Nonylphenol provokes a vesiculation of the Golgi apparatus in three fish epidermis cultures.

The aim of this work was to study the effects of nonylphenol and waste water on the cell ultrastructure of fish skin. Therefore, besides a recently established primary cell culture and a cell line, an epidermal tissue culture of fish was developed and tested. In all three systems a prominent vesiculation of the Golgi apparatus was observed after exposure to nonylphenol, which has not been described before and therefore strongly suggests an effect that might indicate exposure to nonylphenol and/or related substances. The Epithelial papulosum cyprini cell line was the most sensitive to nonylphenol, followed by the primary cell culture of epidermis cells and then the explant tissue culture. The vesiculation of the Golgi apparatus was accompanied by degenerative changes in the two cell cultures only. The lack of degenerative changes in the cells of the tissue culture was discussed with respect to the presence of differentiated cells that might better be able to protect themselves by mucous or by an activated xenobiotic metabolism. In a second type experiment, a waste water sample containing small concentrations of nonylphenol was applied to the cultures. It did not lead to a vesiculation of the Golgi apparatus, probably because the nonylphenol concentrations in the waste water were too low to induce the vesiculation. The cultures exposed to waste water revealed unspecific degenerative cellular changes. Additionally, explant cultures were prepared from fish that had survived a 6-month exposure to polluted river water. In these cultures a higher number of mitochondria containing myelin bodies were observed when compared to control cultures. Consequently, exposure to polluted water containing a mixture of substances in vitro and in vivo was found to lead to degenerative alterations in the ultrastructure of the cells.

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol.

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC50 (24 h) 47.1 µM NP (primary cell culture) and 44.2 µM NP (EPC)) values and apoptotic rate (significantly increased apoptotic rate after exposure to 50 µM NP for 24 h, p < 0.001 (primary cell culture), p = 0.008 (EPC)). The explant culture was slightly less sensitive (increased apoptotic rate after exposure to 50 µM NP for 24 h, but not significant: p = 0.385), which could be due to the capabilities of a differentiated tissue, providing more protective repair mechanisms, compared with single cells. All cultures revealed a concentration-response relationship for the endpoint apoptotic rate after the application of nonylphenol for 24 h. After wastewater exposure, a significant decrease in the apoptotic rate was measured in the primary cell culture (dilution wastewater : medium 1:1:p = 0.018; dilution wastewater : medium 1:2:p = 0.003), whereas the cell line EPC did not reveal any effects. Our results show that the endpoint apoptotic rate is more sensitive than the parameter cell viability for detecting adverse effects of a wastewater sample.

Primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum.

This is the first report on a primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum. These primary cells revealed a low seeding efficiency after 3 days (11.6 +/- 4.6%), whereas subcultured cells had a higher seeding efficiency at the same time point (75.5 +/- 34.0%) and increased in cell number (150-200% of seeded cells after 20 to 30 days). The cells were characterized applying histological, immunocytochemical and ultrastructural methods. The culture consisted of undifferentiated keratinocytes. Mucous cells as well as differentiated epithelial cells were absent. To date the cells were cultured for maximally 9 passages and 402 days and therefore provide the possibility for long-term studies.