Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes
File(s)
Published version
Author(s)
Type
Journal Article
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.
Methodology
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.
Principal Findings
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.
Significance
These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium,
and induce a DNA damage response.
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.
Methodology
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.
Principal Findings
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.
Significance
These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium,
and induce a DNA damage response.
Date Issued
2016-02-11
Date Acceptance
2016-01-11
Citation
PLOS One, 2016, 11 (2)
ISSN
1932-6203
Publisher
Public Library of Science
Journal / Book Title
PLOS One
Volume
11
Issue
2
Copyright Statement
© 2016 Mollet et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
License URL
Sponsor
British Heart Foundation
Grant Number
PG/09/041/27515
Subjects
General Science & Technology
MD Multidisciplinary
Publication Status
Published
Article Number
e0147990