Induction Equivalency Factors Research Articles

Estimation of Per- and Polyfluorinated Alkyl Substance Induction Equivalency Factors for Humpback Dolphins by Transactivation Potencies of Peroxisome Proliferator-Activated Receptors.

The potential risks of per- and polyfluoroalkyl substance (PFAS) accumulation in nearshore dolphins are not well understood. Here, transcriptional activities of 12 PFAS on peroxisome proliferator-activated receptors (PPAR-α, -β/δ, and -γ) in Indo-Pacific humpback dolphins (Sousa chinensis) were evaluated. All PFAS activated scPPAR-α in a dose-dependent manner. PFHpA exhibited the highest induction equivalency factors (IEFs). The order of IEFs for other PFAS was as follows: PFOA > PFNA > PFHxA > PFPeA > PFHxS > PFBA > PFOS > PFBuS ≈ PFDA ≫ PFUnDA and PFDoDA (not activated). The total induction equivalents (∑IEQs, 5537 ng/g wet weight) indicated that more attention should be paid to investigating contamination levels in dolphins, especially in PFOS (82.8% contribution to the ∑IEQs). The scPPAR-β/δ and -γ were not affected by any PFAS, except for PFOS, PFNA, and PFDA. Furthermore, PFNA and PFDA could induce higher PPAR-β/δ and PPAR-γ-mediated transcriptional activities than PFOA. Compared to human beings, PFAS might be more potent PPAR-α activators in humpback dolphins, suggesting that the dolphins may be more susceptible to the adverse effects of PFAS. Our results may be instructive for understanding the impacts of PFAS on marine mammal health due to the identical PPAR ligand-binding domain.

Assessment of Risks of Dioxins for Aryl Hydrocarbon Receptor-Mediated Effects in Polar Bear (Ursus maritimus) by in Vitro and in Silico Approaches

Polar bear (Ursus maritimus) populations accumulate dioxins and related compounds (DRCs) at levels that are of health concern. The toxicities of DRCs are primarily mediated via aryl hydrocarbon receptor (AHR) signaling pathway. To evaluate the sensitivity and responses to DRCs in polar bears, we assessed the activation potencies of polar bear-specific AHR (pbAHR) by DRCs through in vitro and in silico approaches. In vitro assays showed that the pbAHR was as sensitive to DRCs as C3H/lpr mouse AHR, which is well known to be highly sensitive to DRCs. Comparison of pbAHR transactivation potencies indicated that TCDF, 2,3,4,7,8-PeCDF, and BaP exhibited high induction equivalency factors (IEFs). PCB126 was found to be the most active inducer of pbAHR. The in vitro transactivation potencies of ligands of pbAHR showed a significant relationship with in silico ligand docking energies in a pbAHR homology model. The protein ligand interaction fingerprint (PLIF) analysis showed different interaction patterns depending on the ligands. Several amino acids which are highly conserved among mammals, may be involved in species-specific responses via backbone interactions with neighboring amino acid residues which are specific to pbAHR. We document increased susceptibility of polar bears to DRCs, through a mechanistic approach, for the first time.

Aryl hydrocarbon receptor-mediated activity of gas-phase ambient air derived from passive sampling and an in vitro bioassay.

The gaseous fraction of hydrophobic organic contaminants (HOCs) in ambient air appears to be responsible for a significant portion of aryl hydrocarbon receptor (AhR)-mediated activity, but the majority of compounds contributing to this activity remain unidentified. The present study investigated the use of polyethylene passive samplers to isolate gaseous HOCs from ambient air for use in in vitro bioassays and to improve our understanding of the toxicological relevance of the gaseous fraction of ambient air in urban and residential environments. Concentrations of polycyclic aromatic hydrocarbons (PAHs) and organic flame retardants were measured in polyethylene passive sampler extracts. Extracts were also analyzed using an in vitro bioassay to measure AhR-mediated activity. Bioassay-derived benzo[a]pyrene (BaP) equivalents (BaP-Eqbio ), a measure of potency of HOC mixtures, were greatest in the downtown Cleveland area and lowest at rural/residential sites further from the city center. The BaP-Eqbio was weakly correlated with concentrations of 2-ring alkyl/substituted PAHs and one organophosphate flame retardant, ethylhexyl diphenyl phosphate. Potency predicted based on literature-derived induction equivalency factors (IEFs) explained only 2 to 23% of the AhR-mediated potency observed in bioassay experiments. Our results suggests that health risks of gaseous ambient air pollution predicted using data from targeted chemical analysis may underestimate risks of exposure, most likely due to augmentation of potency by unmonitored chemicals in the mixture, and the lack of relevant IEFs for many targeted analytes. Environ Toxicol Chem 2019;38:748-759. © 2019 SETAC.

Alternative In Vitro Approach for Assessing AHR-Mediated CYP1A Induction by Dioxins in Wild Cormorant (Phalacrocorax carbo) Population

Our line of papers revealed that the common (great) cormorant (Phalacrocorax carbo) possesses two isoforms of the aryl hydrocarbon receptor (ccAHR1 and ccAHR2). This paper addresses in vitro tests of the ccAHR signaling pathways to solve two questions: (1) whether there are functional differences in the two ccAHR isoforms, and (2) whether a molecular perturbation, cytochrome P450 1A (ccCYP1A) induction, in the population-level can be predicted from the in vitro tests. The transactivation potencies mediated by ccAHR1 and ccAHR2 were measured in COS-7 cells treated with 15 selected dioxins and related compounds (DRCs), where ccAHR1 or ccAHR2 expression plasmid and ccCYP1A5 promoter/enhancer-linked luciferase reporter plasmid were transfected. For congeners that exhibited dose-dependent luciferase activities, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) and induction equivalency factors (IEFs) were estimated. ccAHR1-IEF profile was similar to WHO avian TCDD toxic equivalency factor (TEF) profile except for dioxin-like polychlorinated biphenyls that showed lower IEFs in ccAHR1-driven reporter assay. ccAHR2-IEF profile was different from WHO TEFs and ccAHR1-IEFs. Notably, 2,3,4,7,8-PeCDF was more potent than TCDD for ccAHR2-mediated response. Using ccAHR1- and ccAHR2-IEFs and hepatic DRC concentrations in the Lake Biwa cormorant population, total TCDD induction equivalents (IEQs) were calculated for each ccAHR-mediated response. Nonlinear regression analyses provided significant sigmoidal relationships of ccAHR1- and ccAHR2-derived IEQs with hepatic ccCYP1A5 mRNA levels, supporting the results of in vitro ccAHR-mediated TCDD dose-response curves. Collectively, our in vitro AHR reporter assay potentially could be an alternative to molecular epidemiology of the species of concern regarding CYP1A induction by AHR ligands.

Potencies of Red Seabream AHR1- and AHR2-Mediated Transactivation by Dioxins: Implication of Both AHRs in Dioxin Toxicity

To evaluate species- and isoform-specific responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in the red seabream ( Pagrus major ), we constructed a reporter gene assay system. Each expression plasmid of red seabream AHR1 (rsAHR1) and AHR2 (rsAHR2) together with a reporter plasmid containing red seabream CYP1A 5'-flanking region were transfected into COS-7 cells. The cells were treated with graded concentrations of seven DRC congeners including 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, and PCB126. Both rsAHR1 and rsAHR2 exhibited dose-dependent responses for all the tested congeners. The rsAHR isoform-specific TCDD induction equivalency factors (rsAHR1- and rsAHR2-IEFs) were calculated on the basis of 2,3,7,8-TCDD relative potency derived from the dose-response of each congener. The rsAHR1-IEFs of PeCDD, HxCDD, TCDF, PeCDF, and HxCDF were estimated as 0.17, 0.29, 2.5, 1.5, and 0.27, respectively. For PCB126, no rsAHR1-IEF was given because of less than 10% 2,3,7,8-TCDD maximum response. The rsAHR2-IEFs of PeCDD, HxCDD, TCDF, PeCDF, HxCDF, and PCB126 were estimated as 0.38, 0.13, 1.5, 0.93, 0.20, and 0.0085, respectively. The rsAHR1/2-IEF profiles were different from WHO toxic equivalency factors for fish. In silico docking simulations supported that both rsAHRs have potentials to bind to these congeners. These results suggest that dioxin toxicities may be mediated by both rsAHRs in red seabreams.

In VitroTransactivation Potencies of Black-Footed Albatross (Phoebastria nigripes) AHR1 and AHR2 by Dioxins To Predict CYP1A Expression in the Wild Population

Our previous studies have detected high levels of dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs), and coplanar PCBs (Co-PCBs) in the black-footed albatross (BFA), Phoebastria nigripes, from the North Pacific region. We have also cloned two aryl hydrocarbon receptors, AHR1 and AHR2, of the BFA. To evaluate the sensitivity to DRCs in the BFA and to assess the status of cytochrome P450 1A (CYP1A) induction in the wild population, this study investigated the mRNA expression levels of BFA AHR1 and AHR2 and also the transactivation potencies of each AHR by 15 selected DRC congeners. Quantitative real-time PCR of BFA AHR mRNAs showed that hepatic AHR1 is more highly expressed than AHR2. Transactivation by graded concentrations of individual DRCs was measured in COS-7 cells, where BFA AHR1 or AHR2 was transiently transfected. For congeners that exhibited AHR-mediated dose-dependent activities, 50% effective concentration (EC(50)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) were estimated. Based on the estimates of the REPs, TCDD induction equivalency factors (IEFs) were determined. For BFA AHR1, PeCDF was equipotent to TCDD, but other congeners exhibited lower IEFs. For BFA AHR2, PCDD/F congeners except OCDD/F showed IEFs ≥ 1.0. Using BFA AHR1- or AHR2-IEFs and hepatic concentrations of DRCs in North Pacific BFAs, TCDD induction equivalents (IEQs) were calculated. We further constructed nonlinear regression models on the relationships between BFA AHR1- or AHR2-IEF derived total IEQ or WHO-TEF derived total TEQ and ethoxyresorufin-O-deethylase activity (EROD) in the liver of wild BFAs. The results indicated that the relationships of BFA AHR1- and AHR2-based IEQs and EROD were predictable from BFA AHR1- and AHR2-mediated transactivation by TCDD, respectively. Collectively, these results suggest that the in vitro assay incorporating the AHR of species of concern would be a useful tool to predict the sensitivity to DRCs in the species and CYP1A induction in the wild population.

Transactivation Potencies of the Baikal Seal (Pusa sibirica) Peroxisome Proliferator-Activated Receptor α by Perfluoroalkyl Carboxylates and Sulfonates: Estimation of PFOA Induction Equivalency Factors

The present study assessed the transactivation potencies of the Baikal seal (Pusa sibirica) peroxisome proliferator-activated receptor α (BS PPARα) by perfluorochemicals (PFCs) having various carbon chain lengths (C4-C12) using an in vitro reporter gene assay. Among the twelve PFCs treated with a range of 7.8-250 μM concentration, eight perfluoroalkyl carboxylates (PFCAs) and two perfluoroalkyl sulfonates (PFSAs) induced BS PPARα-mediated transcriptional activities in a dose-dependent manner. To compare the BS PPARα transactivation potencies of PFCs, the present study estimated the PFOA induction equivalency factors (IEFs), a ratio of the 50% effective concentration of PFOA to the concentration of each compound that can induce the response corresponding to 50% of the maximal response of PFOA. The order of IEFs for the PFCs was as follows: PFOA (IEF: 1)>PFHpA (0.89)>PFNA (0.61)>PFPeA (0.50)>PFHxS (0.41)>PFHxA (0.38)≈PFDA (0.37)>PFBA (0.26)=PFOS (0.26)>PFUnDA (0.15)≫PFDoDA and PFBuS (not activated). The structure-activity relationship analysis showed that PFCAs having more than seven perfluorinated carbons had a negative correlation (r=-1.0, p=0.017) between the number of perfluorinated carbons and the IEF of PFCAs, indicating that the number of perfluorinated carbon of PFCAs is one of the factors determining the transactivation potencies of the BS PPARα. The analysis also indicated that PFCAs were more potent than PFSAs with the same number of perfluorinated carbons. Treatment with a mixture of ten PFCs showed an additive action on the BS PPARα activation. Using IEFs of individual PFCs and hepatic concentrations of PFCs in the liver of wild Baikal seals, the PFOA induction equivalents (IEQs, 5.3-58 ng IEQ/g wet weight) were calculated. The correlation analysis revealed that the hepatic total IEQs showed a significant positive correlation with the hepatic expression levels of cytochrome P450 4A-like protein (r=0.53, p=0.036). This suggests that our approach may be useful for assessing the potential PPARα-mediated biological effects of complex mixtures of PFCs in wild Baikal seal population.

Toxic Effects of Methylated Benz[a]anthracenes in Liver Cells

Monomethylated benz[ a]anthracenes (MeBaAs) are an important group of methylated derivatives of polycyclic aromatic hydrocarbons (PAHs). Although the methyl substitution reportedly affects their mutagenicity and tumor-initiating activity, little is known about the impact of methylation on the effects associated with activation of the aryl hydrocarbon receptor (AhR)-dependent gene expression and/or toxic events associated with tumor promotion. In the present study, we studied the effects of a series of MeBaAs on the above-mentioned end points in rat liver cell lines and compared them with the effects of benz[ a]anthracene (BaA) and the potent carcinogen 7,12-dimethylbenz[ a]anthracene (DMBA). Methyl substitution enhanced the AhR-mediated activity of BaA derivatives determined in a reporter gene assay, as the induction equivalency factors (IEFs) of all MeBaAs were higher than that of BaA. IEFs of 6-MeBaA and 9-MeBaA, two of the most potent MeBaAs, were more than two orders of magnitude higher than the IEF of BaA. Correspondingly, all MeBaAs induced higher levels of cytochrome P450 1A1 mRNA. Both BaA and MeBaAs had similar effects on the expression of cytochrome P450 1B1 or aldo-keto reductase 1C9 in rat liver epithelial WB-F344 cells. In contrast to genotoxic DMBA, MeBaAs induced low DNA adduct formation. Only 10-MeBaA induced apoptosis and accumulation of phosphorylated p53, which could be associated with the induction of oxidative stress, similar to DMBA. With the exception of 10-MeBaA, all MeBaAs induced cell proliferation in contact-inhibited WB-F344 cells, which corresponded with their ability to activate AhR. 1-, 2-, 8-, 10-, 11-, and 12-MeBaA inhibited gap junctional intercellular communication (GJIC) in WB-F344 cells. This mode of action, like disruption of cell proliferation control, might contribute to tumor promotion. Taken together, these data showed that the methyl substitution significantly influences those effects of MeBaAs associated with AhR activation or GJIC inhibition.

Sorptive Behavior of Nitro-PAHs in Street Runoff and Their Potential as Indicators of Diesel Vehicle Exhaust Particles

This is the first report to reveal the particle-water distribution of nitropolycyclic aromatic hydrocarbons (NPAHs) and to discuss their potential risks and utility as indicators of diesel vehicle exhaust particles (DEP). Time-series samples of runoff were collected from a highway, and NPAHs and polycyclic aromatic hydrocarbons (PAHs) were determined by gas chromatography-mass spectrometry (GC-MS) to study their dynamic behavior. The concentrations of total NPAHs ranged from 11 to 73 ng/L in particulate phase (>0.7 mcirom) and from 2.3 to 4.9 ng/L in dissolved phase (<0.7 microm). Like their PAH analogs, most (81-97%) NPAHs were associated with particulate matter. The organic carbon-normalized in situ partition coefficients (Koc') of NPAHs observed in runoff events (10(5.8-6.3) for 2-nitrofluoranthene and 10(5.8-6.2) for 1-nitropyrene [1-NP]) were more than 1 order of magnitude higher than those expected from their Kow, indicating great affinity for particulate matter such as soot. Concentrations of PAHs and NPAHs adjusted by potency equivalency factors and induction equivalency factors showed that the potential risks of NPAHs were smaller than those of PAHs by a factor of more than a hundred for the particulate phase and morethan fourforthe dissolved phase. Comparison of concentrations and compositions of NPAHs and PAHs among runoff, DEP, gasoline vehicle exhaust particles, boiler exhaust particles, and aerosols suggested that the ratio of 1-NP to total PAHs (1-NP/PAH) is a useful indicator of DEP for source apportionment of PAHs among traffic-related sources. Source-apportionment of PAHs in the runoff by 1-NP/PAH and methylphenanthrene/phenanthrene ratios suggested that most PAHs in the runoff except the second flush peak were derived from DEP but that other pyrogenic sources contributed to the particles at the second flush and thus to the overall runoff particles.

A comparison of the concentration–effect relationships of PAHs on CYP1A induction in HepG2 and Mcf7 cells

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants. Some compounds belonging to this group are considered carcinogenic to people. In order to yield carcinogenic properties, these compounds must be metabolically transformed by enzymes of cytochrome P450 family to oxy-derivatives. In this study, the ability of the following six PAHs: anthracene (Ant), benz(a)anthracene (BA), naphthacene (Nap), benzo(a)pyrene (BaP), dibenz(a,c)anthracene (DB(a,c)A) and dibenz(a,h)anthracene (DB(a,h)A) to induce enzymes of cytochrome P450 (CYP450), in particular CYP1A1 and CYP1A2 in Mcf7 and HepG2 cells was studied. The induction of CYP1A enzymes was assessed at the level of enzymatic protein and enzymatic activity. The change in CYP1A1 and CYP1A2 protein level was assessed by means of confocal microscopy. The ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-deethylase (MROD) assays were applied to determine the CYP1A1 and CYP1A2 activity. The Induction Equivalency Factors (IEFs) were also determined. According to EROD and MROD assay and calculated IEFs the following order of the inducing potency was determined in HepG2 cells: DB(a,h)A > BaP > DB(a,c)A approximately BA > Nap > Ant, and in Mcf7 cells: DB(a,h)A > DB(a,c)A > BaP > Nap > BA > Ant. The assessment of the protein levels revealed that DB(a,h)A was also the strongest inducer of protein level, however the correlation between enzymatic activity and protein level induction by other PAHs was not always evident. The EROD and MROD activities were higher in Mcf7 than in HepG2 cells, however the CYP1A2 protein level was shown to be higher in HepG2 cells. The results obtained indicate possible catalytic enzymatic activity alterations induced by PAHs.

Cytotoxicity and aryl hydrocarbon receptor‐mediated activity of N‐heterocyclic polycyclic aromatic hydrocarbons: Structure‐activity relationships

Toxic effects of many persistent organic pollutants (e.g., polychlorinated biphenyls or polychlorinated dibenzo-p-dioxins and furans) are mediated via the aryl hydrocarbon receptor (AhR). Although polycyclic aromatic hydrocarbons (PAHs) and their derivatives also activate AhR, their toxic effects remain to be fully elucidated. In the present study, we used the in vitro H4IIE-luc transactivation cell assay to investigate cytotoxicity and potencies to activate AhR by 29 individual PAHs and their N-heterocyclic derivatives (aza-PAHs). The aza-PAHs were found to be significantly more cytotoxic and more potent inducers of AhR than their unsubstituted analogues. Several aza-PAHs, such as dibenz[a,h]acridine or dibenz[a,i]acridine, activated AhR within picomolar concentrations, comparable to the effects of reference 2,3,7,8-tetrachlorodibenzo-p-dioxin. Ellipsoidal volume, molar refractivity, and molecular size were the most important descriptors derived from the modeling of quantitative structure-activity relationships for potencies to activate AhR. Comparable relative toxic potencies (induction equivalency factors) for individual aza-PAHs are derived, and their use for evaluation of complex contaminated samples is discussed.

Rapid Bioassay for the Determination of Dioxins and Dioxin-like PCDFs and PCBs in Meat and Animal Feeds

Over the past several years, the numerous contamination incidents have raised concerns over the presence of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related chemicals in foods and feeds. Here we applied a sensitive recombinant mouse hepatoma cell (H1L1.1c2) bioassay for the determination of dioxins and dioxin-like polychlorinated dibenzofurans (PCDFs) and biphenyls (PCBs) in meat and animal feeds. These cells responded to TCDD-like chemicals with dose-dependent induction of firefly luciferase activity, and the minimal detection limit of TCDD in the cell was 16 fg. Induction equivalency factors determined for pure TCDD-like polychlorinated dibenzo-p-dioxins (PCDDs), PCDFs, and PCBs in the bioassay were well-correlated with the World Health Organization's toxic equivalency factors. To determine the applicability of the bioassay system to detect those compounds presence in meat and feed samples, cell bioassays for 17 TCDD-like PCDDs and PCDFs congeners-spiked lipid extracted from beef or animal feed were performed. Mean recoveries of TCDD-like chlorinated PCDDs and PCDFs congeners from spiked beef or feed fat ranged from 61.2 to 122.3%. Within-laboratory coefficients of variation for analysis as index of precision were lower than 5.2%, and the calculated limits of detection and quantitation were 0.33 and 1 pg toxicity equivalency quantity (TEQ)/0.5 g fat, respectively. Correlation between bioassay- and high-resolution gas chromatography-mass spectrometry (HR-GC-MS)-determined TEQs for 10 meat samples was 0.85, with 1.2 times higher in bioassay than HR-GC-MS. The correlation between bioassay- and HR-GC-MS-determined TEQs in 10 animal feed products was 0.81, with 2.1 times higher in bioassay than HR-GC-MS. Overall, these results demonstrated that the recombinant cell bioassay can be used for the rapid detection and quantitation of PCDDs and dioxin-like PCDFs and PCBs in meats and animal feeds.

03/02600 New legal regulations for air pollution control and their influence on operation of coke plants: Hein, M. Stahl und Eisen, 2003, 123, (1), 53–58. (In German)

A number of highly toxic environmental pollutants including certain polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and ‘dioxin-like’ polychlorinated biphenyls (PCB) are among the most potent agonists of the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A1 (CYP1A1) in mammalian cell culture is widely used as a functional parameter for AHR activation providing an estimate for ‘dioxin-like’ inducing equivalents in extracts from environmental samples. Since a number of polycyclic aromatic hydrocarbons (PAHs) also act as AHR-agonists, the CYP1A1-inducing potencies, measured as induction of 7-ethoxyresorufin O-deethylase (EROD) activity in rat hepatocyte cultures were analyzed for 16 PAHs frequently present in environmental samples. Among these, seven PAHs including benzo[a]pyrene were relatively potent inducers allowing the determination of Induction Equivalency Factors (IEF). For three PAHs including benzo[k]fluoranthene which acted as weak inducers, IEFs were estimated, while six PAHs including acenaphthylene were classified as inactive. Based on different efficacies the concentration–response characteristics of CYP1A1 induction were analyzed in more detail for benzo[a]pyrene, benzo[k]fluoranthene, and acenaphthylene. Benzo[k]fluoranthene was markedly less effective than benzo[a]pyrene as inducer of EROD activity but even more effective than benzo[a]pyrene as inducer of CYP1A1 protein and mRNA. Acenaphthylene was highly more effective on the level of mRNA than on the levels of protein or EROD activity. Further analysis revealed that the low efficacy of acenaphthylene as inducer of CYP1A1 protein and EROD activity is due to its marked cytotoxicity while no clear-cut explanation was found for the differences in efficacy between benzo[k]fluoranthene and benzo[a]pyrene. The EROD-inducing potency of a mixture of 16 PAH was about 2-fold higher than that calculated on the basis of IEFs of the individual constituents of the mixture.

Naturally produced halogenated dimethyl bipyrroles bind to the aryl hydrocarbon receptor and induce cytochrome P4501A and porphyrin accumulation in chicken embryo hepatocytes

Halogenated dimethyl bipyrroles (HDBPs) recently have been identified as a group of environmentally persistent naturally produced bioaccumulative organohalogens. The ability of these compounds to activate the aryl hydrocarbon receptor (AhR) signaling pathway in vitro was examined. Induction of cytochrome P4501A (CYP1A), measured as ethoxyresorufin-O-deethylase (EROD) activity, and porphyrin accumulation were monitored after exposure of chick embryo hepatocytes to three individual HDBP congeners and two HDBP mixtures. All HDBP congeners and mixtures tested caused induction of EROD activity. Induction equivalency factors, calculated as the ratio of the effective concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce 10 and 50% of the maximal EROD induction to that of the HDBP congener or mixture ranged from 0.0004 to 0.05. Halogenated dimethyl bipyrroles also caused porphyrin accumulation, which increased with exposure time of the hepatocytes and molar bromine content of the HDBP congener or mixture. Heptacarboxylporphyrin III and corproporphyrinogen III were the most abundant porphyrins observed after an exposure period of 48 h. The individual HDBP congeners and mixtures also inhibited binding of [3H]TCDD to chicken and mouse AhR. Binding of [3H]TCDD to chicken AhR was inhibited approximately 20% by tetra- to hexabrominated congeners at concentrations of 20 to 40 microM; binding to mouse AhR was inhibited 40 to 50% by tri- and tetrabrominated dimethyl bipyrroles (DBPs) at 10 microM. The relative affinity of the hexabrominated DBP congener (vs TCDD) for binding to chicken AhR was estimated to be 0.000017. The results of this work illustrate that HDBPs act as agonist ligands for the AhR in the same manner as many anthropogenic organohalogens.

Application of the luciferase recombinant cell culture bioassay system for the analysis of polycyclic aromatic hydrocarbons

An aryl hydrocarbon (Ah) receptor-based luciferase cell culture bioassay developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatics was modified and optimized to detect and quantitate polycyclic aromatics (PAHs). Twenty-four PAHs were analyzed, and subsequent EC50 and EC20 concentrations (based on the median and 20% TCDD maximal response, respectively) and appropriate induction equivalency factors (calculated by comparison to the response obtained with TCDD) were determined from dose-response experiments. Six compounds were shown to be active in the system, with benzo[k]fluoranthene > benz[a,h]anthracene indeno[1,2,3-cd]pyrene > benzo[a]pyrene > benzo[b]fluoranthene > chrysene. A complex mixture of 16 PAHs was also analyzed using this system, and overall induction equivalency (or 1-EQ) of the mixture was shown to be very similar to that predicted from the sum of the activity estimated for each individual PAH. Overall, our results strongly support the use of this system for the detection and relative quantitation of Ah receptor-active PAHs.

In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in The Netherlands.

The ethoxy resorufin dealkylase (EROD) inducing potency of 10 polycyclic aromatic hydrocarbons (PAHs) is measured in the H4IIE in vitro bioassay and the results are compared to those reported in literature. The selected PAHs varied considerably in their potency to induce EROD activity. Anthracene (Ant) and phenanthrene (Phe) showed consistently no response. Naphthalene (Nap) showed no or a very weak response on EROD activity. Fluoranthene (Fla) and benzo[g,h,i]perylene (BghiP) showed weak responses at the highest doses. The other PAHs, including indeno[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), chrysene (Chr) and benzo[k]fluoranthene (BkF), showed full bell shaped dose-response curves. BaP EROD induction equivalency factors (BaP-1EF) were calculated and increased in the order Ant approximately Phe < Fla < Nap < BghiP < IP < BaA < BaP < Chr < BkF. Comparison of BaP-IEFs based on 50% effect concentration (EC50) or lowest effect concentration (LEC), yielded a significant relationship between both methods described by the equation log(BaPIEF(EC50) = 0.55 x log(BaPIEF(LEC)) + 0.07 (r2 = 0.913). BaP-IEFs as derived from our measurements and as reported in literature and measured in other in vitro assays deviated up to a factor of 17 among the different studies, but the potency rankings were comparable. For the PAH mixture as on average present in the human diet an overall tetrachlorodibenzo-p-dioxin (TCDD)-IEF of 1 x 10(-4) was estimated. The total PAH based TCDD induction equivalents (IEQ) intake then was calculated 300 pg/day, which is approximately 2 times higher then the PHAH based TCDD-EQ intake reported for humans.

Aryl hydrocarbon receptor‐mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments

Reproductive dysfunction in wildlife populations can be a result of environmental contaminants binding to aryl hydrocarbon receptor (AhR) or estrogenic receptors. Signaling by both types of receptors can be affected by polycyclic aromatic hydrocarbons (PAHs), which are potential endocrine disruptors. However, our knowledge regarding the effects of oxygenated (oxy)-PAHs and azaarenes on AhR-mediated and estrogenic activities is incomplete. In the present study, we have identified 9-fluorenone, anthrone, anthraquinone, benzanthrone, benz[a]anthracene-7,12-dione, benz[c]acridine, and dibenz[a,h]acridine as prevalent oxy-PAHs and azaarenes found in river sediments. Their concentrations in sediment samples ranged from 2.1 to 165.2 ng g(-1) for oxy-PAHs and up to 27.3 ng g(-1) for azaarenes. Their relative AhR-inducing and estrogenic potencies were quantified in vitro using two cell lines that were stably transfected with a luciferase reporter gene system and expressed as induction equivalency factors (IEFs). The only oxy-PAHs with detectable levels of in vitro AhR-mediated activity were benzanthrone and benz[a]anthracene-7,12-dione. However, their IEFs were approximately three to four orders of magnitude lower than those of benzo[a]pyrene. On the other hand, azaarenes showed a strong AhR-mediated activity, with dibenzo[a,h]acridine being a far more potent inducer of activity than benzo[a]pyrene. Benzanthrone, benz[a]anthracene-7,12-dione, anthraquinone, and benz[a]acridine were weak inducers of in vitro estrogenic activity, with IEFs similar to that of benzo[a]pyrene. Based on concentrations and relative potencies, our results suggest that dibenzo[a,h]acridine can significantly contribute to the overall AhR-mediated activity in river sediments, whereas the remaining compounds do not. No studied compound was found to contribute significantly to estrogen receptor-mediated activity in vitro.

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24 h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[ a]pyrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). In 24 h exposure assay, the highest IEFs were found for benzo[ k]fluoranthene, dibenzo[ a, h]anthracene and dibenzo[ a, k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[ a, j]anthracene, benzo[ j]fluoranthene, indeno[1,2,3- cd]pyrene, and naphtho[2,3- a]pyrene. The 6 h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24 h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[ a, l]pyrene, cyclopenta[ cd]pyrene, and benzo[ a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6 h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[ k]fluoranthene and benzo[ j]fluoranthene, followed by indeno[1,2,3- cd]pyrene, dibenzo[ a, h]anthracene, benzo[ a]pyrene, dibenzo[ a, j]anthracene, chrysene, and benzo[ b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[ a]pyrene, dibenzo[ a, e]pyrene, and naphtho[2,3- a]pyrene and to a lesser extent also for benzo[ a]anthracene, benzo[ b]fluoranthene, indeno[1,2,3- cd]pyrene, benzo[ j]fluoranthene, dibenzo[ a, e]fluoranthene and dibenzo[ a, i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.

Ethoxyresorufin‐O‐deethylase induction in trout exposed to mixtures of polycyclic aromatic hydrocarbons

This study investigated whether ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout exposed to mixtures of polycyclic aromatic hydrocarbons (PAHs) could be predicted from induction equivalency factors (IEF). The test PAHs were classified into strong and weak inducers on the basis of similar exposure-response curves. Induction equivalency factors of strong inducers, based on benzo[k]fluoranthene (BkF) as the reference compound, ranged from 0.03 to 0.16. Trout exposed to mixtures of strong inducers (2, 4, and 6 equipotent parts) at 0.32-, 1.0-, or 3.2-nM BkF-equivalents showed exposure-dependent increases in EROD activity, consistent with an additive interaction. The extent of activity did not vary greatly among mixtures and single PAHs at a given induction equivalent quantity (IEQ). Induction equivalency factors could not be calculated for weak inducers because the range of induction was too low. Hence, each weak inducer was added to mixtures at concentrations that induced EROD activity fivefold. These mixtures appeared additive because binary and quaternary mixtures caused about 10- and 20-fold induction, respectively. Strong inducers mixed the same way also showed additivity. In contrast, EROD induction by mixtures containing both strong and weak PAHs was 800 to 900% greater than expected, suggesting synergistic interactions. Therefore, if mixtures are composed of PAHs that behave similarly, IEFs may be a suitable approach for assessing risk. However, for mixtures that contain PAHs of differing potency and efficacy, bioassays will be a more reliable measure of risk than IEFs.

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL‐W1, and in primary cultures of rainbow trout hepatocytes

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.