Establishment Of Three Fish Cell Lines And Research On The Sensitivity Of Twelve Fish Cell Lines To Heavy Metal Toxicity

Aquatic environmental pollution is one of the major issues of global concern. A large number of chemical pollutants enter the aquatic environment each year from various sources. These pollutants, which include some heavy metals, are known to be potentially cytotoxic to biota and present a health threat to the public. If the prevalence of toxicants is not discovered and their toxicity monitored timely to forecast the potential threat, these pollutants may be at the bottom of severe ecological crisis. Thus, establishment of a sensitive biological monitoring system for early detection and ecotoxicological evaluation is required. Applying cell cultures as in vitro biological model to ecotoxicological assessment offers a number of well-described advantages as compared to in vivo animal tests. Culturing established fish cells in vitro is relatively rapid, cost-effective, readily reproducible, and can be easily adapted to automated high-throughput screening technologies. Establishment of more and more cell cultures from marine and mammalian species has promoted the rapid development of cell cultures as sensitive acute bioassays for the assessment of toxicological risks associated with chemical pollutants worldwide. In the near future, it is anticipated that the utilization of fish cell lines as a biological model for evaluating the cytotoxicity of pollutant chemicals in environmental samples will become a standard practice. To facilitate the use of established in vitro fish cell lines as valuable biological tools to monitor and detect environmental pollutant chemicals, it is necessary to test more cell lines comparatively to identify the more sensitive cell cultures as bio-indicators to common toxic chemicals.In this paper, the sensitivity and applicability of twelve fish cell lines to detect the toxicity of the four common heavy metals cadmium (Cd), chromium (Cr), zinc (Zn) and copper (Cu), which prevail in aquatic environment of China, were investigated. The toxicity and sensitivity of each heavy metal to the different passages, species and tissues of early passaging and permanent fish cell lines were compared to ascertain the relatively sensitive fish cell line against each heavy metal. Then the heavy metal induced cytotoxicity, oxidative damage and genotoxicity on the relatively sensitive fish cell line against each heavy metal were investigated to discuss the foreground of sensitive and applicable in vitro models to investigate the toxicity of heavy metals. The main results are as follows: 1 Establishment of three new fish cell linesThe Chinese sucker muscle cell line (CSM), Chinese sucker snout cell line (CSSN) derived from Chinese sucker (Myxocyprinus asiaticu) and the rare minnow fin cell line (RMF) derived from rare minnow (Gobiocypris rarus) were established and their biological characterizations including the optimal growth conditions (temperature, the content of serum and media), the ability of cryopreservation resuscitation, chromosome and genes from ribosome (12s rRNA/18s rRNA) and cytochrome b were identified. The results are as follows: three newly established cell lines were all fibrillose, homogeneous cells; the new cell lines had the ability of continuum passage in L-15 (CSM in M199 medium) with 10% FBS at 25℃; the new cell lines had the strong ability of cryopreservation resuscitation at the passage 5^th, 10^th, 15^th the viability and the adherent ratio after cryopreservation resuscitation were > 90% and 50% - 80%, respectively; chromosome analysis indicated these cell lines shared a major chromosomal peak of 100 (tetraploid number for CSM and CSSN) and 50 (diploid number for RMF), only small proportion of heteroploidy and aneuploidy; mitochondrial gene analysis indicated the identified sequences from the cells DNA were identical to the respective sequences reported from their fish species. Therefore, the three newly established fish cell lines had the ability of continuum in vitro and no mutation, they could be to detect the toxicity of environmental pollutions as early cell cultures.2 Comparison of the toxical sensitivity to heavy metal among twelve fish cell lines(1) Firstly, the cytotoxicity of cadmium chloride (CdCl₂·2.5H₂O), potassium dichromate (K₂C₂O₇), zinc chloride (ZnCl₂) and copper sulfate (CuSO₄·5H₂O) to grass carp (Ctenopharyngodon idellus) kidney cell line (CIK) was as Cd > Cr > Cu > Zn determined by MTT assay (IC50) and CB assay (IC50). Despite some minor variations, the IC50 - values obtained from MTT assay and CB assay appeared very similar, and no significant difference between these two methods. The genotoxicity of these four heavy metals were also in the aforementioned order determined by SCGE assay (the ratio of DNA damage) and FCM assay (the ratio of cell apoptosis). Comparatively, the MTT assay was relatively rapid, cost-effective, readily reproducible, and could be easily applied to investigate the relatively sensitive fish cell line of each heavy metal.(2) Secondly, to establish the potential use of cell cultures as a simple and sensitive biological tool to detect toxicity of heavy metals, the different passages, species, and tissues of three newly established cell lines and nine permanent cell lines established from different fish species and tissues were tested and compared for their cytotoxic sensitivity by MTT assay. The results indicated that the cytotoxic sensitivity decreased along with the increase of cell passages, but no significant difference amongst 20 passages; the cytotoxic sensitivity were different among the different tissues from the same species; the cytotoxic sensitivity were also different among the different species from the same tissue origin, and the early passaging cell lines were not always more sensitive than the permanent cell lines invariably; CCO cells derived ovary of channel catfish (Ictalurus punctatus) were the most sensitive one to CdCl₂·2.5H₂O, EPC cells derived from epithelioma of common carp (Ctenopharyngodon cyprini) were more sensitive than other cells to K₂Cr₂O₇ and ZnCl₂, while CIK cells derived from kidney of grass carp were the most sensitive cell line to CuSO₄ˉ5H₂O.3 The application of toxically sensitive fish cell line in detecting heavy metal toxicityThe cellular damage including the configuration under inverted microscope, cytotoxicity by MTT and CB assays, oxidative damage by determination the content of MDA and the activities of GPX, SOD and T-AOC, the DNA damage by SCGE, the cell apoptosis and cell cycle by FCM assay of heavy metals on their each toxical sensitive cells were investigated. After the treatment of heavy metals, signicant increases of round, collapsed and desquamated cells, significant decreases in survival, significant decrease in activities of GPX, SOD, T-AOC, an increase in the content of MDA, increases the ratio of DNA damage and apoptosis and the change of cell cycle were observed in a dose-dependent manner. Shortly, it was feasible to confirm the relative sensitive fish cell line to each heavy metal for early detection and ecotoxicological evaluation because of the different sensitivity level of different fish cell lines to the same heavy metal.