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Antibiotics and organophosphate pesticides at maximum residue level: A threat to the safety of animal proteins

Resumen en inglés

Meat is the most consumed animal source food on the planet nowadays. This is due to its nutritional value, which is represented in its high protein content. Therefore, any sort of physicochemical alteration that may be suffered by the structure of meat proteins, could affect greatly the nutritional qualities of this food and even its safety. As evidence, one of these alterations takes place as a consequence of vulnerability present in proteins to suffer oxidation reactions such as carbonylation. This is an irreversible and not enzymatic modification, that is characterized for the incorporation process of the carbonyl’s groups on the side chains of the amino acid residues in the protein. On the other hand, it has been stated that food carbonylation may lead to a substantial loss of solubility and protein functionality, as well as a decrease in all the proteolytic activity (digestibility), factors which favor in this loss of nutritional value and also properties of the meat. Different factors such as internal and external may play a major role in the reactions that lead to the formation of the carbonyl groups in muscle protein of the meat. However, due to the current model of intensive livestock to supply the high demand for meat products, all the different external factors are assessed thoroughly. Such factors associated are the feeding process and slaughtering of the animal (animal husbandry), processing (curing, cooking), and storage (temperature, time). In this case, the farming of these animals plays a major role, keeping in mind that when these animals are in the model of intensive farming are exposed to the appearance of diseases transmitted by microorganisms and/or ectoparasites, which are treated with antibiotics and/or biocides like fluoroquinolones and organophosphate pesticide to preserve the animal's welfare and Doctoral thesis the productivity in the livestock sector. Nevertheless, some experiments in different biological models have proofed that some metabolic reactions are triggered, increasing oxidative stress. As a result, some remnants may help prolonge the outcome because of its presence in muscular tissue even long after the animal has been slaughtered and processed. This specific situation is guarded and regulated by FAO through a group of regulations called Codex Alimentarius, which allows the existence of fluoroquinolones and organophosphate pesticides in the meat, within the maximum residue limits (MRL). These numbers indicate the no toxicity of the residues to human health even when exists a frequent consumption of meat. Yet, the impact of these residues' levels on the muscle protein of the animals is unknown to this day. Therefore, this research proposes was to evaluate the in vitro oxidative effect of fluoroquinolone and organophosphate pesticides residues on beef and chicken meat at concentrations around of its MRLs, as well as the impact on solubility and digestibility. For this, initially, some samples were taken from beef and exposed individually to Enrofloxacin, Danofloxacin, Sarafloxacin, Norfloxacin, and Ciprofloxacin, at concentrations equal to 0.5,1.0 and 1.5 MRL under in vitro conditions. Followed of the extraction and quantification of sarcoplasmic and myofibrillar proteins as well as the determination of carbonyls index. Additionally, the identification of the carbonylated proteins was carried out by the combinations of western blot and mass spectrometry assays. Also, the gastrointestinal digestion of sarcoplasmic and myofibrillar proteins was monitored by SDSPAGE electrophoresis. The results of the first research evidenced the in vitro capability of fluoroquinolones residues to induce carbonylation on sarcoplasmic and myofibrillar proteins even at concentrations below its MLR. Hence, this oxidative damage was correlated to the loss of solubility and digestibility, as a result, being the sarcoplasmic proteins the most impacted. Enrofloxacin and Danofloxacin were the fluoroquinolones with more oxidizing effects, especially on the proteins of glycolytic pathway. The article “Residues of fluoroquinolone antibiotics induce carbonylation and reduce in vitro digestion of sarcoplasmic and myofibrillar beef proteins”, published by Journal Foods de MDPI (https://doi.org/10.3390/foods9020170) provides more detailed information on this investigation. In order to deepen the molecular aspects of oxidation promoted by the pollutants under study, assays were conducted on a myosin pattern, as one of the most abundant proteins in muscle tissue. Solutions of myosin were contaminated with residues of Chlorpyrifos and Diazinon at different exposure times. The degrees of oxidation were determined, later on, this allowing to find a major oxidizing power for Chlorpyrifos in all the times of exposure. Thus, based on these results the investigation led to the contamination of samples of chicken breast with 100 ppb of Chlorpyrifos, followed by extraction of total proteins and the quantification of the carbonyl index. The proteins obtained were separated by size-exclusion chromatography and the resultant fractions were collected and grouped for the quantification of the carbonyls index and the subsequent identification by mass spectrometry. To do this, collected proteins from chromatographic fractioning were digested with trypsin and analyzed by nano-LC-qTOF. Besides, peptidome’s analysis was conducted. For this assay, chicken breast samples contaminated with chlorpyrifos were subjected to simulated gastrointestinal digestion, and the peptides obtained were analyzed by mass spectrometry to identify those which were oxidized. The results showed for proving that the residues of chlorpyrifos were capable to induce in vitro carbonylation not only on the myosin pattern but also on the proteins present in the chicken breast. Moreover, myosin, β-enolase, type M kinase creatine, and actin were identified as the most susceptible to the oxidation triggered by this pollutant. Likewise, in this sort of analysis, conducted by mass spectrometry, oxidized peptides mainly of collagen were identified. The results of this investigation can be found more in detail in two articles. The first one's title is “In vitro oxidation promoted by chlorpyrifos residues on myosin and chicken breast proteins”; published in Food Chemistry (https://doi.org/10.1016/j.foodchem.2020.126922). On the other hand, the resultant data of the proteomic and peptidomics analysis were not included in the previous article, in fact, they were submitted to Data in Brief journal and are currently under review. The title of the investigation is “Peptidomes and proteomics data of oxidized peptides from in vitro gastrointestinal digestion of chicken breast exposed to chlorpyrifos”. In addition, Eisenia fetida biological model was used to confirm the results obtained from in vitro model. The results of this research are detailed in the article " Fluoroquinolone antibiotics and organophosphate pesticides induce protein carbonylation on Eisenia fetida", which was submitted in Science of the Total Environment journal. For this, earthworms were individually exposed to ciprofloxacin, danofloxacin, fethion and diazinon at 0.5, 1.0 and 1.5 MRL values during 28 days. The, earthworms were sacrificed and the region between annus and pre clitelo were cut. Muscle proteins were extracted and its quantification was done by Bradford method. The carbonyl index and oxidative profile were obtained by dot blot and western blot assays, respectively. The identification of carbonylated proteins were realized by mass spectrometry (MALDI-TOF). The results showed that all concentrations evaluated by contaminant induced oxidative damage on muscle proteins of E. fetida. Also, CIPRO and DZN were contaminats that promoted the highest carbonylation on mucle proteins. The actin was identified as the main target of oxidation induced by fluoroquinolones and organophosphare pesticides under study. Thus, these finding supports the observations found in bovine and chicken muscle proteins under in vitro conditions. As a conclusion to this doctoral thesis, it is evidenced for the first time that fluoroquinolones and organophosphate pesticides are capable to induce in vitro carbonylation on beef and chicken breast proteins, even at concentrations considered to be safe to human (MRL) as well as muscle proteins from E. fetida. Besides, such modification demonstrated to have an impact on the solubility of proteins in beef, as well as the digestibility of proteins from both beef and chicken breast. Thus, therefore, these suggests that the presence of residues in meat could have a negative effect on its quality and nutritional value. Additionally, this study contributes to the basis for the creation of a research line focused on the impact of veterinary substances on the nutritional quality of animal and plant origin products, whose purpose will be to promote the need for zero tolerance of chemicals in food.