Abstract
There is increased interest in using high throughput in vitro assays to characterize human population variability in response to toxicants and drugs. Utilizing primary human endothelial colony-forming cells (ECFCs) isolated from blood would be highly useful for this purpose because these cells are involved in neonatal and adult vasculogenesis. We characterized the cytotoxicity of four known toxic chemicals (NaAsO2, CdCl2, tributyltin [TBT], and menadione) and their four relatively nontoxic counterparts (Na2HAsO4, ZnCl2, SnCl2, and phytonadione, respectively) in eight ECFC clones representing four neonatal donors (2 male and 2 female donors, 2 clones per donor). ECFCs were exposed to 9 concentrations of each chemical in duplicate; cell viability was evaluated 48 h later using the fluorescent vital dye fluorescent dye 5-Carboxyfluorescein Diacetate (CFDA), yielding concentration-effect curves from each experiment. Technical (day-to-day) variability of the assay, assessed from three independent experiments, was low: p-values for the differences of results were 0.74 and 0.64 for the comparison of day 2 vs. day 1 and day 3 vs. day 1, respectively. The statistical analysis used to compare the entire concentration-effect curves has revealed significant differences in levels of cytotoxicity induced by the toxic and relatively nontoxic chemical counterparts, demonstrating that donor-specific ECFCs can clearly differentiate between these two groups of chemicals. Partitioning of the total variance in the nested design assessed the contributions of between-clone and between-donor variability for different levels of cytotoxicity. Individual ECFC clones demonstrated highly reproducible responses to the chemicals. The most toxic chemical was TBT, followed by NaAsO2, CdCl2, and Menadione. Nontoxic counterparts exhibited low cytotoxicity at the higher end of concentration ranges tested. Low variability was observed between ECFC clones obtained from the same donor or different donors for CdCl2, NaAsO2, and TBT, but for menadione, the between-donor variability was much greater than the between-clone variability. The low between-clone variability indicates that an ECFC clone may represent an individual donor in cell-based assays, although this finding must be confirmed using a larger number of donors. Such confirmation would demonstrate that an in vitro ECFC-based testing platform can be used to characterize the inter-individual variability of neonatal ECFCs exposed to drugs and/or environmental toxicants.
Highlights
The response of individual humans to hazardous exposures can vary significantly and this variability is thought to underlie individual predispositions to to diseases and/or sensitivity to toxic exposures [1]
Red blood cells were removed by sedimentation with dextran and subsequent centrifugation typically yielding less than 2 ml of plasma containing white blood cells and platelets
Previously we isolated CB Endothelial colony-forming cells (ECFCs) from the mononuclear cell fraction [21], we found that it does not result in a higher ECFC yield compared to unfractionated white blood cells in small size blood samples
Summary
The response of individual humans to hazardous exposures can vary significantly and this variability is thought to underlie individual predispositions to to diseases and/or sensitivity to toxic exposures [1]. In 2012, Lock et al [8] reported on the differential sensitivity of 81 human lymphoblastoid cell lines from 27 Center d’Etude du Polymorphisme Humain trios exposed to 240 chemicals using cytotoxicity and apoptosis as endpoints in a quantitative HT screening platform These investigators concluded that an evaluation of toxicity pathways and the effects of genetic diversity was feasible. The difference in donor-specific cellular responses measured as an EC10 (effective concentration by which control culture cell viability was reduced by 10%) for about half of the tested compounds was found to vary between 10- and 1,000-fold [9] These data were used to develop prediction models for human population responses to toxic chemicals [10], further indicating the value of the approach
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