CELL CULTURE

CATEGORIZATION OF PETROLEUM SUBSTANCES THROUGH HIGH-CONTENT SCREENING OF

INDUCED PLURIPOTENT STEM CELL (iPSC) DERIVED CARDIOMYOCYTES AND HEPATOCYTES

Grimm FA1, Iwata Y1, Sirenko O2, Crittenden C2, Roy T3, Boogaard P4, Ketelslegers H5, Rohde A6, and Rusyn I1

1Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA; 2Molecular Devices LLC, Sunnyvale, CA, USA; 3University of South Carolina, Beaufort, SC, USA; 4SHELL International BV, The Hague, NL; 5EXXON MOBIL Petroleum and Chemicals, Machelen, BE; and 6Concawe, Brussels, BE

PETROLEUM SUBSTANCE EXTRACT PREPARATION

While hazard assessment of data-limited chemicals by chemical structure-based category read across is

sensible for chemically-characterized compounds, it cannot be used to assess complex chemicals, such as

petroleum substances. Therefore, we hypothesized that a biological data-based read across, i.e. safety

evaluation centered on categorizing substances according to similarities in their biological response,

may represent a feasible alternative. To test this, we applied high-content, multi-parametric toxicity screening

of induced pluripotent stem cell-derived (iPSC) cardiomyocytes1,2 and hepatocytes3 that were exposed to

petroleum substances from six distinct product categories in a concentration- and time-response design. Cell-

specific effects were observed and used as high-dimensional “biological” data inputs for evaluation of the

similarities and differences within and across different substance categories in ToxPi Graphical User Interface.

INTRODUCTION

CONCLUSIONS

RESULTS

GOALS OF THE STUDY

Abstract no: 1867

1Sirenko et al. (2013) Toxicol Appl Pharmacol. 273: 500-507 2Sirenko et al. (2013) J Biomol Screen. 18: 39-53 3Sirenko et al. (2015) Assay Drug Dev Technol. 12: 43-52 4Michelmann et al. (2014) J Am Soc Mass Spectrom. 26: 14-24 5Robin et al. (2011) BMC Bioinformatics. 12: 77 6US EPA (2011) Benchmark Dose Technical Guidance 7Reif et al. (2013) Bioinformatics. 29: 402-403

MATERIALS & METHODS

Petroleum substances from six distinct categories (SRGO - Straight Run Gas Oils,

OGO - Other Gas Oils, VHGO - Vacuum & Hydrotreated Gas Oils, Bitumens,

RAE - Residual Aromatic Extracts, and HFO - Heavy Fuel Oils) have been

obtained through a collaboration with Concawe (Brussels, Belgium). Product

samples (5 ml) were extracted into 20 ml DMSO and the DMSO-soluble fraction

was subsequently concentrated by solvent evaporation. Prior to treatment of iPSC

cardiomyocytes and hepatocytes, the concentrated extracts were re-solubilized to

a final volume of either 4 ml or 6 ml in DMSO. Serial dilutions were prepared.

iCell Cardiomyocytes and iCell Hepatocytes 2.0 were purchased from Cellular

Dynamics International (CDI). Cells were plated and maintained in 384-well plates

according to the manufacturers protocols. Plating densities were approximately 5000

cells/well for cardiomyocytes and 25000 cells/well for hepatocytes. Cardiomyocytes

exhibited strong, synchronous contractions, as was routinely confirmed by light

microscopy prior to experimentation.

IN VITRO CARDIOTOXICITY ASSAY

Various effects on cardiophysiology were assessed by monitoring the intracellular

Ca2+-flux of synchronously contracting iPSC cardiomyocytes using the FLIPR Tetra

system (Molecular Devices LLC). Briefly, cells were loaded with EarlyTox™

Cardiotoxicity reagent and incubated for two hours. Following an initial pre-

treatment reading, cells were exposed to extracts of petroleum substances over

five concentration logs and reference chemicals: tetraoctylammonium bromide

(cytotoxic agent), cisapride (K+ channel blocker), isoproterenol (pos. chronotrope),

and propranolol (neg. chronotrope). Readings were acquired at a frequency of

0.125 s-1 for 100 s and recorded at 530 nm following excitation at 475 nm.

HIGH-CONTENT IMAGING / CYTOTOXICITY SCREENING

Nuclei Mitochondria Cytoskeleton Cell Viability

Cytotoxicity screening of iPSC cardiomyocytes and hepatocytes after 24

(cardiomyocytes) or 72 (hepatocytes) hours of treatment with petroleum substance

extracts over five concentrations (10x dilution) was performed using the

ImageXPress Micro XL high-content imaging system (Molecular Devices). Cell

nuclei, viability, mitochondria, and cytoskeletal integrity were assessed following

staining with fluorescent probes Hoechst 33342, Calcein AM, MitoTracker Orange,

and AF488-conjugated phalloidin for 30 minutes prior to image acquisition.

Quantitative analysis of imaging data was performed using the multi-wavelength cell

scoring and granularity application modules in MetaXPress (Molecular Devices).

Hoechst 33258 MitoTracker Phalloidin Calcein AM

DATA PROCESSING & EVALUATION DATA ACQUISITION: HIGH-CONTENT IN VITRO SCREENING

PETROLEUM SUBSTANCE CATEGORIZATION BASED ON THE BIOLOGICAL PROFILES USING ToxPi SOFTWARE

Concentration-dependent effects on cardiomyocyte contractions

Comparison of Ca2+-flux measurements of iPSC cardiomyocytes treated with extracts of

petroleum substances and control chemicals tetraoctyl ammonium bromide (TAB),

cisapride, isoproterenol, and propranolol revealed that petroleum substances were

capable of inducing chronotropic effects (e.g., CON16ii), or of inhibiting cardiomyocyte

contractions (e.g., CON-06). The vehicle control (1% DMSO) did not result in any

alterations of the beating pattern.

High-content imaging of iPSC cardiomyocytes exposed to varying concentrations

of DMSO extracts of petroleum substances. Cell viability was assessed by live cell

staining with Hoechst 33258 (nuclei, blue), Mitotracker Orange (mitochondria, red), and

Calcein AM (cell viability marker, green). Cells were also exposed to the vehicle control

(1% DMSO) and tetraoctyl ammonium bromide (TAB), a cytotoxic agent.

High-content imaging of

cytoskeletal integrity of iCell

hepatocytes exposed to

varying concentrations of

DMSO extracts of petroleum

substances. In addition to live

cell staining as described above,

cell viability of hepatocytes was

assessed by fixed cell staining

with a fluorescent actin probe.

At cytotoxic concentrations,

petroleum substances caused

widespread loss of cytoskeletal

integrity.

Hepatocytes Cardiomyocytes

Substance categorization was performed using the ToxPi approach.7 ToxPi data were integrated to provide a ToxPi score, i.e. a relative score equivalent to the relative toxicity for

each substance. The higher the ToxPi score, the higher the relative toxicity of the substance. Figures for both iPSC cardiomyocytes and hepatocytes indicate strong correlations

between ToxPi scores and substances categories. In most cases, the are considerable similarities between individual phenotypic responses of substances within a certain category.

Moreover, there were also similarities between different substance groups, particularly Straight Run Gas Oils (SRGO) and Vacuum & Hydrotreated Gas Oils (VHGO).

CHARACTERIZATION OF SUBSTANCE COMPOSITION BY 2D-MS

Compositional similarities between Straight Run Gas Oils and Vacuum &

Hydrotreated Gas Oils. Selected Accumulation Ion Mobility Spectrometry coupled to FT-

ICR4 was applied to characterize compositional similarities and/ or differences between

petrochemical extracts. Two-dimensional plots indicate compound distribution according to

m/z ratio and mobility. Samples include three SRGOs (CONCAWE-01, CONCAWE-02,

CONCAWE-03) and three VHGOs (CONCAWE-12, CONCAWE-14, CONCAWE-15) and

demonstrate compositional similarities between these two substance types. Extracts from

other substance categories will be evaluated in future experiments.

Data derived from high-throughput in vitro screenings were initially normalized to negative/

vehicle controls (1 % DMSO in media). Data subsequently underwent statistical evaluation

using a custom script in “R” software to determine benchmark values Point-of-Departure

(POD), EC10, EC50, and EC90.5,6 To be consistent in the interpretation of the results, data in

this presentation were all evaluated using the POD at 1 standard deviation from baseline

level. POD values were then adjusted for differences in original sample concentration, and

normalized to respective minimum and maximum values (excluding “nontoxic” substances)

and visualized in ToxPi.7 Thus, the larger the area of a ToxPi slice (on a 0-1 scale), the

higher the toxicity of a substance on the respective phenotype.

REFERENCES

Mass Spectrometry data were generated in collaboration with the Laboratory

of Biological Mass Spectrometry at Texas A&M (Dr. William Russell).

High-content imaging data on iCell hepatocytes was acquired with assistance

from Dr. Michael Bittner’s laboratory at Texas A&M.

ACKNOWLEDGEMENTS

1. To address the challenge of safety assessment of UVCB (Unknown or Variable composition, Complex reaction products and Biological materials) materials by using a “biological” category read-across and a case study of petroleum substances;

2. To collect toxicological data on in vitro effects of petroleum substances using quantitative high-content screening of iPSC-derived cardiomyocytes and hepatocytes;

3. To use toxicological data as an integrative “biological” high-dimensional matrix for category read-across that can be visualized using Toxicological Priority Index (ToxPi) approach

SAIMS-FT-ICR MASS SPECTROMETRY

Chemical characterization of petroleum substances was achieved by Selected

Accumulation Ion Mobility Spectrometry (SAIMS) coupled to Fourier Transform

Ion Cyclotron Resonance (FT-ICR).4 Prior to sample analysis, DMSO extracts were

diluted 1000-fold in methanol. Both ion mobility chromatograms and mass spectra

were recorded on a Bruker 9.4 T Solari X FT-ICR mass spectrometer. Mass analysis

was preformed at mass resolutions of ~100,000.

2D Plot Ion Mobility Mass Distribution

1. In vitro toxicity testing of petroleum substances, a prototypical example of UVCB, demonstrates appreciable similarities in potential hazard properties of the individual products both within the same category and between related categories;

2. Quantitative high-content imaging using diverse cell-based models provides “biological” means for exercising the similarity principle through category read-across;

3. Effective communication of the complex multi-dimensional datasets comprising of various information streams (e.g., physico-chemical properties, manufacturing process details, toxicity profiling) can be achieved using ToxPi-enabled data integration;

4. Extensions of this approach to additional cell-based model systems representing various tissues, coupled with high-throughput gene expression profiling, will further increase confidence in the “biological” based read-across of UVCB.

This work was performed with support from EPA STAR grant #RD83516601 and

institutional support from Texas A&M University. Fabian Grimm is a recipient of SOT

Colgate-Palmolive postdoctoral fellowship. Concawe provided petroleum substance

extracts used in these studies. ExxonMobil and Shell provided no funding for this work.

FUNDING