An Extended Structure–Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors

Abstract

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca 2þ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [ 3 H]Ryanodine ([ 3 H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the paraposition resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

Enantioselectivity of 2,2′,3,5′,6-Pentachlorobiphenyl (PCB 95) Atropisomers toward Ryanodine Receptors (RyRs) and Their Influences on Hippocampal Neuronal Networks

“…The [ 3 H]Ry binding density of these preparations were approximately 16.4−54-fold lower than those measured with JSR preparations. The high lipid-to-protein content in these less-pure preparations decreased the apparent potencies for PCBs in binding assays, presumably due to lipid partitioning 40 and it was therefore difficult to titrate free PCB 95 to concentrations sufficient to saturate its activating effects on RyR1. Nevertheless, the results obtained from these mouse RyR1 preparations were consistent with those obtained with rabbit JSR, with aR -PCB 95 exhibiting higher potency and efficacy than either rac - or aS -PCB 95 (Figure 3A).…”

“…36−40 Interactions of PCBs with RyRs exhibited stringent structural-activity relationships 39−41 that can adversely influence aspects of neuronal connectivity, 36,37,42,43 activities that appear to be highly stereoselective as demonstrated with PCB 136 enantiomers. 25,26 Using postmortem brain samples and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, exposures to PCB 95 have been recently shown to cause cumulative changes in epigenetic DNA hypomethylation enriched over autism candidate genes, through a mechanism that involves bivalent modification of histone H2A.Z.…”

Interactions of Dichlorodiphenyltrichloroethane (DDT) and Dichlorodiphenyldichloroethylene (DDE) With Skeletal Muscle Ryanodine Receptor Type 1

“…Significant sensitization of RyR1 in our cell-based in vitro assay necessitated 5-10 mM of DDT to facilitate subchronic and chronic impairments in RyR1 channel function and associated changes in Ca 2þ dynamics in cells; although our Ca 2þ imaging experiments with myotubes suggests that lower concentrations (nanomolar range) are sufficient to facilitate muscle dysfunction, because the 1 mM o,p 0 -DDE pretreatment was performed in the presence of 5% serum and DDx is undoubtedly highly protein bound. The high concentration membrane lipid present in microsomal [ 3 H]Ry-binding assays and the incubation conditions are likely contributors to a lower apparent potency of highly lipophilic xenobiotic molecules due to partitioning in the lipid phase, as previously demonstrated with single channel voltage clamp studies with PCB congeners (Holland et al, 2017). Monolayers of myotubes proved to be a very sensitive model to o,p 0 -DDE, eliciting abnormal ECC and fatigability at low micromolar exposures, revealing that ECC may be a particularly sensitive target to DDx.…”

Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus): A phylogenetic and population-based comparison

“…It is thought that NDL PCBs interfere with RyR’s interaction with the accessory protein FK506 binding protein 12kDa and 12.6 kDa (protein name FKBP12 or FKBP12.6; hereafter referred to by their gene name FKBP1A and FKBP1B), which helps maintain the conformational state of the RyR (Zalk et al, 2007). Disruption of the FKBP1-RyR complex causes an increase in channel open probability (Bellinger et al, 2008; Zalk et al, 2007), which is similar to that caused by NDL PCBs (Holland et al, 2016). Additionally, pretreatment with the immunophilin drugs FK506 and rapamycin can eliminate NDL PCB toxicity at the RyR (Wong and Pessah, 1997).…”

“…Several molecular mechanisms proposed to contribute to NDL PCB-induced neurotoxicity include congener potentiation of GABA A receptors (Fernandes et al, 2010), inhibition of dopamine transport via dopamine or vesicular monoamine transporters (Wigestrand et al, 2013), and induced activity of ryanodine receptors (RyR) (Wong and Pessah, 1996; Pessah et al, 2010). RyR Ca 2+ channels are targets of particular interest because they are potentiated at pM to nM NDL PCB concentrations (Holland et al, 2016) and this induced receptor activation leads to altered neuronal growth and plasticity (Kenet et al, 2007; Wayman et al, 2012a; Wayman et al, 2012b; Lesiak et al, 2014) and marked deficits in cognitive function and behavior in mammals (Schantz et al, 1997; Roegge et al, 2006; Yang et al, 2009)…”