The study of the functional features of the P-gp endemic Baikal amphipods using transfected cell lines
February 2020 – December 2021
Massive pollution of freshwater resources remains one of the most urgent problems in a modern world. The monitoring of anthropogenic pressure on reservoirs and maintenance of appropriate drinking water quality demands constant improvements of control methods. Today, in a situation of growing economic development of Baikal region, the creation of fast and effective methods of environmental control is a matter of extreme concern. Furthermore, against the backdrop of climate change, toxic contaminations may cause a serious damage to the welfare of Baikal hydrobionts.
The research was conducted under the leadership of Julia Lubyaga, the winner of the «Baikal Initiative» grant competition for young scientists. Toxicological tests were performed on the amphipods’ cell lines, which would let subsequently apply these technologies in the rapid assessment of toxicity of potentially dangerous substances, that get into the lake’s waters, forecast consequences of anthropogenic impact on Baikal and other water reservoirs.
Goal:
- to study the functional and structural features of the p-glycoprotein of Baikal endemic amphipods using the transfected cell lines.
Targets:
- to provide grant assistance to Irkutsk State University (ISU) for implementing the corresponding project;
- as a part of assistance to conduct the following works in February-December in 2020:
1) to carry out a study on Drosophila melanogaster cell lines;
2) to conduct a detailed analysis of the P-gp sequence of endemic amphipods with further identification of its structure and building a three-dimensional protein model using modern software;
3) to compare comparative the sequences of the P-gp amphipod gene and protein structure with representatives of other species;
4) to identify the localization of the protein in various amphipod tissues using the electrophoresis method followed by western blot analysis;
5) to detect the functional features of amphipods by measuring the absorption of the specific fluorescent substrate in the presence of several chemicals;
6) to determine the IC50 values for a few toxic substances, which are potentially dangerous for Baikal organisms, and to identify the range of permissible concentrations and the degree of toxicity substances in cytotoxicity.
Results:
- The presence and performance of the introduced P-glycoprotein of Baikal aquatic organisms in the cell line was confirmed.
- The topology of the protein was determined, as well as its structure inherent in ABC transporters.
- The excretory activity of P-glycoprotein was analyzed using transfected cell lines and its efficiency was confirmed.
Thanks to the results and conclusions of the study, the understanding of the work of P-glycoprotein, which is an important component of the mechanism of multiple resistance to various toxic xenobiotic substances in Baikal endemic amphipods, is improved. The resulting stable cell lines can be used for further work to identify the features of the functioning of the mechanism of multiple resistance to xenobiotics in other Baikal endemics. Also, these results and proven methods can form the basis of an effective test system using a transfected cell line for ecological monitoring of Lake Baikal and other water bodies.