Enhancement of liposome-mediated gene transfer to human airway epithelial cells by replication-deficient adenovirus.

We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-mediated gene transfer in cultured human airway epithelial cells. Pl/L/Ad complexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a high-molecular-weight, low-mobility complex, the diameter of which was 300 to 350 nm. Compared to pl/L alone, pl/L/Ad enhanced luciferase expression on average by 1 log-fold in human airway epithelial cells which express either mutant or wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Transgene expression was sustained at high levels for up to 7 days following transfection with pl/L/Ad. Using a heat-stable alkaline phosphatase reporter gene, we showed that a larger fraction of cells was transfected by pl/L/Ad compared to pl/L. Finally, cells were exposed to Ad for 0 to 24 hours prior to pl/L or exposed to pl/L prior to Ad. We found that enhancement was significantly greater using pl/L/Ad compared to the simultaneous addition of Ad with the pl/L complexes. In addition, when pl/L was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid DNA remained in a compartment in the cell for several hours and became available for transcription with the addition of Ad. When Ad was added prior to pl/L, enhancement was found suggesting that the effect of the virus on cell membranes may persist for up to 24 hours. We conclude that the tripartite pl/L/Ad complex significantly enhances liposome-mediated transgene expression for a prolonged period of time in human bronchial epithelial cells.

Formation of etheno adducts and their effects on DNA polymerases.

Etheno (epsilon) and related DNA adducts are formed from the reaction of certain bifunctional electrophiles with DNA. Our interest has been focused on oxiranes substituted with leaving groups, e.g. 2-chlorooxirane, the epoxide derived from the carcinogen vinyl chloride. The chemical mechanisms of the formation of the major etheno products derived from adenine, cytosine and guanine have been elucidated by nuclear magnetic resonance analysis and 13C-labelled precursors. The amounts of all major etheno adducts have been quantified in DNA treated with 2-chlorooxirane by coupled high-performance liquid chromatography of nucleoside and base products. 1,N2-epsilon-Gua, its formally hydrated but stable hemiaminal HO-ethanoGua (5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine) and 1,N2-ethanoGua have all been inserted at a single site in oligonucleotides. All three of these bases block polymerases, cause misincorporations and produce some mutations in bacteria. The patterns of blockage and substitution vary among polymerases. In nucleotide excision repair-deficient Escherichia coli, 1,N2-epsilon-Gua yielded a calculated 16% mutation frequency (base-pair substitutions) when the results were corrected for strand usage. 1,N2-epsilon-Gua was also examined in Chinese hamster ovary cells with a stable integration system; the mutants are more complex than observed in bacteria and include rearrangements, deletions and base-pair substitutions other than at the adduct site.

Conjugation of carcinogens by theta class glutathione s-transferases: mechanisms and relevance to variations in human risk.

Conjugation of chemicals with glutathione (GSH) can lead to decreased or increased toxicity. A genetic deficiency in the GSH S-transferase mu class gene M1 has been hypothesized to lead to greater risk of lung cancer in smokers. Recently a gene deletion polymorphism involving the human theta enzyme T1 has been described: the enzyme is present in erythrocytes and can be readily assayed. A rat theta class enzyme, 5-5, has structural and catalytic similarity and the protein was expressed in the Salmonella typhimurium tester strain TA1535. Expression of the cDNA vector increased the mutagenicity of ethylene dibromide and several methylene dihalides. Mutations resulting from the known GSH S-transferase substrate 1,2-epoxy-3-(4'nitrophenoxy)propane were decreased in the presence of the transferase. Expression of transferase 5-5 increased mutations when 1,2,3,4-diepoxybutane (butadiene diepoxide), 4-bromo-1,2-epoxybutane, or 1,3-dichloracetone were added. The latter compound is a model for the putative 1,2-dibromo-3-chloropropane oxidation product 1-bromo-3-chloroacetone. These genotoxicity and genotyping assays may be of use in further studies of the roles of GSH S-transferase theta enzymes in bioactivation and detoxication and any changes in risk due to polymorphism.

Bufuralol hydroxylation by cytochrome P450 2D6 and 1A2 enzymes in human liver microsomes.

Bufuralol 1'-hydroxylation is a prototypical reaction catalyzed by cytochrome P450 (P450) 2D6, an enzyme known to show debrisoquine/sparteine-type genetic polymorphism in humans. In the present study we further examined the roles of several human P450 enzymes, as well as P450 2D6, in the hydroxylation of (+/-)-bufuralol, using liver microsomes from several human samples and human P450 enzymes expressed in human lymphoblastoid cell lines or Escherichia coli. Kinetic analysis of bufuralol 1'-hydroxylation by liver microsomes showed that there were different Km and Vmax values in seven human samples examined; low Km values (approximately 0.05 mM) were observed in four samples (including sample HL-18), high Km values (approximately 0.25 mM) in two samples (including sample HL-67), and an intermediate Km value (approximately 0.1 mM) in one sample. Quinidine and anti-rat P450 2D1 antibody almost completely inhibited bufuralol 1'-hydroxylation in human sample HL-18 at a substrate concentration of 0.4 mM, whereas these effects were not so drastic when liver microsomes from human sample HL-67 were used. In contrast, a very low concentration (< 10 microM) of alpha-naphthoflavone or anti-human P450 1A2 antibody significantly inhibited bufuralol 1'-hydroxylation catalyzed by human sample HL-67, but not HL-18, with 0.4 mM bufuralol. When the relative contents of P450 2D6 and P450 1A2 in 20 human samples were determined, bufuralol 1'-hydroxylation in samples containing large amounts of P450 2D6 tended to be more sensitive to quinidine, whereas the P450 1A2-rich samples were highly susceptible to alpha-naphthoflavone. However, at low substrate concentrations bufuralol 1'-hydroxylation was shown to be catalyzed principally by P450 2D6, based on the inhibitory effects of anti-rat P450 2D1 antibody and quinidine, in both human samples HL-18 and HL-67. At least five other, minor, bufuralol products were formed by human liver microsomes, in addition to 1'-hydroxybufuralol. Two of them were identified as 4- and 6-hydroxybufuralol by 1H NMR spectroscopy and mass spectrometry. The formation of the 4- and 6-hydroxylated products was suggested to be catalyzed by P450 1A2, based on the results of correlation with P450 1A2 contents in 60 human samples and inhibition by anti-P450 1A2 and alpha-naphthoflavone. Purified recombinant P450 1A2 (expressed in E. coli) produced 1'-, 4-, and 6-hydroxybufuralol in a reconstituted system, although P450 2D6 (expressed in human lymphoblast cell lines) was found to catalyze only bufuralol 1'-hydroxylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectroscopic and thermodynamic characterization of the interaction of N7-guanyl thioether derivatives of d(TGCTG*CAAG) with potential complements.

The oligomer d(TGCTGCAAG) corresponds to a region of bacteriophage M13mp18 DNA where mutations have been found to be induced by S-(2-chloroethyl)glutathione (glutathione, GSH) [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., & Guengerich, F. P. (1991) J. Biol. Chem. 267, 6672-6679]. This oligomer was prepared with the central G replaced by S-(2-N7-guanylethyl)-GSH or N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester; these derivatives were purified by HPLC and by affinity chromatography in the latter case. UV mixing and CD spectroscopy studies showed no evidence for preferred pairing of the S-(2-N7-guanylethyl)GSH moiety to any base other than C. UV melting studies of duplexes were performed with complementary strands containing the normal C, as well as the three mismatches (T, A, and G), across from the adducted base. Thermal stabilities were reduced in all cases when G was replaced by either N7-guanyl adduct; the C-containing complement was still the most stable. The reduced stability of the duplex d(TGCTG*CAAG)/d(CTTGCAGCA), where S-(2-N7-guanylethyl)-GSH corresponds to G*, was characterized by an increase in delta G zero of 1.4-2.0 kcal mol-1 (in the range of 25-37 degrees C) relative to the unadducted duplex. van't Hoff analysis of concentration-dependent melting experiments indicated that the delta H zero of the duplex was actually more favorable when this adduct was introduced (delta delta H zero = 13 kcal mol-1), but the decreased thermal stability was due to the entropic component. Similar results were observed when G* was N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester. Under the conditions used, the overall relative stabilities of the oligomeric duplexes containing various base pairs do not indicate that S-(2-N7-guanylethyl)GSH would contribute to a higher frequency of T misinsertion than G. The possibility that ionization at the guanine N1 position may be involved in mutagenesis by N7-guanyl adducts is considered.

Mechanism of formation of ethenoguanine adducts from 2-haloacetaldehydes: 13C-labeling patterns with 2-bromoacetaldehyde.

The mechanism of formation of etheno (epsilon) adducts of nucleic acid bases from 2-haloacetaldehydes is generally assumed to occur via initial Schiff base formation resulting from reaction of the aldehyde with an exocyclic amine. We recently revised the 1H NMR assignments of the epsilon protons of 1,N2-epsilon-Guo (Guengerich, F. P., Persmark, M. P., and Humphreys, W. G. (1993) Chem. Res. Toxicol. 6, 635-648). In that work we also observed a facile and specific exchange of H7 of 1,N2-epsilon-Guo and H5 of N2,3-epsilon-Gua with H2O. These findings raise questions about the mechanistic conclusions reached on the basis of labeling studies with deuterated ClCH2CHO (Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. (1977) J. Org. Chem. 42, 3292-3296). BrCH2-13CHO was prepared from BrCH2(13)CO2H and used to prepare 1,N2-epsilon-Guo (from Guo) and O6-ethyl-N2,3-epsilon-Gua (from O6-ethylGua). The positions of the labels were determined by 1H NMR spectroscopy experiments to be adjacent to the original Gua N2 (exocyclic) atom in both cases, i.e., at C6 in both epsilon products. The labeling patterns are consistent with a mechanism involving initial Schiff base formation from the N2 atom and the aldehyde and subsequent nucleophilic attack of an endocyclic nitrogen on the methylene carbon.

Formation of 1,N2- and N2,3-ethenoguanine from 2-halooxiranes: isotopic labeling studies and isolation of a hemiaminal derivative of N2-(2-oxoethyl)guanine.

Vinyl halides are oxidized to 2-halooxiranes, which rapidly rearrange to 2-haloacetaldehydes. Both of these species can react with DNA to generate a variety of adducts, including the potentially mutagenic etheno (epsilon) products. Evidence was provided through kinetic studies that the epsilon-Gua adducts are formed primarily from 2-haloxiranes; consistent with this view, epoxide hydrolase inhibited the formation of N2,3-epsilon-Gua from vinyl chloride but alcohol dehydrogenase did not. Assignments of the NMR shifts of the etheno protons of 1,N2- and N2,3-epsilon-Gua were made with the use of 15N labeling and nuclear Overhauser effects, in revision of the literature. The H-5 proton of N2,3-epsilon-Gua showed facile exchange in acid or base; the H-7 proton of 1,N2-epsilon-Gua was exchanged at neutral or basic pH but not in acid. Reaction of Br2CHCH2OH (labeled at C1 with 2H or 13C) with Guo yielded 1,N2-epsilon-Gua and N2,3-epsilon-Gua, presumably through the intermediacy of 2-bromooxirane. 1H NMR analysis indicated that the labeled carbon was attached to the original Guo N2 atom in both cases. When N2-(2-oxoethyl)Gua was generated from a diethyl acetal or from a glycol, the major product was the cyclic derivative 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-alpha]purine. This compound was also formed in considerable yield from the reaction of 2-chlorooxirane with Guo, dGuo 5'-phosphate, or DNA and is relatively stable in the presence of acid or mild base. It does not appear to be readily dehydrated to yield the etheno adducts but may be of significance as a DNA adducts in its own right.

Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes.

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

N-nitroso-N-methylvinylamine: reaction of the epoxide with guanyl and adenyl moieties to yield adducts derived from both parts of the molecule.

N-Nitroso-N-methylvinylamine was synthesized and treated with dimethyldioxirane to produce 1-(N-nitrosomethylamino)oxirane. 1-(N-Nitrosomethylamino)oxirane had a t1/2 of < 5 s in buffer at neutral pH and 23 degrees C. This epoxide reacted with Ado to form 1,N6-etheno(epsilon-)Ado. It also reacted with DNA to form products arising from the oxirane portion of the molecule [N7-(2-oxoethyl)Gua,N2,3-epsilon-Gua, and 1,N6-epsilon-dAdo] and the methyl group (N7-methylGua). NADPH-fortified rat liver microsomes oxidized N-nitroso-N-methylvinylamine to form 1,N6-epsilon-Ado in the presence of Ado. Further, 1,N6-epsilon-Ado was also formed in microsomal incubations containing N-nitroso-N-methylethylamine, indicating that desaturation of the ethyl moiety occurs to form a vinyl group and then an epoxide. When NADPH-fortified microsomes were incubated with N-nitroso-N-methylvinylamine, HCHO was formed, and when DNA was included in incubations, 1,N6-epsilon-dAdo and N7-methylGua were isolated from DNA. In the cases of both HCHO and N7-methylGua, product formation was similar to the levels seen with N-nitroso-N,N-dimethylamine and N-nitroso-N-methylethylamine.

Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds.

Erwinia chrysanthemi 3937 possesses a saturable, high-affinity transport system for the ferric complex of its native siderophore chrysobactin, [N-alpha-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine]. Uptake of 55Fe-labeled chrysobactin was completely inhibited by respiratory poison or low temperature and was significantly reduced in rich medium. The kinetics of chrysobactin-mediated iron transport were determined to have apparent Km and Vmax values of about 30 nM and of 90 pmol/mg.min, respectively. Isomers of chrysobactin and analogs with progressively shorter side chains mediated ferric iron transport as efficiently as the native siderophore, which indicates that the chrysobactin receptor primarily recognizes the catechol-iron center. Free ligand in excess only moderately reduced the accumulation of 55Fe. Chrysobactin may therefore be regarded as a true siderophore for E. chrysanthemi.

Iron(III) complexes of chrysobactin, the siderophore of Erwinia chrysanthemi.

The phytopathogenic bacterium Erwinia chrysanthemi produces the monocatecholate siderophore chrysobactin under conditions of iron deprivation. Only the catecholate hydroxyl groups participate in metal coordination, and chrysobactin is therefore unable to provide full 1:1 coordination of Fe(III). The stoichiometry in aqueous solution is a variable dependent on pH and metal/ligand ratio, in addition to being concentration dependent. At neutral pH and concentrations of about 0.1 mM, ferric chrysobactin exists as a mixture of bis and tris complexes. Chrysobactin and its isomers form optically active tris complexes. The dominant configuration depends on the chirality of the amino acid to which the catecholate moiety is attached.

Purification, characterization, and structure of pseudobactin 589 A, a siderophore from a plant growth promoting Pseudomonas.

Under conditions of low-iron stress the plant growth promoting bacterium Pseudomonas putida 589 (DSM 50202) produced a yellow-green fluorescent iron-binding peptide siderophore, which was designated pseudobactin 589 A and had an affinity constant toward Fe3+ of 10(25) at pH 7. Protonated pseudobactin 589 A had the molecular formula C54H78O26N15 and a nominal mass spectral molecular mass of 1353 g/mol. Its structure was determined by a combination of nuclear magnetic resonance, fast atom bombardment mass spectrometry, and Edman degradation. Pseudobactin 589 A consisted of a nonapeptide with the amino acid sequence L-Asp-L-Lys-(D)-beta-OH-Asp-D(L)-Ser-L-Thr-D-Ala-D-Glu-L(D)-Ser-L-N delta-OH- Orn, in which lysine was amide bonded via the carboxy and the N epsilon-amino groups. A quinoline-derived chromophore was connected via an amide bond to the alpha-amino nitrogen of aspartic acid and an L-malamide residue was attached to the chromophore. The three bidentate Fe3+ binding ligands consisted of an o-dihydroxy aromatic group from the quinoline derivative, beta-hydroxyaspartic acid, and an internally cyclized N delta-hydroxyornithine. The structure of pseudobactin 589 A is unique but strikingly similar to that of other pseudobactin-type siderophores from other plant growth promoting and plant deleterious pseudomonads.

Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi.

A catechcol-type siderophore, assigned the trivial name chrysobactin, was isolated from the phytopathogenic bacterium Erwinia chrysanthemi and characterized by degradation and spectroscopic techniques as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine. Chrysobactin, which was also obtained by chemical synthesis, was shown to be active in supplying iron to a group of mutants of E. chrysanthemi defective in biosynthesis of the siderophore.