Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.

Mass spectrometry imaging (MSI) methods and protocols have become widely adapted to a variety of tissues and species. However, the MSI literature contains minimal information on whole-body cryosection preparation for the zebrafish (ZF; Danio rerio), a model organism routinely used in developmental, toxicity, and carcinogenicity studies. The optimal medium for embedding and cryosectioning a whole organism or soft-tissue specimen for histological examination is a synthetic polymer mixture that is incompatible with MSI as a result of ion suppression. We describe the optimal methods and results for embedding and cryosectioning whole-body ZF for MALDI-MSI. We evaluated 13 distinct embedding media formulations and found a supportive hydrogel with the consistency of cartilage to be the optimal embedding medium. The hydrogel medium does not interfere with MSI data collection, aids in tissue stability, is readily available for purchase, and is easy to prepare and handle during cryosectioning. Additionally, we decreased the matrix cluster interference commonly caused by α-cyano-4-hydroxycinnamic acid by adding ammonium phosphate to the solvent spray solution. The optimized methods developed in our laboratory produced high-quality cryosections, as well as high-quality mass spectral images of sectioned ZF.

Development of aquatic toxicity benchmarks for oil products using species sensitivity distributions.

Determining the sensitivity of a diversity of species to spilled oil and chemically dispersed oil continues to be a significant challenge in spill response and impact assessment. We used standardized tests from the literature to develop species sensitivity distributions (SSDs) of acute aquatic toxicity values for several petroleum products and 2 Corexit oil dispersants. Fifth percentile hazard concentrations (HC5s) were computed from the SSDs and used to assess relative oil product toxicity and in evaluating the feasibility of establishing toxicity benchmarks for a community of species. The sensitivity of mysids (Americamysis bahia) and silversides (Menidia beryllina) were evaluated within the SSDs to determine if these common test species were appropriate surrogates for a broader range of species. In general, SSD development was limited by the availability of acute toxicity values that met standardization criteria for a diversity of species. Pooled SSDs were also developed for crude oil and Corexit dispersants because there was only small variability in the HC5s among the individual oil or dispersant products. The sensitivity of mysids and silversides varied across the oil and dispersant products, with the majority of toxicity values greater than the HC5. Application of SSDs appears to be a reasonable approach to developing oil product toxicity benchmarks, but additional toxicity data are needed for a larger range of species conducted under standardized test conditions.

Comparative toxicity of eight oil dispersants, Louisiana sweet crude oil (LSC), and chemically dispersed LSC to two aquatic test species.

The present study describes the acute toxicity of eight commercial oil dispersants, South Louisiana sweet crude oil (LSC), and chemically dispersed LSC. The approach used consistent test methodologies within a single laboratory in assessing the relative acute toxicity of the eight dispersants, including Corexit 9500A, the predominant dispersant applied during the DeepWater Horizon spill in the Gulf of Mexico. Static acute toxicity tests were performed using two Gulf of Mexico estuarine test species, the mysid shrimp (Americamysis bahia) and the inland silversides (Menidia beryllina). Dispersant-only test solutions were prepared with high-energy mixing, whereas water-accommodated fractions of LSC and chemically dispersed LSC were prepared with moderate energy followed by settling and testing of the aqueous phase. The median lethal concentration (LC50) values for the dispersant-only tests were calculated using nominal concentrations, whereas tests conducted with LSC alone and dispersed LSC were based on measured total petroleum hydrocarbon (TPH) concentrations. For all eight dispersants in both test species, the dispersants alone were less toxic (LC50s: 2.9 to >5,600 µl/L) than the dispersant-LSC mixtures (0.4-13 mg TPH/L). Louisiana sweet crude oil alone had generally similar toxicity to A. bahia (LC50: 2.7 mg TPH/L) and M. beryllina (LC50: 3.5 mg TPH/L) as the dispersant-LSC mixtures. The results of the present study indicate that Corexit 9500A had generally similar toxicity to other available dispersants when tested alone but was generally less toxic as a mixture with LSC.

Application of protein expression profiling to screen chemicals for androgenic activity.

Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) coupled with a short term fish assay was used to investigate changes in plasma protein expression as a means to screen chemicals for androgenic activity. Adult gravid female sheepshead minnows (Cyprinodon variegatus) were placed into separate aquaria for seawater control, ethanol solvent control, and the following androgen agonist treatments at 5.0µg/L: dihydrotestosterone (DHT), methyldihydrotestosterone (MDHT), testosterone (T), methyltestosterone (MT) and trenbolone (TB). Treatments of 0.6µg/L endosulfan and 40µg/L chlorpyrifos (CP) served as non-androgenic negative stressor controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus supplying exposure water at 20L/h. Fish were sampled at 7 days, the plasma diluted, processed on weak cation exchange CM10 ProteinChip arrays and analyzed. Spectral processing resulted in 249 individual m/z peak clusters for the androgen exposed fish. Partial least squares-discriminant analysis was used to develop an androgen-responsive model using sample spectra from exposures with DHT and unexposed solvent control fish as the training set. The androgen classification model performed with ≥79% specificity (% true negative) and ≥70% sensitivity (% true positive) for non-aromatizable androgens. The aromatizable androgens T and MT were classified as androgenic with specificities of 42 and 79%, respectively. The reduction in sensitivity observed with T is thought to be caused by its metabolic conversion to an estrogen by aromatase. The results of these studies show diagnostic plasma protein expression models can correctly classify chemicals by their androgenic activity using a combination of high throughput mass spectrometry and multivariate approaches.

Multigenerational exposure of the estuarine sheepshead minnow (Cyprinodon variegatus) to 17 ß-estradiol. I. organism-level effects over three generations.

A 280-d study examined the effects of 17ß-estradiol (E2) on reproduction and development of the sheepshead minnow (Cyprinodon variegatus) exposed from the parental (F0) through three subsequent (F1, F2, and F3) generations and evaluated the need for multigenerational assessments of the risks of endocrine-disrupting chemicals. This first three-generation study exposed adult F0 and F1 fish to measured concentrations of 0.01, 0.04, 0.08, 0.2, and 0.3 µg E2/L; the F2 and F3 generations were exposed to 0.2 µg E2/L or less. The cumulative 21-d production of normal embryos was significantly reduced in the F0 generation at 0.3 µg E2/L and in the F1 and F2 generations at 0.08 µg E2/L or more. The daily reproductive rate was significantly reduced in all three generations at 0.08 µg E2/L or more during spawning days 8 to 14 and 15 to 21. The proportion of infertile eggs from F1 fish was significantly increased above that of the solvent controls at 0.04 and 0.2 µg E2/L and from F2 fish at 0.04 µg E2/L or more. Changes in liver, kidney, and gonadal tissues were seen in the F0 and F1 generations exposed to 0.2 µg E2/L or more. The female gonadosomatic index was significantly decreased at 0.3 µg E2/L in the F0 and F1 generations. Estradiol affected the hepatosomatic index only in female F1 fish, but not in a dose-dependent manner. All F1 fish in 0.3 µg E2/L appeared to be phenotypically female. Our results indicate that life-cycle exposure to E2 significantly decreased embryo production by F1 and F2 fish at concentrations lower than those affecting the F0 generation, and they emphasize the importance of evaluating the impact of an estrogenic chemical on reproduction through a minimum of two (F0 and F1) generations.

Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17beta-trenbolone.

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5m/z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows (Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21-day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17beta-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 microg/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 microg/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 microg/l treatment level which corresponded with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 microg/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.

A simple and rapid matrix-assisted laser desorption/ionization time of flight mass spectrometry method to screen fish plasma samples for estrogen-responsive biomarkers.

In the present study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry coupled with a short-term fish assay. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria consisting of vehicle control and the following estrogen agonist treatments: 17beta-estradiol (0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.5, and 1.0 microg/L, 4-tert-pentylphenol (100 microg/L), methoxychlor (6 and 12 microg/L), and bisphenol A (100 and 1,000 microg/L). Treatments with chlorpyrifos (80 microg/L) and endosulfan (0.6 microg/L) served as nonestrogenic negative controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus. Plasma was obtained from individuals, diluted and applied to an inert surface, and analyzed by MALDI. Multiple protein peaks, ranging from 2.9 to 12.9 kDa, were identified as markers of estrogenic effects when comparing estrogen-treated and control fish using interpercentile reference values. A binary classification tree model was constructed from plasma protein profiles of the vehicle control and the 0.2 microg/L of 17beta-estradiol treatments and then used to evaluate all samples. Treatments with the estrogen agonists 17beta-estradiol, 4-tert-pentylphenol, methoxychlor, and bisphenol-A generated reproducible diagnostic biomarkers based on the presence of specific estrogen-responsive plasma proteins. The controls and nonestrogenic compounds chlorpyrifos and endosulfan did not produce this estrogen-responsive protein profile. A no-observed-effect level for 17beta-estradiol at 0.025 microg/L was estimated from concentration-response exposures. The MALDI method described here provides a straightforward, sensitive, and specific tool to screen chemicals for estrogenic activity.

A proteomic (SELDI-TOF-MS) approach to estrogen agonist screening.

A small fish model and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 17beta-estradiol (E2), methoxychlor (MXC), bisphenol-A (BPA), 4-tert-pentylphenol (TPP), endosulfan (ES), and chlorpyriphos (CP). Fish plasma was applied to weak cation exchange (CM10) ProteinChip arrays, processed, and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen-responsive mass spectral biomarker peaks were identified by comparison of E2-treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1 to 13 kDa were up- or downregulated in E2-treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist, and nonagonist stressor-treated males and normal female fish plasma. One of the biomarkers, mass-to-charge ratio 3025.5, was identified by high-resolution tandem mass spectrometry as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens MXC, BPA, and TPP elicited protein expression profiles consistent with the estrogen expression model. Estrogen-responsive peaks were not detected in plasma from fish in the seawater, vehicle, ES, or CP treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Temporal gene induction patterns in sheepshead minnows exposed to 17beta-estradiol.

Gene arrays provide a powerful method to examine changes in gene expression in fish due to chemical exposures in the environment. In this study, we expanded an existing gene array for sheepshead minnows (Cyprinodon variegatus) (SHM) and used it to examine temporal changes in gene expression for male SHM exposed to 100 ng 17beta-estradiol (E(2))/L for five time points between 0 and 48 hr. We found that in addition to the induction of genes involved in oocyte development (vitellogenin [VTG], zona radiata [ZRP]), other genes involved in metabolism and the inflammatory response are also affected. We identified five patterns of temporal induction in genes whose expression was modified due to E(2) exposure. We validated the gene array data for the expression of VTG 1, VTG 2, ZRP 2 and ZRP 3 and found that with low levels of exogenous E(2) (100 ng E(2)/L) exposure, ZRP expression precedes VTG expression. However, at higher concentrations of E(2) (500 ng E(2)/L), the difference in temporal expression appears to be lost. Exposure to high levels of environmental contaminants may affect the normal ordered expression of genes required for reproduction. Gene expression profiling using arrays promises to be a valuable tool in the field of environmental toxicology. As more genes are identified for species used in toxicological testing, researchers will be better able to predict adverse effects to chemical exposures and to understand the relationships between changes in gene expression and changes in phenotype.

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus).

Several genes normally induced by estradiol (E(2)) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E(2) and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E(2) and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.

Expression profiling of estrogenic compounds using a sheepshead minnow cDNA macroarray.

A variety of anthropogenic compounds are capable of binding to the estrogen receptor (ER) of vertebrate species. Binding of these chemicals to the ER can interfere with homeostasis by altering normal gene expression patterns. The purpose of this study was to characterize the expression of 30 genes using a sheepshead minnow (Cyprinodon variegatus) cDNA macroarray. Many of the genes on the array were previously identified by differential display reverse transcriptase-polymerase chain reaction to be upregulated or downregulated in sheepshead minnows treated through aqueous exposure to known or suspected estrogenic chemicals. The results of this study show that 17 beta-estradiol (E2), 17 alpha-ethinyl estradiol (EE2), diethylstilbestrol (DES), and methoxychlor (MXC) have similar genetic signatures for the 30 genes examined. The genetic signature of fish treated with p-nonylphenol was identical in pattern to that in fish treated with E2, EE2, DES, and MXC except for the additional upregulation of a cDNA clone that shares similarity to ubiquitin-conjugating enzyme 9. Endosulfan produced results that resembled the gene expression patterns of untreated control fish with exception of the upregulation of estrogen receptor alpha and the downregulation of a cDNA clone that shares similarity to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. We show that our estrogen-responsive cDNA macroarray can detect dose-dependent changes in gene expression patterns in fish treated with EE2.

Array technology as a tool to monitor exposure of fish to xenoestrogens.

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).

A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro.

Five natural, pharmaceutical, or xenobiotic chemicals [17beta-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol (NP)] were tested in two in vitro assays [yeast estrogen screen (YES), MCF-7 breast tumor cell proliferation (E-Screen)], and compared with previously reported results from two in vivo male sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 10(7) times less potent than E2 in the YES assay, MXC was 10(5) times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, (Cyprinodon variegatus) after cessation of exposure to 17 beta-estradiol and p-nonylphenol.

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after cessation of exposure to either 17 beta-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 microg E2 per l, and 5.6 and 59.6 microg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2-4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 microg NP per l treatment could not determined. In the 0.089, 0.71 microg E2 per l, and 59.6 microg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 microg E2 per l and 59.6 microg NP per l were not significantly different (P=0.47), however, both demonstrated significantly higher rates of clearance (P<0.02) than observed in the 0.089 microg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.