Development of polyclonal antibodies for detection of protein modification by 1,2-naphthoquinone

Article abstract of DOI:10.1021/tx960014b

Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with naphthalene. To further identify the target proteins of the naphthoquinone, we raised polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 μM at 37 °C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride) microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 μM 1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was observed in the lane loaded with the protein sample incubated with 1.0μM 1,2-naphthoquinone, and many chemiluminescent bands at a wide range of molecular weights were observed in the lane loaded with the protein sample incubated with 10 μM quinone. As expected, no chemiluminescent bands were detected on the membrane blotted with the proteins exposed to vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone cysteine adducts and developed immunostaining to detect protein modification by 1,2-naphthoquinone.

Full text of DOI:10.1021/tx960014b

904
Chem. Res. Toxicol. 1996, 9, 904-909
Develop m en t of P olyclon a l An tibod ies for Detection of
P r otein Mod ifica tion by 1,2-Na p h th oqu in on e
J iang Zheng and Bruce D. Hammock*
Departments of Entomology and Environmental Toxicology, University of California,
Davis, California 95616
Received J anuary 23, 1996X
Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification
is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was
found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with
naphthalene. To further identify the target proteins of the naphthoquinone, we raised
polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high
titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA
showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine
adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as
well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated
mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 µM at 37
°C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western
blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands
developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride)
microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 µM
1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was
observed in the lane loaded with the protein sample incubated with 1.0 µM 1,2-naphthoquinone,
and many chemiluminescent bands at a wide range of molecular weights were observed in the
lane loaded with the protein sample incubated with 10 µM quinone. As expected, no
chemiluminescent bands were detected on the membrane blotted with the proteins exposed to
vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone
cysteine adducts and developed immunostaining to detect protein modification by 1,2-
naphthoquinone.
is proposed to bind covalently to nucleophilic amino acid
residues of cellular proteins. In addition, the resulting
epoxide metabolite is spontaneously and enzymatically
hydrolyzed by epoxide hydrolase to dihydrodiol and
further oxidized to 1,2-dihydroxynaphthalene (1,2-naph-
thohydroquinone) by dihydrodiol dehydrogenase. Alter-
natively, naphthalene oxide is chemically rearranged to
1-naphthol which can then be hydroxylated to 1,2- and
1,4-dihydroxynaphthalenes (naphthohydroquinones).
These hydroquinone metabolites of naphthalene are
readily oxidized to the corresponding naphthoquinones
which possess high chemical reactivity with nucleophiles
of intracellular proteins forming protein adducts. Re-
cently we identified 1,2-naphthoquinone as the major
quinone metabolite of naphthalene which covalently
conjugates to cysteine residues of intracellular proteins
after Clara cells are incubated with naphthalene. In
addition to 1,2-naphthoquinone, epoxide metabolite of
naphthalene was found to bind covalently to cysteine
residues of Clara cell proteins (11).
In tr od u ction
Naphthoquinones, which are known as electrophilic
species, have been reported to be toxic to hepatocytes in
vitro. Blebbing on cellular surface along with a dose-
dependent decrease in intracellular glutathione is ob-
served after isolated hepatocytes are exposed to naph-
thoquinones (1). Arylation of nucleophilic amino acid
residues such as cysteine of cellular proteins by naph-
thoquinones has been suggested to be an important
mechanism of quinone-induced cytotoxicity (2-4). Pes-
sah and co-workers demonstrated that naphthoquinones
caused activation of Ca2+-induced Ca2+ release channel
and suggested that the cytotoxicity of quinones may be
related to alternation of normal cellular Ca2+ regulation
(5, 6). Naphthoquinones have been suggested to be toxic
reactive metabolites of naphthalene. Naphthalene is an
important environmental pollutant (7) and has been
reported to produce selective necrosis of lung Clara cells
in the mouse (8, 9). Covalent binding of cellular proteins
by reactive metabolites of naphthalene is associated with
naphthalene-induced cytotoxicity (10). The pathway of
the formation of reactive metabolites of naphthalene is
proposed as shown in Scheme 1. Naphthalene metabo-
lism is initiated by the formation of naphthalene oxide
mediated by cytochrome P-450. The epoxide metabolite
Immunoblotting of protein adducts of reactive metabo-
lites has been successfully developed to identify target
cellular proteins modified by reactive metabolites and to
investigate the toxicological importance of protein modi-
fication in experimental animals and in humans pre-
exposed to acetaminophen (12) and halothane (13). In
addition, the antibodies prepared for immunoblotting can
be used for the development of enzyme-linked immuno-
sorbent assays (ELISAs)1 to monitor urinary mercapturic
acid metabolites (14) and plasma protein adducts (15)
derived from the reactive metabolite as biomarkers of
* Address correspondence to this author at: Departments of Ento-
mology and Environmental Toxicology, University of California, Davis,
CA 95616. Tel: (916) 752-7519; FAX: (916) 752-1537; E-mail:
bdhammock@ucdavis.edu.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0893-228x(96)00014-8 CCC: $12.00 © 1996 American Chemical Society

Antibody and 1,2-Naphthoquinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 905
Syn th esis. 1,2-Na p h th oqu in on e (1). 1,2-Naphthoquinone
was prepared by the method Fieser (16). Briefly, aminonaphthol
(1.5 g) was dissolved in 100 mL of 0.5 N HCl, and the insoluble
solid was discarded by filtration. To the solution 15 mL of 10%
(w/v) FeCl3 dissolved in 3 N HCl was dropwise added over 10
min, with vigorous stirring at room temperature. The mixture
was stirred at room temperature for 30 min, and the resulting
brownish crystals were recrystallized in hexane and ethyl
acetate, giving bright orange crystals.
Sch em e 1
N-Acetyl-S-(1,2-d ih yd r oxy-4-n a p h th yl)cystein e (2). N-
Acetylcysteine (50 mg, 0.3 mmol) was dissolved in 2.0 mL of
water which had been purged with nitrogen gas. 1,2-Naphtho-
quinone (31 mg, 0.2 mmol) was dissolved in 1.0 mL of nitrogen-
purged acetone and added dropwise to the cysteine solution
under nitrogen. After 30 min stirring at room temperature, the
acetone was removed by rotary evaporation. The resulting
mixture was injected into HPLC for purification. The chroma-
tography was performed using a gradient of 20-80% acetonitrile
in water with 0.1% of trifluoroacetic acid. The desired fractions
were collected and pooled. The acetonitrile was removed by
evaporation with a stream of nitrogen, and the remaining
aqueous fraction was evaporated by lyophylization, giving a
brownish solid. 1H-NMR (DMSO) δ 2.00 (s, 3H, CH3CONH),
3.46 (dd, J ) 8.7, 13.8 Hz, 1H, SCHaHbCH, diastereomers), 3.74
(dd, J ) 4.8, 13.8 Hz, 1H, SCHaHbCH, diastereomers), 4.4 (m,
1H, SCH2CH), 4.9 (b, 2H, H hydroxyl), 6.62 (s, H aromatic), 7.63
(t, J ) 7.5 Hz, 1 H, H aromatic), 7.75 (t, J ) 7.5 Hz, 1H, H
aromatic), 7.93 (t, J ) 7.8 Hz, 1H, H aromatic), 8.12 (d, J ) 7.5
Hz, 1H, H aromatic). ESIMS: 322 (M + 1)+.
N-Acetyl-S-(1,4-d ih yd r oxy-2-n a p h th yl)cystein e (3). The
procedure was similar to the synthesis of 2 as described above.
N-Acetylcysteine (50 mg, 0.3 mmol) was reacted with 1,4-
naphthoquinone (31 mg, 0.2 mmol). The product was purified
by reverse phase HPLC. 1H-NMR (DMSO) δ 1.74 (s, 3H, CH3-
CONH), 2.92 (dd, J ) 8.7, 13.2 Hz, 1H, SCHaHbCH, diastere-
omers), 3.10 (dd, J ) 4.5, 13.2 Hz, 1H, SCHaHbCH, diastereo-
mers), 3.7 (b, 2H, H hydroxyl), 4.2 (m, 1H, SCH2CH), 6.70 (s,
1H, H aromatic), 7.4 (m, 2H, H aromatic), 7.9 (m, 2H, H
aromatic). ESIMS: 322 (M + 1)+.
human exposure to potential toxins. The objective of this
study was to prepare polyclonal antibodies and to develop
immunoblotting used as a probe to detect proteins
modified by 1,2-naphthoquinone. This antibody-based
technique will facilitate our investigation on the mech-
anism of naphthalene-induced cytotoxicity.
N -Ace t yl-S -(1,2-d ih yd r o-1-h yd r oxy-1-n a p h t h yl)cys-
tein e (4). Compound 4 was synthesized by the reaction of
naphthalene oxide with N-acetylcysteine as described by Marco
and co-workers (14).
Keyh ole Lim p et Hem ocya n in -1,2-Na p h th oqu in on e Ad -
d u ct (Im m u n ogen I). Water-soluble keyhole limpet hemocya-
nin (KLH, 20 mg) was dissolved in 1.0 mL of 4.0 M guanidinum
chloride, followed by addition of 20 mg of dithiothreitol. The
mixture was stirred under nitrogen at room temperature in the
dark overnight. The reduced proteins were loaded to a G-25
gel filtration column (1.0 × 10 cm), and the column was eluted
with deionized water. The fractions containing protein were
identified by a UV spectrometer at 254 nm and pooled as
desired. To the protein solution, 1,2-naphthoquinone (3.0 mg)
dissolved in 0.5 mL of acetone was added under nitrogen. After
stirring for 20 min, the mixture was washed with ethyl acetate
and dried by lyophilization.
Exp er im en ta l Section
Ch em ica ls a n d In str u m en ts. Chemical reagents and
solvents were purchased from Aldrich Chemical Co. (Milwaukee,
WI). Mouse albumin, conalbumin, N-acetylcysteine, and im-
munochemicals were obtained from Sigma Chemical Co. (St.
Louis, MO). Water-soluble keyhole limpet hemocyanin (KLH)
and N-succinimidyl (3(2)-pyridyldithio)propionate (SPDP) were
purchased from Pierce (Rockford, IL). HPLC analysis and
purification of synthetic chemicals were performed using a
Varian 9010 solvent delivery system equipped with a Varian
9050 UV-vis detector. A reverse phase C18 chromatography
column (250 × 4.6 mm) purchased from Vydac (Hesperia, CA)
was used for HPLC analysis and purification of synthetic
compounds. 1H-NMR spectra were obtained from a QE-300
spectrometer (General Electric, 300 MHz). Chemicals were
analyzed on a VG/Fisons Quattro-BQ triple quadruple mass
spectrometer (VG Biotech, Altrincham, U.K.) using 1:1 CH3CN/
H2O together with 1.0% formic acid as the mobile phase.
Spectra were obtained in positive mode. Immunoassays were
performed with polystyrene 96-well microtiter plates purchased
from Nunc (Maxisorb, Roskilde, Denmark). The absorbances
were read with a Vmax microplate reader (Molecular Devices,
Menlo Park, CA). The curve fitting was performed using
SigmaPlot.
Th iola ted KLH-1,2-Na p h th oqu in on e Ad d u ct (Im m u -
n ogen II). Water-soluble KLH (20 mg) was dissolved in 1.0
mL of water, followed by addition of 2.0 mg of N-succinimidyl
(3(2)-pyridyldithio)propionate (SPDP) dissolved in 50 µL of
dimethyl sulfoxide. After 1.5 h stirring at room temperature,
25 mg of dithiothreitol dissolved in 50 µL of water was added
to the mixture. The reduction was performed under nitrogen,
with stirring at room temperature for 30 min. The mixture was
loaded onto a G-25 gel filtration column (1.0 × 10 cm), and the
column was eluted with deionized water. The fractions contain-
ing protein were collected and pooled. The reduced protein was
immediately reacted with 3.0 mg of 1,2-naphthoquinone dis-
solved in 0.5 mL of acetone as described above in the preparation
of immunogen I. The resulting protein adducts were washed
with ethyl acetate and lyophilized. The lyophilization of the
protein adducts gave a brownish powder.
1
Abbreviations: ELISAs, enzyme-linked immunosorbent assays;
HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; SPDP,
N-succinimidyl (3(2)-pyridyldithio)propionate.

906 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Zheng and Hammock
Mou se Albu m in -1,2-Na p h th oqu in on e (Coa tin g An ti-
gen ). Mouse serum albumin (20 mg) was reduced by dithio-
threitol as described above to reduce disulfides of the protein.
The reduced mouse albumin was separated from the reducing
agent by gel filtration and immediately reacted with 3.0 mg of
1,2-naphthoquinone using a similar procedure for the arylation
of proteins as described above. The protein adducts were
purified by gel filtration and dried by lyophilization.
Sch em e 2
Im m u n iza tion of th e Ra bbits. Female New Zealand White
rabbits (Herbert’s Rabbitry, Plymouth, CA) weighing 2.5-3.0
kg were immunized with either immunogen I or II. The
immunogen (100 µg) was dissolved in 0.5 mL of PBS buffer (pH
) 7.4), followed by emulsification with 0.5 mL of Freund’s
complete adjuvant. The rabbits were injected subcutaneously
with the emulsion (1.0 mL/rabbit) at multiple sites in the back.
After 4 weeks these animals were boosted several times with a
interval of four weeks by the same procedure except that
Freund’s incomplete adjuvant was used in place of Freund’s
complete adjuvant. These rabbits were boosted until no further
increase in antibody titer was observed.
An a lysis of Titer . The titer of the serum obtained from the
rabbits was determined by measuring the binding of serial
dilutions (1/100 to 1/128 000) to microtiter plates coated with
several concentrations of mouse albumin-1,2-naphthoquinone
adducts. Optimal concentration for the coating antigen and
antisera dilution were determined (17).
Com p etitive En zym e Im m u n osor ben t Assa y. To each
well of 96-well microtiter plates, 100 µL of coating antigen
mouse albumin-1,2-naphthoquinone at 2.0 µg/mL (200 ng/well)
in PBS buffer (pH 7.4) was added and incubated at 4 °C
overnight covered with adhesive plate seals. Serial dilutions
of the analytes were prepared in PBST buffer (0.2 M phosphate-
buffered saline solution containing 0.05% Tween-20, pH 7.4)
with 1% ethanol. The resulting analyte solution was mixed (1:1
v/v) with primary antisera (1/1000 dilution) in 2% nonfat milk
dissolved in PBST buffer, followed by incubation at 4 °C
overnight. The following day the plates were washed 5 times
with PBST buffer and blocked by addition of 1.0% nonfat milk
in PBST buffer (120 µL/well) and incubation for 1.0 h at room
temperature. The plates were washed as described before. The
preincubated antiserum with analytes at various concentrations
was added to the plates (100 µL/well) and incubated at room
temperature for 2.0 h. After the same process of washing, 100
µL of a 1/12 000 diluted goat anti-rabbit IgG-horseradish
peroxidase (HRP) solution was added to each well. The mixture
was incubated for an additional 1 h, and after another wash
step, 100 µL of a substrate solution containing 0.3 mM tetra-
methylbenzidine and 0.1 mM H2O2 in 0.1 M acetate buffer (pH
) 5.5) was added and incubated at room temperature for 30
min, followed by addition of 50 µL of a 4 M sulfuric acid solution
to quench colorimetric development. The absorbance at dual
wavelength (450 - 650 nm) was read.
with horseradish peroxidase-conjugated goat anti-rabbit IgG
antibodies (1/8000 in PBST buffer, Sigma). The blotted protein
bands were detected by chemiluminescence using ECL Western
blotting kits (Amersham International plc, England).
Resu lts a n d Discu ssion
Previous studies (10) have shown that cysteine resi-
dues of cellular proteins were modified by the 1,2-
naphthoquinone metabolite of naphthalene after isolated
mouse Clara cells were exposed to naphthalene (0.5 mM)
at 37 °C for 1.0 h. This finding led to identification of
the cellular proteins chemically modified with the naph-
thoquinone metabolite of naphthalene as a step in
elucidation of the mechanism of naphthalene-induced
cytotoxicity. Western blotting and immunostaining tech-
niques (12) have been used as powerful tools to identify
protein adducts by detecting the hapten moiety of protein
adducts formed during the exposure of cellular proteins
to electrophilic species (haptens). In an attempt to
identify target proteins of the 1,2-quinone metabolite of
naphthalene, we took advantage of immunoblotting as a
detection tool by using polyclonal antibodies which can
recognize the 1,2-naphthohydroquinone moiety of pro-
teins modified with 1,2-naphthoquinone. As the first step
of antibody preparation, immunogens (I and II) were
designed and synthesized. For the preparation of im-
munogen I, free sulfhydryl groups of carrier protein KLH
were generated by reduction with dithiothreitol and
immediately reacted with 1,2-naphthoquinone. Immu-
nogen II was synthesized as shown in Scheme 2. Water-
soluble KLH was thiolated by reacting with SPDP, and
the resulting disulfide products were reduced by dithio-
threitol to thiols, which immediately reacted with 1,2-
naphthoquinone. As shown in Figure 1, immunogen II
is a lysine derivative which mimics the structure of 1,2-
naphthoquinone cysteine residue adducts (immunogen I)
except for the lack of acylamido substitution. The side
In Vitr o Stu d y. Freshly harvested mouse liver which had
been perfused with diluted heparin saline solution to remove
blood was homogenated using a tissue blender. The resulting
homogenates were centrifuged at 1000g for 30 min. The pellet
was discarded, and the supernatants (5.0 mg of protein/mL)
were incubated with 1,2-naphthoquinone at 37 °C at concentra-
tions of 1.0 or 10 µM for 1.0 h, then frozen and stored at -80 °C
for gel electrophoretic separation and immunoblotting.
Electr op h or esis a n d Im m u n oblottin g. The protein bands
resolved by SDS-polyacrylamide gel electrophoresis (Mini-
Protean II, Bio-Rad) according to Laemmli (18) using 3.3%
stacking and 12% resolving gels were transferred to poly-
(vinylidene difluoride) microporous membranes (Immobilon-P
transfer membranes, Millipore) by an electroblotter (LKB
Novablot electrophoretic transfer kit). After 3.0 h transferring
(43 mA), blots were blocked by shaking overnight in 5% nonfat
dry milk in PBST buffer. The blotted poly(vinylidene difluoride)
membranes were incubated for 1.0 h with a 1/4000 dilution of
primary rabbit antiserum in PBST buffer with 3% nonfat milk
in the absence or presence of N-acetyl-S-(1,2-dihydroxy-4-
naphthyl)cysteine. The immunoblots were incubated for 1.0 h

Antibody and 1,2-Naphthoquinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 907
F igu r e 3. Analysis of N-acetyl-S-(1,2-dihydroxy-4-naphthyl)-
cysteine in a competitive ELISA using an antiserum raised
against immunogen I in a microtiter plate coated with the 1,2-
naphthoquinone adduct on mouse serum albumin. Error bars
represent standard deviation of separate runs on different days.
The values are mean ( SD for triplicate assays.
F igu r e 1. Chemical structures of immunogens, coating anti-
gen, and compound 2 used for immunization and ELISAs.
KLH: keyhole limpet hemocyanin; MSA: mouse serum albu-
min.
using heterologous coating antigen for the titration test.
In addition, both antisera showed very low affinity
toward native mouse serum albumin, the carrier protein
of the coating antigen.
Competitive ELISA experiments were conducted to
characterize the antibodies. Both antisera raised against
immunogen I and II were diluted to 1/4000 and used for
the ELISA in plates coated with the same coating antigen
as the one for the titration tests, and N-acetyl-S-(1,2-
dihydroxy-4-naphthyl)cysteine, 2, employed as an ana-
lyte. Optical density measured at 450 nm minus 650 nm
was plotted against the logarithm of the various concen-
trations of the analyte. As demonstrated in Figure 3, a
sigmoid curve was obtained from the competitive ELISA
using the antiserum raised against immunogen I. The
resulting curve was fitted by a four-parameter logistic
equation. An I50 of 2.82 nM was observed with A/D )
61.5, slope ) 0.98, and r ) 0.987 (A: maximal absor-
bance; D: noise absorbance; r: regression coefficient). In
addition, the competition test showed that the presence
of 1.0 µM analyte 2 inhibits 98% of the binding of the
antiserum to the coating antigen. The remaining 2.0%
of the binding unable to be reversed by the analyte may
result from nonspecific binding. This may indicate that
1,2-naphthoquinone reacted with cysteine residues rather
than other nucleophilic amino acid residues of proteins
in the synthesis of the immunogen and the coating
antigen. Interestingly, no competition by N-acetyl-S-(1,2-
dihydroxy-4-naphthyl)cysteine, analyte 2, was observed
using the antiserum generated against immunogen II,
although the antiserum was found to recognize the
coating antigen mouse albumin 1,2-naphthoquinone ad-
duct in the titration tests (Figure 2). It is unknown why
analyte 2 did not reverse the recognition of the coating
antigen by the antiserum raised against immunogen II.
The explanation is likely that the antibodies raised
against immunogen II recognize the thionaphthohydro-
quinone moiety of the coating antigen. However, the
cavity size of the antibody binding sites is not big enough
to hold analyte 2. It thus is unable to displace the coating
antigen from the antibodies.
F igu r e 2. Titration tests of antisera raised against immunogen
I (0 and 9) and immunogen II (O and b) in a microtiter plate
coated with 1,2-naphthoquinone mouse albumin adduct (9 and
b) and native mouse albumin (0 and O). The values are mean
( SD for triplicate assays.
chain of lysine residues supplies a spacer between the
carrier protein KLH and 1,2-naphthohydroquinone.
Mouse albumin 1,2-naphthoquinone adduct used as the
coating antigen was prepared by conjugating 1,2-naph-
thoquinone to mouse albumin which had been treated
with dithiothreitol similarly as described in the prepara-
tion of immunogen I.
Immunizations of rabbits were carried out with im-
munogens I and II. Using a checkerboard titration, the
antisera of each animal were tested against mouse
albumin 1,2-naphthoquinone adduct after every bleeding
until no increase in the titer was observed. As shown in
Figure 2, the antiserum raised against immunogen I
showed a higher titer than the one prepared by the
immunization with immunogen II. The low titer of
antiserum raised against immunogen II may result from

908 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Zheng and Hammock
Ta ble 1. I50 Va lu es, Cr oss Rea ctivities, a n d Slop es for
An a lytes
F igu r e 4. Immunochemical detection of 1,2-naphthoquinone-
proteins in mouse liver homogenates incubated with vehicle
(lane 1), 1.0 µM 1,2-naphthoquinone (lane 2), or 10 µM 1,2-
naphthoquinone (lane 3). Each gel lane was loaded with 2.0 µg
of protein sample.
As shown in Scheme 1, naphthalene oxide, another
reactive metabolite of naphthalene, has been found to
modify cysteine residues of cellular proteins of Clara cells
after exposure to naphthalene (11). Additionally, 1,4-
naphthoquinone has been suggested to be another qui-
none metabolite of naphthalene and likely to react with
cysteine residues to form protein adducts (1, 2). To
determine the cross reactivity of the antibodies toward
naphthalene oxide and 1,4-naphthoquinone modified
proteins, we conducted competitive ELISA of N-acetyl-
S-(1,4-dihydroxy-2-naphthyl)cysteine (3) and N-acetyl-
S-(1,2-dihydro-1-hydroxy-1-naphthyl)cysteine (4) as com-
petitive analytes, using the antiserum raised against
immunogen I. As expected, very weak cross reactivity
(less than 0.3%) was observed to these two N-acetylcys-
teine adducts relative to the hapten (Table 1). Addition-
ally, we ran competitive ELISA of N-acetylcysteine, 5,
as a competitive analyte using the same antiserum, and
only 0.01% of cross reactivity was observed as shown in
Table 1. This suggests that an intact structure of 1,2-
naphthoquinone cysteine adduct is required for hapten
recognition by the antibodies.
Figu r e 5. Competitive immunostaining of 1,2-naphthoquinone-
proteins in mouse liver homogenates incubated with 10 µM 1,2-
naphthoquinone in the presence of vehicle (lane 3) or 1.0 µM
(lane 2) or 10 µM (lane 1) N-acetyl-S-(1,2-dihydroxy-4-naphthyl)-
cysteine, analyte 2. Each lane was loaded with 2.0 µg of protein
sample.
processed in the same manner in the absence of 1,2-
naphthoquinone. This suggests that the antibodies are
able to detect cellular proteins modified by 1,2-naphtho-
quinone and show no cross reaction toward unmodified
native cellular proteins.
In an effort to develop immunostaining for the detec-
tion of 1,2-naphthoquinone protein adducts using the
antiserum described above, protein samples of mouse
liver homogenate were exposed to vehicle or 1,2-naph-
thoquinone at concentrations of 10 µM (20 nmol of
quinone/mg of protein) and 1.0 µM (2.0 nmol of quinone/
mg of protein), and the resulting protein samples were
separated by SDS-PAGE, followed by blotting to an
Immobilon-P transfer membrane. The blotted membrane
was incubated with the antiserum raised against immu-
nogen I and consecutively incubated with goat anti-rabbit
IgG-horseradish peroxidase. The resulting antibody-
incubated membranes were treated with ECL chemilu-
minescence kit. As shown in Figure 4, many chemilu-
minescent bands at a wide range of molecular weights
were observed in the lane loaded with the protein sample
incubated with 10 µM 1,2-naphthoquinone. However,
only one chemiluminescent band at a molecular weight
of 22 kDa was observed in the lane loaded with the
protein sample incubated with 1.0 µM quinone. As
expected, no chemiluminescent bands were observed in
the lane loaded with control protein sample which was
Competitive immunostaining was conducted to deter-
mine the specificity of antibody recognition of the modi-
fication at cysteine residues of proteins by 1,2-naphtho-
quinone. The Immobilon-P transfer membranes blotted
with mouse liver proteins which had been exposed to 10
µM 1,2-naphthoquinone were incubated with a 1/4000
dilution of the antiserum raised against immunogen I
in the absence or presence of N-acetyl-S-(1,2-dihydroxy-
4-naphthyl)cysteine (2) at various concentrations. As
Figure 5 shows, the presence of 1.0 µM analyte 2 in the
incubation of the blotted membrane with the antiserum
partially inhibited the immunostaining of the protein
adducts, and the presence of 10 µM analyte 2 completely
prevented the antibodies from binding to the protein
adducts. This indicates that the binding of the antibodies
to 1,2-naphthoquinone protein adducts attributes the
immunorecognition of the cysteine residue modification
by the naphthoquinone. This antiserum would be a
useful tool to detect protein modification by 1,2-naph-
thoquinone and facilitate the investigation of the mech-
anism of naphthoquinone- and naphthalene-induced
cytotoxicity.

Antibody and 1,2-Naphthoquinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 909
(6) Liu, G., Abramson, J . J ., Zable, A. C., and Pessah, I. N. (1994)
Direct evidence for the existence and functional role of hyper-
reactive sulfhydryls on the ryanodine receptor-triadin complex
selectively labeled by the coumarin maleimide 7-diethylamino-
3-(4′-maleimidylphenyl)-4-methylcoumarin. Mol. Pharmacol. 45,
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(7) Franklin, R. B., Plopper, C. G., and Buckpitt, A. R. (1993)
Naphthalene and 2-methyl naphthalene-induced pulmonary bron-
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toxicity. In Metabolic Activation and Toxicity of Chemical Agents
to Lung Tissues and Cells (Gram, T. E., Ed.) pp 123-144,
Pergamon Press, Oxford, United Kingdom.
In summary, we have successfully raised polyclonal
antibodies to detect 1,2-naphthoquinone cysteine adduct.
These antibodies are able to detect 1,2-naphthoquinone-
modified cellular proteins with no cross reactivity toward
native cellular proteins, providing a powerful tool for the
investigation of the mechanism of cytotoxicity induced
by 1,2-naphthoquinone and naphthalene. In addition,
the antibodies could be used for ELISAs to detect urinary
1,2-naphthoquinone-derived mercapturic acid as a bio-
marker of human exposure to naphthalene.
(8) Plopper, C. G., Suverkropp, C., Morin, D., Nishio, S., and Buckpitt,
A. (1992) Relationship of cytochrome P-450 activity to Clara cell
cytotoxicity. I. Histopathologic comparison of the respiratory tract
of mice, rats and hamsters after parenteral administration of
naphthalene. J . Pharmacol. Exp. Ther. 261, 353-63.
(9) Plopper, C. G., Macklin, J ., Nishio, S. J ., Hyde, D. M., and
Buckpitt, A. R. (1992) Relationship of cytochrome P-450 activity
to Clara cell cytotoxicity. III. Morphometric comparison of changes
in the epithelial populations of terminal bronchioles and lobar
bronchi in mice, hamsters, and rats after parenteral administra-
tion of naphthalene. Lab. Invest. 67, 553-65.
Ack n ow led gm en t. We thank Dr. A. Daniel J ones for
his technical assistance to run mass spectrometry and
Dr. Alan R. Buckpitt for his helpful suggestions. This
work was supported by NIEHS Grant ES02710 and the
UC Davis NIEHS Superfund Basic Research Program
(2P42 ES04699). UC Davis is a NIEHS Health Sciences
Center (ES05707) and an EPA Center for Ecological
Health Research (CR819658).
(10) Warren, D. L., Brown, D. L., J r., and Buckpitt, A. R. (1982)
Evidence for cytochrome P-450 mediated metabolism in the
bronchiolar damage by naphthalene. Chem.-Biol. Interact. 40,
287-303.
(11) Zheng, J ., Cho, M., Chichester, C., Hammock, B., Buckpitt, A.,
Plopper, C., and J ones, D. (1994) Epoxide and quinone metabolites
of naphthalene covalently bound to sulfur nucleophiles of proteins.
The Toxicologist 14, 429.
Su p p or tin g In for m a tion Ava ila ble: Supporting informa-
tion of the analysis of N-acetyl-S-(1,4-dihydroxy-2-naphthyl)-
cyteine (3), N-acetyl-S-(1,2-dihydro-1-hydroxy-1-naphthyl)-
cysteine (4), and N-acetylcysteine (5) in a competitive ELISA
using an antiserum raised against immunogen I in a microtiter
plate coated with the 1,2-naphthoquinone adduct on mouse
serum albumin (3 pages) is available. Ordering information can
be found on any current masthead.
(12) Hinson, J . A., Pumford, N. R., and Roberts, D. W. (1995)
Mechanisms of acetaminophen toxicity: immunochemical detec-
tion of drug-protein adducts. Drug Metab. Rev. 27, 73-92.
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