The role of polycyclic aromatic hydrocarbons in developmental toxicity of petroleum substances

REACH requires prenatal developmental toxicity (PDT) testing for substances registered in the EU at a volume of ≥100 tonnes/year. One of the consequences is that many petroleum substances (PS) will need to be tested for their potential adverse effect on prenatal development according to the current OECD 414 testing guidelines. This will involve a huge number of experimental animals and a considerable amount of resources. Therefore, the application of in vitro alternative testing strategies may reduce the animal experimentation and resources needed to study PDT potencies of PS. Furthermore, since some PS with high concentrations of polycyclic aromatic hydrocarbons (PAH) may induce PDT whilst their gas-to-liquid (GTL) analogues, which are synthetic products completely devoid of aromatics, do not induce PDT, it was hypothesized that PDT observed for some PS is caused by certain types of PAH in these products. This hypothesis was tested in the present thesis using a battery of in vitro alternative assays.

Chapter 1 provided background information and presented the aim of the thesis. In addition, the selected test substances and in vitro alternative assays used in the present thesis were also introduced. In total, 19 samples derived from 6 PS and 2 GTL product categories were tested. These samples were selected because i) they represent a series with a systematic variation in PAH content, being substances containing a range of 3- to 7-ring PAHs including extremes regarding their PAH content (with and without PAHs) and ii) in vivo PDT data for these product categories were available, enabling  in vitro-in vivo comparisons. The selected in vitro alternative assays were presented, including the embryonic stem cell test (EST), the zebrafish embryotoxicity test (ZET), and a panel of CALUX reporter gene assays. Finally, the general outline of the thesis was also provided.

Chapter 2 assessed the applicability of the EST to evaluate in vitro embryotoxic potencies of the DMSO extracts of 9 PS (varying in their PAH content, from 5 PS categories) and 2 GTL products (containing no PAHs) as compared to their in vivo potencies. All DMSO-extracts of PS induced a concentration-dependent inhibition of ES-D3 cell differentiation into beating cardiomyocytes at non-cytotoxic concentrations, and their potency was proportional to their 3- to 7-ring PAH content. In contrast, both GTL extracts, which are completely devoid of PAHs, tested negative in the EST. When the EST results were compared to in vivo PDT data of the corresponding PS, a good correlation was found between in vitro and in vivo results (R2: 0.97). Overall, the EST showed able to evaluate the in vitro embryotoxicity of PS, within and across categories, a result for the in vitro assay that was in line with the in vivo PDT data. The results also supported the hypothesis that PAHs are the primary inducers of the PDT resulting from PS exposure.

In Chapter 3, the role of endocrine- and dioxin-like activity in the developmental toxicity of PS extracts was investigated using a panel of Chemical Activated LUciferase gene eXpression (CALUX) assays. The same set of samples as in Chapter 2 was tested in the panel of CALUX assays that included agonist and antagonist assays for the androgen, estrogen-α, progesterone, and thyroid-β receptor, and also for the aryl hydrocarbon receptor (AhR). All DMSO-extracts of the PS showed strong AhR agonist activity and weak antiprogesterone, antiandrogen, and estrogenic activities. Only minor effects were seen for thyroid-related and antiestrogenic activity with some products. PS that are grouped in the same class induced similar luciferase expression profiles, suggesting a class specific signature of effects. None of the GTL products showed a meaningful interaction with the selected receptors, thus testing negative in all CALUX assays applied. The AhR-mediated activity of the PS correlated best (R2: 0.80) with the in vitro PDT potency of the corresponding PS as quantified previously in the EST, suggesting an important role of the AhR in mediating this effect. In conclusion, a high potential for endocrine and dioxin-like activity of some PS extracts was elucidated, which correlated with their in vitro PDT, and was driven by the type and level of PAHs present in the PS extracts.   The prominent AhR-mediated activity as induced by the PS extracts tested could be one of the underlying mechanisms of PDT by these substances.  

Chapter 4 investigated the usefulness of both the EST and the AhR CALUX assay to evaluate the in vitro PDT potency of an additional series of DMSO-extracts of HFOs, heavy PS containing mainly 3- to 7-ring PAHs, and one HRBO, a highly refined mineral oil that contains no aromatics and no PAHs. All DMSO-extracts of HFOs, but not of the HRBO, resulted in inhibition of ES-D3 cell differentiation in the EST and induced AhR-mediated activity in the AhR CALUX assay, and these potencies were was shown to be proportional to the amount of 3- to 7-ring PAHs they contain. Co-exposure of ES-D3 cells (EST) or H4IIE.luc cells (AhR CALUX assay) with the selected DMSO-extracts of PS and the AhR antagonist trimethoxyflavone (TMF), successfully counteracted the PS-induced inhibition of ES-D3 cell differentiation into cardiomyocytes as well as the AhR-mediated induction of gene expression by these substances. Moreover, also for this series of PS a good concordance was obtained when comparing the EST results with available in vivo PDT data. Altogether, the resulting data corroborate the hypothesis that PS-induced PDT is induced mainly by their 3- to 7-ring PAH content and that the observed PDT is partially mediated via the AhR.

In Chapter 5, the applicability of the ZET to evaluate developmental toxicity potency of the same set of samples as tested in Chapter 2 and 3 (DMSO-extracts of 9 PS and 2 GTL products) was investigated. All PS extracts, varying in PAH level and content, were able to inhibit the development of zebrafish embryos in a concentration-dependent manner and this potency could be associated with the amount of 3-5 ring PAHs they contain. On the contrary, DMSO-extracts of both GTL products, with no aromatics, showed no effect at all in the ZET. The potencies obtained in the ZET moderately correlated with those previously reported for the EST (R2: 0.61) and the AhR CALUX assay (R2: 0.66), while the correlation with potencies reported in in vivo studies were higher for the EST (R2: 0.85) than the ZET (R2: 0.69). Combining the results obtained from the EST (Chapter 2), AhR CALUX assay (Chapter 3), and ZET (Chapter 5) ranked and clustered the test substances in line with their in vivo potencies and chemical characteristics. It was concluded that the ZET did not outperform the EST as a stand-alone assay for testing PDT of PS but confirms the hypothesis that PAHs are the major inducers of PDT by some PS, and that the ZET is a useful addition to a battery of in vitro tests able to predict the in vivo PDT of PS.

In Chapter 6 we combined an exogenous biotransformation system, using hamster liver microsomes, with the EST to compare the in vitro PDT potency with and without bioactivation of two model 5-ring PAHs, benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and of PAH containing PS and GTL base oil (GTLb) extracts. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This indicates that BaP needs metabolic activation to exert its in vitro embryotoxic effect. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for PS to exert their in vitro PDT effects. GTL extracts tested negative in the EST, with and without bioactivation. Altogether, although some PAH constituents require metabolic activation to be able to induce PDT, some do not and this latter also appeared to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.

Chapter 7, first presented an overview of the results and main findings, which was combined with a general discussion of the data obtained and with future perspectives for follow-up studies to be performed in the near future. It was concluded that PAHs present in PS are the major inducers of PDT caused by these substances and that this was successfully and adequately assessed using several in vitro alternative assays, including the EST, ZET, and AhR CALUX assay. The results obtained in Chapter 2, 4, and 5 of the thesis were used in a QSAR (quantitative structure activity relationship) approach to predict the in vivo PDT of  a series of PS based on their PAH content. More PS extracts, ideally from different PS categories than those tested in the present thesis, should be tested to broaden the applicability domain of the proposed assay battery and the related QSAR approach for PDT testing of PS UVCBs in the future.