Phytotoxicity and cytogenetic action mechanism of leaf extracts of Psidium cattleyanum Sabine in plant bioassays / Fitotoxicidade e mecanismo de ação citogenético de extratos foliares de Psidium cattleyanum Sabine em bioensaios vegetais

The search for more environmental friendly herbicides, aiming at the control of agricultural pests, combinated with less harmfulness to human health and the environment has grown. An alternative used by researchers is the application of products of secondary plant metabolism, which are investigated due to their potential bioactivities. Thus, species belonging to the Myrtaceae family are potential in these studies, since this family is recognized for having high biological activity. A species belonging to this genus is Psidium cattleyanum, which has a medicinal effect and its fruits are used in human food. Thus, the objective of this research was to evaluate and compare the phyto-cyto-genotoxicity of aqueous and ethanolic leaf extracts of the specie P. cattleyanum, from plant bioassays, as well as to identify the main classes of compounds present in the extracts. For this, the extracts were prepared, characterized and biological tests were carried out by evaluating, in seeds and seedlings of lettuce and sorghum, the variables: percentage of germination, germination speed index, root growth and aerial growth; and in meristematic lettuce cells the variables: mitotic phases, mitotic index, nuclear alterations and chromosomal alterations. Flavones, flavonones, flavonols, flavononols, flavonoids, alkaloids, resins, xanthones and anthraquinone glycoside were characterized in the ethanolic extract. Both evaluated extracts, in the highest concentration, inhibited the initial plant development. All treatments caused alterations in the mitotic phases and inhibited mitotic index. In addition, the treatments promoted an increase in nuclear and chromosomal alterations. The mechanism of action presented was aneugenic, clastogenic and determined in epigenetic alterations. The ethanolic extract was more cytotoxic, since it had a more expressive effect at a lower concentration. Despite the cytotoxicity of the extracts under study, they promoted alterations at lower levels than the glyphosate positive control.

A busca por herbicidas mais amigáveis ​​ao meio ambiente, visando o controle de pragas agrícolas, aliado a uma menor nocividade à saúde humana e ao meio ambiente tem crescido. Uma alternativa utilizada pelos pesquisadores é a aplicação de produtos do metabolismo secundário de plantas, que são investigados em virtude do seu potencial bioativo. Assim, espécies pertencentes à família Myrtaceae são potenciais nestes estudos, uma vez que esta família é reconhecida por possuir alta atividade biológica. Uma espécie pertencente a este gênero é Psidium cattleyanum, que possui efeito medicinal e seus frutos são utilizados na alimentação humana. Assim, o objetivo desta pesquisa foi avaliar e comparar a fitocitogenotoxicidade de extratos foliares aquosos e etanólicos da espécie P. cattleyanum, a partir de bioensaios vegetais, bem como identificar as principais classes de compostos presentes nos extratos. Para isso, os extratos foram preparados e caracterizados e foram realizados testes biológicos avaliando, em sementes e plântulas de alface e sorgo, as variáveis: porcentagem de germinação, índice de velocidade de germinação, crescimento radicular e crescimento aéreo; em células meristemáticas de alface foram avaliadas as variáveis: fases mitóticas, índice mitótico, alterações nucleares e alterações cromossômicas. Flavonas, flavononas, flavonóis, flavononóis, flavonóides, alcalóides, resinas, xantonas e glicosídeo de antraquinona foram caracterizados no extrato etanólico. Ambos os extratos avaliados, na maior concentração, inibiram o desenvolvimento inicial da planta. Todos os tratamentos causaram alterações nas fases mitóticas e inibiram o índice mitótico. Além disso, os tratamentos promoveram aumento de alterações nucleares e cromossômicas. O mecanismo de ação apresentado foi aneugênico, clastogênico e epigenético. O extrato etanólico foi mais citotóxico, pois teve efeito mais expressivo em menor concentração. Apesar da citotoxicidade dos extratos em estudo, eles promoveram alterações em níveis inferiores ao controle positivo glifosato.

Zootechnical performance, degree of steatosis and the genotoxic potential in yellowtail tetra Astyanax lacustris fed with different levels of L-carnitine / Desempenho zootécnico, grau de esteatose e potencial genotóxico de lambaris Astyanax lacustris alimentados com diferentes níveis de L-carnitina

L-carnitine perform a major role in transporting long-chain fatty acids into the mitochondria, where they are oxidized. It has been used in animal diets to decrease fat and increase muscle protein. The aim of this study was to evaluate the zootechnical performance, degree of steatosis in the liver, and genotoxic potential in Astyanax lacustris fed with different levels of L-carnitine (LC). Yellowtail tetra juveniles (n = 140) were distributed in 20 tanks of 70 L, with seven fish in each, in a water recirculation system with controlled temperature (27±0.1°C). The treatments with different levels of L-carnitine supplementation were: 0 (control), 250, 500, 750, and 1000 mg of LC per kg of food. The diets were provided twice a day for 60 days. The results showed that the different levels of LC did not affect (P>0.05) weight gain, survival, viscerosomatic index, and the liver hepatocytes showed a normal appearance. However, the use of LC supplementation showed genotoxic potential with a significant difference (P<0.05) for cell alterations when compared to the control at concentrations above 500mg kg-1.

A L-carnitina exerce um papel importante no transporte de ácidos graxos de cadeia longa até a mitocôndria para serem oxidados e tem sido incorporada em rações para animais com o objetivo de diminuir a deposição de gordura e aumentar a proteína muscular. O objetivo deste trabalho foi avaliar o desempenho zootécnico, o grau de esteatose no fígado e o potencial genotóxico em Astyanax lacustris alimentados com diferentes níveis de L-carnitina (LC). Juvenis de lambari-do-rabo-amarelo (n=140) foram distribuídos em 20 caixas de 70L, sete peixes em cada, em um sistema de recirculação de água com temperatura controlada (27±0,1°C). Os tratamentos com os níveis de suplementação foram: 0 (controle), 250, 500, 750 e 1000 mg de LC kg-1 de ração. As dietas foram fornecidas duas vezes ao dia, durante 60 dias. Os resultados mostraram que os diferentes níveis de LC não influenciaram (P>0,05) o ganho de peso; a sobrevivência, o índice viscerossomático e os hepatócitos do fígado apresentaram-se com aparência normal. No entanto, a suplementação com LC apresentou potencial genotóxico com diferença significativa (P<0,05) para alterações celulares quando comparada ao controle em concentrações superiores a 500mg kg-1.

Permitted daily exposure from preclinical studies of Ginkgo biloba L. dry extract

Abstract Resolution 658/2022 of the Brazilian Regulatory Agency requires the determination of the permitted daily exposure (PDE) of pharmaceutical agents. Ginkgo biloba L. is used therapeutically to treat memory deficits and other brain diseases. However, published results indicate that more studies are needed to confirm the safety of Ginkgo biloba. This study aimed to evaluate the dry extract of Ginkgo biloba L. leaves PDE as an ingredient in an oral pharmaceutical product in preclinical studies using mice. Acute oral toxicity and repeated dose experiments were performed based on OECD guidelines, as well as genotoxicity tests. The results indicate that Ginkgo biloba L. has low acute toxicity, no liver toxicity, and does not alter blood glucose levels. No changes in weight gain were observed, but food intake decreased in males during the first week of treatment at the highest dose. Hematological parameters were not altered in males, whereas females presented lower leukocyte and lymphocyte counts and higher neutrophil counts at the highest dose. The lipid profile was not altered in males, whereas total cholesterol was increased in females. The estimated PDE was 0.1 mg/day and, when related to the maximum residual concentration, indicates that the cleaning process used is safe and does not require reassessment.

Comprehension of the zinc chloride's ameliorative apoptotic and genotoxic effects on mice with cadmium-induced hepatotoxicity

Cadmium is a typical heavy metal quite dangerous to humans and animals. Zinc supplementation protects the biological system from Cd toxicity and alleviates Cd-induced toxicity. The present study was assessed to evaluate the preventive effects of zinc chloride (ZnCl2) on male mice with liver damage induced by cadmium chloride (CdCl2). Metals accumulation was quantified in the liver. Body weight, liver weight ratio, lipid peroxidation, caspase 3, and DNA damage were determined in the liver of male mice after receiving an intraperitoneal (IP) a single dose of CdCl2 at 1.5 and 3 mg/kg or/and ZnCl2 10 mg/kg during 21 days. The LD50 was 6.023 mg/kg for CdCl2 and 89.05 mg/kg for ZnCl2. The results indicate that mice in control and Zn groups gained body weight at the end of the experiment, while other treated groups significantly decreased. The relative weight of the liver revealed a significant increase in experimental groups. In addition, an increase in malondialdehyde level, Metallothionein concentration, and caspase-3 level was detected in Cd and Zn groups alone or in combination. Strand breaks of DNA of hepatocytes showed a significant increase in tail length of groups treated with cadmium. Co-treatment with zinc reduced these parameters compared to those measured in cells treated with cadmium. The outcome of this study implied that cadmium chloride causes oxidative stress, DNA damage, and elevated apoptosis markers in mice livers at low and medium doses. By pinpointing the target organ involved, the study results have also added some understanding of the impacts of zinc chloride injection to ameliorate cadmium toxicity in a low dose at 10 mg/kg.

Catequinas del té verde: efectos antigenotóxicos y genotóxicos. Revisión sistemática / Green tea catechins: antigenotoxic and genotoxic effects. Systematic review

Las catequinas del té verde (Camellia sinensis) (CTV) presentan efectos benéficos para la salud asociados a su potencial antioxidante. Por otra parte, el estrés oxidante es una de las vías de inducción de daño genotóxico. De ahí que, en la presente revisión se realizó un análisis de los efectos antigenotóxicos y genotóxicos de las CTV, haciendo énfasis en las vías implicadas en estos procesos y sus efectos en la salud. Se realizó una revisión de artículos indexados en las bases de datos de PubMed® y Science Direct® (2021) con las palabras clave "green tea" y "green tea catechins". Se delimitaron los estudios utilizando los operadores booleanos "AND", "OR" y "NOT" ("antigenotoxic", "genotoxic", "antioxidant" y "prooxidant"). En su mayoría se consideraron las publicaciones del 2016 al 2021. Se observó que los efectos benéficos en la salud de las CTV están relacionados con: a) su actividad antioxidante mediante la captura, inhibición y prevención de la formación de las especies reactivas de oxígeno; b) la regulación del sistema antioxidante endógeno; c) la activación de los mecanismos de reparación al contribuir en la eliminación del aducto 8-hidroxi-2'-desoxiguanosina; d) la inducción de apoptosis en células con daño al ADN; y e) la inhibición de la inflamación relacionada con su actividad antiapoptótica. Si bien, en algunos de los estudios se reportaron efectos genotóxicos, estos a su vez contribuyeron en la eliminación de células con daño genético, por lo que, no se puede considerar del todo a la actividad genotóxica de las CTV como perjudiciales para la salud(AU)

The green tea catechins (Camellia sinensis) (CTV) have beneficial effects for health associated with their antioxidant potential. Moreover, oxidative stress is one of the pathways for inducing genotoxic damage. Hence, in this review, an analysis of the antigenotoxic and genotoxic effects of CTV was carried out, emphasizing the pathways involved in these processes and their effects on health. A review of articles indexed in the PubMed® and ScienceDirect® (2021) databases with the keywords "green tea" and "green tea catechins" was carried out. Studies were delimited using the Boolean operators "AND", "OR" and "NOT" ("antigenotoxic", "genotoxic", "antioxidant" and "prooxidant"). For the most part, publications from 2016 to 2021 were considered. It was observed that the beneficial health effects of CTVs are related to: a) their antioxidant activity through the capture, inhibition and prevention of the formation of reactive oxygen species; b) the regulation of the endogenous antioxidant system; c) the activation of the repair mechanisms by contributing to the elimination of the 8-hydroxy-2'-deoxyguanosine adduct; d) the induction of apoptosis in cells with DNA damage; and e) the inhibition of inflammation related to its antiapoptotic activity. Although some of the studies reported genotoxic effects, these in turn contributed to the elimination of cells with genetic damage. Therefore, the genotoxic activity of CTV cannot be considered as harmful to health

Avaliação da citotoxicidade in vitro e genotoxicidade ex-vivo em compostos da Pavonia glazioviana Gürke (Malvaceae / Evaluation of in vitro cytotoxicity and ex-vivo genotoxicity in compounds from Pavonia glazioviana Gürke(Malvaceae)

Introdução: as terapias alternativas que utilizam plantas medicinais e fitoterápicos são bastante comuns no Brasil. Dentre várias espécies vegetais brasileiras utilizadas em terapias destacam-se as espécies da família Malvaceae. Objetivos: o presente estudo teve como objetivo avaliar a citotoxicidade in vitro e a genotoxicidade ex-vivo em compostos da Pavonia glazioviana Gürke espécie brasileira pertencente à família Malvaceae. Metodologia: métodos in vitro foram utilizados para verificar o potencial citotóxico por meio de ensaios hemolíticos e anti-hemolíticos e da análise genotóxica ex-vivo. O Extrato Etanólico Bruto (EEB) e Fração Clorofórmico (FC) foram obtidos na amostra vegetal utilizada neste estudo. Resultados: os produtos EEB-Pg e FC-Pg apresentaram baixo efeito citotóxico apenas nas concentrações de 50 e 100 µg / mL. As amostras expostas às concentrações de 500 e 1000 µg / mL apresentaram índice hemolítico alto a moderado com lise superior a 60%. Foi descrito efeito anti-hemolítico moderado em todas as amostras tratadas com 500 e 1000 µg / mL, com hemólise < 60%. Além disso, os compostos mostraram baixo efeito genotóxico ex-vivo, com um índice geral de células normais superior a 84% em todas as concentrações. Conclusões: os resultados sugerem um baixo perfil tóxico dos compostos obtidos da espécie Pavonia glazioviana, indicando limites seguros para o uso desses produtos naturais.

Introduction: alternative therapies using medicinal plants and herbal medicines are quite common in Brazil. Among several Brazilian plant species used in therapies, the species of the Malvaceae family stand out. Objetctives: the present study aimed to evaluate the in vitro cytotoxicity and ex-vivo genotoxicity in compounds of the Brazilian Pavonia glazioviana Gürke belonging to the Malvaceae family. Methodology: in vitro methods were used to verify the cytotoxic potential through hemolytic and antihemolytic assays and the ex-vivo genotoxic analysis. The Crude Etanolic Extract (CEE) and Cloroformic Fraction (CF) was obtained in vegetal sample used on this study. Results: the CEE-Pg and CF-Pg products only showed a low cytotoxic effect at the concentrations of 50 and 100 µg/mL. The exposure to the concentrations of 500 and 1000 µg/mL showed a high to moderate hemolytic index with lysis higher than 60%. A moderate anti-hemolytic effect was described in all samples treated with 500 and 1000 µg/mL, with hemolysis <60%. In addition, the compounds showed low ex-vivo genotoxic effect with a general index of normal cells greater than 84% at all concentrations. Conclusion: the results suggest a low toxic profile of the compounds obtained from the Pavonia glazioviana Gürke species belonging to the Malvaceae family, indicating safe limits for the use of these natural products.

Assessment of cytotoxicity, genotoxicity, and mutagenicity of the dexchlorpheniramine reference standard and pharmaceutical formula in human peripheral blood mononuclear cells

Abstract Dexchlorpheniramine is a first-generation classical antihistamine, clinically used to treat allergies. The main objective of our study was to evaluate the effects of the dexchlorpheniramine reference standard (DCPA Ref. St) and a pharmaceutical formula on DNA in human peripheral blood mononuclear cells (PBMCs). We exposed PBMCs to five different concentrations (0.5, 2.5, 5, 10, and 50 ng/mL) of DCPA Ref. St DCPA Ref. St and pharmaceutical formula in order to evaluate their cytotoxic, genotoxic, and mutagenic potential. The results showed that both dexchlorpheniramine formulations did not affect PBMC viability and CD3+, CD4+, or CD8+ lymphocyte subpopulations. The DCPA Ref. St and pharmaceutical formula neither induced genotoxic or mutagenic effects nor numerical or structural chromosomal alterations in PBMCs after 24 hours of exposure.

Proteomic analysis of human keratinocytes under UVA-induced stress / Análise proteômica de queratinócitos humanos sob estresse induzido por luz UVA

The solar ultraviolet (UV) radiation that reaches the Earth is composed of 95% of UVA (320 to 400 nm) and 5% of UVB (280 to 320 nm) radiation. UVB is carcinogenic, generating potentially mutagenic DNA lesions. The solar UVA radiation also causes DNA damage, but this fact does not fully account for its biological impact. UVA is absorbed by non-DNA cellular chromophores, generating reactive oxygen species such as singlet oxygen. Knowing the proteome mediates stress responses in cells, here we investigated the cellular effects of a non-cytotoxic dose of UVA radiation, equivalent to about 20 minutes of midday sun exposure, on the proteome of human keratinocytes. Using a combination of mass spectrometry-based proteomics, bioinformatics, and conventional biochemical assays, we analyzed two aspects of UVA-induced stress: spatial remodeling of the proteome in subcellular compartments 30 minutes after stress and long-term changes in protein levels and secretion (24 hours and 7 days postirradiation). In the first part of this thesis, we quantified and assigned subcellular localization for over 3000 proteins, of which about 600 potentially redistribute upon UVA exposure. Protein redistributions were accompanied by redox modulations, mitochondrial fragmentation and DNA damage. In the second part of the work, our results showed that primary human keratinocytes enter senescence upon exposure to a single dose of UVA, mounting antioxidant and inflammatory responses. Cells under UVA-induced senescence further elicit paracrine responses in neighboring premalignant HaCaT epithelial cells via inflammatory mediators. Altogether, these results reiterate the role of UVA radiation as a potent metabolic stressor in the skin

A radiação ultravioleta (UV) solar que atinge a superfície terrestre é composta por 95% de radiação UVA (320 a 400 nm) e 5% de radiação UVB (280 a 320 nm). A radiação UVB é carcinogênica e gera lesões potencialmente mutagênicas no DNA. A radiação UVA solar também gera danos no DNA, mas a genotoxicidade dessa radiação não explica inteiramente o seu impacto biológico. Atualmente, sabe-se que a radiação UVA é absorvida por cromóforos celulares, gerando espécies reativas de oxigênio, como o oxigênio singlete. Sabendo que o proteoma é um mediador de respostas ao estresse celular, nós investigamos os efeitos celulares de uma dose não-citotóxica de radiação UVA, equivalente a cerca de 20 minutos de exposição ao sol, no proteoma de queratinócitos humanos. Utilizando espectrometria de massas, bioinformática e ensaios bioquímicos convencionais, nós analisamos dois aspectos do estresse induzido por radiação UVA: o remodelamento espacial do proteoma 30 minutos depois do estresse e alterações nos níveis e na secreção de proteínas no longo prazo (24 horas e 7 dias depois da irradiação). Na primeira parte desta tese, nós quantificamos e atribuímos classificações de localização subcelular a mais de 3000 proteínas. Dentre essas proteínas, 600 tem potencialmente a sua distribuição subcelular alterada em resposta à radiação. As redistribuições subcelulares são acompanhadas de modulações redox, fragmentação mitocondrial e danos no DNA. Na segunda parte da tese, os nossos resultados mostraram que queratinócitos humanos primários entram em senescência sob exposição a uma única dose de radiação UVA, montando respostas antioxidantes e pró-inflamatórias. Células sob senescência induzida por UVA, por sua vez, desencadeiam respostas parácrinas em queratinócitos pré-tumorais (células HaCaT) por meio de mediadores inflamatórios. Em conjunto, esses resultados reiteram o papel da radiação UVA como um potente estressor metabólico em células da pele

Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

Tartrazina induce genotoxicidad en linfocitos de BALB/c Mus musculus / Tartrazine induces genotoxicity in BALB/c Mus musculus lymphocytes

RESUMEN Objetivos. Determinar el efecto genotóxico de la tartrazina en linfocitos de sangre periférica de Mus musculus BALB/c. Materiales y métodos. Se realizó un estudio experimental, a través de cinco grupos, con cinco ratones en cada uno. Se les registró el peso durante 17 semanas y, en la semana 15 se les administró suero fisiológico (control negativo), dicromato de potasio 25 mg/kg de peso corporal (pc) (control positivo) y tartrazina a dosis de 0,75 mg/kg pc, 7,5 mg/kg pc y 75 mg/kg pc, durante siete días, a excepción del control positivo que fue en dosis única. Luego, cada 24 h se obtuvo una muestra de sangre periférica de la cola y se realizó el frotis, secado y coloración. Posteriormente, se realizó el conteo de 1000 linfocitos por muestra de cada ratón, en todos los tratamientos. Resultados. Los tres tratamientos con tartrazina no causaron diferencias significativas en el peso de ratones a la semana 15, pero sí produjeron diferencias significativas en la frecuencia de linfocitos micronucleados, siendo el tratamiento con tartrazina de 75 mg/kg pc el de mayor efecto genotóxico, induciendo un promedio de 1,63 ± 0,08 linfocitos micronucleados, comparado con el control positivo que generó un promedio de 1,42 ± 0,08 linfocitos micronucleados. Conclusiones. La tartrazina produjo un efecto genotóxico, incrementando el número de linfocitos micronucleados, a dosis de 0,75; 7,5 y 75 mg/kg pc y no afecta el peso corporal durante siete días de administración en M. musculus BALB/c.

ABSTRACT Objectives. To determine the genotoxic effect of tartrazine on peripheral blood lymphocytes of BALB/c Mus musculus. Materials and methods. An experimental study was carried out using five groups, with five mice in each group. Their weight was registered for 17 weeks, and at week 15 they were administered physiological saline solution (negative control), potassium dichromate at 25 mg/kg body weight (bw) (positive control) and tartrazine at doses of 0.75 mg/kg bw, 7.5 mg/kg bw and 75 mg/kg bw, for seven days, with the exception of the positive control which was a single dose. Then, every 24 hours, a peripheral blood sample was obtained from the tail, which was then smeared, dried and stained. Subsequently, 1000 lymphocytes were counted for each sample from each mouse, for all treatment groups. Results. The three tartrazine treatments did not cause significant differences in the weight of mice at week 15, but did produce significant differences in the frequency of micronucleated lymphocytes, with the 75 mg/kg bw tartrazine treatment having the greatest genotoxic effect, inducing an average of 1.63 ± 0.08 micronucleated lymphocytes, compared to the positive control which obtained an average of 1.42 ± 0.08 micronucleated lymphocytes. Conclusions. Tartrazine produced a genotoxic effect, increasing the number of micronucleated lymphocytes, at doses of 0.75; 7.5 and 75 mg/kg bw and did not affect body weight during seven days of administration to BALB/c M. musculus.

Decrease of the DXR-induced genotoxicity and nongenotoxic effects of Theobroma cacao revealed by micronucleus assay / Diminuição da genotoxicidade induzida por DXR e efeitos não genotóxicos de Theobroma cacao revelados pelo ensaio de micronúcleo

This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.(AU)

Este estudo avaliou a genotoxicidade do extrato glicólico liofilizado de sementes de Theobroma cacao Linné (TCL), usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre TCL e doxorrubicina (DXR) foi também analisada. Grupos experimentais foram avaliados 24-48 h após tratamento com N-Nitroso-N-etilureia (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0,5-2 g/kg), e TCL (2 g/kg) em combinação com DXR (ensaio antigenotóxico). As análises de eritrócitos policromáticos micronucleados (EPCMNs) não mostraram diferenças significantes entre todas as doses de tratamento do TCL e o controle NaCl. Camundongos experimentalmente tratados com DXR e NEU induziram significativamente EPCMNs. Contudo, uma redução significante de EPCMNs foi também observada quando TCL foi administrada em combinação com o agente quimioterapêutico DXR. As análises da relação EPC/ENC (eritrócito policromático/eritrócito normocromático) revelaram ausência de diferenças significantes entre o controle NaCl, todas as doses de TCL e DXR. Contudo, houve diferenças significantes na relação EPC/ENC entre o controle positivo NEU e todos os outros tratamento. A relação ECP/ENC observada após o tratamento com TCL e DXR mostrou diferenças significantes e valores intermediários aos controles (NaCl e NEU). Este estudo sugere ausência de genotoxicidade e citotoxicidade de TCL, independentemente da dose, sexo e tempo. TCL reduziu os efeitos genotóxicos induzidos por DXR, sugerindo potencial efeitos antigenotóxicos.(AU)

Genotoxicidad de la acrilamida y la glicidamida en Allium cepa / Genotoxicity of acrylamide and glycidamide in Allium cepa

INTRODUCCIÓN: la formación de acrilamida (AA) en alimentos que fueron sometidos a tratamiento térmico, fue un descubrimiento inesperado en abril del 2002 por la Universidad de Estocolmo y la Administración Nacional de Alimentos de Suecia (NFA). Esta sustancia potencialmente tóxica y su metabolito glicidamida (GA) se forman en muchos alimentos cocidos a temperaturas elevadas. OBJETIVO: en virtud de que no hay estudios de estas sustancias químicas, en células vegetales, el objetivo fue comprobar el efecto genotóxico de la AA y GA en células de Allium cepa. MATERIALY MÉTODO: se determinó el efecto citotóxico a través del crecimiento de la raíz, actividad proliferativa y las aberraciones cromosómicas de la AA y GA en células meristemáticas de Allium cepa, expuestas a diferentes tiempos y concentraciones, evaluando el crecimiento de la raíz, la actividad proliferativa y las aberraciones cromosómicas. RESULTADOS: GA causa una inhibición del crecimiento de la raíz cuya intensidad depende de la concentración en un medio dado y la AA no muestra diferencias estadísticamente significativas respecto al control. La GA bloquea el ciclo de división celular en una etapa previa a la mitosis y no ocurre lo mismo con la AA. En cuanto a los efectos clastogénicos la GA induce estos efectos en las raíces meristemáticas de Allium cepa, en cambio la AA no los produce. CONCLUSIONES: en este estudio, aunque las concentraciones de AA y GA son mucho más altas que lo niveles de exposición alimentaria en humanos, la GA fue claramente genotóxica para células vegetales, a una determinada concentración, con una marcada citotoxicidad. Por lo tanto, nos permitió distinguir las diferencias entre AA y GA.

INTRODUCTION: the formation of acrylamide (AA) in foods that were sometimes heat treated was an unexpected discovery in April 2002, by Stockholm University and the Swedish National Food Administration (NFA). This potentially toxic substance and its metaboliteglycidamide (GA) form in many foods cooked at elevated temperatures. OBJECTIVES: because there are no studies of these chemicals, in plant cells, the objective was to verify the genotoxic effect of AA and GA in Allium cepa cells MATERIAL AND METHODS: to determine the cytotoxic effect through root growth, proliferative activity and chromosomal aberrations of acrylamide and glycidamide in Allium strain meristematic cells exposed to different times and antibodies, to evaluate root growth, proliferative activity and chromosomal aberrations. RESULTS: GA causes an inhibition of root growth whose intensity depends on the concentration in a given medium and AA does not show statistically significant differences with respect to the control. GA blocks the cell division cycle at a stage prior to mitosis, and the same does not occur with AA. Regarding the clastogenic effects, GA induces these effects in the meristematic roots of Allium strain, whereas AA does not produce them. CONCLUSIONS: in this study, although AA and GA concentrations are much higher than levels of dietary exposure in humans, GA was clearly genotoxic to plant cells, at a certain concentration, with marked cytotoxicity. For the therefore, it allowed us to distinguish the differences between AA and GA.

Avaliação do efeito sinérgico entre risedronato sistêmico e genisteína local sobre o reparo peri-implantar em ratas com deficiência de estrógeno e síndrome metabólica / Evaluation of the synergistic effect between systemic Risedronate and local Genistein on peri-implant repair in strogen deficient rats and metabolic syndrome

O objetivo deste estudo foi investigar a ação sinérgica do risedronato de sódio sistêmico e da genisteína administrada localmente, através da funcionalização de implantes, de ratas submetidas a ovariectomia e com hábitos de mimetizam a síndrome metabólica. A parte in vitro deste estudo foi executado em 2 etapas. Na primeira etapa, foi realizada a funcionalização da superfície de discos/implantes com genistína na concentração de 100 µM pela técnica layer by layer (lbl). Na segunda etapa foram feitos testes biológicos em culturas de células, para avaliar as propriedades da superfície funcionalizada, quanto às respostas osteogênicas. Para a cultura de células foram utilizadas células mesenquimais diferenciadas em osteoblastos, isoladas de fêmures de ratos. Após a validação pelos testes executados nas superfícies funcionalizadas, foi realizado estudo in vivo (3ª etapa). Para tanto, no dia 0, as ratas Wistar adultas jovens, fêmeas (n=64) foram divididas em 4 grupos: 1- SHAM (n= 16), animais foram submetidos à ovariectomia (OVX) fictícia e dieta balanceada. 2- SHAM Síndrome Metabólica (SM) (n=16), animais foram submetidos à ovariectomia fictícia e dieta de cafeteria. 3- OVX SM (n=16), animais foram submetidos à ovariectomia bilateral e dieta de cafeteria. 4- OVX SM Risedronato (RIS) (n=16), animais foram submetidos à ovariectomia bilateral, dieta de cafeteria e tratadas com risedronato de sódio. Em cada grupo há 2 subgrupos: A- implantes convencionais e B- implantes funcionalizados com genisteína. No dia 30, foi iniciado o tratamento medicamentoso com risedronato de sódio, na concentração de 0,35mg/kg, ou apenas solução salina, via gavagem, 1 vez por semana. Passados 60 dias da medicação (dia 90), todos os animais foram submetidos à cirurgia para exodontia dos 1os molares superiores bilateralmente e, imediatamente, no alvéolo da raiz mesial, foi instalado os implantes com superfície convencional ou funcionalizada. Os animais foram eutanasiados aos 28 dias (dia 118) após a instalação dos implantes para mensuração do torque de falha na interface osso implante em N/cm. Os dados foram submetidos ao teste de homocedasticidade (Shapiro Wilk). Houve a confirmação de distribuição normal dos dados amostrais e na sequência, foi realizado o teste paramétrico ANOVA One Way or Two Way, seguido do pós teste de Tukey, com o nível de significância de 5% (p< 0,05). Concluiu-se que, a concentração de 100 µM da genisteína manteve a viabilidade celular e resultados favoráveis quanto a genotoxicidade. A dieta de cafeteria e a ovariectomia bilateral mimetizam a síndrome metabólica e a predisposição para osteoporose por deficiência de esteroides gonadais. E, a ação sinérgica entre fármaco sistêmico (risedronato de sódio) e genisteína local foi promissora para a melhora no processo de reparo periimplantar, principalmente no grupo SHAM e OVX SM RIS(AU)

The aim of this study was to investigate the synergistic action of systemic risedronate sodium and locally administered genistein, through implant functionalization, of rats submitted to ovariectomy and with habits mimicking the metabolic syndrome. The in vitro part of this study was performed in 2 steps. In the first step, the surface functionalization of discs/implants was performed with genistein at a concentration of 100 µM by the layer by layer (lbl) technique. In the second step biological tests were performed in cell cultures to evaluate the properties of the functionalized surface for osteogenic responses. For the cell culture, mesenchymal cells differentiated into osteoblasts, isolated from rat femurs, were used. After validation by tests performed on the functionalized surfaces, the in vivo study (third test) was performed. For this purpose, on day 0, young adult female Wistar rats (n=64) were divided into 4 groups: 1- SHAM (n=16), animals were submitted to sham ovariectomy (OVX) and balanced diet. 2- SHAM Metabolic Syndrome (MS) (n=16), animals were submitted to sham ovariectomy and cafeteria diet. 3- OVX SM (n=16), animals underwent bilateral ovariectomy and cafeteria diet. 4- OVX SM Risedronate (RIS) (n=16), animals underwent bilateral ovariectomy, cafeteria diet and treated with risedronate sodium. In each group there are 2 subgroups: A- conventional implants and B- implants functionalized with genistein. On day 30, drug treatment was started with risedronate sodium, at a concentration of 0.35 mg/kg, or just saline solution, via gavage, once a week. After 60 days of medication (day 90), all animals underwent surgery to extract the 1st upper molars bilaterally, and implants with conventional or functionalized surfaces were immediately installed in the mesial root alveolus. The animals were euthanized at 28 days (day 118) after implant installation to measure the failure torque at the implant-bone interface in N/cm. The data were submitted to the homoscedasticity test (Shapiro Wilk). The normal distribution of the sample data was confirmed and then the parametric One Way or Two Way ANOVA test was performed, followed by Tukey's post-test, with a significance level of 5% (p< 0.05). It was concluded that, the concentration of 100 µM of genistein maintained cell viability and favorable results regarding genotoxicity. The cafeteria diet and bilateral ovariectomy mimic the metabolic syndrome and predisposition to osteoporosis by gonadal steroid deficiency. And, the synergistic action between systemic drug (risedronate sodium) and local genistein was promising for the improvement in the periimplant repair process, especially in the SHAM and OVX SM RIS groups(AU)

Citotoxicidade, genotoxicidade e expressão gênica em células-tronco mesenquimais expostas a materiais endodônticos / Cytotoxicity, genotoxicity and gene expression in mesenchymal stem cells exposed to endodontic cements

No mercado encontramos uma grande variedade de materiais endodônticos disponibilizados para uso clínico, mas diversos estudos mostram divergências de opiniões com relação ao comportamento biológico dos diferentes materiais. Este trabalho teve como objetivos investigar a viabilidade celular, a expressão de genes envolvidos na plasticidade celular e a diferenciação celular em culturas de células- tronco recuperadas de polpa dentária humana (hDPSCs) quando em contato com quatro materiais endodônticos (Endofill, Pulp Canal Sealer, Sealer 26, MTA) rotineiramente utilizados na clínica odontológica. Objetivou também, por meio de uma revisão sistemática, analisar a biocompatibilidade de cimentos de uso endodôntico sobre células tronco de origem dental. Para isto, o metabolismo celular das hDPSCs, quando em contato com os capilares contendo ou não os cimentos, foi avaliado pelo ensaio de MTT (24 e 48 horas) e a viabilidade celular foi analisada pelo ensaio de exclusão do azul de tripan (48 horas). A plasticidade celular, na presença dos capilares contendo ou não os cimentos, foi avaliada pela expressão gênica dos marcadores CD34, CD45, Nestin, CD105, Nanog e OCT-4 por PCR. Finalmente, a diferenciação celular frente aos cimentos endodônticos foi verificada pela expressão dos genes RUNX2, ALP, OC/BGLAP e DMP1 por RT-PCR. Os dados foram analisados pelo teste ANOVA com correção de Bonferroni (p<0.05). Observou-se que os cimentos Pulp Canal Sealer e o Endofill reduziram significativamente a viabilidade e o metabolismo celular quando comparados ao controle após 48 horas (p<0.001). O MTA e o Sealer 26 não interferiram na viabilidade celular em ambos os períodos de avaliação (p>0.05). As hDPSCs, quando cultivadas na presença do MTA e Sealer 26, expressaram os marcadores Nestin, CD105, NANOG e OCT-4, e não expressaram CD34 e CD45. Por sua vez, o MTA e o Sealer 26 interferiram positivamente ou negativamente na expressão gênica de DMP1, OC/BGLAP e RUNX2 em relação ao grupo controle (p<0.05), mas não houve diferença significativa em relação à expressão gênica de ALP (p>0.05). Portanto, MTA e Sealer 26 demonstram boa compatibilidade biológica quando na presença das hDPSCs. A revisão sistemática demonstrou que a maioria dos materiais, apresentam boa compatibilidade quando em contato com as células tronco, estando aptos a serem utilizados na prática clínica.

On the market, we found a wide variety of endodontics cements available for clinical use, but several studies show divergences of opinion regarding the biological behavior of these different materials. This work aimed to investigate cell viability and metabolism, an expression of genes involved in cell plasticity and cell differentiation in stem cell cultures recovered from human dental pulp (hDPSCs) when in contact with four endodontic cements (Endofill, MTA, Pulp Canal Sealer, Sealer 26) routinely used in endodontic clinic. It also aimed, through a systematic review, to analyze the biocompatibility of endodontic materials on dental stem cells. For this, the viability and metabolism of hDPSCs, when it comes into contact with capillaries that included or not cements, was assessed by MTT assay (24 and 48 hours) and exclusion of trypan blue assay (48 hours). Cellular plasticity, with the presence of capillaries containing or not sealers, was evaluated by the genetic expression of the markers CD34, CD45, Nestin, CD105, Nanog and OCT-4 by PCR. Finally, cell differentiation from endodontics sealers was verified by the expression of the RUNX2, ALP, OC/BGLAP and DMP1 genes by RT-PCR. The data were analyzed using the ANOVA test with Bonferroni correction (p<0.05). We note that Pulp Canal Sealer and Endofill sealers decrease cell viability and cellular metabolism when compared to control after 48 hours (p<0.001). MTA and Sealer 26 did not interfere with cell viability in the two evaluation periods (p>0.05). hDPSCs, when grown in the presence of MTA and Sealer 26, express the Nestin, CD105, NANOG and OCT-4 markers, and do not express CD34 and CD45. In turn, MTA and Sealer 26 interfered in the gene expression of DMP1, OC/BGLAP and RUNX2 in relation to the control group (p<0.05), but did not find a significant difference in relation to the ALP gene expression (p>0.05). Therefore, MTA and Sealer 26 demonstrate good biological compatibility when in the presence of hDPSCs. The systematic review showed that almost all materials have good compatibility when in contact with stem cells, being able to be used in clinical practice.

Planejamento, síntese, modelagem molecular e avaliação biológica de novos potenciais inibidores duais HDAC/ MMP não-hidroxamatos / Design, synthesis, molecular modeling and biological evaluation of novel potential non-hydroxamate dual HDAC/ MMP inhibitors

A inibição de alvos específicos como metaloproteinase de matriz (MMP) e histona desacetilase (HDAC) é amplamente estudada para impedir o progresso do câncer. Foi estabelecido que a inibição concomitante de MMP e HDAC é eficaz no combate de tumores sólidos e hematológicos. Ambos os alvos possuem um íon Zn2+ em seu sítio ativo, fundamental para a atividade destas enzimas. A alta afinidade dos inibidores conhecidos de MMP e de HDAC é conferida, principalmente, por um potente grupo ligante de zinco (ZBG). O ácido hidroxâmico é o ZBG mais potente conhecido atualmente, entretanto, este apresenta instabilidade farmacocinética, levando a ineficácia e genotoxicidade em testes clínicos. Frente a este contexto, o presente trabalho teve como objetivo o planejamento, síntese, modelagem molecular e avaliação biológica de novos inibidores duais MMP/HDAC não-hidroxamatos. Os compostos foram planejados utilizando estratégias de hibridação molecular, a partir de arcabouços provenientes inibidores de HDAC e MMP, gerando compostos arilsulfonamídicos com variações no tipo de ZBG inserido e na sua respectiva posição relativa na estrutura geral. Foram sintetizados sete análogos, em duas a três etapas reacionais, utilizando métodos de sulfonilação e acoplamento com agentes condensantes, partindo dos ésteres para e meta aminobenzoicos. Os rendimentos globais variaram de 25% a 55% e os produtos obtidos foram caracterizados por RMN 1H e 13C, LC/MS, CLAE e ponto de fusão. Os compostos tiveram sua atividade citotóxica avaliada em células HOG (oligodendroma) e T98G (glioblastoma), dentre os quais o 6a, que possui o ZBG 2-amino anilida, foi o mais promissor, apresentando atividade nas duas linhagens na casa de nM. Ensaios de coordenação com Fe2+ comprovaram a capacidade quelante dos análogos contendo ácido hidroxâmico e dos demais compostos citotóxicos, 4a e 4b (ZBG-2, salicilal-hidrazona), o que não foi observado para o composto 6a. Os estudos de ancoramento molecular permitiram sugerir um modo de interação para todos os ZBG propostos frente aos respectivos alvos (HDAC e MMP), sendo observado que o ZBG 4 (2-amino anilida) faria a interação de modo monodentado com a HDAC, enquanto não seria possível o encaixe no sítio catalítico da MMP. Conclui-se, portanto, que o planejamento proposto permitiu a obtenção de compostos promissores como antitumorais, e que a substituição do ácido hidroxâmico por outros ZBG fornece moléculas ativas frente a células tumorais. Entretanto, a avaliação biológica frente à MMP e HDAC é necessária para confirmar o mecanismo de ação proposto

Inhibition of specific targets such as matrix metalloproteinase (MMP) and histone deacetylase (HDAC) is extensively studied regarding arrest cancer growth. Particularly, concomitant inhibition of MMP and HDAC is effective against solid and hematologic tumors. Both targets have an ion Zn2+ at their catalytic site, which is essential for respective enzymatic activity. High affinity of known MMP and HDAC inhibitors is mainly provided by a potent zinc binding group (ZBG). Hydroxamic acid is the most potent ZBG currently known; however, it presents low pharmacokinetics stability, which results in its ineffectiveness and genotoxicity along clinical trial. So, the aim of this work comprised the design, synthesis, molecular modeling and biological evaluation of novel potential non-hydroxamate dual HDAC/ MMP inhibitors. Compounds were designed by molecular hybridation, employing scaffolds from HDAC and MMP inhibitors, which provided arylsulfonamides with variation about the ZBG type and its respective relative position in the general structure. Seven compounds were synthesized, in two to three reaction steps, through methods that comprise sulfonilation and coupling with condensing agents, using para and meta-aminobenzoic esters as starting material. Compounds showed global yields around 25-55 % and were characterized by 1H and 13C NMR, LC/MS, HPLC and melting point. Compounds were evaluated about their cytotoxicity against HOG (oligodendroma) and T98G (glioblastoma) cells, which 6a, with ZBG 2-aminobenzamide, was the most promising molecules, presenting activity against both cell lines at nM range. Coordination assays with Fe2+ proved the chelating capacity of hydroxamate analogues as well as the cytotoxic compounds, 4a and 4b (ZBG-2, salicylal-hydrazone), which was not observed about 6a. Molecular docking allowed to suggest an interaction model for all proposed ZBG with the respective targets (MMP and HDAC), showing that (ZBG-4) 2-aminobenzamide interacts with HDAC by monodentate way, but does not docks at MMP catalytic site. We conclude that the proposed design allowed obtaining promising compounds as antitumors agents, and the replacement of hydroxamic acid by other ZBG provide active molecules against tumor cells. However, biological evaluation against MMP and HDAC is necessary to confirm the proposed action mechanism

Evaluation of the Genotoxicity of Endodontic Materials for Deciduous Teeth Using the Comet Assay

ABSTRACT Objective: To evaluate genotoxicity of zinc oxide, P. A. calcium hydroxide, mineral trioxide aggregate and an iodoform paste using comet assay on human lymphocytes. Material and Methods: Two positive controls were used: methyl-methanesulfonate for the P.A. calcium hydroxide and mineral trioxide aggregate; and doxorubicin for the iodoform paste and zinc oxide. There were also two negative controls: distilled water for the P.A. calcium hydroxide and mineral trioxide aggregate; and DMSO for the iodoform paste and zinc oxide. Comets were identified using fluorescence microscopy and 100 of them were counted on each of the three slides analyzed per drug test. A damage index was established, taking into consideration the score pattern that had previously been determined from the size and intensity of the comet tail. Analysis of variance, followed by Tukey's test, was used to compare the means of the DNA damage indices. Results: The DNA damage index observed for mineral trioxide aggregate (7.08 to 8.58) and P.A. calcium hydroxide (6.50 to 8.33), which were similar to negative control index. On the other hand, damage index for zinc oxide (104.7 to 218.50) and iodoform paste (115.7 to 210.7) were similar to positive control index. Conclusion: Iodoform paste and zinc oxide showed genotoxicity at all concentrations used.

Cytogenotoxic effects of nitrogen on hydroponic lettuce / Efeitos citogenotóxicos do nitrogênio em alface hidropônica

Nitrogen accumulation in hydroponically-grown lettuce may pose a health risk to consumers. Thus, the objective of this study was to analyze different concentrations of nitrogen applications in hydroponic lettuce cultivation and their effect on toxicity, cytotoxicity and genotoxicity. A nutrient film technique (NFT) hydroponic system was used to grow the lettuce variety "Vanda." The treatments consisted of different concentrations of nitrogen (in the form of calcium nitrate) in Furlani solution (75, 100, 125 and 150%), a negative and a positive control. The following commercial characteristics were measured: plant fresh weight (PFW), root fresh weight (RFW), shoot fresh weight (SFW), shoot diameter (SD), root dry weight (RDW), shoot dry weight (SDW) and leaf nitrogen (LN). Cytogenotoxicity was indicated by toxicity, cytotoxicity and genotoxicity, which were in turn determined by root length, the mitotic index, chromosomal aberrations and the presence of micronuclei. The nitrogen concentrations used in this experiment did not cause phenotypic toxicity or cytotoxicity in lettuce roots. The most severe genotoxicity was observed at the 125% nitrogen concentration, which nevertheless did not affect commercial characteristics. Although nitrogen fertilization provides great benefits to agriculture, such as greater yields, indiscriminate use should be avoided since concentrations above recommended rates may induce genotoxicity.

O acúmulo de nitrogênio em alface cultivada hidroponicamente pode representar um risco à saúde dos consumidores. Assim, o objetivo deste estudo foi analisar diferentes concentrações de aplicações de nitrogênio no cultivo de alface hidropônica e seus efeitos na toxicidade, citotoxicidade e genotoxicidade. Em sistema hidropônico do tipo filme de nutrientes (NFT) foi usado para cultivar a variedade de alface "Vanda". Os tratamentos consistiram em diferentes concentrações de nitrogênio (na forma de nitrato de cálcio) na solução Furlani (75, 100, 125 e 150%), um controle negativo e um positivo. Foram medidas as seguintes características comerciais: peso fresco da planta (PFW), peso fresco da raiz (RFW), peso fresco da parte aérea (SFW), diâmetro da parte aérea (SD), peso seco da raiz (RDW), peso seco da parte aérea (SDW) e nitrogênio foliar (LN). A citogenotoxicidade foi indicada por toxicidade, citotoxicidade e genotoxicidade, que por sua vez foram determinadas pelo comprimento da raiz, índice mitótico, aberrações cromossômicas e presença de micronúcleos. As concentrações de nitrogênio utilizadas neste experimento não causaram toxicidade fenotípica ou citotoxicidade em raízes de alface. A genotoxicidade mais severa foi observada na concentração de nitrogênio de 125%, que, no entanto, não afetou as características comerciais. Embora a fertilização nitrogenada traga grandes benefícios à agricultura, como maiores rendimentos, o uso indiscriminado deve ser evitado, pois concentrações acima das taxas recomendadas podem induzir genotoxicidade.

Modified comet assay with Giemsa staining: a suitable method for assessing genotoxicity in rats / Ensaio cometa modificado com coloração de Giemsa: um método adequado para avaliar a genotoxicidade em ratos

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)

O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)

Modified comet assay with Giemsa staining: a suitable method for assessing genotoxicity in rats / Ensaio cometa modificado com coloração de Giemsa: um método adequado para avaliar a genotoxicidade em ratos

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)

O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)

Evaluación genotóxica de las moléculas con efectos tripanocida y leishmanicida cantin-6-ona y 5-metoxicantin-6-ona en células de médula ósea de ratones / Genotoxic evaluation of the molecules with trypanocidal and leishmanicidal effects, canthin-6-one and 5-methoxycanthin-6-one in mouse bone marrow cells

La tripanosomiasis americana y la leishmaniasis son problemas de salud pública relevantes en Iberoamérica. Las drogas utilizadas actualmente para el tratamiento de estas enfermedades poseen efectos colaterales tóxicos severos. Varios grupos de investigación están abocados a la búsqueda de productos naturales y sintéticos para encontrar nuevos agentes terapéuticos efectivos que no presenten reacciones colaterales adversas. En la evaluación de compuestos de la especie vegetal Zanthoxylum chiloperone (Rutaceae), se demostró que compuestos aislados del extracto presentaban actividad leishmanicida, tripanocida y antifúngica in vivo. Teniendo como antecedentes estos resultados, en el presente estudio se evaluaron los efectos genotóxico y citotóxico del cantín-6-ona y del 5-metoxicantin-6-ona, moléculas aisladas de la planta, en células de médula ósea de animales tratados. El estudio de los efectos genotóxicos se hizo a través del ensayo de modificaciones en la frecuencia de micronúcleos y el efecto citotóxico por modificaciones en la relación entre eritrocitos policromáticos y eritrocitos normocromáticos. Se realizaron 2 ensayos independientes y en cada ensayo los animales fueron divididos en tres grupos de tratamiento: GRUPO I: control negativo que recibió 200 uL de agua y 2.1% de DMSO, vía oral, GRUPO II: compuesto a ser evaluado (canthin-6-ona o 5-methoxicantin-6-ona) con 2.1% de DMSO, y GRUPO III: control positivo que recibió ciclofosfamida 50mg/kg/peso del animal, vía intraperitoneal. El análisis estadístico mostró que ambos compuestos no presentaron efectos genotóxicos ni citotóxicos. Estos resultados permiten proponer a estas moléculas como candidatas a ser sometidas a estudios más detallados como potenciales fármacos contra estas dos enfermedades

American trypanosomiasis and leishmaniasis are relevant public health problems in Latin America. The drugs currently used to treat these diseases have severe toxic side effects. Several research groups are dedicated to the search of natural and synthetic products to find new effective therapeutic agents that do not present adverse collateral reactions. In the evaluation of compounds of the plant species Zanthoxylum chiloperone (Rutaceae), it was shown that isolated compounds of the extract had leishmanicidal, trypanocidal and antifungal in vivo activities. Based on these results, the genotoxic and cytotoxic effects of canthin-6-one and 5-methoxycanthin-6-one, molecules isolated from the plant, on bone marrow cells of treated mice were evaluated in the present study. The study of genotoxic effects was made through the test of modifications in the frequency of micronuclei and the cytotoxic effects by modifications in the relationship between polychromatic erythrocytes and normochromic erythrocytes. Two independent assays were performed and in each assay the animals were divided into three treatment groups: GROUP I: negative control that received 200 µL of water and 2.1% of DMSO, orally, GROUP II: compound to be evaluated (canthin-6 -one or 5-methoxycanthin-6-one) with 2.1% DMSO, and GROUP III: positive control that received cyclophosphamide 50mg /kg animal weight, intraperitoneal. Statistical analysis showed that both compounds had neither genotoxic nor cytotoxic effects. These results allow these molecules to be proposed as candidates to be subjected to more detailed studies as potential drugs against these two diseases