Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun 21;23(6):1076-88.
doi: 10.1021/tx100053n.

The C8-2'-deoxyguanosine Adduct of 2-amino-3-methylimidazo[1,2-d]naphthalene, a Carbocyclic Analogue of the Potent Mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, Is a Block to Replication in Vitro

Affiliations
Free PMC article

The C8-2'-deoxyguanosine Adduct of 2-amino-3-methylimidazo[1,2-d]naphthalene, a Carbocyclic Analogue of the Potent Mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, Is a Block to Replication in Vitro

Plamen P Christov et al. Chem Res Toxicol. .
Free PMC article

Abstract

2-Amino-3-methylimidazo[1,2-d]naphthalene (cIQ) is a carbocyclic analogue of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in which a naphthalene ring system replaces the quinoline unit of IQ. The activity of cIQ in Ames Salmonella typhimurium tester strain TA98 is known to be 4-5 orders of magnitude lower than IQ. cIQ undergoes efficient bioactivation with rat liver microsomes. The C8-dGuo adduct was formed when calf thymus DNA was treated with the N-hydroxy-cIQ metabolite and either acetic anhydride or extracts from cells that overexpress N-acetyl transferase (NAT). These studies indicate that bioactivation, the stability of the N-hydroxylamine ester, and the reactivity of the nitrenium ion with DNA of cIQ are similar to IQ and that none of these factors account for the differences in mutagenic potency of these analogues in Ames assays. Oligonucleotides were synthesized that contain the C8-dGuo adduct of cIQ in the frameshift-prone CG-dinucleotide repeat unit of the NarI recognition sequence. We have examined the in vitro translesion synthesis of this adduct and have found it to be a strong replication block to Escherichia coli DNA polymerase I, Klenow fragment exo(-) (Kf(-)), E. coli DNA polymerase II exo(-) (pol II(-)), and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Previous studies by Fuchs and co-workers identified E. coli pol II as the polymerase responsible for two-base deletions of the C8-dGuo adduct of N-acetyl-2-aminofluorene in the NarI sequence. Our observation that pol II is strongly inhibited by the C8-dGuo adduct of cIQ suggests that one of the other SOS inducible polymerases (E. coli pol IV or pol V) is required for its bypass, and this accounts for the greatly attenuated mutagenicity in the Ames assays as compared with IQ.

Figures

Figure 1
Figure 1
Structure and relative mutagenicity (revertants/μg) of IQ, IQx and analogues in Ames tester strain TA98
Figure 2
Figure 2
Microsomal oxidation products of cIQ. A. ESI-LC-MS chromatogram (m/z 214) of the oxidation of cIQ with rat liver microsomes. B. Mass spectrum of the major (top) and minor (bottom) oxidation product of cIQ C. Relative peak areas of the microsomal oxidation products of cIQ remaining after treatment with K3Fe(CN)6 or NADPH-P450 reductase.
Figure 3
Figure 3
Rates of microsomal oxidation of IQ and cIQ. A solution of rat liver microsomes (0.4 μM of P450), potassium phosphate buffer (100 mM, pH 7.0), NADPH-generating system and varying concentration of IQ or cIQ (0-200 μM) in a total volume of 500 μL were incubated for 10 min at 37 °C.
Figure 4
Figure 4
C8-dGuo adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]naphthelene (cIQ), 2-aminofluorene (AF), and N-acetyl-2-aminoflourene (AAF)
Figure 5
Figure 5
UPLC-ESI/MS/MSn analysis of the reaction of activated IQ and cIQ with calf thymus DNA. A. UPLC-ESI/MS/MS chromatograph of the C8-dGuo-IQ (tr = 2.6 min) and C8-dGuo-cIQ (tr = 4.8 min) adducts formed from the activation of IQ and cIQ by rat liver microsomes and acetic anhydride followed by the modification of calf thymus DNA. MS/MS spectra (M+H−dR) of the two adducts are shown on the right. B. UPLC-ESI-MS/MS3 chromatogram of the reconstructed ion profile at m/z 331 of the C8-dGuo-IQ adducts (left) and its MS3 product ion spectrum (464 → 348 → 331)(right). C. UPLC-ESI-MS/MS3 chromatogram of the reconstructed ion profile at m/z 347 (left) of the C8-dGuo-cIQ adducts and its MS3 product ion spectrum (m/z 463 → 347 → 330) (right). D. UPLC-ESI-MS/MS3 chromatogram of the reconstructed ion profile at m/z 473 (left) of the 13C10-C8-dGuo-IQ standard and its MS/MS and MS3 product ion spectra (right).
Figure 6
Figure 6
A. CD spectra of duplex of 12 containing the C8-dGuo-cIQ adduct (_______) and the unmodified oligonucleotide (-------). B. UV spectra of the C8-dGuo-cIQ containing oligonucleotide 12 in single strand (-------) and duplex (_______).
Figure 7
Figure 7
Tile plots showing NOE cross-peaks between non-exchangeable protons of DNA and cIQ protons in duplex DNA. 1, G19 H1′→cIQ H4A; 2, G17 H2′→cIQ H6A; 3, C18 H2′→cIQ H6A; 4, G17 H2″→cIQ H6A; 5, G19 H5″→ cIQ H5A; 6, G19 H5′→ cIQ H5A; 7, G19 H4′→ cIQ H5A; 8, G19 H1′→ cIQ H5A; 9, G17 H1′→ cIQ H5A; 10, C18 H1′→ cIQ H5A; 11, C18 H1′→ cIQ H7A; 12, G17 H2′→cIQ H7A; 13, G17 H2″→cIQ H7A; 14, G17 H1′→cIQ H7A; 15, G17 H8→cIQ H7A.
Figure 8
Figure 8
Chemical shift changes of (A) aromatic protons H6/H8 and (B) anomeric H1′ protons of the C8-dGuo-cIQ modified duplex in the NarI sequence, relative to the unmodified duplex, where Δδ = [δmodified − δunmodified] (ppm).
Figure 9
Figure 9
Bypass and extension assay of the C8-dGuo-cIQ adduct by Kf, pol II, and Dpo4.
Figure 10
Figure 10
The average refined structures of the C8-dGuo-cIQ (top) and C8-dGuo-IQ (bottom) adducts at the G3-position of the NarI recognition sequence and the flanking base pairs situated (12), looking into the minor groove. The cIQ and IQ adducts are in magenta, the modified Gua (X7) is in gold, and the complementary Cyd (C18) in brown. See Figure 8 for full sequence and residue numbering.
Scheme 1
Scheme 1

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback