Abstract
BackgroundThe biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of experiments e.g. cell number and media volume. Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells. We hypothesize that for many xenobiotics, specifying dose as moles per cell will reduce this ambiguity. Dose as moles per cell can also provide additional information not easily obtainable with traditional dosing metrics.MethodsHere, 1,4-benzoquinone and oligomycin A are used as model compounds to investigate moles per cell as an informative dosing metric. Mechanistic insight into reactions with intracellular molecules, differences between sequential and bolus addition of xenobiotic and the influence of cell volume and protein content on toxicity are also investigated.ResultsWhen the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used (number of cells, volume of medium). When using moles per cell as a dose-metric, direct quantitative comparisons can be made between biochemical or biological endpoints and the dose of xenobiotic applied. For example, the toxicity of 1,4-benzoquinone correlated inversely with intracellular volume for all five cell lines exposed (C6, MDA-MB231, A549, MIA PaCa-2, and HepG2).ConclusionsMoles per cell is a useful and informative dosing metric in cell culture. This dosing metric is a scalable parameter that: can reduce ambiguity between experiments having different physical conditions; provides additional mechanistic information; allows direct comparison between different cells; affords a more uniform platform for experimental design; addresses the important issue of repeatability of experimental results, and could increase the translatability of information gained from in vitro experiments.
Highlights
In the testing of xenobiotics, medicines, and natural products for biochemical and biological responses, the use of laboratory animals is regarded as the best model for providing information to predict effects in humans
Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells
When the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used
Summary
In the testing of xenobiotics, medicines, and natural products for biochemical and biological responses, the use of laboratory animals is regarded as the best model for providing information to predict effects in humans. A critical element in the foundation of scientific research is reproducibility This problem encompasses a wide array of issues ranging from statistical considerations, to laboratory standards, practices, and reporting (Principles and Guidelines for Reporting Preclinical Research at http://www.nih.gov/about/reporting-preclinical-research.htm as accessed 2015.03.31) [1] and references therein. We examine the topic of how to specify dose or exposure to a xenobiotic in cell culture experiments with the goal to address an aspect of the problem of reproducibility in science. This matter may result in more successful translation of information from cell culture studies to whole organisms, thereby addressing the 3R’s, replacement, reduction and refinement, for the use of animals in research [2]. Dose as moles per cell can provide additional information not obtainable with traditional dosing metrics
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