Use of sensitive methods for detection of DNA damage on human lymphocytes exposed to p,p′-DDT: Comet assay and new criteria for scoring micronucleus test

Wide distribution, stability and long persistence in the environment of dichlorodiphenyltrichloroethane (DDT), probably the best-known and most useful insecticide in the world, imposes the need for further examination of the effect of this chemical on human health and especially on the human genome. In this study, peripheral blood human lymphocytes from a healthy donor were exposed to 0.025 mg/L concentration of p,p′-DDT at different time periods (1, 2, 24 and 48 h). For the assessment of genotoxic effect, the new criteria for scoring micronucleus test and alkaline comet assay were used. Both methods showed that p,p′-DDT induces DNA damage in low concentration used in this research. Results of micronucleus test showed a statistically significant (p < 0.05) genotoxic effect of p,p′-DDT on human lymphocytes compared with corresponding control and a different exposure time. A comet assay also showed increased DNA damage caused in p,p′-DDT-exposed human lymphocytes than in corresponding control cells for the tail length. Results obtained by measuring the level of DNA migration and incidence of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) indicate the sensitivity of these tests and their application in detection of primary genome damage after long-term exposure to establish the effect of p,p′-DDT on human genome.     

人外周血淋巴细胞彗星试验检测饮用水水质毒性研究

应用人外周血淋巴细胞彗星试验对无锡充山水厂、中桥水厂,常州西石桥水厂进出水及金坛市拟用水源地长荡湖湖心水样的生物毒性进行了研究．结果表明,各水样的有机浓集物均可对人外周血淋巴细胞DNA产生不同程度的损伤,存在一定的遗传毒性．试验结果与各水样水质污染状况基本一致．同时,季节变化以及常规水处理过程对水样的生物毒性也有影响．研究表明,彗星试验可有效地用于检测饮用水质有机浓集物遗传毒性．     

Susceptibility of the freshwater pulmonate snail Lymnea luteola L. to copper oxide nanoparticle

Copper oxide nanoparticles (CuONP) are among the most widely used engineered nanoparticles and thus likely to permate the environment predominantly in sediments. The present study was designed to examine the adverse effects of CuONP in freshwater snail Lymnea luteola L. (L. luteola) exposed for 5 days. Induction of oxidative stress in digestive gland was evidenced by a decrease in reduced glutathione (GSH) levels, and activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) whereas lipid peroxidation levels were increased at CuONP 7 or 21 µg/L. Superoxide dismutase activity was numerically higher at lower concentration of CuONP at 1 day but significantly decreased at 5 days. Catalase activity was reduced at 2 days but elevated at lower concentration of CuONP at 5 days. DNA impairment was noted in L. luteola based upon comet assay findings and expressed in terms of % tail DNA and olive tail moment. Results indicate that interaction of CuONP with snail produces toxicity, which is mediated by oxidative stress.     

The constitutively active Ah receptor (CA-AhR) mouse as a model for dioxin exposure - effects in reproductive organs

The dioxin/aryl hydrocarbon receptor (AhR) mediates most toxic effects of dioxins. In utero/lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs fetal/neonatal development and the developing male reproductive tract are among the most sensitive tissues. TCDD causes antiestrogenic responses in rodent mammary gland and uterus and in human breast cancer cell lines in the presence of estrogen. Also, more recently an estrogen-like effect of TCDD/AhR has been suggested in the absence of estrogen. A transgenic mouse expressing a constitutively active AhR (CA-AhR) was developed as a model mimicking a situation of constant exposure to AhR agonists. Male and female reproductive tissues of CA-AhR mice were characterized for some of the effects commonly seen after dioxin exposure. Sexually mature CA-AhR female mice showed decreased uterus weight, while an uterotrophic assay in immature CA-AhR mice resulted in increased uterus weight. In immature mice, both TCDD-exposure and CA-AhR increased the expression of the estrogen receptor target gene Cathepsin D. When co-treated with 17β-estradiol no increase in Cathepsin D levels occurred in either TCDD-exposed or CA-AhR mice. In sexually mature male CA-AhR mice the weights of testis and ventral prostate were decreased and the epididymal sperm reserve was reduced. The results of the present study are in accordance with previous studies on dioxin-exposed rodents in that an activated AhR (here CA-AhR) leads to antiestrogenic effects in the presence of estrogen, but to estrogenic effects in the absence of estrogen. These results suggest the CA-AhR mouse model as a useful tool for studies of continuous low activity of the AhR from early development, resembling the human exposure situation.     

Reed beds receiving industrial sludge containing nitroaromatic compounds

Goal, Scope and Background Sweden has prohibited the deposition of organic waste since January, 2005. Since 1 million tons of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a larger degree. One common method in the USA and parts of Europe is the use of wetlands to treat wastewater and sewage sludge. The capacity of reed beds to affect the toxicity of a complex mixture of nitroaromatics in sludge, however, is not fully elucidated. In this study, an industrial sludge containing explosives and pharmaceutical residues was therefore treated in artificial reed beds and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of many pharmaceuticals and explosives, are well known to cause cytotoxicity and genotoxicity. Recently performed studies have also showed that embryos of zebrafish (Danio rerio) are sensitive to nitroaromatic compounds. Therefore, we tested the sludge passing through constructed wetlands in order to detect any changes in levels of embryotoxicity, genotoxicity and dioxin-like activity (AhR-agonists). We also compared unplanted and planted systems in order to examine the impact of the root system on the fate of the toxicants. Methods An industrial sludge containing a complex mixture of nitroaromatics was added daily to small-scale constructed wetlands (vertical flow), both unplanted and planted with Phragmites australis. Sludge with an average dry weight of 1.25%, was added with an average hydraulic loading rate of 1.2 L/day. Outgoing water was collected daily and stored at −20°C. The artificial wetland sediment was Soxhlet extracted, followed by clean-up with multi-layer silica, or extracted by ultrasonic treatment, yielding one organic extract and one water extract of the same sample. Genotoxicity of the extracts was measured according to the ISO protocol for the umu-C genotoxicity assay (ISO/TC 147/SC 5/WG9 N8), using Salmonella typhimurium TA1535/pSK1002 as test organism. Embryotoxicity and teratogenicity were studied using the fish egg assay with zebrafish (Danio rerio) and the dioxin-like activity was measured using the DR-CALUX assay. Chemical analyses of nitroaromatic compounds were performed using Solid Phase Micro Extraction (SPME) and GC-MS. Results Organic extracts of the bed material showed toxic potential in all three toxicity tests after two years of sludge loading. There was a difference between the planted and the unplanted beds, where the toxicity of organic extracts overall was higher in the bed material from the planted beds. The higher toxicity of the planted beds could have been caused by the higher levels of total carbon in the planted beds, which binds organic toxicants, and by enrichment caused by lower volumes of outgoing water from the planted beds. Discussion Developmental disorders were observed in zebrafish exposed directly in contact to bed material from unplanted beds, but not in fish exposed to bed material from planted beds. Hatching rates were slightly lower in zebrafish exposed to outgoing water from unplanted beds than in embryos exposed to outgoing water from planted beds. Genotoxicity in the outgoing water was below detection limit for both planted and unplanted beds. Most of the added toxicants via the sludge were unaccounted for in the outgoing water, suggesting that the beds had toxicant removal potential, although the mechanisms behind this remain unknown. Conclusions During the experimental period, the beds received a sludge volume (dry weight) of around three times their own volume. In spite of this, the toxicity in the bed material was lower than in the sludge. Thus, the beds were probably able to actually decrease the toxicity of the added, sludge-associated toxicants. When testing the acetone extracts of the bed material, the planted bed showed a higher toxicity than the unplanted beds in all three toxicity tests. The toxicity of water extracts from the unplanted beds, detected by the fish egg assay, were higher than the water extracts from the planted beds. No genotoxicity was detected in outgoing water from either planted or unplanted beds. All this together indicates that the planted reed beds retained semi-lipophilic acetone-soluble toxic compounds from the sludge better than the unplanted beds, which tended to leak out more of the water soluble toxic compounds in the outgoing water. The compounds identified by SPME/GC in the outgoing water were not in sufficient concentrations to have caused induction in the genotoxicity test. Recommendations and Perspectives This study has pointed out the benefits of using constructed wetlands receiving an industrial sludge containing a complex mixture of nitroaromatics to reduce toxicity in the outgoing water. The water from planted, constructed wetlands could therefore be directed to a recipient without further cleaning. The bed material should be investigated over a longer period of time in order to evaluate potential accumulation and leakage prior to proper usage or storage. The plants should be investigated in order to examine uptake and possible release when the plant biomass is degraded. This article has been developed on the basis of a presentation given at the Annual meeting of SETAC Europe German Language Branch 2004 in Aachen. ESS-Submission Editor: Dr. Ludek Blaha (blaha@recetox.muni.cz)     

Cytotoxicity of single-walled carbon nanotubes,multi-walled carbon nanotubes,and chrysotile to human lung epithelial cells

Carbon nanotubes (CNTs) have found numerous applications in various industries. Recently, adverse effects of these materials on human and animal cells in vitro have been reported. In the present study, the cytotoxicity of single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), and chrysotile asbestos in human lung epithelial cells has been studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cells were exposed for 6 h and 24 h to between 0.97 and 1500 μg mL^?1 of CNTs and chrysotile fibers prepared in two culture media containing 5% serum and 0.5% dimethylsulfoxide. Dose–response curves were obtained to determine the nonobservable adverse effect concentration and the half-maximum inhibitory concentration (IC₅₀). The way of dispersion affects the cytotoxicity of CNTs. For MWCNT, the toxicological indexes were lower than for SWCNT. Chrysotile fibers were even less cytotoxic than CNTs. Therefore, workplace control measures are recommended as priority for occupational and environmental conditions.     

Screening of textile finishing agents available on the Chinese market: An important source of per- and polyfluoroalkyl substances to the environment

Kendrick mass defect was used for PFASs screening in textile finishing agents (TFAs). Total oxidizable precursor assay provides insight into unknown precursors. Perfluorooctane sulfonate was found as impurity in short ECF technology based TFAs. Perfluorooctanoate was also detected in C6 telomerization based TFAs. Long chain precursors were also observed in both types of TFAs. Organofluorinated surfactants are widely employed in textile finishing agents (TFAs) to achieve oil, water, and stain repellency. This has been regarded as an important emission source of per-and polyfluoroalkyl substances (PFASs) to the environment. China is the biggest manufacturer of clothes, and thus TFA production is also a relevant industrial activity. Nevertheless, to date, no survey has been conducted on PFAS contents in commercially available TFAs. In the present study, TFA products were investigated by the Kendrick mass defect method. The quantification results demonstrated a significant presence of perfluorooctane sulfonate (0.37 mg/L) in TFAs manufactured by electrochemical fluorination technology. The products obtained by short-chain PFAS-based telomerization were dominated by perfluorooctanoic acid (mean concentration: 0.29 mg/L), whose values exceeded the limits stated in the European Chemical Agency guidelines (0.025 mg/L). Moreover, the total oxidizable precursor assay indicated high levels of indirectly quantified precursors with long alkyl chains (C7–C9). Together, these results suggest that there is currently a certain of environmental and health risks in China that originates from the utilization of TFAs, and a better manufacturing processes are required to reduce such risks.     

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural samples

A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC₅₀ and IC₁₀ of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries in six agricultural products were between 79.5% and 118.0%, with the intra-assay coefficient of variation being less than 8 %. The limit of detection for all tested products was 30 ng/g. To the best of our knowledge, this assay has the best specificity among all the published research on ELISAs for chlorpyrifos.     

Determination of the carbon content of biomass—A prerequisite to estimate the complete biodegradation of polymers

This article presents a method to determine the carbon content of biomass, which is formed when degrading biodegradable polymers in an aerobic aqueous test system. Existing methods for determining the carbon content of biomass (e.g., fumigazation, protein assays, dry solids) have several disadvantages when applied for polymer degradation tests. In this work a protein assay based on the Lowry method was used. It was shown that the ratio between protein and carbon content is not constant but depends on the composition of the microbial population, the growth phase, and the substrate supply. This effect was used for the method presented in this article. For determining the carbon content of biomass the absorbance obtained by the Lowry test is correlated directly with the carbon content of biomass in dependence on the duration of the degradation test. The calibration curves are obtained by a mixed population of microorganisms during the course of a degradation test.