Fetal and neonatal iron deficiency but not copper deficiency increases vascular complexity in the developing rat brain.

OBJECTIVES: Anemia caused by nutritional deficiencies, such as iron and copper deficiencies, is a global health problem. Iron and copper deficiencies have their most profound effect on the developing fetus/infant, leading to brain development deficits and poor cognitive outcomes. Tissue iron depletion or chronic anemia can induce cellular hypoxic signaling. In mice, chronic hypoxia induces a compensatory increase in brain blood vessel outgrowth. We hypothesized that developmental anemia, due to iron or copper deficiencies, induces angiogenesis/vasculogenesis in the neonatal brain. METHODS: To test our hypothesis, three independent experiments were performed where pregnant rats were fed iron- or copper-deficient diets from gestational day 2 through mid-lactation. Effects on the neonatal brain vasculature were determined using quantitative real-time polymerase chain reaction to assess mRNA levels of angiogenesis/vasculogenesis-associated genes and GLUT1 immunohistochemistry to assess brain blood vessel density and complexity. RESULTS: Iron deficiency, but not copper deficiency, increased mRNA expression of brain endothelial cell- and angiogenesis/vasculogenesis-associated genes (i.e. Glut1, Vwf, Vegfa, Ang2, Cxcl12, and Flk1) in the neonatal brain, suggesting increased cerebrovascular density. Iron deficiency also increased hippocampal and cerebral cortical blood vessel branching by 62 and 78%, respectively. DISCUSSION: This study demonstrates increased blood vessel complexity in the neonatal iron-deficient brain, which is likely due to elevated angiogenic/vasculogenic signaling. At least initially, this is probably an adaptive response to maintain metabolic substrate homeostasis in the developing iron-deficient brain. However, this may also contribute to long-term neurodevelopmental deficits.

Maternofetal and neonatal copper requirements revealed by enterocyte-specific deletion of the Menkes disease protein.

The essential requirement for copper in early development is dramatically illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by loss-of-function mutations in the gene encoding the copper transporting ATPase ATP7A. In this study, we generated mice with enterocyte-specific knockout of the murine ATP7A gene (Atp7a) to test its importance in dietary copper acquisition. Although mice lacking Atp7a protein within intestinal enterocytes appeared normal at birth, they exhibited profound growth impairment and neurological deterioration as a consequence of copper deficiency, resulting in excessive mortality prior to weaning. Copper supplementation of lactating females or parenteral copper injection of the affected offspring markedly attenuated this rapid demise. Enterocyte-specific deletion of Atp7a in rescued pregnant females did not restrict embryogenesis; however, copper accumulation in the late-term fetus was severely reduced, resulting in early postnatal mortality. Taken together, these data demonstrate unique and specific requirements for enterocyte Atp7a in neonatal and maternofetal copper acquisition that are dependent on dietary copper availability, thus providing new insights into the mechanisms of gene-nutrient interaction essential for early human development.

Erythrocyte copper chaperone for superoxide dismutase is increased following marginal copper deficiency in adult and postweanling mice.

A sensitive and reliable biomarker has yet to be identified for marginal copper deficiency in humans. The need for such a biomarker is critical, because increased cases of human copper deficiency evolve following bariatric surgery and other secondary factors besides diet. Four experiments were devised to induce marginal copper deficiency through copper-deficient (CuD) diets (5 wk for mice and 4 wk for rats). In Expt. 1 and 2, male postweanling mice were raised in either solid-bottom plastic cages (Expt. 1) or stainless steel hanging cages (Expt. 2) and compared. Postweanling rats (Expt. 3) and adult mice (Expt. 4) were also studied using stainless steel cages. Copper-adequate controls were fed a semipurified diet containing 9 mg Cu/kg. CuD rats exhibited the most severe changes in biomarkers due to copper limitation, including major reductions in plasma ceruloplasmin (Cp) and erythrocyte superoxide dismutase (Sod1) and augmentation in copper chaperone for Sod1 (CCS). The CuD mice in Expt. 2 were more deficient than the CuD mice in Expt. 1, likely due to coprophagia differences. In fact, the CuD mice in Expt. 1 had unaltered Sod1 or Cp levels. Importantly though, these marginally deficient mice and CuD adult mice that had no changes in Cp activity or liver copper level had robust augmentation of CCS. Erythrocyte CCS was the only consistent biomarker to change in copper deficiency for all dietary groups, suggesting that CCS may be an excellent biomarker for human confirmation of marginal copper deficiency.

Copper deficiency has minimal impact on ferroportin expression or function.

Interactions between copper and iron homeostasis have been known since the nineteenth century when anemia in humans was first described due to copper limitation. However, the mechanism remains unknown. Intestinal and liver iron concentrations are usually higher following copper deficiency (CuD). This may be due to impaired function of the multicopper oxidases hephaestin or ceruloplasmin (Cp), respectively. However, iron retention could be due to altered ferroportin (Fpn), the essential iron efflux transporter in enterocytes and macrophages. Fpn mRNA is controlled partially by intracellular iron and IRE dependence. CuD should augment Fpn based on iron level. Some argue that Fpn stability is controlled partially by membrane ferroxidase (GPI-Cp). CuD should result in lower Fpn since GPI-Cp expression and function is reduced. Fpn turnover is controlled by hepcidin. CuD results in variable Hamp (hepcidin) expression. Fpn mRNA and protein level were evaluated following dietary CuD in rats and mice. To correlate with Fpn expression, measurements of tissue iron were conducted in several rodent models. Following CuD there was little change in Fpn mRNA. Previous work indicated that under certain circumstances Fpn protein was augmented in liver and spleen following CuD. Fpn levels in CuD did not correlate with either total iron or non-heme iron (NHI), as iron levels in CuD liver were higher and in spleen lower than copper adequate controls. Fpn steady state levels appear to be regulated by a complex set of factors. Changes in Fpn do not explain the anemia of CuD.

Ctr1 is an apical copper transporter in mammalian intestinal epithelial cells in vivo that is controlled at the level of protein stability.

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu(+) importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.

Interactions of peptide amidation and copper: novel biomarkers and mechanisms of neural dysfunction.

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine alpha-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM(+/-) mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM(+/-) mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.

Anemic copper-deficient rats, but not mice, display low hepcidin expression and high ferroportin levels.

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.

Perinatal copper deficiency alters rat cerebellar purkinje cell size and distribution.

Copper is required for activity of several key enzymes and for optimal mammalian development, especially within the central nervous system. Copper-deficient (CuD) animals are visibly ataxic, and previous studies in rats have demonstrated impaired motor function through behavioral experiments consistent with altered cerebellar development. Perinatal copper deficiency was produced in Holtzman rat dams by restricting dietary copper during the last two thirds of gestation and lactation. Male offspring were evaluated at postnatal day 25. Compared to cerebella from copper-adequate pups, the CuD pups had larger Purkinje cell (PC) size and irregularities in the Purkinje cell monolayer. These results suggest that the ataxic behavioral phenotype of CuD rats may result from disrupted inhibitory pathways in the cerebellum. A similar PC phenotype is seen in Menkes disease and in mottled mouse mutants with genetic copper deficiency, suggesting that copper deficiency and not just specific loss of ATP7A function is responsible.

Copper deficiency in rodents alters dopamine beta-mono-oxygenase activity, mRNA and protein level.

Cu is an essential cofactor for at least twelve mammalian enzymes including dopamine beta-mono-oxygenase (DBM), which converts dopamine (DA) to noradrenaline (NA). Previous studies reported that certain Cu-deficient (Cu-) rat tissues have lower NA and higher DA than Cu-adequate (Cu+) tissues, suggesting that DBM function was impaired. However, in vitro studies suggested that DBM activity is higher in Cu- tissue. Experiments were conducted on adrenal glands (AG), medulla oblongata/pons (MO), vas deferens (VD) and heart (HT) from a single rat experiment to provide data to help clarify this puzzling contradiction. In vitro DBM activity assays showed Cu- samples had significantly higher activity than Cu+ samples in both AG and MO, but not VD. Activity data were confirmed by Western immunoblots. Quantitative real-time PCR demonstrated higher DBM mRNA in Cu- tissues but unaltered levels of several other cuproenzymes and Cu-binding proteins. Previous pharmacological data implied that high DBM was associated with low NA. HPLC analyses confirmed that NA and DA levels in Cu- MO, VD and HT were significantly lower and higher, respectively, than in Cu+ tissues. However, the NA content of AG was not statistically lower. Furthermore there was no correlation between higher DBM mRNA and lower NA in four Cu-tissues. Adequate dietary Cu is essential to support DBM function in vivo but additional studies are needed to determine the mechanism for increased DBM transcription associated with Cu deficiency.

Copper deficiency alters the neurochemical profile of developing rat brain.

Copper deficiency is associated with impaired brain development and mitochondrial dysfunction. Perinatal copper deficiency was produced in Holtzman rats. In vivo proton NMR spectroscopy was used to quantify 18 cerebellar and hippocampal metabolites on postnatal day 21 (P21). Copper status was evaluated in male copper-adequate (CuA) and copper-deficient (CuD) brothers at P19 and at P23, 2 days following NMR experiments, by metal and in vitro metabolite data. Compared to CuA pups, CuD pups had lower ascorbate concentration in both brain regions, confirming prior HPLC data. Both regions of CuD rats also had lower N-acetylaspartate levels consistent with delayed development or impaired mitochondrial function similar to prior work demonstrating elevated lactate and citrate. For other metabolites, the P21 neurochemical profile of CuD rats was remarkably similar to CuA rats but uniquely different from iron-deficient or chronic hypoxia models. Further research is needed to determine the neurochemical consequences of copper deficiency.

Augmented cerebellar lactate in copper deficient rat pups originates from both blood and cerebellum.

Copper (Cu) is essential for proper brain development, particularly the cerebellum, and functions as a cofactor for enzymes including mitochondrial cytochrome c oxidase (CCO). Cu deficiency severely limits CCO activity. Augmented lactate in brain of Cu deficient (Cu-) humans and cerebella of Cu- rats is though to originate from impaired mitochondria. However, brain lactate may also originate from elevated blood lactate. The hypothesis that cerebellar lactate originates from elevated blood lactate in Cu- rat pups was tested. Analysis of Cu- and Cu adequate (Cu+) rat pups (experiment I) revealed blood lactate was elevated in Cu- rat pups and cerebellar lactate levels were closely correlated to blood lactate concentration. A second rat experiment (experiment II) assessed Cu- cerebellar lactate without the confounding factor of elevated blood lactate. Blood lactate levels of Cu- rat pups in experiment II were equal to those of controls; however, Cu- cerebellar lactate was still elevated, suggesting mitochondrial impairment by Cu deficiency. Treatment of rat pups with dichloroacetate (DCA), an activator of mitochondrial pyruvate dehydrogenase complex (PDC), lowered Cu- cerebellar lactate to control levels suggesting PDC inhibition is a site of mitochondrial impairment in Cu- cerebella. Results suggest Cu- cerebellar lactate originates from blood and cerebellum.

Iron injection restores brain iron and hemoglobin deficits in perinatal copper-deficient rats.

Copper (Cu) deficiency during perinatal development in rats is associated with anemia, lower plasma iron (Fe), and brain Fe. Experiments were conducted to inject Fe dextran into Cu-deficient (Cu-) rat pups to attempt to reverse these conditions. Previous work with older Cu- rats did not reverse anemia following Fe injection. Dams began Cu-adequate (Cu+) or Cu- dietary treatments starting at embryonic d 7 and lasting through weaning. In Expt. 1, pups from each dietary treatment were given a single dose of Fe, 20 mg Fe/kg, or saline (S) at postnatal d 11 (P11). Plasma Fe and hemoglobin were higher in the Fe-injected groups at P13. Brain Fe deficit and brain transferrin receptor enhancement were eliminated in the Cu- group injected with Fe compared with Cu-S pups, supporting an association between low plasma Fe and low brain Fe. In Expt. 2, Fe treatment was increased to 45 mg Fe/kg. Four injections were given between P5 and P18 (total dose, 5-7 mg Fe). At P20, Fe concentrations in 4 brain regions (cortex, cerebellum, medulla/pons, and hypothalamus) generally were higher in all groups than in Cu-S pups. At P25, impaired vibrissae-elicited foot placement was evident in Cu-S rats and was not improved by Fe injection. However, at P26, the brain Fe deficit in Cu-S pups was eliminated by Fe injection. Fe injections in Cu- pups raised plasma Fe, brain Fe, and hemoglobin but did not reverse low cytochrome c oxidase or abnormal striatal behavior.

Copper deficiency results in AMP-activated protein kinase activation and acetylCoA carboxylase phosphorylation in rat cerebellum.

Copper (Cu) deficiency impairs cerebellar development including biosynthetic processes like myelination and synaptogenesis. The activity of cerebellar mitochondrial cuproenzyme cytochrome c oxidase is markedly lower in Cu deficient rat pups and is accompanied by higher lactate levels indicating mitochondrial inhibition. Cu deficiency impaired energy metabolism is thought to contribute to developmental delays, but specific mechanisms linking these phenomena have remained unexplored. AMP-activated protein kinase (AMPK) is a cellular energy sensor that is activated during mitochondrial inhibition and shuts down biosynthetic processes to help conserve cellular ATP levels. Activated AMPK phosphorylates and inhibits acetylCoA carboxylase (ACC), the first enzyme in fatty acid biosynthesis. We hypothesize that AMPK is activated and ACC inhibited in Cu deficient cerebella. Perinatal copper deficiency was studied in young rats in rapidly frozen cerebella. Compared to copper-adequate (Cu+) pups, copper-deficient (Cu-) pups were hypothermic, had lower brain copper levels and markedly higher cerebellar lactate. Concentration of phosphorylated AMPK (pAMPK), indicating AMPK activation, was robustly higher in Cu- cerebella of rat pups at two ages and in two separate experiments. Compared to Cu+ cerebella, pACC content was significantly higher in all Cu- samples. Mechanisms leading to AMPK activation remain elusive. Higher AMP/ATP ratios and increased reactive nitrogen species (RNS) can lead to AMPK activation. ATP and AMP concentrations were unaltered and nitric oxide metabolites and 3-nitrotyrosine peptide levels remained unchanged in Cu- cerebella. AMPK activation may explain how ATP levels can be maintained even with a severe mitochondrial loss of CCO function.

CCS and SOD1 mRNA are reduced after copper supplementation in peripheral mononuclear cells of individuals with high serum ceruloplasmin concentration.

The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.

Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase.

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy "master-switch" that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu-) rat pups were assessed to evaluate if AMPK activation in Cu- cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu- cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu- cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu- cerebella. Data suggest AMPK activation in Cu- cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu- cerebella than controls.

Plasma peptidylglycine alpha-amidating monooxygenase (PAM) and ceruloplasmin are affected by age and copper status in rats and mice.

In an attempt to identify a sensitive and improved marker of mammalian copper status during neonatal development experiments compared two plasma cuproenzymes, peptidylglycine alpha-amidating monooxygenase (PAM ), an enzyme involved in peptide posttranslational activation, to ceruloplasmin (Cp), a ferroxidase involved in iron mobilization. Dietary Cu deficiency (Cu-) was studied in dams and offspring at postnatal age 3 (P3), P12, and P28. Rodent Cp activity rose during lactation whereas PAM activity fell. Reduction in Cp activity was more severe than reduction in PAM activity in Cu- offspring and dams. Cp activity was greater in rats than mice whereas PAM activity was similar in adults but greater in mouse than rat pups. Both cuproenzymes changed during neonatal development and when dietary copper was limiting. With proper controls, each enzyme can be used to assess copper status.

Cu,Zn-superoxide dismutase is lower and copper chaperone CCS is higher in erythrocytes of copper-deficient rats and mice.

Discovery of a sensitive blood biochemical marker of copper status would be valuable for assessing marginal copper intakes. Rodent models were used to investigate whether erythrocyte concentrations of copper,zinc-superoxide dismutase (SOD), and the copper metallochaperone for SOD (CCS) were sensitive to dietary copper changes. Several models of copper deficiency were studied in postweanling male Holtzman rats, male Swiss Webster mice offspring, and both rat and mouse dams. Treatment resulted in variable but significantly altered copper status as evaluated by the presence of anemia, and lower liver copper and higher liver iron concentrations in copper-deficient compared with copper-adequate animals. Associated with this copper deficiency were consistent reductions in immunoreactive SOD and robust enhancements in CCS. In most cases, the ratio of CCS:SOD was several-fold higher in red blood cell extracts from copper-deficient compared with copper-adequate rodents. Determination of red cell CCS:SOD may be useful for assessing copper status of humans.

Copper, zinc-superoxide dismutase protein but not mRNA is lower in copper-deficient mice and mice lacking the copper chaperone for superoxide dismutase.

Cu, Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. Previous work with mice lacking the Cu chaperone for SOD1 (CCS) indicated a drastic loss of SOD1 activity but not protein, suggesting an accumulation of apo-SOD1. We produced dietary Cu deficiency in mice to clarify this issue. Compared with Cu-deficient rats, reduction in liver SOD1 activity and protein was much less than Cu-deficient mouse dams and offspring. However, after perinatal Cu deficiency, 4-week-old mouse pups had lower levels of SOD1 activity and protein in liver and heart, but not brain, compared with Cu-adequate controls. Reduction in brain Cu was greater than liver. In CCS -/- mice, there was severe reduction in liver, heart, and brain SOD1 activity and protein. In fact, the reduction in activity was similar to the loss of protein. Neither Cu-deficient mouse liver nor CCS -/- mouse liver had altered SOD1 mRNA levels compared with control values. These results in mice are comparable with rats and suggest a posttranscriptional mechanism for reduction of SOD1 protein when Cu is limiting in SOD1.

Impact of copper deficiency in humans.

Humans consume about 1 mg of copper daily, an amount thought adequate for most needs. Genetic, environmental, or physiological alterations can impose a higher copper set point, increasing risk for copper-limited pathophysiology. Humans express about a dozen proteins that require copper for function (cuproenzymes). Limitation in the activity of cuproenzymes can explain the pleiotropic effect of copper deficiency. However, for most of the salient features of human copper deficiency, the precise molecular mechanisms are unknown. This is true for the two most common clinical features, hypochromic anemia and adult onset peripheral neuropathy/ataxia, a condition described frequently in the last decade due to multiple etiologies. The challenge for future scientists will be to identify the mechanisms underlying the pathophysiology of copper deficiency so appropriate screening and treatment can occur. The need for a strong copper biomarker to aid in this screening is critical.

Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression.

Fetal/neonatal iron (Fe) and iodine/TH deficiencies lead to similar brain developmental abnormalities and often coexist in developing countries. We recently demonstrated that fetal/neonatal Fe deficiency results in a mild neonatal thyroidal impairment, suggesting that TH insufficiency contributes to the neurodevelopmental abnormalities associated with Fe deficiency. We hypothesized that combining Fe deficiency with an additional mild thyroidal perturbation (6-propyl-2-thiouracil [PTU]) during development would more severely impair neonatal thyroidal status and brain TH-responsive gene expression than either deficiency alone. Early gestation pregnant rats were assigned to 7 different treatment groups: control, Fe deficient (FeD), mild TH deficient (1 ppm PTU), moderate TH deficient (3 ppm PTU), severe TH deficient (10 ppm PTU), FeD/1 ppm PTU, or FeD/3 ppm PTU. FeD or 1 ppm PTU treatment alone reduced postnatal day 15 serum total T4 concentrations by 64% and 74%, respectively, without significantly altering serum total T3 concentrations. Neither treatment alone significantly altered postnatal day 16 cortical or hippocampal T3 concentrations. FeD combined with 1 ppm PTU treatment produced a more severe effect, reducing serum total T4 by 95%, and lowering hippocampal and cortical T3 concentrations by 24% and 31%, respectively. Combined FeD/PTU had a more severe effect on brain TH-responsive gene expression than either treatment alone, significantly altering Pvalb, Dio2, Mbp, and Hairless hippocampal and/or cortical mRNA levels. FeD/PTU treatment more severely impacted cortical and hippocampal parvalbumin protein expression compared with either individual treatment. These data suggest that combining 2 mild thyroidal insults during development significantly disrupts thyroid function and impairs TH-regulated brain gene expression.