Reaction of <i>cis</i>- and <i>trans</i>-2-Butene-1,4-dial with 2‘-Deoxycytidine to Form Stable Oxadiazabicyclooctaimine Adducts

Reaction of cis- and trans-2-Butene-1,4-dial with 2‘-Deoxycytidine to Form Stable Oxadiazabicyclooctaimine Adduct

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 μM NO along with 186 μM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2‘-deoxyuridine (dU) and 2‘-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 μM NO and 186 μM O2 for 12 h (1260 μM·min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels:  dX, 7 (±1) per 106 nt; dI, 25 (±2.1) per 106 nt; and dU, 40 (±3.8) per 106 nt. dO was not detected above the limit of detection of 6 lesions per 107 nt in 50 μg of DNA. A 12 h exposure to 0.65 μM NO and 190 μM O2 (468 μM·min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a ∼15% (±3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA

Reaction of <i>cis</i>- and <i>trans</i>-2-Butene-1,4-dial with 2‘-Deoxycytidine to Form Stable Oxadiazabicyclooctaimine Adducts

Reaction of cis- and trans-2-Butene-1,4-dial with 2‘-Deoxycytidine to Form Stable Oxadiazabicyclooctaimine Adduct

Summary of findings on N⁶-formyllysine in histones.

<p>N⁶-formyllysine can arise from reaction of lysine with the 3′-formyl phosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with formaldehyde. Furthermore, our data suggest that N⁶-formyllysine is refractory to removal by histone deacetylases, which is consistent with the persistence of this pathological adduct throughout the life of individual histone proteins.</p

Effect of lysine deacetylases on N⁶-formyllysine.

<p>(A) TK6 cells were treated with the class I and class II histone deacetylase inhibitor, SAHA, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05). (B) Treatment of peptide substrates containing N⁶-acetyllysine or N⁶-formyllysine with the class III histone deacetylase, SIRT1.</p

Quantification of lysine modifications in HPLC-purified histone proteins.¹

1<p>Classes of histone proteins resolved by reversed-phase HPLC, with putative isoforms denoted in parentheses.</p>2<p>Column titles denote different N⁶-modifications of lysine.</p>3<p>Data are expressed as modifications per 10⁴ total lysines and represent mean ± SD for 3 biological replicates.</p

Analysis of lysine demethylation as a source of N⁶-formyllysine.

<p>Methyl groups in N⁶-methyllysine species in TK6 cells were labeled using L-methionine-([¹³C,²H₃]-methyl) and N⁶-formyllysine and N⁶-methyllysine species were quantified by LC-MS/MS as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. Panels A and B: N⁶-mono-methyllysine and N⁶-di-methyllysine are predominately labeled (>90%) with heavy isotope methyl groups (mass increase of 4 <i>m/z</i> and 8 <i>m/z</i>, respectively), with <10% of the modifications containing unlabeled methyl groups. Panel C: the level of N⁶-[¹³C, ²H]-formyllysine (177 <i>m/z</i>→114 <i>m/z</i> transition) in histones did not show an increase beyond the natural isotope abundance level of ∼0.7% for [M+2] ion of N⁶-formyllysine.</p

Sources of N⁶-formyllysine.

<p>The adduct can be generated in chromatin proteins from reaction of lysine with 3′-formylphosphate residue derived from 5′-oxidation of 2-deoxyribose in DNA or from reaction of lysine with endogenous or exogenous formaldehyde. Formaldehyde reacts with amines to give a carbinolamine intermediate (N⁶-(hydroxymethyl)-lysine) that is in equilibrium with a Schiff base and that is one oxidation state away from the formamide functional group of N⁶-formyllysine.</p

Different lysine species detected in purified histone H4 from TK6 cells.

<p>Lysine adducts were monitored by tandem mass spectrometry, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. Abbreviations: FK, N⁶-formyllysine; AK, N⁶-acetyllysine; K, lysine; MK, N⁶-mono-methyllysine; M₂K, N⁶-di-methyllysine; M₃K, N⁶-tri-methyllysine.</p

Addition of [¹³C,²H₂]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N⁶-formyllysine.

<p>(A) LC-MS/MS analysis showing signals for the three isotopomeric N⁶-formyllysine species, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. (B) Plot of N⁶-formyllysine levels as a function of exposure to [¹³C,²H₂]-formaldehyde. Data represent mean ± SD for N = 3.</p