Transcriptional changes in innate immunity genes in head kidneys from Aeromonas salmonicida-challenged rainbow trout fed a mixture of polycyclic aromatic hydrocarbons.

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82µg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.

Growth inhibition and stimulation of Shewanella oneidensis MR-1 by surfactants and calcium polysulfide.

Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 µM were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS >> CPS >> NINOL 40-CO>SLES≥CAPB. Dose dependent growth decreases (20-100mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45-7.25 mM CPS). Both SLES (20-100mM) and SDS at lower concentrations (20-500 µM) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface microorganisms. This benchtop system provides a capability to assess adverse microbial-remediation responses and contributes to the development of in situ remedial chemistries before they are deployed in the field.

BDE 49 and developmental toxicity in zebrafish.

The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2',4,5'-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4iµ-32 µM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to aquatic species.

Transcriptional impact of organophosphate and metal mixtures on olfaction: copper dominates the chlorpyrifos-induced response in adult zebrafish.

Chemical exposures in fish have been linked to loss of olfaction leading to an inability to detect predators and prey and decreased survival. However, the mechanisms underlying olfactory neurotoxicity are not well characterized, especially in environmental exposures which involve chemical mixtures. We used zebrafish to characterize olfactory transcriptional responses by two model olfactory inhibitors, the pesticide chlorpyrifos (CPF) and mixtures of CPF with the neurotoxic metal copper (Cu). Microarray analysis was performed on RNA from olfactory tissues of zebrafish exposed to CPF alone or to a mixture of CPF and Cu. Gene expression profiles were analyzed using principal component analysis and hierarchical clustering, whereas gene set analysis was used to identify biological themes in the microarray data. Microarray results were confirmed by real-time PCR on genes serving as potential biomarkers of olfactory injury. In addition, we mined our previously published Cu-induced zebrafish olfactory transcriptional response database (Tilton et al., 2008) for the purposes of discriminating pathways of olfaction impacted by either the individual agents or the CPF-Cu mixture transcriptional signatures. CPF exposure altered the expression of gene pathways associated with cellular morphogenesis and odorant binding, but not olfactory signal transduction, a known olfactory pathway for Cu. The mixture profiles shared genes from the Cu and CPF datasets, whereas some genes were altered only by the mixtures. The transcriptional signature of the mixtures was more similar to that in zebrafish exposed to Cu alone than for CPF. In conclusion, exposure to a mixture containing a common environmental metal and pesticide causes a unique transcriptional signature that is heavily influenced by the metal, even when organophosphate predominates.

Swimming impairment and acetylcholinesterase inhibition in zebrafish exposed to copper or chlorpyrifos separately, or as mixtures.

Pesticides such as chlorpyrifos (CPF) and metals such as copper can impair swimming behavior in fish. However, the impact to swimming behavior from exposure to mixtures of neurotoxicants has received little attention. In the current study, we analyzed spontaneous swimming rates of adult zebrafish (Danio rerio) to investigate in vivo mixture interactions involving two chemical classes. Zebrafish were exposed to the neurotoxicants copper chloride (CuCl, 0.1 µM, 0.25 µM, 0.6 µM, or 6.3, 16, 40 ppb), chlorpyrifos (CPF, 0.1 µM, 0.25 µM, 0.6 µM, or 35, 88, 220 ppb) and binary mixtures for 24h to better understand the effects of Cu on CPF neurotoxicity. Exposure to CPF increased the number of animals undergoing freeze responses (an anti-predator behavior) and, at the highest CPF dose (0.6 µM), elicited a decrease in zebrafish swimming rates. Interestingly, the addition of Cu caused a reduction in the number of zebrafish in the CPF exposure groups undergoing freeze responses. There was no evidence of additive or synergistic toxicity between Cu and CPF. Although muscle AChE activity was significantly reduced by CPF, there was a relatively poor relationship among muscle AChE concentrations and swimming behavior, suggesting non-muscle AChE mechanisms in the loss of swimming behavior. In summary, we have observed a modulating effect of Cu on CPF swimming impairment that appears to involve both AChE and non-AChE mechanisms. Our study supports the utility of zebrafish in understanding chemical mixture interactions and neurobehavioral injury.

Sulfhydryl systems are a critical factor in the zebrafish developmental toxicity of the dithiocarbamate sodium metam (NaM).

Dithiocarbamates (DTCs) are sulfhydryls (thiol)-containing compounds, often associated with metals, and have both antioxidant and pro-oxidant abilities depending on the compound, experimental system and condition. In this study we investigated whether cell death plays a role in the manifestation of DTC-induced notochord distortions in the developing zebrafish and if thiol-containing compounds or antioxidants could modify this developmental toxicity. Sodium metam (NaM) induced notochord distortions could not be protected with the antioxidants ascorbic acid, trolox (synthetic vitamin E) or lipoic acid. However, NaM-induced distortions could be protected with co-exposure to glutathione or N-Acetyl Cysteine. Staggering the NaM and glutathione exposures in consecutive 10h developmental windows also resulted in protection. There were no discernable changes in TUNEL positive cells, a marker of apoptotic cells, at 24h post-fertilization (hpf) in NaM, dimethyl-dithiocarbamate, carbon disulfide, or neocuproine exposed embryos. Live NaM-exposed embryos incubated with acridine orange, a general stain for cell death, for 1h beginning at 11, 18 and 24hpf showed clusters of stained nuclei near the somitogenic front but not in the cells making up the notochord. Overall, induction of apoptotic pathways and widespread cell death are not involved in the manifestation of the adverse developmental outcomes following NaM exposure. However, cellular thiol status or critical sulfhydryl moieties are important considerations in the mechanisms of DTC developmental toxicity.

Exposure to sodium metam during zebrafish somitogenesis results in early transcriptional indicators of the ensuing neuronal and muscular dysfunction.

Exposures to sodium metam (NaM) within the developmental period of somitogenesis (10- to 18-h postfertilization [hpf]) results in easily detectable distortions of the notochord by 24 hpf in the developing zebrafish. We hypothesized that NaM-induced transcriptional changes during somitogenesis would reveal the major molecular targets in the zebrafish embryo. Embryos were exposed to NaM beginning at 4 hpf (1000 cells) and total RNA was isolated from embryos at the 3 somite (11 hpf), 10 somite (14 hpf), 18 somite (18 hpf), and larval (24 hpf) stages of development. Using the Affymetrix zebrafish gene array we observed relatively few mRNAs differentially regulated at least twofold at each time point (11 hpf, 101 genes; 14 hpf, 151; 18 hpf, 154; 24 hpf, 33). The transcriptional profiles reveal neurodevelopment and myogenesis as the two primary targets of NaM developmental exposure. Quantitative PCR of several muscle and neuronal genes confirmed the array response. We also followed the structural development of the peripheral nervous system under NaM exposure using antibodies against neuronal structural proteins. Although there was no change in the onset of antibody staining, profound alterations became apparent during the period in which the notochord becomes distorted (> 18 hpf). Motor neuron development observed with the Tg(NBT:MAPT-GFP)zc1 transgenic zebrafish and a primary motor neuron specific antibody showed similar timing in the structural alterations observed in these cell types. Further study of the interactions of dithiocarbamates with the regulatory elements of fast muscle development and neurodevelopment is warranted.

Transcriptional biomarkers and mechanisms of copper-induced olfactory injury in zebrafish.

Metals such as copper disrupt olfactory function in fish. Unfortunately, little is understood of the molecular consequences of copper olfactory impairment, thus hindering the development of relevant diagnostic tools of olfactory injury. To address this critical data gap, we analyzed gene expression within olfactory tissues of adult zebrafish exposed to CuCl2 (6, 16, 40 ppb) for 24 h. Transcriptional markers of copper impairment within the entire olfactory system were identified and specific genes of interest (e.g., S100a, parvalbumin 8, olfactory marker protein, and calbindin 2-like protein) were confirmed with quantitative real-time PCR. In addition, we performed gene set analysis (GSA) using both a priori and custom pathways of gene sets specifically targeting the olfactory signal transduction (OST) pathway. These analyses revealed down-regulated gene sets related to calcium channels and ion transport, g-proteins, and olfactory receptors. Collectively, these data demonstrate that copper causes a depression of transcription of key genes within the OST pathway and elsewhere within olfactory tissues, likely resulting in an olfactory system less responsive to odorants. Further, these data provide a mechanistic explanation in support of earlier studies of functional olfactory impairment in fish following copper exposure.

Dithiocarbamates have a common toxic effect on zebrafish body axis formation.

We previously determined that the dithiocarbamate pesticide sodium metam (NaM) and its active ingredient methylisothiocyanate (MITC) were developmentally toxic causing notochord distortions in the zebrafish. In this study, developing zebrafish were exposed to isothiocyanates (ITCs), dithiocarbamates (DTCs) and several degradation products to determine the teratogenic relationship of these chemical classes at the molecular level. All dithiocarbamates tested elicited notochord distortions with notochord NOELs from <4 to 40 ppb, while none of the ITCs caused notochord distortions with the exception of MITC. Carbon disulfide (CS(2)), a common DTC degradate, also caused distortions at concentrations >200 times the DTCs. Whole mount in situ hybridization of developmental markers for collagen (collagen2a1), muscle (myoD), and body axis formation (no tail) was perturbed well after cessation of treatment with pyrolidine-DTC (PDTC), dimethyl-DTC (DMDTC), NaM, MITC, and CS(2). Therefore, distinct albeit related chemical classes share a common toxic effect on zebrafish notochord development. To test the responsiveness of the distortion to metal perturbation, five metal chelators and 2 metals were studied. The membrane permeable copper chelator neocuproine (NCu) was found to cause notochord distortions similar to DTC-related molecules. DMDTC and NCu treated animals were protected with copper, and collagen 2a1 and no tail gene expression patterns were identical to controls in these animals. PDTC, NaM, MITC, and CS(2) were not responsive to copper indicating that the chelation of metals is not the primary means by which these molecules elicit their developmental toxicity. Embryos treated with DMDTC, NaM, and NCu were rescued by adding triciaine (MS-222) which abolishes the spontaneous muscle contractions that begin at 18 hpf. In these animals, only collagen 2a1 expression showed a similar pattern to the other notochord distorting molecules. This indicates that the perturbation of no tail expression is in response to the muscle contractions distorting the notochord, while collagen 2a1 is associated with the impact of these molecules on much earlier developmental processes.

Environmental stresses and skeletal deformities in fish from the Willamette River, Oregon.

The Willamette River, one of 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of the Willamette River detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (Newberg pool [NP], rivermile [RM] 26 - 55) and middle (Wheatland Ferry [WF], RM 72 - 74) river, relative to those in the upper river (Corvallis [CV], RM 125-138). The objective of this study was to determine the likely cause of these skeletal deformities. In 2002 and 2003, deformity loads in Willamette River fishes were 2-3 times greater at the NP and WF locations than at the CV location. There were some differences in water quality parameters between the NP and CV sites, but they did not readily explain the difference in deformity loads. Concentrations of bioavailable metals were below detection limits (0.6 - 1 microg/ L). Concentrations of bioavailable polychlorinated biphenyls (PCBs) and chlorinated pesticides were generally below 0.25 ng/L. Concentrations of bioavailable polycyclic aromatic hydrocarbons were generally less than 5 ng/L. Concentrations of most persistent organic pollutants were below detection limits in ovary/oocyte tissue samples and sediments, and those that were detected were not significantly different among sites. Bioassay of Willamette River water extracts provided no evidence that unidentified compounds or the complex mixture of compounds present in the extracts could induce skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large percentage of deformities detected in two Willamette River fishes, and similar deformities were reproduced in laboratoryfathead minnows exposed to cercariae extracted from Willamette River snails. Thus, the weight of evidence suggests that parasitic infection, not chemical contaminants, was the primary cause of skeletal deformities observed in Willamette River fish.

Developmental toxicity of the dithiocarbamate pesticide sodium metam in zebrafish.

Sodium metam (NaM), a dithiocarbamate, is a general agricultural biocide applied prior to planting for the elimination of nematodes, soil pathogens, and weeds. There is a remarkable paucity of information about the mechanism of action and the risk that dithiocarbamates may pose to developing vertebrates. We have characterized NaM toxicity during early life stage exposure in zebrafish. Zebrafish embryos are most sensitive to NaM exposure during gastrulation and early segmentation (4-14 hours post fertilization, hpf). For mortality, the dose response curve is steep with an LC(50) estimate of 1.95 microM (248 ppb) at 48 hpf. The most notable malformation among surviving embryos was a severely twisted notochord, which became evident by 24 hpf. Surprisingly, this notochord defect was not immediately lethal and the animals continued to grow despite delays in hatching, apparent paralysis, and an inability to feed. We have characterized the notochord malformation using histological and in situ hybridization techniques. collagen 2a1 mRNA expression is normally localized to the notochord sheath cells at 24 hpf, whereas in NaM-exposed embryos it is misexpressed in the notochord cells. Histological staining and myoD expression indicate that the myotomes of the NaM-exposed embryos are less defined, compacted and block-shaped compared to controls. The degradation product of NaM, methyl isothiocyanate (MITC), causes similar malformations at similar concentrations as NaM, suggesting that MITC or another common product may be the active toxicant. Our results indicate that developing zebrafish are sensitive to NaM and MITC and we believe that this model is ideal to elucidate the molecular mechanism(s) and etiology of NaM toxicity in vertebrates.

Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input.

The purpose of this study was to survey estrogenic releases from two primarily domestic wastewater treatment plants over three seasons (1996-1999). Mature male channel catfish were maintained at two sites within each WWTP and a reference site for 21 days. Estrogenic activity of effluent was assessed by the Yeast Estrogen Screen (YES) assay (in 1999) and the expression of the female egg yolk precursor protein, vitellogenin (Vtg) in caged male channel catfish (1996-1998). Serum Vtg of animals exposed at WWTP-A was induced 220% above reference values in the Fall of 1996 and 480% in Spring of 1997. In animals exposed to effluent of WWTP-B, serum Vtg was elevated 370% in Spring of 1997 and 480% in Fall of 1997 relative to fish held in a reference location. Serum 17-beta-estradiol (E2) levels were also significantly elevated 13 and 16-fold in the Fall 1997 and Summer 1998 in the fish exposed to WWTP-A effluent. A 13.5-fold increase in serum E2 was observed in fish exposed to WWTP-B during Fall 1997. Utilizing an E2 concentration-Vtg response curve generated in the laboratory, effluent from both plants (in 1997 and 1998) had estrogen equivalent values ranging from 23 to 123 ng/l E2 equivalents. These values were comparable with YES values obtained from 1999, which indicated the presence of 21 to 147 ng/l E2 equivalents. E2 was responsible for 3 (fall) to 100% (summer) of the YES activity. Glucuronides of E2 were also observed in the treated effluent. These studies indicate that variable estrogenic activity is present in municipal wastewater resulting from domestic activities and that this activity may be significantly altered by environmental factors.