Transgenerational radiation genetics: the use of the Japanese Medaka (Oryzias latipes) to investigate adaptive response and genomic instability

Abstract

Over the past decade or so, two new areas of concern that may have an important bearing on the issue of genetic hazard assessment are the so-called adaptive response (AR), meaning a reduction in radiation sensitivity caused by previous low doses of radiation, and genomic instability (GI), meaning an increase in the rate of damage occurring several generations after the generation in which the damage was inflicted. The intent of this project was to initiate research to address both of these issues using an in vivo fish model, the Japanese Medaka (Oryzias latipes). The information generated would be useful to human risk assessment as well as risk assessment for non-human species by challenging the recommended daily dose limits for aquatic species of 1 cGy/day by the International Atomic Energy Agency and the U.S. Department of Energy. To investigate GI an F0 generation was established and starting at 6-hours postfertilization received either no irradiation or chronic gamma irradiation from Cesium-137 (12 cGy/day) for ~ 3 months. During this time the F0 generation underwent critical stages of development such as germ cell formation, and developed from embryo to juvenile and finally to adult stages. At 2 months of age, adult medaka were randomly paired for breeding. Endpoints such as survival at 60-days post-fertilization, wet weight (mg), sex-ratios, fertility and fecundity were measured to determine the effect of the chronic low dose-rate irradiation. Additionally, unexposed F1 embryos generated from these pairs were analyzed by a G2 Chromosome Assay to determine if a GI, caused by damage to parental germ cells and transmitted to subsequent F1 offspring, could be measured. This was done by analysis of cytogenetic damage in F1 offspring, from unirradiated or chronically irradiated F0 parents, receiving no irradiation or a 500 cGy acute challenge dose of gamma rays. Chronic irradiation of F0 embryos from an early embryonic stage to adulthood at 60-days post-fertilization resulted in a 38% greater mortality than the unirradiated F0 fish (P=0.02). There was no indication of an effect on sex-ratios or wet-weight between males and females (P=0.08). Fecundity was marginally affected with the chronically irradiated pairs producing 16% fewer eggs (mean±STD) per female per day than unirradiated females, 16±7 and 19±9 respectively (P=0.05). A statistically significant difference was measured among the two groups (P=0.02) for the end point of fertilization. Cytogenetic analysis of background chromatid-type aberration frequencies in F1 offspring from unirradiated and irradiated F0 parents indicated no statistical differences. The means and standard errors were 0.01±0.01 and 0.02±0.01 respectively (P=0.87). Analysis of F1 embryos receiving the 500 cGy challenge dose showed means and standard errors of 0.18±0.03 and 0.20±0.06 for F1 offspring from unirradiated and irradiated F0 parents. These means when compared to the respective background aberration frequencies were statistically significant indicating the presence of radiation induced aberrations (P=0.003). However, the means when compared against each other were not statistically different (P=0.97). The lack of a measurable statistical difference in both the background and radiation induced aberration frequencies in offspring from unirradiated and irradiated parents indicates that a cytogenetic GI could not be demonstrated in this study. To investigate the adaptive response, chromatid-type aberration frequencies produced from adapting doses of 2 or 21 cGy followed by a 500 cGy challenge dose of Cesium-137 gamma rays were compared with aberration frequencies produced by the 500 cGy challenge dose alone. The adapting doses were delivered in vivo and chronically over periods of 4 and 42 hours, respectively, at a dose-rate of 12 cGy/day The challenge dose was delivered in vivo and acutely at a dose-rate of 390 cGy/min and within 30 minutes of the termination of the adapting dose. To determine if a transgenerational effect was present, F1 offspring from unirradiated and chronically irradiated parents were utilized in the experiment. Results from the G2 chromosome assay, as analyzed by a linear comparison of means, indicated that there were no statistically significant differences in the total aberration frequencies produced with either the 2 or 21 cGy adapting dose followed by the 500 cGy challenge dose versus the 500 cGy challenge dose alone, P=0.93 and P=0.83 respectively, for offspring originating from either unirradiated or irradiated F0 parents. A high degree of variability in the mitotic indices was observed in the medaka embryos which complicated the statistical analyses. More research is obviously needed to clearly understand the total adapting dose required, the timing required for the delivery of the challenge dose after the adapting dose, and the appropriate stage, embryo, juvenile or adult, required to successfully demonstrate any in vivo AR in medaka fish.