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The influence of ethamoxytriphetol on the collagen and calcium contents of the femurs of rabbit fetuses
TOXICOLOGYAND APPLIED PHARMACOLOGY15,185-l 88 (1969)
The Influence of Ethamoxytriphetol Contents of the Femurs
on the Collagen and Calcium of Rabbit Fetusesl-3
RAJA W. ABDUL-KARIM AND LINDA D. MARSHALL Department
of Obstetrics and Gynecology, State University of New Upstate Medical Center, Syracuse, New York 13210 Received
October
York,
IO,1968
The Influence of Ethamoxytriphetol on the Collagen and Calcium Contents of the Femurs of Rabbit Fetuses. ABDUL-KARIM, RAJAW., and MARSHALL, LINDA D. (1969). Toxicol. Appl. Pharmacol. 15,185-188. Ethamoxytriphetol, 1-(P-2-diethylaminoethoxyphenyl)- 1-phenyl-2-methoxyphenylethanol(MER-25), was administered to a group of 6 pregnant rabbits in a daily dose of 25 mg/kg body weight. Eight pregnant rabbits received a daily dose of sesame oil and served as controls. All injections were begun on day 21 of gestation and carried through day 26. All the rabbits were delivered by cesareansection on day 27. The femurs of the liveborn were dissectedfree of soft tissue and the wet and fat-freedry weights were recorded. Thecollagen and calcium contents were analyzed. In the treated group receiving the antiestrogen, MER-25, collagen was significantly reduced when expressed as percent of both the wet weight (2.85 i 0.54 vs. 3.76 f 0.36) and the fatfree dry weight (16.3 + 1.3 vs. 18.2 & 1.1). The calcium to wet weight ratio in the MER-25 treated group waslessthan that of the controls (2.63 y0 & 0.49 vs. 3.42% f 0.50), whereas the calcium as percent of the fat-free dry weight of the bone was not significantly different (15.1 i 2.1 US.16.6 f 2). It is concluded that ethamoxytriphetol retards fetal bone development and produces a decreasein collagen formation in fetal bone, but no disturbance in bone mineralization. Ethamoxytriphetol, I-(P-2-diethylaminoethoxyphenyl)-2-methoxyphenyl ethanol (MER-25), is a nonsteroidal compound capable of inhibiting various effects of both exogenously administered and endogenously produced estrogens. Its effects upon ovulation, implantation and gestation have been studied and several reviews are available on the subject (Lerner et al., 1958; Lerner, 1964; Emmens, 1965; Callantine, 1967). In 1966, Abdul-Karim et al. reported that the administration of MER-25 to pregnant ewes induced a decrease in endochondral new bone formation in the fetuses. Subsequent studies on pregnant rabbits confirmed these findings and demonstrated that 17/3-estradiol prevented the occurrence of the MER-25-induced fetal bone lesions 1 A preliminary report of some of the data was presented at the International Symposium on FoetoPlacental-Unit, Milan, Italy, September 4-6. 1968. 2 Ethamoxykiphetoi was ienkously supphed by Dr. Dorsey Holtcamp, William S. Merrell Company, Cincinnati, Ohio 45215. 3 This study was supported by Institutional Grant No. 1l-8656 from the United States Public Health Service. 185
186
ABDUL-KARIMANDMARSHALL
and that the latter were spontaneously reversible in utero after discontinuation of the drug (Abdul-Karim and Prior, 1968; Abdul-Karim et al., 1968a, b). Since bone matrix is 90-96 % collagen (McLean and Urist, 1968), the present study was designed to show whether MER-25 specifically interfered with collagen formation and whether the observed reduction in bone mineralization reflected the decreased osteoid tissue or was due to a specific disturbance in calcium deposition. METHODS
Fourteen white New Zealand pregnant rabbits with known dates of gestation were divided into two groups. All the animals were housed in the same environment, fed the same diet, and handled by the same personnel. Beginning on day 21 of gestation and carried through day 26, each of the 6 rabbits in group 1 received, daily, 25 mg/kg i.m. of MER-25 in sesame oil. The rabbits in group 2 (8 animals) received the equivalent amount of sesame oil and served as controls. On day 27 of gestation, all the rabbits were delivered by cesarean section, the fetuses were weighed, and their number recorded. The liveborn were sacrificed and their femurs cleanly dissected from soft tissues. The femurs from each litter were pooled together, split, rinsed in distilled water, gently blotted, and weighed. The femurs were then dried to a constant weight, defatted, and their collagen and calcium contents determined according to established techniques (Neuman and Logan, 1950; Leach, 1960; Eastoe, 1965). The results were expressed as percentage of the wet and fat-free dry weights of the bone.
RESULTS
All the does remained healthy throughout the period of study. The fetal data are summarized in Table 1. There was no decrease in the number of liveborns in the MER-25 treated group. The average weight of the latter, however, was significantly less than that of the liveborn in the control group. TABLE
1
THEAVERAGENUMBERAND WEIGHTSOFALLLITTERSINCONTROL AND MER-25 TREATEDGROUPS
Group
Number of litters
Average number of fetuses/ litter i SD”
Average number live births/ litter i SD
Liveborn weights’ (g)
Controls
8
8.8 + 2.09
8.7 I2.11
16.3-37.8
27.9 zk 4.61
6
7.0 i 2.71
6.7 i 2.43
14.9-28.3
22.2 Ik4.11
Range
Average per litter & SD
MER-25*
treated
0 SD = standard deviation. * Dose of 25 mg/kg/day on days21 through 26 of gestation. cLitter obtained by cesareansectionon day 27 of gestation.
ETHAMOXYTRIPHETOLONFETALBONE
187
Collagen and Calcium Contents of the Femurs of the Liveborn When expressed as percentage of wet weight, there was a significant decrease in the dry weight, collagen, and calcium contents of the femurs of the MER-25 treated group (Table 2). The data in Table 2 show further that fetuses in the MER-25 treated group had a significant decrease in the percentage of collagen as related to the fat-free dry weight, whereas the calcium content expressed similarly was the same in both the treated and control groups. The calcium to collagen ratio in the two groups was not significantly different. TABLE
2
MEANSANDSTANDARDDEVIATIONOFTHECALCIUMANDCOLLAGENCONTENTSOF FEMURSOFLIVE~~-DAYFETUSESINCONTROLAND MER-25 TREATEDGROUPS Collagen Group
Wet weight of bone
(%) Dry weight of bone
Calcium
(%)
Wet weight of bone
Dry weight of bone
Calcium to collagen ratio
Dry weight as percent of wet weight
Control
3.76b * 0.36 18.2” * 1.1
3.42d * 0.50
16.6 * 2.0
0.910 3: 0.097
20.6’ i 1.5
MER-25 treateda
2Hb f 0.54
16.3’ i 1.3
2.63d & 0.49
15.1 * 2.1
0.933 3: 0.148
17.4’ * 2.3
a Dose of 25 mg/kg/day on days 21 through 26 of gestation. b Difference between treated and control is significant (P < 0.01). ’ Difference between treated and control is significant (P = 0.01). d Difference between treated and control is significant (P < 0.02). ’ Difference between treated and control is significant (P < 0.02). DISCUSSION
The results of this study indicated that MER-25 administration to pregnant rabbits induced changes in the composition of the femurs of the fetuses. These changes included an increase in the water content as demonstrated by the decreased fat-free dry weight to wet-weight ratio, and a decrease in collagen formation indicated by the decreased collagen to fat-free dry-weight ratio. Despite these changes, however, there appeared to be no disturbances in the mineralization of the bone since the ratio of calcium to the fat-free dry weights, and to the collagen content, were the same in both the control and the MER-25 treated groups. Thus, ethamoxytriphetol retarded normal bone development in the fetal rabbit, making the composition of the femurs at 27 days of gestation resembIe those at an earlier period of pregnancy. A simiIar conclusion was reached by Diener and Hsu (1967) in their studies on rat fetuses. These authors, utilizing the alizarin red stain, showed that ethamoxytriphetol administered to pregnant rats reduced ossification of the fetal skeletons. The latter at 20 days of gestation resembled those of 18-day fetuses. The mechanism by which ethamoxytriphetol induces the preceding fetal skeletal abnormalities appears related, at least in part, to its antiestrogenic properties. This is supported by the morphologic studies showing that 17fi-estradiol prevents the MER-25 induced pathologic bone formation, that the latter are spontaneously reversible in utero, and that 17/&estradiol fails to cause any morphologic disturbances in fetal bone
188
ABDUL-KARIMAND MARSHALL
formation (Abdul-Karim and Prior, 1968; Abdul-Karim ef al., 1968a, c). Furthermore, the fetal bone disturbances resulting from MER-25 were unrelated to any influence the drug may have had on fetal weights. Additional support for this hypothesis is derived from the work of others. Smith and Allison (1966) demonstrated that 17/3estradiol increased the collagen content of rat femurs, and Simmons (1966), working on mice, concluded that the increased collagen synthesis under estrogens was related to the latter’s ability to augment the number of osteoblasts. Whether MER-25 acts directly on the fetuses or indirectly by influencing some maternal and/or placental metabolic process has not as yet been elucidated. ACKNOWLEDGMENT The authors wish to acknowledge the assistanceof Mary E. Haviland in the conduct of this study. REFERENCES ABDUL-KARIM, R. W., and PRIOR,J. T. (1968). Pathologic fetal bone development under the influence of an antiestrogenic compound. Am. J. Pathol. 52, 38a. ABDUL-KARIM, R. W., NESBITT,R. E. L., JR., and PRIOR,J. T. (1966). Study of the effects of experimentally induced endocrine insults upon pregnant and nonpregnant ewes. I. The effects of ethamoxytriphetol in pregnant sheep. Fertility Sterility 17, 637-647. ABDUL-KARIM, R. W., PRIOR,J. T., and MARSHALL,L. D. (1968a). Spontaneous reversibility of fetal pathologic bone development under the influence of an antiestrogenic compound. Lying-In. J. Reprod. Med. 1, 397-405. ABDUL-KARIM,R. W., PRIOR,J. T., and NESBITT,R. E. L., JR. (1968b). Influence of maternal estrogen on fetal bone development in the rabbit. Obstet. Gynecof. 31, 346-353. ABDUL-KARIM, R. W., SHELLEY,T. F., and PRIOR, J. T. (1968~). The influence of Antiestrogenic compounds on fetal bone development in the rabbit. Intern, Symp. FoetoPlacental Unit, 1968, Abstract No. 57. Excerpta Med. Intern. Congr. Ser. 170, 25. CALLANTINE,M. R. (1967). Nonsteroidal estrogen antagonists. Clin. Obstet. Gynecol. 10, 74-87.
DIENER,R. M., and Hsu, B. Y. D. (1967). Effects of certain basic phenolic ethers on the rat fetus. Toxicol. Appl. Pharmacol. 10, 565-576. EASTOE,J. E. (1965). Methods for the determination of phosphate, calcium and protein in small portions of mineralized tissues. In: Calcified Tissues(L. J. Richelle and M. J. Dallemagne, eds.), pp. 265-274. Collection des Colloques de l’universite de Liege. EMMENS,C. W. (I 965). Oestrogenic, anti-oestrogenic, and antifertility activities of various compounds. J. Reprod. Fertility 9, 277-283. LEACH,A. A. (1960). Notes on the modification of the Neuman and Logan method for the determination of the hydroxyproline. Biochem. J. 74, 70-71. LERNER,L. J. (1964). Hormone antagonists. Inhibitors of specific activities of estrogen and androgen. Recent Progr. Hormone Res. 20,435476. LERNER,L. J., HOLTHAUS,F. J.. and THOMPSON,C. R. (1958). A nonsteroidal estrogen antagonist - 1 -(P- 2 - diethylaminoethoxyphenyl)l - phenyl - 2 -P - methoxyphenyl ethanol. Endocrinology 63, 295-3 18. MCLEAN, F. C., and URIST,M. R. (1968). Bone: An Introduction to the Physiology of Skeletal Tissue, 3rd ed., p. 45. Univ. of Chicago Press, Chicago, Illinois. NEUMAN,R. E., and LOGAN,M. A. (1950). The determination of collagen and elastin in tissue. J. Biol. Chem. 186,549-556. SIMMONS,D. J. (1966). Collagen formation and endochondral ossification in estrogen treated mice. Proc. Sot. Exptl. Biol. 121, 1165. SMITH, Q. T., and ALLISON, D. J. (1966). Changes of collagen content in skin, femur and uterus of 17/?-estradiol benzoate-treated rats. Endocrinology 79, 486-492.
The Influence of Ethamoxytriphetol Contents of the Femurs
on the Collagen and Calcium of Rabbit Fetusesl-3
RAJA W. ABDUL-KARIM AND LINDA D. MARSHALL Department
of Obstetrics and Gynecology, State University of New Upstate Medical Center, Syracuse, New York 13210 Received
October
York,
IO,1968
The Influence of Ethamoxytriphetol on the Collagen and Calcium Contents of the Femurs of Rabbit Fetuses. ABDUL-KARIM, RAJAW., and MARSHALL, LINDA D. (1969). Toxicol. Appl. Pharmacol. 15,185-188. Ethamoxytriphetol, 1-(P-2-diethylaminoethoxyphenyl)- 1-phenyl-2-methoxyphenylethanol(MER-25), was administered to a group of 6 pregnant rabbits in a daily dose of 25 mg/kg body weight. Eight pregnant rabbits received a daily dose of sesame oil and served as controls. All injections were begun on day 21 of gestation and carried through day 26. All the rabbits were delivered by cesareansection on day 27. The femurs of the liveborn were dissectedfree of soft tissue and the wet and fat-freedry weights were recorded. Thecollagen and calcium contents were analyzed. In the treated group receiving the antiestrogen, MER-25, collagen was significantly reduced when expressed as percent of both the wet weight (2.85 i 0.54 vs. 3.76 f 0.36) and the fatfree dry weight (16.3 + 1.3 vs. 18.2 & 1.1). The calcium to wet weight ratio in the MER-25 treated group waslessthan that of the controls (2.63 y0 & 0.49 vs. 3.42% f 0.50), whereas the calcium as percent of the fat-free dry weight of the bone was not significantly different (15.1 i 2.1 US.16.6 f 2). It is concluded that ethamoxytriphetol retards fetal bone development and produces a decreasein collagen formation in fetal bone, but no disturbance in bone mineralization. Ethamoxytriphetol, I-(P-2-diethylaminoethoxyphenyl)-2-methoxyphenyl ethanol (MER-25), is a nonsteroidal compound capable of inhibiting various effects of both exogenously administered and endogenously produced estrogens. Its effects upon ovulation, implantation and gestation have been studied and several reviews are available on the subject (Lerner et al., 1958; Lerner, 1964; Emmens, 1965; Callantine, 1967). In 1966, Abdul-Karim et al. reported that the administration of MER-25 to pregnant ewes induced a decrease in endochondral new bone formation in the fetuses. Subsequent studies on pregnant rabbits confirmed these findings and demonstrated that 17/3-estradiol prevented the occurrence of the MER-25-induced fetal bone lesions 1 A preliminary report of some of the data was presented at the International Symposium on FoetoPlacental-Unit, Milan, Italy, September 4-6. 1968. 2 Ethamoxykiphetoi was ienkously supphed by Dr. Dorsey Holtcamp, William S. Merrell Company, Cincinnati, Ohio 45215. 3 This study was supported by Institutional Grant No. 1l-8656 from the United States Public Health Service. 185
186
ABDUL-KARIMANDMARSHALL
and that the latter were spontaneously reversible in utero after discontinuation of the drug (Abdul-Karim and Prior, 1968; Abdul-Karim et al., 1968a, b). Since bone matrix is 90-96 % collagen (McLean and Urist, 1968), the present study was designed to show whether MER-25 specifically interfered with collagen formation and whether the observed reduction in bone mineralization reflected the decreased osteoid tissue or was due to a specific disturbance in calcium deposition. METHODS
Fourteen white New Zealand pregnant rabbits with known dates of gestation were divided into two groups. All the animals were housed in the same environment, fed the same diet, and handled by the same personnel. Beginning on day 21 of gestation and carried through day 26, each of the 6 rabbits in group 1 received, daily, 25 mg/kg i.m. of MER-25 in sesame oil. The rabbits in group 2 (8 animals) received the equivalent amount of sesame oil and served as controls. On day 27 of gestation, all the rabbits were delivered by cesarean section, the fetuses were weighed, and their number recorded. The liveborn were sacrificed and their femurs cleanly dissected from soft tissues. The femurs from each litter were pooled together, split, rinsed in distilled water, gently blotted, and weighed. The femurs were then dried to a constant weight, defatted, and their collagen and calcium contents determined according to established techniques (Neuman and Logan, 1950; Leach, 1960; Eastoe, 1965). The results were expressed as percentage of the wet and fat-free dry weights of the bone.
RESULTS
All the does remained healthy throughout the period of study. The fetal data are summarized in Table 1. There was no decrease in the number of liveborns in the MER-25 treated group. The average weight of the latter, however, was significantly less than that of the liveborn in the control group. TABLE
1
THEAVERAGENUMBERAND WEIGHTSOFALLLITTERSINCONTROL AND MER-25 TREATEDGROUPS
Group
Number of litters
Average number of fetuses/ litter i SD”
Average number live births/ litter i SD
Liveborn weights’ (g)
Controls
8
8.8 + 2.09
8.7 I2.11
16.3-37.8
27.9 zk 4.61
6
7.0 i 2.71
6.7 i 2.43
14.9-28.3
22.2 Ik4.11
Range
Average per litter & SD
MER-25*
treated
0 SD = standard deviation. * Dose of 25 mg/kg/day on days21 through 26 of gestation. cLitter obtained by cesareansectionon day 27 of gestation.
ETHAMOXYTRIPHETOLONFETALBONE
187
Collagen and Calcium Contents of the Femurs of the Liveborn When expressed as percentage of wet weight, there was a significant decrease in the dry weight, collagen, and calcium contents of the femurs of the MER-25 treated group (Table 2). The data in Table 2 show further that fetuses in the MER-25 treated group had a significant decrease in the percentage of collagen as related to the fat-free dry weight, whereas the calcium content expressed similarly was the same in both the treated and control groups. The calcium to collagen ratio in the two groups was not significantly different. TABLE
2
MEANSANDSTANDARDDEVIATIONOFTHECALCIUMANDCOLLAGENCONTENTSOF FEMURSOFLIVE~~-DAYFETUSESINCONTROLAND MER-25 TREATEDGROUPS Collagen Group
Wet weight of bone
(%) Dry weight of bone
Calcium
(%)
Wet weight of bone
Dry weight of bone
Calcium to collagen ratio
Dry weight as percent of wet weight
Control
3.76b * 0.36 18.2” * 1.1
3.42d * 0.50
16.6 * 2.0
0.910 3: 0.097
20.6’ i 1.5
MER-25 treateda
2Hb f 0.54
16.3’ i 1.3
2.63d & 0.49
15.1 * 2.1
0.933 3: 0.148
17.4’ * 2.3
a Dose of 25 mg/kg/day on days 21 through 26 of gestation. b Difference between treated and control is significant (P < 0.01). ’ Difference between treated and control is significant (P = 0.01). d Difference between treated and control is significant (P < 0.02). ’ Difference between treated and control is significant (P < 0.02). DISCUSSION
The results of this study indicated that MER-25 administration to pregnant rabbits induced changes in the composition of the femurs of the fetuses. These changes included an increase in the water content as demonstrated by the decreased fat-free dry weight to wet-weight ratio, and a decrease in collagen formation indicated by the decreased collagen to fat-free dry-weight ratio. Despite these changes, however, there appeared to be no disturbances in the mineralization of the bone since the ratio of calcium to the fat-free dry weights, and to the collagen content, were the same in both the control and the MER-25 treated groups. Thus, ethamoxytriphetol retarded normal bone development in the fetal rabbit, making the composition of the femurs at 27 days of gestation resembIe those at an earlier period of pregnancy. A simiIar conclusion was reached by Diener and Hsu (1967) in their studies on rat fetuses. These authors, utilizing the alizarin red stain, showed that ethamoxytriphetol administered to pregnant rats reduced ossification of the fetal skeletons. The latter at 20 days of gestation resembled those of 18-day fetuses. The mechanism by which ethamoxytriphetol induces the preceding fetal skeletal abnormalities appears related, at least in part, to its antiestrogenic properties. This is supported by the morphologic studies showing that 17fi-estradiol prevents the MER-25 induced pathologic bone formation, that the latter are spontaneously reversible in utero, and that 17/&estradiol fails to cause any morphologic disturbances in fetal bone
188
ABDUL-KARIMAND MARSHALL
formation (Abdul-Karim and Prior, 1968; Abdul-Karim ef al., 1968a, c). Furthermore, the fetal bone disturbances resulting from MER-25 were unrelated to any influence the drug may have had on fetal weights. Additional support for this hypothesis is derived from the work of others. Smith and Allison (1966) demonstrated that 17/3estradiol increased the collagen content of rat femurs, and Simmons (1966), working on mice, concluded that the increased collagen synthesis under estrogens was related to the latter’s ability to augment the number of osteoblasts. Whether MER-25 acts directly on the fetuses or indirectly by influencing some maternal and/or placental metabolic process has not as yet been elucidated. ACKNOWLEDGMENT The authors wish to acknowledge the assistanceof Mary E. Haviland in the conduct of this study. REFERENCES ABDUL-KARIM, R. W., and PRIOR,J. T. (1968). Pathologic fetal bone development under the influence of an antiestrogenic compound. Am. J. Pathol. 52, 38a. ABDUL-KARIM, R. W., NESBITT,R. E. L., JR., and PRIOR,J. T. (1966). Study of the effects of experimentally induced endocrine insults upon pregnant and nonpregnant ewes. I. The effects of ethamoxytriphetol in pregnant sheep. Fertility Sterility 17, 637-647. ABDUL-KARIM, R. W., PRIOR,J. T., and MARSHALL,L. D. (1968a). Spontaneous reversibility of fetal pathologic bone development under the influence of an antiestrogenic compound. Lying-In. J. Reprod. Med. 1, 397-405. ABDUL-KARIM,R. W., PRIOR,J. T., and NESBITT,R. E. L., JR. (1968b). Influence of maternal estrogen on fetal bone development in the rabbit. Obstet. Gynecof. 31, 346-353. ABDUL-KARIM, R. W., SHELLEY,T. F., and PRIOR, J. T. (1968~). The influence of Antiestrogenic compounds on fetal bone development in the rabbit. Intern, Symp. FoetoPlacental Unit, 1968, Abstract No. 57. Excerpta Med. Intern. Congr. Ser. 170, 25. CALLANTINE,M. R. (1967). Nonsteroidal estrogen antagonists. Clin. Obstet. Gynecol. 10, 74-87.
DIENER,R. M., and Hsu, B. Y. D. (1967). Effects of certain basic phenolic ethers on the rat fetus. Toxicol. Appl. Pharmacol. 10, 565-576. EASTOE,J. E. (1965). Methods for the determination of phosphate, calcium and protein in small portions of mineralized tissues. In: Calcified Tissues(L. J. Richelle and M. J. Dallemagne, eds.), pp. 265-274. Collection des Colloques de l’universite de Liege. EMMENS,C. W. (I 965). Oestrogenic, anti-oestrogenic, and antifertility activities of various compounds. J. Reprod. Fertility 9, 277-283. LEACH,A. A. (1960). Notes on the modification of the Neuman and Logan method for the determination of the hydroxyproline. Biochem. J. 74, 70-71. LERNER,L. J. (1964). Hormone antagonists. Inhibitors of specific activities of estrogen and androgen. Recent Progr. Hormone Res. 20,435476. LERNER,L. J., HOLTHAUS,F. J.. and THOMPSON,C. R. (1958). A nonsteroidal estrogen antagonist - 1 -(P- 2 - diethylaminoethoxyphenyl)l - phenyl - 2 -P - methoxyphenyl ethanol. Endocrinology 63, 295-3 18. MCLEAN, F. C., and URIST,M. R. (1968). Bone: An Introduction to the Physiology of Skeletal Tissue, 3rd ed., p. 45. Univ. of Chicago Press, Chicago, Illinois. NEUMAN,R. E., and LOGAN,M. A. (1950). The determination of collagen and elastin in tissue. J. Biol. Chem. 186,549-556. SIMMONS,D. J. (1966). Collagen formation and endochondral ossification in estrogen treated mice. Proc. Sot. Exptl. Biol. 121, 1165. SMITH, Q. T., and ALLISON, D. J. (1966). Changes of collagen content in skin, femur and uterus of 17/?-estradiol benzoate-treated rats. Endocrinology 79, 486-492.