(0.05 p.c. of the diet) without seriously arresting, though affecting, their. growth, development and fertility. Fluorine can be estimated in very

Transcription

1 388 6I2.649:546. i6 MATERNAL TRANSFERENCE OF FLUORINE By MARGARET M. MURRAY (From the Department of Physiology, Bedford College, London) (Received April 23, 1936) IT is often assumed that the placenta acts as an efficient barrier preventing the passage of harmful substances from maternal to foetal blood. To what extent substances, not normally present in the body, yet of a diffusible nature, can be transferred to the offspring via the milk has not been investigated for many substances. If these two points, the transference across the placenta and into the milk are tested by giving the mother some substance capable of taking part in metabolism, the difficulty is to trace its fate. The use of fluorine, it was thought, might get over this difficulty for the following reasons. Fluorine is normally present in animals in very small amounts only, and then chiefly in teeth and bones. It is possible to feed to animals sodium fluoride in suitable doses (0.05 p.c. of the diet) without seriously arresting, though affecting, their growth, development and fertility. Fluorine can be estimated in very small quantities by distillation of the fluorine as H2SiO6, conversion to HF and determination of the latter colorimetrically by its bleaching effect on the red lake of zirconium and alizarin. Since in some cases decomposition products of the perchloric acid, which also bleach the lake, distil over, it is necessary, in order to avoid high values, to do several blank determinations on each sample of the acid. Feeding fluorine compounds is now well known to produce characteristic visible effects in the teeth of rats. At first they become pearly white, losing their natural orange-yellow colour. Later they become opaque, white and very brittle. Frequently the teeth break off and sometimes, because of failure to occlude, they become abnormally long. These are clear signs of fluorosis. The bones also become brittle. Both the teeth and bones contain fluorine. In human beings fluorine in the drinking water produces a characteristic mottling of permanent teeth, first observed by Black and McKay [1916], proved to be due to fluorine by Smith et al. [1931], and

2 MATERNAL TRANSFERENCE OF FLUORINE 389 known to occur so extensively in the United States that it constitutes an important public health question which has received a great deal of attention and produced a mass of scientific work. Maldon in Essex is the only place where this trouble has been recognized in England. Ainsworth [1933] made an investigation on the teeth there. Whether mottling can occur as a result of maternal fluorine ingestion is at the present time debated. Opinions vary as to whether the temporary teeth ever show mottling. Mc Kay [1932], the pioneer on mottled teeth, said he had never seen a clear-cut case of mottling in temporary teeth. Later he [1935] said he had but very rarely seen even slight mottling of temporary molars. Recently Smith and Smith [1935] reported true mottling of the temporary teeth of artificially fed children in a fluorine water area. They maintain that this was not due to an in utero effect of the fluorine but must have been brought about during postnatal calcification when the children were receiving milk foods prepared with fluorine-containing water. They did, however, consider it was possible that the fluorine might be effective in utero at high colcentrations. Brinch and Roholm [1934] described two cases of mottling of the permanent teeth of two children of a woman who had worked in a cryolite factory. These workers stated that the temporary teeth had not been affected. They also then concluded that the placenta had barred the transference of fluorine so that there was no fluorine during the period when the temporary teeth calcify, but that the fluorine passed into the milk and was thereby able to affect the calcification of the permanent teeth. The periods of lactation were 2 and 11 years and the mottling was proportional to the time. On the other hand, Boissevain and Drea [1933], who have worked on the determination of fluorine in teeth, investigated the fluorine content of milk from cows in a fluorine water area. They state that in spite of the intake of fluorine-containing water the milk ash contained no fluorine. They used the spectroscopic method, and because of their negative result advised that children should be given milk in place of water. Smith and Smith [1935] tentatively reported mottlihg in the teeth of breast-fed babies. There is then no agreement of opinion as to the transference of fluorine in utero or through milk. It is hoped that the present experiments will give an answer to these queries. To put the question of transference to the test, a series of experiments was planned. The rats used throughout were all of the same inbred albino stock. The diet of the adults was one used as a stock diet by Coward et al. [1932], which consisted of whole wheat and ground maize,

3 390 M. M. MURRAY dried yeast, casein and salt mixture. Measured additions of cabbage, meat, liver, cod-liver oil and dried, full-cream milk were given. To the salt mixture of 10 g. CaCO3 and 10 g. NaCl, 1 g. KI was added. The animals received 15 g. of diet mixture moistened with 15 c.c. liquid per day to which the adjuncts were added. This diet was considered a complete one. For the control animals the diet was moistened with 15 c.c. water, for the rest the diet was moistened with 15 c.c. of 0.05 p.c. NaF. The bodies of five litters of rats born of normal mothers and five litters from mothers receiving 0 05 p.c. NaF in their diet were killed within 24 hours of birth and analysed for fluorine. Each litter consisting of seven to nine young was weighed (wet weight), ashed and the fluorine determined. The determination was made by distilling the ash in presence of glass beads with perchloric acid, thereby converting the fluorine to H2SiF6, which in the distillate by hydrolysis gave fluorine ions. The estimating of the fluorine was made by the colorimetric method of Elvove [1933]. The results of these analyses are given in Table I. TABLE I Control Fluorine p.c. Fluorine Fluorine p.c. litters of wet weight litters of wet weight S F2, S F21 and F, Ss FM S48 Nil F= 0'00078 S47 Nil F Mean Difference: p.c._ M. The absolute values for the fluorine of some of the controls may be a little too big, but the difference value is quite definite and significant. This quantity works out to a concentration of M, and since Robison and Rosenheim [1934] have shown that a concentration of M NaF in the perfusion liquid inhibits calcification in vitro, it would appear that the quantity of fluorine transferred in utero was in these rats a harmful amount. No doubt the fluorine is not evenly distributed in the bodies of these young rats which gave only 2 p.c. ash, but is concentrated in the developing osseous tissue. Great difficulty was experienced in the estimation of the fluorine content of the blood of the mothers because substances came over in the distillation of the ashed blood which interfered with the colorimetric comparison. We came to the conclusion, however, that the amount was small and probably of the order of p.c. This value was comparable with the fluorine content of the new-born young of these rats.

4 MATERNAL TRANSFERENCE OF FLUORINE To investigate the question of transference during lactation, four groups of young suckling rats were used; the groups were arranged in the following way: Group 00 born of normal mother suckled by normal mother. Group + + born of fluorine mother suckled by fluorine mother. Group 0 + born of normal mother suckled by fluorine foster mother. Group + 0 born of fluorine mother suckled by normal foster mother. The last two groups interchanged mothers within 24 hours of birth. During the first 12 days of lactation the fluorine mothers received the NaF in the diet as described, but after that they were given the same amount of NaF in a more concentrated form by mouth from a pipette. The young were killed at 21 days. It was hoped that there would be distinct and visible signs of the effects of the fluorine on the teeth. The incisor teeth of all very young rats are much paler than in the adult and the tips are white and not so transparent. The teeth of the + + litters were throughout slightly opaque and whiter than the 00 litters. A comparison of the 0 + and the + 0 litters suggested that the teeth of the latter were less pigmented than the former. The bodies were skinned, eviscerated and to a certain extent dismembered. The parts were boiled in water and the flesh picked off the bones as well as possible. After several such pickings a collection of bones with a minimum of soft tissue was obtained. The material was ashed and the fluorine content of the ash determined. Each group contained two litters of six to eight young. The numbers of determinations made on each ash are given in Table II in brackets. Blank deter-- minations were also made. f TABLE II p.c. fluorine p.c. increase! Group in ash over controls 00 (controls) (5) (6) *0208 (10) *0075 (14) The results show that both pre- and postnatal administration of fluorine to the mothers leads to fluorine uptake in the young. The litters of mothers having prenatal fluorine feeding would have been born (see results of determinations on the new-born rats, Table I) with a certain amount of fluorine; it has been calculated from these determinations that the corresponding fluorine in the ash would have been 0-02 p.c. When PH. LXXXVII

5 392 M. M. MURRAY the fluorine is discontinued after birth as in the litters + 0, then, though possibly some of this fluorine acquired before birth is eliminated, a certain amount remains. Continuation of the fluorine after birth of the young gives the highest value and proves clearly that fluorine is transmitted in the milk. When the fluorine is given only after birth of the young, at 3 weeks they contained a considerable percentage of fluorine. It must be pointed out that the sum of the fluorine acquired separately before and after birth by the young of these fluorine rats is of the same order as that acquired by the litters where the mothers had received fluorine both during gestation and lactation. That is, the sum of fluorine of litters + 0 and 0 + is approximately that of fluorine litters + +. The amounts of fluorine fed is many times that likely to be ingested by human beings, but it is most likely accumulative, and considering the time of the gestation and lactation period in human beings the quantities encountered would probably be significant. In any case the results give definite evidence of maternal transference, and even though only a very much smaller concentration arising in a similar way might be found in children, these smaller concentrations would most likely be significant in view of the very low effective concentration of fluorine found by Robison and Rosenheim [1934]. Human beings must either be more sensitive to lower concentrations of fluorine, or small concentrations are more effective in them than in rats, because of the longer time of gestation. It is difficult to obtain evidence of the action of fluorine on rats when given in the concentration found in a water from an area where characteristic mottling of human teeth occurs [Smith and Lantz, 1933]. SUMMARY 1. Determination of the fluorine contents of the new-born young of control and fluorine-fed rats showed that at a level of maternal feeding of 0*05 p.c. NaF the young acquired significant amounts of fluorine. The F2 content of the new-born rats represented p.c. of wet weight. 2. Young rats suckled by mothers receiving 0 05 p.c. fluorine in the diet acquired fluorine through the milk so that their bones contained p.c. F2 as compared with controls p.c. F2. 3. The results suggest that mottling of temporary teeth in humans may result from prenatal maternal fluorosis and also that human or other milk, from regions where fluorine occurs in water, contains fluorine in amounts which may be biologically effective, although, perhaps, too small for detection or determination chemically or spectroscopically.

6 MATERNAL TRANSFERENCE OF FLUORINE 393 Part of the expenses of this work were defrayed by a grant from the Medical Research Council, for which thanks are recorded. The author would also like to thank Miss J. E. Elliot for her help in the care of the animals. REFERENCES Ainsworth, N. J. (1933). Brit. dent. J. 55, 233. Black, G. V. and McKay, F. S. (1916). Dent. Cosmo8, 58, 132. Boissevain, C. H. and Drea, W. F. (1933). J. dent. Res. 13, 495. Brinch, 0. and Roholm, K. (1934). Paradentium, 6, 8. Coward, K. H., Cam bden, M. R. and Lee, E. M. (1932). Biochem. J. 26, 679. Elvove, E. (1933). Publ. ith Rep., Wash., No. 48, p McKay, F. S. (1932). Quoted by Smith, M. C. and Smith, H. V. McKay, F. S. (1935). J. Amer. dent. Ass. 22, 814. Robison, R. and Rosenheim, A. H. (1934). Biochem. J. 28, 684. Smith, M. C., Lantz, E. M. and Smith, H. V. (1931). Univ. Tech. Bull. Ariz. agric. Exp. Sta. No. 32. Smith, M. C. and Lantz, E. M. (1933). Ibid. No. 45. Smith, M. C. and Smith, H. V. (1935). J. Amer. dent. Ass. 22,