Title page
Title Influence of small doses of various vehicles on acetaminophen induced liver
injury
Running title: Vehicles in acetaminophen hepatotoxicity
Authors Tomislav Kelava1, Ivan Ćavar1,2, and Filip Čulo1,2
Affiliations:
1 Department of Physiology, School of Medicine, University of Zagreb, Croatia
2 Department of Physiology, School of Medicine, University of Mostar, Bosnia and
Herzegovina
Corresponding author: Tomislav Kelava, Department of Physiology, School of
Medicine, University of Zagreb, Croatia, Šalata 3b, 10000 Zagreb, Croatia. Phone
number: 00 385 91 34 70 585 Fax Number: 00 385 1 4590-207
E-mails: tkelava@yahoo.co.uk, ivancavarsb@yahoo.com, fculo@mef.hr
All investigations were conducted at Department of Physiology, School of Medicine,
University of Zagreb, Croatia
Source of support: Supported by Research project No. 0108-0000000-0328 by
Croatian Ministry of Science, Higher Education and Sports
Summary:
Background: Organic solvents or detergents are used in biological studies to facilitate solution
of drugs with low solubility. Although they are generally thought of as being relatively inert,
they can induce effects that are often overlooked. Inadequate usage of vehicles is the leading
cause of artifacts in acetaminophen induced liver injury (AILI) investigations. Therefore, we
decided to investigate the effect of dimethylsulfoxide (DMSO), dimethylformamide (DMF),
propylene glycol (PG), ethanol (ETOH) and Tween20 on AILI.
Material and methods: C57BL/6 male mice received intraperitoneally (i.p.) 30 minutes before
acetaminophen (300 mg/kg, i.p., in warm saline) injection of saline, DMSO, DMF, PG or
Tween 20, at 0,6 mL/kg or 0,2 mL/kg. Control groups received only vehicle. After seven
hours blood and livers were taken for analysis. Alanine aminotransferase levels (ALT) in
plasma and reduced glutathione (GSH) levels in liver were determined and histopatological
slides were evaluated.
Results: DMSO and DMF (both doses) almost completely abolished AILI. PG (0,6 mL/kg)
was also protective, but the dose of 0.2 mL/kg was only partially effective. These three
solvent blocked acetaminophen induced GSH depletion. ETOH (0,6 mL/kg) was partially
effective, while the dose of 0,2 mL/kg and TWEEN 20 (both doses) had no influence. In
control groups DMF (0,6 mL/kg) slightly decreased GSH levels, while the dose of 0,2 mL/kg
and any other vehicle had no significant influence on GSH or ALT levels. None of the
vehicles was protective in model of liver injury caused by D-galactosamine and
lipopolysaccharide.
Conclusion: Extreme caution is needed when organic solvents are used in AILI investigations.
Key words: acetaminophen, dimethylsulfoxide, dimethylformamide, propylene glycol,
ethanol, liver injury
Influence of small doses of various vehicles on
acetaminophen induced liver injury
Background
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is considered
safe at therapeutic doses. Overdose of APAP causes liver damage and for the last two decades
this is the leading cause of acute hepatic failure in the western world [1, 2]. The toxicity is
initiated by formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by
liver microsomal enzymes, -which first depletes glutathione and then covalently binds to
cellular proteins [3, 4]. APAP induced liver injury (AILI) in mice is the most extensively used
model for studying mechanisms of drug induced liver injury [5, 6].
Some of the major biases in AILI investigations have occurred because drug solvents, which
have biological effect per se, were used, particularly DMSO. For example El-Hassan et al
reported a protective effect of the pancaspase inhibitor Z-VAD-fmk against APAP toxicity in
mice. As a control, they used cathepsin inhibitor benzyloxycarbonyl-Phe-Alafluoromethylketone (Z-FA-fmk) and found no protection with this compound. However, they
did not add small amount of DMSO to Z-FA-fmk which was used to help Z-VAD-fmk
dilution [7]. Later it was shown that small amount of DMSO and not Z-VAD-fmk protected
mice from APAP [8]. Similarly, it was reported that natural killer cells (NK) have a
pathogenic role in AILI [9], but recently it was shown that this is true only when DMSO is
used to help dissolve APAP (as it was in above mentioned experiments), but not when APAP
was given without DMSO [6].
Vehicles commonly used in AILI experiments include DMSO [7], ethanol (ETOH), corn oil
[10], olive oil [11], Tween 20 [12], Tween 80 [13], propylene glycol (PG) [14] and
methylcellulose [15]. They are used for two purposes; to facilitate solution of some other
substance - investigated for its effect on AILI or to help dissolve APAP itself; in the later case
PG is frequently used. Despite its obvious importance, influence of vehicles on AILI is rarely
investigated; it is usually done when bias already happened. So far, it was shown, more or less
convincible, that DMSO, PG and ETOH, if injected 15-30 minutes before APAP or 1-2 hours
after APAP abolish its toxicity, most probably by preventing APAP conversion to NAPQI by
CYP2e1 inhibition. However, most of these investigations were done for doses of vehicles
larger than 1 mL/kg [16-18] and in AILI experiments smaller doses are usually used. Notable
exception is DMSO, for which good dose dependant study was conducted, which showed a
strong protective effect even when DMSO was administered at dose of 0.2 mL/kg [19]. The
effects of various solvent, to our knowledge, were never compared.
Therefore, we decided to compare effect of three most often used solvent (DMSO, PG and
ETOH) on AILI as well to investigate the effect of Tween 20 and DMF which has not been
investigated. Main object of our study was to show which vehicle at doses of 0,2 and 0,6
mL/kg change the intensity of AILI and which is indifferent. Beside common parameter of
APAP toxicity (serum aminotransferase level) and pathohistology, the influence on GSH
content in liver and microsomal enzyme activity was also investigated.
Materials and Methods
Animals
C57BL/6 mice were raised in an animal colony unit at the Department of Physiology, School
of Medicine, Zagreb. Male mice aged 12-16 weeks and weighing 20-25 g were used in all
experiments. The cages were stored in rooms with a 12 h light period from 6 a.m. to 6 p.m.,
and the temperature and relative humidity in the animal room were 21±2°C and 50±5%,
respectively. The cages were sanitized twice weekly. All mice were given free access to tap
water and standard mouse chow diet (Diete Standard 4 RF21 Mucedola S.r.i., Milan, Italy).
All animal protocols were approved by the Ethics Committee of the University of Zagreb,
School of Medicine (Zagreb, Croatia).
Chemicals
Pure APAP substance was a kind gift from the Belupo Pharmaceutical Company (Koprivnica,
Croatia). Trichloroacetic acid (TCA), Ethanol (96%), DMSO, PG and DMF were purchased
from Kemika, Zagreb, Croatia. Thiopental sodium was from Rotex Medica , Trittau,
Germany). L-Glutathione reduced, D-galactosamine, Bovine serum albumine (BSA) and 5,5dithiobis 2-nitro-benzoic acid (DTNB) were from Sigma-Aldrich, St. Louis, U.S.A.
Acetaminophen induced liver injury
A total of five experiments were conducted. In all experiments mice were fasted overnight (16
hours). Next morning they were randomly divided into groups (n = 6-8) and received i.p. 30
minutes before APAP either saline, DMSO, DMF, PG or Tween 20 (all diluted in saline), at
0,6 mL/kg in first and 0,2 mL/kg in second experiment in a volume of approximately 0,2 mL
per animal. APAP, dissolved in warm saline under mild magnetic stirring, was administered
also i.p. at 300 mg/kg, in a volume of approximately 0,5 mL per animal. Control groups of
animals received only vehicle (i.e. without APAP). In majority of published experiments
APAP and investigated substance were both injected i.p. To exclude the effect of
administration route, and possible interferences of chemicals, we conducted third experiment
which………….. lavage. In fourth experiment saline or DMF (0,6 mL/kg, i.p) were injected 2
hours before or 1, 2.5 and 4 hours after APAP administration. In fifth experiment mice
received sodium phenobarbitone (300 mg/L) in drinking water for 7 days prior to APAP
administration in order to induce liver microsomal enzymes. DMSO (0.2 mL/kg, i.p.) or
saline (i.p.) were injected 30 minutes before APAP. In all experiments food was returned 4
hours after APAP and after additional 3 hours mice were anesthetized with Avertine; blood
was collected by puncture of the medial eye angle with heparinized glass capillary tubes and
the livers were removed for analyses.
Hepatic injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS)
Mice were injected i.p. simultaneously with GalN (300 mg/kg) and LPS (15 g/kg). Saline,
DMSO, DMF, ETOH, PG or Tween 20 were injected i.p. (0,6 mL/kg) 30 minutes before
intoxication with GalN + LPS.
Evaluation of liver injury
Blood was centrifuged and plasma was collected and stored at -80°C. Alanine
aminotransferase (ALT) levels were determined by standard laboratory techniques in clinical
diagnostic laboratory. A portion of the liver was fixed in 10% neutral formalin, processed by
histologic techniques, stained with hematoxylin-eosin, and examined for morphologic
evidence of liver injury.
Hepatic GSH Determination
Reduced glutathione (GSH) in liver tissues was determined according to the Ellman method
[20]. A portion (approximately 100 mg) of liver was homogenized in Tris-HCl buffer
(25mmol/l, pH 7.4) and 0.1 mL of homogenate was mixed with 0.2 mL 5% TCA, followed by
centrifugation at 860 g for 10 minutes. Supernatant was mixed with DTNB. The absorbance
was determined at 412 nm. The GSH level was compared with a standard curve prepared with
six different GSH concentrations. From the rest of homogenate protein concentrations were
determined using the Bradford method with bovine serum albumin as the standard [21] and
GSH content was calculated in nmoles/mg proteins.
Measurement of thiopental sleeping time
Mice (n=4 per group) were injected with saline, DMSO, DMF, PG, ETOH or Tween 20 (0,6
mL/kg, 1l/g, i.p.), and after 30 minutes thiopental sodium (50 mg/kg, i.p.) was injected. The
sleeping time was defined as the sleep-time period from the loss of the righting reflex to its
complete recovery [22, 23].
Statistical analysis
Data are presented as means ± SEM. Statistical significance was evaluated by ANOVA
followed by Dunnetts’ multiple comparison test, and accepted when P < 0.05.
Results
At dose of 0.6 mL/kg DMSO, DMF, and PG almost completely abolished AILI; ALT levels
were 37, 45 and 15 times, respectively, smaller then in control group (which received saline
before APAP) (Table1). The difference was clearly visible histopathologically and also
macroscopically (Figure 1). ETOH only slightly reduced ALT levels (not statistically
significant - NS) and Tween 20 had no influence at all. APAP depleted GSH from liver and
DMF, DMSO and PG inhibited this depletion. When given alone (i.e. without APAP
treatment) DMF reduced slightly but significantly GSH level in comparison to mice given
saline, while other vehicles had no such effect. DMF alone reduced slightly but significantly
GSH level, while other vehicles had no such effect. None of the vehicles alone increased ALT
levels in blood significantly (Table 1). The different protective effect of these substances was
clearly visible histopathologically (Figure 1); centrilobular necrosis seen in mice given saline
(or Tween 20) before APAP Fig. 1A,C), was almost absent in mice given DMF (Fig. 1B,D) or
DMSO before APAP.
At dose of 0.2 mL/kg DMF and DMSO were similarly effective; ALT levels were 15 and 18
times smaller (lower) then in the control group, they also inhibited GSH depletion. At this
dose PG also reduced ALT concentration and blocked GSH depletion, but not significantly.
ETOH and Tween 20 had no influence. DMF alone had no influence on GSH levels (Table 2)
DMF at dose 0.6 mL/kg completely blocked APAP toxicity if given 2 hours before APAP; it
was also effective if given 1 hour after APAP administration, but ineffective not if given 2.5
or 4 hours after APAP (Figure 2).
DMSO at dose 0.2 mL/kg was highly protective even in mice which were given sodium
phenobarbitone in drinking water for 7 days before APAP administration. ALT levels were 30
fold smaller in DMSO group then in control group (Figure 3).
DMSO and DMF increased thiopental sleeping time significantly. PG also prolonged it, but
not significantly, while TWEEN20 or ETOH had no influence (Table 3).
None of the vehicles was able to protect mice from liver injury caused by LPS/GalN
administration (Figure 4).
Discussion
Although solvents are generally thought of as being relatively inert, they can induce
biological effects that are often overlooked. AILI is shown to be very sensitive to bias caused
by solvents [6, 8]. Our results show that DMSO and DMF greatly suppress AILI even at small
doses (0,2 mL/kg), PG is somewhat less protective and it seems that doses of ETOH (0,2
mL/kg) and Tween 20 (0,2 and 0,6 mL/kg) are indifferent. None of them appears to be
hepatotoxic itself at above mentioned doses. The most probable way of protection is
interference with liver microsomal metabolism of APAP. This is supported with our findings
that PG, DMSO and DMF block APAP induced GSH depletion and prolong thiopental
sleeping time. None of them change GSH content if given alone, except DMF at dose of 0,6
mL/kg which caused a small but significant GSH depletion. Yoon ET all proposed that
DMSO inhibits microsomal formation of NAPQI in competitive manner [19]. Therefore, we
hypothesized that experimental model in which microsomal enzymes are induced prior to
APAP administration will be less sensitive to DMSO. However, our results show that model,
in which microsomal enzymes are induced with phenobarbitone for 7 days in drinking water
[24, 25], is equally sensitive to DMSO. Beside its effect on APAP metabolism each of these
vehicles can change several others parameters that play role in AILI. For example, DMSO
posses antioxidative activity [26, 27], and adjuvant-like actions on the immune system [28,
29], the later effect was the cause of bias in already mentioned NK cell investigation and
several antioxidants protect from AILI [30, 31], Since DMSO is so frequently shown to
invalidate investigations, we thought that DMF might be an useful alternative (some of the
substances that we intend to investigate in the future can be dissolved in both DMSO and
DMF). However, DMF showed similar protective effect as DMSO. There are no reports about
its effect on oxidative stress or immunological system. On the other hand, DMF possess
anticoagulant like effect [32] and some anticoagulants are able to protect from AILI [33].
Since this is the first report about the protective effect of DMF we further investigated the
effect of time of its application. It showed typical pattern for substances that interfere with
APAP metabolism - a very strong protection if given before APAP, but no protection if given
2.5 hours after APAP (although it was protective if given 1 hour after APAP). DMF inhibits
CYP2e1 most probably by suicidal enzyme inactivation [34]. Protective effect of PG is well
known; however all experiment were conducted with high doses (between 2 and 4 mL/kg)
[17, 18] and our results demonstrate that even small doses (0.2 and particularly 0.6 mL/kg)
can suppress AILI. For ETOH it is generally accepted that its chronic consumption increase
AILI, but acute administration together with or shortly before APAP suppresses AILI [35].
With dose of 0,2 mL/kg there was no protection, and dose of 0,6 mL/kg only slightly
decreased ALT levels. PG and ETOH can reduce inflammatory response and oppositely to
DMSO, inhibit NK activity [36-39]. Detergents, TWEEN 20 and TWEEN 80, are also
sometimes added to help solubility of lipophilic drugs in AILI experiments. It appears that
doses of TWEEN 20 of 0.2 and 0.6 mL/kg do not change magnitude of AILI and GSH liver
content. There are no reports about its influence neither on immunological response, oxidative
stress, and Cyp2e1 activity nor coagulation system. In another common model of liver injury
(GalN/LPS) none of the vehicles was protective or harmful. Interestingly, there are reports
that show that DMSO is protective against D-galactosamine alone in rats. However, in those
experiments the dose of DMSO was much larger (2.5 mL/kg) [40]
To avoid artifacts in AILI investigations it would be good to use, when it is possible, pure
saline as a vehicle without adding any organic solvent or detergent. This is possible in more
occasions then is actually done. For example, 300 mg/kg of APAP, dissolved in warm saline
under mild magnetic stirring, can be safely administered in a volume of approximately 0.5 mL
per mice and this is more then enough to induce AILI. Since PG and DMSO suppress AILI
when they are used as vehicles for APAP, the dose of APAP must be increased to 500 or even
750 mg/kg [14] in order to cause similar rate of AILI. The result is unnecessary increase of
APAP dose and application of more biologically different organic solvent. Liu et al reported
that 300 mg/kg of APAP is not enough to induce AILI to C57Bl/6 mice and concluded that
this strain is more resistant to APAP [9]. Apparently this was because DMSO was added to
APAP vehicle. In our experience the dose 300 mg/kg of APAP induces severe hepatitis in
CBA or BALB/c mice and similar was shown for some other mice strains [15]. When
substances with low solubility in water are investigated for its effect on AILI the control
groups should be planned more carefully. Typical investigations include group which gets
substance of interest dissolved in organic vehicle and the control group which gets only
vehicle. It would be good to include at least in first experiment one more control group which
would be treated with pure saline or distilled water, in order to better understand effect of
vehicle. The dose of solvent or detergent added to vehicle should be as small as possible and
explicitly quoted. It is probably better to use vehicles that interfere less with APAP toxicity.
This means that Tween 20 and ETOH should be considered first, and then PG. DMSO and
DMF should be used if there is no other option. Alternatively, some water insoluble
substances become soluble if pH of saline is changed or if Tris buffer (pH 7.4) instead DMSO
is used [41], although Tris buffer also possess some biological effects. If some general
hepatoprotective mechanism is investigated it would be helpful to see if it works on
GalN/LPS model of hepatotoxicity also. Since this model appears to be more robust to vehicle
usage.
Conclusion
To avoid possible experimental artifacts with water insoluble drugs, it necessary to reduce the
amount of injected of biologically active organic solvent (or reduces its concentration in vitro
experiments) to possible minimum, use organic solvent with smaller biological effect, try to
dissolve the substance in aqueous solvent with changed pH or different buffer. Finally, most
important is to include control groups in which the effect of vehicle (solvent) alone will be
tested.
Table 1
Effect of DMSO, DMF, ETOH, PG and Tween 20 treatment (0.6 mL/kg) on AILI
Vehicle (0.6
mL/kg)
Saline
DMSO
DMF
ETOH
PG
Tween 20
Saline
DMSO
DMF
ETOH
PG
Tween 20
Normal mice
APAP
(mg/kg)
300
300
300
300
300
300
-
ALT
(U/L)
6747.5 ± 2290.4
178.6 ± 96.9*
135 ± 47.7*
4633.8 ± 2066.4
441.4 ± 317.8*
5268.6 ± 2512.6
20 ± 1.5
30.7 ± 5.7
27 ± 1.5
32.7 ± 5.2
28.3 ± 4.4
37.3 ± 7.1
22 ± 1.1
GSH
(nmol/mg of
proteins)
9.8 ± 1.26
53.2 ± 1.7*
56.1 ± 1.0*
19.6 ± 5.1
46.2 ± 5.7*
9.4 ± 2.5
64.9 ± 0.5
65.8 ± 0.9
57.1 ± 0.28**
63.8 ± 1.3
65.1 ± 0.7
64.5 ± 2.4
64.4 ±0.8
Mice were treated with DMSO, DMF, ETOH, PG, Tween20 or saline intraperitoneally 30 minutes
prior to APAP or only with DMSO, DMF, ETOH, PG, Tween20. Each value represents the mean ±
S.E of six to eight mice.
* Significantly different from mice treated with saline before APAP
** Significantly different from mice given only saline (ANOVA followed by Dunnetts’ multiple
comparison test, p<0.05).
Table 2
Effect of DMSO, DMF, ETOH, PG and Tween20 treatment (0.2 mL/kg) on AILI
Vehicle
(0.2
mL/kg)
Saline
DMSO
DMF
ETOH
PG
Tween 20
Saline
DMSO
DMF
ETOH
PG
Tween 20
APAP
(mg/kg)
300
300
300
300
300
300
-
ALT
(U/L)
8767 ± 1663
585 ± 397*
470 ± 188*
8991 ± 2048
5830 ± 1242
8774 ± 1762
25.6 ± 1.6
29 ± 1.3
34.6 ± 3.5
24.6 ± 1.9
20.3 ± 0.2
29.6 ± 3.3
GSH
(nmol/mg of
proteins)
9.7 ± 0.9
44.9 ± 1.7*
44 ± 2.9*
12.1 ± 4.5
17.2 ± 5.5
11 ± 2.2
67.5 ± 1.8
65.6 ± 0.5
65.7 ± 0.7
65.8 ± 1.9
65.3 ± 1.8
64.1 ± 2.1
Mice were treated with DMSO, DMF, ETOH, PG, Tween20 or saline intraperitoneally 30 minutes
prior to APAP or only with DMSO, DMF, ETOH, PG, Tween20 and saline. Each value represents the
mean ± S.E. of six mice.
* Significantly different from mice treated with saline before APAP (ANOVA followed by Dunnetts’
multiple comparison test, p<0.05).
Table 3
Effect of DMSO, DMF, ETOH, PG, Tween20 treatment (0.6 mL/kg) on thiopental sleeping time
Vehicle
(0.6 mL/kg)
Saline
DMSO
DMF
ETOH
PG
Tween 20
Sleeping time
(min)
1.1 ± 0.3
26.2 ± 2.3**
26 ± 2.2**
1 ± 0.3
3.8 ± 0.7
0.9 ± 0.1
Mice were treated with DMSO, DMF, ETOH, PG, Tween 20 or saline intraperitoneally 30 minutes
prior to thiopental sodium. Each value represents the mean ± S.E. of four mice.
** Significantly different from mice treated with saline (ANOVA followed by Dunnetts’ multiple
comparison test, p<0.01).
A
B
C
D
Figure 1 histopathology
In the typical microphotograph of the liver in the rat treated with acetaminophen, extensive zonal
necrosis of centrilobular hepatocytes was observed (Fig. 5A). In contrast, neither hepatic necrosis
nor abnormality was observed in the hepatic section from the rat treated with ONO-1714 at 100 Ag/
kg in addition to acetaminophen (Fig. 5B).
16000
14000
ALT (U/L)
12000
10000
8000
6000
4000
**
2000
*
0
Saline
DMF (-2h)
DMF(+1h) DMF (+2.5h) DMF (+4h)
Figure 2. Time effect of DMF on ALT plasma levels. Mice were treated with saline or DMF (0.6
mL/kg) at indicated times prior or after the APAP. Each value represents the mean ± S.E of six or
seven mice.
*, ** Significantly different from mice treated with saline and APAP (ANOVA followed by Dunnetts’
multiple comparison test, p<0.05, 0.001 respectively).
ALT (U/L)
15000
10000
5000
**
0
Saline
DMSO
Figure 3. Effect of DMSO on ALT levels in mice treated with phenobarbitone before APAP. Mice
were pretreated with phenobarbitone in drinking water (300 mg/l) and then treated with DMSO (0.2
mL/kg) or saline prior to APAP. Each value represents the mean ± S.E of eight mice.
** significantly different from mice treated with saline before APAP (Student’s t-test, p<0.001).
6000
ALT (U/L)
5000
4000
3000
2000
1000
0
Saline
DMSO
DMF
ETOH
PG
Tween 20
+ GalN/LPS
Figure 4. Effect of DMSO, DMF, ETOH, PG and Tween20 on ALT levels in GalN/LPS model.
Mice were treated with DMSO, DMF, ETOH, PG, Tween20 or saline intraperitoneally 30 minutes
prior to D-galactosamine and lipopolysaccharide administration. Each value represents the mean ± S.E
of six mice.
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