Carcinogenic risk of N-Nitrosamines in Shanghai Drinking Water: Indications for the Use of Ozone Pretreatment.

N-Nitrosamines are drinking water disinfection byproducts that pose a high carcinogenic risk. We hypothesized that raw water treatment processes influence the types and concentrations of nitrosamines in drinking water, thereby posing differential health risks. We compared the finished water of two water treatment plants (WTP-A, WTP-B) serving Shanghai, China. Both plants use the Qingcaosha reservoir as a water source to generate drinking water with conventional but distinct treatment processes, namely preoxidation with sodium hypochlorite (WTP-A) vs ozone (WTP-B). Average nitrosamine concentrations, especially that of the probable human carcinogen (2A) N-nitrosodimethylamine, were higher in finished (drinking) water from WTP-A (35.83 ng/L) than from WTP-B (5.07 ng/L). Other differences in mean nitrosamines in drinking water included N-nitrosodipropylamine (42.62 ng/L) and N-nitrosomethylethylamine (26.73 ng/L) in WTP-A in contrast to N-nitrosodiethylamine (7.26 ng/L) and N-nitrosopyrrolidine (59.12 ng/L) in WTP-B. The estimated adult cancer risk from exposure to mixed nitrosamines was 1.83 times higher from WTP-A than from WTP-B drinking water. Children exposed to nitrosamines had a significantly higher cancer risk than adults ( p < 0.05). Disease burden exceeded 106 person-years. Taken together, these data suggest that use of ozone in the preoxidation step can reduce nitrosamine formation in drinking water and thereby lower the population cancer health risk.

Heavy Exposure of Waste Collectors to Polycyclic Aromatic Hydrocarbons in a Poor Rural Area of Middle China.

Manual collection and open-air incineration of waste materials is a common practice in rural regions of China and beyond. Low-temperature combustion of rubber and plastic waste generates high levels of airborne polycyclic aromatic hydrocarbons (PAHs). We investigated ten urinary hydroxylated PAH metabolites (OH-PAHs), the oxidative damage biomarker (8-hydroxy-deoxyguanosine, 8-OHdG), and four serum biomarkers including gamma-glutamyl transferase (GGT) and alanine aminotransferase (ALT) in 41 waste collectors and 122 control subjects residing in the same or a distant rural village in Henan Province. The level of PAH metabolites in urine (median: 17.24 µg/g Cre) was twice that of controls living in the same area without an occupational history involving waste collection (median: 8.16 µg/g Cre) and thrice that of controls living 30 km away (median: 6.07 µg/g Cre). The concentrations of OH-PAHs were positively associated with urinary 8-OHdG levels (ß = 0.283, p < 0.05). Serum GGT and ALT were slightly increased in waste collectors. Urinary 8-OHdG levels were similar in one-year and longer-term workers, suggesting that rubber and plastic waste collection/incineration carries a high PAH exposure risk. These data provide solid baseline information, emphasizing the importance of monitoring the long-term health outcomes of waste collectors and changes in exposure patterns associated with rural development and regulation of waste disposal.

Iodoacetic acid activates Nrf2-mediated antioxidant response in vitro and in vivo.

Iodoacetic acid (IAA) is an unregulated drinking-water disinfection byproduct with potent cytotoxicity, genotoxicity, and tumorigenicity in animals. Oxidative stress is thought to be essential for IAA toxicity, but the exact mechanism remains unknown. Here we evaluated the toxicity of IAA by examining nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant response, luciferase antioxidant response element (ARE) activity, and intracellular glutathione (GSH) in HepG2 cells. IAA showed significant activation of ARE-luciferase reporter, mRNA, and protein expression of Nrf2 and its downstream genes (GCLC, NQO1, and HO-1). IAA also increased the intracellular GSH level in HepG2 cells in a time- and concentration-dependent manner. Moreover, we verified IAA induced Nrf2-mediated antioxidant response in rats. Subsequently, we confirmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells. Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity and led to an increase of IAA induced micronulei. We also examined the effects of antioxidant on Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly reduced cytotoxicity and genotoxicity (micronuclei formation) IAA in HepG2 cells. Our work here provides direct evidence that IAA activates Nrf2-mediated antioxidant response in vitro and in vivo and that oxidative stress plays a role in IAA toxicity.

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.

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.

Pollution trees: identifying similarities among complex pollutant mixtures in water and correlating them to mutagenicity.

There are relatively few tools available for computing and visualizing similarities among complex mixtures and in correlating the chemical composition clusters with toxicological clusters of mixtures. Using the "intersection and union ratio (IUR)" and other traditional distance matrices on contaminant profiles of 33 specific water samples, we used "pollution trees" to compare these mixtures. The "pollution trees" constructed by neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods allowed comparison of similarities among these samples. The mutagenicity of each sample was then mapped to the "pollution tree". The IUR-distance-based measure proved effective in comparing chemical composition and compound level differences between mixtures. We found a robust "pollution tree" containing seven major lineages with certain broad characteristics: treated municipal water samples were different from raw water samples and untreated rural drinking water samples were similar with local water sources. The IUR-distance-based tree was more highly correlated to mutagenicity than were other distance matrices, i.e., MP/ML methods, sampling group, region, or water type. IUR-distance-based "pollution trees" may become important tools for identifying similarities among real mixtures and examining chemical composition clusters in a toxicological context.

[Investigation on the levels of carbon-, nitrogen-, iodine-containing disinfection by-products in a water plant in Jiangsu province, China].

OBJECTIVE: This work aimed to investigate the carbon-, nitrogen-, iodine-containing disinfection by-products (DBPs) formation and pollution situation in different treatment processes and pipe water of a water plant in Jiangsu province China. METHODS: 12 water samples were collected from raw water, different time points after the addition of chlorine, finished water and pipe water in July, 2011. Trihalomethanes (THM(4)), haloacetic acids (HAA(6)), haloacetonitriles (HAN(s)), chloropicrin (CPs), haloketones (HK(s)), iodoform (IF) and iodoacetic acid (IAA) were detected by gas chromatography with electron capture detector (GC-ECD) after liquid-liquid extraction. Nitrosamines (NAms) were detected by gas chromatography with mass spectrometer (GC-MS) after solid-phase extraction. The concentration of DBPs was represented as the mean of duplicate samples. RESULTS: Most DBPs were found in water treatment processes except dibromochloromethane (0.61 µg/L), chloroform (1.64 µg/L) and N-nitrosodimethylamine (3.06 ng/L), which were detected in raw water. Pre-chlorination formed HAA(6) (5.01 µg/L), HKs (0.66 µg/L), HANs (0.57 µg/L) and NAms (98.09 ng/L). Chlorination and post-chlorination led to a dramatic increase in the levels of THM(4), HAA(6) (70.31 and 43.71 µg/L, respectively), while Nams didn't increase. In finished water, bromodichloromethane (34.12 µg/L) had the highest concentration among THM(4) (70.31 µg/L), and so was trichloroacetic acid (13.45 µg/L) among HAA(6) (43.71 µg/L). Levels of HANs, HKs, and CPs were 14.96, 2.32, 0.96 µg/L, respectively. Levels of N-nitrosodimethylamine and N-nitrosodipropylamine were 21.22, 69.43 ng/L, respectively. IAA, IF and other six NAms including N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosomorpholine, N-nitrosopiperidine and N-nitrosodibutylamine were not detected in all water samples. CONCLUSION: Levels of DBPs in this water plant are relative high.and measures should be taken to control DBPs.

[Determination of anatoxin-a in raw and finished water from two water plants in Shanghai by high performance liquid chromatography].

OBJECTIVE: To establish the HPLC detection method for anatoxin-a in drinking water. METHODS: 1L water was collected, respectively, from raw and finished water in A and B water-plants provided source water by Qingcaosha Reservoir. Water samples were filtered by GF/C membrane, and then intracellular and dissolved anatoxin-a were enriched by weak cation-exchange (WCX) packing, respectively. The optimal ratio of trifluoroacetic acid (TFA) versus methanol for elution was determined. HPLC was employed for qualitative and quantitative analysis of anatoxin-a in water. RESULTS: Methanol containing 0.2% TFA had an optimal elution effect. The linear range of standard curve was 0.01-5 microg/ml and R2 value was 0.9999. The detection limit of anatoxin-a was 2.9 ng/L in water sample. The spiked recovery rates and the relative standard deviation (RSD%) were 75.2% - 100.6% and 6.38% - 8.76%, respectively. When detected continuously for six days, the average values were consistent with the true values, and RSD% of intra- and inter-day was among 1.60% - 8.90%. Anatoxin-a was detectable in raw water of both water-plants. 0.028 microg/L anatoxin-a of A water-plant and 0.137 microg/L anatoxin-a of B water-plant. Anatoxin-a was un-detectable in finished water of both water-plants. CONCLUSION: The current method can satisfy the detection of traces of anatoxin-a in drinking water and be used for continuous batch detection because of the low detection limit, high recovery rate and accuracy, good stability and reliability. More attention should be paid to surveillances of Qingcaosha Reservoir and raw and finished water from it, in order to prevent potential danger of Anatoxin-a on human health.

The genotoxic potential of mixed nitrosamines in drinking water involves oxidative stress and Nrf2 activation.

Nitrosamine by-products in drinking water are designated as probable human carcinogens by the IARC, but the health effects of simultaneous exposure to multiple nitrosamines in drinking water remain unknown. Genotoxicity assays were used to assess the effects of both individual and mixed nitrosamines in finished drinking water produced by a large water treatment plant in Shanghai, China. Cytotoxicity and genotoxicity were measured at 1, 10-, 100- and 1000-fold actual concentrations by the Ames test, Comet assay, γ-H2AX assay, and the cytokinesis-block micronuclei assay; oxidative stress and the Nrf2 pathway were also assessed. Nitrosamines detected in drinking water included NDMA (36.45 ng/L), NDPA (44.68 ng/L), and NEMA (37.27 ng/L). Treatment with a mixture of the three nitrosamines at 1000-fold actual drinking-water concentration induced a doubling of revertants in Salmonella typhimurium strain TA100, DNA and chromosome damage in HepG2 cells, while 1-1000-fold concentrations of compounds applied singly lacked these effects. Treatment with 100- and 1000-fold concentrations increased ROS, GSH, and MDA and decreased SOD activity. Thus, nitrosamine mixtures showed greater genotoxic potential than that of the individual compounds. N-Acetylcysteine protected against the nitrosamine-induced chromosome damage, and Nrf2 pathway activation suggested that oxidative stress played pivotal roles in the genotoxic property of the nitrosamine mixtures.

Air pollution and health studies in China--policy implications.

During the rapid economic development in China, ambient air pollutants in major cities, including PM10 (particulate matter with aerodynamic diameter < or =10 microm) and SO2 have been reduced due to various measures taken to reduce or control sources of emissions, whereas NO2 is stable or slightly increased. However, air pollution levels in China are still at the higher end of the world level. Less information is available regarding changes in national levels of other pollutants such as PM2.5 and ozone. The Chinese Ministry of Environmental Protection (MOEP) set an index for "controlling/reducing total SO2 emissions" to evaluate the efficacy of air pollution control strategy in the country. Total SO2 emissions declined for the first time in 2007. Chinese epidemiologic studies evidenced adverse health effects of ambient air pollution similar to those reported from developed countries, though risk estimates on mortality/morbidity per unit increase of air pollutant are somewhat smaller than those reported in developed countries. Disease burden on health attributable to air pollution is relatively greater in China because of higher pollution levels. Improving ambient air quality has substantial and measurable public health benefits in China. It is recommended that the current Chinese air quality standards be updated/revised and the target for "controlling/reducing total SO2 emissions" be maintained and another target for "reducing total NO2 emissions" be added in view of rapid increase in motor vehicles. Continuous and persistent efforts should be taken to improve ambient air quality.

[Eutrophication of water bodies and pollutions of microcystins in water and sediments in X County in the Huai River Basin].

OBJECTIVE: The purpose of the study is to investigate the status of eutrophication, the polluted situation of algae and microcystins in different water bodies of X County in the Huai River Basin. METHOD: Superficial water and sediment samples were taken from S River, Y River and a representative ditch in May 2010 (median-water period) and August (high water period ). Chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), chlorophyll a (Chl a) and algal density were detected by the recommended methods of national standard microcystin-LF/LR/LW/RR/YR in water (dissolved form), algal cells and sediments were detected by solid phase extraction and HPLC. RESULTS: Water pollutions in rivers and ditches of X County were serious, and total nitrogen and total phosphorus significantly exceed to the national standard for surface water quality. Most indexes of the water quality ranged from grade III to worse than grade V of national standard. Total nitrogen (4.78 mg/L) was most serious pollutions in the mainstream of S river, the water quality in high water period was better than in median-water period (P < 0.05). However, water quality in inner river Y and ditch in high water period was worse than in median-water period. Maximum chlorophyll a in ditch water reached 648.4 mg/ m3. The percent of cyanobacteria in various water bodies was less than 10% in median-water period, increased sharply to 40% and became dominant species in high water period. The maximum percentage of cyanobacteria was 44% in ditch. Microcystin-RR was the main toxin detected in water (dissolved form), algal cells and sediments. Maximum MC-RR in water and sediments reached 17.731 microg/L and 0.802 microg/g, respectively. The detected frequencies and concentrations of MC-LF/LR/LW/YR in most samples were low. CONCLUSION: Water bodies in X County were between middle eutrophication and hyper-eutrophication. Microcystin-RR of sediments in high water period were significant higher than in median-water period (P < 0.05).

[Formation and changes of regulated trihalomethanes and haloacetic acids in raw water of Yangtze River, Huangpu River and different treatment processes and pipelines network].

OBJECTIVES: To investigate the pollutant levels of regulated disinfection by-products trihalomethanes (THMs) and haloacetic acids (HAAs) in raw water from the Huangpu River, the Yangtze River and different treatment processes and finished water, and to explore the changes tendency in transmission and distribution pipeline network. METHODS: A total of 65 ml water samples with two replicates were collected from different raw water, corresponding treatment processes, finished water and six national surveillance points in main network of transmission and distribution, water source for A water plant and B, C water plant was the Huangpu River and the Yangtze River, respectively. Regulated THMs and HAAs above water samples were detected by gas chromatography. RESULTS: The total trihalomethanes (THM(4)) concentration in different treatment processes of A water plant was ND-9.64 µg/L, dichlorobromomethane was the highest (6.43 µg/L). The THM(4) concentration in B and C water plant was ND to 38.06 µg/L, dibromochloromethane (12.24 µg/L) and bromoform (14.07 µg/L) were the highest in the B and the C water plant respectively. In addition to trichloroacetic acid in A water plant from the raw water, the other HAAs came from different treatment processes. The total haloacetic acids (HAA(6)) concentration of different treated processes in A water plant was 3.21 - 22.97 µg/L, mobromoacetic acid (10.40 µg/L) was the highest. Dibromoacetic acid was the highest both in B (8.25 µg/L) and C (8.84 µg/L) water plant, HAA(6) concentration was ND to 27.18 µg/L. The highest and the lowest concentration of THM(4) were found from the main distribution network of C and A water plant respectively, but the concentration of HAA(6) in the main water pipes network of A water plant was the highest, and the lowest in C water plant. The THMs concentration was 21.11 - 31.18 µg/L in C water plant and 6.72 - 8.51 µg/L in A water plant. The concentration of HAA(6) was 25.02 - 37.31 µg/L in A water plant and 18.69 - 23.32 µg/L in C water plant. The highest concentrations of brominated disinfection by-products in B and C water plant were 54.57 µg/L and 45.38 µg/L respectively, those were higher than A water plant (18.98 µg/L), and higher than the chlorinated disinfection by-products in B and C water plants (30.23 µg/L and 30.60 µg/L). CONCLUSIONS: The THM(4) concentrations of finished water treated from Huangpu River was lower than finished water from the Yangtze River, while the HAAs concentrations in finish water from Huangpu River was higher than the two water plants of Yangtze River. The fluctuations of THMs and HAAs concentration in distribution network were low during transmission and distribution process.

[Determination of several herbicides, fungicides and estrogens in water from various treatment processes of different water plants in Shanghai by solid phase extraction-gas chromatography/mass spectrometry].

OBJECTIVE: To establish the solid phase extraction-gas chromatography/mass spectrometry (GC/MS) method for qualitative and quantitative determination of several herbicides, fungicides and estrogens in drinking water. METHODS: Duplicate 1 L water samples were collected from various treatment processes of different water plants. Target pollutants were extracted by XAD-2 resin from water samples and were eluted by 30% acetone-methanol. GC/MS was employed for qualitative and quantitative analysis of target pollutants. RESULTS: The linear ranges of standard curves of 6 target compounds including atrazine, alachlor, 4-cumyphenol, thiabendazole, ß-estradiol and ethylestradiol were 0.1 - 10 µg/ml and the R(2) values were 0.9915 - 0.9995. The detection limits, the recovery rates, and the relative standard deviation (RSD) were separately 0.01 - 0.40 µg/L, 74.0% - 112.0% and 2.3% - 14.8%. Atrazine (0.11 - 0.13 µg/L), 4-cumyphenol (0.20 - 0.35 µg/L), thiabendazole (0.92 - 1.46 µg/L), ß-estradiol (1.02 - 1.32 µg/L) and ethylestradiol (0.96 - 1.66 µg/L)were all detected in raw water, post-coagulation water, post-sedimentation water and finished water using Huangpu River as water source. Alachlor was not detected in any water samples in Huangpu River. The 6 target compounds were not detected in any water samples from the water plants using Yangtse River as water source. CONCLUSION: The methods with high sensitivity and satisfying selectivity are suitable to simultaneously qualitative and quantitative determination of target pollutants in drinking water. Concentrations of above-mentioned pollutants in drinking water coming from Huangpu River are obviously higher than in drinking water coming from Yangtze River. Thiabendazole, ß-estradiol and ethylestradiol at the level of µg/L exist in drinking water coming from Huangpu River and more attention should be paid to surveillance of future.

[Thyroid disruption induced by pentachlorophenol].

OBJECTIVE: To assess thyroid disruption induced by sodium pentachlorophenol (PCP) using Organization for Economic Co-operation and Development (OECD) recommended TG 407 method. METHODS: A total of 30 specific pathogen free (SPF) SD adult male and female rats were randomly divided into 3 groups, and treated with water, 0.33 and 30 mg x kg(-1)x d(-1) of PCP-Na by oral gavage for consecutive 28 days, respectively. After final treatment, histological changes of thyroid were observed by hematoxylin-eosin stain, and the levels of thyroid hormones (total thyroxine (TT(4)), free thyroxine (FT(4)), total triiodothyronine (TT(3)), and free triiodothyronine (FT(3))) were determined by radioimmunoassay. The expression levels of thyroid receptors (TRalpha and TRbeta) mRNA and deiodinases (DioI, DioII and DioIII) mRNA in liver were analyzed by RT-PCR. RESULTS: In high dose group, liver weight coefficient of male and female rats were (4.82 +/- 0.42)% and (4.99 +/- 0.17)%, increased by 36.2% (t = 7.338, P < 0.01) and 41.8% (t = 8.955, P < 0.01), compared to control group ((3.54 +/- 0.14)%, (3.52 +/- 0.19)%), respectively, while the significant changes of kidney or thyroid weight were not observed. In high dose group, the levels of TT(4) and FT(4) in serum of male rats were (64.95 +/- 7.16) nmol/L and (8.16 +/- 2.29) pmol/L, and decreased by 26.6% (t = -3.999, P < 0.01) and 42.3% (t = -4.112, P < 0.01) compared to control group ((88.48 +/- 6.99) nmol/L, (14.13 +/- 1.68) pmol/L). In the same group, FT(4) in serum of female rats was (4.94 +/- 0.89) pmol/L, decreased by 55.5% (t = -3.380, P = 0.012) compared to control group ((11.10 +/- 3.40) pmol/L) and TT(3) and FT(3) in serum of female rats were (1.92 +/- 0.24) nmol/L and (3.05 +/- 0.79) pmol/L, increased by 74.5% (t = 5.263, P < 0.01) and 55.6% (t = 3.495, P < 0.01) compared to control group ((1.10 +/- 0.23) nmol/L, (1.96 +/- 0.32) pmol/L), respectively. PCP-Na didn't affect the expression levels of TRalpha, TRbeta, DioIII mRNA in high dose group, while DioII expression of male rats (0.209 +/- 0.017) down-regulated by 79.2% (t = -5.426, P < 0.01) compared to control group (1.006 +/- 0.137), and DioI expression of female rats (1.844 +/- 0.189) up-regulated by 66.6% (t = 4.359, P < 0.01) compared to control group (1.005 +/- 0.083), indicating DioI and DioII poss different sensitivity to adverse effects induced by PCP-Na between male and female rats. The histopathological results showed that PCP-Na could give rise to hyperplasia of the follicular epithelium cells, and the depletion of colloid. There were no significant changes in serum THs levels and expression of TRalpha, TRbeta, DioI-IIImRNA in low dose group. However, sporadic lymphocytic infiltration, follicles amplification in part and slightly increased in thickness of follicular cells were observed in this group. CONCLUSION: PCP is a kind of thyroid disrupting chemical.

[Study on in vitro isolation and culture method of neural stem cells from fetal rat neocortex].

OBJECTIVE: To establish method for isolation and culture of neural stem cells from fetal rat neocortex in vitro. METHODS: Neocortexes of fetal SD rats were isolated on gestational day 14. Mechanical triturations were used to improve cell dissociation status and conditions of serum-free media were optimized. Three test methods including neural stem cell marker protein (nestin and SOX2), proliferation and clonogenic ability and the multilineage differentiation potential were employed to identify neural stem cells. RESULTS: Nestin of cells cultured with this method was expressed with strong positive, and more than 99% cells were SOX2-positive. BrdU was incorporated into the cells, and the amounts of the cells were 10.55 times as much as the initial cell number after 3 days of culture. The ratios of clonal neurosphere were (33.00 +/- 4.40)% after 6 days of culture. Staining with specific protein markers in the corresponding cells confirmed that neural stem cells could form neurons (MAP2), astrocytes (GFAP) and oligodendrocytes (O4) after differentiation induction. CONCLUSION: Neural stem cells obtained with this method had high purity and cell output, potent self-renewal and multi-lineage differentiation ability, which will provide a good model for toxicological research.

[Determination of trihalomethanes and haloacetic acids in the sample holding time, and their stability in the extract].

OBJECTIVE: The purpose of this study is to investigate the changes of concentration of trihalomethanes (THMs: chloroform, bromodichloromethane, chlorodibromomethane, bromoform) and haloacetic acids (HAAs: monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, dibromoacetic acid, trichloroacetic acid, bromochloroacetic acid) in water samples with holding time and their stability in extract. METHOD: Two water samples from pipe network were collected and added 10 microg/L of THMs mixed standards and 10 microg/L of HAAs mixed standards respectively. The concentrations of THMs and HAAs were measured by U.S. EPA methods 551.1 and 552.3 on the day after sampling, 3 days, 7 days and 14 days, and compared the changes with holding time. The extract with THMs or HAAs mixed standards in the preserving of the day, 7 days, 14 days, 21 days and 30 days were measured by the concentration of THMs and HAAs, compared the changes of targets concentration with holding time. RESULTS: Impact of sample holding time is an important factor in the concentration of the targets which significantly decreased with the extension of the time. The concentration of targets in extract except drichloroactic acid, keep stable in the periods of measurement. CONCLUSION: Sample holding times have a greater impact on the determination of THMs and HAAs, therefore, we suggest that samples be measured or finished pretreatment on the day when sample was collected. In addition to drichloroacetic acid, the concentration of other targets in the extract can be stable for more than 30 days.

Probabilistic health risk assessment of nitrosamines in drinking water of Shaoxing, Zhejiang, China.

Nitrosamines (NAms) are potent genotoxic and carcinogenic but widely detected in drinking water. This study aimed to investigate the occurrence of major types of NAms in drinking water in Shaoxing, China, and to conduct multi-pathway probabilistic cancer risk (CR) assessment to residents based on age-dependent adjustment Chinese exposure factors. Results showed that concentrations of NAms in water varied from not detected (ND) to dozens of nanograms per liter level. N-Nitrosodimethylamine (NDMA) was detected most frequently (93.06%), followed by N-nitrosodiethylamine (NDEA) (64.08%)-with the highest cancer risk among NAms. The CR of NAms came mainly through the oral exposure pathway. The 95th percentile of the total CR of five major NAms was 1.06 × 10-4, exceeding the maximum acceptable lifetime CR (1 × 10-4) recommended by US EPA. Exposure to NDEA contributed the highest to the total CR. The CR of the five NAms through ingestion was 2.5 times higher using the Chinese exposure factors than that of the Americans. The most important variables related to CRs were concentrations of NAms in drinking water, exposure duration, drinking water ingestion rate, and exposure time during bathing. Our findings suggest the urgent need to develop and enforce effective regulatory policies to control the contamination of NAms in drinking water in China. Graphical abstract.

[Effect of anatoxin-A on calcium-mediate signal system of brain tissue in mice].

OBJECTIVE: To explore the mechanism of central neurotoxity induced by anatoxin-A (ANTX-A) from the calcium-mediate signal system. METHODS: Mice were administered by i.p. at different doses of ANTX-A for one month, 0.9% of sodium chloride solution was used as the control. Using fluorescence method and colorimetry method, the intracellular calcium concentration and the activity of calcineurin (CaN, PP2B) and ATPase in brain were determined. RESULTS: Calcium concentrations in brain were increased in 12.5, 50 and 200 microg/kg bw groups compared with the control group, the difference had statistical significance (P < 0.01). The activity of PP2B of brain in 200 microg/kg bw group was enhanced compared with the control group (P < 0.05). In all ANTX-A treated groups, the activity of Na+ -K+ -ATPase of brain in mice had no obvious changes, but the activity of Ca2+ -ATPase in 50 and 200 microg/kg bw groups were significantly reduced compared with the control group ( P < 0.05). CONCLUSION: It is suggested that calcium-mediate signal system could have important regulative action in the central neurotoxity induced by antoxin-A.

[Effects of perinatal exposure to bisphenol A inducing dopaminergic neuronal cell to apoptosis happening in midbrain of male rat offspring].

OBJECTIVE: To investigate the mechanism and effect of rat perinatal exposure to bisphenol A (BPA) resulting in midbrain dopaminergic neuronal cell apoptosis and tyrosine hydroxylase expression of male offspring. METHODS: Rat dams were randomLy divided into 4 groups on gestational day(GD) 10 and given orally the bisphenol A doses as 0, 0.5, 5, 50 mg/kg x d from GD10 to weaning. The brains of male offspring were obtained for detecting, with immunohistochemistry protocol, the Caspase-3, Bcl-2 and tyrosine hydroxylase expression in the midbrain on postnatal day 21 or 30 respectively, and the midbrain apoptotic neuronal cell were detected by TUNEL on PND21. RESULTS: The expression of Caspase-3 in the midbrain of rat male offspring were increased but bcl-2 were decreased on PND21 and 30, respectively. On PND21, apoptotic neuronal cell were found in the midbrain of high and medium doses groups. TH protein expression was decreased. CONCLUSION: Perinatal exposure to bisphenol A can induce the apoptosis of midbrain dopaminergic neuron in the male rat offspring even after weaning, and concomitantly decrease the midbrain TH immunoreactivity, this may cause the abnormal function of dopaminergic pathway of rat male offspring.

[Effects of alcohol on the expression of heat shock protein 70 and super microstructure of astrocytes in rat embryonic brain].

OBJECTIVE: To explore effects of alcohol on the expression of heat shock protein 70 (HSP70) and super microstructure of astrocytes. METHODS: Different dosage of alcohol (1, 5, 25, 50mmol/L) was administered to astrocytes of rat embryos. Effects of alcohol on expression of HSP70 were studied by immunocytochemistry, and changes of super microstructure of astrocytes were observed by electron microscope. RESULTS: The results showed that with increase of alcohol dosage, the expression of HSP70 increased, hepatin granule and survival astrocytes in 25mmol/L and 50mmol/L dosage group decreased, cell membrane and mitochondrion were damaged, and even apoptosis and necrosis occurred. CONCLUSION: It suggested that alcohol could increase expression of HSP of astrocytes, which could depress the expression of normal function protein, and mainly impaired cell membrane and mitochondrion, which could lead to apoptosis and necrosis.