Made available in DSpace on 2014-06-11T19:24:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-15Bitstream added on 2014-06-13T19:10:40Z : No. of bitstreams: 1 oliveira_lrc_me_botfm.pdf: 589752 bytes, checksum: 4d9dc2b0e2961ff5f5d54602fbf367a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A tuberculose permanece como um dos principais problemas de saúde pública no Brasil e no mundo. Tem como principal agente o Mycobacterium tuberculosis, e é uma doença letal sem tratamento, e seu controle é dificultado pela longa duração e ausência de marcadores, para medir o sucesso ou a falha deste. Os TLRs são receptores que após o reconhecimento de produtos microbianos, ativam a resposta inata e adaptativa. Assim, os receptores TLRs e as citocinas possuem um papel chave na defesa contra o bacilo. Considerando a ausência de informações na literatura em relação à resposta imune de pacientes com tuberculose pulmonar durante o tratamento antituberculose, principalmente em relação à interação inicial do M. tuberculosis com as células da imunidade inata e de seu papel, na indução da resposta adaptativa, através dos receptores TLR, estudos neste sentido, são necessários para se compreender melhor como é a resposta imune do hospedeiro contra o bacilo, frente ao tratamento. Assim, o objetivo deste trabalho foi avaliar a expressão dos receptores TLR2 e TLR4, do fator de transcrição Foxp3 e da enzima óxido nítrico sintase induzível (iNOS), produção e expressão de citocinas, e alterações genotóxicas em pacientes durante o tratamento antituberculose. Foram coletadas amostras de sangue total dos controles saudáveis PPD+ em um único momento (G1) e de pacientes (G1) em três momentos diferentes: M1: início; M2: terceiro mês e M3: ao sexto mês de tratamento antituberculose. Após a obtenção das células mononucleares do sangue periférico (PBMC), as concentrações celulares foram ajustadas para a realização da qPCR, da citometria de fluxo e do teste do cometa. Pacientes com tuberculose pulmonar apresentaram expressão gênica, e na superfície célular de linfócitos e... / Tuberculosis (TB) remains a major public health problems in Brazil and worldwide. Its main agent Mycobacterium tuberculosis, and is a lethal disease without treatment, and its control is hampered by the long duration and lack of markers to measure the success or failure of this. The TLRs are receptors that after the recognition of microbial products, activate innate and adaptive response. Thus, TLRs receptors and cytokine have a key role in defense against the bacillus. Considering the absence of information in the literature regarding the immune response of patients with pulmonary tuberculosis during antituberculosis treatment, especially in relation to the initial interaction of M. tuberculosis with the cells of innate immunity and its role in the induction of adaptive response through the TLR receptors, studies in this direction are needed to better understand how the host immune response against the bacillus, towards treatment. The objective of this study was to evaluate the expression of TLR2 and TLR4 receptors, the transcription factor Foxp3 and inducible nitric oxide synthase (iNOS) production and expression of cytokines and genotoxic alterations in patients during TB treatment. Samples were collected from whole blood of healthy PPD + controls in a single moment (G1) and patients (G1) at three different moments: M1: start, M2, M3, and the third month, the sixth month of TB treatment. After obtaining the peripheral blood mononuclear cells (PBMC) and cell concentrations were adjusted to carry out the qPCR, flow cytometry and testing of the comet. Patients with pulmonary tuberculosis showed gene expression and cell surface of lymphocytes and monocytes from TLR2 and TLR4 greater than the control subjects, both in M1, M2 and M3. However, the expression and cell surface receptor TLR2 was... (Complete abstract click electronic access below)

Made available in DSpace on 2016-09-27T13:39:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-07-27. Added 1 bitstream(s) on 2016-09-27T13:45:05Z : No. of bitstreams: 1 000869160_20200801.pdf: 948171 bytes, checksum: b1b99c6dc56613330126eb9e2d642cff (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Danos no DNA, podem ocorrer tanto por agentes genotóxicos endógenos quanto agentes exógenos, que podem promover a instabilidade do genoma e levar diretamente a doenças, como por exemplo, o câncer, alterações neurológicas, imunodeficiências e envelhecimento prematuro. Auxiliando na manutenção da estabilidade, as células apresentam uma série de vias de reparo de DNA, as quais realizam o processo em múltiplas etapas para resolver lesões específicas no DNA e manter a integridade do genoma. A importação nuclear é um pré-requisito para as funções das proteínas de reparo do DNA e dentre os mecanismos responsáveis pela regulação da importação nuclear, a via clássica constituída pelo heterodímero Importina-α/β é um dos principais mecanismos de deslocamento. A Importina-β (Impβ) atua como o transportador enquanto a Importina-α (Impα) atua como adaptador, reconhecendo as sequências de localização nuclear (NLS) presentes nas proteínas que possuem função no núcleo. Esse trabalho trata especificamente dos estudos estruturais de complexos da Impα peptídeos NLSs de proteínas relacionadas ao reparo de DNA utilizando técnicas de cristalografia e ensaios de afinidade pela técnica de calorimetria de titulação isotérmica (ITC). A expressão e purificação da Impα de Mus musculus truncada em sua porção N-terminal foi realizada, bem como a co-cristalização da Impα peptídeos NLSs de proteínas relacionadas ao reparo de DNA, correspondentes as sequências MLH1, PMS2, XPG1 e XPG2. Peptídeos mutados em regiões importantes de reconhecimento nuclear para os peptídeos MLH1 e PMS2 também foram selecionados para o desenvolvimento deste projeto. Dados de difração de raios-X foram coletados dos cristais obtidos e processados no intervalo de 2,0-2,8 Å de resolução. Com esses resultados, as estruturas contendo cNLSs MLH1, PMS2, XPG1 e XPG2 foram elucidadas. As proteínas do complexo MutLα, a MLH1 e... / DNA damage can occur by endogenous and exogenous genotoxic agents, which may promote instability of the genome and directly lead to diseases such as cancer, neurological disorders, immunodeficiencies and even premature aging. Helping in the maintenance of the stability, the cells display a number of DNA repair pathways, which carry out the process in multiple steps to resolve specific DNA damage, and maintain the integrity of the genome. The nuclear import is a pre-requisite for the functions of DNA repair proteins and, among the mechanisms responsible for regulation of nuclear import, the classical pathway constituted by the heterodimer importin-α / β is a major shift mechanisms. Importin-β (Impβ) acts as the carrier while the importin α-(Impα) acts as an adapter recognizing the nuclear localization sequence (NLS) present in proteins that have function into the nucleus.This work concerns specifically the structural studies of complexes with Imp and NLSs peptides from proteins related to DNA repair using crystallographic techniques and binding assays by isotermal titration calorimetry technique (ITC). The expression and purification of Mus musculus Impα truncated at its N-terminal portion was performed, as well as co-crystallization with Impα NLSs peptides of proteins related to DNA repair, the corresponding sequences MLH1, PMS2, XPG1 and XPG2. Peptides mutated in key regions of nuclear recognition for MLH1 and PMS2 peptides were also selected for this project. X-ray diffraction data collected from crystals were obtained and processed in the range of 2.0-2.8 Å resolution. With these results, the structures containing cNLSs MLH1, PMS2, XPG1 and XPG2 were elucidated. The MutLα complex proteins, MLH1 and PMS2, related to the mismatch repair (MMR), bound to the Impα similarly to the T antigen NLS of SV40 in the major binding site. ITC experiments corroborate the crystallographic results, which suggest that both NLSs are classic... / FAPESP: 2011/09905-0

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os bufodienolÃdeos sÃo esterÃides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da famÃlia Bufonidae utilizado na medicina chinesa. Os bufodienolÃdeos possuem grande variedade de atividades biolÃgicas, incluindo atividades antineoplÃsicas. Em relaÃÃo à atividade antitumoral, os bufodienolÃdeos tem demonstrado inibir o crescimento de vÃrias linhagens de cÃlulas cancerÃgenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotÃxico e genetÃxico de seis bufodienolÃdeos em seis linhagens tumorais humanos, trÃs linhagens murinas normais e cÃlulas mononucleadas do sangue perifÃrico (CMSP) humano. Todos os seis bufodienolÃdeos foram citotÃxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 ÂM. Os bufodienolÃdeos testados nÃo apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de aÃÃo envolvido. Uma sequÃncia de experimentos in vitro foram realizados utilizando-se a linhagem leucÃmica HL-60. As cÃlulas foram tratadas em diferentes concentraÃÃes da amostra hellebrigenina (0,03, 0,06 e 0,12 ÂM) por 24 horas. A viabilidade das cÃlulas (nÃmero de cÃlulas viÃveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o nÃmero de cÃlulas reduziu a partir da menor concentraÃÃo (0,03 ÂM) testada e a porcentagem de cÃlulas com membrana integra reduziu a partir da concentraÃÃo 0,06 ÂM. A anÃlise morfolÃgica por citometria de fluxo revelou aumento de cÃlulas com padrÃo apoptÃtico a partir da concentraÃÃo de 0,06 ÂM. Jà a anÃlise do conteÃdo nuclear, nos mostrou aumento de fragmentaÃÃo de DNA sub-G1 indicativo de apoptose e acÃmulo de cÃlulas na fase G2/M a partir das concentraÃÃes de 0,03 e 0,06 ÂM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalizaÃÃo da fosfatidilserina, despolarizaÃÃo mitocondrial, ativaÃÃo da caspase iniciadora 8 e consequente ativaÃÃo das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotÃxico por induÃÃo de mais de uma via apoptÃtica. Hellebrigenina nÃo foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberraÃÃes cromossÃmicas em CMSP. Por meio dos estudos de docking molecular foi possÃvel predizer a ligaÃÃo entre hellebrigenina e topoisomeraseIIα humana, resultado compatÃvel com a possÃvel inibiÃÃo dessa enzima. De forma geral, os resultados apontam o potencial citotÃxico do bufodienolÃdeo hellebrigenina / Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frogâs skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolÃdeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 ÂM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 ÂM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 ÂM) tested and the percentage of cells with reduced membrane integrity from 0.06 ÂM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 ÂM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 ÂM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin

Submitted in fulfilment of the requirements of Master's Degree in Chemistry, Durban University of Technology, 2017. / DNA damage plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) therefore, an innovative ss-DNA/dopamine/TiO2/FTO electrode strategy was developed to detect the genotoxicity upon photocatalytic reactions. This study involves a computational and electrochemical investigation towards the direct measurement of DNA damage. Computational chemistry was useful to resolve the intricate chemistry problems behind electrode constructions. The computational protocols were simultaneously carried out comprising of density functional theory (DFT) calculations, Metropolis Monte Carlo (MC) adsorption studies, and molecular dynamics (MD) simulations. The DFT calculations elucidated the structural, electronics, and vibrational properties of the electrode components resulting in a good agreement with the experimental parameters. The MC simulations carried out using simulated annealing predicted the adsorption process within layer-by-layer electrode as well generating reliable inputs prior to MD simulations. A 100 ns MD simulations were performed using a canonical ensemble provided information on the thermodynamics parameters such as total energy, temperature, and potential energy profiles, including radius of gyrations and atomic density profiles. Binding energies calculated from the MD trajectories revealed increasing interaction energies for the layer-by-layer electrode, in agreement with the electrochemical characterization studies (i.e. gradual decrease of cyclic voltammogram (CV) as well as increasing diameter of electrochemical impedance spectroscopy (EIS) semicircle upon electrode modification). The higher binding energies may lead to smaller changes in the electrochemical polarizability which directly affect to the decreasing of redox peak current and charge transfer resistance enhancement. Instead, HOMO-LUMO DFT levels are also taken into account to explain electron transfer phenomena within layer construction leading to the alteration of CV behaviours. Experimentally, the ss-DNA was electronically linked to TiO2/FTO surface through dopamine as a molecular anchor. Electrochemical measurements using cyclic voltammetry and EIS were employed to characterize the electrode modifications. The square wave voltammetry was subsequently used to measure the DNA damage and the potency of antioxidant treatment using ascorbic acid (AA) due to its ability in protecting the DNA from the damages. The presence of AA significantly protected the DNA from the damage, therefore was able to be used as a potential treatment in AD. Theoretically, guanine residues predicted by DFT as the most reactive sites of the ss-DNA involved in the genotoxic reactions. Overall, the theoretical studies successfully validated the experimental study as well as providing the molecular basis of interaction phenomena towards electrode constructions. Our results highlight the potential application of this methodology to screen the genotoxicity in Alzheimer’s, suggesting the important role of theoretical studies to predict the molecular interaction and validation of the DNA-based sensors and bioelectronics. / M

The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal genes that are mutated in the developmental disorder Primary Microcephaly, where affected individuals have smaller brains than expected for the age- and sex-adjusted mean. Orthologues of CDK5RAP2 in the fruit fly (Centrosomin/Cnn) and in fission yeast (Mod20p) have been well characterised and are known to have important roles in maintaining centrosome structure and in regulating microtubule nucleation. CDK5RAP2 shares two evolutionarily conserved domains with Cnn, known as CNN motif 1 and 2. Using the chicken B-cell line, DT40, I have used gene-targeting methods to disrupt both of these domains in CDK5RAP2. This revealed a function for CDK5RAP2 in attaching centrosomes to mitotic spindle poles. Centrosome attachment to spindle poles is mediated by a binding partner of CDK5RAP2, AKAP450. AKAP450 also localises to centrosomes and provides anchorage sites for spindle poles in the centrosome. Disruption of the CNN1 and CNN2 domains of CDK5RAP2 causes mislocalisation of AKAP450 from the centrosome and detachment of centrosomes from spindle poles. My studies in DT40 and in human cell lines revealed that CDK5RAP2 and AKAP450 also cooperate during interphase to maintain the two centrioles in the centrosome as a pair. In addition to a structural role in the centrosome, I also find that CNN motif 1 of CDK5RAP2 plays a role in the cellular response to DNA damage. In the absence of CNN motif 1, cells no longer efficiently arrest the cell cycle in response to damage. Centrosome-mediated mitotic spindle alignment and the DNA damage response have both been implicated in microcephaly. Therefore, defects in these functions of CDK5RAP2 may explain how mutations in cdk5rap2 may lead to microcephaly.

Gene regulatory networks are dynamic and continuously remodelled in response to internal and external stimuli. To understand how these networks alter cellular phenotype in response towards specific challenges, my first project sought to develop a methodology to explore how the strength of genetic interactions changes according to environmental context. Defined as sensitivity-based epistasis, the results obtained using this methodology were compared to those generated under the conventional fitness-based approach. By integrating this information with gene expression profiles and physical interaction datasets, we demonstrate that sensitivity-based epistasis specifically highlights genetic interactions with a dynamic component. Having investigated how an external stimulus regulates network dynamics, we next sought to understand of how genome positioning impacts transcription kinetics. This feat was accomplished by cloning two gene-reporter constructs, representing contrasting promoter architectures, across 128 loci along chromosome III in S.Cerevisiae. By comparing expression and noise measurements for promoters with “covered” and “open” chromatin structures against a stochastic model for eukaryotic gene expression, we demonstrate that while promoter structure regulates burst frequency (the rate of promoter activation), positional effects in turn appear to primarily modulate burst size (the number of mRNA produced per gene activation event). By integrating these datasets with information describing global chromatin structure, we suggest that the acetylation state of chromatin regulates burst size across the genome. Interestingly, this hypothesis is further supported by nicotinamide-mediated inhibition of Sir2 which would appear to modulate burst size globally across the genome.

Human exposures to polycyclic aromatic hydrocarbons (PAHs) occur as components of complex mixtures. Evaluations of health risks posed by complex mixtures containing PAHs rely on the toxicological knowledge of prioritized PAH mixture components, assuming that these PAHs share a common mode of action and that the sum of the contributions of these PAHs equals the toxic potency of the mixture (i.e., additivity). Traditional toxicity testing methods emphasizing apical endpoints have had limited success at evaluating the validity of these assumptions. Toxicogenomic tools that are able to rapidly generate toxicologically-relevant and mechanistic information have gained acceptance in the regulatory arena for individual chemicals; however, the applicability of these tools for chemical mixtures has been inadequately addressed. This thesis used toxicogenomic tools to (1) improve the understanding of mechanisms underlying the adverse, toxicological responses induced by individual PAHs and (2) evaluate the contention that transcriptional profiles and pathway information can be used to critically examine interactions between individual PAHs in PAH-containing mixtures, and the assumption of additivity. Microarrays were used to profile gene expression changes (transcriptomes) in forestomach, liver, and lung tissues (targets of PAH exposure) from mice orally exposed to three doses of eight individual PAHs, two defined PAH mixtures, and one complex PAH-containing mixture (coal tar). The results revealed that each PAH induced transcriptional changes that were significantly associated with several pathways implicated in carcinogenesis. However, despite a uniform ability to induce DNA damage (i.e., DNA adducts), mutations, and increases in enzyme activity, the pathways differ across PAHs and tissues. A novel strategy that employs single-PAH transcriptome data to models of additivity revealed that the assumption of additivity in PAH mixtures is valid at the pathway level; however, the independent action model of additivity yielded better estimates compared to concentration addition (used in human health risk assessment of PAH mixtures) or generalized concentration addition. Additionally, predicted and observed coal tar-induced transcriptional benchmark doses were comparable to those derived from previously published coal tar-induced murine lung tumour incidence data. Overall, this thesis demonstrates the utility of toxicogenomic data to expand the current understanding regarding the toxic potential of individual PAHs and PAH-containing complex mixtures.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Autophagy is a regulated and evolutionarily conserved catabolic process that serves to degrade superfluous or damaged organelles and recycle their biochemical components for use in energy production and other biosynthetic reactions. It is a crucial cellular response to various stresses including oxidative stress and starvation. Sirtuins are NAD+ dependent deacetylases that link transcriptional regulation to cellular energy homeostasis, DNA damage, ROS response, cell cycle control, apoptosis and autophagy. The role of autophagy in cancer is complex as autophagy exerts both cell protective and damaging functions depending on the circumstances. The purpose of this study is to investigate the role of individual Sirtuin family members in the regulation of gene expression of the known autophagy markers LC3 and Beclin 1 under DNA damage and oxidative stress. To investigate the regulation of autophagy, we followed the gene expression of autophagy genes LC3 and Beclin 1 under diverse stress conditions in human osteosarcoma cells (U2OS). The protein and mRNA levels of LC3-II and the formation of autophagosomes were increased in etoposide and rotenone treated cells. While LC3-II and LC3-1 protein expressions were increased in Sirt1 overexpressing cells, no significant change was observed in Beclin 1 protein level. However, inhibition of Sirt1 by siRNA did not affect the cellular levels of the autophagy markers suggesting the potential involvement of other Sirtuin family members in the regulation of autophagy. Elevated LC3 mRNA was observed in cells overexpressing any of the Sirtuins family members; however etoposide treatment selectively inhibited Sirt1 and Sirt2 dependent upregulation of LC3 mRNA. Induction of LC3-Luc reporter activity was observed in Sirt5 transfected cells, which was further increased by etoposide treatment. Sirt5 carrying mutation in its catalytic domain is not able to induce autophagy and Sirt5 mediating autophagy is under the control of NF-KB transcription factor. These results support the notion that Sirtuins are important regulators of autophagy and the function of each member is to differentially regulate DNA damage and oxidative stress responses.

Irradiamos amostras de DNA com luz UVA em diferentes condições para estudar os possíveis mecanismos envolvidos na indução de lesões de DNA por essa radiação. As lesões de DNA formadas após as irradiações foram quantificadas com enzimas de reparo de DNA que reconhecem e clivam os sítios contendo bases oxidadas e dímeros de pirimidina (CPDs). Complementando essas análises, foram realizados ensaios com anticorpos e HPLC-ED. NaCl e uma maior concentração de DNA são capazes de diminuir a indução de CPDs. Danos gerados por estresse oxidativo são inibidos na presença de azida de sódio e quelantes de metais, indicando o envolvimento de oxigênio singlete e reações de Fenton, na geração dessas lesões. Água deuterada e DNA mais concentrado aumentaram a indução de bases oxidadas. Quanto maior a quantidade de DNA irradiado, mais oxigênio singlete é formado, o que indica um possível mecanismo de fotossensibilização endógeno. / DNA samples were irradiated with UVA light in different conditions for studying the possible mechanisms involved in the induction of DNA lesions by this radiation. DNA lesions formed after irradiation were quantified with DNA repair enzymes, which recognize and cleave the sites containing oxidized bases and pyrimidine dimers (CPDs). Complementing these analyses, tests were performed with antibodies and HPLC-ED. NaCl and more concentrated DNA are capable of reducing the induction of CPDs. Damage caused by oxidative stress is inhibited in the presence of sodium azide and metal chelators, indicating the involvement of singlet oxygen and Fenton reactions, in the generation of these lesions. Deuterated water and more concentrated DNA increased the induction of oxidized bases. The bigger the amount of irradiated DNA, the more singlet oxygen is formed, which indicates a possible endogenous photosensitization mechanism.