Long-Term Hypothyroidism and Hypothalamus-Pituitary-Adrenal (HPA) Response in Adult Mice

Previous studies of the hypothalamus-pituitary-adrenal (HPA) axis in young rodents have found thiouracil-induced hypothyroidism to delay development of the axis past a relative stress non-responsive period. Little attention has been paid to the effects of long-term hypothyroidism on the function of this axis. The present study examined HPA axis response to an acute stress as determined by production of the glucocorticoid corticosterone, in mice made hypothyroid by thouracil exposure from conception to adulthood. The effect of injecting thyroxine (T4) or triiodothyronine (T3) on HPA response in these animals was also determined. Long-term hypothyroidism resulted in depressed body weight, subnormal levels of circulating T4, and elevated levels of T3. However, HPA axis response to an acute stress was normal in hypothyroid mice, and was not augmented by T4 injection for two weeks. On the other hand, two weeks of T3 injection allowed for a 70% increase in stress response as compared to either euthyroid or hypothyroid animals. The basis of the differential effect of the two thyroid hormones on stress response in hypothyroid mice remains to be determined. OHIO J. SCI. 97 (1): 10-13, 1997 INTRODUCTION Early in the study of mammalian physiological response to stress, the hypothalamus-pituitary-adrenal (HPA) axis was described (Selye 1959), and the control of glucocorticoid secretion by this axis was recognized as adaptively advantageous. Subsequent study of ontogeny of the HPA axis in experimental rodents revealed a refractory period during the first two weeks of life that was called the stress non-responsive period (Shapiro et al. 1962). Recent investigators have found the nonresponsiveness to stress to be relative, rather than absolute as was originally thought, depending upon the axis parameter being measured (Walker et al. 1991). Generation of a pronounced response by this axis requires maturation of hypothalamic production and secretion of corticotropin-releasing factor (CRF), of pituitary production and secretion of adrenocorticotropic hormone (ACTH), and of adrenal production and secretion of glucocorticoid. Work in our laboratory has found that hypothyroidism induced perinatally by thiouracil in rats (Meserve and Leathern 1981) and mice (Meserve 1976), by genetic mutation (hyt) in mice (Meserve 1987), and more recently by polychlorinated biphenyl (PCB) in rats (Meserve et al. 1992) delays developmental progression past the relative stress non-responsive period when elevation of circulating glucocorticoid (corticosterone in rats and mice) is taken to indicate HPA response. Fewer data have been collected regarding the influence of prolonged hypothyroidism on the response of the HPA axis to a stressful stimulus. Additionally, the influence, after long-term deficiency, of exogenous thyroid hormone upon HPA function has not been determined. The present study was designed to investigate HPA axis function in mice exposed to dietary thiouracil from conception to adulthood, and to determine whether 'Manuscript received 29 August 1996 and in revised form 7 January 1997 (#96-21). injection of exogenous thyroid hormone modified this function. MATERIALS AND METHODS Adult female Swiss-Webster mice were obtained from the Animal Research Facility at Bowling Green State University, and were mated to males of the same strain. From the day of conception as determined by vaginal plug, pregnant females were housed singly and fed Wayne Lab Blox Mash (Continental Grain Co., Chicago, IL) with or without 2-thiouracil (0.25%, w/w; Sigma Chemical Co., St. Louis, MO) to induce hypothyroidism. Litters were reduced to eight pups at five days of age. Animals were removed from the maternal cage at 25 days of age and were housed in pairs, where they continued to receive the same diet provided to the mother. Animals were between six and eight months of age when assigned to treatment groups. Male and female animals were randomly assigned to treatment groups, since no gender differences were revealed in the parameters tested. A total of 50 mice, 10 per group, were assigned to the following treatment groups: 1. Control Fed no thiouracil, given no thyroid hormone injections (CON). 2. Thiouracil, uninjected fed thiouracil-containing diet, given no thyroid hormone injections (THIONI). 3. Thiouracil, saline-injected fed thiouracil-containing diet, injected with physiological saline (0.9% NaCl) vehicle (0.1 ml, sc) (THIO-SAL). 4. Thiouracil, triiodothyronine (T )-injected fed thiouracil-containing diet, injected with T (25 ng/g b wt, sc, in 0.1 ml saline) (THIO-T3). 5. Thiouracil, thyroxine (T4)-injected fed thiouracilcontaining diet, injected with T4 (50 ng/g b wt, sc, in 0.1 ml salineXTHIO-Tp. Animals receiving either injection vehicle or thyroid OHIO JOURNAL OF SCIENCE L. A. MESERVE AND E. V. RUSS, III 11 hormone were injected with the indicated dose once daily for 14 days. To determine I-IPA axis response, each group of mice was divided into non-stressed and stressed subgroups. Non-stressed animals (five mice per group) were decapitated immediately after removal from the home cage, and stressed animals (five mice per group) were decapitated 15 min after a 1 min exposure to ether fumes. To avoid possible circadian effects on corticosterone concentration, decapitation was always performed between 0800 and 0900 (lights on at 0700; lights off at 1900). Blood was collected at decapitation and allowed to clot at room temperature, and serum was removed by centrifugation for determination of thyroid hormone and corticosterone content. Serum was stored frozen at -20° C. Body weight was determined to the nearest 0.1 g, and liver and thyroid gland weights were determined to the nearest 0.1 mg. Analysis of serum concentrations of T,, Tv and corticosterone was done by radioimmunoassay, using commercially available kits (ICN Biomedicals, Carson, CA). For a given hormone, all samples were assayed in one run. The interassay variation was <5% in each case. Data were statistically analyzed using one-way analysis of variance, with significance ascribed where p <0.05. In cases of significance, multiple comparisons of means were made using the Student-Newman-Keuls test (Zar 1984).