重庆市主城区长江和嘉陵江水中有机污染物对H4IIE细胞CYP1A1表达的诱导

目的以重庆市主城区大溪沟(嘉陵江)和寸滩(长江)两个点为代表,研究2004～2005年度重庆市水源水中有机污染物对H4IIE细胞细胞色素P4501A1(CYP1A1)表达的诱导。方法固相萃取法萃取水中有机污染物,将萃取的有机污染物溶于二甲基亚砜(DMSO)中,对H4IIE细胞进行染毒,用逆转录-聚合酶链式反应(RT-PCR)扩增CYP1A1mRNA,比较不同水样诱导H4IIE细胞CYP1A1mRNA表达的差异,用电泳迁移率实验(EMSA)检测CYP1A1启动子的激活情况。结果CYP1A1引物则可以将4个水样12h和24h处理组细胞的cDNA扩增出条带,48h处理组只有2005年1月寸滩样和2005年1月大溪沟样处理的细胞能扩增出CYP1A1基因的条带,所有样品的72h处理组细胞都没有CYP1A1基因的扩增;4个水样处理H4IIE细胞24h后,均可在细胞核内形成可与标记的XRE探针结合的芳烃受体-芳烃受体核转移蛋白(AHR-ARNT)二聚体,从而在AHR-ARNT二聚体蛋白处呈现发光条带。结论各水样均可激活CYP1A1基因的启动子XRE,也均可诱导H4IIE细胞CYP1A1mRNA的表达,CYP1A1mRNA的表达量随处理时间的差异有所不同。     

多环芳烃受体基因与细胞色素P4501A1/1B1基因表达的调控

目的 在体外人神经细胞瘤细胞株(SK-N-AS)中，以二恶英(TCDD)、三甲基胆蒽(3-MC)作为诱导化合物，探讨多环芳烃受体(AHR、ARNT)基因与细胞色素P4501A1／1B1(CYPlAl、CYPlBl)基因表达的调控，并研究其剂量反应关系和时间反应关系。方法 用常规的细胞培养方法，用二甲基亚砜(DMSO)、5．0、50．0、100．0nmol/L TCDD和0.1、1.0、10．0μmol/L 3-MC处理细胞12h、24h、48h、72h，利用提纯RNA和合成cDNA的药盒，合成cDNA，然后通过逆转录聚合跨反应(RT-PCR)表达AHR、ARNT和CYP1A1,CYP1B1基因，以β-actin作为内对照，分析不同处理剂量、时间时基因表达的强度。结果 4种基因在SK-N-AS中都有基本的表达，TCDD在50．0nmol/L以上，染毒处理48h、72h，对AHR、ARNT及CYPlAl基因表达都有上调作用；5．0nmol/L组在染毒处理72h后对AHR基因表达，50．0和100．0nmol/L组分别在染毒处理72h和48h时对CYPlBl基因表达也有上调作用；3-MCl．0、10.0umol/L在染毒72h后对AHR、ARNT、CYPlAl基因表达有上调作用，其中10．0μmol/L组在染毒48h后就有上调作用，但对CYPlBl基因表达上调仅仅在10．0μmol/L染毒48h组。结论 本研究结果提示，TCDD、3-MC两种多环芳烃类化合物在SK-N-AS中，在一定剂量和一定时间状态下对AHR、ARNT、CYPlAl及CYPlBl都有调控作用。     

细胞色素P4501A1-Wt和Asn~(461)、Val~(462)真核表达载体的构建及人胚肺成纤维细胞转染

目的构建能够高效表达细胞色素P4501A1(CYP1A1)蛋白的细胞株,分析不同基因型蛋白表达的差异。方法首先将已经建立的T载体中CYP1A1-Wt和Asn461、Val462的CYP1A1片断重组至pBABE-neo载体上,转染入人胚肺成纤维细胞(HLF),G418筛选获得阳性细胞,Western-Blot检测CYP1A1蛋白的表达,观察细胞形态,生长曲线,恶变性等生物学特性的改变。结果CYP1A1野生型、变异型转染的HLF细胞均能高效表达CYP1A1蛋白,其生物学特性与正常细胞无显著差异,也不属于恶性细胞。结论通过细胞转染和筛选获得了表达CYP1A1蛋白的细胞株。     

Haplotypes in the GC,CYP2R1 and CYP24A1 Genes and Biomarkers of Bone Mineral Metabolism in Older Adults

Candidate gene studies have analyzed the effect of specific vitamin D pathway genes on vitamin D availability; however, it is not clear whether genetic variants also affect overall bone metabolism. This study evaluated the association between genetic polymorphisms in GC, CYP2R1 and CYP24A1 and serum levels of total 25(OH)D, iPTH and other mineral metabolism biomarkers (albumin, total calcium and phosphorus) in a sample of 273 older Spanish adults. We observed a significant difference between CYP2R1 rs10741657 codominant model and total 25(OH)D levels after adjusting them by gender (p = 0.024). In addition, the two SNPs in the GC gene (rs4588 and rs2282679) were identified significantly associated with iPTH and creatinine serum levels. In the case of phosphorus, we observed an association with GC SNPs in dominant model. We found a relationship between haplotype 2 and 25(OH)D levels, haplotype 4 and iPTH serum levels and haplotype 7 and phosphorus levels. In conclusion, genetic variants in CYP2R1 and GC could be predictive of 25(OH)D and iPTH serum levels, respectively, in older Caucasian adults. The current study confirmed the role of iPTH as one of the most sensitive biomarkers of vitamin D activity in vivo.     

吉林市汉族妇女GSTM1基因和CYP1A1基因Exon7位点多态性与 子宫内膜异位症易感性的研究

目的:探讨在吉林地区汉族妇女中细胞色素P450(CYP1A1)基因Exon7位点多态性即Ile-Val位点的多态性及GSTM1基因多态性和子宫内膜异位症易感性的相关关系。方法:以病例对照的研究方法,采用PCR技术检测216例子宫内膜异位症和216例对照人群的CYP1A1基因Ile-Val位点及GSTM1基因多态性的表达。结果:吉林地区汉族人群中GSTM1空白基因型分布频率0.463,内异症人群中空白基因型分布频率0.667,两组差异有统计学意义(P<0.05),空白基因型患内异症的危险是功能基因型的1.896倍;Ile-Val三种多态基因型在内异症组和对照组分布差异有统计学意义(P<0.05),Ile/Val、Val/Val基因型患内异症的危险分别是Ile/Ile基因型的1.901倍和3.056倍;CYP1A1 Ile/Val联合GSTM1空白基因型个体的OR值为3.409(95%C I 1.897~6.125,P<0.01),而CYP1A1Val/Val联合GSTM1空白基因型个体的OR值增高至7.143(95%C I 2.584~19.742,P<0.01)。结论:CYP1A1 Exon7的Ile/Val、Val/Val基因型及GSTM1空白基因型与内异症的易感性有关,二者联合效应具有协同作用,可望作为内异症易感人群筛选的重要指标。     

Maternal smoking during pregnancy, polymorphic CYP1A1 and GSTM1, and lung-function measures in urban family children

Purpose

Understanding the interplay between genes and in-utero tobacco exposure in affecting child lung development is of great significance. In this study, we tested the hypothesis that tobacco-related lung-function reduction in children differs by maternal polymorphic genes Cytochrome P450 1A1 (CYP1A1) and Glutathione S-transferase Mu 1 (GSTM1).

Materials and methods

Data were collected among 370 children (6–10 years old, 81.6% African-Americans) and their biological mothers visiting a large children’s hospital. Study hypotheses were tested using multiple regression method.

Results

Among the study sample, 143 mothers smoked throughout pregnancy and 72 smoked on a daily basis. Spirometric measures (mean±SD) included were: forced vital capacity (FVC)=1635±431 mL, forced expiratory volume in the first 1 s (FEV₁)=1440 ±360 mL, percent FEV₁/FVC ratio=89±12, and forced expiratory flow between the 25% and 75% of FVC (FEF_25–75)=1745±603 mL. In addition to a tobacco effect on FVC (−131 mL, 95% CI: −245, −17) and FEV₁/FVC ratio (42, 95% CI: 1, 83), regression analysis controlling for covariates indicated that for the subsample of children whose mothers were CYP1A1?2A homozygous, maternal daily smoking was associated with −734 mL (95% CI: −1206, −262) reductions in FEV₁ and −825 mL (95% CI: −909, −795) reductions in FVC; reduced smoking was still associated with −590 mL (95% CI: −629, −551) reductions in FVC. For children of mothers with GSTM1 deletion, persistent daily smoking was associated with −176 mL (95% CI: −305, −47) reductions in FVC.

Discussion and conclusions

Maternal smoking during pregnancy was significantly associated with lung-function reduction in children, particularly for those whose mothers possessed the polymorphic CYP1A1*2A and GSTM1 deletion.     

In utero and lactational exposure to PCBs in mice: adult offspring show altered learning and memory depending on Cyp1a2 and Ahr genotypes

Background: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2)-uninduced basal levels and > 12-fold variability in aryl hydrocarbon receptor (AHR)affinity; because CYP1A2 is known to sequester coplanar PCBs and because AHR ligands include coplanar PCBs, both genotypes can affect PCB response.Objectives: We aimed to develop a mouse paradigm with extremes in Cyp1a2 and Ahr genotypes to explore genetic susceptibility to PCB-induced developmental neurotoxicity using an environmentally relevant mixture of PCBs.Methods: We developed a mixture of eight PCBs to simulate human exposures based on their reported concentrations in human tissue, breast milk, and food supply. We previously characterized specific differences in PCB congener pharmacokinetics and toxicity, comparing high-affinity–AHR Cyp1a2 wild-type [Ahr^b1_Cyp1a2(+/+)], poor-affinity–AHR Cyp1a2 wild-type [Ahr^d_Cyp1a2(+/+)], and high-affinity–AHR Cyp1a2 knockout [Ahr^b1_Cyp1a2(–/–)] mouse lines [Curran CP, Vorhees CV, Williams MT, Genter MB, Miller ML, Nebert DW. 2011. In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes. Toxicol Sci 119:189–208]. Dams received a mixture of three coplanar and five noncoplanar PCBs on gestational day 10.5 and postnatal day (PND) 5. In the present study we conducted behavioral phenotyping of exposed offspring at PND60, examining multiple measures of learning, memory, and other behaviors.Results: We observed the most significant deficits in response to PCB treatment in Ahr^b1_Cyp1a2(–/–) mice, including impaired novel object recognition and increased failure rate in the Morris water maze. However, all PCB-treated genotypes showed significant differences on at least one measure of learning or behavior.Conclusions: High levels of maternal hepatic CYP1A2 offer the most important protection against deficits in learning and memory in offspring exposed to a mixture of coplanar and noncoplanar PCBs. High-affinity AHR is the next most important factor in protection of offspring.