Metabolic activation and DNA adduct detection of PhIP in dogs, rats, and humans in relation to urinary bladder and colon carcinogenesis.

The metabolic activation of the heterocyclic amine carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was examined in dogs and rats as models for urinary bladder and colon carcinogenesis, respectively. The results indicate that unconjugated N-OH-PhIP is not excreted in the urine after oral dosing with PhIP and that the two isomeric N-glucuronides of N-OH-PhIP, which are formed as major metabolites, are stable under acidic conditions. These data suggest that PhIP is unlikely to serve as a urinary bladder carcinogen in either species. Using metabolic inhibitors, bile duct ligation, and intravenous dosing studies, a new hypothesis for colorectal carcinogenesis is proposed involving N-oxidation of PhIP by hepatic cytochrome P-4501A2 (CYP1A2) and O-acetylation by the polymorphic acetyltransferase (NAT2). The resulting N-hydroxy and N-acetoxy metabolites both appear to be transported through the circulation to the colon mucosa, forming covalent DNA adducts. Glucuronidation and reaction with glutathione appear to serve as detoxification pathways. In humans, individuals who are phenotypically rapid metabolizers for both CYP1A2 and NAT2 are significantly higher (p = 0.0015) in colorectal cancer/poly cases vs. controls; and PhIP-DNA adducts can be detected in human colon samples. These studies provide strong evidence that PhIP and other heterocyclic amines play an important role in the etiology of human colorectal cancer.

Metabolic activation and carcinogen-DNA adduct detection in human larynx.

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.

Metabolic activation, DNA adducts, and H-ras mutations in human neoplastic and non-neoplastic laryngeal tissue.

Metabolic activation, DNA adducts, and H-ras mutations were examined in human laryngeal tissue (n = 16) from both smoker and non/ex-smoker patients with laryngeal cancer. DNA adducts detected by 32P-postlabelling were evident only in smokers (n = 13); in fact, smoking cessation for as little as 10 months resulted in no DNA adducts detected (n = 3). Total DNA adduct levels in these samples were significantly correlated with levels of cytochromes P-4502C and 1A1 in laryngeal microsomes. Moreover, the P-4501A1 levels represent the highest yet found in human tissues. In contrast, laryngeal microsomes did not have detectable P-4501A2 activity, while laryngeal cytosols showed appreciable N-acetyltransferase activity for p-aminobenzoic acid (NAT1) but not sulfamethazine (NAT2). DNA was extracted from laryngeal specimens and amplified by PCR. Nylon filter dot or slot blots were hybridized with 32P-labelled probes for codons 12, 13, and 61 of the H-ras gene. Sixty percent of specimens demonstrated mutations in either codon 12, 13, or 61; a single common and specific mutation was a Gln-->Glu transversion in codon 61. This mutation appeared in 5 laryngeal specimens, all from smokers. These results implicate cigarette smoke components, bioactivated by CYP1A1 and/or CYP2C, in DNA adduct formation. These results also demonstrate a probable smoking-related H-ras Gln-->Glu transversion in codon 61.

Advantages and limitations of laboratory methods for measurement of carcinogen-DNA adducts for epidemiological studies.

In molecular epidemiological studies, the measurement of carcinogen-DNA adducts in human tissues can provide direct evidence of current exposure to chemical carcinogens. Moreover, data on steady state DNA adduct levels and the rate of cell proliferation can be related not only to carcinogen-target tissue dosimetry but may also be useful in assessment of human cancer risk. Thus far, laboratory methods for adduct detection have primarily utilized 32P-postlabelling, immunoassays, and mass spectrometry. However, accurate quantitation of DNA adducts requires knowledge of the structural identity and chemical properties of carcinogen-base adducts, the availability of synthetic standards for recovery determinations, and the development of complementary methods to corroborate analytical findings.

Stereoselectivity in the N'-oxidation of nicotine isomers by flavin-containing monooxygenase.

N'-Oxidation of nicotine isomers by porcine liver flavin-containing monooxygenase shows a clear stereoselectivity in the formation of the diastereomeric N'-oxides. (S)-(-)-Nicotine exhibited no stereoselectivity in the formation of cis-1'R,2'S- and trans-1'S,2'S-products, whereas with (R)-(+)-nicotine, only the trans-1'R,2'R-N'-oxide was formed. The concentration of each isomer required for half maximal activity differs significantly, and access of (S)-(-)-nicotine to the active site appears to be more restricted than for (R)-(+)-nicotine as judged from the observed Km values (Km = 181 and 70 microM, respectively, for the (S)-(-)- and (R)-(+)-isomers). These results indicate that a region adjacent to the active site may sterically prohibit binding of (R)-(+)-nicotine when the N'-methyl and pyridyl groups are in a cis-orientation. N-Methylnicotinium ion (both R- and S-isomers) is not a substrate for either porcine flavin monooxygenase, guinea pig liver microsomes, or ram seminal vesicular microsomes.