SHORT COMMUNICATION
EFFECT OF THE NITRIC OXIDE DONOR AND THE NITRIC OXIDE SYNTHASE INHIBITOR ON THE LIVER OF RATS WITH CHRONIC HEPATITIS INDUCED BY
DIMETHYLNITROSAMINE
Oxana Lukivskaya
1, Ruslan Lis
2, Krzysztof Zwierz
3, Vyacheslav Buko
1,#Department of Experimental Hepatology, Institute of Biochemistry, National Academy of Sciences, BLK-50, 230017 Grodno, Belarus; Central Research Laboratory, Grodno Medical University, Gorkogo 2, 230023 Grodno, Belarus;
!Department of Pharmaceutical Biochemistry, Medical University of Bia³ystok, Mickiewicza 2A, PL 15-222 Bia³ystok, Poland
Effect of nitric oxide donor and the nitric oxide synthase inhibitor on the liver of rats with chronic hepatitis induced by dimethylnitrosamine. O. LU- KIVSKAYA, R. LIS, K. ZWIERZ, V. BUKO. Pol. J. Pharmacol., 2004, 56, 599–604.
The present study was designed to examine the effects of the donor of ni- tric oxide (NO), NaNO, and the inhibitor of NO synthase, NM-nitro-L- -arginine (L-NNA), on the development of dimethylnitrosamine (DMNA)- induced chronic hepatitis in rats. L-NNA decreased rat survival and en- hanced the severity of hepatic encephalopathy in the DMNA-treated ani- mals. The aggravation of the morphological signs of hepatitis, the activation of serum alanine aminotransferase and cytosolic superoxide dismutase ac- tivities and the increase in the liver malondialdehyde content were observed in this group. The treatment with NaNO improved liver morphology, decreased serum marker enzyme activities, lowered the activities of a-D- -mannosidase and N-acetyl-b-D-glucosaminidase compared to the DMNA- treated group.
The results of the morphological and biochemical studies suggest that L-NNA increased DMNA-induced liver damage, whereas NaNO partially prevented the development of chronic hepatitis. It is proposed that the oppo- site effects of L-NNA and NaNO are partially explained by a modulation of the free radical-dependent processes in the liver.
Key words: N-nitro-L-arginine, sodium nitrate, rat liver, hepatitis, di- methylnitrosamine
ISSN 1230-6002
Abbreviations: AlAT – alanine aminotransfe- rase,AsAT – aspartate aminotransferase, DMNA – dimethylnitrosamine, D-NNA – N-nitro-D-arginine, L-NNA – N-nitro-L-arginine, NO – nitric oxide, NOS – NO synthase, eNOS – endothelial NO syn- thase, iNOS – inducible NO synthase, SOD – su- peroxide dismutase
INTRODUCTION
Dimethylnitrosamine (DMNA) is a hepatotoxin widely used in animal experiments to induce hepa- tocarcinoma as well as preliminary stages of liver damage: acute hepatitis (frequently in fulminant form), chronic hepatitis, liver fibrosis and cirrhosis.
A development of each of these stages of liver in- jury is dependent on the dosage and duration of DMNA treatment. Moderate DMNA doses admin- istered during 23 weeks lead to chronic hepatitis.
Chronic hepatitis is characterized by necrotic and post-necrotic changes in the liver accompanied by an inflammation. The role of nitric oxide (NO) in development of chronic hepatitis is not quite clear.
In acute hepatitis caused by thioacetamide, methyl ester of N-nitro-L-arginine (L-NAME), the inhibi- tor of NO synthase (NOS), increased liver damage [5]. Liver damage induced by the single DMNA treatment was enhanced by NOS inhibitors, N- nitro-D- and N-nitro-L-arginine (D-NNA and L- NNA), via an activation of intravascular platelet aggregation and adhesion [16].
The aim of this work was to study the effects of sodium nitrite (the NO donor) [7, 12], and L-NNA (the NOS inhibitor), on the morphological and bio- chemical changes in the liver of rats with chronic hepatitis induced by DMNA.
MATERIALS and METHODS
Animal treatment
Four groups (each consisting of 16 animals) of male albino Wistar rats (170–190 g) were used.
Rats were fed a standard diet containing stock pel- lets and vegetables. Eight animals from each group were used for biochemical assays and histological studies were conducted on the remaining rats. Rats (8 animals per cage) were housed in the room with constant temperature and humidity and had free ac- cess to water and food throughout the study. Three groups of rats were treated with 0.5% solution of
DMNA during 3 weeks, 3 times per week (1 ml/kg, ip). The animals from the first DMNA-treated group, received water to drink. The animals from the second DMNA-treated group received a solu- tion of L-NNA, as the only source of water (20 mg/100 ml). To the third DMNA-treated group, so- dium nitrite was administered twice per day, during 3 weeks (30 mg/kg, ip). The dose of NaNO was adopted from the current literature [19]. Rats from the fourth group were treated with ip injection of saline and served as the control.
The Local Ethics Committee for animal experi- ments in Grodno approved the study. The proce- dures involving the animals and their care con- formed to the institutional guidelines and were in compliance with national and international laws and guidelines for the Use of Animals in Biomedi- cal Research [9].
Chemicals
All used chemicals were of analytical grade and were obtained from Sigma Chemical Co. (St. Louis, USA).
Biochemical assays
For the determination of hepatic content of ma- londialdehyde (MDA) and NO-related parameters liver tissue was cut into small pieces using a razor blade, and homogenized after dilution in 1.15%
KCl 1:10 w/v. Homogenates for lysosomal enzyme activity measurements were prepared in 1.15% KCl 1:10 w/v supplemented with 0.2% Triton X-100.
Liver homogenate was centrifuged in 900 r.p.m. for 5 min, and than supernatant was collected and cen- trifuged at 3000 × g for 30 min. The liver cytosol fraction for the measurement of superoxide dismu- tase (SOD) activity was obtained by differential cen- trifugation at 105 000 × g with the use of a VAC- 602 centrifuge (Janetzki, Germany).
NOS activity in the liver was measured by NADPH oxidation, using L-arginine as a substrate [22]. NO content was assessed as nitrate/nitrite concentration determined with Griess reagent after the reduction of nitrate to nitrite by broken cad- mium granules [21] without deproteinization of ho- mogenates.
MDA content was evaluated by thiobarbituric acid reaction [3]. SOD activity was measured by formazane formation in the reaction with nitrotetra- solium blue [20]. Exoglycosidases were deter- mined in liver homogenates by the method of Chat-
terjee et al. [4] in modification of Zwierz et al. [25].
The activity of alkaline phosphatase in the liver, as well as serum aspartate aminotransferase (AsAT) and alanine aminotransferase (AlAT), were meas- ured by routine methods using commercial kits from Lachema (Czech Republic).
Liver histology
The liver was fixed in Bouin’s solution and than embedded into paraffine. Liver slices were stained with hematoxylin-eosin. Azan-Mallory staining was also employed for determination of development of the liver fibrosis.
Statistics
Data are expressed as the means ± SEM. Statis- tical evaluation of differences between sample means was made by the two-tailed Student’s t-test and one-way analysis of variance (ANOVA). The minimal level of significance was set at p < 0.05.
RESULTS
The animal survival was complete in all the groups, except for the group receiving DMNA and L-NNA where 4 of 16 animals died. Before death these rats had a paralysis/paresis of the forelimbs and back extremities, with the severity correspond- ing to 2–3 stages of hepatic encephalopathy [5].
Morphological data are indicative of a typical liver structure in the control and DMNA-treated animals. In all the groups, fibrotic tissue deposition was observed in typical places: in the liver capsule, periportally and in the walls of the central veins. In the DMNA-treated rats, spots of caryolysis and sin- gle necrotic areas were observed (Fig. 1A). In the group treated with DMNA and L-NNA, the picture of hepatitis aggravated (Fig. 1B). We observed manifold centrilobular necrotic areas filled by de- trits and connective tissue, hyperplasia of hepato- cytes, as well as cytoplasmic dystrophy character- ized by small vacuoles tightly packed in the cyto- plasm. Additionally, in this group, sharply dilated central veins were noted. After the application of DMNA + NaNO , caryolysis and significant bal- looned dystrophy were observed (Fig. 1C). Bal- looned dystrophy was usually accompanied by ne- crosis, but no necrosis signs were found in the group treated with DMNA +NaNO .
The activities of serum AlAT and AsAT (Tab. 1) indicate the absence of massive hepatocyte cytoly-
Fig. 1. Histiological examination of the liver. Staining by hematoxilin and eosin. A. DMNA. Threshold necrosis with massive infiltration of the necrotic area by lymphoid cells.
Magnification × 100. B. DMNA + L-NNA. Centrilobular necro- sis. Necrotic areas are filled by detrital elements. Diffuse lym- phoid infiltration. Magnification × 100. C. DMNA + NaNO. Hepatocytes show ballooned dystrophy and caryolysis.
Magnification × 400
sis in animals treated with DMNA alone or with DMNA combined with sodium nitrite. However, the activity of AlAT was significantly elevated in the group administered with DMNA and L-NNA, which suggests significant damage of hepatocyte membranes in this group.
The combination of DMNA and the NOS in- hibitor significantly increased the activities of all the investigated liver lysosomal enzymes, which shows a progression of chronic hepatitis under the influence of L-NNA (Tab. 1). The application of DMNA and sodium nitrite did not change the activ- ity of b-D-galactosidase and alkaline phosphatase in the liver, whereas the activities of a-D-man- nosidase and N-acetyl-b-D-glucosaminidase were significantly lower than in the animals which re- ceived only DMNA.
The treatment of rats with DMNA changed nei- ther NO level nor NOS activity in the liver (Tab. 2).
The administration of L-NNA to the DMNA- treated rats significantly decreased NOS activity and liver NO content, whereas the combination of sodium nitrite (NO donor) and DMNA also signifi- cantly enhanced liver NO level, but markedly in- hibited liver NOS activity. However, the enhanced level of NO in the last group is the result of the ex-
ogenous nitrite administration but not reflects NO production. It is worth noting that activation of free radical-dependent processes evaluated by SOD ac- tivity and MDA content was observed only in the group receiving DMNA and L-NNA. In the liver of animals from this group, the content of products re- acting with thiobarbituric acid and SOD activity in the cytosol were significantly increased (Tab. 2).
DISCUSSION
The results of the morphological studies of the liver suggest that the liver damage in DMNA- treated rats corresponds to chronic hepatitis with accompanying regenerative processes, which usu- ally precede liver fibrosis. The findings indicate that the combined administration of DMN and L- NNA induced neurogenic disorders which can be evaluated as hepatic encephalopathy [6].
Paralysis/paresis of the forelimbs and back ex- tremities in the group treated simultaneously with DMNA and L-NNA suggests the neurogenic rather than the hepatogenic nature of their death. Our ob- servation and morphological data allow us to con- clude that the administration of L-NNA to DMNA-treated rats promoted severe and fatal tox-
Table 1. Activity of serum marker enzymes and lysosomal enzymes in the liver of rats treated with DMNA and its combination with the NO donor and the NOS inhibitor (nmol/mg of protein/h)
Parameters Control DMNA DMNA + L-NNA DMNA + NaNO2
Serum AlAT (mkKat/l) 0.721 ± 0.04 0.771 ± 0.05 0.891 ± 0.03*+ 0.788 ± 0.047 Serum AsAT (mkKat/l) 0.802 ± 0.039 0.848 ± 0.047 0.865 ± 0.043 0.785 ± 0.047
b-D-galactosidase 230.9 ± 9.96 204.6 ± 6.53 259.9 ± 13.08# 207.4 ± 5.73
a-D-mannosidase 234.0 ± 15.99 242.0 ± 5.65 298.2 ± 10.09*# 212.2 ± 5.21#
N-acetyl-b-D-glucosaminidase 397.5 ± 40.51 494.5 ± 16.07 948.4 ± 63.4*# 418.1 ± 16.61# Alkaline phosphatase (nmol/mg of protein/min) 47.6 ± 8.33 52.5 ± 9.13 77.4 ± 12.84*# 50.3 ± 7.28
* p < 0.05, compared to the control group;#p < 0.05, compared to the DMNA-treated group
Table 2. Results of biochemical tests performed in the liver of rats treated with DMNA and its combination with the NO donor and the NOS inhibitor
Parameters Control DMNA DMNA + L-NNA DMNA + NaNO2
NOS activity (nmol/mg of protein/min) 0.86 ± 0.11 0.63 ± 0.11 0.38 ± 0.04*# 0.55 ± 0.07*# NO content (nmol/mg of protein) 25.2 ± 1.03 24.8 ± 1.62 16.65 ± 0.84*# 32.79 ± 1.81*# MDA (nmol/g of tissue) 1.1 ± 0.11 1.05 ± 0.08 1.54 ± 0.1*# 0.91 ± 0.08 SOD (% of inhibition) 3.04 ± 0.55 2.43 ± 0.11 4.28 ± 0.19*# 2.64 ± 0.29
* p < 0.05, compared to the control group;#p < 0.05, compared to the DMNA-treated group
icity in liver tissue which caused the development of neurogenic disorders similar to hepatic encepha- lopathy. Neuronal NOS is involved in development of hepatic encephalopathy [15]. L-NAME in- creased the severity of hepatic encephalopathy in acute hepatitis induced by thioacetamide [5], which points to protective effect of NO in this pathology.
It was reported that NO could protect liver cells against oxidative damage caused by different hepa- totoxins [13]. However, one must take into consid- eration the fact that NO has both prooxidant and antioxidant properties [1]. The data on the effects of NOS inhibitors on free radical-dependent pro- cesses and lipid peroxidation are also controversial, nevertheless, many authors noted a prooxidant ef- fect of NOS inhibitors. It was reported that L-NAME and aminoguanidine enhanced lipid per- oxidation and simultaneously increased liver dam- age caused by CCl" [13] and thioacetamide [2].
Some authors believe that NO is an original chemi- cal barrier for free oxygen radicals, being their scavenger [13]. In our experiment, we showed a significant decrease in the total activity of NOS in the DMNA + L-NNA- and DMNA + NaNO - treated groups. The total liver activity of NOS con- sists of two isoforms. One of them, endothelial NOS (eNOS), is connected with regulation of intra- hepatic hemodynamics, whereas the other isoform, inducible NOS (iNOS), is associated with a pro- gression of liver damage [14]. The literature data suggest that L-NNA preferably inhibits eNOS [10].
Therefore, it may be assumed that the decreases in to- tal NOS in the DMNA + L-NNA- and the DMNA + NaNO-treated groups (Tab. 1) were caused by an inhibition of eNOS.
However, it is very likely that nitrites measured using Griess reagent in the NaNO -treated group originated in large part directly from exogenous source rather than from endogenously formed NO.
The contribution of NOS inhibitors to the liver damage of hepatotoxin-treated rats was not limited by the prooxidant effect of these compounds.
L-NAME induces disturbances of microcirculation in the endotoxemic liver [5]. The inhibitors of NOS can modulate liver damage, promoting platelet ag- gregation and neutrophile adhesion in the vascular channel and increasing vessel resistance in the liver [16]. However, many parameters measured did not change in rats with DMNA alone but only when L-NNA was added. These findings are additionally
required to study effects of L-NNA alone on these parameters in normal rats.
The inhibitors of NOS increased the level of proinflammatory cytokines, in particular, TNF-a, in the liver of rats with acute hepatitis [5]. TNF-a is considered a marker of hepatocellular damage.
Therefore, it can be suggested that the prooxidant effect of NOS inhibitors was secondary, and it was a result of an activation of liver inflammation by these compounds via an increase in TNF-a level in the liver.
The involvement of lysosomes in pathological processes in the liver is well known. The manifold data suggest activation of lysosomal enzymes in the liver and serum in acute and chronic hepatitis induced by hepatotoxins [8, 23]. Some authors con- sidered this activation a marker of inflammatory liver damage [17, 18], associated with an increase in proinflammatory cytokine levels [11]. On the other hand, it was reported that the damage of the liver by DMNA was not accompanied by activation of lysosomal enzymes [24]. In our experiment, the long-term treatment of rats with DMNA did not af- fect the lysosomal enzyme activities in the liver postmitochondrial supernatant (Tab. 2), whereas the single injection of other hepatotoxins, such as thioacetamide or CCl" , significantly increased the activity of these enzymes [8, 24].
In summary, we can conclude that NO plays a protective role in liver damage caused by DMNA, whereas the inhibition of endogenous NO produc- tion intensified liver injury by this hepatotoxin. The damaging effect of L-NNA can partially be ex- plained by the activation of free radical-dependent processes in the liver, but further investigations are required to establish intimate mechanisms of the observed effects.
Acknowledgment. This work was supported by a pro- gram project grant no. B99-121 from the Belarussian Foun- dation for Basic Research and Medical University of Bia³ystok, Poland.
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Received: February 13, 2004; in revised form: September 29, 2004.
