Additional file 1 of Association between maternal blood or cord blood metal concentrations and catch-up growth in children born small for gestational age: an analysis by the Japan environment and children’s study

Supplementary Material

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

<div><p>2,3,7,8-Tetrachlorodibenzo-<em>p</em>-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; <em>i.e.</em>, ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.</p> </div

The LA-specific recovery from a TCDD-induced reduction in the fetal expression of gonadotropins: A–C, pituitary levels of LHβ, FSHβ and LH/FSH α-subunit mRNAs, respectively; and D, serum content of LH.

<p>Gonadotropin mRNAs in the fetuses (GD20), the parents of which were treated with TCDD (GD15) and anti-oxidants (GD15-20), were determined by RT-PCR. The level of gonadotropin mRNA was normalized by β-actin mRNA. Serum LH (GD20 fetuses) was determined by ELISA. In the white bar control, pregnant rats were treated with DMSO alone or anti-oxidant dissolved in DMSO. In the shaded bar control, dams were given aqueous NaCl alone or anti-oxidant dissolved in this solution. The pituitaries and sera of all male fetuses in one dam were pooled to become one analytical unit. Each bar represents the mean value relative to the control ± SEM of 5 (panels A-C) or 7 (panel D) dams. *p<0.05 and **p<0.01, from the respective controls (A-C) or between a pair indicated (<i>D</i>).</p

Fetal brain components showing a significant increase or decrease by maternal exposure to TCDD.

<p>The representative components that were shown by PCA to be significantly changed by TCDD are listed. The data from both positive and negative ion modes were combined. The components of the TCA cycle are shown in bold character.</p

The LA-specific recovery from a TCDD-induced reduction in the fetal expression of testicular StAR and CYP17; A, StAR mRNA; B, StAR protein; C, CYP17 mRNA; and D, CYP17 protein.

<p>The fetal (GD20) testis was analyzed after maternal exposure to TCDD (GD15) and anti-oxidants (GD15-20). The expression of mRNA and protein was analyzed RT-PCR and immunoblotting, respectively. See Materials and Methods for the details of animal treatment and analytical methods. Edaravone is abbreviated as Eda. The levels of StAR and CYP17 mRNAs were normalized by β-actin mRNA. In the white bar control, pregnant rats were treated with DMSO alone or anti-oxidant dissolved in DMSO. In the shaded bar control, dams were given aqueous NaCl alone or anti-oxidant dissolved in this solution. Each bar represents the mean value relative to the control ± SEM of 10 fetuses, each 2 of which were removed from 5 different dams. In processing the data, the values of two fetuses from one dam were averaged to become one analytical unit. Thus, the data are shown as N = 5 dams. *p<0.05 and **p<0.01, from the respective controls (panels A and C) or between a pair indicated (panels B and D).</p

The effects of LA on the induction of fetal brain CYP1A1 and on the acute toxicity of TCDD in pubertal rats.

<p>A, Pituitary and hypothalamic CYP1A1 mRNAs in the fetuses (GD20), the parents of which were treated with TCDD (GD15) and LA (GD15-20). B-D, Change in body weight (B), thymic weight (C) and hepatic EROD activity (D) of male pubertal rats treated orally with TCDD (Day 0) and intravenously with LA (from Day 0 to Day 5). In Experiment (<i>B</i>), the initial body weights of rats in the control, TCDD, LA, and TCDD + LA-treated groups were 219±6, 218±6, 216±7, 216±8 (LA: 20 mg/kg), and 217±5 (LA: 40 mg/kg) g, respectively. The thymus and liver were removed 30 min after the last administration of LA or vehicle. Each bar or plot represents the mean ± SEM (N = 5 or 6). To avoid confusion, the error bar was omitted from panel <i>B</i>. *p<0.05 and **P<0.01, compared with the control, and †P<0.05 compared with the TCDD-treated group. B.D.: below the determination limit (4 pmol/min/mg protein).</p

Summary of TCDD effects on fetal hypothalamic changes in the TCA cycle and amino acid metabolism.

<p>The upward (red) and downward (green) arrows represent a significant increase and reduction produced by maternal exposure to TCDD. Thick arrows indicate a two-fold or more increase, or a reduction to 1/2 or less, compared with the control. The black thick arrows show the steps which are targets for the regulation of the TCA cycle. Regarding gluconeogenesis, dioxins have been reported to down-regulate phosphoenolpyruvate carboxykinase, a key enzyme involved in this process <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040322#pone.0040322-Weber1" target="_blank">[46]</a>.</p

Principal component analysis (PCA) regarding the effect of TCDD on the fetal pituitary and hypothalamic metabolome: the data from negative ion mode analysis.

<p>A and B, TCDD effect on the profile of pituitary (A) and hypothalamic (B) metabolome. Each dot is a different animal (N = 16 dams/group). C and D, Fragment ions in LC-TOF-MS analysis that exhibit an alteration following TCDD treatment in the pituitary (C) and hypothalamus (D). Each dot shows a single ion with a particular mass (<i>m/z</i>). The criteria for selecting ions which were significantly changed by TCDD was set either at more than 0.8 or less than −0.8 of the correlation coefficient. See Experimental Procedures for details.</p

The effect of LA on TCDD-produced alterations in fetal contents of hypothalamic ATP and TCA cycle components.

<p>Each value represents the mean ± S.E.M. (N = 5 dams): the hypothalami of male fetuses in one dam were pooled for assay. Significantly different from the control (*) and from the TCDD-treated group (†): P<0.05.</p

Reduction by TCDD in the fetal content of testosterone and its recovery by LA.

<p>The testes and sera of GD20 fetuses born from the dams treated with TCDD at GD15 were analyzed by LC-TOF-MS. Each bar represents the mean ± SEM (N = 3–5 dams). *p<0.05 between a pair indicated.</p