Download Hepatoprotective Effects of the Shark Bile Salt 5/?

FUNDAMENTAL AND APPLIED TOXICOLOGY 3 3 , 3 1 - 3 7 (1996)
ARTICLE NO. 0140
Hepatoprotective Effects of the Shark Bile Salt 5/?-Scymnol on
Acetaminophen-lnduced Liver Damage in Mice
THEODORE A. MACRIDES,* 1 LEE M. NAYLOR,* NICOLETTE KALAFATIS,* AMAL SHIHATA,* AND PAUL F. A. WRIGHT!
*Biochemistry Unit, Department of Medical Laboratory Science, and ^Key Centre for Applied and Nutritional Toxicology,
RMIT-University, G.P.O. Box 2476V, Melbourne, Victoria, 3001, Australia
Received September 21, 1995; accepted April 15, 1996
hepatic cytochrome P450 mixed-function oxidase system
(Potter et ai, 1973).
The reactive metabolite is normally detoxified through
NAYLOR, L. M., KALAFATIS, N., SHIHATA, A., AND WRIGHT,
conjugation
to glutathione (GSH), but following APAP overP. F. A. (1996). Fundam. Appl. Toxicol. 33, 31-37.
dose GSH is depleted, allowing NAPQI to bind more readily
The hepatoprotective effect of the shark bile salt 5/3-scymnol to hepatic proteins and initiate processes that lead to cytotoxhas been studied in the model of acute hepatotoxicity induced icity (Bartolone et ai, 1989; Albano et ai, 1985). Currently,
by administration of acetaminophen (APAP, paracetamol). 5/3- A'-acetylcysteine (NAC) is widely accepted as an antidote
Scymnol at doses of 20, 35, and 70 mg/kg intraperitoneally (ip) for the prevention of hepatotoxicity after an APAP overdose
decreased significantly the serum activity of alanine aminotrans- (Smilkstein et ai, 1988).
ferase, sorbitol dehydrogenase, and lactate dehydrogenase (p <
AZ-Acetylcysteine protects the liver by a combination of
0.05) caused by APAP treatment (350 mg/kg ip) alone. The highest mechanisms, including direct binding to NAPQI; protection
dose of 5/3-scymnol remained hepatoprotective when administered
of essential thiol groups in functional and structural cellular
4 hr after the APAP overdose. N-Acetylcysteine (NAC) is protective against APAP-induced hepatotoxicity at 250 and 500 mg/kg proteins; enhancement of GSH synthesis in liver cells by
(ip) when administered up to 3 hr after APAP overdose, as shown acting as a GSH precursor; and enhancement of sulfate conby a significant reduction in serum enzyme activity. Coadministra- jugation (Miners et ai, 1984). The use of NAC for APAP
tion of 5/3-scymnol (70 mg/kg) and NAC (250 mg/kg) also reduced overdose in humans is considered fairly safe; however, it is
serum enzyme levels and histopathological effects; however, a sim- a gastrointestinal irritant (Johnston et ai, 1983; Miller and
ilar level of hepatoprotection was conferred by 5/3-scymnol treat- Rumack, 1983). Patients also report an unpleasant body odor
ment alone. In addition, 5/9-scymnol has potent hydroxyl radical after NAC therapy (Loehrer et ai, 1982).
quenching activity as it markedly inhibited deoxyribose degrada5/9-Scymnol [(24/?)-(+)-5/9-cholestane-3a,7a, 12a,24,26,
tion in a ferrous/ascorbate Fenton reaction system. These results 27-hexol] (Fig. 1), found in sharks and rays (Amiet et ai,
indicate a possible role for the use of 5/3-scymnol, either alone
1993; Ishida et ai, 1991; Bridgewater et ai, 1962), is an
or concomitant with NAC, in the prevention of hepatic necrosis
unusual 27-carbon bile alcohol in that the terminal methyl
f o l l o w i n g tOXiC dOSeS Of A P A P .
C 1996 Sodety of Toxicology
groups, C26 and C27, together with C24 are present as alcohols. 5/3-Scymnol is present in shark bile as scymnol sulfate,
which is one of the active constituents of "deep sea shark
Acetaminophen (APAP)2 is an antipyretic and analgesic liver oil," a Japanese folk remedy taken orally for the treatthat, when taken in overdose, may result in characteristic ment of various liver diseases and used topically for the
and sometimes fatal, massive centrilobular necrosis of the treatment of scalds, burns, and acne (Kosuge et ai, 1989).
liver and cortical necrosis of the kidney (Mitchell et ai, Close inspection of the chemical structure of 5/3-scymnol
1973a,b; Newton et ai, 1983). Liver damage results from suggested that the aliphatic trialcohol moiety of this bile sterol
excessive production of a reactive metabolite of APAP, may have potential antioxidant activity. This, together with
A/-acetyl-p-benzoquinoneimine (NAPQI) generated by the its traditional clinical use, indicated that 5/3-scymnol may
have substantial hepatoprotective activity against liver damage induced by free radicals or reactive metabolites. For these
1
reasons we have examined the effects of 5/3-scymnol treatTo whom correspondence should be addressed.
2
Abbreviations used: APAP, acetaminophen; NAPQI, /V-acetyl-p-benzoment on the acute liver toxicity induced by APAP in mice.
Hepatoprotective Effects of the Shark Bile Salt 5/?-Scvmnol on
Acetaminophen-lnduced Liver Damage in Mice. MACRIDES, T. A.,
quinoneimine; NAC, A'-acetylcysteine; LDH, lactate dehydrogenase; SDH,
sorbitol dehydrogenase; ALT, alanine aminotransferase; GSH, reduced glutathione; EDTA, ethylenediaminetetraacetic acid; DMSO, dimethylsulfoxide.
MATERIALS AND METHODS
Chemicals and drugs. Purification of the freeze-dried bile salts was
performed by the Biochemistry Unit's Natural Products Research Labora31
0272-0S9O96 $18.00
Copyright O 1996 by the Society of Toxicology.
All rights of reproduction in any form reserved.
32
MACRIDES ET AL.
of pyruvate to lactate. Serum sorbitol dehydrogenase (SDH) activity was
determined by the method of Gerlach (1963) which also used the oxidation
of NADH during the conversion of fructose to sorbitol.
OH
FIG. 1. Structure of 50-scymnol
12a,24,26,27-hexol].
[(24/f)-(+)-5/9-cholestane-3a,7a,
tory, RMIT-University, Melbourne, Victoria, Australia. 5/)-Scymnol sulfate
was isolated from the bile of two species of sharks, the shovelnose dogfish
(Deania calcea) and Baxter's dogfish (Etmopterus baxtcri), which were
obtained from coastal regions of the South Island, New Zealand. 5a-Cyprinol sulfate was isolated from Cyprinus carpio, which were obtained from
the Victorian Fish Markets, Footscray, Victoria, Australia. 5/?-Scymnol and
5a-cyprinol were obtained from the hydrolysis of their respective sulfates,
which was performed according to the method of Anuet et al (1993).
Acetaminophen, 2,4-dinitrophenylhydrazine, DL-alanine, a-ketoglutaric
acid, sodium pyruvate, triethanolamine, tris(hydroxymethyl)aminomethane,
/9-nicotinamide adenine dinucleotide reduced form (NADH), D (—)-fructose, ethylenediaminetetraacetic acid (EDTA), and W-acetylcysteine were
obtained from Sigma Chemical Company, St. Louis, Missouri. All other
reagents were analytical grade and were purchased from regular commercial
sources.
Animals and treatments.
Male Swiss mice (22-30 g) purchased from
Monash University Animal House, Melbourne, Australia, were used in this
study and were given free access to pelleted laboratory chow and tap water.
Mice were randomized, housed over sawdust beds, and maintained on a
12-hr light/dark cycle in a humidity- and temperature-controlled facility.
Acetaminophen was administered at 350 mg/kg (in 30% glycerol/saline
solution, 10 ml/kg injection volume) via a single intraperitoneal injection.
5/9-Scymnol was administered intraperitoneally (ip) to the mice at 20, 35,
and 70 mg/kg in a single injection volume of 10 ml/kg in physiological
saline either 30 min prior to or 1, 2, 3, or 4 hr after the APAP dose. NAC
dose groups received either 250 or 500 mg/kg ip at the same time points,
and were included for comparison of hepatoprotective effects. An additional
group was administered both NAC at 250 mg/kg and 5/9-scymnol at 70
mg/kg. Three control groups were used in this study and received either
(1) APAP and saline, (2) saline only, or (3) no treatmenL
Hepatoprotection was evaluated 24 hr after APAP injection. The animals
were anesthetized with pentobarbital (60 mg/kg ip) prior to exsanguination
by cardiac puncture. The blood was centrifuged at 5000 rpm for 10 min;
the serum was removed and the sample stored at 4°C and analyzed within
12 hr for serum enzyme activities. Livers were excised from the mice and
assessed for hepatic lesions based on the qualitative procedure developed
by Mitchell et al. (1973a), and were graded as follows: 0, no lesions; 1+,
minimal; 2+, mild to moderate; 3 + , severe. Scores greater than 2+ were
considered significant evidence of damage. A portion of the right lobe was
fixed in 10% formalin for histopathology. Thin sections were prepared from
liver samples embedded in paraffin and stained with hematoxylin and eosin
as described by Culling et al. (1985).
Serum enzyme assays. Serum alanine aminotransferase (ALT; also
termed glutamic-pyruvic transaminase, GPT) levels were determined by
the colonmetric method of Reitman and Frankel (1957) using a manufactured kit protocol (Sclavo Diagnostic S.p.a., Siena, Italy). Serum lactate
dehydrogenase (LDH) levels were measured by the method of Kachmar
and Moss (1976) which used the oxidation of NADH during the conversion
Deoxyribose degradation system. Deoxyribose degradation by hydroxyl radicals generated by a Fenton reaction was performed essentially
using the method of Gutteridge (1981). The hydroxyl radical-generating
system containing (final concentrations) ascorbate (0.1 HIM), ferrous ammonium sulfate (0.22 ITIM) and EDTA (0.23 mM) was added to deoxyribose
(1 mM) in phosphate buffer (20 HIM, pH 7.4) in the presence of 5/3-scymnol,
other bile sterols (scymnol sulfate, 5a-cyprinol, cyprinol sulfate, or cholic
acid) at 0.02, 0.075, 0.1, 0.15, 0.3, 0.5, or 1.0 mM, or hydroxyl radical
scavengers [mannitol, promethazine, or dimethylsulfoxide (DMSO)] at 0.1,
0.5, or 1.0 mM, in a total incubation volume of 3.2 ml. The amount of
deoxyribose degradation following a 30-min incubation period was determined as thiobarbituric acid reactants spectrophotometrically at 532 nm. The
results from triplicate determinations in three experiments were expressed as
percentages of the amount of deoxyribose degradation in the absence of
bile sterols or scavengers. Addition of 5/9-scymnol to the control reaction
mixture after incubation, but prior to the addition of thiobarbituric acid,
showed that it did not directly react with thiobarbituric acid to form adducts.
Statistics. All enzyme data were expressed as means ± SE. Statistical
significance was evaluated by a one-way analysis of variance (ANOVA)
and the Tukey compromise post hoc test, and p values less than 0.05 were
considered to be significant.
RESULTS
Twenty-four hours following a single intraperitoneal injection of APAP (350 mg/kg), serum SDH levels increased
from 5 ± 2 U/liter (in untreated animals) to 646 ± 82
U/liter, as a result of APAP-induced hepatocellular necrosis
(Table 1). This APAP-induced elevation in serum SDH activity was reduced in a dose-dependent manner by 5/3-scymnol. The extent of this hepatoprotection was inversely proportional to the time between the APAP and 5/3-scymnol
treatments, with the 35 and 70 mg/kg doses still providing
significant hepatoprotection when administered as late as 4
hr after APAP overdose.
N-Acetylcysteine, the clinically used hepatoprotective
agent, also reduced serum SDH activity following APAP
treatment. However, NAC was less effective than 5/3-scymnol as (1) both the 250 and 500 mg/kg NAC doses (equivalent to 1.5 and 3 mmol/kg, respectively) conferred the same
amount of hepatoprotection, which was less than that
achieved for the 70 mg/kg (0.15 mmol/kg) dose of 5/3-scymnol, and (2) the need for NAC to be administered within 3
hr of APAP overdose. Coadministration of 70 mg/kg 5/3scymnol and 250 mg/kg NAC was generally more effective
than 5/3-scymnol treatment alone if given « 3 hr after the
APAP dose.
Serum ALT levels were also increased by APAP overdose, from 12 ± 2 U/liter in untreated animals to 463 ± 27
U/liter at 24 hours after the administration of APAP (Table
2). The APAP-induced elevation of serum ALT activity was
also reduced by 5/3-scymnol in a dose-dependent manner,
with the highest dose resulting in a 40% reduction of serum
ALT levels when administered after APAP treatment. As
33
5/3-SCYMNOL HEPATOPROTECTIVITY AGAINST ACETAMINOPHEN
TABLE 1
Effect of 5/3-Scymnol and NAC on Serum SDH Levels When Administered before and after APAP (3S0 mg/kg)
Sorbitol dehydrogenase (U/liter)
Treatment
20 mg/kg scymnol
35 mg/kg scymnol
70 mg/kg scymnol
250 mg/kg NAC
500 mg/kg NAC
70 mg/kg scymnol and
250 mg/kg NAC
-0.5 h r '
142
68
10
32
22
± 13
± 7
± 2C
± 9
± Ac
4 ±
2 hr
1 hr
173 ± 23
122 ± 13
33 ± T
40 ± \°r
37 ± 8C
V
4 ±
Saline-treated and untreated animals
V
5 ± 2
±
±
±
±
±
27
7
17
26
21
80 ±
6
239
196
94
178
169
4 hr
3 hr
436
244
207
397
372
±
±
±
±
±
39
26
21
12
24
129 ± 30
APAP-only-treated animals
561
340
250
498
425
±
±
±
±
±
47'
25
8
42*
62
235 ± 45
646 ± 82
Note. Results are serum SDH activities 24 hr after APAP administration and are expressed as means ± SE of five animals (p < 0.05).
" Treatment given 0.5 hr before APAP administration; remaining treatments were given after the APAP dose
' Not statistically different from value for control group treated with APAP alone.
' Values not statistically different from those for saline-only-treated or untreated animals.
expected, NAC treatment also reduced serum ALT activity
but was also less effective than 5/3-scymnol, i.e., a 20%
reduction by both 250 and 500 mg/kg, when given 4 hr after
the APAP dose. The coadministration of 5/3-scymnol
(70 mg/kg) and NAC (250 mg/kg) produced a 80% reduction
in the elevated serum ALT resulting from APAP overdose.
As with the cellular enzymes previously mentioned, leakage of LDH into the serum was also induced by APAP
overdose, from 32 ± 9 U/liter in untreated animals to 3679
± 433 U/liter 24 hr after APAP treatment (Table 3). Similarly, 5/3-scymnol treatment reduced these elevated serum
LDH levels in a dose-dependent manner. The coadministration of 5/3-scymnol and NAC showed a protective response
similar to that of 5/3-scymnol treatment alone.
The severity of liver necrosis was assessed qualitatively
following inspection of the mouse liver gross morphology
(Table 4). Histopathological examination of hematotoxylin
and eosin-stained liver sections confirmed the APAP-induced hepatotoxicity, with the centrilobular necrosis and
midzonal hydropic degeneration clearly evident (Fig. 2A).
Treatment with 5/3-scymnol (70 mg/kg) 4 hr after APAP
treatment preserved the hepatic architecture (Fig. 2B); however, NAC (250 mg/kg) did not afford protection, as both
necrosis and hydropic degeneration are visible (Fig. 2C).
Coadministration of 5/3-scymnol (70 mg/kg) and NAC (250
mg/kg) is clearly hepatoprotective (Fig. 2D).
5/3-Scymnol and its sulfated form markedly inhibited deoxyribose degradation by a hydroxyl radical-generating sys-
TABLE 2
Effect of 5/9-Scymnol and NAC on Serum ALT Levels When Administered before and after APAP (350 mg/kg)
Alanine aminotransferase (U/liter)
Treatment
20 mg/kg scymnol
35 mg/kg scymnol
70 mg/kg scymnol
250 mg/kg NAC
500 mg/kg NAC
70 mg/kg scymnol and
250 mg/kg NAC
Saline-treated and untreated animals
1 hr
- 0 . 5 hr°
172
117
30
42
23
28
6C
9
12
11
± r
±
±
±
±
69 ±
154
140
71
66
40
±
±
±
±
±
24
24
25
13
5C
10 ± 2cM
12 ± 2
3 hr
2 hr
296
185
150
70
58
± 20
± 24
± 16
± 8
it 5
60 ±
302
233
191
210
219
8^
±
±
±
±
±
21
34
19
52
28
73 ± \&
APAP-only-treated animals
Note. Results are serum ALT activities 24 hr after APAP administration and are expressed as means ± SE of five animals (p < 0.05).
' Treatment given 0.5 hr before APAP administration; remaining treatments were given after the APAP dose.
* Not statistically different from value for control group treated with APAP alone.
' Values not statistically different from those for saline-only-treated or untreated animals.
' Combined 5/9-scymnol and NAC treatment is statistically different from 5/3-scymnol treatment (70 mg/kg) alone.
4 hr
470
443
278
367
365
±
±
it
±
±
25'
26'
11
21
26
92 ±
5'
463 ± 27
34
MACRIDES ET AL.
TABLE 3
Effect of 5^-Scymnol and NAC on Serum LDH Levels When Administered before and after APAP (350 mg/kg)
Lactate dehydrogenase (U/liter)
Treatment
1 hr
-0.5 hr'
20 mg/kg scymnol
35 mg/kg scymnol
70 mg/kg scymnol
250 mg/kg NAC
500 mg/kg NAC
70 mg/kg scymnol and
250 mg/kg NAC
555
405
135
572
155
+
+
+
+
±
53
20
26
20
35
861
706
160
512
122
188 + 18
±
+
+
±
±
71
60
49
36
20
266 ± 81
Saline-treated and untreated animals
30 ± 11
2hr
1061
920
265
474
293
+
+
±
±
±
4 hr
3 hr
59
31
77
74
30
246 ± 93
2452
1571
266
877
874
+
±
±
±
+
330
227
25
158
161
532 ± 131
APAP-only-treated animals
3571
2854
493
1451
1378
±
±
±
±
+
306*
236*
36
214
420
726 + 162
3679 + 433
Note. Results are serum LDH activities 24 hr after APAP administration and are expressed as means ± SE of five animals (p < 0.05).
" Treatment given 0.5 hr before APAP administration; remaining treatments were given after the APAP dose.
* Not statistically different from value for control group treated with APAP alone.
tem using the Fe 2+ EDTA-ascorbate Fenton reaction (Fig.
3), with concentrations that caused 50% inhibition (IC50,
estimated from log dose-response curves) of 0.11 and 0.15
mM, respectively. This bile sterol was an equipotent hydroxyl radical quencher compared with Trolox, the potent
antioxidant analog of a-tocopherol (vitamin E), which exhibited an IC50 of 0.19 mM. Furthermore, 5/3-scymnol was
more potent than the other known free radical scavengers in
this system, i.e., mannitol, promethazine, and DMSO, which
had IC50 values at least twofold greater than the shark bile
sterol. In addition, the other two bile sterols examined in
this system had lower scavenging activity, i.e., 5a-cyprinol
(and its sulfate), a C27 bile sterol that differs chemically
from 5/3-scymnol in that it lacks the C24 chiral alcohol
group; and cholic acid, a C24 bile acid that totally lacks
this trialcohol-substituted aliphatic side chain. These results
suggest that the C24, C26, and C27 alcoholic groups of 5/3scymnol and its sulfated form are all essential to its potent
radical scavenging properties. Unfortunately, 5a-cyprinol is
itself hepatotoxic (Asakawa et ai, 1990), thus precluding the
use of 5a-cyprinol and its sulfate in the in vivo experiments,
despite the sulfated form's having scavenging activity comparable to that of promethazine and DMSO.
DISCUSSION
TABLE 4
Gross Morphological Assessment of Mouse Livers at Euthanasia
Following APAP Overdose (350 mg/kg)
Hepatocellular damage
Treatment
20 mg/kg scymnol
35 mg/kg scymnol
70 mg/kg scymnol
250 mg/kg NAC
500 mg/kg NAC
70 mg/kg scymnol and
250 mg/kg NAC
-0.5
hr°
1 hr
2hr
3 hr
4 hr
0.5+*
0.5 +
0.5 +
1+
0.5 +
0
1+
1+
1+
1.5 +
1+
0.5 +
2+
2+
1.5 +
2+
1+
1+
2.5 +
2+
1.5 +
2.5 +
1.5 +
1.5 +
2.5 +
25 +
1 5+
3+
2.5 +
1.5 +
Note. Results are means of five animals. See Materials and Methods for
the qualitative morphological criteria of hepatoxicity, which was assessed
24 hr after APAP administration. APAP treatment alone resulted in a ranking of 3 + .
" Treatment given 0.5 hr before APAP administration; remaining treatments were given after the APAP dose.
*0, no lesion; 1+, minimal; 2 + , mild to moderate; 3+, severe.
The administration of an APAP overdose (350 mg/kg) to
male Swiss mice causes, after 24 hr, (1) histologically apparent centrilobular hepatic necrosis and midzonal hydropic degeneration, and (2) the elevation of hepatocellular enzymes
in the serum. These acute hepatotoxic effects induced by an
APAP overdose are related to formation of the toxic metabolite NAPQI by cytochrome P450-dependent enzyme systems, in concentrations greater than can be detoxified by
endogenous GSH levels, and the subsequent depletion of
GSH cellular stores results in liver damage (Prescott, 1983).
Our results provide strong evidence that 5/3-scymnol significantly inhibits the acute liver toxicity induced by a high
dose of APAP in mice, as shown by the preservation of the
liver morphology and histopathology, as well as a reduction
of serum liver enzyme activities. The hepatoprotective effects of 5/3-scymnol were clearly dose dependent over the
range of doses employed in this study. The acute 5/3-scymnol
treatment used in this study was well tolerated and did not
alter the liver function parameters measured in this study.
5/3-Scymnol was also a more potent hepatoprotective
'35
5/9-SCYMNOL HEPATOPROTECTIVITY AGAINST ACETAMINOPHEN
B
FIG. 2. (A) Mice were injected with APAP (350 mg/kg), and 24 hr later, a liver section was obtained from the right lobe, fixed, and stained with
hematoxylin and eosin. The tissue shows extensive centrilobular necrosis and midzonal hydropic degeneration. (B) 5/3-Scymnol-injected mice (70 mg/
kg) showing the protective effect of the compound when administered 4 hr after APAP. (C) NAC-injected mice (250 mg/kg) showing extensive
centrilobular necrosis and midzonal hydropic degeneration. The compound was administered 4 hr after APAP. (D) Coadministration of 5/?-scymnol (70
mg/kg) and NAC (250 mg/kg) showing the protective effect of the compounds when administered 4 hr after APAP. C, central vein. X250.
agent than NAC, the clinically accepted APAP antidote. That
is, (1) lower doses of 5/9-scymnol were more effective than
NAC, and (2) 5/3-scymnol was more hepatoprotective when
administered at longer time intervals following the APAP
overdose. The coadministration of 5/3-scymnol and NAC
also ameliorated APAP-induced hepatotoxicity; however,
the improvements in all hepatic parameters (except serum
ALT) were not significantly different from the effects of 5/3scymnol alone, and may therefore be attributable mainly to
the shark bile acid.
The thiol moiety of NAC acts to reduce the depletion of
hepatic GSH stores and NAPQI binding to hepatocellular
macromolecules during APAP overdose (Corcoran et ai,
1985a,b). However, 5/3-scymnol lacks a thiol-containing
radical in its molecular structure and therefore a different
mode of action is expected. Our experimental results show
that 5/3-scymnol is a powerful hydroxyl radical scavenger,
and may therefore act by scavenging free radicals and reactive oxygen species formed during APAP metabolism. Three
possible mechanisms may be postulated for the protective
role shown for 5/3-scymnol against APAP hepatotoxicity:
(1) inhibition of the mixed-function oxidase activity mediated by cytochrome P450, thus reducing the amount of
NAPQI produced; (2) direct scavenging of the reactive
NAPQI (an electrophile) by 5/0-scymnol (a weak nucleophile); or (3) reducing GSH depletion by free radical scavenging, or enhancing other pathways involved in APAP deactivation.
Preliminary studies in our laboratory indicate that the decrease in hepatic reduced GSH levels caused by APAP treatment were not altered by concomitant treatment with 5/3scymnol; i.e., GSH levels were decreased by 39 ± 4%
36
MACRIDES ET AL.
100n
Bile Sterols
—A—
- -A- —•—
- -A- —O—
o
60
U
Q
o
50-
X
OH* Radical
Scavengers
o
&
I
Scymnol
Scymnol Sulphate
Cyprinol
Cyprinol Sulphate
Cholic Acid
25-
o
U
—•—
Mannitol
—•—
Promethazine
— • — DMSO
—A:— Trolox
0
0.25
0.5
0.75
1
Concentration (mM)
FIG. 3. Effect of hydroxyl radical scavengers and bile sterols (at 0.02, 0.075, 0 1, 0.15, 0.3, 0.5, or 1.0 mM) on the degradation of deoxyribose by
a Fenton reaction. Results are means ± SE of triplicate determinations and are expressed as percentages of deoxynbose degradation by the hydroxyl
radical-generating system (Fe 2+ EDTA-ascorbate) in the absence of further additions.
2 hours after APAP treatment, as compared with the 43 ±
4% decrease observed with coadmini strati on of 5/3-scymnol
(n = 4). This would suggest that 5/3-scymnol may not affect
NAPQI formation by inhibiting microsomal activation pathways as this would be expected to spare liver GSH levels.
Future studies will further investigate these proposed mechanisms.
In conclusion, the experimental results indicate a possible
therapeutic role for the shark bile salt 5/?-scymnol, either
alone or concomitant with NAC, in the prevention of hepatic
necrosis following toxic doses of APAP. The apparent ability
of 5/3-scymnol to reduce liver damage when administered 4
hr after an APAP overdose suggests that this marine bile
salt may also have potential as a general therapeutic hepatoprotective agent, in addition to being a more useful APAP
antidote than NAC.
ACKNOWLEDGMENTS
This work was supported by a grant from McFarlane Laboratories, Melbourne, Australia, and a research studentship to L.M N. The authors thank
Dr. W. White and Dr. G. Kweifio-Okai for graciously reviewing the histopathological changes in the liver and the technical assistance of Mrs. K.
Mclntyre for liver section preparation.
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