The aryl hydrocarbon receptor antagonist, 3'methoxy-4'nitroflavone,attenuates 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent regulation of growth factor signaling and apoptosis in the MCF-10A cell line

Previous studies have demonstrated that 2,3,7,8 tetracholorodibenzo-p-dioxin (TCDD) mimics epidermal growth factor receptor (EGFR) signaling in the MCF-10A human mammary epithelial cell line and protects cells from EGF withdrawal-induced apoptosis. These effects appear to be due to the ability of TCDD to increase the expression of transforming growth factor-alpha (TGFalpha), a known EGFR ligand. Because TCDD's effects occurred at concentrations as low as 1 nM, a role for the aryl hydrocarbon receptor (AhR) was hypothesized. In the present study, 3'methoxy-4'nitroflavone (MNF), a known AhR antagonist, was used to analyze AhR signaling in this cell line. MNF suppressed TCDD-dependent dioxin response element (DRE)-driven luciferase activity at concentrations as low as 10 nM. In addition, MNF attenuated TCDD's ability to inhibit apoptosis and to activate Akt and Erk1,2, two EGFR-dependent signaling molecules. Finally, the TCDD-dependent increase in TGFalpha mRNA was also suppressed by MNF. MNF's effects on TCDD action in the MCF-10A cell line occurred at concentrations ranging from 1 nM for Akt phosphorylation and TGFalpha expression to 100 nM for inhibition of apoptosis. Attenuation of TCDD-dependent luciferase activity occurred at concentrations as low as 10 nM, which suggests that TCDD inhibits apoptosis in human mammary epithelial cells by multiple mechanisms.     

腹水中可溶性白介素Ⅱ受体与肿瘤坏死因子的表达研究

目的　探讨良性和恶性腹水中可溶性白介素Ⅱ受体 (SIL 2R)与肿瘤坏死因子 (TNF)水平及其临床意义。方法　采用双抗体夹心ELISA法检测 30例良性、2 4例恶性腹水病人的腹水中SIL 2R与INF表达。结果　良性腹水中SIL 2R与TNF含量分别为 12 10 2 0± 2 6 3 72U/ml、79 32± 2 2 39ng/L ;恶性腹水中SIL 2R与TNF含量分别为 6 10 36± 189 2 3U/ml、2 0 0 1± 2 2 2 5ng/L ;良性腹水组SIL 2R与TNF浓度均明显高于恶性腹水组 ,两组间差异有显著意义 (P <0 0 1) ;联合检测两者诊断恶性腹水的敏感度、特异度、准确度分别为 92 %、83%、90 %。结论　检测腹水中SIL 2R与TNF含量有助于良、恶性腹水的鉴别诊断。     

CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists

1. Cytochrome P450 1B1 (CYP1B1) is highly expressed in testis, but there is conflicting information regarding the inducibility of testicular CYP1B1 by aryl hydrocarbon receptor (AhR) agonists.

2. To assess AhR-mediated regulation, testicular CYP1B1 expression was measured following treatment of adult rats with 3-methylcholanthrene and various dosages of benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The effect of TCDD on CYP1B1 expression in R2C rat Leydig and MA-10 mouse Leydig cells in culture was also determined.

3. Immunoblot analysis showed that treatment with benzo[a]pyrene at dosages up to 200?mg/kg/day and 3-methylcholanthrene at 25?mg/kg/day did not induce testicular CYP1B1 expression. Treatment with TCDD at dosages of 1, 5 or 100 μg/kg had no effect, but testicular CYP1B1 protein levels were increased by approximately 50% at dosages of 10 and 50 μg/kg.

4. CYP1B1 mRNA levels in MA-10 and CYP1B1 protein levels in R2C cells were not induced by exposure to TCDD (10–1000?nM).

5. Overall, the results indicate that rodent testicular CYP1B1 is not inducible by AhR agonists.

     

CD40配基化对肺癌细胞肿瘤坏死因子受体及其相关因子表达的影响

目的 探讨CD40配基化(CD40 ligation)对肺癌细胞株NCI-H460、A549、SPC-A-1的增殖以及对细胞肿瘤坏死因子受体(TNFR)和肿瘤坏死因子受体相关因子(TRAF)表达谱的影响。方法 采用四氮唑盐(MTT)比色法检测CD40配体(CD40 ligand,CD40L)对肺癌细胞增殖的影响,免疫荧光标记和流式细胞术测定细胞表面CD40的表达以及CD40配基化后细胞TNFR和TRAF表达的改变。结果(1)肺癌细胞株NCI-H460、A549和SPC-A-1表面CD40表达分别为(89.2±3.2)％、(42.2±4.5)％、(2.3±0.3)％。(2)CD40配基化后能显著抑制NCI-H460、A549细胞的增殖(P<0.05),而不抑制SPC-A-1的增殖(P>0.05)。(3)CD40配基化可上调NCI-H460、A549细胞TNFRⅠ的表达,下调TNFRⅡ的表达(P<0.05),而不影响SPC-A-1细胞TNFR的表达。(4)CD40配基化可下调NCI-H460、A549细胞中TRAF2、3、5、6的表达(P<0.05),而不影响SPC-A-1中TRAFs的表达。结论 CD40配基化可抑制CD40表达阳性细胞株NCI-H460、A549细胞的增殖,并可引起细胞TNFR、TRAF表达谱的改变,提示CD40信号对CD40表达阳性的肺癌细胞生物学行为具有重要调节作用。     

Effect of biochanin A on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells

Phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. This study examined the action of biochanin A with the carcinogen activation pathway that is mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. A concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and biochanin A markedly reduced the DMBA-inducible EROD activity and CYP1A1 mRNA level. In addition, the biochanin A treatment alone activated the DNA-binding capacity of the AhR for the dioxin-response element (DRE) of CYP1A1, as measured by the electrophoretic-mobility shift assay (EMSA). EMSA revealed that biochanin A reduced the level of the DMBA-inducible AhR-DRE binding complex. Furthermore, biochanin A competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. The biochanin A competitively inhibited the metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. These results suggest that biochanin A may thus be a natural ligand to bind on AhR. Therefore, biochanin A may be due to act an antagonist/agonist of the AhR pathway.     

Effect of mercury on aryl hydrocarbon receptor-regulated genes in the extrahepatic tissues of C57BL/6 mice

The individual toxic effects of aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by mercury (Hg²⁺) has been previously demonstrated. However, little is known about the combined toxic effects of TCDD and Hg²⁺in vivo. Therefore, we examined the effect of exposure to Hg²⁺ (2.5 mg/kg) in the absence and presence of TCDD (15 μg/kg) on the AhR-regulated genes using C57Bl/6 mice. Hg²⁺ alone did not affect kidney, lung, or heart Cyp1a1/1a2/1b1 mRNA levels. On the contrary, Hg²⁺ alone significantly induced kidney Cyp1a1/1a2/1b1 and lung Cyp1b1 protein and catalytic activities. Hg²⁺ also induced Nqo1, Gsta1, and HO-1 at the mRNA, protein, and activity levels in the kidney and heart but not in the lung. Upon co-exposure to Hg²⁺ and TCDD, Hg²⁺ significantly potentiated the TCDD-mediated induction of kidney and lung Cyp1a1/1a2/1b1 mRNA levels, while it decreased their kidney protein and catalytic activity and it increased their lung protein. In addition, Hg²⁺ potentiated the TCDD-mediated induction of Nqo1, Gsta1, and HO-1 at mRNA, protein and activity levels in all tissues. The present study demonstrates that Hg²⁺ modulates the constitutive and TCDD-induced AhR-regulated genes in a time-, tissue- and, AhR-regulated enzyme genes manner.     

TRAF6基因RNA干涉表达载体的构建及鉴定

目的构建针对小鼠肿瘤坏死因子受体相关因子6(TRAF6)的mRNA的siRNA真核表达载体,并鉴定其正确性。方法利用基因重组技术,化学合成用于产生针对TRAF6的发卡状RNA的寡核苷酸,各64个碱基,退火后插入线性化的pSUPER载体的H1启动子下游。重组载体分别用限制性酶切和测序鉴定其正确性。结果限制性酶切和序列测定表明成功构建了可以表达针对TRAF6的发卡状RNA表达载体。结论成功构建了针对TRAF6的发卡状RNA表达载体,为研究TRAF6的功能和进一步的基因治疗提供了实验基础。     

Interactions between polymorphisms in the aryl hydrocarbon receptor signalling pathway and exposure to persistent organochlorine pollutants affect human semen quality

Persistent organic pollutants (POPs) may affect male reproductive function. Many dioxin-like POPs exert their effects by activation of the aryl hydrocarbon receptor (AHR) signalling pathway. We analysed whether gene–environment interactions between polymorphisms in AHR (R554K) and AHR repressor (AHRR P185A) and serum levels of markers of POP exposure 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (p,p′-DDE) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) are associated with 21 parameters of male reproductive function in 581 proven-fertile European and Greenlandic men. In Greenlandic men, AHR variants significantly modified the association between serum levels of both p,p′-DDE and CB-153 and inhibin B levels, sperm chromatin integrity, and seminal zinc levels. In the total cohort, interactions between AHRR variants and serum levels of CB-153 were associated with sperm chromatin integrity and the expression of the pro-apoptotic marker protein Fas. The data indicate that susceptibility to adverse effects of POP exposure on male reproductive function is dependent on polymorphisms in genes involved in AHR signalling.     

血清可溶性肿瘤坏死因子受体Ⅰ、Ⅱ与早期自然流产临床关系的研究

目的：研究早期自然流产妇女血清中可溶性肿瘤坏死因子受体（sTNFR)I和Ⅱ的水平。方法：应用双抗体夹ABC－ELISA法检测孕3月内20例正常妊娠，20例第一次自然流产（spontaneous abortion,SA)和15例反复自然流产（recurrent spontanous abortion,RSA)妇女血清中的sTNFR I与sTNFRⅡ。结果：SA与对照组比较,sTNFR I显著增高（P＜0．05）,sTNFR I与sTNFRⅡ在RSA中的水平较SA均有显著增高（P＜0．01），结论：sTNFR可能与自然流产的发生发展有关，其水平（尤其是sTNFR I)的升高就妊娠而言可能具有自我保护和自我稳定的生理意义。     

二(噁)(口英)致大鼠肺癌与芳香烃受体介导关系的研究

目的用致癌物四氧二苯二氧杂环已二烯(二,TCDD)和苯并(a)芘(B(a)P)联合诱导构建大鼠肺癌的模型,探讨芳香烃受体(AhR)介导TCDD致肺癌的发生机制。方法选Wistar大鼠80只,随机分为ABCD 4组分别代表施以TCDD、TCDD B(a)P、B(a)P染毒的处理组和未施任何染毒处理的对照组,各组在第1.5、3、4.5和6个月,分批系列宰杀,观察肺部癌变情况。结果结果表明,TCDD染毒组在101 d时出现首例肺癌,累计TCDD用量865.94 ng,致癌率为15%,TCDD B(a)P联合染毒组在81 d时出现首例肺癌,TCDD累计用量622.34 ng,B(a)P累计用量为26.83 mg;致癌率30%,B(a)P染毒C组在161 d时,出现首例肺癌,B(a)P累计用量为87.58 mg,致癌率为5%;对照组未出现肺癌。4组之间比较,差异有统计学意义(P<0.05)。结论TCDD和B(a)P成功地诱发了大鼠肺癌,证实了TCDD既是致癌剂又是促癌剂。     

Physiological role of the aryl hydrocarbon receptor in mouse ovary development.

The aryl hydrocarbon receptor (AhR) regulates the toxicity of environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). As the physiological role of the AhR in the ovary is unknown, the purpose of this study was to test the hypothesis that the AhR regulates the appearance and numbers of ovarian follicles. Ovaries were harvested from AhR-deficient (AhRKO) and wild-type mice on gestational day 18 (GD 18) and postnatal days (PND) 2-3, 8, 32-35, and 53. Complete serial sections of ovaries were evaluated histologically for the presence of germ cells and follicles. On GD 18, there was no difference in the number of germ cells per ovary between AhRKO and wild-type fetuses. However, by PND 2-3, AhRKO mice had significantly more fully formed primordial follicles (AhRKO = 38,440 +/- 3632 versus wild-type = 21,120 +/- 2688) and fewer single germ cells than wild-type mice (AhRKO = 12,696 +/- 1192 vs. wild-type = 18,160 +/- 720). On PND 8 and 32-35, there was no difference in the number of follicles between AhRKO and wild-type mice but by PND 53, AhRKO mice had significantly fewer antral follicles than wild-type (AhRKO = 3416 +/- 480 vs. wild-type = 6776 +/- 1024). Taken together, these results suggest that the AhR may play a role in the formation of primordial follicles and the regulation of antral follicle numbers.     

Involvement of aryl hydrocarbon receptor in basal and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced expression of target genes in primary human hepatocytes

Aryl hydrocarbon receptor (AhR) is a drug-sensing receptor activated by environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and is known to drive regulation of target genes in various human cell types. Its involvement in TCDD-mediated regulation of target genes in human hepatocytes however remains to be formally demonstrated. To gain insights into this point, we have analyzed the effects of AhR silencing on the regulation of various genes targeted by TCDD in primary human hepatocytes and highly-differentiated human hepatoma HepaRG cells. Efficient AhR knocking-down was performed through dimethyl sulfoxide-based transfection of small-interfering RNAs targeting AhR (siAhR). SiAhR-transfected human hepatocytes or HepaRG cells, exposed to TCDD, were found to exhibit reduced mRNA expression of various TCDD-responsive genes, i.e. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, IL17RB, FER1L3 and SLC7A5, when compared to TCDD-treated counterparts transfected with non-targeting small-interfering RNAs. AhR silencing was moreover shown to markedly counteract TCDD-mediated induction of CYP1A1/CYP1A2/CYP1B1-related ethoxyresorufin O-deethylase activity in both human hepatocytes and HepaRG cells. It also concomitantly decreased constitutive mRNA expression of some target genes such as CYP1A1, CYP1A2, CYP1B1 and ALDH3A1. Taken together, these data indicate that AhR plays a crucial role in both basal and TCDD-induced expression of target genes in human hepatocytes.     

人肿瘤坏死因子受体75在人肿瘤坏死因子α介导的细胞毒效应中的双重功能

目的：研究人肿瘤坏死因子受体75（hTR75）在hTNFα所引发的细胞毒效应中的功能。方法：用电脉冲将hTR75基因表达载体转入只有内源性hTR55表达的HEp-2细胞中构建了两种TNF受体同时表达的HEp-2-A75细胞株。通过定点突变构建及在大肠杆菌中高效表达,分离纯化出两种分别对hTR55以及hTR75受体具有选择结合活性的hTNFα突变体。野生型hTNFα及其这两种突变体对HEp-2以及HEp-2-A75细胞的细胞毒效应采用比色法测定并进行了比较。结果：RT－PCR以及间接ELISA的结果表明hTR75在RNA转录以及蛋白质翻译水平均获得了表达,其表达量由Scatchard分析确定。HEp-2细胞中hTR75的存在可以明显增强HEp-2细胞对hTNFα的敏感性。结论：hTR75受体在介导hTNFα的细胞毒活性中具有双重功能,即hTR75不但自身可介导部份hTNFα所引发的细胞毒效应,它对hTR55所介导的细胞毒效应还具有相当的协同作用。     

Differences between rats and mice in the involvement of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss

Repeated dosing with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively depletes small pre-antral follicles in the ovaries of rats and mice via apoptosis. The aryl hydrocarbon receptor (AhR) plays a role in mediating the effects of several xenobiotics. Therefore, this study was designed to investigate a potential role of the AhR in VCD-induced ovotoxicity. Female F344 rats, C57BL/6 mice, or AhR-deficient (-/-, AhRKO) mice were dosed daily (15 days) with vehicle, VCD (80 mg/kg, i.p.) and/or the AhR antagonist, alpha-naphthoflavone (ANF; 80 mg/kg, i.p.). Compared with controls, VCD caused a 60% reduction (P < 0.05) in primordial and primary follicles in mice and rats. Concurrent dosing with ANF protected against the VCD-induced follicle loss in rats, but not in mice. As with AhR-intact mice and rats, VCD induced a 70% loss (P < 0.05) of small pre-antral follicles in AhRKO mice. AhR mRNA expression was increased (P < 0.05) by VCD dosing in small pre-antral follicles isolated from ovaries of rats but not mice. AhR protein in rats was increased by VCD dosing in oocyte nuclei in primordial and primary follicles when measured by immunofluorescence and confocal microscopy. In rat small pre-antral follicles, apoptosis-associated caspase-3-like activity was increased (P < 0.05) by VCD treatment, decreased (P < 0.05) by ANF treatment, and unaffected by VCD plus ANF treatment. VCD had no effect on expression of GST Ya1 or GST Ya2 mRNA or CYP 1A1 protein in rats. Taken together, these findings demonstrate a difference between rats and mice in the potential involvement of AhR as related to VCD-induced ovotoxicity. Whereas, AhR appears to be involved in rats, no evidence for a similar role in mice was obtained. Overall, these findings point out that there can be mechanistic species differences in ovarian responses to xenobiotic chemicals.