RESUMO
In vincristine (VCR) chemotherapy the dose-limiting factor is neurotoxicity, a side-effect which probably is caused by the action of the drug on axonal microtubules. VCR transforms them into paracrystalline structures. ORG 2766, an ACTH(4-9)/MSH(4-9), analogue, might have protective properties against this toxicity, because of its potency to induce the formation of microtubules. Therefore, the effects were studied of co-treatment with VCR and ORG 2766 on paracrystals and on microtubules in axons of the cerebral commissure (CC) of the snail Lymnaea stagnalis. In vitro experiments were carried out, incubating cerebral ganglia including the CC, for various periods of time (5-25 h) in Ringer's solution, in ORG 2766 (10(-6) M), in VCR (10(-4) M), or in VCR+ORG 2766. Furthermore, the effects of VCR (5 h) were studied in CC, pre- and/or post-treated with ORG 2766 (10 h). In another experiment VCR was used at a concentration at which no paracrystals were formed (2x10(-6) M). In this experiment VCR-treatment (5, 15 h) was compared to pre treatment and pre- and post-treatment with ORG 2766 (10 h). The numbers of microtubules (per mu m(2) axoplasm) and paracrystals (number encountered along perpendicular axes of the CC) were counted and the size of the paracrystals was measured in cross-sections of the CC, using electron microscopy. Irrespective of the type of additional treatment, significant differences were not observed in the numbers of paracrystals (32+/-10) after VCR-treatment for 5 h. Between 5 and 10 h of incubation the number increased (to 55+/-21) and remained approximately at this level. The number of microtubules decreased during the first 5 h of VCR treatment from 128+/-16 (control) to 37+/-10, and decreased further 19+/-5 between 5 and 10 h of incubation. The size of the paracrystals of all groups treated for 5 h with either VCR or VCR+ORG 2766 were similar (x=1.7 mu m(2)) However, the paracrystals were significantly larger in all groups co-treated for longer periods (10, 15 or 20 h) with ORG 2766 (VCR: x=2.3-3.0 mu m(2); VCR+ORG 2766: x=3.0-3.4 mu m(2)). This indicates that after an initial period of formation of paracrystals, tubulin is added to the existing paracrystals. At the low VCR concentration studied no paracrystals were formed. However, the number of microtubules had decreased significantly (from 159+/-10 to 84+/-17 after 15 h treatment). Pre- as well as pre- and post-treatment with ORG 2766 resulted in microtubule numbers which were not different from control values, indicating the positive effect of ORG 2766. In the groups pre- and/or post-treated with the high concentration of ORG 2766 (especially in the pre-treated group), the remaining number of microtubules was higher than in those co-treated with VCR and ORG 2766, but lower than in the controls. Although translation of the present results into clinical terms is difficult, they suggest that pre-and/or post-treatment with ORG 2766 may have a benificial effect on VCR-induced neurotoxicity. This conclusion is based on the observation that when ORG 2766 is given before VCR administration or after VCR clearance, larger numbers of microtubules were found in the nervous tissue than after treatment with VCR alone.
RESUMO
Modulating effects of WR2721 were studied on cisplatin-induced histological and ultrastructural changes in the ganglia of the central nervous system of the pond snail Lymnaea stagnalis. The relevance of the study was indicated by showing histochemically that alkaline phosphatase, the enzyme converting WR2721 into the active drug WR1065, is present in the snail brain. Central nervous systems of the snail were either preincubated in 5 mM WR2721 or in snail Ringer for 1 h and then postincubated for 10 or 20 h in: (i) Ringer (control), (ii) WR2721 (5 mM), (iii) cisplatin (0.4 mM), or (iv) cisplatin (0.4 mM) plus WR2721 (5 mM). The following parameters were studied: (i) general morphology, (ii) chromatin (number and mean clump size per unit surface ama, clump size frequency distribution), (iii) nucleoli (ratio of granular/fibrillar areas, (iv), Golgi zones (mean surface area, area containing electron dense material), (v) secretory granules (number), (vi) lysosomes (number per unit surface area of cytoplasm). The focus was on two types of identified neuroendocrine cells. Incubation in Ringer alone (controls) caused slight inactivation of the cells between 10 and 20 h of incubation (e.g. relative decrease of the granular part of the nucleoli). WR2721 alone had comparable effects on the tissue. In addition, in this group, a reduction in the electron dense material of the Golgi zones was observed. No major differences in number of secretory granules were observed in any of the groups. Treatment with cisplatin alone for 20 h caused a disappearance of the orange colour of the ganglia, swelling of axons and distension of intercellular spaces. Such changes were not observed in the group treated with cisplatin plus WR2721 or any of the other experimental groups. Both cisplatin alone and cisplatin plus WR2721 caused an increase in the number of chromatin clumps and a decrease in the mean chromatin clump size after 10 and 20 h of incubation. With regard to these parameters no differences were observed between the two groups. The chromatin clump size distribution curves of both groups were significantly different from the curve of the controls. Compared to that of the cisplatin group, the curve of the cisplatin plus WR2721 group, especially after 10 h, had shifted in the direction of that of the controls. Treatment with cisplatin alone induced drastic changes in nucleolar morphology. After 20 h of incubation the nucleoli had transformed into homogeneous dense structures. After 10 h of incubation in cisplatin plus WR2721 nucleoli still had a normal appearance. After 20 h those of the co-treated group had also become electron dense and appeared to contain numerous dark dots. Cisplatin caused a significant decrease in the extent of the Golgi zones. Co-treatment with WR2721 prevented this decrease to a certain degree. In both groups the electron dense material had disappeared from the Golgi zones. Furthermore, cisplatin alone induced an increase in the number of lysosomes. This increase was slightly (but not significantly) prevented by co-treatment with WR2721. The observations on the snail neurons show that WR2721 at the concentration studied induced only minor morphological changes. The drug modulates to some extent the pathological changes caused by cisplatin. The results support clinical data indicating that cisplatin-induced neurotoxicity is reduced by WR2721.