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Effects of cyclopropenoid fatty acids on liver plasma membranes of rainbow trout (Salmo gairdneri)Marino, Donald R. (Donald Robert) 31 October 1988 (has links)
Cyclopropenoid fatty acids (CPFA), which are a group of
fatty acids produced by plants of the order Malvales, are known
to induce adverse physiological effects when administered to a
variety of animal species. A structurally strained cyclopropene
ring is present in all CPFA and is believed responsible for the
toxic action of these fatty acids. Dietary consumption of CPFA
by mammals, poultry and fish has resulted in toxic responses
including hepatic damage, impaired reproductive capabilities and
sizeable alterations in lipid metabolism. Furthermore, CPFA
have been identified as mildly carcinogenic and strongly
cocarcinogenic towards rainbow trout (Salmo gairdneri). The
mechanism by which CPFA enhance carcinogenesis is currently not
understood. The research in this thesis has therefore been
directed toward obtaining a better understanding as to how CPFA
induce toxic responses in rainbow trout.
Hepatic plasma membranes were isolated from both control
trout and trout which had consumed dietary CPFA. The plasma
membranes were then compared via the use of electron microscopy,
chromatographic analysis of phospholipid and fatty acid
content, two dimensional polyacrylamide gel electrophoresis of
proteins, and Western blot analysis of concanavalin A sensitive
glycoproteins. Electron micrographs revealed that control
plasma membranes appeared more homogeneous than CPFA membranes
and were characterized by more membrane sheets and less
vesicularization. The analysis of enzyme activities revealed
that CPFA caused a decrease in whole liver glucose-6-phosphatase
activity and that control plasma membranes expressed slightly
higher glucose-6-phosphatase and 5'-nucleotidase activities as
compared to CPFA membranes. Although dietary CPFA appeared to
have no effect on the phospholipid content of the plasma
membranes, significant alterations in the fatty acid profiles
of ethanolamine and choline phospholipids were observed. CPFA
caused a decrease in palmitic, palmitoleic and oleic acids while
the level of stearic and docosahexaenoic acids subsequently
increased. Differences between the protein content of control
and CPFA plasma membranes were made clear through the analysis
of electrophoretic and Western blotting data. Membranes
isolated from fish fed CPFA contained several proteins of high
molecular weight (above 66,000 daltons) and other proteins of
high isoelectric point that were not present in control plasma
membranes. Additionally, two families of glycoproteins which
had previously been identified as microsomal in origin were detected only in CPFA plasma membranes. A discussion concerning the possible causes and biological ramifications of
the observed subcellular alterations caused by CPFA insult is
also presented in this thesis. / Graduation date: 1989
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Diethylnitrosamine, ethylnitrosourea, and dimethylbenz(a)anthracene DNA binding studies in the rainbow troutVan Winkle, Samina 11 August 1988 (has links)
Dimethylbenz (a) anthracene (EMBA), a carcinogen that requires
metabolic activation to produce active metabolites capable of
binding to DNA, has been studied in the trout and other fish.
Polycyclic aromatic hydrocarbons (PAH) are of importance as they
are ubiquitous in the environment and their carcinogenic effects
in fish from contaminated waters are an important indication of
the pollution risks to man. Since such pollution risk assessment
presents the involvement of multiple agents, the study of the
modulation of PAH-DNA binding produced by other agents is
important. In this study the effect, of dietary pretreatment at
500 ppm, 100 ppm and 2000 ppn, using BNF, Aroclor 1254, or
indole-3-carbinol (I3C) respectively on DMBA-DNA binding was
examined. To study the effect of age on sensitivity to DMBA-DNA
binding, adult trout and fry were used in two separate studies.
The fish were fed treatment diet for at least two weeks. Fry were then injected with [³H] DMBA, at 22.4 μCi/3.9 x 10⁻² μmole/fish and adult trout at 284 μCi/1.58 μmole/fish. Liver DNA
was isolated, purified and binding of radioactivity to DNA was
examined and computed as the covalent binding index (CBI). Mean
CBI for control dietary group vising adult trout was 1000 fold
lower than for fry. Statistical analysis of covalent binding
index for the treatment groups revealed that a statistically
significant (p < 0.05) inhibition in DNA-DMBA binding response
in adult trout and fry was produced fcy the DNF dietary treatment
only.
Diethylnitrosamine (DENA), a potent hepatocarcinogen in
several animal species belongs also to the class of compounds
that require metabolic activation. Dietary treatment and
continuous exposure of trout to the carcinogen in water, have
produced hepatocellular carcinctnas. The water exposure also
produced a dose related DNA ethylation of the O⁶ position of
guanine, believed to be the promutagenic adduct produced after
DENA exposure. This study examines two other routes of exposure
to DENA, in vitro hepatocyte incubations and i.p. injection.
Adult trout and fry were injected with [³H] DENA. Adult fish
received 3.3, 16.5, and 33 mg/kg DENA, and fry received 10, 50
and 100 mg/kg. Hepatocyte incubation was performed with doses up
to 220 μM [³H] DENA, or 1 mM unlabelled DENA. DNA from fish
livers and from hepatocyte pellets was isolated, purified and
examined for radioactive binding of the DENA metabolites or in
the case of the unlabelled DENA, was analyzed for O⁶ and N7
adduct using an HPIC technique with fluorescence detection. O⁶-EtG adduct after DENA exposure, in DNA of hepatocytes obtained
from trout pretreated with beta-naphthoflavone (BNF, a known
inducer of cytodhrcme P-450 dependent enzyme activities involved
in the activation of xenobiotics) was below the limits of
detection of the HPDC-fluorescenoe detection procedure used. To
examine further if the lack of DNA binding and absence of the
O⁶-EtG adduct was due to rapid repair, the persistence of O⁶-EtG
after exposure to 40 nM ethylnitrosourea (ENU, a direct
ethylating agent) was studied in hepatocytes at 2, 4, and 5
hours after treatment. The activity of the alkyltransferase
protein involved in the repair of alkylguanines also was
determined using liver extracts from adult rainbow trout. The
studies did not reveal a significantly high rate of repair. It
is concluded that i.p. injection and in vitro hepatocyte
incubations are not a good method for studying the kinetics of
activation and DNA binding of DENA in the rainbow trout. The
i.p. route may lead to substantial loss of the dose of the
carcinogen administered and hepatocyte incubations are limited
by the toxic effects of increasing carcinogen concentration. The
reasons mentioned above, coupled with low levels of metabolism
of nitrosamines in trout results in the inability to detect and
study the appearance, persistence and repair of the O⁶-EtG
adduct. / Graduation date: 1989
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Cloning, sequencing and aflatoxin B��� metabolism by multiple rainbow trout CYP1A cDNAs expressed in yeastYou, Lijing 20 May 1997 (has links)
Previous reports indicate the presence of multiple CYP1A sequences in rainbow
trout, but their functional differences are unknown. This report describes the cloning and
partial characterization of four trout CYP1A cDNAs, which are given the tentative
designations CYP1A1v2, v3, v4, and v5. Comparison among these four and three
previously reported trout CYP1A sequences reveals that all of the nucleotide and
translated amino acid sequences all are closely related (96.9-99.4% cDNA identity; 95.2-99.4% amino acid sequence identity) but none are identical. Six of these sequences
encode proteins of 522 amino acids, and one encodes a protein of 536 amino acids.
Expression vectors containing the cDNAs for CYP1A1v2, v3, and v4 were transformed
into yeast, yielding microsomal hemoprotein CYP contents (63, 156, 96 pmol/mg)
comparable to those reported for human CYP1A1 (68-156 pmol/mg) expressed in this
system (Eugster et al., 1990, Biochem. Biophys. Res. Commun. 737-744). Kinetic analysis
of CYP1A1v2 and v3 proteins indicated similar but not identical Michaelis constants
(20��3 vs 13��2 ��M) and molar activities (508��47 vs 218��19 pmol/min/nmol P450) for
oxidation of aflatoxin B��� (AFB���) to aflatoxin M a reaction characteristic of human CYP1A2. Trout CYP1A1v2 and v3 exhibited lower activity for production of AFB,-8,9-exo-epoxide, also a human CYP1A2 activity. Kinetic data for ethoxyresorufin 0deethylation, a prototypical mammalian CYP1A1 activity, also revealed modest but distinct differences in which CYP1A1v3 was more active for this substrate (Km=0.07 �� 0.01 ��M, Vm=1398 �� 95 pmol/min/nmol P450) than was CYP1A1v2 (Km=0.15 �� 0.03 ��M, Vm=684 �� 83 pmol/min/nmol p450). Interestingly, CYP1A1v4 showed no catalytic activity towards AFB ethoxyresorufin, or 7,12-dimethylbenzanthracene despite formation of a hemoprotein. These results together with previous studies demonstrate the presence in various rainbow trout populations of at least seven CYPIA cDNAs representing gene duplication or allelic variation. Present results show that one of three such cDNA sequences encodes a CYP1A hemoprotein with no apparent catalytic activity, that two of the encoded proteins possess certain catalytic properties common to both human CYP1A1 and CYP1A2, and that the sequence differences, though small, are reflected in enzymic properties that can be distinguished. / Graduation date: 1998
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Three dimensional computer reconstruction of the rainbow trout (Oncorhynchus mykiss) hepatic tubuleTheis, Lisa C. 30 June 1994 (has links)
Graduation date: 1995
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Recovery from exhaustive exercise in rainbow trout white muscle : a model for studies of the control of energy metabolism in vivoSchulte, Patricia Marita January 1990 (has links)
Recovery from exhaustive exercise in the white muscle of rainbow trout (Oncorhynchus mykiss) was used to examine the role of the adenylates in the control of energy metabolism and to assess the validity of equilibrium models of the behaviour of the high energy phosphates.
The difficulty of obtaining muscle samples from fish makes detailed analysis of the behaviour of the labile high energy phosphates complex. The use of a new sampling procedure, the infusion of a lethal dose of anaesthetic via an indwelling cannula, minimized this problem.
At exhaustion [ATP] and [PCr] were depressed by 75 and 80% respectively relative to the resting values. [ATP] depletion was mirrored by a stoichiometric increase in [IMP]. During recovery [PCr] returned to the resting level within 2 hours, but [ATP] recovery was slow and not complete until 24 hours post exercise. In contrast, energy charge and RATP(the proportion of the free adenylate pool phosphorylated to ATP) were, if anything, higher than the resting values by 2 hours post exercise. Therefore, [ATP] and energy status can be dissociated in tissues like fish white muscle because of the action of the purine nucleotide cycle.
At 2 hours post exercise the calculated free ADP concentration dropped to less than one tenth the value at rest. As a result the [ATP] / [ADP] free ratio increased by nearly 6 fold. This condition may be required for glycogen resynthesis from lactate in muscle.
Several similar equilibrium models of the behaviour of the adenylates and PCr were applied to the fish white muscle system. In general, the models well describe the relationship between the high energy phosphates. However, the definition of the high energy phosphate pool introduces some complications since this includes the total [ATP]. Because of the action of AMP deaminase the [ATP] concentration can change without measurable changes in the energy status, which is not considered in any of the models. As long as the extent of IMP formation is known the models can be applied, but since the formation of IMP may vary from fish to fish or with exercise regime the models lose much of their predictive power. / Science, Faculty of / Zoology, Department of / Graduate
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Dexamethasone Stimulates Release of an ANP-Like Substance From Rainbow Trout CardiocytesPowell, W. H., Miller, Hugh A. 01 August 1992 (has links)
A substance that cross-reacts with antiserum to human atrial natriuretic peptide (ANP) is found in fish hearts. This ANP-like material increases sodium output from the gill and kidney while inhibiting sodium uptake in the gut. Mammalian ANP secretion is stimulated by glucocorticoids, and cortisol injection increases sodium output in salt-loaded fish. Therefore, we wanted to determine if the release of ANP in fish is sensitive to dexamethasone. Ventricle cardiocytes from the rainbow trout Oncorhynchus mykiss were treated with various doses of dexamethasone for 18 or 72 h. Single ventricle cells were then assayed for ANP release using a reverse hemolytic plaque assay and antiserum to human alpha-ANP. Incubation with 100 microM dexamethasone almost doubled the population of ventricle cells committed to ANP release (basal, 15.0 +/- 0.3% vs. Dexamethasone, 28.3 +/- 1.4%; values are percent plaque formation +/- SE). Stimulation of ANP secretion was dependent on dose and time of exposure to dexamethasone. These results suggest that ANP secretion in fish is regulated by glucocorticoids.
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