Xanthine oxidase activity regulates human embryonic brain cells growth

Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22) in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90). XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p ≤ 0.05). On day 12 the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96) and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05). In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.


Introduction.
Neuronal death during the processes of proliferation and cell migration is pronounced in embryonic developing brain [1].
It was demonstrated that in chick embryos, about 45 % of the retinal ganglion cells are lost between E10 and E16 [2], whereas isthmo-optic nucleus loss is about 55 % between E12 and E17 [3].Neuronal death occurs at the time of the formation of connectivity with other neurons, during the development of the synapses.
The mechanism of reactive oxygen species production might be mediated due to the activity of the Xanthine Oxidase (XO; EC1.1.3.22).Activity of this enzyme might be similar at the number of diseases, including myocardial ischemia, stroke, chronic heart failure, hypertension, hypercholesterolemia, atherosclerosis, diabetes etc.
Furthermore, an involvement of XO in cellular proliferation and differentiation has been suggested [4].Moreover, Moriwaki et al. demonstrated the presence of XO in brain, as well as in other organs, by the utility of immunochemical methods [4].
Taking into account that XO is responsible for the ROS formation its involvement into the delayed recovery of injured nerves in old rats as well as into tissue repair might be suggested [5].
We have proposed that XO might play undoubtedly important role during the cell growth, development, maturation as well as in death of human brain derived cell culture.
Materials and methods.Trypan Blue staining.A cell suspension was prepared in BSS (Hank's Balanced Salt Solution, Product No. H9269, «Sigma», USA).After all it was transfered 0.5 ml of 0.1 % Trypan Blue solution to a test tube and 0.3 ml of BSS was added to 0.2 ml of the cell suspension (dilution factor = 5) and mixed thoroughly.The cell suspension-Trypan Blue mixture was kept for at least 5 min [6].The cultured cells were trypsinized with 0.25% trypsine solution and collected from the plates.Cells were stained with the 2% Trypan Blue dye for visualization of the viable/non viable cells.
Cell culturing.All procedures with the utility of biomaterials were carried out in accordance with the Declaration of Helsinki.Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion.Moreover, only fetuses with the age of formation no more than 12 weeks were used as a biomaterial [7].
Xanthine Oxidase activity estimation by determination of the uric acid quantity in the brain tissue [9].Xanthine and Allopurinol were incubated with the biological solution for one hour at 37 °C, after the Specol 2000 estimated all the absorption at 660 nm.
Homogenization of the human embryos brain.For 100 ml of the buffer 0.87 g NaCl, 0.06 g KH 2 PO 4 , 0.09 g Na 2 HPO 4 , 5 mM MgCl 2 , 0.1 M Tris aminomethane, 1 ml of Triton X 100, 200 ug of Trypsine inhibitors, 0.001 M KNaC 4 H 4 O 6 ´ 4H 2 O were added.Glassglass homogenization was performed during 20 min.The mixture was centrifuged at G = 8000 for 20 min.Supernatant was used for the experiments.Detection and quantification of proteins by Bradford.The protein content in samples was determined in the Specol 2000 (Poland) at 590 nm [10].
Statistics.We have used t-test (student) for pair comparison as well as ONE-WAY-ANOVA for estimation of the significance.The results were considered statistically significant when p was lower or equal to 0.05.
Results and discussion.The influence of Allopurinol on the activity of XO.We have measured the XO activity in the wide range of human embryos brains to determine whether there is any correlation between the activity and the age of the latter.According to our results there is no any difference.The activity of XO, disclosed as the formation of total uric acid in the solution, was suppressed by 40 % (Fig. 1).The black column represents an effect of Allopurinol whereas the grey one -the total activity of XO (0.12 ± 0.02; 0.20 ± ± 0.03 respectively, p = 0.05).
The second series of experiments devoted to examination of the XOR inhibitor effect in the cells culture.We used two types of XO inhibitor -Allopurinol.The cells obtained from human E12 embryonic brain were treated with Allopurinol in different concentrations (high and low) as well as in different time points ( 6 th days -as the first period and 6 th to 12 th days -as the second period) to delineate the functional properties and the role of XO activity in the development and survival of the brain cells.
The results were very interesting.Allopurinol in low concentration during the first period of time did initiate the growth of cells (2350.1 ± 199.0), whereas during the second time period the same concentration of the inhibitor had the opposite influence and significantly reduced (1479.6± 103.8) the number of brain cells in the culture in comparison with the Control (2123 ± 96, p < < 0.05) and low dosage treatment during the early period (p < 0.05).The high concentration of Allopurinol had no significant effects (1907.3 ± 194.4; 1992 ± 43) in comparison with the Control group (Fig. 2, see inset ).
It is interesting that all concentrations of Allopurinol at any stage of the cells growth and development decreased the cell death in comparison with the control in statistically significant way (control -3538.33 ± 356.61; early stage of the treatment: low concentration of Allopurinol -2385.20 ± 389.13, high -1033.67±235.15; late stage of the treatment: low concentration of Allopurinol -389.80 ± 66.80, high -876.67± 221.78; p < < 0.05 between all groups vs Control (Fig. 3, see inset).
Our experimental results demonstrated the XO presence in the developing human embryos brain.In accordance with the known data and our results its activity might be inhibited by Allopurinol.
It is well known that XO might initiate the formation of synapses and connections between the neuronal cells [4] and introduce regenerative or developmental activities.
On the other hand it might serve as a source initiating cells' death [11].
Thus, the same enzyme -XO -has a bifacial functional activity -death and proliferation.
In our experiments partial suppression of the XO activity and ROS generation during the early stage of cells growth protects the culture, whereas at the late stages, possibly, activity of the same enzyme and formed products trigger more effective cell development, axonal and dendritic ends outgrowth, formation of the synapses and differentiation.
Further experiments are necessary for detailed delineation of the above-mentioned phenomena.periments and the entire work were performed on the basis of ANSEF-2381 AWARD support.Also, we are thankful to Prof R. A. Abramyan for the providing of the biological material.

Fig. 1 .
Fig. 1.Activity of XO in the human embryos brain.Grey column represents [XO] activity in the brain (n = 16); black column points to the suppressive abilities of Allopurinol.Here are represented standard errors of the mean and p < 0.05 calculated by t-student test