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Manganese is an essential yet potentially toxic metal. Initially reported in 2012, mutations in SLC30A10 are the first known inherited cause of manganese excess. SLC30A10 is an apical membrane transport protein that exports manganese from hepatocytes into bile and from enterocytes into the lumen of the gastrointestinal tract. SLC30A10 deficiency results in impaired gastrointestinal manganese excretion, leading to severe manganese excess, neurologic deficits, liver cirrhosis, polycythemia, and erythropoietin excess. Neurologic and liver disease are attributed to manganese toxicity. Polycythemia is attributed to erythropoietin excess, but the basis of erythropoietin excess in SLC30A10 deficiency has yet to be established. Here we demonstrate that erythropoietin expression is increased in liver but decreased in kidneys in Slc30a10-deficient mice. Using pharmacologic and genetic approaches, we show that liver expression of hypoxia-inducible factor 2 (Hif2), a transcription factor that mediates the cellular response to hypoxia, is essential for erythropoietin excess and polycythemia in Slc30a10-deficient mice, while hypoxia-inducible factor 1 (HIF1) plays no discernible role. RNA-seq analysis determined that Slc30a10-deficient livers exhibit aberrant expression of a large number of genes, most of which align with cell cycle and metabolic processes, while hepatic Hif2 deficiency attenuates differential expression of half of these genes in mutant mice. One such gene downregulated in Slc30a10-deficient mice in a Hif2-dependent manner is hepcidin, a hormonal inhibitor of dietary iron absorption. Our analyses indicate that hepcidin downregulation serves to increase iron absorption to meet the demands of erythropoiesis driven by erythropoietin excess. Finally, we also observed that hepatic Hif2 deficiency attenuates tissue manganese excess, although the underlying cause of this observation is not clear at this time. Overall, our results indicate that HIF2 is a key determinant of pathophysiology in SLC30A10 deficiency.Graphical abstract

Background and Aims Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. Methods We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. Results We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. Conclusions Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.

Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of (54)Mn import and export in mutant cells. While impaired (54)Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired (54)Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated (54)Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease that plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin and other components of the bone morphogenetic protein-signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the nonproteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6-/- mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin-induction pathway, including Hjv, Alk3, ActRIIA, and Hfe, when overexpressed in hepatoma cells. Nevertheless, studies of a murine iron-refractory iron-deficiency anemia-causing mutant (Mt2I286F) in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 domain indicate that Mt2I286F can be activated, but it exhibited a largely compromised ability to suppress hepcidin expression. Coimmunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. In addition, increased expression of a serine protease inhibitor, the hepatocyte growth factor activator inhibitor-2, in the liver failed to alter hepcidin. Together, these observations support the idea that the substrate interaction with Mt2 plays a determinant role and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.

The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

PURPOSE OF REVIEW This review summarizes recent basic science studies on homeostasis of iron, an essential dietary nutrient and potentially toxic metal, and explores the relevance of these studies to our understanding of trauma and related severe, acute events. RECENT FINDINGS Recent studies in experimental models of iron homeostasis have added to our understanding of how iron levels are regulated in the body and how iron levels and iron-dependent biological processes contribute to trauma and related events. Iron deficiency, a common nutritional disorder, can impair critical organ function and wound and injury repair. Iron excess, typically because of genetic defects, can cause toxicity to tissues and, like iron deficiency, impair wound and injury repair. Finally, pharmacologic inhibition of ferroptosis, a novel form of iron-dependent cell death, is beneficial in animal models of cardiac, hepatic, and intestinal injury and intracerebral hemorrhage, suggesting that ferroptosis inhibitors could serve as novel therapeutic agents for trauma and related events. SUMMARY Perturbations in iron homeostasis can contribute significantly to an individual's predisposition to trauma and their ability to recover posttrauma, whereas pharmacologic targeting of ferroptosis may attenuate severity of trauma-induced organ dysfunction.

The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are essential for expression of hepcidin, a key iron regulatory hormone. In mice, hepatocyte-specific Alk2 deficiency leads to moderate iron overload with periportal liver iron accumulation, while hepatocyte-specific Alk3 deficiency leads to severe iron overload with centrilobular liver iron accumulation and a more marked reduction of basal hepcidin levels. The objective of this study was to investigate whether the two receptors have additive roles in hepcidin regulation. Iron overload in mice with hepatocyte-specific Alk2 and Alk3 (Alk2/3) deficiency was characterized and compared to hepatocyte-specific Alk3 deficient mice. Co-immunoprecipitation studies were performed to detect the formation of ALK2 and ALK3 homodimer and heterodimer complexes in vitro in the presence and absence of ligands. The iron overload phenotype of hepatocyte-specific Alk2/3-deficient mice was more severe than that of hepatocyte-specific Alk3-deficient mice. In vitro co-immunoprecipitation studies in Huh7 cells showed that ALK3 can homodimerize in absence of BMP2 or BMP6. In contrast, ALK2 did not homodimerize in either the presence or absence of BMP ligands. However, ALK2 did form heterodimers with ALK3 in the presence of BMP2 or BMP6. ALK3-ALK3 and ALK2-ALK3 receptor complexes induced hepcidin expression in Huh7 cells. Our data indicate that: (I) ALK2 and ALK3 have additive functions in vivo, as Alk2/3 deficiency leads to a greater degree of iron overload than Alk3 deficiency; (II) ALK3, but not ALK2, undergoes ligand-independent homodimerization; (III) the formation of ALK2-ALK3 heterodimers is ligand-dependent and (IV) both receptor complexes functionally induce hepcidin expression in vitro.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

It has been suggested that childhood exposure to neurotoxicants may increase the risk of Parkinson’s disease (PD) or other neurodegenerative disease in adults. Some recessive forms of PD have been linked to loss-of-function mutations in the Park2 gene that encodes for parkin. The purpose of this pilot study was to evaluate whether responses to neonatal manganese (Mn) exposure differ in mice with a Park2 gene defect (parkin mice) when compared with a wildtype strain (C57BL/6J). Neonatal parkin and C57BL/6J littermates were randomly assigned to 0, 11, or 25 mg Mn/kg-day dose groups with oral exposures occurring from postnatal day (PND) 1 through PND 28. Motor activity was measured on PND 19–22 and 29–32. Tissue Mn concentrations were measured in liver, femur, olfactory bulb, frontal cortex, and striatum on PND 29. Hepatic and frontal cortex gene expression of Slc11a2, Slc40a1, Slc30a10, Hamp (liver only), and Park2 were also measured on PND 29. Some strain differences were seen. As expected, decreased hepatic and frontal cortex Park2 expression was seen in the parkin mice when compared with C57BL/6J mice. Untreated parkin mice also had higher liver and femur Mn concentrations when compared with the C57BL/6J mice. Exposure to ≥ 11 mg Mn/kg-day was associated with increased brain Mn concentrations in all mice, no strain difference was observed. Manganese exposure in C57Bl6, but not parkin mice, was associated with a negative correlation between striatal Mn concentration and motor activity. Manganese exposure was not associated with changes in frontal cortex gene expression. Decreased hepatic Slc30a10, Slc40a1, and Hamp expression were seen in PND 29 C57BL/6J mice given 25 mg Mn/kg-day. In contrast, Mn exposure was only associated with decreased Hamp expression in the parkin mice. Our results suggest that the Parkin gene defect did not increase the susceptibility of neonatal mice to adverse health effects associated with high-dose Mn exposure.

High dose manganese (Mn) exposure can result in changes in tissue concentrations of other essential metals due to Mn-induced alterations in metal absorption and competition for metal transporters and regulatory proteins. We evaluated responses in mice with a Parkin gene defect (parkin mice) and a wildtype strain (C57BL/6J) following neonatal Mn exposure. Neonatal parkin and C57BL/6J littermates were randomly assigned to 0, 11, or 25 mg Mn/kg-day dose groups with oral exposures occurring from postnatal day (PND) 1 through PND 28. We report liver, femur, olfactory bulb, striatum, and frontal cortex iron, copper, and zinc concentrations and changes in hepatic gene expression of different metal transporters in PND 29 parkin and C57BL/6J mice. A companion manuscript (Foster et al., 2017) [1] describes the primary study findings. This data provides insights into strain differences in the way Mn interacts with other trace metals in mice. Keywords: Copper, Zinc, Iron, Manganese Toxicity, Mouse

Manganese (Mn), an essential metal and nutrient, is toxic in excess. Toxicity classically results from inhalational exposures in individuals who work in industrial settings. The first known disease of inherited Mn excess, identified in 2012, is caused by mutations in the metal exporter SLC30A10 and is characterized by Mn excess, dystonia, cirrhosis, and polycythemia. To investigate the role of SLC30A10 in Mn homeostasis, we first generated whole-body Slc30a10-deficient mice, which developed severe Mn excess and impaired systemic and biliary Mn excretion. Slc30a10 localized to canalicular membranes of hepatocytes, but mice with liver Slc30a10 deficiency developed minimal Mn excess despite impaired biliary Mn excretion. Slc30a10 also localized to the apical membrane of enterocytes, but mice with Slc30a10 deficiency in small intestines developed minimal Mn excess despite impaired Mn export into the lumen of the small intestines. Finally, mice with Slc30a10 deficiency in liver and small intestines developed Mn excess that was less severe than that observed in mice with whole-body Slc30a10 deficiency, suggesting that additional sites of Slc30a10 expression contribute to Mn homeostasis. Overall, these results indicated that Slc30a10 is essential for Mn excretion by hepatocytes and enterocytes and could be an effective target for pharmacological intervention to treat Mn toxicity.

Manganese is an essential dietary nutrient and trace element with important roles in mammalian development, metabolism, and antioxidant defense. In healthy individuals, gastrointestinal absorption and hepatobiliary excretion are tightly regulated to maintain systemic manganese concentrations at physiologic levels. Interactions of manganese with other essential metals following high dose ingestion are incompletely understood. We previously reported that gavage manganese exposure in rats resulted in higher tissue manganese concentrations when compared with equivalent dietary or drinking water manganese exposures. In this study, we performed follow-up evaluations to determine whether oral manganese exposure perturbs iron, copper, or zinc tissue concentrations. Rats were exposed to a control diet with 10 ppm manganese or dietary, drinking water, or gavage exposure to approximately 11.1 mg manganese/kg body weight/day for 7 or 61 exposure days. While manganese exposure affected levels of all metals, particularly in the frontal cortex and liver, copper levels were most prominently affected. This result suggests an under-appreciated effect of manganese exposure on copper homeostasis which may contribute to our understanding of the pathophysiology of manganese toxicity.

Rodents are commonly used as experimental models in studies of iron homeostasis. They are essential tools for investigating the fundamental mechanisms of iron absorption, tissue distribution, recycling, and excretion. Rodent models with inherited defects in iron homeostasis are particularly valuable. While some models arose serendipitously during routine animal maintenance, most were intentionally generated by irradiation, chemical mutagenesis, or transgenic technologies. In this chapter, we present a survey of most of the currently available mouse and rat models of aberrant iron homeostasis, arranged largely by their respective defects, and discuss the significance of their phenotypes. We also highlight specific areas in need for new or updated models.

In this issue of Blood,Nai et al demonstrate that the serine protease matriptase-2 mediates erythropoietin (EPO)-dependent suppression of hepcidin, a central regulator of iron homeostasis with causal or contributing roles in iron-loading diseases, hemoglobinopathies, and infectious, inflammatory, and neoplastic disorders.1

Manganese is an essential mineral that is found at low levels in virtually all diets (ATSDR, 2012). Ingestion is the principal route by which most people are exposed to manganese, although toxicologically significant inhalation exposures also occur. The gastrointestinal and hepatobiliary systems play an important regulatory role in maintaining tissue manganese concentrations within a relatively narrow physiologic range (Aschner and Aschner, 2005). For example, a moderate increase in dietary manganese intake is accompanied by a compensatory decrease in gut absorption, an increased liver manganese concentration, and an elevated biliary excretion level in order to maintain normal manganese concentrations in the brain and other extrahepatic tissues (Aschner and Aschner, 2005; Dorman et al., 2001). Excessive exposure to manganese and/or hepatic injury can overwhelm these normal homeostatic controls, resulting in elevated tissue manganese and toxicity (Guilarte, 2010). In humans, manganese-induced neurotoxicity is of primary concern and is thought to be the most sensitive endpoint. An extreme case of manganese neurotoxicity is known as manganism, which is usually caused by chronic inhalation of high levels of manganese. Hallmarks of manganism include behavioral changes, extrapyramidal motor dysfunction, and neurochemical and neuropathological changes in the basal ganglia and globus pallidus (Roels et al., 2012). Manganese-induced cognitive deficits have also been reported in workers with exposure to manganese-based welding fumes (Bowler et al., 2006; Chang et al., 2010; Ellingsen et al., 2008; Park et al., 2009). Affected brain regions following high-dose manganese exposure include the striatum, globus pallidus, and other dopaminergic and γ-aminobutyric acid-containing mid-brain structures that control motor functions (Guilarte, 2013). In contrast to the numerous reports describing manganese toxicity following occupational inhalation exposure in humans, there are relatively few reports of manganism arising from water or dietary sources. In part, this trend is due to the relatively low levels of manganese found in these media. For example, most diets in North America result in a manganese intake below the current reference dose of 10 mg/day (Finley and Davis, 1999). Water concentrations of manganese typically range from 1 to 100 µg/l, with most values below 10 µg/l (Keen and Zidenberg-Cherr, 1994). On rare occasions, clinically apparent manganese toxicity can result from ingestion. For example, Kawamura et al. (1941) and Kondakis et al. (1989) documented outbreaks of manganism in Japan and Greece due to the ingestion of water from wells that were contaminated with extremely high levels of manganese (1.8–14 mg Mn/l). There has also been increasing concern regarding the role of environmental manganese exposure and children’s health (Zoni and Lucchini, 2013). For example, several community studies have suggested a link between manganese content in drinking water and a decrease in the IQ of children (Bouchard et al., 2011; Khan et al., 2012; Wasserman et al., 2006), although the toxicological significance of these studies remains to be established (Lucchini et al., 2009). Many questions remain as to the conditions under which ingestion of manganese can result in an increased incidence of human neurological disease (Boyes, 2010). Experimental animal studies remain an important tool for the study of manganese pharmacokinetics and neurotoxicity (Dorman et al., 2012). Animal studies examining the pharmacokinetics and toxicity of ingested manganese often rely on 3 exposure methods: diet, drinking water, and gavage. Most dietary studies rely on the use of a defined rodent diet to which specified amounts of manganese have been added. Drinking water studies often use water combined with manganese chloride (MnCl2), other soluble manganese salts, or insoluble manganese oxides (eg, MnO2). Gavage studies rely on the oral ‘injection’ of a bolus dose of manganese that is typically dissolved in an aqueous media. Both drinking water and dietary exposure studies more closely mimic adult human exposures, as they involve small, repetitive exposures to manganese throughout the day. Although these different methods are used, little data is available to assess the impact of either oral dose rate (eg, gavage versus drinking water) or manganese vehicle (eg, drinking water versus diet) on the pharmacokinetics of manganese exposure. The overall objective of this study was to determine whether dose rate influences the pharmacokinetics of manganese, and evaluate the equivalency of dietary and drinking water manganese intake with regards to tissue manganese concentrations.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

Trace metals are essential for health but toxic when present in excess. The maintenance of trace metals at physiologic levels reflects both import and export by cells and absorption and excretion by organs. The mechanism by which this maintenance is achieved in vertebrate organisms is incompletely understood. To explore this, we chose zebrafish as our model organism, as they are amenable to both pharmacologic and genetic manipulation and comprise an ideal system for genetic screens and toxicological studies. To characterize trace metal content in developing zebrafish, we measured levels of three trace elements, copper, zinc, and manganese, from the oocyte stage to 30 days post-fertilization using inductively coupled plasma mass spectrometry. Our results indicate that metal levels are stable until zebrafish can acquire metals from the environment and imply that the early embryo relies on maternal contribution of metals to the oocyte. We also measured metal levels in bodies and yolks of embryos reared in presence and absence of the copper chelator neocuproine. All three metals exhibited different relative abundances between yolks and bodies of embryos. While neocuproine treatment led to an expected phenotype of copper deficiency, total copper levels were unaffected, indicating that measurement of total metal levels does not equate with measurement of biologically active metal levels. Overall, our data not only can be used in the design and execution of genetic, physiologic, and toxicologic studies but also has implications for the understanding of vertebrate metal homeostasis.

Like iron, manganese is an essential yet potentially toxic metal. While inherited forms of iron excess were discovered long ago, the first disease of inherited manganese excess was only recently reported in 2012. Mutations in a metal transport protein, SLC30A10, were described in patients with manganese excess, polycythemia, liver failure and Parkinsonism. Manganese excess in these patients is attributed to an essential role for SLC30A10 in hepatobiliary manganese excretion. Polycythemia, liver failure and Parkinsonism are attributed to manganese excess although the underlying mechanisms have yet to be established. To explore the role of SLC30A10 in metal biology and disease, we have generated Slc30a10-deficient mice. Characterization of these mice indicates that murine Slc30a10 deficiency recapitulates the human disease. Given that manganese and iron share some transport pathways, we interrogated iron homeostasis in these mutant mice. Intriguingly, mice display hepcidin deficiency and tissue iron excess. While hepcidin deficiency may explain the tissue iron excess, the cause of hepcidin deficiency is unclear. Based on expression profiling of hypoxia-regulated genes, we propose that hepcidin deficiency reflects aberrant activation of hypoxia signaling by manganese excess in the absence of true hypoxia. Overall, this data suggests novel interactions between iron and manganese with implications for our understanding of the mechanisms by which aberrant metal homeostasis leads to human disease. Disclosures No relevant conflicts of interest to declare.