Turk J Vet Anim Sci28 (2004) 591-595 TÜB‹TAK
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
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
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
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
(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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