Genotoxicity of acetaldehyde- and crotonaldehyde-induced 1,N2-propanodeoxyguanosine DNA adducts in human cells

Reaction of crotonaldehyde or two molecules of acetaldehyde with DNA generates 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (2, Scheme 1), which occurs in (6R, 8R) and (6S, 8S) configurations (Fig. 1). These diastereomers were site-specifically incorporated into oligonucleotides, which were then inserted into a double-stranded DNA vector for genotoxicity studies. Modified DNA was introduced into human xeroderma pigmentosum A (XPA) cells to allow replication. Analysis of progeny plasmid revealed that these DNA adducts inhibit DNA synthesis to similar degrees. (6S, 8S)-2 miscodes more frequently than (6R, 8R)-2: 10% versus 5%. For both adducts, major miscoding events were G-->T transversions, but G-->A transitions were also observed at a comparable level for (6R, 8R)-2. G-->C transversions were the second most common events for (6S, 8S)-2. Comparison of these results with those of other 1,N2-propanodeoxyguanosine (PdG) adducts, which were evaluated by the same system, indicates that (i) their synthesis inhibiting potencies are stronger than that of the unsubstituted analog, 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (1, Scheme 1), but weaker than that of 3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (3, Scheme 1); (ii) both isomers of 2 are more miscoding than 1; (iii) the miscoding potency of (6S, 8S)-2 is comparable to those of 3 and a model PdG 4 lacking a hydroxyl and a methyl group (Fig. 1). Therefore, considering the fact that 2 are formed endogenously as well as exogenously, they may play a significant role in aging and cancer in humans.     

Application of the adductome approach to assess intertissue DNA damage variations in human lung and esophagus

Methods for determining the differential susceptibility of human organs to DNA damage have not yet been explored to any large extent due to technical constraints. The development of comprehensive analytical approaches by which to detect intertissue variations in DNA damage susceptibility may advance our understanding of the roles of DNA adducts in cancer etiology and as exposure biomarkers at least. A strategy designed for the detection and comparison of multiple DNA adducts from different tissue samples was applied to assess esophageal and peripherally- and centrally-located lung tissue DNA obtained from the same person. This adductome approach utilized LC/ESI-MS/MS analysis methods designed to detect the neutral loss of 2′-deoxyribose from positively ionized 2′-deoxynucleoside adducts transmitting the [M+H]⁺ > [M+H−116]⁺ transition over 374 transitions. In the final analyses, adductome maps were produced which facilitated the visualization of putative DNA adducts and their relative levels of occurrence and allowed for comprehensive comparisons between samples, including a calf thymus DNA negative control. The largest putative adducts were distributed similarly across the samples, however, differences in the relative amounts of putative adducts in lung and esophagus tissue were also revealed. The largest-occurring lung tissue DNA putative adducts were 90% similar (n = 50), while putative adducts in esophagus tissue DNA were shown to be 80 and 84% similar to central and peripheral lung tissue DNA respectively. Seven DNA adducts, N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG), 1,N⁶-etheno-2′-deoxyadenosine (dA), -S- and -R-methyl-γ-hydroxy-1,N²-propano-2′-deoxyguanosine (1,N²-PdG₁, 1,N²-PdG₂), 3-(2′-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxy-pyrimido[1,2-a]purine-(3H)-one (8-OH-PdG) and the two stereoisomers of 3-(2′-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (6-OH-PdG) were unambiguously detected in all tissue DNA samples by comparison to authentic adduct standards and stable isotope dilution and their identities were matched to putative adducts detected in the adductome maps.     

Formaldehyde-mediated modification of natural deoxyguanosine with amines: one-pot cyclization as a molecular model for genotoxicity

Stable cross-linked adducts, 3-(2-deoxy-beta-D-ribofuranosyl)-7-phenyl-5,6,7,8-tetrahydro[1,3,5]triazino[1,2-a]purin-10(3H)-one and 7-butyl-3-(2-deoxy-beta-D-ribofuranosyl)-5,6,7,8-tetrahydro[1,3,5]triazino[1,2-a]purin-10(3H)-one, that formed chemically from natural deoxyguanosine and aniline or buthyl amine in the presence of formaldehyde were identified. This reaction appears to be a general reaction of deoxyguanosine and primary amines, and it may be a model of DNA modification with carcinogenic aromatic amines without metabolic activation, if formaldehyde is present.     

Synthesis of the minor acrolein adducts of 2(')-deoxyguanosine and their generation in oligomeric DNA

Acrolein, a known mutagen, undergoes reaction in vitro under physiological conditions with both 2(')-deoxyguanosine and native DNA to give rise to exocyclic adducts of the 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)-one class having an hydroxy group at either the 6 or the 8 position. Previously we have shown that the 8-hydroxy derivative in a bacterial system has very low mutagenicity probably because in double-stranded DNA this residue exists in the open-chain aldehydic form [N(2)-(3-oxopropyl)-2(')-deoxyguanosine] (3). To continue our investigation in this area, we needed ample supplies of the 6-hydroxy isomers. This current paper describes high-yield simple methods for the synthesis in bulk of the 6-hydroxy adduct 1 and its incorporation into DNA oligomers. The basic methods for the synthesis of the adduct 1, involve 1-substitution of dG derivatives with a 3-butenyl group, dihydroxylation of the olefin with osmium tetroxide and N-methylmorpholine N-oxide, then diol cleavage with periodate ion after incorporation of the 1-(3,4-diacetoxybutyl)-2(')-deoxyguanosine into oligomeric DNA.     

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in a JNK-dependent manner

The bioactive signaling molecule d-erythro-sphingosine-1-phosphate (S1P) is irreversibly degraded by the enzyme S1P lyase (SPL). The reaction of SPL with C18-S1P generates ethanolamine phosphate and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S1P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac1 and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S1P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling.     

S1P and plasmalogen derived fatty aldehydes in cellular signaling and functions

Long-chain fatty aldehydes are present in low concentrations in mammalian cells and serve as intermediates in the interconversion between fatty acids and fatty alcohols. The long-chain fatty aldehydes are generated by enzymatic hydrolysis of 1-alkyl-, and 1-alkenyl-glycerophospholipids by alkylglycerol monooxygenase, plasmalogenase or lysoplasmalogenase while hydrolysis of sphingosine-1-phosphate (S1P) by S1P lyase generates trans ∆2-hexadecenal (∆2-HDE). Additionally, 2-chloro-, and 2-bromo- fatty aldehydes are produced from plasmalogens or lysoplasmalogens by hypochlorous, and hypobromous acid generated by activated neutrophils and eosinophils, respectively while 2-iodofatty aldehydes are produced by excess iodine in thyroid glands. The 2-halofatty aldehydes and ∆2-HDE activated JNK signaling, BAX, cytoskeletal reorganization and apoptosis in mammalian cells. Further, 2-chloro- and 2-bromo-fatty aldehydes formed GSH and protein adducts while ∆2-HDE formed adducts with GSH, deoxyguanosine in DNA and proteins such as HDAC1 in vitro. ∆2-HDE also modulated HDAC activity and stimulated H3 and H4 histone acetylation in vitro with lung epithelial cell nuclear preparations. The α-halo fatty aldehydes elicited endothelial dysfunction, cellular toxicity and tissue damage. Taken together, these investigations suggest a new role for long-chain fatty aldehydes as signaling lipids, ability to form adducts with GSH, proteins such as HDACs and regulate cellular functions.     

An atom efficient, solvent-free, green synthesis and antimycobacterial evaluation of 2-amino-6-methyl-4-aryl-8-[(E)-arylmethylidene]-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-carbonitriles

An atom efficient, green protocol for the synthesis of fifteen 2-amino-6-methyl-4-aryl-8-[(E)-arylmethylidene]-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-carbonitriles in quantitative yields from the reaction of 1-methyl-3,5-bis[(E)-arylmethylidene]-tetrahydro-4(1H)-pyridinones with malononitrile in presence of solid sodium ethoxide under solvent-free condition is described. The compounds were tested for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant tuberculosis (MDR-TB), and Mycobacterium smegmatis using agar dilution method. 2-Amino-4-[4-(dimethylamino)phenyl]-8-(E)-[4-(dimethylamino)phenyl]methylidene-6-methyl-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]-pyridine-3-carbonitrile was found to be the most potent compound (MIC: 0.43microM) against MTB and MDR-TB, being 100 times more active than standard, isoniazid against MDR-TB.     

Conformations of complexes derived from the interactions of two stereoisomeric bay-region 5-methylchrysene diol epoxides with DNA

The reaction mechanisms of two isomeric bay-region diol epoxides of 5-methylchrysene (trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II) with double-stranded DNA in aqueous solutions were studied utilizing kinetic flow dichroism and fluorescence techniques. As in the case of the previously studied benzo(a)pyrene-7,8-diol-9,10-oxide isomers (BaPDE), both DE-I and DE-II rapidly form intercalation-type complexes (association constants K = 2700 and 1500 M-1 respectively in a neutral 5mM phosphate solution). The physically bound diol epoxide molecules react on time scales of minutes to form predominantly tetraols; a greater fraction (6 +/- 1%) of DE-I than of DE-II (2-3%) molecules react with the DNA to form covalent products. The DE-II isomer is characterized by a greater reactivity than DE-I, and the rates of reaction are markedly accelerated in the presence of DNA in both cases. The linear dichroism spectra of the covalent adducts reveal that the conformations of both types of adducts are similar, with the long axes of the phenanthrenyl chromophores tilted, on the average, at angles of 38-52 degrees with respect to the average orientations of the transition moments (at 260 nm) of the DNA bases. The conformations of the covalently bound DE-I and DE-II molecules resemble those observed in the case of the highly tumorigenic (+) enantiomer of anti-BaPDE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 9-(2-Deoxy-β-D-ribofuranosyl)purine-2-thione

Abstract

A synthesis of 9-(2-deoxy-β-D-ribofuranosyl)purine-2-thione was performed by desulfurization of 2′-deoxy-6-thioguanine to give 2-amino-9-(2-deoxy-β-D-ribofuranosyl)purine, diazotization with chloride replacement to give 2-chloro-9-(2-deoxy-β-D-ribofuranosyl)purine, and the replacement of chloride with sulfur using thiolacetic acid and deacetylation.     

The Synthesis,Structure, and Conformation of 2′-Deoxy Analogues of „Fat“ Xanthine Nucleosides,Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The syntheses of 1-(2-deoxy-β-D-erythro-pentofuranosyl)-4,5,7,8-tetrahydro-6H-imidazo[4,5-e] [1,4]diazepine-5,8-dione (9β), its 3-glycosyl regioisomer (16β), and their respective α anomers (9α and 16α), are reported. Conformational and configurational studies, employing ¹H NMR NOE and CD spectroscopy, are described. The single-crystal X-ray structural analysis of 9β is presented. The attempted enzymic glycosylation of the heterocyclic base 6 with a bacterial purine nucleoside phosphorylase was not successful.     

Metabolism and elimination of the endogenous DNA adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one, in the rat

Endogenously occurring damage to DNA is a contributing factor to the onset of several genetic diseases, including cancer. Monitoring urinary levels of DNA adducts is one approach to assess genomic exposure to endogenous damage. However, metabolism and alternative routes of elimination have not been considered as factors that may limit the detection of DNA adducts in urine. We recently demonstrated that the peroxidation-derived deoxyguanosine adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one (M1dG), is subject to enzymatic oxidation in vivo resulting in the formation of a major metabolite, 6-oxo-M1dG. Based on the administration of [14C]M1dG (22 microCi/kg) to Sprague-Dawley rats (n=4), we now report that 6-oxo-M1dG is the principal metabolite of M1dG in vivo representing 45% of the total administered dose. When [14C]6-oxo-M1dG was administered to Sprague-Dawley rats, 6-oxo-M1dG was recovered unchanged (>97% stability). These studies also revealed that M1dG and 6-oxo-M1dG are subject to biliary elimination. Additionally, both M1dG and 6-oxo-M1dG exhibited a long residence time following administration (>48 h), and the major species observed in urine at late collections was 6-oxo-M1dG.     

The metabolism of tetralin

1. [1-(14)C]Tetralin was synthesized and fed to rabbits. 2. Of the radioactivity, 87-90% was excreted in the urine within two days and 0.5-3.7% on the third day. The faeces contained 0.6-1.8%. No radioactivity was found in the breath and negligible amounts were retained in the tissues. About 90-99% of an administered dose was accounted for. 3. The main metabolite in the urine was the glucuronide of alpha-tetralol (52.4%). Other conjugated metabolites were beta-tetralol (25.3%), 4-hydroxy-alpha-tetralone (6.1%), cis-tetralin-1,2-diol (0.4%) and trans-tetralin-1,2-diol (0.6%). 4. beta-Tetralone, alpha-naphthol, 1,2-dihydronaphthalene and naphthalene, previously reported as metabolites, are artifacts, and tetralin, alpha-tetralone, beta-naphthol, 5-hydroxytetralin, and 6-hydroxytetralin are not metabolites. 5. The major metabolite of tetralin, alpha-tetralol and alpha-tetralone is the glucuronide of alpha-tetralol, which was isolated as methyl (1,2,3,4-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate; the major metabolite of beta-tetralol and beta-tetralone is the glucuronide of beta-tetralol, which was characterized as methyl (1,2,3,4-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 5-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-1-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6-Hydroxytetralin is conjugated with glucuronic acid, and was characterized as methyl (5,6,7,8-tetrahydro-2-naphthyl tri-O-acetyl-beta-d-glucosid)uronate. 6. A metabolic sequence accounting for the observed biological transformation products is proposed.     

Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.     

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.     

Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli

Acrolein, a reactive alpha,beta-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(beta-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (gamma-OH-PdG). The cellular processing and mutagenic potential of gamma-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is approximately 70% in Escherichia coli DeltarecA, uvrA. The block to DNA synthesis can be overcome partially by recA-dependent recombination repair. Targeted G --> T transversions were observed at a frequency of 7 x 10(-4)/translesion synthesis. Inactivation of polB, dinB, and umuD,C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitro primer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite gamma-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across gamma-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct.     

Fluorescent and photoaffinity labeling probes for retinoic acid receptors.

A fluorescent probe for retinoid receptors (RARs) was designed and prepared. The probe consists of a retinoid moiety and a dansyl moiety, i.e., 2-[3-(5-dimethylaminonaphthalene-1-sulfonyl)- aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid: DAM-3. DAM-3 specifically bound RARs. Additionally, a photoreactive RAR fluorescent probe was designed and prepared, i.e., 2-[3-(5-azidonaphthalene- 1-sulfonyl)aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (ADAM-3). ADAM-3 irreversibly and specifically bound RARs using ultraviolet irradiation.     

Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli.

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.     

Metabolic activation of a mutagen, 2-amino-6-methyldipyrido-[1,2-a:3′,2′-d]imidazole. Identification of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and its reaction with DNA

A potent mutagen, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(Glu-P-1), isolated from pyrolysates of L-glutamic acid and casein, was metabolically activated and bound to DNA. An activated form was identified as 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(N-OH-Glu-P-1). Synthetic N-OH-Glu-P-1 reacted with DNA only after O-acetylation to give a modified DNA, which on hydrolysis gave 2-(C⁸-guanyl)amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(gua-Glu-P-1). The same adduct was isolated from DNA modified with Glu-P-1 by microsomes in vitro, as reported earlier.