Recently a report (1) from this laboratory indicated that NaBr in relatively low concentrations accelerated the growth of mouse and egg cultivated tumor tissue. This result occurred when the drug was introduced by way of the drinking water in mice, by injection over the embryonic membranes of eggs inoculated with tumor tissue, and when added to tumor tissue suspensions before implantation into eggs or mice. Preliminary tests with NaI and NaF give evidence that low levels of these halides also accelerated tumor growth.
An extensive investigation of the effect of NaF on growth of mouse and egg cultivated tumor tissue has now been completed.
Materials and methods. Mice of the DBA strain implanted with RC mammary adeno-carcinoma were used in the mouse experiments. Tumor suspensions for transplantation were made by adding 0.4 ml dispersed tumor tissue to 5.0 ml of 0.85% saline, and 0.2 ml of this suspension was injected subdermally in the right inguinal area. NaF was introduced into the mouse-tumor system in 3 different ways.
In one series of tests the compound was added to the tumor tissue suspension prior to inoculation into the mice. Concentrations of the drug were at the levels, 0.000,01-0.005 mg and 0.4-0.8 mg per mouse injection. The controls received the untreated tumor suspension. Control and experimental tumor suspensions were taken from a common pool of tumor-saline preparation.
In another series, NaF was added to the drinking water of the experimental animals immediately after they were inoculated with tumor tissue. Concentrations of the drug varied. In most of these tests, 1-5 mg of NaF per liter of distilled water was utilized. Controls were maintained on untreated distilled water.
A third series involved subdermal injections of NaF into mice bearing tumor transplants. In these tests, 0.25 mg of the compound dissolved in 0.25 ml of saline was injected subdermally on 4 consecutive days, beginning 4 days after tumor transplantation. The tests were terminated one day after the final treatment.
In all the mouse experiments, the animals were sacrificed 8 to 10 days after tumor implantation, at which time the tumor tissue was removed and weighed. The results of a test were evaluated on the basis of the average weight of tumor tissue per mouse in the experimental as compared with the control animals.
A C3H mouse mammary tumor which grows readily in eggs by the yolk sac method (3) was utilized in the egg work. The techniques involved have been described (2,3). Tumor tissue suspensions were made up of 0.4 ml dispersed tissue in 5.0 ml of 0.85% saline, and 0.2 ml of this suspension was injected into the yolk sacs of 4-day incubated embryonated eggs.
Two types of tests were utilized in the experiments with eggs. In one series, NaF was added to tumor suspensions before inoculation into eggs. Concentrations of the drug were used at the levels, 0.000,01-0.000,5 mg and 0.1-0.8 mg per egg injection. The controls received the untreated tumor suspensions. Tumor preparations for control and experimental groups were taken from a common pool of tumor-saline suspension.
In another series, NaF was introduced into tumor-bearing eggs over the chick membranes. A level of 0.05-0.1 mg of the drug in 0.1 ml saline was injected into each egg. The controls were treated with saline. The membrane injection technique has been described (4).
The egg tests were terminated on the 13th day of incubation. The results were evaluated on the basis of the average weight of tumor tissue in experimental as compared with control eggs.
In all the experiments, both with mice and those involving embryonated eggs, every effort was made to randomize as much as possible all proceedings.
A total of 54 tests involving 991 animals was completed with mice bearing tumor transplants, and 58 tests including 1817 eggs were carried out with tumor-bearing eggs. Statistical significance, or P, was obtained by dividing the difference in tumor size for control and experimental tumors of a series by the standard error of the difference.
Results. The results of the various tests are summarized in Tables I-VII. Tumor growth was accelerated in association with NaF, at the concentrations and with the methods of introduction in tumor-bearing mice and eggs, as given in Tables I-V. The degree of growth stimulation was quite similar in tumors grown in mice as compared with tumors cultivated in eggs. In the 3 series of experiments with mice, accelerations of tumor growth averaged 126, 121 and 114% of the control, while in the 2 series of egg experiments, the per cent increase in tumor size was 124 and 121. The difference in average tumor size between control and experimental groups was statistically significant in each of these series of experiments, and in combination this significance is greatly increased.
When the drug was added to tumor suspensions before implantation in mice or eggs at the relatively high levels of 0.4-0.8 mg per mouse, or 0.1-0.8 mg per egg, tumor growth was markedly inhibited.
Discussion. The data appear to establish the association of NaF with acceleration of the growth of tumor transplants in mice and embryonated eggs, at concentrations and under the conditions described here. It has been reported (1) that NaBr likewise has a stimulating effect on tumor growth when administered to tumor-bearing mice or eggs, under conditions similar to the present tests. NaBr is effective at higher levels of concentration as compared with NaF. Concentrations of NaF, as low as 0.000,01 mg per mouse injection, when added to tumor suspensions before implantation in mice, accelerated tumor growth, while levels in the range of 0.000,1 – 0.000,01 mg/injection of NaBr did not affect tumor growth.
In experiments where the compound was added to the drinking water, wide variations in dosage increased tumor growth in the instance of both NaF and NaBr. It was recently reported (5) that the plasma fluoride is regulated in rats. This probably accounts for the similarity of tumor growth acceleration when NaBr is given over a range of 1 to 1,000 mg per liter, and NaF in concentrations of 1 to 55 mg per liter.
Unpublished results of tests with NaI indicate that this halide also stimulates tumor growth when added to tumor suspensions before implantation in eggs or mice at low levels, 0.000,1 – 0.001 mg per mouse or egg.
Summary. In 54 tests involving 991 mice bearing transplanted tumors and 58 tests including 1817 tumor-bearing eggs, data were obtained which indicated a statistically significant acceleration of tumor tissue growth in association with comparatively low levels of NaF.
1. Taylor, A., Taylor, N.C., Cancer Research, 1964, v24, 751.
2. Taylor, A., Carmichael, N., Norris, T., ibid., 1948, v8, 264.
3. Taylor, A., Univ. Texas Publication. 1953, n5314, 13.
4. —–, ibid., 1953, n5314, 27.
5. Singer, L., Armstrong, W.D., Proc. Soc. Exp. Biol. and Med., 1964, v117, 686.