Characterization Of Three Marine Fish Cell Lines FG,SPH And RSBF And Their Application To Toxicity Assay Of Aquatic Pollutants

Cell culture of fish has provided us an important tool to study virology, tumorigenesis, cytotoxicity, genetics and immunology, etc. Firstly, morphology, lactate dehydrogenase (LDH) pattern and random amplified polymorphic DNA (RAPD) fingerprinting of three continuous marine fish cell lines of FG, SPH and RSBF, derived from flounder (Paralichthys olivaceus) gill, sea perch (Lateolabraxjaponicus) heart and red sea bream (Pagrosomus major) fin respectively, were examined and compared with their corresponding tissues of origin. The results obtained indicated that (1) LDH isozyme patterns of these three fish cell lines were markedly different from each other, and could serve as a genetic marker for species identification and detection of cross contamination; (2) In comparison with the corresponding tissues of origin, there was no change in the LDH pattern for cell line FG, but the LDH patterns for SPH and RSBF changed; (3) Morphologically, to their primary cultures, little change was found between the cell line FG and its primary cultures, but the cells of SPH and RSBF differed from those of their primary cultures; (4) Sixty arbitrary primers were tested on these three cell lines and on samples collected from individual fish of origin. The results obtained showed that, these three cell lines could be correlated to their correspondent species on the basis of identical patterns produced by 3 5-48% of the primers tested, but 27-32% of the primers tested produced different RAPD band patterns between cell lines and individuals of their original fish, indicating the existence of genetic variation of these cell lines in relation to the species of their origin; (5) The total mean similarity indices for cell lines vs. correspondent species of individual fish ranged from 0.825 to 0.851, which stand for their homology and could serve as referable data for species identification of other cell lines by RAPD assay; (6) Four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines, detection of cross contamination and characterization of the genetic variation of these cell lines in relation to the species of their origin. Secondly, FG cells after 170 passages had undergone spontaneous neoplastic transformation on the basis of obtaining the ability of focusing and piling up on monolayers. In this study properties of morphology, anchorage-independence (colony formation in semisolid agar) and stability of genomic DNA (RAPD assay) of pre-neoplastic and neoplastic FG cells were further investigated. The results obtained showed that: Accompanied with the loss of contact inhibition and density inhibition, and gain of the ability of focusing and piling up on monolayer, FG cells obtained the ability of growing in semisolid agar (8-14 colonies/106 seeded cells); Comparison of the RAPD fingerprintings of pre-neoplastic and neoplastic FG cells confirmed the existence of genetic variation during the neoplastic transformation. Therefore, in vitro spontaneous neoplastic transformation of the fish cells, like that of mammalian cells, involves a chain of progressive cellular and genetic changes, which eventually lead to the alteration of genomic DNA. In the end, the cytotoxic and genotoxic effects of polyethylenimine (PEI) and nickel chloride (NiCl2) in RSBF cells were examined. The results obtained indicated that: Fish c...