Adducts formed by the food mutagen 2-amino-3-methylimidazo(4, 5-f ) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway
Abstract:The potency of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) adducts to induce -2, -1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines. The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system. We have shown that IQ adducts induce very efficiently both -1 and -2 frameshift mutat… Show more
“…Upon induction of the SOS response, the reversion frequency in the absence of MB+light treatment is increased about 10-fold for both pTc s 6(G) and pTc s 2(GC) plasmids (Figures a and a). The induction of the SOS response by UV irradiation may enhance the spontaneous mutation frequency by promoting a more efficient elongation of the slipped mutagenic intermediates ( , ). 2 MB+light-induced −1 frameshift mutagenesis within a run of six contiguous guanines.…”
By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli. The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively. The SOS and umuDC dependences of these mutagenic processes have been investigated. Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced. However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon. In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis. Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E. coli.
“…Upon induction of the SOS response, the reversion frequency in the absence of MB+light treatment is increased about 10-fold for both pTc s 6(G) and pTc s 2(GC) plasmids (Figures a and a). The induction of the SOS response by UV irradiation may enhance the spontaneous mutation frequency by promoting a more efficient elongation of the slipped mutagenic intermediates ( , ). 2 MB+light-induced −1 frameshift mutagenesis within a run of six contiguous guanines.…”
By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli. The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively. The SOS and umuDC dependences of these mutagenic processes have been investigated. Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced. However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon. In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis. Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E. coli.
“…The mutagenicity of IQ was reported to be SOS independent (66). Thus, the interaction between DNA polymerases and IQ-adducts may share properties with both AF and AAF.…”
Abstract2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is found in cooked meats and forms DNA adducts at the C8-and N 2 -positions of dGuo after appropriate activation. IQ is a potent inducer of frameshift mutations in bacteria and is carcinogenic in laboratory animals. We have incorporated both IQ-adducts into the G 1 -and G 3 -positions of the NarI recognition sequence (5′-G 1 G 2 CG 3 CC-3′), which is a hotspot for arylamine modification. The in vitro replication of the oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo − , E. coli pol II exo − , and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and the extension products were sequenced by tandem mass spectrometry. Replication of the C8-adduct at the G 3 -position resulted in two-base deletions with all three polymerases, whereas error-free bypass and extension was observed at the G 1 -position. The N 2 -adduct was bypassed and extended by all three polymerases when positioned at the G 1 -position, and the error-free product was observed. The N 2 -adduct at the G 3 -position was more blocking and was bypassed and extended only by Dpo4 to produce an error-free product. These results indicate that the replication of the IQ-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct. These results also suggest a possible role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to frameshift mutations in reiterated sequences, whereas noniterated sequences result in error-free bypass.
“…With indirect or inert chemical carcinogens, a bioactivation step is required (mediated primarily by cytochrome P450 mixed function oxidases (CYPs), but also by N -acetyltransferases (NATs) or sulfotransferases) to generate electrophilic intermediates that then give rise to covalent DNA adducts at nucleophilic sites on DNA bases, for example, C8 position of deoxyguanosine [100, 101]. The generation of base substitution or frameshift mutations then occurs via the inaccurate repair of the adducted template, which has been associated with the replication complex performing a mutagenic bypass of the lesion by a slippage mechanism [102, 103]. The question is how does the epigen determine the magnitude of the mutagen or genotoxin effect?…”
Epidemiological studies have consistently supported the notion that environmental and/or dietary factors play a central role in the aetiology of cancers of the breast and prostate. However, for more than five decades investigators have failed to identify a single cause-and-effect factor, which could be implicated; identification of a causative entity would allow the implementation of an intervention strategy in at-risk populations. This suggests a more complex pathoaetiology for these cancer sites, compared to others. When one examines the increases or decreases in incidence of specific cancers amongst migrant populations, it is notable that disease arising in colon or stomach requires one or at most two generations to exhibit a change in incidence to match that of high-incidence regions, whereas for breast or prostate cancer, at least three generations are required. This generational threshold could suggest a requirement for nonmutation-driven epigenetic alterations in the F0/F1 generations (parental/offspring adopting a more westernized lifestyle), which then predisposes the inherited genome of subsequent generations to mutagenic/genotoxic alterations leading to the development of sporadic cancer in these target sites. As such, individual susceptibility to carcinogen insult would not be based per se on polymorphisms in activating/detoxifying/repair enzymes, but on elevated accessibility of crucial target genes (e.g., oncogenes, tumour suppressor genes) or hotspots therein to mutation events. This could be termed a genomic susceptibility organizational structure (SOS). Several exposures including alcohol and heavy metals are epigens (i.e., modifiers of the epigenome), whereas others are mutagenic/genotoxic, for example, heterocyclic aromatic amines; humans are continuously and variously exposed to mixtures of these agents. Within such a transgenerational multistage model of cancer development, determining the interaction between epigenetic modification to generate a genomic SOS and genotoxic insult will facilitate a new level of understanding in the aetiology of cancer.
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