Menglan Chen

Evaluation of Toxicant and Carcinogen Metabolites in the Urine of E-Cigarette Users Versus Cigarette Smokers

INTRODUCTION Electronic cigarettes (e-cigarettes) are rapidly increasing in popularity but little information is available on their potential toxic or carcinogenic effects. METHODS Twenty-eight e-cigarette smokers who had not smoked tobacco cigarettes for at least 2 months provided urine samples which were analyzed by validated methods for a suite of toxicant and carcinogen metabolites including 1-hydroxypyrene (1-HOP), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), 3-hydroxypropylmercapturic acid (3-HPMA), 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1-methylpropylmercapturic acid (HMPMA), S-phenylmercapturic acid (SPMA), nicotine, and cotinine. Levels of these compounds were compared to those found in cigarette smokers from three previous studies. RESULTS Levels of 1-HOP, total NNAL, 3-HPMA, 2-HPMA, HMPMA, and SPMA were significantly lower in the urine of e-cigarette users compared to cigarette smokers. Levels of nicotine and cotinine were significantly lower in e-cigarette users compared to cigarette smokers in one study but not in another. CONCLUSIONS With respect to the compounds analyzed here, e-cigarettes have a more favorable toxicity profile than tobacco cigarettes.

Effects of Smoking Cessation on Eight Urinary Tobacco Carcinogen and Toxicant Biomarkers

We determined the persistence at various times (3, 7, 14, 21, 28, 42, and 56 days) of eight tobacco smoke carcinogen and toxicant biomarkers in the urine of 17 smokers who stopped smoking. The biomarkers were 1-hydroxy-2-(N-acetylcysteinyl)-3-butene (1) and 1-(N-acetylcysteinyl)-2-hydroxy-3-butene (2) [collectively called MHBMA for monohydroxybutyl mercapturic acid] and 1,2-dihydroxy-4-(N-acetylcysteinyl)butane (3) [DHBMA for dihydroxybutyl mercapturic acid], metabolites of 1,3-butadiene; 1-(N-acetylcysteinyl)-propan-3-ol (4, HPMA for 3-hydroxypropyl mercapturic acid), a metabolite of acrolein; 2-(N-acetylcysteinyl)butan-4-ol (5, HBMA for 4-hydroxybut-2-yl mercapturic acid), a metabolite of crotonaldehyde; (N-acetylcysteinyl)benzene (6, SPMA for S-phenyl mercapturic acid), a metabolite of benzene; (N-acetylcysteinyl)ethanol (7, HEMA for 2-hydroxyethyl mercapturic acid), a metabolite of ethylene oxide; 1-hydroxypyrene (8) and its glucuronides (1-HOP), metabolites of pyrene; and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (9) and its glucuronides (total NNAL), a biomarker of exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These biomarkers represent some of the major carcinogens and toxicants in cigarette smoke: 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, polycyclic aromatic hydrocarbons (PAH), and NNK. With the exception of DHBMA, levels of which did not change after cessation of smoking, all other biomarkers decreased significantly after 3 days of cessation (P < 0.001). The decreases in MHBMA, HPMA, HBMA, SPMA, and HEMA were rapid, nearly reaching their ultimate levels (81-91% reduction) after 3 days. The decrease in total NNAL was gradual, reaching 92% after 42 days, while reduction in 1-HOP was variable among subjects to about 50% of baseline. Since DHBMA did not change upon smoking cessation, there appear to be sources of this metabolite other than 1,3-butadiene. The results of this study demonstrate that the tobacco smoke carcinogen/toxicant biomarkers MHBMA, HPMA, HBMA, SPMA, HEMA, 1-HOP, and NNAL are related to smoking and are good indicators of the impact of smoking on human exposure to 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, PAH, and NNK.

Modulation of the metabolism of airborne pollutants by glucoraphanin-rich and sulforaphane-rich broccoli sprout beverages in Qidong, China

Epidemiological evidence has suggested that consumption of a diet rich in cruciferous vegetables reduces the risk of several types of cancers and chronic degenerative diseases. In particular, broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can release the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases. Two broccoli sprout-derived beverages, one sulforaphane-rich (SFR) and the other glucoraphanin-rich (GRR), were evaluated for pharmacodynamic action in a crossover clinical trial design. Study participants were recruited from the farming community of He Zuo Township, Qidong, China, previously documented to have a high incidence of hepatocellular carcinoma with concomitant exposures to aflatoxin and more recently characterized with exposures to substantive levels of airborne pollutants. Fifty healthy participants were randomized into two treatment arms. The study protocol was as follows: a 5 days run-in period, a 7 days administration of beverage, a 5 days washout period and a 7 days administration of the opposite beverage. Urinary excretion of the mercapturic acids of acrolein, crotonaldehyde, ethylene oxide and benzene were measured both pre- and postinterventions using liquid chromatography tandem mass spectrometry. Statistically significant increases of 20-50% in the levels of excretion of glutathione-derived conjugates of acrolein, crotonaldehyde and benzene were seen in individuals receiving SFR, GRR or both compared with their preintervention baseline values. No significant differences were seen between the effects of SFR versus GRR. Intervention with broccoli sprouts may enhance detoxication of airborne pollutants and attenuate their associated health risks.

Urinary Levels of Cigarette Smoke Constituent Metabolites Are Prospectively Associated with Lung Cancer Development in Smokers

Polycyclic aromatic hydrocarbons (PAH) are believed to be among the principal causative agents for lung cancer in smokers, but no epidemiologic studies have evaluated the relationship of PAH uptake and metabolism to lung cancer. In this study, we quantified prediagnostic urinary levels of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), a validated biomarker of PAH uptake and metabolism, as well as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and cotinine and its glucuronides (total cotinine), validated biomarkers of uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and nicotine, respectively, in relation to lung cancer risk among current smokers in a nested case-control study within a cohort of 18,244 Chinese men in Shanghai, China. Urinary levels of PheT, total NNAL, and total cotinine were significantly higher in cases than controls (N = 476 matched pairs). ORs (95% confidence intervals) for lung cancer in the second, third, fourth, and fifth quintiles of PheT were 1.70 (1.00-2.88), 1.07 (0.62-1.84), 1.48 (0.86-2.53), and 2.34 (1.33-4.11), respectively, relative to the lowest quartile (P(trend) = 0.023) after adjustment for self-reported smoking intensity and duration and urinary total NNAL and total cotinine. This study also confirmed that urinary total NNAL and total cotinine are independently related to lung cancer risk.

Comparison of Polymorphisms in Genes Involved in Polycyclic Aromatic Hydrocarbon Metabolism with Urinary Phenanthrene Metabolite Ratios in Smokers

The hypothesis that interindividual differences among smokers in the metabolism of polycyclic aromatic hydrocarbons (PAH) are related to lung cancer risk has been extensively investigated in the literature. These studies have compared lung cancer risk in groups of smokers with or without polymorphisms in genes involved in PAH metabolism. We believe that carcinogen metabolite phenotyping, involving the actual measurement of PAH metabolites, would be a better way to investigate differences in lung cancer risk. With this goal in mind, we have developed methods for quantifying phenanthrene metabolites in urine. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity. The urinary metabolite r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) is a measure of metabolic activation, whereas phenanthrols (HOPhe) are a measure of detoxification. In this study, we quantified urinary PheT/HOPhe ratios in 346 smokers who were also genotyped for 11 polymorphisms in genes involved in PAH metabolism: CYP1A1MspI, CYP1A1I462V, CYP1B1R48G, CYP1B1A119S, CYP1B1L432V, CYP1B1N453S, EPHX1Y113H, EPHX1H139R, GSTP1I105V, GSTP1A114V, and GSTM1 null. The geometric mean molar PheT/3-HOPhe ratio was 4.08 (95% confidence interval, 3.79-4.39). Ten percent of the smokers had PheT/3-HOPhe ratios of ≥9.90. We found a significant association between the presence of the CYP1A1I462V polymorphism and high PheT/3-HOPhe ratios (P = 0.02). This effect was particularly strong in females and in combination with the GSTM1 null polymorphism. In contrast, the CYP1B1R48G and CYP1B1A119S polymorphisms were associated with significantly lower PheT/3-HOPhe ratios, particularly in Blacks. There were no consistent significant effects of any of the other polymorphisms on PheT/3-HOPhe ratios. The highest 10% of PheT/3-HOPhe ratios could not be predicted by the presence of any of the 11 polymorphisms individually or by certain combinations. The effects of the CYP1A1I462 polymorphism observed here, particularly in combination with GSTM1 null, are quite consistent with reports in the literature. However, the results of this study indicate that genotyping is not an effective way to predict PAH metabolism at least as represented by PheT/HOPhe ratios. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1805–11)

Longitudinal study of urinary phenanthrene metabolite ratios: effect of smoking on the diol epoxide pathway.

We have proposed that urinary phenanthrene metabolites could be used in a carcinogen metabolite phenotyping approach to identify individuals who may be susceptible to cancer induction by polycyclic aromatic hydrocarbons (PAH). In support of this proposal, we have developed methods for quantitation of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and phenanthrols (HOPhe) in human urine. PheT is the end product of the diol epoxide metabolic activation pathway of PAH, whereas HOPhe are considered as detoxification products. In this study, we investigated the longitudinal consistency of these metabolites over time in smokers and nonsmokers and compared their levels. Twelve smokers and 10 nonsmokers provided urine samples daily for 7 days, then weekly for 6 weeks. Levels of PheT, HOPhe, and PheT/HOPhe ratios were relatively constant in most individuals, with mean coefficients of variation ranging from 29.3% to 45.7%. There were no significant changes over time in levels of the metabolites or in ratios. These results indicate that a single urine sample should be sufficient when comparing phenanthrene metabolites in different groups. PheT/HOPhe ratios were significantly higher in smokers than in nonsmokers, showing that smoking induces the diol epoxide metabolic activation pathway of phenanthrene. This finding is consistent with previous studies indicating that inducibility of PAH metabolism contributes to cancer risk in smokers. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2969–74)

Children’s Exposure to Secondhand and Thirdhand Smoke Carcinogens and Toxicants in Homes of Hookah Smokers

INTRODUCTION We examined homes of hookah-only smokers and nonsmokers for levels of indoor air nicotine (a marker of secondhand smoke) and indoor surface nicotine (a marker of thirdhand smoke), child uptake of nicotine, the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and the toxicant acrolein by analyzing their corresponding metabolites cotinine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNAL-glucuronides (total NNAL) and 3-hydroxypropylmercapturic acid. METHODS Data were collected at 3 home visits during a 7-day study period from a convenience sample of 24 households with a child 5 years or younger. Three child urine samples and 2 air and surface samples from the living room and the child bedroom were taken in homes of nonsmokers (n = 5) and hookah-only smokers (n = 19) comprised of daily hookah smokers (n = 8) and weekly/monthly hookah smokers (n = 11). RESULTS Nicotine levels in indoor air and on surfaces in the child bedrooms in homes of daily hookah smokers were significantly higher than in homes of nonsmokers. Uptake of nicotine, NNK, and acrolein in children living in daily hookah smoker homes was significantly higher than in children living in nonsmoker homes. Uptake of nicotine and NNK in children living in weekly/monthly hookah smoker homes was significantly higher than in children living in nonsmoker homes. CONCLUSIONS Our data provide the first evidence for uptake of nicotine, the tobacco-specific lung carcinogen NNK, and the ciliatoxic and cardiotoxic agent acrolein in children living in homes of hookah smokers. Our findings suggest that daily and occasional hookah use in homes present a serious, emerging threat to childrens long-term health.

Urinary levels of volatile organic carcinogen and toxicant biomarkers in relation to lung cancer development in smokers

Besides polycyclic aromatic hydrocarbons (PAH) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which are established lung carcinogens, tobacco smoke also contains relatively large quantities of volatile organic carcinogens and toxicants, including 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde. Although animal experiments showed that some of these compounds can induce tumors in multiple organs including the lung, epidemiological studies of their relationship with lung cancer in smokers have not been reported. Therefore, in this study, we quantified urinary mercapturic acid metabolites of 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde in addition to urinary biomarkers for PAH, NNK and nicotine in 343 lung cancer cases and 392 matched controls among a cohort of 18,244 Chinese men in Shanghai, China, followed from 1986 to 2006. Compared with the lowest quartiles, highest quartiles of all measured mercapturic acids were associated with statistically significantly ~2-fold increased risk for lung cancer (all Ps for trend <0.01) after adjustment for smoking intensity and duration. The positive associations between biomarkers of ethylene oxide, benzene or acrolein and lung cancer risk remained statistically significant after adjustment for biomarkers of PAH and NNK, whereas urinary total cotinine completely explained the mercapturic acid metabolites and lung cancer associations (all Ps for trend ≥ 0.39). We conclude that mercapturic acid metabolites of 1,3-butadiene, ethylene oxide, benzene, acrolein and crotonaldehyde may not be independent risk predictors of lung cancer among Shanghai smokers, in contrast to biomarkers of PAH, NNK and nicotine exposure.

Elevated Levels of Volatile Organic Carcinogen and Toxicant Biomarkers in Chinese Women Who Regularly Cook at Home

Background: Epidemiologic studies associate lung cancer in nonsmoking Chinese women with Chinese-style wok cooking. Our goal was to quantify carcinogen and toxicant biomarkers in Chinese women who reported regularly doing home cooking compared with women randomly selected from the Singapore Chinese Health Study as controls. Methods: Biomarkers were quantified by high-performance liquid chromatography–mass spectrometry, high-performance liquid chromatography with fluorescence detection, and gas chromatography–mass spectrometry. Results: Compared with controls, women who engaged in regular home cooking had significantly higher levels of mercapturic acids of acrolein {geometric mean, 1,959 pmol/mg creatinine [95% confidence interval (95% CI), 1,554-2,467] versus 1,370 (95% CI, 1,077-1,742); P = 0.038}, crotonaldehyde [geometric mean, 232 pmol/mg creatinine (95% CI, 193-277) versus 142 (95% CI, 118-171); P = 0.0004], and benzene [geometric mean, 0.58 pmol/mg creatinine (95% CI, 0.44-0.78) versus 0.18 (95% CI, 0.14-0.24); P < 0.0001]. No significant differences were found in levels of mercapturic acids of 1,3-butadiene, metabolites of pyrene and phenanthrene, or acetaldehyde-leukocyte DNA adduct levels between the groups. Levels of the ethylene oxide mercapturic acid were significantly higher in the controls. Conclusions: The higher levels of the mercapturic acid of benzene, a multiorgan carcinogen, in the women who cooked are particularly notable. Overall, the results showing increased exposure to the volatile toxicants and carcinogens acrolein, crotonaldehyde, and benzene in Chinese women who regularly cook provide a plausible lead for further investigating the role of volatile compounds generated during high-temperature cooking with oils as causes of lung cancer. Impact: A new direction for research on lung cancer etiology is suggested. Cancer Epidemiol Biomarkers Prev; 19(5); 1185–92. ©2010 AACR.

High throughput liquid chromatography–tandem mass spectrometry assay for mercapturic acids of acrolein and crotonaldehyde in cigarette smokers’ urine

3-Hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) are urinary metabolites of the toxicants acrolein and crotonaldehyde, respectively. Virtually all human urine samples contain these metabolites, resulting from the action of glutathione-S-transferases on acrolein and crotonaldehyde, which are lipid peroxidation products, environmental and dietary contaminants, and constituents of cigarette smoke. We have developed a high throughput liquid chromatography-tandem mass spectrometry method for quantitative analysis of 3-HPMA and HMPMA in large numbers of small urine samples, as would be required in molecular epidemiology and clinical studies relating levels of these metabolites to cancer risk. Solid-phase extraction on mixed mode reverse phase-anion exchange 96-well plates provided sufficient purification for LC-MS/MS analysis, which was performed by auto-injection using a 96-well format, and resulted in clean, readily interpretable chromatograms, with detection limits of 4.5pmol/mL urine for 3-HPMA and 3.5pmol/mL urine for HMPMA. Accuracy was 92% for 3-HPMA and 97% for HMPMA while inter-day precision was 9.1% (coefficient of variation) for 3-HPMA and 11.0% for HMPMA. The method was applied to more than 2600 urine samples from smokers; mean levels of 3-HPMA and HMPMA were 4800±5358 (S.D.)pmol/mL and 3302±3341pmol/mL, respectively.