Poultry litter-induced endocrine disruption in fathead minnow, sheepshead minnow, and mummichog laboratory exposures.

Animal feeding operations in the United States produce more than 500 million tons of manure annually. Disposal of poultry waste via application as fertilizer results in substantial runoff of poultry litter-associated contaminants (PLAC). Of particular concern are sex steroids, 17ß-estradiol, estrone and testosterone, responsible for sex differentiation and development of reproductive structures. In a series of laboratory assays, mature male and mixed-sex larval/juvenile fish were continuously exposed to environmentally relevant PLAC solutions. Effects on gonads were assessed histologically, and vitellogenin (VTG) induction was measured as a gauge of estrogenicity. Twenty-one-day exposures to laboratory-generated PLAC solutions routinely induced VTG in mature male Pimephales promelas. Vitellogenesis in Fundulus heteroclitus only occurred at the highest tested PLAC concentration, and Cyprinodon variegatus were unresponsive at any tested concentration. All species produced considerable VTG in response to a 17ß-estradiol-positive control. A pronounced feminization was seen in P. promelas when exposed to PLAC as larvae but not when exposed as juveniles. Runoff from a poultry litter-amended field cropped under standard agronomic practices induced significant VTG in male P. promelas. Results indicate that environmentally relevant PLAC concentrations exhibit endocrine activity sufficient to induce VTG production in male fish and possibly affect sex ratios in resident fish populations.

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro.

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Bioindicators of immune function in creosote-adapted estuarine killifish, Fundulus heteroclitus.

Several populations of the estuarine killifish, Fundulus heteroclitus, also known as the mummichog, exhibit characteristics of adaptation to priority pollutants. One such population of mummichog inhabits the Elizabeth River in Virginia at the Atlantic Wood site (AW), a U.S. Environmental Protection Agency (EPA) Superfund site heavily contaminated with creosote containing a mixture of polyaromatic hydrocarbons (PAHs). Although PAHs are known to be immunotoxic in experimental animals, resident AW mummichogs seem to thrive. Mummichogs from the AW site and a reference site were subsequently examined over a 2-yr period for total immunoglobin (IgM), as well as circulating antibody levels against 5 ubiquitous marine bacteria. Expression profiles of circulating and lymphoid lysozyme and lymphoid cyclooxygenase-2 (COX-2) were also examined. Compared to relatively high total IgM and specific antibody responses in reference fish, AW mummichogs had lower circulating IgM and lower specific antibody levels against all bacteria examined, however they had higher levels of circulating lysozyme. Lymphoid cells in the AW mummichogs also expressed higher levels of lysozyme, as well as COX-2, which may indicate a state of macrophage activation. Elevated COX-2 levels may be associated with enhanced metabolism of PAHs through cooxidation-peroxidase pathways. Additional studies attempted to immunize AW mummichogs reared in uncontaminated water to compare their antibody responses to that of reference fish. AW mummichogs did not survive 40 d post culture, while reference fish thrived. Our findings suggest that the chemical environment at the AW site may be vicariously enhancing components of innate immunity, probably through oxidative stress pathways, in resident mummichogs, while actively suppressing humoral immune responses.

Induction of CYP1A and DNA damage in the fathead minnow (Pimephales promelas) following exposure to biosolids.

Biosolids (treated sewage sludge) are increasingly disposed of on land. Thus particle-sorbed and dissolved constituents have the potential to enter nearby watersheds. Although organic contaminants are known to be present in biosolids these are not currently regulated and little data exist on their potential toxicity to aquatic organisms. We exposed Pimephales promelas to two concentrations of biosolids (0.5 and 2.5 g l(-1)) for 28-days (static-renewal) and characterized contaminants present and the extent of CYP1A and DNA damage induction at various time points. Many organic contaminants were detected in the biosolids, with polycyclic aromatic hydrocarbons (PAHs) being the dominant class. Substantial levels of polybrominated diphenyl ethers (PBDEs) and nonylphenols (NPs) were also present. Significant induction of hepatic CYP1A protein compared with controls (P<0.05) was observed in both low (0.5 g l(-1)) and high (2.5 g l(-1)) exposed fish from Day 7. CYP1A levels peaked at Day 21 with 21-fold and 8-fold inductions over controls in high and low dose fish respectively. Induction of DNA damage in hepatocytes (single strand breaks as measured using the COMET assay) was observed in both exposures compared with controls on Days 14 and 28 (P<0.05). A significant correlation was found between CYP1A induction and DNA damage (Pearson correlation index, P<0.05). It is plausible that activation of PAHs may be responsible for the induction of CYP1A and resulting increase in DNA damage. Our data show the potential for detrimental effects in the event of exposure of aquatic organisms to biosolids and the need for further investigations of possible impacts due to constituents not covered by current guidelines.

A universal assay for vitellogenin in fish mucus and plasma.

Expression of vitellogenin (VTG) in male fish has become a widely used biomarker of exposure to environmental estrogens. Vitellogenin is usually measured in blood by immunoassays that require species-specific antibodies. In this paper, we describe a universal assay that is based on the high-molecular weight and extensive phosphoserine content of all VTGs. Plasma and mucosal proteins from Pimephales promelas and Fundulus heteroclitus and mucosal proteins from Gambusia holbrooki were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stained with a commercially available fluropore dye (Pro-Q Diamond), and visualized by ultraviolet transillumination. The method allowed sensitive detection of VTG in females and estradiol-treated males in all species tested. Quantitative analysis indicated that the phosphoprotein assay is at least as sensitive as antibody-based methods but is universal, offering the advantage of VTG measurement in multiple species.

In vitro response of the striped bass natural resistance-associated macrophage protein, Nramp, to LPS and Mycobacterium marinum exposure.

Mycobacteriosis in Chesapeake Bay (USA) striped bass Morone saxatilis is an ongoing disease problem with important economic implications for a large commercial and recreational fishery. Additionally, striped bass serve as a reservoir of potential mycobacterial zoonoses. Recently, we described a striped bass gene homolog of the natural resistance-associated macrophage protein family (MsNramp), which is responsible for resistance to mycobacterial infections in mice. Striped bass MsNramp is strongly induced in peritoneal exudate cells (PE) in vivo after intraperitoneal injection with Mycobacterium spp. The purpose of the present study was to investigate short-term in vitro MsNramp expression and reactive oxygen intermediate (ROI) production in primary cultures of adherent PE after exposure to bacterial lipopolysaccharide (LPS), or live- or heat-killed (HK) Mycobacterium marinum. PE expressed significantly higher levels of MsNramp at 4 and 24 h post-treatment with live and HK M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 h and 20 microg/ml LPS. Treatment of PE with LPS resulted in increased intracellular superoxide anion levels, whereas treatment with live M. marinum caused a significant depression. This study is the first report of induction of a teleost Nramp in vitro by mycobacteria, and supports findings of teleost Nramp induction by LPS.

Mycobacterium-inducible Nramp in striped bass (Morone saxatilis).

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.