Protecve eﬀect of intermient fasng against Acrylamide / Arihan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In a study wistar rats were subjected to IF for 35 d including 18 h of fasng. Histopathological analysis revealed congested central vessels and dilated sinusoids, as well as increased Kupﬀer cell density, were less prominent in the IF groups and were found to resemble a normal hepac appearance [45]. IF has been shown to have correcve eﬀects on hepatotoxicity associated with diabetes (reducing blood sinusoids and inﬂammatory leukocyte inﬁltraon) [46]. TABLE III Liver histopathological ﬁndings C AC IF ACIF Hydropic degeneraon in hepatocytes - ++ - + Hyperemia - ++ - + Bile duct proliferaon - + - - Fat degeneraon - + - + C: Saline, AC: Acrylamide, IF: intermient fasng, ACIF: Acrylamide + intermient fasng (n = 7 in each group). Posive eﬀects of fasng pracces on liver and kidney funcon have also been reported in humans [47]. IF reduces inﬂammaon, oxidave stress, and apoptosis in the senile rat liver, thereby alleviang histological deterioraon [48]. The hepatoprotecve eﬀects of IF are also associated with several molecular mechanisms involving hepac transcripon factors (resisn, SREBP-1c), as well as IL-1β, IL-6, MPO and TNF-α [43]. In the kidneys, the C and IF groups exhibited a normal histological structure (FIG. 2a). Moderate tubular epithelial hydropic degeneraon and congeson was present in the AC group (FIG. 2b and 2c). Mild degenerave changes were observed in the ACIF group (FIG. 2d) (TABLE IV). In the kidneys, while tubular epithelial hydropic degeneraon and congeson were observed in the AC group, only tubular epithelial hydropic degeneraon was observed in the ACIF group, and this parameter was found to be milder compared to the AC group, indicang a protecve eﬀect on kidney tubular epithelial cells. Diﬀerent studies in the literature have also shown histopathological damage caused by AC administraon using various doses and administraon protocols. Histopathological analyses of the kidneys in rats administered 38.27 mg/kg AC have revealed changes indicave of damage [22]. Conversely, IF decreases kidney injury during the progression from acute to chronic renal damage by aenuang ﬁbrosis, inﬂammaon, and oxidave stress [49]. IF has also been shown to reduce age-related renal inﬂammaon, oxidave stress, apoptosis and to modulate autophagy acvaon in aged rats [50]. These histopathological results may be interpreted in light of the mechanisms outlined in the aforemenoned studies. FIGURE 2. Histopathological changes observed in the kidneys. H.E. a) Normal histological structure in the kidneys, C group. b) Tubular epithelial cell hydropic degeneraon in the kidneys, AC group. c) Congeson in the kidneys, AC group. d) Resembling normal structure in the kidneys, ACIF group. C: Saline, AC: Acrylamide, IF: intermient fasng, ACIF: Acrylamide+intermient fasng (n = 7 in each group) TABLE IV Kidney histopathological ﬁndings C AC IF ACIF Hydropic degeneraon - ++ - + Hyperemia - ++ - - C: Saline, AC: Acrylamide, IF: intermient fasng, ACIF: Acrylamide + intermient fasng (n = 7 in each group). CONCLUSION Acrylamide exposure leads to signiﬁcant eﬀects in terms of histopathology, oxidave stress, behavior and serum biochemistry. Intermient fasng cause a complete improvement in ACIF group in liver bile duct proliferaon and kidney hyperemia as well as a paral improvement in CAT, SOD (kidney), CAT, GPX (brain) values, hydropic degeneraon in hepatocytes and hyperemia in liver, hydropic degeneraon in the kidney and rotarod walking period. As a result it can be concluded that intermient fasng exerts a protecve eﬀect on rats in this experimental design. ACKNOWLEDGEMENTS This project is supported by Haceepe University Scienﬁc Research Projects Coordinaon Unit with the Project number THD-2021-19804. Conﬂict of interest Authors declare no conﬂict of interest. 6 of 9

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