Abstract
Despite intensive research over the last two decades, there are still no specific markers of endogenous lipid hydroperoxide-mediated DNA damage. We recently demonstrated that heptanone-etheno-2'-deoxyguanosine adducts are formed in the DNA of rat intestinal epithelial cells that stably express cyclooxygenase-2. Heptanone-etheno adducts can only arise from the reaction of lipid hydroperoxide-derived 4-oxo-2(E)-nonenal with DNA. This raised the possibility that similar adducts would be formed in vivo in settings where cyclooxygenase-2 expression is increased. Therefore, DNA-adduct formation was studied in C57BL/6JAPC(min) mice, a colorectal cancer mouse model in which cyclooxygenase-2 is up-regulated. 15(S)-Hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid is the major lipid hydroperoxide produced endogenously by cyclooxygenase-2. It undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, which forms heptanone-etheno adducts with DNA. A quantitative comparison was made of the heptanone-etheno-DNA adducts present in C57BL/6J and C57BL/6JAPC(min) mice. Using highly specific and sensitive methodology based on stable isotope dilution liquid chromatography/tandem mass spectrometry, we have detected the endogenous formation of heptanone-etheno adducts in mammalian tissue DNA for the first time. In addition, we found that there were statistically significant increased levels of the heptanone-etheno-2'-deoxyguanosine and heptanone-etheno-2'-deoxycytidine adducts in the C57BL/6JAPC(min) mice when compared with the control C57BL/6J mice.
Highlights
Likewise, NSAIDs have demonstrated their efficacy to inhibit carcinoma formation in animal models [5,6,7,8]
COX-2 mRNA is up-regulated in tumor tissue, and COX-2 protein is expressed in high amounts [12]
In rats that have been subjected to carcinogens, COX-2 mRNA and protein levels were increased in the tumors [13]
Summary
Materials and Reagents—Ammonium acetate, activated calf thymus, vitamin C, DNase I, and zinc chloride were purchased from SigmaAldrich. 15(S)-HPETE was obtained from Cayman Chemical (Ann Arbor, MI). LC/ESI/MS analysis for DNA adducts on gradient system 2 was performed using a Hitachi L-2130 Intelligent Pump. For the MRM analysis, parent ions were monitored for the protonated molecular ion of the endogenous adduct and for its 15N5 or 15N3 internal standard. Artifact Formation during Isolation and Analysis—Duplicate small intestine samples from Min mice were spiked with 13C15N-labeled DNA (520 g) before defrosting. Labeled and unlabeled DNA were extracted from the tissues, hydrolyzed in the presence of 15N internal standards using DNase, nuclease P1, and alkaline phosphatase. The samples were analyzed by LC/MRM/MS, monitoring the following transitions: m/z 404 3 m/z 288 (endogenous H⑀dGuo), m/z 419 3 m/z 298 (13C1015N5-labeled H⑀dGuo from labeled DNA), m/z 409 3 m/z 293, FIGURE 8. LC/ESI/MRM/MS analysis of DNA adducts from the small intestine of a Min mouse spiked with 13C15N-labeled DNA It was hydrolyzed as described above, and normal bases were quantified by LC/UV using system 1
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