ACTION OF HYDROGEN SULFIDE DONORS ON NITROSO-OXIDATIVE PROCESSES IN SMALL INTESTINE OF RATS wITH METHOTREXATE-INDUCED ENTEROPATHY

Introduction. Medication-induced enteropathy plays an important part among factors leading to the development of small intestinal injury. There are some evidences indicating a potential preventive action of hydrogen sulfide (H2S) donors against drug-induced enteropathies based on that fact that the use of the most of enterotoxic medications including anti-tumor drugs leads to the suppression of this gaseous mediator production. The aim of the study – to compare the action of H2S donors in small intestine of rats on parameters of NOsynthase system and oxidative stress under condition of methotrexate-induced enteropathy. Research Methods. The experimental procedures were carried out on rats which on the background of methotrexate-induced enteropathy received H2S donors NaHS (1 and 10 mg/kg) and L-cysteine. Following biochemical parameters were measured in small intestinal mucosa: activity of NO-synthases, myeloperoxidase, superoxide dismutase and catalase; concentrations of NOx (nitrite/nitrate) and malonic dialdehyde. H2S concentration was determined in blood serum. Results and Discussion. Administration of methotrexate didn’t cause any visible changes of small intestine surface, however led to serious biochemical changes. NO concentration increased as a result of iNOS activation (more than fivefold (p≤0.01). Simultaneously concentration of H2S decreased in blood serum. Administration of H2.S donors practically returned these parameters to their normal value. Methotrexate-induced enteropathy caused the increase of myeloperoxidase activity by 66 %, p≤0.01, indicating of inflammatory process formation and activation of lipid peroxidation. Administration of NaHS didn’t cause any serious changes in myeloperoxidase activity, however increased SOD activity and practically retuned it to its norm. Conclusions. Nirtoso-oxidative stress plays the key role in enteropathy formation resulted in methotrexate administration. H2S donors modulate parameters of NO-synthase system and activity of SOD.

INTRODUCTION.Increased toxicity of different medications in the gastrointestinal tract is a common and serious medical problem; the number of drugs that can harm the gastrointestinal tract is impressive [1].Enteropathy is known to be one of the most commonly appeared pathologies resulting in the use of medications such as: nonsteroidal anti-inflammatory drugs (NSAIDs), anti-tumor drugs and hypotensive medications [2].Among anti-tumor drugs methotrexate and 5-fluorouracil is known by its ability to damage the small intestinal mucosa by preventing crypt mitotic activity, inhibiting dihydrofolate reductase and leading to the development of malabsorption syndrome and diarrhoea [3].There are some evidences indicating a potential preventive action of hydrogen sulfide (H2S) donors against drug-induced enteropathies [2,4] based on that fact that the use of the most of enterotoxic medications including anti-tumor drugs leads to the suppression of this gaseous mediator production.The most common way to generate H 2 S for experiments is to use common salts such as NaHS and its precursors such as L-cysteine [5].Cytoprotective effects of donors of H 2 S were previously demonstrated on the model of indomethacin-induced small intestinal injury in rats [2,4].However, their effects in methotrexate-induced enteropathy are still poorly studied.
The aim of the study -to evaluate the action of H 2 S donors in small intestine of rats on parameters of NO-synthase system and oxidative stress under condition of methotrexate-induced enteropathy.
RESEARCH METHODS.The structure of this study and the experimental procedures performed on the animals were approved by the Ethical Committee of Lviv National Medical University.The experimental procedures were carried out in accordance with the international guidelines for the use and care of laboratory animals.Male, outbred
Rats were anesthetized with 1 ml of urethane at a dose of 1.1 mg/kg injected intraperitoneally and sacrificed by cervical dislocation.A blood sample from the cervical vessel was immediately collected into vials containing 0.1 ml of heparin.The samples of small intestinal mucosa were homogenised in phosphate buffer pH 6.0 1:4 and centrifuged at 3000 rpm, supernatant was used to determine values of biochemical parameters.Activity of NO-synthase isoenzymes (inducible iNOS and constitutive cNOS) was measured by the method described in detail [7].NOS activity was expressed in nmol L-citrylline/ min×mg of protein.NOx (nitrite/nitrate) concentration in homogenates of small intestine was assayed by the griess reaction-dependent method of [8].In order to determine total (NO 2 /NO 3 ) concentration to deproteinised homogenates (1:100) of zinc for reduction of nitrate to nitrite or manganese sulphate for measurement of nitrate-anion were added.Naphthyl-ethylenediamine was used to perform griess reaction.The absorbance was read in a Statfax at 520-560 (550) nm.Concentration of stable products of NO was expressed as nitrite+nitrate (mmol/g).Myeloperoxidase (MPO) activity in small intestinal homogenates was assayed spectrophoto metrically by the method [9] with some modifications.The MPO activity was analysed spectrophotometrically as follows: 1 ml of homogenate was added to 2.9 ml of 0.1 M K 3 PO 4 buffer (pH 6.0) involving O-dianisidine dihydrochloride (0.167 mg/ml) and 0.005 % hydrogen peroxide of the resection mixture was recorded at a wave length of 450 nm.One unit (U) of activity was defined as that degrading 1 µmole of peroxide/mg of protein.Lipid peroxidation levels were determined as malonic dialdehyde (MDA) concentration in homogenates of gastric mucosa, according to the procedure [10].MDA levels were expressed as mmol/l.Activity of superoxide dismutase (SOD) was determined by the reaction of reduction of nitrotetrazolium blue to nitroformazan [11].SOD activity was expressed in mmol/min×mg of protein.Catalase (CAT) activity was determined by measuring of the decrease in hydrogen peroxide concentration at 410 nm [12].Catalase activity was expressed in mmol H 2 O 2 / min×mg of protein.H 2 S concentration was determined by reaction with para-phenylenediamine [13].
The statistical processing of the data was done by conventional methods for analysis of variance using MS Excel software for Student's test.The difference was considered to be significant at p≤0.05.RESULTS AND DISCUSSION.Administration of methotrexate didn't cause any visible changes of small intestine surface.It should be pointed out that methotrexate-treated animals were suffering from severe enterotoxicosis manifested by diarrhoea and vomiting.
Methotrexate-induced enteropathy was accompanied by significant changes of gaseous mediators production manifested by the decrease of H 2 S concentration in blood serum by 20 % (p≤0.01) and the increase of NO x concentration in small intestinal mucosa by 40 % (p≤0.01) (Fig. 1).In recent years it was shown, that hydrogen sulfide plays an impor- mmol/l.Activity of superoxide dismutase (SOD) was determined by the reaction of reduction of nitrotetrazolium blue to nitroformazan [11].SOD activity was expressed in mmol/min×mg of protein.Catalase (CAT) activity was determined by measuring of the decrease in hydrogen peroxide concentration at 410 nm [12].Catalase activity was expressed in mmol H 2 O 2 /min×mg of protein.H 2 S concentration was determined by reaction with para-phenylenediamine [13].
The statistical processing of the data was done by conventional methods for analysis of variance using MS Excel software for Student's test.The difference was considered to be significant at p ≤ 0.05.RESULTS AND DISCUSSION.Administration of methotrexate didn't cause any visible changes of small intestine surface.It should be pointed out that methotrexate-treated animals were suffering from severe enterotoxicosis manifested by diarrhoea and vomiting.
Methotrexate-induced enteropathy was accompanied by significant changes of gaseous mediators production manifested by the decrease of H 2 S concentration in blood serum by 20 % (p ≤ 0.01) and the increase of NO x concentration in small intestinal mucosa by 40 % (p ≤ 0.01) (fig.1).In recent years it was shown, that hydrogen sulfide plays an important role in promoting resolution of inflammation, and restoration of normal tissue function [14], thus the suppression of its synthesis in medication-induced small intestinal injury may play a crucial role for development of enteropathy.tant role in promoting resolution of inflammation, and restoration of normal tissue function [14], thus the suppression of its synthesis in medication-induced small intestinal injury may play a crucial role for development of enteropathy.H 2 S donor NaHS displayed the tendency to increase of H 2 S concentration whereas L-cysteine administration returned it to its normal level.H 2 S donors dose-dependently decreased NO x concentration in correspondence to the existence of metabolic relationship between both gaseous mediators.
Changes of NO x concentration in group of methotrexate-treated animals were resulted by the increase of iNOS activity (more than fivefold (p≤0.01) as compared with induced of control group) (Fig. 2).Simultaneously cNOS activity decease by 15 % (p≤0.05).All studied inhibitors decreased iNOS activity and practically returned it to its normal levels.
Enterotoxic action of methotrexate was accompanied by the development of oxidative stress in small intestine manifested by the rise of MDA concentration by 61 % (p≤0.01) (Fig. 3) and the significant decrease of antioxidant enzyme SOD (Fig. 4).Simultaneously the activity of MPO was increased by 66 % (p≤0.01) as compared to the control group which suggests neutrophil infiltration resulting in the development of inflammatory process in small intestine.
None of studied H 2 S donors decreased MPO activity as compared to methotrexate action.It should be pointed out that ability of hydrogen sulfide to modulate MPO activity was previously shown [15].However in our study only H 2 S precursor Lcysteine has demonstrated the tendency to de-group 4 -methotrexate+ 10 mg/kg NaSH, group 5 -methotrexate + L-Cys.Mean ± SD, n=8 in each group of animals.* -p≤0.05,* * -p≤0.01, in relation to control animals; # -p≤0.05,## -p≤0.01 as compared to the methotrexate action.
H 2 S donor NaHS displayed the tendency to increase of H 2 S concentration whereas L-cysteine administration returned it to its normal level.H 2 S donors dosedependently decreased NO x concentration in correspondence to the existence of metabolic relationship between both gaseous mediators.
Changes of NO x concentration in group of methotrexate-treated animals were resulted by the increase of iNOS activity (more than fivefold (p ≤ 0.01) as compared with were induced of control group) (fig.2).Simultaneously cNOS activity decease by 15 % (p ≤ 0.05).All studied inhibitors decreased iNOS activity and practically returned it to its normal levels.% (p≤0.01)(fig.3) and the significant decrease of antioxidant enzyme SOD (fig.4).
Simultaneously the activity of MPO was increased by 66 % (p≤0.01) as compared to the control group which suggests neutrophil infiltration resulting in the development of inflammatory process in small intestine.
None of studied H 2 S donors decreased MPO activity as compared to methotrexate action.It should be pointed out that ability of hydrogen sulfide to modulate MPO activity was previously shown [15].However in our study only H 2 S precursor L-cysteine has demonstrated the tendency to decrease of MPO activity in small intestine of methotrexate-treated rats (Fig. 3).
It is well known that H 2 S exhibits strong antioxidant properties.In our study an antioxidant action of H 2 S inhibitor NaHS was manifested by the increase of SOD activity as compared to methotrexate group (Fig. 4).Surprisingly, L-Cys didn't demonstrate such action.However all studied donors didn't decrease MDA concentration.Catalase activity significantly increased as a result of 10 mg/kg NaSH and L-Cys action.(Fig. 4) Thus, although methotrexate is widely used in clinics as an anticancer agent, it's utility is limited by its gastrointestinal toxicity which is one of the most serious side effects in its treatment.However, the mechanism of the toxicity has not been completely clarified [16].On the other hand, the oxidative stress is known to play an important role in various diseases and drug-induced side effects.In our study the administration of methotrexate induced the development of oxidative stress.On the other hand many studies have suggested an im-portant role of nitric oxide in methotrexate-induced injury [17].Our results allow us to suggest the potential role of the decrease of endogenous H 2 S concentration in the mechanisms of methotrexate induced enterotoxicity.Thus, donors of H 2 S may significantly regulate both oxidative stress and iNOS activation in methotrexate models.NaHS is commonly used in in vivo and in vitro experiments as a source of H 2 S to study the possible physiologic functions of endogenous H 2 S. NaHS immediately dissociates and forms the hydrosulfide anion HS − , which then reacts with H + to form H 2 S [18].In our study NaHS displayed the regulatory effect upon iNOS and SOD activity without any significant effect of MDA concentration and MPO activity.
CONCLUSIONS.Nirtoso-oxidative stress plays the key role in small intestine of rats in mechanisms of enteropathy development resulted in methotrexate administration.H 2 S donors modulate parameters of NO-synthase system and activity of SOD of methotrexate-treated rats.Thus, although methotrexate is widely used in clinics as an anticancer agent, it's utility is limited by its gastrointestinal toxicity which is one of the most serious side effects in its treatment.However, the mechanism of the toxicity has not been completely clarified [16].On the other hand, the oxidative stress is known to play an important role in various diseases and drug-induced side effects.In our study the administration of methotrexate induced the development of oxidative stress.On the other hand many studies have suggested an important role of nitric oxide in methotrexate-induced injury [17].

Fig. 1 .
Fig. 1.Concentrations of H 2 S in blood serum and stable products of NO in homogenates of small intestinal mucosa at the background of methotrexate-induced enteropathy of rats of the following groups: group 1 -control group, group 2 -methotrexate-induced enteropathy, group 3 -methotrexate +1 mg/kg of NaSH , group 4 -methotrexate+ 10 mg/kg NaSH, group 5 -methotrexate+L-Cys.Mean±SD, n=8 in each group of animals.* -p≤0.05,** -p≤0.01, in relation to control animals; # -p≤0.05,## -p≤0.01 as compared to the methotrexate action.

Fig. 1
Fig. 1 Concentrations of H 2 S in blood serum and stable products of NO in homogenates of small intestinal mucosa at the background of methotrexate-induced