INVESTIGATION OF ENZYMATIC SCREENING TESTS FOR MUTAGENS
IN ENVIRONMENTAL POLLUTANTS FROM SYNFUEL OPERATIONS
by
J. J. Schmidt-Collerus, N. L. Couse, J. King and L. Leffler
Written and Prepared by: N. L. Couse
Denver Research Institute
University of Denver
Denver, Colorado 80208
Contract No. R8-05671010
Project Officer
Norman Richards
Environmental Research Laboratory
U.S. Environmental Protection Agency
Sabin Island
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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TECHNICAL REPORT DATA
FRI PP 1 5^ (Pleat read Instructions on the reverse before completing)
i. REPORT NO.
EPA-600/4-81-038
2.
ORD Reoort
4. TITLE AND SUBTITLE
Investigation of Enzymatic Screening Tests for Mutagens
in Environmental Pollutants from Synfuel Operations
7. AUTHOH(S)
J.J. Schmidt-Col lerus , N.L
L. Leffler
9. PERFORMING ORGANIZATION NAME Af
Denver Research Institute
University of Denver
Denver, Colorado 80208
. Couse, J. King and
^D ADDRESS
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
3. RECIPIENT'S ACCESSION NO.
----- ?0957 7
5. REPORT DATE
May 1981
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/04
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The objective of this research program was to develop an enzymati
chemical carcinogens based on the selective in vitro stimulation of mi
c screen for
crosomal
biphenyl-2-hydroxylase by known chemical carcinogens. An attempt was made to repeat
published work using a spectrophotofluorometric assay for biphenyl metabolites. It
was found that this assay system is not valid for use with complex mixtures, and that
metabolites must be separated from interfering compounds prior to quantitation. A hiqh
pressure liquid chromatography method was developed which permitted rapid separation of
metabolites. Nanogram quantities of metabolites were detectable using this chromato-
graphic separation in conjunction with a spectrophotofluorometric detector. Using this
method, it was not possible to demonstrate in vitro stimulation of biphenyl -2-hydroxy-
lase by chemical carcinogens. Alternative assays were also- examined. Terphenyl is
metabolized to at least three different compounds by hamster microsomes. Further work
is necessary to validate the utility of this substrate in an enzymatic screen for car-
cinogens. A marine protozooan, Parauronema acutum metabolizes biphenyl in vivo to 2-
and 4-hydroxybiphenyl . This organism may provide a reliable, inexpensive source of
biphenyl hydroxylase for an in vitro enzymatic assay system. This report was submitted
in fulfillment of Contract No. R8-05671010 by Environmental Research Laboratory under
the sponsorship of the U.S. Environmental Protection Agency. This report covers a peri-
od from Nov. 1, 1977 to Dec. 31, 1978, and work was comoleted as of April 11, 1979.
17.
a. DESCRIPTORS
Carcinogens
Synfuel
Enzymatic Screening Test
Mutagens
18. DISTRIBUTION STATEMENT
Release to public
KEY WORDS AND DOCUMENT ANALYSIS
b. IDENTIFIERS/OPEN ENDED TERMS
Biphenyl
Terphenyl
Marine protozoa
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (This page I
Unclassified
c. COSATl Field/Group
21. NO. OF PAGES
22. PRICE
EPA Fofm 2220.1 (R«v. 4-77) PREVIOUS EDITION is OBSOLETE
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NOTICE
This document is a preliminary draft. It has not been for-
mally released by the U.S. Environmental Protection Agency and should
not at this stage be construed to represent Agency policy. It is being
circulated for comments on its technical merit and policy implications.
DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
11
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FOREWORD
The 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
information 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 ecosystems processes and compo-
nents;
. the significance of chemical carcinogens in the estuarine and
marine environments;
Increasing p.ollution of aquatic environments has led to the develop-
ment of biological assays designed to monitor toxic, mutagenic, and carcin-
ogenic effects of contaminating chemicals. This report describes the
investigation of a biochemical (j_n vitro) prescreen test for determining
carcinogenic compounds.
Henry FYEnos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
in
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ABSTRACT
The objective of this research program was to develop an
enzymatic screen for chemical carcinogens based on the selective in
vitro stimulation of microsomal biphenyl-2-hydroxylase by known chemi-
cal carcinogens.
An attempt was made to repeat published work using a spec-
trophotofluorometric assay for biphenyl metabolites. It was found that
this assay system is not valid for use with complex mixtures, and that
metabolites must be separated from interfering compounds prior to
quantitation. A high pressure liquid chromatography method was de-
veloped which permitted rapid separation of metabolites. Nanogram
quantities of metabolites were detectable using this chromatographic
separation in conjunction with a spectrophotofluorometric detector.
Using this method, it was not possible to demonstrate in vitro stimula-
tion of biphenyl-2-hydroxylase by chemical carcinogens.
Alternative assays were also examined. Terphenyl is meta-
bolized to at least three different compounds by hamster microsomes.
Further work is necessary to validate the utility of this substrate in an
enzymatic screen for carcinogens. A marine protozoan, Parauronema
acutum metabolizes biphenyl in vivo to 2- and 4-hydroxybiphenyl. This
organism may provide a reliable, inexpensive source of biphenyl hy-
droxylase for an in vitro enzymatic assay system.
This report was submitted in fulfillment of Contract No.
R8-05671010 by Environmental Research Laboratory under the sponsor-
ship of the U.S. Environmental Protection Agency. This report covers
a period from November 1, 1977 to December 31, 1978, and work was
completed as of April 11, 1979.
iv
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CONTENTS
Page
Foreword iii
Abstract iv
Figures vi
Tables viii
Abbreviations and Symbols ix
Chemicals, Sources and Purity ix
Acknowledgements x
1. Introduction
General 1
Microsomal biphenyl metabolism 3
Program objectives 4
2. Conclusions 6
3. Recommendations 8
4. Analytical Methods
General 9
Spectrophotofluorometry 9
Thin layer chromatography 21
High pressure liquid chromatography 24
5. Enzymatic Hydroxylation Experiments
General 35
Enzymatic methods 36
Biphenyl hydroxylation 41
Terphenyl metabolism 49
6. Metabolism of biphenyl by Parauronema acutum
General 57
Materials and methods 58
Results 60
Discussion 65
References 67
Bibliography
Activation of microsomes by carcinogens
in vivo and in vitro 70
MetKbds of microsome preparation 74
Detoxification of biphenyl by microorganisms 75
Methods for qualitative and quantitative
analysis of hydroxybiphenyls 76
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FIGURES
Number Page
1 Excitation and emission spectra of 2- and
4-hydroxybiphenyl standards 11
2 Extraction procedure for SPF analysis of 2- and
4-hydroxybiphenyl 12
3 Excitation and emission spectra of material extracted
from oil and BaP dissolved in oil 16
4 Excitation and emission spectra of material extracted
from incubation mixtures containing purified hamster
microsomes and biphenyl, BaP, BaP plus biphenyl ... 18
5 Excitation and emission spectra of material extracted
from incubation mixtures containing purified cauliflower
microsomes and biphenyl, BaP, BaP plus biphenyl ... 19
6 Excitation and emission spectra of material extracted
from incubation mixtures containing purified cauliflower
microsomes and MC, MC plus biphenyl 20
7 Separation of 2- and 4-hydroxybiphenyl
standards by HPLC 28
8 Material separated by HPLC and obtained from
reaction mixtures containing purified hamster
microsomes and biphenyl or oil 29
9 HPLC of material extracted from purified
hamster microsomes incubated with biphenyl
and BaP, safrole, 3-methylcholanthrene 30
10 HPLC of material extracted from purified
hamster microsomes incubated with biphenyl
and a-naphthylamine or p-naphthylamine 31
11 HPLC of material extracted from purified hamster
microsomes incubated with BaP in oil 32
VI
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Number Page
12 HPLC of a standard BaP solution exposed to
oxygen and light for several hours 33
13 Material separated by HPLC obtained from reaction
mixtures containing purified hamster microsomes
and m-terphenyl, BaP plus m-terphenyl 51
14 Excitation and emission spectra of an n-heptane
extract of metabolized m-terphenyl 54
15 Excitation and emission spectra of an n-heptane
extract of metabolized p-terphenyl 55
16 Growth of Parauronema acutum in the
presence of biphenyl at 22°C 61
17 Growth of Parauronema acutum at 25°C in the
presence of biphenyl dissolved in DMSO 62
18 Growth of Parauronema acutum at 25°C in the
presence of biphenyl dissolved in Tween 80 63
19 Growth of Parauronema acutum at 25 °C in
the presence of BaP and biphenyl 66
vn
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TABLES
Number Page
1 Separation of biphenyls by TLC 23
2 Separation of biphenyl standards by HPLC 26
3 Protein concentrations in hydroxylation experiments . . 39
4 Incubation mixtures used in hydroxylation reactions . . 40
5 Effect of test compounds on production
of 4-hydroxybiphenyl and 2-hydroxybiphenyl
by liver fractions 42
6 Effect of test compounds on production
of 4-hydroxybiphenyl and 2-hydroxybiphenyl
by plant microsomes 45
7 Effect of test compounds on production
of 4- and 2-hydroxybiphenyl as determined
by quantitative HPLC 48
8 Metabolites of m-terphenyl produced by
purified hamster microsomes 50
9 Metabolites of m-terphenyl produced in
the presence of carcinogens
53
10 Quantities of 2- and 4-hydroxybiphenyl
present in extracts of P. acutum cultures 64
vin
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
BaP ----------- benzo(a)pyrene
DMSO --------- dimethylsulfoxide
HPLC --------- high pressure liquid chromatography
MC ----------- 3-methylcholanthrene
aNA ---------- ot-naphthylamine
PNA ---------- p-naphthylamine
SA ........... Safrole
SPF ---------- Spectrophotofluorometer
SYMBOLS
emission wavelength
excitation wavelength
CHEMICALS, SOURCES AND PURITY
This list does not constitute an endorsement of the manu-
facturers listed. It is included because purity and source may
influence experimental results.
Biphenyl ..................... -J.T. Baker, Ultrex, 99.99%
4,4'-Dihydroxybiphenyl -------- Aldrich, 97%
2,2'-Dihydroxybiphenyl -------- Aldrich, 99%
Dimethylsulfoxide -------------- J.T. Baker, Reagent grade
n-Heptane --------------------- Burdick and Jackson Labs,
UV grade, "distilled in glass"
n-Hexane ----- ........ --------- Burdick and Jackson Labs,
UV grade, "distilled in glass"
4-Hydroxybiphenyl ------------- Aldrich, 97%
2-Hydroxybiphenyl ............. Aldrich, 99+%
Succinic acid .......... - ........ J.T. Baker, 99.4%
p-Terphenyl ................... Aldrich, 99+%
Tetrahydrofuran ........ ------- Burdick and Jackson Labs
UV grade, "distilled in glass"
Tween 80- .................... -Emulsion Engineering, Inc.,
Polysorbate 80, USP
ix
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ACKNOWLEDGEMENTS
We are greatly indebted to F. Krohlow and C. Burdick for
invaluable technical assistance and to K. Gala and M. Shaffron for
assistance with the high performance liquid chromatograph. We also
wish to thank Dr. D. Lindmark for providing the P^ acutum and ad-
vising us as to its growth characteristics.
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SECTION 1
INTRODUCTION
GENERAL
Awareness of increasing chemical pollution of both aquatic and
terrestrial environments has led to the development of biological assays
designed to monitor toxic, mutagenic, and carcinogenic effects of indus-
trial effluents. It is reasonable to assume that the potential hazards to
the biosphere, and especially the marine environment which serves as
the ultimate repository of the majority of pollutants, will continue to in-
crease with expansion of technological productivity and with rapid and
intensified developments in the area of alternate energy sources.
Therefore, biological systems useful for screening effluents should be
simple, sensitive, reliable, rapid and inexpensive. In addition, these
systems should be directly useful for testing the effect of noxious
materials upon the marine environment. Detection of harmful pollutants
will permit detoxification of the material before release into the
biosphere.
Current bioassays use a number of different systems in-
cluding bacteria (Salmonella, Escherichia coli), yeast, Drosophila,
mammalian cell cultures, and mice. A "tier" approach (Epler, 1976) to
biological testing has been designed in order to screen material for
potentially hazardous effects. The rationale of the tier system is to
identify potentially harmful material by initial testing in relatively rapid
and inexpensive assays prior to evaluation of effects in time-consuming
and expensive in vivo mammalian systems. This approach begins with a
microbiological assay and proceeds to other tiers (levels) of increasing
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organismal complexity. The ultimate objective of the tier approach is to
reduce the amount of time and number of tests which must be conducted
in order to assess the potential biological hazard of a given material.
Tests using living organisms or cells suffer from a high
sensitivty to toxic effects caused either by the active compound itself
or by impurities present in complex mixtures. These toxic effects may
thus be secondary, and may mask mutagenic or carcinogenic activities
of the material. Complex mixtures therefore must often be fractionated
prior to testing in order to reduce toxicity, and the number of tests to
be conducted on a given sample may be extremely large. In addition,
the spectrum of substances which can be effectively tested in a given
system is frequently limited.
It would therefore be desirable to have available a rapid
biochemical (in vitro) prescreen test of high sensitivity, broad spectrum
of applicability, and simplicity which would be independent of secondary
toxic effects. This type of system would permit rapid identification of
compounds or fractions to be tested at higher tiers. Such a test would
save considerable time and expense in monitoring effluents and their
fractions for harmful effects.
Oxidative drug-metabolizing enzyme systems appear to fulfill
the requirements for a biochemical assay for carcinogens and mutagens.
These enzymes are associated with the microsomal fraction of a number
of organisms, and have been shown to be selectively affected by known
mutagens and carcinogens. A closer investigation of this effect could
lead to the development of an in vitro system suitable for use as a
rapid prescreen for mutagens and carcinogens.
-2-
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MICROSOMAL BIPHENYL METABOLISM
Microsomal mixed function oxidases of a variety of animals are
able to use biphenyl as a substrate for formation of hydroxybiphenyl
compounds. In general, 4-hydroxybiphenyl is the major metabolite, and
2-hydroxybiphenyl is a secondary metabolite, with ratios of 26:1 to 2:1
being found depending on the specific species examined (Basu et al.,
1971; Creaven et al., 1965; Willis and Addison, 1974).
It has been found by a number of investigators that bi-
phenyl-2-hydroxylase is specifically stimulated by chemical carcinogens.
This effect has been demonstrated in vivo in animals and in vitro using
microsomes from both animals and plants. Administration of chemical
carcinogens and mutagens to test animals resulted in a selective stimu-
lation of biphenyl-2-hydroxylase as measured by an increase of 2- to
20-fold in the amount of 2-hydroxybiphenyl produced (Atlas and
Nebert, 1976; Burke and Bridges, 1975; Burke and Prough, 1976;
Friedman et al., 1972; Nebert et al., 1975; Tredger and Chhabra,
1976). The production of 4-hydroxybiphenyl was not affected by the
treatment. The stimulation of biphenyl-2-hydroxylase by chemical
carcinogens occurred in two phases: enzyme activation followed by
enzyme induction (McPherson et al., 1976; Parke, 1976).
An in vitro system which permits determination of the activity
of biphenyl-2-hydroxylase and biphenyl-4-hydroxylase was developed by
McPherson and coworkers (1976). This system involves preparation of
purified microsomes, incubation with an NADPH-regenerating system in
the presence of biphenyl, extraction and spectrophotofluorometric
determination of the 4- and 2-hydroxybiphenyl metabolites (Creaven
?J §1., 1965).
-3-
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In order to examine the effect of chemical carcinogens and
mutagens on biphenyl hydroxylase in vitro, microsomes from animals or
plants were preincubated with test compounds, biphenyl added, and the
amount of 2- and 4-hydroxybiphenyl compared to control incubation
mixtures. It was reported that in vitro preincubation of microsomes
with known carcinogens resulted in a 60% to 300% increase in
2-hydroxybiphenyl production (Burke and Bridges, 1975; McPherson et
a]., 1976, 1975 a,b,c, 1974 a,b). Noncarcinogens had no effect on the
hydroxylases, and biphenyl-4-hydroxylase was not affected by the
carcinogens.
The biphenyl hydroxylase system which is apparently selec-
tively stimulated by chemical carcinogens would appear to be ideal for
the purpose of providing a preliminary screen for environmental carcin-
ogens. The assay system is simple, the in vitro effects correlate well
i
with the in vivo effects, and the reaction substrate (biphenyl) and
products are themselves noncarcinogenic and easily detectable.
PROGRAM OBJECTIVES
The over-all program objectives include:
1. Compile a bibliography related to the biphenyl
hydroxylase reaction and related subjects.
2. Investigate the utility of the biphenyl hydroxylase
system as an enzymatic prescreen for chemical
carcinogens and mutagens.
a. Repeat the work of McPherson and coworkers
with a small number of known carcinogens and
non-carcinogens in order to validate the in
vitro assay.
b. Refine the test system:
— investigate other sources of microsomes
— examine the metabolism of other
polyphenyls
-4-
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--- study the metabolism of derivatives of
biphenyl
— examine algal biphenyl metabolism
— compare the results of the biphenyl assay
system with the Ames/Salmonella system
for sensitivity to toxic effects and
mutagenesis.
c. Validate the system for use with complex
mixtures such as leachates from retorted oil
shale.
d. Examine the ability of a marine protozoan,
Parauronema acutum, to metabolize biphenyl.
-5-
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SECTION 2
CONCLUSIONS
Attempts to reproduce the experimental data of others (Burke
and Bridges, 1975; McPherson eta]., 1976, 1975 a,b,c, 1974 a,b;
Creaven et al., 1965) as a preliminary step in the development of an
enzymatic screen for chemical carcinogens were negative. In all but
one published report (McPherson et al., 1975a), the spectrophotofluoro-
metric assay of Creaven and coworkers (1965) was used to determine in
vitro production of 2- and 4-hydroxybiphenyl in complex mixtures
without prior separation of the metabolites. It was found that this
method cannot be used with complex mixtures containing fluorescent
metabolites of carcinogens because these metabolites contribute fluores-
cence at the wavelength used to measure 2-hydroxybiphenyl. Our
results are in agreement with the data of Tong and coworkers (1977).
Methods of separating biphenyl metabolites prior to quantifi-
cation were investigated. High pressure liquid chromatography using a
spectrophotofluorometric detector permitted reliable separation and
quantification of metabolites. Using this method, it was not possible to
repeat published work which demonstrated a stimulation of hepatic
microsomal biphenyl-2-hydroxylase by chemical carcinogens. Microsomes
were prepared from the same organisms used in previously published
work (rat, hamster, and mouse liver; avocado mesocarp) as well as new
sources (cauliflower, apple). Further experimentation will be necessary
in order to reconcile the results presented in this report with the data
of McPherson and coworkers (1975a) which demonstrated a 2.5 fold
increase in 2-hydroxybiphenyl using 14C-labeled biphenyl as a
substrate.
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Terphenyl is metabolized to a minimum of three different
compounds by hamster hepatic microsomes. This substrate may prove
useful as a supplement to biphenyl in an enzymatic screen for carcino-
gens. However, further experimentation is necessary to elucidate the
nature of the metabolites and enzymes involved, and the effect of
chemical carcinogens on metabolite production.
The marine protozoan, Parauronema acutum metabolizes bi-
phenyl to 2- and 4-hydroxybiphenyl. It does not metabolize BaP
(Lindmark, 1978). Preliminary experiments indicate that BaP at low
concentrations may protect the organism from the lethal effects of
biphenyl. This sytem may therefore prove useful in the study of
carcinogen-induced membrane changes. In addition, hydroxylases of P_^
acutum are both soluble and membrane-bound (Lindmark, 1978).
Parauronema acutum may prove to be a source of stable, easily re-
covered hydroxylases which could be used in an enzymatic screen for
carcinogens when coupled with a carcinogen activating system.
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SECTION 3
RECOMMENDATIONS
Because the in vivo stimulation of biphenyl-2-hydroxylase by
chemical carcinogens is well documented, it would be useful to develop a
rapid in vitro procedure using this system. Therefore, experiments
should be performed using 14C-labeled substrates and both TLC and
HPLC separation techniques to determine whether or not biphenyl-2-
hydroxylase is selectively stimulated in vitro by chemical carcinogens.
In addition, simpler, more rapid methods of preparing plant or animal
microsomes should be investigated. If the stimulation can be reliably
demonstrated, further work should be performed to allow this system to
be used as a routine assay.
Metabolism of terphenyls should be studied as a possible
alternative or complement to biphenyl metabolism. The enzymatic sys-
tem(s) responsible for terphenyl metabolism may prove to exhibit a
different sensitivity to carcinogens or may be more stable than the
biphenyl hydroxylases. This would prove extremely useful in the
development of an enzymatic screen.
The metabolism of biphenyl by Parauronema acutum should be
confirmed, and an investigation of the nature and stability of the en-
zymes involved should be carried out. Studies should be conducted to
determine the response of these enzymes to activated and unactivated
carcinogens both in vivo and in vitro. This organism could provide an
excellent source of biphenyl-2-hydroxylase and biphenyl-4-hydroxylase
for an enzymatic screen.
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SECTION 4
ANALYTICAL METHODS
GENERAL
A number of different analytical approaches to quantitative
measurement of 2- and 4-hydroxybiphenyl are possible. Initially the
spectrophotofluorometric (SPF) method of Creaven and coworkers (1965)
was employed in an effort to validate the assay system. This method
was used in the majority of the in vitro work published by McPherson
and coworkers, and has the advantage that metabolite separation is not
necessary. It became apparent that the SPF method does not provide a
valid measurement of the amount of 2-hydroxybiphenyl present when
fluorescent carcinogens and their metabolites are present in reaction
mixtures. Therefore two different techniques for separation of the
metabolites were examined: thin layer chromatography (TLC) and high
pressure liquid chromatography (HPLC).
SPECTROPHOTOFLUOROMETRY
Background
Creaven and coworkers (1965) demonstrated that 2-hydroxy-
biphenyl and 4-hydroxybiphenyl can be determined fluorometrically in
mixtures of the two compounds because -the 2-isomer exhibits excited
state ionization whereas the 4-isomer does not. At pH 2-9, 2-hydroxy-
biphenyl absorbs light in the unionized form (excitation wavelength =
nm)( but emits the fluorescence of the anion (emission
-9-
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wavelength = Xr,, = 415 nm) whereas 4-hydroxybiphenyl absorbs
(Xry = 275 nm) and emits (^pM = 338 nm) in the unionized form.
Figure 1 shows the excitation and emission spectra of the pure 2- and
4-hydroxybiphenyls determined using an Aminco Bowman spectrophoto-
fluorometer equipped with a high pressure xenon lamp. The excitation
and emission maxima of 290 nm and 412 nm for 2-hydroxybiphenyl, and
274 nm and 335 nm for 4-hydroxybiphenyl are within the limits of
instrument variability of the values reported by Creaven and coworkers
(1965) given above. The quantities of the two compounds present in a
mixture are therefore determined fluorometrically at an acid pH using
two different combinations of excitation and emission wavelengths.
Materials and Methods
All chemicals were either reagent grade or of the highest
purity available. For detailed information concerning purity, see
page ix.
Incubation of microsomes with substrate and test compounds
was performed as described in Section 5. The method of extracting the
biphenyl metabolites from microsome incubation mixtures used by
Creaven and coworkers (1965) and all subsequent investigators was
followed exactly. It is summarized in Figure 2.
After the incubation period was completed, the reaction was
terminated by addition of 1 ml of 4N HC1, and the mixture immediately
extracted with n-heptane. The incubation procedure and heptane
extraction were carried out in 20 ml glass tubes with teflon lined screw
caps. After the initial centrif ugation , the tubes were stored in the
cold overnight. The samples were extracted and analyzed with a mini-
-10-
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250
350 450
WAVELENGTH (NANOMETERS)
550
Figure 1. Excitation and emission spectra of 2- and 4-hydroxybi-
phenyl standards in 0.1N NaOH buffered to pH 5.5 with
0.1N succinic acid. The solid lines are emission spectra,
and the dashed lines are excitation spectra. The spec-
trum for 2-hydroxybiphenyl is vertically offset and is
shown in the upper portion in this and all following
figures.
-11-
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incubation mixture
aqueous layer
(discard)
n-heptane layer
(discard)
acidify, extract with 10 ml n-heptane by shaking
for 5 minutes at room temperature, centrifuge at
2,000 rpm for 15 minutes to separate the layers
n-heptane layer
2 ml aliquot
extract with 10 ml of 0.1N NaOH for 5 minutes
at room temperature, centrifuge at 2,000 rpm
for 15 minutes to separate the layers
NaOH layer
2 ml aliquot
adjust to pH 5.5 by
adding 0.5 ml of 0.5N
succinic acid
2 ml aliquot
measure fluorescence
nm
nm
(4-hydroxybiphenyl)
measure fluorescence
\£X = 290 nm
\rik, = 413 nm
LM
(2-hydroxybiphenyl
+ 4-hydroxybiphenyl)
figure 2. Extraction procedure for SPF analysis of 2- and 4-hy-
droxybiphenyl (Creaven et al., 1965).
-12-
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mum of exposure to ultraviolet light. Therefore incandescent light
bulbs were used when necessary.
The tubes containing the heptane extract were removed from
storage and allowed to come to ambient temperature. The 2 ml aliquots
of the n-heptane layer were transferred to 14 ml glass tubes with teflon
lined screw caps. The tubes containing the remaining 8 ml of n-hep-
tane were returned to cold storage. After extraction and centrifuga-
uon, the heptane was removed by pipetting and discarded. A 2 ml
aliquot of the NaOH extract was transferred to a 5 ml quartz cuvette,
and' adjusted to pH 5.5 by addition of 0.5 ml of 0.5N succinic acid.
Fluorescence was measured using an Aminco Bowman spectrophotofluoro-
meter. Fluorescence of the 4-isomer was measured first because 4-hy-
droxybiphenyl was less stable in the basic NaOH solution than was
2-hydroxybiphenyl. The instrument was corrected for background
solvent fluorescence using 2 ml of 0.1N NaOH to which 0.5 ml of 0.5N
succinic acid was added.
Calibration curves were prepared using three standard solu-
tions: 24.8 (jg/ml of 4-hydroxybiphenyl, 6.0 ug/ml of 2-hydroxybi-
phenyl, and a mixture containing 24.8 vg/nd of 4-hydroxybiphenyl and
6.0 ug/ml of 2-hydroxybiphenyl. The standards were dissolved in an
aqueous 5% (v/v) ethanol solution. Following HC1 addition, 1 ml of each
standard solution was added to each of three microsome incubation tubes
from which the biphenyl substrate had been omitted. The solutions
were then extracted as described in Figure 2. Three dilutions of each
standard were used to construct quantitative calibration curves. A set
°f standard curves was constructed for each experiment. This method
°' Preparing standard curves allows the calculation of absolute amounts
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of metabolites present in the unknown reaction mixtures without the
need to determine efficiency of extraction because all samples are
treated in an identical manner.
The quantities of the two metabolites present in the extract
were calculated according to the method of Creaven and coworkers
(1965). The 2-isomer does not interfere with fluorometric determination
of the 4-isomer. However, the 4-isomer contributes fluorescence at the
wavelength used to measure 2-hydroxybiphenyl. Therefore, the fol-
lowing measurements and calculations must be performed:
1. Determine the percent fluorescence of the unknown
solution using \EX = 274, \EM = 335 nm. Read the
quantity of 4-hydroxybiphenyl present from the
standard curve.
2. Determine the percent fluorescence of the 4-hy-
droxybiphenyl standard at A.™ = 290, \r-M =
412 nm.
3. Determine the percent fluorescence of the unknown
solution using XEX = 290, \EM = 412 nm. This is
the sum of the emissions of both isomers.
4. Calculate the amount of 2-hydroxybiphenyl present
in the mixture from the equation (Creaven et al.,
1965):
C = B - (Ax/y)
where: C = percent fluorescence of 2-hydroxybi-
phenyl at 412 nm
B = percent fluorescence of the mixture
at 412 nm
A = percent fluroescence of the standard
4-hydroxybiphenyl solution at 412 nm
-14-
-------
x = concentration of 4-hydroxybiphenyl in
the unknown
y = concentration of 4-hydroxybiphenyl in
the standard
The concentration of 2-hydroxybiphenyl in the
unknown mixture can then be determined from the
standard curve.
Results
Because the results obtained using this method were anoma-
lous, excitation and emission spectra of several different mixtures were
examined.
Initially it was thought that the peanut oil present in the
microsome incubation mixture might contribute fluorescence at the
wavelengths used to measure the biphenyls. In addition, it was also
possible that unaltered carcinogens such as benzo[a]pyrene (BaP) could
interfere with the fluorescence measurements. Therefore, 0.5 ml of
peanut oil and 0.5 ml of 1 mM BaP dissolved in peanut oil were dis-
solved in 10 ml each of n-heptane and extracted as described in
figure 2. The excitation and emission spectra of these extracts are
shown in Figure 3. The results show that material extracted from
peanut oil does fluoresce at the wavelength used to measure 2-hydroxy-
fciphenyl. However, the peanut oil is present in all samples, including
the standards, and this effect is therefore corrected for when the
standard curves are constructed. The sample containing BaP in oil
showed the same spectrum as the oil alone. Therefore unaltered BaP
-^s not contribute to fluorescence at the wavelength used to determine
••""• 2-isomer. in addition, BaP is apparently not hydroxylated during
• ••' extraction procedure.
-15-
-------
-------
Figure 4 shows excitation and emission spectra of material
extracted from incubation mixtures containing purified hamster micro-
somes. As expected, both 2- and 4-hydroxybiphenyl were formed from
the biphenyl (Figure 4A). However, the emission peak for 2-hydroxy-
biphenyl was shifted to 418 nm, and 4-hydroxybiphenyl appeared as a
broad shoulder at 335 nm. Incubation mixtures containing BaP (Fig-
ure 4B) had a broad emission peak from 380 to 470 nm when excited at
290 nm. Material which was extracted from mixtures containing BaP and
biphenyl (Figure 4C) again had the 418 nm peak, but lacked the 335 nm
shoulder.
It is apparent that metabolites of BaP formed during incuba-
tion with the microsomes are coextracted with the hydroxylated bi-
phenyls, and contribute significant fluorescence at the wavelength used
to measure 2-hydroxybiphenyl. Determination of 4-hydroxybiphenyl is
relatively unaffected by the fluorescence of these metabolites.
In the case of material extracted from incubation mixtures
containing plant microsomes and biphenyl (Figure 5A), two emission
optima were observed. Excitation at 290 nm gave an emission peak at
405 nm, and excitation at 272 nm gave an emission peak at 367 nm. A
similar pattern was observed in material extracted from microsomes
incubated with BaP in oil (Figure 5B) or methylcholanthrene (MC) in oil
(hgure 6A). Incubation mixtures containing both carcinogen and
biphenyl yielded material which had an increased fluorescence in the 405
10 410 nm region. The emission spectrum obtained by exciting at
-•2 nm showed essentially no discrete peaks in the case of BaP plus
^Phenyl (Figure 5C), and a broad shoulder (350 to 450 nm) in the case
o: MC plus biphenyl (Figure 6B).
-17-
-------
CO
I
INMOUCTOa)
«AVD.EN*TM (•ANOHCTCn)
Figure 4. Excitation and emission spectra of material extracted from incubation mixtures contain-
ing purified hamster microsomes and (A) oil plus biphenyl; (B) BaP in oil; (C) BaP in
oil plus biphenyl. The dashed vertical lines designate 335 and 412 nm, the wave-
lengths at which emission was measured in the fluorometric assay.
-------
to
I
•UKIXMTH (NJUtOHfTDKI
MVCLEIWTH (MAHOMCTCM)
MVCLDMTH (NAHOUETtftS)
Figure 5. Excitation and emission spectra of material extracted from incubation mixtures contain-
ing purified cauliflower microsomes and (A) oil plus biphenyl; (B) BaP in oil; (C) BaP
in oil plus biphenyl.
-------
imunmrrmt
igure 6.
Excitation and emission spectra of material extracted
from incubation mixtures containing purified cauliflower
microsomes and (A) MC in oil; (B) MC in oil plus
biphenyl.
-20-
-------
Discussion
It is obvious that the spectrophotofluorometric method of
Creaven and coworkers (1965) is not useful for determining hydroxybi-
phenyls in the presence of carcinogens whose metabolites fluoresce at
the wavelengths used to measure the hydroxylated biphenyls. This
conclusion agrees with the findings of Tong and coworkers (1977) which
were published after the conclusion of the SPF work presented in this
report.
It is therefore necessary to separate the metabolites of bi-
phenyl from each other and from other fluorescent material extracted
from incubation mixtures prior to making quantitative measurements.
Two such possible separation methods were examined next.
THIN LAYER CHROMATOGRAPHY
Background
McPherson and coworkers (1975a) used thin layer chromato-
graphy (TLC) to separate 14C-labeled metabolites of biphenyl. Initial
experiments therefore employed the same silica gel substrate (HF2s4)
and solvent system (benzene:ethanol, 95:5, v/v) as these investigators
m order to validate the separation technique. Subsequently other
solvent systems were examined as a preliminary step in selecting the
optimal solvents to be used in the high performance liquid chromato-
separation of metabolites on a silica gel column.
-21-
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and Methods
A slurry containing 30 g of silica gel HF254, type 60 (EM Re-
agents), 96 ml deionized water, and 4 ml acetone was mixed in a Virtis
oiendor at high speed for 2 minutes. The slurry was spread using a
Desaga spreader onto 20 x 20 cm glass plates to a thickness of
0.25 mm. The plates were air dried for at least 8 hours. Standards
were dissolved in methanol. Plates were examined under a Mineralight
t'VS-54 lamp at a wavelength of 254 nm. All chemicals were reagent
grade.
Results
Table 1 gives the Rf values obtained using a number of
different solvent systems. As can be seen, several of the solvent
systems gave good separation of the four hydroxylated biphenyls. Both
p- and m-terphenyl were also examined in the benzene : ethanol (95:5)
system, and were found to have the same Rf values as biphenyl.
Use of TLC to quantitate biphenyl metabolite production
requires removal of the spot from the plate and analysis either by
scintillation counting (McPherson et al. 1975a) of labeled material or
iluorescence of unlabeled compounds. In order to examine the possi-
bility of using fluorescence measurements, the silica gel alone was
scraped from the plate, eluted with benzene, and fluorescence deter-
mined. it was found that material was eluted which would interfere
*ith measurement of the hydroxylated biphenyls.
-22-
-------
TABU; i. SKPAHATION or BIPHT.NYLS BY TI.C
Rf values for solvent systems1:
compound
biphenyl
2-hydroxybiphenyl
4-hydroxybiphenyl
2 , 2'-dihydroxybiphenyl
4,4'- dihydroxy biphenyl
benzene:
ethanol3
M9 95:5
14.3
14.1
19.3
16.1
10.3
0.71
0.55
0.38
0.30
0.12
CC14:
acetone
20:1
0.54
0.24
0.12
0.06
0.01
CC14:
methanol
20:1
0.58
0.30
0.15
0.12
0.02
CC14: CHCl3:n-hexane: dioxane:
methanol methanol n-hexane
95:10 45:30:5 20:80
0
0
0
0
0
.63
.39
.27
.24
.08
0
0
0
0
0
.72
.55
.39
.34
.10
0.57
0.26
0.19
0.12
0.04
dioxane :
iso-octane
20:80
0.45
0.19
0.14
0.09
0.03
dioxane:
cyclohexan
20:80
0.49
0.26
0.19
0.12
0.05
to
CO
compound
biphenyl
2-hydroxybiphenyl
4-hydroxybiphenyl
2 , 2 ' -dihydroxybiphenyl
4,4' - dihydroxybiphenyl
THF:
THF cyclohexane
ug 100% 50:50
14.3
14.1
19.3
16.1
10.3
0.63
0.60
0.60
0.60
0.59
0.52
0.45
0.41
0.36
0.35
THF:
cyclohexane
10:90
0
0
0
0
0
.39
.15
.08
.05
.04
THF:
dioxane
10:90
0.63
0.59
0.57
0.59
0.62
THF:
iso-octane
50:50
0.53
0.43
0.39
0.37
0.36
THF:
iso-octane
10:90
0
0
0
0
0
.44
.13
.08
.04
.02
THF:
n-hexane
50:50
0.61
0.52
0.48
0.44
0.37
THF:
n-hexane
10:90
0.55
0.20
0.12
0.07
0.03
2
3
Each compound was placed in a separate spot. Abbreviations:
THF = tetrahydrofuran.
Total amount of material spotted on the plate.
Values are the average of four different determinations.
CC14 = carbon tetrachloride, CC13 = chloroform,
-------
Discussion
Thin layer chromatography is a valid method for separating
hydroxylated biphenyls. However, unless radioactive material is used,
quantitation appears to be difficult. The method is not useful in con-
junction with a routine assay system because the amount of material
present cannot be determined directly on the plate. A more direct
method for separation and quantitation of metabolites is required.
HIGH PRESSURE LIQUID CHROMATOGRAPHY
Background
High pressure liquid chromatography has recently been used
to separate and identify polycyclic aromatic hydrocarbons in complex
mixtures (Burchill et al. , 1978; Dong and Locke, 1976; Thomas et al. ,
1978). This method combined with either an ultraviolet or fluorometric
detection system would appear to provide several advantages as an
analytical means for quantitating biphenyl metabolite production.
Sample handling would be minimal because the n-heptane extract of the
microsomal incubation mixture could be used directly. An SPF detection
system would permit selective quantitative measurement of material at
wavelengths specific for a given compound thus minimizing interfer-
ences. The SPF system also allows detection of very small amounts of
material.
and Methods
A Perkin-Elmer 220 high pressure liquid chromatograph
Quipped with an ultraviolet detector (254 nm) was used. The chroma-
-24-
-------
tograph was attached to the spectrophotofluorometer by means of a
150 M! fl°w thrush cell having a 2 mm path length. Three different
columns (Whatman) were assessed: Partisil 10, Partisil PSX 10/25 PAG,
and Partisil ODS/2. All were 25 cm long x 4.6 mm inside diameter.
The temperature was ambient. The location of compounds in the chro-
matographic fractions was recorded using a Linear Instrument Company
strip chart recorder at a chart speed of 16 inches per hour. Ten
microliters of the n-heptane extract of the microsome incubation mix-
tures was injected directly into the chromatograph. All chemicals were
of the highest purity available, and all solvents were spectrophotometric
grade, distilled in glass.
Results
Table 2 shows that the hydroxybiphenyls can be separated
from each other and from biphenyl using HPLC. The best separation
was obtained using a Partisil PSX 10/25 PAC column and a solvent
system of THF:n-hexane of 15:85. The retention time of BaP under
these conditions was 4.2 minutes.
The optimum fluorescence wavelengths for detection of the
hydroxybiphenyls in this solvent system were determined, and the
following standard conditions were therefore used in all subsequent
analyses:
column: Partisil PSX 10/25 PAC flow rate: 2 ml/min.
solvent: THF:n-hexane, 15:85 pressure: 300 psi
fluorescence detector: \£X = 300 nm range: 0.33
A.,-.., = 335 nm
LM
-25-
-------
TABLE 2. SEPARATION OT BIPHENYL STANDARDS BY
flow
rate
column solvent system (ml/min)
to
CT)
1
Partisil 10 dioxane:n-hexane 10:90
THF:isooctane 15:85
13:87
Partisil PSX THF 100%
10/25 PAC THF:n-hexane 50:50
25:75
15:85
15:85
Partisil ODS/2 me thanol: water 80:203
85:15
90.-103
90:103
1
1
1
1
1
1
1
2
1
1
1
0.5
i ensiiLn
biphenyl
—
—
--
--
—
--
2.2
10.2
7.6
9.2
9.8
DIl Ullie Vl'llliun:
4-hydroxy-
biphenyl
9.9
6.8
8.4
3.8
6.0
11.2
21.4
14. 32
5.7
4.6
6.2
6.5
;o ) yjL •
2-hydroxy-
biphenyl
5.8
4.4
5.0
3.6
5.5
9.7
17.6
10.5
5.9
4.2
4.4
4.4
Except as noted, compounds were monitored with an ultraviolet light detector at 254 nm. A line
indicates that the retention time was not determined.
The retention times for 2- and 4-hydroxybiphenyl are an average of three separate
determinations .
Compounds were monitored fluorometrically at
= 300 nm,
= 335 nm.
-------
An example of the chromatogram of 2- and 4-hydroxybiphenyl standards
obtained using this system is shown in Figure 7.
The separation of 2- and 4-hydroxybiphenyl in an extract
from an incubation mixture containing purified hamster microsomes is
shown in Figure 8. The positions of the 2- and 4-isomers are clearly
defined, and well separated from earlier peaks (Figure 8A). Most of
the early peaks represent material extracted from the complex incuba-
tion mixture as is shown in Figure 8B.
Using this system, 2- and 4-hydroxybiphenyl can be identi-
fied unequivocably, and there is no interference from metabolites of the
carcinogens added to the incubation mixtures (Figures 9 and 10). In
order to be certain that this was true, an extract of an incubation
mixture containing purified hamster microsomes, BaP in oil and biphenyl
was chromatographed using the standard conditions except that \EM was
422 nm. This is the optimum wavelength for detection of potential BaP
metabolites. Figure 11 shows that there are no BaP metabolites at the
retention times of the hydroxybiphenyls.
In order to ascertain whether the supernatant oxygen present
during the extraction of the incubation mixture could produce oxidation
products which would interfere with the detection of the hydroxybi-
phenyl compounds, a standard solution of BaP was exposed to air and
sunlight and subsequently chromatographed. The conditions were
standard with the exception that the emission wavelength was 422 nm.
il can be seen (Figure 12) that even under these extreme conditions,
-"•'V traces of materials have been formed which have retention times
s-T.iiar to those of the hydroxybiphenyls.
-27-
-------
TM
Column - Partisil PAC
Column length - 25 cm
Solvent - n-Hexane:THF (85:15)
Flow Rate - 2 ml/min
Pressure - 300 psi
Fluorescence Detector:
Excitation - 300 nm
Emission - 335 nm
Range 0.33
i
w
z
X
u
Q.
2-OH
_L
10
(MIN.)
15
20
figure 7.
Separation of 2- and 4-hydroxybiphenyl standards by
HPLC. The sample contained 2.4 ng of each standard.
-28-
-------
6 K> 19
(MIH)
\
B
o
(urn I
>gure 8. Material separated by HPLC and obtained from reaction
mixtures containing purified hamster microsomes and
(A) oil plus biphenyl; (B) oil alone.
-29-
-------
I
u>
o
I
4-OH
TV
B
t-OH
1
o
(MN.I
Figure 9. HPCL of material extracted from purified hamster microsomes incubated with biphenyl
and (A) BaP in oil; (B) safrole in oil; (C) 3-methylcholanthrene in oil.
-------
S-OM
iLil
D
I HIM.)
B
Figure 10. HPLC of material extracted from purified hamster micro-
somes incubated with biphenyl and (A) a-naphthylamine
in oil; (B) p-naphthylamine in oil.
-31-
-------
x
<9
UJ
U)
0.
10
(WIN.)
15
20
figure 11.
HPLC of material extracted from purified hamster micro-
somes incubated with BaP in oil. The emission wave-
length was optimum for detection of BaP metabolites.
-32-
-------
U
X
V
Ul
0.
10
(WIN.)
IS
20
Figure 12. HPLC of a standard BaP solution exposed to oxygen and
light for several hours.
-33-
-------
Discussion
HPLC combined with SPF would appear to be the analytical
method of choice for determining the amounts of 2- and 4-hydroxybi-
phenyl produced in complex reaction mixtures. Because of problems
with the Partisil PSX 10/25 PAC column in terms of stability, it may be
necessary to use the Partisil ODS/2 column for long term studies.
-34-
-------
SECTION 5
ENZYMATIC HYDROXYLATION EXPERIMENTS
GENERAL
A second major objective of this work was the duplication of
in vitro stimulation of biphenyl-2-hydroxylase by chemical carcinogens
as described by others.
The in vivo stimulation of microsomal biphenyl-2- and 4-hy-
droxylase by chemical carcinogens is well documented for a number of
animals (Creaven et al., 1965; Atlas and Nebert, 1975; Hook et al.,
1975; Burke and Prough, 1976; Burke and Bridges, 1975). The in
vitro stimulation of biphenyl-2-hydroxylase has also been reported for
both animal and plant microsomes (Creaven et al., 1965; McPherson et
al., 1976, 1975 a,b,c 1974 a,b; Tredger et al., 1976; Tong et al.,
1977).
We have examined the ability of chemical carcinogens to stim-
ulate production of 2-hydroxybiphenyl in vitro using both plant and
animal microsomes. The first series of investigations were carried out
to duplicate the experiments and data reported by McPherson, Bridges
and Parke (1976) on the in vitro effects of benzopyrene and safrole on
biphenyl-2-hydroxylase and other drug-metabolizing enzymes using liver
microsomal extracts and microsomes from avocado pear (Persea
americana) (McPerson et al., 1975b). The method used by these
authors was followed exactly.
The investigation of substrates other than biphenyl was also
initiated. Because of the somewhat labile nature of the biphenyl-2-
-35-
-------
hydroxylase, it was thought that a higher order polyphenyl substrate
such as terphenyl might provide useful information.
ENZYMATIC METHODS
Chemicals
Benzo(a)pyrene (BaP) and safrole (SA) were purchased from
Aldrich Chemical Company, a-naphthylamine (a-NA) and p-naphthylamine
(p-NA) were from Sigma, and 20-methylcholanthrene (MC) was from K&K
Chemicals. All were of the highest purity available. These compounds
were dissolved in peanut oil (Planter's) to provide stock solutions at
1 mM. Biphenyl was dissolved in 1.5% (w/v) Tween 80 and 1.15% (w/v)
KC1 to make a 13 mM stock solution. Stock solutions of the hydroxy-
lated standards were made in 5% (v/v) aqueous ethanol at the following
concentrations: 4-hydroxybiphenyl, 0.146 umoles/ml; 2-hydroxybi-
«
phenyl 0.0342 ^moles/ml; 2-hydroxybiphenyl and 4-hydroxybiphenyl,
0.146 |jmoles/ml and 0.146 pmoles/ml, respectively. A thin layer chro-
matogram of the substrate and standard compounds at 0.1 mg material
per spot showed no impurities in the biphenyl or 2-hydroxybiphenyl,
but trace amounts of biphenyl and 2-hydroxybiphenyl in the 4-hydroxy-
biphenyl standard. Meta-terphenyl was recrystallized, and p-terphenyl
(99+%) was purchased from Aldrich Chemical Co. Stock solutions at
13 mM in Tween 80 and 1.15% KCI were made as for biphenyl.
Animals
Swiss-Webster mice (mean weight 37.9 g), Sprague-Dawley
rats (mean weight 172.5 g) and Syrian hamsters (mean weight 118.7 g)
-36-
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were obtained from commercial breeders. Water and food (Wayne Lab
Blocks) were provided ad libitum. Animals were sacrificed between 8:30
and 10:30 a.m. by decapitation.
Preparation of Hepatic Microsomes
Microsomes were prepared by the method of McPherson and
coworkers (1976). The livers were rapidly removed into cold buffered
KC1 (1.15% w/v KC1, 0.3 M NaH2PO4/ 0.3 M K2HPO4, pH 7.6), blotted,
weighed, and placed in fresh cold buffered KC1. The weighed livers
were homogenized with a motor-driven teflon pestle using 10 strokes of
10 seconds each at 1,200 rpm. The homogenate was diluted with cold
buffered KC1 to 250 mg tissue per ml of homogenate and centrifuged
(2°C) for 10 minutes at 15,000 g. In one case, this low speed pellet
was resuspended in buffered KC1 at 25 mg protein/ml and used in a
hydroxylation experiment. The low speed supernatant was decanted
and centrifuged. The supernatant was discarded and the pellet washed
with cold buffered KC1, resuspended in cold buffered KC1, and again
centrifuged (2°C) for 60 minutes at 104,000 g. The final pellets were
resuspended in cold buffered KC1 at a protein concentration of
10 mg/ml. Protein was determined by the method of Lowry and co-
workers (1951).
Preparation of Plant Microsomes
Plant Microsomes were prepared according to the method of
McPherson and coworkers (1975b). Plant material was obtained
24 hours prior to use and stored in the cold. Cauliflower heads were
soaked in cold water for 1 hour before storage to rehydrate the tissue.
-37-
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The mesocarp portion of both avocado and apple were used; rosettes of
cauliflower were shaved from the head. The tissue was weighed, placed
in cold phosphate buffer (0.1 M NaH2PO4, pH 7.4), and homogenized in
either a Virtis homogenizer or Waring blender. Tissues were homo-
genized at 0.5 to 2 g tissue per ml phosphate buffer. The homogenate
was filtered through muslin and centrifuged (2°C) for 20 minutes at
13,500 g. The supernatant was decanted and centrifuged (2°C) for
90 minutes at 80,000 g. The pellets were resuspended in cold phos-
phate buffer and adjusted to 1-10 mg/ml protein with cold buffered
KC1. Protein was determined by the method of Lowry and coworkers
(1951).
Hydroxylation Reactions
Hydroxylation reactions were performed according to the
method of McPherson and coworkers (1976, 1975c). All reactions were
carried out at 37°C in a shaking water bath at 100 cpm. The micro-
somal mixtures were warmed for 60 seconds after addition of the
NADPH-regenerating system. The tenfold concentrated NADPH-regener-
ating system consisted of: glucose-6-phosphate dehydrogenase,
20 lU/ml; glucose-6-phosphate, 25 mM; NADP, 5 mM; MgSO4 0.5 mM,
dissolved in buffered KC1. In the case of the crude homogenates, low
speed supernatants and pellets, and plant microsomes, 1.8 ml of the
preparation was used directly. The final protein concentration in the
2 ml reaction mixture for these preparations is given in Table 3.
Four-tenths ml of the first high speed pellets and purified animal micro-
somes (second high speed pellets) at 10 mg protein/ml was added to
1.4 ml of cold buffered KC1 to provide a final protein concentration of
2 mg/ml in the 2 ml reaction mixture.
-38-
-------
TABLE 3. PROTEIN CONCENTRATIONS IN
HYDROXYLATION EXPERIMENTS
Preparation Protein Concentration^
(mg/ml)
Mouse Homogenate 29.7
Rat Homogenate 27.0
Hamster Homogenate 29.7; 35.9
Mouse Low Speed Pellet 22.5
Hamster Low Speed Supernatant 13.3
Avocado Microsomes 5.8
Apple Microsomes 1.0
Cauliflower Microsomes 5.0; 9.1
Cauliflower Low Speed Supernatant 2.2
1 Two numbers indicate the concentrations in two separate experi-
ments. Protein is given as the final concentration in 2 ml of
reaction mixture.
Each tube received 0.2 ml of the ten-fold concentrated
NADPH-regenerating system. Five-tenths ml of test compound in oil, or
oil alone, was added and incubated for 10 minutes. Biphenyl or ter-
phenyl (0.3 ml of 13 mM) was added, and incubation continued for an
additional 5 minutes. The reaction was terminated by the addition of
1 ml of 4 M HC1 to each tube.
The incubation mixtures used in each hydroxylation experi-
ment are given in Table 4.
Following the addition of HC1 (and standards where indi-
cated), the tubes were immediately extracted with n-heptane as de-
scribed in a previous section of this report.
Separation and Analysis of Metabolites
In the case of the SPF determinations, standard curves were
contructed for each different biological preparation and for every ex-
-39-
-------
TABLE 4. INCUBATION MIXTURES USED IN
HYDROXYLATION REACTIONS1
Microsomal System2
No. 1 + Oil
No. 2 + Oil
No. 3
No. 4
No. 5
No. 6
+ Test Compound
In Oil
+ Test Compound
In Oil
Oil
+ Biphenyl4
+ Biphenyl
+ Biphenyl
+ HC13
+ HC1
+ HC1
+ HC1
+ HC1
HC1
+ Test Compound
In Oil
+ 2- or 4-Hydroxy-
biphenyl + biphenyl
Materials are listed in order of addition from left to right. The micro-
somal system and HC1 were added to all tubes. A blank space indicates
no addition, but continued incubation.
Microsomes or homogenate fractions plus NADPH-regenerating system.
HC1 was added at the end of the incubation period to terminate the
reaction.
Terphenyl was added in place of biphenyl in some experiments. Tube #6
was omitted in these experiments.
-40-
-------
periment using dilutions from the tubes containing known concentrations
of 2-hydroxybiphenyl or 4-hydroxybiphenyl.
Quantitation of metabolites using the HPLC method was accom-
plished by constructing standard curves using three different concen-
trations of 2- and 4-hydroxybiphenyl in the range of 1 to 5 ng per
injection.
Terphenyl metabolites were examined at several excitation-
emission wavelengths.
BIPHENYL HYDROXYLATION
Experimental Results Using Fluorometric Analysis (SPF)
The results obtained from a relatively large number of exper-
iments carried out to either attempt to duplicate the results published
by others or to obtain an initial evaluation of other microsomal extracts
in conjunction with the biphenyl substrate are summarized in Tables 5
and 6.
Table 5 shows the results of three different hydroxylation
experiments using various fractions of hepatic homogenates from mice,
rats, and hamsters. The quantity of 2-hydroxybiphenyl and 4-
hydroxybiphenyl present in each of the reaction mixtures was deter-
mined fluorometrically by the method of Creaven and coworkers (1965).
Benzo(a)pyrene (BaP) was included in all experiments as a known
carcinogen, and where possible, a-Naphthylamine (a-NA) was included
as an example of a non-carcinogen. All other test compounds are
known carcinogens (McCann, et al., 1975).
-41-
-------
TABLE 5. EFFECT OF TEST COMPOUNDS ON PRODUCTION OF 4-HYDROXYBIPHENYL
AND 2-HYDROXYBIPHENYL BY LIVER FRACTIONS
Fraction Animal
Crude Mouse
Homogenate
Rat
Hamster3
Low Speed Hamster
Supernatant
Low Speed Mouse
Pellet
Reaction
Mixture
Oil
BaP
Oil + Biphenyl
Biphenyl + BaP2
BaP + Biphenyl
Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
Bap + Biphenyl
Oil
BaP
SA
Oil + Biphenyl
Biphenyl + BaP
Biphenyl + SA
BaP + Biphenyl
SA + Biphenyl
Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
BaP + Biphenyl
Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
BaP + Biphenyl
n Mole/Min/mg
4-Hydroxybiphenyl
0.007
0.014
0.026
0.033
0.005
0.016
-0-
0.018
0.024
0.011
0.004
-0-
0.006
0.022
0.018
0.034
0.007
0.021
0.016
0.011
-0-
0.142
0.055
0.005
0.009
0.032
0.045
0.032
Protein
2-Hydroxybiphenyl
1.05
1.41
2.55
l!58
0.19
0.54
0.34
0.86
0.92
0.19
0.37
-0-
0.29
O2
0.005
0.78
-0-
0.008
0.020
0.020
0.122
0.075
0.003
0.008
0.005
0.006
0.008
Corrected1
0.17
0.38
0.41
0.055
-0-
-------
TABLE 5 (continued)
CO
Fraction Animal
First High Hamster
Speed Pellet
Second High Mouse3
Speed Pellet
Rat
Reaction
Mixture
Oil
BaP
SA
MC
Oil + Biphenyl
Biphenyl + BaP
Biphenyl + SA
Biphenyl + MC
BaP + Biphenyl
SA + Biphenyl
MC + Biphenyl
Oil
BaP
SA
MC
aNA
pNA
Oil + Biphenyl
Biphenyl + BaP
Biphenyl + SA
Biphenyl + MC
Biphenyl + aNA
Biphenyl + pNA
BaP + Biphenyl
SA + Biphenyl
MC + Biphenyl
aNA + Biphenyl
PNA + Biphenyl
Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
BaP + Biphenyl
n Mole/Min/mg
4-Hydroxybiphenyl
_ _
-0-
-0-
-0-
0.47
--
1.58
1.32
0.51
0.66
0.56
0.21
0.09
0.82
0.29
0.26
0.07
2.54
2.23
3.85
2.68
3.12
3.38
2.86
1.15
4.00
2.97
1.10
0.54
0.13
1.25
1.10
1.01
Protein
2-Hydroxybiphenyl
. _
0.56
0.07
0.37
0.26
--
0.85
1.43
0.54
0.32
0.65
0.06
0.10
-0-
0.12
0.07
0.29
0.008
0.13
-0-
0.26
0.03
0.04
0.034
0.07
0.03
-0-
0.28
-0-
0.10
0.015
0.044
0.074
Corrected x
-0-
O5
0.28
-0-
OT(57
^P~
-0-
-0-
-0-
-------
TABLE 5 (continued)
Fraction Animal
Second High Hamster4
Speed Pellet
Reaction
Mixture
Oil
BaP
SA
MC
aNA
pNA
Oil + Biphenyl
Biphenyl + BaP
Biphenyl + SA
Biphenyl + MC
Biphenyl + aNA
Biphenyl + pNA
BaP + Biphenyl
SA + Biphenyl5
MC + Biphenyl5
aNA + Biphenyl5
pNA + Biphenyl5
n Mole/Min/mg
4-Hydroxybiphenyl
0.12
0.15
0.62
0.25
0.21
0.71
0.93
1.46
2.08
1.29
1.57
1.72
0.78
1.07
1.03
1.04
0.78
Protein
2-Hydroxybiphenyl
0.008
0.20
0.06
0.42
0.13
0.58
0.17
OO
0.23
0.44
0.68
0.65
0.32
0.18
0.30
0.28
0.56
Corrected1
0.12
OTI2
^~
0.15
^0^
The contribution of test compound metabolites as determined in reaction mixtures containing test com-
pound alone was subtracted from the quantity of 2-hydoxybiphenyl apparently present in the test
compound plus biphenyl reaction mixtures.
When the test compound follows the substrate, it was added to the reaction mixture after the addition
of HC1.
Results are the average of two experiments for the oil, oil and biphenyl, and BaP mixtures.
Results are the average of two experiments, 2 to 3 replicates for the oil, oil and biphenyl, and BaP
mixtures.
Results are the average of two replicates in one experiment.
-------
TABLE 6. EFFECT OF TEST COMPOUNDS ON PRODUCTION OF
4-HYDROXYBIPHENYL AND 2-HYDROXYBIPHENYL BY PLANT MICROSOMES
en
i
Fraction
Low Speed
Supernatant
Purified
Microsomes
Reaction
Plant Mixture
4-Hydroxybiphenyl
Cauliflower Oil 0.53
BaP 0.80
SA 0.80
Oil + Biphenyl 0.27
Biphenyl + BaP2 0749
BaP + Biphenyl 0.43
SA + Biphenyl 0.61
Cauliflower3 Oil 0.18
BaP 0.21
SA 0.25
MC 0.036
Oil + Biphenyl 0.15
Biphenyl + BaP
Biphenyl + SA
Biphenyl + MC
BaP + Biphenyl
SA + Biphenyl
MC + Biphenyl
Avocado Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
BaP + Biphenyl
Apple Oil
BaP
Oil + Biphenyl
Biphenyl + BaP
BaP + Biphenyl
0.065
0.081
0.048
0.12
0.21
0.048
0.102
0.082
0.077
OHI3
0.087
0.34
0.31
0.54
0757
0.43
n Mole/Min/mg Protein
Corrected1 2-Hydroxybiphenyl
0.27
0.39
0.18
0.18
0765
-0- 0.31
-0- 0.12
0.085
0.075
0.032
0.039
0.003
07(525
-0-
0.27
-0- 0.032
-0- 0.012
07512 0.036
0.010
0.031
0.005
Grim
0.005 0.005
-0-
0.31
-0-
0720
0.12 -0-
Corrected1
-0-
-0-
-0-
-0-
-0-
-0-
The contribution of test compound metabolites as determined in reaction mixtures containing test com-
pound alone was subtracted from the quantity of 2- or 4-hydroxybiphenyl apparently present in the
test compound plus biphenyl reaction mixtures.
When the test compound follows the substrate, it was added to the reaction mixture after the addition
of HC1.
All except MC are the average of results from two experiments.
-------
It is apparent that the oil which serves as the solvent for the
test compounds can contribute fluorescence at the wavelengths used to
measure both hydroxylated biphenyls. However, the oil is included in
all reaction tubes, including those containing the standard concentra-
tions of the hydroxylated biphenyls, and is therefore corrected for
when the standard curves are constructed. Of more significance is the
observation that incubation mixtures containing the test compound alone
show fluorescence at the wavelength used to determine 2-hydroxybi-
phenyl, and to a lesser extent at the wavelength used to determine
4-hydroxybiphenyl. In essentially each case, the apparent increase in
the amount of 2-hydroxybiphenyl produced in the presence of the test
compound was accounted for by the contribution of the test compound.
The effect of various test compounds on 4- and 2-hydroxy-
biphenyl production by plant microsomes was also examined fluoro-
metrically because McPherson and coworkers (1975c) had reported a
stimulation of 2-hydroxybiphenyl production by 3,4-benzopyrene in
avocado microsome reaction mixtures. The results are presented in
Table 6. In this case, the test compound contributed significantly to
the determination of both 2- and 4-hydroxybiphenyl.
Experimental Results Using High Pressure Liquid Chromatography (HPLC)
Because of the inconsistent and erratic results obtained using
the fluorometric analysis by Creaven et al. (1965), an alternate method
of analysis was investigated. Quantitative HPLC was used to determine
the amount of 2- and 4-hydroxybiphenyl present in incubation mixtures
°f hamster microsomes (first and second high speed pellet). Figure 7
shows that this method provides a complete separation of the two hy-
-46-
-------
droxylated biphenyls in a known mixture. Figures 9 and 10 show
typical chromatograms obtained from incubation mixtures containing test
compounds and biphenyl. The test compounds did not contribute fluor-
escent material to the 2- and 4-hydroxybiphenyl peaks under the con-
ditions of this assay. Table 7 presents quantitative data obtained from
these chromatograms. It can be seen that using this method, the
amounts of 2- and 4-hydroxybiphenyl produced in the reaction contain-
ing substrate alone agree with the amounts determined using the fluor-
ometric method (Table 5). However, production of 2-hydroxybiphenyl
was not stimulated by preincubation with test compounds.
In the case of BaP, an attempt was made to determine
whether the test compound itself or its metabolites were contributing to
the fluorescence at the 2-hydroxybiphenyl wavelength using the HPLC
method. Samples from reaction mixtures containing BaP were chroma-
tographed on the HPLC and examined at the excitation-emission couples
of: Ex 300, Em 405 and Ex 300, Em 422. Although BaP itself was
detected at these wavelengths (average retention time 5.6 minutes), no
metabolites could be detected. It is possible that the metabolites were
present in undetectable amounts or that they eluted with the early
material.
Discussion
Using a high pressure liquid chromatography system which
permits unequivocal identification of hydroxylated biphenyls, it has
been possible to demonstrate, in contrast to the findings of others
(Creaven et al., 1965; McPherson et al., 1976, 1975 a,b,c, 1974 a,b;
Tredger et al., 1976; Tong et al., 1977), that the amount of 2-
-47-
-------
TABLE 7. EFFECT OF TEST COMPOUNDS ON
PRODUCTION OF 4- AND 2-HYDROXYBIPHENYL
AS DETERMINED BY QUANTITATIVE HPLC
n Mole/Min/mg-Protein Ratio of
Reaction Mixture1 4-Hydroxybiphenyl 2-Hydroxybiphenyl 4-OH/2-OH
First High Speed Pellet
Oil + Biphenyl
BaP + Biphenyl
SA + Biphenyl
MC + Biphenyl
BaP
Second High Speed Pellet
Oil + Biphenyl
BaP + Biphenyl
SA + Biphenyl
MC + Biphenyl
aNA + Biphenyl
PNA + Biphenyl
Oil
BaP
0.64
0.36
0.26
0.55
0.00
0.59
0.26
0.19
0.24
0.30
0.14
0.00
0.04
0.16
0.14
0.20
0.21
0.00
0.22
0.15
0.17
0.16
0.18
0.09
0.00
0.02
4.0
2.6
1.3
2.6
—
2.7
1.7
1.1
1.5
1.7
1.6
—
Hamster microsomes were used.
-48-
-------
hydroxybiphenyl remains constant and the amount of 4-hydroxybiphenyl
decreases in the presence of carcinogens and non-carcinogens using
animal microsome incubation mixtures. Similarly, plant microsome mix-
tures show an apparent total lack of hydroxylation of biphenyl in the
presence of carcinogens. The test compound or its metabolites contri-
bute fluorescence at the wavelengths used to measure the hydroxylated
biphenyls thus causing an apparent increase in 2-hydroxybiphenyl when
the fluorometric method of Creaven and coworkers (1965) is used.
Burke and coworkers (1977) and Tong and coworkers (1977) have
recently suggested that this may be a problem when using this assay.
McPherson, Bridges and Parke (1975a), using radioactive
biphenyl and TLC separation methods reported a 2.5 fold stimulation of
biphenyl-2-hydroxylase by BaP in vitro. Using HPLC methods, this
result was not duplicated and it is not possible at this time to explain
this discrepancy.
TERPHENYL METABOLISM
Results
Purified hamster microsomes were used to examine the in vitro
metabolites of terphenyl and the effect of carcinogens on their produc-
tion. Incubation conditions were identical to those used for biphenyl.
The reaction mixtures were extracted with 10 ml of n-heptane, and
10 pi of this was injected into the HPLC.
Because the fluorescence spectra of possible metabolites was
not known, a number of different excitation-emission wavelengths were
used to examine the chromatograms for possible terphenyl metabolites.
-49-
-------
In each case, unaltered m-terphenyl was observed at an average reten-
tion time of 3.1 minutes. A total of three different m-terphenyl meta-
bolites were detected at two different excitation-emission wavelength
pairs (Table 8).
In an initial experiment, a reaction mixture containing m-ter-
phenyl as a substrate was preincubated for 10 minutes with BaP. The
n-heptane extract was chromatographed and examined at \£X = 270,
\£,. = 350. Figure 13 shows that preincubation with BaP caused a loss
of the metabolite having the 15 minute retention time, and an apparent
decrease in the second (17 minute) metabolite.
This experiment was repeated using several test compounds
and m-terphenyl as the substrate. Because the chemical nature of the
metabolites is not known, it was not possible to construct standard
curves. An auto-oxidation control mixture containing m-terphenyl
without microsomes was included in the experiment. The chromatogram
TABLE 8. METABOLITES OF m-TERPHENYL PRODUCED
BY PURIFIED HAMSTER MICROSOMES
XEX
XEX
AT* v
£j^\.
XEX
\
wavelength
(nm)
= 270, X£M =
= 250, X£M =
= 270, X£M =
= 300, X£M =
= 300, X™, =
Peak heights (mm)1 at retention times of:
360
360
350
360
335
15.1 - 15.3 min.
5.8
2.2
5.4
--
__
17.2 - 17.3 min. 18.9 min.
14.8 -2
5.5
15.0
7.0 9.0
3.0 4.0
1 Peak widths at half-height were identical for each metabolite.
2 Not present.
-50-
-------
r
on'
• o 18 to
IHW.I
B
A—x
a
IMHLI
Figure 13. Material separated by HPLC obtained from reaction
mixtures containing purified hamster microsomes and
(A) oil plus m-terphenyl; (B) BaP in oil plus m-ter-
phenyl. The excitation wavelength was 270 nm and the
emission wavelength was 350 nm.
-51-
-------
of the n-heptane extract of this mixture showed no metabolites. In
addition, incubation mixtures containing test compounds without
terphenyl showed no material at the retention times of the terphenyl
metabolites.
Table 9 presents the results of this experiment. The last
five reaction mixtures in the table are those in which the oil or test
compound in oil was added follwing termination of the reaction with HC1.
In general, metabolite production in these mixtures was higher than
when the oil was added during the incubation. This suggests that the
oil may inhibit the oxidative metabolism to some extent.
The results presented in Table 9 do not substantiate those
obtained in the first experiment. There was apparently no effect, or a
general decrease in metabolite production in the presence of all test
compounds in the second experiment. The major difference between the
two experiments was that the incubation of the microsome reaction
mixtures was conducted in normal laboratory light (fluorescent) in the
first experiment, whereas incubations were performed in dim yellow
light in the second experiment. It is interesting to speculate that these
conditions alter the sensitivity of the enzyme(s) to test compounds.
A preliminary experiment was performed in which p-terphenyl
was incubated with purified hamster microsomes, and an n-heptane
extract prepared. The excitation and emission spectra of n-heptane
extracts of both m- and p-terphenyl substrates incubated with purified
hamster microsomes are shown in Figures 14 and 15. Extracts of mix-
tures containing either substrate without microsomes did not show emis-
sion peaks under these excitation-emission conditions. It can be seen
that p-terphenyl is also metabolized by the microsomes to produce an
emission spectrum very similar to that of the metabolized m-terphenyl.
-52-
-------
TABLE 9. METABOLITES OF m-TERPHENYL
PRODUCED IN THE PRESENCE OF CARCINOGENS
area (nm2) of peaks at retention times (min) of:
\EX=270 nm, \£M=360 nm A£x=300 nm, \EM=360 nm
reaction mixture1 15.0
oil + terphenyl + HC12
BaP/oil +
aNA/oil +
pNA/oil +
terphenyl + HCl
terphenyl + HCl
terphenyl + HCl
MC/oil + terphenyl + HC1
terphenyl
terphenyl
terphenyl
terphenyl
terphenyl
+ HCl + oil3
+ HCl + BaP/oil
+ HCl + aNA/oil
+ HCl + pNA/oil
+ HCl + MC/oil
104
103
88
79
93
112
122
110
140
158
17.1
310
245
200
208
240
298
292
372
435
371
18.9
72
46
38
34
42
44
34
49
66
59
14.9
54
64
53
68
68
75
82
82
88
105
17.0
72
76
68
76
78
94
118
94
106
128
18.6
206
197
188
198
202
280
245
240
312
299
1 Relevant compounds are given in the order of addition to the incubation
mixtures.
2 For the first five reaction mixtures, each number is the average of
results obtained from two different mixtures.
3 For the last five reaction mixtures, each number was obtained from one
incubation mixture.
-53-
-------
3U
>• V)
£ -»J
to
z
ltl
I
9rt
;> tvJ
UJ
CE
ID
Iv
Em 3
,
-
2'
35
/
/
1 ^^
*O 260 21
/•T"\
/ 1 \
i i •
/ i \
' \
' \
i \
i \
i \
i \
i \
, \
t \
i \
i \
i \
i \
\
29S \
i
» 300 3!
X
A
X
>0 34
4OO
Figure 14. Excitation and emission spectra of an n-heptane extract
of purified hamster microsomes incubated with m-ter-
phenyl. (A) Excitation spectrum with emission measured
at 335 nm; (B) emission spectrum with excitation at
295 nm; \ = wavelength in nm.
-54-
-------
2
UJ
UI
>
UJ
o:
Em 3
-
-
-
-
IO
I
/f\
A
/ i
/
/3I3
/ — — 1
A
i
i
i
\
\
\
\
24O 260 280 300 320 34
WAVELENGTH (NANOMETERS)
420
Figure 15.
Excitation and emission spectra of n-heptane extracts of
purified hamster microsomes incubated with p-terphenyl.
(A) Excitation spectrum with emission measured at
340 nm; (B) emission spectrum with excitation at 313 nm;
\ = wavelength in nm.
-55-
-------
Discussion
Meta-terphenyl is metabolized in vitro by purified hamster
microsomes to a minimum of three compounds. Preliminary data sug-
gests that, under some conditions, the production of at least one of
these metabolites may be inhibited by carcinogens such as BaP.
Further experimentation is necessary in order to validate this result
and determine the conditions under which inhibition occurs. Para-
terphenyl is also metabolized by hamster microsomes, but the number
and nature of the metabolites is not known. Further investigation of
this substrate may provide information complementary to that obtained
with the m-terphenyl substrate.
-56-
-------
SECTION 6
METABOLISM OF BIPHENYL BY PARAURONEMA ACUTUM
GENERAL
Previous work (Schmidt-Collerus and Tame, unpublished) has
shown that algae such as Chlorella pyranoidosa exhibit rapid oxidative
metabolism of poly cyclic aromatic hydrocarbons. This indicates that
these organisms possess very effective oxidative enzymes. It is be-
lieved that similar strong activity may be present in marine algae and
possibly ciliates. The use of algae or ciliates as a source of biphenyl
hydroxylases has several advantages: (1) they can be prepared rapid-
ly and inexpensively, (2) because of the large variety of these organ-
isms, hydroxylases specific for various substrates or new enzymes
sensitive to various classes of mutagens or carcinogens may be found,
and (3) the enzymes in these organisms may not be membrane-bound,
and may therefore be more useful in an assay system than microsomal
enzymes.
Parauronema acutum was selected for study because it is a
marine protozoan which can be grown in defined culture in the labora-
tory, and therefore is a possible indicator of pollution in the marine
environment. In addition, P. acutum has been shown to activate 2-
aminofluorene but not BaP, and the enzyme(s) responsible is distributed
among soluble and particulate fractions (Lindmark, 1978). The
hydroxylases of this organism may therefore be more stable than those
which are microsome-associated.
-57-
-------
Preliminary experiments in this laboratory examined the ability
of this protozoan to metabolize biphenyl in vivo.
MATERIALS AND METHODS
Growth Medium
P. acutum was maintained in still culture in 50 ml sterile
baffle flasks containing 10 ml of medium at 22°C in the dark. The
growth medium (Lindmark, D. G., personal communication) consisted of:
20 mg asolectin, 20 mg cephalin, 20 mg Tween 80, 75 ml artificial sea
water (sp. gr. 1.023), 1 g protease peptone, 1 g trypticase, 0.1 g
yeast nucleic acid, and 1 ml vitamin mix per 100 ml final volume. The
pH was adjusted to 7.2, and the medium sterilized by autoclaving for
15 minutes at 121°C. The vitamin mix consisted of: 0.001 pg/ml bio-
tin, 1.0 |jgMl calcium pantothenate, 0.5 \ig/m\ each of folic acid, nico-
tinamide, pyridoxal-HCl and riboflavin, 1.5 vg/ml thiamine-HCl, and
0.01 M9/ml DL-thioctic acid.
Substrate and Test Compounds
A stock solution of biphenyl in dimethylsulfoxide (DMSO) was
prepared at 200 mM. Biphenyl (20 mM) in Tween 80 was prepared by
dissolving 30.8 mg of biphenyl in 0.15 g Tween 80 in a water bath with
heating. P. acutum growth medium (10 ml) was then added slowly to
form an emulsion. For Tween 80 controls in which the biphenyl was
omitted, 0.15 g of Tween 80 was added to 10 ml of growth medium to
provide a stock solution. The DMSO carrier was reagent grade. The
BaP was prepared at 20 mM in DMSO. The 2- and 4-hydroxybiphenyl
-58-
-------
standards were prepared at 24.8 ng/ml each in aqueous 5% (v/v)
ethanol.
Experimental Procedure
Cultures were prepared by adding 0.5 ml of an inoculum
culture to 10 ml of medium in sterile 50 ml baffle flasks. Cultures were
incubated without shaking in the dark at 25°C or 22°C.
Cells were fixed in 10% formaldehyde and counted daily using
a hemocytometer. Viabilities were determined on duplicate, unfixed
samples. Test compounds were added when the cell density was ap-
proximately 1 x io5 cells per ml. Duplicates were made for each treat-
ment. After addition of test compounds, cells were counted daily until
the untreated cultures entered the decline phase of the growth curve.
At that time, 2.5 ml of 8N^ HC1 was added to each flask to terminate the
experiment. Biphenyl, 4-hydroxybiphenyl and 2-hydroxybiphenyl were
added to untreated cultures after HC1 addition. These served as ex-
traction controls. All manipulations following HC1 addition were carried
out in dim yellow light.
Following HC1 addition, a 5 ml aliquot of each culture was
transferred into each of two glass tubes equipped with teflon-lined
screw caps. Microscopic examination of the HCl-treated culture showed
that not all cells were lysed by acidification. Therefore, one of the
replicates of each culture was frozen (-80°C) and thawed twice to
•
ensure disruption of the cells. The solutions were then each extracted
with 10 ml of n-heptane by shaking at room temperature for 10 minutes.
Samples were centrifuged at 2,000 rpm for 10 minutes to separate the
layers, and stored at 4°C in the dark. The heptane layer was removed
and concentrated to approximately 0.5 ml under a nitrogen stream.
-59-
-------
HPLC Analysis
The concentrated n-heptane extracts were analyzed for
biphenyl metabolites using HPLC combined with SPF as described
previously.
RESULTS
The effect of addition of biphenyl on growth of P. acutum
was examined at both 22 and 25°C. In addition, the effect of the
carrier (DMSO or Tween 80) on the ability of the organism to respond
to biphenyl was also examined.
Figure 16 shows that, at 22°C biphenyl dissolved in DMSO at
final concentrations in the culture of above 0.2 mM caused immediate
death and lysis of the cells. The lower concentration allowed normal
growth of the culture in terms of cell counts. The loss in viability of
the cultures was caused by the biphenyl and not the DMSO carrier.
However, when Tween 80 was the carrier, the lethal effect at higher
concentrations of biphenyl was decreased.
Figures 17 and 18 show the effect of biphenyl at 25°C. In
this case there appeared to be no difference between DMSO or
Tween 80, with cultures being unaffected by 0.2 mM concentrations of
biphenyl. Extracts of these cultures were examined by HPLC-SPF in
order to quantitate the metabolites produced. The results are shown in
Table 10. Numbers are provided for those extracts in which metabolites
were detected. It can be seen that both 2- and 4-hydroxybiphenyl
were produced, and that neither carrier nor medium produced material
which interfered with metabolite determination. The results obtained
-60-
-------
o NO ADDITIONS
• O.ZmM biphcnyl
in DMSOl.OI/lOml)
ADMSO (.01/10 ml)
0.9 mM biphcnyl In
DMSO(.02S/IOml)
OMSO 1.025/IOml)
I.OmM blplwnyl in
OMSOl.OS/IOml)
DMSO(.OVWml)
A 0.2 mM biphcnyl In
1.5x10*% Tw««n
A 1.5x10"'% Twatn
• 0.4 mM blplunyl In
3x 10^% T*«an
o 3 * IO"2 % Twe«n
Figure 16.
Growth of Parauronema acutum in the presence of bi-
phenyl at 22°C. The arrows indicate time of addition of
biphenyl or carrier (7 days), and each point is the mean
of the cell counts from at least two different cultures in
one experiment.
-61-
-------
O NO ADDITIONS
< A 0.2 mM blphcnyl In
DMSOl.OI/IOml)
• DMSO(.OI/IOml)
A 1.0 mM blphtnyl in
DMSO(.OVlOml)
A DMSO( 05/IOml)
• 2.0mM bfphtnyl In
DMSO(.OI/IOml)
O DMSOl.OI/lOml)
ui
o
xlO'
Figure 17. Growth of Parauronema acutum at 25°C in the presence
of biphenyl dissolved in DMSO. The arrows indicate
time of addition of biphenyl or carrier (2 days), and
each point is the mean of the cell counts from at least
two different cultures in one experiment.
-62-
-------
O NO ADDITIONS
• Q3mM biphmyl In
1.5x10'*% Tw««n
A I.SxlO'2%Tw«>n
• 1.0 mM blphtnyl In
7.5xUT*%Twt«n
O 7.5x(0'*%Tw«en
A 2 OmM biphcnyl In O.I5%Tw««n
A O.I5%Tw««n
xlO
(A
UJ
O
0 I
Figure 18. Growth of Parauronema acutum at 25°C in the presence
of biphenyl dissolved in Tween 80. The arrows indicate
time of addition of biphenyl or carrier (2 days), and
each point is the mean of the cell counts from at least
two different cultures in one experiment.
-63-
-------
TABLE 10. QUANTITIES OF 2- AND 4-HYDROXYBIPHENYL PRESENT
IN EXTRACTS OF PARAURONEMA ACUTUM CULTURES1
additions to
incubation
mixture
cells + medium2
0.2 mM biphenyl
in DMSO (.01/10)3
1.0 mM biphenyl
in DMSO (.05/10)
DMSO (.01/10)
DMSO (.05/10)
0.2 mM biphenyl
in 1.5~x 10" 2%
Tween2
1.0 mM biphenyl
in 7.5 x 10"2%
Tween2
1.5 x io"2%
Tween
7.5 x 10"2%
Tween
treatment
before
extraction
none
frozen
none
frozen
none
frozen
none
frozen
none
frozen
none
frozen
none
frozen
none
frozen
none
frozen
ng
2-hydroxy-
biphenyl
-0-
-0-
0.43
0.44
0.43
0.40
-0-
-0-
-0-
.10
0.75
0.80
0.34
0.20
-0-
-0-
-0-
0.35
4-hydroxy-
biphenyl
-0-
-0-
1.26
0.98
0.60
3.24
1.06
-0-
-0-
-0-
1.38
3.12
0.40
1.05
-0-
-0-
-0-
0.45
ratio
4-OH/
2-OH
--
--
2.9
2.2
1.4
8.1
--
— —
--
-—
1.8
3.9
1.2
5.2
--
— —
--
Cultures were grown at 25°C. Growth data are presented in
Figures 17 and 18.
Numbers are the average obtained from two different culture
flasks. All others represent one flask.
Numbers in parentheses indicate the volume (ml) of biphenyl in
DMSO or DMSO alone added to the 10 ml of medium.
-64-
-------
using Tween 80 as a carrier seem to indicate that the 4-hydroxybi-
phenyl metabolite may be located intracellularly in a form which is
released by freezing and thawing the cells.
Figure 19 shows the effect of addition of BaP and biphenyl on
culture growth. The data for cultures in which DMSO alone in the
appropriate amounts was added in the place of test compounds were
identical to the data obtained for growth in the absence of additions,
and have been omitted. It is apparent that addition of BaP alone at
5 days has no effect on growth of the cultures. In this experiment,
addition of 0.2 mM biphenyl caused death of the culture after a short
lag. In the case of BaP and biphenyl addition, it appears that the rate
of death of the cultures is decreased. It is known (Lindmark, 1978)
that BaP is not metabolized by P. acutum. However, BaP may interact
with the pellicle in such a way as to allow it to metabolize, and there-
fore detoxify, biphenyl more readily.
DISCUSSION
Parauronema acutum metabolizes biphenyl to form 2- and
4-hydroxybiphenyls. This organism is therefore a likely source of
hydroxylases for in vitro studies of the effects of carcinogens. In
addition, BaP may interact with the pellicle of the organism to allow it
to more efficiently metabolize biphenyl.
-65-
-------
• NO ADDITIONS
O 0.2 mM blphtnyl in DMSOot 6 day*
A 0.2mM BoPlnDMSOo15day*+0.2mM
blph«nyl In DM SO at 6 day«
A DMSOat 5day* + 0.2mM blph. at 6 day*
• 0.2mM BoPinOMSOolSdoy*
O Z mM BaP In OMSO at 5 day*
A 2mM BoPot5doy«,0.2mM blph.
at 6 day*
A IOOXDMSO»0.2mM Mph. at 6 day*
•.O -
Figure 19. Growth of Parauronema acutum in BaP and biphenyl at
25°C. The arrows indicate time of addition of test
compounds, and each point is the mean of the cell counts
from at least two different cultures in one experiment.
-66-
-------
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