ToxSci Advance Access originally published online on June 29, 2006
Toxicological Sciences 2006 93(2):369-381; doi:10.1093/toxsci/kfl049
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Testicular Gene Expression Profiling following Prepubertal Rat Mono-(2-ethylhexyl) Phthalate Exposure Suggests a Common Initial Genetic Response at Fetal and Prepubertal Ages
Division of Biological Sciences, CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
1 To whom correspondence should be addressed at Division of Biological Sciences, CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709. Fax: (919) 558-1300. E-mail: kjohnson{at}ciit.org.
Received April 19, 2006; accepted June 23, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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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.
Key Words: phthalate; testis; gene expression; postnatal; fetal.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Phthalates are industrial chemicals that impart flexibility and resilience to a variety of plastics and consumer goods, including food packaging, medical bags and tubing, and personal care products. Since they are not covalently bound to the product matrix, phthalates can leach from the matrix leading to ubiquitous human exposure (Silva et al., 2004
). Several phthalate diester congeners and their monoester metabolites are testicular toxicants in both fetal and prepubertal male rodents (Boekelheide et al., 2004; Liu et al., 2005).
While testicular responses following fetal and prepubertal phthalate exposure show some similarities, significant differences also are observed. Histopathologically, prepubertal and fetal testes both respond initially to high-dose exposure by a collapse of the Sertoli cell vimentin cytoskeleton and a coincident retraction of Sertoli cell processes (Kleymenova et al., 2005; Richburg and Boekelheide, 1996). These changes and others indicate that, at least for prepubertal exposure, the Sertoli cell may be the primary cellular target (Creasy et al., 1983). Long-term histopathological responses between the fetal and prepubertal testis show significant differences. Phthalate-exposed fetal testes fail to correctly organize seminiferous cords and gonocytes coalesce into multinucleated syncytia (Mahood et al., 2005). Ultimately, high-dose fetal exposure produces a highly abnormal postnatal testis characterized by areas of Leydig cell aggregation and dysgenic seminiferous tubules. Apoptosis is not a significant contributing factor to fetal injury (Kleymenova et al., 2005). Unlike fetal exposure, the main prepubertal cellular response following high-dose phthalate exposure is germ cell sloughing into the seminiferous tubule lumen accompanied by extensive germ cell apoptosis (Creasy et al., 1983; Richburg and Boekelheide, 1996). At the molecular level, few studies have examined correlations between fetal and prepubertal phthalate exposure.
The phthalate testicular injury molecular mechanism remains unknown, and because of the significant differences in morphological outcomes, it is unclear if the molecular mechanisms between fetal and prepubertal exposure are similar or unique. We hypothesized that prepubertal phthalate exposure would show time-dependent alterations in testicular gene expression and that (despite age-dependent phthalate exposure phenotypes) the fetal and prepubertal testicular genetic response would be similar. Here, genome-wide microarray analysis was used to examine testicular gene networks modified in the phthalate-exposed prepubertal rat, and quantitative PCR (qPCR) was employed to determine the phthalate dose-response of select genetic targets. A comparison of the prepubertal and fetal testicular responses showed that similar genetic targets were altered at both ages.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Animals.
Postnatal day (PND) 28 rats were chosen as our model because of its historical use in examination of the postnatal mechanism of phthalate-induced testicular injury and the distinct testicular morphological outcomes at this age compared to fetal phthalate exposure. Male Fisher 344 rats from Charles River Laboratories, Inc (Raleigh, NC) were obtained at PND21. Animals were randomly assigned to a treatment group, with each group containing a minimum of three animals. Animals were housed in the animal facility of the CIIT Centers for Health Research (CIIT, NC), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, in a room with controlled humidity and temperature, and HEPA-filtered, mass air-displacement. The room was maintained on a 12:12 light:dark cycle at approximately 22 ± 4°C with a relative humidity of approximately 30–70%. Animals were identified by ear tags and cage cards, and housed three per cage in polycarbonate cages with Alpha-dri cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum. This study followed federal guidelines for the care and use of laboratory animals and was approved by the Institutional Animal Care and Use Committee at CIIT.
Because of its historical use in postnatal phthalate mechanistic studies from our laboratory and others, mono-(2-ethylhexyl) phthalate (MEHP) was used for these experiments. Rats were gavaged on PND28 with corn oil vehicle (1 ml/kg; Sigma Chemical Co., St Louis, MO) or MEHP (in corn oil) at 10, 100, or 1000 mg/kg. Purity and concentration of dosing solutions were verified using a Hewlett-Packard 5890 gas chromatograph. Rats were sacrificed 1, 2, 3, 6, or 12 h after dosing by carbon dioxide asphyxiation, and the testes were removed and detunicated. The right testis was placed in RNAlater (Ambion, Inc, Austin, TX) and used for RNA extraction. Following puncture of the tunica albuginea with a 28-gauge needle, the left testis was fixed by overnight immersion in 10% neutral buffered formalin, embedded in paraffin, and used to examine histopathology and measure the germ cell apoptotic index using the terminal deoxy-nucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) assay.
TUNEL assay.
TUNEL was performed on 8-mm testis sections using the ApopTag kit (Oncor, Gaithersburg, MD). Briefly, digoxigenin-dUTP end labeled DNA was detected with anti-digoxigenin-peroxidase antibody followed by peroxidase detection with 0.05% diaminobenzidine and 0.02% H2O2. Tissue was counterstained with methyl green. The apoptotic index was determined as described by Richburg and Nanez (2003). At each time point, the slides were blinded, and the number of apoptotic germ cells was counted in 100 randomly selected seminiferous tubules. Only intact, essentially round tubules were counted. The number of tubules having four or more apoptotic germ cells was calculated as a percentage of the total number of tubules examined; this apoptotic index was expressed as average ± SD. Because controls at each time point showed no statistically significant differences, all controls (n = 17) were placed into a single group for statistical analyses. The number of statistical units at each time point for the 10, 100, or 1000 mg/kg MEHP groups was 4, 4, and 3, respectively. Using Prism Graphpad 4 software (San Diego, CA), significance between control and treated groups (p < 0.05) was determined by one-way ANOVA with a Dunnett's post hoc test.
Microarray analysis.
Detunicated testes from individual rats were homogenized in RNA Stat-60 reagent (Tel-Test, Friendswood, TX), and RNA was isolated using a RNeasy Maxi Kit (Qiagen, Valencia, CA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Complementary DNA (cDNA) was synthesized from 2 mg of total RNA and purified using the RiboAmp OA 1 Round RNA Amplification kit (Arcturus, Mountain View, CA) according to the manufacturer's protocol. Equal amounts of purified cDNA per sample were used as the template for subsequent in vitro transcription reactions for complementary RNA (cRNA) amplification and biotin labeling using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Inc, Farmingdale, NY). cRNA was purified and fragmented according to the protocol provided with the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). All GeneChip microarrays (Rat Genome 230 2.0 Array) were hybridized, washed, stained, and scanned using the Complete GeneChip Instrument System according to the Affymetrix Technical Manual. The Rat Genome 230 2.0 microarray contained 31099 probe sets. As on April 2006, analysis of these probe sets using Bioconductor R 2.3.0 software showed that 23250 probe sets were annotated, which mapped to 13,784 unique gene names (Gentleman et al., 2004).
All prepubertal rat microarray primary data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE4514. Primary testicular gene expression data for fetal phthalate exposure were obtained from a previous study of rat gestational day 19 exposure to 500 mg/kg dibutyl phthalate (DBP) (Thompson et al., 2005).
To select significantly expressed genes from Affymetrix probe level data, a linear mixed model was used. The perfect-match only data at the probe level was used, and each array was normalized to a common mean on the log2 scale as previously described (Chu et al., 2002). Each probe set was fit to a linear mixed model. The model used was as follows: 
where yijk is the normalized log2-transformed perfect-match expression of the ith treatment for the jth probe, µ is the overall mean, Ti is the effect of the ith treatment, Pj is the effect of the jth probe, Ak is the effect of the kth chip, and 
ijk is the residual. The Ak's and ijk's are assumed to be independent, and normally distributed, e.g., 
and 
The treatment and probe effects were fixed and the array Ak's were assumed to be random effects. The parameters for the fixed and random effects were estimated using restricted maximum likelihood. Selected genes demonstrated significant changes in expression level following treatment when compared to controls using mixed-model F-tests. The Bonferroni method was used to identify genes with a significance level of 5% from each individual comparison of treatment versus control. SAS Microarray Solution version 1.3 software (Cary, NC) was used to compute the results.
Gene networks were generated using Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood City, CA). A gene network is a graphical representation of the molecular relationships between genes or gene products. Within a network, genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed as various line types that describe the nature of the relationship between the nodes; solid lines are direct interactions while dashed lines represent indirect interactions. Edges are substantiated by at least one reference from the literature, a textbook, or canonical information stored in the Ingenuity Pathways Knowledge Base. The intensity of the node color indicates the level of up- (yellow) or down- (blue) regulation, as determined by microarray analysis.
The qPCR.
Total RNA was isolated from the detunicated testis of control and treated animals using the RNeasy Maxi Kit (Qiagen) following the manufacturer's protocol. The number of animals within each group was controls (n = 11); 10 and 100 mg/kg (n = 4); and 1000 mg/kg (n = 3 or 4). Subsequent reverse transcription reactions, quality control for reverse transcriptase reactions, and qPCR reactions were performed as described (Lehmann et al., 2004). As a template, cDNA prepared from detunicated PND28 testes was used. For the cDNA reaction, samples containing 1 mg of RNA were reverse transcribed using the TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA) and random hexamer primers. PCR amplifications were performed in 20 ml reactions containing 40 ng of template DNA, 1x volume of prevalidated TaqMan Gene Expression Assay mix for each gene (Table 1), and a 1x volume of TaqMan Universal PCR Master Mix. The amplification protocol used was as follows: initial 10-min denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Amplification signals were detected continuously with an Applied Biosystems 7700 Real-Time PCR system.
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For some genes, qPCR was performed using a TaqMan Low Density Array (LDA) preloaded with prevalidated TaqMan gene expression assays (Applied Biosystems). For this assay, 100 ng of cDNA was combined with 47.5 ml RNase/DNase free water and 50 µl TaqMan Universal PCR Master Mix for a total volume of 100 ml and loaded into the fill ports of the TaqMan array. The LDA was centrifuged briefly to load the sample-specific reaction mixes into the individual wells, sealed, and loaded into a 7900 HT Real-Time PCR system (Applied Biosystems). The amplification protocol was as follows: initial 2-min incubation at 50°C and 10-min denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
Analysis of the qPCR data was conducted using the equation set forth by Pfaffl (2001), in which efficiencies were used for both the gene of interest and the calibrator (glyceraldehyde-3-phosphate dehydrogenase or 18-s ribosomal RNA). The average Ct of samples run in duplicate or triplicate was used to establish expression relative to the calibrator. Significance (p < 0.05) of the qPCR data was determined by one-way ANOVA and a Dunnett's post hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Testicular Germ Cell Apoptosis and Neutrophil Infiltration following MEHP Exposure
Germ cell apoptosis following high-dose phthalate exposure of prepubertal rats is documented (Richburg and Boekelheide, 1996
), and a testicular interstitial neutrophil infiltrate has been observed occasionally (Creasy et al., 1983). To compare our phthalate exposure outcome with previous studies and phenotypically anchor the testicular gene expression analyses, germ cell apoptosis and interstitial histology was analyzed. In vehicle-exposed animals, 10% of seminiferous tubules displayed four or more apoptotic germ cells (Fig. 1). At 1, 2, or 3 h following 1000 mg/kg MEHP exposure, no significant change in germ cell apoptosis was seen. Similar to previous observations by others (Richburg and Boekelheide, 1996), a significant increase in germ cell apoptosis was noted at 6 and 12 h. To determine if lower MEHP dose levels produced a similar response, germ cell apoptosis was examined at 6 or 12 h following a single 10 or 100 mg/kg MEHP exposure. At both dose levels, germ cell TUNEL staining was significantly increased at the 6 h time point (Fig. 1). As for a testicular inflammatory response following MEHP exposure, two out of three animals at the 12-h time point (1000 mg/kg) contained a prominent interstitial lymphocytic infiltration. This infiltrate appeared to be composed mainly of neutrophils (polymorphonuclear cells) (Fig. 2) and was not observed in testes of animals from the other treatment groups or the vehicle control group (data not shown). Apart from increased apoptosis, no change in testicular histopathology following 10 or 100 mg/kg MEHP was detected (data not shown).
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Prepubertal Testis Gene Profile following Acute Phthalate Exposure
Using genome-wide Affymetrix microarrays, changes in prepubertal testis gene expression were monitored 1, 2, 3, 6, and 12 h following exposure to 1000 mg/kg MEHP. Of the nearly 23,000 annotated probe sets present on the microarray, approximately 1675 displayed a significant expression change within 12 h of MEHP exposure (Supplementary Table 1). Although more than 1675 Affymetrix probe sets were altered, about 10% were duplicate probe sets that corresponded to a single gene based upon assigned or determined gene identity. For numerical analysis purposes, duplicate genes were culled. The stringent statistical method employed to examine the microarray data resulted in no genes showing significantly altered expression at the 1-h time point. However, expression of the immediate early transcription factors Egr1 and Fos were induced at this time nearly 3- and 1.5-fold, respectively (Table 2). By 2 h after exposure, 26 genes were changed, and all genes displayed an expression increase. At 3, 6, and 12 h, the number of new genes that became significantly altered was 10, 51, and approximately 1595, respectively. Although the large number of gene changes observed at 12 h correlated with a neutrophil infiltrate, it remained uncertain which gene changes solely reflected infiltration of this new cell population. Typically, genes altered in response to the 1000 mg/kg MEHP exposure remained affected through the 12 h time point. The 6 h time point was the first period in which a reduction in gene expression was observed; in general, genes showing reduced expression following phthalate exposure were involved in metabolic processes.
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Significantly changed genes (by microarray analysis) within 6 h of exposure were categorized into general functional groups. Categorization was derived from the Affymetrix website (http://www.affymetrix.com) or a target sequence homology basic local assignment search tool search to identify the gene followed by a Pubmed literature search. The majority of altered genes at 3 h of MEHP exposure were modulators of intracellular signal transduction, extracellular interactions, or gene transcription (Table 3). Additional functional classifications included genes encoding transmembrane or cytoskeletal proteins and those involved in metabolism or response to stress. By 6 h, signal transduction and transcriptional categories remained prominent but a larger number of genes encoding metabolic factors also responded.
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Many of the genes showing the earliest alterations were members of the immediate early response gene family that may regulate NF-
B signaling or downstream targets of NF-B transcriptional activity. Immediate early response genes included Cyr61, Ctgf, Dusp1, 5, and 6, Egr1, Ier3, Jun, and Junb. At 2 h, activation of the NF-B pathway was suggested by an increase in two genes that are downstream targets of NF-B signaling (Nfkbia and Tnfaip3) (Heyninck and Beyaert, 2005). An additional gene encoding a modulator of NF-B activity (Nfkbiz) and a subunit of the NF-B transcriptional complex (Nfkb2) were induced within 6 h.
Among the testicular genes induced within 6 h of prepubertal MEHP exposure were several encoding secreted proteins. This group consisted of chemokines (Cxcl1 and Cxcl10), a pro-inflammatory cytokine (Il1a), and matricellular proteins (Cyr61, Ctgf, and Thbs1). At 12 h, expression of receptors for these secreted ligands also was stimulated significantly. For example, several integrin receptor genes for the secreted matricellular proteins were induced such as Itgav, Itgb1, and Itga6 (Chen et al., 2000; Lau and Lam, 2005). Sdc4, encoding the Cyr61 receptor syndecan-4 (Todorovicc et al., 2005), was increased over fourfold by 12 h.
A plethora of genes potentially mediating MEHP-induced germ cell apoptosis were altered. Some of the significantly induced genes at the 2- and 3-h time points have been associated with apoptosis in various models. Within this list were Thbs1 (Friedl et al., 2002), Edn1 (Cai et al., 2000), Ctgf (Moussad and Brigstock, 2000), Cyr61 (Todorovicc et al., 2005), Ier3 (Kruse et al., 2005), Atf3 (Lu et al., 2006), Egr1 (Kim et al., 2006), Egr3 (Xi and Kersh, 2004), and genes encoding components of the activating protein 1 transcriptional complex (Fos, Jun, and Junb) (Hess et al., 2004). Concomitant with an increase in TUNEL-positive germ cells, genes associated with apoptosis induction via either the c-Jun N-terminal kinase (JNK)–stimulated intrinsic (mitochondrial) pathway or the tumor necrosis factor receptor (TNFR) family–mediated extrinsic pathway were increased. Within the JNK apoptotic pathway, MEHP increased expression of genes encoding a JNK-activating scaffolding protein (Sh3md2), a JNK kinase (Map2k4), and a negative regulator of Map2k4 activity (Gadd45b) (Kukekov et al., 2006; Papa et al., 2004). At 12 h, multiple components of the TNFR apoptotic pathway displayed significantly altered expression: Cflar, Cradd, Ripk1, Ripk2, Ripk3, Tnfrsf1a, Tnfrsf6, Tnfrsf21, Tnf, and Tradd. Caspases, the main executioners of apoptosis, showed significant increases after cell death began; induction of Casp3, 11, and 12 was detected beginning at 12 h.
A deficit in testicular steroidogenesis is a prominent outcome of fetal rat phthalate exposure. While the prepubertal rat testis is less sensitive than the fetal testis regarding phthalate-induced decreases in testosterone levels (Akingbemi et al., 2001; Lehmann et al., 2004), the gene encoding p450 side chain cleavage (Cyp17a1) declined 85% by 12 h and Hsd3b1 was decreased 60% (Supplementary Table 1) after exposure to 1000 mg/kg MEHP. Although not significant by microarray analysis, StAR mRNA levels were reduced after 6 h at this dose level (see below). In addition to these steroidogenic genes, other factors known to modulate Leydig cell androgen production in vivo or in vitro showed altered expression. Among this list were the transcription factors Nr4a1 and Nr0b1, a cholesterol-modifying gene (Ch25h), and the secreted factors Tnf, Il1a, Il1b, and Wnt4 (Hales, 2002; Jo and Stocco, 2004; Jordan et al., 2003; Lukyanenko et al., 2001).
Dose Response of Selected Genes Analyzed by qPCR
To validate the microarray results and identify genes altered at lower doses of MEHP, the expression of 24 genes was examined by Taqman qPCR after acute exposure to MEHP at dose levels of 10, 100, or 1000 mg/kg. The genes chosen for this analysis represented a wide spectrum of cellular pathways and processes. Nearly all genes examined by qPCR showed significant expression changes in the microarray analysis prior to observation of a testicular neutrophil infiltrate. In addition, genes regulating phthalate-induced decreases in fetal steroidogenesis (StAR) and prepubertal germ cell apoptosis (Tnfrsf6) also were analyzed (Lehmann et al., 2004; Richburg et al., 2000).
Overall, the high-dose qPCR data validated the high-dose microarray results, and all genes showed significant expression changes at 10 and/or 100 mg/kg MEHP. In the microarray experiment, a trend of altered expression was noted for numerous genes several hours prior to the differential becoming statistically significant; this earlier trend often became statistically significant using qPCR. Three general observations were noted in the qPCR data: (1) compared to microarray data, the qPCR fold change was higher consistently, which likely reflects different normalization procedures; (2) unlike the 1000-mg/kg dose level, exposure to 100 mg/kg MEHP produced a transient expression change in many genes that were resolved by 12 h following exposure; and (3) compared to higher dose levels, exposure to 10 mg/kg MEHP produced a several hour delayed response. All genes showed altered expression at the 100- and 1000-mg/kg dose levels, and 21 out of the 24 genes were changed at 10 mg/kg. Among the genes examined, the largest expression increase was observed for Thbs1 (encoding thrombospondin 1; Tbs1). Twelve hours after 1000 mg/kg MEHP exposure, Tbs1 testicular mRNA levels were induced 114-fold. For most genes, the direction of expression change was constant for all dose levels. However, a related set of genes involved in cholesterol metabolism or steroidogenesis showed a unique, dose-dependent expression pattern. At 1000 mg/kg MEHP, Cyp17a1, StAR, and Dhcr7 were decreased 6 h following exposure, but lower MEHP dose levels produced an increased expression of these genes by 12 h (Fig. 3). A similar pattern was observed for Nr0b1.
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Comparison of the Fetal- and Prepubertal Phthalate–Exposed Testis Gene Profile
As described in the "Introduction" section, the rat testis is susceptible to injury at both the fetal and postnatal time periods, although the morphological outcomes are disparate. To determine the similarity in genetic response within hours of phthalate exposure between these two ages, a comparison was performed between the prepubertal MEHP gene profile and a DBP-exposure study previously performed on the fetal rat testis (Thompson et al., 2005
). In vivo, DEHP is metabolized quantitatively to its proximal toxicant MEHP, and the effects of DBP and DEHP on fetal testicular gene expression are similar (Liu et al., 2005). For the fetal and prepubertal phthalate exposure comparison, the primary Affymetrix probe level data from the fetal 500 mg/kg DBP exposure were subjected to the same statistical analysis as performed for the prepubertal 1000 mg/kg MEHP exposure. With this analysis applied, approximately 500 unique genes were altered significantly at 1, 3, 6, or 18 h following fetal DBP exposure (Supplementary Table 2). Merging the prepubertal and fetal microarray datasets using either gene symbol or Unigene identifiers produced a subset of genes with modified expression at both ages. Out of the 500 fetal and 1675 prepubertal genes, 176 showed altered expression at both ages (Fig. 4 and Supplementary Table 3). By categorizing the percentage of identical genes at various time points after exposure, the data revealed that genes with changed expression within 3 h of exposure were more likely to be altered in the other age group. For example, nearly 60% of the genes modified up to 3 h of prepubertal MEHP exposure also were modified within the DBP-exposed fetal testis. Genes encoding transcription, intracellular signaling, and secreted factors were prominent functional categories of the common genes (shaded genes in Tables 3 and 4). For nearly all common genes, the direction of change was the same for fetal and prepubertal exposure. At later exposure times (6, 12, and 18 h), the percentage of common genes was reduced. Notably, genes potentially involved in germ cell apoptosis following prepubertal MEHP exposure did not show significant expression changes in the DBP-exposed fetal testis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Despite decades of scrutiny, the phthalate molecular mechanism and testicular target cell leading to adverse effects on testicular function remain unknown (Boekelheide, 2004
). Based upon molecular end points and histopathology (Creasy et al., 1983; Rasoulpour and Boekelheide, 2005; Richburg and Boekelheide, 1996; Thompson et al., 2005), the fetal and prepubertal rat testes clearly respond within hours to high-dose phthalate exposure. The genome-wide transcriptional profiling reported here aimed to reveal the initial genetic response of the phthalate-exposed prepubertal testis and use these data in two ways: (1) to develop testable hypotheses on the molecular mechanism of phthalate testicular toxicity and (2) to compare the molecular response of the prepubertal rat testis with that of the fetal rat testis.
The genes first responding to prepubertal phthalate exposure implied that at least two signal transduction pathways were activated immediately. Because our analysis was based upon a whole testis sample, it was not clear which testicular cell types were responsible for changes in gene expression. Within 3 h of exposure, a number of genes with increased expression fell within the class of immediate early response genes that are induced by numerous stimuli. Immediate activation of NF-B signaling was implied by increased expression of NF-B negative-feedback loop components known to be induced in response to NF-B activation; three such components were Nfkbia (coding for Ikba; inhibitory kappaB alpha), Nfkbiz, and Tnfaip3 (which produces the protein A20) (Heyninck and Beyaert, 2005). These results corroborate previous reports of testicular NF-B activation within 1 h of high-dose phthalate exposure (Rasoulpour and Boekelheide, 2005). Three mRNAs in the sprouty family of receptor tyrosine kinase pathway inhibitors (Spry1, 2, and 4) were induced, which suggests phthalate-induced activation of a mitogen-activated protein kinase (MAPK) pathways (Mason et al., 2006). Additional support for MAPK pathway modulation comes from overexpression of three MAPK phosphatases (Dusp1, 5, and 6).
One potential avenue phthalate exposure may use to induce these signaling pathways is activation of G protein–coupled receptor (GPCR) signaling. It is now clear that downstream GPCR signaling involves stimulation of both MAPK and NF-B (Gao et al., 2004; Luttrell and Lefkowitz, 2002). Three pieces of evidence suggest that phthalate exposure induces a rapid, GPCR-based response. First, altered expression of a variety of GPCR-related genes was observed in the prepubertal testis exposed to MEHP, among them Adcy9, Gnai1, Gpsm1, Gprk5 and, conspicuously, a family of sphingosine-1-phosphate GPCRs (Edg1, 2, 3, and 5). The potential involvement of Edg receptors in fetal testis phthalate exposure was noted previously (Thompson et al., 2005). Second, phthalate exposure of primary Sertoli cells was shown to modulate the signaling activity of the follicle stimulating hormone receptor (a GPCR) (Lloyd and Foster, 1988). Lastly, phthalate exposure modified the phosphorylation status of a transfected GPCR in Hela cells within minutes (Lahousse et al., 2006). Despite these intriguing results, the molecular basis for activation of signaling pathways in response to phthalate exposure remains an enigma.
The apparent cellular stress in the testis caused by phthalate exposure leads to an increase in germ cell apoptosis. Dose-response data showed an increase in germ cell apoptosis at all MEHP levels examined. The prepubertal gene profiling data presented here suggested that the TNFR family–mediated extrinsic apoptotic pathway was activated. Fas receptor signaling activity is known to mediate some of the phthalate-induced germ cell apoptosis (Richburg et al., 2000), and our results support the role for increased expression of the Fas receptor. However, our gene profiling data suggest the involvement of other pathways. A number of genes associated with the canonical tumor necrosis factor (TNF) alpha apoptotic signaling pathway were induced (Fig. 5A). There was a trend toward increasing expression of the gene encoding TNF alpha at 3 h, prior to an increase in TUNEL-positive germ cells. Most other components of the canonical TNF apoptotic pathway, including TNFR family members and effector proteins, were not induced until 12 h. Similar to another report (Kijima et al., 2004), gene profiling suggested that caspase 3 was a major effector caspase executing phthalate-induced germ cell apoptosis.
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Caspase activation leading to cell death also may proceed via a mitochondrial (intrinsic) pathway downstream of JNK activation. Prepubertal phthalate gene profiling also implied that the JNK apoptotic pathway was induced. JNK is activated by phosphorylation mediated via assembly of a protein complex containing scaffolding proteins, mixed lineage kinases, and MAP kinase kinase (MKK) 4 or 7 (both JNK kinases) (Kukekov et al., 2006
). After prepubertal MEHP exposure, induction of genes encoding both MKK4 (Map2k4) and the JNK scaffolding protein POSH (Plenty Of SH3 Domains; Sh3md2) was suggested as early as 2 h. Because overexpression of either MKK4 or POSH can lead to apoptosis (Kukekov et al., 2006), the early increase in MKK4 and POSH mRNA levels suggested that these proteins may be directly involved in phthalate-induced germ cell apoptosis.
A second group of proteins that may be involved in the mitochondrial pathway of germ cell apoptosis is the secreted protein Cyr61 and its receptors syndecan-4 (Sdc4) and integrin alpha6beta 1 (a6b1). A recent report has causally linked the induction of the fibroblast mitochondrial apoptosis pathway and subsequent activation of caspase 3 to interaction of Cyr61 with both Sdc4 and a6b1 (Todorovicc et al., 2005). Like expressions of POSH and MKK4, testicular mRNA levels for Sdc4, Itga6 (integrin alpha6), and Cyr61 appeared induced hours prior to germ cell DNA fragmentation. Numerous genes potentially involved in prepubertal phthalate–induced germ cell apoptosis (Tnf, Tnfrsf1a, Tnfrsf6, Tnfrsf21, Ripk1, Tradd, Cradd, Casp3, Map2k4, Sh3md2, Cyr61, and Sdc4) were not altered significantly following fetal rat phthalate exposure. Because high-dose fetal exposure does not lead to increased apoptosis (Kleymenova et al., 2005), this result lends additional support to their involvement in prepubertal apoptotic induction.
For some prepubertal testis end points, phthalate exposure produces an interesting response at different dose levels or time points. In the data described here, expression of genes involved in cholesterol metabolism (Dhcr7), steroidogenesis (Cyp17a1 and StAR), and one other (Nr0b1) was decreased at 1000 mg/kg MEHP but increased at lower MEHP dose levels. The increase in steroidogenic gene expression at 10 and 100 mg/kg MEHP is corroborated by results showing increased testicular testosterone levels in rats following PND21 to 48 phthalate exposure at comparable dose levels (Akingbemi et al., 2001). A similar biphasic response, but over time and not dose, is observed for phthalate-induced germ cell apoptosis; prior to an induction in apoptosis, a decrease often is apparent (Rasoulpour and Boekelheide, 2005). With the available data, it is unclear if the same molecular pathway is being stimulated at a lower phthalate dose level and repressed at a higher dose level or if the high-dose level perturbs an additional pathway.
Although fetal and prepubertal testes contain cell types at divergent states of differentiation, a strikingly high degree of conservation was observed among genes altered following fetal and prepubertal phthalate exposure. The similar genetic response was evident especially at the early time points of exposure during which approximately 50% of the reactive genes were the same. Over time, the fetal and prepubertal testis gene profiles diverged, and this may reflect the differing histopathology observed; the prepubertal testis initiated germ cell apoptosis and showed a neutrophil infiltrate while these changes were not observed in DBP-exposed fetal rat testis (Kleymenova et al., 2005). Furthermore, the methodologies used in the fetal and prepubertal microarray analyses varied in three ways: (1) use of different phthalate congeners (DBP vs. MEHP); (2) dose levels (500 mg/kg DBP vs. 1000 mg/kg MEHP); and (3) time-point selection. While all time points of prepubertal exposure were examined on a single genome-wide Affymetrix chip, the fetal experiments were performed with two separate Affymetrix chips (RAE230 A and B) and only the 1- and 3-h time points were analyzed on both chips. Given these differences, it is likely that the degree of similarity in genetic response between the phthalate-exposed fetal and prepubertal testis described here is a conservative estimation. Overall, these data following fetal and prepubertal phthalate exposure suggest a common initial molecular and cellular testicular response followed by divergent histopathology due to differences in cellular differentiation state and/or tissue architecture.
To gain insight into the phthalate toxicity mechanism, network analysis of the genes with altered expression following both fetal and prepubertal exposure was performed (Fig. 5B). Because interaction linkages could not be detected for all genes using Ingenuity software, only a subset of genes was included in the network analysis. In the prepubertal testis, a large increase in Tbs1 mRNA levels was noted which began 2 h following 1000 mg/kg MEHP exposure, and by 3 and 12 h, Tbs1 mRNA levels were induced 10- and 114-fold, respectively. A similar increase occurs between 1 and 3 h in the fetal testis following 500 mg/kg DPB exposure (Thompson et al., 2005). This robust and early increase suggested a potentially critical role in downstream adverse testicular outcomes. Tbs1 is a secreted matricellular protein with numerous functional activities, including tissue remodeling and modulation of growth factor/cytokine responses (Chen et al., 2000). Although it binds to several different transmembrane receptors and cell surface proteins, integrins are one of the major Tbs1 receptors. Among Tbs1 integrin receptors, Itga6 expression appeared increased in the testis 3 h after fetal and prepubertal phthalate exposure. Another matricellular integrin ligand (connective tissue growth factor; Ctgf) was induced within hours of fetal and prepubertal phthalate exposure (Heng et al., 2006). Based upon increased expression of integrins and integrin ligands as well as the seminal role of integrins in tissue morphogenesis (De Arcangelis and Georges-Labouesse, 2000), an attractive hypothesis for some of the abnormal fetal testis morphogenesis observed following phthalate exposure is that it results from aberrant integrin-based cell adhesion and/or migration (Mahood et al., 2005).
In addition to a cell adhesion/migration function, Tbs1 can influence growth factor function. In vivo, Tbs1 activates the latent form of transforming growth factor beta 1 (Tgfb1), inducing Tgfb1 signaling events (Crawford et al., 1998). When Tgfb1 was included in the network analysis of common fetal and prepubertal genes, interaction of a large majority of the phthalate-altered genes with Tgfb1 was obvious (Fig. 5B). This network analysis supports the hypothesis that Tbs1 induction after phthalate exposure may produce an increase in active Tgfb1. A phthalate-induced decrease in testicular steroidogenic gene expression and testosterone production is a prominent outcome of exposure (Lehmann et al., 2004), and active Tgfb1 has been shown to decrease steroidogenic enzyme gene expression (Brand et al., 2000) and Leydig cell steroidogenesis (Gnessi et al., 1997). Thus, although Tgfb1 mRNA levels were not changed following phthalate exposure in the microarray analysis, we speculate that phthalate-induced decreases in testicular steroidogenesis may result from induction of Tbs1 protein secretion followed by Tbs1 activation of extracellular latent Tgfb1.
In conclusion, the data reported here showed that a single prepubertal exposure to 1000 mg/kg MEHP resulted in altered expression of many testicular genes related to intracellular signaling, cellular interactions, and gene transcription. At a MEHP dose level of 10 mg/kg, induction of germ cell apoptosis occurred, and nearly all genes tested by qPCR were changed significantly. Finally, the prepubertal testis genetic response appeared similar to that of the fetal testis following DBP exposure. These data suggest a common initial molecular mechanism of phthalate testicular toxicity following both prepubertal and fetal exposure.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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The authors wish to thank Linda Pluta for performing the microarray procedures and Drs. Rusty Thomas and Dave Dorman for reviewing the manuscript. This research was funded through the Long Range Research Initiative of the American Chemistry Council.
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REFERENCES |
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