The effect of nitrilotriacetate on the urinary elimination of sulfated mucopolysaccharides in rats

TOXICOLOGYANDAPPLIEDPHARMACOLOGY42, 617--619 (1977)

The Effect of Nitrilotriacetate on the Urinary Elimination of Sulfated Mucopolysaccharides in Rats

The Effect of Nitrilotriacetate on the Urinary Elimination of Sulfated Mucopolysaccharides in Rats. HILES, R. A. (1977). Toxieol. Appl. Pharmaeol. 42, 617-619. The hypothesis that increased renal excretion of sulfated mucopolysaccharides in rats fed nitrilotriacetate (NTA) could cause the renal toxicity observed in feeding studies was tested. The sulfated mucopolysaccharides were labeled in vivo with 35S. Subsequent exposure to a dietary level of 2% N%NTA.H20 did not cause an increase in the urinary excretion of total or nondialyzable 3% over that of control rats. Thus, the stripping of mucopolysaccharides from the renal system as a cause of NTA toxicity was not supported.

Acute oral exposure to nitrilotriacetic acid (NTA) in dogs causes an elevation in the urinary excretion of chondroitin sulfate, a sulfated mucopolysaccharide (Jarrett and Jaques, 1971; Jaques and Sue, 1975). In rats, high dietary levels (0.15 and 0.5%) of N a 3 N T A . H 2 0 caused kidney damage, primarily in the tubular epithelium (Nixon et al., 1972). Jaques and Sue (1975) hypothesized that this damage (nephritis and nephrosis) could be related to the excretion of sulfated mucopolysaccharides. It seemed reasonable that, if the N T A was disrupting the protective mucopolysaccharide layer from the kidney tubules, then damage to the renal tubules would result. The experiment described here was designed to determine whether a high level of N A 3 N T A . H 2 0 in the diet of rats causes an elevation in urinary sulfated mucot?olysaccharides. Specifically, sulfated mucopolysaccharides were labeled in vivo with: 35S, and the effect of dietary N a 3 N T A - H 2 0 on urinary 3~S was evaluated.

METHODS N a 3 N T A . H 2 0 was obtained from Monsanto Chemical Co,, 1 and Na235SO4 (6.7 gCi/mg) was from New England Nuclear. 2 Twelve male Charles River (CD) 3 rats (200 to 250 g) were housed in individual cages, given free access to water, and fed ground rat chow. 4 Each animal was given a 10-gCi ip dose of Na235SO4. Forty-eight hours after the 35S dose, six animals were placed on a diet of ground rat chow containing 2% (w/w) of N A 3 N T A . H 2 0 , while the other six were kept as controls on chow alone. Total urine was collected for 12 days after the 35S dose. 1Monsanto Chemical Co., St. Louis, Mo. z New England Nuclear Co., Boston, Mass. 3 Charles River Breeding Lab., Wilmington, Mass. 4 Purina Chow, St. Louis, Mo. Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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The amount of 35S in each urine sample was determined using liquid scintillation counting. A portion of each urine was dialyzed in a cellulose bag 5 against running water for 48 hr, and the 35S was determined in the dialyzed urine. The percentage of the dosed 35S in each daily undialyzed and dialyzed urine sample was calculated. RESULTS AND DISCUSSION It is known that SO 2- provides the sulfate for sulfated mucopolysaccharides in rats (Kent, 1961). Rats given an ip or iv injection of 35SO2- fix ~5% of the 35S into mucopolysaccharides during the first 24 hr and eliminate most of the remaining 35S in the 10.0

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FIG. 1. The elimination of 35S in the urine of rats dosed ip with 3sSO42- and fed either control or N%NTA.H20-containing diet. Animals were dosed ip with Na~35SO4 on D a y 0. After 48 hr, half were placed on a 2% Na3NTA. HzO diet. The percentage of the dosed 35S in each 24-hr urine collection was determined both before (circles) and after (squares) dialysis. The figure shows average values of six animals per group with their SE for control (open symbols) and NTA-fed (closed symbols) rats. 35S concentrations beyond the fifth day in the dialyzed urine samples were below detection.

urine as SO 2- (Bostr6m and Gardell, 1953; Bostr6m and Aquist, 1952; Everett and Simmons, 1952; Dziewiatkowski, 1949). Mucopolysaccharides will not pass through a dialysis membrane whereas SO42- will (Scott, 1960). Thus, it is reasonable to expect that an agent which causes an increase in the urinary excretion of sulfated mucopolysaccharides will cause an elevation of urinary 35S in animals previously dosed with Na235SO4. The sensitivity of the test should be increased by looking only at the portion 5 Curtin Matheson Scientific, Cincinnati, Ohio; Catalog No., 077-024.

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of the urine that remains after unmetabolized SO 2- and low-molecular-weight sulfate compounds have been removed by dialysis. Figure 1 shows graphically that the NTA-fed rats and the control rats were identical in their urinary disposition of ip-dosed 35S. There were no significant differences in the 35S concentrations of either the undialyzed or the dialyzed urine. The dialyzed urines after the fifth day contained too little 35S for detection. Thus the urinary 35S assay indicates that N T A does not cause an elevation in urinary mucopolysaccharides, and our results do not support the hypothesis that N T A causes renal tubule damage in ratfeeding studies by removing the protective mucopolysaccharides from the tubular epithelium. ACKNOWLEDGMENTS The technical assistance of D. Caudill, R. Schneider, and H. Lampe was appreciated. REFERENCES BOSTR6M, H., AND AQUIST,S. (1952). Utilization of asS-labeled sodium sulphate in the synthesis of chondroitin sulphuric acid, taurine, methionine and cystine. Aeta Chem. Seand. 6, 15571559. BOSTR6~a, H., AND Gardell, S. (1953). Uptake of sulfates in mucopolysaccharides esterified with sulfuric acid in the skin of adult rats after intraperitoneal injection of 3sS-labeled sodium sulphate. Aeta Chem. Seand. 7, 216-222. DZmWIATKOWSKI,D. D. (1949). Rate of excretion of radioactive sulfur and its concentration in some tissues of the rat after intraperitoneal administration of labeled sodium sulfate. J. Biol. Chem. 178, 197-203. EVERETT, N. B., AND SIMMONS, B. S. (1952). The distribution and excretion of 35S sodium sulfate in the albino rat. Arch. Bioehem. Biophys. 35, 152-156. JAQUES, L. B., AND SUE, T. K. (1975). Mobilization of chondroitin sulphate by nitrilotriacetic acid. ToxieoL AppL Pharmaeol. 34, 521-524. JARRETT, C. L., AND JAQ~ES, L. B. (1971). Effect of iv and oral administration of heparinoids G31150, G31150-A and nitrilotriacetic acid on blood coagulation. Thromb. Diath. 25, 187-200. KENT, P. W. (1961). Some biochemical aspects of sulfate mucosubstances. In The Biochemistry of Mueopolysaeeharides of Connective Tissue (F. Clark and J. K. Grant, eds.), pp. 90-108. Cambridge University Press, England NIXON, G. A., BUEHLER,E. V., AND NIEWENHUIS,R. J. (1972). Two-year rat feeding study with trisodium nitriloacetate and its calcium chelate. Toxieol. Appl. PharmaeoL 21,244--252. SCOTT, J. E. (1960). Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. In Methods of BioehemieaI Analysis (D. Glick, ed.), Vol. 8, pp. 145-197. Interscience, New York. RICHARDA. HILES

The Procter & Gamble Company Miami Valley Laboratories Cincinnati, Ohio 45247 Reeeived May 27, 1977; accepted July 6, 1977