Pathway modeling of microarray data: a case study of pathway activity changes in the testis following in utero exposure to dibutyl phthalate (DBP).

Pathway activity level analysis, the approach pursued in this study, focuses on all genes that are known to be members of metabolic and signaling pathways as defined by the KEGG database. The pathway activity level analysis entails singular value decomposition (SVD) of the expression data of the genes constituting a given pathway. We explore an extension of the pathway activity methodology for application to time-course microarray data. We show that pathway analysis enhances our ability to detect biologically relevant changes in pathway activity using synthetic data. As a case study, we apply the pathway activity level formulation coupled with significance analysis to microarray data from two different rat testes exposed in utero to Dibutyl Phthalate (DBP). In utero DBP exposure in the rat results in developmental toxicity of a number of male reproductive organs, including the testes. One well-characterized mode of action for DBP and the male reproductive developmental effects is the repression of expression of genes involved in cholesterol transport, steroid biosynthesis and testosterone synthesis that lead to a decreased fetal testicular testosterone. Previous analyses of DBP testes microarray data focused on either individual gene expression changes or changes in the expression of specific genes that are hypothesized, or known, to be important in testicular development and testosterone synthesis. However, a pathway analysis may inform whether there are additional affected pathways that could inform additional modes of action linked to DBP developmental toxicity. We show that Pathway activity analysis may be considered for a more comprehensive analysis of microarray data.

Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl phthalate toxicogenomic data set.

An evaluation of the toxicogenomic data set for dibutyl phthalate (DBP) and male reproductive developmental effects was performed as part of a larger case study to test an approach for incorporating genomic data in risk assessment. The DBP toxicogenomic data set is composed of nine in vivo studies from the published literature that exposed rats to DBP during gestation and evaluated gene expression changes in testes or Wolffian ducts of male fetuses. The exercise focused on qualitative evaluation, based on a lack of available dose-response data, of the DBP toxicogenomic data set to postulate modes and mechanisms of action for the male reproductive developmental outcomes, which occur in the lower dose range. A weight-of-evidence evaluation was performed on the eight DBP toxicogenomic studies of the rat testis at the gene and pathway levels. The results showed relatively strong evidence of DBP-induced downregulation of genes in the steroidogenesis pathway and lipid/sterol/cholesterol transport pathway as well as effects on immediate early gene/growth/differentiation, transcription, peroxisome proliferator-activated receptor signaling and apoptosis pathways in the testis. Since two established modes of action (MOAs), reduced fetal testicular testosterone production and Insl3 gene expression, explain some but not all of the testis effects observed in rats after in utero DBP exposure, other MOAs are likely to be operative. A reanalysis of one DBP microarray study identified additional pathways within cell signaling, metabolism, hormone, disease, and cell adhesion biological processes. These putative new pathways may be associated with DBP effects on the testes that are currently unexplained. This case study on DBP identified data gaps and research needs for the use of toxicogenomic data in risk assessment. Furthermore, this study demonstrated an approach for evaluating toxicogenomic data in human health risk assessment that could be applied to future chemicals.

Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption.

BACKGROUND: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. OBJECTIVES: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. METHODS: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. RESULTS: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. CONCLUSIONS: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis.

Molecular alterations underlying the enhanced disruption of spermatogenesis by 2,5-hexanedione and carbendazim co-exposure.

The current study investigated the co-exposure effects of 2,5-hexanedione (HD) and carbendazim (CBZ) on gene expression underlying the enhanced pathology previously observed. Adult male rats were exposed to HD (0.33 or 1%) followed by CBZ (67 or 200 mg/kg), and testis samples were collected after 3 and 24 h. Microarray analysis at 3 h revealed that CBZ and HD interact in an agonistic, or synergistic, way at the gene level. Further analysis of candidate genes by qRT-PCR at both 3 and 24 h after co-exposure, revealed that Loxl1 and Clca2/Clca4l were both decreased in expression. Immunohistochemical analysis of Loxl1 at 24 h revealed that Loxl1 is localized to the seminiferous tubules, with the most intense staining in the basement membrane, blood vessels, and acrosomes, with the relative intensity reflecting the gene level changes at 3 h. These findings provide candidate genes for further investigation of the testicular response to damage.

Uncovering gene regulatory networks during mouse fetal germ cell development.

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.

Suppression of radiation-induced testicular germ cell apoptosis by 2,5-hexanedione pretreatment. II. Gene array analysis reveals adaptive changes in cell cycle and cell death pathways.

Sertoli cells are essential for testicular germ cell maintenance and survival. We made the unexpected observation that x-radiation (x-ray)-induced germ cell loss is attenuated by co-exposure with the Sertoli cell toxicant 2,5-hexanedione (HD). The mechanisms underlying this attenuation of germ cell apoptosis with reduced Sertoli cell support are unknown. The current study was performed to examine alterations in testicular gene expression with co-exposure to HD and x-ray. Adult male rats were exposed to HD (0.33 or 1%) in the drinking water for 18 days followed by x-ray (2 or 5 Gy), resulting in nine treatment groups. Testis samples were collected after 3 h and total messenger RNA was analyzed using Affymetrix Rat Genome 230 2.0 arrays. Normalized log2-expression values were analyzed using LIMMA and summarized using linear contrasts designed to summarize the aggregated effect, in excess of x-ray alteration, of HD across all treatment groups. These contrasts were compared with the overall linear trend expression for x-ray, to determine whether HD effects were agonistic or antagonistic with respect to x-ray damage. Overrepresentation analysis to identify biological pathways where HD modification of gene expression was the greatest was performed. HD exerted a significant influence on genes involved in cell cycle and cell death/apoptosis. The results of this study provide insight into the mechanisms underlying attenuated germ cell toxicity following HD and x-ray co-exposure through the analysis of co-exposure effects on gene expression, and suggest that HD pre-exposure reduces Sertoli cell supported germ cell proliferation thereby reducing germ cell vulnerability to x-rays.

Assessing the relevance of in vitro measures of phthalate inhibition of steroidogenesis for in vivo response.

Human phthalate exposure occurs as mixtures of diesters with varying activity towards testosterone-dependent development. Dibutyl (DBP), diethylhexyl (DEHP) and butylbenzyl (BBP) phthalate disrupt sexual development in the fetal rat. Dimethyl (DMP) and diethyl (DEP) phthalate do not. These differences in potency may result from differential delivery of the monophthalates to the testes or from variation in the abilities of the compounds to alter steroidogenesis. We tested five phthalates in pregnant rats (500mg/kg-d, GD12-19) and analyzed the fetal testes for corresponding monoesters (MMP, MEP, MBP, MEHP, MBeP). Testes MMP and MEP levels were 2-40-fold higher than the active monoesters, MBP and MEHP. BBP exposure led to low concentrations of MBeP, but similar MBP levels to DBP. An in vitro MA-10 cell assay measured the direct effect of monophthalates on testosterone production. MEHP inhibited LH-stimulated testosterone production at 1microM. RT-PCR confirmed down-regulation of genes associated with cholesterol transport and steroid synthesis and metabolism by MEHP. Five additional phthalates were tested for testosterone inhibition. MBP and mono-n-octyl phthalate were similar to MEHP; MMP, MEP and MBeP were poor inhibitors of testosterone production. Based on these results, differences in the phthalates' ability to interfere with sexual development in vivo appears to be more associated with differential potency for testosterone inhibition than differences in tissue doses.

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats.

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offspring's anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring.

Dose-dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n-butyl) phthalate.

UNLABELLED: Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. OBJECTIVE: To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. DESIGN: Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. RESULTS: Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. CONCLUSION: DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

GUDMAP: the genitourinary developmental molecular anatomy project.

In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

Using a comparative in vivo DNase I footprinting technique to analyze changes in protein-DNA interactions following phthalate exposure.

Exposure to environmental chemicals often induces changes in gene expression leading to a variety of developmental and physiological problems. Understanding the underlying mechanism of these changes will aid in assessing human risk to these chemicals. Traditional methods for analyzing protein-DNA interactions include in vivo footprinting and chromatin immunoprecipitation (ChIP). However, ChIP does not provide binding location, and conventional footprinting is too subjective and time consuming for comparing protein binding in toxicological studies. Here, in vivo DNase I footprinting is adapted for use with the automated DNA sequencer to provide a semiquantitative map of changes in DNA-protein interactions in the promoter of steroidogenic acute regulatory (StAR) protein. StAR is the rate-limiting step in testosterone biosynthesis and is downregulated following in utero di-butyl phthalate (DBP) treatment in rats through an unknown mechanism. In vivo footprinting identified three regions of altered DNase digestibility following DBP treatment, and EMSA identified the corresponding transcription factors as SF-1, c/ebp beta, and GATA4. ChIP assays confirmed changes in protein-binding activity of SF-1 and c/ebp beta, but only c/ebp beta gesponds to only DBP. This suggests that c/ebp beta ginding is involved in DBP-induced transcriptional changes. By tailoring in vivo footprinting for toxicological studies, it can provide a detailed and accurate map of protein-DNA interactions and is an excellent first step in determining the changes in the structure of transcriptional machinery following an exogenous chemical treatment.

Mapping gene expression changes in the fetal rat testis following acute dibutyl phthalate exposure defines a complex temporal cascade of responding cell types.

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.

CCAAT/enhancer binding protein beta, but not steroidogenic factor-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis.

Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp beta) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp beta in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp beta, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.

Fetal mouse phthalate exposure shows that Gonocyte multinucleation is not associated with decreased testicular testosterone.

The rat has been explored in detail for its in utero susceptibility to male reproductive tract malformation following phthalate exposure. Few other species have been studied in detail, and it is important for both mechanistic and risk assessment purposes to understand the species specificity of this response. We investigated the response of the fetal mouse testis to phthalate exposure and compared these results with those previously obtained from the rat. Initial experiments using a variety of phthalate congeners (monobutyl phthalate, di-(n-butyl) phthalate, or mono (2-ethylhexyl) phthalate) and exposure paradigms did not reduce fetal mouse testis testosterone levels. Pharmacokinetic data after a single 500 mg/kg di-(n-butyl)-phthalate (DBP) exposure on mouse gestation day (gd) 18 demonstrated that the concentrations and kinetics of the active metabolite monobutyl phthalate (MBP) in fetal and maternal plasma were similar to the rat. After a single 500 mg/kg or multiple day 250 mg/kg fetal mouse DBP exposure, rapid and dynamic changes in testis gene expression were observed, including induction of immediate early genes. Unlike the rat, expression of genes involved in cholesterol homeostasis and steroidogenesis were not decreased and were increased in a few cases. Similar to the rat, however, a 250- or 500-mg DBP/kg/day mouse exposure from gd 16 through 18 significantly increased seminiferous cord diameter, the number of multinucleated gonocytes per cord, and the number of nuclei per multinucleated gonocyte. Together, these results demonstrate that fetal mouse and rat phthalate exposure both induce immediate early gene expression and disrupt seminiferous cord and gonocyte development. This response in the mouse occurs without a measurable decrease in testicular testosterone, suggesting that altered seminiferous cord formation and gonocyte multinucleation may not be mechanistically linked to lowered testosterone.

Testicular gene expression profiling following prepubertal rat mono-(2-ethylhexyl) phthalate exposure suggests a common initial genetic response at fetal and prepubertal ages.

Phthalate chemical plasticizers can damage the fetal and postnatal mammalian testis, but several aspects of the injury mechanism remain unknown. Using a genome-wide microarray, the profile of testicular gene expression changes was examined following exposure of postnatal day 28 rats to a single, high dose (1000 mg/kg) of mono-(2-ethylhexyl) phthalate (MEHP). By microarray analysis, approximately 1675 nonredundant genes exhibited significant expression changes; the vast majority were observed at 12 h. Among the 36 genes significantly altered up to the 3-h time point, prominent functional categories were secreted, transcription, and signaling factors. Using quantitative PCR (qPCR), the dose-response of 24 genes was determined after a single MEHP exposure of 10, 100, or 1000 mg/kg. Increasing 114-fold by 12 h at 1000 mg/kg, Thbs1 (thrombospondin 1) showed the highest level of gene induction. The vast majority of genes analyzed by qPCR exhibited significant expression alterations at the lowest dose level. Interestingly, a unique, dose-dependent expression pattern was observed for the transcription factor Nr0b1, steroidogenic genes (Cyp17a1 and StAR), and a cholesterol metabolism gene (Dhcr7). For these genes, the direction of expression change at 10 or 100 mg/kg was opposite that observed at 1000 mg/kg. Gene profiling data at 1000 mg/kg MEHP were phenotypically anchored to increased germ cell apoptosis (6 and 12 h) and an interstitial neutrophil infiltrate (12 h). At 10 or 100 mg/kg MEHP, no testicular morphological changes were detected, but a significant increase in germ cell apoptosis was seen at 6 h. Finally, comparison of the prepubertal MEHP microarray data to similar data from fetal dibutyl phthalate (DBP) exposure showed conservation in both the identities of testicular genes altered and the direction of expression changes. For example, 60% of the genes altered within 3 h of prepubertal MEHP exposure also were changed following acute fetal DBP exposure, and the direction of expression change was highly preserved. These data demonstrate that similar genetic targets are altered following fetal and prepubertal phthalate exposure, suggesting that the initial mechanism of fetal and prepubertal phthalate-induced testicular injury is shared.

Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets.

The androgen receptor (AR) is expressed in the fetal testis; however, the role of AR in fetal testicular development is poorly understood. Disrupted AR activity and subsequent gene expression alterations may disturb developmental programming of the fetal testis and result in testicular abnormalities later in life. The present study was performed to examine global gene expression patterns in rat fetal testis following in utero exposure to various AR antagonists. Pregnant Sprague-Dawley rats were treated with flutamide (50 mg/kg/day), linuron (50 mg/kg/day), vinclozolin (200 mg/kg/day), p,p'-DDE (100 mg/kg/day) or corn oil vehicle by gavage daily from gestation day (GD) 12-19. Testes were isolated on GD 19, and AR immunostaining, histology, and global changes in gene expression were determined. There were no alterations in the pattern or expression level of AR and no apparent histological changes in the fetal testes in any treatment group. Microarray analysis using Dunnett's test with multiple testing correction revealed no significant gene expression alterations following exposure to flutamide, linuron, vinclozolin, and p,p'-DDE. A less stringent analysis yielded some chemical specific effects on gene expression, and these effects were further evaluated by real-time RT-PCR. Vinclozolin treatment reduced the expression of several genes involved in cholesterol biosynthesis, though the testosterone levels were unchanged in the fetal testes in any treatment group. In flutamide, linuron, and p,p'-DDE treatment groups, the expression of hemoglobin Y, beta-like embryonic chain (Hbb-y) was reduced. Myomesin 2 (Myom2) expression was increased following linuron treatment. Given the lack of a common set of genes and the absence of overt histopathology, we conclude that the fetal testis is not a major target for AR activity at this stage of development although some cell-type specific gene expression changes cannot be ruled out.

Analysis of variation of amplitudes in cell cycle gene expression.

BACKGROUND: Variation in gene expression among cells in a population is often considered as noise produced from gene transcription and post-transcription processes and experimental artifacts. Most studies on noise in gene expression have emphasized a few well-characterized genes and proteins. We investigated whether different cell-arresting methods have impacts on the maximum expression levels (amplitudes) of a cell cycle related gene. RESULTS: By introducing random noise, modeled by a von Mises distribution, to the phase angle in a sinusoidal model in a cell population, we derived a relationship between amplitude and the distribution of noise in maximum transcription time (phase). We applied our analysis to Whitfield's HeLa cell cycle data. Our analysis suggests that among 47 cell cycle related genes common to the 2nd experiment (thymidine-thymidine method) and the 4th experiment (thymidine-nocodazole method): (i) the amplitudes of CDC6 and PCNA, which are expressed during G1/S phase, are smaller in the 2nd experiment than in the 4th, while the amplitude of CDC20, which is expressed during G2/M phase, is smaller in the 4th experiment; and (ii) the two cell-arresting methods had little impact on the amplitudes of the other 43 genes in the 2nd and 4th experiments. CONCLUSION: Our analysis suggests that procedures that arrest cells in different stages of the cell cycle differentially affect expression of some cell cycle related genes once the cells are released from arrest. The impact of the cell-arresting method on expression of a cell cycle related gene can be quantitatively estimated from the ratio of two estimated amplitudes in two experiments. The ratio can be used to gauge the variation in the phase/peak expression time distribution involved in stochastic transcription and post-transcriptional processes for the gene. Further investigations are needed using normal, unperturbed and synchronized HeLa cells as a reference to compare how many cell cycle related genes are directly and indirectly affected by various cell-arresting methods.

Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate.

The phthalate ester di(n-butyl) phthalate (DBP) causes feminization of male rats upon in utero exposure by repressing expression of genes required for testicular steroidogenesis. Previous work in our laboratory has shown that repression of gene expression and steroidogenesis in the fetal testis is apparent within a few hours of DBP exposure. The purpose of this study was to determine the precise timing of DBP-associated gene expression changes in the fetal testis using transcriptional profiling and to determine whether DBP exerts similar effects on steroidogenesis in the fetal adrenal. A DBP time-course experiment showed that testicular steroidogenesis was decreased within 1 h of DBP exposure and that this decrease preceded the repressed transcription of Star (steroidogenic acute regulatory protein); Scarb1 (scavenger receptor class B, member 1; also know as Sr-b1); Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450SCC); and Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17). Gene expression profiling demonstrated rapid (within 1 to 3 h) and transient induction of immediate early genes in the fetal testis after administration of DBP to the pregnant dam. There was a statistically insignificant decrease in corticosterone production by the fetal adrenal after in utero exposure to DBP from Gestation Day 12 to Gestation Day 19. The extent of steroidogenesis diminution was much less in the adrenal than in the testis (approximately 45% decrease in the adrenal versus 87% decrease in the testis) and expression of genes required for steroidogenesis in the adrenal was unaffected by DBP. Together, these studies demonstrate that DBP initiates a rapid and dynamic change in gene expression in the fetal testis that likely plays a role in the reduction in steroidogenesis that is unique to the fetal testis relative to the steroidogenically active fetal adrenal.

Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact.

Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.

Di-n-butyl phthalate activates constitutive androstane receptor and pregnane X receptor and enhances the expression of steroid-metabolizing enzymes in the liver of rat fetuses.

The plasticizer di-n-butyl phthalate (DBP) is a reproductive toxicant in rodents. Exposure to DBP in utero at high doses alters early reproductive development in male rats. Di-n-butyl phthalate also affects hepatic and extrahepatic enzymes. The objectives of this study were to determine the responsiveness of steroid-metabolizing enzymes in fetal liver to DBP and to investigate the potential of DBP to activate nuclear receptors that regulate the expression of liver enzymes. Pregnant Sprague-Dawley rats were orally dosed with DBP at levels of 10, 50, or 500 mg/kg/day from gestation days 12 to 19; maternal and fetal liver samples were collected on day 19 for analyses. Increased protein and mRNA levels of CYP 2B1, CYP 3A1, and CYP 4A1 were found in both maternal and fetal liver in the 500-mg dose group. Di-n-butyl phthalate at high doses also caused an increase in the mRNA of hepatic estrogen sulfotransferase and UDP-glucuronosyltransferase 2B1 in the dams but not in the fetuses. Xenobiotic induction of CYP3A1 and 2B1 is known to be mediated by the nuclear hormone receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). In vitro transcriptional activation assays showed that DBP activates both PXR and CAR. The main DBP metabolite, mono-butyl-phthalate (MBP) did not interact strongly with either CAR or PXR. These data indicate that hepatic steroid- and xenobiotic-metabolizing enzymes are susceptible to DBP induction at the fetal stage; such effects on enzyme expression are likely mediated by xenobiotic-responsive transcriptional factors, including CAR and PXR. Our study shows that DBP is broadly reactive with multiple pathways involved in maintaining steroid and lipid homeostasis.