Halogenated Aromatic Hydrocarbons Research Articles

A comparison of the dose and time response of CYP1A1 mRNA induction in chemically treated Atlantic tomcod from two populations

Quantification of cytochrome P4501A1 (CYP1A1) mRNA levels in environmentally exposed Atlantic tomcod ( Microgadus tomcod) has revealed significantly induced gene expression in fish from contaminated locales including the Hudson River, New York, and the Miramichi River, New Brunswick. In order to calibrate this response, determine its sensitivity and dose-responsiveness, levels of hepatic CYP1A1 mRNA were quantified in depurated Atlantic tomcod intraperitoneally (i.p.) injected with various concentrations of: β-naphthoflavone (β-NF), the PAH benzo[ a]pyrene (B[ a]P), the non- ortho coplanar PCB congener-3,3′,4,4′- tetrachlorobiphenyl (IUPAC: PCB-77), and the dioxin 2,3,7,8-tetrachlorodibenzo -p-dioxin (2,3,7,8-TCDD). Additionally, the rates of CYP1A1 mRNA induction and disappearance were quantified in depurated Atlantic tomcod i.p. injected with single doses of these chemicals and sacrificed at times ranging up to 72 days. Levels of CYP1A1 mRNA were dose-responsive for all four chemicals with maximum induction ranging from 50- to 460-fold and first significant induction being observed in the low mg per kg fish (wet weight) range for β-NF and B[ a]P, μg/kg range for PCB-77 and ng/kg range for 2,3,7,8-TCDD. However, while tomcod from the Miramichi River responded to both PAHs and halogenated aromatic hydrocarbons (HAHs), Hudson River tomcod responded only to PAHs indicating population level differences in CYP1A1 mRNA inducibility in tomcod. Furthermore, differences in the responsiveness to PAHs and HAHs suggest that more than one molecular mechanism mediates CYP1A1 transcription in Atlantic tomcod. Kinetic profiles of CYP1A1 mRNA induction differed greatly between tomcod treated with HAHs and PAHs. Initial induction occurred within hours of treatment with PAHs and peaked after 1–3 days, compared to initial induction 4–7 days after treatment with HAHs, and maximum induction not occurring for up to 72 days after exposure. Quantification of halogenated aromatic hydrocarbons (HAH) in the livers of tomcod caught in the Hudson and Miramichi Rivers confirmed exposure and accumulation of known CYP1A1 inducing chemicals including 2,3,7,8-TCDD at concentrations as high as 1.5 μg/kg lipid (554 ng/kg w.w.) and PCB-77 at concentrations as high as 108 μg/kg lipid (15 μg/kg w.w.). These results suggest that hepatic CYP1A1 mRNA concentration can be a useful bioindicator of exposure to some aromatic hydrocarbon compounds in the aquatic environment and that profiles of gene induction and disappearance may help identify environmental inducers provided that gene responsiveness is also evaluated under controlled laboratory conditions.

Organoclay adsorption of wood‐preserving waste from groundwater: Analytical and toxicological evaluations

Wood‐preserving waste is a complex mixture composed primarily of polycyclic aromatic hydrocarbons (PAHs) and pentachlorophenol (PCP), with trace concentrations of other halogenated aromatic hydrocarbons (HAHs). Although many of these compounds are slightly soluble in water, these substances have been detected in groundwater at high levels. Samples from the aquifers beneath a Superfund site in the northwestern United States were used to determine the efficacy of an organically modified clay adsorbent (CP/LPHM) for PAHs and PCP in the presence of background organics. Groundwater samples were treated with 0.5% (w/w) of CP/LPHM, and compared to samples which were not treated. Determination of the concentrations of the various PAHs was done by GC/MS. Naphthalene concentration, most abundant of all PAHs detected, was reduced by approximately 88% from 261 to 31 ug/ml of the extract. Other PAHs, many of which are known to be genotoxic such as benzo [a]pyrene, were also substantially reduced following treatment with this organoclay. Pentachlorophenol concentration was reduced to non‐detectable levels. A bioassay that is sensitive to the toxic effects of PAHs, the Chick Embryo‐toxicity Screening Test (CHEST), was performed with sample extracts to evaluate the relative toxicity of the samples before and after treatment with the organoclay. The groundwater, before treatment with the CP/LPHM, resulted in 88% mortality of the chick embryos. Following treatment, the mortality was decreased to 21 %, which was not significantly different from solvent controls. These findings suggest that organoclays show potential for application in the treatment of contaminated groundwater as determined by both analytical and toxicological evaluations.

Rainbow trout cell bioassay-derived relative potencies for halogenated aromatic hydrocarbons: Comparison and sensitivity analysis

Rainbow trout hepatoma cells, stably transfected with a luciferase reporter gene under control of dioxin-responsive elements (RLT 2.0 cells) were used to derive relative potencies (RPs) for a variety of halogenated aromatic hydrocarbons (HAHs) that are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This in vitro bioassay utilizes 96-well microplates, which provide high sample throughput and assay efficiency without affecting sensitivity. The RLT 2.0-derived potencies for dioxin and furan congeners, relative to 2,3,7,8-TCDD, ranged from 0.917 for 1,2,3,4,7,8-hexachlorodibenzofuran to 0.208 for 1,2,3,7,8-pentachlorodibenzofuran. All mono- and di-ortho polychlorinated biphenyls (PCBs) tested had RPs that were orders of magnitude less than TCDD, but point estimates could not be determined. The RLT 2.0-derived RPs were found to be comparable to both other rainbow trout-specific RPs and RPs based on mammalian bioassays. Sensitivity analysis suggested that the range of uncertainty associated with TCDD equivalent (TEQ) estimates based on RLT 2.0-derived RPs is approximately 10-fold. Within this degree of uncertainty and the context of this study, the RLT 2.0 bioassay showed no definitive biases or inaccuracies relative to similar mammalian- or fish-specific in vitro bioassays. Thus, the RLT 2.0 bioassay appears to be a useful tool for evaluating dioxinlike potency of HAHs to fish.

Polychlorinated dibenzo-p-dioxins, dibenzofurans and polychlorinated biphenyls in New Zealand cetaceans

Limited information is available on the concentrations of halogenated aromatic hydrocarbons (HAHs) in cetaceans from the Southern Hemisphere. This paper presents data on blubber concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) in Hector's dolphins, dusky dolphins, southern right whale dolphins, blue whales, minke whales, Gray's beaked whales, Cuvier's beaked whales and pygmy right whales stranded in New Zealand. Both HAH concentrations and toxic equivalents (TEQs) are found to be higher in Hector's dolphins, a species with an inshore distribution, than in other odontocetes, which are more oceanic. Baleen whales, which are oceanic and feed at lower trophic levels, present the lowest levels of pollutants, with PCDD and PCDF concentrations usually below detection limits. The PCB profiles of the various species suggest that they are exposed to different PCB sources. Overall, HAH levels detected are lower than those reported for comparable species in the Northern Hemisphere. The relative abundance of low chlorinated PCB congeners in New Zealand cetaceans, as compared to those from northern waters, suggests that the origin of these compounds is mostly atmospheric deposition.

In vitro induction of ethoxyresorufin‐O‐deethylase and porphyrins by halogenated aromatic hydrocarbons in avian primary hepatocytes

AbstractEthoxyresorufin‐O‐deethylase (EROD) and porphyrin induction responses of primary hepatocytes to halogenated aromatic hydrocarbons (HAHs) were examined in newly hatched domestic chickens, herring gulls, ring‐billed gulls, double‐crested cormorants, and Forster's terns. Concentration–response relationships were determined for both biochemical responses in hepatocyte preparations derived from individual avian livers (except for the tern). The choice of vehicle used to dose chicken hepatocytes greatly affected the potencies and efficacies of HAHs. Dimethyl sulfoxide resulted in median effective concentration (EC50) values for EROD induction that were between 10 and 15 times less than isooctane (isooctane was used throughout the study). Neither vehicle induced EROD activity by itself. Concentration‐dependent increases in EROD activity were observed with several HAHs, and their potencies (EC50 values) were compared to that of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) within each hepatocyte preparation to determine relative potency factors (RPFs). Differences in sensitivity to these responses were observed among individuals within each of the species and among species. Median EC50 values (nM) for EROD induction by TCDD were 0.72, 13, 20, 25, and 150 for the chicken, cormorant, ring‐billed gull, herring gull, and tern hatchling, respectively. Relative potency factors for several HAHs were different, in both ranking and potency, from those generally derived in mammalian hepatocytes. Porphyrin accumulation was observed occasionally with the most potent aryl hydrocarbon receptor agonists, but most HAHs were not tested at concentrations sufficiently high to observe a consistent response. This study provides information on interindividual and interspecies differences in responsiveness to TCDD‐like compounds and provides species‐specific RPFs that may prove useful for the purpose of hazard and risk assessment for fish‐eating birds.

TRIBUTYLTIN MODULATES 3,3',4,4',5-PENTACHLOROBIPHENYL (PCB-126)-INDUCED HEPATIC CYP1A ACTIVITY IN CHANNEL CATFISH, ICTALURUS PUNCTATUS

Many harbor estuaries and their tributaries are contaminated with halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Planar congeners of these two classes initiate their toxic effects, including reproductive, developmental, and immunological dysfunction, primarily through the cytosolic arylhydrocabon receptor (Ahr). However, only rarely are aquatic environments contaminated with Ahr-binding contaminants alone. Instead, most are impacted by a variety of pollutants in mixture. Tributyltin (TBT), a common antifouling biocide, is also found in many harbor estuaries and their tributaries. Several reports indicate that TBT inhibits the cytochrome P-4501A system of fish, at least in vitro, and our recent studies with rodents indicate that TBT potentiates PCB-induced CYP1A. However, the effects of TBT on xenobiotic-induced CYP1A activity in aquatic organisms has been virtually unexplored. To this end, channel catfish, Ictalurus punctatus, were exposed to 3,3'4,4',5-pentachlorobiphenyl (PCB-126, PeCB), TBT, or both in combination, with corn oil (CO) serving as the carrier control. Immunoreactive CYP1A protein and ethoxyresorufin O -deethylase (EROD) activity were measured after (1) a single dose of 0.01, 0.1, or 1 mg/kg of each or both in combination, and (2) 6 injections of 0.017, 1.7, or 17 mug/kg of each (or in combination) given every 3 d over a 16-d period to yield a cumulative dose of 0.01, 0.1, or 1 mg/kg. As expected, PeCB alone, but not TBT, greatly induced these two CYP1A parameters. Low and middle doses of TBT (0.01 and 0.1 mg/ kg), but not the high dose, potentiated PeCBinduced activity at these same doses. This effect of TBT was even more pronounced in the repeated exposure study. Furthermore, EROD activity did not always reflect CYP1A protein induction; enzyme activity was inhibited by TBT at doses that potentiated protein induction (0.01 and 0.1 mg/ kg). In summary, TBT potentiates PeCB-induced CYP1A in channel catfish at doses that may be considered environmentally relevant.

Mechanisms of ligand-induced aryl hydrocarbon receptor-mediated biochemical and toxic responses.

The ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a member of a broad group of halogenated aromatic hydrocarbons (HAHs) that is known to induce a wide range of toxic and biochemical responses in laboratory animals and humans. The effects of HAH exposure are mediated by binding to the cytosolic aryl hydrocarbon receptor (AhR), which is expressed in a tissue- and cell type-specific manner. The AhR is a ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-AhR-Arnt-Sim (bHLH/PAS) superfamily of proteins. The mechanism of induction of gene transcription by TCDD involves ligand recognition and binding by the AhR, nuclear translocation, and dimerization with the AhR cofactor, AhR nuclear translocator (Arnt). The nuclear heterodimer interacts with cognate xenobiotic responsive elements (XREs) in promoter/enhancer regions of multiple Ah-responsive genes. Subsequent changes in chromatin structure and/or interaction of the AhR complex with the basal transcriptional machinery play a significant role in AhR-mediated gene expression. Although Arnt is a necessary component of a functional nuclear AhR complex, this protein also forms transcriptionally active heterodimers with other bHLH/PAS factors, including those involved in the transcriptional response to hypoxia. Arnt is ubiquitously expressed in mammalian systems, and results from transgenic mouse studies suggest that this protein plays a vital role in early mammalian embryonic development. Similar experiments suggest that the AhR may be involved in development of various organ systems. Thus, molecular mechanistic studies of TCDD action have contributed significantly to an improved understanding of the role of at least 2 bHLH/PAS proteins, as well as organ- and tissue-specific biochemical and toxic responses to this class of environmental toxins.

Accumulation of halogenated aromatic hydrocarbons and activities of cytochrome P450 and glutathione s‐transferase in CRABS (Eriocheir japonicus) from Japanese Rivers

AbstractThe hepatopancreases of freshwater crabs (Eriocheir japonicus) collected from three Japanese rivers (Barato, Shiribetsu, and Tone) were analyzed for planar halogenated aromatic hydrocarbons (HAHs), including polychlorinated dibenzo‐p‐dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs). The hepatic glutathione S‐transferase (GST)‐dependent enzyme activities in the crab hepatopancreas were also measured to examine their potential as biomarkers for the contaminants. Crabs from the Tone River, which runs through industrial, agricultural, and urban areas, have the highest concentrations of HAHs (4,100 pg/g fat weight), followed by those from the Barato River (2,430‐2,970 pg/g fat weight), whereas crabs from the Shiribetsu River were relatively less contaminated (1,350–1,800 pg/g fat weight). Identification of numerous PCDD and PCDF congeners in crabs from all three rivers provided evidence that one of the major sources of PCDDs and PCDFs was waste incineration. In addition, crabs from the Barato and Shiribetsu Rivers were notably contaminated with 1,3,6,8‐ and 1,3,7,9‐TeCDD congeners, which suggests that a possible source was chlornitrofen, which has been extensively used in paddy fields as a herbicide. Calculation of 2,3,7,8‐tetrachlorinated dibenzo‐p‐dioxin toxic equivalents (TEQs) showed that the causal contaminants of higher TEQs in crabs from the Tone River (94.7 TEQ picograms per gram fat weight) were PCDDs and PCDFs, although the most important contributor to the total TEQs was coplanar PCBs (49.95%). The crab hepatopancreas appeared to have abilities to transfer glutathione to 1‐chloro‐2,4‐nitrobenzene (CDNB) and 3,4‐dichloronitrobenzene (DCNB). The crabs with the highest TEQ levels showed the highest GST activities. The current results and our previous data lead us to conclude that cytochrome P450 and GST‐dependent enzyme activities (benzo[a]pyrene hydroxylation and CDNB and DCNB conjugation) in freshwater crab hepatopancreases are likely to be useful biomarkers for the contamination of planar aromatic hydrocarbons such as benzo[a]pyrene, PCDDs, PCDFs, and coplanar PCBs.

Highly carboxylated porphyrin concentration: a biochemical marker of PCB exposure in herring gulls

Our previous studies showed that the concentration of highly carboxylated porphyrins (HCPs) was higher in livers of herring gulls ( Larus argentatus) from the Great Lakes than in livers of herring gulls from a relatively uncontaminated area of the Atlantic coast of Canada. Since there are relatively few causes of elevated HCPs other than exposure to certain halogenated aromatic hydrocarbons (HAHs), we suggested that HCP concentration might offer promise as a biochemical marker of HAH exposure in herring gulls. We did not confirm if, or identify which, HAHs were the cause of elevated HCPs. Here we provide evidence that polychlorinated biphenyls (PCBs) are a likely cause of HCP accumulation in herring gulls. HCP concentration was measured in livers of adult gulls that were collected from several sites on the Great Lakes and from two reference sites. Non-polar extracts prepared from the livers were assayed for porphyrinogenic potency in a chicken embryo hepatocyte (CEH) bioassay, and analyzed for concentrations of various HAHs, including PCBs. There was a good linear correlation between (a) liver HCP concentration and porphyrinogenic potency of liver extracts, and (b) liver HCP and PCB concentration. Congenerspecific analysis suggested that the mono-ortho substituted PCB congeners 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and 2,3′,4,4′5-pentachlorobiphenyl (PCB 118) were the congeners which contributed most to elevated HCPs in gull livers. We conclude that HCP concentration is a good biochemical marker of PCB exposure in herring gulls.

Cytotoxic Response Profiles of Cultured Renal Epithelial and Mesenchymal Cells toSelected Aromatic Hydrocarbons

The cytotoxic responses of cultured rat renal cortico-tubular epithelial cells (TECs) and glomerular mesangial cells (GMCs) to selected polycyclic and polyhalogenated aromatic hydrocarbons (AHs) were evaluated to further define class specific differences in toxicity potential among these chemicals. Primary cultures of renal cells were exposed to benzo[ a]pyrene (BaP, 0.3–30 μ m); naphthalene (NAPH, 1–1000 μ m); 2-methylnaphthalene (2-MNAPH, 1–1000 μ m), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 0.01–1n m); 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.05–5n m; and pentachlorophenol (PCP, 0.1–10 μ m for 4 or 24 hr. Measurements of mitochondrial membrane fragility in TECs revealed that the polycyclic aromatic hydrocarbons (PAHs) (BaP, NAPH and 2-MNAPH) preferentially injured these cells relative to halogenated aromatic hydrocarbons (HAHs). Extended exposures to all AHs tested were associated with moderate mitochondrial injury in TECs. Challenge of TECs with AHs for 4 hr did not modulate GSH levels, although modest increases in this tripeptide occurred on extended exposures. Similar response profiles were observed in GMCs, where PAHs elicited mitochondrial damage by 4 hr, while extended challenge was associated with injury in response to all AHs. Only aryl hydrocarbon receptor (AhR) ligands (BaP, TCDD and TCDF) depleted intracellular glutathione (GSH) in GMCs, while extended exposures to BaP and TCDD, as well as NAPH and 2-MNAPH, were associated with rebound increases in cellular GSH content. These data indicate that AHs can compromise renal cell function by interference with mitochondrial function and GSH homeostasis and implicate both epithelial and mesenchymal populations in the nephrotoxic response to this heterogeneous class of chemicals.

Development validation and problems with the toxic equivalency factor approach for risk assessment of dioxins and related compounds.

Polychlorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF), and biphenyls (PCB) are industrial compounds or by-products that have been widely identified as environmental contaminants, and residues have been detected in fish, wildlife, and humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is the most toxic member of this class of halogenated aromatic hydrocarbons (HAH); mechanistic studies indicate that the toxic and biochemical effects associated with exposure to TCDD are mediated via initial binding to the cytosolic aryl hydrocarbon (Ah) receptor protein present in target tissues and organs. Several other 2,3,7, 8-substituted PCDD and PCDF and non-ortho substituted PCB also bind to the Ah receptor and induce toxic responses similar to those for TCDD. Moreover, for these HAH there is a rank order correlation between their structure-Ah receptor binding and structure-toxicity relationships, and this supports a role for the Ah receptor in mediating these responses. Thus, the toxic equivalency factor (TEF) approach for HAH is based on the common mechanism of action for TCDD and related compounds in which a TEF value for a "dioxin-like" congener is defined as the potency of the individual (i) congener relative to TCDD ([EC50 [TCDD]/EC50 [test compound]). The toxic or dioxin equivalent (TEQ) for a mixture of HAH is defined by the following equation: TEQ = sigma [PCDDi] x TEFi + sigma [PCDFi] x TEFi. Industrial emissions and environmental and food residues contain complex mixtures of HAH (exodioxins) and the TEF/TEQ approach is used to regulate emissions and estimate the potential exposure and possible adverse health effects of exodioxins. The TEF approach for risk assessment of exodioxins makes a number of assumptions, including response additivity for individual compounds in a mixture of HAH. This review documents some of the following problems and limitations of the TEF approach: 1) environmental and food residues of HAH contain "non-dioxin-like" PCB that exhibit "antidioxin" activity for some responses; 2) the human diet contains endogenous Ah receptor ligands (endodioxins) such as polynuclear aromatic hydrocarbons (PAH), aromatic amines in cooked foods, indole-3-carbinol (I3C), and related hetero-PAH in cruciferous vegetables. Mass balance and mass potency estimates for human dietary intakes suggest that for some responses the effects of natural or endodioxins may be greater than those of exodioxins; and 3) I3C, a weak Ah receptor agonist, also exhibits Ah receptor antagonist activity, and interactions between I3C and endodioxins may inhibit or inactivate some toxic responses and decrease TEQ(Exodioxin).

Halogenated Aromatic Hydrocarbons Suppress CA1 Field Excitatory Postsynaptic Potentials in Rat Hippocampal Slices

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated biphenyls (PCBs) and dibenzo-p-dioxins (PCDDs), alter cognitive function and learning. The cellular basis of HAH-induced alteration of brain function is not well-understood. The hippocampus is a likely site of toxic action because of its well-known roles in learning and memory, as well as its propensity to accumulate environmental neurotoxicants. A hippocampal function that can be measured readily is evoked excitatory postsynaptic potentials (EPSPs), which are an index of excitatory synaptic function. In this study, effects of HAHs on EPSPs were characterized in hippocampal slices from adolescent to adult maleSprague—Dawley rats. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4-TCDD were used because these HAHs are prototypical potent and weak aryl hydrocarbon (Ah) receptor agonists, respectively. 2,2′,5,5′-Tetrachlorobiphenyl (TCB) was used as a prototypical ortho-substituted PCB, which acts through Ah receptor-independent pathways. For each hippocampal slice, peak amplitudes of EPSPs during a 15-min recording period (1 recording/min) were averaged and used as baseline (100%). Subsequent EPSPs were expressed as percentage of baseline. TCDD and 1,2,3,4-TCDD did not alter EPSPs in slices from the middle third of the hippocampus. However, in ventral slices, TCDD significantly decreased EPSPs, whereas 1,2,3,4-TCDD was inactive. TCB decreased EPSPs in both middle and ventral slices at half-maximal stimulation. An unexpected reversal of inhibition was observed within 30 min of continuous application of TCDD or TCB. In ventral slices,l-type calcium channel blocker nifedipine blocked inhibition of EPSPs induced by TCDD but not EPSPs inhibited by TCB. These results suggest that, while TCB-induced inhibition of EPSPs occurs through an unknown mechanism, TCDD-induced inhibition of EPSPs was mediated byl-type calcium channel activity in a congener-specific manner.

Sensitivity of CYP1A1 mRNA Inducibility by Dioxin Is the Same in Cyp1a2(/) Wild-type and Cyp1a2(−/−) Null Mutant Mice

In mammals, the induction of experimental porphyria by halogenated aromatic hydrocarbons (HAHs) seems to be influenced by the levels of hepatic CYP1A2. The pharmacokinetics and relative rates of uptake and storage of HAHs in the liver are correlated with hepatic CYP1A2 concentrations. It is possible that these rates of HAH uptake and storage might affect the expression of other HAH-inducible genes. The differential inducibility of liver CYP1A1 mRNA by dioxin was therefore compared in Cyp1a2(+/+) wild-type mice, Cyp1a2(+/−) heterozygotes, and Cyp1a2(−/−) homozygous null mutants. Using doses of dioxin over eight orders of magnitude (from 10 −12 to 10 −4 g/kg), we could detect no differences in the sensitivity of CYP1A1 mRNA inducibility. These data indicate that the complete absence of the microsomal CYP1A2 enzyme has no measurable effect on hepatic expression of the Cyp1a1, gene, the only other known member of the mammalian CYP1A cytochrome P450 subfamily.

Characterization of the Aromatic Hydrocarbon Receptor Gene and Its Expression in Atlantic Tomcod

Cytochrome P4501A1 (CYP1A1) mRNA is not inducible in Atlantic tomcod from the Hudson River that are treated with halogenated aromatic hydrocarbons (HAHs). In contrast, CYP1A1 mRNA is inducible in Hudson River tomcod that are treated with polycyclic aromatic hydrocarbons (PAHs) and in tomcod that are collected from cleaner rivers and treated with HAHs or PAHs. We hypothesize that CYP1A1 transcription is inhibited in Hudson River tomcod because of down-regulation of the aromatic hydrocarbon receptor (AhR) pathway and that separate molecular pathways modulate CYP1A1 transcription in fish treated with HAHs and PAHs. We initially evaluated levels of hepatic nuclear protein binding at enhancer elements (DREs) in the regulatory region of tomcod CYP1A1. No difference in levels of protein binding was observed between tomcod from the Hudson and Miramichi (cleaner) rivers that were untreated or were treated with benzo[a]pyrene. In contrast, levels of protein binding were lower in tomcod from the Hudson River that were treated with TCB than in similarly treated fish from the Miramichi River, suggesting differences between the populations in the structure or expression of AhR pathway molecules. To address this possibility, AhR DNA sequences were characterized from tomcod cDNA and genomic DNA libraries. In tomcod and mammals, AhR is represented by 11 exons, overall peptide sizes are similar, and amino acid sequences at basic, helix–loop–helix, PAAS A, and PAAS B domains are highly conserved. In contrast, little similarity was observed between tomcod and mammals in the sizes or sequences of AhR exons 10 and 11, including the absence in tomcod of glutamine-rich domains. No differences in levels of hepatic AhR mRNA were observed between the two populations or treatment groups when tomcod were untreated or were treated with aromatic hydrocarbons. In contrast, variation in levels of AhR mRNA expression was observed among tomcod tissues; however, no relationship was observed between levels of AhR mRNAs and CYP1A1 mRNAs in tissues from chemically or vehicle control-treated fish. RFLP analysis revealed extensive variation in exons 10 and 11 of AhR cDNA among tomcod from different rivers. Our results suggest that variation between tomcod populations in CYP1A1 mRNA inducibility is reflected by differences in levels of inducible hepatic protein binding to DREs. However, levels of hepatic AhR mRNA are not down-regulated in the Hudson River population, are not affected by AH treatments, and levels of AhR mRNA expression are not responsible for the differential inducibility of CYP1A1 transcription.

Sensitivity of common tern (Sterna hirundo) embryo HepatocyteCultures to CYP1A induction and porphyrin accumulation by halogenated aromatic hydrocarbons and common tern egg extracts.

Experiments were conducted to compare the sensitivity ofprimary cultures of common tern (Sterna hirundo) and chicken(Gallus domesticus) embryo hepatocytes to cytochrome P4501A (CYP1A)induction and porphyrin accumulation after exposure to halogenated aromatichydrocarbons (HAHs) or tern egg extracts. The HAHs tested were2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD),2,3,7, 8-tetrachlorodibenzofuran (TCDF) 3,3',4,4'-tetrachlorobiphenyl (PCB 77;IUPAC nomenclature), 3,3',4,4',5-pentachlorobiphenyl (PCB 126),3, 3',4,4',5,5'-hexachlorobiphenyl (PCB 169) and Aroclor(R) 1254 (a commercialmixture of PCBs). Extracts were prepared from common tern eggs collected fromthree sites in the Great Lakes basin and one reference site on the east coastof Canada. CYP1A induction was assayed asethoxyresorufin-O-deethylase (EROD) activity and/or immunodetectableCYP1A protein, and total intracellular porphyrin accumulation was measuredfluorometrically. Unlike chicken embryo hepatocyte (CEH) cultures, nointracellular porphyrin accumulation was observed after treatment of ternembryo hepatocyte (TEH) cultures with HAHs or egg extracts. TEH cultures were approximately 50 to >1600 times less sensitive than CEH cultures to HAH-mediated CYP1Ainduction. In contrast, TEH cultures were either approximately equallysensitive or only 3.5-15 times less sensitive than CEH cultures to CYP1Ainduction mediated by tern egg extracts. These data suggest that common ternembryos may be more susceptible to the CYP1A inducing effects mediated bycomplex mixtures of environmental contaminants than indicated by theirresponse to individual HAHs.

Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes

Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), and a commercial mixture of PCBs (Aroclor 1254). For these halogenated aromatic hydrocarbons (HAHs), or mixture, maximal CYP1A activity [measured as ethoxyresorufin- O-deethylase (EROD) activity] and immunodetectable protein were observed at concentrations just prior to, or coincident with, the concentrations at which porphyrin accumulation became evident. Both immunodetectable CYP1A protein and catalytic activity decreased at high concentrations of these compounds, but the rate and extent of decrease of immunodetectable CYP1A protein varied. Time-course studies with PCB 77 indicated a decrease in potency and an increase in maximal CYP1A induction between 24 and 48 hr of exposure which may indicate in vitro metabolism of this HAH. Intracellular accumulation of total porphyrins without CYP1A induction, was observed for 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), 2,2′,6,6′-tetrachlorobiphenyl (PCB 54), 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95), 2,2′,4,5,5′-pentachlorobiphenyl (PCB 101), 2,2′,3,3′,6,6′-hexachlorobiphenyl (PCB 136), and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153). Overall, these results are consistent with a role for CYP1A induction and/or Ah receptor activation in porphyrin accumulation mediated by HAHs with a planar configuration, whereas those that are not planar may mediate porphyrin accumulation by a mechanism not involving induction of CYP1A.

Limitations of the toxic equivalency factor approach for risk assessment of TCDD and related compounds

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs), are industrial compounds or by-products that have been widely identified as environmental contaminants. Hazard and risk assessment of complex HAH mixtures have utilized a toxic equivalency factor (TEF) approach, where the toxic equivalents (TEQs) of any mixture are equal to the sum of the concentration of individual (i) congeners times their potencies (TEFi) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, TEF = 1.0). TEQ = sigma [PCDDi] x TEFi + sigma [PCDFi] x TEFi + sigma [PCBi] x TEFi. The TEQ (or TCDD equivalents) can be readily calculated from analytical data and provides an estimate of the toxicity of any mixture containing HAHs. Several in vivo and in vitro studies with some PCDD/PCDF and PCB mixtures have demonstrated correlations between experimentally determined and calculated TEQs. However, results of several studies have also shown that for specific responses, the TEQ for some HAH mixtures are non-additive. For example, PCB mixtures and individual PCB congeners such as 2,2',4,4',5,5'-hexachlorobiphenyl inhibit toxic and biochemical responses induced by TCDD and related compounds. Another problem associated with hazard and risk assessment of background exposure to HAHs is the relative contribution of trace levels of HAHs (exodioxins) compared to relatively high exposure to naturally occurring aryl hydrocarbon receptor (AhR) agonists, which act through the same mechanistic pathway.

Limitations of the toxic equivalency factor approach for risk assessment of TCDD and related compounds

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated biphenyls (PCBs), dibenzo-ρ-dioxins (PCDDs), and dibenzofurans (PCDFs), are industrial compounds or by-products that have been widely identified as environmental contaminants. Hazard and risk assessment of complex HAH mixtures have utilized a toxic equivalency factor (TEF) approach, where the toxic equivalents (TEQs) of any mixture are equal to the sum of the concentration of individual (i) congeners times their potencies (TEFi) relative to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD, TEF = 1.0). TEQ = Σ [PCDDi] · TEFi + Σ [PCDFi] · TEFi + Σ [PCBi] · TEFi The TEQ (or TCDD equivalents) can be readily calculated from analytical data and provides an estimate of the toxicity of any mixture containing HAHs. Several in vivo and in vitro studies with some PCDD/PCDF and PCB mixtures have demonstrated correlations between experimentally determined and calculated TEQs. However, results of several studies have also shown that for specific responses, the TEQ for some HAH mixtures are non-additive. For example, PCB mixtures and individual PCB congeners such as 2,2′,4,4′,5,5′-hexachlorobiphenyl inhibit toxic and biochemical responses induced by TCDD and related compounds. Another problem associated with hazard and risk assessment of background exposure to HAHs is the relative contribution of trace levels of HAHs (exodioxins) compared to relatively high exposure to naturally occurring aryl hydrocarbon receptor (AhR) agonists, which act through the same mechanistic pathway. Teratogenesis Carcinog. Mutagen. 17:285–304, 1997/98. © 1998 Wiley-Liss, Inc.

AN IN VITRO RAINBOW TROUT CELL BIOASSAY FOR ARYL HYDROCARBON RECEPTOR-MEDIATED TOXINS

Halogenated aromatic hydrocarbons (HAHs) and other chemicals that act as aryl hydrocarbon (Ah) receptor (AhR) agonists cause a variety of toxic effects. In sac fry of many fish species, these effects include blue-sac disease and mortality. Because HAHs occur in complex mixtures, their toxicity in the environment is difficult to predict. A bioassay useful in predicting AhR-mediated toxicity to fish was developed using the RTH-149 rainbow trout hepatoma cell line. Stable transfection of this cell line with the pGudLuc1.1 plasmid, which contains a firefly luciferase reporter gene under the transcriptional regulation of dioxin responsive enhancers, has produced a recombinant cell line designated Remodulated Lightning Trout (RLT 2.0). The RLT 2.0 bioassay method detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is 4 pM. The responses of the RLT 2.0 bioassay to TCDD and several HAH congeners closely matched the responses observed in vivo in fish. The RLT 2.0 bioassay can provide an integrative measure of the total AhR-mediated toxic activity of complex mixtures to fish. The assay will be useful in screening environmental extracts, guiding chemical analysis, and interpreting the AhR-mediated mechanism of toxicity.

Cytochrome P4501 a induction in avian hepatocyte cultures: A promising approach for predicting the sensitivity of avian species to toxic effects of halogenated aromatic hydrocarbons

Concentration-dependent effects of halogenated aromatic hydrocarbons(HAHs) on cytochrome P4501A (CYP1A) induction in primary hepatocyte cultures prepared from embryos of chickens (four breeds), pheasants, turkeys, ducks (three breeds), and herring gulls were determined. CYP1A activity was estimated by measuring ethoxyresorufin O-deethylase (EROD) activity and the concentration of immunodetectable CYP1A was estimated using mouse monoclonal antibody 1-12-3 that was prepared against scup (Stenotomus chrysops) CYP1A1. The HAHs studied were 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 3,4,4′,5-tetrachlorobiphenyl (PCB 81), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105), and 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118). Two general types of comparisons were made: (1) relative potencies of compounds within a species (expressed relative to TCDD as induction equivalency factors, IEFs) and (2) relative sensitivity of each species to EROD induction by each compound. Three methods for estimating potency were compared. These were: (1) the concentration of inducer that produced a half-maximal (EC50) EROD response, (2) the concentration producing a response equivalent to 10% of the maximal response produced by TCDD (ECTCDD10%), and (3) a slope ratio method. For each method, the rank order in potency was TCDD ⩾ TCDF > PCB 126 > PCB 81 > PCB 77 > PCB 169 in chicken, pheasant, and turkey hepatocytes. The rank order was similar in duck and herring gull hepatocytes with the following exceptions: TCDF was approximately 2- to 4-fold more potent than TCDD in duck hepatocytes; PCB 169 induced EROD in gulls, but PCB 77 had no measurable effect in this species. PCB 118 was a relatively weak EROD inducer in most species/breeds, but it did not induce EROD in Pekin ducks or gulls. PCB 105 was a weak inducer in White Leghorn chicken and turkey hepatocytes, but it did not induce EROD in other species. The EC50, ECTCDD10% and slope ratio methods for estimating potencies generally gave similar IEFs for compounds that produced a maximal response that was at least 60% of the maximal response produced by TCDD. For compounds that caused a response that was 50% or lower than that produced by TCDD, EC50-based IEFs were greater (10- to 100-fold) than ECTCDD10%-based IEFs or slope-ratio-based IEFs. Among species, the rank order in sensitivity to EROD induction was chicken > pheasant > turkey ⩾ duck ⩾ herring gull. The relative sensitivity of avian hepatocyte cultures to EROD induction by PCB 77 was similar to the relative sensitivity of these species (reported elsewhere) to lethality after in ovo injection of PCB 77. Chicken hepatocyte cultures were 5–10 times more sensitive to EROD induction by TCDD than were pheasant hepatocyte cultures, which is identical to the difference in sensitivity of these species to the lethal effect of TCDD after in ovo injection. Measuring the sensitivity of hepatocyte cultures to EROD induction might be useful for estimating the sensitivity of avian species (including rare or endangered species, where it is impossible to conduct in vivo studies) to the embryotoxic effects of TCDD, non-ortho substituted PCBs, and other aryl hydrocarbon receptor agonists.