Biomedical subjects

John P Incardona

Publications and source records attributed to John P Incardona.

8 recordsLinked to original sources

PubMed2006

Developmental toxicity of 4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor isoforms and hepatic cytochrome P4501A metabolism.

Polycyclic aromatic hydrocarbons (PAHs) derived from fossil fuels are ubiquitous contaminants and occur in aquatic habitats as highly variable and complex mixtures of compounds containing 2 to 6 rings. For aquatic species, PAHs are generally accepted as acting through either of two modes of action: (1) "dioxin-like" toxicity mediated by activation of the aryl hydrocarbon receptor (AHR), which controls a battery of genes involved in PAH metabolism, such as cytochrome P4501A (CYP1A) and (2) "nonpolar narcosis", in which tissue uptake is dependent solely on hydrophobicity and toxicity is mediated through non-specific partitioning into lipid bilayers. As part of a systematic analysis of mechanisms of PAH developmental toxicity in zebrafish, we show here that three tetracyclic PAHs (pyrene, chrysene, and benz[a]anthracene) activate the AHR pathway tissue-specifically to induce distinct patterns of CYP1A expression. Using morpholino knockdown of ahr1a, ahr2, and cyp1a, we show that distinct embryolarval syndromes induced by exposure to two of these compounds are differentially dependent on tissue-specific activation of AHR isoforms or metabolism by CYP1A. Exposure of embryos with and without circulation (silent heart morphants) resulted in dramatically different patterns of CYP1A induction, with circulation required to deliver some compounds to internal tissues. Therefore, biological effects of PAHs cannot be predicted simply by quantitative measures of AHR activity or a compound's hydrophobicity. These results indicate that current models of PAH toxicity in fish are greatly oversimplified and that individual PAHs are pharmacologically active compounds with distinct and specific cellular targets.

Abnormalities, Drug-Induced↗

PubMed2006

Epithelial trafficking of Sonic hedgehog by megalin.

We present here evidence of in vivo epithelial endocytosis and trafficking of non-lipid-modified Sonic hedgehog (ShhN) when infused into rat efferent ducts via microinjection. Initially, exogenous ShhN is detected in endocytic vesicles and early endosomes located near the apical plasma membrane of non-ciliated cells. Within 30-60 min following infusion, ShhN can be detected in lysosomes and at basolateral regions of non-ciliated cells. Basolaterally, ShhN was observed along the extracellular surfaces of interdigitated plasma membranes of adjacent cells and in the extracellular compartment underlying the efferent duct epithelium. Uptake and subcellular trafficking of infused ShhN by non-ciliated cells could be blocked by either anti-megalin IgG or the megalin antagonist, RAP. Ciliated cells, which do not express megalin, displayed little if any apical internalization of ShhN even though they were found to express Patched-1. However, ShhN was found in coated pits of lateral plasma membranes of ciliated cells as well as in underlying endocytic vesicles. We conclude that megalin-mediated endocytosis of ShhN can occur in megalin-expressing epithelia in vivo, and that the internalized ShhN can be targeted to the lysosome or transcytosed in the plane of the epithelium or across the epithelium. These findings highlight the multiple mechanisms by which megalin may influence Shh morphogen gradients in vivo.

Animals↗

PubMed2006

The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae.

Fipronil is a phenylpyrazole insecticide designed to selectively inhibit insect gamma-aminobutyric acid (GABA) receptors. Although fipronil is often used in or near aquatic environments, few studies have assessed the effects of this neurotoxicant on aquatic vertebrates at sensitive life stages. We explored the toxicological effects of fipronil on embryos and larvae using the zebrafish (Danio rerio) experimental model system. Embryos exposed to fipronil at nominal concentrations at or above 0.7 microM (333 mug/l) displayed notochord degeneration, shortening along the rostral-caudal body axis, and ineffective tail flips and uncoordinated muscle contractions along the body axis in response to touch. This phenotype closely resembles zebrafish locomotor mutants of the accordion class and is consistent with loss of reciprocal inhibitory neurotransmission by glycinergic commissural interneurons in the spinal cord. Consistent with the hypothesis that notochord degeneration may be due to abnormal mechanical stress from muscle tetany, the expression patterns of gene and protein markers specific to notochord development were unaffected by fipronil. Moreover, the degenerative effects of fipronil (1.1 microM) were reversed by coexposure to the sodium channel blocker MS-222 (0.6mM). The notochord effects of fipronil were phenocopied by exposure to 70 microM strychnine, a glycinergic receptor antagonist. In contrast, exposure to gabazine, a potent vertebrate GABA(A) antagonist, resulted in a hyperactive touch response but did not cause notochord degeneration. Although specifically developed to target insect GABA receptors with low vertebrate toxicity, our results suggest that fipronil impairs the development of spinal locomotor pathways in fish by inhibiting a structurally related glycine receptor subtype. This represents an unanticipated and potentially novel mechanism for fipronil toxicity in vertebrates.

Animals↗

PubMed2006

Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish.

Dissolved copper is an increasingly common non-point source contaminant in urban and urbanizing watersheds. In the present study, we investigated the sublethal effects of dissolved copper on the peripheral mechanosensory system, or lateral line, of larval zebrafish (Danio rerio). Zebrafish larvae were exposed to copper (0-65 microg/L), and the cytotoxic responses of individual lateral line receptor neurons were examined using a combination of in vivo fluorescence imaging, confocal microscopy, scanning electron microscopy, and conventional histology. Dissolved copper triggered a dose-dependent loss of neurons in identified lateral line neuromasts at concentrations > or = 20 microg/L. The onset of cell death in the larval mechanosensory system was rapid (< 1 h). When copper-exposed zebrafish were transferred to clean water, the lateral line regenerated over the course of 2 d. In contrast, the lateral line of larvae exposed continuously to dissolved copper (50 microg/L) for 3 d did not recover. Collectively, these results show that peripheral mechanosensory neurons are vulnerable to the neurotoxic effects of copper. Consequently, dissolved copper in non-point source storm-water runoff has the potential to interfere with rheotaxis, schooling, predator avoidance, and other mechanosensory-mediated behaviors that are important for the migration and survival of fish.

Animals↗

PubMed2005

From sensing cellular sterols to assembling sensory structures.

The genome of the nematode C. elegans is peppered with novel genes belonging to a superfamily whose members function in cellular cholesterol homeostasis (Niemann-Pick C1) and Hedgehog signal transduction (Patched) and biogenesis (Dispatched). In this issue of Developmental Cell and an analysis of a pair of Patched- and Dispatched-related proteins in C. elegans extends the superfamily's repertoire to include the formation of tubular organs.

Animals↗

PubMed2005

Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development.

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.

Abnormalities, Multiple↗

PubMed2004

Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons.

Fish embryos exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) from petrogenic sources show a characteristic suite of abnormalities, including cardiac dysfunction, edema, spinal curvature, and reduction in the size of the jaw and other craniofacial structures. To elucidate the toxic mechanisms underlying these different defects, we exposed zebrafish (Danio rerio) embryos to seven non-alkylated PAHs, including five two- to four-ring compounds that are abundant in crude oil and two compounds less abundant in oil but informative for structure-activity relationships. We also analyzed two PAH mixtures that approximate the composition of crude oil at different stages of weathering. Exposure to the three-ring PAHs dibenzothiophene and phenanthrene alone was sufficient to induce the characteristic suite of defects, as was genetic ablation of cardiac function using a cardiac troponin T antisense morpholino oligonucleotide. The primary etiology of defects induced by dibenzothiophene or phenanthrene appears to be direct effects on cardiac conduction, which have secondary consequences for late stages of cardiac morphogenesis, kidney development, neural tube structure, and formation of the craniofacial skeleton. The relative toxicity of the different mixtures was directly proportional to the amount of phenanthrene, or the dibenzothiophene-phenanthrene total in the mixture. Pyrene, a four-ring PAH, induced a different syndrome of anemia, peripheral vascular defects, and neuronal cell death, similar to the effects previously described for potent aryl hydrocarbon receptor ligands. Therefore, different PAH compounds have distinct and specific effects on fish at early life history stages.

Abnormalities, Drug-Induced↗

PubMed2002

Sonic hedgehog induces the segregation of patched and smoothened in endosomes.

BACKGROUND: Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. This suggests that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway. RESULTS: We characterized the dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. We show that, like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting. CONCLUSIONS: These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independently of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway.

Animals↗