Moles of a Substance per Cell Is a Highly Informative Dosing Metric in Cell Culture

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.

Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy

“…1,4-benzoquinone, oligomycin A, and H 2 O 2 ) in cell culture studies as moles of xenobiotic per cell, rather than initial concentration in the medium, yields more consistent results and reduces ambiguity across different physical experimental set-ups [48]. Oxidation of P-AscH − in both in vitro ( i.e.…”

“…Utilizing the quantitative dosing metric (mol cell −1 ) that we previously established for direct-acting compounds that form covalent and tight-binding complexes with their target molecule, we were able to compare the absolute dose that was lethal to 50% of cells (ED 50 ) across five different pancreatic cancer cell lines, without ambiguity resulting from the physical conditions at which the experiments were carried out (Fig. 4) [48]. We observed the k cell for removal of H 2 O 2 across the pancreatic cancer cell lines directly correlated with their sensitivity to P-AscH − (as measured by the ED 50 ) (Fig.…”

Pharmacologic ascorbate (P-AscH−) suppresses hypoxia-inducible Factor-1α (HIF-1α) in pancreatic adenocarcinoma

“…Note that figures display P-AscH − dose in both molarity and on a mole cell − 1 basis. Recent studies have demonstrated that specifying the dose of xenobiotics, including P-AscH − , on a “mole per cell” basis results in improved correlations with toxicity markers as well as greater information content in experimental data [28, 29]. The doubling time of MIA PaCa-2 cells treated with P-AscH − was 59 ± 5 h versus 43 ± 0.2 h (means ± SEM, n = 3, p < 0.05) for controls when treated with the ED 50 dose, 7 mM (7 pmol cell − 1 ) (Fig.…”

Peroxiporin Expression Is an Important Factor for Cancer Cell Susceptibility to Therapeutic H2O2: Implications for Pharmacological Ascorbate Therapy

“…H 2 O 2 exposures of (0 nmol cell -1 –0.30 nmol cell -1 ; representative of 0, 50, 60, 70, 80 and 90 μM) [40] were diluted in the appropriate culture media and cells were exposed for 1 h at 37°C. After exposure, the diluted media was removed, cells were trypsinized and counted with a Moxi Z Mini Automated Cell Counter (ORFLO Technologies, Ketchum, ID, USA) and re-plated at 300 cells mL -1 in triplicates with appropriate media in 6-well culture (Corning, Union City, CA, USA) treated dishes.…”

“…Data were analyzed and plotted using Excel-2007 (Microsoft; Redmond, WA), and SigmaPlot (Systat Software Inc; San Jose, CA, USA) software. Plots of H 2 O 2 exposure doses are represented in the nmol cell -1 instead of concentrations because it serves as a more informative dosing metric for cell culture, as often times variations seen in experimental results arise as these systems are cell density dependent [40]. …”