Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: Role of human CYP1A2 and rat CYP2C11/CYP2E1

Article abstract of DOI:10.1080/00498250050200159

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 μM CuCl₂, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70 %. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 μM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50 % respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

Full text of DOI:10.1080/00498250050200159

xenobiotica, 2000, vol. 30, no. 10, 1005±1017
Metabolic activation of o-phenylphenol to a
major cytotoxic metabolite, phenylhydroquinone:
role of human CYP1A2 and rat CYP2C11/CYP2E1
S. OZAWA‹*, K. OHTA‹Œ, A. MIYAJIMA‹,
H. KUREBAYASHI‹, M. SUNOUCHI‹, M. SHIMIZU‹Œ,
N. MURAYAMA*, Y. MATSUMOTOŒ, M. FUKUOKAŒ
and Y. OHNO‹
‹
Division of Pharmacology, National Institute of Health Sciences, 1±18±1 Kamiyoga,
Setagaya-ku, Tokyo 158±8501, Japan
ŒDepartment of Clinical Pharmacology and Toxicology, Showa Pharmaceutical
University, 3±3165 Higashitamagawagakuen, Machida, Tokyo 194±8543, Japan
Received 31 March 2000
1
. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a
toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in
liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and
human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested.
2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the
presence of 1 lm CuCl , were the most cytotoxic chemicals examined to cultured
#
mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2«-,
4
,4«-, 2,3- and phenylhydroquinone.
3
. Rat and human liver microsomes catalysed the formation of phenylhydroquinone,
but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of
metabolic activation of o-phenylphenol was observed with livers of the male than the female
rats by 5±6- and 2± 6-fold respectively.
4
. Inhibitory antibodies against the male-speci®c CYP2C11 inhibited hepatic
o-phenylphenol p-hydroxylation in the male F344 and Sprague±Dawley rat by " 70%.
Liver microsomes from the isoniazid-treated rats produced 1±8- and 3-fold induction of
o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-depen-
dent activity) respectively.
5
. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems,
exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concen-
trations of 5 (" 5-fold), 50 (" 2-fold) and 500 lm (" 2-fold) than CYP2A, CYP2B,
CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450.
6
. Among various CYP inhibitors tested here, 7,8-benzo¯avone and furafylline, typical
human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol
in human livers most potently by 70 and 50% respectively.
7
. The results thus indicate the involvement of rat CYP2C11}CYP2E1 and human
CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.
Introduction
o-Phenylphenol (OPP) is used as a fungicide for harvested citrus fruits and its
toxicity has been extensively studied, including acute toxicity, mutagenicity and
teratogenic eåects (Luster et al. 1981). Long-term carcinogenicity testing (Hodge et
al. 1952) did not give clear evidence on any carcinogenic eåects by OPP. From these
early results, no signi®cant hazardous eåect was anticipated with the exposure of
*
Author for correspondence; e-mail: sozawa!nihs.go.jp
Xenobiotica ISSN 0049-8254 print}ISSN 1366-5928 online ’ 2000 Taylor & Francis Ltd
http:}}www.tandf.co.uk}journals

1
006
S. Ozawa et al.
man to low doses of OPP. However, in 1981 (Hiraga and Fujii 1981), OPP was ®rst
shown to induce urinary bladder tumours by administration of an OPP-containing
diet to the male F344 rat. Subsequently, a clear sex-related diåerence was observed
in the susceptibility to bladder carcinogenicity by OPP (Hasegawa et al. 1991), with
males being more sensitive than females.
OPP was shown to undergo oxidative metabolism in livers of mouse and rat to
produce a toxic metabolite, 2,5-dihydroxybiphenyl or phenylhydroquinone (PHQ)
(Ushiyama et al. 1992). PHQ is thought to be readily auto-oxidized to phenyl-
benzoquinone (PBQ) via a semiquinone radical (Tayama et al. 1994). A signi®cant
correlation was reported between the incidence of bladder cancer and the level of
reactive intermediates including PBQ in urine after administration of OPP to the
male F344 rat (Morimoto et al. 1987, 1989). Several lines of evidence have
accumulated to support the concept that the OPP metabolite, PHQ, causes oxidative
damage to exposed cells by the induction of DNA damage, to produce 8-hydroxy
deoxyguanosine, adduct formation and DNA strand breakage (Nagai et al. 1990,
1
995, Sasaki et al. 1997, Murata et al. 1999). Among the several forms of xenobiotic-
metabolizing enzymes in rat and mouse, neither phenobarbital- nor 3-methyl-
cholanthrene-induced cytochrome P450 (CYPs) were likely to be the major forms
catalysing the oxidative metabolism of OPP to yield PHQ (Halpaap-Wood et al.
1
981). Furthermore, in spite of use of OPP as a food additive, little is known about
human CYP forms involved in the metabolic activation of OPP. An attempted,
therefore, is made to elucidate which CYPs play major roles in the oxidative
metabolism of OPP to produce the reactive metabolite, PHQ.
In the present study, microsomal oxidation of OPP was quantitated by HPLC
using hepatic microsomes from the male and female rat and human. Inhibition of
PHQ formation catalysed by male rat liver microsomes was examined by the aid of
inhibitory antibodies against various rat CYPs. Also compared was the oxidation of
OPP to produce PHQ catalysed by human CYPs expressed by baculovirus-
mediated systems. Typical inhibitors of human CYPs were used to con®rm the
results obtained from the cDNA-mediated human CYP expression systems. Thus,
rat CYP2C11 and possibly CYP2E1, and human CYP1A2 were shown to be the
major CYPs involved in the metabolic activation of OPP to PHQ in the present in
vitro systems.
Materials and methods
Reagents and hepatic microsomes
All reagents used were of the highest grade available. OPP, p-phenylphenol (PPP), 2,2«-
dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 4,4«-dihydroxybiphenyl, 7,8-benzo¯avone, coumarin
and diethyldithiocarbamate were from Wako Pure Chemical Industries Ltd (Tokyo, Japan).
m-Phenylphenol (MPP) and PHQ were from Tokyo Kasei OrganicChemicals(Tokyo, Japan). The purity
of OPP, PPP, 2,2«-dihydroxybiphenyl, 2,3-dihydroxybiphenyl, 4,4«-dihydroxybiphenyl, MPP and PHQ
was " 99%, since no other hydroxybiphenyl was detected on HPLC with any of the hydroxylated
biphenyls used in the present study. The chemical structures of the hydroxybiphenyls studied and PBQ
are shown in ®gure 1. Diclofenac, troleandomycin, cyclophosphamide and b-naphthol were from Sigma
Chemical Co. Ltd (St. Louis, MO, USA). Furafylline was from Ultra®ne Chemical Co. (Manchester,
UK) through Daiichi Chemicals (Tokyo, Japan), omeprazole was from Astra Japan Ltd (Osaka, Japan),
and quinidine was from Kanto Chemicals (Tokyo, Japan). SKF-525A was from Smith Kline & French
Labs. NADP+ and NADPH were from Oriental Yeast (Tokyo, Japan) and glucose 6-phosphate and
glucose 6-phosphate dehydrogenase were from Boehringer Mannheim. Mixed liver microsomes of
human individuals (six males, six females) and inhibitory antibodies against rat CYP forms were from
GENTEST Corp. (Woborn, MA, USA) through Daiichi Chemicals (Tokyo, Japan). Male and female
rat liver microsomes were prepared from 10 male and 10 female F344 rats (8 weeks old; Japan SLC, Inc.,
Hamamatsu, Japan) respectively, and those from 20 male and 42 female Sprague±Dawley rats were from

Metabolic activation of o-phenylphenol to phenylhydroquinone
1007
Figure 1. Enzymatic reaction of o-phenylphenol p-hydroxylation and the chemical structures of other
related hydroxybiphenyls and phenylbenzoquinone. Metabolic activation of o-phenylphenol
catalysed by cytochrome P450 is depicted as well as the chemical structures of other related
hydroxybiphenyls and phenylbenzoquinone.
Xenotech LLC (Kansas City, KS, USA) to examine sex-related diåerences in hepatic PHQ formation.
Male rat liver microsomes from corn oil- or saline-administered Sprague±Dawley rats (20 each) and
various CYP inducer-treated 15 (phenobarbital-treated), 16 (5,6-benzo¯avone-treated) or 18 (isoniazid-
treated) male Sprague±Dawley rats (8 weeks old) were from Xenotech LLC (Yokohama, Japan).
5
,6-Benzo¯avone (a CYP1A inducer; Sigma) dissolved in corn-oil was administered to 16 male
±
Sprague±Dawley rats at 100 mg.kg " body weight once per day on days 1±4, and liver microsomes were
prepared on day 5. Hepatic 7-ethoxyresoru®n O-dealkylation (the CYP1A-dependent activity) was 3940
pmol.mg " protein.min " in mixed microsomes of the inducer-treated 16 animals and 280 pmol.mg "
protein.min " in corn oil-administered 20 animals. Phenobarbital (a CYP2B inducer; Spectrum Chemical
Mfg. Co.) dissolved in saline was administered to 15 male Sprague±Dawley rats at 80 mg.kg " body
weight once per day on days 1±4, and liver microsomes were prepared on day 5. Hepatic
±
±
±
±
±
±
±
7
-penthoxyresoru®n O-dealkylation (the CYP2B-dependent activity) was 1490 pmol.mg " protein.min "
±
±
in mixed microsomes of the inducer-treated 15 animals and 18 pmol.mg " protein.min " in saline-
administered 20 animals. Isoniazid (a CYP2E1 inducer; Sigma) dissolved in saline was administered to
±
1
8 male Sprague±Dawley rats at 200 mg.kg " body weight once per day on days 1±4, and liver microsomes
were prepared on day 5. Hepatic chlorzoxazone 6-hydroxylation (the CYP2E1-dependent activity) was
±
±
±
1
3±3 nmol.mg " protein.min " in mixed microsomes of the inducer-treated 18 animals and 4± 4 nmol.mg "
±
protein.min " in saline-administered 20 animals.
Cell kill and growth inhibitory assays in cultured mammalian cells
HepG2 cells were cultured in Dulbecco’s MEM supplemented with 10% foetal calf serum (FCS).
Culture media for MIAPaCa-2, V79 and rabbit cornea derived SIRC cells were Eagle’s MEM
supplemented with 10% FCS and that for Morris5123DTC was RPMI1640 with 10% FCS. Cell kill
assays were done according to the inhibition of colony formation induced by OPP and related compounds
using human cancer cell lines, HepG2 (liver) and MIAPaCa-2 (pancreas), rat cancer cell line,
Morris5123DTC (liver) and Chinese hamster ®broblast cell line, V79 (lung). Concentrations of o-, m-,
p-phenylphenol and 2,2«-dihydroxybiphenyl used were 1000, 500, 250, 125, 62±5, 31±3 and 15±6 lm; those
of 4,4«-dihydroxybiphenyl were 1000, 500, 250, 125, 62±5, 31±3 and 15±6 lm; and those of 2,3-
dihydroxybiphenyl and PHQ were 125, 62±5, 31±3, 15±6, 7±8, 3±9, 1±9 and 1±0 lm. Hydroxybiphenyls were
dissolved in H O. Cells (100±4000) were inoculated in 60-mm (diameter) dishes and cultured overnight
#
at 37 °C in a humidi®ed atmosphere of 95 % air and 5% CO . Either OPP or related compounds were
added at various concentrations and further cultured for 48 h. Culture media were changed, and cultured
for 1±2 weeks depending on the growth rates of each cell line. Colonies were counted after 0±4% crystal
#

1
008
S. Ozawa et al.
violet staining. A growth inhibition assay was performed according to Itagaki et al. (1995) and Ohno et
al. (1998). Brie¯y, SIRC cells were inoculated at a cell density of 20 000 cells per well of 96-well plates.
The cells were further cultured with various concentrations of hydroxybiphenyls for 72 h at 37 °C.
Inhibition of cell growth was determined after the cells were stained with 0±4% crystal violet.
In vitro DNA strand scission induced by OPP and related compounds
Covalently closed circular (ccc) DNA (pTrcHisB, Invitrogen, Carlsbad, CA, USA) was treated with
various concentration of OPP and related compounds. The reaction mixture (10 ll) consisted of 50 mm
Tris-HCl (pH 8±0), 1 lm CuCl , 0±4 lg cccDNA and various concentrations of OPP and related
#
compounds. DNA strand breakage was analysed on 1% agarose gel electrophoresis according to mobility
diåerences between cccDNA, open circular DNA and linearized DNA, after incubation performed at
room temperature for 30 min.
Assays of microsomal OPP-hydroxylating activities by rat and human hepatic microsomes
The microsomal oxidation of OPP to PHQ was examined using rat and human liver microsomes. A
typical incubation mixture (250 ll) contained 100 mm potassium phosphate buåer (pH 7± 4), 0±5 mm OPP,
0
±33 mm NADPH and 50 lg hepatic microsomes from rat or 100 lg mixed human microsomes. The
reaction was initiated by the addition of NADPH, incubated for 20±40 min and terminated by the
addition of 250 ll ethyl ether. The reaction was linear with time up to 40 min. OPP and hydroxylated
metabolites were extracted twice with ethyl ether. Amounts of OPP metabolites formed were analysed
together with b-naphthol as an internal standard by reverse-phase HPLC system (Shimadzu LC-VP
series) equipped with a L-column ODS (4±6¬ 250 mm). The initial mobile phase was acetonitrile: 0±5%
±
acetic acid (3:7), and was kept for 2 min with a ¯ow rate of 0±8 ml.min ". The acetonitrile concentration
was then raised as a linear gradient up to acetonitrile: 0±5% acetic acid (8:2) over 22 min. Metabolites
were detected at 312 nm by a UV-VIS detector (SPD-10Avp). The elution times of PHQ, b-naphthol
and OPP were 14±7, 20±3 and 28±6 min respectively.
Inhibitory antisera against rat CYP1A1}2, CYP2B1, CYP2C11, CYP2E1 and CYP3A1 (0, 10, 20 or
0 ll) were incubated with rat hepatic microsomes at room temperature for 30 min as suggested by the
5
manufacturer. Normal goat antisera were added so that total volume of antisera was 50 ll. The mixture
was added to 100 mm potassium phosphate buåer (pH 7±4), 0±5 mm OPP, 0±33 mm NADPH, in a total
volume of 250 ll. The reaction was started by the addition of 0±33 mm NADPH, incubated for 20 min,
and was terminated by the addition of 250 ll ethyl ether. The aqueous layer was extracted twice with ethyl
ether and the PHQ extracted into the organic layer was quantitated by HPLC.
Also measured was OPP hydroxylation in microsomes from rat treated with CYP inducers.
Hydroxylation of OPP by human CYP forms, which had been expressed together with cytochrome
P450 reductase in baculovirus-mediated cDNA-expression systems (SUPERSOMESTM, GENTEST
Corp., Woburn, MA, USA), was examined. The CYP forms used were: CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Insect cell microsomes expressing
cytochrome b and cytochrome P450 reductase were also used. The reaction mixture for OPP
&
hydroxylation (250 ll) consisted of 100 mm potassium phosphate buåer (pH 7±4), either 500 or 5 lm
OPP, 0±33 mm NADP+, 3±3 mm glucose 6-phosphate, 0±4 U}ml glucose 6-phosphate dehydrogenase, 3±3
mm MgCl and insect cell microsomes expressing each human CYP form. Reactions were performed for
#
0 min and metabolites analysed by HPLC.
Eåects of typical CYP inhibitors or substrates on OPP hydroxylation catalysed by human liver
2
microsomes were examined (5 lm OPP). The following substances were used for inhibition of CYP-
dependent enzymatic activities: 7,8-benzo¯avone and furafylline for CYP1A2, coumarin for CYP2A6,
cyclophosphamide for CYP2B6, diclofenac for CYP2C9, omeprazole for CYP2C19, quinidine for
CYP2D6, diethyldithiocarbamate for CYP2E1, troleandomycin for CYP3A4 (Ono et al. 1996, Teramura
et al. 1997). Human microsomes (100 lg) were pre-incubated with various concentrations of CYP
inhibitors or substrates at 37 °C for 15 min. OPP was then added at a concentration of 5 lm. The reaction
was performed at 37 °C for 20 min and terminated by the addition of 250 ll ethyl ether. The aqueous
layer was extracted twice with ethyl ether, and then metabolites formed were quanti®ed by HPLC.
Results
Cell kill and growth inhibitory eåects of OPP and biphenyl-related compounds with
hydroxyl moieties
The potency of cytotoxicity and growth inhibition of OPP and other
hydroxylated biphenyls were compared using established cell lines derived from
various animals. Based on the IC , 2,3-dihydroxybiphenyl and PHQ were the most
&
!
cytotoxic of the hydroxy biphenyls studied (table 1). 2,3-Dihydroxybiphenyl, PHQ

Metabolic activation of o-phenylphenol to phenylhydroquinone
1009
Table 1. Inhibition of colony formation or cell growth of various mammalian cells treated with OPP
and related compounds.
IC (lM)
&
!
Human
Rat
Morris5123DTC
Chinese hamster Rabbit
V79
HepG2 MIAPaCa-2
SIRC
o-Phenylphenol
m-Phenylphenol
p-Phenylphenol
270
210
230
100
70
110
420
250
170
130
160
200
290
250
170
2
4
2
2
,2«Dihydroxybiphenyl
,4«Dihydroxybiphenyl
,3«Dihydroxybiphenyl
,5«Dihydroxybiphenyl
400
42
50
30
310
90
5
560
16
19
21
340
90
9
420
40
70
40
3
9
Inhibition of colony formation of HepG2, MIAPaCa-2, Morris5123DTC and V79 cells was
examined; and growth inhibition of SIRC cells was determined as described by Ohno et al. (1998)
respectively. IC shown are the average of three determinations.
&
!
and 4,4«-dihydroxy biphenyl showed a tendency to be the most potent inhibitors of
colony formation (cell lines listed in table 1 except for SIRC cells) or cell growth
(SIRC cells) among the hydroxylated biphenyl derivatives studied.
Induction of DNA strand breakage by OPP and hydroxylated derivatives of
biphenyl
Induction of DNA strand breakage by OPP and other mono- and di-
hydroxylated biphenyl derivatives was examined in the presence of 1 lm CuCl#.
Both PHQ and 2,3-dihydroxybiphenyl (at 10±20 lm) caused nicks on the plasmid
DNA molecules used, and DNA strand breakage was much weaker with OPP,
MPP, PPP, 2,2«-dihydroxybiphenyl and 4,4«-dihydroxybiphenyl at even 500 lm
(®gure 2).
Microsomal PHQ formation catalysed by rat liver microsomes
Microsomal PHQ formation was compared between livers of the male and
female F344 and Sprague±Dawley rat and a clear male dominance was observed
(table 2), namely 5±64-fold higher in the male F344 rat than in the female, and 2±60-
fold in the male Sprague±Dawley rat than in the female.
Eåects of inhibitory antibodies of rat CYPs on PHQ formation
Inhibitory antibodies against rat CYP forms were used to elucidate the speci®c
rat CYPs involved in PHQ formation by the male F344 and Sprague±Dawley rat
liver microsomes. In both the male F344 and Sprague±Dawley rat, antisera against
the male-speci®c CYP2C11 showed the most eåective and concentration-dependent
inhibition of microsomal PHQ formation from OPP by 88 and 70% respectively
(®gure 3A and B).
Hydroxylation of OPP in liver microsomes from rat treated with various CYP-
inducers
It was examined whether hepatic microsomes from rat treated with CYP
inducers exhibit induction of OPP hydroxylation. 5,6-Benzo¯avone (a CYP1A
inducer) failed to induce microsomal OPP p-hydroxylation in the Sprague±Dawley

1
010
S. Ozawa et al.
Figure 2. DNA single-strand breaks induced by OPP and other hydroxylated biphenyl derivatives.
Covalently closed circular DNA (cccDNA) was treated with various concentration of OPP and
related compounds in the presence of 1 lm CuCl . DNA strand breakage was analysed according
#
to the reduction in the amount of cccDNA and the increase in the amount of nicked open circular
(
(
(
OC) DNA. (A) Concentrations (lm) of PHQ used were: 12±5 (lane 1); 10 (2); 8 (3); 6± 4 (4); 5±1
5); 4±1 (6); 3± 3 (7); 0 (control, 8); 2±6 (9); 2±1 (10); 1± 7 (11); 1±3 (12); 1±1 (13); 0±9 (14); 0±7 (15).
B) Concentrations (lm) of 2,3-dihydroxy-biphenyl used were: 25 (lane 1); 20 (2); 16 (3); 12±8 (4);
1
0±2 (5); 8±2 (6); 6±5 (7); 0 (control, 8); 5±2 (9); 4±2 (10); 3±4 (11); 2± 6 (12); 2±1 (13); 1± 7 (14); 1±3
(
(
15). (C) Biphenyl and other hydroxylated biphenyl (500 lm): biphenyl (lane 1); 2-hydroxy
o-phenylphenol; 2); 3-hydroxy (m-phenylphenol; 3); 4-hydroxy (p-phenylphenol; 4); 2,2«-
dihydroxybiphenyl (5); 4,4«-dihydroxybiphenyl (6); 3-hydroxy (m-phenylphenol; 7); none
control; 8).
(
Table 2. Male-dominant p-hydroxylation of OPP in liver microsomes of the F344 and Sprague±Dawley
rat.
Strain
Male
Female
pa
F344
Sprague±Dawley
3.67³0.40
2.06³0.05
0.65³0.10
0.79³0.02
! 0.001
! 0.001
Hepatic p-hydroxylation of OPP (500 lm, nmol[min­ "[mg microsomal
protein) was determined using mixed liver microsomes from 10 male and 10
female F344 rats and those from 20 male and 42 female Sprague±Dawley rats
as described in the Materials and methods.
Data are the mean³SD of four determinations.
a Degree of statistical signi®cance.

Metabolic activation of o-phenylphenol to phenylhydroquinone
1011
Figure 3. Eåects of antisera against rat CYP forms on OPP p-hydroxylation in rat liver microsomes.
Eåects of inhibitory antibodies on microsomal OPP p-hydroxylation in rat livers were examined
as described in the Materials and methods. The concentration of OPP was 500 lm and OPP was
incubated for 40 min with rat liver microsomes (50 lg protein). Control activities (mean³SD,
±
±
nmol.min ".mg " microsomal protein) in the male F344 rat and male Sprague±Dawley (SD) rat
were 1±17³0±10 and 1±61³0±05 respectively. Data points are the average of four determinations.
Figure 4. OPP p-hydroxylation in microsomes from the male Sprague±Dawley rat pretreated with
CYP inducers. The concentration of OPP was 500 lm and OPP was incubated for 40 min with
microsomes (50 lg protein). Data from 5,6-benzo¯avone-treated (5,6-BZF) and phenobarbital-
treated (PB) rats were compared with those from the corn oil-treated rat as controls. Data from the
isoniazid-treated rat were compared with those from the saline-treated rat as controls. Each
column represents the mean³SD of triplicate determinations. Control activities
±
±
(
nmol.min ".mg " microsomal protein) in the presence of corn oil and saline were 3± 48³0±46 in
quadruplicate determinations and 2± 81³0±15 in triplicate determinations respectively. The
degree of statistical signi®cance is expressed as *p! 0±02 or **p! 0±01.
rat liver, although 7-ethoxyresoru®n O-deethylation, a typical CYP1A-dependent
activity, was induced by 14-fold. Microsomal OPP p-hydroxylation was minimally
induced (1±36-fold) by the administration of phenobarbital (a CYP2B inducer),
whereas 7-pentoxyresoru®n O-depentylation, a typical CYP2B-dependent activity,
was induced by 85-fold. These results indicated that both CYP1A and CYP2B were
not the major isoforms involved in the microsomal of p-hydroxylation OPP (®gure
4). On the other hand, the largest induction (1±8-fold) of OPP p-hydroxylation was
observed by isoniazid-administration.

1
012
S. Ozawa et al.
Figure 5. OPP p-hydroxylation in microsomes from baculovirus-infected insect cells expressing
individual human CYPs. OPP was incubated for 20 min with CYP-expressing microsomes (10
pmol CYP) and the concentration of OPP was 5 lm (A), 50 lm (B) and 500 lm (C). Each column
represents the mean³SD of three separate experiments. ND (not detectable) indicates that
±
metabolic activity was! 0±18 pmol.min "}pmol CYP.
Human CYPs responsible for PHQ formation
Mixed liver microsomes derived from human individuals (n ¯ 12) also catalysed
±
±
the p-hydroxylation of OPP (500 lm) at 2±54³0±48 nmol.min ".mg " protein in
triplicate determinations, which was as high as the rate of formation in male rat liver
microsomes. Forms of human CYP expressed in baculovirus-mediated cDNA
expression systems were used to examine the speci®c CYPs catalysing OPP
hydroxylation. In the reaction mixtures using 10 pmol of each expressed CYP,
CYP1A2 catalysed PHQ formation the most eæciently of the CYP forms tested in
the present study (®gure 5A-C). The PHQ formation was tested at the OPP
concentration of 5 lm (®gure 5A), 50 lm (B) and 500 lm (C). At 5 lm substrate
concentration, CYP1A2 predominantly exhibited OPP p-hydroxylation at 11±94
±
pmol.min "}pmol CYP. The next highest was observed with CYP2B6 (1±26
±
pmol.min "}pmol CYP). CYP2D6, CYP2E1 and CYP3A4 showed lower activities
±
at rates of 0±76, 0±58 and 0±23 pmol.min "}pmol CYP respectively. At 50 lm
substrate concentration, catalytic activities by CYP1A2, CYP2B6, CYP2D6,
±
CYP2E1 and CYP3A4 were 30±6, 15±2, 10±1, 6±7 and 1±7 pmol.min "}pmol CYP
respectively. At 500 lm, CYP1A2, CYP2E1, CYP2D6, CYP2B6 and CYP3A4
±
converted OPP to PHQ at 136±8, 42±4, 27±9, 27±2 and 12±5 pmol.min "}pmol CYP
respectively. In addition, CYP2C19, CYP2C9, CYP2C8 and CYP2A6, which did
not exhibited catalytic activity at 5 lm, showed lower activities at 50 and 500 lm.
Hence, the overall conclusion is that CYP1A2 was the most eæcient isoform for
OPP p-hydroxylation.
Inhibition of OPP hydroxylation catalysed by human liver microsomes
Eåects of typical CYP substrates or inhibitors on human hepatic OPP
hydroxylation were examined to support results obtained using the microsomes of
insect cells expressing diåerent forms of human CYP. 7,8-Benzo¯avone and
furafylline, typical CYP1A inhibitors, exhibited the most potent inhibition of OPP
p-hydroxylation (®gure 6). By contrast, inhibitors of other forms of CYP showed
lesser extent of inhibition of OPP p-hydroxylation. These results supported the
results obtained by the recombinant CYP experiments in that CYP1A2 most
eæciently catalysed OPP p-hydroxylation.

Metabolic activation of o-phenylphenol to phenylhydroquinone
1013
Figure 6. Eåect of CYP inhibitors on OPP p-hydroxylation in human liver microsomes. The
concentration of OPP was 5 lm and each column represents the average of duplicate experiments.
OPP was incubated for 20 min with pooled human microsomes (100 lg protein). SKF-525A,
furafylline, coumarin, cyclophosphamide, diclofenac, omeprazole, quinidine, diethyldithio-
carbamate (DDTC) and troleandomycin were dissolved in distilled water. 7,8-Benzo¯avone (7,8-
±
±
BZF) was dissolved in methanol. Control activities (mean³SD, nmol.min ".mg " protein) in the
presence of distilled water and methanol were 0± 32³0±06 in six determinations and 0±31³0±04 in
triplicate determinations respectively.

1
014
S. Ozawa et al.
Discussion
The involvement of rat CYP2C11 (a male-speci®c form), rat CYP2E1 and
human CYP1A2 has been demonstrated in vitro for the metabolic activation of OPP
to the toxic metabolite, PHQ by hepatic microsomes of the male rat and human.
2
,3-Dihydroxybiphenyl and PHQ were cytotoxic in cell lines derived from
several animal species and also for DNA strand break at the lowest concentration of
the hydroxylated biphenyl derivatives used in this study. 4,4«-Dihydroxybiphenyl
failed to induce eæcient DNA strand break but was relatively cytotoxic, suggesting
diåerent mechanisms of cytotoxicity from those of 2,3-dihydroxybiphenyl and
PHQ (table 1 and ®gure 2).
2
,3-Dihydroxybiphenyl was not detected in the microsomal systems from either
rat or human. Male dominance in the liver microsome-mediated conversion of OPP
into PHQ was observed (table 2), consistent with the fact that a higher incidence in
OPP-induced bladder cancer was also observed in the male F344 rat as compared
with the female (Hasegawa et al. 1991). It was also observed that hepatic OPP
p-hydroxylation catalysed by the male F344 and Sprague±Dawley rat was best
inhibited by inhibitory antibodies against the male-speci®c rat CYP2C11 (®gure 3).
Antibodies against CYP3A seemed to enhance hepatic OPP p-hydroxylation. These
results might suggest a possible interaction of OPP with CYP3A, although the exact
mechanisms are unknown at present (®gure 3). Recently, OPP was reported to
induce cell injury in hepatocytes, associated with a substantial decrease in
intracellular glutathione (Sugihara et al. 1997). Potencies of damage in cultured cells
and induction of DNA strand breaks seemed to correlate among the hydroxy-
biphenyls, including PHQ used in the present study. Renal expression of the male-
speci®c CYP2C11 has been documented (Ryan et al. 1993). Thus, male-speci®c rat
CYP2C11, which has been proposed to be involved in the metabolic activation of
OPP in this study, might play an important role in mediating cell damage in OPP-
exposed rat. Whether the observed male-dominance in hepatic OPP p-hydroxylation
could also be attributed to the bladder cancer incidence in the male rat clearly
depends on the stability of the resultant toxic metabolite (PHQ) in the circulating
blood and uro-epithelial tracts.
PHQ is further oxidized to yield PBQ (®gure 1; Lambert and Eastmond 1994).
CYP-catalysed one-electron reductive bioactivation has been reported as an
important metabolic activation of compounds with a quinone moiety (Goeptar et al.
1
995). The resultant PBQ is likely to undergo reductive bioactivation by CYP
form(s). Elucidation of forms of CYP involved in the PBQ formation and the
reductive bioactivation is required for clari®cation of the complete metabolic
activation pathway of OPP.
Neither phenobarbital- nor 5,6-benzo¯avone-administered rat liver microsomes
exhibited substantially higher OPP p-hydroxylation than those from the corn oil-
administered rat (®gure 4). Failure of 5,6-benzo¯avone to induce OPP
p-hydroxylation is consistent with the results reported by Halpaap-Wood et al.
(1981).
Among various human CYPs expressed in the baculovirus-mediated systems,
CYP1A2 catalysed the OPP p-hydroxylation most eæciently among the CYP forms
which are known to be expressed in human livers, over the OPP concentration
examined (i.e. 5, 50 and 500 lm; ®gure 5). At higher OPP concentrations, CYP2D6
catalysed the OPP p-hydroxylation. Apparently, the K of CYP2D6 for the reaction
was higher than that of CYP1A2 (®gure 5). CYP2D6 is known to catalyse
m

Metabolic activation of o-phenylphenol to phenylhydroquinone
1015
C-hydroxylation of debrisoquin. It has been shown that ring-hydroxylation of
debrisoquin was catalysed by CYP2D6 (Lightfoot et al. 2000). These results may
support our results on OPP being a substrate of CYP2D6 to form PHQ, though
further investigation is required.
It was decided to examine the major hepatic CYP forms catalysing the reaction
at an OPP concentration of 5 lm, since this lowest concentration adopted in the
present study might be of the highest toxicological signi®cance. The results on the
potent inhibition by 7,8-benzo¯avone and furafylline, typical CYP1A inhibitors, on
OPP hydroxylation catalysed by mixed human liver microsomes were quite
consistent with the eæcient catalytic activities by cDNA-expressed CYP1A2. Km
C
for microsomal OPP hydroxylation was 110 lm in human liver systems (data not
±
shown). The approximate hepatic levels (pmol.mg " protein) of CYP forms in
human livers are (Shimada et al. 1996): 55³28 for CYP1A; 83³29 for CYP2C;
1
0³5 for CYP2D6; 33³17 for CYP2E1; and 147³57 for CYP3A. Thus, taking the
substantially high rate of OPP p-hydroxylation by the recombinant CYP1A2 and
the hepatic CYP levels into consideration, CYP1A2 likely plays a major role in PHQ
formation in human livers. It was reported that 7,8-benzo¯avone stimulated human
CYP3A4-mediated phenacetin O-deethylation (Nakajima et al. 1999). 7,8-Benzo-
¯
avone inhibited OPP p-hydroxylation by human liver microsomes at concen-
trations of 5±40 lm (®gure 6), which also enhanced CYP3A4-mediated phenacetin
O-deethylation (data not shown). These results suggested the important role of
CYP1A2 in hepatic OPP-hydroxylation, although CYP3A4 was also shown to
catalyse the reaction (®gure 5A).
CYP1A2-dependent activity is known to show remarkable individual diåerence
and to be polymorphically distributed in the human population (Minchin et al.
1
985, Butler et al. 1992, Nakajima et al. 1994). Thus, the present results on the role
of CYP1A2 in the metabolic activation of OPP raise the possibility of potential toxic
eåects by this food additive in human individuals with high CYP1A2 expression.
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry of Health
and Welfare of Japan and a grant for the research project `Humanised Testing
Systems’ from the Organisation for Pharmaceutical Safety and Research (97-14).
The authors express their deep thanks to Noriaki Shimada of Daiichi Chemical Co.
for all his supports and helpful discussions.
References
Butler, M. A., Lang, N. P., Young, J. F., Caporaso, N. E., Vineis, P., Hayes, R. B., Teitel, C. H.,
Massengill, J. P., Lawsen, M. F. and Kadlubar, F. F., 1992, Determination of CYP1A2 and
NAT2 phenotypes in human populations by analysis of caåeine urinary metabolites. Pharmaco-
genetics, 2, 116±127.
Goeptar, A. R., Scheerens, H. and Vermeulen, N. P. E., 1995, Oxygen and xenobiotic reductase
activities of cytochrome P450. Critical Reviews in Toxicology, 25, 25±65.
Halpaap-Wood, K., Horning, E. C. and Horning, M. G., 1981, The eåect of phenobarbital and
b-naphtho¯avone induction on the metabolism of biphenyl in the rat and mouse. Drug
Metabolism and Disposition, 9, 97±102.
Hasegawa, R., Fukuoka, M., Takahashi, T., Yamamoto, A., Yamaguchi, S., Shibata, M.-A.,
Tanaka, A. and Fukushima, S., 1991, Sex diåerence in o-phenylphenate rat urinary bladder

1
016
S. Ozawa et al.
carcinogenesis: urinary metabolites and electrolytes under conditions of aciduria and alkaliuria.
Japanese Journal of Cancer Research, 82, 657±664.
Hiraga, K. and Fujii, T., 1981, Induction of tumours of the urinary system in F344 rats by dietary
administration of sodium o-phenylphenate. Food and Cosmetic Toxicology, 19, 303±310.
Hodge, H. C., Maynard, E. A., Blanchet, H. J., Jr, Spencer, H. C. and Rowe, V. K., 1952,
Toxicological studies of orthophenylphenol (Dowcide 1). Journal of Pharmacology and Ex-
perimental Therapeutics, 104, 202±210.
Itagaki, H., Shibata, M., Tani, N., Kinoshita, S., Kakishima, H., Seyama, Y., Ohuchi, J., Kasai, Y.,
Okada, J., Kojima, H., Okamoto, Y., Kotani, M., Ohno, Y., Miyajima, A., Takanaka, A.,
1
995, First-phase inter-laboratory validation of the in vitro eye irritation tests for cosmetic
ingredients. X-Evaluation of cytotoxicity tests on SIRC cells. AATEX, 3, 201±209.
Lambert, A. C. and Eastmond, D. A., 1994, Genotoxic eåects of the o-phenylphenol metabolites
phenylhydroquinone and phenylbenzoquinone in V79 cells. Mutation Research, 322, 243±256.
Lightfoot, T., Ellis, S. W., Mahling, J., Ackland, M. J., Blaney, F. E., Bijioo, G. J., De Groot,
M. J., Vermeulen, N. P., Blackburn, G. M., Lennard, M. S. and Tucker, G. T., 2000,
Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active
site modelling. Xenobiotica, 30, 219±233.
Luster, M. I., Dean, J. H., Boorman, G. A., Archer, D. L., Lauer, L., Lawson, L. D., Moore, J. A.
and Wilson, R. E., 1981, The eåect of orthophenylphenol, tris(2,3-dichloroporpyl)phosphate,
and cyclophosphamide onthe immune system and host susceptibility of mice following subchronic
exposure. Toxicology and Applied Pharmacology, 58, 252±261.
Minchin, R. F., McManus, M. E., Boobis, A. R., Davies, D. S. and Thorgeirsson, S. S., 1985,
Polymorphic metabolism of the carcinogen 2-acetylamino¯uorene in human liver microsomes.
Carcinogenesis, 6, 1721±1724.
Morimoto, K., Fukuoka, M., Hasegawa, R., Tanaka, A., Takahashi, A. and Hayashi, Y., 1987, DNA
damage in urinary bladder epithelium of male F344 rats treated with 2-phenyl-1,4-benzoquinone,
one of the non-conjugated urinary metabolites of sodium o-phenylphenate. Japanese Journal of
Cancer Research (Gann), 78, 1027±1030.
Morimoto, K., Sato, M., Fukuoka, M., Hasegawa, R., Takahashi, T., Tsuchiya, T., Tanaka, A.,
Takahashi, A. and Hayashi, Y., 1989, Correlation between the DNA damage in urinary bladder
epithelium and the urinary 2-phenyl-1,4-benzoquinone levels from F344 rats fed sodium
o-phenylphenate in the diet. Carcinogenesis, 10, 1823±1827.
Murata, M., Moriya, K., Inoue, S. and Kawanishi, S., 1999, Oxidative damage to cellular and isolated
DNA by metabolites of a fungecide ortho-phenylphenol. Carcinogenesis, 20, 851±857.
Nagai, F., Ushiyama, K., Satoh, K. and Kano, I., 1990, DNA cleavage by phenylhydroquinone: the
major metabolite of a fungicide o-phenylphenol. Chemico-Biological Interactions, 76, 163±179.
Nagai, F., Ushiyama, K., Satoh, K., Kasai, H. and Kano, I., 1995, Formation of 8-hydroxy-
deoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major
metabolite of o-phenylphenol. Carcinogenesis, 16, 837±840.
Nakajima, M., Kobayashi, K., Oshima, K., Shimada, N., Tokudome, S., Chiba, K. and Yokoi, T.,
1
999, Activation of phenacetin o-deethylase activity by a-naphtho¯avone in human liver
microsomes. Xenobiotica, 29, 885±898.
Nakajima, M., Yokoi, T., Mizutani, M., Shin, S., Kadlubar, F. F. and Kamataki, T., 1994,
Phenotyping of CYP1A2 in Japanese population by analysis of caåeine urinary metabolites:
absence of mutation prescribing the phenotype in the CYP1A2 gene. Cancer Epidemiology
Biomarkers Prevention, 3, 413±421.
Ohno, Y., Miyajima, A. and Sunouchi, M., 1998, Alternative methods for mechanistic studies in
toxicology. Screening of hepatotoxicity of pesticides using freshly isolated and primary cultured
hepatocytes and non-liver-derived cells, SIRC cells. Toxicology Letters, 102±3, 569±573.
Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F. J. and Tsutsui, M., 1996, Speci®city of
substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using
cDNA-expressed human P450s and human liver microsomes. Xenobiotica, 26, 681±693.
Ryan, D. E., Thoma, P. E., Levin, W., Maines, S. L., Bandiera, S. and Reik, L. M., 1993, Monoclonal
antibodies of diåerentiating speci®cities as probes of cytochrome P450h (2C11). Archives in
Biochemistry and Biophysics, 301, 282±293.
Sasaki, Y. F., Saga, A., Akasaka, M., Yoshida, K., Nishidate, E., Su, Y. Q., Matsusaka, N. and
Tsuda, S., 1997, In vivo genotoxicity of ortho- phenylphenol, biphenyl, and thiabendazole
detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay. Mutation
Research, 395, 189±198.
Shimada, T., Yamazaki, H., Mimura, M., Wakamiya, N., Ueng, Y.-F., Guengerich, F. P. and Inui,
Y., 1996, Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of
xenobiotic chemicals in human fetal livers and adult lungs. Drug Metabolism and Disposition, 24,
5
15±522.
Sugihara, N., Shimomichi, K. and Furuno, K., 1997, Cytotoxicity of food preservatives in cultured rat
hepatocytes loaded with linolenic acid. Toxicology, 120, 29±36.

Metabolic activation of o-phenylphenol to phenylhydroquinone
1017
Tayama, S. and Nakagawa, Y., 1994, Eåect of scavengers of active oxygen species on cell damage caused
in CHO-K1 cells by phenylhydroquinone, an o-phenylphenol metabolite. Mutation Research,
3
24, 121±131.
Teramura, T., Fukunaga, Y., van Hoogdalem, E. J., Watanabe, T. and Higuchi, T., 1997, Examin-
ation of metabolic pathways and identi®cation of human liver cytochrome P450 isozymes
responsible for the metabolism of barnidipine, a calcium channel blocker. Xenobiotica, 27,
8
85±900.
Ushiyama, K., Nagai, F., Nakagawa, A. and Kano, I., 1992, DNA adduct formation by o-phenylphenol
metabolite in vivo and in vitro. Carcinogenesis, 13, 1469±1473.