Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

BackgroundEndocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies.ObjectivesWe fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species.MethodsWe used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data.ResultsMicroarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas.ConclusionsThe agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish

CRTH2 antagonism significantly ameliorates airway hyperreactivity and downregulates inflammation-induced genes in a mouse model of airway inflammation

Prostaglandin D2, the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD2 and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD2-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation

Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in southern california using a second generation multi-species microarray.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences

Q-PCR validation of Multi-Species Endocrine Microarray.

<p>Multi-species SYBR green Q-PCR validation of multi-species microarray for (A) <i>CYP3A</i> (B) <i>Vit1</i> (C) <i>Vit2</i> (D) ESR1/<i>ERα</i> (E) ESR2/<i>ERβ</i> specific transcripts. <i>GAPDH</i>-normalized fold changes (based on triplicate measurements) of gene expression in turbot liver from selected impacted sites with respect to reference fish are presented. Each fold change was derived from the mean log₂ ratio between each fish and a reference derived from two control fish. <i>Vit1</i> and <i>Vit2</i> transcripts were strongly up-regulated in all fish. <i>ERα</i> was down-regulated in one fish and up-regulated in two others relative to control fish. <i>ERβ</i> was down-regulated in all fish examined relative to control fish. <i>CYP3A</i> was up-regulated in eight and down-regulated in two fish.</p

Differential expression and signal intensity measurements for impacted turbot livers.

<div><p>A: Gene expression changes were investigated in male turbot liver collected from exposed fish sampled from sanitation districts in San Diego, Orange County, Dana Point, Los Angeles City and Los Angeles County in California that are considered impacted. Control fish were obtained from a separate monitoring station in Dana Point, a relatively non-impacted area and maintained in a clean-water laboratory for four weeks. The control reference sample consisted of a pool of labeled cRNA from three control fish, designated <i>A</i>, <i>B</i>, and <i>C</i>.</p> <p>M (Y-axis) is a measure of differential gene expression (log2 (exposed /control) in the samples in the first three panel rows of plots or absence of significant differential gene expression in the self-self plots (log₂ (control / control intensity) in the bottom panel row of plots. A (X-axis) is a measure of signal intensity (0.5 log₂ exposed intensity + 0.5 log₂ control intensity) in the first three panel rows of plots or (0.5 log₂ control intensity + 0.5 log₂ control intensity) in the bottom panel row of plots. Careful analysis of the heavy tail of outliers for the individual fish <i>A</i> (second panel from right, bottom row), revealed these data points in the tail are not real and are derived from an array artifact.</p></div

Multi-species microarray gene expression profiling of turbots for exposure to endocrine disruptors.

<p>The mRNA level fold changes observed between exposed and control fish are depicted as a diagnostic heat map representative of all the genes present on the multi-species array. The log₂ ratio for each gene is defined as the mean of all representations of that gene on the microarray; these include probes from different regions of the gene, as well as from different species of fish (individual log₂ ratios for each unique probe are calculated as the median of 46 replicates present on the array). The range of colors is between -8-fold and +8-fold and preserves qualitative relationships among individual values. All fold changes outside of this range have been truncated to ± 8.</p

Quality control specifications for the second generation multi-species endocrine microarray.

<p>Comparison of the performance characteristics of on both prototype (spotted oligonucleotide) and second generation (Agilent ink jet oligonucleotide) endocrine multi-species microarray platforms; Panels A and B: scatter plots of log₂ signal intensities of an individual fish from the Los Angeles Sanitation District versus the control fish pool. Panels A and B depict prototype and second generation platforms respectively. Panel C; direct comparison of platform sensitivity defined as log₂ ratio between treatment and ABC control. Panel D: Analysis of signal concordance. Panel E: Analysis of variance with both platforms. To measure internal consistency (variance of internal replicates), log₂ transformed expression value was plotted on the x-axis. Standard deviation is plotted on the y-axis, with a range of plus or minus one standard deviation from the mean expression value. The y-axis is a measure of the deviation of individual replicate probes stray from the mean value. The second generation platform has smaller variance of the internal replicates. Probe variance on both platforms is independent of the signal. Panel F: Comparison of the probe signal intensity distribution (plotted as log₂ transformed) for 149 probes that were present on both platforms.</p

Molecular Analysis of Endocrine Disruption in Hornyhead Turbot at Wastewater Outfalls in Southern California Using a Second Generation Multi-Species Microarray

Sentinel fish hornyhead turbot ( Pleuronichthys verticalis ) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences