Aging Of Japanese Medaka Fish Under Normal And Radiation Exposure Conditions

BackgroundAging is the accumulation of changes in an organism or object over time resulted in degeneration of organismal structures, deterioration of the physiological functions, decrease of adaptability and increase of mortality. Radiation is a known environmental toxigen. Understanding the biological effects of radiation is important because human live and work in radiation environments. With the increasing environmental radiation pollution induced by the human activities, there is an increased risk of radiation exposure of human and all organisms on the earth. There is considerable overlap between cellular and molecular changes that occur in response to ionizing radiation and those that occur during normal aging. Both processes are characterized by constant existing of oxidative stress, increased levels of oxidative biomolecules, damage of cellular function. All these changes occur to the biomolecules lead to the loss of regenerative capacity at the cellular and tissue level resulted in all kinds of pathologic changes including decreased immunity, tumorigenesis, cognitive decline and organ failure.Japanese medaka fish (Oryzias latipes) is small laboratory fish that share many anatomical and histological characteristics with other vertebrates. Because of their well-developed genetics, tolerance to the outside circumstances and low incidence of the spontaneous cancer and other disease, and relatively lower mortality and easier maintaince under laboratory conditions, and because it is feasible and relatively low cost to perform lifetime studies using large numbers of individuals make it a useful model organism for investigating the effects of ionizing radiation and other genotoxic agents, and it has the potential to be used for aging research.Here we characterize biomarkers associated with normal aging in the medaka fish under normal laboratory conditions, and quantified some of the aging associated biomarkers in medaka fish during aging process which will facilitate tracing the aging changes in the following research.γirradiation is a widely used environmental stressor, and is used for treatment of tumors in clinic. In this study, y irradiation was used as a genotoxic stressor and was delivered into early development stage of medaka embryos, then investigated the changes of life-span and aging process of major organs of medaka fish population. We explored the effects of early embryo stage radiation exposure on medaka fish normal aging process and its possible mechanisms, provides basis for medaka fish as a useful organism for radiation and ecology research.Part I Quantifiable Biomarkers of Normal Aging in the Japanese Medaka Fish (Oryzias latipes)Purpose:To characterize a set of the biomarkers to trace the process of normal aging in Japanese medaka fish that can be used for evaluating the effects of environmental stressors.Methods:CAB wild type Japanese medaka fish population were maintained under laboratory conditions started from fertilization until the natural mortality reached 100%. We did a Kaplan-Meier analysis based on the survival data. Three different age groups of male medaka fish were used in this study:6 months old (young adults),16 months old (mature adults), and 24 month old (adults that had survived beyond the median lifespan). Male fish from each age group were labeled with 5-Bromo-2’-deoxyuridine (BrdU) then sacrificed for further study. We did an anti-BrdU immunofluorescent staining to detect the proliferative cells in the skin and skeletal muscle, and X-gal staining (pH6.0) to detect the aging associatedβ-galactosidase (SAβ-gal) activity in the skin and skeletal muscle. We did H.E staining of liver, heart and eye sections, observed the pathologic changes in these tissues. Based on the above experiments, we further investigated the ultrastructural changes, quantified the apoptotic cells and accumulation of lipofuscin granules in liver tissue. We also detected the protein oxidation level of livers using Western Blot.Results:The median lifespan of medaka fish was approximately 22 months under normal laboratory conditions. We observed aging-related changes in the medaka fish liver, skin, heart and retina. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain. Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits, in the number of TUNEL-positive apoptotic cells and in the protein oxidation level. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in SAβ-gal activity. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium from the 16-month old and 24-month old group to some extent.Conclusion:Aging associated changes occur to a variety of Japanese medaka fish organs to different extent during normal aging process under laboratory conditions, the morphologic changes, accumulation of lipofuscin granules, apoptotic cells, protein oxidation level in the liver, the proliferation rate and SA P-gal activity of the skin and the retinal pigment epithelial cell changes are more sensitive, constant and quantifiable. These are reliable biomarkers for Japanese medaka fish aging process.Part II The Effects of Early Embryonic Radiation Exposure on Life-span and Major Organ and Tissue Aging Process of Japanese Medaka FishPurpose:To investigate the influence of early embryo stage acute ionizing radiation exposure on the life span and major organ and tissue aging process of Japanese medaka fish.Methods:We surveyed biological and biochemical markers on aging using CAB wild type Japanese medaka fish that received total absorbed doses ranging from 0-15 Gy (1.87 Gy min-1) of y radiation delivered at an early stage of development (12 hours past fertilization,12hpf). Fish were maintained and observed under normal laboratory conditions after exposure for 36 days or till the mortality rate reached 100%. Kaplan-Merier survival analyses were performed to analyze the influence of radiation on survival or life span. The other five dose groups (0 Gy,0.5 Gy,1.5 Gy,5 Gy and 15 Gy) of fish embryos were irradiated using the same method.16 months after irradiation, male fish from each dose group were selected and used for analysis of aging-related changes in major organ and tissue using the same methods illustrated in the part one.Results:1. The influence of different doses of radiation on the survival and lifespan of Japanese medaka fishAcute toxicity of radiation:36 days past radiation exposure the Kaplan-Meier Analysis showed dramatically increased mortality in 15 Gy group (p<0.05). Long term effects of radiation:life-time Kaplan-Meier analysis showed changed survival curve. The mean and median survival were significantly decreased in the 15 Gy group but not in the lower dose groups (0.5-5 Gy) compare to the unirradiated control group.2. The influence of different doses of radiation on the major organ or tissue aging process of Japanese medaka fishThe radiated fish livers exhibited extensive morphology changes, including spongiosis hepatis, steatosis, balloon degeneration, inflammatory infiltration, pyknosis nucleus, cyst and fibrosis; X-gal staining showed increased SA (3-gal activity in the radiated fish basal layer of skin and satellite cells of skeletal muscle. These changes didn’t show dose-dependence.Conclusion:Early embryonic stage one acute exposure to high dose ionizing radiation (15 Gy) accelerates major organ and tissue aging process of Japanese medaka fish, resulted in significantly shortened life expectancy. Low dose ionizing radiation (0.5-5 Gy) exposure accelerates aging process of some organ or tissue but not change the life expectancy.