Long-lived epigenetic interactions between perinatal PBDE exposure and Mecp2308 mutation.

The widespread use of persistent organic polybrominated diphenyl ethers (PBDEs) as commercial flame retardants has raised concern about potential long-lived effects on human health. Epigenetic mechanisms, such as DNA methylation, are responsive to environmental influences and have long-lasting consequences. Autism spectrum disorders (ASDs) have complex neurodevelopmental origins whereby both genetic and environmental factors are implicated. Rett syndrome is an X-linked ASD caused by mutations in the epigenetic factor methyl-CpG binding protein 2 (MECP2). In this study, an Mecp2 truncation mutant mouse (Mecp2(308)) with social behavioral defects was used to explore the long-lasting effects of PBDE exposure in a genetically and epigenetically susceptible model. Mecp2(308/+) dams were perinatally exposed daily to 2,2',4,4'-tetrabromodiphenyl ether 47 (BDE-47) and bred to wild-type C57BL/6J males, and the offspring of each sex and genotype were examined for developmental, behavioral and epigenetic outcomes. Perinatal BDE-47 exposure negatively impacted fertility of Mecp2(308/+) dams and preweaning weights of females. Global hypomethylation of adult brain DNA was observed specifically in female offspring perinatally exposed to BDE-47 and it coincided with reduced sociability in a genotype-independent manner. A reversing interaction of Mecp2 genotype on BDE-47 exposure was observed in a short-term memory test of social novelty that corresponded to increased Dnmt3a levels specifically in BDE-47-exposed Mecp2(308/+) offspring. In contrast, learning and long-term memory in the Morris water maze was impaired by BDE-47 exposure in female Mecp2(308/+) offspring. These results demonstrate that a genetic and environmental interaction relevant to social and cognitive behaviors shows sexual dimorphism, epigenetic dysregulation, compensatory molecular mechanisms and specific behavioral deficits.

Bioaccumulation and behavioral effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in perinatally exposed mice.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that have become pervasive environmental contaminants and may contribute to adverse health outcomes. We evaluated in mice the developmental neurotoxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), one of the most abundant PBDE congeners detected in animal and human tissues. Female C57BL/6J mice were exposed to daily doses of 0, 0.03, 0.1 or 1mg/kg beginning 4 weeks prior to conception, continuing through gestation and lactation, and ending at weaning on postnatal day (PND) 21. Levels of BDE-47 in blood, brain, liver and adipose tissues of dams were markedly increased after 4 weeks of exposure, around the time of mating, and continued to increase through the time of parturition. Blood levels of BDE-47 in the dosed dams were within the range reported in humans. BDE-47 tissue levels in the dams decreased between parturition and weaning, possibly reflecting mobilization during lactation. Brain BDE-47 levels in the offspring at PND 1 approached those of the dams at parturition. Perinatal exposure to BDE-47 resulted in significant dose dependent growth retardation, slower motor performance in several behavioral tests, and mice exposed to 1mg/kg/day BDE-47 showed altered performance in the Morris water maze. There were no differences between groups in the numbers of pyramidal neurons in hippocampus CA1. These results document accumulation of BDE-47 in several organ systems following exposure to low-levels of BDE-47, and provide evidence that such exposure is associated with early behavioral deficits in exposed neonates.

Ryanodine receptor type 1 (RyR1) possessing malignant hyperthermia mutation R615C exhibits heightened sensitivity to dysregulation by non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95).

Malignant hyperthermia (MH) susceptibility is conferred by inheriting one of >60 missense mutations within the highly regulated microsomal Ca(2+) channel known as ryanodine receptor type 1 (RyR1). Although MH susceptible patients lack overt clinical signs, a potentially lethal MH syndrome can be triggered by exposure to halogenated alkane anesthetics. This study compares how non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95), a congener identified in environmental and human samples, alters the binding properties of [(3)H]ryanodine to RyR1 in vitro. Junctional sarcoplasmic reticulum (SR) was isolated from skeletal muscle dissected from wild type pigs ((Wt)RyR1) and pigs homozygous for MH mutation R615C ((MH)RyR1), a mutation also found in humans. Although the level of (Wt)RyR1 and (MH)RyR1 expression is the same, (MH)RyR1 shows heightened sensitivity to activation and altered regulation by physiological cations. We report here that (MH)RyR1 shows more pronounced activation by Ca(2+), and is less sensitive to channel inhibition by Ca(2+) and Mg(2+), compared to (Wt)RyR1. In a buffer containing 100nM free Ca(2+), conditions typically found in resting cells, PCB 95 (50-1000nM) enhances the activity of (MH)RyR1 whereas it has no detectable effect on (Wt)RyR1. PCB 95 (2microM) decreases channel inhibition by Mg(2+) to a greater extent in (MH)RyR1 (IC(50) increased nine-fold) compared to (Wt)RyR1 (IC(50) increased by 2.5-fold). PCB95 reduces inhibition by Ca(2+) two-fold more with (MH)RyR1 than (Wt)RyR1. Our data suggest that non-coplanar PCBs are more potent and efficacious toward (MH)RyR1 than (Wt)RyR1, and have more profound effects on its cation regulation. Considering the important roles of Ca(2+) and Mg(2+) in regulating Ca(2+) signals involving RyR channels, these data provide the first mechanistic evidence that a genetic mutation known to confer susceptibility to pharmacological agents also enhances sensitivity to an environmental contaminant.

Structure-activity relationship for noncoplanar polychlorinated biphenyl congeners toward the ryanodine receptor-Ca2+ channel complex type 1 (RyR1).

Ryanodine receptor isoforms are expressed in both excitable and nonexcitable tissues where they form microsomal Ca2+ release channels broadly involved in shaping cellular signaling. In this report, we provide a detailed structure-activity relationship (SAR) for polychlorinated biphenyl (PCB) congeners and metabolites necessary for enhancing ryanodine receptor type 1 (RyR1) activity using [3H]ryanodine ([3H]Ry) binding analysis. The 2,3,6-Cl PCB configuration is most important for optimal recognition by the RyR1 complex and/or critical for sensitizing its activation. Para substitution(s) diminishes the activity with para-chloro having a higher potency than the corresponding para-hydroxy derivative. The addition of a more bulky para-methyl-sulfonyl group eliminates the activity toward RyR1, supporting the importance of the para positions in binding RyR1. The requirement for an intact major T cell immunophilin FKBP12-RyR1 complex was observed with each of 12 active PCB congeners indicating a common mechanism requiring an immunophilin-regulated Ca2+ release channel. An excellent correlation between the relative potencies for doubling [3H]Ry binding and the corresponding initial rates of PCB-induced Ca2+ efflux indicates that [3H]Ry binding analysis provides a measure of dysregulation of microsomal Ca2+ transport. The SAR for activating RyR1 is consistent with those previously reported in several in vivo and in vitro studies, suggesting that a common mechanism may contribute to the toxicity of noncoplanar PCBs. A practical application of the receptor-based screen developed here with RyR1 is that it provides a quantitative SAR that may be useful in predicting biological activity and risk of mixtures containing noncoplanar PCB congeners that have low or a lack of aryl hydrocarbon receptor activity.

Hydroxylated xestospongins block inositol-1,4,5-trisphosphate-induced Ca2+ release and sensitize Ca2+-induced Ca2+ release mediated by ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) often coexist within the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane and coordinate precise spatial and temporal coding of Ca(2+) signals in most animal cells. Xestospongin C (XeC) was shown to selectively block IP(3)-induced Ca(2+) release and IP(3)R-mediated signaling (Gafni et al., 1997). We have further studied the specificity of xestospongin structures possessing ring hydroxyl (-OH) substituents toward IP(3)R, RyR, and ER/SR Ca(2+)-ATPase (SERCA) activities. XeC potently inhibits IP(3)R, weakly inhibits RyR1, and lacks activity toward SERCA1 and SERCA2. XeD (9-OH XeC), 7-OH-XeA, and araguspongin C isolated from the marine sponge Xestospongia species also inhibit IP(3)-mediated Ca(2+) release and lack activity toward SERCA. However, these hydroxylated derivatives possess a unique activity in that they enhance Ca(2+)-induced Ca(2+) release from SR vesicles by a mechanism involving the sensitization of RyR1 channels within the same concentration range needed to block IP(3)-induced Ca(2+) release. These results show that xestospongins and related structures lack direct SERCA inhibitory activity, as suggested by some previous studies. A new finding is that XeD and related structures possessing a hydroxylated oxaquinolizidine ring are IP(3)R blockers that also enhance Ca(2+)-induced Ca(2+) release mediated by RyRs. In intact cells, the actions of XeD are blocked by ryanodine pretreatment and do not interfere with thapsigargin-mediated Ca(2+) mobilization stemming from SERCA block. Hydroxylated bis-oxaquinolizadine derivatives isolated from Xestospongia species are novel bifunctional reagents that may be useful in ascertaining how IP(3)Rs and RyRs contribute to cell signaling.

Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (

(+)-7S-Hydroxyxestospongin A from the marine sponge Xestospongia sp. and absolute configuration of (+)-xestospongin D.

The structure of the title compound, (+)-7S-hydroxyxestospongin A was solved by single-crystal X-ray diffraction analysis and the absolute stereochemistry obtained by analysis of the derived R and S Mosher's esters. The absolute configuration of xestospongin D was determined for the first time by analysis of anomalous scattering from the X-ray crystal diffraction data set. Xestospongins A, C, and D, araguspongine C, and demethylxestospongin B exhibited modest antifungal activity (MIC 30-100 g/mL) against various fluconazole-resistant Candida spp., but 7S-hydroxyxestospongin A was inactive.