Fast quantification of short chain fatty acids and ketone bodies by liquid chromatography-tandem mass spectrometry after facile derivatization coupled with liquid-liquid extraction.

Short chain fatty acids (SCFA) and ketone bodies recently emerged as important physiological relevant metabolites because of their association with microbiota, immunology, obesity and other metabolic states. They were commonly analyzed by GC-MS with long run time and laborious sample preparation. In this study we developed a novel LC-MS/MS method using fast derivatization coupled with liquid-liquid extraction to detect SCFA and ketone bodies in plasma and feces. Several different derivatization reagents were evaluated to compare the efficiency, the sensitivity and chromatographic separation of structural isomers. O­benzylhydroxylamine was selected for its superior overall performance in reaction time and isomeric separation that allowed the measurement of each SCFAs and ketone bodies free from interferences. The derivatization procedure is facile and reproducible in aqueous-organic medium, which abolished the evaporation procedure hampering the analysis of volatile short chain acids. Enhancement in sensitivity remarkably improved the detection limit of SCFA and ketone bodies to sub-fmol level. This novel method was applied to quantify these metabolites in fecal and plasma samples from lean and DIO mouse.

Hepatic GALE Regulates Whole-Body Glucose Homeostasis by Modulating Tff3 Expression.

Transcripts of key enzymes in the Leloir pathway of galactose metabolism in mouse livers are significantly increased after chronic high-fat/high-sucrose feeding. UDP-galactose-4-epimerase (GALE) is the last enzyme in this pathway that converts UDP-galactose to UDP-glucose and was previously identified as a downstream target of the endoplasmic reticulum (ER) stress effector spliced X-box binding protein 1, suggesting an interesting cross talk between galactose and glucose metabolism in the context of hepatic ER stress and whole-body metabolic fitness. However, its specific role in glucose metabolism is not established. Using an inducible and tissue-specific mouse model, we report that hepatic overexpression of Gale increases gluconeogenesis from pyruvate and impairs glucose tolerance. Conversely, genetic reduction of Gale in liver improves glucose tolerance. Transcriptional profiling identifies trefoil factor 3 (Tff3) as one of the downstream targets of GALE. Restoration of Tff3 expression corrects glucose intolerance in Gale-overexpressing mice. These studies reveal a new link between hepatic GALE activity and whole-body glucose homeostasis via regulation of hepatic Tff3 expression.

Endogenous formation and repair of oxidatively induced G[8-5 m]T intrastrand cross-link lesion.

Exposure to reactive oxygen species (ROS) can give rise to the formation of various DNA damage products. Among them, d(G[8-5 m]T) can be induced in isolated DNA treated with Fenton reagents and in cultured human cells exposed to γ-rays, d(G[8-5m]T) can be recognized and incised by purified Escherichia coli UvrABC nuclease. However, it remains unexplored whether d(G[8-5 m]T) accumulates in mammalian tissues and whether it is a substrate for nucleotide excision repair (NER) in vivo. Here, we found that d(G[8-5 m]T) could be detected in DNA isolated from tissues of healthy humans and animals, and elevated endogenous ROS generation enhanced the accumulation of this lesion in tissues of a rat model of Wilson's disease. Additionally, XPA-deficient human brain and mouse liver as well as various types of tissues of ERCC1-deficient mice contained higher levels of d(G[8-5 m]T) but not ROS-induced single-nucleobase lesions than the corresponding normal controls. Together, our studies established that d(G[8-5 m]T) can be induced endogenously in mammalian tissues and constitutes a substrate for NER in vivo.

Systematic evaluation of supported liquid extraction in reducing matrix effect and improving extraction efficiency in LC-MS/MS based bioanalysis for 10 model pharmaceutical compounds.

In past a few years, there has been a large increase in the application of supported liquid extraction (SLE) for LC-MS/MS based bioanalysis due to its distinct practical advantage in reduced time cost, ease of operation and the feasibility for automation. The main purpose of this study was to systematically evaluate supported liquid extraction in reducing matrix effect and improving extraction efficiency/recovery under various extraction conditions with 10 model pharmaceutical compounds in liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) analysis. Selected compounds have diverse physicochemical properties where logP ranges from 0.1 to 6.24 and pK(a) ranges from 4.0 to 11.1. The factors that may have the impact on the recovery of analytes and phospholipids (PL) were assessed. Over 75% recovery was achieved for every analyte under its respectively optimized extraction conditions where the selection of the polarity of extraction solvent and buffered pH can be critical for efficient recovery. Furthermore, the matrix effect was assessed by postextraction spike and postcolumn infusion method. The matrix effect was considerably reduced for all analytes under most extraction conditions evaluated for SLE, compared with protein precipitation (PPT) method. The correlation between matrix effect and residual phospholipids in sample extract was clearly shown. Although analyte-dependent matrix effect was observed prominently in sample extract prepared by PPT, it was minimized by SLE sample preparation process that effectively removes the majority of phospholipids. Sample extracted by ethyl acetate contained more phospholipids and demonstrated stronger matrix effect than by other organic solvents. Water-miscible organic content, such as methanol and acetonitrile in samples prior to loading has significant impact on PL recovery when eluting with methyl tert-butyl ether. However, isopropanol does not enhance the recovery of PL when adding to dichloromethane for elution. In addition, the compromise between improved extraction efficiency by SLE and reduced matrix effect is sometimes necessary to yield clean extract with acceptable recovery. The effective removal of phospholipids and reduction of matrix effect, while achieving good recovery for all pharmaceutical compounds with diverse physicochemical properties, demonstrated that SLE is a valuable alternative technique to liquid-liquid extraction (LLE) in high throughput LC-MS/MS based bioanalysis.

High-throughput analysis of the mutagenic and cytotoxic properties of DNA lesions by next-generation sequencing.

Human cells are constantly exposed to environmental and endogenous agents which can induce damage to DNA. Understanding the implications of these DNA modifications in the etiology of human diseases requires the examination about how these DNA lesions block DNA replication and induce mutations in cells. All previously reported shuttle vector-based methods for investigating the cytotoxic and mutagenic properties of DNA lesions in cells have low-throughput, where plasmids containing individual lesions are transfected into cells one lesion at a time and the products from the replication of individual lesions are analyzed separately. The advent of next-generation sequencing (NGS) technology has facilitated investigators to design scientific approaches that were previously not technically feasible or affordable. In this study, we developed a new method employing NGS, together with shuttle vector technology, to have a multiplexed and quantitative assessment of how DNA lesions perturb the efficiency and accuracy of DNA replication in cells. By using this method, we examined the replication of four carboxymethylated DNA lesions and two oxidatively induced bulky DNA lesions including (5'S) diastereomers of 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG) and 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) in five different strains of Escherichia coli cells. We further validated the results obtained from NGS using previously established methods. Taken together, the newly developed method provided a high-throughput and readily affordable method for assessing quantitatively how DNA lesions compromise the efficiency and fidelity of DNA replication in cells.

Quantification of N2-carboxymethyl-2'-deoxyguanosine in calf thymus DNA and cultured human kidney epithelial cells by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope dilution method.

Glyoxal is generated endogenously from the degradation of glucose and the oxidation of carbohydrates, lipids, and the 2-deoxyribose moieties of DNA. Glyoxal is also widely used in industry and is present in cigarette smoke and food. Glyoxal can conjugate with nucleobases and proteins to give advanced glycation end products. N(2)-Carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) and the cyclic 1,N(2)-glyoxal-dG are the major glyoxal adducts formed in DNA. In this study, we first assessed the stabilities of these two adducts. It turned out that 1,N(2)-glyoxal-dG was very unstable, with more than 70% of the adduct being decomposed to dG upon a 24 h incubation at 37 degrees C in phosphate-buffered saline. However, N(2)-CMdG was very stable; less than 0.5% of the lesion was degraded to dG after a 7 day incubation under the same conditions. We further developed a sensitive capillary liquid chromatography-electrospray ionization-tandem mass spectrometry coupled with a stable isotope dilution method and quantified the formation of N(2)-CMdG in calf thymus DNA and 293T human kidney epithelial cells that were exposed to glyoxal and in calf thymus DNA treated with d-glucose. Our results showed that N(2)-CMdG was produced at 2-134 lesions per 10(6) nucleosides in calf thymus DNA when the surrounding glyoxal concentration was increased from 10 to 500 microM and approximately 3-27 lesions per 10(7) nucleosides, while the D-glucose concentration changed from 2 to 50 mM. Furthermore, N(2)-CMdG was induced endogenously in 293T human kidney epithelial cells and exposure to glyoxal further stimulated the formation of this lesion; the level of this adduct ranged from 7 to 15 lesions per 10(8) nucleosides, while the glyoxal concentration increased from 10 microM to 1.25 mM. Collectively, our results suggested that N(2)-CMdG might serve as a biomarker for glyoxal exposure.

Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA.

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C -->T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C -->T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37 degrees C were 37.4 +/- 1.6 and 27.4 +/- 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2'-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H(2)O(2)/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2'-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C -->T transition mutation at mCpG dinucleotide site.

Fragmentation of isomeric intrastrand crosslink lesions of DNA in an ion-trap mass spectrometer.

The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand crosslink-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. A Dimroth-like rearrangement, which was reported for protonated 2'-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5-8]G, C[5-2]A, and C[5-8]A, but not C[5-N(2)]G or C[5-N(6)]A. In these two sets of crosslinks, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N(2)]G, C[5-N(6)]A, and unmodified cytosine, but not C[5-8]G, C[5-2]A, or C[5-8]A. In addition, ammonia could be lost more readily from C[5-N(2)]G and C[5-N(6)]A than from C[5-8]G, C[5-2]A, and C[5-8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand crosslink lesions.

Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and UVA irradiation.

Upon exposure to UVA light, psoralens can induce DNA interstrand cross-links (ICLs), which can block DNA replication and transcription. Among the psoralen derivatives, 8-methoxypsoralen (8-MOP) is conventionally applied for psoriasis therapy, and amotosalen S59 is used to inactivate bacterial and viral pathogens in blood components. In addition to the ICL formation, psoralens also readily form various monoadducts (MAs) with thymidine residues in DNA when exposed to UVA light, and the biological implications for these monoadducts remain unclear. Here, we reported a method that encompassed digestion with a single enzyme (nuclease P1) and LC-MS/MS, for the simultaneous quantification of ICL and MAs induced in human cells exposed with 8-MOP or S59 and UVA light. Our results showed that the yield of ICL induced by S59, which increased from 3.9 to 12.8 lesions/10(3) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2), was approximately 100 fold more than that induced by 8-MOP. In addition, three and five products were identified as 8-MOP- and S59-MAs, respectively, and the yields of MAs were significantly lower than that for ICL. The yields of the three 8-MOP-MAs were 7.6-2.2, 1.9-9.9, and 7.2-51 per 10(6) nucleotides and those of the five S59-MAs were 215-19, 106-39, 25-21, 32-146, and 22-26 per 10(6) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2). Although the yields of MAs induced by 8-MOP and S59 were lower than those of the respective ICLs under the same exposure conditions, the formation of appreciable amounts of MAs might account for some of the mutations induced by psoralens.

Efficient and accurate bypass of N2-(1-carboxyethyl)-2'-deoxyguanosine by DinB DNA polymerase in vitro and in vivo.

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N(2)-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) as the major stable DNA adduct. Here, we found that N(2)-CEdG could be detected at a frequency of one lesion per 10(7) nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N(2)-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N(2)-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N(2)-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase kappa), opposite the N(2)-CEdG is both accurate and efficient. Taken together, our data support that N(2)-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.

LC-MS/MS for the detection of DNA interstrand cross-links formed by 8-methoxypsoralen and UVA irradiation in human cells.

DNA interstrand cross-links (ICLs) are induced by many carcinogens and anitcancer drugs. ICL is a covalent linkage of both strands of DNA, preventing DNA strand separation during transcription and replication; thus, it is extremely cytotoxic in vivo. Psoralen and its derivatives are widely applied for the clinical treatment of several skin diseases and cutaneous T cell lymphoma, and they are also commonly used as model compounds for the study of ICL. Upon UVA photoactivation, 8-methoxypsoralen alkylates both strands of DNA at the 5,6-double bond of thymidines at the 5'-TpA-3' site, generating monoadducts and ICLs. Here we developed a method utilizing HPLC-tandem mass spectrometry, combined with nuclease P1 digestion, to assess the formation of ICL in DNA of human skin melanoma cells exposed to 500 ng/mL 8-methoxypsoralen and UVA irradiation. We were able to quantify ICL, in the form of tetranucleotide, at the level of 1 lesion/10(6) unmodified nucleobases using a low-microgram quantity of DNA. In addition, our results revealed that the formation of ICL increased linearly with the UVA dose. The yield of ICL increased by 15-fold from 4.5 to 76 lesions/10(6) nucleotides when the UV dose was increased from 0.5 to 5 J/cm2. This is the first report of an LC-MS assay for the quantification of DNA interstrand cross-links. The specificity and accuracy of this high-throughput approach are advantageous over other methods for the detection of ICLs formed in vitro and in vivo.

In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion.

G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.

Formation and genotoxicity of a guanine-cytosine intrastrand cross-link lesion in vivo.

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/gamma-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine-cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to gamma-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 10(9) nucleosides per Gy, which was approximately 300 times lower than that of 5-formyl-2'-deoxyuridine (0.011 lesions per 10(6) nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G-->T and a 1.2% G-->C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.

Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine.

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.

Quantification of oxidative single-base and intrastrand cross-link lesions in unmethylated and CpG-methylated DNA induced by Fenton-type reagents.

Methylation of cytosine at CpG sites in mammalian cells plays an important role in the epigenetic regulation of gene expression. Here, we assessed the formation of single-nucleobase lesions and intrastrand cross-link lesions (i.e. G[8-5]C, C[5-8]G, mC[5m-8]G, and G[8-5m]mC, where 'mC' represents 5-methylcytosine) in unmethylated and the corresponding CpG-methylated synthetic double-stranded DNA upon treatment with Fenton-type reagents [i.e. H2O2, ascorbate together with Cu(II) or Fe(II)]. Our results showed that the yields of oxidative single-nucleobase lesions were considerably higher than those of the intrastrand cross-link lesions. Although no significant differences were found for the yields of single-base lesions induced from cytosine and mC, the G[8-5m]mC cross-link was induced approximately 10 times more efficiently than the G[8-5]C cross-link. In addition, the mC[5m-8]G was induced at a level that was approximately 15 times less than G[8-5m]mC, whereas the corresponding C[5-8]G intrastrand cross-link lesion was not detectable. Moreover, Cu(II) is approximately 10-fold as effective as Fe(II) in inducing oxidative DNA lesions. These results suggest that oxidative intrastrand cross-link lesions formed at methylated-CpG sites may account for the previously reported mCG-->TT tandem double mutations induced by Fenton-type reagents.

Collisionally activated dissociation of protonated 2'-deoxycytidine, 2'-deoxyuridine, and their oxidatively damaged derivatives.

We examined the collisionally activated dissociation (CAD) pathways of protonated 2'-deoxycytidine (dC), 5-formyl-2'-deoxycytidine (5-FmdC), 5-hydroxy-2'-deoxycytidine (5-OHdC), 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MSn) results showed that protonated cytosine, its 5-hydroxyl- and 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H2O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry.

Facile photocyclization chemistry of 5-phenylthio-2'-deoxyuridine in duplex DNA.

We report here the synthesis of 5-phenylthio-2'-deoxyuridine (d(PhS)U), its incorporation into oligodeoxynucleotides (ODNs), and its photocyclization chemistry. Irradiation of dinucleoside monophosphate d((PhS)UG) and d(PhS)U-bearing duplex ODNs with 254 nm light results in the facile formation of a cyclic product where the C6 of uracil is covalently bonded to the C2 of the phenyl ring. The chemistry reported here may serve as the basis for the efficient preparation of a new class of duplex DNA with an extended pi system. [reaction: see text]

Identification and quantification of a guanine-thymine intrastrand cross-link lesion induced by Cu(II)/H2O2/ascorbate.

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may arise from Fenton reaction, which also induces the formation of *OH; the structures of the proposed intrastrand cross-link lesions, however, have not been determined. Here, we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of an intrastrand cross-link lesion, i.e., G wedge T, where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G wedge T. In addition, the yield of the intrastrand cross-link was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-formyl-2'-deoxyuridine. The induction of intrastrand cross-link lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Complex formation between a DNA duplex and lipid-like spermine derivatives: hydrophobic protection of DNA from one-electron oxidation.

The one-electron oxidation of DNA leads to formation of a nucleo-base radical cation that can migrate to a distant site where it is trapped by H2O or O2 to form a "damaged" guanine that is revealed as strand cleavage when the irradiated sample is treated with piperidine. We prepared a series of alkyl-substituted spermine derivatives and assessed their effect on the oxidative reactions of DNA induced by photoexcitation of a covalently linked anthraquinone derivative. The spermine compounds are polycations and bind nonselectively to DNA. When the spermine derivative is substituted with C8 alkyl chains, it shows lipid-like properties. The reaction of the radical cation at guanine is affected by this lipid-like spermine. The distance dependence of the migration process becomes weaker, and the efficiency of strand cleavage is reduced. These results are attributed to the formation of a hydrophobic layer on the DNA that restricts access of H2O to the nucleo-base radical cation.