RESUMO
Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.
Assuntos
Telefone Celular , Citocinese , Mucosa Bucal , Humanos , Mucosa Bucal/efeitos da radiação , Mucosa Bucal/citologia , Adulto , Masculino , Citocinese/efeitos da radiação , Morte Celular/efeitos da radiação , Adulto Jovem , Feminino , Aberrações Cromossômicas/efeitos da radiação , Testes para Micronúcleos , Campos Eletromagnéticos/efeitos adversos , Micronúcleos com Defeito Cromossômico/efeitos da radiaçãoRESUMO
Cervical cancer (CC) is the fourth most common cancer in women; the survival rates depend strongly on its early detection. The Pap test is the most frequently used diagnostic tool, but due to its limited sensitivity/specificity, additional screening tests are needed. Therefore, we evaluated the use of micronucleus (MN) assays with cervical cells for the prediction and diagnosis of CC. MN reflects structural and numerical chromosomal aberrations. A search was performed in Pubmed, Scopus, Thomson ISI and Google Scholar. Subsequently, meta-analyses were performed for different grades of abnormal findings in smears and biopsies from patients which were diagnosed with CC. Results of 21 studies in which findings of MN experiments were compared with data from Pap tests show that higher MN frequencies were found in women with abnormal cells that are indicative for increased cancer risks. MN frequency ratios increased in the order inflammation (2.1) < ASC-US and ASC-H (3.3) < LGSIL (4.4) < HGSIL (8.4). Furthermore, results are available from 17 investigations in which MN were scored in smears from patients with neoplasia. MN rates increased with the degree of neoplasia [CIN 1 (4.6) < CIN 2 (6.5) and CIN 3 (10.8)] and were significantly higher (8.8) in CC patients. Our meta-analysis indicates that the MN assay, which is easy to perform in combination with Pap tests, may be useful for the detection/prediction of CC. However, standardization (including definition of the optimal cell numbers and stains) and further validation is necessary before the MN test can be implemented in routine screening.
Assuntos
Testes para Micronúcleos , Neoplasias do Colo do Útero/diagnóstico , Feminino , Humanos , PrognósticoRESUMO
During the harvest period, tobacco workers are exposed to nicotine and it is known that absorption of the alkaloid via the leaves causes green tobacco sickness (GST). We investigated if GST and its symptoms are associated with DNA damage and alterations of the redox status. DNA damage was measured in lymphocytes of tobacco workers and controls (n = 40/group) in single cell gel electrophoresis assays. Exposure to nicotine was determined by plasma cotinine measurements, alterations of the redox status by quantification of the total antioxidant capacity (TEAC) and of thiobarbituric acid reactive substances (TBARS). The symptoms of GTS included nausea, abdominal cramps, headache, vomiting and dizziness, and 50% of the workers had more than one symptom. Cotinine levels were enhanced in the workers (111 ng/mL); furthermore, the extent of DNA damage was ca. 3-fold higher than in the controls. This effect was more pronounced in participants with GST compared to healthy nicotine exposed workers and increased in individuals with specific symptoms (range 22-36%). TBARS levels did not differ between workers and unexposed controls, while TEAC values were even increased (by 14.3%). Contact with nicotine present in tobacco leaves causes GTS and leads to damage of the DNA; this effect is more pronounced in workers with GTS symptoms and is associated with alterations of the redox status. Damage of the genetic material which was found in the workers may lead to adverse long-term effects that are caused by genomic instability such as cancer and accelerated ageing.
Assuntos
Doenças dos Trabalhadores Agrícolas/induzido quimicamente , Dano ao DNA , Fazendeiros , Nicotiana/crescimento & desenvolvimento , Nicotina/toxicidade , Exposição Ocupacional/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Doenças dos Trabalhadores Agrícolas/genética , Doenças dos Trabalhadores Agrícolas/metabolismo , Brasil , Estudos de Casos e Controles , Cotinina/sangue , Feminino , Instabilidade Genômica/efeitos dos fármacos , Humanos , Masculino , Nicotina/metabolismo , Exposição Ocupacional/análise , Oxirredução , Estresse Oxidativo/genética , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Nicotiana/metabolismo , Adulto JovemRESUMO
PURPOSE: Aim of the study was to find out if gallic acid (GA), a common phenolic in plant foods, prevents obesity induced DNA damage which plays a key role in the induction of overweight associated cancer. METHODS: Male and female C57BL6/J mice were fed with a low fat or a high fat diet (HFD). The HFD group received different doses GA (0, 2.6-20 mg/kg b.w./day) in the drinking water for 1 week. Subsequently, alterations of the genetic stability in blood and inner organs were monitored in single cell gel electrophoresis assays. To elucidate the underlying molecular mechanisms: oxidized DNA bases, alterations of the redox status, lipid and glucose metabolism, cytokine levels and hepatic NF-κB activity were monitored. RESULTS: HFD fed animals had higher body weights; increased DNA damage and oxidation of DNA bases damage were detected in colon, liver and brain but not in blood and white adipose tissue. Furthermore, elevated concentrations of insulin, glucose, triglycerides, MCP-1, TNF-α and NF-κB activity were observed in this group. Small amounts of GA, in the range of human consumption, caused DNA protection and reduced oxidation of DNA bases, as well as biochemical and inflammatory parameters. CONCLUSIONS: Obese animals have increased DNA damage due to oxidation of DNA bases. This effect is probably caused by increased levels of glucose and insulin. The effects of GA can be explained by its hypoglycaemic properties and indicate that the consumption of GA-rich foods prevents adverse health effects in obese individuals.
Assuntos
Dano ao DNA/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Ácido Gálico/farmacologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cannabidiol (CBD) and cannabidivarin (CBDV) are natural cannabinoids which are consumed in increasing amounts worldwide in cannabis extracts, as they prevent epilepsy, anxiety, and seizures. It was claimed that they may be useful in cancer therapy and have anti-inflammatory properties. Adverse long-term effects of these drugs (induction of cancer and infertility) which are related to damage of the genetic material have not been investigated. Therefore, we studied their DNA-damaging properties in human-derived cell lines under conditions which reflect the exposure of consumers. Both compounds induced DNA damage in single cell gel electrophoresis (SCGE) experiments in a human liver cell line (HepG2) and in buccal-derived cells (TR146) at low levels (≥ 0.2 µM). Results of micronucleus (MN) cytome assays showed that the damage leads to formation of MNi which reflect chromosomal aberrations and leads to nuclear buds and bridges which are a consequence of gene amplifications and dicentric chromosomes. Additional experiments indicate that these effects are caused by oxidative base damage and that liver enzymes (S9) increase the genotoxic activity of both compounds. Our findings show that low concentrations of CBD and CBDV cause damage of the genetic material in human-derived cells. Furthermore, earlier studies showed that they cause chromosomal aberrations and MN in bone marrow of mice. Fixation of damage of the DNA in the form of chromosomal damage is generally considered to be essential in the multistep process of malignancy, therefore the currently available data are indicative for potential carcinogenic properties of the cannabinoids.
Assuntos
Canabinoides/toxicidade , Aberrações Cromossômicas , Dano ao DNA , Animais , Canabidiol/toxicidade , Linhagem Celular , Células Hep G2 , Humanos , Masculino , Testes para Micronúcleos , Mutagênicos/toxicidade , Ratos Sprague-DawleyRESUMO
Road markers are exposed to various chemicals and particles. The aim of this study was to determine whether road worker exposure induceschromosomal damage which is indicative for increased cancer risks. Micronucleus (MN) cytome assays were thus conducted with exfoliated nasal and buccal cells collected from 42 workers and 42 matched controls. The frequencies of MN (reflecting chromosomal aberrations), nuclear buds (NBuds; reflecting gene amplifications) and binucleated cells (BN; reflecting disturbed mitosis) were scored. Further, the rates of nuclear anomalies indicative of acute cytotoxicity (condensed chromatin, karyorrhexis, karyolysis, pyknosis) were evaluated. Data demonstrated marked induction of MN, NBuds, and BN by 1.34-fold, 1.24-fold and 1.14-fold in buccal cells. In nasal cells, only MN frequencies were elevated, 1.23-fold. These effects were paralleled by increased rates of condensed chromatin, karyorrhexis and karyolysis in both cell types. The effects were more pronounced in individuals who had worked for more than 10 years while smoking did not produce synergistic responses. This is the first investigation concerning the induction of genetic damage in road markers and the results are suggestive for enhanced cancer risks. It is conceivable that exposure to silica dust (known to induce cancer and genetic damage) and/or benzoyl peroxide which forms reactive radicals may be associated with the observed genetic damage in road workers. Further investigations of the cancer risks of these workers are warranted.
Assuntos
Peróxido de Benzoíla/toxicidade , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mucosa Bucal/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Neoplasias/induzido quimicamente , Exposição Ocupacional/efeitos adversos , Dióxido de Silício/toxicidade , Adulto , Biomarcadores , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição de RiscoRESUMO
Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.
Assuntos
Colite/complicações , Neoplasias Colorretais/etiologia , Instabilidade de Microssatélites , Mutagênese/fisiologia , Neutrófilos/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Colite/metabolismo , Citocinas/metabolismo , Mutação da Fase de Leitura , Humanos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.
Assuntos
Fígado/diagnóstico por imagem , Testes de Mutagenicidade/métodos , Mutagênicos/análise , Aflatoxina B1/toxicidade , Benzo(a)pireno/toxicidade , Linhagem Celular Tumoral , Dimetilnitrosamina/toxicidade , Humanos , Peróxido de Hidrogênio/toxicidade , Imidazóis/toxicidade , Inativação Metabólica , Fígado/citologia , Quinolinas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Results of a number of studies indicate that electroplaters have increased cancer risks as a consequence of exposure to genotoxic metals such as chromium (VI) and nickel. These effects may be due to induction of damage of the genetic material which plays a key role in the etiology of cancer, and it was found that workers in galvanization factories exhibited increased levels of DNA damage. The aim of the present study was to investigate genetic stability in workers of a bright plating factory who are exposed to chromium (Cr) and cobalt (Co). Exfoliated cells were collected from the buccal and nasal mucosa of workers (n = 42) and matched controls (n = 43) and analyzed for induction of micronuclei (MN) which are formed as a consequence of chromosomal aberrations. In addition, other nuclear anomalies namely nuclear buds (Nbuds) which are formed as a consequence of gene amplification and markers indicating different stages of cell death (condensed chromatin, karyorrhexis, karyolysis, and pyknosis) were also assessed. No evidence was noted for induction of MN, but significantly increased rates of Nbuds in cells from both, buccal and nasal mucosa, were found. Parameters which are indicative for cytotoxic effects were more pronounced in nasal cells and rose with duration of employment period. Overall, our findings indicated that no apparent chromosomal damage occurred in bright electroplaters. However, data demonstrated that acute cytotoxic effects may lead to inflammations and/or lesions in epithelia of the respiratory tract of the workers.
Assuntos
Cromo/toxicidade , Cobalto/toxicidade , Galvanoplastia , Mucosa Bucal/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Exposição Ocupacional/efeitos adversos , Adulto , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Feminino , Humanos , Masculino , Micronúcleos com Defeito Cromossômico/induzido quimicamenteRESUMO
Synthetic cannabinoids (SCs) are marketed worldwide as legal surrogates for marihuana. In order to predict potential health effects in consumers and to elucidate the underlying mechanisms of action, we investigated the impact of a representative of the cyclohexylphenols, CP47,497-C8, which binds to both cannabinoid receptors, on protein expression patterns, genomic stability and on induction of inflammatory cytokines in human lymphocytes. After treatment of the cells with the drug, we found pronounced up-regulation of a variety of enzymes in nuclear extracts which are involved in lipid metabolism and inflammatory signaling; some of the identified proteins are also involved in the endogenous synthesis of endocannabinoids. The assumption that the drug causes inflammation is further supported by results obtained in additional experiments with cytosols of LPS-stimulated lymphocytes which showed that the SC induces pro-inflammatory cytokines (IL12p40 and IL-6) as well as TNF-α. Furthermore, the proteome analyses revealed that the drug causes down-regulation of proteins which are involved in DNA repair. This observation provides an explanation for the formation of comets which was seen in single-cell gel electrophoresis assays and for the induction of micronuclei (which reflect structural and numerical chromosomal aberrations) by the drug. These effects were seen in experiments with human lymphocytes which were conducted under identical conditions as the proteome analysis. Taken together, the present findings indicate that the drug (and possibly other structurally related SCs) may cause DNA damage and inflammation in directly exposed cells of consumers.
Assuntos
Canabinoides/toxicidade , Cicloexanóis/toxicidade , Citocinas/biossíntese , Dano ao DNA , Leucócitos Mononucleares/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Proteínas Nucleares/biossíntese , Fenóis/toxicidade , Adulto , Células Cultivadas , Cromatografia Líquida , Ensaio Cometa , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Espectrometria de Massas , Análise Serial de Proteínas , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/imunologia , Frações SubcelularesRESUMO
Aim of this study was the investigation of the genotoxic properties of XLR-11 [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone, a widely consumed synthetic cannabinoid (SC), and of the benzoyl indole RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone). We characterized the DNA-damaging properties of these drugs in different experimental systems. No evidence for induction of gene mutations was detected in bacterial (Salmonella/microsome) tests, but clear dose-dependent effects were found in in vitro single cell gel electrophoresis (SCGE) assays with human lymphocytes and with buccal- and lung-derived human cell lines (TR-146 and A-549). These experiments are based on the determination of DNA migration in an electric field and enable the detection of single- and double-strand breaks and apurinic sites. Furthermore, we found that both drugs induce micronuclei which are formed as a consequence of chromosomal aberrations. The lack of effects in SCGE experiments with lesion-specific enzymes (FPG, Endo III) shows that the DNA damage is not caused by formation of oxidatively damaged bases; experiments with liver enzyme homogenates and bovine serum albumin indicate that the drugs are not converted enzymatically to DNA-reactive intermediates. Furthermore, results with buccal- and lung-derived human cells show that gaseous treatment of the cells under conditions which reflect the exposure situation in drug users may cause damage of the genetic material in epithelia of the respiratory tract. Since DNA instability is involved in the etiology of cancer, these findings can be taken as an indication that consumption of the SCs may cause tumors in the respiratory tract of consumers.
Assuntos
Canabinoides/toxicidade , Dano ao DNA , Drogas Desenhadas/toxicidade , Mutagênicos/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Neoplasias do Sistema Respiratório/induzido quimicamente , Células A549 , Biotransformação , Canabinoides/metabolismo , Linhagem Celular , Células Cultivadas , Ensaio Cometa , Drogas Desenhadas/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Testes para Micronúcleos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Mutagênicos/metabolismo , Mutação/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Absorção pelo Trato Respiratório , Neoplasias do Sistema Respiratório/metabolismo , Neoplasias do Sistema Respiratório/patologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologia , Salmonella typhimurium/metabolismoRESUMO
Cotton workers in small weaving household factories (power looms) in Pakistan are typically exposed to high levels of cotton dusts. Working in the textile manufacturing industry has been classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer. The study set out to determine potential cytotoxic and genotoxic effects of occupational exposure to cotton dusts in exfoliated buccal cells of exposed cotton workers. Nuclear anomalies reflecting cytotoxic and genotoxic effects were evaluated in a representative sample of 51 exposed male cotton weavers and in the same number of age-matched male non-exposed subjects applying the micronucleus cytome assay. Nuclear anomalies reflecting cytotoxicity (karyolysis, karyorrhexis, condensed chromatin and pyknosis) were significantly elevated in exposed cotton workers. The frequency of micronucleated cells increased significantly with increasing years of work in power looms (odds ratio = 1.043 per year; 95% confidence interval: 1.012-1.076, P = 0.007). Results were consistent with the typical inflammatory pattern and injury in epithelia due to unprotected occupational exposure to cotton dusts and other toxic, allergic and infectious substances in the working areas of the cotton industry. Occupational exposure in power looms induces cytotoxic effects and, upon chronic exposure, DNA damage. This may eventually result in typical obstructive patterns of pulmonary symptoms and in a clinical condition called byssinosis in exposed cotton workers. Long exposure may lead to chronic inflammation and cumulative damage of DNA in buccal stem cells that may indicate an increased risk of oropharyngeal cancer.
Assuntos
Poeira , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mucosa Bucal/ultraestrutura , Exposição Ocupacional/efeitos adversos , Indústria Têxtil , Adulto , Humanos , Masculino , Testes para Micronúcleos , PaquistãoRESUMO
The LEM domain (for lamina-associated polypeptide, emerin, MAN1 domain) defines a group of nuclear proteins that bind chromatin through interaction of the LEM motif with the conserved DNA crosslinking protein, barrier-to-autointegration factor (BAF). Here, we describe a LEM protein annotated in databases as 'Ankyrin repeat and LEM domain-containing protein 1' (Ankle1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. Although most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus. Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 is an unusual LEM protein with a GIY-YIG-type endonuclease activity in higher eukaryotes.
Assuntos
Clivagem do DNA , Endonucleases/química , Endonucleases/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Dano ao DNA , Endonucleases/análise , Endonucleases/genética , Perfilação da Expressão Gênica , Sistema Hematopoético/metabolismo , Humanos , Imunoprecipitação , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de SinaisRESUMO
Cannabicyclohexanol (CP-47,497-C8) is a representative of a group of cannabimimetic cyclohexylphenols which is added to herbal mixtures as a cannabis substitute since 2008. Although in the beginning CP-47,497-C8 was the main ingredient of "Spice" and similar products, it was partly replaced by aminoalkylindole-type cannabinoid receptor agonists like JWH-018, JWH-073 or JWH-250, but never completely disappeared from the market. Since information on its toxicological properties is scarce, we investigated the effects of the drug in human derived cell lines. The cytotoxic effects were studied in a panel of assays (SRB, XTT, LDHe and NR tests) in a buccal derived (TR146) and a liver derived (HepG2) cell line. The strongest effects were seen in the two former assays at levels ≥ 7.5 µM indicating that the compound interferes with protein synthesis and causes membrane damage. In additional comet assays, DNA damage was detected at levels ≥10 µM. Experiments with lesion specific enzymes showed that these effects are not due to oxidative damage of DNA bases. The negative findings obtained in Salmonella/microsome assays and the positive results of micronucleus tests with the cell lines indicate that the compound does not cause gene mutations but acts on the chromosomal level. In contrast to other synthetic cannabinoids, no indication for estrogenic/antiestrogenic properties was seen in a luciferase assay with bone marrow derived U2-OS cells. In conclusion, our findings show that the drug has only weak cytotoxic properties. However, the induction of chromosomal damage indicates that it may cause adverse effects in users due to its impact on the stability of the genetic material.
Assuntos
Canabinoides/toxicidade , Cicloexanóis/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Genes Reporter , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Testes para Micronúcleos , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Mutação , Biossíntese de Proteínas/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Medição de Risco , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , TransfecçãoRESUMO
This study aimed to compare the frequencies of nuclear anomalies in buccal cells between diabetic and non-diabetic individuals and to assess the impact of a 'healthy diet'-a cornerstone in the treatment of diabetes. Seventy-six diabetic and 21 non-diabetic individuals participated in this parallel, randomised, intervention trial. All participants received information about the importance of a healthy diet, while participants randomly assigned to the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids (PUFA) per day for 8 weeks. Cytogenetic damage in buccal cells was assessed at baseline and after 8 weeks using the buccal micronucleus cytome assay. Micronucleus (MN) frequency at baseline was significantly higher in participants with diabetes (0.58±0.30) compared with non-diabetic individuals (0.28±0.29). Further analysis of baseline data revealed significantly higher MN levels in participants of the highest tertile of waist circumference (+40%), fasting plasma glucose (+55%), glycated haemoglobin (+41%) and cardiovascular disease risk (+39%) relative to participants of the lowest tertile. The dietary intervention had no effect on MN frequencies. Glycated haemoglobin and biomarkers reflecting cytokinetic defect or acute cell death were reduced in both the intervention and 'information only' groups. The results of this study suggest a strong impact of abdominal obesity and glucose metabolism on genomic stability. Similar effects on nuclear anomalies were observed in the 'information only' group and the intervention group receiving vegetables and PUFA-rich plant oil.
Assuntos
Núcleo Celular/genética , Núcleo Celular/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Dieta , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Idoso , Feminino , Humanos , Masculino , Micronúcleos com Defeito Cromossômico , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Micronucleus (MN) assays with buccal cells are at present widely used to investigate occupational exposures to genotoxic carcinogens. This article describes their use for the monitoring of metal exposed workers. We found in total 73 relevant articles, in the majority (97â¯%) increased MN and/or other nuclear anomalies were reported. Most studies were realized in South East Asia and South America. A variety of different occupations was studied including welders, electroplaters, painters, workers in battery recycling and production, tannery workers, dental technicians, miners, workers in foundries and smelters, and also subjects working in waste recycling, glass, aluminum and steel production. In many investigations the effects increased with the duration of the working period. The quality of individual studies was evaluated with a quality score tool. The number of cells was in most studies sufficient and DNA-specific stains were used. However, many studies have shortcomings, e.g. they focused solely on MN formation and did not evaluate anomalies, which provide additional information about the stability of the genetic material and acute cytotoxic effects. Only 35â¯% of the investigations contain quantitative information about exposures to metals and other toxicants. In 6 of these studies, correlations were observed between the concentrations of specific metals (As, Pb, Cr, Cd) in body fluids and MN frequencies. Taken together, the available data indicate that the MN assay can be used to detect chromosomal damage in metal exposed groups; furthermore, it enables also comparisons between subgroups differing in regard to their exposure and allows an estimation of the efficiency of protective measures. The exposure of workers to metals is currently controlled with chemical analytical measurements only, MN assays with buccal cells could contribute to further improve the safety at workplaces as they reflect the biological consequences including synergistic and antagonistic interactions between toxicants.
RESUMO
Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Flavonoides/farmacologia , Propiofenonas/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias da Mama/metabolismo , Adesão Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocromo P-450 CYP1A1/metabolismo , Relação Dose-Resposta a Droga , Selectina E/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células MCF-7 , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Esferoides Celulares , TransfecçãoRESUMO
The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.
Assuntos
Dano ao DNA , Dímeros de Pirimidina , Animais , Humanos , Ensaio Cometa/métodos , Células Eucarióticas , DNA/genéticaRESUMO
Aim of this study was to investigate the cytotoxic and genotoxic properties of inorganic and organic mercury compounds, i.e., HgCl(2) and methylmercury (MeHg). In addition, the DNA-protective and antioxidant effects of the flavonoid quercetin (QC) were studied. All experiments were conducted with human-derived liver cells (HepG2), which possess antioxidant and drug-metabolizing enzymes in an inducible form. 8-Hydroxydeoxyguanosine (8-OHdG) and comet formation were monitored as endpoints of DNA damage. The impact of the metal compounds on the redox status was also investigated, since it is assumed that their toxic effects are due to oxidative damage. A number of biochemical parameters related to oxidative stress, namely glutathione, malondialdehyde, protein carbonyl and formation of reactive oxygen species (ROS) were measured after treatment of the cells with the mercury compounds in the presence and absence of quercetin. To elucidate the mechanisms that underlie the effects of QC, three protocols (pre-, simultaneous and post-treatment) were used. Both mercury compounds (range 0.1-5.0µM) caused induction of DNA migration and formation of 8-OHdG. In combination with the flavonoid (range 0.1-5.0µM), DNA-protective effects of QC were observed after pre- and simultaneous treatment but not when the flavonoid was added after treatment with the metal compounds. Exposure to the metal compounds led also to substantial changes of all parameters of the redox status and co-treatment experiments with QC showed that these alterations are reversed by the flavonoid. Taken together, the results of our experiments indicate that these two mercury compounds cause DNA damage and oxidative stress in human-derived liver cells and that the flavonoid reduces these effects. Since the concentrations of the metals and of the flavonoids used in the present work reflect human exposure, our findings can be taken as an indication that QC may protect humans against the adverse effects caused by the metal.
Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Compostos de Metilmercúrio/toxicidade , Oxirredução/efeitos dos fármacos , Quercetina/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Sobrevivência Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Células Hep G2 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation of oxidatively damaged DNA in humans.