Received: 23/01/2026 Accepted: 23/04/2026 Published: 22/05/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 10 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI hps://doi.org/10.52973/rcfcv-e362908 The inﬂuence of alive or heat - killed Limosilactobacillus reuteri administraon on some serum anoxidant and cytokine levels in rats exposed to cadmium Efecto de la administración de Limosilactobacillus reuteri vivo o muerto por calor en los niveles de algunos anoxidantes y citocinas en la suero de ratas expuestas al cadmio İhsan Kisadere 1,* , Hasan Susar 2 , Tevhide Elif Güner 3 ¹ Balıkesir University, Faculty of Veterinary Medicine, Department of Physiology. Balıkesir, Türkiye ² Balıkesir University, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Balıkesir, Türkiye ³ Balıkesir University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology. Balıkesir, Türkiye Correspondıng author: ihsan.kisadere@balikesir.edu.tr ABSTRACT This study aimed to examine the eﬀects of alive or heat - killed Limosilactobacillus reuteri (L. reuteri) administraon on some blood anoxidant and cytokine levels in rats exposed to chronic cadmium toxicity. For this purpose, rats (n = 48) were randomly separated into six equal groups : cadmium, alive L. reuteri, cadmium + alive L. reuteri, dead L. reuteri, cadmium + dead L. reuteri, and control. cadmium was administered orally to the cadmium, cadmium + alive L. reuteri, and cadmium + dead L. reuteri groups three mes a week for four weeks. Also, alive or heat - killed L. reuteri were administered orally to alive L. reuteri, dead L. reuteri, cadmium + alive L. reuteri, and cadmium + dead L. reuteri groups at the same periods. Aﬅer the treatment period, serum samples were obtained from the animals and analyzed for serum cadmium and glutathione peroxidase levels, catalase and superoxide dismutase enzyme acvies, malondialdehyde concentraon, and tumor necrosis factor - alpha (TNF - α), interleukin - 6 (IL - 6), IL - 10, and IL - 1beta (β) levels. The administraon of alive or heat - killed L. reuteri suspension decreased the cadmium accumulaon in serum samples of alive L. reuteri and dead L. reuteri (especially) group animals compared to the cadmium group (P < 0.05). While the administraon of alive L. reuteri lowered serum superoxide dismutase enzyme acvity, the dead L. reuteri decreased glutathione peroxidase levels in comparison to the control group (P < 0.05). In conclusion, alive and dead L. reuteri strains had varied protecve eﬀects on the serum cytokine levels of rats, but their eﬀects on oxidave stress indicators were limited. Key words: Cadmium; cytokines; L.reuteri; rat. RESUMEN El propósito de este estudio fue examinar cómo la administración de Limosilactobacillus reuteri (L. reuteri), ya sea en su forma viva o muerta por calor, inﬂuye sobre los niveles de ciertos anoxidantes y citocinas en la sangre de ratas expuestas a la toxicidad crónica del cadmio. Para ello, se separó aleatoriamente a las ratas (n = 48) en seis grupos homogéneos : cadmio, L. reuteri vivo, cadmio + L. reuteri vivo, L. reuteri muerto, cadmio + L. reuteri muerto y grupo de control. Se administró cadmio por vía oral a los grupos cadmio, cadmio + L. reuteri vivo y cadmio + L. reuteri muerto tres veces por semana durante cuatro semanas. Se dio L. reuteri, ya sea vivo o muerto por calor, por vía oral a los grupos L. reuteri vivo, L. reuteri muerto, cadmio + L. reuteri vivo y cadmio + L. reuteri muerto durante el mismo empo. Tras el periodo de tratamiento, se obtuvieron muestras de suero de los animales. Se estudiaron los niveles de cadmio y glutaón peroxidasa en la sangre, así como las acvidades de las enzimas catalasa y superóxido dismutasa. Se evaluaron también los niveles de malondialdehído (MDA) y los niveles de factor de necrosis tumoral alfa (TNF - α), interleucina - 6 (IL - 6), IL - 10 e IL - 1β. La administración de L. reuteri, ya sea vivo o muerto por calor, redujo la acumulación de cadmio en muestras de suero de los animales de los grupos L. reuteri vivo y L. reuteri muerto (especialmente) en comparación con el cadmio (P < 0,05). La administración de L. reuteri vivo redujo la acvidad de la enzima superóxido dismutasa en suero, mientras que, el L. reuteri muerto disminuyó los niveles de glutaón peroxidasa en comparación con el grupo control (P < 0,05). En conclusión, las cepas vivas y muertas de L. reuteri tuvieron diversos efectos protectores sobre los niveles séricos de citocinas en ratas, pero sus efectos sobre los indicadores de estrés oxidavo se limitaron. Palabras clave: Cadmio; citocinas; L. reuteri; rata.

The Protecve Eﬀect of Limosilactobacillus reuteri in Cadmium Toxicity / Kisadere et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Cadmium (Cd) toxicity is one of the most common cases of toxic exposure to heavy metals worldwide, parcularly in industrialized countries. Worldwide, it has a detrimental eﬀect on human health, animals, and the ecosystem [1]. Cd is released into the environment through natural causes such as volcanic erupons, forest ﬁres, soil erosion, sea salt aerosols, and the chemical / physical breakdown of rocks containing Cd [2 ,3]. The classiﬁcaon of Cd as a toxic, carcinogenic, and smulant element has resulted in extensive study of its biological signiﬁcance over the past few decades [4]. The widespread availability of Cd in air, food, feed, and water is an important problem regarding human and animal health. Previous studies have esmated that heavy metals pollute 13 % of farmland and 40 % of the world’s rivers and lakes [5]. The binding of Cd to blood erythrocytes (RBC) and albumine (Alb) following its entry into an organism through inhalaon, food, feed, or water results in the reaching of diﬀerent important ssues and organs. Then, it triggers the producon of metallothionein mRNA by binding to low molecular weight proteins to form metallothioneins. It enters the bloodstream following renal glomerular ﬁltraon processes and is reabsorbed by proximal epithelial cells [6]. Numerous studies suggest that prolonged exposure to Cd can result in numerous diseases that aﬀect humans, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, mulple sclerosis, diabetes, cardiovascular disease, decreased kidney funcon, pancreac damage, and cancer [7 ,8, 9 , 10]. Prior research on the topic has revealed that exposure to Cd results in the producon of reacve oxygen species (ROS), which subsequently causes oxidave damage to cells. Addionally, it has been demonstrated that Cd mostly binds to anoxidant enzymes that include thiols, which lowers the amounts of these enzymes in the living organism [11]. It has also been observed that Cd exposure aﬀects the malondialdehyde (MDA) concentraons and some anoxidant enzyme acvies, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), in living beings [12 , 13]. The process of inﬂammaon is complicated and mulfaceted. It is an organism’s defense mechanism against injury (from physical smuli, chemicals, etc.) or infecon (from microbes). Its funcons include removing the agents that cause harm, prevenng ssue damage, starng the healing process, and reestablishing the physiological funcons of the ssue or organ that is impacted by the inﬂammatory process [14]. It has been found that oxidave stress (OS) and inﬂammaon are inmately associated with each other. Previous studies have indicated that Cd causes OS and also aﬀects certain cytokine levels such as tumor necrosis factor alpha (TNF - α), interleukin - 6 (IL - 6), IL - 10, and IL – 1 - beta (β) in diﬀerent ssues of the body [15 , 16]. A variety of approaches have been invesgated to reduce the toxicity of Cd, with an increasing amount of focus being placed on the use of chemical agents, herbal remedies, and detoxiﬁcaon strategies [17 , 18]. Heavy metals can be eliminated from the body cost-eﬀecvely and in an environmentally friendly way using microbiological methods. Microbiological procedures indicate that numerous beneﬁts encompass enhanced processing ability, high environmental adaptability, cost - eﬀecveness, and ecological sustainability. The cell walls of fungi contain polyphosphoric acid, chin and other ingredients that can react with elements such as heavy metals. Also, fungi are microorganisms that have the capacity to eliminate heavy metals that are present in the natural environment [19 , 20]. In addion to fungi, certain beneﬁcial microorganisms of food origin have aracted aenon for their detoxiﬁcaon capabilies. Lacc acid bacteria (LAB), parcularly L. reuteri, are frequently employed as probiocs in fermented milk and non - dairy foods. These microorganisms not only help to preserve food and increase nutrional quality, but they also improve food safety by lowering hazardous chemicals such as mycotoxins and heavy metals. Therefore, it is important to invesgate the ability of L. reuteri to regulate cadmium toxicity in the context of public health and food safety [21 , 22]. In recent years, researchers have been reported that speciﬁc strains of LAB possess the capability to bind and detoxify Cd in both in - vitro and in - vivo circumstances [23]. It was suggested that Lactobacillus and Biﬁdobacterium probioc strains that decrease systemic absorpon by binding Cd ions in the gastrointesnal tract [24 , 25]. Limosilactobacillus reuteri (L. reuteri), which was previously classiﬁed within the Lactobacillus genus, is naturally found in various systemic areas, including the digesve system, urinary system, skin, and mammary ssue. The amount of L. reuteri has been shown to vary from person to person and depending on individual circumstances. Many L. reuteri strains have been shown to play diﬀerent roles in various diseases, such as hypercholesterolemia, skin infecons, allergy - induced asthma, periodons, and ausm spectrum disorders. For example, in allergy-related asthma, L. reuteri has been reported to reduce airway inﬂammaon by modulang the immune response. This is reportedly enhancing an - inﬂammatory pathways and inﬂuencing cytokine balance. It has been suggested that L. reuteri supports the immune system in treang skin infecons by producing bioacve compounds that exhibit anmicrobial acvity against pathogenic microorganisms. It has been reported that, in condions of periodons, it may contribute to the control of dysbiosis by producing anmicrobial compounds such as 3 - hydroxypropionaldehyde, thereby balancing the oral bioﬁlm and improving the balance between pathogenic and commensal microbiota, as well as reducing plaque formaon. In addion, it is suggested that it modulates the immune response by reducing levels of the pro-inﬂammatory cytokines IL - 1β, TNF - α and IL - 6 while increasing the an - inﬂammatory cytokine IL - 10; furthermore, it may reduce the inﬂammatory response by inhibing the Nuclear Factor kappa B (NF – κB) signalling pathway. It is thought that these mechanisms may result in a reducon in periodontal inﬂammaon and ssue degeneraon. In addion, they have been found to act as crical probiocs, parcularly in gastrointesnal disorders [26 , 27 , 28]. It has been reported that L. reuteri may be eﬀecve in the prevenon of Cd toxicity in living beings [29]. However, the eﬀect of L. reuteri on speciﬁc serum cytokine and anoxidant levels in rats (Raus norvegicus) chronically exposed to Cd has not been determined. In this context, the aim of this study was to determine whether L. reuteri (alive or dead) had a protecve 2 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico eﬀect on certain serum cytokine and anoxidant levels in rats exposed to chronic Cd toxicity. MATERIAL AND METHODS Ethical commiee approval In the present study, all research procedures carried out on animals were approved by the Experimental Animal Ethics Commiee of Balıkesir University (Approval no: 2024 / 11 – 11). Assessment of resistance to Cd The L. reuteri strain exhibing the strongest resistance to Cd was ulized in this study. Minimum inhibitory concentraon (MIC) and minimum lethal concentraon (MLC) were deter- mined for four diﬀerent L. reuteri strains. Each strain was grown in de Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) supplemented with diﬀerent concentraons of cad- mium chloride (CdCl₂), following the method described by Zhai et al. [30] with minor modiﬁcaons. Cadmium concentraons ranging from 0.1 to 200 mg / L were used in order to cover a wide range including environmen- tally relevant levels and concentraons previously reported to aﬀect lacc acid bacteria growth and cadmium interacons with probioc bacteria [31 , 32]. All required concentraons were prepared by ten - fold serial diluons from a 1.000 mg / L CdCl₂ stock soluon, resulng in progressively decreasing Cd concen- traons. Each diluon was inoculated with L. reuteri strains and incubated at 37 °C for 24 - 48 h in an incubator (Memmert, mod- el E412.0361, Schwabach, Germany). Bacterial growth was monitored by visual turbidity and op- cal density measurements at 600 nm (OD600) using a microplate spectrophotometer (SPECTROstar Nano, BMG LABTECH GmbH, Ortenberg, Germany). MIC was deﬁned as the lowest Cd con- centraon that inhibited observable bacterial growth, whereas MLC was determined as the lowest concentraon resulng in no growth aﬅer subculturing onto fresh Cd - free MRS agar plates. Each experiment was performed in triplicate. At the end of the incubaon period, L. reuteri DSM 17938, which showed growth at the highest Cd concentraon, was idenﬁed as the most Cd - resistant strain and was selected for use in subsequent experiments Preparaon of live and heat - inacvated L. Reuteri L. reuteri was culvated in 10 mL of de MRS broth and incubated (Memmert, model E412.0361, Schwabach, Germany) at 37 °C for 24 h. At the end of the incubaon period, bacterial cultures were centrifuged (Hermle Z380, Germany) at 4200 × g for 5 minutes (min) at 4 °C using a refrigerated centrifuge (Hermle Z380, Germany). The supernatants were discarded and the bacterial pellets were resuspended in 10 mL of sterile skim milk soluon (Merck, 115363, Darmstadt, Germany). For preparaon of live L. reuteri, the bacterial suspension was adjusted to obtain a ﬁnal concentraon of approximately 10 9 - 10 10 cfu/mL and directly used as the experimental treatment. The selected bacterial dose (10 9 - 10 10 cfu/mL) was chosen based on previous studies evaluang the detoxiﬁcaon and binding capacity of Lactobacillus strains and represents a concentraon commonly used in probioc intervenon experiments [31 , 33]. For preparaon of heat - inacvated L. reuteri, the bacterial suspension was subjected to heat treatment at 121 °C for 15 min using an autoclave (Hirayama, Hiclave HV85, Japan). Following heat treatment, the suspensions were cooled to room temperature and used as the dead - cell treatment. The absence of bacterial growth aﬅer heat treatment was conﬁrmed by plang on MRS agar. Animals and study design Study design and administraon of Cd were performed according to the method described before by Kısadere et al. [17], Almenara et al. [34], and Wardani et al. [35]. In the present study, 48 healthy male Wistar albino rats (~ 12 - 16 weeks old, weighing (Kern, EW 620 - Germany) 200 ± 30 g) obtained from Balıkesir Experimental Animal Producon Center, were used. The rats were kept under convenonal laboratory condions (standard plasc rat cages, 12 hours light / 12 hours dark, 23 ± 2 °C, 55 ± 10 % relave humidity, ad libitum feeding and watering) during the experiment. The rats were randomly divided into six equal groups : Cd, the alive L. reuteri (ALR), the Cd + ALR, the dead L. reuteri group (DLR), Cd + DLR, and the control (C) group. 1. Cd (n = 8) : CdCl₂ (2 mg / kg / d) was administered to animals via gastric gavage three mes a week for four weeks [17 , 25]. 2. ALR (n = 8) : Alive L. reuteri soluon (containing ~ 10 9 - 10 10 cfu / mL) was administered to animals via gastric gavage three mes a week for four weeks [31 , 33]. 3. Cd + ALR (n = 8) : CdCl₂ (2 mg / kg / d) and alive L. reuteri soluon (containing ~ 10 9 - 10 10 cfu / mL) were administrated to animals via gastric gavage three mes a week for four weeks [17 , 25 , 31 , 33]. At this stage, the rats were ﬁrst administered CdCl₂ and then, without any waing me, ALR. 4. DLR (n = 8): Dead L. reuteri soluon (containing ~ 10 9 - 10 10 cfu / mL) was administered to animals via gastric gavage three mes a week for four weeks [31 , 33]. 5. Cd + DLR (n = 8) : CdCl₂ (2 mg / kg / day) and dead L. reuteri soluon (containing ~ 10 9 - 10 10 cfu / mL) were administrated to animals via gastric gavage three mes a week for four weeks [17 , 25 , 31 , 33]. At this stage, the rats were ﬁrst administered CdCl₂ and then, without any waing me, DLR. 6. C (n = 8) : The physiological saline soluon was administered to animals via gastric gavage three mes a week for four weeks. The rats were given the treatment by way of an oral gavage, with a volume of 1 mL/kg body weight (b.w.). Following the 28 - d experiment, a cardiac puncture was performed to obtain blood samples from the rats’ hearts for the study. Rats were euthanised under anaesthesia using an intramuscular injecon of Ketamine / Xylazine (0.1 mL / 100 g b.w.) at the end of the 4 - week experimental period. Five milliliters of blood were drawn and placed in tubes without ancoagulants. Centrifugaon was used to separate the serum samples (Hermle Z380, Germany, 3500 rpm, 15 min). Before analysis, serum samples were stored at -80 °C (Arçelik 270530EB, Türkiye). 3 of 10

The Protecve Eﬀect of Limosilactobacillus reuteri in Cadmium Toxicity / Kisadere et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Assessment of Cd levels in serum samples The determinaon of serum Cd levels was carried out using an Inducvely Coupled Plasma Opcal Emission Spectroscopy (ICP-OES) (Perkin Elmer Opma 7300, USA) device in accordance with the methodologies established by Altundag and Tüzen [36] and Tokay and Bağdat [37]. Serum samples (1 mL) were taken into teﬂon containers. Aﬅer, H₂O₂ (2 mL) and HNO₃ (5 mL) soluons were added to these containers. Then, the teﬂon containers were heated to 90 °C. Clear soluons were obtained by connuous mineralizaon. Clear samples were transferred to balloon vials and diluted to 10 mL with ultrapure water. Assessment of serum cytokine and anoxidant levels The serum concentraons of TNF - α [(Bioassay Technology Laboratory (BT Lab), E0764Ra, China)], IL -1β [(BT Lab, E0119Ra, China)], IL - 6 [(BT Lab, E0135Ra, China)], and IL - 10 (BT Lab, E0108Ra, China) were quanﬁed ulising commercially available ELISA kits in line with the protocol outlined by the manufacturer’s by using an ELISA reader (SPECTROstar Nano, Germany). The concentraons of MDA (BT Lab, E0156Ra, China), GPX values (BT Lab, E1172Ra, China), SOD (BT Lab, E1444Ra, China), and CAT (BT Lab, E0869Ra, China) enzyme acvies were quanﬁed ulizing commercially available ELISA kits in accordance with the manufacturer’s instrucons and an ELISA reader (SPECTROstar Nano, Germany). Stascal analysis The data was stascally analyzed using SPSS 26 (IBM SPSS Stascs for Windows, Version 22.0, Armonk, New York: IBM Corporaon). The mean ± SEM was used to show all data. The Shapiro - Wilk test was employed to assess the distribuon characteriscs of variables and the homogeneity of variance. Parameters were assessed using analyses of variance (ANOVA) and subsequently evaluated with Duncan’s test. A P -value of less than 0.05 indicated stascal signiﬁcance for the collected data. RESULTS AND DISCUSSION In the Cd group, serum Cd levels were found to be signiﬁcantly higher than in the other groups (P < 0.05). On the other hand, Cd + ALR showed signiﬁcantly higher serum Cd levels than Cd + DLR in the present study (P < 0.05). Serum concentraons of Cd were not signiﬁcantly diﬀerent within the C, ALR, and DLR groups (P > 0.05). The serum Cd levels of the animals are shown in FIG 1. FIGURE 1. Serum Cd levels in rats. a, b, c, d The bars exhibit a signiﬁcant diﬀerence (P < 0.05) in the superscripts. Cd : Cadmium; µg : Microgram; L : Liter. Tumor necrosis factor alpha (TNF – α) levels were determined to be higher in the Cd group compared to the C group (P < 0.05). Although the administraon of ALR and DLR alone increased TNF - α levels when compared to the C group, this increase was lower than that observed in the Cd group. In addion, the Cd + ALR and Cd + DLR groups observed a decrease in TNF - α levels; however, this difference was not considered statistically significant (P > 0.05). When compared to the C group, IL - 6 levels were signiﬁcantly higher in the Cd group (P < 0.05). DLR group showed higher IL - 6 levels than ALR group. On the other hand, when compared to the Cd group, the Cd + ALR and Cd + DLR groups showed a decrease in IL - 6 levels that was stascally signiﬁcant (P < 0.05). In this study, IL - 10 levels were determined higher in the C group compared to other experimental groups (P < 0.05). The IL - 1β level in the Cd group was lower than that in the C and ALR groups. On the other hand, IL - 1β levels in the Cd + ALR group were elevated compared to the Cd group and comparable to those in the C group (P < 0.05). In contrast, Cd + DLR did not signiﬁcantly improve IL - 1β levels in rats. The levels of serum cytokines are presented in TABLE I. TABLE I Some serum cytokine levels of the rats Groups n TNF - α (ng / L) IL - 6 (ng / L) IL - 10 (pg / mL) IL - 1β (pg / mL) C 8 1.52 ± 0.24 c 0.66 ± 0.11 d 267.82 ± 72.09 a 47.55 ± 2.80 a ALR 8 2.31 ± 0.02 ab 0.95 ± 0.09 c 144.86 ± 4.63 b 50.04 ± 7.53 a DLR 8 2.04 ± 0.11 b 1.23 ± 0.05 bc 100.17 ± 18.60 b 35.03 ± 5.33 ab Cd 8 2.56 ± 0.12 a 2.02 ± 0.11 a 116.75 ± 8.82 b 21.42 ± 3.26 b Cd + ALR 8 2.32 ± 0.16 ab 1.08 ± 0.06 bc 88.17 ± 12.42 b 42.79 ± 4.53 a Cd + DLR 8 2.31 ± 0.04 ab 1.30 ± 0.12 b 86.82 ± 8.05 b 25.02 ± 4.80 b It means that diﬀerent leers in the same column are signiﬁcantly diﬀerent (P < 0.05). C: Control, ALR: Alive L. reuteri group, DLR : Dead L. reuteri group, Cd : Cadmium, Cd + ALR: Cadmium + Alive L. reuteri group, Cd + DLR : Cadmium + Dead L. reuteri group. TNF – α : Tumor Necrosis Factor - Alpha; IL – 6 : Interleukin - 6; IL – 10 : Interleukin - 10; IL - 1β : Interleukin 1 – Beta; n : Number of animals; ng : Nanograms; L : Liter; pg : Pictograms; mL : Mililiter. 4 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In terms of serum MDA and CAT levels, no signiﬁcant diﬀerences were observed between the groups (P > 0.05). In contrast, the group that obtained Cd administraon showed a signiﬁcant decrease in GPx levels, although group C presented the highest levels The GPx levels in the Cd + ALR and Cd + DLR groups were similar to those in the Cd group and did not show stascally signiﬁcant increases. The C group showed the highest SOD levels, while the Cd group exhibited the lowest, with stascally signiﬁcant diﬀerences observed in comparison to the other groups. Furthermore, when compared to the C group, the ALR administraon demonstrated signiﬁcantly lower levels, but when compared to the Cd group, it showed signiﬁcantly higher levels. Serum oxidant - anoxidant levels are presented in TABLE II. TABLE II MDA concentraons and some serum anoxidant levels in the experimental groups Groups n MDA (nmol / mL) GPX (ng / mL) SOD (ng / mL) CAT (ng / mL) C 8 1.32 ± 0.14 2000.00 ± 488.25 a 22.56 ± 3.37 a 12.43 ± 5.65 ALR 8 1.25 ± 0.16 1385.00 ± 287.36 ab 15.30 ± 1.63 b 10.73 ± 3.05 DLR 8 1.48 ± 0.02 975.00 ± 15.08 b 12.76 ± 1.48 bc 7.71 ± 0.26 Cd 8 1.69 ± 0.22 898.75 ± 42.71 b 6.77 ± 0.43 c 7.36 ± 2.11 Cd + ALR 8 1.67 ± 0.23 1042.50 ± 27.17 b 12.43 ± 3.20 bc 9.53 ± 0.58 Cd + DLR 8 1.57 ± 0.08 992.50 ± 54.23 b 8.74 ± 2.08 bc 7.45 ± 1.14 It means that diﬀerent leers in the same column are signiﬁcantly diﬀerent (P < 0.05). C : Control, ALR : Alive L. reuteri group, DLR : Dead L. reuteri group, Cd : Cadmium, Cd + ALR : Cadmium + Alive L. reuteri group, Cd + DLR : Cadmium + Dead L. reuteri group. MDA : Malondialdehyde; SOD : Superoxide Dismutase; GSH : Glutathione; CAT: Catalase; n : Number of animals; nmol : Nanomol; ng : Nanograms; mL : Mililiter. It has been suggested that Cd exacerbates metabolic dysfuncon and ssue damage by upregulang ROS producon and encouraging chronic inﬂammatory responses, which upset cellular homeostasis [38]. In the present study, serum Cd levels considerably elevated in the Cd group (2 mg / mL for 4 weeks, peros (p.o.)) compared to the other experimental groups. Similarly, Kısadere et al. [25] suggested that Cd administraon (2.04 mg / mL for 28 days, p.o.) increased the serum Cd levels (83.96 ± 8.48 μg / L) in female Wistar rats. In addion, it was reported by Andjelkovic et al. [39] that serum Cd levels rose to 5.42 ± 0.88 and 8.97 ± 1.16 μg / L as a result of a single oral dose of Cd (15 and 30 mg / kg) administraon. The outcomes demonstrate that the ulized dosage regime raises blood Cd levels in a manner consistent with ﬁndings from prior invesgaons. On the other hand, serum Cd levels decreased in the Cd + ALR and Cd + DLR groups aﬅer the alive or dead L. reuteri administraons in this study. This reducon was observed signiﬁcantly greater in Cd + DLR group than Cd + ALR group. Zhai et al. [40] conﬁrmed that increased blood Cd levels (6.32 ± 4.44 μg / L) following the Cd administraon (100 mg / L CdCl₂) were ameliorated by using the probioc L. plantarum CCFM8610 (1 × 10⁹ cfu, administered orally for eight weeks) in rats. In a previous study, it was reported that L. plantarum administraon led to a decrease of serum Cd levels in female Wistar rats [25]. The changes may have been observed due to the metal - binding acvity of DLR (especially) and ALR. Recently, researchers have reported that Cd administraon alters the levels of various pro - and an - inﬂammatory cytokine levels in the blood [41]. In the immune system, IL - 6 is a mulfunconal cytokine that exhibits both pro - and an - inﬂammatory acvies. It typically exists in the bloodstream in small quanes, but when the organism is exposed to harmful substances, toxic chemicals, infecons, or ssue injury, its levels rise dramacally [42 , 43]. The levels of IL - 6 were determined to be the highest in the Cd group (2.02 ± 0.11 ng / L) when compared to the C group (0.66 ± 0.11 ng / L) in the present study. The results were in line with the results of past studies on IL - 6 levels [29 , 44 , 45 , 46 , 47]. On the contrary, Kısadere [41] found that administraon of Cd did not change the blood IL - 6 levels in male Wistar rats, interesngly. These diﬀerences may be inﬂuenced by factors such as gender, dose, me, or administraon method [23]. In this study, increased IL - 6 levels (2.02 ± 0.11 ng / L) due to Cd administraon were ameliorated by using alive or dead L. reuteri in the Cd + ALR (1.08 ± 0.06 ng / L) and Cd + DLR (1.30 ± 0.12 ng / L) groups. Furthermore, this results aligned with the ﬁndings of previous studies [44 , 45 , 46 , 47]. The protecve eﬀects of L. plantarum and L. reuteri administraons on IL - 6 levels in the other ssues have also been demonstrated in previous studies [29 , 48]. It could be aributed to the immune system - smulang acons of probioc - derived structural components. TNF - α, a significant proinflammatory cytokine, is primarily produced by macrophages, monocytes, T lymphocytes, and natural killer (NK) cells. It induces an acute inflammatory response in circumstances of infection or tissue damage [49]. Besides, IL-1β (a proinﬂammatory cytokine that increases the inﬂammatory response with TNF - α) plays a crucial role in the body›s initial immune response against infection or tissue damage. It is generally produced by monocytes and macrophages, and is crucial for beginning and connuing inﬂammaon [50]. In this study, the administraon of Cd increased TNF - α levels but decreased IL - 1β levels in Wistar rats. Interesngly, Li et al. [46] suggested that Cd administraon (5 mg / kg for 28 d) caused to increase in both TNF - α and IL - 1β levels in rats. Conversely, it was reported that TNF - α and IL - 1β levels were not aﬀected by Cd administraon in female rats [25]. On the other hand, Kısadere [41] also concluded that TNF - α levels increased in Wistar rats after CdCl₂ administraon (at a dose of 2 mg / kg / day for four weeks). The dose and duraon 5 of 10

The Protecve Eﬀect of Limosilactobacillus reuteri in Cadmium Toxicity / Kisadere et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico of Cd administraon, and the use of diﬀerent commercial kits may explain the diﬀerences in the results [51 , 52]. This study demonstrated that L. reuteri administraon, as a probioc, exhibited a tendency to decrease TNF - α levels in the Cd + ALR and Cd + DLR groups. In addion, TNF - α levels decreased after the administration of DLR alone in the present study. Also, it was determined that administration of ALR and Cd + ALR increased IL - 1β levels, thereby eliminang the inhibitory eﬀect of Cd. However, it was found that neither DLR nor Cd + DLR administraon increased IL - 1β levels in the present study. Moreover, Sajjad et al. [29] reported that increased levels of TNF - α and IL - 1β depended on the Cd administraon (2 mg / kg) were ameliorated by the L. reuteri administraon in the breast cancer - detected mice (Mus musculus). Similarly, Li et al. [53] found that a novel polysaccharide derived from Lactobacillus - fermented Nostoc commune Vauch. reduced IL - 6, IL - 1β, and TNF - α levels and mitigated Cd - induced kidney injury in rats. These obtained results indicate that L. reuteri has a limited inhibitory eﬀect on inﬂammaon caused by Cd. In addion, alive probiocs can play a regulatory and balancing role in the immune system. The main funcon of the cytokine IL - 10 is to reduce inﬂammaon. This characterisc prevents the producon of pro - inﬂammatory cytokines such IL - 1β, IL - 6, and TNF - α. It protects the organism from tissue damage and stops an overabundance of immune response activation [54]. In this study, the C group’s higher IL - 10 levels (267.82 ± 72.09 pg / mL) in comparison to the other groups is suggesve of the system’s natural responses that reduce inﬂammaon in physiological condions. On the other hand, Cd administraon led to a decrease of rat’s IL - 10 levels in the present study. Similarly, Cd exposure signiﬁcantly suppressed the IL - 10 secreon of rats in previous studies [25]. These outcomes might have been brought on by Cd’s immunosuppressive funcon. On the other hand, administraon of either the alive (144.86 ± 4.63 pg / mL) or dead forms (100.17 ± 18.60 pg / mL) of L. reuteri resulted in a signiﬁcant decrease of IL - 10 levels in rats compared to the C group (267.82 ± 72.09 pg / mL) in this study. Sajjad et al. [29] reported that IL - 10 levels (60.2 ± 2.0 pg / mL) decreased with the administraon of cadmium + tamoxifen + Lactobacillus reuteri (20.8 ± 1.1 pg / mL) in rats. Also, Kısadere et al. [25] reported that L. plantarum administraon decreased the increased serum IL - 10 levels aﬅer Cd administraon in female Wistar rats. It may be expressed that probiocs have immunomodulatory properes in both their alive and dead forms. It has been reported that Cd exposure increases ROS and OS parameters by reducing intracellular glutathione (GSH) levels and inhibing the acon of anoxidant enzymes such as SOD and CAT in living beings [55]. MDA, one of the important markers of lipid peroxidaon, concentraon was not aﬀected by the diﬀerent administraons in the present study. Conversely, Cd administraon increased the serum MDA concentraons in rats in the previous studies [25 , 56 , 57]. The ﬁndings may have varied depending on the Cd dose, administraon period, individual characteriscs, or ulized commercial kits. Similar to Cd administraon, alive or dead L. reuteri treatments did not cause any changes in the serum MDA concentraon of rats in the present study. This result may indicate that the eﬀect of L. reuteri administraon on lipid peroxidaon products is limited. In addion, Cd administraon led to a decrease in serum GPx levels (898.75 ± 42.71 ng / mL) of Cd group animals compared to C group (2000.00 ± 488.25 ng / mL) in this study. Galażyn-Sidorczuk et al. [58] and Poli et al. [59] also determined similar results to this study regarding GPX levels in rats’ serum and kidney ssues exposed to Cd. On the other hand, administraon of neither alive nor dead L. reuteri caused any crucial change in GPX levels of the Cd + ALR (1042.50 ± 27.17 ng / mL) and Cd + DLR (992.50 ± 54.23 ng / mL) groups compared to the Cd group (898.75 ± 42.71 ng / mL) in the present study. On the contrary, Banwo et al. [23] and Li et al. [53] observed protecve eﬀects of diﬀerent types of Lactobacillus administraons (Lacplanbacillus plantarum ML05 and Lactobacillus - fermented Nostoc commune Vauch) in the ssue GPX and GSH levels in rats exposed to Cd. Furthermore, Kısadere et al. [25] reported that the administraon of L. plantarum did not aﬀect the serum GSH levels in female rats. Although serum CAT enzyme acvity tended to decrease in the Cd group compared to the other groups, it did not cause signiﬁcant change in this study. Similarly, it was suggested that administraon of Cd (2.04 mg / mL for 28 days, p.o.) did not inﬂuence the serum CAT enzyme acvies in female rats [25]. In contrast, serum CAT acvies in Sprague Dawley female rats were found to be reduced as a result of Cd administraon by Xue et al. [60]. Also, numerous studies demonstrated that Cd adversely aﬀected the CAT enzyme acvity in various ssues and organs [59 , 61]. The dose of Cd administraon, diﬀerent genders, or individual properes may explain the diﬀerent results. On the other hand, alive or dead L. reuteri administraon could not aﬀect the serum CAT enzyme acvies in the Cd + ALR and Cd + DLR groups compared to only Cd treated group in the present study. In a previous study, Tian et al. [48] suggested that Lp TW1 - 1 ameliorated the enzymac acvies of CAT in the serum of diethylhexylphthalate - administered male mice. These ﬁndings are compable with Kisadere et al. [25]’s results regarding to serum CAT enzyme acvity. The diﬀerent anoxidant properes of probioc strains may explain the various results. In the present study, Cd administraon decreased the serum SOD enzyme acvity in male Wistar rats. On the contrary, Kisadere et al. [25] conﬁrmed that Cd administraon did not lead to an alteraon in serum SOD levels in female Wistar rats. Suhartono et al. [62] also found similar results to this study regarding to SOD enzyme acvity in male Sprague - Dawley male rats exposed to Cd. In other invesgaons, it has also been established that Cd suppresses the acvity of the SOD enzyme in diﬀerent ssues [45 , 63 , 64]. In this study, SOD enzyme acvity was found markedly lower in the ALR group (15.30 ± 1.63 ng/mL) when compared to C group (22.56 ± 3.37 ng / mL), interesngly. It was found that SOD acvity was signiﬁcantly reduced in the CD group (6.77 ± 0.43 ng/mL) compared to group C (22.56 ± 3.37 ng/mL). It is thought that this is due to the suppression of the anoxidant defence system resulng from cadmium-induced oxidave 6 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico stress. Addionally, the decrease in SOD enzyme acvies caused by Cd could not be ameliorated in the Cd + ALR and Cd + DLR groups by the administraon of live or dead L. reuteri. Moreover, it was determined that L. plantarum administraon did not cause any inﬂuence on the serum SOD levels of rats exposed to Cd [25]. In contrast, Lacplanbacillus plantarum ML05 and Lactobacillus - fermented Nostoc commune Vauch administraons showed a posive reversal impact on SOD enzyme acvies in animals (rats and mice) whose kidney and liver ssues were exposed to Cd toxicity [23 , 53]. This may be due to the low binding rate of Cd ions by L. reuteri in the gastrointesnal tract compared to other bacterial strains (Lactobacillus and Biﬁdobacterium probioc strains) [24]. Due to the fact that L. reuteri strains are food - derived and are frequently employed as probiocs in the fermentaon of dairy products, the Cd binding capacity and eﬀects on cytokine modulaon observed in this study indicate a potenal role in the enhancement of the safety of food contaminated with heavy metals, as well as in the maintenance of health. These probioc applicaons can be regarded as a biotechnological approach to reducing toxic metal residues in the food chain that is both environmentally friendly and food compable. CONCLUSIONS The invesgaon of the beneﬁcial eﬀects of probiocs can be extended by using cadmium as a toxic substance. The ﬁndings of this invesgaon showed that dead L. reuteri can bind to Cd especially in the bloodstream. Although the eﬀect on markers of oxidave stress is limited, when cytokine levels are taken into account, the protecve eﬀect of both live and dead L. reuteri is clear. Further studies at both the molecular and cellular levels are necessary to beer understand the protecve eﬀect of L. reuteri strains in Cd toxicity. L. reuteri, a food - derived bacterium frequently used in funconal and fermented foods, shows Cd binding capacity and immunomodulatory eﬀects, suggesng potenal applicaons in the development of probioc foods and food safety. Funding The Scienﬁc Research Unit of Balıkesir University provided the ﬁnancial support for this study (BAP Project No : 2024 / 032). Conﬂicts of interest The authors of this arcle do not have potenal conﬂicts of interest. BIBLIOGRAPHIC REFERENCES [1] Soni S, Jha AB, Dubey RS, Sharma P. Migang cadmium accumulaon and toxicity in plants: The promising role of nanoparcles. Sci. Total Environ. [Internet]. 2024; 912:168826. doi: hps://doi.org/gzwvbd [2] Kubier A, Wilkin RT, Pichler T. Cadmium in soils and groundwater: A review. Appl. Geochem. 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