An assessment of physiological and biochemical characteristics of cells based on the respiratory activity under substrates and toxic substances impact

Abstract

BACKGROUND: Metabolic profiling of cells is an essential aspect of studies of intracellular metabolism. One of the parameters which can be used for this purpose is a rate of oxygen consumption during degradation or transformation of substrates by cells.

AIM: This study was aimed at the development of a method for assessment of cell physiology and biochemistry based on the metabolic profiling via registration of the cell respiratory activity, and use of this method for evaluation of inhibition and recovery of liver parenchyma cells affected by different compounds.

MATERIALS AND METODS: The method used a Clark-type oxygen electrode with the cells located at its working area, in order to measure dissolved oxygen concentration in the immediate surrounding of living intact cells thereby ensuring their metabolic profiling and physiological status assessment. The experiments were performed using Wistar rat liver parenchyma fragments. Three experimental series were performed: first (series A) was aimed at metabolic profiling of the liver parenchyma; the second (series B) determined the stability of parenchyma’s response to sodium citrate over time; and the third (series C) ensured real-time recording and assessment of the effect of hepatotoxicants on the liver tissue. Solutions of glucose, fructose, sucrose, sodium citrate, sodium pyruvate, L-glutamine, ethanol and hydroquinone were used as the substrates for liver tissue metabolic profiling, and solutions of methanol and clarithromycin as the toxic agents.

RESULTS: During the experiments, a metabolic profiling of tissue and real-time monitoring of toxicity effect on liver parenchyma cells have been demonstrated.

CONCLUSION: The developed method for assessing the physiological and biochemical characteristics of cells can be used to track the metabolic activity, inhibition and restoration of liver parenchyma cells when exposed to substrates and toxic compounds.

Keywords

cell biochemistry and physiology / metabolic profile / respiratory activity / toxicity

References

[1] Wang Z, Yip LY, Lee JHJ, et al. Methionine is a metabolic dependency of tumor-initiating cells. Nat Med. 2019;25(5):825–837. doi: 10.1038/s41591-019-0423-5

[2] Wang Z., Yip L.Y., Lee J.H.J., et al. Methionine is a metabolic dependency of tumor-initiating cells // Nat Med. 2019. Vol. 25, N 5. P. 825–837. doi: 10.1038/s41591-019-0423-5

[3] Fenaille F, Barbier Saint-Hilaire P, Rousseau K, et al. Data acquisition workflows in liquid chromatography coupled to high resolution mass spectrometry-based metabolomics: where do we stand? J Chromatogr A. 2017;1526:1–12. doi: 10.1016/j.chroma.2017.10.043

[4] Fenaille F., Barbier Saint-Hilaire P., Rousseau K., Junot C. Data acquisition workflows in liquid chromatography coupled to high resolution mass spectrometry-based metabolomics: where do we stand? // J Chromatogr A. 2017. Vol. 1526. P. 1–12. doi: 10.1016/j.chroma.2017.10.043

[5] Kumar M, Tanoj N, Saran S. A modified, efficient and sensitive pH indicator dye method for the screening of acid-producing acetobacter strains having potential application in bio-cellulose production. Appl Biochem Biotechnol. 2020;191(2):631–636. doi: 10.1007/s12010-019-03211-x

[6] Kumar M., Tanoj N., Saran S. A modified, efficient and sensitive pH indicator dye method for the screening of acid-producing acetobacter strains having potential application in bio-cellulose production // Appl Biochem Biotechnol. 2020. Vol. 191, N 2. P. 631–636. doi: 10.1007/s12010-019-03211-x

[7] Mihooliya KN, Nandal J, Swami L, et al. A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms. Enzyme Microb Technol. 2017;107:72–81. doi: 10.1016/j.enzmictec.2017.08.004

[8] Mihooliya K.N., Nandal J., Swami L., et al. A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms // Enzyme Microb Technol. 2017. Vol. 107. P. 72–81. doi: 10.1016/j.enzmictec.2017.08.004

[9] Schneider S, Ettenauer J, Pap IJ, et al. Main metabolites of pseudomonas aeruginosa: a study of electrochemical properties. Sensors (Basel). 2022;22(13):4694. doi: 10.3390/s22134694

[10] Schneider S., Ettenauer J., Pap I.J., et al. Main metabolites of pseudomonas aeruginosa: a study of electrochemical properties // Sensors (Basel). 2022. Vol. 22, N 13. P. 4694. doi: 10.3390/s22134694

[11] Yu Y, Wang Y, Li M. Reliable method for the detection of horseradish peroxidase activity and enzyme kinetics. Analyst. 2019; 144(4):1442–1447. doi: 10.1039/c8an02072h

[12] Yu Y., Wang Y., Li M. Reliable method for the detection of horseradish peroxidase activity and enzyme kinetics // Analyst. 2019. Vol. 144, N 4. P. 1442–1447. doi: 10.1039/c8an02072h

[13] Zhang J, Cui J, Liu Y, et al. A novel electrochemical method to determine α-amylase activity. Analyst. 2014;139(13):3429–3433. doi: 10.1039/c3an01839c

[14] Zhang J., Cui J., Liu Y., et al. A novel electrochemical method to determine α-amylase activity // Analyst. 2014. Vol. 139, N 13. P. 3429–3433. doi: 10.1039/c3an01839c

[15] Ziegler FD, Strickland EH, Anthony A. Oxidative phosphorylation and respiratory regulation in rat liver homogenates measured with the oxygen electrode. Rep US Army Med Res Lab. 1962;1–25.

[16] Ziegler F.D., Strickland E.H., Anthony A. Oxidative phosphorylation and respiratory regulation in rat liver homogenates measured with the oxygen electrode // Rep US Army Med Res Lab. 1962. P. 1–25.

[17] Voss DO, Cowles JC, Bacila M. A new oxygen electrode model for the polarographic assay of cellular and mitochondrial respiration. Anal Biochem. 1963;6:211–222. doi: 10.1016/0003-2697(63)90128-3

[18] Voss D.O., Cowles J.C., Bacila M. A new oxygen electrode model for the polarographic assay of cellular and mitochondrial respiration // Anal Biochem. 1963. Vol. 6. P. 211–222. doi: 10.1016/0003-2697(63)90128-3

[19] Holtzman D, Moore CL. A micro-method for the study of oxidative phosphorylation. Biochim Biophys Acta. 1971;234(1):1–8. doi: 10.1016/0005-2728(71)90122-8

[20] Holtzman D., Moore C.L. A micro-method for the study of oxidative phosphorylation // Biochim Biophys Acta. 1971. Vol. 234, N 1. P. 1–8. doi: 10.1016/0005-2728(71)90122-8

[21] Gaylor JL, Miyake Y, Yamano T. Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes. J Biol Chem. 1975;250(18):7159–7167.

[22] Gaylor J.L., Miyake Y., Yamano T. Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes // J Biol Chem. 1975. Vol. 250, N 18. P. 7159–7167.

[23] Pitts OM, Priest DG. A steady-state kinetic investigation of the uricase reaction mechanism. Arch Biochem Biophys. 1974;163(1):359–366. doi: 10.1016/0003-9861(74)90487-1

[24] Pitts O.M., Priest D.G. A steady-state kinetic investigation of the uricase reaction mechanism // Arch Biochem Biophys. 1974. Vol. 163, N 1. P. 359–366. doi: 10.1016/0003-9861(74)90487-1

[25] Weetman DF, Sweetman AJ. Realistic estimations of kinetic constants for the oxidation of naturally occurring monoamines by monoamine oxidase. Anal Biochem. 1971;41(2):517–521. doi: 10.1016/0003-2697(71)90174-6

[26] Weetman D.F., Sweetman A.J. Realistic estimations of kinetic constants for the oxidation of naturally occurring monoamines by monoamine oxidase // Anal Biochem. 1971. Vol. 41, N 2. P. 517–521. doi: 10.1016/0003-2697(71)90174-6

[27] Clark JB, Greenbaum AL, Slater TF. Effects of Tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria. Biochem J. 1965;94(3):651–654. doi: 10.1042/bj0940651

[28] Clark J.B., Greenbaum A.L., Slater T.F. Effects of tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria // Biochem J. 1965. Vol. 94, N 3. P. 651–654. doi: 10.1042/bj0940651

[29] Haugaard N, Lee NH, Kostrzewa R, et al. The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochrondia. Biochim Biophys Acta. 1969;172(2):198–204. doi: 10.1016/0005-2728(69)90063-2

[30] Haugaard N., Lee N.H., Kostrzewa R., et al. The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochrondia // Biochim Biophys Acta. 1969. Vol. 172, N 2. P. 198–204. doi: 10.1016/0005-2728(69)90063-2

[31] Tedjo W, Chen T. An Integrated biosensor system with a high-density microelectrode array for real-time electrochemical imaging. IEEE Trans Biomed Circuits Syst. 2020;14(1):20–35. doi: 10.1109/TBCAS.2019.2953579

[32] Tedjo W., Chen T. An integrated biosensor system with a high-density microelectrode array for real-time electrochemical imaging // IEEE Trans Biomed Circuits Syst. 2020. Vol. 14, N 1. P. 20–35. doi: 10.1109/TBCAS.2019.2953579

[33] Rajendran ST, Huszno K, Debowski G, et al. Tissue-based biosensor for monitoring the antioxidant effect of orally administered drugs in the intestine. Bioelectrochemistry. 2021;138:107720. doi: 10.1016/j.bioelechem.2020.107720

[34] Rajendran S.T., Huszno K., Debowski G., et al. Tissue-based biosensor for monitoring the antioxidant effect of orally administered drugs in the intestine // Bioelectrochemistry. 2021. Vol. 138. P. 107720. doi: 10.1016/j.bioelechem.2020.107720

[35] Cai Y, Wang M, Xiao X, et al. A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase. Biosens Bioelectron. 2022;207:114197. doi: 10.1016/j.bios.2022.114197

[36] Cai Y., Wang M., Xiao X., et al. A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase // Biosens Bioelectron. 2022. Vol. 207. P. 114197. doi: 10.1016/j.bios.2022.114197

[37] Emelyanova EV, Antipova TV. Biosensor approach for electrochemical quantitative assessment and qualitative characterization of the effect of fusaric acid on a culture-receptor. J Biotechnol. 2022;357:1–8. doi: 10.1016/j.jbiotec.2022.08.004

[38] Emelyanova E.V., Antipova T.V. Biosensor approach for electrochemical quantitative assessment and qualitative characterization of the effect of fusaric acid on a culture-receptor // J Biotechnol. 2022. Vol. 357. P. 1–8. doi: 10.1016/j.jbiotec.2022.08.004

[39] Hiramoto K, Yasumi M, Ushio H, et al. Development of oxygen consumption analysis with an on-chip electrochemical device and simulation. Anal Chem. 2017;89(19):10303–10310. doi: 10.1021/acs.analchem.7b02074

[40] Hiramoto K., Yasumi M., Ushio H., et al. Development of oxygen consumption analysis with an on-chip electrochemical device and simulation // Anal Chem. 2017. Vol. 89, N 19. P. 10303–10310. doi: 10.1021/acs.analchem.7b02074

[41] Rejmstad P, Johansson JD, Haj-Hosseini N, et al. A method for monitoring of oxygen saturation changes in brain tissue using diffuse reflectance spectroscopy. J Biophotonics. 2017;10(3):446–455. doi: 10.1002/jbio.201500334

[42] Rejmstad P., Johansson J.D., Haj-Hosseini N., et al. A method for monitoring of oxygen saturation changes in brain tissue using diffuse reflectance spectroscopy // J Biophotonics. 2017. Vol. 10, N 3. P. 446–455. doi: 10.1002/jbio.201500334

[43] Thews O, Vaupel P. Spatial oxygenation profiles in tumors during normo- and hyperbaric hyperoxia. Strahlenther Onkol. 2015;191(11):875–882. doi: 10.1007/s00066-015-0867-6

[44] Thews O., Vaupel P. Spatial oxygenation profiles in tumors during normo- and hyperbaric hyperoxia // Strahlenther Onkol. 2015. Vol. 191, N 11. P. 875–882. doi: 10.1007/s00066-015-0867-6

[45] Lau JC, Linsenmeier RA. Oxygen consumption and distribution in the Long-Evans rat retina. Exp Eye Res. 2012;102:50–58. doi: 10.1016/j.exer.2012.07.004

[46] Lau J.C., Linsenmeier R.A. Oxygen consumption and distribution in the Long-Evans rat retina // Exp Eye Res. 2012. Vol. 102. P. 50–58. doi: 10.1016/j.exer.2012.07.004

[47] Sakr Y. Techniques to assess tissue oxygenation in the clinical setting. Transfus Apher Sci. 2010;43(1):79–94. doi: 10.1016/j.transci.2010.05.012

[48] Sakr Y. Techniques to assess tissue oxygenation in the clinical setting // Transfus Apher Sci. 2010. Vol. 43, N 1. P. 79–94. doi: 10.1016/j.transci.2010.05.012

[49] Sekine K, Kagawa Y, Maeyama E, et al. Oxygen consumption of human heart cells in monolayer culture. Biochem Biophys Res Commun. 2014;452(3):834–839. doi: 10.1016/j.bbrc.2014.09.018

[50] Sekine K., Kagawa Y., Maeyama E., et al. Oxygen consumption of human heart cells in monolayer culture // Biochem Biophys Res Commun. 2014. Vol. 452, N 3. P. 834–839. doi: 10.1016/j.bbrc.2014.09.018

[51] McDonald JM, Ramsey JJ, Miner JL, et al. Differences in mitochondrial efficiency between lines of mice divergently selected for heat loss. J Anim Sci. 2009;87(10):3105–3113. doi: 10.2527/jas.2009-1935

[52] McDonald J.M., Ramsey J.J., Miner J.L., et al. Differences in mitochondrial efficiency between lines of mice divergently selected for heat loss // J Anim Sci. 2009. Vol. 87, N 10. P. 3105–3113. doi: 10.2527/jas.2009-1935

[53] Tanumihardja E, Slaats RH, van der Meer AD, et al. Measuring both pH and O2 with a single on-chip sensor in cultures of human pluripotent stem cell-derived cardiomyocytes to track induced changes in cellular metabolism. ACS Sens. 2021;6(1):267–274. doi: 10.1021/acssensors.0c02282

[54] Tanumihardja E., Slaats R.H., van der Meer A.D., et al. Measuring both pH and O2 with a single on-chip sensor in cultures of human pluripotent stem cell-derived cardiomyocytes to track induced changes in cellular metabolism // ACS Sens. 2021. Vol. 6, N 1. P. 267–274. doi: 10.1021/acssensors.0c02282

[55] Godsman N, Kohlhaas M, Nickel A, et al. Metabolic alterations in a rat model of Takotsubo syndrome. Cardiovasc Res. 2022;118(8):1932–1946. doi: 10.1093/cvr/cvab081

[56] Godsman N., Kohlhaas M., Nickel A., et al. Metabolic alterations in a rat model of Takotsubo syndrome // Cardiovasc Res. 2022. Vol. 118, N 8. P. 1932–1946. doi: 10.1093/cvr/cvab081

[57] Pandya JD, Sullivan PG, Leung LY, et al. Advanced and high-throughput method for mitochondrial bioenergetics evaluation in neurotrauma. Methods Mol Biol. 2016;1462:597–610. doi: 10.1007/978-1-4939-3816-2_32

[58] Pandya J.D., Sullivan P.G., Leung L.Y., et al. Advanced and high-throughput method for mitochondrial bioenergetics evaluation in neurotrauma // Methods Mol Biol. 2016. Vol. 1462. P. 597–610. doi: 10.1007/978-1-4939-3816-2_32

[59] Divakaruni AS, Rogers GW, Murphy AN. Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a clark-type oxygen electrode. Curr Protoc Toxicol. 2014;60:25.2.1–25.2.16. doi: 10.1002/0471140856.tx2502s60

[60] Divakaruni A.S., Rogers G.W., Murphy A.N. Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a clark-type oxygen electrode // Curr Protoc Toxicol. 2014. Vol. 60. P. 25.2.1–25.2.16. doi: 10.1002/0471140856.tx2502s60

[61] Vial G, Guigas B. Assessing mitochondrial bioenergetics by respirometry in cells or isolated organelles Methods Mol Biol. 2018;1732:273–287. doi: 10.1007/978-1-4939-7598-3_18

[62] Vial G., Guigas B. Assessing mitochondrial bioenergetics by respirometry in cells or isolated organelles // Methods Mol Biol. 2018. Vol. 1732. P. 273–287. doi: 10.1007/978-1-4939-7598-3_18

[63] Silva AM, Oliveira PJ. Evaluation of respiration with clark type electrode in isolated mitochondria and permeabilized animal cells. Methods Mol Biol. 2012;810:7–24. doi: 10.1007/978-1-61779-382-0_2

[64] Silva A.M., Oliveira P.J. Evaluation of respiration with clark type electrode in isolated mitochondria and permeabilized animal cells // Methods Mol Biol. 2012. Vol. 810. P. 7–24. doi: 10.1007/978-1-61779-382-0_2

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