Free Radical Biology & Medicine, Vol. 36, No. 5, pp.

618 – 631, 2004

Copyright D 2004 Elsevier Inc.
Printed in the USA. All rights reserved
0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2003.11.014

Original Contribution
UNCOUPLING-MEDIATED GENERATION OF REACTIVE OXYGEN
BY HALOGENATED AROMATIC HYDROCARBONS IN MOUSE
LIVER MICROSOMES

HOWARD G. SHERTZER , COREY D. CLAY, MARY BETH GENTER, MARK C. CHAMES,

SCOTT N. SCHNEIDER, GREG G. OAKLEY, DANIEL W. NEBERT, and TIMOTHY P. DALTON
Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center,
Cincinnati, OH, USA

(Received 26 August 2003; Revised 10 November 2003; Accepted 20 November 2003)

Abstract—Studying liver microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced or vehicle-treated

(noninduced) mice, we evaluated the in vitro effects of added chemicals on the production of reactive oxygen due to
substrate/P450-mediated uncoupling. The catalase-inhibited NADPH-dependent H2O2 production (luminol assay) was
lower in induced than noninduced microsomes. The effects of adding chemicals (2.5 AM) in vitro could be divided into
three categories: Group 1, highly halogenated and coplanar compounds that increased H2O2 production at least 5-fold in
induced, but not in noninduced, microsomes; Group 2, non-coplanar halogenated biphenyls that did not affect H2O2
production; Group 3, minimally halogenated biphenyls and benzo[a]pyrene that decreased H2O2 production. Molar
consumption of NADPH and O2 and molar H2O2 production (o-dianisidine oxidation) revealed that Group 1 compounds
mostly increased, Group 2 had no effect, and Group 3 decreased the H2O2/O2 and H2O2/NADPH ratios. Microsomal
lipid peroxidation (thiobarbituric acid-reactive substances) was proportional to H2O2 production. Although TCDD
induction decreased microsomal production of H2O2, addition of Group 1 compounds to TCDD-induced microsomes in
vitro stimulated the second-electron reduction of cytochrome P450 and subsequent release of H2O2 production. This
pathway is likely to contribute to the oxidative stress response and associated toxicity produced by many of these
environmental chemicals. D 2004 Elsevier Inc. All rights reserved.

Keywords—Cytochrome P450, Free radicals, Hydrogen peroxide, Polyhalogenated aromatic hydrocarbons, Superoxide,
Uncoupling

INTRODUCTION
Formation of reactive oxygen species (ROS) has been
implicated in susceptibility to numerous complex dis-
eases (including neurodegenerative disorders, cancer,
type II diabetes, cataracts, and AIDS) and aging, as
well as toxicity of many environmental chemicals and
certain heavy metal ions [1– 4]. Greater understanding
of the mechanisms underlying the oxidative stress re-
sponse at the molecular level should provide insight into
Address correspondence to: Dr. H. G. Shertzer, Department of
Environmental Health, University of Cincinnati Medical Center, 3223
Eden Avenue, Cincinnati, OH 45267-0056, USA. Fax: (513) 558-0522;
E-mail: shertzhg@ucmail.uc.edu.
intervention, as well as prevention, of some of these
disorders.
Many polyhalogenated aromatic hydrocarbons
(PHAHs), including dibenzodioxins, dibenzofurans and
biphenyls, are toxic and carcinogenic in laboratory ani-
mals and humans [5 –8]. Highly halogenated compounds,
such as TCDD (dioxin, 2,3,7,8-tetrachlorodibenzo-p-
dioxin) and polychlorinated biphenyls (PCBs), are persis-
tent in the environment, bioconcentrate, and bioaccumu-
late, and have been categorized as dangerous pollutants,
especially in waterways such as the Great Lakes [9].
Because many such compounds are poorly metabolized
and do not react directly with macromolecules such as
DNA and protein, they are believed to act via signal
transduction pathways, often involving cellular oxidative

618
 Microsomal reactive oxygen production
stress [10 –20]. We have previously described one such
oxidative stress pathway that involves the TCDD-induc-
ible succinate-dependent mitochondrial production of
reactive oxygen [21,22]. Other studies have shown that
PHAHs may contribute to a cytochrome P450 (CYP)-
dependent microsomal generation of reactive oxygen, by
binding to the CYP active site but not being metabolized,
hence allowing electrons to leak into the subcellular
milieu; this process is termed uncoupling [23,24].
The toxicity and carcinogenicity of TCDD are related
primarily to its ability to alter the expression of genes that
are under the control of the aromatic hydrocarbon receptor
(AHR) [25,26]. These genes encode many proteins, some
of which are upregulated, such as those in the Ahr gene
battery, including CYP1A1, CYP1A2, and CYP1B1 [27].
Other genes are downregulated, and there appears to be
hundreds of genes controlled by the activated AHR [28].
Although TCDD itself is typically a minor component of
environmental PHAHs, its high affinity for the AHR,
coupled with its environmental and biological persistence,
cause it to be a prototype for the capacity of other PHAHs
to bind to the AHR and exert toxicity [29].
The pathways leading to TCDD-induced oxidative
stress are beginning to be understood. The well-estab-
lished pathway of NADPH-dependent production of
ROS in liver microsomes [30] is now known to involve
CYPs [31]. Although CYP-catalyzed reduction and acti-
vation of O2 are required for CYP monooxygenase
reactions, certain CYPs, including CYP1A1, CYP2E1,
CYP2B4, and CYP3A, can release reactive oxygen in the
process known as uncoupling. CYP1A1 uncoupling and
microsomal oxidative stress are enhanced in vivo and in
vitro by highly chlorinated PCBs [23,24,32].
In the present study, we examined the capacity of a
series of coplanar and non-coplanar PHAHs (mostly
polyhalogenated biphenyls) to generate ROS in liver
microsomes from vehicle-treated (noninduced) and
TCDD-treated (induced) mice. It was expected that
PHAHs might alter the balance of the flow of electrons,
either in favor of or against pro-oxidant CYPs, depending
on their capacity to bind and/or be metabolized by CYPs.
We found a distinct pattern for the PHAH-stimulated
production of hepatic microsomal oxidative stress, which
was dependent on TCDD induction and the structure of the
specific PHAH.
MATERIALS AND METHODS
Chemicals
TCDD, 2,4,6,2V,4V,6V-hexachlorobiphenyl (246-HCB,
PCB 155), 3,4,5,3V,4V,5V-hexachlorobiphenyl (345-HCB,
PCB 169), 3,4,3V,4V-tetrachlorobiphenyl (34-TCB, PCB
77), and 3,5,3V,5V-tetrachlorobiphenyl (34-TCB, PCB 80)
619
were purchased from AccuStandard, Inc. (New Haven,
CT, USA) and used without further purification. 4-Mono-
chlorobiphenyl (MCB, PCB 3), 2,4,4V-trichlorobiphenyl
(TriCB, PCB 28) and 4,4V-dichlorobiphenyl (DCB, PCB
15) [33], and 2,4,6,2V,4V,6V-hexabromobiphenyl and
3,4,5,3V,4V,5V-hexabromobiphenyl [34] were synthesized
and purified as described. All other chemicals and reagents
were obtained from Sigma –Aldrich Chemical Company
(St. Louis, MO, USA) as the highest available grades.
Animals and treatment
All experiments involving mice were conducted in
accordance with the National Institutes of Health stand-
ards for care and use of experimental animals and the
University of Cincinnati Institutional Animal Care and
Use Committee (IACUC). Five-week-old male mice of
the inbred strain C57BL/6J were purchased from The
Jackson Laboratory (Bar Harbor, ME, USA) and allowed
to acclimate to the animal facility for 1 week before
experiments. Animals were group-housed and main-
tained on a 12-h light/dark cycle, and had access to
standard rodent chow and water ad libitum.
For the current study, mice were administered TCDD
(15 Ag/kg body wt) in corn oil by intraperitoneal injection;
control mice were given equivalent volumes of corn oil.
One week after treatment, the mice were killed by carbon
dioxide asphyxiation, followed by cervical dislocation.
The liver was excised and washed in ice-cold 0.9% NaCl.
A 10% whole homogenate was prepared in 70 mM
sucrose, 220 mM mannitol, 2 mM EDTA, 2 mM EGTA,
2 mM Hepes (pH 7.4), using a motor-driven (1000 rpm)
Potter – Elvehjem homogenizer. The homogenate was
centrifuged for 10 min at 1000g, and the supernatant
fraction was centrifuged again for 15 min at 10,000g.
The resulting supernatant fraction was centrifuged at
105,000g for 60 min, and the resulting microsomal pellet
was washed and then suspended in 0.1 M potassium
phosphate (KPi) buffer (pH 7.4).
Microsomal oxygen and NADPH consumption
Microsomal oxygen consumption was measured po-
larographically with a Clark-type oxygen electrode
(Hansatech Instruments; Norfolk, England). Briefly sum-
marized, 0.6 ml of 0.1 M KPi (pH 7.4) and 200 Ag of
microsomal protein were equilibrated at 37jC with stir-
ring. Following the in vitro addition of DMSO or 2.5 AM
test compound, an NADPH-regenerating system was
added (NADPH-RS, with final concentrations of 240
AM NADP, 5 mM glucose 6-phosphate, 1 mM MgCl2,
and 1 U/ml glucose-6-phosphate dehydrogenase). O2
consumption was monitored for 10 min, during which
time it remained linear. NADPH consumption was fol-
lowed spectrophotometrically at 340 nm at 37jC, in a
620 H. G. SHERTZER et al.
reaction mixture consisting of 0.1 M KPi (pH 7.25) and
200 Ag of microsomal protein. Following the addition of
DMSO or 2.5 AM test compound, NADPH was added
and NADPH consumption was monitored for 10 min,
during which time the reaction remained linear.
Measurement of H2O2 and superoxide
H 2O 2-induced luminol (5-amino-2,3-dihydro-1,4-
phthalazinedione) luminescence and superoxide-induced
lucigenin (bis-N-methylacridinium) chemiluminescence
were measured as previously described [21,22], using a
Berthold Autolumat Plus luminometer. The in vitro reac-
tion mixture consisted of 50 Ag of mitochondrial protein
in a final volume of 1.0 ml 0.1 M KPi (pH 7.25)
containing additions described in the figure legends. We
first determined that the vehicle used in adding chemicals
to the luminol reaction mixture did not affect luminol
luminescence; we found that up to 1% DMSO or meth-
anol had no effect on luminescence under any conditions.
In contrast, acetone, hexane, or acetonitrile stimulated
NADPH-dependent microsomal luminescence, whereas
xylene inhibited and tetrahydrofuran greatly inhibited the
reaction (accompanying article). DMSO was therefore
used as vehicle for the in vitro addition of all test
compounds. Luminescence was determined in the pres-
ence of 5 AM luminol plus 2.5 U/ml horseradish perox-
idase, 50 Ag microsomal protein, 2.5 Al DMSO, or 2.5
Al of 1 mM test chemical (2.5 AM final concentration), in
0.1 M KPi buffer (pH 7.25), in a final volume of 1.0 ml.
The reaction was initiated by the addition of 12.5
Al NADPH-RS and monitored at 37jC. For lucigenin
chemiluminescence, 20 AM lucigenin replaced luminol
and horseradish peroxidase, with all the other components
remaining the same.
Hydrogen peroxide was quantified using o-dianisidine
(3,3V-dimethoxybenzidine). The reaction mixture con-
sisted of 100 Ag o-dianisidine, 2.5 U/ml HRP, 1.25
Al DMSO or 1.25 Al of 1 mM test chemical (2.5 AM final
concentration), and 0.1 M KPi buffer (0.5 ml reaction
volume at pH 7.25). Samples were incubated at 37jC for
5 min, and the reaction was started by addition of 12.5
Al NADPH-RS. The reaction was stopped at zero time or
at 60 min by the addition of 0.5 ml ethanol and centri-
fuged at 10,000g for 10 min. The absorbance of the
supernatant was determined at 450 nm 750 nm, and
compared against hydrogen peroxide standards. The
results are expressed as chemiluminescence units / per
minute per milligram of protein.
Measurement of lipid peroxidation by means of
thiobarbituric acid-reactive substances
The reaction mixture consisted of 0.9 ml 0.1 M KPi
buffer (pH 7.25), 2.25 Al DMSO or 2.25 Al of 1 mM test
chemical (2.5 AM final concentration), and 180 Ag micro-
somal protein, in a 0.9 ml reaction volume. Samples were
incubated at 37jC for 5 min, and the reaction was started
by the addition of 12.5 Al NADPH-RS. The reaction was
stopped at zero time or at 60 min by the addition of 0.1 ml
50% trichloroacetic acid and centrifuged at 10,000g for 10
min. Butylated hydroxytoluene (10 Al of 0.5 M in DMSO)
was added, and the samples were heated at 100jC for 10
min. After centrifugation, the absorbance of the superna-
tant was determined at 532 nm 600 nm. Using malo-
naldehyde diacetal standards, the results are expressed as
nanomoles per minute protein.
Protein and CYP determinations
Protein was measured by the bicinchoninic acid
method (Pierce Chemical Co., Rockford, IL, USA),
according to details provided by the manufacturer. Total
CYP levels were assayed as the reduced CYP – carbon
monoxide binding pigment [35] and NADPH – cyto-
chrome c oxidoreductase was assayed by following the
reduction of cytochrome c in the presence of NADPH as
described [36].
Statistics
Statistical differences between group mean values were
determined by one-way ANOVA, followed by the Stu-
dent – Newman – Keuls test for pairwise comparison of
means. Statistical analysis was performed using SigmaStat
Statistical Analysis software (SPSS Inc., Chicago, IL,
USA).
Biohazard precaution
TCDD and BaP are highly toxic and likely human
carcinogens. Other halogenated biphenyls are toxic. All
personnel were instructed as to safe handling procedures.
Lab coats, gloves, and masks were worn at all times, and
contaminated materials were collected separately for dis-
posal by the Hazardous Waste Unit or by independent
contractors. TCDD-treated mice were housed separately,
and their carcasses were treated as contaminated biolog-
ical materials.
RESULTS
Previous studies have used the chemiluminescent
probes luminol and lucigenin for the determination of
H2O2 and superoxide, respectively [21,22,37,38]. To
determine the suitability of luminol and lucigenin for
use in the current study, they were examined for specificity
in formation of their respective ROS. Figure 1 shows that
each probe was more than 99% specific for the targeted
ROS. Catalase was specific for inhibiting H2O2-stimulat-
 Microsomal reactive oxygen production 621

Fig. 1. Specificity of luminol and lucigenin for H2O2 and superoxide, respectively. Left: H2O2 was generated by addition of increasing
amounts of glucose oxidase, in the presence of 20 mM glucose and 0.1 M KPi buffer (pH 7.25); Closed circles, luminol alone; closed
squares, luminol + SOD; closed triangles, luminol + catalase; open circles, lucigenin. Right: Superoxide was generated by addition of
increasing amounts of xanthine oxidase in the presence of 1 mM hypoxanthine and 0.1 M KPi buffer (pH 7.25); Open circles, lucigenin
alone; open squares, lucigenin + catalase; open triangles, lucigenin + SOD; solid circles, luminol. The activities of SOD and catalase
used were 100 and 500 U/ml, respectively. The results illustrated represent averages of two trials.

ed luminol luminescence, whereas SOD was specific for
inhibiting superoxide-stimulated lucigenin chemilumines-
cence. At 20 AM lucigenin there was no apparent redox
cycling-induced chemiluminescence, as was shown to
occur at higher concentrations of this probe [37].
A group of PHAHs plus the polycyclic hydrocarbon
BaP—potent substrate for CYP1A1 and CYP1B1, much
less so for CYP1A2—were used in this study (Fig. 2).
These chemicals were mostly chlorinated and brominated
biphenyls, with which we compared compounds (1)

Fig. 2. Structures and abbreviations used for the compounds used in this study. TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; 34-TCB,
3,4,3V, 4V-tetrachlorobiphenyl; 3,5,3V, 5V, -tetrachlorobiphenyl; 345-HCB, 3,4,5,3V,4V,5V-hexachlorobiphenyl; 345-HBB, 3,4,5,3V,4V,5V-
hexabromobiphenyl; TriCB, 2,4,4V-trichlorobiphenyl; DFCH, dodecafluorocyclohexane; 246-HCB, 2,4,6,2V,4V,6V-hexachlorobiphenyl;
246-HBB, 2,4,6,2V, 4V, 6V-hexabromobiphenyl; DCB, 4,4V-dichlorobiphenyl; MCB, 4-monochlorobiphenyl; BaP, benzo[a]pyrene.
622 H. G. SHERTZER et al.

3
Luminescence units  10 Ratio (induced/noninduced)
Noninduced Induced
DMSO 112 F 4 35 F 1 0.31
TCDD 107 F 2 122 F 3 1.13
34-TCB 106 F 2 145 F 5 1.36
35-TCB 107 F 4 141 F 2 1.32
345-HCB 107 F 2 128 F 3 1.20
345-HBB 104 F 2 127 F 3 1.22
TriCB 110 F 3 103 F 5 0.94
DFCH 98 F 3 123 F 4 1.26
246-HCB 112 F 2 45 F 2 0.40
246-HBB 99 F 3 35 F 1 0.36
DCB 103 F 3 8 F 3 0.07
MCB 46 F 2 18 F 2 0.38
BaP 82 F 3 12 F 1 0.15
 Microsomal reactive oxygen production 623

Fig. 3. (A) Effects of test compounds on NADPH-dependent luminol chemiluminescence. In the presence of 2.5 AM test compound, the
reaction was started by injection of an NADPH-regenerating system containing 240 AM NADPH. Liver microsomes from vehicle-
treated (noninduced, open circles) or TCDD-treated (induced, closed circles) C57BL/6J mice were used, as described under Materials
and Methods. Results shown are average values for four experiments. The areas under the curves were integrated over the times shown.
The averages for these integrals F SEM are listed in the table; the ratios of integrated luminescence values for induced/noninduced
microsomes are included. (B) Fold changes elicited by test compounds in NADPH-dependent luminol luminescence relative to DMSO
alone. The average luminescence values at each time point, and under each condition, were divided by the average value obtained in the
presence of DMSO alone (upper most panel of (A)), for vehicle-treated (noninduced, open circles) or TCDD-treated (induced, closed
circles) microsomes.

highly halogenated with compounds minimally haloge- In vitro H2O2 production

nated, (2) coplanar with non-coplanar, and (3) chlorinated
with brominated. Based on the type of ROS generation
and related effects, we could divide the compounds into
three functional groups, as described below.
The concentration of 2.5 AM test compound was
chosen, following the titration of 34-TCB into the
luminol test system for hydrogen peroxide production.
34-TCB was among the most potent agents for stim-
ulating hydrogen peroxide, which increased linearly
with 34-TCB concentration to about 1 AM 34-TCB,
leveled off to about 6 AM, then decreased about 40%
at 10 AM (data not shown).
The compounds were evaluated for their capacity to
affect the NADPH-dependent production of H2O2 (Fig.
3A). In the absence of NADPH or microsomes, there was
no luminescence under any condition. Furthermore, the
redox-inactive iron chelator deferoxamine mesylate (1
mM) had no effect on luminol luminescence, and, for
each data set, catalase completely abolished lumines-
cence (data not shown). For each curve, the lumines-
cence was integrated over time, and the integrated
luminescence values are listed (Fig. 3A). In addition,
the ratios of the mean integrated luminescence in induced
624 H. G. SHERTZER et al.

Noninduced Induced Ratio (I/N-I) N-I/P450 Induced/P450

DMSO 600 F 31 1290 F 39 2.14 769 710
TCDD 555 F 54 1190 F 61 2.14 712 657
34-TCB 583 F 23 1510 F 68 2.58 747 833
35-TCB 578 F 21 1380 F 66 2.38 741 761
345-HCB 602 F 34 1340 F 58 2.22 772 740
345-HBB 601 F 44 1310 F 52 2.17 701 722
TriCB 547 F 31 1340 F 26 2.44 701 739
DCFH 551 F 32 1360 F 69 2.47 706 753
246-HCB 553 F 31 1220 F 31 2.21 709 675
246-HBB 627 F 34 1330 F 78 2.11 804 732
DCB 221 F 14 27 F 2 0.12 283 15
MCB 121 F 7 29 F 3 0.24 155 16
BaP 470 F 14 28 F 1 0.06 603 15
CYP 0.78 F 0.1 1.81 F 0.3 2.32 – –
POR 47.0 F 2.6 65.5 F 4.9 1.39

Fig. 4. Effects of test compounds on NADPH-dependent lucigenin chemiluminescence. In the presence of 2.5 AM test compound, the
reaction was started by injection of a NADPH-regenerating system. The results shown (luminescence units  10 4) are average values
for four experiments. The areas under the curves were integrated over the times shown. The averages for these integrals F SEM are
listed in the table; also listed are total CYP concentrations (nmol/mg protein) and NADPH – cytochrome c oxidoreductase activity (POR,
nmol/min/mg protein). The ratios of integrated luminescence values for induced/noninduced microsomes (I/N-I) are included, as are
luminescence ratios relative to the microsomal content of P450.
 Microsomal reactive oxygen production 625

microsomes to that in noninduced microsomes are In vitro superoxide production

shown.
In the absence of test compound, TCDD-induced
microsomes displayed a 69% decrease in H2O2 produc-
tion relative to noninduced microsomes (Fig. 3). This
effect was attributed to the CYP1A2 induction by
TCDD and the capacity of CYP1A2 to act as an
electron sink for NADPH-dependent pro-oxidant CYPs,
primarily CYP2E1 (accompanying article). The addition
of Group 1 compounds, consisting of highly halogenated
coplanar aromatics (DFCH, 345-HCB, 345-HBB, 34-
TCB, 35-TCB, TCDD, and TriCB), increased H2O2
production in induced microsomes, but not in noninduced
microsomes. Group 2 compounds, consisting of highly
halogenated non-coplanar biphenyls (246-HCB and 246-
HBB), had little effect on H2O2 production. Group 3
compounds, consisting of minimally halogenated biphen-
yls (DCB and MCB) and BaP, decreased NADPH-depen-
dent H2O2 production in both noninduced and induced
microsomes, with a greater degree of inhibition in the
latter group.
The Fig. 3A data are more easily visualized by
examining the ratios of luminescence for each in vitro
compound added, relative to the luminescence obtained
when vehicle alone was added (Fig. 3B). It is clear that
Group 1 compounds stimulated H2O2 production in a
sustained fashion, but only in induced microsomes.
Although we did not pursue further the slight rebound
in luminescence with induced microsomes in the pres-
ence of in vitro MCB, we believe that this effect is
likely due to the generation of MCB metabolites.
We also used the probe lucigenin in an attempt to
assess superoxide generation (Fig. 4). The integrated
luminescence over time was quantified and tabulated
(Fig. 4 legend). Lucigenin chemiluminescence was
entirely dependent on NADPH and microsomes, and
was not affected by 1 mM deferoxamine mesylate (data
not shown). In the presence of the vehicle DMSO in
vitro, induced microsomes produced more than twice
the luminescence generated by noninduced microsomes.
The addition of Group 1 (highly halogenated coplanar)
or Group 2 (highly halogenated non-coplanar) test
compounds did not alter the luminescence produced
by either induced or noninduced microsomes. In the
presence of Group 3 compounds (minimally halogenat-
ed and easily metabolized), however, lucigenin lumi-
nescence was largely abolished, especially in the
induced microsomes. Most surprisingly, lucigenin lumi-
nescence was not inhibited at all by superoxide dis-
mutase, at the same superoxide dismutase activity that
completely abolished xanthine/xanthine oxidase-stimu-
lated superoxide production (Fig. 1, right). If superoxide
is released, then SOD should scavenge, as it does for
xanthine/xanthine oxidase-generated superoxide. If su-
peroxide is not released, but lucigenin produces chemi-
luminescence, the lucigenin would appear to react
between NADPH and the one-electron reduced form
of CYP. Since lucigenin does not react with NADPH in
the absence of microsomal protein, the lack of the effect
of SOD on lucigenin chemiluminescence suggests that

Table 1. Molar Utilization of O2 and NADPH and the Production of H2O2

O2 (nmol/min/mg) NADPH (nmol/min/mg) H2O2 (nmol/min/mg)

Noninduced Induced Noninduced Induced Noninduced Induced

No NADPH 0 0 0 0 0 0
NADPH +
a a
DMSO 22.4 F 1.1 35.8 F 2.1 20.6 F 1.4 34.3 F 4.0 0.41 F 0.05 0.29 F 0.04a
TCDD 17.6 F 1.4b 37.1 F 2.0a 21.3 F 0.6 45.7 F 4.1a,b 0.45 F 0.07 1.02 F 0.06a,b
34-TCB 15.3 F 1.0b 36.8 F 1.8a 23.7 F 1.3 52.0 F 4.9a,b 0.38 F 0.03 1.57 F 0.10a,b
35-TCB 15.9 F 0.9b 35.5 F 1.8a 22.8 F 1.0 48.3 F 4.5a,b 0.41 F 0.06 1.53 F 0.20a,b
345-HCB 15.6 F 1.4b 32.6 F 2.6a 21.8 F 1.5 54.3 F 3.8a,b 0.41 F 0.06 1.15 F 0.08a,b
345-HBB 15.6 F 1.5b 35.4 F 3.2a 23.0 F 1.1 59.3 F 6.3a,b 0.44 F 0.06 1.12 F 0.06a,b
TriCB 20.1 F 2.0 28.9 F 1.4a,b 23.8 F 1.4 39.0 F 3.9a 0.55 F 0.11 0.61 F 0.07b
DFCH 19.6 F 1.1b 33.4 F 2.2a 23.0 F 1.6 46.4 F 3.3a,b 0.43 F 0.06 0.79 F 0.05a,b
246-HCB 21.6 F 0.9 30.8 F 1.0a,b 23.2 F 1.3 37.3 F 2.9a 0.46 F 0.08 0.61 F 0.06a,b
246-HBB 20.1 F 1.1 29.4 F 2.4a,b 21.9 F 1.6 34.4 F 3.2a 0.54 F 0.09 0.71 F 0.04a,b
DCB 24.1 F 2.1 82.7 F 5.1a,b 22.8 F 1.4 86.1 F 3.5a,b 0.29 F 0.06b 0.16 F 0.05a,b
MCB 27.0 F 2.0b 80.5 F 5.6a,b 28.8 F 1.3b 77.5 F 9.4a,b 0.22 F 0.07b 0.13 F 0.03a,b
BaP 23.3 F 1.1 74.8 F 3.3a,b 21.9 F 1.3 80.0 F 5.3a,b 0.28 F 0.08 0.55 F 0.08a,b

H2O2 was quantified using o-dianisidine. DMSO (2.5 Al) or 2.5 Al test compound in DMSO (2.5 AM final concentration) was added as indicated. The
reactions were started by addition of an NADPH-regenerating system containing 240 AM NADPH. The results are average values for four experiments F
SEM. Significance at the p < .05 level is indicated.
a
Significantly different from noninduced microsomes when same compound is added in vitro.
b
Significantly different from values obtained when DMSO alone is added to microsomes.
626 H. G. SHERTZER et al.

lucigenin is reacting directly either with an iron – oxo
species of CYP or with NADPH – cytochrome P450
oxidoreductase (POR).
To examine these possibilities, CYP levels and POR
activities were quantified in induced and noninduced
microsomes (Fig. 4 legend). For Group 1 or Group 2
compounds, the integrated levels of lucigenin chemilu-
minescence were proportional to the amount of total
CYP in both induced and noninduced microsomes. The
increase in lucigenin luminescence that occurred in the
induced microsomes (2.1- to 2.6-fold) was proportional
to the increase in total microsomal CYP concentrations
(2.3-fold), and far exceeded the smaller increase in POR
activity (1.4-fold). These results are consistent with a
pathway whereby lucigenin accepts electrons from
CYPs, rather than directly from POR.

Fig. 5. Ratios of H2O2 production to O2 and NADPH consumption and of NADPH production to O2 consumption. These ratios were
calculated from the primary data that were used to generate Table 1. Values obtained for H2O2 production and for O2 and NADPH
consumption were paired for each set (induced, noninduced) of microsomes to obtain ratios. The results shown represent noninduced
microsomes (open bars) and induced microsomes (closed bars). The mean ratios for four experimental determinations F SEM are
shown. *Significantly different ( p < .05) from mean value obtained when adding DMSO alone, using corresponding microsomes (non-
induced or induced).
 Microsomal reactive oxygen production
NADPH and O2 consumption and H2O2 production
To examine further the pathways for electron transfer,
we determined the molar consumption of NADPH and
O2 and the molar production of H2O2 (Table 1). For
every condition, induced microsomes had greater
NADPH and O2 consumption and greater H2O2 produc-
tion, relative to noninduced microsomes. With nonin-
duced microsomes, Group 1 compounds slightly
decreased O2 consumption, but did not affect NADPH
utilization or H2O2 production. Group 2 compounds did
not affect any parameter measured in noninduced micro-
somes, whereas MCB (Group 3) increased O2 and
NADPH consumption and decreased H2O2 generation.
The results were more striking with induced micro-
somes than noninduced microsomes. For O2 consumption,
Group 1 compounds had no effect, Group 2 compounds
were slightly inhibitory, whereas Group 3 compounds
were highly stimulatory. Regarding NADPH utilization,
Group 1 compounds stimulated, Group 2 compounds had
no effect, while Group 3 compounds greatly increased this
parameter. For H2O2 production, Group 1 compounds
were generally highly stimulatory, Group 2 compounds
and BaP (Group 3) stimulated to a lesser extent, whereas
DCB and MCB (Group 3) were inhibitory.
The Table 1 results can be evaluated better when
calculated as ratios that show the percentage of elec-
trons from NADPH resulting in H2O2 generation and
Fig. 6. NADPH-dependent lipid peroxidation production. The reaction mixture for determining TBARS was described under Materials
and Methods. The reaction was initiated with an NADPH-regenerating system containing 240 AM NADPH, and incubated at 37jC for
60 min. The results shown represent noninduced microsomes (open bars) and induced microsomes (closed bars). *Significantly different
( p < .05) from mean value obtained using noninduced microsomes.
627
the H2O2 produced relative to O2 consumption (Fig.
5). H2O2 production, relative to NADPH used and O2
consumed, was lower in induced microsomes than in
noninduced microsomes. In the presence of the highly
halogenated coplanar Group 1 compounds, these ratios
were significantly elevated, whereas for Group 1 com-
pounds having fewer halogens (TriCB and DFCH) and
for Group 2 compounds (highly halogenated non-
coplanar), the ratios were the same in the induced as
in the noninduced microsomes. All Group 3 compounds
(minimally halogenated plus BaP) significantly de-
creased the percentage of electrons needed to produce
H2O2.
Lipid peroxidation
To determine whether H2O2 production was capable
of producing an oxidative stress response in micro-
somes, lipid peroxidation was evaluated (Fig. 6). Lipid
peroxide formation was lower in the induced micro-
somes than in the noninduced microsomes, and this
pattern was essentially unchanged in the presence of
Group 2 compounds. Group 1 compounds greatly
stimulated lipid peroxidation in induced microsomes,
but had little effect in the noninduced microsomes. For
Group 3 compounds, excluding BaP, lipid peroxide
formation was lower in the induced, relative to the
noninduced, microsomes.
628 H. G. SHERTZER et al.
DISCUSSION
Exposure to PHAHs may produce a variety of dele-
terious consequences, including neurobehavioral deficits,
environmental estrogenicity, immunosuppression, and
cancer [5,6,29,39– 41]. Many PHAHs of greatest toxico-
logical concern are highly halogenated and relatively
stable, both environmentally and metabolically. Due to
their inability to be readily metabolized, highly haloge-
nated PHAHs are thought to exert their toxicity via
receptor-mediated pathways, possibly involving oxida-
tive stress. Because of this, we evaluated a representative
group of PHAHs having varying degrees of halogenation
(both chloro- and bromo- substitutions), and with copla-
nar versus non-coplanar configurations.
Three categories of PHAHs defined
We were able to categorize the in vitro effects of these
PHAHs into three groups, based on their capacity to
stimulate NADPH-dependent production of H2O2, in
induced versus noninduced liver microsomes. Group 1
compounds generated a strong oxidative stress response;
Group 1 consisted of coplanar (not ortho-substituted)
highly halogenated compounds, containing three or more
halogens. (Within this group, trichlorobiphenyl was the
least active member.) Group 2 had no effect on the
oxidative stress response and consisted of non-coplanar
(ortho-substituted) and highly halogenated (six halogens)
biphenyls. The electron-deactivating properties of the
multiple halogens and the steric hindrance of the hydrat-
ed halogens render both Group 1 and 2 compounds
recalcitrant to metabolism. Steric effects due to halogen
hydration at the ortho position are also responsible for
torsion of the biphenyl rings, producing the non-coplanar
configuration for ortho-substituted biphenyls. Group 3
compounds, consisting of minimally halogenated
PHAHs and BaP, are rapidly metabolized. CYP-cata-
lyzed oxidation of minimally halogenated PCBs forms
mono- and dihydroxy metabolites, which give rise to
catechols and hydroquinones [42]. These metabolites
have the potential to generate ROS by redox cycling
[43]. When tested in rats, however, minimally haloge-
nated biphenyls, including MCB, did not produce an
oxidative stress response [18].
Which CYP enzyme is likely involved?
The results obtained in the present study, as well as
any biological and toxicological effects, are dependent
on both the specific CYP enzymes induced by TCDD
and the chemical configurations and exposure levels of
the PHAH compounds. Using small subsets of PCB
congeners, a coplanar configuration appeared to be
required for generating an oxidative stress response in
3-methylcholanthrene- or h-naphthoflavone-induced rat
liver microsomes (CYP1A1 and CYP1A2 induction),
whereas non-coplanar PCBs were preferentially active
in phenobarbital-induced microsomes (CYP2B1 induc-
tion) [44]. Thus, the microsomal oxidative stress re-
sponse observed in this study may be applicable to
PHAH bioaccumulation and exposure conditions that
result in activation of the AHR. In this context, the
enzyme induction profile for AHR agonists may pro-
duce different patterns of CYP expression. Such pat-
terns that produce different relative expression ratios of
CYP1A1/CYP2E1/CYP3A to CYP1A2 may determine
the levels of hepatic microsomal oxidative stress (ac-
companying article).
Even related PHAH compounds may differentially
alter the expression ratios for hepatic CYP1A1- versus
CYP1A2-mediated monooxygenase activities, as well as
the CYP1A1 activity profiles for different tissues [45], or
even regionally within one tissue such as liver [46].
Differential CYP expression profiles for related PHAHs
may be explained in part, by varying affinities for the
AHR, as well as differing pharmacokinetics for any
particular inducing agent [47,48]. The pharmacokinetic
profile for AHR – ligand PHAHs is mostly a function of
the compound’s hydrophobicity, coplanarity, rates of
metabolism and elimination, and dosing regimen [49].
Regardless of the factors controlling AHR-mediated gene
expression, CYP expression profiles may regulate tissue-
specific oxidative stress responses and resulting toxicity.
For example, subchronic exposure of rats to PCBs and
TCDD produces a greater oxidative stress response in
brain than in liver [12], consistent with the well-estab-
lished neurotoxicity of environmental PHAHs.
Differences in the pharmacokinetic profiles for
PHAHs in different tissues, and cell types within tissues,
are important considerations in evaluating the potency of
Group 1 compounds used in this study for generating an
oxidative stress response in vivo. The concentrations of
TCDD and other PHAHs in the membranes of isolated
microsomes would be less than the concentrations that
occur in hepatic endoplasmic reticulum of the intact
animal. Thus, in TCDD-induced microsomes, the addi-
tion of TCDD or other Group 1 compounds in vitro can
stimulate H2O2 production more than 7-fold. This in-
crease should represent an upper limit to the stimulation
of ROS generation in vivo in TCDD-treated mice. In this
context, a single high dose or multiple sequential smaller
doses of TCDD are most effective in producing oxidative
stress [16]. Such a dosing regimen would be optimal for
inducing CYP1A1 and CYP1A2 and for maintaining
high tissue concentrations of TCDD. Similarly, multiple
sequential small doses of TCDD are required for maximal
production of hepatic foci that are positive for glutathione
S-transferase and hepatocarcinogenesis in rats [50].
 Microsomal reactive oxygen production
The results in Table 1 and Fig. 5 illustrate that the molar
ratios of H2O2 production, relative to the consumption of
NADPH and O2, increased in the presence of Group 1
compounds in the induced, but not in the noninduced,
microsomes. Concomitantly, we observed a slight increase
in the ratio of NADPH to O2 utilization. These results
suggest that electrons from NADPH are diverted from O2
to H2O2. Because the NADPH utilization-to-O2 consump-
tion ratios were the same for induced and noninduced
microsomes—in the presence or absence of any chemicals
added in vitro—it appears that the pathway for electron
flow was the same for each condition. We find (accom-
panying article) that H2O2 in noninduced microsomes is
most likely generated by CYP2E1.
Proposed electron flow pathway
Our postulated pathway for electron flow, therefore, is
illustrated in Fig. 7. We show (accompanying article) that
when additional compounds are not added to micro-
somes, CYP2E1 is the major pro-oxidant CYP, and
CYP1A2 reduces CYP2E1-generated H2O2 production.
The addition of Group 1 PHAHs to uninduced micro-
somes did not alter the ROS production, suggesting that
PHAH exposure alters the relative production of H2O2 by
different CYPs. Since TCDD exposure decreases micro-
Fig. 7. Scheme representing the electron transport pathways under investigation. NML, nonmetabolizable ligand; RH, metabolizable
substrate.
629
somal H2O2 production in the absence of PHAHs, the
measured amount of PHAH-stimulated H2O2 production
in TCDD-induced microsomes must represent an under-
estimation of ROS production by TCDD-induced pro-
oxidant CYPs. Since TCDD does not induce CYP2E1,
CYP1A1 is the most likely candidate for mediating the
TCDD-induced and Group 1 compound-stimulated pro-
duction of H2O2; this is in keeping with previous reports
showing that CYP1A1 is instrumental for the PHAH-
stimulated production of reactive oxygen [23,24].
CONCLUSIONS
In summary, we have shown that highly halogenated
and coplanar aromatic hydrocarbons increase microsom-
al NADPH-dependent H2O2 production up to more than
7-fold in TCDD-induced mouse liver microsomes. Non-
coplanar (o-substituted) halogenated biphenyls do not
affect H2O2 production, whereas minimally halogenated
biphenyls and benzo[a]pyrene decrease H2O2 produc-
tion. The one-electron-reduced CYP –iron-oxo complex
does not appear to release superoxide, whereas the two-
electron-reduced CYP –iron-oxo complex does release
H2O2, which causes microsomal lipid peroxidation. The
results from this study suggest that the capacity of
630 H. G. SHERTZER et al.
PHAHs to generate an oxidative stress response, and
associated toxicity, is a function of the induction state of
CYP1A1, CYP2E1, and CYP1A2; this postulate is
pursued in the accompanying article. The tissue-specific
levels of pro-oxidant and anti-oxidant CYPs must be
considered with respect to the potential of PHAHs to
generate a tissue-specific oxidative stress response.
Acknowledgements—We thank our colleagues for a careful reading of
the manuscript. This work was funded, in part, by NIH Grants R01
ES10133, R01 ES06321, R01 ES08147, T32 ES07250, and P30
ES06096.
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ABBREVIATIONS
AHR — aromatic hydrocarbon receptor
BaP — benzo[a]pyrene
CYP — cytochrome P450
DCB — 4,4V-dichlorobiphenyl, PCB 15
DFCH — dodecafluorocyclohexane
246-HBB — 2,4,6,2V,4V,6V-hexabromobiphenyl
246-HCB — 2,4,6,2V,4V,6V-hexachlorobiphenyl, PCB
155
345-HBB — 3,4,5,3V,4V,5V-hexabromobiphenyl
345-HCB — 3,4,5,3V,4V,5V-hexachlorobiphenyl, PCB
169
KPi — 0.1 M potassium phosphate buffer
MCB — 4-monochlorobiphenyl, PCB 3
NADPH-RS — NADPH regenerating system
PCB — polychlorinated biphenyl
PHAH — polyhalogenated aromatic hydrocarbon
POR — NADPH – cytochrome P450 oxidoreductase
ROS — reactive oxygen species
SOD — superoxide dismutase (SOD1, cytosolic; SOD2,
mitochondrial; SOD3, extracellular)
TBARS — thiobarbituric acid-reactive substances
34-TCB — 3,4,3V,4V-tetrachlorobiphenyl, PCB 77
35-TCB — 3,5,3V,5V-tetrachlorobiphenyl, PCB 80
TCDD — 2,3,7,8-tetrachlorodibenzo-p-dioxin
TriCB — 2,4,4V-trichlorobiphenyl, PCB 28