[CANCER RESEARCH 48, 4513-4519, August 15, 1988]
Specificity of Rabbit Cytochrome P-450 Isozymes Involved in the Metabolic
Activation of the Food Derived Mutagen 2-Amino-3-methylimidazo[4,5-/]
quinoline
Michael E. McManus,1 Wendy Burgess, Elizabeth Snyderwine, and leva Stupans
Department of Clinical Pharmacology, School of Medicine, Flinders University of South Australia, Bedford Park, 5042, Australia [M. E. M., W. B., I. S.J, and Laboratory
of Experimental Carcinogenesis, NIH, Bethesda, Maryland 20205 [E. S.]
forms of rabbit cytochrome P-450, Forms 4 and 5, have been
primarily implicated in these reactions. In previous studies we
The involvement of rabbit liver cytochromes P-450 in the activation of
have shown that of seven highly purified forms of rabbit cyto
the food derived heterocyclic amine mutagen, 2-amino-3-methylimichrome P-450 only Form 4 W-hydroxylated the carcinogenic
dazo[4,5-/]quinoline (IQ), was assessed using the Ames/Salmonella test.
aromatic amide, 2-acetylaminofluorene (6, 7). Norman et al.
The number of revertants induced by IQ per UKof control rabbit liver
microsomes was 1872 ±50 (SD, n = 3), and this increased to 3690 ± (8) showed Form 4 to be involved also in the metabolic activa
tion of 2-aminoanthracene, while Robertson et al. (9, 10) have
239 when microsomes from 2,3,7,8-tetrachlorodibenzo-/»-dioxin(TCDD)
animals were used as the metabolic activation source. Microsomes from concluded that the metabolism of aromatic amines to mutagenic
products by rabbit hepatic microsomes is dependent on the
phénobarbital,rifampicin, and acetone pretreated rabbits were less effi
relative composition of both Forms 4 and 5 of cytochrome Pcient than controls at activating IQ to a mutagen. Cytochrome P-450
450.TV-Hydroxylation is also a prerequisite for the metabolic
Forms 4 and 6, which are induced by TCDD, were found to be efficient
activators of IQ to a mutagen in reconstitution experiments. Form 4 was activation of the food derived heterocyclic amine mutagens
7.7-fold more active than Form 6 and produced 1702 revertants/0.125
(11). These amines are formed during pyrolysis of amino acids,
pmol with a 20-min preincubation step in the Ames test. Anti-Form 4
peptides, proteins, and intact foods and at least 11 of these
IgG inhibited the activation of IQ in control and TCDD induced micro
compounds
have been isolated and identified (12, 13). In the
somes by 78 and 79%, respectively. The contents of Cytochrome P-450
present
study
we have investigated the mutagenic activation of
Form 4, determined by Western blot analysis, in control and phénobar the food derived
heterocyclic amine, IQ,2 by purified forms of
bital, acetone, rifampicin, and TCDD pretreated microsomes were 0.55
rabbit
cytochrome
P-450 and induced rabbit liver microsomes.
±0.19, 0.63 ±0.34, 0.5 ±0.27, 0.28 ±0.16, and 2.19 ±0.43 (n = 3)
IQ in the presence of Aroclor pretreated rat liver microsomes
nmol/mg protein, respectively. A highly significant statistical correlation
existed between the capacity of the above microsomes to activate IQ to has been shown to be one of the most highly mutagenic com
a mutagen and their cytochrome P-450 Form 4 content (r = 0.96; r2 =
pounds in the Ames/Salmonella test (12, 13). The present
0.92). The content of cytochrome P-450 Form 6 in the above microsomes
results show that both Forms 4 and 6 of cytochrome P-450
was also highly correlated with their capacity to activate IQ (r = 0.92;
metabolic-ally activate IQ to a mutagen in the Ames/Salmonella
r2 = 0.85). Based on these results and the tissue distribution of cyto
test and that the former is approximately 7 times more active.
chrome P-450 Forms 4 and 6, the former obviously contributes most
ABSTRACT
toward the activation of IQ in the liver, whereas Form 6 would be
expected to be primarily involved in this process in extrahepatic tissues.
INTRODUCTION
The cytochrome P-450 monooxygenase system has been
shown to be intimately involved in the metabolic activation of
numerous chemical carcinogens (1-3). This enzyme system is
composed of a family of hemoproteins, and in the rabbit at
least eight forms of cytochrome P-450 have been purified and
extensively characterized (2). The different isozymes of cyto
chrome P-450 involved in the metabolism of both endogenous
and xenobiotic substrates have been shown to exhibit distinct
substrate specificities and differ markedly in their response to
enzyme inducers and inhibitors (1-3). In addition, these hem
oproteins exhibit distinct tissue distributions and their expres
sion in hepatic and extrahepatic tissues appears in certain cases
to be under different regulatory control mechanisms (2, 4, 5).
These observations have led to the speculation that the relative
composition of cytochrome P-450 isozymes may be a major
determinant of individual susceptibility to cancer as well as of
target organ selection upon exposure to chemical carcinogens.
The first step in the metabolic activation of many aromatic
amines and amides usually involves ,/V-hydroxylation (1). Two
Received 12/15/87; revised 5/9/88; accepted 5/19/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' To whom requests for reprints should be addressed, at Department of Clinical
Pharmacology. Flinders Medical Centre, Bedford Park. 5042 Australia.
MATERIALS
AND METHODS
NADPH, cytochrome c, Tween 20, bovine serum albumin, /3-naphthoflavone, molecular weight markers, and diaminobenzidine were
obtained from Sigma Chemical Co., St. Louis, MO. Nitrocellulose
paper (0.45 /¿m)and calcium phosphate gel were purchased from BioRad (Australia), anti-goat IgG peroxidase was obtained from Silenus
Laboratories (Australia), and horse serum was from Commonwealth
Serum Laboratories (Australia). DEAE-Sepharose CL-6B and carboxymethyl-Sepharose were obtained from Pharmacia (Australia) and UItragel HA was from LKB (Australia). Randomly labeled [3H]-2-acetylaminofluorene (18 Ci/mmol) was purchased from Moravek Biochemicals (City of Industry, CA) and unlabeled 2-acetylaminofluorene and
its metabolites plus 2-aminofluorene, IQ, and TCDD were kindly
supplied by Dr. S. S. Thorgeirsson, NIH, Bethesda, MD. Salmonella
typhimurium TA98 was a kind gift from Dr. Robert Baker, School of
Public Health and Tropical Medicine, University of Sydney, Sydney,
Australia. All other chemicals were of analytical reagent grade.
Animals. Adult male and female New Zealand White rabbits were
obtained from the Institute of Medical and Veterinary Science (Ade
laide, Australia), housed in plastic cages, and allowed free access to
food and water. Male rabbits were treated with various compounds at
the following dosages: sodium phénobarbital,70 mg/kg in 0.15 M NaCl
administered i.p. once daily for S days; rifampicin, 50 mg/kg in 10 HIM
sodium phosphate (pH 8.2) containing 0.15 M NaCl given i.p. once
daily for 4 days; ß-naphthoflavone,40 mg/kg in corn oil i.p. once daily
for 4 days; acetone, 1% (v/v) in drinking water for 9 days. Four female
2 The abbreviations used are: IQ, 2-amino-3-methylimidazo[4,5-/]quinoline;
TCDD, 2,3,7,8-tetrachlorodibenzo-^-dioxin;
PBS, 10 mM potassium phosphate
buffer containing 0.15 M sodium chloride; FCMO, flavin-containing monooxy
genase.
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ACTIVATION OF IQ BY CYTOCHROMES
rabbits were given one injection each of 10 jig/kg TCDD dissolved in
dimethyl sulfoxide i.p. 5 days prior to sacrifice. Control animals re
ceived no treatment. Animals were killed by cervical dislocation and
liver and lung microsomes were prepared as described previously (7).
Knzymes. Rabbit liver cytochrome P-450 Form 4 and NADPHcytochrome P-450 reducÃ-asewere purified from /3-naphlhoflavone and
phénobarbitalinduced rabbit liver microsomes, respectively, as de
scribed by McManus et al. (7). Cytochrome P-450 Form 6 was prepared
from TCDD induced rabbit liver microsomes by monitoring the elution
of this protein from columns using an antibody raised against cyto
chrome P-450 Form 6 and the Western blot procedure. The polyclonal
antibody used for this procedure was a kind gift from Dr. E. F. Johnson,
Scripps Clinic and Research Foundation, La Jolla, CA. All procedures
were carried out at 0-4°C.For the purification of Form 6, 2000 mg of
microsomal protein suspended in 0.1 M potassium phosphate buffer
(pH 7.4) containing 20% glycerol were solubilized with 25 ml of 5%
(w/v) sodium cholate, and 25 ml of 10% (w/v) Nonidet P-40 were
added dropwise over 30 min. The mixture was stirred for a further 30
min and then centrifuged at 105,000 x g for 60 min to remove
undissolved material. The supernatant fraction was then diluted with
an equal volume of 5 mM potassium phosphate buffer (pH 7.4) con
taining 0.1 mM dithiothreitol and 0.1 mM EDTA, 0.1% w/v Nonidet
P-40, and 20% glycerol (Buffer A) and applied to a DEAE-Sepharose
column (2.6 x 30 cm) that had been equilibrated with Buffer A. After
being washed with 450 ml of Buffer A Form 6 was eluted with a linear
0-0.3 M potassium chloride gradient in Buffer A. Fractions with the
highest Form 6 content, determined by the intensity of the preoxidase
stain on Western blots, were pooled and dialyzed against Buffer A. The
pooled fractions which contained 140 mg of protein were applied to a
carboxymethyl-Sepharose column (2.6 x 10 cm) previously equilibrated
with Buffer A. After being washed with 200 ml of Buffer A cytochrome
P-450 Form 6 was eluted using a linear 0-0.5 M potassium chloride
gradient in Buffer A. Western blots were again used to monitor the
elution of Form 6. The fractions containing the highest amounts of
Form 6 were pooled (35 mg of protein in total), dialyzed against Buffer
A overnight, and then applied to a Ultragel HA (2.6- x 22-cm) adsorp
tion column previously equilibrated with Buffer A. The column was
washed sequentially with 10, 40, and 60 mM potassium phosphate in
Buffer A and Form 6 was eluted by increasing the phosphate concen
tration to 90 mM. The fractions containing Form 6 (4.5 mg protein)
determined by Western blotting were pooled and dialyzed against two
changes of Buffer A overnight and then adsorbed onto 80 mg of calcium
phosphate gel. The calcium phosphate gel was washed four times with
5 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol to
remove detergents. Form 6 was eluted from this gel in 10 ml of a 300
mM potassium phosphate buffer (pH 7.4) containing 20% glycerol.
Cytochrome P-450 Form 3b was isolated from control rabbit liver
microsomes according to the method of Dieter and Johnson (14). The
protein isolated exhibited an oxidized spectrum typical of a low spin
cytochrome with a Soret maximum at 417 nm and had aniline hydroxylase and aminopyrine /V-demethylase activities of 2.3 and 34 nmol
product formed/min/nmol P-450, respectively. Based on these criteria
plus a molecular weight of 51,000 this protein was called cytochrome
P-450 Form 3b (2).
Antibodies. Antibodies against cytochrome P-450 Form 4 and
NADPH-cytochrome P-450 reducÃ-asewere raised in adult female goals,
and ami-Form 4 IgG, anti-NADPH-cylochrome P-450 reducÃ-aseIgG,
and preimmune IgG were isolated as described previously (15, 16). The
final protein concentrions
of anti-Form 4 IgG and anli-reduclase IgG
were 13.9 and 51 mg/ml, respectively. The prolein concenlration of the
anti-Form 6 antiserum provided by Dr. E. F. Johnson was 84 mg/ml.
Polyacrylamide Gel Electrophoresis and Immunoblolting. Polyacrylamide gel eleclrophoresis in Ihe presence of sodium dodecyl sulfate was
performed essentially as described by Laemmli (17). Prior to electrophoresis, microsomal samples and molecular weighl markers were
healed at 100°Cfor 3 min while purified proteins received the same
P-450
bovine serum albumin (M, 69,000), and phosphorylase b (M, 94,000)
were electrophoresed in a separale well. Where appropriale gels were
slained for prolein by soaking for at least 2 h in water:methanol:acetic
acid (40:50:10) conlaining 0.2% (w/v) Coomassie Blue R-250. Gels
were deslained by washing in waier:melhanol:acetic acid (67.5:25:7.5)
overnight. Gels run for immunoquantitation experiments were applied
directly to nilrocellulose paper and Ihe "Western blotling" assay was
carried out essentially as described by Towbin et al. (18). Transfer of
proteins from polyacrylamide gels to nilrocellulose paper was accom
plished in 16 h al 30 mA in a Trans Blol Cell (Bio-Rad). Following the
transfer of proleins, Ihe nilrocellulose sheels were rinsed in (pH 7.4)
and then incubated at 37°Cfor l h in a solution containing 1.5% (w/v)
bovine serum albumin and 5% (v/v) heat inactivated horse serum to
block reactive sites. Blocked sheets were then incubaled with anlibody
(Form 4, 1:40,000; Form 6, 1:10,000) in blocking solution for 2 h.
Sheets were then washed with PBS containing 0.05% (v/v) Tween 20
(4 changes in 30 min) and incubated with anti-goat IgG peroxidase
conjugate (1:500) in blocking solution for 1 h. Following Iwo further
rinses in PBS, Ihe sheels were incubated (5-15 min) in 20 mM imidazole
buffer, pH 7.0, conlaining 0.05% (w/v) diaminobenzidine lelrahydrochloride and 0.05% (v/v) hydrogen peroxide lo visualize immunoreaclive bands. The immunoslained nitrocellulose was scanned with a
Camag densitometer (Switzerland) and the area of each peak was
determined by triangulation.
Enzyme Assays. Cytochrome P-450 content and NADPH-cyto
chrome P-450 reducÃ-aseaclivily of microsomes were delermined as
described previously (15). 2-Acetylaminofluorene metabolism was
measured according lo Ihe method of McManus et al. (19). During
antibody inhibition experiments the concentration of IgG protein in
each incubation was kept constanl by the addition of preimmune IgG.
Aminopyrine A'-demethylation and aniline hydroxylation were deter
mined as described previously (15).
Mutation Assay. The mulagenicily of IQ was lesled on S. typhimurÃ-umTA98 by the method of Marón and Ames (20) with a 20-min
incubation step prior to pouring the plates. A standard 0.5 ml incuba
tion volume contained 2 Mg microsomal protein, 1-2 x IO8 cells of
bacleria, 1.2 ^mol NADPH, and 5 Mg 'Q or 10 Mg 2-aminofiuorene
dissolved in 5 M!of dimelhyl sulfoxide. For reconslitution experiment,
varying amounts of either cytochrome P-450 Forms 3b, 4, or 6, 1.0
unit of reducÃ-ase,and 75 Mgof dilauroyl-L-a-lecithin were substituted
for Ihe microsomal prolein unless otherwise indicaled. Reaclion mixlures were preincubaled al 37°Cfor 3 min prior lo adding bacleria and
NADPH. The reactions were terminaled by the addition of 0.1 ml of 5
mM cytochrome <•
and then placed on ice. Following the addilion of 2
ml of million lop agar al 45°CIhe incubaiion mixiures were poured
onto Petri dishes conlaining 30 ml of minimal agar and a limited
amount of L-histidine. The colonies on each piale were scored after a
48-h incubation period at 37°C.When anlibody inhibilion sludies were
conducted the concentraron of protein in each incubation was kept
conslant by the addilion of preimmune IgG. All protein and lipid
solutions used in these experimenls were filler sierili/ed ihrough 0.22MinMillex-GV filler units (Millipore Corporation, Bedford, MA) prior
to use. The concenlralions of prolein or lipid in Ihe fÃ-ltraleswere
determined alter filtration to estÃ-malelosses during ihis procedure.
Prolein concenlralions were determined by Ihe melhod of Lowry et al.
(21) using bovine serum albumin as a slandard and lipid was delermined
by the method of Barile«(22).
RESULTS
Fig. 1 shows the effect of microsomal protein concentration
on the activation of IQ by control rabbit liver microsomes. The
number of revenants formed decreased substantially when more
than 10 Mg of microsomal protein was used per incubation.
Therefore, when comparing the capacity of pretreated micro
somes to activate IQ, 2 fig of microsomal protein were used per
incubation, except in the case of TCDD pretreated microsomes
where 1 Mgof microsomal protein was used. Fig. 2 shows the
capacity of control and induced rabbit liver microsomes to
treatment for 1 min. The amounts of purified proleins and microsomal
prolein were applied to the indicated wells and for Fig. 3 0.5 Mgeach
of the molecular weight slandards aldolase (M, 40,000), ovalbumin (M,
43,000), glulamate dehydrogenase (M, 53,000), catalase (M, 58,000),
4514
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ACTIVATION OF IQ BY CYTOCHROMES
8000 r
10
15
20
jjg MICROSOMAL PROTEIN
Fig. I. Effect of control rabbit liver microsomal protein on the mutagenic
activation of IQ. The results represent the mean ±SD (bars), n = 3.
4000
co0 Oo
-D
AF
P
<
Å’ 2000
ILI
I
i
—
00o
1
1
1!
I
RIF
ACET B-NF TCDD
CONT
Pb
Fig. 2. Mutagenic activation of IQ and 2-aminofluorene (AF) by control
(CONT) and phénobarbital(Pb). rifampicin (RIF). acetone (ACET), 0-naphthoflavone (B-NF) and TCDD pretreated rabbit liver microsomes. The results for IQ
and 2-aminofluorene are expressed as the number of revenants per 1 and 10 >ig
microsomal protein, respectively. Values represent the mean ±SD (bars) of three
determinations on microsomes from a single rabbit.
activate IQ to a mutagen. For comparison, the model mutagen
2-aminofluorene, which Robertson et al. (9, 10) have shown to
be activated mainly by rabbit cytochrome P-450 Form 5, has
also been included. As expected, phénobarbitalpretreated mi
crosomes exhibited the highest capacity to activate 2-aminoflu
orene. Phénobarbital pretreatment caused a 61% increase in
the number of revenants formed per 10 ng of microsomal
protein compared to controls. The number of revenants pro
duced by microsomes from animals pretreated with rifampicin,
acetone, 0-naphthoflavone, or TCDD either remained un
changed or decreased slightly. In contrast to these results,
microsomes from 0-naphthoflavone and TCDD pretreated an
P-450
imals exhibited the greatest capacity to activate IQ to a muta
gen. The number of revenants induced by IQ per ng of control
rabbit liver microsomes was 1872 ±50 (SD; n = 3) and this
was increased to 3149 ±198 and 3690 ±239, respectively,
when microsomes from /3-naphlhoflavone and TCDD pre
treated rabbits were used as the metabolic activation source.
Liver microsomes from phénobarbital,rifampicin, and acetone
pretreated rabbits were less efficient than control rabbit liver
microsomes at activating IQ to a mutagen (Fig. 2). The capacity
of control rabbit lung microsomes which predominantly contain
cytochrome P-450 Forms 2 and 5 (2, 10) and lung microsomes
from TCDD induced rabbits that have elevated levels of Form
6 (2) to metabolically activate IQ was also assessed. The number
of revenants produced by control and TCDD induced rabbit
lung microsomes per 50 ^g microsomal protein were 333 ±28
and 1994 ±542 (n = 3), respectively.
These data strongly suggested the involvement of cytochrome
P-450 Form 4 and/or Form 6 in the metabolic activation of IQ
by rabbit liver microsomes. To test this hypothesis, the effects
of purified preparations of cytochrome P-450 Forms 4 and 6
were investigated. A constitutive form of cytochrome P-450,
Form 3b, was also tested for its ability to activate IQ. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis showed that
the cytochrome P-450 Forms 3b, 4, and 6 and NADPH-cytochrome P-450 reducÃ-asemigrated as single electrophoretic ho
mogeneous bands (Fig. 3) with molecular weights of 51,000,
53,000, 57,000, and 77,000, respectively. The specific cyto
chrome P-450 contents of each purified hemoprotein were 12,
11, and 10 nmol/mg protein for Forms 3b, 4, and 6, respec
tively. Rabbit liver cytochrome P-450 reducÃ-asehad a specific
activily of 53,000 nmol cylochrome c reduced/mg/min.
The
oplimal reaclion condilions for ihe metabolic activation of IQ
lo a mulagen by purified cylochrome P-450 Form 4 are shown
in Fig. 4. The subslrale concenlralion of IQ used was 5 /ig
which, in a preliminary experimenl, had been shown lo be
saturaling for Ihe aclivalion of this compound by TCDD in
duced rabbit liver microsomes. The aclivalion of IQ by Form
4, as delermined by Ihe number of reverlanls formed, increased
up lo a concenlration of 0.75 unii of reducÃ-aseand up lo 20 /ig
of dilauroyl-L-a-lecilhin per 0.5-ml incubation. Therefore when
comparing ihe capacily of Ihe purified forms of cylochrome P450 lo aclivale IQ, l unii of reducÃ-aseand 75 ¿igof lipid were
3b
6
Red
94k
69k
58k
53k
43k
40k
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel of 2 «igeach of purified
rabbit liver cytochrome P-450 Forms 3b, 4, and 6 and NADPH-cytochrome P450 reducÃ-ase(Red). The standard proteins (0.5 ng each) for relative molecular
mass indicated by numbers are: aldolase (40,000), m allunimi (43,000), glutamate
dehydrogenase (53,000), catalase (58,000), bovine serum albumin (69,000), and
phosphorylase b (94,000). A, thousands.
4515
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ACTIVATION OF IQ BY CYTOCHROMES P-»50
2000
1500
1000
500
500
05
1.5
1O
UNITS OF REDUCÕASE
25
50
75
100
Mg LIPID
6000
FORM 4
4000
2000
FORM 6
0.25
pmol PURIFIED
05
075
CYTOCHROME
1.0
P450
Fig. 4. Effect of reducÃ-ase(A) and lipid (III concentrations on the mutagenic
activation of IQ by 0.125 pmol of purified cytochrome P-450 Form 4. C, effect
of cytochrome P-450 Form 4 and 6 concentrations on the mutagenic activation
of IQ. The amount of reducÃ-aseand lipid per O.S-ml incubation volume were I
unit and 75 Mg, respectively. See "Materials and Methods" for experimental
conditions.
used. Under these conditions, the capacity of Forms 4 and 6 to
activate IQ increases linearly up to 0.25 and 1.0 pmol of
cytochrome P-450, respectively, no activation was observed
with Form 3b. Form 4 was 7.7-fold more active than Form 6
when both were compared at 0.125 pmol of protein.
The above studies strongly suggest that cytochrome P-450
Forms 4 and 6 are the major enzymes involved in the mutagenic
activation of IQ. However, since the flavin-containing monooxygenase has been shown to activate primary aromatic amines
such as 2-aminofluorene (23), the capacity of this enzyme to
mutagenically activate IQ was investigated. Since the FCMO
has been shown to be very thermolabile (15, 24) especially in
the absence of NADPH, the effect of heating rabbit liver microsomes to 45°Cfor 10 min in the absence and presence of
NADPH on their capacity to activate IQ was studied. This
treatment had no effect on the capacity of either control or
TCDD pretreated rabbit liver microsomes to metabolically
activate this heterocyclic amine. For example, the number of
revenants produced by control microsomes heated in the pres
ence and absence of NADPH were 2031 ±37 (n = 3) and 2077
±162 per /ug protein and 3715 ±220 and 3739 ±250 per fig
protein with TCDD pretreated microsomes. Heat treatment
nearly completely abolished the capacity of these same micro
somes to A-oxidize dimethylaniline, a metabolic activity asso
ciated with the FCMO (25). Control rabbit liver microsomes
heated to 45°Cfor 10 min in the presence and absence of
NADPH exhibited dimethylaniline ./V-oxidase activities of 17.3
±3.5 and 2.85 ±1.07 nmol TV-oxide formed/min/mg microsomal protein (n = 3), respectively. The effect of heat treatment
on the capacity of TCDD pretreated microsomes to A'-oxidize
dimethylamine resulted in activities of 8.54 ±1.74 (n = 4) and
1.56 ±1.09 nmol A'-oxide formed/min/mg microsomes heated
in presence and absence of NADPH, respectively. The purified
hog liver FMCO (kindly supplied by Dr. D. M. Ziegler, Uni
versity of Texas at Austin) did not activate IQ to a mutagen
inasmuch as no increases in revertants over background were
observed (data not shown).
The role of cytochrome P-450 in the activation of IQ was
further investigated utilizing antibodies against NADPH-cytochrome P-450 reducÃ-aseand cytochrome P-450 Forms 4 and 6
as inhibitors (Fig. 5) and to immunoquantify Forms 4 and 6
(Fig. 7). Anti-NADPH-cytochrome
P-450 reducÃ-asecaused ap
proximately 90% inhibition of cytochrome c reduction and the
metabolism of 2-acetylaminofluorene to its N-, 7-, and 1-hydroxylated metabolites by polycyclic hydrocarbon induced rab
bit liver microsomes (Fig. 5, A and B). The same antibody
inhibited the mutagenic activation of IQ by control and TCDD
induced rabbit liver microsomes by 78 and 72%, respectively
(Fig. 5Q. Anti-cytochrome P-450 Form 4 IgG caused a similar
level of inhibition of IQ mutagenesis with the number of re
vertants being reduced 78 and 79%, respectively, in control and
TCDD induced microsomes, respectively (Fig. 5D). Since cy
tochrome P-450 Forms 4 and 6 share 62% homology at the
amino acid level (2), it is possible in the microsomal membrane
that anti-Form 4 IgG is inhibiting both Forms 4 and 6. To test
this the capacity of anti-Form 4 IgG to inhibit the mutagenesis
of IQ by Forms 4 and 6 in the reconstituted system was assessed.
One hundred ng of anti-Form 4 IgG inhibited totally the
capacity of Form 4 to metabolically activate IQ, while approx
imately 1 mg of antibody was required to inhibit the activation
of IQ by Form 6 (Fig. 6). The anti-Form 6 used was a noninhibitory antibody inasmuch as it had no effect on the mutagenic
activation of IQ or the capacity of TCDD and /3-naphthoflavone
induced rabbit liver microsomes to metabolize benzopyrene.
Fig. 7 shows typical Western blots used to quantify the levels
100iiÕ
A
75\A
50\-*•04
SIv*~*"----,05
>
^->100755025t
0.8
1.2i
1.0
1.5B'--42.0
0.2
0.3
mg ANTI-REDUCTASE IgG
Z
C
100t
100«
ö
oc
° 75
75
50
50
25
25
02
04
mg ANTI-REDUCTASE IgG
0.6
V¿:
0.1
0.4
mg ANTI-FORM 4 IgG
Fig. 5. Effect of anti-rabbit NADPH-cytochrome P-450 reducÃ-aseIgG on (A)
the reduction of cytochrome c; (B) 2-acetylaminofluorene l-hydroxylation (A), 7hydroxylation (•),and jV-hydroxylation (•)by polycyclic hydrocarbon induced
rabbit liver microsomes. C, effect of anti-reductase IgG on the mutagenic activa
tion of IQ by control (•)and polycyclic hydrocarbon pretreated (A) rabbit liver
microsomes. D, effect of anti-rabbit cytochrome P-450 Form 4 IgG on the
mutagenic activation of IQ by control (•)and polycyclic hydrocarbon (A) pre
treated rabbit liver microsomes. The numbers of revertants in the presence of
preimmune IgG in control and pretreated microsomes were 1514 and 4352,
respectively.
4516
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ACTIVATION OF IQ BY CYTOCHROMES
P-450
100-
0.5
1.0
1.5
1 2
3
4
5
6 7
8
9 10 11 12 13 14
1 2
3
4
5
6
7 8
9 10 11 12 13 14
mg ANTIFORM 4 IgG
Fig. 6. Effect of anti-rabbit cytochrome P-450 Form 4 IgG on the mutagenic
activation of IQ by cytochrome P-450 Form 4 (•;0.08 pmol) and Form 6 (A; 1
pmol). The amounts of reducÃ-aseand lipid per 0.5-ml incubation volume were 1
unit and 75 jig, respectively. The numbers of revertants produced by cytochrome
P-450 Forms 4 and 6 in the presence of 2 mg preimmune IgG were 1236 and
1546, respectively. See "Materials and Methods" for experimental conditions.
of Forms 4 and 6 in control and induced rabbit liver microsomes. To negate variability between blots a standard curve of
either Form 4 or Form 6 was run on the same blot as the
microsomal samples. Also for estimating the concentration of
these hemoproteins in microsomes at least two levels of micro
somal protein were used. Anti-Form 4 IgG recognized a single
protein with a molecular weight identical with that of rabbit
cytochrome P-450 Form 4 in all rabbit liver microsomes tested
(Fig. 1A). At the concentration of this antibody used (1:40,000),
only a slight cross-reactivity with cytochrome P-450 Form 6
occurred on some blots (data not shown). Anti-Form 6 at a
dilution of (1:10,000) recognized both Form 6 and Form 4 in
all the rabbit liver microsomes tested (Fig. IB). Immunoquantitation of both cytochromes using Western blots was linear up
to 2 pmol cytochrome P-450. The concentrations of cyto
chromes P-450 Form 4 and 6 and the effect of inducing agents
on their microsomal concentration are shown in Table 1. The
spectrophotometrically
determined total cytochrome P-450
concentrations of each set of microsomes are included for
comparison. The mutagenic activation of IQ by the above
microsomes was highly correlated with both Form 4 [r = 0.96
(Fig. 8,4)] and Form 6 [r = 0.92 (Fig. SB)] content, respectively.
Consistent with polycyclic hydrocarbon inducible cytochromes
P-450 being under similar regulatory control (2), Form 4 con
tent of microsomes was highly correlated with Form 6 content
(r = 0.96). The correlation of mutagenicity with total cyto
chrome P-450 content although significant was weaker air =
0.61 (P>0.01).
Fig. 7. Typical quantitative Western blots of purified rabbit cytochrome P450 Forms 4 and 6 and control and pretreated rabbit liver microsomes with antiForm 4 (A) and anti-Form 6 (H) IgG, respectively. Electrophoresis and ¡nummo
blotting were carried out as described under "Materials and Methods." The
migration of proteins was from lop to bottom of the gel. A, Lanes 1-6 contained
0.2-2 pmol of purified cytochrome P-450 Form 4. The remaining lanes contained
two amounts of microsomal protein from individually pretreated rabbits as
follows: Lanes 7 and 8, 5 and 2.5 /ig rifampicin; Lanes 9 and 10, 2 and 1 Mg
phénobarbital;Lanes 11 and 12, 2 and 1 jig phénobarbital;and Lanes 13 and 14,
0.5 and 0.25 ¿igTCDD. B, Lanes 1-6 contained 0.2-2 pmol of purified cyto
chrome P-450 Form 6. The remaining lanes contained two amounts of microso
mal protein from individually pretreated rabbits as follows: Lanes 7 and 8, 1 and
0.5 jig TCDD; Lanes 9 and 10, 1 and 0.5 ¿ig
TCDD; Lanes 11 and 12, 7.5 and 5
ng rifampicin; and Lanes 13 and 14, 7.5 and 5 ¿igrifampicin.
Table 1 Effect of inducing agents on the concentration of cytochrome P-450
Forms 4 and 6 in rabbit liver microsomes
proteinInducing
cytochrome
P-4501.35
40.55
60.27
±0.19 (4)°
+ 0.38(4)
±0.13 (4)
Phénobarbital
0.63 ±0.34 (3)
0.24 ±0.11 (3)
2.64 ±0.08 (3)
1.18 + 0.35(3)
Acetone
0.51 ±0.27(3)
0.22 ±0.12 (3)
1.87 + 0.38(3)
Rifampicin
0.28 ±0.16 (3)
0.13 ±0.07 (3)
/3-Naphthoflavone
1.83(1)
1.10(1)
0.61 (1)
TCDDForm
2. 19 ±0.43 (4)Form0.84 ±0.11 (4)Total2.64 + 0.31 (4)
" Mean ±SD; the number of animals used is shown in parentheses.
agentNone
DISCUSSION
In the liver, both the cytochrome P-450 (1-3, 9, 10) and
flavin-containing monooxygenase (23, 25, 26) enzyme systems
have been implicated in the oxidative activation of aromatic
and heterocyclic amines and their derivatives (1-3). For these
compounds W-hydroxylation is considered the first and obliga
tory step in their metabolic activation (1, 11). Eight forms of
rabbit cytochrome P-450 have been isolated and extensively
characterized and indications are that there are at least 13
forms (27). The rabbit flavin-containing monooxygenase system
is also composed of more than one form (28,29). An important
property of the cytochrome P-450 system is the differential
induction of different isozymes by certain chemicals. In the
nmol/mg
rabbit, TCDD has been shown to induce cytochrome P-450
Forms 4 and 6 in the liver whereas phénobarbitalinduces Forms
2 and 5 (2). In a previous study we showed that of six highly
purified forms of cytochrome P-450, only Form 4 yV-hydroxylated the carcinogen 2-acetylaminofluorene (7). Form 4 has also
been shown to be involved in the metabolic activation of 2aminoanthracene (8), acetaminophen (30), 2-aminofluorene (9),
and to a lesser extent jY-nitrosodimethylamine (31). Polycyclic
4517
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ACTIVATION OF IQ BY CYTOCHROMES
s
tfì
10
20
30
pmol FORM a / W9MICROSOMALPROTEIN
025
050
075
100
P-450
inconclusive. This is due to the fact that this antibody inhibits
the activation of IQ by both Forms 4 and 6 of cytochrome P450 in reconstituted experiments, which is not unexpected since
these proteins share 62% homology (2). It can also be concluded
from these observations that the remaining uninhibited activity
in control and polycyclic hydrocarbon induced rabbit liver mi
crosomes is not due to cytochrome Form 6.
The possibility existed that the activity not inhibited by antiForm 4 IgG could in part be due to the FCMO. This enzyme
has been shown previously to contribute in a minor way to the
activation of 2-aminofluorene (23) but to be the major enzyme
involved in the jV-hydroxylation of jY-methyl-4-aminoazobenzene (26). However, failure of heating control and TCDD
induced rabbit liver microsomes to 45°Cin the absence of
pmol FORM6 / ÌÌQ
MICROSOMALPROTEIN
NADPH to reduce their capacity to activate IQ to a mutagen
strongly suggests that the FCMO is not involved in this process
(15, 28). Lack of FCMO involvement is further supported by
the fact that the same treatment nearly completely destroyed
the capacity of the above microsomes to /V-oxidize dimethyling symbols represent control (A), phénobarbital(A), acetone (•),rifampicin (D).
aniline. This activity in rabbit liver microsomes has been shown
f)-naphlhoflavone (x), and 2,3,7,8-tetrachlorodibenzo-p-dioxin(*)
pretreated rab
to be primarily mediated by the FCMO (36). Further support
bit liver microsomes. The line through the points was drawn using linear regression
analysis.
for the noninvolvement of the FCMO comes from the obser
vation that anti-NADPH-cytochrome
P-450 reducÃ-asesignifi
hydrocarbon inducible forms of rat cytochrome P-450 have also cantly inhibited the mutagenicity of IQ, which infers this proc
ess is mediated by cytochrome P-450. Interestingly this anti
been shown to be involved in the metabolic activation certain
body was less efficient at inhibiting the activation of IQ than
heterocyclic amines (32-34). In the present study, an increase
in the activation of the food derived heterocyclic amine IQ to a inhibiting the /V-hydroxylation of 2-acetylaminofluorene (Fig.
5, B and C) which has been shown previously to be selectively
mutagen was observed in TCDD and ß-naphthoflavone induced
liver microsomes but not with rifampicin, acetone, or phéno carried out by cytochrome P-450 Form 4 (6, 7). Guengerich et
al. (37) has previously observed a differential effect of antirebarbital induced microsomes. These data strongly implicate
ductase on the metabolism of different substrates by human
either Form 4 or 6 in this process. Indeed with liver microsomes
from animals pretreated with other inducing agents such as liver microsomes. The above observations plus the inability of
phénobarbital (Forms 2 and 5), rifampicin (Form 3c), and the purified hog liver enzyme to activate IQ to a mutagen
indicate that this process is cytochrome P-450 mediated.
acetone (Form 3a) (2), a decrease in the number of revenants
For 2-aminofluorene and 2-aminoanthracene Robertson et
produced by IQ was observed. Robertson et al. (9, 10) have
al. (9) have indicated that the activation of these compounds to
strongly implicated cytochrome P-450 Form 5 as the major
mutagens by rabbit microsomes depends on the relative concen
form responsible for the metabolic activation of 2-aminoflutration of cytochrome P-450 Forms 4 and 5. To ascertain
orene. The results from the present study support this obser
whether the levels of Forms 4 and 6 are critical to the activation
vation, because only liver microsomes from phénobarbitalpre
treated animals increased the mutagenesis of 2-aminofluorene
of IQ we estimated the content of these isozymes in control
above control values (Fig. 2). However, Form 5 is unlikely to and induced rabbit liver microsomes and correlated this with
contribute significantly to the activation of IQ because phéno their capacity to activate IQ. Using Western blots for immunoquantitation, Form 4 and 6 contents of control microsomes
barbital pretreated liver microsomes exhibited a reduced capac
were 0.55 ±0.19 and 0.27 ±0.13 nmol/mg protein (Table 1),
ity to activate IQ to a mutagen compared to control micro
somes. Further, in control and TCDD induced rabbit lung respectively. The value in this study for Form 4 is lower than
those of 0.89-1.61 nmol/mg protein reported by Bon til s et al.
microsomes where cytochrome P-450 Forms 5 and 6 comprise
approximately 50% of total cytochrome P-450 content, respec
(38) who used a radial immunodiffusion assay and 0.75 nmol/
tively (10, 35), the latter microsomes were 6-fold more efficient
mg protein reported by Chiang et al. (39) who used rocket
in activating IQ to a mutagen. Collectively the above liver and immunoelectrophoresis to estimate the content of this enzyme.
lung microsomal data support a major role for cytochrome P- When Form 4 content is expressed as a percentage of total
cytochrome P-450 content the value in this study of 41% is
450 Forms 4 and 6 in the activation of IQ.
In reconstitution experiments cytochrome P-450 Form 4 was approximately twice that reported by Guengerich et al. (40) of
considerably more active in IQ activation than Form 6. The 22%. However, both the Form 4 and Form 6 content of control
constitutive isozyme, Form 3b, was totally inactive. This is microsomes in this study compare favorably with values re
ported by Philpot et al. (35). As expected in TCDD and ßconsistent with the work of Yamazoe et al. (33), who showed
that a rat cytochrome P-450 termed P-448-lla was the most
naphthoflavone pretreated rabbit liver microsomes a significant
increase in the content of both cytochrome P-450 Forms 4 and
active in metabolizing IQ to a mutagen. The authors state that
this form is thought to correspond to rat cytochrome P-450d, 6 was observed and the degree of induction is consistent with
previously reported values (2, 35). Phénobarbitalpretreatment
which is the orthologue of rabbit Form 4 (2). The rat orthologue
of Form 6 was also less active than its Form 4 equivalent in caused no induction of Forms 4 or 6 which is also consistent
activating IQ to a mutagen. While reconstitution data show with previous studies (35). To our knowledge the effect of
that cytochrome P-450 Form 4 is considerably more efficient
acetone and rifampicin induction on the synthesis of rabbit liver
than Form 6 in activating IQ to a mutagen, anti-Form 4 IgG cytochromes P-450 Forms 4 and 6 have not been reported. In
inhibition of IQ activation in the microsomal membrane is this study both these pretreatments caused no statistically sigFig. 8. Correlation of the mutagenic activation of IQ with immunochemically
determined cytochrome P-450 Form 4 (A) and Form 6 (B) content in control and
pretreated rabbit liver microsomes. Microsomal samples were prepared from at
least three animals per treatment group. The Ames test and Western blotting
assays were carried out as described under "Materials and Methods." The follow
4518
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ACTIVATION OF IQ BY CYTOCHROMES
nificant changes in the microsomal content of cytochrome P450, Forms 4 and 6 (Table 1). In the case of TCDD induced
microsomes the combined total of cytochromes P-450 Forms 4
and 6 exceeded the spectrophotometrically determined value of
total cytochrome P-450. This is probably because the antibodies
recognize both an apoprotein and holocytochrome P-450.
When cytochrome P-450 Forms 4 and 6 content of control
and induced rabbit liver microsomes were correlated with their
capacity to mutagenically activate IQ, highly statistically sig
nificant correlations were obtained. This is further proof that
these two polycyclic hydrocarbon inducible hemoproteins are
primarily responsible for the metabolic activation of IQ. How
ever, Form 4 was the major isozyme involved in the activation
of IQ in all liver microsomes tested, since it is present at a
higher concentration than Form 6 and is approximately 8 times
more active than this isozyme. In in vivo carcinogenicity tests,
IQ has been shown to induce tumors in both hepatic and
extrahepatic tissues (12). Based on the results of this study and
the tissue distribution of Forms 4 and 6 (2), the former ob
viously contributes the most towards the activation of this
heterocyclic amine in the liver, whereas Form 6 would be
expected to be primarily involved in this process in extrahepatic
tissues.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
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4519
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Specificity of Rabbit Cytochrome P-450 Isozymes Involved in
the Metabolic Activation of the Food Derived Mutagen
2-Amino-3-methylimidazo[4,5-Β ƒ] quinoline
Michael E. McManus, Wendy Burgess, Elizabeth Snyderwine, et al.
Cancer Res 1988;48:4513-4519.
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