Thyroidal dysfunction and environmental chemicals--potential impact on brain development.

Certain polyhalogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCBs) and dibenzo-p-dioxins (dioxins, 2,3,7, 8-tetrachlorodibenzo-p-dioxin) have been shown to have neurotoxic effects and to alter thyroid function during critical periods of thyroid hormone-dependent brain development. This has led to the suggestion that some of the neurotoxic effects of these compounds could be mediated through the thyroid system. Thyroid hormones are essential for normal brain development during a critical period beginning in utero and extending through the first 2 years postpartum. They regulate neuronal proliferation, migration, and differentiation in discrete regions of the brain during definitive time periods. Even transient disruption of this normal pattern can impair brain development. Thyroid hormones are necessary for normal cytoskeletal assembly and stability and the cytoskeletal system is essential for migration and neuronal outgrowth. In addition, they regulate development of cholinergic and dopaminergic systems serving the cerebral cortex and hippocampus. Animals perinatally exposed to certain environmental organohalogens such as many of the PCBs and dioxins have abnormal thyroid function and neurologic impairment. Although there are both species and congener variabilities, most reports show exposure results in thyroid enlargement and reduced serum T(4) levels with normal T(3) levels. Initial research concentrated on studying the direct actions of xenobiotics on the thyroid; however, some of these compounds bear a structural resemblance to the natural thyroid hormones and have high affinity with thyroid hormone-binding proteins such as transthyretin. These compounds could act as agonists or antagonists for receptors of the thyroid/steroid/retinoic acid superfamily. These structurally similar organohalogens could act at multiple points to alter thyroid hormone action. The similarity of the neurologic impairment seen in thyroid disorders to that seen following PCB or dioxin exposure suggests that one mechanism of neurotoxicity of these compounds could involve interaction with the thyroid system.

Impact of PCBs on thyroid hormone directed brain development.

Thyroid hormones regulate neuronal proliferation, migration, process outgrowth, synaptic development, and myelin formation in specific brain regions. Because brain development occurs during discrete windows of time, inappropriate levels of thyroid hormones in definitive periods can produce permanent damage, the nature of which depends upon the timing and magnitude of the insult. Thyroid hormones cross the placenta and enter the brain primarily as thyroxine (T4); therefore, conditions selectively lowering serum T4 levels alter brain hormone availability. Triiodothyronine (T3) is the predominant form of the hormone that binds to the receptor. T3 is produced from T4 in the brain by the enzyme type II, 5'-deiodinase. Polychlorinated biphenyls (PCBs) are synthetic environmental toxicants that bear a striking structural resemblance to the active thyroid hormones and can, depending upon the species, dosage, and congener used, act as agonists, antagonists, and partial agonists to thyroid hormones.

Ribosomal protein synthesis in 16 and 19 day gestation fetuses of hypothyroid mothers.

Thyroid hormones are transported across the placenta. Thyroid hormone receptors are present in the midgestation rat fetus and fetal tissues selectively accumulate thyroid hormones prior to the onset of fetal thyroid function. It has not been demonstrated adequately that maternal thyroid hormones are essential for early fetal physiologic functions and neurological development. The present study compares the effects of maternal thyroid hormone versus fetal thyroid hormone on the regulation of rRNA and ribosomal protein synthesis in the developing fetus. This was accomplished by first comparing 16-day gestation (just prior to the onset of fetal thyroid function) fetuses of control and hypothyroid mothers. Then 19-day gestation (fetal thyroids are functional) fetuses of control and hypothyroid mothers were compared as development of fetal thyroid function lags in hypothyroid mothers. Rats made hypothyroid (Tx) by radiothyroidectomy were given replacement doses of thyroxine (T4) until the day that they were placed with a male for mating. Control, Tx and growth hormone (GH)-treated Tx dams and their fetuses were sacrificed on either the 16th or 19th day of gestation. Ribosomes (r) were isolated from placentas and from fetal brains and livers and rRNA, total 14C-leucine incorporation, and 14C-leucine incorporation into ribosomal protein per microgram of rRNA were determined. On the 16th day of gestation, prior to the onset of fetal thyroid function, all three of the above metabolic parameters were reduced significantly below control levels in the placentas of Tx rats and in the brains and livers of their fetuses. This was true also for the fetal brains and livers of GH-treated Tx mothers. Development of fetal thyroid function lags in the Tx mother. rRNA, total 14C-leucine incorporation, and 14C-leucine incorporation into ribosomal protein per microgram of rRNA continue to be significantly depressed in these fetal tissues and placentas of Tx rats on the 19th day of gestation. In fact, brain protein synthesis falls further behind in fetuses of Tx dams at this gestational age when compared with control fetal brains. These data support the hypothesis that maternal thyroid hormones are important, by at least midgestation, for normal fetal physiologic development and support the concept that appropriate maturation of the fetal pituitary-thyroid plays a role in fetal brain protein synthesis and therefore neurological development.

Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system.

Neurologic development follows orderly patterns that can be severely disturbed when thyroid hormones are deficient or excessive. Should this occur at appropriate development periods, irreversible neurologic damage can result. The nature of the deficits depends upon the specific development period and the severity of the thyroid disturbance. PCBs and dioxins are structurally similar to the thyroid hormones. Their binding characteristics are similar to those of thyroid hormones and all three groups bind to the cytosolic Ah receptor, the thyroid hormone receptor and the serum thyroid hormone binding protein transthyretin. Depending upon the dose of toxin and the congener used, the toxins either decrease or mimic the biological action of the thyroid hormones. Either effect, if occurring during brain development, can have disastrous consequences. Children and animals exposed to PCBs or dioxins in utero and/or as infants can exhibit varying degrees of behavioral disorders. These disorders resemble those seen in children exposed to thyroid hormone deficiencies in utero and/or in infancy. The mechanism of developmental neurotoxicity of PCBs and dioxins is not known but data suggest it could be partially or entirely mediated by alterations in availability and action of thyroid hormones during neurological development. It is possible that transient exposure of the mother to doses of toxins presently considered nontoxic to the mother could have an impact upon fetal or perinatal neurological development. If the toxins act via their effect on thyroid hormone action, it is possible that doses of toxins that would normally not alter fetal development, could become deleterious if superimposed on a pre-existing maternal/or fetal thyroid disorder.

Serum growth hormone levels in hypothyroid and GH-treated thyroidectomized rats and their progenies.

Growth hormone (GH) was measured in the sera of control, hypothyroid (thyroidectomized [Tx]) and GH-treated Tx rats and their fetuses on Days 19, 20, 21, and 22 of gestation and in their progenies on postnatal Days 1, 5, 30, and 75. Maternal endogenous serum GH increased dramatically between the 19th and 20th days of gestation and remained elevated through the 22nd day in control rats, but was depressed significantly in Tx and GH-treated Tx rats during this period. GH was not always detected in the sera of 19-day-old fetuses. On Day 20, GH was depressed in fetuses of Tx mothers as compared with those form controls or GH-treated Tx mothers. GH was elevated in sera of fetuses from GH-treated Tx rats over fetuses of control and Tx only rats on the 22nd day of gestation. In postnatal rats, those from GH-treated mothers continued to show elevated serum GH on Day 1 as compared with those from Tx only mothers. On postnatal Days 5 and 30, progenies of Tx mothers had significantly elevated GH as compared with progenies of control mothers. At 75 days of age, the GH levels of these progenies had normalized. We have shown previously that the hormonal secretions of the pituitary-thyroid axis are badly disrupted in the progenies of Tx and GH-treated Tx mothers and that even as adults these animals have tissue (brain and liver) deficits of active thyroid hormones. Although the onset of GH secretion is mildly delayed in fetuses of Tx but not GH-treated Tx mothers, the serum GH levels of both groups of progenies are elevated during most of the neonatal period through the time of puberty. It is, therefore, concluded that GH in the absence of adequate levels of thyroid hormones is ineffective in preventing many of the learning and memory deficits induced in the progenies of Tx mothers.

Tissue iodothyronine levels in fetuses of control and hypothyroid rats at 13 and 16 days gestation.

Some investigators have reported that there is minimal placental transport of thyroid hormones in humans and rats. Consequently, it was thought that thyroid hormones were not present in the fetal brain before fetal thyroid hormone synthesis and, hence, were not important for brain development before fetal thyroid hormonogenesis. Recently, however, thyroid hormones have been detected by 14 days postconception (dpc) in the rat fetus and by 11 dpc in the rat embryotrophoblast. Thyroid hormone receptors have been shown in the fetal rat by 14 dpc. The present experiments were designed to determine if T4, T3, and their metabolites can be detected in rat fetuses at 13 and 16 dpc and if iodothyronines are selectively accumulated in fetal brain and liver. Furthermore, one group of dams was radiothyroidectomized before breeding to ascertain the effect of maternal hypothyroxinemia on fetal tissue iodothyronine concentrations. Tissue iodothyronines were extracted and measured by HPLC. T4, T3, rT3, and 3,5-diiodothyronine were well within the limits of detection by this procedure at both fetal ages. The only possible source of these hormones is the mother. In addition, if maternal serum T4 levels are low, fetal tissue T4 and T3 levels are low. The presence of high intracellular T3 levels, even at 13 dpc, shows that 5'-monodeiodination occurs in the midgestational fetus. Intracellular hormone measurements show that T3, rather than rT3, is the predominant intracellular iodothyronine in the rat fetus. Both brain and liver selectively accumulate T4 and T3, supporting the observations of others that fetal thyroid hormone receptors are present in midgestation. The presence of thyroid hormones in fetal rat brain by 13 dpc coupled with the observation that hormone receptors are present by 14 dpc suggests that thyroid hormones do play a role in midgestational brain development. These data show that normal maternal serum thyroid hormone levels are important during midgestation to provide adequate thyroid hormones to the fetus.

Method for the quantitation of iodothyronines in body tissues and fluids using high-performance liquid chromatography.

The separation and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-, 3,3' and 3',5'-diiodo-L-tyronines, 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine and 3,3',5,5'-tetraiodo-L-thyronine (T4)] from animal tissues (brain, liver and serum) by a new high-performance liquid chromatographic (HPLC) method is described. Rats were infused with iso-osmotic sodium chloride containing 100 microM phloretin to block deiodination. The tissues were extracted using differential pH values to separate other amines from the amine containing iodothyroid hormones. Aliquots of tissue extracts (25-100 microliters) were reacted overnight with 5-dimethylaminonaphthalene-1-sulfonyl chloride and their iodotyrosine and iodothyronine content determined by HPLC utilizing fluorimetric detection. Resolution of the individual compound peaks was achieved by gradient elution with a 3.0 mM H3PO4 buffer. Greater sensitivity has been achieved (less than 1.0 pmol/g) utilizing fluorescence rather than ultraviolet absorbance for the quantitation of these iodinated compounds. The method is superior also to other methods in that recoveries, based on those of 125I-labelled T4 and T3, were 89-97%.

Distribution of free amino acids in streptozotocin-induced diabetic pregnant rats, their placentae and fetuses.

Amino acid levels in the non-pregnant streptozotocin (STZ)-induced diabetic rat have been shown to be abnormal. Our preliminary studies showed that placental transport, fetal serum levels and tissue uptake of the non-metabolizable amino acid alpha-amino isobutyric acid (AIB) were decreased in STZ-diabetic pregnant rats. In the present experiments, amino acid concentrations were measured in maternal (MS) and fetal (FS) sera and placentae (PL) by high performance liquid chromatography (HPLC) after triple extraction in 80% ethanol. Control (C), STZ-diabetic (D) and insulin-treated diabetic (DI) animals were studied at 22 days gestation. Pregnant diabetic rats had low serum levels of Gln, Lys, and Ser and insulin treatment corrected Gln and Ser but not Lys levels. Branched-chain amino acids did not show the large elevation characteristic of the non-pregnant diabetic rat. Placental levels of Tau, Gln, HPr, Thr and Lys were depressed in the diabetic animals and insulin treatment only partially improved these amino acid profiles. Placental amino acid levels did not always reflect maternal serum levels. Serum levels of most amino acids were lower in the fetus of the diabetic rat than in the fetus of the control rat. The notable exception was Ala which was higher in the fetuses of the diabetic animals. Insulin treatment of the mother did not correct many of the fetal amino acid levels even though maternal and fetal serum glucose levels at the time of autopsy were normal. The ability to maintain normal serum levels of many amino acids is impaired in the fetus of the diabetic rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodothyronine-5'-deiodinase activity in progenies of hypothyroid rats.

Iodothyronine-5'-deiodinase activity (I-5'DA) was measured in the progenies of control rats, hypothyroid (Tx) rats, and hypothyroid treated with ovine GH (Tx + GH) during gestation. The enzyme was measured in cerebral cortex and cerebellum at 22 days gestation and at 5, 10, 30 and 60 days postpartum. In addition, the pituitary I-5'-DA was assessed in the postnatal animals. The experiments were undertaken because the tissues of the progenies of rats that were hypothyroid during pregnancy appeared in many ways to resemble those of hypothyroid animals, even at ages when serum thyroxine (T4) and triiodothyronine (T3) levels were normal. It was found that the progenies of Tx mothers had low liver 5'-deiodinase activities. This is a likely cause of the low serum T3 levels with normal T4 levels seen in these progenies in the neonatal period. Cerebral and cerebellar 5'-deiodinase activities were low in these progenies during the thyroid hormone-dependent perinated period of brain development. The progenies of GH-treated Tx dams had higher enzyme activities than the progenies of untreated Tx dams. These pups from GH-treated Tx mothers have been shown previously to have significantly less neurological impairment than the progenies of untreated Tx mothers. As most of the brain intracellular T3 is produced in situ, a functional thyroid deficiency could result from such a 5'-deiodinase deficiency. As the deiodinase deficiency was still seen in the progenies of Tx mothers at 60 days of age, such a deficiency could explain why, even though serum T4 and T3 levels were normal, brain metabolism was in many ways characteristic of hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of streptozotocin-induced diabetes in pregnant rats on placental transport and tissue uptake of alpha-amino-isobutyric acid.

Placental transport and tissue uptake of amino acids were studied in streptozotocin (STZ)-induced diabetic rats by using the non-metabolizable amino acid [U-14C]-alpha-amino-isobutyric acid (AIB). Fifteen minutes prior to autopsy, animals of each group, control (C), diabetic (D), diabetic-insulin treated (DI) and diabetic-T4 followed by 3-5-Dimethyl-3'-isopropyl-L-thyronine (DIMIT) treated (DTD), received an injection of the [U-14C]-AIB SC. Disintegrations per minute (DPM) were measured in serum and tissues subsequent to autopsy. There were no differences in maternal serum DPM/ml among groups. Fetal serum DPM, however, were lower in D and DTD groups than in the C group. The whole fetal tissue homogenate radioactivity was lower in the D, DTD, and DI groups than in the C group. In general, more AIB was taken up by fetal tissues of C than D animals. Maternal liver AIB uptake was reduced in D, DI, and DTD from C animals and net placental transport of AIB was less in D and DTD than C animals. Fetal liver protein concentrations were depressed in D and DTD animals from C and DI, but fetal brain protein concentrations showed no significant differences. Furthermore, the lower organ and fetal body weights of the D and DTD groups compared with the C and DI groups support the proposal that fetal anabolism is impaired. Maternal and fetal serum T4 concentrations were lower in D and DTD than in C and DI animals. Insulin therapy improved serum T4 levels in both mother and fetuses. It did not, however, correct all other measured parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal exposure of the fetal rat to excessive L-thyroxine or 3,5-dimethyl-3'-isopropyl-thyronine produces persistent changes in the thyroid control system.

Studies were conducted to determine if brief exposure, in utero, to high levels of T4 or to the synthetic thyromimetic agent 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) can produce permanent disruption of the thyroid control system in a manner analogous to the changes in the "set point" reported to occur due to neonatal T4 exposure in the "neo-T4 syndrome". If such a change were to occur, it could explain the persistent thyroid disturbances seen in the progeny of hypothyroid mother rats. These latter progeny are exposed in utero to both low and high serum T4 levels. Maternal T4 treatment produced a 4-fold elevation in fetal serum T4 accompanied by a large decrease in serum TSH levels. The brief treatment in utero with high doses of T4 or of DIMIT resulted in higher neonatal mortality and the T4-treatment produce subsequent growth stunting. These treatments resulted in suppression of the fetal/neonatal thyroid which was very apparent at 5 days of age. At 30 days post-partum, the thyroid control system of the progeny of the T4 and DIMIT-treated animals was still abnormal with low serum T4 levels accompanied with normal serum TSH and T3 levels. At 60 days of age, serum T4 levels remained low in the progeny of the T4-treated animals and the TSH response to TRH was subnormal in both the progeny of the T4-treated and the DIMIT-treated animals. However, serum and pituitary TSH and serum T3 were normal. The thyroid control system of the rat is sensitive to prenatal exposure to hyperthyroxinemia as it is to postnatal exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral testing of progenies of Tx (hypothyroid) and growth hormone-treated Tx rats: an animal model for mental retardation.

Virgin Sprague-Dawley Holtzman rats were rendered Tx (hypothyroid) by radiothyroidectomy and maintained on 1.0 microgram T4 (thyroxine) per 100 g BW until pregnant. One-half of these Tx animals were administered 0.5 IU of growth hormone (GH) during the last 10-11 days of gestation as GH secretion is especially deficient in Tx rats. Untreated, food restricted to the level consumed by the Tx-only rats, GH-treated euthyroid, and T4-treated until pregnant animals served as controls. The animals were allowed to go through parturition and each litter was reduced to no more than 6 pups by removing pups for tissue weights and protein analyses at 1 and 5 days of age. The pups were weaned at 22 days of age and 2 animals per litter were utilized for behavioral testing between 40 and 60 days of age. At the end of the behavioral testing period the 60-day-old offspring were sacrificed to obtain tissue weights and protein concentrations. The behavioral tests were based on the ability of the animals to learn a Lashley's type 3 enclosed alley maze and their spontaneous activity was measured in stabilimeter cages. The animals were fasted overnight on alternate days and then given a food reward upon traversing the maze. This allowed for 10 separate trials in both the Lashley maze and the stabilimeters over the 20-day period from 40 to 60 days of age. Our previous studies have shown the fetuses and progenies of Tx-only mothers to have multiple metabolic defects including reduced rates of protein synthesis and tissue protein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of serum thyroxine, triiodothyronine, and thyrotropin in the progeny of hypothyroid rats.

Thyroid function in the progeny of hypothyroid rats was studied from 19 days of gestation until adulthood. Total T4, T3, and TSH were measured by RIA in the sera of control, surgically thyroidectomized (Tx), and GH-treated Tx pregnant rats and their progeny from the late fetal period through adulthood. By the 22nd day of gestation, the fetuses of Tx mothers had T4 and TSH levels significantly above those of their controls. This condition, whereby fetal serum T4 and TSH levels were elevated, was aggravated by GH treatment of the Tx mothers. The progeny of Tx- and Tx and GH-treated mothers also showed alterations in serum T4, T3, and TSH. While the T4 levels of the progeny of Tx mothers were significantly below those of th control progeny only at 30 days of age, T3 levels were significantly depressed at 1, 5, and 30 days of age. These same animals had lower serum TSH levels than the control progeny at 1 and 5 days of age and higher TSH levels at 30 and 75 days of age. Prenatal GH treatment of the mother did not consistently correct thyroid function of the progeny. As adults the progeny of the Tx mothers had normal serum concentrations of T4 and T3 but TSH was high. High serum TSH values were not seen in the progeny of Tx plus GH-treated mothers. These results suggest that maternal hypothyroidism during pregnancy may result in permanent alterations in the thyroidal response to TSH in their progeny.

The effects of growth hormone, thyroxine and insulin on the activities of reduced nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase in fetal rat liver.

Growth hormone (GH), thyroxine (T4) and insulin were injected, in utero into 20.5 day-old rat fetuses to study the effects of these hormones on the activities of liver NADPH dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase. It was found that at 21.5 days of gestation, GH increases the fetal liver glucose-6-phosphatase activity and decreases the liver glycogen phosphorylase activity. T4 treatment augments the activity of NADPH dehydrogenase even at 0.3% of the dose shown previously to produce premature elevation of activity. Prior to this experiment T4 in large doses has been shown to be capable of elevating glucose-6-phosphatase. However, at the lower T4 dose used, no treatment effect was observed. The fetal rat liver is responsive to insulin at 21.5 days and insulin was able to depress glucose-6-phosphatase activity. Thereby, showing that the influence of insulin on this enzyme begins prior to birth instead of just subsequent to birth.