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[MZZ41-02] Effect of diphacinone on immortalized cells from green sea turtle (Chelonia mydas)
Introduction
To accurately evaluate the effect of pollutants on wildlife, experiments should be performed on wild animals; however, it is difficult to investigate wildlife compared with experimental animals. In this study, we focused on cultured cells, which are an important bioresource for the investigation of wildlife because they can be obtained from dead wildlife. Hepatocytes are the first choice for the evaluation of the effects of pollutants on wildlife, but they are difficult to obtain. Dead wildlife-derived somatic cells are limited to fibroblasts derived from the skin or muscle since they are in better condition than other organ-derived cells. In this study, we used fibroblasts for the assessment of pollutants.
Several environmental pollutants have been reported in Japan, with rodenticides being one of the major environmental pollutants. Rodenticides are chemicals used to kill rodents; their effects on non-target species have not been substantially investigated. This study focused on green sea turtles (Chelonia mydas) because the Ogasawara Islands are famous as a green sea turtle habitat and have been extensively exposed to rodenticides used for killing rats. Therefore, the green sea turtle is listed as a high-risk non-target animal for rodenticides in the Ogasawara area. In this study, we established an immortalized cell line of green sea turtles and exposed these cells to rodenticides to compare the response with in vivo results.
Material and methods
In this study, we collected muscle samples of dead green sea turtles and established primary and immortalized fibroblast cell lines. To establish immortalized cells (K4DT cells), genes encoding CDK4R24C, cyclinD1, and TERT were transfected into primary cells. Moreover, we analyzed the growth of K4DT cells after sequential passage. We also performed karyotype analysis. We exposed green sea turtle and rat K4DT cells to diphacinone and analyzed the expression of vitamin K epoxide reductase (VKOR) mRNA to elucidate the effects of rodenticides.
Results and discussion
First, we optimized the culture conditions for green sea turtle primary fibroblast cell line. We observed that as a basal medium, RPMI 1640 was better than DMEM and DMEM/F12. Moreover, cell proliferation and live-cell ratio were higher at 30°C than at 33°C and 37°C.
Next, we established green sea turtle immortalized cells using genes encoding CDK4R24C, cyclinD1, and TERT. Green sea turtle K4DT cells could actively proliferate until more than the PD150 stage. In the late-passage stage, K4DT cells maintained a live-cell ratio of over 90%. Moreover, these cells did not stain for senescence-associated beta-galactosidase. Therefore, the green sea turtle K4DT cells escaped senescence. Moreover, karyotyping of the green sea turtle K4DT cells revealed normal karyotypes (2n=56); therefore, green sea turtle K4DT cells maintained the karyotype of primary cells unlike the cells obtained using other methods, such as SV40T.
Next, we evaluated the effects of diphacinone on green sea turtle K4DT cells. We compared the expression of VKOR mRNA in green sea turtle and rat K4DT cells, which revealed that VKOR mRNA expression decreased in rat K4DT cells, but it was maintained in green sea turtle K4DT cells. This result was in agreement with the coagulation time obtained after exposure to diphacinone in vivo. Moreover, we are conducting a more detailed transcriptome analysis to understand the effect of diphacinone green sea turtle K4DT cells and will publish the results in our next opportunity.
To accurately evaluate the effect of pollutants on wildlife, experiments should be performed on wild animals; however, it is difficult to investigate wildlife compared with experimental animals. In this study, we focused on cultured cells, which are an important bioresource for the investigation of wildlife because they can be obtained from dead wildlife. Hepatocytes are the first choice for the evaluation of the effects of pollutants on wildlife, but they are difficult to obtain. Dead wildlife-derived somatic cells are limited to fibroblasts derived from the skin or muscle since they are in better condition than other organ-derived cells. In this study, we used fibroblasts for the assessment of pollutants.
Several environmental pollutants have been reported in Japan, with rodenticides being one of the major environmental pollutants. Rodenticides are chemicals used to kill rodents; their effects on non-target species have not been substantially investigated. This study focused on green sea turtles (Chelonia mydas) because the Ogasawara Islands are famous as a green sea turtle habitat and have been extensively exposed to rodenticides used for killing rats. Therefore, the green sea turtle is listed as a high-risk non-target animal for rodenticides in the Ogasawara area. In this study, we established an immortalized cell line of green sea turtles and exposed these cells to rodenticides to compare the response with in vivo results.
Material and methods
In this study, we collected muscle samples of dead green sea turtles and established primary and immortalized fibroblast cell lines. To establish immortalized cells (K4DT cells), genes encoding CDK4R24C, cyclinD1, and TERT were transfected into primary cells. Moreover, we analyzed the growth of K4DT cells after sequential passage. We also performed karyotype analysis. We exposed green sea turtle and rat K4DT cells to diphacinone and analyzed the expression of vitamin K epoxide reductase (VKOR) mRNA to elucidate the effects of rodenticides.
Results and discussion
First, we optimized the culture conditions for green sea turtle primary fibroblast cell line. We observed that as a basal medium, RPMI 1640 was better than DMEM and DMEM/F12. Moreover, cell proliferation and live-cell ratio were higher at 30°C than at 33°C and 37°C.
Next, we established green sea turtle immortalized cells using genes encoding CDK4R24C, cyclinD1, and TERT. Green sea turtle K4DT cells could actively proliferate until more than the PD150 stage. In the late-passage stage, K4DT cells maintained a live-cell ratio of over 90%. Moreover, these cells did not stain for senescence-associated beta-galactosidase. Therefore, the green sea turtle K4DT cells escaped senescence. Moreover, karyotyping of the green sea turtle K4DT cells revealed normal karyotypes (2n=56); therefore, green sea turtle K4DT cells maintained the karyotype of primary cells unlike the cells obtained using other methods, such as SV40T.
Next, we evaluated the effects of diphacinone on green sea turtle K4DT cells. We compared the expression of VKOR mRNA in green sea turtle and rat K4DT cells, which revealed that VKOR mRNA expression decreased in rat K4DT cells, but it was maintained in green sea turtle K4DT cells. This result was in agreement with the coagulation time obtained after exposure to diphacinone in vivo. Moreover, we are conducting a more detailed transcriptome analysis to understand the effect of diphacinone green sea turtle K4DT cells and will publish the results in our next opportunity.