Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

Rabbit N-acetyltransferase 2 genotyping method to investigate role of acetylation polymorphism on N- and O-acetylation of aromatic and heterocyclic amine carcinogens.

The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 (NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine (p = 0.0014), benzidine (p = 0.0257), 4-aminobiphenyl (p = 0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p = 0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly (p > 0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens.

Relative Contributions of CYP1A2 and CYP2E1 to the Bioactivation and Clearance of 4-Aminobiphenyl in Adult Mice.

4-Aminobiphenyl (ABP), a prototypical aromatic amine carcinogen in rodents and humans, requires bioactivation to manifest its toxic effects. A traditional model of ABP bioactivation, based on in vitro enzyme kinetic evidence, had postulated initial N-hydroxylation by the cytochrome P450 isoform CYP1A2. This is followed by phase 2 O-conjugation and hydrolysis to form a reactive nitrenium ion that covalently binds to DNA and produces tumor-initiating mutations. However, Cyp1a2(-/-) mice still possess significant liver ABP N-hydroxylation activity, DNA damage, and incidence of ABP-induced liver tumors, and in vivo induction of CYP1A2 paradoxically reduces levels of ABP-induced DNA damage. Competing ABP detoxification pathways can include N-acetylation by arylamine N-acetyltransferase 1 (NAT1) and/or NAT2; however, wild-type and Nat1/2(-/-) mice have similar in vivo ABP clearance rates. Together, these studies suggest the existence of novel ABP bioactivating and clearance/detoxification enzymes. In the present study, we detected similar reductions in Vmax for ABP N-hydroxylation by liver microsomes from Cyp1a2(-/-) and Cyp2e1(-/-) mice when compared with wild-type mice. In addition, recombinant mouse CYP1A2 and CYP2E1 were both able to N-hydroxylate ABP in mouse hepatoma cells. However, the in vivo clearance of ABP was significantly reduced in Cyp1a2(-/-) but not in Cyp2e1(-/-) mice. Our results support a significant role for CYP2E1 as a novel ABP N-oxidizing enzyme in adult mice, and suggest a more important contribution of CYP1A2 to the in vivo plasma clearance and thus detoxification of ABP.

NATb/NAT1*4 promotes greater arylamine N-acetyltransferase 1 mediated DNA adducts and mutations than NATa/NAT1*4 following exposure to 4-aminobiphenyl.

N-acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ABP). NAT1 catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5'-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea, and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5'-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3'-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (P < 0.05). Consistent with NAT1 expression and activity, ABP-induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (P < 0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5'-UTRs are differentially regulated.

Biomonitoring of smoke constituents: exposure to 4-aminobiphenyl and 4-aminobiphenyl hemoglobin adduct levels in nonsmokers and smokers.

Public health authorities worldwide have concluded that exposure to environmental tobacco smoke (ETS) causes diseases, including cancer, in adult nonsmokers. The arylamine, 4-aminobiphenyl (4-ABP), has been identified as a human carcinogen. Some publications have suggested that 4-ABP hemoglobin (4-ABP-Hb) adduct levels in nonsmokers are a result of exposure to ETS, whereas others could not confirm these observations. Toxicokinetic and exposure models proposed in this work are used to estimate the concentration of 4-ABP-Hb adducts resulting from ETS exposure that is based on experimental values for respirable suspended particulates (RSP) concentration. Monte Carlo methods were used to obtain estimates of population distributions of 4-ABP-Hb adduct levels resulting from indoor ETS exposure in homes, workplaces, and hospitality environments. It is found that the mean, median, and 95th percentile 4-ABP-Hb adduct steady-state levels of 0.4-1.4, 0.2-1.0, and 0.97-4.63 pg/g Hb, respectively, are estimated from ETS exposure. These 4-ABP-Hb adduct levels from ETS exposure account for approximately 1-4% of the median levels reported for nonsmokers, explaining, in part, contradictory literature data on 4-ABP-Hb adduct levels in nonsmokers. No risk assessment of ETS or 4-ABP was conducted in this work, consequently the known health effects of ETS are neither confirmed or challenged and our conclusions are limited to the determination that ETS is not a major source of 4-ABP-Hb adduct levels in non-smokers.

Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants.

Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and beta-naphthylamine (beta-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H(2)O(2) or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

Primary aromatic amines and cancer: Novel mechanistic insights using 4-aminobiphenyl as a model carcinogen.

Aromatic amines are an important class of human carcinogens found ubiquitously in our environment. It is estimated that 1 in 8 of all known or suspected human carcinogens is or can be converted into an aromatic amine, making the elucidation of their mechanisms of toxicity a top public health priority. Decades of research into aromatic amine carcinogenesis revealed a complex bioactivation process where Phase I and Phase II drug metabolizing enzymes catalyze N-oxidation and subsequent conjugation reactions generating the highly electrophilic nitrenium intermediate that reacts with and forms adducts on cellular macromolecules. Although aromatic amine-DNA adducts were believed to be the main driver of cancer formation, several studies have reported a lack of correlation between levels of DNA adducts and tumors. Using genetically modified mouse models, our laboratory and others observed several instances where levels of conventionally measured DNA adducts failed to correlate with liver tumor incidence following exposure to the model aromatic amine procarcinogen 4-aminobiphenyl. In this review we first provide a historical overview of the studies that led to a proposed mechanism of carcinogenesis caused by aromatic amines, where their bioactivation to form DNA adducts represents the central driver of this process. We then highlight recent mechanistic studies using 4-aminobiphenyl that are inconsistent with this mechanism which suggest novel drivers of aromatic amine carcinogenesis.

Recent technical and biological development in the analysis of biomarker N-deoxyguanosine-C8-4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) which is primarily formed during tobacco combustion and overheated meat is a major carcinogen responsible for various cancers. Its adducted form, N-deoxyguanosine-C8-4-aminobiphenyl (dG-C8-4-ABP), has long been employed as a biomarker for assessment of the risk for cancer. In this review, the metabolism and carcinogenisity of 4-ABP will be discussed, followed by a discussion of the current common approaches of analyzing dG-C8-4-ABP. The major part of this review will be on the history and recent development of key methods for detection and quantitation of dG-C8-4-ABP in complex biological samples and their biological applications, from the traditional 2P-postlabelling and immunoassay methods to modern liquid chromatography-mass spectrometry (LC-MS) with the latter as the focus. Many vital biological discoveries based on dG-C8-4-ABP have been published by using the nanoLC-MS with column switching platform in our laboratory, which has also been adopted and further improved by many other researchers. We hope this review can provide a perspective of the challenges that had to be addressed in reaching our present goals and possibly bring new ideas for those who are still working on the frontline of DNA adducts area.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

4-Aminobiphenyl hemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.

Maternal-fetal exchange of a potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in smoking (n = 14) and nonsmoking (n = 38) pregnant women. N-Hydroxy-4-aminobiphenyl, the active metabolite of 4-aminobiphenyl, forms chemical addition products (adducts) with hemoglobin. Levels of 4-aminobiphenyl hemoglobin adducts were measured in maternal-fetal paired blood samples obtained from smoking and nonsmoking women during labor and delivery. Carcinogen-hemoglobin adducts were detected in all maternal and fetal blood samples. Levels of such adducts were significantly higher (P less than .001) in maternal and fetal blood samples from smokers: the mean 4-aminobiphenyl hemoglobin adduct level was 92 +/- 54 pg/g of hemoglobin in blood samples from fetuses of smokers, and 17 +/- 13 pg/g of hemoglobin in blood samples from fetuses of nonsmokers; the mean maternal 4-aminobiphenyl hemoglobin adduct level was 183 +/- 108 pg/g of hemoglobin in smokers, and 22 +/- 8 pg/g of hemoglobin in nonsmokers. Fetal carcinogen-adduct levels were consistently lower than maternal levels: the mean maternal to fetal ratio was 2.4 +/- 1.1 in smokers and 1.9 +/- .98 in nonsmokers. Fetal 4-aminobiphenyl hemoglobin adduct levels were strongly associated (correlation coefficient [r2] = .51, P = .002) with maternal 4-aminobiphenyl hemoglobin adduct levels when paired samples from smoking mothers were analyzed. A measure of third-trimester tobacco smoke exposure based on number of cigarettes smoked per day, amount of each cigarette smoked, and depth of inhalation was associated (r2 = .59, P = .029) with maternal 4-aminobiphenyl levels but not with fetal 4-aminobiphenyl levels. This study demonstrates that a potent tobacco-related carcinogen, 4-aminobiphenyl, or its active metabolite, N-hydroxy-4-aminobiphenyl, crosses the human placenta and binds to fetal hemoglobin in concentrations that are significantly higher in smokers than in nonsmokers.

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)

DNA adducts in target and nontarget tissues of 3,2'-dimethyl-4-aminobiphenyl in rats.

3,2'-Dimethyl-4-aminobiphenyl (DMAB) is a potent carcinogenic aromatic amine which demonstrates multiorgan tropism in rats. Using polyclonal antibodies against DMAB-DNA adducts, an immunohistochemical procedure as well as an ELISA were applied to investigate the relationship between DMAB-DNA adduct formation and tumorigenicity. Dose-related nuclear staining was observed 24 hr after application of the carcinogen but specificity in terms of sites of tumor development was lacking. No observable decrease in staining intensity was evident in most organs by 168 hr after administration of DMAB. Specific DNA lesions which could be responsible for carcinogenesis were not detected by the 32P-postlabeling method. The tumorigenic response of the ventral prostate in five strains of rats was roughly paralleled by DMAB-DNA adduct levels generated in the tissue. Strong enhancement of bladder tumor development by combined administration of the antioxidants, butylated hydroxyanisole, or butylated hydroxytoluene, with DMAB, was well correlated with an increase in DNA adducts. Our findings so far suggest that DNA adduct formation itself does not determine the carcinogenic organotropism of DMAB. Other factors (including cell proliferation and promotion by exogenous agents) may play important additional roles. For individual target organs or tissues, however, there seems to be a correlation between adduct levels and carcinogenic potential.

Neonatal ontogeny of murine arylamine N-acetyltransferases: implications for arylamine genotoxicity.

Age-related changes in the expression of xenobiotic biotransformation enzymes can result in differences in the rates of chemical activation and detoxification, affecting responses to the therapeutic and/or toxic effects of chemicals. Despite recognition that children and adults may exhibit differences in susceptibility to chemicals, information about when in development specific biotransformation enzymes are expressed is incomplete. N-acetyltransferases (NATs) are phase II enzymes that catalyze the acetylation of arylamine and hydrazine carcinogens and therapeutic drugs. The postnatal expression of NAT1 and NAT2 was investigated in C57Bl/6 mice. Hepatic NAT1 and NAT2 messenger RNAs (mRNAs) increased with age from neonatal day (ND) 4 to adult in a nonlinear fashion. The presence of functional proteins was confirmed by measuring NAT activities with the isoform selective substrates p-aminobenzoic acid and isoniazid, as well as the carcinogens 2-aminofluorene and 4-aminobiphenyl (4ABP). Neonatal liver was able to acetylate all of the substrates, with activities increasing with age. Protein expression of CYP1A2, another enzyme involved in the biotransformation of arylamines, showed a similar pattern. The genotoxicity of 4ABP was assessed by determining hepatic 4ABP-DNA adducts. There was an age-dependent increase in 4ABP-DNA adducts during the neonatal period. Thus, developmental increases in expression of NAT1 and NAT2 genes in neonates are associated with less 4ABP genotoxicity. The age-related pattern of expression of biotransformation enzymes in mice is consistent with human data for NATs and suggests that this may play a role in developmental differences in arylamine toxicity.

HPLC and GC/MS determination of 4-aminobiphenyl haemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.

Maternal-fetal exchange of the potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in women nonsmokers and in women smokers as well as in the corresponding fetuses during pregnancy. Smoking status of the women in the study was assessed via questionnaire and measurement by immunoassay of serum cotinine in maternal and fetal blood samples. 4-Aminobiphenyl was extracted from both maternal and fetal blood samples using organic solvent extractions and the released amine was qualitatively and quantitatively characterized by analysis of the samples by high pressure liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC/MS). Background levels (pg 4-aminobiphenyl/g haemoglobin) of 4-aminobiphenyl-haemoglobin adducts were detected in maternal smokers (mean +/- S.D; 29.6 +/- 16.2 (GC/MS); 23.7 +/- 13.5 (HPLC) and in fetal samples (14.0 +/- 6.5 (GCMS); 10.0 +/- 4.6 (HPLC). Elevated levels of 4-aminobiphenyl-haemoglobin adducts were found in maternal smokers (488 +/- 174 (GC/MS); 423 +/- 154 (HPLC). as well as in the corresponding fetal blood samples (244 +/- 91 (GC/MS); 197 +/- 77 (HPLC). This study confirms that a potent tobacco-related carcinogen, 4-aminobiphenyl, crosses the human placenta and binds to fetal haemoglobin in significantly higher concentrations in smokers when compared to nonsmokers.

Identification of an unintended consequence of Nrf2-directed cytoprotection against a key tobacco carcinogen plus a counteracting chemopreventive intervention.

NF-E2-related factor 2 (Nrf2) is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. Although Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2(+/+) mice than in Nrf2(-/-) mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. Although glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, although higher liver UGT activity may protect the liver against ABP, it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further show that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues but does not seem to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2.

CYP2A13 expressed in human bladder metabolically activates 4-aminobiphenyl.

Cigarette smoking is the predominant risk factor for bladder cancer. Aromatic amines such as 4-aminobiphenyl (ABP) is the major carcinogens found in tobacco smoke. Although it is generally accepted that ABP is metabolically activated via N-hydroxylation by CYP1A2 in human liver, previous studies using Cyp1a2-null mice indicated the involvement of other enzyme(s). Here we found that CYP2A13 can metabolically activate ABP to show genotoxicity by Umu assay. The K(m) and V(max) values for ABP N-hydroxylation by recombinant CYP2A13 in E. coli were 38.5 +/- 0.6 microM and 7.8 +/- 0.0 pmol/min/pmol CYP, respectively. The K(m) and V(max) values by recombinant CYP1A2 were 9.9 +/- 0.9 microM and 39.6 +/- 0.9 pmol/min/pmol CYP, respectively, showing 20-fold higher intrinsic clearance than CYP2A13. In human bladder, CYP2A13 mRNA, but not CYP1A2, is expressed at a relatively high level. Human bladder microsomes showed ABP N-hydroxylase activity (K(m) = 34.9 +/- 4.7 microM and V(max) = 57.5 +/- 1.9 pmol/min/mg protein), although the intrinsic clearance was 5-fold lower than that in human liver microsomes (K(m) = 33.2 +/- 2.0 microM and V(max) = 293.9 +/- 5.8 pmol/min/mg protein). The activity in human bladder microsomes was prominently inhibited by 8-methoxypsoralen, but not by fluvoxamine, anti-CYP1A2 or anti-CYP2A6 antibodies. CYP2S1, which is expressed in human bladder and has relatively high amino acid identities with CYP2As, did not show detectable ABP N-hydroxylase activity. In conclusion, although the enzyme responsible for ABP N-hydroxylation in human bladder microsomes could not be determined, we found that CYP2A13 metabolically activates ABP.

In vivo and in vitro metabolism of arylamine procarcinogens in acetyltransferase-deficient mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of which are procarcinogenic agents. Interestingly, these enzymes are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play in either preventing or enhancing toxic responses. The ultimate direction may be substrate-specific and dependent on its tissue-specific metabolism by competing, but genetically variable, drug-metabolizing enzymes. To investigate the effect of N-acetylation on the metabolism of some classical procarcinogenic arylamines, we have used our double knockout Nat1/2(-/-) mouse model to test both in vitro activity and the in vivo clearance of some of these agents. As expected, N-acetylation activity was undetectable in tissue cytosol preparations from Nat1/2(-/-) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), whereas significant levels were measured in all wild-type tissue cytosols tested, indicating the widespread metabolism of these agents. Nat1/2(-/-) mice displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3- to 4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype, signifying a dependence on other drug-metabolizing enzymes. The elucidation of the role that N-acetylation plays in the clearance of procarcinogenic agents is the first step in attempting to correlate metabolism by NATs to toxic outcome prevention or augmentation.

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 of the N-hydroxy (N-OH) metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP) was investigated in vitro with human tissue cytosols. Using an enzymatic assay consisting of a PAPS-regenerating system, [3H]N-OH-ABP, calf thymus DNA and tissue cytosols, the sulfotransferase-mediated metabolic activation of N-OH-ABP was determined as the PAPS-dependent covalent binding of the N-OH substrate to DNA. With cytosols prepared from various tissues, we found that the sulfotransferase(s) in human liver, and to a lesser extent colon, can readily metabolize N-OH-ABP. No PAPS-dependent metabolic activation was detected with cytosols prepared from human pancreas or from the carcinogen target tissue, the urinary bladder epithelium. The N-OH-ABP sulfotransferase activities of liver and colon cytosols from different individuals were highly 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 thermolabile phenol sulfotransferase (TL-PST; liver, r = 0.29; colon, r = 0.53), or for the dehydroepiandrosterone sulfotransferase (DHEA-ST; liver, r = 0.32; colon, negligible activity). N-OH-ABP sulfotransferase activity was highly sensitive to inhibition by a selective TS-PST inhibitor, 2,6-dichloro-4-nitrophenol (IC50 = 0.7 microM), and by p-nitrophenol, but was unaffected by competitive inhibitors of TL-PST (dopamine) or DHEA-ST (DHEA, DHEA-sulfate). The N-OH-ABP sulfotransferase activity also exhibited thermostability properties similar to that of the TS-PST. From these data, we conclude that human liver TS-PST but not TL-PST or DHEA-ST can metabolically activate the proximate human carcinogen N-OH-ABP to a reactive sulfuric acid ester intermediate that binds covalently to DNA. In addition, in view of the putative role of N-OH-ABP as a major transport form of the carcinogen to the urinary bladder and of the absence of sulfotransferase activity in this tissue, we hypothesize that sulfotransferase activation in the liver may actually decrease the bioavailability of N-OH-ABP toward extrahepatic tissues and thus serve as an important overall detoxification mechanism for the urinary bladder.

Metabolism of 4-aminobiphenyl and 4-acetylaminobiphenyl in perfused guinea pig liver.

To obtain further knowledge of the metabolic fate of carcinogenic 4-aminobiphenyl (ABP) and 4-acetyl-aminobiphenyl (AABP) in intact guinea pig liver, we performed in situ liver perfusion using a recirculation method. Following a biexponential disappearance of ABP from the perfusate, not only AABP but also its secondary metabolites 4'-hydroxy AABP (4'-OH AABP) and 4-glycolylaminobiphenyl (GABP) appeared as the major metabolites. 4'-Hydroxy ABP (4'-OH ABP) was also detected in the perfusate as another predominant metabolite, while N-hydroxy ABP (N-OH ABP) as a minor one. Most of these metabolites, except for AABP and GABP, found in both perfusate and bile were conjugated with glucuronic acid. In addition, considerable amounts of all the metabolites were also detected in either unconjugated or conjugated form in a homogenate of the liver tissue after perfusion. When AABP was infused, an almost similar metabolic profile to that of ABP was obtained, but the amount of ABP was quite small and N-OH AABP rather than N-OH ABP was found. These results demonstrate that both ABP and AABP are rapidly metabolized by a combination of N-acetylation, C- and N-hydroxylations as well as glucuronidation in guinea pig liver.

Preparation and characterization of antibodies against 3,2'-dimethyl-4-aminobiphenyl-modified DNA.

Polyclonal antibodies for 3,2'-dimethyl-4-aminobiphenyl (DMAB)-DNA adducts were obtained from the sera of rabbits immunized with N-OH-DMAB-modified DNA. Using the enzyme linked immunosorbent assay (ELISA), these antibodies were shown to recognize DNA modified by N-OH-DMAB and N-hydroxy-4-aminobiphenyl, but not unmodified DNA, 2-acetylaminofluorene- or 4-nitroquinoline-1-oxide-modified DNA, free aminobiphenyl or DMAB derivatives. Using competitive ELISA with DMAB-modified denatured DNA, a 50% inhibition of antibody antigen binding was caused by 7 fmol of DMAB adducts applied to each assay well as an inhibitor. Native DNA bearing adducts was associated with 50% inhibition in the range of 22-90 fmol/asay well, depending on the levels of modification. The adducts recognized by the antibody were shown to be stable against treatment of heat or alkali denaturation. Optimal conditions for the detection of adducts in DNA from the rat organs exposed to DMAB were established by means of competitive ELISA, using alkaline-denatured DNA as an inhibitor. Thus, as little as 5 fmol adducts in 1 microgram of DNA could be detected. Indirect immunofluorescence staining indicated that anti-DMAB-DNA antibodies bound specifically to nuclear components of rat fibroblast 3Y1 cells treated with N-OH-DMAB. The intensity of the fluorescence was proportional to the dose of carcinogen administered. The antibodies should be helpful for use in studies on the formation of adducts and their removal in cells and tissues after DMAB administration.