Measurement of the extracellular release of adenosine triphosphate in cultured primary rat hepatocytes

Commonly used acronym: ATP measurement

Scope of the method

The Method relates to
  • Animal health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
rat
Type of cells/tissues/organs
primary hepatocytes

Description

Method keywords
  • ATP
  • cytotoxicity
  • bioluminescent determination
  • extracellular ATP
Scientific area keywords
  • Hepatotoxicity
  • liver
  • cholestasis
  • Steatosis
Method description

ATP transports chemical energy within cells by serving as a substrate for kinases and as such fulfills a vital function in numerous cellular processes such as cell injury and subsequent cell death. ATP is therefore a crucial player in these events that are results of intracellular stress. Hepatotoxic chemical compounds can cause intracellular stress. The general cytotoxicity of compounds can be estimated through the biolouminescent assessment of extracellular release of ATP. As such, this procedure relies on two reactions in which firefly luciferase catalyzes the oxidation of luciferin to oxyluciferin and whereby ATP is consumed and light becomes emitted. The latter can be measured and is proportional to the amount of ATP present outside cells.

Lab equipment
  • Multiplate reader (Victor, 1420 Multilabel counter, PerkinElmer, Belgium)
Method status
  • History of use

Pros, cons & Future potential

Advantages
  • Easy to apply method to quantitatively characterize extracellular ATP release and hence cell injury in primary hepatocyte cultures.
Challenges

Increased extracellular levels of ATP do not specifically indicate cell death by either apoptosis or necrosis. Ideally this method should be combined with established tests, such as the monitoring of cell proliferation potential and mitochondrial function, which can be done by measurement of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) during DNA syntehsis and by adressing an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively.

References, associated documents and other information

References

Eguchi Y., Shimizu S. and Tsujimoto Y. (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Research 57:1835-1840

Elaut G., Henkens T., Papeleu P., Snykers S., Vinken M., Vanhaecke T. and Rogiers V. (2006) Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Current Drug Metabolism 7: 629-660

Golstein P. and Kroemer G. (2007) Cell death by necrosis: towards a molecular definition. Trends in Biochemical Sciences 32: 37-43

Gómez-Lechón M.J., Lahoz A., Gombau L., Castell J.V. and Donato M.T. (2010) In vitro evaluation of potential hepatotoxicity induced by drugs. Current Pharmaceutical Design 16: 1963-1977

Jaeschke H., Gores G.J., Cederbaum A.I., Hinson J.A. Pessayre D. and Lemasters J.J. (2002) Mechanisms of hepatotoxicity. Toxicological Sciences 65: 166-176. McKim J.M. (2010) Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance. Combinatorial Chemistry and High Throughput Screening 13: 188-206

Papeleu P., Vanhaecke T., Henkens T., Elaut G., Vinken M., Snykers S. and Rogiers V. (2006) Isolation of rat hepatocytes. Methods in Molecular Biology 320: 229-237

Seglen P.O. (1976) Preparation of isolated rat liver cells. Methods in Cell Biology 13: 29-83

Tsujimoto Y. (1997) Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes. Cell Death and Differentiation 4: 429-434

Van Calsteren V. (2010) Analyse van de herhaalde toedieningstoxiciteitstesten met cosmetische ingrediënten bestudeerd op Europees niveau. Dissertation Master in Pharmaceutical Sciences - Vrije Universiteit Brussel, Rogiers V. (promotor) and Pauwels M. (co-promotor)

Vinken M., Decrock E., De Vuyst E., Leybaert L., Vanhaecke T. and Rogiers V. (2009) Biochemical characterisation of an in vitro model of hepatocellular apoptotic cell death. Alternatives to Laboratory Animals 37: 209-218

Associated documents

Contact person

Axelle Cooreman

Organisations

Vrije Universiteit Brussel
Pharmaceutical and Pharmacological Sciences (FARM)
In Vitro Toxicology and Dermato-cosmetology
Belgium
Brussels Region