THE INTERCHANGEABILITY of the ELEMENTS CHLORINE and IODINE in CULTURE MEDIA for HUMAN THYROID FIBROBLASTS Among Those Elements W

THE INTERCHANGEABILITY OF THE ELEMENTS CHLORINE AND IODINE IN CULTURE MEDIA FOR HUMAN THYROID FIBROBLASTS

JOHANNES P. M. VOGELAAR, D.M.S., AND ELEANOR ERLICHMAN, B.S. (From the Institute of Cancer Research, Columbia University, and St. Luke's Hospital, New York City')

Among those elements which occur only .in minor amounts in the human body, none has such an interesting history or has been the subject of so much research as iodine. Although there can be no doubt concerning its physiologi- cal significance, actually very little is known about the requirement of various cells and tissues for this element in both its organic and inorganic forms. In view of the fact that the human body not only contains chlorine and iodine, but also fluorine and bromine, the question naturally arises whether all are vital elements and to what extent they can replace one another. The experiments reported in this article were undertaken to determine the interchangeability of chlorine and iodine in culture media for human thyroid fibroblasts. The effect of iodine and iodides on tissue cultures has been studied by various investigators. Pitini ( 12) observed a stimulating effect of potassium iodide on the growth of spleen and sciatic nerve of young guinea-pigs. Glass capillaries filled with a 10 per cent solution of the iodide were placed in close proximity to the cultures. In the interpretation of the results obtained with such a method it must be borne in mind that divers uncontrollable factors may affect the tissue growth, such as local changes in the osmotic pressure and in the pH of the culture medium. Lambert (8), in an investigation of the comparative resistance of bacteria and human tissue cells to certain common antiseptics, determined the effect of iodine on pieces of tissue taken from human spleen and lymph nodes. After having been immersed for some time in iodine solutions of various concentrations, the pieces were washed with a salt solution and then placed in a culture medium. A good growth occurred after exposure to a 1:2000 solution of iodine for one hour, a strength sufficient to sterilize the tissue completely in most instances. Lambert and Meyer (9) made similar experiments with pieces of rabbit spleen which were exposed for only twenty minutes to the antiseptic. The tissue cells survived an exposure to iodine in the concentration 1:2500, but were killed by iodine in the concentration 1: 1000. In interpreting their results the writers remark that the surviving tissue cells may have come from the center of the small fragments, where they were partially protected. This seems plausible and makes it difficult to formulate a definite statement as to which concentration can be endured by the cells. Salle and Lazarus (13) also studied the resistance of bacteria and embryonic tissue to germicidal substances. They found that iodine in a concentration 1:1800 or higher completely inhibits the growth of embryonic chick

1 With the assistance of a grant from the Anna Fuller Fund, 242 INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 243 tissue. The iodine was applied in the form of a Lug01 solution, which was mixed with chick embryonic fluid and placed over tissue fragments ,embedded in plasma. Cervello and Levi (3) studied the effect of iodine on tissues from various organs of chicks and chick embryos. This was done either by adding an iodine-potassium iodide solution to the culture medium or by using plasma taken from a chicken previously injected with the salt solution. The writers found that both connective-tissue cells and epithelium grow well in a medium containing a considerable amount of iodine (0.125% I,, 1.25% KI). The presence of free iodine in the medium was demonstrated by the addition of starch. It should be kept in mind that an iodine-iodide solution has iodine partly in the form of a tri-iodide, a fact which necessarily complicates the interpretation of the results. Olivo (1 1) used the heart and skin of chick embryos for his experiments with iodine. Small pieces of tissue were kept for several hours in isotonic solutions of sodium chloride, potassium iodide, and several mixtures of these before being placed in the culture medium. No appreciable difference between these cultures and the controls was observed except that the emigration of cells began sooner in cultures previously treated with potassium iodide. In other experiments cultures were grown in media containing different amounts of the iodide. Cultures grew well in the presence of 0.25 per cent potassium iodide, whereas in a medium with 0.45 per cent potassium iodide the outgrowth of the explants was decidedly less than that of the controls. Buchsbaum and Bloom (2) observed an approximately normal growth of embryonic chick fibroblasts in a culture medium containing iodine in a concentration 1:4000 or,less. Strickler and Fowler (14) determined the effect of iodine vapor on the growth of chick fibroblasts and malignant cells of the rat. The cultures were exposed to vapors arising from iodine crystals or from an iodine-potassium iodide solution. The iodine vapor did not stimulate the growth of the fibroblasts and was frequently harmful. Tumor cultures treated with iodine always grew less than the controls and showed a more extensive degeneration. How much iodine reached the growing cells, and in what form could not be ascertained. Zepponi and Crainz (17) used a novel method for studying the effect of iodine on chick fibroblasts, placing some gold previously exposed to iodine vapor'in the vicinity of the explants. The gold thus treated exerted a strongly stimulating effect, probably caused by a direct action of the iodine on the cells, since untreated gold did not influence the cells in any way. Hirashima (6) secured a vigorous growth of heart fibroblasts from the chick embryo in a medium containing sodium iodide tantamount to 25 X M (0.037 per cent). With an increasing concentration of the iodide, the growth was correspondingly less. When a value of 1/10 M (1.5 per cent) was reached, a majority of the cultures did not show any growth. Baker and Ebeling (1) maintained cultures from a pure strain of chick embryo fibroblasts in vital condition over a period of 56 days in a medium containing 130y per cent potassium iodide. This is equal to 783 X lo-* M potassium iodide. 244 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN

Crainz (4) studied the effect of iodine on heart fibroblasts and liver epithelium of the chick embryo. The iodine was added to the culture medium in the form of Lugol’s solution. The addition of a small amount of iodine to the culture medium was advantageous, whereas with greater quantities an inhibi- tory effect on the growth activities was noticed. The latter, however, was lessened when the cultures had been treated previously with a lower concentration of the halogen. Iodine was applied in concentrations ranging from 1:4000 to 1 :20,000. With the latter value a strongly stimulating action was observed, whereas with the former the cultures showed slight activity. Crainz exposed some cultures to iodine vapor arising from Lugol’s solution and, like Strickler and Fowler, noted a harmful effect on the cells. The epithelial cells showed a lesser sensitivity to iodine than the fibroblasts. Lambert (7, 8) and German (5) exposed cultures to the action of potassium mercuric iodide using, respectively, human fibroblasts and those of the chick embryo. The composition of this compound, so different from the chemicals used by the aforementioned authors, makes a comparison of the results with those of others impossible.

EXPERIMENTAL Fibroblasts from fresh human thyroid tissue were grown for fourteen days in a mixture of equal parts of citrated beef plasma (16), calcium Ringer (15), feeding solution (15), and magnesium Ringer (15). After liquefying the plasma with trypsin (16) the pieces of tissue were equally divided over four culture media containing different amounts of iodine. In the preparation of three of the media, modifications of calcium Ringer, magnesium Ringer, and feeding solution were used. These were made by replacing part or all of the chlorides of the original solutions by equivalent amounts of the corresponding iodides. The composition of the various salt solutions follows : Ca. Ringer solution A Ca. Ringer solution R Ca. Ringer solution C 73.8 C.C. 1 M NaCl 73.8 C.C. 1 M NaI 73.8 C.C. 1 M NaI 121.6 C.C. M/100 KCl 121.6 C.C. M/100 KC1 121.6 C.C. M/100 KI 162.2 C.C. M/lO CaClz 162.2 C.C. M/10 CaCl2 162.2 C.C. M/10 CaIz 142.4 C.C. dist. water 142.4 C.C. dist. water 142.4 C.C. dist. water

500.0 C.C. Ca. R. A 500.0 C.C. Ca. R. B 500.0 C.C. Ca. R. C Salt solution A Salt solution R 70.8 C.C. 1 M NaCl 70.8 C.C. 1 M NaI 22.0 C.C. M/lO KCl 22.0 C.C. M/10 KI 12.5 C.C. M/10 CaCl2 12.5 C.C. M/10 CaI2 51.4 C.C. M/100 MgClz 51.4 C.C. M/100 MgI2 11.7 C.C. M/100 KaSOa 11.7 C.C. M/100 KZSO, 50.0 C.C. M/100 NaZHP04 50.0 C.C. M/lOO Na2HP04 281.6 C.C. dist. water 281.6 C.C. dist. water

500.0 C.C. Salt sol. A 500.0 C.C. Salt sol. B The preparation of feeding solution A has already been described (15). Feeding solution B is primarily the same as A except that iodides replace the chlorides entering into the composition of the latter. INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 245 It is easy to calculate the amount of chlorine and iodine in the above solutions, especially if we express them in suitable units. If we assume that one unit is the atomic weight of an element expressed in milligrams, it is clear that 1 C.C. of a 1 M NaCl solution at 20" C. contains 1 unit of chlorine. From the formula above it follows that 500 C.C. of Ca. Ringer solution A at 20" C. contains the following amount of the halogen: 73.8 + 121.6/100 + 2(162.2)/10 or 107.46 units

100 C.C. Ca. Ringer solution A contains 21.49 C1 100 C.C. Ca. Ringer solution B contains 6.73 C1 and 14.76 I 100 C.C. Ca. Ringer solution C contains 21.49 I 100 C.C. Salt solution A contains 15.31 C1 100 C.C. Salt solution B contains 15.31 I 100 C.C. Feeding solution A contains 13.46 C1 100 C.C. Feeding solution B contains 13.46 I

The amount of chlorine contained in 100 C.C. of the citrated beef plasma used in our experiments may be estimated at about 10.1 units. This value was attained by using figures from the well known blood analyses by Abderhalden. The fact that citrated beef blood has a lower concentration of chlorides than the non-citrated blood was taken into consideration. The four culture media in which the fibroblasts were grown had the following composition :

Medium A: equal parts of plasma, Ca. Ringer A, Feeding sol. A, Salt sol. A Medium B: equal parts of plasma, Ca. Ringer A, Feeding bol. A, Salt sol. B Medium C: equal parts of plasma, Ca. Ringer B, Feeding sol. A, Salt sol. B Medium D: equal parts of plasma, Ca. Ringer C, Feeding sol. B, Salt sol. B

100 C.C. Medium A contains 15.09 C1 (100: 0) 100 C.C. Medium B contains 11.26 C1 and 3.83 I ( 75:25) 100 C.C. Medium C contains 7.57 C1 and 7.52 I ( 50:50) 100 C.C. Medium D contains 2.52 C1 and 12.57 I ( 17533) The values between the parentheses denote the relative amounts of chlorine and iodine expressed in atomic weight units. For example, the " chlorine- iodine ratio " 17 :83 means that the relative amounts of chlorine and iodine in medium D are 17 X 35.457 and 83 X 126.932 weight units, the figures 35.457 and 126.932 being, respectively, the atomic weights of the halogens chlorine and iodine. The absolute amounts of chlorine and iodine present in 100 C.C. of medium D are, of course, 2.52 X 35.457 and 12.57 X 126.932 milligrams. The ratio 17:83 theoretically also means that if the same fraction of the total amounts of chlorine and of iodine of medium D were present in the form of chlorine and iodine ions, the medium would contain for every 17 chlorine ions no less than 83 iodine ions. This, however, is not the case, since chlorine and iodine compounds, as for example NaCl and NaI, do not have the same dis- sociation constants. It must be understood that the given CI:I ratios are ap- proximate values only, because neither the iodine in the beef plasma was considered nor the exact amount of chlorine in the plasma known. The amount of iodine in the plasma is so small that it does not affect the values in the last column in a material way. Even if the blood were obtained from an animal 246 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN

which had access to iodized salt, the amount of iodine in the blood would still be negligible and hardly affect the ratios given above. The tissue obtained from thyroid specimens probably contained some inorganic iodine which no doubt may be neglected in view of the considerable amounts present in the test media. The same may be said in regard to traces of iodine possibly present as a contamination of the salts (Merck) with which the divers solutions were prepared. Only fresh iodide solutions were used, inasmuch as they may change chemically, resulting in the formation of free iodine. The salt solutions were sterilized in a steam sterilizer, the feeding solutions by filtering through a Chamberland-Pasteur candle.

OBSERVATIONS At the time that the experiments were begun, it was thought that the addition of iodine in any appreciable amount to the culture medium would render the latter less suitable for the growth of fibroblasts. The amount of iodine in human blood is extremely low (5 - 10 y per cent) (lo), and it might be ex- pected, therefore, that culture media containing the element in concentrations ranging from 0.486 gm.per cent (medium B) to 1.595 gm. per cent (medium D> would be entirely unsuitable for the growth of fibroblasts. This, however, is not the case. It was found that the subcultures showed about the same activity in the culture media containing iodine as in the control medium. In other words, a change in the composition of the medium, consisting of a re- placement of up to 80 per cent of the chlorides by an equivalent amount of the corresponding iodides, had hardly any effect on the growth activities of the explants. Occasionally, however, with’ an increase in the amount of iodine, the cultures neither looked as healthy as those in the control medium nor grew quite as well. The cells may show signs of degeneration, such as an abnormal form or a coarsely granulated cytoplasm. They never have a tendency to form fat droplets, an inclination so pronounced in chick fibroblasts under the same circumstances, as observed by Hirashima. It is a fact, however, that frequently a good growth of fibroblasts has been obtained with culture medium D, which had the largest amount of iodine among the media used in our experiments. The cultures were kept in the various media for two weeks, after which time they were treated with trypsin and the tissue was further grown in fresh media of the same composition. In these cases the cultures were exposed to the same concentration of iodine over a period of four weeks. In a few instances, cultures grown for fourteen days in medium C (0.955 gm. per cent I), were then placed in medium D (1.595 gm. per cent I). No apparent difference was noted. The cultivation of the fibroblasts was discontinued after a four week growth in the test media. In view of the results obtained, it does not seem beyond possibility that fibroblasts could be grown in a medium containing only iodides instead of chlorides. Such experiments could not be performed since the chlorine in the beef plasma naturally could not be replaced by iodine. Experiments as indi- cated above will, be carried out when the cultivation technic for human tissue 2This concentration seems to be extraordinarily high. However, one must consider the fact that a NaI solution containing 1.595 gm. per cent of iodine merely has a molarity equal to that of a 0.73 per cent NaCl solution. INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 247 will have advanced to the stage where artificial culture media of known composition can be used. Our experiments were not always attended with success, since frequently the test. media liquefied in the immediate vicinity of the explants within 24 hours. In these cases the. experiments of necessity had to be discontinued. The liquefaction of the culture media very probably was favored by a swelling of the fibrin network under influence of the iodine ions. It should be remarked here that Lambert, who used iodine, also noted an extreme tendency of the culture media to liquefy.

DISCUSSIONOF RESULTS In comparing our results with those of the authors mentioned above, it should be emphasized that a comparison is made difficult, not only because tissues from different species were used (man, guinea-pig, rabbit, chicken), but also several methods of experimentation were employed. The table on pages 248-249 gives a survey of the work done and of the results obtained by several authors. The experiments can be conveniently divided into three groups; those in which only iodine was used, those in which the iodine was made available in the form of an iodine-potassium iodide solution (I' Lugol ") , and those in which the iodine was added in the form of one or more iodides. As far as the first group is concerned, the various statements do not con- flict. There is, however, a great divergence of opinion among the authors of the second group, who all studied the effect of an iodine-potassium iodide solution on heart fibroblasts of the chick embryo. Although further experiments would be necessary to clarify the existing discrepancy, it must be remarked here that the results thus far obtained are hard to interpret because of the complicated chemical composition of the aforementioned solution. This solution contains not only the iodine and the iodide used in its preparation, but also iodine in the form of a tri-iodide. Any deviation of growth activity from that of control cultures is necessarily caused by the combined action of iodine, iodide, and tri-iodide, aside from the fact that in most experiments a change in the osmotic pressure of the culture medium and, inevitably, a change in the relative amounts of potassium and sodium are made. In other words, so many factors are altered that a correct interpretation of the results is well-nigh impossible. In comparing the first and second groups, the prevailing discord seems hardly explainable by the fact that tissues from various species were used and that an iodine-iodide solution will necessarily affect the tissue 'cells in a different way from pure iodine. Concerning the third group, the results of Pitini and Olivo may seem ir- reconcilzible. However, in the experiments of the former it must be considered that although the concentration of the iodide in the capillary was 10 per cent, the concentration in which the iodide actually reached the cells was much lower. It should also be kept in mind that whereas Olivo worked with chick embryo tissue, Pitini utilized that of the cavy. It seems worthwhile to compare the results of Olivo with those of Cervello and Levi. In some experiments, the latter added Lugol's solution to the plasma in the ratio 1 :3, in which case the 248 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN

Experimental Procedures and Results

Author Technic Concentration Buchsbaum and Bloom. .Iodine added directly to the culture I 1:2000 to medium. 1 :150OO

Lambert ...... Tissue treated with iodine solution I 1:2000 before being placed in culture me- for 1 hour dium. Hanging drop cultures. I I 1:5000 for 1 hour

Lambert and Meyer...... Same technic. I 1:lOOO for 20 min. I 1:2500 for 20 min.

Cervello and Levi ...... I-KI solution added directly to the I 1:800 culture medium. Hanging drop cul- I 1:2200 i tures. I 1:3200

Crainz ...... Same technic. Subcultures made I 1:4000 every 3-4 days. I 1:6000 I1 I 1:20000

1 Salle and Lazarus ...... I-KI solution placed over cultures embedded in plasma. Carrel flask cultures. I 1:1800 ...... Iodide solution in capillary near ex- plant. Hanging drop cultures. 0.6 M KI

...... Iodide solution added directly to the 0.015 M KI culture medium. 0.027 M KI Tissue treated with iodide solution 4-5 hours before being placed in culture medium. 0.054 M KI I Hirashima...... Iodide solution added directly to the 0.0025 M NaI culture medium, Hanging drop cul- 0.0100 M NaI I11 tures subcultured every 2 days. 0.0250 M NaI 0.1000 M NaI

Vogelaar and Erlichman. ..Chlorides of control medium ex- Iodine content changed for equivalent amounts of of medium the corresponding iodides. equivalent to that of 0.038 M NaI 0.075 M NaI 0,126 M NaI INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 249

Obtained by Different Workers

Tissue Results Fibroblasts of the chick embryo I 1:4000, the greatest concentration in which cells show approximately normal growth. Observation period 2 days. Human lymph nodes and spleen Cells killed with I 1:1250. Good growth with I 1:2000 and I 1:5000

Rabbit spleen Cells killed with I 1:lOOO. Survive treatment with I 1:1250. Fibroblasts and large mono- nuclear cells about equally resistant to the iodine.

Heart, skin, intestine, and kidney of the chick embryo

Heart fibroblasts and liver epithelium The addition of some iodine is advantageous of the chick embryo to fibroblasts. I 1:20000 stimulates growth. With I 1:4G00 less growth than controls. .I1 Liver epithelium cells are less sensitive than fibroblasts. Cells adapt themselves to action of iodine. Observation period up to 47 days. Heart fibroblasts of the chick embryo

Spleen and sciatic nerve of the guinea- Potassium iodide has a growth stimulating pig embryo and young animals effect on spleen and nervous tissue.

Heart and skin of the chick embryo Normal growth obtained with 0.015 M KI. Subnormal growth with 0.027 M KI. Treat- ment of tissue for 4-5 hours with 0.054 M KI (0.9ojO) has no harmful effect.

Heart fibroblasts of the chick embryo. A vigorous growth obtained with 0.0025 M NaI although less than that of control cultures. With higher concentrations the growth .I11 is less. 0.1 M NaI is very harmful. Obser- vation period covering four generations of cultures. Fibroblasts of adult human thyroid A normal growth obtained in media containing gland iodine equal to that of 0.038 M and 0.075 M NaI solutions. In a medium corresponding with 0.126 M NaI the growth is sometimes normal, sometimes less than normai. Obser- vation period 4 weeks. 250 JOHANNES P. M. VOGELAAR AND ELEANOR ERLICHMAN

potassium iodide concentration in the culture medium must have been as high as 0.075 M, if we choose to neglect the formation of tri-iodide. This concentration, with which a normal growth activity of guinea-pig tissue was secured, is about three times as high a concentration as that with which Olivo noted a subnormal growth of chick embryo tissue. It is no less than thirty times as high as the concentration with which Hirashima observed a good growth of chick fibroblasts, although less than that of control cultures. This discrepancy certainly cannot be ascribed to the fact that different iodides were used. In regard to our own work, it is clear from our formulae that the media were prepared by employing not only one iodide, but no less than four, namely, sodium iodide, potassium iodide, magnesium iodide, and calcium iodide. This may seem an unnecessary complication, unless it is realized that by replacing the chlorides of the control medium by equivalent amounts of the corresponding iodides, culture media are obtained which differ only in the relative amounts of chlorine and iodine, whereas the sodium, potassium, magnesium, and calcium content of all the media remains the same, The use of more than one iodide is imperative if a culture medium is desired with a larger amount of iodine than that attained when all the sodium chloride of the control medium is replaced by an equivalent amount of sodium iodide. From this it follows that any appreciable difference in growth activity must be attributed to the presence of the iodine and not to a disturbance of the ratio of other elements. The media made up with iodine have a slightly higher osmotic pressure than the control medium, a variance which could be neglected because a satisfactory growth was achieved with the medium containing the greatest displacement of the chlorine by the iodine, technically possible. If we take into account that sometimes the growth in medium D was less than that of control cultures, we may make the generalization that a medium containing iodine in an amount equal to that of a 0.075 M sodium iodide solution is suitable for a normal growth of human fibroblasts. This molarity is identical with that of the potassium iodide in the medium used by Cervello and Levi. There is a considerable dissimilarity between our results and those of Hirashima. However, he studied chick fibroblasts by employing the hanging drop method, which necessitated subculturing every forty-eight hours, whereas we cultivated human thyroid fibroblasts in Gabritschewsky dishes and subcultured at fourteen-day intervals. It is quite possible that during this long period of undisturbed growth the fibroblasts had the opportunity to adapt themselves better to the changes in the culture medium. It is also conceivable that the thyroid fibroblasts have a special resistance to iodine since they come from an organ so intimately bound up with the iodine metabolism of the body. However, Cervello and Levi also secured a good growth with a very high concentration of iodide, although they used fibroblasts of non-thyrogenic origin. Our experiments should be repeated with several strains of human fibroblasts obtained from other organs of the body in order to determine whether the thyroid fibroblasts have a greater resistance. It is obvious that the experiments reported in this article do not answer the important question whether iodine is an element absolutely necessary for the well-being of human thyroid fibroblasts. Only when cultures can be grown over a long period in media of known composition will the answer be possible. INTERCHANGEABILITY OF CHLORINE AND IODINE IN CULTURE MEDIA 251

Our results, however, do prove that chlorine and iodine in the culture medium are to a large extent interchangeable, Whether all the chlorine can be replaced by iodine is a problem, the solution of which is left to the future.

SUMMARY (1) The literature dealing with the effect of iodine and iodine compounds on the growth of various tissues in vitro is critically discussed. (2) Formulae of salt solutions for the preparation of culture media containing different amounts of iodine are given. (3) By ustng these solutions it is possible to replace approximately 80 per cent of the chlorides of the control medium by an equivalent amount of the corresponding iodides, without changing the sodium, potassium, magnesium, and calcium content of the medium. (4) A good growth of human thyroid fibroblasts is obtained in a culture medium containing iodine tantamount to that of a 0.075 M sodium iodide solution, whereas in a medium equal to 0.126 M NaI the growth is either normal or less than normal. (5) Since thyroid fibroblasts come from an organ so intimately bound up with the iodine metabolism of the body, experiments should also be performed with strains of human fibroblasts obtained from other organs. (6) The elements chlorine and iodine are to a very large extent interchangeable in a culture medium for human thyroid fibroblasts. NOTE: It gives us great pleasure to acknowledge the cooperation received from the staffs of the operating rooms of St. Luke’s Hospital and the Presbyterian Hospital. We especially wish to express our sincere gratitude to Miss Florence Manne of the Presbyterian Hospital for her considerable assistance in obtaining thyroid specimens for us.

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