Made available in DSpace on 2016-07-18T17:53:09Z (GMT). No. of bitstreams: 1 Jose Luiz Parizi.pdf: 1222714 bytes, checksum: 854c90d0c15e3aa2b272185565785d0e (MD5) Previous issue date: 2012-09-17 / Tilapia is the common name of a large number of species of cichlid fish. In aquaculture, this is one of the most profitable fish, since it presents lower feed conversion than other fish. The chromium oxide has been used as a marker for digestibility because it is inert and not alter the biological process of dietary intake. The use of radioactive form of chromium as a marker of digestibility saves time and labor. The aim of this study was to evaluate the genotoxic potential of 51Cr2O3, used as a biological marker in studies of nutrition and physiology, through the frequency of nuclear abnormalities. We used 40 adult, male Nile tilapia (Oreochromis niloticus L.) (01 animal / tank) in a randomized design. The experimental period lasted 21 days, with 14 days to adapt to the installations and receiving encapsulated feed, free of the marker, with 30% crude protein (CP) and 2,800 Kg-1Kcal of digestible energy (DE)/kg. After this period, two capsules containing chromium oxide, 51Cr2O3 (radioactive form), were provided daily (experimental group) and to satiety were administered capsules containing the diet without the marker. We evaluated seven times sampling. The marker was not significantly absorbed by the digestive tract, but the micronucleus test and the frequency of nuclear abnormalities demonstrated that there was significant genotoxic effect on the erythrocytes of juvenile Nile tilapia, exposed to concentrations of 66mCi.g-1 of 51Cr in the form of chromium oxide, for the seven sampling times. / Tilápia é a denominação comum de um grande número de espécies de peixes ciclídeos. Dentro da aquicultura, este é um dos peixes mais rentáveis, pois apresenta conversão alimentar menor que os demais peixes. O óxido de crômio tem sido usado como marcador de digestibilidade por ser inerte e não alterar o processo biológico de absorção alimentar. O uso da forma radioativa do crômio como marcador de digestibilidade economiza tempo e trabalho. O objetivo deste estudo foi avaliar o potencial genotóxico do 51Cr2O3, utilizado como marcador biológico em estudos de nutrição e fisiologia, por meio da frequência de anormalidades nucleares. Foram utilizadas 40 tilápias do Nilo (Oreochromis niloticus L.) adultas, machos (01 animal/aquário), em um delineamento experimental casualizado. O período experimental foi de 21 dias, sendo 14 dias para adaptação às instalações e recebendo ração encapsulada, isenta do marcador, com 30% de proteína bruta (PB) e 2.800 Kg-1Kcal de energia digestível (ED)/kg de ração. Após este período, foram fornecidas diariamente duas cápsulas contendo óxido de crômio, 51Cr2O3 (forma radioativa) (grupo experimental) e cápsulas contendo a ração sem o marcador até a saciedade. Foram avaliados sete tempos amostrais. O marcador não foi absorvido significativamente pelo trato digestório, porém o teste do micronúcleo e a frequência de anormalidades nucleares demonstraram que houve efeito genotóxico significante sobre os eritrócitos de juvenis de tilápias do Nilo, expostos à concentração de 66 mCi.g-1 de 51Cr, na forma de óxido de crômio, para os sete tempos amostrais.

Made available in DSpace on 2016-07-18T17:53:14Z (GMT). No. of bitstreams: 1 Francijane Ferreira Paixao.pdf: 342552 bytes, checksum: 6c3ba89009e18cc2c819a6a07ee738d6 (MD5) Previous issue date: 2014-03-27 / This study aimed to evaluate the possible genotoxic effects of supplementation with capsiate extracted Pepper Capsicum annuum (sweet pepper), and the phenolic compound resveratrol extracted from grapes in Wistar rats by the micronucleus test in bone marrow. Sixty-four male rats, adult Wistar were divided into four groups (n = 16) and supplemented with: negative control group (GCN) - 0.5 ml of sodium chloride 0.9%; positive control group (GCP) - 50mg/kg cyclophosphamide; capsiate group (CG) - capsiate 10mg/kg diluted in 0.5 mL of 0.9% sodium chloride; resveratrol group (GR) - resveratrol 30mg/kg diluted in 0.5 mL of 0.9% sodium chloride. Only the PCM received intraperitoneal administration. The other groups were supplemented by gavage once a day for six weeks. At the end of six weeks the animals were anesthetized, sacrificed and the micronucleus test performed in bone marrow. The frequency of micronuclei in polychromatic erythrocytes of the BCM, PCM, GC and GR were 2.5, respectively, ± 0.53, 5.7 ± 1.03, 2.5 ± 0.73 and 2 ± 0.73 in a thousand cells. When comparing both groups there was statistically significant difference only GCP compared to other groups. It is concluded that supplementation at a dose of 10mg/kg capsiate and resveratrol at a dose of 30mg/Kg in daily frequency for 06 weeks did not produce genotoxicity in Wistar rats. / Este estudo teve como objetivo avaliar os possíveis efeitos genotóxicos da suplementação com o capsiate, extraído da pimenta Capsicum annuum (pimenta doce), e do composto fenólico resveratrol, extraído de uvas, em ratos Wistar através do teste de micronúcleo em medula óssea. Sessenta e quatro ratos machos, adultos de linhagem Wistar, foram distribuídos em quatro grupos (n=16) e suplementados com: grupo controle negativo (GCN)- 0,5ml de solução de cloreto de sódio 0,9%; grupo controle positivo (GCP)- 50mg/kg de ciclofosfamida; grupo capsiate (GC)- 10mg/kg de capsiate diluído em 0,5 mL de solução de cloreto de sódio 0,9%; grupo resveratrol (GR)- 30mg/kg de resveratrol diluído em 0,5 mL de solução de cloreto de sódio 0,9%. Somente o GCP recebeu a administração por via intraperitoneal. Os demais grupos foram suplementados por gavagem uma vez ao dia, durante seis semanas. Ao final das seis semanas os animais foram anestesiados, sacrificados e realizado o teste de micronúcleo em medula óssea. A frequência de micronúcleos em eritrócitos policromáticos do GCN, GCP, GC e GR foram, respectivamente, 2,5±0,53, 5,7±1,03, 2,5±0,73 e 2±0,73 em mil células. Na comparação entre os grupos observou-se diferença estatística significativa somente do GCP em relação aos demais grupos. Conclui-se que a suplementação de capsiate na dose de 10mg/Kg e resveratrol na dose de 30mg/Kg, em frequência diária, durante 06 semana, não produziu genotoxicidade em ratos Wistar.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-08-15T19:37:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eloisa.pdf: 351801 bytes, checksum: 5d8bb006aa699c1f75f27ffc6a85479b (MD5) / Made available in DSpace on 2017-08-15T19:37:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eloisa.pdf: 351801 bytes, checksum: 5d8bb006aa699c1f75f27ffc6a85479b (MD5) Previous issue date: 2017-05-23 / L-arginine has been used as a dietary supplement to minimize the side effects of chemotherapy, but it is not fully elucidated its actions and nor the ideal dose that can minimize the side effects. The objective of this study was to evaluate the effect of L-arginine supplementation on the formation of micronuclei in 70 Wistar rats after chemotherapy with 5-FU divided into 7 batches (10 male rats weighing 200 g/batch): LC water ad libitum; LArg2% and LArg4% mice fed commercial feed and 2% and 4% of L-arginine in water ad libitum, respectively; LC rats fed commercial feed, water ad libitum and a dose of 50 mg cyclophosphamide/kg; L5-FU rats fed commercial feed, water ad libitum and a dose of 200 mg of 5-FU/kg was applied; LArg2%+5-FU and LArg4%+5-FU rats fed commercial feed, 2% and 4% L-arginine were added in water ad libitum, respectively, and a dose of 200 mg of 5-FU/Kg. The mice were sacrificed 72 hours after the application of the chemotherapeutics to evaluate the formation of micronuclei. In the data analysis, one-way ANOVA was applied and followed by the Tukey test at 5%. Mice supplemented ad libitum with 2% and 4% L-arginine showed a reduction (P < 0.05) in the formation of micronuclei in bone marrow cells after chemotherapy, indicating that these supplements minimize the mutagenic effect of 5-FU. / A suplementação com L-arginina tem sido utilizada para minimizar os efeitos colaterais da quimioterapia, no entanto, ainda não está totalmente elucidado seus benefícios e nem determinado uma dose ideal de suplementação que pode minimizar esses efeitos. Objetivou avaliar o efeito da suplementação com L-arginina na formação de micronúcleos em 70 ratos Wistar após a aplicação da 5-FU. Estes ratos foram divididos em sete lotes (10 ratos/lote): Lc ratos alimentados com ração comercial e água ad libitum; LArg2% e LArg4% ratos alimentados com ração comercial e adicionou-se 2% e 4% de L-arginina na água ad libitum, respectivamente; Lciclo ratos alimentados com ração comercial, água ad libitum e aplicou-se uma dose de 50 mg de ciclofosfamida/Kg; L5-FU ratos alimentados com ração comercial, água ad libitum e aplicou-se uma dose de 200 mg de 5-FU/Kg; LArg2%+5-FU e LArg4%+5-FU ratos alimentados com ração comercial, adicionou-se 2% e 4% de L-arginina na água ad libitum, respectivamente, e aplicou-se uma dose de 200 mg de 5-FU/Kg. A contagem de micronúcleos nos eritrócitos policromáticos dos ratos foi realizada 72 horas após a aplicação dos quimioterápicos. Os resultados obtidos foram analisados pela ANOVA one-way seguido do teste de Tukey a 5%. Os ratos dos lotes LArg2%+5-FU e LArg4%+5-FU apresentaram redução (P < 0,05) na formação de micronúcleos nos eritrócitos policromáticos após a quimioterapia, indicando que estas suplementações com L-arginina minimizou o efeito mutagênico da 5-FU.

O objetivo do presente estudo foi avaliar os efeitos genotóxicos/citotóxicos ocasionados por disjuntores de Haas em células epiteliais esfoliadas da mucosa bucal de pacientes submetidos a tratamento ortodôntico, por meio do ensaio de micronúcleos. Participaram do estudo 22 pacientes entre 06 a 12 anos de idade, de ambos os gêneros, que necessitaram de disjuntores de Haas para correção de mordida cruzada posterior. Foi efetuada a coleta de células epiteliais da mucosa da bochecha, por meio de raspagem suave com escova científica. As células foram coletadas antes (T0), um mês após a instalação do aparelho (T1) e 3 meses após o travamento dos disjuntores (T2). As células foram processadas para obtenção de lâminas, as quais foram coradas com o método de Feulgen/Fast Green para quantificação do número de células normais, cariolíticas, picnóticas, brotos nucleares, bi/trinucleadas e com a presença de micronúcleos, em microscospia de luz. Os resultados foram submetidos à analise estatistica por meio do teste ANOVA, seguido do pós-teste de Tukey. O nível de significância adotado foi de 5%. Os resultados demonstraram que não houve diferença estatisticamente significante com relação à preença de micronúcleos, nos períodos avaliados (p>0,05). Os brotos nucleares sofreram aumento em T1 (p>0,05), com retorno aos níveis baseline em T2. Em relação às outras anormalidades (células cariolíticas, picnóticas e bi/trinucleadas), houve aumento significante em T1 e T2 (p<0,0001). Concluindo, este estudo demonstrou que os disjuntores de Haas não ocasionaram aumento de micronúcleos em células da mucosa bucal. Entretanto, foram observadas aumento estatisticamente significante das células cariolíticas, picnóticas e bi/trinucleadas durante o tratamento, sugerindo possíveis efeitos citotóxicos. / The aim of the present study was to evaluate the genotoxic and cytotoxic effects caused by Haas appliance through micronuclei assay in buccal mucosa epithelial cells of patients submitted to orthodontic treatment. The study included 22 patients between 06 and 12 years of age, both genders, who required Haas appliance for correction of posterior crossbite. Epithelial cells from the cheek mucosa were collected performed by gentle scraping scientific toothbrush. The cells were collected before (T0), one month after the device was installed (T1) and 3 months after the appliance immobilization (T2). The cells were processed to obtain slides and Feulgen/Fast Green was used as staining method for counting the number of normal, karyolytic, pyknotic, nuclear buds, binucleated and micronucleus cells under light microscopy. The cellular abnormalities were evaluated with the parametric tests for comparison of the means by ANOVA test followed by the Tukey post-test. The significance level was 5%. The results of this study showed that there were no statistically significant results for the micronuclei in the evaluated periods (p>0,05). Nuclear buds increased in T1 (p<0,05), returning to baseline levels in T2. In relation to the other abnormalities (cariolytic, pyknotic and bi/trinucleated cells), there were a significant increase in T1 and T2 (p<0.0001). In conclusion, this study demonstrated that Haas appliance did not cause micronuclei increase in cells of buccal mucosa. However, a statistically significant increase in cariolytic, pyknotic and bi/trinucleated cells were observed during treatment, suggesting possible cytotoxic effects.

Childhood sexual abuse (CSA) is a stressful life experience with lasting/far-reaching health and psychopathological consequences. Our laboratory recently identified a significantly increased frequency of acquired chromosomal anomalies (assessed using the cytokinesis-blocked micronucleus assay) in adult female twins exposed to CSA when compared to their unexposed co-twin. The primary aim of this study was to evaluate potential mechanism(s) underlying the observed increases in levels of micronuclei in an expanded group of 90 female identical twins (61 CSA+ females and 29 CSA- females [including a total of 27 MZ co-twin pairs]) using fluorescence in situ hybridization (FISH) methodologies, with PNA probes specific for the centromeric and telomeric regions of all chromosomes coupled with the standard CBMN assay, we were able to characterize the chromosomal contents of MN and, thus, gain insight into the mechanisms underlying MN formation. By scoring 100 MN per study participant for the number of centromeric signal(s) and/or telomeric signal(s) present, we categorized the MN as harboring either: (1) terminal fragments (only a telomeric signal); (2) acentric interstitial fragments (no telomeric or centromeric signal); (3) centric interstitial fragments (only a centromeric signal); or (4) an intact chromosome(s) or chromatid(s). We identified elevated frequencies of intact chromosome-derived MN in CSA+ women as compared to CSA- women (P=0.014), implicating chromosome loss as a mechanism potentially underlying the increased frequencies of MN identified in adult females with a history of CSA. MN containing fragmented chromosomes were also observed in all of the study participants evaluated; however MN containing terminal fragments and MN containing acentric interstitial fragments were seen less frequently in CSA+ women compared to CSA- women. This study represents the first time that the chromosomal contents of MN have been evaluated in individuals in the context of a psychosocial factor. As chromosomal loss and breakage contributes to the development of age-related health problems, these observations provide important insight into the biological mechanisms that may underlie the latent morbidity and psychopathology associated with childhood adversity. Future studies aimed at understanding the biological impact of early-life trauma could determine if the observed increase in acquired chromosomal abnormalities results in detectable somatic clonal mosaicism. This knowledge could ultimately be used to develop screening tools to identify individuals “at risk” for negative health outcomes in adulthood.

Background Evaluating potential genotoxicity of pharmaceutical drug candidates is important during drug development. A method that can be used for this purpose is the micronucleus assay (MN- assay) which can identify agents that induce chromosomal damage. One of the most commonly used cell lines in an in vitro MN-assay is Chinese Hamster Ovarian K1 (CHO-K1) cells. Propolis, a natural substance produced by honeybees from exudates of different types of plant, is used in folk medicine to improve health and prevent diseases and was the evaluated substance in this study. Aim The major aim of this thesis project was to evaluate the potential cytotoxic and genotoxic effects of propolis in CHO-K1 cells by in vitro MN-assay. Method Two solutions of propolis were prepared in different ways and CHO-K1 cells were cultured. The two different ethanolic extracts of propolis were evaluated with the cytochalasin B protocol of the MN-assay, using mitomycin C as a positive control. Results Ethanolic extract number 1 had a statistically significant increase of genotoxicity at 50 μg/ml and 100 μg/ml. It was also found to induce a statistically significant increase of cytotoxicity at all concentrations tested (5-100 μg/ml). Ethanolic extract number 2 had a statistically significant increase of genotoxicity at 50 μg/ml but no statistically significant increase of cytotoxicity. General conclusion Rather surprisingly, the present study showed that propolis induced chromosomal damage in CHO-K1 cells, and one of the extracts tested was also found to be cytotoxic using the cytochalasin B version of the in vitro MN-assay.

In the investigation of radiation accidents, it is of great importance to estimate the dose absorbed by exposed persons in order to plan their therapy. Although occasionally in these situations physical dose measurements are possible, most often biological methods are required for dose estimation. The aim of this investigation was to assess the suitability of the cytokinesis blocked (CB) micronucleus assay as a biodosimetric method using lymphocytes irradiated in vivo. The approach adopted to achieve this was to estimate whole body doses by relating micronuclei yields in patients undergoing radiotherapy treatment with an in vitro radiation dose-response curve. These biologically derived estimates were then compared with the corresponding doses obtained by physical measurement and calculation. As a first approach a study was performed of the in vitro dose-response of gamma-ray induced micronuclei following cytokinesis-block in the lymphocytes of peripheral blood samples obtained from 4 healthy donors. The results indicated that the distribution of the induced micronuclei were overdispersed. Furthermore, a linear dose-response relationship was established when a curve was fitted to the data by an iteratively reweighted least squares method. By means of an analysis of covariance it was demonstrated that this result is in agreement with the dose-response relationships found by various other workers (Fenech et al., 1985; Fenech et al., 1986; Fenech et al., 1989; Balasem et al., 1992, and Slabbert, 1993). To assess the suitability and accuracy of dose assessment using the CB micronucleus assay for in vivo exposure of lymphocytes, blood samples obtained from 8 patients undergoing radiotherapy before, during and after treatment were examined. The physical doses of these patients were determined according to conventional radiation treatment plans and cumulative dose-volume histograms. The dose-volume histograms permitted calculation of integral doses and subsequently the estimate of equivalent whole-body doses. The results of the CB micronucleus assay applied to peripheral blood lymphocytes of 6 patients undergoing fractionated partial-body irradiation showed a dose-related increase in micronucleus frequency in each of the patients studied. This demonstrated that micronuclei analysis may serve as a quantitative biological measure of such exposures. The pooled data of these patients compared to the pooled data of the healthy donors show that there was no statistically significant difference between in vitro and in vivo results, however a slightly lower induced micronuclei frequency was observed after in vivo exposure. When the biological dose estimates for equivalent whole-body doses obtained from the in vitro dose response curve were compared with calculated physical doses, it was found that: biologically estimated dose = 0.936 physical dose. However, there was inadequate statistical evidence to discard the hypothesis that the gradient of the equation was equal to one. Therefore, the analysis of micronuclei induced in lymphocytes in vivo yields highly quantitative information on the equivalent whole-body dose. The negative binomial method was used for analysing the micronucleus data from two patients who received single, relatively larger tumour doses of 10 Gy each, with the objective to obtain estimates of the exposed body fraction and the dose to this fraction. The dose estimates to the irradiated volume were found to be within 30% of the physical tumour dose. The irradiated volume estimates seemed to be higher than the physically calculated volumes but by discarding the correction for the loss of cells due to interphase death the agreement was good between the physically and biologically determined integral doses. This study has revealed that the CB micronucleus assay appears to offer a reliable, consistent and relatively rapid biological method of whole body dose estimation. It is recognised that further corroborative work using the techniques described in this thesis is required for estimating localized exposure.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21696号 / 医科博第100号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 清水 章, 教授 Shohab YOUSSEFIAN / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / The present study investigated the genoprotective and genotoxic effects of myricetin bulk (10 μM) and nano forms (20 μM) in the lymphocytes from pre-cancerous, monoclonal gammopathy of unknown significance (MGUS) patients and healthy individuals using the Comet and micronucleus assays. The study also evaluated the effect of myricetin on P53 expression levels, using the Western blot technique. Results showed that throughout the in-vitro treatment, lymphocytes from the patients group had higher levels of baseline DNA damage compared to the healthy group. Myricetin in both forms induced significant DNA damage, only at higher concentrations (>40 μM). The micronucleus assay showed a significant reduction (P < 0.01) in the frequency of micronuclei in mono-nucleated cells in the patient group treated with the nano form of myricetin at the non-toxic dose of 20 μM. There was a significant increase in both gene and protein P53 levels in lymphocytes isolated from healthy individuals and pre-cancerous patients. These results suggested a protective effect of myricetin and indicated its nutritional supplement potential for protection against cancer development among patients suffering from MGUS. / This study was kindly funded by Mr Nasir Qayyum, Bradford, UK.

In the present study DNA damage was measured in peripheral blood lymphocytes from polyposis coli and colorectal cancer patients, treated with different dietary and environmental compounds and compared with lymphocytes from healthy individuals. In addition, confounding factors such as age, gender, alcohol intake and smoking habits were taken into consideration. The assays used in this study included the Comet assay, the Micronucleus assay, the Micronucleus ¿ FISH assay and the sister chromatid exchange assay. The food mutagens, PhIP and IQ, as well as titanium dioxide nanoparticles (TiO2 NPs) induced a dose dependent increase in the DNA damage and chromosomal abnormalities in all tested groups regardless of confounding factors. Prior to experiments physicochemical characterisation of nanoparticles was conducted. In the presence of the flavonoids, quercetin and rutin that were acting in an antioxidant manner, the DNA damage resulting from the highest doses of food mutagens was significantly reduced. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protection against oxidative stress. The polyposis coli and colon cancer patients were more susceptible to food mutagens, PhIP and IQ, as well as TiO2 NPs, and in the majority of cases had a higher level of DNA damage in the Comet assay and higher cytogenetic damage in the Micronucleus assay. In the final project, twelve frequently encountered (NewGeneris) chemical compounds were evaluated to establish their damaging potential in lymphocytes and spermatozoa from healthy donors. The highest damage was produced by DNA reactive aldehydes, food mutagens and benzo[a]pyrene when assessed with the neutral and alkaline Comet assay with and without metabolic activation. / EU NewGeneris Programme and United Kingdom - India Education and Research Initiative (UKIERI)