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Fang J, Rietjens IMCM, Carrillo JC, Boogaard PJ, Kamelia L. Evaluating the in vitro developmental toxicity potency of a series of petroleum substance extracts using new approach methodologies (NAMs). Arch Toxicol 2024; 98:551-565. [PMID: 38085275 PMCID: PMC10794406 DOI: 10.1007/s00204-023-03645-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Accepted: 11/16/2023] [Indexed: 01/18/2024]
Abstract
The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.
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Affiliation(s)
- Jing Fang
- Division of Toxicology, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands.
| | - Ivonne M C M Rietjens
- Division of Toxicology, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands
| | | | - Peter J Boogaard
- Division of Toxicology, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands
| | - Lenny Kamelia
- Shell Global Solutions International B.V, 2596 HR, The Hague, The Netherlands
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2
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Fang J, Wang D, Kramer NI, Rietjens IMCM, Boogaard PJ, Kamelia L. The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity. J Appl Toxicol 2022; 43:845-861. [PMID: 36585251 DOI: 10.1002/jat.4428] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Revised: 12/11/2022] [Accepted: 12/22/2022] [Indexed: 01/01/2023]
Abstract
The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 μM BaA and the developmental retardations induced by 15 μM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.
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Affiliation(s)
- Jing Fang
- Division of Toxicology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands
| | - Danlei Wang
- Division of Toxicology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands
| | - Nynke I Kramer
- Division of Toxicology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands
| | - Ivonne M C M Rietjens
- Division of Toxicology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands
| | - Peter J Boogaard
- Division of Toxicology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands
| | - Lenny Kamelia
- Shell Global Solutions International B.V., The Hague, The Netherlands
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3
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Trung NT, Anh HQ, Tue NM, Suzuki G, Takahashi S, Tanabe S, Khai NM, Hong TT, Dau PT, Thuy PC, Tuyen LH. Polycyclic aromatic hydrocarbons in airborne particulate matter samples from Hanoi, Vietnam: Particle size distribution, aryl hydrocarbon ligand receptor activity, and implication for cancer risk assessment. CHEMOSPHERE 2021; 280:130720. [PMID: 33964743 DOI: 10.1016/j.chemosphere.2021.130720] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Revised: 04/21/2021] [Accepted: 04/24/2021] [Indexed: 06/12/2023]
Abstract
Concentrations and profiles of unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs and Me-PAHs) were analyzed in airborne particulate matter (PM) samples collected from high-traffic roads in Hanoi urban area. Levels of PAHs and Me-PAHs ranged from 210 to 660 (average 420) ng/m3 in total PM, and these pollutants were mainly associated with fine particles (PM2.5) rather than coarser ones (PM > 10 and PM10). Proportions of high-molecular-weight compounds (i.e., 5- and 6-ring) increased with decreasing particle size. Benzo[b+k]fluoranthene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene were the most predominant compounds in the PM2.5 samples. In all the samples, Me-PAHs were less abundant than unsubstituted PAHs. The PAH-CALUX assays were applied to evaluate aryl hydrocarbon receptor (AhR) ligand activities in crude extracts and different fractions from the PM samples. Benzo[a]pyrene equivalents (BaP-EQs) derived by the PAH-CALUX assays for low polar fractions (mainly PAHs and Me-PAHs) ranged from 300 to 840 ng/m3, which were more consistent with theoretical values derived by using PAH-CALUX relative potencies (270-710 ng/m3) rather than conventional toxic equivalency factor-based values (22-69 ng/m3). Concentrations of PAHs and Me-PAHs highly correlated with bioassay-derived BaP-EQs. AhR-mediated activities of more polar compounds and interaction effects between PAH-related compounds were observed. By using PAH-CALUX BaP-EQs, the ILCR values ranged from 1.0 × 10-4 to 2.8 × 10-4 for adults and from 6.4 × 10-5 to 1.8 × 10-4 for children. Underestimation of cancer risk can be eliminated by using effect-directed method (e.g., PAH-CALUX) rather than chemical-specific approach.
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Affiliation(s)
- Nguyen Thanh Trung
- Faculty of Environmental Science, University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam
| | - Hoang Quoc Anh
- Center of Advanced Technology for the Environment (CATE), Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Japan
| | - Nguyen Minh Tue
- Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, 790-8577, Japan; Centre for Environmental Technology and Sustainable Development (CETASD), University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam
| | - Go Suzuki
- Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies (NIES), Tsukuba, 305-8506, Japan
| | - Shin Takahashi
- Center of Advanced Technology for the Environment (CATE), Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Japan
| | - Shinsuke Tanabe
- Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, 790-8577, Japan
| | - Nguyen Manh Khai
- Faculty of Environmental Science, University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam
| | - Tran Thi Hong
- Faculty of Environmental Science, University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam
| | - Pham Thi Dau
- Faculty of Biology, University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam
| | - Pham Chau Thuy
- Faculty of Environment, Vietnam National University of Agriculture, Trau Quy, Gia Lam, Hanoi, 12400, Viet Nam
| | - Le Huu Tuyen
- Centre for Environmental Technology and Sustainable Development (CETASD), University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai, Hanoi, 11400, Viet Nam.
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Hoang AQ, Suzuki G, Michinaka C, Tue NM, Tuyen LH, Tu MB, Takahashi S. Characterization of unsubstituted and methylated polycyclic aromatic hydrocarbons in settled dust: Combination of instrumental analysis and in vitro reporter gene assays and implications for cancer risk assessment. THE SCIENCE OF THE TOTAL ENVIRONMENT 2021; 788:147821. [PMID: 34029822 DOI: 10.1016/j.scitotenv.2021.147821] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/14/2021] [Revised: 04/19/2021] [Accepted: 05/12/2021] [Indexed: 06/12/2023]
Abstract
Concentrations of 34 unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs and Me-PAHs) and AhR-mediated activities in settled dust samples were determined by a combination of gas chromatography-mass spectrometry and an in vitro reporter gene assay (PAH-CALUX). The levels of Σ34PAHs and bioassay-derived benzo[a]pyrene equivalents (CALUX BaP-EQs) were significantly higher in workplace dust from informal end-of-life vehicle dismantling workshops than in common house dust and road dust. In all the samples, the theoretical BaP-EQs of PAHs (calculated using PAH-CALUX relative potencies) accounted for 28 ± 19% of the CALUX BaP-EQs, suggesting significant contribution of aryl hydrocarbon receptor (AhR) agonists and/or mixture effects. Interestingly, the bioassay-derived BaP-EQs in these samples were significantly correlated with not only unsubstituted PAHs with known carcinogenic potencies but also many Me-PAHs, which should be included in future monitoring and toxicity tests. The bioassay responses of many sample extracts were substantially reduced but not suppressed with sulfuric acid treatment, indicating contribution of persistent AhR agonists. Cancer risk assessment based on the CALUX BaP-EQs has revealed unacceptable level of risk in many cases. The application of bioassay-derived BaP-EQs may reduce underestimation in environmental management and risk evaluation regarding PAHs and their derivatives (notably Me-PAHs), suggesting a consideration of using in vitro toxic activity instead of conventional chemical-specific approach in such assessment practices.
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Affiliation(s)
- Anh Quoc Hoang
- Center of Advanced Technology for the Environment (CATE), Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan; Faculty of Chemistry, University of Science, Vietnam National University, 19 Le Thanh Tong, Hanoi 11000, Viet Nam
| | - Go Suzuki
- Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan
| | - Chieko Michinaka
- Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan
| | - Nguyen Minh Tue
- Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan; Centre for Environmental Technology and Sustainable Development (CETASD), University of Science, Vietnam National University, 334 Nguyen Trai, Hanoi 11400, Viet Nam
| | - Le Huu Tuyen
- Centre for Environmental Technology and Sustainable Development (CETASD), University of Science, Vietnam National University, 334 Nguyen Trai, Hanoi 11400, Viet Nam
| | - Minh Binh Tu
- Faculty of Chemistry, University of Science, Vietnam National University, 19 Le Thanh Tong, Hanoi 11000, Viet Nam
| | - Shin Takahashi
- Center of Advanced Technology for the Environment (CATE), Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
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Barhoumi B, Tedetti M, Heimbürger-Boavida LE, Tesán Onrubia JA, Dufour A, Doan QT, Boutaleb S, Touil S, Scippo ML. Chemical composition and in vitro aryl hydrocarbon receptor-mediated activity of atmospheric particulate matter at an urban, agricultural and industrial site in North Africa (Bizerte, Tunisia). CHEMOSPHERE 2020; 258:127312. [PMID: 32947663 DOI: 10.1016/j.chemosphere.2020.127312] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2020] [Revised: 05/17/2020] [Accepted: 06/01/2020] [Indexed: 06/11/2023]
Abstract
As recognized risk factor to pose a health threat to humans and wildlife globally, atmospheric particulate matter (PM) were collected from a North African coastal city (Bizerte, Tunisia) for one year, and were characterized for their chemical compositions, including mercury (HgPM), as well as organic contaminants (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs)), organic carbon (OC) and organic nitrogen (ON), determined in a previous study. Then, we applied an in vitro reporter gene assay (DR-CALUX) to detect and quantify the dioxin-like activity of PM-associated organic contaminants. Results showed that average HgPM concentration over the entire sampling period was found to be 13.4 ± 12 pg m-3. Seasonal variation in the HgPM concentration was observed with lower values in spring and summer and higher values in winter and autumn due to the variation of meteorological conditions together with the emission sources. Principal component analysis suggested that fossil fuel combustion and a nearby cement factory were the dominant anthropogenic HgPM sources. Aryl hydrocarbon receptor (AhR)-mediated activities were observed in all organic extracts of atmospheric PM from Bizerte city (388.3-1543.6 fg m-3), and shows significant positive correlations with all PM-associated organic contaminants. A significant proportion of dioxin-like activity of PM was related to PAHs. The dioxin-like activity followed the same trend as PM-associated organic contaminants, with higher dioxin-like activity in the cold season than in the warm season, indicating the advantage and utility of the use of bioassays in risk assessment of complex environmental samples.
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Affiliation(s)
- Badreddine Barhoumi
- Laboratory of Hetero-Organic Compounds and Nanostructured Materials (LR18ES11), Department of Chemistry, Faculty of Sciences of Bizerte, University of Carthage, 7021, Zarzouna, Tunisia.
| | - Marc Tedetti
- Aix Marseille Univ., Université de Toulon, CNRS, IRD, MIO UM 110, 13288, Marseille, France
| | | | - Javier A Tesán Onrubia
- Aix Marseille Univ., Université de Toulon, CNRS, IRD, MIO UM 110, 13288, Marseille, France
| | - Aurélie Dufour
- Aix Marseille Univ., Université de Toulon, CNRS, IRD, MIO UM 110, 13288, Marseille, France
| | - Que Thi Doan
- Laboratory of Food Analysis, FARAH-Veterinary Public Health, University of Liège, Liège, 4000, Belgium
| | - Samiha Boutaleb
- Laboratory of Food Analysis, FARAH-Veterinary Public Health, University of Liège, Liège, 4000, Belgium
| | - Soufiane Touil
- Laboratory of Hetero-Organic Compounds and Nanostructured Materials (LR18ES11), Department of Chemistry, Faculty of Sciences of Bizerte, University of Carthage, 7021, Zarzouna, Tunisia
| | - Marie-Louise Scippo
- Laboratory of Food Analysis, FARAH-Veterinary Public Health, University of Liège, Liège, 4000, Belgium
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Wang L, Zhao Y, Yi X, Wang Z, Yi Y, Huang T, Gao H, Ma J. Spatial distribution of atmospheric PAHs and their genotoxicity in petrochemical industrialized Lanzhou valley, northwest China. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2017; 24:12820-12834. [PMID: 28364205 DOI: 10.1007/s11356-017-8808-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/25/2016] [Accepted: 03/13/2017] [Indexed: 06/07/2023]
Abstract
The present study investigated the spatial and seasonal variations and sources of 16 priority polycyclic aromatic hydrocarbons (PAHs) in Lanzhou, a petrochemical industrialized and the capital city of Gansu province, northwest China. The human health risks to these PAHs were assessed using an in vitro genotoxic bioassay technique. Associations among direct genotoxic potency, atmospheric PAH concentrations, and potential carcinogen risks were examined. Due to high PAH emissions from fossil fuel combustion and petrochemical industries, considerable higher PAH levels in the atmosphere were observed in Xigu district, a suburb featured by heavy petrochemical industry, compared with those collected at downtown and rural sampling sites. Ambient PAH levels at all sampling sites during the wintertime were higher than that in the summertime due to the winter domestic heating. BaP equivalent (BaPeq) concentrations in winter (41 ng/m3) and summer (28 ng/m3) exceeded the China's new national daily BaPeq standard. The average excess inhalation cancer risks (ECR) due to human exposure to PAHs during winter and summer sampling periods were 45-3540 cancer cases and 31-2451 cases per million people, respectively. The average ECR in the industrial area of Lanzhou valley was 1.97 (winter) and 1.88 times (summer) higher than that in other sampling areas. The higher ECR in the industrial area was resulted primarily by industrial activities and insufficient emission control measures. Extracts from passive air samples in genotoxicity SOS/umu test demonstrated that the genotoxic effect of atmospheric PAHs in Lanzhou was seasonal dependent. PAH air samples collected in winter showed more statistically significant genotoxicity, as manifested by a strong correlation between in vitro genotoxicity and atmospheric PAH concentrations. This indicates that the local residents were under higher potential cancer risk through the inhalation of ambient PAH air concentrations in Lanzhou valley during the wintertime.
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Affiliation(s)
- Li Wang
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Yuan Zhao
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Xin Yi
- State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, 510640, China
| | - Zhanxiang Wang
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Yayi Yi
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Tao Huang
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Hong Gao
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China.
| | - Jianmin Ma
- Key Laboratory for Environmental Pollution Prediction and Control, Gansu Province, College of Earth and Environmental Sciences, Lanzhou University, Lanzhou, 730000, China.
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Bakand S, Hayes A, Winder C, Khalil C, Markovic B. In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media. Toxicol Ind Health 2016; 21:147-54. [PMID: 16149729 DOI: 10.1191/0748233705th223oa] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The purpose of this study was to identify a suitable sampling model for on-site toxicity assessment of soluble air contaminants such as formaldehyde, a well known industrial and indoor air contaminant. The in vitro cytotoxicity of formaldehyde, the selected model for soluble air contaminants, was studied using the MTS (tetrazolium salt) assay in two carcinoma cell lines, A549 epithelial lung and HepG2 hepatocarcinoma, and in skin fibroblasts. The cytotoxic effects of airborne formaldehyde were evaluated using test atmospheres in concentrations below 10 ppm (12.3 mg/m3), generated by a dynamic diffusion method and bubbled (0.3 L/min) through serum-free culture media for one or four hours. Human cells were treated with formaldehyde air samples, and cell viability was determined after four hours incubation. In parallel, the concentration of airborne formaldehyde was monitored, using the 3500 NIOSH method. Cell viability of the HepG2 cells exposed to formaldehyde air samples (8.75 ppm-4 h) was reduced to less than 50% (31.69/1.24%). The HepG2 cell lines were found to be more sensitive (IC50=103.799/23.55 mg/L) to formaldehyde than both A549 cell lines (IC50=198.369/9.54 mg/L) and skin fibroblasts (IC50=196.689/36.73 mg/L) (PB/0.01). An average of 96.8% was determined for collection efficiency of formaldehyde in serum-free culture media. The results of this study suggest that absorption of soluble air contaminants, such as formaldehyde, in serum-free culture media can be used as a suitable sampling model for on-site toxicity assessments.
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Affiliation(s)
- S Bakand
- Chemical Safety and Applied Toxicology Laboratories, School of Safety Science, The University of New South Wales, Sydney 2052, Australia.
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8
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Eichbaum K, Brinkmann M, Buchinger S, Reifferscheid G, Hecker M, Giesy JP, Engwall M, van Bavel B, Hollert H. In vitro bioassays for detecting dioxin-like activity--application potentials and limits of detection, a review. THE SCIENCE OF THE TOTAL ENVIRONMENT 2014; 487:37-48. [PMID: 24762647 DOI: 10.1016/j.scitotenv.2014.03.057] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Revised: 03/14/2014] [Accepted: 03/14/2014] [Indexed: 05/05/2023]
Abstract
Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology. While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs. This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples.
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Affiliation(s)
- Kathrin Eichbaum
- Institute for Environmental Research, Department of Ecosystem Analysis, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
| | - Markus Brinkmann
- Institute for Environmental Research, Department of Ecosystem Analysis, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
| | - Sebastian Buchinger
- Federal Institute of Hydrology (BFG), Department G3: Biochemistry, Ecotoxicology, Am Mainzer Tor 1, 56068 Koblenz, Germany
| | - Georg Reifferscheid
- Federal Institute of Hydrology (BFG), Department G3: Biochemistry, Ecotoxicology, Am Mainzer Tor 1, 56068 Koblenz, Germany
| | - Markus Hecker
- School of the Environment & Sustainability and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, SK S7N 5B3 Saskatoon, Canada
| | - John P Giesy
- School of the Environment & Sustainability and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, SK S7N 5B3 Saskatoon, Canada; Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, SK S7N 5B3 Saskatoon, Canada; Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA; Department of Biology and Chemistry, State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China; School of Biological Sciences, University of Hong Kong, Hong Kong, China
| | - Magnus Engwall
- Man-Technology-Environment Research Centre, Deptartment of Natural Sciences, Örebro University, 70182 Örebro, Sweden
| | - Bert van Bavel
- Man-Technology-Environment Research Centre, Deptartment of Natural Sciences, Örebro University, 70182 Örebro, Sweden
| | - Henner Hollert
- Institute for Environmental Research, Department of Ecosystem Analysis, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Key Laboratory of Yangtze River Environment of Education Ministry of China, College of Environmental Science and Engineering, Tongji University, Shanghai, China; College of Resources and Environmental Science, Chongqing University, Chongqing 400030, China; School of Environment, Nanjing University, China.
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9
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Novák J, Hilscherová K, Landlová L, Čupr P, Kohút L, Giesy JP, Klánová J. Composition and effects of inhalable size fractions of atmospheric aerosols in the polluted atmosphere. Part II. In vitro biological potencies. ENVIRONMENT INTERNATIONAL 2014; 63:64-70. [PMID: 24263139 DOI: 10.1016/j.envint.2013.10.013] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2013] [Revised: 10/21/2013] [Accepted: 10/22/2013] [Indexed: 05/20/2023]
Abstract
Exposure to particulate matter (PM) in ambient air has been shown to lead to adverse health consequences. Six size fractions of PM with aerodynamic diameter smaller than 10μm (PM10) and gas phase were collected at six localities with different major pollution sources. Extracts of samples were assessed for AhR-mediated toxicity, (anti-)estrogenicity, (anti-)androgenicity and genotoxicity. The biological responses were interpreted relative to chemical characterization. Historically, for regulatory purposes, evaluation of air pollution was based mainly on assessment of the sum of PM10. In the case of AhR-mediated activity, PM1 was responsible for more than 75% of the activity of the particulate fraction from all localities. The assessed effects were correlated with concentrations of polycyclic aromatic hydrocarbons (PAH), organic carbon content and specific surface area of the PM. A significant proportion of biologically active chemicals seems to be present in the gas phase of air. The results suggest that an average daily exposure based just on the concentrations of contaminants contained in PM10, as regulated in EU legislation so far, is not a sufficient indicator of contaminants in air particulates and adoption of standards more similar to other countries and inclusion of other parameters besides mass should be considered.
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Affiliation(s)
- Jiří Novák
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic.
| | - Klára Hilscherová
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - Linda Landlová
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - Pavel Čupr
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - Lukáš Kohút
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic
| | - John P Giesy
- Dept. Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Zoology Department, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI 48823, United States; Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, China
| | - Jana Klánová
- Masaryk University, RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Kamenice 753/5, 625 00 Brno, Czech Republic
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Pieterse B, Felzel E, Winter R, van der Burg B, Brouwer A. PAH-CALUX, an optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic hydrocarbons (PAHs) as individual compounds and in complex mixtures. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2013; 47:11651-11659. [PMID: 23987121 DOI: 10.1021/es403810w] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
Polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitously occurring environmental compounds that are implicated in a wide range of toxicological effects. Routine measurement of PAH contamination generally involves chemical analytical analysis of a selected group of representatives, for example, EPA-16, which may result in underestimation of the PAH-related toxicity of a sample. Many high molecular weight PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a nuclear receptor that mediates toxic effects related to these compounds. Making use of this property we developed a PAH CALUX assay, a mammalian, H4IIe- cell-based reporter assay for the hazard identification of total PAH mixtures. The PAH CALUX reporter cell line allows for specific, rapid (4 h exposure time) and reliable quantification of AhR-induced luciferase induction relative to benzo[a]pyrene (BaP), which is used as a positive reference PAH congener. Full dose response relationships with inductions over 100-fold were reached within only 2 h of exposure to BaP. The PAH CALUX is highly sensitive, that is, using a 4 h exposure time, a limit of detection (LOD) of 5.2 × 10(-11) M BaP was achieved, and highly accurate, that is, a repeatability of 5.9% and a reproducibility of 6.6% were established. Screening of a selection of PAHs that were prioritized by the European Union and/or the U.S. Environmental Protection Agency showed that the PAH CALUX bioassay has a high predictability, particularly for carcinogenic PAHs. Experiments with synthetic mixtures and reference materials containing complex PAH mixtures show the suitability of the assay for these types of applications. Moreover, the presented results suggest that application of the PAH CALUX will result in a lower risk of underestimation of the toxicity of a sample than chemical analytical approaches that focus on a limited set of prioritized compounds.
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Affiliation(s)
- B Pieterse
- BioDetection Systems BV. , Science Park 406. Amsterdam, The Netherlands
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11
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Olivares A, van Drooge BL, Casado M, Prats E, Serra M, van der Ven LT, Kamstra JH, Hamers T, Hermsen S, Grimalt JO, Piña B. Developmental effects of aerosols and coal burning particles in zebrafish embryos. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2013; 178:72-79. [PMID: 23542445 DOI: 10.1016/j.envpol.2013.02.026] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Revised: 02/15/2013] [Accepted: 02/21/2013] [Indexed: 06/02/2023]
Abstract
Embryo toxicity of particles generated by combustion processes is of special concern for human health. A significant part of these toxic effects is linked to the binding of some pollutants (like polycyclic aromatic hydrocarbons or PAHs) to the Aryl hydrocarbon Receptor (AhR) and the activation of target genes, like the cytochrome P4501A. This activity was analyzed for ambient air and coal-combustion particle extracts in zebrafish embryos (the cyp1aDarT assay) and in two single-cell bioassays: the yeast-based YCM-RYA and the DR-luc (rat cells) assay. Observed AhR ligand activity of samples generally correlated to the predicted toxic effect according to their PAH composition, except for one of the coal combustion samples with an anomalously high activity in the cyp1aDarT assay. This sample induced deformities in zebrafish embryos. We concluded that the combination of morphological and molecular assays may detect embryonic toxic effects that cannot be predicted from chemical analyses or single-cell bioassays.
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Affiliation(s)
- Alba Olivares
- Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona 18, 08034 Barcelona, Catalonia, Spain
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12
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Vrabie CM, Sinnige TL, Murk AJ, Jonker MTO. Effect-directed assessment of the bioaccumulation potential and chemical nature of Ah receptor agonists in crude and refined oils. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2012; 46:1572-1580. [PMID: 22257214 DOI: 10.1021/es2036948] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Recent studies have indicated that in addition to narcosis certain chemicals in crude oils and refined petroleum products may induce specific modes of action, such as aryl hydrocarbon receptor (AhR) agonism. The risks these toxic compounds pose to organisms depend on internal exposure levels, as driven by the chemicals' bioaccumulation potential. Information on this potential however is lacking, as the chemicals' identity mostly is unknown. This study showed that AhR agonists bioaccumulate from oil-spiked sediments into aquatic worms and persist in the worms for at least several weeks. Chemical fractionations of eight pure oils into saturates, aromatics, resins, and asphaltenes (SARA), followed by effect-directed analyses using in vitro reporter gene assays revealed that the agonists predominantly are aromatic and resin-like chemicals. Some of the compounds were easily metabolized in vitro, while others were resistant to biotransformation. HPLC-assisted hydrophobicity profiling subsequently indicated that the AhR-active chemicals had a high to extremely high bioaccumulation potential, considering their estimated logK(ow) values of 4 to >10. Most of the AhR agonism, however, was assigned to compounds with logK(ow) of 5-8. These compounds were present mainly in the mid to high boiling point fractions of the oils (C(14)-C(32) alkane range), which are usually not being considered (the most) toxic in current risk assessment. The fractionations further revealed considerable oil and fraction-dependent antagonism in pure oils and SARA fractions. The results of this study clearly demonstrate that crude oils and refined petroleum products contain numerous compounds that can activate the AhR and which because of their likely persistence and extremely high bioaccumulation potential could be potential PBT (persistent, bioaccumulative and toxic) or vPvB (very persistent and very bioaccumulative) substance candidates. Many chemicals were identified by GC-MS, but the responsible individual compounds could not be exactly identified in the complex mixtures of thousands of compounds. Because this obstructs a classical PBT risk assessment, our results advocate an adapted risk assessment approach for complex mixtures in which low concentrations of very potent compounds are responsible for mixture effects.
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Affiliation(s)
- Cozmina M Vrabie
- Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80177, 3508 TD Utrecht, The Netherlands
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13
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Andrysík Z, Vondráček J, Marvanová S, Ciganek M, Neča J, Pěnčíková K, Mahadevan B, Topinka J, Baird WM, Kozubík A, Machala M. Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: the role of polycyclic aromatic hydrocarbons. Mutat Res 2011; 714:53-62. [PMID: 21762708 DOI: 10.1016/j.mrfmmm.2011.06.011] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2011] [Revised: 06/07/2011] [Accepted: 06/28/2011] [Indexed: 11/19/2022]
Abstract
Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.
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Affiliation(s)
- Zdeněk Andrysík
- Department of Cytokinetics, Institute of Biophysics AS CR, Královopolská 135, 61265 Brno, Czech Republic
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Diagnostic Tools for Effect-Directed Analysis of Mutagens, AhR Agonists, and Endocrine Disruptors. ACTA ACUST UNITED AC 2011. [DOI: 10.1007/978-3-642-18384-3_4] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/26/2023]
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15
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Dabrowska H, Murk AJ, van den Berg HJ. Bioassay-derived dioxin equivalent concentrations in gonads and livers of the Atlantic cod females from the Baltic Sea. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2010; 73:1829-1834. [PMID: 20863569 DOI: 10.1016/j.ecoenv.2010.08.030] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2010] [Revised: 08/19/2010] [Accepted: 08/22/2010] [Indexed: 05/29/2023]
Abstract
The DR-H4IIE.Luc bioassay is based on the ability of dioxin and dioxin-like contaminants to activate the AhR and its signal transduction pathway, a mechanism through which these contaminants elicit their toxic effects. The bioassay was used to examine the total dioxin-equivalent (TEQ) toxicity in gonads and livers of cod females from the southern Baltic Sea. The bioassay-derived TEQ-luc was measured after 24-h and 48-h exposure periods. Mean concentrations in the 24-h bioassay were 95 and 35 pg TEQ-luc g(-1) lipid in gonads and livers, respectively, and 58 and 38 pg TEQ-luc g(-1) lipid in the 48-h bioassay, respectively. The 48-h TEQ-luc levels displayed significant relationships with ΣPCB(7) and selected PCB congeners but not with the TEQ(DLPCB-REP). Levels in gonads approached 10% of the LC50 for developing larvae of other marine fish, yet the impact on survival of the cod during its early life remains to be assessed in a future.
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Affiliation(s)
- Henryka Dabrowska
- Sea Fisheries Institute in Gdynia, Department of Food and Environmental Chemistry, 81-332 Gdynia, Poland.
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16
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Schipper CA, Rietjens IMCM, Burgess RM, Murk AJ. Application of bioassays in toxicological hazard, risk and impact assessments of dredged sediments. MARINE POLLUTION BULLETIN 2010; 60:2026-2042. [PMID: 20850157 DOI: 10.1016/j.marpolbul.2010.07.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/21/2009] [Revised: 07/16/2010] [Accepted: 07/16/2010] [Indexed: 05/29/2023]
Abstract
Given the potential environmental consequences of dumped dredged harbour sediments it is vital to establish the potential risks from exposure before disposal at sea. Currently, European legislation for disposal of contaminated sediments at sea is based on chemical analysis of a limited number of well-known contaminants for which maximum acceptable concentrations, action levels (ALs), have been set. The present paper addresses the issue of the applicability of in vitro and in vivo bioassays for hazard, risk and local impact assessment of dredged polluted sediments to be disposed of at sea. It discusses how and to what extent selected bioassays can fill in the gaps left open by chemical analysis and the way in which the bioassays may contribute to the present licensing system for disposal. Three different purposes for application were distinguished: the most basic application (A) is a rapid determination of the hazard (potential toxicity) of dredged sediments which is then compared to ALs in a licensing system. As with chemical analysis on whole sediment extracts, the bioavailability of the chemicals is not taken into account. As in vitro assays with sediment extracts are not sensitive to matrix effects, a selection of specific in vitro bioassays can be suitable fast and standardized additions for the licensing system. When the outcome of (A) does not convincingly demonstrate whether the sediment is clean enough or too polluted, further bioanalysis can help the decision making process (B). More aspects of the mostly unknown complex chemical mixtures are taken into account, including the bioavailability and chronic toxicity focusing on ecologically relevant endpoints. The ecotoxicological pressure imposed by the dredged sediments can be quantified as the potentially affected fraction (PAF) based on chemical or biological analysis of levels of contaminants in sediment or biota. To validate the predicted risk, the actual impact of dumped harbour sediments on local ecosystems (C) can be determined using a dedicated set of in vitro and in vivo bioassays as well as bio-indicators selected based on the information obtained from (A) and (B) and on the characteristics of the local ecosystem. Conversely, the local sediment impact assessment (C) can direct fine-tuning of the selection of chemical and bioassay analyses and for setting safe levels in the licensing system. It is concluded that in vitro and in vivo bioassays and biological indicators are useful tools in the process of hazard, ecotoxicological risk and impact assessment of dredged harbour sediments, provided they are consciously chosen and quality criteria for assay performance are defined.
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Affiliation(s)
- C A Schipper
- Deltares, P.O. Box 177, 2600 MH Delft, The Netherlands.
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Whyte JJ, Schmitt CJ, Tillitt DE. The H4IIE Cell Bioassay as an Indicator of Dioxin-like Chemicals in Wildlife and the Environment. Crit Rev Toxicol 2010; 34:1-83. [PMID: 15000436 DOI: 10.1080/10408440490265193] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.
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Affiliation(s)
- J J Whyte
- Columbia Environmental Research Center, U.S. Geological Survey-Biological Resources Division, Columbia, Missouri 65201, USA
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18
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Bakand S, Hayes A. Troubleshooting methods for toxicity testing of airborne chemicals in vitro. J Pharmacol Toxicol Methods 2010; 61:76-85. [DOI: 10.1016/j.vascn.2010.01.010] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2009] [Revised: 01/18/2010] [Accepted: 01/20/2010] [Indexed: 01/01/2023]
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Kennedy K, Macova M, Bartkow ME, Hawker DW, Zhao B, Denison MS, Mueller JF. Effect based monitoring of seasonal ambient air exposures in Australia sampled by PUF passive air samplers. ATMOSPHERIC POLLUTION RESEARCH 2010; 1:50-58. [PMID: 21552507 PMCID: PMC3087176 DOI: 10.5094/apr.2010.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
There has been relatively little bioanalytical effect based monitoring conducted using samples derived from polyurethane foam (PUF) passive air samplers. Combining these techniques may provide a more convenient and cost effective means of monitoring the potential for biological effects resulting from exposure to complex mixtures in a range of scenarios. Seasonal polycyclic aromatic hydrocarbon (PAH) levels were monitored at sites around Australia using direct chemical analysis. In addition, both indirect acting genotoxicity (umuC assay) and aryl hydrocarbon receptor (AhR) activity (chemically activated fluorescent gene expression [CAFLUX assay]), which are effects potentially relevant to subsequent carcinogenesis for these compounds, were measured. The levels of PAHs as well as genotoxicity and AhR activity were all higher in winter compared to summer and for sites in urban capital cities compared to other locations. Statistically significant relationships were found between the levels of PAHs and both genotoxicity and AhR activity. The dominant contributors to the total AhR activity, were found to be for compounds which are not resistant to H(2)SO(4)/silica gel treatment and were relatively rapidly metabolised that is consistent with a PAH type response. Relative potency estimates for individual PAHs determined for the first time on the CAFLUX assay were used to estimate the proportion of total AhR activity (≤ 3.0%) accounted by PAHs monitored. Observed responses are thus largely due to non-quantified AhR active compounds.
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Affiliation(s)
- Karen Kennedy
- The University of Queensland, Entox (The National Research Centre for Environmental Toxicology), Brisbane QLD 4108, Australia
| | - Miroslava Macova
- The University of Queensland, Entox (The National Research Centre for Environmental Toxicology), Brisbane QLD 4108, Australia
| | - Michael E. Bartkow
- The University of Queensland, Entox (The National Research Centre for Environmental Toxicology), Brisbane QLD 4108, Australia
| | - Darryl W. Hawker
- School of Environment, Griffith University, Nathan QLD 4111, Australia
| | - Bin Zhao
- Department of Environmental Toxicology, University of California, Davis CA 95616, USA
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Centre for Eco-Environmental Sciences, Chinese Academy of Science, Beijing, China
| | - Michael S. Denison
- Department of Environmental Toxicology, University of California, Davis CA 95616, USA
| | - Jochen F. Mueller
- The University of Queensland, Entox (The National Research Centre for Environmental Toxicology), Brisbane QLD 4108, Australia
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Anezaki K, Yamaguchi K, Takeuchi S, Iida M, Jin K, Kojima H. Application of a bioassay using DR-EcoScreen cells to the determination of dioxins in ambient air: a comparative study with HRGC-HRMS analysis. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2009; 43:7478-7483. [PMID: 19848164 DOI: 10.1021/es901003b] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
There is a strong need for the development of relatively rapid and low-cost bioassays for the determination of dioxins (polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls) in environmental and food samples. In this study, we applied a bioassay system using highly sensitive DR-EcoScreen cells (DR-cell assay) to the determination of low levels of dioxins in ambient air samples. The dioxins from 80 ambient air samples were extracted, purified by cleanup procedure, and applied to both DR-cell assay and high-resolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS) analysis. Interestingly, the toxic equivalents (TEQ) values obtained from the DR-cell assay were closely correlated with those from the HRGC-HRMS analysis (r2 = 0.957). Although the cell-based TEQ values were relatively (3.66-fold) higher than the TEQ values from the HRGC-HRMS analysis, this discrepancy was thought to be due to the difference between the toxic equivalency factor (TEF) and the relative potency (DR-REP) determined for individual dioxins. Therefore, we multiplied the TEQ values from the DR-cell assay by a conversion coefficient (0.273, the reciprocal of 3.66) and could approximate the values from the HRGC-HRMS analysis. These results clearly suggest at the DR-cell assay might be a promising method for the rapid and low-cost screening of dioxins in ambient air samples.
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Affiliation(s)
- Katsunori Anezaki
- Hokkaido Institute of Environmental Sciences, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan
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22
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Kennedy K, Macova M, Leusch F, Bartkow ME, Hawker DW, Zhao B, Denison MS, Mueller JF. Assessing indoor air exposures using passive sampling with bioanalytical methods for estrogenicity and aryl hydrocarbon receptor activity. Anal Bioanal Chem 2009; 394:1413-21. [PMID: 19430962 PMCID: PMC2864012 DOI: 10.1007/s00216-009-2825-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2008] [Revised: 04/13/2009] [Accepted: 04/24/2009] [Indexed: 10/20/2022]
Abstract
Passive air sampling was undertaken using polyurethane foam passive air samplers at three types of locations, including indoors (six offices) at buildings in the central business district (CBD) and at a private suburban home (indoor and outdoor) located 9 km from the CBD in Brisbane, Queensland, Australia. Estrogenic (E-SCREEN--MCF7-BOS) and aryl hydrocarbon receptor (AhR) (CAFLUX--H4G1.1c2) activity were assessed for samples collected from each of these locations. The samples were tested either as crude extracts ("untreated") or were subjected to H2SO4 silica gel ("treated") for each location in order to determine whether chemicals, which are not resistant to this treatment like polycyclic aromatic hydrocarbons, potentially account for the observed activity. In most cases, H2SO4 treatment resulted in a statistically significant reduction of potency for both endpoints, suggesting that chemicals less resistant to treatment may be responsible for much of the detected biological activity in these locations. Estrogenic potency measurements (<0.22-185 pg m(-3)) were highest in the indoor offices, followed by the indoor suburban home and finally the outdoor suburban home (which was not estrogenic). Total AhR activity for crude extracts (1.3-10 pg m(-3)) however was highest for the outdoor suburban home site. Levels of polycyclic aromatic hydrocarbons were monitored indoors and outdoors at the suburban home. At that location, polycyclic aromatic hydrocarbon air concentrations were on average approximately two times higher outdoor than indoor, while AhR potency was five times higher outdoor than indoor. No significant correlation was found between the estrogenic and AhR activity (P = 0.88) for the sites in this study.
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Affiliation(s)
- Karen Kennedy
- EnTox (The National Research Centre for Environmental Toxicology), The University of Queensland, Brisbane, QLD 4108, Australia.
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23
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Novák J, Jálová V, Giesy JP, Hilscherová K. Pollutants in particulate and gaseous fractions of ambient air interfere with multiple signaling pathways in vitro. ENVIRONMENT INTERNATIONAL 2009; 35:43-9. [PMID: 18678411 DOI: 10.1016/j.envint.2008.06.006] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2008] [Revised: 06/23/2008] [Accepted: 06/24/2008] [Indexed: 05/20/2023]
Abstract
Traditionally, contamination of air has been evaluated primarily by chemical analyses of indicator contaminants and these studies have focused mainly on compounds associated with particulates. Some reports have shown that air contaminants can produce specific biological effects such as toxicity mediated by the aryl hydrocarbon receptor (AhR) or modulation of the endocrine system. This study assessed the dioxin-like toxicity, anti-/estrogenicity, anti-/androgenicity and anti-/retinoic activity of both the particulate and gas phase fractions of air in two regions with different types of pollution sources and a background locality situated in an agricultural area of Central Europe. The first region (A) is known to be significantly contaminated by organochlorine pesticides and chemical industry. The other region (B) has been polluted by historical releases of PCBs, but the major current sources of contamination are probably combustion sources from local traffic and heating. Samples of both particle and gas fractions produced dioxin-like (AhR-mediated) activity, anti-estrogenic and antiandrogenic effects, but none had any effect on retinoid signaling. AhR-mediated activities were observed in all samples and the TEQ values were comparable in both fractions in region A, but significantly greater in the particulate fraction in region B. The greater AhR-mediated activity corresponded to a greater coincident antiestrogenicity of both phases in region B. Our study is the first report of antiestrogenicity and antiandrogenicity in ambient air. Anti-androgenicity was observed in the gas phase of all regions, while in the particulate phase only in one region due to the specific type of pollution in that area. Even though based on concentrations of individual compounds, except for the OCPs, the level of contamination of the two regions was similar, there were strong differences in responses in the bioassays between the two regions. Moreover, AhR-mediated activity and antiestrogenic potencies were greater in region B, where the pollution level according to the chemical analysis was similar or less than in the other region, which indicates the presence of other atmospheric pollutants with specific effects. The results document the advantage and utility of the simultaneous use of bioassays and chemical analysis in risk assessment of complex environmental samples.
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Affiliation(s)
- Jirí Novák
- Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, 625 00 Brno, Czech Republic
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24
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Bakand S, Winder C, Khalil C, Hayes A. Toxicity Assessment of Industrial Chemicals and Airborne Contaminants: Transition fromIn VivotoIn VitroTest Methods: A Review. Inhal Toxicol 2008; 17:775-87. [PMID: 16195213 DOI: 10.1080/08958370500225240] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.
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Affiliation(s)
- S Bakand
- Chemical Safety and Applied Toxicology (CSAT) Laboratories, School of Safety Science, University of New South Wales, Sydney, Australia.
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25
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Wenger D, Gerecke AC, Heeb NV, Zennegg M, Kohler M, Naegeli H, Zenobi R. Secondary effects of catalytic diesel particulate filters: reduced aryl hydrocarbon receptor-mediated activity of the exhaust. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2008; 42:2992-2998. [PMID: 18497156 DOI: 10.1021/es071827x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
Diesel exhaust contains numerous toxic substances that show different modes of action such as triggering aryl hydrocarbon receptor (AhR)-mediated pathways. We investigated AhR-mediated activity of exhaust generated by a heavy-duty diesel engine operated with or without iron- or copper/iron-catalyzed diesel particulate filters (DPFs). AhR agonists were quantified using the DR-CALUX reporter gene assay (exposure of cells for 24 h). We found 54-60 ng 2,3,7,8-tetrachlorodibenzo-p-dioxin CALUX equivalents (TCDD-CEQs) per m3 of exhaust in unfiltered samples and 6-16 ng TCDD-CEQ m3 in DPF-treated samples. DPF applications decreased TCDD-CEQ concentrations by almost 90%. Concentrations of known AhR agonists were determined with GC/HRMS and converted to TCDD-CEQ concentrations using compound-specific relative potency values. The analyzed nine polycyclic aromatic hydrocarbons (PAHs) and the 172,3,7,8-chlorinated dibenzodioxins/furans (23,7,8-PCDD/Fs) contributed only marginally (0.6-1.6%) to the total agonist concentration. However, both DPFs also decreased concentrations of individual PAHs by 7(0-80%. Variation of the assay exposure time (8, 24, 48,72, and 96 h) revealed that AhR-mediated activity decreased over time and reached a plateau after 72 h, which was most likely due to biotransformation of AhR agonists by the exposed H4IIE cells. At the plateau, we measured 1-2 ng TCDD-CEQ m(-3) in both an unfiltered and a filtered exhaust sample. Our findings show that DPFs are a promising technology to detoxify diesel exhaust regarding compounds with AhR-mediated activity.
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Affiliation(s)
- Daniela Wenger
- Laboratory for Analytical Chemistry, Swiss Federal Laboratories for Materials Testing and Research, Uberlandstrasse 129, CH-8600 Dübendorf, Switzerland
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Skarek M, Janosek J, Cupr P, Kohoutek J, Novotná-Rychetská A, Holoubek I. Evaluation of genotoxic and non-genotoxic effects of organic air pollution using in vitro bioassays. ENVIRONMENT INTERNATIONAL 2007; 33:859-66. [PMID: 17512055 DOI: 10.1016/j.envint.2007.04.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/28/2006] [Revised: 03/11/2007] [Accepted: 04/03/2007] [Indexed: 05/15/2023]
Abstract
In this study, organic extracts of total suspended particles (TSP) and the particulate matter (PM) with the size below 2.5 microm (PM(2.5)) combined with organic extracts of the gas phase (GP) collected at two urban and two background localities were analyzed with a bacterial genotoxicity test, SOS chromotest, and an in vitro test for the dioxin toxicity determination, using a modified cell-line of rat hepatoma H4IIE.luc. In addition, the samples of TSP and GP were analyzed for PAHs contents. The PAHs concentrations and both of the toxic activities at the urban localities were much higher than ones at the background localities. Predominantly, traffic was a source of the urban air pollution there which was also confirmed by the evaluation of portions of certain PAHs (BaP/BPE, PYR/BaP) at the localities. On the other hand, the background localities were apparently affected by a long-distance transport of the pollutants from urban and industrial centers. The results of the bioassays indicated potential health risks for the population exposed to the organic air pollutants, especially at the urban localities. Based on the collected samples, distribution of the organic pollutants with the toxic effects in the air was evaluated. The significant portion of the direct genotoxins was bound to the particles larger than 2.5 microm. On the contrary, the indirect genotoxins were bound predominantly to the particles with the size below 2.5 microm. However, in the urban air they may be also bound to the larger particles, as well. While the direct genotoxicity may be related with the presence of PAH-derivatives as well as some polar organic pollutants, the indirect genotoxicity is related with the detected carcinogenic PAHs. But besides the above specified pollutants it is also necessary to consider the presence of other toxic components of the complex organic air pollution mixture that may also show potential health risks. This study demonstrates application of the combination of the screening bioassays for the evaluation of organic air pollution and identification of its health risks.
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Affiliation(s)
- M Skarek
- Research Center for Environmental Chemistry and Ecotoxicology (RECETOX), Faculty of Science, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic
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Skarek M, Cupr P, Bartos T, Kohoutek J, Klánová J, Holoubek I. A combined approach to the evaluation of organic air pollution - a case study of urban air in Sarajevo and Tuzla(Bosnia and Herzegovina). THE SCIENCE OF THE TOTAL ENVIRONMENT 2007; 384:182-93. [PMID: 17675217 DOI: 10.1016/j.scitotenv.2007.06.040] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/22/2006] [Revised: 06/12/2007] [Accepted: 06/21/2007] [Indexed: 05/16/2023]
Abstract
Organic pollution is a complex mixture where besides usually discussed polycyclic aromatic hydrocarbons (PAHs) a lot of other toxic or potentially toxic compounds occur. In this case, the organic air pollution in two important industrial cities, Sarajevo and Tuzla, in Bosnia and Herzegovina (part of former Yugoslavia) was assessed with the emphasis placed on genotoxic risks using both chemical (PAHs analyses) and biological approaches (genotoxicity testing with a screening bacterial genotoxicity test - SOS chromotest). The study was performed as a part of the APOPSBAL project (ICA2-CT2002-10007). So far there has not been any information either about the PAHs pollution or the genotoxic activity of the organic air pollution for the localities under the study. Therefore, the presented information is considered absolutely unique. Both used approaches made possible to identify the localities with the highest pollution level and genotoxic risks in both cities. Generally, higher levels of both parameters were determined in Tuzla, which is much more industrialized than Sarajevo, and especially at localities close to city centers and affected by traffic emissions, but also at localities polluted by emissions from industry and household heating. Even if benzo(a)pyrene concentrations exceeded the maximum permitted levels for this pollutant at some localities in Tuzla, the PAHs concentrations were fully comparable with the levels determined in other industrial European cities. Significant genotoxicity of the organic extracts was detected for almost all of the urban localities in the test both without (-S9; direct genotoxicity) and with the addition of metabolic activation (+S9; indirect genotoxicity). The observed direct genotoxic activities were discussed in relation to a potential presence of PAHs derivatives in the air. The indirect genotoxic activities were apparently higher at the localities with higher contents of carcinogenic PAHs. The significant relationship between the determined genotoxic activities and the PAHs pollution was also confirmed by a regression analysis. However, the correlations were not absolute because the observed genotoxic activity was also dependent on the presence of other organic pollutants than the PAHs. It concerns predominantly direct genotoxicity which is not related with the PAHs, but with their nitro-, oxi-, and hydroxy-derivatives and also other unknown polar organic pollutants. However, the concentrations of the direct genotoxins apparently correlated with the PAHs contents in the air. The study showed that screening genotoxicity tests, such as the SOS chromotest, could be effectively used for the identification of localities with increased genotoxic risks. In comparison with the health risk assessment which is usually based on the chemical analyses of only a small part of the pollution mixture, the bioassays enable us to evaluate the risks of all the mixture. The localities with the highest detected human health risks according to the screening bioassays may then be analyzed in detail with specific chemical methods to identify their causes.
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Affiliation(s)
- M Skarek
- Research Center for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Kamenice, Brno, Czech Republic
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Hwang DY, Chae KR, Kim CK, Kim BG, Shim SB, Jee SW, Lee SH, Sin JS, Jang MK, Seo SJ, Kim MS, Cho JS, Sheen YY, Choi SY, Kim YK. Differential effect of 7,12-dimethylbenz[a]anthracene on human and mouse CYP1B1 from livers of castrated transgenic mice. Int J Toxicol 2007; 26:71-80. [PMID: 17365149 DOI: 10.1080/10915810601120640] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time-and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.
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Affiliation(s)
- Dae Y Hwang
- Laboratory Animal Resources Team, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea
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Cupr P, Klánová J, Bartos T, Flegrová Z, Kohoutek J, Holoubek I. Passive air sampler as a tool for long-term air pollution monitoring: Part 2. Air genotoxic potency screening assessment. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2006; 144:406-13. [PMID: 16533550 DOI: 10.1016/j.envpol.2005.12.045] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/22/2005] [Accepted: 12/20/2005] [Indexed: 05/07/2023]
Abstract
The capability of passive air sampling to be employed in the evaluation of direct genotoxicity of ambient air samples was assessed. Genotoxic effects of the total extracts from the polyurethane foam filters exposed for 28 days during a regional passive air sampling campaign were investigated. Twenty sampling sites were selected in Brno city on the area of approximately 20x20 km in October and November 2004. Brno is the second largest city of the Czech Republic, highly industrialized with approximately 370,000 of permanent inhabitants. The levels of PAHs, PCBs, and chlorinated pesticides were determined in all samples. Fraction of each extract was also assayed in the bacterial genotoxicity test using Escherichia coli sulA::lacZ. Complete dose-response relationships of the air extracts were determined. The statistical analysis showed significant correlation between observed biological effects and PAHs concentrations in samples.
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Affiliation(s)
- Pavel Cupr
- RECETOX-Research Center for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3/126, 625 00 Brno, Czech Republic.
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Hamers T, van den Berg JHJ, van Gestel CAM, van Schooten FJ, Murk AJ. Risk assessment of metals and organic pollutants for herbivorous and carnivorous small mammal food chains in a polluted floodplain (Biesbosch, The Netherlands). ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2006; 144:581-95. [PMID: 16530312 DOI: 10.1016/j.envpol.2006.01.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/01/2005] [Revised: 01/04/2006] [Accepted: 01/11/2006] [Indexed: 05/07/2023]
Abstract
A risk assessment was made for a carnivorous and a herbivorous food chain in a heavily polluted natural estuary (Biesbosch), by determining the most critical pollutants and the food chain most at risk. Exposure of food chains to metals, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) was assessed by analyzing dietary concentrations, internal concentrations, and biomarkers of exposure. Common shrew (Sorex araneus) and bank vole (Clethrionomys glareolus) were selected as representative small mammal species for the carnivorous and herbivorous food chain, respectively, and earthworms (Lumbricus rubellus) and snails (Cepaea nemoralis) as representative prey species for the carnivorous food chain. Metals contributed most to the total risk for small mammals and earthworms. PCBs, but not PAHs, contributed to the overall risk for S. araneus at regularly flooded locations. The carnivorous food chain appeared most at risk given the higher exposure levels and bioaccumulating potency found for contaminants in S. araneus.
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Affiliation(s)
- Timo Hamers
- Wageningen University, Toxicology Group, PO Box 8000, 6700 EA Wageningen, The Netherlands.
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Xiao RY, Wang Z, Wang CX, Yu G, Zhu YG. Genotoxic risk identification of soil contamination at a major industrialized city in northeast China by a combination of in vitro and in vivo bioassays. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2006; 40:6170-5. [PMID: 17051817 DOI: 10.1021/es0607335] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
The present study evaluated the genotoxicity of field soils in the Tianjin area, one of the most industrialized contaminated areas in northeast China. The genotoxicity of organic extracts of 41 soils was assayed by an in vitro SOS/ umu bioassay with Salmonella typhimurium TA 1535/pSK 1002. From the 41 soil samples, 11 samples were selected to confirm the genotoxic effect by in vivo single-cell gel electrophoresis (comet assay) using earthworms (Eisenia fetida). The results obtained demonstrated that, in the in vitro assay, genotoxicity expressed as induction ratios (IR) ranged from 1.00 to 4.60, and in the in vivo assay, the genotoxicity expressed as tail moment (TM) varied from 14.6 to 57.8 microm. All samples with high genotoxicity assessed by the SOS/umu bioassay possessed significantly high genotoxic effects in the comet assay, and there was a correlation (R2 = 0.736, p < 0.05) between IR and TM in both bioassays. It is concluded that soils in the Tianjin area were seriously contaminated by organic genotoxicants and higher levels of genotoxic effects existed in soils in the urban area of Tianjin as well as in areas near the coastal towns in the northeast part of the city. It can be concluded that a combination of in vivo and in vitro bioassays as a powerful and efficient genotoxicity-assessing tool could facilitate the assessment of genotoxic risk at a regional scale.
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Affiliation(s)
- Rui-Yang Xiao
- State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, People's Republic of China
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Klein GP, Hodge EM, Diamond ML, Yip A, Dann T, Stern G, Denison MS, Harper PA. Gas-phase ambient air contaminants exhibit significant dioxin-like and estrogen-like activity in vitro. ENVIRONMENTAL HEALTH PERSPECTIVES 2006; 114:697-703. [PMID: 16675423 PMCID: PMC1459922 DOI: 10.1289/ehp.8496] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/13/2005] [Accepted: 12/29/2005] [Indexed: 05/09/2023]
Abstract
Several adverse health effects, such as respiratory and cardiovascular morbidity, have been linked to exposure to particulate matter in ambient air; however, the biologic activity of gas-phase ambient organic air contaminants has not been examined as thoroughly. Using aryl hydrocarbon receptor (AHR)-based and estrogen receptor (ER)-based cell bioassay systems, we assessed the dioxin-like and estrogenic activities of gas-phase organic ambient air contaminants compared with those of particulate-phase contaminants using samples collected between seasons over 2 years from an urban and a rural location in the Greater Toronto Area, Canada. The concentration of the sum (Sigma) of polycyclic aromatic hydrocarbons, which was highest in the gas phase, was 10-100 times more abundant than that of Sigmapolychlorinated biphenyls, Sigmanitro-polycyclic aromatic hydrocarbons, and Sigmaorganochlorine pesticides, and 10(3) to 10(4) times more abundant than Sigmapolychlorinated dibenzo-p-dioxins/dibenzofurans. Gas-phase samples induced significant AHR- and ER-dependent gene expression. The activity of the gas-phase samples was greater than that of the particulate-phase samples in the estrogen assay and, in one case, in the AHR assay. We found no strong associations between either summer or winter seasons or urban or rural locations in the relative efficacy of the extracts in either the ER or AHR assay despite differences in chemical composition, concentrations, and abundance. Our results suggest that mechanistic studies of the health effects of ambient air must consider gas and particulate phases because chemicals present in both phases can affect AHR and ER signaling pathways.
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Affiliation(s)
- Gail P Klein
- Department of Pharmacology, Centre for Urban Health Initiatives, University of Toronto, and Program in Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada
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Windal I, Denison MS, Birnbaum LS, Van Wouwe N, Baeyens W, Goeyens L. Chemically activated luciferase gene expression (CALUX) cell bioassay analysis for the estimation of dioxin-like activity: critical parameters of the CALUX procedure that impact assay results. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2005; 39:7357-64. [PMID: 16245802 DOI: 10.1021/es0504993] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/02/2023]
Abstract
The chemically activated luciferase gene expression (CALUX) in vitro cell bioassay is a bioanalytical tool that is increasingly being used by research and commercial laboratories for the screening and relative quantification of dioxins and dioxin-like compounds in sample extracts. Since CALUX analyses provide a biological response to all aryl hydrocarbon receptor active compounds present in a given sample extract containing a complex mixture of chemicals, interpretation of results is significantly more complexthan of chemical analyses. Operators in the laboratory can adjust many parameters when performing CALUX analyses, and the applied procedure strongly affects the result and, hence, the interpretation of the results. This paper examines critical methodological parameters and aspects of the CALUX bioassay that can affect the quality and accuracy of the analyses. Moreover, the study aims to identify the ways that alteration of these parameters influences CALUX measurements. A greater understanding of these characteristics will lead to increased accuracy, precision, and reproducibility of the widely used CALUX bioassay within and between research laboratories.
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Affiliation(s)
- Isabelle Windal
- Scientific Institute of Public Health, rue J. Wytsman 14, B-1050 Brussels, Belgium.
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Janosek J, Hilscherová K, Bláha L, Holoubek I. Environmental xenobiotics and nuclear receptors--interactions, effects and in vitro assessment. Toxicol In Vitro 2005; 20:18-37. [PMID: 16061344 DOI: 10.1016/j.tiv.2005.06.001] [Citation(s) in RCA: 122] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2004] [Revised: 05/19/2005] [Accepted: 06/13/2005] [Indexed: 11/18/2022]
Abstract
A group of intracellular nuclear receptors is a protein superfamily including arylhydrocarbon AhR, estrogen ER, androgen AR, thyroid TR and retinoid receptors RAR/RXR as well as molecules with unknown function known as orphan receptors. These proteins play an important role in a wide range of physiological as well as toxicological processes acting as transcription factors (ligand-dependent signalling macromolecules modulating expression of various genes in a positive or negative manner). A large number of environmental pollutants and other xenobiotics negatively affect signaling pathways, in which nuclear receptors are involved, and these modulations were related to important in vivo toxic effects such as immunosuppression, carcinogenesis, reproduction or developmental toxicity, and embryotoxicity. Presented review summarizes current knowledge on major nuclear receptors (AhR, ER, AR, RAR/RXR, TR) and their relationship to known in vivo toxic effects. Special attention is focused on priority organic environmental contaminants and experimental approaches for determination and studies of specific toxicity mechanisms.
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Affiliation(s)
- J Janosek
- RECETOX, Masaryk University Brno, Kamenice 3, 625 00 Brno, Czech Republic.
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Windal I, Van Wouwe N, Eppe G, Xhrouet C, Debacker V, Baeyens W, De Pauw E, Goeyens L. Validation and interpretation of CALUX as a tool for the estimation of dioxin-like activity in marine biological matrixes. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2005; 39:1741-8. [PMID: 15819233 DOI: 10.1021/es049182d] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Among the different analytical tools proposed as an alternative to the very expensive gas chromatography high-resolution mass spectrometry (GC-HRMS) analyses of polychlorodibenzo-p-dioxin and polychlorodibenzofurans, Chemically Activated LUciferase gene eXpression (CALUX) in vitro cell bioassay is very promising. It allows the analyses of a high number of samples since it is relatively fast, inexpensive, and sensitive. However, this technique is not yet widely applied for screening or environmental monitoring. The main reasons are probably the lack of validation and the difficulty in interpreting the global biological response of the bioassay. In this paper, the strict quality control criteria set up for the validation of CALUX are described. The validation has shown good repeatability (relative standard deviation (RSD) = 9%) and good within-lab reproducibility (RSD = 15%) of the results. The quantification limit, in the conditions applied in this paper, is 1.25 pg CALUX-TEQ/g fat. Comparison of CALUX and GC-HRMS analysis was made forvarious marine matrixes (fishes, mussels, starfishes, sea birds, and marine mammals). Good correlations are usually observed, but there are systematic differences between the results. Attempts are made to identify the origin of the discrepancy between the two methods.
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Affiliation(s)
- Isabelle Windal
- Scientific Institute of Public Health, 1050 Brussels, Belgium.
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Abstract
The delicate and dynamic balance of the physiological steady state and its maintenance is well characterized by studies of bacterial stress response. Through the use of genetic analysis, numerous stress regulons, their physiological regulators and their biochemical processes have been delineated. In particular, transcriptionally activated stress regulons are subjects of study and application. These regulons include those that respond to macromolecular damage and toxicity as well as to nutrient starvation. The convenience of reporter gene fusions has allowed the creation of biosensor strains, resulting from the fusion of stress-responsive promoters with a variety of reporter genes. Such cellular biosensors are being used for monitoring dynamic systems and can report the presence of environmental stressors in real time. They provide a greater range of sensitivity, e.g. to sub-lethal concentrations of toxicants, than the simple assessment of cell viability. The underlying physiological context of the reporter strains results in the detection of bioavailable concentrations of both toxicants and nutrients. Culture conditions and host strain genotypes can be customized so as to maximize the sensitivity of the strain for a particular application. Collections of specific strains that are grouped in panels are used to diagnose targets or mode of action for unknown toxicants. Further application in massive by parallel DNA and gene fusion arrays greatly extends the information available for diagnosis of modes of action and may lead to development of novel high-throughput screens. Future studies will include more panels, arrays, as well as single reporter cell detection for a better understanding of the population heterogeneity during stress response. New knowledge of physiology gained from further studies of novel systems, or using innovative methods of analysis, will undoubtedly yield still more useful and informative environmental biosensors.
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Affiliation(s)
- Amy Cheng Vollmer
- Department of Biology, Swarthmore College, 500 College Avenue, Swarthmore, PA 19081, USA.
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Behnisch PA, Hosoe K, Sakai SI. Brominated dioxin-like compounds: in vitro assessment in comparison to classical dioxin-like compounds and other polyaromatic compounds. ENVIRONMENT INTERNATIONAL 2003; 29:861-877. [PMID: 12850102 DOI: 10.1016/s0160-4120(03)00105-3] [Citation(s) in RCA: 239] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Recently, several countries agreed to adopt the Stockholm convention on persistent organic pollutants (POPs). One future obligation will be to add other POPs as new evidence becomes available. In vitro cell-based bioassays offer a rapid, sensitive, and relatively inexpensive solution to screen possible POP candidates. In the present study, we investigated the aryl hydrocarbon (Ah)-receptor activity of several dioxin-like POPs by using the Micro-EROD (Ethoxy-Resorufin-O-Deethylase) and DR-CALUX (Dioxin-Responsive-Chemical Activated Luciferase gene eXpression) bioassays, which are two state-of-the-art methods. The Micro-EROD system used in our study utilizes a wild-type rat liver cell line (rat liver H4IIEC3/T cells), while the DR-CALUX bioassay consists of a genetically modified rat hepatoma H4IIE cell line that incorporates the firefly luciferase gene coupled to dioxin-responsive elements (DREs) as a reporter gene. In the case of the DR-CALUX bioassay, we used an exposure time of 24 h, whereas we used a 72-h exposure time in the Micro-EROD bioassay. The aim of this study was to compare conventional dioxin-like POPs (such as polychlorinated dibenzodioxins and -furans, PCDD/Fs and coplanar polychlorinated biphenyls, PCBs) with several other classes of possible candidates to be added to the current toxicity equivalent factor (TEF) model in the future. Therefore, this study compares in vitro CYP1A1 (Micro-EROD bioassay) and firefly luciferase induction (DR-CALUX bioassay) in several mixed polyhalogenated dibenzodioxins and -furans (PXDD/Fs; X=Br, Cl, or F), alkyl-substituted polyhalogenated dibenzodioxins and -furans (PMCDD/Fs; M=methyl), polyhalogenated biphenyls (PXBs, X=Br, Cl ), polybrominated diphenyl ethers (PBDEs), pentabromophenols (PBPs), and tetrabromobisphenol-A (TBBP-A). We also evaluate congener-specific relative potencies (REPs) and efficacies (% of TCDD(max)) and discuss the dose-response curves of these compounds, as well as the dioxin-like potency of several other Ah-receptor agonists, such as those of the polyaromatic hydrocarbons (PAHs) and polychlorinated naphthalenes (PCNs). The highest REP values were found for several PXDD/F congeners, followed by some coplanar PXBs, trichlorinated PCDD/Fs, PAHs, PBDE-126, 1-6-HxCN, and some brominated flame retardants (TBBP-A). These in vitro investigations indicate that further research is necessary to evaluate more Ah-receptor agonists for dioxin-like potency.
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Affiliation(s)
- Peter Alexander Behnisch
- Life Science Research Laboratories, Kaneka Corporation, 1-8 Miyamae-Machi, Hyogo Takasago 676-8688, Japan.
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Zhang ZR, Xu SQ, Sun X, Xu YJ, Cai XK, Liu ZW, Tan XL, Zhou YK, Zhang JY, Yan H. Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals. World J Gastroenterol 2003; 9:1460-4. [PMID: 12854141 PMCID: PMC4615482 DOI: 10.3748/wjg.v9.i7.1460] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs.
METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL3-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG2, both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG2 cells stably transfected by the recombinant vector (HepG2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG2 cells (HepG2-wt).
RESULTS: The inducible luciferase expression of HepG2-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG2-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG2-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 × 10-12 to 5 × 10-9 mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG2-luc and HepG2-wt cells. The correlation between TCDD doses from 1.1 × 10-13 to 1 × 10-8 mol/L and luciferase activities was also found to be significant in HepG2-luc cells (r = 0.997, P < 0.001), but not in their HepG2-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG2-luc cells were both better than that of EROD in HepG2-wt cells, the former was at 1.1 × 10-13 mol/L and 3.5 × 10-12 mol/L, and the coefficients of variation (CV) of the latter was 15%-30% and 22%-38%, respectively.
CONCLUSION: The luciferase expression of HepG2-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.
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Affiliation(s)
- Zhi-Ren Zhang
- Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
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Aït-Aïssa S, Pandard P, Magaud H, Arrigo AP, Thybaud E, Porcher JM. Evaluation of an in vitro hsp70 induction test for toxicity assessment of complex mixtures: comparison with chemical analyses and ecotoxicity tests. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2003; 54:92-104. [PMID: 12547640 DOI: 10.1016/s0147-6513(02)00026-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The aim of this study was to assess the potential of a human cell line containing the hsp70 promoter linked to the chloramphenicol acetyltransferase reporter gene in evaluating the toxic potential of complex mixtures. Cells were exposed to eluates of industrial wastes and the cellular responses were compared with the metal contents of the samples and with standardized aquatic (microalgal growth inhibition, daphnia Immobilization, bacterial luminescence inhibition, Ceriodaphnia dubia reproduction inhibition) and terrestrial (earthworm lethality, plant growth inhibition) tests. The hsp70 promoter was significantly induced by 11 of 14 samples, with different dose-response patterns. Significant correlations of in vitro induction potency with aquatic ecotoxicity, especially with chronic tests, and with the metal contents of the samples were observed. Our study provides new information on the relevance of hsp70 gene induction as a criterion of toxicity and suggests its usefulness for the detection of toxicity associated with metallic pollution in complex mixtures.
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Affiliation(s)
- Sélim Aït-Aïssa
- Ecotoxicological Risks Assessment Unit, INERIS, BP2, F-60550 Verneuil-en-Halatte, France.
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Behnisch PA, Hosoe K, Sakai S. Combinatorial bio/chemical analysis of dioxin and dioxin-like compounds in waste recycling, feed/food, humans/wildlife and the environment. ENVIRONMENT INTERNATIONAL 2001; 27:495-519. [PMID: 11800431 DOI: 10.1016/s0160-4120(01)00029-0] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
The present review describes international activities using bioassays/biomarkers in combination with chemical analysis to measure the effects of dioxin and dioxin-like compounds (DLCs) in the environment. The above authors reviewed already the state-of-art bioanalytical detection methods (BDMs) for dioxins and DLCs [Environ Int (2001)]. The aim of this study will be to review applications of these bioassays/biomarkers to evaluate potential dioxins and DLCs. The present literature study lists relative potencies (REPs) of polyhalogenated dibenzo-p-dioxins and -furans (PXDD/Fs; X = Cl, Br, F), their thio analogues polychlorinated dibenzothiophenes (PCDTs) and thianthrens (PCTAs), polyhalogenated biphenyls (PXBs), polychlorinated naphthalenes (PCNs) and other Ah receptor agonists measured by several biodetectors (Tier 3 screening). The authors will discuss some examples of the applications of some of these biodetectors in biomonitoring programmes and recently occurred dioxin crisis in feed/food. The diagnosis of the biopotency of these pollutants in technical processes like thermally treated waste, waste water treatment, landfill leachate treatment, commercial PCB-mixtures, the release into the environment (soil, air and water) and the final intake into wildlife and humans will be reviewed.
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Affiliation(s)
- P A Behnisch
- Life Science Research Laboratories, Kaneka Corporation, Takasago, Japan.
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Behnisch PA, Hosoe K, Sakai S. Bioanalytical screening methods for dioxins and dioxin-like compounds a review of bioassay/biomarker technology. ENVIRONMENT INTERNATIONAL 2001; 27:413-39. [PMID: 11757855 DOI: 10.1016/s0160-4120(01)00028-9] [Citation(s) in RCA: 167] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Determination of environmental pollutants utilizing biodetectors such as bioassays, biomarkers, enzyme immunoassays (EIAs), or other bioanalytical tools is a continuously growing area. The present literature review describes the principles and advantages/limitations of several bioanalytical detection methods (BDMs) for the screening and diagnosis of dioxin and dioxin-like compounds. This study characterizes briefly the family of dioxin and dioxin-like compounds, discusses potential Ah receptor (AhR) ligands and cytochrome P-450 (CYP) 1A1-enzyme-inducing compounds. 'Milestones' in the development of BDMs are summarized and explained in detail for a number of bioanalytical tools that can be used to detect these classes of dioxin-like persistent bioaccumulative toxicants (PBTs). The design of a screening profile with a battery of bioassays/biomarkers coupled with the chemical analysis is evaluated. The relative potencies (REPs) to 2,3,7,8-TCDD for dioxin-like compounds are reviewed for various BDMs and the differences are noted.
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Affiliation(s)
- P A Behnisch
- Life Science Research Laboratories, Kaneka Corporation, Takasago, Japan.
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