RELATIONSHIP BETWEEN CELLULAR ADENINE NUCLEOTIDE CONCENTRATIONS IN ERYTHROCYTES AND SERUM FLUORIDE LEVELS IN TWO BREEDS OF SHEEP

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1 60 60 RELATIONSHIP BETWEEN CELLULAR ADENINE NUCLEOTIDE CONCENTRATIONS IN ERYTHROCYTES AND SERUM FLUORIDE LEVELS IN TWO BREEDS OF SHEEP Maria Suska, a Mariola Janiak Szczecin, Poland SUMMARY: The relationship between cellular adenine nucleotide (ATP, ADP, AMP) concentrations in erythrocytes and fluoride (F) in serum was studied in 20 Polish Merino sheep and 18 Blackhead Mutton sheep living in an industrial environment contaminated by F compounds. The influence of F contamination on the total adenine nucleotide (TAN) content and adenylate energy charge (AEC) of red cells was determined. The concentrations of F in serum as well as ATP, ADP, AMP, TAN contents, and AEC of erythrocytes were also determined. Compared to results from 24 control sheep living in a relatively uncontaminated environment, a significant increase in ATP concentration (p 0.01) and a significant increase in AMP concentration (p 0.01) in both breeds of sheep were observed. The values of AEC of erythrocytes in both breeds were significantly lower (p 0.01). A linear negative correlation (r = 0.719) was found between the erythrocyte ATP concentration and serum F levels in the Blackhead Mutton breed, and a similar negative correlation (r = 0.522) was found between the TAN level and that of serum F. Moreover, in both breeds of sheep, there was also a negative correlation between AEC and the concentration of F in serum (r = and r = 0.468, respectively). Keywords: Adenine nucleotides; Adenylate energy charge (AEC); Blackhead Mutton sheep; Erythrocytes; Fluoride pollution; Polish Merino sheep; Serum fluoride; Total adenylate nucleotides (TAN); Sheep. INTRODUCTION Studies on erythrocyte energy metabolism conducted under various conditions in vivo confirm that the fluoride ion (F) is strongly reactive due to its high negative charge density. 1,2 F may alter the distribution of electrons in the molecules of various compounds, including enzymes and membrane transport proteins, thereby affecting the metabolism of many cells, tissues and organs. 3-5 Usually, the F concentration in blood is low because F is excreted by the kidneys and accumulated in the bones. 6,7 However, when the concentration of F in urine and blood reaches twice the value of control populations, one may observe the inhibitory influence of F upon the magnesium-dependent enzymes of the glycolytic process and the pentose phosphate pathway Inhibition of glycolysis regulatory enzyme activity may result in changes in ATP, ADP, and AMP concentrations, total adenine nucleotide (TAN) level, and adenylate energy charge (AEC) in erythrocytes. The aim of this study was to determine the influence of F ion concentration in serum on the erythrocyte levels of ATP, ADP, AMP, and on the concentration of TAN and AEC levels in sheep living in an environment contaminated with fluorides. Two different breeds of sheep living under the same conditions were studied to determine which was more sensitive to equivalent levels of F in the environment. We wanted to know if there were any genetically conditioned differences that influence erythrocyte sensitivity in animals of the same species. a Dr hab. Maria Suska, University of Szczecin, Al. Piastów 40B St., Szczecin, Poland. E- mail: suska@univ.szczecin.pl

2 61 61 Despite the existence of a large amount of data concerning the influence of F compounds on erythrocyte metabolism in various animal species and humans living in a contaminated environment, 2,11-14 no comparative studies appear to have been carried out on the relation between F concentration in serum and the level of organic adenine nucleotides in erythrocytes within different races or breeds of the same species. MATERIALS AND METHODS The present study was conducted in October The sheep were 2 3 years old and belonged to two breeds (Polish Merino and Blackhead Mutton). The F- exposed animals (38 in all) came from private farms situated in an area of high industrial F contamination (1 2 km away from the Police chemical plant). This plant annually releases some 46.5 metric tonnes of F compounds into the atmosphere. 15 In veterinary studies the sheep demonstrated various degrees of fluorosis due to their absorption of F not only from the air or water (0.2 mm F), but also from F-contaminated fodder originating from F-polluted fields. 15 The control group comprised 24 sheep of both breeds, living in an area considered relatively clean ecologically situated 150 km away from the contaminated area. Potassium ion (K + ) concentration in erythrocytes did not exceed 27 mmol/l, since the sheep belonged to the LK genetic variation (low potassium level and high sodium concentration in erythrocytes). 16 Blood was extracted in the morning hours from the exterior jugular vein into two test tubes (in different volumes). Seven ml of blood was collected into a heparinized test-tube (Heparinum Polfa 250 JU) and 5 ml (for clotting) into a plastic test-tube. The blood samples were transported to the laboratory in a thermos flask with ice and analyzed immediately. The purine nucleotide and F ion concentrations were determined and calculated using procedures described previously. 14 Results are expressed in SI units compared statistically with Student s t-test as well as the Mann-Whitney test (Statistica v.5.1 software). Spearman s rank correlation coefficient (r) was determined for F levels in serum vs. erythrocyte parameters in each study group. Significance levels of 0.05 and 0.01 were used to accept or discard hypotheses derived from the analytical data. RESULTS In both groups of sheep exposed to the action of F a significant decrease in erythrocyte ATP concentration (p 0.01) and a significant increase in erythrocyte AMP concentration (p 0.01) compared to control groups were observed (Figure 1a and 1c). The TAN concentration (sum of [ATP], [ADP], and [AMP]) in erythrocytes of sheep exposed to F was significantly lower in the Blackhead Mutton sheep (p 0.01) than in the control group, whereas no differences were observed in the Polish Merino sheep (Figure 1d). The level of AEC shown in the equation below 14 was significantly lower (p 0.01) in both groups (Figure 1e). ACE = ½ {[ADP] + 2[ATP]} [TAN]

3 62 62 Legend: mean ± SD 95% confidence interval of mean Figure 1. ATP (Figure 1a), ADP (Figure 1b), and AMP (Figure 1c) concentrations and calculated TAN (Figure 1d) and AEC (Figure 1e) in erythrocytes in Polish Merino and Blackhead Mutton sheep in the control group (C) and the F-exposed groups. As seen in the Table, the serum F concentration of both exposed groups was significantly higher (p 0.01) than in the corresponding controls. Table. Concentration of serum F (µmol/l) in Polish Merino and Blackhead Mutton sheep in the F-exposed and control groups Group N SD Polish Merino F-exposed group * 0.82 control group Blackhead Mutton F-exposed group * 0.58 control group *Compared to control, p 0.01.

4 63 63 In the Polish Merino sheep, the serum F concentration in the exposed group was nearly 8.0 µm, whereas in the control group it was 6.24 µm. In the Blackhead Mutton sheep, it was just over 7.0 µm, and in the control group it was 5.5 µm. Although plots of the data are not shown, a linear negative correlation (r = 0.719) between the erythrocyte ATP concentration and the serum F concentration was observed in the Blackhead Mutton group. A similar correlation (r = 0.522) was found between the TAN level and the serum F concentration in this group. On the other hand, no such relationships were observed in the Polish Merino group, which exhibited a positive correlation (r = 0.671) between the ADP concentration and the serum F concentration. However, the AEC level and serum F concentration correlated negatively in both groups (r = in the Polish Merino sheep and r = in the Blackhead Mutton sheep). DISCUSSION Adenine nucleotides play a key role in the energy metabolism of cells. Levels of ATP, ADP, and AMP reflect the rate of energy transformations in erythrocytes, fueled mainly in the glycolytic process and the pentose phosphate pathway. Their concentration in the cells is influenced by various physiological and toxicological factors. 8,19,20 This study involved LK type sheep (low serum potassium level) in which polymorphism HK/LK is determined by one autosomal gene with two alleles, and where the LK allele is dominant. 16 The erythrocytes of the sheep have a very low K + /Na + ratio (not exceeding 1), a genetically conditioned low ATP concentration, low Na +, K + -ATPase activity and a few times higher than normal K + /Cl cotransporter activity. 21 ATP-dependent mechanisms regulating membrane transport and high K + /Cl activity enable a rapid loss of electrolytes and the release of water from a cell. Therefore, it seems likely that LK sheep may be more sensitive to the toxic influence of F than animals of the HK type. The LK type may therefore be a better population for environmental studies, as they have lower activity values of various enzymes, both in membrane transport and intracellular metabolism. 16,21 The significant increase in serum F concentration observed in both breeds of sheep resulted in significant changes in adenine nucleotide content, especially ATP and AMP. A lower concentration of these nucleotides decreased the value of AEC in both breeds. It also seems likely that the increase in serum F concentration inhibits magnesium-dependent enzymes in glycolysis and hence affects energy metabolism balance. Therefore the significant decrease in AEC may be a good indicator of toxin induced disorders of intracellular erythrocyte homeostasis. In conclusion, Polish Merino sheep appear to be more sensitive to the toxic effects of F than Blackhead Mutton sheep, suggesting a connection with an LK type animal. Such breeds are probably better avoided at stock-farms close to areas where high levels of toxins like F are present.

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