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Genome-wide screening of temporal responsive genes induced by a low concentration of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine in a normal human cell line

ABSTRACT: The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct mutagen and carcinogen, causing DNA damage and other comprehensive alterations that lead to chromosomal aberrations, mutations, tumor initiation, and cell death. Our previous study revealed that MNNG at different concentrations could induce extensive changes in gene expression at an early stage of exposure. To further understand the dynamic cellular responses and hazardous effects caused by this environmental carcinogen, we used a whole-genome time-course screening methods to find out the gene expression changes induced by a low concentration of MNNG in human normal amnion epithelial FL cells. The cells were exposed to 1.0 µM MNNG, and differential gene expression profiles at 3, 12, and 24 h after MNNG treatment were obtained by use of Affymetrix HG-U133 Plus 2.0 oligonucleotide microarray technology, followed by quantitative real-time RT-PCR validation. The results showed that the low-dose MNNG exposure triggered extensive but moderate changes in gene expression at these three experiment time points after exposure. The responsive genes encode important proteins, including cell cycle regulators, transcription factors and signal transducers that determine cell cycle progression, cell fate and other activities associate with pro-oncogenic potentials. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after MNNG exposure. These results will aid our understanding of the complicated mechanisms of MNNG-induced cellular responses. Keywords：N-methyl-N'-nitro-N-nitrosoguanidine; Cellular response; Temporal gene expression change; Oligonucleotide microarray; Quantitative real-time RT-PCR

ORGANISM(S): Homo sapiens

PROVIDER: GSE21270 | GEO | 2013/07/01

SECONDARY ACCESSION(S): PRJNA126257

REPOSITORIES: GEO

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Project description:The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct mutagen and carcinogen, causing DNA damage and other comprehensive alterations that lead to chromosomal aberrations, mutations, tumor initiation, and cell death. Our previous study revealed that MNNG at different concentrations could induce extensive changes in gene expression at an early stage of exposure. To further understand the dynamic cellular responses and hazardous effects caused by this environmental carcinogen, we used a whole-genome time-course screening methods to find out the gene expression changes induced by a low concentration of MNNG in human normal amnion epithelial FL cells. The cells were exposed to 1.0 M-BM-5M MNNG, and differential gene expression profiles at 3, 12, and 24 h after MNNG treatment were obtained by use of Affymetrix HG-U133 Plus 2.0 oligonucleotide microarray technology, followed by quantitative real-time RT-PCR validation. The results showed that the low-dose MNNG exposure triggered extensive but moderate changes in gene expression at these three experiment time points after exposure. The responsive genes encode important proteins, including cell cycle regulators, transcription factors and signal transducers that determine cell cycle progression, cell fate and other activities associate with pro-oncogenic potentials. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after MNNG exposure. These results will aid our understanding of the complicated mechanisms of MNNG-induced cellular responses. KeywordsM-oM-<M-^ZN-methyl-N'-nitro-N-nitrosoguanidine; Cellular response; Temporal gene expression change; Oligonucleotide microarray; Quantitative real-time RT-PCR Human amnion epithelial FL cells were exposed to vehicle control (dimethyl sulfoxide) and a low concentration (1.0 M-BM-5M) MNNG for 2 h, respectively. The differential gene expression profiles at 3, 12 and 24 h post MNNG treatment were obtained using Affymetrix HG-U133 Plus 2.0 oligonucleotide microarrays. The transcriptomic changes at different time points post MNNG treatment would provide insight into the dynamic processes of cellular response to this genotoxic agent.

Project description:N-nitroso compounds (NOC) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Since it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using ESR spectroscopy, we investigated radical generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosurea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon centered radicals which was further stimulated in presence of Caco-2 cells. N-methyl-N-nitrosurea exposure resulted in a small ROS signal, and formation of nitrogen centered radicals (NCR), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns which may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation. Keywords: Nitrosamines, nitrosamides, N-nitroso compounds, free radicals, toxicogenomics, colon carcinogenesis The study investigated differential gene expression in Caco-2 cell line mRNA following 1, 6 or 24 hours of exposure to six different N-nitroso compounds. Two biological replicates per sample compound. One compound per array, hybridized against vehicle control. Dye-swap between biological replicates.

Project description:N-nitroso compounds (NOC) are genotoxic compounds and animal carcinogens, and may play a role in human cancer development. Since the gastro-intestinal tract is an important route of exposure, we hypothesize that NOC exposure targets genetic processes relevant in colon carcinogenesis. To investigate this, we analysed the transcriptomic effects of genotoxic concentrations of two nitrosamides, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1ÂµM) and N-methyl-N-nitrosurea (MNU, 1mM), and four nitrosamines, N-nitrosodiethylamine (NDEA, 50mM), N-nitrosodimethylamine (NDMA, 100mM), N-nitrosopiperidine (NPIP, 40mM), and N-nitrosopyrrolidine (NPYR, 100mM), in the human colon carcinoma cell line Caco-2. Gene Ontology gene group, consensus motif gene group and biological pathway analysis revealed that nitrosamides had little effect on gene expression after 24 hours of exposure, whereas nitrosamines had a strong impact on the transcriptomic profile. Analyses showed modifications of cell cycle regulation and apoptosis pathways for nitrosamines which was supported by flow cytometric analysis. We found additional modifications in gene groups and pathways of oxidative stress and inflammation, which suggest an increase in oxidative stress and pro-inflammatory immune response upon nitrosamine exposure, although less distinct for NDMA. Furthermore, NDEA, NPIP and NPYR most strongly affected several developmental motif gene groups and pathways, which may influence developmental processes. Many of these pathways and gene groups are implicated in the carcinogenic process and their modulation by nitrosamine exposure may therefore influence the development of colon cancer. In summary, our study has identified pathway modifications in human colon cells which may be associated with cancer risk of nitrosamine exposure in the human colon. Keywords: Comparison of genome-wide gene expression between different conditions The study investigated differential gene expression in Caco-2 cell line mRNA following 24 hours of exposure to six different N-nitroso compounds. Four biological replicates per sample compound. One compound per array, hybridized against vehicle control. Dye-swap between biological replicates 1 and 2, and 3 and 4.

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Genome-wide screening of temporal responsive genes induced by a low concentration of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine in a normal human cell line

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