ReviewDioxin-like activity in environmental and human samples from Greenland and Denmark
Highlights
► Some pesticides, plasticizers and phytoestrogens can activate the AhR. ► The combined effect of plasticizers with no or weak AhR potency can be additive. ► Dioxin-like activity in wastewater effluents shows insufficient plant treatment. ► The combined serum POP induced dioxin-like activity reflects the mixture profile. ► The AhR reporter assay is a cost-effective screening tool for dioxin-like compounds.
Introduction
Humans are exposed to diverse harmful environmental contaminants such as persistent organic pollutants (POPs). Among POPs, dioxins and dioxin-like compounds (DLCs) are some of the most toxic chemicals that are highly persistent in the environment. It has been documented that exposure to dioxins and DLCs may cause a series of negative effects both in animal experiments and in human epidemiological studies such as carcinogenicity (Steenland et al., 2004), immunotoxicity and adverse effects on reproduction, neurobehaviour (Lindstrom et al., 1995). The most famous events about dioxins and their toxicity are the use of Agent Orange in the Vietnam War (1961–1971) and the industrial disaster in Seveso, Italy (1976) where dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was the primary toxic component and caused high exposure of the residential populations. Dioxins and DLCs have never been produced for commercial purposes and are made inadvertently during many processes involving chlorine by burning chlorine-based chemical compounds with hydrocarbons. The major source of dioxins in the environment comes from waste-burning incinerators of various sorts and also from backyard burn-barrels. Paper bleaching, production of Polyvinyl Chloride plastics and production of chlorine and organochlorine chemicals (e.g. pesticides) are also the sources of dioxins and DLCs in the environment. Human exposure to dioxins and DLCs is mainly through the consumption of contaminated food. Since these compounds are fat-soluble, they do bioaccumulate through the food chain and found in e.g. meat, fish, milk, dairy products, fats, and oils that are the main sources of exposure for adults, and newborns get an additional exposure via breast feeding.
The classical dioxins and DLCs include 7 polychlorodibenzo-p-dioxins (PCDDs), 10 polychlorodibenzofurans (PCDFs), and 12 dioxin-like polychlorinated biphenols (DL-PCBs) (non-ortho coplanar PCBs: PCB 77, 81, 126, and 169 and mono-ortho-substituted PCBs: PCB 105, 114, 118, 123, 156, 157, 189) (Van den Berg et al., 1998, van den Berg et al., 2000) (Supplementary Fig. 1). The biological and toxicological effects of dioxins and DLCs are mediated via the aryl hydrocarbon receptor (AhR) (Poland and Knutson, 1982, Schmidt and Bradfield, 1996).
The AhR is an intracellular ligand-dependent transcriptional factor. The AhR binds dioxins and DLCs and is transactivated and mediates the toxicity of these compounds by cellular processes. Most tissues in mammals express the AhR constitutively at low levels (Dolwick et al., 1993, Carver et al., 1994).
Mechanistically, the function of AhR is similar to that of the steroid hormone receptors (Hankinson, 1995). Upon binding to the ligand, the cytosolic ligand-AhR complex translocates into the nucleus and dimerizes with the AhR translocator (Arnt). The ligand-AhR-Arnt complex then binds to specific DNA sequences, the dioxin-responsive elements (DREs), and thus stimulates the transcription of adjacent genes including e.g. CYP1A1 and CYP1B1 (Hankinson, 1995, Whitlock, 1999), and in reporter gene assays the transcription of the luciferape (Fig. 1).
Classical AhR ligands are planar and can occupy a hydrophobic pocket within AhR, such as PCDDs, PCDFs and DL-PCBs mentioned as well as polycyclic aromatic hydrocarbons e.g. benzo[a]pyrene (Poland and Knutson, 1982). TCDD is the most potent known AhR ligand. However, more recent studies reveal that compounds of diverse structure and lipophility can bind AhR and induce gene expression, such compounds can be synthetic (e.g. methylenedioxybenzenes) or naturally occurring (e.g. indoles) (Denison and Heath-Pagliuso, 1998, Ciolino et al., 1999). Therefore humans are exposed to a mixture of man-made and natural AhR-modulation compounds. It is necessary to identify and characterize possible AhR ligands in the environment and biota and monitor the actual concerted action of these compounds on the AhR function in humans as well as elucidate their effects in vitro and ex vivo.
Due to different origin, solubility, volatility and metabolic stability, dioxins and DLCs occur as complex mixtures with concentrations of the individual compounds differing substantially. This complexity, along with differences in toxicity, increases the difficulty of risk evaluation. To estimate the total toxicity of dioxins and related compounds, the concept of the Toxic Equivalency Factor (TEF) has been developed. TEF is a number representing the toxic potency of a particular compound to induce AhR mediated effects related to the reference substance, TCDD which TEF value is set to 1.0 (Van den Berg et al., 2006) (Supplementary Table 1). The concept of TEQ (TCDD toxic equivalent) has thus been introduced to simplify risk assessment and regulatory control (Van den Berg et al., 1998). The classical TEQs are calculated by multiplying the concentration of individual PCDDs/PCDFs/PCBs by their respective TEFs.
Previous studies emphasize that assessment of the toxicological potential of a chemical mixture is much more complex than can be deduced by a given TEF dependent calculated TEQ value (Van Overmeire et al., 2001, Bonefeld Jorgensen and Ayotte, 2003). There are several drawbacks using the TEF concept for risk assessment of mixtures of POPs such as the very expensive, high volume requirement and time consuming gas chromatography mass spectrometry (GC-MS) determinations, small concentrations of individual congeners (below detection limits), presence of compounds not routinely measured or unknown substances with AhR affinity, the lack of TEF values for several POPs, and possible antagonistic or synergistic interactions between POPs (Safe, 1994, Aarts et al., 1995, Long et al., 2003). Thus there is a need for an integrated risk assessment of dioxins and DLCs.
A variety of different in vitro assays for studies of AhR-mediated toxicities were suggested (Behnisch et al., 2001). Since AhR acts as a transcription factor, reporter gene assays for assessment of its activity have become widespread during the last decade. The AhR-mediated transactivation bioassay is a mechanistically based technique that can detect all the compounds which can activate the AhR and AhR-dependent gene expression (i.e. AhR agonists). To facilitate the identification of AhR agonists, recombinant cell lines have been established by transient or stable transfection of a wide of type cell lines with the firefly luciferase reporter gene under transcriptional control of the DREs. These constructed cell lines still contain the complete machinery which is involved in the mode of action of DLCs. They are capable of quantifying compounds that have the potency to transactivate the AhR, resulting in the production of the reporter luminescent enzyme luciferase. The cellular response can be measured by adding suitable reagents (e.g., the substrate luciferin and ATP), and quantifying the produced luminescence emission by an automated luminometer (Fig. 1). The measured luminescence is then converted into a relative potency value for pure compounds or an AhR-TEQ value for the actual mixtures found in environmental or biological sample (Long et al., 2003, Long et al., 2006). The rapidity and lower cost of the AhR-mediated transactivation bioassay is attractive for analyses of high number of samples required for epidemiological studies, and as a tool to monitor and ensure reduction of contamination by DLCs in the food chain. The key application role of the AhR-mediated transactivation bioassay is in screening and relative quantification of DLCs in samples such as blood, sediments, food matrices and milk (Windal et al., 2005b). The AhR-mediated transactivation bioassay have been utilized in an array of projects to study the AhR -mediated activities of individual chemicals and mixtures (Safe, 1994, Aarts et al., 1995, Garrison et al., 1996, Long et al., 2003, Anderson et al., 2006, Bonefeld-Jorgensen et al., 2007, Bonefeld-Jorgensen, 2010, Kruger et al., 2008a) as well as for epidemiological purposes (Windal et al., 2005a, Long et al., 2006, Long et al., 2007a, Bonefeld-Jorgensen and Long, 2010).
In this review, a series of studies carried out in our research group regarding the dioxin-like (DL) potential of single compounds such as pesticides, plasticizers and phytoestrogens and their mixtures, and the DL-activity of complex compound mixtures in environmental and human samples are summarized.
Section snippets
Pesticides
Pesticides comprise a large number of different substances with dissimilar structures and diverse toxicity. The AhR potential of the persistent organochlorine insecticide Dieldrin and 22 pesticides currently or previously used in Denmark were assessed by using the AhR-mediated transactivation bioassay in the human TV101L hepatoma cells and the rat H4IIE hepatoma cells (Long et al., 2003). The pesticides were analyzed by cell exposure alone reflecting the agonistic potential and by co-exposure
Wastewater
Wastewater contains multiple chemical substances, some of which have the potential to disrupt endocrine processes in living organisms (Ma et al., 2005, Murata and Yamauchi, 2008, Van der Linden et al., 2008, Ishihara et al., 2009). In industrialized countries most of the sewage produced is treated in a sewage treatment plant (STP) before it is discharged to receiving environmental water streams. Nevertheless some natural hormones and a number of man-made chemicals with the potential to disrupt
Dioxin-like activity in the serum/plasma of Greenlandic Inuit
Laboratory studies on the effects of single chemicals or chemical mixtures in vitro in cell cultures and laboratory animals cannot fully elucidate the human health risks. Integration of epidemiological biomarker studies on humans from exposed populations is needed in order to obtain information about the real health risks resulting from exposures to the accumulated complex mixtures of contaminants. The burden of POPs in the Arctic people has been monitored since 1991, and a program for
Conclusions and perspectives
The in vitro result showed that some pesticides, plastic components and phytoestrogens might affect the AhR function, and the combined effect of compounds with no or weak AhR potency cannot be ignored, and that the combined effect of the complex mixture of compounds present in human blood must be taken into consideration for risk assessment.
The results from the wastewater study indicated that the AhR-mediated transactivity can be used for a more sensitive biomonitoring of effects of effluents
Acknowledgements
The authors thank all those have contributed to the reported data. These include researchers associated with the Centre for Arctic Environmental Medicine & Unit of Cellular and Molecular Toxicology, Department of Public Health, Aarhus University: postdoctoral researchers Tanja Kruger and Mandana Ghisari, laboratory assistant Birgitte Sloth Andersen and Dorte Olsson. Also thanked are Associate Professor Bente Deutch, Associate Professor Jens C. Hansen, Dr. Henning S. Pedersen, senior scientist
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