Ecological Research Series
BENZO(a)PYRENE METABOLISM IN THE
AMERICA OYSTER CRASSOSTREA VIRGINICA
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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BENZO[a]PYRENE METABOLISM IN THE
AMERICAN OYSTER CRASSOSTREA VIRGINICA
by
Robert S. Anderson
Sloan-Kettering Institute for Cancer Research
Donald S. Walker Laboratory
Rye, New York 10580
Grant No. R804435
Project Officer
John A. Couch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory—
Gulf Breeze, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the intro-
duction of these compounds into the environment be formulated on a sound
scientific basis. Accurate information describing dose-response relation-
ships for organisms and ecosystems under varying conditions is required. The
Environmental Research Laboratory, Gulf Breeze, contributes to this informa-
tion through research programs aimed at determining:
' the effects of toxic organic pollutants on individual species
and communities of organisms;
' the effects of toxic organics on ecosystem processes and components;
' the significance of chemical carcinogens in the estuarine and
marine environments.
The present report deals with a subject of great potential significance
in the area of early detection of carcinogens in the environment. The action
of carcinogens on enzyme systems of aquatic species should provide the basis
for sensitive early warning detection tests. Further, the susceptibility of
aquatic species to carcinogens or mutagens may depend on the ability of these
species to metabolize xenobiotics to proximal or active intermediate
compounds. This report provides data on the metabolic responses of oysters
to known carcinogens and mutagens.
Thomas W. Duke
Director
Environmental Research Laboratory -
Gulf Breeze
ill
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ABSTRACT
This research program was initiated with the overall objective of deter-
mining the role of NADPH-dependent microsomal mono-oxygenase in the metabo-
lism of the widespread environmental carcinogen benzo[a]pyrene (BP) by the
oyster Crassostrea virginica. This enzyme system is important in detoxifying
various xenobiotics and in activating polycyclic aromatic hydrocarbon onco-
gens such as BP.
A sensitive radioisotopic system was developed to permit the quantifi-
cation of alkali-soluble and water-soluble BP metabolites produced by oyster
mono-oxygenase. An NADPH- and Oa-dependent aryl hydrocarbon hydroxylase
(AHH) was shown to be located in the digestive glands of these bivalves
associated with the microsomal subcellular fraction. The specific activity
of oyster AHH was considerably lower than that of laboratory mice, but was
consistently demonstrable. The BP metabolites produced were primarily water-
soluble derivatives.
There was some indication that oyster AHH was induced by chronic ex-
posure of the animals to the environmental carcinogens BP and 3-methyl-
cholanthrene. There was strong evidence that exposure to polychlorinated
biphenyls (PCB) caused AHH induction.
High-pressure liquid chromatography was used to identify BP metabolites
produced by oyster AHH. The generation of various dihydrodiol, quinone, and
hydroxy BP derivatives was shown; this production was augmented in PCB-
exposed oysters. Quinones were the major metabolites in normal oysters;
the production of hydroxy derivatives was particularly stimulated by PCB
induction. There is no evidence as yet for production of the suspected
ultimate carcinogenic BP metabolite (7,8 diol-9,10-epoxide); the 7,8-diol
and the mutagenic 4-5 oxide derivatives are present in the oyster. However,
identification of all the metabolites is not known.
This report was submitted in fulfillment of Grant No. R804435 by the
Sloan-Kettering Institute for Cancer Research under the partial sponsorship
of the U. S. Environmental Protection Agency. This report covers the period
from July 1, 1976 to June 30, 1977, and work was completed as of August 31,
1977.
xv
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CONTENTS
Foreword iii
Abstract iv
1. Introduction 1
2. Conclusions and Recommendations 2
3. Experimental Procedures 4
General procedures 4
Enzyme preparation „ 4
Enzyme assay 5
4. Results 6
Optimal assay conditions 6
Baseline AHH activity in oysters 7
Time-course of BP metabolism 7
Induction of oyster AHH by carcinogens ... 8
Induction of oyster AHH by PCB's ........... 9
Resolution of oyster BP metabolites by high-pressure
liquid chromatography .... 9
5. Discussion 12
Methodological considerations 12
The fate of PAH in bivalves 13
Comparison of AHH activity in oysters and other animals 13
Tentative identification of BP metabolites produced by
oysters 14
References 16
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SECTION 1
INTRODUCTION
As a result of industrialization, a wide variety of xenobiotics is
constantly introduced into the environment. One of the most ubiquitous of
these pollutants is the carcinogenic polycyclic aromatic hydrocarbon benzo-
[ajpyrene (BP). It is thought that certain BP oxide metabolites are
responsible for its mutagenicity and carcinogenicity. These BP derivatives
are produced by aryl hydrocarbon hydroxylase (AHH), which is associated with
the smooth endoplasmic reticulum of hepatic cells in mammals and digestive
gland cells in invertebrates.
Filter-feeding mollusks are known to concentrate BP and other hydro-
carbon pollutants of their aquatic environment. A considerable portion of
these hydrocarbons can be eliminated by depuration; however, the question of
BP metabolism by mollusks has not been thoroughly studied. It is important
to assess the capacity of marine mollusks to metabolize BP as a step in our
understanding of invertebrate chemical carcinogenesis; also, the presence
of potentially carcinogenic metabolites in shellfish may have public health
implications. We have developed a sensitive in vitro method to quantify the
production of radiolabeled BP derivatives by oyster AHH. The results of
these studies are included in this report.
We present evidence that oyster AHH is inducible by commercial poly-
chlorinated biphenyl mixtures (PCB's), which represent an important class of
environmental contaminants. If future work proves that bivalve AHH activity
levels are directly proportional to the levels of pollutants to which the
animals are exposed, it might be possible to develop a sensitive bio-
indicator system for monitoring environmental pollution.
Aryl hydrocarbon hydroxylase catalyzes the production of a variety of
biologically active and inactive BP metabolites. It is essential to identify
the BP derivatives actually generated under particular conditions in order to
speculate on carcinogenicity. In the present study, high-pressure liquid
chromatography was used to resolve and tentatively identify the BP
metabolites generated by AHH from normal and PCB-exposed oysters.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
Technical difficulties have been resolved for development of an assay
system to measure aryl hydrocarbon hydroxylase (AHH), using the carcinogenic
substrate benzo[a]pyrene (BP) in the oyster Grassestrea virginica. It is
possible to show a low, but consistent, level of AHH activity in oysters.
This activity is primarily reflected in the production of water-soluble
metabolites, although other more nonpolar (NaOH-soluble) metabolites are
also produced. The enzyme is 02- and NADPH-dependent, and greatest specific
activity is associated with the microsomal subcellular fraction of digestive
gland homogenates. Whereas some information concerning cofactor requirements,
temperature and pH optima, and enzyme kinetics is now known, some very basic
facts need to be determined. For example, although cytochromes play a major
role in AHH reactions in higher animals, their involvement in oyster BP
metabolism has not been studied. The comparatively high percentage of water-
soluble BP metabolites produced suggests that conjugation of metabolites to
sulfate, glucuronic acid, etc. is an important pathway of BP metabolism in
oysters; sulfotransferase and other conjugating enzymes have not as yet been
studied.
There is strong evidence that oyster AHH is readily inducible by poly-
chlorinated biphenyls (PCB's); PCB's are quite stable and are major environ-
mental pollutants in many geographical areas. This observation should be
followed up; it is important to know such parameters of the inductive
response as dose-dependency, duration, and influence degree of chlorination
and isomeric composition of the various commercial PCB mixtures. If indeed
AHH activity can be shown to be directly proportional to pollutant concen-
trations in the aqueous environment, it might be possible to use its level
of activity in shellfish as a sensitive bioindicator of pollution. Labora-
tory studies should be coordinated with field studies of AHH levels in
mollusks collected from waters of varying, known concentrations of con-
tamination.
The ability of oysters to metabolize BP has now been established. Such
oncogen metabolism is thought to be required to produce active derivatives
that are able to react covalently with biological macromolecules such as
DNA. This alkylation reaction Is probably responsible for mutagenicity and
carcinogenicity of BP. Both active and inactive (detoxified) BP metabolites
are produced; therefore, it is necessary to identify the specific metabolites
generated by oyster AHH. We have separated six major BP metabolites produced
by the oyster, using high-pressure liquid chromatography. These include
dihydrodiols, quinones, and hydroxylated derivatives. Most of these
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metabolites are probably poor carcinogens, based on their lack of muta-
genicity for bacteria and mammalian cells. However, the 7,8-dihydrodiol may
be present; this is the precursor for the suspected ultimate carcinogen, whirh
is the 9-10 epoxide of this compound. Also, there is evidence for the
presence of the 4,5-oxide metabolite which is moderately mutagenic in several
assay systems. It is very important to establish definitively the identity
of each of the BP metabolites produced by oyster AHH; an indication of this
can be obtained by coelution studies using known metabolite standards.
Those metabolites not commercially available can be obtained through the
generosity of our collaborators. Furthermore, these metabolites should be
tested for mutagenicity with the Ames bacterial system.
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SECTION 3
EXPERIMENTAL PROCEDURES
GENERAL PROCEDURES
The presence of aryl hydrocarbon hydroxylase (AHH) in oyster tissues was
determined in vitro by measuring the formation of labeled derivatives of 7,
10- 4C-benzo[a]pyrene (BP) during incubation with homogenates of microsomes.
Mixed-function oxidases, including AHH, require the cofactor NADPH (nicotin-
amide adenine dinucleotide phosphate, reduced form) and oxygen. Oyster AHH
also conforms to these requirements; however, it was soon shown that co-
factor concentrations optimal for mammalian systems were inappropriate for
the study of molluscan enzymes. For example, the NADPH concentration recom-
mended by Nebert and Gelboin (1) is inhibitory for oyster AHH but excellent
for use with preparations from laboratory rodents.
The reaction is stopped after incubation of enzyme and substrate under
appropriate conditions, and the reaction mixture is extracted by using a
modification of the method of Abramson and Hutton (2). The parent compound
and a number of metabolites are extracted in hexane; the metabolites are
separated from BP by additional extraction with NaOH. Other metabolites re-
maining in the aqueous phase include water-soluble derivatives and metabo-
lites that have been conjugated with glucuronic acid and/or sulfate. Both
NaOH-soluble and water-soluble metabolites are quantified by using a liquid
scintillation spectrometer.
ENZYME PREPARATION
Fresh oysters (Crassostrea virginica) were opened, and the visceral mass
excised and immediately placed on ice. The digestive gland was dissected as
free as possible from surrounding tissues and a 25% by weight homogenate
prepared in ice-cold 0.839 M sucrose. Homogenates were first centrifuged at
10,886 g Zor 10 min in a Sorvall RC2-B automatic refrigerated centrifuge
using an SS-34 rotor. Homogenate supernatants were recentrifuged at 10,886 g.
The resulting supernatants were ultracentrifuged at 100,000 g in a Beckman
Model L preparative ultracentrifuge using a 40 rotor. The microsomal pellets
were resuspended in 0.839 M sucrose at 1°C. All digestive gland homogenates
and/or microsomes were held in an ice bath until the enzyme assays were per-
formed; this period never exceeded 30 min. An aliquot of each homogenate or
microsomal preparation was frozen for subsequent protein determination by the
method of Lowry et al. (3).
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ENZYME ASSAY
The incubation conditions described here were found to be optimal for
the demonstration of AHH activity in oyster digestive gland. Because no pro-
cedure for measuring AHH activity in bivalves exists in the literature, con-
siderable effort was expended in determining the optimal cofactor concen-
trations, time, temperature, buffer system, etc.
One milliliter reaction mixtures were prepared in 10-ml Erlenmeyer flasks
and held on ice until the incubation stage. The final concentrations of the
components of the reaction mixture were 0.1 mM NADPH (reduced nicotinamide
adenine dinucleotide phosphate), 5 mM MgSO^, and 1.6 mg/ml albumin (bovine
serum albumin, fraction V) in 0.4 M HEPES (N-2-hydroxyethylpiperazine-N'-2-
ethane sulfonic acid) buffer, pH 7.8. These compounds were purchased from
the Sigma Chemical Company, St. Louis, Missouri. Digestive gland homogenate
(0.3 ml) or microsomes (0.05 ml) then were added to each flask, and the re-
action was initiated by the addition of 80 nmoles (0.1 yCi) 7, lO-^C-benzo-
[a]pyrene, purchased from Amersham/Searle Corporation, Arlington Heights,
Illinois. The desired amount of 14C-BP was introduced into the reaction
mixture dissolved in 0.025 ml acetone; this amount of acetone had no effect
on AHH activity.
Experimental flasks were incubated 60 min at 30°C in an Eberbach shaking
water bath; the reaction was stopped by the addition of 1 ml cold acetone.
Reactions in control (TQ) flasks were stopped immediately after the addition
of lt+C-BP. Once the reactions were terminated, both T60 and TQ samples were
subjected to the same methodological protocols. Reaction mixtures were ex-
tracted with hexane 15 min at 30°C in a shaking water bath. A 1-ml aliquot
of the hexane layer was removed and vigorously mixed with an equal volume of
IN NaOH; a 0.5 ml sample of the NaOH extract was placed in a scintillation
vial. Additional NaOH extractions of the hexane layer showed that all of the
NaOH-soluble metabolites had been removed in the initial extract. The aqueous
layer was further extracted with 2 ml of hexane to assure that all the labeled
parent compound had been eliminated. A 0.5 ml aliquot of the aqueous layer
was pipetted into a scintillation vial; the protein in this sample was solu-
bilized by overnight incubation at room temperature in 0.25 ml Protosol (New
England Nuclear, Boston, Massachusetts).
The radioactivity in the NaOH and aqueous aliquots was quantified by
using a Packard Model 3255 liquid scintillation spectrometer. Appropriate
control (T0) values were subtracted from experimental (T6Q) results. The
total amount of radioactivity, representing total metabolites produced, in
the NaOH and aqueous layers from a given sample was calculated, taking into
account quenching, machine efficiency, partitioning, and dilution. The nmoles
of lltC-BP metabolized during the course of the experiment were determined
based on the specific activity of the parent compound. The activity of benzo-
[ajpyrene hydroxylase was expressed as nmoles 14C-BP metabolized/mg protein/
60 min.
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SECTION 4
RESULTS
OPTIMAL ASSAY CONDITIONS
The experimental conditions given in the procedure for AHH assay were
determined by measuring the influence of several factors on the enzymatic
reaction in vitro. Some of those studies are summarized below.
Cofactor Concentration
The cofactor NADPH is required for AHH activity. In this system the
presence of 0.1 mM NADPH usually enhanced AHH activity; however, it should be
noted that considerable activity was measured in homogenates in the absence
of added NADPH. This presumably was due to the natural presence of the co-
factor in homogenate. NADPH requirement was most easily shown with washed
microsome preparations. The addition of NADPH in excess of 0.5 mM severely
inhibited the metabolism of BP by oyster AHH. Various concentrations of NADH
(nicotinamide adenine dinucleotide, reduced form) failed to alter the rate of
reaction; it would appear that NADPH is the preferred electron donor. The
addition of an NADPH-generating system composed of NADP, glucose-6-phosphate,
and glucose-6-phosphate dihydrogenase was shown not to enhance AHH activity.
Results from a series of experiments using various concentrations of each of
these components indicated that the generating system was not useful in this
system.
Temperature
The incubation temperature selected for these studies was 30°C. This
temperature, while higher than physiological for these poikilothermic
animals, was shown to favor the activity of BP hydroxylase. However, it was
noted that considerable enzymatic activity could be demonstrated at 15°C and
22°C. A temperature of 30°C is reached in the estuaries of the Gulf Coast
during July and August. The data indicate that the enzyme is heat-stable at
temperatures in excess of those likely to exist in vivo. In a typical series
of four determinations at each temperature, the following mean AHH-specific
activities ± SD (standard deviation) were found: 0°, 0.063 ± 0.008; 15°,
0.301 ± 0.21; 22°, 0.348 ± 0.030; 30°, 0.422 ± 0.052; 37°, 0.219 ± 0.056.
Little BP hydroxylase activity was measured at temperatures above 60°C.
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Oxygen Requirement
Oxygen dependency of oyster AHH was shown by N2 inhibition of enzyme
activity. Nitrogen was bubbled through the reaction mixture for 3 min prior
to the addition of ^C-BP. Subsequently, the reaction vials were gassed with
N?, capped, and incubated following the standard protocol. Untreated samples
were run simultaneously as controls. Benzo[a]pyrene metabolism was inhibited
36.3 ± 12.5% (n = 4) in the N2-treated aliquots as compared to identical un-
treated homogenates. It is probable that some 02 remained dissolved in the
medium after gassing with N2, permitting the reaction to proceed at a reduced
rate.
Dithiothreitol
It has been shown that maximal mammalian AHH activity is obtained when
DTT (DL-dithiothreitol) is present in medium (2). This reagent is useful for
maintaining -SH groups in the reduced state (4). Accordingly, we assayed the
effect of 1-10 ymoles/ml DTT on Crassostrea BP hydroxylase activity. The
procedure resulted in slight, unpredictable fluctuations in activity; there-
fore, we abandoned its use in these studies.
BASELINE AHH ACTIVITY IN OYSTERS
The activity of benzo[a]pyrene mono-oxygenase in normal Crassostrea
virginica digestive gland homogenate, as measured by the assay described
above, was 0.465 ± 0.128 nmoles/mg protein/60 min (n = 20). This represents
the sum of the NaOH-soluble BP metabolites (0.041 ± 0.039) and the water-
soluble BP metabolites (0.424 ± 0.104). The removal of the nuclei and cell
debris from the homogenate by low-speed centrifugation (11,000 g) only
slightly reduced total AHH activity to 0.436 ± 0.082 (n = 5). It was shown
by further centrifugation (100,000 g) that the activity was primarily
associated with the microsomal fraction, the AHH specific activity of which
was at least twice that of the whole digestive gland homogenate.
TIME-COURSE OF BP METABOLISM
After an initial burst of oyster AHH activity, there is a steady pro-
duction of BP metabolites for at least 60 min in this system. The rate of
reaction is constant during this period (Figure 1); therefore, 60 min was
selected for the length of incubation. The T6o sample contains comparatively
high amounts of BP metabolites, and 60 min is on the linear portion of the
curve. The data are best represented by a straight line because the corre-
lation coefficient equals 0.992.
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100
E
o
T> *
Si
si
f -g 60
:r o
D O>
Ji
e
0- ^
CD -D
D
5
_ 20
o
o
20 40
Time in Minutes
60
Figure 1. The production of BP metabolites by oyster homogenate during 60
min incubation. Values are expressed as percentage of total metabolites gen-
erated in 60 min; mean values of two experiments are indicated. Line was
produced by calculating least-squares linear regression.
INDUCTION OF OYSTER AHH BY CARCINOGENS
As a part of a study of the oyster as an indicator of environmental
carcinogens (5), exposed oysters were periodically sent to this laboratory
for AHH analysis. These animals had been exposed to 1.0 pg/1 BP, or 3-
methylcholanthrene (MC), or a combination of both carcinogens. The usual
exposure regimen was 8 hr/day, 5 days/week. Representative carcinogen-
exposed and control oysters were periodically quick-frozen and shipped in dry
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ice for AHH assay. All data reported in this section are the means of AHH
activity in two animals from each group.
After 5 months of exposure, the following AHH activity was measured in
digestive gland homogenates: BP-exposed = 0.183 nmoles BP products/mg/10 min;
MC-exposed = 0.206; unexposed control animals had no detectable activity.
These data suggest possible AHH induction by the carcinogens. The compara-
tively low apparent enzyme specific activity may be explained by the short
incubation period (thought to be optimal at the time of these assays) and by
the fact that some AHH activity is lost during freezing and thawing. After
9 months of exposure, the following AHH activity was measured: BP-exposed =
0.107; MC-exposed = 0.081; BP + MC-exposed = 0.121; unexposed control = 0.035
nmoles BP products/mg/10 min. After 13 months of exposure to these carcino-
gens, significant levels of AHH were detected in all exposed and control
animals; however, enzyme induction was not seen in either BP- or MC-exposed
animals. Induction in the BP + MC-exposed oysters was minimal and probably
not significant.
INDUCTION OF OYSTER AHH BY PCB's
Commercial mixtures of polychlorinated biphenyls (PCB's) were kindly
supplied by the Monsanto Company, St. Louis, Missouri. Those used in this
study included Capacitor 21, Aroclor 1016, and Aroclor 1242, which contain
21, 41, and 42% chlorine by weight, respectively. These PCB's were applied
topically by introducing 0.3-0.5 ml directly into the mantle cavity of the
oysters. The animals were maintained at 5 C in a moist chamber for the
duration of these experiments. Oysters kept under these conditions are
viable and none of the administered PCB dose can be lost by exposure to
water.
Preliminary results indicate that oyster AHH, like mammalian MFC, is
inducible by PCB's. Administration of Aroclor 1242 produced an approximate
2-fold (2.29 ± 0.6, n = 5) increase in AHH activity by 48 hr. Similar levels
of induction were produced by Capacitor 21 (218% increase in activity, n = 4)
and Aroclor 1016 (304% increase, n = 4).
RESOLUTION OF OYSTER BP METABOLITES BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY
Recently, a technique for efficient separation of BP metabolite* using
high-pressure liquid chromatography (HPLC) has been developed by Selkirk
et al. (6). In collaboration with Dr. George B. Brown of this Institute, we
applied this method to separate BP metabolites produced by oyster AHH.
Flasks containing 1 ml reaction mixtures, each containing 0.3 ml oyster
digestive gland homogenate, albumin, NADPH, etc. (as described above), were
prepared. The reaction was initiated by the addition of 80.5 nmoles (0.625
yCi) 3H-BP; the use of higher specific activity BP permitted more accurate
quantification of metabolites. Each run consisted of 10 To and 10 T6Q
flasks. At the appropriate time the reactions were terminated by the
addition of 1 ml cold acetone, and each flask was extracted with 2 ml ethyl
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acetate. The ethyl acetate layers from the T0 flasks were pooled with each
other; the ethyl acetate layers from the T60 flasks were also combined in a
separate pool. Anhydrous MgSO^ (1.0 g) was added to each pooled extract and
filtered out, after vigorous mixing. The ethyl acetate samples were dried
under N2 and redissolved in 0.2 ml absolute methanol. The samples were in-
jected into a 2.1 x 1000 mm permaphase (1-m ODS) column. The column was
eluted with a reverse phase gradient system with methanol and water (30:70
initially). The gradient rate of change was 3%/min; column temperature was
45°C; the pressure was about 600 pounds/in2; flow rate was 0.6 ml/min. Ultra-
violet absorption was continuously monitored, and samples from the fraction
collector were assayed for radioactivity by liquid scintillation spectrometry.
Peaks corresponding to the major classes of BP metabolites produced by
mammalian AHH were also produced by oyster BP hydroxylase. The identifi-
cation of metabolites was made by comparing Rfs of unknown peaks with those
of known standards. This information is summarized in Table 1.
TABLE 1. BP METABOLITES PRODUCED BY OYSTER AHH, RESOLUTION BY HPLC
Peak number* Rf^ Probable identity
1 0.059
2 0.175
3 0.500
4 0.668
5 0.794
6 1.000
± 0.015
± 0.013
± 0.028
± 0.034
± 0.022
9,10-dihydrodiol
4,5-dihydrodiol (or 7, 8-dihydrodiol)
1,6-quinone (or 3,6-quinone)
3-hydroxybenzo [a] pyrene
Unknown monohydroxylated derivative
Benzo [a]pyrene
Peaks common to all oyster preparations analyzed
Mean Rf ± SD, n = 18
The exact identification of many of these metabolites awaits coelution
experiments with known standards. However, as indicated, these radiolabeled
metabolites are consistently seen in all oyster preparations after incubation
with 3H-BP- Furthermore, the peaks are distinct and do not overlap. Al-
though there is some uncertainty concerning their precise identity, it may be
said that peaks 1 and 2 represent dihydrodiols; peak 3 represents quinones,
and peaks 4 and 5 are hydroxylated BP metabolites.
The percentage increase observed in each of these metabolites was
measured by comparing T60 with TQ results. Whereas the actual percentages of
10
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of increase varied considerably, there was general production of all 5 major
metabolites with time, as shown in Table 2. In control oysters, the quinones
were the major metabolites produced, followed by the dihydrodiols and the
hydroxylated products. The metabolite profiles in control oysters were com-
pared to those in oysters 24 hr after exposure to Aroclor 1242, according to
the protocol described above. In AHH-induced oysters, again the quinones
were the major metabolites, followed by the hydroxy- and dihydrodiol metabo-
lites. Aroclor treatment produced AHH induction as evidenced by an increased
production of BP metabolites. After 60-min incubation of oyster AHH and 3H-
BP, metabolites accounted for 1.196% of the total radioactivity present in
ethyl acetate extracts. This percentage increased to 2.205% in the case of
Aroclor-exposed oysters. It appeared that the production of hydroxy deriv-
atives and quinones is more pronounced in PCB-induced oysters. It must be
remembered that this procedure measures only those BP metabolites that are
soluble in organic solvents.
TABLE 2. PERCENT TOTAL CPM's IN BP METABOLITES FROM CONTROL
AND AROCLOR 1242-INDUCED OYSTERS
Peak number Control Induced
1 0.198 ± 0.205 0.264 ± 0.045
2 0.266 ± 0.299 0.125 ± 0.093
3 0.520 ± 0.602 1.095 ± 0.417
4 0.098 ± 0.097 .318 ± 0.635
5 0.114 ± 0.163 .403 ± 0.488
6 98.804 97.795
11
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SECTION 5
DISCUSSION
Microsomal MFO activity has been described in many vertebrates and in
various lower animals. This enzyme system represents a major pathway for
detoxification of a host of xenobiotic chemicals. This paper presents, for
the first time, evidence for this enzyme system in a marine mollusk
Crasspstrea virginica. Aryl hydrocarbon hydroxylase activity was measured in
the oyster by a sensitive radioisotopic technique, as well as by high-pressure
liquid chromatography. Since other investigators have not demonstrated the
presence of AHH in mollusks, it is important to explain these findings by
comparing the methodologies used in the various assays.
METHODOLOGICAL CONSIDERATIONS
Most laboratories report the metabolism of BP in terms of fluorescent
equivalents, in which the results are compared to the fluorescence of 3-OH-BP
which is described as the major metabolite. This assay tells nothing of the
total number or the concentration of the metabolites produced; it only shows
that one or more of the metabolites formed have a certain fluorescence. This
method is not as sensitive as the method described in this paper, in which
the production of both organic solvent-soluble and water-soluble ^C-BP
metabolites is quantified. Since nothing was known about the BP metabolite
profile in oysters, the usefulness of a method which depended heavily upon
the production of 3-OH-BP was considered questionable.
A major difficulty in demonstrating oyster AHH resulted from the use of
an excellent assay system developed for mammalian cells (1) for similar
assays in bivalves (7). It would be unlikely that the ideal medium compo-
sition for mammalian cells would be useful in work with oysters. Indeed,
this work shows that oyster AHH is effectively inhibited by NADPH concen-
trations commonly used in mammalian MFO studies. As already mentioned, it
was necessary to modify the salt composition, buffer system, and the strength
of the buffer to optimize conditions for oyster AHH.
Several other reasons can be suggested to explain the apparent lack of
AHH in bivalves reported in the literature. The use of whole animal homoge-
nates makes detection of AHH activity unlikely. In oysters, AHH activity is
high only in the digestive gland, as in the case of mammals where activity
is predominantly found in the liver. In whole animal homogenates, digestive
gland tissue is diluted with all the other components of the organism. In
such preparations AHH is exposed to proteolytic digestive enzymes, which
12
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would probably denature it. In addition, AHH could be exposed to endogenous
MFO inhibitors, such as those associated with the gut contents and certain
natural pigments in other invertebrates (8,9). Furthermore, most studies of
polycyclic aromatic hydrocarbon (PAH) metabolism concentrate only on quanti-
fying metabolites that are soluble in organic solvents; the water-soluble
metabolites are ignored. Our data indicate that the majority of BP metabo-
lites produced by the oyster are water-soluble.
THE FATE OF PAH IN BIVALVES
There is no doubt that bivalves can remove from seawater and concentrate
within their tissues a variety of PAH. In an early study, Zechmeister and
Koe (10) showed that barnacle extracts contained PAH, including BP, which
probably originated "from tarry materials floating along the southern
California coast." The ability of mussels to take up environmental hydro-
carbons is well established (11,12); it has been suggested that these animals
could be useful in estimating PAH contamination in the marine environment
(5,13,14). It has been generally held that bivalves are able to dispose of
much of the hydrocarbon load by depuration processes, but are not able to
metabolize PAH by mixed-function oxygenase enzymes (7,15-18).
However, there was a report, prior to this one, that indicated that
mollusks have some MFO activity. Mollusks can synthesize sterols (19), a
process in which squalene is converted into the 2,3-oxide as an intermediate
via a microsomal NADPH-dependent oxidase (20).
COMPARISON OF AHH ACTIVITY IN OYSTERS AND OTHER ANIMALS
The basic similarity between the characteristics of AHH in oysters and
other animals has already been described in this paper. In this section the
level of AHH activity in oysters is compared to that in another invertebrate
and laboratory mice. This work was done in the laboratory using the same
technique for separating BP metabolites; however, the conditions for the
reaction were optimal for each species studied.
As indicated in Table 3, AHH activity can be consistently recorded in
the oyster. However, the level of activity is only about 1/13 of that seen
in laboratory mice and also is lower than that reported for an insect (21) .
The NaOH-extracted metabolites comprise less than 1/10 of the total*BP
metabolites produced by oyster AHH. This is not the case for the other
animals studied, in which about equal quantities of metabolites were
recovered from the water and NaOH phases.
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TABLE 3. COMPARISON OF AHH ACTIVITY* IN VARIOUS ANIMALS AS DETERMINED
BY THE GENERATION OF RADIOISOTOPIC BP METABOLITES
Animal
Water-soluble NaOH-soluble
n Total activity derivatives derivatives
Mouse
0* C57BL/6
2.12
1.05 ± 0.24 1.07 ± 0.18
Mouse
& Camm Swiss-Webster
2.37
1.32 ± 0.37 1.05 ± 0.30
Insect 15 0.398
Spodoptera eridania
0.169 ± 0.044 0.229 ± 0.131
Oyster 20 0.163
Crassostrea virginica
0.149 ± 0.036 0.014 ± 0.013
* AHH activity in uninduced, normal digestive gland (or liver) homogenates.
nmoles BP metabolites/mg protein/10 min.
TENTATIVE IDENTIFICATION OF BP METABOLITES PRODUCED BY OYSTERS
It is thought that metabolic activation of PAH, such as BP, is required
before they can express biological activity. BP does not bind covalently to
biological macromolecules, including DNA; its active intermediates are capable
of this interaction, which probably results in subsequently observed muta-
genicity and carcinogenicity. This metabolic activation is mediated by AHH,
which also catalyzes the detoxification of oncogens. The final physiological
result of BP metabolism depends on the balance between the detoxifying and
activating pathways, as well as on target tissue sensitivity.
The primary BP metabolites are epoxides (22). BP epoxides are trans-
formed to phenols by spontaneous rearrangement, to dihydrodiols by epoxide
hydrase, to glutathione conjugates by GSH-epoxide-transferase, or to deriva-
tives that are bound covalently to tissue macromolecules. Phenolic deriva-
tives can be further conjugated with glucuronic acid or sulfate. Dihydro-
diols may be converted to diol-epoxides via AHH and cytochrome P-450 or P-448.
14
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The susceptibility of invertebrates to chemical carcinogens is largely
unknown. An aim of this study was to determine if the potent mammalian
carcinogen and environmental contaminant BP could be considered as a possible
bivalve carcinogen. To this end, the ability of the oyster to metabolize BP
was shown in a sensitive radioisotopic assay. Next an attempt was made to
identify specific BP metabolites using high-pressure liquid chromatography.
Aside from possible carcinogenic effects on the oyster, the presence of
carcinogenic BP metabolites in shellfish, common in the human diet, might have
public health implications. It has been suggested tentatively that a corre-
lation may exist between PAH pollution and neoplasia in certain bivalves (23,
24).
The vast literature on the cytotoxicity, mutagenicity, and carcino-
genicity of BP derivatives cannot be reviewed here. However, it is generally
thought that epoxides, produced via the NADPH-dependent microsomal mono-
oxygenases, are the important reactive metabolites responsible for carcino-
genesis. This idea was supported by studies of the properties of synthetic
K-region epoxides, which are alkylating agents that bind covalently to
nucleic acids. However, more recent work suggests that diol-epoxides,
particularly 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene 9,10-oxide, are the most
likely carcinogenic forms of BP (25). These epoxides would be in the form
of the corresponding dihydroxydiols in the chromatographed extracts, due to
the action of epoxide hydrase. The ultimate carcinogenic BP metabolite would
be tetradiol, which would chromatograph with a retention time less than the
first diol to appear (9,10-diol-BP). Recently, 29 BP derivatives were tested
for mutagenic activity against bacterial cells (the Ames assay) and mammalian
cells (Chinese hamster V79) by Wislocki et al. (26). BP-4,5-oxide was the
most mutagenic compound tested in this report, but BP-7,8-diol-9,10-epoxide
was shown to be 40-60 times more mutagenic and cytotoxic than BP-4,5-oxide
(27). All the other arene oxides, phenols, quinones and dihydrodiols were
not (or weakly) mutagenic, with the exception of BP-ll,12-oxide, and 6-OH BP
mutagenic to hamster cells, and 6-OH BP and 12-OH BP moderately mutagenic for
bacteria.
According to our interpretation of HPLC separation of BP metabolites
produced by oyster AHH, there is little evidence as yet for the production
of highly mutagenic or carcinogenic BP derivatives. The first labeled peak
in these chromatographs corresponds to the 9,10-diol. The most suspect
carcinogenic BP derivative should have a shorter retention time; however,
there is no indication of such a peak in these preparations. Both normal
and Aroclor-induced oysters produce BP-4,5-oxide which is somewhat mutagenic
for both bacteria and mammalian cells. The production of BP-4,5-oxide is
apparently not induced by PCB exposure. The major BP metabolites produced
by oyster AHH are quinones, which are rather inactive. Hydroxy BP deriv-
atives (such as 3-OH BP), which are generated particularly in PCB-induced
oysters, are generally inactive. However, there is evidence for the muta-
genicity of certain other hydroxy BP metabolites, and peak 5 contains an
unidentified monohydroxylated derivative.
15
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TECHNICAL REPORT DATA
(Please read Iiislnictions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-009
3. RECIPIENT'S ACCESSI ON- NO.
4. TITLE AND SUBTITLE
BENZO[a]PYRENE METABOLISM IN THE AMERICAN OYSTER
CRASSOSTREA VIRGINICA
5. REPORT DATE
November 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Robert S. Anderson
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Sloan-Kettering Institute for Cancer Research
Donald S. Walker Laboratory
Rye, New York 10580
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA Grant R804435
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory, Gulf Breeze
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final 7/1/76 - 6/30/77
14. SPONSORING AGENCY CODE
EPA/600/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This research program was initiated with the overall objective of determining the role
NADPH-dependent microsomal mono-oxygenase in the metabolism of the widespread envi-
ronmental carcinogen benzo[a]pyrene (BP) by the oyster Crassostrea v±rgjLnica. This
enzyme system is important in detoxifying various xenobiotics and in activating poly-
cyclic aromatic hydrocarbon oncogens as BP-
sensitive radioisotopic system was developed to permit the quantification of alkali-
oluble and water-soluble BP metabolites produced by oyster mono-oxygenase. An NADPH-
and 02-dependent aryl hydrocarbon hydroxylase (AHH) was shown to be located in the di-
estive glands of these bivalves associated with the microsomal subcellular fraction.
The specific activity of oyster AHH was considerably lower than that of laboratory mice,
out was consistently demonstrable. The BP metabolites produced were primarily water-
oluble derivatives.
There was some indication that oyster AHH was induced by chronic exposure of the ani-
nals to the environmental carcinogens BP and 3-methyl-cholanthrene. There was strong
evidence that exposure to polychlorinated biphenyls (PCB) caused AHH induction.
iigh-pressure liquid chromatography was used to identify BP metabolites produced by
oyster AHH. The generation of various dihydrodiol, quinone, and hydroxy BP derivatives
s shown; this production was augmented in PCB-exposed oysters. This r%port covers the
period from July 1, 1976 to June 30, 1977, and work was completed as of August 31,1977.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Carcinogens
benzo[a]pyrene
American oyster
enzyme induction
mono-oxygenase
PCB's
Mixed-function oxidases
oysters
Induction enzymes in
Carcinogen metabolism in
Metabolism of carcinogens
Benzo[ajpyrene in oyster
Oyster enzyme systems
Mixed-function oxidases
in oysters
quatic
DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
19
20. SECURITY CLASS (This pageJ
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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