Laboratory Experiments

The growing concern over the health state of the sea has attracted the attention of many laboratories to the use of aquatic invertebrates, e.g. echino-derms, as tools for monitoring environmental hazards. However, only few investigations report the effects of pollution in terms of cellular and biochemical modifications on adults of higher marine invertebrates, such as the sea urchin. Most of the reports present in the literature describe in fact the toxic/teratogenic effects of various chemical agents and drugs on the development of sea urchin embryos (Kobayashi 1980; Ozretic and Krajnovic-Ozretic 1985; Sconzo et al. 1995; Morale et al. 1998; Russo et al. 2003; Geraci et al. 2004; Roccheri et al. 2004). The studies described below were aimed at finding a biological indicator to be used as a stress marker and to characterize the response to induced or accidental stresses by the use of molecular markers using sea urchin coelomocytes as biosensors.

It was already known that sea urchin embryos respond to temperature stress by the activation of specific genes (Giudice 1989), whose sequence for the P. lividus species has been identified and described (Sconzo et al. 1992). However, there were no studies on the presence of constitutive and/or inducible hsp70 protein in adult sea urchin cells at that time. In a first attempt, we tested the hypothesis that coelomocytes play a role in defence mechanisms activated by adverse external conditions, challenging whole P. lividus sea urchin to low and high temperature stress. For our purpose, adult individuals were kept at 16 °C, the control temperature (CT), with subsequent 35 °C heat stress (HS) or 4 °C cold stress (CS) for 4 h. After a 1-h recovery at 16 °C, coelomocytes were gently collected at 4 °C to prevent handling stress as much as possible, lysed, and analyzed by Western blotting. The anti-hsp70 McAb used for immunodetection, previously shown to cross-react with other invertebrate hsp70s (Koziol et al. 1997), recognized both the constitutive and the inducible forms. We found an overexpression of the hsp70 protein in coelomocytes obtained from temperature-stressed urchins, with a two- and a five-fold increase in the protein levels for heat and cold stress respectively (Matranga et al. 2000). The inductive response was time-dependent, i.e. individuals kept for 60 min at the non permissive temperature had twice the hsp70 levels as those exposed to the stress for 30 min. No increases in hsp70 expression were found for periods between 1 and 4 h, and periods longer than 4 h under temperature stress led to death of the animals (Matranga et al. 2000).

The presence and function of acetylcholinesterase (AChE) in vertebrate blood cells and plasma have been elucidated, suggesting its pivotal role in ion exchange (for a review of AchE activity in sea urchin embryos see Angelini et al., this Vol.); in addition, its activity has been found to be associated with the perinuclear region of leucocytes (Falugi 1985). Bearing in mind the similarity between coelomocytes and vertebrate blood cells, we investigated the presence of AChE activity in coelomocytes exposed to cold stress, with the aim of assessing the possibility of using the AChE activity present in these cells as a biomarker for both field and laboratory studies on environmental stress. Our results showed that cholinesterase activity increased with the time of exposure to cold stress and it was detected in petaloid phagocytes and colourless amoebocytes (Angelini et al. 2003). In both cases, the function might be linked to cytoskeletal dynamics, which follows changes in intracellular ion concentrations. This has been observed in sea urchin zygotes, as a result of the action of molecules related to the cholinergic system (Harrison et al. 2002).

In our experiments on whole sea urchins, we found a discrete variability in the basal levels of hsp70 as well as a different inductive response when comparing coelomocytes that were obtained from different individuals. This can be explained by the fact that different sea urchins possess a different personal history, thus reflecting different physiological conditions. We cannot in fact avoid the possibility that individuals utilized for the experiment were already under stress pressure caused, for example, by pathogens or other foreign agents (drugs, pollutants). We now know that pollution can be one of such factors increasing the hsp70 expression (Matranga et al. 2000; see following section).

It was then important to repeat these experiments using the same cell population taken from one individual and confirm the results obtained with whole adults as object of study. Then, using in vitro short-term cultures (1-4 h) of sea urchin coelomocytes, an identical number of cells, having an identical cell type composition, could be exposed to the same stress conditions, giving us also the possibility of monitoring their morphology. When their response to temperature, acidic pH, and heavy metals was tested, using again the hsp70 protein as a stress marker, results recapitulated those obtained with whole sea urchins (Matranga et al. 2002). This was an important achievement since, for the first time, coelomocyte cultures were used to "sense" heavy metal pollution, induced in our case experimentally by the addition of cadmium chloride to the medium. Moreover, since a dose-dependent increase in the hsp70 levels was shown, this could have been conveniently used for studies applied to environmental monitoring.

However, a concern for the applicability of the measurements was given by the fact that in order to elicit a response in a short period of time (4 h), high concentrations of cadmium were used, in the micromolar range. We know that such high cadmium levels are never reached in the marine environment, even in highly polluted areas. Then it was important to keep cells in culture for longer periods of time and to test lower cadmium concentrations, in the nanomolar range. This was possible thanks to the definition of a coelomocyte culture medium, LMCC medium, by which cells could be easily maintained for 3-8 days in culture (see previous section). The hsp70 expression was then monitored in total coelomocytes continuously exposed to CdCl2 for 3 days, after plating the cells for 5 days in LMCC medium in 24-well plates. Under these conditions cells were responsive to the heavy metal and showed an increase in hsp70 levels. However, higher expression levels were observed at the lowest CdCl2 doses, probably due to damage of cells exposed to the heavy metal for long periods of time. Figure 6 shows the results of a representative experiment.

It is well known that UV radiation causes damage to DNA and/or protein in a variety of organisms (for more information see Schroder et al., this Vol.). Many invertebrates, including sea urchins, spawn and/or develop in the 1-m zone of the sea and thus are susceptible to genetic damage. The effects on sea urchin embryo development have already been recently described by a few authors (Epel et al. 1999; Lesser et al. 2003; Bonaventura et al. 2005).

To test whether sea urchin coelomocytes respond to UV-B radiation (312 nm) (1,000 J/m2) alone, or in combination with different concentrations of CdCl2, by activating the expression of hsp70, in vitro cultures have been used as follows. Sea urchins were bled through a cut in the peristomal mem-

Fig. 6. Densitometric analysis of Western blot with anti-hsp70 antibody of lysates from coelomocytes exposed for 3 days in culture to different CdCl2 concentrations
Fig. 7. Western blot analysis of coelomocytes exposed to different CdCl2 concentrations for 2 h,with (+) or without (-) UV-B irradiation (1,000 J/m2), probed with anti-hsp70 antibody. Histogram shows the densitometric scanning of the filter

brane. The fluid was poured onto ISO-EDTA and the cell suspension was divided into a certain number of Petri dishes. Cultures of coelomocytes were kept under different stress (cadmium) conditions for 2 h; then the cells were or were not exposed to UV-B, collected, centrifuged and the pellet lysed as previously described (Matranga et al. 2002). Equal amounts of protein were loaded onto SDS-PAGE and analysed by Western blotting for the expression of hsp70 using a commercially available antiserum (Fig. 7). A two-fold increase in the expression of hsp70 is found in UV-B-stressed coelomocytes as compared to the control. Similarly, a three-fold increase in the hsp70 level is observed in coelomocytes exposed to 10-4M CdCl2.When coelomocytes were exposed to both 10-4M CdCl2 and UV-B radiation, we did not observe an addi tive effect, the increase in hsp70 level being about 2.5-fold. We observed the same trend for coelomocytes exposed to both 10-5 M CdCl2 and UV-B. This apparent paradox could be explained by the experimental conditions used: i.e., cells were first exposed to cadmium for 2 h and then UV-B treated. We can hypothesize that, in agreement with what is known about the protective effect on apoptosis produced by hsp70 expression (Samali and Cotter 1996), under these conditions the first stress is somehow protecting cells from the second one (UV-B).

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