[CANCER RESEARCH 45, 4838-4843,
October 1985]
Metabolism of Benzo(a)pyrene by Subcellular Fractions of Rat Liver: Evidence
for Similar Patterns of Cytochrome P-450 in Rough and Smooth
Endoplasmic Reticulum but not in Nuclei and Plasma Membrane
F. Oesch, P. Bentley, M. Golan, and P. Stasiecki
Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-6500 Mainz, Federal Republic of Germany
ABSTRACT
Since our earlier work (P. Stasiecki, F. Oesch, G. Bruder, E. D.
Jarasch, and W. W. Franke, Eur. J. Cell Biol., 21: 79-92, 1980)
had shown that carcinogen-metabolizing monooxygenase activ
ity was present in almost all investigated cellular membranes,
the possibility of differential control of the various metabolic
pathways in the individual cellular membranes arose. Using high
pressure liquid chromatography we have now studied the
benzo(a)pyrene metabolites formed by rough and smooth endoplasmic reticulum, nuclei, and plasma membrane as well as
mitochondrial fractions and investigated the metabolic coopera
tion between the monooxygenases and epoxide hydrolase in
these fractions. Since various cytochrome P-450 isozymes cat
alyze the oxidative attack on the benzo(a)pyrene molecule at
defined preferential sites, this analysis also provides an indirect
trace of potential differences in the pattern of cytochrome P-450
isozymes present in the individual membranes.
The metabolic profiles produced by the two most active frac
tions, smooth and rough endoplasmic reticulum, were very sim
ilar to each other but different from those produced by the other
three preparations. The metabolite pattern produced by incuba
tions containing nuclear fractions differed slightly from that pro
duced by the fractions of endoplasmic reticulum, but plasma
membrane and mitochondria produced markedly different pat
terns.
Since the similarity of the benzo(a)pyrene metabolite pattern
produced by the smooth and rough endoplasmic reticulum sug
gested similar cytochrome P-450 isozyme patterns in these two
subfractions, they were further investigated by the use of selec
tive ¡nducersas well as a broad spectrum substrate, 7-ethoxycoumarin, in the absence and presence of selective inhibitors.
Treatment of animals with frans-stilbene oxide or phénobarbital
were similar in the smooth and rough endoplasmic reticulum
fraction.
These findings indicate that the composition of cytochrome P450 isozymes is different in nuclei, plasma membrane, mitochon
drial fractions, and the endoplasmic reticulum but that the isoenzyme pattern of smooth and rough endoplasmic reticulum is
similar even after enzyme induction.
The total amount of benzo(a)pyrene metabolites produced by
smooth and rough endoplasmic reticulum fractions from controls
and from the animals treated with the various inducers correlated
well with 7-ethoxycoumarin O-deethylase activities in absence
of inhibitors and in presence of metyrapone or tetrahydrofuran (r
= +0.983, +0.995, +0.990) but not in the presence of anaphthoflavone (r = -0.225), indicating that the monooxygenase
forms inhibited by a-naphthoflavone were major contributors to
benzo(a)pyrene metabolism in all these fractions. No good cor
relation was found between dihydrodiol formation and epoxide
hydrolase activity. In line with the relatively high ratio of epoxide
hydrolase to monooxygenase in rat liver microsomes, this indi
cates that in all these fractions epoxide hydration is not the ratedetermining step in the formation of the dihydrodiols studied.
INTRODUCTION
The metabolism of carcinogenic polycyclic aromatic hydrocar
bons is mediated by a complex mixture of enzymes which are
mainly localized in membranous fractions of mammalian hepatocytes. These enzymes are responsible for the formation of
ultimate carcinogens and for the detoxification of reactive metab
olites (11,17, 20, 24, 25, 27, 28, 33, 34, 37). The monooxygen
ase system, which consists of NADPH-cytochrome P-450 reductase and several cytochrome P-450 isoenzymes with overlapping
substrate specificities as well as stereo- and regioselectivities (9,
(a) increased the total amount of metabolites per protein mass
16, 23, 24, 36, 41 ), is responsible for the conversion of aromatic
and time, (b) changed the pattern of metabolites, but (c) induced
hydrocarbons to epoxides, phenols, and quiñones (5, 12, 18a pattern of metabolites which was again very similar in rough
20, 33). The monooxygenase products are then further trans
and smooth endoplasmic reticulum. Even more distinct changes
were found following treatment with 3-methylcholanthrene or ß- formed for example by epoxide hydrolase (1). Its products can
be further processed by the cytochrome P-450-dependent mon
naphthoflavone. Both of these compounds (a) preferentially in
ooxygenase system (33, 36, 43), and some of these reaction
duced the activity of rough endoplasmic reticulum, (b) changed
products appear to be extremely mutagenic and carcinogenic
the profile of metabolites, but (c) again did not disturb the
(19,21,26,42).
similarities of the benzo(a)pyrene metabolite pattern between
We have recently observed that the cytochrome P-450-deboth fractions. Selective inhibitors (metyrapone, a-naphthoflapendent monooxygenase system is present in almost all inves
vone, tetrahydrofuran) had characteristically different effects on
tigated cellular membranes (34). Individual cytochrome P-450
7-ethoxycoumarin 0-deethylase activity in preparations from un
isoenzymes possess defined preferential sites of oxidative attack
treated versus frans-stilbene oxide (or phenobarbital)-treated
on
larger molecules such as benzo(a)pyrene. The composition
versus /3-naphthoflavone (or 3-methylcholanthrene)-treated rats,
of individual cytochrome P-450 isoenzymes may vary in different
but in any of these situations the effects of the selective inhibitors
cellular membranes, so that the control of reactive metabolites
Received 6/28/83; revised 12/5/84, accepted 6/3/85.
and thereby the pattern of the more stable metabolites may also
CANCER RESEARCH VOL. 45 OCTOBER
1985
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BENZO(/4)PYRENE
METABOLISM
FRACTIONS
13) using a Spinco 35 rotor. The resuspended rough ER2 pellet and the
vary in different subcellular compartments.
Besides the obvious interest of information on metabolic con
trol in individual subcellular compartments, the study of the
metabolite pattern generated from benzo(a)pyrene in various
subcellular fractions provides an indirect trace of the various
cytochrome P-450 isoenzymes present in these fractions, since
the lack of highly nucleo- or electrophilic regions in the reso
nance-stabilized polycyclic aromatic hydrocarbons leads to a
great number of metabolites rather than to a single or very few
favored products.
In the present study we have therefore investigated the
benzo(a)pyrene metabolite pattern generated by smooth and
rough endoplasma reticulum, mitochondria, nuclei, and plasma
membrane fractions, which were characterized chemically, mor
phologically, and biochemically. The characteristic features which
were observed in the subfractions of the endoplasmic reticulum
were investigated more closely by the use of selective inducers1
smooth ER interface were centrifugea separately on a second discontin
uous gradient (same conditions as for the first centrifugation) to improve
the separation. Mitochondria were isolated according to the method of
Guerra (15). Isolation of nuclei was performed as described by Blobel
and Potter (2). Plasma membranes were isolated by the method of
Doriing and LePage (10). Preparations were controlled routinely by
biochemical, chemical, and morphological characterization as already
described (34) (data are given in Table 1).
Chemical Determinations and Enzyme Assays. Proteins were mea
sured by the method of Lowry et al. (22) with bovine serum albumin as
a standard. RNA was determined according to the microassay described
by Boer (3). Monooxygenase activity was measured as the O-deethylation of 7-ethoxycoumarin (38). Epoxide hydrolase was assayed fluorimetrically with benzo(a)pyrene 4,5-oxide (7).
Determination of the Benzo(a)pyrene Metabolite Pattern by High
Pressure Liquid Chromatography. Benzo(a)pyrene and subcellular ma
terial were incubated under conditions modified from those used by
Selkirk ef al. (32). The incubation mixture (total volume, 10 ml) consisted
of 50 mm potassium phosphate (pH 7.4), 5 ITIMMgCI2, 0.62 ÕTIM
NADPH,
0.36 mM NADH, 8 HIM glucose 6-phosphate, 2.5 Komberg units of
glucose-6-phosphate dehydrogenase, 5 mg of bovine serum albumin,
and inhibitors (4, 31, 39).
MATERIALS AND METHODS
and approximately 3 mg of endoplasmic protein. The protein content of
freshly prepared biological material was determined before the incuba
tion. When non-ER fractions were assayed the protein concentration
Chemicals. 7-Hydroxycoumarin (E. Merck, Darmstadt, Germany) was
recrystallized and the sodium salt was alkylated with ethyl iodide in
absolute ethanol under reflux for 8 h. After two recrystallizations white
crystals with a melting point of 91 °Cwere obtained. Benzo(a)pyrene 4,5-
was increased: plasma membrane, 15 mg; nuclei, 12 mg; and mitochon
dria, 60 mg. These amounts were limited either by the yield of the
preparation or the requirement of linearity with protein concentration.
The reaction was started by the addition of 0.95 /¿molbenzo(a)pyrene
(G-3H-labeled material diluted with the cold compound and purified as
oxide was prepared as described by Oansette and Jerina (8), and the
frans-4,5-dihydrodiol was prepared by incubating the 4,5-oxide with rat
liver microsomes as described (30). All other metabolites of benzo(a)-
described above) which was dissolved in ethanol. After 20 min incubation
at 37°Cin the dark, the reaction was stopped by addition of 30 ml chilled
pyrene were obtained from the Standard Chemical Carcinogen Reference
Repository, National Cancer Institute, Bethesda, MD.
The following chemicals were purchased: Boehringer, Mannheim,
Germany, glucose 6-phosphate, glucose-6-phosphate
dehydrogenase;
EGA-Chemie, Steinheim, Germany, benzo(a)pyrene, irans-stilbene oxide;
Fluka, Buchs, Switzerland, 3-methylcholanthrene, a-naphthoflavone; E.
Merck, sodium phénobarbital; Riedel-de-Haen, Seelze, Germany, 0-
ethyl acetate:acetone
was repeated twice.
drous Na2S04, they
dissolved in 100 /il
(2:1, v/v) and extracted for 10 min. The extraction
After the combined extracts were dried with anhy
were evaporated to dryness. The products were
ethanol and stored at -18°C under an argon
atmosphere prior to HPLC analysis. The analysis was carried out at
30°C with a Spectra Physics SP 3500 B high performance liquid Chro
naphthoflavone; Sigma, Munich, Germany, bovine serum albumin, rRNA;
Zinsser, Frankfurt, Germany, Unisolve I.
Chemicals which were used as inducers or inhibitors were purchased
at the best quality available and in addition vigorously purified by repeated
recrystallization. Other chemicals not mentioned in the above list were
analytical grade reagents from E. Merck, C. Roth (Karlsruhe, Germany),
or Serva (Heidelberg, Germany).
Radiochemicals. |G-3H|Benzo(a)pyrene (15 to 30 Ci/mmol) was pur
matograph fitted with a Spherisorb ODS 10-tim column (2100 theoretical
plates). The calibration was performed with standards [diols, quiñones,
and phenols of benzo(a)pyrene]. The initial concentration of the acetonitrile:water mixture was 55% acetonitrile (v/v); the final one was 85%.
The gradient was 0.25%/min; the elution flow was 0.4 ml/min.
Benzo(a)pyrene and its metabolites were detected photometrically at
280 nm. In the elution sequence diols, quiñones, and phenols was
identical with that observed in other studies (cf. Réf.32). The concentra
tions of metabolites of the incubation mixture were determined continu
ously on a flow counter using Unisolve I as scintillator.
chased from Amersham Buchler (Braunschweig, Germany). The com
pound was purified on a Spectra Physics SP 3500 B high performance
liquid Chromatograph fitted with a Spherisorb ODS 10-Mtn column (final
purity, >99%). Acetonitrile was used as an elution liquid.
Animals and Membrane Isolation Procedures. Male Sprague-Daw-
RESULTS AND DISCUSSION
ley rats purchased from SüddeutscheVersuchstierfarm, Tuttlingen, Ger
many, weighing approximately 130 to 180 g were used routinely. For
induction studies the animals were given ¡.p.injections of either sodium
phénobarbital(80 mg/kg body weight) in buffered saline solution or frans stilbene oxide (400 mg/kg body weight) in sunflower oil once daily for 3
days before death. Inductions with 3-methylcholanthrene and 0-naphtrioflavone were performed by a single i.p. application (80 mg/kg body
weight, finely suspended in sunflower oil) on the third day before death.
Animals were starved for 14 h before being killed. Livers were minced
into small pieces. The homogenization was performed using a Teflon
homogenizer in a volume twice that of the liver. All preparation steps
were carried out at 0-4°C. Rough and smooth endoplasmic reticulum
were separated on a discontinuous
IN SUBCELLULAR
CsCI-containing sucrose gradient (6,
Studies on the Metabolism of Benzo(a)pyrene by Subcel
lular Fractions. The criteria for purity of the subcellular fractions
used are shown in Table 1. The estimated cross-contamination
by other fractions was always less than 10%.
The metabolite profiles obtained following incubation of
benzo(a)pyrene with the different subcellular fractions are shown
in Table 2. When fractions were isolated from control animals
the smooth endoplasmic reticulum was most active, metabolizing
about twice as much benzo(a)pyrene as does rough endoplasmic
reticulum per time and protein amount. Nuclei, plasma membrane
fractions, and mitochondria were less active. A good correlation
(r = 0.98) was found between the total amounts of metabolites
1The term induction is used in this study in its broad sense to denote an increase
2The abbreviation used is: ER, endoplasmicreticulum.
in enzyme activity.
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RESEARCH
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1985
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BENZO(/»)PYRENE METABOLISM
IN SUBCELLULAR
FRACTIONS
Table1
Purity of the fractions
The purity of the fractions was assessed by determinations of cross-contamination using morphometry and marker enzyme activities. Characteristic weight ratios of
macromolecule classes in the fractions are also presented. Enzyme activities are based on the phospholipid contents and were determined in fractions which (except the
nuclei) had been treated with sonication and extracted with high salt concentrations. The specific activity of glucose-6-phosphatase in rough ER was 0.89 ±0.12 «mol/
min/mg phospholipid and that of 5'-nucteotidase in plasma membranes was 3.1 ±0.5 (SD) ^mol/min/mg phospholipid. The concentration of cytochrome aa3 in
mitochondrial membranes was 2.3 ±0.35 nmol/mg phospholipid. Values represent means or ranges from 5 or more experiments.
membrane<4<533-452-3<10.320.05Total
ER68-8010-2080-906-82-30.300.400.11NDRough
ER90-946-81006-9<10.280.370.120.04Smooth
ER5-1083-85"65-805-82-30.320.480.030.02Golgi
apparatus6-88-157-106-103-50.320.500.0150.010Plasma
membrane2-48-102-41002-30.400.55NO"0.007Mitoch
Portion
membranearea
of total
asRoughclassified
(%)TotalER
smoothmembranes
unidentified
(%)Glucose-6-phosphatase
(%specific
ofrough activity
ER)5
' -Nucieotidase
specificactivity
(%
of plasma mem
brane)Cytochrome
aa3 (% concen
mitochondrialmembrane)Phospholipid:protein
tration of
(w/w)Before
treatmentAfter
high salt
treatmentRNA:
high salt
(w/w)Before
protein
treatmentAfter
high salt
high salt treatmentNuclei3-5<530-361-31-30.050.09Nuclear
* Contained 85-90% smooth-surfaced membrane area, 2-5% of which was identified as of dictyosomal, plasma membrane, and mitochondrial origin.
" ND, not determined.
Table 2
and the monooxygenase activity of the fractions measured with
Metabolites of [G-3H]benzo(a)pyrene in subcellular tractions
7-ethoxycoumarin as substrate (data not shown).
Subcellular fractions were incubated with |G-'H]benzo(a)pyrene. and high pres
The metabolites formed may be roughly classified as dihydro- sure liquid Chromatographie analyses were performed as described in "Materials
diols, quiñones,and phenols. The group of dihydrodiols and that and Methods." Data are expressed in percentage of total metabolites."
Rough
Smooth
Plasma
Mitochonof quiñoneseach accounted for about 30 to 40% of the metab
ER
ER
Nuclei membrane
dria
olites produced by smooth and rough endoplasmic reticulum and
Metabolites
nuclei. Phenols contributed about 20% of the metabolites in
"Tetrols"cII.
I.
these incubations. Plasma membrane fractions produced mainly
gjO-Diot"III.
'IV.
phenols, while dihydrodiol formation was relatively low in mito
4,5-DtolV.
chondrial fractions. More detailed analysis of phenol formation
7.8-DiolVI.
"VII.
shows that rough and smooth endoplasmic reticulum and plasma
membrane fractions produced about twice as much 3-hydroxy1,6-Quinone
VIII.
3,6-QuinoneIX.
29.95.214.0100.03.712.63.311.83.73.19.6
30.27.514.5100.04121856305201005101322182030100414523
benzo(a)pyrene as 9-hydroxybenzo(a)pyrene. With nuclear frac
9-PhenolX.
229.0
tions this situation was more marked [4-fold more 3-hydroxy3-PhenolTotal
10100
benzo(a)pyrene|. However, mitochondrial preparations produced
amount3.113.04.112.94.24.88.8
twice as much 9-hydroxybenzo(a)pyrene as 3-hydroxybenzo" The total activities of the fractions (pmol metabolites/20 min/mg protein) were:
(a)pyrene. These differences in the regioselectivity of benzorough ER, 13,200; smooth ER, 24,000; nuclei, 390; plasma membrane, 330; and
(a)pyrene oxidation are also reflected in the ratio of 4,5-dihydro- mitochondria, 90.4.
" Numbers in order of elution.
diol to 9,10-dihydrodiol produced by the various fractions.
c This fraction is composed of tetrols and other high hydroxylated products.
"9,10-Diol, frans-9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene;
4,5-diol, transThe different metabolic patterns produced by the fractions
7,8-diol, frans-7,8-dihydroxy-7,8-dihyindicate the presence of a different pattern of monooxygenase 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene;
drobenzo(a)pyrene;
1,6-quinone, benzo(a)-pyrene 1,6-quinone; 3,6-quinone,
isoenzymes within the various membranes and show that the benzo(a)pyrene 3,6-quinone; 9-phenol, 9-hydroxybenzo(a)pyrene; 3-phenol, 3-hyactivities in nuclei, mitochondria, and plasma membrane are not droxybenzo(a)pyrene.
'' Metabolites not identified.
simply the result of contamination by endoplasmic reticulum. The
' These fractions were not homogeneous and are named after the predominant
low activities in mitochondrial and plasma membrane fractions constituent.
would argue against a significant contribution of these subcellular
compartments to the total cellular benzo(a)pyrene metabolism.
The activity measured in nuclear fractions was slightly less than and smooth endoplasmic reticulum suggested that these two
that observed in a previous study (29). This difference can mainly fractions contain a similar pattern of cytochrome P-450 forms.
be accounted for by the low level of phenols detected in our This prompted us to investigate the effect of enzyme induction
upon benzo(a)pyrene metabolism by these preparations. The
nuclear incubations.
Effect of Enzyme Induction upon Metabolism of results are shown in Tables 2 and 3. All four inducers increased
the total amount of benzo(a)pyrenemetabolites by both fractions
Benzo(a)pyrene by Rough and Smooth Endoplasmic Reticu
« 3-methyllum. The similarityof the metabolitepatternsproducedby rough (induction by frans-stilbene oxide < phénobarbital
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1985
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BENZOWPYRENE
METABOLISM
IN SUBCELLULAR
FRACTIONS
Tabte3
Rough endoplasma reticulum: total metabolismand metaboliteprofile of [G-3H¡benzo(a)pyrene
in subfractions of liver which were isolated from control and
induced rats
Induction with phénobarbital, frans-stilbene oxide, 3-methylcholanthrene, and .¡-naphthoflavone. incubations with [G-3H]benzo(a)pyrene,
Chromatographie analyses were performed as described in "Materials and Methods." Data are expressed as percentage of total metabolites.8
oxide%
Stilbene
Metabolites"1.
9,10-Dk/'e4,5-Diol7,8-DiOle1
II.
III.IV.V.VI.VII.
cholanthrene%6.3
and high pressure liquid
flavone%5.7
Ind.6.1
(%)3.1
3.2
7.0
0.9
13.0
6.5
1.0
2.7
4.112.94.24.88.81.734.84.72.89.7
0.85.32.11.12.11.034.2
4.23.24.17.5
34.112.512.61.016.53.014.0Ind.21
269.8292.175.8100-Naphtho33.613.412.01.018.43.012.9Ind.20
2711312.28.46.29.8
,6-Quinone
3,6-Quinone'9-Phenol3-PhenolControl
29.95.214.0Phénobarbital%8.7
16.23.711.2Ind.95.6
Vili.IX.X."Tetrols"c
1.11.4.1.6frans-19.84.111.3.2.3.4
.1.3.33-Methyl-
Total amount
100.0
100.0
2.0 1051 100.0
1.6
100.0
10.1
100.0
10.7
* Total activities in pmol/min/mg protein were: control, 659; phenobarbital-induced, 1290; frans-stilbene oxide-induced, 1051 ; 3-methyteholanthrene-induced, 6640; 0phthoflavone-induced, 7086.
"^ For Footnotes b-f see Table 2.
" Ind., apparent induction factor: treated:control ratio (taking into account increased contribution and increase in total metabolism).
Table 4
Smooth endoplasmi reticulum: data on the total metabolite and metaboliteprofile of [G-*H]benzo(a)pyrenein subfractions of liver which were isolated from control and
induced rats
The subcellular material was derived from the same isolation procedures as the rough endoplasmi reticulum in Table 2. Incubations with |G-3H|benzo(a)pyrene in
both compartments were performed simultaneously. Data are expressed as percentage of total metabolites.8
oxide%7.98.71.431.12.63.96.7
Siberie
Metabolites"I.
flavone%5.730.40.616.115.71.017.53.010.0
(%)3.712.63.311.83.73.19.6
"Tetrols"0II.
9,10-Diol"III.
•IV.
4,5-DiolV.
7.8-DiolVI.
•VII.
1,6-Quinone
VIII.
3,6-Quinone'IX.
30.27.514.5Phénobarbital%8.89.71.826.23.44.07.9
22.34.411.5Ind."4.51.414.21.82.41.6
1.41.11.5trans-20.84.012.9Ind.3.31.10.64.01.11.91.1
1.00.81.33-Methyl-cholanthrene%8.035.01.010.913.71.715.42.312.0Ind.10131.44.2172.61.81.
9-PhenolX.
3-PhenolControl
Total amount
100.0
100.0
1.9
100.0
1.5
100.0
4.7
100.0
4.9
8 The total metabolism (pmol total metabolites/min/mg protein) in the fractions was: control, 1200; phénobarbital,
2260; frans-stilbene oxide, 1811; 3-methylcholan
threne, 5610; and ii-naphthoflavone, 5870.
""' For Footnotes b-f see Table 2.
9 Ind., apparent induction factor: treated/control ratio.
cholanthrene = .¿-naphthoflavone).Activity in rough and smooth
cases the qualitative changes induced in smooth endoplasmi
endoplasmic reticulum was induced to similar extents following
treatment with frans-stilbene oxide or phénobarbital(1.5- to 2.0fold). 3-Methylcholanthrene and 0-naphthoflavone were more
reticulum were similar to those induced in rough endoplasmic
reticulum, such that the metabolite patterns which were substantially different following enzyme induction were in all these
potent inducers in both fractions, but preferentially in the rough
endoplasmic reticulum (approximately 5-fold in the smooth and
approximately 10-fold in the rough endoplasmic reticulum), such
that in contrast to the control situation following this treatment
the total amounts of metabolites per protein mass and time by
rough endoplasmic reticulum were greater than by smooth endoplasmic reticulum.
Tables 3 and 4 show that all inducers caused marked changes
in the pattern of benzo(a)pyrene metabolites when compared to
control incubations. Phénobarbitaland frans-stilbene oxide markedly induced the formation of 4,5-dihydrodiol, while 3-methylcholanthrene and 0-naphthoflavone preferentially stimulated formation of 7,8-dihydrodiol and 9,10-dihydrodiol as shown previously (for phénobarbital and 3-methylcholanthrene) (18). In all
situations almost identical in smooth and rough endoplasmic
reticulum. The analysis of benzo(a)pyrene metabolites is a sensitive method of demonstrating differences in the pattern of
monooxygenase isoenzymes within different membrane preparations. Thus these results indicate that, in contrast to the
relationship between endoplasmic reticulum, plasma membrane,
and mitochondria (see preceding section and Table 2), similar
patterns of cytochrome P-450 forms occur in rough and smooth
endoplasmic reticulum even after enzyme induction,
Ethoxycoumarin 0-Deethylase and Epoxide Hydrolase Activities in Smooth and Rough Endoplasmic Reticulum. These
findings were confirmed by studies of the ethoxycoumarin Odeethylase activities of rough and smooth endoplasmic reticulum
(Table 5). 0-Deethylase activity was induced by all four com-
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1985
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BENZO(A)PYRENE
METABOLISM
IN SUBCELLULAR
FRACTIONS
Tables
Data of the chemical and biochemical characterization of SER* and RERwhich was performed in parallel to the high pressure liquid chromatographystudies
Conditions of the inductions with phénobarbital,
Ã-rans-stilbene
oxkJe,3-methylcholanthrene,and /3-naphthoflavoneare described in "Materials and Methods."
ControlRNA/protem"
oxideSER0.06
Stilbene
7-Ethoxycoumarin0-deethylasec
+ Metyrapone (10"4 M)
-f-a-Naphthoflavone(2 x IO'6 M)
+ Tetrahydrofuran (10~2M)
Epoxide hydrolase''SER0.05
1.6
0.8
6.4
2.2
1.0
0.6
2.1
0.8
1.3
0.7
6.4
2.2
0.9
0.2
0.1
2.8
34.0RER0.15
12.0rrans9.8RER0.14
5.5PhénobarbitalSER0.06
cholanthreneSER0.08
5.6
2.0
12.0
14.0
12.0
13.6
1.8
0.8
8.5
10.4
8.3
9.8
5.5
2.0
1.3
1.8
1.2
1.4
2.2
10.3
0.9
12.5
10.8
10.9
46.0RER0.15
21.03-Methyl13.0RER0.22
15.00-Naphtho-llavoneSER0.07
11.0RER0.18
13.0
SER, smooth endoplasmicreticulum; RER, rough endoplasmic reticulum.
nmol product/min/mg protein.
Table 6
Effect of the inducers on the relative distribution of membraneprotein between
the rough and smooth endoplasmic reticulum
Data are basedon yieldsobtainedfrom separationsof the endoplasmicreticulum
on discontinuous sucrose gradients containing a 0.25-1.3 M sucrose step. Induc
tions were performed as described in "Materials and Methods."
Relative amounts of total ER
protein (%)
InducerNone
(control)
Phénobarbital
frans-Stilbeneoxide
3-Methyteholanthrene
0-NaphthoflavoneRough
' Mean ±SD.
ER59
±5*
36 ±3
31 ±2
58 ±6
60±5Smooth
ER41
±3
64±7
69 ±6
42 ±3
40 ±5
pounds, but this effect was most marked following treatment
with 3-methylcholanthrene and /3-naphthoflavone. These two
compounds showed the greatest effect upon the activity of rough
endoplasmic reticulum [as was the case with benzo(a)pyrene
metabolism], in marked contrast to phénobarbital and fransstilbene oxide. The behavior of frans-stilbene oxide is similar to
that of phénobarbital. The two groups of inducers differed in
another independent parameter. frans-Stilbene oxide and phén
obarbital preferentially increased the protein content of smooth
endoplasmic reticulum in contrast to 3-methylcholanthrene and
/8-naphthoflavone (Table 6). This is in accordance with the
changes in liver cell morphology induced by these compounds
(35).
Inhibition of ethoxycoumarin 0-deethylase activity by three
inhibitors with markedly different specificities, metyrapone, anaphthoflavone, and tetrahydrofuran (39), also showed differ
ences in the pattern of monooxygenase activities following treat
ment with the various enzyme inducers (Table 6). When the
identical standard concentrations of inhibitors were used for all
preparations, in both subfractions, rough and smooth endo
plasmic reticulum, tetrahydrofuran inhibited the activity of control
fractions to the greatest extent, while following induction with
frans-stilbene oxide or phénobarbitalactivity was most affected
by metyrapone. a-Naphthoflavone was the most potent inhibitor
following induction by 3-methylcholanthrene or /3-naphthoflavone
as expected from the literature (40). However, despite these
differences each inhibitor had similar effects upon the activities
of smooth and rough endoplasmic reticulum. The inhibition stud
ies show that different induction regimens resulted in alterations
of the pattern of cytochrome P-450 within the endoplasmic
reticulum. However, it appears that the pattern is very similar in
rough and smooth fractions in all cases.
CANCER
RESEARCH
The results given in Tables 3, 4, and 5 show that, although
the induction of the investigated monooxygenase activities was
consistently somewhat higher with phénobarbitalthan with fransstilbene oxide, the pattern of benzo(a)pyrene metabolites was
similar after treatment with these two inducers. On the other
hand frans-stilbene oxide clearly induced epoxide hydrolase to a
higher degree than did phénobarbital.
A good correlation was found between the ethoxycoumarin
O-deethylase activity measured in the absence of inhibitors (r =
+0.983) or in the presence of tetrahydrofuran (r = +0.990) or
metyrapone (r = +0.995) and the total amount of benzo(a)pyrene
metabolites produced by smooth and rough endoplasmic retic
ulum fractions from controls and from the animals treated with
the inducers used. This is not surprising since the oxidation of
benzo(a)pyrene and ethoxycoumarin is known to be catalyzed
by several cytochrome P-450 forms ("broad spectrum sub
strates"). In contrast the total amount of benzo(a)pyrene metab
olites did not correlate with 7-ethoxycoumarin deethylase activity
in the presence of a-naphthoflavone (r = -0.225), indicating that
in all these fractions the enzymes inhibited by a-naphthoflavone
contribute greatly to total benzo(a)pyrene metabolism. The epox
ide hydrolase activity did not correlate with total metabolism (r
= -0.209); with total diol formation (r = -0.133); with formation
of 4,5-, 7,8-, or 9,10-dihydrodiols (r = -0.036, +0.114 and
+0.184, respectively); or with the ratio of 9,10-dihydrodiol to 9hydroxybenzo(a)pyrene (r = -0.317). This indicates that in all
these fractions the rate-determining step in the formation of the
dihydrodiols is not epoxide hydration. This finding may seem
surprising in view of the fact that the covalent binding of
benzo(a)pyrene metabolites to DMA and the mutagenicity of
benzo(a)pyrene is profoundly influenced by modulation of microsomal epoxide hydrolase activity of the metabolizing systems (1,
14). The mutagenicity of benzo(a)pyrene is proportional to the
steady state concentration of the responsible reactive interme
diates such as epoxides and diol-epoxides. At any one time only
a small proportion of the total benzo(a)pyrene and its metabolites
will be in this reactive metabolite form. Consequently large
changes in the steady-state epoxide concentration need not be
reflected as measurable changes in the amount of diols formed.
ACKNOWLEDGMENTS
We thank Dr. K. L. Platt and A. Sparrow from our laboratory for the preparation
of 7-ethoxycoumarin and for the purification of inhibitors; the National Cancer
Institute Standard Chemical Reference Repository, Bethesda, MD, for
benzo(a)pyrenederivatives;and the Stiftung Volkswagenwerk for financialsupport.
VOL. 45 OCTOBER
1985
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BENZO(¿)PYRENE METABOLISM
IN SUBCELLULAR
REFERENCES
1. Bentley, P., Oesch, F., and Glatt, H. R. Dual role of epoxkte hydratase in both
activation and inactivation of benzo(a)pyrene. Arch. Toxicol., 39: 65-75,1977.
2. Blobel, G., and Potter, V. R. Nuclei from rat liver: isolation method that
combines purity with high yield. Science (Wash. DC), 154:1662-1665,1965.
3. Boer, G. J. A simplified microassay of DMA and RNA using ethidium bromide.
Anal. Biochem.. 65: 225-231,1975.
4. Conney, A. H. Pharmacological implications of microsomal enzyme induction.
Pharmacol. Rev., 79: 317-366,1967.
5. Cray, R. G., Selkirk, J. K., Harvey, R. G., Engel, J. F., and Gelboin. H. V.
Separation of ten benzo(a)pyrene phenols by recycle high pressure liquid
chromatography and identification of four phenols as metabolites. Biochem.
Pharmacol., 25: 227-230,1976.
6. Dallner, G. Isolation of rough and smooth microsomes. Methods Enzymol., 37:
191-201,1974.
7. Dansette, P. M., DuBois, G. C., and Jerina, D. M. Continuous fluorometric
assay of epoxide hydrase activity. Anal. Biochem., 97: 340-345,1979.
8. Dansette, P. M., and Jerina, D. M. A facile synthesis of arene oxides at the Kregions of polycyclic hydrocarbons. J. Am. Chem. Soc., 96:1224-1225,1974.
9. Deutsch, J., Leutz, J. C., Yank, S. K., Gelboin, H. V., Chiang, Y. L, Vatsis, K.
P., and Coon, M. J. Regio- and stereoselectivity of various forms of purified
cytochrome P-450 in the metabolism of benzo(a)pyrene and (-Xrans 7,8dihydroxy-7,8-dihydrobenzo(a)pyrene
as shown by product formation and
binding to DNA. Proc. Nati. Acad. Sci. USA, 75: 3123-3127,1978.
10. Doriing, P. R., and LePage, R. N. A rapid yield method for the preparation of
rat liver plasma membrane. Biochim. Biophys. Acta, 378:33-44,1973.
11. Estabrook, R. W., Baron, J., Franklin, M., Mason, J., Waterman, M., and
Peterson, J. Cytochrome P-450—panacea or plague. In: J. Schultz and B. F.
Cameron (eds.), The Molecular Basis of Electron Transport, pp. 197-230. New
York: Academic Press, 1972.
12. Gillette, J. R. Biochemistry of drug oxidation and reduction by enzymes in
hepatic endoplasmic retteulum. Adv. Pharmacol., 4: 219-261,1966.
13. Gram, T. E. Separation of hepatic smooth and rough microsomes associated
with drug-metabolizing enzymes. Methods Enzymol., 37: 225-237,1974.
14. Guenthner, T. M., and Oesch, F. The effects of modulation of microsomal
epoxide
hydrolase
activity
on microsome-catalyzed
activation
of
benzo(a)pyrene and its covalent binding to DNA. Cancer Lett., 77: 175-183,
1981.
15. Guerra, F. C. Rapid isolation techniques for mitochondria: technique for rat
liver mitochondria. Methods Enzymol., 37: 299-305,1974.
16. Gustafsson, J. A., and Ingelman-Sundberg, M. Multiple forms of cytochrome
P-450 in rat liver microsomes. Separation and some properties of different
hydroxylases active on free and sulphoconjugated steroids. Eur. J. Biochem.,
64: 35-43,1976.
17. Heidelberger, C. Chemical carcinogenesis. Annu. Rev. Biochem., 44: 79-121,
1975.
18. Holder, G. M., Yagi. H., Jerina, D. M., Levin, W., Lu, A. Y. H., and Conney, A.
H. Metabolism of benzo(a)pyrene. II. Effect of substrate concentration and 3methylcholanthrene pretreatment on hepatic metabolism by microsomes from
rats and mice. Arch. Biochem. Biophys., 770: 557-566,1975.
19. Huberman, E., Sachs, L., Yang, S. K., and Gelboin, H. V. Identification of
mutagenic metabolites of benzo(a)pyrene in mammalian cells. Proc. Nati. Acad.
Sci. USA, 73:607-611,1976.
20. Jerina, D. M., and Daly, J. W. Arene oxides: a new aspect of drug metabolism.
Science (Wash. DC), 785: 573-582,1974.
21. Kapitulnik, J., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M.
Benzo(a)pyrene-7.8-dihydrodio!
is more carcinogenic than benzo(a)pyrene in
newborn mice. Nature (Lond.), 266: 378-380, 1977.
22. Lowry, O. H., Rosebrough, N. J., Fan-, A. L., and Randall, R. J. Protein
measurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275,
1951.
23. Lu, A. Y. H., Levin, W., West, S. B., Jacobson, M., Ryan, D., Kutzman, R.,
and Conney, A. H. Reconstituted liver microsomal enzyme system that hy-
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
FRACTIONS
droxylates drugs, other foreign compounds, and endogenous substrates. VI.
Different substrate specificities of the cytochrome P450 fractions from control
and phenobarbital-treated rats. J. Biol. Chem., 248: 456-460,1973.
Nebert, D. W., and Negishi, M. Multiple forms of cytochrome P-450 and the
importance of motecular biology and evolution. Biochem. Pharmacol., 37:
2311-2317,1982.
Nemoto, N., and Gelboin, H. V. Enzymatic conjugation of benzo(a)pyrene
oxides phenols, and dihydrodiols with UDP-glucuronte acid. Biochem. Phar
macol., 25:1221-1226,1977.
Newbdd, R. F., and Brookes, P. Exceptional mutagenicity of a benzo(a)pyrene
did epoxide in cultured mammalian cells. Nature (Lond.), 267: 52-54,1976.
Oesch, F. Mammalian epoxide hydrases: inducÃ-aleenzymes catalyzing the
inactivation of carcinogenic and cytotoxic metabolites derived from aromatic
and définiecompounds. Xenobiotica, 3: 395-340,1973.
Parke, D. V. The Biochemistry of Foreign Compounds, pp. 1-269. Oxford:
Pergamon Press, 1968.
Pezzuto, J. M., Yang, C. S., Yang, S. K., McCourt, D. W., and Gelboin, H. V.
Metabolism
of benzo(a)pyrene
and (-Xrans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver nuclei and microsomes. Cancer Res., 38: 12411245,1978.
Schmassmann, H. U., Glatt, H. R., and Oesch, F. A rapid assay for epoxide
hydratase activity with benzo(a)pyrene 4,5-(K-region)-oxide as substrate. Anal.
Biochem., 74:94-104,1976.
Schmassmann, H. U., and Oesch, F. frans-Stilbene oxide: a selective inducer
of rat liver epoxide hydratase. Mol. Pharmacol., 74: 834-847,1978.
Selkirk, J. K., Cray, R. G., and Gelboin, H. V. Benzo(a)pyrene metabolites:
efficient and rapid separation by high pressure liquid chromatography. Science
(Wash. DC), 784:169-171,1974.
Sims, P., and Graver, P. L. Epoxides in polycyclic aromatic hydrocarbon
metabolism and carcinogenesis. Adv. Cancer Res., 20:165-274,1974.
Stasiecki, P., Oesch, F., Bruder, G., Jarasch, E. D., and Franke. W. W.
Distribution of enzymes involved in metabolism of polycyclic aromatic hydro
carbons among rat liver endomembranes and plasma membranes. Eur. J. Cell
Bid., 27:79-92,1980.
Staubli, W., Hess, R., and Weibel, E. R. Correlated morphometric and bio
chemical studies of the liver cell. II. Effects of phénobarbitalon rat hepatocytes.
J. Cell Bid., 42: 92-119,1969.
Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W.,
Wood, A. W., Conney, A. H., and Jerina, D. M. Metabolism of benzo(a)pyrene.
VI. Stereoselective metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8dihydrodiol to did-epoxktes. Chem.-Biol. Interact., 76: 281-300,1977.
Ullrich, V., Hildebrandt, A., Roots, I., Estabrook, R. W., and Conney. A. H.
(eds.). Microsomes and Drug Oxidation. Oxford: Pergamon Press, 1977.
Ullrich, V., and Weber, P. The O-deethylation of 7-ethoxycoumarin by liver
microsomes. Hoppe-Seyter's Z. Physid. Chem., 353:1171-1177,1972.
39. Ullrich, V., Weber, P., and Wdtenweber, P. Tetrahydrofurane—an inhibitor for
ethand-induced liver microsomal cytochrome P-450. Biochem. Biophys. Res.
Commun., 64: 808-813,1975.
40. Wiebel, F. J., Leutz, J. C., Diamond, L., and Gelboin, H. V. Aryl hydrocarbon
(benzo(a)pyrene) hydroxylase in microsomes from rat tissues: differential inhi
bition and stimulation by benzoflavones and organic sdvents. Arch. Biochem.
Biophys.. 744:78-87,1971.
41. Wiebel, F. J., Selkirk, J. K., Gelboin, H. V., Haugen, D. A., van der Hoeven, T.
A., and Coon, M. J. Position-specific oxygénationof benzo(a)pyrene by differ
ent forms of purified cytochrome P-450 from rabbit liver. Proc. NatJ. Acad. Sci.
USA, 72: 3917-3920,1975.
42. Wislocki, P. G., Wood, A. W., Chang, R. L., Levin, W., Yagi, H., Hernandez,
O., Jerina, D. J., and Conney. A. H. High mutagenicity and toxicity of a did
epoxide derived from benzo(a)pyrene. Biochem. Biophys. Res. Commun., 68:
1006-1012,1976.
43. Yang, S. K., McCourt, D. W., Roller, P. P., and Gelboin, H. V. Enzymatic
conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t8-dihydroxy-f-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene
through a single
enantiomer of f-7,f-8-dihydroxy-7,8-dihydro-benzo(a)pyrene.
Proc. Nati. Acad.
Sci. USA, 73: 2594-2598,1976.
CANCER RESEARCH VOL. 45 OCTOBER
1985
4843
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research.
Metabolism of Benzo(a)pyrene by Subcellular Fractions of Rat
Liver: Evidence for Similar Patterns of Cytochrome P-450 in
Rough and Smooth Endoplasmic Reticulum but not in Nuclei
and Plasma Membrane
F. Oesch, P. Bentley, M. Golan, et al.
Cancer Res 1985;45:4838-4843.
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