Journal of Toxins
Mutagenicity Analysis of C8-Phenoxy-Guanine in the NarI Recognition DNA Sequence
Anne MR Verwey1, Aaron A Witham1, Mei Li2 and Richard A Manderville1*
- 1Departments of Chemistry and Toxicology, University of Guelph, Canada
- 2State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, PR China
*Address for Correspondence: Richard A Manderville, Departments of Chemistry and Toxicology, University of Guelph, Canada N1G 2W1, Tel: (519)-824-4120, x53963; Fax: (519)-766-1499; E-mail: firstname.lastname@example.org
Citation: Verwey AMR, Witham AA, Li M, Manderville RA, et al. Mutagenicity Analysis of C8-Phenoxy-Guanine in the NarI Recognition DNA Sequence. J Toxins. 2014;1(1): 6.
Copyright © 2014 Manderville et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Toxins | ISSN: 2328-1723 | Volume: 1, Issue: 1
Submission: 31 July 2014 | Accepted: 06 August 2014 | Published: 11 August 2014
Reviewed & Approved by: Dr. Guangming Xiong, Head of the Pharmacology and Toxicology, University Kiel, Germany.
AbstractPhenoxyl radicals can covalently attach to the C8-site of 2′-deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To determine the mutagenicity of an O-linked C8- dG adduct, C8-phenoxy-dG (PhOdG) was incorporated into the G3 position (X) of the NarI recognition sequence within a 22-mer oligonucleotide template (5′-CTCGGCX-CCATCCCTTACGAGC, where X = dG, or PhOdG) using solid-phase DNA synthesis. The NarI(22) template was annealed to a 15-mer primer and in vitro mutagenicity was assessed using primer-extension assays with a high-fidelity replicative polymerase, Escherichia coli pol I Klenow fragment exo− (Kf−), and a lesion-bypass Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). These studies predict that the O-linked C8-dG lesion PhOdG will have a low mutagenic effect, and is unlikely to contribute strongly to phenol toxicity.
KeywordsIron; Copper; Zinc; Manganese; Meat; Meat products; ICP-OES
AbbreviationsdG: 2′-deoxyguanosine; PhOdG: C8-(phenoxy)-2′-deoxyguanosine; ROS: Reactive oxygen species; Kf−: Escherichia coli pol I Klenow fragment exo−; Dpo4: Y-family lesion-bypass DNA polymerase IV
IntroductionPhenols are ubiquitous compounds that possess many biological properties including toxicity [1-3]. Human exposure to phenolic toxins occurs predominately through industrial activities, tobacco smoke, and inhalation of polluted air [4,5]. Phenol toxicity stems from their oxidative metabolism by peroxidase or cytochrome P450 enzymes to generate hydroquinone/quinone redox pairs, reactive oxygen species (ROS) and phenoxyl radicals that are well-known DNA-damaging agents [1,6-8]. Human exposure to damaging radicals [9,10] and quinone electrophiles [6,7] are associated with cancer and aging.
23]. The NarI sequence is a hot-spot for frameshift mutations mediated by the N-linked C8-dG adduct of N-acetyl- 2-aminofluorene (AAF) [19-21]. Within the NarI(12) duplex, the PhOdG lesion was shown to adopt the major groove B conformation opposite C, with minimal disruption to the duplex structure . These findings correlated with the conformational preference for the corresponding single-ringed N-linked C8-dG adduct produced by aniline . On the basis of this comparison, the PhOdG lesion was predicted to be weakly mutagenic .
Materials and MethodsMaterials
Results and DiscussionPrimer extension by Klenow fragment exo− (Kf−) Single nucleotide insertion assays using Kf− were carried out using the NarI(22):15mer template:primer (Figure 2). On the unmodified NarI(22) template (X = dG), Kf− mainly incorporated the correct base C (~ 82%, Figure 2b). At 20 nM enzyme concentration, significant amounts of G (~37%) and T (~30%) were also incorporated opposite dG during the 60 min reaction time. On the modified NarI(22) template (X = PhOdG), the polymerase again mainly inserted the correct base C (~70%, Figure 2b). However, a higher relative frequency of a second C (10%) and a slight increase in the insertion of A compared to the unmodified template was observed. The PhOdG adduct also inhibited misincorporation of G and T compared to the unmodified template (Figure 2b).
In the presence of all four dNTPs (Figure 3), extension past PhOdG was stalled after the incorporation of one nucleotide (presumably C across from PhOdG), though the full length extension product was clearly obtained. The observed stalling at position 1 (numbering on gel in Figure 3) is in agreement with previous reports that extension past bulky C8-dG adducts is more difficult than base insertion opposite the lesion . An 8th band for one base additional extension beyond the template strand was also observed, which is a typical non-template-dependent extension from a blunt end .
ConclusionsThe current study has allowed us to conclude the following: (1) the single-ringed oxygen-linked C8-phenoxy-dG adduct (PhOdG) does not strongly impede the progress of DNA replication by either Kf− or Dpo4 when inserted into the NarI(22) template and annealed to a 15-mer primer. Both the high-fidelity polymerase Kf− and the lesion-bypass polymerase Dpo4 are able to fully extend the 15-mer primer in the presence of the PhOdG lesion, although PhOdG causes some stalling after the first incorporation opposite the adduct. (2) In single nucleotide insertion assays, the PhOdG adduct does not strongly alter the relative frequency of dNTP incorporation compared to insertion opposite dG. Overall, these results suggest that PhOdG is a weakly mutagenic lesion, which correlates with our earlier prediction for PhOdG based on its structural characteristics within the NarI(12) duplex. These findings suggest that the O-linked PhOdG adduct will not strongly contribute to phenol toxicity. Our results are the first to report the in vitro mutagenicity of an oxygen-linked biaryl ether C8-dG adduct and provide a basis for comparison to other O-linked C8-dG adducts derived from phenolic toxins.
AcknowledgementsSupport for this research was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation, the Ontario Innovation Trust Fund.
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