Effects of Oxidative Stress and Genotoxic Stress on Zebrafish Aging and Senescence

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Several lines of evidence suggest that genotoxic or oxidative stress promote the aging process (Campisi, 2005; Finkel and Holbrook, 2000; Lombard et al., 2005). It is well known that the aging process diminishes the capability to adapt to environmental stresses. We hypothesized that genotoxic and oxidative stress leads to mutations and general declines in reproductive ability, ultimately producing the spectrum of age-related characteristics in zebrafish. Support for this idea comes from the fact that radiation increases genetic mutations and leads to premature aging in animals, such as mice and Medaka fish (Oryzias latipes) (Curtis, 1963; Egami and Eto, 1973; Ferbeyre and Lowe, 2002; Trifunovic et al., 2004; Tyner et al., 2002; Celeste et al., 2002). Therefore, we have subjected embryos and adult zebrafish to genotoxic or oxidative stress and measured aging markers over the lifespan of the fish. To determine the effects of genotoxic and oxidative stresses on zebrafish aging, we have analyzed the effects of ionizing radiation (IR) and hydrogen peroxide (HP)/t-butyl hydroperoxide (tBH) on both embryos and adults using the markers already described. For quantitative aging markers we expect to see changes in activity that correlate with age; the rate of change should also correlate with the stress applied. In this manner, we can detect changes in aging in fish in a much shorter time intervals than would be required to measure mortality rates.

Typical features of cells derived from humans with premature aging syndromes include hypersensitivity to genotoxic stress and DNA damage such as IR (Cheng et al., 2004; Naka et al., 2004; Nove et al., 1986; Smith and Paterson, 1980; Thacker, 1994). It has been reported that IR during embryogenesis shortens the lifespan in

Medaka fish (Egami and Eto, 1973). Although shortened lifespan was demonstrated, no other phenotypic markers of aging were reported at that time. An aging phenotype induced by IR was also observed in mice (Curtis, 1963). IR has been used to perform mutagenesis in zebrafish, whereas the biological effects of IR on the fish themselves have not been examined in detail. To address this question, we initially exposed zebrafish adults and embryos to several doses of IR and observed the short-term as well as long-term biological effects. We have already examined radiation doses of 5 to 40 Gy for embryos (6-24 hours post fertilization, hpf) and adult fish (3 to 30 months old). Overall cell-cycle profiles and the level of apoptotic cells were studied in whole embryos by flow cytometry at 48 hpf. When embryos were irradiated at 6 hpf with 20 Gy, we observed an aberrant increase in apoptotic cells at 48 hpf, followed by subsequently causing malformation of the entire embryos later on, with decreased survival by 5 days post fertilization (dpf). In contrast, the survival rate is greater than 90% at 5 dpf in 10 Gy-irradiated animals, without obvious malformation as well as abnormal apoptosis induction in early development. Moreover, no embryos survived for more than 10 days following 20 Gy of irradiation, which may be due to an inability to start eating. Intriguingly, these embryos exposed to IR induced SA-^-gal activity by 6 dpf, indicating genotoxic stress-induced premature senescence, can be observed during the early development of zebrafish (Figure 28.2). We have raised group of embryos irradiated with 10 Gy at 6 hpf in groups at 6, 18, 30, and 42 months of ages. Each age group of embryos need to be raised to adulthood to examine the radiation effects late in life, as compared with irradiated control animals. These experiments are currently in progress.

It is noteworthy that 25-40 Gy of irradiation killed a large number of adult fish in a dose-dependent manner within 3 months after IR exposure, with possible acute lesions, as recently reported in the literature (Traver et al., 2004). On the other hand, 20 Gy did not significantly affect survival rates even after 6 months postirradiation. Therefore, based on these pilot studies, we intend to examine the chronic effects of 20 Gy on adult fish during aging.

There are several lines of evidence that suggest that oxygen free radicals can contribute in an undetermined way to the aging process (Balaban et al., 2005). Physiologically, superoxide is generated by the mitochon-drial respiratory chain. The transformation of superoxide into HP and then, under certain conditions, into hydroxyl radicals appears to play an important role in various respiratory chain diseases (Taylor et al., 2003). These may influence the aging process through mutagenesis of mitochondrial DNA (mtDNA) and an increased rate of shortening of telomeric DNA. Therefore, we are interested in the relationship between ROS production and telomere metabolism resulting from genotoxic and oxidative stress in zebrafish by measuring protein oxidation, lipid peroxidation, and the extent of oxidized DNA. The effects of ROS on telomeres seem to be mediated through the susceptibility of the telomeric GGG sites to DNA damage. ROS actively attack these telomeric regions, predisposing to DNA strand breaks and damage leading to increased telomere shortening. Importantly, fibroblasts from donors of several premature aging syndromes, such as ataxia-telengiectasia (A-T) with mutations in the ataxia telangiectasia mutated (ATM) gene and Hutchinson-Gilford progeria syndrome (HGPS) with mutations in the lamin AC gene (lmna), have short telomeres (Allsopp et al., 1992; Metcalfe et al., 1996; Smilenov et al., 1997), consistent with reduced cell division potential in vitro. Recently, it has been suggested that ATM functions in the cellular response to oxidative damage (Ito et al., 2004; Reliene et al., 2004; Rotman and Shiloh, 1997; Watters, 2003). Support for this hypothesis comes from observations that ATM-deficient cells are very sensitive to the toxic effects of hydrogen peroxide, nitric oxide and superoxide treatment, as well as to exposure of IR (Green et al., 1997; Takao et al., 2000; Ziv et al., 2005). Moreover, ATM-deficient mice have elevated markers of oxidative stress, particularly in organs such as the cerebellum, which are consistently affected in individuals with A-T (Barlow et al., 1999a). In addition, elevated levels of Cu/Zn superoxide dismutase exacerbate specific features of the murine ATM-deficient phenotype, including abnormalities in hematopoiesis and radiosensitivity (Peter et al., 2001). Accordingly, we have employed both hydrogen peroxide (HP) and t-butyl hydroperoxide (tBH) as sources of oxidative stress to zebrafish to establish baseline information. Because tBH is poorly hydrolyzed by catalase, we can examine the effect of oxidative damage irrespective of variation and difference in catalase activities in organisms. We hace exposed embryos to HP (100 mM) or tBH (300 mM) from 6 hpf for 3 days, and then raised them for 6, 18, 30, and 42 months, to observe late-onset aging phenotypes by oxidative stress early in life. These stress exposure studies examining multiple aging biomarkers already described above are currently underway.

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