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We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family 'zyklopen.' J. Nutr. 140: 1728-1735, 2010. doi: 10.3945/jn.109.117531.

Iron metabolism during pregnancy is biased toward maintaining the fetal supply, even at the cost of anemia in the mother. The mechanisms regulating this are not well understood. Here, we examine iron deficiency and supplementation on the hierarchy of iron supply and the gene expression of proteins that regulate iron metabolism in the rat. Dams were fed iron-deficient diets for 4 wk, mated, and either continued on the deficient diet or an iron-supplemented diet during either the first half or the second half of their pregnancy. A control group was maintained on normal iron throughout. They were killed at 0.5, 12.5, or 21.5 days of gestation, and tissues and blood samples were collected. Deficiency and supplementation had differential effects on maternal and fetal hematocrit and liver iron levels. From early in pregnancy, a hierarchy of iron supply is established benefiting the fetus to the detriment of the mother. Transferrin receptor, transferrin receptor 2, and hepcidin mRNA expression were regulated by both iron deficiency and supplementation. Expression patterns showed both organ and supplementation protocol dependence. Further analysis indicated that iron levels in the fetal, and not maternal, liver regulate the expression of liver transferrin receptor and hepcidin expression in the mother. placenta; deficiency; supplementation

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 [+ or -] 0.5% of the total dose was transported after 8 h, equivalent to 38.8 [+ or -] 2.1 pmol x [cm.sup.-2] x [h.sup.-1]. Transfer of Tf across the cell layer was much more limited; 2.4 [+ or -] 0.2% of the total dose was transported after 8 h, equivalent to 5.0 [+ or -] 0.4 pmol x [cm.sup.-2] x [h.sup.-1]. Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta. placenta; fetus; mineral metabolism

Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [[sup.14]C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 [micro]M. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus. transcellular transport; intrauterine growth retardation; placenta; system A transporter 2 doi:10.1152/ajpendo.00359.2005

Imbalances in nutrition during pregnancy can lead to long-, as well as short-term consequences, a phenomenon known as fetal programming. However, there is little information about when the fetus is most sensitive to its environment during gestation. We hypothesize that different fetal systems are most vulnerable to nutritional stress during periods of maximal growth and differentiation. We used iron (Fe) deficiency, which causes hypertension in the offspring, to test this hypothesis. We examined development between embryonic day (E) 10.5 and 12.5, when cardiovascular development is maximal, using whole embryo culture. Female rats were fed Fe-deficient or control diet for 4 wk before mating and up to E10.5. The embryos were cultured for 48 h in 95% rat serum collected from males fed either a control or Fe-deficient diet. Growth was impaired and heart size increased in embryos taken from Fe-deficient mothers and cultured in deficient serum compared with control embryos cultured in control serum. To test whether restoring normal Fe levels could reverse these effects, we cultured embryos from control and deficient dams in either control or deficient medium. The yolk sac circulation of embryos from dams fed either diet cultured in deficient medium was less developed, with a thinner and less branched network than that in all embryos cultured in control serum. The heart was enlarged in embryos of deficient dams cultured in deficient serum compared with the heart size of those cultured in control serum. Culturing embryos in control serum reversed these changes. We conclude, therefore, that this period of cardiovascular organogenesis is one of the sensitive windows during which optimal Fe status is critical for normal development. KEY WORDS: * fetal programming * developmental origins * embryo culture * micronutrients

We previously reported an endogenous, membrane-bound Cu oxidase with homology to ceruloplasmin in BeWo cells, a placental choriocarcinoma cell line. In this previous study, ceruloplasmin immunoreactivity was localized to the perinuclear region and non-brush-border membranes. Here, we show that azide-sensitive oxidase activity is enriched in the same fractions, correlating subcellular localization of enzyme activity with ceruloplasmin immunoreactivity. Expression of the placental Cu oxidase is inversely proportional to Fe status and directly proportional to Cu status at enzyme and protein levels. To identify a role for the Cu oxidase, cells were exposed to [sup.59]Fe-transferrin for 18 h in an environment of 20% [O.sub.2] or 5% [O.sub.2]. At 5% [O.sub.2], Cu-deficient cells retain significantly more [sup.59]Fe than control cells. This excess in [sup.59]Fe accumulation is caused by a significant decrease in [sup.59]Fe release. These results indicate that downregulation of the placental Cu oxidase in BeWo cells impairs Fe release. This effect is only apparent in an environment of limited [O.sub.2]. BeWo; hephaestin; hypoxia; placenta; human; ferroxidase

It has been shown the the interactions of Cp(sub.2)TiCl(sub.2) with human serum transferrin (hTF) and that of Ti(sub.2)-hTF with adenosine triphosphate (ATP) may make it possible for transferrin to be a mediator for titanium delivery to tumor cells. Cp(sub.2)TiCl(sub.2), an organometallic cancer agent, is in phase II clinical trials as an anticancer drug. The mechanism of action, however, is not well understood. It appears that titanium transferrin may be recognized by the transferrin receptor and be taken into cancer cells.

The copper transport system by the microvillar vesicles of the human placenta is studied in vitro by measuring administered radiolabeled copper. Human placental microvillar vesicles can accumulate copper from maternal copper- dihistidine complexes. and is mediated by a carrier-mediated process, dependent on saturation kinetics and sensitive to low temperature and osmotic pressure. Ceruloplasmin can directly block the uptake of copper from its dihistidine complex to the placental vesicles. Similar to this, histidine itself can also inhibit transport of copper through the placental barrier by depleting the amount of substrate.

During pregnancy, nutrients are transferred from mother to fetus across the placenta. The mechanisms whereby this occurs, and the adaptations that occur in response to deficiency or overload of iron (Fe) and copper (Cu) are examined in this review. Fe deficiency during pregnancy is common and has serious consequences both in the short and the long term such as fetal growth retardation and cardiovascular problems in the adult offspring. Similarly, Cu deficiency, although not so common, also has deleterious effects. The placenta minimizes the effect of the deficiency by up-regulating the proteins involved in Fe transfer. For example, transferrin receptor levels increase inversely to maternal Fe levels. Divalent metal transporter 1 (DMT1) mRNA in the iron-responsive element (IRE) regulated, but not the non-IRE regulated form is increased, as is the placenta Cu oxidase. Conversely, iron-regulated gene 1 (IREG1) expression is not affected. Fe deficiency increases Cu levels in maternal liver, serum and placenta, but has much less effect in the fetal serum and liver. Apart from maternal ceruloplasmin, mRNA levels of Cu-related proteins are not changed. The Cu oxidase, which we suggest fulfils the function of hephaestin in placenta, is regulated by Cu as well as by Fe. Fe deficiency also has marked effects on cytokine levels in the placenta. Tumor necrosis factor [alpha] (TNF[alpha]) and TNF[alpha] receptor 1 (TNF[alpha]R1) levels both increase. The data show that altering Fe status has a marked effect on metabolism of other metals and of other important mediators of cell function. This is particularly important during pregnancy, when the developing fetus is very vulnerable to inappropriate micronutrient status. KEY WORDS: * BeWo cells * micronutrients * placental transfer * deficiency * rat * human