Journal Article

Excretion of the <i>N</i><sup>2</sup>-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-<i>b</i>]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans

W.G. Stillwell, R. Sinha and S.R. Tannenbaum

in Carcinogenesis

Volume 23, issue 5, pages 831-838
Published in print May 2002 | ISSN: 0143-3334
Published online May 2002 | e-ISSN: 1460-2180 | DOI:
Excretion of the N2-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans

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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic aromatic amine formed in meat products during cooking. The genotoxity of PhIP requires an initial cytochrome P450-mediated N-oxidation followed by N-O-esterification catalyzed generally by N-acetyltransferases and sulfotransferases. This study examined the urinary excretion of N2-(β-1-glucos-iduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine—the major human urinary N-oxidation metabolite of PhIP—and determined its relationship to individual activity levels of cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT2). The subjects (33 males and 33 females) in the dietary study were phenotyped for their CYP1A2 and NAT2 activity prior to consumption of meat-based diet, and urine collections were obtained 0–12 and 12–24 h after ingestion of the meal. Acidic hydrolysis of N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its d3-analog to form their respective deaminated products 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-OH-PhIP) was used in the assay. The products after derivatization were analyzed by capillary gas chromatography-negative ion chemical ionization mass spectrometry with selective ion monitoring. The amount of N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine measured as the acid hydrolysis product 2-OH-PhIP in the 0–12 h urine was 20.2 ± 8.0% (mean ± SD) of the ingested dose; the median was 18.8% and the range varied from 5.4 to 39.6% within the group. In a subset (n = 18) of samples from individual urine collected from the 12–24 h period, an average value of 4.4 ± 2.5% (± SD) of the dose was recovered. The excretion of N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the 0–12 h urine was significantly related to the quantity of PhIP ingested for all subjects (r = 0.52, P <0.0001). Linear regression analysis of the relationship between the excretion level of N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, adjusted for meat intake and CYP1A2 activity in the combined group of males and females showed a low association (r = 0.25, P = 0.05). There was no association between the amount of N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimid-azo[4,5-b]pyridine in urine and NAT2 activity levels of the subjects nor with the age of the subjects. N2-(β-1-glucosi-duronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine comprised a significant proportion of the ingested dose in some individuals; however, considerable variation was found within the group. The results indicate that interindividual differences in the rates of N-oxidation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, as well as phase II glucuronidation reactions regulate the formation of this metabolite in humans.

Keywords: CYP1A2, cytochrome P4501A2; GC/MS, gas chromatography/mass spectrometry; HAA, heterocyclic aromatic amine; HPLC, highperformance liquid chromatography; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NAT2, N-acetyltransferase; NCI, negative ion chemical ionization; N-OH-PhIP, 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; N-OH-PhIP-N2-glucuronide, N2-(β-1-glucosiduronyl)-2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 2-OH-PhIP, 2-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine;; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine;; SIM, selective ion monitoring

Journal Article.  7282 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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