| Monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a widely spread environmental mutagen and carcinogen that targets DNA and proteins to generate adducts. Among the adducts, O6-alkyl guanine is the predominant mutagenic lesion because of its mispairing properties, which 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. However, DNA damage has been ruled out as the prerequisite for alkylating agents-induced mutagenesis. For example, somatic hypermutation, which occurred around the variable region in the immunoglobin gene of B cells, is driven by antigen activation not by DNA damage and is considered a kind of active mutation. Environmental mutagens can also induce mutations at undamaged sites and result in the so-called nontargeted mutation. This kind of mutation may be not only induced by DNA damage but also through the epigenetic pathway other than the nuclear origin including the activation of various signal transduction pathways and altered expressions of many genes. Similarly, the cellular response to the environmental mutagens is not always initiated by DNA damage. Exposure to genotoxic agents would trigger a series of comprehensive and complex responses in cells to counteract the abnormal conditions, which include the rapid activation of signal transduction pathways independent of the nuclear damage signal and late alteration of gene expression.In our laboratory, a unique mutation detection system using a shuttle vectorplasmid has been established to demonstrate that a low concentration of MNNG (0.2 M) can induce nontargeted mutation in mammalian cells: the mammalian cells were exposed to 0.2M MNNG for 2.5h, 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 analysis showed 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 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 the alteration. An expression vector 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.Currently there are many techniques available to screen the gene expression at thetranscriptional levels, such as mKNA differential display and cDNA microarray. Although these methods can provide high-throughput information about the differential gene expressi...
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