Metabolism and nucleic acid binding of N-hydroxy-4-acetylaminobiphenyl and N-acetoxy-4-acetylaminobiphenyl by cultured human uroepithelial cells.

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP), the proximate carcinogenic metabolites of the human bladder carcinogen 4-aminobiphenyl (ABP), was examined in human uroepithelial cells (HUC). Bioconversion was studied by incubating HUC cultures with [3H]N-OAc-AABP or [3H]N-OH-AABP. Three organo-soluble metabolites, N-OH-AABP, 4-acetylaminobiphenyl (AABP), and ABP were identified in ethyl acetate extracts from cultures exposed to N-OAc-AABP. Similarly, AABP and ABP were characterized as the major metabolites from cultures treated with N-OH-AABP. Incubation of N-OAc-AABP with HUC microsomes in vitro yielded primarily the O-deacetylation product N-OH-AABP. The HUC microsomes also catalyzed the N-deacetylation of N-OAc-[14C]AABP, N-OH-[14C]AABP, and [3H]AABP. The O- and N-deacetylase activities for N-OAc-AABP were 55.9 and 38.2 nmol/mg/min, respectively. These O- and N-deacetylase activities were both blocked by paraoxon. Incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes and tRNA or DNA showed that 23.0 and 8.0 nmol of N-OAc-AABP and 74.5 and 25.2 pmol of N-OH-AABP were bound per mg protein/mg RNA or DNA, respectively. In comparison, the acetyl CoA-dependent HUC cytosol-mediated bindings of [3H]N-OH-ABP to RNA and DNA were 801 and 447 pmol/mg nucleic acid/mg protein. The HUC microsome-mediated bindings of N-OAc-AABP and N-OH-AABP to nucleic acids were inhibited by paraoxon, whereas the cytosol-mediated binding of N-OH-ABP was insensitive to paraoxon inhibition. Chromatography of the DNA hydrolysate obtained from the in vitro incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes showed N-(deoxyguanosine-8-yl)-4-aminobiphenyl as the major adduct, based on comparison with authentic synthetic standard. These results show that human uroepithelia contain microsomal acetyl transferases that are capable of converting the proximate metabolites N-OAc-AABP and N-OH-AABP of the human bladder carcinogen ABP, to reactive electrophiles that bind to DNA. The occurrence of these acetyl transferases in the target organ of the human bladder carcinogen ABP suggests that metabolic activation of some proximate metabolites of ABP could occur directly in HUC and could play a pivotal role in susceptibility to aryl-amine/acetamide induced human bladder cancers.     

Tumorigenic transformation and neoplastic progression of human uroepithelial cells after exposure in vitro to 4-aminobiphenyl or its metabolites.

A multistep in vitro/in vivo transformation system was used to test the transforming effect(s) of the human bladder procarcinogen 4-amino-biphenyl (ABP) and two putative proximate carcinogenic metabolites, N-hydroxy-4-aminobiphenyl (N-OH-ABP) and N-hydroxy-4-acetylamino-biphenyl (N-OH-AABP), on a clonally derived nontumorigenic SV40-immortalized human uroepithelial cell line, SV-HUC. SV-HUC were exposed in vitro to concentrations of ABP, N-OH-ABP, or N-OH-AABP that caused a range of cytotoxicity from 5 to 76%. Tumorigenic transformation of SV-HUC, as assessed by the ability of the exposed cells to form carcinomas when inoculated s.c. into athymic nude mice, was achieved after a single exposure to ABP, N-OH-ABP, or N-OH-AABP. In the tumorigenic transformation experiments, 28 of 45 mice representing all 15 carcinogen-exposed observation groups formed carcinomas, whereas none of 9 mice from control groups formed carcinomas (P = 0.001). Neoplastic progression of a low grade regressive squamous cell carcinoma, MC-T11, was also achieved in this system after in vitro exposure to ABP, N-OH-ABP, or N-OH-AABP. In these progression experiments, 11 of 33 mice representing 7 of 12 carcinogen-exposed observation groups formed persistent, high grade nongressing tumors, while only 1 of 19 untreated MC-T11 controls spontaneously progressed on reinoculation (P = 0.022). Forty independent carcinomas generated in athymic nude mice recapitulated diverse cancer phenotypes (including different growth kinetics and histopathological subtypes and grades) represented in clinical bladder cancers. These results demonstrate for the first time the transforming effects of the potent human carcinogen ABP and two of its proximate N-hydroxy metabolites on a prime human target cell type, HUC.     

Acetyl transferase-mediated metabolic activation of N-hydroxy-4-aminobiphenyl by human uroepithelial cells.

Metabolism and nucleic acid binding of N-hydroxy-4-aminobiphenyl (N-OH-ABP), a proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), was investigated using cultured normal human uroepithelial cells (HUC). HPLC and TLC of the ethyl acetate extract of the media from cultured HUC after 4 h exposure to N-OH-ABP revealed the formation of two major metabolites, ABP and 4-acetylaminobiphenyl (AABP), suggesting the presence of N-acetyl transferase(s) in HUC. This was further confirmed by the formation of AABP, during the incubation of ABP with acetyl coenzyme A (AcCoA) and HUC cytosol. To test whether these enzymes also catalyze the AcCoA-dependent O-acetylation, we examined the metabolic activation of N-OH-ABP using cytosolic preparations. Cytosol from HUC catalyzed AcCoA-dependent binding of [3H]N-OH-ABP to RNA; the amount of binding was 757 pmol/mg RNA/mg protein. Binding with DNA was quantitatively similar to RNA. HPLC and TLC analyses of the enzymatic hydrolysate of [3H]N-OH-ABP-bound DNA revealed the major adduct to be N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the radioactivity in comparison with the authentic synthetic standard. 32P-Post-labeling analysis of the DNA from the cytosol-mediated binding of N-OH-ABP revealed four radioactive spots. In contrast, post-labeling analysis of the DNA from intact HUC exposed to N-OH-ABP showed five adducts, including two of the adducts observed with HUC cytosols, suggesting the possible involvement of additional activation pathway(s) in intact HUC. These results suggest that bioactivation of N-OH-ABP could occur within the HUC, the target organ for ABP, and that cytosolic acetyl transferase(s) may play a critical role in susceptibility to arylamine-induced bladder carcinogenesis.     

Quantitative assessments of the cytotoxicity of bladder carcinogens towards cultured normal human uroepithelial cells

Chemical carcinogens are known to exert cytotoxic effects on cells. The survival of cultured human uroepithelial cells (HUC) after exposure to several important classes of human and experimental animal bladder carcinogens has been quantitatively assessed in vitro using reduction in cell number and/or colony forming efficiency as the endpoint(s). Cells were treated with different carcinogens or various metabolites of a procarcinogen and the responses were analyzed with respect to the cell type used and to the donor source of the cells. The cytotoxic responses of HUC to the stable bladder procarcinogens tested [4-aminobiphenyl (ABP), 4-nitrobiphenyl, N-[4-(5-nitro-2-furyl)-2-thiazole]formamide and 2-amino-4-(5-nitro-2-furyl)thiazole] were dependent on both the concentration of chemical used and the duration of exposure. The survival of HUC after exposure to several metabolites of ABP differed. The N-hydroxylated derivatives of ABP (N-hydroxy-4-amino-biphenyl and N-hydroxy-4-acetylaminobiphenyl) were considerably more cytotoxic toward HUC than ABP or 4-acetylaminobiphenyl. The survival of HUC from different individuals after treatment with the direct acting carcinogen N-nitro-N-methylurea was very similar. In contrast, the survival of HUC from different donors varied considerably after treatment with the procarcinogen 3-methylcholanthrene which requires metabolic activation. However, significant heterogeneity in the survival of HUC from five donors after exposure to the human bladder procarcinogen ABP was not observed in this study. Cultures of normal human fibroblasts from four donors showed an unexpected heterogeneous response to the cytotoxic effects of ABP. These results demonstrate that many variables affect the cytotoxic response of normal cells to bladder carcinogens.     

Microsome-mediated transacetylation and binding of N-hydroxy-4-aminobiphenyl to nucleic acids by hepatic and bladder tissues from dog.

Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes were investigated. HPLC analysis of the ethyl acetate extracts of hepatic microsomal incubates of [3H]ABP in the presence of 4-acetylaminobiphenyl (AABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), or acetyl coenzyme A (AcCoA) as acetyl donors showed the formation of [3H]AABP, suggesting that microsomes catalyze N-acetylation of ABP involving transacetylation. Dog hepatic microsomes also catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of AABP, N-OH-AABP or AcCoA, and the binding was blocked by paraoxon, an inhibitor of microsomal deacetylases. Binding of [3H]N-OH-ABP to DNA was catalyzed also by dog hepatic microsomes, and the extent of binding was 266, 156 and 135 pmol/mg DNA for AABP, N-OH-AABP and AcCoA as acetyl donors respectively. HPLC analyses of the DNA hydrolysates showed that the major adduct formed was N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the adduct in comparison with the synthetic standard. The acetyl adduct N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl was not detected in the DNA hydrolysates. Adduct profiles obtained from 32P-postlabeling of DNA samples from the microsome-mediated binding of [3H]N-OH-ABP showed similarities to the profile obtained previously from the chemical interaction of N-OH-ABP with DNA under acidic conditions, suggesting that the microsome-mediated binding of N-OH-ABP may proceed via formation of aryl nitrenium ions as the ultimate electrophilic species. Microsomes from dog bladder also catalyzed the binding of [3H]N-OH-ABP to RNA and DNA in the presence of AABP, N-OH-AABP or AcCoA as acetyl donors, though the levels of binding were less than those observed with hepatic microsomes. The prevalence of these acetyl transferases in the target organs for ABP and AABP carcinogenesis raises the possibility that metabolic activation of the proximate metabolite N-OH-ABP could occur directly in these tissues and these reactions could play a critical role in the initiation of cancers.     

Induction of DNA repair synthesis in human urothelial cells by the N-hydroxy metabolites of carcinogenic arylamines

Urinary N-hydroxy metabolites of carcinogenic arylamines were investigated for their abilities to induce unscheduled DNA synthesis (UDS) in human urothelial cell lines HCV 29, HU 1734, and HU 1752, and in a primary culture of human urothelial cells. N-Hydroxy-2-aminofluorene (CAS: 53-94-1; N-OH-AF), N-hydroxy-2-acetylaminofluorene (CAS: 53-95-2; N-OH-AAF), and the N-glucuronide of N-OH-AF induced UDS in HCV 29, HU 1734, and HU 1752. N-Hydroxy-4-aminobiphenyl (CAS: 6810-26-0; N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (CAS: 4463-22-3; N-OH-AABP), N-hydroxy-2-aminonaphthalene (CAS: 613-47-8; N-OH-AN), N-hydroxy-2-acetylaminonaphthalene (CAS: 2508-23-8; N-OH-AAN), and the N-glucuronide of N-OH-ABP induced UDS in HCV 29. However, the N-glucuronide of N-OH-AN did not. The O-glucuronide of N-OH-AAF induced UDS in HCV 29 only when beta-glucuronidase was present. Paraoxon inhibited the induction of UDS in HCV 29 by N-OH-AAF and N-acetoxy-2-acetylaminofluorene (CAS: 6098-44-8), but not by N-OH-AF. When examined in a primary culture of human urothelial cells, N-OH-AF, N-OH-AAF, N-OH-ABP, and N-OH-AABP were active, but N-OH-AN, N-OH-AAN, 2-aminonaphthalene (CAS: 91-59-8), 2-aminofluorene (CAS: 153-78-6;), and 4-aminobiphenyl (CAS: 92-67-1) were not. These results demonstrate that human urothelial cells are able to activate both acetylated and non-acetylated N-hydroxy metabolites of carcinogenic arylamines, and they suggest that O-glucuronidation may be a detoxification mechanism for N-arylacethydroxamic acids.     

Two-dimensional electrophoretic protein profile analysis following exposure of human uroepithelial cells to occupational bladder carcinogens

Protein biomarkers to occupational carcinogens were investigated using a transformable human uroepithelial cell system, SV-HUC.PC. SV-HUC.PC was treated with N-hydroxy-4,4'-methylene bis (2-chloroaniline) (N-OH-MOCA) or N-hydroxy-4 aminobiphenyl (N-OH-ABP). Two-dimensional gel electrophoresis of cell lysates compared protein changes across treatments. Increasing N-OH-MOCA resulted in a dose-related increase in protein spots altered. Comparing cell profiles treated with either carcinogen revealed alterations in the expression of nine proteins, identified using the TagIdent database. These demonstrated isoelectric point shift (1) or quantity change (8). Our investigation may be useful in identifying biomarkers of effects of exposure to bladder carcinogens.     

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl by cultured human breast epithelial cell line MCF 10A

Metabolism and nucleic acid binding of the mammary gland carcinogenN-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) was investigatedusing the human mammary epithelial cell line MCF 10A. Chromatographicanalysis of the ethyl acetate extract of the media from culturedMCF 1OA after 24 h exposure to N-OH-AABP revealed the formationof two metabolites, 4-aminobiphenyl (ABP) and 4-acetylamlnobi-phenyl (AABP). Incubation of [³H]N-OH-AABP with calf thymusDNA in the presence of the cytosols or microsomes revealed abinding of 0.21 and 2.36 nmol/mg DNA/mg protein respectively.In contrast to cytosol-mediated binding, the microsome-mediatedbinding of [³H]N-OH-AABP to DNA was inhibited by paraoxon. Furthermore,exogenous addition of non-labelled N-hydroxy-4-aminobiphenyl(N-OH-ABP) to the incubation mixture blocked the binding of[³H]N-OH-AABP to DNA, suggesting that the metabolic activationprocess involves inter-molecular transacetylation. Cytosolsfrom MCF 10A also catalyzed acetyl coenzyme A (AcCoA) dependentbinding of [³H]N-OH-ABP to DNA; the amount of binding was 0.51nmol/mg DNA/mg protein. HPLC of the DNA hydrolysate obtainedafter incubation of [³H]N-OH-AABP and [³H]N-OH-ABP with theMCF 10A microsomes and cytosols showed N-(deoxyguanosin-8-yl)-4-aminobiphenyl(dG-ABP) as the primary adduct, based on the mobility of theradioactive peak in comparison with the synthetic standard.³²P-postlabelllng of adducted DNA obtained on incubation withN-OH-ABP or N-OH-AABP showed similar adduct profiles, with themajor adduct corresponding with the bisphospho derivative ofdG-ABP and a minor adduct corresponding with N-(deoxyadenosin-8-yl)-4-aminobiphenyl(dA-ABP). Additionally, the cellular DNA isolated from MCF 10Afollowing exposure to N-OH-AABP also revealed a major spot correspondingwith the dG-ABP derivative. These results suggest that the mammarygland carcinogen N-OH-AABP is activated to reactive electrophilicspecies in the target human mammary tissues by acetyl transferase(s)enzyme systems.     

Neoplastic progression by EJ/ras at different steps of transformation in vitro of human uroepithelial cells.

The biological effects of expression of mutant ras at different stages of human uroepithelial cell (HUC) tumorigenesis were tested after transfection of EJ/ras into nonestablished HUC and three isogeneic cell lines representing different steps in HUC transformation in vitro. Transfection with EJ/ras failed to immortalize diploid HUC and also failed to cause tumorigenic conversion of a near-diploid SV40-immortalized HUC line (SV-HUC) except at one of six nude mouse inoculation sites. In contrast, EJ/ras-transfected aneuploid low-grade squamous cell carcinoma cells formed undifferentiated, invasive carcinomas at four of six inoculation sites. Furthermore, EJ/ras accelerated tumor growth in MC-ppT11-HA2, an aneuploid high-grade transitional cell carcinoma line, as determined by decreased tumor latent periods and doubling times. These results suggest that EJ/ras contributes to progression, possibly by accelerating tumor growth, but does not in itself cause tumorigenic transformation of uroepithelial cells. To test whether chromosome losses accompanied EJ/ras transformation of SV-HUC, the karyotype of the one SV-HUC tumorigenic transformant obtained (above) was examined. This tumor cell line showed losses of chromosome arms 3p, 10p, 11p, and 18, all of which have been hypothesized to contain genes that suppress cancer development. Therefore, these results also provide new evidence suggesting that genetic losses may be required for mutant ras to contribute to HUC tumorigenic progression.     

Characterization of human uroepithelial cells immortalized in vitro by simian virus 40

Normal human uroepithelial cells (HUC) were transformed with simian virus 40 (SV40) in vitro. SV40-transformed HUC (SV-HUC) were selected by their ability to survive senescence which normally occurs in HUC between passages 4 and 6. At passage 6, 100% of SV-HUC stained positive for SV40 T-antigen. The epithelial nature of SV-HUC was confirmed by positive staining for human cytoplasmic keratins in all cells. SV-HUC have altered growth characteristics compared to HUC including the capacity to grow on plastic, independent of a collagen-gel substrate; loss of the dependence on medium supplements for optimal growth, loss of the dependence on feeder cells for growth at clonal density, and an apparently unlimited lifespan in culture (greater than 2 years). Although SV-HUC have an increased percentage of viable cells and increased saturation density compared to HUC, the generation time of SV-HUC during log phase is similar to that of HUC. Cultures of SV-HUC are epithelial in appearance and show some morphological heterogeneity in cell size and shape. At the ultrastructural level, SV-HUC have numerous alterations such as, irregularly shaped nuclei and nucleoli, pleomorphic microvilli, and the lack of a glycocalyx on the cell surface. In addition, SV-HUC does not stratify in culture, suggesting an inability to differentiate. Unlike HUC, SV-HUC are capable of growth in soft agarose, a property which increased with serial passage. Yet, through at least P50, SV-HUC remained nontumorigenic as determined by the inability to form tumors in athymic nude mice. This cell line of human epithelial origin may be suitable for studying the conversion of cells to tumorigenicity by subsequent treatment with another oncogenic agent.     

Sulfation-dependent formation of N-acetylated and deacetylated DNA adducts of N-hydroxy-4-acetylaminobiphenyl in male rat liver in vivo and in isolated hepatocytes.

Administration of 3H-labeled N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to male Wistar rats with or without prior partial hepatectomy (PH) resulted in covalent binding of 3H activity to liver macromolecules. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before administration of the arylhydroxamic acid strongly decreased the covalent binding. Analysis of aminobiphenyl adducts after TFA hydrolysis of DNA and RNA showed that PCP decreased the formation of both the N-acetylated adduct N-[deoxy)guanosin-8-yl)-4-acetylaminobiphenyl [(d)G-C8-AABP] and the deacetylated adduct N-[deoxy)-guanosin-8-yl)-4-aminobiphenyl [(d)G-C8-ABP] by 60-80%. In incubations with hepatocytes from male Wistar or Sprague-Dawley rats, omission of inorganic sulfate also strongly decreased the covalent binding of 3H-labeled N-OH-AABP to RNA and protein. Analysis of RNA adducts showed a 70-80% decrease in the formation of G-C8-ABP in the absence of sulfate. Another, as yet unidentified, adduct was only slightly decreased. Similar results were obtained with the structurally related carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP). Pretreatment with PCP decreased the incidence of gamma-glutamyltranspeptidase-positive foci in the liver of male rats when analyzed 30 days after a single injection of N-OH-AABP or N-OH-FAABP by 60 and 80% respectively. Thus, both N-acetylated and deacetylated RNA and DNA adducts of N-OH-AABP in rat liver are formed by sulfation and this metabolic activation pathway is responsible for the formation of genotoxic metabolites involved in the generation of preneoplastic cells.     

Altered glycosylation of membrane glycoproteins in human uroepithelial cell lines

Total cellular glycopeptides of 7 human uroepithelial cell lines that differ in the grade of transformation (TGr) were analysed by gel filtration and affinity chromatography on immobilized lectins. The 4 cell lines that are tumorigenic in nude mice and invasive in vitro (TGr III) possess more highly branched, tri- and tetraantennary N-acetyllactosaminic glycans, with less biantennary glycans than the 2 non-tumorigenic, noninvasive (TGr II) cell lines examined. The only exception to this general pattern is the third cell line, which is classified as TGr II. The cellular glycopeptide distribution pattern in this cell line is similar to that of the TGr III cells. The possible relationship between altered glycosylation of membrane glycoproteins and the expression of a malignant phenotype is discussed.     

Frequency of urination and its effects on metabolism, pharmacokinetics, blood hemoglobin adduct formation, and liver and urinary bladder DNA adduct levels in beagle dogs given the carcinogen 4-aminobiphenyl

The human urinary bladder carcinogen, 4-aminobiphenyl (ABP), is known to undergo hepatic metabolism to an N-hydroxy arylamine and its corresponding N-glucuronide. It has been proposed that these metabolites are both transported through the blood via renal filtration to the urinary bladder lumen where acidic pH can facilitate the hydrolysis of the N-glucuronide and enhance the conversion of N-hydroxy-4-aminobiphenyl (N-OH-ABP) to a reactive electrophile that will form covalent adducts with urothelial DNA. Blood ABP-hemoglobin adducts, which have been used to monitor human exposure to ABP, are believed to be formed by reactions within the erythrocyte involving N-OH-ABP that has entered the circulation from the liver or from reabsorption across the urothelium. To test these hypotheses directly, experimental data were obtained from female beagles given [3H]ABP (p.o., i.v., or intraurethrally). [3H]N-OH-ABP (i.v. or intraurethrally), or [3H]N-OH-ABP N-glucuronide (i.v.). Analyses included determinations of total ABP in whole blood and plasma, ABP-hemoglobin adducts in blood erythrocytes, ABP and N-OH-ABP levels (free and N-glucuronide) in urine, urine pH, frequency of urination (controlled by urethral catheter), rates of reabsorption of ABP and N-OH-ABP across the urothelium, and apparent volumes of distribution in the blood/tissue compartment. The major ABP-DNA adduct, N-(guan-8-yl)-4-aminobiphenyl, was also measured in urothelial and liver DNA using a sensitive immunochemical method. An analog/digital hybrid computer was then utilized to construct a multicompartmental pharmacokinetic model for ABP and its metabolites that separates: (a) absorption; (b) hepatic metabolism and distribution in blood and tissues; (c) ABP-hemoglobin adduct formation; (d) hydrolysis and reabsorption in the urinary bladder lumen; and (e) excretion. Using this model, cumulative exposure of the urothelium to free N-OH-ABP was simulated from the experimental data and used to predict ABP-DNA adduct formation in the urothelium. The results indicated that exposure to N-OH-ABP and subsequent ABP-DNA adduct formation are directly dependent on voiding frequency and to a lesser extent on urine pH. This was primarily due to the finding that, after p.o. dosing of ABP to dogs, the major portion of the total N-OH-ABP entering the bladder lumen was free N-OH-ABP (0.7% of the dose), with much lower amounts as the acid-labile N-glucuronide (0.3% of the dose).(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of deoxyadenosine-4-aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis

To characterize the DNA adducts in human uroepithelial cells(HUC) exposed to 4-aminobiphenyl and its proximate N-hydroxymetabolites, we used ³²P-analyses following butanol extractionof the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-aminobiphenyl (pdGp-ABP) as a major adductand N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4- aminobiphenyl(pdAp-ABP) as a minor adduct in an immortalized non-tumorigeniccell line of HUC following exposure to N-hydroxy-4-aminobiphenyl(N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeledthe synthetic N-(deoxyguanosin-3', 5'-bisphospho-8-yl)-4-aminobi-phenyl (dAp-ABP) adduct to generate pdAp-ABP and determinedits chromatographic (TLC and HPLC) proper ties and sensitivityto nuclease P1 digestion. In contrast to pdGp-ABP, which wascleaved to the corresponding 5' monophosphate by nuclease P1,the pdAp-ABP adduct was unaffected when incubated with nucleaseP1 under similar conditions. To test whether nuclease P1 digestioncould be adopted for enrichment of the dAp-ABP adduct in HUCsamples, postlabeling analyses were carried out after but anolextraction following nuclease P1 digestion of the DNA hydrolysate.Under these conditions, the pdAp-ABP adduct was detected inDNA from HUC E7 cells treated with N OH-ABP and in calf thymusDNA reacted with N-OH ABP under acidic (pH 5.0) conditions.These data indicate that pdGp-ABP and pdAp-ABP adducts are generatedin HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichmentmay provide a method for qualitative and quantitative analysesof the pdAp-ABP adduct in DNA.     

32P-Postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA

³²P-Postlabeling analysis of the bisphosphate derivatives wasconducted to characterize the DNA adducts generated from theperoxidase-mediated activation of N-hydroxy-4-acetylaminobiphenyl(N-OH-AABP). Autoradiography of the D1 chromatogram of the postlabeledDNA hydrolysate revealed a major adduct (adduct 1) that migratedat R_f 0.15. An adduct with similar chromatographic characteristicswas also obtained by postlabeling the products generated bychemical interaction of: (i) 2', 6'-dichloro-benzoyloxy-4-acetylaminobiphenylwith the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylamino-biphenyl(N-OAc-AABP) with calf thymus DNA. The adduct derived from chemicalreaction exhibited the same mobilities on two-dimensional TLCas that obtained from the peroxidase-mediated DNA binding ofN-OH-AABP. Moreover, on HPLC analyses, these bisphosphate derivativesexhibited identical retention times, suggesting that structurallythey might be the same. Furthermore, adduct 1 was insensitiveto digestion with nuclease P1. In addition to adduct 1, anotherminor adduct (adduct 2) was also detected in the peroxidase-mediatedDNA binding of N-OH-AABP. The adduct 2 in D1 exhibited an R_fof 0.66. Adduct 2 was also observed in the DNA sample chemicallyinteracted with N-OAc-AABP. Both these adducts retained theacetyl moiety, which was confirmed by the presence of radioactivityin the hydrolysate of DNA derived by interaction with N-OAc-[¹⁴C-acetyl]AABP(labeled at the N-acetyl group). Based on proton NMR and MSanalyses of the 5'-phospho analogs of adducts 1 and 2, the structuresof these have been identified as 3-(deoxyguanosin-N²-yl)-4-acetylaminobiphenyl(dG-N2-AABP) and N-(deoxyguanosin-8-yl)-4-acetylaminobiphenyl(dG-C8-AABP). Analyses of the DNA samples obtained from humanuroepithelial cells following exposure to N-OH-AABP revealedprimarily the non-acetylated derivative N-(deoxyguanosin-8-yl)-4-aminobiphenyl(dG-C8-ABP) with trace amounts of dG-N2-AABP. These resultssuggest that in the target cells for 4-aminobiphenyl carcinogenesis,the prevalence of the peroxidase mediated activation reactionof N-OH-AABP is relatively minor compared to the acetyltransferasepathway.     

Correlation between clastogenicity and promotion activity in liver carcinogenesis by N-hydroxy-2-acetylaminofluorene, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl and N-hydroxy-4-acetylaminobiphenyl

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were compared for their initiation and promotion activity in the rat liver using a modified Solt-Farber system. N-OH-AAF, N-OH-FAABP and N-OH-AABP showed comparable initiation capacity when administered to male Wistar rats at a dose of 30, 120 and 120 mumol/kg respectively, 24 h after a two-thirds partial hepatectomy (PH). In contrast, only N-OH-AAF was very effective as promoter when administered to rats previously initiated with diethylnitrosamine. This was evidenced by a high number of large gamma-glutamyltranspeptidase-positive (GGT+) foci occupying a high percentage (22%) of liver volume. N-OH-FAABP was a much weaker promoter, resulting in smaller foci and lower percentage (4%) of GGT+ liver volume. The incomplete carcinogen N-OH-AABP was totally ineffective as promoter in our model. A similar difference was seen in the clastogenicity of these carcinogens in rat liver in vivo as measured by the formation of micronuclei: N-OH-AAF was far more clastogenic than N-OH-FAABP, which in turn was more clastogenic than N-OH-AABP. We have recently shown that N-acetylated deoxyguanosine adducts are responsible for clastogenicity of N-OH-AAF and may be important for promotion. DNA adduct analysis after injection of 120 mumol/kg of tritium-labeled N-OH-FAABP or N-OH-AABP, 24 h after PH, showed that N-acetylated adducts to C8 of deoxyguanosine are also formed from these structurally related liver carcinogens. However, the formation of these adducts from N-OH-FAABP and N-OH-AABP was approximately 8 and approximately 5% of the formation of dG-C8-AAF after injection of 25 mumol/kg N-OH-AAF. These data show that for the structurally related liver carcinogens N-OH-AAF, N-OH-FAABP and N-OH-AABP, clastogenicity does not predict initiating efficacy but correlates with promotion activity. Possibly, N-acetylated adducts to C8 of deoxyguanosine are involved in both clastogenicity and promotion.     

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.     

Role of SV40 T antigen binding to pRB and p53 in multistep transformation in vitro of human uroepithelial cells

In the present study, we examined the sufficiency of SV40 Tantigen (Tag) binding to pRB and p53 to substitute for alterationsin RB and TP53 at all stages of human uroepi-thelial cell (HUC)transformation in vitro. Two independent SV40 immortalized HUCs(SV-HUC and SV-HUC/CK2) and 17 independent derivative carcinogen-inducedor spontaneous tumors (T-SV-HUCs and T-SV-HUC/CK2) representingdifferent stages of urothelial tumorigenesis were examined.Although five of 17 T-SV-HUCs and SV-HUC/CK2 and its derivativetumor showed 13q chromosome deletion and loss of heterozygosity(LOH), this did not reflect functional loss of pRB because Tag/pRBbinding was unaltered and sequencing showed a normal RB genein all these tumors. No genetic alterations involving 17p orTP53 were detected in any tumors in this study using the sametechniques. These results indicate that Tag/pRB and Tag/p53binding apparently abrogate requirements for/or a selectiveadvantage of RB and TP53 mutations in HUC tumorigenic transformationand progression, as well as in HUC immortalization. These dataalso provide new evidence that more than one suppressor genemay be located on chromosome 13q.     

Mutational signature of the proximate bladder carcinogen N-hydroxy-4-acetylaminobiphenyl: inconsistency with the p53 mutational spectrum in bladder cancer

We studied the mutagenicity of the proximate bladder carcinogen, N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) in embryonic fibroblasts of the Big Blue mouse. Treatment of these cells with increasing concentrations of N-OH-AABP for 24 h resulted in a dose-dependent increase in mutation frequency of the cII transgene up to 12.8-fold over the background. Single base substitutions comprised 86% of the N-OH-AABP-induced mutations and 74% of the spontaneous cII mutations (sequenced number of mutant plaques, 141 and 145, respectively). Of these, 63 and 36%, respectively, occurred at guanine residues along the cII gene. Whereas G to T transversions predominated in the induced cII mutations (47%), insertion was the most spontaneously derived cII mutation (19%). Mapping of N-OH-AABP-induced DNA adducts along the cII gene by terminal transferase-dependent PCR showed the formation of DNA adducts at specific nucleotide positions. Five preferential DNA adduction sites were established, of which four were major mutation sites for N-OH-AABP, especially for G to T transversions. This unique mutational signature of N-OH-AABP in the cII gene was, however, in sharp contrast with the mutational spectrum of the p53 gene in human bladder cancer. G to A transitions are the dominant type of p53 mutations (53%), being also prevalent in almost all of its five mutational hotspots (codons 175, 248, 273, 280, and 285). In addition, the majority of mutations in three of these hotspots (codons 175, 248, and 273) are at a methylated CpG site, whereas in the cII gene neither the preferential N-OH-AABP DNA adduction sites nor the induced mutational hotspots are biased toward methylated CpG dinucleotides. We conclude that N-OH-AABP leaves a characteristic mutational signature in the cII transgene, which is consistent with its preferential DNA adduction profile. However, the pattern of mutation induced by N-OH-AABP in the cII gene is largely at odds with the mutational spectrum of the p53 gene in human bladder cancer.     

N-acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded phi X174 in Escherichia coli.

Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP). The modified DNAs were used as templates for in vitro DNA synthesis. DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types of adducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase. In addition, all three types of adducts completely blocked replication of ss phi X174 in an E. coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage. Polyacrylamide gel electrophoresis of DNA fragments synthesized by E. coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template. However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-trifluoroacetyl-N-acetoxy esters) were frequently bypassed during replication of ss phi X174 in E. coli, though with different efficiencies: 1 out 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation. Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.