The Oxidative Stress Response In Erythroid Precursors Is Mediated By tp53

Oxidative stress plays a key role in acute and especially chronic anemia as well red blood cell storage. Recent findings suggest that both erythroid precursors as well as mature red cells have increased sensitively to oxidative stress. Modeling oxidative stress in animals has been a challenge as many of the central genes in the oxidative stress response pathway are necessary for life and knockout animals die of overwhelming oxidative stress early in life. The zebrafish model allows pro-oxidant exposure early in hematopoietic development, and gata1^DsRed1 transgenic animals allow clear identification of erythroid precursors. We capitalized on these advantages of the zebrafish to interrogate the effects of oxidative stress on erythroid precursors. After 72 hours of exposure to the strong pro-oxidant naphthol at 10 – 30 µg, embryonic zebrafish up-regulated several anti-oxidant genes including: hypoxia-inducible factor (hif1a), nuclear factor (erythroid derived)-like 2 (nrf2), ferritin heavy chain (fth1a), thioredoxin (txn), and heme oxygenase 1 (hmox1); 1.5, 2.3, 2.5, and 3.0-fold respectively (p < 0.05) as shown by qRT-PCR. To understand if a common pathway was driving anti-oxidant gene expression, we performed an in silico promoter analysis of these genes and discovered several tp53 binding sites within 4 kb upstream of the first exon in each gene. We showed naphthol was able to induce tp53 expression 3-fold over baseline by qRT-PCR. We next took advantage of the tp53 mutant line tp53^M214K which has a mutation in the DNA binding region of tp53 rendering it non-functional similar to a complete knockout. We found that tp53^M214K fish were highly sensitive to pro-oxidant exposure with 80% of embryos showing severe to moderate anemia and cardiac edema after 72 hours of exposure to naphthol (versus 25% in wild-type control animals, n = 100/group; p < 0.001). There was also a 3-fold decrease in the number of hemoglobin staining cells in naphthol treated tp53^M214K animals as shown by o-dianisidine staining (versus control animals, n = 10/group; p < 0.01). A dose-response between the amount of pro-oxidant exposure and severity of anemia/edema also existed as determined by correlation of pro-oxidant concentration to an edema severity scale. We next measured the amount of reactive oxygen species (ROS) generated after naphthol exposure using CellROX detection assays and found that tp53^M214K animals showed a doubling in ROS generated compared to wild-type (n = 30/group, p < 0.01) in whole animals. To disable tp53 by an alternative manner, we employed a known tp53 inhibitor, pifithrin, to inactivate tp53. Exposure of animals to pifithrin simultaneous with naphthol recapitulated the finding that inhibition of tp53 increased sensitivity to ROS as 90% of exposed embryos displayed moderate to severe anemia induced cardiac edema (versus 30% in controls, n = 100/group; p < 0.01). Although pifithrin combined with naphthol exposure caused no increase in ROS above that seen with naphthol alone. Our hypothesis is that the anemic phenotype is largely caused by hemolysis in erythroid precursors. The gata1^DsRed1 zebrafish has labeled erythroid precursors, and using our experimental system we were able to specifically measure a dose responsive induction of ROS in erythroid precursors after naphthol exposure. Furthermore, gata1^DsRed1 animals harboring tp53^M214K showed a 20-fold increase in ROS after naphthol exposure (versus tp53^+/+ animals, n = 10/group; p < 0.01). Accompanying the increase ROS is apoptosis of erythroid precursors as shown by flow cytometry for gata1^DsRed1 cells. In conclusion, we show that amongst the many functions of tp53, providing an anti-oxidant response is also a mechanism though which erythroid precursors metabolize ROS after exposure to pro-oxidants. Understanding the mechanisms by which the anti-oxidant response is regulated will allow us to potentially find more effective drug-able targets to treat the oxidative stress that accompanies acute and chronic anemia’s.