Turk J Vet Anim Sci28 (2004) 591-595 TÜB‹TAK Research Article
The Effect of Acute Fluoride Poisoning on Nitric Oxide and
Methemoglobin Formation in the Guinea pig
Department of Physiology, Faculty of Veterinary Medicine, Ankara University, Ankara - TURKEY Department of Physiology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon - TURKEY Abstract: To study the effect of acute fluoride poisoning on nitric oxide and methemoglobin formation, 250 mg/kg bw sodium
fluoride was applied alone and verapamil was applied together with fluoride. Blood nitric oxide (Griess reaction) and calcium levels;
hemoglobin, methemoglobin and hematocrit values; and erythrocyte counts were determined and compared with those of the
controls. After the fluoride application it was found that there was a relative relationship between the increase in nitric oxide and
methemoglobin levels and the decrease in calcium, hemoglobin and hematocrit levels and erythrocyte count. It was concluded that
the increase seen in blood nitric oxide levels as a result of the ionophore effect of fluoride could come from cNOS, as that increase
is related to the decrease in calcium amount.
Key Words: Fluoride poisoning, nitric oxide, methemoglobin, guinea pig. Kobaylarda Akut Flor Zehirlenmesinin Nitrik Oksit ve Methemoglobin Oluflumu Üzerine Etkisi
Özet: Araflt›rmada, akut flor zehirlenmesinin nitrik oksit ve methemoglobin oluflumu üzerine etkisinin belirlenmesi amac›yla, 250
mg/kg dozunda sodyum florür yaln›z ve flor varl›¤›nda verapamil uyguland› ve kontrol grubuna göre kan nitrik oksit (Griess
reaksiyonu), kalsiyum düzeyleri ve hemoglobin, methemoglobin, hematokrit de¤erleri ile alyuvar say›lar› belirlendi. Florun
uygulanmas› sonras›nda kan nitrik oksit ve methemoglobin düzeyindeki yükselme ile rölatif iliflkili olarak kalsiyum, hemoglobin,
hematokrit ve alyuvar de¤erlerinde azalma belirlendi. Akut flor zehirlenmesinde florun iyonafor etkisi ile kan nitrik oksit düzeyinde
flekillenen yükselmenin kalsiyum miktar›ndaki düflüflle iliflkili olmas›ndan dolay› cNOS kaynakl› olabilece¤i kan›s›na var›lm›flt›r.
Anahtar Sözcükler: Flor zehirlenmesi, nitrik oksit, methemoglobin, kobay Introduction
also enzyme level changes tending to increase the calcium Sodium fluoride is currently used in many areas, like metabolism, and that hypoxia forms following the as an insecticide and in anti-helmintic drugs, and most inhibition of the enzyme system in general (2).
commonly in rodenticide drugs. In smaller amounts it It has been reported that histamine is released prevents cavities in teeth, and it is used in the treatment following the stimulation of isolated rat mast cells and of osteoporosis in humans (1). Cases of poisoning with hypoxia is formed. Related to this, there are reports that organic and inorganic fluorides, which are definitely the formation of nitric oxide (NO), which is considered a considered poisonous in high dosages, are seen rather free radical, is stimulated by uncontrolled increases in histamine in the body (3), and the effect of NO increases Fluorine is an anion with a rather small molecular the permeability of the blood-brain barrier (4). On the weight, and it shows its effect on the organism by other hand, hypoxia that forms in conditions resembling combining with calcium (Ca+2) and causing a severe the effects of fluoride poisoning and increasing NO levels hypocalcemia picture. On the other hand, it is named a due to hypoxia in the circulation, and several internal and calcium ionophore, as it enhances the transport of Ca+2 external factors that enhance calcium ion transport and into cells. In studies on fluoride poisoning, it is generally thus result in intracellular Ca+2 increase are also mentioned that there are increases in calcium affinity and The Effect of Acute Fluoride Poisoning on Nitric Oxide and Methemoglobin Formation in the Guinea pig NO is a molecule that easily passes through the cell membrane, and that rapidly reacts with the ferro part of Serum from blood samples that were taken in the proteins containing heme and also reacts with oxygen, experimental tubes not containing anti-coagulants were and it is synthesized from the amino-acid L-arginine in stored at –18 ºC. In serum samples, calorimetry was used many cells of the body. Nitric oxide synthase (NOS) is to determine calcium levels using SIGMA-diagnostic kits involved in the formation of NO, and it is a de-oxygenase (587-A), while diazotization (Griess reaction) was used that is dependent on nicotine amide dinucletide phosphate when measuring nitric oxide amounts in serum samples.
(NADPH) (6,7). NOS has 2 isomers, 1 inducible (iNOS) According to the procedure, sulfanilamide was solved in and the other constitutional (cNOS) (8,9). In a study in to the concentration 1%, and N-(1-naphthyl) which the mechanism of the fluorine effect was examined ethylenediamine dihydrochloride (NEDD) was solved in the activation of NADPH required in NO formation (10) the same solvent to the concentration 1%. On the other was mentioned, and, on the other hand, it was reported hand, solutions of sodium nitrite were prepared with that extra cellular calcium was needed for the effects of concentrations between 0.25 and 0.50 µM and calibration curves were determined. Then 1.5 ml serum In this study, we took into consideration the previous samples from each of the 3 groups were taken, and 0.75 reports stating that the substances known as calcium ml of sulfanilamide solution and 0.75 ml of NEDD ionophores increase NO levels, that hypoxia causes NO solution were added to these samples. After waiting for production, and thus the body develops a defense 15 min at room temperature, absorption of the samples mechanism, and we studied the possible effects of acute fluoride poisoning on NO levels, and also methemoglobin formation and anemia that is thought to be related to thehypoxic picture following the poisoning.
Erythrocyte counts (16) and hematocrit (17), Materials and Methods
hemoglobin (18), and methemoglobin (19) values were determined in blood samples collected from all 3 groups Thirty albino male guinea pigs were used in this study, and taken into test tubes containing EDTA.
the weights of which differed between 280 and 320 g.
One week was allowed for the guinea pigs to get familiar with the environment, and during this period they werefed ad libitum.
Statistical significance was assessed using one-way ANOVA followed by the Newman-Keuls multiplecomparison test (P < 0.05) (20).
The animals were divided into 3 groups: 1 control (group I), and 2 experimental groups (groups II and III).
For the first experimental group (group II) 250 mg/kg The statistical comparison (Table) between the control (live weight) sodium fluoride was applied subcutaneously and the experimental groups showed that there were to cause acute fluoride poisoning (12) and blood samples significant decreases in erythrocyte counts and were collected from the heart 8 h later. To the other hemoglobin and hematocrit values of the experimental experimental group (group III), 7 h following the groups compared to those of the control group (P < application of sodium fluoride in the same dosage, 0.05). The decreases in erythrocyte counts were about intramuscular verapamil (isoptin), which is a calcium 50% for both experimental groups. The mean value of channel blocker, was applied at a dosage of 0.8 mg/kg methemoglobin increased in the fluoride and verapamil (live weight), and blood samples from the heart were combined group. This was statistically significant (P < 0.05) compared to the values of the control and onlyfluoride applied groups, although the increase in the fluoride applied group was about 3-fold that in the sodium fluoride was chosen as 250 mg/kg, and the control group, which was not statistically significant due waiting period after the dosage was 8 h.
to the high error values. On the other hand, there was a The NO level in animals after the application of sodium statistically significant decrease (P < 0.05) in the level of fluoride was higher than that obtained before (P < 0.05).
calcium after applying fluoride alone (Table and Figure 1).
This finding apparently supports the studies that report Using fluoride with verapamil returned the value near to histamine release following fluoride treatment (3).
that of the control group, which was not obviously Likewise, histamine is mentioned in the literature among significant. In contrast, applying fluoride alone caused a the factors that enhance NO formation (11,21-23).
significant increase (P < 0.05) in the level of nitric oxide Authors state that hypoxia also affects NO formation at a rate of 125% (Table and Figure 2) and this value (24), and, likewise, hypoxia takes place as a result of NO statistically decreased (P < 0.05) at a rate of 73% when poisoning (5). This hypoxic picture shows itself with low using fluoride with verapamil, which was statistically iron levels, and can be explained with the inactivation of lower than the value of the control group too (P < 0.05).
hemoglobin and other iron components (25), and thereare reports on anemia resulting from excess NO levels Discussion
(26). Although the stimulation of NO production as adefense mechanism of the body, following the decrease in This study aimed to determine how acute fluoride oxygen content of blood indirectly to below-normal levels poisoning occurs and the effects of fluoride on the in the anemic hypoxia picture seen when hemoglobin formation of NO. With this purpose, the dosage of levels are below normal can be considered an expected Hematological values of control and experimental groups (fluoride and fluoride + verapamil groups). Mean values were given with ±standard errors (n = 10).
a, b: Different letters in the line indicate significant difference (P < 0.05).
Figure 1. Blood calcium levels of control and experimental groups Figure 2. Blood nitric oxide levels of control and experimental groups (fluoride and fluoride + verapamil).
(fluoride and fluoride + verapamil).
The Effect of Acute Fluoride Poisoning on Nitric Oxide and Methemoglobin Formation in the Guinea pig effect, findings in studies on fluoride poisoning are Super oxide dismutase (SOD) is among the protective considered results of hypoxia in the circulation (5).
enzymes against the toxic effects of free radicals (29).
However, in this study, significant decreases in Rzeuski et al. (10) reported that SOD is inhibited in hemoglobin and hematocrit values and erythrocyte counts higher concentrations of fluorine, and also reported that seen in experimental groups relative to the control group the said enzyme is found in low concentrations in people after sodium fluoride application, and the relative living in areas of endemic fluorosis. Researchers have increase in methemoglobin values indicate an anemic stated that the formation of methemoglobin in guinea hypoxia picture. Likewise, the findings obtained with the pigs occurs at high levels compared to the low activity of measurements based on milliliters are findings of anemic super oxide dismutase, which is an enzyme that hypoxia. Of course, the uncontrolled stagnation in transforms the excess NO to nitroxyl ions (30). The high circulation, slowing down of blood in veins, and hypoxia level of methemoglobin in group II compared to the due to circulatory reasons are the inevitable final findings control group (P < 0.05) in our study can be explained by of poisoning after NO formation. In this context, after the increase originating from the suppression of SOD respiratory distress, cardiac failure and myocardial with high concentrations of fluorine. However, verapamil infarction arising from an increase in the work load of the application in the third group after sodium fluoride was heart are among the last findings of fluoride poisonings insufficient to prevent methemoglobin formation.
Likewise, Raikhlin-Eisenkraft et al. (27) reported that In this study, the hypocalcemia formed after the verapamil therapy was not successful in fluoride application of sodium fluoride is explained by the calcium poisonings. Fahey and Isaacson (31) found that calcium ionophore property of fluorine, and the calcium- channel blockers given 2 h before poisoning could result dependent increase in cNOS levels can be interpreted as in a decrease in methemoglobin formation. In our study, an expected effect of the increase in intracellular calcium the higher levels of methemoglobin in the third group as levels. Again, the increase in intracellular levels appears to compared to the control group in a statistically significant cause an inevitable increase in NO production originating manner can be explained by the application of verapamil from cNOS. Likewise, increases in blood NO levels are in the last hour, and by the failure of the reductive system found in the group II along with hypocalcemia after to operate. On the other hand, while the normal levels of NO can be explained by the sufficient levels of Ca+2channel blockers that prevent the transport of calcium, The data obtained from the third group, to which the the high levels of methemoglobin can be explained by the calcium channel blocker verapamil was applied after insufficiency of the preventive factors effective in the sodium fluoride application, support the findings of group transformation of methemoglobin back to oxy- II with decreases in blood NO levels and increases in hemoglobin following intoxication. Likewise, the high levels of methemoglobin after verapamil application, and It has been reported in studies on the mechanisms of the low levels of blood hemoglobin and hematocrit, and the effects of fluorine that intracellular calcium is erythrocyte counts in the same group in a similar and required for it to increase cyclic guanosine related fashion (P < 0.05) appear to be findings monophosphate (cGMP) levels at the cellular level, and NO has the same effect, and that intracellular calcium is In conclusions, an increase in NO levels with the required for the effects of both to take place.
ionophore effect of fluorine in acute fluorine intoxication Furthermore, the necessity of activation of NADPH for the effects of NO to take place is stated (10), and it isinteresting that NO formation is NADPH-dependent.
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