INT. J. IMMUNOTHERAPY XIX(2-4) 129-134 (2003)
PROTECTIVE EFFECT OF UKRAIN AGAINST ACUTE ACETAMINOPHEN-
LEVINA O.A.,1 GONCHAROVA I.A.,2 FILATOVA T.G.,1 NADEEV A.P.,3 NOWICKY W.,4
SUKHENKO T.G.,5 KOLESNIKOVA O.P.,5 KOROLENKO T.A.1
1) Laboratory of Cellular Biochemistry, Institute of Physiology, Russian Academy of Medical Sciences,
2) First Hospital of Municipal Infectious Diseases, Novosibirsk, Russia.
3) Novosibirsk Medical Academy, Novosibirsk, Russia.
4) Ukrainian Anti-Cancer Institute, Vienna, Austria.
5) Institute of Clinical and Experimental Immunology, Russian Academy of Medical Sciences, Novosibirsk,
Summary: A high dose of acetaminophen (AAP; 1000 mg/kg, single, p.o) administered to rats induced acuteliver failure with centrolobular degeneration and necrosis. We studied the role of hepatic macrophages (Kupffercells) in the modulation of the acetaminophen-induced damage to the liver. Ukrain, which is known to possessimmunomodulatory characteristics, has been shown to exhibit a positive hepatoprotective effect in human viralhepatitis C, significantly preventing liver cell injury. The selective inhibitor of Kupffer cells, gadolinium chloride(GdCl , 7.5 mg/kg i.v., administered 24 h before AAP), and the macrophage stimulator carboxymethylated (1→ 3)-
mg/kg i.p., administered 48 h before AAP), were used to modulate the activity of liver
macrophages. It was shown that preliminary administration of Ukrain, CMG or GdCl to rats produced in each
normalizing liver functional tests and reducing damage to the liver. The role of tumor
necrosis factor (TNF)-alpha secretion in the protective effect of these agents in AAP-induced hepatitis is dis-cussed.
Address for correspondence: T.A. Korolenko, Laboratory ofCellular Biochemistry, Institute of Physiology, Russian
Ukrain has been shown to exhibit a significant
Academy of Medical Sciences, 4 Timakov Street, Novosi-birsk 630117, Russia.
hepatoprotective effect in human viral hepatitis C,
Tel: +7 383 233 4872 Fax: +7 383 232 4254
preventing liver cell injury (1-3). We suggested that
Ukrain could prevent liver damage in other types of
hepatitis, in particular those induced by drugs that
24 h after AAP administration (group 2). Ukrain was
have side effects on liver structure and function.
injected at a dose of 2 mg/kg i.v. to intact rats (group
Acetaminophen (AAP) is a well known medical
3), or 48 h prior to administration of AAP (group 6).
drug, widely used in the therapy of numerous inflam-
CMG was administered at a dose of 25 mg/kg i.p. to
matory diseases (4-6). It has been shown that high
intact rats (group 4), or 48 h prior to administration of
doses of AAP can induce toxic hepatitis and liver fail-
AAP (group 7); GdCl was administered in the tail
ure in humans and experimental animals, accompa-
vein at a dose of 7.5 mg/kg to intact rats (group 5), or
nied by the development of centrolobular necrosis
24 h prior to administration of AAP (group 8).
(6-8). It has been suggested that AAP toxicity is theresult of the interaction of its metabolite N-acetyl-p-
Liver functional tests. Functional assays of serum
benzoquinone with liver proteins and of the depletion
transaminase activity (alanine transaminase [ALT],
of glutathione in liver cells (8). In this study, we tested
aspartate transaminase [AST]) were carried out using
the hypothesis that the functional activity of liver non-
a Cobas Mira biochemical analyzer (Cobas Mira La
parenchymal cells (macrophages, endothelial cells)
Roche Diagnostic System, Basel, Switzerland) and Bio-
can play an important role in the development of toxic
con Diagnostik kits (Biocon Diagnostik GmbH, Vöhl-
hepatitis induced by high doses of AAP (4, 7, 9-11).
Marienhagen, Germany). The activity of enzymes wasexpressed in U/L.
TNF-alpha. TNF-alpha level was measured in the
supernatant of peritoneal macrophages by the bio-logical method according to Flick (12), using the
Study design. Male Wistar rats weighing 200-250 g
TNF-alpha sensitive cell line L-929 purchased from the
(Institute of Cytology and Genetics, Russian Academy
Russian Ministry of Public Health State Research Center
of Sciences, Novosibirsk, Russia) were used. Rats were
for Virology and Biotechnology Vector (Koltsovo, Rus-
with Ukrain (Nowicky Pharma, Austria) at a
sia). For the standard curve, murine recombinant TNF-
dose of 2 mg/kg i.v. administered 48 h before admin-
alpha was used, the results being expressed in pg/ml.
istration of AAP. As positive and negative controls ofmacrophage stimulation, water-soluble carboxymeth-
Light microscopy study of the liver. For the prepa-
ylated (13)-beta-D-glucan (CMG; Institute of Chemis-
ration of histological samples, livers were removed
try, Slovak Academy of Sciences, Bratislava, Slovakia)
and fixed in 10% buffered formalin (ALTEY Labor-
at a dose of 25 mg/kg i.p. 48 h before administration
atory, Moscow, Russia) and paraffin-embedded sec-
of AAP, and a macrophage activity suppressing agent,
tions were stained with hematoxylin-eosin (ALTEY
gadolinium chloride (GdCl ; kind gift of Prof. Hardonk,
Laboratory). Statistical analysis of the results was
Department of Pharmacokinetics and Drug Delivery,
carried out on Statistica for Windows (release 5.1 A,
University of Groningen, The Netherlands) at a dose
1984-1996, Tulsa, OK, USA), using Students t-test;
of 7.5 mg/kg i.v. 24 h before administration of AAP,
values were considered as significant at p < 0.05.
As control, intact rats were used (group 1). Toxic
hepatitis was induced by AAP (ICN Biomedicals,Irvine, CA, USA) administered to the rats in a single
TNF-alpha production by isolated peritoneal macro-
dose of 1,000 mg/kg p.o. (7); animals were sacrificed
phages. In the first series of experiments it was re-
Protective effect of Ukrain against acute acetaminofen-induced hepatitis in rats
vealed that CMG (25 mg/kg, single dose) increased
animals with AAP hepatitis, preliminary administra-
TNF-alpha production by isolated peritoneal macro-
tion of the immunomodulator Ukrain or the macro-
phages. The TNF-alpha level in the macrophage
phage stimulator CMG prevented the development of
supernatant of intact animals was 333 ± 30 pg/ml.
centrolobular necrosis and the dystrophy of hepato-
Following CMG administration this increased to 831
± 80 pg/ml. The value in the positive control group inthis part of study after treatment with zymosan
Serum ALT and AST activity. Serum ALT and AST
(Sigma, St. Louis, MO, USA), a known macrophage
activity was studied in rats with AAP-induced hepati-
stimulator, at a dose of 100 mg/kg i.p. 6 h after
tis and in the groups receiving a combined adminis-
administration of CMG, was 1,560 ± 200 pg/ml. This
tration of AAP and the macrophage function modula-
showed that CMG-induced macrophage stimulation
tor. The treatment of the rats with AAP resulted in a
was followed by increased TNF-alpha production,
10- to 13-fold increase in serum ALT and AST activi-
but the effect was less than that of the macrophage
ty, indicating the development of cytolytic syndrome
and damage to liver cells (Table II). The elevation ofALT and AST activity was significantly reduced by
Liver weight. Liver weight increased in the AAP
pretreatment with Ukrain and CMG (Table II). Ad-
hepatitis group and in the AAP groups receiving pre-
ministration of Ukrain or CMG alone to intact rats had
liminary administration of Ukrain or CMG. There were
no effect on serum transaminase activity. Positive,
no changes in spleen weight in any group studied
but less significant, effects were observed in rats with
Light microscopic study of the liver. AAP adminis-
White blood cells. It was shown that AAP de-
Table I Effect of Ukrain, during acetaminophen
creased the total number of white blood cells com-
acute toxic hepatitis with centrolobular necroses.
pared with the control animals (Table III). CMG ad-
Vacuolar dystrophy occurred in some hepatocytes,
ministration to intact animals was followed by relative
and proliferation of sinusoidal cells was observed. In
neutropenia and a slight tendency to lymphocytosis.
carboxymethylated (13)-beta-D-glucan (CMG) and gadolinium chloride (GdCl ) treatment on rat liver and spleen weight
Liver weight (g)Relative liver weight (g)
Table II Effect of Ukrain, carboxymethylated (13)-beta-D-glucan (CMG) and gadolinium chloride (GdCl ) treatment on the serum alanine
transaminase (ALT) and aspartate transaminase (AST) activity of rats with acetaminophen (AAP)-induced hepatitis
There was also a tendency to neutropenia in the case
kines), among which the most important are TNF-
of administration of Ukrain to intact rats (Table III).
alpha, interleukin (IL)-6 and IL-8 (9, 10, 13). Cytokines
Combined administration of Ukrain and AAP pro-
induce the neutrophils to release the biologically
duced no changes in white blood cell count or differ-
active substancesH O , free oxygen radicals and
ential count as compared with intact rats, nor were
polymorphonuclear cell elastasewith a simultane-
there any changes in these indexes in the groups re-
ous decrease in peroxisomal catalase activity in liver
ceiving CMG with AAP and GdCl with AAP (Table III).
cells (5, 14). This is probably why preliminary appli-
cation of the known peroxisome activator clofibrate,resulting in a decrease in H O synthesis, consider-
ably reduced toxic damage to the liver by AAP (15).
At 24 h after its administration, AAP administered
Table III Effects differential count Page Proofs
known that inflammation is followed by the
to rats at a dose of 1,000 mg/kg leads to acute liver
secretion of primary inflammatory mediators (cyto-
failure accompanied by centrolobular degeneration
of gadolinium chloride (GdCl ), carboxymethylated (13)-beta-D-glucan (CMG) and Ukrain on the white blood cell (WBC) and
in rats with acetaminophen (AAP)-induced hepatitis
PMN: polymorphonuclear leucocyte; * percent of total leucocytes; **p < 0.05, compared to control.
Protective effect of Ukrain against acute acetaminofen-induced hepatitis in rats
and necrosis, with a considerable increase in serum
and IL-6 by macrophages, reducing their participa-
transaminase activity (ALT and AST). The pretreat-
tion in the inflammatory process (6). GdCl both
ment of rats with the immunomodulator Ukrain, the
selectively depletes the population of large macro-
macrophage stimulator CMG and the selective in-
phages in the liver, and decreases nitric oxide pro-
hibitor of liver macrophages GdCl , prevented in each
duction and the formation of peroxide oxidation prod-
case the development of AAP-induced hepatitis, nor-
ucts (4). It appears that the mechanism of protective
malizing functional and structural changes to liver cells.
action in AAP hepatitis of the various compounds
Changes in TNF-alpha secretion by macrophages
studied is connected with the secretory activity of
due to CMG can modify the degree of liver injury
macrophages and requires further analysis.
caused by hepatotropic agents. Ukrain was shown toexhibit a hepatoprotective effect in experimentalhepatitis, and may be useful in the treatment of drug-
We found that in acute toxic hepatitis induced by
(1) Boyko V.N., Belskiy S.N. The influence of the novel drug
Ukrain on hemo- and immunopoiesis at the time of its maximum
AAP, Ukrain and CMG each had a protective effect via
radioprotective effect. Drugs Exp. Clin. Res., 24(5/6), 335, 1998.
the stimulation of liver macrophages (with increased
(2) Voltchek I., Sologub T., Nowicky J.W., et al. Preliminary
TNF-alpha production in isolated macrophages in
results of individual therapy of chronic hepatitis C by Ukrain and inter-
the case of CMG), while the protective effect of GdCl
feron-alpha. Drugs Exp. Clin. Res., 26(5/6), 261, 2000.
was via the suppression of liver macrophages.
(3) Zhalilo L.I., Susak Y.M., Zemskov S.V., Susak I.A. Influence of
Ukrain on the redox processes of hepatocyte. Drugs Exp. Clin. Res.,
According to previous results, preliminary stimulation
of liver macrophages by lipopolysaccharide (LPS)
(4) Michael S.L., Pumford N.R., Niesman M.R., Hinson J.A.
has a protective effect in toxic liver injury (7). A posi-
Pretreatment of mice with macrophage inactivators decreases aceta-
tive effect from macrophage stimulation was also
minophen hepatotoxicity and the formation of reactive oxygen and
nitrogen species. Hepatology, 30(1), 186, 1999.
(5) Mirochnichenko O., Neisborot-Lefkowitz M., Yang C., Inouye
acute blood loss. It is probable that previously primed
M. Acetaminophen toxicity. Opposite effects of two forms of gluta-
macrophages are not able to secrete significant
tione peroxidase. J. Biol. Chem., 274(15), 10349, 1999.
amounts of cytokines, and as a result such macro-
(6) Vengerobskii A.I., Saratikov A.S. The mechanisms of the
phages are less involved in the development of toxic
hepatotoxicity of paracetamol. Farmakol. Toxicol., 54(1), 76, 1991.
(7) Laskin D.L., Gardner C.R., Price V.F., Jollow D.J. Modulation
of macrophage functioning abrogates the acute hepatotoxicity of
The protective effect of Ukrain in experimental
acetaminophen. Hepatology, 21(4), 1045, 1985.
AAP hepatitis opens up the possibility of its use in the
(8) Matthews A.M., Roberts D.W., Hinson J.A., Pumford N.R.
treatment of hepatitis induced as a side effect by
Acetaminophen-induced hepatotoxicity. Drug Metab. Dispos.,
some medical drugs. There is a significant possibility
(9) Ivashkin V.T. Cellular and molecular biology of liver inflamma-
that Ukrain may exhibit a hepatoprotective effect in
tion. Rus. J. Gastroent. Hepat. Coloproct., 14(5), 13, 1998 (in
human viral hepatitis C, especially in cases of drug-
(10) Hinson J.A., Michael S.L., Ault S.G., Pumford N.R. Western
We also observed a protective effect of GdCl
blot analysis of nitrotyrosine protein addicts in livers of saline-treated
against experimental AAP hepatitis, depressing liver
and acetaminophen-treated mice. Toxicol. Sci., 53(2), 467, 2000.
(11) Shiratory Y., Hongo S., Hikuba Y., Omata M., et al. Role of
macrophages in vivo (17). It is known that GdCl ad-
macrophages in regeneration of liver. Dig. Dis. Sci., 41(10), 1939,
ministration to rats decreases secretion of TNF-alpha
(12) Flick D.A., Gifford G.E. Comparison of in vitro cell cytotoxic
(15) Nickolls-Grzemski F.A., Calder I.C., Priestly B.C., Burchan
assays for tumor necrosis factor. J. Immunol. Methods, 68, 167,
P.C. Clofibrate-induced in vitro hepatoprotection against aceta-
minophen is not due to altered glutathione homeostasis. Toxicol.
(13) De Leve L.D., Wang X., Kaplowitz N., Van der Hoek A.
Sinusoidal endothelial cells as target for acetaminophen. Direct
(16) Korolenko T.A., Poteryaeva O.N., Li X., Uchkina T.V. Acute
phase proteins as biological markers of addictive disorders in
action versus requirement for hepatocyte in different mouse strains.
teenagers and children. Int. J. Circumpolar Health, 60, 288, 2001.
Biochem. Pharmacol., 53(9), 1339, 1997.
(17) Hardonk M.J., Dijkhuis F.W., Hulstaert C.E., Koudstaal J.
(14) Lores-Arnaiz S., Lesuy S., Cutrin J.C., Boveris A. Oxidative
Heterogeneity of rat liver and spleen macrophages in gadolinium
stress by acute acetaminophen administration in mouse liver. Free
chloride-induced elimination and repopulation. J. Leukoc. Biol.,
Radical. Biol. Med., 19(3), 303, 1995.
Table 1. Referral numbers, sources, patient diagnoses and candidacy for ChEI/memantine treatment before and after NICE217 effective. Before NICE217 effective After NICE217 effective (1 June 2010–31 May 2011) (1 June 2011–31 May 2012) Letters not directly related to articles published in Clinical Medicine and pre-senting unpublished original data should be submitted for publ
Wednesday, 1st December Welcome of participants and registration Introduction Lavandier M. (Director of the C2RMF) and Menu M. (Head of the research department) Address to the workshop participants Presentation of the ArBoCo ANR programme (LC2RMF, Paris) Opening lecture 14:30-15:15 Steve Atomic Disorder in Bone Mineral: Formation Processes and Diagenesis Diagenesis a