Metabolic fate of endogenous molecular damage: Urinary glutathione conjugates of DNA-derived base propenals as markers of inflammation

Abstract

Although mechanistically linked to disease, cellular molecules damaged by endogenous processes have not emerged as significant biomarkers of inflammation and disease risk, due in part to poor understanding of their pharmacokinetic fate from tissue to excretion. Here, we use systematic metabolite profiling to define the fate of a common DNA oxidation product, base propenals, to discover such a biomarker. Based on known chemical reactivity and metabolism in liver cell extracts, 15 candidate metabolites were identified for liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) quantification in urine and bile of rats treated with thymine propenal (Tp). Analysis of urine revealed three metabolites (6% of Tp dose): thymine propenoate and two mercapturate derivatives of glutathione conjugates. Bile contained an additional four metabolites (22% of Tp dose): cysteinylglycine and cysteine derivatives of glutathione adducts. A bis-mercapturate was observed in urine of untreated rats and increased approximately threeto fourfold following CCl 4 -induced oxidative stress or treatment with the DNA-cleaving antitumor agent, bleomycin. Systematic metabolite profiling thus provides evidence for a metabolized DNA damage product as a candidate biomarker of inflammation and oxidative stress in humans.DNA damage | metabolism | biomarker | mass spectrometry | oxidative stress E ndogenous DNA damage has long been considered a potentially useful source of biomarkers of diseases associated with inflammation and oxidative stress given the strong mechanistic links to the pathophysiology of cancer and the broad spectrum of damage chemistries, such as alkylation, oxidation, deamination, nitration and halogenation (1-6). Major limitations to the development of endogenous DNA damage products as biomarkers involve the choice of a sampling compartment, with invasive sampling of tissues preventing large-scale human studies, and a lack of appreciation for the biological fate of endogenous DNA lesions following their formation in a tissue. The latter problem is reflected in the potential for rapid DNA repair to maintain relatively low steady-state levels of DNA damage even in severely inflamed tissues (1). Although there have been attempts to characterize endogenous DNA damage products released from cells into accessible compartments such as urine, blood or feces (7,8), the biotransformational fates of the damage products, such as the metabolism after release by DNA repair, have not been rigorously assessed, which has the potential to allow important biomarker candidates to escape detection. One notable exception arises in the recent studies of the metabolic fate of the 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1, 2-α]purin-10(3H)-one (M 1 dG) adduct arising from reaction of 2-deoxyguanosine (dG) with the endogenous electrophiles malondialdehyde and base propenals. When released from cells presumably by nucleotide excision repair, M 1 dG is found at low levels (10-20 fmol/kg) in human urine (9). However, Marnett and coworkers disco...

Urinary DNA adductomics – A novel approach for exposomics

“…The literature consensus appears to be that DNA adducts in urine are a consequence of DNA repair (Cooke et al 2005; Rozalski et al 2005), with the general assumption that base excision repair (BER) is the source of nucleobase adducts (Rozalski et al 2004), and global genome- and transcriptional coupled-nucleotide excision repair (GG-NER, and TCR-NER, respectively), generate 2’-dN adducts (Evans et al 2010; Jaruga et al 2012; Jumpathong et al 2015) (Fig. 1).…”

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

“…Several laboratories have developed sensitive and specific methods for measuring a wide array of DNA base adducts; however, such methods would require either enzymatic or acid hydrolysis prior to analysis (11)(12)(13)(14)(15)(16).…”

“…Several laboratories have developed sensitive and specific methods for measuring a wide array of DNA base adducts; however, such methods would require either enzymatic or acid hydrolysis prior to analysis ( 11 , 12 , 13 , 14 , 15 , 16 ). The mutagenic significance of the deaminated cytosine adducts is a consequence of residing in a base mispair with guanine.…”

Sensitive mass spectrometric analysis of carbonyl metabolites in human urine and fecal samples using chemoselective modification

“…6 Furthermore, many carbonyl-containing metabolites are excreted through the kidney and have been detected in urine samples. 7 Metabolomics aims at investigating all metabolites present in any biological sample. [8][9][10] Metabolite extracts can either be analyzed by NMR or diverse mass spectrometric techniques.…”