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1.
Environ Int ; 174: 107917, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37062159

RESUMO

Diisocyanates are highly reactive substances and known causes of occupational asthma. Exposure occurs mainly in the occupational setting and can be assessed through biomonitoring which accounts for inhalation and dermal exposure and potential effects of protective equipment. However the interpretation of biomonitoring data can be challenging for chemicals with complex kinetic behavior and multiple exposure routes, as is the case for diisocyanates. To better understand the relation between external exposure and urinary concentrations of metabolites of diisocyanates, we developed a physiologically based kinetic (PBK) model for methylene bisphenyl isocyanate (MDI) and toluene di-isocyanate (TDI). The PBK model covers both inhalation and dermal exposure, and can be used to estimate biomarker levels after either single or chronic exposures. Key parameters such as absorption and elimination rates of diisocyanates were based on results from human controlled exposure studies. A global sensitivity analysis was performed on model predictions after assigning distributions reflecting a mixture of parameter uncertainty and population variability. Although model-based predictions of urinary concentrations of the degradation products of MDI and TDI for longer-term exposure scenarios compared relatively well to empirical results for a limited set of biomonitoring studies in the peer-reviewed literature, validation of model predictions was difficult because of the many uncertainties regarding the precise exposure scenarios that were used. Sensitivity analyses indicated that parameters with a relatively large impact on model estimates included the fraction of diisocyanates absorbed and the binding rate of diisocyanates to albumin relative to other macro molecules.We additionally investigated the effects of timing of exposure and intermittent urination, and found that both had a considerable impact on estimated urinary biomarker levels. This suggests that these factors should be taken into account when interpreting biomonitoring data and included in the standard reporting of isocyanate biomonitoring studies.


Assuntos
Exposição Ocupacional , Tolueno 2,4-Di-Isocianato , Humanos , Monitoramento Biológico , Isocianatos/análise , Tolueno 2,4-Di-Isocianato/efeitos adversos , Causalidade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/análise
2.
Arch Toxicol ; 90(9): 2215-2229, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26525393

RESUMO

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.


Assuntos
Carcinógenos/toxicidade , Biologia Computacional , Perfilação da Expressão Gênica , Túbulos Renais Proximais/efeitos dos fármacos , Ensaio de Proficiência Laboratorial , Fígado/efeitos dos fármacos , Toxicogenética/métodos , Transcriptoma/efeitos dos fármacos , Carcinógenos/classificação , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Humanos , Túbulos Renais Proximais/metabolismo , Fígado/metabolismo , Variações Dependentes do Observador , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Tempo , Fluxo de Trabalho
3.
Regul Toxicol Pharmacol ; 71(3): 601-23, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25604881

RESUMO

Carbon capture and storage (CCS) technologies are considered vital and economic elements for achieving global CO2 reduction targets, and is currently introduced worldwide (for more information on CCS, consult for example the websites of the International Energy Agency (http://www.iea.org/topics/ccs/) and the Global CCS Institute (http://www.globalccsinstitute.com/)). One prominent CCS technology, the amine-based post-combustion process, may generate nitrosamines and their related nitramines as by-products, the former well known for their potential mutagenic and carcinogenic properties. In order to efficiently assess the carcinogenic potency of any of these by-products this paper reviews and discusses novel prediction approaches consuming less time, money and animals than the traditionally applied 2-year rodent assay. For this, available animal carcinogenicity studies with N-nitroso compounds and nitramines have been used to derive carcinogenic potency values, that were subsequently used to assess the predictive performance of alternative prediction approaches for these chemicals. Promising cancer prediction models are the QSARs developed by the Helguera group, in vitro transformation assays, and the in vivo initiation-promotion, and transgenic animal assays. All these models, however, have not been adequately explored for this purpose, as the number of N-nitroso compounds investigated is yet too limited, and therefore further testing with relevant N-nitroso compounds is needed.


Assuntos
Compostos de Anilina/toxicidade , Sequestro de Carbono , Transformação Celular Neoplásica/induzido quimicamente , Neoplasias/induzido quimicamente , Nitrobenzenos/toxicidade , Nitrosaminas/toxicidade , Compostos de Anilina/química , Animais , Testes de Carcinogenicidade/métodos , Dose Letal Mediana , Camundongos Transgênicos , Modelos Biológicos , Estrutura Molecular , Testes de Mutagenicidade , Nitrobenzenos/química , Nitrosaminas/química , Relação Quantitativa Estrutura-Atividade , Medição de Risco
4.
Regul Toxicol Pharmacol ; 70(1): 392, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24462580

RESUMO

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

5.
Xenobiotica ; 38(6): 574-86, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18570159

RESUMO

1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.


Assuntos
Hidroxitolueno Butilado/farmacologia , Curcumina/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/enzimologia , Galato de Propila/farmacologia , Tiabendazol/farmacologia , Idoso , Anti-Helmínticos/farmacologia , Antioxidantes/farmacologia , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/metabolismo
6.
Toxicol In Vitro ; 21(5): 892-901, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17336492

RESUMO

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.


Assuntos
Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Actinas/biossíntese , Actinas/genética , Animais , Biotransformação , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Técnicas Citológicas , Interpretação Estatística de Dados , Hepatócitos/efeitos dos fármacos , Hidroxilação , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , RNA/biossíntese , RNA/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/metabolismo
7.
Eur J Cancer Prev ; 14(5): 439-57, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16175049

RESUMO

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/análise , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Genes Neoplásicos , Adenocarcinoma/química , Adenocarcinoma/prevenção & controle , Adulto , Biópsia , Linhagem Celular Tumoral , Neoplasias do Colo/química , Neoplasias do Colo/prevenção & controle , Feminino , Mucosa Gástrica/patologia , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Proteínas de Neoplasias/análise , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/análise
8.
Rev Environ Health ; 19(2): 83-139, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15329008

RESUMO

Recent developments in hazard identification and risk assessment of chemical mixtures are reviewed. Empirical, descriptive approaches to study and characterize the toxicity of mixtures have dominated during the past two decades, but an increasing number of mechanistic approaches have made their entry into mixture toxicology. A series of empirical studies with simple chemical mixtures in rats is described in some detail because of the important lessons from this work. The development of regulatory guidelines for the toxicological evaluation of chemical mixtures is discussed briefly. Current issues in mixture toxicology include the adverse health effects of ambient air pollution; the application of such modern, sophisticated methodologies as genomics, bioinformatics, and physiologically based pharmacokinetic modeling; and databases for mixture toxicity. Finally, the state of the art of our knowledge on the potential adverse health effects of combined exposures to chemicals and non-chemical stressors (noise, heat/cold, microorganisms, immobilization, restraint, or transportation), research initiatives in these fields, and the development of an indicator for the cumulative health impact of multiple environmental exposures are discussed.


Assuntos
Exposição Ambiental/efeitos adversos , Toxicologia/métodos , Xenobióticos/toxicidade , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Modelos Estatísticos , Medição de Risco , Segurança , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Xenobióticos/farmacocinética
9.
Mutat Res ; 475(1-2): 45-56, 2001 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-11295153

RESUMO

Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD(+) (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD(+) is the sole substrate. The enzyme PARP-1 is highly activated by DNA strand breaks during the cellular genotoxic stress response, is involved in base excision repair, plays a role in p53 expression and activation, and hence, is thought to be important for genomic stability. In this review, first the absorption, metabolism of niacin to NAD(+), as well as the assessment of niacin status are discussed. Since NAD(+) is important for PARP-1 activity, various aspects of PARP-1 in relation to DNA synthesis and repair, and regulation of gene expression are addressed. This is followed by a discussion on interactions between dietary methyl donor deficiency, niacin status, PARP-1 activity and genomic stability. In vitro studies show that PARP-1 function is impaired and genomic stability decreased when cells are either depleted from NAD(+) or incubated with high concentrations of NAM which is a PARP-1 inhibitor. In vitro as well as animal studies indicate that niacin deficiency increases genomic instability especially in combination with genotoxic and oxidative stress. Niacin deficiency may also increase the risk for certain tumors. Preliminary data suggest that niacin supplementation may protect against UV-induced tumors of the skin in mice, but data on similar preventive effects in humans are not available. NAM has been shown in vitro to have an antioxidant activity comparable to that of ascorbic acid. Data on niacin status and genomic stability in vivo in humans are limited and yield ambiguous results. Therefore, no firm conclusions with respect to optimal niacin intake are possible. As a consequence of oral niacin supplementation, however, NAM levels in the body may increase, which may result in inhibition of PARP-1 and increased genomic instability. More studies are needed to define an optimal level of niacin nutriture in relation to genomic stability and tumorigenesis.


Assuntos
Dano ao DNA , Niacina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , DNA/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Humanos , Niacina/farmacologia
10.
Nutr Metab Cardiovasc Dis ; 11(4 Suppl): 94-8, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11894763

RESUMO

Functional Food Ingredients Against Colorectal Cancer is one of the first European Union funded Research Projects at the cross-road of functional genomics [comprising transcriptomics, the measurement of the expression of all messengers RNA (mRNAs) and proteomics, the measurement of expression/state of all proteins], nutrition and human health. The goal of Functional Food Ingredients Against Colorectal Cancer is to develop a colon epithelial cell line-based screening assay for nutrients with presumed anti-colorectal carcinogenic properties. Genes involved in colon carcinogenesis are identified at the RNA and protein level, using a variety of methods (subtractive hybridisation, DNA microarray, proteomics) in combination with models for colorectal cancer development (human biopsies, rat model for colorectal carcinogenesis, colorectal cancer epithelial cell lines). Secondly, colorectal cancer epithelial cell lines are selected, in terms of their capacity to undergo gene/protein expression changes representing different phases in the colorectal carcinogenesis. Thirdly, these cell lines are used to determine the effects of nutrients with presumed anti-carcinogenic properties (e.g. resveratrol, flavonoids) on functional genomics-derived endpoints. Once validated against the effects of these nutrients in in vivo animal models and classical biomarkers for colorectal carcinogenesis, these cell line models combined with functional genomics represent useful tools to study colorectal carcinogenesis and screen for nutrients with anti-carcinogenic properties.


Assuntos
Neoplasias Colorretais/prevenção & controle , Alimentos Orgânicos , Fenômenos Fisiológicos da Nutrição/fisiologia , Animais , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Epitélio/patologia , Genômica , Humanos , Células Tumorais Cultivadas
11.
Mutat Res ; 434(3): 137-48, 1999 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-10486588

RESUMO

DNA repair mechanisms are fairly well characterized for nuclear DNA while knowledge regarding the repair mechanisms operable in mitochondria is limited. Several lines of evidence suggest that mitochondria contain DNA repair mechanisms. DNA lesions are removed from mtDNA in cells exposed to various chemicals. Protein activities that process damaged DNA have been detected in mitochondria. As will be discussed, there is evidence for base excision repair (BER), direct damage reversal, mismatch repair, and recombinational repair mechanisms in mitochondria, while nucleotide excision repair (NER), as we know it from nuclear repair, is not present.


Assuntos
Reparo do DNA , DNA Mitocondrial , Envelhecimento/genética , Alquilação , Animais , Núcleo Celular/metabolismo , Modelos Biológicos , Mutação , Estresse Oxidativo , Ratos , Recombinação Genética , Uracila/metabolismo
12.
Nucleic Acids Res ; 27(18): 3712-9, 1999 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10471741

RESUMO

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.


Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , Mitocôndrias Hepáticas/enzimologia , Nucleotídeos de Uracila/genética , Animais , Afidicolina/farmacologia , Sequência de Bases , DNA Ligases/metabolismo , DNA Polimerase gama , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta a Droga , Etilmaleimida/farmacologia , Cinética , Masculino , Mitocôndrias Hepáticas/genética , Inibidores da Síntese de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ratos , Ratos Wistar , Mapeamento por Restrição , Temperatura , Nucleotídeos de Uracila/metabolismo
13.
J Biol Chem ; 274(11): 7128-36, 1999 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-10066771

RESUMO

Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of approximately 37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein.


Assuntos
Endodesoxirribonucleases/isolamento & purificação , Proteínas de Escherichia coli , Mitocôndrias Hepáticas/enzimologia , Animais , Sequência de Bases , Catálise , Cromatografia em Gel , Cromatografia por Troca Iônica , DNA/efeitos dos fármacos , Dano ao DNA , Primers do DNA , Desoxirribonuclease (Dímero de Pirimidina) , Eletroforese em Gel de Poliacrilamida , Endodesoxirribonucleases/metabolismo , Membranas Intracelulares/enzimologia , Peso Molecular , Tetróxido de Ósmio/farmacologia , Ratos , Especificidade por Substrato
14.
Nucleic Acids Res ; 26(2): 662-8, 1998 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9421531

RESUMO

Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture. The primary product of this reaction is the DNA lesion 7-hydro-8-oxo-deoxyguanosine (8-oxo-dG). The DNA damage was quantitated using Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) in a gene-specific damage and repair assay. Assay conditions were refined to give incision at all enzyme-sensitive sites with minimal non-specific cutting. Cultured fibroblasts were exposed to photoactivated methylene blue under conditions that would produce an average of three oxidative lesions per double-stranded mitochondrial genome. Within 9 h, 47% of this damage had been removed by the cells. This removal was due to repair rather than to replication, cell loss or degradation of damaged genomes. The rate of repair was measured in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region. Fpg-sensitive alkali-resistant oxidative base damage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional activity.


Assuntos
Dano ao DNA , Reparo do DNA , DNA Mitocondrial/efeitos dos fármacos , Proteínas de Escherichia coli , Oxigênio/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Linhagem Celular , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , DNA-Formamidopirimidina Glicosilase , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Embrião de Mamíferos , Escherichia coli/enzimologia , Fibroblastos , Humanos , Luz , Azul de Metileno/química , Azul de Metileno/farmacologia , N-Glicosil Hidrolases/metabolismo , Oxigênio Singlete
15.
Nutr Cancer ; 32(2): 113-20, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9919621

RESUMO

As a substrate for poly(ADP-ribose) polymerase (PARP; EC, 2.4.2.30), an enzyme that is activated by DNA strand breaks and is thought to facilitate efficient DNA repair, NAD+ and its precursor nicotinic acid (niacin) are involved in the cellular defense against DNA damage by genotoxic compounds. In this study, the effect of nicotinic acid supplementation on cytogenetic damage and poly(ADP-ribosylation) was evaluated in a human population that is continuously exposed to genotoxic agents, e.g., smokers. By use of a placebo-controlled intervention design, 21 healthy smokers received supplementary nicotinic acid at 0-100 mg/day for 14 weeks. An increased niacin status, as assessed from blood nicotinamide concentrations and lymphocyte NAD+ concentrations, was observed in groups supplemented with 50 and 100 mg/day. This effect was most pronounced in subjects with lower initial NAD+ levels. An increased niacin status did not result in decreased hypoxanthine guanine phosphoribosyltransferase variant frequencies and micronuclei induction in peripheral blood lymphocytes (PBLs). Sister chromatid exchanges in PBLs, however, were increased after supplementation with nicotinic acid. This increase was positively associated with the daily dose of nicotinic acid. No effects of nicotinic acid supplementation were found for ex vivo (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced poly(ADP-ribosylation), although the small number of samples that could be analyzed (n = 12) does not allow firm conclusions. Because no evidence was found for a decrease in cigarette smoke-induced cytogenetic damage in PBLs of smokers after nicotinic acid supplementation of up to 100 mg/day, it is concluded that supplemental niacin does not contribute to a reduced genetic risk in healthy smokers.


Assuntos
Dano ao DNA/efeitos dos fármacos , Linfócitos/metabolismo , Niacina/administração & dosagem , Niacina/sangue , Poli Adenosina Difosfato Ribose/sangue , Fumar/efeitos adversos , Adulto , Humanos , Masculino , NAD/sangue , Niacinamida/sangue , Estado Nutricional , Placebos
16.
Cancer Epidemiol Biomarkers Prev ; 6(11): 943-8, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9367068

RESUMO

Chemical or physical modification of DNA may cause an increase in genomic mutations or other genetic alterations, which may ultimately result in the onset of cancer. To avoid these deleterious effects of DNA damage, humans possess DNA repair mechanisms. Decreased DNA repair, induced ex vivo by UV light or ionizing radiation in human peripheral blood lymphocytes (PBLs), has been associated with aging. The aim of this study was to investigate whether repair of DNA damage, after ex vivo exposure of PBLs obtained from smokers (n = 20) to (+/-)-anti-benzo(a)pyrene diolepoxide [(+/-)-anti-BPDE], which is a mixture of reactive metabolites from the environmental carcinogen benzo(a)pyrene, is also associated with age. Furthermore, age-related associations between ex vivo (+/-)-anti-BPDE-induced DNA repair and the frequency of endogenous cytogenetic damage (sister chromatid exchange frequencies and micronuclei frequencies) in PBLs were evaluated. A statistically significant negative association was observed between ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and age of the donors. Also, parameters of endogenous lymphocytic cytogenetic damage were negatively associated with ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and positively associated with age in this population. It is concluded that, with increasing age, a decrease in lymphocytic excision repair capacity may be responsible for increased lymphocytic DNA damage in smokers.


Assuntos
Envelhecimento/genética , Benzopirenos/farmacologia , Carcinógenos Ambientais/farmacologia , Dano ao DNA , Reparo do DNA , Fumar/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , Adulto , Benzopirenos/efeitos adversos , Carcinógenos Ambientais/efeitos adversos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Masculino , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/genética , Pessoa de Meia-Idade , Troca de Cromátide Irmã/genética
17.
Carcinogenesis ; 16(11): 2765-71, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7586197

RESUMO

In response to DNA damage, in particular DNA strand breaks, the proposed roles for normal tumour suppressor protein p53 are to increase the period of time available for DNA repair prior to replication, or to direct damaged cells into programmed cell-death. Since treatment of mammalian cells with (+/-)-anti-benzo[a]pyrene diolepoxide [(+/-)-anti-BPDE] --a mixture of metabolites comprising the most reactive (+)-anti-enantiomer of the full environmental carcinogen benzo[a]pyrene--has been shown to result in induction of DNA repair processes and consequently in DNA strand break formation, the aim of the present study was to investigate whether p53 accumulation is induced in (+/-)-anti-BPDE-treated phytohaemagglutinin-stimulated human peripheral blood lymphocytes (PBLs). Both immunocytochemical and immunoblot analysis indicated that treatment of PBLs with (+/-)-anti-BPDE results in p53 accumulation. Optimal accumulation was observed at 2.5 microM, while no increase of p53 levels was observed at concentrations < 2.5 microM and > 10 microM. Further, (+/-)-anti-BPDE-induced p53 accumulation in PBLs was found to be time-dependent with accumulation up to 24 h after the onset of treatment. Treatment of PBLs with 2.5 microM of (+/-)-anti-BPDE and 1 mM of 3-aminobenzamide, an inhibitor of the DNA strand break-dependent enzyme poly(ADP-ribose) polymerase, resulted in increased p53 levels, in comparison to cells treated with (+/-)-anti-BPDE alone. This combination also potentiated the frequency of (+/-)-anti-BPDE-induced micronuclei. These findings suggest that (+/-)-anti-BPDE-induced DNA strand break formation is responsible for the observed p53 accumulation. It is unlikely that poly(ADP-ribose) polymer formation is a prerequisite in the process of p53 accumulation, as triggered by DNA strand-break inducing agents like (+/-)-anti-BPDE. It is hypothesized that p53-dependent pathways may be activated in phytohaemagglutinin-stimulated human peripheral blood lymphocytes exposed ex vivo to (+/-)-anti-BPDE.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Carcinógenos/toxicidade , Linfócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Animais , Células Cultivadas , Dano ao DNA , Humanos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Masculino , Inibidores de Poli(ADP-Ribose) Polimerases , Coelhos
18.
Mutat Res ; 325(1): 31-7, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7521010

RESUMO

In order to study the relative importance of endogenous and environmental factors for the individual relation between DNA damage and DNA excision repair, a method was developed for measuring quantitatively the persistence of N2-deoxyguanosine adducts formed in non-stimulated isolated human peripheral blood lymphocytes after in vitro incubation with 0.2 microM (+/-)anti-BPDE, applying 32P-postlabeling. Total binding of radiolabeled (+/-)anti-BPDE to DNA and its removal has been studied previously in human peripheral blood lymphocytes, but the method presented here enables the direct investigation of repair of the main (+/-)anti-BPDE-DNA adduct, which is implicated in benzo[a]pyrene-induced mutagenesis. Using this method, it was found that in lymphocytes, obtained from 5 individuals, most (+/-)anti-BPDE-N2-dG adducts are removed within the first 24 h after treatment, while interindividual differences appear to exist in both adduct formation and rate and extent of removal.


Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/análogos & derivados , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Adutos de DNA , Dano ao DNA , Reparo do DNA/fisiologia , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Análise Mutacional de DNA/métodos , Desoxiguanosina/metabolismo , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Radioisótopos de Fósforo
19.
Carcinogenesis ; 15(4): 745-51, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8149490

RESUMO

Poly(ADP-ribose) polymerase, which catalyzes the formation of poly(ADP-ribose) polymers, is an enzyme involved in cell proliferation, differentiation and transformation as well as in recovery from DNA damage. Poly(ADP-ribose) polymers are rapidly synthesized from the ADP-ribose moieties from intracellular NAD+, which, as a consequence, is depleted. It has been shown that DNA strand breaks are required for enzyme activation and it is suggested that one of the functions of poly(ADP-ribosylation) is to improve accessibility of damaged sites to other DNA repair enzymes. The aim of this study was to investigate whether poly(ADP-ribosylation) is involved in repair of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE]-induced DNA damage in human lymphocytes in vitro. Results show that (+/-)-anti-BPDE is capable of inducing poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Also, repair of (+/-)-anti-BPDE induced DNA damage was confirmed by both unscheduled DNA synthesis and (+/-)-anti-BPDE-deoxyguanosine adduct removal. Based on these findings, it is concluded that poly(ADP-ribosylation) is involved in (+/-)-anti-BPDE-induced DNA repair in these cells. In addition, these results confirm the possible relation between poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation, after induction of DNA damage.


Assuntos
Reparo do DNA , Linfócitos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/toxicidade , Dano ao DNA , Humanos , Técnicas In Vitro , Ativação Linfocitária , NAD/metabolismo
20.
Mutat Res ; 319(4): 245-55, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7504197

RESUMO

In a previous study we found increased SCE frequencies in peripheral blood lymphocytes (PBLs) of workers occupationally exposed in a coal fly ash processing industry, as compared to a non-exposed control population. Shortly after this study, measures were taken in this plant to reduce fly ash levels. The objective of the present study, conducted 2 years later in the same plants, was to evaluate the effect of these measures with respect to genotoxic risk. A group of 18 male workers of the coal fly ash processing industry agreed to participate in the study. The control population consisted of 18 male workers from a flour processing industry, who were matched for age and smoking behavior. In contrast to our previous study, no increased SCE frequencies were found in PBLs of workers potentially exposed to coal fly ash when compared to the control group (mean SCEs: 6.4 +/- 1.2 and 7.0 +/- 0.9, respectively). In addition, no differences were observed between the exposed and control groups for frequencies of gene mutations at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus in PBLs, for micronucleus frequencies using the cytokinesis block method, or for urinary mutagen excretion measured with Salmonella typhimurium tester strains TA98 and TA97 with and without metabolic activation. In smokers, however, SCE frequencies in PBLs were significantly increased in comparison to non-smokers (7.1 +/- 1.1 vs. 6.1 +/- 0.5; P < 0.005), as was 24-h urinary mutagen excretion measured with strain TA98 with S9 mix (2373 +/- 1870 vs. 156 +/- 211; P < 0.001) and with TA98 with S9 mix and beta-glucuronidase/arylsulfatase (2361 +/- 1958 vs. 538 +/- 396; P < 0.005). In addition, hprt variant frequencies in PBLs were higher in smokers than in non-smokers (15.0 +/- 23.5 x 10(-6)6 vs. 2.6 +/- 2.8 x 10(-6); P < 0.05). No differences were observed for micronucleus induction between smokers and non-smokers. It is concluded that the protective measures taken in the coal fly ash processing plant appear to have been sufficient, since an effect of exposure to coal fly ash on parameters of genetic risk was not found any longer.


Assuntos
Carbono/efeitos adversos , Carvão Mineral/efeitos adversos , Resíduos Industriais/efeitos adversos , Mutagênicos/efeitos adversos , Exposição Ocupacional/prevenção & controle , Adulto , Cinza de Carvão , Humanos , Hipoxantina Fosforribosiltransferase/genética , Linfócitos/efeitos dos fármacos , Masculino , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mutagênicos/metabolismo , Material Particulado , Análise de Regressão , Risco , Troca de Cromátide Irmã/efeitos dos fármacos , Fumar/genética , Urina/química
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