Reaction of trichloroethylene oxide with proteins and DNA: Instability of adducts and modulation of functions

Article abstract of DOI:10.1021/tx000185n

Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N⁶-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t_1/2 of ～1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), α-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts (～2%) was detected in the direct reaction of TCE oxide with 2′-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t_1/2 of 30 min at pH 8.5. These results indicate the transient nature of the adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.

Full text of DOI:10.1021/tx000185n

54
Chem. Res. Toxicol. 2001, 14, 54-61
Rea ction of Tr ich lor oeth ylen e Oxid e w ith P r otein s a n d
DNA: In sta bility of Ad d u cts a n d Mod u la tion of
F u n ction s
Hongliang Cai† and F. Peter Guengerich*
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-0146
Received August 23, 2000
Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result
of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed
N6-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and
Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to
measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein
amino acid adducts were directly observed by mass spectrometry during the reaction of TCE
oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues
1-24). The majority (80%) of the protein adducts were unstable under physiological conditions
and had a collective t1/2 of ∼1 h, suggesting that they are ester type adducts formed from
reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis.
Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0,
respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct
formation on catalytic activities were examined with five model enzymes. No recovery of
catalytic activity was observed during the reaction of TCE oxide with two model enzymes for
which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate
dehydrogenase. However, in the cases of papain (essential Cys residue in the active site),
R-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues),
time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide
or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride),
paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts
(∼2%) was detected in the direct reaction of TCE oxide with 2′-deoxyguanosine, but not with
the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic
8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However,
the adducts had a t1/2 of 30 min at pH 8.5. These results indicate the transient nature of the
adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.
(3-5). Three major hypotheses have been developed to
account for the rodent carcinogenicity: genotoxicity of
TCE oxidation products (6), genotoxicity of GSH conju-
gates (4, 7), and peroxisome proliferation by the oxidation
products Cl2CHCO2H and Cl3CCO2H (8, 9). Recently,
high TCE exposure (either occupationally or environ-
mentally) has been suggested to be related to the
development of immune system dysfunction and autoim-
mune disorders in humans (10-12). Currently, TCE is
undergoing a thorough evaluation and risk assessment
by the National Center for Environmental Assessment
of the United States Environmental Protection Agency
(13).
In tr od u ction
1,1,2-Trichloroethylene (TCE)1 is a volatile organic
solvent that is widely used as a degreasing agent, a
polymer precursor, and a dry cleaning agent. It has been
estimated that approximately 34% of the drinking water
supplies tested in the United States have TCE contami-
nation (1). Because of its widespread industrial use and
improper disposal, TCE has become a major environ-
mental pollutant and is one of the most abundant organic
contaminants found in Superfund sites (1, 2). TCE, like
other chlorinated ethylenes, can generate toxic effects
and induces liver, lung, and kidney tumors in rodents
TCE is metabolized via two general pathways, an
oxidative pathway in which the first step is catalyzed by
P450 and a GSH-dependent pathway in which the initial
step is catalyzed by GSH transferase (14). Only a small
fraction of TCE is metabolized by the GSH conjugation
pathway, primarily by GSH transferase in the liver,
followed by further metabolism in the kidney (14). Key
oxidative metabolites of the P450 pathway, including
Cl3CCHO [chloral (hydrate)], Cl3CCO2H, and Cl2CHCO2H,
are thought to be critical for development of toxicity or
* To whom correspondence should be addressed: Department of
Biochemistry and Center in Molecular Toxicology, Vanderbilt
University School of Medicine, Nashville, TN 37232-0146. Tele-
phone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@
toxicology.mc.vanderbilt.edu.
† Current address: Pharmacokinetics, Dynamics, and Metabolism,
Pfizer Global R&D, 2800 Plymouth Rd., Ann Arbor, MI 48105.
1 Abbreviations: TCE, trichloroethylene; ACTH, adrenocorticotropic
hormone (fragment consisting of residues 1-24); MOPS, 3-(N-mor-
pholino)propanesulfonic acid; Tris, tris(hydroxymethyl)aminomethane;
BTEE, benzoyl-L-Tyr ethyl ester; L-BAPNA, N2-benzoyl-L-Arg-p-nitro-
anilide. The nucleoside abbreviations are standard for the journal.
10.1021/tx000185n CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/07/2000

Trichloroethylene-Protein and -DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 55
carcinogenicity (13, 14). Metabolites of TCE derived from
the GSH conjugation pathway that may be responsible
for the renal toxic effects of TCE include S-(1,2-dichlo-
rovinyl)glutathione, S-(1,2-dichlorovinyl)-L-Cys, and S-(1,2-
dichlorovinyl)-L-Cys sulfoxide (13).
The metabolism of TCE has been summarized in detail
(14), and the toxicity and mutagenicity of TCE and its
metabolites have been revisited (13, 15). However, there
is much research yet to be done to assess the risk of TCE
to human health. Clewell et al. reported a physiologically
based pharmacokinetic model for risk assessment of TCE
and its metabolites (16-18). This model was based on
the view that all Cl3CCHO is generated from rearrange-
ment of an oxygenated P450-TCE intermediate. Previ-
ously, physiologically based pharmacokinetic modeling
was also based on the concept that all Cl3CCHO is formed
from TCE oxide rearrangement (19, 20).
analysis of modified proteins as a means of characterizing
total adduct levels. The methodology was extended to the
reaction of TCE oxide with nucleosides and oligonucle-
otides. We also considered the transient nature of the
inhibition of enzymes by TCE oxide in the context of
adduct lability.
Exp er im en ta l P r oced u r es
Ca u tion : Care should be exercised in handling TCE oxide,
dichloroacetyl chloride, and formic acetic anhydride, all of which
are reactive electrophiles. Use adequate skin protection and avoid
inhalation.
En zym es. Rabbit muscle aldolase, Leuconostoc mesenteroides
glucose-6-phosphate dehydrogenase, R-chymotrypsin, D-amino
acid oxidase, papain, catalase, trypsin, proteinase K, super
oxidase dismutase, insulin, and adrenocorticotropic hormone
(ACTH, residues 1-24) were obtained from Sigma (St. Louis,
MO).
In the 1970s, controversy existed over whether an
epoxide intermediate is the only route of TCE oxidation
(21, 22). Although we found evidence for TCE oxide
formation, TCE oxide was found not to be an obligate
intermediate in the formation of chloral, and we con-
cluded that Cl3CCHO formation proceeds via chloride
migration in an oxygenated P450-TCE intermediate
(23). We also suggested that TCE oxide itself may not be
the intermediate responsible for irreversible binding to
protein and DNA (23). Consistent with our conclusion,
Green and Prout (24) concluded that there was evidence
in favor of only low levels of TCE oxide formation in the
oxidative metabolism of TCE in rat or mouse liver
microsomes. Recently, we elucidated the roles of acyl
halides derived from TCE oxide hydrolysis in the modi-
fication of protein Lys groups (25, 26).
Previously, we demonstrated that TCE-protein ad-
ducts are much more abundant than any DNA adducts
(27). Consistent with our observation, Kautianinen et al.
(28) also found that the level of protein binding (1.4 ng/g
of protein) was 100 times higher than the level of DNA
binding (1.5 pg/g of DNA) in B6C3F1 male mice exposed
to TCE at low doses (relevant to human exposure).
Pumford and his associates used a polyclonal antibody
to detect putative dichloroacetylated proteins in livers
of mice exposed to TCE (2). Immunoblots revealed the
presence of two major TCE adducts at 50 and 100 kDa
in liver microsomal fractions from male B6C3F1 mice.
One of the adducts has been reported to be P450 2E1,
an enzyme known to oxidize TCE (1, 29-31). However,
further characterization of adducts and quantitation has
not been reported. TCE oxide has been postulated to be
responsible for the covalent binding of TCE with protein
and, to a much lesser extent, DNA (32-34). Previously,
we had not found a good correlation between conversion
of TCE to Cl3CCHO, TCE oxide, and protein adducts in
rat, mouse, and human liver microsomes (27). Therefore,
a more detailed reinvestigation of TCE oxide and its
protein and possible DNA adducts is needed.
Sp ectr oscop y. 1H and 13C NMR spectra were recorded on a
Bruker AM 400 spectrometer (operating at 400.13 MHz in the
1H mode, Bruker, Billerica, MA) at 25 °C in the Vanderbilt
facility. Samples were prepared in either D2O or CD3OD (100.0
at. % D grade, Aldrich Chemical Co., Milwaukee, WI). UV/visible
spectroscopy was carried out using a Cary 14/OLIS spectropho-
tometer equipped with a circulating water bath for temperature
control (On-Line Instrument Systems, Bogart, GA). Mass
spectrometry was carried out with a Waters 2690 separations
module (Alliance) (Waters Corp., Milford, MA) and a Finnigan
MAT TSQ/SSQ 7000 series mass spectrometer (Finnigan,
Sunnyvale, CA). MS studies were performed using either the
positive ion mode (for analysis of insulin, ACTH, and nucleoside
adducts) or negative ion mode (for analysis of oligonucleotide
adducts) with an electrospray needle voltage of 4.5 kV. N2 was
used as the sheath gas (70 psi) to assist nebulization and as
the auxiliary gas (10 psi) to assist with desolvation. The
stainless steel capillary was heated to 200 °C, and the electro-
spray interface and mass spectrometer parameters were opti-
mized to obtain maximum sensitivity. The tube lens and the
heated capillary were operated at 75 and 20 V, respectively, and
the electron multiplier was set at either 1900 or 2000 V.
Ch em ica ls. Solvents for HPLC and HPLC/MS were HPLC
grade (EM Science, Gibbstown, NJ ). An 8-mer oligonucleotide
(5′-GCCTAGAT-3′) was purchased from Midland Certified Re-
agent Co. (The Woodlands, TX).
TCE was distilled (atmospheric pressure) to remove inhibitors
prior to epoxide synthesis. Reagents for synthesis were ACS
grade or better, unless otherwise specified.
Syn th eses. (1) TCE Oxid e. TCE oxide was synthesized by
m-chloroperbenzoic acid treatment of TCE and partially distilled
(25). Concentrations (in residual TCE) were determined colo-
rimetrically using the 4-(4-nitrobenzyl)pyridine reagent (23, 25,
35).
(2) Acetic F or m ic An h yd r id e (36). The synthesis has been
described previously (26).
(3) O-Acetyl-L-Ser . O-Acetyl-L-Ser was synthesized by a
published method (37). The general experimental procedure is
as follows. The amino acid (10 mmol) was dissolved in 6 N HCl
(2 mL). CH3CO2H (2 mL) was added, and the solution was cooled
to 0 °C in an ice bath. Acetyl chloride (20 mL) was then added
slowly to the beaker. After 30 min, 3 volumes of (CH3CH2)2O
was added and the O-acetyl-L-Ser‚HCl precipitated. The com-
pound was recovered by filtration, washed with (CH3CH2)2O,
dried in vacuo, and recrystallized from C2H5OH (75% yield): 1H
NMR (D2O) δ 2.07 (s, 3H, COCH3), 4.36 (t, 1H, CHCH2), 4.50
(m, 2H, CHCH2); 13C NMR (D2O) δ 20.4 (COCH3), 52.6 (CHCH2),
62.3 (CHCH2), 169.6 (COCH3), 173.6 (COOH).
(4) O-Acet yl-L-Tyr . O-Acetyl-L-Tyr was synthesized by
published methods (37, 38) with a yield of 45%: 1H NMR (D2O)
δ 2.19 (s, 3H, COCH3), 3.12 (m, 2H, CHCH2), 4.12 (t, 1H, CH-
CH2), 6.75 [d, 2H, CH2C(CHCH)2CO], 7.03 [d, 2H, CH2C(CHCH)2-
CO]; 13C NMR (D2O) δ 20.7 (COCH3), 35.1 (CHCH2), 54.6
We have characterized the reaction of TCE oxide with
proteins and found that both N6-formyl-Lys and N6-
(dichloroacetyl)-Lys are formed, with the level of the
former exceeding the latter (26). However, our prelimi-
nary studies on the stability of O-acyl amino acids
indicated that these adducts were unstable under the
prolonged conditions needed for protease digestion, de-
rivatization, and HPLC/MS analysis. Because of our
concerns about the contributions of unstable adducts to
the estimate of the total level, we utilized direct MS

56 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Cai and Guengerich
(CHCH2), 116.3 [CH2C(CHCH)2CO], 122.7 [CH2C(CHCH)2CO],
126.0 [CH2C(CHCH)2CO], 131.2 [CH2C(CHCH)2CO], 169.6
(COCH3), 173.6 (COOH).
(2) Glu cose-6-P h osp h a te Deh yd r ogen a se (41). L. mesen-
teroides glucose-6-phosphate dehydrogenase (100 µL of a 0.5 mg/
mL solution in 100 mM potassium phosphate buffer, pH 7.8)
was reacted with TCE oxide (12 mM final concentration, added
in CH3CN, <2.5 µL). Aliquots of the reaction mixture (0.5 µL)
were used for activity assays at different times. A mixture of
0.9 mL of 55 mM Tris-HCl buffer (pH 7.8) containing 0.33 mM
MgCl2, 0.2 mM NADP+, and 3.3 mM glucose 6-phosphate was
incubated at 30 °C for 7 min to achieve temperature equilibrium.
An enzyme solution (0.5 µL) was added, and the increase in A340
was recorded for 6 min. ∆A340/min was calculated from the initial
linear phase (ꢀ340 ) 6.22 × 103 M-1 cm-1 for NADPH). The
catalytic activity of unreacted glucose-6-phosphate dehydroge-
nase was measured at different times in control experiments.
(3) Ra bbit Mu scle Ald ola se (42-45). Rabbit muscle aldo-
lase (100 µL, 0.5 mg of rabbit muscle aldolase/mL in 100 mM
potassium phosphate buffer, pH 7.4) was reacted with TCE
oxide (1.0 mM final concentration, added in CH3CN, <2.5 µL).
At different times, aliquots of the reaction mixture (10 µL) were
added to a mixture of 1.0 mL of 12 mM fructose 1,6-bisphosphate
(pH 7.5) and 2.0 mL of 0.1 mM EDTA (pH 7.5) containing 3.5
mM hydrazine sulfate. ∆A240 was recorded for 7 min at 25 °C
[and also for a blank of 2.0 mL of 0.1 mM EDTA (pH 7.5)
Rea ction s. (1) Rea ction of TCE Oxid e w ith In su lin or
ACTH. The indicated amounts of TCE oxide (10 µL of dry
CH3CN) were added to a solution of insulin or ACTH (1 mg/mL
in 200 µL of 10 mM NH4CH3CO2, pH 7). After 5 min, 20 µL of
the reaction mixture was analyzed directly by HPLC/MS using
a syringe pump (flow rate of 0.1 mL/min) with a Vydac C4 guard
column (Vydac, Hesperia, CA) (placed between the injection loop
and the mass spectrometer). The HPLC/MS analysis procedure
is described as follows. Before the 20 µL sample was loaded and
injected, the HPLC system was washed thoroughly with 20 mM
NH4CH3CO2 for 5 min. The sample was then loaded, injected,
and washed with 20 mM NH4CH3CO2 for 2 min. Subsequently,
the eluting buffer was changed to H2O/CH3OH (50/50, v/v,
containing 0.1% CH3CO2H, v/v), which eluted the insulin or
ACTH. In an alternative experiment, the mixture of insulin or
ACTH reacted with TCE oxide (100 µL, after reaction for 5 min)
was treated with NaOH (adjusted to pH 12) for 5 min. The pH
was then adjusted to 5 by addition of 0.1 N HCl. Subsequently,
the mixture (20 µL) was subjected to HPLC/MS for analysis of
adducts. Control experiments were also carried out with unre-
acted insulin or ACTH following the same procedure.
containing 3.5 mM hydrazine sulfate and 1.0 mL of H2O]. ∆A240
/
min was calculated by subtracting ∆A240/min (before the addi-
tion of enzyme) from ∆A240/min (after the addition of enzyme).
The activity (units/mg) was calculated using eq 1 (46):
Quantitation of the adducts was carried out with the assump-
tion that all modified polypeptides yield a response similar to
that of the unmodified polypeptide. Peak heights were used,
along with knowledge of the number of formyl or dichloroacetyl
modifications derived from the presence of multiples of 28 or
110/112 added to the MH+ of the unmodified polypeptide.
Normalization was on the basis of the sum of all peak heights,
including the unmodified polypeptide.
∆A240/min
units/mg )
(1)
mg of enzyme/mL of reaction mixture
The catalytic activity of unreacted aldolase was measured at
different times in control experiments.
(2) Rea ction of TCE Oxid e w ith 8-m er Oligon u cleotid e
(5′-GCCTAGAT-3′). TCE oxide (15 mM) was added to a solution
of the 8-mer oligonucleotide (0.78 mM, 100 µL in H2O). After
15 min, a 0.10 µM oligonucleotide solution in H2O/2-propanol
(50/50, v/v, containing 2.5 mM imidazole and 2.5 mM piperidine)
was made from the reaction mixture (39), and subsequently,
20 µL of the solution was analyzed by HPLC/MS using a syringe
pump (flow rate of 20 µL/min) with a Vydac C4 guard column
(placed between the injection loop and the mass spectrometer).
The solution used for HPLC/MS was H2O/2-propanol (50/50, v/v).
A control experiment was carried out with the unmodified 8-mer
oligonucleotide.
(4) r-Ch ym otr yp sin (47). R-Chymotrypsin (150 µL of a 1.0
mg/mL solution in 100 mM potassium phosphate buffer, pH 7.4)
was reacted with TCE oxide (8 mM final concentration), acetic
formic anhydride (1.5 mM final concentration), or dichloroacetyl
chloride (1.5 mM final concentration) (added in CH3CN, 1.5 µL).
An aliquot of the reaction mixture (10 µL) was used for activity
assays at different times. A mixture of 1.5 mL of 80 mM Tris-
HCl buffer (pH 7.8) containing 100 mM CaCl2 and 1.4 mL of
1.1 mM benzoyl-L-Tyr ethyl ester (BTEE) (in H2O/CH3OH, 50/
50, v/v) was incubated at 25 °C for 4 min to achieve temperature
equilibrium. An enzyme solution (10 µL) was added, and the
increase in A256 was recorded for 4 min. ∆A256/min was calcu-
lated from the the initial linear phase. The activity (units/mg)
was calculated using an ꢀ256 of 964 M-1 cm-1 for BTEE. The
catalytic activity of unreacted R-chymotrypsin was measured
in control experiments.
(5) D-Am in o Acid Oxid a se (48). D-Amino acid oxidase (100
µL of a 1.0 mg/mL solution in 100 mM potassium phosphate
buffer, pH 7.4) was reacted with TCE oxide (1.5 mM final
concentration), acetic formic anhydride (1.5 mM final concentra-
tion), or dichloroacetyl chloride (1.5 mM final concentration)
(added in CH3CN, 1.5 µL). An aliquot of the reaction mixture
(5 µL) was used for activity assays at different times. A mixture
of 0.9 mL of 50 mM D-Ala in 20 mM K2P2O7 buffer (pH 8.3)
containing 0.27 mM 3,3′,5,5′-tetramethylbenzidine and 2 µL of
a solution of 1 mg/mL horseradish peroxidase in 20 mM K2P2O7
(pH 8.3) was incubated at 37 °C for 5 min to achieve tempera-
ture equilibrium. The enzyme solution (5 µL) was added, and
the increase in A426 was recorded for 5 min. ∆A426/min was
calculated from the initial linear phase. The activity (units/mg)
was calculated using eq 2 (48):
(3) Rea ction of TCE Oxid e w ith Nu cleosid es. TCE oxide
was added (to a final concentration of 15 mM) to a solution of
either dAdo, dCyd, dGuo, or dThd (1 mM, 100 µL in H2O). After
reaction for 15 min, HPLC/MS analysis was performed to
identify possible nucleoside adducts. HPLC conditions (Zorbax
Rx-C8 column, 2.1 mm × 150 mm, Mac-Mod, Chadds Ford, PA)
were as follows: flow rate of 0.2 mL/min; solvent A, 10 mM
NH4CH3CO2 in H2O with 2% CH3OH, v/v (pH 4.6); solvent B,
10 mM NH4CH3CO2/CH3OH, pH 7.4, 5/95, v/v; 100% A and 0%
B at time zero, 90% A and 10% B after 10 min, 70% A and 30%
B after 20 min, 0% A and 100% B after 30 min, 0% A and 100%
B after 35 min, 100% A and 0% B after 40 min, and 100% A
and 0% B after 50 min. In control experiments, TCE oxide was
added to 100 µL of H2O and, after 15 min, nucleosides (1.0 mM,
100 µL) were added to the mixture; the remainder of the
analytical procedure was carried out as described for the direct
reaction with TCE oxide.
En zym e Assa ys. (1) Kin etic Assa ys of O-Acetyl-L-Ser
a n d O-Acetyl-L-Tyr Sta bility. O-Acetyl-L-Ser or O-acetyl-L-
Tyr (final concentration of 2 mg/mL) was dissolved in 100 mM
tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 8.0).
At varying times, 1 µL of the solution was spotted onto a TLC
plate. The TLC plate was developed in n-butanol/CH3CO2H/H2O
(v/v/v, 80/20/20) (40) to separate the acetyl derivatives from the
amino acids. The quantitation of O-acetyl-L-Ser and O-acetyl-
L-Tyr was performed after colorimetric reaction with ninhydrin
(in acetone, 1%, w/v) (40) and analyzed using a NucleoTech
GelExpert system (San Mateo, CA).
∆A426/min × 3.0 × dilution
units/mg )
(2)
8.1 × 0.1 × (mg/mL)
The catalytic activity of unreacted D-amino acid oxidase was
measured at different times in control experiments.
(6) P a p a in (49). Papain (200 µL of a 1.0 mg/mL solution in
100 mM potassium phosphate buffer, pH 6.0, with 71 mM

Trichloroethylene-Protein and -DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 57
Ta ble 1. P r otein Ad d u cts F or m ed d u r in g th e Rea ction of
In su lin or ACTH w ith TCE Oxid ea
protein adducts
insulin
ACTH
before treatment with base (nmol of
amino acid adducts/nmol of protein)
2.8 ( 0.4
2.7 ( 0.3
ratio of formyl/dichloroacetyl adducts 2.6
3.6
after treatment with base (nmol of
amino acid adducts/nmol of protein)
0.67 ( 0.01 0.90 ( 0.10
ratio of formyl/dichloroacetyl adducts 2.5
1.7
a
Treatment was with 4 mM TCE oxide. Raw data are from
Figures 1 and 2. All results are presented as means ( SD of
triplicate determinations.
F igu r e 1. Mass spectra of the reaction of insulin with TCE
oxide: (A) insulin, (B) reaction of insulin with TCE oxide (4 mM),
and (C) base-treated (pH 12, 5 min) reaction mixture of insulin
with TCE oxide, subsequently neutralized (pH 5).
F igu r e 3. Time dependence of release of TCE oxide adducts
of insulin and ACTH at pH 7, monitored by MS (Figures 1 and
2): (A) ACTH and (B) insulin.
sis (25). All of the major peaks in Figures 1 and 2 can be
assigned to an adducted insulin or ACTH with either one
or multiple formyl or dichloroacetyl groups attached (with
H+ or ammonia attached). In the case of the reaction of
insulin with TCE oxide, 2.8 nmol of amino acid adducts
per nanomole of protein (with a ratio of formyl adducts/
dichloroacetyl adducts of 2.6) was observed before treat-
ment with base (Table 1). After base treatment, only 0.67
nmol of amino acid adduct per nanomole of protein
(formyl/dichloroacetyl ratio of 2.5) was detected. For
ACTH, similar results were observed (Table 1). Before
base treatment, 2.7 nmol of amino acid adducts per
nanomole of protein formed (formyl/dichloroacetyl ratio
of 3.6); after treatment with base, the level decreased to
0.9 nmol of amino acid adducts per nanomole of protein
(formyl/dichloroacetyl ratio of 1.7).
The time courses of insulin and ACTH amino acid
adduct stability were examined at pH 7 using MS (Figure
3, concentration of TCE oxide of 2 mM in both cases).
The t1/2 of either insulin or ACTH amino acid adducts
was ∼1 h at pH 7 (Figure 3) [the t1/2 of TCE oxide is 12
s (25), and the resulting acyl chlorides are also short-
lived, so the t1/2 of stability is not influenced by continued
adduct formation]. After 2 h, approximately 80% of the
amino acid adducts had decomposed.
Kinetic assays with O-acetyl-L-Ser and O-acetyl-L-Tyr
indicated that these two esters had half-lives of 1 h and
10 min at pH 8.0, respectively. At pH 8.4, O-acetyl-L-Ser
had a half-life of 15 min.
Recover y of En zym a tic Activity. The effects of
stable protein Lys adducts on enzyme activities were
performed with two model enzymes for which the litera-
ture suggests roles of Lys residues, rabbit muscle aldolase
(50) and glucose-6-phosphate dehydrogenase (51). In our
previous work with TCE oxide (26), we showed that these
enzymes are sensitive to inhibition by TCE oxide and that
Lys adducts are formed. No recovery occurred after TCE
oxide reacted with either aldolase or glucose-6-phosphate
F igu r e 2. Mass spectra of the reaction of ACTH with TCE
oxide: (A) ACTH, (B) reaction of ACTH with TCE oxide (4 mM),
and (C) base-treated (pH 12, 5 min) reaction mixture of ACTH
with TCE oxide, subsequently neutralized (pH 5).
HSCH2CH2OH, activated for 30 min) was reacted with TCE
oxide (8 mM final concentration), acetic formic anhydride (1.5
mM final concentration), or dichloroacetyl chloride (1.5 mM final
concentration) (added in CH3CN, 1.5 µL). An aliquot of the
reaction mixture (10 µL) was used for activity assays at different
times. A solution of 1.0 mL of 100 mM potassium phosphate
buffer (pH 6.5) containing 1.15 mM N2-benzoyl-L-Arg-p-nitro-
anilide (L-BAPNA)-HCl and 2 mM EDTA was incubated at 25
°C for 5 min to achieve temperature equilibrium. The enzyme
solution (10 µL) was added, and the increase in A410 was
recorded for 4 min. ∆A410/min was calculated from the initial
linear phase. The activity (units/mg) was calculated using an
ꢀ410 of 8800 M-1 cm-1 for L-BAPNA. The catalytic activity of
unreacted papain was measured at different times in control
experiments.
Resu lts
F or m a tion of P r otein Ad d u cts w ith TCE Oxid e.
Formation of TCE oxide-derived protein amino acid
adducts was observed directly by MS with the model
polypeptides insulin (Figure 1) and ACTH (Figure 2).
After treatment with mild base (pH 12 for 5 min), large
decreases in the amounts of amino acid adducts were
observed (Figures 1 and 2). The decreases could be
attributed to unstable ester adducts formed from reaction
of Cys, Ser, Thr, and Tyr (which have either a free -OH
or -SH group) with intermediates of TCE oxide hydroly-

58 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Cai and Guengerich
F igu r e 4. Time dependence of catalytic activities of enzymes following treatment with TCE oxide (0), acetic formic anhydride (4),
and dichloroacetyl chloride (b): (A) rabbit muscle aldolase, (B) glucose-6-phosphate dehydrogenase, (C) chymotrypsin, (D) D-amino
acid oxidase, and (E) papain. The uninhibited enzyme activities were (A) 8.8 units/mg, (B) 590 units/mg, (C) 19 units/mg, (D) 1800
units/mg, and (E) 0.17 unit/mg.
dehydrogenase (Figure 4). The effects of unstable ester
type amino acid adducts on enzyme activities were also
studied. Three model enzymes were chosen, including
papain [containing a catalytic essential Cys residue (49)],
R-chymotrypsin [containing a sensitive Ser (47)], and
D-amino acid oxidase [with essential Cys and Tyr resi-
dues (48)]. Concentrations of acylating agents were added
that had been shown to decrease the activity of each
enzyme to ∼50% in preliminary experiments. In all three
cases, TCE oxide and the model acylating agents dichlo-
roacetyl chloride and acetic formic anhydride decreased
the enzyme activities rapidly (Figure 4), suggesting
formation of adducts at the active sites. However, enzy-
matic activities all recovered after 4-5 h at neutral pH,
indicating the lability of the putative ester adducts.
F igu r e 5. Mass spectra of TCE oxide-derived 8-mer oligonucle-
TCE Oxid e-Der ived DNA Ad d u cts. Individual reac-
tions of the four nucleosides with TCE oxide were
analyzed using HPLC/MS. Only dGuo formed adducts
[2% formyl adduct (MH+ 296.2, λmax ) 243 and 272 nm)]
with TCE oxide (see the Supporting Information). No
adducts were detected during the reactions of TCE oxide
with dAdo, dCyd, or dThd. The UV spectrum of the dGuo
adduct was unusual, and comparison of spectra at
neutral, acidic, and basic pH indicated that it did not
match those of N1-methyl-Guo, O6-methyl-Guo, N7-meth-
yl-Guo, N2-methyl-Guo, or N2-acetyl-Guo. The formyla-
tion is presumably not on the deoxyribose hydroxyls,
because the UV spectrum is perturbed. Further attempts
to identify the site of attachment were unsuccessful due
to the lability of the adduct.
otide adducts: (A) reaction of TCE oxide with 8-mer oligonucle-
otide and (B) control.
was observed (Figure 5). However, the 8-mer oligonucle-
otide adducts were unstable, with a t1/2 of 30 min at pH
8.5 [conditions used for HPLC/MS analysis (39)] (Figure
6).
Discu ssion
F or m a tion of P r otein Ad d u cts w ith TCE Oxid e.
We previously reported the formation of stable protein
Lys adducts derived from TCE oxide (26). Synthetic
O-acetyl-L-Ser and O-acetyl-L-Tyr (model compounds for
O-formyl-L-Ser and O-formyl-L-Tyr) were found to be
unstable during the analysis procedure, which included
proteinase digestion and phenyl isothiocyanate deriva-
tization (26). The results indicated that O-formyl-L-Ser
and O-formyl-L-Tyr would not be able to survive the
analysis procedure even if they were formed in the
A model 8-mer oligonucleotide (5′-GCCTAGAT-3′) was
reacted with 15 mM TCE oxide. Subsequently, the
reaction mixture was analyzed for possible adducts using
HPLC/MS (Figure 5) (39). Formation of the adducted
8-mer oligonucleotide with two formyl groups attached

Trichloroethylene-Protein and -DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 59
lung following. Stott et al. (55) reported similar findings,
with more protein binding of [14C]TCE metabolites
detected in the liver than in the kidneys of male B6C3F1
mice and Osborne-Mendel rats. We also reported the
formation of protein adducts (in vitro) in human, mouse,
and rat liver microsomes (27). Recently, at doses more
relevant to humans than those used previously (27, 53,
55), Kautiainen et al. (28) detected 1.4 pg of adduct per
gram of liver protein (in vivo) in male B6C3F1 mice using
accelerator mass spectrometry. Despite the available data
on TCE binding to protein, covalent binding of TCE oxide
with protein has not been explored in detail except in
our initial study on Lys adducts (26). Most (∼80%) of the
TCE oxide-derived protein adducts were unstable under
physiological conditions (vide supra). The findings indi-
cate that the level of total TCE binding to protein has
been underestimated in the literature because of the
presence of unstable protein esters/thioesters. The results
also suggest that the highest level of protein binding
should be observed at short times (1 h) after a single
exposure, although the technical difficulty in doing
analyses rapidly may obscure the time course.
Recover y of En zym a tic Activity. We reported pre-
viously that the enzymatic activities of two model en-
zymes for which the literature suggests roles of Lys
residues, rabbit muscle aldolase (50) and glucose-6-
phosphate dehydrogenase (51), were decreased following
addition of TCE oxide or model acylating agents (dichlo-
roacetyl chloride and acetic formic anhydride) (26). Here
we further investigated the time-dependent nature of
TCE oxide inhibition of the two enzymes, in light of the
transient nature of adduct stability with insulin or
ACTH. No recovery occurred after TCE oxide reacted
with either rabbit muscle aldolase or glucose-6-phosphate
dehydrogenase (Figure 4), presumably because protein
Lys conjugates are very stable (26). However, following
reaction with the electrophiles TCE oxide, dichloroacetyl
chloride, and acetic formic anhydride, the inhibition was
reversible for papain, R-chymotrypsin, and D-amino acid
oxidase, indicating that the putative protein esters/
thioesters are unstable. Furthermore, similarities be-
tween TCE oxide inhibition patterns and those observed
for the model acylating agents (dichloroacetyl chloride
and acetic formic anhydride) are consistent with the view
that acyl chlorides are most likely to be the intermediates
involved in TCE oxide derivatization of proteins (25), as
opposed to the possibility that the unstable adducts might
be the result of any gem-substituted products derived
from direct attack of nucleophiles on the epoxide itself.
DNA Ad d u cts. [14C]TCE was reported to bind co-
valently to hepatic RNA (27, 56) and DNA (27, 57, 58) in
vitro in the presence of a metabolic activation system.
Bergman observed a small amount of radioactivity that
coeluted with 1,N6-ethenoadenine in hydrolysates of the
DNA, suggesting the reaction of dGuo with a TCE
metabolite (59). However, no further experiments were
carried out regarding the identity of the adducts or
individual reactions of TCE metabolites. Moreover, the
valence state of TCE is incorrect for an etheno adduct,
because vinyl chloride yields this etheno adduct. Very
limited evidence has been available to support the view
that [14C]TCE binds to DNA in vivo, because of possible
protein contamination due to a high level of protein
labeling and a very low level of putative DNA labeling
(55). Recently, with the help of the advanced technology,
i.e., accelerator mass spectroscopy with detection of
F igu r e 6. Time-dependent loss of TCE oxide-derived 8-mer
oligonucleotide adducts at pH 8.6.
incubation of TCE oxide with protein. Therefore, we
developed a different analytical procedure (without any
workup procedure or extensive column separation) by
using HPLC/MS, with only a syringe pump and C4 guard
column to avoid destroying unstable protein ester/
thioester adducts, and allowing direct observation of
formation of protein amino acid adducts.
(1) Ma ss Sp ectr om etr ic Detection . Formation of
TCE oxide-derived insulin and ACTH adducts was de-
tected (Figures 1 and 2), and adduct formation was
quantitated (Table 1). Approximately 70% of the protein
(or peptide) adducts hydrolyzed after treatment with mild
base (pH 12 for 5 min). We attribute the loss to unstable
ester/thioester adducts which could be formed by reaction
of amino acid residues such as Cys (containing -SH), Ser/
Thr (-OH), or Tyr (-OH) with reactive intermediates
formed during TCE oxide hydrolysis, e.g., acyl chlorides
(26). Amino acids containing either the -SH or -OH
group are reactive enough to trap nucleophiles, including
aliphatic halides (52, 53). It is of interest to note that
larger amounts of formyl adducts were formed than
dichloroacetyl adducts (Table 1). The findings are con-
sistent with previously reported ratios of N6-formyl-Lys
to N6-(dichloroacetyl)-Lys during the reaction of TCE
oxide with other proteins (26).
(2) In sta bility of Ad d u cts. Unstable protein adducts
were further investigated by MS. The putative ester/
thioester adducts were the major adducts (∼80%) formed
during the reaction of TCE oxide with proteins and had
a collective t1/2 of 1 h under physiological conditions
(Figure 3). Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr
had t1/2 values of 1 h and 10 min at pH 8.0, respectively,
which are similar to those of the protein adducts (Figure
3). The t1/2 values of O-acetyl esters decreased with
increasing pH, indicating that O-acetyl ester hydrolysis
is most likely OH--dependent. This observation also
suggests that nucleophiles (which exist in biological
systems, such as GSH) could shorten the t1/2 of O-acyl
esters by nucleophilic attack through the same pathway
as hydroxide anion and decrease TCE toxicity, although
we have not carried out detailed kinetic studies on this
point.
TCE has been shown to bind to proteins in vitro as
well as in vivo (1, 26-28, 32, 52-54). In 1977, Bolt and
Filser studied irreversible protein-bound metabolites of
[14C]TCE in different tissues of male Wistar rats after
exposure for 5 h (53). The largest amount of protein-
bound metabolites was found in liver, with kidney and

60 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Cai and Guengerich
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radiocarbon with attomole sensitivity, the detection of
DNA adducts at the level of 1 adduct/1011 nucleosides was
reported (28). However, identification of any TCE-DNA
adducts has not been performed. In the work presented
here, we directly observed formation of an 8-mer oligo-
nucleotide adduct (with two formyl groups attached)
using HPLC/MS (Figure 5). The formyl groups were most
likely attached to the two dGuo residues of the 8-mer
oligonucleotide because only dGuo adducts (∼2% yield)
were detected during the reaction of TCE oxide with the
four individual nucleosides. The 8-mer oligonucleotide
adducts were unstable (Figure 6), which may explain the
low level of DNA adducts observed previously in the
literature.
Con clu sion s. Direct mass spectrometric analysis of
proteins and oligonucleotides treated with TCE oxide
indicates that a large fraction of the adducts are unstable,
presumably because of the instability of ester/thioester
products derived from the acyl chlorides (26). The exist-
ence of a large fraction of unstable adducts raises new
questions about protein adducts. One view might be that
toxicological risk from TCE is low because the recovery
of many modified proteins is rapid (Figure 4). An opposite
conclusion regarding risk assessment could be that TCE
is more likely to inactivate important proteins tran-
siently, because the level of protein binding is much
higher than estimated previously and presumably most
studies with [14C]TCE have underestimated the amount
of initial binding. Ultimately, answers to the dilemna will
need to involve measurement of levels of not only adducts
in surrogate (or total) protein fractions but also critical
targets, because of the diversity of recovery of function
(Figure 4). Nevertheless, these results and our previous
work (26) suggest that relatively high concentrations of
TCE oxide must be generated to inactivate proteins and
that TCE oxide is probably only weakly genotoxic,
possibly because of adduct instability (Figure 6). The
methods used for these analyses may be applied to other
electrophiles to study their modification of macromol-
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(26) Cai, H., and Guengerich, F. P. (2000) Acylation of protein lysines
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Ack n ow led gm en t. We thank Prof. D. Hachey for
helpful suggestions regarding the HPLC/MS analyses.
This work was supported in part by U.S. Public Health
Service Grants R35 CA44353 and P30 ES00267. H.C. was
supported in part by U.S. Public Health Service Post-
doctoral Fellowship F32 ES05919.
Su p p or tin g In for m a tion Ava ila ble: UV spectrum of the
TCE oxide-dGuo adduct and comparison to that of dGuo and
tables of m/z assignments of peaks observed in Figures 1 and
2. This material is available free of charge via the Internet at
http://pubs.acs.org.
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