Elucidating the roles of gut neuropeptides on channel catfish feed intake, glycemia, and hypothalamic NPY and POMC expression.

Both intrinsic and extrinsic factors modulate food intake and glycemia in vertebrates, in part through interactions with hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons. The objective of this project was to elucidate the effects of ghrelin (GHRL), gastrin-releasing peptide (GRP), cholecystokinin (CCK), glucagon-like peptide (GLP), pancreatic polypeptide (PP), and peptide YY (PYY) on appetite, glycemia, and hypothalamic expression of NPY and POMC in channel catfish. Catfish were injected intraperitoneally with a single peptide at concentrations of either 0 (control), 50, 100, or 200 ng/g body weight (BW), respectively. Fish were allowed to recover for 30 min, and then fed to satiation over 1 h. Feed intake was determined 1h post-feeding. Catfish injected with GHRL at 50 and 100 ng/g BW and GRP at 200 ng/g BW consumed significantly (P<0.05) less feed compared to controls. A tendency (P<0.1) to suppress feed intake was also observed in the 200 ng/g BW GHRL and PP treatments. PYY, CCK, and GLP had no effects on feed intake. Glycemia was not affected by GHRL, GRP, PP, and PYY treatments, but was suppressed by CCK. A tendency toward lower plasma glucose concentrations was observed in fish administered GLP at 50 ng/g BW. Hypothalamic NPY expression was highly variable and not significantly affected by treatment. POMC expression was also variable, but tended to be reduced by the highest concentration of CCK. These results provide new insight into the roles and regulation of gut neuropeptides in catfish appetite and glycemia.

Aroclor 1248 exposure leads to immunomodulation, decreased disease resistance and endocrine disruption in the brown bullhead, Ameiurus nebulosus.

The brown bullhead Ameiurus nebulosus is a species of the family Ictaluridae commonly used as a sentinel of environmental contamination. While these fish have been utilized for this purpose in areas contaminated with polychlorinated biphenyls (PCBs), few controlled, laboratory-based studies have been designed to document the effects of PCB mixtures in this species. Here, brown bullhead were exposed to the PCB mixture, Aroclor 1248, via intraperitoneal injection and the effects on immune function, plasma hormones and disease resistance were evaluated. Exposure to this mixture led to a decrease in bactericidal activity and circulating antibodies to Edwardsiella ictaluri present from a previous exposure to this pathogen. A subsequent E. ictaluri disease challenge led to significantly higher mortality in A1248 treated fish compared to vehicle-control fish. The mitogenic response to the T-cell mitogen, phytohemaglutinin-P, was increased compared to vehicle-control fish. The steroid hormone, cortisol, and the thyroid hormone, T3, were also significantly lower in A1248 exposed fish. In summary, we have validated a number of functional immune assays for application in brown bullhead immunotoxicity studies. Additionally, we have demonstrated that the PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead. Such data may compliment the interpretation of data yielded from applied field studies conducted in PCB contaminated aquatic ecosystems.

Interaction of xenobiotics with estrogen receptors alpha and beta and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus).

Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ERalpha and beta were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol (3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (+/-SE) of 1.9+/-0.14 nM and a Bmax of 14.3+/-2.4 pmol/mg protein ( n = 3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ERalpha and beta at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ERbeta. The xenobiotics tested generally showed equivalent or greater affinity for ERalpha than ERbeta, whereas natural estrogens had much greater affinity for ERbeta than ERalpha. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine disruption caused by environmental contaminants.

Irreversible inhibition of [3H]glycine transport into channel catfish erythrocytes by thiol group modifiers.

Erythrocytes can take up amino acids from the blood by using a variety of transport systems. GLYT is a key transport protein in the plasma membrane responsible for the Na(2+)-dependent uptake of glycine needed for glutathione biosynthesis. Certain cysteine-specific compounds, particularly mercuric chloride and 5,5'-dithiobis(2-nitrobenzoate), irreversibly inhibited the [(3)H]glycine transport via GLYT by red blood cells isolated from channel catfish. Bimolecular rate constants (k(2)) of 0.556 (mmol/l)(-1) min(-1) and 0.032 (mmol/l)(-1) min(-1), respectively, were calculated for the two inhibitors. Addition of 2-mercaptoethanol 1 min after the initiation of inactivation by mercuric chloride stopped further inactivation, but did not reverse the inhibition. The presence of glycine, but not Na(+) ions, during the preincubation of the cells with each inhibitor markedly reduced the degree of inhibition. Thus cysteinyl residues within the transport protein appear to be vital for the binding and uptake of glycine by channel catfish erythrocytes.

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.

High plasma buffering and the absence of a red blood cell beta-NHE response in brown bullhead (Ameiurus nebulosus).

The high non-bicarbonate buffer capacity of brown bullhead (Ameiurus nebulosus) plasma was postulated to function as an alternative mechanism for the protection of red blood cell (RBC) intracellular pH (pHi) in the absence or attenuation of a RBC adrenergic response. The requirement for protecting RBC pHi arises from the presence of a Root effect haemoglobin in bullhead. In support of this hypothesis, bullhead RBCs incubated in vitro with isoproterenol (10(-8)-10(-5) mol l(-1)) or forskolin (10(-4) mol l(-1)) exhibited significant cyclic AMP accumulation, but failed to exhibit cell swelling or significant Na(+) or Cl(-) accumulation; plasma pH (pHe) was also unaffected. Similarly, no significant effect on RBC water content, Na(+) or Cl(-) concentration, or pHe was detected in bullhead blood incubated with 8-bromo cyclic AMP (10(-4)-10(-2) mol l(-1)) in vitro. These results suggest that while bullhead RBCs possess a beta-adrenoreceptor linked to cyclic AMP formation, stimulation of this adrenergic receptor does not result in measurable activation of a Na(+)/H(+) exchanger.

Molecular and functional characterization of channel catfish (Ictalurus punctatus) neutrophil collagenase.

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.

Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures.

In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus).

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish serum. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genus Aeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.