Wen-Qing Lu

Elevated levels of urinary 8-hydroxy-2 -deoxyguanosine, lymphocytic micronuclei, and serum glutathione S-transferase in workers exposed to coke oven emissions.

To investigate associations among occupational exposure to coke oven emissions (COEs), oxidative stress, cytogenotoxic effects, change in the metabolizing enzyme glutathione S-transferase (GST), and internal levels of polycyclic aromatic hydrocarbons (PAHs) in coke oven workers, we recruited 47 male coke oven workers and 31 male control subjects from a coke oven plant in northern China. We measured the levels of 1-hydroxypyrene (1-OHP) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine, micronucleated binucleated cells (BNMNs) in peripheral blood lymphocyte, and GST in serum. Our results showed that the group exposed to COEs had significantly increased levels of 1-OHP [median 5.7; interquartile range (IQR), 1.4–12.0 μmol/mol creatinine] compared with the control group (3; 0.5–6.4 μmol/mol creatinine). In addition, the median levels (IQR) of 8-OHdG, BNMNs, and GST were markedly increased in the exposed [1.9 (1.4–15.4) μmol/mol creatinine; 6 (2–8) per thousand; 22.1 (14.9–31.2) U/L, respectively] compared with controls [1.3 (1.0–4.0) μmol/mol creatinine, 2 (0–4) per thousand; and 13.1 (9.5–16.7) U/L, respectively]. These results appeared to be modified by smoking. However, multivariate logistic regression analysis revealed that exposure to COEs had the highest odds ratio among variables analyzed and that smoking was not a significant confounder of the levels of studied biomarkers. Overall, the present findings suggest that COE exposure led to increased internal PAH burden, genetic damage, oxidative stress, and GST activity. The consequences of the changes in these biomarkers, such as risk of cancer, warrant further investigations.

Phthalate exposure and human semen quality: Results from an infertility clinic in China

Exposure to phthalates has been demonstrated to have adverse effects on male reproduction in animal studies, but findings in human studies have been inconsistent. We recruited 1040 men from the Reproductive Center of Tongji Hospital in Wuhan, China from March to June 2013. Each man provided one semen sample and two urine samples. Semen quality parameters and the urinary concentrations of eight phthalate metabolites were determined. After multivariable adjustments, the urinary concentrations of monobutyl phthalate (MBP) were found to be positively associated with the below-reference sperm concentration and total sperm count, and the odds ratios (ORs) comparing extreme MBP quartiles were 2.01 (95% CI: 1.07, 3.79; p for trend=0.06) and 1.80 (95% CI: 1.05, 3.08; p for trend=0.02), respectively. The associations were confirmed by multivariable linear regression analysis, which showed that the MBP concentration was significantly associated with decreasing trends in the sperm concentration and total sperm count (both p for trend <0.05). Additionally, we found significant dose-dependent relationships of the urinary level of mono-(2-ethylhexyl) phthalate (MEHP) and the percentage of di-(2-ethylhexyl)-phthalate metabolites (DEHP) excreted as MEHP (%MEHP) with an increased percentage of abnormal heads (both p for trend <0.01). Our findings suggest that environmental exposure to di-n-butyl phthalate (DBP) and DEHP may contribute to a decline in semen quality.

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay.

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM)>dibromochloromethane (DBCM)>tribromomethane (TBM)>trichloromethane (TCM) of the four THMs; iodoacetic acid (IA)>bromoacetic acid (BA)>dibromoacetic acid (DBA)>dichloracetic acid (DCA)>trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN)approximately dichloroacetonitrile (DCN)>trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs.

Semen phthalate metabolites, spermatozoa apoptosis, and DNA damage: a cross-sectional study in China.

Toxicological studies have shown that phthalates, a class of widely used chemicals, can impair male reproductive function, but epidemiological evidence is inconsistent. This study aimed to investigate the associations of semen phthalate metabolites with sperm apoptosis and DNA damage in a Chinese population. We assessed sperm apoptosis markers with Annexin V/PI analysis and sperm DNA integrity with comet assay before measuring eight phthalate metabolites in semen by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) among 463 men from Wuhan, China. We found a suggestive dose-response relationship between semen mono-(2-ethylhexyl) phthalate (MEHP) and an increased percentage of Annexin V+/PI- sperm (p for trend of <0.10). We also observed that semen monomethyl phthalate (MMP) and monoethyl phthalate (MEP) were associated with significant dose-related increases in tail length of the comet (both p for trend of <0.01). In conclusion, our data indicate that semen MEHP is associated with increased sperm apoptosis and that semen MMP and MEP are associated with increased sperm DNA damage in a Chinese population.

Assessment of the cytotoxicity and genotoxicity of haloacetic acids using microplate-based cytotoxicity test and CHO/HGPRT gene mutation assay

Haloacetic acids (HAAs) are the second most prevalent class of disinfection byproducts found in drinking water. The implications of HAAs presence in drinking water are a public health concern due to their potential mutagenic and carcinogenic effects. In the present study, we examined the cytotoxic and genotoxic effects of six common HAAs using a microplate-based cytotoxicity test and a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene mutation assay in Chinese hamster ovary K1 (CHO-K1) cells. We found that their chronic cytotoxicities (72h exposure) to CHO-K1 cells varied, and we ranked their levels of toxicity in the following descending order: iodoacetic acid (IA)>bromoacetic acid (BA)>dibromoacetic acid (DBA)>chloroacetic acid (CA)>dichloroacetic acid (DCA)>trichloroacetic acid (TCA). The toxicity of IA is 1040-fold of that of TCA. All HAAs except TCA were shown to be mutagenic to CHO-K1 cells in the HGPRT gene mutation assay. The mutagenic potency was compared and ranked as follows: IA>DBA>BA>CA>DCA>TCA. There was a statistically significant correlation between cytotoxicity and mutagenicity of the HAAs in CHO-K1 cells. The microplate-based cytotoxicity assay and HGPRT gene mutation assay were suitable methods to monitor the cytotoxicity and genotoxicity of HAAs, particularly for comparing the toxic intensities quantitatively.

Studies on the in vivo and in vitro mutagenicity and the lipid peroxidation of chlorinated surface (drinking) water in rats and metabolically competent human cells

In the present study, DNA damaging and mutagenic effects of chlorinated drinking water (CDW) extracts obtained from polluted raw water resources were examined in metabolically competent human Hep G2 hepatoma cells using the in vitro micronucleus assay and the single cell gel electrophoresis (SCGE, comet assay). Additionally, the in vivo induction of micronuclei (MN) was studied in polychromatic erythrocytes (PCEs) derived from bone marrow of CDW-treated Wistar rats. Furthermore, we examined the influence of CDW on the lipid peroxidation (LpO) in blood, liver, kidney and testicle of rats. The results demonstrated significant increases of micronucleated PCEs in the bone marrow of rats fed with relatively low CDW doses (33.3ml/kg body weight per day). Similar effects, i.e. increases of MN frequencies, were found in Hep G2 hepatoma cells after CDW treatment (41 MN/1000 binucleated cells (BNCs) for 167ml CDW) in comparison to the vehicle control (24 MN/1000 BNC). Additionally, DNA damages caused by CDW were observed in the comet assay. As a product of LpO, the levels of malondialdehyde (MDA) were significantly enhanced almost in all animals and organs tested after CDW treatment. In livers and serum of rats dose-dependent increases of MDA were observed. The data indicated that extracts from CDW obtained from polluted raw water were able to cause oxidative damages and to induce various biological effects in mammalian cells in vivo and in vitro, i.e. clastogenicity and/or aneugenicity, DNA strand breaks and/or alkali-labile damages. The consistency of the results among the various biological systems and endpoints led to the conclusion that the consumption of chlorinated drinking water obtained from polluted raw water may enhance the body burden with mutagenic and/or carcinogenic substances and therefore, means a potential genetic hazard for human health.

Phthalate exposure in association with serum hormone levels, sperm DNA damage and spermatozoa apoptosis: A cross-sectional study in China

Exposure to phthalates has been demonstrated to cause reproductive toxicity in animals, but evidence of the association between phthalates and markers of male reproductive function have been inconsistent in human studies. Here we examined whether environmental exposure to phthalates contributes to altered reproductive hormone levels, sperm DNA damage and spermatozoa apoptosis in a Chinese population. From March to June 2013, repeated urine samples collected from male partners of couples attending an infertility clinic in Wuhan, China were analyzed for 8 phthalate metabolites. Associations of the urinary phthalate metabolites with serum hormone levels (n=483), sperm DNA damage parameters (n=509) and spermatozoa apoptosis measures (n=467) were assessed using multivariable linear regression models. After adjusting for potential confounders, mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of di-(2-ethylhexyl)-phthalate (DEHP), was inversely associated with serum levels of estradiol, total testosterone (T) and free T (all P for trend<0.05). Additionally, we found positive dose-response relationships between the percentage of DEHP metabolites excreted as MEHP (%MEHP) and percentages of tail DNA (P for trend<0.05) and between three metabolites of DEHP [MEHP, mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP)] and percentages of Annexin V+/PI- spermatozoa (all P for trend<0.05). Our findings strengthen the emerging evidence that exposure to DEHP may alter hormone levels, disrupt sperm DNA integrity and induce spermatozoa apoptosis.

Variability of Metal Levels in Spot, First Morning, and 24-Hour Urine Samples over a 3-Month Period in Healthy Adult Chinese Men.

Background: Metals in single spot urine samples are often used to estimate individual exposure in human studies. However, measurements in urine could vary greatly over time due to variable exposure, potentially leading to exposure misclassification. Objective: We examined the variability of metal levels in the urine of 11 men who provided 529 samples on 8 days during a 3-month period, which corresponds to the duration of spermatogenesis. Method: The urinary levels of arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), lead (Pb), molybdenum (Mo), and nickel (Ni) were measured using inductively coupled plasma–mass spectrometry. We calculated the intraclass correlation coefficients (ICCs) to assess the reproducibility of metal measures and computed the sensitivity and specificity to evaluate how well spot urine samples determined the individuals’ 3-month average exposure. Results: Fair to good reproducibility was observed for the serial measurements of Cd (ICC = 0.53) in spot samples collected during the 3-month period, whereas the serial measurements of As, Co, Cu, Pb, Mo, and Ni showed poor reproducibility (ICCs = 0.01–0.29). Use of single spot urine samples to classify the high (top 33%) 3-month average metal levels had uniformly high specificities (0.70–0.84) but relatively low sensitivities (0.40–0.57), except for Cd (0.77). The minimum number of specimens (k) required to estimate the participant-specific mean for the seven metals within 20% of the “true” values ranged from 3 for Cd to 27 for Ni. Conclusions: The high variability observed in the urinary levels of As, Co, Cu, Pb, Mo, and Ni suggests that a single measurement provides only a brief snapshot in time of the exposure levels of an individual, which can result in a moderate degree of exposure misclassification. Citation: Wang YX, Feng W, Zeng Q, Sun Y, Wang P, You L, Yang P, Huang Z, Yu SL, Lu WQ. 2016. Variability of metal levels in spot, first morning, and 24-hour urine samples over a 3-month period in healthy adult Chinese men. Environ Health Perspect 124:468–476; http://dx.doi.org/10.1289/ehp.1409551

Benzo(a)pyrene induced micronucleus formation was modulated by persistent organic pollutants (POPs) in metabolically competent human HepG2 cells

Due to their bioaccumulation and their biological properties persistent organic pollutants (POPs) attract wide attention. In the present study we investigated the genotoxicity, cogenotoxicity and antigenotoxicity of three selected POPs (DDT, aroclor-1254 and toxaphene) in the HepG2 micronucleus assay. Exposure of HepG2 cells to DDT (17.8-60 microM) and aroclor-1254 (23-184 microM) alone did not increase the micronucleus-frequencies. A slight genotoxic effect could be observed after exposure to toxaphene (20-40 microM). Additionally, the ability of POPs to enhance/decrease the benzo(a)pyrene (BaP)-induced micronucleus formation was investigated. Exposure of HepG2 cells to 50 microM BaP alone led to a more than 2-fold increase of micronuclei (MN) compared with the background frequency. But when the cells were pretreated with 23-181 microM aroclor-1254 or 10-20 microM toxaphene, BaP exposure caused significantly more MN than BaP alone. In contrast, DDT (17.8-60 microM) reduced BaP-induced micronucleus induction by 6-38%. Mechanisms of action are discussed.

Drinking-Water Disinfection By-products and Semen Quality: A Cross-Sectional Study in China

Background: Exposure to disinfection by-products (DBPs) has been demonstrated to impair male reproductive health in animals, but human evidence is limited and inconsistent. Objective: We examined the association between exposure to drinking-water DBPs and semen quality in a Chinese population. Methods: We recruited 2,009 men seeking semen analysis from the Reproductive Center of Tongji Hospital in Wuhan, China, between April 2011 and May 2012. Each man provided a semen sample and a urine sample. Semen samples were analyzed for sperm concentration, sperm motility, and sperm count. As a biomarker of exposure to drinking-water DBPs, trichloroacetic acid (TCAA) was measured in the urine samples. Results: The mean (median) urinary TCAA concentration was 9.58 (7.97) μg/L (interquartile range, 6.01–10.96 μg/L). Compared with men with urine TCAA in the lowest quartile, increased adjusted odds ratios (ORs) were estimated for below-reference sperm concentration in men with TCAA in the second and fourth quartiles (OR = 1.79; 95% CI: 1.19, 2.69 and OR = 1.51; 95% CI: 0.98, 2.31, respectively), for below-reference sperm motility in men with TCAA in the second and third quartiles (OR = 1.46; 95% CI: 1.12, 1.90 and OR = 1.30; 95% CI: 1.00, 1.70, respectively), and for below-reference sperm count in men with TCAA in the second quartile (OR 1.62; 95% CI: 1.04, 2.55). Nonmonotonic associations with TCAA quartiles were also estimated for semen parameters modeled as continuous outcomes, although significant negative associations were estimated for all quartiles above the reference level for sperm motility. Conclusion: Our findings suggest that exposure to drinking-water DBPs may contribute to decreased semen quality in humans. Citation: Zeng Q, Wang YX, Xie SH, Xu L, Chen YZ, Li M, Yue J, Li YF, Liu AL, Lu WQ. 2014. Drinking-water disinfection by-products and semen quality: a cross-sectional study in China. Environ Health Perspect 122:741–746; http://dx.doi.org/10.1289/ehp.1307067