April Feswick

Distribution of silver nanoparticles in pregnant mice and developing embryos

Abstract The objective of this study was to evaluate the distribution of silver nanoparticles (NPs) in pregnant mice and their developing embryos. Silver NPs (average diameter 50 nm) were intravenously injected into pregnant CD-1 mice on gestation days (GDs) 7, 8, and 9 at dose levels of 0, 35, or 66 μg Ag/mouse. Mice were euthanised on GD10, and tissue samples were collected and analysed for silver content. Compared with control animals injected with citrate buffer vehicle, silver content was significantly increased (p < 0.05) in nearly all tissues from silver NP-treated mice. Silver accumulation was significantly higher in liver, spleen, lung, tail (injection site), visceral yolk sac, and endometrium compared with other organs from silver NP-treated mice. Furthermore, silver NPs were identified in vesicles in endodermal cells of the visceral yolk sac. In summary, the results demonstrated that silver NPs distributed to most maternal organs, extra-embryonic tissues, and embryos, but did not accumulate significantly in embryos.

Uptake, retention and internalization of quantum dots in Daphnia is influenced by particle surface functionalization.

Nanomaterials are a diverse group of compounds whose inevitable release into the environment warrants study of the fundamental processes that govern the ingestion, uptake and accumulation in aquatic organisms. Nanomaterials have the ability to transfer to higher trophic levels in aquatic ecosystems, and recent evidence suggests that the surface chemistry of both the nanoparticle and biological membrane can influence uptake kinetics. Therefore, our study investigates the effect of surface functionalization on uptake, internalization and depuration in Daphnia spp. Uncharged (polyethylene glycol; PEG), positively charged (amino-terminated: NH2) and negatively charged (carboxyl-modified; COOH) cadmium selenide/zinc sulfide quantum dots were used to monitor ingestion, uptake and depuration of nanometals in Daphnia magna and Ceriodaphnia dubia over 24h of exposure. These studies demonstrated that particles with higher negative charge (COOH quantum dots) were taken up to a greater extent by Daphnia (259.17±17.70 RFU/20 Daphnia) than either the NH2 (150.01±18.91) or PEG quantum dots (95.17±9.78), however this is likely related to the functional groups attached to the nanoparticles as there were no real differences in zeta potential. Whole body fluorescence associates well with fluorescent microscopic images obtained at the 24h timepoint. Confocal and electron microscopic analysis clearly demonstrated that all three types of quantum dots could cross the intestinal epithelial barrier and be translocated to other cells. Upon cessation of exposure, elimination of all three materials was biphasic with rapid initial clearance that likely represents elimination of material remaining in the GI tract followed by a much slower elimination phase that likely represents elimination of internalized material. These studies demonstrate that daphnids can take up intact nanomaterial from the water column and that this uptake is strongly influenced by particle surface functionalization. In addition, the usefulness of using quantum dots as a proxy for other nanometals (no acute toxicity, clear visualization in electron microscopy), in conjunction with several different imaging techniques in assessing uptake and accumulation of nanoparticles in daphnids was demonstrated.

Effects of acute dieldrin exposure on neurotransmitters and global gene transcription in largemouth bass (Micropterus salmoides) hypothalamus

Exposure to dieldrin induces neurotoxic effects in the vertebrate CNS and disrupts reproductive processes in teleost fish. Reproductive impairment observed in fish by dieldrin is likely the result of multiple effects along the hypothalamic-pituitary-gonadal axis, but the molecular signaling cascades are not well characterized. To better elucidate the mode of action of dieldrin in the hypothalamus, this study measured neurotransmitter levels and examined the transcriptomic response in female largemouth bass (LMB) to an acute treatment of dieldrin. Male and female LMB were injected with either vehicle or 10 mg dieldrin/kg and sacrificed after 7 days. There were no significant changes in dopamine or DOPAC concentrations in the neuroendocrine brain of males and females after treatment but GABA levels in females were moderately increased 20-30% in the hypothalamus and cerebellum. In the female hypothalamus, there were 227 transcripts (p<0.001) identified as being differentially regulated by dieldrin. Functional enrichment analysis revealed transcription, DNA repair, ubiquitin-proteasome pathway, and cell communication, as biological processes over-represented in the microarray analysis. Pathway analysis identified DNA damage, inflammation, regeneration, and Alzheimers disease as major cell processes and diseases affected by dieldrin. Using multiple bioinformatics approaches, this study demonstrates that the teleostean hypothalamus is a target for dieldrin-induced neurotoxicity and provides mechanistic evidence that dieldrin activates similar cell pathways and biological processes that are also associated with the etiology of human neurological disorders.

Queen conch (Strombus gigas) testis regresses during the reproductive season at nearshore sites in the Florida Keys.

Background Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. Methodology/Principal Findings Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. Conclusions/Significance Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure.

Transcriptomic profiling of progesterone in the male fathead minnow (Pimephales promelas) testis.

P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin.

Defining the role of omics in assessing ecosystem health: Perspectives from the Canadian environmental monitoring program

Scientific reviews and studies continue to describe omics technologies as the next generation of tools for environmental monitoring, while cautioning that there are limitations and obstacles to overcome. However, omics has not yet transitioned into national environmental monitoring programs designed to assess ecosystem health. Using the example of the Canadian Environmental Effects Monitoring (EEM) program, the authors describe the steps that would be required for omics technologies to be included in such an established program. These steps include baseline collection of omics endpoints across different species and sites to generate a range of what is biologically normal within a particular ecosystem. Natural individual variability in the omes is not adequately characterized and is often not measured in the field, but is a key component to an environmental monitoring program, to determine the critical effect size or action threshold for management. Omics endpoints must develop a level of standardization, consistency, and rigor that will allow interpretation of the relevance of changes across broader scales. To date, population-level consequences of routinely measured endpoints such as reduced gonad size or intersex in fish is not entirely clear, and the significance of genome-wide molecular, proteome, or metabolic changes on organism or population health is further removed from the levels of ecological change traditionally managed. The present review is not intended to dismiss the idea that omics will play a future role in large-scale environmental monitoring studies, but rather outlines the necessary actions for its inclusion in regulatory monitoring programs focused on assessing ecosystem health.

Progesterone increases ex vivo testosterone production and decreases the expression of progestin receptors and steroidogenic enzymes in the fathead minnow (Pimephales promelas) ovary

Progesterone (P4) is a metabolic precursor for a number of steroids, including estrogens and androgens. P4 also has diverse roles within the vertebrate ovary that include oocyte growth and development. The objectives of this study were to measure the effects of P4 on testosterone (T) and 17β-estradiol (E2) production in the fathead minnow (FHM) ovary and on the mRNA abundance of transcripts involved in steroidogenesis and steroid receptor signaling. Ovary explants were treated with P4 (10(-6)M) for 6 and 12h. P4 administration significantly increased T production ∼3-fold at both 6 and 12h, whereas E2 production was not affected, consistent with the hypothesis that excess P4 is not converted to terminal estrogens in the mature ovary. Nuclear progesterone receptor mRNA was decreased at 6h and membrane progesterone receptor gamma-2 mRNA was significantly down-regulated at both 6 and 12h; however there was no change in membrane progesterone receptor alpha or beta mRNA levels. Androgen receptor (ar) and estrogen receptor 2a (esr2a) mRNA were significantly reduced at 6h with P4 treatment, but there was no change in esr2b mRNA at either time point. Transcripts for enzymes in the steroid pathway (star, hsd11b2) were significantly lower at 6h compared to controls, whereas cyp17a and cyp19a mRNA abundance did not change with treatments at either time point. These data suggest that P4 incubation can lead to increased T production in the FHM ovary without a concomitant change in E2, and that the membrane bound progestin receptors are differentially regulated by P4 in the teleost ovary. As environmental progestins have received increased attention due to their suspected role as endocrine disruptors, mechanistic data on the role of exogenous P4 treatments in the male and female gonad is warranted.

Transcriptomics profiling and steroid production in mummichog (Fundulus heteroclitus) testes after treatment with 5α-dihydrotestosterone

5α-Dihydrotestosterone (DHT) is a potent androgen in mammals with multiple roles; however the physiological actions of DHT in male fishes are not well known. To address this knowledge gap, male mummichog (Fundulus heteroclitus) were continuously exposed to 0, 5, and 50 μg/L DHT for 21 days. Following exposure, testes were separated for histology, ex vivo incubation to measure steroidogenic capacity, and gene expression analyses (real-time PCR and microarray). DHT significantly decreased ex vivo 11-ketotestosterone (11KT) production in males exposed to 50 μg/L DHT but not 5 μg/L DHT, and DHT exposure did not affect ex vivo testosterone production. Histological examination revealed that the amount of interlobular and connective tissue present in the testes was increased in the 50 μg/L DHT treatment. Despite reductions in the production of 11KT, DHT did not affect the expression of targeted genes in the steroidogenic pathway such as steroidogenic acute regulatory protein (star), P450 side chain cleavage (cyp11a1) and 11β-hydroxysteroid dehydrogenase (hsd11b3). Microarray analysis in the testes of individuals from control and 50 μg/L DHT revealed that males exposed to 50 μg/L DHT showed regulated transcriptional sub-networks that were related to immunity, regulation of blood flow, lipids and xenobiotic clearance, suggesting that DHT may be involved in the physiological regulation of these processes in the fish testes. A second objective of this study was to determine the feasibility of measuring mRNA levels in tissues used for ex vivo steroid production by comparing RNA integrity and transcript levels in testes of both immediately flash frozen tissue and incubated tissue. There was no significant difference in RNA quality between the two time points, indicating RNA integrity can remain intact for at least 18 h in ex vivo assays, thereby providing a viable option for researchers assessing multi-level biological reproductive endpoints when limited tissue is available. While the gene expression levels of actb, efla, rps12, rps18, star, and hsd11b3 remained unchanged, esr2a (esrba), esr2b (esrbb) and cyp11a1 were significantly lower in incubated tissue compared to flash frozen tissue. Therefore caution must be used as the steady-state levels of select genes may change over time. This study improves our understanding of DHT action in the teleostean testis and generates new hypotheses regarding cell processes that are regulated by this underexplored and potent androgen.

Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells.

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.

Investigation of acute nanoparticulate aluminum toxicity in zebrafish.

In freshwater fish, aluminum is a well‐recognized gill toxicant, although responses are influenced by pH. Aluminum nanomaterials are being used in diverse applications that are likely to lead to environmental release and exposure. However, it is unclear if the effects of nanoparticulate aluminum are similar to those of other forms of aluminum or require special consideration. To examine the acute toxicological effects of exposure to aluminum nanoparticle (Al‐NP)s, adult female zebrafish were exposed to either Al‐NPs or aluminum chloride for up to 48 hours in moderately hard fresh water. Al‐NPs introduced into test water rapidly aggregated and up to 80% sedimented from the water column during exposures. No mortality was caused by concentrations of Al‐NP up to 12.5 mg/L. After exposure, tissue concentrations of aluminum, effects on gill morphology, Na+, K+ ‐ATPase (NKA) activity, and global gene expression patterns were examined. Exposure to both aluminum chloride and nanoparticulate aluminum resulted in a concentration dependent decrease in sodium potassium ATPase activity, although Al‐NP exposure did not alter gill morphology as measured by filament widths. Decreased ATPase activity coincided with decreases in filamental NKA staining and mucous cell counts. Analysis of gill transcriptional responses demonstrated that exposure to 5 mg/L Al‐NP only resulted in significant changes in expression of two genes, whereas aluminum chloride exposure significantly affected the expression of 105 genes. Taken together, these results indicate that nanoparticulate aluminum has little acute toxicity for zebrafish in moderately hard freshwater.