Chemoprotection against the formation of colon DNA adducts from the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat.

The mutagenic heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), is a pyrolysis product in cooked foods that has been shown to be a rat colon carcinogen and has been implicated in the etiology of human colon cancer. In order to identify chemoprotection strategies that could be carried out in humans, a pilot study was conducted in which PhIP-DNA-adduct levels were quantified in the colons of male F344 rats that had been subjected to 16 different putative chemoprotection regimens, followed by a gavage of PhIP (50 mg/kg) and sacrifice 24 h later. The 16 treatments (Oltipraz, benzylisothiocyanate, diallyl sulfide, garlic powder, ethoxyquin, butylated hydroxyanisole, glutathione, indole-3-carbinol, alpha-angelicalactone, kahweol/cafestol palmitates, quercetin, green tea, black tea, tannic acid, amylase-resistant starch, and physical exercise) comprised sulfur-containing compounds, antioxidants, flavonoids, diterpenes, polyphenols, high dietary fiber, etc. The strongest inhibition of PhIP-DNA adduct formation in the colon was observed upon pretreatment with black tea, benzylisothiocyanate, and a mixture (1:1) of kahweol:cafestol palmitates, which resulted in 67, 66, and 54% decreases in colon PhIP-DNA adduct levels, as compared with controls. Preliminary studies on their mechanism of action indicated that only kahweol:cafestol caused a substantial induction of glutathione S-transferase isozymes (GSTs) that are thought to be important in the detoxification of PhIP. Notably, this induction occurred in the liver rather than in the colon.

Metabolic activation of aromatic amines by human pancreas.

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

Metabolic polymorphisms and carcinogen-DNA adduct formation in human populations.

Metabolic polymorphisms have long been recognized as important determinants of carcinogen susceptibility and recent efforts have shown that interindividual differences in specific cytochromes P450, acetyltransferases, sulfotransferases and glutathione S-transferases are often disproportionately represented in epidemiological studies between cancer cases and controls. Concomitantly, biomonitoring of carcinogen-DNA adducts in human tissues using immunochemical, 32P-postlabelling, fluorescence, and mass spectrometric methods have recently provided direct evidence of human exposure to genotoxic aromatic and heterocyclic amines, polycyclic hydrocarbons, alkylating agents, and endogenous products. However, a combined approach is now needed in order to assess the relevance of these findings to cancer etiology, to identify high-risk individuals, and to provide better health monitoring, earlier diagnosis, and cancer prevention. Using this paradigm, results are presented that implicate specific aromatic amines, heterocyclic amines, and polycyclic aromatic hydrocarbons in the etiology of human urinary bladder, colon, and laryngeal cancers.

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes.

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes.

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues.

The food-borne mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumors in colon of male rats and has been implicated in the etiology of human cancers, particularly colorectal cancer. This study was conducted to examine: (1) the biliary and/or circulatory transport of N-hydroxy-PhIP and its N-glucuronides, N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximate and ultimate carcinogenic metabolites of PhIP; (3) the potential role of glutathione in modulating PhIP-DNA adduct formation. PhIP-DNA adducts, measured by the 32P-postlabeling method, were highest in the pancreas (361 adducts/10(8) nucleotides or 100%), followed by colon (56%), lung (28%), heart (27%) and liver (2%), at 24 h after a single oral dose of PhIP (220 mumol/kg) to male rats. In each tissue examined, we observed two major adducts, each of which accounted for 35-45% of the total, and one minor adduct, which represented about 10-20% of the total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine by chromatographic comparisons with an authentic standard. The major urinary metabolites of PhIP in these rats were 4'-hydroxy-PhIP and its glucuronide and sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide, N-hydroxy-PhIP N2-glucuronide and unchanged PhIP. In bile duct-ligated rats, the urinary excretion of the N-OH-PhIP N3-glucuronide was increased two-fold, but there was no effect on PhIP-DNA adduct formation in the colon, heart, lung, pancreas or liver. 2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediated DNA binding in vivo, had no effect on PhIP-DNA adduct levels in liver or in extrahepatic tissues. Pretreatment of rats with buthionine sulfoximine, which results in hepatic glutathione depletion, caused a five-fold increase in adduct formation in the liver. Intravenous administration (10 mumol/kg) of N-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels of PhIP-DNA adducts in each of the extrahepatic tissues examined. Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP) and four- to 28-fold higher (for N-acetoxy-PhIP) as compared to that after an i.v. dose of the parent compound, indicating that these two bioactivated derivatives of PhIP are sufficiently stable to be transported through the circulation to extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

32P-postlabelling analysis of DNA adducts of 4,4'-methylenebis(2-chloroaniline) in target and nontarget tissues in the dog and their implications for human risk assessment.

4,4'-Methylenebis(2-chloroaniline) (MOCA) has considerable human occupational exposure and it induces urinary bladder tumors in the dog, a species that has been often used as a model for aromatic amine-induced urinary bladder carcinogenesis in humans. Metabolic activation and formation of DNA adducts are considered to be critical steps in this process; and two major C8-adenine adducts have been shown to be formed in vitro by reaction with the proximate carcinogenic metabolite N-hydroxy-MOCA. MOCA-DNA adducts have also been detected in vivo in treated rats and in exfoliated urothelium of a worker accidentally exposed to MOCA. Thus, the aim of this study was to detect and quantify DNA adducts in the urinary bladder of dogs exposed to MOCA and thereby provide data that could be useful for risk assessment after human exposure to MOCA. Beagle dogs were treated with single and multiple doses of MOCA and DNA adduct levels were determined in liver and bladder epithelium. After a single dose, adduct levels in the liver were 1.5-fold higher than that in the bladder epithelium. Adduct levels in these two organs increased 3- to 5-fold after 10 doses and adducts in the liver were then 2.8-fold higher than that in the bladder epithelium. The levels found in these two organs after single exposures were compared, per unit exposure dose, with that reported for other carcinogenic aromatic amines. The comparison showed that MOCA was as effective in DNA adduct formation as most other potent urinary bladder carcinogens. These results suggest that MOCA may have high carcinogenic potential in humans and are consistent with the recent classification of MOCA as a probable human carcinogen.

4,4'-Methylene-bis(2-chloroaniline)-DNA adduct analysis in human exfoliated urothelial cells by 32P-postlabeling.

4,4'-Methylene-bis(2-chloroaniline) (MOCA) is an aromatic amine used widely in industry, and human exposure to this compound is well documented. MOCA induces lung and liver tumors in rodents and urinary bladder tumors in dogs, and it is regarded as a suspect urinary bladder carcinogen in humans. In this pilot study, we investigated the occurrence of MOCA-DNA adducts in exfoliated urothelial cells of a MOCA-exposed worker by 32P-postlabeling analysis. Urine samples were collected from the worker at various times after accidental acute exposure to MOCA. DNA isolated from exfoliated urothelial cells collected from each urine sample was enzymatically digested and postlabeled with excess [32P]ATP. Thin-layer chromatographic analysis of the labeled digests revealed the presence of a single, major DNA adduct that cochromatographed with the major N-hydroxy-MOCA-DNA adduct, N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, formed in vitro. The MOCA-DNA adduct was detected in samples obtained between 4 and 98 h after initial exposure but not in samples collected at later times. The level of DNA adducts 4 h after exposure was determined to be 516 adducts/10(8) nucleotides. A 5-fold decrease in adduct level was observed 14 h later, followed by a gradual decrease over subsequent days. The results indicate that MOCA is potentially genotoxic to human urinary bladder in vivo and that 32P-postlabeling analysis of exfoliated urothelial cells provides a noninvasive means for biomonitoring the formation of MOCA-DNA adducts resulting from occupational exposure.

Polymorphisms for aromatic amine metabolism in humans: relevance for human carcinogenesis.

The metabolic pathways associated with carcinogenic aromatic amines in humans provide an excellent example of polymorphisms that appear to be relevant to human carcinogenesis. In this regard, the N-acetylation of arylamines and the O-acetylation of their N-hydroxy metabolites are catalyzed preferentially by a genetically polymorphic acetyltransferase, high activity of which has been correlated with decreased risk for urinary bladder cancer and increased susceptibility to colorectal cancer. Cytochrome P450IA2, the principal liver enzyme involved in aromatic amine N-oxidation, exhibits a wide interindividual variation that appears trimodal in several populations and is clearly inducible by cigarette smoking and probably other host factors as well. UDP-Glucuronosyltransferases, which catalyze the N-glucuronidation of N-hydroxyarylamines and are likely to be responsible for their transport to the colon, show widely varied but unimodal distributions in humans. In contrast, human liver sulfotransferase activity for N-hydroxyarylamines, which would be expected to decrease their transport through the circulation, is catalyzed by a polymorphic enzyme(s) that is expressed at higher levels in blacks, as compared to whites, and could contribute to their relatively lower incidence of urinary bladder cancer. Peroxidative activation of aromatic amines can also occur, especially from prostaglandin H synthase in the urinary bladder and myeloperoxidase in the lungs of cigarette smokers, and both show considerable individual variability, apparently due to the extent of tissue inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine as the major adduct formed by the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, with DNA.

The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)