Sponge Primmorphs

In the experiments described below, the marine sponge S. domuncula was used to monitor the response to UV-A and UV-B radiation and visible light. A special cell culture system was used to study the effects of exposure to these radiations on DNA integrity of sponge cells (Fig. 7). The sponge cell culture

Fig. 7a-d. Formation of primmorphs from single cell suspensions of the sponge S. domuncula. Sponge tissue (a) (x0.7) was dissociated into single cells (b) using Ca2+-and Mg2+-free artificial seawater containing EDTA (Rottmann et al. 1987) (x100); after transfer into seawater/antibiotics, aggregates are formed (c) (x100), which finally resulted in the formation of prim-morphs after an incubation period of 3-4 days (d) (x20)

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starts from dissociated cells of S. domuncula, which subsequently form aggregates; in those aggregates the cells start DNA synthesis and proliferate. The aggregates, termed primmorphs, show a tissue-like appearance and can be cultured for more than 5 months.

UV Radiation

The primmorphs from S. domuncula cells were used as a model to analyze the effects of UV-A and UV-B on DNA integrity in sponge cells in vitro. The Fast Micromethod was adapted to work with low-integrity sponge DNA; unwinding was performed at pH 11.6. At this pH only strand breaks and cross-links could be measured. At higher pH (>12.0), alkali-labile sites (enzyme binding sites, apurinic and apyrimidinic sites) are the predominant effects of DNA denaturation.

Sponge cells (primmorphs) were irradiated with a SOL 500 lamp, which covered the whole solar spectrum using either H1 (wavelengths >320 nm; UV-A and visible light) or H2 (wavelengths >295 nm; UV-B, UV-A and visible light) filters. Subsequently, aliquots were analyzed for DNA single-strand breaks using Fast Micromethod. As in sea urchin coelomocytes, a dose-dependent increase in DNA damage was observed following exposure of sponge cells to 80-940 J/m2 of UV-A or 5-60 J/m2 of UV-B (Fig. 8). The UV-A and UV-B doses were monitored using a UV radiometer with an A or B sensor respectively. Again, the effects of UV-B (plus UV-A and visible light; filter H2) were much (several-fold) stronger than those observed with UV-A (plus visible light; filter H1). The extent of UV-A-induced DNA single-strand breaks

Fig. 8. UV-induced DNA damage in S. domuncula cells. Cells (1.5X105 cells/ml) were exposed to the indicated doses of UV-A and UV-B using a SOL 500 lamp with filter H1 (UV-A and visible light; wavelengths >320 nm) or filter H2 (UV-B, UV-A, and visible light; wavelengths >295 nm). Control (Co): non-irradiated cells

Fig. 8. UV-induced DNA damage in S. domuncula cells. Cells (1.5X105 cells/ml) were exposed to the indicated doses of UV-A and UV-B using a SOL 500 lamp with filter H1 (UV-A and visible light; wavelengths >320 nm) or filter H2 (UV-B, UV-A, and visible light; wavelengths >295 nm). Control (Co): non-irradiated cells

Co 470 470 470 470 470 940 940 940 940 940 30 30 30 30 30 60 60 60 60 60 Dos« (rrvl/cm) 0 10 20 30 GO 0 10 20 30 60 0 1 0 20 30 60 0 10 20 30 60 Repair (mn)

UV-B

Fig. 9. DNA damage and repair in S. domuncula cells after exposure of cells (1.5X105 cells/ml) to the indicated doses of UV-A and UV-B using a full solar spectrum SOL 500 lamp with filter H1 (UV-A and visible light; wavelengths >320 nm) or filter H2 (UV-B, UV-A, and visible light; wavelengths >295 nm). DNA repair was allowed to occur for 0,10, 20,30 and 60 min at 16 °C. Control (Co): non-irradiated cells

Co 470 470 470 470 470 940 940 940 940 940 30 30 30 30 30 60 60 60 60 60 Dos« (rrvl/cm) 0 10 20 30 GO 0 10 20 30 60 0 1 0 20 30 60 0 10 20 30 60 Repair (mn)

UV-B

Fig. 9. DNA damage and repair in S. domuncula cells after exposure of cells (1.5X105 cells/ml) to the indicated doses of UV-A and UV-B using a full solar spectrum SOL 500 lamp with filter H1 (UV-A and visible light; wavelengths >320 nm) or filter H2 (UV-B, UV-A, and visible light; wavelengths >295 nm). DNA repair was allowed to occur for 0,10, 20,30 and 60 min at 16 °C. Control (Co): non-irradiated cells increased during a post-irradiation repair period at 16 °C for 1.5 h (Fig. 9), due to the formation of transient breaks by the action of repair enzymes. The apparent decrease in strand breaks observed at higher UV doses (Fig. 9) may be caused by UV-induced formation of DNA cross-links resulting in lower SSF X (-1) values.

Induction of DNA single-strand breaks was also measured following irradiation of sponge cells (primmorphs) using a monochromatic UV-B lamp (peak at 312 nm; results not shown). DNA repair was accompanied by a time-dependent increase in the number of single-strand breaks; after a repair period of 2 h, the extent of DNA single-strand breaks decreased, reaching similar levels as in non-irradiated control cells after an incubation period of 18 h. The production of DNA single-strand breaks strongly depended on the medium used; the SSF X (-1) values were lower (negative) in Ca2+-/Mg2+-free seawater containing EDTA, but increased in Ca2+-/Mg2+-containing seawater. The decrease in SSF X (-1) value in EDTA-containing, Ca2+-/Mg2+-free seawa-ter may be caused by a higher radiosensitivity (induction of formation of DNA cross-links) of the sponge single cells present under these conditions, compared to sponge primmorphs formed in the presence of calcium (Ca-/Mg-containing seawater).

The Fast Micromethod has also been applied for measuring the extent of DNA single-strand breaks in whole tissue samples of the marine sponge Geo-dia cydonium, irradiated with UV light under controlled experimental conditions, or from specimens collected in the field (Batel et al. 1998). The tissue samples were irradiated with 0-300 mJ/cm2 UV-B (monochromatic UV-B lamp). One hour later, samples were analyzed for DNA strand breaks. In the untreated controls, which remained in the dark, the SSF was set to zero (0.00±0.04; n=5). Irradiation at a dose of 10 mJ/cm2 significantly increased the number of strand breaks [SSF x (-1)] to 0.14±0.02. Higher doses of UV-B further increased the extent of DNA damage; at 300 mJ/cm2 the SSF x (-1) reached a value of 0.32±0.08.

Cadmium

DNA damage in sponges is also induced by cadmium chloride. Exposure of whole specimen of marine sponges, e.g., S. domuncula, to cadmium resulted in a strong accumulation of the metal in the sponge (Schröder et al. 1999b).As a consequence a strong increase in the extent of DNA single-strand breaks was found using Fast Micromethod. The maximal increase was observed after an incubation period of 12 h in the presence of 1 mg/l of cadmium chloride and after an incubation period of 1-3 days in the presence of 10 or 100 |g/l of cadmium chloride; after a prolonged incubation period, the number of damages decreased, most likely due to DNA repair (Schröder et al. 1996).

Field experiments using S. domuncula collected from five stations in the northern Adriatic Sea, characterized by a gradient of pollution, revealed significant differences in the cadmium levels between these stations (Müller et al. 1998). The frequency of DNA strand breaks roughly paralleled the gradient of pollution (cadmium levels) at these sites.

Combined Effects

Results of the effect of cadmium on DNA repair in S. domuncula cells after irradiation with full solar spectrum lamp with filter H1 (wavelengths >320 nm) or filter H2 (wavelengths >295 nm) did not indicate an inhibition of DNA repair processes as found in sea urchin coelomocytes (results not shown). Possibly some mechanism(s) preventing cadmium toxicity despite its high accumulation in sponge tissue (a 17,500 enrichment has been determined under field conditions; Müller et al. 1998), such as expression of metal-lothionein-like proteins (Schröder et al. 2000b), are responsible for the finding that an inhibitory effect of cadmium on DNA repair could not be detected in the sponge system.

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