Spotlight Cross-Link Quantification Interstrand cross-links (ICLs) are a highly toxic form of DNA damage. Difficult to repair, they are a block to both replication and transcription and often lead to cell death. A number of carcinogens and antitumor agents exert their effects by ICL formation; yet, the absence of reliable methods to quantify these lesions has made it difficult to determine their exact role in toxicity. Cao et al. [( 2008) Anal. Chem. 80, 12932] now report an LC-MS/MS method for the absolute quantification of ICLs formed from the exposure of 8-methoxypsoralen (8MOP)-treated DNA to UVA irradiation. 8MOP is used for the treatment of a number of skin diseases, including psoriasis. It intercalates into DNA, usually at 5′-TA-3′ sites. Upon irradiation, reaction between 8MOP and the 5,6-double bonds of the opposing thymidines yields the ICL. Cao et al. synthesized the ICL in an oligonucleotide containing a single
The “No Damage” Response Damage to genomic DNA initiates a rapid repair response, beginning with assembly of the MRN complex, comprised of MRE11, Rad50, and NBS1 proteins. Next, the transducer proteins MDC1 and 53BP1 bind to the site, followed by the kinases ATM, DNAPK, and ATR. Phosphorylation of the histone H2AX is mediated by one or more of these kinases, which are also responsible for activation of downstream effectors, such as Chk1 and Chk2. Although it is clear that this DNA damage response (DDR) is initially directed by the physical presence of DNA damage, the degree to which damage per se is required for the full assembly of the repair complex is not known. Soutoglou and Misteli [( 2008) Science, 320, 1507] now address this question by showing that the simple accumulation of DDR proteins in the absence of DNA damage is enough to trigger the assembly of the full complex and initiate the DDR. Soutoglou and Misteli conducted their experiments in an NIH-3T3 cell line bearing a cluster of 256 repeats of the lac operon (lacO) sequence stably incorporated into chromosome 3. Transfection of a DDR protein fused to the E. coli lac-repressor (lacR) resulted in binding of the lacR portion of the fusion protein to the lacO repeats and an accumulation of the protein on chromosome 3. The fusion proteins were also tagged with Cherry-Red fluorescent protein, so that their accumulation at the site could be detected by fluorescence microscopy. Soutoglou and Misteli transfected the NIH-3T3 cells with labeled fusion proteins of MRE11, NBS1, MDC1, ATM, Chk1, or Chk2. Microscopy confirmed the accumulation of each of the proteins at the lacO repeat region, and multiple controls Published online 08/18/2008 • DOI: 10.1021/tx800231m © 2008 American Chemical Society
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5′-TA-3′ dinucleotide within the sequence. The ICL’s structure was confirmed by mass spectrometry. Nuclease P1 digestion rendered the ICL-containing tetranucleotide bearing 5′-phosphates. HPLC using 1,1,1,3,3,3-hexafluoro-2-propanol as an ion-pairing reagent, coupled with negative ion, selected reaction monitoring mass spectrometry with a deuterated internal standard allowed quantification of the tetranucleotide ICL with a limit of detection of 1 lesion/106 nucleotides in a 1 µg DNA sample. The method successfully detected the ICL in the DNA of cells treated with 8MOP and UVA. This approach will allow a quantitative correlation between 8-MOP treatment, ICL formation, and clinical results and sets a standard for the development of methods for quantification of ICLs generated by other agents. • Carol A. Rouzer
ruled out DNA damage at the site. However, despite the absence of damage, the accumulation of MRE11, NBS1, MDC1, or ATM, but not Chk1 or Chk2, resulted in phosphorylation of H2AX, indicating initiation of the DDR. Soutoglou and Misteli went on to show that accumulation of NBS1 or MRE11 recruited downstream DDR proteins, whereas MDC1 accumulation recruited upstream MRN proteins. In contrast, ATM accumulation led only to the recruitment of MDC1, while Chk1 and Chk2 accumulation resulted in recruitment of no additional proteins. These results reinforce what is already known about the pivotal role of the MRN proteins in DDR complex initiation and suggest a feedback reinforcement role for MDC1.
Reproduced from Science [Soutoglou and Misteli (2008) Science 320, 1507].
A key component of the DDR is cell cycle arrest, which prevents the cell from replicating and perpetuating the damaged DNA. Soutoglou and Misteli showed that accumulation of NBS1, MRE11, MDC1, or ATM, but not Chk1 or Chk2, led to arrest of cells at G2. Together, these results suggest that DNA damage serves only as a focus for initial MNR complex binding but is not needed for any subsequent aspect of the DDR. • Carol A. Rouzer Vol. 21,
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Spotlight Biomarking Oxidative Stress Oxidative stress results when the generation of reactive oxidants exceeds a cell’s ability to detoxify them. Whether they arise from endogenous or exogenous sources, such oxidizing species can be a major source of damage to cellular constituents. Because many disease states and toxic exposures result in oxidative stress, considerable interest has been directed toward developing biomarkers of oxidant damage. To date, the most reliable method of detection has been monitoring urinary levels of isoprostanes, a complex mixture of products resulting from the peroxidation of polyunsaturated fatty acids, predominantly arachidonic acid. However, lipid peroxidation also yields mixtures of reactive aldehydes of which 4-hydroxy-2-nonenal (HNE) has been the most thoroughly studied. HNE arises from 4-hydroperoxy-2-nonenal, which can also dehydrate to form 4-oxo-2-nonenal (ONE). Both HNE and ONE are cytotoxic and have been shown to react with DNA and proteins. They are also subject to phase-2 metabolism, including oxidation or reduction of the aldehyde to a carboxylic acid or alcohol, respectively, and/or adduction with glutathione, which serves as the first step toward mercapturic acid (MA) formation. Kuiper et al. [(2008) J. Biol. Chem. 283, 17131] proposed that urinary levels of MA metabolites of HNE and ONE could serve as new biomarkers of oxidative stress. Using LC-MS/MS and synthetic standards, Kuiper et al. first developed methods for the detection of HNE-MA, ONEMA, ONO-MA (formed from reduction of the aldehyde of ONE), DHN-MA (formed from reduction of the aldehyde of HNE), ONA-MA (formed from oxidation of the aldehyde of ONE), and HNA-MA and its lactone (formed from oxidation of the aldehyde of HNE). They went on to show that all of these metabolites could be detected in the urine of normal rats. Furthermore, treatment of the rats with CCl4, a standard model of oxidative stress, resulted in statistically significant increases in ONE-MA, DHN-MA, ONAMA, and HNA-MA lactone. Nonsignificant increases were observed in the other metabolites. These results confirmed the formation of both HNE and ONE plus their phase-2 metabolites in vivo and suggested that the phase-2 metabolites may prove to be valuable biomarkers for oxidative stress. Although this work was only semiquantitative, the strong signals for ONA-MA and HNA-MA lactone also suggest that oxidation of the aldehyde of both HNE and ONE is a major route of phase-2 metabolism for these two reactive aldehydes. • Carol A. Rouzer
Protein Nitration Protection Because of the growing evidence of the role of oxidative stress in the pathogenesis of many disease states and toxic responses, it seems reasonable that antioxidants should help to protect against the sequelae of oxidant damage.
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However, use of antioxidants in animal models or in the clinic to ameliorate conditions associated with oxidative stress has often yielded disappointing results. Vaz and Augusto [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 8191] propose that this may be explained by an inadequate understanding of exactly how antioxidants work in vivo. They are working to address this problem by exploring the mechanism of action of tempol, a stable cyclic nitroxide radical that has been shown to be beneficial in a number of animal models of oxidative stress. Specifically, they have investigated the effects of tempol on peroxidase-mediated nitration of proteins, a reaction that results from the oxidative generation of reactive nitrogen species.
Reproduced with permission from Vaz and Augusto [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 8191]. Copyright 2008 National Academy of Sciences U.S.A.
Vaz and Augusto incubated ribonuclease in the presence of myeloperoxidase (MPO), H2O2, and NO2-. Their reaction conditions generated 290 µM nitrotyrosine residues in the ribonuclease protein. Tempol (10 µM) inhibited this reaction by 90%. Tempol (TPNO•) can be reversibly reduced to a hydroxylamine (TPNOH), oxidized to an oxammonium cation (TPNO+), or irreversibly quenched by reaction with a second radical. Partial consumption of tempol in the MPO/ribonuclease reaction mixtures indicated that inhibition of nitrotyrosine formation was not totally catalytic in nature. This finding was explained at least in part by the competition of tempol with 3,5-dibromo-4-nitrosobenzenesulfonate for reaction with tyrosine radicals formed by MPO in its reaction with ribonuclease, suggesting a direct irreversible reaction of tempol with the tyrosine radicals. However, additional studies showed that tempol almost completely blocked the consumption of NO2-, while increasing the consumption of H2O2 and evolution of O2 during the reaction of MPO with ribonuclease. These results suggested that tempol’s major mechanism of action is reaction with NO2• to form NO2- and TPNO+. TPNO+ then reacts with H2O2, peroxynitrite, or other cellular reductants to regenerate TPNO•. In these reactions, tempol reversibly prevents the reaction of NO2• with tyrosine radicals, allowing effective protection at low micromolar concentrations. Nitration reactions are a significant byproduct of the production of reactive nitrogen species, which occurs under many conditions of oxidative stress. Tempol’s ability to prevent protein nitration may be an important mechanism by which it exerts its protective effects in models of oxidative stress in vivo. • Carol A. Rouzer TX800231M
Published online 08/18/2008 •
DOI: 10.1021/tx800231m $40.75 © 2008 American Chemical Society
