Histological Pattern

The effects observed at the level of general morphology can be correlated with relevant atypical features in terms of microscopic anatomy of both the stump and the regenerating arm. These alterations are the most sensitive index of ecologically relevant, individual-level responses such as growth and development (Hinton 1997). With all the test compounds employed so far, the histological sections, in spite of a general good preservation of tissue integrity in all the exposed samples,showed clear signs of anomalies,which were detectable since the early regenerative phase and became more and more evident at the more advanced regenerative stages (1 and 2 weeks post-amputation). It is significant that these histological alterations always involved the same target structures, but showed rather characteristic aspects that could be considered specific effects of the exposure to the individual pollutant (Candia Carnevali et al. 2001a,b,2003; Barbaglio et al. 2004).Apart from a number of other minor atypical features, the following anomalies appeared to be particularly relevant:

1. Development of an atypical blastema (pseudo-blastema), flattened and/or ectopic, often including foreign non-blastemal elements (myocytes and/or skeletal spicules) (in 4-NP- and TPT-exposed samples; Fig. 5a,b).

2. Unusual hypertrophy and swelling of the coelomic canals of both the stump and the regenerate (particularly in PCB- and 4-NP-exposed samples; Fig. 5d,e,f,i).

3. Pronounced abnormal development of the skeletal components in the regenerate (particularly in TPT-exposed samples; Fig. 5 c).

4. Enhanced and prolonged phenomena of cell migration (coelomocytes, amoebocytes, phagocytes and granulocytes) evident at the level of both coelomic canals and tissues (in all the exposed samples; Fig. 5b,i,k). In particular, this intense migratory activity tended to involve a large number of granulocytes: their diffuse presence in the tissues, even at the advanced regenerative stages, and the massive unusual occurrence of degranulation phenomena in the stump tissues, particularly close to the coelomic canals, were distinctive features of all the exposed samples. Under standard conditions, in fact, the granule cells are extensively but limitedly employed during the repair processes, their degranulation processes occurring specifically at the level of the amputation surface.

5. Massive recruitment of dedifferentiated or semi-dedifferentiated cells, particularly myocytes, that were frequently involved in migration phenomena, particularly in the coelomic canals close to the amputation area (in all the exposed samples, but mostly in 4-NP and TPT samples; Fig. 5b,i,k). The muscles of the stump contributed extensively to this cell recruitment/ migration through phenomena of tissue rearrangement and dedifferentiation during which the muscle fibres were massively replaced by other elements (coelomocytes and phagocytes; Fig. 5g,h,i,j). At TEM (Fig. 6a) many individual myocytes appeared to progressively dedifferentiate to acquire the features of undifferentiated coelomocytes actively involved in cell division (see below), whereas other myocytes were obviously involved in apop-tosis (Fig. 6b). These processes of muscular dedifferentiation and turnover were always accompanied by related phenomena of massive cell migration/proliferation in the adjacent coelomic canals (Fig. 6 c). This is strongly in contrast to what was observed under standard conditions, where regeneration is typically accomplished by the contribution of undifferentiated stem cells which are responsible for the blastema formation.

6. Enhanced or inhibited cell proliferation. The first effect was particularly evident in PCB-exposed samples, the second in 4-NP- and in TPT-exposed samples. As indicated by our BrdU incorporation experiments, although cell proliferation in both exposed and control samples was always localized in the usual specific cytopoietic sites, mainly at the level of the apical blastema and the coelomic epithelium of both the regenerate and the stump, there was a substantial difference in labelling intensity and distribution among the different samples: the labelling in fact was much stronger and more widely distributed in PCB samples (Fig. 6f,h), whereas it was

Fig. 5a-k. LM sagittal sections of control and exposed regenerating arms of Antedon mediterranea at different stages. Exposure concentrations: PCB: 14 ng/l; 4-NP: 2 |g/l; TPT-Cl: 100 or 225 ng/l. a-c Details of the blastema regions at 72 h pa (post-amputation): control (a) and TPT-Cl samples (b, c). In b, an atypical pseudo-blastema which mainly consists of ectopic migratory myocytes (arrows) is evident; c shows the unusual presence of well-

Fig. 5a-k. LM sagittal sections of control and exposed regenerating arms of Antedon mediterranea at different stages. Exposure concentrations: PCB: 14 ng/l; 4-NP: 2 |g/l; TPT-Cl: 100 or 225 ng/l. a-c Details of the blastema regions at 72 h pa (post-amputation): control (a) and TPT-Cl samples (b, c). In b, an atypical pseudo-blastema which mainly consists of ectopic migratory myocytes (arrows) is evident; c shows the unusual presence of well-

rather modest and less diffuse in 4-NP and TPT samples (Fig. 6g) with respect to the controls. Unexpectedly, in PCB-exposed specimens, the labelling involved to a relevant extent also the muscles of the stump (Fig. 6h). This seems to indicate that, at least in the PCB-exposed samples, the muscles can directly provide a significant contribution to regeneration also in terms of cell proliferation.

7. Recycling and turnover of the stump tissues, namely the endoskeleton and the connective tissue (mostly in PCB- and 4-NP-exposed samples; Fig. 6d). These phenomena were significantly different from those related to the muscles and involved extensive degenerative phenomena which led to vac-uolization/vesiculation of both the extracellular matrix and the cells (fibroblasts or scleroblasts respectively). The massive presence of phagocytes at advanced regenerative stages of 2 weeks indicates that these tissues are employed as a secondary indirect source of reserve materials for new synthesis.

8. Atypical ultrastructural features of specific cell types, including the blastemal cells (mostly in PCB- and 4-NP-exposed samples; Fig. 6e). In particular, a marked development of endoplasmic reticulum (both RER and SER) and Golgi complexes and an unusual abundance of lipid granules and empty vacuoles were typical features of the exposed samples and could be correlated with a cytological pattern of steroid dysfunction. Interestingly, in the exposed samples, besides these histological and cytological features, the integrity/preservation of other tissues of both the stump and regenerate (epithelia, nerve tissue, etc.) was rather good, even in the long-tem exposed samples of 2 weeks post-amputation, without any significant morphological variation with respect to the standard conditions seen in normal regeneration;

9. Possible induction of protective biochemical responses. Immunocyto-chemical results showed, in fact, that in PCB- and 4-NP-exposed samples there was an appreciable increase in the expression pattern of specific developed skeletal spicules in the blastema (arrows). a and b, bar 100 |m; c 20 |m. d-f Exposed regenerating samples of 1 week pa: d PCB; e TPT-Cl; f 4-NP.Abnormal growth ofthe regenerate is evident in PCB samples. In contrast, the regenerative blastema is heterogeneous and poorly developed in TPT-Cl and 4-NP specimens. In all the exposed samples the coelomic canals (cc) are hypertrophic. b Blastema; ne nerve. Bars 200 |m. g-i Exposed and control samples at 1 week pa: g PCB; h control; i 4-NP. Details of the muscle bundles of the stump. In contrast to the compact structure of the control muscle, the PCB- and 4-NP-exposed samples show extensive muscle rearrangement/dedifferentiation. cc Coelomic canal; mu muscle. Bars 30 |m. j, k Details of 4-NP-exposed samples of 1 week pa. j The muscle bundle consists of semi-dedifferentiated myocytes and undifferentiated coelomocytes (arrows); k the coelomic canals of the stump are involved in extensive cell proliferation/ migration (arrows).A number of myocytes are released through the coelomic wall into the lumen. Bars 20 |m

Fig. 6. TEM and ICC sections of control and exposed regenerating arms of Antedon mediterranea at different stages. Exposure concentrations: PCB: 14 ng/l; 4-NP: 2 |g/l; TPT-Cl: 100 or 225 ng/l. a-d TEM details of 1 and 2 weeks exposed samples. Bars 2 |m. PCB (a, e, f), 4-NP (b, c). a Detail of the muscle bundle of the stump. b Myocyte showing a typical apoptotic nucleus. c Migratory semi-dedifferentiated myocytes inside the coelomic canal. d Connective tissue of the stump showing extensive processes of cellular rearrangement/ vacuolization. e Blastemal cell showing unusual development of endoplasmic reticulum and Golgi complexes. Bar 1 |m. f-h ICC for BrdU. The number of BrdU-labelled cells is massive in the blastema of PCB samples (f) and rather limited in that of 4-NP samples (g). A strong and extensive labelling is also found in the coelomic epithelium (cc) of PCB samples (h). i ICC for cytochrome P450. The details show an intense immunoreaction in many cells (arrows) at the level of the tissues of the stump, particularly in connective and skeletal tissue. Bars: f 25 |m; g 30 |m; h 10 |m; i 30 |m enzymes such as the microsomal cytochrome P-450 mono-oxygenase system, i.e. the main enzymes responsible for biotransformation and metabolism of the majority of lipophilic xenobiotics (den Besten 1998). Interestingly, the reaction was intense and diffuse in the tissues of the stump more involved in turnover activity (Fig. 6i).

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