RESUMO
With the rapid development of nanotechnology there has been a corresponding increase in the application of titanium dioxide nanoparticles (TiO2-NPs) in various consumer and industrial products, consequently their potential health hazards and environmental effects are considered an aspect of great concern. In the present study, in order to assess the impact of TiO2-NPs in the marine environment, the biological effects of TiO2-NPs on a sea bass cell line (DLEC) were investigated. Cells were exposed for 24 h to different concentrations of TiO2-NPs (1, 8, 40, 200 and 1000 µg/ml) or co-exposed with CdCl2 (Cd). The effects of UV light irradiation were also investigated in cells treated with TiO2-NPs and/or Cd. The internalization of TiO2-NPs and the morphological cell modifications induced by the treatments were examined by transmission and scanning electron microscopy, this latter coupled with energy dispersive X-ray spectroscopy (EDS) for particle element detection. In addition, the effects of controlled exposures were studied evaluating the cytotoxicity, the DNA damage and the expression of inflammatory genes. Our study indicates that TiO2-NPs were localized on the cell surface mainly as agglomerates revealed by EDS analysis and that they were uptaken by the cells inducing morphological changes. Photoactivation of TiO2-NPs and/or co-exposure with Cd affects ATP levels and it contributes to induce acute cellular toxicity in DLEC cells dependent on Ti concentration. The inflammatory potential and the DNA damage, this latter displayed through a caspase-3 independent apoptotic process, were also demonstrated. Overall our data suggest that the interaction of TiO2-NPs with marine water contaminants, such as cadmium, and the UV irradiation, may be an additional threat to marine organisms.
Assuntos
Bass/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Titânio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Cloreto de Cádmio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Microscopia Eletrônica de Varredura/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Espectrometria por Raios X/veterinária , Titânio/metabolismo , Raios Ultravioleta , Poluentes Químicos da Água/metabolismoRESUMO
Hospitalized preterm infants are exposed to stressful stimuli and early parental separation, which can undermine their long-term development and mother-infant bonding. Family-centered music therapy can enable positive mother-infant interactions, mediated by maternal infant-directed singing. This study aimed to investigate the effects of music therapy on preterm infant's signs of engagement, namely Eye Opening (EO) and Smiling (SM), and maternal vocalizations. Participants were 30 mother-preterm infant dyads in a Brazilian Neonatal Intensive Care Unit (NICU), divided into a Music Therapy Group (MTG) and a Comparison Group (CG). The MTG participated in 6 sessions of the Music Therapy Intervention for the Mother-Preterm Infant Dyad (MUSIP), with the aim of supporting maternal singing with the infant. Prior to discharge, all mothers were filmed during a Non-singing (NS) and Singing (S) interactional condition; in the S condition, mothers were explicitly asked to address their infants by singing. Results of video and audio analysis showed that infants in the MTG displayed greater Eye Opening (EO) frequency compared to CG, but only when they were in an initial awake state at test, suggesting that music therapy can potentialize infants' alertness, by increasing their disposition and chances of being engaged in the interaction with the mother. Non-religious mothers appeared to sing significantly more in the MTG than in the CG. These preliminary findings indicate that music therapy in the NICU could promote infant's signs of engagement during interactions and can sustain maternal singing, especially with non-religious mothers in Brazil.
Assuntos
Musicoterapia , Canto , Feminino , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Unidades de Terapia Intensiva Neonatal , Relações Mãe-Filho , MãesRESUMO
Polycyclic aromatic hydrocarbons (PAH) such as dibenzo[a,l]pyrene (DBP) are wide-spread environmental pollutants most probably mutagenic and carcinogenic to humans. Detailed data on the cytogenetic effects of anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) in mammalian cells are not available in the literature. The aim of this study is to elucidate the mechanisms involved in the induction of chromosomal aberrations and sister chromatid exchanges (SCEs) by DBPDE in mammalian cells. In order to achieve this a parental (AA8) and different DNA repair-deficient Chinese hamster ovary cell lines such as UV4, UV5, UV61 (nucleotide excision repair, NER), EM9 (base excision repair, BER), irs1SF (homologous recombination repair, HRR) and V3-3 (non-homologous end joining, NHEJ) were used. The most sensitive cell lines for DBPDE-induced chromosome aberrations were EM9 and irs1SF, while EM9 and V3-3 cell lines were the most sensitive in terms of SCEs induction. It can be suggested that the BER pathway plays an important role in the repair of lesions induced by DBPDE, affecting both chromosomal aberrations and SCEs induction. Moreover, the HRR pathway seems to play a role in cellular resistance to DBPDE mainly in terms of chromosomal aberration induction while the NHEJ pathway takes part affecting only the induction of SCEs.
Assuntos
Benzopirenos/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Poluentes Ambientais/farmacologia , Compostos de Epóxi/farmacologia , Animais , Células CHO , Aberrações Cromossômicas , Cricetinae , CricetulusRESUMO
G(0) human peripheral blood lymphocytes were X-irradiated to determine whether there is a direct relationship between radiation-induced dicentric chromosomes and the triggering of apoptosis. Immediately after X-ray exposure, control and irradiated lymphocytes were analyzed for viability, apoptosis and chromosome damage using the premature chromosome condensation technique. A batch of lymphocytes was kept in liquid holding for 48 h and then loaded on Ficoll-Paque medium to separate apoptotic (high-density) and normal (normal-density) cells. Then the same end points were analyzed in high-density and normal-density fractions of control and irradiated lymphocytes. After 48 h of liquid holding, the majority of apoptotic cells contained dicentric chromosomes. These results demonstrate that in human lymphocytes, the type of chromosome damage influences the induction of programmed cell death and provide direct evidence that cells bearing dicentrics are eliminated by apoptosis. G0 lymphocytes are the most common tissue used in biodosimetry studies, and the amount of chromosomal damage detected depends on the time between exposure and sampling. Since the radiation-induced apoptotic cells show the presence of dicentrics, radiation-induced damage can be underestimated. These results may have relevance in evaluations of the efficacy of radiotherapy based on the frequencies of chromosomal aberrations.
Assuntos
Apoptose/fisiologia , Apoptose/efeitos da radiação , Aberrações Cromossômicas/efeitos da radiação , Linfócitos/fisiologia , Linfócitos/efeitos da radiação , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Humanos , Linfócitos/citologia , Doses de RadiaçãoRESUMO
PURPOSE: To determine the relationships between the frequencies of radiation-induced chromosomal alterations and the extent of apoptosis in G0 human lymphocytes. MATERIAL AND METHODS: G0 human peripheral blood lymphocytes (HPBL) were X or gamma-irradiated, in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). Directly after irradiation, a part of the lymphocytes were stimulated to grow while the rest were stimulated 48 h after irradiation. These lymphocyte cultures were analysed for induction of chromosomal aberrations. A subset of lymphocytes was kept in G0 and analysed for cell viability, apoptosis and p53 expression. RESULTS: The fraction of cells bearing dicentrics was reduced in lymphocytes stimulated to grow 48 h post irradiation as compared to lymphocytes stimulated immediately after irradiation. The decrease in the frequency of dicentrics correlated with the increase in the number of apoptotic cells. The operative apoptotic pathway in irradiated Go lymphocytes was dependent on the expression of p53. CONCLUSIONS: The radiation-induced apoptotic response of G0 lymphocytes is p53 dependent and increases with the time they are held in G0. When mitogen was added 48 h after irradiation, cells with dicentrics were either preferentially eliminated or did not enter mitosis. Thus the radiation-induced damage can be underevaluated depending on the time between radiation exposure and the induction of proliferation. These results may have relevance for biodosimetry studies or for evaluations of the efficacy of radiotherapy which are based on the frequencies of chromosomal aberrations.
Assuntos
Apoptose/efeitos da radiação , Aberrações Cromossômicas/efeitos da radiação , Proteína Supressora de Tumor p53/biossíntese , Técnicas de Cultura de Células , Sobrevivência Celular , Relação Dose-Resposta à Radiação , Genes p53 , Humanos , Linfócitos , Radiação Ionizante , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Telomerase plays a key role in the maintenance of chromosomal stability in tumours, and the ability of anti-cancer agents to inhibit telomerase activity is under investigation. In this study, we evaluated the effect of etoposide and taxol, on the telomerase activity and telomere length in human leukaemia p53 null cells and human bone marrow cells, as well as apoptosis and cell cycle modulation. Results showed that after exposure to the drugs, HL-60 cells as well as the human progenitors underwent a block in G2 and subsequently apoptosis, whereas stromal cells from bone marrow did not undergo a block in G2 or enter apoptosis after etoposide exposure. Telomere length increased in stromal cells after treatment with both etoposide and taxol whereas in HL-60 cells only after etoposide treatment with. Bax, bcl-2 and bcl-x change their expression in stromal cells, whereas bcl-x was induced after drug treatment and bcl-2 down regulated in progenitor cells. Our data suggest that telomerase activity and apoptosis are correlated and they seem to be modulated by a common gene, bcl-2.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Inibidores Enzimáticos/farmacologia , Telomerase/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologia , Anexina A5/metabolismo , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , DNA/biossíntese , DNA/genética , Citometria de Fluxo , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Genes bcl-2/genética , Células HL-60 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sondas RNA , Telômero/efeitos dos fármacos , Telômero/ultraestrutura , Proteína X Associada a bcl-2RESUMO
PURPOSE: To investigate whether unstable types of chromosomal aberrations are more effective in priming apoptotic cell death in comparison with stable ones. Also, to highlight the phase of the cell cycle at which apoptosis occurs and the mechanism of its execution. MATERIALS AND METHODS: G0 human peripheral blood lymphocytes were X-irradiated in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). After irradiation, the lymphocytes were analysed for induction of dicentrics, translocations, apoptosis, p53 and survivin expression at various recovery times. RESULTS: A preferential elimination of cells bearing dicentrics with respect to those with balanced translocations was observed. There was a time-dependent correlation between the decrease in the frequency of dicentrics and the increase in the per cent of apoptotic cells. Most of the apoptotic cells were labelled with bromodeoxyuridine and were mononucleated in cytochalasin B-treated cells cultures (blocked cytokinesis). However, after continuous colcemid treatment, the apoptotic pathway was not induced. Moreover, in the G2/M-phase, an increase in p53 and a decrease in survivin occurred that were X-ray and Ara-C dose dependent. CONCLUSIONS: The apoptotic process is primed when the dicentric-bearing human peripheral blood lymphocytes attempt to exit from metaphase. It is possible that unstable aberrations generate changes in the mitotic spindle causing mechanical tension at the kinetochore, activating the mitotic checkpoint and the execution of p53/survivin-dependent apoptosis.
Assuntos
Apoptose/efeitos da radiação , Aberrações Cromossômicas/classificação , Aberrações Cromossômicas/efeitos da radiação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos da radiação , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios X , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Células Cultivadas , Cromossomos Humanos/classificação , Cromossomos Humanos/efeitos da radiação , Citarabina/farmacologia , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Proteínas Inibidoras de Apoptose , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Proteínas de Neoplasias , Doses de Radiação , SurvivinaRESUMO
PURPOSE: Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes. METHODS AND MATERIALS: Unstimulated G0 lymphocytes were pretreated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique. RESULTS: The results show that sodium butyrate pretreatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G0 lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations. CONCLUSIONS: Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB.
Assuntos
Ácido Butírico/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Animais , Células CHO , Cricetinae , Humanos , Técnicas In Vitro , Linfócitos/citologia , Linfócitos/metabolismo , Fase de Repouso do Ciclo CelularRESUMO
The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.
Assuntos
Cromossomos/efeitos dos fármacos , Hidrazida Maleica/toxicidade , Piridazinas/toxicidade , Animais , Medula Óssea/efeitos dos fármacos , Ciclo Celular , Linhagem Celular , Aberrações Cromossômicas , Cricetinae , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Camundongos , Troca de Cromátide Irmã/efeitos dos fármacosRESUMO
An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.
Assuntos
Carcinógenos , Mutagênicos , Troca de Cromátide Irmã , Animais , Biotransformação , Carcinógenos/farmacocinética , Linhagem Celular , Cricetinae , Cricetulus , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Testes de MutagenicidadeRESUMO
Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency. The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Aberrações Cromossômicas/efeitos dos fármacos , Etoposídeo/farmacologia , Linfócitos/efeitos dos fármacos , Tiazóis/farmacologia , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Benzotiazóis , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Técnicas In Vitro , Cariotipagem , Linfócitos/metabolismo , Fatores de Tempo , Tolueno/análogos & derivados , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: During convective techniques, a replacement fluid (R) is necessary that is sterile and pyrogen-free. Using an integrated absorption cartridge, the ultrafiltrate (UF) can be "regenerated"; and used as R. This method is hemodiafiltration with reinfusion (HFR). This study aimed to evaluate the real UF composition after "regeneration" by the resin-charcoal integrated absorption cartridge. METHODS: In eight uremic patients treated with HFR the UF was evaluated at 5, 15, 30, 60, 120, 180 and 240 min after HFR start at the inlet and the outlet resin-charcoal cartridge using the following parameters: urea, creatinine (Cr), uric acid, phosphates, glucose, Beta 2-microglobulin (beta2-m), Na+, K+, Ca++, pH, pCO2, and HCO3-. RESULTS: Blood (%): urea -61.2 +/- 9.7; Cr -55.4 +/- 8.1; uric acid -69.8 +/- 9.3; phosphates -31.8 +/- 15.7; glucose -8.4 +/- 20.5; Beta2-m -60.3 +/- 11.1; pH +0.76 +/- 0.58; pCO2+ 3.3 +/- 8.5; HCO3- +18.1 +/- 13.5. In UF (outlet vs inlet): urea was not adsorbed; Cr and uric acid were adsorbed; phosphates were not adsorbed; glucose was partially adsorbed (only in the 1st 90 min); Beta2-m was almost totally adsorbed; Na+ and K+ were not adsorbed; for pH, pCO2, and HCO3- there were no significant variations between the inlet and the outlet. CONCLUSIONS: HFR seems to be an easy-to-perform hemodiafiltration (HDF) technique, capable of resolving the typical problems of availability and the production of sterile and ultrapure reinfusion solution.
Assuntos
Hemodiafiltração/métodos , Soluções para Hemodiálise/administração & dosagem , Uremia/terapia , Gasometria , Feminino , Soluções para Hemodiálise/análise , Humanos , Masculino , Pessoa de Meia-Idade , Uremia/sangueRESUMO
Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges (ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment, in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due to structural restrictions within the diplochromosome.
Assuntos
Cromossomos/fisiologia , Troca de Cromátide Irmã , Animais , Bromodesoxiuridina , Linhagem Celular , Cromossomos/efeitos dos fármacos , Cromossomos/ultraestrutura , Colchicina/farmacologia , Cricetinae , Cricetulus , Diploide , Embrião de Mamíferos , Pulmão , Metáfase , Mitomicina/farmacologia , Modelos Genéticos , Troca de Cromátide Irmã/efeitos dos fármacosRESUMO
Human peripheral blood G(0) lymphocytes were X-irradiated and allowed to recover for different periods both in the presence and absence of phytohemagglutinin (PHA). For each experimental condition the induction of apoptosis was investigated by nuclear morphology and formation of both DNA laddering and high molecular weight DNA fragments by pulsed field gel electrophoresis. The results showed that PHA cell growth stimulation could rescue peripheral blood lymphocytes (PBLs) from X-ray-induced apoptotic cell death. Instead, most X-irradiated lymphocytes held in G(0) phase, once they were committed to apoptosis, inexorably executed the process. These data indicate that the proliferative status of PBLs can influence apoptotic cell death: PHA-stimulated PBLs appear to be more radioresistant as a result of a less efficient apoptotic process. Therefore, in current tests for mutagenicity or cytotoxicity and in biodosimetrical studies the possible role of apoptosis has to be considered.
Assuntos
Apoptose/efeitos da radiação , Linfócitos/efeitos da radiação , Fito-Hemaglutininas/farmacologia , Divisão Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Fragmentação do DNA/efeitos da radiação , Humanos , Ativação Linfocitária/efeitos da radiação , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Raios XRESUMO
Peripheral blood lymphocytes from normal human volunteers or from Down syndrome patients were pre-treated with sodium butyrate (a compound which is known to induce structural modifications in the chromatin through hyperacetylation of nucleosomal core histones) and exposed to X-irradiation or treated with bleomycin in vitro in the G0 and/or G1 stage(s) of the cell cycle. The frequencies of chromosomal aberrations in the first mitosis after treatment were scored. The results show an enhancement in the yield of aberrations in the butyrate pre-treated groups. However, the absolute frequencies of chromosomal aberrations as well as the relative increases with butyrate pre-treatment varied between blood samples from different donors suggesting the existence of inter-individual variations. There is a parallelism between the effects of X-irradiation or of combined treatments in G0 and G1 stages and between effects observed in the X-ray and bleomycin series. The increase in the yields of chromosomal aberrations in butyrate-treated and X-irradiated lymphocytes (relative to those which received X-irradiation alone) is interpreted as a consequence of the inhibition of repair of DNA damage by butyrate.
Assuntos
Bleomicina/farmacologia , Butiratos/farmacologia , Aberrações Cromossômicas , Síndrome de Down/genética , Ácido Butírico , Células Cultivadas , Cromossomos/efeitos dos fármacos , Cromossomos/efeitos da radiação , Humanos , Interfase , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiaçãoRESUMO
Cytogenetic and molecular analyses were performed in G1 Chinese hamster ovary cells treated with 254 nm UV light, to test the hypothesis that UV may affect the recognition site of specific restriction endonucleases (RE). Since short-wavelength UV light induces mainly cyclobutane dimers (CPD) at TT and CT sequences, RE were selected according to the presence or absence of thymine at the recognition site (DraI TTT/AAA, AluI AG/CT and HaeIII GG/CC). A drastic reduction of DraI-and AluI-induced chromosome-type aberrations was found in cells pretreated with UV. Conversely, such a reduction was not observed with HaeIII. To better understand this phenomenon, a molecular analysis was carried out at both the genome level and at the hypoxanthine phosphoribosyl transferase gene level, showing that the cutting pattern of DraI on isolated DNA from UV-irradiated cells was strongly reduced compared with an untreated sample, whereas HaeIII was not able to modify the cutting pattern of irradiated cells. Our data demonstrate a good correlation between the results obtained with cytogenetic and molecular approaches, suggesting that cyclobutane dimers are the main lesions responsible for the observed reduction of the cleaving activities of RE, at both the chromatin and naked DNA levels.
Assuntos
Cromossomos/efeitos da radiação , Dano ao DNA/efeitos da radiação , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Raios Ultravioleta , Animais , Sítios de Ligação , Southern Blotting , Células CHO/metabolismo , Células CHO/efeitos da radiação , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , Aberrações Cromossômicas , Cricetinae , DNA/química , DNA/efeitos da radiação , Eletroforese em Gel de ÁgarRESUMO
We investigated the contribution of the global and the transcription-coupled nucleotide excision repair pathway to the removal of structurally different DNA lesions. The repair kinetics of UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) were determined in an active and inactive gene in normal human fibroblasts and in xeroderma pigmentosum group C (XP-C) fibroblasts. Previously we have shown that in normal human cells exposed to a UV dose of 10 J/m2 repair of CPDs takes place via two pathways: global repair and transcription-coupled repair, the latter being responsible for accelerated repair of CPDs in the transcribed strand of active genes. So far, no clear evidence for transcription-coupled repair of 6-4PPs has been presented. Here we demonstrate that 6-4PPs really form a target for transcription-coupled repair. In XP-C cells, exposed to 30 J/m2 and only capable of performing transcription-coupled repair, CPDs as well as 6-4PPs are removed selectively and with similar kinetics from the transcribed strand of the adenosine deaminase (ADA) gene. The non-transcribed strand of the ADA gene and the inactive 754 gene are hardly repaired. In contrast to XP-C cells, normal cells exposed to 30 J/m2 lack strand-specific repair of both 6-4PPs and CPDs, suggesting that transcription-coupled repair is overruled by global repair, probably due to severe inhibition of transcription at this high UV dose. The much more rapid repair of 6-4PPs compared with CPDs in normal cells may be related to higher affinity of the global repair system for the former lesion. In XP-C cells the similarity of the rate of repair of both 6-4PPs and CPDs in the transcribed strand at 30 J/m2 indicates that transcription-coupled repair of photolesions takes place in a sequential way. Our results strongly suggest that the significance of transcription-coupled repair for removal of lesions depends on the type of lesion and on the dose employed.
Assuntos
Reparo do DNA , DNA/genética , Proteínas de Escherichia coli , Dímeros de Pirimidina , Transcrição Gênica , Xeroderma Pigmentoso/genética , Células Cultivadas , DNA/efeitos da radiação , Endodesoxirribonucleases/metabolismo , Fibroblastos/metabolismo , Humanos , Fotoquímica , Raios Ultravioleta , Xeroderma Pigmentoso/patologiaRESUMO
BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is the most common porokeratosis and is characterized by multiple keratotic lesions which tend to occur at sun-exposed sites. A mild hypersensitivity to X-rays has been reported for DSAP-derived fibroblasts and frequent over-expression of p53 has been found in lesional epidermis. OBJECTIVES: In order to clarify whether genome maintenance mechanisms might be compromised in this disease the following approaches were undertaken: (i) primary cultured keratinocytes and fibroblasts from DSAP patients were characterized for ultraviolet (UV) B and X-ray response; (ii) 15 lesions were studied for p53 mutations, and (iii) the differentiation status of DSAP-derived keratinocytes was evaluated. METHODS: Primary cultures of keratinocytes and fibroblasts were established from lesional and nonlesional skin biopsies of two subjects with DSAP. p53 mutations were analysed by DNA sequencing of the conserved region of the TP53 gene. Differentiation was evaluated both in stratified epithelial sheets from confluent keratinocyte cultures and in organotypic skin cultures. RESULTS: The cytotoxic and apoptotic response to UVB or X-irradiation was similar in DSAP-derived keratinocytes and fibroblasts when compared with normal cells. Two of 15 lesions examined presented p53 mutations located at nondipyrimidine sites. A strikingly decreased expression of filaggrin was observed both in reconstructed epidermis and in reconstructed skin. CONCLUSIONS: The UVB and X-ray response of DSAP-derived keratinocytes and fibroblasts indicates that the actinic character of this skin pathology is not due to radiation hypersensitivity. In agreement with this finding, mutations in the p53 gene, which are often associated with UV-related skin carcinogenesis, were rarely detected in DSAP lesions and were not UV-specific. Reconstructed epidermis and reconstructed skin models successfully reproduced the main features of this genodermatosis, showing that DSAP-derived keratinocytes bear an inherent defect in the terminal differentiation programme.
Assuntos
Epiderme/efeitos da radiação , Poroceratose/patologia , Raios Ultravioleta , Adulto , Idoso , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Criança , Relação Dose-Resposta à Radiação , Epiderme/patologia , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Proteínas Filagrinas , Genes p53 , Humanos , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Pessoa de Meia-Idade , Mutação , Poroceratose/genéticaRESUMO
Ultraviolet (UV)-induced repair and mutational spectra were analyzed in an inducible marker gene, the metallothionein-l/guamine-xanthine phosphoribosyl transferase (gpt) fusion gene, carried by an Epstein-Barr virus-derived shuttle vector episomically maintained in human cells. The repair rate of UV photodimers from the shuttle-vector molecules was typical of transcriptionally active sequences, 70% of the dimers being removed within 8 h after irradiation. The spectrum obtained under basal gene transcription was compared with that obtained under induced transcription. In both cases, base substitutions at dipyrimidine sequences predominated. Multiple mutations and deletions probably due to recombinational events induced by UV damage were also observed. Most of the UV-mutated dipyrimidine sites were located in the transcribed strand and were independent of the transcriptional activity of the target gene. In contrast, the distribution of mutations throughout the coding region of the gpt gene was affected by transcription, with a preferential clustering of mutations occurring in the 3' half of the gene after transcription induction. The strand bias observed in the UV spectra most likely reflects selection for nonfunctional gpt protein.