Oxidative Stress, Dna Damage And P53 Expression In The Larvae Of Atlantic Cod (Gadus Morhua) Exposed To Ultraviolet (290–400 nm) Radiation

The decrease in stratospheric ozone levels resulting from anthropogenic inputs of chlorinated fluorocarbons has resulted in an increase in the amount of harmful ultraviolet-B (UVB, 290–320 nm) radiation reaching the sea surface. Particular attention has been focused on the Antarctic, and more recently the Arctic (Hofmann and Deschler, 1991), where the autocatalytic destruction of stratospheric ozone (‘the ozone hole’) leads to enhanced fluxes of UVB radiation (Hofmann, 1996; Smith and Baker, 1989) and a decrease in primary productivity (Smith et al., 1992). Recent data describing global decreases in stratospheric ozone levels show a highly variable trend in the loss of ozone over equatorial regions in the last decade. In addition, ozone depletion has resulted in an increase in UVB radiation reaching temperate latitudes (30–50°N) of the Northern Hemisphere (Blumthaler and Ambach, 1990; Kerr, 1992; Kerr and McElroy, 1993), with a negative trend of ozone depletion of as much as −6.6 to −7.6 % and as little as −2.6 to −3.0 % per decade (Stolarski et al., 1992; Madronich, 1992; Madronich, 1994).

These temperate latitudes cover areas that include many productive coastal estuarine, intertidal and shallow subtidal systems such as those found in the Gulf of Maine, and current trends in stratospheric ozone loss suggest that irradiances of UVB radiation could increase in the future. Of particular concern at any latitude is that ozone depletion results in an increase in damaging UVB wavelengths without a proportional increase in longer ultraviolet-A (UVA; 320–400 nm) radiation and blue wavelengths involved in photoreactivation and photorepair of DNA damage (Smith, 1989).

In the Gulf of Maine, UVB radiation can penetrate to depths of 7–12 m (Banaszak et al., 1998) depending on the optical properties of the water column. The dynamic changes in the stability and optical properties of the water column (due to seasonal changes in solar irradiation, phytoplankton blooms or increases in dissolved organic matter) are significant contributors to changes in the attenuation of UVR in the Gulf of Maine. As a result of these changes in the optical properties of the water column, changes in the attenuation of ultraviolet radiation (UVR) could have dramatic effects on the mortality and morbidity of planktonic life-history phases of marine organisms. The harmful effects of UVR include damage to DNA, proteins and membrane lipids that will in turn compromise the physiology, biochemistry and organismal performance (i.e. growth and reproduction) for those organisms exposed to UVR. These processes have important implications for the ecology of the affected organisms.

There is also a growing awareness of the separate and interacting effects of solar UVR and oxidative stress on marine organisms. Reactive oxygen species (ROS) are formed by the univalent reduction of molecular O₂ owing to spin restrictions on its valence electrons, yielding superoxide radicals (O₂⁻) and, by further reduction of superoxide, to hydrogen peroxide (H₂O₂) and hydroxyl radicals (HO^•) (Asada and Takahashi, 1987). Also, the transfer of absorbed energy, especially within the ultraviolet wavelengths, to ground-state O₂ (photodynamic action) from activated photosensitizers produces highly reactive singlet oxygen (Halliwell and Gutteridge, 1989). Exposure to UVR and ROS can act synergistically to cause extensive DNA damage and lead to apopotosis or programmed cell death.

Oxidative stress is known to play a role in programmed cell death (i.e. apoptosis) through several cell cycle genes such as p53. The principal function of p53 is to promote survival or deletion of cells exposed to agents that cause DNA damage, such as hypoxia, UVR, ROS or mutagens (Graeber et al., 1996; Renzing et al., 1996; Clarke et al., 1997; Griffiths et al., 1997). The p53 protein is involved in complex cellular responses to DNA damage. These responses involve DNA editing and repair followed either by normal cell division (Polyak et al., 1997) or by apoptosis (Hale et al., 1996). Cells damaged by UVR and oxidative stress can survive but often remain in the G1-phase of the cell cycle for long periods or even remain in a permanent state of cellular growth arrest (Evan and Littlewood, 1998).

Groundfish populations, especially of Atlantic cod (Gadus morhua), in the western North Atlantic and Gulf of Maine have declined dramatically over the last several years. One of the principal uncertainties in fisheries models used to assess year class strength is larval mortality. Embryo and larval mortality rates are routinely estimated to be over 99 %, and any additional increase in mortality as a result of exposure to UVR may further affect recruitment and year class strength. Atlantic cod are known to spawn in deep water and have buoyant embryos and larvae that reach the upper 0–25 m of the water column (Solemdal and Sundby, 1981). UVR, specifically UVB radiation, can penetrate up to 12 m in the Gulf of Maine (Banaszak et al., 1998), and UVB radiation is known to affect the mortality of Atlantic cod embryos (Kouwenberg et al., 1999) and the embryos of other species of marine fishes (Hunter et al., 1979; Hunter et al., 1981). Damage to DNA in marine fish species has also been observed during exposure to UVB radiation (Malloy et al., 1997; Vetter et al., 1999).

Here, we report on the results of laboratory experiments examining the direct and indirect effects of UVR on DNA damage in the Atlantic cod Gadus morhua. We find that DNA damage leads to the expression of p53 which, in turn, causes either DNA repair or apoptosis. Any delay in the cell cycle may lead to prolonged developmental times and a smaller size at hatching for cod larvae. If the DNA damage is not repairable, this would lead to the alternative p53 pathway, resulting in apoptosis and potential mortality of the developing embryo. We present the results of our laboratory experiments on exposures of UVR simulating the upper 10 m of Gulf of Maine waters and its potential for enhanced larval mortality.

Eggs and sperm of Atlantic cod (Gadus morhua L.) were collected from several freshly caught fish on Jeffreys Ledge, Gulf of Maine (42°54′N latitude, 70°05′W longitude), during the month of June. Eggs were fertilized at sea and maintained at 4–6 °C until they arrived at the University of New Hampshire, where experimental exposures of embryos began 3 days after fertilization. Gastrulating embryos were maintained on a 12 h:12 h light:dark cycle at 7 °C and 33 ‰ salinity. Three treatment groups (UVO, 400–700 nm; UVA, 320–700 nm; UVT, 290–700 nm) were established using glass bowls (N=3 for each treatment) containing approximately 250 ml of aerated, filtered (0.22 μm) sea water and embryos at a density of approximately 1–2 ml⁻¹. Ultraviolet-opaque (50 % cutoff approximately 385 nm; UVO) Plexiglas, UVA (50 % cutoff approximately 315 nm)=UVT Plexiglas and Mylar-D, and ultraviolet-transparent (50 % cutoff approximately 295 nm; UVT) Plexiglas coverings were used. These treatment groups correspond to specific portions of the spectrum as follows: UVT is the total ultraviolet spectrum (from approximately 290 to 400 nm plus visible radiation), UVA is the UVA portion of the spectrum (approximately 320–400 nm plus visible radiation) and UVO is the visible (400–700 nm) portion of the spectrum only. Visible radiation was from two Vita-Lite full-spectrum fluorescent lamps providing an irradiance of 50 μmol quanta m⁻² s⁻¹ (photosynthetically active radiation, 400–700 nm). Ultraviolet radiation, predominantly UV-B but including all UV-A wavelengths, came from two aged fluorescent lamps (FS20 T12-UVB) covered with cellulose acetate film (0.13 mm thickness). The ultraviolet and visible radiation sources were suspended 25 cm above the experimental bowls.

Spectral irradiance from the ultraviolet and visible lamps was measured with a calibrated (using National Institute of Standards and Technology; NIST) traceable-standards, diodearray spectroradiometer system (S2000, Ocean Optics, Inc.). The irradiance and total dose for each treatment were calculated, and the biologically effective irradiance and dose were also calculated using the Atlantic cod mortality weighting (with irradiance-independent mortality and photorepair) function of Kouwenberg et al. (Kouwenberg et al., 1999). A weighting function defines the wavelength-dependence for some function of interest (e.g. DNA damage) and, when multiplied by a solar or laboratory spectrum, provides the biologically effective irradiance or dose. The experiment ran under these conditions for 10 days, during which samples were taken for analysis and water changes were conducted every 2 days.

The percentage survival of developing embryos was determined during the experiment by counting dead versus live embryos in each bowl. Cod embryos become opaque upon dying and were removed after counts had been completed. Developmental state was also recorded during these analyses using the Nordahl series at 6–8 °C as described (Hardy, 1978). At the end of the experiment, a series of analyses was conducted. Experiments ended just as yolk-sac larvae were hatching, and the standard size at hatching was determined for each treatment (N=30) by measuring the standard length of yolk-sac larvae to the nearest 0.1 mm using Vernier calipers.

At the end of the experiment, samples of newly fertilized embryos and yolk-sac larvae from each treatment group were analyzed for the presence of ultraviolet-absorbing compounds known as mycosporine-like amino acids (MAAs) according to the procedures described (Shick et al., 1992). Samples were extracted overnight in 100 % high-performance liquid chromatography (HPLC)-grade methanol at 4 °C. The extracts were centrifuged, and the supernatant was used for MAA and protein analysis. Individual MAAs were separated by reversephase, isocratic HPLC on a Brownlee RP-8 column (Spheri-5, 4.6 mm i.d × 250 mm) protected with an RP-8 guard column (Spheri-5, 4.6 mm i.d. × 30 mm). The mobile phase consisted of 40–55 % methanol (v:v), 0.1 % glacial acetic acid (v:v) in water and was run at a flow rate of 0.6 ml min⁻¹. Detection of MAA peaks was by ultraviolet absorbance at 313 and 340 nm. Standards were available for seven MAAs (mycosporine-glycine, shinorine, porphyra-334, palythine, asterina-330, palythinol and palythene). The identities of peaks were confirmed by co-chromatography with standards and by the ratios of absorbances at 313 nm and 340 nm. Peaks were integrated, and quantification of individual MAAs was accomplished using HPLC peak areas and calibration factors determined by analysis of the standards listed above. All MAAs were normalized to soluble protein content from a portion of the methanol-extracted sample and concentrations are expressed in nmol MAA mg⁻¹ protein. Protein concentrations were determined using the procedure of Bradford, 1976.

Cyclobutane pyrimidine dimer (CPD) formation was measured using the procedures and monoclonal antibody (TDM-2) of Mori et al., 1991. Briefly, DNA was isolated from individual yolk-sac larvae (N=3) from each treatment group using commercially available kits (Invitrogen, Inc.), and 50 ng from each larva was then used in an enzyme-linked immunosorbent assay (ELISA) technique with TDM-2 as the primary antibody. An affinity-purified goat anti-mouse IgG secondary antibody conjugated with peroxidase was used with the appropriate substrate, and the color development was read in a microtiter plate reader as described by Mori et al. (Mori et al., 1991).

Individual larvae (N=3) were homogenized with a tissue homogenizer in 10 mmol l⁻¹ Hepes buffer (pH 7.5) with dithiothreitol and phenylmethylsulfonyl fluoride to prevent protein oxidation and protease activity respectively. The homogenate was then centrifuged at 500 g for 20 min, and the supernatant was saved for analysis of protein (Bradford, 1976) and enzymes. The supernatant was sonicated while on ice and then centrifuged for 30 min at 16 000 g to obtain a cell-free extract for enzyme measurements. Superoxide dismutase activity was assayed spectrophotometrically as described (Elstner and Heupel, 1976; Oyanagui, 1984). Standards of bovine erythrocyte Cu-Zn SOD (Sigma) were used. All enzyme assays were performed at 7 °C using a water-jacketed cuvette, and values are expressed as units of enzyme activity (1.0 unit=1 μmole of substrate converted per minute) per milligram of soluble protein.

From the experiment described above, protein extracts of samples (N=3) of equivalent biomass were separated on SDS gradient polyacrylamide (4–15 %) gels using a modified Laemmli buffer system and transferred to nitrocellulose as described by Lesser et al. (Lesser et al., 1996). The transferred proteins were immunoblotted using polyclonal antibodies against cytsolic superoxide dismutase (SOD) and a monoclonal antibody to human p53 (CM-1, Novacastra Laboraories, Inc.) and visualized using secondary antibodies with a peroxidase label. The immunoblots were scanned, and the optical density of the positive bands was measured using the gel-scanning procedures described in NIH Image (ver. 1.61) and based on a calibrated gray scale. Optical densities were log-transformed before analysis.

Significant treatment effects for the analyses described above were determined using an analysis of variance (ANOVA) at a significance level of 0.05 %. Any significant treatment effects were then analyzed using a Student– Neuman–Keuls (SNK) post-hoc multiple-comparison test at a significance level of 0.05 %.

Table 1 shows the UVR dose rates for each treatment together with the total dose for the duration of the experiment. In addition, the biologically effective irradiance for each treatment was calculated using an Atlantic cod mortality weighting function from Kouwenberg et al. (1999). The biologically effective irradiance of UVR for the UVT treatment (see Table 1) is equivalent to that measured in the Gulf of Maine at a depth of approximately 8 m.

The percentage survival of cod embryos in the UVA and UVT treatments declined rapidly compared with the UVO treatments (Fig. 1). Both time and treatment were significant (two-way ANOVA, P=0.0001 on log-transformed data) factors contributing to lower survival. Multiple comparison testing showed that all treatment groups were significantly different from one another (SNK, P<0.05, Fig. 1) and that all pairwise day (except between day 6 and 8) comparisons were significantly different (SNK, P<0.05) from one another. In addition, there was a significant interaction term between time and treatment (ANOVA, P=0.0001) indicating a dose-dependence for cod embryo survival when exposed to UVR. On day 8, all treatments contained yolk-sac larvae, and by day 10 of the experiment the UVT treatment showed a precipitous and significant (SNK, P<0.05) decline in survival compared with the other treatments. There was no apparent lag in developmental rate for any of the treatments over time. On each day of analysis, all treatments contained 85–90 % of developing embryos at the same developmental stage (Table 2).

At the end of the experiment, yolk-sac larvae showed a small, but significant (ANOVA, P=0.032), effect of UVR on standard length at hatching (Fig. 2). Multiple comparison testing revealed that UVT-treated larvae were significantly (SNK, P<0.05) smaller than UVO-but not UVA-treated larvae, and that UVA-and UVO-treated larvae were similar in size. This suggests that UVR, and specifically the UVB component of UVR, has a subtle effect on growth in the surviving larvae.

The concentrations of MAAs in yolk-sac larvae from the UVA and UVT treatments were significantly (ANOVA, P=0.0001) lower than those of early embryos or of larvae from the UVO treatments (SNK, P<0.05, Fig. 3). Of the MAAs detected, 52–73 % of the total consisted of the MAA mycosporine-glycine.

In addition to decreased survivorship, smaller size at hatching and decreased MAA concentrations, yolk-sac larvae at the end of the experimental period exhibited significantly (ANOVA, P=0.007 on log-transformed values) greater damage to their DNA measured as CPD formation (Fig. 4). All treatment groups were significantly different from one another (SNK, P<0.05, Fig. 4).

Yolk-sac larvae exposed to UVR also expressed significantly (ANOVA, P=0.001) higher activities of the antioxidant enzyme SOD. All treatment groups were significantly different from one another (SNK, P<0.05), with UVT animals having the highest enzyme activities (Fig. 5). These results were supported by the western blots for Cu-Zn SOD protein (ANOVA, P=0.006 on log-transformed values). Western blots revealed a single band at 17 kDa that corresponded to a Cu-Zn SOD standard (bovine erythrocytes; Sigma, Inc.). Densitometer scans of SOD immunoblots showed that larvae in the UVT treatments had significantly greater amounts of SOD protein (SNK, P<0.05, Fig. 6A). In addition, densitometer scans of western blots for the cell cycle gene p53 revealed a trend of increasing p53 protein (53 kDa) content with the addition of UVR (Fig. 6B). There was a significant treatment effect (ANOVA, P=0.012 on log-transformed values), with levels of p53 protein in larvae in the UVT treatment being significantly (SNK, P<0.05) higher than those in larvae in the UVA and UVO treatments.

Several studies have clearly shown enhanced mortality of pelagic embryos or larvae of temperate marine fishes when exposed to UVB radiation (Hunter et al., 1979, 1981; Kaupp and Hunter, 1981; Kouwenberg et al., 1999; Vetter et al., 1999). The life-history of the Atlantic cod is of particular interest because of its ecological importance as a link between tropic levels in several temperate pelagic food webs and as a commercial resource. Female Atlantic cod spawn at depths greater than 200 m, and the demersal eggs are positively buoyant and can reach surface waters several days later: 10–30 % of embryos can occur in the upper 0–25 m of the water column (Solemdal and Sundby, 1981; Ouellet, 1997). In the Gulf of Maine, many populations of Atlantic cod spawn in May and June when irradiances of UVR are seasonally high for temperate latitudes. Attenuation coefficients for UVB can be low in places such as the Wilkinson Basin, K_d (UVB^300−320 nm)=0.30 m⁻¹ (Banaszak et al., 1998), with 10 % of ambient UVB radiation reaching a depth of 7.6 m (2.3/K_d; Kirk, 1994).

The experiments described above simulate a weighted (using the weighting function for cod mortality, with irradiance-independent mortality and photorepair, as described by Kouwenberg et al., 1999) UVB irradiance similar to those measured in the Gulf of Maine in June at a depth of 7–8 m (M. P. Lesser, unpublished observations). At these irradiances, Atlantic cod embryos in the laboratory exhibit high rates of mortality. Mortality is greater than 50 % in the first 2 days of UVR exposure and continues at a more moderate pace until day 10 of the experiment, when yolk-sac larvae exhibit significantly higher mortalities in treatments exposed to UVB as well as UVA radiation. The higher mortalities at the end of the experiment are potentially the result of the loss of the protective chorion at hatching. These controlled exposures should not be extrapolated directly to embryos and larvae in their natural environment. Rates of vertical mixing, the depth of the mixed layer and the distribution of embryos and larvae in the mixed layer will need to be incorporated into models estimating the effects of UVR on mortality in these planktonic life-history phases. A physical model for UVR effects on cod embryos is available for the Gulf of Saint Lawrence, and it predicts no discernible effects of UVR above that expected for losses due to predation and other factors (Kuhn et al., 2000). The measured attenuation coefficients (Kuhn et al., 1999) for UVR in the Gulf of Saint Lawrence are, however, higher for those stations used in the cod embryo simulations than those observed in the Gulf of Maine (M. P. Lesser, unpublished observations).

The mortality results of our laboratory experiments are very similar to those obtained by Kouwenberg et al., 1999 for UVR exposures of Atlantic cod embryos using a solar simulator despite the much lower total dose of UVR in their experiments. In our experiments, larvae that survived exposure to UVR, especially the UVB portion of the spectrum, also exhibited smaller standard lengths at the end of the experiment. Similar results have been observed in laboratory experiments using Northern anchovy (Engraulis modax) larvae (Hunter et al., 1981). A smaller size at hatching, as a non-lethal effect, might have ecological consequences such as susceptibility to predation and could potentially contribute further to the high rates of mortality in pelagic fish embryos and larvae.

Exposure to UVA or UVA plus UVB radiation resulted in significant DNA damage in the surviving yolk-sac larvae. It is reasonable to assume that many of the embryos and larvae that succumbed during the experiment also sustained significant DNA damage. Vetter et al. (Vetter et al., 1999) observed diurnal cycling of DNA damage by both UVB and UVA radiation, also measured as CPD formation, in experiments with Northern anchovy larvae that tracked solar irradiances. Experiments on cod embryos by Browman et al. (2000) showed a significant effect of UVB, but not UVA, radiation on DNA damage. In those experiments, cod embryos were given only a single 1 h exposure of UVR, with the resulting dose of UVA being much lower than in the experiments described here.

Experiments in the Antarctic on embryos of the ice fish Chaenocephalus aceratus showed that CPD formation was significantly correlated with cumulative daily UVB dose (Malloy et al., 1997). DNA damage can be caused directly by exposure to UVR as a result of absorption of photons of UVR or indirectly through photodynamic action and the production of ROS (Imlay and Linn, 1988; Valenzeno and Pooler, 1987). The absorption of excitation energy in the presence of oxygen leads to the production of active oxygen (singlet oxygen ¹O₂, superoxide radicals O₂⁻ and hydrogen peroxide H₂O₂) for which there are many cellular targets (Asada and Takahashi, 1987). In cod larvae exposed to UVA and UVB radiation, there are significantly higher activities of SOD and concentrations of SOD protein as shown in immunoblots. This is strong evidence that these animals are responding to high concentrations of ROS produced during exposure to UVR. In addition, at the end of the experiment, larvae exposed to UVR have significantly lower levels of UVR-absorbing compounds (MAAs). The suite of MAAs in cod larvae is dominated by mycosporine-glycine, an MAA known to have antioxidant activity and to be consumed during reactions with ROS (Dunlap and Yamamoto, 1995).

In addition, oxidative stress is known to play a role in apoptosis or programmed cell death through p53. Exposure to UVR results in the production of ROS, damage to DNA, activation (up-regulation) of the p53 pathway and arrest of cell division in the G1-phase. Activation of the p53 pathway allows those cells affected to undergo DNA editing and repair while arrested in G1-phase, followed either by apoptosis or by normal cell division (Hale et al., 1996; Renzing et al., 1996; Polyak et al., 1997). Damaged cells can be repaired and survive but are often retained in the G1-phase of the cell cycle for long periods or even permanently arrested (Evan and Littlewood, 1998). Our results show that the expression of p53 is significantly increased in cod larvae exposed to UVR and oxidative stress. This enhanced expression of p53 not only delays cell division, and potentially the total number of cells a larva may have, but also decreases the amount of time that G1-arrested and repaired cells will have to grow in the context of a temporally fixed developmental program. Therefore, expression of the p53 pathway, when not leading to apoptosis that causes larval mortality, may result in the smaller size at hatching observed for cod larvae exposed to UVR. The principal function of p53 is to promote survival or deletion of cells exposed to agents that cause DNA damage, such as hypoxia, UVR, ROS or mutagens (Graeber et al., 1996; Renzing et al., 1996; Clarke et al., 1997; Griffiths et al., 1997).

For those larvae that survive the effects of UVR at the biochemical and molecular levels, there must be an energetic cost at the organismal level. Some of that cost is probably manifest through the slower growth rates observed in these laboraotry experiments. The sub-lethal energetic costs of repairing DNA damage or responding to oxidative stress may contribute to poor individual growth performance in surviving larvae. If the small, but significant, decrease in size at hatching observed in these experiments occurs in nature, it may result in a longer planktonic period for these fish and increase the probability of being predated upon. In addition to predicting mortality, modeling efforts on the effects of UVR on cod and other fish and invertebrate larvae should also include the energetic costs of sub-lethal effects and how these costs affect other life-history phases in addition to predicting direct mortality.

This project would not have been possible had it not been for the efforts of Debra Bidwell in obtaining gamete material at sea. The authors also want to thank Dr Toshio Mori for generously supplying the monoclonal antibodies to CPD. This project was supported by the National Science Foundation (Biological Oceanography Program, OCE-9818918).