Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: Significance of cytochrome P450 peroxygenase

Article abstract of DOI:10.1021/tx960144k

We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3- methylimidazo[4,5-f]quinoline (IQ) to genetoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3- methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2- containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His⁺) revertants. This increase was inhibited, in a concentration-dependent manner, by α-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) =0.1 μM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'- deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.

Full text of DOI:10.1021/tx960144k

582
Chem. Res. Toxicol. 1997, 10, 582-588
Hyd r ogen P er oxid e Su p p or ts Hu m a n a n d Ra t
Cytoch r om e P 450 1A2-Ca ta lyzed
2-Am in o-3-m eth ylim id a zo[4,5-f]qu in olin e Bioa ctiva tion to
Mu ta gen ic Meta bolites: Sign ifica n ce of Cytoch r om e
P 450 P er oxygen a se
M. Reza Anari,† P. David J osephy,‡ Tracey Henry,‡ and Peter J . O’Brien*,†
Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada, and Department
of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada
Received August 13, 1996X
We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective
in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the
heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic me-
tabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain
YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholan-
threne-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes
(hP4501A2). The mutagenic response was dependent on the concentration of microsomal
protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide
to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase
was inhibited, in a concentration-dependent manner, by R-naphthoflavone, a P450 1A inhibitor.
Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2
(Km ) 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates
which bound to 2′-deoxyguanosine; LC/MS analysis of the adducts revealed the same major
(protonated) adduct at m/ z ) 464.4 as previously reported for the DNA adduct formed (in
vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-
catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen
peroxide in the physiological/pathological concentration range may be able to support the
metabolic activation of arylamines to genotoxic products through the cytochrome P450
peroxygenase pathway.
when electrons are supplied to cytochrome P450 by
NADPH via NADPH-cytochrome P450 reductase in the
P450 monooxygenase pathway (eqs 1 and 2). In the
overall reaction, equimolar amounts of substrate, O2, and
NADPH are consumed and equimolar amounts of oxi-
dized substrate, H2O, and NADP+ are formed (11).
In tr od u ction
Heterocyclic aromatic amines (HAAs1) are formed in
proteinaceous foods as a result of pyrolysis during cooking
(1). Seventeen HAAs have been identified so far in a
variety of foods at parts per billion levels (1, 2). IQ and
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)
are abundant HAAs, potent bacterial mutagens, and
rodent carcinogens (2, 3); IQ is also a liver carcinogen in
monkeys (4). HAAs, like many carcinogens, must be
metabolically activated in order to exert their genotoxic
effects. Metabolic activation occurs through N-oxidation
of the exocyclic amine group catalyzed primarily by
cytochrome P450 1A2 (5-8).
P450-FeIII + O2 + 2NADPH + 2e- f
P450•+(FeIVdO) + H2O + 2NADP+ (1)
P450•+(FeIVdO) + X-H f P450-FeIII + X-OH (2)
Cytochrome P450 can also utilize reduced oxygen
equivalents, e.g., hydroperoxides, to support the oxidation
of various substrates, in a reaction that is independent
of molecular oxygen, NADPH, and NADPH-cytochrome
P450 reductase (12-18). Cytochrome P450 functions as
a “peroxygenase” in this reaction and forms iron-oxygen
species identical to the iron-oxene complex in the P450
monooxygenase pathway and analogous to peroxidase
compound I, as shown in eqs 3 and 4 (12, 19, 20).
Cytochrome P450 (P450) enzymes are extraordinarily
versatile hemoproteins that catalyze the oxidation of
physiological substrates and foreign chemicals, such as
drugs, pesticides, polycyclic aromatic hydrocarbons, and
HAAs (9, 10). The versatility also extends to the oxygen
donor. Molecular oxygen serves as the natural donor
* Corresponding author. Phone: (416) 978-2716. Fax: (416) 978-
8511. E-mail: peter.obrien@utoronto.ca.
† University of Toronto.
‡ University of Guelph.
P450-FeIII + ROOH f P450•+(FeIVdO) + ROH (3)
P450•+(FeIVdO) + X-H f P450-FeIII + X-OH (4)
X Abstract published in Advance ACS Abstracts, April 15, 1997.
1
Abbreviations: Hepes, 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; P450, cy-
tochrome P450; 3-MC, 3-methylcholanthrene; HAAs, heterocyclic
aromatic amines; His+, histidine-prototrophic; MROD, methoxyresoru-
fin O-demethylation; hP4501A2, human P450 1A2-containing mi-
crosomes; rP4501A, 3-MC-induced rat liver microsomes; tBHP, tert-
butyl hydroperoxide.
Analysis of the microsomal hydroperoxide-supported
oxidation reaction has provided valuable insights into the
S0893-228x(96)00144-0 CCC: $14.00 © 1997 American Chemical Society

P450 Peroxygenase Bioactivation of Arylamines
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 583
Dr. T. Nohmi, National Institute of Hygienic Sciences, Tokyo,
J apan. This strain expresses elevated levels of acetyl-CoA:
arylamine N-acetyltransferase and has high sensitivity for
detection of mutagenic nitroarenes and aromatic amines (29,
30). The mutagenicity assay was performed as described
previously (31-33) except that arachidonic acid and ram
seminal vesicle microsomes were replaced by hydroperoxides
and rP4501A or hP4501A2, respectively. In brief, IQ, mi-
crosomes, and bacteria were preincubated in 0.5 mL of potas-
sium phosphate buffer (100 mM, pH 7.4) at 37 °C for 2 min and
then incubated for an additional 30 min following hydroperoxide
addition. The mixture was plated on minimal glucose medium
and incubated at 37 °C for 72 h, prior to scoring of His+
revertant colonies with the Optimas (Microsoft Windows-based)
videoimage analysis system.
mechanism of oxygen activation by P450 (12, 19, 21), but
the significance of this pathway in intact cells remains
unresolved. Recently, we reported that cytochrome P450-
catalyzed xenobiotic metabolism in, and acute cytotoxicity
to, isolated rat hepatocytes could be enhanced markedly
by the addition of a nontoxic concentration of the alkyl
hydroperoxide tert-butyl hydroperoxide (tBHP) (22). The
peroxidase or peroxygenase nature of xenobiotic oxidation
was not studied in this preliminary report, although
further studies with intact hepatocytes showed that the
P450-catalyzed homolytic cleavage of hydroperoxides
initiates the formation of cytotoxic radical species (23).
The objective of the present study was to determine
whether hydrogen peroxide or fatty acid hydroperoxides,
which are being produced in vivo particularly during the
course of oxidative cell stress (24), can support the
bioactivation of promutagens/procarcinogens to genotoxic
metabolites through the cytochrome P450 peroxygenase
pathway. Previously it was shown that hydrogen per-
oxide was much less effective that alkyl hydroperoxides
in supporting cytochrome P450 peroxygenase activities
(25-27). However in the following,2 low concentrations
of hydrogen peroxide were shown to effectively support
the cytochrome P450 1A2-catalyzed metabolic bioactiva-
tion of a heterocyclic arylamine to DNA-reactive mu-
tagenic metabolites.
F a tty Acid Hyd r op er oxid es P r ep a r a tion . Linoleic and
linolenic acid hydroperoxides were prepared by the enzymatic
peroxygenation of the corresponding fatty acid by soybean
lipoxidase and purified as described (34). The P450 peroxidase
activity for each fatty acid hydroperoxide was determined by
measuring the rate of oxidation of tetramethylphenylenediamine
as described previously (14). A rate of 8.30 and 2.54 µmol of
oxidized tetramethylphenylenediamine min-1 (mg of uninduced
rat liver microsomal protein)-1 was detected for linoleic and
linolenic acid hydroperoxides, respectively.
En zym a tic Assa ys. Microsomal protein was determined by
the method of Lowry et al. (35), and the total cytochrome P450
content was assayed as described by Omura and Sato (36).
Methoxyresorufin O-demethylation (MROD), an enzymatic probe
for the P450 1A2 activity (37), was determined as follows.
Briefly, microsomes were diluted with 0.1 M potassium phos-
phate buffer (pH 7.4) to a concentration of 50 µg/mL for rP4501A
and 0.1 mg/mL for hP4501A2, which provided a linear rate of
reaction for at least 10 min. Methoxyresorufin (in DMSO) was
then added (0.5 µM final concentration), and the mixture was
incubated at 37 °C for 3 min. A stable baseline was established,
and the reaction was initiated by the addition of various
concentrations of hydroperoxides. Due to the high microsomal-
associated catalase activity, the catalase inhibitor sodium azide
(1 mM) was added to the incubation mixture just before
hydrogen peroxide addition. The increase of resorufin fluores-
cence was recorded using a Shimadzu RF5000U spectrofluoro-
photometer, with excitation and emission wavelengths of 530
and 586 nm, respectively. The fluorometer was calibrated with
100 pmol of resorufin in each experiment, and the Km and Vmax
for each hydroperoxide were obtained using a Lineweaver-Burk
plot of the reciprocal rate of reaction (1/V) versus the reciprocal
hydroperoxide concentration (38).
Exp er im en ta l P r oced u r es
Ch em ica ls. IQ (67730-10-3) and nitro-IQ were purchased
from Toronto Research Chemicals (Downsview, ON). tert-Butyl
hydroperoxide and 3-methylcholanthrene were obtained from
Aldrich Chemical Co. Inc. (Milwaukee, WI). Hydrogen peroxide,
linolenic acid, linoleic acid, resorufin, methoxyresorufin, sodium
azide, and soybean lipoxygenase were obtained from Sigma
Chemical Co. (St. Louis, MO). HPLC grade solvents were
purchased from Calden (Georgetown, Ontario). Ca u tion : IQ,
nitro-IQ, and their metabolites are carcinogenic to rodents and
should be handled with the appropriate safety precautions.
Micr osom a l P r ep a r a tion s. Adult male Sprague-Dawley
rats, 250-300 g, were obtained from Charles River Canada
Laboratories (Montreal, Quebec), fed ad libitum, and allowed
to acclimatize for 1 week on clay chip bedding. Animals were
pretreated by daily injection of 3-methylcholanthrene (3-MC)
(25 mg/kg of body wt in corn oil, ip) or vehicle (0.5 mL of corn
oil, ip) for 3 days. The animals were anesthetized by sodium
pentobarbital (60 mg/kg of body wt) on day 4. Livers were
removed under sterile condition and perfused with KCl solution
(1.18%, w/v, 4 °C). Hepatic microsomes were prepared as
described (28). The microsomal pellet was suspended and
homogenized in sterile potassium phosphate buffer:KCl solution
(50 mM KH2PO4 and 0.23% (w/v) KCl, pH 7.4) and stored at
-80 °C. Microsomal preparations containing human P450 1A2
(hP4501A2) (from AHH-1 TK +/- cell line expressing human
P450 1A2 cDNA) and control vector microsomes (from AHH-1
TK +/- cell line transfected with l-histidinol-resistance vector
without human P450 1A2 cDNA) were obtained from Gentest
Corp. (Woburn, MA). hP4501A2 and its control vector mi-
crosomes were sterilized by γ radiation (680 Gy) which had no
effect on P450 1A2 enzymatic activity. The total cytochrome
P450 content was 880 and 41 pmol of P450/mg of protein for
the 3-MC-induced rat liver microsomes (rP4501A) and hP4501A2,
respectively. The ethoxyresorufin O-deethylation activities of
rP4501A, control rat liver microsomes, hP4501A2, and control
vector microsomes were 12 500, 253, 0.6, and 155 pmol of
resorufin min-1 (mg of microsomal protein)-1, respectively.
Mu ta gen icity Assa ys. Salmonella typhimurium strain
YG1012 (TA1538 1,8-DNP pYG213) was graciously provided by
LC/MS An a lysis of IQ Met a b olit e:2′-Deoxygu a n osin e
Con ju ga tes. For identification of the deoxyguanosine:IQ me-
tabolite adducts, a reaction mixture containing 3-MC-induced
microsomes (0.2 mg/mL), 2′-deoxyguanosine (5 mM), and IQ (1
mM) in potassium phosphate buffer (pH 7.4) was preincubated
for 2 min at 37 °C. Hydroperoxides (5 mM) were added in four
equal portions during the 30 min incubation, and the reaction
was stopped by the addition of equal volumes of acetonitrile. In
the case of hydrogen peroxide, 1 mM sodium azide was added
to inhibit microsomal catalase activity. Samples were centri-
fuged at 15000g for 10 min to pellet the proteins. The
supernatants were concentrated under a stream of nitrogen at
room temperature. The residues were dissolved in 100 µL of
water:methanol (50%, v/v) and analyzed by LC/MS and selective
ion monitoring (SIM), as previously described (22). Briefly,
narrow-bore C18 Phenomenex columns (Torrance, CA) packed
with 5 µm i.d. Ultracarb ODS 30 were used. This packing does
not require use of triethylamine, which prevents the “tailing”
of amines but severely inhibits ionization in mass spectral
analysis. The dimensions of the columns used were 2 × 100
mm with a 2 × 30 mm guard column. A mobile phase of
acetonitrile:water:acetic acid (30:70:1, v/v) including 1 mM
ammonium acetate was used.
2
Part of this work was presented at the 87th Annual Meeting of
Sta tistica l An a lysis. One- and two-way ANOVA followed
by the Scheffe’s test was used for comparison among the
the American Association for Cancer Research, Washington, DC, April
1996.

584 Chem. Res. Toxicol., Vol. 10, No. 5, 1997
Anari et al.
A
F igu r e 2. Effectiveness of various hydroperoxides at support-
ing the mutagenic activation of IQ. The frequency of His+
revertants was determined using rP4501A (25 µg/plate) in the
presence of IQ (10 pmol/plate) and various concentrations of
hydrogen peroxide (0), tBHP (O), linoleic acid hydroperoxide
(]), or linolenic acid hydroperoxide (4) as described in Experi-
mental Procedures. Values represent means ( SE of three
separate experiments.
B
F igu r e 1. Hydrogen peroxide-supported P450-catalyzed mu-
tagenic activation of IQ. (A) Enzyme concentration-response
curve: The frequency of His+ revertants was determined using
various concentrations of rP4501A in the presence of hydrogen
peroxide (50 nmol/plate) and IQ (10 pmol/plate) as described in
Experimental Procedures. (B) Mutagen concentration-response
curve: The frequency of His+ revertants was determined using
rP4501A (25 µg/plate) in the presence of hydrogen peroxide (50
nmol/plate) and various concentrations of IQ as described in
Experimental Procedures. Values represent means ( SE of
three separate experiments.
F igu r e 3. Hydrogen peroxide-supported P450-catalyzed mu-
tagenic activation of IQ: effect of R-NF. The frequency of His+
revertants was determined using rP4501A (25 µg/plate) in the
presence of IQ (10 pmol/plate), hydrogen peroxide (50 nmol/
plate), and various concentrations of R-NF as described in
Experimental Procedures. Values represent means ( SE of
three separate experiments.
multiple-treated groups and the relevant controls. Results
represent the mean ( standard error of the mean of triplicate
samples.
Ta ble 1. Hyd r ogen P er oxid e- a n d NADP H-Su p p or ted
Hu m a n P 450 1A2-Ca ta lyzed Meta bolic Activa tion of IQ in
S. typh im u r iu m Str a in YG1012a
Resu lts
The bioactivation system used in the Ames assay
usually comprises rat liver S9 fraction and an NADPH-
generating system (39). We used 3-MC-induced rat liver
microsomes or human P450 1A2-containing microsomes
(40) but replaced the NADPH-generating system with
hydrogen peroxide or an organic hydroperoxide. The
complete system of microsomes, IQ, and hydroperoxide
gave a strong mutagenic response, which was dependent
on the concentrations of microsomal protein (Figure 1A),
IQ (Figure 1B), and hydroperoxide (Figure 2). Using a
concentration of microsomal protein within a linear range
(25 µg/plate), the effect of various concentrations of
naturally occurring hydroperoxides (fatty acid hydro-
peroxides and H2O2) as well as tert-butyl hydroperoxide
on the mutagenicity of IQ (10 pmol/plate, 5 nM) was
investigated (Figure 2). Hydrogen peroxide showed the
highest efficacy in this system with a maximum mu-
tagenic response of 1650 His+ revertants/plate, whereas
tert-butyl hydroperoxide showed a maximum mutagenic
response of 500 His+ revertants/plate. Linoleic and
linolenic acid hydroperoxides were not effective in sup-
porting the P450 1A2-catalyzed mutagenic bioactivation
of IQ (P > 0.05, compare to control group without
hydroperoxide). R-NF, a competitive inhibitor of P450
treatment
hP4501A2
IQ
cofactor
His+ revertants
+
+
+
-
+
-
+
+
H2O2
H2O2
H2O2
-
H2O2
NADPH
NADPH
2200 ( 83b
44 ( 5
c
-
+
+
+
+
23 ( 4
225 ( 37
33 ( 3
1186 ( 75
25 ( 3
c
-
a
The Ames assay was performed with IQ (10 pmol/plate, 5 nM),
human P450 1A2-containing microsomes (2.05 pmol of P450/plate,
1.025 nM), and hydrogen peroxide (50 nmol/plate, 25 µM) or
NADPH (1 mM) as described in Experimental Procedures. In the
absence of an activation system, nitro-IQ (0.2 pmol/plate) gave
b
1570 ( 22 revertants/plate. Data represent the means of three
separate experiments ( SE. c Microsomes from the AHH-1 TK +/-
cell line transfected with vector without human P450 1A2 cDNA
were used as a negative control.
1A isoforms (40), inhibited the H2O2-dependent mutage-
nicity of IQ, in a concentration-dependent manner (Fig-
ure 3). Human P450 1A2-containing microsomes were
also effective in the H2O2-dependent system (Table 1).
Again, all three components of the bioactivation mixture
(P450 1A2, IQ, and hydrogen peroxide) were required for

P450 Peroxygenase Bioactivation of Arylamines
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 585
A
A
B
B
C
F igu r e 5. Analysis of IQ:deoxyguanosine adducts. (A) LC/MS
analysis: Adducts were formed by the microsomal metabolism
(rP4501A, 0.2 mg/mL) of IQ (1 mM) in the presence of deox-
yguanosine (5 mM). Major protonated adduct peaks (MH+)
occurred at m/ z ) 464.4, 480.6, 496.5, and 512.5 and reached
up to 14.8% of the total ion current. (B) Selective ion monitoring
at m/ z ) 464.4: Experimental conditions were as in panel A;
tR of major peak ) 2.49 min.
F igu r e 4. Kinetic analysis of hydroperoxide-supported meth-
oxyresorufin O-demethylation: (A) hydrogen peroxide and hu-
man P450 1A2, (B) hydrogen peroxide and rat liver microsomal
P450 1A2, (C) tBHP and rat liver microsomal P450 1A2.
Incubation mixtures contained methoxyresorufin (0.5 mM),
rP4501A (50 mg/mL) or hP4501A2 (0.1 mg/mL), and hydroper-
oxides. Reciprocal rate of MROD is plotted against reciprocal
hydroperoxide concentration. The linear regression equations
were (A) y ) 0.103x + 0.001, r2 ) 0.9821, Vmax ) 1000 pmol
min-1 mg-1, Km ) 103 µM; (B) y ) 2.416x + 0.002, r2 ) 0.9868,
Vmax ) 500 pmol min-1 mg-1, Km ) 1208 µM; (C) y ) 0.316x +
0.002, r2 ) 0.9895, Vmax ) 500 pmol min-1 mg-1, Km ) 158 µM.
Values represent the average of two separate experiments.
oxide (MROD activity of less than 1 pmol of resorufin
min-1 (mg of protein)-1). The formation of reactive
metabolites from IQ was examined by trapping with the
deoxyribonucleoside 2′-deoxyguanosine; adducts were
analyzed by LC/MS. The major deoxyguanosine adduct
was characterized on the basis of its protonated molecular
ions and is probably either N-(deoxyguanosin-8-yl)-2-
amino-3-methylimidazo[4,5-f]quinoline or (deoxyguanosin-
N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (Figure
5). Selective ion monitoring (m/ z ) 464.4) confirmed the
formation of a single major adduct (tR ) 2.49 min; Figure
5). Distinguishing between the two possible isomeric
forms of the adduct will require further investigation.
Adduct yield was dependent on hydroperoxide (Table 2),
in a manner which paralleled the results of the mutage-
nicity and enzyme assay experiments. Neither the
NADPH-supported microsomal P450 1A2 monooxygenase
system nor the hydrogen peroxide-supported horseradish
peroxidase system produced deoxyguanosine adducts.
the induction of the mutagenic response, and the mu-
tagenic response was almost double that obtained by the
NADPH-supported monooxygenase system under similar
conditions.
We also evaluated hydroperoxide-supported P450 ac-
tivity by direct enzyme assay (MROD; Figure 4). In the
human P450 1A2-enriched microsomal system, MROD
activity was efficiently supported by hydrogen peroxide
(Vmax ) 1000 pmol of resorufin min-1 (mg of protein)-1
,
Km ) 103 µM H2O2). With a rat liver microsomal system,
MROD was also supported by hydrogen peroxide (Vmax
Discu ssion
) 500 pmol of resorufin min-1 (mg of protein)-1, Km
)
1208 µM) or tBHP (Vmax ) 500 pmol of resorufin min-1
(mg of protein)-1, Km ) 158 µM). MROD activity however
was not supported by linoleic or linolenic acid hydroper-
Following our finding that P450 peroxidase/peroxyge-
nase can bioactivate xenobiotics in isolated hepatocytes
(22), we have investigated whether naturally occurring

586 Chem. Res. Toxicol., Vol. 10, No. 5, 1997
Ta ble 2. Hyd r op er oxid e-Dep en d en t F or m a tion of IQ:Deoxygu a n osin e Ad d u ctsa
treatment IQ:deoxyguanosine conjugates at m/ z
Anari et al.
rP4501A
IQ
cofactorb
464.4
480.4
496.2
512.4
+
+
+
+
+
+
+
+
+
H2O2, 5 mM
1.0 × 105 c
1.9 × 105
2.2 × 105
1.7 × 105
-
NDd
ND
ND
ND
e
-
H2O2, 5 mM
ND
ND
ND
ND
+
+
+
+
tBHP, 1 mM
linoleic HP, 0.5 mM
linolenic HP, 0.5 mM
NADPH, 1 mM
2.4 × 104
4.4 × 104
8.3 × 104
7.5 × 104
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
a
Incubations contained rat liver microsomal preparation (0.2 mg/mL), IQ (1 mM), 2′-deoxyguanosine (5 mM), and hydroperoxides, as
b
indicated. Adducts were measured by LC/MS analysis, as described in Experimental Procedures. Fatty acid hydroperoxide cofactors:
linoleic HP ) linoleic acid hydroperoxide; linolenic HP ) linolenic acid hydroperoxide. c Data represent the average of two measurements
(arbitrary units) obtained from the area-under-curve of each adduct peak and should be used only for comparison of values for a given
adduct. Not detectable. e Microsomes from control rat livers (vehicle-treated) were used as a negative control.
d
hydroperoxides can support the P450-catalyzed metabolic
activation of promutagens to DNA-reactive and genotoxic
metabolites. Both human and rat P450 1A2 can utilize
hydroperoxides and bioactivate IQ to mutagenic metabo-
lites. The mutagenic response was dependent on each
component of the bioactivation system (enzyme, peroxide,
and promutagen). Hydrogen peroxide was much more
efficient than tert-butyl hydroperoxide, though fatty acid
hydroperoxides were ineffective.
found by others may be attributed to catalase associated
with the microsomal preparations and the possibility that
it is the peroxide anion (HO2-) that is the reactive species
interacting with other cytochrome P450 isoforms (27).
The unusual high affinity of P450 1A2 for hydrogen
peroxide warrants further investigation, particularly as
such hydrogen peroxide concentrations may be formed
in vivo by various oxidases and hydrogen peroxide
formation could be higher during oxidative stress (24).
Site-directed mutagenesis studies of rat P450 1A2 have
demonstrated that the putative distal amino acid resi-
dues Glu 318 and Thr 319 are essential for high peroxy-
genase activity (41). Optical absorption spectroscopy
studies indicated that the hydrogen peroxide O-O bond
was cleaved heterolytically by the wild type rat P450 1A2,
resulting in the formation of an oxo-P450-heme complex
analogous to horseradish peroxidase compound I (41).
The amino acid residues Glu 318 and Thr 319, in the
oxygen-binding pocket, as well as neighboring residues
are highly conserved between the rat and human 1A2
(41, 42). Our results show for the first time that the
human form of P450 1A2 can effectively catalyze the
hydrogen peroxide-supported IQ bioactivation.
P450 isoforms which lack oxygen-binding pockets and
utilize physiological lipid hydroperoxides as oxygen sources
have recently been characterized in plants (43), platelets
(44), and endothelial cells (45, 46). The homolytic cleav-
age of the hydroperoxide O-O bond is involved in the
catalytic cycle of these novel cytochrome P450 enzymes.
Naturally occurring lipid hydroperoxides can also support
the microsomal-catalyzed oxidation of retinoic acid (47)
and diethylstilbestrol (48). However, linoleic and lino-
lenic acid hydroperoxides did not support the mutagenic
bioactivation of IQ by rat or human P450 1A2. It is
known that the dioxygen bond in unsaturated fatty acid
hydroperoxides is primarily metabolized homolytically by
certain cytochrome P450 enzymes and P420 to free
radicals which catalyze the above oxidations (12). There-
fore, these hydroperoxides, although excellent peroxidase
cofactors, cannot efficiently support the P450 peroxyge-
nase-catalyzed metabolism of P450 substrates.
Covalent modification of DNA by genotoxins may be
an initiating event in carcinogenesis (49). HAAs form
DNA adducts primarily at the C-8 and N2 atoms of
guanine and to a lesser extent at the C-8 and N6 positions
of adenine (50-52). The activated metabolite (2-(hy-
droxyamino)-3-methylimidazo[4,5-f]quinoline, NHOH-IQ)
formed by monooxygenase-dependent metabolism of IQ
reacts with DNA primarily to form N-(deoxyguanosin-8-
yl)-2-amino-3-methylimidazo[4,5-f]quinoline, with 5-(deox-
yguanosin-N2-yl)-IQ as a minor adduct (53, 54) (Scheme
1). 32P-Postlabeling analysis of DNA adducts of IQ
formed in vivo (rodents, monkey) identified the same two
2′-deoxyguanosine adducts (51, 52, 55). We identified,
by LC/MS, a major adduct which may be identical to
these adducts. The formation of the deoxyguanosine
adduct and its oxidized derivatives was dependent on the
presence of all components of the bioactivation system.
Again the order of potency for hydrogen peroxide and
tBHP at supporting adduct formation was similar to that
obtained in supporting the MROD reaction as well as
enhancing IQ mutagenicity. Whether or not the deox-
yguanosine adducts formed under these in vitro condi-
tions are responsible for the mutagenic properties of IQ
requires further investigation.
The N-hydroxy metabolites of IQ and related com-
pounds require O-acetylation to generate a metabolite
sufficiently reactive to form 2′-deoxyguanosine adducts
(56). The bacterial strain used in this study expresses
high levels of acetyl-CoA:arylamine N-acetyltransferase,
which also catalyzes acetyl CoA-dependent O-acetylation.
Prostaglandin H synthase metabolizes IQ to mutagenic
products, via the peroxidase reaction; however, this
metabolism proceeds by one-electron oxidation to form,
ultimately, nitro metabolites (30, 57, 58). HPLC analysis
of the hydrogen peroxide-supported P450 1A2-catalyzed
IQ metabolites did not indicate the formation of nitro or
nitroso metabolites.3
Hydrogen peroxide was also an efficient cofactor for
another P450 1A2-catalyzed activity, i.e., methoxyresoru-
fin O-demethylation (Km ) 0.103 mM for human P450
1A2 and 1.2 mM for 3-MC-induced rat liver microsomes).
This was surprising as other investigators have reported
that hydrogen peroxide was several orders of magnitude
less effective that alkyl hydroperoxides in supporting
microsomal P450-catalyzed benzphetamine N-demethy-
lation (Km ) 250 mM) (25) or aniline hydroxylation (Km
) 18 mM) (26). The high concentrations of hydrogen
peroxide required as a peroxygenase cofactor for P450
In conclusion, hydrogen peroxide and alkyl hydroper-
oxides can support the rat and human P450 1A2-
catalyzed metabolic bioactivation of arylamines to reac-
3
M. R. Anari and P. J . O’Brien, unpublished observations.

P450 Peroxygenase Bioactivation of Arylamines
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 587
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a
(1) Cytochrome P450 1A2 monooxygenase/peroxygenase oxida-
tion of IQ to hydroxylamine metabolite; (2) acetyl CoA:arylamine
N-acetyltransferase-dependent formation of nitrenium- and carbon-
centered cations.
tive genotoxic metabolites. P450 1A2 may be
a
particularly active peroxygenase with hydrogen peroxide,
as a result of its distal active site structure, which favors
the heterolytic cleavage of hydroperoxides. The activities
of other human P450 enzymes toward hydroperoxide-
supported oxidation reactions also require investigation.
Ack n ow led gm en t. The authors acknowledge Nasir
Zahid’s assistance with LC/MS analysis and Dr. J ack P.
Uetrecht for assistance in interpretation of mass spectral
data and review of the manuscript. This work was
supported by grants from the Natural Sciences and
Engineering Research Council of Canada (P. J . O’Brien,
P. D. J osephy).
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