Differential methylation of the TRPA1 promoter in pain sensitivity.

Chronic pain is a global public health problem, but the underlying molecular mechanisms are not fully understood. Here we examine genome-wide DNA methylation, first in 50 identical twins discordant for heat pain sensitivity and then in 50 further unrelated individuals. Whole-blood DNA methylation was characterized at 5.2 million loci by MeDIP sequencing and assessed longitudinally to identify differentially methylated regions associated with high or low pain sensitivity (pain DMRs). Nine meta-analysis pain DMRs show robust evidence for association (false discovery rate 5%) with the strongest signal in the pain gene TRPA1 (P=1.2 × 10(-13)). Several pain DMRs show longitudinal stability consistent with susceptibility effects, have similar methylation levels in the brain and altered expression in the skin. Our approach identifies epigenetic changes in both novel and established candidate genes that provide molecular insights into pain and may generalize to other complex traits.

Effects of estrogens and xenoestrogens on androgen production by Atlantic croaker testes in vitro: evidence for a nongenomic action mediated by an estrogen membrane receptor.

The short-term effects of estrogens and xenoestrogens on testicular androgen production were investigated in an in vitro incubation bioassay system using testicular tissue from the Atlantic croaker (Micropogonias undulatus). Incubation of testicular tissue fragments with estradiol over the concentration range of 37 nM to 37 microM caused concentration-dependent decreases in gonadotropin-stimulated 11-ketotestosterone (11-KT) production. The effect was specific for estrogens; progesterone, cortisol, and the synthetic androgen mibolerone did not significantly alter 11-KT production at similar concentrations. Diethylstilbestrol, the antiestrogen ICI 182,780, and several xenoestrogens including Kepone (chlordecone), 4-nonylphenol, and a hydroxylated polychlorinated biphenyl metabolite also significantly decreased gonadotropin-stimulated 11-KT production. The action of estradiol was rapid (<5 min) and was not blocked by actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively. Moreover, estradiol conjugated to BSA, which cannot pass through the cell membrane, also caused a decrease in 11-KT production. In addition, an estrogen-binding moiety was identified in testicular membrane preparations that had a single class of high-affinity (K(d) 1.6 nM), saturable (1.2 nM), displaceable, finite (B(max) 0.03 nM, 26 fmol/g testis) binding sites specific for estrogens and exhibited rapid association (t(1/2) = 5 min), characteristics typical of steroid membrane receptors. Overall the relative binding affinities of estrogens, other steroids, antiestrogens, and xenoestrogens for the membrane preparation correlated with their activities in the androgen production bioassay, thereby satisfying the final criteria for the designation of this estrogen-binding moiety as a steroid membrane receptor. The results demonstrate that estrogens and also probably xenoestrogens can act on the cell surface via a nongenomic mechanism to alter testicular androgen production in this vertebrate species.

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol >> estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.