Genotoxicity Evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus Essential Oils by a Combination of Micronucleus and Comet Assays in Rats

foods Article Genotoxicity Evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus Essential Oils by a Combination of Micronucleus and Comet Assays in Rats Antonio Cascajosa-Lira 1, María Puerto 1, Ana I. Prieto 1, Silvia Pichardo 1, Leticia Díez-Quijada Jiménez 1, Alberto Baños 2, Enrique Guillamón2, Rosario Moyano 3, Verónica Molina-Hernández 3,Ángeles Jos 1 and Ana M. Cameán1,*   Citation: Cascajosa-Lira, A.; Puerto, M.; Prieto, A.I.; Pichardo, S.; Díez-Quijada Jiménez, L.; Baños, A.; Guillamón, E.; Moyano, R.; Molina-Hernández, V.; Jos, Á.; et al. Genotoxicity Evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus Essential Oils by a Combination of Micronucleus and Comet Assays in Rats. Foods 2021,10, 989. https://doi.org/10.3390/ foods10050989 Academic Editor: Jean-Paul Vernoux Received: 26 March 2021 Accepted: 28 April 2021 Published: 1 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García González n 2, 41012 Seville, Spain; [email protected] (A.C.-L.); [email protected] (M.P.); [email protected] (A.I.P.); [email protected] (S.P.); [email protected] (L.D.-Q.J.); [email protected] (Á.J.) 2DMC Research Center, Camino de Jayena, 82, 18620 Alhendín, Spain; [email protected] (A.B.); [email protected] (E.G.) 3Department of Anatomy and Comparative Pathology and Toxicology, Faculty of Veterinary Medicine, University of Cordoba, Campus de Rabanales, 14014 Cordoba, Spain; r[email protected] (R.M.); [email protected] (V.M.-H.) *Correspondence: [email protected]; Tel.: +34-954-556-762 Abstract: Propyl-propanethiosulfinate (PTS) is a component of Allium essential oils. This organosulfur molecule can be used as a feed additive to decrease the appearance of bacterial resistances caused by the residues of antibiotics. In previous in vitro genotoxicity studies, contradictory results were reported for PTS. In this work, the in vivo genotoxicity of PTS in male and female rats was assessed for the first time, following OECD (Organisation for Economic Co-operation and Development) guidelines. After oral administration (doses: 5.5, 17.4, and 55.0 mg/kg PTS body weight), a combination of the micronucleus (MN) assay (OECD 474) in bone marrow and the standard and enzyme-modified comet assay (OECD 489) was performed. After necropsy, histopathological studies were also carried out. The results did not show the in vivo genotoxicity of PTS at any doses assayed, revealed by the absence of increased MN, and DNA strand breaks or oxidative DNA damage in the standard and enzyme-modified comet assays. The histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, the present work confirms that PTS is not genotoxic at the doses assayed, and it is a promising natural alternative to synthetic preservatives and antibiotics in animal feed. Keywords: micronucleus; comet assay; genotoxicity; organosulfur compounds; propyl-propanethio sulfinate 1. Introduction Allium species and their essential oils have been extensively used for medical applications and as a flavoring agent. Vegetables of the genus Allium (e.g., garlic or onion) are well known for their antioxidant and anticancer properties in humans [ 1 ]. In addition, other properties such as antimicrobial, antispasmodic, antiasthmatic, anti-amnesic, anti-inflammatory, hepatoprotective, neuroprotective, hypotensive, hypoglycemic, immunomodulatory urease/xanthine oxidase inhibitory, and prebiotic properties have also been described [2–6]. Organosulfur compounds (OSCs) are secondary metabolites of plants from the Allium genus (e.g., thiosulfinates and ajoenes) with biological activity, and they have a characteristic aroma. These molecules are generated when tissues of these plants are damaged. If so, alliinase degrades the initial compounds into new ones exerting biological activity [ 7 ] Foods 2021,10, 989. https://doi.org/10.3390/foods10050989 https://www.mdpi.com/journal/foods Foods 2021,10, 989 2 of 15 (Figure 1). Some properties of these OSCs can be valuable for the agri-food industry, including their antioxidant, antibacterial, antifungal, and anti-yeast activities [8–13]. Considering the increasing interest of consumers in the use of natural active compounds as an alternative of chemical agents [ 14 ], products made from the essential oil of garlic and rich in OSC have been released into the market. One of the most promising products is ProAllium ® (DOMCA, Granada, Spain), which is composed of a mixture of several compounds, mainly propyl thiosulfinate oxide (PTSO) and, in a lower proportion, of propyl-propanethiosulfinate (PTS). ProAllium ® has been successfully incorporated into films to produce active packaging, able to expand the shelf life of ready-to-eat food and to control their spoilage [ 11 , 15 , 16 ]. In addition, several uses of PTS have been investigated and patented in relation to the treatment of farm animals. In this sense, it could be used against parasites in cattle, sheep, and poultry [ 17 ], for the reduction of apicomplexa in aquatic animals [ 18 ], and as an antimethanogenic additive in ruminant feed [ 19 ]. Therefore, the effectiveness of the PTS has been verified in many uses, and it could be a good natural alternative to synthetic agents. Foods 2021, 10, x FOR PEER REVIEW 2 of 16 Organosulfur compounds (OSCs) are secondary metabolites of plants from the Allium genus (e.g., thiosulfinates and ajoenes) with biological activity, and they have a characteristic aroma. These molecules are generated when tissues of these plants are damaged. If so, alliinase degrades the initial compounds into new ones exerting biological activity [7] (Figure 1). Some properties of these OSCs can be valuable for the agri-food industry, including their antioxidant, antibacterial, antifungal, and anti-yeast activities [8–13]. Considering the increasing interest of consumers in the use of natural active compounds as an alternative of chemical agents [14], products made from the essential oil of garlic and rich in OSC have been released into the market. One of the most promising products is ProAllium® (DOMCA, Granada, Spain), which is composed of a mixture of several compounds, mainly propyl thiosulfinate oxide (PTSO) and, in a lower proportion, of propyl-pro- panethiosulfinate (PTS). ProAllium® has been successfully incorporated into films to produce active packaging, able to expand the shelf life of ready-to-eat food and to control their spoilage [11,15,16]. In addition, several uses of PTS have been investigated and patented in relation to the treatment of farm animals. In this sense, it could be used against parasites in cattle, sheep, and poultry [17], for the reduction of apicomplexa in aquatic animals [18], and as an antimethanogenic additive in ruminant feed [19]. Therefore, the effectiveness of the PTS has been verified in many uses, and it could be a good natural alternative to synthetic agents. Figure 1. Scheme of the chemical structures and biochemical pathways of organosulfur compounds. ACOs: Alk(en)yl cysteine sulfoxides. However, its use for these applications requires the characterization of its toxicological profile considering consumer exposure to this compound. In this regard, the European Food Safety Authority (EFSA) panel on Food Contact Materials, Enzymes, Flavorings, and Processing Aids (CEF) has published the “Recent developments in the risk assessment of chemicals in food and their potentials impact on the safety assessment of substances used in food contact materials” [20]. Additionally, the panel on additives and products or substances used in Animal feed (FEEDAP) has published “Guidance on the assessment of safety of feed additives for the consumers” [21]. In these publications the EFSA explains that it is mandatory to study genotoxicity, even in the predictable case of low exposure. Toxicological data about Allium extracts and essential oil are still scarce, especially in vivo. However, subchronic toxicity has been studied through a 90-day oral assay of Proal- Figure 1. Scheme of the chemical structures and biochemical pathways of organosulfur compounds. ACOs: Alk(en)yl cysteine sulfoxides. However, its use for these applications requires the characterization of its toxicological profile considering consumer exposure to this compound. In this regard, the European Food Safety Authority (EFSA) panel on Food Contact Materials, Enzymes, Flavorings, and Processing Aids (CEF) has published the “Recent developments in the risk assessment of chemicals in food and their potentials impact on the safety assessment of substances used in food contact materials” [ 20 ]. Additionally, the panel on additives and products or substances used in Animal feed (FEEDAP) has published “Guidance on the assessment of safety of feed additives for the consumers” [ 21 ]. In these publications the EFSA explains that it is mandatory to study genotoxicity, even in the predictable case of low exposure. Toxicological data about Allium extracts and essential oil are still scarce, especially in vivo . However, subchronic toxicity has been studied through a 90-day oral assay of Proallium [ 22 ], whose main component is PTSO. In this study, the extract demonstrated a complete absence of signs of toxicity. In addition, the pure PTSO has also been administered orally to rats for 90 days in a study published by Lira et al. [23]. Previous in vitro genotoxicity and mutagenicity studies for PTS carried out in our laboratory reported contradictory results [ 24 ]. Negative results were observed in the Ames test and mouse lymphoma (MLA) assay for PTS. In contrast, the micronucleus (L5178Y Tk ±cells) and comet assays (Caco-2 cells) revealed that PTS produced positive results at Foods 2021,10, 989 3 of 15 the highest concentrations assayed. These discrepancies in the results obtained raise the concern about PTS genotoxicity and make it necessary to clarify its genotoxic potential. For this purpose, EFSA suggests three different assays to assess the genotoxicity: the in vivo MN test, the in vivo comet assay, or the transgenic rodent gene mutation assay. Following the 3Rs principles (Replaces, Reduce, and Refine), Bowen et al. [ 25 ] suggested a combined multi-end point in vivo assay, and they evaluated the combination of a bone marrow MN test with the application of the comet assay, and actually, this in vivo combination assay in several organs has been proposed for international genotoxicity testing groups [26]. Considering all these facts, in order to clarify the contradictory results obtained in in vitro genotoxicity studies with PTS, the present study aimed to carry out the in vivo genotoxic assessment for PTS by using the combined MN-comet assay in rats for the first time. The tissues selected were bone marrow for the MN assay [ 27 ], and stomach, liver, and blood cells for the comet assay [ 28 ]. Moreover, the potential oxidative DNA damage was evaluated by the enzyme-modified comet assay with the bacterial repair enzymes formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), which release damaged purines and pyrimidines, respectively [ 29 ]. Finally, a histopathological study was carried out on the stomach and liver to obtain further information for the genotoxic assessment and to provide useful information to perform the risk assessment required by EFSA for PTS before its approval as an additive. 2. Materials and Methods 2.1. Chemical and Reagents PTS (Cod. 0200026, batch 31/19, 91% purity) was developed, chemically isolated, and provided by DMC RC (Granada, Spain). Sodium chloride (NaCl), potassium chloride (KCl), ethylenediaminetetraacetic acid (EDTA), sodium ethylenediaminetetraacetate (Na 4 EDTA), HEPES, Giemsa, and Trizma ® base were purchased from Sigma Aldrich (Madrid, Spain). Bovine serum was purchased from Gibco (Biomol, Seville, Spain). Endonuclease III and formamidopyrimidine DNA Glycosylase were purchased from Biolabs Inc. Sybr Gold was purchased from Invitrogen. 2.2. Animal Hosting and Nourishing Conditions The Ethics Committee on Animal Experimentation of the University of Sevilla approved this in vivo experiment (09/03/2016/026). Animals were humanely cared for in compliance with the guidelines for the protection of animals used for scientific purposes (Directive 2010/63 UE). Eight-week-old Wistar female and male rats, strain RjHan:WI (type outbred rats), were obtained from the Centre for Animal Production and Experimentation of the University of Sevilla (Spain). Upon arrival, animals were weighted and then accommodated into polycarbonate cages with stainless steel covers. Later, the animals were acclimatized to the environmental conditions with a 12 h dark/light cycle, temperature of 23 ±1◦C, and relative humidity of 55 ± 10% for 1 week before the experiment. During this time, the animals were provided certified pellet food (Harlan, 2014; Harlam Laboratories, Barcelona, Spain) and tap water ad libitum, free from any type of chemical contamination. 2.3. Treatment Schedules and Dose Levels The selection of PTS doses was calculated while taking into account a preceding acute toxicity study carried out by our research group, in which rats (8–10 weeks old) were administrated decreasing doses of PTS to establish the maximum tolerated dose (MTD). According to the OECD guidelines for testing chemicals [ 30 ], the initial dose tested was 175.0 mg of PTS/kg b.w., which induced the death of one of the three animals, so the next dose was established at 55.0 mg of PTS/kg b.w. In this case, three consecutive animals survived; therefore, the MTD of PTS was set at 55.0 mg of PTS/kg. At that dose, no remarkable injury was detected in the treated rats, and no evidence of study-limiting toxicity was observed. Then, considering the ICH S2 Guidelines and the OECD protocols Foods 2021,10, 989 4 of 15 474 [ 27 ] and 489 [ 28 ], the limit dose or MTD of 55.0 mg/kg b.w. was chosen as the highest dose, and two additional properly spaced dose levels (divided by √ 10) were also calculated. Consequently, the doses were set at 55.0, 17.4, or 5.5 mg of PTS/kg b.w. After acclimation time, 28 male and 28 female rats were randomly divided into control or treatment groups: the negative control (C − ) (five male and five female rats), the solvent control (C sol) (five male and five female rats) treated with water with 0.3% Tween 80, and the positive control (C+) (three male and three female rats) treated with 200 mg/kg b.w. of ethylmethanesulfonate (EMS). The three PTS-treated groups (five male and five female rats per group) were exposed to 55.0, 17.4, and 5.5 mg of PTS/kg b.w. The number of animals was selected according to OECD 474 [ 27 ] and OECD 489 [ 28 ], which state five animals per sex and per group, and which also allow the use of at least three animals of both sexes treated with a positive control. Doses were dissolved in water with Tween 80 (final concentration 0.3%) in a final volume of 1.5 mL. Tween 80 was previously used in genotoxicity in vivo studies by Mellado- Garcia et al. [ 31 ] with the aim of better conveying alliaceous compounds with an oily nature. All groups were dosed at 0, 24, and 45 h via gavage using a stomach tube (Vygon, Ecouen, France) and were sacrificed 3 h after the final dose administration for the combined comet assay and MN endpoints. The time points of PTS administrations, as well as the timing of sacrifice, were selected following the criteria of OECD guideline 489 and Bowen et al. [ 19 ]. After treatment, each rat was observed twice daily for clinical signs such as changes in the eye, fur, or skin, changes in gait and posture, and secretions. 2.4. Sample Collection Samples of the liver and stomach were extracted, and a small part of each tissue was immediately handled, as described in Section 2.6, for the standard and enzyme-modified comet assay. Moreover, blood samples were extracted and preserved in Vacutainer ® sodium Heparin Tubes (Becton, Dickinson, Rutherford, NJ, USA). Furthermore, samples from the bone marrow of both femurs of each animal were gathered for the MN assay and were rapidly used to prepare smears (Figure 1). For the histopathologic study, sections of the liver and stomach were excised, rinsed with cold saline solution, and preserved in 10% buffered formalin for light microscopy, according to Llana-Ruiz-Cabello et al. [32]. 2.5. Micronucleus Assay The MN test was performed according to the OECD guideline 474 [ 27 ] and Diez- Quijada et al. [ 33 ]. The bone marrow of the femurs of the rats were mixed with a drop of fetal bovine serum. Therefore, slides were immersed in methanol, dried, and stained with Giemsa (Figure 1). The polychromatic erythrocytes (PCE) among total erythrocytes (polychromatic and normochromic erythrocytes (NCE)) ratio and PCE for every NCE ratio were calculated by counting 500 erythrocytes per rat. The number of micronuclei found in immature erythrocytes was calculated by counting a total of 5000 micronucleated immature erythrocytes (MNPCE) per rat, and it was expressed as %MN. 2.6. Standard and Enzyme-Modified Comet Assay Single cell suspensions from the liver and stomach were isolated following Diez Quijada et al. [ 33 ], and blood samples were isolated following Medrano-Padial et al. [ 25 ]. Both samples were placed on microscope slides. At first, slides were in lysis buffer (2.5 M of NaCl, 0.1 M of EDTA, 8 mM of Na 4 EDTA, and 9.9 µ M of Trizma ® base) at 4 ◦ C for at least 1 h. After this step, microscope slides intended for enzyme treatment were washed 3 times for 5 min with an enzyme buffer (buffer F) (pH 8; 0.2 mg/mL of bovine serum albumin; 0.5 mM of EDTA; 0.1 M of KCl; 40 mM of HEPES) Afterward, the agarose gels in slides were exposed to 30 µ L of lysis buffer, buffer F, and buffer F comprising Endo III or FPG, and they were incubated in a metal box for 30 min at 37 ◦ C. All microscope slides were subjected to electrophoresis for 20 min, at 0.81 V/cm up to 400 mA. The DNA released Foods 2021,10, 989 5 of 15 from the nucleus of the cells was immersed in PBS, water, 70% ethanol, and absolute before being stained with SYBR Gold (Figure 1). At least 150 randomly selected nuclei per rat were analyzed with the image-analysis- software Comet Assay IV (Perceptive Instruments, UK). The %DNA in the tail, automatically acquired by the software, were used to define each of the comets analyzed. Endo III and Fpg sensitive sites were established by deducting the % of DNA in the tail after repair enzymes incubation. The medians of the scored comets were taken to describe each animal, and the means of each group of doses and controls are represented in Figure 2. Foods 2021, 10, x FOR PEER REVIEW 6 of 16 Figure 2. Micronucleus and comet assay schematic diagram on the same animal and its visualization under the optical microscope (fluorescence and bright field). 2.7. Histopathological Analysis Liver and stomach samples were processed and observed under the microscope according to Diez-Quijada et al. [33]. A semiquantitative evaluation of the severity of lesions was scored as follows: 0, no significant lesions (0%); 1, minimal (<10%); 2, mild (11–25%); 3, moderate (26–50%); 4, severe (51–75%); and 5: very severe (>75%). Samples were individually examined by two unbiased and experienced observers, a veterinary pathologist, and an investigator (V.M-H. and R.M.). 2.8. Statistical Analysis The results of the MN test are presented as the mean ± standard deviation (SD) for each group of animals, and a statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The results of the standard and enzyme-modified comet assays were calculated for each group as the mean ± SD Figure 2. Micronucleus and comet assay schematic diagram on the same animal and its visualization under the optical microscope (fluorescence and bright field). 2.7. Histopathological Analysis Liver and stomach samples were processed and observed under the microscope according to Diez-Quijada et al. [ 33 ]. A semiquantitative evaluation of the severity of lesions was scored as follows: 0, no significant lesions (0%); 1, minimal (<10%); 2, mild Foods 2021,10, 989 6 of 15 (11–25%); 3, moderate (26–50%); 4, severe (51–75%); and 5: very severe (>75%). Samples were individually examined by two unbiased and experienced observers, a veterinary pathologist, and an investigator (V.M-H. and R.M.). 2.8. Statistical Analysis The results of the MN test are presented as the mean ± standard deviation (SD) for each group of animals, and a statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The results of the standard and enzyme-modified comet assays were calculated for each group as the mean ± SD of the medians. The distribution of the results was checked for normality utilizing the Kolmogorov–Smirnov test, and the total scores of the different groups were compared using the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. For the histopathological study, the Kolmogorov–Smirnov test was used to assess if distributions were parametric. Groups were compared by applying the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. pvalues < 0.05 were considered statistically significant. Analyses were carried out using Graph-Pad Prism software (Graph-Pad software Inc., La Jolla, San Diego, CA, USA). 3. Results No clinical signs were observed during the entire course of the study after treating the rats with all levels of doses of PTS, water, or water with added Tween 80. However, rats treated with EMS presented piloerection and an attenuation in their normal physical activity. The results obtained in the positive and negative control groups in others and recent studies [ 29 ] were similar to those obtained in the present work for comet and micronucleus assays. 3.1. Micronucleus Assay In Table 1, the results of the in vivo MN assay of rats exposed to PTS are shown. When the results were compared to their respective controls, significant differences in PCE/NCE ratio were found, showing a decrease at the highest dose assayed (55 mg/kg) in males and females (p< 0.05). Considering the PCE/Total ratio, a significant reduction was also observed in males and females at 55 mg/kg (p< 0.05), and in females at 17.4 mg/kg (p< 0.05). Moreover, the % of MN in immature erythrocytes was not significantly changed in any groups of either sex. Rats exposed to 200 mg/kg of EMS showed remarkable reductions in PCE/NCE and PCE/total ratios, and a significant increase in the frequency of MN (positive control). 3.2. Standard and Enzyme-Modified Comet Assay The %DNA in the tail results found in the standard and enzyme-modified comet assays are represented in Figure 3. No observed DNA stand breaks were recorded in the liver, stomach, and blood in rats treated with PTS (Figure 3A) at any doses assayed in either sex. Likewise, no remarkable changes were found in the frequency of Endo III or FPG-sensitive sites in any tissue assayed (Figure 3B,C). In contrast, significant elevations were clearly observed in rats treated with 200 mg/kg of EMS (positive control). The distribution of comet results with each treatment by presenting data for individual comet data is available as Supplementary Material Tables S1–S3. Foods 2021,10, 989 7 of 15 Table 1. Results of the micronucleus assay in rats exposed to PTS. Bone marrow cytotoxicity expressed as polychromatic erythrocytes (PCE) among total erythrocytes (normochromic erythrocytes (NCE) + PCE), ratio PCE among NCE, and the micronuclei induction expressed as % micronucleus (MN). Statistical analysis was performed using the analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The values are expressed as the mean ± SD. Significant difference from the negative and solvent control (* p< 0.05; ** p< 0.01; *** p< 0.001). Groups Sex N Doses PCE/NCE PCE/Total %MN Negative Control ♂5 - 1.72 ±0.85 0.57 ±0.04 0.57 ±0.23 ♀5 - 1.27 ±0.07 0.61 ±0.05 0.86 ±0.16 Solvent Control ♂5 - 1.74 ±0.63 0.60 ±0.09 0.49 ±0.37 ♀5 - 1.16 ±0.34 0.53 ±0.07 0.75 ±0.24 Positive Control (EMS 1) ♂3200.0 mg/kg 0.54 ±0.12 *** 0.35 ±0.05 ** 1.46 ±0.72 * ♀3 0.53 ±0.29 ** 0.33 ±0.12 ** 2.53 ±0.38 * PTS ♂55.5 mg/kg 1.31 ±0.30 0.56 ±0.05 0.46 ±0.21 ♀5 1.03 ±0.43 0.49 ±0.10 0.86 ±0.09 ♂517.4 mg/kg 1.14 ±0.34 0.52 ±0.08 0.72 ±0.30 ♀5 0.68 ±0.36 0.38 ±0.13 * 0.74 ±0.06 ♂555.0 mg/kg 0.89 ±0.21 * 0.46 ±0.06 * 0.46 ±0.15 ♀5 0.54 ±0.28 * 0.33 ±0.11 ** 1.03 ±0.21 1EMS = ethylmethanesulfonate; ♂= Males; ♀= Females. 3.3. Histopathological Study The histopathological study of the stomach and liver did not show differences between males and females (data not shown). The scores obtained after the pathological evaluation of both the stomach and liver are shown in Figure 4. Under light microscopy, the negative and solvent controls stomach samples showed no pathological signs, displaying a minimal flaking epithelium of the gastric mucosa (Figure 5A,B). The positive controls displayed lesions scored as mild, showing an evident flaking epithelium with focal necrosis of the apical gastric glands (Figure 5C). This group showed significant differences (p< 0.05) in the damage recorded in comparison to the negative and solvent control group (Figure 4). Occasionally, there were minimal inflammatory infiltrates with neutrophils and macrophages within the connective tissue of the submucosa layer. The PTS 55.0 mg/kg dose group presented minimal to mild pathological lesions in the glandular stomach, such as hypertrophy of the mucus cells and flaking epithelium (Figure 5), these changes being statistically significant (p< 0.05) compared to the negative and solvent controls (Figure 4). However, the PTS 17.4 and 5.5 mg/kg doses presented statistically significant minimal histopathological changes (Figure 4E,F). Occasionally, in the rats exposed to PTS 17.4 mg/kg, minimal hyperemia of the apical mucosa glands was observed (Figure 5E). Moreover, in the positive control and PTS 55.0 mg/kg groups, lesions were found in the nonglandular stomach characterized by vacuolation of the squamous epithelium in the mucosa associated with a mild to moderate pyogranulomatous inflammatory infiltrate and by a moderate pyogranulomatous inflammatory infiltrate in the submucosa (Figure 5H,I). Foods 2021,10, 989 8 of 15 Foods 2021, 10, x FOR PEER REVIEW 8 of 15 liver, stomach, and blood in rats treated with PTS (Figure 3A) at any doses assayed in either sex. Likewise, no remarkable changes were found in the frequency of Endo III or FPG-sensitive sites in any tissue assayed (Figures 3B,C). In contrast, significant elevations were clearly observed in rats treated with 200 mg/kg of EMS (positive control). The distribution of comet results with each treatment by presenting data for individual comet data is available as Supplementary Material Tables S1, Tables S2 and Tables S3. Figure 3. The % of DNA in the tail is representative of damage produced in the liver, stomach, and blood of male and female rats exposed to PTS and EMS (positive control). (A) Damage by the standard comet assay as strand breaks (SBs), (B) oxidative damage detected by the FPG enzymes, and (C) oxidative damage detected by Endo III enzyme. All values are expressed as the mean ± SD. The statistical test used was ANOVA or Kruskal–Wallis based on the normality test. Significant differences from the negative control were: * p < 0.05; ** p < 0.01; *** p < 0.001. Figure 3. The % of DNA in the tail is representative of damage produced in the liver, stomach, and blood of male and female rats exposed to PTS and EMS (positive control). ( A ) Damage by the standard comet assay as strand breaks (SBs), ( B ) oxidative damage detected by the FPG enzymes, and ( C ) oxidative damage detected by Endo III enzyme. All values are expressed as the mean ± SD. The statistical test used was ANOVA or Kruskal–Wallis based on the normality test. Significant differences from the negative control were: * p< 0.05; ** p< 0.01; *** p< 0.001. Foods 2021,10, 989 9 of 15 Foods 2021, 10, x FOR PEER REVIEW 9 of 16 3.3. Histopathological Study The histopathological study of the stomach and liver did not show differences between males and females (data not shown). The scores obtained after the pathological evaluation of both the stomach and liver are shown in Figure 4. Figure 4. Mean ± standard deviation (SD) of the histopathological score recorded in the liver and stomach of female and male rats treated with PTS. * Statistical difference (p < 0.05) compared to the negative and solvent controls using the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. Under light microscopy, the negative and solvent controls stomach samples showed no pathological signs, displaying a minimal flaking epithelium of the gastric mucosa (Figure 5A,B). The positive controls displayed lesions scored as mild, showing an evident flaking epithelium with focal necrosis of the apical gastric glands (Figure 5C). This group showed significant differences (p < 0.05) in the damage recorded in comparison to the negative and solvent control group (Figure 4). Occasionally, there were minimal inflammatory infiltrates with neutrophils and macrophages within the connective tissue of the submucosa layer. The PTS 55.0 mg/kg dose group presented minimal to mild pathological lesions in the glandular stomach, such as hypertrophy of the mucus cells and flaking epithelium (Figure 5), these changes being statistically significant (p < 0.05) compared to the negative and solvent controls (Figure 4). However, the PTS 17.4 and 5.5 mg/kg doses presented statistically significant minimal histopathological changes (Figure 4E,F). Occasionally, in the rats exposed to PTS 17.4 mg/kg, minimal hyperemia of the apical mucosa glands was observed (Figure 5E). Moreover, in the positive control and PTS 55.0 mg/kg groups, lesions were found in the nonglandular stomach characterized by vacuolation of the squamous epithelium in the mucosa associated with a mild to moderate pyogranulomatous inflammatory infiltrate and by a moderate pyogranulomatous inflammatory infiltrate in the submucosa (Figure 5H,I). Figure 4. Mean ± standard deviation (SD) of the histopathological score recorded in the liver and stomach of female and male rats treated with PTS. * Statistical difference (p< 0.05) compared to the negative and solvent controls using the nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test. Foods 2021, 10, x FOR PEER REVIEW 10 of 16 Figure 5. Histopathological changes in the stomach of rats exposed to PTS. (A–C) Glandular stomach of controls (negative, solvent, and positive, respectively); (D–F) Glandular stomach of PTS doses (55.0, 17.4, and 5.5 mg/kg, respectively); (G–I) Nonglandular stomach of negative control, positive control, and PTS 55.0 mg/kg dose, respectively. Hematoxylin and eosin stain. *: flaking epithelium of the gastric mucosa; Nc: focal necrosis of the apical gastric glands; arrow-head: hypertrophy of the mucus cells; arrow: occasional hyperemia of the apical mucosa glands; V: vacuolation of the squamous epithelium in the mucosa; Inf: inflammatory infiltrates in the mucosa and submucosa. Livers from the negative and solvent controls showed no pathology findings apart from a normal physiological deposition of glycogen (Figure 6A,B). The lesions found in the positive controls were a mild multifocal distribution of hepatocyte degenerated in the surroundings of the central vein with occasional necrosis of hepatocytes (Figure 6C). Occasionally, there were areas filled with plasma and congestion. These lesions were significantly different (p < 0.05) in comparison to negative and solvent controls (Figure 4). Moreover, PTS doses of 55.0 mg/kg displayed minimal inflammatory infiltrates apart from a minimal centrilobular degeneration of hepatocytes (Figure 6D) showing statistically significant differences (p < 0.05) compared to the negative and solvent controls (Figure 4). However, PTS 17.4 and 5.5 mg/kg doses displayed minimal changes similar to the negative and solvent controls (Figure 6E,F). Figure 5. Histopathological changes in the stomach of rats exposed to PTS. ( A – C ) Glandular stomach of controls (negative, solvent, and positive, respectively); ( D – F ) Glandular stomach of PTS doses (55.0, 17.4, and 5.5 mg/kg, respectively); ( G – I ) Nonglandular stomach of negative control, positive control, and PTS 55.0 mg/kg dose, respectively. Hematoxylin and eosin stain. *: flaking epithelium of the gastric mucosa; Nc: focal necrosis of the apical gastric glands; arrow-head: hypertrophy of the mucus cells; arrow: occasional hyperemia of the apical mucosa glands; V: vacuolation of the squamous epithelium in the mucosa; Inf: inflammatory infiltrates in the mucosa and submucosa.