Constance M. Harris

The aflatoxin B1 formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B1 (AFB1). Hypotheses have been put forth that AFB1, in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB1 is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB1-formamidopyrimidine (AFB1-FAPY) adduct. AFB1-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB1 exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB1-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB1-N7-Gua, and (ii) one proposed rotamer of AFB1-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.

Evaluation of the Mutagenic Potential of the Principal DNA Adduct of Acrolein

Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

Biosynthesis of swainsonine in the diablo locoweed (Astragalus oxyphyrus)

Abstract In addition to the toxic indolizidine alkaloid swainsonine, the diablo locoweed (Astragalu oxyphysus) has been found to produce [1,8a-trans]-1-hydroxyindolizidine and [1,8a-trans-1,2-cis]-1,2-dihydroxyindolizidine, known precursors of swainsonine in the fungus Rhizoctonia leguminicola. As in the fungus, [G-3H]-pipecolic acid is incorporated into all three alkaloids. It is proposed that swainsonine biosynthesis in A. oxyphysus occurs by a pathway very similar to the fungal route.

Biomimetic syntheses of pretetramides. 2. A synthetic route based on a preformed D ring

A route to pretetramides has been developed on the basis of tandem condensations of homophthalate esters with the dianion of methyl acetoacetate to give aromatic bis(diketo esters), which cyclize spontaneously to anthracene diesters. A further condensation of one of the ester groups with acetamide synthons gives anthracene ester-keto amides, which can be cyclized to naphthacenecarboxamides. By this technique dimethyl 3-methoxyhomophthalate (2b) was condensed with the dilithium salt of methyl acetoacetate to give anthracene diester (4b). Protection of the hydroxyl groups followed by treatment with the dilithium salt of N-(trimethy1silyl)acetamide gave anthracene ester-keto amide 6, which cyclized to pretetramide (1) on treatment with HBr in acetic acid. The procedure for synthesis of pretetramide was modified to permit separate addition of the ketide chains. By use of the aliphatic mono ester (7b) of 3-methoxyhomophthalic acid, chain extension was brought about at the aliphatic carboxyl group by condensation with the dilithium salt of tert-butyl acetoacetate. This product was cyclized to isocoumarin 9b by treatment with acetic anhydride. Completion of the backbone was achieved in a single condensation by treatment of 9b with trianion 18 of 3,5-dioxohexanenitrile (prepared from the nitrile or indirectly by cleavage of isoxazole 17 with LDA) to give anthrone 32. A variety of other condensations of the trianion 18 were performed with electrophiles to demonstrate the utility of the reagent. The synthesis of pretetramide from anthrone 32 was completed by treatment with HI/phenol. Pretetramide (1) is the fully aromatic tetracyclic precursor of the tetracycline antibiotics.] The first paper in this series2 described a synthesis of pretetramide on the basis of tandem condensations of dianions of acetoacetic esters with glutarate derivatives to give a naphthalene diester after spontaneous cyclization. Further condensations with (a) tert-butyl lithioacetate and (b) the dianion of an isoxazole completed the carbon skeleton and after another spontaneous cyclization gave an anthraceneisoxazole, which could be transformed to pretetramide by treatment with a mixture of hydriodic acid and red phosphorus in acetic acid. The sequence circumvents the formidable problem of assembling a full decacarbonyl species prior to closure of the four rings; this segmented method for assembly of the polyketide intermediates is termed a [3 + (2 X 2) + 1 + 21 strategy (Scheme Ia). Alternative routes are described in this paper, which also skirt the preparation of large arrays of p carbonyl groups by employing homophthalate esters as pentacarbonyl equivalents; polyketide chains are extended from this central core by Claisen condensations with the two ester groups. The first of these routes is a [ 5 + (2 X 2) + I ] approach (Scheme Ib) in which tandem condensations of acetoacetate dianions with the two homophthalate ester groups yield a nonacarbonyl equivalent, which cyclizes to an anthracene. The skeleton is completed with an acetamide synthon followed by acid-catalyzed Claisen cyclization. The second synthesis, a [ 5 + 2 + 31 approach (Scheme IC) involves addition of a diketide chain followed by a triketide chain to the homophthalate nucleus. Results and Discussion The 3-methoxy derivative of homophthalic acid is required as the central unit for the preparation of pretetramide by the [ 5 + (2 X 2) + 11 strategy; but pilot studies were carried out with homophthalic acid because of its commercial availability. Tandem chain extensions were achieved by Claisen condensation of dimethyl homophthalate (2a) with methyl acetoacetate dianion (see Scheme 11). The dilithium salt was employed for the condensation to minimize the risk that the dianion, which is highly basic, would ionize the a-methylene group of homophthalate in preference to making nucleophilic attacks on the two ester groups. Prior studies of the condensation of aliphatic esters with the dianion of ace(1) McCormick, J. R. D. In Biogenesis o f h f i b i o f i c Substances; Vanek, (2) Gilbreath, S. G.; Harris, C. M.; Harris, T. M. J . Am. Chem. SOC., Z., Hostalek, Z., Eds.; Academic: New York, 1965; Chapter 8. preceding paper in this issue. Scheme I -m [3 (2 x 2) + 1 + 21 M M a c M /

Mutagenic potential of adenine N6 adducts of monoepoxide and diolepoxide derivatives of butadiene

To determine the biological effects of specific DNA adducts resulting from the interaction of 1,3‐butadiene metabolites with DNA, deoxyoligonucleotides have been synthesized with four different adducts at the N6 position of adenine, centrally located within the human N‐ras codon 61. The adducts are those arising from adduction by either the R or S stereoisomer of the monoepoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE). The diolepoxide can arise from partial hydrolysis of the diepoxide (BDO2) or from epoxidation of hydrolyzed monoepoxide. These adducted oligonucleotides were used in in vivo and in vitro assays designed both to determine their mutagenic potency and to examine specific interactions with Escherichia coli polymerases. Each adducted oligonucleotide was ligated into a single‐stranded vector M13mp7L2 that was subsequently used to transfect E. coli. The resulting mutagenic spectrum for these modified DNAs was stereoisomer specific. Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for the modified DNAs containing BDE adducts were stereoisomer specific. The mutations generated by adducts of the R,R enantiomer of the diolepoxide were exclusively A → G, whereas adducts of the S,S enantiomer of the diolepoxide yielded exclusively A → C mutations. None of the four modifications resulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque‐forming ability. Consistent with these data, when each of three purified E. coli polymerases was used to replicate DNAs containing these adducted deoxyoligonucleotides, the individual polymerases appeared to be virtually unaffected, such that all lesions were readily bypassed. Whereas previous animal model studies identified the mutagenic spectrum related to butadiene exposure, these studies begin to establish the specific lesions responsible for mutagenesis. This is the first report of stereoselectivity related to butadiene‐induced mutagenesis. Environ. Mol. Mutagen. 35:48–56, 2000

1-Epiastraline, a new pyrrolizidine alkaloid from Castanospermum australe

Abstract A new pyrrolizidine alkaloid [ 6a ] has been isolated from the legume Castanospermum australe A. Cunn. (Leguminosae) and identified as (1 S , 2 R , 3 R , 7 S , 7a R )-3-hydroxymethyl-1,2,7-trihydroxypyrrolizidine on the basis of 1 H NMR nuclear Overhauser effects and other spectroscopic studies.

Formation of Deoxyguanosine Cross-Links from Calf Thymus DNA Treated with Acrolein and 4-Hydroxy-2-nonenal

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

Condensations of dehydroacetic acid at the 6-methyl position

Abstract Dehydroacetic acid in the presence of two equivalents of sodium amide in liquid ammonia underwent methylation at the acetyl methyl position. In contrast, treatment of dehydroacetic acid with three equivalents of sodium amide converted it to a trianion which underwent condensations with electrophiles predominantly at the 6-Me position. The condensations included alkylation with alkyl halides, carbonyl addition with benzophenone, conjugate addition with chalcone, and acylation with aromatic esters. The benzoylation product was converted by acid treatment into 3,4-dihydroxy-6-methylbenzophenone. Methylation of both the 6-methyl and the acetyl methyl positions was accomplished by treatment of dehydroacetic acid trianion with excess methyl iodide.

Non-biomimetic route to deoxyadenosine adducts of carcinogenic polycyclic aromatic hydrocarbons

Aminotriols are prepared by direct aminolysis of the diol epoxides of polycyclic aromatic hydrocarbons, providing a substantial improvement over literature methods. The condensation of aminotriols with 6-halopurine deoxyribonucleosides provides a regio- and stereospecific synthesis of deoxyadenosine N6 adducts.

Structural Perturbations Induced by the α-Anomer of the Aflatoxin B1 Formamidopyrimidine Adduct in Duplex and Single-Strand DNA

The guanine N7 adduct of aflatoxin B1exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between α and β anomers. The β anomer is highly mutagenic in Escherichia coli, producing G → T transversions; it thermally stabilizes the DNA duplex. The AFB-α-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-α-FAPY adduct has been elucidated in 5′-d(C1T2A3T4X5A6T7T8C9A10)-3′·5′-d(T11G12A13A14T15C16A17T18A19G20)-3′ (X = AFB-α-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5′ face of the pyrimidine moiety at the damaged nucleotide between base pairs T4·A17 and X5·C16, placing the FAPY C5−N5 bond in the Ra axial conformation. Large perturbations of the ε and ζ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5′ neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3′-neighbor adenine stabilizes the E conformation of the formamide moiety. NMR studies reveal a similar 5′-intercalation of the AFB moiety for the AFB-α-FAPY adduct in the tetramer 5′-d(C1T2X3A4)-3′, involving the Ra axial conformation of the FAPY C5−N5 bond and the E conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-β-FAPY adduct also intercalates on the 5′ side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5′-stacking leads to the Ra conformational preference about the C5−N5 bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-α-FAPY adduct explain its lower stability as compared to the AFB-β-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3′-neighboring adenine stabilizing the E configuration of the formamide moiety is also observed for the AFB-β-FAPY adduct, and suggests that the identity of the 3′-neighbor nucleotide modulates the stability and biological processing of AFB adducts.