Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.

Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (> 97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a "spindle cell," consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

Detection of proliferating cell nuclear antigen in tissues of three small fish species.

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.

Immunohistochemical detection of P-glycoprotein in teleost tissues using mammalian polyclonal and monoclonal antibodies.

Mammalian P-glycoprotein is a highly conserved 170-kD integral plasma membrane protein functioning as an energy-dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. In this study, the tissue specificities of one polyclonal (pAb) and three monoclonal (mAbs) antibodies to mammalian P-glycoprotein were identified in paraffin-embedded, parasagittal whole-body sections of the guppy Poecilia reticulata. Polyclonal antibody mdr(Ab-1) and mAbs C219, C494, and JSB-1 demonstrated differential staining patterns in the following tissues: bile canaliculi in the liver, exocrine pancreas, lumenal surface of the intestinal epithelium, renal tubules, interrenal tissue, branchial blood vessels, gas gland, pseudobranch, and the gill transverse septa. Positive P-glycoprotein expression in P. reticulata correlates well with published results for homologous mammalian tissues of secretory and excretory function. These data indicate that one or more highly conserved members of the P-glycoprotein transporter family exist in a teleost species and can be detected using commercially available mammalian antibodies.

Early life-stage effects in medaka (Oryzias latipes) following in ovo exposure to polyamine biosynthetic inhibitors.

Medaka, Oryzias latipes, were exposed in ovo to the polyamine (PA) biosynthesis inhibitors alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG). In an additional group, spermine, the end product of the PA pathway, was added with DFMO and MGBG for a "rescue" treatment. At 4 days posthatch, length, DNA and RNA content, and swimming endurance were measured. The only parameter affected by treatment was swimming endurance which revealed decreased latent time to fatigue with increased dose, although not statistically significant. The rescue group, however, did demonstrate a statistically significant decrease in fatigue latency as compared to controls.