Genome-wide CRISPR Screen Reveal Targets of Chiral Gold(I) Anticancer Compound in Mammalian Cells.

Metal-based drugs, such as cisplatin and auranofin, are used for the treatment of cancer and rheumatoid arthritis, respectively. Auranofin and other gold-derived compounds have been shown to possess anticancer, anti-inflammatory, antimicrobial, and antiparasitic activity in preclinical and clinical trials. Unlike platinum agents which are known to target DNA, the target of gold is not well elucidated. To better understand the targets and effects of gold agents in mammalian cells, we used a targeted CRISPR (ToxCRISPR) screen in K562 cancer cells to identify genes that modulate cellular sensitivity to gold. We synthesized a novel chiral gold(I) compound, JHK-21, with potent anticancer activity. Among the most sensitizing hits were proteins involved in mitochondrial carriers, mitochondrial metabolism, and oxidative phosphorylation. Further analysis revealed that JHK-21 induced inner mitochondria membrane dysfunction and modulated ATP-binding cassette subfamily member C (ABCC1) function in a manner distinct from auranofin. Characterizing the therapeutic effects and toxicities of metallodrugs in mammalian cells is of growing interest to guide future drug discovery, and cellular and preclinical/clinical studies.

Identifying Toxicity Mechanisms Associated with Early Lanthanide Exposure through Multidimensional Genome-Wide Screening.

Lanthanides are a series of elements essential to a wide range of applications, from clean energy production to healthcare. Despite their presence in multiple products and technologies, their toxicological characteristics have been only partly studied. Recently, our group has employed a genomic approach to extensively characterize the toxicity mechanisms of lanthanides. Even though we identified substantially different behaviors for mid and late lanthanides, the toxicological profiles of early lanthanides remained elusive. Here, we overcome this gap by describing a multidimensional genome-wide toxicogenomic study for two early lanthanides, namely, lanthanum and praseodymium. We used Saccharomyces cerevisiae as a model system since its genome shares many biological pathways with humans. By performing functional analysis and protein-protein interaction network analysis, we identified the main genes and proteins that participate in the yeast response to counter metal harmful effects. Moreover, our analysis also highlighted key enzymes that are dysregulated by early lanthanides, inducing cytotoxicity. Several of these genes and proteins have human orthologues, indicating that they may also participate in the human response against the metals. By highlighting the key genes and proteins in lanthanide-induced toxicity, this work may contribute to the development of new prophylactic and therapeutic strategies against lanthanide harmful exposures.

Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

Organochlorine Pesticide Dieldrin Suppresses Cellular Interferon-Related Antiviral Gene Expression.

Organochlorine pesticides (OCPs) are persistent pollutants linked to diverse adverse health outcomes. Environmental exposure to OCPs has been suggested to negatively impact the immune system but their effects on cellular antiviral responses remain unknown. Transcriptomic analysis of N27 rat dopaminergic neuronal cells unexpectedly detected high level expression of genes in the interferon (IFN)-related antiviral response pathways including the IFN-induced protein with tetratricopeptide repeats 1 and 2 (Ifit1/2) and the MX Dynamin Like GTPases Mx1 and Mx2. Interestingly, treatment of N27 cells with dieldrin markedly downregulated the expression of many of these genes. Dieldrin exterted a similar effect in inhibiting IFIT2 and MX1 gene expression in human SH-SY5Y neuronal cells induced by an RNA viral mimic, polyinosinic: polycytidylic acid (poly I:C) and IFIT2/3 gene expression in human pulmonary epithelial cells exposed to human influenza H1N1 virus. Mechanistically, dieldrin induced a rapid rise in levels of intracellular reactive oxygen species (iROS) and a decrease in intracellular glutathione (GSH) levels in SH-SY5Y cells. Treatment with N-acetylcysteine, an antioxidant and GSH biosynthesis precursor, effectively blocked both dieldrin-induced increases in iROS and its inhibition of poly I:C-induced upregulation of IFIT and MX gene expression, suggesting a role for intracellular oxidative status in dieldrin's modulation of antiviral gene expression. This study demonstrates that dieldrin modulates key genes of the cellular innate immune responses that are normally involved in the host's cellular defense against viral infections. Our findings have potential relevance to understanding the organismal effects of environmentally persistent organochlorine contaminants on the mammalian cellular immune system.

Applying genome-wide CRISPR to identify known and novel genes and pathways that modulate formaldehyde toxicity.

Formaldehyde (FA), a ubiquitous environmental pollutant, is classified as a Group I human carcinogen by the International Agency for Research on Cancer. Previously, we reported that FA induced hematotoxicity and chromosomal aneuploidy in exposed workers and toxicity in bone marrow and hematopoietic stem cells of experimental animals. Using functional toxicogenomic profiling in yeast, we identified genes and cellular processes modulating eukaryotic FA cytotoxicity. Although we validated some of these findings in yeast, many specific genes, pathways and mechanisms of action of FA in human cells are not known. In the current study, we applied genome-wide, loss-of-function CRISPR screening to identify modulators of FA toxicity in the human hematopoietic K562 cell line. We assessed the cellular genetic determinants of susceptibility and resistance to FA at 40, 100 and 150 µM (IC10, IC20 and IC60, respectively) at two time points, day 8 and day 20. We identified multiple candidate genes that increase sensitivity (e.g. ADH5, ESD and FANC family) or resistance (e.g. FASN and KDM6A) to FA when disrupted. Pathway analysis revealed a major role for the FA metabolism and Fanconi anemia pathway in FA tolerance, consistent with findings from previous studies. Additional network analyses revealed potential new roles for one-carbon metabolism, fatty acid synthesis and mTOR signaling in modulating FA toxicity. Validation of these novel findings will further enhance our understanding of FA toxicity in human cells. Our findings support the utility of CRISPR-based functional genomics screening of environmental chemicals.

Functional Pathway Identification With CRISPR/Cas9 Genome-wide Gene Disruption in Human Dopaminergic Neuronal Cells Following Chronic Treatment With Dieldrin.

Organochlorine pesticides, once widely used, are extremely persistent and bio-accumulative in the environment. Epidemiological studies have implicated that environmental exposure to organochlorine pesticides including dieldrin is a risk factor for the development of Parkinson's disease. However, the pertinent mechanisms of action remain poorly understood. In this study, we carried out a genome-wide (Brunello library, 19 114 genes, 76 411 sgRNAs) CRISPR/Cas9 screen in human dopaminergic SH-SY5Y neuronal cells exposed to a chronic treatment (30 days) with dieldrin to identify cellular pathways that are functionally related to the chronic cellular toxicity. Our results indicate that dieldrin toxicity was enhanced by gene disruption of specific components of the ubiquitin proteasome system as well as, surprisingly, the protein degradation pathways previously implicated in inherited forms of Parkinson's disease, centered on Parkin. In addition, disruption of regulatory components of the mTOR pathway which integrates cellular responses to both intra- and extracellular signals and is a central regulator for cell metabolism, growth, proliferation, and survival, led to increased sensitivity to dieldrin-induced cellular toxicity. This study is one of the first to apply a genome-wide CRISPR/Cas9-based functional gene disruption screening approach in an adherent neuronal cell line to globally decipher cellular mechanisms that contribute to environmental toxicant-induced neurotoxicity and provides novel insight into the dopaminergic neurotoxicity associated with chronic exposure to dieldrin.

Obese Individuals with and without Type 2 Diabetes Show Different Gut Microbial Functional Capacity and Composition.

Obesity and type 2 diabetes (T2D) are metabolic disorders that are linked to microbiome alterations. However, their co-occurrence poses challenges in disentangling microbial features unique to each condition. We analyzed gut microbiomes of lean non-diabetic (n = 633), obese non-diabetic (n = 494), and obese individuals with T2D (n = 153) from German population and metabolic disease cohorts. Microbial taxonomic and functional profiles were analyzed along with medical histories, serum metabolomics, biometrics, and dietary data. Obesity was associated with alterations in microbiome composition, individual taxa, and functions with notable changes in Akkermansia, Faecalibacterium, Oscillibacter, and Alistipes, as well as in serum metabolites that correlated with gut microbial patterns. However, microbiome associations were modest for T2D, with nominal increases in Escherichia/Shigella. Medications, including antihypertensives and antidiabetics, along with dietary supplements including iron, were significantly associated with microbiome variation. These results differentiate microbial components of these interrelated metabolic diseases and identify dietary and medication exposures to consider in future studies.

Genome-Wide CRISPR Screening Identifies the Tumor Suppressor Candidate OVCA2 As a Determinant of Tolerance to Acetaldehyde.

Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.

Functional Profiling Identifies Determinants of Arsenic Trioxide Cellular Toxicity.

Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (AsIII) is also an effective chemotherapeutic agent. The current use of AsIII in chemotherapy is limited to acute promyelocytic leukemia (APL). However, AsIII was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPR-based approach to identify modulators of AsIII toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased AsIII tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to AsIII whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to AsIII. Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to AsIII. Our data suggest a model in which an intracellular interaction between selenium and AsIII may impact intracellular AsIII levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of AsIII in CML cells. Targeting such processes simultaneously with AsIII treatment could potentiate AsIII in CML therapy.

A comparison between whole transcript and 3' RNA sequencing methods using Kapa and Lexogen library preparation methods.

BACKGROUND: 3' RNA sequencing provides an alternative to whole transcript analysis. However, we do not know a priori the relative advantage of each method. Thus, a comprehensive comparison between the whole transcript and the 3' method is needed to determine their relative merits. To this end, we used two commercially available library preparation kits, the KAPA Stranded mRNA-Seq kit (traditional method) and the Lexogen QuantSeq 3' mRNA-Seq kit (3' method), to prepare libraries from mouse liver RNA. We then sequenced and analyzed the libraries to determine the advantages and disadvantages of these two approaches. RESULTS: We found that the traditional whole transcript method and the 3' RNA-Seq method had similar levels of reproducibility. As expected, the whole transcript method assigned more reads to longer transcripts, while the 3' method assigned roughly equal numbers of reads to transcripts regardless of their lengths. We found that the 3' RNA-Seq method detected more short transcripts than the whole transcript method. With regard to differential expression analysis, we found that the whole transcript method detected more differentially expressed genes, regardless of the level of sequencing depth. CONCLUSIONS: The 3' RNA-Seq method was better able to detect short transcripts, while the whole transcript RNA-Seq was able to detect more differentially expressed genes. Thus, both approaches have relative advantages and should be selected based on the goals of the experiment.

Severe Iron Metabolism Defects in Mice With Double Knockout of the Multicopper Ferroxidases Hephaestin and Ceruloplasmin.

Background & Aims: Multicopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice. Methods: Mice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped. Results: Heph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver. Conclusions: These studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis.

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed Mytilus edulis to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 µg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an M. edulis microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field.

Ceruloplasmin and hephaestin jointly protect the exocrine pancreas against oxidative damage by facilitating iron efflux.

Little is known about the iron efflux from the pancreas, but it is likely that multicopper ferroxidases (MCFs) are involved in this process. We thus used hephaestin (Heph) and ceruloplasmin (Cp) single-knockout mice and Heph/Cp double-knockout mice to investigate the roles of MCFs in pancreatic iron homeostasis. We found that both HEPH and CP were expressed in the mouse pancreas, and that ablation of either MCF had limited effect on the pancreatic iron levels. However, ablation of both MCFs together led to extensive pancreatic iron deposition and severe oxidative damage. Perls' Prussian blue staining revealed that this iron deposition was predominantly in the exocrine pancreas, while the islets were spared. Consistent with these results, plasma lipase and trypsin were elevated in Heph/Cp knockout mice, indicating damage to the exocrine pancreas, while insulin secretion was not affected. These data indicate that HEPH and CP play mutually compensatory roles in facilitating iron efflux from the exocrine pancreas, and show that MCFs are able to protect the pancreas against iron-induced oxidative damage.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Multi-Copper Ferroxidase-Deficient Mice Have Increased Brain Iron Concentrations and Learning and Memory Deficits.

Background: The accumulation of iron occurs in the central nervous system (CNS) in several neurodegenerative diseases. Although multi-copper ferroxidases (MCFs) play an important role in cellular iron metabolism and homeostasis, the mechanism of MCFs in the CNS remains unclear. Objective: The aim was to study the role of MCFs in CNS iron metabolism and homeostasis by using hephaestin/ceruloplasmin (Heph/Cp) double knockout (KO) mice. Methods: Heph/Cp double KO male mice were generated by crossing both single KO mice. In Heph/Cp KO and wild-type (WT) control mice at 4 wk and 6 mo of age, iron concentrations of selected brain regions were measured by atomic absorption spectrophotometry, and gene expressions of Heph, Cp, ferroportin 1 (Fpn1) [+ iron responsive element (IRE)], L-ferritin, H-ferritin, transferrin receptor 1 (Tfrc), and divalent metal transporter 1 (Dmt1) (+IRE) were quantitated by quantitative reverse transcriptase-polymerase chain reaction. Brain region L-ferritin protein concentration, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and malondialdehyde (MDA) concentration were also determined. Learning and memory abilities in Heph/Cp KO and WT control mice at 6 mo of age were tested by the IntelliCage system (New Behavior). Results: Iron concentration was significantly higher in Heph/Cp KO mice than in WT control mice at 4 wk of age in the cortex (50%), hippocampus (120%), brainstem (35%), and cerebellum (220%) and at 6 mo of age in the cortex (140%), hippocampus (420%), brainstem (560%), and cerebellum (340%). L-Ferritin and MDA concentrations were significantly higher and SOD and GPx activities were significantly lower in the cortex, hippocampus, brainstem, and cerebellum of KO mice than in those of WT controls at both 4 wk and 6 mo of age. Iron-related gene expressions also differed significantly between groups. Learning and memory deficits occurred in Heph/Cp KO mice at 6 mo of age. Conclusion: Mutation of both MCFs in mice induces iron accumulation in brain regions, oxidative damage, and learning and memory defects.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.

Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice.

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin (Hamp1) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels.NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.

Intestinal hephaestin potentiates iron absorption in weanling, adult, and pregnant mice under physiological conditions.

Regulation of intestinal iron absorption is crucial to maintain body iron levels because humans have no regulated iron-excretory system. Elucidating molecular events that mediate intestinal iron transport is thus important for the development of therapeutic approaches to modify iron absorption in pathological states. The process of iron uptake into duodenal enterocytes is relatively well understood, but less is known about the functional coupling between the iron exporter ferroportin 1 and the basolateral membrane iron oxidase hephaestin (Heph). Initial characterization of intestine-specific Heph knockout (Hephint) mice demonstrated that adult male mice were mildly iron deficient; however, the specific role of intestinal Heph has not been determined in weanling mice, in female mice, or during physiological states which stimulate iron absorption. Furthermore, because ferroportin 1-mediated iron export from some tissues (eg, liver) is impaired in the absence of the Heph homolog, ceruloplasmin, we hypothesized that Heph is rate limiting for intestinal iron absorption, especially when iron demands increase. Our experimental approach was to assess various physiological parameters and iron (59Fe) absorption and tissue distribution in weanling, adult, and pregnant Hephint mice (and controls) under physiological conditions and in adult Hephint mice after dietary iron deprivation or acute hemolysis. Results demonstrate that intestinal Heph is essential for optimal iron transport in weanlings and adults of both sexes and during pregnancy, but not in adult mice with iron-deficiency or hemolytic anemia. Moreover, activation of unidentified, intestinal ferroxidases was noted, which may explain why intestinal Heph is not always required for optimal iron absorption.

Hephaestin and ceruloplasmin facilitate iron metabolism in the mouse kidney.

Multicopper ferroxidases (MCFs) play an important role in cellular iron homeostasis. However, the role of MCFs in renal metabolism remains unclear. We used Hephaestin (Heph) and Ceruloplasmin (Cp) single or double (Heph/Cp) knockout (KO) mice to study the roles of MCFs in the kidney. Renal iron levels and the expression of iron metabolism genes were examined. The non-heme iron content both in the renal cortex and medulla of Heph/Cp KO mice was significantly increased. Perls' Prussian blue staining showed iron accumulation on the apical side of renal tubular cells in Heph/Cp KO mice. A significant increase in ferritin protein expression was also observed in the renal medulla and cortex of Heph/Cp KO mice. Both DMT1 and TfR1 protein expression were significantly decreased in the renal medulla of Heph/Cp KO mice, while the expression of DMT1 protein was significantly increased in the renal cortex of these animals. Significant increase in proteinuria and total urinary iron was observed in the double knockout mice, and this was associated with compromised structural integrity. These results suggest that KO of both the HEPH and CP genes leads to kidney iron deposition and toxicity, MCFs could protect kidney against a damage from iron excess.

Functional Toxicogenomic Profiling Expands Insight into Modulators of Formaldehyde Toxicity in Yeast.

Formaldehyde (FA) is a commercially important chemical with numerous and diverse uses. Accordingly, occupational and environmental exposure to FA is prevalent worldwide. Various adverse effects, including nasopharyngeal, sinonasal, and lymphohematopoietic cancers, have been linked to FA exposure, prompting designation of FA as a human carcinogen by U.S. and international scientific entities. Although the mechanism(s) of FA toxicity have been well studied, additional insight is needed in regard to the genetic requirements for FA tolerance. In this study, a functional toxicogenomics approach was utilized in the model eukaryotic yeast Saccharomyces cerevisiae to identify genes and cellular processes modulating the cellular toxicity of FA. Our results demonstrate mutant strains deficient in multiple DNA repair pathways-including homologous recombination, single strand annealing, and postreplication repair-were sensitive to FA, indicating FA may cause various forms of DNA damage in yeast. The SKI complex and its associated factors, which regulate mRNA degradation by the exosome, were also required for FA tolerance, suggesting FA may have unappreciated effects on RNA stability. Furthermore, various strains involved in osmoregulation and stress response were sensitive to FA. Together, our results are generally consistent with FA-mediated damage to both DNA and RNA. Considering DNA repair and RNA degradation pathways are evolutionarily conserved from yeast to humans, mechanisms of FA toxicity identified in yeast may be relevant to human disease and genetic susceptibility.