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Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.
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Fenda Labial , Fissura Palatina , Displasia Ectodérmica , Fenda Labial/diagnóstico , Fissura Palatina/diagnóstico , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Humanos , Inibidores da Agregação Plaquetária , Células-TroncoRESUMO
Ionising radiation damages DNA directly and indirectly through increased production of reactive oxygen species. Although telomeres have been reported as indicators of radiosensitivity, their maintenance in response to occupational exposure to low radiation doses is still a matter of debate. In this work we aimed to investigate telomere length and structure in hospital workers occupationally exposed to X-rays and to relate these findings to oxidation of biomolecules and chromosome aberrations. Blood samples of exposed participants and matching controls were taken during periodical check-ups. Chromosome aberrations and telomere length and structure were analysed in peripheral blood lymphocytes using Q-FISH, whereas oxidative stress parameters [pro/antioxidant balance (PAB), lipid peroxidation, and 8-oxo-dG] were measured in plasma samples. Based on the CA findings we divided the exposed group into two subgroups, of which one had chromosome aberrations in the first division metaphases and the other did not. There was no significant difference in telomere length between any of the groups. However, both subgroups showed significantly higher rate of fragile telomeres and higher lipid peroxidation product and 8-oxo-dG levels than controls. The rate of fragile telomeres significantly correlated with plasma levels of 8-oxo-dG, which suggests that continuous exposure to low radiation doses induces oxidative base damage of guanine resulting in telomere fragility.
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Exposição Ocupacional , Radiologia , 8-Hidroxi-2'-Desoxiguanosina , Aberrações Cromossômicas , Humanos , Exposição Ocupacional/efeitos adversos , Radiação Ionizante , TelômeroRESUMO
BACKGROUND: Gentiana lutea (GL), commonly known as yellow gentian, bitter root, and bitterwort, belongs to family Gentianaceae. GL belongs to genus Gentiana, which is a rich natural source of iridoids, secoiridoids, xantones, flavonoids, triterpenoids, and carbohydrates. Medicinal plants from Gentiana species have anti-oxidant, anti-inflammatory, anti-mitogenic, anti-proliferative, and lipidlowering effects, as well as a cardioprotective, hypotensive, vasodilator and anti-platelet activities. OBJECTIVE: We reviewed the recent literature related to the effects of Gentiana species, and their active components on vascular diseases. METHODS: Data used for this review were obtained by searching the electronic database [PUBMED/ MEDLINE 1973 - February 2020]. The primary data search terms of interest were: Gentiana lutea, Gentienacea family, phytochemistry, vascular diseases, treatment of vascular diseases, antioxidant, anti-inflammatory, anti-atherogenic. CONCLUSION: Gentiana species and their constituents affect many different factors related to vascular disease development and progression. Therefore, Gentiana-based therapeutics represent potentially useful drugs for the management of vascular diseases.
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Gentiana , Fitoterapia , Raízes de Plantas , Doenças Vasculares , Humanos , Resultado do Tratamento , Doenças Vasculares/tratamento farmacológicoRESUMO
BACKGROUND: Recurrent pregnancy loss (RPL) is a heterogeneous condition affecting up to 5% of women of reproductive age. Inherited thrombophilia have been postulated as one of the causes of RPL. Here we examined the prevalence of nine thrombophilic gene polymorphisms among women with history of recurrent miscarriages and fertile controls. METHODS: The study included 70 women with history of at least three early pregnancy losses and 31 fertile controls with no miscarriages. We investigated mutations in genes responsible for clotting and fibrinolysis, including factor V (FV) Leiden, FV H1299R, factor II (FII) G20210A, methylene tetrahydrofolate reductase (MTHFR) C677T and A1298C, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G and endothelial protein C receptor (EPCR) H1 and H3 haplotypes using reverse polymerase chain reaction ViennaLab cardiovascular disease StrippAssays. RESULTS: Our results showed no significant increase in prevalence of tested polymorphisms in women with RPL. However, relative risk for PRL among women heterozygous for FXIII V34L was 2.81 times increased (OR 2.81, 95% CI 1.15-6.87, P=0.023). Haplotype analysis showed that combined presence of high-risk genotypes for FXIII and PAI-1 significantly increases risk for RPL (OR 13.98, CI 95% 1.11-17.46, P=0.044). CONCLUSIONS: This is the first study in Serbian population that investigated prevalence of FVR2, A1298C, FXIII V34L and EPCR gene variants. Compound heterozygosity for FXIII V34L and PAI-1 4G is significant risk factor for recurrent miscarriage. Our results should be viewed in context of small case-control study, so further large prospective studies are need for confirmation of our findings.
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Yellow gentian (Gentiana lutea L.), a medicinal plant widely used in traditional medicine, displays multiple biological effects, ranging from beneficial to toxic. Since many promising applications have been reported so far, our aim was to evaluate its potential concentration- and time- dependent cytotoxic and genotoxic effects in vitro. To that end we exposed human peripheral blood mononuclear cells to 0.5, 1, and 2 mg/mL of yellow gentian root extract (YGRE) to determine its effects on oxidative stress parameters [pro/antioxidant balance (PAB) and lipid peroxidation], DNA damage (alkaline comet assay and chromosome aberrations), and cell viability (trypan blue exclusion test). Cell viability decreased with increasing concentrations and treatment duration. Only the lowest YGRE concentration (0.5 mg/mL) increased oxidative stress but produced minor DNA damage and cytotoxicity. At higher concentrations, redox parameters returned to near control values. The percentage of chromosome aberrations and percentage of DNA in the comet tail increased with increased YGRE concentration after 48 h and declined after 72 h of treatment. This points to the activation of DNA repair mechanism (homologous recombination), evidenced by the formation of chromosomal radial figures after 72 h of treatment with the highest YGRE concentration of 2 mg/mL. Our results suggest that YGRE, despite induction of cytotoxic and genotoxic effects, activates cell repair mechanisms that counter oxidative and DNA lesions and induce cell death in highly damaged cells. Therefore, observed protective effects of yellow gentian after longer exposure could be a result of activated repair and removal of cells with irreparable damage.
Assuntos
Gentiana , Leucócitos Mononucleares , Extratos Vegetais , Ensaio Cometa , Dano ao DNA , Humanos , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND AND OBJECTIVES: The present study was undertaken to ascertain whether the modulatory effects of blueberries on cell proliferation induced by Swedish snus in the rat forestomach epithelium is mediated via abrogation of the PI3K/Akt/NFκB signaling axis that regulates cell fate decision. METHODS: The transcript and protein expression of genes involved in cell cycle progression and apoptosis, as well as canonical PI3K/Akt/NF-κB signaling pathways, were analyzed by qRT-PCR, immunoblotting and ELISA. Expression profiling of noncoding RNAs (ncRNAs) that influence PI3K/Akt/NF-κB signaling was undertaken. TUNEL assay was performed using flow cytometry. RESULTS: Administration of snus induced basal cell hyperplasia in the rat forestomach with increased cell proliferation and inhibition of apoptosis. This was associated with the activation of PI3K/Akt/NFκB signaling. Coadministration of blueberries significantly suppressed snus-induced hyperplasia. Analysis of the molecular mechanisms revealed that blueberries suppress the phosphorylation of Akt, NF-κB and IKKß, prevent nuclear translocation of NF-κB and modulate the expression of microRNAs that influence PI3K/Akt/NF-κB signaling. CONCLUSION: Taken together, the results of the current study provide compelling evidence that blueberries exert significant protective effects against snus-induced soft tissue changes in the rat forestomach epithelium mediated by inhibiting key molecular players in the PI3K/Akt/NF-κB signaling axis. Long-term studies on the impact of snus exposure on various cellular processes, signaling pathways, and the interplay between genetic and epigenetic mechanisms are however warranted. The results of this investigation may contribute to the development of protection against soft tissue changes induced by smokeless tobacco in the human oral cavity.
Assuntos
Mirtilos Azuis (Planta)/química , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estômago/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Substâncias Protetoras/química , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Suécia , Tabaco sem Fumaça/efeitos adversosRESUMO
BACKGROUND: Obesity, diabetes, and associated diseases are increasing all over the world, and pose a great burden on public health. According to the latest reports, 440 million people are suffering from diabetes. Diabetes is caused by impaired ability to produce or respond to the hormone insulin consequently resulting in hyperglycemia. METHODS: Data used for this review was obtained by using PUBMED/MEDLINE (1987-2018). The main data search terms were: Gentiana lutea, Gentiana lutea extract, Gentiana lutea constituents, obesity, diabetes mellitus, diabetic complications. RESULTS: In the present review, we describe the potential of root powder of yellow gentian (Gentiana lutea) for the prevention of obesity and diabetes including complications related to this disease. CONCLUSION: Reasonably effective, low-cost alternatives could fulfill an important role for a large part of the human population and could be of great value for the food market. Even a modest reduction of morbidity and mortality with respect to this disease translates into millions of lives saved.
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Gentiana/química , Obesidade/tratamento farmacológico , Fitoterapia , Diabetes Mellitus/tratamento farmacológico , Humanos , Raízes de Plantas/químicaRESUMO
Long term exposure to oral smokeless tobacco may induce lesions in the oral cavity characterized by a hyperplastic epithelium. The possible role of nicotine and the physical properties of oral tobacco for developing these lesions, as well as of dysplasia and neoplasia is unclear. Low nitrosamine Swedish snus as well as non-genotoxic butylated hydroxyanisole induces increased cellular proliferation in the rat forestomach epithelia. Using this model, we report here on the effects of nicotine, pH, and particle size. Snus with different properties had no impact on oxidative stress as determined by 8-oxo-7,8-dihydro-2'-deoxyguanosine, or on interleukin IL-1b. Whereas BHA boosted IL-6, probably due to the presence of nicotine. there was no significant enhancement of cell divisions with increasing particle size, although in individual samples the variations in proliferation rates increased greatly with increasing particle size. Conforming to human experience, the enhanced cell proliferation caused by snus was found to be completely reversible. A cacao bean extract had a protective action similar to that previously found for blueberries. The main cause of the observed tobacco induced cell proliferation could be mechanical irritation, possibly in combination with nicotine, whereas within the studied range, pH did not affect the rate of cell division.
Assuntos
Hiperplasia/induzido quimicamente , Nicotina/toxicidade , Estômago/efeitos dos fármacos , Tabaco sem Fumaça/toxicidade , Administração Oral , Animais , Proliferação de Células/efeitos dos fármacos , Hiperplasia/patologia , Masculino , Nicotina/administração & dosagem , Ratos , Ratos Wistar , Estômago/patologia , SuéciaRESUMO
BACKGROUND: Fanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization. RESULTS: We found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage. CONCLUSIONS: FA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value.
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Deletions within chromosome 11q22-23, are considered among the most common chromosomal aberrations in chronic lymphocytic leukemia (CLL), and are associated with a poor outcome. In addition to the ataxia telangiectasia mutated (ATM) gene, the baculoviral IAP repeat-containing 3 (BIRC3) gene is also located in the region. BIRC3 encodes a negative regulator of the non-canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) protein. Disruption of BIRC3 is known to be restricted to CLL fludarabine-refractory patients. The aim of the present study was to determine the frequency of copy number changes of BIRC3 and to assess its association with two known predictors of negative CLL outcome, ATM and tumor protein 53 (TP53) gene deletions. To evaluate the specificity of BIRC3 alterations to CLL, BIRC3 copy numbers were assessed in 117 CLL patients in addition to 45 B-cell acute lymphocytic leukemia (B-ALL) patients. A commercially available multiplex ligation dependent probe amplification kit, which includes four probes for the detection of TP53 and four probes for ATM gene region, was applied. Interphase-directed fluorescence in situ hybridization was used to apply commercially available probes for BIRC3, ATM and TP53. High resolution array-comparative genomic hybridization was conducted in selected cases. Genetic abnormalities of BIRC3 were detected in 23/117 (~20%) of CLL and 2/45 (~4%) of B-ALL cases. Overall, 20 patients with CLL and 1 with B-ALL possessed a BIRC3 deletion, whilst 3 patients with CLL and 1 with B-ALL harbored a BIRC3 duplication. All patients with an ATM deletion also carried a BIRC3 deletion. Only 2 CLL cases possessed deletions in BIRC3, ATM and TP53 simultaneously. Evidently, the deletion or duplication of BIRC3 may be observed rarely in B-ALL patients. BIRC3 duplication may occur in CLL patients, for which the prognosis requires additional studies in the future. The likelihood that TP53 deletions occur simultaneously with BIRC3 and/or ATM aberrations is low. However, as ATM deletions may, but not always, associate with BIRC3 deletions, each region should be considered in the future diagnostics of CLL in order to aid treatment decisions, notably whether to treat with or without fludarabine.
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In this work we present biological effects of silver nanoparticles (AgNPs) synthesized by picosecond laser ablation of silver in deionized water. We examined induction of chromosomal aberrations, lymphocyte micronuclei, appearance and recovery of double strand breaks (DSBs) of DNA, cell proliferation potential, concentration of lipid peroxidation products and insulin-like growth factor 1 (ILGF-1). We found that AgNPs sized from 3 nm to 8 nm induce cell cytostasis, which is accompanied with its clastogenic action on DNA, while AgNPs, sized 2 nm behaves contrary stimulating cell proliferation by enhancing ILGF-1 concentration.
Assuntos
Proliferação de Células/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Tamanho da Partícula , Prata/toxicidade , Aberrações Cromossômicas/efeitos dos fármacos , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Testes para Micronúcleos , Mutagênicos/toxicidade , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The aim of this study was to identify palatable additives which have a significant protective action against soft tissue changes in the oral cavity caused by Swedish smokeless tobacco ("snus"), and that satisfy existing legal requirements. Although the cancer risk from snus is extremely low, long term use may result in highly undesirable keratotic lesions and associated epithelial abnormalities in the oral cavity. The rat forestomach, which is vulnerable to the irritative action of non-genotoxic compounds like butylated hydroxyanisole, propionic acid as well as snus, was chosen as an experimental model. Studied toxicological endpoints included histopathology and cellular proliferation based on DNA incorporation of bromodeoxyuridine. After 6 weeks' exposure, blueberries (bilberries) and an extract from the common milk thistle were found to exert a highly significant inhibition of cell proliferation induced by snus in the rat forestomach epithelium, indicating a potential protection with respect soft tissue changes in the human oral cavity.
Assuntos
Mirtilos Azuis (Planta)/química , Proliferação de Células/efeitos dos fármacos , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Silybum marianum/química , Silimarina/farmacologia , Estômago/efeitos dos fármacos , Tabaco sem Fumaça/toxicidade , Animais , Citoproteção , Replicação do DNA/efeitos dos fármacos , Frutas , Hiperplasia , Masculino , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Substâncias Protetoras/isolamento & purificação , Ratos Wistar , Silimarina/isolamento & purificação , Estômago/patologia , Fatores de TempoRESUMO
Genomic instability tends to occur at specific genomic regions known as common fragile sites (FS). FS are evolutionarily conserved and generally involve late replicating regions with AT-rich sequences. The possible correlation between some FS and cancer-related breakpoints emphasizes on the importance of understanding the mechanisms of chromosomal instability at these sites. Although about 230 FS have already been mapped cytogenetically, only a few of them have been characterized on a molecular level. In this chapter, we provide a protocol for mapping of common FS using bacterial artificial chromosome (BAC) probes in fluorescence in situ hybridization (FISH) and suggest the usage of lymphocytes from Fanconi anemia patients as a model system. In the latter, rare FS are expressed much more frequently than in, for example, aphidicolin-induced blood lymphocyte preparations. Knowing the exact location of FS enables the molecular comparison of their location and breakpoints that appear during evolution, cancer development and inherited disorders.
Assuntos
Sítios Frágeis do Cromossomo , Cromossomos Artificiais Bacterianos/química , Anemia de Fanconi/genética , Genoma Humano , Hibridização in Situ Fluorescente/métodos , Sondas Moleculares/química , Afidicolina/toxicidade , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Instabilidade Genômica , Humanos , Linfócitos/química , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Sondas Moleculares/genética , Mutagênicos/toxicidadeRESUMO
Na(+)/K(+)-ATPase is in charge of maintaining the ionic and osmotic intracellular balance by using ATP as an energy source to drive excess Na(+) ions out of the cell in exchange for K(+) ions. We explored whether three representative cytotoxic gold(III) compounds might interfere with Na(+)/K(+)-ATPase and cause its inhibition at pharmacologically relevant concentrations. The tested complexes were [Au(bipy)(OH)2][PF6] (bipy=2,2'-bipyridine), [Au(py(dmb)-H)(CH3COO)2] (py(dmb)-H=deprotonated 6-(1,1-dimethylbenzyl)-pyridine), and [Au(bipy(dmb)-H)(OH)][PF6] (bipy(dmb)-H=deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine). We found that all of them caused a pronounced and similar inhibition of Na(+)/K(+)-ATPase activity. Inhibition was found to be non-competitive and reversible. Remarkably, treatment with cysteine resulted in reversal or prevention of Na(+)/K(+)-ATPase inhibition. It is very likely that the described effects may contribute to the overall cytotoxic profile of these gold complexes.
Assuntos
Antineoplásicos/farmacologia , Ouro/química , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Antineoplásicos/química , Humanos , Técnicas In Vitro , Cinética , Linfócitos/efeitos dos fármacos , Masculino , Testes para Micronúcleos , Espectrofotometria UltravioletaRESUMO
PURPOSE: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells. MATERIALS AND METHODS: Analyses of phosphorylated histone H2AX (γ-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h). RESULTS: The accumulation of γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were γ-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as γ-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis. CONCLUSION: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Mitose/efeitos da radiação , Núcleo Celular/efeitos da radiação , Células Cultivadas , Criança , Citocinese/genética , Citocinese/efeitos da radiação , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Histonas/metabolismo , Humanos , Fosforilação/efeitos da radiação , Tolerância a Radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.
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Proliferação de Células/efeitos dos fármacos , Gentiana/química , Extratos Vegetais/farmacologia , Animais , Apigenina/farmacologia , Becaplermina , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinase Quinase 1/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Raízes de Plantas/química , Proteínas Proto-Oncogênicas c-sis/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Carbon nanotubes are unique one-dimensional macromolecules with promising application in biology and medicine. Since their toxicity is still under debate, here we describe an investigation of genotoxic properties of purified single-walled carbon nanotubes (SWCNT), multiwall carbon nanotubes (MWCNT), and amide-functionalized purified SWCNT. We used two different cell systems: cultured human lymphocytes where we employed cytokinesis-block micronucleus test and human fibroblasts where we investigate the induction of DNA double-strand breaks (DSBs) employing H2AX phosphorylation assay.
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Testes de Mutagenicidade , Nanotubos de Carbono/toxicidade , Linhagem Celular , HumanosRESUMO
The in vitro influence of gold(III) complexes, H[AuCl4], [Au(DMSO)2Cl2]Cl and [Au(bipy)Cl2]Cl (bipy = 2,2'-bipyridine), upon commercially available Na(+)/K(+) ATPase activity, purified from porcine brain cortex, was investigated. Additionally, the complexes were tested on human lymphocytes, and incidence of micronuclei and cell proliferation index was determined. Concentration-dependent inhibition of the enzyme for all three compounds was obtained, but with differing potencies. Calculated IC50 from Hill analysis were (in M): 5.75×10(-7), 5.50×10(-6) and 3.98×10(-5), for H[AuCl4], [Au(DMSO)2Cl2]Cl and [Au(bipy)Cl2]Cl, respectively, while Hill coefficient values, n, were above 1 in all cases. This inhibition can be prevented using -SH donating ligands such as L-Cys and glutathione, and these ligands can also cause a recovery of the enzyme activity after the induced inhibition. Kinetic analysis demonstrated that each of the studied gold(III) complexes affects Na(+)/K(+) ATPase reducing maximum enzymatic velocity, Vmax, but not significantly changing the affinity for the substrate (KM value), implying a noncompetitive mode of the interaction. Furthermore, among investigated gold(III) complexes, the [Au(bipy)Cl2]Cl complex exhibits a strong cytotoxic effect on human lymphocytes, which suggests its potential for use in antitumor therapy.
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Proliferação de Células/efeitos dos fármacos , Citotoxinas/farmacologia , Compostos de Ouro/farmacologia , Linfócitos/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Linfócitos/citologia , Masculino , SuínosRESUMO
Exposure of the general population to polycyclic aromatic hydrocarbons (PAH) is ubiquitous. The aim of this study was to analyze biomarkers associated with the uptake of PAH in 428 non-smoking women from Lodz (Poland), Viterbo (Italy), Belgrade (Serbia) and from the Pancevo area, where the petrochemical complex was destroyed by the air raids in 1999. Urinary excretion of PAH metabolites was lowest in Italian women, intermediary for Serbian and highest in Polish women, who predominantly excreted hydroxy phenanthrenes as metabolites of phenanthrene. Bulky DNA adduct levels were highest in Italian and Polish women. Genotype or PAH ambient air levels could not explain the dissimilarities between the study groups with respect to biomarker patterns, which probably reflected differences in life style-associated factors.
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Dieta , Poluentes Ambientais/urina , Hidrocarbonetos Policíclicos Aromáticos/urina , Adulto , Biomarcadores/urina , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/genética , Adutos de DNA/sangue , Dano ao DNA , Poluentes Ambientais/farmacocinética , Poluentes Ambientais/toxicidade , Feminino , Frutas/química , Genótipo , Técnicas de Genotipagem , Humanos , Itália , Pessoa de Meia-Idade , Polônia , Hidrocarbonetos Policíclicos Aromáticos/farmacocinética , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Sérvia , Verduras/químicaRESUMO
BACKGROUND: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. RESULTS: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. CONCLUSION: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.