Identification Of Potential Biomarker For N-methyl-N'-nitro-N-nitrosoguanidine Exposure In Mammalian Cells

Alkylating agents especially the N-Nitroso alkylating agents are widely present in the environment and DNA alkylation seems to be a common event. The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a model chemical of .W-Nitroso alkylating agent is a mutagen/carcinogen that targets cellular DNA and proteins to generate adducts. Among the adducts, O~6-alkyl guanine is the predominantly mutagenic lesion because of its mispairing properties, and these adducts can eventually lead to chromosomal aberrations, point mutations, and cell death. This lesion also appears to be involved in tumor initiation, particularly in gastric carcinogenesis.In our laboratory, a unique mutation detection system using a shuttle vector plasmid has been established to demonstrate that a low concentration of MNNG (0.2 μ M) can induce nontargeted mutation in mammalian cells: the mammalian cells were exposed to 0.2 μM MNNG for 2.5 h, then a shuttle plasmid pZ189 carrying supF tRNA gene was transfected into cells after 24h culture. We found a 5-fold higher mutation frequency of the plasmid replicated in pretreated cells than the spontaneous mutation frequency of the plasmid replicated in control cells. This kind of mutation did not occur immediately after MNNG exposure. Time-course analysisshowed that the frequency of MNNG induced nontargeted mutation increased gradually, reached the peak at 12 h after MNNG treatment, and then declined. The specific nontargeted mutation spectrum is different from that of targeted mutation, whereas the mutation occurs at damaged DNA site.Furthermore, we have demonstrated that low concentration MNNG exposure induced comprehensive cellular responses. For example, we have found the clustering of EGFR (epidermic growth factor receptor) and TNFR (tumor necrosis factor receptor) and the activation of endoplasmic reticulum stress and cAMP-PKA-CREB and JNK/SAPK pathways after MNNG treatment. It is even more interesting that the activation of these pathways seems to be independent of DNA damage, because these events can still occur in enucleated cells. In addition, more than 30 differential expressed sequence tags (EST) have been isolated by mRNA differential display. Among them, fragment 9 showed enhanced expression after MNNG exposure and protein synthesis inhibitor couldn't inhibit its expression. An expression plasmid with fragment 9 in antisense orientation was constructed to block the expression of the relevant gene (fragment 9 related gene, FNR gene) in vero cells. Interestingly, we found that the nontargeted mutation frequency induced by MNNG was increased significantly, implicating that the product of the blocked gene may be involved in the inhibition of nontargeted mutation. Therefore, it is important to study the profiles of gene expression, which will help understand the global cellular stress responses to chemical carcinogens, and further elucidate the mechanisms of nontargeted mutagenesis.Biomarkers play an important role in risk assessment, disease prediction, early detection, diagnosis, prognosis, disease monitoring, and evaluation of therapeutic response. The field of biomarker discovery is currently in an exciting and productive phase of development. Currently, there are many techniques available to screen the gene expression at the transcriptional levels, such as mRNA differential display andcDNA microarray, etc. Although these methods can provide high-throughput information about the differential gene expression, there were no cost-effective biomarkers for early detection and evaluation of prognosis developed. There are several reasons why the numerous and extensive previous transcriptomic analyses of disease may not have revealed all disease-associated proteins. These include (1) a lack of linear correlation between transcription and disease-associated protein levels;(2) translocation of a protein in the disease state rather than simply differential levels of the transcript;(3) levels of transcript cannot predict post-translational modification and interaction with other proteins.The analysis of cellular proteins expressed by a genome, by a cell or by a tissue, termed proteomics, represents a powerful analytic technology to enhance the study of the diagnosis, treatment and prevention of human disease. Combination of techniques including two dimensional gel electrophoresis (2-DE), mass spectrometry (MS), and bioinformatics, proteomics can be expected to show the changes in the protein expression profile during disease development and progression, thus leading to the identification of new molecular markers and potential therapeutic targets. Therefore, we used proteomic analysis to determine the effect of MNNG treatment on the protein expression profile of human amnion FL cells. Among the protein spots with altered intensity, finally 36 proteins were identified by MALDI-TOF MS. It is quite possible that some of the proteins identified could be used as biomarkers for MNNG exposure. However, in a practical sense, the most common sample types for population monitoring are obtained through noninvasive procedures, thus the best biomarkers should be peptide/protein appeared in the biofluids which may be shed from the membrane or secreted/outpoured from the exposed cells. Body fluid such as blood, urine, gastric juice and saliva has the advantage of being more easily accessible than tissue because procuring body fluid is a non-invasive procedure. Taking practical application into account, candidate biomarkers in body fluid wouldbenefit patients enormously by a greater availability of such effective molecular indicators that can be monitored noninvasively from readily accessible bodily fluids. However, most of the known biomarkers in blood occur at very low abundance and would not be revealed directly by proteomic technology. One strategy to solve such problem involves using a secondary tissue or fluid of interest to identify potential candidates, followed by screening the complementary plasma sample to detect those candidates.Therefore, in an effort to search for such biomarker and further probe the molecular mechanism of mutagenesis, the culture medium for MNNG exposed cells, which might mimic the biofluid to some degree, was subjected to 2-DE analyses. Proteins of serum-free culture medium were concentrated and subjected to isoelectric focusing (IEF) on an IPG strip (pH 3-10 NL, 24cm). The second separation was carried out on 12.5% SDS slab gels and silver-staining was performed. The digitized images then were analyzed with Phoretix 2D 6.01 software in order to establish the differential expression profiles between the MNNG-treated and control. In the results, there were 12 protein spots appeared and 4 protein spots up-regulated after MNNG treatment as determined by spot volume (.PO.05).Differential expressed protein spots were cut from the gels and subjected to in-gel digestion with trypsin. Peptide mixtures analyzed with a voyager-DE STR MALDI-TOF mass spectrometer using a delayed ion extraction and ion mirror reflector mass spectrometer (Applied Biosystems). Proteins were identified using the MASCOT software. The database (NCBInr) searches were performed using the following parameters: homo sapiens (human), trypsin digest (allowed up to 1 missed cleavage), cysteine as carbamidomethylated, methionine as oxidized, mass tolerance of 100 ppm using internal calibration. A total of 13 proteins were successfully identified with high confidence. These proteins include nuclear isoform of dUTP pyrophosphatase (DUT-N), phosphoglycerate mutase 1 (PGAM1), heparin sulfateproteoglycan perlecan (HSPG), etc, which are involved in multiple cellular functions. Interestingly, 2-DE and MS analysis of cell lysate exposed to MNNG revealed that DUT-N was down-regulated. To further confirm the 2-DE results, the appearance of DUT-N in culture medium and its down-regulation in cell lysate was confirmed by Western blot. These data suggested that these proteins, especially DUT-N, could be used as candidate biomarkers for monitoring MNNG exposure.Conclusion:2-DE coupled with MS is an effective technique to screen the differentially expressed proteins of culture medium between MNNG-treated and control. These proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens and offer new insights into the mechanisms of low concentration MNNG-induced nontargeted mutagenesis.