Chiung-Wen Hu

Characterization of multiple airborne particulate metals in the surroundings of a municipal waste incinerator in Taiwan

Abstract Heavy metals are one of the concerned pollutants emitted by the municipal waste incineration system (MWIs). The objective of this study was to evaluate the potential impact on local airborne metals from the emissions of an MWI. Aerosol samples were simultaneously collected at eight different sites around the municipal waste incinerator using PS-1 sampler. The concentrations of 16 elements (Mg, Al, Fe, Cu, Zn, Pb, Ti, V, Cr, Mn, Co, Ni, As, Cd, Ba and Hg) were quantified by inductively coupled plasma-mass spectrometry (ICP–MS) and atomic absorption spectrometer (AA). The profiles of the 16 metals in the surroundings of a municipal incinerator in central Taiwan were compared with those of the emission sources. The results showed that the profiles of multiple metals obtained at all sampling sites were similar to those emitted from the MWI stack. These findings suggested that the local airborne metal pollutants might probably derive from the stack emission of the MWI. Using cadmium as an index metal, it was found that the metals like Mg, Ti, V, Cr, Mn, Co, Ni, As, and Hg are highly influenced by the stack emission from the municipal incinerator. Moreover, the ratio of other metals to Cd that were increased with the distance from the incinerator. This might be due to the additional sources contributed to airborne metals following the emission from the incinerator and a difference in particle size of each particle-bound metal.

Comparative syntheses of tetracycline-imprinted polymeric silicate and acrylate on CdTe quantum dots as fluorescent sensors.

The amphoteric drug molecule tetracycline, which contains groups with pKa 3.4-9.9, was used as a template for conjugating molecularly imprinted polymers (MIPs) and as a quencher for CdTe quantum dot (QD) fluorescence. Two MIP-QD composites were synthesized by a sol-gel method using a silicon-based monomer and a monomer linker between the MIP and QD, i.e., tetraethoxylsilane/3-mercaptopropyltriethoxysilane (MPS) and tetraethoxylsilane/3-aminopropyltriethoxysilane (APS). Another MIP-QD composite was synthesized by the chain-growth polymerization of methacrylic acid (MAA) and an allyl mercaptan linker. The prepared MIP-QDs were characterized by FTIR and SEM and utilized at 0.33 mg/mL to determine the tetracycline content in phosphate buffers (pH 7.4, 50mM) through the Perrin and Stern-Volmer models of quenching fluorometry. The Perrin model was applied to tetracycline concentrations of 7.4 μM-0.37 mM for MIP-MPS-QD, 7.4 μM-0.12 mM for MIP-APS-QD, and 7.4 μM-0.10mM for MIP-MAA-QD (R(2)=0.9988, 0.9978, and 0.9931, respectively). The Stern-Volmer model was applied to tetracycline concentrations of 0.12-0.37 mM for MIP-APS-QD (R(2)=0.9983) and 0.10-0.37 mM for MIP-MAA-QD (R(2)=0.9970). The detection limits were 0.45 μM, 0.54 μM, and 0.50 μM for MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively. Equilibrium times, differences between imprinted and nonimprinted polymers, and MIP-QD quenching mechanisms were discussed. Finally, specificity studies demonstrated that MIP-MAA-QD exhibited optimal recoveries of 96% from bovine serum albumin (n=5, RSD=3.6%) and 91% from fetal bovine serum (n=5, RSD=4.8%).

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction A cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.

Repeated measurements of urinary methylated/oxidative DNA lesions, acute toxicity, and mutagenicity in coke oven workers

We conducted a repeated-measures cohort study of coke oven workers to evaluate the relationships between the traditional exposure biomarker, urinary 1-hydroxypyrene (1-OHP), and a series of biomarkers, including urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), N7-methylguanine (N7-MeG), acute toxicity, and mutagenicity. A total of eight spot urine samples were collected from each high-exposed (at topside oven area) and low-exposed workers (at side oven area) during the whole working cycle, which consisted of 6 consecutive days of working followed by 2 days off. Our results showed that the high-exposed workers had significantly higher urinary levels of 1-OHP, 8-oxodG, and N7-MeG compared with the low-exposed workers. Acute toxicity and mutagenicity of urine were also found to be markedly increased in the high-exposed workers, as determined by Microtox assay and Ames test, respectively. Multivariate regressions analysis revealed that the urinary 8-oxodG, N7-MeG, or acute toxicity was significantly correlated with 1-OHP concentrations. Overall, the present study showed that exposure to coke oven emissions increased oxidatively damaged DNA products and mutagenicity of urine, and for the very first time, such exposure was also found to increase DNA methylation and urinary acute toxicity. The potential source of methylating agents in coke oven emissions warrants further investigation. Additionally, with repeated measurements, the pattern of time course for urinary 1-OHP was found to be different from those of 8-oxodG and N7-MeG, as well as acute toxicity and mutagenicity. This finding implies that the single measurement that was often conducted in occupational healthy investigations should be used with certain precautions, because single measurement may fail to provide the proper information of interest. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3381–9)

Simultaneous determination of N7-alkylguanines in DNA by isotope-dilution LC-tandem MS coupled with automated solid-phase extraction and its application to a small fish model

In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.

High-Throughput Simultaneous Analysis of Five Urinary Metabolites of Areca Nut and Tobacco Alkaloids by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction

Background: Areca nut and tobacco are commonly used drugs worldwide and have been frequently used in combination. We describe the use of on-line solid-phase extraction and isotope-dilution liquid chromatography-tandem mass spectrometry for the simultaneous measurement of five major urinary metabolites of both areca nut and tobacco alkaloids, namely, arecoline, arecaidine, N-methylnipecotic acid, nicotine, and cotinine. Methods: Automated purification of urine was accomplished with a column-switching device. After the addition of deuterium-labeled internal standards, urine samples were directly analyzed within 13 minutes. This method was applied to measure urinary metabolites in 90 healthy subjects to assess areca nut/tobacco exposure. Urinary time course of arecoline, arecaidine, and N-methylnipecotic acid was investigated in five healthy nonchewers after oral administration of areca nut water extracts. Results: The limits of detection were 0.016 to 0.553 ng/mL. Interday and intraday imprecision were <10%. Mean recoveries of five metabolites in urine were 97% to 114%. Mean urinary concentrations of arecoline, arecaidine, N-methylnipecotic acid, nicotine, and cotinine in regular areca nut chewers also smokers were 23.9, 5,816, 1,298, 2,635, and 1,406 ng/mg creatinine, respectively. Time course study revealed that after administration of areca nuts extracts, the major urinary metabolite was arecaidine with a half-life of 4.3 hours, followed by N-methylnipecotic acid with a half-life of 7.9 hours, and very low levels of arecoline with a half-life of 0.97 hour. Conclusions: This on-line solid-phase extraction liquid chromatography-tandem mass spectrometry method firstly provides high-throughput direct analysis of five urinary metabolites of areca nut/tobacco alkaloids. Impact: This method may facilitate the research into the oncogenic effects of areca nut/tobacco exposure. Cancer Epidemiol Biomarkers Prev; 19(10); 2570–81. ©2010 AACR.

Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products.

Areca nut is a carcinogen to humans and has been strongly associated with oral premalignant and malignant diseases. Previous studies speculated the presence of unknown direct-acting mutagens present in aqueous extracts of areca nut. We hypothesized whether any direct-acting alkylating agents are present in areca nut and its commercial products. In this study, calf thymus DNA was treated with four different aqueous extracts obtained from unripe and ripe areca nuts or their commercial products, namely, pan masala (without tobacco) and gutkha (with tobacco). Three N-alkylated purines including N7-methylguanine (N7-MeG), N3-methyladenine (N3-MeA), and N7-ethylguanine (N7-EtG) were detected using sensitive and specific isotope-dilution liquid chromatography-tandem-mass spectrometry (LC-MS/MS) methods. The results showed that four types of aqueous extracts significantly induced the formation of N7-MeG and N3-MeA in a linear dose-response manner. Extracts from unripe areca nut exhibited higher methylating potency than those of ripe areca nut, while gutkha had higher methylating potency than pan masala. Meanwhile, gutkha made with areca nut and tobacco, was the only extract found to induce the formation of N7-EtG. Overall, this study first demonstrated that the presence of direct-acting alkylating agents in areca nut and its commercial products exist at a level that is able to cause significant DNA damage. Our findings may provide another mechanistic rationale for areca nut-mediated oral carcinogenesis and also highlight the importance and necessity of the identification of these direct-acting alkylating agents.

Modification of urinary N7-methylguanine excretion in smokers by glutathione-S-transferase M1 polymorphism

Tobacco smoke contains many carcinogens which can lead to DNA methylation. Glutathione-S-transferases (GSTs) are detoxifying enzymes, and the effects of the genes GSTM1, GSTT1, and GSTP1 on cigarette smoke-induced methylated adducts have not been elucidated. Therefore, we investigated the association of the GSTM1, GSTT1, and GSTP1 genes and N7-methylguanine (N7-MeG) adducts in smokers. Urinary N7-MeG concentrations were measured by using liquid chromatography-tandem mass spectrometry in 112 smokers and 89 non-smokers, and GSTM1, GSTT1, and GSTP1 genotypes were identified by polymerase chain reaction. Smokers had higher N7-MeG concentrations than did non-smokers (3238+/-305 ng/mg creatinine [standard error] vs. 2386+/-153 ng/mg creatinine; P=0.01). Higher N7-MeG concentrations were observed with the GSTM1 null genotype than with the GSTM1 non-null genotype (3230+/-292 ng/mg creatinine vs. 2336+/-153 ng/mg creatinine; P=0.007), particularly in smokers (3775+/-483 ng/mg creatinine vs. 2468+/-228 ng/mg creatinine; P=0.02). However, the GSTT1 and GSTP1 genotypes were not associated with urinary N7-MeG concentrations. Therefore, the susceptible GSTM1 genotype may modulate the concentrations of N7-MeG adducts in the DNA of smokers.

Urinary nitrite/nitrate ratio measured by isotope-dilution LC–MS/MS as a tool to screen for urinary tract infections

Urinary tract infections (UTIs) are the most common type of nosocomial infection. Traditionally, the presence of white blood cells and microorganisms in the urine provides objective evidence for UTI diagnosis. Here, we describe the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the nitrite and nitrate levels in urine and investigate the potential of this method for UTI diagnosis. LC-MS/MS analysis was performed in positive electrospray ionization mode. After adding (15)N-labeled internal standards and derivatizing with 2,3-diaminonaphthalene (DAN), the urinary nitrite content was directly analyzed by LC-MS/MS, whereas the urinary nitrate was first reduced to nitrite before derivatization and LC-MS/MS analysis. The derivatization of nitrite and enzymatic reduction of nitrate were optimized. This method was then applied to 241 healthy subjects and 73 UTI patients. Optimization tests revealed that 1 mL of crude urine required at least 6.25 μmol of DAN to completely derivatize nitrite and 2.5 U of nitrate reductase to completely reduce nitrate to nitrite. Urinary analysis showed that the urinary concentration of nitrite and the nitrite/nitrate ratio were higher in UTI patients than in healthy subjects. Compared with the dipstick-based urinary nitrite test and using LC-MS/MS to determine the nitrite concentration (sensitivity: 23-25%), the nitrite/nitrate ratio was significantly more sensitive (95%) and exhibited a satisfactory specificity (91%) in the screening of UTIs. Taken together, the nitrite/nitrate ratio, which reflects the reducing ability of pathogenic bacteria, could be a better method for the diagnosis of UTIs that is not subject to variations in urine specimen quality.

Inhalation exposure of children to indoor PM10 and associated metals during river-dust episodes

Three exposure groups (high exposure, low exposure, and control), using six elementary schools in Yulin County, were selected to study the impacts of aeolian river-dust on school children’s exposure to PM10 and associated metals. One classroom and about five school-aged children’s houses for each school were chosen to collect indoor PM10 during the river-dust episodes (RDEs) and non-river-dust episodes (NRDEs). The results indicated that the river-dust episodes had significant impacts on the school-aged children’s exposure to concentrations of PM10 and metals, especially in the high exposure group. For the Ni and Mn metals, the 8-h school exposure concentrations during RDEs were both higher than the standards suggested by the California Environmental Protection Agency. Three interventions for protecting school children from being affected by the river dust during RDEs are suggested in this study. Among the three interventions, children who have a day off of school during RDEs can result in the greatest decrease in the exposure levels of PM10 and associated metals.