Long-Term Effects of Ionizing Radiation on Gene Expression in a Zebrafish Model

<div><p>Understanding how initial radiation injury translates into long-term effects is an important problem in radiation biology. Here, we define a set of changes in the transcription profile that are associated with the long-term response to radiation exposure. The study was performed <i>in vivo</i> using zebrafish, an established radiobiological model organism. To study the long-term response, 24 hour post-fertilization embryos were exposed to 0.1 Gy (low dose) or 1.0 Gy (moderate dose) of whole-body gamma radiation and allowed to develop for 16 weeks. Liver mRNA profiles were then analyzed using the Affymetrix microarray platform, with validation by quantitative PCR. As a basis for comparison, 16-week old adults were exposed at the same doses and analyzed after 4 hours. Statistical analysis was performed in a way to minimize the effects of multiple comparisons. The responses to these two treatment regimes differed greatly: 360 probe sets were associated primarily with the long-term response, whereas a different 2062 probe sets were associated primarily with the response when adults of the same age were irradiated 4 hours before exposure. Surprisingly, a ten-fold difference in radiation dose (0.1 versus 1.0 Gy) had little effect. Analysis at the gene and pathway level indicated that the long-term response includes the induction of cytokine and inflammatory regulators and transcription and growth factors. The acute response includes the induction of p53 target genes and modulation of the hypoxia-induced transcription factor-C/EBP axis. Results help define genes and pathways affected in the long-term, low and moderate dose radiation response and differentiate them from those affected in an acute response in the same tissue.</p></div

Probe sets associated with long-term and acute responses.

<p>A. Inclusion criteria used to define probe sets as primarily or exclusively associated with long-term or acute radiation responses. Statistical comparisons and their abbreviations are as diagrammed in Fig. 1. A “1” indicates that expression of the first set in the comparison was greater than the second; a “−1” indicates that expression in the second set was greater, and a “0” indicates that the difference in expression was not significant. B. Heat map of probe sets primarily associated with long-term response. Red, higher expression; green, lower expression. Each column represents a biological replicate, and each row represents a probe set. C. Heat map of probe sets primarily associated with acute response.</p

Validation of microarray data by quantitative polymerase chain reaction.

<p>Scatter plot shows fold change on logarithmic scale as measured for selected genes by microarray and qPCR. Each point represents mean value for one gene and experimental group. Shape and color denotes gene; letter indicates experimental group as indicated.</p

Genes increased or decreased as part of the acute response to radiation.

<p>Top 10 genes that were increased and top 10 genes that were decreased in association with the acute response to radiation, drawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone.0069445.s002" target="_blank">Table S2</a>. Gene symbols, gene names, fold change, and <i>P</i> values (in the “time comparison”, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g001" target="_blank">Fig. 1C</a>) are given. Expression of the genes listed here was either unchanged, or changed less, in the long-term response (as indicated in column labeled “Long-term”). Some gene names and functions have been edited, and some have been omitted, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-t001" target="_blank">Table 1</a>.</p

Genes increased or decreased as part of the long-term response to radiation.

<p>Top 10 genes that were increased and top 10 genes that were decreased in association with the long-term response to radiation, drawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone.0069445.s001" target="_blank">Table S1</a>. Gene symbols, gene names, fold change, and <i>P</i> values (in the “time comparison”, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g001" target="_blank">Fig. 1C</a>) are given. Expression of the genes listed here was either unchanged, or changed less, in the acute response (as indicated in column labeled “Acute”). Some gene names and functions have been edited for clarity or brevity. Genes without a substantive common name, for which biological process is not annotated, have been omitted. Genes that are represented by more than probe set are listed only once.</p

Experimental design and overview of results.

<p>A. Design. There are five experimental groups, which were established as detailed in Materials and Methods. Group A, non-irradiated control, Groups B and C, long-term response, Groups D and E, acute response. B. Multidimensional scaling representation. Three-dimensional plot shows three biological replicates per experimental group. PC, Principal Coordinates. Color key as shown. C. Inter-group comparisons. The first three comparisons, which were set up in a way to minimize the effects of multiple testing, evaluate the effect of time of irradiation, dose, and time-dose interaction. Three further comparisons identify genes that are significant comparing long-term response samples as a group versus control samples, acute response samples as a group versus control samples, and all irradiated samples as a group versus control. The number of probe sets identified as significant in each comparison is indicated.</p

Analysis of apoptosis pathway.

<p>A. Multidimensional scaling representation of results for apoptosis pathway, based on 42 probe sets for which data are available, symbols as in Fig. 4. B. Responses for 20 probe sets that showed significant differences in one or more statistical comparisons, depicted and labeled as in Fig. 4. For details of KEGG apoptosis pathway – Danio rerio (zebrafish): (<a href="http://www.genome.jp/kegg-bin/show_pathway?dre04210" target="_blank">http://www.genome.jp/kegg-bin/show_pathway?dre04210</a>). Note that the name of badb has been corrected to “BCL2-agonist of cell death” (not “antagonist”) in agreement with direct experimental evidence in the zebrafish <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone.0069445-Hsieh1" target="_blank">[34]</a> and the known function of the mammalian ortholog <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone.0069445-Jiang1" target="_blank">[35]</a>.</p

Analysis of p53 signaling pathway.

<p>A. Multidimensional scaling representation of results for the p53-signaling pathway, based on 36 probe sets for which data are available. Each symbol represents one biological replicate. Color denotes treatment group as indicated. B. Response for 23 probe sets that showed statistically significant differences in one or more comparisons. (Results of comparisons are expressed as “1”, “0” or “−1” using the same convention as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g002" target="_blank">Figure 2</a>). One gene (gadd45ba) was included because it was significant in an overall F test, although not in any of the individual comparisons. Left, results of statistical comparisons, notation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g002" target="_blank">2</a>. Center, heat map, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069445#pone-0069445-g002" target="_blank">Figure 2</a>. When more than one row has the same gene symbol, it indicates different probe sets directed against the same mRNA. Right, gene symbols, whether gene is a direct target of the p53 transcription factor, and gene name. In some cases, an alternative name or description of gene function is given in parentheses. For details of KEGG p53 signaling pathway – Danio rerio (zebrafish): <a href="http://www.genome.jp/kegg-bin/show_pathway?dre04115" target="_blank">http://www.genome.jp/kegg-bin/show_pathway?dre04115</a>.</p

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding

<div><p>The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of <i>ALKBH7</i>, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7’s ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.</p></div

Difference absorption spectra of WT ALKBH7 and its R191Q variant.

<p>The spectra of the anaerobic proteins (0.3 mM) were recorded in the presence of 2 mM α-kg and 100 μM Fe(II). The difference spectra were obtained by subtracting the spectra for proteins with α-kg, but without the metal. <b>A</b>, WT ALKBH7; <b>B</b>, R191Q ALKBH7.</p