Immunohistochemical localization of trichloroacylated protein adducts in tetrachloroethene-treated mice

Article abstract of DOI:10.1080/15287390151126487

Tetrachloroethene (PCE), a common industrial solvent and environmental contaminant, is primarily used in the dry-cleaning industry. The toxicity of PCE has been linked to vision disorders, renal and hepatic cancer, and autoimmune diseases. Although the mechanism of toxicity is not fully understood, PCE forms trichloroacylated protein adducts in tissues where toxicity is known to occur. These adducts may be responsible for toxicity by altering the function of cellular proteins. Using Western blot analysis, formation of trichloroacylated protein adducts has been reported. To determine the localization of the adducts in a specific zone of a tissue, immunohistochemical staining was used in the study. An antiserum to trichloroacylated proteins was raised in rabbits and its specificity was established by enzyme-linked immunosorbent assay (ELISA). Female MRL-Ipr/Ipr and MRL +/+ mice were treated with PCE using a single 5-mmol/kg dose over 24 h or on every fourth day for 6 wk (total 20 doses). Formation of trichloroacylated protein adducts was observed in the liver, and localized to the centrilobular zones. Intensity and circumference of the staining around the central vein were much greater in subchronically treated mice than in acutely treated mice. No immunochemical reactivity was observed in any of the other tissues examined. This study shows that hepatic trichloroacylated protein adducts are localized in a region of the liver where PCE-mediated toxicity is known to occur. Immunohistochemical localization of these adducts and its association with PCE-induced toxicity support the contention that adducts may contribute to toxicity.

Full text of DOI:10.1080/15287390151126487

Journal of Toxicology and Environmental Health, Part A, 63:145–157, 2001
Copyright© 2001 Taylor & Francis
1528-7394/01 $12.00 + .00
IMMUNOHISTOCHEMICAL LOCALIZATION
OF TRICHLOROACYLATED PROTEIN ADDUCTS
IN TETRACHLOROETHENE-TREATED MICE
Steven M. Green
Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, Texas, USA
M. Firoze Khan, Bhupendra S. Kaphalia, G. A. S. Ansari
Department of Pathology, University of Texas Medical Branch,
Galveston, Texas, USA
Tetrachloroethene (PCE), a common industrial solvent and environmental contaminant, is
primarily used in the dry-cleaning industry. The toxicity of PCE has been linked to vision
disorders, renal and hepatic cancer, and autoimmune diseases. Although the mechanism
of toxicity is not fully understood, PCE forms trichloroacylated protein adducts in tissues
where toxicity is known to occur. These adducts may be responsible for toxicity by alter-
ing the function of cellular proteins. Using Western blot analysis, formation of trichloro-
acylated protein adducts has been reported. To determine the localization of the adducts
in a specific zone of a tissue, immunohistochemical staining was used in the study. An
antiserum to trichloroacylated proteins was raised in rabbits and its specificity was estab-
lished by enzyme-linked immunosorbent assay (ELISA). Female MRL-lpr/lpr and MRL
+/+ mice were treated with PCE using a single 5-mmol/kg dose over 24 h or on every
fourth day for 6 wk (total 20 doses). Formation of trichloroacylated protein adducts was
observed in the liver, and localized to the centrilobular zones. Intensity and circumfer-
ence of the staining around the central vein were much greater in subchronically treated
mice than in acutely treated mice. No immunochemical reactivity was observed in any
of the other tissues examined. This study shows that hepatic trichloroacylated protein
adducts are localized in a region of the liver where PCE-mediated toxicity is known to
occur. Immunohistochemical localization of these adducts and its association with PCE-
induced toxicity support the contention that adducts may contribute to toxicity.
Tetrachloroethene (perchloroethene, PCE) is widely used as a solvent
in commercial and domestic applications. PCE is the most common sol-
vent used in the dry-cleaning industry, and is also found in spot removers
and other household cleaning products (ATSDR, 1997). Due to its wide-
spread use, PCE is a prevalent environmental contaminant, and is classified
as a known animal carcinogen and a probable human carcinogen (ATSDR,
Received 30 August 2000; sent for revision 5 October 2000; accepted 14 November 2000.
The project described was supported by grant ES08347 from the National Institute of Environ-
mental Health Sciences (NIEHS), NIH, and its contents are the sole responsibility of the authors and
do not necessarily represent the official view of NIEHS, NIH. We would like to acknowledge Drs.
Joachim Liehr, Alfred Nichols, and Neil Pumford for their advice on thioester synthesis, and Ed Ezell
at the University of Texas Medical Branch for assistance with the NMR spectra.
Current address for Steven M. Green is Dow Corning Corporation, Midland, MI, USA.
Address correspondence to G. A. S. Ansari, PhD, Professor, Department of Pathology, University
of Texas Medical Branch, Galveston, TX 77555-0609, USA. E-mail: sansari@utmb.edu
1
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S. M. GREEN ET AL.
1
997). PCE and other chlorinated ethenes have been implicated in auto-
immune diseases, including systemic lupus erythematosus and scleroderma
Veltman et al., 1975; Sparrow, 1977; Saihan et al., 1978; Lockey et al.,
987). PCE has also been shown to cause hepatocellular degradation and
(
1
fat accumulation in the centrilobular zone of the liver (Kylin et al., 1962),
produces jaundice and hepatomegaly in infants when present in breast
milk (Schreiber, 1997), and produces protein droplet nephropathy and
proteinurea in the kidney (Goldsworthy et al., 1988; Mutti et al., 1992).
It has been demonstrated that trichloroacylated protein adducts are
formed in the liver and kidney of rats (Birner et al., 1994) and in the plasma
of rats and humans (Pahler et al., 1999) after exposure to PCE. Exposure to
a single dose of PCE in male rats resulted in formation in the liver of an ad-
duct identified as n-e-trichloroacetyl-L-lysine, while adducts in the kidney
were identified as n-e-dichloroacetyl- L-lysine. The differences in adduct
production were attributed to the type of metabolites formed (Birner et al.,
1
994). In the liver, PCE is oxidized by cytochrome P-450 to trichloroacetyl
chloride, which can directly acylate proteins (Ikeda et al., 1972). In the
kidney, direct oxidation of PCE is minimal, but trichlorovinyl-L-cysteine,
formed from the glutathione conjugate trichlorovinylglutathione, can be
further activated by renal cysteine conjugate b-lyase to form dichlorothio-
ketene, which can acylate proteins (Dekant & Henschler, 1999).
The presence of protein adducts in tissues where toxicity occurs has led
to speculation that adduct formation may be related to the toxicity. How-
ever, the presence of adducts in a tissue does not necessarily reflect the
adducts’ presence in cells/zones of a tissue that are adversely affected by
the chemical. The purpose of this study was to determine localization of
the protein adducts using immunohistochemistry in tissues of PCE-treated
mice where PCE toxicity is known to occur.
MATERIALS AND METHODS
Chemicals
Ethanethiol, ethyl disulfide, and trichloroacetyl chloride were purchased
from Aldrich (Milwaukee, WI). Tetramethylbenzidine (TMB), lysine hydro-
chloride, bovine serum albumin (BSA) (fraction V), biotinylated goat anti-
rabbit immunoglobulin G (IgG), peroxidase-conjugated goat anti-rabbit IgG,
and EDAC [(1-ethyl-3-(dimethylaminopropyl)carbodiimide)] were from
Sigma Chemical Co. (St. Louis, MO). Imject keyhole limpet hemocyanin
(
KLH), peroxidase inhibitor, and metal-enhanced diaminobenzidine were
purchased from Pierce Chemical Co. (Rockford IL). ABC reagents, avidin/
biotin blocking kit, and Gills formula hematoxylin were from Vector labora-
tories (Burlingame, CA). Antigen-retrieval fluid was from Accurate Chemical
(
Westbury, NY). All other chemicals used were of analytical grade.
Synthesis of Acylation Reagents and Specificity of the Antisera
Since antisera to detect trichloroacylated proteins are not available
commercially, an antiserum to trichloroacylated KLH (TCA-KLH) was raised

PROTEIN ADDUCTS IN PCE-TREATED MICE
147
in rabbits. Trichlorothioacetate was synthesized and used as a trichloroacy-
lation agent, while trichloroacylated lysine and glycine were synthesized to
establish the specificity of the antisera.
Trichlorothioacetate was synthesized by mixing ice-cold trichloroacetyl
chloride with a three-fold excess of ice-cold ethanethiol under a dry nitro-
1
13
gen atmosphere. The reaction product was characterized by H and C nu-
clear magnetic resonance (NMR) and gas chromatography/mass spectrome-
try (GC/MS). Samples for NMR were run on a JEOL GX-270 pulsed Fourier
1
transform unit at 6.34 Tesla with a H resonance frequency of 270 MHz and
13
a C frequency of 67.8 MHz. GC/MS analysis of the samples was performed
using a Finnigan Incos 50 GC/MS equipped with an LGC Galaxy 2000
data system. Gas chromatographic separation was achieved using a 15 m ×
0
.32 mm ID DB-5 fused silica capillary column (temperature programmed
from 108 to 120°C at 11°C/min followed by 120–210°C at 35°C/min), with
helium as the carrier gas. Spectra were recorded by scanning the mass range
m/z 90–290 at 1 s/scan at an ionization voltage of 70 eV.
Trichloroacylated lysine (TCA-LYS) was synthesized according to
Schallenberg and Calvin (1955) by substituting trichlorothioacetate for
trifluorothioacetate. The reaction product was characterized by thin-layer
chromatography (TLC) using Analtech 250-µm plates and acetone/butanol/
ammonium hydroxide/water (37:37:19:4, v/v) as a mobile phase. NMR
spectra were run on a Varian Unity+ 400 pulsed Fourier transform NMR unit
1
13
operating at 9.4 Tesla with a H resonance frequency of 400 MHz and a C
frequency of 100 MHz. Fast atom bombardment (FAB) mass spectra were
run on a Kratos MS 50-RF double-focusing mass spectrometer attached to a
Mach 3 data system and scanned the mass range m/z 150–1000.
Trichloroacylated (TCA-GLY) glycine was synthesized by a modification
of Pahler et al. (1998). Glycine (20 g) was mixed with cold trichloroacetyl
chloride (30 ml) in tetrahydrofuran (150 ml) and refluxed for 1 h. The reac-
tion product was precipitated, washed with petroleum ether, and the pre-
cipitate was collected by vacuum filtration. The product was recrystal-
1
13
lized twice with benzene and characterized by TLC, followed by H and C
NMR, and FAB mass spectrometry. Conditions for TLC and FAB mass spectra
were the same as described for trichloroacylated lysine, while NMR condi-
tions were as described for trichlorothioacetate.
Trichloroacylated glycine was coupled to BSA (TCA-GLY BSA) using the
method of Pahler et al. (1998). Trichloroacetic anhydride-modified BSA was
prepared by the method of Halmes et al. (1996), using trichloroacetic an-
hydride instead of dichloroacetic anhydride, while trichlorothioacetate mod-
ification of BSA and KLH (TCA-BSA, TCA-KLH) was achieved using the
method of Birner et al. (1994) substituting trichlorothioacetate for dichloro-
thioacetate.
Antisera Production
Antiserum to trichloroacylated proteins was raised at Alpha Diagnostics
International (San Antonio, TX) using TCA-KLH as the immunogen. Two
New Zealand White female rabbits were given intramuscular (im) and sub-

1
48
S. M. GREEN ET AL.
cutaneous (sc) injections of TCA-KLH in Freund’s complete adjuvant after a
preimmune bleed (2 ml). Booster shots were given every 2 wk in Freund’s
incomplete adjuvant. Bleeds were obtained at 7, 9, and 11 wk.
Animals and Treatments
Female MRL-lpr/lpr and MRL +/+ mice, 4 wk (~26 g) or 10 wk (~35 g)
old, were purchased from the Jackson Laboratory (Bar Harbor, ME) and
acclimatized in our animal facility for 1 wk prior to treatment. Mice were
gavaged with 5 mmol/kg PCE in 100 µl corn oil, while control mice re-
ceived vehicle only. Subchronically treated mice received PCE every fourth
day for 6 wk (a total of 20 doses), while acutely treated mice received 1
dose of PCE. All mice were euthanized under ether anesthesia 24 h after
the last dose. Seven mice were used in each group.
At necropsy the heart, lung, liver, kidneys, thymus, adrenals, brain, pan-
creas, and spleen were removed, fixed in 10% neutral buffered formalin,
and processed for routine histological examination. Separate unstained sec-
tions were processed for immunohistochemistry.
Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay (ELISA) method used in this
study was a modification of Kenna et al. (1984), with Corning flat-bottom,
high-protein-binding 96-well plates and TMB as a chromogen. Color was
read on a Bio-Rad 3550-UV microplate reader at 450 nm. For competitive
ELISA, a fixed dilution of antiserum (1:16,000) that gave an OD of ~2.0 was
used with varying concentrations of soluble competing compounds.
Immunohistochemistry
Immunohistochemistry was performed using standard methods (Harlow
Lane, 1999). Antigen retrieval was done with antigen retrieval fluid fol-
&
lowed by Pierce peroxidase inhibitor to block endogenous peroxidase activ-
ity. Nonspecific binding sites were blocked with 10% normal goat serum,
followed by blockade of endogenous biotin with a biotin-blocking kit. The
slides were incubated in preimmune or TCA-KLH sera (1:1000 dilution) fol-
lowed by secondary antisera (goat anti-rabbit IgG, biotinylated, 1:1500 dilu-
tion). The slides were then incubated in Vector Elite ABC reagent, and color
was developed with metal-enhanced diaminobenzidine.
RESULTS
Structural Characterization of Synthesized Compounds
Gas chromatographic analysis of trichlorothioacetate showed a single
peak at 10 min, while trichloroacetyl chloride and ethyl disulfide controls
(
parent compound and a putative contaminant) eluted at 6.5 and 5.5 min,
1 13
respectively. H and C NMR and GC/MS analysis were consistent with the

PROTEIN ADDUCTS IN PCE-TREATED MICE
149
1
structure of the product. H NMR: (400 MHz; CDCl ) d = 3.1 (q, CH CH S),
3
3
2
13
1
.4 (t, CH CH S). C NMR: (100 MHz, CDCl ) d = 188.9 (C= O), 94.7
3
2
3
(
CCl ), 26.0, 13.5 (SCH CH , unassigned). Mass spectral analysis gave a
3 2 3
•+
molecular ion at m/z 206/208/210, 1:1:0.4 (M ), followed by fragmenta-
tion ions at m/z 162/164/166, 1:1:0.4 (Cl CCOOH), 143/145/147, 1:0.7:0.1
3
•
+
+
(
Cl C(OH)CS) , 117/119/121, 1:0.9:0.3 (Cl C ).
2
3
TLC analysis of trichloroacylated lysine had an R of 0.45 (R of lysine =
f
f
1
13
0
.13). H and C NMR and FAB mass spectral analysis were consistent with
1
6
the structure of the product. H NMR: (400 MHz, D-DMSO) d = 3.6 (t, CH),
13
3
.2 (t, CH NH), 1.8, 1.5, 1.3 (m, p, m, CHCH CH CH , unassigned).
C
2
2
2
2
6
NMR: (100 MHz, D-DMSO) d = 170.2, 161.0 (C= O, unassigned) 92.8
CCl ), 66.7 (CH), 53.7 (CH NH), 30.5, 27.7, 22.1 (CHCH CH CH , unas-
(
3
2
2
2
2
signed). Mass spectral analysis gave a protonated molecular ion at m/z 291/
+
2
93/295, 1:0.9:0.3 (M+H ), followed by loss of chloride to give a daughter
+ –
ion at m/z 256/258/260, 1:0.8:0.4 [(M+H )-Cl ].
TLC analysis of trichloroacylated glycine had an R of 0.58 (R of gly-
f
f
1
13
cine = 0.12). H and C NMR and FAB mass spectral analysis were consis-
1
tent with the structure of the product. H NMR: (270 MHz, D O) d = 4.1 (s,
2
1
3
CH ). C NMR: (100 MHz, D O) d = 169.5, 162.2 (C= O, unassigned), 39.7
2
2
(
CH ), 88.4 (CCl ). Mass spectral analysis gave an adducted molecular ion at
2 3
+
m/z 237/239/241, 0.9:1:0.3 (M+ NH ), a protonated molecular ion at 220/
4
+
2
1
22/224, 1:0.9:0.3 (M+H ), followed by daughter ions at m/z 185/187/189,
+
–
+
:0.7:0.1 [(M+H )-Cl ], and 174/176/178, 1:1:0.3 [M -COOH].
Characterization of Antiserum Specificity for the Antigen
The antiserum to trichloroacylated proteins (TCA-KLH antisera) was
analyzed for its specificity by ELISA, using TCA-BSA or BSA alone as solid-
phase antigens. A dose-dependent response was observed when TCA-BSA
was used (Figure 1), while no reactivity was observed with unmodified
BSA. When preimmune serum was used as the primary antiserum with
TCA-BSA, no reactivity was found (Figure 1).
Further specificity of the antiserum was established using three differ-
ent methods of synthesis for TCA-BSA to ensure that the reactivity ob-
served was due to specific binding of the antiserum, and not reactivity
with an irrelevant epitope formed during acylation. This was necessary
because the same trichloroacylating reagent was used to make the KLH
immunogen and BSA solid-phase antigen. Therefore, an irrelevant epitope
formed during synthesis could produce a false positive result by ELISA.
The two additional synthetic methods used were trichloroacylation of
BSA using trichloroacetic anhydride, or by coupling trichloroacylated
glycine to BSA with the bifunctional cross-linking agent EDAC. As shown
in Figure 2, strong reactivity was observed against trichloroacylated BSA
that was acylated by trichlorothioacetate. Similarly, trichloroacylated glycine
coupled to BSA displayed strong reactivity, although somewhat less than

1
50
S. M. GREEN ET AL.
FIGURE 1. ELISA for TCA-KLH antisera using serial dilutions. TCA-BSA was used as a solid-phase
antigen for TCA-KLH antisera and preimmune sera. As a control, BSA was used as a solid-phase anti-
gen for TCA-KLH antisera.
FIGURE 2. ELISA for titer of TCA-KLH antisera using different synthesis methods for TCA-BSA. Methods
included trichloroacylation by trichlorothioacetate, by trichloroacetic anhydride, and by coupling
trichloroacylated glycine to BSA. Bovine serum albumin was used as a solid-phase antigen control.

PROTEIN ADDUCTS IN PCE-TREATED MICE
151
trichlorothioacetate. Positive results were obtained in only the first two
dilutions with trichloroacetic anhydride-modified BSA.
As an additional control, a competitive ELISA was done with TCA-LYS
and lysine alone as competing agents. As seen in Figure 3, at 5 mM concen-
tration, TCA-LYS inhibited the binding of the antisera to TCA-BSA by ~95%,
while lysine inhibited binding by ~5%.
Immunohistochemical Localization of Protein Adducts
Immunohistochemical results are shown in Figure 4. Liver sections from
PCE-treated mice using preimmune serum or TCA-KLH antiserum pre-
absorbed with 5 mM TCA-LYS served as method controls. In both cases,
no staining was observed and the immunoreactivity was identical to the
vehicle-treated control; thus for brevity only a representative section of the
different controls is shown. All PCE-treated mice gave consistent binding
patterns. Intense staining was found in the centrilobular regions of the liver,
but no portal staining was observed. The intensity of staining and the cir-
cumference of stained hepatocytes around the central vein were greater
in subchronically treated mice compared to acutely treated mice for both
strains, although the lobular distribution of the adducts was the same in
all treated mice. There were no strain differences in staining at the same
time points. Immunochemical reactivity was not found in any other
tissue examined.
FIGURE 3. Competitive ELISA to evaluate the specificity of the TCA-KLH antisera. A 1:16,000 dilu-
tion of antisera was made up in different concentrations of either trichloroacylated lysine or lysine.
TCA-BSA was used as the solid-phase antigen.

1
52
S. M. GREEN ET AL.
A
B
FIGURE 4. Immunohistochemistry of liver sections from MRL-lpr/lpr and MRL +/+ mice (60×). Mice
were treated with either 1 dose for 24 h or 20 doses of 5 mmol/kg PCE (po) over 6 wk. (A)
Representative control panel for vehicle-treated control mice, and for sections stained with preim-
mune serum or preabsorbed with TCA-LYS. (B) Female MRL-lpr/lpr mouse acutely treated with PCE.

PROTEIN ADDUCTS IN PCE-TREATED MICE
153
C
D
FIGURE 4. (Continued) Immunohistochemistry of liver sections from MRL-lpr/lpr and MRL +/+ mice
(
(
60×). Mice were treated with either 1 dose for 24 h or 20 doses of 5 mmol/kg PCE (po) over 6 wk.
C) Female MRL +/+ acutely treated with PCE. (D) Female MRL lpr/lpr mouse subchronically treated
with PCE.

1
54
S. M. GREEN ET AL.
E
FIGURE 4. (Continued) Immunohistochemistry of liver sections from MRL-lpr/lpr and MRL +/+ mice
(
60×). Mice were treated with either 1 dose for 24 h or 20 doses of 5 mmol/kg PCE (po) over 6 wk. (E)
Female MRL +/+ mouse subchronically treated with PCE.
DISCUSSION
PCE is one of the most common environmental contaminants (Schreiber,
997). A wide range of toxicities has been attributed to PCE, including
1
visual defects, renal and hepatic cancers, and autoimmune diseases. For
both cancer (Hinson & Roberts, 1992) and chemical-induced autoimmune
disease (Uetrecht, 1988), binding of reactive compounds to cellular macro-
molecules has been proposed as an important step in the pathogenesis of
several diseases. Published studies have used Western blotting to demon-
strate that trichloroacylated protein adducts are formed in the liver and
kidney of rats (Birner et al., 1994) and in the plasma of rats and humans
after PCE exposure (Pahler et al., 1999). The present study was conducted
to determine localization of adducts in tissues of PCE-exposed mice.
To achieve this, an antiserum was raised in rabbits that recognized tri-
chloroacylated proteins, and its specificity was established by ELISA, which
confirmed that the antiserum recognized trichloroacylated proteins, but not
native proteins. Further control ELISA assays were done to ensure that the
antiserum was not recognizing an irrelevant epitope generated during syn-
thesis. This step was necessary because both BSA and KLH had been made
into adducts with the same trichloroacylating agent; thus, any irrelevant epi-
topes would be present on both proteins and lead to false positives
by ELISA. Trichloroacylation by two different methods was used to demon-

PROTEIN ADDUCTS IN PCE-TREATED MICE
155
strate the specificity. Strong reactivity was observed with trichloroacylated
glycine coupled to BSA, although the reactivity was somewhat lower than
when BSA was acylated with trichlorothioacetate, even though the degree
of adduction was approximately equal. This could be due to the two-car-
bon spacer arm of glycine interfering with binding of some of the antibody
clones to the antigen. Trichloroacylation with trichloroacetic anhydride re-
sulted in positive results (OD greater than 3× BSA alone) in the first two di-
lutions only, apparently due to the minimal adduction by this method (~8%
compared to ~40% for trichlorothioacetate).
A competitive ELISA was done to confirm that the antisera recognized
trichloroacylated proteins. Using TCA-LYS at 5 mM concentration, the OD
decreased by ~95%, while the LYS alone only decreased the OD by ~5%.
Overall, the ELISA results show that the antiserum does specifically recog-
nize trichloroacylated proteins with only minimal recognition of unmodified
lysine residues.
Autoimmune-prone female MRL mice have been used in this laboratory
as a model of a population susceptible to autoimmune disease (Khan et al.,
1
995, 1997), and were used in this study due to ongoing studies explor-
ing the involvement of PCE and trichloroacylated protein adducts with
chemical-induced autoimmune disease. PCE treatment did not result in any
striking gross morphology at necropsy, with the exception of the livers of
treated animals having a slight grayish hue. No significant histopathological
changes were observed in the organs examined (liver, lung, heart, thymus,
spleen, kidney, brain, adrenal, and pancreas), except in the liver, where
centrilobular hepatocytes appeared to be slightly swollen, and scattered
cells had microvesicular fat droplets (data not shown).
Immunohistochemistry was performed with unstained tissue sections
and TCA-KLH antiserum. Control samples were first run to establish the suit-
ability of the antiserum for immunohistochemistry. Using sections from
PCE-treated animals, no reactivity was observed with TCA-KLH antiserum
preabsorbed with 5 mM trichloroacylated lysine, or using preimmune sera
used in place of TCA-KLH antisera. In all PCE-treated mice, staining was
only observed in the liver, and was centrilobular in origin, with some possi-
ble midzonal staining. It could not be determined if the midzonal staining
was real, because the midzonal area is defined by the lack of a portal tract
or central vein, and not the presence of an identifiable structure. Therefore,
the staining observed may be due to the presence of a central vein above or
below the plane of the tissue slice.
There was no difference in staining intensity between the MRL-lpr/lpr
and MRL +/+ mice at either time point; however, there was a difference be-
tween acutely and subchronically treated animals, where more intense
staining was observed in subchronically treated mice. An explanation may
involve the high lipid solubility of PCE, which may result in a gradual re-
lease from lipid stores, resulting in adduct formation over several days. This
effect would be minimal in the 24-h group. To establish this hypothe-

1
56
S. M. GREEN ET AL.
sis, adduct formation could be evaluated at multiple time points after a
single dose. Another explanation for the increased staining could be the
retention of adducted proteins. Recent observations suggest that modified
proteins may be more resistant to degradation than native proteins (Cohn
et al., 1996). Odum et al. (1988) demonstrated that administration of PCE
to B6C3F1 mice resulted in fat-droplet accumulation in centrilobular re-
gion. The presence of vacuoles in centrilobular hepatocytes corresponds
with localization of the adducts, and may reflect a cause-effect relationship.
No adducts were found in the kidneys of treated mice. However, using
Western blotting and amino acid analysis, several investigators (Birner et al.,
1
994; Pahler et al., 1998, 1999) have shown that trichloroacylated proteins
are found in the kidneys of PCE-treated rats. The sensitivity of the different
methods used for these studies and the current study may explain the differ-
ences. Species differences may also play a role for kidney adduct produc-
tion. Glutathione conjugation is necessary for renal adduct formation with
PCE, so rates of oxidative metabolism versus glutathione conjugation will
affect the numbers of adducts formed. Dekant et al. (1998) have shown that
hepatic GST activity for PCE is four times greater in male rats than either sex
of mouse, although neither species of MRL mice was used in the study, and
their GST activity has not been determined.
In conclusion, the present studies demonstrate that treatment of mice
with PCE results in the formation of trichloroacylated protein adducts that
can be detected by immunohistochemical techniques. While there were
no detectable strain differences between MRL-lpr/lpr and MRL +/+ mice,
staining intensity was greater in the subchronically treated mice than in
the acutely treated mice. Further studies into the identity of the protein
adducts may provide a mechanism by which PCE can elicit an autoimmune
response.
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