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The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

Preservatives deter microbial growth, providing crucial functions of safety and durability in composite materials, formulated products, and food packaging. Concern for human health and the environmental impact of some preservatives has led to regulatory restrictions and public pressure to remove individual classes of compounds, such as parabens and chromated copper arsenate, from consumer products. Bans do not address the need for safe, effective alternative preservatives, which are critical for both product performance (including lifespan and therefore life cycle metrics) and consumer safety. In this work, we studied both the safety and efficacy of a series of phenolic preservatives and compared them to common preservatives found in personal care products and building materials. We quantified antimicrobial activity against Aspergillus brasiliensis (mold) and Pseudomonas aeruginosa (Gram negative bacteria), and we conducted a hazard assessment, complemented by computational modeling, to evaluate the human and environmental health impacts of these chemicals. We found that octyl gallate demonstrates better antimicrobial activity and comparable or lower hazards, compared to current-use preservatives. Therefore, octyl gallate may serve as a viable small-molecule preservative, particularly in conjunction with low concentrations of other preservatives that act through complementary mechanisms.

BackgroundDifferences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.ObjectivesGrantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.MethodsConsortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.ResultsThe overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.ConclusionsThe results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

BackgroundIn humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMAIII), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase.ObjectiveWe investigated the potential role of N6AMT1 in arsenic-induced toxicity.MethodsWe measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma-mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAsIII or MMAIII.ResultsN6AMT1 was able to convert MMAIII to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAsIII and MMAIII. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo.ConclusionsConsidering that MMAIII is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMAIII to DMA and other unknown mechanisms.

Abstract Background The delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses. Results Exposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 μg.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations. Conclusions The results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes. hereditary hemochromatosis; liver disease; 2D-PAGE doi:10.1152/ajpgi.00455.2006

A subtractive hybridization approach is utilized to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. A membrane protein named HCP1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner.

Abstract Background The delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses. Results Exposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 μg.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations. Conclusions The results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofiuorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined. divalent metal transporter-1; Ireg1; absorption; intestine

Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 μM desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (−604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency. [ABSTRACT FROM AUTHOR]