Agency
Research and Develn
Juresto
Assessment <
Clinical Proc<
Evaluate Liver
Intoxication in Fish
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3 Ecological Research
4, Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-.Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technicaTt>asis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-79-088
August 1979
ASSESSMENT OF CLINICAL PROCEDURES TO EVALUATE
LIVER INTOXICATION IN FISH
by
William H. Gingerich
and
Lavern J. Weber
Department of Fisheries and Wildlife
Oak Creek Laboratory of Biology
Oregon State University
Corvallis, Oregon 97331
Grant No. R 803090
Project Officer
James M. McKim
Physicological Effects of Pollution Section
Environmental Research Laboratory
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.
11
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FOREWORD
The ability to generalize from the results of research on specific
environmental toxicants and test organisms to classes of toxicants and
organisms depends to a large extent on knowledge of the mechanism of
toxicant action at the physiological level and the comparison of these
mechanism access taxonomic lines. The research reported here evaluates
the effect on liver function in the rainbow of several model liver
toxicants used in mammalian tests, as measured by existing clinical
diagnostic tests of liver dysfunction.
J. David Yount, Ph.D.
Deputy Director
Environmental Research Laboratory-Duluth
111
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ABSTRACT
Procedures were developed to clinically evaluate liver damage
and liver function in rainbow trout following either acute intra-
peritoneal (i.p.) treatment or subacute bath exposure to selected
mammalian hepatotoxic agents. Elevations in serum of liver specific
enzymes such as aspartate aminotransferase (GOT), alanine amino-
transferase (GPT) and alkaline phosphatase (AP) were investigated
as potential indicators of hepatocellular damage. An exogenous test
of liver function, plasma clearance of the organic anion sulfobromo-
phthalein (BSP), also was investigated as a potentially useful test
of overall liver function in the trout.
Histological damage was apparent in the livers of rainbow trout
treated by i.p. injection with either carbon tetrachloride (CC14
or monochlorobenzene (MCB), however this damage could not be corre-
lated consistently with indications of liver injury suggested by
either the endogenous or exogenous tests of liver damage. Total plasma
protein concentration decreased only in response to CC1* treatment
but GPT activity in the plasma was significantly increased following
treatment with both toxicants. Results also suggested that dietary
factors may have influenced the response of fish to treatment by CC14.
Plasma clearance of BSP was impaired following acute treatment with both
toxicants.
Unlike acute i.p. treatment with MCB, treatment with CC1,
resulted in significant weight gain in rainbow trout that was related
to water retention. Anuria or severe oliguria with attendant proteinuria
was evident as early as one hour after treatment and persisted for at
least 24 h. Histological examination of the kidney revealed incon-
sistent damage to the proximal tubules of some treated fish. No
detectable lesions were observed in the kidney of any fish earlier
than 36 h after treatment indicating that direct effects of CC14 on
the kidney probably were not responsible for the altered urine flow
rates observed early in the course of the intoxication.
In a separate study trout were exposed continuously to two sub-
lethal concentrations of monochlorbenzene (2.6 and 3.9 ppm) for 15
and 30 days. Fish treated with both concentrations of toxicant were
anorexic during the first 15 days of exposure and a dose-related weight
loss was observed in treated fish when compared to their paired controls.
Total serum protein concentration was decreased and activity of serum
GPT was increased in a dose-dependent manner at this time. Serum
BSP concentrations in treated and non-fed control fish were similar
but were significantly greater than BSP concentrations in the serum
IV
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of a separate group of fed control fish suggesting that food deprivation
might greatly influence the disposition of this compound by the trout.
Fish at both toxicant concentrations accepted food again between 15
and 30 days and appeared to tolerate better their exposure during this
time period. After 30 days of exposure serum activities of GPT and
AP from treated fish were elevated but were not significantly differ-
ent from their paired controls. The impaired plasma clearance of BSP
in treated fish observed at this time could not be related to MCB
exposure.
The application of clinical tests to diagnose liver dysfunction in
fishes following their exposure to environmental toxicants may be
practical in controlled laboratory facilities Despite the considerable
variation that exists between groups of fish, significant differences
could be demonstrated between control and treated fish. Variation among
groups of fish make intergroup comparison of the field populations
increasingly difficult by these methods,. Therefore, the use of such
techniques should be employed to evaluate liver toxicity under precisely
controlled laboratory studies„ Their application to field studies
does not seem advisable.
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CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables xi
1. Introduction 1
2. Recommendations and Conclusions 3
3. Materials and Methods 4
Holding Facilities and Culture Techniques 4
Development of Clinical Procedures to Evaluate
Liver Toxicity in Rainbow Trout 4
Acute Exposure Studies: Carbon Tetraehloride 10
Acute Exposure Studies: Monochlorobenzene 13
Subacute Exposure Studies: Monochlorobenzene 13
Gross Pathology and Histology 16
Statistical Methods 16
4. Results 17
Development of Clinical Procedures to Evaluate
Liver Toxicity in Rainbow Trout 17
Acute Exposure Studies: Carbon Tetraehloride 36
Acute Exposure Studies: Monochlorobenzene 57
Gross Pathology and Histology 63
5. Discussion 75
6. Publications Resulting from Project 94
7. References 95
VI1
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FIGURES
Number Page
1 Toxicant solution generator 14
2 Alanine aminotransferase (GPT) specific activity in rainbow
trout liver at increasing assay temperature.
20
3 Arrhenius plot of alanine aminotransferase (GPT) activity
in rainbow trout liver 22
4 Alanine aminotransferase (GPT) specific activity in rainbow
trout liver with increasing assay pH
5 Lineweaver-Burke plot of alanine aminotransferase (GPT)
activity in liver and kidney tissue from rainbow trout at
increasing assay concentrations of alanine
6a Alkaline phosphatase (AP) activity with increasing assay
temperature
27
b Alkaline phosphatase (AP) activity with increasing assay pH. .
7 Arrhenius plot of alkaline phosphatase (AP) activity in
rainbow trout liver .....................
8 Liver BSP content and plasma BSP concentrations in
surgically treated rainbow trout ..............
9 Biliary excretion of BSP during prolonged, graded infusion
in rainbow trout . . . . ..................
10 Representative chromatograms of BSP and BSP metabolites
appearing in the bile of rainbow trout during prolonged
infusion of the dye ..................... 35
11 Alanine aminotransferase (GPT) activity in plasma from rainbow
trout fed two different commercial fish diets and treated
with CCl4(1.0 ml/kg, i.p.) ...... ............ 38
12 Alanine aminotransferase (GPT) activity in plasma from rainbow
trout fed the Donaldson Diet and treated with CC14
(1.0 or 2.0 ml/kg, i.p.) ................... 39
Vlll
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Number Page
13 Plasma disappearance curve for BSP in control trout and
trout treated 24 h earlier with CC1.(0.2 or 2.0 ml/kg
i.p.) ............... 7 .......... 40
14 Plasma BSP retention in rainbow trout with time following
CC14 treatment (2.0 ml/kg i.p.) ............. 41
15 Plasma hemoglobin concentrations in control fish and fish
receiving CC14 12, 24, 96 and 120 h earlier ....... 42
16 Percent of a single dose of BSP (10.0 mg/kg) appearing in
the liver of trout 15, 30, 60 and 120 min after injection
in control fish and fish treated with CC14 24 h earlier. 45
17 Biliary excretion of BSP by control trout and trout treated
with CC14 24 h prior to the start of BSP infusion. 48
18 Total metabolized BSP appearing in the bile of control fish
or fish treated with CC1. during continuous, graded
infusion of BSP ..... ................ 49
19 Urine flow rate of Cort land- treated (2.0 ml/kg, i.p.)
control rainbow trout for 24 h pre- treatment and 24 h
post- treatment ..................... 53
20 Urine flow rate for CC1, treated rainbow trout (2.0 ml/kg,
i.p.) for 24 h pre-treatment and 24 h post- treatment . . 54
21 Plasma GPT activity ratios for rainbow trout with time after
treatment with monochlorobenzene (1.0 ml/kg, i.p.) ... 59
22 Mean plasma BSP concentrations in corn oil injected control
fish (1.0 ml/kg, i.p.) or in fish receiving MCB (0.5 or
1.0 ml/kg, i.p.) 24, 48, 72 h earlier .......... 60
23 Mean plasma BSP concentrations in corn oil treated control
trout (1.0 ml/kg, i.p.)°r in trout receiving MCB
(1.0 ml/kg, i.p.) 3, 12, 24, 36, 48, 72 h earlier. ... 61
24 Mean plasma GPT activities from baseline, fed and paired
control trout and trout exposed to two subacute concen-
trations of MCB (2.6 ppm and 3.9 ppm) for 15 and 30 days. 64
25 Mean plasma BSP concentrations from baseline, fed and
paired control trout and trout exposed to two subacute
concentrations of MCB (2,6 ppm and 3.9 ppm) for 15 and
30 days ......................... 65
IX
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Number Page
26 Relative weight gain in spinal transected control trout and
spinal transected trout receiving CC14(2.0 ml/kg, i.p.)
12, 24, 48, 96 and 120 h earlier 68
27 Photomicrographs of liver sections from rainbow trout .... 69
28 Relative weight change in rainbow trout following treatment
with either MCB (0.5 or 1.0 ml/kg, i.p.) or CCl.fl.O or
2.0 ml/kg i.p.) 70
29 Relative weight change in baseline, fed and paired control
trout and in trout exposed to two concentrations of MCB
(2.6 and 3.9 ppm) for 15 and 30 days 72
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TABLES
Number Page
1 Alanine Aminotransferase (GPT) and Aspartate Aminotransferase
(GOT) Activities in Plasma and Liver from Rainbow Trout. . . 18
2 Ratio of Plasma Alanine Aminotransferase (GPT), Aspartate
Aminotransferase (GOT) Activities, and Plasma Hemoglobin
Concentration after in vitro CCL^Induced or Physically-
Induced Hemolysis of Blood from Rainbow Trout 19
3 Alanine Aminotransferase (GPT) Activity and Protein Concentration
in Liver and Kidney Tissue from Rainbow Trout 24
4 Alanine Aminotransferase (GPT), Aspartate Aminotransferase (GOT)
Activities in Plasma and Liver and Protein Concentration in
Liver from Four Members of the Family Salmonidae 26
5 Serum Activity of Alkaline Phosphatase (AP) in Rainbow Trout
Following Ligation of the Cystic Duct and Common Bile Duct.
Each Value Represents the Mean ±SE of Five Fish 29
6 Serum Activity of Alkaline Phosphatase (AP) in Rainbow Trout
Following Treatment with ANIT (400 mg/kg I.P.). Values
are the Mean ±SE of Five Fish 29
7 Liver and Plasma Concentrations, Percent of Injected Dose and
Liver:Plasma Concentration Ratio of BSP Following a Single
i.v. Injection to Spinal Transected Rainbow Trout 31
8 A Comparison of Plasma and Liver Alanine Aminotransferase
(GPT), Aspartate Aminotransferase (GOT) Activities and
Liver Protein Concentration for Rainbow Trout Fed Two
Commercial Fish Diets. 37
9 Liver and Plasma BSP Concentrations Following its Administration
(10 mg/kg i.v.) to Control Fish and Fish Receiving CC14
(2.0 ml/kg i.p) 24 Hours Earlier. 44
10 Bile Flow, Bile BSP Concentration and Rate of Biliary BSP
Excretion 12 Hours After Beginning Infusion of BSP in
Control Fish and Fish Receiving CC14 (2.0 ml/kg i.p.)
37 Hours Earlier 46
XI
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Number Pase
11 Plasma Protein and Albumin Concentrations for Rainbow Trout
at 24 Hours Post-treatment with CC14, . . . . 51
12 Plasma Protein Concentration, Relative Body Weight Change and
Plasma Osmolality for Rainbow Trout Post-Treatment with CC14. 52
13 Urine Osmolality for Rainbow Trout Treated with Cortland
Saline or CC14 (2.0 ml/kg, i0p.) Determined Every 12 Hours
for 24 Hours Pre-treatment and 24 Hours Post-treatment. ... 55
14 Protein Concentration in Urine from Rainbow Trout Treated with
Cortland Saline or CC1. (2.0 ml/kg, i.p.) 56
15 Plasma GPT Activity, Percent of Animal Responding to Treatment
and Total Plasma Protein Concentration After i.p. Adminis-
Administration of Monochlorobenzene 58
16 Serum GPT and AP Activities, Serum Total Protein, BSP and
Hemoglobin Concentrations in Control Rainbow Trout and
Trout Exposed to Two Concentrations of MCB for 15 and 30 Days 62
17 Electrophoretic Distributions of Serum Proteins (gm/100 ml:
Mean ± SE) from Trout after 15 Days of Exposure to
Concentrations of Monochlorobenzene 66
18 Liver Weight to Body Weight and Spleen Weight to Body Weight
Ratios in Fish Exposed to Subacute Concentrations of
Monochlorobenzene for 15 and 30 Days . . ,
19 A Comparison of Alanine Aminotransferase (GPT) and Aspartate
Aminotransferase (GOT) Activities in Plasma and Liver from
Selected Species of Fishes 76
20 Dependence of Biliary Excretion of a Single Intravenous
Injection of BSP on the Bile Flow Rate in Different Species. 80
21 Comparison of Doses of Model Hepatotoxic Agents Used in
Mammalian and Fish Toxicology Studies 90
XII
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SECTION I
INTRODUCTION
Biologists now realize the need for technical capabilities to
measure the sublethal effects on aquatic organisms resulting from
exposure to water-borne environmental toxicants. Short-term exposure
tests designed to estimate the median tolerance limit (TLm) of chemicals
to aquatic life yield little understanding of the effects of chronic
exposure to low concentrations of these materials. On the other hand,
while long-term exposure studies may be generally useful in establishing
bioaccumulation rates and pharmacokinetic properties of certain
toxicants in aquatic species, little is understood of how best to
detect the subtle changes in physiological processes which result
in the intoxication of the animal.
The application of existing diagnostic tests of organ function to
the field of aquatic toxicology should shorten the search for fine
indicators of pollutant-induced physiological dysfunction in fishes.
Before this technology can be applied successfully to fishes however, a
more fundamental understanding of specific physiological processes
in these animals is essential. This understanding provides the
investigator with a point of reference from which to compare and contrast
similar physiological functions between divergent species and, in
so doing, to identify appropriate tests that may find useful application
in fisheries research.
The liver, one of the vital organs of a fish, has received limited
study in this regard, even though morphological damage to this organ is
most consistently reported in fish which have been exposed to a variety
of halogenated hydrocarbons (Johnson, 1968). Because the liver is
physiologically important to fishes for the maintenance of their
metabolic homeostasis, this organ may be particularly sensitive to
long-term sublethal exposure to a variety of toxicants. For this
reason the development of techniques to evaluate liver function in
fishes may be especially useful in detecting subtle pollutant-induced
changes in physiological function.
Clinical diagnostic tests of liver dysfunction generally have been
grouped into two broad categories, endogenous tests and exogenous
tests. Endogenous tests usually require the measurement of specific
enzymes in the plasma or serum or the estimation of serum concentrations
of other biological chemicals such as bilirubin that are routinely
handled by the liver. These biological chemicals generally increase
or decrease in the serum when liver injury occurs. Exogenous tests
usually involve the prior administration of an exogenous material
that is specifically taken up or metabolized by the liver. Examples
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of these tests are plasma clearance of liver specific dyes such as
sulfobromophthalein (BSP) or the conjugation by the liver of exogenous
benzoic acid with glycine and its subsequent urinary excretion as
hippuric acid. The latter tests generally measure the true function
of the organ in that the animal is given a quantity of material and
the ability of the liver to process this load is evaluated.
In the present studies we have evaluated both endogenous and
exogenous liver function tests in a representative cold water fish
species, the rainbow trout (Salmo gairdneri). Endogenous tests used
in these investigations included determination of changes in the serum
activity of the enzymes alanine-aminotransferase (SGPT) and
aspartate-aminotransferase (SGOT) and alkaline phosphatase (AP). Serum
elevations in the activity of the two former enzymes are useful indica-
tors of necrotic processes in the liver of mammals while elevations
in the serum activity of the latter generally are associated with
either an intra or extrahepatic pathological process (Plaa, 1968). The
exogenous test evaluated in these studies was plasma clearance of the
anionic dye sulfobromophthalein (BSP). Aspects of the functional
capacity of the liver to transport this dye from the plasma to the
bile also were studied.
In order to assess the usefulness of these tests in identifying
liver dysfunction, trout were treated by intraperitoneal (i.p.) injec-
tion with two chemicals, carbon tetrachloride (€014) and mono-
chlorobenzene (MCB). Both of these agents produce consistent necrotic
hepatotoxicity in mammals, including man, and in this respect they are
commonly used to model liver toxicity in experimental animals (Raisfeld,
1974). In some studies a third model liver toxicant, alpha-napthy1-
isothiocynate (ANIT), was used to produce liver dysfunction in the
trout. This agent is used to model intrahepatic cholestasis in
laboratory mammals (Plaa and Priestly, 1977). Finally, liver function
tests were evaluated in a group of fish exposed to subacute concen-
trations of monochlorobenzene in the water for 15 and 30 days.
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SECTION 2
RECOMMENDATIONS AND CONCLUSIONS
1) The tests used in these studies to clinically evaluate liver
dysfunction in rainbow trout can be successfully applied to carefully
controlled laboratory experiments. The variability in the values for
these clinical tests between and among the different groups of
test fish used in these studies suggest that their application to field
studies would be impractical.
2) The evaluation of liver function through endogenous indicators such
as change in the serum activity of liver specific enzymes was the most
discriminating indicator of liver toxicity in trout used in acute and
subacute exposure studies. Serum or plasma elevations in the activity
of alanine aminotransferase were consistently observed following
exposure to the model mammalian hepatotoxic agents carbon tetrachloride
and monochlorobenzene. Furthermore, unlike serum aspartate amino trans-
ferase activity, alanine aminotransferase activity was not influenced by
high concentrations of serum hemoglobin that resulted following acute
exposure of trout to either carbon tetrachloride or monochlorobenzene„
3) Diagnosis of hepatic dysfunction in rainbow trout using serum
alkaline phosphatase activity as a clinical indicator of bile stasis
does not appear to be feasible. No differences in the serum activities
of alkaline phosphatase were evident following either acute extrahepatic
cholestasis created by ligation of the common bile duct and cystic
duct or by treatment with the mammalian cholestatic agent alpha-
napthylisothiocynate. Increased serum alkaline phosphatase activity was
apparent in fish fed a high ration suggesting that diet may greatly
influence the serum activity of this enzyme.
4) The plasma clearance rate of the organic anion sulfobromophthalein
(BSP) can be used as a sensitive test of liver dysfunction in rainbow
trout following their acute exposure to toxicants. This test appears
to provide a direct method of detecting liver dysfunction in the trout,
however it is relatively non-specific and may be influenced by factors
not related to the liver. Our studies indicate that food deprivation
can significantly influence the results of this clearance test in trout.
Therefore it is recommended that this test not be used in long term
studies where food consumption is reduced or lacking. Furthermore,
the method requires a modest amount of technical expertise in the
injection of the dye before it can be used successfully. The difficulties
associated with this test may preclude its routine use as a diagnostic
aid.
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SECTION 3
MATERIALS AND METHODS
HOLDING FACILITIES AND CULTURE TECHNIQUES
Yearling rainbow trout (50-70 gin) were purchased from Roaring
River fish hatchery, Scio, Oregon and transported to the Smith farm
hatchery facilities of the Department of Fisheries and Wildlife where
they were held until of a proper size for use in experiments (100-450
gin). Fish were held at densities of 25 kg per tank in 6000 liter
circular tanks supplied with a continuous flow of well water (35 1/min).
The well water temperature was constant at 13°C, the pH was 7D3 and
the total alkalinity of the water as calcium carbonate was 80 mg/1.
Other fish used in some experiments included Skamania River steelhead
trout (Roaring River fish hatchery), kokanee salmon and brook trout.
These fish were held at Smith faun under conditions similar to those
described for rainbow trout.
Prior to use in experiments fish were transferred to laboratory
holding facilities at either Nash Hall on the OSU campus or at Oak
Creek Laboratory and acclimated to these facilities for at least one
week. Fish taken to Nash Hall were held in a constant temperature room
in 130 1 aquaria supplied with continuously flowing (3 1/min) dechlorinated
tap water (12.0°C ± 0.5°). A 12 h light:12 h dark photoperiod was
maintained constantly. Fish were held at Oak Creek Laboratory in 500 1
aquaria supplied with a continuous flow (10 1/min) of well water (11°C or
15°C). The photoperiod in the laboratory was adjusted every other
week to conform to the natural photoperiod.
During the course of these studies diets from several sources were
fed to the fish. In general, however, individual groups of fish were
fed only one diet. Diets used in these studies included Purina
Trout Chow (Ralston-Purina, St. Louis, Mo.), Donaldson Diet (Ore-Aqua,
Inc., Newport, Oregon) and Siever Cup Diet (Murray Elevators, Murray,
Utah). In all instances fish were fed a diet estimated to be slightly
above their maintenance requirements.
DEVELOPMENT OF CLINICAL PROCEDURES TO EVALUATE LIVER TOXICITY IN RAINBOW
TROUT
Endogenous Tests
Plasma and Serum Enzymes—
Alanine Aminotransferase (GPT) and Aspartate Amino Transferase (GOT)
activities were determined in the liver and plasma from rainbow trout.
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Fish were fed Purina Trout Chow daily but food was withheld the day
before sampling. Blood was collected with tuberculin syringes, previously
rinsed with sodium heparin, and deposited into small glass test tubes
that had been rinsed with 10% potassium oxalate. Livers were removed and
held on ice until homogenized.
The"activity of GPT in kidney and liver tissue was compared in a separate
group of fish acclimated to 15°C ± 1.0° and fed Purina Trout Chow daily.
Liver and kidney tissues were removed and held on ice until homogenized.
The activities of GPT and GOT from the plasma and livers of rainbow
trout, kokanee salmon and brook trout fed Donaldson Diet were compared.
In another study a comparison of plasma and liver activities was made between
steelhead trout held under laboratory conditions for 60 days or held in a fine
meshed cage for 60 days in the Willamette River. Both groups of animals were
fed Purina Trout Chow throughout the study. Liver and blood samples were
collected as previously described.
A Gilford model 2400 recording spectrophotometer fitted with a
Haake constant temperature recirculating water bath was used for all
clinical assays. A constant temperature of 25°C was maintained in
all cases except where otherwise indicated. Reagents were purchased
from Sigma Chemical Co., St. Louis, Mo. and ammonia-free lactate dehydro-
genase was obtained from Boehringer-Mannheim, San Diego, Calif. Whenever
possible the procedures, concentrations and volumes as described in
the appropriate Sigma Technical Bulletin were used for these tests.
Plasma or serum alanine aminotransferase (GPT) activities were
measured by the method of Wroblewski and LaDue (1956), however tissue
GPT assays were modified to accommodate the presence of glutamate
dehydrogenase (GDH) as described by Bergmeyer and Bernt (1974). The
final reaction mixture for GPT assays contained 2.0 ml NADH, 0.1 mg/ml;
0.1 ml lactate dehydrogenase, 28.8 yM/ml in 50% ammonia-free glycerol;
0.5 ml alanine, 0.4 M in 0.35 M phosphate buffer, pH 7.5; 0.2 ml
alpha-ketoglutarate, 0.1 M in 0.1 M phosphate buffer, pH 7.5; and 0.2
ml of serum or diluted tissue homogenate. The enzyme reaction was
initiated with alpha-ketoglutarate (AKG) after ensuring that the
preliminary reaction had depleted extraneous substrates. The loss of
absorbance at 340 nm was recorded for 5 min.
The GPT activities of blood and tissue were determined on the
day that fish were sampled even though preliminary experiments indicated
that serum GPT activity was stable for at least one week if the sample
was stored under refrigeration (4°C). Tissue GPT activities were
measured in the liver and middle or trunk portion of the kidneys.
Whole organs were homogenized (10% u/v) in 0.25 M ice cold sucrose
buffered at pH 7.4 with 0.05 M Tris-HCl, 0.025 M KC1, and 5mM MgCl2
(TKM buffer, Cousins, et al., 1970). The homogenate was centrifuged
for 15 min (600 xg; 0-4°C) to remove blood cells and cellular debris.
The supernatant was used directly to determine the alanine-Km but was
diluted five fold (1:4) with sucrose-TKM buffer for standard assays
of GPT activity. The concentration of AKG used for the alanine-Km
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experiments (7mM) was 10-20 times the known Km value for AKG in human
plasma or pig heart GPT activities (Bergmeyer, 1978).
Plasma, serum and tissue (GOT) activities were determined by the
method of Karmen (1955) and modified in a manner similar to that for
GPT assays to account for the presence of endogenous GDH activity.
Sample preparation and tissue handling techniques were the same as
described for GPT analyses. The final reaction mixture for the GOT
assay was 2.0 ml NADH, 0.1 mg/ml; 0.1 ml lactate dehydrogenase, 28.8
u M/ml in 50% ammonia-free glycerol; 0.5 ml aspartate, 0.4 M in 0.35 M
phosphate buffer, pH 7.5; AKG, 0.1 M in 0.1 M phosphate buffer; pH 7.5;
and 0.2 ml of serum or diluted tissue homogenate.
Alkaline phosphatase (AP) activities were determined by modifying
the method of Bessey et al., (1946) to accommodate the use of 2-amino-
2-methyl-l-propanal buffer (1.5 M, pH 10.3). The final reaction mixture
consisted of 1.0 ml buffer, 1.0 ml substrate solution (p-nitrophenyl-
phosphate, disodium salt 6^0, 0.4 mg/ml), and 50 yl of serum
or diluted tissue homogenate. The buffer and substrate were mixed in
a quartz cuvette, equilibrated to assay temperature, and the enzyme
source added. Enzyme activity was determined by recording the change
in absorbance at 410 run and comparing this with a previously established
calibration curve. Serum AP activity was stable at least 2 weeks
when the samples were stored under refrigeration. Liver tissue was
homogenized in chilled 0.15 M KC1 (1:10, w/v) and centrifuged for
15 min. The resulting supernatant was used directly for protein deter-
mination and diluted (1:3, u/v) with 0.15 M KC1 for determination of AP
activity.
The influence of acute extrahepatic biliary obstruction on the
serum activity of AP was determined in one group of five trout. Fish
were prepared by ligation of the cystic duct and common bile duct and
a blood sample was drawn from each animal after 3, 24, 48 and 72 h.
In a second experiment the effect on a mammalian cholestatic agent
(alpha-napthylisothiocynate, ANIT) was determined on the activity of
AP in the serum in rainbow trout. A group of five fish was given ANIT
400 mg/kg i.p.) dissolved in salmon oil and after 3, 6 and 24 h a single
blood sample (0.5 ml) was drawn from each fish by cardiac puncture.
Control fish received an equivalent volume of salmon oil.
Other Endogenous Tests—
Plasma, serum and tissue protein concentration were determined by the
biuret reaction (Gornall, et al., 1949) using Sigma kit no. 540. Volumes
were adjusted to use 1.0 ml biuret reagent and 0.02 ml sample. A reagent
blank was included with each protein determination to account for erron-
eously high protein concentrations resulting from the influence of
turbidity in tissue samples or from increased absorbance due to high
concentrations of bromosulfophthalein (BSP) in the sample. Serum albumin
concentrations were measured by the bromocresol green method (Doumas
and Biggs, 1972) as described in Sigma Technical Bulletin No. 630.
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Serum proteins were separated by electorphoresis on cellulose
acetate strips. Serum (3 ul) was applied to each strip, placed in an
electrophoresis chamber containing 0.5 M tris-sodium barbital buffer
(pH 8.8) and the separation was conducted for 20 min (4 milliamp per
strip, 180 Vdc). Strips were stained with Ponceau S and then scanned,
traced.and the resulting peaks integrated using a scanning densitometer.
A linear relationship was assumed for all protein bands between color
intensity and protein concentration.
Serum hemoglobin concentrations were measured as cyanmethemoglobin
using 0.5 ml of modified Drabkins solution (Richterich, 1969) and 50 PI
of serum. Because intra-peritoneal administration of both carbon tetra-
chloride (CC14) and monochlorobenzene (MCB) resulted in increased
intra-vascular hemolysis, serum hemoglobin values were used to correct
for total serum or plasma protein concentrations as indicated in Sigma
Technical bulletin 540. The concentration of bilirubin in the plasma
was determined by the diazotization procedure of Malloy and Evelyn
(1937).
Disposition of BSP by Rainbow Trout—
Animals used in all experiments were immobilized by transection
of the spinal cord. This method of immobilization simplifies the technical
difficulties associated with estimating biliary BSP excretion rates and
does not appear to significantly alter either the rates of plasma clearance
or biliary excretion of the dye relative to those of free swimming fish
(Schmidt and Weber, 1973). After immobilization, animals were weighed and
placed into individual troughs of a plastic coated wire frame support
within a plexiglas aquarium and allowed to recover at least 18 h.
Plasma clearance and hepatic accumulation of BSP were determined
in two groups of five fish (220-290g). In experiments requiring timed
serial sampling of blood from a single fish, a cannula was inserted
into the caudal vein at a point just ventral to the lateral line and
immediately above the anterior insertion of the adipose fin. The cannula
consisted of PE tubing of known volume (50 Pi). The shaft of a 23 gauge
needle was attached to one end with the hub of the needle fitted to
the other end. A suture in the caudal peduncle secured the cannula
to the fish. A solution of BSP in physiological saline (either 5.0
or 10.0 mg/kg) was injected by the caudal vein cannula and 002 ml blood
samples were taken every 15 min for one hour. Plasma volume was maintained
by reinjecting an equivalent volume of heparinized (100 U»S.P. units/ml)
saline following the withdrawal of each blood sample. The plasma half
life of BSP was estimated from the slope of a line visually fit to a plot
of the points of the log of plasma BSP concentration vs time. The frac-
tional turnover rate (ft) of BSP was c = 0.693/T 1/2 where T 1/2 is the
plasma half life of BSP in min and 0.693 is In of 1/2.
To determine the concentration of BSP in the liver and plasma,
fish were sampled 15, 30, and 60 min after a single dose of BSP (10.0
rog/kg) had been injected into the caudal vein. Each fish was stunned
by a blow to the head, a blood sample taken by cardiac puncture, and
-------
the liver removed. Livers were perfused with 10 ml of chilled
physiological saline by the hepatic portal vein and then placed on
absorbent paper pads on ice.
The effects of impaired hepatic blood flow or bile flow on plasma
clearance and hepatic accumulation of BSP were determined in three
groups of five fish after preparation by the following surgical treat-
ments. The cystic ducts and common bile ducts of the fish in the
first group were ligated with 5-0 silk sutures. In the second group
the cystic duct, common bile duct and hepatic portal vein were ligated.
Sham surgery involving isolation of the ducts and vessels without
ligation was performed in the fish of the third group. The incisions
were closed with 4-0 surgical silk sutures and the trout were allowed
a 10 h recovery period. Surgically prepared fish were used in experiments
to determine either plasma clearance or hepatic accumulation of BSP as
previously described.
To determine the rate of biliary excretion of a single dose of BSP
the common bile duct was cannulated with PE 10 tubing of known volume
(40 id) and the cystic duct ligated (Schmidt and Weber, 1973). Bile duct
cannulation was accomplished in anesthetized trout that had been fasted
for at least 18 h. The fish was placed in dorsal recumbancy on a V-
shaped trough positioned such that the head and gills were immersed
in free flowing water. An incision (5 cm) was made along the linea alba
and the abdomen retracted with butterfly forceps. The liver was identified
at the anterior center of the incision and the gall bladder and common bile
duct generally were found in the posterior right quadrant of the liver.
Viscera were retracted to the left and the cystic duct was identified and
ligated with a 5-0 silk suture. The common bile duct was isolated
from the surrounding vasculature by careful blunt dissection with
glass probes and exposed. Two ligatures of 4-0 silk were positioned
along the bile duct, one proximal and one distal to the liver.
A lateral incision was made in the common bile duct between the two
ligatures with iris scissors and a length of polyethylene tubing (PE 10) was
inserted through the incision toward the liver. The tubing was secured
to the common bile duct by the two ligatures. The catheter was passed
out through the posterior end of the incision and was fixed to the skin
with 5-0 silk sutures. The incision was closed with 5-0 silk sutures
and the animal allowed to recover for 12 h. With practice this procedure
could be accomplished in 30-40 min.
After a 12 h recovery period, a single dose of BSP (10.0 mg/kg)
was injected into the caudal vein and then bile flow was determined every
half hour for six hours. Bile was collected into PE 90 tubing which was
volume calibrated in 10 vl intervals and attached to the bile duct cannula
by a collar of PE 50 tubing. Bile flow rates were determined by recording
the progress of the bile in the collecting cannula. The bile produced in
each half hour period was obtained by cutting the tubing into segments
corresponding in length to the volume of bile produced during each period.
The biliary transport maximum (Tm) for BSP and metabolites was
determined in spinal transected trout in a manner similar to that described
by Schmidt and Weber (1973). These fish received BSP by graded infusion
-------
over a 12 h period. The initial infusion rate (20 yg/kg/min) was maintained
for 4 h and then the rate was increased to 40 and then 60 y g/kg/min in two
ensuing 4 h periods. After 12 h the infusion was discontinued and the trout
received a single dose of BSP (5.0 mg/kg) by the caudal vein to insure that
the excretory capacity of the liver for BSP had been exceeded. Bile was
collected.into a lengthof ffi 90 tubing as described earlier. Bile flow rates
were determined every hour by recording the progress of the bile in the
collecting cannula. Collecting cannulae were changed after 6, 12, and
15 h to prevent longitudinal mixing of the BSP in the tubing. No attempt
was made to replace bile salts lost during these experiments.
The concentration of BSP in the bile and plasma was estimated
colorimetrically after appropriate dilution of each sample with alkaline
buffer solution (Richterich, 1969). Absorbance was read at 578 nm on
a Beckman DB spectrophotometer and converted to units of concentration
by comparison with BSP reference standards,, A blank for each sample
was obtained by acidifying the sample with acid buffer solution (Richterich,
1969). The extinction coefficients of BSP and its metabolites in the
bile and liver of trout were assumed to be equal (Combes, 1965; Whelan,
et al., 1970).
The concentration of BSP in the liver was determined by a modifica-
tion of the method of Whelan et al. (1970). Livers were weighed, minced,
and then homogenized on ice in Potter-Elvehjem tissue homogenizers.
Approximately 0.5 g (±0,02g) of the homogenate was weighed into a tared
screw cap test tube and extracted twice with 10 ml volumes of 75%
methanol in water (V/V)„ After each addition of solvent the homogenates
were shaken and then centrifuged for 10 min (1850 x g)„ The methanol
supernates were combined and brought to a final volume of 25 ml with 75%
methanol-water. Concentrations of BSP were determined from 100 yl samples
of this final extract in a manner identical to that described for plasma
and bile BSP. Recoveries of BSP using this method were greater than 97%.
Liver extracts were prepared for chromatographic separation of
free and metabolized BSP by the method of Whelan and Combes (1971).
The residue containing the dye was reconstituted with equal volumes
(20 yl) of distilled water and 75% methanol in water (V/V). A portion
(10 yl) of the reconstituted extract was applied to TLC strips and
chromatographed. Samples of bile (205 or 5.0 yl), collected 1, 2, 4, 6,
8, 10, 12 and 14 h after the start of BSP infusion, were applied directly
to TLC strips and chromatographed.
Free BSP and its metabolites were separated by thin layer chromato-
graphy on precoated microcrystalline cellulose TLC.strips (Baker-flex,
J. T. Baker Chemical Co., New Jersey) following the procedure of Whelan
and Plaa (1963). Ninhydrin reagent (Nutritional Biochemicals Co., Cleveland,
Ohio) was sprayed on thin layer strips to detect amino acid conjugates
of BSP while aniline diphenylamine reagent (Sigma Chemical Co., St. Louis,
Mo.) was used to detect carbohydrate conjugates. Standards of BSP were
prepared by adding a solution of BSP in physiological saline to freshly
collected plasma, bile or to liver homogenates. BSP fractions not having
relative mobility (Rf) values similar to those of the BSP standards
-------
were considered to be metabolites of the dye.
The proportion of metabolized dye that appeared in liver extracts or
bile was determined by eluting either free or metabolized BSP fractions
from the TLC strips into separate test tubes with alkaline buffer (Richterich,
1969) and the optical density read at 578 nm. The relative contribution of
metabolized BSP was determined as the ratio of the optical density of
the metabolized BSP to the sum of the optical densities of both free and
metabolized BSP. The optical densities of all samples were within the
linear portion of the calibration curve prepared for BSP.
ACUTE EXPOSURE STUDIES: CARBON TETRACHLORIDE
Determination of Median Lethal Dose
The median lethal dose of carbon tetrachloride to rainbow trout
was estimated by the method of Brownlee et al. (1953). Basically this
method involves treating the animal with a given dose of toxicant and if
the animal dies the dose is decreased by a fraction of the original dose
and the trial repeated. This procedure is replicated three succes-
sive times after obtaining one positive and one negative response
with two successive doses. The dosage increment used in this study was
a 25% increase in the first negative response obtained and the dosage
interval ranged from 1.6 to 5.0 ml/kg (i.p.) of undiluted CC14.
Control fish received a comparable dose of physiological saline (Wolf,
1963). Mortality was recorded every 24 h for 96 h. The LD50 value
was estimated by dividing by five the sum of the last four consecutive
doses of toxicant plus that dose of toxicant that would have been given
as the sixth dose.
Dose and Time Response Studies; GPT
The effect of CC14 intoxication on the activity of GOT and GPT in
olasma and liver was determined in fish fed either Purina Diet (Ralston
Purina St. Louis, Mo.) or Donaldson Diet (ORE-AQUA, Newport, Ore.). Fish
in all studies were weighed, marked with an identifying fin clip and
allowed to recover in a 200 1 aquarium supplied with continuously flowing
well water (11.0°C or 15.0°C). After 24 h fish were given either undiluted
CC14 (1.0 ml/kg or 2.0 ml/kg, i.pi or an equivalent volume of Cortland's
saline (Wolf, 1963). Blood and tissue samples were taken after 3, 6,
12, 18 or 24 h from fish fed the Purina diet or after 3, 6, 12, 18,
24, 36 or 48 h from fish fed the Donaldson diet.
Dose and Time Response Studies; BSP
Animals used in all experiments were immobilized by transection of the
spinal cord. This method of immobilization simplifies the technical diffi-
culties associated with estimating biliary BSP excretion rates and does not
appear to significantly alter either the rates of plasma clearance or
biliary excretion of the dye relative to those of free swimming fish
(Schmidt and Weber, 1973). After immobilization animals were weighed and
placed into individual troughs of a plastic coated wire frame support
10
-------
within a plexiglas aquarium and were allowed to recover at least
18 h.
The effect of CC1, intoxication on plasma clearance of BSP was
determined following administration of undiluted CC14 (0.2 or 2.0
ml/kg} or an equivalent volume of physiological saline. After 24 h
a cannula was placed in the caudal vein and a single dose of
BSP (5.0 mg/kg) in physiological saline was injected into the caudal vein.
Blood samples (0.2 ml) were taken from the cannula every 15 min for one hour
and plasma volume was maintained by reinjecting an equivalent volume of
heparinized (100 U.S.P. units/ml) physiological saline following withdrawal
of each blood sample. The plasma half life of BSP was estimated from the
slope of a line visually fitted to a plot of the points of log plasma BSP
concentration vs time.
Fish used in time-response studies received either undiluted CC14
(2.0 ml/kg i.p.) or an equivalent volume of physiological saline 12, 24, 48,
96, and 120 h prior to BSP administration. BSP (5.0 mg/kg) was injected into
the caudal vein and after 45 min a 0.2 ml blood sample was taken by cardiac
puncture. Immediately prior to administration of the dye a blood sample was
taken from the caudal vin for estimation of the plasma hemoglobin concentra-
tion.
The effect of high plasma concentrations of bilirubin or hemoglobin
on plasma BSP clearance was determined in two groups of seven fish. Bili-
rubin was dissolved in a solution of 0.5 g Na2COj and 0.5 g NaCl per 100 ml
water (Weinbren and Billing, 1956) and stabilized with 25 mg/100 ml of bovine
serum albumin. Solutions, of appropriate concentration for each fish, were
prepared in a darkened laboratory with the aid of a photographic dark room
light and held overnight at 4°C in foil wrapped injection vials.
Animals were prepared for infusion experiments by exposing the ventral
intestinal vein at a point between the pelvic fins and the anus and inserting
an infusion cannula (PE 10 tubing). The wound was tightly closed with 4-0
surgical silk sutures and the fish were allowed a 30-60 min recovery period.
A cannula was inserted into the caudal vein and a loading dose of bilirubin
(7.0 mg/kg) was administered by this cannula immediately prior to the start
of infusions. Bilirubin was infused (40 ug/kg/min) for 4 h using a Sage
model 341 variable speed syringe pump and 3 h after the infusion began BSP
(5.0 mg/kg) was injected by the caudal vein cannula and serial blood samples
were taken every 15 min for one hour. Control fish received bilirubin
vehicle in a similar manner over the same time period.
In experiments requiring the infusion of hemoglobin, a hemolysate
was prepared from the blood of donor trout in a manner similar to that
described by Ostrpw et al. (1962). The tonicity of the hemolysate was brought
to 300 milliosmol/kg with 5% (W/V) NaCl solution, the pH was adjusted to 7.3
with 0.15 M phosphate buffer and the hemoglobin content was adjusted with
physiological saline to a concentration appropriate for each fish. A loading
dose of hemoglobin (40 mg/kg) was administered and hemoglobin was infused
(250 yg/kg/min) for 4 h. After 3 h animals received a single i.v.
11
-------
injection of BSP (500 rag/kg) and blood samples were withdrawn by the caudal
vein cannula every 15 rain for one hour.
The influence of CC14 intoxication on the distribution of BSP between
the liver and plasma was determined in four groups of five fish. Animals
received either CC14 (2.0 ml/kg i.p.) or an equivalent amount of physiologi-
cal saline. After 24 h BSP (10.0 mg/kg) was injected into the caudal vein
and fish were sampled after 15, 30, 60, and 120 min. Each fish was stunned
by a blow to the head, a blood sample taken by cardiac puncture, and the
liver removed. Livers were perfused with 10 ml of chilled physiological
saline by the hepatic portal vein and placed on absorbent paper pads over
ice.
The influence of CCl^ intoxication on the biliary excretion of BSP was
determined in five control and three treated animals. Fish received either
undiluted CCl^ or an equivalent volume of physiological saline (2.0 ml/kg
i.p.) and after 12 h the common bile duct was cannulated with PE 10 tubing of
known volume (40 ul) and the cystic duct was ligated (Schmidt and Weber,
1973). An infusion cannula (PE 10 tubing) was inserted into the ventral
intestinal vein and the incision closed with 4-0 surgical silk sutures. Free
CC14 was not apparent in the peritoneal cavities of animals treated 12 h
earlier. No attempt was made to replace bile salts lost during the experiment,
Animals were administered BSP by graded infusion over a 12 h period as
indicated in a previous section of this report.
Effect of Acute CCla Intoxication on Plasma Protein
Concentration and Water Balance
In experiments to determine the effect of CCl^ on plasma pro-
tein concentration and wet whole body weight change fish were weighed,
given an identifying fin clip and held in 200 1 aquaria supplied with
continuously flowing well water. After 24 h undiluted CC14 (1.0 or
2.0 ml/kg, i.p.) or an equivalent volume of Cortland's saline (Wolf, 1963)
was given. Blood samples were drawn and wet whole body weights were
determined in individual groups of fish 0, 3, 6, 12, 18, 24, 36 and 48 h
after treatment.
In a second group of experiments fish were weighed, fin clipped
and held in 200 1 aquaria and after 24 h individual groups of fish were
treated with 0.25, 0.5, 1.0 or 2.0 ml/kg of undiluted CC14. Wet
whole body weights were taken and blood samples drawn 24 h after
treatment.
For experiments testing the influence of CC14 intoxication on urine
flow rates fish were anesthetized in a solution of MS-222 (50 mg/1), weighed
and a urinary catheter inserted into the urinary bladder and secured by
sutures to the base of the anal fin. The fish was placed in a plexiglas
restraining chamber within a larger aquarium supplied with continuously
flowing well water (15°C ± 1.0°). Urine was collected into conical graduate
centrifuge tubes positioned in a linear fraction collector. After 24 h fish
12
-------
were removed from the aquaria, reanesthetized, reweighed and given a dose of
undiluted CC14 (2.0 ml/kg, i.p.) or an equivalent volume of Cortland's saline
(Wolf, 1963). Fish were repositioned in the restraining chamber and urine
again was collected for hourly intervals over a 24 h period. A separate
group of nine fish were catheterized and not disturbed for 48 h. Urine from
this group was collected into separate tubes for hourly intervals for the
entire 48 h period.
ACUTE EXPOSURE STUDIES: MONOCHLOROBENZENE
Determination of Median Lethal Dose
Four groups of 3 trout each were treated with MCB using a dose range of
from 1.0-3.0 ml/kg. MCB was diluted with corn oil (1:1, vol:vol) and admin-
istered by i.p. injection. The number of dead animals per group was recorded
daily for 3 days. The median lethal dose was calculated for 24, 48 and 72
h by the method of Weil (1952).
Time Response Studies: GPT
Experimental fish were weighed, marked with an identifying fin clip and
treated with MCB (1.0 ml/kg, i.p.) diluted with an equal volume of corn oil.
Control animals received a similar volume of corn oil. Fish from individual
treatment groups were placed in separate 150 1 aquaria supplied with con-
tinuously flowing (10 1/min) well water of constant temperature (15°C ± 1°).
Blood samples were drawn into heparinized tuberculin syringes from
separate groups of fish 3, 8, 12, 24, 36, 48, 72 and 96 h after treatment and
plasma was held on ice until enzyme analyses were performed.
Dose and Time Response Studies: BSP
Experimental fish used in dose and time response studies with BSP were
handled in a manner similar to those used in studies of plasma GPT activity.
Treated fish were weighed, marked with an identifying fin clip and were given
either 0.5 or 1.0 ml/kg MCB, dissolved in corn oil, by" i.p. injection. Fish
treated with 0.5 ml/kg were sampled 24, 48 and 72 h after treatment and
groups treated with 1.0 ml/kg were sampled after 3, 12, 24, 36, 48 and 72 h.
Prior to sampling fish were administered a dose of BSP (5.0 mg/kg, i.v.) and
after 45 min each fish was stunned by a blow on the head, a blood sample
drawn from the caudal vein and the BSP concentration in the serum determined.
SUBACUTE EXPOSURE STUDIES: MONOCHLOROBENZENE
Preparation and Introduction of Toxicant
A saturated solution of monochlorobenzene was continuously produced by
means of a self-regulating stock solution generator. The entire generator
apparatus consists of a dilution water head box and a toxicant stock solution
reservoir (Fig. 1). In principle of operation, the dilution water headbox
functions to establish a constant head pressure which ensures a regulated
13
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Dilution Water Headbox
TOXICANT SOLUTION GENERATOR
Dilution System
_
Valve <•
T
1
i
1
"s^
1 **•
^
Magnetic Drive Pumps
(1/20 HP)
Dilution
Water
Manifold
Variable
Voltage
Transformer
Toxicant Toxicant-Water
Reservoir Mixing Flask
Figure 1. Toxicant solution generator
-------
flow of water to both the main diluter apparatus and to the toxicant reser-
voir. The stock solution reservoir consists of a 200 liter glass aquarium
separated into an affluent and effluent compartment by a glass partition.
Water enters the affluent compartment by gravity flow and is pumped from this
compartment by a small magnetic drive pump through a volume of monochloro-
benzene and into the effluent compartment. The solution is pumped at a rate
in excess of its rate of withdrawal from the reservoir and the excess solu-
tion flows over the glass partition and recirculates within the toxicant
reservoir. The rate of recirculation of solution within the reservoir is
adjusted by a variable voltage transformer connected to the recirculating
pump. The pattern of water recirculation and inflow within the reservoir
provides a constant and self-regulated renewal of stock toxicant solution.
Diluent water and stock toxicant solution were delivered to test aquaria by a
diluter system as described by Chadwick et al. (1972). Individual fish were
held in separate glass aquaria that contained an exposure volume of 28 1 and
received a continuous flow (400 ml/min) of constant temperature (15°C ± 1.°)
well water. Under these conditions a volume of diluted toxicant equal to
the aquarium volume entered each test aquaria every 1.18 h and the 99 percent
replacement time for toxicant solution in individual aquaria was estimated
to be five hours.
Determination of Median Lethal Concentration
The 96 h median lethal dose of MCB was determined by bath exposure
of five groups of 4 trout in a continuous flow bioassay. The 96 h median
lethal concentration was determined by the logit linear regression method of
Ashton (1972). Confidence interval estimates were made using a computer
program designed by D. A. Pierce (Department of Statistics, Oregon State
University). The concentration of MCB in the water was determined by direct
injection of a water sample into a Waters Associates high performance liquid
chromatograph fitted with a C18 microbond column and a model 444 UV absor-
bance detector.
Effects of Prolonged Subacute Exposure
Two groups of 8 trout were exposed continuously to two concentrations of
MCB (2.6 or 3.9 ppm) for either 15 or 30 days and two separate groups of 8
fish served as individual controls for treated animals. All fish were held
in separate aquaria and control fish were paired randomly with treated fish.
Food was offered to all treated fish daily during the experiment and each
control animal was fed a ration equivalent to the relative amount of food
consumed by the exposed member of the pair. A second group of 8 control fish
was held under conditions similar to those of the paired control animals but
these fish were fed daily as much food as they would consume in a 15 min
feeding period. Baseline control values were determined from two groups of
10 fish that were held in a community tank and were sampled either before or
after the 30 day exposure study. Data for both baseline groups of fish were
pooled after a test for homogeneity between variances of the two groups
indicated that no differences existed between the two sample populations.
15
-------
Half of the fish .exposed to both concentrations of toxicant, their
paired controls and half of the fish maintained on the unrestricted ration
were sampled 15 days after the beginning of the exposure and the remaining
fish were sampled after 30 days of exposure. Food was withheld and fish were
weighed 24 h prior to sampling.
On the day of sampling, fish were injected with BSP (10 rag/kg, i.v.) and
after 45 min they were stunned by a blow to the head, the wet whole body
weight recorded and a blood sample was drawn from the caudal vein. A lapa-
rotomy was performed, observation of the gross pathology of the peritoneal
cavity recorded and the liver and spleen removed, weighed and sections of
each tissue fixed in Bouin's solution for histological examination.
GROSS PATHOLOGY AND HISTOLOGY
Observations of gross pathological changes in the peritoneal cavity were
made and tissue samples were taken for histological studies in every experi-
ment. Liver weight to body weight ratios were determined in spinal tran-
sected rainbow trout treated with CC14 (2.0 ml/kg, i.p.) or in free swimming
trout treated with MCB (1.0 ml/kg, i.p.). Liver weight to body weight and
spleen weight to body weight ratios were determined in trout exposed to both
sublethal concentrations of MCB for 15 and 30 days.
Liver sections were taken from free swimming or spinal transected
trout every six hours for 24 h following treatment with CC14 (2.0 ml/kg
i.p.). Sections of trunk kidney were taken 24 h after treatment with CC1,
(1.0 ml/kg) in conjunction with studies of water balance in trout. Liver
slices were taken from trout 24, 48, 72 h after acute treatment with mono-
chlorobenzene (1.0 ml/kg i.p.) and from fish both exposed to sublethal con-
centrations of MCB for either 15 or 30 days.
Tissue slices (generally 2-3 mm) were fixed in Bouin's fixative, embedded
in paraffin and 6 urn sections were cut and stained with hemotoxylin and
eosin. Some liver slices were fixed in Carnoy's #1 solution and 6 pm sections
stained with Best's carmine stain for resolution of glycogen deposits.
STATISTICAL METHODS
Treatment group means of plasma BSP concentration, plasma osmolality,
total plasma or serum protein concentration, serum hemoglobin concentration,
whole wet body weight change, liver and spleen to body weight ratios and
urine flow rates were compared by student's t test for independent sample
means (Steel and Torrie, 1960). The variation among treatment groups was
assessed for significance by Bartlett's test for homogeneity of variance
(Sokal and Rohlf, 1969). Plasma or serum enzyme activity data were converted
to a quantal form and analyzed by the Fischer Exact Probability Test (Seigel,
1954). Values greater than 2 standard deviations (P < 0.05) from the control
values were chosen to indicate a positive response in treated fish.
1.6
-------
SECTION 4
RESULTS
DEVELOPMENT OF CLINICAL PROCEDURES TO EVALUATE LIVER TOXICITY
IN RAINBOW TROUT
Endogenous Tests
Plasma and Serum Enzymes--
The activity of alanine aminotransferase (GPT) and aspartate amino
transferase (GOT) in plasma and liver were determined in the fall and winter
from a group of fish held at 11.0°C ± 1.0°C and maintained on a ration of
Purina Trout Chow. Plasma GOT activity was greater than that of GPT but
liver GPT activity was greater than liver GOT activity (Table 1).
The effect of in_ vitro hemolysis on plasma GPT and GOT activity was
determined in the spring on a group of fish (150-300 g) held at 15°C ± 1.0°
and maintained on Donaldson Diet. Physically induced hemolysis did not tend
to increase the activity of GPT or GOT in the plasma even though the plasma
hemoglobin concentration was between 7 and 16 times greater than corresponding
control plasma (Table 2). Carbon tetrachloride added to whole blood caused a
dose-dependent increase in the hemoglobin concentration in the plasma and yet
did not cause significant elevation in either GPT or GOT activity. At the
highest dose of CC14 used, the plasma hemoglobin concentration was more than
430 times greater than that of the control and yet the activity of plasma GPT
was the same as that of the control and plasma GOT activity was 1.4 times
that of the control plasma.
A slight degree of correlation was evident between the plasma hemoglobin
concentration and the plasma activity of both GPT and GOT. The correlation
coefficient between plasma hemoglobin and GPT and GOT activities in physi-
cally induced hemolysis were 0.29 and 0.36 respectively. In the case of
hemolysis induced by CC14 the correlation coefficients were 0.13 for GPT
activity and 0.89 for GOT activity.
The effects of assay temperature and pH on GPT and GOT activity were
determined in liver tissue from fish held at 11.0°C ± 1.0° and maintained on
Purina Trout Chow. Liver GPT activity was measured at 7.5°C, 15°C, 25°C,
30°C and 37°C at pH 705 and at pH values of 6.5, 700, 7.5, 8,0 and 8.5 at
25°C. The mean liver GPT activity increased linearly from 7.5°C to 25°C and
the GPT activity was four times greater at 37°C than at 7.5°C (Fig. 2). The
change in reaction velocity with 10°C increase in temperature (Qio) was
estimated to be 2.4. The greatest variability in activity was found at assay
temperatures above 25°C. An Arrhenius plot of this data suggested that
structural and conformational changes in enzyme structure from thermal
17
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TABLE 1. ALANINE AMINOTRANSFERASE (GPT) AND ASPARTATE AMINOTRANSFERASE (GOT)
ACTIVITIES IN PLASMA AND LIVER FROM RAINBOW TROUT.
Enzyme Plasma Activity**
N (U/l)
GPT 12 9.3 ± 1.3C
GOT 7 113.4 ±27.4
Tissue Activity^
(U/mg x
N CU/g) 1
-------
TABLE 2. RATIO3 OF PLASMA ALANINE AMINOTRANSFERASE (GPT), ASPARTATE
AMINOTRANSFERASE (GOT) ACTIVITIES, AND PLASMA HEMOGLOBIN CONCENTRATION AFTER
IN_ VITJ
TROUT.
IN VITRO CCL4-INDUCED OR PHYSICALLY-INDUCED HEMOLYSIS OF BLOOD FROM RAINBOW
Plasma Enzyme and Hemoglobin Ratio
GPT HbGOT Hb"
Physically ,
Induced 0.93 6.9 1.0° 16.6
(6) (7)
CC1. Induced
lOpl
20pl
30yl
40pl
50pl
1.2
1.5
0.8
0.8
1.0
261
328
386
233
430
1.2
1.3
1.3
1.1
1.4
261
328
386
233
430
*3
Ratio was determined as follows:
_., . .... . , , Mean hemolyzed value
Physcially induced:
Mean non-hemolyzed value (control
„„. . , , CC14 value
CC1. induced: 4
Cortland control
There was an apparent sensitivity difference in the erythrocyte viability
of the fish used for GPT versus those used for GOT determination, there-
fore Hb values are reported separately. The mean values for GPT for
the hemolyzed group were 3.9 ± 1.6 and for non-hemolyzed 4.2 ± 1.5. The
Hb for the GPT group was 173.6 ± 58.3 and 25.2 ± 2 for the hemolyzed and
non-hemolyzed respectively. The mean values for GOT were 92.2 ± 12.2 and
89.7 ± 10.3, while Hb was 544.7 ± 174 and 32.8 ± 4.4 hemolyzed and non-
hemolyzed respectively.
Ratio is mean of 6 fish
£
Ratio is mean of 7 fish.
volume of CC1 added to ml whole blood
eControl Values x ± SD used to calculate ratios
19
-------
30
N)
O
CM
i
O
X
o
k_
Q.
C7>
E
20
t 10
h-
o
Q_
O
1
1
10 15 20 25
TEMPERATURE (°C)
30
35
40
Figure 20 Alanine aminotransferase (GPT) specific activity in rainbow trout liver at increasing
assay temperature. Values are the mean ±SEM for three fish. Assay pH was 7.5.
-------
instability occurred at temperatures above 20°C (Fig. 3)0 The pH optimum for
GPT activity was found to be 7.5 (Fig. 4).
The relative activities and enzyme characteristics of liver and kidney
GPT were determined from a group of fish (175-300g) held at 15°C ± 1.0°-and
maintained on Purina Trout Chow. The specific activity of liver GPT was 30
percent greater than that in the kidney while the liver protein concentration
was nearly 50% greater than that of the kidney (Table 3). The apparent Km
and Vmax of liver and kidney GPT were determined from a Lineweaver-Burke plot
of reciprocal reaction velocity vs reciprocal substrate concentration (Fig.
5)o Optimum enzyme activity was measured with alanine concentrations between
50 mM and 100 mM and GPT activity was inhibited by alanine concentrations
greater than 100 mM. The Vmax for GPT in liver and kidney were 20 mg x
10 and 12.5 mg x 10~2 respectively while the apparent Km values for ala-
nine of the liver and kidney GPT catalyzed reactions were 5.6 mM and 5.0 mM
respectively.
Plasma and liver activities were determined in four different
salmonid species: rainbow trout (58-138g), steelhead trout (24-58g), brook
trout (140-182g), and kokanee salmon (62-178g). Generally the plasma GOT
activity was greater than the plasma GPT activity and, except for the rainbow
trout, this relationship was similar in the liver tissue as well (Table 4).
Differences (P < 0.01) in the mean liver protein concentration also were
noted between steelhead reared in the laboratory (13.5 ± 0.6 mg/100 mg tissue)
and those in holding cages in the Willamette River (11.2 ± 0.4 mg/100 mg
tissue).
The effects of assay temperature and pH on alkaline phosphatase (AP)
activity were determined from serum samples taken from sexually mature rain-
bow trout (170-250 g) maintained on Silver Cup Diet,, The temperature optimum
for serum AP was determined by comparing enzyme activity over a temperature
range of from 8°C to 50 °C at a pH of 10.3. The optimum pH range was deter-
mined by measuring enzyme activity in pH buffers ranging in value from 8.5 to
11.5 at 25°C. The mean serum AP activity increased linearly from 8°C to 25°C
and enzyme activity at 35°C was three times greater than at 8°C (Fig 6a)0
The QIO for serum AP activity was estimated to be 2.0 ,and increased variability
in estimated enzyme activity was observed at temperatures above 25°C. The pH
optimum for AP was found to be 10.3 (Fig 6b). An Arrhenius plot of enzyme
activity and temperature indicated that the enzyme was stable in the range of
temperatures between 8°C and 23°C (Fig. 7).
The mean activity of AP in the serum was estimated to be 30.56 ± 0.61
IU/1 when calculated on a volume basis and the specific activity was 0.977
± .019 lU/g serum protein. These values are considerably less than those
calculated for the liver on either a per gram of liver basis (1.45 lU/g 1
± 0.0361 or t>er eram of liver nrotein (21.38 lU/g liver nrotein ± 0.89)0
Serum activity of AP was not influenced by surgically created choles-
tasis in rainbow trout (Table 5) or by prior treatment with the mammalian
cholestatic agent ANIT (Table 6).
21
-------
N)
3.0 3.1 3.2 3.3 3.4 3.5 3.6
I/TEMP x I03 K '
Figure 3. Arrhenius plot of alanine aminotransferase (GPT) activity in rainbow trout liver.
Values are the mean of three fish. Assay pH was 7.5.
-------
35
_ 30
CVI
r
o>
E
X,
Z>
25
20
15
o 10
Q_
CD
5 -
0
6.0 6.5
7.0
7.5
pH
8.0
8.5
9.0
Figure 4. Alanine aminotransferase (GPT) specific activity in rainbow
trout liver with increasing assay pH. Values are the mean ±SEM for fish
at each pH. Assay temperature was 25°C.
23
-------
TABLE 3. ALANINE AMINOTRANSFERASE (GPT) ACTIVITY AND PROTEIN CONCENTRATION
IN LIVER AND KIDNEY TISSUE FROM RAINBOW TROUT.
Parameter Liver Kidney
Tissue Activity3 . 26.2b± 3.5 14.3 ±1.5
CU/g)
Protein Concentration 13.6 ± 0.5 9.3 ±0.3
(mg/100 rag tissue)
Specific Activitya_2 19.4 ± 2.8 14.8 ±1.5
(U/mg protein x 10" )
International Units of activity at 25°C, pH 7.5.
^Values are mean ±S.E.M. for 4 fish.
24
-------
100
80
60
Ol
• LIVER
A KIDNEY
-0.4
0
0.4 0.8 1.2 1.6
I/[S] mM~' ALANINE
Figure 5. Lineweaver-Burke plot of alanine aminotransferase (GPT) activity in liver and kidney tissue
from rainbow trout at increasing assay concentrations of alanine. The Vmax for liver and kidney GPT
are 20 U/rag x 10-2 and 1205 U/mg x 10-2, respectively, and the apparent Km values for alanine in the
liver and kidney GPT catalyzed reactions are 5.6 mM and 5,0 mM, respectively.
-------
N)
TABLE 4. ALANINE AMINOTRANSFERASE (GPT), ASPARTATE AMINOTRANSFERASE (GOT) ACTIVITIES IN PLASMA AND
LIVER AND PROTEIN CONCENTRATION IN LIVER FROM FOUR MEMBERS OF THE FAMILY SALMONIDAE.
Parameter
GPT Plasma Activity
CU/1)
GOT Plasma Activity
CU/1)
Liver Protein concen.
(mg/100 mg tissue)
GPT Specific Activity
(U/mg protein x 10~2)
GOT Specific Activity
(U/mg protein x 10" 2)
Rainbow
Trout
C8)a
9.7C
±1.0
113.8
±10.2
15.7
±0.5
22.3
±2.6
17.9
±1.5
Steelhead
Trout
(lab)
(14)
9.6
±0.9
188.9
±19.3
13.5**
±0.6
23.4
±1.9
34.6
±2.8
Steelhead
Trout
(river)
(16)
11.0
±0.7
-
11.2
±0.4
23.0
±2.1
Brook
Trout
(6)
4.7
±1.4
291.4
±17.5
13.9
±0.7
30.3
±3.3
53.5
±5.6
Kokanee
Salmon
(15)
12.5
±1.9
130.9
±14.1
17.1
±0.6
8.3
±0.8
14.9
±1.2
wumber of fish.
International Units of activity at 25°C, pH 7.5.
°Values are mean ±S.E.M.
Significantly different from Willamette River Steelhead (P < 0.01)
**
-------
40
30
>-
^20
I-
O
10
0
a)
I
1
0
10 20 30 40 50
TEMPERATURE (degrees C)
60
40
30
H 20
o
< 10
Q_
b)
7.0
8.0
9.0
10.0
PH
11.0
12.0
13.0
Figure 6a. Alkaline phosphatase (AP) activity with increasing assay
temperature. Assay pH was 10.3. 6b. Alkaline phosphatase (AP) activity
with increasing assay pH. Assay temperature was 25°C.
27
-------
ts)
00
I
TEMP.
x I03 K"1
Figure 7. Arrehnius plot of alkaline phosphatase (AP) activity in rainbow trout liver. Assay PH
was 10.3.
-------
TABLE 5. SERUM ACTIVITY OF ALKALINE PHOSPHATASE (AP) IN RAINBOW TROUT
FOLLOWING LIGATION OF THE CYSTIC DUCT AND COMMON BILE DUCT. EACH VALUE
REPRESENTS THE MEAN ±SE OF FIVE FISH.
Time After Serum AP Activity
Surgery (lU/gm serum protein)
(Hr)
3 0.339 ± 0.066
24 0.255 ± 0.057
48 0.270 ± 0.051
72 0.291 ± 0.031
TABLE 6. SERUM ACTIVITY OF ALKALINE PHOSPHATASE (AP) IN RAINBOW TROUT
FOLLOWING TREATMENT WITH ANIT (400 MG/KG I.P.). VALUES ARE THE MEAN +SE
OF FIVE FISH.
Time After Serum AP Activity
Treatment (lU/gm serum protein)
(Hr)
3 0.478 ± 0.0247
6 0.519 ± 0.035
24 0.487 ± 0.021
Control (24 h) 0.547 + 0.082
29
-------
Exogenous Tests
Disposition of BSP --
BSP disappeared from the plasma of trout which received doses of either
5.0 or 10.0 mg/kg at nearly equal rates. The half life and fractional turn-
over rate of BSP were estimated to be 11 min and 6.3%/min respectively in
animals receiving either dose of BSP0 Assuming that the plasma volume of
trout was four percent of the wet body weight (Houston and DeWilde, 1969) the
mean percentages of the initial dose of BSP remaining in the plasma compart-
ment after 60 min were estimated to be 2.43% ± SE 0.35 (6 fish) and 3.5%
± SE 0.31 (5 fish) in groups of animals receiving 5.0 and 10.0 mg/kg of BSP
respectively.
Following its injection BSP accumulated rapidly in the liver of trout*
After 15 min the hepatic content of BSP was at its highest level (0.55 mg/lOOg
body weight) and represented more than half of the injected dose of the dye
(Table 7). Thereafter both hepatic content and plasma concentration of BSP
declined. Proportionately greater decreases in the plasma concentration
between 15 and 60 min resulted in a steady increase in the apparent liver to
plasma concentration ratio of the dye. The absolute concentrations of BSP in
the liver after one hour were from 38 to 49 times greater than those found in
the plasma. Because the liver homogenates included residual BSP within the
intrahepatic biliary space it was not possible to determine the actual
hepatocyte to plasma concentration gradient of the dye. However, even when
it was assumed that volume of this space was one percent of the wet liver
mass (Peterson et al., 1976) and that the BSP concentration in that space was
8.5 rag/ml, the corrected ratio of BSP in liver to plasma was not less than
20:1 in any fish sampled 60 min after the dye had been administered.
To further establish the importance of normal liver function in trout
for the disposition of BSP, plasma clearance and hepatic accumulation of BSP
were determined in fish having hepatic blood flow or bile flow occluded by
experimental ligation. The influence of surgical impairment of hepatic blood
flow and/or bile flow on the rate of plasma BSP clearance was dramatic. The
concentration of BSP in the plasma of cystic-common bile duct ligated
animals was more than four times that of sham operated animals after 60 min
and plasma concentrations of the dye were significantly higher (P < 0.01)
than controls after 30, 45 and 60 min. The added effects of impaired hepatic
blood flow were even more striking since the estimated plasma half life of
BSP from this group (42 min) was nearly four times that of sham treated
animals (11 min) and almost one and one-half times that of animals having
only ligated bile and cystic ducts (28 min). Ligation of the hepatic portal
vein as well as the cystic-common bile duct resulted in (P < 0.05) in these
animals after 30, 45 and 60 min than in animals having only ligated cystic
and common bile ducts. These results suggested that decreased plasma clear-
ance rates in the former group could be attributed to decreased hepatic blood
flow.
Experimental ligation of the hepatic portal vein and/or the cystic and
common bile ducts greatly influenced the distribution of BSP between plasma
and liver (Figure 8)«, In fish of both surgically treated groups the
30
-------
TABLE 7. LIVER AND PLASMA CONCENTRATIONS, PERCENT OF INJECTED DOSE AND
LIVER:PLASMA CONCENTRATION RATIO OF BSP FOLLOWING A SINGLE IV INJECTION3
TO SPINAL TRANSECTED RAINBOW TROUT.
BSP Time
(min)
15 30 60
Liver BSP ,
concentration 0.37 ± 0.02D 0.40 ± 0.03b 0.35 ± 0.01
(mg/g liver)
Hepatic BSP
content 0.55 ± 0.02 0.53 ±0.06 0.44 ± 0.02
(mg/lOOg BW)
Percent of
injected dose 54.7 ± 2.1 53.0 ± 5.6 44.0 ± 1.7
of BSP in
liver
Plasma BSP
concentration 9.33 ± 0.31 3.40 ± 0.80 0.88 ± 0.03
(mg/100 ml)
Liver:plasma
ratio
uncorrected0 4.0 ± 0.14 13.8 ± 2.54 42.1 ± 2.00
correctedd - - 10.9 ± 1.93 32.0 ± 1.71
S10 mg BSP/kg.
bM
:ean ±SE of 5 fish-
mean liver:plasma BSP concentration ratio not corrected for BSP in
intrahepatic biliary space.
'Mean liver:plasma BSP concentration ratio corrected for BSP remaining
in biliary tree. See text for details.
31
-------
o
<^r
H- 5
Z :>>
UJ-o
CL2
co\
CD 0»
UJ
0.6
0.4
0.2
30 60
TIME (min)
Z
O
<
o:
K
Z —
uj-E
o fc
zo
oo
•
O
- 10.0
Q_
CO
GO
CO
TIME (min)
Figure 8. Liver BSP content and plasma BSP concentrations in sham treated
control trout (a) and trout having either cystic duct-common bile duct
ligation (b), or hepatic portal vein and cystic-common bile duct ligation
(c) 30 and 60 min after a single i.v. dose of BSP (10 mg/kg). Values are
the mean ±SE of five animals. Asterisks denote values which are
significantly different (P < 0.05) from controls.
32
-------
hepatic content of BSP was significantly lower (P < 0.05) than in fish on
which sham surgery was performed,, Plasma BSP concentrations were signifi-
cantly higher (P < 0.05) in all surgically treated fish.
Two BSP fractions were separated from liver extracts of surgically
treated rainbow trout by thin layer chromatography. The R£ value of the
fastest migrating fraction was similar to that of BSP standards (0»7) and did
not react with ninhydrin. The Rf value of the slower migrating fraction
(0.38) was similar to that of fraction IV isolated from the bile and reacted
with ninhydrin. Surgical impairment of bile flow or hepatic blood flow did
not significantly influence the relative proportion of metabolized BSP in the
liver homogenates. The mean percent of metabolized BSP in liver homogenates
from five sham treated fish was 27.9% ± S.E. 2.3. In cystic-common bile
duct ligated and cystic-common bile duct and hepatic portal ligated groups
these values were 23.7% ± 1.5 and 24.6 ± 1.9 respectively.
When administered as a single intravenous injection (10..0 mg/kg), BSP
was detected in the bile within 15 min even though maximum bile concentra-
tions of the dye were not apparent until between 1.5 and 3 h after dosing.
In general, the rate of bile flow was inversely proportional to the concen-
tration of BSP in the bile. Maximum bile flow rates during the experiments
ranged from 1.29 to 2.3 ul/kg/min and maximum BSP concentrations ranged from
7.28 to 11.50 mg/ml. Maximum rates of biliary BSP excretion occurred between
2 and 2.5 h after fish received the dye. By 6 h the accumulative excretion
of BSP into the bile approached 50% of the injected dose of the dye. Maximum
rates of biliary excretion ranged from 12.1 to 14.4 yg/kg/min0
The mean rates of biliary BSP excretion for five fish during prolonged,
graded infusion were plotted with time (Figure 9). The rate of BSP excretion
increased during the first nine hours of infusion and then remained relatively
unchanged for the remainder of the infusion period. The maximum rate of
biliary BSP excretion was considered to be the Tm for the dye and was esti-
mated to be 12.1 ± 2.5 yg/kg/min (mean + S.E.; 5 fish). Bile flow rates and
bile BSP concentrations also remained relatively constant after 9 h and were
found to be 1.24 ± 0.35 yl/kg/min and 11.8 ± 1.84 mg BSP/ml respectively.
The number of separable fractions of BSP in the bile of each fish
increased with the time of dye infusion. Individual bands appeared in the
order and at approximately the times indicated in Figure 10. The three BSP
fractions having slowest mobility on the chromatograms reacted with ninhydrin
indicating a probable association with amino acids. None of the BSP fractions
reacted with aniline diphenylamine and therefore were probably not associated
with carbohydrates as conjugates. Prolonged infusion of BSP also resulted in
a steady increase in the proportion of metabolized dye which appeared in the
bile. After a one-hour infusion period, metabolized BSP comprised only 24%
of the total dye content of the bile but after 14 hours this value had
increased to nearly 40%.
33
-------
l6
-T 14
.2
c 10
JO
•*~ a
o> 8
k.
o
CD
20 fig/kg/mln 40 pg/kg/mfn
\
60 fig/kg/mln
I
5.0 mg/kg L v.
\
I
I
I
I
I
I
6 7 8 9 10 II
Time (hrs)
12 13 14 15 16
Figure 9. Biliary excretion of BSP during prolonged, graded infusion. Time on abscissa corresponds to
time after the beginning of infusion. Each point is the mean ±SEM of samples from five fish.
-------
Solvent
front
I
H
tn
Origin
R,
.8
.6
in
rz .4
3ZE
BILE BSP
Ninhydrin
Reaction
Std. I
4 6 8 10
TIME (hrs)
12 14
Figure 10. Representative chromatograms of BSP and BSP metabolites appearing in the bile of rainbow
trout during prolonged infusion of the dye. Time on abscissa corresponds to the time after the
beginning infusion. See text for details.
-------
ACUTE EXPOSURE STUDIES: CARBON TETRACHLORIDE
Determination of LD50
The 24 h median lethal dose of undiluted CC14 administered intraperi-
toneally to rainbow trout was estimated to be 4.75 ml/kg. While observations
of mortality were made at 24 h intervals for 72 h, all deaths occurred within
the first 24 h.
Dose and Time Response Studies: GPT
Activities of GPT and GOT in the plasma were similar for fish maintained
on either Donaldson or Purina Diets, however the specific activities of liver
GPT and GOT, respectively were 38% and 118% greater in fish maintained on
Donaldson Diet than those maintained on the Purina Diet (Table 8). Addi-
tionally, the mean liver protein concentration of fish mainted on the Purina
Diet was 13% greater than that of fish maintained on the Donaldson Diet.
Plasma GPT activity was elevated in all fish treated with CC14
(1.0 ml/kg), however dramatic differences were apparent in the magnitude
of this response between groups of fish maintained on different diets.
The mean plasma GPT activities from trout fed the Purina Diet consistently
were higher (P < 0.01) at all sampling times from 3 to 18 h after treatment
than from trout maintained on the Donaldson diet (Fig. 11). Variability of
response to treatment with CC14 was greater in fish fed the Purina Diet.
Plasma GPT activity increased in CC14 treated trout in a dose-related
manner. Significant differences (P < 0.05) in mean plasma GPT activity were
evident between fish treated with 1.0 ml/kg and 2.0 ml/kg at all periods
after dosing from 3 h to 24 h (Fig. 12). A biphasic response pattern was
noted in plasma GPT activity from fish treated with 2.0 ml/kg CC14 with
activity maxima occurring at both 3 and 36 h post treatment. Plasma GPT
activity in fish treated with 1,0 ml/kg CC14 increased steadily and was
highest 36 h after treatment.
Dose and Time Response Studies: BSP
Significant (P < 0.05) retention of BSP was evident 30, 45, and 60 rain
after its administration of fish treated with 0.2 and 2.0 ml/kg of CC14
24 h earlier (Figure 13). The plasma half life of BSP was estimated to be
11 min in control animals and 15 and 32 min in animals receiving 0.2 and 2.0
ml/kg CC14 respectively, indicating some degree of dose dependence.
Significant (P < 0.05) retention of BSP was evident as early as 12 h
after CC14 treatment and was still apparent after 120 h. Highest observable
plasma retention of BSP was found after 48 h whereupon it slowly declined
(Figure 14). Levels of BSP in the plasma of control animals were relatively
constant.
The apparent hemolytic action of CC^ was reflected in sharply increased
levels of hemoglobin in the plasma (Figure 15). Twelve hours after
36
-------
TABLE 8. A COMPARISON OF PLASMA AND LIVER ALANINE AMINOTRANSFERASE (GPT),
ASPARTATE AMINOTRANSFERASE (GOT) ACTIVITIES AND LIVER PROTEIN CONCENTRATION
FOR RAINBOW TROUT FED TWO COMMERCIAL FISH DIETS.
Parameter
Purina Diet
Donaldson Diet
GPT Plasma Activity
(U/l)
GOT Plasma Activity
(U/l)
Liver Protein Concentration
(mg/100 mg)
GPT Specific Activity
(U/mg protein x
1(T2)
GOT Specific Activity
(U/mg protein x
1(T2)
9.3 ± 1.3
(12)
113.4 ±27.4
C 7)
17.8 ± 0.9
(12)
16.1 ± 1.5
(12)
8.2 ± 0.5
(12)
9.7 ± 1.0
C8)
113.8 ±10.2
(8)
15.7 ± 0.5
(8)
22.3 ± 2.6
(8)
17.9 + 1.5
(8)
International Units of activity at 25°C, pH 7.5.
Values are mean +S.E.M. for the number of fish in parentheses.
37
-------
IOW
170
160
150
140
^ 130
~ 120
£ no
> 100
o 90
«- 80
CL
0 70
S 60
3 50
CL
40
30
20
10
n
~~
—
—
(3)
(7)
—
—
—
—
—
—
—
—
(10)
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(
IB)
fa,
1
t
f/2
1
**
7
W
i
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V
f/i
[
%
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**
V
~] PURINA DIET
\ DONALDSON DIET ~
—
-
—
—
7>
—
—
•M
—
jlfjk 1 ' ' /
(9) ,4,T
j J !/sr-
% % ~m
3 6 12
TIME (hours)
18 24 MEAN
EXPERIMENTAL
CONTROL
Figure 11. Alanine aminotransferase (GPT) activity in plasma from rainbow
trout fed two different commercial fish diets and treated with 0014(1.0
ml/kg, i.p.)« Control fish received Cortland saline. Zero time mean
values are for non-fed control fish. Values are the meaniSEM for
the number of fish in parentheses. Asterisks denote values for Donaldson
diet fish that are significantly different (P < 0.01) from Purina diet fish.
38
-------
4
.Oml/kg
2.0 ml/kg
0
12 18 24 36
TIME (hours)
48 MEAN
EXPERIMENTAL
CONTROL
Figure 12. Alanine aminotransferase (GPT) activity in plasma from rainbow trout fed the Donaldson diet
and treated with CCl4(1.0 or 2.0 ml/kg, i.p.). Control fish received Cortland saline. Zero time mean
values are for non-treated control fish. Values are the mean ±SEM of the number of fish in parentheses.
Asterisks denote values for fish given 2.0 ml/kg i.p. that are significantly different (*P < 0.05, **P <
0.01) from fish given 1.0 ml/kg.
-------
10
£
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0.5
0.2
15
2.O ml /kg (7)
0.2 ml /kg (5)
Control (7)
I
I
30 45
Time (min)
60
Figure 13. Plasma disappearance curve for BSP in control trout and trout
treated 24 h earlier with CCl4(0.2 or 2.0 ml/kg, i,p.) Each point represents
the mean ±SEM of the number of fish in parentheses. Asterisks indicate
values which are significantly different (P < 0005) from controls.
40
-------
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c
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(7)
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£,
p
v/
^
^
* ^7;
r
^
W f • /
T
||*
4
n
48 96 120
Time (hrs)
Figure 14. Plasma BSP retention in rainbow trout following CCl^ treatment
(2.0 ml/kg i.p.). Plasma dye concentrations were determined 45 min after
a single dose of BSP (5.0 mg/kg, i.Vo) was administered. Values represent the
mean ±SEM of the animals in parentheses. Asterisks denote values which are
significantly different (P <0.05 ) from controls.
41
-------
— 350
E
o
2 300
o
c
o
o
250
200
o* ,50
E
o>
o
E
_o
Q.
100
50
(5)
V//\ s* s* i
—
(7)
I*
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y
X
^
1 1 Control
*
/~7\
r
i » /
? ^
1
it
(7)
i <»
//
12
24 48 96
Time ( hrs)
120
Figure 15. Plasma hemoglobin concentrations in control fish and fish
receiving CCl^ 12, 24, 96 and 120 h earlier. Values represent the
mean ±SEM of the number of fish in parentheses. Asterisks denote values
which are significantly different (P < 0.05) from controls.
42
-------
treatment the concentration of hemoglobin in the plasma was nearly 300 mg/ml
but these levels slowly declined to those of the controls by 120 h. Despite
the apparent increase in total body water following CC14 intoxication, dif-
ferences in plasma osmolality between treatment groups were not evident after
24, 48, 96, and 120 h.
Previous studies have established that bilirubin can reduce the rate
of plasma BSP clearance in rats (Hunton, et al., 1961; Dragstedt and Mills,
1936), presumably by competing for some process involved with its hepatic
elimination (Clarenburg and Kao, 1973). Because preliminary studies indi-
cated that bilirubin was the major bile pigment excreted by rainbow trout
(unpublished observations), it is possible that BSP retention was caused in
part by competition for excretion with large quantities of endogenous bili-
rubin derived from hemolyzed red cells. To test this hypothesis fish were
administered either bilirubin or an equivalent volume of bilirubin vehicle
prior to BSP administration and the rate of plasma clearance determined.
Animals receiving bilirubin tended to retain more BSP in their plasma than
controls, but the difference in plasma BSP concentrations was significant (P<
0.05) only 60 min after BSP administration. The plasma half life of BSP in
control fish was 14 min while that of animals receiving bilirubin was 18
min. In a similar study it was found that high levels of hemoglobin in the
plasma had no significant effect on the rate of plasma BSP clearance. The
plasma half life of BSP was estimated to be 14 min in both groups.
The hepatic content of BSP (rag BSP/100 g body weight) in animals receiv-
ing CC14 was significantly different (P < 0.05) from those of controls 15,
60, and 120 min after the dye was given while plasma BSP concentrations in
treated animals were significantly higher (P < 0.01) than those of controls
at all times (Table 9). Concentrations of BSP in the plasma and liver of
control animals declined uniformly throughout the experimental period. In
treated animals the hepatic content of BSP appeared to increase until at
least 60 min after the dye had been injected even though plasma BSP concen-
trations decreased during the entire period.
BSP accumulated in the livers of control animals at a faster rate than
in animals receiving CC14 (Figure 16). After 15 min the amount of BSP found
in the livers of control animals was more than twice that found in the livers
of treated animals and represented approximately 55% of the injected dose of
the dye. Even though as much as 57% of the injected dye eventually was found
in the livers of animals treated with CC14, this level was not attained until
60 min after animals had received the dye. The levels of BSP in both groups
of animals decreased uniformly between 60 and 120 min.
Bile flow rates, bile BSP concentrations and the rates of biliary BSP
excretion were not significantly different between treated and control groups
at any time during the experiment. Twelve hours after the infusion began,
bile flow rates, bile BSP concentrations and biliary BSP excretory rates were
stable in both groups and are presented for comparison in Table 10. The
concentration of BSP in the bile of both groups was highest at this time and
remained at these levels for the duration of the experiment.
43
-------
TABLE 9. LIVER AND PLASMA BSP CONCENTRATIONS FOLLOWING ITS ADMINISTRATION
(10 MG/KG IV) TO CONTROL FISH AND FISH RECEIVING CCL4 (2.0 ML/KG I.P.)
24 HOURS EARLIER. VALUES ARE THE MEAN ±SE OF 5 ANIMALS. ASTERISKS DENOTE
VALUES WHICH ARE SIGNIFICANTLY DIFFERENT (P < 0.05).
Time after BSP injection
(min)
15 30 60 120
Control
Liver
(mg/gl)a 0.37 ± 0.02 0.40 ± 0.03 0.35 ± 0.01 0.14 ± 0.02
(rag/100 g bw)b 0.55 ± 0.02 0.53 ± 0.06 0.44 ± 0.02 0.21 ± 0.01
Plasma
(mg/100 ml) 9.33 ± 0.31 3.40 ± 0.80 0.88 ± 0.33 0.52 ± 0.01
Carbon tetrachloride
Liver
(rag/gl) 0.19 ± 0.03* 0.40 ± 0.04 0.40 ± 0.03 0.27 ± 0.02*
(mg/100 g bw) 0.25 ± 0.04* 0.51 ± 0.09 0.56 ± 0.04* 0.36 ± 0.03*
Plasma
(mg/100 ml) 17.14 ± 1.46* 8.40 ± 1.19* 1.65 ± 0.17* 1.45 ± 0.18*
amg/g liver
mg/g body weight
44
-------
en
Q_
CO
00
•D
(D
w.
O
E
TJ
C
-------
TABLE 10. BILE FLOW, BILE BSP CONCENTRATION AND RATE OF BILIARY BSP EXCRETION
12 HOURS AFTER BEGINNING INFUSION OF BSP IN CONTROL FISH AND FISH RECEIVING
CCL4 (2.0 ML/KG I.P.) 36 HOURS EARLIER. VALUES ARE THE ±SE OF THE NUMBER
OF ANIMALS IN PARENTHESES.
Bile BSP
Bile Flow Bile BSP Excretion
(yl/kg/min) (mg/ml) (yg/kg/min)
Control 1.24 ± 0.35 11.8 ± 1.84 12.1 ± 2.49
(5)
CC1. 0.67 ± 0.19 11.0 ± 1.76 6.8 ± 2.49
(3)
46
-------
The mean rates of biliary BSP excretion in control and intoxicated
fish were not significantly different at any time during the course of the
infusion (Figure 17). However, when these rates were integrated over the 15
h infusion period, the total amount of BSP excreted in the bile was estimated
to be 8.36 mg/kg and 6.88 mg/kg in control and treated animals respectively.
The apparent decrease in the rate of biliary BSP excretion in treated animals
after 11 h was due to a decrease in the rate of bile flow rather than to a
decrease in the concentration of BSP in the bile. The bile flow rates in
both groups of animals declined during the infusion period. Bile flow in
control animals dropped approximately 35% from 1.92 yl/kg/min to 1.24 yl/kg/
min over a 7 h period and this lower rate was maintained for the remainder of
the experiment. Over a similar 7 h period bile flow in treated fish dropped
64% from 1.97 yl/kg/min after 5 h to 0.7 pl/kg/min at 12 h. This bile flow
was maintained for the remainder of the experiment. The peak sustained rate
of biliary BSP excretion was considered to be the biliary transport maximum
(Tin) for the dye. This value was estimated to be 12.1 yg/kg/min in control
fish, however it was not possible to demonstrate a sustained rate of biliary
BSP excretion in animals receiving CC14 due to the variable rates of biliary
BSP excretion (Figure 17).
Chromatography of plasma and liver extracts and bile indicated that th.6
separable fractions of BSP were qualitatively similar in control and treated
fish. A single BSP fraction was found on chromatograms of plasma extracts
that did not react with ninhydrin and this fraction migrated with mobility
similar to that of the plasma BSP standard. Two BSP fractions "ere present
on chromatograms of liver extracts; the fastest of which had an Rf value similar
to that of the liver BSP standard. The slowest migrating fraction reacted
with ninhydrin and was assumed to be an amino acid conjugate of the dye.
The mean percent of metabolized BSP present in liver extracts of control
animals represented 19.6% of the total amount of BSP in the liver and ranged
from 18.5% to 20.5%. In treated animals the mean value was 18.9% and ranged
from 17.6% to 21.0%. The number of separable fractions of BSP in the bile of
fish from both groups increased with the time of infusion. No qualitative
differences in these fractions were evident between treated and control fish
and the pattern of metabolites that appeared in the bile was similar to that
previously described (Gingerich et al. 1977). The BSP fraction which
demonstrated the greatest mobility had an Rf value similar to that of the
bile BSP standard and did not react with ninhydrin. The three fractions
exhibiting lowest mobility reacted with ninhydrin while the two fractions of
intermediate mobility did not. None of the BSP fractions reacted with ani-
line diphenylamine and therefore were probably not associated with carbohy-
drates.
Prolonged infusion of BSP resulted in a steady increase in the propor-
tion of total metabolized dye which appeared in the bile of both treated and
control fish (Figure 18). The relative amount of total metabolized BSP
increased by 32% over initial levels in the bile of CC14 treated fish and by
39% in the bile of control animals between 1 and 14 h after infusion of the
dye began. The bile of CC14 treated trout contained a slightly higher pro-
portion of metabolized dye throughout the infusion period than did that of
control animals; however, these differences were not significant. Some
47
-------
oo
C
e
\
o»
C
o>
t.
o
X
UJ
d.
C/)
CD
16
14
2 10
8
4Ojjg/kg/min 6O jjg/kg/min
I I
5,0 mg/kg i.v.
I
6789
Time (hrs)
10 II 12 13 14 15 16
17 Biliary excretion of BSP by control trout and trout treated with CC14 24 h prior to the
stt of BSP infusion ?ime on the abscissa corresponds to time after the begimung of infUsion.
Each point is the mean ±SEM of the number of animals in parentheses.
-------
50
10
40
Q_
(f)
m 30
o
o>
\ 20
o
O
c
0)
o 10
w.
CD
O.
•—•• Control
20 jjg/kg/min 40yg/kg/min
Jf I I I I I I
6O jjg/kg/min
I
I
I
I
I
5.O mg/kg i.v.
I
I I I I
6 7 8 9 10
Time (hrs)
II 12 13 14 15
Figure 18. Total metabolized BSP appearing in the bile of control fish or fish treated with CC14 during
continuous, graded infusion of BSP. Time on abscissa represents time after the start of infusion. Each
value represents the mean ±SEM of at least three fish,,
-------
error may be associated with these percentage estimates however, because it
was assumed that the extinction coefficients of metabolized BSP in the trout
bile were similar to that of the unconjugated dye. While there is no experi-
mental evidence to support this assumption for BSP metabolites in trout
bile, Combes (1965) and Whelan et al. (1970) have reported that the extinction
coefficients of the major BSP metabolites in rat bile are similar to free
BSP.
Effect of CC14 Intoxication on Plasma Protein Concentration
and Water Balance
A dose-dependent decrease in the plasma total protein concentration was
observed in rainbow trout treated with CCl^ 24 h earlier. The plasma total
protein concentrations of trout treated with 1.0 and 2.0 ml/kg CC14 (i.p.)
were 80 percent and 59 percent respectively of control fish treated with
Cortland's saline (Table 11). No differences in the mean plasma total
protein concentration were evident in groups of fish receiving either
0.25 or 0.5 ml/kg CC14. A significant decrease (P < 0.01) in plasma
total protein concentration was apparent as early as 12 h after treatment
with CC14 (2.0 ml/kg i.p.) and protein concentrations remained depressed
for at least 36 h (Table 11).
The pattern of whole wet body weight change in fish treated with
was considerably different from those treated with Cortland's saline. Fish
treated with CC1. (2.0 ml/kg) either lost less weight than control fish or
tended to gain weight. Differences (P < 0.05) in this pattern of weight
change were evident as early as 12 h after treatment and continued for at
least 36 h (Table 12). Plasma osmolality of treated fish also was less (P <
0.05) than that of control fish as early as 3 h after treatment and remained
depressed for at least 48 h (Table 12).
Urine flow rates (UFR) in control trout treated with Cortland's saline
had a tendency to be elevated however these increases were never significant
(Fig. 19). The hourly mean UFR increased from 4.1 ml/kg/h to 4.3 ml/kg/h and
the mean urine output for 24 h increased from 98.1 ml/kg to 104 ml/kg.
Additionally, a diurnal pattern in the mean UFR remained evident following
treatment with Cortland's saline. Conversely, urine flow rates in trout
treated with CC1. (2.0 ml/kg i.p.) were decreased dramatically within 1 h
after treatment (Fig. 20). The mean, hourly UFR decreased from the pre-
treatment rate of 4.1 ml/kg/h to 1.0 ml/kg/h within one hour post-treatment
and the mean pre- and post-treatment urine outputs for 24 h were 98.7 ml/kg
and 23.2 ml/kg, respectively. The diurnal pattern of urine flow evident in
control fish was abolished in fish receiving CC14. When compared to control
fish the urine osmolality of trout treated with CC14 was increased (Table
13), in part by the resultant proteinuria (Table 14).
50
-------
TABLE 11. PLASMA PROTEIN AND ALBUMIN CONCENTRATIONS FOR RAINBOW TROUT AT
24 HOURS POST-TREATMENT WITH CCL4 (0.25 ML TO 2.0 ML/KG, I.P.)
Dose of N
cci4
Control5 12
(non-treated)
Control0 6
(treated)
Plasma
Protein
(mg/ml)
27.9 ± 0.8d
28.3 ± 1.2
Plasma
Albumin
(mg/ml)
13.7 ± 0.4
12.4 ± 0.6
n
Albumin /
Plasma
Protein
49.1
43.8
CC14 (ml/kg)
0.25
0.5
1.0
2.0
5
5
10
9
27.
29.
22.
16.
5 ±
2 ±
7 ±
7 ±
1.
2.
2.
1.
3
6
2*
6**
12.
12.
11.
8.
3 ±
5 ±
5 ±
0 ±
0.
1.
1.
0.
5
1
0
7**
44.
42.
, 50.
47.
7
8
7
3
\alue is ratio of albumin/total plasma protein x 100.
Non-treated control fish were sampled from holding tank
°Treated control received 0.25 ml to 2.0 ml/kg of Cortland saline, i.p.
values are mean tS.E.M. for N fish.
*Significantly different from Cortland control (P < 0.05).
**
Significantly different from Cortland control (P< 0.01).
51
-------
N)
TABLE 12. PLASMA PROTEIN CONCENTRATION, RELATIVE BODY WEIGHT CHANGE AND PLASMA OSMOLALITY FOR RAINBOW
TROUT POST-TREATMENT WITH CCL4 (2.0 ML/KG, I.P.)
Parameter
3
6
12
Time8
18
24
36
48
Plasma Protein (rag/ml)
Control 28
Treated 23
Weight Change (g/100
Control -3
Treated -1
Plasma Osnolality (m
Control
Treated
.Ob t 1.9
(3)
.3 t 2.2
(9)
g BW)
.2 t 1.3
(3)
.2 ±0.4
(9)
Os/kg)
306 t 2
(3)
293 ± 4*
(13)
28.6 t 3.2
(3)
20.8 i 1.6
(7)
-3.1 i 0.6
(3)
-1.7 i 0.7
(7)
297 t 4
(3)
283 i 6
(12)
30.8 t 3.2
(3)
18.8 t 1.2*
(5)
-2.8 i 1.3
(3)
1.1 i 0.7
(6)
285 < 13
(3)
287 i 4
(6)
30.9 ± 1.2
(3)
18.2 t 0.8
(4)
-4.7 * 0.3
(3)
4.6 « 0.8*
(4)
296 i. 7
(3)
280 i 9
(4)
30.8 i 1.2
(3)
14.4 * 1.2*
(S)
-3.6 + 0.8
(3)
4.5 t 0.8*
(5)
290 t 8
(3)
273 i 2
(S)
30.8 ± 2.0
(3)
1 3.7i 2.1
(8)
-5.3 * 1.0
(3)
6.4 i 1.5**
(8)
289 t 3
(3)
268 + 4**
(8)
25.1 i 3.6
(3)
14.4 i 1.2
(4)
-5.1 ± 0.6
(3)
1.6 i 0.9*
(4)
295 i 2
(3)
270 + 5*
(4)
Hours post-treatment.
Values are mean tSEM for number of fish in parentheses.
Significantly different from Cortland control (P < 0.05).
*
Significantly different from Cortland control (P < 0.01).
-------
en
o>
cc
o
u.
UJ
(E
7
6
5
4
3
2
I
0
PHOTOPERIOD
LIGHT
T
LIGHT
DARK-
DARK
DARK or
LIGHT
T
T
24-19 18-13 12-7 6-1
HOURS BEFORE TREATMENT
1-6 7-12 13-18 19-24
HOURS AFTER TREATMENT
Figure 19. Urine flow rate of Cortland-treated (2.0 ml/kg, i.p.) control rainbow trout for 24 h
pre-treatment and 24 h post-treatment. Values are the mean ±SEM for eight fish during each 6 h time
periodo Asterisks denote value that is significantly different (P < 0.05) from non-treated control
fish during a similar time period post catheterization.
-------
JC
o»
\ 7
1 6
u s
h- 3
2 4
0 3
-J °
u.
LJ 2
E I
0
- T
T
T
24-19 18-13 12-7 6-
T
PHOTOPERIOD
LIGHT
LIGHTS DARK or
DARK —> LIGHT
DARK
**
** _
**
T
-6 7-12 13-18 19-24
HOURS BEFORE TREATMENT HOURS AFTER TREATMENT
Figure 20o Urine flow rate for CC14 treated rainbow trout (2.0 ml/kg, i.p.) for 24 h pre-treatment and 24
h post-treatment. Values are the hourly mean ±SEM for 10 fish during each 6 h time period. Asterisks
denote values that are significantly different (P < 0.01) from Cortland-treated controls during the same
time periods post-treatment.
-------
X.
100
80
I 60
I-
o
Q.
ON
Ol
u
40
20 -
*
Baseline Fed Control Low
Control CB
K /5 days
High Fed Control Low High
CB Control CB CB
H K 30 days >
DArS OF TREATMENT
Figure 25, Mean plasma GPT activities from baseline, fed and paired control trout and trout exposed to
two subacute concentrations of MCB (2.6 ppm and 3.9 ppm) for 15 and 30 days. Values are the mean ±SEM
of the number of animals in parentheses„
-------
TABLE 17. ELECTROPHORETIC DISTRIBUTIONS OF SERUM PROTEINS (GM/100 ML: MEAN ± SE) FROM TROUT AFTER
15 DAYS OF EXPOSURE TO CONCENTRATIONS OF MONOCHLOROBENZENE.
Fraction
I
Zone I II
III
IV
V
Zone II VI
VII
VIII
Ratio0
Fed Control
(2)a
.406 ± 1.
.763 ± 0.
.907 ± .
.681 ± .
0.200 ± 0.
0.432 ± 0.
0.203 ± 0.
0.025 ± 0.
1.3420
55b
343
123
149
051
059
010
012
Control
(2)
0.209
0.521
0.513
0.472
0.201
.185
.225
.095
1
± .020
± .008
± .127
± .154
± .063
± .034
± .122
± .050
.1515
Low
CB
CD
.52*
.579
.841
.607
.342
.593
.324
.131
0.9497
High
CB
(3)
.241
.273
. 507 .
0.559
.204
.390
.233
.064
0.
± .142
± .079
± .166
± .154
± .044
± .103
± .086
± .029
6810
Number of fish sampled
Mean ± SE
Mean ratio of the sum of proteins in Zone I to sum of proteins in Zone II.
-------
weight of 15 fish treated with CC14 was 1,31% of body weight and values
ranged from 0.98% to 1.81%. In control animals, the mean liver weight of
15 animals was 1.37% of body weight and values ranged from 1.09% to 1.76%.
These values were somewhat misleading however since animals receiving
CC14 gained significantly (P < 0.05) more weight 24 h after treatment,
presumably as water, and maintained this weight for a longer time than did
controls (Figure 26)„ Thus, even though livers of treated animals were
enlarged, the concomitant increase in body weight negated demonstration of
this effecto
After 24 h, fish receiving 0.2 ml/kg CC14 exhibited slight inflamma-
tion of the peritoneal cavity around the site of injection but thrombi
were not observed in any of the major vessels of the splanchnic drainage.
In addition, there was no evidence of hemoglobinuria during the first 24 h
after intoxication. Livers of animals in this group were not taken for
histological examination.
The livers from transected and ncn-transected control trout were similar
histologically to those described by Weinbreb and Bilstad (1955).
Slight vacuolization was evident in some hepatocytes, however the majority
of cells displayed a normally granular cytoplasm (Figure 27a). Morpho-
logical changes were evident in the liver taken from non-transected fish
6 h after CC14 treatment. Necrosis was apparent both in the sub-
capsular region and in well defined areas surrounding the central veins
(pericentral regions) (Figure 27b). Damage in the subcapsular region was
characterized by coagulative necrosis and pyknosis (Figure 27c).
Pathological changes in pericentral regions were characterized by liquifac-
tive necrosis and karyolysis and necrotic areas were surrounded by a zone
of swollen hepatocytes (Figure 27d)0 The essential aspects of the peri-
central lesion were similar in only one spinal transected animal 18 h
after treatment.
It was not possible to assess the development of liver damage with
time following treatment. Pericentral liver necrosis was evident in
one free swimming fish after 6 h and in one spinal transected animal after
18 ho Eosinophilic degeneration and areas of slight hydropic degenera-
tion were noted in sections of liver taken from non-transected fish after
12, 18, and 24 h while similar degenerative changes were noted after
6, 12, and 24 h in spinal transected fish. Cellular regeneration was not
evident in livers from transected or non-transected individuals.
Marked tissue damage was not evident in the kidneys of trout receiving
CCl4o In the sections of kidney examined no glomerular damage was
observed at any of the times following treatment and evidence of proximal
tubule damage was found in one one fish treated 36 h earlier with CCl^.
Gross Pathology and Histology: MCB
Unlike trout receiving CC14, no significant wet whole body weight
changes were observed in trout treated with monochlorobenzene (Fig 28).
Slight inflammation of the peritoneal cavity and in sections of both the
67
-------
T3
O
.O
14.0
12.0
o>
O 10.0
O
o>
c
"o
8.0
6.0
> 4.0
^
-------
• '
r
<>^jj"**v.
Figure 27. Liver sections from rainbow trout. Hemotoxylin and eosin
stain. (a). Control liver 128x. (b) Peripheral and pericentral
necrosis in trout liver 6 h after CC14 treatment 20x. (c) Peripheral
necrosis 128 x. (d) Pericentral necrosis 128 x.
6<
-------
6.0
LL|
O
< 4.0
5?
f-CQ 2.0
II
x^
<
£! -4.0
HOURS:
TREATMEN
DOSE(ml/k
(6)
*T (5)
1 1 '
(5) "y> //(4)
V ** / / ' //
- {IO) (5) (sJ ^ ^J! -
r*, T 15) /^ V/^^
' Fl (5J Tirn ^ /X^y "
1 (IOK5) (5) T^^ ^.^x^tS) (3)(3) (5) ^/ (3)(3) y/y/>
^Si JL ^ KkJ
1 1
T i
24 48 72 24 48 72 24 48 72 24 48 24 48 24 48 24 48
T: Control MCB MCB Control CCI4 Control CCI4
g): - 0.5 1.0 - 1.0 - 2.0
Figure 28. Relative weight change in rainbow trout following treatment with either MCB (0.5 of 1.0 ml/kg,
i.p.) or CC14 (1.0 or 2.0 ml/kg i.p.). Values represent the mean ±SEM of the number of animals in
parentheses. Asterisks denote values that are significantly different (P < 0.01) from controls.
-------
large and small intestine were observed in all fish from all treatment
groups. Enlargement of the spleen was noted in 3 of 5 fish after 24 h
and 48 h and in 2 of 5 fish after 72 h. Even though the liver weight to
body weight ratios were higher in all treatment groups than in the
respective controlgroups, these differences were never significant. The
mean liver weight in 7 control fish was 0.76 percent of body weight while
in four groups of five treated fish the mean liver weight comprised 0.86,
1.07, 0.99, and 0.94 percent of the final body weight after 24, 36, 48,
and 72 h respectively. Mild hydropic degeneration of hepatocytes surrounding
central veins was noted in 1 of 2 fish taken from histological examination
at 8, 24, and 48 h and moderate pericentral necrosis was observed in one
fish 8 h after treatment. The glycogen content of the livers of treated
fish generally was reduced relative to that of the controls.
Fish exposed to subacute concentrations of MCB were irritable and
non- excitable throughout the experimental period. Respiratory rates,
as opercular beats per minute, of resting fish were increased in response
to the dose of MCB. The mean number of opercular beats per minute in both
the high concentrations (11.4 BPM ± SE 4.0) and low concentration (91 BPM ±
SE 2.8) of MCB was nearly twice that of the paired control fish (67 BPM +
SE 2.1). Oxygen consumption rates for fish exposed to MCB were variable.
The mean oxygen consumption of trout exposed to the low concentration of
MCB (0.60 mg02/kg/min ± 0.15) was actually less than that calculated for
the pooled control group (0.72 mg02/kg/min ± .OS) while the mean 02
consumption of fish held in the high MCB concentration was nearly 4 times
that of the control (2.86 mg02/kg/min ± SE 0.30). In addition, fish
exposed to MCB were anorexic for at least the first 23 days of the experiment
and consequently negative whole body weight changes were recorded for
experimental fish after 15 and 30 days of exposure (Fig. 29). Fish sampled
after 15 days had lost weight in a dose-dependent manner and mean weight
loss in both treatment groups was less (P < 0.05) than that of the pooled
mean of the non-fed control group. A similar trend in weight'change was
observed in fish sampled after 30 days but these results were complicated
by the fact that fish in both treatment groups began to accept food during
the second 15 days of exposure. One half of the fish (2/4) in the low MCB
exposure group and 1/4 in the high MCB exposure group accepted food before
the experiment was terminated on day 30 of exposure. The mean relative
food consumption rates of fish held in the high and low MCB concentrations
were 1.0 gm/100 gm body weight/day and 1.5 gm/100 gm body wt/day, respectively.
In contrast, fish offered an unrestricted ration consumed an average of
3.5 gm/100 gm body weight/ day.
At the times of sampling, fish in both treatment groups appeared
to be tetanic and emaciated relative to their controls and a dramatic
decrease in the white muscle mass of treated fish was particularly evident.
Inspection of the peritoneal cavities of exposed fish revealed few remarkable
differences when compared to their paired controls. The livers and spleens
of fish in all groups except the fed control fish were reduced in size but
otherwise were unremarkable. The liver weight to body weight ratios of
non-fed fish were less than those fed an unrestricted ration and relative
spleen weights of treated fish were smaller and less variable than were
71
-------
--4
N)
£ +35
o>
| +30
>» . «^
-g +25
jQ
E +20
o>
0 + 1*5
o 1- 10
^ +10
O>
UJ * 5
1 - 5
H*
£ - 10
£ -.5
i-
< -20
—
—
•~
^"
-
(17)
—
ffl
W
f
1
(8)
(4)
^
(4)
\
('
4)
(8) (4) (4)
I
*
UJ
o:
Baseline
Fed Control Low High
Control CB CB
Fed Control Low High
Control CB CB
K-^—50
DAYS OF TREATMENT
Figure 29. Relative weight change in rainbow trout following treatment with either MCB (0.5 of 1.0
ml/kg, i.p.) or CC14(1.0 or 2.0 ml/kg i0p.}. Values represent the mean ±SEM of the number of animals
in parentheses. Asterisks denote vlaues that are significantly different (P < 0.01) from controls.
-------
those of unexposed control animals (Table 18). No histopathological
alterations were observed in livers or spleens taken from treated or
control fish.
73
-------
TABLE 18. LIVER WEIGHT TO BODY WEIGHT AND SPLEEN WEIGHT TO BODY WEIGHT RATIOS IN FISH EXPOSED TO
SUBACUTE CONCENTRATIONS OF MONOCHLOROBENZENE FOR 15 AND 30 DAYS.
Liver wt/
Body wt ratio
(gm/100 gm bw)
Spleen wt/
Body wt ratio
(gm/100 gm bw)
Baseline
(19)
1.153a'b
±0.025
0.101b
± .008
Fed
Control
(4)
1.675b
±0.138
0.120b
± 0.042
15 days
Control
(8)
0.6961
±0.0135
0.222
± .050
30 days
Low
CB
(4)
0.670
±0.037
0.119*
±0.019
Higfr
CB
(4)
0.931*
±0.059
0.137*
±0.011
Fed
Control
(4)
1.358b
±0.121
0.065b
±0.004
Control
(8)
0.755
±0.052
0.130
±0.029
Low
CB
(4)
0.865
±0.149
0.077*
+0.007
High
CB
(4)
0.873
±0.066
0.105
±0.024
*
Denotes values significantly different (P < 0.05) from paired control
b
3 Mean ±SE
Denotes values significantly different from non-fed control
-------
SECTION 5
DISCUSSION
Endogenous Tests; Plasma, Serum and Tissue Enzymes
A comparison of plasma and liver GPT and GOT activities found in
these studies with those activities in rainbow trout reported by other
investigators demonstrates the variability of these enzyme activities in
normal fish (Table 19). Blood and biochemical parameters for fishes
are known to depend on the strain, diet, sex, age, time of year, holding
conditions and disease states (Barnhart, 1969; Blaxhall, 1972; Hickey,
1976). Plasma activities for both enzymes in this study were considerably
lower than values reported by Gaudet et al. (1975), which were 54 IU/1 and
259 IU/1 for GPT and GOT, respectively for rainbow trout at 15°C. On the
other hand, in a sequel study Racicot et al. (1975) found plasma activities
for GPT of 26.6 IU/1 and 196 IU/1 for GOT, in one group of control fish,
and 15.6 IU/1 and 141 IU/1 for plasma GPT and GOT, respectively, in another
group of control fish. In both of these studies all fish were fed Purina
Diet and, presumably, were genetically similar, disease-free and maintained
under identical conditions. Nevertheless, considerable variation exists
in values for mean plasma GPT and GOT activities of rainbow trout. It
should be noted that different transaminase assay kits were used in their
studies and may have accounted in part for some of the variability in the
plasma GPT and GOT activities.
Statham et a!0 (1978a) reported mean serum activities of 22.7 IU/1
for GPT and 169 IU/1 for GOT in control rainbow trout maintained at 12 °C.
The type of diet was not specified. In a recent study, Sauer and Haider
(1977) measured GPT and GOT activities in plasma from rainbow trout,
which had been acclimated to different water temperatures. At 12.5°C
plasma GPT and GOT activities were 10 IU/1 and 250 IU/1, respectively,
and increased to 15 IU/1 and 300 IU/1, respectively, at 15°C. The fish
were fed Fukosalm, a commercial fish diet.
In the measurement of GPT and GOT activities, ammonia (NH^) containing
reagents, e.g. the LDH solution in Sigma kits for GPT and GOT, can give
higher apparent activities due to the concurrent measurement of GDH activity.
In control fish, plasma GDH activity should be negligible, but liver
homogenates and pathologic plasma may have considerable GDH activity.
Our primary purpose in these investigations was to develop a repro-
ducible analytical assay for GPT and GOT in plasma and liver from rainbow
trout. As long as the precision of the measurements was relatively consistent,
i.e. within 2 S.D., the concentrations of the assay reactants were not changed.
These enzyme assay kits and reagents are designed primarily for measuring
75
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TABLE 19. A COMPARISON OF ALANINE AMINOTRANSFERASE (GPT) AND ASPARTATE AMINOTRANSFERASE (GOT)
ACTIVITIES IN PLASMA AND LIVER FROM SELECTED SPECIES OF FISHES.
linzyme
GPT
GOT
GPT
GOT
GPT
GPT
GOT
GOT
GOT
GPT
GOT
GPT
COT
GPT
GOT
Common Name
Water Temperature
CO
Rainbow trout (IS)
Rainbow trout
Rainbow trout (15)
Rainbow trout
Rainbow trout (IS)
Rainbow trout (12. S)
Rainbow trout (IS)
Rainbow trout (12.S)
Sockeyc salmon( ?)
Native channel catfish ( ?)
Native channel catfish ( ?)
Cultured channel catfish ( ?)
Cultured channel catfish ( ?)
Rainbow trout (10)
Pink salmon ( ?)
Plasma Activity4
(U/l)
54
259
26.6
IS. 6
196
141
IS
9
300
250
299
309
(17}b
(10)
(10)
( 7)
(10)
( 7)
(20)
(12)
(21)
(14)
( 7)
( ?)
Liver Activity
(U/g) (U/mg)
41 ( 2) 0.3S ( 2)
33 ( 2) 0.30 ( 2)
31.4 i S) O.i7 ( S)
45.8 ( S) 0.3S ( 5)
42.6 (10) 0.31 (10)
57.0 (10) 0.41 (10)
30.1 (20)
Reference
Caudct et al. (1975)
Caudct ct al. (1975)
Racicot et al. (1975)
Racicot ct al. (1975)
Sauer and Haider (1977)
Sauer and Haider (1977)
Sauer and Haider (1977)
Sauer and Haider (1977)
Bell (1968)
Wilson (1973)
Wilson (1973)
Wilson (1973)
Wilson (1973)
Smith et al. (1974)
Marquez (1976)
non-spawning
GOT
COT
COT
GOT
GPT
CPT
COT
CPT
COT
Pacific herring ( ?)
Uogfish ( ?)
Lingcod ( ?)
Itainbow trout (15)
Brook trout (15)
tcl (20)
tcl (20)
Rainbow trout (12)
Rainbow trout
1778
128
28
22.7
169.0
( ?)
( ?)
( ?)
(49)
(51)
91.2 ( 5)
411.0 (10)
22.3 ( 7)
196.7 ( 7)
Marquez (1976)
Marquez (1976)
Marquez (1976)
freeman and Idler (1973)
Freeman and Idler (1973)
Inui (1969)
Inui (19691
Statham et al. (1978)
Statham et al. (19.78)
a
International Units of activity under the assay conditions.
c ? r Kater temperature or number of fish not specified.
'' Values arc the mean for the number of fish in parentheses.
-------
enzyme activity in human plasma, and it is doubtful that the concentra-
tions in the assay mixture are optimum for rainbow trout plasma or liver
enzymes. Bergmeyer and Bernt (1974) determined the optimum conditions
and concentrations for GPT and GOT activities in human serum but
emphasized that these conditions and concentrations do not necessarily
apply to sera or organ tissues from other species.
Carbon tetrachloride can cause in^ vitro and in_ vivo hemolysis of
mammalian erythrocytes (Von Oettingen, 1955) and this~hemolysis does
add significantly to the transaminase activity in the plasma (Caraway,
1962). The results of this study indicate that plasma GOT activity was
increased by CC14-induced hemolysis but not by physical disruption of
erythrocytes while plasma GPT activity was not consistently elevated
by either treatment,, These results appear to support the findings of
Gaudet et al. (1975).
The hemolytic effect of CC14 and the resultant release of intra-
cellular enzymes appears to be different from the physically induced
hemolysis. CC14 has a high affinity for lipids, as does its metabolite,
chloroform. The chemical structure and properties of membranes can vary
from one tissue to another, and yet all cells, including erythrocytes,
have membranes with certain common constituents. The membranes consist
primarily of protein and lipids, e.g. phospholipids such as phosphatidyl
choline. In vitro studies with mammalian red blood cells have shown
that CC14 is 10 times more active than chloroform in its hemolytic
effect (Von Oettingen, 1955). The reactive compound can bind covalently
and selectively to unsaturated fatty acid double bonds, displaying a
great affinity for microsomal lipid, particularly cholesterol esters
and phosphatidylcholine (Reynolds, 1967). Presumably, the direct contact
by CC14 on the surface and intracellular erythrocyte membranes results
in the disruption of membrane structural and functional integrity and
thereby causes loss of the intracellular components, including enzymes,
into the plasma.
Ill vitro studies can only approximate the physiological conditions
in the intact organism,, It is not known, for example, what concentration
of CC14 is present in the blood of rainbow trout after i.p. injection
or how long the CC14 remains in contact with the erythrocytes. Our
in vitro experiments indicate that CC14 has the potential to cause
Hemolysis in vivo that may influence plasma enzyme measurements in the
rainbow trout„
The first two experiments indicated: 1) that plasma GOT activity
is greater and more variable than plasma GPT activity; 2) that in vitro,
CCl4-induced hemoglobinemia appears to alter plasma GOT activity to a
greater extent than plasma GPT activity. Since the simultaneous measurement
of both plasma transaminases provided little relevant information concerning
the nature of the pathological response of trout to CC14 intoxication
only plasma GPT activity was measured.
77
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The enzyme assay temperature may have little similarity to the environ-
mental temperature of the organism; however, this 111 vitro temperature is
important in comparative analysis of enzyme activitTes^Bell (1968) found
that purified GOT from the liver of an adult coho salmon, Oncorhynchus
kisutch, had activity which increased linearly from 10°C to 30°C.The
International Union of Biochemistry and the International Federation
of Clinical Chemistry has recommended a standard enzyme assay temperature
of 30°C, but Bergmeyer (1978) suggests that 25°C would be more practical
for most situations. He contends that adequate reaction rates can still
be achieved and there would be less reagent and cuvette temperature fluctu-
ation. In the measurement of enzyme activities in fishes, the assay
temperature should approximate the environmental temperature of the animal,
however this is not always practical. Our studies indicate that 25°C was
the maximum assay temperature to measure practically the liver GPT activity
in these fish, and this temperature therefore was used in subsequent
experiments. When enzyme activity is measured at a "non-physiological"
temperature, it should be understood that this is an artificial situation
and may lead to erroneous assumptions about the scope of enzyme activity
in the natural environment.
Carbon tetrachloride has been shown to be hepatotoxic to many verte-
brate species (Diaz Gomez et al., 1975), including fishes (Bell, 1968;
Gingerich et al., 1978a; Inui, 1969; Racicot et al., 1975; Statham et al.,
1978a). Moon (1950) and Strieker et al. (1968) reported that CC14 also
is nephrotoxic to humans and laboratory mammals. GPT activity has been
found in the liver, kidney and heart tissues from rainbow trout (Gaudet
et al., 1975), however no information is currently available regarding
the nephrotoxic effect of CC14 in fishes. It is conceivable that measurable
GPT activity could occur in the plasma from kidney damage in fish following
CC14 intoxication. The kidney GPT activity obtained in this experiment
was considerably less than the activity reported for rainbow trout by
Gaudet et al. (1975); however, they also found that specific liver GPT
activity was approximately 40% of the specific kidney GPT activity.
The Michaelis constant (Km) is an important and useful character-
istic of the enzyme and is fundamental to the mathematical description
of enzyme kinetics and also to the quantitative assay of enzyme activity
in different tissues. Isoenzymes catalyze the same reaction in different
tissues but can differ significantly in their Km requirements. Although
there have been no isoenzymes reported for GPT in mammals or fish, the
original intent of the Km studies with trout was to characterize liver and
kidney GPT by their apparent Km-alanine values for future application in
CC14 toxicity experiments. If the Km values were different for the
liver and kidney GPT, any GPT present in the plasma from tissue damage
could similarly be characterized, and the damaged tissue identified. The
apparent Km-alanine for liver and kidney GPT was 5.6 mM and 5.0 mM,
respectively and, therefore too similar to be applicable to the problem
of identifying specific organ damage by CC14. No attempt was made to
determine the apparent Km values for a-ketoglutarate, the other substrate
for GPT, in liver and kidney tissue. This was primarily because the GPT
was not in a purified form, and the presence of any glutamate dehydrogenase
78
-------
and ammonia in the tissue homogenate could appreciably interfere with
the measurement of GPT activity,,
Diseases of the liver, bone, small intestine, kidney and placenta
all may contribute to increased activity of serum AP (Zimmerman and Henry,
1969)o Serum alkaline phosphatase activity particularly is increased
during both intra and extrahepatic cholestasis (Steiner et al. 1965), and
for "this reason it has been a useful diagnostic index of liver function in
mammalian toxicology,. In the present series of investigations serum
alkaline phosphatase activity was not found to be useful as a diagnostic
aid in assessing liver dysfunction in rainbow trout. Unlike mammals,
(Baker et al., 1978) serum alkaline phosphatase activity in experimental
trout was not increased following either acute extrahepatic cholestasis
(ligation of cystic and common bile ducts) or by treatment with a mammalian
cholestatic agent (alpha-napthyisothiocynate) even though the time course
for these experiments was at least 48 h.
Exogenous Tests; Disposition of BSP by Rainbow Trout
Comparison of hepatic uptake and accumulation of BSP with its biliary
excretion indicates that, as in mammals (Klaassen and Plaa, 1967) the
latter is probably the rate limiting step in the transfer of this com-
pound from plasma to bile in the trout. Biliary excretion-appears to
be dependent both on the rate of bile secretion and the capacity of the
membrane systems to actively transport the compound into the bile. Thus,
the rate of biliary excretion may be limited either by a reduced rate of
bile secretion or a reduced capacity for active transport of the compound„
Differences in rates of biliary excretion between trout and rats are not
well explained by assuming the latter possibility since the concentration
of BSP in trout bile at the transport maximum (11.8 mg BSP/ml bile) is
similar to that of rat bile under similar experimental conditions (15.6 mg
BSP/ml; Klaassen and Plaa, 1968). Therefore, differences in the inherent
rates of bile flow between individual species are more likely to explain
differences in the rates of biliary BSP excretion,, The dependence of the
rate of canalicular bile secretion on biliary excretory rate of BSP has
been established in rats (O'Maille et al., 1966)„ Comparison of bile flow
rates with the percent of a single dose of BSP excreted in the bile by
several species suggests that inherent rates of bile secretion are most
responsible for interspecific differences in biliary BSP excretory capacity
(Table 20) 0 The percent of a single dose of BSP secreted by each species
after six hours was well correlated with the log of relative bile flow
based on wet liver weights among three species (r = 0.996). Thus, differ-
ences in the biliary excretory capacity of BSP between these species may
be explained as differences in the inherent rates of bile secretion rather
than as differences in canalicular transport processes.
The relative importance of conjugation to the overall process of
biliary BSP excretion has not been established in fishes. If dye conjugation
was the important prerequisite for this process in the trout that it
appears to be in the rat (Whelan et al., 1970; Priestly and Plaa, 1970b),
a much higher proportion of metabolized dye should be expected in fish
79
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TABLE 20. DEPENDENCE OF BILIARY EXCRETION OF A SINGLE INTRAVENOUS INJECTION
OF BSP ON THE BILE FLOW RATE IN DIFFERENT SPECIES.
Species
Dose
Bile Flow
(mg/kg) (yl/kg/min) (ul/100 g
liver/min)
Percent Dose Excreted
in Bile after 6 h
Dogfish
1.0
Rainbow Trout 10.0
Rat 37.5
1.23'
1.5
64.0*
1.12'
11.1
142.0
10.0
43.4 8.6'
84.6 3.2
From Boyer et al (1976a)
Calculated from Boyer et al. (1976 b)
Mean ±SE
4 From Klassen (1975)
From Klassen and Plaa (1967)
Based on estimate of bile flow in ref. 5 and liver mass of 4.5% of
body weight (Klaassen, 1973).
80
-------
bile. Metabolized BSP initially represented approximately 25 percent of
the total dye concentration of the bile in rainbow trout. Even though
this value is nearly twice that which has been reported previously in the
bile of several cartilagenous fishes (Boyer et al., 1976b,c), it represents
only about one third of the amount of conjugated BSP which appears in the
bile of rats (Whelan et al., 1970; Shultz and Czok, 1974). Studies comparing
the relative rates of biliary excretion for free and conjugated BSP would
be useful in determining the relative importance of conjugation for
biliary excretion of this particular compound in the trout.
The apparent increase in the percent of metabolized BSP which was
observed in trout bile during prolonged dye infusion was not expected.
Infusion of BSP above the biliary Tm in rats results in a decrease
in the relative amount of glutathione conjugate and an increase in the
relative amount of free BSP appearing in the bile (Schulz and Czok, 1974).
The increased number of BSP fractions, as well as the increased proportion
of metabolized BSP in trout bile, may be the result of anomalies in hepatic
blood flow which do not permit immediate and uniform distribution of the
dye to all sinusoidal surfaces or the result of incorporation of minor
pathways of BSP metabolism after major pathways have become saturated.
Identification of the separable fractions of BSP in trout bile would prove
useful in understanding more fully the nature of the processes responsible
for biliary excretion of this dye by the trout.
The decrease in the rate of plasma BSP clearance which was observed
in trout 24 h after experimental ligation of the cystic and common bile
ducts confirms the results of similar studies by Schmidt and Weber (1975).
In addition, the present studies indicate that the rate of hepatic BSP
accumulation also is severely reduced by this surgical procedure„ Consider-
ing the relative efficiency of hepatic uptake and accumulation of BSP in
the trout it is not immediately clear why surgically created cholestasis
should impede these processes. Differences in the hepatic BSP content of
the livers of sham and cystic-common bile duct ligated fish do not seem to
be the result of differences in the amount of dye that was transferred
into the canalicular and ductular biliary space. If this were the case
more than one quarter of the injected dose of BSP would need to have been
actively transported into the bile of sham treated fish within the initial
15 min period. Even if the net rate of transport of BSP had equaled the
maximum biliary excretory rate (12.1 yg/kg/min) less than five percent of
the injected dose of BSP could have been transported into the bile during
this time. Thus, the differences in hepatic BSP content are more likely
the result of altered uptake or storage capacities of the livers following
experimental ligation. Such impairment may be due to cell wide biochemical
and/or morphological changes in the hepatocytes which might reduce their
functional capacity to take up and store BSP. Decreased activity of the
membrane bound enzymes Mg+2-ATPase and 5-nucleotidase has been demon-
strated in rat liver 24 h after experimental ligation of the common bile
duct (Simon and Arias, 1973). Further, Vial et al. (1976) have shown
recently that prolonged bile stasis results in a loss of microvilli on the
bile canalicular surface and other ultrastructural alterations on the
surfaces of rat hepatocytes. Similar biochemical and morphological altera-
tions of trout hepatocytes following experimental bile duct ligation may
81
-------
be responsible in part for the impaired plasma clearance and hepatic accumu-
lation of BSP observed in this study.
These observations indicate that efficient mechanisms for hepatic
uptake and biliary excretion of the organic anion BSP are present in the
rainbow trout. Furthermore, the processes associated with the transfer
of this compound from the plasma to bile in the trout appear to be
relatively similar to those described for mammals. The results suggest
that, as in mammals, hepatic excretory function in this fish may be an
equally important route of elimination for certain classes of foreign
compounds„
Acute Exposure Studies; Carbon Tetrachloride
The temporal pattern of plasma GPT activity in the Purina or Donaldson
Diet fed fish treated with CC14 (1.0 ml/kg) was different from responses
reported in previous studies. Racicot et al. (1975) found maximum GPT
activity in plasma from rainbow trout fed Purina Trout Chow at 6 hours and
18 hours post-treatment with CC14 (1.33 ml/kg, i.p.). The enzyme
activity at these times was approximately five times greater than control
GPT activity and similar to the maximum plasma GPT activity measured in
our Donaldson Diet fish treated with nearly twice the dose of CC14 (2.0
ml/kg). Statham et al. (1978a) measured maximum plasma GPT activity in
rainbow trout at 2 hours and 72 hours post-injection with CC14 (1.0
ml/kg i.p.). Plasma enzyme activity at these times was nine times greater
than control activity and greater than those plasma activities reported by
Racicot et al. (1975) or those found in our studies. In laboratory rats
plasma GPT activity has been shown to reach maximum activity at 36 hours
post-treatment with CC14 (1.0 ml/kg, i.p.) (Koeferl, 1972; Zimmerman
et al., 1965). In addition, Koeferl found a biphasic temporal pattern
for both GPT and GOT in rats with peak activities at 12 hours and 36
hours. The significance of this biphasic plasma enzyme pattern in rainbow
trout and laboratory rats treated with CC14 is unknown.
The results suggest that the diet of rainbow trout may have significantly
altered the plasma GPT activity response to treatment with CC14. Pre-
vious studies with mammals and fish have demonstrated a variation in the
hepatotoxic response to organochlorine compounds due to changes in dietary
protein concentration (Korsrud et al., 1976; McLean and McLean, 1967).
The protein quality or quantity in the Purina and Donaldson Diets was
probably not a factor in the response of our trout to 0014. Purina
Trout Chow (Large Fingerling Size #5105) contains not less than 40% total
protein, primarily from herring fish meal. The Donaldson Diet is approxi-
mately 40% total protein, which is obtained from herring fish meal (30%)
and other fish sources (10%). Forty percent total dietary protein is
considered to be the minimum concentration required by rainbow trout to
insure normal metabolic homeostasis (Personal Communication, Dr. W. Stott;
Department of Food Science, Oregon State University).
Campbell and Hayes (1974) reviewed the effects of lipotropes on
biotransformation mechanisms. Lipotropes are compounds which function
82
-------
as methyl donors or assist in methyl group transfer during synthesis
of the phospholipids necessary for normal mixed function oxidase (info)
activity (Cooper and Feuer, 1973). The amino acid methionine is the
principal methyl donor in mammalian lipotropic metabolism, Mehrle et al.
(1977) reported that when dietary methionine concentration was increased
from 0.96 to 2.2%, the toxicity of DDT and Dieldrin to rainbow trout
significantly increased and decreased, respectively.
If an increase in the concentration of dietary methionine can increase
the activity of the microsomal MFO enzymes in rainbow trout, the biotrans-
formation of CC14 to its active metabolite would similarly be enhanced.
Purina Trout Chow is fortified with 10 amino acids, and methionine is
present in a concentration of 1.4%. The concentration of methionine in
the Purina Diet may have been sufficient to increase the hepatotoxic response,
i.e. plasma GPT activity, of the trout to CC14.
An alternate explanation for these findings involves the possible
presence of trace contaminants in commercially formulated diets and dietary
componentso Schoettger and Mehrle (1972) reported that the occurrence of
organochlorine contaminants was widespread in commercial fish diets and
dietary constituents. Although these workers did not find organochlorine
contaminants in Purina Trout Chow, they indicated that chemical residues
can vary considerably between feed lots (Personal communication, Dr. P.
Mehrle, Fish-Pesticide Research Laboratory, Fish and Wildlife Service,
Columbia, Missouri). Low level exposure to many compounds, including
organochlorine derivatives, has been shown "to induce MFO enzyme activities
in laboratory mammals (Remmer, 1972)„ Induction of the MFO system in fish
varies with the species of fish and type of inducing agent; however, recent
studies indicated the MFO system of trout liver is inducible by xenobiotics
(Chambers and Yarbrough, 1976; Lidman et al., 1976; Payne and Penrose,
1975; Pedersen et al., 1974; Statham et al., 1976). If a chemical inducing
agent was present as a contaminant in the Purina diet, the hepatotoxic
response to treatment with CC14 would have been greater in our fish,
Another hypothesis to explain the variation in hepatotoxic response
between the two groups of fish is the effect of dietary constituents
on the glutathione concentration in the liver. In mammalian systems
glutathione is a nucleophile that acts to break down intracellular hydroper-
oxides in reactions catalyzed by glutathione peroxidase in the cytoplasmic
fraction of the hepatocyte (O'Brien, 1969). This mechanism protects the
intracellular organelles, e.g. endoplasmic reticulum and mitochondrial
membranes, from the peroxidative effects of free radicals, e.g. the active
metabolite of CC14. Laboratory rats that were given glutathione prior
to treatment with CC14 were protected against polysome disturbances and
had improved amino acid incorporation into liver microsomal proteins
(Gravela and Dianzani, 1970). Moreover, the prior administration of cysteine,
which is required for glutathione synthesis, to laboratory mice decreased
both the covalent binding of an active metabolite of acetaminophen to
hepatocyte macromolecules and the severity of the resulting liver necrosis.
DeFerreyra et al. (1974) showed that cysteine pretreatment in laboratory
rats prevented the development of CCl4-induced liver necrosis by an
83
-------
unknown process. At present no information is available regarding the
effects of dietary cysteine or glutathione on the response of fish to
hepatotoxic chemicals.
Visceral adipose tissue may have influenced the absorption and distri-
bution of CC14 given by i.p. injection. Statham et al. (1978a) found
that adipose tissue of rainbow trout treated with 14CC14 (bath
exposure; 1 mg/1 for 2 h) had the highest concentration of 14C, which
increased until 1.75 hours post-treatment. Peak liver concentration of
14C occurred at 0.5 hours post-treatment followed by a slow elimination
phase (tj/2 = 39 h). In our studies, variable quantities of visceral
adipose tissue may have provided a storage depot for the CC14> reducing
the availability to the liver and causing the variability in plasma GPT
activity.
Results of our experiments with BSP suggest that plasma clearance
of this dye may be a useful criterion by which to evaluate liver dysfunction
in fish following acute exposure to toxicants. A significant decrease
in plasma clearance was detected in fish receiving as little as 0.2 ml/kg
i.p. of 0014. When plasma retention of BSP was used as an index of liver
dysfunction, elevated levels of BSP were found in the plasma as long as
120 h after treatment. It is also apparent that plasma BSP clearance is
not influenced by abnormally high levels of plasma hemoglobin which might
develop after prolonged exposure to certain classes of toxicants. Studies
by Hallesy and Benitz (1963) and Cutler (1974) have established the usefulness
of BSP plasma clearance as a test to predict liver dysfunction in laboratory
animals. Yet it was pointed out in both of these investigations that
morphological changes are more discriminating of liver damage in long term
studies than are functional changes. This may also be true in fish. In
the present histological studies some form of degenerative change was
evident in the livers of all trout receiving CC1. in acute doses. This
is not to imply that in chronic exposure studies a similar relation between
functional impairment and morphological alteration would be as readily
apparent.
As in mammals, intoxication of rainbow trout with CC14 results
in demonstrable morphological damage to the liver and plasma retention
of BSP (Gingerich et al., 1978a). Because the processes of hepatic accumula-
tion, metabolism and biliary excretion of this organic anion in the trout
appear to conform to those of mammals (Schmidt and Weber, 1973; Gingerich
et al., 1977, 1978b), it was of interest to investigate which of these
processes in trout were most affected by CC14 treatment.
The accumulation of more than half of the dose of BSP in the livers
of control animals 15 rain after its injection indicates that
egress of the dye from the plasma compartment was primarily the result
of its uptake and accumulation by the liver. In contrast, the
hepatic BSP content of treated fish was less than half that of the
controls after this time. Furthermore, the apparent net rate of
hepatic BSP accumulation was slower in treated fish despite plasma
BSP concentrations that should have favored its hepatic uptake.
84
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Maggio and Fujimoto (1966) similarly found that the concentration of
BSP in the livers of mice treated with CC14 was less than that of
controls following a single injection of BSP and concluded that impairment
of uptake or storage was most responsible for the decrease in plasma BSP
clearance. From the present study it appears that BSP plasma clearance in
treated fish may be retarded in part by impaired uptake or storage; processes
which were not differentiated by the methods used in this study.
While extracts of whole livers would be expected to be contaminated
with residual BSP in the canalicular and ductular spaces, it does not
seem likely that differences in hepatic BSP concentrations could result
from differences in the amounts of BSP within these spaces. Bile BSP
concentrations and rates of bile secretion in treated and control trout
were similar during the first nine hours of BSP infusion. Even if the
rate of biliary BSP transport in these animals had equalled the maximum
rate of biliary BSP excretion as determined during the infusion experi-
ments (12.1 yg BSP/kg/min), less than five percent of the injected dose of
BSP would have been transported into the bile after 15 min. The difference
in the total amount of hepatic BSP found in livers of treated and control
fish 15 min after injection of the dye was more than 25 percent of the
injected dose. Therefore, it appears that differences in the amount of
BSP in the intrahepatic biliary space cannot adequately account for differences
in the amount of BSP found in liver extracts of treated and-control fish.
Results of the infusion experiments suggest that the excretory capacity
of the liver was not greatly reduced 24 h after CC14 treatment. Bile
flow rates, bile BSP concentration and total metabolized BSP appearing in
the bile of treated fish were similar to those of control animals for at
least 9 h after the start of BSP infusion and for 33 h after CC14 treatment.
These results are in contrast to similar studies which have demonstrated
that impaired biliary excretory function is the factor which most contributes
to plasma BSP retention in rats following CC14 treatment (Klaassen and
Plaa, 1968; Priestly and Plaa, 1970a). The possibility that impairment of
biliary excretory function in treated fish occurs at a time later in
the course of the intoxication cannot be excluded. The decrease in the
rate of BSP excretion nine hours after the beginning of the infusion
was the result of decreased bile flow. Furthermore, the greatest plasma
BSP retention observed in treated animals during a time-course study
was found 48 h after CC14 was administered (Gingerich et al., 1978a).
Therefore, it may be possible that a decrease in bile flow may have contributed
in part, to plasma BSP retention at some time beyond the temporal limits
which were chosen for the biliary excretion studies.
The relative importance of BSP conjugation to the overall process
of BSP excretion in fish has not been established. If metabolism of
this compound was the important prerequisite for its biliary excretion
in -fishes that it appears to be in mammals (Whelan et al., 1970; Priestly
and Plaa, 1970b), a higher proportion of metabolized BSP would be expected
in their bile. The proportion of metabolized BSP in the bile of both
treated and control trout was approximately 25 percent during the first
hour of the infusion. This value is only one-third that reported for rat
85
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bile (Schulz and Czok, 1974). However, despite these quantitative differences,
the biliary excretory capacity of the trout liver does not appear to be
overtly deficient. The concentration of BSP in the bile of trout during
maximum rates of biliary dye excretion (11.8 mg/ ESP/ml) is not greatly
different from that found in rat bile under similar experimental conditions
(15.6 rag BSP/ml, Klaassen and Plaa, 1968). Therefore, it appears that
even if CC14 intoxication had resulted in decreased hepatic BSP metabolism,
it is unlikely that this would have affected biliary excretion of this
compound sufficiently to cause its plasma retention.
It is possible that a decrease in hepatic blood flow following exposure
to CC14 may be responsible for both impaired plasma clearance and hepatic
accumulation of BSP by limiting its transport to the liver. Impairment of
hepatic blood flow by ligation of the hepatic portal vein has been shown
to decrease plasma clearance and hepatic accumulation of BSP in rainbow
trout (Gingerich et al., 1977). Intraperitoneal administration of undiluted
CC14 is known to result in general inflammation of the peritoneal cavity
and the formation of thrombii in the ventral intestinal vein (Gingerich et
al., 1978a). It is possible that these changes may have altered blood
flow in the splanchnic drainage sufficiently to influence blood flow to
the liver.
The use of function tests incorporating the organic anion BSP to
evaluate liver dysfunction in trout may be useful providing that limi-
tations of the technique are recognized. Thus, measurement of hepatic
BSP concentrations must be interpreted not only in terms of the processes
of uptake and accumulation; but also in terms of hepatic excretory function.
Further, the techniques which have been successfully applied to study
hepatic excretory function and storage capacity in small mammals (Klaassen
and Plaa, 1967) do not seem practical in the trout because of the toxic
effects of high plasma BSP concentrations (Schmidt and Weber, 1973) and
the length of time necessary to establish a maximal rate of biliary excretion.
The increase in body weight and decrease in plasma osmolality in
CCl4~treated fish suggested that body water was retained in these animals
and that the reduction in plasma protein concentration was due in part to
an increase in the plasma volume. An in_ vitro dilution of trout plasma,
of known protein concentration and osmolality, established that approximately
20% of the total protein decrease at 24 hours could be attributed to dilution
of the plasma proteins with water retained in the fish.
Plasma albumin concentration in fish treated with CC14 was lower
than that of controls at 24 hours posttreatment, but the increase in the
albumin/total plasma protein ratio indicated that a part of the reduction
in plasma proteins presumably was due to a loss of some globular fraction.
Erickson et al. (1938) found that the plasma proteins in CCl4-poisoned
dogs were reduced mainly by a decrease in the albumin fraction. Berryman
and Bo11man (1943) reported a reduction of total plasma proteins, chiefly
albumin, and a relative and absolute increase in the globulin fraction in
CCl4~treated laboratory rats. The implication that a specific protein
fraction accounted for the plasma protein reduction in the treated fish is
difficult because of the complex homeostatic mechanisms (in higher vertebrates)
86
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which control the level of plasma proteins (Zilva and Pannall, 1972). The
loss of a particular fraction, e.g. albumin, is followed by an increased
synthesis of that fraction, suggesting a feed-back stimulation of hepatic
synthesis. The nature of this feed-back control is unknown and apparently
non-specific since there is a concurrent increase in hepatic synthesis of
other plasma proteins.
Intoxication with CC1, (2.0 ml/kg, i.p.) also produced an oliguria
or anuria in rainbow trout as early as one hour after treatment which was
still apparent 24 hours after treatment. CC14 poisoning in humans,
whether by inhalation or ingestion, has been reported to produce oliguria
or anuria within 1 to 3 days following exposure (Guild et al., 1958; Moon,
1950; Sirota, 1949). Cornish and Ryan (1964), on the other hand, found a
two-fold increase in urine volume during the first 24 hours after exposing
rats to CCl^ vapors. Also using rats, Strieker et al. (1968) showed an
increase in urine volume for the first 24 hours after an oral dose of
CC14 (2.5 ml/kg).
Previous studies with salmonid fishes demonstrated that the stress
of handling, exposure to sub-lethal concentrations of chemicals or hypoxic
conditions, increased UFR (Hunn, 1969; Hunn and Allen, 1975; Lloyd and
Orr, 1969; Swift and Lloyd, 1974). Lloyd and Orr (1969) attributed the
diuretic response in rainbow trout exposed to sub-lethal levels of ammonia
to an increase in gill permeability to water, rather than a direct action
on the kidney. Gingerich et al. (1978a) reported a significant weight
gain in rainbow trout 24 hours post-treatment with CC14 (2.0 ml/kg,
i.p.). The results of these studies indicate that the weight gain experienced
by CCl4-treated trout was related to impaired water clearance as reflected
by a decrease in UFR and 24 h urine output.
Urine osmolality increased in the trout treated with CC14, A
decrease in the ability of the proximal or distal tubules to absorb electrolytes
from the glomerular filtrate would have been reflected as an increase in
urine osmolarity and may have been due to subtle changes in the integrity
of the tubule epithelial cells.
In mammalian toxicology, proteinuria is frequently indicative of
renal dysfunction and is associated with glomerular renal tubular damage.
This condition may occur even in the absence of demonstrable histopathology
(Foulkes and Hammond, 1975). The proteins in the urine that are associated
with glomerular damage are of relatively high molecular weight, e.g.
albumin, while those associated with tubular damage tend to be of lower
molecular weight. We have found that CCl4-treated trout experienced a
reduction in total plasma protein concentration after 24 hours, in
part due to dilution by retained body water. The presence of protein in
the urine suggests that some proteins also may have been lost through
damaged glomeruli or renal tubules.
The significance of this proteinuria is difficult to establish since
this test does not differentiate between structural and plasma proteins.
For example, CC14 could directly damage the kidney tubules and release
87
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structural proteins into the urine. Quantitative and qualitative measurements
of urinary protein should be considered in toxicity studies before much
significance is placed on proteinuria. These tests should be interpreted
with adequate knowledge of specific organ toxicity of the compound and
should be used in conjunction with histopathological evaluations.
Acute Exposure Studies: Monochlorobenzene
Elevated plasma GPT activity (Pfeifer et al.,1977; Racicot et al.,
1975; Statham et al., 1978a) and attenuated clearance of plasma BSP
(Gingerich et al., 1978a) both have been used as diagnostic criteria by
which to assess liver dysfunction in rainbow trout. In the present study
MCB intoxication produced elevations in plasma enzyme activity at 8 and 72 h
post-treatment. While a significant increase in plasma enzyme activity
occurred only at 72 h, it is interesting to note the secondary increase in
enzyme activity at 8 h. Biphasic responses in plasma GPT activity have
been reported previously in both rats (Koeferl, 1972) and in rainbow trout
treated with CC14 (Pfeifer et al., 1977; Racicot et al., 1975; Statham
et al., 1978a). A similar biphasic pattern of plasma enzyme activity may
be associated with MCB intoxication in the trout and may be indicative of
the pathological processes occurring during the intoxication.
Significant retention of BSP was evident in the plasma of fish at 3, 12,
and 24 h after MCB treatment. While it is possible that anesthetic effects
associated with the MCB intoxication may have influenced plasma clearance
of BSP by altering total hepatic blood flow, it is not likely that this
effect was solely responsible for the significant plasma retention of the
BSP observed in this study. Histological studies confirmed the presence
of some degenerative changes in the hepatocytes as early as 8 h after
treatment and evidence of minor degenerative changes were observed in
livers of fish sampled at 24 and 48 h post treatment.
A comparison of the relative liver toxicities of MCB and CC14
following their i.p. administration of trout suggest that CC14 may
be more hepatotoxic than is MCB. Significant changes in both the plasma
GPT activity and concentration of BSP from plasma were apparent following
treatment of trout with CC14 at all doses of this toxicant employed and
at all sample periods used in these studies. In addition, CC14 did
produce histological alterations in the livers of exposed trout including
necrosis of hepatocytes surrounding central veins. In contrast, the results
of studies with MCB were variable and inconclusive. Significant alterations
of the clinical indicators of liver dysfunction were evident only at the
highest doses of MCB used and only at specific times either early or late
in the course of the intoxication. These results suggest that the
liver of the rainbow trout may be more sensitive to intoxication by CC14
than by MCB.
The seemingly greater effect of CC14 in producing liver damage
in trout may be attributed to several factors. Because CC1, was not
administered in a corn oil vehicle it may have been absorbed more readily
and had greater access to body compartments than did MCB. However, when
88
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That serum GPT activity increased in a dose-dependent manner in
trout both exposed to MCB is consistent with our impression that elevations
in serum activity of this enzyme do reflect some specific organ toxicity.
In laboratory mammals, halogenated benzene compounds, administered orally,
produce consistent hepatic lesions presumably through highly reactive
intermediate metabolites (Jerina and Daly, 1974). This hepatotoxicity, as
well as that mediated through CCl^, does cause an elevation in the serum
activity of GPT and in most instances this elevated enzyme activity does
correlate closely with the pathological state of the organ. We have not
been able to demonstrate consistently these correlations in trout treated
either acutely or subacutely with known mammalian hepatotoxic agents and
therefore we can only speculate that the source of the increased GPT activity
is from the liver.
Serum alkaline phosphatase activity of trout exposed to subacute
levels of MCB was variable. A significant increase in AP activity was
observed in fish exposed to low MCB concentrations after 15 days but this
increase could not be related to dose. The serum AP activity after 30
days was not different from that of the paired controls. Fed control fish
consistently had higher serum AP activity than either the treated or
non-fed control group suggesting that serum AP activity may be mediated
either by diet or nutritional state of the animal.
The relative decrease in the concentration of serum proteins noted in
the subacute exposure study was related to the dose of MCB to which the
fish were exposed. While these changes were not statistically significant
the results suggest that some specific alteration in the constituent serum
proteins had occurred. This is supported by the decrease observed in the
ratio of fast to slow migrating zones identified on the serum electro-
pherograms of fish sampled after 15 days of exposure. Because the liver
functions to synthesize albumin decrease in the albumin to globulin ratio
has been used as an index of liver function in mammalian toxicology and
clinical medicine (Harper, 1975). Similarly, serum electrophoresis has
been used to evaluate the responses of fish to such general conditions of
stress as hypoxia (Bouck and Ball, 1965) and disease (Pesch, 1970) and to
stress induced by exposure to sublethal levels of such pollutants as pulp
mill effluent and industrial chemicals (Fujiya, 1961) and copper (Thurston,
1967). In all cases the response has been a decrease in the rapidly
migrating fraction or fractions of the serum protein constituents. Whether
this response is the result of a decrease in the rate of synthesis of
these fractions or a preferential utilization of these fractions by the
fish during food deprivation is not known.
Gross Pathology and Histology
The development of pericentral liver necrosis in trout following
acute CC14 intoxication is not unlike the centrilobular liver necrosis
that routinely develops in mammals after treatment with this toxicant.
In mammals, it is not clear whether these lesions result from the irreversible
binding of active intermediates of CC14 metabolism to critical cellular
elements (Castro, et al., 1972; Klaassen and Plaa, 1969) or whether these
91
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active intermediates precipitate a peroxidative attack on lipid structural
elements (Rechnagel, 1967). In either case, it is generally felt that the
hepatotoxicity associated with CC14 intoxication is related to metabolism
of the compound. In view of recent reports which indicate that components
of the mixed function oxidase system are present in various fish, including
rainbow trout (Stanton and Khan, 1975; Chan, et al . , 1967; Ludke, et al.,
1972), and that rainbow trout are capable of hepatic biotrans format ions by
this mixed function oxidase system (Petersen et al . , 1976), it is conceivable
that the pericentral liver necrosis that develops in trout following
CC14 treatment is the result of its metabolism to an active intermediate
or intermediates.
Only one animal in four from both the transected and non-transected
fish treated with CCl^ developed necrotic lesions in the pericentral
regions of the liver, even though minor degenerative changes were
found in all treated animals. The reason for this variability is not
known. Differences in the nutritional status among fish used in this
experiment may be responsible in part, since diet is known to greatly
influence both metabolism of CC1. and the degree of hepatotoxicity it
produces in rats (Seawright and McLean, 1967). In addition, unequal rates
of uptake or differences in distribution of the toxicant may have contributed
to this variability. Statham et al.(197Sa) have reported that rainbow
trout dosed with undiluted 14CC14 (1.0 ml/kg i.p.) accumulated highest
levels in the mesenteric fat surrounding the G.I. tract followed by intermediate
concentrations in the heart, Ivier, and gills. In the present study,
large amounts of visceral fat may have reduced the effective dose of
by providing a storage depot for the toxicant.
Necrosis in the subcapsular region was probably caused by direct
contact of CC14 with the liver. Conversely, it is not likely that the
pericentral necrosis was caused by direct contact with high concentrations
of the toxicant. If this were the case, one would expect that periportal
hepatocytes also would be damaged since cells in this region should be
exposed to levels of CC1. sooner and in higher concentrations than those
in the pericentral region. No evidence of periportal necrosis was found
in the liver of any animal receiving
Intoxication of laboratory mammals with CC14 results in vacuoliza-
tion of hepatocytes and triglyceride accumulation (Cornish, 1975). Previous
studies with trout have demonstrated intense vacuolization of hepatocytes
in both control fish and fish treated with CC14 (Racicot et al., 1975).
These observations were similar to those made in this study, however histo-
chemical staining confirmed that the vacuoles contained no lipid material
but did contain glycogen. In support of these findings, Statham et al.
(1978a) found that CC14 had no effect on liver triglyceride accumulation
in rainbow trout and Sakaguchi and Hamaguchi (1975) reported no effect of
CC14 on the hepatopancreas lipids of the yellowtail (Seriola dorsalis) .
Additionally, Statham et al. (1978a) noted vacuolization in control trout
and intense vacuolization as well as focal and laminar necrosis in CC14-
treated trout at 6 h post treatment (1.0 ml/kg i.p.).
92
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Histopathological examination of liver and kidney sections of trout
treated with CCl^ indicate that there was no definitive correlation
between the degree of hepatocyte or nephron damage and the dose of CC14
given, the diet fed or the time after treatment. Furthermore, it was not
possible to correlate plasma GPT activity with the degree of hepatocelluar
damage. The central vein necrosis, a characteristic lesion in mammals
given .CC1,, was not a consistent histopathological feature of CC14
intoxication in the trout used in these studies.
The results of acute exposure studies suggest that monochlorobenzene
intoxication may cause some hepatotoxicity in the rainbow trout. After
8 h moderate pericentral necrosis was evident in the liver of one treated
fish and less dramatic degenerative changes were seen in pericentral hepato-
cytes of several other treated fish sampled at 24 and 48 h. The development
of such lesions in pericentral regions of the trout liver is a response
not unlike the centrilobular necrosis observed in laboratory mammals
following MCB intoxication (Reid and Krishna, 1973; Brodie et al., 1971).
In view of reports indicating that active metabolites are responsible for
the hepatotoxicity observed in mammals following MCB intoxication (Reid
and Krishna, 1973) and that rainbow trout are capable of hepatic biotrans-
formations by a mixed function oxidase system (Petersen et al. 1976), it
is possible that the morphological changes observed in the pericentral
hepatocytes of the trout are mediated through active intermediates.
The value of assessing the effects of subacute or chronic exposure
to pollutants on the function and performance of individual organ systems
in fish in some sense lies in the successful adaptation of these techniques
to field studies. Because of the considerable variation that exists in
the clinical indices of liver function between individual fish and groups
of fish these methods seem somewhat impractical for general field use.
Such variations make inter-group comparison of field populations, increasingly
difficult. Observations of acutely and subacutely treated fish revealed
gross behavioral changes even though alterations of clinical indices were
not evident. For significant alterations to be apparent in these indices
treated animals were severely stressed in most cases, suggesting that fish
in the field would have to be similarly incapacitated before differences
could be detected. The investment of large amounts of time and effort
to describe such obviously deleterious effects seems impractical. Therefore,
while these methods do have value in comparative toxicological research
their relevance seems restricted to precisely controlled laboratory studies.
93
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PUBLICATIONS RESULTING FROM PROJECT
1. Gingerich, W. H., L. J. Weber and R. E. Larson. 1977. Hepatic Accumu-
lation, Metabolism and Biliary Excretion of Sulfobromophthalein by
Rainbow Trout (Salmo gairdneri). Comp. Biochem. Physiol. 58:113-120.
2. Gingerich, W. H,, Weber, L. J. and Larson, R. E. 1978. The Effect of
Carbon Tetrachloride on Hepatic Accumulation, Metabolism, and Biliary
Excretion of Sulfobromophthalein in Rainbow Trout. Tox. Appl. Pharmacol.
43:159-167.
3. Gingerich, W. H., L. J. Weber and R. E. Larson. 1978. Carbon Tetra-
chloride- Induced Retention of Sulfobromophthalein in the Plasma of
Rainbow Trout. Tox. Appl. Pharmacol. 43:147-158.
4. Gingerich, W. H. and L. J. Weber. 1976. Carbon Tetrachloride Induced
Plasma Retention of Sulfabromophthalein in Rainbow Trout (Salmo gaird-
neri) . Fed. Proc. 35:585.
5. Gingerich, W. H., L. J. Weber and R. E. Larson. 1977. Hepatic Accumu-
lation, Metabolism and Biliary Excretion of Sulfobromophthalein by
Rainbow Trout (Salmo gairdneri). Proc. West. Pharmacol. Soc. 20:83-84.
6. Pfeifer, K. F., L. J. Weber and R. E. Larson. 1977. Alanine Amino-
transferase (GPT) in Rainbow Trout: Plasma Enzyme Levels as an Index
of Liver Damage. Proc. West. Pharmacol. Soc. 20:431-437.
7. Pfeifer, K. F. and L. J. Weber. 1978. Plasma Protein Changes in
Rainbow Trout after Carbon Tetrachloride Intoxication. Twenty-first
Annual Meeting Western Pharmacology Society.
8. Pfeifer, K. F. and L. J. Weber. The Effect of Carbon Tetrachloride
on Total Plasma Protein Concentration of Rainbow Trout, Salmo gairdneri.
Comp. Biochem. Physiol. (In press)
9. Pfeifer, K. F. and L. J. Weber. The Effect of Carbon Tetrachloride on
Urine Flow Rate of the Rainbow Trout, Salmo gairdneri. Tox. Appl.
Pharmacol. (In press)
10. Weber, L. J., W. H. Gingerich and K. F. Pfeifer. Alterations in Rainbow
Trout Liver Function and Body Fluids Following Treating with Carbon
Tetrachloride or Certain Chlorinated Benzenes. Amer. Chem. Soc. (In
press)
94
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-79-088
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Assessment of Clinical Procedures to Evaluate Liver
Intoxication in Fish
5. REPORT DATE
August 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William H. Gingerich and Lavern J. Weber
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Fisheries and Wildlife
Oak Creek Laboratory of Biology
Oregon State University
Corvallis, Oregon 97331
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
R803090
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT ~~ ———
Procedures were developed to clinically evaluate liver damage and liver function in
rainbow trout following either acute intraperitoneal (i.p.) treatment or subacute
bath exposure to selected mammalian hepatotoxic agents. Elevations in serum of liver
specific enzymes such as aspartate aminotransferase (GOT), alanine aminotransferase
(GPT) and alkaline phosphatase (AP) were investigated as potential indicators of
hepatocellular damage. An exogenous test of liver function, plasma clearance of the
organic anion sulfobromophthalein (BSP), also was investigated as a potentially
useful test of overall liver function in the trout.
The application of clinical tests to diagnose liver dysfunction in fishes following
their exposure to environmental toxicants may be practical in controlled laboratory
facilities. Despite the considerable variation that exists between groups of fish,
significant differences could be demonstrated between control and treated fish.
Variation among groups of fish make intergroup comparison of the field populations
increasingly difficult by these methods. Therefore, the use of such techniques should
be employed to evaluate liver toxicity under precisely controlled laboratory studies.
Their application to field studies does not seem advisable.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Biochemistry
Physiology
Fishes
Aquatic biology
Enzymes
Rainbow trout
Liver toxicity
Clinical tests
Hepatic
06/A
06/C
06/P
06/T
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
118
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Fora 2230-1 (R.v. 4-77)
106
» US GOYBWICXTm>ITl«OmC£:I979 -657-060/5419
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