RESUMO
BACKGROUND: Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. AIM: The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. METHODS: In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 microL/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 microM). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. RESULTS: In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 microL/mL WCE, 3.7-fold 1 microM PEITC) and SOD2 (12.1-fold, 10 microL/mL WCE, 7.3-fold, 10 microM PEITC), and increased SOD2 activity (1.9-fold, 10 microL/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. CONCLUSION: The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.
Assuntos
Anticarcinógenos/farmacologia , Glutationa Peroxidase/metabolismo , Leucócitos Mononucleares/enzimologia , Nasturtium/química , Neoplasias/prevenção & controle , Extratos Vegetais/farmacologia , Superóxido Dismutase/metabolismo , Células Cultivadas , Estudos Cross-Over , Relação Dose-Resposta a Droga , Expressão Gênica , Genótipo , Glutationa Peroxidase/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isotiocianatos/farmacologia , Polimorfismo Genético , Superóxido Dismutase/genética , Glutationa Peroxidase GPX1RESUMO
An extract of the Mediterranean carob (Ceratonia siliqua L.) pod (carob fibre extract), products formed after its fermentation by the gut flora and the major phenolic ingredient gallic acid (GA), were comparatively investigated for their influence on survival and growth parameters of colon adenocarcinoma HT29 cells and adenoma LT97 cells. Hydrogen peroxide (H2O2) formation in the cell culture media was quantified. After 1h 97+/-4 microM or 70+/-15 microM were found in HT29 medium and 6+/-1 microM or 3+/-3 microM in LT97 medium for carob fibre extract or GA, respectively. After 72 h carob fibre extract reduced survival of rapidly proliferating HT29 cells (by 76.4+/-12.9%) whereas metabolic activity and DNA-synthesis were only transiently impaired. Survival of slower growing LT97 cells was less decreased (by 21.5+/-12.9%), but there were marked effects on DNA-synthesis (reduction by 95.6+/-7%, 72 h). GA and fermented carob fibre did not have comparable effects. Thus, carob fibre extract resulted in H2O2 formation, which, however, could not explain impairment of cell growth. The differently modulated growth of human colon cell lines was more related to proliferation rates and impairment of DNA-synthesis than to H2O2 formation.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Antineoplásicos Fitogênicos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Fibras na Dieta/farmacologia , Fabaceae/química , Adenocarcinoma/tratamento farmacológico , Adenoma/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/tratamento farmacológico , Meios de Cultura , DNA de Neoplasias/biossíntese , Fermentação , Células HT29 , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Fenóis , Espectrometria de Massas por Ionização por Electrospray , Sulfóxidos , Xilenos/químicaRESUMO
The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Anticarcinógenos/farmacologia , Colo/citologia , Neoplasias do Colo/patologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Neoplasias/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Adenocarcinoma/enzimologia , Adenoma/enzimologia , Butiratos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Colo/enzimologia , Neoplasias do Colo/enzimologia , Regulação para Baixo , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Flavonoides/farmacologia , Humanos , Lisofosfolipídeos/farmacologia , Malus/química , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Polifenóis , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/farmacologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Chá/química , Transcrição Gênica/efeitos dos fármacos , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
Iron could be a relevant risk factor for carcinogenesis since it catalyses the formation of reactive oxygen species (ROS), which damage DNA. We previously demonstrated genotoxic effects by ferric iron using the human colon cancer cell line HT29. Here we investigated ferric iron in primary non-transformed colon cells and in a preneoplastic colon adenoma cell line (LT97), which both are suitable models to study effects of carcinogens during early stages of cell transformation. Genetic damage was determined using the Comet assay. Comet FISH (fluorescence in situ hybridization) was used to assess specific effects on TP53. Fe-NTA (0-1000 microM, 30 min, 37 degrees C) significantly induced single strand breaks in primary colon cells (500 microM Fe-NTA: Tail intensity [TI] 22.6%+/-5.0% versus RPMI control: TI 10.6%+/-3.9%, p<0.01) and in LT97 cells (1000 microM Fe-NTA: TI 26.8%+/-7.3% versus RPMI control: TI 11.1%+/-3.7%, p<0.01). With the Comet FISH protocol lower concentrations of Fe-NTA significantly increased DNA damage already at 100 and 250 microM Fe-NTA in primary colon and LT97 adenoma cells, respectively. This damage was detected as an enhanced migration of TP53 signals into the comet tail in both cell types, which indicates a high susceptibility of this tumor relevant gene towards Fe-NTA. In conclusion, Fe-NTA acts genotoxic in non-transformed and in preneoplastic human colon cells, in which it also enhances migration of TP53 at relatively low concentrations. Translated to the in vivo situation these results suggest that iron overload putatively contributes to a genotoxic risk during early stages of colorectal carcinogenesis on account of its genotoxic potential in non-tumorigenic human colon cells.
Assuntos
Colo/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Compostos Férricos/toxicidade , Nitratos/toxicidade , Adenoma/genética , Adenoma/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ensaio Cometa , Relação Dose-Resposta a Droga , Humanos , Hibridização in Situ Fluorescente , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/análiseRESUMO
Interest in the beneficial effects of green tea has led to investigations on activities by the main catechin (-)-epigallocatechin-3-gallate (EGCG). This antioxidative compound could contribute to cancer chemoprevention by acting antigenotoxic. To further explore this hypothesis we investigated antigenotoxic potentials of low EGCG concentrations in human peripheral leucocytes. Leucocytes isolated from whole blood were (1) stimulated with phytohaemagglutinin, (2) damaged with genotoxic bleomycin, and (3) post-incubated to allow DNA repair. After each phase DNA integrity was measured with the comet assay. EGCG (2, 20, 100 microM) was added either during phases 1, 2 or 3 or during the whole process (1-3), to delineate mechanisms of antigenotoxicity reflecting induction of detoxification (phase 1), scavenging of radicals (phase 2), stimulation of repair (phase 3), respectively. Bleomycin induced breaks and endonuclease III specific damage, but EGCG did not affect damage or repair of these lesions when added during phases 1, 2 or 3. However, the application of EGCG during phases 1 and 2 significantly reduced both bleomycin-induced breaks and endonuclease III sensitive sites. EGCG added during all phases impaired persistence of damage. Our studies show that the continuous presence of EGCG can reduce radical-induced DNA damage in primary leucocytes, possibly due to a combination of different mechanisms. Together the findings support the hypotheses that EGCG acts protective in human cells.
Assuntos
Antibióticos Antineoplásicos/antagonistas & inibidores , Bleomicina/antagonistas & inibidores , Catequina/análogos & derivados , Dano ao DNA/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Catequina/farmacologia , Ensaio Cometa , Desoxirribonucleases de Sítio Específico do Tipo III/antagonistas & inibidores , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Humanos , Técnicas In Vitro , Fito-Hemaglutininas/químicaRESUMO
Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.
Assuntos
Aldeídos/toxicidade , Neoplasias do Colo/etiologia , Dano ao DNA , Compostos Férricos/toxicidade , Ácido Nitrilotriacético/análogos & derivados , Espécies Reativas de Oxigênio/metabolismo , Proliferação de Células/efeitos dos fármacos , Glutationa/metabolismo , Células HT29 , Humanos , Ácido Nitrilotriacético/toxicidadeRESUMO
Ingestion of viable probiotics or prebiotics is associated with anticarcinogenic effects, one mechanism of which is the detoxification of genotoxins in the gut. This mechanism was shown experimentally in animals with use of the rat colon carcinogen 1,2-dimethylhydrazine and by determining endpoints that range from tumorigenesis to induction of DNA damage. Because of the complexity of cancer initiation, cancer progression, and the exposure of cancer in the gut, many types of interactions may be envisaged. Notably, some of our newer studies showed that short-lived metabolite mixtures isolated from milk that was fermented with strains of Lactobacillus bulgaricus and Streptococcus thermophilus are more effective in deactivating etiologic risk factors of colon carcinogenesis than are cellular components of microorganisms. Ingestion of prebiotics results in a different spectrum of fermentation products, including the production of high concentrations of short-chain fatty acids. Gut flora, especially after the ingestion of resistant starch, induces the chemopreventive enzyme glutathione transferase pi in the colon of the rat. Together, these factors lead to a reduced load of genotoxic agents in the gut and to an increased production of agents that deactivate toxic components. Butyrate is one such protective agent and is associated with lowering cancer risk. It was recently shown that buytrate may inhibit the genotoxic activity of nitrosamides and hydrogen peroxide in human colon cells. In humans, the ingestion of probiotics leads to the excretion of urine with low concentrations of components that are genotoxic in human colon cells and high concentrations of components that induce oxidized DNA bases.
Assuntos
Colo/microbiologia , Neoplasias do Colo/prevenção & controle , Lactobacillus , Probióticos/uso terapêutico , Streptococcus , Animais , Butiratos/metabolismo , Quimioprevenção , Colo/metabolismo , Colo/fisiologia , Dano ao DNA , Modelos Animais de Doenças , Fermentação , Glutationa Transferase/metabolismo , Humanos , Mutação , RatosRESUMO
A previous intervention study had shown that consumption of carotenoid-containing vegetable juices reduces oxidative DNA damage in lymphocytes of 23 male subjects. It was the aim of this study to elucidate the potential mechanisms involved. Specifically, we studied the modulation of protein expression and determined susceptibility factors. Cryopreserved lymphocytes from the study were analyzed for genetic polymorphisms of glutathione S-transferase (GSTM1, GSTP1, and GSTT1) using multiplex PCR, GSTP1-protein with an ELISA, total protein by a colorimetric enzyme reaction, and DNA-repair enzymes with the Comet Assay. Analyses of the genotoxicity data revealed a more steady state of protection for GSTM1*+ than for GSTM1*0 (15 and 8 of 23, respectively) genotypes. Increased expression of cytosolic protein was observed in 11 of 23 subjects, increased expression of GSTP1 in 6 of 23 subjects, and capacity of repair of oxidized DNA bases in 9 of 21 subjects. GSTP1 induction was independent of the GSTP1 genotype (GSTP1a or GSTP1b/c alleles). Kinetics of induction of cytosolic protein and of GSTP1 were compared in one GSTM1*+ and one GSTM1*0 subject and showed an efficacy of tomato and carrots, but not of spinach. Reduced genetic DNA damage in lymphocytes may be due to the enhancement of cytosolic GSTP1, and DNA-repair proteins by tomato and carrot juices. Enhancement of cytosolic proteins may be indicative of increased gene expression by vegetable juices, some of which may be associated with protective activities.
Assuntos
Antioxidantes/uso terapêutico , Carotenoides/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dieta , Linfócitos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Verduras , Adulto , Bebidas , Predisposição Genética para Doença/genética , Genótipo , Glutationa Transferase/genética , Humanos , Masculino , Testes de Mutagenicidade , Polimorfismo Genético/genéticaRESUMO
The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 microM) or hydrogen peroxide (75 microM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.
Assuntos
Colo/patologia , Dano ao DNA/genética , Glutationa Transferase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Biomarcadores Tumorais/metabolismo , Biópsia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Células HT29 , Humanos , Fatores de RiscoRESUMO
Multicolor chromosome banding (MCB) using one single chromosome-specific MCB probe set per experiment was previously reported as powerful tool in molecular cytogenetics for the characterization of all kinds of human marker chromosomes. However, a quick analysis of karyotypes with highly complex chromosomal changes was hampered by the problem that up to 24 MCB experiments were necessary for a comprehensive karyotype description. To overcome that limitation the 138 available region-specific microdissection-derived libraries for all human chromosomes were combined to one single probe set, called multitude MCB (mMCB). A typical fluorescence banding pattern along the human karyotype is produced, which can be evaluated either by transforming these profiles into chromosome region-specific pseudo-colors or more reliably by studying the fluorescence profiles. The mMCB probe set has been applied on chromosomes of normal male and female probands, two primary myelodysplastic syndromes and two solid tumor cell lines. Additionally, a cell line of Gorilla gorilla (GGO) studied previously by single chromosome-specific MCB was reevaluated by the mMCB method. All results were in concordance with those obtained in parallel or by other cytogenetic and molecular cytogenetic approaches indicating that mMCB is a powerful multicolor FISH banding tool for fast characterization of complex karyotypes.
Assuntos
Aberrações Cromossômicas , Bandeamento Cromossômico/métodos , Hibridização in Situ Fluorescente/métodos , Animais , Linhagem Celular Tumoral , Cor , Feminino , Gorilla gorilla , Humanos , Cariotipagem , Masculino , Metáfase , Síndromes Mielodisplásicas/genética , Neoplasias/genéticaRESUMO
Molecular orbital calculations with aristolochic acid I (AAI) and the model compounds 8-nitro-1-naphthoic acid (1,8NNA) and 3-nitro-2-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups in these molecules. The ortho isomer 2,3NNA is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic activity similar to AAI in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation via a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic acids.
Assuntos
Ácidos Aristolóquicos , Mutagênicos/química , Fenantrenos/química , Testes de Mutagenicidade , Mutagênicos/toxicidade , Fenantrenos/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
A. bisporus has been reported to be carcinogenic to mice [Toth et al. (1986) Cancer Res 38:177-180] and mutagenic in Salmonella typhimurium [Sterner et al. (1982) Mutat Res 101:269-281]. The effects of different heat treatments on the mutagenicity of raw, cooked (boiled) and fried A. bisporus extracts in the S. typhimurium test is reported. The spectrum of potential mutagenic activity of A. bisporus extracts was tested in vitro in Syrian hamster embryo cells for selective DNA amplification and in primary rat hepatocytes for DNA single-strand breaks. DNA single-strand breaks were also determined in liver cells of rats and micronuclei were measured in bone marrow cells of mice in vivo following oral application of A. bisporus extracts. It was shown that the complex A. bisporus extracts per se are not detectably mutagenic in S. typhimurium and that the previously observed increase in number of colonies per plate is probably due to a histidine artefact. No indication of genotoxicity was seen in the two in vitro assays with primary mammalian cells with two different end points. No evidence of in vivo genotoxic effects was observed in the rat liver cells. Finally, A. bisporus was not genotoxic in the micronucleus assay of mouse bone marrow cells in contrast to its previously reported carcinogenicity in mice.
Assuntos
Agaricus , Extratos Vegetais/farmacologia , Animais , Cricetinae , Dano ao DNA , DNA de Cadeia Simples/efeitos dos fármacos , Temperatura Alta , Fígado/efeitos dos fármacos , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes de Mutagenicidade , Ratos , Ratos Endogâmicos , Salmonella typhimurium/efeitos dos fármacosRESUMO
Human lymphocytes (HL) as well as lymphocytes (RL), hepatocytes (RH), and gastric mucosa cells (GM) of Sprague-Dawley rats were treated in vitro for 1 h with methylmercury chloride (MMC, 0.5-4 micrograms/ml) and dimethylmercury (DMM, 5-40 micrograms/ml). The cytotoxicity of the two organic mercury compounds was assessed by dye exclusion, and the extent of induced DNA fragmentation was measured with a single-cell microgel electrophoresis assay. Both MMC and DMM induced DNA damage and cytotoxicity in a dose-related manner in HL, RL, and GM. MMC was more effective in causing a significant increase in median DNA migration than DMM at doses yielding approximately the same degree of cytotoxicity. In rat hepatocytes the MMC-induced DNA damage was, however, lower than in the other cells. An analysis of repair kinetics following exposure to 2 micrograms/ml MMC was carried out in human lymphocytes obtained from an adult male donor. The bulk of DNA repair occurred 90 min after in vitro exposure, and it was about complete by 120 min following cessation of exposure. Finally, in order to have a basis for extrapolating to the human situation, in vivo studies were performed with Sprague-Dawley rats, also assessing the DNA damage and cytotoxicity in the lymphocytes and gastric mucosa cells. These in vivo results after oral exposure may be directly compared to the in vitro data obtained in the same cells.
Assuntos
Dano ao DNA , Compostos de Metilmercúrio/toxicidade , Mutagênicos/toxicidade , Adulto , Análise de Variância , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia em Gel , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/ultraestrutura , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Masculino , Testes de Mutagenicidade , Ratos , Ratos Sprague-Dawley , Análise de RegressãoRESUMO
To assess genotoxic burdens from chemicals, it is necessary to relate observations in experimental animals to humans. The success of this extrapolation would be increased by including data on chemical activities in human tissues. Therefore, we have developed techniques to assess DNA damage in human gastric and nasal mucosa (GM, NM) cells. Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach or from healthy nasal epithelia during surgery. The specimens were incubated for 30-45 min at 37 degrees C with a digestive solution. We obtained 1.5-8 x 10(6) GM cells and 5-10 x 10(5) NM cells per donor, both with viabilities of 80-95%. The cells were incubated in vitro for 1 hr at 37 degrees C with the test compounds added in their appropriate solvents. In GM cells, we studied N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium dichromate (Na2Cr2O7), nickel sulphate (NiSO4), cadmium sulphate (CdSO4), and lindane. In NM cells, lindane was investigated. Each compound was assessed for DNA damaging activity in cells of at least three different human donor samples using the microgel single cell assay. Similar studies were performed with GM and NM cells obtained from Sprague-Dawley rats. We have found human GM cells to be more sensitive to the genotoxic activity of MNNG than rat GM cells (low effective concentration [LEC] = 0.16 and 0.625 micrograms/ml for human and rat, respectively). Human cells were also more sensitive to the cytotoxic/genotoxic activity of NiSO4 (LEC = 5 and 19 mumoles/ml for human and rat, respectively). CdSO4 was genotoxic in human GM cells (LEC = 0.03-0.125 mumoles/ml), whereas no dose-related genotoxicity was observed in rat GM at concentrations up to 0.5 mumoles/ml. In contrast, approximately equal responses regarding genotoxicity and cytotoxicity were observed in rat and human GM for Na2Cr2O7 (0.25-1 mumoles/ml). Lindane, however, was genotoxic in three out of four rat GM but not in human GM cells (0.5-1 mumoles/ml), whereas it was active in both rat and human NM cells. Together with other recently published in vivo findings, our results with lindane can be interpreted according to a parallelogram approach. In view of possible human exposure situations and the sensitivities of the two target tissues from both species, the data imply that lindane will pose a health risk to humans by inhalation but not by ingestion.
Assuntos
Compostos de Cádmio , Dano ao DNA , Mucosa Gástrica/efeitos dos fármacos , Mutagênicos/toxicidade , Mucosa Nasal/efeitos dos fármacos , Sulfatos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Cádmio/toxicidade , Células Cultivadas , Cromatos/toxicidade , Feminino , Mucosa Gástrica/patologia , Hexaclorocicloexano/toxicidade , Humanos , Masculino , Metilnitronitrosoguanidina/toxicidade , Pessoa de Meia-Idade , Mucosa Nasal/patologia , Níquel/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology. METHODS: We have measured H2O2-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H2O2 and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis ("Comet") Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H2O2-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage. RESULTS: A dose-response relationship was found for the H2O2-induced dye decomposition in NIH3T3 cells (7.8-125 microM H2O2) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 M H2O2). Fluorescence was significantly increased at 62microM H2O2 in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H2O2 than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells. CONCLUSIONS: Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H2O2-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H2O2.
Assuntos
Colo/metabolismo , Microscopia Confocal , Estresse Oxidativo , Células 3T3 , Animais , Biópsia , Células CACO-2 , Colo/patologia , Neoplasias do Colo , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Aflatoxin B1 (AFB1) is toxic to the systemic immune system in various animal species, whereas little is known about its effect on the gut-associated lymphoid tissue (GALT). It may be hypothesized that the toxicity of AFB1 and its locally generated metabolites in the intestinal tissue may result in a disturbed intestinal integrity and, subsequently, in an impaired immune response towards dietary proteins. The objective of our study was to investigate the toxic effect of short-term moderate AFB1 exposure on the intestinal epithelium and on the immune cells associated with the intestinal tract. The toxicological potential of AFB1 and its metabolites to the intestinal epithelium was determined by measuring viability and genotoxic damage in isolated jejunal epithelial cells (comet assay) after 30 min incubation in vitro. In vivo toxicology studies were carried out with Brown Norway (BN) rats, which were exposed orally once a week with AFB1 (1 x 100 microg/kg body weight (b.w.)/week) for 5 consecutive weeks. Viability and genotoxicity were measured in explanted jejunal epithelial cells. For studying the effectiveness of AFB1 on immunological parameters BN rats were treated with a high (study 1: 1 x 1 mg/kg b.w./week) or a low (study 2: 1 x 100 microg/kg b.w./week) AFB1 dose for 5 consecutive weeks with or without ovalbumin (OVA). Mesenteric lymphocytes were isolated and proliferative responsiveness, secretion of interferon-gamma, and changes in lymphocyte subpopulations as well as mucosal mast cell specific protease and anti-OVA specific antibody concentrations were measured. In vitro, AFB1 ( >30 microM) induced genotoxicity in rat jejunal epithelial cells. The oral administration of AFB1 (1 x 100 microg/kg b.w./week) did not induce DNA damage in jejunal epithelial cells. The high AFB1 dose increased the number of CD8+ and CD8/CD71 + cells in mesenteric lymph nodes. The immune response towards OVA was not affected. The low AFB1 dose only reduced the proliferative responsiveness of mesenteric lymphocytes (P < 0.05). Serum concentrations of anti-OVA specific IgE antibody, of RMCPII, and the capacity of mesenteric lymphocytes to produce interferon-gamma were not impaired by AFB1. In conclusion, exposure to moderate doses of AFB1 does not damage the intestinal epithelium and has only minor effects on the GALT. The low exposure, as it may predominantly occur in western countries, does not appear to increase the risk for sensitization to dietary antigens.
Assuntos
Aflatoxina B1/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Aflatoxina B1/imunologia , Aflatoxina B1/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imunofluorescência , Imunoglobulina E/análise , Imunoglobulina E/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Interferons/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Mastócitos/enzimologia , Mesentério , Metaloendopeptidases/metabolismo , Testes de Mutagenicidade , Ovalbumina/imunologia , Ratos , Ratos Endogâmicos BNRESUMO
In order to increase the understanding of the factors responsible for causing human colon cancer, a technique was developed to detect genotoxic effects of chemicals in human colon cells. Risk factors suspected to be associated with the aetiology of human colon cancer were subsequently investigated: the method is based on the measurement of DNA damage in primary cells freshly isolated from human colon biopsies with the single cell microgel ectrophoresis technique ('Comet Assay'). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methyl-3H-imidazo[4,5f]quinoline (IQ), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), dinitrosocaffeidine (DNC) lithocholic acid (LCA), hydrogen peroxide (H2O2) and benzo[a]pyrene (B[a]P) were investigated for their genotoxic and cytotoxic effects following 30 min incubation with colon cells of human, and for comparative purposes also of the rat colon. The nitrosamides (MNNG, DNC) were very genotoxic in human colon cells. MNNG was more genotoxic in human than in rat colon cells. In contrast, the rat colon carcinogens PhIP and IQ were not genotoxic in human colon cells. PhIP did induce DNA damage in rat colon cells, which correlates to its capacity of inducing tumors in this animal tissue. LCA was toxic (rat > human) and concomitantly caused DNA damage in higher concentrations. The widespread contaminant B[a]P was not genotoxic in colon cells of either species using this system. H2O2 was found to be a potent genotoxic agent to both rat and human colon cells (human > rat). In summary, those compounds chosen as representatives of endogenously formed risk factors (MNNG, H2O2, LCA) have a higher toxic and/or genotoxic potency in human colon tissue than in rat colon. They are also more effective in this system than the contaminants tested so far (B[a]P, PhIP, IQ). The newly developed technique is rapid and yields relevant results. It is a novel and useful approach to assess different chemical compounds for genotoxic activities in tumour target tissues of the human.
Assuntos
Dano ao DNA , Mucosa Intestinal/citologia , Mutagênicos/farmacologia , Adulto , Idoso , Animais , Benzo(a)pireno/farmacologia , Biópsia , Células Cultivadas , Colo , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Ácido Litocólico/farmacologia , Masculino , Metilnitronitrosoguanidina/farmacologia , Pessoa de Meia-Idade , Testes de Mutagenicidade/métodos , Quinolinas/farmacologia , Ratos , Fatores de RiscoRESUMO
Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.
Assuntos
Apoptose , Ensaio Cometa , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Quebra Cromossômica , Dano ao DNA , Fragmentação do DNA , Humanos , Peróxido de Hidrogênio/toxicidade , Metilnitronitrosoguanidina/toxicidade , Mutagênicos/toxicidade , Estaurosporina/toxicidade , Células Tumorais CultivadasRESUMO
DNA amplification is a frequently observed event in continuous cell lines and in tumors. It is likely that a common mechanism underlies the amplification of specific DNA sequences which confer drug resistance and genes which give a growth advantage to the tumor. To find a correlation between the induction of DNA amplification by chemicals and morphological cell transformation we treated Syrian hamster embryo (SHE) cells with diverse antineoplastic agents of different classes. Analysis of these agents seems to be important since they are potentially carcinogenic and resistance inducing. For the measurement of DNA amplification we established a new system using adeno-associated virus type 2 (AAV)-infected primary SHE cells as target cells and amplification of viral DNA as marker of DNA amplification. Simultaneously we determined morphological cell transformation in SHE cells. Our findings demonstrate that there is only a limited correlation between the induction of AAV DNA amplification and the morphological cell transformation in SHE cells. The newly established system of AAV DNA amplification appears to be a useful tool for the investigation of drug resistance in target cells of choice.
Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/genética , Amplificação de Genes , Animais , Linhagem Celular Transformada , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Viral/genética , Cricetinae , DNA Viral/metabolismo , Dependovirus/genética , Embrião de Mamíferos , Mesocricetus , Vírus 40 dos SímiosRESUMO
INTRODUCTION: Cells other than lymphocytes may be preferable as surrogate biomarkers during exposure monitoring. In nutritional toxicology, cells from colorectal tissues are particularly relevant for studying associations between food and cancer. Thus, we have previously shown that colonic cells of males have higher levels of DNA damage than females, which (among other factors) could be due to a higher consumption of alcoholic beverages by males. To test this hypothesis, we have performed a first exploratory study to compare DNA damage in rectal cells from biopsies of male patients with alcohol abuse and of male and female controls. Peripheral blood lymphocytes were additionally monitored to assess systemic exposure loads. METHODS: Cells were isolated and subjected to microgelelectrophoresis +/- endonuclease III to measure DNA breaks and oxidized pyrimidine bases ("comet-assay"). Cell aliquots were treated with H(2)O(2) for 5min in suspension culture and processed immediately or after 60min to determine induced damage and its persistence. RESULTS: Pooled data from subjects of all groups revealed that oxidative DNA damage in rectal cells directly correlated to damage in lymphocytes. Female controls had lower levels of DNA damage than male controls, confirming the previous studies. An unexpected result was that male alcohol abusers had significantly less genetic damage than male controls. Also, repair was detected in lymphocytes of male alcohol abusers and female controls, but not in male controls. CONCLUSION: This is the first time the comet-assay has been used to detect genotoxicity in human rectal cells as a biomonitoring tool. Our pilot study confirms earlier reports on sex differences and indicates a good correlation between damage in rectal cells and damage in lymphocytes and implies that alcohol exposure enhances endogenous defence.