Development and Characterization of a New Model of

Tacrine-Induced Hepatotoxicity: Role of the Sympathetic

Nervous System and Hypoxia-Reoxygenation
1
ROBERT F. STACHLEWITZ, GAVIN E. ARTEEL, JAMES A. RALEIGH, HENRY D. CONNOR, RONALD P. MASON and
RONALD G. THURMAN
Department of Pharmacology (R.F.S., R.G.T.), Curriculum in Toxicology (G.E.A., J.A.R., R.G.T.) and Department of Radiation Oncology (J.A.R.),
University of North Carolina at Chapel Hill, Chapel Hill and Laboratory of Molecular Biophysics (H.D.C., R.P.M.), NIEHS, NIH, Research Triangle
Park, North Carolina
Accepted for publication May 23, 1997
ABSTRACT
Tacrine is an acetylcholinesterase inhibitor approved for the
treatment of Alzheimers disease. Unfortunately, reversible
hepatotoxicity in about 30% of patients at therapeutic doses
limits clinical use. The purpose of this study was to develop and
characterize a model of tacrine hepatotoxicity to begin to un-
derstand the mechanisms of injury. Rats were given tacrine
(1050 mg/kg, intragatrically) and killed 24 hr later. An increase
in serum aspartate aminotransferase was observed up to 35
mg/kg and histology revealed pericentral necrosis and fatty
changes. Aspartate aminotransferase was increased from 12 to
24 hr and returned to control values by 32 hr. Livers were
perfused in a nonrecirculating system to measure oxygen up-
take and trypan blue was infused at the end of each experiment
to evaluate tissue perfusion. Time for trypan blue to distribute
evenly throughout the liver 3 hr after tacrine treatment was
significantly increased (6.9 1.3 min) compared to controls
(1.0 0.3 min) reflecting decreased tissue perfusion. Tacrine
also significantly increased the binding of a hypoxia marker,
pimonidazole, in pericentral regions almost 3-fold, and in-
creased portal pressure in vivo significantly. It is hypothesized
that tacrine, by inhibiting acetylcholine breakdown in the celiac
ganglion, increases sympathetic activity in the liver leading to
vascular constriction, hypoxia and liver injury. To test this hy-
pothesis, the hepatic nerve was severed and animals were
allowed to recover before tacrine treatment. This procedure
significantly reduced serum aspartate aminotransferase, time
of dye distribution, pimonidazole binding and portal pressure.
Furthermore, a free radical adduct was detected with spin
trapping and electron spin resonance spectroscopy 8 hr after
tacrine treatment, providing evidence for reoxygenation. When
catechin (100 mg/kg, i.p.), a free radical scavenger, was given
before tacrine, injury was decreased by about 45%. Further-
more, feeding 5% arginine in the diet significantly reduced
portal pressure and time of dye distribution. These data are
consistent with the hypothesis that tacrine hepatotoxicity is a
hypoxia-reoxygenation injury mediated through the sympa-
thetic nervous system.
Alzheimers disease affects about 4 million people in the
United States and is characterized by a degradation of cho-
linergic nerves in the cerebral cortex and hippocampus lead-
ing to a decrease in cholinergic transmission (Owens, 1993).
Drug therapy to increase cholinergic transmission has been
one strategy used to combat the symptoms of Alzheimers
disease (Starr, 1992; Volger, 1991). Tetrahydroaminoacri-
dine (also known as THA, tacrine, and Cognex) is the first
agent approved by the Food and Drug Administration for
treatment of Alzheimers disease. Tacrine acts as an acetyl-
cholinesterase inhibitor to block the degradation of acetyl-
choline in the neurons of cerebral cortex thereby increasing
cholinergic transmission (Wu and Yang, 1989; Hunter et al.,
1989). Preclinical trials showed that many Alzheimers pa-
tients had a significant improvement in symptoms when
tacrine (23 mg/kg/day) was administered on a daily basis
(Farlow et al., 1992; Gamzu et al., 1990; Summers et al.,
1981). However, prolonged use of tacrine has proven to be
hepatotoxic in about 30% of the patients (Elinder et al., 1989;
Ames et al., 1990; Forsyth et al., 1989b; Hammel et al., 1990;
Marx, 1987; OBrien et al., 1991; Summers et al., 1981; Sum-
mers et al., 1989) by unknown mechanisms.
Liver injury due to tacrine is characterized by an elevation
of the liver-specific enzyme AST (Summers et al., 1989).
Histopathology, documented by liver biopsy, revealed peri-
central necrosis and fat accumulation in midzonal regions as
well as a few cases of drug-induced granulomatous hepatitis
(Waktkins et al., 1994; OBrien et al., 1991; Ames et al., 1990;
Received for publication January 10, 1997.
1
This work was supported, in part, by a grant from NIH (ES-04325), a
generous gift from Parke-Davis and a traineeship to R.S. (GM- 070402).
ABBREVIATIONS: AST, aspartate aminotransferase; 4-POBN, -(4-pyridyl-1-oxide)-N-tert-butylnitrone; ESR, electron spin resonance.
0022-3565/97/2823-1591$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3
Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 282:15911599, 1997
1591
Forsyth et al., 1989a). Because liver biopsy is not a practical
alternative to monitor hepatotoxicity, blood must be drawn
weekly to determine if liver enzyme levels are within the
normal range. If AST levels are greater than two times nor-
mal, the patient is removed from tacrine for 4 to 6 wk or until
AST levels decline to values within normal limits (Cognex
insert, Parke-Davis, Ann Arbor, MI, 1993).
The mechanism by which tacrine causes hepatotoxicity has
not been elucidated, in part, because of the lack of an animal
model to study the injury. In safety studies using rats, mice
and dogs, chronic tacrine treatment failed to produce liver
injury (Fitten et al., 1987; Woolf et al., 1993). From these
studies, it was suggested that these species are less vulner-
able to tacrine hepatotoxicity because they produce fewer
toxic metabolites (Hsu et al., 1990; Madden et al., 1993; Woolf
et al., 1993). However, this hypothesis is not likely because
hepatotoxicity of tacrine in humans correlates directly with
the concentration of tacrine in the blood and not with the
concentration of tacrine metabolites (Ford et al., 1993).
Our first goal was to develop an acute animal model in the
rat to characterize the hepatotoxicity of tacrine by complet-
ing dose and time course studies of tacrine toxicity. Because
tacrine caused a midzonal and pericentral injury, we hypoth-
esized that it produced hypoxia in the liver leading to peri-
central cell death. Accordingly, the second goal of these ex-
periments was to use this new model to study the mechanism
of acute tacrine hepatotoxicity. Preliminary accounts of this
work have appeared elsewhere (Stachlewitz and Thurman,
1996).
Materials and Methods
Chemicals. Tacrine (9-amino-1,2,3,4-tetra-hydroacridine hydro-
chloride, catalog #A3773), trypan blue (#T0776), and catechin
(#C1251, 98% pure) were purchased from Sigma Chemical Company
(St. Louis, MO). -(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN,
#P-0020) was purchased from OMRF Spin Trap Source (Oklahoma
City, OK). Pimonidazole hydrochloride was synthesized according to
published procedures (Smithen and Hardy, 1982) and characterized
using standard chromatographic, elemental analysis and spectro-
graphic techniques. Chemicals for the preparation of formalin-fixed,
paraffin-embedded tissue sections were reagent grade purity from
local suppliers. Vector Laboratories Inc. (Burlingame, CA) supplied
the ABC peroxidase Vectastain kit, avidin-biotin blocking kit, rat
adsorbed horse-antimouse antibodies and the DAB peroxidase sub-
strate. The antibody to pimonidazole (Raleigh and Koch, 1990) was
isotyped using a Clonotyping System/AP kit purchased from Fisher
Scientific Company (Pittsburgh, PA).
Experimental animals. Female Sprague-Dawley rats (weight
range 175225 g) were given food and water ad libitum and treated
intragastrically with up to 50 mg/kg tacrine dissolved in saline.
Animals were anesthetized with sodium pentobarbital (50 mg/kg) or
methoxyflurane before all surgical procedures. Blood was drawn
from the descending vena cava for enzyme analysis and the liver was
perfused as described below. All animals received humane care in
accordance with institutional guidelines.
Microsurgical sympathectomy in the liver. Rats were anes-
thetized and maintained with methoxyflurane. The peritoneum was
opened and the portal vein, hepatic artery and bile duct were ex-
posed. A 3-mm wide region of tissue around the vessels and bile duct
was removed to cut the sympathetic nerve (Cucchiaro et al., 1990).
Additionally, ligaments surrounding the liver were also severed to
remove sympathetic nerves entering via this route. Sham operations
were also performed by opening the abdomen and exposing the portal
vein for 10 min. The incision was closed and the rat was allowed to
recover at least 72 hr before tacrine treatment.
Liver perfusion. Livers were perfused with Krebs-Henseleit-
bicarbonate buffer (pH 7.4, 37C) saturated with an oxygen-carbon
dioxide mixture (95:5) in a nonrecirculating system as described
elsewhere (Scholz et al., 1973). Perfusate leaving the liver was al-
lowed to flow past a Clark-type oxygen electrode shielded by Teflon
(Instech Laboratories, Plymouth Meeting, PA) to measure oxygen
content in the perfusate. Oxygen uptake was calculated from the
difference between oxygen concentration in the perfusate entering
and leaving the system, the flow rate and the liver weight. Oxygen
uptake values were used to assess tissue viability during perfusion.
Trypan blue distribution. Trypan blue infusion has been used
to assess changes in tissue perfusion (Beckh et al., 1985; Ji et al.,
1984; Zhong et al., 1996). A 0.5 mM trypan blue solution (Sigma) in
Krebs-Henseleit-bicarbonate buffer saturated with an oxygen-car-
bon dioxide mixture (95:5) was infused into the perfused liver for up
to 15 min at the end of each perfusion experiment (Belinsky et al.,
1984). The time for trypan blue to distribute evenly to all regions of
the liver was recorded to assess changes in tissue perfusion.
Use of pimonidazole to detect hypoxia in the liver. One hour
after treatment with tacrine (35 mg/kg) or saline, animals received
pimonidazole (120 mg/kg i.p.) (Arteel et al., 1995, 1996). Pimonida-
zole detects hypoxia in vivo by binding in cells at oxygen concentra-
tions of 14 M or less (Raleigh and Koch, 1990). After 2 hr, animals
were anesthetized with pentobarbital (50 mg/kg i.p.) and livers were
isolated surgically. The liver was perfusion-fixed with 10% formalin,
and tissue sections were collected from the right lobe for immuno-
histochemical analysis of pimonidazole adduct binding.
Analysis of tissue-bound pimonidazole by immunohisto-
chemistry. Paraffin blocks of formalin-fixed liver tissue were sec-
tioned at 6 m and pimonidazole adducts were detected with a
biotin-streptavidin-peroxidase indirect immunostaining method
modified for rat livers (Arteel et al., 1995). Sections were hydrated
and treated briefly with 0.01% protease (pronase E) and exposed to
mouse antipimonidazole IgG
1
antibody in PBS-Tween for 2 hr at
37C. Rat adsorbed horse anti-mouse antibody was then applied to
the sections for 30 min. Once the antibody-biotin-peroxidase complex
was formed, DAB chromogen was added as the peroxidase substrate.
After the immunostaining procedure, a light counterstain of hema-
toxylin was applied followed by mounting with crystal mount solu-
tion.
Image analysis of immunohistochemistry. A Universal Imag-
ing Corp. image acquisition and analysis system (Chester, PA) in-
corporating an Axioskop 50 microscope (Carl Zeiss, Inc., Thornwood,
NY) was used to capture and analyze the immunostained tissue
sections at 40 magnification as described previously (Arteel et al.,
1995). Five pericentral fields were chosen randomly from each tissue
section and positioned so that vessel lumenae were in the center of
each field. Color detection thresholds were set for the red-brown color
the DAB chromogen based on an intensely labeled point; subse-
quently, a default color threshold range was assigned. The percent-
age of pericentral area positive for pimonidazole was determined
from the percentage of the field labeled intensely with DAB chromo-
gen minus the acellular space. For uniformity, comparison of label-
ing was restricted to lumenae whose diameters fell in the range from
5 to 8 m. To avoid the effects of nonspecific staining, regions near
the edge of tissue sections were not evaluated.
In vivo portal pressure. A piece of tygon tubing (i.d. 3/16 inch)
was attached to a meter stick that was positioned vertically as a
manometer and a 22-gauge needle was placed at the end. The tubing
was filled with Krebs-Heinseliet buffer and clamped to keep the
fluid from escaping. The needle was inserted into the portal vein and
the clamp was removed. After the buffer stopped moving in the tube,
a measurement was read from the meter stick in centimeters. This
measurement represents the portal pressure in vivo in centimeters
of water. The apparatus was routinely calibrated with a manometer.
1592 Stachlewitz et al.
Vol. 282
Detection of free radical adducts. To assess free radical for-
mation after tacrine treatment, the spin trapping reagent 4-POBN
(250 mg/kg, dissolved in saline) was injected into the tail vein 7 hr
after tacrine treatment. The abdomen was opened while the rat was
under ether anesthesia and bile was collected for 1 hr via a cannula
(polyethylene tubing #50) placed in the common bile duct into 50 l
dipyridyl on ice to prevent ex vivo free radical formation and stored
on dry ice until analysis (Knecht et al., 1990). Bile samples were then
thawed, placed in a quartz ESR cell, and scanned. Free radical
adducts were detected with a Bruker ESP 106 ESR spectrometer.
Instrument conditions were as follows: 20-mW microwave power;
1.007-G modulation amplitude and 80-G scan range.
Histology and serum enzymes. One centimeter thick sections
of liver were placed in 1% paraformaldahyde for at least 24 hr. The
fixed tissue was embedded in paraffin, prepared for light microscopy
and stained with hemtatoxylin and eosin. Blood was collected from
the descending vena cava or tail vein, placed in 1.5-ml plastic tubes
and allowed to clot. Serum was separated by centrifugation (500 g
and stored at -20C until AST activity was measured by standard
enzymatic methods (Bergmeyer, 1983).
Statistics. All data are presented as mean S.E.M. Comparisons
between groups were performed by Students t test, analysis of
variance or repeated measures analysis of variance followed by Bon-
ferronis post hoc test for multiple comparisons. The criterion for
significance of P .05 was selected before the study.
Results
Characterization of tacrine-induced liver injury. An-
imals were treated with 10 to 50 mg/kg tacrine or a compa-
rable amount of saline intragastrically 24 hr before serum
was collected for measurement of AST as described in Ma-
terials and Methods. Tacrine caused a significant increase
in serum AST activity at 35 mg/kg (table 1). Animals treated
with a higher dose of tacrine (50 mg/kg) died within 4 hr of
acute cholinergic toxicity as evidenced by salivation, tremor
and difficult breathing (Taylor, 1990) without any liver pa-
thology; therefore, 35 mg/kg was selected for all subsequent
studies.
Twenty four hours was selected as an end point for dose-
response experiments described above out of convenience. To
determine the complete time course, rats were sacrificed
every 4 hr for up to 32 hr after tacrine treatment and serum
AST measured (fig. 1). From 0 to 8 hr, there was a steady
increase in AST. At 12 hr, AST reached a plateau (120
U/liter) which remained about double the control value for up
to 24 hr. By 32 hr, values had returned to control levels (50
U/liter).
Histology from animals treated with 35 mg/kg tacrine for
24 hr and saline-treated controls is shown in figure 2. Livers
from untreated rats were normal (fig. 2A). In livers from the
tacrine-treated rats (fig. 2B), necrosis was detected in mid-
zonal and pericentral regions of the liver lobule accompanied
by fatty changes.
Role of hypoxia and the sympathetic nervous system
in tacrine-mediated hepatotoxicity. To determine if ta-
crine caused cell death, livers were perfused and then infused
with trypan blue at the end of the perfusion. About 10% of the
cells in the pericentral region stained positive for trypan blue
in histology. Yet, during perfusion, it was observed that the
distribution of trypan blue in the circulation of the tacrine-
treated liver was significantly impaired. Not only can trypan
blue be used in histologically to stain dead cells in the liver
(Belinsky et al., 1984), but time for vital dye to distribute
evenly can be used to detect changes in intrahepatic perfu-
sion (Beckh et al., 1985; Ji et al., 1984; Zhong et al., 1996).
Therefore, the time for trypan blue to distribute evenly in the
perfused liver was used as an index of changes in intrahe-
patic distribution of flow (fig. 3). Overall, there was a signif-
icant, nearly 7-fold increase in the time for trypan blue to
distribute when tacrine-treated animals (6.9 1.7 min) were
compared to controls (1.0 0.3 min), showing decreased
Fig. 1. Time course of AST release after tacrine administration. Blood
was drawn from the tail vein every 4 hr for 32 hr after tacrine (35 mg/kg)
treatment for determination of AST as described in Materials and
Methods. Values are the mean S.E.M. (n 4). Representative error
bars are shown.
TABLE 1
Serum AST levels in rats 24 hr after treatment with tacrine
a
Tacrine (mg/kg) AST (U/liter)
0 39 14
10 60 9
30 66 20
35 116 16
b
a
Rats were given 0 (control), 10, 30 or 35 mg/kg tacrine intragastrically. Blood
was drawn, serum was collected and AST levels were determined 24 hr after
treatment as described in Materials and Methods. Values are the mean
S.E.M. (n 6, 35 mg/kg tacrine treatment, n 4 all other groups). Statistical
comparisons were made using analysis of variance and Bonferronis post hoc
test.
b
P .05 compared to control.
1997
Acute Tacrine Hepatotoxicity 1593
tissue perfusion. However, there was no significant differ-
ence in the oxygen uptake of perfused livers ex vivo from
tacrine-treated animals (112 10 mol/g/hr) compared to
controls (105 7 mol/g/hr). When the hepatic nerve, which
is known to control liver microcirculation, was severed 72 hr
before tacrine treatment, the increase in trypan blue distri-
bution time by tacrine was prevented.
To test the hypothesis that redistribution of hepatic flow
caused by tacrine leads to hypoxia, the hypoxia marker pi-
monidazole was injected and 2 hr later livers were harvested
and pimonidazole adduct binding was quantitated (Arteel et
al., 1995, 1996). Tacrine significantly increased pimonidazole
binding nearly 3-fold in pericentral regions of the liver lobule
(fig. 4). To determine if the hepatic nerve is involved in the
mechanism of hypoxia, some rats underwent hepatic sympa-
thectomy or sham operation and were allowed to recover for
72 hr before tacrine administration. Sham operation alone
had no significant effect on the binding of pimonidazole after
tacrine treatment; however, sympathectomy significantly re-
duced pimonidazole binding. Most important, in animals
treated with tacrine, sympathectomy 72 hr before tacrine
treatment significantly decreased pericentral binding of pi-
monidazole compared to treated sham-operated animals.
There was a significant increase in portal pressure after
treatment with 35 mg/kg tacrine in unoperated and sham-
operated animals compared to saline-treated controls (fig. 5).
Cutting the sympathetic hepatic nerve completely blocked
the tacrine-mediated increase in portal pressure in vivo.
The ultimate test of the hypothesis that the hepatic nerve
is involved in tacrine-mediated hepatotoxicity is to show that
severing the nerve blocks damage. Accordingly, blood was
drawn 24 hr after tacrine treatment for measurement of AST
(fig. 6). As before, tacrine caused a significant increase in
AST compared to controls. Sham operation had no effect on
serum AST; however, cutting the sympathetic nerve before
tacrine treatment prevented the increase in serum AST.
Prevention of tacrine hepatotoxicity by hepatic den-
ervation is not due to inability to regulate body tem-
perature. Because tacrine is extensively metabolized by the
liver and hepatotoxicity has been proposed to be caused by
production of toxic metabolites, one possible explanation for
the observations in these studies is that severing the hepatic
nerve may decrease body temperature and inhibit metabo-
lism of tacrine. Accordingly, the time course of changes in
body temperature was measured after tacrine treatment (fig.
7). As with other acetylcholinesterase inhibitors (Kooka et
al., 1987), tacrine treatment caused a significant decrease in
body temperature of about 3C at 4 and 8 hr that was unaf-
fected by prior surgery. Body temperature returned to near
pretreatment values at 24 hr in all groups.
Evidence for free radical production and reperfu-
sion injury after tacrine treatment. It is possible that the
injury due to tacrine treatment is not the result of prolonged
hypoxia but instead occurs upon reperfusion when the vas-
Fig. 2. Liver histology from rats 24 hr after treatment with tacrine.
Animals were given saline (A and B) or 35 mg/kg tacrine intragastrically
(C and D) Livers were removed 24 hr later and prepared for light
microscopy as described in Materials and Methods. Representative
morphology from six rats per group is shown. A and C are at low (100
power) and B and D are at high (400) power.
Fig. 3. Time for trypan blue to fully distribute in the perfused liver. Rats
received tacrine (35 mg/kg) or a comparable volume of saline. Some
animals had hepatic sympathectomy or an appropriate sham operation
72 hr before treatment with tacrine (35 mg/kg). Livers were perfused as
in figure 3 and the time for trypan blue to fully distribute in the liver was
recorded. Values are mean S.E.M. (n 4). Statistical comparisons
were made using analysis of variance and Bonferronis post hoc test. a,
different from saline-treated control. b, different from sham operated
animal receiving tacrine.
1594 Stachlewitz et al.
Vol. 282
cular space increases (i.e., after tacrine is metabolized) and
oxygen reenters previously hypoxic cells leading to the pro-
duction of free radicals that cause cell injury. Using the spin
trapping technique, a free radical adduct was detected in the
bile of tacrine-treated animals 8 hr after treatment (fig. 8).
The radical spectrum simulated as a mixture of two radical
species (species I: a
N
15.8 and a
H
2.1, species II: a
N

15.6 and a
H
3.4). The coupling constants of species I is
consistent with a carbon-centered free radical while species
II is consistent with an oxygen-centered free radical.
Dietary arginine has been shown to prevent reperfusion
injury in the perfused rat liver (Jones and Thurman, 1996). A
diet high in the arginine (5% w/w), which is known to im-
prove the hepatic microcirculation most likely by increasing
nitric oxide production, or a control diet was fed to rats for 3
days before tacrine treatment. Dietary arginine prevented
the increases in portal pressure and time of vital dye distri-
bution caused by tacrine (table 2).
To determine if free radical production plays a role in
tacrine hepatotoxicity, the free radical scavenger catechin
(100 mg/kg) or saline vehicle was administered 1 hr before
tacrine treatment (fig. 9). Catechin prevented the increase in
AST observed with tacrine treatment (P .05).
Discussion
Development and characterization of a newmodel of
tacrine-induced liver injury. In this study, a new model
has been developed in the rat to study the hepatotoxicity of
tacrine. Tacrine caused a significant increase in serum AST,
a specific marker for liver toxicity (table 1), and injury in
midzonal and pericentral regions of the liver lobule as well as
fatty changes (fig. 2) at 35 mg/kg. Lower doses, as reported by
others (Hunter et al., 1989), do not cause significant liver
injury. This pattern of injury 24 hr after 35 mg/kg tacrine is
Fig. 4. Pimonidazole binding in pericentral regions of the liver lobule.
Animals received 35 mg/kg tacrine or a comparable volume of saline
intragastrically. Some animals had hepatic sympathectomy or a sham
operation 72 hr before treatment with tacrine. One hour after tacrine,
the animals received the hypoxia marker pimonidazole (120 mg/kg, i.p.)
and the liver was perfusion-fixed 2 hr later with formalin. Pimonidazole
binding was detected with immunohistochemistry and the average
region of binding around the pericentral vein was determined by image
analysis as described in Materials and Methods. Values are mean
S.E.M. (n 4). Statistical comparisons were made using analysis of
variance and Bonferronis post hoc test. a, different from saline-treated
control. b, different from sham operated animal receiving tacrine.
Fig. 5. Effect of tacrine on portal pressure in vivo. Animals were
treated with 35 mg/kg tacrine or a comparable volume of saline intra-
gastrically. Some animals had hepatic sympathectomy or a sham op-
eration 72 hr before treatment with tacrine. Portal pressure was mea-
sured 3 hr after tacrine treatment as described in Materials and
Methods. Values are the mean S.E.M. (n 4). Statistical compari-
sons were made using analysis of variance and Bonferronis post hoc
test. a, different from saline-treated control. b, different from sham
operated animal receiving tacrine.
1997
Acute Tacrine Hepatotoxicity 1595
similar to that observed with hypoxia (Lemasters et al.,
1981). Additionally, it resembles the pathology observed
in the clinic when patients are given tacrine chronically
(Waktkins et al., 1994; OBrien et al., 1991; Ames et al., 1990;
Forsyth et al., 1989a; Hammel et al., 1990). Furthermore,
tacrine decreased tissue perfusion (fig. 3), increased portal
pressure in vivo (fig. 5) and increased pimonidazole adduct
binding in the pericentral region of the lobule (fig. 4) provid-
ing evidence that tacrine causes hypoxia in the liver by
altering intrahepatic blood flow and decreasing tissue perfu-
sion. Collectively, these data support the hypothesis that
acute tacrine treatment causes hypoxia.
Role of the sympathetic nervous system in the mech-
anism of tacrine-induced hepatotoxicity. It is well
known that the sympathetic nervous system plays an impor-
tant role in control of the microcirculation in the liver (Lautt,
1980; Gardemann et al., 1992). The pathway by which the
liver is innervated is shown schematically in figure 10. The
sympathetic nerve controlling the liver vasculature origi-
nates from the cholinergic celiac ganglion. Acetylcholine is
released in the celiac ganglion and causes an action potential
that propagates through the hepatic nerve, which is the
afferent, sympathetic pathway to the liver. It is postulated
that norepinephrine is released directly onto endothelial cells
and stellate cells in periportal regions of the liver, decreasing
sinusoidal vascular space and tissue perfusion (Gardemann
et al., 1992). Infusion of noradrenaline or electrical stimula-
tion of the sympathetic pathway in the perfused liver alters
the distribution of trypan blue in the liver and decreases
surface oxygen tension (Beckh et al., 1985; Ji et al., 1984).
Prolonged hypoxia is known to cause cell death; therefore, it
was hypothesized that tacrine, by increasing acetylcholine at
the celiac ganglion, would increase sympathetic activity via
the hepatic nerve and release norepinephrine in the liver
causing injury. To test this hypothesis, the sympathetic af-
ferent to the liver was severed. Hepatic sympathectomy pre-
vented the decrease in tissue perfusion (fig. 3), the increase
in portal pressure (fig. 5), and the increase in binding of the
hypoxia marker pimonidazole in pericentral regions of the
Fig. 6. Prevention of acute, tacrine-induced liver injury by severing the
hepatic nerve. Animals were treated with 35 mg/kg tacrine or a com-
parable volume of saline intragastrically. Some animals had hepatic
sympathectomy or a sham operation 72 hr before tacrine. AST levels in
serum were measured 24 hr later as described in Materials and Meth-
ods. Values are the mean SEM (n 6, 35 mg/kg tacrine treatment,
n 4 all other groups). Statistical comparisons were made using
analysis of variance and Bonferronis post hoc test. a, different from
saline treated control. b, different from sham operated animal receiving
tacrine.
Fig. 7. Time course of changes in body temperature after tacrine
treatment. Basal body temperature was determined at 0 hr and animals
were given 35 mg/kg tacrine. Some animals had hepatic sympathec-
tomy or comparable sham operation 72 hr before tacrine. Body tem-
perature was measured every four hr. Values are mean S.E.M. (n
4). Statistical comparisons were made using repeated measures anal-
ysis of variance and Bonferronis post hoc test. a, all points at this time
are different from respective controls at 0 hr. b, denotes point is
different from control at 0 hr.
1596 Stachlewitz et al.
Vol. 282
liver (fig. 4). The increase in AST (fig. 6) caused by tacrine
was also reduced significantly by cutting the sympathetic
hepatic nerve. These data support the hypothesis that the
sympathetic hepatic nerve plays a central role in the mech-
anism of tacrine-induced liver injury.
Several years ago, it was shown that severing the spinal
cord just above the branch to the celiac ganglion, which
controls the sympathetic hepatic nerve, prevented carbon
(CCl
4
) tetrachloride toxicity (Calvert and Brody, 1960; Brody
et al., 1961). It was proposed that CCl
4
toxicity occurred
through the sympathetic nervous system by altering liver
hemodynamics. In an elegant study, however, Larson and
Plaa (1965) showed that cordotomy decreased body temper-
ature in the rat thereby decreasing bioactivation of CCl
4
explaining why injury was prevented (Larson et al., 1964).
Therefore, it was possible that cutting the sympathetic he-
patic nerve would decrease core body temperature thereby
decreasing bioactivation of tacrine and preventing hepatotox-
icity by a similar mechanism. However, the decrease in ta-
crine-induced liver injury after hepatic sympathectomy was
not due to the inability of animals to regulate body temper-
ature (fig. 7). There was no difference in body temperature in
any group before tacrine treatment. Moreover, tacrine treat-
ment caused only about a 3C decrease in core body temper-
ature that was independent of prior surgery. In the studies
with CCl
4
, there was about a 10C decrease in body temper-
ature when the drug was administered. Therefore, it is con-
cluded that hepatic denervation has little effect on body
temperature compared to cordotomy. Furthermore, it is con-
cluded that the decrease in tacrine toxicity after hepatic
denervation is not due to inability of animals to regulate body
temperature.
The data generated in these studies are consistent with the
hypothesis that increased stimulation of the sympathetic
pathway in the liver, mediated through the action of tacrine
at the celiac ganglion, causes hypoxia in the liver that plays
a major role in the mechanism of tacrine hepatotoxicity.
Reperfusion injury occurs after tacrine. As tacrine is
metabolized, it is proposed that resistance in the sinusoids
decreases because of diminishing stimulation of the sympa-
thetic hepatic nerve. Blood flow to the liver increases, restor-
ing oxygen to previously hypoxic cells (fig. 10). Reperfusion of
the liver after hypoxia could result in production of free
radicals by activated Kupffer cells or xanthine oxidase lead-
ing to parenchymal cell injury (Lemasters and Thurman,
1993). Indeed, we have detected two free radical species 8 hr
after tacrine treatment by spin trapping and ESR spectros-
copy (fig. 8). Furthermore, the free radical scavenger catechin
Fig. 8. Detection of free radical adducts in the bile after tacrine treat-
ment. Tacrine (35 mg/kg) was given intragastrically. After 7 hr, rats were
anesthetized and 4-POBN (250 mg/kg) was injected i.v. Bile was col-
lected for the next hour into dipyridyl to prevent ex vivo radical forma-
tion, and free radical adducts were detected by ESR as described in
Materials and Methods. Data shown are of a typical spectrum.
Fig. 9. Effects of catechin treatment on tacrine-induced liver injury.
Catechin (100 mg/kg, i.p.) or a comparable amount of saline was
administered 1 hr before treatment with tacrine (35 mg/kg, intragastri-
cally). Sixteen hours after drug treatment, blood was collected and AST
determined as described in Materials and Methods. Values are
mean S.E.M. (n 4). Statistical comparisons were made using
analysis of variance and Bonferronis post hoc test. a, different from
saline-treated control. b, different from tacrine-treated animals.
TABLE 2
The effect of dietary arginine on in vivo portal pressure and vital
dye distribution in perfusion after tacrine treatment
a
Diet Treatment
Portal pressure
(cm Water)
Time of vital dye
distribution (min)
Control Tacrine 7.9 0.7 5.6 2.2
Arginine Tacrine 5.4 0.7
b
1.0 0.1
b
a
Rats were fed a semisynthetic powdered diet containing 5% arginine or
isonitrogenous control diet for 3 days (Jones and Thurman, 1996). Animals were
given 35 mg/kg tacrine intragastrically and in vivo portal pressure and time of vital
dye distribution during perfusion were determined three hours later as described
in Materials and Methods. Values are the mean S.E.M. (n 4).
b
P .05 compared to control diet by Students t test.
1997
Acute Tacrine Hepatotoxicity 1597
was shown to decrease hepatotoxicity resulting from tacrine
treatment (fig. 9). Collectively, these data are consistent with
the hypothesis that a significant component of tacrine-medi-
ated hepatotoxicity may not be prolonged hypoxia per se, but
rather the result of increased free radical formation upon
reperfusion of previously hypoxic tissue.
Even though sympathectomy before tacrine treatment pre-
vents hepatotoxicity in this model, surgery is not a viable
alternative to prevent tacrine-induced liver injury in the
clinic. The hypoxia-reperfusion mechanism of tacrine-in-
duced liver injury proposed here predicts that drugs which
block increases in cholinergic transmission in the ganglion or
prevent constriction of the microvasculature in the liver
could be useful in preventing tacrine-induced liver injury.
Indeed, arginine, which is known to provide substrate for
nitric oxide synthesis and cause vasodilatation (Jones and
Thurman, 1996), blunts increases in portal pressure and
time of vital dye distribution due to tacrine (table 2). These
data suggest that dietary interventions and radical scaven-
gers would minimize the hepatotoxic effects of cholinomi-
metic agents in Alzheimers patients.
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Send reprint requests to: Dr. Ronald G. Thurman, CB# 7365, Department
of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC
27599-7365.
1997
Acute Tacrine Hepatotoxicity 1599