[CANCER RESEARCH 51, 5284-5291. October 1. 1991]
Activation of Amino-a-carboline, 2-Amino-l-methyl-6-phenylimidazo[4,5-A]pyridine,
and a Copper Phthalocyanine Cellulose Extract of Cigarette Smoke Condensate
by Cytochrome P-450 Enzymes in Rat and Human Liver Microsomes1
Tsutomu Shimada2 and F. Peter Guengerich3
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
ABSTRACT
The ability of cigarette smoke condensate to induce a genotoxic re
sponse has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450)
enzymes, as determined by unni gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-a-carboline and 2amino-l-methyl-o-phenylimidazo|4,5-A)pyridine (PhIP), two compounds
known to be present at considerable levels in cigarette smoke condensate,
were also determined and compared with regard to genotoxicity. Aminoa-carboline and PhIP are activated principally by P-450 1A2 enzymes in
human and rat liver microsomes: (a) activation of both compounds was
catalyzed efficiently by liver microsomes prepared from rats treated with
5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl
mixture Aroclor 1254, and the activities could be considerably inhibited
by antibodies raised against P-450 1A1 or l A2; (b) the rates of activation
of these compounds were correlated with the amount of human P-450
1A2 and of phenacetin O-deethylation activity in different human liver
microsomal preparations, and these activities were inhibited by anti-P450 l A2; (c) reconstituted enzyme systems containing P-450 1A enzymes
isolated from rats and humans showed the highest rates of activation of
amino-a-carboline and PhIP. In rat liver microsomes PhIP may also be
activated by P-450 3A enzymes; activity was induced in rats treated with
pregenenolone 16a-carbonitrile and was inhibited by anti-human P-450
3A4. However, in humans the contribution of P-450 3A enzymes could
be excluded as judged by the very low effects of anti-P-450 3A4 on the
microsomal activities and poor correlation with P-450 3A4-catalyzed
activities in various liver samples. Cigarette smoke condensate strongly
inhibited the activation of several potent procarcinogens by human liver
microsomes, particularly the reactions catalyzed by P-450 1A2, but was
not so inhibitory of the activation reactions catalyzed by P-450 3A4 and
of P-450 2D6-catalyzed bufuralol I'-hydroxylation. Genotoxic compo
nents of the cigarette smoke condensate were extracted by using copper
phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was
observed only after activation by P-450, and the inhibition of P-450 l A2
activities by these extracts was slight. The following results collectively
indicate that P-450 IA2 may be the major catalyst for the activation of
the components in this blue cotton extract: (a) isosafrole and Aroclor
1254 induced the activities seen in rat liver microsomes; (b) in different
human liver preparations the activation of the extract was correlated with
activities catalyzed by P-450 1A2 and was inhibited by 7,8-benzoflavone
or a human autoimmune antiserum that strongly and specifically recog
nizes human P-450 1A2; (c) reconstituted systems containing purified
human, rat, or rabbit P-450 1A2 catalyzed the activation of the extract.
The participation of the human P-450 2D6 enzyme in the activation of
these chemicals does not seem as likely because the activities were not
inhibited by quinidine or by antibodies raised against rat P-450 2D1.
Thus, the work does not provide evidence for the activation of genotoxic
components of tobacco smoke by P-450 2D6 as being the basis for the
reported association between debrisoquine 4-hydroxylation and incidence
Received 4/4/91; accepted 7/19/91.
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.
1Supported in part by USPHS Grants CA 44353 and ES 00267 (F. P. G.)
and by a Grant in Aid for Cancer Research from the Ministry of Education,
Science, and Culture of Japan (T. S.).
2 Present address: Osaka Prefectura! Institute of Public Health, Nakamichi,
Higashinari-ku. Osaka 537. Japan.
3 To whom requests for reprints should be addressed.
of lung cancer. The present results show for the first time that cigarette
smoke condensate contains both inhibitors of P-450 enzymes and procar
cinogens capable of being activated by P-450 enzymes and that the P450 1A2 enzymes appear to be the most important catalysts for the
(hepatic) activation reaction of a copper phthalocyanine cellulose extract
of cigarette smoke condensate.
INTRODUCTION
A positive relationship between ingestion of tobacco smoke
and cancers of the lung and other tissues has been established
in epidemiological investigations; further, most cancer scien
tists agree that approximately one-third of all cancers in the
United States may be attributed to tobacco use (1,2). However,
not all smokers acquire cancer and there is reason to believe
that genetic factors are operative (2-4).
Chemical carcinogens often produce their effects by modify
ing DNA and causing tumor initiation (5). Indeed, there is a
reasonable correlation between the carcinogenicity of chemicals
and their mutagenicity in simple bacterial systems (6), at least
in the absence of dramatic cell proliferation and tumor pro
motion. Indeed, cigarette smoke condensate has been shown to
be mutagenic after activation by rat liver microsomes (7) and
direct mutagens are excreted in the urine of smokers (8). Ciga
rette smoke and even tobacco itself are extremely complex
mixtures and contain thousands of individual chemicals [>4000
have reportedly been identified (9)], many of which may have
the capability to induce cancer. Exactly which chemicals in the
tobacco and smoke are most responsible for causing cancer is
unknown. Some classes of compounds under consideration are
polycyclic aromatic hydrocarbons, arylamines, nitrosamines,
vinyl monomers, radioisotopes, benzene, and catechols (9-12).
The work of Kier et al. (7) suggests that P-4504 enzymes are
involved in the activation of chemicals in cigarette smoke
condensate to mutagens. However, there are many different P450 enzymes and most of these show rather independent pat
terns of regulation and catalytic specificity (23, 24). Epidemi
ological studies suggest that human P-450 1Al and P-450 2D6
may have roles in the etiology of tobacco-related cancer (25,
26). However, the complexity of the chemical mixtures in
4 The abbreviations used are: P-450, microsomal cytochrome P-450; BNF, ßnaphthoflavone (5,6-benzoflavone); ISF, isosafrole; PCN, pregnenolone-16n-carbonitrile: Aroclor, Aroclor 1254, a polychlorinated biphenyl mixture containing
~54% chlorine; A«C,amino-n-carboline; PhIP, 2-amino-l-methyl-6-phenylimidazo[4,5-e]pyridine; MelQ, 2-amino-3,5-dimethylimidazo[4,5-/]quinoline;
IQ, 2amino-3-methylimidazo[4,5-/]quinoline;
6-AC, 6-aminochrysene. The P-450 en
zymes characterized in this laboratory are sometimes referred to with abbrevia
tions based upon the substrates which were utilized during purification. See
Nebert et al. (13) for a categorization of the P-450 genes and Guengerich (14) for
a review of individual human P-450 enzymes; both references describe related
preparations derived in other laboratories. For reference, P-450 3A4 (P-450NF>is
the nifedipine oxidase (15-17), P-450 2D6 (P-450DB) is the debrisoquine 4hydroxylase/bufuralol l'-hydroxylase (18), P-450 1A2 (P-450PA) is the highaffinity phenacetin O-deethylase (18, 19), and P-450MP refers to a group of
enzymes (P-450MP.„P-450MP-2,P-450MP.j, P-450TB: CYP 2C8, CYP 2C9, CYP
2C10, and other genes in the 2C family), two of which are (S)-mephenytoin 4'hydroxylases (20, 21). P,-450 is the product of the CYP I Al gene (22) and is
referred to as P-450 1A1 here.
5284
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
tobacco and cigarette smoke condensate has made in vitro
investigations difficult.
In this work we utilized the so-called umu test for genotoxicity, where bacterial DNA damage induces the SOS response
(27). Bioactivation of chemicals in an extract of cigarette smoke
condensate, as measured by this assay, appears to be catalyzed
primarily by P-450 1A2. The results are also consonant with a
major role for arylamines in such extracts of cigarette smoke.
MATERIALS
AND METHODS
Chemicals. AaC, PhIP, and 6-AC were gifts from Dr. F. F. Kadlubar,
National Center for Toxicological Research (Jefferson, AR). Polycyclic
hydrocarbon derivatives were obtained from the National Cancer Insti
tute Repository through Midwest Research Institute (Kansas City,
MO). Other procarcinogens and chemicals used were from the same
sources described previously (28).
Cigarette Smoke Condensate and Extraction of Carcinogens by Blue
Cotton. Cigarette smoke condensate (standard 2R1 cigarette) was ob
tained from the University of Kentucky Tobacco and Health Research
Institute (Lexington, KY) and dissolved in (CH3)2SO or acetone at a
concentration of 5 mg/ml. Extraction of carcinogens with blue cotton
(copper phthalocyanine cellulose, Sigma Chemical Co., St. Louis, MO)
was carried out with the methods described (29). Briefly, about 2 g of
cigarette smoke condensate was dissolved in a small amount of
(CH3)2SO and then diluted with H2O to 400 ml. To this solution was
added about l g of blue cotton (which had been prewashed with
(CH3)2SO, 2% concentrated NH4OH (v/v) in CH,,OH, and then exten
sively with H2O). The extraction was carried out at room temperature
with vigorous shaking for 1 h, and elution was done with 400 ml of
0.1% concentrated NH4OH in CH3OH (v/v) after extensive washing of
the blue cotton with H2O. The overall recovery was about 3% (w/w),
and the materials were dissolved in acetone to give a concentration of
5 mg/ml; this is referred to as "blue cotton extract." It was stored under
argon at -70°C.
Enzyme Preparations and Antibodies. Human liver samples were
obtained from organ donors through Tennessee Donor Services and
microsomal fractions were prepared as described elsewhere (30). Male
rats (Sprague-Dawley strain, weighing about 250 g; HarÃ-anIndustries,
Indianapolis, IN) were treated i.p. with each of several P-450 inducers:
phénobarbital(80 mg/kg, daily for 3 days), BNF (50 mg/kg, daily for
3 days), ISF (100 mg/kg, daily for 3 days), PCN (80 mg/kg, daily for
3 days), or Aroclor (500 mg/kg, once). Rats were starved overnight
before killing. Microsomes were prepared as described elsewhere (30).
Human P-450Mp (31), P-450 3A4 (15), and P-450 1A2 (18) were
prepared as described elsewhere. Rat P-450s 1A1, 1A2, and 2B1 (32)
and rabbit P-450 l A2 (33) were purified to electrophoretic homogeneity
as described previously. Rabbit anti-human P-450MP (31), anti-human
P-450 3A4 (15), anti-rat P-450 1A1 (32), and anti-rat P-450 1A2 (32)
preparations have been characterized previously. The anti-rat P-450
1Al antisera was cross-absorbed with microsomes prepared from livers
of phenobarbital-treated rats (immobilized on gels) to remove antibod
ies that react with P-450 1A2 (34). The anti-human P-450 3A4 prepa
ration appears to recognize most or all of the human and rat P-450 3A
enzymes (13, 14). IgG fractions were prepared and used to inhibit
catalytic activities in microsomes (35). Rabbit liver NADPH-P-450
reducÃ-asewas purified by using the general procedure described by
Yasukochi and Masters (36).
Assays. Bufuralol 1'-hydroxylation (18, 37) and phenacetin O-deethylation (38) were determined as described elsewhere. Protein concen
trations were estimated by using the Pierce bincinchonic acid procedure
according to the manufacturer's directions (Pierce Chemical Co., Rockford, IL). P-450 was estimated spectrally from Fe2+-CO versus Fe2+
spectra as described by Omura and Sato (39). Protein immunoblotting
was done as described elsewhere (34).
Details of the umu gene expression assay for DNA damage are
described elsewhere (27, 28, 40); in general, human and rat liver
microsomes containing 10 to 100 pmol of P-450 were used with 0-25
Mgof cigarette smoke condensate or blue cotton extract (or 5 nmol of
each procarcinogen when noted) in a volume of 1.0 ml of 50 mM
potassium phosphate buffer (pH 7.4) containing a NADPH-generating
system (consisting of 5 mM glucose-6-phosphate, 0.5 mM NADP+, and
1 IU yeast glucose-6-phosphate dehydrogenase/ml) and a suspension
of bacterial tester strain Salmonella typhimurium TA1535/pSK1002 as
described previously (27, 28, 40). In initial experiments we also used a
new bacterial strain, S. typhimurium TA1535/pSK1002/pNM12,
which overexpresses /V-acetyltransfer activity; the strain was kindly
donated by Dr. Y. Oda, Osaka Prefectural Institute of Public Health
(Osaka, Japan). Optimal conditions for the bioactivation of MelQ, IQ,
2-amino-6-methyldipyrido[l,2-a:3',2'-i/]imidazole,
aflatoxin B,, and 6AC have already been reported in this laboratory (28) and were deter
mined for the assays with A«Cand PhIP in the present study. In the
reconstituted systems, microsomes were replaced with a premixed
complex of (final concentrations in assay) 0.01-0.20 UM P-450, 0.05
MMrabbit NADPH-P-450 reducÃ-ase,and 7.5 nM L-a-dilauroyl-sn-glycero-3-phosphocholine. The expression of the umu gene was monitored
by measuring fi-galactosidase activity (the product of a fused
umuC'lacZ gene), and the bioactivation is presented as units of ßgalactosidase activity per min per mg protein or nmol P-450 (41).
RESULTS
Activation of Cigarette Smoke Condensate and Blue Cotton
Extract by P-450 Enzymes. In order to optimize the reaction
catalyzed by P-450 enzymes, we first examined the activation
of cigarette smoke condensate by liver microsomes and recon
stituted P-450 systems. The ability of the condensate to induce
umu gene expression after metabolism by human and rat liver
microsomes was very low (results not shown). However, when
the reconstituted system containing human P-450 1A2 was
used for the activation, considerable genotoxicity produced by
crude cigarette smoke condensate was observed (Fig. 1). The
genotoxic components of cigarette smoke condensate were ex
tracted with blue cotton as described under "Materials and
Methods" and were used in the umu assay in conjunction with
P-450 enzymes. This extraction procedure has previously been
shown to yield excellent recoveries of arylamines and other
hydrophobic carcinogens (>60% from very crude and dilute
solutions) (29,42-44). No response was observed in the absence
of the enzymatic activation system. The genotoxic activity of
the blue cotton extract was increased dramatically compared
with that of the condensate, and the reaction was dependent on
both the concentrations of P-450 1A2 and the condensate or
extract (Fig. 1). However, when a high substrate concentration
was included in the reaction mixture less activity was the result,
probably reflecting the presence of inhibitors in the extract.
Although the results are not presented here, it should be men-
0
100
200
Human P-4501A2
(pmol)
Condensate or extract
(ng/ml)
Fig. 1. Dependence of umu gene response on amount of human P-450 IA2 (A)
and on concentration of cigarette smoke condensate or blue cotton extract (B) in
reconstituted monooxygenase systems. The basic incubation systems contained
0.05 nmol of P-450 and 5 »ig
of either cigarette smoke condensate (•)or blue
cotton extract (O) per ml. P-450 concentrations were varied as indicated in A. In
B, the P-450 1A2 concentration was held constant and the amount of cigarette
condensate or blue cotton extract was varied.
5285
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
.5
150
o
•¿
~
150
(AFB1)
Effects of Cigarette Smoke Condensate or Blue Cotton Extract
on Activation of Several Procarcinogens in Human Liver Micro
somes. In order to examine the possibility that the cigarette
smoke condensate contains some inhibitors of P-450 reactions
as well as procarcinogens, we examined the effect of cigarette
smoke condensate or blue cotton extract on the activation of
several procarcinogens and bufuralol 1'-hydroxylation by liver
(6-AC)
100
50
50
microsomes (Fig. 2). Cigarette smoke condensate caused a
slight inhibition of the activation of aflatoxin B,, 6-AC, and
7,8-dihydroxy-7.8-dihydrobenzo(a)pyrene
and bufuralol 1'-hy
c
o
u
u
12
Q.
150
C
24
12
24
150
(B(a)P-7,8-diol)
o
100
50
o.
O
12
24
Condensate
o
or extract
12
(ng ml)
Fig. 2. Effects of cigarette smoke condensate (•)and blue cotton extract (O)
on the activation of (.4) aflatoxin B, (AFB,); (B) 6-AC; (C) 7,8-dihydroxy-7.8dihydrobenzo(a)p>Tene [B(a)P-7,8-diol\\ and (D) bufuralol I '-hydroxylation cat
alyzed by human liver microsomes (sample HL-I01). Control activities without
the chemicals were 1201. 259, and 122 (umu) units/min/nmol P-450 for the
bioactivation of aflatoxin BI, 6-AC, and 7.8-dihydroxy-7,8-dihydrobenzo(o)pyrene, respectively, and 0.33 nmol product formed/min/nmol P-450 for bufuralol
1'-hydroxylation.
100
24
(IQ)
100
12
24
Condensate
0
or extract
12
24
(ng/ml)
Fig. 3. Effects of cigarette smoke condensate (•)and blue cotton extract (O)
on the activation of (A) A«C.(A) PhlP. (C) MelQ. and (O) IQ catalyzed by
human liver microsomes (sample HL-101). Control activities without the chem
icals were 37, 67, 1301, and 826 units/min/nmol P-450 for the bioactivation of
A«C,PhlP, MelQ, and IQ, respectively.
droxylation; the former three reactions are known to be cata
lyzed by P-450 3A4 and the latter hydroxylation by P-450 2D6
in human liver microsomes (18, 28, 45). Blue cotton extract,
on the other hand, showed a much less inhibitory effect on
these reactions.
The effects of cigarette smoke condensate and blue cotton
extract on the activation of A«C,PhlP, MelQ, and IQ catalyzed
by human liver microsomes are shown in Fig. 3. The latter two
reactions have been shown in this laboratory to be catalyzed
mainly by P-450 1A2 in human liver, and the former two
reactions are reported in this study to be catalyzed principally
by the same enzyme (see below; see also Ref. 46). Cigarette
smoke condensate showed potent inhibition of these activation
reactions and blue cotton extract caused less but still consider
able inhibition.
On the basis of these results subsequent experiments were
done by using 5 ^g of blue cotton extract per ml of incubation
mixture; in this concentration range the inhibition of P-450
1A2 reactions was not so extensive and the activation of these
components could readily be measured in the system.
Activation of AaC, PhlP, and Blue Cotton Extract by Rat
Liver Microsomes. The roles of P-450 enzymes in the activation
of AivC, PhlP, and the blue cotton extract of cigarette smoke
condensate were examined by using liver microsomes from
variously pretreated rats (Table 1). The activation of A«Cand
PhlP by liver microsomes could be enhanced by about 10-fold
and 6-fold in rats treated with BNF and Aroclor, respectively,
on the basis of units/min/nmol P-450. ISF was also an ¡mincer
for the two reactions although not as potent as BNF. PCN also
produced some increase in the activation of A«C(2-fold) and
PhlP (4-fold). The trend for the activation of the blue cotton
extract by rat liver microsomes seems to be similar to that of
the activities of A«Cand PhlP activation, except that ISF was
the strongest inducer in the case of the blue cotton extract and
PCN did not cause an increase in the activation of the blue
cotton extract.
Activation of AaC, PhlP, and Blue Cotton Extract by Human
Liver Microsomes. The genotoxic activities of A«C,PhlP, and
the blue cotton extract were also determined in 9 human liver
Table 1 Activation of AaC. PhlP, ana hlue cotton extract hy rat liver microsomes
AnC (5 MM).PhlP (5 /IM). or blue cotton extract (5 tig/mi) was incubated with
rat liver microsomes (0.05 MMP-450) in the presence of an NADPH-generating
system and bacterial tester strain 5. typhimurium TA1535/pSK1002; the induced
umu gene expression was determined as described in "Materials and Methods."
Data presented are means of duplicate determinations with combined microsomal
preparations from 3 rats ±SD (range).
umu response (units/min/nmol P-450)
cotton
extract3.8
tioned that the use of a newly developed bacterial strain, S.
typhimurium TA1535/pSK1002/pNM12,
increased the genotoxic response seen by cigarette smoke condensate or the blue
cotton extract but the enhanced response relative to the control
was not overly dramatic.
Control
Phénobarbital
BNF
ISF
PCN
AroclorAaC2.3
2.3
23.3
10.4
5.4
13.0
±1.2
12.4
±0.6
±3.7
64.1
±1.4
37.1
±1.6
29.7
40.5
±2.0PhlP6.8
±0.9
+ 4.3
±11.6
±6.2
±5.4
±7.1Blue
5.4
8.1
11.6
4.0
14.0
±2.9
±2.2
±3.2
+ 2.9
±1.9
±4.1
5286
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
Table 2 .Miration of AaC, PhIP, and hlue cotton extract hy human liver
microsomes
Experimental conditions are similar to those of Table 1, except that human
liver microsomes from 9 individual samples were used. The results are presented
as means of duplicate determinations.
umu response (units/min/nmol P-450)
(the activity was not as high when reconstituted in the presence
of human cytochrome h*). Among the rat P-450 enzymes, P450s 1A1 and 1A2 catalyzed the activation of these compounds;
rat P-450 2B1 was a poor catalyst. Rabbit P-450 1A2 could
also catalyze the activation of AaC, PhIP, and the blue cotton
extract.
extract7.39.34.03.14.93.66.96.08.05.9
cotton
SampleHL-99HL-101HL-102HL-105HL-107HL-110HL-112HL-113HL-124Mean
Effects of Anti-P-450 IgG Preparations on Activation of AaC,
PhIP, and Blue Cotton Extract Catalyzed by Rat and Human
Liver Microsomes. The above results support the view that P450 1A enzymes may be the principal ones involved in the
activation of these chemicals in liver microsomes. The effects
of anti-P-450 IgG preparations on the microsomal activities in
rats and humans were examined: anti-rat P-450 1A1 and antirat P-450 1A2 inhibited the activation of AaC catalyzed by
±2.1
±SDAnC17.435.710.35.39.54.36.914.718.513.6
±9.7PhIP37.860.721.76.011.88.523.919.737.325.3
±17.5Blue
liver microsomes from Aroclor-treated rats, and anti-human P450 1A2 caused about 50% inhibition of the human liver
microsomal activation of AaC (Table 5). The effects of anti-PTable 3 Correlation of activities ofmonooxygena.se activities and amounts of
microsomal components in human liver microsomal preparations
450 IgG preparations on the activation of PhIP catalyzed by
Correlation coefficient (r)
liver microsomes prepared from a human sample (HL-99) and
Aroclor-treated rats are shown in Fig. 4. The activation of PhIP
A«C PhIP
Blue cotton extract
was inhibited by anti-P-450 1A2 but not by anti-P-450 3A4 or
0.74
0.92
anti-P-450 2C IgG in human liver microsomes (Fig. 4/1). In
0.85
0.92
Blue cotton extract"
0.74
0.85
liver microsomes prepared from Aroclor-treated rats the en
Glu P-1"'*
0.85
0.89
0.64
zymes catalyzing PhIP activation were inhibited by anti-rat PPhenacetin O-deethylation'
0.80
0.89
0.91
P-450 \\2d
0.81
0.90
0.78
450 1Al, anti-rat P-450 1A2, and anti-human P-450 3A4 IgG
P-450Mp (2C)"
0.09
0.04
0.03
preparations (Fig. 4B).
P-450 3A4*
0.43
0.05
0.03
Total P-450''
The effect of a human autoimmune antiserum on the activa
0.03
0.06
0.40
" Activation of procarcinogens (umu units/min/mg of protein).
tion of the blue cotton extract by human liver microsomes is
*Glu P-1. 2-amino-6-methyldipyrido(1.2-a:3'.2'-</)imidazole.
shown in Fig. 5/1; these antibodies have been shown to selec
' Microsomal hydrox) lation (nmol product/min/mg of protein).
tively recognize and inhibit human P-450 1A2 strongly (47).
J P-450 content (nmol/mg of protein or blotting ¡mensity/mgof protein).
The antiserum caused significant inhibition of activities of the
blue cotton extract by liver microsomes. Anti-rat P-450 2D1
microsomal preparations (Table 2). The activities for A«Cand showed no effect on the activation of the blue cotton extract
PhIP were found to be relatively high in human liver micro
catalyzed by human liver microsomes (Fig. 5B); this particular
somes as compared with rat liver microsomes. The mean activ
antibody preparation had been shown to inhibit >90% of the
ities (13.6 and 25.3 units/min/nmol P-450 for A«Cand PhIP,
bufuralol 1'-hydroxylation in human liver microsomes when
respectively) were greater than the activities obtained with the
control rat preparations. The activation of the blue cotton
Table 4 Activation of AaC, PhIP, and hlue cotton extract by reconstituted
monooxygenase systems containing purified P-450 enzymes
extract was also seen in human liver microsomal samples; the
activities were always lower than those in A«Cand PhIP. In
Experimental conditions were as in the legend to Table 1. except that recon
order to identify the possible roles of P-450 enzymes in the stituted monooxygenase systems were used. Data are presented as means of
duplicate or triplicate determinations.
activation of these chemicals in human liver microsomes, we
umu response (units/min/nmol P-450)
determined several other parameters (catalytic activities and
formsHuman
P-450
amounts of individual P-450 enzymes) and made comparisons
cotton
extract34<1<1IS23418
P-4501A22C
(Table 3). Good correlation could be seen between the rates of
activation of A«C,PhIP, and 2-amino-6-methyldipyridol[l,2(P-450MP)3A4Rat
a:3',2'-</]imidazole, phenacetin O-deethylation activity, and the
P-4501A11A22B1Rabbit
amount of P-450 1A2 in the 9 different human samples. The
activation of the blue cotton extract was also correlated with
the other activities and the amount of P-450 1A2. However,
P-4501A2AaC741454034332PhIP125<144675171Blue
other components, including the amounts of P-450\ip, P-450
3A4, or total P-450 showed poor correlation with the activation
of A«C,PhIP, or the blue cotton extract.
Reconstitution of Activation of AaC, PhIP, and Blue Cotton
Table 5 Effects of anti-P-450 IgC preparations on the activation of AaC
catalyzed hy liver microsomes of Aroclor-treated rats and a human sample
Extract in Systems Containing Purified Forms of P-450. The
Experimental conditions were described in "Materials and Methods." Data
activation of AaC, PhIP, and the blue cotton extract were
are presented as means of duplicate determinations ±SD (range).
measured in reconstituted systems containing NADPH-P-450
geneexpression (units/min/nmol
reducÃ-ase and several P-450 enzymes isolated from humans,
P-450)Aroclor-treated
rats, and rabbits (Table 4). Among the three human P-450
Anti-P-450None
P-450III
rats33.5
(HL-99)20.9
enzymes examined the rate measured with P-450 l A2 was the
±
8.2
±2.8
highest for the activation of AaC, PhIP, and the blue cotton
Preimmune
35.2 ±4.9
19.9
2.69.5
±
extract. The human P-450 2C enzyme (P-450MP) had consid
Anti-rat P-450 1A1
10
8.4 ±1.9
Anti-rat P-450 1A2
102umu
16.1 ±4.2Human
erable activity for AaC but not for PhIP or the blue cotton
Anti-human P-450 1A2mg/nmol
±1.9
extract. P-450 3A4 showed some reactivity with AaC and PhIP
5287
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
>.
*«
'>
U
a
100
•¿a
a>
50
5
u
I
0
5
10
Anti-P-450
o
(mg
5
IgG/nmol
10
P-450)
Fig. 4. Effects of anti-P-450 IgG preparations on the activation of PhIP
catalyzed by liver microsomes from a human sample (HL-99) and from Aroclortreated rats. (A) Activation of PhIP by human liver microsomes in the presence
of anti-P-450Mp (A). anti-P-450 3A4 (•).and anti-P-450 1A2 (O); (B) activation
of PhIP by rat liver microsomes in the presence of anti-rat-P-450 1A1 (•),antirat-P-450 1A2 (O), anti-rat-P-450 2B1 (A), and anti-human P-450 3A4 (A) are
shown. Control activities in the absence of the IgGs were 35 units/min/nmol P450 for human liver microsomes and 44 units/min/nmol P-450 for rat liver
microsomes.
S-,
—¿150
150 -
C
o
o
*M
(0
100'
o
50
a.
M
9
0>
e
01
3
3
a
E
0.0
0.5
1.0
Human serum (pi)
3
)
5
10
Anti-P-450 2D1
(mg IgG/nmol P-450)
Fig. 5. Effects of (A) human autoimmune antisera and (B) anti-P-450 2D1
IgG on the activation of blue cotton extract catalyzed by liver microsomes of a
human sample, HL-IOI. In I. control sera (O) or autoimmune antisera (•)was
included. Control activities without the serum or IgG were the same as in the
legend to Fig. 5.
used at the same concentration in parallel experiments (results
not presented).
Effects of 7,8-Benzoflavone and Quinidine on the Bioactivation
of Blue Cotton Extract by Human Liver Microsomes. The effects
of 7,8-benzoflavone and quinidine on the metabolic activation
of the blue cotton extract catalyzed by liver microsomes were
examined (Fig. 6). 7,8-Benzoflavone, a selective inhibitor of P450 1A2 (19), caused significant inhibition of the activities.
Quinidine, a strong inhibitor of P-450 2D6 (48,49) was without
effect on these reactions catalyzed by human liver microsomes.
associated with low levels of bacterial mutagenesis (7, 52).
Although some studies have suggested that bacterial mutage
nesis is seen only in frameshift-dependent systems and is not
dependent upon the SOS response (7), other studies have shown
that base pair substitutions are also induced (8, 53) and that
the mutagenicity is responsive to the SOS response (53). How
ever, the level of mutagenicity is still relatively low in all of
these systems. After considerable effort with S. typhimurium
TA98 and the YG1006 and YG1024 strains containing overexpressed yV-acetyltransfer activity (54), we decided that the use
of the induction of the umu response in an established test
system was most appropriate. The use of such a system which
overexpressed ¿V-acetyltransferasedid not dramatically enhance
the response.
A standard cigarette smoke condensate preparation was
found to be highly inhibitory of the P-450-catalyzed oxidative
activation of many promutagens (Fig. 3). This inhibition was
particularly notable in the case of reactions catalyzed by P-450
l A2. The exact nature of this inhibition is unknown, and it is
not clear that the inhibition is caused by the same chemicals
which are genotoxic. A number of procedures have been re
ported in the literature for the concentration of mutagens from
tobacco-related mixtures and other crude carcinogens (8, 11,
51, 55). We found the so-called blue cotton extraction proce
dure (29, 42-44) to be more efficient than polystyrene beads
(8) or alumina chromatography (10, 56, 57). It is difficult to
quantify the recovery of potentially mutagenic material because
of the poor and variable response seen in the crude cigarette
smoke condensate, but the apparent recovery was >100%,
probably because of the removal of inhibitors.
Consonant with previous reports, we found that the ability
to activate cigarette smoke condensate (actually the blue cotton
extract) was markedly less in human liver microsomes than in
liver microsomes prepared from rats treated with P-450 inducers such as BNF, ISF, or Aroclor (Table 1) (7, 52). There was
also some variation among individual human samples (Table
1), although not to the extent seen with some other P-450
reactions (24, 28). P-450 1A2 appears to be a major hepatic
enzyme involved in the bioactivation of cigarette smoke con
densate, or at least the blue cotton extract, as judged by (a) the
inducibility of the response in rat liver (Table 1); (¿>)
the corre
lation seen between the response and several marker activities
(Table 3); (c) inhibition of the activation by 7,8-benzoflavone
and by antibodies raised against P-450 1A enzymes (Figs. 4 and
DISCUSSION
The purpose of these studies was to characterize the enzy
matic activation of cigarette smoke condensate to genotoxic
materials. This is difficult in light of the complexity of the
material and the large number of carcinogens known to be
present (9). One of the initial concerns was the choice of an
assay system. While urine from cigarette smokers is known to
be directly mutagenic to bacteria (8, 50, 51 ), the in vitro acti
vation of cigarette smoke condensate itself to genotoxins is
o 100
'co
O)
o
o.
X
o
o
C
0)
O)
E
3
Inhibitor
(uM)
Fig. 6. Effects of 7,8-bcnzoflavone (•)and quinidine (O) on the bioactivation
of blue cotton extract catalyzed by liver microsomes (sample of HL-IOI). The
activity in the absence of added inhibitor was 11.2 units/min/nmol P-450.
5288
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
5); and (d) the demonstration of activity with P-450 1A2
enzymes isolated from rats, rabbits, and humans (Table 4).
Cigarette smoking is known to induce human liver P-450 1A2
(58). It should be emphasized, however, that there is consider
able evidence that P-450 1A2 is not expressed to a measureable
degree in extrahepatic tissues (14). Indeed, McLemore et al.
(59) did not detect P-450 1A2 in human lung samples, using
an oligonucleotide probe and conditions of stringency that
should have been appropriate (60) [the P-450 1A2 mRNA can
be distinguished from the P-450 1A1 mRNA by its size (61)].
However, a wide variety of evidence indicates that P-450 1A1
is usually not expressed in human liver (14); the significance of
tissue localization of P-450s is not totally clear in this regard,
since mutagens may distribute from the liver after activation
(62, 63).
The matter of P-450 2D6 may be considered. Several inves
tigations have linked the occurrence of the debrisoquine 4hydroxylation "poor metabolizer" phenotype with decreased
risk of tobacco-associated lung cancer (2, 25, 64, 65). There
have also been reports of similar associations with cancers in
other tissues (66, 67), but there have been references to lack of
association as well (68, 69). One hypothesis for the association
has been that P-450 2D6 is catalyzing the bioactivation of a
genotoxin in cigarette smoke to cause tumor initiation; indeed,
certain epidemiológica! associations have been specifically cited
to support such a view (2). However, with our in vitro screens
we have been unable to find evidence to support such a view.
For instance, the genotoxicity of the blue cotton extract was
not inhibited by quinidine or anti-rat P-450 2D1 under condi
tions known to extensively block bufuralol 1'-hydroxylation in
human liver microsomes (Tables 2 and 3 and associated data).
Qualitatively similar findings resulted from our assays involving
mutations in 5. typhimurium TA98 (data not shown). It should
also be emphasized that of all of the carcinogens examined in
recent years, none has been found to be activated primarily by
human P-450 2D6 (28, 70, 71). If there really is a relationship
between P-450 2D6 and tumor initiation, then, what can it be
due to? One possibility relates back to early studies on debri
soquine 4-hydroxylation, when it was thought that the same
enzyme was involved in phenacetin O-deethylation (72, 73).
This view was subsequently disproven with the separation and
purification of the two enzymes (18), and it is now finally clear
that P-450 1A2 is the phenacetin 0-deethylase (13, 14, 19).
Although the P-450 1A2 and 2D6 structural genes are on
separate chromosomes (13), there is still in vitro and in vivo
data suggesting some correlation in expression (18, 72). The
basis of the relationship is speculatory, but it may be possible
that the altered incidence of cancer may be related to P-450
1A2 and not to P-450 2D6 itself. In this regard, it would be of
interest to know if the caffeine 3-demethylation phenotype (P450 1A2) (19) is associated with any of the cancers of interest.
What is the major class of genotoxic material present in
cigarette smoke condensate? This is not an easy question to
answer in view of the many potentially tumorigenic materials
in the mixture and the synergism and inhibition that might be
expected (Figs. 2 and 3) (9, 10, 56, 57). Although polycyclic
hydrocarbons and their dihydrodiol epoxides in particular have
long been of concern in this regard, there is actually some
evidence against them in the etiology of lung cancer induced by
cigarette smoke, at least with regard to DNA adduct formation
resulting from the stereoisomeric epoxides derived from 7,8dihydroxy-7,8-dihydrobenzo(a)pyrene.
For instance, in the
work of Randerath et al. (74), no "P-labeled DNA adducts
corresponding to these and some related compounds were detected in smokers' lungs or placenta. Further, in rats treated
with cigarette smoke condensate no such adducts were found
under conditions where benzo(a)pyrene did yield the products
(75). There is a considerable body of evidence to support the
view that arylamines may be highly significant in smokinginduced cancer, at least at the level of tumor initiation. For
instance, Kier et al. (7) reported the highest mutagenicity for a
slightly basic fraction of cigarette smoke condensate, and other
work has shown a correlation between the content of the
arylamine A«C and the mutagenicity of different cigarette
preparations (76). Peluso et al. (50) have presented evidence
that a major fraction of the mutagenicity in smokers' urine is
related to Phi P and its products. However, the significance of
this conclusion must be considered in terms of the unusual
stability of Ar-hydroxy-2-amino-l-methyl-6-phenylimidazo[4,5¿>]pyridine
and the high activity of bacterial /V-acetyltransferases
for some aryl hydroxylamines. Many arylamines have been
detected in cigarette smoke condensate, some at high levels (11,
76, 77). Of these, A«Cseems to be present at the highest level
(76) and, in our work, it had only somewhat less genotoxicity
than PhIP (Table 1). Both rat P-450 1Al and P-450 1A2 can
activate A«C(Table 4). While McManus et al. (46) have shown
that recombinant human P-450 1A1 (primarily extrahepatic) is
much inferior to human P-450 1A2 in activating IQ, MelQ,
and MelQx, the activities toward PhIP are similar (46). This
point may be of significance in consideration of the tissue
distribution of these enzymes. However, the paradigm remains
to be better clarified in light of the low concentrations of some
of the arylamines that have exceptional genotoxicity in these
assays (27, 28). Further, there is probably a considerable degree
of variation among cigarette preparations (76, 78). Indeed,
preliminary work indicates that Glu P-2, which has genotoxicity
similar to A«Cor PhIP (28), is the main arylamine identified
in the particular cigarette smoke condensate we have worked
with.5 Another point is that the (rat) P-450 1Al enzymes have
also been shown to detoxicate PhIP by C-hydroxylation (79).
On the other hand, the slightly acidic fractions of cigarette
smoke condensate have also been shown to possess tumor
initiating and promoting properties (10, 57). Indeed, catechol
has been identified as a cocarcinogen in this fraction (10).
Nitrosamines cannot be ignored, for several are tobacco specific
and present at levels as high as some of the arylamines in
tobacco (9). The P-450s principally involved in the bioactivation
of these remain to be elucidated. Further, the nitrosamines are
notorious for not being detected readily in bacterial genotoxicity
assays, as well as the umu test used here (28, 71), and the
reason is probably not lack of the appropriate P-450s, for the
P-450 2E1 substrates can be activated but still show a poor
response (71). Thus, these might be overlooked in the in vitro
work we have done here. Further, it is impossible to completely
rule out a role for any enzyme (including P-450 2D6) in tumor
initiation because the substrate may not produce an appropriate
response in a particular set of assays.
Finally, the caveat should be presented that tumor initiation
may not be the only aspect of cancer related to cigarette smok
ing. Epidemiological studies indicate that the cancer risk to
individuals who stop smoking drops, even after many packyears of exposure. However, some of the undefined DNA ad
ducts in exsmokers' lungs have been reported to persist for up
to 14 years after smoking cessation (74), and the biologically
relevant lesions are unknown. Further, the risk of urinary
5 F. F. Kadlubar. personal communication.
5289
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
bladder cancer has been shown to be correlated with the inher
ent mutagenicity of different types of tobacco (78).
In summary, our work indicates that cigarette smoke conden
sate is highly inhibitory of human P-450 catalytic activities,
particularly those of P-450 1A2, and that in human liver
microsomes much of the activation of components of the blue
cotton extract to genotoxic materials can be attributed to P450 1A2 in liver microsomes of humans and experimental
animals. The results are consonant with roles of arylamines
such as AaC, PhIP, and 2-aminodipyrido[l,2-a:3',2'-rf]imidazole in mutagenicity associated with cigarette smoking. No
positive evidence for a role of P-450 2D6 in the activation of
genotoxins was obtained. Further studies will be required to
establish the significance of these findings to tissue-specific
cancers related to smoking.
ACKNOWLEDGMENTS
We thank Dr. D-H. Kim, T. Cheng, and A. Mower for carrying out
preliminar)' studies. Dr. P. H. Beaune for the human anti-P-450 1A2
serum. Dr. M. Iwasaki for some of the reagents. Dr. Y. Oda for the use
of a newly developed bacterial strain S. typhimurium TA1535/
pSK1002/pNM12, and Dr. F. F. Kadlubar for chemicals and helpful
discussions.
REFERENCES
1. Stjernsward, J. Battle against tobacco. J. Nati. Cancer Inst.. 81: 1524-1525,
1989.
2. Caporaso. N. E., Tucker, M. A., Hoover, R. N.. Hayes, R. B.. Pickle. L. W.,
Issaq, H. J., Muschik, G. M., Greengallo, L., Buivys, D.. Aisner. S., Resau.
J. H., Trump. B. F., Tollerud. D.. Weston, A., and Harris. C. C. Lung cancer
and the debrisoquine metabolic phenotype. J. Nati. Cancer. Inst., 82: 12641272. 1990.
3. Reif, A. E. Effect of cigarette smoking on susceptibility to lung cancer.
Oncology (Basel), 38: 76-85, 1981.
4. Sellers, T. A.. Bailey-Wilson, J. E., Elston, R. C., Wilson. A. F., E|ston, G.
Z., Ooi, W. L.. and Rothschild, H. Evidence for Mendelian inheritance in
the pathogenesis of lung cancer. J. Nati. Cancer Inst., 82: 1272-1279. 1990.
5. Miller. E. C., and Miller, J. A. Searches for ultimate chemical carcinogens
and their reactions with cellular macromolecules. Cancer (Phila.), 47: 23272345, 1981.
6. McCann, J.. Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcino
gens as mutagens in the Salmonella/mkrosome test: assay of 300 chemicals.
Proc. Nati. Acad. Sci. USA, 72: 5135-5139. 1975.
7. Kier, L. D.. Yamasaki. E., and Ames, B. N. Detection of mutagenic activity
in cigarette smoke condensâtes.Proc. Nati. Acad. Sci. USA, 7/.-4159-4163.
1974.
8. Yamazaki, E., and Ames, B. N. Concentration of mutagens from urine by
adsorption with the nonpolar resin XAD-2: cigarette smokers have mutagenic
urine. Proc. Nati. Acad. Sci. USA, 74: 3555-3559, 1977.
9. Hecht, S. S., and Hoffmann, D. The relevance of tobacco-specific nitrosamines to human cancer. Cancer Surv., 8: 273-294, 1989.
10. Hecht, S. S., Cannella, S., Mori, H., and Hoffman. D. A study of tobacco
carcinogenesis. XX. Role of catechol as a major carcinogen in the weakly
acidic fraction of smoke condensate. J. Nati. Cancer Inst., 66: 163-169.
1981.
11. Manabe. S.. and Wada. O. Carcinogenic tryptophan pyrolysis products in
cigarette smoke condensate and cigarette smoke-polluted indoor air. Environ.
Pollut.. 64: 121-132, 1990.
12. Cerna, M., Kocisova, J., Kodytkova, !.. Kopecky, J., and Sram. R. J. Muta
genic activity of oxiranecarbonitrile (glycidonitrile). In: I. Gut, M. Cikrt, and
G. L. Plaa (eds.). Industrial and Environmental Xenobiotics. Metabolism
and Pharmacokinetics of Organic Chemicals and Metals, pp. 251-254.
Berlin: Springer-Verlag, 1981.
13. Nebert, D. W., Nelson, D. R.. Coon, M. J., Estabrook, R. W.. Feyereisen.
R.. Fujii-Kuriyama. Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus. I. C.,
Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., and Waxman, D.
J. The P450 superfamily: update on new sequences, gene mapping, and
recommended nomenclature. DNA Cell Biol., 10: 1-14. 1991.
14. Guengerich. F. P. Characterization of human microsomal cytochrome P-450
enzymes. Annu. Rev. Pharmacol. Toxicol., 29: 241-264, 1989.
15. Guengerich, F. P., Martin, M. V.. Beaune. P. H.. Kremers, P., Wolff. T..
and Waxman, D. J. Characterization of rat and human liver microsomal
cytochrome P-450 forms involved in nifedipine oxidation, a prototype for
genetic polymorphism in oxidative drug metabolism. J. Biol. Chem., 261:
5051-5060. 1986.
16. Beaune, P. H., Umbenhauer, D. R., Bork. R. W., Lloyd. R. S.. and Guenger
ich, F. P. Isolation and sequence determination of a cDNA clone related to
human cytochrome P-450 nifcdipine oxidase. Proc. Nati. Acad. Sci. USA,
«J:8064-8068, 1986.
17. Bork, R. W., Muto. T.. Beaune. P. H.. Srivastava. P. K., Lloyd, R. S., and
Guengerich, F. P. Characterization of mRNA species related to human liver
cytochrome P-450 nifedipine oxidase and the regulation of catalytic activity.
J. Biol. Chem.. 264: 910-919. 1989.
18. Distlerath. L. M., Reilly. P. E. B.. Martin, M. V.. Davis. G. G.. Wilkinson.
G. R., and Guengerich, F. P. Purification and characterization of the human
liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin 0-deethylation, two prototypes for genetic polymorphism in oxidative
drug metabolism. J. Biol. Chem., 260: 9057-9067, 1985.
19. Butler, M. A., Iwasaki. M., Guengerich. F. P.. and Kadlubar. F. F. Human
cytochrome P-450PA (P-450IA2), the phenacetin O-deethylase. is primarily
responsible for the hepatic 3-demethylation of caffeine and A"-oxidation of
carcinogenic arylamines. Proc. Nati. Acad. Sci. USA, 86: 7696-7700, 1989.
20. Brian, W. R.. Srivastava, P. K., Umbenhauer, D. R.. Lloyd, R. S., and
Guengerich, F. P. Expression of a human liver cytochrome P-450 protein
with tolbutamide hydroxylase activity in Saccharomyces cererisiae. Biochem
istry, 28: 4993-4999. 1989.
21. Srivastava, P. K., Yun, C-H.. Beaune. P. H.. Ged, C., and Guengerich, F. P.
Separation of human liver tolbutamine hydroxylase and (5)-mephenytoin 4'hydroxylase cytochrome P-450 enzymes. Mol. Pharmacol., 40:69-79, 1991.
22. Jaiswal, A. K., Gonzalez, F. J., and Nebert. D. W. Human dioxin-inducible
cytochrome P,-450: complementary DNA and amino acid sequence. Science
(Washington DC), 22«:80-83. 1985.
23. Gonzalez, F. J. The molecular biology of cytochrome P450s. Pharmacol.
Rev., 40:243-288, 1989.
24. Guengerich. F. P., and Shimada. T. Oxidation of toxic and carcinogenic
chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol., 4:
391-407, 1991.
25. Law. M. R., Hetzel, M. R., and Idle. J. R. Debrisoquine metabolism and
genetic predisposition to lung cancer. Br. J. Cancer, 54: 686-687, 1989.
26. Kellerman, G., Shaw, C. R., and Luyten-Kellerman, M. Aryl hydrocarbon
hydroxylase inducibility and bronchogenic carcinoma. N. Engl. J. Med., 298:
934-937, 1973.
27. Nakamura, S., Oda, Y., Shimada, T., Oki, L, and Sugimoto, K. SOS-inducing
activity of chemical carcinogens and mutagens in Salmonella typhimurium
TA1535/pSK1002: examination with 151 chemicals. Mutât.Res., 192: 239246, 1987.
28. Shimada, T., Iwasaki. M.. Martin, M. V.. and Guengerich, F. P. Human
liver microsomal cytochrome P-450 enzymes involved in the bioactivation of
procarcinogens detected by umu gene response in Salmonella typhimurium
TA1535/pSK1002. Cancer Res., 49: 3218-3228, 1989.
29. Kikugawa, K., Kalo, T., and Takahashi, S. Possible presence of 2-amino-3,4dimethylimidazo[4,5-/|quinoline
and other heterocyclic amine-like mutagens
in roasted coffee beans. J. Agrie. Food Chem., 37: 881-886. 1989.
30. Guengerich. F. P. Analysis and characterization of enzymes. In: A. W. Hayes
(ed.). Principles and Methods of Toxicology, pp. 777-814. New York: Raven
Press. 1989.
31. Shimada, T., Misono, K. S., and Guengerich, F. P. Human liver microsomal
cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic poly
morphism in oxidative drug metabolism. Purification and characterization
of two similar forms involved in the reaction J. Biol. Chem., 261: 909-921.
1986.
32. Guengerich, F. P.. Dannan. G. A., Wright. S. T.. Martin, M. V., and
Kaminsky, L. S. Purification and characterization of liver microsomal cyto
chromes P-450: electrophoretic. spectral, catalytic, and immunochemical
properties and inducibility of eight isozymes isolated from rats treated with
phénobarbitalor fi-naphthoflavone. Biochemistry. 21: 6019-6030. 1982.
33. Alterman, M. A., and Dowgii, A. I. A simple and rapid method for the
purification of cytochrome P-450 (form LM4). Biomed. Chromatogr., 4:
221-222, 1990.
34. Guengerich, F. P., Wang, P.. and Davidson, N. K. Estimation of isozymes
of microsomal cytochrome P-450 in rats, rabbits, and humans using immu
nochemical staining coupled with sodium dodecyl sulfate-polyacry lamide gel
electrophoresis. Biochemistry. 21: 1698-1706. 1982.
35. Kaminsky, L. S., Fasco, M. J., and Guengerich. F. P. Production and
application of antibodies to rat liver cytochrome P-450. Methods Enzymol.,
74:262-212, 1981.
36. Yasukochi, Y., and Masters, B. S. S. Some properties of a detergentsolubilized NADPH-cytochrome c (cytochrome P-450) reducÃ-asepurified by
biospecific affinity Chromatograph). J. Biol. Chem., 257: 5337-5344, 1976.
37. Haefelfinger. P. Determination of bufuralol and its major metabolites in
plasma by high-performance liquid Chromatograph). J. Chromatogr., 227:
327-335. 1980.
38. Larrey. D., Distlerath. L. M., Dannon. G. A., Wilkinson, G. R., and Guen
gerich, F. P. Purification and characterization of the rat liver microsomal
cytochrome P-450 involved in the 4-hydroxylation of debrisoquine. a proto
type for genetic variation in oxidative drug metabolism. Biochemistry. 23:
2787-2795, 1984.
39. Omura. T., and Sato. R. The carbon monoxide-binding pigment of liver
microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239:
2370-2378, 1964.
40. Shimada. T.. and Nakamura. S. Cytochrome P-450-mediated activation of
procarcinogens and promutagens to DNA-damaging products by measuring
5290
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
P-450 ACTIVATION OF CIGARETTE SMOKE
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
expression of umu gene in Salmonella typhimurium TA1535/pSK1002.
Biochem. Pharmacol., 36: 1979-1987, 1987.
Miller, J. H. Experiments in Molecular Genetics, p. 352. Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory, 1972.
Hayatsu, H., Kasai, H., Yokoyama, S., Miyazawa, T., Yamaizumi, Z., Sato.
S., Nishimura. S., Arimoto. S., Hayatsu, T., and Ohara, Y. Mutagenic
metabolites in urine and feces of rats fed with 2-amino-3.8-dimethyIimidazo[4,5-/|quinoxaline,
a carcinogenic mutagen present in cooked meat.
Cancer Res., 47: 791-794, 1987.
Hayatsu. H., Matsui, Y., Ohara, Y., Oka, T., and Hayatsu, T. Characteriza
tion of mutagenic fractions in beef extract and in cooked ground beef. Use
of blue-cotton for efficient extraction. Gann. 74:472-482, 1983.
Hayatsu, H.. Oka, T., Wakata, A., Ohara. Y., Hayatsu. T., Kobayashi. H.,
and Arimoto, S. Adsorption of mutagens to cotton bearing covalently bound
trisulfocopper-phthalocyanine.
Mutât.Res., 119: 233-238, 1983.
Shimada, T., and Guengerich, F. P. Evidence for cytochrome P-450NF, the
nifedipine oxidase, being the principal enzyme involved in the bioactivation
of aflatoxins in human liver. Proc. Nati. Acad. Sci. USA, «6:462-465, 1989.
McManus, M. E., Burgess, W. M., Veronese, M. E., Huggett, A., Quattrochi,
L. C., and Tukey, R. H. Metabolism of 2-acetylaminofluorene and
benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens
by human cytochromes P-450. Cancer Res.. 50: 3367-3376, 1990.
Bourdi, M., Larrey, D., Nataf, J.. Berunau. J., Pessayre, D., Iwasaki, M.,
Guengerich, F. P., and Beaune, P. H. A new anti-liver endoplasmic reticulum
antibody directed against human cytochrome P-450 IA1: a specific marker
of dihydralazine-induced hepatitis. J. Clin. Invest.. 85: 1967-1973, 1990.
Otton, S. V., Inaba. T., and Kalow. W. Competitive inhibition of sparteine
oxidation in human liver by fi-adrenoceptor antagonists and other cardiovas
cular drugs. Life Sci., 34: 73-80, 1984.
Guengerich, F. P., Muller-Enoch, D.. and Blair. I. A. Oxidation of quinidine
by human liver cytochrome P-450. Mol. Pharmacol.. 30: 287-295. 1986.
Peluso. M.. Castegnaro, M., Malaveille, C., Friesen, M., Garren, L., Hautefeuille, A., Vineis, P., Kadlubar, F., and Bartsch, H. "P-Postlabelling analysis
of urinary mutagens from smokers of black tobacco implicate 2-amino-lmethyl-6-phenylimidazo[4.5-o)pyridine
(PhIP) as a major DNA-damaging
agent. Carcinogenesis (Lond.). 12: in press, 1991.
Williams, R. W.. Pasley, T., Watts, R., Inmon, J., Fitzgerald, J.. and Claxton,
L. Comparative yields of mutagens from cigarette smokers' urine obtained
by using solid-phase extraction techniques. Environ. Mol. Mutagen., 14: 2026, 1989.
Beaune. P., Lemestre-Cornet. R.. Kremers, P., Albert, A., and Gielen, J. The
5a/mon?//a/mammalian microsome mutagenicity test: comparison of human
and rat livers as activating systems. Mutât.Res.. 756: 139-146, 1985.
Doolittle, D. J., Lee, C. K., Ivett, J. L., Mirsalis, J. C., Riccio. E.. Rudd. C.
J., Burger, G. T., and Hayes, A. W. Comparative studies on the genotoxic
activity of mainstream smoke condensate from cigarettes which burn or only
heat tobacco. Environ. Mol. Mutagen., /5: 93-105. 1990.
Watanabe. M., Nohmi. T.. and Ishidate, M., Jr. New tester strains of
Salmonella typhimurium highly sensitive to mutagenic nitroarenes. Biochem.
Biophys. Res. Commun., 147: 974-979, 1987.
Williams, R. W., Watts. R.. Inmon. J.. Pasley. T., and Claxton. L. Stability
of the mutagenicity in stored cigarette smokers' urine and extract. Environ.
Mol. Mutagen., 16: 246-249, 1990.
Wynder, E. L, and Wright, G. A study of tobacco carcinogenesis. 1. The
primary fractions. Cancer (Phila.), 10: 255-271, 1957.
Hoffmann, D., and Wynder, E. L. A study of tobacco carcinogenesis. XI.
Tumor initiators, tumor accelerators, and tumor promoting activity of con
densate fractions. Cancer (Phila.), 27: 848-864, 1971.
Sesardic. D., Boobis. A. R., Edwards. R. J., and Davies, D. S. A form of
cytochrome P450 in man, orthologous to form d in the rat. catalyses the Odeethylation of phenacetin and is inducible by cigarette smoking. Hi. J. Clin.
Pharmacol., 26: 363-372, 1988.
McLemore, T. L., Adelberg, S.. Liu, M. C., McMahon, N. A., Yu, S. J.,
Hubbard. W. C.. Czerwinski. M.. Wood. T. G.. Storeng. R.. Lubet, R. A.,
Eggleston, J.C., Boyd, M. R., and Hiñes,R. N. Expression of CYP1 Al gene
in patients with lung cancer: evidence for cigarette smoke-induced gene
expression in normal lung tissue and for altered gene regulation in primary
pulmonary carcinomas. J. Nati. Cancer Inst.. 82: 1333-1339. 1990.
Iversen, P. L., Heiger, W. J., Bresnick, E., and Hiñes.R. N. Structural details
of the human cytochrome P-450c gene. Arch. Biochem. Biophys.. 256: 397401, 1987.
61. Quattrochi, L. C., Okino. S. T., Pendurthi, U. R., and Tukey, R. H. Cloning
and isolation of human cytochrome P-450 cDNAs homologous to dioxininducible rabbit mRNAs encoding P450 4 and P450 6. DNA (NY), 4: 395400, 1985.
62. Levin, W., Chang, R. L., Wood. A. W.. Thakker, D. R.. Yagi, H., Jerina, D.
M., and Conney, A. H. Tumorigenicity of optical isomers of the diastereomeric bay-region 3,4-diol-1.2-epoxides of benzo(f)phenanthrene in murine
tumor models. Cancer Res., 46: 2257-2261. 1986.
63. Wall, K. L., Gao, W., te Koepple, J. M., Kwei, G. Y., Kauffman, F. C., and
Thurman, R. G. The liver plays a central role in the mechanism of chemical
carcinogenesis due to polycyclic aromatic hydrocarbons. Carcinogenesis
(Lond.). 12: 783-786. 1991.
64. Ayesh, R.. Idle, J. R., Ritchie, J. C., Crothers, M. J.. and Hetzel, M. R.
Metabolic oxidation phenotypes as markers for susceptibility to lung cancer.
Nature (Lond.), 312: 169-170, 1984.
65. Caporaso, N., Hayes, R. B., Dosemeci. M., Hoover, R.. Ayesh, R.. Hetzel,
M., and Idle, J. Lung cancer risk, occupational exposure, and the debrisoquine metabolic phenotype. Cancer Res., 49: 3675-3679, 1989.
66. Kaisary, A., Smith. P., Jaczq, E., McAllister. C. B., Wilkinson, G. R., Ray,
W. A., and Branch, R. A. Genetic predisposition to bladder cancer: ability to
hydroxylate debrisoquine and mephenytoin as risk factors. Cancer Res., 47:
5488-5493, 1987.
67. Cartwright, R. A.. Philip. P. A.. Rogers, H. J., and Glashan, R. W. Geneti
cally determined debrisoquine oxidation capacity in bladder cancer. Carci
nogenesis (Lond.). 5: 1191-1192. 1984.
68. Drakoulis. N., Minks. T.. Ploch, M., Otte, F., Heinemeyer. G., Kampf. D.,
Loddenkemper, R., and Roots. I. Questionable association of debrisoquine
hydroxilator phenotype and risk for bronchial carcinoma. Acta Pharmacol.
Toxicol., 59: (Supp. 5): 220, 1986.
69. Boobis, A. R., and Davies, D. S. Debrisoquine oxidation phenotype and
susceptibility to lung cancer. Br. J. Clin. Pharmacol.. 30: 653-656. 1990.
70. Wolff. T., Distlerath, L. M., Worthington, M. T., Groopman, J. D.. Hammons, G. J., Kadlubar. F. F.. Prough, R. A., Martin, M. V.. and Guengerich,
F. P. Substrate specificity of human liver cytochrome P-450 debrisoquine 4hydroxylase probed using immunochemical inhibition and chemical model
ing. Cancer Res.. 45: 2116-2122. 1985.
71. Guengerich. F. P., Kim, D-H., and Iwasaki, M. Role of human cytochrome
P-450 IIE1 in the oxidation of several low molecular weight cancer suspects.
Chem. Res. Toxicol., 4: 168-179, 1991.
72. Boobis, A. R., Kahn, G. C., Murray. S., Brodie, M. J.. and Davies, D. S.
Evidence for a common defect in the impaired oxidation of substrates in the
debrisoquine oxidation polymorphism. Br. J. Pharmacol., 14: 602P-603P.
1982.
73. Kahn, G. C., Boobis, A. R., Brodie. M. J., Toverud, E. L., Murray, S., and
Davies, D. S. Phenacetin O-deethylase: an activity of a cytochrome P-450
showing genetic linkage with that catalysing the 4-hydroxylation of debriso
quine? Br. J. Clin. Pharmacol.. 20:67-76, 1985.
74. Randerath. E., Miller, R. H., Mittal, D., Avitts, T. A., Dunsford, H. A., and
Randcrath, K. Covalent DNA damage in tissues of cigarette smokers as
determined by 32P-postlabeling assay. J. Nati. Cancer Inst., 81: 341-347,
1989.
75. Bjelogrlic, N., Iscan, M., Raunio, H.. Pelkonen. O., and Vahakangas, K.
Benzo(a]pyrene-DNA-adducts and monooxygenase activities in mice treated
with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate. Chem.Biol. Interact., 70:51-61, 1989.
76. Yoshida, D., and Matsumoto, T. Amino-«-carbolines as mutagenic agents in
cigarette smoke condensate. Cancer Lett., 10: 141-149, 1980.
77. Peluso, M., Castegnaro. M., Malaveille, C., Talaska. G.. Vineis, P., Kadlubar.
F., and Bartsch. H. '2P-Postlabelling analysis of DNA adducted with urinary
mutagens from smokers of black tobacco. Carcinogenesis (Lond.), //: 13071311, 1990.
78. Malaveille, C, Vineis, P., Estéve,J.. Ohshima, H., Brun, G., Haulefeuille,
A., Gallet. P., Ronco. G.. Terracini. B., and Bartsch, H. Levels of mutagens
in the urine of smokers of black and blond tobacco correlate with their risk
of bladder cancer. Carcinogenesis (Lond.), 10: 577-586. 1989.
79. Wallin. H., Mikalsen, A.. Guengerich. F. P.. Ingelman-Sundberg, M.. Solberg. K. E., Rossland, O. J., and Alexander. J. Different rates of metabolic
activation and detoxication of the food mutagen 2-amino-l-methyl-6-phenylimidazo(4.5-/)pyridine by different cytochrome P-450 enzymes. Carcino
genesis (Lond.), //: 489-492, 1990.
5291
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.
Activation of Amino-α-carboline,
2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine, and a
Copper Phthalocyanine Cellulose Extract of Cigarette Smoke
Condensate by Cytochrome P-450 Enzymes in Rat and Human
Liver Microsomes
Tsutomu Shimada and F. Peter Guengerich
Cancer Res 1991;51:5284-5291.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/51/19/5284
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1991 American Association for Cancer Research.