Preparation of a methylated DNA standard, and its stability on storage

Several methods are available for the determination of low levels of O(6)-methylguanine in DNA; this base arises after methylation by environmental carcinogens. The reliability of these assays is much improved by the use of a standard. We have prepared such a standard by treating calf thymus DNA with [(3)H-methyl]-N-methyl-N-nitrosourea. We characterized the methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatography and liquid scintillation counting of the tritium content. The level of O(6)-methylguanine was 0.6 per one million nucleotides. This base is stable, and its level was unchanged after storage for 5 years at -20 degrees C. The methylated DNA also contained 7-methylguanine and 3-methyladenine, which are slowly cleaved from the DNA on standing. The half-life for loss of 7-methylguanine at neutral pH was estimated to be 70 h at 39 degrees C, 460 h at 22 degrees C, 3800 h at 10 degrees C, and about 4 years at -20 degrees C.     

Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine

2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.     

A 2-iminohydantoin from the oxidation of guanine

The nucleobase guanine was oxidized with dimethyldioxirane (DMDO) to explore the role of epoxidizing agents in oxidative DNA damage. Treatment of guanine with 10% molar excess DMDO in aqueous solution at 0 degrees C and pH 7.5 followed by workup under mild conditions gave 5-carboxamido-5-formamido-2-iminohydantoin (1) as the sole isolable product in 71% yield. The structure of 1 was established on the basis of mass spectrometry and NMR studies on 1 and its isotopomers generated by the oxidation of [4-(13)C] and [7-(15)N]guanine, which yield [5-(13)C]1 and [7-(15)N]1. The distribution of 13C and 15N labels in the isotopomeric products supports initial epoxidation of the C4-C5 bond of guanine followed by a 1,2-acyl migration of guanine C6. Compound 1 is suggested as a possible primary DNA lesion from putative epoxidizing agents, including hydroperoxides present during biological processes such as lipid peroxidation.     

Influence of pH and temperature on kinetics of ceftiofur degradation in aqueous solutions.

The objective of this study was to evaluate the stability of ceftiofur (1 mg mL(-1)) in aqueous solutions at various pH (1, 3, 5, 7.4 and 10) and temperature (0, 8, 25, 37 and 60 degrees C) conditions. The ionic strength of all these solutions was maintained at 0.5 M. Ceftiofur solutions at pH 5 and 7.4 and in distilled water (pH = 6.8) were tested at all the above temperatures. All other solutions were tested at 60 degrees C. Over a period of 84 h, the stability was evaluated by quantifying ceftiofur and its degradation product, desfuroylceftiofur, in the incubation solutions. HPLC was used to analyse these compounds. At 60 degrees C, the rate of degradation was significantly higher at pH 7.4 compared with pH 1, 3, 5 and distilled water. At both 60 degrees C and 25 degrees C, degradation in pH 10 buffer was rapid, with no detectable ceftiofur levels present at the end of 10 min incubation. Degradation rate constants of ceftiofur were 0.79+/-0.21, 0.61+/-0.03, 0.44+/-0.05, 1.27+/-0.04 and 0.39+/-0.01 day(-1) at pH 1, 3, 5, 74 and in distilled water, respectively. Formation of desfuroylceftiofur was the highest (65%) at pH 10. The rate of degradation increased in all aqueous solutions with an increase in the incubation temperature. At pH 7.4 the degradation rate constants were 0.06+/-0.01, 0.06+/-0.01, 0.65+/-0.17, and 1.27+/-0.05 day(-1) at 0, 8, 25, 37 and 67 degrees C, respectively. The energy of activation for ceftiofur degradation was 25, 42 and 28 kcal mol(-1) at pH 5, 7.4 and in distilled water, respectively. Desfurylceftiofur formation was the greatest at alkaline pH compared with acidic pH. Ceftiofur degradation accelerated the most at pH 7.4 and was most rapid at pH 10. The results of this study are consistent with rapid clearance of ceftiofur at physiological pH.     

Stability of the new antileukemic 4-pyranone derivative, BTMP, using HPLC and LC-MS analyses

The stability of the new antileukemic kojic acid derivative, 5-benzyloxy-2-thiocyanatomethyl-4-pyranone (BTMP) was investigated. The degradation of BTMP was studied using specific and reproducible HPLC and LC-MS methods. Accelerated stability studies of BTMP were conducted in 0.1 M hydrochloric acid solution, physiological phosphate buffer solution (pH 7.5) and basic phosphate buffer solution (pH 9.0) at 30, 40 and 60 degrees C, respectively. The degradation of BTMP was found to follow pseudo-first order kinetics. In basic solution (pH 9.0) BTMP underwent rapid hydrolysis at a degradation rate constant (0.183-0.638 h-1) and degradation half-life (3.67-1.06 h) depending on the temperature setting. On the other hand, BTMP was significantly stable in 0.1 M hydrochloric acid solution (kdeg: 0.0017-0.0052 h-1; degradation half-life t1/2: 408.6-135.7 h), whereas in physiological phosphate buffer solution (pH 7.5), BTMP was only moderately stable (kdeg: 0.006-0.231 h-1; degradation half-life: 117.7-3.0 h). Arrhenius plots were constructed to predict the degradation kinetic parameters of BTMP at 25 degrees C and 4 degrees C. LC-MS analyses confirmed the degradation of BTMP in basic solutions and indicated at least two degradation products; namely 5-benzyloxypyran-2-ol-4-one (m/z 217.8) and 2-thiocyanatomethylpyran-5-ol-4-one (m/z 181.6).     

Comparative toxicity of ifosfamide metabolites and protective effect of mesna and amifostine in cultured renal tubule cells.

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde contributes to this nephrotoxicity. The present study examined the effects of chloroacetaldehyde and acrolein, another ifosfamide metabolite, on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medications sodium 2-mercaptoethanesulfonate (mesna) and amifostine to prevent chloroacetaldehyde- and acrolein-induced renal cell injury was also assessed. Chloroacetaldehyde and acrolein (25-200 M) produced dose-dependent declines in neutral red dye uptake, glucose transport and glutathione content. Chloroacetaldehyde was a more potent toxin than acrolein. Pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of both chloroacetaldehyde and acrolein while co-administration of mesna or amifostine prevented metabolite toxicity. These results support the hypothesis that chloroacetaldehyde is responsible for ifosfamide-induced nephrotoxicity. The protective effect of mesna and amifostine in vitro contrasts with clinical experience showing that these medications do not eliminate ifosfamide nephrotoxicity.     

Synthesis of N2,3-ethenodeoxyguanosine, N2,3-ethenodeoxyguanosine 5'-phosphate, and N2,3-ethenodeoxyguanosine 5'-triphosphate. Stability of the glycosyl bond in the monomer and in poly(dG,epsilon dG-dC)

The synthesis of a new modified etheno-2'-deoxyguanosine is reported. N2,3-Ethenodeoxy-guanosine (epsilon dGuo) is a product in double-stranded DNA treated with the carcinogen vinyl chloride in vivo or its metabolite chloroacetaldehyde in vitro. The lability of its glycosyl bond has, however, interfered with its isolation from DNA. The synthesis, starting with O6-benzyl-2'-deoxyguanosine 5'-phosphate, reacted with bromoacetaldehyde, could only be accomplished in slightly alkaline media, which prevented significant loss of the sugar. The 5'-phosphate also decreased the lability of the glycosyl bond. The resulting compound, when deprotected, was converted to N2,3-ethenodeoxyguanosine 5'-triphosphate, as well as the corresponding nucleoside. Fluorescence, UV, and 1H NMR data were consistent with the assigned structures and almost identical with those of the previously synthesized much more stable ribo analogues [Ku?mierek et al. (1987) J. Org. Chem. 52, 2374-2378]. A systematic study of the pH-dependent glycosyl bond cleavage gave a t1/2, 37 degrees C, pH 6, of approximately 3.5 h for the nucleoside and 7-10 h for the nucleotides. Comparison, under the same conditions, of stability of the glycosyl bond in poly(dG,epsilon dG-dC) showed an increased stability of 2 orders of magnitude, t1/2 = approximately 600 h. The rate of sugar loss was, in all cases, greatly decreased at higher pH's, over the range of pH 5-9. These stability data indicate that when slightly alkaline conditions can be used, studies on incorporation of epsilon dGuo into polymers for in vitro mutagenesis studies are possible.     

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.     

Synthesis of nucleosides and oligonucleotides containing adducts of acrolein and vinyl chloride

Vinyl chloride and acrolein are important industrial chemicals. Both form DNA adducts, vinyl chloride after enzymatic oxidation to chlorooxirane and acrolein by direct reaction. Reaction at the N(2) position of guanine is a major pathway. The resulting 2-oxoethyl and 3-oxopropyl adducts cyclize spontaneously to hydroxyethano and hydroxypropano derivatives, respectively. The two cyclic adducts have been detected in DNA exposed to these mutagens. A new method has been developed for the synthesis of deoxyguanosine adducts of chlorooxirane and acrolein, as well as oligonucleotides containing these adducts. Reaction of O(6)-[(trimethylsilyl)ethyl]-2-fluoro-2'-deoxyinosine with the appropriate aminodiol followed by oxidative cleavage of the diol with NaIO(4) gave the adducts in excellent yields. Reaction of oligonucleotides containing the halonucleoside with the aminodiols followed by NaIO(4) efficiently created the nucleosides in the oligonucleotides. Deoxyadenosine adducts were created similarly using 6-chloropurine 9-(2'-deoxyriboside).     

Site specific synthesis and polymerase bypass of oligonucleotides containing a 6-hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one base, an intermediate in the formation of 1,N2-etheno-2'-deoxyguanosine

The reaction of DNA with certain bis-electrophiles such as chlorooxirane and chloroacetaldehyde produces etheno adducts. These lesions are highly miscoding, and some of the chemical agents that produce them have been shown to be carcinogenic in laboratory animals and in humans. An intermediate in the formation of 1,N2-ethenoguanine is 6-hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one (6-hydroxyethanoguanine), which undergoes conversion to the etheno adduct. The chemical properties and miscoding potential of the hydroxyethano adduct have not been previously studied. A synthesis of the hydroxyethano-adducted nucleoside was developed, and it was site specifically incorporated into oligonucleotides. This adduct had a half-life of between 24 and 48 h at neutral pH and 25 degrees C at the nucleoside and oligonucleotide levels. The miscoding potential of the hydroxyethano adduct was examined by primer extension reactions with the DNA polymerases Dpo4 and pol T7-, and the results were compared to the corresponding etheno-adducted oligonucleotide. Dpo4 preferentially incorporated dATP opposite the hydroxyethano adduct and dGTP opposite the etheno adduct; pol T7- preferentially incorporated dATP opposite the etheno adduct while dGTP and dATP were incorporated opposite the hydroxyethano adduct with nearly equal catalytic efficiencies. Collectively, these results indicate that the hydroxyethano adduct has a sufficient lifetime and miscoding properties to contribute to the mutagenic spectrum of chlorooxirane and related genotoxic species.     

Degradation of berenil (diminazene aceturate) in acidic aqueous solution

The trypanocide berenil was assessed for chemical stability over the pH range 1-8 at 37 degrees C and 0.2 M ionic strength. It was found to be sufficiently unstable under acid conditions that its therapeutic efficacy is most likely severely compromised when administered orally. At pH 3, the half-life was 35 min, decreasing to 1.5 min at pH 1.75. Reaction rate constants were corrected for the effects of buffer catalysis and were found to range from 2.00 min(-1) at pH 1 to 6.1 x 10(-6) min(-1) at pH 8. The pH-rate profile displayed a region (pH 1-4) where specific acid catalysis was dominant, followed by a transitional region (pH 5-7), and finally a region (pH >7) where uncatalysed degradation was most important. It is recommended that berenil be enteric coated for formulations to be used in treating Third World parasitic diseases.     

N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis,Characterization, and Decomposition Profile Under Physiological Conditions

(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2′S,3′S)-N-7-(3′,4′-epoxy-2′-hydroxybut-1′-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2′S,3′S)-N-7-(2′3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a β-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.     

1,N2-ethenodeoxyguanosine: properties and formation in chloroacetaldehyde-treated polynucleotides and DNA.

1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.     

Analysis of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human leukocyte DNA from smokers and nonsmokers

Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N(2)-propanodeoxyguanosine adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H(2)O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10(9) nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10(9) nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.     

On the hydrolytic behavior of tinidazole,metronidazole, and ornidazole

Using two UV-spectrophotometric methods, the hydrolysis of tinidazole was studied at pH 1.00-8.45 at 80 degrees C. The reaction followed apparent first-order kinetics throughout the studied range. No kinetic salt effect was detected, indicating that at least one of the reacting partners forming the transition state has a charge of 0. The reaction rate macro constants M(1)-M(4) were calculated to be 3.35 x 10(-2) M(-1) h(-1), 1.45 x 10(-2) h(-1), 3.76 x 10(-6) M h(-1), and 2.85 x 10(-11) M(2) h(-1), respectively. At pH >or= 7, the uncharged tinidazole was decomposed by the hydroxide ion; the reaction was found out to involve a proton transfer from the ethylsulfonylethyl side chain. At around pH 4.5, the degradation of the uncharged tinidazole was due to the solvent. In more acidic conditions, the reaction mechanism could not be fully resolved. The alkaline hydrolysis of metronidazole was studied on the basis of literature data. A general reaction mechanism was proposed, but an unequivocal explanation for the inflection point in the pH rate profile at pH 6 could not be found. The implications of the proposed reaction mechanism for the hydrolytic behavior of ornidazole were discussed.     

Synthesis, stability and pharmacological evaluation of a novel codrug consisting of lamivudine and ursolic acid

A novel codrug (LMX) was obtained from lamivudine (LMV) and ursolic acid (UA) coupled with ethyl chloroacetate through an amide and ester linkage. The structure of LMX was confirmed by 1H NMR, 13C NMR, IR and HRMS. Herein, the in vitro non-enzymatic and enzymatic hydrolysis and in vivo pharmacological activities of LMX were studied. The kinetics of hydrolysis of LMX was studied in aqueous solution of pH 1-10, 80% buffered human plasma and in the presence of lipase from Porcine pancreas (EC 3.1.1.3) at 37°C. It is found that LMX hydrolysis rate was significantly faster in lipase with half-life of 1.4h compared to pH 7.4 phosphate buffer (t(1/2) 11.2h) and buffered human plasma (t(1/2) 5.4h). The decomposition rates in aqueous solution (pH 1-10) showed a U-shaped curve. LMX was comparatively stable between pH 3 and 6 (half-life >40 h). Pharmacological studies indicated that LMX had the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury. These findings suggest that LMX could be a promising candidate agent for the treatment of hepatitis.     

Kinetics of O(6)-methyl-2'-deoxyguanosine repair by O(6)-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences

O(6)-Methyl-2'-deoxyguanosine (O(6)-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O(6)-Me-dG is directly repaired by the specialized DNA repair protein, O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O(6)-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O(6)-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5'-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3', where G5, G6, G7, G8, G9, G10, or G11 was replaced with O(6)-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G --> A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O(6)-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of O(6)-Me-G remaining in DNA. The relative extent of demethylation for O(6)-Me-dG adducts located at G5, G6, G7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O(6)-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G6G7T-3') were similar (1.4 x 10(7) M(-1) s(-1) vs 7.4 x 10(6) M(-1) s(-1), respectively), suggesting that O(6)-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.     

Mass spectrometry based approach to study the kinetics of O6-alkylguanine DNA alkyltransferase-mediated repair of O6-pyridyloxobutyl-2'-deoxyguanosine adducts in DNA

O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).     

A new LC-MS/MS method for the quantification of endogenous and vinyl chloride-induced 7-(2-Oxoethyl)guanine in sprague-dawley rats

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [(13)C(2)]-7-OEG from the reaction of calf thymus DNA with [(13)C(18)]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10(6) guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days was 104.0 ± 23.0 adducts per 10(6) guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10(6) guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [(13)C(2)]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10(6) guanine in liver. Studies on the persistence of [(13)C(2)]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [(13)C(2)]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.     

Inhibition of the hepatic O6-alkylguanine-DNA alkyltransferase in vivo by pretreatment with antineoplastic agents

The mammalian DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) is inactivated during the repair process and its activity can only be restored by de novo synthesis. We have made use of this property to determine whether and to what extent various chemotherapeutic agents alkylate DNA in the O6-position of guanine, ie. produce lesions susceptible to AT repair. Adult female Fischer rats received a single i.p. injection of a high dose (LD50) of the respective agent and, 5 hr later, a chasing dose of N-nitroso-[14C]dimethylamine (0.2 mg/kg; 4 hr survival). The amount of 7-[14C]methylguanine formed was approximately 95 mumol/mol guanine and not significantly altered by pretreatment with any of the drugs. The ratio of O6-[14C]methylguanine/7-[14C]methylguanine was 0.019 for control animals, indicating that during the observation period of 4 hr, 83% of the O6-[14C]methylguanine produced had been removed by the hepatic AT. Little or no effect was found in rats that received spirohydantoin mustard, hexamethylmelamine, cis-platinum or mitomycin C. A significant increase in the O6-/7-[14C]methylguanine ratio was found after pretreatment with AZQ (0.026) and cyclophosphamide (0.028), agents for which lesions involving the O6-position of guanine have not yet been identified. N-(2-Hydroxyethyl)-N-nitrosourea and the cytostatic haloethylinitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), and N-chloroethyl-N-hydroxyethylnitrosourea (HECNU) inhibited the hepatic AT, producing a ratio of 0.025-0.035. Considerably higher ratios of 0.059 and 0.101 were observed after administration of the methylating agents procarbazine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), respectively. Complete saturation of the repair system (O6-/7-[14C]methylguanine ratio, 0.11) was only achieved with N-methyl-N-nitrosourea.