In Vitro Bioavailability of the Hydrocarbon Fractions of Dimethyl Sulfoxide Extracts of Petroleum Substances

Abstract

Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

Grouping of UVCB substances with new approach methodologies (NAMs) data

“…It is acknowledged that using a DMSO extract of the substance means these biological activity data are not representative of the full substance. Even though DMSO selectively extracts lipophilic constituents including PACs from test substances, the chemical profiles of PACs across molecular classes remain consistent after extraction ( Luo et al, 2020 ). Modelling results from in vivo testing of a range of petroleum substances indicated that the higher-tier toxicological profile of high-boiling petroleum substances is related to the types and levels of PAC ( Nicolich et al, 2013 ).…”

“…Finally, studies on the experimental approaches that may aid in integration of biokinetic information in the grouping of complex substances are needed. For example, a previous study of bioavailability of the hydrocarbon fractions showed that extraction procedure, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the bioavailable concentrations of bioactive constituents of petroleum substances ( Luo et al, 2020 ).…”

Risk Characterization and Probabilistic Concentration–Response Modeling of Complex Environmental Mixtures Using New Approach Methodologies (NAMs) Data from Organotypic in Vitro Human Stem Cell Assays

“…Another issue, which may partially explain the apparent “synergy” in the mixture, has to do with bioavailability, because it is possible that there is a greater freely available fraction of each chemical in a mixture in comparison with single chemical experiments. This phenomenon has been recently demonstrated for complex mixtures and petroleum substances ( Luo et al. 2020 ); it is thought to be due to saturation of binding sites in the presence of multiple compounds.…”

Challenges in the regulatory use of water-accommodated fractions for assessing complex substances

“…This is because when the solubility of different constituents varies the composition of the test medium will depend on the method of preparation. This is for the reasons described above and has more recently been indirectly demonstrated in in vitro systems in which DMSO extraction of petroleum substances was shown to select for certain polycyclic aromatic compounds, with different dilutions leading to different chemical profiles and bioavailabilities [24]. Chemical analysis of WAF solutions during toxicity testing can therefore mechanistically aid interpretation of the data.…”