CytoChrome P 450 2 e 1 PartiCiPation in the Pathogenesis of exPerimental metaboliC syndrome in guinea Pigs

In this work the experimental metabolic syndrome on the basis of protamine sulfate modeling in guinea pigs was reproduced and pathological processes in the liver of experimental animals were studied. We determined the level of free radicals and markers of liver damage in the blood of experimental animals. We investigated the liver glycogen content and k+,Na+-aTPase activity in the liver of experimental animals as well as measured the cytochrome P450 2E1 (cyP2E1) expression – one of the main factors of oxidative stress. Evidence of development of hepatotoxic processes, increasing of the cyP2E1 level as well as of the free radical level in the animals with metabolic syndrome were found. Using of cyP2E1 inhibitors had shown that the free radical level in the blood of experimental animals depended on the level of the enzyme expression and activity. The obtained results suggest that the changes in the cyP2E1 expression play an important role in the development of hepatotoxic processes upon experimental metabolic syndrome. It was assumed that pharmacological correction of the enzyme expression may be an important mechanism for the influence on the metabolic syndrome clinical course.

In this work the experimental metabolic syndrome on the basis of protamine sulfate modeling in guinea pigs was reproduced and pathological processes in the liver of experimental animals were studied.We determined the level of free radicals and markers of liver damage in the blood of experimental animals.We investigated the liver glycogen content and k + ,Na + -aTPase activity in the liver of experimental animals as well as measured the cytochrome P450 2E1 (cyP2E1) expression -one of the main factors of oxidative stress.Evidence of development of hepatotoxic processes, increasing of the cyP2E1 level as well as of the free radical level in the animals with metabolic syndrome were found.Using of cyP2E1 inhibitors had shown that the free radical level in the blood of experimental animals depended on the level of the enzyme expression and activity.The obtained results suggest that the changes in the cyP2E1 expression play an important role in the development of hepatotoxic processes upon experimental metabolic syndrome.It was assumed that pharmacological correction of the enzyme expression may be an important mechanism for the influence on the metabolic syndrome clinical course.k e y w o r d s: cyP2E1; diabetes; experimental metabolic syndrome; oxidative stress; protamine sulfate.M etabolic syndrome (MS) is a complex and multifactorial etiology medical condition.Specialists do not consider any of MS symptoms as a main symptom including abdomi nal obesity, since new data have been reported that abdominal obesity has ethnic related characteristics (for example, the eastern nations), and is not always associated with development of the metabolic syn drome.The development of MS diagnostic criteria is one of the most important tasks, however to consider the diversity of all processes in the human body at the development of this syndrome in one characteris tic is an extremely difficult problem.Currently, in clinical practice several definitions of MS are used [1].Thus, to develop new MS and DM (diabetes mel litus) models, the necessity of reconstruction of a broad spectrum of pathological processes that occur in humans upon these diseases must be considered.
It is known that guinea pigs, owing to certain organism peculiarities, such as blood lipid composi tion and the absence of endogenous vitamin C, are the optimal model for the study of carbohydrate and lipid metabolism disorders [2,3].When modeling of MS in these animals, there is no extraneous influen ce of enhanced antioxidants secretion and blood atherogenic properties on the development of patho logical processes.Upon prolonged intramuscular administration of protamine sulfate to guinea pigs a number of symptoms, indicating the development of systemic homeostasis disorders, were observed.In particular, carbohydrate metabolism disorder (hyperglycemia and reduction of glycogen store in the liver) and lipid metabolism disorder (increase in choleste rol level) as well as inflammatory processes in the liver and kidneys (increased level of ALT, AST, creatinine and microalbuminuria), disorder of insulin synthesis, degradation of βcells and accu mulation of free radicals were observed [4,5].The symptoms mentioned above are typical in the devel opment of MS and DM in humans.
It is known that oxidative damage to tissues has been implicated in the etiology and pathogene sis of chronic complications in several diseases [6].It has been shown that the increased production of reactive oxygen species induced by hyperglycemia upon metabolic syndrome and subsequent glucose toxici ty lead to the development of oxidative stress accompanied by a lesion of the pancreatic βcells and hepato cytes [7].Recent studies have suggested that the development of the pathological conditions, doi: http://dx.doi.org/10.15407/ubj88.02.098 such as alcoholic and non alcoholic steatohepatitis, type 1 dia betes, hepatitis C are associated with the increased level of the CYP2E1 expression (CYP2E1 is one of the cytochrome P450 isoforms) [8].It has been explained by the fact that CYP2E1 is a power ful source of reactive oxygen species (ROS) [9], which makes it one of the substantial factors in the development of oxidative stress.It can be assumed that the development of the metabolic syndrome will also lead to an increase in the CYP2E1 expres sion and in the degree of hepatocytes damage, and the regulation of the enzyme expression will affect the pathological processes which accompany this disease [10].
In our work, using the specific protamine sul fate model for metabolic syndrome, we studied the development of hepatotoxic processes and changes in the level of the CYP2E1 expression in the liver of guinea pigs with MS.The correlation between the enzyme expression and activity and the develop ment of pathological processes in experimental animals using specific CYP2E1 inhibitors was also investi gated.

materials and methods
Experimental animals and diet.Male four monthold guinea pigs (30 animals) weighing 450 g (on the average) from the vivarium of the Institute of Pharmacology and Toxicology of NAMS of Ukraine were used in the study.Most reagents and substrates were purchased from SigmaAldrich (St. Louis, MO).All animal experiments were performed in ac cordance with the rules and regulations of The Euro pean Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes [11].
Animals were housed in individual stainless steel cages with a controlled 12 h light/12 h dark cy cle at 2023 °C and fed on a standard rodent diet.Blood sampling was carried out from the ear vein.Glucose level was measured using a glucometer Ac cuChek Active (AccuChek, Germany).
Induction of metabolic syndrome.The meta bolic syndrome was induced by intramuscular injec tion of protamine sulfate 15 mg/kg twice a day for 5 weeks, followed by keeping animals under normal conditions for 2 weeks.Such scheme was selec ted given the results of our previous studies which showed that experimental animals exhibited clear signs of the metabolic syndrome two weeks after the last injection of protamine sulfate [12].Intact ani mals (n = 5) during this period were kept under nor mal conditions.Thereafter the blood of intact ani mals and animals with MS (n = 5) under light ether anesthesia was taken by dissection of the femoral vein.The animals were then decapitated.
The remaining animals were divided into 4 groups.The animals of the first group were adminis tered intramuscularly quercetin at a dose of 20 mg/ kg for 2 weeks The animals of the second group were administered intramuscularly 4methylpyra zole at a dose of 20 mg/kg for 2 weeks.The animals of the third group were orally administered disulfi ram at a dose of 25 mg/kg.The control group (MS*) was maintained under normal conditions for 2 weeks to control the develop ment of pathological processes.Thereafter, the animals were euthanized according to the protocol described above.
Preparation of microsomal fraction and determination of cyP2E1 activity.The animals were decapitated at the same time of a day to avoid diur nal changes in the microsomal enzymes activity.Microsomal fraction was obtained according to the protocol described in [13].Pnitrophenol hydroxylase activity was determined as the rate of 4nitrokatehol formation per time unit per 1 mg of protein [14].The obtained values were expressed as nmol of nitrokate hol/min/mg of protein.
Western blot analysis of cyP2E1.The total pro teins were isolated from liver homogenate according to a standard protocol for membrane proteins prepa ration (Abcam, UK).The obtained proteins were separated by polyacrylamide gel electrophoresis (50 mg/track) followed by transfer to nitrocellulose membrane (Biorad, USA).After treatment with 2% skimmed dry milk solution, the membrane was incu bated with antiCYP2E1 antibodies (1/400, by volu me) for 1 hour.Primary antibodies were produced in the Department of Molecular Oncogenetics, Institute of Molecular Biology and Genetics, NAS of Ukraine.The membrane was washed and then incubated with antiRabbit IgGHRP secondary antibodies (Sigma, 1/5000, by volume) for 1 h.Proteins were visualized by chemiluminescence.Protein concentration was normalized relative to amount of the control βactin protein on the same gel track.Densitometry analy sis was performed using ImageJ software (Wayne Rasband, NIH).The results of Western blot analysis were presented in relative units of the luminescence intensity of the target protein.
histological analysis.The liver was fixed in neutral buffered formalin solution (10%) and de hydrated in a graded ethanol series (50, 70, 80, 90, 95 and 100%).Tissues were placed in Paraplast and cut to 4 µmthick sections.Glycogen content and K + ,Na + ATPase activity were determined in the ob tained samples [15].The obtained tissue samples were examined under optical microscope Leica DM 1000.
The assessment of free radical level and blood biochemical parameters.The level of ROS was measured in blood of experimental animals by spec trofluorimetry using 2,7dichlorofluorescein diace tate (2,7DCFHDA) probe as described previously [16].The results are presented in arbitrary units of the fluorescence intensity of dichlorofluorescein (oxidized 2,7DCFHDA).Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase activities, as well as content of serum cholesterol, creatinine and glucose were measured using an automatic biochemical analyzer Tokyo Boe ki Prestige 24i (Japan) and sets of specific reagents.
Statistical analysis.The data are presented as average values ± error of the arithmetic mean for 5 animals per group.Statistical processing was per formed using Student's ttest (for 2 groups) [17].Values with P < 0.05 were considered statistically significant.

results and discussion
Biochemical and histological analysis.It is known that during development of metabolic syn drome, changes in tissue biochemical and morpho logicalfunctional parameters occur.The results of serum biochemical analysis of animals with MS are presented in Table 1.It was found that the typical MS symptoms such as hyperglycemia (nearly 2fold increase in serum glucose level) and hyperlipidemia (more than 2fold increase in cholesterol) occurred at the early stages of MS.It was also found that the development of MS in experimental animals was accompanied by hepatotoxicity, as evidenced by an increase in the content of cytolysis markers and hepatocytes necrosis markers, in particular more than 2fold increase in ALT and alkaline phos phatase, and also 6fold increase in AST compared to control.An increase in serum creatinine, the end product of nitrogen metabolism, which indicates acute or chronic renal failure, was observed.
Immunohistrochemical analysis of the experi mental animals' liver was carried out to determine glycogen content and K + ,Na + ATPase activity, by the PASreaction according to MacManus and by Vah shtein and Meisel methodology, respectively.Carbo hydrate metabolism disorder is considered to be the main pathogenetic mechanism of the MS develop ment.Normal liver cells contain a large amount of glycogen (Fig. 1, a, pink granules).It is known that type 2 diabetes is accompanied by elevated glucagon level and activated hepatic glycogenolysis.Decrease in the stain intensity of the slide (Fig. 1, B) indicates a decrease in the amount of glycogen, which is a typical pathological process upon MS and type 2 diabetes [18].
It is known that insulin activates the liver K + ,Na + ATPase.Insulin facilitates potassium inflow into the liver cells and this effect is not related to hormone influence on glucose transport.A fall in activity of this enzyme could indicate insulin defi ciency.Changes in the K + ,Na + ATPase activity is a reversible process, since the insulin administration to animals with insulin deficiency completely re stores the activity of sodiumpotassium pump [19].
The obtained results showed that the enzyme activity significantly decreased in the liver tissue of experimental animals (Fig. 2, B) compared to the in tact animals (Fig. 2, a).This may also indicate insu lin deficiency and development of MS.
Hepatic glycogen level and K + ,Na + ATPase ac tivity were slightly lower under inhibitors action,

Biochemical parameters
Intact The data are presented as average value ± error, n = 5, * P < 0.05 relative to intact animals (see text for explanation).
though significantly higher than in the MS animals (Fig. 1, c, 2, c).Study of the cyP2E1 expression level.Cyto chrome P450 system is a powerful source of free radical generation [20,21].First of all it concerns the CYP2E1 isoform, which is characterized by a high level of oxidase activity and the ability to produce free radicals, even in the absence of the substrate [22].It was shown that increased production of free radicals upon some diseases owing to increase in the CYP2E1 expression can cause oxidative stress.This leads to the development of various complications, such as micro and macroangiopathy, retinopathy, nephropathy, and others.This is why the study of the mechanisms for CYP2E1dependent oxidative stress and correction of some manifestations of this pathology by regulating the CYP2E1 activity are ex tremely important.
To investigate the changes in CYP2E1 expres sion during the MS development, CYP2E1 activity and protein content were evaluated.It was found that CYP2E1 activity in the liver of experimental ani mals in the early stages of MS was slightly higher compared to control, however it increased nearly 3fold in the further disease development (MS*) (Fig. 3, a).The changes in the CYP2E1 protein level during the MS development in experimental animals are shown in Fig. 3, B. A slight increase in the CY P2E1 protein content was observed in animals with MS, and CYP2E1 level in liver cells of experimental (MS*) animals increased almost 3fold in progres sion of MS.Thus, it can be asserted that increasing of the enzyme activity is caused by an increase in the cells CYP2E1content.

Fig. 1. histochemical assessment of the glycogen content in liver cells (PaS-reaction according to Mac-Manus). Leica DM 1000, 400 x. a -intact animals, B -control group (MS*), c -metabolic syndrome + quercetin
Some studies indicated the important role of CYP2E1 in the ROS production and oxidative tis sue damage [23,24].Therefore we investigated the ROS level in the blood of experimental animals with metabolic syndrome and relationship between ROS production and the level of CYP2E1 expression.It was shown that the development of pathological pro cesses in experimental animals was accompanied by an increase in free radical level, which was almost Investigating CYP2E1dependent processes in experimental animals with MS, we compared blood biochemical parameters at various levels of the CYP2E1 activity depending on the effect of enzyme inhibitors, namely disulfiram, 4methylpyrazole and quercetin.As it is seen in Fig. 4, a, the inhibitors re duce (in varying degree) the liver CYP2E1 activity.Quercetin exhibited the most inhibitory effect at the given doses, 4methylpyrazole -the least inhibitory effect.A significant decrease in the enzyme activi ty under the quercetin action confirms the results of computer modeling, concerning its efficacy in CY P2E1 inhibition.

Fig. 4. changes in cyP2E1 expression and activity in liver of experimental animals with MS in the presen ce of cyP2E1 inhibitors: a -cytochrome P450 2E1 activity; B -protein cyP2E1 level; caccumulation of free radicals in leukocytes of experimental animals. * P < 0.05 relative to control group, n = 5
The assessment of CYP2E1 content in liver cells revealed that protein level decreased propor tionally to its activity.As it is seen in Fig. 4, B, 4methylpyrazole affects more protein CYP2E1 content than its activity, whereas quercetin, on the contrary, is a potent inhibitor of the enzyme activity.
The inhibitors prevent the increase in free radical level, thereat quercetin and disulfiram lead to a slight decrease in the free radical level compared to that at the beginning of the administration (Fig. 4, c).
The results of biochemical blood tests (Table 2) showed that the administration of CYP2E1 inhibi tors to MS animals was accompanied by a tendency to normalization of blood biochemical parameters.Quercetin showed the most pronounced effect, in particular, 2fold decrease in the cholesterol level and decrease in ALT, AST and ALP levels to the values in intact animals were observed.At the same time, the level of creatinine, one of the integral in dicators of kidney condition, remained almost un changed.It is worth noting that the administration of disulfiram was accompanied by less pronounced changes in biochemical parameters.This indicates the need for further studies of the disulfiram dose dependent effect.
It should be noted that a decrease in the pro tein content and CYP2E2 activity, as well as free radical level led to a decrease in the degree of oxi dative damage to hepatocytes.This was evidenced by normalization of functional parameters of the liver, in cells of which the CYP2E1 expression was the highest .This suggests that liver damage in MS animals might be reversible.Thus liver functional parameters at the CYP2E1 inhibition by quercetin restored almost to the norm (Fig. 1, c, Table 12).Quercetin may possibly have such pronounced hepatoprotective effect owing also to its antioxidant properties.It was confirmed by the fact that a de crease in the levels of ALT, AST and ALP under the influence of 4methyl pyrazole and disulfiram was less pronounced than that of quercetin.
Thus in our work, hepatotoxic processes in guinea pigs during MS development were studied.The increase in blood free radical level, ALT, AST, alkaline phosphatase activities, as well as the de crease in glycogen content and the K + ,Na + ATPase activity in hepatocytes indicated a disorder of the structuralfunctional state of liver cells.In experi mental animals two weeks after the onset of MS symptoms (group MS*), there were no significant changes in biochemical parameters that indicate the resistance in the symptoms and the formation of the systemic pathological processes inherent to MS.
It has been shown that the induction of CYP2E1 expression is one of the factors in the development of systemic oxidative stress in some diseases [25].In crease in CYP2E1 expression and free radicals pro duction results in oxidative damage to the liver cells.This leads to progressive carbohydrate metabolism disorders and progressive tissue and organ dama ges [26].Thus the regulation of CYP2E1 expression might be an effective mechanism for the correction of systemic CYP2E1dependent oxidative stress upon metabolic syndrome.
We have studied the relationship between changes in CYP2E1 expression and hepatotoxicity using three specific inhibitors.These inhibitors af fect CYP2E1 through different mechanisms.Thus disulfiram works through prior conversion to die thyldithiocarbamate, which directly inhibits enzyme activity [27].This inhibition of CYP2E1 activity can be attributed to disulfiram hepatoprotective proper ties in case of paracetamol liver damage.
CYP2E1 inhibition by 4methylpyrazole [28] leads to a significant reduction of the manifestations of oxidative stress, hepatotoxicity and the formation of an aldehydeprotein adducts.Such properties are used in the development of drugs for the prevention of hepatotoxicity in cases of methanol and ethylene glycol poisoning.Literature data have shown results similar to ours, that is, 4methylpyrazole exhibits relatively slight effect on CYP2E1 activity.Though it The data are presented as average value ± error, n = 5, # P < 0.05 relative to control (MS+).See text for explanation.

T a b l e 2. Biochemical parameters of MS animals blood serum under the action of the cyP2E1 inhibitors
V. V. Rushchak, M. O. chashchyn has been shown that 4methylpyrazole significantly reduced free radical level in hepatocytes cell cul ture, preincubated for one day in the medium with 100 mM ethanol [29,30].This indicates that the an tioxidant potential of 4methylpyrazole is realized by reducing the CYP2E1 protein level and free radicals production.
Quercetin is widely used as an antioxidant and angioprotector [31].Moreover, as we reported previous ly [32], quercetin can be highly effective in hibitor of CYP2E1.
Simultaneous changes in CYP2E1 expression and oxidative stress level suggests that, in particu lar, increased CYP2E1 expression and activity are key factors of liver cells oxidative damage upon the development of metabolic syndrome.
Thus it has been shown that the protamine sulfate model (with a high degree of similarity) re produces liver pathologic processes, which is typi cal symptom of the MS development.It enables to use this model in pharmacological and toxicological studies of drugs for diabetes therapy.The obtained results have indicated the correlation between in creased SYP2E1 expression in liver cells, increased free radicals production in blood and hepatotoxic ity in animals with metabolic syndrome.It has been shown that a decrease in CYP2E1 activity under the influence of specific inhibitors leads to impairment of severity of hepatotoxicity in experimental ani mals with MS.Thus pharmacological regulation of CYP2E1 functions and through them the pathologi cal processes which accompany metabolic syndrome may be quite possible.This approach can be useful for correction of pathological processes accompa nied by increased CYP2E1 level.К л ю ч е в ы е с л о в а: CYP2E1, диабет, экспериментальный метаболический синдром, оксидативный стресс, протаминсульфат.

Fig. 2 .
Fig. 2. histochemical assessment of k + ,Na + -aTPase in liver cells.Leica DM 1000, 400 x. a -intact animals, B -control group (MS*), c -metabolic syndrome + quercetin with intact animals in the early stages of MS and 4fold higher at further de velopment of pathology (MS*) (Fig 3, B).

acknowledgments
The authors would like to thank A. K. Voronina and G. M. Shayakhmetova (Department of General Toxicology of Institute of Pharmacology and Toxi cology NAMS of Ukraine); O. V. Maksymchuk and I. V. Rosokhatska (Department of Molecular Onco genetics of Institute of Molecular Biology and Ge netics) for their support.