The fluorescence emission properties of dGuo-C8-AAF, dGuo-C8-AF and the imidazole ring-opened products of dGuo-C8-AF indicate a different dynamic structure for the various compounds

The reactive metabolites of the carcinogenic N-acetyl-2-aminofluorene (AAF) form adducts with the guanine base of DNA. The fluorescence emission characteristics of N-(deoxy-guanosin-8-yl)-N-acetyl-2-aminofluorene (dGuo-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF) and the two N7 = C8 imidazole ring-opened products of dGuo-C8-AF (ro-dGuo-C8-AF I + II) were investigated and related to their conformational properties. The dGuo-C8-AF adduct (phi F congruent to 4-5 X 10(-4) shows a broad and structureless emission band, which is attributed to the formation of an excited-state complex. In contrast, the emission spectra of dGuo-C8-AAF (phi F congruent to 1.10(-4] and both ro-dGuo-C8-AF compounds [phi F(ro-dGuo-C8-AF I) congruent to 4.10(-3); phi F(ro-dGuo-C8-AF II) congruent to 4.10(-4)] are narrow. This indicates that dGuo-C8-AAF and ro-dGuo-C8-AF I + II do not decay into an exciplex as occurs in dGuo-C8-AF. The spectroscopic features are discussed in terms of the differences in the dynamic structure of the various compounds.     

The biological activity of single-stranded {Phi}X174 DNA, modified by N-hydroxy-2-aminofluorene, is inhibited by guanine imidazole ring-opening of the major, non-lethal aminofluorene-DNA adduct

The major aminofluorene-DNA derivative, found in the liver ofrats after administration of the hepatocarcinogen N-acetyl-2-aminofluoreneand identified as N-(deoxyguanosin-8-yI)-2-aminofluorene (dGuo-C8-AF),was introduced in different amounts in single-stranded øX174DNA by reacting the DNA with tritium labeled N-hydroxy-2-aminofluorene.The modified DNA was subsequently incubated in 0.1 M NaOH at37°C for increasing periods of time to convert the dGuo-C8-AFresidues into their guanine imidazole ring-opened forms. Thedegree of conversion was determined by measuring the amountof residual N-(guanin-8-yI)-2-aminofluorene in trifluoroaceticacid hydrolyzates of the alkali-treated DNA by h.p.l.c. In addition,the effect of ring opening on the biological activity of theDNA was monitored by transfecting the DNA to Escherichia coliwild-type spheroplasts. The results indicate that the majoraminofluorene-DNA adduct formed initially, which contributeslittle to inactivation, becomes lethal when its guanine imidazolering is opened.     

Excision of imidazole ring-opened N7-hydroxyethylguanine from chloroethylnitrosourea-treated DNA by Escherichia coli formamidopyrimidine-DNA glycosylase.

Alkylkation of the N7 of guanine residues in DNA favours the opening of the imidazole ring, yielding a formamidopyrimidine (Fapy). This Fapy residue blocks DNA replication and is actively excised by a DNA glycosylase. We have cloned and sequenced the Escherichia coli gene responsible for synthesis of the enzyme, which has also been purified to homogeneity. It was found to have associated apurinic/apyrimidinic (AP) lyase activity, nicking DNA at AP sites. Chloroethylnitrosoureas are used in cancer chemotherapy. The lesions induced in DNA by these compounds, including N7-chloro- and hydroxyethylguanine, are excised by E. coli 3-methyladenine DNA glycosylase II, and we report that the corresponding imidazole ring-opened forms are repaired by Fapy-DNA glycosylase. Human cells have the counterpart to these enzymes, which could contribute to the repair of these lesions during chemotherapy.     

Binding efficiency of antibodies to DNA modified with aromatic amines and polycyclic aromatic hydrocarbons: implications for quantification of carcinogen-DNA adducts in vivo

Antibodies raised against the bovine serum albumin conjugates of N-(guanosin-8-yl)-N-2-acetylaminofluorene (Guo-8-AAF), the imidazole ring-opened form of N-(guanosin-8-yl)-2-aminofluorene (roGuo-8-AF) and the methylated bovine serum albumin complex of DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) have been employed in a highly sensitive enzyme-linked immunosorbent assay (ELISA) to determine their affinity for DNA modified with the corresponding carcinogens at various levels of modification. All antibodies recognized highly modified DNA more efficiently than DNA of low modification. This property, which may be common to all antibodies raised against carcinogen-DNA adducts, has to be taken into account when these antibodies are used to quantify carcinogen-DNA adducts in biological samples. Appropriate DNA preparations of low modification have to be used as reference compounds in immunoassays to allow reliable quantification of adduct levels in DNA from animals and human cells.     

Adducts from in vivo action of the carcinogen 4-hydroxyaminoquinoline 1-oxide in rats and from in vitro reaction of 4-acetoxyaminoquinoline 1-oxide with DNA and polynucleotides

In vivo 4-hydroxyamino[2-3H]quinoline 1-oxide-modified DNA and in vitro 4-acetoxyamino[2-3H]quinoline 1-oxide-modified DNA were enzymatically hydrolyzed, and the hydrolysates were analyzed by high-performance liquid chromatography. The two patterns were compared, and we showed that all of the high-performance liquid chromatography peaks which were recovered from in vivo-modified DNA were present in the hydrolysate of in vitro-modified DNA. Therefore, we used the in vitro 4-acetoxyamino[2-3H]quinoline 1-oxide-modified DNA to investigate the quinoline-purine adducts which are characteristics of the mode of action of the carcinogen 4-nitroquinoline 1-oxide. By comparison with the enzymatic hydrolysates of 4-acetoxyamino[2-3H]quinoline 1-oxide-modified covalent poly(deoxyadenylate-deoxythymidylate) X poly(deoxyadenylate-deoxythymidylate) and covalent poly(deoxyguanylate-deoxycytidylate) X poly(deoxyguanylate-deoxycytidylate) three nitroquinoline adducts were enumerated on the modified DNA. One of them was previously characterized as a C8-guanyl adduct. We proved that the two other are a guanine and an adenine adduct, respectively. A quinoline derivative was identified in the hydrolysates of the in vivo- and in vitro-modified DNAs as 4-aminoquinoline 1-oxide, the origin of which was postulated to be a degradation compound of one (or more) adduct(s). Moreover, the presence of two degradation compounds of the C8-guanyl adduct was shown in mild alkaline conditions. We suspected an imidazole ring-opened form.     

Detection of N-hydroxy-2-acetylaminofluorene--DNA adducts in rat liver measured by radioimmunoassay

Antibodies to imidazole ring opened derivatives of alkali treatedN-(guanosin-8-yl)-2-aminofluorene (1-[6-(2, 5-diamino-4-oxopyrimidinyl-N⁶-riboside)]-3-(2-fluorenyl)ureawere elicited in rabbits by immunization with a conjugate betweenthe ring-opened derivative and bovine serum albumin. The specificityof the antibodies was studied by radio-immunoassay. These antibodiesand antibodies to N-(guanosin-8-yl)-N-acetyl-2-aminofluorenewere used to titrate the adducts formed in liver DNA of ratstreated with N-hydroxy-2-acetylaminofluorene. The ring-openedderivatives represent 10% of the dGuo-C8-adducts.     

Fpg protein releases a ring-opened N-7 guanine adduct from DNA that has been modified by sulfur mustard

Transfection of the Escherichia coli fpg gene into Chinese hamster ovary cells has been reported to enhance survival after exposure to aziridine (C.Cussac and F.Laval, 1996, Nucleic Acids Res., 24, 1742- 1746). This result suggests that Fpg protein protects cells from toxicity by removing ring-opened N-7 guanine adducts from DNA, and raises the possibility that Fpg protein would offer protection from other agents that alkylate the N-7 position of guanine. Since the major adduct formed by sulfur mustard in DNA is 7-hydroxyethyl- thioethylguanine (HETEG), we have investigated the action of Fpg protein on the ring-opened form of this adduct (ro-HETEG). A substrate containing ro-HETEG was prepared by alkaline treatment of DNA modified by [14C]sulfur mustard. Fpg protein purified from an over-producing strain of E. coli released ro-HETEG from this substrate in an enzyme- and time-dependent manner, and at a rate that is similar to that at which it releases ring-opened 7-methylguanine. Thus, Fpg protein acts efficiently on ro-HETEG, and may offer some protection against the toxic action of sulfur mustard.      

Sequence specificity of DNA repair by Escherichia coli Fpg protein.

The sequence specificity of guanine methylation in DNA by N-methyl-N-nitrosourea and the subsequent repair of ring-opened N7-methylguanine was studied using oligonucleotides of defined sequence. It was found that the methylation of TAGGGGCCCCTA was less than 2-fold greater than that occurring in TAGAGATCTCTA or TATGTGCACATA and 6-fold greater than in TACGCGCGCGTA. This is consistent with the concept that guanine methylation is least when the 5' preceding base is a pyrimidine and greatest when the 5' base is another guanine. These dodecamers were used to study repair by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) after the 7-methyl-guanine present in them was converted to the ring-opened form by alkaline treatment. The repair of ring-opened 7-methylguanine was much faster in self-complementary double-stranded 12mer substrates and was twice as rapid at 37 degrees C in TAGGGGCCCCTA compared with TACGCGCGCGTA. However, at 15 degrees C, the relative rates were reversed since TACGCGCGCGTA was repaired at the same rate as at 37 degrees C, whereas the repair of TAGGGGCCCCTA was much slower at 15 degrees C. The repair of TAGGGGCCCCTA at 37 degrees C was also much faster than the repair of TAGAGATCTCTA and was slightly more rapid than repair of TATGTGCACATA. Ligation of the dodecamer substrates to form 24mers or 36mers slightly reduced the initial rates of repair but did not abolish these differences. These results indicate that under physiological conditions, the Fpg protein is more active against adducts in guanine-rich regions and such regions may be the most likely sites of adduct formation at the N7-position of guanine which can then give rise to derivatives attacked by this enzyme.     

Formation of unique arylamine:DNA adducts from 2-aminofluorene activated by prostaglandin H synthase

Prostaglandin H synthase in the presence of arachidonic acid catalyzes the peroxidative metabolism of 2-aminofluorene (2-AF) to an electrophile(s) which binds covalently to calf thymus DNA in vitro. Moreover, this electrophile(s) appears distinct from the classical 2-AF-derived electrophiles, N-hydroxy-2-AF and the 2-AF nitrenium ion. Both the prostaglandin H synthase:arachidonic acid and horseradish peroxidase:hydrogen peroxide systems were used to investigate the binding of [3H]-2-AF to DNA and the nature of the DNA adducts formed from peroxidative activation of 2-AF. Modification of DNA by N-hydroxy-2-AF under mildly acidic conditions was used as a reference system in these studies and yielded a single 2-AF:nucleoside adduct, identified as N-(deoxyguanosin-8-yl)-2-AF (C8-dGuo-AF). Enzymatic hydrolysis of DNA modified by 2-AF activated in either of the peroxidase systems liberated 2-AF:nucleoside adducts that differed considerably from C8-dGuo-AF in chromatographic and extraction properties. C8-dGuo-AF from DNA hydrolysates was easily extracted into n-butyl alcohol and adsorbed by Sephadex LH-20 columns. In contrast, the peroxidase-derived adducts were poorly extracted into n-butyl alcohol and were not retained on Sephadex LH-20 columns. Experimental evidence suggests the peroxidase-derived adducts may possess a negative charge at neutral pH. Since C8-dGuo-AF is the only 2-AF:nucleoside adduct formed when 2-AF is activated via N-hydroxylation, these new adducts represent a marker unique to peroxidative activation of 2-AF. Therefore, 2-AF:DNA adducts can be used as a differential end point with which to assess the relative roles of N-hydroxylation and peroxidation in the metabolic activation of 2-AF in cell culture and in target tissues in vivo.     

Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest becauseit is formed in vivo by reaction of DNA with ethylene oxide(EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine,can be converted to this adduct by reduction. Two monoclonalantibodies (9E2, 4A5) recognizing 7HEG have been developed fromBALB/c mice immunized with the adduct coupled to keyhole limpethemocyanin. In addition, another antibody (8E10) was developedagainst the imidazole ring-opened form of the adduct (ro-7HEG).ELISAs were used to determine the sensitivity and specificityof these antibodies. With antibody 9E2, 50% inhibition of antibodybinding in the competitive ELISA was at 54 pmol of the modifiedbase 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5,the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well.Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well.Neither antibody 9E2 nor 8E10 cross-reacted with unmodifiedDNA or with the normal nudeosides at the highest concentrationtested. However, antibody 4A5 had a low affinity for deoxyguanosine(50% inhibition at 31 000 pmol). Sensitivity of adduct measurementcan be increased 3- to 10-fold using an ELISA with fluorescenceendpoint detection. These antibodies have been used to determinethe level of adducts in DNA modified in vitro with [³H]- or[¹⁴C]EO. Because of the cross-reactivity of the most sensitiveantibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassaymethod was developed to quantitate 7HEG in DNA. The limit ofsensitivity of this method is dependent upon the amount of DNAavailable for analysis. Using 30 fmol as the lowest detectableamount (20% inhibition) in the fluorescent ELISA with antibody4A5 and 100 µg of DNA assayed per well, adduct levelsof 1/10⁷ nucleotide can be determined. This method was appliedto DNA adduct detection in EO-treated myeloma cells and wholeblood. Antibody 8E10 was also used in immunohistochemical studiesto visualize ring-opened adducts in cells treated with EO followedby high pH. These antibodies will be used for the detectionand quantitation of adducts in human samples.     

Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.     

An optical study of conformation of the aminofluorene-DNA complex

Calf thymus DNA was modified with 2-aminofluorene (AF) to differentextents by treatment with N-hydroxy-2-amino-fluorene. The AF-modifiedDNAs together with free AF, the AF-modified guanine (Gua-C8-AF)and the AF-modified deoxyguanosine (dGuo-C8-AF) were subsequentlystudied by u.v. absorbance, linear dichroism and fluorescencespectroscopy. The emission and absorption properties of doublestrandedDNA-AF and single-stranded DNA-AF closely resemble those ofdGuo-C8-AF. The emission spectra of these three compounds showa broad, red-shifted emission, characteristic for exdplex formation.The linear dichroism and circular dichroism spectra of double-strandedDNA-AF show that the AF moiety forms a well-defined, regularstructure. The dichroic ratio in the 310–340 nm regionis constant, which indicates the presence of only one type ofadduct. The long-wavelength transition moment of this adductmakes an angle of 72–74° with the DNA helix axis.The binding of AF to double-stranded is DNA is accompanied bya destabilization of the DNA helix structure, a strong quenchingof the AF emission quantum yield, intense AF circular dichroismand an apparent immobilization of the dGuo-C8-AF complex. Insingle-stranded DNA-AF, the AF conformation appears more random,although the interactions between AF and the surrounding basespersist. The strong interactions between AF and the surroundingbases which dominate the optical properties of the studied complexes,the significant destabilization of the DNA double helix aftermodification with AF, and the relatively small angle betweenAF and the base planes support a model in which the adduct isinserted into the DNA helix.     

Reaction of the N2-acetoxy derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with 2'-deoxyguanosine and DNA. Synthesis and identification of N2-(2'-deoxyguanosin-8-yl)-PhIP.

The direct acting mutagenic N2-hydroxylated metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) does not react with DNA. Upon acetylation of the N2-hydroxy-PhIP with acetic anhydride two products could be detected. Mass spectrometric analysis showed that both products were monoacetyl derivatives of N2-hydroxy-PhIP. One of the products did not show any reactivity towards DNA and is probably the N-acetyl derivative of N2-hydroxy-PhIP. The other product which is most likely to be N2-acetoxy-PhIP reacted with DNA and 2'-deoxyguanosine but not with 2'-deoxycytidine, 2'-deoxyadenosine or 2'-deoxythymidine. The PhIP-2'-deoxyguanosine adduct was purified and characterized by mass spectral, 1H and [13C]NMR analysis, showing that PhIP like the other cooked food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline, had reacted with C-8 of guanine forming N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC analysis of enzymatically hydrolyzed calf thymus DNA which had been reacted with N2-acetoxy-PhIP showed one adduct which was chromatographically and spectroscopically identical to N2-(2'-deoxyguanosin-8-yl)-PhIP. HPLC separation followed by liquid scintillation counting of hydrolyzed liver DNA from a rat dosed with [3H]PhIP showed that radioactivity coeluted with the hydrolysis product of the synthetic PhIP-2-deoxyguanosine adduct, indicating that PhIP in vivo also forms an N2-(2'-deoxyguanosin-8-yl)-PhIP adduct.     

A spectroscopic study of the conformation of poly d(G -- C){dot}poly d(G -- C) modified with the carcinogenic 2-aminofluorene

The conformational properties of both the B- and Z-form helixof the 2-aminofluorene (AF) modified synthetic polynucleotidepoly d(G - C).poly d(G - C) were extensively studied with avariety of optical techniques (UV absorbance, linear dichroism,circular dichroism and fluorescence spectroscopy). The spectroscopicresults show, that: (i) the presence of the AF adduct in polyd(G-C).poly d(G-C) facilitates the conversion of the right-handedB-form polynucleotide to the left-handed Z-type poly d(G-C).polyd(G-C) duplex; (ii) in B- as well as in Z-form poly d(G-C).polyd(G-C)-AF the carcinogenic residue is situated in a specificand ordered complex with the DNA, in which the AF chromophoreis significantly immobilized and the long axis of the fluorenering system is positioned in a parallel mode to the DNA baseplanes; (iii) in B-type poly d(G-C).poly d(G-C)-AF the carcinogenicresidue exhibits strong stacking interactions with the adjacentDNA bases, which are coupled to an important destabilizationof the AF containing B-duplex; (iv) in contrast to the B-formcomplex, the AF-containing Z-type DNA is highly stabilized anda remarkable reduction of the AF-base interactions is observed;(v) in Z-form poly d(G-C).poly d(G-C)-AF the AF chromophoreresides at a medium-exposed position. The combined data supporta conformational model, in which the planar AF is inserted inthe B-type polynudeotide helix, while the carcinogenic residuein the Z-form poly d(G-C).poly d(G-C)-AF structure is placedin an outside position.     

Excision of aflatoxin B1-imidazole ring opened guanine adducts from DNA by formamidopyrimidine-DNA glycosylase

This investigation has confirmed the earlier reports that whenaflatoxin B₁ - DNA adducts are stored under physiological conditionssome aflatoxin B₁ - guanine adducts are converted to a secondaryproduct in which fission of the imidazole ring of the adductguanine has occurred. Incubation of DNA containing aflatoxinB₁ - guanine adducts for an increasing number of hours underphysiological conditions resulted in a progessive increase inthe number of adducts in which the imidazole rings of guaninesunderwent fission. It was shown that the Escherichia coli enzyme,formamidopyrimidine-DNA glycosylase exercises from the 6-dayincubated DNA, an amount of imidazole ring opened guanines equivalentto 40% of the aflatoxin B₁ - guanine adducts present in theDNA. The enzymatic excision of imidazole ring opened aflatoxinB₁ - guanine adducts is inhibited by Cibacron Blue F3GA a stronginhibitor of formamidopyrimidine-DNA glycosylase. Treatmentof afaltoxin B₁ - DNA with mild alkali (pH 9.6), resulted ina 2-fold increase in the amount of aflatoxin B₁ - guanines withopened imidazole rings; this was revealed by enzyme assays usingthis alkaline treated DNA substrate as well as by analysis ofacid hydrolysates of the alkaline treated DNA.     

Measurement by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA of healthy individuals and cancer patients treated with dacarbazine and procarbazine. Human data and method development for 7-alkylguanines

A ³²P-postlabelling method was developed to measure 7-methylguaninein human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-mono-phosphate(7-me-dGMP) was isolated from normal nucleotides using stronganion-exchange column chromatography. Overall the method gave35–45% yield as measured with DNA methylated with tritiateddimethyl suffate. Total white blood cell DNA from healthy non-smokers(n = 17) contained from 2.5 7-methylguanine residues/10⁷ nucleotides,corrected for the losses in preparation. Among four patientssampled immediately after a total dose of 1050–2800 mgof dacarbazine or procarbazine, the mean adduct level was 577-methylguanine residues/10⁷ nucleotides. As further methoddevelopment, we also investigated the phosphorylation reactionby T4 polynucleotide kinase using dinucleotides containing 7-methylguanineand corresponding imidazole ring-opened products as substrates.We found that imidazole ring-opened dTpdG-Me is resistant todigestion with deoxyribonuclease I, snake venom phosphodiesteraseand prostatic acid phosphatase. It is quantitatively phosphorylatedat femtomolar levels. This method is shown to be suitable forthe detection of 7-methylguanine in DNA, and is suggested tobe the approach most suited to postlabelling large and labile7-alkylguanines in DNA.     

Oxidation of 5'-site guanine at GG and GGG sequences induced by a metabolite of carcinogenic heterocyclic amine PhIP in the presence of Cu(II) and NADH

Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with an electrochemical detector coupled to a high-performance liquid chromatograph, we revealed that N-hydroxy metabolite of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in the presence of Cu(II). Addition of an endogenous reductant NADH enhanced the 8-OH-dG formation. Experiments with (32)P-labeled DNA fragments showed that this metabolite [PhIP(NHOH)] caused 8-hydroxylation of guanines in the presence of Cu(II) and NADH, and subsequent treatment with formamidopyrimidine-DNA glycosylase led to chain cleavages at the 5'-site guanine of GG and GGG sequences. Interestingly, antioxidant enzyme SOD enhanced the intensity of DNA damage, and thymine residues were appended to its guanine-predominant cleavage sites. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). A UV-visible spectroscopic study indicated that Cu(II) and SOD catalyze the autoxidation of PhIP(NHOH). These results suggest that Cu(II)-dependent autooxidation of PhIP(NHOH) coupled with NADH-mediated reduction of its oxidized product form redox cycle, resulting in oxidative DNA damage by low concentrations of PhIP(NHOH). We conclude that in addition to DNA adduct formation, oxidative DNA damage may be involved in the carcinogenic process of PhIP.     

Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo

Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1). The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms. Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.     

Direct O-acetylation of N-hydroxy arylamines by acetylsalicylic acid to form carcinogen-DNA adducts.

Acetylsalicylic acid (aspirin) has been shown to acetylate a number of drugs and biological macromolecules. Since enzymatic O-acetylation of N-hydroxy arylamines is regarded as an important activation step for DNA adduct formation, we initially examined the ability of aspirin to serve as an acetyl donor for this reaction, using rabbit liver cytosol. Instead, a direct non-enzymatic reaction was observed. Arylamine-DNA binding was enhanced from 3- to 25-fold at pH 7 by addition of aspirin to reactions containing the N-hydroxy derivatives of 2-aminofluorene (AF), 4-aminobiphenyl, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, but not for 2-amino-3-methylimidazo[4,5-f]quinoline or 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. Further studies with N-hydroxy-AF showed that reaction rates were first order with respect to both aspirin (0.1-10 mM) and N-hydroxy-AF (0.01-0.1 mM) concentrations. In contrast, aspirin had no effect on reactions conducted at pH 5 where N-hydroxy-AF is known to undergo protonation and react with DNA to form high levels of N-(deoxyguanosin-8-yl)-AF. N-Acetylation of AF by aspirin under these conditions was also negligible. However, the formation of the adduct from N-hydroxy-AF occurred at high yield (64-82%) at pH 7 with either DNA or 2'-deoxyguanosine. HPLC analyses showed only an aspirin-dependent loss of N-hydroxy-AF and concomitant adduct formation, with no detectable formation of solvolysis products. This indicated that the reaction proceeds to a significant extent only upon addition of the nucleophile, and suggests the formation of an O-tetrahedral intermediate that is in equilibrium with both the N-hydroxy derivative and the reactive N-acetoxy arylamine. Thus, the apparent O-acetylation of certain N-hydroxy arylamines selectively by aspirin offers a convenient route for the synthesis of arylamine-DNA adducts. The potential biological significance of this reaction in vivo is also discussed.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.