Connexin gap junctions play an important role in hearing function, but the mechanism by which this contribution occurs is unknown. Connexins in the cochlea are expressed only in supporting cells; no connexin expression occurs in auditory sensory hair cells. A gap junctional channel is formed by two hemichannels. Here, we show that connexin hemichannels in the cochlea can release ATP at levels that account for the submicromolar concentrations measured in the cochlear fluids in vivo. The release could be increased 3-to 5-fold by a reduction of extracellular Ca 2؉ or an increase in membrane stress, and blocked by gap junctional blockers. We also demonstrated that extracellular ATP at submicromolar levels apparently affected outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining cochlear sensitivity to sound stimulation in mammals. ATP reduced OHC electromotility and the slope factor of the voltage dependence and shifted the operating point to reduce the active amplifier gain. ATP also reduced the generation of distortion products. Immunofluorescent staining showed that purinergic receptors P2x2 and P2x7 were distributed on the OHC surface. Blockage of P2 receptors eliminated the effect of ATP on the OHC electromotility. The data revealed that there is a hemichannel-mediated, purinergic intercellular signaling pathway between supporting cells and hair cells in the cochlea to control hearing sensitivity. The data also demonstrated a potential source of ATP in the cochlea.active cochlear mechanics ͉ cochlear supporting cells ͉ connexin ͉ outer hair cell electromotility ͉ P2 receptor

a b s t r a c t A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in ␤-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with elevated concentrations in waters that may also experience hypoxia. Previous research has shown interactions between hypoxia and some PAHs (fluoranthene, α-naphthoflavone) but no interaction with others (benzo[a]pyrene (BaP), β-naphthoflavone). Here we examine how hypoxia (7.4% oxygen, ~35% of normoxia) affects the embryotoxicity of PAHs that act through different mechanisms and the role that CYP1A inhibition may play in these interactions. 500 μg/L BaP and 1-200 μg/L benzo[k]fluoranthene (BkF) interacted synergistically with hypoxia to induce pericardial edema in developing zebrafish (Danio rerio). Hypoxia protected from the embryotoxicity of pyrene (PY) and had no effect on the toxicity of polychlorinated biphenyl-126. Despite previous reports of other CYP1A inhibitors interacting with hypoxia, up to 2000 μg/L dibenzothiophene, 2-aminoanthracene (AA), and carbazole (CB) all failed to induce embryotoxicity under normoxic or hypoxic conditions. The toxicity of PAH mixtures—including binary mixtures of BaP/AA and BaP/CB and two environmentally relevant, complex mixtures—were exacerbated severely by hypoxia to induce or worsen pericardial edema and cause mortality. The interactions between hypoxia and BkF and PY were closely mimicked by morpholino knockdown of CYP1A, indicating a potential role for metabolism of these compounds in their toxicity. Our results indicate that various PAHs may exhibit synergistic, antagonistic or additive toxicity with hypoxia. The enhanced toxicity of environmental mixtures of PAHs under hypoxia suggests that risk assessments that do not take into account potential interactions with hypoxia may underestimate the threat of PAHs to fish in contaminated sites.

Hypoxic events often occur in waters contaminated with toxic chemicals, including agonists of the aryl hydrocarbon receptor (AhR). HIF-1α, 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% O2) or normoxia (21% O2) 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 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-1α is able to sequester ARNT from AhR and limit the activity of the AhR pathway, but suggests that the converse is not true.

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