Prenatal developmental toxicity testing of petroleum substances: Application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo

Abstract

Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3-7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R=0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS.

The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon‐containing extracts of petroleum substances

“…The fact that microsomal bioactivation of PAH‐containing PS may not be a prerequisite for their PDT may be linked to the fact that the underlying modes of action may require non‐covalent receptor or enzymes interactions rather than the chemical (DNA) reactivity that is necessary to exert the mutagenic and carcinogenicity potency of these substances (Blackburn et al, ; Siddens et al, ). Corroborating this notion, our recent observation showed that the in vitro PDT of the PS extracts tested in the present study was at least in part mediated via the aryl hydrocarbon receptor (AhR) (Kamelia et al, ). It is generally accepted that three‐ to seven‐ring PAHs are able to activate the AhR (Pieterse et al, ; Puga, Tomlinson, & Xia, ), and the AhR‐mediated activity of PAH‐containing PS has been linked to the developmental toxicity potency of these groups of substances (Billiard et al, ; Goodale et al, ; Incardona, Linbo, & Scholz, ).…”

“…Recently we reported on the use of the EST to study PDT potency of DMSO extracts of PS and GTL products (Kamelia et al, , ). A highly significant correlation was found between the EST results and in vivo data ( R 2 = 0.97; Kamelia et al, ).…”

“…The ES-D3 cell viability and differentiation assays of the mouse EST were performed as described in previous published studies (Kamelia et al, 2019(Kamelia et al, , 2017).…”

“…In the EST, the ability of test substances to inhibit the differentiation process of ES‐D3 cells is used as the endpoint to study PDT potency of test substances in vitro (Genschow et al, , ; Seiler & Spielmann, ). Recent studies in our lab showed the ability of the ES‐D3 cell differentiation assay of the EST to assess the PDT potencies of dimethyl sulfoxide (DMSO) extracts of PS (within and across categories with variable polycyclic aromatic hydrocarbon [PAH] content) and GTL products (containing no PAHs) (Kamelia et al, ; Kamelia et al, ). PDT potencies of different PS extracts, as evaluated in the ES‐D3 cell differentiation assay of the EST, appeared to be in line with their potencies observed in vivo (Kamelia et al, , ).…”

“…Recent studies in our lab showed the ability of the ES‐D3 cell differentiation assay of the EST to assess the PDT potencies of dimethyl sulfoxide (DMSO) extracts of PS (within and across categories with variable polycyclic aromatic hydrocarbon [PAH] content) and GTL products (containing no PAHs) (Kamelia et al, ; Kamelia et al, ). PDT potencies of different PS extracts, as evaluated in the ES‐D3 cell differentiation assay of the EST, appeared to be in line with their potencies observed in vivo (Kamelia et al, , ). GTL extracts tested negative in the EST, while all PAH‐containing PS extracts inhibited the differentiation of ES‐D3 cells into beating cardiomyocytes in a concentration‐dependent manner at non‐cytotoxic concentrations with a potency that was proportional to their three‐ to seven‐ring PAH content (Kamelia et al, , ).…”