NOAA
EPA
National Oceanic and
Atmospheric Administration
Seattle WA 98112
United States
Environmental Protection
Agency
Office of Environmental
Engineering and Technology
Washington DC 20460
EPA-600/7-81-128
July 1981
Research and Development
Interactive Effects of
•*• \
Aromatic Hydrocarbons,
Their Derivatives, and
Heavy Metals in
Marine Fish
Interagency
Energy/Environment
R&D Program
Report
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EPA-600/7-81-128
July 1981
INTERACTIVE EFFECTS OF AROMATIC HYDROCARBONS
THEIR DERIVATIVES, AND HEAVY METALS IN MARINE FISH
w
by
Edward H. Gruger, Jr., Joyce W. Hawkes, and Donald C. Mai ins
Environmental Conservation Division
Northwest and Alaska Fisheries Center
National Marine Fisheries Service
National Oceanic and Atmospheric Administration
2725 Montlake Boulevard East
Seattle, Washington 98112
NOAA Project Officer: Douglas A. Wolfe (NOAA/Boulder, CO)
This study was conducted as part of the Federal Interagency
Energy/Environment Research and Development Program
Prepared ^or
OFFICE OF ENERGY, MINERALS, AND INDUSTRY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
U.S. Environmental Protect;?
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Prepared for the NOAA Energy Resources Project #3
in partial fulfillment of the
Environmental Interagency Agreement #EPA-AIG-E693
Work Unit #3-3-2
DISCLAIMER
This work is the result of research sponsored by the Environmental
Drotection Agency and administered by the Environmental Research Labora-
tories of the National Oceanic and Atmospheric Administration.
The Environmental Research Laboratories do not approve, recommend
or endorse any proprietary product or proprietary material mentioned in
this publication. No reference shall be made to the Environmental
Research Laboratories or to this publication in any advertising or
sales promotion which would indicate or imply that the Environmental
Research Laboratories approve, recommend, or endorse any proprietary
product or proprietary material mentioned herein.
This report has been reviewed by the Office of Research and Develop-
ment, U. S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the il.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
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CONTENTS
Foreword v
Abstract vi
Figures ix
Tables xii
Acknowledgment xv
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Experimentation 6
Effects of chlorinated biphenyls and petroleum
hydrocarbons on the uptake and metabolism of
aromatic hydrocarbons in salmonids 6
Materials 6
Procedures 7
Results and Discussion 9
Summary 28
The effects of petroleum hydrocarbons and chlor-
inated hiphenyls on the morphology of tissues
of chinook salmon. I 29
Methods 29
Results 29
Discussion 34
Summary 37
Interactions of polycyclic aromatic hydrocarbons
and heavy metals on activities of aryl hydrocarbon
hydroxylase 37
Methods 37
Results and Discussion 38
Determination of activities of aryl hydrocarbon mono-
oxygenase using three different substrates 39
Materials 39
111
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Procedures 41
Results and Discussion 42
Summary 46
Organic syntheses of 2,6-dimethylnaphthalene
derivatives 46
Methods 46
Mutagenesis assays of 2,6-dimethylnaphthalene
derivatives 49
Methods 49
Results and Discussion 49
Summary 52
In vitro metabolism of 2,6-dimethylnaphthalene
by coho salmon liver microsomes 52
Methods 52
Results 55
Discussion 58
Summary 59
Effects of naphthalene and p-cresol, separately and
together in foodpath exposures, in starry flounder
on the in vivo metabolism of 2,6-dimethylnaphthalene. 59
Materials 59
Methods and Procedures ....... 60
Results 64
Discussion 68
Summary 71
Effects of naphthalene and p-cresol on in vivo
synthesis of lipids in starry flounder 72
Methods 72
Results 73
Discussion 76
Summary 77
Effects of 2,6-dimethylnaphthalene and p-cresol on
the morphology of the liver of coho salmon 84
Methods 84
Results 84
Discussion 90
Summary 93
Bibliography 94
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Along with the accelerated development of petroleum resources on
the continental shelf of the United States and continued importation
of petroleum of foreign origin, we can expect increased transfer and
refinement activities in coastal areas, with an associated incidence
of oil pollution. In order to develop an adequate understanding of the
potential marine environmental consequences of such pollution, the
National Oceanic and Atmospheric Administration has conducted studies
on the Fate and Effects of Petroleum Hydrocarbons and Toxic Metals in
Selected Marine Organisms and Ecosystems under Interagency Agreement
with the Environmental Protection Agency. The overall objectives of
this effort have been to study experimentally specific processes
underlying the distribution, transport and biological effects of petro-
leum hydrocarbons in coastal marine ecosystems. The results are
expected to facilitate the assessment of impacts of petroleum releases,
and thereby to improve the basis for developing regulatory measures for
suitable protection of the marine environment. A primary concern
expressed consistently during early considerations of study priorities
was that different classes of xenobiotic compounds, introduced into the
environment as a result of various human activities, might act
synergistically to produce effects of greater magnitude than would be
predicted from experimental results with individual compounds. The
report that follows presents results directed at this experimentally
difficult area of concern.
Douglas A. Wolfe
Office of Marine Pollution Assessment
NOAA
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ABSTRACT
Marine organisms living in environments containing toxic chemicals
are often exposed simultaneously to many different classes of compounds,
which collectively pose a different threat of toxicological effects
than is posed separately by the individual compounds. The present
research was directed toward elucidating the effect of xenobiotics
which alter the metabolism and toxicity of aromatic hydrocarbons by
marine fish, as evinced through biochemical changes and altered cellular
morphology. The xenobiotics used included petroleum aromatic hydro-
carbons, chlorinated biphenyls, p-cresol, cadmium and lead.
Coho salmon (Oncorhynchus kisutch), maintained in seawater at 7°C
and fed model mixtures containing chlorobiphenyls, petroleum hydro-
carbons, and a mixture of the two classes of compounds, were examined
for uptake of these chemicals in liver, kidney, and residual body
tissues comprised of eviscerated, headless and tailless carcass; and
for possible changes in the activities of hepatic microsomal aryl
hydrocarbon hydroxylase (AHH). Analyses of the coho salmon tissues
indicated that the concentrations of individual chlorobiphenyls were
highly variable; the concentrations of the hydrocarbons were below
detection limits, suggesting that they were metabolized and/or excreted
by the fish. In addition, the activities of hepatic AHH were induced
by mixtures of the chlorobiphenyls and petroleum hydrocarbons, but not
by the chlorobiphenyls alone or hydrocarbons alone. An apparent
synergism, reflected by increased AHH activities, occurred when the
chlorobiphenyls and hydrocarbons were administered together.
Chinook salmon (Oncorhynchus tshawytscha), maintained in seawater
at 13°C and fed mixtures of chlorobiphenyls and petroleum hydrocarbons
separately and together, were examined for changes in hepatic microsomal
AHH activity and for alterations in the morphology of liver, kidney,
intestine, gill and skin tissues. The data suggested that the AHH
activity was affected differently for chinook than for coho salmon.
Using chinook salmon microsomes, AHH activities were found depressed
for all fish treated with either the chlorobiphenyls or the hydrocarbon
mixture. In addition, morphological changes relating to inclusions in
the cells of the intestinal mucosa were observed for chinook salmon
fed either hydrocarbons or chlorobiphenyls; whereas, considerable
sloughing of the mucosal epithelium occurred in fish treated with the
combined mixtures. The latter finding also indicated an interactive
effect of the two classes of xenobiotics. Additional alterations were
found in some hepatocytes, but not in the other tissues.
The differences for the coho and chinook salmon in terms of the
responses of the hepatic AHH systems to petroleum hydrocarbons and
chlorobiphenyls may have been caused by differences due to seasonal
parameters between the two experiments. Hence, depending on the temp-
eratures, normal biotransformations of petroleum in exposed marine
VI
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organisms may be enhanced or retarded, assuming no species differences.
These xenobiotic effects maybe influential factors in marine
environments.
Coho salmon and starry flounder (P1 a t i c h t hy s s t el 1 at us), exposed
to 200 ppb of cadmium or lead in seawater at 10°C, were fed a model
mixture of polycyclic aromatic hydrocarbons (PAH's) consisting of
2-methylnaphthalene, 2,6-dimethylnaphthalene, and phenanthrene. The
effects of the metals were determined on the AHH activity of liver
microsomes. (Concentrations of cadmium and lead in livers were not
determined.) The results suggest that hepatic hydroxylations of PAH's,
using naphthalene as a substrate, are not affected by exposure of fish
to these metals at 200 ppb. Addition of 4-5 ppm cadmium to reaction
mixtures containing liver microsomes caused 82-98% inhibition of AHH
activity; lead at 10 ppm caused 84% inhibition. Thus, the possibility
exists that alterations in PAH metabolism may occur if cadmium or lead
accumulates in salmon and flounder livers. Such alterations may result
in changes in the toxic effects of PAH's in organisms exposed to petro-
leum in the environment.
Benzo[a]pyrene, 2,6-dimethylnaphthalene, and naphthalene were used
as substrates for comparisons of AHH activities in a preparation from
coho salmon liver microsomes. The apparent Michael is constants (Km; the
concentration of substrate required to obtain one-half of the maximum
reaction velocity with a given concentration of enzyme) were determined,
and the results indicated that salmon microsomal mixed-function oxidase
systems have a higher affinity for benzo[a]pyrene and 2,6-dimethyl-
naphthalene than for naphthalene. Because results indicate that
microsomal preparations from livers have a high affinity for 2,6-
dimethylnaphthalene, and that the latter compound is more soluble in
water and less hazardous to handle than is benzo(a)pyrene, 2,6-dimethyl-
naphthalene is a useful substrate for research on evaluating effects
and dispositions of xenobiotics introduced into marine environments
through petroleum-related activities.
Microbial tests for mutagenicity were performed on 2,6-dimethyl-
naphthalene, on four oxygenated derivatives of 2,6-dimethylnaphthalene,
and on the naphthalene derivative, trans-1,2-dihydroxy-1,2-dihydronaph-
thalene. All tests for mutagenicity were negative; however, 2,6-
dimethyl-3,4-naphthoquinone was lethal to the test- organism, Salmonella
typhimurlum. As far as can be generalized from microbial bioassays,
mutagenic reactions are unlikely in fish due to exposures to these
naphthalenic chemicals.
The hepatic microsomes from coho salmon were used in the eluci-
dation of the metabolism of 2,6-dimethylnaphthalene. Major metabolites
produced were 6-methyl-2-naphthaldehyde and 6-methyl-2-naphthalenemethanol.
Products of naphthyl-ring oxidations included a quinone, two naphthols,
and a dihydrodiol of 2,6-dimethylnaphthalene. Studies of PAH metabolism
in vitro provided a basis for preparing metabolites for chemical analyses,
VI 1
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which are necessary for elucidating the metabolic fate of PAH's in
marine organisms in this and future work.
Other studies were performed to determine the effects of exposing
starry flounder to naphthalene, p-cresol, or a mixture of both compounds.
The work included (1) in vivo synthesis of metabolites from
2,6-dimethylnaphthalene, and (2) the biosynthesis of lipids. Biliary
metabolites of 2,6-dimethylnaphthalene consisted primarily of con-
jugates, namely, glucuronides and glucosides. Exposures of flounder
to naphthalene, p-cresol, or both indicated a significant reduction of
the glucuronide of 6-methyl-2-naphthalenemethanol in the bile and a
corresponding increase in the glucuronide of trans-3,4-dihydroxy-3,4-
dihydro-2,6-dimethylnaphthalene. Such a shift in metabolite com-
position, from products of methyl-group oxidation to those of aromatic-
ring oxidation, may result in metabolites which are more toxic.
In the study of xenobiotic-induced alterations in lipid biosyn-
thesis, significant decreases were observed in the incorporation of
acetate into free fatty acids in liver of flounder exposed to naphth-
thalene alone (Pj<0.05) or together with p-cresol (P^O.Ol), compared to
that of controls. Free fatty acid synthesis was apparently not altered
upon exposure of animals to p-cresol alone; however, all exposure
groups experienced changes in the biosynthesis of triglycerides, as
reflected by decreases in the incorporation of oleate into triglycerides.
No evidence was found to indicate that phospholipid biosynthesis was
altered. Because triglycerides are a source of energy for fish,
xenobiotic-induced alterations in biosynthesis of these compounds may
pose a threat to the viability of exposed fish.
A study of the effects of 2,6-di-methylnaphthalene and p-cresol on
liver-cell morphology of coho salmon has established that these chemicals,
separately or together at 10-20 ppm, damaged the sinusoids and certain
organelles of the hepatocytes. The morphological changes, particularly
those involving the sinusoids, are similar to hepatic changes seen in a
variety of other animals afflicted with conditions such as hepatitis
and toxic-chemical injury.
vm
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FIGURES
Number
1-4 Micrographs of intestine from control salmon. 30
1 Light micrograph of intestinal villi. SOX. 30
2 Tip of villus with normal mucous cells (m) and brush border
(arrow). 1100X. 30
3 Electron micrograph of columnar epithelial cells and mucous
cell (m). 3500X. 30
4 Microvillar surface and upper fourth of a typical columnar
cell. 14,OOOX. 30
5-7 Micrographs of intestine from Chinook salmon fed a model
mixture of 5 ppm petroleum hydrocarbons for 28 days. 32
5 Light micrograph of intestinal villi with numerous
inclusions (arrow). SOX. 32
6 Higher magnification of columnar cells of the mucosa. 1100X. 32
7 Electron micrograph of upper third of a portion of the intest-
inal mucosa. 17,OOOX. 32
8-10 Micrographs of intestine from Chinook salmon fed 5 ppm
chlorinated biphenyl mixture for 28 days. 33
8 Light micrograph of the intestinal mucosa with exfoliation
(arrow). SOX. 33
9 Two types of inclusions (a,b) are evident in this light
micrograph. 1100X. 33
10 Electron micrograph of the upper portion of mucosal cells
with brush border and exfoliation (arrow). 3500X. 33
11-14 Micrographs of intestine from Chinook salmon fed 5 ppm
each of a- petroleum hydrocarbon mixture and a chlorin-
ated biphenyl mixture. 35
11 Light micrograph of intestinal villus with several sites of
exfoliation (arrows). SOX. 35
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12 Mucous cells (m) and columnar cells in a region of sloughing.
1100X. ' 35
13 Electron micrograph of columnar cell with inclusions from
an area of the mucosa in which there was no evidence of
exfoliation. 480QX. 35
14 Higher magnification of the base of the microvilli, terminal
web, and subadjacent region. 13,OOOX. 35
15 Hofstee plot of benzol a]pyrene metabolism by liver microsomes
of coho salmon. 44
16 Hofstee plot of 2,6-dimethylnaphthalene metabolism by
liver microsomes of coho salmon. 45
17 Hofstee plot of naphthalene metabolism by liver microsomes
of coho salmon. 45
18 Oxygenated derivatives of 2,6-dimethylnaphthalene. 47
19 Induct test response-to-background ratios for naphthalenic
compounds (100 ug/plate) and reference compounds (1 to
10 ug/pllate) with E. coli K12 cells. 53
20 In vitro metabolism of 2,6-dimethylnaphthalene. 57
21 HPLC profile of metabolites of 2,6-dimethylnaphthalene-4-14C
from livers of starry flounder exposed to naphthalene
and p-cresol. 66
22 Hepatocytes from control coho salmon held for five weeks.
Sinusoidal space (arrows). 680X. 87
23 Hepatocytes from coho salmon exposed to 10 ppm 2,6-dimethyl-
naphthalene plus 10 ppm p-cresol for five weeks. Sinusoidal
space (arrows); Vacuolated cytoplasm (*). 850X. 87
24 Hepatocytes from control coho salmon held for five weeks.
Sinusoidal border (arrow). 2,400X. 87
25 Hepatocytes from control coho salmon held for five weeks.
Space of Disse (D). 5,600X. 87
26 Hepatocytes fropi coho salmon after five weeks exposure to
10 ppm 2,6-dimethylnaphthalene. Sinusoidal border (arrow).
2.900X 88
27 Hepatocytes from coho salmon after five weeks exposure to
10 ppm 2,6-dimethylnaphthalene. Sinusoidal border (arrow).
6,OOOX. 88
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28 Hepatocytes from coho salmon exposed to 20 ppm 2,6-dimethyl-
naphthalene for five weeks. Sinusoidal border (arrow);
Vacuolated cytoplasm (*). 3,OOOX. 88
29 Hepatocytes from coho salmon exposed to 20 ppm 2,6-dimethyl-
naphthalene for five weeks. Sinusoidal border (arrow);
Vacuolated cytoplasm (*). 6,750X. 88
30-33 Hepatocytes from coho salmon after five weeks exposure to
10 ppm or 20 ppm p-cresol. 89
30 Exposures to 10 ppm p-cresol. Sinusoidal border (arrow).
2,800X. 89
31 Exposure to 10 ppm p-cresol. Sinusoidal border (arrow).
6,OOOX. 89
32 Exposure to 20 ppm p-cresol. Sinusoidal border (arrow).
2,800X. 89
33 Exposure to 20 ppm p-cresol. Sinusoidal border (arrow);
granular endoplasmic reticulum (ger). 5,800X. 89
34 Hepatocytes from coho salmon exposed to 10 ppm 2,6-dimethyl-
naphthalene and 10 ppm p-cresol for five weeks. Sinusoidal
border (arrow). 6,600X. 91
35 Hepatocytes from coho salimon exposed to 10 ppm of 2,6-di-
methylnaphthalene and 10 ppm of p-cresol for five weeks.
Sinusoidal border (arrow); Vacuolated cytoplasm (*).
5,600X. 92
XI
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TABLES
Number Page
1 Concentrations of chlorobiphenyls in coho salmon body tissues 10
2 Concentrations of chlorobiphenyls in coho salmon kidney
tissues 11
3 Concentrations of chlorobiphenyls in coho salmon liver
tissues 12
4 Time effects of tissue storage at -60QC on microsomal aryl
hydrocarbon hydroxylase (AHH) activity 14
5 Intracellular distribution of AHH activity 15
6 Effects of substrate storage time on AHH activity blank
values: Storage of a H-benzo[a]pyrene solution 15
7 Effects of NADPH concentration in benzol a]pyrene hydroxylase
reactions of chinook salmon hepatic microsomes 16
8 AHH activity influenced by conditions of NADPH in enzyme
reactions 16
9 Cofactor requirements for benzo[a]pyrene hydroxylase reactions
with chinook salmon liver preparations 17
10 Benzo[a]pyrene hydroxylase activity of coho salmon hepatic
microsomes at various temperatures of the enzyme analysis
reaction 17
11 AHH activity of coho salmon hepatic microsomes as a function
of time of enzyme analysis reaction 18
12 Exposures to chlorobiphenyls and hydrocarbons: Influence on
optimum pH of AHH activities for chinook salmon hepatic
microsomes 19
13 Specific activities of hepatic microsomal aryl hydrocarbon
hydroxylase (AHH) in coho salmon fed a control diet and
diets containing test compounds 20
14 Specific activities of hepatic microsomal aryl hydrocarbon
hydroxylase (AHH) in chinook salmon fed a control diet and
diets containing test compounds 21
xii
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15 Body weights of coho salmon taken for AHH analyses 24
16 Body weights of chinook salmon taken for AHH analyses 25
17 Percentage incidence of fin rot disease observed for chinook
salmon taken for AHH analyses 26
18 Hematocrits for chtnook salmon taken for AHH analyses 27
19 Naphthalene hydroxylase activity in vitro of liver microsomes
of PAH-fed fish exposed to cadmium and lead in seawater 38
20 Inhibition of hepatic microsomal aryl hydrocarbon hydroxylase
(AHH) activity by cadmium chloride and lead nitrate i_n_ vitro 40
21 Activities of coho salmon liver monooxygenases"with three
polycyclic aromatic hydrocarbon substrates 44
22 Tests for mutagenic activity in S. typhimurium TA-98 cells 50
23 Tests for mutagenic activity in S. typhimurium TA-100 cells 51
24 Time course of metabolite formation from 2,6-dimethyl-
naphthalene 56
25 Metabolites of 2,6-dimethylnaphthalene from in vitro
incubations of 15-minute reaction with liver microsomes 56
26 R.p values for reference compounds analyzed by thin-layer
chromatography using three solvent systems 61
27 Percent of C-2,6-dimethylnaphthalene metabolized in starry
flounder livers as determined by HPLC analysis 65
28 Analysis of 14C-2,6-dimethylnaphthalene metabolites by HPLC
of liver extracts from starry flounder fed p-cresol and
naphthalene, separately and together: Radioactivity in
HPLC fractions. 65
29 2,6-Dimethylnaphthalene-4- C metabolites in bile of starry
flounder exposed to naphthalene and p-cresol 69
30 Metabolites of 2,6-dimethylnaphthalene-4- C from enzymatic
hydrolysis of the glucuronide fraction of starry flounder
bile samples
31 Starry flounder used for study of lipids 74
32 Solvent distributions of carbon-14 labeled substances from
tissues of starry flounder at 24 hours after final intra?,
peritoneal injections of 1- C-naphthalene and p-cresol(CH-) 75
xm
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33 Activity of hepatic 2,6-dimethylnaphthalene monooxygenase
for starry flounder treated with naphthalene and p-cresol
by intraperitoneal injections, 24 hours after two
injections 24 hours apart 77
34 Lipid content of starry flounder livers used to determine the
incorporation of lipid precursors at 4-hour and 10-hour
intervals 78
35 Four-hour incorporation of ( H-9,10)oleate and 1- C-acetate
into lipids of livers from starry flounder treated with
naphthalene and p-cresol 80
o
36 Incorporation of ( H-9,10)oleate into sterol esters and
triglycerides of liver lipids from starry flounder treated
with naphthalene and p-cresol 81
37 Incorporation of 1- C-acetate into free fatty acids,
triglycerides, and sterol esters of liver lipids from
starry flounder treated with naphthalene and p-cresol 82
38 Incorporation of ( H-9,10]oleate and 1- C-acetate into
phospholipids of liver lipids from starry flounder treated
with naphthalene and p-cresol 83
39 Morphological abnormalities in liver tissue from echo salmon
exposed to 2,6-dimethylnaphthalene and p-cresol 85
xiv
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ACKNOWLEDGMENT
We wish to acknowledge several scientists and research associates,
whose creativity and dedicated work helped to consummate the present
research; namely. Dr. Marleen M. Wekell, Paul A. Robisch, John S. Finley,
Neil Stewart, Dr. Jerome V. Schnell, Suzyann Gaz,arek, Dr. Peter Fraser,
0. Paul Olson, Donald W. Brown, Dr. William L. Reichert, Lucinda Grant,
and Marianne Y. Uyeda. Technical consultations, advice, and assistance
are also acknowledged, namely, for Dr. Harold 0. Hodgins, Dr. Lawrence
Thomas, Patty Prohaska, Victor Henry, Scott Ramos, Robert C. Clark, Jr.,
Conrad Mahnken, Earl Prentice, William Waknitz, and Douglas Weber. We
also gratefully acknowledge the analyses by nuclear magnetic resonance
spectroscopy performed by Bernard J. Mist, of the University of Washington,
and mutagenicity tests performed by Dr. Richard Pelroy, of the Battelle-
Northwest Laboratories. Much appreciation is expressed to Frank J.
Ossiander and Dr. Russell F. Kappenman for their assistance with stat-
istics. Also, we extend our appreciation to those persons whose efforts
have aided in producing this and other reports and manuscripts, namely,
Gail Siani, Frank Piskur, Marge Morey, Ethel Terashita, Susan DeBow,
Marianne Tomita, Annette Hodgson, Marci Worlund, and Ethel Zweifel.
Finally, we thank Drs. Usha Varanasi and Harold Hodgins for their reviews
of this report.
xv
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SECTION 1
INTRODUCTION
The toxicity of petroleum or pretroleum products toward marine
animals will result from the combined effects of a complex mixture of
organic and inorganic chemicals. Observed toxic effects may not be
simply a sum of the effects of the individual components, but rather
the influence of interactions between different chemicals.
Although it is important to assess the toxicity of single
chemicals, interactive effects cannot be inferred from such research.
Direct chemical interactions between xenobiotics may give rise to new
chemicals that will have greatly enhanced or reduced toxicities. Also,
in vivo potentiation, addition, or antagonism may result from simulta-
neous exposure to mixtures of xenobiotics and their metabolites (Conney
and Burns, 1972; Krieger and Lee, 1973; Lichtenstein et al., 1973;
Livingston et al., 1974; O'Brien, 1967). Most laboratory investigations
have involved studies of accumulation, depuration, and toxicity of
individual classes of contaminants. This is particularly true for
chlorobiphenyls, petroleum-related hydrocarbons, and heavy metals
(Hansen, et al., 1971; Malins, 1977, Stalling and Mayer, 1972; Waldichuk,
1974). Prior to this research, there was little information on the
combined effects of chlorobiphenyls and polycyclic aromatic hydrocarbons
(PAH's) on marine fish. Interactive effects of mixtures of chlorobiphenyls
and PAH's or of mixtures of PAH's had not been assessed in marine
organisms prior to the preliminary work by Gruger et al. (1977a).
Fish possess xenobiotic-metabolizing enzyme systems which mediate
oxidations of compounds such as aromatic hydrocarbons (Adamson, 1967;
Ahokas et al., 1975; Buhler, 1966; Buhler and Rasmusson, 1968; Pohl
et al., 1974). These enzyme systems, present in microsomes prepared
from liver at usually higher concentrations than from other tissues,
have served as an index for evaluating potential toxicities of xenobiotics
in various animals (Gelboin et al., 1970; Franke, 1973). In addition,
the activities of these enzymes from fish have been proposed as indicators
of pollution of aquatic environments (Ahokas et al ., 1976; Payne, 1976
Payne and Penrose, 1975). In other words, when fish are exposed to, for
example, PAH's from petroleum, the activities of the liver enzymes, aryl
hydrocarbon hydroxylases (AHH), are useful in evaluating changes that
may occur in the metabolism and toxicity of the PAH's (Kurelec et al.,
1979).
The principal objective of the present research, between 1975 and
1978, was to provide information about the effects of biochemical inter-
actions of chlorinated hydrocarbons and PAH's on the accumulation,
metabolism, and potential toxicities of PAH's in marine fish. The
-------
objective was expanded in 1977 to include information about interactive
effects of heavy metals on the metabolism of PAH's. From 1978 to
October 1980, the work was further expanded to provide information about
possible interactive effects of two xenobiotics on the metabolism of a
third xenobiotic, namely, the combined effects of a representative PAH
and an oxygenated PAH on the metabolism of an alkyl-substituted PAH.
In addition, a study was conducted on possible effects of the two
former classes of compounds on lipid biosynthesis in fish. Related
studies of possible alterations in cellular morphology of experimental
fish were also an integral part of the research.
The research had the following specific objectives over the five
year period: (1) To determine changes that may occur in the accumula-
tion, metabolism, and disposition of mixtures of chlorobiphenyls and
PAH's in target tissues of coho salmon (Oncorhynchus kisutch) exposed
to those compounds singly and together in food; (2) to determine the
effects of mixtures of chlorobiphenyls and PAH's in coho salmon and
chinook salmon (Oncorhynchus tshawytscha) on the activities of hepatic
microsomal AHH; (3) to determine whether those mixtures cause possible
alterations in gross pathology and cellular morphology of salmon;
(4) to determine possible effects of a mixture of PAH's and cadmium
or lead in marine fish on the activities of hepatic AHH; (5) to determine
and compare activities of AHH from coho salmon liver microsomes toward
three PAH's (i.e., naphthalene, 2,6-dimethylnaphthalene, and benzo[a]-
pyrene); (6) to elucidate the in vitro metabolism of 2,6-dimethyl-
naphthalene by coho salmon liver microsomes; (7) to synthesize a series
of oxygenated derivatives of 2,6-dimethylnaphthalene for use as analytical
standards and for tests of mutagenicity; (8) to elucidate the effects of
naphthalene and p-cresol, singly or together, on the metabolism of 2,6-
dimethylnaphthalene in starry flounder (Platichthys stellatus); (9) to
determine whether 2,6-dimethylnaphthalene and p-cresol, singly or together
in coho salmon, cause alterations in the cellular morphology; and (10) to
determine whether naphthalene and p-cresol, singly or together in starry
flounder, alter lipid biosynthesis.
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SECTION 2
CONCLUSIONS
The findings from this study lead to the following conclusions:
1. Simultaneous exposure of salmonids to chlorinated biphenyls and
aromatic hydrocarbons significantly influence the biotransformations of
aromatic hydrocarbons. In chinook salmon, exposures to chlorinated
biphenyls and aromatic hydrocarbons alone also cause alterations in
metabolism of aromatic hydrocarbons. These interactions of xenobiotics
appear to be influential in modifying toxic effects in marine organisms
exposed to petroleum.
2. Exfoliation of intestinal epithelium can occur in chinook
salmon exposed to chlorinated biphenyls or to a mixture of petroleum
hydrocarbons and chlorinated biphenyls; the combined effect of the two
types of xenobiotics appears to be much greater than with the chloro-
biphenyls alone, suggesting an interaction. These compounds singly or
together had no effect on the morphology of liver, kidney, gills or
skin from exposed chinook salmon.
3. In coho salmon and starry flounder, no marked alterations
occur in metabolism of polycyclic aromatic hydrocarbons (PAH's) when
fish are exposed to waterborne cadmium or lead for prolonged periods.
However, alterations may result if very high concentrations of these
metals accumulate in livers.
4. The aryl hydrocarbon monooxygenase system of coho salmon liver
microsomes has an affinity for 2,6-dimethylnaphthalene that is near to
that for benzo[a]pyrene and much greater than that for naphthalene.
2,6-Dimethylnaphthalene is a convenient and useful compound for
evaluating the biochemical fate and effects of low-molecular weight
alkyl-substituted PAH's from petroleum.
5. Mutagenic reactions, as far as can be generalized from
microbial bioassays, are unlikely in fish due to exposures to
2,6-dimethylnaphthalene, 6-methyl-2-naphthalenemethanol, trans-3,4-
dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene, trans-1,2-dihydroxy-
1,2-dihydronaphthalene, 2,6-dimethyl-3-naphthol or 2,6-dimethyl-3,4-
naphthoquinone.
-------
6. The composition of metabolites of 2,6-dimethylnaphthalene
shifts from products of alkyl-group oxidation to naphthyl-ring
oxidation in starry flounder exposed to naphthalene, p-cresol, or
both xenobiotics together. Such a shift in metabolism of alkyl-substi-
tuted PAH's implies that different toxicities of these compounds may
occur in fish exposed to mixtures of different xenobiotics.
7. Triglyceride biosynthesis is altered in starry flounder
exposed to naphthalene, p-cresol, or a mixture of the two xenobiotics.
In addition, biosynthesis of fatty acids is altered in flounder exposed
to naphthalene with or without p-cresol. Such xenobiotic-induced
changes in the biosynthesis of neutral lipids may pose a threat to the
viability of exposed fish which must rely on lipids as energy sources
and for other essential physiological functions.
8. Alterations were observed in the morphology of hepatocytes of
coho salmon exposed to 2,6-dimethylnaphthalene and p-cresol. These
alterations, indicating toxic-chemical injury, would predictably
be harmful to fish exposed to such compounds from petroleum
pol lution.
-------
SECTION 3
RECOMMENDATIONS
The findings from this research suggest that exposure of marine
fish to mixtures of xenobiotics produces quite different results at
both the biochemical and morphological levels from results obtained
from exposures to the single compounds of the mixtures. Clearly,
more work is needed to examine the effects of combinations of
xenobiotics. For example, additional research is needed to elucidate
to what extent chlorinated hydrocarbons, in a wide range of concentr-
ations in the environment, can possibly change the composition of
metabolites of PAH's and alter toxicities in different marine species.
Eventually, every effort should be directed toward a better under-
standing of the combined effects of multiple contaminant systems
characteristic of polluted marine environments.
Future studies of the combined effects could include areas of
marine biology such as predator-prey relationships, reproductive
behavior and success, and feeding behavior. Studies of these kinds
should be supported by an interdisciplinary approach involving
physiology, morphology, biochemistry, and analytical chemistry.
-------
SECTION 4
EXPERIMENTATION
EFFECTS OF CHLORINATED BIPHENYLS AND PETROLEUM HYDROCARBONS ON THE
UPTAKE AND METABOLISM OF AROMATIC HYDROCARBONS IN SALMONIDS
Materials
Selection of Experimental Fish--
Approximately 2,400 coho salmon were obtained from seawater facilities of
Domsea Farms, Gorst, Washington. Groups of 20-30 fish were anesthesized with
tricaine methanesulfonate (MS-222) and their lengths and weights were
measured. After the weighings, the fish were distributed and maintained in
outdoor floating net pens. The pens were located in seawater at an
aquaculture facility of the Northwest and Alaska Fisheries Center on Puget
Sound, near Manchester, Washington. Each pen (4 ft x 7 ft x 6 ft deep with
1.5 ft above water) contained 150 fish. For these experiments, 2,100 fish
were held in 14 pens for duplicate exposures. The initial range of mean
weights +S.D. for the 14 groups was 184+41 to 204+41 g.
Approximately 600 chinook salmon were obtained from the aquaculture
facility of the Northwest and Alaska Fisheries Center. These fish were
treated in the same manner as the coho salmon, but were distributed in four
net pens in seawater. The initial weights of chinook salmon in the four
groups ranged from 72+14 g to 76+16 g.
Preparation of Control and Test Diets--
The basal diet for all fish was Oregon moist pellets (Hublou, 1963),
which were prepared from ingredients obtained from Moore-Clark Co., La Conner,
Washington. In order to minimize extraneous organic contaminants such as
PCB's in the diet, a fish protein concentrate (25.2% wt/wt) was substituted
for herring meal and extra food-grade soybean oil was added to the
preparations. The basal diet served as the control diet. Test diets were
prepared from the basal diet with chlorobiphenyls and hydrocarbons added as
separate mixtures in soybean oil solutions. For coho salmon fed diets with
chlorobiphenyls, the mixture of chlorobiphenyls consisted of 17.8% (wt/wt)
biphenyl, 11.6% 2-chlorobiphenyl, 19.8% 2,2'-dichlorobiphenyl, 9.6% 2,4'-
dichlorobiphenyl, 23.8% 2,5,2'-trichlorobiphenyl, and 17.3% 2,5,2',5'-
tetrachlorobiphenyl. For chlorobiphenyl diets fed to chinook salmon, the
mixture of chlorobiphenyls consisted of 20.2% biphenyl, 19.1% 2-
chlorobiphenyl, 20.9% 2,2'-dichlorobiphenyl, 20.2% 2,5,2'-trichlorobiphenyl,
and 19.8% 2,5,2',5'-tetrachlorobiphenyl. Coho salmon were fed a hydrocarbon
mixture that consisted of 12.1% 2,3-benzothiophene, 9.8% n-pentadecane, 12.2%
2,6-dimethylnaphthalene, 12.6% 2,3,6-trimethylnaphthalene, 12.6% fluorene,
12.7% 1-phenyldodecane, 12.4% phenanthrene, and 15.5% heptadecylcyclohexane.
Chinook salmon were fed a mixture of hydrocarbons that consisted of 14.8%
-------
benzothiophene, 12.6% 2-methylnaphthalene, 10.9% 2,6-dimethylnaphthalene,
11.4% 2,3,6-trimethylnaphthalene, 10.7% 1-phenyldodecane, 14.1% fluorene,
13.4% phenanthrene, and 12.1% heptadecylcyclohexane. The chlorobiphenyls
(Analabs Inc., North Haven, Conn.; RFR Corp., Hope, Rhode Is.) and the
hydrocarbons (Chemical Samples Co., Columbus, Ohio; Chem. Service, Inc.,
Westchester, Penn.; Aldrich Chemical Co., Inc., Milwaukee, Wis.) were obtained
from commercial sources and used without further purifications.
The pelleted diets were prepared in lots of 4.5 kg, using aliquots (10 g
and 50 g) of stock solutions of mixtures of the test compounds in food-grade
soybean oil. The test diets consisted of 1 and 5 ppm (wt/wt) of
chlorobiphenyls, 1 and 5 ppm of hydrocarbons, and 2 and 10 ppm of 50-50
mixtures of the two types of compounds. The pellets were stored at -15°C
until used.
Procedures
Feeding Schedules--
Fish were acclimated for three weeks before the initiation of the
exposure experiments. Coho salmon were fed 1 ppm and 5 ppm hydrocarbons and
chlorobiphenyls alone. In addition, coho salmon designated to receive the 2
and 10 ppm of the 50-50 mixtures of xenocompounds were fed the diets
containing 1 and 5 ppm of chlorobiphenyls, respectively, for one week before
beginning the feeding of the mixtures. The coho were fed three to four times
daily at a daily rate of 0.75% of the biomass, which was adjusted for the
weights of fish removed from the pens for analysis. (Examination of stomach
contents revealed food pellets, indicating that the food was ingested.) The
experiments were carried out from February to April, 1976, when ambient
seawater temperature was low (7°C), so that a feeding rate of 0.75% of biomass
was a maintenance ration. Test coho salmon were fed their respective diets
for four weeks, followed by four weeks with control diets.
The four groups of chinook salmon were fed the control diet, and diets
containing 5 ppm chlorobiphenyls, 5 ppm hydrocarbons, and 10 ppm of the 50-50
mixtures of chlorobiphenyls and hydrocarbons, respectively. The experiment
with the chinook salmon was performed during July and August, 1976, when
ambient seawater temperature was about 6°C greater (13°C) than during the
forementioned experiments with coho salmon. In a manner of feeding similar to
that for coho salmon, the chinooks were fed twice daily at a rate of 2.5% of
the biomass per day; that rate was chosen to provide a minimum growth to the
fish during July and August. Test fish were fed diets containing the
xenocompounds for four weeks, followed by a return to control diets for the
remainder of the time.
Handling of Samples—
Ten coho salmon for AHH analyses and six coho for chemical uptake
analyses were taken from half of duplicate exposure groups at weekly
intervals, alternating between duplicates. Only ten chinook salmon were taken
periodically from each group for AHH analyses. In addition, fish were taken
for microscopic examinations with sampling and handling protocols different
than for chemical and AHH analyses (cf., below). Fish were sacrified by
cervical dislocation, and lengths and weights were measured. For chemical
-------
analyses, fish were quickly placed over dry ice and transferred to laboratory
storage at -60°C. For AHH analyses, livers were excised, sex determined, and
inspections were made for abnormalities in physical characteristics. Excised
livers were rinsed with chilled 0.25 M-sucrose, immediately placed into screw-
capped vials, and frozen in liquid nitrogen. In the case of chinook salmon,
however, the livers were frozen over dry ice. The livers were then
transferred to the laboratory on dry ice and held at -60°C until analyses for
AHH activities were performed. Hematocrits were measured with blood taken
from the caudal vein before removal of the liver.
Chemical Analyses of Chlorobiphenyls and Hydrocarbons—
The procedures for hydrocarbon analyses of fish tissues were essentially
those reported by Warner (1976). Chlorobiphenyls in diets were analyzed by
gas-liquid chromatography (GLC) with electron-capture detection by the
procedures of Gruger et al. (1975), and in fish tissues by an adaptation of
the Warner method for hydrocarbons. Tissue samples consisted of kidney,
liver, and eviscerated, headless, tailless carcass; the latter being
essentially muscle tissue, bone, skin, and fat.
The analyses of tissues were performed in cooperation with the NOAA
National Analytical Facility (NAF). The modifications developed by NAF for
adapting the procedures of Warner (1976) were done in the following respects:
1. Tissue digestions were carried out in alkali, overnight at 30°C and
in Teflon-lined screw-capped centrifuge tubes. This manner of digestion
minimized losses of volatile compounds.
2. Silica gel chromatography was carried out with 100-200 mesh silica
gel (MC/B Manufacturing Chemists No. SX0144-06), which is a coarser, more
uniform grade than called for by the Warner procedure (Warner, 1976).
3. High-resolution capillary columns, rather than packed columns, were
used in GLC analyses. A 20 m by 0.26 mm WCOT glass capillary coated with
SE-30 stationary phase was operated at 2 ml helium per min flow, with
temperature programming from 60° to 250°C. Detection was by hydrogen-flame
ionization. It was possible to resolve the test compounds, both
Chlorobiphenyls and the hydrocarbons together, under optimum GLC conditions.
Analyses of Enzyme Activities in Livers--
AHH activity was determined by a modification of the procedure of
DePierre et al. (1975). Nonenzymatic background conversions were measured
with either boiled microsomes or by using reactions that contained a stop
solution before the addition of the substrate. Microsomal protein was
determined by an automatic system (Technicon Instruments Corp., Tarrytown,
N.Y.) adapted to the method of Lowry et al. (1951). Bovine serum albumin was
used as a protein standard.
For optimum specific activity of AHH, reaction mixtures contained the
following: 0.8-1.0 mg microsomal protein, 3 mM-MgCl2» 1.1 mM-NADPH,
62.5 mM-tris hydrochl oride buffer, and 66 yM-tritiated-benzo[a]pyrene
(12.5 mCi/mmole). The final volume was 1 ml. The pH of 7.5 was used in
reactions for coho salmon, while pH 7.8 was used initially for chinook salmon
-------
AHH reactions but changed to pH 7.5 for analyses of reactions at the seventh
day and thereafter (cf., Results and Discussion). The reaction mixtures were
preincubated for 10 min in a shaking water bath at 25+0.2°C before the
initiation of the reactions by the addition of 20 1 of acetone containing the
benzo[a]pyrene. Duplicate reactions were incubated in 15 x 125 mm open
culture tubes in subdued light. After 20 min at 25°C, reactions were stopped;
reaction products were extracted and analyzed by the method of DePierre et al.
(1975). Radioactivities of 0.3 ml aliquots of neutralized aqueous phase in 15
ml "PCS" (Amersham/Searle Corp., Arlington Heights, 111.) were measured in a
scintillation spectrometer (Packard Tri-Carb model 3003), with corrections
determined for background and quenching. AHH activities were calculated as
nmoles products/mg microsomal protein/20 min. The Student's t test was
applied to AHH activity values to compare differences between control and test
fish.
Tritiated benzo[a]pyrene was purified according to DePierre et al.
(1975), except the hexane solution of substrate was dried over sodium sulfate
prior to making an acetone stock solution. The specific activity of the
acetone solution was measured weekly. Purified benzo[a]pyrene was stored at
-20°C under nitrogen for up to four weeks.
Results and Discussion
Chemical Analyses--
The analyses for chlorobiphenyls and petroleum hydrocarbons, which were
contained in the diets, in liver, kidney, and eviscerated, headless and
tailless carcass from the coho salmon revealed that chlorobiphenyls readily
accumulated in each tissue after the fish were maintained for 1 to 4 or
5 weeks on those diets containing the chlorobiphenyls (Tables 1, 2, 3).
Moreover, no hydrocarbons clearly originating from the mixtures in the diets
were detected in liver, kidney, and the eviscerated carcass of fish which were
maintained for up to 4 weeks on diets containing the hydrocarbon mixture.
Incidentally, n-pentadecane and 1-phenyldodecane were found in all tissues
irrespective of whether the diets contained the hydrocarbons. In the
analyses, the limit of detection for each chlorobiphenyl and hydrocarbon was
found to be 20 ng/g dry weight of tissue.
Radiochemical tracer studies showed that 17% of an administered dose of
naphthalene is accumulated as parent hydrocarbon in coho salmon fingerlings,
in 14°C freshwater, in 24 hr (Roubal et al., 1977). If hydrocarbons
accumulate in 200-g coho salmon, which are maintained in 7°C seawater, to the
same degree that naphthalene accumulates in coho fingerlings, which are
maintained in 14°C freshwater, then the analyses should have revealed the
presence of the hydrocarbons originating from the diets. The apparent absence
of the dietary hydrocarbons suggests that those compounds were either poorly
absorbed in the stomach and intestines, or readily metabolized to form other
products in the body, or readily excreted from the body. In any case, any
amount that may have accumulated was below the detection limit.
Reaction parameters for analyses of AHH activities—It was recognized
that reaction conditions for determining specific activities of AHH from
mammals cannot necessarily be applied directly for fish. Thus, the reaction
-------
TABLE 1. CONCENTRATIONS OF CHLOROBIPHENYLS IN COHO SALMON BODY TISSUES
o
Diet Time
Week
5 ppm
Chlorobiphenyls
10 ppm of
50-50 Mixture,
Chlorobiphenyls
& hydrocarbons
1
3
5
6
8
1
2
3
5
6
8
BP 2-C1-BP 2,2'-Cl2-BP
36.1
44.0
Tr
ND
ND
28.7
ND
28
Tr
ND
NO
40.4
91.4
37.0
240
ND
32.8
79.9
32.0
41.0
139
ND
107
175
104
167
48.7
125
173
165
239
172
55.4
2,4'-Cl2-BP 2
y weight of tiss
125
182
146
267
134
89.5
143
68.6
203
207
54.5
,5,2'-Cl3-BP
120
190
209
345
226
182
320
168
525
708
124
2,5,2',5'-Cl4-BP
80.3
104
234
218
414
27.9
203
112
796
736
66.6
ND = not detected; Tr = trace. Limit of detection was 20 ng/g dry weight. Abbreviations:
BP, biphenyl; 2-C1-BP, 2-chlorobiphenyl; 2,2'-Cl2-BP, 2,2'-dichlorobiphenyl; 2,4'-Cl2-BP,
2,4'-dichlorobiphenyl; 2,5,2'-Cl3-BP, 2,5,2'-trichlorobiphenyl; 2,5,2'5'-Cl4-BP,
2,5,2'5'-tetrachlorobiphenyl. The body tissue samples consisted of the residual tissues
remaining after removal of heads, tails, and viscera.
-------
TABLE 2. CONCENTRATION OF CHLOROBIPHENYLS IN COHO SALMON KIDNEY TISSUES
Diet
5 ppm
Chlorobiphenyl
10 ppm of
50-50 Mixture,
Chlorobiphenyl
& hydrocarbons
Time
Week
1
3
s5
6
8
1
2
3
s 5
6
8
BP
Tr
25.0
ND
ND
ND
Tr
ND
ND
ND
46.8
ND
2-C1-BP
45.9
74.4
ND
ND
ND
55.5
ND
ND
ND
33.8
ND
2,2'-C!2-BP
ny/y ury
61.8
59.9
37.8
ND
ND
102
Tr
ND
71.7
86.2
59.3
2,4'-Cl2-BP
weight of ti
40.2
149
59.2
ND
Tr
62.5
ND
ND
88.1
80.6
51.2
2,5,2'-Cl3-BP
47.7
36.2
71.8
Tr
70.9
112
Tr
Tr
198
155
99.9
2,5,2'5'-Cl4BP
20.6
210
Tr
Tr
Tr
Tr
ND
ND
ND
78.4
Tr
ND = not detected; Tr = trace, Limit of detection was 20 ng/g dry wt.
Abbreviations of biphenyls same as for Table 1.
-------
TABLE 3. CONCENTRATION OF CHLOROBIPHENYLS IN COHO SALMON LIVER TISSUES
Diet Time
Week
5 ppm'
Chlorobiphenyls
10 ppm of
50-50 Mixture,
Chlorobiphenyls
& hydrocarbons
1
3
5
6
8
1
2
3
5
6
8
BP 2-C1-BP 2,2'-Cl2-BP
41.6
37.8
ND
ND
ND
44.1
ND
Tr
ND
ND
ND
133
155
ND
ND
ND
103
ND
ND
ND
39.7
ND
80.5
108
45.6
ND
33.9
156
ND
219
156
38.7
68.4
2,4'-Cl2-BP 2
ry weight of tis
81.6
159
78.1
Tr
103
101
ND
109
141
102
74.8
,5,2'-Cl3-BP 2
69.3
120
92.3
ND
146
183
ND
221
313
246
161
,5,2',5'-Cl4-BP
ND
Tr
Tr
ND
20.8
Tr
ND
Tr
ND
184
Tr
a ND = not detected; Tr = trace. Limit of detection was 20 ng/g dry wt.
Abbreviations of biphenyls same as for Table 1.
-------
parameters for measuring activities of hepatic AHH were determined for coho
and chinook salmon. Also, it was necessary to determine the conditions for
handling and storing tissues prior to their use in assays of AHH activity.
The data in Tables 4 and 5 show that activities of hepatic AHH in coho
salmon and rainbow trout (Salmo gairdneri), respectively, are greatest in
microsomal fractions. Rainbow trout microsomes served as a reliable reference
material for periodic validation of the method for measuring the AHH activity.
There was essentially no loss in AHH activity when microsomes were suspended
in 0.25 M-sucrose and stored at -60°C for up to 5 months (Table 4). This
information was important because samples of suspended microsomes were held
frozen for 2-4 months before final analyses. Also, repeated thawing and
refreezing of suspended microsomes apparently resulted in a loss of one-third
of the AHH activity (compare 3.4 and 2.1 nmoles/mg for 2-day stored salmon
parr microsomes, in Table 4); therefore, hepatic AHH assays were performed
only with once-frozen microsomes.
Information about the stability of the PAH substrate used in AHH assay
reactions was important to the reproducibility~of the assay procedure. Thus,
the effects of storage and exposure to air of H-benzo[a]pyrene in acetone,
for up to 65 days, were investigated. The results given in Table 6 show that
oxidation products are progressively formed upon repeated intermittent
exposure of the substrate to air. In between the exposures, the solutions
were held at -20°C. Protection with nitrogen did not entirely prevent
oxidation of benzo[a]pyrene in solution. It was important to prepare fresh
benzo[a]pyrene substrate after 10-14 days; otherwise, the nonenzymatic
analytical blank values became undesirably large.
It was important to establish the optimum concentrations of cofactors,
NADPH and magnesium ion, in the AHH reactions. Data for a Lineweaver-Burk
calculation from AHH reaction rates, for 0.05 to 5 mM NADPH, are given in
Table 7. The concentration of NADPH employed throughout the experiments
compares well with a calculated Michaelis constant of 1.33 mM. In the AHH
reaction employed in this study, higher specific activities were obtained
(Table 8) when fresh preformed NADPH was used, rather than when a NADPH-
generating system was employed in situ. In Table 9, NADPH and MgC^ are shown
for comparisons in reactions to indicate the requirements for higher AHH
activities. Manganese was without effect, in spite of its use in the method
adapted from mammal studies (DePierre et al., 1975).
A study of incubation temperatures of the enzymatic reactions revealed
that 25°C is optimum for the salmon hepatic microsomal AHH (Table 10). The
optimum temperature generally is lower than for other animals (Adamson, 1967).
Optimum reaction times were determined. The data in Table 11 indicate that
the AHH reaction rate is linear to 20 min.
The pH optimum for determinations of hepatic microsomal AHH activity for
the coho salmon was the same (pH 7.5 in Tris buffer) as with microsomes from
mammalian livers (Nebert and Gelboin, 1968; DePierre et al., 1975). By
comparison, the same determinations for chinook salmon showed the optimum pH
13
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TABLE 4. TIME EFFECTS OF TISSUE STORAGE AT -60°C ON MICROSOMAL
ARYL HYDROCARBON HYDROXYLASE (AHH) ACTIVITY a
Fish
Tissue preparation Time
AHH activity
Days
nmoles products/rug
microsomal protein/
20 min.
Coho salmon
parr
Coho salmon
parr
Whole minced liver
Liver homogenate
9,000 x g supernate
microsomes
Microsomes
Microsomes
Microsomes
Microsomes,
3 x thaw-refreeze
14
14
14
14
0
1
2
2
1.6"
1 fib
1.8.
2.7°
2.3
4.9
4.5
3.4
2.1
Rainbow
trout
Microsomes
Microsomes
Microsomes
Liver
Microsomes
Microsomes
Microsomes
Microsomes
0
3
6
6
13
38
79
149
5.1
5.1
4.8
4.0
4.5
4.7
5.0,5.7
4.6
Incubation mixture contained 3 pinoles MgCl2> 1.1 umoles NADPH (reduced
nicotimamide adenine dinucleotide^ phosphate), 62.5 ^imoles tris hydro-
chloride, pH 7.5, 66 nmoles 3H-benzo[a ]pyrene (12.5 mCi/mmole), and 0.8 mg
microsomal protein (from 100,000 g pellet resuspended in 4 ml of 0.25
M-sucrose/g liver), in 1 ml, at 25°C in air.
AHH activity based on proteins of tissue preparation, rather than on micro-
somal proteins.
-------
TABLE 5. INTRACELLULAR DISTRIBUTION OF AHH ACTIVITY a
Cellular fraction Fish # 969 Fish # 995 Fish #1004 Fish #1008
Whole homogenate
9,000 x g pellet
Microsomes b
Solubles c
- - - nmoles products formed/mg protein/20 min - - -
2.91 2.14 3.66 2.46
1.70 2.00 5.34 1.96
6.77 4.82 8.67 5.64
nil nil nil nil
Preparations from rainbow trout livers. Reaction conditions like those
reported in Table 4.
b 100,000 x g pelleted material.
c 100,000 x g supernatant fraction.
TABLE 6. EFFECTS OF SUBSTRATE STORAGE TIME ON AHH ACTIVITY BLANK VALUES:
STORAGE OF A 3H-BENZO[A]PYRENE SOLUTION
Storage time
Days
0
8
10
16
17
23
24
24
24
31
33
39
40
40
46
65
Blank values a
nmole oxidation
0.4
0.45
0.36
0.62
0.53
0.45
0.63
0.50
0.58
0.94
0.82
0.90
1.36
1.53
0.9lb
2.3
products
Storage container opened to air on each day measured. Stored at -20°C
in between times. Tritium activity, O.OljuCi/nmole.
A sample stored unopened under nitrogen at -20°C for 46 days.
15
-------
TABLE 7. EFFECTS OF NADPH CONCENTRATION IN BENZO[A]PYRENE
HYDROXYLASE REACTIONS OF CHINOOK SALMON HEPATIC MICROSOMES
NADPH Benzo[a]pyrene hydroxylase activity a
umoles nmoles products/mg protein/20 min
5 0,823
3 0.816
2 0.792
1 0.686
0.5 0.667
0.05 0.061
a Km for NADPH is 1.33 mMolar. Reaction conditions like those reported
in Table 4, except at pH 7.8.
TABLE 8. AHH ACTIVITY INFLUENCED BY CONDITIONS OF NADPH IN ENZYME REACTIONS
NADPH-system Benzo[a]pyrene hydroxylase activity
Coho salmon Rainbow trout
- - -nmoles products/mg protein/20 min - -
Generating system a 1.63 (88%) 3.36+1.4 (72%)
1.1 umoles, 1.84 (100%) 4.69+0.4 (100%)
fresh solution
1.1 umoles, -- 2.91 (58%)
48 hr frozen solution
a NADPH generated by method of DePierre et al. (1975) using isocitrate and
isocitrate dehydrogehase with NADP.
16
-------
TABLE 9. COFACTOR REQUIREMENTS FOR BENZOtAJPYRENE HYDROXYLASE REACTIONS
WITH CHINOOK SALMON LIVER PREPARATIONS
Reaction system Benzotalpyrene hydroxylase activity
nmoles products formed/nig protein/20 min
Complete3 ' 1.38
Minus MgCl2 0.93
Minus NADPH 0.34
Reaction conditions were the same as reported in Table 4, except at
pH 7.74.
TABLE 10. BENZOfA]PYRENE HYDROXYLASE ACTIVITY OF COHO SALMON HEPATIC
MICROSOMES AT VARIOUS TEMPERATURES OF THE ENZYME ANALYSIS REACTION a
Temperature Benzo[a]pyrene hydroxylase activity
C nmoles products formed/mg protein/20 min
10 1.29
15 2.14
20 2.29
25 2.23
40 0.63
Reaction conditions were the same as reported in Table 4.
17
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TABLE 11. AHH ACTIVITY OF COHO SALMON HEPATIC MICROSOMES AS A FUNCTION
OF TIME OF ENZYME ANALYSIS REACTION a
Time
min
2
5
8
12
16
20
25
30
57
74
AHH activity
nmoles products/mg protein
0.27
0.56
0.90
2.1
2.5
3.9
3.3
3.6
5.2
6.1
Reaction conditions were the same as reported in Table 4.
to be 7.8; however, following exposures of chinook to petroleum hydrocarbons
the optimum appeared to shift to pH 7.5 (cf., Table 12). This shift, which
occurred after one week, was examined only at the 5 ppm concentration of test
compounds. A shift of pH 7.8 to pH 7.5 can result in significantly different
AHH activities. The pH shift was not observed for the coho salmon.
Observed activities of liver microsomal AHH in relation to test
compounds—The specific activities of AHH, as benzo[a]pyrene hydroxylase,
found for various groups of fish are presented in Table 13 (coho) and Table 14
(chinook).
Control coho salmon (Table 13) exhibited gradual increases in mean
specific activities of AHH during the first four weeks of feeding. However,
only the maximum specific activity at the fourth week and the minimum specific
activities at the sixth and seventh weeks are significantly different (P<0.05)
from each other; activities at other times were statistically the same as for
the controls. These differences from maximum to minimum may be associated
with the condition of the fish at later experimental periods, as will be
discussed below in the section on condition of fish.
The coho fed 1 ppm chlorobiphenyls exhibited no significant differences
in AHH activities.compared to controls. At the 5 ppm concentration in food,
the chlorobiphenyls produced a significant (P<0.05) decrease in AHH activities
after three weeks, but thereafter there were no differences compared to
controls.
-------
TABLE 12. EXPOSURES TO CHLOROBIPHENYLS AND HYDROCARBONS: INFLUENCE ON
OPTIMUM pH OF AHH ACTIVITIES FOR CHINOOK SALMON HEPATIC MICROSOMES
Treatment a
Control, #1
zero time
Control, #2
zero time
5 ppm chlorobi-
phenyls, 3 days
5 ppm chlorobi-
phenyls, 7 days
5 ppm chlorobi-
phenyls, 14 days
5 ppm hydrocarbons,
3 days
5 ppm hydrocarbons,
7 days
5 ppm hydrocarbons,
14 days
10 ppb mixed chloro-
biphenyls and
hydrocarbons,
14 days
AHH activity
nmoles products/
mg protein/20 min
0.44
0.48
0.66
0.70
0.56
1.02
1.38
1.75
1.44
0.44 + 0.24 (n=4)
0.64 1 0.25 (n=6)
0.43
0.46
0.56
0.26
0.30 + 0.18 (n=5)
0.43 + 0.34 (n=5)
0.18 + 0.17 (n=6)
0.54 + 0.29 (n=5)
0.25
0.26
0.07
0.06
0.02
0.42 + 0.22 (n=10)
0.30 + 0.19 (n=10)
0.78 + 0.42 (n=5)
0.91 + 0.54 (n=5)
Observed pH Optimum pH
7.29 7.9
7.75
7.81
7.96
8.42
7.32 7.8
7.74
7.82
8.01
7.5 7.8
7.8
7.23 7.8
7.43
7.76
7.96
7.5 7.8
7.8
7.5 7.8
7.8
7.27 7.5
7.50
7.84
8.05
8,53
7.5 7.5
7.8
7.5 7.8
7.8
Chinook salmon fed various diets and sampled at 0, 3, 7, and 14 days, as
described in text.
pH measurements made with a research pH meter (Radiometer-Copenhagen, Type
PHM-25, London Co., Westlake, Ohio).
19
-------
t\D
O
TABLE 13. SPECIFIC ACTIVITIES OF HEPATIC MICROSOMAL ARYL HYDROCARBON HYDROXYLASE (AHH) IN
COHO SALMON FED A CONTROL DIET AND DIETS CONTAINING TEST COMPOUNDS a
Time,
week
1
2
3
4
5
6
7
8
Control
b
0.58 +
0.61 +
0.72 +
0.89 +
0.56 +_
0.37 +
0.37 +
0.44 +_
0.35
0.32
0.31
0.59
0.25
0.18
0.14
0.24
Chlorobiphenyls
1 ppm
0.52 + 0.29
0.72 + 0.29
0.76 + 0.37
0.68 + 0.41
0.70 + 0.50
0.32 + 0.27
--
0.38 + 0.21
5 ppm
0.90 +
0.74 +
0.43 +
0.59 +
0.51 +
0.36 +
0.49 +_
0.48 +
0.54
0.19
0.29d
0.24
0.36
0.34
0.30
0.13
Hydrocarbons
1 ppm
0.79 + 0.48
0.70 + 0.55
0.80 +_ 0.50
0.63 + 0.33
0.32 + 0.23
0.22 + 0.13
—
0.44 + 0.46
5 ppm
0.60 + 0.41
1.09 + 0.69
0.71 + 0.30
0.88 +_ 0.46
0.60 +_ 0.34
0.33 +0.18
0.69 +_ 0.46
0.57 +_ 0.23
Mixed Chlorobiphenyls
and hydrocarbons
2 ppm
0.73 +
1.06 +
0.81 +
0.72 +
0.74 +
0.42 +_
0.54 +_
0.27 +
0.42
0.45d
0.54
0.56
0.36
0.22
0.29
0.19
10
1.09 +
0.73 +
0.61 +_
0.70 +
0.71 +
0.45 +
0.47 +
0.39 +_
ppm
0.25C
0.26
0.41
0.56
0.43
0.25
0.33
0.26
a Ten fish per group per time period. Mean +_S.D.; AHH activity units as nmoles of benzo[a]pyrene
hydroxylated products per mg of microsomal proteins per 20 minutes.
Weeks 1-4 for feeding Chlorobiphenyls and hydrocarbons; weeks 5-8 for depurations by feeding
control diet to all groups.
c Statistically different from controls (P £0.01)
d Statistically differentTrom controls (P < 0.05)
-------
TABLE 14. SPECIFIC ACTIVITIES OF HEPATIC MICROSOMAL ARYL HYDROCARBON HYDROX-
YLASE (AHH) IN CHINOOK SALMON FED A CONTROL DIET AND DIETS CONTAINING TEST
COMPOUNDS
Time
AHH activity'
Control5 ppm5 ppm
Chlorobiphenyls Hydrocarbons
10 ppm
Chlorobiphenyls
and Hydrocarbons
Day
0 1.09 + 0.
0
1
2
3
4
7
14
21
0.96 + 0.
(1.22 + 0.
(1.22 + 0.
(1.22 + 0.
(1.22 + 0.
1.39 + 0.
1.08 + 0.
1.18 +_ 0.
45b
56
55)C
55)C
55)C
55)C
63
25
56
_
0.76 + 0.24d
0.96 +0.22
0.72 +_ 0.37d
0.44 + 0.28f
0.83 + 0.49d
0.96 + 0.45
0.60 + 0.35d
0.
0.
0.
0.
0.
0.
0.
82
77
86
50
63
65
73
_
+ 0.
+ 0.
+ 0.
+ 0.
+ 0.
+ 0.
+ 0.
4
35d
48
27e
36e
32e
42d
0.
0.
0.
0.
0.
1.
0.
67
85
62
91
52
27
65
_
+ 0.23
+ 0.38
+ 0.47
+ 0.48
+ 0.34
+ 0.66
+ 0.45
d
e
e
d
a
b
c
d
e
f
Ten fish per group AHH
/mg microsomal
activity (mean
+ S,
D.
) as
nmoles
products
formed
protein/20 min.
Activity related to fish before the standard
Average value
Significantly
Significantly
Significantly
of controls at 0 and 7
different
different
different
from controls
fron controls
from controls
days
(P <
(P <
(P <
0
0
0
exposures
.05)
.01]
,001)
to MS-222
The coho fed 1 ppm hydrocarbons alone .exhibited no increase in the AHH
activity. In addition, the 5 ppm hydrocarbon feeding resulted in no
significant increases in AHH activity in coho hepatic microsomes (Table 13).
In Table 13, the data indicate that fish fed the mixtures of Chlorobiphenyls
and hydrocarbons together, i.e., 50-50 in 2 ppm and 10 ppm concentrations,
respectively, exhibited AHH induction in one to two weeks. Those increases in
AHH activities were significant (P<0.01 and P<0.05). No other significant
differences were found at other times for the fish fed mixtures of
Chlorobiphenyls and hydrocarbons.
21
-------
Inasmuch as the results with the test compounds in coho salmon suggested
AHH induction within one to two weeks, the experiment with chinook salmon
included analyses of AHH activities more frequently in the first week of
exposures in an effort to determine more precisely the time that significant
change occurs in AHH activity. Only the effect of a 5 ppm concentration of
test compounds was studied (Table 14), because that concentration caused the
most significant change (increase) in AHH activity in coho in the shortest
time.
The control chinook salmon exhibited no significant change in hepatic AHH
activity in three weeks of feeding on the basal diet. Table 14 includes data
for chinook salmon before and after exposure to the anesthetic MS-222. The
data indicate no effect of anesthesia on the hepatic AHH activity. The
anesthetic was necessary for weighing and sorting fish, so its possible effect
on the enzymatic system was important.
Examination of the data in Table 14 reveals significant decreases in
chinook hepatic AHH activities in relation to the three examined test diets.
Significant differences compared to controls were generally consistent, but
varied in degree of confidence (P<0.001 to P<0.05). No induction of hepatic
AHH activity was observed in the chinook salmon.
Conditions of Coho Salmon--
An understanding of the general health of experimental animals was
considered to be important; therefore, the fish were monitored for visible
signs of pathological abnormalities. By the fourth week of exposures to the
dietary test compounds, it appeared that many coho salmon were developing
health problems.
Some mortalities were observed throughout the experiments, but these
could not be attributed to the composition of the diets. At the beginning of
the study, all fish appeared healthy. However, the test coho salmon exhibited
random and progressive signs of fin lesions (fin rot disease), kidney
lesions, enlarged spleens, and orange pigmentations of livers during the
course of the study. Moreover, the control salmon appeared generally
healthier than the salmon in the test groups. At the final eighth week
sampling, all fish exhibited the abnormal health signs. Hematocrits of coho
salmon from control and test groups, measured during the fourth and fifth
weeks, ranged from 4% to 50%, with mean +S.D. values of 34+12 and 32+14,
respectively.
On the whole, average values of hematocrits appear to be poor indicators
of the presence of pathology; however, individual hematocrits of 20% and less
may be indicative of a forthcoming disease condition in the fish. More
hematocrit information relating to healthy salmon is needed.
The body weight of fish can be useful in judging effects of diet and test
compounds on fish. Table 15 presents the weight data for coho salmon used for
AHH analyses: No correlation is evident between diets and weight. No overall
growth occurred among controls or test coho salmon, which was consistent with
the feeding plan.
22
-------
A recalculation of hepatic AHH activities for coho salmon based on
visible signs of good health versus pathological symptoms did not change the
results and interpretations discussed above for the effects of test compounds
on the enzyme system.
Conditions of Chinook Salmon--
The data is Table 16 presents the body weights of chinook salmon taken
for AHH analyses. Comparisons of mean weights over a 49-day period indicate
slow growth rates for all four groups, which is according to the feeding
schedule and does not reflect obvious differential effects of xenocompounds.
The incidence of physical signs of lesions found in the chinook salmon
used for analysis of AHH activities are summarized in Table 17. All fish were
in excellent condition at the start and no signs of pathology were evident
until after one week of feeding the test compounds. It was not until four
weeks of exposures to the test compounds that kidney lesions were observed.
In general, the control chinook salmon were healthier than the test fish.
Measurements of hematocrits were made for the chinook salmon. These
measurements were for all chinook salmon taken for the analyses of AHH
activity, because of our concern for the condition of the fish after the
experience with the coho salmon. The values for the hematocrits are presented
in Table 18. Hematocrits ranged randomly from 25+11% to 40+10% with no
apparent relationship to pathology.
Many factors affect hematocrits or blood cell counts in fish, such as
water temperature, state of maturity, spawning cycle, metabolic activity, and
sampling techniques (Katz, 1949; Barnhart, 1969). If we assume that values of
32% to 52% found by Wedemeyer and Chatterton (1971) for juvenile hatchery coho
salmon (10-15 g size) are reasonable values for mature salmon in seawater,
then perhaps salmon with hematocrits of about 30% may be regarded as bordering
on developing a health problem.
Interpretation of Results--
The highest activities of hepatic AHH were observed when the hydrocarbons
together with the chlorobiphenyls were fed to the coho salmon. Results
suggest that the concentration of these xenobiotics influenced the induction
of AHH in coho salmon; i.e., the induction occurred one week later when the
fish were fed 2 ppm of the 50-50 mixture of chlorobiphenyls and hydrocarbons
than when the fish were fed 10 ppm of the mixtures of compounds.
Chlorobiphenyls alone did not cause increased AHH activity in coho
salmon; however, the results suggest that these compounds did influence the
hepatic AHH enzyme system when administered together with hydrocarbons. The
data showed that salmon treated with 10 ppm of the 50-50 mixture exhibited
higher AHH activity after one week of feeding than did the controls. Fish
that were fed only 5 ppm of hydrocarbons or 5 ppm of chlorobiphenyls, for one
week, exhibited no change in AHH activity. In addition, induction of AHH
activity occurred when the fish were fed 2 ppm of the 50-50 mixture for two
weeks, but not when either chlorobiphenyls or hydrocarbons were administered
23
-------
TABLE 15. BODY WEIGHTS OF COHO SALMON TAKEN FOR AHH ANALYSES
INJ
Time
Week
1
2
3
4
5
6
7
8
Control
222 + 41
222 + 56
194 + 38
196 + 31
199 + 36
199 +_ 38
172 + 34
209 + 21
Chlorobiphenyjs
1 ppm
196 + 64
216 + 44
230 + 34
220 + 47
253 + 19
198 + 51
--
178 + 37
5 ppm
218 + 37
210 i 49
184 + 32
210 + 45
189 + 33
210 +_ 37
227 +_ 33
213 +_ 56
Hydrocarbons
1 ppm
198 +_ 56
176 + 38
200 i 23
209 + 51
213 +41
191 +_ 39
--
185 + 84
5 ppm
225 + 24
211 + 32
182 + 42
213 + 43
210 +_ 41
172 + 30
224 + 26
236 + 48
Mixed chlorobiphenyls
and hydrocarbons .
2 ppm
215 + 40
238 +_ 50
208 +_ 31
224 + 38
209 + 41
170 + 34
194 + 54
212 + 39
10 ppm
242 + 38
204 1 36
181 + 39
180 +_ 36
207 + 46
208 + 44
210 + 45
203 + 53
Ten fish per group for mean weights +_ standard deviations.
-------
Table 16. BODY WEIGHTS OF CHINOOK SALMON TAKEN FOR AHH ANALYSES a
-J-
Time
Day
0
1
2
3
4
7
14
21
28
31
36
42
49
Control
75
72
86
75
95
86
88
96
99
+
-
+
+
+
+
+
+
+
±
15
17
18
11
19
15
13
17
19b
5 ppm
Chlorobiphenyls
82
66
81
81
90
71
104
102
109
92
100
98
+
+
+
+
+
+
+
+
+
+
+
+
17
10
18
18
18
8
39
21
17
11
39
27
5 ppm 10 ppm mixed
Hydrocarbons Chlorobiphenyls
and hydrocarbons
79 +_
73 +_
74 +
76 +
83 1
79 +_
79 1
91 1
90 1
97 +_
-
116 +
18
15
10
16
14
15
15
13
18
17
24C
76
70
82
82
89
89
78
108
97
103
105
100
± 16
+ 13
+ 14
+ 14
+ 15
+ 19
+ 8
+ 25
+ 14
+ 16
+ 15
+ 23C
a Ten fish per group for mean weights ± standard deviations, except as
noted specifically.
n = 59 for mean +_ S.D.
c n = 19 for mean + S.D.
25
-------
TABLE 17. PERCENTAGE INCIDENCE OF FIN ROT DISEASE OBSERVED FOR CHINOOK
SALMON TAKEN FOR AHH ANALYSES a
5 ppm 5 ppm 10 ppm mixed
Time Control chlorobiphenyl hydrocarbons chlorobiphenyls and
hydrocarbons
Day
0
1
2
3
4
7
14
21
28
31
36
42
49
0
0
0
0
0
0
10
40
20
10 (20)
20 (10)
20 (10)
10
0
0
0
0
0
50
50
90
70 (20)
100 (20)
70 (20)
20
20 (10)
0
0
0
0
0
30
30
40
70 (30)
50 (20)
20 (20)
-
20 (30)
0
0
0
0
0
40
80
80
70
80 (10)
50 (30)
10 (10)
40 (10)
Ten fish per group. Numbers in parentheses are percentage incidence
of observed kidney disease. No kidney disease observed before 28 days.
alone at 1 ppm in the diets for the same time. These findings imply that
chlorobiphenyls and aromatic hydrocarbons act together synergistically in
inducing hepatic AHH activity in coho salmon.
Results with Chinook salmon showed that their hepatic AHH system
responded negatively to the test compounds. Perhaps a possible cause
for a lowered AHH activity for the Chinook salmon enzyme system is
linked to the fact that the fish were in seawater in the summer, when
temperatures of the water rose to about 13°C. This factor together with
other seasonal parameters may have altered xenobiotic metabolism and the
AHH activities. Additional research is required to answer questions about
26
-------
TABLE 18. HEMATOCRITS FOR CHINOOK SALMON TAKEN FOR AHH ANALYSES
Time
Control
5 ppm
Chlorobiphenyls
5 ppm
Hydrocarbons
10 ppm mixed
Chlorobiphenyls and
hydrocarbons
Day
1
2
3
4
7
14
21
28
31
36
42
49
_ _ _
-
-
-
-
33 +
37 +
35 +
41 +_
40 +
39 +
37 +
37 +
. _ _ _ .
6
3
7
3
9
11
9
5
- -Percent
25 +
33 +
30 +_
29 +
31 +
34 +
39 +
31 +
36 +
40 +
37 +
33 +
packed-cell vo'
5
7
4
5
5
7
7
16
13
5
8
9
31
35
35
24
33
38
32
39
40
32
± 6
+ 10
± 4
+ 5
± 5
-
± 8
+ 14
± 4
+ 10
-
± 12
26
29
33
30
34
40
37
41
38
38
25
29
+
+
+
+
+
+
+
+
+
+
+
+
8
4
10
6
4
2
10
4
11
10
11
10
Ten fish per group for mean values +_ standard deviations.
the effects of Chlorobiphenyls and petroleum hydrocarbons together in Chinook
salmon in the winter or spring, when seawater temperatures are near to 7°C or
less.
At this point it is premature to suggest what effect a decreased hepatic
AHH activity has in chinook salmon exposed to the test xenobiotics; however,
it is reasonable to conclude that lower activity of the AHH system may result
in decreased metabolism of xenobiotics such as petroleum PAH's. The
possibility of decreased metabolism of PAH's may affect the toxicity of these
compounds in the species.
A brief statement of findings with other species will illustrate
additional complexities of the AHH system. For instance, many xenobiotics may
not induce AHH activity in animals or, if so, the extent to which aromatic
hydroxylation occurs with different compounds varies considerably among
species, as work by Williams (1971) has shown. Benzo[a]pyrene hydroxylase is
not especially responsive to many inducers of drug-metabolizing enzymes,
27
-------
including PCB's and certain chlorine-containing pesticides (Fouts, 1973).
Furthermore, some compounds exert an opposite effect of inducers. For
example, Ahokas et al. (1976) found that pike exposed to domestic and
industrial wastes exhibited lower AHH activity than controls.
Our results are consistent with others which show that activities of
xenobiotic metabolizing enzyme systems are often lower in fish compared to
those in higher animals (Adamson, 1967; Buhler and Rasmusson, 1968).
Moreover, while the microsomal xenobiotic metabolizing enzyme systems of fish
appear to be similar in many respects to that of mammals (Stanton and Khan,
1975), there are species differences such as the lack of mixed function
oxidase induction by phenobarbital in marine fishes (Philpot et al., 1976).
Thus, there is no reason to assume that the effects of xenobiotics on hepatic
AHH activity for fish will parallel changes brought about by these compounds
in mammals. The nature and extent of induction of AHH activity within a given
species of fish can also vary among different strains and is related to the
geographic origin of the fish (Pedersen et al., 1976). Furthermore, induction
of AHH in fish appears to be inversely affected by both water temperature
(Dewaide, 1970) and salinity (Gruger et al., 1977). These, and perhaps other
factors, make it difficult to make absolute comparisons of results of
experiments on the AHH systems in fish between laboratories.
Summary
Coho salmon and Chinook salmon were exposed to two mixtures of pure
chlorobiphenyls and petroleum hydrocarbons, separately and together, at 1 ppm
and 5 ppm of each mixture in Oregon moist pellets. Chemical analyses of coho
salmon tissues indicated that the chlorobiphenyls accumulated in the liver,
kidney, and residual tissues composed of eviscerated, headless and tailless
carcasses; although, hydrocarbons were not detected in these organs and
tissues.
Aryl hydrocarbon (benzo[a]pyrene) hydroxylase activities of liver
microsomes were compared among test and control salmon of both species in
order to determine whether alterations in enzyme activities occurred in
relation to the exposures. Optimal reaction conditions were established for
measurements of aryl hydrocarbon hydroxylase activities in salmon hepatic
microsomes; the conditions indicated temperature differences for reactions of
salmon microsomes compared to mammalian microsomes and differences in pH for
the reactions of microsomes from coho salmon compared to those from chinook
salmon.
Induction of the aryl hydroxylase enzyme occurred in coho salmon,
maintained in 7°C seawater, within two weeks of exposure to the hydrocarbons;
the induction was also potentiated by the presence of the chlorobiphenyls,
which by themselves had no effect on the enzyme activity. A synergistic
effect was indicated for the test compounds on the induction of aryl
hydrocarbon hydroxylase in coho salmon liver. In chinook salmon maintained in
13°C seawater, the activity of aryl hydrocarbon hydroxylase in hepatic
microsomes was depressed by the chlorobiphenyls and hydrocarbons administered
singly or together. No interaction of these two classes of xenobiotics on the
hydroxylase activity was found with chinook salmon.
28
-------
THE EFFECTS OF PETROLEUM HYDROCARBONS AND CHLORINATED BIPHENYLS
ON THE MORPHOLOGY OF TISSUES OF CHINOOK SALMON
Methods
Samples of skin, gills, intestine, liver, and kidney were taken from
three chinook salmon randomly selected from each test group, described in the
previous (above) experiments, and the control group at 14, 28, and 49 days
(Hawkes et al., 1980). The tissues were fixed in 0.75% glutaraldehyde, 3%
formalin, 0.5% acrolein in 0.1 M sodium cacodylate buffer (pH 7.4) with 0.02%
CaClg'HoO, and 5.5% sucrose. After a buffer wash (0.1 M sodium cacodylate,
0.02% CaClp'HoO, 5.5% sucrose), specimens were postfixed in 1% osmium
tetroxide in buffer. Dehydration with ethanol and embedding in Spurr medium
(Spurr, 1969) completed the preparation of the tissues for sectioning with
either glass or diamond knives. Sections were prepared with Richardson's
stain for light microscopy, and thin sections were triple stained with lead
citrate, uranyl acetate, and lead citrate for electron microscopy.
Several staining techniques were used to identify the subcellular
inclusions in the intestinal mucosa. For glycoproteins, a modification of the
periodic acid-Schiff (PAS) technique for use with plastic-embedded tissue was
employed (Nevalainen et al., 1972), and for lipid, Sudan black and Aparicio's
stain (Aparicio and Marsden, 1969) were used.
Results
Morphological changes that deviated from the normal appearance of the
controls were observed only in liver and intestine. In some of the salmon
exposed to chlorinated biphenyls, the rough endoplasmic reticulum was
vesiculated and there were degenerating membranes in hepatocytes throughout
these liver sections (Hawkes, 1980).
Examination of the intestinal mucosa from 32 of the 36 fish was
completed. In samples from four fish the sections passed below the mucosa and
were not included in the study. There were minor variations among
individuals, but there were uniform patterns of normal morphology among fish
of the control group and consistent alterations among the exposed animals.
These findings were similar after both the 14- and the 28-day exposures with
no apparent increased damage in the 28-day-exposed group. In addition,
structural changes in exposed animals persisted at the same level of severity
after 21 days of depuration.
Control Group—
The general organization and histology of the intestine of chinook salmon
have been described by Greene (1913) and will not be repeated here. Because
structural changes occurred in the mucosa of contaminant-fed fish, the normal
ultrastructure of that area of the intestine is briefly described (Figs. 1-4).
The mucosa contains mucous cells, cylindrical epithelial cells, rodlet cells,
and leukocytic cells similar to those reported in rainbow trout (Yamamoto,
1966). Mucous cells in several stages of maturation are present: the most
immature are located near the basal regions of the mucosa, and the more mature
cells are near the surface and are filled with typical mucin inclusions
29
-------
4B» JRWW- ' •
FIGURES 1-4. Micrographs of intestine from control chinook salmon.
Fig. 1. Light micrograph of intestinal villi. 30 x. Fig. 2. Tip
of villiis with normal mucous cells (m) and brush border (arrows).
1100 x. Fig. 3. Electron micrograph of columnar epithelial cells
and mucous cell (m). 3500 x. Fig, 4. Microvillar surface and
upper fourth of a typical columnar cell, Note the aggregation of
mitochondria and the few profiles of endoplasmic reticulum. 14,300 x.
30
-------
sequestered in discrete membrane-bound vesicles. The columnar epithelial
cells are stratified and have aggregations of mitochondria near the luminal
surface and the nucleus is in the basal region. Granular endoplasmic
reticulum, mitochondria, and agranular cytoplasm occupy the midportion of
these cells. Multivesiculate bodies and other vesicles are present in the
mid- or luminal regions of some of the mucosal cells.
Petroleum-Exposed Group--
Chinook salmon exposed to the mixture of petroleum hydrocarbons
maintained overall integrity of the intestinal mucosa and underlying regions.
There was little exfoliation of cells and little focal necrosis (Figs. 5, 6);
however, varying degrees of subcellular changes occurred in eight of nine fish
examined. Three aberrations were noted: (1) presence of unusual cellular
inclusions in the columnar cells of the mucosa, (2) vesiculation of cytoplasm
near the luminal surface of columnar cells, and (3) cytoplasmic changes in
basal cells of the mucosa. A flocculent or finely granular material, enclosed
in membrane-bound vesicles of varying size and electron density, filled the
luminal half of columnar cells of the intestine of some fish (Figs. 6, 7). In
other individuals, normal-appearing and vesicle-packed cells were
interspersed. In all the petroleum-exposed fish, typical goblet cells were
present throughout the affected regions. Immediately below the luminal
surface of the mucosa, there was a 13-ym zone that contained small vesicles
with the flocculent inclusions. The zone also contained profiles of agranular
endoplasmic reticulum not observed elsewhere in the cell (Fig. 7).
In tissue from animals with the greatest amount of vesiculation, the
basal mucosal cells had an unusually electron-transparent cytoplasm and an
increase in granular endoplasmic reticulum.
Chlorinated Biphenyl-Exposed Group--
Although the majority of the intestinal cells were intact, there was a
noticeable increase in the amount of exfoliation (Figs. 8,9) of the mucosa of
the chlorinated biphenyl-treated chinook compared with the control or the
petroleum-treated groups. The brush border was intact over most of the
mucosa, but was reduced or absent in some areas, even though the cells showed
no signs of impending lysis and sloughing. Clusters of columnar cells with
abnormal cytoplasmic inclusions were interspersed among normal-appearing
cells.
Inclusions in the mucosal cells could be grouped into at least two and
possibly three different structural types (Fig. 10). The first consisted of
irregularly shaped and variably sized vesicles of relatively electron-
transparent material of a finely fibrillar consistency. It was difficult to
determine if a limiting membrane was present on the large masses of this
material, even though small vesicles were clearly membrane bound. Membranes
of nearby mitochondria and granular endoplasmic reticulum were well defined
and appeared normal. The second type of inclusion was much more electron
dense; the small vesicles were obviously membrane bound. Large aggregates of
this material frequently included small foci of electron-transparent material.
A possible third type of inclusion was intermediate in density and resembled
the second type of vesicle in having small areas of quite clear material.
31
-------
FIGURES 5-7. Micrographs of intestine from chinook salmon fed a
model mixture of 5 ppm petroleum hydrocarbons for 28 days.
Fig. 5. Light micrograph of intestinal villi with numerous inclu-
sions (arrow), 30 x. Fig. 6. Higher magnification of columnar
cells of the mucosa. The mucous cells (m) appear to be normal,
The inclusions (arrow) are restricted to the upper third of the
columnar cells. 1100 x. Fig. 7, Electron micrograph of upper
third of the portion of the intestinal mucosa. Mucous cell (m),
inclusion (i)« 5300 x, The insert, a high magnification of the
region below the microvillar terminal web, has an abundant net-
work of agranular endoplasmic reticulum. 17,000 x.
32
-------
.^TfC^ - ,
FIGURES 8-10. Micrographs of intestine from chinook salmon fed
5 ppm chlorinated biphenyl mixture for 28 days.
Fig. 8. Light micrograph of the intestinal mucosa with exfolia-
tion (arrow). 30 x. Fig. 9. Two types of inclusions (a,b) are
evident in this light micrograph. 1100 x. Fig. 10. Electron
micrograph of the upper portion of mucosal cells with damaged brush
border and exfoliation (arrow). The inclusions correspond to the
(a) and (b) types in Fig. 9. The finely granular material of the
type (a) granule may coalesce to form type (b) with (b1) being an
intermediate. 3500 x.
33
-------
Near the luminal surface of these intestinal mucosal cells, there were
clusters of small vesicles containing all of the above types of inclusions as
well as numerous profiles of agranular endoplasmic reticulum. Nuclei
maintained the typical basal location but the mitochondria were distributed
throughout the cell. Normal-appearing mucous and rodlet cells were commonly
observed.
Petroleum- and Chlorinated Biphenyl-Exposed Group--
The amount of exfoliation of the surface mucosal cells of intestine
appeared to increase in seven of eight combined-contaminant-treated fish
(Figs. 11,12) compared to the other groups. The brush border was reduced in
areas where exfoliation had not occurred, and this reduction may have been a
harbinger of impending cell loss.
Cellular changes ranged from slight to pronounced in areas where the
mucosa was intact. The columnar shape of affected cells was maintained as was
the basic polarity of the cell (i.e., the nucleus remained in the basal
region). Mitochondria, however, were interspersed throughout the cytoplasm
rather than being clustered near the luminal surface as in the controls. The
cytoplasm exhibited several major changes, including alterations in the
vesiculation near the brush border, presence of inclusions throughout the
cell, an increase in endoplasmic reticulum, and reduction of cytoplasmic
density (Fig. 13). A region of agranular endoplasmic reticulum near the
luminal surface extended deeply into the cell (Fig. 14). The surface
microvilli had deep intertwining extensions in the upper quarter of this
region.
Membrane-bound vesicles of varying size and electron density occurred
throughout the cells, but were located primarily in the luminal half of the
columnar cells. The inclusions were membrane-bound vesicles of widely ranging
sizes and electron densities and had a finely fibril lar structure. The
cytoplasmic ground substance frequently appeared much less electron dense,
lacked free ribosomes, and had increased profiles of granular endoplasmic
reticulum. Columnar cells with these modifications were located near or
adjacent to normal-appearing mucuous, rodlet, and columnar cells.
Histochemistry--
Inclusions in the intestinal mucous cells of both normal and contaminant-
exposed intestine showed a positive PAS reaction; however, cells with
inclusions from all of the contaminant-exposed animals were clearly PAS
negative. The inclusions did not stain with Sudan black. Aparicio's staining
technique was used to delineate the red mucin from blue cytoplasm of columnar
cells and from greenish lipid droplets. The inclusions stained a bluish hue
with this technique.
Discussion
Structural changes in the intestine of salmon exposed to the chlorinated
biphenyls and petroleum hydrocarbons used in this study involved marked
subcellular alterations in intact cells compared with cells of controls
(Hawkes et al., 1980). Exfoliation of the intestinal epithelium occurred when
the fish were exposed to chlorinated biphenyls or to the mixture of petroleum
34
-------
'&•/**? «.%"Wt;^f' i
;;X ? -„;*, ^^ ,f , ,
1
FIGURES 11-14. F^Iicrographs of intestine from chinook salmon fed
5ppm each of a petroleum hydrocarbon mixture and a chlorinated
biphenyl mixture. FIG. 11. Light micrograph of intestinal villus
with several sites of exfoliation (arrows). 30 x. FIG. 12. Mucous
cells (m) and columnar cells in a region of sloughing, 1100 x.
FIG. 13. Electron micrograph of columnar cell with inclusions from
an area of the mucosa in which there was no evidence of exfoliation.
4800 x. FIG,. 14. Higher magnification of the base of the micro-
villi, terminal web, and subadjacent region. Note the abundance of
agranular endoplasmic reticulum. 13,000 x.
35
-------
and chlorinated biphenyls; the combined effect of the two contaminants on
epithelial exfoliation appeared much greater than with chlorobiphenyls alone,
suggesting an exacerbated interaction. A very clear case of interactive
effects was reported by Itokawa et al. (1975) on the effects of
polychlorinated biphenyls and alkylbenzene sulfonic acid on rats. Testicular
damage occurred only when both contaminants were administered. The rats also
showed liver pathology with exposure to the individual contaminants and an
increased pathology with combined exposure; this increase appeared to be an
additive effect. Therefore, in the same animal and within the same time
frame, one tissue showed a clearly enhanced interactive effect from two
contaminants whereas another tissue showed an additive change.
The presence of inclusions in the Chinook salmon intestine represents
distinct changes from the normal columnar cell morphology. We do not know
whether the substances were incorporated into the cell from material in the
intestinal lumen, were produced by the cell, or can be solely attributed to
degenerative changes. The appearance of the inclusions suggests that they
were not composed of absorbed food material. Moreover, some of the cells were
observed in the process of releasing the material into the lumen of the gut.
Histochemical tests clearly demonstrated that the inclusions were not stained
by either the lipid-specific or the glycoprotein-specific reagents used, but
the mucuous (goblet) cells of all reference and exposed groups were PAS
positive. Although PAS is highly reactive with glycoproteins, not all of
these compounds give a positive reaction (Jones and Reid, 1978). In addition,
different cell types synthesize various types of mucous glycoproteins which,
in turn, respond differently to a variety of stimuli, from both hormones and
xenobiotics (Parke, 1978). If rapid synthesis of abnormal mucus occurs, the
typical staining reactions could be absent, because it has been shown that
only when all the constituent residues are finally assembled in the
glycoprotein moiety do they produce the colored PAS-reaction product (Reid and
Clamp, 1978).
Changes in the chemical and physical properties of mucins in pathological
states have been reported by Schrager and Dates (1978). They found that human
gastrointestinal mucus from ulcer and cancer patients had altered rheological
properties that may reduce the effectiveness of the mucous barrier against
bacterial and enzymatic attack.
The persistence of the inclusions beyond the time of ingestion of the
contaminants is consistent with the "limited return toward preexposure
morphology" in the liver of rats exposed to polychlorinated biphenyls (Kasza
et al., 1978) and may indicate degenerative changes beyond any possibility of
recovery. Retention of chlorinated biphenyls beyond the time of exposure has
also been reported in rainbow trout (Lieb et al., 1974) and in coho salmon
(Gruger et al., 1975); information on the structure of the intestine from
these fish is not available for comparison with the chinook salmon.
Based on the -observation of similar weight gains of all groups during
this study (Gruger et al., 1977b), functional impairment was not detected in
the chinook salmon exposed to petroleum or chlorinated biphenyls. In other
work, Gruger et al. (1976) showed that juvenile coho salmon gained weight when
fed 1-12 ppm of a mixture of three chlorinated biphenyls in OMP. Mayer et al.
36
-------
(1977) also found continued growth in coho salmon fed Aroclor 1254, a far more
complex mixture of chlorinated biphenyl isomers than we used; however, all the
fish fed a dose of 14,500 yg/kg body wt per day died after 265 days' exposure.
The cause of death was not ascertained. Therefore, the observation in our
studies of weight gain by the group of exposed fish showing obvious intestinal
damage does not rule out the possibility that a degree of functional
impairment existed which may have reduced long-term survival.
Summary
Sections of skin, gill, intestine, liver, and kidney were examined from
juvenile Chinook salmon exposed in parallel experiments to a model mixture of
petroleum hydrocarbons, chlorinated biphenyls, and the combined contaminants.
Morphological changes were observed only in liver and intestine, with changes
in liver limited to salmon treated with chlorinated biphenyls. The most
consistent and notable changes were in the mucosal cells of the intestine.
The mucosa was intact in the control and petroleum hydrocarbon-exposed fish;
some exfoliation was observed in the group exposed to chlorinated biphenyls.
The group fed the combined contaminants (hydrocarbons and chlorinated
biphenyls) had considerably increased sloughing indicating an interactive
effect. The "goblet" or mucous cells appeared normal in all groups, but in
the contaminant-exposed groups the columnar cells of the mucosa had distinct
subcellular inclusions. The inclusions were not stained by carbohydrate-
specific (PAS) or lipid-specific (Sudan black) reagents. Ultrastructurally,
the inclusions that appeared after exposure to the hydrocarbons were variable
in size and contained a flocculent, finely granular material. In the
hydrocarbon-exposed group the inclusions differed from both the chlorinated
biphenyl and the combined-contaminant groups. The inclusions in the latter
groups included large, irregularly shaped vesicles with relatively electron-
transparent material and other vesicles with a range of electron densities.
The cellular alterations were consistent within the exposed group and
presented a distinct change from normal morphology. Since the average gain in
weight for all groups was similar, we could not conclude, however, that
functional impairment from intestinal damage had occurred.
INTERACTIONS OF POLYCYCLIC AROMATIC HYDROCARBONS AND HEAVY
METALS ON ACTIVITIES OF ARYL HYDROCARBON HYDROXYLASE
Methods
Coho salmon and starry flounder were initially held for 10-14 days in
10°C seawater to which was added 200 ppb of cadmium or lead, in order to allow
time for heavy metals to interact with the hepatic AHH systems in the exposed
fish. During the subsequent final two weeks, while the metal exposures
continued, the fish were fed OMP which contained a model mixture of PAH's.
The PAH mixture in the OMP consisted of 107 ppm (w/w) phenanthrene, 128 ppm
2-methylnaphthalene, and 99 ppm 2,6-dimethylnaphthalene. After the final two
weeks, the fish were sacrificed and livers taken for analyses of AHH
activities in microsomal preparations. The controls were fish that received
an OMP diet without the addition of the PAH mixture. The experiment was
carried out twice with the salmon and once with the flounder. The AHH
37
-------
activities were determined on six groups of seven to ten fish per group for
each species. Activities of the AHH, as naphthalene hydroxylase, were
determined by the method of Nilsson et al. (1976).
An assessment of the effects of the heavy metals on the microsomal AHH
activities in an in vitro system was made. Cadmium and lead concentrations
were separately varied from 0.05 to CO. 0 ppm in enzymic reactions. The sources
of microsomes were pooled samples of the liver fractions from the coho salmon
which were fed chlorobiphenyls and hydrocarbons in the chronic exposure
studies (discussed above) and from hatchery-reared rainbow trout.
Results and Discussion
The study of possible effects of the three dietary PAH's in coho salmon
and starry flounder exposed to cadmium and lead in seawater showed little or
no significant alterations in hepatic aryl hydrocarbon (naphthalene)
hydroxylase activity for either species. The results given in Table 19
indicate that metabolic hydroxylations of aromatic hydrocarbons may not be
TABLE 19. NAPHTHALENE HYDROXYLASE ACTIVITY IN VITRO OF LIVER MICROSOMES OF
PAH-FED FISH EXPOSED TO CADMIUM AND LEAD IN SEAWATER a
Naphthalene hydroxylase activity ^
Exposure group Coho salmon Starry flounder
Expt. 1 Expt. 2 Expt. 3r
Control 0.50+0.22 (7) 0.20+0.13 (8) 0.30+0.25 (10)
Cadmium 0.82+_0.24 (4) 0.26+_0.13 (8) 0.45+0.41 (6)
Lead 0.52+0.14 (10) 0.28+0.13 (9) 0.22+0.14 (9)
Fish fed Oregon moist pellets (OMP) containing 128 ppm (w/w) of
2-methylnaphthalene, 99 ppm 2,6-dimethylnaphthalene, and 107 ppm phen-
anthrene, while 200 ppb of the metals were added to the seawater. The
fish were exposed to the metals, as cadmium chloride and lead nitrate,
for 14 days following a period of 10-14 days of preconditioning in the
test aquaria. Control group received OMP without the three aromatic
hydrocarbons.
Napthalene hydroxylase activity, as nmoles of products/mg microsomal
protein, in 20 min incubations at 25°C, with other conditions according
to Nilsson et al. (1976). Number of samples in parentheses.
38
-------
affected by cadmium or lead exposures under the test conditions. For
instance, duplicate experiments with coho salmon, i.e., Expt. 1 and 2 in
Table 19, indicated a slight (0.05
-------
TABLE 20. INHIBITION OF HEPATIC MICROSOMAL ARYL HYDROCARBON HYDROXYLASE
(AHH) ACTIVITY BY CADMIUM CHLORIDE AND LEAD NITRATE IN VITRO a
Concentration Inhibition of AHH activity b
of metal Cadmium exposure Lead exposure
Coho salmon Rainbow trout Rainbow trout
DDin fw/w)
0.05
0.10
0.50
1.00
2.00
3.00
4.00
5.00
10.0
50.0
0
__
7
39
—
--
--
98
97
100
0
13
23
39
60
75
84
82
--
72
0
0
22
--
__
--
42
84
57
AHH as naphthalene hydroxylase. Microsomal preparations used were from
coho salmon which were fed 10 ppm of 1:1 mixture of chlorobiphenyls and
petroleum hydrocarbons (Gruger et al. 1977b), and rainbow trout.
Inhibition determined as percent of AHH activity without added metal.
by elution from a column of silica gel (Yang et al., 1976). After this
purification step, all three substrates were found to_be greater than 99%
pure. The purity of the [ H]benzo[a]pyrene and the [ H]naphthalene was
verified by HPLC; the purity of the 2,6-dimethyl [^naphthalene was checked by
TLC. A Packard model 3255 liquid scintillation spectrometer was used to
measure the radioactivity of the labeled substrates and metabolic products.
Stock solutions of [ H]naphthalene were?prepared in ethanol; stock solutions
of [^HlbenzoCalpyrene and 2,6-dimethyl [^naphthalene were prepared in
acetone.
Preparation of Microsomes--
Coho salmon, weighing 200-500 g, were used for these studies. The fish
were killed by a blow to the head. The livers were removed immediately and
transported to the laboratory on crushed ice. All subsequent steps were
40
-------
conducted in the cold. The combined livers were weighed, minced with
scissors, and then homogenized with a Potter-Elvehjem homogenizer, using 4 ml
of 0.25 M sucrose solution per gram of liver. The homogenate was centrifuged
at 2600 x g for 10 min. The resulting supernatant was centrifuged at 15,000 x
g for 20 min. Microsomes were sedimented from the second supernatant at
105,000 x g for 1 hr. The surface of each microsomal pellet was rinsed gently
with 1 ml of 0.25 M sucrose, and the rinse was discarded. The microsomes were
suspended with a Bounce homogenizer in a volume of 0.25 M sucrose equal to the
original wet weight of the livers. The microsomal suspension was stored at
-60°C. When maintained at this temperature, there was no loss in
monooxygenase activity over 8 months. Microsomes were thawed only once,
immediately before use. Protein concentration was measured by the method of
Lowry et al. (1951).
Procedures
Enzyme Assays--
A single set of incubation conditions was employed for the assay of the
three substrates. The conditions used were those found to be optimum for
determination of benzo[a]pyrene monooxygenase activity of coho salmon
microsomes (Gruger et al., 1977b). Our incubation conditions were similar to
those used by other investigators for fish liver monooxygenases (Pedersen et
al., 1976; Chambers and Yarbrough, 1976; Pohl et al., 1974). Our assays were
conducted at 25°C for 20 min; in other respects the conditions were similar to
those developed for mammalian enzymes (Hansen and Fouts, 1972; Nilsson et al.,
1976; DePierre et al., 1975).
The total reaction volume was 1.12 ml, and the final concentrations were:
tris buffer, pH 7.5, 55 mM; MgClo, 2.6 mM; and NADPH, 1.2 mM. Microsomal
protein concentration was 0.7 mg/ml. Substrate concentrations used are given
with the results. The labeled substrate solutions had the following specific
activities: benzo[a]pyrene, 14.6 Ci/mol; 2,6-dfmethylnaphthalene, 3.9 Ci/mol;
and naphthalene, 2.0 Ci/mol. The volume of substrate solution added was 20 yl
for all assays.
Blanks, which contained all reagents except NADPH, were included for each
substrate level. For benzo[a]pyrene assays, the tests and blanks were run in
triplicate. For assays of naphthalene and 2,6-dimethylnaphthalene
monooxygenases, duplicate tests and a single blank gave reliable results.
Enzyme activity represents the difference between product formation in the
tests and that observed in the blank(s). All components, except the labeled
substrate, were added to the reaction tubes and incubated for 10 min at 25°C.
Then the substrate was added and the reaction was run for 20 min at 25°C with
shaking at a rate of 130 oscillations per min. After the reaction was
stopped, unreacted substrate was extracted from the reaction mixture. All
three assays are based on determination of radioactivity remaining in the
aqueous phase after the unreacted substrate had been extracted. Unmetabolized
naphthalene or 2,6-dimethylnaphthalene was adsorbed onto polyethylene as
described by Nilsson et al. (1976). Our results indicated that using a series
of three, rather than two, polyethylene vials gave better removal of unreacted
substrate. The effectiveness of the polyethylene adsorption procedure for
removal of [JH]naphthalene has been established (Nilsson et al., 1976). We
41
-------
have verified that the procedure is equally effective for removal of 2,6-
dimethyl[:?H]naphthalene. We found that 95% of unreacted 2,6-
dimethyl[ H]naphthalene was removed by the first adsorption step; 99.8% was
removed after the second step; and 99.98% was removed after the third step.
Recovery of total label ed metabolites of 2,6-dimethylnaphthalene was better
than 85%. For the benzo[a]pyrene assays, the unreacted substrate was
extracted into hexanes after stopping the reaction with ethanolic base. The
validity of this extraction procedure has been established (DePierre et al.,
1975). We found that two extractions with hexanes gave lower and more
reproducible blank values. In all of the assays 0.3 ml of the aqueous phase
was used for liquid scintillation counting.
Calculation of Vma and Apparent Km Values--
The results of the kinetics experiments were plotted and constants were
calculated using a Wang 720C calculator with an automatic data plotter. All
of the kinetic data reported here were plotted by both the double-reciprocal
method attributed to Lineweaver and Burk (1934) and by the method of Hofstee
(1952) as recommended by Dowd and Riggs (1965). For most assays, the
Lineweaver-Burk graphs yielded excellent straight lines but considerable
errors in estimations of maximum velocity (Vmax) and Michaelis constant (Km).
The alternate plots (v against v/s) demonstrated greater departure from
linearity, but proved to be more useful for judging the nature of variations
from Michaelis kinetics. The Hofstee plots were used to calculate values for
vmax and Km tn^ are Presented below. The square of the correlation
coefficient, r^, is cited in the text to indicate the degree of reliability of
linear correlation for the graphs presented.
Results and Discussion
Benzo[a]pyrene has been used to assess aromatic hydrocarbon metabolism in
fish (Gruger et al., 1977a, b; Payne and Penrose, 1975; Pedersen et al.,
1976), but there is evidence suggesting that it is only a minor constituent of
crude oils (Brown and Weiss, 1978) and, therefore, is not the most appropriate
choice for research on the effects of petroleum pollution on marine animals.
Metabolism of 2-methylnaphthalene in vivo by rainbow trout has been reported
(Melancon and Lech, 1978). Dimethyl naphthalenes are abundant in crude oils
and refined petroleum products (Clark and Brown, 1977). Moreover, alkylated
naphthalenes can be accumulated and metabolized by marine animals (Anderson et
al., 1974; Roubal et al., 1978).
For these experiments, reaction rates observed for the coho salmon liver
monooxygenases with the three substrates are presented in Table 21. The table
includes data at the lowest and highest substrate concentrations employed, and
at the substrate level which is closest to the apparent Km. In the case of
naphthalene, the highest substrate concentration used and the apparent Km
coincided at 300 yM. Reaction rates which we obtained when benzo[a]pyrene was
used as substrate were closely comparable to those previously obtained in this
laboratory (Gruger .et al., 1977a, b) and to results reported for a species of
lake trout (Ahokas et al., 1975). However, the maximum reaction rate we
obtained for benzo[a]pyrene monooxygenase is one third of that reported by
DePierre et al. (1975) and only one tenth of the level observed in rainbow
trout (Pedersen et al., 1976). Similarly, our data for monooxygenase towards
42
-------
naphthalene indicate that our enzyme preparation was one third as active as
the rat liver microsomes assayed by Nilsson et al. (1976).
Figure 15 is the plot of v against v/s for benzo[a]pyrene concentration
ranging from 0.16 to 80 yM. Calculations from the data of figure 15
(r =0.974) yielded a value of 122 pmol/mg per min for V and 2.1 yM for the
apparent K . This value is comparable to data obtained by others: 1.2 yM
(Robie et al., 1976), 2.48 yM (Cumps et al., 1977), and 2.95 yM (Gurtoo and
Campbell, 1970) for rat liver benzo[a]pyrene monooxygenase and 2.8 yM for the
Chambers Creek strain of rainbow trout (Pedersen et al., 1976). In other
instances our value is lower than some reported values (Schenkman et al.,
1977).
Figure 16 (r =0.953) is the plot of v against v/s for 2,6-dimethyl-
naphthalene over a concentration range of 0.7 to 144 yM. Calculations from
Fig. 16 gave v"max=244 pmol/mg per min and an apparent Km=15.3 yM. The assay
obeyed Michael is kinetics over the broad concentration range, and the K
indicated that the enzyme preparation has a lower affinity for 2,6-dimethyl-
naphthalene than it has for benzo[a]pyrene. We are unaware of comparable data
for dimethyl naphthalenes used as substrates for monooxygenases.
Naphthalene was tested as substrate for the fish liver microsomes over a
concentration range of 1.5 to 300 yM. The data indicated that naphthalene
concentrations below 15 yM produced low reaction velocities: i.e., the graph
of 1/v against 1/s gave a negative intercept at the ordinate. This kinetic
behavior has been noted by other researchers (Cumps et al., 1977; Hansen and
Fouts, 1972; Robie et al., 1976) for benzo[a]pyrene monooxygenase activity of
microsomes prepared from livers of induced rats. When the data for
naphthalene over the concentration range of 15 to 300 yM were plotted, values
for V and Km could be calculated. Figure 17 (r^=0.956) is the graph of v
against v/s over this 20-fold concentration range. V flx from figure Sis
314 pmol/mg per min, and the apparent K is 300 yM. Tne apparent Km is
appreciably different from the figure of 70 yM reported for rat liver
microsomes (Nilsson et al., 1976). It is possible that the discrepancy
between the two values is a reflection of limitations in the assay system, but
a species difference cannot be ruled out.
The apparent Km values for the three substrates indicate that our
preparation of coho salmon liver microsomes had a high affinity for
benzo[a]pyrene, intermediate affinity for 2,6-dimethylnaphthalene, and low
affinity for naphthalene. The affinity of the enzyme for naphthalene was
sufficiently low that the Km of 300 yM approaches the maximum solubility of
this substrate in the reaction medium. This characteristic limits the
usefulness of naphthalene as an alternative to benzo[a]pyrene for
monooxygenase assays.
The use of 2,6-dimethylnaphthalene as a substrate for fish microsomal
monooxygenases appears very attractive. The apparent K of 15.3 yM indicates
good affinity of the enzyme for this substrate, and Michaelis kinetics applied
over a 200-fold concentration range. Methylated naphthalenes are interesting
substrates for research on monooxygenases since oxidative metabolism can occur
at the methyl substituent(s) or on the aromatic rings (Kaubisch et al., 1972).
43
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TABLE 21. ACTIVITIES QF COHO SALMON LIVER MONOOXYGENASES
WITH THREE POLYCYCLIC AROMATIC HYDROCARBON SUBSTRATES
Substrate
Benzo[a]pyrene
Concentration
Activity
Naphthalene
uMol
0.16
1.6
80
2,6-Dimethylnaphthalene 0.7
14.4
144
1.5
300
pmol products/mg per min
8.8
46.8
127
10.4
84.2
212
1.2
156
160 r
120
80
40
20
40
v/s
60
80
FIGURE 15. Hofstee plot of benzo[a]pyrene metabolism by liver
microsomes of coho salmon. Abscissa represents the reaction
velocities expressed as pmol products per mg protein per min
divided by the benzo[a]pyrene concentration (0.16 to 80 uM)
and the ordinate represents reaction velocities.
44
-------
200 r
160 -
120 -
40 -
0.4 0.6
v/s
0.8
1.0
FIGURE 16. Hofstee plot of 2,6-dimethylnaphthalene metabolism by
liver microsomes of echo salmon. Abscissa represents the reaction
velocities expressed as pmol products per mg protein per min
divided by 2,6-dimethylnaphthalene concentrations (0.7 to 144 yM)
and the ordinate represents reaction velocities.
250 p
200
150
100
50
8
V/S
12 16
FIGURE 17. Hofstee plot of naphthalene metabolism by liver microsomes
of coho salmon. Abscissa represents reaction velocities expressed
as pmol products per mg protein per min divided by naphthalene
concentrations (15 to 300 yM) and the ordinate represents reaction
velocities.
45
-------
Summary
Benzo[a]pyrene, 2,6-dimethylnaphthalene, and naphthalene were used as
substrates for a coho salmon liver microsomal preparation.
The apparent Michael is constants (K ) were as follows: benzo[a]pyrene,
2.1 yM; 2,6-dimethylnaphthalene, 15.3 yM; and naphthalene, 300 yM.
The results indicate that the microsomal preparation had a high affinity
for 2,6-dimethylnaphthalene. We conclude that dimethyl naphthalenes are
important substrates for research on metabolism of alkyl-substituted PAH's in
marine fish, and that such studies are relevant to problems of petroleum
pollution of the oceans.
ORGANIC SYNTHESES OF 2,6-DIMETHYLNAPHTHALENE DERIVATIVES
Methods
Instrumental Analyses—
Proton NMR spectra were recorded on a Varian model CFT-20 spectrometer
using tetramethysilane as an internal standard. Gas chromatography/mass
spectrometry (GC/MS) analyses were performed on a Finnigan model 3200
automated gas chromatography/mass spectrometer operated at 70 eV. Other GC
analyses were done on a Nuclear-Chicago model series 5000 gas chromatograph
using a packed column with OV-101, 10% on 80-100 mesh Chromosorb G. High-
pressure liquid chromatography (HPLC) was performed on a Spectra-Physics Model
3500 chromatograph equipped with a UV absorbance detector. Melting points
were determined on a Fisher-Jones apparatus and are corrected. Elementary
analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. 2,6-
Dimethylnaphthalene was purchased from both Aldrich Chemical Co. (Milwaukee,
WI) and Chemical Samples Co. (Columbus, OH). Fast Blue Salt B (azoicdiazo
component 48) (Matheson Coleman and Bell) was used as a spray reagent for the
detection of naphthols (Roubal et al., 1977).
Organic Syntheses--
The preparation and characterization of compounds (1-7, Fig. 18) are
described below.
2,3-Dimethy1-3-naphtho1 (l)--Crystalline potassium 2,6-dimethylnaph-
tha! ene-3-sulfonate was prepared in 39% yield from 2,6-dimethyl naphthalene
(Fieser and Seligman, 1934). Fusion of the potassium sulfonate with KOH
(Fieser and Seligman, 1934; Weissgerber and Kruger, 1919) gave the white
crystalling naphthol (1) in 50% yield, melting point 170-174° [literature mp
169-170° (Cassebaum, 1957) and 173-174° (Weissgerber and Kruber, 1919)]. TLC
showed the presence of two trace contaminants. The purity of the naphthol (1)
was estimated by gas chromatography (GC) to be 98%. The mass spectrum of the
98% component was in accord with the assigned structure.
46
-------
OH H3C
(1)
(3)
OH
CH2OH
(6)
HOH2C
CH2OH
FIGURE 18. Oxygenated derivatives of 2,6-dimethylnaphthalene.
(1), 2,6-dimethyl-3-naphthol; (2), 2,6-dimethyl-3,4-naphthoquinone;
(3), trans-3,4-di hydroxy-3,4-dihydro-2,6-dimethylnaphthalene;
(4), 2,6-dimethyl-4-naphthol; (5), 2,6-dimethylnaphthalene 3,4-oxide;
(6), 6-methyl-2-naphthalenemethanol; (7), 2,6-naphthalenedimethanol„
2,6-Dimethy1-3,4-naphthoquinone (2)--The naphthoquinone (2) was prepared
batchwise from (1) through the use of potassium nitrosodisulfonate (Fremy
salt) (Cassebaum, 1957). Average yield of (2) was 79%. All batches had
sufficient purity (98.1 to 99.6% by GC on OV-101) for use in the next
synthetic step.
A batch was prepared, for use in Ames tests for mutagenicity (see next
Section), by recrystallization from ethanol. Highly crystalline orange
needles were obtained, mp 151.0-151.4° (mp 151°, Cassebaum, 1957). This
sample was chromatographically homogeneous (GC on OV-101), and had a mass
spectrum in accord with the assigned structure. Subsequently, a sizeable
impurity (ca. 7%) was observed to be present by HPLC analysis. The
accompanying product could not be removed by repeated recrystallization of the
sample from ethanol. It is probably very closely related structurally to the
quinone, since even on HPLC its retention time is nearly like that of the
quinone. The accompanying product is a potential metabolite, and further
investigation of its structure is important for the interpretation of the HPLC
metabolic profiles and future Ames test results.
trans-3.4-Dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene (3)—The
naphthoquinone (2) was reduced by an established synthetic route (Booth et
al., 1950) with lithium aluminum hydride and gave, after workup and
recrystallization, a 34% yield of (3) as white needles, mp 107.2-108.2°.
47
-------
Chromatographic homogeneity was demonstrated on HPLC and on TLC. The
elemental analysis, mass spectrum, and NMR spectrum were all consistent with
the assigned structure. NMR spectral features were furthermore supportive of
the assignment of a trans rather than a cis structure (Jerina et al., 1971).
2,6-Dimethyl-4-naphtho1 (4)--Different procedures were tried on a small
scale to find a workable method for the conversion of the dihydrodiol (3) to
the mixture of the naphthols (1) and (4). It was found that the naphthol (4),
the predominant product, could be sublimed more readily than the naphthol (1)
and thus separated from it by successive sublimations. Analytical TLC
procedures were worked out for the separation of the two naphthols. Both
naphthols were found to be quite labile in the presence of air and light. All
of the preliminary small scale work for the preparation of the naphthol (4)
has been completed; a larger scale synthesis is yet to be carried out.
2,6-Dimethylnaphthalene 3,4-Oxide (5)—A preliminary small scale
synthesis of (5) from the dihydrodiol (3) was carried out with the use of
dimethylformamide dimethylacetal (Harvey et al., 1975). Mass spectral
confirmation of the formation of (5) was obtained. In the presence of an
added acid catalyst, (5) very rapidly rearranged to a naphthol. With careful
exclusion of traces of acid, spontaneous rearrangement and loss of (5) still
took place (in solution at -20"), but the process was slowed.
6-Methy1-2-naphthalenemethanol (6)—White solid 2-bromomethyl-6-
methylnaphthalene was prepared from 2,6-dimethylnaphthalene according to
literature procedures (Buu-Hoi and Lecocq, 1946; Bullpitt et al., 1976). The
bromide obtained was highly impure. Attempts to upgrade the purity of the
somewhat unstable sample by recrystallization did not work well. Thus, the
impure bromide was reacted with acetone, water, and silver nitrate to give (6)
in addition to several by-products. Compound (6) was purified by preparative
TLC and had mp 131.7-132.7° (mp 128-130; Julia et al., 1960). NMR and mass
spectra were in accord with the assigned structure. With any exposure to air
and light, a major decomposition product (faster migrating on TLC) was
observed to arise directly from the alcohol (6). This conversion is of
interest, since the decomposition product may be involved metabolically.
2,6-Naphthalenedimethanol (7)--White solid 2,6-bis(bromomethyl)naphtha-
lene was prepared from 2,6-dimethylnaphthalene according to literature
procedures (Ried and Bodem, 1958; Golden, 1961). The dibromide was to be
converted to 2,6-bis(acetoxymethyl)naphthalene, which may then be converted to
(7) (Storms and Taussig, 1966); however, low yields and impurities in the
intermediates resulted in postponing further work on the dimethanol (7).
Preparation of Samples for Mutagenesis Tests--
Compounds (l)-(3) and (6) were prepared in sufficient quantity (125 mg
required) and purity for use in the Ames test for mutagenesis (Ames et al.,
1975; cf., next Section).
48
-------
MUTAGENESIS ASSAYS OF 2,6-DIMETHYLNAPHTHALENE DERIVATIVES
Methods
The Ames assays for mutagenicity were carried out in duplicates on the
following chemicals: 2,6-dimethylnaphthalene, 2,6-dimethyl-3-naphthol, 2,6-
dimethyl-3,4-naphthoquinone, trans-3,4-dihydroxy-3,4-dihydro-2,6-
dimethylnaphthalene, 6-methyl-2-naphthalenemethanol, and trans-l,2-dihydroxy-
1,2-dihydronaphthalene. Tests on known carcinogens, 2-aminoanthracene and
benzo[a]pyrene, were carried out for comparison with the naphthalenic
compounds.
Agar plate assays were carried out essentially as decribed by Ames et al.
(1975). Dimethylsulfoxide was used as solvent for all of the chemicals, which
were tested at eight concentrations in the range of 2-1,000 yg/plate. To
obtain maximum expression of mutagenicity, agar plates were preincubated to a
temperature of 37°C before adding the components of the Ames assay to the
semisolid agar. After the top-agar layer was poured onto the 37°C plates,
they were incubated in the upright position for several hours to allow
hardening of the semisolid top-agars. The assay plates were then inverted and
incubated for 24-36 hr to allow expression of mutant colonies. Colonies
originated from TA98 and TA100 strains of Salmonella typhirnurium. Colonies
were counted using a Biotran II automated colony counter (New Brunswick
Scientific Co., Inc., Edison, NJ).
Liver homogenates used in the mutagenicity assays were prepared from male
Wistar rats, which had been injected intraperitoneally with 250 mg/kg body
weight of a polychlorinated biphenyl (Aroclor 1254, Monsanto Chemical Corp.)
to provide the necessary induced mixed-function oxidase system. After
sacrifice, the livers were perfused with ice-cold 0.154 M KC1 and removed for
preparation of an S9 homogenate (9,000 x g supernate), following standard
procedures (Ames et al., 1975).
Another series of tests for mutagenesis were carried out with phage-
induction procedures described by Moreau et al. (1976). The tests used E.
coli K12 permeable (envA) tester bacteria, which is deficient in DNA repair
(uvrB). These "induct tests" were carried out on the chemicals at 20, 40, and
100 yg/plate, in quadruplicates with and without the S9 liver homogenate.
Results and Discussion
The responses of TA98 and TA100 cells, in numbers of revertant colonies
per plate, for the naphthalenic compounds were essentially all negative. The
data for the three largest concentrations of each compound are presented in
Tables 22 and 23. Only 2,6-dimethyl-3,4-naphthoquinone showed an effect by
killing the cells at 100 yg/plate with 810 yg S9 homogenate; killings were
observed also at 500 yg/plate with 135 yg of S9 homogenate. The reference
compounds, 2-aminoanthracene and benzo[a]pyrene, which gave positive responses
at 0.5 yg/plate and 5 yg/plate, respectively, were not tested at other
concentrations to determine if similar killings were possible.
49
-------
TABLE 22. TESTS FOR MUTAGENIC ACTIVITY IN S. TYPHIMURIUM TA-98 CELLS
C71
o
Compound tested Amount
tested
ug/plate
2, 6-Dimethyl naphthalene 100
500
1,000
2, 6-Dimethyl -3-naphthol
1
2, 6-Dimethyl -
3,4-naphthoquinone
trans-3,4-Dihydroxy-3,4-dihydro-
2, 6-dimethyl naphthalene
1
6-Methyl -2-naphthaleriemethanol
1
trans-1 ,2-Dihydroxy-l ,2-
dihydronaphthalene
1
2-aminoanthracene
Benzo[a]pyrene
None
100
500
,000
20
100
500
100
500
,000
100
500
,000
100
500
,000
0.5
0,5
5
5
—
Ratio of response-to-background (Response) a
0 jug S-9
0.97 (114)
1,17 (132)
0.98 (116)
1.03 (122)
0,92 (109)
0.98 (116)
0.87 (103)
0.99 (117)
0.86 (102)
0,97 (114)
1.10 (130)
1.07 (126)
0.87 (103)
0.86 (101)
1.23 (145)
0.85 (100)
1.07 (126)
1.00 (118)
135 jug S-9
1.29 (27)
0.62 (13)
1.33 (28)
0.67 (14)
0.71 (14)
1.24 (26)
0.86 (18)
(killing)
(killing)
0.62 (13)
1.52 (32)
1.19 (25)
1.48 (31)
0.90 (19)
0.90 (19)
0.76 (16)
1.48 (31)
1.50 (22)
49,7 (1,416)
38.8 (814)
—
1.00 (21)
810 jjg S-9
0.53 (15)
0.67 (19)
0.46 (13)
0.98 (25)
1.12 (32)
0.67 (19)
1.47 (42)
(killing)
0.74 (21)
0.81 (23)
0.84 (24)
0.56 (16)
0,95 (27)
0.56 (16)
0.91 (26)
1.30 (37)
0,49 (14)
—
28.9 (675)
25.7 (606)
1,00 (28)
Response, in parentheses, is the average number of histidine revertant colonies per plate.
-------
TABLE 23, TESTS FOR MUTAGENIC ACTIVITY IN S. TYPHIMURIUM TA-100 CELLS
Compound tested
2, 6-Dimethyl naphthalene
2,6-Dimethyl-3-naphthol
2, 6-Dimethyl -
3,4-naphthoquinone
trans-3,4-Dihydroxy-3,4-dihydro-
2, 6-dimethyl naphthalene
6-Methyl -2-naphthalenemethanol
trans-l,2-Dihydrox.y-l,2-
dihydronaphthalene
2-ami noanthracene
Benzo[a]pyrene
None
Amount
tested
ug/plate
100
500
1,000
100
500
1,000
20
100
500
100
500
1,000
100
500
1,000
100
500
1,000
0.5
0,5
5
5
--
Ratio of response-to-background (Response) a
135 uq S-9
0.83 (119)
0.93 (133)
0.56 (80)
0.76 (108)
0.81 (116)
0.92 (131)
0.44 (63)
0.49 (70)
(killing)
0.92 (132)
0.52 (75)
0.76 (108)
0.71 (102)
0.63 (90)
0.44 (63)
0.83 (119)
0.59 (81)
0,52 (75)
6.85 (980 + 136)
10.81 (1081 + 260)
_ _ _
1.00 (143 + 17)
810 ug S-9
1.42 (142)
1.13 (113)
1.06 (106)
0.85 ( 85)
1.27 (127)
0.90 (90)
0.65 (65)
(killing)
(killing)
1.05 (105)
0.92 (92)
0.65 (65)
1.14 (114)
1.28 (128)
0.69 (69)
0.86 (86)
0,91 (91)
0.84 (84)
_ _ _
4.99 (714 +
4.70 (470 +
1.00 (100 +_
213)
100)
21)
Response, in parentheses, is the average number of histidine revertant colonies per plate,
-------
The results of the induct tests with E. coli K12 cells and the
naphthalenic compounds are illustrated by Figure 19, where test-response to
background-response ratios for these compounds can be compared with results
with reference carcinogens. Again, 2,6-dimethylnaphthalene and the
naphthalenic derivatives gave negative results from the induct test.
Assuming that the microbiological test data can be extrapolated to fish,
then none of the naphthalenic compounds which were tested would be likely to
cause changes that are mutagenic in fish. Of those compounds, 2,6-dimethyl-
3,4-naphthoquinone appears to be the most potentially lethal to isolated cells
at the concentrations employed in the assays. Work on toxic effects of
naphthalenic quinones in fish should be considered for the future. Quinones
are conversion products of phenolic compounds, which can be formed by free-
radical autoxidations of various substituted aromatic hydrocarbons in the
presence of oxygen (Uri, 1961); consequently, quinones in fish may arise from
exogenous as well as endogenous reactions of petroleum aromatic hydrocarbons.
Summary
Tests were conducted on several naphthalenic compounds for their
potential mutagenic activities. An Ames test with Salmonella was performed on
each of the following: 2,6-dimethylnaphthalene, 2,6-dimethyl-3-naphthol, 2,6-
dimethyl-3,4-naphthoquinone, 6-methyl-2-naphthalenemethanol, trans-3,4-
dihydroxy-3,4-dihydro-2,6-dimethyl naphthalene, and trans-l,2-dihydroxy-l,2-
dihydronaphthalene. 2-Aminoanthracene and benzo[a]pyrene, as references, were
the only compounds that gave positive results with the Ames test. A lethal
effect upon the Salmonella caused by 2,6-dimethyl-3,4-naphthoquinone was the
only effect found for the naphthalenics tested. The research results suggest
that, as far as can be generalized from microbial bioassays, mutagenesis is an
unlikely event in fish due to exposures to these naphthalenic substances.
IN VITRO METABOLISM OF 2,6-DIMETHYLNAPHTHALENE BY COHO SALMON LIVER
MICROSOMES
Methods
Microsomal Reactions JJT_ Vitro and Analyses—
Microsomes from normal coho salmon livers were prepared by homogenization
and differential centrifugation. The microsomes were incubated at 25°C with
2,6-dimethylnaphthalene (2,6-DMN) in a reaction mixture consisting of Tris
buffer pH 7.5, MgC^, and reduced nicotinamide adenine dinucleotide phosphate
(NADPH). Controls contained everything except the NADPH. The concentration
of 2,6-DMN was usually 30.6 yM. This value is twice the Michaelis constant
for this substrate (cf. Table 21, above) (Schnell, Gruger and Malins, 1980).
Unreacted substrate and nonconjugated metabolites were extracted into ethyl
acetate from the reaction mixture at pH 7.5. Conjugated metabolites were then
extracted into ethyl acetate after the aqueous phase was adjusted to pH 1 with
hydrochloric acid.
52
-------
With S9 homogenate
13
O
O
CD
0)
05
o
Q.
OJ
DC
0
0)
c
c
CO
cn
O
c
OJ
o
03
CD
o
c
CM
CL
^. CD
I ^
c I
CD CM
Without S9 homogenate
Q.
CD —
c. o
CM
11
•p OJ
0> C
2-2
ro
•References
Naphthalenic compounds
FIGURE 19. Induct test response-to-background ratios for
naphthalenic compounds (100 yg/plate) and reference compounds
(1 to 10 yg/plate) with E. coli K12 cells.
53
-------
For preparation of the glucuronides of oxidized products of 2,6-
dimethylnaphthalene, we employed conditions similar to those reported by
Nilsson et al. (1976). After 30 min. incubation, Triton-X-100 and uridine
diphosphoglucuronic acid were added. The incubation was then resumed for an
additional 30 minutes. An equal volume of ethyl acetate was added to stop the
reaction and to extract neutral metabolites. A total of three extractions
with ethyl acetate were conducted at neutral pH. Then, the aqueous phase was
adjusted to pH 1 with 1M HC1, and three more extractions with ethyl acetate
were performed. TLC on silica gel was used to establish a profile of the
metabolites in the two organic solvent extracts.
The metabolites, which were extracted at pH 1, were subjected to
hydrolysis with limpet s-glucuronidase (containing aryl sulfatase activity) as
described by Dodgson et al. (1953). Inhibition of sulfatase was accomplished
by the method of Dodgson and Spencer (1953). The hydrolysis was conducted at
pH 4.0 in acetate buffer at 37 C for one hour. The reaction was stopped and
hydrolysis products were extracted with ethyl acetate. The ethyl acetate
extracts were subjected to TLC on silica gel using a solvent system of
toluene-.ethyl acetate (50:10; v:v). Potential metabolites of 2,6-
dimethylnaphthalene, prepared by Dr. Peter Fraser (cf., section on organic
syntheses, above), were run in the same solvent system and their Rf values
were determined.
Analyses for metabolites of 2,6-DMN were conducted by gas
chromatography/mass spectrometry (GS/MS), thin-layer chromatography on silica
gel, and high-performance liquid chromatography (HPLC) using ultraviolet
fluorescence spectrometry (Krahn et al., 1980). Liquid scintillation
spectrometry was used to assay for 2,6-dimethyl[ H]naphthalene.
Analyses of 2,6-dimethylnaphthalene metabolites by glass-capillary
GC/MS—Samples were injected into a Hewlett-Packard 5840A gas chromatograph
interfaced to a Finnigan 3200 mass spectrometer. The GC column was a 30 m x
0.25 mm (i.d.) WCOT capillary column coated with SE-54 (Supelco, Inc.;
Bellafonte, PA). The injector temperature was 320°C and column temperature
was programmed at 4°C/min from 50-280°C. Mass spectrometer/electron impact
conditions were: electron energy, 70eV; filament emission current, 500 A; and
scan, 34-534 Amu/sec.
Detection of metabolites of 2,6-dimethylnaphthalene by thin-layer
chromatography—TLC was used to monitor metabolite formation on a routine
basis. We employ silica gel plates and a dual solvent system for development.
The plates are first developed with ethyl acetate to a distance of 1 cm above
the origin. This serves to move the dihydrodiol of 2,6-DMN cleanly away from
the origin. After a brief drying, the plate is then developed with
toluene:ethyl acetate (40:1, v:v). This system provides good resolution of
the nonconjugated metabolites. Conjugated metabolites and highly-polar
metabolites (e.g., more polar than dihydrodiol and monocarboxylic acids) will
remain at the origin in this system.
54
-------
Time Course for In Vitro Metabolism—
For a time course experiment, the incubation procedure was modified
slightly. The usual volumes were increased to allow for analyses of
sufficient numbers of reaction samples.
Test and control were run simultaneously; the control had no NADPH. At
selected times (Table 24), an aliquot was removed from each reaction mixture
and immediately extracted with ethyl acetate. The extracts and marker
compounds were analyzed by TLC. Metabolites were located on TLC plates by
scraping segments and counting them by liquid scintillation spectrometry. The
radioactivity in the metabolite fractions from TLC was corrected for the
slight activity found in the zero time sample.
Results
GC/MS analyses confirmed the presence of several metabolites prepared by
in vitro incubation with microsomes. We have obtained confirmation of the
substrate 2,6-DMN and the following metabolites: 6-methyl-2-
naphthalenemethanol, two naphthols of 2,6-DMN, and a quinone of 2,6-DMN.
Also, we have a tentative confirmation of the presence of 6-methyl-2-
naphthaldehyde. None of the metabolites were detected in the control sample.
Thin-layer chromatography of neutral metabolites indicates that 6-methyl-
2-naphthalenemethanol is a significant metabolite of 2,6-DMN by coho salmon
hepatic microsomes. Conditions designed to prepare glucuronides of the polar
metabolites indicate that we are able to form a product, which is extracted
into ethyl acetate at pH 1 but not at pH 7. Hydrolysis of the acidic
metabolites with e-glucuronidase (sulfatase inhibited with phosphate)
liberates at least three significant metabolites of 2,6-[
Data from the time-course experiment are given in Tables 24 and 25. The
results provided considerable information on the nature of 2,6-DMN metabolism
by fish liver microsomes. In Table 25, data are given that summarize the
metabolite distribution at 15 minutes of incubation with the microsomes, and
the Rf values for the marker compounds. These data indicate that the
principal metabolite formed during the incubation was 6-methyl-2-naphthalene-
methanol. Also, an aldehyde of 2,6-DMN was apparently a major metabolite.
Lesser amounts of a quinone, the dihydrodiol, and a naphthol(s) were also
detected. The Rf values indicate that the TLC system provides good separation
of the compounds.
In A and B of Figure 20, plots are shown for the time course of the
formation of total metabolites and individual metabolites. It can be seen
that oxidation of the substrate occurs in a linear fashion for the first
twenty minutes. After this time, the rate decreases, but metabolite formation
still proceeds at a significant rate after 60 minutes. Also, there are
important differences in the rates of formation of the identified metabolites.
At all time points, the principal metabolites were the 6-methyl-2-naphthalene-
methanol and the corresponding aldehyde. The results for the formation of the
dihydrodiol were not plotted, because its formation followed an irregular time
course. The formation of the naphthol(s) of 2,6-DMN was not very extensive in
this experiment and seemed to cease after the first 15 minutes.
55
-------
TABLE 24. TIME COURSE OF METABOLITE FORMATION FROM 2,6-DIMETHYLf3H]NAPHTHALENE
Time
min
5
10
15
20
30
45
60
Total Arylmethyl Aldehyde Dihydrodiol
metabolites alcohol
-------
5,342
9,341
15,302
19,089
23,066
28,076
33,573
- - - dpm /
2,420
3,746
5,551
6,871
8,638
10,048
12,249
unit volume
1,227
2,412
3,570
4,593
5,422
7,656
9,014
of reacti
271
447
1,227
1,325
2,004
2,238
3,235
Quinone
on mixture
371
659
1,015
1,340
1,494
1,811
2,072
Naphthol
_____
222
392
767
772
664
621
851
TABLE 25. METABOLITES OF 2,6-DIMETHYLNAPHTHALENE FROM IN VITRO INCUBATIONS
OF 15-MINUTE REACTION WITH LIVER MICROSOMES
Compound
2, 6-Dimethyl naphthalene
Aldehyde
Naphthol
Quinone
Arylmethyl alcohol
Dihydrodiol
Percentage of
total metabolites
—
23.3
5.0
6.6
36.3
8.0
Percentage of
identified metabolites
—
29.4
6.3
8.4
45.8
10.1
Rfb
0.94
0.73
0.56
0.47
0.32
0.12
21% of dpm in areas of TLC bands were not positively identified.
Rf values for marker compounds.
56
-------
30,000 -
20,000 -
0>
•*-<
D
.£ 10,000
E
O
2000 -
1000 -
15
30
Time (minutes)
45
FIGURE 20, j£ yjjtro metabolism of 2,6-dimethylnaphthalene.
A. Formation of total metabolites (———), 6-methyl-2-
naphthalenemethanol ( ), and 6-methyl-2-naphthaldehyde
(•———). B. Formation of 2,6-dimethylnaphthoquinone ( ••••")
and 2,6-dimethylnaphthol ( ).
57
-------
Discussion
The results of the GC/MS analyses verified some of our preliminary
conclusions based on the results of TLC of H-2,6-DMN metabolites formed jn_
vitro. The results have provided proof that in vitro metabolism of 2,6-DMN
results in the formation of 6-methyl-2-naphthaTenemethanol. Ring oxidation of
2,6-DMN is indicated by confirmation of the presence of a quinone and two
naphthols of the substrate. (The hydroxyl group could be on the 1, 3, or 4
position of the naphthalene moiety.) The quinone does not correspond to our
standard of 2,6-dimethyl-3,4-naphthoquinone. In addition to ring-oxidation
products, we believe an aldehyde is formed in the in vitro systems. Although
GC/MS did not provide clear confirmation of the formation of the 6-methyl-2-
naphthaldehyde, this compound is a logical product of oxidation of the 6-
methyl-2-naphthalenemethanol, and it is of considerable interest. In other
work, we found that ultraviolet irradiation of 2,6-DMN in air gave rise to a
compound that was identified as 6-methyl-2-naphthaldehyde.
Thin-layer chromatography in two different solvent systems indicates that
6-methyl-2-naphthalenemethanol is an important metabolite of 2,6-DMN.
Metabolites generated when uridine diphosphoglucuronic acid is added are
readily separated from neutral metabolites by different extractions at pH 7
and pH 1. The acidic metabolites can be hydrolyzed by g-glucuronidase to
yield several oxidized forms of 2,6-DMN. In the presence of saccharo-1,4-
lactone, this hydrolysis is inhibited. One of the products released by
hydrolysis with p-glucuronidase has the same Rf values as 6-methyl-2-
naphthalenemethanol. At least two other metabolites of 2,6-DMN are also
released by the 3-glucuronidase hydrolysis.
Our results suggest that an aldehyde of 2,6-DMN is apparently an
important metabolite. An aldehyde may be formed by further enzymatic
oxidation of the 6-methyl-2-naphthalenemethanol.
Oxidation of the methyl group(s) of 2,6-DMN represents an important j_n
vitro metabolic pathway involved in coho salmon hepatic microsomes. Oxidation
of methylated naphthalenes at the alkyl substituents has been reported in
bacteria (Starovoitov et a!., 1976) and by guinea pig microsomes (Kaubisch et
al., 1972). Thus, oxidation of alkyl substituents of methylated naphthalenes
is a common metabolic pathway in a variety of organisms.
Interproject Collaborative Studies—
Metabolites of 2,6-DMN and benzo[a]pyrene, which were obtained from j_n_
vitro reactions with coho salmon liver microsomes, were employed by our
National Analytical Facility, notably Dr. Margaret Krahn, in studies of HPLC.
An aim of that work was to find out whether it was possible to detect the
presence of the metabolites of 2,6-DMN among the metabolites of
benzo[a]pyrene. The findings from this interproject collaboration have been
submitted for publication (Krahn et al., 1981).
58
-------
Summary
Our results indicate that coho salmon liver microsomes actively
metabolize 2,6-DMN i_n vitro. The results indicated that methyl-group
oxidation is the principal metabolic pathway for 2,6-DMN. An aldehyde and an
alcohol arising from methyl-group oxidation are principal products of the
metabolism in vitro; the former is believed to be 6-methyl-2-naphthaldehyde
and the latter is the 6-methyl-2-naphthalenemethanol. Metabolic products of
naphthyl-ring oxidations were isolated from the in vitro reactions, namely a
quinone, two naphthols and a dihydrodiol of 2,6-DMN. A time-course study of
the metabolites formed in vitro revealed limited formation of the naphthols.
EFFECTS OF NAPHTHALENE AND P-CRESOL, SEPARATELY AND TOGETHER IN FOODPATH
EXPOSURES, IN STARRY FLOUNDER ON THE IN VIVO METABOLISM OF
2,6-DIMETHYLNAPHTHALENE
Materials
2,6-Dimethylnaphthalene-4-14C (2.1 mCi/mmol) was synthesized by
California Bionuclear Corp., Sun Valley, Calif., and found to be 98%
radiochemically pure by gas-liquid chromatography. The following chemicals
were obtained from commercial sources: 2,6-DMN (Aldrich Chemical Co.,
Milwaukee, Wise., and Chemical Samples Co., Columbus, Ohio); potassium
nitrosodisulfonate (K & K Laboratories, Plainview, N.Y.); Fast Blue Salt B
(Matheson, Coleman and Bell, Norwood, Ohio); Amberlite XAD-2 and XAD-4 resins
(Rohm & Haas Co., Philadelphia); limpet e-glucuronidase, a-glucosidase,
3-glucosidase, a-naphthyl sulfate, 3-naphthyl-a-D-glucoside and ot-naphthyl-S-
glucuronide (Sigma Chemical Co., St. Louis, Missouri); p-cresol (Aldrich
Chemical Co.); and naphthalene (Baker and Adamson, Morristown, N.J.). All
other chemicals were reagent grade, pesticide grade, or highest-available
purity.
Four derivatives of 2,6-DMN were synthesized for use as reference
standards. These compounds are described in a previous section; cf.
derivatives 1, 2, 3, and 6 in Figure 18.
Starry flounder were netted from near the mouth of the Columbia River and
acclimated to seawater holding facilities for six weeks. For an initial
experiment, five fish were used. For a major experiment, forty-eight fish
were selected for uniformity in size and randomly divided into four groups of
twelve each. Of the latter fish, mean lengths and weights were as follows:
group 1, 139+6 mm, 24+4 g; group 2, 150+10 mm, 27+6 g; group 3, 150+9 mm,
29+5 g; and group 4, T47+7 mm, 27+4 g. "There were no significant differences
(P>0.05) in length and weight among the four groups. The temperature of the
water was 12.7+_0.7°C and the salinity was 28.3+1.0°/oo. The flounder were
given minced herring and hake ad libitum as a cfaily diet throughout the
experiment.
59
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Methods and Procedures
Instrumental Analyses--
Radioactivity was determined with a Packard Tri-Carb model 3255 liquid
scintillation spectrometer (Packard Instrument Co., Downers Grove, 111.) using
ScintiVerse scintillation fluid (Fisher Scientific Co., Fair Lawn, N.J.).
HPLC of 2,6-DMN metabolites was performed on a Hewlett-Packard model 1084B
chromatograph (Hewlett Packard Co., Palo Alto, Calif.) equipped with a 0.4 cm
i.d. x 25 cm steel column packed with 10-ym diameter Lichrosorb RP-18
(E. Merck, Darmstadt, Germany). Gas chromatography/mass spectrometry was
performed using a Hewlett-Packard 5840A gas chromatograph equipped with a
glass capillary column (SE-54 wall coated), 30 meters in length, and
interfaced with a Finnegan (Sunnyvale, Calif.) model 3200 automated mass
spectrometer. Other GC analyses were performed on a Nuclear Chicago 5000
series gas chromatograph, using a column packed with 10% OV-101 on 80-100 mesh
Chromosorb G (Applied Science Lab., State College, Penn.).
Thin-layer Chromatography—
Metabolites of 14C-2,6-DMN were separated by TLC utilizing three
different systems. For system A, silica gel 60 F-254 (E. Merck, Darmstadt,
Germany) plates were developed with toluenerethanol (92:8, v/v) in an unlined
tank. For system B, silica gel Q6F (Kontes Glass Co., Vineland, N.J.) plates
were first developed with toluenerethyl acetate (85:15, v/v) to 15 cm in an
unlined tank and then developed with ethanol:acetonitrile:acetic acid
(90:10:1, v/v/v) to 8 cm in a filter paper-lined tank. For system C, Q6F
plates were developed with toluene:acetone:acetonitrile (80:10:10, v/v/v) to
15 cm in a lined tank and then developed with n-butyl alcohol:water:ammonium
hydroxide (85:13:2, v/v/v) to 8 cm in a lined tank.
Reference compounds consisted of 2,6-DMN, the four derivatives of 2,6-DMN
(described above), a-naphthyl sulfate, a-naphthyl-g-glucuronide, and
g-naphthyl-a-D-glucoside. Following TLC separations, the reference compounds
were located on TLC plates using short wavelength ultraviolet light. The Rf
values for the reference compounds in the three TLC systems are given in Table
26. Solvent system A was used for gross separations of conjugated metabolites
from unconjugated metabolites; conjugated metabolites of 2,6-DMN remain at the
origin. TLC system B resolved all of the reference compounds except for
a-naphthyl sulfate and trans_-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphtha-
lene. These two compounds could be effectively separated by the use of system
C. All reference compounds were stable during the course of TLC analyses,
although certain chemical changes were observed on TLC plates after 12-18 hrs.
Recovery of radiolabeled metabolites from TLC plates was greater than 90%.
Exposure of Starry Flounder—
Two exposure experiments were conducted: the first was a small-scale
experiment to determine the time intervals to be employed for the major,
large-scale experiment. The initial experiment involved the administration of
14C-2,6-DMN to five starry flounder. Each of these fish was fed 9.6 yCi
(0.71 mg) of the-labeled 2,6-DMN in a gelatin capsule containing Oregon moist
pellet food. At 6, 12, 24, 48, and 120 hours after administering the
capsules, a fish was killed, and the liver and gall bladder were removed for
analysis. Analysis of radioactivities in the latter tissue indicated that
60
-------
TABLE 26. Rf VALUES FOR REFERENCE COMPOUNDS ANALYZED BY THIN-LAYER
CHRQMATOGRAPHY USING THREE SOLVENT SYSTEMS
Solvent system a
Compound <
2 ,6-Dimethyl naphthalene
2,6-Dimethyl~3-naphthol
2,6-Dimethy1-3,4-naphthoquinone
6-Methyl -2-naphthalenemethanol
system A
0.82
0.66
0.71
0.52
System B
0.92
0.67
0.60
0.47
System C
0.74
0.66
0.43
0.59
trans_-3,4-Dihydroxy-3,4-di hydro-
2, 6-dimethyl naphthalene
a-Naphthyl sulfate
3-Naphthyl-a-D- glucoside
a-Naphthyl-3- glucuronide
0.35
0
0
0
0.41
0.41
0.29
0.09
0.48
0.20
0.15
0.05
System A, toluene:ethanol, 92:8 (v/v) in an unlined tank. System B,
developed with toluene:ethyl acetate, 85:15 (v/v) to 15 cm in an unlined
tank; then developed with ethanol:acetonitrile:acetic acid, 90:10:1 (v/v/v)
to 8 cm in a filter paper-lined tank. System C, developed with toluene:
acetone:acetonitrile, 80:10:10 (v/v/v) to 15 cm in a lined tank; then
developed with n-butyl alcohol .-water: ammonium hydroxide, 85:13:2 (v/v/v)
to 8 cm in a lined tank.
within 24 hours much of the labeled DMN had been absorbed and metabolized
the fish. Thus, it was concluded that a 24-hour exposure of fish to the
2,6-DMN was suitable for the major experiment.
For the major experiment, the xenobiotics were dissolved in peanut oil
and administered in doses of 0.2 ml/fish. The appropriate peanut oil solution
was deposited into the stomach, via the mouth, using a stainless steel
cannula. The experiment was conducted over a period of 9 days. On each of
the first 6 days the fish were given the following: group 1, peanut oil only;
group 2, 12 yg (0.09 ymol) of naphthalene (0.44 mg/kg body wt.); group 3,
9.7 yg (0.09 ymol) of p-cresol (0.33 mg/kg); group 4, 12 yg of naphthalene
(0.44 mg/kg) and 9.7 yg of p-cresol (0.36 mg/kg). On day 7, no treatment took
place. On day 8, each..fish was given an oral dose of 0.2 ml of peanut oil
containing 4.8 yCi of 14C-2,6-DMN (0.35 mg/fish; average dose 12-15 mg/kg).
-------
On day 9 the fish were sacrificed, and enlarged gall bladders were
removed and frozen at -60°C.
Bile samples from four fish were pooled to yield three samples, in
amounts ranging from 51 to 201 nig, for each of the four groups. Total bile
for the four groups ranged from (mean +_S.D.) 96+52 mg to 134+59 mg.
The livers from the 48 starry flounder which received the C-2,6-DMN
were excised and frozen immediately after sacrifice. Four livers were
combined in each case to provide a total of three pooled samples per group.
Each pooled liver sample was divided into two portions. One portion was used
for preparation of microsomes and assay for activity of benzo[a]pyrene
monooxygenase (DePierre et a!., 1975). The other portion was homogenized and
extracted for HPLC analysis of metabolites of 2,6-DMN.
Analysis of 2,6-DMN Metabolites in Liver--
Metabolites of 2,6-DMN were extracted from the 12 pooled liver samples.
The livers were weighed, minced, and homogenized in isopropyl alcoholrwater
(25:2, v/v) using three parts of solvent for one part of liver. Two 60-yl
aliquots were removed to measure total radioactivity and radioactivity as
unmetabolized 2,6-dimethylnaphthalene. Then, 100 yl of the 2,6-
dimethyl[ H]naphthalene-labeled rat urine extract (see sub-section below) were
added as an internal standard, so as to estimate recoveries of metabolites.
The homogenates were filtered through filter paper, and the residue on the
paper was extracted three times with 8 ml portions of boiling methylene
chloride:isopropyl alcohol:water (75:25:2, v/v/v). The residue was next
extracted three times with 8 ml portions of boiling ethanol:ethyl acetate
(50:50, v/v). The original filtrate and the extracts were combined and
concentrated under a stream of nitrogen over an ice bath. When the extract
volume reached less than 1 ml, 40 ml of anhydrous methanol were added and the
concentration step was repeated. This solvent exchange was repeated, and the
final methanol extract was concentrated to about 1.5 ml and used for HPLC
analysis.
Tritiated metabolites of 2,6-DMN from rat urine—The procedures of Thomas
et al. (1978), for naphthalene metabolites, were employed to assess the
recoveries of 14C-2,6-DMN metabolites from the liver and bile of the starry
flounder. Accordingly, a 200 g male rat was injected intraperitoneally with
2,6-dimethyl[3H]naphthalene dissolved in peanut oil. Urine was collected for
3-4 days following the injection. One week after the injection, the rat was
reinjected with a second dose of the tritiated substrate. The urine samples
containing the highest total radioactivity were combined prior to extraction
of tritiated metabolites.
Our initial attempts to prepare the urine extracts consisted of
saturating the urine with sodium chloride, followed by multiple extractions
with ethyl acetate. We found this procedure yielded poor recoveries of total
radioactivity. Much better results were obtained by applying the procedures
of Horning et al. (1974). The urine was saturated with powdered ammonium
carbonate and then extracted with ethyl acetate. The aqueous phase was
carefully acidified to pH 1 and reextracted with ethyl acetate. The ethyl
acetate fractions were combined, evaporated to near-dryness under nitrogen,
62
-------
and the residue taken up in a small volume of absolute methyl alcohol. This
procedure allowed 60-80% recovery of the radioactivity in the urine samples.
HPLC of the rat urine extracts indicated that they contained sufficient H for
liquid scintillation counting and also enough UV absorbing peaks for use in
assessing the recoveries of 4C-2,6-DMN and its metabolites in starry flounder
tissues.
HPLC analysis of metabolites from liver—Separation of 2,6-DMN
metabolites was accomplished by HPLC.Metabolites were eluted with a 60-min
linear gradient beginning with 100% aqueous 10% monobasic potassium phosphate
(5xlO~4 M) and proceeding to 100% methanol. Fractions from the HPLC were
collected at one-half minute intervals from zero to 70 minutes. Each fraction
volume was 0.5 ml. Seven ml of scintillation fluid were added to each
fraction, and the samples were counted for both 14C and^H in a liquid
scintillation spectrometer.
Separation and Identification of 2,6-DMN Metabolites from Bile--
Work on methods of analysis of biliary metabolites was directed at
comparisons of procedures employing techniques with Amber!ite XAD-2 and XAD-4
resins and preparative TLC. The procedures with XAD resins were patterned
after those reported by Statham et al. (1976, 1978), while the TLC procedures
included those reported by Roubal et al. (1977) as well as new procedures
(described above). The methods were evaluated for optimum conditions for the
recovery and separation of both conjugated and nonconjugated metabolites of
2,6-DMN from bile. Semi-quantitative analysis of recovery and evaluations of
separations by the XAD-resin method and TLC method employed T-labeled
compounds in bile taken from the flounder in the initial small-scale
experiment.
In the major experiment, labeled metabolites of the bile samples were
separated by TLC. TLC system A was used to estimate the relative proportions
of metabolites recovered as conjugated or nonconjugated products. Individual
conjugated and nonconjugated metabolites were assayed using either TLC system
B or system C. Labeled compounds were quantitated from resultant data of
liquid scintillation counting of scraped TLC bands. Results of liquid
scintillation counting are expressed as mole-% of total metabolites.
The glucuronides, i.e., compounds in a TLC band having an Rf
corresponding to that of the standard a-naphthyl-3-glucuronide, were recovered
from the silica gel by extraction with methanol. The glucuronides were
treated with limpet 6-glucuronidase (2,140 Fishman units) in 0.1 M citrate-
phosphate buffer, pH 4.0, at 37° for 3 hr (Dodgson et al., 1953). Blanks
contained either the e-glucuronidase-inhibitor D-saccharo-l,4-lactone (Levvy,
1952) or heat-denatured B-glucuronidase. Following hydrolysis, the reaction
mixtures were acidified with 0.1 ml of 6M-HC1, and the products were extracted
into peroxide-free ethyl ether. After drying with NapSO^ and concentration
under nitrogen, the extracts were analyzed by TLC. The efficacy of the
3-glucuronidase action was verified by the hydrolysis of a-naphthyl-B-
glucuronide to yield a-naphthol (detected by spraying with Fast Blue Salt B;
Boyland and Solomon, 1956), and by autoradiography of TLC plates using Kodak
X-Omat R film. More than 90% of the material in the glucuronide fraction was
hydrolyzed by 3-glucuronidase.
63
-------
The glucosides (TLC section with Rf corresponding to e-naphthyl-a-
glucoside) were extracted from silica gel with methanol and hydrolyzed at
pH 6.8 (citrate-phosphate) first with a-glucosidase (13 units) for 3 hr at
37 C, then with 6-glucosidase (23 units) for 2 hr at 37°. The blanks were run
at pH 10, which inhibited the glucosidases. The products of the hydrolysis
were recovered and analyzed as described for the glucuronides. More than 50%
of the material in the glucoside fraction was hydrolyzed by mixed a- and
B-glucosidases.
Statistics—
Statistical analyses involving two-sided comparisons of treatment groups
versus the control group (Dunnett, 1955; Dunnett, 1964) were performed on data
from replicate samples. Student's t-test was used to compare sizes of fish.
Results
2,6-DMN Metabolites from Livers--
14 Tables 27 and 28 present the qualitative results of the HPLC analyses of
C-2.6-DMN metabolites extracted from the starry flounder liver samples. For
these analyses, five compounds were utilized as external standards: 2,6-DMN,
trans-3.4-dihydroxy-3t4-dihydro-2.6-dimethv1naphthalene. 2,6-dimethyl-3,4-
naphthoquinone (containing a persistent contaminant), 2,6-dimethyl-3-naphthol,
and 6-methyl-2-naphthalenemethanol. Table 27 presents the distributions of
total metabolites and residual substrate from the four experimental groups.
The data in Table 27 are further resolved and presented in Table 28.
The results presented in Table 28 indicate three significant peaks for
metabolites in the liver samples from the control groups, the p-cresol-exposed
groups and the naphthalene-exposed group. The liver samples from the group of
fish exposed to both p-cresol and naphthalene exhibited five significant peaks
of radioactivity which we attribute to metabolites of 2,6-dimethylnaphthalene.
Figure 21 is a graphical representation of the metabolite profile for the
livers of the group of fish exposed to both p-cresol and naphthalene. The
graph also indicates the retention times of the external standards.
2,6-DMN Metabolites from Bile--
The results of experiments with XAD-2 and XAD-4 resins showed no benefit
of one resin over the other in analyses of biliary metabolites. Reference
standards of 2,6-DMN, 2,6-dimethyl-3-naphthol, and the trans-3,4-dihydrodio1
of 2,6-dimethylnaphthalene were retained on the resins; the latter two
derivatives elute with methanol and the parent compound elutes with acetone.
In another experiment where bile containing ^C-labeled metabolites was passed
through XAD-2 resin, the initial water eluate removed 2.5% radioactive
substances in relation to those substances removed with methanol. The water
eluate is normally discarded according to procedures of Statham et al. (1976,
1978), so metabolites are lost by the resin technique. Also, losses can occur
during evaporation of solvents, e.g., when 50 ml of a methanol extract are
concentrated.
Preparative TLC procedures offered advantages over the procedures with
XAD resins by allowing less sample handling and providing separations in a
much shorter time. Preparative TLC and liquid scintillation spectrometry of
64
-------
TABLE 27. PERCENT OF 14C 2,6-DIMETHYLNAPHTHALENE METABOLIZED IN STARRY
FLOUNDER LIVERS AS DETERMINEDLY HPLC ANALYSIS a
% as Metabolites
% as Substrate
group
23.5
76.4
p-Cresol
26.5
73.5
Naphthalene
17.0
82.9
Exposure group
p-Cresol and naphthalene
31.0
68.9
14
Percentages calculated on the basis of C radioactivity recovered from
HPLC as parent compound or as metabolites. Livers of 12 fish per group
were pooled and extracted, the extracts were concentrated to 0.75-1.5 ml,
and 0.1-0.2 ml of the concentrates were analyzed by HPLC.
TABLE 28. ANALYSIS OF C 2,6-DIMETHYLNAPHTHALENE METABOLITES BY HPLC OF
LIVER EXTRACTS FROM STARRY FLOUNDER FED p-CRESOL AND NAPHTHALENE,
SEPARATELY AND TOGETHER: RADIOACTIVITY IN HPLC FRACTIONS
Retention
time
min
12.5
16.5
20.0
25.0
30.0
35.0
40.0
50.0
- 16.5
- 20.0
- 25.0
- 30.0
- 35.0
- 40.0 a
- 50.0
- 70.0 b
Control
group
135
nil
551
nil
nil
16
nil
2277
Exposure group
p-Cresol
107
nil
532
nil
nil
57
nil
1934
Naphthalene p-Cresol and naphthalene
164
nil
1135
nil
nil
91
nil
6749
49
171
1110
173
167
97
nil
3920
Corresponds to trans-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene
Corresponds to 2,6-dimethylnaphthalene
65
-------
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FIGURE 21. HPLC profile of metabolites of 2,6-dimethylnaphthalene-4-
from livers of starry flounder exposed to naphthalene and p-cresol.
Elutions of reference standards are indicated by arrows.
14,
66
-------
TLC fractions (bands) were used to assess the optimum formation of biliary
metabolites from fish in the short-term exposure experiment discussed
previously. The biliary metabolites appeared to reach maximum concentrations
between 12 and 48 hr. Also, analyses showed that the metabolites were nearly
all conjugated derivatives.
Other TLC analyses of biliary metabolites from 12- and 48-hr samples,
using the forementioned reference standards, showed that the conjugated
metabolites corresponded to standards for naphthylglucoside and
naphthylglucuronide.
The concentrations of metabolites of 2,6-DMN found in the four groups of
starry flounder in the major experiment are presented in Table 29. Three
nonconjugated products were isolated and quantitated: 2,6-dimethyl-3-
naphthol; 2,6-dimethyl-3,4-naphthoquinone; and 6-methyl-2-naphthalenemethanol.
The method employed (TLC system B) did not resolve trans-3,4-dihydroxy-3,4-
dihydro-2,6-dimethylnaphthalene and aryl sulfate conjugates. Conjugated
metabolites of 2,6-DMN constituted 54.6 mole-% to 77 mole-% of metabolites.
Comparisons of the controls with the xenobiotic-exposed fish indicated no
statistically significant differences (P>0.05) between the amounts of the
glucoside fraction (Table 29). Enzymatic hydrolysis of the glucoside fraction
was incomplete; therefore, this fraction was not analyzed further. The
concentrations of glucuronides in groups 2 and 3 are not significantly
different from those in group 1. However, the concentration of glucuronides
in group 4 (naphthalene and p-cresol, 19.9 mole-%) is significantly lower
(P<0.05) than that in group 1 (controls, 34.8 mole-%).
When the glucuronides of 2,6-DMN metabolites were hydrolyzed, more than
98% of the radioactivity was accounted for by two products: 6-methyl-2-
naphthalenemethanol (alcohol) and trans-3,4-dihydroxy-3,4-dihydro-2,6-
dimethylnaphthalene (dihydrodiol). The results are presented in Table 30.
There was a significant reduction (P_<0.01) in the concentrations of the
alcohol metabolite in groups 2, 3, and 4 relative to group 1.
Correspondingly, there was an increase in the amount of the glucuronide of the
dihydrodiol. This increase, relative to the control group, was more
significant for groups 2 and 3 (P<0.01) than it was for group 4 (P_<0.05).
Measurements of radioactivity in aliquots of bile samples showed
variations of 0.26% to 0.72% of the administered dose present in the bile
after 24 hr. There were no statistical differences in the percentages of dose
present in bile between treatment groups and controls.
Microsomal Benzo[a]pyrene Monooxygenase--
Microsomes, prepared from the 12 pooled liver samples from each group,
were assayed for benzo[a]pyrene monooxygenase activity. The low limit of
measurement of the monooxygenase activity in our assay system is 0.2 nmol of
products formed per mg of microsomal protein for a 20-min incubation (Gruger
et al., 1977a, 1977b). In all enzyme assays, the microsomes possessed the
same low activities of <0.2 nmol/mg; therefore, no effect on the monooxygenase
activity could be attributed to the xenobiotics fed to the fish.
67
-------
Discussion^
The experiments were designed to ascertain whether exposure of starry
flounder to naphthalene and/or p-cresol alters the in vivo metabolism of 2,6-
DMN. A detailed description of the metabolism of this alkylated naphthalene
in fish has not been reported previous to the present experiments (Gruger et
al., 1981).
The results demonstrate that these flatfish can oxidize 2,6-DMN to at
least four products: a naphthol, a quinone, a dihydrodiol and an alcohol.
The bile contained both nonconjugated and conjugated metabolites, with the
conjugated derivatives predominating in all four groups. The most important
feature of the data on the nonconjugated metabolites is the finding that fish
metabolize 2,6-DMN by either aromatic-ring or side-chain oxidation. Another
methylated polycyclic aromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene,
has been shown to be metabolized by rats by either aromatic-ring or side-chain
oxidation (Jellinck and Goudy, 1967).
In all four groups of fish (Table 29), the glucoside fraction of 2,6-DMN
metabolites constituted the largest class of identified conjugates (range =
34.7 to 42.5 mole-%). In a closely comparable experiment (Varanasi et al...
1979), it was found that in 24 hrs, starry flounder, which were force-fed H-
naphthalene, yielded sulfate/glucoside conjugates as 1.4% of total biliary
metabolites of naphthalene. Comparison of these two experiments indicated
that the presence of the alkyl substituents on the naphthalene nucleus leads
to increased proportions of biliary glucoside conjugates.
The data of Table 29, indicating that simultaneous exposure to
naphthalene and p-cresol decreased the proportion of 2,6-DMN metabolites
recovered as biliary glucuronides, suggest an interactive effect; i.e., the
two xenocompounds act together to elicit the change in composition of 2,6-DMN
metabolites. This conclusion, however, must be tempered by two facts. First,
the total molar xenobiotic dose in group 4 (0.18 ymole/fish) is twice that in
either group 2 or group 3 (0.09 ymole/fish). The effect observed in group 4
might have occurred if either naphthalene or p-cresol had been tested at a
dose of 0.18 pmole/fish; a more complicated experimental protocol would be
needed to resolve this question. The second fact which must be considered is
that the reduction in the glucuronide fraction of group 4 was accompanied by
an increase in the 2,6-DMN metabolites recovered by TLC as "unidentified
migrating fraction" (Table 29). Therefore, although there was a statistically
significant reduction (P<0.05) in the glucuronide fraction for group 4, the
nature of the metabolic changes is presently unknown.
The results (Table 30) support the conclusion that exposure to
naphthalene alone had a greater effect on the metabolism of 2,6-DMN than did
exposure to p-cresol alone. This conclusion is reasonable in view of the
physical and chemical properties of the two compounds. p-Cresol is both more
volatile and more soluble in water than is naphthalene. Thus, p-cresol can
probably be excreted more effectively. Furthermore, p-cresol could undergo
direct conjugation at its hydroxy group without the need for oxidative
metabolism; this may also favor rapid excretion by the fish.
68
-------
TABLE 29. 2,6-DIMETHYLNAPHTHALENE-4-14C METABOLITES IN BILE OF
STARRY FLOUNDER EXPOSED TO NAPHTHALENE AND P-CRESOL3
Exposure jjroup
Metabolite
fraction
2,6-Dimethyl-3-naphtho1b
2,6-Dimethy1-3,4-b
Controls Naphthalene
(Group 1) (Group 2)
- - — Mol-% in total me1
4.4 +_ 4.9 5.4 + 2.9
0.60+ 0.53 1.3 + 0.64
p-Cresol Naphthalene
and p-Cresol
(Group 3) (Group 4)
5.4 +_ 3.1 7.1 +_ 4.7
0.87+ 0.21 3.5 + 4.8
6-Methyl-2-naphthalene-
methanol 1.2+0.21 4.1+2.0 2.6+0.62 2.7+1.5
trans-3,4-Dihydroxy-
3,4-dihydro-2,6-
dimethylnaphthalene 1.9 + 0.87 5.1+_0.62 2.0 + 0.60 3.5 + 0.40
and sulfates
Glucosides
Glucuronides
Unidentified migrating
fractions
Unidentified, Rf= 0
Total
42.5 + 4.7
34.8 + 4.4
6.8 + 2.5
7.8 + 1.9
100
39.1 + 2.3
26.8 + 6.4
6.4 + 4.9
11.7 + 3.8
99.9
37.1 + 6.4
35.5 + 7.4
8.3 + 2.9
8.2 + 0.90
100
34.7 + 4.2
19.9 + 3.8C
19.0 + 2.4
9.5 + 1.9
99.9
a Biliary metabolites 24 hours after a force-fed dose of C-2,6-dimethyl-
naphthalene (12-15 mg/kg, 4,8 uCi/fish, 2.1 mCi/mmole). Mean values t S.D.
(n = 3); pooled samples from 4 fish; 12 fish/group.
Metabolites separated and identified by TLC System B (cf., Table 26).
c Significantly different (P _< 0.05) from control group.
69
-------
TABLE 30. METABOLITES OF 2,6-DIMETHYLNAPHTHALENE-4-14C FROM ENZYMATIC HYDROLYSIS OF THE
GLUCURONIDE FRACTION OF STARRY FLOUNDER BILE SAMPLES a
Group
Number Treatment
1 Control
2 Naphthalene
3 p-Cresol
0 4 Naphthalene and p-cresol
6-Methyl-2-naph-
thalenemethanol
31.4 + 4.0
10.4 + 2.5b
19,5 + 4.1b
8.25+ 2.1b
t^-3,4-Dihydroxy-3,4-
di hydro-2 , 6-dimethyl -
naphthalene
_ _ _ Mnlp °/ _______
3.43 + 0.43
16.4 + 3.9b
15.9 + 3.3b
11.7 + 1.8C
Ratio
6-Me-2-naphthalene
methanol
Dihydrodiol
9.15
0.63
L23
0.71
a Biliary metabolites 24 hours after a force-fed dose of 14C-2,6-DMN (4,8 uCi/fish, 2.1 mCi/mmole)
Mean values +_ S.D. (n = 3); pooled bile samples from 4 fish; 12 fish/group.
Significantly different (P _< 0.01) from control group.
c Significantly different (P <_ 0.05) from control group.
-------
The glucuronide fraction of 2,6-DMN metabolites from each group of fish
was subjected to further analysis (Table 30). Only two products resulted when
the glucuronides were hydrolyzed with 3-glucuronidase. One of these products,
the dihydrodiol, represents hydroxylation of the aromatic rings of 2,6-DMN;
the other, the alcohol, is produced by oxidation of one of the methyl
substituents. The most intriguing aspect of the data (Table 30) is the change
in the relative proportion of the dihydrodiol and the alcohol following
exposure to either xenobiotic or to the mixture of the two pollutants. In
each exposure group, there was a shift from methyl-group oxidation to
aromatic-ring oxidation (hydroxylation). This alteration was most pronounced
for the fish of group 2 (exposure to naphthalene only). An analogous result
has been reported in rats where it was demonstrated that exposure to
polycyclic aromatic hydrocarbons altered hepatic microsomal metabolism of
7,12-dimethylbenz(a)anthracene from side-chain oxidation to ring hydroxylation
(Jellinck and Goudy, 1967).
It is well established that the oxidation of naphthalene to dihydrodiols
is catalyzed by aryl hydrocarbon monooxygenases (Jerina et al., 1970); however,
far less is known about the enzymes responsible for oxidation of alkyl side
chains of methylated naphthalenes (Kaubisch et al., 1972). Thus, it is not
possible to interpret our results in terms of specific changes in the levels
or specificities of identified enzymes. Nevertheless, the principal finding
is clear: in starry flounder, previous exposure to an aromatic compound
alters the nature of the metabolism of 2,6-DMN i_n viv_o_.
Our demonstration that exposure to naphthalene results in an increased
proportion of dihydrodiol formation from 2,6-DMN could mean that exposure to
similar aromatic compounds may lead to a different formation of products, and
thus alter the potential toxicity of products, upon subsequent exposure to
other aromatic chemicals. This type of interactive effect of two (or more)
chemicals does not necessarily require the simultaneous presence of both (or
all) chemicals. The present results have shown that exposure of a demersal
fish to xenobiotics, especially repeated exposures to aromatic hydrocarbons,
may affect the subsequent metabolism and potential toxicity of aromatic
xenobiotics.
Summary
Juvenile starry flounder were force-fed naphthalene, p-cresol, or a
mixture of naphthalene and p-cresol in daily doses of 0.3-0.4 mg/kg body
weight, for six consecutive days. On the eighth day, each fish was force-fed
a dose of 12-15 mg/kg of 14C-2,6-dimethvlnaphthalene (2,6-DMN). Twenty-four
hours later, the fish were killed and ^C-labeled metabolites in the bile
were isolated and identified by TLC.
Most of the biliary metabolites were recovered as conjugates, principally
as glucosides and glucuronides. Analyses of the nonconjugated metabolites and
compounds resulting from enzymatic hydrolysis of the conjugate metabolites
provided identification of four metabolites of 2,6-DMN: 2,6-dimethyl-3-
naphthol; 2,6-dimethyl-3,4-naphthoquinone; trans-3,4-dihydroxy-3,4-dihydro-
2,6-dimethylnaphthalene (dihydrodiol); and 6-methyl-2-naphthalenemethanol
(alcohol). Enzymatic hydrolysis of the glucuronides yielded two compounds:
71
-------
the alcohol, representing metabolism at a methyl substituent, and the
dihydrodiol, representing oxidation of an aromatic ring. Exposure to
naphthalene and/or p-cresol led to a significant reduction (P<0.05) in
the proportion of the alcohol product and a corresponding increase in
the proportion of the dihydrodiol. This perturbation, which favors the
formation of potentially damaging epoxides, may alter the nature of
toxicological effects of such aromatic hydrocarbons.
EFFECTS OF NAPHTHALENE AND P-CRESOL ON IN VIVO SYNTHESIS OF LIPIDS
IN STARRY FLOUNDER
The purpose of the experiment was to study the effect of xenobiotics
on the in vivo lipid biosynthesis in starry flounder.
Method
Exposure Protocol --
The xenobiotics were administered by intraperitoneal injections.
Each fish of the control group was injected with the solvent (80%
aqueous ethanol; 1 ml/kg). Each fish of the naphthalene group received
5 mg/kg of naphthalene (5 mg/kg in 80% ethanol) per injection. Each fish
of the p-cresol group received 5 mg/kg of p-cresol (5 mg/kg in 80%
ethanol) per injection. Each fish of the fourth group received 5 mg/kg
of naphthalene and 5 mg/kg of p-cresol per injection.
Immature starry flounder (1 1/2-2 yr old) were used for this ex-
periment. There were 20 fish for each of the three xenobiotic-treatment
groups and 24 fish for the control group. On the first day all fish
were injected with the appropriate xenobiotic solution. Twenty-four hr
later the injections were repeated. Forty-eight hr after the initial
injection, four fish from the control group were sacrificed and samples
were taken for histological examinations. At the same time, four fish
from each group were sacrificed, and tissue samples were taken for
analyses of uptake of xenobiotics and activities of liver microsomal
2,6-DMN monooxygenase.
Four fish from each of the three treatment groups received labeled
xenobiotics [1- C-naphthalene and/or p-cresol( CHo), each with a
specific radioactivity of 10 yCi/mg]. Forty-eight hr after the initial
injection, four fish from each xenobiotic-treatment group were sacrificed,
and liver, gill, intestine, and muscle samples were taken. All of these
tissues were assayed for distribution of '^C. Liver homogenates were
prepared and the microsomes were assayed for 2,6-DMN monooxygenase
activity.
Forty-eight hr after the initial injections, each of the remaining
fish (16 per group; total of 64) was given a single intraperitoneal
injection of labeled lipid precursors [l-'^C-acetate, 59 mCi/mmole;
(3H-9,10)oleate, 6.4 Ci/mmole; both dissolved together in 0.9% aqueous
sodium chloride made pH 9.5 with KOH]. Four fish from each group were
72
-------
sacrificed at intervals of 1 , 2, 4, and 10 hr after administration of
the acetate and oleate. The fish were rapidly frozen on dry ice and
held frozen until samples could be taken and analyzed. Mean lengths
and weights of all 80 fish are presented in Table 31.
Analyses --
Monooxygenase activity of hepatic microsomes was assayed using
2,6-DMN as the substrate (Schnell et al . , 1980). Distribution of
ue to 1- C-naphthalene and/or p-cresol (
radioactivity due to 1- C-naphthalene and/or p-cresol (CHg) was
measured by digestion of tissues in 4 N NaOH and extraction into
n-hexane (Collier, 1978; Varanasi et al., 1978).
Liver lipids were extracted by the procedure of Hanson and Olley
(1963). The method of Hornstein et al.(1967) was used to separate
lipid classes. TLC separations of phospholipids were performed on
silica gel using chloroform:methanol rwater (65:25:4, v/v/v) according
to Wagner et al.(1961). Phosphorus content of the total phospholipids,
phosphatidylethanolamines, and phosphatidylcholines was determined by
the method of Bartlett (1959) as modified by Parker and Peterson (1965).
Weights of extracted total lipids, fractions of neutral lipids (with
free fatty acids), and phospholipids were determined by microgravimetry,
Results
Histological examinations of four control fish indicated no morpho-
logical abnormalities; thus, the starry flounder were healthy specimens.
Distributions of^C-labeled compounds in fish tissues following in-
jections with l-I4C-naphthalene and/or p-cresol( CH3) are presented in
Table 32. All tissues examined showed significant radioactivity
indicating that the xenobiotics were distributed throughout the bodies
of the fish. In the ^C-naphthalene group, the radioactivity in the
alkaline phase of the extracts exceeded that in the hexane layer; this
was seen in all four tissues assayed. These results indicate that ex-
tensive metabolism of the ^c_naphthalene had taken place.
Monooxygenase activities of liver microsomes toward 2,6-DMN are
presented in Table 33. These data indicate that the naphthalene-treated
group had significantly higher monooxygenase activity than did the con-
trol group. Enzyme activities for the p-cresol-treated group and the
group of fish which received both xenobiotics were not significantly
different from the controls.
Only the 4-hr and 10-hr liver samples were evaluated for lipid
content and lipid biosynthesis. Lipid contents of the livers are
summarized in Table 34; a range of 2.9 to 6.8 mg-% was found.
Data for the 4-hr incorporation of the lipid precursors into
neutral lipids and phospholipids are presented in Table 35. Neutral
lipids were more highly labeled than were phospholipids, whether the
precursor was tritiated oleate or ^C-labeled acetate. There were no
73
-------
TABLE 31. STARRY FLOUNDER USED FQR STUDY OF LIPIDS
Treatment group Sampling time
hr
1
2
Control 4
10
oa
1
2
Naphthalene 4
10
Oa
1
2
p-Cresol 4
10
Oa
1
Naphthalene 2
and p-cresol ^
10
Oa
Fish weight
g
92 + 25
89 i 81
126 + 58
87 + 54
130 ±. 58
114 + 61
148 + 43
88 + 33
92 + 40
122 +_ 30
82 + 74
112 + 69
74 + 38
92 + 35
120 +_ 12
62 +_ 13
135 1 84
88 +. 55
100 + 85
139 + 42
Fish length
mm
173 t 10
159 ± 41
184 + 29
166 ±. 26
230 ± 37
174 + 31
184 + 17
160 + 15
157 + 25
231 +_ 16
153 + 42
176 + 32
158 +_ 29
171 + 21
231 + 22
151 + 6
178 + 38
166 + 29
163 + 34
238 + 14
Used for enzyme assays and uptake analyses. Mean + S.D. (n = 4)
74
-------
TABLE 32. SOLVENT DISTRIBUTIONS OF CARBON-14 LABELED SUBSTANCES FROM TISSUES OF
STARRY FLOUNDER AT 24 HOURS AFTER FINAL INTRAPERITONEAL INJECTIONS OF
1-14C-NAPHTHALENE AND P-CRESOL(14CH3)
Treatment
group
Naphthalene
p-Cresol
Naphthalene
and p-cresol
Solvent
Hexane
Aqueous NaOH
Hexane
Aqueous NaOH
Hexane
Aqueous NaOH
Liver
3,210
7,990
310
27,090
6,350
19,870
Radioactivity
Gill
570
1,180
20
27,800
840
13,800
in tissues
Intestine
1,190
3,180
240
56,000
1,490
47,000
Muscle
240
630
100
3,630
540
2,930
Treatments were made by i.p. injections on each of two consecutive days of single doses of
naphthalene and/or p-cresol at 5 mg/kg body weight. Samples were taken at 24 hours after
the second injections. Specific activity of the injection fluids were 50 juCi/5mg/ml of each
compound in 80% ethanol.
Values are the averages for four fish. Distribution is between 4N-NaOH (after digestion)
and n-hexane, following extractions according to Varanasi et al. (1978).
-------
statistically significant differences among the four experimental
groups. The incorporation of tritiated oleate into sterol esters and
triglycerides is summarized in Table 36. Those data indicate a
significant decrease in incorporation of (3H-9,10)oleate into tri-
glycerides (at 4 hr) as a result of prior exposure of the fish to
p-cresol and/or naphthalene. Differences between the xenobiotic-
treatment groups and the control group were not statistically
significant in incorporation of 3H-oleate into sterol esters.
The incorporation of ^C-acetate into free fatty acids, trigly-
cerides, and sterol esters is presented in Table 37. Significant
decreases in acetate incorporation into fatty acids were seen due to
the prior treatment with naphthalene (P < 0.05) or with both naphthalene
and p-cresol (P < 0.01). Treatment with p-cresol alone did not produce
a statistically significant change in fatty acid synthesis from acetate.
Table 38 is a summary of the results obtained from incorporation
of both 3H-oleate and ^C-acetate into total phospholipids, phosphatidyl-
ethanolamines, and phosphatidylcholines. There was greater radio-
activity in all products at 4 hr than at 10 hr, but there were no
significant differences among the four groups of fish.
Discussion
The results presented for the distribution of 1-^C-naphthalene
and p-cresol(' CH3) indicate that there was significant uptake of
the xenobiotics during the course of this experiment. Also, it was
evident that ^C-naphthalene was extensively metabolized by the starry
flounder. The data of Table 33 demonstrates that the fish livers
possess highly active mixed-function oxidases capable of metabolizing
PAH's (Schnell et a!., 1980). The elevation of mixed-function oxidase
activity in the naphthalene-treated group compared to the control group
suggests that some induction of monooxygenase activity had taken place
during the 48-hr treatment with naphthalene. Thus, it is clear that
the xenobiotics had ample opportunity to effect metabolic changes in
the experimental animals.
Two significant findings resulted from the investigation of effects
of naphthalene and p-cresol on in vivo lipid biosynthesis. At 4 hr,
there was a demonstrable decrease in triglyceride synthesis from
3H-oleate due to treatment with naphthalene, p-cresol, or both organic
perturbants. Precisely how these chemicals exert this effect is un-
certain. A detailed study of the enzymatic steps involved might be
required to ascertain the location of this change in the overall pathways
of triglyceride biosynthesis.
The second notable finding is that naphthalene diminished the
biosynthesis of fatty acids from the 14C-acetate precursor. p-Cresol
treatment alone did not elicit the same effect. It would be interesting
to ascertain whether other PAH's could produce the same result as
76
-------
naphthalene. Naphthalene is certainly less polar than p-cresol and, thus,
may have greater access to the cellular organelles and processes which
are responsible for the control of lipid biosynthesis from a two-carbon
precursor of fatty acids.
Summary
Starry flounder exposed for 48 hours to naphthalene, p-cresol, or
a 50-50 mixture of the two compounds, by intraperitoneal injections,
were found to decrease the incorporation of tritiated oleic acid into
triglycerides of hepatic lipids. Only naphthalene was found to decrease
the incorporation of 1-^C-acetate into fatty acids. No significant
effects were demonstrated with phospholipid biosynthesis. Naphthalene-
treated flounder exhibited enhanced activity of hepatic microsomal
aryl hydrocarbon (2,6-dimethylnaphthalene) monooxygenase. The data
suggest that measurements of incorporation of fatty acids into trigly-
cerides in liver lipids may be a sensitive indicator of xenobiotic
exposures.
TABLE 33. ACTIVITY OF HEPATIC 2,6-DIMETHYLNAPHTHALENE MONOOXYGENASE
FOR STARRY FLOUNDER TREATED WITH NAPHTHALENE AND p-CRESOL BY INTRA-
PERITONEAL INJECTIONS, 24 HOURS AFTER TWO INJECTIONS 24 HOURS APART
Treatment Percent of
group Monooxygenase activity9 control
pmole/mg protein/min
Control 15.2 +_ 16.4 100
Naphthalene 50.7 _+ 28.6^ 334
p-Cresol 22.2 +_ 5.8 146
Naphthalene 26.7 ± 11.7 176
and p-cresol
a Hepatic microsomes analyzed according to the procedures of Schnell
et al. (1980).
b P _< 0.05 relative to control group.
77
-------
TABLE 34. LIPID CONTENT OF STARRY FLOUNDER LIVERS USED TO DETERMINE THE
INCORPORATION OF LIPID PRECURSORS AT 4-HOUR AND 10-HOUR INTERVALS
Treatment Time
group interval
hr
4
4
4
Control .
10
10
10
10
4
4
4
Naphthalene ^
10
10
10
10
Fish
body weight
g
191
60
100
155
165
54
84
46
58
119
99
75
114
69
50
137
Liver weight
Total Pooled
g g
0.796
1.428
0.632
0,462
1.712
1.250
1.250
1.449
0.199
0.784
1,075
0.291
0,325
1.344
1.019
0.987
1.609
0.622
0.697
1.254
0.557
0.336
1.306
0.970
Lipid
content
mg-%
4.28
6.75
6.46
5.98
5.01
5.06
4.84
4.87
78
(continued)
-------
TABLE 34 (continued)
Treatment
group
p-Cresol
Naphthalene
and p-cresol
Time
interval
hr
4
4
4
4
10
10
10
10
4
4
4
4
10
10
10
10
Fish
body weight
9
46
120
38
91
71
140
94
63
83
57
166
43
55
60
227
57
Liver weight
Total Pooled
g g
0.332
1,419
1.087
0.216
0.903
0.687
0.472
1.639
1.167
0.838
1.309
0.471
0.948
1.675
0.727
1.198
1.451
0.253
0.810
1.332
0.522
1.934
2.417
0.483
Lipid
content
mg-%
3.82
3.57
2.91
3.86
5.15
4.74
5.68
4.75
79
-------
TABLE 35. FOUR-HOUR INCORPORATION OF (3H-9,10)OLEATE AND 1-14C-ACETATE INTO LIPIDS OF
LIVERS FROM STARRY FLOUNDER TREATED WITH NAPHTHALENE AND P-CRESOL a
Specific activity in liver
Treatment group Neutral lipid fraction Phospholipid fraction
3H-labeled 14C-labeled 3H-labeled 14C-labeled
______________ picomoles/g liver ----------
Control 31.4+ 9.0 1,870 +_ 1,390 5,2 +_ 1.8 179 + 81
Naphthalene 25.8 +_ 11.1 2,080+ 686 4,8 +_ 1.6 204 + 22
oo
0 p-Cresol 29.0 +_ 5.4 1,760 +_ 212 6.3 ± 0.3 152 + 37
Naphthalene 17.0+ 8.1 1,340 +_ 584 3.9+1.7 118+11
and p-cresol
a Specific_activity of lipid precursors: ( H-9,10)oleate, 6.4 Ci/mmole;
1 4
1- C-acetate, 59.0 mCi/mmole.
Includes free fatty acids.
-------
CO
TABLE 36. INCORPORATION OF (3H-9,10)OLEATE INTO STEROL ESTERS AND TRIGLYCERIDES
OF LIVER LIPIDS FROM STARRY FLOUNDER TREATED WITH NAPHTHALENE AND P-CRESOL
Treatment
Control
Naphthalene
p-Cresol
Naphthalene
and p-cresol
Time of
incorpor-
ation
hr
4
10
4
10
4
10
4
10
Radioactivity
in lipids
Sterol esters
Dpm/mg total lipids Dpm/g li
192
51
61
98
154
136
70
52
+
+
+
+
+
+
+
+
95
28
27
3
28
60
41
21
9,776
3,130
3,050
4,759
5,700
4,398
3,540
2,617
+ 1
+_ 1
+ 1
±
+ 1
+ 1
+ 2
±
ver
,901
,568
,324
168
,316
,102
,244
769
Triglycerides
Dpm/mg total lipids Dpm/g liver
442
164
78
138
166
97
85
203
1 11
+_ 165
+ 36a
1 74
+ 30a
+ 22
+ 64a
+_ 40
24,300
9,944
3,892
6,716
6,186
3,224
4,290
10,716
+ 7,142
+ 9,685
+_ l,785b
+3,647
+ l,405b
+ 94
+ 3,392b
+ 3,375
P 1 0.01 compared to controls at 4 hr (n = 2).
P 5. 0-05 compared to controls at 4 hr (n = 2).
-------
TABLE 37. INCORPORATION OF 1-14C-ACETATE INTO FREE FATTY ACIDS, TRIGLYCERIDES, AND STEROL ESTERS
OF LIVER LIPIDS FROM STARRY FLOUNDER TREATED WITH NAPHTHALENE AND P-CRESOL
oo
no
Treatment
group
Control
Naphthalene
p-Cresol
Naphthalene
and p-cresol
Time o
incorp
ation
hr
4
10
4
10
4
10
4
10
„„ Radioactivity
or-
Free fatty acids
in 1
ipids
Triglycerides
Dpm/mg Dpm/g Dpm/mg
total lipids liver total lipids
1,383+130 75,150+16,990
445 + 358 27,080+20,770
677 + 157a 34,100+ 8,160a
635 + 48 30,820+ 2,475
1,100+156 40,800+7,700
948 + 49 31,930+ 4,707
288 +_ 70b 14,380+ 4,282b
426 + 36 22,120+_ 905
180
59
35
79
67
29
15
44
+ 34
+_ 63
± 3
+ 43
+ 66
+ 5
+ 16
+ 4
6
3
1
3
2
2
Dpm/g
liver
,260+3
,536+3
,768+
,843+2
,529+2
988+
784+
,305+
Sterol
Dpm/mg
total lipids
,779
,688
162
,077
,584
370
834
481
731 +
188 +
324 +
390 +
710 +
483 +
466 +_
274 +
153
176
271
77
199
204
446
96
esters
Dpm/g
liver
41,650+21
11,400+10
16,370+13
18,920+_ 3
26,080+ 6
17,040+10
23,700+23
14,610+ 6
,190
,320
,740
,825
,081
,140
,420
,828
a
b
P < 0.05 compared to controls
P ^0.01 compared to controls
at
at
4 hr (n
4 hr (n
= 2)
= 2)
•
-------
03
GO
TABLE 38. INCORPORATION OF (3H-9,10)OLEATE AND 1-14C-ACETATE INTO PHOSPHOLIPIDS OF LIVER LIPIDS
FROM STARRY FLOUNDER TREATED WITH NAPHTHALENE AND P-CRESOL a
Treatment Time of
incorporation
Control
Naphthalene
p-Cresol
Naphthalene
and p-cresol
hr
4
10
4
10
4
10
4
10
Radioactivity in phospholipi
Total
phospholipids
3H-dpm
M9
158 +
104 +
214 +
118 +
268 +
191 +
124 +
108 +
Pi
58
59
125
26
112
49
52
18
14C-dpm
M9
45 +
24 +
86 +
26 +
52 +
36 +
34 +
24 +_
Pi
27
17
20
1
29
2
4
1
Phosphatidyl-
ethanol amines
3H-dpm
ug Pi
130 + 16
54 +_ 15
104 + 8
51+6
142 +_ 78
74 + 62
66 + 23
56 + 19
14C-dpm
jug
27 +
10 +
33 +
8 +
23 +
11 +
9 +
10 +
Pi
4
6
7
0
23
10
3
2
ds b
Phosphatidyl-
cholines
3H-dpm
ug Pi
394 +_ 92
33 + 21
819 +_ 525
25 +_ 2
669 + 252
82 + 65
478 + 324
28 + 18
14C-dpm
|jg Pi
136 +
8 +
332 +
5 +
116 +
16 +
102 +
7 +
90
5
111
1
95
10
23
4
Total phospholipid (PL) fractions of liver lipids were separated by TLC into
phosphatidylethanolamines and phosphatidylcholines, as well as other components.
Duplicate TLC fractions were used for determining contents of phosphorus in
each phospholipid fraction.
-------
EFFECTS OF 2,6-DIMETHYLNAPHTHALENE AND P-CRESOL ON THE MORPHOLOGY
OF THE LIVER OF COHO SALMON
Methods
Coho salmon, each weighing an average of 30g(range of 25-34g),
were obtained from Domsea Farms, Scatter Creek, WA. They were acclimated
to laboratory holding tanks for two months. The stock supply of salmon
was fed Oregon Moist Pellet (Hublou, 1963) and was maintained in flowing
dechlorinated Seattle City water that slowly increased in temperature
from 16.5°C at the beginning of the experiment to 19.5°C at the termina-
tion. Initially, 120 salmon were anesthetized, weighed, measured, and
separated into 6 groups of 20 fish, none of which were fed. Three
times per week each group of salmon was anesthetized with MS-222(ethyl
m-aminobenzoate) and injected intraperitoneally with 0.05 ml of sockeye
salmon (£. nerka) oil containing one of the following contaminants:
0.30mg 2,6-dimethylnaphthalene (DMN), 0.60mg 2,6-DMN~, 0.30mg p-cresol,
O.SOmg p-cresol, and 0.30mg 2,6-DMN in combination with 0.30mg p-cresol.
The dose to each fish was either 10 or 20ppm(mg/kg) of contaminants.
A control group was injected with salmon oil in the same manner but
without any contaminant. Five fish from each group were sampled after
a 5-wk exposure, and the remaining salmon were sacrificed after an
additional 3-wk depuration period during which they received neither
injections nor food.
The sampling procedure was the same for all fish: they were
anesthetized, weighed, measured, killed by severing the spinal cord,
and examined to determine the sex and the general condition of the
external and internal organs. Portions of tissue from the tip of the
posterior lobe of the liver were excised and placed in a fixative
solution containing 0.7% glutaraldehyde, 3% formalin, and 0.5% acrolein
in sodium cacodylate buffer (0.1M sodium cacodylate, 0.02% CaCl2«H20,
5.5% sucrose pH7.4) (Hawkes, 1974). After a cacodylate buffer wash
the tissues were post-fixed in 1% osmium tetroxide in buffer.
Dehydrating with ethanol and embedding in Spurr's medium (Spurr, 1969)
completed the preparation of the tissue for sectioning with either
glass or diamond knives. Semi-thin sections (1.0 ym) were stained
with Richardson's stain for light microscopic examination. Thin sections
were successively stained with lead citrate, uranyl acetate and again
with lead citrate for electron microscopy. The stained sections were
coded such that the sources of samples were unknown to an independent
observer.
Results
After five week's exposure, fish in all groups including the
controls seemed normal in behavior and general appearance. The
average lengths and weights were the same in all groups at the end
of the experiment'as at the beginning and there were few mortalities.
84
-------
Liver Morphology of 5-Week Exposure Group--
The livers of five fish in each group were examined by both light
microscopy and transmission electron microscopy (Table 39; Figs. 22, 23).
Sections of livers from starved control fish and fed control (from the
stock supply) fish were compared and the liver morphology was normal in
both groups except that there were fewer glycogen deposits and very few
lipid vacuoles in the starved fish. Hepatocytes in all control salmon
had a well developed microvillar surface facing the sinusoids (Fig. 24),
and there were numerous polyribosomes and whorls of granular endoplasmic
reticulum in the cytoplasm. Also, within the cytoplasm, there were
numerous mitochondria, particularly in the portion of the cell
adjacent to the space of Disse, as well as numerous lysosomal vesicles
distributed throughout the hepatocytes. The nuclei typically occupied
a major proportion of the hepatocytes and appeared to have a normal
distribution of condensed chromatin (Fig. 25).
The liver morphology of many of the fish exposed to 10 ppm 2,6-DMN
for five weeks appeared normal; however, forty percent had some damage
to the endothelial cells and to the surface membranes of the hepatocytes
facing the sinusoids (Table 39; Figs. 26, 27). Cytoplasmic organelles
such as the mitochodria appeared normal but the cytoplasm in 40% of the
fish was vacoulated and cytosol coagulation was evident in all the fish.
In the group exposed to 20 ppm 2,6-DMN, all of the five fish examined had
severely damaged sinusoidal borders (Table 39; Figs. 28, 29) and the
endothelial cells were disrupted. In some cases, only fragments of the
cell surface membranes remained and the space of Disse was no longer
distinguishable. The cytosol of hepatocytes from this group of animals
appeared coagulated (Table 39). Eighty percent of the livers from this
group had clusters of necrotic cells with marginated chromatin in the
nuclei and an equal number were found with vacuolated cytoplasm.
Damage to the sinusoidal borders was noted in all of the fish
treated with 10 ppm p-cresol (Table 39; Figs. 30, 31): the endothelial
cells and membranes of the adjacent portions of the hepatocytes were
disrupted with consequent loss of the space of Disse. The cytoplasm of
the hepatocytes in this group was quite different from the cytoplasm of
either the 10 ppm or the 20 ppm 2,6-DMN treated groups. The configura-
tion of the endoplasmic reticulum near the damaged sinusoidal borders
was unusual: there were short stacks of granular endoplasmic reticulum
interspersed in the cytosol in 75% of the fish treated with 10 ppm
cresol and in 80% of the fish treated with 20 ppm cresol (Table 39).
All salmon in the group treated for five weeks with 20 ppm p-cresol
showed sinusoidal damage (Table 39; Figs. 32, 33). In many of these
fish, the sinusoids were nearly occluded, border membranes were in-
distinguishable and there was no evidence of the space of Disse. The
arrangement of the endoplasmic reticulum was similar to the 10 ppm
p-cresol group with short fragments of the membranes occurring through-
out the cells (Fig. 33), and there were vacuoles in the cytoplasm,
although the cytosol did not appear coagulated.
85
-------
TABLE 39. PERCENT COHO SALMON WITH MORPHOLOGICAL ABNORMALITIES IN LIVER TISSUE AFTER EXPOSURE TO
2,6-DIMETHYLNAPHTHALENE (DMN) AND p-CRESOLa
oo
en
Exposure
0
10 ppm 2, 6- DMN
20 ppm 2,6-DMN
10 ppm p-Cresol
20 ppm p-Cresol
10 ppm DMN + 10 ppm p-Cresol
Sinusoidal
border
damage
Ob
40
100
100
100
100
oc
100
100
100
80
100
Marginated
chromatin
Ob
20
80
20
40
40
0C
40
20
0
60
60
Vacuolated
chtoplasm
Ob
40
80
0
100
40
Oc
60
40
0
60
40
Coagulated
cytosol
Ob
60
100
40
20
100
Fragmented
Endoplasmic
Reticulum
Ob
0
0
80
80
100
Data based on five fish in each group
After 5-week exposure
c After 3-week depuration in addition to the previous 5-week exposure
-------
FIGURE 22. Hepatocytes from control coho salmon held for five weeks,
Sinusoidal space (arrows). 680X.
FIGURE 23. Hepatocytes from coho salmon exposed to 10 ppm 2,6-di-
methylnaphthalene plus 10 ppm p-cresol for five weeks, Sinusoidal
space (arrows); Vacuolated cytoplasm (*). 850X.
FIGURE 24, Hepatocytes from control coho salmon held for five weeks,
Sinusoidal border (arrow). 2,400X.
FIGURE 25. Hepatocytes from control coho salmon held for five weeks,
Space of Disse (D). 5,600X,
87
-------
r,,«
^S" *, ^ * & t^ rf *V 3*aw» >o? j ., 1 ^^^tfK^f yJs™ tiit "' x-^ "OiWr ifi^^» Hs^A P'
FIGURE 26. Hepatocytes from coho salmon after five weeks exposure to
10 ppm 2,6-dimethylnaphthalene. Sinusoidal border (arrow). 2,900X,
FIGURE 27. Hepatocytes from coho salmon after five weeks exposure to
10 ppm 2,6-dimethylnaphthalene. Sinusoidal border (arrow). 6,OOOX.
FIGURE 28. Hepatocytes from coho salmon exposed to 20 ppm 2,6-di-
methylnaphthalene for five weeks. Sinusoidal border (arrow);
Vacuolated cytoplasm (*). 3,OOOX.
FIGURE 29. Hepatocytes from coho salmon exposed to 20 ppm 2,6-di-
methylnaphthalene for five weeks. Sinusoidal border (arrow);
Vacuolated cytoplasm (*). 6,750X.
88
-------
•MMi \'-:
^Jfe^^A.^:*.
:
1 ^
FIGURES 30-33. Hepatocytes from coho salmon after five weeks exposure
to 10 ppm or 20 ppm p-cresol.
FIG. 30. Exposures to 10 ppm p-cresol. Sinusoidal border (arrow).
2,800X. FIG. 31. Exposure to 10 ppm p-cresol» Sinusoidal border
(arrow). 6,OOOX, FIG. 32. Exposure to 20 ppm p-cresol, Sinusoidal
border (arrow). 2,800X. FIG. 33. Exposure to 20 ppm p-cresol.
Sinusoidal border (arrow); granular endoplasmic reticulum (ger).
5,800X.
89
-------
Livers from all the salmon treated with 10 ppm 2,6-DMN in combina-
tion with 10 ppm p-cresol were found to have damaged sinusoidal borders
(Table 39; Figs. 34, 35); however, in some of the fish, portions of
the space of Disse could be distinguished. Many sinusoids were partial-
ly occluded. The cytoplasm of the hepatocytes in the combined group
underwent similar changes to those seen in the groups treated with the
single contaminants; the cytoplasm was coagulated and appeared similar
to the cytoplasm in the hepatocytes of the 2,6-DMN treated group. The
unusual arrays of short portions of the endoplasmic reticulum were
observed in the region of the hepatocytes adjacent to the sinusoids,
as was observed in the p-cresol treated groups.
Liver Morphology of 3-Week Depurated Groups--
Liver tissues from some groups appeared to have recovered slightly
and in others become slightly more necrotic (Table 39), but the types of
morphological changes in the liver tissue were the same as in the 5-wk-
exposed groups. The incidence of sinusoidal damage remained high
(80-100%) in all exposed groups, and remained 0 in the control.
Discussion
Disruption of the sinusoids of the liver of salmon exposed to
either p-cresol or 2,6-DMN or both contaminants included loss of the
endothelial cell lining of the sinusoids, loss of the microvillar
portion of the hepatocytes, and compression of the peri sinusoidal
spaces (spaces of Disse). In some tissue sections, the lumina of the
sinusoids were also compressed and appeared occluded. Similar sinusoidal
changes have been reported in the liver tissue of experimental animals
(primarily rats) and in humans that were chronically exposed to toxic
substances (Tanikawa, 1979). In experiments conducted to determine
the acute responses of rats to carbon tetrachloride, the same type of
sinusoidal damage occurred within 30-120 minutes after exposure (Motta
et al., 1978). This change, indicative .of ischemia in the liver, has
also been reported in the mummichog, Fundulus heteroclitus, chronically
exposed to naphthalene (DiMichele and Taylor, 1978).In many examples
of toxic injury to the liver, sinusoidal damage was the first pathologic
change observed and this was postulated to result in the restriction
of exchange of respiratory gases and nutrients, leading to hypoxia and
further damage to liver cells (Miyai, 1979). Necrotic changes within
the salmon hepatocytes, such as vacuolated cytoplasm and chromatin
margination may, therefore, not only be related to direct toxic effects
of the contaminants or their metabolites, but to a combination of
toxicity and hypoxia.
Microscopic evidence indicated that 2,6-DMN and p-cresol affected
the morphology of different cytoplasmic components of the liver cells.
The changes in the endoplasmic reticulum in the hepatocytes from the
p-cresol groups resembled that configuration reported in regenerating
liver cells, rapidly proliferating cells, and pre-neoplastic hepatocytes
(Miyai, 1979), but those changes were not observed in the liver cells of
salmon treated with 2,6-DMN. Salmon from groups exposed simultaneously
90
-------
''•^-:- -
l ••' v
FIGURE 34. Hepatocytes from coho salmon exposed to 10 ppm 2,6-di-
methylnaphthalene and 10 ppm p-cresol for five weeks. Sinusoidal
border (arrow). 6,600X.
91
-------
FIGURE 35. Hepatocytes from coho salmon exposed to 10 ppm of
2,6-dimethylnaphthalene and 10 ppm of p-cresol for five weeks.
Sinusoidal border (arrow); Vacuolated cytoplasm (*). 5,600X.
92
-------
to both contaminants seemed to have a combination of cytopathology
reflecting both contaminants. The sinusoidal damage was similar to
that of the groups exposed to the highest doses of single contaminants
and was also similar to numerous cases of nonspecific toxic injury of
the liver (Miyai, 1979). The hepatocellular changes, however, appeared
to be a combination of cytosol coagulation, similar to that observed
in the 2,6-DMN treated groups, and fragmentation of the endoplasmic
reticulum, as in the p-cresol treated groups. This pattern of sub-
cellular changes suggests that the contaminants interact with different
cytoplasmic components.
Summary
We have demonstrated similar morphological changes in liver
sinusoids of coho salmon exposed to p-cresol or 2,6-DMN. In addition,
there are suggestions of specific types of hepatocellular changes
distinctive to each contaminant. When the contaminants were administered
in combination, cytopathological changes reflective of both contaminants
were observed. Therefore, the contaminants appeared to affect the
same hepatocytic organelles whether the exposure was to the individual
or combined compounds. Although we have no direct measure of liver
impairment as a result of the contaminant exposures, the morphological
changes, particularly those involving the sinusoids, were similar to
hepatic changes seen in other organisms with functionally detrimental
conditions such as hepatitis, toxic injury, and hypoxia (Tanikawa,
1979).
93
-------
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