IMPACT OF FEEDING MALE RATS F 2 WITH DIFFERENT DOSES OF GERMANIUM CITRATE ON THE CONTENT OF TRACE ELEMENTS IN THEIR TISSUES AND ORGANS

It is known that the uptake and incorporation of trace elements in the organism of humans and animals is determined by a number of physiological mechanisms, including synergetic or antagonistic interactions. These interactions have been rmly established for most macroand trace elements, and they are normalized in nutrition [1–3]. The regulatory impact of physiologically active, but insuf ciently studied elements, among which germanium takes a prominent place, on organtissue distribution of other minerals, is presently actively studied in biology, medicine, and veterinary science [3, 4]. Organic formulations of macroand trace elements (nanoaquachelates) as obtained by nanotechnology methods receive special attention [5, 6]. The ISSN: 2312-3370, Agricultural Science and Practice, 2018, Vol. 5, No. 3


INTRODUCTION
It is known that the uptake and incorporation of trace elements in the organism of humans and animals is determined by a number of physiological mechanisms, including synergetic or antagonistic interactions.These interactions have been ¿ rmly established for most macro-and trace elements, and they are normalized in nutrition [1][2][3].The regulatory impact of physiologically active, but insuf¿ ciently studied elements, among which germanium takes a prominent place, on organtissue distribution of other minerals, is presently actively studied in biology, medicine, and veterinary science [3,4].Organic formulations of macro-and trace elements (nanoaquachelates) as obtained by nanotechnology methods receive special attention [5,6].The biological role of these organo-compounds and their interaction with other macro-and trace elements and their impact on their distribution in the organism and its organs are actively studied as well.In particular, our earlier research [7,8] showed that the administration of germanium citrate, obtained via the erosive and explosive ablation with electro-impulse, caused a number of biological effects in the organism of rats.This may have found its cause in the fact that germanium (Ge) has an immuno-stimulating effect, enhances transport and transfer of Ɉ 2 and ensures the decrease of hypoxia at tissue level [3,4].Certain organic forms of Ge have negatively charged oxygen ions that can scavenge free damaging hydrogen ions and minimize their damage to cells and tissues [9].A notable characteristic of organic forms of Ge is that they are removed fast with urine from the organism, which indicates its low accumulation in tissues [4,10].Different concentrations and duration of Ge intake affect physiological-biochemical processes in the organism differently, including their inÀ uence on the level of macro-and trace elements in tissues [3,6,7].Recent experimental studies on physiological mechanisms of the effect of different doses of germanium citrate on organ-tissue and systemic level [6,7,9] and preparations, including Astrogerm, Germatranol, Germavit, elaborated on the basis of this chelate complex, stimulated a profound investigation of this compound on the intake of such vital elements as Cu, Co, Mn, Fe, Zn in the organism, the results of which are reported in this article.

MATERIALS AND METHODS
The studies were conducted using white laboratory male F 2 rats, divided into one control (I) and three experimental (II, III, IV) groups, 4 animals in each.Contrary to the control group, the rats of experimental groups daily received the addition of nanogermanium citrate (NGeC), manufactured by the nanotechnology method [11,12], with drinking water, calculated as 10 (experimental group II ), 20 (III) and 200 (IV) g Ge/kg of the bodyweight.Feeding female rats of generations F 0 and F 1 with germanium citrate in the mentioned doses during their ontogenesis and pregnancy, and feeding young rats of respective groups F 1 and F 2 with milk of the germanium-fed mothers was conducted.The effect of germanium citrate on the organism of young F 2 rats was revealed at the stages of embryonic, fetal and preweaning period of development via mothers' blood and milk as well as via absorption in the digestive tract after the start of independent consumption of feeds and water.At the age of 4-4.5 months, 4 male rats from each group were decapitated after narcosis to study their internal organs.The content of Fe, Zn, Cu, Mn, Co in homogenates of tissues of liver, kidneys, lungs and femoral muscle was determined after dry ashing in the mufÀ e furnace at 450-500 °ɋ and dissolving the mineral residue in 10 % HCl.The trace elements were detected during the period of burning their acid solutions in the acetylene À ame, using an atomic absorption spectrophotometer SF-115 PC (Selmi, Ukraine) with the software for concentration calculation, as described in [13].The obtained results were statistically processed using MS Excel and determining the mean values (M), and their deviations, where standard deviation = standard error of the mean (± m SD), and the probability degree by Student's coef¿ cient (Ɋ 0.05).The obtained mean results of the experimental groups were compared against those of the control group.

RESULTS AND DISCUSSION
The analysis of the obtained results indicated unevenly directed changes in the content of the investigated trace elements in the tissues of internal organs and muscle tissue of F 2 males depending on the dose of NGeC.In particular, a higher content of Cu was 38.7 and 51.6 % detected in liver tissues of rats at (Ɋ < 0.05) and 200 (Ɋ < 0.01) g Ge/kg of the bodyweight, and the elevated Fe of 21.3 % at 10 g (Ɋ < 0.05) with the preservation of this tendency for Fe also for males receiving 20 -16.7 % and less so for those receiving 200 g -5.3 % (Figure ).
The content of Co decreased by 23.3 (Ɋ < 0.05) and 50 % (Ɋ < 0.001) in liver tissues of males receiving and 200 g.The content of Mn decreased also by and 20 % at the dose of 10 and 20 g Ge.No signi¿ cant differences in Mn content were detected in the tissues of liver, kidneys and lungs of F 2 rats with all dose rates applied, which was also noted by other researchers [2,3].It is known that Mn is found in all the tissues and liquids of the organism without considerable organ-, species-or age-related differences.The increase in Mn concentration in the liver of rats was noted at the effect of 5 pg Ge in the form of sodium germanate [3].There was a con¿ rmed impact of this compound on the mineral exchange via functioning of the main regulatory enzymes, activated by Mn -hydrolases, kinases, decarboxylases.
In the processes of absorption from the intestines, Mn competes with Co for the binding sites, whereas the mechanisms of absorption of Mn and Fe are similar and there is no competition [2].The differences in Co content in the tissues of internal organs, noted for F 2 rats, were also established for male F 1 rats at the age of 2-2.5 months.In particular, the content of Co in liver tissues of male F 1 rats at the doses of 20 and 200 g Ge was 40-50 % lower as compared to the control group [7].The mentioned regularity of Co content in liver tissues of F 1 and F 2 rats at the effect of these doses of NGeC in male F 1 rats was also preserved for Co content in lung tissue of animals of generation F 2 .In particular, the effect of germanium was revealed in the decrease by 40.3 and 70.4 % in the content of Co in lung tissues of rats of groups II (P < 0.05) and III (P < 0.01) and the decrease by 32 % -group IV.
A signi¿ cantly lower content of Co (by 29.4; 50.3 and 39.2 %) was found in the tissues of kidneys of male F 2 rats of all the experimental groups, Cu and Feat the effect of 10 and 20 g Ge at the background of a higher level of Mn (Ɋ < 0.001) in groups III and IV and Cu (Ɋ < 0.01) and Zn (Ɋ < 0.05) in male rats of group IV, which received 200 g Ge.NGeC had a more visible inhibiting effect on the content of Fe and Co in the tissues of kidneys and on the content of Co in liver tissues at the effect of 20 and 200 g Ge, and as for Mn -10 and 20 g Ge in liver, which may impact the hematopoietic ability of the organism of male F 2 rats as follows: it is known that Co enhances the intake of Fe and synthesis of hemoglobin, stimulating erythropoiesis.Co negatively affects the synthesis of proteins and repair of S-S group sulphur bridges, that operate in the processes of blocking and detoxi¿ cation of poisonous elements in the organism [2,3].
A less clear regulatory effect of NGeC compared to the liver tissue on the content of the investigated elements was noted for the tissues of lungs, but the content of Zn and Cu in lung tissue was also 66.9 and 175 % higher (P < 0.05; P < 0.01) for male rats of group IV.It is remarkable that the content of Cu in lung tissue of male rats of group IV was 27.5 % higher (P < 0.01) against the control compared to the tissues of liver, kidneys and muscles.It may indicate a stimulating effect of NGeC in the dose of 200 g Ge on the intensity of Cu metabolism in the organism and as a result, the level of this microelement in the internal organs and muscles of rats.
The increase in the content of Cu in the tissues of liver, kidneys, lungs and muscles of rats may condition enhanced redox processes and supply of these tissues with O 2 , which was also noted by other researchers [3,14,15,16].It is known that a high level of Cu in the tissues of the organism stimulates the processes of antioxidant protection with the participation of its incorporation into enzymes involved in these processes.
The content of trace elements in the tissues of femoral muscle of rats was found to be 2-10 times different from that of the tissues of internal organs.For instance, a higher content (by 27.7; 74.0 and 23.4 %) of Mn for males of all the experimental groups was noted in the The content of trace elements in the tissues of male F 2 .Note: Statistically signi¿ cant differences from the control group I in samples of muscle tissues at the effect of low (10 g), medium (20 g) and high (200 g) doses of Ge, and Zn by 56.3 and 46.7 % -10 (P < 0.001) and 200 g (P < 0.01), see Table .The content of Fe was also considerably higher in the muscle tissues at the effect of 10 and 20 g Ge, and as for Cu -200 g (P < 0.01).
The absolute content of the trace elements Cu, Co, Mn, Fe and Zn was also studied in the liver, kidneys and lungs in relation to the weight of these organs.The weight of the organs, except for kidneys in group IV, in males of all experimental groups showed a tendency to decrease in the range from 7.3 to 22.6 % which was statistically signi¿ cant at least for liver in group II (P < 0.01) and III (P < 0.05), and for lungs in group III (P < 0.05) (Table ).In particular, a lower content (from 35.7 to 56.7 %) of Co was found in the liver of rats in groups III (P < 0.01) and IV (P < 0.001) and for Mn in groups II and III (P < 0.001).To the contrary, a higher level of Zn was found in animals of group IV (P < 0.05).The absolute content of Cu in the liv-er of male rats of group IV increased from 34.9 % (P < 0.05).
The absolute content of the investigated trace elements in the kidneys of rats mainly preserved the direction of differences between the control and experimental groups relative to their level in mg/kg of the weight of the tissue (Figure).The absolute content of Mn in the kidneys of males of group II at the effect of a low (10 g) dose of NGeC, however, was 15.5 % lower as compared to that of the control.It may be conditioned by the impact of lower indices of Mn content in these tissues and a smaller weight of this organ by 7.3 % in rats of group II.The absolute content of Mn in kidneys of males of groups III and IV was 47.8 and 54.8 % higher respectively (P < 0.001) at an insigni¿cantly increased level (by 10.7 %) of the weight of this organ as compared to that of the control.A statistically signi¿ cant higher absolute content of Mn in the kidneys of males of group III and IV was demonstrated at the expressed impact of 20 and 200 g Ge of NGeC The absolute content of trace elements in some internal organs of male F 2 rats at the age of 4 months, (mean, M and standard deviation, ± m; number of rats, n = 4)

Groups
Organ on the increase in Mn content in kidneys.This effect is also con¿ rmed by the increase (P < 0.001) in Mn content in the tissues of kidneys (in mg/kg) of males from groups III and IV (Figure).
Statistically signi¿ cant differences in the absolute content of the investigated trace elements were preserved in the lungs of rats of experimental groups similarly to those as per one unit of tissue weight in mg/kg, presented in Figure .The detected changes demonstrated that the absolute content of Mn in the lungs of group II males showed a signi¿ cant decrease similarly to the liver and kidneys.It was conditioned both by the lower level of this element in the lung tissue and by the tendency to the decrease in the weight of this organ (12.1 %) compared to the control.However, a lower index of the weight of lungs in animals of group IV had no considerable impact on the signi¿ cant increase in the absolute content of Zn in this organ, as the content of Zn in the lung tissues of rats of this group was 46.9 % higher (P < 0.01).It should be noted that a high (200 g) dose of Ge conditioned a signi¿ cant increase in the absolute content of Zn in the liver, kidneys and lungs, Cu -in the kidneys and lungs, Mn -in the kidneys.

CONCLUSIONS
Thus, the introduction of germanium citrate for 120-135 days, obtained via electric impulse ablation and its administration with drinking water in the amounts of 10, 20 and 200 g Ge/kg bodyweight into F 2 rats is characterized by the changes in the content of Cu, Co, Mn, Fe, Zn both per unit of soft tissue weight and their absolute content in the entire internal organs.The biological effect of germanium citrate is more expressed in the high dose of 200 g Ge/kg than in the lower doses conditioning the increase in the content of Cu from 51 to 95 % and Zn from 22 to 78 % in all the investigated tissues of rats.There is a decrease of Co by 40.3 % in the liver at 20 and by 70 % at 200 g Ge, and at all the administered doses from 27 to 50 % in kidneys and 33-310 % in lungs.The level of Mn increased from 23.4 to 74 % respectively in the muscle tissues of male F 2 rats at all administered doses of Ge, for Fe it was respectively for the doses of 10 (P < 0.001) and 20 g (P < 0.05), and ¿ nally for Zn respectively for the doses of 10 (P < 0.001) and 200 g Ge (P < 0.01).There was a statistically signi¿ cant increase in the content of Cu and Zn in all the investigated tissues and organs of F 2 rats at the effect of 200 g Ge, which may indicate enhanced accumulation of Cu and Zn in the organism of rats at long-term (F 0 -F 1 -F 2 ) intake of germanium citrate with drinking water.