DNA Damage in Coelomocytes

Sea urchin coelomocytes were used as a bioindicator to study the effects of exposure to (solar) UV-A and UV-B radiation, heavy metals (cadmium chloride), and combinations on the occurrence of DNA strand breakage using the Fast Micromethod.

UV Radiation

Irradiation of sea urchin coelomocytes using the full solar spectrum lamp (SOL 500) at wavelengths >320 nm (filter H1) or >295 nm (filter H2) resulted in a dose-dependent increase in DNA damage, as determined by Fast Micromethod (Fig. 1). The increase in frequency of DNA single-strand breaks (expressed as SSF) was much higher if a filter was used that excludes only UV-C but not UV-B (filter H2). In the experiment shown in Fig. 1, the cells were irradiated for the same time periods (10 and 60 s) with filters H1 and H2, and the UV-A (filter H1) and UV-B (filter H2) doses were determined (UV-radiometer with a UV-A and UV-B sensor respectively). Interestingly, the SSF

Fig. 1. DNA damage induced by irradiation of sea urchin coelomocytes 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). DNA single-strand breaks were determined using Fast Micromethod and are expressed as strand scission factor (SSF) value. Control (Co): non-irradiated cells values were found to increase after incubation of the cells following irradiation due to the formation of transient breaks during repair of DNA damage (see Figs. 3 and 4).

Similar results were obtained after irradiation of coelomocytes using a monochromatic (312 nm) UV-B lamp (results not shown).

Cadmium

The effects of mixed pollution on marine invertebrates have been intensively studied in the past (Müller et al. 2000), but less attention has been paid to the combined effects of exposure to UV radiation and environmental pollutants. This problem has become increasingly important due to depletion of the UV-B protective ozone layer. Therefore, we determined the effects of cadmium (as a model pollutant) and its combined effects with UV-B (see Sect. 2.5.3) on DNA integrity in sea urchin coelomocytes.

Analysis of DNA damage in coelomocytes treated with various concentrations of cadmium chloride (10-6 to 10-12 M) for 2 h revealed a dose-dependent effect of the heavy metal on frequency of single-strand breaks (Fig. 2). Even at the low concentration of cadmium chloride of 10-12 M, an induction of DNA strand breaks in coelomocytes was found (Fig. 2).

Combined Effects

In the following, we determined the effects of different combinations of UV and cadmium exposure on DNA integrity in sea urchin coelomocytes. Unexpectedly, we found that after exposure of the cells to radiation emitted by full

Fig. 2. Effect of sublethal concentrations of cadmium chloride on DNA integrity in sea urchin coelomocytes. The coelomocytes (4.8X105 cells/ml) were exposed to various concentrations of cadmium chloride (10-6 to 10-12 M) for 2 h at 16 °C. For further details, see Fig. 1

Fig. 2. Effect of sublethal concentrations of cadmium chloride on DNA integrity in sea urchin coelomocytes. The coelomocytes (4.8X105 cells/ml) were exposed to various concentrations of cadmium chloride (10-6 to 10-12 M) for 2 h at 16 °C. For further details, see Fig. 1

Fig. 3. Effect of cadmium on DNA repair in sea urchin coelomocytes after irradiation using a SOL 500 lamp with filter H1 (UV-A and visible light; wavelengths >320 nm). Coelomocytes (4.8X105 cells/ml) were irradiated with 90 or 560 mJ/cm2 UV-A in the absence or presence of 10-6 M cadmium chloride; cadmium chloride was present throughout the repair period of 30 or 60 min at 16 °C. For further details, see Fig.1

Fig. 4. Effect of cadmium on DNA repair in sea urchin coelomocytes after irradiation using a SOL 500 lamp with filter H2 (UV-B, UV-A, and visible light; wavelengths >295 nm). Coelomocytes (4.8X105 cells/ml) were irradiated with 6.5 or 40 mJ/cm2 UV-B in the absence or presence of 10-6 M cadmium chloride; cadmium chloride was present throughout the repair periods of 30 or 60 min at 16 °C. For further details, see Fig. 1

solar spectrum lamp at wavelengths >320 nm (UV-A and visible light; filter H1), lower levels of DNA single-strand breaks [decrease in SSF X (-1) value] were present in coelomocytes if cadmium chloride was added during a postirradiation repair period of 30 or 60 min at 16 °C (Fig. 3), even at a concentration (10-6 M) that has a strong DNA damaging effect. The same result was observed after exposure of the cells to wavelengths >295 nm (UV-B, UV-A, and visible light; filter H2) and subsequent recovery in the presence of 10-6 M cadmium chloride (Fig. 4). These effects are most likely caused by a cadmium-induced inhibition of the enzymes (endonucleases) involved in DNA excision repair occurring after UV damage of DNA (Hartwig 1994).

Expression ofHSP70

Sea urchin coelomocytes also respond to UV-B radiation by an induction of expression of the heat shock protein HSP70 (see Matranga et al., this Vol.). Similarly, an increase in HSP70 level was observed in coelomocytes exposed to 10-4M cadmium chloride (Matranga et al. 2002).

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