Permanent Alterations in the Hypothalamic-Pituitary-Thyroid Axis in the Rat Following Phenytoin Exposure In Utero T. J. Theodoropoulos, M. A. Pappolla, O. S. Goussis, J. C. Zolman and D. M. Benson Departments of Medicine and Pathology, VA Medical Center, Montrose, New York New York Medical College, Valhalla, New York University of Texas, Galveston, Texas, U. S. A.

Phenytoin exposure in utero results in permanent alterations of the hypothalamic-pituitary-thyroid axis in the rat. The DPH exposed animals have decreased weight gain, thyroxine and triiodothyronine concentrations. In addition, they have blunted thyroid-stimulating hormone responses to thyrotropin-releasing hormone, propylthiouracil challenge or thyroidectomy. The diminished pituitary response in these animals is similar to that reported in neonatal thyrotoxicosis in the rat. This may be due, in part, to structural similarities between phenytoin and the thyroid hormone. Key words Phenytoin and Thyrotropin-Releasing Hormone - Phenytoin and Thyroid-Stimulating Hormone — Phenytoin and Thyroid Function

Introduction Phenytoin (Diphenylhydantoin, DPH) is one of the most commonly used anticonvulsants. Perinatal exposure to anticonvulsants may result in syndromes (fetal hydantoin syndrome) characterized by poor growth and development, associated with multiple neurological, craniofacial and skeletal abnormalities. These teratogenic effects are more pronounced at birth (Phelan, Pellock and Nance 1982). Most antiepileptics cross the placental barrier and are detected in similar concentrations in maternal and cord blood. Since they are excreted in colostrum, they can be ingested by neonates during breast feeding (Bossi 1982). The metabolism of these drugs is different in maternal and fetal tissues. Neonates have a slower drug disposition rate which results in high serum concentrations of active metabolites. Furthermore, anticonvulsants cross the blood brain barrier with much greater ease in the perinatal period.

ported following intra-uterine exposure to phenobarbital (Gupta, Sonawane and Yaffe 1980). We report that permanent alterations of the hypothalamic-pituitary-thyroid axis follow D P H exposure in utero. Material and Methods Female Sprague-Dawley rats weighing 200—250 g were used in all experiments. The day of conception was accurately assessed by obtaining vaginal smears and identification of spermatozoa. Pregnant rats were treated with daily intra-peritoneal injections of DPH (50 mg/kg body weight) or diluent (40% propylene glycol) throughout pregnancy. Term fetuses were removed through cesarian section and fetal blood collected from the trunk after decapitation. Pooled blood from fetal siblings was centrifuged and was frozen until assayed. Additional blood samples (0.2—0.4 ml) were collected and placed immediately into 4 ml of 90 % methanol and frozen after nitrogen evaporation. The methanolic extracts were reconstituted in 2 ml 0.01 M phosphate buffer just before determining thyrotropin releasing hormone (TRH) levels. Fetal thyroids, pituitaries and hypothalami were dissected and pooled. Thyroids were weighed. Anterior pituitaries were placed on dry ice immediately upon removal, weighed, homogenized in 1 ml 0.01 M phosphate buffer, pH 7.8, centrifuged and the supernatants quickly frozen at - 20 °C until assayed for thyrotropin stimulating hormone (TSH) content. Hypothalami were removed according to the method of Glowinski and Iverson (1966), placed on dry ice, weighed, homigenized in 1 ml of 90% methanol and centrifuged. The supernatants were collected, evaporated under nitrogen and kept frozen. They were reconstituted in 2 ml of 0.01 M phosphate buffer just before determining TRH content. Additional experiments were done in neonatal rats delivered from DPH treated and control rats at various stages of their development. Body weight gain was followed closely in the experimental and control animals. Several hormonal parameters of hypothalamic-pituitary-thyroid axis were again assessed, as stated above. At the age of 60 days (d=, the functional capacity of the axis was challenged with TRH tests, propylthiouracil (PTU) administration and thyroidectomy (Tx). An indwelling venous catheter was placed in the femoral vein under light ketamine anesthesia and blood obtained. TRH (1 ug/kg body weight) was administered intravenously, and blood obtained by decapitation 10 minutes later. A different group of 60 d old rats was fed with a low iodine-PTU diet (LID-PTU) (Theodoropoulos, Braverman and Vagenakis 1979), for seven days and another was subjected to Tx. Blood was collected seven days post LID-PTU or Tx.

DPH-induced alterations of the neuroendocrine system have been investigated to a very limited extent. Alterations in the female reproductive function in rats were re-

Sera from individual experiments were assayed for T4, T3 and TSH concentrations by radioimmunoassay (RIA). Pituitary TSH content, hypothalamic and blood TRH were also measured by

Horm.metab.Res.22(1990)521-523 © Georg Thieme Verlag Stuttgart -New York

Received: 22 Aug. 1989

Accepted: 28 March 1990

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Horm. metab. Res. 22 (1990)

T. J. Theodoropoulos, M. A. Pappolla, O. S. Goussis, J. C. Zolman and D. M. Benson

Fig. 3 Serum TSH levels in rats exposed to DPH in utero. NS : significant.


Fig. 4 TSH 10 min. post TRH in male rats exposed to DPH in utero.

given to the 60 d rats, the increment in serum TSH at 10 min RIA (Theodoropoulos, Braverman and Vagenakis 1979; Theodoropoulos, Fang, Azizi, Ingbar, Vagenakis and Braverman 1980).was significantly lower in the D P H exposed than in the control All values were expressed as the mean + SE. Statistical analyses were rats (paired t). Similarly, blunted TSH responses were obcarried out by the Student's and paired t-tests. served in the D P H groups after a LID-PTU challenge or Tx (Fig. 4). In all experiments, six rats were used in each group of Results animals (n = 6). The administration of D P H had no effect on litter size and all neonates appeared normal on gross examination. Body weights were similar to the controls during the neonatal period, however, they were significantly (P < 0.05) decreased at 60 d of age. No significant differences in the thyroid weights were observed between ages. Serum T4 and T3 concentrations were undetectable in the serum of term fetuses in DPH exposed and control groups, which is similar to findings reported previously {Fisher, Dussault, Salk and Chopra 1977; Theodoropoulos, Braverman and Vagenakis 1979). In older ages, both T4 and T3 were significantly decreased in the D P H groups as compared to controls (Figs. 1 and 2). The TSH levels appeared to be lower in the D P H groups, however, these differences were not statistically significant (Fig. 3). Similarly, pituitary TSH contents were slightly, but not significantly, lower in the DPH groups. No differences in blood or hypothalamic TRH contents were observed. Blood TRH levels were detectable only in the immature rats and this finding is consistent with previous observations (Theodoropoulos, Braverman and Vagenakis 1979). When synthetic TRH was

Discussion These findings demonstrate that prenatal exposure to D P H produced long term effects in the hypothalamic-pituitary-thyroid axis of the rat. It has been reported that perinatal maturation of the axis is critical for its future functional integrity {Fisher et al. 1977). The body weight data from this study suggest that growth may be decreased in the D P H treated animals. Growth is affected after weaning, particularly at 60 d of age. Serum and pituitary TSH levels were lower in the D P H groups (although inappropriately low for the observed low T4 and T3 concentrations). The blunted serum TSH responses to TRH, PTU challenge or Tx and the normal blood hypothalamic TRH levels suggest that the hypothyroidism observed in these animals is secondary (pituitary) and probably partial. However, a tertiary (hypothalamic) defect cannot be excluded from these data. This partial pituitary dysfunction raises some interesting questions. On one hand, this defect may be

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Phenytoin in Pregnancy and Hypothalamic-Pituitary-Thyroid


Horm. metab. Res. 22 (1990)


Dussault, J. H., P. Coulcombe, P. Walke: Effects of neonatal hyperthyroidism on the development of hypothalamic-pituitary-thyroid axis in the rat. Endocrinology 110:1037-1043 (1982) El-Zaheri, M. M., L. E. Braverman, A. G. Vagenakis: Enhanced conversion of T4 to T3 by the neonatal rat pituitary. Endocrinology 106:1735-1739(1980) Franklin, J. A., J. R. Davis, D. B. Rausden, M. C. Sheppard: Phenytoin and thyroid hormone action. J. Endocrinol. 104:201 —204 (1985) Fisher, D. A., J. H. Dussault, J. Salk, I. Chopra: Ontogenesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheep and rat. Recent Prog. Horm. Res. 33:59-116 (1977) Glowinski, J., L. Iverson: Regional studies of catecholamines in the rat brain. J. Neurochem. 13:655-669 (1966) Gupta, C, B. R. Sonawane, S. J. Yaffe: Phenobarbital exposure in utero: Alterations in female reproductive function in rats. Science 208:508-510(1980) Structural similarities between DPH and the Hufner, M., M. Knopfle: Pharmacological influences onT4to T3 conthyroid hormones (diphenyl-group) (Oppenheimer and Taverversion in rat liver. Clin. Chim. Acta 72:377-381(1976) netti 1962) play a significant role in the pathogenesis of these Mann, D. N., M. I. Surks: 5,5'-Diphenylhydantoin decreases specific alterations. We have reported that DPH, like the thyroid hor3,5,3'-triiodothyronine (T3) binding by rat hepatic nuclear T3 remone, has suppressive effects on TSH synthesis/release in the ceptors. Endocrinology 112: 1723-1731 (1983) adult rat (Theodoropoulos et al. 1980). These findings were Oppenheimer, J. H., R. R. Tavernetti: Displacement of thyroxineconfirmed later in rats and humans (Mann and Surks 1983; binding globulin by analogues of hydantoin. Steric aspects of the thyroxine-binding site. J. Clin. Invest. 41:2213-2220 (1962) Surks, Ordene, Mann and Kumara-Siri 1983), suggesting that Phelan, M. C, J. M. Pellock, W. E. Nance: Discordant expression of DPH may be a thyroid hormone agonist. To this end, some stufetal hydantoin syndrome in heteropaternal dizygotic twins. N. dies have shown that DPH inhibits T3 binding to nuclear and Eng. J.Med. 307:99-101 (1982) cytosol fractions of pituitary and liver cells, suggesting a speReichlin, S., J. B. Martin, M. A. Mitnick, R. L. Boshans, Y. Grimm, J. cific interaction between DPH and T3 binding sites (Surks, KuBollinger, J. Gordon, J. Malacara: The hypothalamus in pituitarymara-Siri, Mann, Ordene and DeFesi 1981; Surks et al. 1983; thyroid regulation. RecentProg. Horm. Res. 28:229-286 (1972) Surks, M. I, M. H. Kumara-Siri, D. N. Mann, K. W. Ordene, C. R. Franklin, Davis, Rausden and Sheppard 1985; Zemel, Smith, DeFesi: Effect of diphenylhydantoin on TSH secretion in man and Shapiro and Surks 1988). Studies on the kinetic parameters of cellular T3 binding in rat tissues. Endocrine Society, 63 Annual T3 binding sites may help clarify these issues. Meeting (1981) #420 Surks, M. I, K. W. Ordene, D. N. Mann, M. H. Kumara-Siri: It has been suggested that DPH increases the Diphenylhydantoin inhibits the thyrotropin response to thyconversion of T4 to T3 in man (Cullen and Burger 1972), and rat rotropin-releasing hormone in man and rat. J. Clin. Endocrinol. (Hufner and Knopfle 1976), but its effect on intrapituitary conMetab. 56:940-945 (1983) Theodoropoulos, T., L. E. Braverman, A. G. Vagenakis: Thyrotropin version has not been studied. However, intrapituitary converreleasing hormone is not required for thyrotropin secretion in the sion of T4 to T3 is increased in normal neonates (El-Zaheri, perinatal rat. J. Clin. Invest. 63: 588-594 (1979) Braverman and Vagenakis 1980), and some data (Walker and Theodoropoulos, T., L. E. Braverman, A. G. Vagenakis: Iodide-inDussault 1982), suggest that neonatal thyrotoxicosis may reduced hypothyroidism: a potential hazzard during perinatal life. sult in increased intrapituitary conversion in the adult animals. Science 205:502-503(1979) Theodoropoulos, T, S. L. Fang, F. Azizi, S. H. Ingbar, A. G. Vagenakis, In summary, DPH exposure in utero results in L. E. Braverman: Effect of diphenylhydantoin on hypothalamicpituitary-thyroid function in the rat. Amer. J. Physiol. 239: E468— a diminished pituitary response in the offspring rat, similar to E473 (1980) that reported in neonatal thyrotoxicosis. The underlying Walker, P., J. H. Dussault: Neonatal hyperthyroidism in the rat permechanisms are still unclear. However, the changes in pituimanently increases pituitary thyroxine 5'-deiodinase activity. tary-thyroid function herein reported suggest that this drug Clin. Res. 30: 686A (1982) should be used with caution in pregnant humans. Zemel, L. R., P. J. Smith, L. E. Shapiro, M. I. Surks: 5,5'-Diphenylhydantoin decreases the entry of 3,5,3'-triodo-L-thyronine, but not Lthyroxine in cultured GH producing cells. Acta Endocrinol. 117: References 392-398(1988) Azizi, F., A. G. Vagenakis, J. Bollinger, S. Reichlin, L. E. Braverman, S. H. Ingbar: Persistent abnormalities in pituitary function following neonatal thyrotoxicosis in the rat. Endocrinology 94: 1631 — 1685 Requests for reprints should be addressed to: (1974) Bossi, L.: Epilepsy, Pregnancy and the Child. Raven Press, new York T. J. Theodoropoulos, M. D. (1982), p. 327 Cullen, M. J., A. G. Burger: Effects of diphenylhydantoin (DPH) on Department of Medicine peripheral thyroid hormone economy and the conversion of thyNew York Medical College roxine (T4) to triiodothyronine (T3). (Abstract) Prog. Am. Thyroid VA Medical Center Assoc. (1972), p. 21 Montrose, N. Y. 10548 (U. S. A.)

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ascribed as to inability to synthesize (or release) TSH and on the other, to increased pituitary thyrotrope sensitivity to feedback inhibition by the thyroid hormones. It is possible that DPH leads to a permanent "resetting" of the regulatory set point for pituitary TSH secretion manifested as a permanent defect in maturation. These findings are similar to those reported following neonatal thyrotoxicosis in the rat (Azizi, Vagenakis, Bollinger, Reichlin, Braverman and Ingbar 1974; Dussault, Coulcombe and Walker 1982), to the adult rat with hypothalamic lesions {Reichlin, Martin and Mitnick 1972) and to the normal immature perinatal rat (Theodoropoulos, Braverman and Vagenakis 1979).

Permanent alterations in the hypothalamic-pituitary-thyroid axis in the rat following phenytoin exposure in utero.

Phenytoin exposure in utero results in permanent alterations of the hypothalamic-pituitary-thyroid axis in the rat. The DPH exposed animals have decre...
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