Analysis of endocrine disruption in Southern California coastal fish using an aquatic multispecies microarray - PubMed (original) (raw)

. 2009 Feb;117(2):223-30.

doi: 10.1289/ehp.11627. Epub 2008 Aug 28.

Barbara Ruggeri, L James Sprague, Colleen Eckhardt-Ludka, Jennifer Lapira, Ivan Wick, Laura Soverchia, Massimo Ubaldi, Alberta Maria Polzonetti-Magni, Doris Vidal-Dorsch, Steven Bay, Joseph R Gully, Jesus A Reyes, Kevin M Kelley, Daniel Schlenk, Ellen C Breen, Roman Sásik, Gary Hardiman

Affiliations

Analysis of endocrine disruption in Southern California coastal fish using an aquatic multispecies microarray

Michael E Baker et al. Environ Health Perspect. 2009 Feb.

Abstract

Background: Endocrine 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.

Objectives: We 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.

Methods: We 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.

Results: Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas.

Conclusions: The 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.

Keywords: Danio rerio; Pleuronichthys verticalis; endocrine disruptors; flatfish; hornyhead turbot; microarray; multispecies array; nonylphenol; xenobiotics; xenoestrogens; zebrafish.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Flatfish (Pleuronectiformes) in an evolutionary context. Adapted from the phylogeny (Coleman 2004; Helfman et al. 1997). Tetraodontiformes (Fugu, Tetraodon) and Perciformes (cichlid, tilapia, sea bass, sea bream, perch) are close phylogenetic relatives of Pleuronectiformes (turbot, halibut, sole) (box 1). Cypriniformes (zebrafish) are distant phylogenetic relatives (box 2).

Figure 2

Figure 2

Schematic representation of the design and application of a multispecies microarray-based test to monitor xenoestrogen exposure for environmental monitoring.

Figure 3

Figure 3

MA plots of differential expression and signal intensity measurements using the multispecies endocrine microarray for control and exposed fish (loess normalization). (A–D) Individual LACSD-exposed fish versus pooled controls. (E–G) Individual OCSD-exposed fish versus pooled controls. (H) Pooled controls versus pooled controls. (I–K) Individual control fish versus pooled control. Each point represents data from a single 65mer oligonucleotide probe. M is a measure of differential gene expression [log2 (exposed /control)] in A–G or absence of significant differential gene expression in the self–self plots [log2 (control/control intensity)] in H_–_K. A is a measure of signal intensity [(0.5 log2 exposed intensity + 0.5 log2 control intensity) in A–G or (0.5 log2 control intensity + 0.5 log2 control intensity) in _H–K_].

Figure 4

Figure 4

Normal q-q plots of multispecies endocrine microarray data. The q-q plots were constructed to determine whether control and exposed fish data sets derived from populations have a common distribution. (A–D) Individual LACSD-exposed fish versus pooled controls. (E–G) Individual OCSD-exposed fish versus pooled controls. (H) Pooled controls versus pooled controls. (I–K) individual control fish versus pooled control. The q-q plots show the distribution of the log2 (exposed/control) fold changes and the deviation, if any, from a normal Gaussian distribution. When the two data sets derive from a population with the same distribution, the points fall approximately along this straight line, as is the case with the control sample data populations, both pooled and individual (I–K). When the two data sets derive from populations with different distributions, the data deviate from this straight line (A–G). Exposed samples differ from the control, with a sharp rise observed in the quantile curve at log2 ratios of 2, indicating the presence of large log2 ratios and true differences in gene expression.

Figure 5

Figure 5

Gene expression profiling of male turbot liver collected at OCSD and LACSD (contaminated) and control fish from a nonaffected area. This heat map depicts fold changes observed between exposed and control fish. LACSD and OCSD data derived from four and three independent biologic replicate microarray experiments, respectively.

Figure 6

Figure 6

Multispecies SYBR Green qRT-PCR validation of multispecies endocrine microarray for Vtg1 (A), Vtg2( B), thyroid hormone receptor β (C), and _CYP3A_-specific transcripts (D) in livers from control (Con) and exposed (Exp) hornyhead turbot (each bar represents one fish). 18S rRNA served as an internal control for normalization. Plots are mean fold changes from triplicate measurements, relative to control fish. Vtg1 (A) and Vtg2 (B) transcripts were strongly up-regulated (> 15 fold) in one exposed fish. TRβ was down-regulated in two control fish. CYP3A was up-regulated in three fish and down-regulated in one fish.

Figure 7

Figure 7

Cross-species applicability of the Multispecies endocrine microarray: detection of alterations in gene expression in zebrafish liver after a 2-week exposure to either 4-nonylphenol or estradiol. This heat map depicts fold changes between exposed and control fish. Data derived from four independent biological replicate experiments.

Similar articles

Cited by

References

    1. Ahel M, Conrad J, Giger W. Persistent organic chemicals in sewage effluents. 3. Determinations of nonylphenoxy carboxylic acids by high-resolution gas chromatography/mass spectrometry and high-performance liquid chromatography. Environ Sci Technol. 1987;21:697–703. - PubMed
    1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. - PubMed
    1. Atanasov AG, Nashev LG, Tam S, Baker ME, Odermatt A. Organotins disrupt the 11beta-hydroxysteroid dehydrogenase type 2-dependent local inactivation of glucocorticoids. Environ Health Perspect. 2005;113:1600–1606. - PMC - PubMed
    1. Baker ME. Adrenal and sex steroid receptor evolution: environmental implications. J Mol Endocrinol. 2001;26:119–125. - PubMed
    1. Baker ME. Evolutionary analysis of 11beta-hydroxysteroid dehydrogenase-type 1, -type 2, -type 3 and 17beta-hydroxy-steroid dehydrogenase-type 2 in fish. FEBS Lett. 2004;574:167–170. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources