TOXICOLOGY AND APPLIED PHARMACOLOGY
ARTICLE NO.
141, 394–402 (1996)
0305
In Vivo CHCl3 Bioactivation, Toxicokinetics, Toxicity, and Induced
Compensatory Cell Proliferation in B6C3F1 Male Mice
SIMONETTA GEMMA, SILVIA FACCIOLI,* PASQUALE CHIECO,* MARIA SBRACCIA,
EMANUELA TESTAI, AND LUCIANO VITTOZZI1
Department of Comparative Toxicology and Ecotoxicology, Biochemical Toxicology Unit, Istituto Superiore di Sanità, Rome, Italy;
and *Oncologic Institute ‘‘F. Addarii’’, Policlinico S. Orsola, Bologna, Italy
Received June 12, 1995; accepted July 29, 1996
In Vivo CHCl3 Bioactivation, Toxicokinetics, Toxicity, and Induced Compensatory Cell Proliferation in B6C3F1 Male Mice.
GEMMA, S., FACCIOLI, S., CHIECO, P., SBRACCIA, M., TESTAI, E.,
AND VITTOZZI, L. (1996). Toxicol. Appl. Pharmacol. 141, 394–402.
Chloroform carcinogenicity has often been associated with acute
tissue damage and consequent compensatory cell proliferation.
However, available data do not fully support this hypothesis, and
other biological factors may play a role in the tumor induction by
chloroform. The purpose of this study was to characterize the in
vivo CHCl3 metabolism and the time course of toxic effects and
of cell proliferation in the liver and kidney of B6C3F1 male mice
dosed ip or by gavage with 150 mg CHCl3/kg body wt. Microsomal
phospholipid adducts attributed to 14CHCl3 metabolism by both
oxidative and reductive pathways were detected in both liver and
kidney. The levels and composition of the adducts were similar in
the liver and kidney of treated animals. In the liver, although no
necrosis was histologically detectable, a transient cell proliferation
was found starting at 24 and peaking at 48 hr post-treatment.
Kidney toxicity was evident by biochemical and cytochemical
methods at 5 hr after dosing and progressed to severe necrosis at
48 and 96 hr. An intense kidney cell regeneration began 48 hr after
CHCl3 treatment, became maximal at 96 hr, and was sustained for
at least the following 3 days. These observations raise questions
about the purely epigenetic action of chloroform in tumor induction since bioassays have found tumors in liver but not kidneys of
CHCl3-treated B6C3F1 mice. q 1996 Academic Press, Inc.
Chloroform is an established rodent carcinogen
(USDHHS, 1994). At dose levels close to the maximum
tolerated dose, chloroform was found to be carcinogenic for
the liver of male and female B6C3F1 mice and for the kidneys of male Osborne Mendel (OM) rats (NCI, 1976; Jorgenson et al., 1985). At lower doses, chloroform exposure induced kidney tumors in male ICI mice, but was not carcinogenic for Sprague–Dawley (S.D.) rats, beagle dogs, or
C57Bl, CBA, and CF/1 mice (Roe et al., 1979; Heywood
1
To whom correspondence should be addressed at the Department of
Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità,
Viale Regina Elena, 299 I-00161 Rome, Italy. Fax: //39-6-49387139.
0041-008X/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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et al., 1979; Palmer et al., 1979). Hepatic tumors were found
in B6C3F1 mice treated by gavage with chloroform dissolved in corn oil but not when administered chloroform in
drinking water (NCI, 1976; Jorgenson et al., 1985).
High doses of chloroform also rapidly induce acute liver
damage in female and male mice and kidney necrosis in
male mice (Eschenbrenner and Miller, 1945; Moore et al.,
1982; Larson et al., 1993, 1994). Although DNA alkylation
has been recently found in liver and kidneys after [14C]chloroform administration to Wistar rats and BALB/c mice
(Colacci et al., 1991), the genotoxic properties of chloroform
are not yet clearly established (Rosenthal, 1987). A few
authors have suggested that a causal association exists between tumors and chloroform-induced acute damage followed by compensatory cell proliferation (Eschenbrenner
and Miller, 1945; Reitz et al., 1982, 1990; Larson et al.,
1993). However, B6C3F1 male mice treated with chloroform
develop acute kidney damage followed by intense compensatory renal cell proliferation but not kidney tumors (Larson
et al., 1994). Moreover, acute kidney damage has not been
assessed in CHCl3-treated OM rats (NCI, 1976; Jorgenson
et al., 1985; Reuber, 1979). Observations of CHCl3-induced
kidney damage and compensatory cell proliferation in rats
were reported in a strain (F-344; Larson et al., 1993, 1995)
not used in the CHCl3 carcinogenicity bioassays.
Chloroform can be activated by liver and kidney microsomes through oxidative and reductive pathways (Mansuy
et al., 1977; Pohl et al., 1977; Testai and Vittozzi, 1986;
Testai et al., 1992; Smith et al., 1983; Ade et al., 1994).
Metabolic formation of reactive electrophilic species is a
plausible basis for both acute cellular toxicity and genotoxicity of chloroform (Ilett et al., 1973; Pohl et al., 1980;
Rosenthal, 1987). Therefore a study of chloroform metabolism could yield key information relevant to the processes
of initiation and promotion of chloroform-induced tumors.
The well known oxidative pathway of chloroform activation
produces phosgene in vivo as a major determinant for the
acute liver and kidney toxicity (Pohl et al., 1980; Branchflower et al., 1984). However, the species-specific hepatocarcinogenic effect of this halomethane is not fully explained
394
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CHCl3 METABOLISM AND CELL PROLIFERATION
by the biotransformation of CHCl3 to phosgene nor to its
terminal metabolite CO2 (Brown et al., 1974; Reitz et al.,
1978, 1990; Testai et al., 1992). Activation by the reductive
pathway produces a dichloromethyl radical in several experimental systems (Tomasi et al., 1985; Testai et al., 1995).
In vitro bioactivation to this radical by hepatic and renal
microsomes from selected rodent strains produces adducts
to phospholipids (PL), which are distinctly different from
those of phosgene (Testai and Vittozzi, 1986; Testai et al.,
1990, 1992; De Biasi et al., 1992; Ade et al., 1994). No
information on the occurrence and toxicological relevance
of the reductive pathway in vivo is available.
We have therefore undertaken a study to determine if
different pathways of chloroform biotransformation associated with toxicity and cell proliferation could account for
the different susceptibility of the liver and the kidneys to
CHCl3-induced carcinogenicity in B6C3F1 male mice.
MATERIALS AND METHODS
Chemicals. [14C]chloroform (about 3.0 mCi/mmol, radiochemical purity 99%) was obtained from New England Nuclear Corp. (Boston, MA).
Unlabeled chloroform (IR purity) was from Merck (Darmstad, Germany).
Tanax was supplied by Hoechst A.G. (Frankfurt am Main, Germany). Liquid scintillation cocktails, Aqualuma, Lipoluma, Lumasolve, Lumagel, and
the CO2 trapper, were purchased from Lumac System A.G. (Basel, Switzerland). All substrates used in cytochemical studies and 5*-bromo-desoxyuridine (BrdU) were purchased from Sigma Chemical (St. Louis, MO). Mouse
monoclonal anti-BrdU antibodies were obtained from Becton & Dickinson
(San Jose, CA). Commercial kits used to measure serum alanine transaminase (ALT), creatinine, triglycerides, and blood urea nitrogen (BUN) were
from Boehringer GmBh (Mannheim, Germany). Reduced glutathione
(GSH) and defatted bovine serum albumin were purchased from Serva,
Feinbiochemicals (Heidelberg, Germany). All other reagents used were of
the highest purity commercially available.
Animals. Male B6C3F1 mice (20–25 g body wt) from Charles River
(Calco, Italy) were used. Mice were housed in solid-bottom shoebox-type
polycarbonate cages with stainless steel wire-bar lids, using a wooden dustfree litter (Ditta Mucedoli, Italy) as bedding material. Mice were maintained
on a 12-hr light cycle and provided with contaminant-free food (Ditta
Mucedoli, Italy) and water ad libitum. Animals were treated at 9:00 AM
with 14CHCl3 (ip) or unlabeled chloroform (by gavage) in corn oil at 150
mg/kg body wt, equivalent to 1.26 mmol/kg body wt. Control animals were
treated with the same volume of corn oil. In toxicokinetics experiments and
covalent adduct determinations, the 14CHCl3 specific radioactivity used was
0.049 and 0.29 mCi/mmol, respectively.
Toxicokinetics of [14C]chloroform. During the period of treatment,
mice were maintained in all-glass metabolic cages Metabowls MK III (Jencons Scientific Ltd.). A constant dry and CO2-free air flow of 500 ml/min
was maintained in the cage by a vacuum pump. The outgoing air was drawn
into a series of three traps; the first two traps contained toluene for 14CHCl3
trapping and the third was filled with Lumasorb II to collect expired 14CO2 .
No significant levels of 14C label were found when a fourth trap containing
Lumasorb II was added to the system. In long term (24, 48, and 72 hr)
studies, the 14C-trapping liquids were renewed at 3–6 hr after the treatment
and every 24 hr.
At different times after 14CHCl3 injection (10, 20, and 30 min; 1–6, 8, 24,
48, and 72 hr), samples were collected from the traps and dissolved in 10 ml
of the appropriate scintillation liquids to measure the volatile 14C compounds
expired. Aliquots of 0.1-ml urine samples were dissolved in 10 ml Aqualuma.
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395
Feces were weighed, homogenized in 10 vol (w/v) of ethyl acetate for 5 min,
and centrifuged at 3000 rpm for 10 min; 1 ml of the supernatant was transferred into a plastic vial containing 10 ml Lipoluma. The pellet was digested
in 10 vol (w/v) Lumasolve (607C, overnight) and centrifuged (3000 rpm for
5 min) and then two aliquots of the digest (1 ml each) were taken. In order
to reduce optical quenching, samples were bleached by adding a few drops
of H2O2 . Samples were then dissolved in 10 ml Lumagel:0.5 N HCl (9:1, v/
v), stored overnight in the dark, and then counted.
At each time point, 3 or 4 animals were euthanized with an ip injection of
Tanax (5 ml/kg body wt). Blood was immediately drawn from the heart;
aliquots of 100 ml were extracted for 5 min with 3 ml of toluene:1 mM NaOH
(2:1, v/v), and centrifuged at 3000 rpm for 3 min. Then, 1 ml organic extract
was counted in 10 ml Lipoluma to measure toluene soluble 14C label in blood;
aliquots of the aqueous phase (300 ml) were decolored with a few drops of
NaClO, neutralized, and counted in 10 ml Aqualuma, to measure radioactivity
due to [14C]chloroform-derived alkali-soluble metabolites. Toluene-extractable
radioactivity present in liver and kidneys was measured after tissue homogenization in 5 vol (w/v) of cold toluene for 5 min using an Ultraturrax homogenizer. The homogenate was centrifuged at 3000 rpm for 10 min, and 10 ml
Lipoluma was added to 1 ml supernatant. Total body radioactivity was measured after digesting the carcasses of four animals with 1.5 M KOH in 20%
ethanol (v/v) for 5 days. Samples of the digest (10 ml) were centrifuged (3000
rpm, 10 min); two aliquots of the supernatant (1 ml each) were decolored with
a few drops of H2O2 and added to 10 ml Lumagel:1.5 N HCl (9:1, v/v).
Samples were kept in the dark overnight and then counted.
Covalent binding of [14C]chloroform metabolites. Mice were sacrificed
5 hr after 14CHCl3 dosing with an ip injection of Tanax (5 ml/kg body wt).
Microsomal preparations were obtained from liver and kidneys as previously described (Testai and Vittozzi, 1986; Ade et al., 1994). Microsomal
lipids were extracted from aliquots of 250 ml of microsomal suspension
(equivalent to 1 g wet tissue), according to the method of Folch et al.
(1957). Covalent binding of [14C]chloroform metabolites to microsomal PL
moieties was measured by the method of De Biasi et al. (1992).
Serum enzymes and tissue GSH. Treated and control animals were
sacrificed by means of cervical dislocation at 5 or 24 hr after CHCl3 treatment. Serum ALT, creatinine, triglycerides, and BUN were measured with
commercial kits and serum sorbitol-dehydrogenase activity (SDH) according to Rose and Henderson (1975). Liver and kidneys were rapidly
removed and their cytosolic GSH contents were measured as described by
Ellman (1959).
Histology and BrdU immunohistochemistry. CHCl3-treated and control animals were injected twice with BrdU (100 mg/kg body wt) in 0.1 M
phosphate buffer saline (pH 7.2) 2 and 1 hr before sacrifice. They were
sacrificed in an ether-saturated chamber 5, 8, 12, 24, 48, and 96 hr or 1
week after CHCl3 treatment. Liver and kidneys were rapidly removed, fixed
in 70% ethanol, and processed for paraffin embedding. Serial sections were
cut at 4 mm nominal thickness. One set of sections was stained with hematoxylin and eosin (H&E) for morphological analysis. A second set was
immunohistochemically stained using a standard protocol to reveal BrdU
incorporation in ethanol-fixed tissues (Schutte et al., 1987). Sections were
incubated with an anti-BrdU mouse monoclonal antibody, followed by a
biotin/peroxidase-labeled streptavidin method (Biospa Division, Milano,
Italy) with 3,3*-diamino-benzidine as the chromogen. Stained sections were
counterstained with hematoxylin. Cells with incorporated BrdU were easily
identified by their brown stained nucleus and were counted in the entire
liver and kidney sections. The area of each section was measured with a
calibrated image cytometer (see following) and BrdU labeling was expressed as the number of labeled cells per square millimeter of tissue. The
average areas evaluated per animal were 30 and 27 mm2 for liver and
kidney sections, respectively. In animals with high proliferative activity,
labeled nuclei were counted in two circular areas of 1.5 mm diameter
randomly positioned on the section. BrdU labeling was then expressed as
the number of positive nuclei per unit area.
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GEMMA ET AL.
TABLE 1
Recovery of 14C Radioactivity in Male B6C3F1 Mice
after [14C]Chloroform Administration
Dose fraction (%)
Sample
0–24 hr
0–48 hr
Expired 14CHCl3
Expired 14CO2
Urine
Feces
Carcass
Total recovery
30.8 { 5.0
48.2 { 6.1
2.3 { 1.0
n.d.
n.d.
31.2 { 5.0
55.5 { 6.7
2.8 { 1.0
n.d.
n.d.
0–72 hr
31.0
56.7
3.0
0.2
1.2
92.1
{
{
{
{
{
{
5.4
6.4
1.1
0.01
0.3
9.2
Note. n.d., not determined. Groups of four male mice were dosed ip with
CHCl3 (150 mg/kg, equivalent to 1.26 mmol/kg body wt). Results represent means { SD of data obtained from at least three different groups of
treated mice.
14
Cytochemistry. Small samples of liver and kidneys were attached to a
piece of cork and quickly frozen in liquid nitrogen. Serial sections (8 mm
nominal thickness) were cut with a motorized cryostat and collected on a
glass coverslip precoated with 1004% poly-L-lysine. The sections were airdried and stored at 0807C in air-tight plastic boxes. Unfixed liver and kidney
sections were stained for glucose-6-phosphatase (G6Pase; EC 3.1.3.9) and
lactate dehydrogenase (LDH; EC 1.1.1.27) as previously described (Chieco
et al., 1988). Intracellular content of calcium was examined with alizarin red
S (McGee, 1985). Unfixed kidney sections were also stained for succinate
dehydrogenase (SuDH; EC 1.3.99.1), glycerophosphate dehydrogenase
(GPODH; EC 1.1.99.5), reduced nicotinamide adenine dinucleotide
(NADH)-dehydrogenase (NADH-DH; EC 1.6.99.3), reduced nicotinamide
adenine dinucleotide phosphate (NADPH)-dehydrogenase (NADPH-DH;
EC 1.6.2.4) as previously described (Chieco et al., 1988), for naphthyl
acetate esterase (NACES; EC 3.1.1.1, EC 3.1.1.2, EC 3.1.1.6) with a hexazotized pararosaniline method (Pearse, 1991) and for dipeptidyl peptidase
IV (DPP IV; EC 3.4.14.5) following Lojda (1979).
Cytometry. Measurements on histological sections were performed with a
computerized image analysis system (Cytometrica C&V, Bologna, Italy) using
a CCD video camera (XC77ce, Sony, Kangara-Ken, Japan) connected to a
Bravado frame grabber board (Truevision Indianapolis, IN). The system was
calibrated for morphometric and absorbance measurements as previously described (Chieco et al., 1994). GPODH, LDH, and NADPH-DH were measured
at a wavelength of 575 nm in three fields randomly selected in the cortex
using a 101 objective. For NACES the absorbance of five fields in the cortical
region was measured with a 101 objective at a wavelength of 520 nm. Threshold levels were set at 0.4 absorbance units (A.U.) for GPODH and LDH, at
0.1 A.U. for NADPH-DH, and at 0.2 A.U. for NACES. Section thickness was
corrected by measuring a predetermined area on three different serial sections
and reporting the original measure to the average of these. Measurements were
expressed as integrated absorbance per square micrometer of tissue.
Statistical analysis. The ANOVA test was used to determine significant
differences in cell proliferation and cytometry results between control and
treated animals. p õ 0.05 was considered significant. Data for biochemical
markers of CHCl3-induced toxicity from control and treated mice were
compared using Student’s t test.
stant up to 1 hr after dosing, being equal to 232 { 18 (90%
confidence interval) mmol/kg body wt/hr. By 2 hr the mice
had exhaled about 85% of total 14CHCl3 expired during the
first 24 hr post-treatment. 14CO2 expiration was slow during
the first 30 min, and then it increased reaching a rate of
102.5 { 7.0 (90% confidence interval) mmol/kg body wt/hr
which remained constant for about 3 hr. By 5 hr, the mice
had exhaled 73% of the total 14CO2 expired during the first
72 hr post-treatment. The cumulative recovery of 14CO2 increased significantly until 24 hr post-treatment, attaining
90% of the total expired 14CO2 . Most [14C]chloroform-derived radioactivity excreted in the urine was detected within
24 hr. The percentage of 14C radioactivity expired as 14CHCl3
and 14CO2 or excreted in the urine by 24, 48, and 72 hr is
reported in Table 1, together with the estimated total recovery of 14C label.
Figure 1 shows the levels of 14CHCl3 (toluene-extractable
14
C label) in the liver, kidneys, and blood after a single
ip injection of 14CHCl3 . Alkali-soluble radioactivity due to
14
CHCl3-derived metabolites (14CO2) in blood is also reported in Fig. 1. The halomethane was rapidly distributed
to these tissues, as indicated by the peak levels detected at
10 min after 14CHCl3 injection in liver, kidneys, and blood.
The kinetics of 14CHCl3 elimination were also similar in the
liver, kidneys, and blood, with significant levels of toluenesoluble radiolabeled material no longer detectable after 3 hr.
Blood level of 14CO2 increased during the first hour after
dosing, remained almost stable from 1 to 4 hr, and then
slowly decreased.
Similar amounts of covalent adducts with PL were recovered in hepatic and renal microsome fractions at 5 hr after
[14C]chloroform injection. Specifically, the amounts of adducts to PL polar heads were 4.20 { 1.30 and 3.95 { 1.57
RESULTS
Toxicokinetics and in Vivo Covalent Binding of
[14C]Chloroform
[14C]Chloroform expiration rate in B6C3F1 male mice
treated ip with 14CHCl3 (1.26 mmol/kg body wt) was con-
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FIG. 1. Blood and tissue levels of 14CHCl3 and 14CO2 in B6C3F1 male
mice dosed with a single ip injection of 1.26 mmol/kg body wt (equivalent
to 150 mg/kg body wt). Results represent means { SD of data obtained
from at least three groups of four or five animals.
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CHCl3 METABOLISM AND CELL PROLIFERATION
TABLE 2
Serum Parameters and Tissue GSH in Control and CHCl3-Treated Male B6C3F1 Mice
CHCl3 dose
(mg/kg body wt)
Hepatic GSH
(mmol/g)
ALT
(U/liter)
SDH
(U/liter)
Triglycerides
(mg/100 ml)
Renal GSH
(mmol/g)
Creatinine
(mg/100 ml)
BUN
(mg/100 ml)
0
150
0
150
0
150
0
150
0
150
0
150
0
150
5 hr
3.1
3.3
144
266
126
2069
80
72
3.0
1.4
0.9
1.1
61
103
{
{
{
{
{
{
{
{
{
{
{
{
{
{
0.4
1.2
54
130
18
484
18
9
0.5
0.3
0.05
0.1
13
6
24 hr
(6)
(6)
(8)
(7)
(6)
(4)*
(6)
(6)
(5)
(5)*
(6)
(5)*
(6)
(5)*
3.2
3.2
94
2269
240
2716
181
170
2.7
1.4
1.1
2.0
97
336
{
{
{
{
{
{
{
{
{
{
{
{
{
{
1.2
1.0
10
1267
112
163
37
42
0.4
0.4
0.3
0.2
18
52
(7)
(6)
(3)
(3)*
(3)
(3)*
(3)
(3)
(3)
(3)*
(3)
(3)**
(3)
(3)*
Note. Results are expressed as means { SD of data obtained from groups of five animals. Numbers in parentheses represent the number of independent
determinations. Asterisks indicate results significantly different from their respective controls at p õ 0.002 (*) and p õ 0.02 (**).
nmol PL-bound 14C/g tissue (means { SD) and the amounts
of adducts to the PL fatty acyl chains were 1.64 { 0.52 and
1.37 { 0.67 nmol PL-bound 14C/g tissue (means { SD) in the
liver and in the kidneys, respectively. If reactive metabolites
(phosgene or dichloromethyl radicals) are necessary intermediates for the metabolism of chloroform to CO2 and covalent
adducts to cell components, then our data indicate that a
very small percentage of reactive intermediates binds to hepatic and renal microsomal PL (about 0.03% of administered
dose), while most intermediates are transformed to CO2
(about 57% of the dose) (Table 1). Based on other reports
of covalent binding to microsomal protein and to other subcellular fractions (Uehleke and Werner, 1975; Hill et al.,
1975), we estimate that the total amount of radioactivity
covalently bound to the liver and the kidneys represents less
than 0.12% of the administered dose of 14CHCl3 .
changes spread all over the cortex (Figs. 2A and 2B). Necrotic tubules accounted for 15, 40, 25, and 10% of total
tubules at 12, 24, 48, and 96 hr, respectively. At 48 hr, wellrecognizable basophilic regenerating cells were scattered
among necrotic areas. The extent of regeneration was higher
at 96 hr and 1 week after exposure. Regenerating tubules
were enriched in BrdU-positive nuclei. The time course of
compensatory cell regeneration in the kidney is shown in
Fig. 3B.
No morphological alterations were found in the liver of
treated mice at any time point (5, 8, 12, 24, 48, 96, or
168 hr). An increased hepatocyte proliferation was observed
starting at 24 hr after treatment and peaking at 48 hr
(Fig. 3A).
Serum Chemistry and Tissue GSH
Cytochemical analysis was carried out at 5, 8, and 12
hr after CHCl3 administration to detect damage preceding
necrotic changes in the kidneys and the presence of subtle
changes in the liver preceding increased proliferation. The
enzymes selected are located in the cytoplasm (LDH) or in
different parts of cell membranes, and changes in their activities are indicative of damage in the surrounding membrane
or of cell metabolism alterations. Succinate dehydrogenase
and GPODH are located in the internal and external leaflets
of the inner mitochondrial membrane, respectively; G6Pase
and NADPH-DH are present in the smooth endoplasmic
reticulum; NACES is present mostly in mitochondrial and
endoplasmic reticulum membranes; and DPP IV is located
in the hepatocyte plasma membrane lining bile canaliculi
and in the brush border membrane of renal tubular cells.
Serum SDH, creatinine and BUN were increased at 5 hr
after ip administration of 150 mg/kg body wt CHCl3 (Table
2). At 24 hr all serum parameters, except triglycerides, significantly differed from control values. A significant GSH
depletion (50%) was found in the kidney at 5 and 24 hr
post-treatment, but not in the liver.
Morphological and Immunohistochemical Analysis
On histological examination, chloroform was found to
cause extensive cortical renal damage. At 5 hr after CHCl3
treatment, scattered proximal tubules lined by necrotic epithelium were observed in one of three treated mice. At later
time points all treated animals had similar pathologic
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Cytochemical Analysis
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GEMMA ET AL.
FIG. 2. Morphology and cytochemistry of kidney lesions in CHCl3-treated and control B6C3F1 mice. (A) Control kidney, H&E (1601). (B) At 24
hr after treatment with CHCl3 several proximal tubules are lined by necrotic epithelium (arrows), H&E (1601). (C) Calcium accumulation is not observed
in control mice. Darker dots in the figure are cell nuclei, Alizarin red staining (2601). (D) Starting 5 hr after CHCl3 treatment, scattered tubules (arrows)
display an increase of calcium content, Alizarin red staining (2601). (E) NACES activity is uniformly distributed in the kidneys of control mice,
Pararosaniline method (2601). (F) A strong increase in NACES activity is observed in most tubules at 5 hr after treatment, Pararosaniline method
(2601).
Kidneys of CHCl3-treated animals showed increased cellular calcium in scattered tubules starting at 5 hr (Figs. 2C–
2D). Microscopic areas with decreased LDH activity were
observed in the cortex at 8 and 12 hr. A transient increase
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in NADPH-DH was observed in medullary rays at 5 hr after
CHCl3 administration, but not at 8 and 12 hr. NACES (Figs.
2E–2F) and GPODH were definitely increased in several
cortical tubules already at 5 hr. The time course of enzymatic
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CHCl3 METABOLISM AND CELL PROLIFERATION
kg body wt compare well with the results of a previous study
(Mink et al., 1986). At this dose level, most 14CO2 was
already expired, 14CHCl3 was no longer detectable in the
expired air, and very low levels of the halomethane were
found in the metabolically competent tissues, by 5 hr. Therefore, the measurement of the adducts at 5 hr after the treatment provides representative information on chloroform bioactivation, while minimizing the possible occurrence of confounding secondary effects (Stevens and Anders, 1981).
We monitored 14CHCl3 conversion to reactive metabolites
in the liver and kidneys of male B6C3F1 mice using an
assay which exploits the regioselectivity of binding to microsomal PL. Specifically, the reactive species formed by the
reductive and the oxidative pathways of chloroform metabolism (namely, dichloromethyl radical and phosgene) were
reported to produce adducts with PL fatty acyl chains and
PL polar heads, respectively (De Biasi et al., 1992). This
assay was also reported to selectively detect the formation
of electrophiles and radicals produced from halomethanes
in vivo (De Curtis et al., 1994). Our data indicate that the
proportion of 14CHCl3-derived radioactivity associated with
the PL fatty acyl chains (29.0 { 6.0% of total bound radioactivity in the liver and 21.6 { 2.5% in the kidneys, means {
SE) was higher than that measured in vitro (10.5 { 1.0 and
12.9 { 1.7%) in air-equilibrated incubations of 14CHCl3 with
hepatic and renal microsomes, respectively (De Biasi et al.,
1992; Ade et al., 1994), and in vivo in the liver of S.D. rats
(10.4 { 3.6%; Gemma et al., 1994). Therefore, a significant
formation of •CHCl2 radical adducts did occur in both organs,
although formation of adducts predominantly occurred from
the oxidative pathway of chloroform metabolism. Notice,
however, that the total amount of reactive metabolites
formed from chloroform, as calculated by the expired 14CO2 ,
is extremely high, although we detected only a tiny fraction
as covalent adducts. It is likely that an effective chemical
or enzymatic scavenging occurs for reactive metabolites of
chloroform (Testai et al., 1990; Ade et al., 1994). Therefore,
the levels of covalent adducts cannot be considered a quantitative measure of chloroform activation in vivo. Neverthe-
FIG. 3. Cell regeneration in liver (A) and kidneys (B) of male B6C3F1
mice treated by gavage with a single dose of chloroform in corn oil (150
mg/kg body wt) (dotted line) or with corn oil alone (solid line). Results are
expressed as number of nuclei/mm2, incorporating BrdU. Results represent
the mean { SE of data obtained from at least three animals. Maximum cell
proliferation corresponded to a labeling index (labeled nuclei/100 cells,
means { SD) of 2.6 { 0.8 in the liver and 3.9 { 1.1 in the kidneys. Results
differing significantly from control values (with p õ 0.05) are marked by
asterisks.
changes is summarized in Table 3. Activities of SuDH,
NADH-DH, G6Pase, and DPP IV were unchanged.
Liver of treated animals showed activity levels of LDH
and G6Pase similar to controls and no hepatocytes showed
increased staining for calcium.
DISCUSSION
The observed kinetics of 14CHCl3 and 14CO2 expiration
by the B6C3F1 mice after ip injection of 1.26 mmol 14CHCl3/
TABLE 3
Cytochemical Parameters of CHCl3-Induced Nephrotoxicity in Control and Treated Male B6C3F1 Mice
Treated mice
Control mice
LDH
NADPH-DH
NACES
GPODH
253.1
78.6
242.6
231.1
{
{
{
{
32.2
15.9
34.3
53.6
5 hr
238.1
123.0
350.3
339.6
{
{
{
{
8 hr
38.1
32.3*
58.0*
69.5*
144.5
80.9
452.1
333.9
{
{
{
{
61.7*
12.6
61.1*
49.1*
12 hr
119.9
87.2
363.7
324.6
{
{
{
{
23.0*
17.6
38.9*
37.3*
Note. Treated mice (4 or 5 animals per group) were dosed by gavage with CHCl3 in corn oil (150 mg/kg body wt). Results are expressed as the
integrated absorbance per square micrometer (Ar103/mm2) and represent means { SD of data obtained from three different sections of each kidney.
Asterisks indicate results significantly different from their respective controls with p õ 0.05.
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GEMMA ET AL.
less, our results indicate similar amounts of both types of
PL adducts are formed in the liver and kidneys of B6C3F1
male mice.
In spite of the metabolic similarity of the liver and the
kidneys, overt toxic effects were detected only in the kidneys
at 150 mg/kg body wt, which is close to the bioassay dose
(NCI, 1976). Early tissue alterations include GSH depletion,
as is typical for CHCl3 acute nephrotoxicity (Smith et al.,
1983; Docks and Krishna, 1976). At 5 hr calcium accumulation also was evident in a few tubules. Spotty LDH release
from tubule cells was evident at 8 hr, and necrotic cells were
morphologically detectable at 12 hr. These initial alterations
progressed to massive renal necrosis, which was highest at
24 hr, accompanied by a reduced creatinine clearance. Since
the activities of the membrane enzymes examined with cytochemical methods were not decreased at early times after
chloroform treatment, the tubule cell necrosis does not seem
to be preceded by specific damage to the mitochondrial,
microsomal, or plasma membranes. No definitive explanation can be given for the early and remarkable increase of
NACES in tubule cells of CHCl3-treated mice. Since NACES
is a marker of nonspecific esterases, its increase might be
related to the processing of phosgene adducts through the
cleavage of their ester and amide bonds. We also observed
increased activity of mitochondrial GPODH in the kidneys
of chloroform-treated mice. This enzyme is involved in PL
synthesis and its activity could have been stimulated by the
high Ca2/ cellular influx (Fisher et al., 1973; Wernette et
al., 1981), which occurred early after chloroform exposure.
Renal damage was followed by a high rate of cellular proliferation which started at 48 hr post-treatment and was sustained for longer than 5 days.
No histological nor cytochemical signs of cellular injury
were detected in the livers of chloroform-treated mice at
early time points (5, 8, or 12 hr). No evidence of GSH
depletion, which is an early and sensitive alteration associated with acute chloroform hepatotoxicity, was found (Docks
and Krishna, 1976; Brown et al., 1974; Ekstrom and Högberg, 1980). No hepatic injury was histologically detectable
at later time points (24, 48, 96, and 168 hr). The normal
serum levels of triglycerides throughout the experiment are
consistent with a lack of liver dysfunction. The increases of
serum SDH and ALT were the only alterations observed
suggestive of hepatic damage. Such marked changes (Table
2) are not consistent with a histologically undetectable or
limited damage of the liver. Nevertheless it is possible that
these enzymes leak from liver cells due to membrane permeability changes. On the other hand, serum ALT is not liver
specific and may reflect also renal injury (Zimmerman, 1978;
Plaa and Hewitt, 1982), while SDH increase may be caused
by CHCl3-induced lysis of red cells (Belfiore and Zimmerman, 1970), which contain high SDH activity in mice (Agar,
1979). The weight of the evidence, therefore, favors the
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conclusion that no appreciable liver necrosis occurred in
B6C3F1 mice treated with 150 mg CHCl3/kg body wt. Such
results are in agreement with previous reports that no hepatic
necrosis occurred in male mice treated similarly (Eschenbrenner and Miller, 1945; Moore et al., 1982; Bull et al.,
1986; Larson et al., 1994).
A transient cell proliferation was observed in the liver
starting at 24 and peaking at 48 hr. Because of the lack of
histologically detectable liver necrosis, this proliferation may
represent a response of the liver to a mild cellular insult
caused by CHCl3 , to products from the damaged kidney, or
to circulating mediators produced to stimulate kidney cell
regeneration.
Although extrapolations from single dose to multiple dose
experiments are of limited value, some cautious comments
might be appropriate. Tumor development in B6C3F1 mice
has been related to chloroform-induced cytolethality followed by increased cell replication (Larson et al., 1993).
However, weak hepatocyte proliferation was observed in
the livers of male B6C3F1 mice after single or repeated
chloroform treatments at the lowest carcinogenic dose (Larson et al., 1994; present results). On the other hand, renal
tubule cells of male B6C3F1 mice underwent massive necrosis followed by intense proliferation (Larson et al., 1994;
present results), but no tumors were reported in the kidney
(NCI, 1976). Therefore, the role of cell proliferation in chloroform-induced kidney tumors is unclear.
Biological models (Cohen and Ellwein, 1990a,b) point
out the importance of tissue necrosis-mediated cell proliferation in carcinogenesis. These models indicate that DNA mutations and tumors may also occur as a consequence of increased cell replication (epigenetic mechanism). Our observations raise questions about the role of cell proliferation as
the only determinant of chloroform-induced tumor development, since bioassays have found tumors in liver but not
kidneys of chloroform-treated B6C3F1 mice (NCI, 1976).
Based on the present results, together with findings of DNA
adducts of chloroform metabolites in the liver and kidneys
in vivo (Pereira et al., 1982; Colacci et al., 1991), we suggest
that a weak genotoxic action plus cell proliferation may
result in tumor formation (as is the case of the liver), while
massive necrosis, eliminating cells with damaged DNA, has
an antagonistic effect resulting in no tumor formation (as is
the case of the kidneys). The organ-specific carcinogenesis
of chloroform in B6C3F1 mice seems therefore regulated
by a balance between its cytotoxic and weak genotoxic effects. Such a balance is likely dependent mainly on the presence of differently effective cellular repair and protection
systems (e.g., GSH) in hepatocytes and tubular cells, since
reactive metabolite formation appeared similar in the two
organs.
ACKNOWLEDGMENTS
We thank Professor H. M. Mehendale for his helpful discussion on experimental design and results. This work has been partially supported by the
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CHCl3 METABOLISM AND CELL PROLIFERATION
National Research Council, within the frame of the Targeted Project ‘‘Clinical Applications of Oncologic Research,’’ Grant No. 95.00488.39.
Ade, P., Guastadisegni, C., Testai, E., and Vittozzi, L. (1994). Multiple
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