title: Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes.
abstract: BACKGROUND
  Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron.
  
  
  METHODS
  Confluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types.
  
  
  RESULTS
  Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology (GO) performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively), and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR) foci, and p53 stabilization.
  
  
  CONCLUSIONS
  These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium, and induce a DNA damage response.
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Inês G
authors: IG
authors: Mollet
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Dilipkumar
authors: D
authors: Patel
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Fatima S
authors: FS
authors: Govani
authors: Medical Research Council Clinical Sciences Centre, Imperial College London, London, United Kingdom.
authors: Adam
authors: A
authors: Giess
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Koralia
authors: K
authors: Paschalaki
authors: Department of Surgery & Cancer, Imperial College London, London, United Kingdom.
authors: Manikandan
authors: M
authors: Periyasamy
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Elaine C
authors: EC
authors: Lidington
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Justin C
authors: JC
authors: Mason
authors: Medical Research Council Clinical Sciences Centre, Imperial College London, London, United Kingdom.
authors: Michael D
authors: MD
authors: Jones
authors: Medical Research Council Clinical Sciences Centre, Imperial College London, London, United Kingdom.
authors: Laurence
authors: L
authors: Game
authors: Department of Surgery & Cancer, Imperial College London, London, United Kingdom.
authors: Simak
authors: S
authors: Ali
authors: NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
authors: Claire L
authors: CL
authors: Shovlin
mesh_terms: D002453:Cell Cycle; D002951:Citrates / Q000008:administration & dosage; D016000:Cluster Analysis; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D005091:Exons; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007501:Iron / Q000008:administration & dosage*; D008833:Microcirculation; D010641:Phenotype; D010766:Phosphorylation; D017423:Sequence Analysis, RNA; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D002951:Citrates; C468783:H2AX protein, human; D006657:Histones; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D007501:Iron