Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking

Abstract

2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and during the high-temperature cooking of meats. Human enzymes biotransform AαC and PhIP into reactive metabolites, which can bind to DNA and lead to mutations. We sought to understand the relative contribution of smoking and diet to the exposure of AαC and PhIP, by determining levels of AαC, its ring-oxidized conjugate 2-amino-9H-pyrido[2,3-b]indole-3-yl sulfate (AαC-3-OSO3H), and PhIP in urine of smokers on a free-choice diet before and after a six week tobacco smoking cessation study. AαC and AαC-3-OSO3H were detected in more than 90% of the urine samples of all subjects during the smoking phase. The geometric mean levels of urinary AαC during the smoking and cessation phases were 24.3 pg/mg creatinine and 3.2 pg/mg creatinine, and the geometric mean levels of AαC-3-OSO3H were 47.3 pg/mg creatinine and 3.7 pg/mg creatinine. These decreases in the mean levels of AαC and AαC-3-OSO3H were, respectively, 87% and 92%, after the cessation of tobacco (P < 0.0007). However, PhIP was detected in < 10% of the urine samples, and the exposure to PhIP was not correlated to smoking. Epidemiological studies have reported that smoking is a risk factor for cancer of the liver and gastrointestinal tract. It is noteworthy that AαC is a hepatocellular carcinogen and induces aberrant crypt foci, early biomarkers of colon cancer, in rodents. Our urinary biomarker data demonstrate that tobacco smoking is a significant source of AαC exposure. Further studies are warranted to examine the potential role of AαC as a risk factor for hepatocellular and gastrointestinal cancer in smokers.

Metabolism of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole (AαC) in Primary Human Hepatocytes

“…The AαC-6-OH and AαC-3-OH reference compounds produced by human liver microsomes were characterized by 1 H-NMR, which unambiguously identified the sites of ring-oxidation of AαC. 17,34 Taken together these results lead us to identify M2 as AαC-6- O -Gluc and M3 as AαC-3- O -Gluc.…”

“…16 Recently, a tobacco smoking cessation study conducted in the United States revealed that AαC was present in urine during the smoking phase in greater than 90% of the subjects, and the geometric mean urinary level of AαC decreased by 87% six weeks after cessation of tobacco usage. 17 These urinary biomarker data demonstrate that tobacco smoking is a significant source of AαC exposure. The data reported in the literature on urinary level of AαC in human is restricted to few reports where the mean level of AαC in urine of subjects who smoked greater than 20 cigarette per day range between 11.9 and 3511.9 pg/mg creatinine.…”

“…AαC-3-OH and AαC-6-OH were prepared with human liver microsomes or rat liver microsomes and spectroscopically characterized as previously reported. 17,34 N -(deoxyguanosin-8-yl)-2-amino-9 H -pyrido[2,3- b ]indole (dG-C8-AαC) and [ 13 C 10 ]-dG-C8-AαC were prepared as previously described. 27 …”

“…The data reported in the literature on urinary level of AαC in human is restricted to few reports where the mean level of AαC in urine of subjects who smoked greater than 20 cigarette per day range between 11.9 and 3511.9 pg/mg creatinine. 15-17 …”

Bioactivation of Heterocyclic Aromatic Amines by UDP Glucuronosyltransferases

“…68 The amount of AαC formed in well-done cooked meats has often not been measured and the estimates of daily exposure to AαC are uncertain; 69–72 however, urinary biomarker data reveal that the exposure to AαC through tobacco smoke or well-done cooked meat can be appreciable. 73–76 Our biochemical data on UGT1A9 activity offers an alternative interpretation for the association among the high/intermediate UGT1A9 genotype and colorectal cancer risk. HAA, such as AαC and PhIP, undergo N-oxidation by P450 1A2 in the liver, followed by O -glucuronidation of the N -hydroxy-HAA metabolites by UGT1A9 and transport of the HAA-HN- O -Gluc via bile to the gastrointestinal tract, where ensuing binding to colonic DNA occurs.…”