Rosa roxburghii, also known as "Burr Rose" or "Chestnut Rose", originated in southwest China and was introduced to the botanic garden in Calcutta around 1824. It was named after William Roxburgh who was the superintendent. The extract of fruit of the Rosa roxburghii plant is the base ingredient of a range of products that is commercially sold under the Cili Bao label. The extract is composed of a wide range of substances of nutritional value, in particular a relatively high amount of antioxidants such as ascorbate and plant phenols. It has been reported before that supplementation with the fruit extract resulted in increased red blood cell superoxide dismutase, catalase and the reduced form of glutathione. An enhancement of the antioxidant status could contribute to the protection against several diseases where oxidative stress is a major factor in the pathology, such as atherosclerosis, cancer and immunity stress. Several anecdotal reports with little (published) scientific support claim that human supplementation of the Rosa roxburghii extract to the diet has a protective effect against several diseases, including the above mentioned. Medicinal and herbal plants are used in large sections in developing countries for primary care and there is now also an increase in the use of natural therapies in developed countries. However, plant extracts can also consist of anti-nutritional and possible toxic components, such as oxalic acid and nitrates, which could express cytotoxic and genotoxic activities. Therefore, understanding the health benefits but also the potential toxicity of these plants is important. The objective of this study was to investigate the beneficial properties of Rosa roxburghii extract from an antioxidant potential perspective and in particular to investigate the safety of the product for human consumption. For this purpose in vitro evaluation of the cellular toxicity, mutagenicity and genotoxicity was performed. In addition, specific biochemical parameters relating to the antioxidant status of the product, i.e. antioxidant capacity, oxidative stress prevention and glutathione redox state profiles were investigated in vitro as well as in vivo. The results indicated that Rosa Roxburghii fruit extract was not mutagenic when tested with Salmonella typhimurium strains TA 98, TA 100 and TA 102 in the Ames test. The results, however, pointed towards an antimutagenic effect of the extract in these strains against metabolic activated mutagens 2- acetylaminoflurorene (2-AAF) and aflatoxin B1, and the direct-acting mutagen, methanesulfonate (MMS). In primary rat hepatocyte, Rosa roxbughii extract did not elicit double or single strand DNA damage and cell viability loss using the single cell gel electrophoresis (Comet assay), lactate dehydrogenase leakage test or the mitochondria1 conversion test of MTT to formazan (MTT test). Again the opposite effect was observed: pre-treatment of hepatocytes with Rosa roxbughii extract significantly reduced the effect of oxidative stress-induced cellular- and genotoxicity. These results point to a protective effect against oxidative stress which is reflected in an increase of the antioxidant capacity and glutathione redox state (GSH/GSSG) in vitro (lymphoblasts) and in vivo (humans) reported in this study. This study underlines the previously suggested potential of this plant extract as a natural and safe antioxidant supplement. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.

Rosa roxburghii, also known as "Burr Rose" or "Chestnut Rose", originated in southwest China and was introduced to the botanic garden in Calcutta around 1824. It was named after William Roxburgh who was the superintendent. The extract of fruit of the Rosa roxburghii plant is the base ingredient of a range of products that is commercially sold under the Cili Bao label. The extract is composed of a wide range of substances of nutritional value, in particular a relatively high amount of antioxidants such as ascorbate and plant phenols. It has been reported before that supplementation with the fruit extract resulted in increased red blood cell superoxide dismutase, catalase and the reduced form of glutathione. An enhancement of the antioxidant status could contribute to the protection against several diseases where oxidative stress is a major factor in the pathology, such as atherosclerosis, cancer and immunity stress. Several anecdotal reports with little (published) scientific support claim that human supplementation of the Rosa roxburghii extract to the diet has a protective effect against several diseases, including the above mentioned. Medicinal and herbal plants are used in large sections in developing countries for primary care and there is now also an increase in the use of natural therapies in developed countries. However, plant extracts can also consist of anti-nutritional and possible toxic components, such as oxalic acid and nitrates, which could express cytotoxic and genotoxic activities. Therefore, understanding the health benefits but also the potential toxicity of these plants is important. The objective of this study was to investigate the beneficial properties of Rosa roxburghii extract from an antioxidant potential perspective and in particular to investigate the safety of the product for human consumption. For this purpose in vitro evaluation of the cellular toxicity, mutagenicity and genotoxicity was performed. In addition, specific biochemical parameters relating to the antioxidant status of the product, i.e. antioxidant capacity, oxidative stress prevention and glutathione redox state profiles were investigated in vitro as well as in vivo. The results indicated that Rosa Roxburghii fruit extract was not mutagenic when tested with Salmonella typhimurium strains TA 98, TA 100 and TA 102 in the Ames test. The results, however, pointed towards an antimutagenic effect of the extract in these strains against metabolic activated mutagens 2- acetylaminoflurorene (2-AAF) and aflatoxin B1, and the direct-acting mutagen, methanesulfonate (MMS). In primary rat hepatocyte, Rosa roxbughii extract did not elicit double or single strand DNA damage and cell viability loss using the single cell gel electrophoresis (Comet assay), lactate dehydrogenase leakage test or the mitochondria1 conversion test of MTT to formazan (MTT test). Again the opposite effect was observed: pre-treatment of hepatocytes with Rosa roxbughii extract significantly reduced the effect of oxidative stress-induced cellular- and genotoxicity. These results point to a protective effect against oxidative stress which is reflected in an increase of the antioxidant capacity and glutathione redox state (GSH/GSSG) in vitro (lymphoblasts) and in vivo (humans) reported in this study. This study underlines the previously suggested potential of this plant extract as a natural and safe antioxidant supplement. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.

Orientador: Maria Aparecida Marin-Morales / Banca: Jonas Contiero / Banca: Edson Luis Maistro / Banca: Mário Sérgio Mantovani / Banca: Silvia Regina Batistuzzo de Medeiros / Resumo: O ramnolipídio é um biossurfactante da classe dos glicolipídios, produzido pela bactéria Pseudomonas aeruginosa. Estudos têm mostrado que, mesmo com um grande potencial de uso do ramnolipídio na indústria cosmética e na área da saúde, este composto é, principalmente, promissor para uso na indústria do petróleo. Apesar dos biossurfactantes serem considerados menos tóxicos e mais biodegradáveis que os surfactantes sintéticos, estes compostos ainda não foram investigados quanto a sua ação genotóxica direta ou indireta sob o material genético de organismos expostos. Desta maneira, o objetivo deste trabalho foi investigar os possíveis danos celulares promovidos pela ação do biossurfactante ramnolipídio e suas implicações no material genético quando utilizado como agente biorremediador de águas impactadas por petróleo, por meio dos bioindicadores Allium cepa, Oreochromis niloticus e as células humanas mantidas em cultura - HepG2. Para a realização da investigação, o biossurfactante ramnolipídio foi produzido em condições de laboratório e a partir da concentração indicada para o uso em processos de biorremediação (1g/L), foram estabelecidas as concentrações utilizadas no estudo. Para avaliar o potencial tóxico, genotóxico e mutagênico das concentrações de ramnolipídio, assim como seu feito sinérgico com o petróleo, foram realizados os testes de germinação de sementes, aberrações cromossômicas e teste do micronúcleo em A. cepa; teste do micronúcleo em eritrócitos circulantes, ensaio do cometa com sangue periférico e células de brânquias e análise ultraestruturais de eritrócitos circulantes em O. niloticus; e teste do MTT, teste do micronúcleo e ensaio do cometa em células HepG2. Nossos resultados mostraram que as concentrações 1g/L e 2g/L de ramnolipídio são tóxicas para A. cepa. No entanto, quando as concentrações 1g/L e 10g/L... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rhamnolipids belong to the glycolipid class of biosurfactants, which are produced by the bacterium Pseudomonas aeruginosa. Studies have shown that, even being potentially used in the cosmetics industry, this compound is especially promising in the petroleum industry. Although the biosurfactants are considered less toxic and more biodegradable than the synthetic surfactants, these compounds have not yet been investigated when it comes to their either direct or indirect genotoxic action upon the genetic material of exposed organisms. Thus, the purpose of this study was to investigate the possible cell damage promoted by the action of rhamnolipid biosurfactants and their implications for the genetic material when they are used as a bioremediation agent of petroleum-impacted water, by using Allium cepa, Orechromis niloticus and human cells kept in culture - HepG2. In order to carry out this investigation, the rhamnolipid biosurfactant was produced under laboratory conditions, and the recommended concentration for use in bioremediation processes (1g/L) was also used; the concentrations used in the study were established. To assess the toxic, genotoxic and mutagenic potential of rhamnolipid concentrations, as well as their synergy with petroleum, seed germination, chromosome aberration tests and micronucleus test in A. ceppa; micronucleus test in circulating red blood cells, comet assay in the peripheral blood and gill cells, and ultrastructural analysis of circulating blood cells in O. niloticus; and MTT test, micronucleus test and comet assay in HepG2 cells. Our studies showed that the 1 g/L and 2 g/L rhamnolipid concentrations are toxic to A. cepa. However, when 1 g/L and 10 g/L concentrations were used in the bioremediation process, these concentrations did not cause toxic, genotoxic or mutagenic damage besides the ones observed for the cells exposed to soluble petroleum... (Complete abstract click electronic access below) / Doutor

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Made available in DSpace on 2014-06-11T19:27:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:36:16Z : No. of bitstreams: 1 brianezi_g_me_botfm.pdf: 2870031 bytes, checksum: 61bf38a3fd318eca76bd3e6eab217c9f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O teste do cometa é utilizado para estimativa de dano ao DNA nos protocolos de avaliação do risco toxicológico. É rápido, de baixo custo, de fácil realização, seguro e pode ser aplicado em diversos tecidos. Corantes fluorescentes são os mais empregados para o ensaio. É também descrita a utilização de sais de prata com vantagens operacionais, carecendo de sistemas automatizados validados para esta análise. Neste trabalho, desenvolveram-se dois algoritmos automatizados para a análise do cometa corado pela prata. Foram realizados ensaios do cometa para sete grupos com dois camundongos Balb/c machos cada, submetidos ao tratamento intraperitoneal com soro fisiológico (G1), e três doses crescentes de genotóxicos conhecidos: MNU (N-nitroso-N-metilurea) (G2 – 5 μg/g, G3 – 25 μg/g, G4 – 50 μg/g) e DEN (N-nitrosodietilamina) (G5 – 20 μg/g, G6 – 80 μg/g, G7 – 180 μg/g). As lâminas dos testes do cometa foram confeccionadas a partir do sangue total e coradas pela prata e SYBR Gold. As coradas pela prata foram analisados por três avaliadores e pelos algoritmos automatizados. Aqueles corados pela fluorescência foram analisados pelo software Comet IV®. Os avaliadores apresentaram alta correlação de suas estimativas. Os algoritmos se correlacionaram fortemente com a análise dos avaliadores, principalmente com os escores obtidos pela classificação visual. Houve alta concordância na avaliação da sua repetitividade. Os algoritmos apresentaram resultados semelhantes, não indicando superioridade entre si para a análise do teste. Todos os métodos avaliados se mostraram capazes de diferenciar as concentrações dos genotóxicos utilizados. Porém, os algoritmos desenvolvidos não apresentaram correlação com os resultados obtidos pelo sistema do teste do cometa corado pela fluorescência, sugerindo que os métodos de coloração discriminem diferentes estruturas... / The comet assay is used to estimate DNA damage in the protocols for toxicological risk assessment. It's fast, inexpensive, easily performed, safe and can be applied in most tissues. Fluorescent dyes are frequently used for testing. Silver salts have been reported with some operational advantages; however automated systems for its analysis were not sufficiently validated for this staining. In this study, we have developed two algorithms for automated analysis of the comet assay silver stained. The assay was performed for seven groups of two (Balb/c) mice each, treated with intraperitoneal saline (G1) and three different genotoxic doses: MNU (N-nitroso-N-methylurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) and DEN (N-nitrosodiethylamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). The slides of the comet assay were made with total blood and were stained with silver and SYBR Gold. The silver stained slides were analyzed by three evaluators and the automated algorithms. Those stained by fluorescence were analyzed through software Comet IV®. Evaluators showed high correlation of their estimates. Both algorithms were correlated strongly with the evaluators' analysis mainly on the scores obtained by visual classification. Moreover, their agreement was high when assessed by repetitivity. The algorithms showed similar results, demonstrating no superiority to each other for the analysis of the test. All methods evaluated proved able of differentiating genotoxic concentrations used. However, the algorithms were not correlated with the results obtained by the automated system of comet assay fluorescence stained, suggesting that the staining methods linkage different genomic structures or with different affinities. The automated algorithms developed proved valid for the direct analysis of comet assay silver stained, allowing their use in genotoxicity research

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Alpinia zerumbet, conhecida popularmente como ColÃnia, no Nordeste do Brasil, à uma planta medicinal usada amplamente na medicina popular na forma de chÃ, sendo tradicionalmente utilizada para o tratamento da hipertensÃo arterial. O objetivo desse estudo foi avaliar a seguranÃa e o potencial genotÃxico do chà de A. zerumbet em voluntÃrios sadios. O ensaio clÃnico consistiu de um estudo duplocego, controlado por placebo, randomizado e paralelo, com 36 voluntÃrios (18 homens e 18 mulheres), adultos. Os quais foram aleatoriamente distribuÃdos em dois grupos: ColÃnia, constituÃdo por 24 voluntÃrios, e Placebo, composto por 12 sujeitos. Os voluntÃrios foram tratados durante 28 dias ininterruptos com 540 mL de chà de ColÃnia ou Placebo. AvaliaÃÃes clÃnica e laboratorial foram realizadas no prÃ-estudo, durante o perÃodo de tratamento, bem como apÃs o encerramento do estudo. A genotoxicidade do chà de colÃnia, por sua vez, foi investigada mediante o emprego do teste do cometa. O Ãndice de massa corpÃrea (IMC) foi de 25,300Â2,918 Kg/ cm2 para o grupo ColÃnia no prÃ-estudo e 25,289Â2,965Kg/ cm2 no pÃs-estudo. No grupo Placebo o IMC foi de 25,179Â2,564 no prÃ-estudo e de 24,961Â2,409 no pÃs-estudo. As funÃÃes hematolÃgica, hepÃtica, renal e metabÃlica foram analisadas, antes, durante (14 e 28 dia) e apÃs o estudo atravÃs dos exames laboratoriais, os quais nÃo evidenciaram sinais de toxicidade. CefalÃia, sonolÃncia, dor abdominal, vÃmito, flatulÃncia e poliÃria foram os eventos adversos atribuÃdos a ingestÃo do chà nos dois grupos. Pelo teste do cometa, nÃo foram observados danos (p<0,05) nos linfÃcitos perifÃricos dos voluntÃrios tratados com o chà de colÃnia. Os estudos de toxicologia clÃnica e genotoxicidade nÃo evidenciaram nenhuma toxicidade nos voluntÃrios tratados por 28 dias ininterruptos com o chà de ColÃnia. / Alpinia zerumbet, popularly known as Colonia in the Northeast of Brazil, is a medicinal plant widely used in the popular medicine as a tea, being traditionally used to treat arterial hypertension. The objective of this study was to evaluate the safety and genotoxic potential of the A. zerumbet tea in healthy volunteers. Clinical trial consisted of a double-blind study, placebo controlled, randomized and parallel, with 36 adult volunteers (18 men and 18 women), which were randomly distributed in two groups: Colonia, consisted of 24 volunteers, and Placebo, consisted of 12 subjects. The volunteers were treated for 28 consecutive days, with 540 mL of Colonia or Placebo tea. Clinical and laboratorial evaluations were performed during the pre-study and treatment, as well as during the closure of the study. The genotoxicity of the Colonia tea was investigated by the comet assay. The body mass index (BMI) was 25.300Â2.918 Kg/ cm2 for the Colonia group in the pre-study and 25.289Â2.965Kg/ cm2 in the end of the study. For the Placebo group, BMI was 25.179Â2.564 in the pre-study and 24.961Â2.409 after the study. The blood, liver, kidney and metabolic functions were analyzed before, during (14th and 28th day) and after the study by the evaluation of laboratorial exams, which did not evidence signs of toxicity. Headache, sleepiness, abdominal pain, vomit, flatulence and polyuria were the adverse events attributed to the ingestion of the tea in both groups. The comet assay did not show damage in peripheral lymphocytes of the volunteers treated with the Colonia tea (p<0.05). The clinical toxicology and genotoxicity studies did not evidence any toxicity in the volunteers treated for 28 consecutive days with the Colonia tea.

Corantes são utilizados na coloração de diferentes substratos, incluindo papel, couro e plásticos, mas o uso mais importante é o têxtil e 1 a 5% destes corantes podem ser descartados no ambiente. Em geral, os corantes do tipo azo são tóxicos para os organismos aquáticos e alguns tipos de corantes podem ser mais tóxicos que outros. Mas, embora estes compostos e seus produtos de transformação reduzidos e/ou clorados podem ser encontrados no ecossistema aquáticos, não existem dados sobre genotoxicidade em organismos aquáticos até o momento. Muitos estudos têm demonstrado que avaliar danos ao DNA representa um biomarcador de exposição muito sensível em espécies aquáticas, que pode ser estudado utilizando ensaios in vivo e in vitro, como no caso das linhagens de células de peixe. Os objetivos deste trabalho foram: avaliar a ocorrência de corantes dispersos em amostras ambientais; avaliar a mutagenicidade dessas amostras utilizando o ensaio de Salmonella/microssoma com as linhagens TA98 e YG1041, e a genotoxicidade com o ensaio do cometa em culturas celulares de peixe RTL-W1. HPLC-MS/MS foi utilizada para verificar a ocorrência de corantes em amostras do Rio Piracicaba à montante e à jusante do Ribeirão Quilombo e do descarte de uma Estação de Tratamento de Efluentes (ETE), localizados no Estado de São Paulo, Brasil. Foram detectados seis corantes dispersos nas amostras de águas superficiais e efluentes. O corante Disperse Red 1 foi o composto mais frequente, detectado em 8 das 16 amostras, porém sua contribuição para a mutagenicidade total foi baixa; os corantes Disperse Blue 373 e Disperse Violet 93 foram os que mais contribuíram. A genotoxicidade do Rio Piracicaba, avaliada pelo ensaio de Salmonella/microssoma e ensaio do cometa, aumentou após o lançamento do Ribeirão Quilombo e do efluente ETE, mostrando uma possível contribuição destes na genotoxicidade do Rio Piracicaba. / Dyes are used in the coloration of different substrates, including paper, leather and plastics, but the most important use is on textiles and 1 to 5% of these dyes might be lost into the environment. Azo dyes are the most important class, accounting for over 50% of all commercial dyes, and this class has been the most studied. In general, azo dyes are toxic to aquatic organisms and some types of dyes are more toxic than others. But although these compounds as well as their reduced/chlorinated transformation products can be found in aquatic ecosystems, no mutagenicity data are available until now in aquatic organisms. This remark remains of value, as well, regarding genotoxicity potential of such dyes towards aquatic organisms. Many studies have demonstrated that DNA damage measurement represents a very sensitive biomarker of exposure in aquatic species that can be studied both in vivo and in vitro using for example fish cell lines. The objectives of this work were evaluate the occurrence of disperse dyes in environmental samples; evaluate the mutagenicity of this samples using the Salmonella/microsome assay with strains TA98 and YG1041; evaluate the genotoxicity using the comet assay with fish cell lines RTL-W1. HPLC-MS/MS was used to verify the occurrence of dyes in samples of Piracicaba River upstream and downstream the discharge of Quilombo River and Wastewater Treatment Plant (WWTP) effluent, located in São Paulo State, Brazil. Six dyes were detected in samples of water and effluents. Disperse Red 1 dye was detected in 8 of 16 samples, but its contribution for the mutagenicity was low. Disperse Blue 373 and Disperse Violet 93 were the major contributors for the mutagenicity found in the samples. The genotoxicity of Piracicaba River, evaluated with Salmonella/microsome assay and comet assay, increased after the discharges of Quilombo River and the effluent of WWTP, showing a contribution of this discharges on the river genotoxicity.

This study evaluated the potential toxicity, genotoxicity, and cellular uptake of various sizes and concentrations of chitosan (CS) nanoparticles cultured with normal human dental pulp cells. Normal human dental pulp cells (hDPCs) were derived from human dental pulp tissues and cultured with (50–67) nm and (318–350) nm CS-nanoparticles in concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL as study groups and 0 mg/mL as a control group for time intervals of 16 hours, 24 hours, 3 days, 7 days and 14 days. Attachment efficiency and proliferation rate were assessed by measuring the optical density of crystal violet-stained cells. Cell viability was determined by the activity of mitochondrial dehydrogenase enzymes. Genotoxicity was assessed using the cytokinesis-block micronucleus method and by measuring the fluorescent intensity of phosphorylated H2AX nuclear foci. Cellular uptake was determined by tagging chitosan nanoparticles with FITC stain and then measuring the fluorescence intensity of FITC-tagged chitosan nanoparticles using a spectrophotometer. Statistical analysis was performed using chi-square, one-way ANOVA, and post-hoc Tukey tests. All concentrations of the (50–67) nm group significantly reduced attachment efficiency in comparison with control (P< 0.01) and with (318–350) nm group (p<0.01). Proliferation rate and cell viability were significantly reduced in cells exposed to various concentrations of (50-67) nm chitosan when compared to (318-350) nm group (P<0.05) and control group (P<0.05). For both size groups, higher concentrations significantly showed lower proliferation rate and cell viability when compared to lower concentration (P< 0.01). CS-nanoparticles were able to internalize hDPCs and significantly induced micronuclei, nuclear buds, and pH2AX at concentrations of 0.5 mg/mL and 2 mg/mL as compared to 0.1 mg/mL (P<0.01) and control groups (P< 0.01). At both the 0.5 mg/mL and 2 mg/mL concentrations, (50–67) nm chitosan significantly induced higher proportions of micronuclei (P= 0.001), nuclear buds (P= 0.009), and pH2AX nuclear foci (P= 0.00004) compared to (318–350) nm chitosan. In conclusion, CS-nanoparticles at sizes (50–67) nm and (318–350) nm at a concentration of (0.5–4) mg/mL internalized hDPCs and exhibited cytotoxic and genotoxic effects in dose-dependent and size-associated manners.

Genotoxic effects such as mutations and chromosome abnormalities can augment the risk of adverse health effects such as cancer and heritable genetic diseases; chemicals in commerce must be screened for genotoxic activity. To this end, Toxys B.V. developed the in vitro ToxTracker® assay, which detects (geno)toxicity by monitoring the activity of six reporter genes in cultured mES cells (murine embryonic stem cells), i.e., Rtkn, Bscl2, Btg2, Srxn1, Blvrb and Ddit3. The reporters respond to genotoxic stress, oxidative stress, and endoplasmic reticulum stress characterized by protein unfolding; reporter induction is monitored using flow cytometry. The ToxTracker® assay generates large amounts of multivariate concentration-response data; this study employed innovative quantitative methods to scrutinize ToxTracker® assay results. The work (i) defined a fold-change threshold for identification of a significant positive response, (ii) used two analytical approaches to define endpoint-specific Benchmark Response (BMR) values, (iii) used the BMD (Benchmark Dose) combined-covariate approach for potency ranking of assay validation compounds, and (iv) used PCA (Principal Component Analysis) to investigate functional and statistical relationships between the reporters. The results revealed fold-change cut-offs of 1.5 and 1.7 for identification of weak and strong positive responses, respectively. 1.5-fold is consistent with the value advocated by Toxys B.V.; 1.7-fold is more conservative than the Toxys-advocated 2-fold value. Potency ranking of the validation compounds permitted comparative identification of the most potent inducers of each reporter. The most potent compounds consistently included clastogens used for cancer chemotherapy. BMR values determined using the Zeller et al. (2017) approach ranged from 2.2% for Blvrb and Rtkn, to 7.0% for Ddit3, with an average of 3.9% across all the reporters. The Slob (2016) approach yielded values that ranged from 30% for Ddit3, to 52% for Rtkn, with an average of 43%. The PCA results indicated the Rtkn, Bscl2 and Btg2 reporters are functionally redundant; collectively indicative of genotoxic stress. The Blvrb and Ddit3 reporters are orthogonal indicators of oxidative stress and protein unfolding, respectively; they are essential for toxicological profiling using the ToxTracker® assay. PCA axis scores reflect the toxicological MOA (Mode of Action) of the tested compounds; hitherto unknown MOAs can be inferred using PCA axis-plot proximity to well-studied compounds. Like most in vitro (geno)toxicity assessment assays, ToxTracker® employs a material known as S9 to simulate mammalian hepatic metabolism. S9 is prepared from the livers of rats exposed to an inducer of microsomal CYP (Cytochrome P450) isozymes; the most common CYP inducer is the PCB (polychlorinated biphenyl) mixture known as Aroclor-1254. Due to restrictions in the availability of Aroclor-1254, this study also evaluated the utility of Phenobarbital (PB)/β-Naphthoflavone (BNF)-induced S9, a proposed substitute for Aroclor-induced S9. The results indicate that, despite differences in enzymatic profiles, a 24-hr protocol using 0.40% v/v PB/BNF-induced S9 yields results that are comparable to those obtained using 0.25% v/v Aroclor-induced S9. This study constitutes a significant step towards augmenting the utility of the ToxTracker® assay; it provides a foundation for eventual adoption of high-throughput reporter assays for routine regulatory screening of new and existing chemicals.

Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients. Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels). Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage. The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk. / In-vitro-Genotoxizitätsstudien mit hohen Konzentrationen von Insulin und die Kombination mit Adrenalin zeigten keinen additiven Anstieg der Mikrokernzahl. Der Insulinrezeptor und der AKT-Signalweg waren in den insulinvermittelten Genomschaden involviert. Die endogenen ROS-Quellen, Mitochondrien und NOX, waren an dem insulinvermittelten DNA-Schaden beteiligt. Hohe Konzentrationen von mitochondrialen ROS alleine, verursacht durch einen Komplex III Mitochondrien-Inhibitor, führten zu Zytotoxizität, aber nicht zu einer Zunahme des Genomschadens. Daher ist die durch das NOX-Enzym vermittelte ROS-Produktion wahrscheinlich der gemeinsame Faktor des genotoxischen Signalweges von Insulin und Adrenalin. Die Überstimulation des NOX-Enzyms führte zu einer Sättigung der zellulären biologischen Effekte und fehlender Additivität bei der Induktion von Genomschaden. Dies könnte jedoch unter physiologischen Bedingungen anders sein, da die Hormonspiegel niedriger sind und die ROS-Quellen nicht durch jedes einzelne der Hormone bereits maximal genutzt und daher erschöpft werden. Damit könnte die Möglichkeit eines additiven Genomschadens in vivo bestehen. Die Rolle des AKT-Signalwegs bei der Insulin-vermittelten genomischen Schädigung ist bereits etabliert und hier wurde nun die Funktion des negativen Regulatorproteins PTEN untersucht. Die Ergebnisse zeigten, dass die PTEN Inhibierung nicht nur zu einer erhöhten Genotoxizität durch MN-Induktion führte, sondern auch zur Beeinträchtigung der mitochondrialen Funktion. Obwohl kein Anstieg von ROS nach PTEN-Inhibierung beobachtet wurde, könnte die mitochondriale Dysfunktion zur metabolischen Imbalance sowie zur Zunahme des Genomschadens führen. Dies könnte insbesondere bei Patienten mit bestimmten PTEN-assoziierten Syndromen und Krebserkrankungen, die eine defekte PTEN-vermittelte Tumorsuppressorfunktion, DNA-Reparaturdefekte und kompromittierte antioxidative Abwehrmechanismen aufweisen, eine wichtige Rolle spielen. Wenn diese Patienten zusätzlich von Hyperinsulinämie betroffen sind, könnte eine Akkumulation von Genomschaden erfolgen und das Risiko zur Krebsentstehung wäre erhöht. Der Mechanismus der Genomschadensinduktion durch Insulin wurde bisher mit einer ROS-vermittelten DNA-Oxidation in Verbindung gebracht, aber noch nicht mit der mitogenen Signalgebung. Bei dieser beschleunigte das mitogene Potential des Insulins die Zellteilung und verursachte einen leichten replikativen Stress. Der milde replikative Stress könnte der Kontrolle durch die mitotischen Checkpoint-Proteine entgehen und zu Chromosomen-Fehlverteilungen und Chromosomenbrüchen führen. Dieser Effekt wurde in der Krebszelllinie Hela in Form von multipolaren Spindeln und Mikronuklei beobachtet und es ist nicht klar ob normale Zellen mit effizienterer Kontrolle dies verhindern könnten. Insgesamt könnte ein durch hohe Insulinspiegel vermittelter Schaden im Kontext anderer Komorbiditäten wie etwa PTEN Syndromen, metabolischem Syndrom oder Adipositas zu einer Akkumulation von DNA-Schäden führen. Schließlich zeigte die Analyse von Proben adipöser Patienten eine Zunahme von DNA-Schaden und oxidativem Stress im Vergleich zu den gesunden Kontrollen. Der Anstieg des DNA-Schadens war am höchsten in der Untergruppe der Patienten mit Insulinresistenz. Hoher Insulinspiegel bedeutet somit ein Risiko vom erhöhten oxidativen Stress und Genomschaden, insbesondere in Kombination mit Komorbiditäten. Erschwert wird das Verständnis dieser multifaktoriellen Zusammenhänge durch das komplexe Zusammenspiel von oxidativem Stress und seiner zellulären Regulation in vielen physiologischen sowie pathophysiologischen Prozessen. Daneben ist es eine Herausforderung, Genomschäden bei den geringen Wirkspiegeln hormoneller Effekte zu detektieren. Weitere Untersuchungen der komplexen Insulin-vermittelten Genomschadenswege werden notwendig sein, um mögliche Risiken der Hyperinsulinämie bei Erkrankungen wie Stoffwechselkrankheiten, Diabetes Typ 2 und Adipositas besser zu charakterisieren.