Iminohydantoin lesion induced in DNA by peracids and other epoxidizing oxidants.

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-chloroperbenzoic acid (m-CPBA) and dimethyldioxirane (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 has been 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 [(18)O(2)]-m-CPBA and H(2)(18)O 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.

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.

Synthesis of chlorinated and non-chlorinated biphenyl-2,3- and 3,4-catechols and their [2H3]-isotopomers.

A synthetic scheme is described for chlorinated biphenyl-2,3- and 3,4-catechols to be used as standards for structural assignment of metabolites and protein adducts of 2,2',5,5'-tetrachlorobiphenyl in which both rings retain chlorine substituents. The scheme has general applicability to the synthesis of chlorinated biphenyl catechols. Dimethyl catechol ethers are coupled to dichloroaniline via the Cadogan reaction to give a library of isomers, followed by demethylation of the ethers with BBr3 to yield the target catechols. Separation of pure isomers is accomplished by TLC or HPLC prior to or following demethylation, depending on the isomer mixture. [2H3]-Isotopomers are generated using 2,5-dichloroaniline-d3 as the starting arylamine in the coupling reaction. The dichloroaniline-d3 hydrochloride is obtained as the sole product from nitration of p-dichlorobenzene-d4 followed by Pd/C-catalyzed hydrogenation under strongly acidic conditions. This hydrogenation procedure provides a simple and convenient approach to selective reduction of aryl nitro groups in the presence of halogen ring substituents.

Synthesis and characterization of peptides containing a cyclic Val adduct of diepoxybutane, a possible biomarker of human exposure to butadiene.

1,3-Butadiene, a potential human carcinogen widely used in industry, is oxidized by cytochrome P450 to diepoxybutane (DEB), which is the most mutagenic of the known butadiene metabolites. Assessment of the toxicological significance of DEB formation in humans and animals requires identification of a biomarker uniquely associated with DEB for use in molecular dosimetry studies. We wished to develop a specific and sensitive assay for one such suitable marker, the cyclic adduct 2-(3,4-dihydroxypyrrolidin-1-yl)-3-methylbutyramide (pyr-V), which is formed from addition of DEB to the terminal Val of the alpha- and beta-chains of hemoglobin. We needed to prepare a pure, rigorously characterized DEB-modified N-terminal oligopeptide for raising antibodies both to use in an immunoaffinity purification step and to standardize the assay. In addition, we needed a pure isotopomer to serve as an internal standard for quantitation by LC-MS. Direct modification of the globin sequences by reaction with DEB in vitro proved to be unproductive. We therefore opted to synthesize the cyclic Val adduct and incorporate it by FMOC chemistry into the appropriate oligopeptide sequences. In vitro and in vivo, butadiene is oxidized to enantiomeric and meso forms of DEB. A priori, all three DEB isomers are expected to form pyr-V adducts, resulting in three diastereomeric N-terminal peptides. We therefore synthesized a mixture of the cyclic Val diastereomers as their methyl esters by reaction of DEB with l-Val methyl ester hydrochloride. After protection as the di-O-tert-butyl derivatives, the mixture of 2-(3,4-di-t-butoxypyrrolidin-1-yl)-3-methylbutyric acid diastereomers was incorporated as the N-terminal residue into the 1-11 human globin alpha-chain sequence VLSPADKTNVK. The presence of the three diastereomers was confirmed by two-dimensional correlation NMR spectroscopy and temperature-dependent (1)H NMR. This strategy enabled us to obtain pure, rigorously characterized haptens in quantity for the preparation of polyclonal antibodies. Use of FMOC-protected (2)H(3)-Leu in the automated oligopeptide synthesis provided the required isotopomers for use as internal standard.

Fluoranthene-2,3- and -1,5-diones are novel products from the bacterial transformation of fluoranthene.

Fluoranthene is one of the predominant compounds found in soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAH). Four bacterial strains isolated from PAH-contaminated soils transformed fluoranthene to a number of products during growth on phenanthrene, including the novel metabolites fluoranthene-2,3-dione (F23Q) and fluoranthene-1,5-dione (F15Q). Given the known toxicity and mutagenicity of F23Q, we focused on characterizing this metabolite with respect to its effects on the metabolism of other PAH. The yield of F23Q from fluoranthene ranged from 2% for Sphingomonas yanoikuyae R1 to greater than 20% for Pseudomonas stutzeri P16 and Bacillus cereus P21. None of the strains appeared capable of metabolizing F23Q any further. F23Q strongly inhibited phenanthrene removal by strain R1 but had a negligible to minor effect on phenanthrene degradation by the other organisms. At a concentration of 6.8 microM, F23Q also substantially inhibited the mineralization of benz[a]anthracene, benzo[a]pyrene (BaP), and chrysene by strain R1 as well as BaP mineralization by Pseudomonas saccharophila P15. Inhibition of BaP mineralization by strain P15 was still evident at an F23Q concentration of 0.68 microM. The inhibition of strain R1 by F23Q was explained in part by a cytotoxic effect, but results with strain P15 indicate that other mechanisms of inhibition occur. These findings suggest that quinones such as F23Q and F15Q have the potential to accumulate in PAH-contaminated systems and can inhibit the degradation of other PAH.

Simultaneous quantitation of N(2),3-ethenoguanine and 1,N(2)-ethenoguanine with an immunoaffinity/gas chromatography/high-resolution mass spectrometry assay.

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.

Formation of quinonoid-derived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2,2',5, 5'-tetrachlorobiphenyl.

A possible role for metabolic activation of 2,2',5, 5'-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound to liver proteins. The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4-(2', 5'-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone (Cl(4)PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dose-dependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2. 03 +/- 0.131 days (mean +/- SE), which is approximately 2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability. Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days. The levels of Cl(4)PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 +/- 13.2 vs 45.7 +/- 3. 73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism. A single cysteinyl adduct, inferred to be 5-cysteinyl-3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry method for analysis of endogenous and exogenous N7-(2-hydroxyethyl)guanine in rodents and its potential for human biological monitoring.

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.

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure.

The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.

Quantitation of 1,N6-ethenoadenine in rat urine by immunoaffinity extraction combined with liquid chromatography/electrospray ionization mass spectrometry.

A fast, highly specific analytical method was developed to quantify 1,N6-ethenoadenine (epsilonA) in urine of rats. epsilonA is a highly mutagenic DNA adduct generated by vinyl chloride (VC) exposures as well as endogenously from lipid peroxidation. epsilonA was concentrated through extraction from rat urine by immunoaffinity chromatography and quantitated by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The average epsilonA recovery by immunoaffinity extraction was 66%. The LC/ESI-MS selected-ion monitoring (SIM) of the response ratio of epsilonA to its isotopically labeled internal standard [15N5]epsilonA was linear (r2 = 0.999) and reproducible from 0.15 to 30 pmol/injection. The detection limit obtained in the routine analysis of urine of unexposed rats was 270 fmol/sample with a signal-to-noise ratio (S/N) 3:1. The concentration of endogenous epsilonA was determined to be 21.6 +/- 14.8 pmol/mL (3 rats). Following portal injection of chloroethylene oxide (CEO; the putative active metabolite of VC), the rate of epsilonA excretion in urine was greatest from 0 to 24 h, with approximately 90% of the CEO-induced epsilonA excreted. By 132 h, the excretion of epsilonA was similar to pretreatment amounts. The accuracy of the quantitation was 107 +/- 6% (n = 4), established by analyzing urine of an unexposed rat spiked with authentic epsilonA. These data indicate that the LC/ESI-MS with immunoaffinity extraction method is precise and accurate for epsilonA quantification. The measurement of epsilonA in urine provides a potential biomarker for exposure to chemicals and processes that form this adduct.

Synthesis, characterization, and in vitro quantitation of N-7-guanine adducts of diepoxybutane.

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.

Identification and quantitation of DNA adducts from calf thymus DNA exposed to 3,4-epoxy-1-butene.

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.

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

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.

Macromolecular adducts of butadiene.

Butadiene (BD) is an important industrial chemical classified as a probable human carcinogen. Marked species differences in susceptibility to the carcinogenic effects of BD have been observed, possibly due to the differences in its metabolism. In this work, guanine and adenine adducts formed by the reactive metabolites of BD in vitro were isolated and structurally characterized by UV spectroscopy, liquid secondary ion mass spectrometry and tandem mass spectrometry, electrospray mass spectrometry and nuclear magnetic resonance spectroscopy. The adducts were prepared by reacting purine nucleobases or nucleosides with epoxybutene (EB) or diepoxybutane (DEB) followed by HPLC separation. The reaction of guanine (Gua) with EB resulted in two isomeric products, N7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I) and N7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II). The reaction of adenine at N3 led to the formation of N3-(2-hydroxy-3-buten-1-yl)adenine (EB-Ade I) and N3-(1-hydroxy-3-buten-2-yl) (EB-Ade II). The major guanine adduct with DEB was identified as N7-(2',3', 4'-trihydroxybutyl)guanine (DEB-Gua-I). Three products formed from the reaction of DEB with adenine at pH 7 were identified as N3, N7 and N9-(2',3',4'-trihydroxybutyl)adenines (DEB-Ade I, II and III, respectively). Our results indicate that nucleophilic nitrogens of guanine and adenine first attack one of the epoxy groups of DEB giving (2'-hydroxy-3',4'-epoxybutane-1-yl) intermediates which can be rapidly hydrolyzed to the corresponding (2',3',4'-trihydroxybutyl) adducts or form cross-links with DNA or proteins. N7 and N3 adducts of Ade and Gua are expected to undergo spontaneous depurination and repair by methylpurine glycosylase and therefore may be useful as biomarkers of exposure in urine. The preliminary data on quantification of EB-induced N-terminal valine hemoglobin adducts in red blood cells of exposed mice and rats using modified Edman degradation followed by GC-NI MS was investigated. The amount of EB-N-terminal valine adducts in mouse globin was about 3 times greater than that in rats which may be explained by higher rates of the formation and/or limited detoxification of EB in mice. Female rats and mice had greater amounts of hemoglobin adducts than males.

Role of O-acetyltransferase in activation of oxidised metabolites of the genotoxic environmental pollutant 1-nitropyrene.

The genotoxic environmental contaminant 1-nitropyrene is metabolised in mammalian systems by pathways more complex than the straightforward nitroreduction which accounts for most of its biological activity in bacteria. In order to evaluate the role of O-acetyltransferase (OAT) activity in generation of genotoxic intermediates from 1-nitropyrene, the mutagenicity of the major primary oxidised metabolites of 1-nitropyrene was characterised in the Ames Salmonella typhimurium plate incorporation assay with strain TA98, and with variants of TA98 deficient (TA98/1,8-DNP6) or enhanced (YG1024) in O-acetyltransferase. 1-Nitropyren-3-ol was more mutagenic in the absence than in the presence of S9, while 1-nitropyren-4-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol required S9 for maximum expression of mutagenicity. 1-Nitropyren-4-ol (176 rev/nmol without S9, 467 rev/nmol with S9 in TA98) and 1-nitropyren-6-ol (13 rev/nmol without S9, 266 rev/nmol with S9 in TA98) were overall the most potent nitropyrenol isomers assayed. 1-Acetamidopyren-8-ol and 1-acetamidopyrene 4,5-quinone were only minimally active. 1-Acetamidopyren-3-ol exhibited direct-acting mutagenicity. 1-Acetamidopyren-6-ol, previously shown to be a major contributor to mutagenicity in the urines of rats dosed with 1-nitropyrene (Ball et al., 1984b), was confirmed as a potent (359 rev/nmol) S9-dependent mutagen. Both the direct-acting and the S9-dependent mutagenicity of all the compounds studied was enhanced in the OAT-overproducing strain and much diminished (though not always entirely lost) in the OAT-deficient strain, showing that OAT amplifies expression of the genotoxicity of these compounds. 1-Acetamidopyren-6-ol required both S9 and OAT activity in order to exhibit any mutagenicity; this finding strongly implicates N-hydroxylation followed by O-esterification, as opposed to further S9-catalyzed ring oxidation, as a major route of activation for urinary metabolites of 1-nitropyrene.

Synthesis of [4,5,6,8-(13)C4]guanine, a reagent for the production of internal standards of guanyl DNA adducts.

Using readily available labeled compounds, [4,5,6,8-(13)C4]guanine was synthesized in high overall yield. Intermediates as well as the final product were characterized by 1H NMR, 13C NMR, and high resolution mass spectrometry. The labeled guanine was used to generate [13C4]-labeled analogs of the guanine adducts, N2,3-ethenoguanine and 7-(2-hydroxyethyl)guanine. The application of such adducts in isotope dilution mass spectrometry was illustrated with DNA samples from rats exposed to two different mutagenic compounds, vinyl chloride and ethylene oxide.

Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fused polycyclic aromatic hydrocarbon. CPP is highly genotoxic in bacterial and mammalian systems inducing gene mutations, sister chromatid exchanges and morphological transformation. CPP is a mouse skin carcinogen, a mouse skin tumor initiator and induces pulmonary tumors in newborn mice. We have examined the tumorigenic activity of CPP in strain A/J mice, have determined the formation and persistence of CPP-induced DNA adducts in lung tissue, and analyzed the mutational spectrum in the Ki-ras oncogene from CPP-induced tumors. CPP dissolved in tricaprylin was administered by i.p. injection to male A/J mice (20 mice/dose) at 0, 10, 50, 100 and 200 mg/kg. Animals were killed 8 months later and the lungs removed, fixed, and surface adenomas enumerated. CPP proved to be highly tumorigenic in A/J mice in terms of inducing lung adenomas. The observed tumor multiplicities (lung adenomas/mouse) were: 97.7 +/- 28.7 at 200 mg/kg, 32.8 +/- 15.4 at 100 mg/kg, 4.63 +/- 2.11 at 50 mg/kg and 0.58 +/- 0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60 +/- 0.58 lung adenomas/mouse. Groups of mice treated under the same dosing conditions as those in the tumor studies were killed 1, 3, 7, 14 and 21 days after treatment. The lungs were removed, and the DNA was subjected to DNA adduct analysis by the 32P-postlabeling method. Total CPP-DNA adducts in mouse lung peaked at day 3 with 5870 amol CPP adducts/micrograms DNA after a single dose of 200 mg/kg. DNA adduct levels decreased to 1800 amol CPP adducts/micrograms DNA at day 21. Qualitative DNA adduct analysis revealed four major adducts and one minor adduct. Co-chromatography of the lung DNA from CPP-treated mice with calf thymus DNA treated with CPP-3,4-oxide indicated that all DNA adducts were oxide derived and comparison with CPP-3,4-oxide-treated polydeoxyguanylic acid suggests that almost all of these adducts are CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12 mutation analysis of the DNA from tumors taken from the 100 and 200 mg/kg CPP dose groups demonstrated the following patterns: GGT-->CGT (50%); GGT-->GTT (15%); GGT-->TGT (25%); GGT-->GAT (10%). We conclude that CPP is highly tumorigenic in the A/J mouse lung adenoma model, being five times more active than benzo[a]pyrene. This is unlike the result of CPP as a mouse skin tumorigen or tumor initiator in which CPP is considerably less potent than benzo[a]pyrene.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphological transformation of C3H10T1/2CL8 cells by cyclopenta-fused derivatives of benzo[a]pyrene and benzo[e]pyrene.

Cyclopenta-fused homologs of polycyclic aromatic hydrocarbons (PAH) have proven to be more genotoxic and tumorigenic than their parent PAHs. In an effort to uncover their mechanisms of metabolic activation, the morphological transforming activities of dibenzo[k,mno]acephenanthrylene (CP(3,4)B[a]P), dibenzo[j,mno]acephenanthrylene (CP(1,12)B[a]P) and naphtho[1,2,3,4-mno]acephenanthrylene (CPB[e]P) were studied in C3H10T1/2CL8 mouse embryo fibroblasts. CP(3,4)B[a]P, a PAH with a blocked K region and unblocked bay region, was highly active inducing an average of 1.1 Type II and III foci/dish at 5 micrograms/ml with an average of 67% of the dishes containing foci. This activity was similar to that of benzo[a]pyrene. CP(1,12)B[a]P and CPB[e]P were inactive. The relative positions of the cyclopenta-ring and bay region may play an essential role in the metabolic activation of these PAHs and their biological activities.

Mouse skin tumor-initiating activity of benz[j]aceanthrylene in SENCAR mice.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused derivative of benz[a]anthracene, has been reported to be an active bacterial cell and mammalian cell gene mutagen, a morphological transforming agent in C3H10T1/2CL8 mouse embryo fibroblasts and a mouse lung tumorigen in strain A/J mice. B[j]A was evaluated as a skin tumor initiator in female SENCAR mice and was found to induce papilloma formation in the range of 40-400 micrograms/mouse. B[j]A was found to be extremely active, inducing 8.7 papillomas/mouse after an initiating dose of 40 micrograms/mouse. At this dose, 100% of the mice bore tumors. Comparison with four other cyclopenta-fused polycyclic aromatic hydrocarbons suggests that B[j]A is extremely potent.

Activation and metabolism of benz[j]aceanthrylene-9,10-dihydrodiol, the precursor to bay-region metabolism of the genotoxic cyclopenta-PAH benz[j]aceanthrylene.

Benz[j]aceanthrylene, a cyclopentafused polycylic aromatic hydrocarbon produced in combustion emissions, possesses a bay region and an etheno bridge which may both contribute to the overall genotoxicity of the compound. In order to assess the role of activation at the bay region, the precursor epoxide benz[j]aceanthrylene 9,10-oxide, its dehydration product 10-hydroxybenz[j]aceanthrylene, the key dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene and the bay-region diol-epoxide 7,8-epoxy-9,10-dihydroxy-7,8,9,10- tetrahydrobenz[j]aceanthrylene were evaluated in the bacterial histidine-reversion plate incorporation assay (Ames assay) with Salmonella typhimurium strain TA98. The diol-epoxide alone showed direct-acting mutagenicity (10 revertants per nmole), which was decreased by addition of exogenous metabolic activation (Aroclor 1254-treated rat-liver S9), whereas all the other compounds tested were activated by increasing concentrations of S9. The potency of the diol-epoxide was not sufficient to account for the activity of the parent compound. Identification by proton nuclear magnetic resonance and mass spectrometry of the major products of further metabolism by Aroclor 1254-treated rat-liver S9 of the bay region precursor dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene indicated that oxidation occurred predominantly at the etheno bridge, to give 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene-2(1H)-one, arising by (non-enzymic) rearrangement of the etheno bridge epoxide and the tetrol 1,2,9,10-tetrahydroxy-1,2,9,10- tetrahydrobenz[j]aceanthrylene. The bay region tetrol 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenz[j] aceanthrylene was observed, implying further bay-region metabolism; re-aromatization of the benzo ring to benz[j]aceanthrylene-9,10-diol also occurred. Thus oxidation at the etheno bridge accounts for the majority of the activity of benz[j]aceanthrylene and its derivatives when Aroclor 1254-treated rat-liver S9 is used for exogenous metabolic activation.