3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential.

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).

Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

Analysis of nonylphenol and nonylphenol ethoxylates in environmental samples by mixed-mode high-performance liquid chromatography-electrospray mass spectrometry.

A new method is described based on mixed-mode high-performance liquid chromatography with electrospray mass spectrometry detection for comprehensive quantitative analysis of nonylphenol (NP) and nonylphenol ethoxylates (NPEOs) in wastewater and sediment. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing a methanol-water gradient on a mixed-solvent gel filtration column designed for MS interfacing. Quantitative accuracy and precision of the method were improved by the use of custom-synthesized [13C6]NPEO analogs as isotope-dilution surrogate standards. Method detection limits for NP and individual NPEOs ranged from I to 55 pg injected on column.

Characterization of 2'-deoxycytidine and 2'-deoxyuridine adducts formed in reactions with acrolein and 2-bromoacrolein.

The products of the reaction of the mutagenic aldehydes, acrolein and 2-bromoacrolein, with 2'-deoxycytidine and 2'-deoxyuridine have been determined. These products, formed at physiological conditions, were isolated by reverse-phase HPLC and characterized by UV, 1H NMR, fast atom bombardment MS, electrospray MS, and chemical transformation. The reaction of 2'-deoxycytidine with acrolein and 2-bromoacrolein produced the exocyclic compounds 3-(2'-deoxyribosyl)-7,8,9-trihydro-7-hydroxypyrimido[3,4- c]pyrimidin-2-one and 3-(2'-deoxyribosyl)-7,8,9-trihydro-7-hydroxy-8-bromopyrimido [3,4-c] pyrimidin-2-one, respectively. In addition to the chiral centers of deoxyribose, one new chiral center was formed from C-1 of acrolein and two new chiral centers were formed from C-1 and C-2 of 2-bromoacrolein, creating a mixture of diastereomers for each product. These compounds are not stable in basic solution and undergo ring opening and hydrolytic deamination, resulting in 2'-deoxyuridine adducts. The N3-alkylated 2'-deoxyuridines were also synthesized by permitting 2'-deoxyuridine to react with 2-bromoacrolein and acrolein. An unstable intermediate, N3-(2"-bromo-3"-oxopropyl)-2'-deoxyuridine, was also isolated and characterized from the reaction with 2-bromoacrolein. The reaction of 2'-deoxyuridine with acrolein gave N3-(3"-oxopropyl)-2'-deoxyuridine as the major product, which was reduced to its corresponding alcohol with NaBH4. Reactions of 2'-deoxycytidine with 2-bromoacrolein and acrolein proceed most rapidly at acidic or neutral pH; however, 2'-deoxyuridine reacts most rapidly at neutral or basic pH.

A chemical ionization selected ion monitoring assay for methylphenidate and ritalinic acid.

A methane chemical ionization quantitative assay for methylphenidate and its major metabolite, ritalinic acid, is described. Methylphenidate and the internal standard, ethylphenidate, were extracted from plasma samples and derivatized to prevent thermal decomposition in the gas chromatography. Ritalinic acid was esterified with diazomethane and extracted as methylphenidate. The intensity of the protonated molecular ion of the derivatized drug and internal standard was measured by selected ion monitoring. Calibration curves were prepared from drug standards dissolved in drug-free plasma, and the lower limit of the curves extended to 0.5 ng methylphenidate per ml plasma. The method was used to generate plasma decay curves for pediatric patients undergoing methylphenidate therapy.

Characterization of thymidine adducts formed by acrolein and 2-bromoacrolein.

Thymidine was permitted to react with the known mutagens acrolein and 2-bromoacrolein under physiological conditions. The products of these reactions were separated by HPLC and characterized by UV, FAB/MS, electrospray MS, 1H NMR and chemical transformation. The reaction with acrolein gave one major product, N3-(3''-oxopropyl)thymidine, which is unstable in aqueous solution and was reduced with sodium borohydride to the corresponding alcohol. Reaction with 2-bromoacrolein yielded the unstable intermediate, N3-(2''-bromo-3''-oxopropyl)thymidine, and two stable products, the diastereomers of N3-(2''-hydroxy-3''-oxopropyl)thymidine, which are slowly transformed to N3-(2''-oxo-3''-hydroxypropyl)thymidine. Reactions with both mutagens proceed most rapidly at pH 9.2, less rapidly at pH 7.4, and no products are found at pH 4.2. Stable adducts found in the reaction of 2-bromoacrolein were also identified in reactions with single-strand oligodeoxynucleotides using a sensitive, selected ion monitoring GC/MS procedure.

Analysis of alkylphenol ethoxylate metabolites in the aquatic environment using liquid chromatography-electrospray mass spectrometry.

A quantitative method is described for the analysis of the metabolites of alkylphenol ethoxylate (APEO) surfactants in estuarine water and sediment samples using reversed-phase high-performance liquid chromatography with electrospray mass spectrometry detection. Nonyl- and octylphenols, nonyl- and octylphenol mono-, di-, and triethoxylates, halogenated nonylphenols, and nonylphenol ethoxycarboxylates were concentrated from water samples using a C18 solid-phase extraction procedure. A novel, continuous-flow, high-temperature, sonicated extraction system was developed to isolate APEO metabolites from sediment samples. Quantitative LC-MS was performed in the negative ion mode for nonylphenols, octylphenols, and halogenated nonylphenols and in the positive ion mode for nonyl- and octylphenol ethoxylates using selected ion monitoring with isotopically labeled surrogate standards. Recoveries for sediment and water analyses ranged between 78 and 94%, and detection limits for APEO metabolites were between 1 and 20 pg injected on column. This is a significant improvement over previously reported methods. Suppression of analyte response was encountered in the presence of matrix components in sediment samples, but this effect was eliminated by careful selection of surrogate and internal standards. Individual APEO metabolite concentrations of 1-320 ng/L and 5-2000 ng/g are reported for water and sediment samples, respectively, from Jamaica Bay, NY.

Characterization of the alkaline degradation products of an oligodeoxynucleotide containing 8-oxo-7,8-dihydro-2'-deoxyguanosine by electrospray ionization mass spectrometry.

Oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine exhibit alkaline sensitivity and undergo cleavage of the phosphodiester backbone. Identification of the major degradation products and unstable intermediates formed in concentrated ammonia was accomplished by HPLC isolation and characterization by electrospray ionization mass spectrometry. Unstable intermediates were reduced in situ with NaBH4 prior to isolation and mass analysis. This technique produced accurate mass data for an oligonucleotide intermediate containing an abasic site, a strand cleavage, product containing the 3'-terminus, and two products with the 5'-terminus. 8-Oxoguanine was not present in the product HPLC chromatogram, suggesting rearrangement or degradation of this moiety prior to glycosidic bond cleavage. A scheme for the decomposition of 7,8-dihydro-2'-deoxyguanosine-containing oligonucleotides in 28% ammonia solution is presented.

Distribution and fate of neutral alkylphenol ethoxylate metabolites in a sewage-impacted urban estuary.

The distribution and fate of neutral metabolites of the alkylphenol ethoxylate (APEO) surfactants in an urbanized estuarine environment were examined utilizing a recently developed, highly sensitive LC-MS method. Results indicated that short ethoxyl-chain APEOs and alkylphenols (APs) were present in surficial sediments throughout the estuary at concentrations roughly correlated to the organic carbon content of the sediment and that the APEO mixture was dominated by nonylphenol ethoxylate (NPEOs) metabolites (0.05-30 microg/g), with lesser amounts of octylphenol ethoxylate metabolites (OPEOs)(<0.005-0.09 microg/ g) and halogenated nonylphenols (<0.001-0.03 microg/g). NPEO metabolites in surface water (0.22-1.05 microg/L) were also present at higher concentrations than OPEO metabolites (0.007-0.040 microg/L). APEO metabolite concentrations in both sediment and water showed a strong correlation with conventional sewage tracers, affirming a wastewater source of these contaminants. APEO distributions in surface waters within the estuary could be explained by a combination of post-discharge degradation and mixing with a seawater end-member enriched in OPEO metabolites. Measured in situ Koc values of APEO metabolites were comparable to previously reported values derived from field experiments but higher than Kow and Koc values derived from laboratory experiments. Results from the present work indicate that the fate of APEO metabolites entering the estuarine environment through discharge of wastewater is directed primarily by scavenging onto particles and subsequent burial in sediments, degradation during residence in the water column, and transport out of the estuary through advective and dispersive processes.

Determination of steroid estrogens in wastewater by immunoaffinity extraction coupled with HPLC-electrospray-MS.

A new method, based on immunoaffinity extraction coupled with liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) is described for the determination of the steroid estrogens beta-estradiol (E2), estrone (E1), and alpha-ethynylestradiol (E2) in wastewater. The use of highly selective immunosorbents in sample preparation prior to analysis allows the removal of interfering sample matrix compounds present in the wastewater extracts that would otherwise cause severe ionization suppression of the estrogens during the electrospray process. In addition, immunoextraction removes much of the isobaric noise from the selected ion monitoring chromatograms, increasing the signal-to-noise ratios for analytes, and contributing to the low detection limits (0.18 and 0.07 ng/L for E2 and E1, respectively) achieved by the current method. The method was applied to analysis of estrogens in two wastewater effluents. Recoveries of E2 and E1 were excellent (>90%), while the nonimmunogen (but structurally related) analyte EE2 was not retained (recovery <2%) from effluent extracts by the immunosorbent. This illustrates the extreme selectivity of the immunoextraction purification step. Precision of the method was high, with relative standard deviations below 5%. Concentrations of E2 in wastewater varied from 0.77 to 6.4 ng/L, while concentrations of E1 were greater (1.6-18 ng/L).

NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein.

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Analysis of synthetic oligodeoxynucleotides containing modified components by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI/MS) has been used to confirm the synthesis of oligodeoxynucleotides containing modified structures and to identify products of unstable oligomer degradation. Negative ion mass spectra of oligomers and dimethoxytrityl-protected oligomers exhibited a distribution of multiply charged molecular ions with few adduct ions present. Molecular masses may be determined from the array of peaks with excellent accuracy and serve to corroborate the synthetic sequence and the inclusion of a modified structure within the sequence. Incorporation of modified deoxynucleotides, stable isotopes, and other novel structures such as a terminal biotin moiety and a phosphorothioate linkage can be rapidly verified by ESI/MS. This technique has also proved useful for the identification of products of unstable oligomers formed during synthesis, deprotection, or purification, including the major products formed under basic conditions from 5'-dimethoxytrityl-protected oligomers containing 8-oxo-2'-deoxyguanosine.

Synthesis of 1,N2-(1,3-propano)-2'-deoxyguanosine and incorporation into oligodeoxynucleotides: a model for exocyclic acrolein-DNA adducts.

2'-Deoxyguanosine (3) and native DNA both give rise to exocyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts 6 and 7 upon treatment with acrolein (1), a known mutagen, in vitro under physiological conditions. The use of synthetic deoxyoligonucleotides containing adduct 6 or 7 could shed light on the mechanism of the mutagenicity of 1 and on the nature of the structural perturbations present in DNA duplexes where they are present. Unfortunately, this is precluded by the instability of 6 and 7 to the conditions of automated DNA synthesis. We have prepared 1,N2-(1,3-propano)-2'-deoxyguanosine (PdG) (8) as a stable model for 6/7. The structure of 8 has been verified by magnetic resonance, ultraviolet spectroscopy, and mass spectrometry. This moiety has been incorporated into oligodeoxynucleotides via solid-state synthesis technology. Negative ion fast atom bombardment (FAB) mass spectrometry of the pentaoligodeoxynucleotide 5'-GT(PdG)CG-3' verified the identity and position of the modified base. The validity of 8 as a model system for the adduct pair 6/7 in structural and biological studies of DNA duplexes is discussed.

Synthesis of enzymatically noncleavable carbocyclic nucleosides for DNA-N-glycosylase studies.

Carbocyclic nucleosides have been of great interest as antiviral agents and in studies in the area of antisense technology. The recent finding that the replacement of a single 2'-deoxynucleoside in DNA by a carba analogue does not alter the Watson-Crick base pairing, yet at the same time provides a chemically and enzymatically stable "glycosidic" linkage, led us to examine this class of compound as enzyme inhibitors of the DNA-repair enzymes involved in oxidative damage. We now report the synthesis and incorporation into oligomeric DNA via suitable derivatives, the carbanucleosides 8-oxo-7,8-dihydro-2'-deoxycarbainosine, 8-oxo-7,8-dihydro-2'-deoxycarbaguanosine, and 2'-deoxyaristeromycin. Aristeromycin (1) was deoxygenated at the 2'-position as follows. Treatment of 1 with TPDSCl2 gave the 3',5'-protected derivative 3 (76%) which on phenylthiocarbonylation at the 2'-position gave 4 in 51% yield. The latter compound on reduction with Bu3SnH led to the 2'-deoxy derivative 5 (90%). Benzoylation followed by deprotection with TBAF in THF then gave the desired intermediate (6) in 65% yield. N2-Isobutyryl-8-oxo-7,8-dihydro-2'-deoxycarbaguanosine (16) was synthesized from 3-chloro-2'-deoxycarbainosine (9). Treatment of 9, either with hydrazine followed by catalytic reduction of the 2-hydrazino derivative or with 1-(2-nitrophenyl)ethylamine followed by photolysis of the resulting 2-substituted derivative, in both instances gave the desired 2'-deoxycarbaguanosine (12) in approximately 50% overall yield in each case. Bromination of 12 gave 13 (90%) which, when treated with BnONa in DMSO at 65 degrees C, led to the 8-benzyloxy derivative 14 (46%). Isobutyrylation of 14 followed by catalytic reduction then afforded 16. 8-Oxo-7,8-dihydro-2'-deoxycarbainosine (23) was prepared in four steps. Bromination of 2'-deoxyaristeromycin (19) at the 8-position gave 20 (> 95%) which was converted to the 8-benzyloxy derivative 21 (61%) using BnONa/DMSO at 80 degrees C. Reductive debenzylation of 21 then led to 8-oxo-7,8-dihydro-2'-deoxyaristeromycin (approximately 100%) which, when treated with adenosine deaminase, provided the desired carbainosine derivative 23 in quantitative yield. Compounds 6, 16, and 23 were converted to their respective 5'-O-DMT, 3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine] derivatives (8, 18, and 25) in excellent overall yields. The latter were then used to synthesize a series of DNA oligomers by automated procedures.

Suppression of adventitious formation of 8-oxoguanine(TMS)4 from guanine during trimethylsilylation.

GC/MS quantitation of 8-oxoguanine derived from DNA may yield artificially high values when trimethylsilylation is used as the technique to form a volatile derivative. Significant quantities of the tetrakis trimethylsilyl-derivative of 8-oxoguanine may be formed from guanine during the derivatization reaction at elevated temperatures. We have screened eight antioxidants in an attempt to identify a substance that will reduce the spurious formation of this product. Whereas several compounds were active, N-phenyl-1-naphthylamine was the most effective agent. Moreover, this compound was sufficiently basic to act as a catalyst for the derivatization reaction. Our data, which are based on the use of an isotopically labeled internal standard, show that the level of the tetrakis trimethylsilyl derivative of 8-oxoguanine remained constant as a function of derivatization heating time when N-phenyl-1-naphthylamine was added to a reaction in which guanine was present. In contrast, the level in control samples increased linearly with reaction time indicating formation of 8-oxoGua during derivatization. The level of 8-oxoguanine was measured in both a synthetic oligodeoxynucleotide containing a single 8-oxo-2'-deoxyguanosine moiety and commercially available calf thymus DNA using this technique.

Formation of s-triazines during aerial oxidation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in concentrated ammonia.

After automated DNA synthesis, oligodeoxynucleotides containing 8-oxoguanine are sensitive to aerial oxidation when subjected to the basic conditions necessary for deprotection and release of the oligomer from the control pore glass support. The major oxidation products of this heterocyclic moiety have been characterized by permitting 8-oxo-7,8-dihydro-2'-deoxyguanosine to react with oxygen in the presence of 28% aqueous ammonia at room temperature. Products were isolated by reverse phase HPLC and analyzed by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry of the trimethylsilyl-derivatives. 2-Amino-4-hydroxy-s-triazine-6-carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine were identified by these techniques and standards were synthesized. In addition, GC-MS analysis revealed other oxidation products, including urea, guanidine and 2-deoxyribose, which were not observed by HPLC because these compounds are transparent in the UV region of the spectrum. Both s-triazines were also observed when a purified, synthetic oligodeoxynucleotide containing a single 8-oxoguanine moiety was exposed to the same conditions. Oxidation of 8-oxoguanine appears to parallel the uric acid oxidation pathway, and a mechanistic scheme is proposed to account for the products of degradation.

8-Amino-2'-deoxyguanosine incorporation into oligomeric DNA.

During the incorporation of 8-amino-dG into oligomeric DNA, the deprotection conditions previously recommended (28% ammonia at room temperature) do not effect complete removal of the dimethylaminomethylene protecting groups. At elevated temperatures oxidative degradation of the oligomer and exchange of ammonia with dimethylamine in the protecting group at C8 occurred. The resolution of these problems and a method to obtain a series of homogeneous oligomers in reasonable yield containing 8-amino-dG located site-specifically are described.

Tiered exposure-assessment strategy in the Semiconductor Health Study.

The Semiconductor Health Study included 14 U.S. companies with 82 silicon-based wafer-fabrication rooms and a complex array of processes and chemicals. Its epidemiologic components were a historical examination of spontaneous abortion rates among 902 women, a prospective evaluation of reproductive outcomes in 483 women, and a cross-sectional review of male fertility and of respiratory, ergonomic, neurologic, or gastrointestinal problems among 3,175 men and women. Designing an exposure assessment strategy presented unique problems, and multiple agents had to be evaluated. A three-tiered approach to exposure assessment was developed to reflect increasing specificity of exposures. At the first tier, employees were divided into fabrication and nonfabrication groups. At the second tier, work groups with qualitatively different exposures were determined. At the third tier, intensity of exposures to specific chemical, physical, and ergonomic agents was evaluated. Evaluations were based on worker reports of tasks performed, moderated by fabrication-specific factors observed by study industrial hygienists during site visits.