In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.     

Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen

The sensitivity of various methods suitable for biomonitoring the exposure to genotoxicants was compared in an animal model. The results were related to the presence of genotoxic effects in the target organ. Groups of male Wistar rats were given one oral dose of 0, 0.1, 10 or 200 mg 2-acetylaminofluorene (2-AAF)/5 ml dimethyl sulphoxide/kg body weight. Peripheral blood cells, excreta, liver and spleen were collected at different time intervals after dosing. Mutagenicity in urine and extracts of faeces was determined using the Ames test with Salmonella typhimurium TA98 with and without S9 and with and without beta-glucuronidase. Genotoxic effects were studied by measuring DNA-adduct formation in lymphocytes, liver and spleen, and sister-chromatid exchanges (SCEs) in lymphocytes. DNA adducts were measured with immunochemical techniques and postlabelling methods. Mutagenicity in urine and faeces, collected during the first 24 h after treatment, was detected at 2-AAF doses of 1 mg/kg b.w. and higher. At these doses DNA adducts also became apparent in the liver, the main target organ for tumour induction by 2-AAF. The adduct detected appeared to be the N-(deoxyguanosin-8-yl)-2-AAF adduct. There was no evidence of the presence of any other types of DNA adducts. At doses of 1 and 10 mg/kg b.w. no mutagenicity was detected in excreta collected during the second and third day after dosing. The DNA-adduct level in liver cells of the 1 mg/kg b.w. group was maximal 24 h after dosing. At 200 mg/kg b.w. a delay in excretion of mutagenicity with urine and faeces was seen and at 10 and 200 mg/kg b.w. the amount of DNA adducts continued to increase with time after dosing. At 24 and 48 h after treatment with 10 mg, the adduct levels were of the same order of magnitude as those found after the 20-fold higher dose. This points to overloading of the metabolizing system which in combination with the enterohepatic circulation, may lead to an increased retention of 2-AAF in the body. A slightly increased incidence of SCEs of doubtful significance was seen in lymphocytes, but only at the very high dose of 200 mg/kg b.w. No DNA adducts could be detected in blood lymphocytes or spleen cells at any of the dose levels applied, either with the immunochemical or with the postlabelling method.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction products of the carcinogen N-hydroxy-4-acetylamino-4'-fluorobiphenyl with DNA in liver and kidney of the rat

The binding of AABP4'F and ABP4'F residues to rat liver and kidney DNA in vivo was studied at different periods of time after administration of N-[G-3H]hydroxy-AABP4'F at dose levels of 5 and 25 mg/kg body weight. DNA preparations from both organs were hydrolyzed enzymatically at pH 8--9 with mixtures of DNAase, snake venom phosphodiesterase and alkaline phosphatase from Escherichia coli. The enzymatic digests were analysed by Sephadex LH-20 chromatography using synthetic N-([G-14C] deoxyguanosin-8-yl)-AABP4'F as marker. Elution with 30% ethanol gave three major peaks of tritium activity. The first peak consisted largely of N-(deoxyguanosin-8-yl)-ABP4'F decomposition products, which were not further characterized. The second product has similar chromatographical and chemical properties as 3-(deoxyguanosin-N2-yl)-AAF; and was also persistent in liver as well as in kidneys. The third peak of tritium activity co-chromatographed with the marker compound N-([G-14C] deoxyguanosin-8-yl)-AABP4'F. Kinetic studies revealed that the latter product was removed rapidly from liver and kidney DNA at equal rates (t1/2 = 2 days). Approximately 80% of the total radioactivity bound to DNA consisted of deacetylated material, which was removed at a much slower rate (t1/2 = 10 days) in both organs. An initial rapid removal of all products in kidney during the first 7 days (t1/2 = 3.3 days) at dose levels of 25 mg/kg is probably due to toxic effects on the kidneys, because this phenomenon was not observed at dose levels of 5 mg/kg. The synthetic ester N-OSO3K-AABP4'F was at least twice as reactive towards L-methionine and guanosine as compared to the corresponding AABP derivative, but had 40% of the reactivity of N-acetoxy-AAF under similar conditions. The new compounds 3-methylmercapto-4-acetylamino-4'-fluorobiphenyl and N-(deoxyguanosin-8-yl)-4-acetylamino-4'-fluorobiphenyl have been characterized by means of their NMR and mass spectra. Attempts to devise an unambiguous synthesis for 3-(deoxyguanosin-N2-yl)arylamides have been unsuccessful.     

The formation of 2-aminofluorene-DNA adducts in vivo: evidence for peroxidase-mediated activation

Formation of DNA adducts in various tissues of dogs fed a single dose of the carcinogen 2-aminofluorene was investigated. Adduct analysis was performed using a technique that allows measurement of both N-(deoxyguanosin-8-yl)-2-amino-2-aminofluorene-DNA adduct formed by reaction of N-hydroxy-2-aminofluorene with DNA, as well as the polar 2-aminofluorene-DNA adducts formed when 2-aminofluorene is activated by prostaglandin H synthase-peroxidase in vitro. Two male beagle (A and B) dogs were examined and a different DNA adduct profile was observed with each dog. For the dog A, N-(deoxyguanosin-8-yl)-2-aminofluorene was the major adduct found in hepatic DNA; no peroxidase-derived adducts were detected in this tissue. In contrast, adducts eluting similarly to peroxidase-derived adducts were found in urinary tract tissues of this dog with the relative abundance of these adducts in the order urothelium greater than renal medulla greater than renal cortex, which correlates with the respective tissues' prostaglandin H synthase activity. N-(Deoxyguanosin-8-yl)-2-aminofluorene was detected in the renal tissues, but not in urothelium. For dog B, only the N-(deoxyguanosin-8-yl)-2-aminofluorene adduct was observed in all tissues examined, including the urothelium. However, total binding to liver, kidney, and bladder were two-, two-, and four-fold lower, respectively, than dog A. These data indicate that both prostaglandin H synthase-mediated activation and N-hydroxylation of 2-aminofluorene occur in vivo and may be subjected to pharmacodynamic considerations. Furthermore, the tissue distribution of the peroxidase-mediated 2-aminofluorene adducts suggests this process may also be of importance in the bladder-specific carcinogenicity of aromatic amines.     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

1-Nitrosopyrene: an intermediate in the metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538

Incubation of Salmonella typhimurium TA1538, in suspension culture, with 1.5 or 23 microM 1-nitropyrene resulted in a time-dependent increase in reversions for up to 7 h. In contrast, when the bacteria were exposed to 1.5 microM 1-nitrosopyrene, a reduction product of 1-nitropyrene, maximum reversion induction occurred after 1 h and a much higher mutation frequency was detected. Examination of DNA isolated from Salmonella typhimurium incubated with 4.1 microM [4,5,9,10-3H]1-nitrosopyrene indicated the presence of one major adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, the same adduct observed previously when the bacteria were exposed to 1-nitropyrene. When calf thymus DNA was treated with 1-nitrosopyrene in the presence of ascorbic acid, 1-aminopyrene was formed concomitant with the production of N-(deoxyguanosin-8-yl)-1-aminopyrene. In the absence of ascorbic acid, a 20-fold reduction in DNA binding was observed and 1-aminopyrene was not detected. The observations that 1-nitrosopyrene forms the same DNA adduct and is more mutagenic than 1-nitropyrene, suggest that 1-nitrosopyrene is an intermediate in the mutagenic activation of 1-nitropyrene in Salmonella typhimurium TA1538. Since reduction of 1-nitrosopyrene was necessary to get appreciable DNA binding in vitro, further reduction of 1-nitrosopyrene to N-hydroxy-1-aminopyrene is probably required in the activation pathway.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.     

Influence of flanking sequence context on the mutagenicity of acetylaminofluorene-derived DNA adducts in mammalian cells

Site-specifically modified oligodeoxynucleotides were used to explore the influence of neighboring base sequence context on the mutagenic potential of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) in mammalian cells. Oligodeoxynucleotides ((5)(')TCCTCCTNXNCTCTC, where X is dG-AAF, dG-AF, or dG and N is C, A, G, or T) with different bases flanking the lesion were incorporated into a single-strand shuttle plasmid vector and used to establish the mutational frequency and specificity of dG-AAF and dG-AF adducts in simian kidney (COS-7) cells. Vectors containing dG-AAF promote preferential incorporation of dCMP at the site of the lesion; misincorporation of dAMP and dTMP also was observed. Mutational frequencies range from 11 to 23%. High mutational frequencies (18-23%) were observed when G or T was positioned 5' to dG-AAF and a lower frequency (11%) when C was 5' to the lesion. dCMP was predominantly incorporated opposite the dG-AF adduct when C, A, or T was 5' to the lesion; dAMP and dTMP were misincorporated at a frequency of 2-4%. With G 5' to the lesion, the overall mutational frequency for dG-AF ranged between 11 and 70%; the highest value occurred when C was the 3' flanking base, and the predominant mutation event was G --> T transversion (59%). We conclude from these experiments that dG-AAF and dG-AF promote G --> T transversions and G --> A transitions in mammalian cells. The mutational frequency and specificity of dG-AF vary significantly, depending on the nature of the bases flanking the lesion.     

An examination of the weak mutagenic response of 1-nitropyrene in Chinese hamster ovary cells

Incubation of suspension cultures of Chinese hamster ovary (CHO) cells with 1-nitropyrene for as long as 2.5 h failed to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus, while incubation with 1-nitrosopyrene, a reduced derivative of 1-nitropyrene, resulted in a strong mutagenic response. Examination of the metabolites produced during these incubations indicated that 1-nitrosopyrene was rapidly reduced to 1-aminopyrene while 1-nitropyrene was not detectably metabolized. Both compounds produced a single major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, in the CHO cells and a strong linear relationship was found between mutation induction and the extent of DNA binding. The low level of adducts produced by 1-nitropyrene was consistent with the weak mutagenic response produced by this compound. These results indicate that both 1-nitropyrene and 1-nitrosopyrene are reduced to a reactive electrophile, presumably N-hydroxy-1-aminopyrene, which produces potentially mutagenic DNA damage in CHO cells. Comparison of the relationship between N-(deoxyguanosin-8-yl)-1-aminopyrene formation and mutation induction in CHO cells with the levels of 1-nitropyrene-induced DNA damage associated with positive responses in other assays of genetic toxicity and with the number of mutations associated with the DNA adducts produced by other agents in CHO cells suggests that the CHO/HGPRT assay may be relatively insensitive to 1-nitropyrene-induced DNA damage. The poor capability of CHO cells in reducing 1-nitropyrene and the relative insensitivity of the assay to the DNA damage produced by this compound may contribute to the weak mutagenic response of 1-nitropyrene in CHO cells.     

Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites

Two related carcinogen adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) or N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into the lacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA. The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct. These site-specifically modified templates were transfected into competent wild-type Escherichia coli cells (JM103) and a uvrA strain (SMH12). The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective beta-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region. DNA sequencing was used to characterize 179 of the mutants obtained. We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct. A specific frameshift (+1G) was also observed at a displaced site. All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide. Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts. The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment)

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.     

Differential effects induced by N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene in DNA excision-repair-deficient Chinese hamster cells

The direct-acting cytotoxic properties of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-2-aminofluorene (N-OH-AF) have been determined in repair-proficient (AA8-4) and repair-deficient (UV-5) Chinese hamster ovary cells. Cytotoxicity comparisons indicate that UV-5 cells are considerably more sensitive to exposure to N-OH-AAF than is the parental AA8-4 cell line, i.e., concentrations needed to obtain a D37 for survival of AA8-4 is greater than 5-fold higher than for UV-5. Mutation analysis at the HGPRT locus also indicates the increased sensitivity of UV-5 cells to N-OH-AAF as witnessed by an enhanced induction of 6-thioguanine-resistant colonies at equitoxic doses. Conversely, N-OH-AAF, did not induce a 'UV-mimetic' response when comparing genotoxicity between these two cell lines. Our data coupled with previously published model-building and adduct removal studies (Broyde and Hingerty, 1983; Fuchs and Daune, 1974; Grunberger and Weinstein, 1976; Yamasaki et al., 1977) suggest that the minor DNA adduct species, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene, may be responsible for the hypermutagenicity witnessed in DNA excision-repair-deficient cells treated with N-OH-AAF.     

Synthesis and characterization of nucleoside derivatives,n-(benzoyl)-n-(deoxyguanosin-8-yl)4-aminobiphenyl and n-(2'-deoxyguanosin-8-yl)4-aminobiphenyl via alpha-phenyl-n-(4-biphenyl)nitrone

Lead tetraacetate (LTA) oxidation of alpha-Phenyl-N-(4-bipheny])nitrone (8) to give a new ultimate carcinogen, N-acetoxy-N-benzoyl-4-aminobiphenyl (9) which was reacted with deoxyguanosine (dG) at pH 6.9 to give nucleoside derivative, N-(benzoyl)-N-(deoxyguanosin-8-yl)-4-aminobiphenyl (10). Following debenzoylation with sodium carbonate-methanol leads to N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (11).     

Enzymatic basis for changes in fucoganglioside during chemical carcinogenesis. Induction of a specific alpha-fucosyltransferase and status of an alpha-galactosyltransferase in precancerous rat liver and hepatoma

Previous studies indicated that accumulation of alpha-fucosyl-GM1 (IV2FucII3NeuAcGgOse4Cer) and alpha-galactosyl-alpha-fucosyl-GM1 (IV3GalIV2FucII3NeuAcGgOse4Cer) occurs in precancerous livers of rats fed the chemical carcinogen N-2-acetylaminofluorene, before development of hepatoma. Both fucogangliosides were completely absent in normal rat liver as well as in livers of rats fed a nonhepatic carcinogen and tumor promoters (Holmes, E.H., and Hakomori, S. (1982) J. Biol. Chem. 257, 7698-7703). The enzymatic basis of the chemical changes described above is reported in this paper. The alpha-L-fucosyltransferase activity toward GM1 (II2NeuAcGgOse4Cer) as well as asialo-GM1 (GgOse4Cer) was almost undetectable in extracts from normal rat liver, but significant activity of this enzyme was detected in extracts of rat livers after 4 weeks of feeding a diet containing N-2-acetylaminofluorene. The same enzyme activity in cultured rat hepatoma cells was 18- to 47-fold higher than in N-2-acetylaminofluorene-fed rat liver. In contrast, alpha-galactosyltransferase activity with a broad substrate specificity was detected in normal as well as in N-2-acetylaminofluorene-fed liver, although the specific activity of this enzyme in Golgi membranes in precancerous liver was significantly higher than that of normal rat liver. Thus, the appearance of alpha-fucosyl-alpha-galactosyl-GMI in precancerous liver is due to an induction of synthesis of alpha-fucosyl-GMI which is the substrate for the normally existing alpha-galactosyltransferase. The activity of alpha-fucosyltransferase was highly specific toward a substrate structure Gal beta 1 leads to 3GalNAc beta 1 leads to R in GMI or asialo-GMI and showed an anomalous inhibition by a large variety of detergents tested. In contrast, the alpha-galactosyltransferase showed a wide substrate specificity, activated by detergents and Mn2+ ion. Membrane alterations in precancerous and malignant transformation of rat liver is associated with an induction of an unusual alpha-fucosyltransferase which is the key step in synthesis of both fucogangliosides.     

2-(Allylthio)pyrazine inhibition of aflatoxin B1-induced hepatocarcinogenesis in rats.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has hepatoprotective effects against toxicants. Effect of 2-AP on hepatic tumorigenesis in association with glutathione S-transferase (GST) induction was examined in rats exposed to aflatoxin B1 (AFB1). Both AFB1-DNA adduct formation in the liver and urinary elimination of 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-N7-guanine) adduct were also determined. Male Sprague Dawley rats were treated with 2-AP at the daily oral doses of 10, 25 and 50 mg/kg for 16 consecutive days, during which four repeated doses of AFB1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-thirds of hepatectomy, followed by administration of phenobarbital (PB). Focal areas of hepatocellular alteration were identified after 44 days and preneoplastic foci expressing the placental form of glutathione S-transferase P (GST-P) were quantified by immunostaining of liver sections. 2-AP reduced the volume of liver occupied by GST-P foci by 65-96%. Under these experimental conditions, 2-AP treatment resulted in significant elevations in GST activity in the liver. Levels of radiolabeled AFB1 covalently bound to hepatic DNA, RNA and proteins were significantly reduced in rats treated with 2-AP for 5 days. 2-AP pretreatment also caused a 45% reduction in the urinary elimination of AFB1-N7-guanine adduct over the 24-h postdosing period. The present findings demonstrated that 2-AP exhibited protective effects against AFB1-induced hepatocarcinogenesis in rats with a marked decrease in the level of AFB1-DNA adduct. Reduction of hepatic DNA adducts might result from elevations of activity of GST, which catalyzes detoxification of the carcinogen.     

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N²-yl)-MAB(N²-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N²-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [³H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N²-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N²-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M⁺ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M⁺ = 528; M-N(CH₃)(CD₃) = 481) indicated the structure to be a deoxyadenosin-N⁶-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz ¹H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N⁶-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N⁶-amino and N⁶-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.     

Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

Mutation induced in vitro on a C-8 guanine aminofluorene containing template by a modified T7 DNA polymerase

We reacted uracil-containing M13mp2 DNA with N-hydroxy-2-aminofluorene to produce a template with N-(deoxyguanosin-8-yl)-2-aminofluorene adducts. This template was hybridized to a non-uracil-containing linear fragment from which the lac z complementing insert had been removed to produce a gapped substrate. DNA synthesis using this substrate with the modified T7 DNA polymerase Sequenase led to an increase in the number and frequency of lac- mutations observed. Escherichia coli DNA polymerase I (Kf) did not yield a comparable increase in mutation frequency or number even though both Sequenase and the E. coli polymerase had similar, low, 3'----5' exonuclease activities as compared to T4 DNA polymerase. We did not observe an increase in mutations when synthesis was attempted on a template reacted with N-acetoxy-2-(acetylamino)fluorene to give N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts. Both E. coli and T7 enzymes terminate synthesis before all (acetylamino)fluorene lesions. Only some of the putative aminofluorene adducts produced strong termination bands, and there was a difference in the pattern generated by Sequenase and E. coli pol I (Kf) using the same substrate. Analysis of the mutations obtained from Sequenase synthesis on the aminofluorene-containing templates indicated a preponderance of -1 deletions at G's and of G----T transversions.