Determination of in vitro relative potency (REP) values for mono-ortho polychlorinated biphenyls after purification with active charcoal

Abstract

The TEF system for dioxin-like compounds has included assignment of TEF values for monoortho polychlorinated biphenyls (MO-PCBs). Small traces of aryl hydrocarbon receptor (AhR)-active impurities could result in artifactually higher relative potency (REP) values. were purified on an active charcoal column to remove AhR agonists that could be present as impurities. Activation or inhibition of AhR-dependent gene expression by purified MO-PCBs was studied in stably transfected cell lines (H1G1.1c3 mouse, H4G1.1c2 rat hepatoma), containing an AhR-responsive (AhR-EGFP) reporter gene. In addition, EROD activity was used as marker for CYP1A1 activity in these cell lines. MO-PCBs -105, -118, -156 induced AhR-EGFP expression in both rodent cell lines, with PCB-156 (10 μM) being most effectively; inducing gene expression to ~27% of TCDD (mouse cells) and 62.5 ± 3.4% (rat cells) of TCDD. This concurred with increased EROD activity in both cell lines to maxima of 20.5 ± 1.5% and 68 ± 3.2% of TCDD, respectively. No induction was observed for PCB-167. In the H1G1.1c3 mouse cells, PCB-105, -118 and -156 (10 μM) significantly reduced TCDD-induced AhR-EGFP expression to 50.9 ± 2.9%, 58.3 ± 2.2% and 70.8 ± 1.3% of TCDD. Reduced EROD activity was also observed, of 39.3 ± 2.8%, 67 ± 5% and 48.3 ± 4% compared to TCDD. PCB-167 did not result in significant reduction. In rat cells, only PCB-156 resulted in significant decrease in TCDD-induced AhR-EGFP expression of 35%, suggesting species differences play a role. Our results suggest that purification of MO-PCBs is an essential step in determining accurate REP values, and could very likely lead to lower TEF values than those presently assigned by the WHO.

Direct Assessment of Cumulative Aryl Hydrocarbon Receptor Agonist Activity in Sera from Experimentally Exposed Mice and Environmentally Exposed Humans

“…These assays have been shown to produce similar results, with the advantage that the EGFP provides a nondestructive readout (Nagy et al 2002; Nording et al 2007; Peters et al 2006). We calculated AhR agonist activity values, presented as picograms TCDD equivalents (eq) per milliliter, by interpolation from TCDD standard curves.…”

“…Mouse serum was applied directly to mouse hepatoma cells stably transfected with an AhR-driven enhanced green fluorescent protein (EGFP)-expressing reporter construct with a promoter similar to that in the AhR–CALUX assay ( Nagy et al 2002 ). These assays have been shown to produce similar results, with the advantage that the EGFP provides a nondestructive readout ( Nagy et al 2002 ; Nording et al 2007 ; Peters et al 2006 ). We calculated AhR agonist activity values, presented as picograms TCDD equivalents (eq) per milliliter, by interpolation from TCDD standard curves.…”

Neurodevelopmental toxicity of prenatal polychlorinated biphenyls (PCBs) by chemical structure and activity: a birth cohort study

“…Also notable is a recent effort to develop a Neurotoxic Equivalence Scheme that would take into account multiple mechanisms [ 94 ]. Another concern about the calculation of dioxin-like potencies in epidemiologic studies relates to the difficulty in extrapolating from rat to human, given that large differences in AhR-mediated gene expression and EROD activity are observed even comparing rats with mice [ 95 ].…”

Identification of Sex-Specific Transcriptome Responses to Polychlorinated Biphenyls (PCBs)

“…This lack of overlap might be a consequence of the species, tissue and dose differences between our study and those reported in the literature. However, the different pathways shown in the results in association with different PCBs suggest a variety of ways that each PCB interacts with genes and also independently of the classification (dioxin-like or non dioxin-like) relevant to the AhR-mediated activation 37,38 . Other toxic effects associated with PCBs are endocrine disruption by blocking the thyroid system functioning and neurotoxicity.…”