Metabolic activation of PCBs to quinones: Reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase

Article abstract of DOI:10.1021/tx950117e

Polychlorinated biphenyls (PCBs) may undergo cytochrome P-450-catalyzed hydroxylations to form chlorinated dihydroxybiphenyl metabolites. When the hydroxyl groups are ortho or para to each other, oxidation to a quinone may be catalyzed by peroxidases present within the cell. In order to study the reactivity of PCB-derived quinones, selected chlorophenyl 1,2- and 1,4- benzoquinones were synthesized and characterized, including their reduction potentials against a saturated calomel electrode. Two quinones, 4-(4'- chlorophenyl)-1,2-, and 4-(3',4'-dichlorophenyl)-1,2-benzoquinone, were obtained via the oxidation of the corresponding dihydroxybiphenyls with 2,3- dichloro-5,6-dicyano-1,4-benzoquinone. Six 1,4-benzoquinones were synthesized via the Meerwein arylation: 2-(2'-chlorophenyl)-1,4-, 2-(3'-chlorophenyl)1,4- , 2-(4'-chlorophenyl)-1,4-, 2-(2',5'-dichlorophenyl)-1,4-, 2-(3',4'- dichlorophenyl)-1,4-, and 2-(3',5'-dichlorophenyl)-1,4-benzoquinone. As a model study, the rate of reactivity of 2-(4'-chlorophenyl)-1,4-benzoquinone toward the nitrogen nucleophiles glycine, L-arginine, L-histidine- and L- lysine was determined under pseudo-first-order conditions at pH 7.4. The rate constants ranged from 0.45 to 0.75 min^-1 M^-1. Higher rates were obtained under conditions of higher pH. Two reaction products were identified as the 5- and 6-ring addition products in the ratio of 1:4. In contrast, the reaction of 2-(4'-chlorophenyl)-1,4-benzoquinone with the sulfur nucleophiles glutathione or N-acetyl-L-cysteine was instantaneous. The major product of the reaction of glutathione with 2-(4'-chlorophenyl)-1,4-benzoquinone was also the 6-ring addition product. The hydroquinone thioether could be enzymatically reoxidized to the quinone thioether. Also, the influence of atmospheric oxygen and superoxide dismutase on the rates of the following horseradish peroxidase/H₂O₂-catalyzed oxidations was investigated: 3,4- dichloro-2',5'-dihydroxybiphenyl to 2-(3',4'-dichlorophenyl)-1,4-benzoquinone and 3,4-dichloro-3',4'-dihydroxybiphenyl to 4-(3',4'-dichlorophenyl)-1,2- benzoquinone. While the presence or absence of atmospheric oxygen did not alter the rates of the oxidation reactions, the presence of superoxide dismutase significantly increased the rates of both oxidation reactions, having the greater effect on the oxidation of the 1,4-hydroquinone. These data show that PCB-derived quinones react with both nitrogen and sulfur nucleophiles of the cell and may explain, in part, the toxic effects of individual PCBs and PCB formulations, such as glutathione depletion, oxidative stress, and cell death.

Full text of DOI:10.1021/tx950117e

Chem. Res. Toxicol. 1996, 9, 623-629
623
Meta bolic Activa tion of P CBs to Qu in on es: Rea ctivity
tow a r d Nitr ogen a n d Su lfu r Nu cleop h iles a n d In flu en ce
of Su p er oxid e Dism u ta se
Anthony R. Amaro,† Greg G. Oakley,† Udo Bauer,‡ H. Peter Spielmann,§ and
Larry W. Robertson*,†
Graduate Center for Toxicology, University of Kentucky Medical Center, 306 Health Sciences
Research Building, Lexington, Kentucky 40536-0305, and Department of Biochemistry, University of
Kentucky Medical Center, Lexington, Kentucky 40536
Received J une 29, 1995X
Polychlorinated biphenyls (PCBs) may undergo cytochrome P-450-catalyzed hydroxylations
to form chlorinated dihydroxybiphenyl metabolites. When the hydroxyl groups are ortho or
para to each other, oxidation to a quinone may be catalyzed by peroxidases present within the
cell. In order to study the reactivity of PCB-derived quinones, selected chlorophenyl 1,2- and
1,4-benzoquinones were synthesized and characterized, including their reduction potentials
against a saturated calomel electrode. Two quinones, 4-(4′-chlorophenyl)-1,2-, and 4-(3′,4′-
dichlorophenyl)-1,2-benzoquinone, were obtained via the oxidation of the corresponding
dihydroxybiphenyls with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Six 1,4-benzoquinones
were synthesized via the Meerwein arylation: 2-(2′-chlorophenyl)-1,4-, 2-(3′-chlorophenyl)-
1,4-, 2-(4′-chlorophenyl)-1,4-, 2-(2′,5′-dichlorophenyl)-1,4-, 2-(3′,4′-dichlorophenyl)-1,4-, and
2-(3′,5′-dichlorophenyl)-1,4-benzoquinone. As a model study, the rate of reactivity of 2-(4′-
chlorophenyl)-1,4-benzoquinone toward the nitrogen nucleophiles glycine, L-arginine,
L-histidine- and L-lysine was determined under pseudo-first-order conditions at pH 7.4. The
rate constants ranged from 0.45 to 0.75 min-1 M-1. Higher rates were obtained under conditions
of higher pH. Two reaction products were identified as the 5- and 6-ring addition products in
the ratio of 1:4. In contrast, the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone with the sulfur
nucleophiles glutathione or N-acetyl-L-cysteine was instantaneous. The major product of the
reaction of glutathione with 2-(4′-chlorophenyl)-1,4-benzoquinone was also the 6-ring addition
product. The hydroquinone thioether could be enzymatically reoxidized to the quinone
thioether. Also, the influence of atmospheric oxygen and superoxide dismutase on the rates
of the following horseradish peroxidase/H2O2-catalyzed oxidations was investigated: 3,4-
dichloro-2′,5′-dihydroxybiphenyl to 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone and 3,4-dichloro-
3′,4′-dihydroxybiphenyl to 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone. While the presence or
absence of atmospheric oxygen did not alter the rates of the oxidation reactions, the presence
of superoxide dismutase signficantly increased the rates of both oxidation reactions, having
the greater effect on the oxidation of the 1,4-hydroquinone. These data show that PCB-derived
quinones react with both nitrogen and sulfur nucleophiles of the cell and may explain, in part,
the toxic effects of individual PCBs and PCB formulations, such as glutathione depletion,
oxidative stress, and cell death.
biphenyl are more resistant to chemical and biologic
attack, PCBs with fewer halogens may have measurable
rates of metabolism (3).
In tr od u ction
Polychlorinated biphenyls (PCBs)1 were large scale
industrial chemicals which were used in diverse applica-
tions, such as in dielectric fluids, in transformers and
capacitors, in hydraulic fluids, and as sealants (1). The
physical properties which made PCBs ideal for industry
have also resulted in worldwide environmental contami-
nation (2). While PCBs with multiple halogens per
Cytochrome P-450 metabolism of aromatic hydrocar-
bons, including halogenated benzenes and halogenated
biphenyls, often involves a hydroxylation reaction, the
introduction of an OH functionality into the molecule.
When two hydroxyl groups are introduced ortho or para
to each other, further oxidation by peroxidases within the
cell will lead to quinone metabolites.
* Address correspondence to this author at the Graduate Center
for Toxicology, 306 Health Sciences Research Bldg., University of
Kentucky, Lexington, KY 40536-0305. Telephone: (606) 257-3952;
Fax: (606) 323-1059; E-mail: LWROBE01@UKCC.UKY.EDU.
† The Graduate Center for Toxicology.
Considerable data support the occurrence of catechols
and hydroquinone metabolites of PCBs. These have been
detected in in vivo (4,5) and in vitro (6,7) studies. We
have recently shown that three peroxidases (myelo- and
horseradish peroxidase and prostaglandin (H) synthase)
(8) will catalyze the further oxidation of such PCB-
derived catechols and hydroquinones to quinoid species,
which bind to deoxynucleotides (9) and DNA (8). The
occurrence of PCB quinone metabolites and their poten-
tial reactivity toward cellular nucleophiles is therefore
of current interest.
‡ Current address: Department of Chemistry, University of Oulu,
Fin-90570 Oulu, Finland.
§ Department of Biochemistry.
X Abstract published in Advance ACS Abstracts, March 15, 1996.
1
Abbreviations: PCB, polychlorinated biphenyl; SOD, superoxide
dismutase; HRP, horseradish peroxidase; GSH, glutathione; NAC,
N-acetyl-L-cysteine; FAB/MS, fast atom bombardment mass spectrom-
etry; EI-MS, electron impact mass spectrometry; SCE, saturated
calomel electrode; TEAP, tetraethylammonium perchlorate; NMR,
nuclear magnetic resonance.
0893-228x/96/2709-0623$12.00/0 © 1996 American Chemical Society
+
+

624 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Amaro et al.
diazotized chloroanilines (Aldrich Chemical Co.). The 1H and
13C NMR spectra reported in this section were recorded on either
a Varian VXR-400S or a Varian Gemini-200 spectrometer by
using CDCl3 and acetone-d6 (Aldrich Chemical Co.) as solvents
and tetramethylsilane (Aldrich Chemical Co.) as an internal
standard. GC/MS spectra were recorded on a Finnigan INCOS
50 using a fused silica capillary column (DB-5MS 15 m × 0.25
mm and OV-1, 25 m, J &W Scientific, Folsom, CA).
The PCB-1,2-benzoquinones were unstable, so their deriva-
tives with ethylenediamine were synthesized (14). Ethylene-
diamine (1 µL) was added to the quinone 1 (1 mg) dissolved in
absolute ethanol (500 µL) and was stirred at room temperature
for 1 min. After the addition of ethylenediamine, the yellow-
colored solution turned to a dark orange-brown color. This
procedure was repeated for quinone 2. The samples were
submitted for GC/EI-MS analysis.
4-(4′-Chlorophenyl)-1,2-benzoquinone from 4-chloro-3′,4′-di-
hydroxybiphenyl (1). 1H NMR (400 MHz, acetone-d6): δ )
6.48-6.62 (m, 3H), 7.24-7.62 (m, 4H); MS (EI) m/ z (relative
intensity) ) 243 (100) [M+ + 1], 208 (5) [M+ - Cl] 152 (25), [M+
- Cl, C2H4N2]. Mass spectral data reported for ethylenediamine
derivative of quinone.
4-(3′,4′-dichlorophenyl)-1,2-benzoquinone from 3,4-dichloro-
3′,4′-dihydroxybiphenyl (2). 1H NMR (400 MHz, acetone-d6): δ
) 6.52 (dd, J ) 10.4, 0.9 Hz , 1H, 6′-H), 6.59 (dd, J ) 2.4, 0.9
Hz , 1H, 3′-H), 7.40 (dd, J ) 10.4, 2.4 Hz , 1H, 5′-H), 7.48 (dd,
J ) 8.5, 2.4 Hz , 1H, 6′-ArH), 7.60 (d, J ) 8.5 Hz , 1H, 5′-ArH),
7.74 (d, J ) 2.4 Hz , 1H, 2′-ArH); 13C NMR (100 MHz, acetone-
d6): δ ) 126.32, 128.89, 131.04, 131.67, 133.96, 135.73, 136.38,
140.09, 140.10, 148.26, 179.83, 179.94; MS (EI) m/ z (relative
intensity) ) 277 (100) [M+ + 1], 186 (8), [M+ - Cl, C2H4N2],
151(5) [M+ - 2 Cl, C2H4N2]. Mass spectral data reported for
ethylenediamine derivative of quinone.
2-(2′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone
and 2-chloroaniline (3) mp 80-81 °C (acetone). 1H NMR (200
MHz, CDCl3): δ ) 6.78-6.81 (m, 1H), 6.86-6.90 (m, 2H), 7.19-
7.51 (m, 4H); 13C NMR (50 MHz, CDCl3): δ ) 126.82, 129.86,
130.64, 130.75, 132.41, 133.12, 135.03, 136.41, 136.82, 146.16,
185.05, 187.24; MS (EI) m/ z (relative intensity) ) 218 (50) [M+],
190 (5) [M+ - CO], 183 (100), [M+ - Cl], 155 (20) [M+ - CO,
Cl], 127 (8) [M+ - 2 CO, Cl].
2-(3′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone
and 3-chloroaniline (4) mp 144-145 °C (acetone). 1H NMR (200
MHz, CDCl3):δ ) 6.81-6.92 (m, 3H), 7.34-7.49 (m, 4H); 13C
NMR (50 MHz, CDCl3): δ ) 127.35, 129.24, 129.78, 130.11,
133.15, 134.27, 134.53, 136.33, 136.99, 144.60, 186.04, 187.20;
MS (EI) m/ z (relative intensity) ) 218 (61) [M+], 190 (12), [M+
- CO], 183 (100) [M+ - Cl], 155 (25) [M+ - CO, Cl], 127 (15)
[M+ - 2 CO, Cl].
2-(4′-Chlorophenyl)-1,4-benzoquinone from 1,4-benzoquinone
and 4-chloroaniline (5) mp 130-132 °C (acetone). 1H NMR (200
MHz, CDCl3): δ ) 6.80-6.92 (m, 3H), 7.43 (s, 4H); 13C NMR
(50 MHz, CDCl3):δ ) 128.90, 130.56, 131.07, 132.69, 136.37,
136.63, 137.05, 144.82, 186.28, 187.25; MS (EI) m/ z (relative
intensity) ) 218 (82) [M+], 190 (12), [M+ - CO], 183 (100) [M+
- Cl], 155 (19) [M+ - CO, Cl], 127 (15) [M+ - 2 CO, Cl].
2-(2′,5′-Dichlorophenyl)-1,4-benzoquinone from 1,4-benzo-
quinone and 2,5-dichloroaniline (6) mp 114-116 °C (acetone).
1H NMR (200 MHz, CDCl3): δ ) 6.78-6.81 (m, 1H), 6.83-6.95
(m, 2H), 7.21-7.24 (m, 1H), 7.33-7.43 (m, 2H); 13C NMR (50
MHz, CDCl3): δ ) 130.55, 130.70, 130.99, 131.55, 132.88,
133.83, 135.33, 136.55, 136.77, 144.97, 184.56, 186.81; MS m/ z
(relative intensity) ) 252 (49) [M+], 217 (100), [M+ - Cl], 189
(25) [M+ - CO, Cl], 161 (8) [M+ - 2 CO, Cl], 126 (12) [M+ - 2
CO, 2 Cl].
F igu r e 1. Structures of the synthetic chloroquinones.
Quinones make up a large class of compounds with
diverse biological activity. They can be found in many
animal and plant cells and are widely used as anticancer,
antibacterial, or antimalarial drugs as well as fungicides.
The cytotoxicity of quinones can be attributed to their
binding with cellular nucleophiles such as protein and
nonprotein sulfhydryls and/or their ability to redox cycle
with the creation of oxidative stress (10). We are
currently investigating the metabolic activation of lower
chlorinated biphenyls to dihydroxy compounds and their
subsequent oxidation to the quinone/semiquinones. In
an effort to better understand the reactivity of PCB
quinone metabolites, we have synthesized a series of
PCB-1,4-benzoquinones and two 1,2-benzoquinones for
our studies (Figure 1). These compounds have allowed
us to determine their reactivity with nitrogen and sulfur
nucleophiles at physiological pH. We have also studied
the role of oxygen and superoxide dismutase (SOD) on
the horseradish peroxidase (HRP)/H2O2-catalyzed oxida-
tion reactions of lower chlorinated dihydroxybiphenyls
to PCB quinones.
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d Rea gen ts. Horseradish peroxidase (type
6) (EC 1.11.1.7, Type VI), superoxide dismutase (EC 1.15.1.1),
glutathione, and N-acetyl-L-cysteine were purchased from Sigma
Chemical Co. (St. Louis, MO). Ethylenediamine was pur-
chased from Aldrich Chemical Co. All other reagents were of
the highest purity available and were from Fisher Chemical
(Cincinnati, OH). Ca u tion : Synthetic PCBs and metabolites
should be considered potentially toxic and hazardous and
therefore should be handled in an appropriate manner.
Dih yd r oxybip h en yl a n d Qu in on e Syn th eses. We have
previously reported the synthesis, isolation, and characterization
of the chlorinated dihydroxybiphenyls (11). The chlorophenyl-
substituted 1,2-benzoquinones were synthesized by oxidation
of the respective catechols (12) using 2,3-dichloro-5,6 dicyano-
1,4-benzoquinone (Aldrich Chemical Co.), while the 1,4-benzo-
quinones were synthesized using the Meerwein arylation as
described by Brassard et al. (13) from 1,4-benzoquinone and
2-(3′,4′-Dichlorophenyl)-1,4-benzoquinone from 1,4-benzo-
quinone and 3,4-dichloroaniline (7) mp 163-164 °C (acetone).
1H NMR (200 MHz, CDCl3): δ ) 6.82-6.94 (m, 3H), 7.31 (dd, J
) 8.3, 2.0 Hz, 1H, 6′-ArH), 7.53 (d, J ) 8.3 Hz, 1H, 5′-ArH),
7.61 (d, J ) 2.0 Hz 1H, 2′-ArH); 13C NMR (50 MHz, CDCl3): δ
) 128.41, 130.62, 131.11, 132.47, 133.07, 133.15, 134.78, 136.48,
137.03, 143.69, 185.84, 186.96; MS m/ z (relative intensity) )
252 (52) [M+], 217 (100), [M+ - Cl], 189 (15) [M+ - CO, Cl],
161 (7) [M+ - 2 CO,Cl], 126 (12) [M+ + 2 CO, 2 Cl].
+
+

Reactivity of PCB Quinones
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 625
2-(3′,5′-Dichlorophenyl)-1,4-benzoquinone from benzoquinone
and 3,5-dichloroaniline (8) mp 102-104 °C (acetone). 1H NMR
(200 MHz, CDCl3): δ ) 6.82-6.94 (m, 3H), 7.35-7.48 (m, 3H);
13C NMR (50 MHz, CDCl3): δ ) 127.62, 130.00, 133.63, 135.31,
135.36, 136.47, 136.99, 143.53, 185.53, 186.82; MS m/ z (relative
of the major glutathione adduct of 4′-chloro-1,1′-biphenyl-2,5-
quinone (125.7 MHz, D2O, 25 °C) δ ) 30.51, 35.79, 46.17, 57.78,
58.42, 122.75, 124.58, 125.38, 133.14, 134.34, 135.26, 137.71,
140.56, 150.84, 153.73, 176.77, 177.10, 178.17, 179.17. Fast
atom bombardment (FAB/MS) spectra were determined on a
Kratos Concept 1H double focussing mass spectrometer.
Rea ction of 2-(4′-Ch lor op h en yl)-1,4-ben zoqu in on e (5)
with Glycin e. 2-(2′-Chlorophenyl)-1,4-benzoquinone (4.5 mmols),
dissolved in 5 mL of tetrahydrofuran, was added dropwise to
2.5 mL of 0.1 M phosphate (pH 9.0) containing 4.5 mmol of
glycine. A dark purple color developed immediately. After 1 h
the pH of the reaction mixture was adjusted to 7, and the
mixture was extracted with diethyl ether. The ether phase was
discarded. The aqueous layer, after the pH was adjusted to 2,
was extracted again with ether, which efficiently extracted the
purple product. The product was washed by extraction into
water (pH 7) and into ether (pH 2) two additional times. The
product was dried and further purified on a sephadex G-10
column (2 × 55 cm) in methanol. The dark purple product,
which eluted in 5 mL fractions (11-25), showed two peaks in
reverse-phase HPLC using the conditions described (Rt ) 18.0
min and Rt ) 20.0 min). These were also characterized by 500
MHz NMR.
1H NMR of the major glycine adduct of 4′-chloro-1,1′-biphenyl-
2,5-quinone (500 MHz, D2O, 10 °C) δ ) 3.88 (s, overlapped with
minor isomer, 2H, CH2), 5.41 (d, J ) 3 Hz, 1H, CH), 6.69 (d, J
) 3 Hz, 1H, CH), 7.47 (m, 4H, CH). 13C NMR of the major
glycine adduct of 4′-chloro-1,1′-biphenyl-2,5-quinone (125.7 MHz,
CD3OD/D2O 3:1, 0 °C) δ 48.29, 98.82, 130.66, 130.85, 132.88,
133.52, 133.84, 137.84, 138.41, 144.21, 150.69, 176.35, 184.43,
188.80.
1H NMR of the minor glycine adduct of 4′-chloro-1,1′-biphenyl-
2,5-quinone (500 MHz, D2O, 10 °C) δ ) 3.88 (s, overlapped with
major isomer, 2H, CH2), 5.44 (s, 1H, CH), 6.70 (s, 1H, CH),
7.44 (m, 4H, CH).
intensity) ) 252 (72) [M+], 217 (40), [M+ - Cl], 189 (8) [M+
-
CO, Cl], 161 (5) [M+ - 2 CO,Cl], 126 (11) [M+ + 2 CO, 2 Cl].
Deter m in a tion of Oxid a tion /Red u ction P oten tia ls. All
quinone biphenyls (5 mM in DMSO) were diluted to 1 mM
solutions with dry DMSO to a final volume of 5 mL. Tetra-
ethylammonium perchlorate (TEAP, 0.1 mM) was added to the
solution as a supporting electrolyte. The working electrode was
glassy carbon and platinum wire was used as the counter
electrode. All peak potentials were measured against a satu-
rated calomel electrode (SCE). The solutions were purged with
N2 for 10 min prior to recording the cyclic voltammogram at a
sweep rate of 100 mV/s on
Analyzer.
a BAS 100A Electrochemical
Rea ctivity w ith Nu cleop h iles. A. Kinetic analysis of
amino acid addition to 2-(4′-chlorophenyl)-1,4-benzoquinone. The
rates for selected amino acids were determined following the
decrease in absorbance of the 2-(4′-chlorophenyl)-1,4-benzo-
quinone chromophore at 380 nm (ꢀ ) 1430 M-1 cm-1). Reactions,
run under pseudo-first-order conditions, were initiated by
adding 5 µL of a 25 mM solution of the quinone in DMSO to
995 µL of a buffer solution (12 mM amino acid in 50 mM
phosphate, pH 7.4) in a quartz cuvette. The absorbance of each
reaction was monitored on a Shimadzu MPS-2000 UV-vis
spectrophotometer at 25 °C for 15 min. All plots of ln At versus
time were linear, indicating that each reaction followed pseudo-
first-order kinetics. Pseudo-first-order rate constants were
estimated for each reaction from the slope of the regression lines
fit to each plot. Rate constants were normalized to second-order
with units of min-1 M-1 after dividing by the amino acid
concentration (12 mM).
Hor ser a d ish P er oxid a se-Ca ta lyzed Oxid a tion of 3,4-
Dich lor o-3′4′- a n d 3,4-Dich lor o-2′,5′-d ih yd r oxybip h en yls).
Reactions were performed in 100 mM sodium citrate (pH 7.4)
at 37 °C under the following conditions: 0.05 mM 3,4-dichloro-
3′4′-dihydroxybiphenyl or 3,4-dichloro-2′,5′-dihydroxybiphenyl;
2.3 nM HRP; 0.5 mM H2O2; ( 300 units of SOD. The formation
of 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone or 4-(3′,4′-dichlo-
rophenyl)-1,2-benzoquinone was monitored by measuring ab-
sorbance changes at 365 (ꢀ ) 2716 M-1 cm-1) or 323 nm (ꢀ )
5604 M-1 cm-1), respectively. Anaerobic conditions (oxygen
exclusion) were achieved by bubbling N2 through the solution
for 10 min before the reaction while a constant flow of N2 was
blown over the cuvette during the reaction.
B. Analysis of nucleophilic addition of thiols to 2-(4′-chlo-
rophenyl)-1,4-benzoquinone and subsequent reoxidation of the
thiol-hydroquinone adducts. Reactions were initiated under
second-order conditions with the addition of 10 µL of 25 mM
glutathione or N-acetyl-L-cysteine to 10 µL of 25 mM 2-(4′-
chlorophenyl)-1,4-benzoquinone in 970 µL of 50 mM phosphate
buffer solution. The reaction was monitored over the range of
650 nm to 350 nm. Horseradish peroxidase (5 µL of 0.25 units/
µL) and 0.1 M H2O2 (5 µL) were added to the cuvette and the
reaction was monitored over the same wavelength range and
recorded.
Rea ction of 2-(4′-Ch lor op h en yl)-1,4-ben zoqu in on e (5)
w ith Glu ta th ion e (GSH). A procedure was employed similar
to that described by Eckert et al. (15). To a solution of 2-(4′-
chlorophenyl)-1,4-benzoquinone (4.5 mmol), dissolved in 2.5 mL
of tetrahydrofuran, 120 mmol of glutathione was added in 10
mL of 0.1 M sodium acetate, pH 9.0. The reaction, which was
allowed to run for 5 min at room temperature, was accompanied
by a slight color change from orange to light yellow. Purification
of the crude product was achieved with a Sephadex G-10 column
(1 × 25 cm) using water as the solvent. The glutathione adducts
were eluted from the column in two faint but distinct yellow
bands. The purity of the fractions from the column was
determined by HPLC chromatography (Shimdzu HPLC system
fitted with a 3 mm × 25 cm Upchurch C-18 column) and UV-
Vis spectroscopy. The major glutathione conjugate was sepa-
rated using a linear gradient of 40-100% acetonitrile containing
0.1% trifluoroacetic acid over 30 min at a flow rate of 0.3 mL/
min (Rt ) 11.5 min).
1H NMR spectra were obtained with a Varian Inova 500 MHz
spectrometer and are reported in part per million referenced to
the residual HDO peak in the spectra. 1H NMR of the major
glutathione adduct of 4′-chloro-1,1′-biphenyl-2,5-quinone (500
MHz, D2O, 25 °C) δ ) 2.11 (m, 2H, CH2), 2.46 (t, J ) 7.5 Hz,
2H, CH2), 3.23 (dd, J ) 8.3, 14.3 Hz, 1H, CH2), 3.38 (dd, J ) 5,
14.3 Hz, 1H, CH2), 3.76 (t, J ) 6.5 Hz, 1H, CH), 3.82 (s, 2H,
CH2), 4.48 (dd, J ) 5, 8.3 Hz, 1H, CH), 6.81 (d, J ) 2.9 Hz, 1H,
CH), 7.01 (d, J ) 2.9 Hz, 1H, CH), 7.48 (m, 4H, CH). 13C NMR
Resu lts a n d Discu ssion
Syn th esis. A modified version of the Meerwein ary-
lation was used to synthesize a series of mono- and
dichlorophenyl-substituted-1,4-benzoquinones (13). The
benzoquinones were recrystallized from acetone in 60-
1
70% yields and characterized using H NMR, 13C NMR,
and GC/MS. Three of these compounds, 4-(2′,5′-dichlo-
rophenyl)-1,4-, 4-(3′,4′-dichlorophenyl)-1,4-, and 4-(3′,5′-
dichlorophenyl)-1,4-benzoquinone have not been previ-
ously reported. The 1,2-benzoquinones, 4-(4′-chloro-
phenyl)-1,2- and 4-(3′,4′-dichlorophenyl)-1,2-benzoquino-
ne, were synthesized from the corresponding dihydroxy-
biphenyls using 2,3-dichloro-5,6-dicyano-1,4-benzoquino-
ne as the oxidizing agent. The yields ranged from
20-30%, and attempts to recrystallize these compounds
resulted in a loss of product due to polymerization and/
or decomposition (i.e., insoluble black precipitate).
The 1,2-benzoquinones were analyzed with GC/MS, but
the molecular ion peak was two mass units higher than
expected. Apparently, these compounds were reduced on
the GC stationary phase, and the eluted compound was
the reduced (catechol) species. We derivatized the
+
+

626 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Amaro et al.
Ta ble 2. P ea k P oten tia ls of Ch lor in a ted Qu in on e
Bip h en yls vs a Sa tu r a ted Ca lom el Electr od e
Ep,c1
Ep,a1
Ep,c2
Ep,a2
benzoquinones
(mV)a (mV)a
(mV)b
.(mV)b
4-(4′-chlorophenyl)-1,2-
4-(3′,4′-dichlorophenyl)-1,2- -228
-262
-185 -1018
-160 -983
-890
-865
2-(2′-chlorophenyl)-1,4-
2-(3′-chlorophenyl)-1,4-
2-(4′-chlorophenyl)-1,4-
2-(2′,5′-dichlorophenyl)-1,4- -366
2-(3′,4′-dichlorophenyl)-1,4- -362
2-(3′,5′-dichlorophenyl)-1,4- -380
-405
-384
-392
-315 -1229 -1103
-317 -1136 -1061
-327 -1081 -1012
-293 -1159 -1068
-293 -1090
-993
-311 -1135 -1059
a
Ep,c1 and p,a1 - semiquinone + e- ) (hydroquinone)2- b Ep,c2 and p,a2
.
- quinone + e- ) semiquinone.
to 340 nm occurred within the first 10 min of the reaction.
A new absorbance also appeared at about 500 nm which
was attributed to the formation of a new product.
The glycine-quinone adduct was isolated as a 4:1
mixture of isomers. There were only two protons present
on the quinone ring of both of the isomers. This forces
the conclusion that the glycine added 1,4 to the ring and
did not form the expected 1,2 imino-quinone addition
product. The major isomer was identified as the 1,2,3,5-
substituted product by the presence of a 3 Hz coupling
between the protons on the quinone ring. This is
consistent with an aromatic meta coupling between the
two protons. The minor product shows no resolvable
coupling between the two protons on the quinone ring.
This is consistent with a 1,2,4,5-substitution pattern
where the two remaining protons are para to each other.
F igu r e 2. UV-visible spectrum of 2-(4′-chlorophenyl)-1,4-
benzoquinone (A), the reaction product(s) of 2-(4′-chlorophenyl)-
1,4-benzoquinone with glutathione at pH 7.4 (B), and the
reoxidation of the 2-(4′-chlorophenyl)-1,4-benzoquinone thioether
with horseradish peroxidase and H2O2 at pH 7.4 (C).
Ta ble 1. Ra tes of Rea ctivity of
2-(4′-Ch lor op h en yl)-1,4-ben zoqu in on e w ith Selected
Nu cleop h iles a t p H 7.4
nucleophile
L-arginine
glycine
L-histidine
L-lysine
K (min-1 M-1
)
0.45
0.62
0.75
0.64
glutathione
N-acetyl-L-cysteine
instantaneousa
instantaneousa
On the basis of (1) extraction procedure which would
favor a carboxylic acid, (2) NMR data showing ring
addition, with a 1,2,3,5- and a 1,2,4,5-substitution pat-
terns for the major and minor products, respectively, (3)
quinone carbon chemical shifts, and (4) UV/vis spectra
consistent with ortho-iminoquinones, we have assigned
an iminoquinone structure to the products. Similar
reactions involving both ortho- and para-quinones have
also produced iminoquinone products (16-20).
a
Instantaneous reaction. The rate could not be measured under
pseudo-first-order or second-order conditions. Results represent
means of three experiments.
quinones with ethylenediamine to obtain a stable product
(15). The GC showed only one product for each reaction,
and a corresponding [M+ + 1] peak was obtained for each
compound using electron impact mass spectrometry. The
[M+ + 1] peak may be a result of the protonation (i.e.,
self-chemical ionization) of the basic sites on the deriva-
tives.
Absor p tion Sp ectr a l Ch a n ges d u r in g th e Glu -
ta th ion e-Red u ctive Ad d ition to P CB Qu in on es. The
nucleophilic addition of the sulfur nucleophile glutathione
(GSH) to 2-(4′-chlorophenyl)-1,4-benzoquinone proceeded
rapidly and was over within seconds. The spectral
changes corresponding to this reaction under aerobic
conditions involved a decrease in the absorbance due to
2-(4′-chlorophenyl)-1,4-benzoquinone at 380 nm (Figure
2; spectrum A) and the formation of a new peak at 310
nm (Figure 2, spectrum B). The latter absorption peak
reached its maximal intensity within the first minute and
was attributed to glutathionyl-2-(4′-chlorophenyl)-1,4-
hydroquinones. The products from this reaction were
analyzed using FAB/MS. The data confirm the presence
of monoglultathionyl-hydroquinone adducts with a mo-
lecular ion peak at [M - 1 ) 524] (Figure 4). Crude
reaction products also showed a molecular ion peak
corresponding to diglutathionyl-hydroquinone adduct(s)
(data not shown). The products from spectrum B were
subsequently treated with HRP/H2O2, and within 15 s
the clear solution had acquired an orange color. The
absorbance at 310 nm rapidly decreased, and the forma-
tion of a new peak was observed at 365 nm (Figure 2,
spectrum C); this latter absorption peak is attributed to
the oxidation of the glutathionyl-hydroquinones to cor-
responding glutathionyl-quinone adducts. The reaction
with N-acetyl-L-cysteine gave similar spectral changes.
Kin etics a n d Absor p tion Sp ectr a l Ch a n ges d u r -
in g Am in o Acid Ad d ition to P CB Qu in on es. The
quinone 2-(4′-chlorophenyl)-1,4-benzoquinone was se-
lected as a model compound to study the reactivity of
PCB quinones with nitrogen nucleophiles. This quinone
has a distinct absorption maximum at 380 nm (Figure
2, spectrum A) which facilitated the monitoring of the
reactions with glycine, L-arginine, L-histidine, and
L-lysine. All reactions were conducted in the presence
of a 99-fold excess of the amino acid, and all reactions
followed pseudo-first-order kinetics. The rate constants
for each reaction are rather small (Table 1) signifying
the lack of nucleophilicity of the amino acids at pH 7.4;
the protonated form of the amino group(s) will predomi-
nate under these conditions. A hypochromic shift of the
quinone absorbance at 380 nm was the only significant
change observed in the UV-vis spectrum at pH 7.4.
When the ratio of amino acid/quinone was reduced to 10:1
and the pH of the reaction was increased to 9.0, the
absorbance changes were more pronounced as shown in
Figure 3. The reaction of 2-(4′-chlorophenyl)-1,4-benzo-
quinone and glycine was monitored every 5 min for 45
min. The original quinone absorbance (380 nm) de-
creased while a concomitant hypsochromic shift from 380
+
+

Reactivity of PCB Quinones
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 627
F igu r e 3. UV-visible spectra depicting the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone with glycine at pH 9.0. The reaction
was monitored every 5 min for 45 min. The concentration of the quinone was 125 µM, while the concentration of glycine was 1250
µM, giving a 1:10 ratio. Arrows (at 340, 380, and 500 nm) indicate the wavelengths at which the greatest change in absorbance was
seen. The structures represent tautomers of the two major products of the reaction.
cycling with formation of reactive oxygen species. Both
effects would result in an increased oxidative stress (due
to depletion of radical scavengers and/or production of
free radicals in exposed cells) with possible damage to
cellular macromolecules (i.e., lipids, protein, or DNA) and
even cell death. Several studies have reported a reduc-
tion in hepatic GSH levels following PCBs administration
(22,23).
Hor ser a d ish P er oxid a se-Med ia ted Qu in on e F or -
m a tion in th e Absen ce a n d P r esen ce of SOD. Chlo-
rinated biphenyls may be hydroxylated to dihydroxy
compounds and subsequently oxidized to quinones by
various peroxidases present in cells (cf. Introduction).
F igu r e 4. Fast atom bombardment mass spectrum of the
Peroxidases are one-electron transfer enzymes which
6-glutathionyl-2-(4′-chlorophenyl)-1,4-hydroquinone.
oxidize catechols and hydroquinones with the abstraction
of 1 e- to yield semiquinones (24). Further oxidation to
Under reaction conditions optimized for the mono
quinones occurs by one of two routes. The semiquinone
can disproportionate to give the quinone and hydro-
quinone (eq 1) or semiquinones can react directly with
adducts (cf. Materials and Methods), the major product
of the reaction of 2-(4′-chlorophenyl)-1,4-benzoquinone
with glutathione shows only two protons present on the
quinone ring of the major isolated product leading to the
conclusion that a 1,4-Michael addition has taken place
on the quinone ring. The product exhibits a 2.9 Hz
coupling between the two protons on the quinone ring.
This is consistent with a 1,2,3,5-substitution pattern
where the two protons are meta- to each other.
PCB-hydroquinone/glutathionyl adduct(s) formed after
1,4-Michael addition behave chemically similar to other
GSH-hydroquinone adducts; these compounds are also
susceptible to oxidation and the formation of quinone-
thioethers (21). The reaction of PCB-derived quinoid
metabolites with GSH and the further oxidation of the
glutathionyl-hydroquinone adducts may result in a deple-
tion of GSH levels in exposed cells. Alternatively, (PCB-
quinone)-glutathione conjugates may participate in redox
2 semiquinone a quinone + hydroquinone (1)
oxygen (autoxidation), generating both the quinone and
superoxide anion radicals (eq 2).
semiquinone + O2 a quinone + O2- + H+ (2)
The oxidation of the 3,4-dichloro-3′,4′-dihydroxybiphe-
nyl and 3,4-dichloro-2′,5′-dihydroxybiphenyl by HRP/
H2O2 was monitored on the UV-vis spectrometer by
following the formation of the quinone over a 50 s period
(Figures 5 and 6, respectively). The initial experiment
was conducted under anaerobic conditions (exclusion of
oxygen) to establish a baseline rate of quinone formation.
+
+

628 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Amaro et al.
F igu r e 6. Formation of 4-(3′,4′-dichlorophenyl)-1,2-benzo-
quinone from 3,4-dichloro-3′,4′-dihydroxybiphenyl in the pres-
ence and absence of superoxide dismutase. The reaction was
monitored for 50 s. Results represent means + SD of three
experiments.
F igu r e 5. Formation of 2-(3′,4′-dichlorophenyl)-1,4-benzo-
quinone from 3,4-dichloro-2′,5′-dihydroxybiphenyl in the pres-
ence and absence of superoxide dismutase and oxygen. The
reaction was monitored for 50 s. Results represent means + SD
of three experiments.
In conclusion, these studies demonstrate that PCB
quinones react slowly with nitrogen nucleophiles, such
as glycine, L-arginine, L-histidine, and L-lysine, at physi-
ological pH. The sulfur nucleophiles, N-acetyl-L-cysteine
and glutathione, react instantaneously to form the cor-
responding hydroquinone adducts. These adducts may
be reoxidized and add a second moiety. Through these
mechanisms, PCB metabolites may bind to cellular
macromolecules and cause GSH depletion, oxidative
stress, and other quinone-mediated toxicities.
The experiment was repeated in the presence of atmo-
spheric oxygen, and there was no significant rate change.
This indicates that the forward reaction in eq 2 is
negligible unless SOD is added.
It has been previously reported that the rate of 1,4-
benzoquinone formation increased when SOD was added
(25) while there was no increase in 1,2-benzoquinone
formation from catechol in the presence of SOD (26). We
found a significant increase in the rate of chlorophenyl-
substituted 1,4-benzoquinone formation after SOD was
added under aerobic conditions as well as an increase in
1,2-benzoquinone formation using alkyl- and chlorophe-
nyl-substituted catechols as substrates. Apparently alkyl
or phenyl substituents on catechols enhance the rate of
1,2-benzoquinone formation. This trend has also been
described for alkyl-substituted 1,4-benzoquinones (27).
The reduction of oxygen is thermodynamically favor-
able for both the 1,2-benzoquinones and 1,4-benzoquino-
nes considering the reduction potential of O2/O2•- ) -179
mV at neutral pH (28) and the Ey2 of the Q/Q•- couple
for the 4-(3′,4′-dichlorophenyl)-1,2-benzoquinone and the
2-(3′,4′-dichlorophenyl)1,4-benzoquinone are -924 and
-1042 mV, respectively. However, the rate of 1,4-
benzoquinone formation was significantly increased,
whereas only a minor increase in 1,2-benzoquinone
formation was observed in the presence of SOD. If the
equilibrium in eq 1 favors disproportionation versus
comproportionation, then the overall rate should not be
significantly altered once SOD is added. This could
explain why the 1,2-benzoquinone formation was not
drastically altered during the first 30 s of the reaction
(Figure 6). Thus, 1,2-benzoquinones might undergo
disproportionation more readily, whereas comproportion-
ation of the hydroquinone and quinone is favored for the
1,4-benzoquinones.
Ack n ow led gm en t. A.R.A. and G.G.O. have contrib-
uted equally to this project, and both should be consid-
ered as first authors. This work is supported by NIH
Grant CA 57423. A.R.A. and G.G.O. are supported by
NIEHS Training Grant ES 07266. The authors thank
Dr. A. Daniel J ones from the Facility of Advanced
Instrumentation at the University of California, Davis
for his helpful comments and suggestions, Dr. J an Pyrek
of the University’s Life Sciences Mass Spectrometry
facility for the mass spectral analyses, Dr. J ohn P.
Selegue, Department of Chemistry, for helpful discus-
sions, and Wendell Neeley, Amanda Varner, Timothy
Twaroski, and Chris Girard for technical assistance.
Refer en ces
(1) Silberhorn, E., Glauert, H. P., and Robertson, L. W. (1990)
Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs.
Crit. Rev. Toxicol. 20, 439-496.
(2) J ensen, S. (1966) A new chemical hazard. New Sci. 32, 612.
(3) Mills, R. A., Millis, C. D., Dannan, G. A., Guengerich, F. P. and
Aust, S. D. (1985) Studies on the structure-activity relationships
for the metabolism of polybrominated biphenyls by rat liver
microsomes. Toxicol. Appl. Pharmacol. 78, 96-104.
(4) Koga, N., Beppu, M., Ishida, C., and Yoshimura, H. (1989) Further
studies on metabolism in vivo of 3,4,3′,4′-tetrachlorobiphenyl in
rats: identification of minor metabolites in rat feces. Xenobiotica
19, 1307-1318.
In separate experiments we have observed that 1,4-
and 1,2-quinones (HRP/H2O2 generated) participate in a
redox cycle with the production of O2-. The 1,4-benzo-
quinones exhibited the highest rate of reduction of
acetylated cytochrome c, whereas the 1,2, benzoquinones
showed only a slight or no redox activity.2 This would
agree with the fact that 1,2-benzoquinones do not readily
react with O2 while 1,4-benzoquinones can react directly
with O2, generating both the quinone and superoxide
anion radicals.
(5) Goto, M., Sugiura, K., Hattori, M., Miyagawa, T., and Okamura,
M. (1973) Hydroxylation of Dichlorobiphenyls in Rats. In New
Methods in Environmental Chemistry and Toxicology (Coulston,
F., Korte, F., and Goto, M., Eds.) pp 299-302, International
Academic Printing C., Ltd., Tokyo.
(6) Ariyoshi, N., Yoshimura, H., and Oguri, K. (1993) Identification
of in Vitro metabolites of 2,4,6,2′,4′,6′-Hexachlorobiphenyl from
Phenobarbital-Treated Dog Liver Microsomes. Biol. Pharm. Bull.
16, 852-857.
(7) Borlakoglu, J . T., Haegele, K. D., Reich, H. J ., Dils, R. R., and
Wilkins, J . P. G. (1991) In Vitro Metabolism Of [14C]4-Chlorobi-
phenyl and [14C] 2,2′,5,5′-Tetrachlorobiphenyl by Hepatic Mi-
crosomes from Rats and Pigeons. Evidence Against an Obligatory
Arene Oxide in Aromatic Hydroxylation Reactions. Int. J . Bio-
chem. 23, 1427-1437.
2
Unpublished results from our laboratory.
+
+

Reactivity of PCB Quinones
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 629
(8) Oakley, G. G., Robertson, L. W., and Gupta, R. C. (1996) Analysis
of polychlorinated biphenyl-DNA adducts by 32P-postlabeling.
Carcinogenesis 17, 109-114.
(9) McLean, M. R., Robertson, L. W., and Gupta, R. C. (1996)
Detection of PCB-adducts by the 32P-postlabeling technique.
Chem. Res. Toxicol. 9, 165-171.
(10) O’Brien, P. J . (1991) Molecular Mechanisms Of Quinone Cyto-
toxicity. Chem.-Biol. Interact. 80, 1-41.
(11) Bauer, U., Amaro, A. R., and Robertson, L. W. (1995) A New
Strategy for the Synthesis of Polychlorinated Biphenyl Metabo-
lites. Chem. Res. Toxicol. 8, 92-95.
(20) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988)
Spectroscopic Identification of ortho-Quinones as the Products of
Polycyclic Aromatic trans-Dihydrodiol Oxidation Catalyzed by
Dihydrodiol Dehydrogenase. J . Biol. Chem. 263, 1814-1820.
(21) Monks, T. J ., and Lau, S. S. (1992) Toxicology of Quinone-
Thioethers. Crit. Rev. Toxicol. 22, 243-270.
(22) Dogra, S., Filser, J . G., Cojocel, C., Greim, H., Regel, U., Oesch,
F., and Robertson, L. W. (1988) Long-term effects of commercial
and congeneric polychlorinated biphenyls on ethane production
and malondialdehyde levels, indicators of in vivo lipid peroxida-
tion. Arch. Toxicol. 62, 369-374.
(12) Achenbach, H., Waibel, R., Hefter-Bu¨bl, U., and Constelna, M.
A. (1993) Constituents of Fevillea Cordifolia: New Norcucurbi-
tacin And Cucurbitacin Glycosides. J . Nat. Prod. 56, 1506-1519.
(13) Brassard, P., and L’Ecuyer, P. (1958) L’Arylation Des Quinones
Par Les Sels De Diazonium. Can. J . Chem. 36, 700-708.
(14) J ellinck, P. H., and Irwin, L. (1963) Interaction of oestrogen
quinones with ethylene diamine. Biochim. Biophys. Acta 78, 778-
780.
(15) Eckert, K. G., Eyer, P., Sonnenbichler, J ., and Zetl, I. (1990)
Activation and detoxication of aminophenols. III. Synthesis and
structural elucidation of various glutathione addition products
to 1,4-benzoquinone. Xenobiotica 20, 351-361.
(16) Ononye, A. I., Graveel, J . G., and Wolt, J . D. (1989) Kinetic and
spectroscopic investigations of the covalent binding of benzidine
to quinones. Environ. Toxicol. Chem. 8, 303-308.
(17) Ononye, A. I., and Graveel, J . G. (1994) Modeling the reactions
of 1-naphthylamine and 4-methylaniline with humic acids: Spec-
troscopic investigations of the covalent linkages. Environ. Toxicol.
Chem. 13, 537-541.
(23) Garthoff, L. H., Friedman, L., Farber, T., Locke, K., Sobotka, T.,
Green, S., Hurlen, N. E., Peters, E., Story, G., Moreland, F. M.,
Graham, C., Keys, J ., Taylor, M. J ., Rothlein, J ., and Sporn, E.
(1977) Biochemical and cytogenetic effects in rats caused by short-
term ingestion of Aroclor 1254 or Firemaster BP-6. Toxicol. and
Environ. Health 3, 769-796.
(24) Hollenberg, P. F. (1992) Mechanisms of Cytochrome P450 and
Peroxidase-catalyzed Xenobiotic Metabolism. FASEB J . 6, 689-
694.
(25) Sawada, Y., Iyanagi, T., and Yamazaki, I. (1975) Relation between
Redox Potentials and Rate Constants in Reactions Coupled with
the System Oxygen-Superoxide. Biochemistry 14, 3761-3764.
(26) Sadler, A., Subrahmanyam, V. V., and Ross, D. (1988) Oxidation
of Catechol by Horseradish Peroxidase and Human Leukocyte
Peroxidase: Reactions of o-Benzoquinone and o-Benzosemiquino-
ne. Toxicol. Appl. Pharmacol. 93, 62-71.
(27) Brunmark, A., and Cadenas, E. (1988) Reductive addition of
glutathione to p-benzoquinone, 2-hydroxy-p-benzoquinone, and
p-benzoquinone epoxides. Effect of the hydroxy- and glutathionyl
substituents on p-benzohydroquinone autoxidation. Chem.-Biol.
Interact. 68, 273-298.
(18) Murty, V. S., and Penning, T. M. (1992) Polycyclic Aromatic
Hydrocarbon (PAH) Ortho-Quinone Conjugate Chemistry: Kinet-
ics of Thiol Addition to PAH Ortho-Quinones and Structures of
Thioether Adducts of Naphthalene-1,2-Dione. Chem.-Biol. Inter-
act. 84, 169-188.
(19) Tabakovic, K., and Abul-Hajj, Y. J . (1994) Reaction of Lysine with
Estrone 3,4-o-Quinone. Chem. Res. Toxicol. 7, 696-701.
(28) Wardman, P. (1991) The reduction potential of benzyl viologen:
an important reference compound for oxidant/radical redox
couples. J . Free Radicals Res. Commun. 14, 57-67.
TX950117E
+
+