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  • To learn what specific genes and biological pathways are


    To learn what specific genes and biological pathways are involved in the response to pro-oxidant exposure, we performed RNA-seq analysis on 72-hpf embryos after 48 hr of pro-oxidant exposure; the heatmap analysis of unsupervised clustering is shown in Figure 3D. We found 316 genes upregulated (increased >2-fold, Table S1) in response to pro-oxidant exposure and 56 genes downregulated (decreased >2-fold), as shown in Figure 3E. To determine if there was a common regulatory pathway governing the pro-oxidant-induced genes, we performed Ingenuity Pathway Analysis (IPA; Ingenuity Systems) to identify networks of specific upstream transcription factor regulators and found that the tp53 pathway was the most significant regulator involved in the oxidative stress response (p = 6.17 × 10−9, Figure 3F). This was not surprising given our prior findings of DNA modification and DNA damage that occurred after pro-oxidant exposure. We performed qRT-PCR to determine if tp53 expression was changed after 1-naphthol exposure and found a 2- to 3-fold induction at the highest amounts of pro-oxidant (Figure 3G). In addition, qRT-PCR of fluorescence-activated cell sorted stressed Gata1+ erythroid 69 8 showed induction of tp53 as well as known downstream tp53 targets, including sestrin 2 (sens2), nuclear Factor Erythroid 2-Related Factor 2 (nrf2), heme oxygenase 1 (hmox), and thioredoxin (txn) that are upregulated by ROS (Figures S2C–S2F). To study the effect of tp53 inhibition and oxidative stress, we utilized pifithrin-alpha, a small molecule inhibitor of TP53 activity (Duffy and Wickstrom, 2007; Rocha et al., 2003). Pifithrin is thought to block the transcription factor activity of TP53, as well as protein-protein interactions, and is able to protect cells from TP53-dependent apoptosis (Culmsee et al., 2001). We found that exposure to pifithrin from 24 to 72 hpf led to a slight (but not significant) increase in ROS generated by Gata1+ erythroid cells during simultaneous pro-oxidant exposure (p = 0.50, Figure 3H). Given the relatively short half-life of pifithrin (approximately 4 hr), it is possible that Tp53 was not sufficiently inhibited (Gary and Jensen, 2005), although exposure of stressed embryos to pulses of pifithrin every 4 hr also did not significantly alter ROS (Figure S2G). Therefore, we decided to employ an alternative approach using a well-characterized zebrafish with a tp53 mutation that disables Tp53 DNA-binding activity. The tp53 zebrafish has a tp53 mutation (methionine-214 to lysine, M214K) known to be analogous to the human TP53 M246K mutation commonly found in human cancers (Berghmans et al., 2005). The M214K mutation disrupts the DNA-binding domain of Tp53, rendering it unable to upregulate p21 after UV irradiation and animals also show cellular resistance to apoptosis. Furthermore, zebrafish harboring this mutation develop malignancies early in life (starting at 8 months of age) (Berghmans et al., 2005). We crossed tp53 animals to the gata1:DsRed transgenic line, followed by in-crossing to generate offspring in which tp53 was mutated in DsRed-positive Gata1+ erythroid cells. Pro-oxidant exposure of tp53 heterozygous zebrafish generated 3-fold higher levels of ROS in Gata1+ erythroid cells, in comparison with wild-type counterparts (p = 0.0002, Figure 3I). At the same time, total-body ROS level was also significantly increased (data not shown). Similarly, using tp53 homozygous animals, the ROS levels increased 5-fold above that of wild-type (p < 0.0001). To understand if TP53 played a similar role in mammalian erythroid cells, we evaluated ROS production in erythroid precursors from wild-type and Tp53 mice. Tp53 mice harbor the Tp53 mutation arginine-270 to histidine (R270H), which abolishes TP53 DNA-binding activity (Olive et al., 2004). Like the tp53 zebrafish, Tp53 69 8 mice develop tumors early in life, and their thymocytes show partial resistance to gamma-induced apoptosis (Olive et al., 2004). While zebrafish and other teleosts are uniquely sensitive to 1-naphthol, we used acrolein (a metabolite of cyclophosphamide) as a strong pro-oxidant to induce ROS in mammalian cells. We identified erythroid precursors using antibodies to the transferrin receptor (CD71) and gating on marrow cells with the highest CD71 expression; we ensured that cells would be at an immature stage (erythroblasts), as opposed to mature erythrocytes that have dim to zero CD71 expression (Chen et al., 2009; Marsee et al., 2010; Peslak et al., 2012). Analysis of nucleated CD71+ erythroid precursors after short-term (5 hr) acrolein exposure indicated significantly higher levels were generated in erythroid precursors from TP53R270H/+mice (Figures 3J and S2H, p < 0.0001). These data indicate that TP53 plays a role in the metabolism of oxidative stress and that haploinsufficiency of TP53 allows for increased ROS generation in mammalian and zebrafish erythroid precursors. In zebrafish, homozygous tp53 disruption increased ROS to an even greater extent.