Extended fasting does not affect the liver innate immune response in rainbow trout.

Activation of immune response pathway is energy demanding. We tested the hypothesis that negative energy balance will curtail the liver's capacity to evoke an immune response in rainbow trout (Oncorhynchus mykiss). Fish were either fed or fasted for 118 d and challenged with lipopolysaccharide (LPS) to determine the liver capacity to elicit an immune response. Fasting led to negative specific growth rate, reduced tissue metabolite levels, and higher transcript abundance of SOCS-2. LPS treatment increased the liver transcript abundances of IL-1ß and IL-8 and serum amyloid protein A, while SOCS-2 was reduced. LPS lowered plasma cortisol level only in the fasted fish, but did not affect liver glucocorticoid or mineralocorticoid receptor protein expressions. Extended fasting did not suppress the liver capacity to evoke an immune response. Upregulation of liver SOCS-2 may be playing a key role in the energy repartitioning, thereby facilitating immune response activation despite extended fasting in trout.

Chemical contaminants in fish feeds used in federal salmonid hatcheries in the USA.

Recent studies have demonstrated that fish feeds contain significant concentrations of contaminants, many of which can bioaccumulate and bioconcentrate in fish. Organochlorine (OC) contaminants are present in the fish oils and fish meals used in feed manufacture, and some researchers speculate that all fish feeds contain measurable levels of some contaminants. To determine the concentration of contaminants in feeds used in US Fish and Wildlife Service's National Fish Hatcheries, we systematically collected samples of feed from 11 cold-water fish hatcheries. All samples (collected from October 2001 to October 2003) contained at least one polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), polychlorinated biphenyl (PCB) congener, or dichlorodiphenyltrichloroethane (DDT) metabolite. Of the 55 samples in which they were analyzed 39 contained PCDDs, 24 contained PCDFs and 24 contained DDT or its metabolites. There were 10- to 150-fold differences in concentrations of total PCBs, PCDDs, PCDFs and DDT. Although PCBs were the most commonly detected contaminant in our study, concentrations (range: 0.07-10.46 ng g(-1) wet weight) were low compared to those reported previously. In general, we also found lower levels of OCs than reported previously in fish feed. Perhaps most notable was the near absence of OC pesticides - except for DDT or its metabolites, and two samples containing hexachlorocyclohexane (HCH). While contaminant concentrations were generally low, the ecological impacts can not be determined without a measure of the bioaccumulation of these compounds in the fish and the fate of these compounds after the fish are released.

Biomarker response and hypothalamus-pituitary-interrenal axis functioning in Arctic charr from Bjørnøya (74°30' N), Norway, with high levels of organohalogenated compounds.

The populations of Arctic charr (Salvelinus alpinus) residing in Lake Ellasjøen at Bjørnøya Island in the Norwegian Arctic (74° 30'N, 19° 00'E) possess substantially higher levels of organohalogenated compounds (strongly dominated by polychlorinated biphenyls, PCBs) than conspecifics residing in other, proximate lakes on the island. In the present study we sampled large (<400g), immature charr from Lake Ellasjøen (high PCB levels) and Lake Laksvatn (reference lake, low PCB levels) by hook and line for an immediate blood sampling, and blood and tissue sampling after a 1h confinement stressor. This was done in order to investigate possible effects of pollutants on an acute stress performance in a high-latitude fish species by comparing muscle PCB levels, hepatic cytochrome P4501A (CYP1A) biomarker activation and functioning of the hypothalamus-pituitary-interrenal (HPI) axis between these two populations of Arctic charr. As expected sum PCB muscle levels were 8-fold higher on a wet weigh basis, and 19-fold higher on a lipid weight basis, in charr from Ellasjøen than in charr from Laksvatn. This was accompanied by a 3.5-fold higher liver cyp1a mRNA abundance in the Ellasjøen charr compared to Laksvatn charr. Brain transcript levels encoding glucocorticoid receptor 1 and 2 (GR2) and corticotropin-releasing factor, and pituitary transcript levels encoding GR2 and proopiomelanocortin A1 and A2 were higher in Ellasjøen charr than in Laksvatn charr, while interrenal transcript levels encoding melanocortin 2 receptor and steroidogenic acute regulatory protein were lower. There were no differences in plasma cortisol concentration between the two charr populations immediately after capture and one hour after confinement. The strong biomarker response to OHCs and altered mRNA abundances of key genes related to HPI axis functioning in the Ellasjøen charr suggest endocrine disruptive effects of OHCs in this charr population. Possible ecological implications are not known, but it cannot be excluded that a slower growth rate in Ellasjøen charr compared to Laksvatn charr due to an increased metabolic demand associated with the activation of xenobiotic defense and detoxification systems may have contributed to the lower body mass of Ellasjøen charr compared to Laksvatn charr.

Fasting augments PCB impact on liver metabolism in anadromous arctic char.

Anadromous arctic char (Salvelinus alpinus) undertake short feeding migrations to seawater every summer and accumulate lipids, while the rest of the year is spent in fresh water where the accumulated lipid reserves are mobilized. We tested the hypothesis that winter fasting and the associated polychlorinated biphenyls' (PCBs) redistribution from lipid depots to critical tissues impair the liver metabolic capacity in these animals. Char were administered Aroclor 1254 (0, 1, 10, and 100 mg/kg body mass) orally and maintained for 4 months without feeding to mimic seasonal winter fasting, while fed groups (0 and 100 mg Aroclor 1254/kg) were maintained for comparison. A clear dose-related increase in PCB accumulation and cytochrome P4501A (CYP1A) protein content was observed in the livers of fasted fish. This PCB concentration and CYP1A response with the high dose of Aroclor were 1.5-fold and 3-fold greater in the fasted than in the fed fish, respectively. In fed fish, PCB exposure lowered liver glycogen content, whereas none of the other metabolic indicators were significantly affected. In fasted fish, PCB exposure depressed liver glycogen content and activities of glucose-6-phosphate dehydrogenase, alanine aminotransferase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase and elevated 3-hydroxyacylcoA dehydrogenase activity and glucocorticoid receptor protein expression. There were no significant impacts of PCB on heat shock protein 70 (hsp70) and hsp90 contents in either fed or fasted fish. Collectively, our study demonstrates that winter emaciation associated with the anadromous lifestyle predisposes arctic char to PCB impact on hepatic metabolism including disruption of the adaptive metabolic responses to extended fasting.

Effect of long-term fasting and a subsequent meal on mRNA abundances of hypothalamic appetite regulators, central and peripheral leptin expression and plasma leptin levels in rainbow trout.

Knowledge about neuroendocrine mechanisms regulating appetite in fish, including the role of leptin, is inconclusive. We investigated leptin mRNA abundance in various tissues, plasma leptin levels and the hypothalamic gene expression of putative orexigenic (neuropeptide Y and agouti-regulated peptide) and anorexigenic (melanocortin receptor, proopiomelanocortins (POMCs), cocaine- and amphetamine-regulated transcript and corticotropin-releasing factor) neuropeptides in relation to feeding status in rainbow trout (Oncorhynchus mykiss). Blood and tissues were first (Day 1) sampled from trout that had been fed or fasted for 4 months and the day after (Day 2) from fasted fish after they had been given a large meal, and their continuously fed counterparts. The fasted fish ate vigorously when they were presented a meal. There were no differences between fed, fasted and re-fed fish in hypothalamic neuropeptide transcript levels, except for pomca1 and pomcb, which were higher in fasted fish than in fed fish at Day 1, and which, for pomcb, decreased to the level in fed fish after the meal at Day 2. Plasma leptin levels did not differ between fasted, re-fed and fed fish. A higher leptina1 transcript level was seen in the belly flap of fasted fish than in fed fish, even after re-feeding on Day 2. The data do not reveal causative roles of the investigated brain neuropeptides, or leptin, in appetite regulation. It is suggested that the elevated pomc transcript levels provide a satiety signal that reduces energy expenditure during prolonged fasting. The increase in belly flap leptin transcript with fasting, which did not decrease upon re-feeding, indicates a tissue-specific role of leptin in long-term regulation of energy homeostasis.

Evidence of detrimental effects of environmental contaminants on growth and reproductive physiology of white sturgeon in impounded areas of the Columbia River.

This study sought to determine whether wild white sturgeon from the Columbia River (Oregon) were exhibiting signs of reproductive endocrine disruption. Fish were sampled in the free-flowing portion of the river (where the population is experiencing reproductive success) and from three reservoirs behind hydroelectric dams (where fish have reduced reproductive success). All of the 18 pesticides and almost all of the 28 polychlorinated biphenyls (PCBs) that were analyzed in livers and gonads were detected in at least some of the tissue samples. Metabolites of p,p -dichlorodiphenyltrichloroethane (DDT) [p,p -dichlorodiphenyldichloroethylene (DDE) and p,p -1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD)]were consistently found at relatively high levels in fish. Some males and immature females showed elevated plasma vitellogenin; however, concentrations were not correlated with any of the pesticides or PCBs analyzed. Negative correlations were found between a number of physiologic parameters and tissue burdens of toxicants. Plasma triglycerides and condition factor were negatively correlated with total DDT (DDD + DDE + DDT), total pesticides (all pesticides detected - total DDT), and PCBs. In males, plasma androgens and gonad size were negatively correlated with total DDT, total pesticides, and PCBs. Fish residing in the reservoir behind the oldest dam had the highest contaminant loads and incidence of gonadal abnormalities, and the lowest triglycerides, condition factor, gonad size, and plasma androgens. These data suggest that endocrine-disrupting chemicals may be accumulating behind dams over time. Overall, results of this study indicate that exposure to environmental contaminants may be affecting both growth and reproductive physiology of sturgeon in some areas of the Columbia River.

Aroclor 1254 exposure reduces disease resistance and innate immune responses in fasted Arctic charr.

To examine the immunological impacts of polychlorinated biphenyls (PCBs) in an environmentally relevant way, we orally contaminated Arctic charr (Salvelinus alpinus) with Aroclor 1254. After contamination, fish were either fed (0 and 100 mg Aroclor 1254 kg(-1) fish wt) or fasted (0, 1, 10, and 100 mg kg(-1)) to mimic cycles of feeding-fasting experienced by Arctic animals. After four months, PCB concentrations in muscle were the same in fasted and fed fish; however, PCBs in kidneys of fed fish were 33 to 50% of those in fasted fish. Arctic charr were exposed to Aeromonas salmonicida, the bacteria responsible for furunculosis, by cohabitation with infected conspecifics. Fasted fish had a significant trend toward lower survival with higher dose of PCBs--from 68% in controls to 48% in treatment involving 100 mg kg(-1). Independent of PCB contamination, fed fish had the lowest survival; we attribute this to stress associated with establishing and maintaining feeding hierarchies. A significant decrease in the activity of lysozyme was observed in skin mucus, as was hemagglutination ability of a putative rhamnose lectin in fasted, but not in fed, PCB-treated fish. These results demonstrate the immunosuppressive effects of PCBs on Arctic charr, and they illustrate the importance of considering environmentally relevant nutritional status in ecotoxicological studies.

Tissue-specific molecular immune response to lipopolysaccharide challenge in emaciated anadromous Arctic charr.

Anadromous Arctic charr (Salvelinus alpinus) undergo voluntary winter fasting for months in the Arctic. We tested the hypothesis that extended fasting will compromise the ability of this species to evoke an immune response. Charr were either fed or fasted for 85 days and challenged with lipopolysaccharide (LPS), and the molecular immune response in the liver and spleen assessed at 8 and 96 h post-injection. LPS increased IL-1ß, IL-8, and serum amyloid protein A (SAA) mRNA levels in both groups, but the liver IL-1ß and IL-8, and spleen IL-8 responses were reduced in the fasted group. Fasting upregulated SOCS-1 and SOCS-2 mRNA abundance, while LPS stimulated SOCS-3 mRNA abundance and this response was higher in the fasted liver. Collectively, extended fasting and emaciation does not curtail the capacity of charr to evoke an immune response, whereas upregulation of SOCS may be a key adaptation to conserve energy by restricting the inflammatory response.

Correlation of gene expression and contaminant concentrations in wild largescale suckers: a field-based study.

Toxic compounds such as organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether flame retardants (PBDEs) have been detected in fish, birds, and aquatic mammals that live in the Columbia River or use food resources from within the river. We developed a custom microarray for largescale suckers (Catostomus macrocheilus) and used it to investigate the molecular effects of contaminant exposure on wild fish in the Columbia River. Using Significance Analysis of Microarrays (SAM) we identified 72 probes representing 69 unique genes with expression patterns that correlated with hepatic tissue levels of OCs, PCBs, or PBDEs. These genes were involved in many biological processes previously shown to respond to contaminant exposure, including drug and lipid metabolism, apoptosis, cellular transport, oxidative stress, and cellular chaperone function. The relation between gene expression and contaminant concentration suggests that these genes may respond to environmental contaminant exposure and are promising candidates for further field and laboratory studies to develop biomarkers for monitoring exposure of wild fish to contaminant mixtures found in the Columbia River Basin. The array developed in this study could also be a useful tool for studies involving endangered sucker species and other sucker species used in contaminant research.

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.

PCB disruption of the hypothalamus-pituitary-interrenal axis involves brain glucocorticoid receptor downregulation in anadromous Arctic charr.

We examined whether brain glucocorticoid receptor (GR) modulation by polychlorinated biphenyls (PCBs) was involved in the abnormal cortisol response to stress seen in anadromous Arctic charr (Salvelinus alpinus). Fish treated with Aroclor 1254 (0, 1, 10, and 100 mg/kg body mass) were maintained for 5 mo without feeding in the winter to mimic their seasonal fasting cycle, whereas a fed group with 0 and 100 mg/kg Aroclor was maintained for comparison. Fasting elevated plasma cortisol levels and brain GR content but depressed heat shock protein 90 (hsp90) and interrenal cortisol production capacity. Exposure of fasted fish to Aroclor 1254 resulted in a dose-dependent increase in brain total PCB content. This accumulation in fish with high PCB dose was threefold higher in fasted fish compared with fed fish. PCBs depressed plasma cortisol levels but did not affect in vitro interrenal cortisol production capacity in fasted charr. At high PCB dose, the brain GR content was significantly lower in the fasted fish and this corresponded with a lower brain hsp70 and hsp90 content. The elevation of plasma cortisol levels and upregulation of brain GR content may be an important adaptation to extended fasting in anadromous Arctic charr, and this response was disrupted by PCBs. Taken together, the hypothalamus-pituitary-interrenal axis is a target for PCB impact during winter emaciation in anadromous Arctic charr.

Lateral line pore diameters correlate with the development of gas bubble trauma signs in several Columbia River fishes.

Gas bubble trauma (GBT) caused by gas supersaturation of river water continues to be a problem in the Columbia River Basin. A common indicator of GBT is the percent of the lateral line occluded with gas bubbles; however, this effect has never been examined in relation to lateral line morphology. The effects of 115, 125 and 130% total dissolved gas levels were evaluated on five fish species common to the upper Columbia River. Trunk lateral line pore diameters differed significantly (P<0.0001) among species (longnose sucker>largescale sucker>northern pikeminnow>/=chinook salmon>/=redside shiner). At all supersaturation levels evaluated, percent of lateral line occlusion exhibited an inverse correlation to pore size but was not generally related to total dissolved gas level or time of exposure. This study suggests that the differences in lateral line pore diameters between species should be considered when using lateral line occlusion as an indicator of gas bubble trauma.

PCB impairs smoltification and seawater performance in anadromous Arctic charr (Salvelinus alpinus).

The impacts of polychlorinated biphenyl (PCB) exposure on smoltification and subsequent seawater performance were investigated in hatchery-reared, anadromous Arctic charr (Salvelinus alpinus). The fish were subjected to a 2-month summer seawater residence, after which they were orally dosed with 0 (Control, C), 1 (Low Dose, LD) or 100 mg Aroclor 1254 kg(-1) body mass (High Dose, HD) in November. They were then held in fresh water, without being fed (to mimic their natural overwintering in freshwater), until they had smolted in June the next year. The smolts were then transferred to seawater and fed to mimic their summer feeding residence in seawater, followed by a period without food in freshwater from August until maturation in October. Compared with C and LD charr, the HD charr had either a transient or a permanent reduction in plasma growth hormone, insulin-like growth factor-1, and thyroxin and triiodothyronine titers during the period of smoltification. These hormonal alterations in the HD charr corresponded with impaired hyposmoregulatory ability in May and June, as well as reduced growth rate and survival after transference to seawater. Consequently, fewer fish in the HD group matured in October compared to the other two treatments. The HD fish had a liver PCB concentration ranging between 14 and 42 mg kg(-1) wet mass, whereas there were similar, and very low, liver PCB concentrations in LD and C fish throughout the smolting period. Our findings suggest that PCB might compromise mechanisms important for fitness in a fish species living in an extreme environment.