TOXICITY OF MESOXALATES AND HISTOLOGICAL OBSERVATIONS AFTER CONTINUOUS ADMINISTRATION

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1 TOXICITY OF MESOXALATES AND HISTOLOGICAL OBSERVATIONS AFTER CONTINUOUS ADMINISTRATION YOSHITO KOBAYASHI, SHIGERU OHASHI AND SETSUYA TAKEUCHI Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo YOSHIO IKEDA National Hygienic Laboratory, Tokyo Received for publication August 24, 1954 The results of experiments on the antidiabetic activity of calcium and sodium me soxalate were reported (1). This is a report on the acute and chronic toxicity of these drugs. EXPERIME-, i AL Acute Toxicity in th, Rabbits. For oral administration, both the calcium and, so dium mesoxalate used in the experiment were ground into a very fine power and syring ed into the stomach with a small amount of water through a No. 10 catheter. Obser vations for several days after administration revealed no unusual change although single doses as large as log were given. It was clear that an acute oral toxic dose could not be determined practically. For intravenous administration, a 3?0 (solubility: 4 g/100 cc at 23 C), was injected into the ear-vein. With single doses as large as 3 g no unusual signs were noted. Since it is impossible to inject more than 3 g intravenously without causing overhydration, acute toxicity could not be evaluated. Chronic Oral Toxicity in the Rabbits. Thirty rabbits, weighing about 1.6 kg each, were selected at random and divided into 4 groups. Three groups, each composed of 8 rabbits, were used for this experiment, while the other 6 rabbits formed a control group. The 3 experimental groups were fed daily amounts of 50 mg/kg, 100 mgjkg and 200 mg/kg of calcium mesoxalate respectively, mixed with a small amount of carrot. Body weights and general condition were noted every 2 weeks, and from each group 1 or 2 animals were sacrificed every 3 months for histological examination of the pancreas. Observation and experiment were continued for 15 months at the longest with each group. Animals that died incidentally during the test were excluded from histological study. None of the 3 expe: imental groups showed any difference in the rate of growth and general health as compared with the control group. Toxic effects from continued oral

2 administration of the drug were not apparent. In each group, from second week through the fifteenth month, 3 specimens of pan creatic tissue were taken from the duodenal-, gastric and splenic-portions of the gland. Fixation was made with Bouin's fluid, and Gomori's chrome alum hematoxylin stain was used to differentiate between A and B cells in the islets. In each instance 50 islets of Langerhans were selected from the tissue specimens, comprised of 20 from the duodenal part, 20 from the gastric and 10 from the splenic part. In each islet two dia meters were measured at right angles to each other. In this way one hundred diameters were determined, from which a mean diameter was calculated for each animal. For each of the 50 selected islets, the total number of A and B cells were counted. From this the average A and B cell count for the 50 islets was expressed. Any other histo logic changes were also noted. The detailed results of these findings are summarized in Table 1. It will be seen that the mean islet diameters ran from 72.2,u to 81.9,u in the control group, while in the experimental groups, there is a mean diameter of more than 85,u in 9 animals and less than 70,u in 2 animals. An increase is observable especially after the 4th month. The A and B cell count increased considerably in all the rabbits of the test group given the drug for 4 months. In A cell count, 3 animals showed a decrease. In two of these, the decrease took place in the 2nd and 4th week. An increase in A cell count appeared in 4 animals, all after the 8th month of drug feeding. In B cell count, 2 ani TABLE 1. Observation of islets of Langerhans in rabbits

3 mals showed a decrease, and 6 an increase which was most marked in the 4th month. The proportion' of B to A cells increased in the 4th week and in the 4th month of drug administration. The increase at the early stage is due mainly to the decrease in A cell` count, while that at the later stage to the increase in B cell count. The A cell count returns to the normal level by the 7th week, hence no change is seen in the proportion of B to A cells at this stage : the drop in the proportion of B to A cells after the 8th month is due more to the increase in A cells than to the decrease in B cells. The greatest cellular changes were most perceptible in the 4th month. There was marked proliferation of B cells with nuclear swelling and appearance of giant nuclear cells. In some areas B cells were seen to form a characteristic alveolar struc ture and were closely clustered on the periphery of the islet. Normally the histological picture of the islets of Langerhans shows a funicular structure (Figures 1 and 2). These histological changes parallel closely the change in the cell count. After the third month, hydropic degeneration, pyknotic nuclei, and decrease in granules were discerned in some of the experimental groups, but similar cellular changes were also seen in the control group. However, hydropic degeneration was more frequent and more -:severe in the experimental groups after the eighth month. decrease in granules of the A cells was seen in some cases after the third month. Acute Toxicity in the Rat. Calcium or sodium mesoxalate in the form of fine powder was suspended in tragacanth solution and syringed by catheter into the stomach administered calcium mesoxalate for various periods

4 FIG. 1. Islet of Langerhans in a normal rab bit (control). Stained by Gomori's method ; x 400. The isl t of Langerhans shows fu nicular or reticular structure, abound ing in B cells and scant in A cells strewn sporadically 'on the periphery of the islet. FIG. 2. Islet of Langerhans from a rabbit ad ministered 100,ng/kg of calcium mes oxalate continuously for 4 months. Stained by Gomori's method ; x 400. The islet is large and is made up almost exclusively of B cells in strik ing contrast with Fig. 1. Note the marked proliferation of B cells forming alveolar structures. of each of 16 rats, weighing more than 300 g. I)osings were performed several times a day, but no significant changes were detected even after a dose as large as 3 g a day. Also the sodium salt injected subcutaneously up to the maximum in single doses as large as 1 g did not result in any signs of poisoning. The injection of more than 1 g subcutaneously into the rat was not possible and determination of the minimum toxic dose could not be made. Chronic Oral Toxicity in th. Ra!. Sixteen rats, each weighing about 70g, were se lected at random and divided into 4 equal groups. One group was used for control and the other 3 groups were offered doses of 50 mg-kg, 200 mg;kg and 500 mg/kg of calcium mesoxalate mixed with a small amount of wheat flour every morning. After the animals had completely consumed the drug, they were fed wheat flour and green vegetables. Body weight and general condition were carefully observed and histologi cal examination of several internal organs was made on the 3rd, 7th, 12th, and 14th months in each group. None of the experimental groups showed any unusual differ ence in rate of growth as compared with the control group (Fig. 3). Specimens taken from the pancreas were fixed by Zenker's solution and formalin, dehydrated, embedded in paraffin, and stained with hematoxylin and 'eosin. The is lets from experimental animals in the third month showed little difference from those of the cotrols. On the other hand, considerable cellular change was observed after the seventh month : viz. the size and number of the pancreatic islets increased in all ex perimental animals but one, this increase being relatively marked in the 50 mg;'kg and 200 mg/kg groups, in which several islets were seen to merge to form a very large island of Langerhans. Sometimes small islets of Langerhans, presumably newly formed, were observed.

5 FIG. 3. Average weight curves of rats receiv ing 50, 200 and 500 mg/kg per d-y of Ci mesoxalate orally, without force feeding. A cells showed a general tedency to decrease and B cells to increase, with a notice able proliferation of B cells on the periphery of the islets. On the other hand, some of the animals which were administered 500 mg/kg and 200 mg/kg for a long period of time showed degenerative changes in some of the B cells : viz. pyknotic nuclei and hy dropic degenerations. However, in the 500 mg kg group there appeared what seemed to be several points of acino-insular transformation, of which the other groups were entirely free. In the 50 mg,'kg group, such degenerative changes in B cells were all but negligible. Further, dilatation of capillaries was noted in many cases. There were no consistent cellular alterations discovered when such organs as liver, kidney and spleen were examined after prolonged mesoxalate feeding. After the chro nic administration of large doses, some changes were detected, but in moderate doses, given for as long as 14 months, no significant pathology appeared. When the daily dietary dose was as large as 200 mg/kg, cloudy swelling, slight necrosis, vacuolization, slight edema and some nuclear changes were noted in the cells of the hepatic paren chyma and of the renal tubules. DISCUSSION There have been many reports dealing with the effects of a variety of drugs on the pancreas. Florentin and Watrin (1) found that thyroxin caused hyperplasia of the islet tissue in guinea pigs. Anselmino, Herold and Hoffmann(3), Richardson and Young (4) reported the same effect in rats with pituitary extract, while Vazquez-Lopez (5) also observed this in mice after oestrogenic hormone. Other investigators have found cellular proliferation and hydropic degeneration in individual cells of the pancreatic islets follow ing various procedures `Richardson and Young (6); _ Brown, Dohan, Freedman, De Moor and Lukens (7) ; Wissler, Findley and Frazier (8) ; Toreson (9)D. Johnson (10) reported

6 that alloxan effects hypertrophy of insulin-producing tissue through the formation of small islets composed mainly of B cells. Recovery from this drug effect requires about 4 days after the alloxan administration. We discovered that administration of calcium mesoxalate causes a marked proliferation of B cells. When calcium mesoxalate was administered in large doses for prolonged periods to rabbits (200 mg/kg a day for 8 months) or to rats (500 mg/kg a day for 12 months), pykno tic nuclei and hydropic degeneration, in addition to islet hyperplasia appeared. These changes occurred in the same islet, i.e. a large hyperplastic islet might contain degranulat ed B cells or B cells possessing pyknotic nuclei. These degenerative changes are not seen when small doses are administered for a long period. It is clear that administration of suitable amounts of mesoxalate produce B cell proliferation and that continued large doses result in both cellular proliferation and hydropic degeneration. From previous experiments demonstrating elevated glucose tolerance, together with the present histological findings, we conclude that the mesoxalate in appropriate dose acts on the islets of Langerhans to stimulate the secretion of insulin. As mentioned previously, there is no doubt that the mesoxalates do not directly affect carbohydrate metabolism. Therefore, from the results of our previous and present findings, we are of the opinion that a state of islet hyperfunction precedes the marked proliferative changes seen in the B cells. SUMMARY 1. Both calcium and sodium mesoxalates were administered to rabbits in doses as large as 10 g orally and 3 g intravenously, without any toxic effects noted. 2. Observations on rabbits given daily oral doses of 50 to 200 mg/kg of calcium mesoxalate, and rats given similar daily oral doses for a period of one year revealed no significant deviations from the normal growth pattern. 3. Histological observations were made on rabbits given daily oral doses of 50 to 200 mg/kg of calcium mesoxalate for 15 months. Proliferation of B cells and an increase in the total count of pancreatic islet cells was noted by the third and forth months of drug administration. Also, degenerative changes and pyknosis in some of the B cells were seen when the doses were increased and the periods of administration lengthened. 4. Three groups of normal rats were given daily oral doses of 50 to 500 mg/kg of calcium mesoxalate continously for 14 months. After the sixth month, each experi mental group showed hypertrophy and neogenesis of the islets of Langerhans and a marked increase of B cells inside the islets. The proliferative changes in rats were more marked than those in the rabbits. However, the degenerative changes in B cells, after large and prolonged doses, were not as pronounced as in rabbits. 5. Histological examinations were also made of the liver, kidney, and spleen, but compared with, the control group no substantial histological changes were noted.

7 6. There results, in conjunction with previous work, provide an important experi mental basis for the conclusions that the antidiabetic activity of the mesoxalates is due to an action on the B cells of the islets. This action is considered to stimulate the secretion of insulin. REFERENCES 1) KOBAYASHI, Y., OHASHI, S. AND TAKEUCHI, S.: Jap. j. Pharmacol. 1, 9 (1951) 2) FLORENTIN, P. AND WATRIN, J.: Compt. rend. Soc. biol., Paris 107, 372 (1931) 3) ANSELMINO, K.J., HEROLD, L. AND HOFFMANN, F.: Klin. Wschr. 12,1245 (1933) 4) RICHARDSON, K. C. AND YOUNG, F.G.: J. Physiol. 91, 352 (1937) 5) VAZQUEZ-LOPEZ, E.: Nature 146, 589 (1940) 6) RICHARDSON, K. C. AND YOUNG, F. G.: Lancet 234, 1098 (1038) 7) BROWN, E. M., DOHAN, F. C.,FREEDMAN, L. R., DE MOOR, P. AND LUKENS, F. D. W.: Endo crinology 50, 644 (1952) 8) WISSLER, R. W., FINDLEY, J.W. AND FRAZIER, I. F.: Proc. Soc. Exper. Biol. & Med. 71, 308 (1949) 9) TORESON, W. E.: Am. J. Path. 27, 327 (1950) 10) JOHNSON, D. D.: Endocrinology 46, 135; 47, 393 (1950)