Peter A. Van Veld

Induced cytochrome P-450 in intestine and liver of spot (Leiostomus xanthurus) from a polycyclic aromatic hydrocarbon contaminated environment

Abstract Levels of total cytochrome P-450, of specific P-450 (determined immunologically with MAb 1-12-3 and referred to as P-450E) and ethoxyresorufin O-deethylase (EROD) were elevated in intestine and liver microsomes of spot (Leiostomus xanthurus) collected from the Elizabeth River, a polycyclic aromatic hydrocarbon (PAH) contaminated tributary of Chesapeake Bay. Fish were collected over a sediment PAH concentration gradient that ranged from 9 to 96 000 μg PAH/kg dry sediment. Intestinal P-450E was near the lower limits of detection in fish collected at the relatively clean sites but was elevated 80- to 100-fold in fish collected from contaminated sites. Intestinal EROD activity exhibited a similar trend. Liver P-450E and associated EROD activity was detectable in all samples and was induced approximately eight-fold at the most heavily contaminated site. Despite the sensitivity of the intestine to PAH inducing agents, intestinal P-450E levels did not correlate well with sediment PAH, whereas liver P-450E did. Instead, the intestinal enzyme was induced to similar and high levels at all contaminated sites. The results suggest that the intestine plays an important role in the absorption and metabolism of dietary PAH and/or other PAH-type inducing agents and that intestinal P-450E may be a useful indicator of exposure to these compounds via the diet.

An elevated glutathione S-transferase in creosote-resistant mummichog (Fundulus heteroclitus)

A population of mummichog (Fundulus heteroclitus) inhabit a site (Atlantic Wood) in the Elizabeth River, VA, heavily contaminated with creosote. Although chronic effects including hepatic neoplasms have been observed in adult mummichog, these fish are resistant to the acute effects (mortality within a few days of exposure) of the creosote-contaminated sediments while fish from a reference site are not. Increased levels and activity of glutathione S-transferases (GST) in Atlantic Wood fish may play a role in this resistance. In the present study, hepatic GSTs from Atlantic Wood and reference site fish were isolated by S-hexylglutathione affinity chromatography. A monoclonal antibody was produced that recognizes a GST that is elevated approximately six-fold in Atlantic Wood fish and two-fold in fish from a moderately contaminated site relative to fish from the reference site. GST activity towards 1-chloro-2,4-dinitrobenzene was elevated approximately four-fold and two-fold in these fish relative to fish from the reference site. The elevated GST has a molecular mass of approximately 27.2 kDa and isoelectric point of 8.1. The N-terminus of the isoform was blocked, preventing N-terminal amino acid determination.

Glutathione S-transferase in intestine, liver and hepatic lesions of mummichog (Fundulus heteroclitus) from a creosote-contaminated environment

Cytosolic glutathione S-transferase (GSH transferase) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was elevated approximately three to four-fold in intestine and liver of mummichog (Fundulus heteroclitus) collected from a creosote-contaminated site in the Elizabeth River, Virginia. Intestinal GSH transferase activity at the most heavily contaminated site, at a moderately contaminated site and at a relatively clean site averaged 3.64, 2.83 and 1.11µmoles/min/mg respectively, while values for liver at these sites averaged 2.84, 1.75 and 0.93µmoles/min/mg. In addition, densitometric tracings of sodium dodecylsulfate-polyacrylamide gels of intestine and liver cytosol revealed a similar trend in the staining intensity of a 25.8 kD protein band, which lies within the molecular weight range of GSH transferase subunits. Activity in putative preneoplastic and neoplastic hepatic lesions of fish collected from the creosote-contaminated site was not significantly different from that of adjacent normal tissue. In the laboratory, dietary betanaphthoflavone (ßNF) treatment resulted in a three-fold increase in intestinal GSH transferase. Hepatic GSH transferase activity in the same fish was not affected by dietary ßNF although hepatic monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), was. The results of this study indicate a response of the intestinal detoxification system to environmental contaminants and supports previous studies on the importance of intestinal metabolism of foreign compounds. Further, our results indicate the trend towards elevated GSH transferase in liver of feral fish could not be attributed to a cancerous disease state in these fish but indicates chemical induction in this organ as well.

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.

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.

Toxicity of sediments contaminated with fractions of creosote

Creosote, a mixture of polycyclic aromatic hydrocarbons (PAH), typically becomes depleted of low molecular weight compounds once in the environment. Previous studies indicated that PAH toxicity increased with increasing molecular weight up to phenanthrene; heavier compounds were less toxic than phenanthrene, possibly due to their limited solubility. A high molecular weight fraction (HMWF) of creosote, with a composition similar to environmentally weathered creosote, and a low molecular weight fraction (LMWF) were obtained by distillation. Fish were exposed to suspended sediments contaminated with each fraction for 10 days. Samples of fish were removed on days 2, 4, 7 and 10, observed for gross pathological abnormalities, weighed, measured, and the livers analyzed for ethoxyresorufin O-de-ethylase (EROD) activity. Mortality, epidermal lesions, fin erosion, and temporary induction of EROD activity occurred in fish exposed to the HMWF. No mortality, fin erosion, or induction of EROD activity occurred in fish exposed to the LMWF or uncontaminated sediment. Fish exposed to the LMWF did develop lesions, but only in the area surrounding the mouth, nares, and opercula. These results suggest that the environmental toxicity of creosote is due to the high molecular weight compounds.

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.

Mycobacterium-Inducible Nramp in Striped Bass (Morone saxatilis)

ABSTRACT 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.

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, 17β-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 17β-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.

Uptake, distribution, metabolism and clearance of chlordecone by channel catfish (Ictalurus punctatus)☆

Abstract Channel catfish ( Ictalurus punctatus ) were exposed to a [ 14 C]chlordecone-contaminated diet for a period of 90 days, followed by a chlordecone-free diet for 30 days. The uptake and clearance rate constants were 0.237 day −1 and 0.080 day −1 , respectively. The half-life of chlordecone in these fish was 8.7 days. The dietary accumulation factor (DAF), defined as the equilibrium concentration of pesticide in fish/daily dietary dosage level was calculated to be 3.0. Highest concentrations of chlordecone in channel catfish were found in the blood and brain. White catfish exposed to the [ 14 C]chlordecone-contaminated diet contained highest concentrations in the brain. In both species lowest concentrations were measured in the mesenteric adipose and carcass. Chlordecone may be eliminated with bile via the intestinal contents. There is evidence for an extrabiliary source of fecal chlordecone in channel catfish. Chlordecone may be excreted across the gills and sloughed off with epidermal mucus. Urinary excretion is negligible. Channel catfish are capable of reducing chlordecone to chlordecone alcohol as shown by the presence of this metabolite in the bile. Enzymatic digestion of bile with β-glucuronidase did not significantly affect the recovery of chlordecone or chlordecone alcohol.