[CANCER
RESEARCH
37, 3427-3433,
September
1977]
The Role of Microsomes and Nuclear Envelope in the Metabolic
Activation of Benzo(a)pyrene Leading to Binding with Nuclear
Macromolecules1
John M. Pezzuto, Michael A. Lea, and Chung S. Yang2
Department
of Biochemistry,
College of Medicine
and Dentistry
of New Jersey, New Jersey Medical School,
SUMMARY
In an attempt to resolve existing conflicting reports and
further substantiate the roles of microsomes and the
nuclear envelope
in the metabolic
activation
of
benzo(a)pyrene (BP), factors affecting the binding of BP to
the DNA, RNA, histone, and nonhistone proteins of isolated
nuclei were investigated. Examination of the spectra and
catalytic properties of the mixed-function oxidase systems
of nuclei and microsomes indicated that they are similar.
Regard less of the BP concentration used, microsomes from
control or 3-methylcholanthrene-treated rats increased the
binding of BP to the components of control nuclei. With 30
IJLMBP, microsomes enhanced the binding to the nuclei
from 3-methylcholanthrene-treated rats. With lower BP con
centrations (1 to 2 /J.M),addition of microsomes reduced the
binding. A reduction was also observed when denatured
microsomes were added. It was shown that the reduction
was due to physical binding rather than the metabolism of
BP by microsomes, and in fact the latter contributed to the
binding of BP to nuclear components. With incubation sys
tems containing microsomes and nuclei, the results indi
cated that microsomes can (a) activate BP leading to bind
ing with nuclear macromolecules; and (o) physically bind
BP and reduce the effective BP concentration around the
nuclei. Both the microsomes and nuclear envelope are po
tentially important in the activation of carcinogens. The
endoplasmic reticulum may play a more important role than
the nuclei in the activation of BP when the carcinogen is
present in high concentrations. When the concentration of
the carcinogen is low, the endoplasmic reticulum should
still contribute to the metabolic activation of BP, although it
would also physically bind BP and lower the concentration
of BP available for nuclear metabolism.
INTRODUCTION
It is generally agreed that chemicals are a major causative
factor in the initiation of cancer (13, 22). BP3 is a wideReceived March 8, 1977; accepted June 10, 1977.
1 This work was supported by Grants CA-16788, CA-12933, and CA-16274
from the National Cancer Institute and Grant 472 from the Nutrition Founda
tion. Some preliminary results have appeared in an abstract (26).
2 Recipient of Faculty Research Award PRA-93 from the American Cancer
Society. To whom requests for reprints should be addressed.
3 The abbreviations
used are: BP, benzo (a) pyrene; AHH, aryl hydrocar-
Newark, New Jersey 07013
spread atmospheric pollutant that has also been shown to
be a component of cigarette smoke (7). Like many chemical
carcinogens, unmetabolized BP does not covalently react
with macromolecules (10, 12). Metabolic activation results
in binding with several cellular nucleophilic centers (13,14,
22, 33), and such binding will presumably lead to carcinogenesis. Recent evidence suggests that metabolism by the
cytochrome
P-450-containing
mixed-function
oxidase
(AHH) in conjunction with epoxide hydrase results in the
formation of a diol-epoxide that is probably the ultimate
carcinogen (4, 8, 34, 36, 38, 41). The relatively short lifetime
of this species (36, 38) and the existence of cytoplasmic
detoxification
mechanisms (35) raise the question of
whether metabolic activation by the endoplasmic reticulum
can lead to covalent reactions with macromolecules within
the cell nucleus. The presence and inducibility of AHH in the
nuclear envelope have been well documented (17,19, 31). It
has been suggested that carcinogen metabolism at this site
might more readily lead to binding with nuclear compo
nents and therefore is critical in the process of carcinogenesis (29). Carcinogens have been shown to bind to nuclear
macromolecules with experiments in vivo (5, 6, 8, 11, 16,
27), but the subcellular site of carcinogen activation cannot
be determined in such experiments.
We have recently described conditions in which metabolically activated BP covalently binds to the DMA, RNA, his
tone, and nonhistone proteins of nuclei isolated from rat
liver or lung (25). Incubation of isolated nuclei with NADPH
in the presence of molecular oxygen resulted in binding.
Treatment of the animals with MC increased the level of
nuclear AHH and the binding of BP to nuclear components.
Similar results have also been reported by several other
laboratories (1, 15, 29, 30, 37). The addition of liver micro
somes to the incubation system greatly enhanced the level
of BP bound to the components of liver or lung nuclei. The
nuclei were isolated from either control or MC-treated rats,
and the maximal levels of bound carcinogen were similar.
On the basis of this analysis, it was concluded that both the
endoplasmic reticulum and nuclear envelope were poten
tially important sites of carcinogen activation. Consistent
with this concept, increased binding of BP to nuclear DMA
in the presence of microsomes has also been reported by
bon hydroxylase; MC, 3-methylcholanthrene;
TKM, 50 mM Tris-HCI buffer,
ph 7.5, containing 25 mM KCI, and 5 mM MgCI..; MC microsomes or MC
nuclei, respectively,
isolated from animals that were pretreated with 3methylcholanthrene.
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3427
J. M. Pezzato et al.
Alexandrovef
al. (1) and Jernström et al. (15). On the other
hand, Rogan and Cavalieri (29) and, more recently, Vaught
and Bresnick (37) have observed that upon addition of
microsomes to isolated nuclei, the level of bound BP was
reduced. It may be inferred from the last 2 reports that the
metabolic role of microsomes is one of detoxification.
In order to resolve the existing conflict and further sub
stantiate the roles of microsomes and the nuclear envelope
in carcinogen activation, we have undertaken the present
investigation.
We have examined the mixed-function
oxi
dase system of the nuclear envelope and studied the effect
of added microsomes on BP binding to nuclear macromolecules under a variety of conditions.
The results of these
studies and a discussion of the cellular sites of carcinogen
activation are included in this report.
MATERIALS
AND METHODS
Chemicals and Biochemicals. BP, NADPH, DL-isocitric
acid, isocitric dehydrogenase,
Triton X-100, and protease
(Streptomyces griseus, type VI) were obtained from Sigma
Chemical Co., St. Louis, Mo. MC was from Mann Research
Laboratories, New York, N. Y. Bovine pancreatic RNase and
DNase were obtained from Worthington Biochemical Corp.,
Freehold, N. J. 3-Hydroxybenzo(a)pyrene
was supplied by
the National Cancer Institute,
Bethesda, Md. All other
chemicals were of reagent grade and were used as supplied
by commercial sources.
Radiochemical. Generally labeled [3H]BP was obtained
from Amersham/Searle,
Arlington Heights, III., with a spe
cific activity of 8.3 Ci/mmole.
Prior to use, the benzene
solvent was removed by a stream of nitrogen, and unlabeled
BP was added in acetone to a specific activity of 0.5 Ci/
mmole. During the course of these studies, the radiochemical purity was not less than 96% as analyzed by thin-layer
chromatography
with benzene as the solvent (32).
Treatment of Animals. Male Long-Evans rats with a body
weight of about 100 g were obtained from Marland Farms,
Hewitt, N. J. Prior to use, the rats were given a daily i.p.
injection of MC (25 mg/kg, in corn oil), for 4 days or no
treatment. For each study, between 3 and 10 rats were used
in each group. The animals were given a commercial labo
ratory chow and water ad libitum and kept in air-condi
tioned quarters with a 12-hr light-dark cycle.
Isolation of Microsomes and Nuclei. The rats were sacri
ficed by decapitation, and the livers were excised, placed in
ice cold 0.14 M NaCI, and rinsed 4 to 6 times. All subsequent
procedures were performed at 0-4°unless otherwise indi
cated. Microsomes
were isolated by differential
centrifuga-
tion as previously described (40) and stored frozen in small
portions at -90°. Metabolically inactive (denatured) micro
somes were prepared as follows. The microsomal suspen
sion was brought to pH 12 with 1 N NaOH and left at room
temperature for 10 min. The pH was then adjusted to neu
trality with 1 N HCI, followed by the addition of buffer (40 rriM
Tris-HCI, pH 7.4; 0.25 M sucrose; 6 mM MgCI2; and 2 mM
EDTA) and sonic disruption (25).
Nuclei were isolated by the method of Kasper (18). This
entails homogenizing the minced liver in 2 volumes (v/w) of
0.25 M sucrose-TKM.
Following filtration through 2 and 4
3428
layers of cheesecloth,
20 ml of 2.3 M sucrose-TKM were
added to 10 ml of the homogenate
in a 40-ml cellulose
nitrate tube and were thoroughly mixed. This was underlayered with 5 ml of 2.3 M sucrose-TKM and centrifuged at
24,000 rpm for 1 hr in a Beckman SW-27 rotor. The nuclear
pellets were washed with 1.0 M sucrose-TKM, 0.25 M su
crose-TKM, and finally suspended in 20 mw Tris-HCI buffer,
pH 7.4, containing 0.25 M sucrose, 3 mw MgCU, and 1 mw
EDTA (Buffer A). Total nuclear protein was measured by the
method of Lowry ef al. (20). When binding studies were to
be performed, the preparation was used immediately.
Assay of AHH. This was done by the fluorimetrie method
of Nebert and Gelboin (23) with some modifications (39, 40).
Duplicate determinations
were made with either 0.2 to 0.4
mg of nuclear protein or 0.05 to 0.1 mg of microsomal
protein and an incubation time of 5 to 10 min. The fluores
cence of the phenolic products was measured with a Farrand spectrofluorometer,
and the amount
uct was quantitated
by comparison
with
of the prod
a 3-hydroxy-
benzo(a)pyrene
standard. The activity was expressed as
pmoles of product formed per min.
Measurement of Cytochrome P-450. The carbon monox
ide difference spectra of reduced microsomes
or nuclei
were recorded with a Gary Model 17 spectrophotometer.
An
extinction coefficient of 91 mivrVcrrr1
for A45„_,90nm
was
used for cytochrome P-450 (24).
Binding of [3H]BP to Nuclear Components. The method
utilized has been described in detail (25). The nuclei were
incubated at 37°with microsomes, [3H]BP, and 1 /¿moleof
NADPH in a total volume of 2.0 ml. After 2 washes with 1%
Triton X-100 in Buffer A, h ¡stoneswere extracted with 0.24 N
HCI and repeatedly precipitated with acetone. RNA, DMA,
and nonhistone proteins were then selectively extracted by
enzymic digestions and treated with ether to remove noncovalently bound BP. In some experiments, the microsomes
were omitted or replaced with denatured
microsomes.
Background levels of radioactivity associated with the macromolecules were determined by omitting NADPH from the
incubation. The data are expressed as pmoles of BP metab
olites bound per mg of DNA, RNA, or protein. All proce
dures were performed in dim light. All binding studies were
repeated at least 1 time, and the results were qualitatively
similar.
Gross Binding of [3H]BP to Nuclei and Microsomes. To
determine the overall distribution of [3H]BP under our assay
conditions,
nuclei and microsomes were incubated at 37°
for 30 min with [3H]BP. The reaction mixture was then
placed on ice, and 2 ml of cold Buffer A were added.
Following homogenization,
the nuclei were sedimented by
centrifugation
at 800 x g for 10 min, washed with 4 ml of
Buffer A, and digested with 1 ml of Protosol (New England
Nuclear, Boston, Mass.). Microsomes were recovered from
the supernatant by centrifugation
at 105,000 x g for 1 hr.
The microsomal pellet was homogenized in 4 ml of Buffer A,
recentrifuged,
and digested with Protosol.
RESULTS
In considering
ogen activation,
the role of nuclei or microsomes in carcin
the problem of possible microsomal con-
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BP Metabolism and Binding to Nuclei
lamination in the nuclear preparation is of great impor
tance. There is no biochemical marker for checking microsomal contamination in a nuclear preparation, and the pres
ence of disrupted nuclei in the sample may complicate the
interpretation of electron micrographs. We have examined
several methods of nuclear isolation. The method of Kasper
(18) was found to be most suitable and was adopted in this
study. The yield of this method is lower than that of our
previous method (25), but the nuclei appear to be more pure
as judged by the lack of a contaminating 420 nm peak as
well as the lower cytochrome P-450 content and AHH activ
ity (data not shown). In order to test the purity of the nuclear
preparation, a dilute sample of isolated nuclei was subject
to a 2nd discontinuous sucrose gradient centrifugation; the
cytochrome P-450 content and AHH activity of the nuclei
were not decreased. The nuclear preparation is similar to
that of Berezneyef al. (3) in terms of yield, AHH activity, and
cytochrome P-450 content (data not shown). The carbon
monoxide difference spectra of nuclei and microsomes are
shown in Chart 1. Control nuclei and control microsomes
both have absorption peaks at 450 nm, whereas MC nuclei
and MC microsomes have peaks at 448 nm. This is in agree
ment with previous reports (15, 30) that treatment of the
animals with MC induces nuclear and microsomal cyto
chrome P-448, a form of cytochrome P-450 that has higher
AHH activity than does the cytochrome P-450 of control
animals. The extent of induction for microsomes and nuclei
was 2.0- and 2.3-fold, respectively. The spectra of NaOHtreated MC microsomes is also shown; the peak at 420 nm
and the absence of a 450 nm peak indicating complete
denaturation of cytochrome P-450.
ÛA = 004
NciOH
Denatured
MC Microtomes
MC
Microsomes
Control
MC
Control
Microsomes
Nuclei
Nuclei
450
Nanometers
Chart 1. The carbon monoxide difference spectra of nuclei and micro
somes. The respective protein concentrations (mg/ml) were: control nuclei,
3.8; MC nuclei, 4.0; control and MC microsomes, both 0.40; and NaOH
denatured MC microsomes, 0.28.
SEPTEMBER
1977
Table 1
Cytochrome P-450 content and AHH activity of nuclei and
microsomes
AHH activity and cytochrome P-450 were determined as de
scribed in "Materials and Methods." The values given for control
and MC nuclei are an average of 2 and 6 preparations, respectively.
The microsomal data are from 1 preparation, and they are within
the range of values obtained with other microsomal preparations in
our laboratory.
AHH (pmoles product/
min)
SampleControl
P450 (pmoles/
mg protein)36
nuclei
MC nuclei
84
Control microsomes
850
MC microsomesCytochrome1,730Per
pmole
mg cytochrome
P-4500.306
protein11
92
470
2,090Per
1.10
0.553
1.21
The cytochrome P-450 contents and the AHH activities of
the samples are shown in Table 1. In agreement with pre
vious reports (1, 15, 19, 30) MC treatment resulted in an
8.4-fold increase in activity when expressed on the basis of
protein. On the same basis, microsomal activity was in
duced only 4.4-fold. Comparison of the turnover numbers
(pmoles of product formed per min per pmole cytochrome
P-450), however, indicates that the hydroxylation of BP by
microsomes and nuclei is similar.
The effect of BP concentration on the binding to the
macromolecules of MC nuclei is shown in Table 2. A 30-min
incubation time was used in this experiment. In agreement
with the results of Vaught and Bresnick (37), the addition of
microsomes reduced the levels of BP binding when 1 or 2
/UMBP were used. This inhibition was not observed with 30
fj.M BP, consistent with our previous observation (25). Fur
thermore, when equal amounts of denatured microsomes
were added to the incubation systems, the levels of binding
were reduced in all the experiments except the DMA and
histone samples in Experiment 4. These reduced levels of
binding were also reflected in control values (no NADPH
added). These results suggest that due to the physical bind
ing of BP to microsomes, the quantity of BP available for
metabolism by the nuclei was reduced. This resulted in a
lower covalent binding of BP to nuclear macromolecules
with the addition of denatured microsomes or with the
addition of active microsomes when lower BP concentra
tions were used. A comparison between the levels of bind
ing observed with denatured and active microsomes indi
cates that the metabolism of BP by active microsomes con
tributed to the total BP binding to nuclear components.
Such an effect is not apparent with low BP concentrations
but became clear as the concentration of BP increased.
Table 3 shows the binding of BP metabolites to the mac
romolecules of control nuclei, again with a 30-min incuba
tion time. At all the BP concentrations tested, control mi
crosomes were able to increase the binding. Addition of MC
microsomes resulted in much higher levels of binding, com
parable to those obtained with MC nuclei. It is therefore
apparent that metabolism by microsomes can increase the
level of BP metabolites bound to nuclear components, even
3429
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J. M. Pezzato et al.
Table 2
Effect of BP concentration
on the binding to MC nuclear macromolecules
Each incubation
contained
MC nuclei (5 mg protein) or MC nuclei plus native or
denatured MC microsomes (4 mg protein). [3H]BP was added in 0.02 ml of acetone, and
the incubation was at 37°for 30 min. Binding is expressed as pmoles of [3H]BP bound per
mg of macromolecules.
Unless indicated,
the incubation
contained
NADPH.
Experi
mentno.1234BP
Microsomes
(/UM)None added to nuclei
tone63.124.126.569.232.644.7106.937.6107.823.77.1
1DenaturedMC
inducedNone
2DenaturedMC
inducedNone
4DenaturedMC
inducedNone
30MC (-NADPH)
(-NADPH)NoneDenaturedMC
induced
inducedDMA25.110.79.944.517.417.662.425.433.915.99.862.564.6152.0RNA249.874.4101.8345.9104.9187.0309.9141.4355.01
Effect of BP concentration
Control nuclei (5 mg protein)
protein). Control microsomes
same as those in Table 2.
Table 3
on the binding to control nuclear macromolecules
were incubated at 37°for 30 min with microsomes
were denatured
for some studies. Other conditions
Experi
mentno.1234BP
Microsomes
(/<M)Denatured
added to nuclei
(4 mg
were the
tone1.63.18.72.34.120.22.63.924.48.912.914.3110.9
1ControlMC
inducedDenatured
2ControlMC
inducedDenatured
4ControlMC
inducedControl
30DenaturedControlMC
(-NADPH)
inducedDMA1.52.56.62.12.912.62.13.625.84.67.910.9122.7RNA12.123.287.015.826.6157.021.841.6348.146.181.7100.3984.1
when low concentrations of BP are used in the incubation
system.
The effect of incubation time on the binding of [3H]BP
metabolites to nuclear macromolecules is shown in Table 4.
MC nuclei, MC or denatured microsomes, 2 ¿IM
[3H]BP, and
NADPH were incubated for the indicated time at 37°.With
short incubation times (2 to 8 min) the level of binding was
greater when native MC microsomes were included. The
difference diminished with an incubation time of 12 min and
disappeared with 30 min of incubation. Microsomes also
increased the binding of BP to nuclear components with
short incubation times when the experiment was repeated
with 1 H.MBP.
The distribution of [3H]BP physically bound to the compo
nents in the incubation system is shown in Table 5. With 2
/nM BP, a large portion of BP (68%) bound to microsomes,
22% bound to nuclei, and 10% was in the supernatant. In
3430
the presence of NADPH, the fraction of BP physically asso
ciated with microsomes decreased to 34% and that with
nuclei decreased to 14%. About half of the BP molecules
were found in the supernatant. This is probably due to the
production of hydroxylated BP metabolites that are more
soluble in the aqueous media. Similar results were also
observed with 30 /nM [3H]BP.
DISCUSSION
The existence of cytochrome P-450 and AHH activity in
the nuclear envelope has been observed in several laborato
ries (1,15,17-19, 25, 29-31). Removal of components of the
nuclear envelope by detergent treatment (9) abolishes the
enzyme-dependent binding of BP to nuclear DNA (30). The
proximity of the enzyme system to possible carcinogen
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BP Metabolism and Binding to Nuclei
Effect of incubation
time on the binding
Table 4
of [3H]BP metabolites
to nuclear macromolecules
MC nuclei (5 mg protein) were incubated with native or denatured MC microsomes (4
mg protein) at 37°for the indicated time. The concentration
of [3H]BP was 2 pM. The zero
time samples were kept on ice. Other conditions
added
Experiment
nucleiDenatured
to
no.123456Microsomes
Distribution
Table 5
of [3H]BP in incubations
of incuba
tion02481230DMA1.5
1.42.0
11.818.6
0.30.9
2.14.1
inducedDenatured
MC
6.03.4
63.131.4
5.31.7
12.06.2
inducedDenatured
MC
7.26.3
105.454.8
8.74.5
19.010.6
inducedDenatured
MC
8.712.0
163.6118.0
11.29.9
17.621.3
inducedDenatured
MC
10.813.0
117.1180.8 14.716.6
MC inducedMinutes
12.2RNA11.2
210.2Histone0.4
15.9Nonhis- 32.7
containing
nuclei and
added to the incubation.
Unbound is that which was recovered in
the supernatant after centrifuging
at 105,000 x g for 60 min. Wash
ing the nuclei or microsomes with Buffer A removed 2 to 3% of the
radioactivity.
Nuclei22+
2
30
30NADPH
+
14
34
52
26
6335Unbound101146
19Microsomes68
binding sites in the nucleus has led to the suggestion that
the nuclear enzyme may play an important role in carcino
gen activation (29). It has been reported that MC treatment
preferentially induces the AHH activity in nuclei (19, 30), in
contrast to phénobarbital, which induces microsomal but
not nuclear cytochrome P-450(17,19). These results further
suggest the uniqueness of the nuclear envelope mixedfunction oxidase system. In the present study, MC treatment
caused a 2.3-fold induction in nuclear cytochrome P-450
that is slightly higher than the induction of microsomal
cytochrome P-450, and an 8-fold increase in nuclear AHH
activity that is significantly higher than the increase in mi
crosomes. The latter difference may be a reflection of the
low AHH activity of the control nuclei, rather than any differ
ence between the cytochrome P-448 in MC nuclei and MC
microsomes, since both have a similar turnover number in
catalyzing the AHH reaction. The pattern of BP metabolites
produced with the nuclear enzyme also resembles that ob
tained with microsomes (1, 15). Moreover, treatment of the
animals with phénobarbital results in approximately a 2.5and 2-fold increase in cytochrome P-450 contents of micro
somes and nuclei, respectively (unpublished results). Over
SEPTEMBER
1977
tone1.8
inducedDenatured
MC
microsomes
MC nuclei (5 mg protein) were incubated with MC microsomes (4
mg protein) at 37°for 30 min with 2 or 30 /¿MBP in duplicate.
Averaged values are given as the percentage of the total [3H]BP
BP (MM)2
were the same as those in Table 2.
22.434.2
all, it appears that the mixed-function oxidase system in the
nuclear envelope is similar to that in microsomes.
The present study confirmed our previous results (25)
that, in an incubation system containing isolated nuclei,
[3H]BP, and NADPH, the addition of microsomes increases
the binding of BP to nuclear macromolecules. This is appar
ent in experiments with 30 /U.MBP, and with 15 to 112 /nMBP
as shown previously (25). With a BP concentration of about
3 fj.M, Vaught and Bresnick (37) observed that the added
microsomes decreased the binding. We now report that
with BP concentrations as low as 1 /AM, the addition of
microsomes to MC nuclei does not cause a reduction in
binding when compared with an appropriate control, the
addition of denatured microsomes (Table 2). The apparent
reduction of carcinogen bound to the macromolecules of
MC nuclei is probably due to physical binding of BP by the
microsomes. This interpretation is consistent with the ob
served high affinity of BP for microsomes (Table 5). The
microsomes, therefore, have 2 opposite effects on the me
tabolism of BP: Effect A, the metabolic activation of BP by
microsomes; and Effect B, the physical binding of BP to
microsomes causing a decrease in the effective concentra
tion of BP to nuclear enzymes. The Kmof the MC-induced
mixed-function oxidase system has been reported to have
values ranging from 0.3 to 2.4 /*M BP (2, 21, 28). With 30 ^M
BP added to incubations containing microsomes, the de
creased BP concentration should still be much higher than
the Km; therefore, Effect A outweighs Effect B, and an
enhanced level of binding is seen. With lower BP concen
trations, when the metabolism is substrate limiting, e.g., 2
fj,M, Effect B outweighs Effect A, and decreased binding is
seen. Under these conditions, the enzymes in the micro
somes should contribute to the binding. This is demon
strated by the experiments with shorter incubations in Table
4. Such an effect is less apparent with longer incubations,
due possibly to the exhaustion of the substrate, BP (Tables
2 and 4). With control nuclei, which have very low AHH
activity, Effect A outweighs Effect B even at low BP concen3431
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J. M. Pezzuto et al.
trations when either control or MC microsomes are used
(Table 3).
Jernströmet al. (15) have suggested that the microsomes
metabolize BP to a form that the nuclei can convert more
efficiently than BP to a DNA-binding species. Our results
however, suggest that such a 2-step process may not be
needed. The metabolism-dependent binding of BP to the
components of control nuclei (which contain little AHH
activity) is very low in the absence of microsomes but is
greatly enhanced when microsomes are added. The ob
served levels of binding are similar to those attained when
MC nuclei are incubated with microsomes. Further investi
gation of this aspect of carcinogen activation is needed.
The fact remains that the AHH system of isolated nuclei
can activate BP and cause binding to nuclear components.
The active metabolites of BP produced by microsomes can
also enter into the nuclei and react with nuclear macromolecules. Since most of the AHH activity is associated with the
endoplasmic reticulum, it appears that the endoplasmic
reticulum may play a more important role than nuclei in the
activation of BP when the carcinogen is present in high
concentrations, e.g., 15 to 30 ¿¿M.
When the cellular con
centration of BP is 2 fj.M or lower, a condition that may more
closely approximate the situation in carcinogenesis, the
endoplasmic reticulum should contribute to the metabolic
activation of BP, although it would also physically bind BP
and decrease the effective carcinogen concentration
around the nucleus. The latter effect should also be mani
fested by other organelles and cellular membranes. The
relative importance of the endoplasmic reticulum and the
nuclear envelope in carcinogen activation remains to be
unequivocally defined, since it may also depend upon cytoplasmic detoxification reactions (35) and the mechanisms
of carcinogen transport.
ACKNOWLEDGMENTS
We thank L. P. Kicha and F. S. Strickhart for technical assistance and C.
Sheffield for secretarial assistance.
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The Role of Microsomes and Nuclear Envelope in the Metabolic
Activation of Benzo( a)pyrene Leading to Binding with Nuclear
Macromolecules
John M. Pezzuto, Michael A. Lea and Chung S. Yang
Cancer Res 1977;37:3427-3433.
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