Time-course changes of mixture effects on AhR binding-dependent luciferase activity in a crude extract from a compost sample

Abstract

Crude extracts of environmental samples contain stable and labile aryl hydrocarbon receptor (AhR) ligands, and show huge activities in the cell-based bioassay, and these activities are higher than the chemically calculated induction equivalent values. It is thought that not only unidentified AhR ligands but also mixture effect among compounds might contribute to these activities. In the previous work, we have indicated that hydrophobic compounds in household sewage sludge (HSS) compost may interact synergistically with 2,3,7,8-TCDD in the CALUX (DR-CALUX ®: Dioxin-Responsive Chemical-Activated Luciferase gene eXpression) assay [Suzuki, G., Takigami, H., Kushi, Y., Sakai, S., 2004. Evaluation of mixture effects in a crude extract of compost using the CALUX bioassay and HPLC fractionation. Environ. Int. 30, 1055–1066]. In this study, we focused on co-existing stable compounds such as halogenated aromatic hydrocarbons (HAHs) and labile compounds such as polyaromatic hydrocarbons (PAHs) in the crude extract and investigated the time-course changes of mixture effects among compounds in environmental samples using the CALUX assay and normal-phase high-performance liquid chromatography (NP-HPLC) fractionation. We confirmed that CYP1A-inducing PAHs and HAHs could be separated by NP-HPLC on a nitrophenylpropylsilica (NITRO) column. To determine whether the activities of AhR ligands in environmental samples (including the HSS compost) could be assessed by the additivity theory, we compared the CALUX activity of the crude extract with the arithmetical sum of the activities of all the fractions separated by NP-HPLC. We confirmed a potentiation of CALUX activity at 12-, 24- and 48-h exposure durations. In contrast, CALUX activity increased additively at 6- and 72-h exposure durations. CALUX activity was potentiated when the CALUX activity of the HPLC fractions showed a remarkable reduction resulting in a change of activity profiles. In contrast, additivity was observed at a 72-h exposure duration when the CALUX activity of the HPLC fractions showed neither remarkable reduction nor a change in profile. Our results suggest that differences in the metabolic decomposition of compounds affected mixture effects on CALUX activity in a crude extract from HSS compost.