Lung cancer is one of the main causes of death worldwide, with most patients suffering from an advanced unresectable or metastatic non-small cell lung cancer. The mortality trends are mostly related to patterns of tobacco use, specifically from cigarettes. Tobacco is the basic etiological agent found as a consequence of the inhalation of tobacco smoke. Published data show the use of interferons (IFNs) in the treatment of lung tumours due to their potential in displaying antiproliferative, anti-angiogenic, immunoregulatory, and proapoptotic effects. Type1 IFNs have been employed as treatments for many types of cancer, both for haematological cancers and solid tumours. The IFN-γ (naked) functions as an anticancer agent against various forms of cancer. Hence, this study aimed to investigate the genoprotective and genotoxic effects of IFN-γ liposome (nano) on 42 blood samples from lung cancer patients, compared to the same sample size from healthy individuals. The effectiveness of IFN- γ liposome against oxidative stress was also evaluated in this study. A concentration of 100U/ml of IFN-γ liposome was used to treat the lymphocytes in: Comet and micronucleus assays, Comet repair, Western blotting and real-time polymerase chain reaction (qPCR) were based on a preliminary test for the optimal dose. The lymphocytes from lung cancer patients presented with higher DNA damage levels than those of healthy individuals. IFN-γ liposome was not found to induce any DNA damage in the lymphocytes. Also, it caused a significant reduction in DNA damage in the lymphocytes from lung cancer patients in; Comet, Comet repair and micronucleus assays. Furthermore, the 100U/ml of IFN-γ liposome significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and micronucleus assays. Results from; Comet, Comet repair and micronucleus assays were consistent. The data obtained indicated that IFN-γ in both forms (naked INF-γ and INF-γ nano-liposome) may potentially be effective for the treatment of lung cancer and showed the ability of IFN-γ liposome to reduce the DNA damage more than the naked form. The IFN-γ in both forms has also shown anti-cancer potential in the lymphocytes from lung cancer patients by regulating the expression of p53, p21, Bcl-2 at mRNA and protein levels by up-regulating the p53 and p21 to mediate cell cycle arrest and DNA repair in lung cancer patients. The findings of this study are consistent with the view that the naked IFN-γ and liposome could have a significant role in cancer treatment, including lung cancer. / Mutah University in Jordan

Atualmente, a utilização de azo corantes pelas indústrias de tingimento constitui um problema ambiental e de saúde, considerando o lançamento de quantidades elevadas para o meio ambiente e a falta de dados toxicológicos dos corantes disponíveis para as indústrias. Vários estudos têm demonstrado o potencial genotóxico de diversos corantes azóicos, porém para os corantes Disperse Red 1, Disperse Orange 1 e o Disperse Red 13, não foram encontrados dados na literatura relativos à sua capacidade de dano ao material genético. Considerando que esses corantes são empregados em processos de tingimento no Brasil, esse trabalho teve como objetivo a avaliação de sua atividade mutagênica, utilizando o teste de micronúcleo (MN) em linfócitos humanos e em células HepG2. Os resultados obtidos no teste com linfócitos, demonstram que na menor concentração testada (0,2 µg/mL), o número de micronúcleos presentes foi semelhante ao controle negativo, mas esse número aumenta à medida que eleva-se a concentração. No entanto, a partir da concentração de 1,0 µg/mL, este valor começa a decair. Isso provavelmente se deve à citotoxidade dos corantes, levando à morte celular ou redução da divisão celular e, conseqüentemente, não há a formação de micronúcleo. Embora o perfil de mutagenicidade dos três corantes seja semelhante, o corante Disperse Red 13 parece ter maior potencial de dano sobre os linfócitos em relação aos demais, seguido pelo Disperse Red 1 e Disperse Orange 1, respectivamente. Os resultados obtidos para o teste de MN em células HepG2 foram semelhantes aos obtidos no teste feito em linfócitos. O aumento do número de micronúcleos em relação ao aumento da concentração dos corantes, ocorreu até o limite de 2,0 µg/mL em células HepG2, excetuando-se o corante Disperse Red 13, para o qual o limite foi de 1,0 µg/mL. E a partir desses pontos, considerados como limites, ocorreu uma redução no número de MN. Para este sistema celular, os três corantes parecem ter potencial mutagênico bastante semelhante. Portanto, a análise dos resultados mostrou que os corantes Disperse Red 13, Disperse Red 1 e Disperse Orange 1 são mutagênicos para sistemas celulares diferentes. Foi também avaliado Índice de Proliferação do Bloqueio de Citocinese (IPBC), que permite a avaliação de toxicidade celular ou atraso no ciclo celular por meio da determinação da proliferação celular nas culturas. Porém, neste estudo não foram observadas diferenças estatísticas entre o controle negativo e as concentrações testadas. Nossos resultados confirmam que os azo corantes constituem uma importante classe de contaminantes ambientais e devem ser avaliados e utilizados de forma cautelosa. / Currently, the use of azo dyes for the textile industries can causes direct and/or indirect effects on human health and on the environment, considering the discharge of industrial effluents that contain toxic dyes and the lack of reports in the literature about the toxic effects of these compounds. Several studies have been demonstrated the genotoxic effect of diverse azo dyes, however for the dyes Disperse Red 1, Disperse Orange 1 and Disperse Red 13 no information about their capacity to cause DNA damage was found in the literature. Considering that these dyes are used for dying processes in Brazil, the main of this work was the evaluation of the mutagenic activity of Disperse Red 1, Disperse Orange 1 and Disperse Red 13, using the micronucleus assay (MN) in human lymphocytes and HepG2 cells. For the lymphocytes assay, we observed that the number of micronucleus induced by the lowest concentration of each dye (0,2 µg/mL) was similar to the negative control. For the other concentrations we observed a dose response micronucleus formation, until 1,0 µg/mL. Above this concentration, the number of micronucleus has decreased, probably because of the cytotoxic effects of the dyes, which leads to cellular death or reduction of cellular division and, consequently, does not have the micronucleus formation. Although the mutagenicity profile of the three dyes is similar, Disperse Red 13 seems to be the strongest for the lymphocytes, followed by Disperse Red 1 and Disperse Orange 1, respectively. For the HepG2 cells the results were similar to the lymphocytes. For the three dyes we noted a dose dependent increase in the frequency of micronuclei. However, for the HepG2 the threshold for this increase was 2,0 µg/mL, except for Disperse Red 13, which the limit was at 1 µg/ml, after this point a reduction in the MN number occurred. For this cellular system, the three dyes seem to have similar mutagenic potential. Therefore, our results suggest that the dyes Disperse Red 13, Disperse Red 1 and Disperse Orange 1 are potentially mutagenic for different cellular systems. Besides, cytokinesis-block proliferation index (CBPI) was calculated, in order to evaluate cellular toxicity or delay in the cellular cycle through of the determination of the cellular proliferation in the cultures. No statistical difference was detected between the tested concentrations and the negative control. Our results confirmed that azo dyes constitute an important class of environmental contamination and they should be evaluated and used carefully.

<p>Generation of DNA damage is regarded to be an important initial event in the development cancer. Consequently, a battery of tests have been developed to detect different types of genotoxic effects in order to be able to predict the potential genotoxicity and mutagenicity of chemicals, including both pharmaceutical drugs and various types of environmental and occupational agents, as well as dietary factors. The aim of this thesis was to evaluate whether the combination of the comet assay and the extended-term cultures of human lymphocytes (ETC) can be used as an alternative <i>in vitro</i> system to more commonly used transformed mammalian cell lines, and primary cell cultures from humans, when testing the potential genotoxicity of chemicals. </p><p>Using the comet assay, a panel of reference compounds showed that the ETC were found to detect the DNA-damaging effects with no remarkable difference to what has been reported in other cell types. Moreover, in comparison with a well-established rodent cell line, the mouse lymphoma L5178Y cells, the ETC showed similar sensitivity to the DNA damaging effects of the genotoxic agents hydrogen peroxide and catechol. Although there was an interindividual variation in induced DNA damage and the subsequent repair when using ETC from different blood donors, it did not seem to be of crucial importance for the identification of DNA-damaging agents. The demonstrated difference in sensitivity to catechol-induced DNA damage between freshly isolated peripheral lymphocytes and ETC may very well be due to their different proliferative status but despite this difference, both <i>in vitro</i> systems were able to identify catechol as a DNA-damaging agent at the same concentration.</p><p>Based on these results, it is proposed that the ETC and the comet assay are a useful combination when testing for the potential DNA damaging effects of chemicals. Representing easily cultivated cells possessing the normal human karyotype, where one blood sample can be used for numerous experiments performed over a long time, extended-term cultures appear to be a useful alternative, both to transformed mammalian cell lines, and primary cell cultures from humans. In fact, the extended-term lymphocytes, with or without S9 and/or lesion specific DNA repair enzymes, should be used more frequently when screening for the potential genotoxicity of chemicals.</p>

Generation of DNA damage is regarded to be an important initial event in the development cancer. Consequently, a battery of tests have been developed to detect different types of genotoxic effects in order to be able to predict the potential genotoxicity and mutagenicity of chemicals, including both pharmaceutical drugs and various types of environmental and occupational agents, as well as dietary factors. The aim of this thesis was to evaluate whether the combination of the comet assay and the extended-term cultures of human lymphocytes (ETC) can be used as an alternative in vitro system to more commonly used transformed mammalian cell lines, and primary cell cultures from humans, when testing the potential genotoxicity of chemicals. Using the comet assay, a panel of reference compounds showed that the ETC were found to detect the DNA-damaging effects with no remarkable difference to what has been reported in other cell types. Moreover, in comparison with a well-established rodent cell line, the mouse lymphoma L5178Y cells, the ETC showed similar sensitivity to the DNA damaging effects of the genotoxic agents hydrogen peroxide and catechol. Although there was an interindividual variation in induced DNA damage and the subsequent repair when using ETC from different blood donors, it did not seem to be of crucial importance for the identification of DNA-damaging agents. The demonstrated difference in sensitivity to catechol-induced DNA damage between freshly isolated peripheral lymphocytes and ETC may very well be due to their different proliferative status but despite this difference, both in vitro systems were able to identify catechol as a DNA-damaging agent at the same concentration. Based on these results, it is proposed that the ETC and the comet assay are a useful combination when testing for the potential DNA damaging effects of chemicals. Representing easily cultivated cells possessing the normal human karyotype, where one blood sample can be used for numerous experiments performed over a long time, extended-term cultures appear to be a useful alternative, both to transformed mammalian cell lines, and primary cell cultures from humans. In fact, the extended-term lymphocytes, with or without S9 and/or lesion specific DNA repair enzymes, should be used more frequently when screening for the potential genotoxicity of chemicals.

Atualmente, a utilização de azo corantes pelas indústrias de tingimento constitui um problema ambiental e de saúde, considerando o lançamento de quantidades elevadas para o meio ambiente e a falta de dados toxicológicos dos corantes disponíveis para as indústrias. Vários estudos têm demonstrado o potencial genotóxico de diversos corantes azóicos, porém para os corantes Disperse Red 1, Disperse Orange 1 e o Disperse Red 13, não foram encontrados dados na literatura relativos à sua capacidade de dano ao material genético. Considerando que esses corantes são empregados em processos de tingimento no Brasil, esse trabalho teve como objetivo a avaliação de sua atividade mutagênica, utilizando o teste de micronúcleo (MN) em linfócitos humanos e em células HepG2. Os resultados obtidos no teste com linfócitos, demonstram que na menor concentração testada (0,2 µg/mL), o número de micronúcleos presentes foi semelhante ao controle negativo, mas esse número aumenta à medida que eleva-se a concentração. No entanto, a partir da concentração de 1,0 µg/mL, este valor começa a decair. Isso provavelmente se deve à citotoxidade dos corantes, levando à morte celular ou redução da divisão celular e, conseqüentemente, não há a formação de micronúcleo. Embora o perfil de mutagenicidade dos três corantes seja semelhante, o corante Disperse Red 13 parece ter maior potencial de dano sobre os linfócitos em relação aos demais, seguido pelo Disperse Red 1 e Disperse Orange 1, respectivamente. Os resultados obtidos para o teste de MN em células HepG2 foram semelhantes aos obtidos no teste feito em linfócitos. O aumento do número de micronúcleos em relação ao aumento da concentração dos corantes, ocorreu até o limite de 2,0 µg/mL em células HepG2, excetuando-se o corante Disperse Red 13, para o qual o limite foi de 1,0 µg/mL. E a partir desses pontos, considerados como limites, ocorreu uma redução no número de MN. Para este sistema celular, os três corantes parecem ter potencial mutagênico bastante semelhante. Portanto, a análise dos resultados mostrou que os corantes Disperse Red 13, Disperse Red 1 e Disperse Orange 1 são mutagênicos para sistemas celulares diferentes. Foi também avaliado Índice de Proliferação do Bloqueio de Citocinese (IPBC), que permite a avaliação de toxicidade celular ou atraso no ciclo celular por meio da determinação da proliferação celular nas culturas. Porém, neste estudo não foram observadas diferenças estatísticas entre o controle negativo e as concentrações testadas. Nossos resultados confirmam que os azo corantes constituem uma importante classe de contaminantes ambientais e devem ser avaliados e utilizados de forma cautelosa. / Currently, the use of azo dyes for the textile industries can causes direct and/or indirect effects on human health and on the environment, considering the discharge of industrial effluents that contain toxic dyes and the lack of reports in the literature about the toxic effects of these compounds. Several studies have been demonstrated the genotoxic effect of diverse azo dyes, however for the dyes Disperse Red 1, Disperse Orange 1 and Disperse Red 13 no information about their capacity to cause DNA damage was found in the literature. Considering that these dyes are used for dying processes in Brazil, the main of this work was the evaluation of the mutagenic activity of Disperse Red 1, Disperse Orange 1 and Disperse Red 13, using the micronucleus assay (MN) in human lymphocytes and HepG2 cells. For the lymphocytes assay, we observed that the number of micronucleus induced by the lowest concentration of each dye (0,2 µg/mL) was similar to the negative control. For the other concentrations we observed a dose response micronucleus formation, until 1,0 µg/mL. Above this concentration, the number of micronucleus has decreased, probably because of the cytotoxic effects of the dyes, which leads to cellular death or reduction of cellular division and, consequently, does not have the micronucleus formation. Although the mutagenicity profile of the three dyes is similar, Disperse Red 13 seems to be the strongest for the lymphocytes, followed by Disperse Red 1 and Disperse Orange 1, respectively. For the HepG2 cells the results were similar to the lymphocytes. For the three dyes we noted a dose dependent increase in the frequency of micronuclei. However, for the HepG2 the threshold for this increase was 2,0 µg/mL, except for Disperse Red 13, which the limit was at 1 µg/ml, after this point a reduction in the MN number occurred. For this cellular system, the three dyes seem to have similar mutagenic potential. Therefore, our results suggest that the dyes Disperse Red 13, Disperse Red 1 and Disperse Orange 1 are potentially mutagenic for different cellular systems. Besides, cytokinesis-block proliferation index (CBPI) was calculated, in order to evaluate cellular toxicity or delay in the cellular cycle through of the determination of the cellular proliferation in the cultures. No statistical difference was detected between the tested concentrations and the negative control. Our results confirmed that azo dyes constitute an important class of environmental contamination and they should be evaluated and used carefully.

The head and neck cancers (HNC) are a group of cancers that begin in the squamous cells that line the mucosal surfaces of the head and neck. Therefore, they are commonly known as squamous cell carcinoma of the head and neck. Squamous cell carcinoma of the maxillary sinus (MC) is a rare type of HNC, and it is a very aggressive tumour. This cancer is typically diagnosed at a very advanced stage and most patients have a poor survival rate and prognosis. This study is based on the synthesis and applications of gold nanoparticles (AuNPs) conjugated with hesperidin (Hsp) as a targeted drug delivery system. AuNPs are ideal for loading different drugs and delivering them to targets sites due to their stability, small size, substantial surface area, non-cytotoxic and inert nature. Hsp is a naturally occurring substance with anti-inflammatory and antioxidant capabilities. The main aim of this research was to develop a highly efficient and safer method to deliver Hsp to the target sites. The Hsp with poor solubility and bioavailability render it only slightly absorbed, requiring a delivery system to reach its therapeutic target. This study focused on the effects of 15μg/ml Hsp loaded on gold nanoparticles (Hsp-AuNPs) on 20 healthy individuals’ lymphocytes as compared to 20 HNC patients’ lymphocytes using the alkaline Comet assay. While enzyme-based Comet repair was performed on 5 healthy individuals’ lymphocytes as compared to 5 HNC patients’ lymphocytes. The Hsp-AuNPs reduced the DNA damage in HNC patients’ lymphocytes compared to the healthy lymphocytes (***p<0.001). Furthermore, the 15μg/ml of Hsp-AuNPs significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and Comet repair assay. Results from Comet and Comet repair assay were consistent. The human squamous cells of maxillary sinus (MC) were also treated with 5μg/ml of Hsp-AuNPs. The alkaline Comet assay results showed that Hsp-AuNPs induced DNA damage in MC cells (***p<0.001). Therefore, Hsp-AuNPs demonstrated the most substantial genotoxic effects and confirmed a possible anticancer agent. The Hsp was also used to treat lymphocytes from healthy individuals as compared to HNC patients’ lymphocytes they reduced the DNA damage, but they were less effective as compared to Hsp-AuNPs. Published data shows that using the AuNPs as a drug carrier has a more potent therapeutic effect against different diseases including cancer. Also, this study investigated the gene protective and genotoxic impact of bulk Hsp in Maxillary sinus carcinoma cells. The data obtained indicated that Hsp-AuNPs might possibly be effective for the treatment of MC and demonstrated the ability of Hsp-AuNPs to increase the DNA damage more than the bulk form of Hsp (***p<0.001). The outcomes of this study are consistent with the viewpoint that the Hsp-AuNPs might have a substantial role in cancer treatment, including MC. The concentration of 5μg/ml Hsp-AuNPs was used to treat the MC cells in Western blotting, and real-time polymerase chain reaction (qPCR) was based on a preliminary test for the optimal dose. The data obtained indicated that Hsp-AuNPs might potentially be effective for the treatment of HNC and showed the ability of Hsp-AuNPs to reduce DNA damage more than the bulk form of Hsp. Hsp-AuNPs has also shown anti-cancer potential in the MC cells by up-regulating the expression of p53, p21, PPAR gamma, and Caspase 3, at mRNA and protein levels by up-regulating the p53, PPAR gamma, Caspase 3, and p21 to mediate apoptosis and DNA repair in MC cells. The findings of this study are consistent with the view that the Hsp-AuNPs could have a significant role in cancer treatment, including HNC and MC.

Les adduits exocycliques à l’ADN paraissent être la conséquence indirecte, sous la médiation de la peroxydation lipidique, du stress oxydant cellulaire induit par les composés aromatiques nitrés (CANs) de l’environnement. Ces derniers, formés le plus souvent in situ dans les environnements complexes sont un sujet de préoccupation croissante en santé environnementale. Le but étant de comprendre les mécanismes de formation et de dégradation de ces adduits afin d’en apprécier leur place dans la toxicité des CANs et leur intérêt en tant que biomarqueurs du stress oxydant induit par l’environnement.Nous avons réalisé la synthèse de deux adduits exocycliques : le 1,N²-éthéno-2’-déoxyguanosine (εdG) et le 1,N²-propano-2’-déoxyguanosine (pdG-HNE) et étudié leurstabilité en présence d’une oxydation radicalaire. Il est apparu que le pdG-HNE semble être lemeilleur candidat en tant que biomarqueur du stress oxydant.Pour approcher au mieux la chimie du vivant, nous nous sommes posés la question dela stabilité de ces adduits en milieu cellulaire. Une étude préliminaire de génotoxicité a étéréalisée et montre que seuls les hydrocarbures aromatiques polycycliques nitrés présententune potentialité mutagène significative. Nous avons donc étudié le pyrène et ses dérivés nitrés(1-Nitropyrène/1,3-Dinitropyrène/1,6-Dinitropyrène/1,8-Dinitropyrène) afin d’étudier leurcapacité à générer des adduits dans les lymphocytes humains.Nos résultats montrent que le 1-Nitropyrène génère in vitro des adduits stables dans letemps ; ce qui pose la question de leur réparabilité par les systèmes cellulaires et de leurspotentialités cancérogènes pour l’homme. / Exocyclic DNA adducts seem to be the indirect consequence, mediated by lipidperoxidation, of oxidative stress induced by nitro aromatic compounds (NACs). The latterusually formed in situ in environmental complex mixtures are a matter of concern inenvironmental health. The aim is to understand the mechanisms of formation and degradationof these adducts to assess their place in toxicity of NACs and their importance as oxidativestress biomarkers induced by the environment.We synthesized two exocylic adducts: 1, N²-etheno-2’-deoxyguanosine (εdG) and 1,N²-propano-2'-deoxyguanosine (pdG-HNE) and studied their stability when a radicaloxidation is present. It appeared that pdG-HNE seems a suitable biomarker of oxidative stress.To come close to life chemistry, we were wondering whether these adducts are stablein cellular environment. A preliminary study of genotoxicity was carried out and showed thatonly nitro polycyclic aromatic hydrocarbons have a significant mutagenic potency. Thereforewe studied pyrene and nitropyrenes (1-Nitropyrene/1,3-Dinitropyrene/1,6-Dinitropyrene/1,8-Dinitropyrene) to examine their ability to produce adducts in human lymphocytes.Our results show that 1-Nitropyrene give rise to stable adducts in vitro, which raisesthe question of their repairability by cellular systems and their potential carcinogenic tohumans.

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