antioxidants Article Changes in Biomarkers of Redox Status in Saliva of Pigs after an Experimental Sepsis Induction María José López-Martínez 1 , Damián Escribano 1,2 , Alba Ortín-Bustillo 1 , Lorena Franco-Martínez 1 , Luis Guillermo González-Arostegui 1 , José Joaquín Cerón 1 and Camila Peres Rubio 3, * 1 Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence “Mare Nostrum”, University of Murcia, 30100 Murcia, Spain; mariajose.lopezm96@gmail.com (M.J.L.-M.); det20165@um.es (D.E.); alba.ortinb@um.es (A.O.-B.); lorena.franco2@um.es (L.F.-M.); luisgarostegui@gmail.com (L.G.G.-A.); jjceron@um.es (J.J.C.) 2 Department of Animal Production, Regional Campus of International Excellence “Mare Nostrum”, University of Murcia, 30100 Murcia, Spain 3 Department of Animal and Food Science, School of Veterinary Science, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain * Correspondence: camila.peres@uab.cat Abstract: Saliva from pigs is gaining attention as an easy sample to obtain, being a source of biomarkers that can provide information on animal health and welfare. This study aimed to evaluate the changes that can occur in salivary biomarkers of the redox status of pigs with an experimentally induced sepsis. For that, the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC), advanced oxidation protein products (AOPP), ferrous oxidation-xylenol orange (FOX), peroxide activity (POX-Act), and reactive oxygen- derived compounds (d-ROMs) were measured in the saliva of pigs with experimentally induced sepsis by endotoxin lipopolysaccharide (LPS), non-septic inflammation induced by turpentine, and Citation: López-Martínez, M.J.; in healthy individuals before and after 3 h, 6 h, 24 h, and 48 h. AOPP, POX-Act, and d-ROMs in Escribano, D.; Ortín-Bustillo, A.; the sepsis group were higher than in the control from 3 h to 24 h after the inoculation. CUPRAC, Franco-Martínez, L.; González-Arostegui, L.G.; Cerón, J.J.; FRAS, and TEAC were higher in sepsis than the control group at 24 h. These changes were of higher Rubio, C.P. Changes in Biomarkers of magnitude than those that occurred in the turpentine group. In conclusion, our findings reveal that Redox Status in Saliva of Pigs after an sepsis produces changes in salivary biomarkers of redox status, which opens the possibility of using Experimental Sepsis Induction. them as potential biomarkers in this species. Antioxidants 2022, 11, 1380. https:// doi.org/10.3390/antiox11071380 Keywords: antioxidants; cupric; ferric; inflammation; oxidative stress; peroxides Academic Editor: Stanley Omaye Received: 10 June 2022 Accepted: 14 July 2022 1. Introduction Published: 16 July 2022 The use of saliva for the determination of biomarkers has gained special attention Publisher’s Note: MDPI stays neutral in the last decade due to its easy collection, being of particularly high interest in pigs, in with regard to jurisdictional claims in which blood collection is highly stressful. In addition, it has been shown that the saliva published maps and institutional affil- matrix is appropriate for health monitoring and disease diagnosis [1,2]. iations. Infectious diseases in pigs are possibly the most important health concern in swine production [3,4]. Depending on factors, such as the immune status of the pig, pathogen involved, and environment, the disease can progress to sepsis and death. In addition, the intensification and globalization of the swine industry are being accompanied by a global Copyright: © 2022 by the authors. spread of pathogens at local and international scales, which, together with the need for Licensee MDPI, Basel, Switzerland. appropriate use of antibiotics and the withdrawal in the European Union of zinc oxide This article is an open access article supplementation, highlights the importance of the correct detection and control of sepsis in distributed under the terms and conditions of the Creative Commons pigs [5,6]. Attribution (CC BY) license (https:// The endotoxin lipopolysaccharide (LPS) challenge is a validated model of sepsis creativecommons.org/licenses/by/ in pigs [7]. In addition, porcine models of LPS-induced sepsis have been developed to 4.0/). simulate human sepsis [8,9]. The LPS is an outer-membrane component of Gram-negative Antioxidants 2022, 11, 1380. https://doi.org/10.3390/antiox11071380 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 1380 2 of 8 bacteria that provokes a systemic inflammatory response, which includes the activation of free-radical-generating molecules in various types of cells that initiate tissue damage [10,11]. Free-radical molecules and other oxidants are produced constantly as a result of the respiratory chain, and the antioxidant system works continually to maintain the oxidant levels under control and without damage to the cells [12]. The imbalance between both the oxidant and antioxidant system is involved in the etiology of various diseases. Several studies have already shown that biomarkers of oxidative status can be measured in the saliva of pigs [13] and other species, such as bovine [14], canine [15], ovine [16], as well as humans [17]. To investigate the effects of a natural antioxidant compound during human sepsis, the LPS-induced model in piglets was used and various redox biomarkers were evaluated in serum [18]. However, to the author’s knowledge, no information is available yet about the changes that occur in the oxidative status of the saliva of pigs during induced sepsis. The objective of this study was to evaluate the changes that can occur in biomarkers of redox status in the saliva of pigs with sepsis. For this purpose, an experimental trial using LPS was made. In addition, an experimentally induced non-septic inflammation by turpentine administration was performed in order to compare sepsis with a non-septic in- flammatory condition. In these trials, a profile, including four antioxidant (cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC) and uric acid) and four oxidant biomarkers (advanced oxida- tion protein products (AOPP), ferrous oxidation-xylenol orange (FOX), peroxide activity (POX-Act), and reactive-oxygen-derived compounds (d-ROMs)) was measured. 2. Materials and Methods 2.1. Animals, Housing, and Experimental Design The experimental protocol was approved by the Ethical Committee on Animal Experi- mentation (CEEA) of the University of Murcia (CEEA 563/2019) according to the European Council Directives considering the protection of animals used for experimental purposes. In addition, this study complies with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the care and use of animals. In total, 15 growing male pigs were included in this study. They were in the mid- fattening period and belonged to the Experimental Farm of the University of Murcia (Murcia, Spain). All of them had water and a balanced diet ad libitum and were housed with a minimum space of 0.65 m2 per animal (Council Directive 2001/88/CE of 23 October 2001 amending Directive 91/630/CEE concerning minimum standards for the protection of pigs) and a standard temperature of 24 ± 2 ◦ C. During the study, the pigs were 14 weeks old and had a median weight of 51.5 kg (interquartile range 48–53 kg). Before starting the experiment, the animals were adapted to the experimental con- ditions for a week and then randomized and divided into three groups. The first group (n = 5; control group) received saline treatment (2 mL) by intramuscular route. The sec- ond group (n = 5; LPS group) received a single dose of 30 ug/kg LPS from Escherichia Coli (LPS; O55:B5, Sigma-Aldrich) reconstituted in sterile saline solution by intramuscular injection [19,20]. In the third group (n = 5, TURP group), 8 mL of TURP (oil of turpentine purified, Sigma–Aldrich) was given by two 4 mL subcutaneous injections in each front flank per animal. All injections were completed between 8 and 9 am. In some of these saliva samples, procalcitonin was measured for a previous study [21]. 2.2. Sampling Procedure Saliva samples were obtained 24 h before (baseline) the saline, LPS, or TURP injections and at 3, 6, 24, and 48 h after. Saliva was collected using a polypropylene sponge clipped to a metal rod. The pigs were allowed to chew the sponge without forcing them. Then the sponges were placed in Salivette tubes (Sarstedt, Aktiengesellschaft & Co. D-51588 Nümbrecht, Germany) and centrifuged at 3000× g for 10 min. The saliva samples were collected and stored in Eppendorf tubes at −80 ◦ C until analysis. Antioxidants 2022, 11, 1380 3 of 8 2.3. Assessment of Salivary Biomarkers of Oxidative Status The salivary concentration of CUPRAC, FRAS, TEAC, uric acid, and AOPP was determined using automated techniques previously described and validated for the saliva of pigs [13]. The measurement of FOX was based on the oxidation of ferrous to ferric ions by lipid hydroperoxides in the sample as previously published [22] and results were expressed in µmol of peroxide hydrogen per L of the sample (µmol/L). The POX-Act determination was based on the assay described by Tatzber et al. [23] in which the oxidation of 3,5,30 50 -Tetramethylbenzidine (TMB) by peroxides in the sample is monitored. The results were also expressed in µmol of peroxide hydrogen per L of the sample (µmol/L). Salivary d-ROMs levels were determined based on monitoring the N,N-Diethyl-p- phenylenediamine radical cation concentration as previously described [24] with results expressed in Carratelli units (Carr units). The assays were performed using the Olympus AU400 (AU400 Automatic Chemistry Analyser, Olympus Europe GmbH, Hamburg, Germany) and showed lower than 15% imprecision. More details about the assays are described in Table S1. 2.4. Statistical Analysis Data (Spreadsheet S1) were analyzed using GraphPad Prism software Inc. (GraphPad Prism, version 8 for Windows, Graph Pad Software Inc., San Diego, CA, USA). The normal- ity of the distribution of the results was assessed by using the D’Agostino and Pearson test, giving a nonparametric distribution; therefore, all data were normalized by logarithmic transformation (Y = Log[Y]) before analysis. A two-way analysis of variance (ANOVA) mixed model of repeated measures was performed, and time and treatment were used as random factors to account for multiple observations. Sidak’s multiple comparison test was used to compare the groups over time. Tukey’s multiple comparison test was applied to the data considering the sampling time as a repeated measure, with fixed effects of treatment. The effects were considered as significant if p-values were <0.05. 3. Results 3.1. Antioxidant Biomarkers LPS-treated pigs had significant increased concentrations of CUPRAC at 24 h when compared to basal time (p = 0.039, Figure 1a). TEAC showed a tendency to increase (p = 0.056, Figure 1b) at 24 h in comparison to basal time in this group. When compared to healthy controls, the LPS group showed significantly higher concentrations of CUPRAC (p = 0.004; Figure 1a), FRAS (p = 0.028; Figure 1b), and TEAC (p = 0.001; Figure 1c) 24 h after injection. TURP-treated pigs showed no changes in antioxidant biomarkers throughout the study, except for uric acid (Figure 1d), which was decreased at 48 h in relation to baseline (p = 0.044). There was no significant difference comparing control and TURP-treated pigs at any time point (p > 0.05) (Figure 1). Antioxidants 2022, 11, x FOR PEER REVIEW 4 of 9 Antioxidants 2022, 11, 1380 4 of 8 (a) (b) (c) (d) Figure Figure 1. 1. (a)(a) Cupric Cupric reducing reducing antioxidant antioxidant capacity capacity (CUPRAC), (CUPRAC), (b)(b) ferric ferric reducing reducing ability ability of of saliva saliva (FRAS), (c) Trolox equivalent antioxidant capacity (TEAC), and (d) uric acid concentrations in con- (FRAS), (c) Trolox equivalent antioxidant capacity (TEAC), and (d) uric acid concentrations in control trol (▲), lipopolysaccharide (LPS)-treated pigs (●), and turpentine (TURP)-treated pigs (■) before (N), lipopolysaccharide (LPS)-treated pigs (•), and turpentine (TURP)-treated pigs () before (basal) (basal) and after 3 h, 6 h, 24 h, and 48 h the inoculations. The results are presented as median with and anafter 3 h, 6 h,range. interquartile 24 h, and 48 h the inoculations. #, significantly The different from theresults are control presented group as median (p < 0.05; one-waywith ANOVAan interquartile with Sidak’s range. #, significantly multiple comparisons different test). from the control Differences group between (p < 0.05; times one-way by are indicated ANOVA withthe bars and Sidak’s multiple obtained comparisons p-value (one-way test). ANOVA Differences between with Tukey’s times are multiple indicatedtest). comparison by bars and the obtained p-value (one-way ANOVA with Tukey’s multiple comparison test). 3.2. Oxidant Biomarkers 3.2. Oxidant Biomarkers LPS-treated pigs presented significantly higher POX-Act (Figure 2c) and d-ROMs LPS-treated pigs presented significantly higher POX-Act (Figure 2c) and d-ROMs (Figure 2d) than control at 3 h (p < 0.039), 6 h (p < 0.033) and 24 h (p < 0.001), reaching the (Figure 2d) than control at 3 h (p < 0.039), 6 h (p < 0.033) and 24 h (p < 0.001), reaching highest values at 6 h. They also showed higher POX-Act than TURP-treated pigs at 3 h (p the highest values at 6 h. They also showed higher POX-Act than TURP-treated pigs at < 0.036). When compared to control, the LPS group showed significantly higher concen- 3 h (p < 0.036). When compared to control, the LPS group showed significantly higher trations of AOPP (p = 0.004, Figure 2a) 24 h after the injections. concentrations of AOPP (p = 0.004, Figure 2a) 24 h after the injections. There was no significant difference in FOX concentrations between groups and be- There was no significant difference in FOX concentrations between groups and be- tween time points (p > 0.05) (Figure 2b). tween time points (p > 0.05) (Figure 2b). Antioxidants 2022, 11, x FOR PEER REVIEW 5 of 9 Antioxidants 2022, 11, 1380 5 of 8 (a) (b) (c) (d) Figure 2. Salivary (a) advanced oxidation protein products (AOPP), (b) ferrous oxidation-xylenol Figure 2. Salivary (a) advanced oxidation protein products (AOPP), (b) ferrous oxidation-xylenol orange (FOX), (c) peroxide activity (POX-Act), and (d) reactive-oxygen-derived compounds (d- orange (FOX), (c) peroxide activity (POX-Act), and (d) reactive-oxygen-derived compounds (d- ROMs) concentrations in control (▲), lipopolysaccharide (LPS)-treated pigs (●), and turpentine ROMs) concentrations (TURP)-treated inbefore pigs (■) control (N), lipopolysaccharide (basal) and after 3 h, 6 h, 24(LPS)-treated h, and 48 h the pigs (•), and turpentine inoculations. The results (TURP)-treated  are presented as median with an interquartile range. #, significantly different fromThe pigs ( ) before (basal) and after 3 h, 6 h, 24 h, and 48 h the inoculations. theresults control are presented group; as median different *, significantly with an interquartile range. #, pigs from TURP-treated significantly (p < 0.05;different one-way from the control ANOVA with group; Sidak’s *, multiple comparisons significantly test). TURP-treated different from Differences between pigs (p