Evaluación toxicológica in vitro e in vivo de compuestos organosulfurados con potencial uso en envasado activo en contacto con alimentos

Abstract

Currently, the food industry is betting on the incorporation of natural substances into food packaging in order to increase the durability of food in the market. These practices are intended to satisfy the needs of consumers so that their use entails technological advantages and benefits for the consumer. However, in order to admit a substance as an additive, it must be well characterized chemically and must overcome the toxicological assessment established by the corresponding health agencies, due to the lack of knowledge of the use of these components and their possible consequences. Among the natural substances used for this purpose are Essential Oils (EOs), which have traditionally been used for their pharmacological properties. In our case, both an extract of the genus Allium, commercially known as PROALLIUM AP®, as well as some of its components (PTSO and its analog PTS) are intended to be used as antimicrobials in the food industry as part of active packaging. As a preliminary step to the study of its safety, a bibliographical review of the toxicity data available so far was made in the scientific literature. Afterwards, the lack of studies required in different compounds with interest in food preservation and the disparity of available results have been evidenced. The toxicological evaluation carried our in the present Thesis started with a battery of tests both in vitro, with cells that would be in contact with these substances when ingested, and in vivo, in rats after acute and chronic consumption, in order to investigate possible toxic effects, mainly genotoxicity, which may be triggered upon exposure to PROALLIUM AP®, PTSO and PTS. Tre relevance of the toxicological information of these substances is essential for their future use since it is a regulatory requirement by the competent authorities prior to their commercialization. All these experiments has led to the following publications: GENOTOXICITY ASSESSMENT OF PROPYL THIOSULFINATE OXIDE, AN ORGANOSULFUR• COMPOUND FROM ALLIUM EXTRACT, INTENDED TO FOOD ACTIVE PACKAGING. (Mellado-García y cols., 2015), Food and Chemical Toxicology 86, 365-373. The most important route of exposure in our context is the oral route because once the PROALLIUM AP® extract is incorporated into films in active packaging, PTSO could be ingested by consumers. In this work, Caco-2 cells (colon adenocarcinoma) were exposed to different concentrations depending on the maximum concentration that would migrate from the film to the consumer in the worst possible scenario. Experiments carried out in our laboratory included the evaluation of the mutagenicity of PTSO in different strains of S. typhimurium (0-20 μM) each one with different genetically altered characteristics to present mutations in genes involved in histidine synthesis to cover a wide range of possible mutations. Mutagenicity in mammalian L5178Y TK +/- cells (MLA assay) was also studied following a previous cytotoxicity study, in which the exposure concentrations were determined at 4h (0-30 μM) and at 24h ( 0-20 μM). In the case of the Ames test, there were no significant differences in any of the strains studied in the absence or presence of S9. However, at 24 h of exposure in the MLA assay, significant differences were observed in the revertant colonies in the range of 2.5-20 μM. On the other hand, PTSO did not induce increases in the percentage of MN (0-40 μM) in the absence of S9 at any of the concentrations tested, but it did in the presence of the microsomal fraction S9 from 15 μM, indicating the genotoxicity of its metabolite. Finally, the comet assay (0-50 μM) showed that PTSO did not induce DNA strand breaks or oxidative damage in the DNA of Caco-2 cells exposed. Afterwards, considering the contradictory results of PTSO obtained in genotoxicity in vitro, following the recommendations of the EFSA (EFSA 2011), the in vivo genotoxicity of PTSO in Wistar rats was studied in the following work: GENOTOXICITY OF A THIOSULFONATE COMPOUND DERIVED FROM ALLIUM sp.• INTENDED TO BE USED IN ACTIVE FOOD PACKAGING: IN VIVO COMET ASSAY AND MICRONUCLEUS TEST. (Mellado-García y cols., 2016), Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 800-801, 1-11. Genotoxicity was studied by the MN test in the bone marrow in Wistar rats, and with the comet assay in stomach and liver of rats, being the first contact organ in the ingestion of PTSO and the main metabolism organ of xenobiotics, respectively. This study was performed by administering to the rats doses of 55; 17.4; 5.5 mg/kg b.w. of PTSO. After necropsy, a histopathological study of both organs was performed, with no evidence of genotoxicity under optical microscopy or electron microscopy in rats exposed to the control. Only at the highest concentration tested an increase in glycogen storage in liver and degenerative processes in the stomach with vacuolization of cell membranes were observed. The study was completed with an analysis to determine the presence of PTSO by UHPLC-MS/MS Orbitrap chromatography in the blood of treated rats, with no PTSO detected. Finally, in order to corroborate the absence of genotoxicity in the organs previously studied, the evaluation of the presence of PTSO in the tissues using the analytical pyrolysis technique by gas chromatography combined with a mass detector (Py-GC-MS) was carried out. It was demonstrated the existence in liver of derivatives of the main component and two possible metabolites, which confirmed the phenomenon of metabolism of PTSO in the organism. Following the acute toxicity study of PTSO in rats, and again following the recommendations of the EFSA (EFSA, 2011), in order to complete the results of genotoxicity obtained, and given the lack of in vivo information, a test of chronic toxicity for 90 days with PROALLIUM AP® was carried out. TOXICOLOGICAL EVALUATION OF AN ALLIUM-BASED COMMERCIAL PRODUCT IN• A 90-DAY FEEDING STUDY IN SPRAGUE-DAWLEY RATS. (Mellado-García y cols., 2015), Food and Chemical Toxicology 90, 18-29. PROALLIUM AP® is an extract of different components present in Allium sp. This extract will be incorporated into the films mentioned above for their antimicrobial activity. In this regard, following the previous tests, due to the lack of information on this component and following the recommendations of the Scientific Committee on Food of the European Union, which requires the evaluation of substances used in food contact materials. An oral subchronic toxicity study was performed on Sprague-Dawley rats for a 90-day oral exposure period. The doses: 0, 25, 100 and 400 mg/kg/day PROALLIUM AP® were used. Rats were sacrificed and their organs (liver, kidney, intestine, brain, thymus, epididymis, adrenal gland, heart, testicles/ovaries, lungs and spleen) and blood by cardiac puncture were extracted. In addition, rats were monitored weekly for weight, water and food consumption, and a histopathological, clinical and hematological study of the exposed rats was added. Rats showed no clinical signs of dose-related mortality. The results did not show significant differences at any of the concentrations exposed to the control, in any of the parameters studied. In this way, PROALLIUM AP® NOAEL was determined at 400 mg/kg/day, a value 500 times higher than exposure derived from its potential use in active packaging. In addition, in order to study another OS component with potential application in the food industry, PTS, to which antimicrobial properties are also attributed, its in vitro cytotoxicity, mutagenicity and genotoxicity evaluation were performed in the following publication: IN VITRO TOXICOLOGICAL ASSESSMENT OF AN ORGANOSULFUR COMPOUND• FROM ALLIUM EXTRACT: CYTOTOXICITY, MUTAGENICITY AND GENOTOXICITY STUDIES. (Mellado-García y cols., 2016), Food and Chemical Toxicology (in press) The objective of this study was to study for the first of cytotoxicity of PTS in Caco-2 cells at 24 and 48h, determining its CE50 (280 μM) through different biomarkers of viability (RN, MTS, CP). Moreover, an evaluation of the mutagenicity in the Ames test (0-280 μM) was carried out, in which 5 strains of S. typhimurium were used. In this test, no significant differences were observed at any of the concentrations tested, in the presence or absence of S9. On the other hand, in the MLA assay, no significant differences were observed in the assay either after 4 or 24 hours of exposure, thus demonstrating the absence of mutagenicity. In addition to these assays, an assessment of genotoxicity was performed by the MN test on L5178Y TK+/- cells both in the absence (0-17.25 μM) and in the presence (0-25 μM) of S9. In this assay, an increase in the frequency of binucleated cells with MN at the highest concentration tested without S9 (17.25 μM) was detected, and at the two highest concentrations with S9 (20-25 μM), showing that the metabolites as well as the original component produce genotoxicity. Finally, genotoxicity was studied using the standard comet assay (0-280 μM) and modified with restriction enzymes in Caco-2 cells. In this case, only DNA damage at the highest concentration tested in the standard comet assay was observed, whereas no oxidative damage was observed in the modified comet assay at any concentration. For the accomplishment of this doctoral thesis, the Phd student realized an international stay in the department of "SEBIO, Stress Environnementaux et Biosurveillance de milieux aquatiques" in the University of Reims, Champagne-Ardennes, (France). This stay was conducted under the direction of Dr. Stéphane Bettoulle and Alain Geffard (department director) and with the collaboration and supervision of Dr. Hakim Samai. • "DETERMINATION OF CELL MORTALITY, OXIDATIVE STRESS AND PHAGOCYTOSIS IN THP-1 CELL MACROPHAGES BY FLOW CYTOMETRY", Revista Española de Toxicología (pending publication). In this work, the behavior of PTSO in THP-1 cells, human monocytic leukemia cells, was studied by means of cell death, oxidative stress and phagocytosis activity by flow cytometry. For this purpose, THP-1 cells were activated into a macrophage state, resulting in a significant increase in cell death from 60 μM PTSO. On the other hand, no significant production of reactive oxygen species was observed at any concentration. Finally, phagocytosis activity was studied using fluorescent latex microspheres, which showed significant differences at 60 μM of PTSO and the highest concentration tested (150 μM PTSO). The mean number of microspheres phagocytosed per cell was determined, with significant differences obtained at the two highest concentrations tested against the negative control (100 and 150 μM) for a total of 6 microspheres/cell. Taking into account these results, we could say that the concentrations that can produce toxic effects in vitro in lymphocytic cells are lower than those expected to reach the consumer in the worst case scenario (37.5 μM PTSO).