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.

Figure 1

Chemical structures of AαC, AαC-3-OSO₃H, and PhIP.

Figure 2

UPLC/MS² analysis of AαC during the baseline smoking phase and 6 weeks after cessation of tobacco and product ion spectra of analyte and synthetic AαC standard.

Figure 3

UPLC/MS² analysis of AαC-3-SO₃H in urine of a subject who ate well-done cooked meat. (A) negative ion mode (278.1 → 198.1), (B) positive ion mode (280.1 → 200.1), and (C) positive ion mode, following in-source CID to fragment AαC-3-SO₃H to AαC-3-OH, and monitoring the transition 200.1 → 155.1, attributed to the loss of CO and NH₃ from protonated AαC-3-OH.

Figure 4

UPLC/MS² analysis of AαC-3-SO₃H during the baseline smoking phase and 6 weeks after cessation of tobacco, and the product ion spectra of the analyte and synthetic AαC-3-OSO₃H standard, following in-source CID to fragment AαC-3-OSO₃H to AαC-3-OH

Figure 5

Linear regression curve (dashed lines 95% CI) of the levels of AαC and AαC-3-OSO₃H during the baseline smoking phase.

Figure 6

Urinary levels of AαC and AαC-3-SO₃H during the baseline smoking phase and 6 weeks after cessation of tobacco.

Figure 7

Scatter dot plots of the levels of cotinine and AαC (showing the geometric mean and 95% CI) during the baseline smoking phase and 6 weeks after cessation of tobacco.

Scheme 1

Mechanism of AαC-3-OSO₃H formation through sulfation of 3-HO-AαC or by rearrangement of N-sulfooxy-AαC.