Yuxin Zheng

Simultaneous determination of urinary 1- and 2-naphthols, 3- and 9-phenanthrols, and 1-pyrenol in coke oven workers

A method was developed for simultaneous quantification of urinary 1- and 2-naphthols, 3- and 9-phenanthrols and 1-pyrenol using gas chromatography with mass spectrometry (GC-MS). This method was applied to urine samples from coke oven workers (n =28) and controls (n =22) from Northern China. Geometric mean levels of urinary 1-naphthol (58.8 μg l−1), 2-naphthol (34.1 μg l−1), 3-phenanthrol (7.35 μg l−1), 9-phenanthrol (1.28 μg l−1) and 1-pyrenol (25.4 μg l−1) were significantly higher among coke oven workers than controls. All the substances tested were highest among top-of-oven workers, who had 15-fold higher 1-naphthol, eight-fold higher 2-naphthol and 20-fold higher 1-pyrenol levels compared with controls. Using multiple linear regression models, 72.5% of the variation in 1- and 2-naphthol and 82.8% of the variation in 1-pyrenol were explained by the concentration of naphthalene or pyrene in the urine, the work category and the smoking intensity. Cigarette consumption significantly contributed to levels of urinary 1-pyrenol and naphthols, particularly 2-naphthol. A negative relationship between work category and the ratio of naphthols/1-pyrenol was observed among smokers. Our results suggest that urinary naphthols and phenanthrols reflect polycyclic aromatic hydrocarbon (PAH) exposure as well as the widely used 1-pyrenol, and that interactions between cigarette smoking and PAH exposure result in different patterns of metabolism for individual PAHs.

Determination of polycyclic aromatic hydrocarbons in urine of coke oven workers by headspace solid phase microextraction and gas chromatography–mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) represent a complex mixture of toxic compounds that are ubiquitous in the environment. We investigated the utility of head space-solid phase microextraction (HS-SPME) to measure the following surrogate PAHs in urine: naphthalene (NAP), phenanthrene (PHE), pyrene (PYR), and benzo(a)pyrene (BAP), representing classes of 2-, 3-, 4- and 5-ring compounds, respectively. We then applied the method to urine from 28 coke oven workers (median levels (microg/l) were: NAP=3.65, PHE=1.51, PYR=0.003, BAP not detected) and 22 controls (median (microg/l) NAP=0.859, PHE=0.062, PYR=0.001, BAP not detected). Urinary levels of NAP, PHE, and PYR were all associated with exposure category (controls, side- and bottom-workers, and top-workers) but not with smoking status. Strong correlations were observed between urinary levels of NAP, PHE, and PYR in coke-oven workers. Our results indicate that unmetabolized 2-, 3- and 4-ring PAHs can be measured in urine by HS-SPME. Such measurements can be used to investigate the uptake and metabolism of complex PAH mixtures in humans.

Urinary naphthalene and phenanthrene as biomarkers of occupational exposure to polycyclic aromatic hydrocarbons

Objectives: The study investigated the utility of unmetabolised naphthalene (Nap) and phenanthrene (Phe) in urine as surrogates for exposures to mixtures of polycyclic aromatic hydrocarbons (PAHs). Methods: The report included workers exposed to diesel exhausts (low PAH exposure level, n = 39) as well as those exposed to emissions from asphalt (medium PAH exposure level, n = 26) and coke ovens (high PAH exposure level, n = 28). Levels of Nap and Phe were measured in urine from each subject using head space-solid phase microextraction and gas chromatography-mass spectrometry. Published levels of airborne Nap, Phe and other PAHs in the coke-producing and aluminium industries were also investigated. Results: In post-shift urine, the highest estimated geometric mean concentrations of Nap and Phe were observed in coke-oven workers (Nap: 2490 ng/l; Phe: 975 ng/l), followed by asphalt workers (Nap: 71.5 ng/l; Phe: 54.3 ng/l), and by diesel-exposed workers (Nap: 17.7 ng/l; Phe: 3.60 ng/l). After subtracting logged background levels of Nap and Phe from the logged post-shift levels of these PAHs in urine, the resulting values (referred to as ln(adjNap) and ln(adjPhe), respectively) were significantly correlated in each group of workers (0.71⩽ Pearson r⩽0.89), suggesting a common exposure source in each case. Surprisingly, multiple linear regression analysis of ln(adjNap) on ln(adjPhe) showed no significant effect of the source of exposure (coke ovens, asphalt and diesel exhaust) and further suggested that the ratio of urinary Nap/Phe (in natural scale) decreased with increasing exposure levels. These results were corroborated with published data for airborne Nap and Phe in the coke-producing and aluminium industries. The published air measurements also indicated that Nap and Phe levels were proportional to the levels of all combined PAHs in those industries. Conclusion: Levels of Nap and Phe in urine reflect airborne exposures to these compounds and are promising surrogates for occupational exposures to PAH mixtures.

Albumin Adducts of Naphthalene Metabolites as Biomarkers of Exposure to Polycyclic Aromatic Hydrocarbons

We investigated the utility of adducts formed by the reaction of the naphthalene metabolites naphthalene-1,2-oxide, 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) with serum albumin (Alb) as biomarkers of exposure to polycyclic aromatic hydrocarbons. Cysteinyl serum Alb adducts of 1,2- and 1,4-NPQ (1,2-NPQ-Alb and 1,4-NPQ-Alb, respectively) but not of naphthalene-1,2-oxide were detected in 28 coke oven workers and 22 controls from the steel industry of northern China. The median level of 1,2-NPQ-Alb in coke oven workers (76.6 pmol/g) was significantly higher than that observed in controls (44.9 pmol/g; P = 0.0027). However, the median level of 1,4-NPQ-Alb in exposed subjects was not significantly different from that of controls (48.6 versus 44.2 pmol/g; P = 0.296). Levels of 1,2-NPQ-Alb were significantly correlated with exposure category (controls, side and bottom workers, and top-of-oven workers) as well as with previously measured levels of urinary naphthalene, 1- and 2-naphthol, and 1-pyrenol in these subjects. Multiple linear regression analysis revealed that 35% of the variation in 1,2-NPQ-Alb could be explained by the work category and age. A negative relationship between 1,2-NPQ-Alb and age was observed, suggesting that cytochrome P450 c metabolism diminished with age at ∼3%/year of life.

Drinking Water Disinfection Byproduct Iodoacetic Acid Induces Tumorigenic Transformation of NIH3T3 Cells

Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 μg/L IAA and 0.86 μg/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 μM exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 μM). IAA, but not IF, induced a concentration-dependent DNA damage measured by γ-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern.

A sandwich enzyme-linked immunosorbent assay for adducts of polycyclic aromatic hydrocarbons with human serum albumin

Adducts of benzo[a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive enzyme-linked immunosorbent assay (ELISA) that employs monoclonal antibody 8E11 to detect benzo[a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we used 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1-mg samples of HSA or 20 microl of serum/plasma. The assay employs an anti-HSA antibody for detection, and this is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is approximately 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high PAH exposures (coke oven workers), medium PAH exposures (steel factory control workers), and low PAH exposures (volunteer subjects) (n=30). The respective geometric mean levels of BPDE-HSA adducts--67.8, 14.7, and 1.93 ng/mg HSA (1010, 220, and 28.9 fmol BPDE equiv/mg HSA)--were significantly different (P<0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available.

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

Abstract The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers

Water pollutant fingerprinting tracks recent industrial transfer from coastal to inland China: a case study.

In recent years, China’s developed regions have transferred industries to undeveloped regions. Large numbers of unlicensed or unregistered enterprises are widespread in these undeveloped regions and they are subject to minimal regulation. Current methods for tracing industrial transfers in these areas, based on enterprise registration information or economic surveys, do not work. We have developed an analytical framework combining water fingerprinting and evolutionary analysis to trace the pollution transfer features between water sources. We collected samples in Eastern China (industrial export) and Central China (industrial acceptance) separately from two water systems. Based on the water pollutant fingerprints and evolutionary trees, we traced the pollution transfer associated with industrial transfer between the two areas. The results are consistent with four episodes of industrial transfers over the past decade. Our results also show likely types of the transferred industries - electronics, plastics, and biomedicines - that contribute to the water pollution transfer.

Combined exposure to 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone and microsytin-LR increases genotoxicity in Chinese hamster ovary cells through oxidative stress.

The disinfection byproducts 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX) and microcystins-LR (MC-LR), which are common contaminants in drinking water, often occur together in water sources in areas with high gastrointestinal tract cancer risks. While often studied alone, combination effects of these compounds are unknown. Here, we examine combined genotoxic responses to mixtures of MX and MC-LR using the Ames test, a cytokinesis-block micronuclei assay, and the comet assay with analysis for interactions by fractional analysis. We also evaluated a possible mechanism of genotoxicity by examining effects of the compounds on markers of oxidative stress. MX and MC-LR administrated jointly at noncytotoxic concentrations demonstrated significant interactions in the Ames test, the micronuclei assay, and the comet assay showing responses greater than those expected for additivity. Moreover, coexposure to MX and MC-LR significantly increased luciferase antioxidant response element activity, intracellular superoxide dismutase, catalase, glutathione, and reactive oxygen species production. In comparison with exposure to either compound alone, the mixtures of MX and MC-LR caused a less than additive effect on oxidative stress. Taken together, these results indicate that MC-LR exacerbates MX genotoxicity in low-dose combined exposure. This interaction may be enhanced by oxidative stress in the combined exposures.

Predicting cytotoxicity of complex mixtures in high cancer incidence regions of the Huai River Basin based on GC–MS spectrum with partial least squares regression

Complex mixture exposures, such as those associated with water sources, are an important issue in health risk assessment. This study assessed the cytotoxicity of chemical mixtures extracted from water sources in regions of the Huai River Basin with high cancer incidences and built statistical models of cytotoxicity based on pollution profiles that were measured with gas chromatography-mass spectrometry (GC-MS). Both surface and ground waters were collected from rural water sources of Shenqiu County, Henan Province of China from 2008 to 2011 and extracted with XAD-2 resigns. Cytotoxicity was evaluated with Chinese hamster ovary K1 (CHO-K1) cells and compared against the pollution profiles of the extracts. IC50 of water samples ranged from 0.023 to 0.338L-eq/mL. The pollutants in waters determined by GC-MS are complex and some of the compounds that contributed to cytotoxicity lack toxicity data. A partial least squares (PLS) regression model of cytotoxicity was built based on linear aggregation of predictor variables (i.e., peaks for single compounds in the gas chromatograms). The PLS model contains 2 PLS factors extracted from 141 variables. The model was validated internally with training data permutation and externally with a test sample. The model explained 92% of the cytotoxicity in the training samples and 40% in the test sample. This approach provides a general, rapid method for relating water toxicity to GC-MS chromatograms and for predicting the compounds that contribute most to toxicity.