Immediate and long-term consequences of vascular toxicity during zebrafish development.

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Identification of estrogen-responsive vitelline envelope protein fragments from rainbow trout (Oncorhynchus mykiss) plasma using mass spectrometry.

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17ß-estradiol (E2) were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Specifically, plasma peptides of interest were fractionated and subsequently identified via spectra obtained by MALDI QqTOF MS/MS and LC-MALDI TOFTOF MS/MS analysis, de novo sequencing and database matching. The two peptide masses were identified as significant matches for fragments of the C-terminal propeptides from rainbow trout vitelline envelope protein (VEP)α and VEPγ isoforms. Our findings document the presence of the C-terminal propeptides from rainbow trout VEPα and VEPγ proteins in the bloodstream of juvenile male rainbow trout exposed to E2 via MALDI-TOF-MS detection. We provide three possible explanations for the presence of C-terminal propeptides in the bloodstream, as well as compare previously obtained hepatic transcriptomic results with the plasma proteomic results obtained in the present study.

Multiple responses in gene expression in fish treated with estrogen.

During the last decade there has been a significant body of research conducted on environmental estrogens. These include industrial, agricultural and pest-control chemicals that bind to the estrogen receptor and induce biological changes during development or reproduction. Most of these changes are probably due to modified gene expression, since estrogen receptors function at this level. We have mapped qualitative gene expression responses (by differential display reverse transcriptase polymerase chain reaction, DD) in adult male sheepshead minnows (Cyprinidon variegatus) receiving high dose injections (5 mg/kg), or constant flow-through aquatic exposures to environmentally relevant concentrations (100 ng/l) of estradiol-17beta, and found them nearly identical. We have observed both up-regulation and down-regulation of transcripts, which fit into known responses to estradiol. Among the genes up-regulated are vitellogenin and several vitelline envelope proteins indicating that genes for proteins involved in egg development and maturation are susceptible to environmental estrogen exposure. While physiological changes caused by estradiol treatment are not totally explained by changes at the mRNA level, those changes can nevertheless be used as fingerprints to characterize an in vivo estrogenic response.

Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17beta-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation.

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17beta-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.

Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus).

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is activating estrogen-responsive genes, such as vitellogenin (Vtg). Primers to vitellogenin mRNA have been used to amplify a portion of the coding sequence in sheepshead minnow (SHM) (Cyprinodon variegatus). Two Vtg cDNA fragments from SHM were isolated exhibiting 72% sequence homology and corresponding to the two Vtg genes identified in the mummichog, Fundulus heteroclitus. Using these Vtg cDNA fragments as sensitive genetic probes, we evaluated the initial estrogenic response of fish exposed to natural or anthropogenic chemicals. These probes were used to study in vivo gene induction in SHM exposed to 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)) under controlled laboratory conditions. Hepatic Vtg mRNA was upregulated and plasma Vtg synthesis in estrogen-induced SHM was assessed. Two in vivo time-course experiments were conducted; a single injection of E(2) followed over 72 h and a double E(2) injection examined for 12 days. These two protocols provided evidence for differential hepatic Vtg mRNA regulation resulting from a single or a double injection. In a separate experiment using an aqueous flowthrough system, constant exposures to low doses of E(2) (200 ng/L) and EE(2) (100 ng/L) induced hepatic Vtg mRNA and plasma Vtg to levels comparable with the E(2) injections. Larger aqueous exposure doses (2000 ng/L E(2) or 1000 ng/L EE(2)) in the flowthrough experiment resulted in greater responses of hepatic Vtg mRNA and plasma Vtg at 7 days. Constant aqueous exposure to E(2) (2000 ng/L) or EE(2) (1000 ng/L) may thus be more effective than a single large-dose injection (5 mg/kg) to stimulate Vtg gene activation and synthesis.

Dieldrin induces cytosolic [3H]7, 12-dimethylbenz[a]anthracene binding but not multidrug resistance proteins in rainbow trout liver.

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Comparison of three histological fixatives on the immunoreactivity of mammalian P-glycoprotein antibodies in the sheepshead minnow, Cyprinodon variegatus.

Mammalian P-glycoprotein is a highly conserved integral membrane protein functioning as an energy-dependent efflux pump which decreases the concentration of certain lipophilic aromatic compounds entering the cell by diffusion. Expression of such a transporter in teleost species could play a significant role in conferring resistance to fish populations exposed to xenobiotic stressors and may serve as a potential indicator of species at risk for certain environmental contaminants. In previous studies we demonstrated that a strong correlation existed between corresponding mammalian and teleost tissues showing immunoreactivity to specific mammalian P-glycoprotein antibodies. In the present study, comparisons of staining pattern, intensity, and tissue specificity between tissues treated in Bouin's, Dietrich's and Lillie's histological fixatives were determined in the sheepshead minnow, Cyprinodon variegatus, using monoclonal antibodies C219, C494, JSB-1 and polyclonal antiserum MDR(Ab-1). Immunoreactivity of these antibodies was found to be fixative-dependent. Results are presented illustrating the differential staining patterns and tissue specificity observed for each tissue type, fixative, and antibody combination. Our data indicate tissue fixation has a significant impact on P-glycoprotein antibody immuno-reactivity in teleost tissues and must be considered in the comparison and interpretation of results.

Immunohistochemical detection of P-glycoprotein in teleost tissues using mammalian polyclonal and monoclonal antibodies.

Mammalian P-glycoprotein is a highly conserved 170-kD integral plasma membrane protein functioning as an energy-dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. In this study, the tissue specificities of one polyclonal (pAb) and three monoclonal (mAbs) antibodies to mammalian P-glycoprotein were identified in paraffin-embedded, parasagittal whole-body sections of the guppy Poecilia reticulata. Polyclonal antibody mdr(Ab-1) and mAbs C219, C494, and JSB-1 demonstrated differential staining patterns in the following tissues: bile canaliculi in the liver, exocrine pancreas, lumenal surface of the intestinal epithelium, renal tubules, interrenal tissue, branchial blood vessels, gas gland, pseudobranch, and the gill transverse septa. Positive P-glycoprotein expression in P. reticulata correlates well with published results for homologous mammalian tissues of secretory and excretory function. These data indicate that one or more highly conserved members of the P-glycoprotein transporter family exist in a teleost species and can be detected using commercially available mammalian antibodies.