Metabolites of acetaminophen trigger Ca2+ release from liver microsomes

Article abstract of DOI:10.1016/S0378-4274(99)00009-0

Release of mitochondrial calcium is believed to play a key role in the toxicity of acetaminophen in biological systems. Elevated cytosolic Ca²⁺ may also result from activation of calcium releasing channels. The major metabolites of acetaminophen, benzoquinone imine and 1,4-benzoquinone, induced Ca²⁺ release in isolated rat liver microsomes. The 1,4-benzoquinone-induced release of calcium was suppressed by ryanodine and fully inhibited by reduced glutathione. Concentrations of 1,4-benzoquinone that induced Ca²⁺ release did not affect the activity of the microsomal Ca²⁺, Mg²⁺-APTase. The binding of [³H]ryanodine to liver microsomes, however, was significantly decreased by 1,4-benzoquinone, suggesting a direct interaction of this metabolite with the ryanodine-binding protein (ryanodine receptor). These results suggest that cellular Ca²⁺ levels may be elevated by acetaminophen by pathways involving, in part, activation of Ca²⁺ releasing channels such as the ryanodine receptor. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0378-4274(99)00009-0

Toxicology Letters 106 (1999) 23–29
2
+
Metabolites of acetaminophen trigger Ca
microsomes
release from liver
Detcho A. Stoyanovsky, Arthur I. Cederbaum *
Department of Biochemistry, Box 1020, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place New York, NY 10029, USA
Received 2 November 1998; received in revised form 4 January 1999; accepted 5 January 1999
Abstract
Release of mitochondrial calcium is believed to play a key role in the toxicity of acetaminophen in biological
2
+
systems. Elevated cytosolic Ca
metabolites of acetaminophen, benzoquinone imine and 1,4-benzoquinone, induced Ca
microsomes. The 1,4-benzoquinone-induced release of calcium was suppressed by ryanodine and fully inhibited by
may also result from activation of calcium releasing channels. The major
2
+
release in isolated rat liver
2
+
reduced glutathione. Concentrations of 1,4-benzoquinone that induced Ca
microsomal Ca , Mg –APTase. The binding of [ H]ryanodine to liver microsomes, however, was significantly
decreased by 1,4-benzoquinone, suggesting a direct interaction of this metabolite with the ryanodine-binding protein
ryanodine receptor). These results suggest that cellular Ca
involving, in part, activation of Ca
Ireland Ltd. All rights reserved.
release did not affect the activity of the
2
+
2+
3
2
+
(
levels may be elevated by acetaminophen by pathways
2
+
releasing channels such as the ryanodine receptor. © 1999 Elsevier Science
2
+
Keywords: Metabolites; Acetaminophen; Ca -release; Liver microsomes
dergo decomposition to 1,4-benzoquinone (BQ)
Dahlin and Nelson, 1982). BQ is a metabolite
1. Introduction
(
The hepatotoxicity of acetaminophen APAP is
believed to result largely from its metabolic acti-
vation to N-acetyl-p-aminobenzoquinone imine
NAPQI). In aqueous solutions NAPQI can un-
produced from benzene and phenol, and is a
useful model for studies on the interaction of
quinones with protein thiols. Both NAPQI and
BQ are highly reactive compounds that can pro-
duce a dose-dependent depletion of intracellular
glutathione and protein thiols (Dahlin and Nel-
(
*
Corresponding author. Tel.: +1-212-241-7285; fax: +1-
2
12-996-7214.
son, 1982; Moore et al., 1987). Experiments with
E-mail address: acederbacederb@smtplink.mssm.edu (A.I.
Cederbaum)
14
[
C]acetaminophen aimed to elucidate the cellular
0
378-4274/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378-4274(99)00009-0

2
4
D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
distribution of covalent protein adducts of
APAP, as well as immunochemical quantifica-
tion of 3-(cystein-S-yl)-acetaminophen protein
adducts in subcellular liver fractions following
hepatotoxic doses of acetaminophen, have
shown the presence of adducts in the cytosolic,
mitochondrial and microsomal fractions (Pum-
ford et al., 1990; Dai and Cederbaum, 1995).
There is, however, less information suggesting
the cellular targets of covalent binding, or the
functional consequences that occur after interac-
tion of the metabolites of APAP with key cellu-
lar macromolecules.
2. Methods and materials
2.1. Reagents
All reagents used were purchased from Sigma
(
St. Louis, MO). The solutions used in the ex-
periments were prepared in deionized and
Chelex-100 treated water or potassium phos-
3
phate buffer. [ H]ryanodine (70.8 Ci/mmol) was
from DuPont (Boston, MA).
2.2. Preparation of hepatic microsomes
Male Sprague–Dawley rats weighing 120–150
There is substantial evidence that the ac-
etaminophen-dependent alteration of the thiol
status in cells is followed by perturbation of cal-
cium homeostasis (Moore et al., 1987). In in
vitro experiments Moore et al. (1987, 1988) have
shown that both NAPQI and BQ can induce
g were sacrificed and liver microsomes were pre-
pared as described previously (Stoyanovsky and
Cederbaum, 1996), resuspended in 0.125 M
KCl–0.01 M potassium phosphate or 0.1 M
Hepes (pH 7.4), stored at −70°C, and used
within 1 month. All solutions used during the
preparation contained 2 mM DTT, 0.15 mM
desferrioxamine, leupeptin (0.001 mg/ml) and
PMSF (0.1 mM).
2
+
release of Ca
from intact mitochondria. The
localization of high levels of protein adducts in
the mitochondria suggest that APAP-dependent
release of mitochondrial calcium may play a
critical role in acetaminophen toxicity (Pumford
et al., 1990). Besides release of mitochondrial
2.3. Preparation of NAPQI
2
+
Ca
etaminophen, increased cytosolic Ca
by reactive metabolites produced from ac-
NAPQI was prepared by oxidation of
2
+
levels
acetaminophen with silver oxide (Dahlin and
Nelson, 1982). Acetaminophen (0.5 g) and silver
oxide (1 g) were stirred in dry chloroform (30 ml)
for 1 h at room temperature. Subsequently, the
reaction mixture was treated for 5 min with
activated charcoal (0.2 g) and filtered. The filtrate
was stabilized with butylated hydroxytoluene (1
mg/25 ml filtrate), concentrated to 1 ml (rotor
evaporator; 30°C) and transferred into a 3×15 cm
silica gel (70–325 mesh) column. The NAPQI was
eluted with dry diethyl ether and collected as a
distinct yellow fraction. The organic solvent from
the NAPQI fraction was evaporated, the remaining
residue was re-dissolved in tetrahydrofurane and
the concentration of NAPQI was determined
may result from activation of the calcium chan-
nels, which are responsible for mobilization of
stored calcium. Increasing evidence suggests that
a thiol-dependent calcium channel exists in the
liver endoplasmic reticulum, which is sensitive to
ryanodine (Shoshan-Barmatz, 1990; Lilly and
Gollan, 1995). The existence of a liver isoform
of the ryanodine receptor is interesting since the
skeletal and cardiac isoforms have cysteine
sufhydryls whose conversion to disulfide(s)
2
+
opens the channels for Ca
(Zaidi et al., 1989).
If similar reactions occur for the ryanodine re-
ceptor of the liver endoplasmic reticulum, in-
2
+
creased release of Ca
from storage sites may
4
play a role in the hepatotoxic mechanism(s) of
APAP. In the present study, we evaluated
spectrophotometrically (m263 nm=3.3×10 /M/cm)
(Dahlin and Nelson, 1982).
2
+
whether NAPQI and BQ can induce Ca
re-
2
+
lease from rat liver microsomes via activation of
the ryanodine-sensitive calcium-releasing chan-
nel.
2.4. Measurement of Ca
transport
2
+
Ca
uptake and release from the liver mi-

D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
25
crosomes was monitored using a double beam
double wavelength spectrophotometer (Perkin-
Elmer 557) through the differential absorption
changes of antipyrylazo III (AP-III) at 720–790
nm. In a typical experiment, uptake and release of
with 5 ml ice-cold washing buffer (10 mM Hepes,
0.2 M NaCl, pH 7.4). The remaining radioactivity
on the filters was determined in a LKB liquid
scintillation counter (1209 Rackbeta; Wallac Oy,
Finland). The specific binding was determined as
2
+
Ca
0.2 or 0.5 mg protein/ml) in 0.1 M phosphate
buffer (pH 7.0), containing KCl (0.1 M), Hepes
was measured in microsomal suspensions
the
difference
between
total
binding
3
(
([ H]ryanodine alone) and non-specific binding
3
1
([ H]ryanodine+0.2 mM [ H]ryanodine). In our
2
+
3
(
20 mM), Mg
(0.5 mM), ATP (0.5 mM), and
experiments, the [ H]ryanodine binding to the
an ATP-regenerating system consisting of creatine
phosphokinase (30 U/ml) and creatine phosphate
control microsomes was 275924 fmol/mg
protein, a value that is in agreement with the
studies of Shoshan-Barmatz et al. (1991).
(
8 mM). The experiments were initiated by the
2
+
addition of CaCl (final Ca
concentration of
2
0
.03 or 0.05 mM).
The Ca , Mg –ATPase activity was deter-
3
. Results
2
+
2+
mined by measuring the accumulation of inor-
ganic phosphate during the hydrolysis of ATP
3.1. NAPQI- and BQ-dependent release of
calcium from rat li6er microsomes
(
Srivastava et al., 1990). The liver microsomes (0.5
2
+
mg/ml) were incubated with BQ (0–1 mM) for 15
min in Hepes (0.05 M, pH 7.4 or pH 7.0, 37°C).
Subsequently, an aliquot of 0.05 ml was trans-
ferred into 1 ml Hepes (0.05 M, pH 7.0), contain-
The microsomal stores from which Ca
is
released consist of three major components: a
Ca , Mg –ATPase that sequesters calcium;
Ca -binding proteins that store calcium (calse-
2
+
2+
2
+
ing ATP (1 mM), ouabain (0.2 mM), CaCl (0.06
questrin and calreticulin); and the specific IP
2
3
+
2
mM) and MgCl2 (0.5 mM), and the resulting
solution was incubated for 20 min at 37°C. The
reaction was stopped by addition of 0.3 ml 20%
and/or ryanodine receptors that release Ca
2
+
2+
back to the cytosol. The Ca , Mg –ATPase
and both the IP3 and ryanodine receptors are
thiol-containing proteins whose activity depend
on the redox status of their thiol groups (Zaidi et
al., 1989; Kaplin et al., 1994). Since both NAPQI
and BQ readily arylate protein thiols, experiments
were conducted to elucidate whether these
metabolites can cause release of microsomal cal-
(
3
w/v) TCA and, after centrifugation (5000×g for
min), an aliquot of 0.1 ml was transferred into a
solution of 0.034% malachite green (0.8 ml, in 1
M HCl). The color development was fixed by
addition of 0.1 ml 1% ammonium molybdate and
absorbance was determined at 640 nm.
2
+
2+
cium, alter the hepatic Ca , Mg –ATPase
3
3
2
.5. [ H]ryanodine binding
and/or the binding of [ H]ryanodine to liver
microsomes.
The liver microsomes were incubated with BQ
The addition of CaCl (0.03 mM) to a microso-
2
(
0
0–1 mM) for 20 min (0.1 M phosphate buffer,
.1 M KCl, 20 mM Hepes, pH 7.0, 37°C). Subse-
mal suspension (0.2 mg protein/ml), containing
ATP initially resulted in a rapid increase of the
2
+
quently, an addition of GSH (2 mM; to remove
absorbance due to the formation of a Ca –AP-
III complex (Fig. 1A). Subsequently, the ab-
sorbance slowly decreased to its initial value as a
unreacted BQ by thiolation) was made and the
3
[
H]ryanodine binding was determined as de-
2
+
2+
scribed in Shoshan-Barmatz et al. (1991). Briefly,
a mixture containing NaCl (0.5 M), Hepes (20
mM), EGTA (1 mM), [ H]ryanodine (20 nM) and
microsomal suspension (1 mg protein/ml) was in-
cubated for 15 min at 20°C in 0.1 M phosphate
buffer (pH 7.0). Aliquots of 0.15 ml were filtered
through 0.22 m Millipore filters and washed twice
result of the Ca , Mg –ATPase-driven trans-
port of Ca
2
+
into the microsomal compartment.
3
No decrease in the absorbance was observed in
the absence of ATP (data not shown). Once accu-
2
+
mulated, the Ca was stably maintained and not
released back to the incubation medium (Fig. 1A
curve 1). The addition of NAPQI to the micro-

2
6
D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
2
+
somes already loaded with Ca
resulted in a fast
2
+
release of the accumulated Ca ; the rate of
2
+
Ca
release was dependent upon the concentra-
tion of NAPQI used over the range 0.025–0.10
mM (Fig. 1A). The NAPQI-induced release of
2
+
Ca
was affected by variations in the protein
content in the incubation suspension, with in-
creasing release by a fixed concentration of
NAPQI occurring at lower protein concentrations
(
Data not shown). This suggests that NAPQI may
also bind to other microsomal proteins, which are
not involved in the releasing process.
Fig. 2. Effect of glutathione and ryanodine on BQ (0.05
2+
mM)-induced release of Ca
from liver microsomes. Incuba-
While both metabolites, NAPQI and BQ, ap-
pear to have similar reactivity, experiments were
conducted to evaluate the potential of BQ to
tion conditions were the same as indicated in the legend to Fig.
1. Final concentration of additions was (n=2–3): BQ, 0.05
mM (curve 5); BQ plus ryanodine, 0.1 mM (curve 4); BQ plus
glutathione, 2 mM (curve 3); NAPQI (0.05 mM) plus glu-
tathione, 2 mM (curve 2); and curve 1, no additions.
2
+
release microsomal Ca . Fig. 1B illustrates the
2
+
effect of BQ on Ca -containing liver micro-
2
+
somes. BQ triggered a fast release of Ca
from
tion of GSH after arylation would not be protec-
tive. In view of the immediate release of calcium
from the liver microsomes by NAPQI (Fig. 1A), it
is likely that this release is directly mediated by
NAPQI itself and not by BQ produced from the
hydrolysis of NAPQI.
the liver microsomes in a dose-dependent manner.
2
+
The release of Ca
induced by either NAPQI or
BQ was fully prevented by reduced glutathione
when added to the system prior to the addition of
NAPQI or BQ (Fig. 2 curves 2 or 3); however,
glutathione was ineffective when added to a reac-
tion system already in the midst of either NAPQI-
2
+
2+
2
+
3.2. Effects of BQ on Ca , Mg –APTase and
ryanodine binding to rat li6er microsomes
or BQ-induced Ca
release (data not shown).
This probably reflects the irreversible arylation of
critical thiols on the RyR by BQ such that addi-
Effective concentrations of BQ that induced
release of calcium (Fig. 1B) strongly inhibited the
3
[
H]ryanodine binding to liver microsomes (Fig.
3
3
). The [ H]ryanodine binding to liver microsomes
decreased with increasing concentrations of BQ
0.025–0.100 mM). We did not conduct experi-
(
3
ments for the determination of the [ H]ryanodine
binding to NAPQI-treated microsomes because of
the considerable decomposition of the latter to
BQ during the time course required for the analyt-
ical procedure (Dahlin and Nelson, 1982). In con-
centrations up to 0.1 mM, BQ did not affect the
2
+
2+
microsomal Ca , Mg –ATPase; at higher con-
centrations of BQ, however, the activity of the
calcium pump progressively decreased (Fig. 3).
Since at relatively low concentrations of BQ there
Fig. 1. NAPQI and BQ-induced release of liver microsomal
calcium. Incubation conditions were liver microsomes (0.2 mg
protein/ml), 0.1 M phosphate buffer (pH 7.0) at 37°C, KCl
(
0.1 M), Hepes (0.02 M), CaCl (0.03 mM), MgCl (0.5 mM),
2+
2+
2
2
is insignificant inhibition of the Ca , Mg
–
ATP (0.5 mM), phosphocreatine kinase (30 U/ml), and phos-
phocreatine (8 mM). The following concentrations of NAPQI
or BQ were added (mM): (1) 0, (2) 0.025, (3) 0.05, (4) 0.075,
and (5) 0.1.
ATPase, the observed calcium release can likely
be attributed to a BQ-dependent activation of the
ryanodine-sensitive calcium-releasing channel. In-

D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
27
deed, ryanodine partially prevented or slowed
down the release of calcium induced by BQ (Fig.
by perturbation of the microsomal calcium se-
questration. Numerous in vitro studies have
shown that the major metabolites of APAP,
NAPQI and BQ, can induce release of mitochon-
2, curve 4 compared to curve 5).
2
+
drial Ca , most likely via arylation of critical
protein thiols (Moore et al., 1987, 1988; Burcham
and Harman, 1990; Henry and Wallace, 1995;
Parmar et al., 1995; Wallace et al., 1997). How-
4. Discussion
Although the role of altered calcium homeosta-
2
+
ever, the effects of NAPQI and BQ on the Ca
sis in acetaminophen hepatotoxicity is somewhat
controversial (Grewal and Racz, 1993), many
studies have shown that acetaminophen hepato-
stores in the hepatic endoplasmic reticulum have
not been studied.
2
+
There is considerable evidence that the microso-
toxicity is a process characterized by Ca
dereg-
2
+
mal Ca
stores are critically dependent on
ulation. In experiments in vivo, APAP has been
shown to affect the hepatic calcium homeostasis
at various levels, depending on doses and time
variables. Tirmenstein and Nelson (1989) have
reported that APAP affects the plasma membrane
protein sulfhydryl groups. Thor et al. (1985) have
shown that cystamine and alkylating agents in-
hibit the calcium sequestration in rat liver micro-
somes. Zhang et al. (1990) have reported that in
2
+
2+
2
+
2+
hepatic microsomal vesicles, Cd , Cu , and
Ca , Mg –ATPase, depletes the mitochon-
2
+
2+
2
+
Zn cause a prompt efflux of Ca in a dose-de-
pendent manner, most likely via chelation of criti-
cal thiol groups. Recently, we have reported that
thiol oxidants and radical species derived from the
drial GSH and alters the mitochondrial Ca
sequestration, while Tsokos-Kuhn et al. (1985)
have observed a pronounced inhibition of the
2
+
2+
plasma membrane Ca , Mg –ATPase without
any changes in the mitochondrial homeostasis. In
contrast, Burcham and Harman (1988) have ob-
served an APAP-dependent increase of mitochon-
drial and cytosolic calcium, which was paralleled
NADPH-dependent metabolism of CCl or iron
4
2
+
redox cycling can induce release of Ca
from
ryanodine-sensitive calcium stores in liver micro-
somes, apparently without damaging the hepatic
2
+
2+
Ca , Mg –ATPase or the lipid component of
the microsomal membranes (Stoyanovsky and
Cederbaum, 1996, 1998).
Since NAPQI and BQ are highly reactive SH-
arylating agents, experiments were conducted to
answer whether these metabolites can cause re-
2
+
lease of Ca
from rat liver microsomes. A
2
+
NAPQI- and BQ-induced Ca release was found
with liver microsomes. The BQ-induced release of
calcium was decreased by ryanodine, a compound
known to form complexes with the skeletal and
cardiac isoforms of the calcium gated calcium
releasing channels (ryanodine receptors). The re-
leasing process was also inhibited by reduced glu-
tathione, which is known to form covalent
adducts with quinones. It appears that inhibition
Fig. 3. Effect of BQ on Ca2+, Mg2+ –ATPase activity and
3
[
H]ryanodine binding to liver microsomes. The assays for
2
+
2+
2+
2+
determination of the Ca , Mg –ATPase activity and the
of the Ca , Mg –ATPase is not the major
mechanism by which low concentrations of BQ
3
[
H]ryanodine binding are described in Section 2. The activity
2
+
2+
of the Ca , Mg –ATPase in control microsomes was
2+
impair the sequestration of Ca in liver micro-
9
[
.290.4 nmol of P/min/mg of protein (“), while the
H]ryanodine binding was 275924 fmol/mg protein (ꢀ). The
3
somes. Effective concentrations of BQ that in-
2
+
duced Ca
release did not affect the activity of
data presented are the mean9S.E.M. of three independent
experiments (n=3).
3
the calcium pump. The binding of [ H]ryanodine

2
8
D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
to the liver microsomes, however, was signifi-
cantly decreased by BQ, suggesting a direct inter-
action of BQ with the ryanodine-binding
Lilly, L.B., Gollan, J.L., 1995. Ryanodine-induced calcium
release from hepatic microsomes and permeabilized hepa-
tocytes. Am. J. Physiol. 268, 1017–1024.
3
Moore, G.A., Rossi, L., Nicotera, P., Orrenius, S., O’Brien,
protein(s). Modulation of the [ H]ryanodine bind-
P.J., 1987. Quinone toxicity in hepatocytes: studies on
ing to the skeletal sarcoplasmic reticulum and
induction of calcium release via the skeletal ryan-
odine receptor by a series of cyclic quinones was
reported by Abramson et al. (1988). Future stud-
ies are required, however, to focus on the nature
and extent of the microsomal injury mediated by
APAP metabolites in intact cells and/or in vivo,
and on the relationship between such membrane
damage and alterations in the calcium
sequestration.
2+
mitochondrial Ca
release induced by benzoquinone
derivatives. Arch. Biochem. Biophys. 259, 283–295.
Moore, G.A., Weis, M, Orrenius, S, O’Brien, P.J., 1988. Role
2
+
of sulfhydryl groups in benzoquinone-induced Ca
lease by rat liver mitochondria. Arch. Biochem. Biophys.
67, 539–550.
Parmar, D.V., Ahmed, G., Khandkar, M.A., Katyare, S.S.,
995. Mitochondrial ATPase: a target for paracetamol-in-
re-
2
1
duced hepatotoxicity. Eur. J. Pharmacol. 293 (19), 225–
229.
Pumford, N.R., Roberts, D.W., Benson, R.W., Hinson, J.A.,
1
990. Immunochemical quantitation of 3-(cystein-S-
yl)acetaminophen protein adducts in subcellular liver frac-
tions following a hepatotoxic dose of acetaminophen.
Biochem. Pharmacol. 40, 573–579.
Acknowledgements
Shoshan-Barmatz, V., 1990. High affinity ryanodine binding
sites in rat liver endoplasmic reticulum. FEBS Lett. 263,
317–320.
Shoshan-Barmatz, V., Pressley, T.A., Higham, S., Kraus-
Friedmann, N., 1991. Distinct ryanodine- and inositol
This work was supported by USPHS Grant
AA-06610 from The National Institute on Alco-
hol Abuse and Alcoholism.
1
,4,5-trisphosphate-binding sites in hepatic microsomes.
Biochem. J. 276, 41–46.
References
Srivastava, S.P., Chen, N.Q., Holtzman, J.L., 1990. The in
vitro NADPH-dependent inhibition by CCl4 of the ATP-
dependent calcium uptake of hepatic microsomes from
male rats. Studies on the mechanism of the inactivation of
Abramson, J.J., Buck, E., Salama, G., Casida, J.E., Pessah,
I.N., 1988. Mechanism of anthraquinone-induced calcium
release from skeletal muscle sarcoplasmic reticulum. J.
Biol. Chem. 263, 18750–18758.
the hepatic microsomal calcium pump by the CCl .radical.
J. Biol. Chem. 265, 8392–8399.
3
Burcham, P.C., Harman, A.W., 1988. Effect of acetaminophen
hepatotoxicity on hepatic mitochondrial and microsomal
calcium contents in mice. Toxicol. Lett. 44, 91–99.
Burcham, P.C., Harman, A.W., 1990. Mitochondrial dysfunc-
tion in paracetamol hepatotoxicity: in vitro studies in
isolated mouse hepatocytes. Toxicol. Lett. 50 (15), 37–48.
Dahlin, D.C., Nelson, S.D., 1982. Synthesis, decomposition
kinetics, and preliminary toxicological studies of pure N-
acetyl-p-benzoquinone imine, a proposed toxic metabolite
of acetaminophen. J. Med. Chem. 25 (1), 85–87.
Dai, Y., Cederbaum, A.I., 1995. Cytotoxicity of ac-
etaminophen in human cytochrome P4502E1-transfected
HepG2 cells. J. Pharmacol. Exp. Ther. 273, 1497–1505.
Grewal, K.K., Racz, W.J., 1993. Intracellular calcium disrup-
tion as a second event in acetaminophen-induced hepato-
toxicity. Can. J. Physiol. Pharmacol. 71, 26–33.
Stoyanovsky, D.A., Cederbaum, A.I., 1996. Thiol oxidation
and cytochrome P450-dependent metabolism of CCl trig-
4
2
+
gers Ca
release from liver microsomes. Biochemistry 35,
1
5839–15845.
Stoyanovsky, D.A., Cederbaum, A.I., 1998. Redox-cycling of
iron ions triggers calcium release from liver microsomes.
Free Radic. Biol. Med. 24, 745–753.
Thor, H., Hartzell, P., Svensson, S.A., et al., 1985. On the role
2
+
of thiol groups in the inhibition of liver microsomal Ca
sequestration by toxic agents. Biochem. Pharmacol. 34,
717–3723.
3
Tirmenstein, M.A., Nelson, S.D., 1989. Subcellular binding
and effects on calcium homeostasis produced by ac-
etaminophen and a nonhepatotoxic regioisomer, 3%-hydrox-
yacetanilide, in mouse liver. J. Biol. Chem. 264,
9
814–9819.
Henry, T.R., Wallace, K., 1995. Differential mechanisms of
induction of the mitochondrial permeability transition by
quinones of varying chemical reactivities. Toxicol. Appl.
Pharmacol. 134, 195–203.
Tsokos-Kuhn, J.O., Todd, E.L., McMillin-Wood, J.B.,
Mitchell, J.R., 1985. ATP-dependent calcium uptake by rat
liver plasma membrane vesicles. Effect of alkylating hepa-
totoxins in vivo. Mol. Pharmacol. 28, 56–61.
Kaplin, A.I., Ferris, C.D., Voglmaier, S.M., Snyder, S.H.,
1
994. Purified reconstituted inositol 1,4,5-trisphosphate re-
Wallace, K.B., Eells, J.T., Madeira, V.M., Cortopassi, G.,
Jones, D.P., 1997. Mitochondria-mediated cell injury. Sym-
posium overview. Fundam. Appl. Toxicol. 38, 23–37.
ceptors. Thiol reagents act directly on receptor protein. J.
Biol. Chem. 269, 28972–28978.

D.A. Stoyano6sky, A.I. Cederbaum / Toxicology Letters 106 (1999) 23–29
29
Zaidi, N.F., Lagenaur, C.F., Abramson, J.J., Pessah, I.,
Zhang, G.H., Yamaguchi, M., Kimura, S., Higham, S., Kraus-
Friedmann, N., 1990. Effects of heavy metal on rat liver
2
+
Salama, G., 1989. Reactive disulfides trigger Ca
release
2
+
from sarcoplasmic reticulum via an oxidation reaction. J.
Biol. Chem. 264, 21725–21736.
microsomal Ca2(+)–ATPase and Ca
sequestering. Re-
lation to SH groups. J. Biol. Chem. 265, 2184–2189.
.