PAH-CALUX, an optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic hydrocarbons (PAHs) as individual compounds and in complex mixtures.

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.

[1]  H. Segner,et al.  Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio. , 2012, Reproductive toxicology.

[2]  M. Bird,et al.  Preliminary evaluation of the human relevance of respiratory tumors observed in rodents exposed to naphthalene. , 2012, Regulatory toxicology and pharmacology : RTP.

[3]  A. Kozubík,et al.  The interplay of the aryl hydrocarbon receptor and β-catenin alters both AhR-dependent transcription and Wnt/β-catenin signaling in liver progenitors. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[4]  Thomas Braunbeck,et al.  Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-CALUX assay and the EROD assay with RTL-W1 cells , 2011, Environmental science and pollution research international.

[5]  B. Kaina,et al.  The aryl hydrocarbon receptor (AhR) in the regulation of cell-cell contact and tumor growth. , 2010, Carcinogenesis.

[6]  Yi-Wen Liu,et al.  Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor. , 2009, Toxicology.

[7]  H. Takigami,et al.  Evaluation of Toxic Activities of Polycyclic Aromatic Hydrocarbon Derivatives Using In Vitro Bioassays , 2009 .

[8]  I. Pongratz,et al.  Endocrine disruptive chemicals: mechanisms of action and involvement in metabolic disorders. , 2009, Journal of molecular endocrinology.

[9]  U. Stenius,et al.  Exposure of HepG2 cells to low levels of PAH‐containing extracts from contaminated soils results in unpredictable genotoxic stress responses , 2009, Environmental and molecular mutagenesis.

[10]  Henner Hollert,et al.  AhR agonist and genotoxicant bioavailability in a PAH-contaminated soil undergoing biological treatment , 2009, Environmental science and pollution research international.

[11]  Alvaro Puga,et al.  The aryl hydrocarbon receptor cross-talks with multiple signal transduction pathways. , 2009, Biochemical pharmacology.

[12]  A. Seidel,et al.  Determination of polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with confirmation by chemical analysis. , 2008, Journal of food protection.

[13]  Abraham Brouwer,et al.  Glucocorticoid-enhanced expression of dioxin target genes through regulation of the rat aryl hydrocarbon receptor. , 2007, Toxicological sciences : an official journal of the Society of Toxicology.

[14]  A. Masih,et al.  Polycyclic aromatic hydrocarbons (PAHs) concentrations and related carcinogenic potencies in soil at a semi-arid region of India. , 2006, Chemosphere.

[15]  C. Tohyama,et al.  The 2005 World Health Organization reevaluation of human and Mammalian toxic equivalency factors for dioxins and dioxin-like compounds. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[16]  Mary Beth Genter,et al.  Naphthalene toxicity in mice and aryl hydrocarbon receptor-mediated CYPs. , 2006, Biochemical and biophysical research communications.

[17]  A. Puga,et al.  Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis , 2005, Journal of cellular biochemistry.

[18]  Alvaro Puga,et al.  Ah receptor signals cross-talk with multiple developmental pathways. , 2005, Biochemical pharmacology.

[19]  M. Machala,et al.  A combined chemical and bioassay analysis of traffic-emitted polycyclic aromatic hydrocarbons. , 2004, The Science of the total environment.

[20]  Y. Fujii‐Kuriyama,et al.  Dibenzo[A,L]pyrene‐induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor‐deficient mice , 2004, International journal of cancer.

[21]  Y. Fujii‐Kuriyama,et al.  Functional role of AhR in the expression of toxic effects by TCDD. , 2003, Biochimica et biophysica acta.

[22]  M. Gallo,et al.  Ah receptor and NF-κB interactions: mechanisms and physiological implications , 2002 .

[23]  P. Roos.  Differential induction of CYP1A1 in duodenum, liver and kidney of rats after oral intake of soil containing polycyclic aromatic hydrocarbons , 2002, Archives of Toxicology.

[24]  J. Vondráček,et al.  Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay. , 2001, Mutation research.

[25]  A. Kozubík,et al.  Monitoring river sediments contaminated predominantly with polyaromatic hydrocarbons by chemical and in vitro bioassay techniques , 2001, Environmental toxicology and chemistry.

[26]  T. Hamers,et al.  The application of reporter gene assays for the determination of the toxic potency of diffuse air pollution. , 2000, The Science of the total environment.

[27]  Y. Fujii‐Kuriyama,et al.  Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Tohyama,et al.  Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells. , 1999, Biochemical and biophysical research communications.

[29]  D W Gaylor,et al.  Tumors and DNA adducts in mice exposed to benzo[a]pyrene and coal tars: implications for risk assessment. , 1998, Environmental health perspectives.

[30]  G V Alexeeff,et al.  Potency equivalency factors for some polycyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbon derivatives. , 1998, Regulatory toxicology and pharmacology : RTP.

[31]  S. Safe,et al.  Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene. , 1998, Biochemical pharmacology.

[32]  M. Mumtaz,et al.  Atsdr Evaluation of Health Effects of Chemicals. Iv. Polycyclic Aromatic Hydrocarbons (PAHs): Understanding a Complex Problem , 1996, Toxicology and industrial health.

[33]  L. Ehrenberg,et al.  Relevance of different biological assays in assessing initiating and promoting properties of polycyclic aromatic hydrocarbons with respect to carcinogenic potency. , 1996, Mutation research.

[34]  J. Giesy,et al.  Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. , 1996, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[35]  J. Stegeman,et al.  Aminoanthracene is a mechanism-based inactivator of CYP1A in channel catfish hepatic tissue. , 1995, Toxicology and applied pharmacology.

[36]  I. Nisbet,et al.  Toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs). , 1992, Regulatory toxicology and pharmacology : RTP.

[37]  Kevin C. Jones,et al.  ORGANIC CONTAMINANTS IN WELSH SOILS: POLYNUCLEAR AROMATIC HYDROCARBONS , 1989 .

[38]  B. Mahadevan,et al.  Carcinogenic polycyclic aromatic hydrocarbon‐DNA adducts and mechanism of action , 2005, Environmental and molecular mutagenesis.

[39]  F Kalberlah,et al.  Cancer risk assessment for oral exposure to PAH mixtures , 2002, Journal of applied toxicology : JAT.

[40]  J. H. Jansen,et al.  Development of improved DR-CALUX bioassay for sensitive measurement of aryl hydrocarbon receptor activating compounds , 2002 .