RESUMO
An emerging vision for toxicity testing in the 21st century foresees in vitro assays assuming the leading role in testing for chemical hazards, including testing for carcinogenicity. Toxicity will be determined by monitoring key steps in functionally validated molecular pathways, using tests designed to reveal chemically-induced perturbations that lead to adverse phenotypic endpoints in cultured human cells. Risk assessments would subsequently be derived from the causal in vitro endpoints and concentration vs. effect data extrapolated to human in vivo concentrations. Much direct experimental evidence now shows that disruption of epigenetic processes by chemicals is a carcinogenic mode of action that leads to altered gene functions playing causal roles in cancer initiation and progression. In assessing chemical safety, it would therefore be advantageous to consider an emerging class of carcinogens, the epigenotoxicants, with the ability to change chromatin and/or DNA marks by direct or indirect effects on the activities of enzymes (writers, erasers/editors, remodelers and readers) that convey the epigenetic information. Evidence is reviewed supporting a strategy for in vitro hazard identification of carcinogens that induce toxicity through disturbance of functional epigenetic pathways in human somatic cells, leading to inactivated tumour suppressor genes and carcinogenesis. In the context of human cell transformation models, these in vitro pathway measurements ensure high biological relevance to the apical endpoint of cancer. Four causal mechanisms participating in pathways to persistent epigenetic gene silencing were considered: covalent histone modification, nucleosome remodeling, non-coding RNA interaction and DNA methylation. Within these four interacting mechanisms, 25 epigenetic toxicity pathway components (SET1, MLL1, KDM5, G9A, SUV39H1, SETDB1, EZH2, JMJD3, CBX7, CBX8, BMI, SUZ12, HP1, MPP8, DNMT1, DNMT3A, DNMT3B, TET1, MeCP2, SETDB2, BAZ2A, UHRF1, CTCF, HOTAIR and ANRIL) were found to have experimental evidence showing that functional perturbations played "driver" roles in human cellular transformation. Measurement of epigenotoxicants presents challenges for short-term carcinogenicity testing, especially in the high-throughput modes emphasized in the Tox21 chemicals testing approach. There is need to develop and validate in vitro tests to detect both, locus-specific, and genome-wide, epigenetic alterations with causal links to oncogenic cellular phenotypes. Some recent examples of cell-based high throughput chemical screening assays are presented that have been applied or have shown potential for application to epigenetic endpoints.
Assuntos
Testes de Carcinogenicidade , Epigênese Genética , Epigenômica , Trifosfato de Adenosina/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Carcinógenos/farmacologia , Carcinógenos/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Metilação de DNA , Epigenômica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Histonas/metabolismo , Humanos , Modelos Biológicos , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA não Traduzido/genética , Medição de Risco , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. Furthermore, many microarrays lack control features that can be used for quality assurance (QA). Here, we describe a novel external control series integrated with a design feature that addresses the above issues. RESULTS: An EC dilution series that involves spike-in of a single concentration of the A. thaliana chlorophyll synthase gene to hybridize against spotted dilutions (0.000015 to 100 microM) of a single complimentary oligonucleotide representing the gene was developed. The EC series is printed in duplicate within each subgrid of the microarray and covers the full range of signal intensities from background to saturation. The design and placement of the series allows for QA examination of frequently encountered problems in hybridization (e.g., uneven hybridizations) and printing (e.g., cross-spot contamination). Additionally, we demonstrate that the series can be integrated with a LOWESS normalization to improve the detection of differential gene expression (improved sensitivity and predictivity) over LOWESS normalization on its own. CONCLUSION: The quality of microarray experiments and the normalization methods used affect the ability to measure accurate changes in gene expression. Novel methods are required for normalization of small focused microarrays, and for incorporating measures of performance and quality. We demonstrate that dilution of oligonucleotides on the microarray itself provides an innovative approach allowing the full dynamic range of the scanner to be covered with a single gene spike-in. The dilution series can be used in a composite normalization to improve detection of differential gene expression and to provide quality control measures.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Animais , Arabidopsis/genética , Artefatos , Canadá , Reações Falso-Negativas , Reações Falso-Positivas , Perfilação da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Controle de Qualidade , RNA Complementar/genética , RNA Mensageiro/genética , Ratos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The ability of tumour promoters to alter DNA stability within regions that contain tandemly repeated sequences (TRSs), was studied in a cell culture model of multi-stage carcinogenesis. Non-cytotoxic concentrations of TPA (12-O-tetradecanoyl-phorbol-13-acetate) and xanthine oxidase with xanthine substrate, sufficient to promote morphological transformation in C3H/10T1/2 cultures, were tested for their effects on mutation frequencies in TRSs by a DNA fingerprinting approach. Specifically, restriction digests of genomic DNA samples from randomly selected, non-transformed clones, isolated from cultures after several days exposure to promoters, were visualized by Southern hybridizations with the multi-locus pentamer repeat sequence probe, Ms6-Hm (Pc-1). Basal and promoter-induced frequencies of sub-clone TRS fingerprint polymorphisms were estimated in five cell populations: an uncloned stock culture, three populations established from normal-appearing sub-clones, and one clonal population established from a 3-methylcholanthrene (MCA)-transformed focus. Basal variant fingerprint frequencies spanned a range from 0.0 to 0.43% mutants/band among cells from the four untransformed populations. Both TPA and xanthine oxidase treatments significantly increased recorded mutation frequencies, 2.3- and 2.7-fold, respectively, using combined data from the progenitor population and three untransformed clones. The untreated MCA-transformed clonal population appeared to contain a single, pre-existing mutant restriction fragment, but additional mutations were induced thereafter, in response to the promoting treatments. Taken together, the measured increases in mutations were highly significant and suggest that promoters of cell transformation in the C3H/10T1/2 cell line might induce a genome-wide instability targeted to regions containing Ms6-Hm sequence motifs.
Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , DNA/efeitos dos fármacos , Animais , Sequência de Bases , Carcinógenos/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais , DNA/química , Impressões Digitais de DNA , Primers do DNA , Camundongos , Camundongos Endogâmicos C3H , Estresse Oxidativo/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaAssuntos
Sondas de DNA/genética , Sondas de DNA/efeitos da radiação , Hibridização In Situ/métodos , Nylons/química , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Sondas RNA/genética , Raios Ultravioleta , Reagentes de Ligações Cruzadas , Sondas de Oligonucleotídeos/química , Sondas RNA/químicaRESUMO
Mitogen-regulated protein/proliferin (mrp/plf) gene family transcripts rise in abundance as a response to diverse chemical and physical agents that promote morphological transformation in the murine C3H/10T1/2 cultured cell model of multi-step carcinogenesis. To determine if proliferin genes respond to tumor promoters in vivo, RNA was extracted from the whole skin of SENCAR mice after single applications of 2 or 20 microg 12-O-tetradecanoylphorbol-13-acetate (TPA); 3.2 or 32 nmole), 20 or 40 mg benzoyl peroxide (BPO; 83, 165 micromole), or acetone vehicle alone (2.72 mmole). RNA samples were prepared from treated skin areas, 2-48 h after painting. Mrp/plf-mRNA was not detected in Northern blot hybridizations, but large increases in mRNAs for ornithine decarboxylase gene and mRNA (odc), v-jun oncogene-related transcription factor gene and mRNA (junB), egr1 (early growth response protein gene and mRNA) were measured relative to beta 2 microglobulin gene and mRNA (b2m) mRNA in response to TPA. BPO induced small relative changes in these mRNAs. Reverse transcriptase (RT)-polymerase chain reactions (PCR) detected fully-processed MRP/plf-mRNA 16-48 h after TPA treatments in five of six animals, and in three of six BPO-treated animals. The MRP/plf-mRNA species expressed in the skin were predominantly plf1 and mrp3 as determined by gene-specific restriction enzyme sites within the RT-PCR products. Expression was either undetectable or found at low levels in acetone-painted controls and was not detected during the anagen phase of the normal hair growth cycle in unpainted animals. These results demonstrate that mrp/plf-mRNA is differentially expressed in murine skin in response to mechanistically distinct tumor promoters and has potential utility as a short-term biomarker for tumor promoting effects in chemical carcinogenesis.
Assuntos
Carcinógenos/toxicidade , Glicoproteínas/genética , RNA Mensageiro/genética , Pele/patologia , Acetona , Animais , Peróxido de Benzoíla/toxicidade , Northern Blotting , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Camundongos , Camundongos Endogâmicos C3H , Prolactina , Regiões Promotoras Genéticas , Pele/efeitos dos fármacos , Acetato de Tetradecanoilforbol/toxicidade , Transcrição Gênica/efeitos dos fármacosRESUMO
Increases in proliferin (PLF) gene family mRNA abundance and promotional effects in cell transformation assays are paired responses that follow exposures to diverse chemical and physical agents in the C3H/10T1/2 in vitro model of multi-stage carcinogenesis. This study measured PLF mRNA abundance changes over 1 to 3 d in response to several types of promoters that were previously unassessed for this effect. Saccharin is a known promoter of cell transformation in C3H/10T1/2 cell cultures, but unlike 12-O-tetradecanoylphorbol 13-acetate (TPA) or mezerein, PLF mRNA abundance increases were inconsistently detected following simple addition of saccharin to the culture medium. Consistent effects occurred when pretreatments with promoting concentrations of saccharin or sodium saccharin (1-13 mM) were combined with subsequent additions of serum or complete medium changes. When added at or near their lowest observed effect levels (LOELs) for transformation, other promoters of 10T1/2 cells such as formaldehyde (50-100 microM), diethylstilbesterol (DES) (0.5-30 microM), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (4-40 pM) were shown to be inducers of both basal and serum-induced PLF mRNA levels. Acetaldehyde (300-900 microM) was comparable to formaldehyde as an inducer. In contrast to these various promoters, pretreatment with phenobarbital or methanol, both non-promoters in these cells, did not affect serum-induced PLF mRNA levels at concentrations up to 3 mM and 2 M, respectively. The published values for the LOELs of 17 promoters of cell transformation and the LOELs determined to date for PLF mRNA induction were highly correlated over a 1 billion - fold concentration range. The response of PLF mRNA is a short-term marker sensitive to the active concentration ranges of diverse chemical agents with promotional activity in C3H/10T1/2 cell transformation system.
Assuntos
Carcinógenos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Glicoproteínas/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , RNA Mensageiro/biossíntese , Sacarina/farmacologia , Edulcorantes/farmacologia , Teratogênicos/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Glicoproteínas/sangue , Glicoproteínas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , ProlactinaRESUMO
Hundreds of thousands of people worldwide live or work in close proximity to steel mills. Integrated steel production generates chemical pollution containing compounds that can induce genetic damage (1, 2). Previous investigations of herring gulls in the Great Lakes demonstrated elevated DNA mutation rates near steel mills (3, 4) but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden. To address these issues experimentally, we exposed laboratory mice in situ to ambient air in a polluted industrial area near steel mills. Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated steel mills was 1.5- to 2.0-fold elevated compared with those at a reference site 30 km away. This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germline. Our results indicate that human and wildlife populations in proximity to integrated steel mills may be at risk of developing germline mutations more frequently because of the inhalation of airborne chemical mutagens.