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1.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200473

RESUMO

The interactions of compounds with DNA have been studied since the recognition of the role of nucleic acid in organisms. The design of molecules which specifically interact with DNA sequences allows for the control of the gene expression. Determining the type and strength of such interaction is an indispensable element of pharmaceutical studies. Cognition of the therapeutic action mechanisms is particularly important for designing new drugs. Owing to their sensitivity, simplicity, and low costs, electrochemical methods are increasingly used for this type of research. Compared to other techniques, they require a small number of samples and are characterized by a high reliability. These methods can provide information about the type of interaction and the binding strength, as well as the damage caused by biologically active molecules targeting the cellular DNA. This review paper summarizes the various electrochemical approaches used for the study of the interactions between pharmaceuticals and DNA. The main focus is on the papers from the last decade, with particular attention on the voltammetric techniques. The most preferred experimental approaches, the electrode materials and the new methods of modification are presented. The data on the detection ranges, the binding modes and the binding constant values of pharmaceuticals are summarized. Both the importance of the presented research and the importance of future prospects are discussed.


Assuntos
DNA/química , Interações Medicamentosas/fisiologia , Técnicas Eletroquímicas/métodos , Preparações Farmacêuticas/química , Técnicas Biossensoriais/métodos , Dano ao DNA/efeitos dos fármacos , Eletrodos , Ácidos Nucleicos/química , Reprodutibilidade dos Testes
2.
Chem Biol Interact ; 346: 109580, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34280354

RESUMO

Dichloromethane (DCM), a widely used chlorinated solvent, is classified by IARC (2017) as probably carcinogenic to humans. Exposure to DCM has been associated with increased incidence of cholangiocarcinoma (CCA) in humans. This study aimed to investigate how DCM could contribute to CCA development by investigating the effects of DCM on DNA damage and cell transformation in cholangiocytes (MMNK-1) and on metastatic potential as measured by invasion and cell migration in malignant CCA cell lines (HuCCA-1 and RMCCA-1). MMNK-1 cells treated with the non-cytotoxic concentration of DCM (25 µM, 24 h) significantly increased the levels of mutagenic DNA adducts including 8-hydroxydeoxyguanosine, 8-OHdG, (1.84-fold, p < 0.01) and 8-nitroguanine (1.96-fold, p < 0.01) and enhanced cell transformation by 1.47-fold (p < 0.01). In addition, the expression of various genes involved in carcinogenesis, namely, NFE2L2 (antioxidative response), CXCL8 (inflammation), CDH1 (cell adhesion), MMP9 (tissue remodeling) and MKI67 (cell proliferation) were altered in cholangiocytes treated with DCM. When MMNK-1 cells were transformed by DCM, the expression of all the aforementioned genes was also increased. In malignant cell lines (HuCCA-1 and RMCCA-1), DCM treatment resulted in increased CXCL8 and MMP9 transcription and decreased CDH1 transcription accompanied by increased invasion and migration capabilities of these cells. Taken together, this study demonstrated that DCM exposure could be linked to the development of CCA.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Cloreto de Metileno/toxicidade , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Adutos de DNA/análise , Adutos de DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Cloreto de Metileno/química , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo
3.
Nat Commun ; 12(1): 4373, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272385

RESUMO

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Animais , Camptotecina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , RNA Interferente Pequeno , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação
4.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298882

RESUMO

Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.


Assuntos
Apoptose/fisiologia , Micropartículas Derivadas de Células/fisiologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Daunorrubicina/farmacologia , Células THP-1/fisiologia , Apoptose/efeitos dos fármacos , Plaquetas , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo
5.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202329

RESUMO

The interactions between pharmaceuticals and nanomaterials and its potentially resulting toxicological effects in living systems are only insufficiently investigated. In this study, two model compounds, acetaminophen, a pharmaceutical, and cerium dioxide, a manufactured nanomaterial, were investigated in combination and individually. Upon inhalation, cerium dioxide nanomaterials were shown to systemically translocate into other organs, such as the liver. Therefore we picked the human liver cell line HuH-7 cells as an in vitro system to investigate liver toxicity. Possible synergistic or antagonistic metabolic changes after co-exposure scenarios were investigated. Toxicological data of the water soluble tetrazolium (WST-1) assay for cell proliferation and genotoxicity assessment using the Comet assay were combined with an untargeted as well as a targeted lipidomics approach. We found an attenuated cytotoxicity and an altered metabolic profile in co-exposure experiments with cerium dioxide, indicating an interaction of both compounds at these endpoints. Single exposure against cerium dioxide showed a genotoxic effect in the Comet assay. Conversely, acetaminophen exhibited no genotoxic effect. Comet assay data do not indicate an enhancement of genotoxicity after co-exposure. The results obtained in this study highlight the advantage of investigating co-exposure scenarios, especially for bioactive substances.


Assuntos
Acetaminofen/efeitos adversos , Cério/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Nanopartículas , Acetaminofen/administração & dosagem , Transporte Biológico , Linhagem Celular Tumoral , Cério/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Metaboloma , Metabolômica/métodos , Nanopartículas/química , Tamanho da Partícula , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
J Dermatol Sci ; 103(1): 41-48, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34147320

RESUMO

BACKGROUND: Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. OBJECTIVE: To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. METHODS: The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. RESULTS: The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. CONCLUSION: Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.


Assuntos
Cálcio/metabolismo , Queratinócitos/efeitos da radiação , Fagocitose/efeitos da radiação , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Linhagem Celular , Quelantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melaninas/biossíntese , Melanossomas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/efeitos da radiação , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Espécies Reativas de Oxigênio/metabolismo
7.
Nutrients ; 13(6)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063915

RESUMO

The present report was designed to determine the antioxidant and antigenotoxic effects of phaseolin (isolated from Phaseolus vulgaris) against mouse colon and liver damage induced by azoxymethane (AOM) and its colon chemopreventive effect. Eight groups with 12 mice each were utilized for an eight-week experiment: the control group was intragastrically (ig) administered 0.9% saline solution; the positive control group was intraperitoneally (ip) injected with 7.5 mg/kg AOM twice a week (weeks three and four of the experiment); three groups were ig administered each day with phaseolin (40, 200, and 400 mg/kg); and three groups were ig administered phaseolin daily (40, 200, and 400 mg/kg) plus 7.5 mg/kg AOM twice a week in weeks three and four of the experiment. The results showed that phaseolin did not produce oxidative stress, DNA damage, or aberrant crypts; in contrast, 100% inhibition of lipoperoxidation, protein oxidation, and nitrites induction generated by AOM was found in both organs, and DPPH radical capture occurred. The two highest phaseolin doses reduced DNA damage induced by AOM in both organs by more than 90% and reduced the AOM-induced aberrant crypts by 84%. Therefore, our study demonstrated the strong in vivo antioxidant, antigenotoxic, and chemopreventive potential of phaseolin.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colite/prevenção & controle , Phaseolus/química , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Focos de Criptas Aberrantes/prevenção & controle , Animais , Antioxidantes , Azoximetano , Quimioprevenção , Colite/induzido quimicamente , Colo , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sementes/química
8.
Mol Cell ; 81(14): 3018-3030.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34102106

RESUMO

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.


Assuntos
Reparo do DNA/genética , DNA/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Animais , Linhagem Celular , Quebras de DNA de Cadeia Simples , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos
9.
Methods Mol Biol ; 2326: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097270

RESUMO

Cyantraniliprole can effectively control lepidopteran pests and has been used all over the world. In general, the risk of cyantraniliprole seems low for fish, but the toxicity selectivity among different fish species was not clear. Here, we present the methods for the acute toxicity and chronic effects of cyantraniliprole by using juvenile tilapia (Oreochromis mossambicus). Based on this test, 96 h LC50 of cyantraniliprole to tilapia was 38.0 mg/L. After exposed for 28 days, specific growth rates of the blank control, solution control, and the treatments of 0.037, 0.37 and 3.7 mg/L of cyantraniliprole were 1.14, 0.95, 0.93, 0.82, and 0.70% per day, respectively. The results of micronucleus experiment and single cell gel electrophoresis showed that cyantraniliprole damaged DNA in liver cells of tilapia larvae. Quantitative PCR results showed that cyantraniliprole could induce the upregulation of Rpa 3 that is responsible for the DNA repair. The significant downregulation of Chk 2 gene was related to p53 pathway. It is therefore proposed that cyantraniliprole causes DNA damage in liver cells of tilapia and activates DNA damage and repair pathways.


Assuntos
Dano ao DNA/efeitos dos fármacos , Inseticidas/toxicidade , Pirazóis/toxicidade , Tilápia , Poluentes Químicos da Água/toxicidade , ortoaminobenzoatos/toxicidade , Animais , Larva/citologia , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Testes para Micronúcleos/métodos , Análise de Célula Única/métodos , Tilápia/crescimento & desenvolvimento , Tilápia/metabolismo , Testes de Toxicidade/métodos
10.
Exp Parasitol ; 226-227: 108121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097889

RESUMO

Cystic echinococcosis (CE), a parasitic larval cystic stage of a small taeniid-type tapeworm (Echinococcus granulosus), causes illness in intermediate hosts and has become a threat to global public health. Currently, chemical compounds recommended by the WHO targeting CE are albendazole and mebendazole, however, none of them shows enhanced efficacy. Novel molecular compounds are urgently required to treat this disease. Our group uncover a drug, termed harmine (HM), that may be capable of treating CE. In this study, we aim to evaluate the anti-parasitic efficacy and the mechanism of DNA damage of HM against E. granulosus. In vitro, the results indicated that, within two and three days of treatment, ABZ killed 30.4% and 35.3% of protoscoleces, whereas HM killed 52.7% and 100% of protoscoleces, respectively. Furthermore, the presence of abnormalities in the internal structure of protoscoleces was examined by ultrastructural images of TEM, and the result showed that there were scattered nucleoli and heterochromatin margination phenomenon by HM treatment. DNA damage of protoscoleces was examined by using the comet assay, and results showed the DNA of protoscoleces was damaged. Moreover, EgATM, EgP53, EgTopo2a and EgRad54 genes were used to support the DNA damage by HM treatment, and results showed that all four genes were upregulated expression. In further, the result of HM treatment was tested by using designed siRNA to inhibit the expression of EgTopo2a and EgRad54. The results demonstrated that the viability was 88.75 ± 2.11% after suppressing the expression of EgTopo2a, which was significantly higher than that for HM alone group (P < 0.01). The viability was 10.11 ± 2.60% after transfected with EgRad54 siRNA, which was significantly lower compared with the HM alone group (P < 0.01). Based on our preliminary data, HM demonstrated significant parasiticidal activity against E. granulosus in vitro without obvious toxicity towards its host cells, suggesting that HM can be a potential anti-echinococcosis drug. HM was found to induce DNA damages of CE by activating the EgATM-EgP53-EgTopo2a signaling pathway. We therefore surmise that DNA damage response may be one of the mechanisms of HM against the parasite.


Assuntos
Antiparasitários/farmacologia , Dano ao DNA/efeitos dos fármacos , Equinococose/tratamento farmacológico , Echinococcus granulosus/efeitos dos fármacos , Harmina/farmacologia , Animais , Antiparasitários/uso terapêutico , Ensaio Cometa , Echinococcus granulosus/genética , Echinococcus granulosus/ultraestrutura , Harmina/uso terapêutico , Microscopia Eletrônica de Transmissão , Inibidores da Monoaminoxidase/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Ovinos
11.
Nat Commun ; 12(1): 3636, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140467

RESUMO

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ratos , Mutações Sintéticas Letais/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
12.
Expert Opin Drug Metab Toxicol ; 17(7): 857-865, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34102941

RESUMO

BACKGROUND: Acrylamide (AA) is a water-soluble toxic chemical that is considered one of the most important food contaminants. Furthermore, AA is considered a major public health risk. METHODS: This study was designed to evaluate the effects of AA on cytotoxicity, oxidative damage and genotoxicity in human lymphocytes and also to evaluate the protective effects of the chrysin (CH). Lymphocytes after isolation from the blood were treated with AA (50 µM), AA (50 µM) plus CH (10, 25, 50 µM) and CH (50 µM), and parameters such as cell viability, mitochondrial and lysosomal damage, as well as oxidative damage to DNA were examined. RESULTS: The results showed that CH was able to reduce cytotoxicity, reactive oxygen species (ROS) levels, lipid peroxidation (LPO) level, collapse in mitochondrial membrane potential (MMP) and oxidative damage of DNA caused by AA in human lymphocytes. Also, co-treatment of the AA-exposed human lymphocytes with CH increases the glutathione (GSH) levels. CONCLUSION: Results suggest that CH (10, 25, 50 µM) shows a protective role in AA-induced cytotoxicity, oxidative stress, mitochondrial damage and DNA oxidative damage.


Assuntos
Acrilamida/toxicidade , Flavonoides/farmacologia , Linfócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
13.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068728

RESUMO

To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.


Assuntos
Epitélio/efeitos dos fármacos , Nanopartículas Metálicas/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo/genética , Transcrição Genética/efeitos dos fármacos , Células A549 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Cobre/química , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
14.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071702

RESUMO

Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Genoma/efeitos dos fármacos , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
15.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065593

RESUMO

Interest in graphene oxide nature and potential applications (especially nanocarriers) has resulted in numerous studies, but the results do not lead to clear conclusions. In this paper, graphene oxide is obtained by multiple synthesis methods and generally characterized. The mechanism of GO interaction with the organism is hard to summarize due to its high chemical activity and variability during the synthesis process and in biological buffers' environments. When assessing the biocompatibility of GO, it is necessary to take into account many factors derived from nanoparticles (structure, morphology, chemical composition) and the organism (species, defense mechanisms, adaptation). This research aims to determine and compare the in vivo toxicity potential of GO samples from various manufacturers. Each GO sample is analyzed in two concentrations and applied with food. The physiological reactions of an easy model Acheta domesticus (cell viability, apoptosis, oxidative defense, DNA damage) during ten-day lasting exposure were observed. This study emphasizes the variability of the GO nature and complements the biocompatibility aspect, especially in the context of various GO-based experimental models. Changes in the cell biomarkers are discussed in light of detailed physicochemical analysis.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Grafite/química , Grafite/toxicidade , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Gryllidae/efeitos dos fármacos , Nanopartículas/química , Nanopartículas/toxicidade , Oxirredução/efeitos dos fármacos , Óxidos/metabolismo
16.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067339

RESUMO

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.


Assuntos
Apoptose/efeitos dos fármacos , Sangue Fetal/efeitos dos fármacos , Histonas/metabolismo , Linfócitos/efeitos dos fármacos , Toxinas Marinhas/farmacologia , Oxazóis/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Sangue Fetal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos/metabolismo , Microscopia de Fluorescência/métodos , Fosforilação/efeitos dos fármacos
17.
Nucleic Acids Res ; 49(10): 5779-5797, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048572

RESUMO

Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dano ao DNA , Reparo do DNA , Espécies Reativas de Oxigênio/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Desmetilação/efeitos dos fármacos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metilação/efeitos dos fármacos , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Sumoilação/efeitos dos fármacos , Espectrometria de Massas em Tandem , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
18.
Aging (Albany NY) ; 13(9): 12334-12358, 2021 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-33934090

RESUMO

Sirtuins have been shown to regulate the aging process. We have previously demonstrated that Sirt6 blocks the pressure overload-induced cardiac hypertrophy in mice. Here, we show that Sirt6 can also mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. We found that aging is associated with altered Sirt6 activity along with development of cardiac hypertrophy and fibrosis. Compared to young mice (4-months), the hearts of aged mice (24-months) showed increased levels of mitochondrial DNA damage, shortened telomere length, and increased accumulation of 8-oxo-dG adducts, which are hallmarks of aging. The aged hearts also showed reduced levels of NAD+ and altered levels of mitochondrial fusion-fission proteins. Similar characteristics were observed in the hearts of Sirt6 deficient mice. Additionally, we found that doxorubicin (Dox) induced cardiomyocyte senescence, as measured by expression of p16INK4a, p53, and ß-galactosidase, was associated with loss of Sirt6. However, Sirt6 overexpression protected cardiomyocytes from developing Dox-induced senescence. Further, compared to wild-type mice, the hearts of Sirt6.Tg mice showed reduced expression of aging markers, and the development of aging-associated cardiac hypertrophy and fibrosis. Our data suggest that Sirt6 is a critical anti-aging molecule that regulates various cellular processes associated with aging and protects the heart from developing aging-induced cardiac hypertrophy and fibrosis.


Assuntos
Envelhecimento/fisiologia , Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Sirtuínas/metabolismo , Animais , Cardiomegalia/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sirtuínas/genética , Encurtamento do Telômero
19.
J Med Chem ; 64(11): 7617-7629, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34008967

RESUMO

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or ++++), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 µM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.


Assuntos
Benzofenantridinas/química , DNA Topoisomerases Tipo I/metabolismo , Inibidores de Fosfodiesterase/síntese química , Diester Fosfórico Hidrolases/metabolismo , Inibidores da Topoisomerase I/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzofenantridinas/metabolismo , Benzofenantridinas/farmacologia , Benzofenantridinas/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/metabolismo , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase I/uso terapêutico
20.
Toxicology ; 457: 152806, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33961948

RESUMO

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1-100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.


Assuntos
Carcinógenos/toxicidade , Neoplasias Colorretais/metabolismo , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidade , Carcinógenos/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Neoplasias Colorretais/patologia , Dano ao DNA/fisiologia , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Necrose Tumoral alfa/administração & dosagem
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