Hypoxia inhibits induction of aryl hydrocarbon receptor activity in topminnow hepatocarcinoma cells in an ARNT-dependent manner.

Hypoxic events often occur in waters contaminated with toxic chemicals, including agonists of the aryl hydrocarbon receptor (AhR). HIF-1alpha, the mediator of cellular responses to hypoxia, shares a dimerization partner (ARNT) with AhR and reciprocal crosstalk may occur. Studies addressing AhR/hypoxia crosstalk in mammalian cells have produced contradictory results regarding whether reciprocal crosstalk actually occurs between these pathways and the role ARNT plays in this interaction. We assessed hypoxia-AhR crosstalk in fish cells (PLHC-1) treated with hypoxia (1% O(2)) or normoxia (21% O(2)) and AhR agonists (benzo[a]pyrene (BaP), 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and benzo[k]fluoranthene (BkF)) with and without overexpression of ARNT. Hypoxia limited the induction of a transiently transfected AhR reporter by all three of the AhR agonists; overexpression of ARNT eliminated this effect. PCB-126 had no effect on induction of a transiently transfected hypoxia reporter. BkF caused a minor increase in basal and induced hypoxia reporter activity. BaP decreased basal and induced hypoxia reporter activity; overexpression of ARNT did not alter this effect indicating that this interference with hypoxia pathway activity occurs through an alternate mechanism. Reduced hypoxia pathway activity with BaP treatment may be the result of a metabolite. This study supports the hypothesis that HIF-1alpha is able to sequester ARNT from AhR and limit the activity of the AhR pathway, but suggests that the converse is not true.

Nonadditive effects of PAHs on Early Vertebrate Development: mechanisms and implications for risk assessment.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Traditionally, much of the research has focused on the carcinogenic potential of specific PAHs, such as benzo(a)pyrene, but recent studies using sensitive fish models have shown that exposure to PAHs alters normal fish development. Some PAHs can induce a teratogenic phenotype similar to that caused by planar halogenated aromatic hydrocarbons, such as dioxin. Consequently, mechanism of action is often equated between the two classes of compounds. Unlike dioxins, however, the developmental toxicity of PAH mixtures is not necessarily additive. This is likely related to their multiple mechanisms of toxicity and their rapid biotransformation by CYP1 enzymes to metabolites with a wide array of structures and potential toxicities. This has important implications for risk assessment and management as the current approach for complex mixtures of PAHs usually assumes concentration addition. In this review we discuss our current knowledge of teratogenicity caused by single PAH compounds and by mixtures and the importance of these latest findings for adequately assessing risk of PAHs to humans and wildlife. Throughout, we place particular emphasis on research on the early life stages of fish, which has proven to be a sensitive and rapid developmental model to elucidate effects of hydrocarbon mixtures.

The role of the aryl hydrocarbon receptor pathway in mediating synergistic developmental toxicity of polycyclic aromatic hydrocarbons to zebrafish.

Planar halogenated aromatic hydrocarbons (pHAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), show strong binding affinity for the aryl hydrocarbon receptor (AHR) and are potent inducers of cytochrome P4501A (CYP1A). It is widely accepted that dioxin toxicity is largely AHR mediated; however, the role of CYP1A activity in causing that toxicity is less clear. Another class of AHR agonists of increasing concern because of their known toxicity and ubiquity in the environment is the polycyclic aromatic hydrocarbons (PAHs). Like dioxin, some PAHs also cause toxicity to early life stages of vertebrates. Symptoms include increased cardiovascular dysfunction, pericardial and yolk sac edemas, subcutaneous hemorrhages, craniofacial deformities, reduced growth, and increased mortality rates. Although developmental effects are comparable between these two types of AHR agonists, the roles of both the AHR and CYP1A activity in PAH toxicity are unknown. As observed in previous studies with killifish (Fundulus heteroclitus), we demonstrate here that coexposure of zebrafish (Danio rerio) embryos to the PAH-type AHR agonist beta-naphthoflavone (BNF) and the CYP1A inhibitor alpha-naphthoflavone (ANF) significantly enhanced toxicity above that observed for single-compound exposures. In order to elucidate the role of the AHR pathway in mediating synergistic toxicity of PAH mixtures to early life stages, we used a morpholino approach to knock down expression of zebrafish AHR2 and CYP1A proteins during development. We observed that while knock down of AHR2 reduces cardiac toxicity of BNF combined with ANF to zebrafish embryos, CYP1A knockdown markedly enhanced toxicity of BNF alone and BNF + ANF coexposures. These data support earlier chemical inducer/inhibitor studies and also suggest that mechanisms underlying developmental toxicity of PAH-type AHR agonists are different from those of pHAHs. Identifying the pathways involved in PAH toxicity will provide for more robust, mechanistic-based tools for risk assessment of single compounds and complex environmental mixtures.

Altering cytochrome P4501A activity affects polycyclic aromatic hydrocarbon metabolism and toxicity in rainbow trout (Oncorhynchus mykiss).

The polycyclic aromatic hydrocarbons (PAHs) phenanthrene and retene (7-isopropyl-1-methyl phenanthrene) are lethal to rainbow trout (Oncorhynchus mykiss) larvae during chronic exposures. Phenanthrene is a low-toxicity, non-cytochrome P4501A (CYP1A)-inducing compound that accumulates in fish tissues during exposure to lethal concentrations in water. Retene is a higher toxicity CYP1A-inducing compound that is not detectable in tissue at lethal exposure concentrations. The metabolism, excretion, and toxicity of retene and phenanthrene were examined in juvenile and larval rainbow trout during coexposure to the model CYP1A inducer beta-naphthoflavone (betaNF), or to the inducer-inhibitor piperonyl butoxide to determine if modulating CYP1A activity affected PAH metabolism and toxicity. Phenanthrene metabolism, excretion rate, and toxicity increased with coexposure to betaNE Piperonyl butoxide inhibited phenanthrene metabolism and reduced the excretion of all phenanthrene metabolites. As a consequence, embryo mortality rates increased but rates of sublethal effects did not. Coexposure of trout to retene and betaNF caused no change in retene metabolism and excretion, but retene toxicity increased, perhaps due to additivity. Piperonyl butoxide inhibited retene metabolism, decreased the excretion of some retene metabolites while increasing the excretion of others, and increased the toxicity of retene. These results support the role of CYP1A activity in PAH metabolism and excretion, and the role ofthe CYP1A-generatedmetabolites of PAHs in chronic toxicity to larval fish.

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.