Studies on the Metabolism of Thioacetamide-S35 in the Intact Rat*

ODDVARNYGAARD,LORENTZELDJARN,ANDKARLFORDNAKKEN (Norsk Hydro's Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway)

Through the work of Fitzhugh and Nielson (5) ing of TAA with radioactive sulfur on a micro and Ambrose et al. (1), it has been established that scale were studied, and the procedure finally thioacetamide (TAA) in moderate doses (25 worked out was as follows : mg/kg/day) induces liver cirrhosis in rats, when One hundred mg. of barium sulfate-S35 was re administered over a period of several months. Ac duced with carbon monoxide to barium sulfide, as cording to Rather (7), the earliest morphological described elsewhere (3). The barium sulfide was effect is an increase in the size of the nucleoli and transferred to the centrally suspended cup A of the nuclei of the parenchymal cells. These changes the diffusion apparatus shown in Chart 1. In the are well developed within a few days and are outer compartment was placed 200 mg. of un- largely reversible. labeled TAA dissolved in 9.5 ml. of "super dry" From measurements of nuclear volume and ethyl alcohol1 and 0.5 ml. of 0.05 N sodium alco analyses of desoxyribonucleic acid (DNA), Carnes hÃ³late in absolute alcohol. The apparatus was and co-workers (2) have found a proportionality flushed with dry oxygen-free nitrogen and stop between nuclear volume and DNA content in the pered, leaving a gas phase of approximately 20 ml. hepatic cells of TAA-treated rats. However, Through the greased rubber stopper was intro Laird (6) and Thomson et al. (8) have failed to find duced a hypodermic needle, and the barium sul an increase in the average content of DNA per fide was acidified with 1.5 ml. of 75 per cent nucleus after administration of TAA, although phosphoric acid by means of a syringe. The needle they observed the morphological changes de was carefully withdrawn to avoid loss of hydrogen scribed by Rather. On the other hand, Laird found sulfide. The vessel was kept in the dark at room a considerable increase in nuclear protein and temperature and with magnetic stirring in the pentosenucleic acid (PNA) which reached a maxi outer compartment for 4-7 days. After this time mum after 7-11 days. The liver is the only organ two hypodermic needles were introduced through that appears to be affected by the administration the stopper. Hydrogen chloride in absolute ethyl of TAA (1,5,7). alcohol was added in an amount sufficient to To study the metabolism of TAA, the com neutralize the sodium alcohÃ³late. The hydrogen pound was labeled with S35.In this paper we shall sulfide was flushed out with nitrogen and trapped present some data on the gross metabolism of in sodium hydroxide. The TAA solution was TAA and on the distribution of TAA and total evaporated to dryness under reduced pressure and sulfur radioactivity in various tissues after sub the TAA recovered and purified by recrystalliza- cutaneous injection of the labeled compound into tions from benzene. The recovery of pure TAA rats. (m.p. 107.5Â°-108.5Â°C.)was 50-60 per cent of the EXPERIMENTAL amount initially added. The compound traveled as a single spot in two different paper chromato- The synthesis of thioacetamide-S36.â€”The ex graph systems.2 change reaction At equilibrium the radioactive sulfur will be CH3CSNH2 + SH- ^ CH3CSNH2 + SH~ distributed between the TAA and the hydrogen 1According to Lund and Bjerrum. See A. I. Vogel, A which we found to take place in absolute ethyl Textbook of Practical Organic Chemistry, 2d ed., p. 166. alcohol was used because of its simplicity and London: Longmans, Green & Co., 1951. relatively high yield. The conditions for the label- 1The chromatograms were run on Whatman No. 1, grade 1 * This project was supported in part by a grant from paper, in one system with isopropanol:ethanol:N HC1 (3:3:1) Landsforeningen mot Kreft, Oslo, Norway. on untreated paper, and in the other with isopropano] :ethanol: water (2:2:1) on paper pretreated with 0.05 M phosphate Received for publication April 1, 1954. buffer at pH 7.5 in 0.5 MKC1. 625

sulfide according to their absolute sulfur contents, TAA, evaporation of the solution to dryness under and the maximum obtainable specific activity can reduced pressure, and extraction of the residue be calculated accordingly. In Chart 2 is shown the with hot benzene. The isolated TAA was recrystal- percentage of maximum specific activity obtained lized from hot benzene several times after the cor in TAA after various equilibration times. After 4 rect melting point was obtained. Since wet oxida days 80 per cent of the maximum activity was tion with copper nitrate and sodium perchlorate reached. With the amounts of TAA and barium gave low recoveries of TAA sulfur as sulfate, the sulfide used the over-all yield of radioactive sulfur samples for TAA and total sulfur were oxidized as TAA was about 40 per cent. When the reaction by fusion with sodium peroxide in a steel bomb. The aliquots for the determination of total sulfur were evaporated to dryness under reduced pres sure, after the addition of a known amount of sucrose to provide bulk for the residue. A weighed amount of the total residue was transferred to the bomb and oxidized. All samples were assayed for radioactivity as previously described (4). Distribution of injected thioacetamide in various rat tissues.â€”A male rat, weighing 260 gm., was given a subcutaneous injection of 6.5 mg. (ap proximately 50 /ic.) of labeled TAA. The animal was killed after 85 minutes by a blow on the head, and oxalated blood was immediately collected by

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Chart 1.â€”Sectionthrough diffusion apparatus. A.â€”Central cup, suspended by thin steel wires. 20 it.â€”Magnet stirring bar. O S.â€”Greased rubber stopper with injection needle insert ed. The stopper is kept firmly clamped during the Ã• equilibration phase to prevent leakage of hydrogen 123*5 sulfide. DAYS OF EQUILIBRATION CHART2.â€”Production of TAA-S" from exchange of sulfur mixture was exposed to light, the solution became between unlabeled TAA and radioactive HiS under the condi turbid, and the recovery of TAA was lower. The tions described. The solid line represents the theoretical curve omission of sodium alcohÃ³late decreased the re for a first-order reaction. action rate considerably. High sodium alcohÃ³late concentrations also greatly lowered the recovery of heart puncture. The tissues listed in Table 1, TAA. Experiment 1, were removed, washed quickly with Gross metabolism of tkioacetamide in the rat.â€” cold physiological saline, blotted with a filter Four male rats, weighing 200-235 gm., were each paper, and weighed. The larger tissue samples were given a subcutaneous injection of 6 mg. of labeled forced through a tissue press, and suitable aliquots TAA in 1 ml. of physiological saline. The animals were homogenized with physiological saline in a were kept in metabolism cages, and the urine was glass "Potter-Elvehjem" homogenizer. The collected after 1, 2, 3, 4, and 8 days. Care was smaller organs were homogenized directly. For the taken to avoid contamination with feces. The emulsification of fat tissue, the addition of 0.1 gm. urine samples were analyzed for total sulfur, total of a detergent ("Teepol") proved advantageous. sulfate, free sulfate, and TAA radioactivity. The TAA was isolated from aliquots of the homoge- methods for the analysis of free and total sulfate nates after the addition of carrier TAA and are described elsewhere (4). TAA-S36 was isolated analyzed as described for the urine. For the de from the acidified urine by the addition of carrier termination of total sulfur radioactivity, other

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1954 American Association for Cancer Research. NYGAARDet al.â€”Metabolism of Thioacetamide-S^ 627 aliquots were dried at 40Â°-60Â°C.to minimize losses frequent or continuous administration appears due to sublimation of TAA. The residues were preferable when the effect of prolonged treatment finely ground in an agate mortar and oxidized by is to be studied. Addition of TAA to the diet, as sodium peroxide fusion. The radioactivity of the originally used by other workers (1, 2, 5, 7), thus barium sulfate recovered was determined as de seems advantageous. scribed for urine. In Table 1 are recorded the data obtained for A second male rat, weighing 260 gm., was the distribution of TAA and the total sulfur radio given a subcutaneous injection on 7 consecutive activity in various tissues. Experiment 1 shows days of a total of 40.7 mg. (approximately 350 nc.) that, 85 minutes after injection of TAA, the com of radioactive TAA. The animal was sacrificed 2 pound was highly concentrated in the bone mar days after the last injection. The tissues listed in row, the thyroid, and the adrenals. The liver, on TABLE1 DISTRIBUTIONOFSÂ»INRATAFTERSUBCUTANEOUSINJECTIONOFTAA-SÂ»

1TAA-S"counts/min/gmof2TotÂ»l SÂ»counta/min/gmof SÂ«'counts oforgan(gm.)0.0370.0450.500.600.711.171.732.6S7.041.74EXPERIMENTorgan(gm.)0.0270.0360.850.701.181.522.418.771.45Total/min/gmof TISSUEBone tissue69,00044,00032,0007,7005,7003,7002,7502,6602,1402,0302,0101,7601,5101,130EXPEBIUENTtissue*55,0004,3008,1004,2008,7004,5009,2003,0001,0704,500Wt.oftissue1,5703,1002,1209706101,7903001,4001,000710650 marrowThyroid glandAdrenalsSpleenEpididymisHeart

muscleLungsStriated muscleKidneysBlood serumTestesBlood corpusclesLiverBrainWt.

Experiment 1.â€”SÃ•min. after the injection of a single dose of TAA-SÂ»corresponding to 0.87 X 10* counts/min. Experiment Ã•.â€”After7 daily injections of TAA-S" followed by 2 days during which no TAA was administered. Total dose corresponds to 4.S X 10Â«counts/min. * Some of the total SÂ»values are obviously too low. See text for explanation.

Table 1, Experiment 2, were collected and pre pared as for the above rat, with the exception that only total sulfur radioactivity was determined. Total S RESULTS AND DISCUSSION The results from the study on the gross metabo lism of TAA, expressed as per cent of administered radioactivity, are shown in Chart 3. It is apparent Total so. that the elimination of the injected radioactivity is very rapid, the major share being excreted Frt* during the first 24 hours. The transformation of TAA to other metabolites must also proceed at a very high rate, since very little S36was excreted as

TAA after the 1st day. Sulfate was the main Organic S metabolic product of TAA-sulfur, and the sulfate was distributed between esterified and free sulfate in proportion to their absolute amounts. The excretion of sulfur-containing metabolic products of TAA, other than sulfate, did not exceed 3 per cent of the injected S36.Eleven per cent of the in jected radioactivity has not been accounted for in 2 3 + S the urine and is presumably present in the tissues DAYS AFTER INJECTION or feces. CHART3.â€”Recoveryof S" in urine following a single sub Because of the rapid metabolism of TAA a cutaneous injection of TAA-S34in rats.

the other hand, does not appear to have an excep which will normally cause liver changes, and from tional affinity for the compound, since the concen the work of Rather (7), who injected TAA directly tration was very similar to that found in blood and into various rat organs other than liver. In both in most tissues. The values recorded for "total cases the liver was the only organ exhibiting the S36" under Experiment 1 are probably too low. typical signs of TAA poisoning. The striking Thus, the values for "total S36"in liver and spleen specific action of TAA may depend on "a special were lower than those found for TAA in the same metabolic characteristic of the parenchymal liver organs. This, in all probability, is due to sublima cell" as suggested by Rather (7). It is possible that tion of TAA from the samples during the drying TAA or a metabolite of TAA interferes with period. However, the "total S35" values give an enzymatic reactions specific for the liver, or that a upper limit for the concentrations of S35-contain- toxic metabolite of TAA is formed in the liver ing compounds other than TAA. It is of interest only and exerts its effect at the site of formation. that for the kidneys the ratio of total sulfur to TAA radioactivity is of the same order as that, SUMMARY found in the urine. 1. A method for the labeling of thioacetamide Experiment 2, Table 1, shows the data obtained (TAA) with radioactive sulfur has been described. for total sulfur radioactivity after seven daily in 2. When TAA-S36 was injected subcutaneously jections of TAA-S35, followed by a 2-day period into rats, more than 80 per cent of the radioactiv during which the animals received no TAA. Ac ity was excreted in the urine during the first 24 cording to the data in Chart 3 one should expect hours. Approximately 25 per cent was recovered the bulk of the TAA and sulfate radioactivity to as unaltered TAA, while most of the remainder have been eliminated by this time. If a tissue were was accounted for as free and esterified sulfate. able to specifically incorporate TAA-sulfur into 3. Following injection of TAA-S36 the bone cell constituents, and the metabolism of the marrow, thyroid, and the adrenals of the rat con products were not too rapid, this tissue should centrated the compound temporarily to a con show a greater retention of S36 than would the siderable degree. However, no tissue showed any others. The data do not indicate such a specific exceptionally great retention of radioactive sulfur retention of S36.The thyroid and adrenals again after repeated injections of TAA-S36. had the highest concentrations, but the values are 4. The liver, although it is the only organ show not much higher than those found for the other ing morphological changes after treatment with tissues. The value for the liver falls well in line TAA, did not concentrate TAA-S36 nor retain S'5 with those for the other tissues. The morpho to any greater extent than the other tissues. logical changes described by Rather (7) were readily demonstrated in the liver of this rat.3 REFERENCES 1. AMBROSE,A.M.; DEEDS, F.; and RATHER,L. J. Toxicity Some of the possible mechanisms by which TAA of Thioacetamide in Rats. J. Ind. Hyg. & Tox., 31:158-61, might affect the liver cells have been discussed by 1949. Rather (7). From the data in the present report it 2. CARNES,W. H.; BERNSTEIN,D. S.; and RATHER,L. J. is apparent that the high sensitivity of the liver to Effect of Thioacetamide on the Desoxyribonucleic Acid TAA is not due to a specific ability of the liver to Content of Liver Cells. Fed. Proc., 12:385-86, 1953. concentrate this compound. Moreover, other 3. ELDJARN,L. An Improved Method for the Synthesis of Potassium Thiocyanate Labeled with Radioactive Sulphur. tissues, such as the thyroid and the adrenals, show Acta. Chem. Scandinav., 7:343-46, 1953. a marked ability to concentrate TAA but are not 4. ELDJABN,L., and NYGAABD,0. Comments on Methods in similarily affected. The lack of effect on these Biological Work with S36-Labeled Compounds. Scandinav. tissues also renders it unlikely that the suscepti J. Clin. & Lab. Invest., 6:160-67, 1954. 5. FITZHUOH,O.G., and NIELSON,A. A. Liver Tumors in Rats bility of the liver to TAA poisoning is due to a Fed Thiourea or Thioacetamide. Science, 108:626-28, 1948. lower critical concentration for TAA in this 6. LAIRD,A. K. Nuclear Changes Induced in Rat Liver Cells organ. Further evidence against a "critical con by Thioacetamide. Arch. Biochem. & Biophys., 46:119-27, centration" hypothesis is obtained from the work 1953. of Fitzhugh and Nielson (5), who gave TAA to 7. RATHER,L. J. Experimental Alteration of Nuclear and Cytoplasmic Components of the Liver Cell with Thio rats in dietary concentrations far exceeding that acetamide. I. Early Onset and Reversibility of Volume 3Since Laird (6) has shown that there is an increase of Changes of the Nucleolus, Nucleus and Cytoplasm. Bull. PNA in the nuclei of TAA-treated animals, a search was made Johns Hopkins Hosp., 88:38-58, 1951. for the possible occurence of anomalous, sulfur-containing PNA 8. THOMSON,R. Y.; HEAGT,F. C.; HUCHISON,W. C.; and produced under the influence of TAA. PNA isolated from vari DAVIDSON,J.N. The Deoxyribonucleic Acid Content of the ous cell particulates showed the presence of some S36,but the Rat Cell Nucleus and Its Use in Expressing the Results of amounts were so low that contamination with radioactivity Tissue Analyses, with Particular Reference to the Composi from other sources seems the most likely explanation. tion of Liver Tissue. Biochem. J., 63:460-74, 1953.

Oddvar Nygaard, Lorentz Eldjarn and Karl Ford Nakken

Cancer Res 1954;14:625-628.

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