trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis-and trans-N ⑀ -3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis-and trans-HHP-lysine and confirmed that the 2-nonenallysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

DNA adducts are produced both exogenously and endogenously via exposure to various DNA-damaging agents. Two lipid peroxidation (LPO) products, 4-oxo-2(E)-nonenal (4-ONE) and 4-oxo-2(E)-hexenal (4-OHE), induce substituted etheno-DNA adducts in cells and chemically treated animals, but the adduct levels in humans have never been reported. It is important to investigate the occurrence of 4-ONE- and 4-OHE-derived DNA adducts in humans to further understand their potential impact on human health. In this study, we conducted DNA adductome analysis of several human specimens of pulmonary DNA as well as various LPO-induced DNA adducts in 68 human autopsy tissues, including colon, heart, kidney, liver, lung, pancreas, small intestine, and spleen, by liquid chromatography tandem mass spectrometry. In the adductome analysis, DNA adducts derived from 4-ONE and 4-OHE, namely, heptanone-etheno-2'-deoxycytidine (HεdC), heptanone-etheno-2'-deoxyadenosine (HεdA), and butanone-etheno-2'-deoxycytidine (BεdC), were identified as major adducts in one human pulmonary DNA. Quantitative analysis revealed 4-ONE-derived HεdC, HεdA, and heptanone-etheno-2'-deoxyguanosine (HεdG) to be ubiquitous in various human tissues at median values of 10, 15, and 8.6 adducts per 10(8) bases, respectively. More importantly, an extremely high level (more than 100 per 10(8) bases) of these DNA adducts was observed in several cases. The level of 4-OHE-derived BεdC was highly correlated with that of HεdC (R(2) = 0.94), although BεdC was present at about a 7-fold lower concentration than HεdC. These results suggest that 4-ONE- and 4-OHE-derived DNA adducts are likely to be significant DNA adducts in human tissues, with potential for deleterious effects on human health.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.