Uncertainties in risk assessment of dioxin-like compounds : A focus on systemic relative potencies and species differences

This thesis describes experimental work undertaken to reduce uncertainties in the risk assessment of dioxin-like compounds. The toxic equivalency factor (TEF) approach is the most commonly used method for assessing the risk of complex mixtures of dioxin-like compounds. Consequently, accurate estimates of TEF values are crucial for human risk assessment. The TEF-concept is mainly based on animal experiments with oral dosage as the principal route of exposure, but human risk assessment is often assessed based on systemic (plasma) concentration. The major objectives of this thesis were to establish if there is a need for development of specific “systemic” TEFs and to evaluate whether there are consistent species-specific differences in relative potencies. The research described in this thesis shows that for some congeners the current TEF might under- or over- estimate the risk for humans based on plasma concentrations. This is due to either congener-specific toxicokinetics or species differences in response. Taking these congener- and species- specific differences into account can help to improve human risk assessment of dioxin-like compounds

Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade

Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX® bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptor agonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX®. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptor agonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discusse

Relative effective potencies of dioxin-like compounds in rodent and human lung cell models

© 2018 Elsevier B.V. Toxicity of dioxin-like compounds (DLCs), such as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls, is largely mediated via aryl hydrocarbon receptor (AhR) activation. AhR-mediated gene expression can be tissue-specific; however, the inducibility of AhR in the lungs, a major target of DLCs, remains poorly characterized. In this study, we developed relative effective potencies (REPs) for a series of DLCs in both rodent (MLE-12, RLE-6TN) and human (A549, BEAS-2B) lung and bronchial epithelial cell models, using expression of both canonical (CYP1A1, CYP1B1) and less well characterized (TIPARP, AHRR, ALDH3A1) AhR target genes. The use of rat, murine and human cell lines allowed us to determine both species-specific differences in sensitivity of responses to DLCs in lung cellular models and deviations from established WHO toxic equivalency factor values (TEF) values. Finally, expression of selected AhR target genes was determined in vivo, using lung tissues of female rats exposed to a single oral dose of DLCs and compared with the obtained in vitro data. All cell models were highly sensitive to DLCs, with murine MLE-12 cells being the most sensitive and human A549 cells being the least sensitive. Interestingly, we observed that four AhR target genes were more sensitive than CYP1A1 in lung cell models (CYP1B1, AHRR, TIPARP and/or ALDH3A1). We found some deviations, with strikingly low REPs for polychlorinated biphenyls PCBs 105, 167, 169 and 189 in rat RLE-6TN cells-derived REPs for a series of 20 DLCs evaluated in this study, as compared with WHO TEF values. For other DLCs, including PCBs 126, 118 and 156, REPs were generally in good accordance with WHO TEF values. This conclusion was supported by in vivo data obtained in rat lung tissue. However, we found that human lung REPs for 2,3,4,7,8-pentachlorodibenzofuran and PCB 126 were much lower than the respective rat lung REPs. Furthermore, PCBs 118 and 156 were almost inactive in these human cells. Our observations may have consequences for risk assessment. Given the differences observed between rat and human data sets, development of human-specific REP/TEFs, and the use of CYP1B1, AHRR, TIPARP and/or ALDH3A1 mRNA inducibility as sensitive endpoints, are recommended for assessment of relative effective potencies of DLCs

Relative effective potencies of dioxin-like compounds in rodent and human lung cell models

© 2018 Elsevier B.V. Toxicity of dioxin-like compounds (DLCs), such as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls, is largely mediated via aryl hydrocarbon receptor (AhR) activation. AhR-mediated gene expression can be tissue-specific; however, the inducibility of AhR in the lungs, a major target of DLCs, remains poorly characterized. In this study, we developed relative effective potencies (REPs) for a series of DLCs in both rodent (MLE-12, RLE-6TN) and human (A549, BEAS-2B) lung and bronchial epithelial cell models, using expression of both canonical (CYP1A1, CYP1B1) and less well characterized (TIPARP, AHRR, ALDH3A1) AhR target genes. The use of rat, murine and human cell lines allowed us to determine both species-specific differences in sensitivity of responses to DLCs in lung cellular models and deviations from established WHO toxic equivalency factor values (TEF) values. Finally, expression of selected AhR target genes was determined in vivo, using lung tissues of female rats exposed to a single oral dose of DLCs and compared with the obtained in vitro data. All cell models were highly sensitive to DLCs, with murine MLE-12 cells being the most sensitive and human A549 cells being the least sensitive. Interestingly, we observed that four AhR target genes were more sensitive than CYP1A1 in lung cell models (CYP1B1, AHRR, TIPARP and/or ALDH3A1). We found some deviations, with strikingly low REPs for polychlorinated biphenyls PCBs 105, 167, 169 and 189 in rat RLE-6TN cells-derived REPs for a series of 20 DLCs evaluated in this study, as compared with WHO TEF values. For other DLCs, including PCBs 126, 118 and 156, REPs were generally in good accordance with WHO TEF values. This conclusion was supported by in vivo data obtained in rat lung tissue. However, we found that human lung REPs for 2,3,4,7,8-pentachlorodibenzofuran and PCB 126 were much lower than the respective rat lung REPs. Furthermore, PCBs 118 and 156 were almost inactive in these human cells. Our observations may have consequences for risk assessment. Given the differences observed between rat and human data sets, development of human-specific REP/TEFs, and the use of CYP1B1, AHRR, TIPARP and/or ALDH3A1 mRNA inducibility as sensitive endpoints, are recommended for assessment of relative effective potencies of DLCs