Tobacco use is the single largest preventable cause of disease and death in the United States, and tobacco smoking is responsible for more than 480,000 deaths each year in the United States (1). A significant body of evidence accumulated over several decades suggests that tobacco smoke exposure is related to development of lung cancer and other cancers (1–4). Various carcinogens have been identified in tobacco smoke (4–6).

Heterocyclic aromatic amines (HAAs) are a group of hazardous substances produced during burning of tobacco or high-temperature cooking of meats (7–10). HAAs are amines that contain at least one heterocyclic ring and one aromatic ring (Supplementary Figure S1). HAAs are strongly mutagenic and carcinogenic in various in vitro and in vivo models. HAAs can induce tumors in various organs, including mammary glands, prostate, lungs, colon, skin, bladder, and liver (11–14). Epidemiological studies associate frequent HAA exposure with elevated cancer risk (15–17). The International Agency for Research on Cancer (IARC) categorized a number of HAAs as possible (Group 2B) and probable (Group 2A) human carcinogens (18).

Among more than 25 HAAs so far identified (19), 2-amino-9H-pyrido[2,3-b]indole (AαC, Group 2B carcinogen) is one of most abundant carcinogenic HAAs in tobacco smoke (10). AαC levels in tobacco smoke can reach as high as 260 ng per cigarette (7,10,20,21). This approaches the levels of other well-known carcinogens in tobacco smoke, such as N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[α]pyrene (BaP) (5,22,23).

Another carcinogenic HAA detected in tobacco smoke is 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC, Group 2B carcinogen) (Supplementary Figure S1). MeAαC is a methyl homolog of AαC. MeAαC levels in tobacco smoke are about 10-fold lower than AαC levels (7,8,10). These two HAAs are carcinogenic in animal models (5,24–28), and IARC classifies AαC and MeAαC as suspected human carcinogens (29). Additionally, the U.S. Food and Drug Administration (FDA) lists AαC and MeAαC as harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke (30). Besides tobacco smoke, AαC and MeAαC can form in foods prepared at high temperatures (approximately 200°C–300°C), such as barbecued, fried, or broiled meats, poultry, and fish (8,31–34).

Metabolic activation has been proposed as a mechanism for the carcinogenicity of AαC and MeAαC (9,35). Cytochrome P450 enzymes are involved in the oxidation of the exocyclic amine group of AαC and MeAαC (35–37). Sequentially, these N²-hydroxylated metabolites of AαC and MeAαC can be further catalyzed by acetyltransferases, sulfotransferases, or glucuronosyltransferases to generate reactive O-esters that could bind covalently to DNA and elicit genotoxicity (38–43).

Although the potential for AαC and MeAαC to harm human health is well documented, few studies have evaluated human exposure to these two carcinogens. A small-scale study (170 subjects) conducted in China documented that AαC was consistently detected in smokers’ urine. The number of cigarettes smoked per day was positively associated with urinary levels of AαC in study participants (44). A U.S. study (30 subjects) detected AαC in most of the urine samples of smokers, but those levels had dropped 87% after 6 weeks of not smoking (45). Even fewer studies have measured MeAαC in human biospecimens. The aim of this report is to characterize human exposure to AαC and MeAαC in the general population. We measured AαC and MeAαC as urinary biomarkers of exposure to these carcinogenic HAAs as part of the National Health and Nutrition Examination Survey (NHANES), which conducts biomonitoring of the United States civilian, non-institutionalized population.

Sample-weighted demographic distributions of NHANES 2013–2014 participants (AαC: N = 1,792, MeAαC: N = 1,793) were summarized in Table 1. Among these participants, AαC was detected in 61.5 percent of participants, but only in 30.6 percent for MeAαC, so MeAαC was excluded from multiple regression analysis. By comparison, AαC and MeAαC were detected at much higher rates (98.9% and 75.6%, respectively) in exclusive tobacco smokers than those in non-users (39.5 % for AαC and 4.2% for MeAαC). Among exclusive smokers, urinary AαC and MeAαC concentrations were significantly correlated, with a sample-weighted Pearson correlation of 0.788. The sample-weighted median of urinary AαC among exclusive smokers was 40 times higher than non-users (31.90 vs. 0.79 ng/g creatinine, respectively; Table 2). Geometric means and selected percentiles of urinary AαC concentrations for U.S. population were summarized in Supplementary Table S2 and S3. The difference in urinary concentrations of MeAαC in exclusive tobacco smokers and non-users at selected percentiles was summarized in Supplementary Table S4 and S5. The percent-distribution of urinary AαC and MeAαC depicted these differences among exclusive smokers and non-users (Figure 1).

The sample-weighted multiple regression model among exclusive smokers (Table 3) found that urinary AαC was positively associated with serum cotinine (0.113 pg AαC/mL per ng cotinine/mL), controlling for sex, age, race/Hispanic origin, BMI, fasting time, urinary creatinine, diet, and impoverishment. Mexican-American exclusive smokers had significantly lower AαC in comparison with non-Hispanic White exclusive smokers. Urinary AαC level among impoverished exclusive smokers was significantly higher than among non-impoverished participants.

Among non-users of tobacco products (Table 4), urinary AαC was positively associated with serum cotinine (0.140 pg AαC/mL per ng cotinine/mL), presumptively attributable to secondhand smoke exposure, controlling for sex, age, race/Hispanic origin, BMI, fasting time, urinary creatinine, diet, and impoverishment. Non-Hispanic Black and Other/Multi-Racial non-users had lower AαC than non-Hispanic White non-users. Urinary AαC was also positively associated with the amount consumed (kg) of beef cooked under high temperature (15.5 pg/mL per kg), whereas the association with consumption of fish cooked at high temperature was not significant. Consumption of vegetables (−0.264 pg/mL per kg) was a significant negative predictor of urinary AαC. Fasting time also was negatively associated with AαC.

In the un-stratified, sample-weighted CPD regression model for non-users and exclusive smokers (Supplementary Table S6), each CPD exposure group had significantly higher urinary AαC levels compared with those of non-users whose serum cotinine levels were <0.05 ng/mL, controlling for sex, age, race/Hispanic origin, BMI, fasting time, urinary creatinine, diet, and impoverishment. Figure 2 displays the sample-weighted least-square means of urinary AαC from the CPD model by exposure category, where it can be seen that urinary AαC increased in a dose-dependent manner across exposure categories.

From a national, population-based study, we show that tobacco smoke and diet are significantly associated with AαC exposure. Sample-weighted linear regression models showed that serum cotinine was a significant positive predictor of urinary AαC among both exclusive smokers and non-users. Moreover, we found a significant dose-dependent association between exclusive smokers’ urinary AαC levels and CPD smoked. Our findings for tobacco smoke exposure among both exclusive smokers and non-users is consistent with the fact that AαC is the most abundant carcinogenic HAAs in tobacco smoke (7,10,20,21), and indicated that tobacco smoke is a major source of AαC exposure in the U.S. population.

Only three small-scale studies previously reported human AαC exposure levels. A study in China (78 subjects) found that the mean of total urinary concentrations of AαC (free and conjugated forms) for smokers (20 ng/g creatinine) was significantly higher than that for nonsmokers (7 ng/g creatinine) (54). Compared with above study, median urinary AαC was slightly higher for exclusive smokers (31.90 ng/g creatinine) and much lower for non-users (0.79 ng/g creatinine) in the NHANES 2013–2014 samples. The lower median urinary AαC of non-user in our study could be related to lower levels of second-hand smoke exposure in the U.S. than in China. Another study in China measured free urinary AαC in 170 volunteers (44). The mean urinary AαC among light (1–19 CPD) smokers was 7.50 ng/g creatinine (9.2 pg/mL urine) and among heavy (>20 CPD) smokers it was 11.92 ng/g creatinine (13.8 pg/mL urine), while mean urinary AαC among nonsmokers was 2.54 ng/g creatinine. In contrast to Turesky et al. 2007, we observed higher exposure levels to AαC at corresponding CPD (least squared means 1-10 CPD: 14.0 pg/mL; 11-20 CPD: 37.2 pg/mL; >20 CPD: 64.1 pg/mL). These differences may be explained, at least in part, to differences in sample preparation. Turesky et al. 2007 measured free AαC in non-hydrolyzed samples, whereas our measurements were preceded by hydrolysis, which enables measurement of both free and base-sensitive conjugated species of AαC, thereby leading to systematically higher measured concentrations. In addition, there is large variation of AαC in cigarettes between manufacturers that would increase the observed variation in urinary AαC at the same CPD consumed in the two countries (Manabe et al. 1991; Smith et al. 2004; Zhang et al. 2011). Nonetheless, both studies corroborated that urinary AαC followed a dose response pattern with CPD. In one small U.S. study (30 subjects) where urinary AαC measurements comprised both free and base-sensitive conjugated species (45), the measurements had magnitudes closer to ours. Konorev et al. 2015 observed that mean urinary AαC among smokers decreased from 24.3 ng/g creatinine to 3.2 ng/g creatinine after subjects had stopped smoking tobacco for six weeks.

In the NHANES 2013-2014 population, urinary AαC in non-Hispanic White smokers was around 7-fold higher than that in Mexican American smokers (Table 2).The effect of race/Hispanic origin cannot be completely explained by differences in tobacco smoke exposure alone because serum cotinine was included in the model. Difference in toxicokinetics may partly contribute to the race/Hispanic origin-related differences. CYP1A2 is one of the major P450 isoforms responsible for AαC metabolism (35,55–57). N-hydroxylation of the exocyclic amine group results in HONH-AαC, and ring oxidation of AαC at the C-3 and C-6 positions results in AαC-3-OH and AαC-6-OH, respectively (35,56). Various factors such as sex, race/Hispanic origin, and smoking status can affect CYP1A2 activity (58–60). Unfortunately, standards for these compounds and their corresponding stable isotope-labeled internal standards are not commercially available, so hydroxylated metabolites of AαC were not included in our assay. Therefore, the influence of race/Hispanic origin on AαC metabolism remains unclear and awaits future investigation.

We also found that certain diet components are significantly associated with AαC exposure. Beef and fish cooked at high temperature have been regarded as the most likely sources of AαC from foods eaten in the United States (34). Based on the formation mechanism, AαC is produced from pyrolysis of proteins or amino acids heated at high temperature (8). In the non-user and CPD regression models, eating beef cooked at high temperature was shown to be significantly associated with increased urinary AαC. In contrast, we found no significant association between urinary AαC and the amount of “meat, poultry, fish, and mixture” eaten without also specifying meat types or cooking temperature, which is consistent with a process requiring high temperature to contribute to dietary AαC exposure.

Eating vegetables was associated with significantly diminished urinary AαC levels in nonsmokers. Either decreased HAA absorption or enhanced hepatic elimination of HAA via induced CYP2A1 activity from eating vegetables has been proposed to explain reduced urinary HAA in persons who ate cooked meat (61,62). Because the hydroxylated AαC metabolites formed by CYP2A1 were not measured in our assay, we cannot say whether the influence of eating vegetables on urinary HAA levels in previous studies could be directly used to interpret our findings.

Fasting time was a significant negative predictor of urinary AαC levels in non-users of tobacco products, indicating that urinary excretion of AαC declined with time after the last meal. Because the AαC half-life is around 3.0 hr (63), urinary AαC levels would be expected to decline rapidly after complete AαC absorption from the food matrix.

We compared the potential influence of diet vs. smoking on urinary AαC by estimating the 99^th percentile of beef consumption cooked at high temperature for the United States population, which we found to be 0.22 kg/day. At this level of consumption, we estimated the likely increase in urinary AαC ΔY 95%CI to be 0.99 [0.16, 2.29]pg AαC/mL, compared to the increase associated with smoking one-half pack of cigarettes per day of 23.6 [18.7, 29.6] pg AαC/mL. Under these assumptions, the likely level of dietary exposure is considerably lower than from exclusive cigarette smoking.

Dietary exposure was based on 24-hour recall data collected in the MEC, thus reflecting dietary consumption for a single day. Although interviewers elicited recall with structured questions and standardized techniques, retrospective responses are generally susceptible to recall bias.

So far, no population-based study has identified tobacco smoke as a source of MeAαC exposure. A study in China lacked sufficient sensitivity of the urinary MeAαC assay to detect MeAαC in smokers (54). With the improved sensitivity of MeAαC measurement in our assay, our analysis provides the first population-based data on MeAαC. MeAαC was found to co-occur with AαC at about 10-fold lower content in tobacco smoke (7,8,10). As with AαC, MeAαC was mostly detected in smokers at around 20 times lower levels than those of AαC. Moreover, urinary AαC and MeAαC concentrations were significantly correlated among exclusive smokers with a sample-weighted Pearson correlation of 0.788. Therefore, MeAαC could serve as a valuable ancillary biomarker to assess AαC exposure.

In the United States, urinary AαC is significantly associated with serum cotinine levels among both exclusive smokers and non-users of tobacco, and tobacco smoke is a major source of AαC exposure in the general population. Much smaller increases in urinary AαC were associated with secondhand smoke exposure and eating beef cooked at high temperature.