Daniel P. Selivonchick

Effects of dietary vegetable, animal and marine lipids on muscle lipid and hematology of rainbow trout (Oncorhynchus mykiss)

The effects of dietary vegetable and animal fats on the growth, nutrition, and tissue lipid profiles of rainbow trout raised to market size on a commercially available diet were examined. Rainbow trout of 80 g mean initial weight were fed pellets prepared according to Oregon Moist Pellet (OMP) specifications for 20 weeks. Salmon oil (OMP-1), soybean oil (OMP-2), linseed oil (OMP-3), chicken fat (OMP-4), pork lard (OMP-5) and beef tallow (OMP-6) were used for the lipid component of the diets. No differences in feed conversion or growth rate were observed across diet groups. Hematological tests revealed oxidative stress caused by the OMP-1 diet, as measured by hemolysis, while hematocrit and percent hemoglobin values fell within a narrow range across diet groups. Only 10–12% of lipid in trout on diets OMP-2–6 was deposited as 20:5n−3 plus 22:6n−3 despite the availability of dietary precursor (18:3n−3) to further elongate and desaturate to 20:5n−3 and/or 22:6n−3. Dietary ratios of 20:120:1, ranging from 0.6 to 0.8, also were modified to 0.5 in the muscle tissue of trout on all diets. This consistent pattern suggests that a physiologically optimum level of long chain fatty acids is maintained in rainbow trout.

The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate, a zwitterionic detergent, has been shown to exhibit superior membrane protein solubilizing characteristics as compared to nonionic detergents. Replacement of NP-40 with CHAPS in isoelectric focusing of rainbow trout liver microsomes has increased resolution markedly. The two-dimensional electrophoretic system described will allow effective resolution of up to 300 micrograms of crude microsomal protein. CHAPS exhibits no effect on the stability or type of pH gradient when compared to NP-40 during isoelectric focusing in the presence of urea.

The effect of prolonged administration of bovine insulin in rainbow trout (Salmo gairdneri R.)

Effects on growth parameters and tissue composition of rainbow trout were investigated following injection of bovine insulin at two dose levels every 48 hr for 56 days. In addition, [14C]leucine incorporation into plasma, liver, and skeletal muscle was studied for the two insulin treatments and a group of the saline-injected controls given a single shock-dose of insulin (5.0 IU/kg). Hypoglycemic responses were observed with all insulin treatments. In comparison to controls, high insulin treatment gave a significant body weight increase. At both levels, insulin increased the content of protein, lipid, and also the incorporation of [14C]leucine activity in skeletal muscle. Simultaneous decreases in specific activity of plasma and liver tissue indicated a net movement of [14C]leucine toward the peripheral musculature. No effect of the hormone was seen on the glycogen content of liver or muscle tissue over the 56-day period.

The effect of high-protein and high-carbohydrate diets on [I]iodoinsulin binding in skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout ( Salmo gairdneri )

[125I]iodoinsulin-binding studies in the presence of a concentration range of bovine insulin were conducted to establish specific insulin-binding levels in skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout (Salmo gairdneri) reared on control, high-protein or high-carbohydrate diets. Negative co-operativity was observed and receptor concentrations and apparent dissociation constants established for each preparation. No differences of specific binding attributed to diet were detected in skeletal muscle plasma membrane preparations; however, the receptor concentration of isolated hepatocytes from high-carbohydrate-reared trout was increased. This contrasted to comparable mammalian studies.

The use of isolated hepatocytes from rainbow trout (salmo gairdneri) in the metabolism of acetaminophen

Abstract 1. 1. Hepatocytes were isolated from rainbow trout. Cytrochrome P-450 and glutathione (GSH) were determined and found to be considerably lower than values reported for rat hepatocytes. 2. 2. Acetaminophen was incubated with rainbow trout hepatocytes; and glucuronide, glutathione and sulfate conjugates were analyzed. 3. 3. The formation of GSH conjugates were lower than that of glucuronide conjugates and both were several fold lower than those reported for rat hepatocytes. 4. 4. In contrast to rat hepatocytes, sulfate conjugates were not detected. 5. 5. These results demonstrate the ability of isolated fish hepatocytes to metabolize xenobiotics and further illustrates differences in the formation of metabolites between trout and mammals.

The effect of bovine insulin on [14C]glucose and [3H]leucine incorporation in fed and fasted rainbow trout (Salmo gairdneri)☆

The effect of bovine insulin on tissue incorporation of [14C]glucose and [3H]leucine was investigated in fed and fasted rainbow trout reared on a control and high-protein diet. Insulin produced marked hypoglycemia and mobilization of liver glycogen in all treatments. Although insulin gave no evidence of glycogenic stimulation it did appear to promote oxidative clearance of [14C]glucose. Compared to [14C]glucose much greater tissue incorporation of [3H]leucine was observed in fasted fish; insulin stimulated the incorporation of [3H]leucine into skeletal muscle protein. In plasma, liver, and skeletal muscle of all treatments, the summed specific activities of [3H]leucine was considerably greater than that of the summed values of [14C]glucose following insulin administration. Four weeks of fasting apparently lowered basal metabolism but no changes were observed in plasma glucose and glycogen stores. There was some evidence of gluconeogenic activity in the high protein-fasted fish and the data indicated in all fasted treatments a stimulation of [14C]glucose and [3H]leucine metabolism following insulin administration.

Uptake and metabolism of lipid vesicles from seawater by juvenile Pacific oysters (Crassostrea gigas)

Abstract Ingestion and metabolism of liposomes by juvenile Pacific oysters ( Crassostrea gigas ) were studied by several methods in order to assess their potential as encapsulating agents. Liposomes composed of egg phosphatidylcholine/cholesterol/stearylamine formed readily and appeared stable in synthetic seawater. Radiotracer studies with liposomes containing [4- 14 C]cholesterol or [1- 14 C]dipalmitoyl phosphatidylcholine (DPPC) showed uptake of up to 40% of the available dose in 24 h. Most label appeared in epithelial cells of ducts and tubules of the digestive diverticula as shown by autoradiography. Fluorescence microscopic examination of oysters fed fluorescein isothiocyanate (FITC)-labeled liposomes or liposomes with encapsulated FITC-albumin revealed tissue distribution similar to that observed with radiotracer studies. Label from [1- 14 C]DPPC was redistributed into triplycerides, phosphatidylethanolamine, and in a tentatively identified choline phosphonolipid, and appeared to involve transacylation rather than de novo synthesis. Radioactivity from encapsulated 14 C-glucose or amino acids appeared predominantly in solvent-insoluble and acid-precipitable materials, indicating incorporation into polysaccharides or protein. The uptake of liposomes followed by redistribution and metabolism of liposomal lipids and encapsuled materials indicate the potential for liposomes as a nutrient delivery system for filter-feeding organisms.

Mechanisms of Dietary Modification of Aflatoxin B1 Carcinogenesis

Trout were fed a range of dietary components which altered their carcinogenic response to aflatoxin B1 (AFB1). Dietary protein at levels substantially exceeding nutritional requirements were synergistic with AFB1. Cyclopropene fatty acids (CPFA) were carcinogenic when fed alone at 20 or 55 ppm, and synergistic when fed with AFB1. In contrast, several flavonoid and indole compounds, especially beta-naphthoflavone (beta-NF) and indole-3-carbinol, inhibited the carcinogenic response when fed prior to and along with AFB1. The mechanisms by which some of these dietary factors modulate AFB1 carcinogenesis were investigated. Dietary beta-naphthoflavone was shown to substantially induce the levels of mixed function oxidase (MFO) activities assayed in vitro. These changes were accompanied by alterations in AFB1 metabolism and binding in freshly isolated hepatocytes. AFB1 incubated in hepatocytes freshly isolated from fish fed beta-NF diet was metabolized more rapidly, showed enhanced rates of detoxication reactions, and decreased accumulation of AFB1-DNA adducts compared to control hepatocytes. These results suggest that beta-NF inhibits AFB1 carcinogenesis at least in part by altering MFO activities such that detoxication is enhanced and initial DNA damage by AFB1 is reduced. In contrast, high dietary protein is a synergist for AFB1 carcinogenesis, and this appears to occur primarily by enhancing the transformation probability for AFB1-initiated genome damage. Fish treated with AFB1 as embryos and then reared on high protein diets had substantially higher incidences of hepatocellular carcinoma (86%) than similarly treated fish fed normal protein diet (44%) or high protein controls without AFB1 exposure (0-2%). The synergistic behavior of dietary CPFAs also appears to partially involve enhanced transformation following DNA damage by AFB1. Fish exposed as embryos to AFB1 and then fed CPFA-containing diets are known to show promotion effects similar to the high protein results (Hendricks, J.D., Proc. 11th Int. Symp. of the Princess Takamatsu Cancer Research Fund, in press.) However, factors other than promotion are involved in the synergism between CPFA and AFB1. Preliminary studies indicate that dietary CPFAs repress MFO activities and depress DNA damage by AFB1 in vitro. If this occurs in vivo, then the net synergistic effect of dietary CPFAs would involve depression of initial AFB1-induced DNA damage, but highly efficient promotion of transformation from the remaining lesions.

Cytochrome P-450 isozymes in salmonids determined with antibodies to purified forms of P-450 from rainbow trout

Abstract Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM4b)∗ appears to be a P-448 type of cytochrome. A minor form (LM4a), having properties very similar to LM4b, was also obtained. In addition, a P-450 form (LM2) was isolated, with properties quite different from LM4a or LM4b, including a high rate of aflatoxin B1 (AFB1) metabolism (Williams & Buhler, 1983c). Antibodies to all three forms were obtained from rabbits. The IgGs prepared against LM4a and LM4b both cross-reacted (forming lines of identity) equally well with both antigens on Ouchterlony plates. Rat P-448 cross-reacted (without lines of identity) with both LM4a- and LM4b-IgG. LM4b-IgG was much more effective than LM4a-IgG for inhibition of LM4a or LM4b reconstituted benzo[a]pyrene (BP) hydroxylase, suggesting that these two antibodies recognize different antigenic sites. The LM2-IgG did not cross-react with any of the other rat or trout cytochromes P-450 examined. Levels of LM2 and LM4b in microsomes from untreated, polychlorinated biphenyl (PCB), phenobarbital (PB) or BNF-treated trout were estimated with an immunological technique involving electrophoresis on SDS-PAGE, followed by transfer to nitrocellulose and staining with either LM 2 - or LM4b -IgG. The ratio of LM 4b LM 2 in microsomes from PCB- or BNF-treated rainbow trout was much higher than 1, whereas the reverse was true with microsomes from untreated rainbow trout. These results are consistent with previous observations (Vodicnik et al., 1982) that pretreatment with BNF induced the synthesis of a P-448 type cyytochrome, presumably responsible for the great increase in the metabolism and activation of BP seen in these fish. Conversely, pretreatment with PB did not affect the levels of either LM2 or LM4b. This specific immunological technique should make it possible to assay the levels of these P-450 and P-448 isozymes in various strains of rainbow trout and other species of fish. In addition, the effect of age, sex, diet and exposure to P-450 and P-448 inducers could be examined and, perhaps, utilized to predict the relative risk of certain populations to pollutants activated by these different isozymes.

Turnover of label from [1-14C]linolenic acid in phospholipids of coho salmon, Oncorhynchus kisutch.

Juvenile coho salmon were injected intraperitoneally with [1-14C] linolenic acid, and sampled at 24, 120, and 240 hr. Liver, heart, and gill lipids were extracted, analyzed, and halflives of individual liver glycerophospholipids and n-3 fatty acids determined from rates of loss of radioactivity. Incorporation of label into gill was much less than into either heart or liver. Total acyl halflife was shorter for the choline phospholipids than for the ethanolamine phospholipids, as were the halflives of all individual n-3 fatty acids. Eicosapentaenoic acid (20∶5n-3) had the shortest halflife in both phospholipids (50–60 hr), while docosapentaenoic acid (22∶5n-3) and docosahexaenoic acid (22∶6n-3) had much longer halflives. Specific activities of the shorter chain n-3 fatty acids were much greater than the longer, more unsaturated homologs at all times, suggesting possible differences in their mechanisms of incorporation into phospholipids. Diacylglycerol analysis indicated that de novo synthesis could be responsible for the incorporation of only a small portion of the labeled long chain fatty acids found in phospholipids. The fatty acid halflives reported here for salmon are in general agreement with those found previously in mammals.