Embryonic ionizing radiation exposure results in expression alterations of genes associated with cardiovascular and neurological development, function, and disease and modified cardiovascular function in zebrafish

Abstract

The relationship between ionizing radiation (IR) and carcinogenesis is long established, but recently the association between IR and other diseases is starting to be recognized. Currently, there is limited information on the genetic mechanisms governing the role of IR in non-cancer related adverse health effects and in regards to an early developmental exposure. In this study, zebrafish embryos were exposed to a range of IR doses (0, 1, 2, 5, 10 Gy) at 26 h post fertilization (hpf). No significant increase in mortality or hatching rate was observed, but a significant decrease in total larval length, head length, and eye diameter was observed in the 10 Gy dose. Transcriptomic analysis was conducted at 120 hpf to compare gene expression profiles between the control and highest IR dose at which no significant differences were observed in morphological measurements (5 Gy). 253 genes with well-established function or orthology to human genes were significantly altered. Gene ontology and molecular network analysis revealed enrichment of genes associated with cardiovascular and neurological development, function, and disease. Expression of a subset of genetic targets with an emphasis on those associated with the cardiovascular system was assessed using Quantitative PCR (qPCR) to confirm altered expression at 5 Gy and then to investigate alterations at lower doses (1 and 2 Gy). Strong correlation between microarray and qPCR expression values was observed, but zebrafish exposed to 1 or 2 Gy resulted in a significant expression alteration in only one of these genes (LIN7B). Moreover, heart rate was analyzed through 120 hpf following IR dosing at 26 hpf. A significant decrease in heart rate was observed at 10 Gy, while a significant increase in heart rate was observed at 1, 2, and 5 Gy. Overall these findings indicate IR exposure at doses below those that induce gross morphological changes alters heart rate and expression of genes associated with cardiovascular and neurological functions.

Early embryonic exposure of ionizing radiations disrupts zebrafish pigmentation

“…Our initial experiment showed that <3 hpf embryos were extremely sensitive to IR (data not shown), 1 Gy IR increased mortality and a dose-dependent mortality rate was observed (Figure 1a-c). Our findings are consistent with other reports on the early development stages and IR toxicity (Freeman, Weber, Peterson, & Nie, 2014;Geiger et al, 2006;T. C. Pereira, Rico, et al, 2011), where the lethal effects of IR were inversely related to embryonic age.…”

Zebrafish exposure to environmentally relevant concentration of depleted uranium impairs progeny development at the molecular and histological levels

“…Target primer sequences were forward , reverse . Similarly to previous studies β-actin was found to be the most consistent and least variable control for this analysis [31]. The primer sequence for β-actin was the same as used in a previous study [32] forward and reverse .…”

Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis

“…Fluorescence signal intensities were normalized using quantile normalization (Bolstad et al 2003) and gene calls generated using the Robust Multichip Average algorithm (Irizarry et al 2003) following manufacturer recommendations. Array image data was analyzed as previously described (Freeman et al 2014; Peterson et al 2011; Weber et al 2013). A robust and reproducible list of differentially expressed genes for each atrazine treatment using recommendations from the Microarray Quality Consortium (Guo et al 2006; Shi et al 2006) was first determined by genes consistently expressed using an ANOVA in SAS software (p<0.05) and substantially altered with a mean absolute log 2 expression ratio of at least 0.585 (50% increase or decrease in expression).…”

“…A robust and reproducible list of differentially expressed genes for each atrazine treatment using recommendations from the Microarray Quality Consortium (Guo et al 2006; Shi et al 2006) was first determined by genes consistently expressed using an ANOVA in SAS software (p<0.05) and substantially altered with a mean absolute log 2 expression ratio of at least 0.585 (50% increase or decrease in expression). Each gene list was imported into Ingenuity Pathway Analysis (IPA) for gene ontology and molecular pathway analysis following similar parameters as described previously (Freeman et al 2014; Peterson et al 2011; Weber et al 2013). Genes referred to in the results and discussion sections are represented as the human homologs of the genes identified to be altered by microarrays.…”

“…Probes specific to target genes were designed using the Primer3 website (Table 1). qPCR was performed following similar methods as described previously (Freeman et al 2014; Peterson et al 2011; Weber et al 2013) following MIQE guidelines (Bustin et al 2009). Similar to as performed in previous studies in our laboratory (Peterson et al 2011, 2013; Weber et al 2013; Wirbisky et al 2014), several genes were assessed to determine the best reference gene to be used for this data set (data not shown).…”