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Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure.

ABSTRACT: The presence of endogenous and exogenous N(2)-hydroxymethyl-dG adducts in DNA from the nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [(13)CD(2)]-formaldehyde for 6 h a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a limit of detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogenous adducts observed. In contrast, only endogenous adducts were detectable in the bone marrow, even though ?10 times more DNA was analyzed.

SUBMITTER: Moeller BC

PROVIDER: S-EPMC3273041 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure.

Moeller Benjamin C BC Lu Kun K Doyle-Eisele Melanie M McDonald Jacob J Gigliotti Andrew A Swenberg James A JA

Chemical research in toxicology 20110111 2

The presence of endogenous and exogenous N(2)-hydroxymethyl-dG adducts in DNA from the nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [(13)CD(2)]-formaldehyde for 6 h a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a limit of detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogen ...[more]

PMID: 21222454

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Project description:Over the last two decades, enormous effort has been focused on developing virus-based gene delivery vectors to target the respiratory airway epithelium as a potential treatment for cystic fibrosis (CF) lung disease. However, amongst other problems, the efficiency of gene delivery to the differentiated airway epithelial cells of the lung has been too low for clinical benefit. Although not a target for CF therapy, the nasal epithelium exhibits cellular morphology and composition similar to that of the lower airways, thus representing an accessible and relevant tissue target for evaluating novel and improved gene delivery vectors. We previously reported that replication-competent human parainfluenza virus (PIV)-based vectors efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to sufficient numbers of cultured CF airway epithelial cells to completely correct the bioelectric function of CF cells to normal levels, resulting in restoration of mucus transport. Here, using an in vitro model of rhesus airway epithelium, we demonstrate that PIV mediates efficient gene transfer in rhesus epithelium as in the human counterpart. Naive rhesus macaques were inoculated intranasally with a PIV vector expressing rhesus macaque ?-fetoprotein (rhAFP), and expression was monitored longitudinally. rhAFP was detected in nasal lavage fluid and in serum samples, indicating that PIV-mediated gene transfer was effective and that rhAFP was secreted into both mucosal and serosal compartments. Although expression was transient, lasting up to 10 days, it paralleled virus replication, suggesting that as PIV was cleared, rhAFP expression was lost. No adverse reactions or signs of discomfort were noted, and only mild, transient elevations of a small number of inflammatory cytokines were measured at the peak of virus replication. In summary, rhAFP proved suitable for monitoring in vivo gene delivery over time, and PIV vectors appear to be promising airway-specific gene transfer vehicles that warrant further development.

Project description:Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.

Dataset Information

Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure.

Publications

Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure.

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