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INTRODUCTION |
Acetaminophen is one of the most commonly used
analgesics/antipyretics worldwide. Although generally considered a safe
drug, it continues to be a cause of death either through overdose,
idiopathic reaction, or synergism with alcoholic liver disease. Death
from acetaminophen overdose is thought to be secondary to liver
failure, which is caused by massive hepatic necrosis, the hallmark
pathological feature of acetaminophen toxicity. In addition to liver,
however, many organ systems may fail under acute overdose such as
renal, cardiac, and central nervous systems (1). It is thought that the
liver is the target organ for acetaminophen toxicity because this is
primarily where the drug is detoxified. Under normal conditions, acetaminophen is mainly metabolized by undergoing sulfation and glucuronidation (2). It has been proposed that a small amount of drug
goes through the cytochrome P450 mixed function oxidase system and is
metabolized into the reactive intermediate
N-acetyl-P-benzoquinoneimine (NAPQI),1 which is in turn
detoxified by reaction with glutathione (3, 4). When large quantities
of acetaminophen are consumed, the three detoxification pathways become saturated.
The precise mechanism by which acetaminophen causes cell death remains
unknown, although there are two prevailing theories that are
controversial today. The first theory, the oxidative stress theory,
maintains that acetaminophen metabolites cause oxidative stress in the
cell ultimately leading to its demise. The second theory, the covalent
binding theory, states that the binding of the highly reactive
acetaminophen metabolites to cell macromolecules causes cell death.
There is much evidence to substantiate both theories, and the question
may be to what extent each plays a role in acetaminophen toxicity (5).
The depletion of cellular glutathione, a natural antioxidant, leaves
the cell particularly vulnerable to oxidative insults following
acetaminophen overdose. The oxidative stress theory has gained
increased recognition as a result of a number of studies, which
indirectly and directly demonstrate the presence of reactive oxygen
species in cells following acetaminophen administration. Several
antioxidants have been shown to protect against acetaminophen toxicity
such as
-carotene (6) and
-tocopherol (7). In addition to the
studies involving these chemicals, the exogenous administration of
antioxidant enzymes such as catalase and superoxide dismutase (8) has
been shown to protect dramatically against acetaminophen toxicity.
Although it seems clear that oxidative stress plays some role in
acetaminophen toxicity, the exact source(s) of the oxidative stress is
not known, and the mechanism of the resultant cytotoxicity is also the
subject of speculation.
We are using transgenic animals overexpressing the human antioxidant
enzymes glutathione peroxidase, intracellular (GPI) and extracellular
(GPP) forms, as well as Cu,Zn-superoxide dismutase (SOD) to investigate
their ability to influence acetaminophen toxicity. We report here that
GPP and SOD transgenic mice are equally protected against the lethality
of an overdose of acetaminophen, whereas GPI transgenic mice showed
significantly increased sensitivity to acetaminophen as compared with
normal animals. We demonstrate that metabolism of acetaminophen in GPI
mice leads to a significant decrease in the replenishment of GSH in
liver and blood, in particular, in comparison with the other animal
groups. In contrast, GPP mice were able to sustain an elevated level of
GSH during acetaminophen intoxication. The results also indicate
that GP overexpression influences the synthesis of several oxidized
acetaminophen metabolites.
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EXPERIMENTAL PROCEDURES |
Transgenic Mice--
Transgenic mice with human GP or Cu,Zn-SOD
genes were produced as described previously (9, 10). Normal and
transgenic animals for the experiments were obtained by breeding
heterozygous transgenic founders with C57BL/6 × CBA/J F1 mice. SOD and
GPI transgenic mice show increased activities of Cu,Zn-SOD and GPI, respectively, in most of the tissues, as reported earlier. For example
Cu,Zn-SOD activity in liver of SOD mice is 2.2-fold higher, whereas
activity of GP in liver of GPI mice is 1.3-fold higher than that of
nontransgenic mice (11). GPP mice have 50% increased activity of GP in
blood plasma, and the same activity in liver as nontransgenic mice.
Animals were maintained with 12 h light/dark cycling at 25 °C
and fed ad libitum. Nontransgenic littermates were used as
controls. Experimental animals were matched for age and gender.
Experimental protocol has been approved by the University of Medicine
and Dentistry Animal Care and Use Committee.
Plasma Enzymes and Histopathology--
Lactate dehydrogenase and
alanine aminotransferase were measured in plasma samples using an assay
kit (Sigma) according to the kit's protocol. Thin sections were cut by
microtome and stained with hemotoxylin and eosin and examined by light
microscope. Damage was assessed by expert pathologist on a scale from 0 to 4 based on qualitative and quantitative criteria.
Drug Treatments--
For survival studies, 425 mg/kg
acetaminophen (Sigma) were injected intraperitoneally from a 15 mg/ml
solution in sterile PBS. Animals were followed for 72 h for
survival. To evaluate the effect of intravenously administered GP on
animal sensitivity to acetaminophen, bovine GPI (Sigma) was dissolved
in sterile PBS to a concentration of 500 units/ml and 1.4 milliunits/kg
injected into the tail veins of 11 nontransgenic mice. Approximately
equal volumes of sterile PBS were injected into tail veins of 10 additional nontransgenic mice. Intravenous injections were followed
immediately by intraperitoneal injections of acetaminophen, 350 mg/kg.
The dosage was based on a dose response study in males used in this analysis, that are known to be more sensitive to acetaminophen toxicity.
Glutathione and Lipid Peroxidation Measurements--
Total (GSH + GSSG) and oxidized (GSSG) glutathione were measured using the
recycling method (12). Tissue was homogenized in 10% perchloric acid
and frozen at
70 °C until time of assay. Frozen homogenate was
then centrifuged for 20 min at 6,000 × g. Acid
supernatant was used for GSH assay, and acid supernatant reacted with
10 mM N-ethylmaleimide (Sigma) and run through
Sep-Pak P18 cartridge (Waters) was used for GSSG determination.
Reaction mixtures contained 0.15 mM NADPH and assay buffer
(0.6 mM 5,5'-dithio-bis(2-nitro-benzoic acid) in phosphate
buffer (0.1 M, pH 7.4) containing 1 mM EDTA, and glutathione reductase (80 milliunits/ml)). Plasma was mixed with
equal volumes of 5-sulfosalicylic acid for plasma glutathione measurements (10%, w/v) (13). Proteins were removed by centrifugation and the supernatant assayed using reagents in the above proportions. Tissue lipid peroxidation was assayed using thiobarbituric acid as
previously published (14). 2.5 ml of 20% trichloroacetic acid solution
and 1 ml of 0.67% thiobarbituric acid solution were added to 0.5 ml of
liver homogenate. The mixtures were incubated at 100 °C for 30 min.
After cooling to room temperature, the reaction mixture was extracted
with 4 ml of butanol and centrifuged for 10 min at 3,000 × g. Optical density of the upper phase was measured at 532 nm. 1,1,3,3-tetraethoxypropane was used as a standard for malonaldehyde.
Analysis of Acetaminophen Metabolism--
Quantitative
determination of the acetaminophen metabolites in plasma was carried
out by a modified high pressure liquid chromatography method as
previously published (15).
Detection of NAPQI in Vitro--
Fluorescence spectra of NAPQI
(Sigma) in 50 mM sodium phosphate buffer (pH 7.7) and of
solutions containing 0.05-0.5 mM acetaminophen, 36 mM cetyltrimethylammonium bromide, 0.2-1 mM
H2O2, 1-10 units of GPI (Sigma) in 50 mM sodium phosphate buffer (pH 7.7) were recorded on a
Perkin Elmer spectrofluorometer using
exc 306 nm as
reported by Schmitt and Cilento (16). H2O2 was
added last and the fluorescence emission (360-560 nm) was monitored at
5 min intervals.
NADPH Oxidation--
The ability of GP to stimulate NADPH
oxidation by acetaminophen in the presence of
H2O2 was determined as described by Keller et al. (17). The absorbance of the reaction mixture
containing 50 mM sodium phosphate buffer (pH 7.2), 0.38 mM EDTA, 0.12 mM NADPH, 0.1-5 units of GPI
(Sigma) and 0.1-10 mM H2O2 was
continuously monitored at 340 nm. Acetaminophen was added at final
concentrations of 0.01-1 mM.
Statistics--
All results are expressed as mean ± S.E.
Statistical differences between means were evaluated using the
Student's t test.
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RESULTS |
Effect of GP Overexpression on Survival and Hepatotoxicity after
Acetaminophen Administration--
Transgenic mice overexpressing SOD,
GPI, GPP, and nontransgenic controls received 425 mg/kg acetaminophen
intraperitoneally in a single dose. SOD mice were dramatically
protected from toxicity, with 25% mortality as compared with 75%
mortality in nontransgenic controls (Fig.
1A). Similar protection has
been reported for exogenous administration of liposome-encapsulated SOD
to rats immediately before acute acetaminophen overdose (18). Animals
overexpressing extracellular GP in the blood also demonstrated dramatic
protection against acetaminophen, again suffering only 25% mortality.
Surprisingly, animals overexpressing GP in the liver tissue itself
displayed a completely opposite phenotype-enhancement of acetaminophen
toxicity. All GPI animals receiving acetaminophen died within 5.5 h of treatment. Control mortality was only 75% and occurred over a
more extended period of time, with the last death occurring at 36 h post-injection. The behavior of the animals corresponded to survival
pattern, with more sensitive animals feeding poorly and displaying less spontaneous locomotor activity.

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Fig. 1.
Survival following lethal doses of
acetaminophen. A, SOD, GPP, GPI, and Ntg mice received
an intraperitoneal injection of acetaminophen (425 mg/kg). Survival was
followed for 72 h. n = 8 per group. B,
Ntg animals received 20 units/ml glutathione peroxidase or PBS
intravenously immediately followed by an intraperitoneal injection of
acetaminophen (350 mg/kg). n = 11 for GP-injected,
n = 10 for PBS-injected.
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To confirm that the response of the GPP transgenic mice was caused by
increased GP activity in the blood, 1.4 milliunits/kg GP was injected
into tail veins of 11 nontransgenic mice and equal volumes of PBS into
tail veins of 10 additional animals, immediately before an
intraperitoneal injection of a lethal dose of acetaminophen. The dose
of GP was selected because it achieves roughly the same activity of GP
in the blood as is normally found in GPP transgenic mice. Almost 90%
of GP-injected animals survived beyond 72 h, whereas none of the
PBS-injected animals survived beyond 6 h (Fig. 1B).
Increased GP activity in the blood appears to be responsible for the
reduced mortality seen in both GP-injected as well as GPP transgenic mice.
By histopathologic criteria there were no differences between groups at
4 h, with all animals sustaining equally severe damage. At 8 h, histopathology generally agreed with survival data, with SOD and GPP
animals displaying less damage than controls and GPI displaying more
severe damage (Fig. 2). Liver necrosis
was also evaluated by measurement of blood alanine aminotransferase
activity. Levels of alanine aminotransferase at 8 h were as
follows: 5,477 ± 2,035 units/liter, 802 ± 465 units/liter
and 3,119 ± 1,594 units/liter for GPI, GPP, and nontransgenic
mice, respectively; baseline alanine aminotransferase values were the
same in all groups and equal to 21 ± 1.3 units/liter for
nontreated mice. Data obtained indicate that GPI mice had the highest
increase in alanine aminotransferase, whereas GPP mice had the smallest
elevation of alanine aminotransferase compared with nontransgenic
mice.

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Fig. 2.
Liver histology of SOD, GPP, GPI transgenic,
and nontransgenic mice 8 h after acetaminophen overdose (350 mg/kg). Hematoxylin-eosin-stained sections of livers of animals
displaying representative hepatocellular damage characteristic of a
given group at a given time point. Original magnification × 200. A, SOD: rim of swollen and vacuolated hepatocyes surrounding
the central venule. An occasional necrotic hepatocyte is observed in
these regions. Hepatocytes surrounding the portal triad are
unremarkable. B, GPP: cytoplasm of pericentral hepatocytes
is slightly condensed and sinusoidal channels are compressed. No
hepatocellular necrosis is apparent. C, GPI: significant
hemorrhage and necrosis centered around the central venule. The
sinusoidal architecture in the effected zones is effaced, and only
Kupffer cell nuclei and cellular debris remains. The periportal
hepatocytes are essentially unaffected. D, Ntg: mild
congestion and focal hepatocellular vacuolization. The affected
hepatocytes are clustered around the central venule. Hepatocytes
elsewhere in the lobule are unremarkable.
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Studies of Glutathione Metabolism and Lipid
Peroxidation--
Hepatic glutathione has a well established pattern
of depletion and recovery under conditions of acetaminophen overdose.
All animal groups suffered approximately 95% depletion of total GSH at
1 h, as seen in Fig. 3A.
At 8 h, when total GSH content returned to baseline in all other
groups, GPI mice had significantly lower total GSH than SOD, controls,
and GPP mice (37, 32, and 26% lower, respectively). This difference
might indicate the inability of GPI mice to efficiently replenish liver
GSH, leading to an increase of the sensitivity of these animals to
acetaminophen toxicity. However, we could not exclude another
possibility that the effect observed in GPI mice might be the result of
earlier necrosis developed in these animals, because liver damage in
GPI mice was higher than in all other groups at this time point.
Interestingly, a comparison of liver GSH content among the different
groups reveals a significantly lower proportion of GSH (higher
proportion of GSSG) in SOD mice relative to all other groups at 1 h (Fig. 3B). Because GPP animals overexpress the plasma form
of GP, plasma levels of total glutathione were measured to determine
the degree of oxidative stress in the blood. Similar to what was
observed in the liver, total glutathione in the plasma of SOD, GPI, and control animals was almost completely depleted by 1 h (Fig.
3C). In contrast to the liver, however, plasma glutathione
levels at 4 h remained low in all groups, except for GPP, in which
clear tendencies for recovery were observed. By 8 h, levels of
plasma glutathione in SOD and GPP animals returned to baseline, whereas control animals were still at 73% and GPI at 60% of baseline levels. Significantly, although depleted from baseline levels, plasma total GSH
was substantially higher in GPP animals than in all other groups at 1 and 4 h and higher than both GPI and controls at 8 h.

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Fig. 3.
Measurements of glutathione and TBARS in SOD,
GPP, and GPI transgenic, and nontransgenic mice following acetaminophen
overdose (350 mg/kg). Each point represents the mean ± S.D.
of data obtained from 6 to 8 mice. A, total hepatic
glutathione; *, p < 0.05 compared with GPI group.
B, percentage of the reduced form of total glutathione; *,
p < 0.05 compared with GPP, GPI, and Ntg groups.
C, total plasma glutathione; *, p < 0.05 compared with GPP, GPI, and Ntg groups; **, p < 0.05 compared with Ntg group; ***, p < 0.05 compared with
GPI and Ntg groups; ****, p < 0.05 compared with GPP,
SOD, and Ntg groups. D, hepatic TBARS in SOD, GPP, GPI
transgenic, and nontransgenic mice following acetaminophen overdose; *,
p < 0.05 compared with Ntg group.
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Lipid peroxidation was assessed by measurement of thiobarbituric acid
reactive substances (TBARS). After 4 h, TBARS were elevated in all
groups (Fig. 3D), but after 8 h, they returned to
baseline, except for SOD mice. The significant increases in lipid
peroxidation in the livers of SOD mice following an overdose of
acetaminophen are most likely the result of increased production of
H2O2. This observation correlates with the
decreased GSH content in these animals compared with nontransgenic and
GP mice at earlier time points. Importantly, we did not observe the
expected decrease in hepatic lipid peroxidation in GP transgenic mice
compared with nontransgenic animals following acetaminophen
administration. These data indicate absence of correlation between
survival of different group of animals and the level of liver lipid
peroxidation as well as inability of both types of GPs to decrease the
level of liver peroxidation measured as TBARS. The data are consistent with a number of studies that have found a dissociation between lipid
peroxidation and acetaminophen toxicity (19).
Acetaminophen Metabolism in GP Transgenic Mice--
Altered
metabolism of acetaminophen in the presence of an increased level of GP
may explain the seemingly paradoxical response of GPI transgenic mice.
For example, acetaminophen has been reported to be oxidized to NAPQI
during peroxidative metabolism of horseradish peroxidase (20),
mammalian lactoperoxidase (21), thyroid peroxidase (22), and
prostaglandin H synthase (23).
Acetaminophen metabolism was evaluated by measurements of specific
metabolites in the serum of animals following administration of
acetaminophen. Overall there are significant similarities in the
patterns of changes of acetaminophen metabolites in all animals tested
(Fig. 4). Nevertheless, the concentration
of free acetaminophen was somewhat higher in the serum of GPP mice at
30 min, and the concentration of acetaminophen-glucuronide and
mercapturate were lower in GPP mice at 50 min compared with normal and
GPI mice (Fig. 4, A, B, and F).
Furthermore, the concentrations of the mercapturic acid metabolites of
acetaminophen were much higher in GPI animals at 25-60 min
concomitantly with a lower concentration of acetaminophen-GSH. These
data indicate that GP overexpression influences the level of several
acetaminophen metabolites. The rate of acetaminophen oxidation by
cytochrome P450 in liver microsomal fractions from normal and GPI mice
was the same (data not shown).

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Fig. 4.
Analysis of acetaminophen metabolites in
plasma of SOD, GPP, GPI transgenic, and nontransgenic mice following
acetaminophen overdose (350 mg/kg). The concentrations of free
acetaminophen (A) as well as its conjugates
(B-F) were determined at various times after
drug administration. Each point represents the mean ± S.D. of
data obtained from four mice. *, p < 0.05 compared
with GPI and Ntg groups; **, p < 0.05 compared with
GPP and Ntg groups; ***, p < 0.05 compared with Ntg
group.
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Acetaminophen Oxidation in Vitro--
Based on the above results,
it was hypothesized that intracellular GP could use acetaminophen as
electron donor, thus converting acetaminophen to acetaminophen free
radical. The latter could be reduced back to acetaminophen by a variety
of processes, including potentially toxic reactions such as abstracting
hydrogen atoms from critical cellular molecules. This process in liver
overexpressing GPI might lead to the increase in toxicity and
exacerbation of damage in transgenic mice. To test this hypothesis, we
exposed acetaminophen to the purified intracellular glutathione
peroxidase in the presence and absence of H2O2.
Two approaches were used to test the ability of GPI to form NAPQI from
acetaminophen. In the first series of experiments, a mixture of
acetaminophen and GPI was treated with H2O2,
and NAPQI formation was measured by fluorescence spectroscopy. As shown
in Fig. 5A, commercially
available NAPQI exhibits readily detectable fluorescence emission with
a maximum at 440-450 nm using 306 nm as the excitation wavelength (16). Prolonged exposure of acetaminophen to
H2O2 leads to the appearance of a similar, but
weaker fluorescence emission, probably reflecting autooxidation of the
drug (Fig. 5B). Addition of 0.1-10 units of GPI to the
reaction mixture did not increase but decreased the spontaneous
oxidation (Fig. 5C). Though we do not know the exact reason
for this effect at present, GPI at least was not able to augment NAPQI
formation as other peroxidases. In a second indirect approach, GP
activity was analyzed by following oxidation of NADPH to NADP in the
presence of acetaminophen and H2O2 according to
the method of Keller and Hinson (17). We did not observe any effect of
acetaminophen (0.01 to 1 mM) on the rate of NADPH oxidation.

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Fig. 5.
Fluorescence spectrum of NAPQI
(A) and reaction mixtures containing 50 mM
sodium phosphate (pH 7.7), 0.5 mM acetaminophen, 36 mM cetyltrimethylammonium bromide, 0.2 mM
H2O2 in the absence (B) and
presence (C) of 2 units of GPI.
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DISCUSSION |
Acetaminophen toxicity is one of many human disease processes
widely believed to involve reactive oxygen species. The success in
using various antioxidants to protect against acetaminophen toxicity is
important not only for its therapeutic application but because it may
shed light on the mechanism of hepatotoxicity. Because of the severity
and high costs of poor clinical outcomes of acetaminophen poisoning and
because acetaminophen may serve as a model for other diseases involving
oxidative stress, research into mechanisms of toxicity and candidates
for therapeutic intervention is quite important.
GP is a critical antioxidant enzyme for the detoxification of
peroxides. Its particular kinetics and relatively low substrate specificity makes it a very efficient reducer of peroxides (24), far
more so than catalase, which is the other enzyme that detoxifies H2O2. The importance of GP has been shown in
many studies including several that demonstrate marked protection
against oxidative damage in cells by overexpression of GP through
transfection (25, 26). GP has been largely overlooked despite its
promise as a candidate for therapeutics in diseases involving oxidative
stress, in favor of studies involving SOD, the enzyme which converts
O
2 into H2O2. One of the current
prevailing theories regarding the toxicity of acetaminophen maintains
that increased oxidative stress in the liver is a critical factor in
acetaminophen-induced injury. As both SOD and GP are antioxidant
enzymes that reduce oxidative stress, we were interested in the effects
of overexpression of these enzymes on hepatocellular damage and
mortality as compared with one another and to controls. Because
acetaminophen is transported to the liver through the blood and
post-detoxification events are unclear and may also involve secretion
of by-products into the blood, we were also interested in the effects
of overexpression of the plasma form of GP on acetaminophen toxicity.
Overexpression of the plasma form of GP and of intracellular SOD are
equally protective against a lethal overdose of acetaminophen. That SOD
is protective, both in this and a previous study (10), implicates the
superoxide anion in the mechanism of acetaminophen toxicity. Indeed,
O
2 was found to be increased more than 2-fold in mouse liver
microsomes following a large dose of acetaminophen in the studies of
Lores Arnaiz et al. (27). The enhanced sensitivity of GPI
transgenic mice to acetaminophen toxicity was an unexpected finding in
our study. Intracellular overexpression of GPI, which is able to
detoxify the final products of oxidative stress and is significantly
more efficient in protecting cells in vitro against reactive
oxygen species than SOD and catalase (28), would be expected to confer
protection on the animals. Nevertheless, overexpression of GPI was
unable to protect against liver damage, and moreover, significantly
increased mortality of animals. The explanation of this effect most
likely lies in a unique situation, i.e. depletion of the
important intracellular antioxidant GSH by the antioxidant defense
enzyme GPI, an action that compounds GSH depletion by acetaminophen
metabolites and reactive oxygen species and could account for the slow
recovery of GSH in GPI mice. We did not observe any difference in GSH
depletion even at a short period of time, such as 5, 10, and 30 min
after acetaminophen administration (data not shown). There were
significant differences in GSH recovery in the liver and blood in GPI
mice compared with all other groups of mice at a later time point.
Importantly, this time period (4-8 h) correlates with the highest
lipid peroxidation, which requires active antioxidant enzymatic
activity for detoxification. Insufficient amounts of intracellular GSH
might explain why the level of lipid peroxidation in the liver was not
affected by GPI overexpression as well.
The protection of GPP animals is surprising in view of the traditional
belief that the major target organ of acetaminophen toxicity is liver
and that metabolism and thus toxic by-products would be localized
there. Several possibilities may contribute to acetaminophen resistance
in GPP animals. First, toxic metabolites of acetaminophen and/or
peroxides may be released from the liver into the blood and these are
efficiently detoxified by the increased plasma levels of GP.
Circulation of toxic metabolites could explain why, in some cases of
acetaminophen toxicity, organs besides the liver and kidney are
affected (sometimes even in the absence of severe hepatic necrosis)
(31). Another possibility is that secondary factors following
acetaminophen toxicity are influenced by overexpression of plasma GP.
For example, overexpression of extracellular GP can protect against
damage by increased scavenging of the released reactive oxygen species
as well as by inhibition of activation of inflammatory leukocytes,
which play an important role in acetaminophen-induced hepatotoxicity
(30). An additional important factor could be related to the difference
in electron donors utilized by GPP and GPI. It was recently shown that
GPP, in contrast to GPI, uses thioredoxin and glutaredoxin
significantly more efficiently than GSH (31). Thus, GPP mice were able
to detoxify blood reactive oxygen species under conditions of severe
oxidative stress caused by scavenging of GSH. On the other hand,
increased levels of lipid peroxides will not lead to increased GSH
oxidation by elevated levels of GPP. Indeed, GPP animals showed the
least depletion of glutathione in the plasma (Fig. 3C) and
thus were experiencing less oxidative stress in blood than any of the
other three groups. Details of the critical role of GSH presence in
extracellular pools, including blood, in detoxification and protection
against chemical and oxidant-induced injuries are described in a recent review by Smith et al. (32).
In summary, SOD and GPP transgenic mice demonstrated marked resistance
to acetaminophen overdose. In contrast, GPI animals showed
significantly increased sensitivity to acetaminophen as compared with
controls. These animals had a delay in restoration of the level of
glutathione, whereas GPP mice were characterized by least depletion and
most efficient restoration. Our study indicates that the phenotype may
be independent of lipid peroxidation with regard to acetaminophen
toxicity, consistent with a number of studies that have found no
correlation between toxicity and lipid peroxidation (24). Increased
toxicity most likely does not involve elevated peroxidative activity of
GPI using acetaminophen as cofactor, because this enzyme was not able
to form NAPQI from acetaminophen in vitro. At this point we
do not know the mechanism by which GPs were able to affect the profile
of oxidized acetaminophen metabolites, especially mercapturate. The
blood levels of these metabolites are the result of several processes
that might be affected by the level of GP expression. Our data also
suggest that in addition to hepatocellular damage, which has been the accepted hallmark feature of acetaminophen toxicity, events in the
blood are also crucial for organismal well being and survival in the
condition of an acetaminophen overdose. This finding may have important
implications for therapeutic intervention in patients suffering from
acetaminophen toxicity.