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.     

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.     

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.     

Metabolic activation of the N-hydroxy derivative of the carcinogen 4-aminobiphenyl by human tissue sulfotransferases

The role of human sulfotransferase(s) in the bioactivation ofthe N-hydroxy (N-OH) metabolite of the human bladder carcinogen4-aminobiphenyl (ABP) was investigated in vitro with human tissuecytosols. Using an enzymatic assay consisting of a PAPS-regeneratingsystem, [³H]N-OH-ABP, calf thymus DNA and tissue cytosols, thesulfotransferase-mediated metabolic activation of N-OH-ABP wasdetermined as the PAPS-dependent covalent binding of the N-OHsubstrate to DNA. With cytosols prepared from various tissues,we found that the sulfotransferase(s) in human liver, and toa lesser extent colon, can readily metabolize N-OH-ABP. No PAPS-dependentmetabolic activation was detected with cytosols prepared fromhuman pancreas or from the carcinogen target tissue, the urinarybladder epithelium. The N-OH-ABP sulfotransferase activitiesof liver and colon cytosols from different individuals werehighly correlated with their thermostable phenol sulfotransferase(TS-PST) activity (liver, r = 0.99, P < 0.01; colon, r =0.88, P < 0.01), but not with activities for the thermolabilephenol sulfotransferase (TL-PST; liver, r = 0.29; colon, r =0.53), or for the dehydroepiandrosterone sulfotransferase (DHEAST;liver, r = 0.32; colon, negligible activity). N-OH-ABP sulfotransferaseactivity was highly sensitive to inhibition by a selective TS-PSTinhibitor, 2,6-dichloro-4-nitrophenol (IC₅₀ = 0.7µM),and by p-nitrophenol, but was unaffected by competitive inhibitorsof TL-PST (dopamine) or DHEA-ST (DHEA, DHEA-sulfate). The N-OHABPsulfotransferase activity also exhibited thermostability propertiessimilar to that of the TS-PST. From these data, we concludethat human liver TS-PST but not TL-PST or DHEA-ST can metabolicallyactivate the proximate human carcinogen N-OH-ABP to a reactivesulfuric acid ester intermediate that binds covalently to DNA.In addition, in view of the putative role of N-OH-ABP as a majortransport form of the carcinogen to the urinary bladder andof the absence of sulfotransferase activity in this tissue,we hypothesize that sulfotransferase activation in the livermay actually decrease the bioavailability of N-OH-ABP towardextrahepatic tissues and thus serve as an important overalldetoxification mechanism for the urinary bladder.     

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.     

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.     

Effect of ovariectomy on the in vitro and in vivo activation of carcinogenic N-2-fluorenylhydroxamic acids by rat mammary gland and liver

N-Hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and its benzamideanalogue N-OH-2-FBA are mammary gland carcinogens in the femaleSprague—Dawley rat. Ovariectomy inhibits tumorigenicityof topically applied N-OH-2-FAA suggesting modulation of carcinogen-activatingenzymes in the gland. This study concerned the activation ofN-OH-2-FBA and N-OH-2-FBA by the mammary gland and liver, achief site of metabolism, from 50-day-old female rats and effectson the activation of ovariectomy performed at 22 days of age.The levels of N-debenzolyation of N-OH-2-FBA to N-hydroxy-N-2-fluor-enamine(N-OH-2-FA), catalyzed by microsomal carboxyl-esterases in mammarygland and liver were similar and increased 1.5- and 1.7-fold,respectively, by ovariectomy. N-Debenzoylating activity in cytosolsof both tissues appeared to be partially of microsomal origin.Mammary gland cytosol contained N-, O- and N,O-acyltransferaseactivities at levels 40–50% those of liver. N-Acyltransferaseactivity was determined via acetyl coenzyme A (AcCoA)-dependentacetylation of 2-FA and a new assay, N-OH-2-FAA-dependent acetylationof 9-oxo-2-FA. The latter activity was decreased in mammarygland by ovariectomy. Microsomal N-acyltransferase activitieswere <36% those of cytosols. AcCoA-dependent binding of N-OH-2-[ring-³H]FBAto DNA, catalyzed by cytosol, was consistent with a two-stepactivation of N-OH-2-FBA involving esterase catalyzed N-debenzoylationto N-OH-2-FA and its O-acyl-transferase-catalyzed acetylationto the electrophilic N-acetoxy-2-FA. O-Acetyltransfer by mammarygland appeared to be rate-limiting since ovariectomy-dependentincreases in N-debenzoylation did not increase binding withS9 fraction. Little or no sulfotransferase-catalyzed bindingof N-OH-2-[ring-³H]FBA-derived N-OH-2-[ring-³H]FA was detectedin the liver or mammary gland cytosol, respectively. The levelof binding of N-OH-2-[ring-³H]FAA to DNA catalyzed by cytosolicN, O-acyltransferase was decreased 23% in mammary gland andincreased 1.2-fold in liver by ovariectomy. ³²P-Postlabelinganalyses indicated a single adduct N-(deoxyguanosin-8-yl)-2-fluorenaminein DNA of both tissues 24 h after one intraperitoneal injectionof N-OH-2-FBA or N-OH-2-FAA. Respective levels were 3.6- and5.5-fold greater in liver than mammary gland. After ovariectomy,the adduct levels from N-OH-2-FBA increased 1.8-fold in mammarygland and from N-OH-2-FAA decreased 50% in both tissues. Thus,the ovariectomy-dependent changes in levels of enzymes activatingN-OH-2-FBA and N-OH-2-FAA were consistent with in vivo DNA adductlevels in the target mammary gland, but not in the liver.     

32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats

The ³²P-postlabeling technique was used to qualitatively establishthe pattern of DNA adduct formation in mammary tissue and liverfollowing administration of 1-nitropyrene to female Sprague-Dawleyrats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavagein trioctanoin and the rats were sacrificed 24 h later. DNAwas isolated from mammary fat pads and liver, enzymaticallyhydrolyzed to deoxy ribonucleoside-3'-monophosphates and thenconverted to [5'-³²P]3',5'-bisphosphates. The polyethyleneimine-cellulose(PEI-cellulose) TLC ³²P-fingerprints revealed the presence ofmultiple putative adducts in the mammary fat pads and in thelivers. To investigate the role of nitroreduction in the formationof these adducts, calf thymus DNA was incubated with [³H]1-nitropyrenein vitro in the presence of xanthine oxidase. The DNA was isolatedand analyzed by the ³²P-postlabeling technique. A major adductspot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-amino-pyrene.This adduct cochromatographed with a minor in vivo adduct ofDNA obtained from mammary fat pads and livers. However, themajor adducts detected in vivo did not appear to originate fromsimple nitroreduction of 1-nitropyrene. The results of thisstudy suggest that other metabolic pathways, such as ring oxidation,or ring oxidation followed by nitroreduction, may be responsiblefor the putative 1-nitropyrene—DNA adducts observed inmammary fat pads and livers of female Sprague-Dawley rats.     

32P-Postlabeling analysis of DNA adducts in human and rat mammary epithelial cells

The etiology of hwnan breast cancer is currently undefined.However, it has been hypothesized that exposure to chemicalcarcinogens may be an important factor. Extrapolation from rodentmodels for chemically-induced mammary cancer suggests the possibilitythat human mammary epithelial cells insitu might contain DNAadducts due to exposure to environmental chemicals. We havetherefore screened breast epithelial cells from 10 donors forthe existence of DNA adducts using the ³²P-postlabeling assay.In order to validate this analysis technique, we also examinedthe DNA adducts formed in human mammary cells exposed to benzo[a] Pyrene (B[a]P)in vitro, and adducts formed in rat mammaryepithelial cells exposed to B[a]P in vitro and in vivo. Confirmingprevious results using HPLC analysis of [³H]B[a]P-DNA adducts,the major B[a]P-adduct formed by human mammary epithelial cellsin vitro was (+)-anti-B[a]P-7, 8-dihydrodiol-9, 10-epoxide (BPDE):deoxyguanoslne.This adduct did not appear to be formed b rat mammary cellsexposed to B[a]P in vitro. However, ³²P-postlabeling analysisof mammary epithelial cell DNA from rats exposed to B[a]P invivo indicated that (+)-anti-BPDE-deoxyguanosine was a majorB[a]P-DNA adduct under these exposure conditions. When the mammaryepithelial cells from 10 human donors were screened for DNAadducts formed in situ, cells from three donors exhibited distinctadduct patterns. None of these adducts appeared to be (+)-anti-BPDE-deoxyguanosine.The existence of DNA adducts in human mammary epithelial cellsin situ, coupled with the data indicating that rat mammary cellsform different B[a]P adducts in vitro and in situ, suggeststhe need for further study of human breast cell adducts.     

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)     

Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen withbenzo[a]pyrene (BaP) and with (?)-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BaP-7, 8-diol) in mouse skin. The effects of catechol on themetabolic activation of (+)- and (–)- [³H]BaP-7,8-diolsand on epidermal DNA adduct formation of racemic and enantiomeric[³H]BaP-7, 8-diols were examined by applying the tritlated diolsto mouse skin. The major metabolite of the (+)- [³H]BaP-7, 8-diolswas the hydrolysis product of (–)- [³H]-7,8ß-diolsepoxy-9ß,10ß-epoxy-7,8,9, 10-tetrahydroheiizo[a]pyrene (anti-BPDE).This suggests that a peroxyl radical-mediated pathway Is predominantlyresponsible for the epoxidation of this diol. Formation of (–)-anti[³H]BPDEfrom (+)-[³H]BaP-7,8-diol was greater than that of (+)-anti-BPDEfrom (–)-[³H]BaP-7,8-diol Co-application of catechol with[³H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomerto a greater extent than that of the (–) enantiomer. Catecholdecreased the total DNA-binding and the formation of the majoradduct with (+)-[³H]BaP-7, 8-diols metabolites but catecholhad no significant effect on the binding and formation of (+)-anti-[³H]BPDE-deoxyguanosine the major DNA adduct derived from (–)-[³H]BaP-7,8-diolsCo-administration of catechol with (?)-[³H]BaP-7,8-diols increasedthe ratio of (–)- to (+)-[³H]BaP-7, 8-diols major DNAadducts in mouse skin suggesting that catechol selectively inhibitscertain pathways of metabolic activation of (? )-[³H]BaP-7,8-diols Thus, catechol modifies the tumorigenic activity of(?)- BaP-7 ,8.-diol either by alteration of the relative proportionof various hydrocarbon:DNA adducts or by a totally differentas yet unexplored mechanism.     

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.     

Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts

Polycyclic aromatic hydrocarbon (PAH) o-quinones are productsof the dihydrodiol dehydrogenase-catalyzed oxidation of trans-dihydrodiolswhich are proximate carcinogens. The PAH o-quinones are highlyreactive molecules and have the potential to alkylate DNA. Inthis study, the reactivity of [³H](+/–)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene([³H](+/–)-anti-BPDE), [³H]benzo[a]pyrene-7,8-dione ([³H]BPQ)and [³H](+/–)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ([³H](+/–)-B[a]P-diol) with DNA were compared.(+/–)-anti-BPDE reacted equally well with native, deproteinatedand deproteinated/sheared calf thymus DNA. In each case DNAadducts were formed which upon digestion to deoxyribonucleosidescomigrated on reverse-phase (RP)-HPLC with adducts synthesizedby reacting (+/–)-anti-BPDE with oligo-p(dG)₁₀. (+/–)-anti-BPDEalso reacted with plasmid (pGEM-3) DNA to yield multiple adductsone of which comigrated with the (+)-anti-BPDE-deoxyguanosineadduct. Under identical conditions [³H]BPQ reacted preferentiallywith native calf thymus DNA but displayed low reactivity withdeproteinated and deproteinated/sheared calf thymus DNA. RP-HPLCanalysis of deoxyribonucleoside—BPQ adducts indicatedthat the predominant adduct formed comigrated with a standardsynthesized by reacting BPQ with oligo-p(dG)₁₀. BPQ also reactedwith pGEM-3 DNA to yield multiple adducts one of which comigratedwith the BPQ—deoxyguanosine adduct. Reactions between[³H]BPQ and poly(dA), poly(dT), poly(dC) and oligo-p(dG)₁₀ indicatedthat BPQ preferentially formed deoxyguanosine adducts. In thisstudy, [³H]BPQ and [³H](+/–)-anti-BPDE covalently labelednative calf thymus DNA to an equal extent, however, less [³H]BPQwas recovered as deoxyguanosine adducts. By contrast, no covalentmodification of calf thymus DNA, pGEM-3 DNA or oligonucleotideswas observed with [³H](+/–)-B[a]P-diol. These studiesindicate that BPQ has the potential to be genotoxic in vitro;that reactivity is heightened in the presence of protein orcircular DNA and that the major adduct formed is a deoxyguanosineadduct.     

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats

2-Amino-l-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), themost abundant heterocyclic amine in fried beef, is a mammarygland carcinogen in rats. Using the ³²PP-postlabeling method,PhIP-DNA adduct levels were measured in mammary epithelial cellsisolated from female Sprague-Dawely rats given 10 daily dosesof PhIP (75 mg/ kg, p.o.) according to a protocol previouslyshown to induce mammary gland cancer. At 24 h, 48 h, 1 weekand 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels[relative adduct labeling (RAL) x 10⁷, mean ± SD] were10.2 ± 0.7, 7.9 ± 2.7, 2.2 ± 0.6 and 0.9± 0.03 respectively. When isolated rat mammary epithelialcells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP(45 µM, 1 h, 37°C), PhIP-DNA adducts were detectedin cell DNA (RAL =     

DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo

The presence of relatively high levels of prostaglandin H synthase(PHS) in the dog urinary bladder and its ability to mediatethe activation of carcinogenic arylamines to DNA-bound productsin vitro suggests the involvement of this enzyme in arylamine-inducedbladder carcinogenesis. Since the PHS-dependent metabolism of2-naphthylamine (2-NA) had been shown to yield both ring- andN-oxidation products in vitro, we compared the reactivity of³H-labeled N-hydroxy-2-naphthylamine (N-OH-2-NA), 2-nitrosonaphthalene,and 2-amino-1-naphthol (2-AN) toward DNA and protein. In thePHS-incubation system, all three derivatives bound at high levelsto protein, but only N-OH-2-NA and 2-AN bound appreciably toDNA. Though ring-oxidation has usually been considered a detoxificationpathway, the covalent binding of [³H]2-AN to DNA was found tooccur readily under aerobic conditions and was enhanced at acidicpH. At pH 5 in air, the reactivity of [³H]2-AN with nucleicacids and protein was in the order: serum albumin > tRNA> poly G > poly C > DNA > poly A > rRNA >poly U. Enzymatic hydrolysis of DNA reacted with [³H]2-AN andsubsequent analysis by h.p.l.c. indicated the presence of severalcarcinogen-nucleoside adducts. The major product was characterizedas N⁴-(deoxyguanosin-N²-yl)-2-amino-1,4-naphthoquinoneimine;and two minor products were tentatively identified as N⁴-(deoxyadenosin-N⁶-yl)-2-amino-1,4-naphthoquinoneimineand a deoxyguanosin-N²-yl adduct of a naphthoquinoneimine dimer.These adducts accounted for 60% of the total DNA binding obtainedby incubation of [³H]2-NA with PHS in vitro and for 20% of the[³H]2-NA bound to dog urothelial DNA in vivo. The remainingadducts were identical to those previously reported as productsof the reaction of N-OH-2-NA with DNA. These results suggestthat a minor proportion of the DNA adducts found in vivo maybe formed by PHS-activation of 2-NA in the target tissue. Furthermore,the reactivity of 2-AN with cellular nucleophiles, presumablythrough formation of 2-imino-1-naphthoquinone or a protonated4-naphthocarbenium ion, indicates that ring-oxidation productsof arylamines and of other carcinogenic aryl compounds shouldbe evaluated as proximate carcinogenic metabolites.     

DNA adducts of the carcinogen, 15, 16-dihydro-11-methylcyclo-penta(a)phenanthren-17-one, in vivo and in vitro: high pressure liquid chromatographic separation and partial characterization

The 11-methyl derivative (11-methyl ketone) is the most carcinogenicof the series of methylated derivatives based on 15, 16-dihydro-cyclopenta[a]phenanthren-17-one.The nudeoside adducts derived from [³H]-11-methyl ketone-modifiedmouse skin DNA have been separated by both Sephadex LH-20 chromatographyand reverse-phase h.p.l.c and compared to those derived fromDNA modified in vitro with the [¹⁴C]-11-methyl ketone usingrat liver microsomes. The in vivo modified DNA separated togive 6 adducts (designated I- VI) on h.p.l.c. The major in vivoadduct (80% total adducts) co-chromatographed with the majorin vitro adduct. The metabolites of the 11-methyl ketone (designateda-g) have been separated by h.p.l.c, and the adducts derivedfrom each of these individual metabolites determined by furthermetabolism in the presence of DNA. H.p.l.c. separation of theseadducts has allowed characterization of the in vivo adducts.The major adduct (V) and possibly one of the minor adducts (IV)were derived from the 3, 4-dihydro-3, 4-diol of the 11-methylketone (metabolite e). Adducts II and III were derived fromthe 16- and 15-monohydroxylated derivatives of the 11-methylketone and also from their corresponding 3, 4-diols and thereforeare likely to be the 16- and 15-hydroxy derivatives, respectively,of adduct V. Adduct VI, however, although derived from the 15-hydroxy-3,4-diol had a late retention time on h.p.l.c, suggesting eithera non-diol-epoxide adduct or a deoxyadenosine adduct. The useof [³H-G]DNA has established that the major adduct (V) and the16-hydroxy-derived adduct (II) contain deox-yguanosine. Reactionof the carcinogen with [³H-A]poly(dA-dT) gave adduct VI whichwas the only adduct peak shown to contain [³H]deoxyadenosine.     

Metabolism and DNA binding of aflatoxicol and aflatoxin B1 in vivo and in isolated hepatocytes from rainbow trout (Salmo gairdneri)

The purpose of this study was to compare the metabolism andDNA binding of aflatoxicol (AFL) with that of aflatoxin B₁ (AFB₁)in vivo and in isolated hepatocytes from Mt Shasta strain rainbowtrout (Salmo gairdneri). Maximum total binding of [³H]AFL toliver DNA from trout exposed by intraperitoneal injection was38–47% of that of [³H]AFB₁ over a 1–7 day period.The average AFL/AFB₁ DNA binding ratio in 1-h incubations withisolated hepatocytes was 0.67±0.36 (n=13). In freshlyisolated hepatocytes, substantial interconversion between AFB₁and AFL via reductase and dehydrogenase enzymes was observed.Total in vivo excretion of conjugates in bile over 4 days wasgreater for [³H]AFL substrate than for [³H]AFB₁. To determineif AFL binding was due to direct activation or to prior metabolismto AFB₁ followed by activation, AFL with a tritium atom on thecarbon containing the cyclopentenol function ([1-³H]AFL, wassynthesized and incubated with hepatocytes. Binding of [1-³H]AFLwas 3% that of [³H]AFB₁ and represents only direct binding ofthe intact cyclopentenol epoxide molecule before transformationto AFB₁ and consequent loss of ³H. H.p.l.c. analysis of DNAhydrolyzed after incubation with [1-³H]AFL resulted primarilyin production of non-radioactive 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB₁ (AFB₁-N⁷-guanine). A radioactive peak estimated to be 1%as abundant as the AFB₁-N⁷-guanine was also observed. The overallbinding of generally labeled [³H]AFL to trout liver DNA in vivoand in freshly prepared hepatocytes correlates well with availabletumor incidence and mutagenicity data. Conclusions from thesefindings are that direct interaction of AFL-8,9-epoxide withDNA is of relatively minor quantitative importance in rainbowtrout hepatocytes and that the major adduct results from conversionof AFL to AFB₁ prior to epoxide formation.     

Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice

Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) is a carcinogenmetabolized by hepatic cytochrome P4501A (P4501A). This studyshowed that there was a highly selective solvent-resistant bindingof radioactive substance in endothelial cells of heart and kidney1 day following injection of [³H]-Try-P-1 (0.1 or 1.5 mg/kg)in NMRI mice treated with the P450-inducing agent ß-naphthoflavone(BNF). In the heart, the binding was highest in capillariesand coronary vessels. In the kidney, the binding was highestin afferent and efferent arterioles and glomerular and peritubularcapillaries. A corresponding localization of radioactivity didnot occur in corn oil-treated mice injected with [³H]-Trp-P-1.On incubation of heart and kidney slices with [³H]-Trp-P-1,there was a binding of radioactivity in endothelial cells ofBNF-treated mice, but not in corn oil-treated mice. The P4501Ainhibitor effipticine abolished the BNF-induced endothelialbinding of [³H]-Trp-P-1 in vivo and in vitro, whereas the effectsof     

Evidence for N-(deoxyguanosin-8-yl)-1-aminopyrene as a major DNA adduct in female rats treated with 1-nitropyrene

[³H]1-Nitropyrene was administered at a dose of 25 mg/kg byi.p. injection to female Wistar rats. Animals were killed 24hlater and DNA was isolated from kidney, liver and mammary gland,enzymically hydrolysed and analysed by reversephase h.p.l.c.A major adduct peak was detected in DNA from each of the threeorgans. Enzymic hydrolysates of DNA, which had been reactedin vitro with 1-nitropyrene in the presence of xanthine oxidase,were similarly analysed by h.p.l.c. One major adduct peak wasobtained which had the same retention time as the in vivo product.Confirmatory evidence that the in vivo adduct and the in vitroadduct were structurally similar was obtained from the determinationof the pH-dependent solvent partitioning profiles. Further,treatment of the in vivo adduct from liver, kidney or mammarygland DNA hydrolysates and the in vitro adduct with sodium hydroxideresulted in the formation of a more polar product which elutedearlier on h.p.l.c. This behaviour is consistent with scissionof the imidazole ring of a deoxyguanosine adduct.The major DNAadduct formed in vitro following xanthine oxidase reductionof 1-nitropyrene has previously been identified by others asN-(deoxyguanosin-8-yl)-1-amino-pyrene. The present data suggestthat the in vivo 1-nitro-pyrene-DNA adduct has the same structure.     

Short Communication: Interaction of aflatoxin B2 with rat liver DNA and histones in vivo

The interaction of aflatoxin B₂ (AFB₂) in vivo with rat livernuclear macromolecules was examined in an attempt to correlatethis binding with biological potency. The incorporation of [³H]AFB₂residues into rat liver his tones and DNA was determined 2,24 and 48 h following administration of a single i.p. dose of1 mg (³H)AFB₂/kg body weight. At each time point, hist one H1and the total histone fraction contained 5–30-fold more[³H]AFB₂ moieties than did DNA on a weight basis. Analyticalreversed-phase h.p.l.c. of the acid hydrolysis products resultingfrom AFB² binding to DNA revealed that 85% of the radioactivityco-chromatographed with the major aflatoxin B₁-DNA adduct, 2,3-dinydro-2-(N⁷-guanyl)-3-hydroxyaftatoxinB₁. These studies revealed an apparent correlation between AFB₂derived binding to DNA in vivo in rats and its potency as atoxin and carcinogen in this species.