Chih-Chun Jean Huang

Rapid and sensitive on-line liquid chromatographic/tandem mass spectrometric determination of an ethylene oxide-DNA adduct, N7-(2-hydroxyethyl)guanine, in urine of nonsmokers

Ethylene oxide (EtO) is classified as a known human carcinogen. The formation of EtO-DNA adducts is considered as an important early event in the EtO carcinogenic process. An isotope-dilution on-line solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry method was then developed to analyze one of the EtO-DNA adducts, N7-(2-hydroxyethyl)guanine (N7-HEG), in urine of 46 nonsmokers with excellent accuracy, sensitivity and specificity. The merits of this method include small sample volume (only 120 microL urine required), automated sample cleanup, and short total run time (12 minutes per sample). This method demonstrates its high-throughput capacity for future molecular epidemiology studies on the potential health effects resulting from the low-dose EtO exposure.

Comparative analysis of urinary N7-(2-hydroxyethyl)guanine for ethylene oxide- and non-exposed workers.

Ethylene oxide (EO), a direct alkylating agent and a carcinogen, can attack the nucleophilic sites of DNA bases to form a variety of DNA adducts. The most abundant adduct, N7-(2-hydroxyethyl)guanine (N7-HEG), can be depurinated spontaneously or enzymatically from DNA backbone to form abasic sites. Molecular dosimetry of the excised N7-HEG in urine can serve as an EO exposure and potential risk-associated biomarker. This study was to analyze N7-HEG in urine collected from 89 EO-exposed and 48 nonexposed hospital workers and 20 exposed and 10 nonexposed factory workers by using our newly developed on-line solid-phase extraction isotope-dilution LC-MS/MS method. Statistical analysis of data shows that the exposed factory workers excreted significantly greater concentrations of N7-HEG than both the nonexposed factory workers and hospital workers. Multiple linear regression analysis reveals that the EO-exposed factory workers had a significantly greater post-shift urinary N7-HEG than their nonexposed coworkers and hospital workers. These results demonstrate that analysis of urinary N7-HEG can serve as a biomarker of EO exposure for future molecular epidemiology studies to better understand the role of the EO-induced DNA adduct formation in EO carcinogenicity and certainly for routine surveillance of occupational EO exposure for the study of potential health impacts on workers.

Potential Association of Urinary N7-(2-Carbamoyl-2-hydroxyethyl) Guanine with Dietary Acrylamide Intake of Smokers and Nonsmokers.

Acrylamide (AA), a rodent carcinogen, is widely used in industry and present in cigarette smoke as well as in foods processed at high temperatures. The metabolic activation of AA to glycidamide (GA) could be critical for AA carcinogenicity since GA causes DNA adduct formation in vivo. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct of AA, is subjected to spontaneous and enzymatic depurination and excreted through urine. Urinary N7-GAG analysis can confirm AA genotoxicity and identify active species of AA metabolites in humans, thereby serving as a risk-associated biomarker for molecular epidemiology studies. This study aimed to develop an isotope-dilution solid-phase extraction liquid chromatography tandem mass spectrometry method to comparatively analyze urinary N7-GAG levels in nonsmokers and smokers. Urinary N-acetyl-S-(propionamide)-cysteine (AAMA), a metabolite of AA, was also analyzed as a biomarker for current AA exposure. Urinary N7-GAG was quantified by monitoring m/z 239 → 152 for N7-GAG and m/z 242 → 152 for (13)C3-labeled N7-GAG under positive electron spray ionization and multiple reaction mode. The median urinary N7-GAG level was 0.93 μg/g creatinine in nonsmokers (n = 33) and 1.41 μg/g creatinine in smokers (n = 30). Multiple linear regression analysis of data revealed that N7-GAG levels were only significantly associated with AAMA levels. These results demonstrate that urinary N7-GAG of nonsmokers and smokers is significantly associated with a very low level of dietary AA intake, assessed by analyzing urinary AAMA.

Feasibility of using urinary N7-(2-carbamoyl-2-hydroxyethyl) Guanine as a biomarker for acrylamide exposed workers

Acrylamide (AA), a probable human carcinogen, is a widely-used industrial chemical but is also present in tobacco smoke and carbohydrate-rich foods processed at high temperatures. AA is metabolized to glycidamide (GA) to cause the formation of DNA adducts. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct induced by GA, was recently detected in urine of smokers and non-smokers. In this study, we assessed the variability of AA exposure and biomarkers of AA exposure in urine samples repeatedly collected from AA-exposed workers and explored the half-life of N7-GAG. A total of 8 AA-exposed workers and 36 non-exposed workers were recruited. Pre-shift and post-shift urine samples were collected from the exposed group in parallel with personal sampling for eight consecutive days and from the control group on day 1 of the study. Urinary N7-GAG and the mercapturic acids of AA and GA, namely N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R,S)-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-l-cysteine (GAMA) were analyzed using on-line solid phase extraction-liquid chromatography-electrospray ionization/tandem mass spectrometry methods. We found that N7-GAG levels in urine were significantly higher in exposed workers than in controls and that N7-GAG level correlated positively with AAMA and GAMA levels. Results from this study showed that AAMA and GAMA possibly remain the more preferred biomarkers of AA exposure and that N7-GAG levels could be elevated by occupational exposures to AA and serve as a biomarker of AA-induced genotoxicity for epidemiological studies.