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.     

Detection of N-(deoxyguanosin-8-yl)-2-fluorenamine in DNA of peritoneal serosa and liver after intraperitoneal exposure of rats to N-hydroxy-iy-2-fluorenylbenzamide or yV-hydroxy-N-2-fluorenylacetamide

DNA adduct formation was examined in rat peritoneal serosa,a tumor target for i.p. administered aqueous suspensions ofW-hydroxy-N-fluorenylbenzamide (N-OH-2-FBA) and N-hydroxy-W-2-fluorenylacetamide(N-OH-2-FAA), and compared to that in the liver, which is atumor target for W-OH-2-FAA in the male rat ³²P-Postlabelinganalyses showed the presence of a single adduct, N-(deoxyguanosin-8-yl)-2-fluorenamine(dG-C8-FA), from activation of both hydroxamic acids by theserosa and liver in vitro and in vivo. The relatively low levelsof dG-C8-FA (60–80 fmol/(µg DNA) from N-OH-2-FBAin vitro were increased 2.7- and 35-fold upon the addition ofacetyl coenzyme A (AcCoA) to the serosal cytosol and hepaticcytosol or microsomes respectively. By contrast, addition ofAcCoA led to a decrease (34%) in the high level of dG-C8-FA(4330 fmol/µg DNA) from activation of N-OH-2-FAA by hepaticcytosol and did not alter the levels from activation by hepaticmicrosomes and serosal cytosols (530 and 783 fmol/µg DNArespectively). These data and the previously reported hydroxamicacid activation enzyme activities in the serosa and liver indicatedthat the precursor of dG-C8-FA, Nacetoxy-Af-2-fluorenamine,was formed from N-OH-2-FAA chiefly via an intramolecular N,O-acetyltransferand from N-OH-2-FBA via a two-step sequence of W-debenzoylationand AcCoA-dependent O-acetylation. The levels of dG-C8-FA were2- to 3-fold higher in the serosal DNA (up to 515 and 1012 fmol/µgDNA) after one (30 (imol/rat) and ten or eleven (cumulativedose of 275 (imol/rat) injections of NOH-2-FBA or N-OH-2-FAAthan in the hepatic DNA. This correlated with the carcinogenicitiesof the hydroxamic acids, but was inversely proportional to therates and extents of their activation in vitro. Multiple injectionsaffected hepatic enzyme activities related to the activationof the hydroxamic acids in that the cytosolic W-debenzoylationof N-OH-2-FBA increased (1.7-fold) whereas NOH-2-FAA acetyltransferaseand sulfotransferase activities decreased. The effect of treatmentwith N-OH-2-FBA was greater than that with N-OH-2-FAA and wasgreater on the sulfotransferase activity (88% decrease). Thelatter suggested that N-OH-2-FBA, although a poor acceptor foran enzymatic sulfate transfer, may be carcinogenic for the ratliver. This study provides first evidence on the DNA adductformation in vitro and in vivo from N-OH-2-FBA in the peritonealserosa and liver and from N-OH-2-FAA in the serosa.     

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide(N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes(PMNL), chiefly neutrophils, elicited with intraperitoneal injectionsof proteose peptone, was examined. At 10⁶ PMNL/ml in media containinghalide (X^–), 0.14 M Cl^– ± 0.1 mM Br^–(without Ca⁺⁺ and Mg⁺⁺), addition of 10 nM phorbol myristateacetate (PMA) resulted in generation of superoxide anion andH₂O₂. Subsequent cetyltrimethylammonium Cl^– (Cetac) additionat 0.002% effected myeloperoxidase (MPO) activity release. PMNLtreated with PMA and/or Cetac did not metabolize N-OH-2-FAA(30 µM). However, 1–2 pulses of H₂O₂ (50 µM)after Cetac addition resulted in oxidation of N-OH-2-FAA toN-acetoxy-2-FAA (<0.5 µM) and 2-nitrosofluorene (2-NOF)(1–2 µM). In the presence of Br^– 2-NOF wasincreased (3–5 µM). The results are consistent withoxidation of N-OH-2-FAA by MPO/H₂O₂ and MPO/H₂O₂/X^– viatwo pathways: one electron oxidation leading to N-acetoxy-2-FAAand 2-NOF, and X^–-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA(10 µM) incubated with PMNL under similar conditions wasconverted non-enzymatically to 4-OH-2-FAA (5 µM) and enzymaticallyto N-OH-2-FAA (3 µM). In the presence of H₂O₂, smalleramounts of these products were formed. Formation of N-OH-2-FAAwas prevented by paraoxon (0.1 mM) suggesting O-deacetylaseactivity. However, accountability for N-acetoxy-2-FAA decreasedwith time, presumably because of binding to cellular macromolecules.With H₂O₂ addition, 2-NOF (10 µM) was converted to 0.5or 0.25 µM 2-nitrofluorene by active PMNL or heat-inactivatedcell lysates, respectively. Low recoveries of 2-NOF were alsoattributed to binding. The results suggest that PMNL may beinvolved in activation of the carcinogenic N-arylhydroxamicacids in vivo.     

Interaction of the synthetic ultimate carcinogens, N-sulfonoxy- and N-acetoxy-2-acetylaminofluorene, and of enzymatically activated N-hydroxy-2-acetylaminofluorene with nucleophiles

The interaction of four cellular nucleophiles with the putativeultimate carcinogens N-sulfonoxy-2-[ring-³H]acetylamino-fluorene(N-sulfonoxy-2-AAF) and N-acetoxy-2-[ring-³H] acetylaminofluorene(N-acetoxy-2-AAF), and with N-hydroxy-2-[ring-³H]acetylaminofluorene(N-hydroxy-2-AAF) activated to the ultimate carcinogens by enzymaticsulfonation or trans-acetylation was determined. The adductswere isolated and adduct formation was quantified by isotopedilution. The order of nucleophilicity of the acceptors wasguanosine > tRNA polyguanylic acid (poly G) > N-acetyl-L-methioninewhen N-sulfonoxy-2-AAF, N-acetoxy-2-AAF or N-hydroxy-2- AAFactivated by transacetylation were the electrophiles. In thecase of N-hydroxy-2-AAF activated by enzymatic sulfonation,the order of nucleophilicity was N-acetyl-L-methionine >guanosine tRNA > poly G. The increase in the reactivityof N-acetyl-L-methionine is hypothesized to be due to cytosolicenzyme(s) which facilitate transfer of the methionine residuefrom the nitrogen to carbon atoms 3 and 1 of the fluorene moiety.Of the two synthetic esters, N-sulfonoxy-2- AAF exhibited greaterelectrophilicity than N-acetoxy-2-AAF. The rate of adduct formationof N-sulfonoxy-2-AAF and of N-acetoxy-2-AAF with each nucleophilewas a function of nucleophile concentration, indicative of abimolecular reaction mechanism. The interaction is thought toinvolve attack of the nucleophile on the uncharged ultimatecarcinogen, although interaction with an ion pair cannot beeliminated. The mutagenicity of N-sulfonoxy-2-AAF, N-acetoxy-2-AAFand of enzymatically activated N-hydroxy-2-AAF was evaluatedby the Ames test. N-Sulfonoxy-2-AAF was virtually inactive,while N-acetoxy-2-AAF exhibited weak mutagenicity. N-Hydroxy-2-AAFactivated by enzymatic sulfonation exhibited greater mutagenicitythan synthetic N-sulfonoxy-2-AAF. The mutagenicity and reactivityof ultimate carcinogens derived from N-hydroxy-2-AAF by enzymaticactivation do not necessarily coincide with the mutagenicityand reactivity of the synthetic ultimate carcinogens.     

Sulfation of aromatic hydroxamic acids and hydroxylamines by multiple forms of human liver sulfotransferases

Sulfation activity towards various heterocyclilc and homocyclicaromatic hydroxamic acids and hydroxylainines was determinedin adult human liver cytosol and with partially purified humanliver sulfofransferases (STs). In adult human liver cytosolscomparable ST activities towards N-hydroxy-2-acetyl-amino-5-phenylpyridine(N-OH-2AAPP), N-hydroxy-4-acetyl-aminobiphenyl (N-OH-4AABP)and N-hydroxy-4'fluoro-4-acetylaminobiphenyl (N-OH-4FAABP) werefound, while the sulfation rates towards N-hydroxy-2-acetylaminofluorene(N-OH-2AA N-hydroxy-2-acetylaminonaphthalene (N-OH 2AAN), N-hydroxy-2-acetylamlnophenanthrene(N-OH-2AAP) and N-hydroxy-4-acetylaminostilbene (N-OH-4AAS)were two- to five-fold lower. In adult liver cytosol ST activitywas found towards all hydroxylamines tested. No significantdifferences were found for the various hydroxylainines. In general,the ST activities towards the various hydroxamic acids and hydroxylamineswere comparable to phenol ST activity using adult liver cytosols.Partial purification of adult human liver STs was achieved byDEAE-Sepharose chromatography followed by anion exchange FPLC.Two separated protein peaks showing both N-OH-2AAPP and N-OH-2APPST activities were observed and were designated human hydroxylamine/hydroxamicacid sulfotransferase (hHST) 1 and 2. Iimmunoblot analysis usingan anti-rat estrogen ST antibody demonstrated cross reactivitywith both hHSTs at a subunit mol. wt of 32 kDa correspondingto the phenol-sulfating form of phenol ST (P-PS ST activitytowards dopamine was low with both hHSTs, but hHST1 also containedsignificant capacity to sulfate dehydroepiandrosterone. Thehighest ST activity towards N-OH-2AAPP and N-OH-2APP was measuredat pH 5.5 with both hHSTs. The K_m values of the two hHSTs forsulfation of N-OH-2AAPP     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide

We determined ring- and N-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 1-hydroxy-2-FAA was induced. In mammary gland microsomes,the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased,but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

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.     

Autoradiographic studies on in vivo covalent binding of N-hydroxy-2-acetylaminofluorene in rat liver containing {gamma}-glutamyltranspeptidase-positive foci. The effect of the sulfation-inhibitor pentachlorophenol

The role of sulfation in the covalent binding of [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) in rat liver containing -glutamyltranspeptldase-positive(GGT⁺) foci was studied in vivo by autoradiography. GGT⁺ fociwere induced by initiation with diethylnitrosamine, followedby a selection protocol consisting of a 2-week exposure to 2-aminofluorenein the drinking water and a single administration of CCl₄. Bothsurrounding (‘normal’) liver tissue and, to a lesserextent, GGT⁺ foci covalently bound N-OH-AAF, administered 10days after selection. The sulfation inhibitor, pentachlorophenol(PCP), reduced the covalent binding of N-OH-AAF in cells surroundingthe foci. However, PCP had no effect on binding of radiolabelin GGT⁺ foci. Thus, reduced sulfotransferase activity may contributeto the resistance of GGT⁺ preneoplastic lesions to carcinogencytotoxicity. The results suggest also, that cells in GGT⁺ fociare able to bind N-OH-AAF covalently by a sulfotransferase-independentpathway.     

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.     

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases(NAT1^* and NAT2^*) from rat and hamster were cloned and expressedin Escherichia coli. Active clones were selected by a simplevisual test for their ability to decolorize 4-aminoazobenzenein bacterial medium by acetylation. These recombinant acetyltransferaseswere analyzed for: (i) N-acetyltransferase, which was assayedby the rate of acetyl coenzyme A-dependent N-acetylation of2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamicacid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene.Both NAT2s showed first order increases in N-acetylation rateswith increasing 2-AF or AAB concentrations between 5 and 100µM, with apparent K_m values of 22–32 and 62–138µM respectively. Although under the same conditions theN-acetylation rates for the two NAT1s declined by >50%, below5 µM 2-AF or AAB, the NAT rate data fit Michaelis-MentenKinetics, and the apparent K_m values were 0.2–0.9 µM.For N, O-acetyltransferase, the apparent K_m values of the NAT1swere 6 µM, while the K_m values of the NAT2s were 20- to70-fold higher. SDS-PAGE/Western blot analysis of the recombinantacetyltransferases gave apparent relative molecular weights(MW_r) of 31 kDa for both NAT1s and rat NAT2 and 29 kDa for hamsterNAT2. Comparable MW_r values were observed for native hamsterliver NAT1 and NAT2 and for rat NAT1 under the same conditions.Although we did not detect NAT2-like activity in rat liver cytosolpreviously, the present data show that the rat NAT2^* gene doescode for a functional acetyltransferase, with properties similarto those of hamster liver NAT2. The data also indicate thatat low substrate concentrations, NAT1 would apparently playthe predominant role in vivo in N-acetylation and N, O-acyltransferof aromatic amine derivatives, including their metabolic activationto DNA-reactive agents.     

Detection of cyclic 1, N2-propanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde

Cyclic 1, N²-propanodeoxyguanosine adducts are formed in vitroin DNA treated with -acetoxy-N-nitrosopyrrolidine or its metabolite,crotonaldehyde. However, the in vivo formation of these cyclicadducts in DNA has not been demonstrated due to the lack ofa sensitive detection method. In this study, a ³²P-postlabelingmethod specific for the detection of 1, N²-propanodeoxyguanosineadducts was developed by using the corresponding 3'-monophosphatesas standards. This method was validated by using DNA modifiedin vitro. It was then applied for the in vivo experiments inwhich hepatic DNA of rats treated with N-nitosopyrrolidine (NPYR)(total dose, 1.0 mmol) in drinking water or skin DNA of Sencarmice treated topically with crotonaldehyde (1.4 mmol) was isolatedand subjected to ³²P-postlabeling analysis. 1, N²-Propanodeoxyguanosineadducts were detected in these DNA samples. The minimal levelsof adducts from liver DNA and skin DNA detected were estimatedto be 0.06 and 0.24 µmol/mol guanine respectively. Interestingly,a background adduct spot chromatographically indistinguishablefrom the 1, N²-cyclic adducts was observed in the liver DNAof untreated rats. However, no such background adduct was detectedin skin DNA of mice. This method demonstrated for the firsttime the in vivo formation of the cyclic 1, N²-propanodeoxyguanosineadducts.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. II. Glucuronidation of ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide

We determined UDP-glucuronyltransferse (UDP-GT) activities ofhepatic and mammary gland microsomes of female rats with p-nitrophenoland the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamlde(2-FAA) and the effects of hepatic inducers of UDP-GT's on theseglucuronidations. Pre-treatment of non-lactating (NL)and lactating(L) rats with ß-naphthoflavone (ß-NF) significantlyincreased glucuronidations, of p-nitrophenil, aphenolic metabolitesof 2-FAA, especially of 5-hydroxy 2-FAA and also of N-hydrosy-2-FAAby hepatic microsomes. Pre-treatment of L rats with ß-NFor 3-methyl cholanthrene (3-MC) significantly incresed glucuronidationsof these compounds by mammary gland microsomes suggesting thatboth liver and mammary gland of L rats possess similar UDP-GTactivities. In NL rats, UDP-GT activities of mammary microsomestoward phenols were greater than in L rats, and except for thatof 5- and 7- hydroxy-2-FAA, were not inducible with ß-NF.The data obtained with L rats, the greater magnitude of stimulationof the hepatic UDP-GT of NL rats by ß-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT ofmammary microsomes suggested that the phenolic metab olitesof 2-FAA and N-hydroxy-2-FAA share chiefly the characteristicsof substrates for group 1 UDP-GT activities (i.e., those induciblewith ß-NF or 3-MC). Neither inducer increased glucuronidationof 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT ofmammary or hepatic micro somes. In contrast to hepatic microsomeswhich formed considerable amounts of the glucuronide of N-hydroxy-2-FAA,mammary gland microsomes glucuronidated this substrate to aminor extent only. This suggested that glu curonide of N-hydroxy-2-FAAmay play a role in systemic, but not in local mammary tumorigenesisby N-hydroxy-2-FAA.     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

Formation and persistence of O6-(2-hydroxyethyl)-2'-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O⁶-(2-hydroxyethyl)-2'-deoxyguanosine(O⁶-HEdG) were used to develop a highly sensitive immuno-slot-blotassay for this promutagenic base which enabled the quantitationof 3.6 µmol O⁶-HEdG/mol deoxy-guanosine, correspondingto 5 fmol in a 3-µg DNA sample. This assay was used tostudy DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea(HENU) in adult male F344 rats. Initial amounts of O⁶-HEdG 2h after a single i.v. dose of 50 mg/kg were highest in kidney(81 µmol O⁶HEdG/mol deoxyguanosine), followed by lungand liver (67 and 55 µmol/mol dG respectively). Formationof O⁶-HEdG in cerebral DNA was considerably lower (18 µmolO⁶-HEdG/mol deoxyguanosine), probably reflecting delayed crossingof the blood—brain barrier by HENU due to its hydrophilicity.The formation of O⁶-HEdG in liver and kidney was strictly proportionalto dose over a range of 5–50 mg HENU/kg. Repair of O⁶-HEdGwas very rapid in liver (apparent half-life, 12 h), and somewhatslower in kidney and lung (approximate half-life, 40 h and 48h respectively). In contrast, 62% of the initial amount of O⁶-HEdGin cerebral DNA was still present after 7 days. Saturation ofthe hepatic O⁶-alkyl-guanine-DNA alkyltransferase by pretreatmentwith N-nitrosodimethylamine (20 mg/kg) almost completely inhibitedthe removal of O⁶-HEdG, indicating that O⁶-HEdG is predominantlyrepaired by this repair enzyme.     

Metabolism of 4,4'-methylene-bis-2-chloroaniline (MOCA) by rats in vivo and formation of N-hydroxy MOCA by rat and human liver microsomes

The metabolism of 4,4'-methylene-bis-2-chloroaniline (MOCA)was investigated because it is an animal carcinogen to whichhumans have been exposed. In CD rats, where MOCA is a hepatocarcinogen,0.2% of an oral dose of [¹⁴ C]MOCA was recovered unchanged inthe urine; enzymatic hydrolysis and extraction of urinary radioactivityindicated the presence of glucuronide and sulfate conjugates.In rat bile, the predominant metabolite was N-glucuronyl MOCA.Liver microsomes from male CD rats or human males (surgicalspecimens) were incubated in vitro with [¹⁴C] MOCA. Metaboliteformation, which was dependent upon reduced pyridine nucleotidesand intact microsomes, was quantitated by TLC and HPLC usingappropriate chemically synthesized standards. ^N-Hydroxylationof MOCA occurred at a rate of 335 ? 119 pmol/min/mg rat microsomalprotein (n=3) versus 230 or 765 (n=2) with microsomes from humans;the product was identified by isotopic dilution for both species.The rates of 5-hydroxy-MOCA (o-aminophenol) formation were 92? 33 (rats) and 7, 35 (human); rates for the benzhydrol derivativewere 82 ? 12 (rats) and 60, 160 (human). In rats, all threerates were elevated 4- to 8-fold by pretreatment with phenobarbital,which also enhanced the formation of partially characterizedpolar derivatives that appeared to result from oxidation andcleavage at the methylene carbon. The latter pathway typicallyamounted to 50–100% of the 4,4'-diamino-3,'-dichlorobenzhydrolvalue in control or pretreated animals. Thus, rats metabolizeMOCA extensively and the pathways include N-hydroxlation, whichis regarded as an obligatory step in metabolic activation ofarylamines. The presence of MOCA N-hydroxylase in human liversupports the hypothesis that exposure of humans to MOCA entailsa carcinogenic risk.     

Free radicals in arylamine carcinogenesis

Free radical processes and their involvement in carcinogenesis is an unresolved question but one subjected to intense investigation recently. Using in vitro systems, we demonstrated that certain heme compounds combined with hydroperoxides catalyzed the oxidation of N-hydroxy-2-fluorenylacetamide (N-OH-2-FAA) to nitroxyl free radical intermediate which dismutated to form 2-nitrosofluorene (2-NF) and N-acetoxy-2-fluorenylacetamide (N-AcO-2-FAA). Ascorbate and certain antioxidants inhibited this reaction. Lipid hydroperoxides were effective substrates for this reaction, especially in target tissue, rat mammary gland parenchymal cells. Cytochrome P420 in the high-spin state catalyzed the reaction effectively but low-spin cytochrome P420 or P450 were ineffective. Recently, we found that 2-NF added covalently to unsaturated carbon-carbon double bonds of membrane lipids to form an adduct termed 2-nitroso-fluorene lipid adduct (N-O-LAF), which, in it oxidized state, exists in the membrane as a nitroxyl free radical. This N-O-LAF undergoes reduction-oxidation changes in the natural membrane. Formation of N-O-LAF occurred in rat liver microsomal membranes by deacylation of N-OH-2-FAA, but the esterase inhibitor paraoxon, prevented its formation from N-OH-2-FAA. The mutagenicity of 2-NF was enhanced in Salmonella typhimurium TA98 if the bacteria were cultured to contain more unsaturated membrane lipids. However, synthesized adducts were only slightly mutagenic.     

N- and O-deacetylation of N-acetoxy-N-arylacetamides by mammalian hepatic microsomes

The present study demonstrates that hepatic microsomes fromguinea pigs, rats, mice, hamsters, rabbits and dogs catalyzeN-deacetylation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF)and N-hydroxy-3, 2'-dimethyl-4-acetylaminobiphenyl and O-deacetylationof N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) and N-acetoxy-3,2'-dimethyl-4-acetylamino-biphenyl(N-AcO-DMAABP). Gel filtrationresolves el filtration resolves the solu-bilizedolu-bilizedguinea pig microsomal enzymes into two deacetylases. The largermolecular weight enzyme catalyzes N-deacetylation of N-OH-AAF,whereas the smaller one cannot. Both enzymes catalyze O-deacetylationof N-AcO-AAF and N-AcO-DMAABP, but the activity is mainly dueto the larger enzyme. Guinea pig and rat liver microsomes alsocatalyze N-deacety-lation of N-AcO-AAF resulting in the bindingof 2-amino-fluorene to nucleic acids, but the activities aremuch less than that of the O-deacetylation of N-AcO-AAF. Thus,the amino-fluorene-DNA adducts which have been found in intactcells treated with N-AcO-AAF may result directly from N-deacety-lationor indirectly from N,O-acetyltransfer following O-deacetylation.     

Enzymatic acetylation and sulfation of N-hydroxyarylamines in bacteria and rat livers

In mammalian hepatic cytosol both acetyltransferase and sulfotransferaseare involved in the activation of N-hydroxy derivatives of arylaminesand arylamides. The role of acetyltransferase is also shownin Salmonella, whereas no rigid evidence- is provided on therole of sulfotransferase in Salmonella. In Ames mutagenesistest without S9-mix, the number of revertants of Salmonellatyphimurium TA98 induced was 10-fold higher with 2-hydroxyamino-3-methyl-imidazo[4,5-f]quinoune(N-hydroxy-IQ) than with 2-hydroxy-amino-6-niethvldipyrido[l,2-a:3',2'-d]imidazole(N-hydroxy-Glu-P-1). The extents of the binding to calf thymusDNA of N-hydroxy-Glu-P-1 were, however, 3.9 to 8.6-fold higherthan that of N-hydroxy-IQ in both acetyl CoA- and PAPS-fortifiedrat hepatic cytosol systems. To understand the mechanism causingthe apparent discrepancy between the results of the mutationand DNA binding, the activating capacities of cytosols of S.typhimuriumTA98 and TA98/1,8-DNP₆ strains on the binding of N-hydroxy-Ghu-P-1and N-hydroxy-IQ have been examined in comparison with thoseof rat livers. Although both N-hydroxyarylamines were activatedby hepatic cytosols in the presence of PAPS, no significantDNA binding of these N-hydroxyarylamines was detected in thepresence of PAPS and either one of the two strains of bacterialcytosols. In addition, both cytosols of TA98 and TA98/1,8-DNP₆strains showed no measurable activity on the sulfation of p-nitrophenol,suggesting no capacity for sulfotransferase-mediated activationof N-hydroxyarylamines in Salmonella. On the contrary, the extentsof the acetyl CoA-dependent binding of N-hydroxy-IQ in cytosolsof TA98, but not of TA98/1,8-DNP₆, were respectively 6- andWold higher than those in hepatic cytosols of male and femalerats, although the extents of the binding of N-hydroxy-Glu-P-1were rather higher in hepatic than in bacterial cytosols. Inaddition, the covalent binding of N-hydroxy-2-acetylaminofhioreneto DNA was detected in hepatic, but not in bacterial cytosob,although the binding of N-hydroxy-2-aminofluorene was detectablein both hepatic and bacterial cytosols in the presence of acetylCoA. These results indicate that the metabolic activating capacitiesof Salmonella and rat liver cytosols differ qualitatively, andthe difference in the substrate specificity of acetyltransferasebetween Salmonella and rat livers may be involved, in part,in the difference of then- DNA damage in bacteria and mammals.     

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder

N-Acetoxyarylamines are reactive metabolites that are implicatedin the initiation of the carcinogenic process by some N-substitutedaryl compounds. The objective of this study was to explore therelationship between the production of these reactive speciesand N-acetylation (NAT), a reaction previously demonstratedto be polymorphic in the human. Human liver and urinary bladdermucosa samples were frozen within 4–8 h post mortem. Thesetissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-di-methyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii)intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene(N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoicacid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF.Cytosolic AHAT and OAT showed partial inhibition by paraoxon.The ratio of paraoxon insensitive AHAT to OAT to NAT of PABAto NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosolsfrom frozen livers. Freezing of the cytosol resulted in extensiveloss of activities. All four of these cytosolic enzyme activitiesexhibited a similar polymorphic response. Microsomal deacetylationshowed a monomorphic response. Similar to the liver, urinarybladder epithelial cells also catalyzed the same reactions.However, the OAT and AHAT activities were detected mainly inmicrosomes. These data suggest that phenotypically rapid acetylatorshave a greater biochemical potential for the metabolic activationof aromatic amines by pathways that involve O-acetylation.     

Dibenz[a,j]acridine: distributions of metabolites formed by liver and lung microsomes from control and pretreated rats

The structures of many dibenz[a,j]acridine (DBAJAC) metabolitesformed in vitro in incubations with liver microsomes preparedfrom 3-methyicholanthrene-pretreated male Wistar rats have previouslybeen determined; they were trans-DBAJAC-3,4-dihydrodiol, trans-DBAJAC-5,6-dihydro-diol,DBAJAC-5,6-oxide, 3-hydroxy-DBAJAC, 4-hydroxy-DRAJAC and severalmultiply oxidized secondary metab olites. Herein are reported[14³]DBAJAC metabolite distributions obtained by h.p.l.c. separationof products pro duced in incubations with liver and lung microsomesprepared from untreated, phenobarbital-pretreated and 3-methyl-cholanthrene-pretreatedmale Wistar rats. Liver microsomal metabolites were also quantitatedin preparations from trans stilbene oxide-pretreated rats. Forall preparations trans DBAJAC-3,4-dihydrodiol, the candidateproximate car cinogen according to the bay-region theory ofcarcinogenesis, was the major metabolite (30–40%) whileDBAJAC-5,6-oxide and phenols were also quantitatively important.In incuba tions conducted in the presence of 3,3,3-trichloropropene-1,2-oxide (1.5 mM) formation of dihydrodiol was inthibited byabout 85%. DBAJAC-N-oxide was also identified as a minor metabolite(1%) formed in incubations with pheno barbital-induced and controlliver microsomes.