Biomedical subjects

Thomas M Harris

Publications and source records attributed to Thomas M Harris.

41 records · Page 3Linked to original sources

PubMed2002

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines.

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon >> 3-carbon > 4-carbon.

Base Sequence↗

PubMed2002

Reaction of aflatoxin B(1) oxidation products with lysine.

Aflatoxin (AF) B(1) exo-8,9-epoxide hydrolysis yields AFB(1) dihydrodiol, which undergoes base-catalyzed rearrangement to, and is in equilibrium with, AFB(1) dialdehyde. We investigated the reaction of AFB(1) dialdehyde with albumin to generate a Lys adduct, previously characterized by others [Sabbioni, G., Skipper, P. L., Büchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis8, 819-824; Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15]. Pronase digestion of bovine albumin serum treated with AFB(1) dialdehyde and HPLC yielded the adduct, identified by its characteristic UV and mass spectra. The structure of the Lys-AFB(1) dialdehyde adduct is concluded to be (S)-alpha-amino-2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-hexanoic acid, structure B of the former paper and 8 of the latter, based on work with the methylamine adduct described in the following paper in this issue [Guengerich, F. P., Voehler, M., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 793-798]. The time course of product formation at varying concentrations of AFB(1) dialdehyde could be described by complexation with albumin with a K(d) of 1.5 mM and a first-order reaction rate with the N6-amino group of Lys of 0.033 min(-)(1). The reaction of AFB(1) dialdehyde with N(2)-acetylLys was monitored by UV spectroscopy and yielded a final spectrum similar to that of the described Lys adduct. Kinetic analysis of the changes at pH 7.2 was best described with a scheme involving equilibrium of the dialdehyde with dihydrodiol and a rate-limiting reaction of AFB(1) dialdehyde with the N6 atom of N(2)-acetylLys, with an apparent second-order rate constant of 2.6 x 10(3) M(-)(1) min(-)(1), followed by putative carbinolamine formation and rearrangement, collectively described by a first-order rate constant of 7.6 min(-)(1). Competition experiments with the hydrolysis of AFB(1) exo-8,9-epoxide indicate that N2-acetylLys also reacts with the epoxide at pH 7.2 (k = 350 M(-)(1) min(-)(1)) and 9.5 (k = 1.8 x 10(3) M(-)(1) min(-)(1)). This reaction might contribute to the formation of protein Lys adducts, depending upon the local concentration of free or protein Lys. Mass spectral analysis of trypsin digests of bovine serum albumin modified with AFB(1) dialdehyde indicated selective modification of Lys455 and Lys548. Collectively, these results provide more insight into the mechanism of formation of AFB(1) dialdehyde-protein adducts and indicate that the formation of Lys adducts is a moderately efficient process. The binding of AFB(1) dialdehyde to albumin or the protonation of the N6-amino group retards the reaction with Lys residues.

Aflatoxin B1↗

PubMed2002

DNA adducts of acrolein: site-specific synthesis of an oligodeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine.

3-(2-Deoxy-beta-D-erythro-pentofuranosyl)-6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one is formed in low yield by the reaction of acrolein with 2'-deoxyguanosine. The nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation of the nucleoside 2'-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene to give 1-(3-butenyl)-2'-deoxyguanosine. Oxidation with OsO(4) and N-methylmorpholine-N-oxide to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO(4) gave the 1-(3-oxopropyl) adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing mixture of two diastereomers. Reduction of the nucleoside with NaBH(4) gave the unfunctionalized compound plus 1-(3-hydroxypropyl)-2'-deoxyguanosine showing that epimerization was occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH(3) gave exclusively unfunctionalized 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucleotides was prepared from 1-(3-butenyl)-2'-deoxyguanosine by glycolation after protection of the 3' and 5' hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups and the exocyclic amino group followed by removal of silyl protection gave the protected nucleoside. Protection of the 5' hydroxyl group as the 4,4'-dimethoxytrityl ether followed by phosphitylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite gave the prosphoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.

Acrolein↗

PubMed2002

Structure of the aflatoxin B(1) dialdehyde adduct formed from reaction with methylamine.

Amine conjugates of activated aflatoxin (AF) B(1) are formed in various systems, e.g., buffer amines and with protein lysine groups. Structures have been published in the literature, but the evidence is indirect in that (i) halogenated AFB(1) was usually used as the precursor and (ii) the assignment of the structure of the five-membered ring formed by cyclization is based on NMR chemical shifts. To better define these adducts and distinguish among several possibilities, we synthesized AFB(1) dialdehyde and reacted this with the surrogate methylamine at neutral pH, to simplify the system. The isolated product had the expected molecular ion (mass spectrometry) and showed pH-dependent UV spectra similar to those published for a lysine conjugate. Nuclear Overhauser enhanced spectroscopy (two-dimensional NMR, 800 MHz) of the sample (2H(2)O) showed proximity of the N-CH(3) protons only with a singlet at delta 4.10, assigned to the methylene of the added five-membered ring, but not to a delta 6.53 singlet assigned as the vinylic proton of that ring. All protons in the coumarin-furanone portion of the system were correlated to each other but not to those in the added five-membered ring. These experiments establish the structure as 2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-methane. The similarity of the reaction to that occurring in the reaction of AFB(1) dialdehyde with lysine and the agreement of the UV spectra suggest that this structure is applicable for the lysine analogue. The NMR results support the possible structure B of Sabbioni et al. [Sabbioni, G., Skipper, P. L., Buchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis 8, 819-824] and the proposed structure 8 of Sabbioni [Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15] but not alternative proposals. Kinetic and mechanistic considerations of the reaction of lysine with AFB(1) dialdehyde are presented in the previous article in this issue [Guengerich, F. P., Arneson, K. O., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 780-793].

Aflatoxin B1↗

PubMed2002

Mutagenic spectrum of butadiene-derived N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand cross-links in mammalian cells.

Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-diepoxybutane (BDO(2)), and 3,4-epoxy-1,2-butanediol (BDE), form both stable and unstable base adducts in DNA and have been implicated in producing genotoxic effects in rodents and human cells. N1 deoxyadenosine adducts are unstable and can undergo either hydrolytic deamination to yield N1 deoxyinosine adducts or Dimroth rearrangement to yield N(6) adducts. The dominant point mutation observed at AT sites in both in vivo and in vitro mutagenesis studies using BD and its epoxides has been A --> T transversions followed by A --> G transitions. To understand which of the butadiene adducts are responsible for mutations at AT sites, the present study focuses on the N1 deoxyinosine adduct at C2 of BDO and N(6),N(6)-deoxyadenosine intrastrand cross-links derived from BDO(2). These lesions were incorporated site-specifically and stereospecifically into oligodeoxynucleotides which were engineered into mammalian shuttle vectors for replication bypass and mutational analyses in COS-7 cells. Replication of DNAs containing the R,R-BDO(2) intrastrand cross-link between N(6) positions of deoxyadenosine yielded a high frequency (59%) of single base substitutions at the 3' adducted base, while 19% mutagenesis was detected using the S,S-diastereomer. Comparable studies using the R- and S-diastereomers of the N1 deoxyinosine adduct gave rise to approximately 50 and 80% A --> G transitions with overall mutagenic frequencies of 59 and 90%, respectively. Collectively, these data establish a molecular basis for A --> G transitions that are observed following in vivo and in vitro exposures to BD and its epoxides, but fail to reveal the source of the A --> T transversions that are the dominant point mutation.

Animals↗