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Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes
Inês G Mollet1, Dilipkumar Patel1, Fatima S Govani1
1NHLI Cardiovascular Sciences, Imperial College London, London, United Kingdom.
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 10micromol/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 10micromol/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 10microM 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.
Insights
Conventional iron treatments can harm vascular endothelium. Low-dose iron induces DNA damage and response in human endothelial cells, suggesting a need for safer iron therapies.
Area of Science:
- Vascular Biology
- Cellular Response to Iron
- Genomics
Background:
- Patient reports suggest vascular issues from iron treatments.
- Serum non-transferrin bound iron can reach high levels post-infusion.
- Hypothesis: Conventional iron treatments may cause acute vascular injury.
Purpose of the Study:
- Investigate if low-dose iron exposure affects human endothelial cells.
- Examine cellular responses to iron (II) citrate in vitro.
- Identify molecular changes in endothelial cells exposed to iron.
Main Methods:
- Primary human endothelial cells (EC) treated with iron (II) citrate.
- Directional RNA sequencing for transcript profiling.
- Comet assays and DNA damage response (DDR) foci analysis.
Main Results:
- 10 micromol/L iron (II) citrate rapidly altered EC RNA profiles.
- Genes involved in vesicle transport, protein catabolism, and cell cycle were affected.
- Iron treatment induced DNA damage and a DNA damage response (DDR) within 1 hour.
Conclusions:
- Low-dose iron is sufficient to alter vascular endothelium.
- Iron exposure triggers a significant DNA damage response in EC.
- Findings suggest potential risks of conventional iron treatments.
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