Faculty Bibliography

Simple epoxides such as ethylene oxide, propylene oxide, epichlorohydrin and glycidol are mutagenic and carcinogenic compounds that are important industrial chemicals. Mutagenic and carcinogenic epoxides can also be formed metabolically from Industrially important compounds such as alkenes (ethylene, butadiene, propylene and styrene), vinyl halides (vinyl chloride and vinyl bromide) and other vinyl monomers (acrylonitrile and acrylamide). Simple epoxides react with nucleosides and DNA predominantly by the SN2 mechanism at the most nucleophilic sites (ring nitrogens) in DNA to form 2-hydroxy-2-alkyl adducts. The major hydroxyalkyl adducts that form at N7 of deoxyguanosine and N3 of deoxyadenosine are chemically unstable owing to the presence of a charged quaternary nitrogen at the site of alkylation, and they depurinate spontaneously to remove the charge, forming potentially mutagenic abasic sites. Hydroxyalkylation at N1 of deoxyadenosine and N3 of deoxycytidine also results in the production of charged, unstable species because the pKa increases dramatically after alkylation. The charge can be lost from these adducts by the formation of cyclic adducts, which occurs when there is a good leaving group on the hydroxyalkyl side-chain. Most simple epoxides remove the charge on hydroxyalkyl adducts at N1 of deoxyadenosine and N3 of deoxyxytidine by competitive rearrangements, such as hydrolytic deamination, to form 1-hydroxyalkyl-deoxyinosine and 3-hydroxyalkyl-deoxyuridine adducts and Dimroth rearrangement to form N6-hydroxyalkyl-deoxyadenosine adducts. These rearrangements are facilitated intramolecularly by the formation of cyclic intermediates, with the participation of the hydroxyl group of the hydroxyalkyl side-chain. These adducts are uncharged, stable and potentially mutagenic and are likely to contribute to the biological activity of simple epoxides

3-Hydroxyalkyl-dU lesions are produced by several aliphatic epoxides after initial alkylation at N3 of dC followed by rapid hydrolytic deamination. The mutagenic and carcinogenic epoxides epichlorohydrin (ECH) and glycidol (GLY) produce the same adduct 3-dihydroxypropyl-dU (3-DHP-dU). The mutagenic potential of 3-DHP-dU was investigated by in vitro DNA replication studies of this lesion placed at a single site in a DNA template. In the presence of the natural metal cofactor Mg++, 3-DHP-dU blocked DNA synthesis 3' to the lesion and after incorporating a nucleotide opposite 3-DHP-dU. Postlesion synthesis was negligible (1%). In the absence of polymerase proofreading activity, bypass (52%) at 3-DHP-dU occurred which was enhanced (91%) by substitution of Mg++ with Mn++. Sequencing revealed that dA and dT are incorporated opposite 3-DHP-dU during postlesion synthesis. Since 3-DHP-dU is derived from dC alkylation by ECH and GLY incorporation of dA and dT opposite 3-DHP-dU implicates this lesion in GC to AT and GC to TA mutagenesis by these epoxides. These results together with our DNA replication studies of ethylene oxide-induced 3-hydroxyethyl-dU and propylene oxide-induced 3-hydroxypropyl-dU suggest that 3-hydroxylalkyl-dU may be critical premutagenic lesions produced by these environmentally important epoxides

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance and thus has the potential for human exposure in the workplace. It has been shown to be genotoxic in several systems and is a compound capable of reacting with biological nucleophiles. This study details the products formed from the reaction of ECH with 2'-deoxynucleosides at pH 7 and 37 degrees C for 6 h. Reaction with 2'-deoxyguanosine yielded 7-(3-chloro-2-hydroxypropyl) guanine (7-CHP-Gua) resulting from alkylation at N-7 of 2'-deoxyguanosine followed by depurination. Two unusual adducts were also partially characterized which resulted from further reaction of 7-CHP-Gua with another molecule of ECH to yield 1,7-bis(3-chloro-2-hydroxypropyl)guanine (1,7-bis-CHP-Gua) which could then cyclize with the exocyclic amino group to yield 1,N2-(2-hydroxypropano)-7-(3-chloro-2-hydroxypropyl) guanine (1,N2-HP-7-CHP-Gua). Reaction with 2'-deoxyadenosine gave only one product, namely 1,N6-(2-hydroxypropano)-2'-deoxyadenosine (1,N6-HP-dAdo). The reaction of 2'-deoxythymidine with ECH also yielded one product which was identified as 3-(3-chloro-2-hydroxypropyl)-2'-deoxythymidine (3-CHP-dThd). A 3-(3-chloro-2-hydroxypropyl)-2'-deoxyuridine (3-CHP-dUrd) product was isolated from the reaction of ECH with 2'-deoxycytidine. This product most likely resulted from the deamination of an initially formed 3-(3-chloro-2-hydroxypropyl) -2'-deoxycytidine (3-CHP-dCyd), a phenomenon which we have previously reported to occur during the reaction of 2'-deoxycytidine with other aliphatic epoxides. Evidence is also presented that 3-CHP-dUrd is converted to 3-(2,3-dihydroxypropyl)-2'deoxyuridine (3-DHP-dUrd) under physiological conditions, with a half-life of 213 h. Reaction of ECH with calf thymus DNA (pH 7.0, 37 degrees C, 3 h) resulted in the formation of 7-CHP-Gua (200 nmol/mg DNA

Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hydroxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of dC, followed by rapid hydrolytic deamination. The significance of formation of 3-HE-dU in DNA was investigated by in vitro DNA replication of 3-HE-dU. A 55-nucleotide DNA template, containing 3-HE-dU at a single site, was constructed. DNA products, synthesized on the site-modified template, were analyzed and mutagenic bypass at 3-HE-dU estimated. The 3-HE-dU lesion blocked DNA replication by the Klenow fragment of Escherichia coli polymerase I (Kf Pol I) and bacteriophage T7 polymerase (T7 Pol) 3' to 3-HE-dU and after incorporating a nucleotide opposite 3-HE-dU. DNA synthesis past 3-HE-dU was negligible (< 3%). Substitution of Kf Pol I (exo-) and T7 Pol (exo-), polymerases lacking 3'-->5' exonuclease proofreading activity, for Kf Pol I and T7 Pol, respectively, facilitated DNA synthesis past 3-HE-dU. The bypass synthesis by Kf Pol I (exo-) was 60% and 90% by T7 Pol (exo-). These results suggest that the 3-HE-dU lesion could be bypassed, but that the extension at 3-HE-dU is rate-limiting. In the absence of proofreading, the nucleotide incorporated opposite 3-HE-dU is not excised and remains in position long enough for extension to occur. During post-lesion synthesis, both dA and dT were incorporated opposite 3-HE-dU. Since 3-HE-dU is derived from dC alkylation by ethylene oxide, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G.C-->A.T and G.C-->T.A mutagenesis

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or inaccessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior UV irradiation of delta recA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVM (UV modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase III, we have examined mutagenesis at an epsilon C residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in UV-irradiated recA+, and in delta recA cells. These data indicate that UV irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap-filling DNA synthesis as well as during normal DNA replication

A variety of alkylating mutagens and carcinogens produce pyrimidine adducts in DNA that block DNA synthesis in vitro. Since DNA synthesis past the lesion is a necessary step to produce mutations, we investigated the role of the mutagenic metal ion Mn++ in facilitating DNA synthesis past alkylpyrimidines. In the presence of the natural metal activator Mg++, N3-ethyldeoxythymidine (N3-Et-dT) and O2-ethyldeoxythymidine (O2-Et-dT), present at a single site in DNA, blocked in vitro DNA synthesis 3' to the lesion and after incorporating dA opposite each lesion. The presence of Mn++ permitted postlesion synthesis with dT misincorporated opposite N3-Et-dT and O2-Et-dT, implicating these lesions in A.T-->T.A transversion mutagenesis. The DNA synthesis block by O4-ethyldeoxythymidine (O4-Et-dT) in the presence of Mg++ was partial and was also removed by Mn++. Consistent with in vivo studies, dG was incorporated opposite O4-Et-dT during postlesion synthesis, leading to A.T-->G.C transition mutagenesis. We also have discovered a new class of DNA adducts, N3-hydroxyalkyldeoxyuridine (3-HA-dU) lesions, which are produced by mutagenic and carcinogenic aliphatic epoxides. 3-HA-dU is formed after initial alkylation at the N3 position of dC followed by a rapid hydrolytic deamination. As observed with the analogous mutagenic N3-Et-dT, the ethylene oxide-induced 3-hydroxyethyldeoxyuridine (3-HE-dU) blocked in vitro DNA synthesis, which could be by-passed in the presence of Mn++. The nucleotide incorporated opposite 3-HE-dU during postlesion synthesis is being identified. These studies suggest a role for Mn++ in mediating mutagenic and carcinogenic effects of environmentally important ethylating agents and aliphatic epoxides

Propylene oxide (PO) is a widely used industrial reagent which is mutagenic and carcinogenic. We have recently shown that a variety of aliphatic epoxides, including propylene oxide, can react with DNA to form hydroxyalkyl adducts at N-3 of cytosine which rapidly undergo hydrolytic deamination to produce uracil adducts. These 3-hydroxyalkyl uracil adducts are stable in DNA and are postulated to be an important class of potentially mutagenic lesions. Mutagenesis at cytosine residues due to PO modification of single-stranded M13mp2/C141 DNA was studied by transfection of modified DNA into SOS and non-SOS induced E. coli host cells. Mutations of the proline (CCC) codon at C141 which result in reversion of the lacZ phenotype (blue plaques) were scored. It was found that PO treatment of single-stranded DNA results in dose-dependent mutagenesis that is highly SOS dependent. The spectrum of base-substitution mutations found at this site differed when PO-modified DNA was transfected into E. coli with different DNA repair backgrounds. These results indicate that propylene oxide induced DNA adducts at template cytosine residues are mutagenic in E. coli and that this mutagenesis is greatly increased by SOS processing. They also show that these lesions may be repaired by one or more mechanisms

2-cyanoethylene oxide (CEO) is a direct-acting mutagen and the postulated proximate carcinogenic form of acrylonitrile (AN). We have studied the reactions of CEO with 2'-deoxyribonucleosides and in vitro with calf thymus DNA at pH 7.0-7.5 and 37 degrees C for 3 h. Reaction of CEO with dAdo gave 2 adducts, N6-(2-hydroxy-2-carboxyethyl)-dAdo (N6-HOCE-dAdo) (2% yield) and 1,N6-etheno-dAdo (epsilon-dAdo) (11%); reaction with dCyd resulted in the isolation of 3-HOCE-dUrd (22%); reaction with dGuo gave 7-(2-oxoethyl)-Gua (7-OXE-Gua) (31%) and reaction with dThd yielded 3-OXE-dThd (3%). Structural elucidation of adducts was accomplished by ultraviolet spectroscopy, high-field proton NMR spectroscopy and mass spectrometry. Structural confirmation was provided by an accurate mass measurement technique where diagnostic ions in the electron impact mass spectra of trimethylsilyl derivatives were measured to within 0.0007 atomic mass units. The facile Dimroth rearrangement of 1-HOCE-dAdo to N6-HOCE-dAdo and hydrolytic deamination of a dCyd adduct to 3-HOCE-dUrd is postulated to be catalyzed by the hydroxyl group on the 3-carbon side chain of the adduct. Reaction of CEO with calf thymus DNA yielded (nmol/mg DNA) N6-HOCE-dAdo (2); epsilon-dAdo (11); 3-HOCE-dUrd (80); 7-OXE-Gua (110) and 3-OXE-dThd (1). Thus CEO, like its metabolic precursor AN, directly alkylates DNA in vitro but at a much more rapid rate