Effect of prolactin on DNA methylation in the liver and kidney of rat

Prolactin is an important growth modulatory hormone in fetal and adult tissues. It stimulates DNA synthesis and enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. In this study the effects of prolactin on 5-methyl cytosine content in liver and kidney of rats was studied using HPLC. Prolactin treatment caused hypomethylation of DNA in the liver and kidney of immature rats at 48 h after treatment and the effect remained even at 72 h. Prolactin also caused hypomethylation of DNA in the kidney and liver of adult rats at 48 h after treatment. These results indicate that prolactin probably regulates DNA methylation in the liver and kidney of immature and adult rats.     

The effect of morphine upon DNA methylation in ten regions of the rat brain

Morphine is one of the most effective analgesics in medicine. However, its use is associated with the development of tolerance and dependence. Recent studies demonstrating epigenetic changes in the brain after exposure to opiates have provided insight into mechanisms possibly underlying addiction. In this study, we sought to identify epigenetic changes in ten regions of the rat brain following acute and chronic morphine exposure. We analyzed DNA methylation of six nuclear-encoded genes implicated in brain function (Bdnf, Comt, Il1b, Il6, Nr3c1, and Tnf) and three mitochondrially-encoded genes (Mtco1, Mtco2, and Mtco3), and measured global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5?hmC) levels. We observed differential methylation of Bdnf and Il6 in the pons, Nr3c1 in the cerebellum, and Il1b in the hippocampus in response to acute morphine exposure (all P value < 0.05). Chronic exposure was associated with differential methylation of Bdnf and Comt in the pons, Nr3c1 in the hippocampus and Il1b in the medulla oblongata (all P value < 0.05). Global 5mC levels significantly decreased in the superior colliculus following both acute and chronic morphine exposure, and increased in the hypothalamus following chronic exposure. Chronic exposure was also associated with significantly increased global 5hmC levels in the cerebral cortex, hippocampus, and hypothalamus, but significantly decreased in the midbrain. Our results demonstrate, for the first time, highly localized epigenetic changes in the rat brain following acute and chronic morphine exposure. Further work is required to elucidate the potential role of these changes in the formation of tolerance and dependence.     

Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study

Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM_2.5 exposure was calculated for each participant''s home address using a dispersion model. An interquartile range (IQR) increment in PM_2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM_2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM_2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.     

The mysterious presence of a 5-methylcytosine oxidase in the Drosophila genome

5-methylcytosine is an important epigenetic modification involved in gene control in vertebrates and many other complex living organisms. Its presence in Drosophila has been a matter of debate and recent bisulfite sequencing studies of early-stage fly embryos have concluded that the genome of Drosophila is essentially unmethylated. However, as we outline here, the Drosophila genome harbors a well-conserved homolog of the TET protein family. The mammalian orthologs TET1/2/3 are known to convert 5-methylcytosine into 5-hydroxymethylcytosine. We discuss several possible explanations for these seemingly contradictory findings. One possibility is that the 2 modified cytosine bases are generated in Drosophila only at certain developmental stages and in a cell type-specific manner during neurogenesis. Alternatively, Drosophila Tet and its mammalian homologs may carry out catalytic activity-independent functions, and the possibility that these proteins may oxidize 5-methylcytosine in RNA created by the methyltransferase Dnmt2 should also be strongly considered.     

Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme variants carrying E-->A, E-->D or E-->Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis. Since the glutamic acid residue contacts the flipped base, its main function could be to hold the target base at a position that supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does not. Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.     

Bayesian distributed lag models: estimating effects of particulate matter air pollution on daily mortality

Summary .  A distributed lag model (DLagM) is a regression model that includes lagged exposure variables as covariates; its corresponding distributed lag (DL) function describes the relationship between the lag and the coefficient of the lagged exposure variable. DLagMs have recently been used in environmental epidemiology for quantifying the cumulative effects of weather and air pollution on mortality and morbidity. Standard methods for formulating DLagMs include unconstrained, polynomial, and penalized spline DLagMs. These methods may fail to take full advantage of prior information about the shape of the DL function for environmental exposures, or for any other exposure with effects that are believed to smoothly approach zero as lag increases, and are therefore at risk of producing suboptimal estimates. In this article, we propose a Bayesian DLagM (BDLagM) that incorporates prior knowledge about the shape of the DL function and also allows the degree of smoothness of the DL function to be estimated from the data. We apply our BDLagM to its motivating data from the National Morbidity, Mortality, and Air Pollution Study to estimate the short-term health effects of particulate matter air pollution on mortality from 1987 to 2000 for Chicago, Illinois. In a simulation study, we compare our Bayesian approach with alternative methods that use unconstrained, polynomial, and penalized spline DLagMs. We also illustrate the connection between BDLagMs and penalized spline DLagMs. Software for fitting BDLagM models and the data used in this article are available online.     

5-azaC对小麦幼苗生长及盐胁迫后DNA甲基化变异的影响

DNA甲基化是调节植物生长发育,调控逆境基因表达的表观遗传机制之一。该研究采用不同浓度的DNA甲基化抑制剂5-azaC处理耐盐性不同的春小麦种子,分析其对种子萌发及盐胁迫后叶片基因组DNA甲基化的影响,探究DNA甲基化与小麦耐盐性之间的相关性。结果表明:(1)5-azaC显著抑制幼苗根长伸长,降低根系鲜重和干重。(2)甲基化敏感扩增多态性(MSAP)分析发现,单独盐胁迫后甲基化水平上升, 5-azaC预处理材料经盐胁迫后甲基化水平呈下降趋势。(3)盐胁迫后基因组同时发生DNA去甲基化和DNA甲基化。敏盐品种‘新春6号’DNA去甲基化比率上升,DNA甲基化增加的比率下降;耐盐品种‘新春11号’DNA去甲基化比率和DNA甲基化增加的比率均上升,但去甲基化比率大于DNA甲基化增加的比率,说明盐胁迫引起的基因组DNA去甲基化为主,5-azaC预处理提高了盐胁迫下DNA去甲基化的比率。(4)DNA甲基化修饰位点序列分析发现,在核糖体亚基蛋白、蛋白激酶和转座子序列均存在DNA甲基化修饰现象,说明存在多种代谢途径共同参与了盐胁迫调控。     

5-Aza-CdR对膀胱癌细胞生长及hsa-miR-203表达的影响

许多研究表明,miRNAs在肿瘤中失活与特定的遗传和表观遗传机制改变有关,hsa-miR-203在膀胱癌组织和细胞中表达下调并扮演着抑癌基因的角色。为了验证hsa-miR-203在膀胱癌细胞中是否受DNA甲基化抑制,采用去甲基化抑制剂5-Aza-CdR(5-氮-2'-脱氧胞苷)处理5637和BIU-87膀胱癌细胞,MSP和RT-PCR检测表明,hsa-miR-203的启动子在5637和BIU-87细胞中存在完全的甲基化,而5-Aza-CdR能逆转hsa-miR-203启动子的甲基化状态,恢复hsa-miR-203的表达。MTT法测定显示,5-Aza-CdR使5637和BIU-87膀胱癌细胞增殖受到明显抑制,并呈时间和剂量依赖性。同时,流式细胞仪检测显示,5-Aza-CdR使5637和BIU-87膀胱癌细胞周期阻滞于G_0/G_1期。因此,5-Aza-CdR能抑制膀胱癌细胞5637和BIU-87增殖并干扰其细胞周期。hsa-miR-203启动子异常甲基化是其在膀胱癌细胞中低表达的重要机制,5-Aza-CdR能逆转hsa-miR-203基因的甲基化,恢复hsa-miR-203的表达,为hsa-miR-203作为膀...     

Novel properties of statins: suppression of the systemic and bone marrow responses induced by exposure to ambient particulate matter (PM10) air pollution

Exposure to ambient particulate matter (PM(10)) elicits systemic inflammatory responses that include the stimulation of bone marrow and progression of atherosclerosis. The present study was designed to assess the effect of repeated exposure of PM(10) on the turnover and release of polymorphonuclear leukocytes (PMNs) from the bone marrow into the circulation and the effect of lovastatin on the PM(10)-induced bone marrow stimulation. Rabbits exposed to PM(10) three times a week for 3 wk, were given a bolus of 5'-bromo-2'-deoxyuridine to label dividing cells in the marrow to calculate the transit time of PMNs in the mitotic or postmitotic pool. PM(10) exposure accelerated the turnover of PMNs by shortening their transit time through the marrow (64.8 ± 1.9 h vs. 34.3 ± 7.4 h, P < 0.001, control vs. PM(10)). This was predominantly due to a rapid transit of PMNs through the postmitotic pool (47.9 ± 0.7 h vs. 21.3 ± 4.3 h, P < 0.001, control vs. PM(10)) but not through the mitotic pool. Lovastatin delayed the transit time of postmitotic PMNs (38.2 ± 0.5 h, P < 0.001 vs. PM(10)) and shifted the postmitotic PMN release peak from 30 h to 48 h. PM(10) exposure induced the prolonged retention of newly released PMNs in the lung, which was reduced by lovastatin (P < 0.01). PM(10) exposure increased plasma interleukin-6 levels with significant reduction by lovastatin (P < 0.01). We conclude that lovastatin downregulates the PM(10)-induced overactive bone marrow by attenuating PM(10)-induced systemic inflammatory responses.     

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.     

5'-Aza-CdR 对B细胞淋巴瘤的抑制作用及对瘤细胞CHFR 表达和甲基化的影响

目的:探讨5-氮杂-2'-脱氧胞苷(5'-Aza-Cd R)对人伯基特淋巴瘤Raji裸鼠移植瘤的抑制作用,以及对瘤组织中CHFR表达和甲基化的影响。方法:1皮下接种Raji细胞悬液构建荷瘤裸鼠模型18只,并随机分为实验组和对照组,分别予腹腔内注射等体积的5'-Aza-Cd R(1μg/g)及生理盐水,计算肿瘤体积大小,绘制肿瘤生长曲线,24天后比较两组移植瘤体积大小和重量。2分别从移植瘤组织中提取DNA及RNA,应用实时荧光定量PCR检测CHFR m RNA的表达,甲基化特异性聚合酶链反应(MSP)检测CHFR基因甲基化情况。结果:1实验组移植瘤生长速度较缓慢。实验结束时,实验组移植瘤体积、重量与对照组相比,差异有显著统计学意义(P0.01)。2实验组瘤细胞中CHFR m RNA的表达水平(3.69±1.20)与对照组(1.04±0.30)相比,差异有显著统计学意义(t=3.94,P0.001)。3实验组中的甲基化条带与对照组相比明显变暗,同时出现非甲基化条带,对照组中CHFR基因启动子只有甲基化条带被扩增。结论:5'-Aza-Cd R能抑制人伯基特淋巴瘤的生长,诱导瘤细胞CHFR m RNA表达增加,其机制可能为5'-Aza-Cd R使甲基化的CHFR启动子发生去甲基化。     

Effects of metabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: results from the EXPAH study

Data from the EXPAH project on PAH exposure and intermediary biomarkers were analyzed with respect to individual genotypes at seven metabolic gene loci. The GSTM1 null allele was associated with significantly higher levels of two biomarkers, malondialdehyde-2′-deoxyguanosine and benzo[a]pyrene DNA adducts in the total population from three Central and Eastern European countries. The CYP1B1 Leu/Val variant demonstrated effects on both markers of oxidative DNA damage in opposite directions, producing a higher level of M₁dG with a trend from wild type (Leu/Leu) to heterozygotes to homozygous (Val/Val) variants, whereas the effects of these variants were reversed for 8-oxodG. Cluster Analysis was used to group composite genotypes in order to determine if combined genotypes of multiple loci could explain some of the variation seen with the biomarkers, expressed per unit of exposure, referred to as a sensitivity index. This analysis revealed two closely related genotypes each involving four of the loci (GSTM1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1 and GSTT1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1.) that conferred significant resistance to the DNA damaging effects of benzo[a]pyrene, measured as the level of a benzo[a]pyrene-like adduct per unit of benzo[a]pyrene exposed.     

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor,target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.     

Mines over matter: Effects of foliar particulate matter on the herbivorous insect,Helicoverpa armigera

Coalmines, which are major contributors of particulate matter in the form of coal dust, are expanding globally into rural environments. However, ecological effects on organisms interacting with coal‐dusted foliage in mining landscapes are unknown. We tested how the behaviour, development and survival of a polyphageous insect herbivore, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is affected by consuming tomato leaves Lycopersicon esculentum laden with coal dust. We tested (a) feeding site establishment preference of neonates, (b) feeding behaviour and leaf consumption of late‐instar larvae, (c) survival of neonates and (d) survival and development of late‐instar larvae. We found that coal dust consumption increased the mortality of late‐instar larvae but did not influence their development. Despite long‐term implications for survival, late‐instar larvae did not adjust their feeding behaviour or the amount of leaf material consumed in response to foliar coal dust. Contrastingly, when neonate H. armigera were given a choice, they avoided establishing themselves on the coal‐dusted adaxial surface of leaves. Neonate mortality was 99% within 7 days, with no effect of coal dust. Our study provides the first data on the impact of coal dust on an insect herbivore. This has implications for ecological interactions in landscapes adjoining coalmines.     

Hierarchical bivariate time series models: a combined analysis of the effects of particulate matter on morbidity and mortality

In this paper we develop a hierarchical bivariate time series model to characterize the relationship between particulate matter less than 10 microns in aerodynamic diameter (PM10) and both mortality and hospital admissions for cardiovascular diseases. The model is applied to time series data on mortality and morbidity for 10 metropolitan areas in the United States from 1986 to 1993. We postulate that these time series should be related through a shared relationship with PM10. At the first stage of the hierarchy, we fit two seemingly unrelated Poisson regression models to produce city-specific estimates of the log relative rates of mortality and morbidity associated with exposure to PM10 within each location. The sample covariance matrix of the estimated log relative rates is obtained using a novel generalized estimating equation approach that takes into account the correlation between the mortality and morbidity time series. At the second stage, we combine information across locations to estimate overall log relative rates of mortality and morbidity and variation of the rates across cities. Using the combined information across the 10 locations we find that a 10 microg/m3 increase in average PM10 at the current day and previous day is associated with a 0.26% increase in mortality (95% posterior interval -0.37, 0.65), and a 0.71% increase in hospital admissions (95% posterior interval 0.35, 0.99). The log relative rates of mortality and morbidity have a similar degree of heterogeneity across cities: the posterior means of the between-city standard deviations of the mortality and morbidity air pollution effects are 0.42 (95% interval 0.05, 1.18), and 0.31 (95% interval 0.10, 0.89), respectively. The city-specific log relative rates of mortality and morbidity are estimated to have very low correlation, but the uncertainty in the correlation is very substantial (posterior mean = 0.20, 95% interval -0.89, 0.98). With the parameter estimates from the model, we can predict the hospitalization log relative rate for a new city for which hospitalization data are unavailable, using that city's estimated mortality relative rate. We illustrate this prediction using New York as an example.