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.

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.

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.

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.

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.

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.