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Plasma fluoride concentrations in rats acutely poisoned with sodium fluoride
TOXICOLOGYAND APPLIED PHARMACOLOGY37, 75-83 (1976)
Plasma Fluoride Concentrations in Rats Acutely Poisoned with Sodium Fluoride OFELIA H . DE LdPEZ, 1 FRANK A . SMITH, AND HAROLD C. HODGE 2
Departments of Pharmacology and Toxicology, and Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received September 9, 1975; accepted March 10, 1976 Plasma Fluoride Concentrations in Rats Acutely Poisoned with Sodium Fluoride. DELdPEZ, O. H., SMITH,F. A., ANDHODGE,H. C. (1976). Toxicol. Appl. Pharmacol. 37, 75-83. Female rats of average body weight 250 g proved to be approximately twice as susceptible to the acute toxic effects of sodium fluoride given po as were female rats weighing on the average 80 or 150 g. The 24-hr oral LD50 values were 31, 54, and 52 mg of F/kg, respectively. Following single doses of 25 mg of F/kg for 250-g rats or 50 mg of F/kg for rats weighing 80 or 150 g, absorption of fluoride from the gastrointestinal tract was approximately 50 ~ complete in less than 90 min for the 250-g rats and in about 2 hr for the 80- and 150-g animals. In treated rats given 50 mg of F/kg, plasma fluoride concentrations (normally 0.3 pg/ml or less) increased to 10/tg/ml or more in 15 min in 80- or 150-g rats. Quantitatively similar increases were noted in 250-g rats given 25 mg of F/kg. Plasma fluoride concentrations of 8-10/tg/ml or higher were often associated with death, regardless of dose or body weight (age), but were achieved more rapidly in the heaviest rats. In surviving rats, plasma fluoride concentrations declined rapidly from maximal values reaching concentrations at 24 hr of 3 and 0.3/~g/ml, respectively, for the 150- and 250-g rats, and of approximately 4/tg/ml at 8 hr for the 80-g rats. Plasma fluoride concentrations after near lethal doses were dose-dependent. The greater resistance of the 80- and 150-g rats to the lethal effects of fluoride may reflect the greater efficiency of the younger skeletons in removing fluoride from the circulation. In spite of long acquaintance with acute fluoride poisoning, there is still uncertainty as to the relative importance in the lethal process of the several fluoride actions. These may include interference with the role of calcium, inhibition of enzymes, induction of shock and electrolyte imbalance due to loss of fluids by vomiting and diarrhea, and vascular or respiratory depression. Presumably, these actions can be related to fluoride concentrations in the circulating plasma, concentrations which are the resultant of several processes including absorption from the gastrointestinal tract and removal from the blood by the skeletal system and by the kidneys into the urine. The importance of these processes in governing the magnitude of the circulating plasma fluoride elevation is suggested in the observation of Maynard (1949) that intraperitoneally injected 1 Present address: Ministerio de Sanidad y Asistencia Social, Division de Higiene de Los Alimentos, Edificio Sur Oficina 417, Centro Simon Bolivar, Caracas, Venezuela. 2 Present address: Pharmacology Department, University of California, San Francisco, CA 94143. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
75
76
DE LOPEZ, SMITH AND HODGE
NaF is approximately twice as toxic for rats weighing 200-300 g as it is for 100-200-g rats. The younger animals may have a much greater skeletal availability and a corresponding rapidity of fluoride storage, which lower plasma fluoride concentrations more effectively if absorptions were comparable. It is the intent of the experiments described here to confirm the lower toxicity of NaF for young rats, and to seek an explanation by examining (a) the plasma fluoride concentrations associated with the LD50 for young and old animals, and (b) absorption of fluoride from the gastrointestinal tract. METHODS Three populations of female Sprague-Dawley white rats were used 3 of mean body weights and ranges 80 (50-108), 150 (l 12-184), and 250 (200-359) g, respectively. Prior to and following dosing, the animals were maintained on Purina Rat Chow which contained approximately 39 ppm of fluoride, and on fluoridated-tap water containing approximately 1 ppm. The LD50 for each population was determined in groups of 8-15 rats per dose that had been fasted 24 hr and were then given NaF in aqueous solution (0.2-1.6 ml/dose) by stomach tube. At least seven dose levels were used for each population. The 24-hr LD50 values were calculated according to the method of Litchfield and Wilcoxon (1949). Blood samples were taken at various time intervals by heart puncture under chloroform anesthesia; the plasma, separated in a refrigerated centrifuge, was frozen until analyzed. Heart blood samples from dead animals were removed as promptly as possine (never more than 30 min after death). Initially, plasma fluoride was measured by the diffusion method of Taves (1968) as modified by Hall et al. (1972a), and later by the known single-addition method of Bruton (1971) as adapted by Hall et al. (1972b). Absorption of fluoride from the gastrointestinal tract was measured indirectly by subtracting from the initial dose the amount of fluoride remaining at various times after administration. At preselected intervals, the rats were anesthetized with chloroform, the stomach, small and large intestines removed, and analyzed separately. The tissues were burned to ashes with calcium oxide fixative, the fluoride separated from the ash by steam distillation, and titrated in an aliquot of the distillate using thorium nitrate and alizarin indicator (Smith and Gardner, 1955). RESULTS The LD50 for the 80-g rats was 54 mg/kg, with upper and lower confidence limits of 54 and 49 mg/kg, respectively; the slope function was 1.26 (upper and lower confidence limits, 1.35 and 1.18, respectively). For the i150-g rats, the LD50 was 52 mg/kg with upper and lower confidence limits of 57 and 48 mg/kg; this LD50 did not differ significantly from that of the 80-g rats. The slope function was 1.32 with confidence limits of 1.52 and 1.15. The LD50 for the 250-g rats was significantly lower, being 31 mg/kg with confidence limits of 38 and 25 mg/kg. The slope function in this instance was 1.73, with confidence limits of2.13 and 1.40. Female rats approximately 30- to 45-days-old (80 or 150 g) were equally resistant compared to rats 90-days-old (250 g). These results are 3 Obtained from Blue Spruce Farm, Inc., Gardner Road, Altamont, N.Y. 12009.
PLASMA FLUORIDE IN ACUTE POISONING
77
TABLE 1 CUMULATIVE PERCENTAGE MORTALITIESOCCURRING WITH TIME IN RATS RECEIVINGDOSESEQUAL TO OR LARGER THAN THE LD50 Interval
Population according to average body weight
(hr)
80 g
150 g
250 g
0-1
11.4
--
12.5 27.5 50.0 75.0 77.5 77.5 77.5 100.0
1-2 2-3 3-4 4-5 5-6 6-7 7-24
11.4 34.2 39.9 48.6 51.5 71.5 100.1
2.3 6.9 13.8 18.4 27.6 32.2 99.7
similar to M a y n a r d ' s (1949) finding for ~NaF given ip; L D 5 0 values were 21 m g o f F / k g for 100-200-g versus 11 m g / k g for 200-300-g female rats. R a p i d i t y o f d e a t h is characteristic o f fluoride (Table 1), the older rats dying m o r e r a p i d l y t h a n the y o u n g e r ones. W h e n doses equal to o r greater t h a n the L D 5 0 were administered, h a l f o f the 250-g rats died within 3 hr whereas only a third o f the 150-g rats died within 7 h r a n d h a l f o f the 80-g rats within 6 hr. To determine p l a s m a fluoride concentrations associated with the oral LD50, g r o u p s o f the three p o p u l a t i o n s o f rats were fasted; the 80-g a n d 150-g rats were given 50 m g / k g a n d the 250-g rats 25-30 m g o f F / k g , all po. G r o u p s o f f o u r rats were sacrificed at selected time intervals (Table 2) a n d b l o o d samples were t a k e n f r o m each rat for fluoride TABLE 2 PLASMAFLUORIDE (/tg F/ml) IN RATS SACRIFICEDAT VARIOUSTIMES AFTERRECEIVINGN a F BY STOMACH TUBEa Average body weight (g):
80
150
250
Dose (mg of F/kg):
50
50
25
Hours to sacrifice
Mean _+ SE
Mean + SE
Mean + SE
Control 0.25 0.5 1 1.5 2 3 4
0.27 + 0.011
8
12 24
10.5 + 0 . 8 7.1 + 0 . 9 5.1 + 0.3 4.7
+0.7
2.5 +0.3
a Each value is the mean of three or four rats.
0.21 9.1 10.9 8.3 10.0 10.0 10.2 6.9 6.8 6.4 1.8
+ 0.022 + 0.94 + 2.39 ___1.1 + 1.4 +0.8 + 1.6 + 1.2 +0.4 + 1.6 + 0.3
0.09 + 0.014 9.8 + 3.05 7.6 + 1.15 7.2 +0.33 5.2 + 1.25 5.9 + 1.28 7.64 + 0.98 4.3 + 1.6 1.5 +0.16 1.6 +0.08 0.25 + 0.007
78
DE LOPEZ, SMITH AND HODGE
analysis. Mean plasma F concentrations rose rapidly, e.g., to 9-10 #g/ml in 15 min. Mean concentrations during the first 1.5 hr consistently reached 5-11 #g/ml with individual samples as high as 14-16 #g/ml in these surviving rats. During the first 3 hr, the average plasma concentrations were 5 #g/ml for the 80-g rats, 10 #g/ml for the 150-g rats, and nearly 8 #g/ml for the 250-g rats (given half the dose). Plasma fluoride values were generally lower after 4 hr. Although no consistent rate of decrease appeared, in only one rat did a plasma fluoride value exceed 9 #g/ml at 4 hr. The 80- and 150-g rats tended to maintain plasma concentrations of 5 to 10 #g/ml from 3 to 12 hr, whereas the plasma samples of the 250-g rats contained 1.6 #g/ml or less from 8 hr on. Additional data on the lethal plasma fluoride concentration were gained from analyses of the blood of seventeen 150- and 250-g rats dying from these doses. In 6 of the 17, plasma fluorides were 8-10 #g of F/ml, and in the other 11 were 12-19 #g of F/ml, as shown in Table 3. None of the 80-g rats died. A certainly lethal threshold for plasma concentrations was not established. TABLE 3 PLASMA FLUORIDE CONCENTRATIONS IN RATS FATALLY POISONED BY ORAL DOSES OF SODIUM FLUORIDE
Average body weight Dose (g) (mg of F/kg)
250
25 30
150
50
Number of rats dying with plasma F concn, in the indicated range 8-10/~g/ml I/4 2/7
Totals
3/6
12-19 ,ug/ml 3/4 5/7 12-16 #g/ml 3/6
6/17
11/17
Evidence was sought of a dose dependence for plasma fluoride concentrations by giving groups of fasted rats of each age single oral doses of (a) approximately the respective LD50, (b) ~, and (c) 3 of these amounts. Plasma fluoride concentrations measured 3 hr later increased linearly with the logarithm of dose (Fig. 1). The data for the 250- and 150-g rats followed parallel lines; the older rats given half the dose showed lower plasma fluoride values as expected. Plasma fluoride values for the 80-g rats were about half those for 150-g rats and the increase with dose was also less marked. The percentages of the doses remaining in the stomach, in the small, and in the large intestine at successive time intervals after dosing rats orally with 25 or 50 mg of F/kg are shown in Table 4. The percentages remaining at each time interval are shown graphically in Fig. 2A. The absorption patterns of the three populations did not differ greatly; the stomachs of the heaviest rats may have emptied more rapidly. Absorption is rapid; 50 ~ of the dose was absorbed in approximately 2 hr or less and absorption is practically complete within 24 hr. Fluoride did not accumulate in the lower segments of the digestive tract, indicating neither impairment of passage through the gastrointestinal tract, nor serious interference with uptake of fluoride by the intestinal epithelium.
79
PLASMA FLUORIDE IN ACUTE POISONING • 12-
/"
"- 10-
.~4-
'
0
i 15
10
I i i i 25 35 40 50 DOSE, mgF/kg BODYWEIGHT
FIG. 1. Dose dependence of plasma fluoride concentration. • 150 g average body wt; • • , 250 g average body wt.
i 70
• , 80 g average body wt; •
I00:
A
~0
{
0
2
4
6
~ i0 12 14 ~6 j8 ~0 5
~4
HOURS FIG. 2. (A) Percentage of dose remaining in the gastrointestinal tract after fluoride administration po. A single line has been fitted visually to the points. • • , 80 g average body wt, 50 mg of F/kg; • • , 150 g average body wt, 50 mg of F/kg; • o, 250 g average body wt, 25 mg of F/kg. (B) Plasma fluoride concentrations. A single line has been fitted visually to the data for the 80- and 150-g rats, and another line to the data for the 250-g animals. Symbols as before.
Plasma fluoride concentrations (Table 4) measured at the same intervals at which gastrointestinal absorption was determined, are shown in Fig. 2B. Taken together, the data indicate that in the two younger populations, an initial rapid absorption of fluoride
80
DE L6PEZ, SMITH AND HODGE
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PLASMA FLUORIDE IN ACUTE POISONING
81
promptly produces high plasma fluoride concentrations. The F concentration soon declines as the absorption approaches 100 700,and as urinary excretion and skeletal deposition remove fluoride from the plasma (Hodge and Smith, 1965). For the heaviest rats, plasma fluoride continued to rise at least through the first 3 hr, after which it declined as the increments of absorbed fluoride became smaller. Gross examinations of the gastrointestinal tracts 3 hr after dosing showed the stomachs to be distended with a yellow liquid and with gas, and the intestines to be swollen with liquid, presumably in response to local irritation and osmotic stress. The pH of the gastric fluids of the fluoride-treated rats was about 8, as was also the p H of the fluoride solution, whereas the gastric fluids in control rats and in rats given a NaC1 solution (pH 4) of the same osmolality had a pH of about 2. The microscopic appearance of sections of the gastrointestinal tracts of two rats given 50 mg of F/kg po was not different from that seen in rats receiving the NaC1 solution.
DISCUSSION The lethal doses of N a F given either po or ip for older (heavier) rats are about half those for younger rats. The two important means whereby fluoride in the organism is "detoxified" are (1) excretion into the urine and (2) deposition in the bone. Inasmuch as the oldest rats were only 90 days of age, the kidneys in all the groups were surely functionally normal. Deposition in the skeletal systems may have been less efficient in the oldest rats either because less mineral is "available" to the circulation or because the mineral is less reactive with fluoride. The latter possibility is doubtful: Ericsson (1966) found no relation between preexisting femur F concentrations and the uptake of lSF in small single doses. The log dose-mortality curves for N a F in rats characteristically have steep slopes. The limited human data available indicate that in man, soluble fluoride doses onequarter to one-half those certainly lethal are no longer lethal. For example, in unsuccessful chemotherapeutic trials, Black et aL (1949) gave one leukemic patient as much as 23 mg of F/kg iv daily for 9 days without apparent toxic effects. Each dose presumably exceeded a quarter of the acute lethal dose given by mouth. The rapidity of death in rats is noteworthy; 50 ~ died in 3 to 6 hr depending on age. The course of fatal poisoning is usually rapid in man also; deaths frequently occur in 2 to 4 hr and if the victim survives for 24 hr, the prognosis is favorable. Plasma fluoride concentrations of 5-10 #g of F/ml were found in many of the younger rats receiving 50 mg of F/kg and sacrificed during the first 4 hr after dosing; this range of concentrations was also achieved in older rats given 25 mg of F/kg. Among all three populations, spontaneous death was associated with concentrations as low as 8-10 #g of F/ml. The ranges of plasma fluoride concentrations in the dying rats were not appreciably different in the 150- and 250-g populations, but the deaths occurred earlier in the heavier rats. Plasma F concentrations prior to death may well have been higher. Hall et al. (1972a) located a minimal lethal plasma fluoride concentration for rabbits; if concentrations exceeded 28 ~tg/ml 1 hr after dosing, death ensued within 12 hr. Gettler and Ellerbrook (1939) found concentrations of 3.5-15.6/~g/ml in the blood of :five human victims who ingested unknown but lethal amounts of fluoride. In three of
82
DE LOPEZ, SMITH AND HODGE
these cases the concentrations were greater than 10 #g/ml blood. Lethal blood concentrations in man and in the rat thus appear to be considerably lower than in the rabbit. Plasma fluoride concentrations were proportional to fluoride doses near the LD50 (Fig. 1). For a given dose of fluoride, concentrations were highest in the heaviest (oldest) rats and lowest in the youngest rats. These findings are consistent with the suggestion that the young skeleton is more efficient in removing fluoride from the plasma. The data in Table 4 indicate that the 250-g rats cleared the fluoride from the stomach more rapidly than did the other two groups. Absorption throughout the tract of the 250-g rats was not necessarily more rapid; the fractions remaining in the small and large intestines generally were greater in the 250-g animals. The apparent near cessation of absorption by the 250-g rats in the 1.5-3-hr interval cannot be explained. The observation that 57 ~ of the dose was absorbed by these animals after 1.5 hr would be consistent with higher early plasma concentrations. The 80- and 150-g populations (Table 4) each received doses of fluoride twice those given the 250-g animals. The lesser absorption following the larger doses may have resulted from damage to the absorption surfaces. Fifty percent of much smaller doses (1.7 mg of F/kg) were absorbed in 30 min (Zipkin and Likins, 1957).
ACKNOWLEDGMENTS This article is based on the thesis submitted by Ofelia H. de Ldpez to the School of Medicine and Dentistry of the University of Rochester in partial fulfillment of the requirements for the M.S. degree. The research was supported in part by USPHS NIH Training Grant No, 5-T01-GM-017181, and by the Atomic Energy Commission at the University of Rochester Atomic Energy Project under Contract No. AT(11-1)3490 and assigned Report No. UR-825. After completion of the degree requirements Mrs. L6pez was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnol6gicas (CONICET) of Caracas, Venezuela. The authors are indebted to Dr. C. L. Yuile for histological evaluation of the tissues, and to Mr. D. E. Gardner for analysis of the gastrointestinal tract samples.
REFERENCES BLACK,M. M., KLEINER,I. S., ANDBOLKER,H. (1949). The toxicity of sodium fluoride in man. N. Y. State J. Med. 49, 1187-1188. BRUTON, L. G. (1971). Known addition ion selective electrode technique for simultaneously determining fluoride and chloride in calcium halophosphate. Anal. Chem. 43, 579-581. ERICSSON, Y. (1966). Blood fluoride clearance in rats differing in age or previous fluoride exposure. Investigations using radioactive fluoride. Acta Odontol. Scand. 24, 393-404. GETTLER, A. O., AND ELLERBROOK,L. (1939). Toxicology of fluorides. Amer. J. Med. Sci. 197, 625-638. HALL, L. L., SMITH,F. A., AND HODGE,H. C. (1972a). Plasma fluoride levels in rabbits acutely poisoned with sodium fluoride. Proc. Soc. Exp. BioL Med. 139, 1007-1009. HALL, L. L., SMITH,F. A., L6PEZ, O. H., ANDGARDNER,D. E. (1972b). Direct determination of ionic fluoride in biological fluids. Clin. Chem. 18, 1455-1458. HODGE, H. C., AND SMITH, F. A. (1965). In Fluorine Chemistry (J. H. Simons, Ed.), Vol. 4, p. 145. Academic Press, New York. LITCHFIELD,J. T., JR., AND WILCOXON,F. (1949). A simplified method of evaluating doseeffect experiments. J. Pharmacol. Exp. Ther. 96, 99-113.
PLASMAFLUORIDEIN ACUTEPOISONING
83
MAYNARD, E. A. (1949). Quoted by H. E. Stokinger in Chap. 17. In Pharmacology and Toxicology of Uranium Compounds (C. Voegtlin and H. C. Hodge, Eds.), Part 2, pp. 1054-1056, McGraw-Hill, New York. SMITH, F. A., AND GARDNER,D. E. (1955). Determination of fluoride in urine. Amer. Ind. Hyg. Assoc. Quart. 16, 215-220. WAVES,D. R. (1968). Determination of submicromolar concentrations of fluoride in biological samples. Talanta 15, 1015-1023. ZIPKIN, I., AND LIKINS, R. C. (1957). Absorption of various fluorine compounds from the gastrointestinal tract of the rat. Amer. J. Physiol. 191, 549-550.
Plasma Fluoride Concentrations in Rats Acutely Poisoned with Sodium Fluoride OFELIA H . DE LdPEZ, 1 FRANK A . SMITH, AND HAROLD C. HODGE 2
Departments of Pharmacology and Toxicology, and Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received September 9, 1975; accepted March 10, 1976 Plasma Fluoride Concentrations in Rats Acutely Poisoned with Sodium Fluoride. DELdPEZ, O. H., SMITH,F. A., ANDHODGE,H. C. (1976). Toxicol. Appl. Pharmacol. 37, 75-83. Female rats of average body weight 250 g proved to be approximately twice as susceptible to the acute toxic effects of sodium fluoride given po as were female rats weighing on the average 80 or 150 g. The 24-hr oral LD50 values were 31, 54, and 52 mg of F/kg, respectively. Following single doses of 25 mg of F/kg for 250-g rats or 50 mg of F/kg for rats weighing 80 or 150 g, absorption of fluoride from the gastrointestinal tract was approximately 50 ~ complete in less than 90 min for the 250-g rats and in about 2 hr for the 80- and 150-g animals. In treated rats given 50 mg of F/kg, plasma fluoride concentrations (normally 0.3 pg/ml or less) increased to 10/tg/ml or more in 15 min in 80- or 150-g rats. Quantitatively similar increases were noted in 250-g rats given 25 mg of F/kg. Plasma fluoride concentrations of 8-10/tg/ml or higher were often associated with death, regardless of dose or body weight (age), but were achieved more rapidly in the heaviest rats. In surviving rats, plasma fluoride concentrations declined rapidly from maximal values reaching concentrations at 24 hr of 3 and 0.3/~g/ml, respectively, for the 150- and 250-g rats, and of approximately 4/tg/ml at 8 hr for the 80-g rats. Plasma fluoride concentrations after near lethal doses were dose-dependent. The greater resistance of the 80- and 150-g rats to the lethal effects of fluoride may reflect the greater efficiency of the younger skeletons in removing fluoride from the circulation. In spite of long acquaintance with acute fluoride poisoning, there is still uncertainty as to the relative importance in the lethal process of the several fluoride actions. These may include interference with the role of calcium, inhibition of enzymes, induction of shock and electrolyte imbalance due to loss of fluids by vomiting and diarrhea, and vascular or respiratory depression. Presumably, these actions can be related to fluoride concentrations in the circulating plasma, concentrations which are the resultant of several processes including absorption from the gastrointestinal tract and removal from the blood by the skeletal system and by the kidneys into the urine. The importance of these processes in governing the magnitude of the circulating plasma fluoride elevation is suggested in the observation of Maynard (1949) that intraperitoneally injected 1 Present address: Ministerio de Sanidad y Asistencia Social, Division de Higiene de Los Alimentos, Edificio Sur Oficina 417, Centro Simon Bolivar, Caracas, Venezuela. 2 Present address: Pharmacology Department, University of California, San Francisco, CA 94143. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
75
76
DE LOPEZ, SMITH AND HODGE
NaF is approximately twice as toxic for rats weighing 200-300 g as it is for 100-200-g rats. The younger animals may have a much greater skeletal availability and a corresponding rapidity of fluoride storage, which lower plasma fluoride concentrations more effectively if absorptions were comparable. It is the intent of the experiments described here to confirm the lower toxicity of NaF for young rats, and to seek an explanation by examining (a) the plasma fluoride concentrations associated with the LD50 for young and old animals, and (b) absorption of fluoride from the gastrointestinal tract. METHODS Three populations of female Sprague-Dawley white rats were used 3 of mean body weights and ranges 80 (50-108), 150 (l 12-184), and 250 (200-359) g, respectively. Prior to and following dosing, the animals were maintained on Purina Rat Chow which contained approximately 39 ppm of fluoride, and on fluoridated-tap water containing approximately 1 ppm. The LD50 for each population was determined in groups of 8-15 rats per dose that had been fasted 24 hr and were then given NaF in aqueous solution (0.2-1.6 ml/dose) by stomach tube. At least seven dose levels were used for each population. The 24-hr LD50 values were calculated according to the method of Litchfield and Wilcoxon (1949). Blood samples were taken at various time intervals by heart puncture under chloroform anesthesia; the plasma, separated in a refrigerated centrifuge, was frozen until analyzed. Heart blood samples from dead animals were removed as promptly as possine (never more than 30 min after death). Initially, plasma fluoride was measured by the diffusion method of Taves (1968) as modified by Hall et al. (1972a), and later by the known single-addition method of Bruton (1971) as adapted by Hall et al. (1972b). Absorption of fluoride from the gastrointestinal tract was measured indirectly by subtracting from the initial dose the amount of fluoride remaining at various times after administration. At preselected intervals, the rats were anesthetized with chloroform, the stomach, small and large intestines removed, and analyzed separately. The tissues were burned to ashes with calcium oxide fixative, the fluoride separated from the ash by steam distillation, and titrated in an aliquot of the distillate using thorium nitrate and alizarin indicator (Smith and Gardner, 1955). RESULTS The LD50 for the 80-g rats was 54 mg/kg, with upper and lower confidence limits of 54 and 49 mg/kg, respectively; the slope function was 1.26 (upper and lower confidence limits, 1.35 and 1.18, respectively). For the i150-g rats, the LD50 was 52 mg/kg with upper and lower confidence limits of 57 and 48 mg/kg; this LD50 did not differ significantly from that of the 80-g rats. The slope function was 1.32 with confidence limits of 1.52 and 1.15. The LD50 for the 250-g rats was significantly lower, being 31 mg/kg with confidence limits of 38 and 25 mg/kg. The slope function in this instance was 1.73, with confidence limits of2.13 and 1.40. Female rats approximately 30- to 45-days-old (80 or 150 g) were equally resistant compared to rats 90-days-old (250 g). These results are 3 Obtained from Blue Spruce Farm, Inc., Gardner Road, Altamont, N.Y. 12009.
PLASMA FLUORIDE IN ACUTE POISONING
77
TABLE 1 CUMULATIVE PERCENTAGE MORTALITIESOCCURRING WITH TIME IN RATS RECEIVINGDOSESEQUAL TO OR LARGER THAN THE LD50 Interval
Population according to average body weight
(hr)
80 g
150 g
250 g
0-1
11.4
--
12.5 27.5 50.0 75.0 77.5 77.5 77.5 100.0
1-2 2-3 3-4 4-5 5-6 6-7 7-24
11.4 34.2 39.9 48.6 51.5 71.5 100.1
2.3 6.9 13.8 18.4 27.6 32.2 99.7
similar to M a y n a r d ' s (1949) finding for ~NaF given ip; L D 5 0 values were 21 m g o f F / k g for 100-200-g versus 11 m g / k g for 200-300-g female rats. R a p i d i t y o f d e a t h is characteristic o f fluoride (Table 1), the older rats dying m o r e r a p i d l y t h a n the y o u n g e r ones. W h e n doses equal to o r greater t h a n the L D 5 0 were administered, h a l f o f the 250-g rats died within 3 hr whereas only a third o f the 150-g rats died within 7 h r a n d h a l f o f the 80-g rats within 6 hr. To determine p l a s m a fluoride concentrations associated with the oral LD50, g r o u p s o f the three p o p u l a t i o n s o f rats were fasted; the 80-g a n d 150-g rats were given 50 m g / k g a n d the 250-g rats 25-30 m g o f F / k g , all po. G r o u p s o f f o u r rats were sacrificed at selected time intervals (Table 2) a n d b l o o d samples were t a k e n f r o m each rat for fluoride TABLE 2 PLASMAFLUORIDE (/tg F/ml) IN RATS SACRIFICEDAT VARIOUSTIMES AFTERRECEIVINGN a F BY STOMACH TUBEa Average body weight (g):
80
150
250
Dose (mg of F/kg):
50
50
25
Hours to sacrifice
Mean _+ SE
Mean + SE
Mean + SE
Control 0.25 0.5 1 1.5 2 3 4
0.27 + 0.011
8
12 24
10.5 + 0 . 8 7.1 + 0 . 9 5.1 + 0.3 4.7
+0.7
2.5 +0.3
a Each value is the mean of three or four rats.
0.21 9.1 10.9 8.3 10.0 10.0 10.2 6.9 6.8 6.4 1.8
+ 0.022 + 0.94 + 2.39 ___1.1 + 1.4 +0.8 + 1.6 + 1.2 +0.4 + 1.6 + 0.3
0.09 + 0.014 9.8 + 3.05 7.6 + 1.15 7.2 +0.33 5.2 + 1.25 5.9 + 1.28 7.64 + 0.98 4.3 + 1.6 1.5 +0.16 1.6 +0.08 0.25 + 0.007
78
DE LOPEZ, SMITH AND HODGE
analysis. Mean plasma F concentrations rose rapidly, e.g., to 9-10 #g/ml in 15 min. Mean concentrations during the first 1.5 hr consistently reached 5-11 #g/ml with individual samples as high as 14-16 #g/ml in these surviving rats. During the first 3 hr, the average plasma concentrations were 5 #g/ml for the 80-g rats, 10 #g/ml for the 150-g rats, and nearly 8 #g/ml for the 250-g rats (given half the dose). Plasma fluoride values were generally lower after 4 hr. Although no consistent rate of decrease appeared, in only one rat did a plasma fluoride value exceed 9 #g/ml at 4 hr. The 80- and 150-g rats tended to maintain plasma concentrations of 5 to 10 #g/ml from 3 to 12 hr, whereas the plasma samples of the 250-g rats contained 1.6 #g/ml or less from 8 hr on. Additional data on the lethal plasma fluoride concentration were gained from analyses of the blood of seventeen 150- and 250-g rats dying from these doses. In 6 of the 17, plasma fluorides were 8-10 #g of F/ml, and in the other 11 were 12-19 #g of F/ml, as shown in Table 3. None of the 80-g rats died. A certainly lethal threshold for plasma concentrations was not established. TABLE 3 PLASMA FLUORIDE CONCENTRATIONS IN RATS FATALLY POISONED BY ORAL DOSES OF SODIUM FLUORIDE
Average body weight Dose (g) (mg of F/kg)
250
25 30
150
50
Number of rats dying with plasma F concn, in the indicated range 8-10/~g/ml I/4 2/7
Totals
3/6
12-19 ,ug/ml 3/4 5/7 12-16 #g/ml 3/6
6/17
11/17
Evidence was sought of a dose dependence for plasma fluoride concentrations by giving groups of fasted rats of each age single oral doses of (a) approximately the respective LD50, (b) ~, and (c) 3 of these amounts. Plasma fluoride concentrations measured 3 hr later increased linearly with the logarithm of dose (Fig. 1). The data for the 250- and 150-g rats followed parallel lines; the older rats given half the dose showed lower plasma fluoride values as expected. Plasma fluoride values for the 80-g rats were about half those for 150-g rats and the increase with dose was also less marked. The percentages of the doses remaining in the stomach, in the small, and in the large intestine at successive time intervals after dosing rats orally with 25 or 50 mg of F/kg are shown in Table 4. The percentages remaining at each time interval are shown graphically in Fig. 2A. The absorption patterns of the three populations did not differ greatly; the stomachs of the heaviest rats may have emptied more rapidly. Absorption is rapid; 50 ~ of the dose was absorbed in approximately 2 hr or less and absorption is practically complete within 24 hr. Fluoride did not accumulate in the lower segments of the digestive tract, indicating neither impairment of passage through the gastrointestinal tract, nor serious interference with uptake of fluoride by the intestinal epithelium.
79
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Plasma fluoride concentrations (Table 4) measured at the same intervals at which gastrointestinal absorption was determined, are shown in Fig. 2B. Taken together, the data indicate that in the two younger populations, an initial rapid absorption of fluoride
80
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PLASMA FLUORIDE IN ACUTE POISONING
81
promptly produces high plasma fluoride concentrations. The F concentration soon declines as the absorption approaches 100 700,and as urinary excretion and skeletal deposition remove fluoride from the plasma (Hodge and Smith, 1965). For the heaviest rats, plasma fluoride continued to rise at least through the first 3 hr, after which it declined as the increments of absorbed fluoride became smaller. Gross examinations of the gastrointestinal tracts 3 hr after dosing showed the stomachs to be distended with a yellow liquid and with gas, and the intestines to be swollen with liquid, presumably in response to local irritation and osmotic stress. The pH of the gastric fluids of the fluoride-treated rats was about 8, as was also the p H of the fluoride solution, whereas the gastric fluids in control rats and in rats given a NaC1 solution (pH 4) of the same osmolality had a pH of about 2. The microscopic appearance of sections of the gastrointestinal tracts of two rats given 50 mg of F/kg po was not different from that seen in rats receiving the NaC1 solution.
DISCUSSION The lethal doses of N a F given either po or ip for older (heavier) rats are about half those for younger rats. The two important means whereby fluoride in the organism is "detoxified" are (1) excretion into the urine and (2) deposition in the bone. Inasmuch as the oldest rats were only 90 days of age, the kidneys in all the groups were surely functionally normal. Deposition in the skeletal systems may have been less efficient in the oldest rats either because less mineral is "available" to the circulation or because the mineral is less reactive with fluoride. The latter possibility is doubtful: Ericsson (1966) found no relation between preexisting femur F concentrations and the uptake of lSF in small single doses. The log dose-mortality curves for N a F in rats characteristically have steep slopes. The limited human data available indicate that in man, soluble fluoride doses onequarter to one-half those certainly lethal are no longer lethal. For example, in unsuccessful chemotherapeutic trials, Black et aL (1949) gave one leukemic patient as much as 23 mg of F/kg iv daily for 9 days without apparent toxic effects. Each dose presumably exceeded a quarter of the acute lethal dose given by mouth. The rapidity of death in rats is noteworthy; 50 ~ died in 3 to 6 hr depending on age. The course of fatal poisoning is usually rapid in man also; deaths frequently occur in 2 to 4 hr and if the victim survives for 24 hr, the prognosis is favorable. Plasma fluoride concentrations of 5-10 #g of F/ml were found in many of the younger rats receiving 50 mg of F/kg and sacrificed during the first 4 hr after dosing; this range of concentrations was also achieved in older rats given 25 mg of F/kg. Among all three populations, spontaneous death was associated with concentrations as low as 8-10 #g of F/ml. The ranges of plasma fluoride concentrations in the dying rats were not appreciably different in the 150- and 250-g populations, but the deaths occurred earlier in the heavier rats. Plasma F concentrations prior to death may well have been higher. Hall et al. (1972a) located a minimal lethal plasma fluoride concentration for rabbits; if concentrations exceeded 28 ~tg/ml 1 hr after dosing, death ensued within 12 hr. Gettler and Ellerbrook (1939) found concentrations of 3.5-15.6/~g/ml in the blood of :five human victims who ingested unknown but lethal amounts of fluoride. In three of
82
DE LOPEZ, SMITH AND HODGE
these cases the concentrations were greater than 10 #g/ml blood. Lethal blood concentrations in man and in the rat thus appear to be considerably lower than in the rabbit. Plasma fluoride concentrations were proportional to fluoride doses near the LD50 (Fig. 1). For a given dose of fluoride, concentrations were highest in the heaviest (oldest) rats and lowest in the youngest rats. These findings are consistent with the suggestion that the young skeleton is more efficient in removing fluoride from the plasma. The data in Table 4 indicate that the 250-g rats cleared the fluoride from the stomach more rapidly than did the other two groups. Absorption throughout the tract of the 250-g rats was not necessarily more rapid; the fractions remaining in the small and large intestines generally were greater in the 250-g animals. The apparent near cessation of absorption by the 250-g rats in the 1.5-3-hr interval cannot be explained. The observation that 57 ~ of the dose was absorbed by these animals after 1.5 hr would be consistent with higher early plasma concentrations. The 80- and 150-g populations (Table 4) each received doses of fluoride twice those given the 250-g animals. The lesser absorption following the larger doses may have resulted from damage to the absorption surfaces. Fifty percent of much smaller doses (1.7 mg of F/kg) were absorbed in 30 min (Zipkin and Likins, 1957).
ACKNOWLEDGMENTS This article is based on the thesis submitted by Ofelia H. de Ldpez to the School of Medicine and Dentistry of the University of Rochester in partial fulfillment of the requirements for the M.S. degree. The research was supported in part by USPHS NIH Training Grant No, 5-T01-GM-017181, and by the Atomic Energy Commission at the University of Rochester Atomic Energy Project under Contract No. AT(11-1)3490 and assigned Report No. UR-825. After completion of the degree requirements Mrs. L6pez was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnol6gicas (CONICET) of Caracas, Venezuela. The authors are indebted to Dr. C. L. Yuile for histological evaluation of the tissues, and to Mr. D. E. Gardner for analysis of the gastrointestinal tract samples.
REFERENCES BLACK,M. M., KLEINER,I. S., ANDBOLKER,H. (1949). The toxicity of sodium fluoride in man. N. Y. State J. Med. 49, 1187-1188. BRUTON, L. G. (1971). Known addition ion selective electrode technique for simultaneously determining fluoride and chloride in calcium halophosphate. Anal. Chem. 43, 579-581. ERICSSON, Y. (1966). Blood fluoride clearance in rats differing in age or previous fluoride exposure. Investigations using radioactive fluoride. Acta Odontol. Scand. 24, 393-404. GETTLER, A. O., AND ELLERBROOK,L. (1939). Toxicology of fluorides. Amer. J. Med. Sci. 197, 625-638. HALL, L. L., SMITH,F. A., AND HODGE,H. C. (1972a). Plasma fluoride levels in rabbits acutely poisoned with sodium fluoride. Proc. Soc. Exp. BioL Med. 139, 1007-1009. HALL, L. L., SMITH,F. A., L6PEZ, O. H., ANDGARDNER,D. E. (1972b). Direct determination of ionic fluoride in biological fluids. Clin. Chem. 18, 1455-1458. HODGE, H. C., AND SMITH, F. A. (1965). In Fluorine Chemistry (J. H. Simons, Ed.), Vol. 4, p. 145. Academic Press, New York. LITCHFIELD,J. T., JR., AND WILCOXON,F. (1949). A simplified method of evaluating doseeffect experiments. J. Pharmacol. Exp. Ther. 96, 99-113.
PLASMAFLUORIDEIN ACUTEPOISONING
83
MAYNARD, E. A. (1949). Quoted by H. E. Stokinger in Chap. 17. In Pharmacology and Toxicology of Uranium Compounds (C. Voegtlin and H. C. Hodge, Eds.), Part 2, pp. 1054-1056, McGraw-Hill, New York. SMITH, F. A., AND GARDNER,D. E. (1955). Determination of fluoride in urine. Amer. Ind. Hyg. Assoc. Quart. 16, 215-220. WAVES,D. R. (1968). Determination of submicromolar concentrations of fluoride in biological samples. Talanta 15, 1015-1023. ZIPKIN, I., AND LIKINS, R. C. (1957). Absorption of various fluorine compounds from the gastrointestinal tract of the rat. Amer. J. Physiol. 191, 549-550.