Article pubs.acs.org/est
Activation of Avian Aryl Hydrocarbon Receptor and Inter-species Sensitivity Variations by Polychlorinated Diphenylsulfides Rui Zhang,† Xiaowei Zhang,*,† Junjiang Zhang,† Ruijuan Qu,† Jiamin Zhang,† Xing Liu,‡ Jun Chen,‡ Zunyao Wang,*,† and Hongxia Yu† †
State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, People’s Republic of China ‡ Taizhou Environmental Monitoring Central Station, Taizhou 225300, People’s Republic of China S Supporting Information *
ABSTRACT: It was hypothesized that polychlorinated diphenyl sulfides (PCDPSs) can potentially interact with an aryl hydrocarbon receptor (AHR) and thereby cause adverse effects in wildlife like birds. A recently developed avian AHR1-luciferase report gene (LRG) assay was used to assess the interaction between avian AHR1 and 18 PCDPSs and to compare the interspecies sensitivity among chicken, ring-necked pheasant, and Japanese quail by PCDPSs. Most of the tested PCDPSs could activate the AHR1-mediated pathways in avian species, and the relative potency (ReP) of the PCDPSs increased with the increasing number of substituted Cl atoms. The rank orders of PCDPSs potency were generally similar among birds, although the ReP varied. In addition, not all the sensitivity rank orders of avian AHR1 constructs for PCDPSs were consistent with that of TCDD. ReP values of PCDPSs suggested that some PCDPSs like 2,3,3′,4,5,6-hexa-CDPS and 2,2′,3,3′,4,5,6-hepta-CDPS are higher than the avian WHO-TEFs of OctaCDD, OctaCDF, and most of the coplanar PCBs. Our results report for the first time the activation of an AHR1-mediated molecular toxicological mechanism by PCDPSs, and provide the ranking of ReP and relative sensitivity values of different congeners, which could guide the further toxicity test of this group of potential high priority environmental pollutants.
■
Toxicological knowledge about PCDPSs is very limited.1−6 Several PCDPSs are known to have antimicrobial and pesticidal activity.9,10 While for vertebrates, a tetra-PCDPS (3,3′,4,4′tetra-CDPS) had acute mortality in rats, rabbits, and freshwater fish.11,12 Enzymatic assays showed that exposure to PCDPSs modulated the hepatic antioxidant defense system, including superoxide dismutase, catalase, and in the levels of malondialdehyde, in mice and freshwater goldfish.11,13 However, the underlying toxicological mechanism by PCDPSs is not clear. The recently developed “adverse outcome pathway” (AOP) concept represents a new paradigm of toxicological assessment of chemicals, which uses a pathways-based approach to assess toxicity and potential risks caused by chemicals.14 One key aspect of applying the AOP framework is the establishment of well-characterized assays that can be used to evaluate the perturbation at the molecular initiating event by chemicals. Binding to and activation of nuclear aryl hydrocarbon receptor (AHR) is the molecular initiating event by dioxins and many
INTRODUCTION
Polychlorinated diphenyl sulfides (PCDPSs) are a group of chlorinated aromatic compounds comprising possibly 209 homologues. They have recently been ranked as high priority environmental pollutants because of their considerable persistence and environmental mobility properties.1,2 These chemicals have a wide range of industrial applications, such as high-temperature resistant lubricants in gas turbines and steam machines,3 or as additives in fire-preventing or insulating media.4 In the environment, PCDPSs have been detected in dust from metal recycling plants,5 gas and fly ash from waste incineration,6 pulp and paper mill wastewater,6 and water and sediments in Elbe and Yangtze rivers.7,8 Their structures resemble that of polychlorinated diphenyl ether, with the thioether bond replacing the ether bond. Gaussian-based models indicated that PCDPSs are lipophilic and tend to accumulate in organisms at high trophic levels.2 Multimedia modeling of environmental fate and transport indicated that intensive bioaccumulation of almost all of PCDPSs, as well as high overall persistence of all of them and long-range transport potential of some of them.1 These data suggest that PCDPSs could cause severe harm to living organisms in the environment. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 10948
May 30, 2014 August 20, 2014 August 20, 2014 August 20, 2014 dx.doi.org/10.1021/es502641f | Environ. Sci. Technol. 2014, 48, 10948−10956
Environmental Science & Technology
Article
Figure 1. Structural formulas of 18 PCDPSs tested in bioassays.
PCDPSs on AHR1 activity could be caused by the genetic variation on the AHR1 sequence, especially on the LBD. The objectives of this study were two-fold: (1) to determine the dioxin-like activities of 18 PCDPSs in birds using the avian AHR1-LRG assays; and (2) to compare the avian species sensitivities and to derive species-specific ReP values of PCDPSs.
dioxin-like compounds (DLCs), which can further lead to oxidative stress, embryo mortality, and carcinogenesis in vertebrates like mammals and birds.15−17 In mammalian species, only one AHR isoform has been identified.18 Whereas in avian species, two AHR isoforms (AHR1 and AHR2) have been identified to be orthologous to the mammalian AHR.19 It has previously been shown that both AHR1 and AHR2 might be involved in the toxicity of DLCs,20 but AHR1 is the dominant isoform that is involved in dioxin-induced toxicity in avian species,19 Luciferase report gene (LRG) assay is an effective cell-based assay for monitoring nuclear receptormediated molecular initiating event. For example, AHR-LRG assay has been shown to be useful for testing chemical-induced effects on AHR-mediated activity in mammalian and avian species.20−24 Furthermore, genetic variation on the AHR sequence, especially on the ligand-binding domain (LBD), among wildlife could lead to the interspecies sensitivity variation on activity. LRG assay has also been used for predicting the avian species-specific relative potency (ReP) values of DLCs and the relative sensitivity (ReS) values of avian species to the effects of the compounds.22,25−28 Here we hypothesized that PCDPSs could mimic the activity of dioxin to activate avian AHR1 due to their dioxin-like structure. Furthermore, the interspecies sensitivity variation to
■
MATERIALS AND METHODS Chemicals and Solutions. The PCDPSs were synthesized previously by the palladium-catalyzed carbon−sulfur bond formation method.29 The purity (>99%) of all test PCDPSs has been identified by high pressure liquid chromatography (Agilent 1200) and 1H NMR (Bruker Avance-400 MHz) elsewhere.29 In addition, six randomly selected PCDPSs were analyzed to confirm the absence of dioxin-like PCDDs/PCDFs using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) (Thermo DFS; Thermo Fisher Scientific, Bremen, Germany) following the HJ 77.4 method.30 For the PCDPSs (Figure 1), series of desired nominal concentrations ranging from 3 × 103 to 1 × 107 nM were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, U.S.A.) for LRG assays (no cytotoxic effect could be observed by MTS cytotoxicity assay). As the reference 10949
dx.doi.org/10.1021/es502641f | Environ. Sci. Technol. 2014, 48, 10948−10956
Environmental Science & Technology
Article
Table 1. Relative Sensitivity (ReS) Values for the Chicken, Ring-Necked Pheasant, and Japanese Quail AHR1 Constructs Exposed to TCDD or PCDPSsa AHR1 constructb,c compounds 2,3,7,8-tetra-chlorodibenzodioxin
TCDD
2,3,3′,4,5,6-hexa-chlodiphenylsulfides
2,3,3′,4,5,6-hexa-CDPS
2,2′,3,3′,4,5,6-hepta-chlodiphenylsulfides
2,2′,3,3′,4,5,6-hepta-CDPS
2,2′,3′,4,5-penta-chlodiphenylsulfides
2,2′,3′,4,5-penta-CDPS
2,4,4′,5-tetra-chlodiphenylsulfides
2,4,4′,5-tetra-CDPS
2,3,3′,4,4′,5,6-hepta-chlodiphenylsulfides
2,3,3′,4,4′,5,6-hepta-CDPS
2,3,4,4′,5,6-hexa-chlodiphenylsulfides
2,3,4,4′,5,6-hexa-CDPS
2,2′,4,5-tetra-chlodiphenylsulfides
2,2′,4,5-tetra-CDPS
4,4′-dichlodiphenylsulfudes
4,4′-di-CDPS
2,2′,4,4′,5-penta-chlodiphenylsulfides
2,2′,4,4′,5-penta-CDPS
2,3,4,5,6-pentac-hlodiphenylsulfides
2,3,4,5,6-penta-CDPS
2,3′,4,5-tetra-chlodiphenylsulfides
2,3′,4,5-tetra-CDPS
3,4′-dichlodiphenylsulfudes
3,4′-di-CDPS
2,2′,3,3′-tetra-chlodiphenylsulfides
2,2′,3,3′-tetra-CDPS
2,3-dichlodiphenylsulfudes
2,3-di-CDPS
2,2′,3-trichlodiphenyl sulfides
2,2′,3-tri-CDPS
2,4′,5-trichlodiphenylsulfides
2,4′,5-tri-CDPS
2,4′,6-trichlodiphenylsulfides
2,4′,6-tri-CDPS
2,3,3′-trichlodiphenylsulfides
2,3,3′-tri-CDPS
ReS
chicken
pheasant
quail
ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10
1.0a 1.0a 1.0 1.0a 1.0a 1.0a 1.0 1.0a 1.0 1.0a 1.0a 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0 1.0 1.0a 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
0.031a 0.093b NC 0.31a NC 1.2a NC NC NC 0.094b 0.98b 0.72b NC 1.5a NC NC NC 0.12b NC 0.20b NC 0.43b NC NC NC NC NC 1.1a NC NC NC NC
Activation of Avian Aryl Hydrocarbon Receptor and Inter-species Sensitivity Variations by Polychlorinated Diphenylsulfides Rui Zhang,† Xiaowei Zhang,*,† Junjiang Zhang,† Ruijuan Qu,† Jiamin Zhang,† Xing Liu,‡ Jun Chen,‡ Zunyao Wang,*,† and Hongxia Yu† †
State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, People’s Republic of China ‡ Taizhou Environmental Monitoring Central Station, Taizhou 225300, People’s Republic of China S Supporting Information *
ABSTRACT: It was hypothesized that polychlorinated diphenyl sulfides (PCDPSs) can potentially interact with an aryl hydrocarbon receptor (AHR) and thereby cause adverse effects in wildlife like birds. A recently developed avian AHR1-luciferase report gene (LRG) assay was used to assess the interaction between avian AHR1 and 18 PCDPSs and to compare the interspecies sensitivity among chicken, ring-necked pheasant, and Japanese quail by PCDPSs. Most of the tested PCDPSs could activate the AHR1-mediated pathways in avian species, and the relative potency (ReP) of the PCDPSs increased with the increasing number of substituted Cl atoms. The rank orders of PCDPSs potency were generally similar among birds, although the ReP varied. In addition, not all the sensitivity rank orders of avian AHR1 constructs for PCDPSs were consistent with that of TCDD. ReP values of PCDPSs suggested that some PCDPSs like 2,3,3′,4,5,6-hexa-CDPS and 2,2′,3,3′,4,5,6-hepta-CDPS are higher than the avian WHO-TEFs of OctaCDD, OctaCDF, and most of the coplanar PCBs. Our results report for the first time the activation of an AHR1-mediated molecular toxicological mechanism by PCDPSs, and provide the ranking of ReP and relative sensitivity values of different congeners, which could guide the further toxicity test of this group of potential high priority environmental pollutants.
■
Toxicological knowledge about PCDPSs is very limited.1−6 Several PCDPSs are known to have antimicrobial and pesticidal activity.9,10 While for vertebrates, a tetra-PCDPS (3,3′,4,4′tetra-CDPS) had acute mortality in rats, rabbits, and freshwater fish.11,12 Enzymatic assays showed that exposure to PCDPSs modulated the hepatic antioxidant defense system, including superoxide dismutase, catalase, and in the levels of malondialdehyde, in mice and freshwater goldfish.11,13 However, the underlying toxicological mechanism by PCDPSs is not clear. The recently developed “adverse outcome pathway” (AOP) concept represents a new paradigm of toxicological assessment of chemicals, which uses a pathways-based approach to assess toxicity and potential risks caused by chemicals.14 One key aspect of applying the AOP framework is the establishment of well-characterized assays that can be used to evaluate the perturbation at the molecular initiating event by chemicals. Binding to and activation of nuclear aryl hydrocarbon receptor (AHR) is the molecular initiating event by dioxins and many
INTRODUCTION
Polychlorinated diphenyl sulfides (PCDPSs) are a group of chlorinated aromatic compounds comprising possibly 209 homologues. They have recently been ranked as high priority environmental pollutants because of their considerable persistence and environmental mobility properties.1,2 These chemicals have a wide range of industrial applications, such as high-temperature resistant lubricants in gas turbines and steam machines,3 or as additives in fire-preventing or insulating media.4 In the environment, PCDPSs have been detected in dust from metal recycling plants,5 gas and fly ash from waste incineration,6 pulp and paper mill wastewater,6 and water and sediments in Elbe and Yangtze rivers.7,8 Their structures resemble that of polychlorinated diphenyl ether, with the thioether bond replacing the ether bond. Gaussian-based models indicated that PCDPSs are lipophilic and tend to accumulate in organisms at high trophic levels.2 Multimedia modeling of environmental fate and transport indicated that intensive bioaccumulation of almost all of PCDPSs, as well as high overall persistence of all of them and long-range transport potential of some of them.1 These data suggest that PCDPSs could cause severe harm to living organisms in the environment. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 10948
May 30, 2014 August 20, 2014 August 20, 2014 August 20, 2014 dx.doi.org/10.1021/es502641f | Environ. Sci. Technol. 2014, 48, 10948−10956
Environmental Science & Technology
Article
Figure 1. Structural formulas of 18 PCDPSs tested in bioassays.
PCDPSs on AHR1 activity could be caused by the genetic variation on the AHR1 sequence, especially on the LBD. The objectives of this study were two-fold: (1) to determine the dioxin-like activities of 18 PCDPSs in birds using the avian AHR1-LRG assays; and (2) to compare the avian species sensitivities and to derive species-specific ReP values of PCDPSs.
dioxin-like compounds (DLCs), which can further lead to oxidative stress, embryo mortality, and carcinogenesis in vertebrates like mammals and birds.15−17 In mammalian species, only one AHR isoform has been identified.18 Whereas in avian species, two AHR isoforms (AHR1 and AHR2) have been identified to be orthologous to the mammalian AHR.19 It has previously been shown that both AHR1 and AHR2 might be involved in the toxicity of DLCs,20 but AHR1 is the dominant isoform that is involved in dioxin-induced toxicity in avian species,19 Luciferase report gene (LRG) assay is an effective cell-based assay for monitoring nuclear receptormediated molecular initiating event. For example, AHR-LRG assay has been shown to be useful for testing chemical-induced effects on AHR-mediated activity in mammalian and avian species.20−24 Furthermore, genetic variation on the AHR sequence, especially on the ligand-binding domain (LBD), among wildlife could lead to the interspecies sensitivity variation on activity. LRG assay has also been used for predicting the avian species-specific relative potency (ReP) values of DLCs and the relative sensitivity (ReS) values of avian species to the effects of the compounds.22,25−28 Here we hypothesized that PCDPSs could mimic the activity of dioxin to activate avian AHR1 due to their dioxin-like structure. Furthermore, the interspecies sensitivity variation to
■
MATERIALS AND METHODS Chemicals and Solutions. The PCDPSs were synthesized previously by the palladium-catalyzed carbon−sulfur bond formation method.29 The purity (>99%) of all test PCDPSs has been identified by high pressure liquid chromatography (Agilent 1200) and 1H NMR (Bruker Avance-400 MHz) elsewhere.29 In addition, six randomly selected PCDPSs were analyzed to confirm the absence of dioxin-like PCDDs/PCDFs using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) (Thermo DFS; Thermo Fisher Scientific, Bremen, Germany) following the HJ 77.4 method.30 For the PCDPSs (Figure 1), series of desired nominal concentrations ranging from 3 × 103 to 1 × 107 nM were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, U.S.A.) for LRG assays (no cytotoxic effect could be observed by MTS cytotoxicity assay). As the reference 10949
dx.doi.org/10.1021/es502641f | Environ. Sci. Technol. 2014, 48, 10948−10956
Environmental Science & Technology
Article
Table 1. Relative Sensitivity (ReS) Values for the Chicken, Ring-Necked Pheasant, and Japanese Quail AHR1 Constructs Exposed to TCDD or PCDPSsa AHR1 constructb,c compounds 2,3,7,8-tetra-chlorodibenzodioxin
TCDD
2,3,3′,4,5,6-hexa-chlodiphenylsulfides
2,3,3′,4,5,6-hexa-CDPS
2,2′,3,3′,4,5,6-hepta-chlodiphenylsulfides
2,2′,3,3′,4,5,6-hepta-CDPS
2,2′,3′,4,5-penta-chlodiphenylsulfides
2,2′,3′,4,5-penta-CDPS
2,4,4′,5-tetra-chlodiphenylsulfides
2,4,4′,5-tetra-CDPS
2,3,3′,4,4′,5,6-hepta-chlodiphenylsulfides
2,3,3′,4,4′,5,6-hepta-CDPS
2,3,4,4′,5,6-hexa-chlodiphenylsulfides
2,3,4,4′,5,6-hexa-CDPS
2,2′,4,5-tetra-chlodiphenylsulfides
2,2′,4,5-tetra-CDPS
4,4′-dichlodiphenylsulfudes
4,4′-di-CDPS
2,2′,4,4′,5-penta-chlodiphenylsulfides
2,2′,4,4′,5-penta-CDPS
2,3,4,5,6-pentac-hlodiphenylsulfides
2,3,4,5,6-penta-CDPS
2,3′,4,5-tetra-chlodiphenylsulfides
2,3′,4,5-tetra-CDPS
3,4′-dichlodiphenylsulfudes
3,4′-di-CDPS
2,2′,3,3′-tetra-chlodiphenylsulfides
2,2′,3,3′-tetra-CDPS
2,3-dichlodiphenylsulfudes
2,3-di-CDPS
2,2′,3-trichlodiphenyl sulfides
2,2′,3-tri-CDPS
2,4′,5-trichlodiphenylsulfides
2,4′,5-tri-CDPS
2,4′,6-trichlodiphenylsulfides
2,4′,6-tri-CDPS
2,3,3′-trichlodiphenylsulfides
2,3,3′-tri-CDPS
ReS
chicken
pheasant
quail
ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10 ReSEC50 ReSPC10
1.0a 1.0a 1.0 1.0a 1.0a 1.0a 1.0 1.0a 1.0 1.0a 1.0a 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0a 1.0 1.0 1.0 1.0a 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
0.031a 0.093b NC 0.31a NC 1.2a NC NC NC 0.094b 0.98b 0.72b NC 1.5a NC NC NC 0.12b NC 0.20b NC 0.43b NC NC NC NC NC 1.1a NC NC NC NC
