Melatonin protects on toxicity by acetaminophen but not on pharmacological effects in mice.

Abstract

The pineal gland and its main hormone, melatonin (MLT), are involved in a variety of physiological processes. MLT is a member of the indolamine family and has significant antioxidative activity. Acetaminophen (AA) is the most widely used medication in the world, both by prescription and over the counter. In large doses, AA is hepatotoxic causing oxidative stress and lipid peroxidation. Therefore, antioxidants have been used to protect against the toxicity of AA. Here, we examined in vitro and in vivo the protective effects of MLT against AA-induced toxicity in mice. MLT (100 microM) had a significant protective effect on the AA (7 mM)-induced loss of cell viability in mouse primary cultured hepatocytes as determined using the 3H-thymidine incorporation assay and MTT assay. The AA-induced generation of reactive oxygen species (ROS) peaked at 6 h and was followed by an increase in lipid peroxidation at 12 h in hepatocytes. MLT (0.1, 1, 10 or 100 microM) dose-dependently attenuated the increase in both production of ROS and lipid peroxidation by AA. Similarly, in vivo, AA (400, 600 or 800 mg/kg, intraperitoneally)-induced mortality and hepatotoxicity were significantly decreased by MLT (10 mg/kg, subcutaneously). Pretreatment with MLT had a greater protective effect on the hepatotoxicity of AA than post-treatment. However, MLT had no protective effect on the antipyretic effect or antinociception caused by AA. These results suggest that MLT is potentially useful for preventing AA-induced toxicity, but not the antipyretic effect or antinociception caused by AA.

Full text

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The pineal gland and its main hormone, melatonin (MLT),
are involved in a variety of physiological processes including
the regulation of endocrine rhythms,1)antigonadotropic
effects,2)neuroprotective effects,3)and stimulation of the im-
mune function.4)In addition, recent studies in vitro have
shown that MLT functions as an antioxidant, i.e. a scavenger
of the hydroxy radical and peroxy radical.5,6)It has also been
shown that when animals and tissues are subjected to lipid
peroxidation, MLT affords substantial protection against the
oxidative destruction of lipids.7,8)
Acetaminophen (AA) is the most widely used medication
in the world, both by prescription and over the counter. In
large doses, however, AA produces centrilobular hepatic
necrosis in humans and experimental animals.9,10)The AA-
induced hepatotoxicity involves a change in cellular redox
status toward a state of oxidative stress.11,12)Whereas deple-
tion of intracellular gluthathione (GSH) by the active AA
metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), is an
early event leading to oxidative stress, reactive oxygen and
nitrogen species generated by hepatic nonparenchymal cells
and infiltrating phagocytes contribute to the persistence of
this response.13,14)The concept that oxidative stress is impor-
tant in the pathogenic process is supported by findings that
antioxidants abrogate AA-induced hepatotoxicity.15,16)
Since AA is included in cold remedies as an analgestic or
antipyretic and frequently used long-term, there are numer-
ous opportunities for its concomitant use with other drugs.
Although not licensed as a drug, MLT is widely sold as a
nutritional supplement in many countries for its purported
sleep-promoting and antiageing properties.17)Therefore, it is
important to evaluate the interaction between AA and MLT.
Recently, Sener et al. has shown to protect by MLT on AA-
induced hepatotoxicity in mice.18)The aim of this study was
to examine the effects of MLT on AA-induced hepatotoxicity
in vitro and in vivo or pharmacological action by AA using
mice.
MATERIALS AND METHODS
Animals and Chemicals
(25—30g) obtained from Japan SLC (Hamamatsu, Japan)
were maintained on a 12h light/dark cycle in a temperature-
and humidity-controlled room. The experiments were con-
ducted in accordance with the standards established by the
Japanese Pharmacological Society. The animals were allowed
free access to laboratory pellet chow (CE-2; CLEA Japan
Inc., Tokyo, Japan) and water before the experiments. AA
was purchased from Junsei Pharmaceutical Co., Ltd.
(Nagano, Japan). Melatonin (MLT), N-acetyl-L-cysteine
(NAC), and other drugs of the highest grade available were
purchased from Wako Pure Chemical Industries (Osaka,
Japan). All cell culture reagents were obtained from Invitro-
gen Corp. (Carlsbad, CA, U.S.A.). For the experiments
in vitro, AA was dissolved in medium, and MLT was
dissolved in dimethylsulfoxide (DMSO) to make a concen-
tration of 1 M as a stock solution. It was used after dilution of
the stock solution with DMSO. DMSO at concentrations
lower than 0.25% had no effect on cell growth. For the exper-
iments in vivo, AA was dissolved in 0.5% Tween 80 in
saline. MLT was initially dissolved in DMSO and diluted
with saline to produce a 0.25% DMSO solution. Exposure to
light was kept to a minimum for all drugs used.
Hepatocyte Culture
Hepatocytes were isolated from the
mice by a modified version of the two-step collagenase per-
Adult male C57BL/6mice
472Vol. 29, No. 3
Biol. Pharm. Bull. 29(3) 472—476 (2006)
Melatonin Protects on Toxicity by Acetaminophen But Not on
Pharmacological Effects in Mice
Syu-ichi KANNO,*,aAyako TOMIZAWA,aTakako HIURA,aYuu OSANAI,aMai KAKUTA,aYasue KITAJIMA,a
Kimiko KOIWAI,aTakaharu OHTAKE,bMayuko UJIBE,aand Masaaki ISHIKAWAa
aDepartment of Pharmacology and Toxicology, Cancer Research Institute, Tohoku Pharmaceutical University; 4–4–1
Komatsushima, Aoba-ku, Sendai 981–8558, Japan: and bTohoku Employee’s Pension Welfare Hospital; 1–12–1 Hukumuro,
Miyagino-ku, Sendai 983–0005, Japan.
Received October 3, 2005; accepted November 24, 2005; published online December 5, 2005
The pineal gland and its main hormone, melatonin (MLT), are involved in a variety of physiological
processes. MLT is a member of the indolamine family and has significant antioxidative activity. Acetaminophen
(AA) is the most widely used medication in the world, both by prescription and over the counter. In large doses,
AA is hepatotoxic causing oxidative stress and lipid peroxidation. Therefore, antioxidants have been used to pro-
tect against the toxicity of AA. Here, we examined in vitro and in vivo the protective effects of MLT against AA-
induced toxicity in mice. MLT (100m mM) had a significant protective effect on the AA (7mM)-induced loss of cell
viability in mouse primary cultured hepatocytes as determined using the 3H-thymidine incorporation assay and
MTT assay. The AA-induced generation of reactive oxygen species (ROS) peaked at 6h and was followed by an
increase in lipid peroxidation at 12h in hepatocytes. MLT (0.1, 1, 10 or 100m mM) dose-dependently attenuated the
increase in both production of ROS and lipid peroxidation by AA. Similarly, in vivo, AA (400, 600 or 800mg/kg,
intraperitonealy)-induced mortality and hepatotoxicity were significantly decreased by MLT (10mg/kg, subcuta-
neously). Pretreatment with MLT had a greater protective effect on the hepatotoxicity of AA than post-treat-
ment. However, MLT had no protective effect on the antipyretic effect or antinociception caused by AA. These
results suggest that MLT is potentially useful for preventing AA-induced toxicity, but not the antipyretic effect or
antinociception caused by AA.
Key words
melatonin; acetaminophen; antioxidant; hepatotoxicity
© 2006 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: syu-kan@tohoku-pharm.ac.jp

Page 2

fusion method of Seglen.19)Briefly, the liver of a mouse was
perfused for 5min with prewarmed (37°C) Ca2?- and Mg2?-
free Hank’s balanced salt solution, pH 7.2, containing 10mM
HEPES, 0.5mM EGTA, and 4.0mM NaHCO3. This was fol-
lowed by a 15min perfusion with prewarmed (37°C) Hank’s
balanced salt solution, pH 7.5, containing collagenase
(0.05%) and buffered with 10mM HEPES and 4.0mM
NaHCO3. After the second step of perfusion, isolated cells
were centrifuged (50?g for 1min, 4 spins) in minimum
essential medium (MEM) to remove nonparenchymatous and
dead cells. Then the medium was changed to Williams’ E
medium (WE) containing 5% fetal bovine serum, 10?7M in-
sulin, 10?7M dexamethasone, 100units/ml penicillin and
100mg/ml streptomycin. This procedure routinely yielded
over 90% viability based on the trypan blue exclusion test.
Approximately 1?106/ml parenchymatous cells were plated
on 35-mm Falcon collagen-coated type I dishes. After incu-
bation at 37°C for 2h in a humidified environment of 5%
CO2–95% air, the cultures were rinsed with warmed phos-
phate-buffered saline (PBS) to remove free cells and debris,
and then serum-free WE containing 0.12mg/ml aprotinin was
added and the cells were incubated with AA and MLT or
NAC.
Cell Viability
Cell viability was evaluated by measuring
the level of 3H-thymidine incorporation or using a modifica-
tion of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay.20)Briefly, 3H-thymidine at a final
concentration of 0.05mCi/ml was added to cultured hepato-
cytes 30min before the measurement time points. The cul-
tured hepatocytes were washed with PBS two times, and then
the cells were harvested from the dishes with a cell scraper.
Samples were emulsified in scintillation fluid and measured
in a Beckman LS 6000TA beta scintillation counter. 3H-
thymidine incorporation (%) was calculated according to
{cpm (test groups)/cpm (control groups)}?100. For MTT
assay, following exposure of hepatocytes to AA and MLT or
NAC, 50ml of MTT (10mg/ml saline) was added, and sam-
ples were incubated for 1h at 37°C. The cells were lysed and
solubilized by addition of 500ml of dimethyl sulfoxide
(DMSO) for 2h at 37°C, and the absorbance of 100-ml
aliquots was determined at 590nm using an Inter-med model
NJ-2300 Microplate Reader. Cell viability (%) was calcu-
lated relative to the control.
Generation of Reactive Oxygen Species (ROS)
generation of intracellular reactive oxygen species (ROS)
was evaluated based on the intracellular peroxide-dependent
oxidation of 2?,7?-dichlorodihydrofluorescein diacetate
(DCFH-DA) to form the fluorescent compound, 2?,7?-dichlo-
rofluorescein (DCF), as previously described by us.21)After
the treatment with drugs, the cells were incubated with
20mM DCFH-DA (from a stock solution of 4mM DCFH-DA
in ethanol) at 37°C for 10min. The culture medium was
removed, the cells were washed with PBS, and 2ml of PBS
was added to each well. The fluorescence intensity of the cell
suspension was determined using a fluorescence spectropho-
tometer (RF-1500, Shimadzu, Kyoto, Japan) with excitation
at 488nm and emission at 525nm. The untreated groups
were used as the control. The results are expressed as a per-
centage of the fluorescence intensity with respect to the con-
trol.
Lipid Peroxidation
The lipid peroxide level was assayed
The
by fluorimetrically measuring the level of malondialdehyde
(MDA) in hepatocytes using the method of Yagi.22)Cells
were exposed to drugs, and then washed with PBS, scraped
and homogenized in ice-cold 1.15% KCl. Samples contain-
ing 20ml of cell lysate were combined with 20ml of 8.1%
sodium dodecyl sulfate (SDS), 150ml of 20% acetic acid ad-
justed to pH 3.5, and 150ml of 0.8% thiobarbituric acid
(TBA). The mixture was heated at 95°C for 60min. After
cooling to room temperature, 100ml of distilled water and
2.0ml of a mixture of n-butanol and pyridine (15:1, v/v)
were added to each sample and the mixture shaken was vig-
orously. After centrifugation at 1200rpm for 10min, the su-
pernatant was isolated. The fluorescence intensity was meas-
ured using the RF-1500 (Shimadzu) with excitation at
515nm and emission at 553nm. A MDA solution made
freshly by the hydrolysis of 1,1,3,3-tetraethoxypropane
(TEP) was used as the standard. The results are expressed as
nmol MDA/mg protein.
Hepatotoxicity and Mortality in Vivo
MLT on the hepatotoxicity of AA in vivo was estimated by
measuring levels of serum glutamyl oxaloacetic transaminase
(GOT) and glutamyl pyruvic transaminase (GPT) activity.
These activities were monitored with a commercial kit from
Wako Pure Chemical Industries (Osaka, Japan). Acute mor-
tality was recorded for 72h after the intraperitoneal (i.p.) in-
jection of AA.
Temperature Measurement
and a thermocouple-monitoring thermometer were obtained
from Natume Industries (Tokyo, Japan). The probe was in-
serted into the rectum to a depth of 2cm. All experiments
began with the i.p. administration of AA at 10 am, and tem-
perature measurements were made 1h thereafter.
Nociceptive Assessment
Mice were assessed for noci-
ceptive sensitivity using the writhing test as described by
Koster et al.23)All mice were acclimatized to the procedure
room for at least 1h prior to testing. All testing occurred near
midphotoperiod (10:00—16:00). Mice were placed on a
glass surface within transparent Plexiglas cylinders and al-
lowed 30min to habituate to the cylinder. At this point, the
mice were weighed, injected with drugs or vehicle, and then
placed back into the cylinder. Thirty minutes later, 0.9%
acetic acid in saline was injected i.p. (10ml/kg). Mice were
placed in the cylinders once again and observed continuously
for 30min. Stereotypical writhes (lengthwise constrictions of
the torso with a concomitant concave arching of the back)
were counted over this period in 5-min bins. Four mice were
observed and scored at a time. In all cases, the experimenter
was kept blind to the drug/vehicle solutions. Antinociception
was quantified by reference to the vehicle-treated control
group.
Statistical Analysis
Statistical analysis of the results
was performed with a one-way analysis of variance
(ANOVA) followed by Scheffe’s F test. A p-value of less
than 0.05 was considered significant.
The Effect of
A rectal probe for mice
RESULTS AND DISCUSSION
Cell Viability
(MLT) on the acetaminophen (AA)-induced loss of cell via-
bility in mouse primary cultured hepatocytes (Fig. 1A). MLT
(100mM) and AA (7mM) were administered simultaneously
First, we examined the effect of melatonin
March 2006473

Page 3

to the hepatocytes. The concentration of AA (7mM) used in
the experiments was reported to reduce cell viability in our
previous study.21)As shown in Fig. 1A, MLT had a signifi-
cant protective effect on cell viability from 12h in the 3H-
thymidine incorporation assay and MTT assay. MLT alone at
more than 5mM caused a reduction in viability (data not
shown). We also examined the dose-dependent effect of MLT
on the AA-induced loss of cell viability in the hepatocytes
(Fig. 1B). A typical antioxidant, N-acetyl-L-cysteine (NAC)
is a cysteine precursor known to scavenge free radicals and
replenish tissue glutathione (GSH) levels. Treatment of hu-
mans and animals with NAC ameliorates the hepatotoxicity
of AA.24,25)Based on these reports, we used NAC as a posi-
tive control in this experiment. The AA-induced decline in
cell viability was dose-dependently reduced by NAC and
MLT. Interestingly, MLT had a protective effect at a lower
dose than NAC. MLT is lipophil product than NAC. It is pos-
sible to effect of cellular uptake of NAC and MLT depend on
their difference of lipophilia. MLT was reported to stimulate
the activities of several antioxidant enzymes including super-
oxide dismutase (SOD) and glutathione peroxidase (GSH-
Px) in hepatocytes.26)Transgenic mice over-expressing SOD
or GSH-Px are protected from the hepatotoxicity of AA.27)
This may be due to the greater efficacy of MLT in scavenging
free radicals and also its ability to stimulate antioxidant
enzymes, which is not observed with NAC.
Generation of Reactive Oxygen Species (ROS) and
Lipid Peroxidation
A significant amount of evidence
points to the potential involvement of oxidative stress in the
toxicity of AA. We attempted to confirm the effect of MLT
(0.1, 1, 10 or 100mM) on the AA (7mM)-induced generation
of reactive oxygen species (ROS) and lipid peroxidation
(Figs. 2A, B). The AA-induced generation of ROS peaked at
6h (Fig. 2A), followed by a maximal increase in lipid peroxi-
dation at 12h (Fig. 2B). These results suggest that after ex-
posure to AA, hepatocytes undergo a sequence of events, an
increase in the generation of ROS, followed by lipid peroxi-
dation, and ultimately a loss of cell viability. MLT dose-de-
pendently attenuated the increase in both the generation of
ROS and lipid peroxidation by AA. When the co-incubation
with 100mM MLT, it reduced the ROS and lipid peroxidation
until near the levels of control. Cuzzocrea and Reiter sug-
gested that MLT is a better antioxidant against lipid peroxi-
dation than vitamins.6)Thus MLT has a potential antioxida-
tive effect on the AA-induced generation of reactive oxygen
species (ROS) and lipid peroxidation.
Mortality and Hepatotoxicity in Vivo
Figs. 1 and 2, the AA-induced loss of cell viability, genera-
tion of ROS, and increase in lipid peroxidation were signifi-
cantly abolished by MLT in mouse primary cultured hepato-
cytes. Next, we examined in vivo the effect of MLT
As shown in
474 Vol. 29, No. 3
Fig. 1.
duced Loss of Cell Viability in Mouse Primary Cultured Hepatocytes
(A) Time-dependent effect of MLT (100mM) on AA (7mM)-induced reduction of cell
viability. Open circles: AA treatment groups. Closed circles: MLT plus AA treatment
groups. (B) Dose-dependent effect of N-acetyl-L-cysteine (NAC) and MLT on AA
(7mM)-induced loss of cell viability after treatment for 24h. The reduction in cell via-
bility was estimated using the 3H-thymidine incorporation assay and MTT assay, as de-
scribed in Materials and Methods. Each value represents the mean?S.E. for three dif-
ferent experiments performed in triplicate. ∗p?0.05 vs. AA (7mM) treatment groups.
The Effect of Melatonin (MLT) on the Acetaminophen (AA)-In-
Fig. 2.
Generation of Reactive Oxygen Species (ROS) and Increase in Lipid Peroxi-
dation in Mouse Primary Cultured Hepatocytes
(A) Left panel: Time-dependent effect of MLT (100mM) on AA (7mM)-induced gen-
eration of ROS. Open circles: AA treatment groups. Closed circles: MLT plus AA
treatment groups. Right panel: Dose-dependent effect of MLT on AA -induced genera-
tion of ROS for 6h. The generation of ROS was evaluated using DCFH fluorescence, as
described in Materials and Methods. (B) Left panel: Time-dependent effect of MLT
(100mM) on AA (7mM)-induced increase in lipid peroxidation. Open circles: AA treat-
ment groups. Closed circles: MLT plus AA treatment groups. Right panel: Dose-de-
pendent effect of MLT on AA-induced increase in lipid peroxidation for 12h. The lipid
peroxidation level was evaluated by fluorimetrically measuring the level of malondi-
aldehyde (MDA), as described in Materials and Methods. Each value represents the
mean?S.E. for three different experiments performed in triplicate. ∗p?0.05 vs. AA
(7mM) treatment groups.
The Effect of Melatonin (MLT) on Acetaminophen (AA)-Induced

Page 4

(10mg/kg, subcutaneously; s.c.) on mortality and hepatotoxi-
city in AA-treated mice (Figs. 3A, B). MLT and AA were ad-
ministered simultaneously. The mortality for AA at 400, 600,
or 800mg/kg (i.p.) alone (n?10) was 15?5, 50?10, and
80?10%, respectively (Fig. 3A). A single treatment with
MLT (10mg/kg, s.c.) has never shown the mortality. Co-
treatment with MLT significantly reduced the mortality (AA
400mg/kg, 0%; AA 600mg/kg, 20?5%; AA 800mg/kg,
55?5%). The hepatotoxicity of AA was estimated by meas-
uring transaminase activity, such as GPT and GOT, after
treatment for 18 h (Fig. 3B). This was the time point at which
the increase in transaminase activity caused by AA peaked in
our previous report.28)AA (400mg/kg, i.p.) alone remarkably
increased levels of GPT (8125?1923KU/l) and GOT
(8028?1743KU/l) activity compared to the control group
(33.8?4.8 and 108?8.8KU/l, respectively). MLT (10mg/kg,
s.c.) alone treatment group has no change the control group
(GPT, 35.6?5.2KU/l; GOT, 107?11.8KU/l). We examined
the timing of the effect of MLT on the hepatotoxicity of AA.
Pretreatment for 3d, or treatment 2h before, simultaneously,
or 2h after all significantly ameliorated the AA-induced rise
in GPT and GOT activity, 102.8?24.8 and 142.7?37.6KU/l,
82.5?13.8 and 136.7?29.4KU/l, 345.4?55.8 and 336.5?
63.4KU/l, and 944.6?210.1 and 882.6?145.3KU/l, respec-
tively. Pretreatment had a greater effect on the hepatotoxicity
of AA than post-treatment. The results suggest that MLT pre-
vents the hepatotoxicity and which does not mean direct ac-
tion to toxicity caused by AA. The depletion of liver tissue
GSH was restored by MLT and/or NAC treatment.29)Grewal
and Racz reported that late treatment with NAC had no effect
on the toxicity of AA,30)however, NAC is used for detoxifi-
cation in a variety of diseased states including AA toxicity.
Pierrefiche et al. suggested that the hepatic metabolite of
MLT, 6-hydroxymelatonin sulfate, may also be a free radical
scavenger since it was shown to be capable of resisting lipid
peroxidation.31) These results suggest that MLT may act not
only to prevent AA toxicity but also as a detoxification medi-
cine.
Pharmacological Effect
In general, it is thought that the
pharmacological effect is weakened if the toxicity is attenu-
ated. We also examined the effect of MLT on the pharmaco-
logical action of AA as an antipyretic effect and antinocicep-
tion (Figs. 4A, B). MLT and AA were administered simulta-
neously. Body temperature in the non-treatment (control)
group and MLT (10mg/kg, s.c.) treatment group was 37.41?
0.19 and 37.23?0.14°C, respectively. AA at 25, 50, 100,
or 200mg/kg (i.p.) caused hypothermia, which increased
dose-dependently at 36.77?0.18, 37.51?0.14, 36.32?0.19,
and 34.82?0.19°C, respectively (Fig. 4A). MLT slightly in-
creased the antipyretic effect of AA (AA 25mg/kg, 36.81?
0.22°C; AA 50mg/kg, 37.06?0.14°C; AA 100mg/kg,
35.69?0.16°C; AA 200mg/kg, 34.45?0.19°C). Similarly,
the antinociceptive effect of AA at 25, 50, 100, or 200mg/kg
(i.p.) dose-dependently increased 80.51?10.12, 65.51?9.18,
39.66?7.14, or 20.34?5.32%. MLT caused a slight increase
in AA-induced antinociception (AA 25mg/kg, 79.32?13.64%;
AA 50mg/kg, 55.63?3.89%; AA 100mg/kg, 29.41?5.69%;
AA 200mg/kg, 12.44?3.20%). MLT had no protective effect
on either the antipyretic effect or antinociception caused by
March 2006475
Fig. 3.
Hepatotoxicity and Mortality in Mice
(A) The effect of MLT on mortality in AA-treated mice. Acute mortality was
recorded over the 72h after the intraperitoneal injection of AA. (B) The effect of MLT
on the hepatotoxicity of AA in mice. Hepatotoxicity was estimated by measuring levels
of serum glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase
(GPT) activity for 18h, as described in Materials and Methods. Each value represents
the mean?S.E. for three different experiments (n?10). ∗p?0.05 vs. AA alone treat-
ment groups.
The Effect of Melatonin (MLT) on Acetaminophen (AA)-Induced
Fig. 4.
Antipyretic Effect (A) and Antinociception (B) in Mice
Open column: AA treatment groups. Closed column: MLT (10mg/kg, s.c.) plus AA
treatment groups. The experimental procedures were described in Materials and Meth-
ods. Each value represents the mean?S.E. for five different experiments (n?4).
The Effect of Melatonin (MLT) on Acetaminophen (AA)-Induced

Page 5

AA (Figs. 4A, B). There are a number of points at which
MLT may interfere with the inflammatory process.6)
Prostaglandin (PG) levels in exudates and cyclooxygenase-2
expression in carrageenan-treated rats were found to be com-
pletely suppressed by MLT.32)There was no significant dif-
ference in pharmacological action between the control group
and MLT alone treatment group, but MLT may potentiate the
effects of other antiinflammatory drugs.
We have used MLT at 10mg/kg for in vivo studies and
100mM for in vitro studies, which dosage too high as com-
pared with the concentration of MLT under physiological
conditions. However, half-life of MLT in the blood is rela-
tively short (20—40min), tissue levels of MLT remain
higher than physiological levels when blood MLT concentra-
tion fall after administration of a pharmacological dose of
MLT.33) The mechanisms of MLT action in physiological and
pharmacological concentration may be different.
In conclusion, our results suggest that MLT has a poten-
tially useful preventive effect on the toxicity of AA, but no
effect on the antipyretic effect and antinociception caused by
AA.
Acknowledgments
NISHINOMIYA Basic Research Fund (Japan).
This work was supported in part by
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