Atropisomers toward Ryanodine Receptors (RyRs) - ACS Publications

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Enantioselectivity of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) atropisomers toward ryanodine receptors (RyRs) and their influences on hippocampal neuronal networks Wei Feng, Jing Zheng, Gaelle Robin, Yao Dong, Makoto Ichikawa, Yoshihisa Inoue, Tadashi Mori, Takeshi Nakano, and Isaac N Pessah Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04446 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Environmental Science & Technology REVISION manuscript for EST

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Enantioselectivity of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) atropisomers toward

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ryanodine receptors (RyRs) and their influences on hippocampal neuronal networks

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Wei Feng1, ^, Jing Zheng1,2, ^, Gaëlle Robin1, Yao Dong1, Makoto Ichikawa3, Yoshihisa Inoue3,

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Tadashi Mori3, Takeshi Nakano4 and Isaac N. Pessah1,*

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USA; 2China Pharmaceutical University, Jiangsu Provincial Key Laboratory for TCM

Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA,

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Evaluation and Translational Development, China; 3 Graduate School of Engineering, Osaka

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University, Japan ; 4 Research Center for Environmental Preservation, Osaka University,

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Japan

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^ Equally contributed to this work.

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* To whom correspondence may be addressed. Email: [email protected]. Telephone:

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+1-(530)-752-6696.

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ABSTRACT

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Nineteen ortho-substituted PCBs are chiral and found enantioselectively enriched in

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ecosystems. Their differential actions on biological targets are not understood. PCB 95

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(2,2',3,5',6-pentachlorobiphenyl), a chiral PCB of current environmental relevance, is among

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the most potent towards modifying ryanodine receptors (RyR) function and Ca2+ signaling.

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PCB 95 enantiomers are separated and assigned aR- and aS-PCB 95 using 3 chiral-

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columns HPLC and circular dichroism spectroscopy. Studies of RyR1-enriched microsomes

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show aR-PCB 95 with >4X greater potency (EC50=0.20±0.05µM), ~1.3X higher efficacy

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(Bmax=3.74±0.07µM) in [3H]Ryanodine-binding and >3X greater rates

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(R=7.72±0.31nmole/sec/mg) of Ca2+ efflux compared with aS-PCB 95, whereas racemate

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has intermediate activity. aR-PCB 95 has modest selectivity for RyR2, and lower potency

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than racemate toward the RyR isoform mixture in brain membranes. Chronic exposure of

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hippocampal neuronal networks to nanomolar PCB 95 during a critical developmental period

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shows divergent influences on synchronous Ca2+ oscillation (SCO): rac-PCB 95 increasing

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and aR-PCB 95 decreasing SCO frequency at 50nM, although the latter’s effects are non-

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monotonic at higher concentration. aS-PCB95 shows the greatest influence on inhibiting

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responses to 20Hz electrical pulse trains. Considering persistence of PCB 95 in the

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environment, stereoselectivity towards RyRs and developing neuronal networks may clarify

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health risks associated with enantioisomeric enrichment of PCBs.

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INTRODUCTION PCBs are a group of 209 man-made chlorinated hydrocarbons. Because of their

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chemical, thermal stability, non-flammability and electrical insulating properties, they were

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broadly included in numerous industrial and commercial applications

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(https://www.epa.gov/pcbs). Although production and use of PCBs have been banned since

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late 1970s, exposures continue through proximity to durable end products such as chalks in

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schools, dredging of PCB contaminated sediments, and inadvertent leaks from storage

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sites1.The chemical stability and bioaccumulation of PCBs in food animals and humans has

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been unquestionably documented2-4. Several epidemiological studies of human populations

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have linked exposures to PCBs and chemically related persistent organic pollutants (POPs)

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to increased risk of certain cancers, prompting the International Agency of Research on

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Cancer (IARC) to classify PCBs as Group 1 carcinogens5. Importantly, pre- and/or postnatal

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PCB exposures have been linked to a number of cognitive, behavioral, psychomotor and

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metabolic deficits in humans and animal models6-13. Mechanistic links between cancer

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promotion and neurological disorders has not been established.

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Nineteen of the 209 PCB that possess 3 chlorine substituents confer chiral

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asymmetry about the biphenyl bond axis, and thus they exist as stable atropisomer or

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enantiomers14. It is the Ortho-substituted noncoplanar PCBs, including many of the chiral

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PCB congeners that originated out of the manufacturing process as racemic mixtures

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continue to predominate15. Although physico-chemical processes do not promote

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enantiomeric enrichment of racemic PCBs16-18, enrichment has been demonstrated in living

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organisms through mechanisms of differential absorption, distribution, biotransformation and

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excretion19. More contemporary analytical results show ample evidence of enantioselective



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dispositions of PCBs and related POPs not only in sediments and soils, but also in tissues of

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wildlife, laboratory animal, and humans20-23. However, much less information is available

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about the consequences of enantioselective enrichments, their differential interactions with

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the biological targets whose physiological role, when modified, are known to induce and

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promote neurotoxicity. A major factor limiting our understanding of the molecular

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mechanisms underlying stereoselective influences of PCB enantiomers is the analytical

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challenges in enantiomeric separation and assignment of absolute conformation24-29.

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PCB 95, one of 19 chiral PCB congeners, is not only one of the more prevalent legacy

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congeners found in aging schools30-32, but there is also evidence of contemporary sources

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from the pigment industry33-35. Rac-PCB 95 is also among the most potent and efficacious

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PCB congeners that target and modify ryanodine receptor (RyRs), microsomal Ca2+

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channels that regulate the spatial and temporal fidelity of Ca2+ dynamics in a broad number

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of cell types, including striated muscle and neurons36-40. Interactions of PCBs with RyRs

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exhibit stringent structural-activity relationships39-41 that can adversely influence by aspects

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of neuronal connectivity36, 37, 42, 43, activities that appear to be highly stereoselective as

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demonstrated with PCB 136 enantiomers25, 26. Using postmortem brain samples and a

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neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, exposures to PCB

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95 have been recently shown to cause cumulative changes in epigenetic DNA

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hypomethylation enriched over autism candidate genes, through a mechanism that involves


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bivalent modification of histone H2A.Z44,45. Whether such effects of PCB 95 are

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stereoselective at the levels of RyR engagement or cellular functions have not been

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demonstrated.

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The present study specifically addresses the separation of PCB 95 enantiomers and

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demonstrates their enantioselective actions towards RyR isoforms and their differential

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influences on neuronal network activity in vitro.

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MATERIALS AND METHODS

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Chemicals. PCB 95 (racemic) was purchased neat from AccuStandard (Lot: 010610KS; 99–

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100%), and aR-PCB 95 and aS-PCB 95 were separated from this racemic stock. [9,21-3H]

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Ryanodine specific activity 50-100Ci/mmol was purchased from PerkinElmer (Bellerica, MA).

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Separation of enantiomers PCB 95. A detailed description of the separation of PCB 95

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enantiomers is presented in RESULTS AND DISCUSSION.

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Preparation of RyR1-enriched junctional sarcoplasmic reticulum (JSR). All procedures with

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animals were approved by the UC Davis IACUC Committee. JSR membrane fraction

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enriched in RyR1 and SR/ER Ca2+ ATPase (SERCA) was isolated from fast-twitch skeletal

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muscles of 98% (Publication in Chinese; Abstract in

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English)61. Utilizing online optical rotation detector (IBZMESSTECHNIK, Germany), they

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assigned the absolute configurations for enantiomers of first elute as (-)-PCB 95 and the


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second (+)-PCB9561. We therefore propose aR is equivalent to (-)-PCB 95 and aS-PCB 95 is

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equivalent to (+)-PCB 95.

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Enantiomeric enrichments is expressed as EF (enantiomeric fraction), the

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concentration of one enantiomer divided by the total concentration of both enantiomers

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(EF=A1/(A1+A2), with 0.5 corresponding to racemic mixture)62. Enantiomeric enrichment of

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PCB 95 was found with an EF value as high as 0.693±0.172 in the serum samples of E-

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waste recycling workers in China58; 0.64-0.76 in the samples from human milk in

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Switzerland 56; 0.51-0.75 from liver, 0.50-0.57 from kidney in Belgium54.

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PCB 95 enantiomers differentially sensitize [3H]Ry binding to RyRs.

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[3H]Ryanodine ([3H]Ry) binds with nanomolar affinity to RyRs that are anchored within

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the endoplasmic/sarcoplasmic reticulum (ER/SR) of muscle and neurons, respectively46, 63-65.

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Since [3H]Ry interactions with RyRs have been shown to be highly specific, and exhibit

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binding kinetics that were highly sensitive to the conformational states of the Ca2+ channel

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complex, [3H]Ry binding has been broadly used to identify and quantify the influences of

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ligands that allosterically modulate channel function, positively and/or negatively. It has been

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used to develop a quantitative structure-activity relationship among PCB congeners with RyR

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preparations from mammals and fish38, 40, 41, although the stereoselectivity of chiral PCBs of

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environmental relevance has remained poorly understood. In previous studies, racemic PCB

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95 was shown to significantly increases maximal [3H]ryanodine binding capacity (Bmax) and



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affinity51 by stabilizing the high affinity state of RyR1, an effect demonstrated with the purified

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channel protein66. To determine whether and to what degree PCB 95 stereoisomers

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influence the conformation of RyR1, we used concentrations of [3H]ryanodine that itself does

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not influence receptor conformation (ie, 2-fold greater receptor occupancy

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compared to aS- and rac-PCB 95, respectively (Fig 2A insert). These data showed a clear

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stereoselectivity for interaction of PCB 95 enantiomers toward RyR1.

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To test our hypothesis whether the stereoselectivity observed towards RyR1

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extended to preparations that express only RyR2 or complex mixtures of RyRs present in

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brain tissue, membrane homogenates from 110,000xg pellets were assayed as described in

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Supporting Information and Methods. For consistency, western blot and [3H]Ry binding

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analyses were performed to compare homogenates prepared from mouse skeletal muscle,


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heart and brain cortex in parallel. Cortical preparations were probed with selective antibodies

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at saturating levels by western blot analysis and skeletal and cardiac membranes served as

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positive controls for RyR1 and RyR2. All three isoforms (RyR1, RyR2, and RyR3 were

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detected in brain cortical preparations (SI_Figure 1), and qualitatively the levels followed the

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rank order in abundance RyR2>RyR1>RyR3. The relative abundance of RyR isoforms

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expressed cerebellum, cortex and hippocampus has not been clearly established69. Although

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RyR1 has not been consistently shown to be expressed in cortex70-72, our results clearly

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affirm its presence (SI_Figure 1). The [3H]Ry binding density of these preparations were

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approximately 16.4 to 54 -fold lower than those measured with JSR preparations. The high

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lipid-to-protein content in these less-pure preparations decreased the apparent potencies for

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PCBs in binding assays, presumably due to lipid partitioning40 and it was therefore difficult to

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titrate free PCB 95 to concentrations sufficient to saturate its activating effects on RyR1.

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Nevertheless, the results obtained from these mouse RyR1 preparations were consistent

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with those obtained with rabbit JSR, with aR-PCB 95 exhibiting higher potency and efficacy

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than either rac- or aS-PCB 95 (Fig 3A). Although RyR2 preparations showed much narrower

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selectivity among the three PCB 95 forms, aR-PCB 95 exhibited 15.4% (*p≤0.05 vs. rac-

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PCB95) higher activity towards RyR2 at 1.5µM compared to racemate, while no significance

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was found between the activity of aS and racemate towards RyR2, and aR-PCB 95

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possessing higher potency than rac- and aS-PCB 95(Fig 3B), consistent with the



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observations from in RyR1 binding analyses. Unexpectedly, the combination of all three RyR

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isoforms known to be expressed in the brain, resulted is a distinct pattern of activity with rank

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order rac>aS-PCB 95>aR-PCB 95, with aR-PCB 95 and aS-PCB 95 exhibiting 43.1% (***p≤

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0.001 vs. rac-PCB95) and 22.4% (*p≤0.05 vs. rac-PCB95) lower activity towards brain RyRs

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compared with racemate, respectively (Fig 3B).

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PCB 95 enantiomers differentially alter microsomal Ca2+ transport.

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Consistent with their direct RyR-activating properties, noncoplanar PCBs have been

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shown to selectively trigger net Ca2+ release from JSR49, 73 and from brain microsomes49

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without inhibiting SERCA activity, in a manner prevented with RyR blockers. In this study, we

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interrogated the possible stereoselectivity of aR- and aS- compared to rac-PCB 95 on Ca2+

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fluxes using the metallochromic Ca2+ dye Arsenazo III and in the presence of active SERCA

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pump-dependent vesicular Ca2+ uptake as depicted in the Figure 4A. After 180nmole Ca2+

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were sequentially loaded into the JSR (Fig 4B), addition of DMSO (vehicle, 0.5%) did not

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elicit a release of actively accumulated Ca2+ (Fig 4B, grey trace). However, addition of aR-

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PCB 95 (5µM) triggered a nearly 3-fold higher rate of Ca2+ efflux compared to aS-PCB 95,

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whereas the rate elicited by rac-PCB 95 was intermediate (Fig 4B and C). Compared to

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racemate, the initial rate of Ca2+ release elicited by aR-PCB 95 and aS-PCB 95 are 70.9%

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(***p≤0.001 vs. rac-PCB95) faster and 38.6% (***p≤0.001 vs. rac-PCB95) slower,

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respectively (Fig 4D).


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RyR isoforms in combination with inositol 1,4,5-trisphosphate receptor (IP3R) have

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been shown to mediate a physiological process CICR, representing a fundamentally

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important Ca2+ signaling mechanism in a broad numbers of cell types38, 74-77. Previous study

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has shown that racemic PCB 95 enhances CICR by increasing the sensitivity of RyR1 to

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Ca2+51. We therefore used JSR to test whether PCB 95 enantioselectively influenced CICR.

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Figure 5 showed that JSR actively loaded with Ca2+ to ~70% of their capacity retain

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accumulated Ca2+ when either DMSO (0.5%) or a low concentration (250nM) of PCB 95 is

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introduced 2.5min prior to eliciting CICR by addition of 90nmol Ca2+ to the vesicle medium

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(Fig 5A). Their presence enhanced CICR with the rank potency: aR-PCB>rac>aS-PCB

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95>DMSO (Fig 5B).

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In general, the stereoselectivities observed with aR- over aS-PCB 95 in both [3H]Ry

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binding and Ca2+ flux assays were in very good agreement with our previous results obtained

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with enantiomers of the less environmentally relevant congener PCB 13625. In this regard,

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aR-PCB 95, which we presume is (-)-PCB 95 based on the results of Xu and coworkers 61, is

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more active towards the major isoforms RyR1 and RyR2 when expressed individually,

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consistent with the stereoselectivity assigned for PCB 136 enantiomers25. However, it should

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be emphasized that; (1) the separation in potency and efficacy of (-)- over (+)-PCB 136

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toward RyR1 and RyR2 was significantly more pronounced compared to our current findings

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with aR- over aS-PCB 95, and (2) the greater relative potency of rac-PCB 95 compared to



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either enantiomeric form where multiple RyR isoforms are expressed, as they are in brain

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and several other tissues. Nevertheless, the stereoselectivity of PCB 95 enantiomers

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identified here was significant because PCB 95 has been shown to predominate in recent

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sampling of many grade schools32, human breast milk, blood and other tissues, and some

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found enantioselectively enriched54,58, 78-80. Typically noncoplanar PCBs were found to be

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more persistent with half-lives in human body ranging from 0.4-3 years78, 81 and metabolic

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elimination rates averaging about 6 years82.

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PCB 95 enantiomers differentially alter Ca2+ dynamics in hippocampal neurons

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How the observed pattern of stereoselectivity towards RyRs for PCB 95 translated to

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their ability to alter Ca2+ signals is known to be important for regulating excitability, but the

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proper development of neuronal networks83 has not been previously investigated. Single cell

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real-time Ca2+ imaging techniques were therefore used to measure both acute responses in

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terms of neuronal network excitability, as well as the consequences of chronic exposure (12

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days in vitro; from 2 to 14 DIV). Two functional endpoints were measured in neuronal

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cultures from mouse hippocampus, (1) patterns of SOCs known to be important for long-term

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activity-dependent neuronal plasticity, and (2) properties of Ca2+ transients evoked by

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electrical stimulation. For acute responses, SCO patterns were analyzed from mature

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networks at 14 DIV 10min before and after focal perfusion of vehicle or PCB 95 (500nM)(Fig

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6A). At 14 DIV, neurons maintained well-defined SCO activity and addition of DMSO did not


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significantly alter SCO frequency (Fig 6B) or Ca2+ transient amplitudes by electrical pulses at

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20Hz (Fig 6D), although DMSO did reduce SCO amplitude by ~15% (Fig 6C; prac (Fig 6C; reductions of 8.5, 21, and 39%, respectively),

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whereas only aS-PCB 95 caused a 35% (p

Atropisomers toward Ryanodine Receptors (RyRs) - ACS Publications

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