Association between methylmercury exposure from fish consumption and child development at five and a half years of age in the Seychelles Child Development Study: an evaluation of nonlinear relationships.

Studies to date of the developmental effects of pre- and postnatal methylmercury exposure from fish consumption in the Seychelles Islands, using linear regression models for analysis, have not shown adverse effects on neurodevelopmental test scores. In this study we evaluated whether nonlinear effects of methylmercury exposure were present, using scores on six tests administered to cohort children in the Seychelles Child Development Study at 66 months of age. Prenatal exposure was determined by measuring mercury in a segment of maternal scalp hair representing growth during pregnancy. Postnatal exposure was measured in a segment of the child's hair taken at 66-months of age. Generalized additive models (GAMs), which make no assumptions about the functional form of the relationship between exposure and test score, were used in the analysis. GAMs similar to the original linear regression models were used to reanalyze the six primary developmental endpoints from the 66-month test battery. Small nonlinearities were identified in the relationships between prenatal exposure and the Preschool Language Scale (PLS) Total score and Child Behavior Check List (CBCL) and between postnatal exposure and the McCarthy General Cognitive Index (GCI) test scores. The effects are best described graphically but can be summarized by computing the change in the predicted test score from 0 to either 10 or 15 ppm and then above this point. For the PLS the trend involved a decline of 0.8 points between 0 and 10 ppm followed by an increase (representing improvement) of 1.3 points above 10 ppm. For the CBCL there was an increase of 1 point from 0 to 15 ppm, and then a decline (improvement) of 4 points above 15 ppm. The GCI increased by 1.8 points through 10 ppm and then declined 3.2 points (representing worse performance) above 10 ppm. These results are not entirely consistent. Two of the trends involve what appear to be beneficial effects of prenatal exposure. The one possibly adverse trend involves postnatal exposure. In every case the trend changes direction, so that an effect in one direction is followed by an effect in the opposite direction. Because of the descriptive nature of GAMs it is difficult to provide a precise level of statistical significance for the estimated trends. Certainly above 10 ppm there is less data and trends above this level are estimated less precisely. Overall there was no clear evidence for consistent (across the entire range of exposure levels) adverse effects of exposure on the six developmental outcomes. Further nonlinear modeling of these data may be appropriate, but there is also the risk of fitting complex models without a clear biological rationale.

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.

Semiparametric modeling of age at achieving developmental milestones after prenatal exposure to methylmercury in the Seychelles child development study.

Controversy exists concerning the fetal risk associated with exposure to low-dose methylmercury from maternal fish consumption. Previous studies of the effects of acute prenatal mercury exposure identified delays in achieving developmental milestones among exposed children. This led to public health concern that prenatal low-dose exposure from fish consumption could adversely affect the fetus. We evaluated the effects of prenatal methylmercury exposure (through maternal fish consumption) on the age that children walked and first said words in the main study cohort of the Seychelles Child Development Study. We used semiparametric generalized additive models to identify nonlinearities in the relationships between prenatal exposure and developmental outcomes, after adjusting for covariates, and to evaluate their importance. Very slight delays (<1 day) in walking were seen as mercury levels increased from 0 to 7 ppm, but this effect did not persist at the higher exposure levels represented by the cohort, making it difficult to conclude that a cause and effect relationship existed at the exposure levels seen in this cohort. There was no evidence for any association between prenatal exposure and age at talking.

Signaling pathways involved in GnRH secretion in GT1 cells.

The GT1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the role of the cyclic AMP/protein kinase A, cyclic GMP/protein kinase G and Ca2+/protein kinase C signaling pathways in the regulation of GnRH secretion. Superfusion of GT1-1 cells with the cyclic AMP analog 8-Br-cyclic AMP (0.5 and 2.5 mM) or the adenylate cyclase activator forskolin (1 and 10 microM) for 100 min increased the amplitude of GnRH secretion 2- to 35-fold. The cyclic GMP analog 8-Br-cyclic GMP (2.5 mM) also stimulated the amplitude of GnRH release from superfused GT1-1 cells, although to a much lesser extent (1.5- to 3-fold). The amplitude of GnRH pulses was also stimulated (5- to 50-fold) by the protein kinase C activator TPA (1 microM). Increasing intracellular Ca2+ with an ionophore (ionomycin, 1 microM) or by the Ca2+ channel activator Bay K 8644 (10 microM) also stimulated GnRH release, while secretion was markedly decreased and spontaneous pulsatility abolished by the L-type Ca2+ channel blocker methoxyverapamil (10 microM). These results demonstrate that in GT1 cells the protein kinase A, protein kinase G and protein kinase C pathways are functionally coupled to regulation of GnRH secretion. Furthermore, pulsatile GnRH secretion is coupled to the entry of extracellular Ca2+ via L-type Ca2+ channels.

Biphasic gabaergic regulation of GnRH secretion in GT1 cell lines.

The mouse GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines exhibit highly differentiated properties of GnRH neurons. This report investigates the direct effect of gamma-aminobutyric acid (GABA) and subtype selective GABA agonists on GnRH secretion by GT1 cells in perifusion. Treatment of GT1-1 cells with GABA (10 microM) for 100 min resulted in a biphasic release of GnRH. A rapid and sharp stimulation of GnRH secretion was followed by a sustained inhibition of GnRH secretion. During the inhibitory phase, pulses of GnRH assessed by 'cluster analysis' were totally suppressed. The GABAA receptor agonist muscimol (10 microM) stimulated a rapid but transient release of GnRH. On the other hand, treatment of GT1-1 cells with the GABAB receptor agonist baclofen (10 microM) resulted in the prolonged inhibition of GnRH secretion which returned to normal after the treatment stopped. These results demonstrate a direct biphasic effect of GABA upon GnRH release. The initial stimulation appears to be mediated via GABAA receptors, while the sustained inhibition of GnRH secretion appears to involve the activation of GABAB receptors.

Dopaminergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via D1-receptors positively coupled to adenylate cyclase.

The release of GnRH evoked by dopamine (DA) was studied in the GT1 GnRH neuronal cell lines. Superfusion of GT1-1 cells with DA or the D1-dopaminergic agonist SKF 38393, but not with the D2-dopaminergic agonist bromocriptine, increased 2-fold the amplitude of the spontaneous GnRH pulses. Treatment with DA for 30 min also stimulated GnRH release from static cultures of GT1-7 cells. This effect was mimicked by the selective D1-dopaminergic agonist SKF 38393 and blocked by the D1-dopaminergic antagonist SCH 23390. However, the D2-dopaminergic agonist bromocriptine had no effect, and the stimulation of GnRH release by DA was not blocked by the D2-dopaminergic antagonist spiroperidol. In parallel to the stimulation of GnRH release, DA also rapidly increased (first observed at 120 sec) in a dose-dependent fashion, the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-7 cells. The pharmacological profile of the increase in cAMP was identical to that for GnRH release. The cAMP responses to DA and norepinephrine were lost after long term treatment with SKF 38393, i.e. heterologous desensitization. GT1 cells also express the mRNA for the dopamine- and cAMP-regulated phospho-protein (mol wt, 32,000; DARPP-32) only seen in cells expressing DA D1-receptors. These results demonstrate a direct stimulatory effect of DA on GnRH release via DA D1-receptors positively coupled to adenylate cyclase in GnRH neuronal cell lines.

Beta 1-adrenergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via receptors positively coupled to adenylate cyclase.

The release of GnRH evoked by norepinephrine (NE) was studied in GT1 GnRH neuronal cell lines in superfusion and static cultures. GnRH release from static cultured GT1-7 cells was stimulated by NE in a dose-dependent fashion. This effect was mimicked by the nonsubtype-selective beta-adrenergic agonist isoproterenol and blocked by the beta-adrenergic antagonist propranolol and the beta 1-adrenergic subtype-specific antagonist CGP 20712A. However, the stimulation of GnRH release by NE was not affected by the beta 2-, alpha-, alpha 1-, or alpha 2-adrenergic antagonists ICI 118.551, phentolamine, prazosin, or yohimbine, respectively. Superfusion of GT1-1 cells with NE for 60-100 min resulted in rapid and sustained increases in GnRH secretion. The NE-stimulated GnRH release showed a higher amplitude and longer duration than the spontaneous GnRH pulses characteristic of GT1-1 cells. In parallel to the stimulation of GnRH release, NE also rapidly increased (first observed at 60 sec) the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-1 and GT1-7 cells in a dose-dependent fashion. The stimulation of intracellular cAMP concentration was also mimicked by isoproterenol and blocked by propranolol and CGP 20712A. In addition, GT1 cells express beta 1- but not beta 2-adrenergic receptor mRNA, as probed by Northern blot analysis. These results demonstrate a direct stimulatory effect of NE on GnRH neurons. The pharmacological evidence and the mRNA analysis are consistent with NE acting through a beta 1-adrenergic receptor positively coupled to adenylate cyclase.

Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line.

The immortalized neuronal cell line GT1-1 was used to investigate the endogenous pattern of GnRH release. The GT1-1 cell line was derived from a GnRH-secreting tumor in a transgenic mouse induced by genetically targeted expression of the potent simian virus 40 oncogene encoding tumor antigen. Cells attached to coverslips were superfused in Sykes-Moore chambers with Locke's medium, Ca(2+)-free Locke's medium, or Opti-MEM (another defined medium) for 2 hr, and samples were collected at 4-min intervals. Release of GnRH in 17 of 18 superfusion chambers was seen to be pulsatile when data were analyzed by cluster analysis. No significant differences were observed whether only one or both of the coverslips forming the chamber were coated with cells. Pulses exhibited a mean interpulse interval of 25.8 +/- 1.5 min, a mean duration of 18.8 +/- 1.4 min, and a mean amplitude of 150.5 +/- 6.0% above preceding nadir. The removal of Ca2+ from the Locke's medium resulted in the progressive reduction of the amplitude and eventually in the absence of identifiable pulses. Pulses reappeared after the return of Ca2+ to the medium. It is concluded that the GT1-1 cell line secretes GnRH in a rhythmic pattern. These findings suggest that the pulsatile release of GnRH (GnRH pulse generator) may be an intrinsic characteristic of the GnRH neurons. Synchronization of pulsatile release from individual neurons could be mediated via numerous cell-to-cell contacts observed in the cultured cells on coverslips. Synchronization of GnRH release from cells on two physically separated coverslips forming a chamber would appear to be accomplished by a diffusible mediator.