Activities of xanthine oxidase , nitric oxide synthase , aromatase and level of cytochrome P 450 1 A 1 , 1 A 2 and IBl isoforms in rat upon parenteral genistein injections

It has been established that genistein inhibits the xanthine oxidase and nitric oxide synthase activity in the rat liver, lung and brain and decreases the aromatase activity and the enzyme level in the rat ovaries and uteri. Genistein has been also established as the inductor of the rat liver cytochrome P450 IAl, 1A2 and IBl isoforms. It has been shown in vitro that genistein is both the xanthine oxidase allosteric inhibitor, decreasing the superoxide generation, and the aromatase competitive inhibitor.

Introduction.Reactive oxygen species as well as NO may induce such processes as cancerogenesis and apoptosis [1 ].It is well-known that the apoptosis may be induced either by superoxide or NO, but not by peroxynitrite ONOO", the product of their condensation [1].It was established that unsteroid estrogens, poly chlorinated biphenyls and polychlorodibenzodioxins, are xanthine oxidase allosteric inhibitors that strongly decrease the superoxide production by this enzyme [2,3].Also these compounds being structurally related to steroids may inhibit the expression of inducible nitric oxide synthase genes [1].On the other hand, poly chlorinated biphenyls and polychlorodibenzodioxins are powerful inductors of CYPlAl, CYPlA2 and CYPlBl expression, performing this through Ah-receptors [4 ].
In the last years it was established that fitoestrogens such as isoflavonoids were powerful antioxidants [5].Different studies revealed the superoxide scavenging activity of isoflavonoids with estrogen activity, such as genistein, based on their reductive properties [5 ].However, the complete mechanisms of isoflavonoids antioxidative activity are not so far © V. V.

SUMBAYEV, I. M. YASINSKA, 2001
investigated.Analysing the isoflavonoids structure quantitatively, we assumed that these compounds may be the allosteric xanthine oxidase inhibitors such as corticosteroids, polychlorinated biphenyls and polychlorodibenzodioxins.It is a well-known fact that xanthine oxidase produces free superoxide that induces the peroxidation processes [6].So the antioxidative effect of some flavonoids may be based on their xanthine oxidase inhibiting activity.Structurally related to corticosteroids, fitoestrogens, namely genistein may inhibit the inducible nitric oxide synthase genes expression.Earlier it was established that some flavonoids possessed the aromatase inhibiting activity (the inhibition was isosteric) [7].Aromatase (cytochrome P450 XIX Al) is the enzyme that turns testosterone into estradiol and androstendione into estrone [8,9].So fitoestrogens such as genistein may be the aromatase isosteric inhibitors and such as the estrogens may inhibit the expression of the aromatase gene.Genistein and some other isoflavons are structurally related to the polychlorinated biphenyl's and polychlorodibenzodioxins.So these compounds are probably may interact with the Ah-receptors and induce the CYPlAl, CYP1A2 and CYPlBl expression.
The aim of the presented work was to study xanthine oxidase, nitric oxide synthase and aromatase activities as well as cytochrome P450 IAl, 1A2 and IBl level in rat organism under conditions of parenteral genistein injections and the in vitro genistein effects on the xanthine oxidase, nitric oxide synthase and the aromatase activities.Materials and Methods.The in vivo studied were performed on 10 Vistar female rats (age 3 months, weight 100 ± 20 g) from one generation were used for the investigations.Animals were divided into two groups.The first group (control, five rats) received 0.2 ml of the peach oil with intraperitoneum injections and the another group (five rats) received 500 μ% of genistein per 1 kg of weight (genistein was dissolved in the peach oil) per 1 kg of weight with intraperitoneum injections.All injections were performed during three days with the interval in 24 hours.In two hours after the last injection animals were mortified and the liver, lung, brain, ovaries and uteri were immediately isolated and homogenized in the 0.25 M saccharose (1:5).
Measurement of xanthine oxidase activity.Xanthine oxidase activity was measured in the liver, lung and brain as we described earlier [10].
Nitric oxide synthase activity measurement.Nitric oxide synthase activity was measured in the liver, lung and brain in the reaction system that consisted of 0.1 ml of homogenate, 2.5 ml of 0.1 M tris-HCl buffer (pH 7.4), 0.3 ml of arginine (320 μ M solution in the bidistilled water) and 0.1 ml of 1 mM NADPH + H + water solution by the method described in [11].The products of the aerobic NO oxidation were also measured in the liver, lung and brain by the method, based on the Griess reaction [11 ].
The aromatase activity and quantity measurement.The aromatase activity was measured in the rat ovaries and uteri as we described earlier [12].The aromatase level was quantified with the help of diskelectrophoresis.The microsomes were isolated from the described organs by the differential centrifugation.The microsomal proteins were eliminated with the sodium cholate [13] and divided by disk-electrophoresis [14].The electrophoregrams were put at the reaction system (10 ml) that consisted of 0.1 M tris-HCl buffer (pH 7.4), 1 mM aqueous solution of NADPH+ H + and 6 mM 17-methyltestosterone solution in 0.01 M HCl (1:25:1) and incubated during 1 hour.In 1 hour 2 ml of 0.2 % blue tetrazolium solution in 7 % NaOH was added to the reaction systems.After 24 h incubation the electrophoregrames were isolated and the blue sticks formed by the aromatase bound 17-methyltestosterone were eluted by 7 % CH 3 COOH.The aromatase quantity was measured by differential spectrophotometry [15].

Quantification of cytochromes P450 IAl y IAl and JBL
The cytochromes P450 IAl, IAl and IBl in rat liver were measured as we described earlier [14].
Study of the genistein effect on the xanthine oxidase activity.These investigations were performed on the highly purified homogeneous xanthine oxidase preparation obtained from rat liver by the affinity precipitation on xanthine-agarose after previous purification (purificational degree-1100 times, the enzyme outlet -73.5 %) [16].Then the xanthine oxidase activity was measured as we described earlier [10] but the reaction systems included also 0.1-1.0μ M of genistein.A probable genistein binding with xanthine oxidase was studied as follows.The quantities of free genistein were measured in the reaction systems after 15 min incubation (t = 40 °С) by the described method [3,14] before and after HCl destruction [2,3] of probable genistein-xanthine oxidase interactions.Also the quantities of free genistein in the reaction systems without xanthine oxidase were measured.The binding was determined as the number of genistein molecules, bound with one molecule of xanthine oxidase.
The genistein effect on the superoxide generation by xanthine oxidase was also studied according to the method described [17].
Study of genistein effect on the nitric oxide synthase activity.These studies were performed on rat liver homogenates.The nitric oxide synthase activity was measured in the reaction systems that also included 0.1-1.0μΜ of genistein as we described.
Study of genistein effect on the aromatase activity.These investigations were performed on highly purified rat uterine aromatase.The aromatase was purified by the affinity precipitation on 17-methyltestosterone-agarose after previous purification, that included the material homogenization, microsomes isolation, microsomal proteins elimination by sodium cholate and aromatase precipitation by the acetone.The aromatase activity was studied in the reaction systems that also included 25-500 μM of genistein.
Statistics.The results were statistically validated according to the Student's ^-criterion.The results were considered as significant at ρ < 0.05.
Results and Discussion.The xanthine oxidase activity in liver, lung and brain of genistein injected rats decreased in comparison with the control group as well as the nitric oxide synthase activity and the level of the nitric oxide aerobic oxidation products (table 1).Both the aromatase activity and level decreased in the rat ovaries and uteri compared to the control group (table 2).The level of the cytochrome P450 1A1, 1A2 and IBl isoforms in the liver of genistein injected animals strongly increased in comparison with the control group (table 1).
As we established earlier unsteroid estrogens, polychlorinated biphenyls and polychlorodibenzodioxins, are xanthine oxidase allosteric inhibitors.So it was suggested that genistein as the compound that structurally related to the described unsteroid estrogens may be xanthine oxidase allosteric inhibitor.In the case of inhibition of the nitric oxide synthase activity, genistein may influence the nitric oxide synthase directly or regulate the expression of the inducible NO-synthase genes.The aromatase inhibition may be caused by the inhibition of its gene expression.But the direct enzyme inhibition may also take place because, as we indicated, it was established that some flavonoids are the aromatase isosteric inhibitors [7].The level of cytochrome P450 IAl, 1A2 and IBl is controlled only by the Ah-receptors [4 ].It is well-known that these isoforms are inducible by the polychlorinated aromatic compounds [4].According to the quantitative analysis of the structure-activity relationship [18] the orbital structures of the polychlorinated aromatic unsteroid estrogens and genistein are display major relationships.So genistein interacts with Ah-receptors as their well-known

Table 2 The aromatase activity and quantity in the ovaries and uteri of intact and genistein injected rats (n-5)
The ligands and induces the CYP1A1, CYP1A2 and CYPlBl genes expression.It was shown in vitro that genistein is a powerful xanthine oxidase inhibitor (fig. 1) and according to the kinetic studies the inhibition is not competitive (fig.2).After incubation of the reaction systems icluding genistein of any concentration the amount of free genistein was less than injected.But after HCl destruction in all cases the free genistein level became equal to that of injected.In the reaction systems without xanthine oxidase the amount of free genistein was the same as injected.These results allow to conclude that genistein binds to xanthine oxidase.The dependence of the bound genistein quantity on its concentration in the reaction systems was reflected in the reverse coordinates (fig.3).The dissociation constants (K d ) and В тях were: calculated as K 6 = = 10 μ M, В тях = 101 molecules per 1 enzyme molecule.
Therefore, genistein inhibits xanthine oxidase through binding to the enzyme.According to the quantity of genistein and its electron structure it may form a steady complex with the amino acids in the xanthine oxidase allosteric center -one histidine, one serine, two tyrosine and two phenylalanine residues [2].We have also established that xanthine oxidase dependent superoxide production strongly decreases in the reaction systems with genistein (fig.4).The inhibiting effect of xanthine oxidase dependent superoxide formation may be the main base of the genistein antioxidant activity.
It has been established that genistein does not change the nitric oxide synthase activity in vitro.So the most reasonable explanation may be that genistein like many steroids and structurally related compounds [1 ] is the inhibitor of the inducible nitric oxide synthase genes expression.The aromatase inhibiting activity of genistein has been also revealed in vitro (fig.5).The inhibition is competitive according to the kinetic studies (fig.6).Therefore genistein is the direct inhibitor of aromatase as well as the inhibitor of its gene expression.The results presented demonstrate the novel data concerning the antioxidative activity and inhibiting effect of genistein on nitric oxide and estrogens formation.It was also established genistein is the powerful cytochrome P450 IAl, 1A2 and IBl inductor.

Fig. 2 .Fig 3 .
Fig. 2. Reverse dependence of the xanthine oxidase activity on the substrare concentration in reaction systems in the Lineweaver's and Burk's coordinates

The group of rats Organs Xanthine oxidase activity, nmo(/min per 1 mg of proteins Nitric oxide synthase activity, nmoi/min per 1 mg of proteins NO aerobic oxidation products, nmol per 1 mg of proteins Cytochrome P450 IAl, 1A2 and IBl level, nmol per 1 mg of protein
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