Organ differences in the impact of p27kip1 deficiency on carcinogenesis induced by N‐methyl‐N‐nitrosourea

To evaluate the impact of p27 on carcinogenesis in various organs, N‐methyl‐N‐nitrosourea (MNU), a direct‐acting alkylating agent, was given to p27 knock‐out mice. Groups of 20–40 male and female mice with null, hetero‐ or wild‐type p27 alleles were given drinking water containing 240 ppm MNU or distilled water every other week for five cycles. The incidence and multiplicity of the induced proliferative lesions were then histologically evaluated at weeks 14 and 20. MNU treatment induced various lesions including squamous hyperplasia and squamous cell carcinoma in the forestomach, atypical hyperplasia and adenocarcinomas in the fundic and pyloric glands, adenomas and adenocarcinomas in the duodenum, malignant lymphomas in the thymus, liver, kidney and spleen and alveolar hyperplasia, adenomas, adenocarcinomas and malignant lymphomas in the lung. Although the incidences of the lesions in the forestomach, fundic and pyloric glands did not differ among the p27 genotypes, those of alveolar hyperplasia of the lung and malignant lymphoma of the thymus were significantly increased in p27‐null males as compared with both wild‐ and hetero‐type animals. Moreover, in both p27^+/+ and p27^+/? cases, the rates for p27‐positive cells were obviously increased in proliferative lesions of the pyloric gland and the lung. However, an increased rate of p27‐positive cells was not observed in malignant lymphoma of the thymus. These findings suggest that p27 does not control the cell cycle equally in all organs affected by MNU‐induced carcinogenesis. Copyright © 2011 John Wiley & Sons, Ltd.     

Increased expression of miR‐34a in mouse spleen one day after exposure to N‐ethyl‐N‐nitrosourea

MicroRNAs (miRNAs) are a class of single‐stranded small RNA molecules (~22 nucleotides) that are not translated into proteins and function as regulators of gene expression. Many miRNAs are involved in carcinogenesis. One of them, miR‐34a, is associated with various p53‐initiated biological processes and may act as a tumor suppressor miRNA. Its expression is generally down‐regulated in tumor tissues and up‐regulated in tissues exposed to carcinogens chronically or subchronically. However, the response of this miRNA to acute exposure of a genotoxic carcinogen is little known. In this study, miR‐34a expression was evaluated in spleen tissues of mice treated with a dose of 120 mg kg^?1 body weight N‐ethyl‐N‐nitrosourea (ENU), a potent mutagenic carcinogen. Real‐time PCR analysis showed that the ENU exposure resulted in a 5.5‐fold increase of miR‐34a expression over the control one day after the treatment. The result suggests that miR‐34a expression responds sensitively to genotoxic insults within a short period after exposure of the mutagen, and therefore, this gene has the potential to be used as an indicator for genotoxin exposure.     

埃他卡林对重要器官心脑肝基因表达的影响

目的利用BioStarR40s cDNA芯片,检测经全新结构类型化合物埃他卡林3mg·kg-1灌胃2wk处理后的大鼠心脏、脑、肝脏组织基因表达的变化,初步判断埃他卡林的基因作用谱;同时探讨埃他卡林给药2wk后,其心脏血流动力学的变化、心肌形态学和超微结构的变化,为阐述埃他卡林的体内生物学作用特征和评价其长期用药安全性提供实验依据。方法①给予埃他卡林(3mg·kg-1)2wk后取大鼠心脏、脑、肝脏组织,抽提总RNA,反转录合成cDNA探针;将荧光标记的cDNA探针与BioStarR-40S芯片杂交,对荧光信号扫描分析。结合RT-PCR方法对芯片结果进行验证。②Wistar♂大鼠随机分为5组:正常对照组、埃他卡林1、3、9mg·kg-1组以及9mg·kg-1撤药组。采用直接插管法,观察麻醉大鼠的血压、心率、收缩参数以及舒张参数的变化;采用HE染色和电镜技术观察不同组对心肌的形态学和超微结构的变化。结果①埃他卡林对心、脑和肝脏基因表达的调节作用具有选择性。采用4096个具有重要功能的基因序列的芯片,埃他卡林3mg·kg-1灌胃2wk,大鼠心脏236个基因的表达发生变化,其中100个基因表达上调,136个基因表达下调;肝脏6个基因表达上调;脑组织的基因表达未发生变化;埃他卡林调节心脏组织的基因表达包括与肌肉收缩相关的肌球蛋白、肌动蛋白和微管蛋白等;与信号转导相关的G蛋白、腺苷酸环化酶和钙调结合蛋白等;②埃他卡林1、3、9mg·kg-1灌胃2wk,在体心脏功能未发现有药理学意义的变化,也未发现心肌组织及其超微结构有病理学意义的变化。结论该实验体系下埃他卡林对重要器官、心、脑和肝脏基因表达的调节作用具有选择性;反复给予埃他卡林未发现其对心脏的不良反应。     

Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.     

四氯化碳损伤小鼠肝脏基因表达谱的变化四氯化碳损伤小鼠肝脏基因表达谱的变化

目的研究CCl₄肝损伤小鼠肝脏的基因表达谱，筛选与CCl₄肝损伤相关的基因网络，探讨其肝损伤机制。方法提取小鼠的肝组织总RNA，经反转录分别用Cy3和Cy5荧光标记，制备用于芯片杂交的cDNA探针；cDNA探针与联合基因公司BioStarM-141S小鼠基因表达谱芯片进行杂交，结果由扫描仪扫描并用软件进行分析统计。结果 在CCl₄肝损伤小鼠的基因表达谱中，有379条基因发生了差异性表达(2.69％)。其中，163条基因表达量明显上调，另外216条基因表达量明显下调。结论CCl₄引起小鼠肝脏基因表达谱变化，利用小鼠基因表达谱芯片能大规模、高通量筛选与CCl₄肝损伤相关基因，对进一步阐明CCl₄及类似的化学肝毒物对肝脏的损伤机制有十分重要的意义。     

Differential gene expression in human peripheral blood mononuclear cells induced by cigarette smoke and its constituents.

In current molecular epidemiology studies, a wide range of methods are used to monitor early biological effects after exposure to xenobiotic agents. Gene expression profiling is considered a promising tool that may provide more sensitive, mechanism-based biomarkers. As a first step toward obtaining information on the applicability of gene expression profiles as a biomarker for early biological effects of carcinogen exposure, we conducted in vitro studies on human peripheral blood mononuclear cells (PBMC). We used cigarette smoke condensate (CSC) and a selection of its genotoxic constituents as model agents, applying cDNA microarray technology to investigate modulated gene expression. In independent experiments using cells from several donors, quiescent PBMC were exposed for 18 h, followed by gene expression analyses on a microarray containing 600 toxicologically relevant genes. The search for candidate biomarker genes was binomial: first we looked for genes responding similarly to all agents; second, for agent-specific genes. Many genes were significantly deregulated by all compounds, but as the direction of deregulation frequently differed per agent, they are not useful as generic biomarkers. Cigarette smoke condensate modulated the expression of many more genes than any of its constituents, with the largest effect in SERPINB2. The affected genes are involved in immune or stress responses, but surprisingly no genes involved in DNA damage response were modulated, and only a few in DNA repair. In conclusion, several genes have been identified as potential biomarkers for population studies on early biological effects caused by cigarette smoke exposure, but no genes were identified that represent a generic biomarker.     

Regulation of cardiac angiotensin‐converting enzyme and angiotensin AT1 receptor gene expression in Npr1 gene‐disrupted mice

1. Understanding of the regulatory mechanisms of gene expression in the control of blood pressure and fluid volume is a key issue in cardiovascular medicine. Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signalling antagonizes the physiological and pathophysiological effects mediated by the renin–angiotensin–aldosterone system (RAAS) in the regulation of cardiovascular homeostasis. 2. The targeted‐disruption of the Npr1 gene (coding for GC‐A/PRA) leads to activation of the cardiac RAAS involved in the hypertrophic remodelling process, which influences cardiac size, expression of pro‐inflammatory cytokine genes and the behaviour of various hypertrophy marker genes. The Npr1 gene‐knockout (Npr1^?/?) mice exhibit 35–40 mmHg higher systolic blood pressure and a significantly greater heart weight to bodyweight ratio than wild‐type (Npr1^+/+) mice. 3. The expression of both angiotensin‐converting enzyme (ACE) and angiotensin II AT_1a receptors are significantly increased in hearts from Npr1^?/? mice compared with hearts from Npr1^+/+ mice. In parallel, the expression of interleukin‐6 and tumour necrosis factor‐α is also markedly increased in hearts from Npr1^?/? mice. 4. These findings indicate that disruption of NPRA/cGMP signalling leads to augmented expression of the cardiac RAAS in conjunction with pro‐inflammatory cytokines in Npr1‐null mutant mice, which promotes the development of cardiac hypertrophy and remodelling.     

双环醇对刀豆蛋白A引起肝损伤小鼠肝脏基因表达谱的影响

本实验研究双环醇对刀豆蛋白A(concanavalin A，Con A)静脉注射引起免疫性肝损伤小鼠肝脏基因表达谱变化的影响，探讨双环醇肝保护作用的分子机制。小鼠于注射Con A 26.5 mg·kg^-1前24、 8及1 h分别口服双环醇250 mg·kg^-1。测定血清丙氨酸转氨酶(alanine aminotransferase，ALT)及天冬氨酸转氨酶(aspartate aminotransferase，AST)水平，提取小鼠肝脏总RNA，经反转录用Cy3-dUTP和Cy5-dUTP分别标记制备cDNA探针。将cDNA探针与BiostarM-40S小鼠基因表达谱芯片进行杂交，经ScanArray 4000扫描仪扫描芯片并用GenePix Pro 3.0软件进行分析。双环醇可显著抑制刀豆蛋白A引起的血清ALT和AST升高。与刀豆蛋白A对照组相比，双环醇给药组有287条基因发生差异表达，占芯片基因总数的7.00%。其中166条基因表达量明显下调，121条基因表达量明显上调。表达变化的基因主要涉及代谢与细胞色素P450、应激与炎症凋亡、细胞周期调控、信号传导以及再生等相关功能。双环醇对刀豆蛋白A引起小鼠肝损伤肝脏基因表达谱变化具有一定的影响，此结果对今后深入研究双环醇的肝脏保护作用特点和临床应用具有重要意义。     

单次大剂量水杨酸钠注射所致大鼠耳蜗基因表达谱改变

目的:研究大剂量水杨酸钠耳毒性的作用机制。方法:运用寡核苷酸基因芯片技术分析大剂量水杨酸钠注射对大鼠耳蜗基因表达谱的影响。联用TRIzol和RNA纯化柱分别从13周正常大鼠和水杨酸钠注射后的大鼠耳蜗内容物提取总RNA,将总RNA逆转录为cDNA,再将cDNA体外转录为cRNA,在此过程中同时进行生物素标记,将cRNA片段化后作为探针与质量检测芯片杂交,确定无问题后再与寡核苷酸基因芯片杂交扫描,用Affymetrix Microarray Suite software 5.0分析处理扫描结果,获得两种情况下耳蜗的基因表达谱。结果:表达上调2倍以上的基因和EST有42个,下调2倍以上的有49个。结论:水杨酸钠影响各种离子通道和突触蛋白基因,可能有助于解释水杨酸钠所致的耳聋和耳鸣作用机制,而其他变化基因亦有助于解释水杨酸钠广泛的药理作用。     

IL-23诱导人外周血单个核细胞对MUTZ-1细胞增殖、凋亡及相关基因表达的影响

目的 白介素-23(IL-23)诱导人外周血单个核细胞对骨髓增生异常综合征(MDS)细胞系MUTZ-1增殖、凋亡及相关基因表达的影响.方法 采用密度梯度离心法分离获得正常人外周血单个核细胞(PBMNC),细胞分4组:阴性对照组(未加IL-23),3个浓度IL-23组(2、10、50 ng/ml),体外诱导72 h,并与MUTZ-1共培养.MTT实验检测MUTZ-1细胞增殖.流式细胞仪检测MUTZ-1细胞凋亡和细胞周期变化.Real time-PCR和Western-blot检测MUTZ-1细胞Bax、Bcl-2、caspase-3、survivin mRNA和蛋白表达.结果 2、10、50 ng/ml IL-23诱导的PBMNC与MUTZ-1细胞共培养后,MUTZ-1细胞增殖速度、S期细胞比例明显降低,细胞凋亡率、G0/G1期细胞比例明显增加,呈时间和浓度依赖性,与阴性对照组比较差异均有统计学意义(P<0.05);同时Bax、caspase-3 mRNA和蛋白表达明显上调,Bcl-2、survivin mRNA和蛋白表达明显下调,呈时间和浓度依赖性,与阴性对照组比较差异均有统计学意义(P<0.05).结论 IL-23诱导PBMNC能够明显抑制MUTZ-1细胞增殖,促进MUTZ-1细胞凋亡,其机制与上调Bax、caspase-3表达、下调Bcl-2、survivin表达有关.     

Glutathione peroxidase‐1 gene rescues cocaine‐induced conditioned place preference in mice by inhibiting σ‐1 receptor expression

The aim of this study was to investigate whether the glutathione peroxidase‐1 gene (GPx‐1) affects cocaine‐induced conditioned place preference (CPP) using a mouse model. Cocaine‐induced CPP was accompanied by an increase in the level of σ‐1 receptor in the nucleus accumbens (NAc). This phenomenon was more pronounced in the GPx‐1 gene knockout (GPx‐1 KO) than in wild type (WT) mice. In contrast, the CPP and expression of σ‐1 receptor were much less pronounced in GPx‐1‐overexpressing transgenic (GPx‐1 TG) mice than non‐transgenic (non‐TG) mice. Treatment of the mice with BD1047 , a σ‐1 receptor antagonist, significantly attenuated both cocaine‐induced CPP and c‐Fos‐immunoreactivity (c‐Fos‐IR) in WT and GPx‐1 KO mice, although the effects were more evident in the latter group. Despite the protective effects of BD1047 on cocaine‐induced CPP and c‐Fos in non‐TG mice, there were no additional protective effects in cocaine‐treated GPx‐1 TG mice, indicating that the σ‐1 receptor is a critical target for GPx‐1‐mediated psychoprotective activity. Overall, our results suggest that GPx‐1 attenuates cocaine‐induced CPP via inhibition of σ‐1 receptor expression.     

Systems toxicology of chemically induced liver and kidney injuries: histopathology‐associated gene co‐expression modules

Organ injuries caused by environmental chemical exposures or use of pharmaceutical drugs pose a serious health risk that may be difficult to assess because of a lack of non‐invasive diagnostic tests. Mapping chemical injuries to organ‐specific histopathology outcomes via biomarkers will provide a foundation for designing precise and robust diagnostic tests. We identified co‐expressed genes (modules) specific to injury endpoints using the Open Toxicogenomics Project‐Genomics Assisted Toxicity Evaluation System (TG‐GATEs) – a toxicogenomics database containing organ‐specific gene expression data matched to dose‐ and time‐dependent chemical exposures and adverse histopathology assessments in Sprague–Dawley rats. We proposed a protocol for selecting gene modules associated with chemical‐induced injuries that classify 11 liver and eight kidney histopathology endpoints based on dose‐dependent activation of the identified modules. We showed that the activation of the modules for a particular chemical exposure condition, i.e., chemical‐time‐dose combination, correlated with the severity of histopathological damage in a dose‐dependent manner. Furthermore, the modules could distinguish different types of injuries caused by chemical exposures as well as determine whether the injury module activation was specific to the tissue of origin (liver and kidney). The generated modules provide a link between toxic chemical exposures, different molecular initiating events among underlying molecular pathways and resultant organ damage. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.     

Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of alpha1- or beta-adrenoceptors, a DNA microarray was used. 2. First, the existence of alpha1- and beta-adrenoceptors in A7r5 aortic smooth muscle cells was confirmed by radioligand binding. Then, the inhibitory effects of phenylephrine (an alpha1-adrenoceptor agonist) and isoproterenol (a beta-adrenoceptor agonist) on the proliferation of A7r5 cells were determined by [3H]-thymidine incorporation. 3. The A7r5 cells were treated with 10 micromol/L phenylephrine or 1 micromol/L isoproterenol for 24 h and changes in gene expression were detected with the DNA microarray. Only 14 and 20 genes were identified after treatment of cells with phenylephrine and isoproterenol, respectively, and most genes displayed decreased expression. The changed genes could be grouped into five major functional categories: cell signalling/communication, cell structure/motility, cell/organism defence, gene/protein expression and metabolism. The gene expression profile in response to the activation of alpha1-adrenoceptors was very different from that following activation of beta-adrenoceptors. Interestingly, many phenylephrine-responsive genes were associated with metabolism, whereas many isoproterenol-responsive genes encoded cell signalling and structure proteins. This means that adrenoceptors may modulate multiple aspects of biological function in vascular smooth muscle cells. 4. Collectively, the activation of both alpha1-adrenoceptors (with phenylephrine) and beta-adrenoceptors (with isoproterenol) inhibited the proliferation of A7r5 cells, but microarray data revealed that the mechanisms may be different: the activation of alpha1-adrenoceptors could induce the expression of metabolic genes, resulting in the inhibition of proliferation, whereas activation of beta-adrenoceptors altered the expression of genes that encoded cell signalling and structure proteins to inhibit cell proliferation.     

昆明种小鼠细胞色素氧化酶CYP1A基因表达的昼夜节律变化

目的探究昆明种小鼠细胞色素氧化酶1A1(CYP1A1)的昼夜节律、性别差异以及对肝毒物毒性变化的影响。方法昆明小鼠在环境控制的SPF饲养室内适应性饲养2周后,于06:00,10:00,14:00,18:00,22:00和次日02:00处死取肝,用逆转录PCR方法检测24h内核激素孤儿受体α(Rev-erbα),时钟基因(Per)1,Per2和CYP1A1,CYP1A2及其调控核受体基因AhR的表达。另取小鼠于6:00和18:00 ip给予对乙酰氨基酚500mg·kg-1,12h后检测血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性。结果细胞色素氧化酶基因CYP1A1,CYP1A2及其调节基因AhR在18:00左右表达最高,且雌鼠的表达高于雄鼠,在6:00左右表达最低,表达峰谷差为4～7倍,其节律变化的差异与Rev-erbα,Per1,Per2的节律差异大致相符。与此节律相对应,对乙酰氨基酚引起的肝毒性在18:00给药高于6:00给药。结论昆明种小鼠细胞色素氧化酶CYP1A1基因表达存在昼夜节律及性别差异,该差异可影响肝毒物如对乙酰氨基酚的代谢和毒性。