[ 13 C 6 ]salicylate, [U-13 C]naphthalene, and [U-13 C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the "heavy" and "light" DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [ 13 C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate-and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2-and 7-day incubations with naphthalene, so secondary utilization of the 13 C during the incubation did not appear to be an issue in this experiment.

Uncultivated bacteria associated with the degradation of pyrene in a bioreactor treating soil contaminated with polycyclic aromatic hydrocarbons (PAH) were identified by DNA-based stable-isotope probing (SIP) and quantified by real-time quantitative PCR. Most of the 16S rRNA gene sequences recovered from (13)C-enriched DNA fractions clustered phylogenetically within three separate groups of beta- and gamma-Proteobacteria unassociated with described genera and were designated "Pyrene Groups 1, 2 and 3". One recovered sequence was associated with the Sphingomonas genus. Pyrene Groups 1 and 3 were present in very low numbers in the bioreactor but represented 75% and 7%, respectively, of the sequences recovered from 16S rRNA gene clone libraries constructed from (13)C-enriched DNA. In a parallel time-course incubation with unlabelled pyrene, there was between a 2- and 4-order-of-magnitude increase in the abundance of 16S rRNA genes from Pyrene groups 1 and 3 and from targeted Sphingomonas spp. over a 10 day incubation. Sequences from Pyrene Group 2 were 11% of the SIP clone libraries but accounted for 14% of the total bacterial 16S rRNA genes in the bioreactor community. However, the abundance of this group did not increase significantly in response to pyrene disappearance. These data indicate that the primary pyrene degraders in the bioreactor were uncultivated, low-abundance beta- and gamma-Proteobacteria not previously associated with pyrene degradation.

3,4-Epoxy-1-butene (EB) is the major mutagenic metabolite of butadiene (BD), an important industrial chemical classified as a probable human carcinogen. Although the mechanism of carcinogenicity of EB is not known, its reactions with nucleophilic sites of DNA giving pro-mutagenic lesions are likely to constitute the early crucial step in multistage carcinogenesis. This study was conducted to characterize the adducts formed from reactions of EB with the most nucleophilic DNA nucleobases, adenine (Ade) and guanine (Gua), as free nucleobases, 2'-deoxyribonucleosides and constituents of calf thymus DNA (CT DNA) in order to provide insight into the nature of DNA modification by EB. The adducts were isolated using HPLC separation coupled with diode array detection (DAD) and structurally characterized from their electronic, mass- and nuclear magnetic resonance spectra. Four EB-adenine products were identified as N-1-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade I), N-1-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade II), N-3-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade III) and N-3-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade IV). Two previously reported guanine adducts: N-7-(2-hydroxy-3-buten-1-yl) guanine (EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-Gua II) were also collected. The purified adducts were used as reference compounds to detect and quantitate the corresponding adduct species formed in calf thymus DNA incubated with EB. All six adducts were detected in treated DNA. The N-7 position of guanine was the most reactive in DNA followed by N-3 of adenine and N-1 of adenine. The formation of N-1 and N-3-adenine adducts (EB-Ade I, 1.2 +/- 0.36; EB-Ade II, 0.8 +/- 0.27; EB-Ade III, 2.7 +/- 0.38; EB-Ade IV, 5.9 +/- 0.68 nmol/micromol Ade) in CT DNA was approximately one-tenth that of EB-guanine adducts (50.7 +/- 2.37 and 47.9 +/- 3.6 nmol/micromol Gua, respectively). The N-1-EB-Ade adducts detected in this study are likely to be the precursors of previously reported N6-EB-adenine adducts (Koivisto et al., 1995) through Dimroth rearrangement. Since BD and EB induce significant numbers of point mutations at A:T base pairs, the EB-adenine adducts may represent important lesions involved in BD-induced mutagenesis and carcinogenesis.

Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene (BD), a high-volume industrial chemical classified as a probable human carcinogen. Rodent inhalation studies show strikingly high sensitivity of mice to carcinogenic effects of butadiene compared to rats, which has been linked to differences in metabolism. Both species convert BD to 3,4-epoxy-1-butene (EB), but mice further oxidize a significantly greater part of EB to DEB. DEB is a potent bifunctional genotoxic agent which is 100-fold more mutagenic than EB and is likely to be involved in BD-induced carcinogenesis. Identification of specific BD-induced DNA adducts is critical to understanding the mechanism of its biological activity. We have previously described reactions of EB with guanine and adenine as nucleobases, nucleosides, and constituents of DNA. In this work, DEB-induced guanine adducts were isolated and structurally characterized by UV spectroscopy, mass spectrometry, and nuclear magnetic resonance. When guanosine was reacted with DEB in glacial acetic acid followed by hydrolysis in hydrochloric acid, three products were isolated: N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (DEB-Gua I, major adduct), N-7-(2',4'-dihydroxy-3'-chlorobut-1'-yl)guanine (DEB-Gua II), and N-7-(2',3'-dihydroxy-4'-acetoxybut-1'-yl)guanine (DEB-Gua III). We suggest initial formation of the N-7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine intermediate followed by nucleophilic substitution at the 3',4'-epoxy ring with hydroxide, chloride, or acetate anions to give DEB-Gua I, II, or III, respectively. DEB-Gua I and the epoxy intermediate were also isolated from hydrolysates of DEB-exposed calf thymus DNA (CT DNA). N-7-Guanine adducts are known to undergo spontaneous and enzymatic depurination producing apurinic sites. If not repaired before DNA replication, apurinic sites can give rise to mutations and ultimately cancer. The extent of alkylation at the N-7 of guanine in DEB-exposed DNA (58.7 +/- 1.1 adducts/10(3) normal guanines) was similar to that previously reported for CT DNA exposed to EB at the same molar ratio. Since EB and DEB appear to induce comparable levels of overall DNA alkylation at the conditions applied in this work, other factors, such as formation of DNA cross-links by DEB but not EB or differences in repair of EB and DEB adducts, may be responsible for the differences in mutagenicity.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-CPBA and DMDO as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT)·(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism probed by oxidation of the monomeric species dGuo, dGMP and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with 18O2-m-CPBA and H218O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker.

We have previously described an immunoaffinity/gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay for quantitation of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N(2)-ethenoguanine (1,N(2)-epsilonGua), in the same DNA sample. 1,N(2)-epsilonGua and N(2),3-epsilonGua were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect the [M - 181](-) fragments of 3,5-(PFB)(2)-N(2),3-epsilonGua and 3,5-(PFB)(2)-[(13)C(4),(15)N(2)]-N(2),3-epsilonGua and the [M - 201](-) fragments of 3,5-(PFB)(2)-1,N(2)-epsilonGua and 3,5-(PFB)(2)-[(13)C(3)]-1,N(2)-epsilonGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N(2),3-epsilonGua and 15 fmol of 1,N(2)-epsilonGua in approximately 250 microg of DNA, which corresponded to 5.0 N(2),3-epsilonGua and 8.7 1,N(2)-epsilonGua adducts/10(8) unmodified Gua bases, respectively. 1,N(2)-epsilonGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N(2)-epsilonGua to N(2),3-epsilonGua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N(2),3-epsilonGua was detected in DNA treated with the vinyl chloride (VC) metabolite 2-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks (6 h/day for 5 days/week). These data suggest that 1,N(2)-epsilonGua plays a minor role relative to N(2),3-epsilonGua in VC-induced carcinogenesis, but that 1,N(2)-epsilonGua may be formed to a larger extent from endogenous oxidative processes.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-(13)C(4)]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C(21)H(9)N(4)O(3)F(10), resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C(4)]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C(4)]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 microg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7-HEG varied from 0.2 to 0.3 pmol/micromol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 microg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.