ANGELICA BUENO BARBEZAN

ANGELICA BUENO BARBEZAN

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Evaluation of cytotoxicity through MTS test of 2-ACBs (2-dDCB AND tDCB) after exposure to hepatic cells
2019 - BARBEZAN, A.B.; CARVALHO, L.R.; VIEIRA, D.P.; MACHADO-SANTELLI, G.M.; MARTINS, R.; VILLAVICENCIO, A.L.C.H.
Food irradiation has been approved in more than 60 countries for many applications in a wide variety of foods. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products formed when foods containing fatty acids are irradiated. Despite the importance of food irradiation, the toxicological potential of 2-ACBs in irradiated food is still no fully understood. In this study we investigated the cytotoxic effects of irradiated palmitic and stearic fatty acids byproducts, 2-dDCB and 2-tDCB, in hepatic cell lines (HepG2, BRL3A and HTC). The cytotoxic effects of 2-dDCB and 2-tDCB were evaluated at 100, 300 and 500 μM for 24 and 48 hours and the cell viability was measured using the MTS assay. While no toxicity was observed for 2-tDCB in all cells for all tested conditions, 2-dDCB was found to be toxic to BRL3A cells (at 100 μM after 48 hours) and HTC cells (at 24 hours in all tested concentrations). HepG2 cells on the other hand, were found to be resistant to 2-dDCB-induced toxicity. Overall our data shows that the byproduct 2-tDCB is not toxic for hepatic cells while 2-dDCB can be used has a potential marker for food irradiation-induced toxicity.
Avaliação do potencial citotóxico da 2-tetradecilciclobutana em células hepáticas linhagem HepG2
2016 - BARBEZAN, A.B.; SALES, B.R.; MARTINS, R.; BUENO, J.B.; SANTELLI, G.M.M.; VILLAVICENCIO, A.L.C.H.
Introdução: A irradiação de alimentos é um método eficaz e seguro para a preservação e armazenamento em longo prazo, é aprovado para utilização em mais de 60 países para diversas aplicações em uma ampla variedade de produtos alimentares (Agric. Food Chem. 51; 927, 2003. Food Chem. 201; 52-58, 2016). Este processo é realizado através da utilização de feixes de elétrons acelerados, raios-X ou radiação gama (60Co ou 137Cs). As 2-Alcilciclobutanonas (2-ACBs) são os únicos produtos radiolíticos conhecidos gerados a partir de alimentos que possuem ácidos graxos (Triglicérides) e são submetidos à irradiação (J. Food Protc. 67; 142, 2004. T. Food Scie.Tech. 44; 66-78, 2015). O ácido analisado neste estudo é o esteárico que quando irradiado forma 2-Tetradecilciclobutanona (2-tDCB). Desde a década de 1990 estudos toxicológicos de segurança das 2-ACBs tem sido conduzido extensivamente através de compostos sintéticos. Testes de mutagenicidade das 2-ACBs realizados indicam claramente que nenhuma evidência foi observada, enquanto estudos de viabilidade apresentaram citotoxicidade notada através da morte celular (Food Scie. Tech. 44; 66-78, 2015). Parte das 2-ACBs ingeridas é excretada através das fezes e parte ficam depositadas em tecidos adiposos. Estudos realizados até o momento foram somente em células de cólon. A linhagem escolhida para este trabalho é derivada de células hepáticas uma vez que o acumulo de gordura neste órgão é bastante comum. Objetivo: Avaliar possíveis danos citotóxicos, através do teste de viabilidade celular MTT observando a influência de diversas concentrações da 2-tDCB em diferentes tempos de incubação em células hepáticas da linhagem HepG2. Métodos: O composto 2-tDCB foi solubilizado em etanol a 2%. A linhagem celular escolhida é derivada de hepatocarcinoma humano (HepG2) e foi cultivada em meio de cultura suplementado com 10% de soro fetal bovino. As células foram plaqueadas na densidade de 5x103 cél/poço em uma placa de 96 poços. O efeito citotóxico da 2-tDCB foi avaliado nas concentrações de 100, 300 e 500μM, durante 24 e 48 horas. Os testes foram realizados de acordo com instruções do kit CellTiter96 Aqueus Non-radioactive Cell Proliferation Assay, em triplicatas (biológica e experimental) e os resultados foram analisados pelo programa Prisma GraphPad. Resultados: A linhagem tratada com 2-tDCB em 24 e 48h não apresentou citotoxicidade em nenhuma das concentrações avaliadas. Conclusão: Não houve inviabilidade causada pelo composto 2-tDCB na linhagem de células hepática estudadas, nenhum dano foi observado em nenhuma das variações pesquisadas. Estudos mais aprofundados são necessários para identificar os mecanismos moleculares pela qual o composto em questão atua.
Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2
2019 - BARBEZAN, ANGELICA B.; SANTOS, CARLA J.B.; CARVALHO, LUMA R.; VIEIRA, DANIEL P.; SANTELLI, GLAUCIA M.M.; VILLAVICENCIO, ANNA L.C.H.
Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2- Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line.
In vivo evaluation of genotoxic potential of liver cells from rats fed with irradiated diet using flow cytometry
2017 - MARTINS, R.; CARVALHO, L.R. de; VIEIRA, D.P.; BARBEZAN, A.B.; VILLAVICENCIO, A.L.C.H.
Radiation sources became widely available since 1960’s, and between its main uses are the applications in food irradiation and research of effects of ionizing radiation on food products. Despite some public concern, the process is safe, free from chemical residues and presents advantages for preservation and storage. Nevertheless, safety dose parameters must be adopted in irradiation procedures to inhibit formation of undesirable and/or toxic products, for example, 2-ACB’s (2-alkylcyclobutanones) that are cyclic compounds containing four carbon rings that can be formed in food when its fat content is irradiated through breakdown of fatty acids. 2-ACBs are considered a unique class of compounds due to divergences between results of its mutagenicity potential collected from different studies. In this study, a cell population collected from rat livers were chosen for in vivo genotoxicity analysis because the importance of the liver in the metabolization of compounds. Analysis was performed using the micronuclei test using flow cytometry, allowing faster analysis, use of few materials and reduction in the number of animals, what is a subject much addressed currently in research. Irradiated rat diet did not show any genotoxic effect on liver cell populations. The improvement of the techniques is important for the future of the research since the irradiation process is already consolidated.
Estudos in vitro da genotoxicidade e citotoxicidade em células hepáticas da formação de 2-alcilciclobutanonas resultantes da irradiação de alimentos que contenham gordura
2017 - BARBEZAN, ANGÉLICA B.
A irradiação de alimentos já foi aprovada e vem sendo utilizada em diversos países para aplicações e finalidades de uma ampla variedade de alimentos. Seus benefícios abrangem o aumento do prazo de validade, melhoria de higiene dos alimentos e consequentemente menor deterioração e perdas se comparado com alimentos que não sofrem radiação. Além disto, os alimentos após irradiados apresentam-se seguros em termos nutritivos e de redução de patógenos. Porém, alimentos que contem de médio a alto teor de gordura induzem a formação de um subproduto denominado 2-Alcilciclobutanonas, a qual sabemos que parte destes compostos ingeridos são normalmente excretados através das fezes, porém parte permanece depositada nos tecidos adiposos. Trabalhos realizados com estes compostos anteriormente apresentaram efeitos citotóxicos e genotóxicos em células de cólon. Desta forma, o objetivo do presente trabalho foi investigar os efeitos citotóxicos realizados em testes de viabilidade celular, testes genotóxicos em micronúcleo e testes mutagênicos com a técnica de Ames em condições experimentais in vitro dos compostos 2-dDCB e 2-tDCB. Para isso, o fígado foi o órgão de escolha para avaliar os possíveis efeitos destes compostos, uma vez que este órgão é geralmente acometido pelo acumulo de gordura. Foram utilizadas três linhagens hepáticas: HepG2, BRL3A e HTC. A análise dos resultados da viabilidade celular, revelou que as 2-dDCBs apresentaram discreto efeito citotóxico na concentração de 500 μM e as 2-tDCBs apresentaram danos baixos a partir de 100 μM e maiores em 500 μM, mostrando ser dose dependentes. Nos resultados de mutagenicidade, os compostos não apresentaram quaisquer efeitos mutagênicos nas concentrações e doses utilizadas, detectados pelo teste de Ames. Por fim, o ensaio de micronúcleo correspondeu às expectativas não demonstrando efeitos genotóxicos na linhagem, doses e tempos testados. Com base nos resultados atingidos, as 2 ACBs podem ser consumidas com relativa segurança, sob a ótica de possíveis efeitos mutagênicos e genotóxicos nas concentrações avaliadas.
Information on the presence of 2- Alkylcyclobutanones in animal feed after ionizing radiation treatment
2017 - CAMPOS, A.M.; BARBEZAN, A.B.; VILLAVICENCIO, A.L.C.H.
In agribusiness, it is related to the development of the activities of creation, production and commercialization of pets in the Pet segment. This market grew 7.6% between 2014/2015, generated R$ 18 million in Brazil and 67.3% of this value refers to products for animal feed, commonly called pet food. In animal feed, the raw materials are chosen according to the method (s) of processing (s) in which the product is submitted, its stability in the process and its nutritional source during the validity of the product. In food preservation, the irradiation process is a treatment that seeks to reduce the microbial load of foods, however, it can also alter the composition of the present ingredients and form radiolytic products in the formulation that are still under study. In products containing the presence of fat, the main concern is 2-Alkylciclobutanones (2-ACBs), which are radiolytic products formed exclusively post-processing with ionizing radiation. The formation of 2-ACBs is directly related to the lipid concentration and the dose of irradiation. The objective of this work is to describe the possible radiolytic by-products formed in canine rations containing extruded fat after irradiation and to determine if the formation of 2-ACBs depends on the increase of the radiation dose and, as a consequence, verify their cytotoxicity and genotoxicity.
Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2
2017 - BARBEZAN, ANGELICA B.; SANTOS, CARLA J.B.; CARVALHO, LUMA R.; VIEIRA, DANIEL P.; SANTELLI, GLAUCIA M.M.; VILLAVICENCIO, ANNA L.C.H.
Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line.
Ames test to detect mutagenicity of 2-alkylcyclobutanones: a review
2017 - BARBEZAN, ANGELICA B.; MARTINS, REGIANE; BUENO, JENNIFER B.; VILLAVICENCIO, ANNA L.C.H.
Food irradiation is an effective and safe method for preservation and long-term storage, and it is approved for use in over 60 countries for various applications in a wide variety of food products. This process is performed by use of accelerated electron beams, X-rays, or gamma radiation (60Co or 137Cs). 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that have fatty acids (triglycerides) and are subjected to irradiation. Since the 1990s toxicological safety studies of 2-ACBs have been conducted extensively through synthetic compounds, then and tests to determine if the compounds have any mutagenic activity are strictly necessary. The Ames test was chosen by many researchers to assess the mutagenicity of 2-ACBs. The test uses distinct bacterial cell lines Salmonella typhimurium to detect point mutations at sites guanine–cytosine (G–C) and Escherichia coli to detect point mutations at sites adenine–thymine (A–T). This bibliographic research aims to bring together all the results obtained and a comparison and cell lines used, type of plates, and solvents. This research showed that no mutagenic activity was observed in any of the cell lines and concentrations evaluated by the works of authors, so the 2-ACBs compounds showed no mutagenic substance in concentrations detectable by the Ames test.
Avaliação do potencial citotóxico da 2-tetradecilciclobutana em células hepáticas linhagem HepG2– estudos in vitro
2016 - BARBEZAN, A.B.; SALES, B.R.; MARTINS, R.; BUENO, J.B.; SANTELLI, G.M.M.; VILLAVICENCIO, A.L.C.H.