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The effect of acetylsalicylic acid on the development of the skeletal system in rats
Exp. Pathol. 1991; 43: 33-36 Gustav Fischer Verlag lena
National Institute of Occupational Health (General Director: EVA ZSOGON, M. D.), Budapest, Hungary
The effect of acetylsalicylic acid on the development of the skeletal system in rats By E. TATRAI and Gv. UNGVARY With 4 figures Received: November 17, 1989; Revised: February IS, 1990; Accepted: March 26, 1990
Address for correspondence: ERZSEBET TATRAI, M.D., Ph.D., National Institute of Occupational Health, POB 22, H - 1450 Budapest, Hungary Key words: acetylsalicylic acid; mucopolysaccharides; micromelia; phocomelia; sulphation; glycosaminoglycans; skeletal system; development
Summary The authors wished to clarify the cause of micro- and phocomelia developing in rat fetuses following administration of acetylsalicylid acid. It has been proved by histological examinations that the positive ground substance of the epiphyseal cartilage, detected by Rivanol reaction, became disarranged or negative. The sulphation of glycosaminoglycans was diminished or inhibited by acetylsalicylic acid in the chondrocytes depending on the dose applied. The conclusion has been drawn that the malformation developed is due to the reduced production of sulphated mucopolysaccharides.
Introduction Acetylsalicylic acid (ASA) and salicylates cause different skeletal malformations in rats depending on the dose, introduced between the 9th and 12th day of gestation. In addition to the malformation of ribs, vertebrae and sternum ASA caused micro- and phocomelia (6, 11, 12). It also proved to be an inhibitor of chondrogenesis in in vitro limb bud cell culture (9). In these investigations we wished to decide how ASA inhibited chondro- and osteogenesis.
Materials and Methods Adult Wistar rats (LATI-G6d6116) were used. The animals were mated in a harem system. The pregnant animals were treated daily between the 9th and 12th day of pregnancy with 500 mg/kg b.wt. acetylsalicylic acid (CHINOIN, Budapest) through gastric tube (copulation was evidenced by the presence of sperms in estrous vaginal smear, which was considered the 3
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34
Exp . Pathol. 43 (1991) 1- 2
1st day of gestation). The fetuses were removed after Caesarean section on the 21st day of pregnancy and fixed in neutral formalin. The femurs of the fetuses of control pregnants and the shortened femurs of treated pregnants (1 fetus from both 5 litters groups) were processed histologically: hematoxilin and eosin, picrosirius red F3BA stainings were made, alkaline and acidic phosphatase activities were estimated (2). The sulphated mucopolisaccharides were detected by Rivanol reaction between crossed polars (5, 8).
Results No change could be detected in the controls and shortened femurs by means of HE and picrosirius red staining (figs. 1, 2) neither was there any difference in the activities of alkaline and acidic phosphatase. By means of the Rivanol reaction, however a significant difference has been found in the ground substance of the epiphyseal cartilages: the ground substance of the control femurs was a regular network (fig. 3), whereas in treated animals this regular structure could hardly be detected, the ground substance slackened and could not be visualized (fig. 4).
Discussion ASA has a wide mode of action with a well-known role in coagulation (4,7). It inhibits the synthesis of prostaglandin by blocking prostaglandin synthetase, and by uncoupling oxidative phosphorylation (1,3). According to SAITO et al. the resulting malformations may also be partly attributed to the effect of ASA leading to hypocalcaemia (10). We do not think, on the basis of our above results, that ASA has influenced calcification. Rivanol is bound strongly to the sulphated mucopolysaccharides (5,8). Irregularity and decrease in the Rivanol positivity structures pointed to reduced production of the ground substance in the epiphyseal cartilage, i.e., to a damage in the function of chondrocytes. Our studies also proved that acetylsalicylic acid did not damage the collagen structure of the bones and probably did not influence the function of osteoblasts and osteoclasts. This inhibitory effect on the sulphated mucopolysaccharides is due to the inhibition of ATP dependent sulphate ion transport by the uncoupling of ATP-ADP resynthesis and the sulphation of glycosaminoglycans inside the chondrocytes (13). The inhibition of ATP-ADP
Fig. I. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w, of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). The chondrocytes show regular, columnar arrangement. HE staining, X 200.
Fig. 2. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w. of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). Picrosirius red F3BA staining. The appearance of collagen fibres is normal, fibres detected in the periosteum and at the border of the degeneration zone. x 200. Fig.3. Distal epiphyseal cartilage of the femur in a 21-day-old control rat embryo. Rivanol reaction between crossed polars. The ground substance seems to be an oriented network among the chondrocytes. X 200.
Fig. 4. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w. of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). Rivanol reaction between crossed polars. The ground substance was slackened, its positivity diminished in the area of the epiphyseal cartilage (---+). x 200. 3*
Exp. Pathol. 43 (1991) 1-2
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resynthesis, as an essential biochemical process, caused embryolethality in high doses (above 500 mg/kg b.w .). In the dosis applied, in our experiment , the damaging effect manisfested itself in selectivity on the chondrocytes between the 9th- 12th day of pregnancy. The consequence is "malformation" of the epiphyseal cartilage and micro- and phocomelia , depending on the free salicylic acid level.
Acknowledgements The authors express thanks to Mrs. BAJUSZ, Mrs . GYURIK and Mrs. KRAUSZ for skillful technical assistance .
References I. BEYNEN, A. C.; BUECH LER , K. F. , MOLEN, A. J., VAN DER , GEELEN, M . J. H.: Inhibit ion of hepatic lipoge nesis by Salic ylate. To xicology 1982 ; 24 : 33 -43 . 2 . BURSTONE, M. S .: Histochemical comparison of naphtho l AS phosphates for the demonstration of phosphatases. J . Natl. Cance r Inst. 1958 ; 20 : 60 1- 6 16. 3. GURA, F.: Orvosi Biokernia, 2nd Edition, Med icina , Budapest 1988 ; pp. 386-387. 4. FEKETE, GY. , BRAUN, P.: A terapia aktualis kerde sei. Medicina, Budapest 1964 ; pp. 51-74 . 5. KAWIAK , J ., KOWALSKI, H.: Evaluation of the method of rivanol precip itation of [35SJ sulph ated mucopolysaccharides present in the material obtained from a small group of cells . Experientia (Base l) 1964 ; 20 : 397 . 6. KIMMEL , C. A. , WILSON, J. G., SCH UMACH ER, H. J.: Studies on metabolism and identification of the causa tive agent in aspirin teratogenesis in rats. Teratology 1971 ; 4 : 15- 24 . 7. LEWIS, R. B. , SCHULMAN, J. D.: Influence of acetylsalicylic acid , an inhibitor of prostagla ndin synthesis , on the duration of human gestation and labour. Lancet 1973 ; 2 : 1159-11 61. 8 . NEMETH-Cs6KA, M. : A turbid imetric method for the determination of chondroitinsulphuric acid by rivano l. Bioch im. Bioph ys . Acta 1961 ; 50 : 585 -588. 9. RENAULT, J .-Y. , MELCION, C.; CORDI ER, A.: Limb bud cell cu lture for in vitro teratogen screening: Validation of an improved assess ment method using 5 1 compounds tera togen . Carcinoge n. Mut. 1989,9 : 83-96. 10. SAITO, H. , YOKOYAMA, A., TAKENO, S., SAKAI, T., VEND, K., MASUMURA, H. , KITAGAWA , H.: Fetal toxicity and hypocalcaemia induced by acetylsalicylic acid analogues. Res. Comm. Chern . PathoI. Pharmacol. 1982 ; 38 : 209 -220 . II. VNGVARY , GY., TATRAI , E. , L6RINCZ, M. , BARCZA, Gv .: Com bined emb ryotoxic action of toluene, a widely used industrial chemical, and acety lsalicylic acid (aspirin). Teratology 1983 ; 27 : 26 1-269. 12. WARKANY, 1. , TAKACS, E : Expe rime ntal production of congenital malform ations in rats by salicylate poisoning Am. J . Pathol . 1959 ; 35 : 3 15-331. 13. WHITEHOUSE , M . W . , BOSTROM, H .: Stud ies on the action of some anti-inflamm atory agents in inhib iting the biosynthesis of mucopolysaccharid e sulphates. Biochem ical Pharmacology, Vol. 7., pp. 135- 150. Pergam on Press Ltd. , Oxford 196 1.
36
Exp. Patho!. 43 (199 1) J- 2
National Institute of Occupational Health (General Director: EVA ZSOGON, M. D.), Budapest, Hungary
The effect of acetylsalicylic acid on the development of the skeletal system in rats By E. TATRAI and Gv. UNGVARY With 4 figures Received: November 17, 1989; Revised: February IS, 1990; Accepted: March 26, 1990
Address for correspondence: ERZSEBET TATRAI, M.D., Ph.D., National Institute of Occupational Health, POB 22, H - 1450 Budapest, Hungary Key words: acetylsalicylic acid; mucopolysaccharides; micromelia; phocomelia; sulphation; glycosaminoglycans; skeletal system; development
Summary The authors wished to clarify the cause of micro- and phocomelia developing in rat fetuses following administration of acetylsalicylid acid. It has been proved by histological examinations that the positive ground substance of the epiphyseal cartilage, detected by Rivanol reaction, became disarranged or negative. The sulphation of glycosaminoglycans was diminished or inhibited by acetylsalicylic acid in the chondrocytes depending on the dose applied. The conclusion has been drawn that the malformation developed is due to the reduced production of sulphated mucopolysaccharides.
Introduction Acetylsalicylic acid (ASA) and salicylates cause different skeletal malformations in rats depending on the dose, introduced between the 9th and 12th day of gestation. In addition to the malformation of ribs, vertebrae and sternum ASA caused micro- and phocomelia (6, 11, 12). It also proved to be an inhibitor of chondrogenesis in in vitro limb bud cell culture (9). In these investigations we wished to decide how ASA inhibited chondro- and osteogenesis.
Materials and Methods Adult Wistar rats (LATI-G6d6116) were used. The animals were mated in a harem system. The pregnant animals were treated daily between the 9th and 12th day of pregnancy with 500 mg/kg b.wt. acetylsalicylic acid (CHINOIN, Budapest) through gastric tube (copulation was evidenced by the presence of sperms in estrous vaginal smear, which was considered the 3
Exp. Pathol. 43 (1991) 1-2
33
34
Exp . Pathol. 43 (1991) 1- 2
1st day of gestation). The fetuses were removed after Caesarean section on the 21st day of pregnancy and fixed in neutral formalin. The femurs of the fetuses of control pregnants and the shortened femurs of treated pregnants (1 fetus from both 5 litters groups) were processed histologically: hematoxilin and eosin, picrosirius red F3BA stainings were made, alkaline and acidic phosphatase activities were estimated (2). The sulphated mucopolisaccharides were detected by Rivanol reaction between crossed polars (5, 8).
Results No change could be detected in the controls and shortened femurs by means of HE and picrosirius red staining (figs. 1, 2) neither was there any difference in the activities of alkaline and acidic phosphatase. By means of the Rivanol reaction, however a significant difference has been found in the ground substance of the epiphyseal cartilages: the ground substance of the control femurs was a regular network (fig. 3), whereas in treated animals this regular structure could hardly be detected, the ground substance slackened and could not be visualized (fig. 4).
Discussion ASA has a wide mode of action with a well-known role in coagulation (4,7). It inhibits the synthesis of prostaglandin by blocking prostaglandin synthetase, and by uncoupling oxidative phosphorylation (1,3). According to SAITO et al. the resulting malformations may also be partly attributed to the effect of ASA leading to hypocalcaemia (10). We do not think, on the basis of our above results, that ASA has influenced calcification. Rivanol is bound strongly to the sulphated mucopolysaccharides (5,8). Irregularity and decrease in the Rivanol positivity structures pointed to reduced production of the ground substance in the epiphyseal cartilage, i.e., to a damage in the function of chondrocytes. Our studies also proved that acetylsalicylic acid did not damage the collagen structure of the bones and probably did not influence the function of osteoblasts and osteoclasts. This inhibitory effect on the sulphated mucopolysaccharides is due to the inhibition of ATP dependent sulphate ion transport by the uncoupling of ATP-ADP resynthesis and the sulphation of glycosaminoglycans inside the chondrocytes (13). The inhibition of ATP-ADP
Fig. I. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w, of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). The chondrocytes show regular, columnar arrangement. HE staining, X 200.
Fig. 2. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w. of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). Picrosirius red F3BA staining. The appearance of collagen fibres is normal, fibres detected in the periosteum and at the border of the degeneration zone. x 200. Fig.3. Distal epiphyseal cartilage of the femur in a 21-day-old control rat embryo. Rivanol reaction between crossed polars. The ground substance seems to be an oriented network among the chondrocytes. X 200.
Fig. 4. Distal epiphyseal cartilage of the femur in a 21-day-old rat embryo. Treatment: 500 mg/kg b.w. of acetylsalicylic acid (between the 9th-12th day of pregnancy, by gastric tube). Rivanol reaction between crossed polars. The ground substance was slackened, its positivity diminished in the area of the epiphyseal cartilage (---+). x 200. 3*
Exp. Pathol. 43 (1991) 1-2
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resynthesis, as an essential biochemical process, caused embryolethality in high doses (above 500 mg/kg b.w .). In the dosis applied, in our experiment , the damaging effect manisfested itself in selectivity on the chondrocytes between the 9th- 12th day of pregnancy. The consequence is "malformation" of the epiphyseal cartilage and micro- and phocomelia , depending on the free salicylic acid level.
Acknowledgements The authors express thanks to Mrs. BAJUSZ, Mrs . GYURIK and Mrs. KRAUSZ for skillful technical assistance .
References I. BEYNEN, A. C.; BUECH LER , K. F. , MOLEN, A. J., VAN DER , GEELEN, M . J. H.: Inhibit ion of hepatic lipoge nesis by Salic ylate. To xicology 1982 ; 24 : 33 -43 . 2 . BURSTONE, M. S .: Histochemical comparison of naphtho l AS phosphates for the demonstration of phosphatases. J . Natl. Cance r Inst. 1958 ; 20 : 60 1- 6 16. 3. GURA, F.: Orvosi Biokernia, 2nd Edition, Med icina , Budapest 1988 ; pp. 386-387. 4. FEKETE, GY. , BRAUN, P.: A terapia aktualis kerde sei. Medicina, Budapest 1964 ; pp. 51-74 . 5. KAWIAK , J ., KOWALSKI, H.: Evaluation of the method of rivanol precip itation of [35SJ sulph ated mucopolysaccharides present in the material obtained from a small group of cells . Experientia (Base l) 1964 ; 20 : 397 . 6. KIMMEL , C. A. , WILSON, J. G., SCH UMACH ER, H. J.: Studies on metabolism and identification of the causa tive agent in aspirin teratogenesis in rats. Teratology 1971 ; 4 : 15- 24 . 7. LEWIS, R. B. , SCHULMAN, J. D.: Influence of acetylsalicylic acid , an inhibitor of prostagla ndin synthesis , on the duration of human gestation and labour. Lancet 1973 ; 2 : 1159-11 61. 8 . NEMETH-Cs6KA, M. : A turbid imetric method for the determination of chondroitinsulphuric acid by rivano l. Bioch im. Bioph ys . Acta 1961 ; 50 : 585 -588. 9. RENAULT, J .-Y. , MELCION, C.; CORDI ER, A.: Limb bud cell cu lture for in vitro teratogen screening: Validation of an improved assess ment method using 5 1 compounds tera togen . Carcinoge n. Mut. 1989,9 : 83-96. 10. SAITO, H. , YOKOYAMA, A., TAKENO, S., SAKAI, T., VEND, K., MASUMURA, H. , KITAGAWA , H.: Fetal toxicity and hypocalcaemia induced by acetylsalicylic acid analogues. Res. Comm. Chern . PathoI. Pharmacol. 1982 ; 38 : 209 -220 . II. VNGVARY , GY., TATRAI , E. , L6RINCZ, M. , BARCZA, Gv .: Com bined emb ryotoxic action of toluene, a widely used industrial chemical, and acety lsalicylic acid (aspirin). Teratology 1983 ; 27 : 26 1-269. 12. WARKANY, 1. , TAKACS, E : Expe rime ntal production of congenital malform ations in rats by salicylate poisoning Am. J . Pathol . 1959 ; 35 : 3 15-331. 13. WHITEHOUSE , M . W . , BOSTROM, H .: Stud ies on the action of some anti-inflamm atory agents in inhib iting the biosynthesis of mucopolysaccharid e sulphates. Biochem ical Pharmacology, Vol. 7., pp. 135- 150. Pergam on Press Ltd. , Oxford 196 1.
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Exp. Patho!. 43 (199 1) J- 2