Towards a realistic risk characterization of complex mixtures using in vitro bioassays

This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments. In Chapter 1 the importance of complex mixture characterization in modern society is introduced. The methods available, their current advantages and their disadvantages for complex mixture testing are described. With the shift from policy oriented chemical testing towards the inclusion of in vitro bioanalysis, important challenges have to be overcome to ensure a relevant and reliable quantification of the toxic potency of complex mixtures. These challenges are explained in the introduction, including the status of development and validation of those aspects for reliable testing. One of the main advantages that in vitro bioanalysis has to offer is the possibility to quantify the toxic potency of compounds for which chemical analytical methods have not or hardly been developed, for example because standards do not yet exist. Hydroxylated metabolites of POPs are an example of a toxicologically relevant group of compounds that can exert endocrine disrupting effects, but they cannot yet be routinely analysed. A selection of yet unsolved issues are further studied and discussed in this thesis, as outlined in the “approach and structure of the thesis”. In Chapter 2 a meta-analysis is performed to study the occurrence and relevance of hydroxylated (OH) compounds in humans and wildlife. Reported body burdens of halogenated phenolic contaminants (HPCs), including OH-POP in different tissues from humans and wildlife species, are reviewed in relation to the concentration of their putative parent compounds to be able to reveal relevant exposure routes and sub-populations at risk. Highest OH-POP levels were found in blood plasma, and highly perfused and fetal tissues. Plasma concentrations of analysed known HPCs ranged from 0.1-100 nM in humans and up to 240, 454, 800 and 7650 nM for birds, fish, cetaceans and other mammals, respectively. Reported metabolite blood plasma levels also are compared with relevant toxicological threshold concentrations from toxicological studies, and appeared to fully fall within the in vitro (0.05–10000 nM) and in vivo (3-940 nM) effect concentrations reported for OH-POPs. Given the sensitivity of early developmental stages, and information lacking about the general population, it is advisable to determine HPC background blood levels in children and fetal tissue . Given the toxicological relevance of the OH-POPs, Chapter 3 aims at providing solutions to the long standing problem of the in vitro production and analysis of OH-POP metabolite thyroid hormone disrupting (THD) potency via binding to plasma thyroid hormone binding proteins (THBPs). In sediments and for example seafood, the POPs occur as parent compounds that would only become THD after metabolisation (hydroxylation). Several methods have shown the competitive thyroxine (T4) T4 displacement potency of pure metabolites. However, in vitro metabolization of, among others, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers(PBDEs) followed by in vitro quantification of their potency has encountered drawbacks related to the co-extraction of compounds disturbing the T4-TTR competitive binding assay. The present study identifies and quantifies the major co-extractants, cholesterol and saturated and non-saturated fatty acids (SFA and NSFA), at levels above 20 μM (20 nmol per mg protein in the incubation mixture) following various extraction methods. A new method is presented to in vitro metabolise parent compounds into OH-metabolites followed by selective extraction of metabolites while four-fold reducing co-extraction of the disturbing compounds. In addition a microplate-format non-radioactive fluorescence displacement assay was developed to quantify the TTR binding potency of the metabolites formed. The effectiveness of the in vitro metabolism and extraction of the OH-metabolites of the model compounds CB 77 and BDE 47 was chemically quantified with a newly developed chromatographic method analyzing silylated derivatives of the OH-metabolites and co-extractants. Due to the mentioned improvements, it is now possible to make a dose-response curve up to 50% inhibition with OH-metabolites extracted from bioactivated CB 77 and BDE 47. Without taking the toxic potencies of bio-activated POPs into account with bioanalysis, the hazard and risk posed by POPs will be seriously underestimated. The chapters 4 and 5 are committed to tackle the issues of supramaximal (SPMX) responses and sample extract concentration which are crucial to reliably quantify of the toxic potencies of complex mixtures with in vitro bioassays. A SPMX effect is the phenomenon that compounds induce a maximum response in an assay that is significantly higher than that of the positive control. As the positive control is used to quantify the toxic potency of a sample, this could result in over-estimation of its toxic potency. As this has been most elaborately reported for in vitro estrogenicity assays, a meta-analysis was performed of such assays, compounds and conditions in which the effect is observed (Chapter 4a).For the 21 natural and industrial chemicals that could be identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Relevant information on assay characteristics, however, sometimes lacked. Diethylstilbestrol (DES), genistein (GEN) and bisphenol A (BPA) were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects, could be related to a number of specific assay characteristics: 1) the type and concentration of the serum used to supplement the exposure medium; 2) the endpoint used to quantify the estrogenic potency (endogenous or transfected reporter gene), 3) the number of EREs (estrogen responsive elements) used before the reporter gene, and 4) the nature of the promoter’s. There were no indications that solvent concentration in culture, exposure period or cell model influenced the occurrence of SPMX. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts. Several SPMX inducers also have been reported to block cellular efflux pumps in vivo and in vitro (Anselmo et al. 2012; Georgantzopoulou et al. 2013). Therefore it was hypothesized that efflux pump blockers present in environmental matrices could increase the internal concentration of bioassay agonists and thus cause the SPMX. In Chapter 4b this hypothesis was tested by adapting a 96-well plate cellular efflux pump inhibition assay (CEPIA) to the H4IIE rat hepatoma cell line used for the DR.Luc reporter gene assay for dioxin-like compounds. The influence of various environmentally relevant efflux pump inhibitors on the 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) response was tested. Under the DR.Luc assay conditions there was no evidence that P-gp efflux pump inhibitors modified or potentiated the activity of TCDD. Neither genistein nor quercetin, two potent SPMX inducers on ER-mediated assays, induced any signal on the DR.Luc assay, nor influenced the luciferase induction by TCDD. Future work should be focused on testing the consequences of efflux pump inhibition with an AhR-agonist which is a P-gp substrate, as this could result in intracellular accumulation of this AhR-agonist. It is standard practice to use a high single stock concentration of extracts to further dilute test concentrations from and perform the analysis. However, a high contaminant load in an extract may oversaturate the solubility of the extracted compounds in carrier solvents and overload the clean-up columns which may reduce the efficiency of polyaromatic hydrocarbons (PAHs) elimination from the extract. These problems may cause respectively under- or over-estimation of the quantified dioxin-like toxic potency. Therefore Chapter 5 focuses on the effects of initial stock concentrations, including sonication assisted dissolution and exposure time, on the quantified dioxin-like potency of cleaned nonpolar sediment extracts. Indeed, more than 20 g sediment equivalents (SEQ)/mL DMSO) as initial stock concentrations resulted in underestimation of bio-TEQ levels in the sediments as observed for cleaned nonpolar sediment extracts from various locations in Luxembourg. An overload of extract on clean-up columns caused an over-estimation of the dioxin-like potency at 24 hours of exposure, probably due to limited removal of PAHs that can induce false positive responses in the in vitro assays. Sonication assisted dissolution of the stock before serial dilution strongly reduced the standard variation of the outcomes. Taking into account the aspects revealed in this study, in addition to already described important issues for quality control, the in vitro bioassays based bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment. For the generally applied sediment quality criteria, advices are given about maximum initial stock concentrations to achieve reliable bioassay outcomes. The methods and concepts developed for metabolic activation of compounds in non-polar sediment extracts and in in vitro analysis of the TTR-competitive binding are applied in Chapter 6 to extracts from highly or less contaminated sediments collected in Luxembourg. Nonpolar fractions of sediment extracts were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the non-radioactive 96-well plate transthyretin (TTR) competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). Without bioactivation, medium polar and polar fractions also contained potent TTR-binding compounds with potencies from 1.6 to 17 nmol T4-Eq/g SEQ. This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing ofboth polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions. Without bioactivation THD potency is not observed in nonpolar sediment extracts, although in in vivo experiments PCBs and PBDEs, and not with dioxins or PAHs, have shown to be thyroid hormone disrupting (THD), demonstrating this bio-activation is toxicologically relevant and therefore required for sediment hazard characterisation. Chapter 7 discusses the implications of our results to improve the relevance and reliability of in vitro bioassay applied for risk characterisation of complex mixtures from sediments and other matrices. The evidence obtained to support the relevance of POP bio-activation is considered both from the exposure perspective as well as the toxicity perspective. Various features of the newly developed methods and knowledge acquired within this PhD project are discussed in relation to in vitro bioassay risk characterization of sediments towards a realistic in vitro bioassay-based risk characterization of complex mixtures. Some important aspects for the inclusion of metabolizing systems within in vitro bioassay are discussed. In addition, alternatives to deal with the SPMX effect and the definition of suitable sample amounts to improve in vitro bioassay reliability are offered. The suitability of the developed approach application is considered for the risk characterization of sediments. Furthermore, an analysis is made to decide whether this thesis have made in vitro bioassays more reliable and relevant for risk characterization of complex mixtures. Finally, it provides some concluding remarks and aspects for further applications and research.