Integrative analysis of mRNA and microRNA expression of a human alveolar epithelial cell(A549) exposed to water and organic‐soluble extract from particulate matter (PM)2.5

MicroRNA (miRNA) is now attracting attention as a powerful negative regulator of messenger RNA(mRNA) levels, and is implicated in the modulation of important mRNA networks involved in toxicity. In this study, we assessed the effects of particulate matter 2.5 (PM_2.5), one of the most significant air pollutants, on miRNA and target gene expression. We exposed human alveolar epithelial cell (A549) to two types of PM_2.5[water (W‐PM_2.5) and organic (O‐PM_2.5) soluble extracts] and performed miRNA microarray analysis. A total of 37 miRNAs and 62 miRNAs were altered 1.3‐fold in W‐PM_2.5 and O‐PM_2.5, respectively. Integrated analyses of miRNA and mRNA expression profiles identified negative correlations between miRNA and mRNA in both W‐PM_2.5 and O‐PM_2.5 exposure groups. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses showed that the 35 W‐PM_2.5 target genes are involved in responses to nutrients, positive regulation of biosynthetic processes, positive regulation of nucleobase, nucleoside, and nucleotide, and nucleic acid metabolic processes; while the 69 O‐PM_2.5 target genes are involved in DNA replication, cell cycle processes, the M phase, and the cell cycle check point. We suggest that these target genes may play important roles in PM_2.5‐induced respiratory toxicity by miRNA regulation. These results demonstrate an integrated miRNA‐mRNA approach for identifying molecular events induced by environmental pollutants in an in vitro human model. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 302–310, 2017.     

Benzo[a]pyrene induced p53‐mediated cell cycle arrest,DNA repair,and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus)

The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45β, igfbp‐3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p < 0.05), but gadd45β and igfbp‐3 expression was not significantly changed. Microphotographs revealed enlargement of the cell nuclei and cellular degeneration in males, while atrophy and vacuolization of hepatocytes were observed in females (10 µg/L). These results suggested that BaP induced liver DNA repair and apoptosis pathways and caused adverse pathological changes in rare minnow. The strongly responsive p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979–988, 2017.     

m6A修饰调控的COL6A5对肺腺癌细胞侵袭能力的影响

目的 明确6型胶原蛋白α5（COL6A5）在肺腺癌中的表达及意义。方法 通过在线数据库及实时荧光定 量PCR（qPCR）法检测COL6A5在肺腺癌细胞和组织中的表达。采用GEPIA数据库分析COL6A5的表达量与肺腺癌 患者预后的相关性。生物信息学预测结合 RNA 甲基化免疫沉淀-实时荧光定量 PCR（MeRIP-qPCR）实验检测 COL6A5转录本上的m6A位点。qPCR法检测RNA去甲基酶ALKBH5在肺腺癌组织中的表达量,并分析ALKBH5与 COL6A5在27例肺腺癌组织中表达的相关性。qPCR和Western blot检测ALKBH5对COL6A5的抑制作用。生物信息 学预测结合Transwell实验和Western blot验证COL6A5在肺腺癌中的作用。结果 在线数据库Oncomine分析显示, COL6A5 在肺癌中低表达,GEPIA 数据库分析显示肺腺癌组织中 COL6A5 mRNA 表达水平显著低于正常组织（P＜ 0.05）。qPCR 结果显示,COL6A5 mRNA 在 27 例肺腺癌组织中的表达量低于癌旁组织;与正常肺上皮细胞相比, COL6A5 mRNA在肺腺癌细胞中的表达量明显受到抑制（P＜0.05）。生物信息学预测结合MeRIP-qPCR 实验证实, COL6A5 mRNA上存在m6A位点。肺腺癌组织中ALKBH5 mRNA的表达水平高于癌旁正常组织,且与COL6A5 mRNA 表达呈负相关（r=-0.599,P＜0.01）。在H1299细胞中敲低ALKBH5,可显著上调COL6A5的表达。分析与COL6A5共 表达的基因,结果显示 COL6A5 可能参与调控肺腺癌细胞上皮间质转换、止血和细胞表面相互作用等生命活动。 Transwell和Western blot证实,过表达COL6A5可显著抑制H1299细胞的侵袭转移能力。结论 ALKBH5在肺腺癌中 下调COL6A5,过表达COL6A5可明显抑制肺腺癌细胞的侵袭转移能力。     

基于数据挖掘分析GPR87在非小细胞肺癌中的表达及临床意义

目的 探索G蛋白偶联受体87（GPR87）在非小细胞肺癌（NSCLC）中的表达及意义。方法 通过GE-IA在线工具分析TCGA数据库中GPR87 mRNA在NSCLC和正常肺组织中的表达情况,并进一步分析GPR87基因表达与NSCLC患者TNM分期和总生存期（OS）之间的关系。利用String在线工具分析与GPR87相互作用的蛋白。结果 GPR87 mRNA在肺腺癌（LUAD）和肺鳞癌（LUSC）组织中的表达水平均明显高于正常肺组织（P<0.01）。GPR87 mRNA高表达倾向于与NSCLC患者临床进展期有关,但差异无统计学意义（P>0.05）。TCGA数据库中,GPR87 mRNA高表达组LUAD患者的OS明显短于低表达组（P=0.006 5）,并在Kaplan-Meier Plotter数据库中得到了进一步验证（P=0.029）。然而,在TCGA数据库中,GPR87 mRNA高表达组LUSC患者的OS明显长于低表达组（P=0.036）,但在Kaplan-Meier Plotter数据库中差异无统计学意义（P=0.35）。String分析显示,LPAR1、LPAR2、LPAR3、SCIN、OR56A3、OR52W1、OR52L1、OR56B4、OR56A1、OR52B2等蛋白与GPR87有明显的相互作用。结论 基于肿瘤基因数据库信息挖掘,GPR87在NSCLC组织中呈高表达,并与LUAD患者预后不良相关,可能是LUAD的潜在分子标志物。     

Gene expression profile changes induced by acute toxicity of [C16mim]Cl in loach (Paramisgurnus dabryanus)

Ionic liquids (ILs) are widely used as reaction media in various commercial applications. Many reports have indicated that most ILs are poorly decomposed by microorganisms and are toxic to aquatic organisms. In this study, differential gene expression profiling was conducted using a suppression subtraction hybridization cDNA library from hepatic tissue of the loach (Paramisgurnus dabryanus) after exposure to 1‐hexadecyl‐3‐methylimidazolium chloride ([C₁₆mim]Cl), a representative IL. Two hundred and fifty‐nine differentially expressed candidate genes, whose expression was altered by >2.0‐fold by the [C₁₆mim]Cl treatment, were identified, including 127 upregulated genes and 132 downregulated genes. A gene ontology analysis of the known genes isolated in this study showed that [C₁₆mim]Cl‐responsive genes were involved in cell cycle, stimulus response, defense response, DNA damage response, oxidative stress responses, and other biological responses. To identify candidate genes that may be involved in [C₁₆mim]Cl‐induced toxicity, 259 clones were examined by Southern blot macroarray hybridization, and 20 genes were further characterized using quantitative real‐time polymerase chain reaction. Finally, six candidate genes were selected, including three DNA damage response genes, two toxic substance metabolic genes, and one stress protein gene. Our results indicate that these changes in gene expression are associated with [C₁₆mim]Cl‐induced toxicity, and that these six candidate genes can be promising biomarkers for detecting [C₁₆mim]Cl‐induced toxicity. Therefore, this study demonstrates the use of a powerful assay to identify genes potentially involved in [C₁₆mim]Cl toxicity, and it provides a foundation for the further study of related genes and the molecular mechanism of [C₁₆mim]Cl toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 404–416, 2017.     

Screening Feature Genes of Venous Thromboembolism with DNA Microarray

We aimed to explore the potential genes or pathways related to venous thromboembolism (VTE) and expected our findings could contribute to the development of new target drugs for VTE. The gene expression profile of GSE19151 was downloaded from Gene Expression Omnibus (GEO) database. The bioinformatics methods were applied to screen the feature genes and pathways related with VTE. A total of 115 DEGs were identified, including 25 downregulated genes and 90 upregulated genes. Function enrichment analysis showed that upregulated genes of VTE were mainly enriched in ribosome and translation‐related pathways, while downregulated genes were mainly enriched in cytoskeletal protein binding and non‐membrane‐bounded organelle‐related pathways. MCL1, TP53, and RERE were three outstanding genes involved in the interaction network. The most significant pathways enriched by module genes were ribosome and oxidative phosphorylation. Moreover, all the products of the 18 genes enriched in ribosome (hsa03010) were ribosomal proteins. Ribosome, translation, actin binding, and non‐membrane‐bounded organelle pathways were closely related to the development of VTE. Moreover, MCL1, TP53, and RERE might play key roles in the process of VTE.     

Increased expression of TTC21A in lung adenocarcinoma infers favorable prognosis and high immune infiltrating level

BackgroundLung adenocarcinoma (LUAD) is a crucial pathological type of lung cancer. Immune-infiltration of the tumor microenvironment positively associated with overall survival in LUAD. TTC21A is a gene has not reported in cancer, and the mechanism behind it is still unclear. Our study assesses TTC21A role in LUAD, via TCGA data.MethodsGEPIA was utilized to analyze the expression of TTC21A in LUAD. We evaluated the influence of TTC21A on survival of LUAD patients by survival module. Then, data sets of LUAD were downloaded from TCGA. The correlations between clinical information and TTC21A expression were analyzed using logistic regression. Clinicopathologic characteristics associated with overall survival in TCGA patients using Cox regression. In addition, we explored the correlation between TTC21A and cancer immune infiltrates using CIBERSORT and “Correlation” module of GEPIA.ResultsThe univariate analysis using logistic regression, wherein TTC21A expression served as a categorical dependent variable (with a median expression value of 2.5), indicated that increased TTC21A expression is significantly correlated with pathological stage, tumor status and lymph nodes. Moreover, multivariate analysis revealed that the up-regulated TTC21A expression, negative results of pathological stage and distant metastasis are independent prognostic factors for good prognosis. Specifically, a positive correlation between increased TTC21A expression and immune infiltrating level of B cells, Neutrophils, Mast cells and T cells was established using CIBERSORT analysis. Furthermore, we confirmed it in “correlation” module of GEPIA.ConclusionTogether with all these findings, increased TTC21A expression correlates with favorable prognosis and increased proportion of immune cells, such as B cells, Neutrophils, Mast cells and T cells in LUAD. These conclusions indicate that TTC21A could serve as a potential biomarker to assess prognosis and immune infiltration level in LUAD.     

Pathway analysis of gene expression in local lymph nodes draining skin exposed to three different sensitizers

Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT‐PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway‐based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top‐ranked functions (P < 0.01) identified as being affected by the sensitizers were associated with aspects of cell growth, such as DNA replication, cell cycle regulation and pyrimidine metabolism. All the sensitizers tested (DNCB, OXA and TDI) induced significant up‐regulation of Psme4, which is associated with DNA replication; Tfdp1, which is related to cell cycle regulation; and Dut, which is involved in pyrimidine metabolism. Specific changes were also shown in functional categories related to the immune response, including cytokines and their receptors. Genes identified in these functional categories, such as Ccl21c, Cxcl9, Cxcl10, Ifng and Il12rb1, were found to have functional relevance. These findings may enhance our understanding and assessment of chemical sensitizers, and enable us to distinguish sensitizers from irritants and to classify chemicals as contact sensitizers. Copyright © 2011 John Wiley & Sons, Ltd.     

NUF2基因表达与肺腺癌免疫浸润及临床预后的相关性分析

目的： 研究NUF2与肺腺癌（lung adenocarcinoma，LUAD）免疫细胞浸润及预后的关系。方法： 采用Oncomine数据库分析NUF2在LUAD和肺鳞癌（lung squamous cell carcinoma，LUSC）组织中的表达水平；用GEPIA和PrognoScan数据库评估NUF2对LUAD和LUSC患者预后的影响；采用TIMER数据库分析LUAD中NUF2表达和免疫细胞浸润水平的相关性；采用LinkedOmics数据库进行NUF2相关差异表达基因的GO功能注释和KEGG通路分析。RT-qPCR和Western Blot检测NUF2在肺腺癌细胞中的表达，并采用集落形成实验研究NUF2对肺腺癌细胞增殖的影响。结果： NUF2在LUAD和LUSC组织中的表达水平显著高于正常组织（P<0.01）。NUF2高表达与LUAD患者的总生存率和无疾病生存率负相关（P<0.05），但与LUSC患者的总生存率无相关性。NUF2表达与LUAD组织中B细胞、CD4⁺ T细胞、巨噬细胞和树突状细胞的浸润水平显著负相关（P<0.01）。NUF2相关基因富集在氧化磷酸化等通路，参与的生物学过程有细胞周期检查点和DNA重组等。NUF2在人肺腺癌细胞中高表达，且敲减NUF2可抑制肺腺癌细胞集落形成（P<0.01）。结论： NUF2高表达与LUAD免疫细胞浸润水平降低和不良预后相关，有望成为LUAD潜在的预后标志物和免疫相关治疗靶点。     

Effects of decabromodiphenyl ether (BDE‐209) on mRNA transcription of thyroid hormone pathway and spermatogenesis associated genes in Chinese rare minnow (Gobiocypris rarus)

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants, which are ubiquitous environmental contaminant found in both abiotic and biotic environmental samples. Deca‐BDE (BDE‐209) is the principal component, which is currently used worldwide. In this study, the effect of BDE‐209 on the mRNA levels of thyroid hormone (TH) related genes and spermatogenesis associated genes were determined from larvae and adult rare minnow (Gobiocypris rarus) exposed to concentrations 0.01, 0.1, 1, and 10 μg/L for 21 days. The results showed that the type II deiodinase (dio2) and sodium iodide symporter (nis) mRNA levels were significantly up‐regulated in the larvae at 10 μg/L treatment. In adult, histopathological observations showed that liver of female fish were degenerated at 10 μg/L treatment, and inhibition of spermatogenesis were observed in testis of male fish. In addition, the thyroid hormone receptor α (trα), dio2, and nis mRNA levels in the liver of male and female fish were significantly up‐regulated, whereas dio2 and nis mRNA levels were significantly down‐regulated in the brain. These results indicate that exposure to BDE‐209 could result in tissue‐specific alternations of TH‐related genes expression in adults. Moreover, the mRNA levels of the testis‐specific apoptosis genes, the spermatogenesis‐associated 4 (spata4) and spermatogenesis‐associated 17 (spata17), were down‐regulated at 10 μg/L treatment in testis of male fish. Our results suggest that BDE‐209 may pose threat to normal thyroid and reproductive function in fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 1–9, 2014.     

人脐带间充质干细胞外泌体的鉴定及其作用于肺动脉高压的生物信息学分析

目的 探讨人脐带间充质干细胞外泌体(human umbilical cord mesenchymal stem cell exosomes,hUCMSC-Exo)缓解肺动脉高压(pulmonary hypertension,PH)的潜在分子作用机制。方法 对hUCMSC-Exo进行提取、鉴定及高通量测序。利用GeneCards搜索PH作用靶点,在GEO数据库下载PH相关芯片(GSE53408、GSE113439),经过批次校正后筛选出差异基因。GeneCards和GEO数据库以及hUCMSC-Exo所含miRNAs预测的靶向调控的mRNAs取交集,构建蛋白互作网络筛选出hUCMSC-Exo作用于PH的关键靶点,并进行GO和KEGG富集分析。结果 测序得到hUCMSC-Exo中43个高表达的miRNAs,其中17个miRNAs可作用于PH,发现其作用机制可能与前列腺癌、HIF-1信号通路、PI3K-Akt信号通路、癌症的蛋白多糖和癌症通路等相关。结论 本研究可为hUCMSC-Exo防治PH的临床研究提供新思路。     

Genotoxic effects of the cyanobacterial hepatotoxin cylindrospermopsin in the HepG2 cell line

The cyanobacterial alkaloid cylindrospermopsin (CYN) is being increasingly identified in drinking water supplies worldwide. It is a potent protein synthesis inhibitor and causes human intoxications and animal mortality. The few genotoxicity studies available indicate that CYN is genotoxic, generally implying that it is pro-genotoxic. We evaluated CYN genotoxicity in the human hepatoma cell line, HepG2, analyzing the induction of DNA strand breaks, with the alkaline comet assay, and micronuclei (MNi), nuclear bud (NBUD), and nucleoplasmic bridge (NPB) formation, with the cytokinesis block micronucleus (CBMN) assay. In addition, changes in the expression of genes involved in the response to DNA damage (P53, CDKN1A, GADD45α, and MDM2) and genes presumably involved in CYN metabolism (genes from the Cytochrome P450 family: CYP1A1 and CYP1A2) were determined, using quantitative real-time PCR. Non-cytotoxic concentrations of CYN induced increased DNA damage after 12 and 24 h of exposure and increased the frequency of MNi, NBUDs, and NPBs after 24 h exposure. Moreover, CYN up-regulated the expression of the CYP1A1 and CYP1A2 genes. Although no changes in the expression of the P53 tumor-suppressor gene were found, CYN up-regulated the expression of the P53 downstream-regulated genes CDKN1A, GADD45α, and MDM2. Our results provide new evidence that CYN is genotoxic and strongly suggest that it needs to be considered in the human health risk assessment.     

Fluoxetine-induced hepatic lipid accumulation is mediated by prostaglandin endoperoxide synthase 1 and is linked to elevated 15-deoxy-Δ12,14PGJ2

Major depressive disorder and other neuropsychiatric disorders are often managed with long-term use of antidepressant medication. Fluoxetine, an SSRI antidepressant, is widely used as a first-line treatment for neuropsychiatric disorders. However, fluoxetine has also been shown to increase the risk of metabolic diseases such as non-alcoholic fatty liver disease. Fluoxetine has been shown to increase hepatic lipid accumulation in vivo and in vitro. In addition, fluoxetine has been shown to alter the production of prostaglandins which have also been implicated in the development of non-alcoholic fatty liver disease. The goal of this study was to assess the effect of fluoxetine exposure on the prostaglandin biosynthetic pathway and lipid accumulation in a hepatic cell line (H4-II-E-C3 cells). Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes (Ptgs1, Ptgs2, and Ptgds), PPAR gamma (Pparg), and PPAR gamma downstream targets involved in fatty acid uptake (Cd36, Fatp2, and Fatp5) as well as production of 15-deoxy-Δ^12,14PGJ₂ a PPAR gamma ligand. The effects of fluoxetine to induce lipid accumulation were attenuated with a PTGS1 specific inhibitor (SC-560), whereas inhibition of PTGS2 had no effect. Moreover, SC-560 attenuated 15-deoxy-Δ^12,14PGJ₂ production and expression of PPAR gamma downstream target genes. Taken together these results suggest that fluoxetine-induced lipid abnormalities appear to be mediated via PTGS1 and its downstream product 15d-PGJ₂ and suggest a novel therapeutic target to prevent some of the adverse effects of fluoxetine treatment.     

Transcript profiling and DNA damage in the European eel (Anguilla anguilla L.) exposed to 7,12-dimethylbenz[a]anthracene

The molecular responses induced during and after an acute exposure to 7,12-dimethylbenz[a]anthracene (DMBA) were analysed in liver, gill and blood cells of juvenile Anguilla anguilla with the aim of developing molecular biomarkers of environmental PAH pollution. Changes in the mRNA expression levels of the cell cycle checkpoint-related rad1 gene and the mRNAs of differentially expressed genes by suppression subtractive hybridization (SSH) were analysed in the liver, and related to well-established biomarkers: cyp1A1 mRNA expression and assessment of the DNA integrity using the comet assay and flow cytometry. DMBA exposure resulted in increased cyp1A1 mRNA levels, suggesting that cyp1A1 might be involved in the metabolism of DMBA. Global DNA damage, detected by the comet assay, was observed in the three tissues analysed but only blood cells showed chromosomal lesions as analysed by flow cytometry. Although DNA damage was found in the liver, no induction in rad1 gene was observed in this organ. The global SSH approach revealed that mRNAs of genes related to xenobiotic metabolism, immune processes and cytoskeleton dynamics were differentially expressed in DMBA-exposed eel livers, highlighting the complexity in the response observed in fish exposed to a genotoxic agent and providing directions for new biomarker development.     

柔性脂质体在经皮给药系统中的研究进展

目的 采用响应面法优化北豆根脂肪油的提取工艺，分析其成分组成，并研究北豆根脂肪油的体外抗氧化活性。方法 以北豆根脂肪油提取率为指标，在单因素试验基础上，采用响应面法考察提取温度、提取时间、料液比对提取工艺的影响；采用气相色谱-质谱联用法 (GC-MS) 分析脂肪油的组成；运用清除1,1-二苯基-2-三硝基苯肼（DPPH）自由基实验，评价北豆根脂肪油的体外抗氧化活性。结果 经响应面优化，确定最佳提取工艺为提取温度温度90 ℃，回流提取3次，每次2.3 h，料液比为1:21。在这些条件下，提取率为1.820 %；采用GC-MS从北豆根脂肪油中鉴定出32 个成分；北豆根脂肪油总抗氧化活性随着浓度的增加而逐渐升高，在浓度达到0.833 mg/mL后对DPPH自由基清除率的增幅变缓，当浓度为1.333mg/mL时，清除率为70.1%。结论 优化的工艺方便、稳定、可行，可为后续研究提供依据，北豆根脂肪油中含有多种重要脂肪油成分, 并具有一定的体外抗氧化能力。     

Sex-biased ceRNA networks reveal that OSCAR can promote proliferation and migration of lung adenocarcinoma in women

Lung adenocarcinoma (LUAD) is one of several malignant tumours with the highest incidence rates. Currently, there is an urgent need for effective diagnostic and therapeutic targets for LUAD in clinical practice. Numerous studies have shown that there may be differences in the development pattern of LUAD between male and female patients, leading to the need for differential treatment. At the same time, previous studies have shown that competitive endogenous (ce)RNA plays an important role in the development of LUAD, but there is no relevant research on whether there is a gender difference in the ceRNA network of LUAD. In this study, we constructed gender-independent, male-specific, and female-specific ceRNA networks using RNA sequencing results from TCGA database. Subsequently, through analysis of the core genes of the ceRNA network, we determined that the male and female ceRNA networks indeed display different features. In addition, we also found that the osteoclast-associated receptor (OSCAR) gene was a potential diagnostic target for detecting LUAD in females, and that increased expression of this gene promoted the proliferation and migration of A549 and H1975 LUAD cell lines; more specifically, A549 and H1975 are male and female LUAD cell lines, respectively. This suggests that the OSCAR gene has the potential to serve as target molecule for the diagnosis and treatment of female-specific LUADs.     

Prenatal exposure to di-n-butyl phthalate (DBP) differentially alters androgen cascade in undeformed versus hypospadiac male rat offspring

This study was to compare the alterations of androgen cascades in di-n-butyl phthalate (DBP)-exposed male offspring without hypospadias (undeformed) versus those with hypospadias. To induce hypospadias in male offspring, pregnant rats received DBP via oral gavage at a dose of 750 mg/kg BW/day during gestational days 14–18. The mRNA expression levels of genes downstream of the androgen signaling pathway, such as androgen receptor (AR) and Srd5a2, in testes of undeformed rat pups were similar to those in controls; in hypospadiac rat pups these levels were significantly lower than those of control pups. In contrast, both undeformed and hypospadiac rats had decreased serum testosterone levels, reduced mRNA expression of key enzymes in the androgen synthetic pathway in the testes, and ablated genes of developmental pathways, such as Shh, Bmp4, Fgf8, Fgf10 and Fgfr2, in the genital tubercle (GT) as compared to those in DBP-unexposed controls, albeit hypospadiac rats had a more severe decrement than those of undeformed rats. Although other possibilities cannot be excluded, our findings suggest that the relatively normal levels of testosterone-AR-Srd5a2 may contribute to the resistance to DBP toxicity in undeformed rats. In conclusion, our results showed a potential correlation between decreased testosterone levels, reduced mRNA expression of AR and Srd5a2 and the occurrence of hypospadias in male rat offspring prenatally exposed to DBP.