Mono-(2-ethylhexyl) Phthalate Treatment Research Articles

Inhibition of Claudin‐11 and Occludin Expression in Rat Sertoli Cells by Mono‐(2‐Ethylhexyl) Phthalate Through p44/42 Mitogen‐Activated Protein Kinase Pathway

Our objectives were to evaluate the effects of mono(2-ethylhexyl) phthalate (MEHP) on tight junctions (TJ) in cultured rat Sertoli cells (SC) and to investigate changes in the signal transduction pathways in SCs following MEHP treatment. SCs were isolated and purified from the testes of 18-day-old Sprague Dawley rats and incubated at 34°C for 3 days. After treatment of SCs with either the vehicle or MEHP for 0.5, 1, 3, 6 and 24 hours, whole cell lysates were isolated from each replicate to prepare RNA and protein. Expression levels of claudin-11, occludin, and zonula occludens-1 (ZO-1) mRNA were evaluated by quantitative real-time reverse transcription-polymerase chain reaction and changes in signal transduction pathways possibly induced by MEHP treatment were assessed by Western blot analyses. MEHP treatment led to significant decreases in the expression of claudin-11 and occludin mRNA, but not that of ZO-1, in rat SCs. Exposure of rat SCs to MEHP resulted in the marked induction of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), whereas other pathways examined in this study were not activated by MEHP. Furthermore, treatment of rat SCs with a specific inhibitor of p44/42 MAPK prevented the MEHP-induced down-regulation of claudin-11 and occludin. These findings demonstrate that MEHP exposure inhibited the expression of claudin-11 and occludin mRNA in rat SCs through the p44/42 MAPK pathway, suggesting the possible involvement of MEHP in spermatogenic function by regulating major components of TJs in SCs.

Mono-(2-Ethylhexyl) Phthalate (MEHP) Promotes Invasion and Migration of Human Testicular Embryonal Carcinoma Cells1

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

Mono-2-ethylhexyl phthalate induced loss of mitochondrial membrane potential and activation of Caspase3 in HepG2 cells

L02 and HepG2 cells were exposed to mono-(2-ethylhexyl) phthalate (MEHP) at concentrations of 6.25–100μM. After 48h treatment, MEHP decreased HepG2 cell viability in a concentration-dependent manner and L02 cell viability in the 50 and 100μM groups (p<0.01). Furthermore, at 24 and 48h after treatment, MEHP decreased the glutathione levels of HepG2 cells in all treatment groups and in the ΔΨm in L02 and HepG2 cells with MEHP≥25μM (p<0.05 or p<0.01). At 24h after treatment, MEHP induced activation of caspase3 in all treated HepG2 and L02 cells (p<0.05 or p<0.01) except the 100μM MEHP treatment group. The increase in the Bax to Bcl-2 ratio suggests that Bcl-2 family involved in the control of MEHP-induced apoptosis in these two cell types. The data suggest that MEHP could induce apoptosis of HepG2 cells through mitochondria- and caspase3-dependent pathways.

Evaluation of ovarian toxicity of mono-(2-ethylhexyl) phthalate (MEHP) using cultured rat ovarian follicles

Mono-(2-ethylhexyl) phthalate (MEHP) is the most toxic metabolite of di-(2-ethylhexyl) phthalate (DEHP). It has been reported that DEHP causes abnormal reproductive development in women, and suppresses estradiol synthesis and ovulation in female rats with diminished size of preovulatory follicles. The present study was conducted to evaluate the ovarian toxicity of MEHP using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with MEHP (0, 10, 30, and 100 µg/ml). At 0, 24, and 48 hr of MEHP treatment, follicular diameters were measured. After the culture, viability and apoptosis of follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At 100 µg/ml, suppression of follicular development was observed, which is associated with decreased viability of follicles and apoptosis of granulosa cells. At this concentration, progesterone level increased markedly, whereas androstenedione, testosterone, and estradiol levels decreased. At 10 and 30 µg/ml, follicular development was not suppressed, no apoptotic change was observed, and the levels of all measured steroid hormones tended to increase. The combined levels of all steroid hormones increased at all concentrations of MEHP, and the increase implies that MEHP activates the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. However, the progesterone/androstenedione ratio increased extremely at 100 µg/ml, and the increase implies that MEHP inhibits the conversion of progesterone to androstenedione. In conclusion, MEHP induces ovarian toxicity via suppression of follicular development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.

Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1

Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.

Mono-(2-ethylhexyl) phthalate impairs neurodevelopment: Inhibition of proliferation and promotion of differentiation in PC12 cells

Di-(2-ethylhexyl)-phthalate (DEHP) is the most widely used plasticizers in daily-life products. In this study we evaluated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, on the neurodevelopment in vitro. After neuronotypic PC12 cells were exposed to MEHP (0.25, 2.5, 25, and 250 μM), the effects of which on cell proliferation and differentiation were investigated. In undifferentiated PC12 cells, MEHP inhibited cell proliferation in a dose-dependent manner. After 24 h of MEHP treatment, there was a dose-dependent G2/M cell cycle arrest as well as a sharp drop of DNA synthesis. During the process of NGF-induced differentiation of the cell line, 4 days of MEHP exposure (2.5–250 μM) increased membrane and cytoskeletal protein contents, enhanced NGF-induced neurite outgrowth, up-regulated the choline acetyl transferase (ChAT) mRNA and down-regulated tyrosine hydroxylase (TH) mRNA levels, which suggested the promoted differentiation towards the acetylcholine (ACh) phenotype at the expense of the dopamine (DA) phenotype. Take together, our results indicate that MEHP has a potential to disturb neurodevelopment by suppressing cell proliferation and promote cell differentiation in neurocytes.

FasL Gene–Deficient Mice Display a Limited Disruption in Spermatogenesis and Inhibition of Mono-(2-ethylhexyl) Phthalate–Induced Germ Cell Apoptosis

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene–deficient mice (FasL−/−) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL−/− mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 ± 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 ± 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL−/− mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL−/− mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL−/− mice. However, PND 44 FasL−/− mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL−/− mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.

Phthalates Impair Germ Cell Development in the Human Fetal Testis in Vitro without Change in Testosterone Production

BackgroundSeveral studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat.ObjectivesUsing the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate—mono-2-ethylhexyl phthalate (MEHP)—an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function.MethodsHuman fetal testes were recovered during the first trimester (7–12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH).ResultsWhatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10−4 M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10−4 M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3–positive germ cells, without modification of their proliferation.ConclusionsThis is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.

An Endogenous Prostaglandin Enhances Environmental Phthalate-Induced Apoptosis in Bone Marrow B Cells: Activation of Distinct but Overlapping Pathways

Phthalate esters are ubiquitous environmental contaminants that are produced for a variety of common industrial and commercial purposes. We have shown that mono-(2-ethylhexyl) phthalate (MEHP), the toxic metabolite of di-(2-ethylhexyl) phthalate, induces bone marrow B cell apoptosis that is enhanced in the presence of the endogenous prostaglandin 15-deoxy-Delta((12, 14))-PGJ(2) (15d-PGJ(2)). Here, studies were performed to determine whether 15d-PGJ(2)-mediated enhancement of MEHP-induced apoptosis represents activation of an overlapping or complementary apoptosis pathway. MEHP and 15d-PGJ(2) induced significant apoptosis within 8 and 5 h, respectively, in a pro/pre-B cell line and acted cooperatively to induce apoptosis in primary pro-B cells. Apoptosis induced with each chemical was accompanied by activation of a combination of initiator caspases (caspases-2, -8, and -9) and executed by caspase-3. Apoptosis induced with MEHP and 15d-PGJ(2) was reduced in APAF1 null primary pro-B cells and accompanied by alteration of mitochondrial membranes, albeit with different kinetics, indicating an intrinsically activated apoptosis pathway. Significant Bax translocation to the mitochondria supports its role in initiating release of cytochrome c. Both chemicals induced Bid cleavage, a result consistent with a truncated Bid-mediated release of cytochrome c in an apoptosis amplification feedback loop; however, significantly more Bid was cleaved following 15d-PGJ(2) treatment, potentially differentiating the two pathways. Indeed, Bid cleavage and cytochrome c release following 15d-PGJ(2) but not MEHP treatment was profoundly inhibited by Z-VAD-FMK, suggesting that 15d-PGJ(2) activates apoptosis via two pathways, Bax mobilization and protease-dependent Bid cleavage. Thus, endogenous 15d-PGJ(2)-mediated enhancement of environmental chemical-induced apoptosis represents activation of an overlapping but distinct signaling pathway.

Mono‐(2‐ethylhexyl) Phthalate Increases Spermatocyte Mitochondrial Peroxiredoxin 3 and Cyclooxygenase 2

Mono-(2-ethylhexyl) phthalate (MEHP), the biologically active metabolite of the plasticizer di-(2-ethylhexyl) phthalate, is a member of a class of chemical compounds with known adverse effects on the male reproductive system. Recent studies showed that oxidative stress and mitochondrial dysfunction in germ cells may contribute to phthalate-induced disruption of spermatogenesis. To determine whether the redox-protein mitochondrial thioredoxin-dependent peroxidase, peroxiredoxin 3 (Prx3), may be a component of germ cell homeostasis mechanisms, this study first examined the physiologic relevance of Prx3 in the rodent testis by determining its cell-specific expression. Our findings show that prx3 mRNA is expressed in a developmental, cell-specific manner in rat Leydig cells, Sertoli cells, and germ cells; among mouse germ cells, prx3 expression was highest in spermatocytes, findings consistent with those in rat. In mouse meiotic spermatocytes, Prx3 was strikingly localized at the nuclear perimeter and cytoplasm, findings suggestive of a direct role for Prx3 in determining spermatocyte response to toxicants. To better define the mechanisms involved in male germ cell dysfunction following phthalate exposure, an immortalized mouse spermatocyte-derived germ cell line, GC-2spd(ts), was exposed to MEHP (24 hours; 100 and 200 microM). We determined whether Prx3 and cyclooxygenase-2 (COX-2), pivotal proteins involved in oxidative stress responses in spatially restricted subcellular domains, were affected. Mitochondrial Prx3 and mitochondrial and cytosolic COX-2 significantly increased following 200 microM MEHP treatment; proliferation was inhibited without inducing cell death. Using this germ cell model, the data suggest that changes in cellular oxidation-reduction (redox) homeostasis in the germline can accompany MEHP exposure, disrupting mitochondrial antioxidant defenses, despite absence of phthalate-induced apoptosis.

Effect of mono‐(2‐ethylhexyl) phthalate (MEHP) on resumption of meiosis, in vitro maturation and embryo development of immature mouse oocytes

The purpose of this study was to evaluate the effect of MEHP on in vitro maturation of mouse oocytes and resulting embryo development. Denuded oocytes (DO) were cultured in maturation medium supplemented with 0, 50, 100, 200 and 400 microM levels of MEHP for 24 h. The matured oocytes then were fertilized and cultured for 4 days. The percentage of Germinal Vesicle (GV) stage oocytes were significantly higher in 200 and 400 microM MEHP treatment comparing to the control (P < 0.05). The proportion of oocytes that progressed to the metaphase II (MII) stage was significantly decreased by adding of MEHP in a dose related pattern. The 2-cell embryo formation was significantly decreased with 400 microM treatments than the control. Moreover with further culture in experimental groups none of the embryos comparing to that of the control group were developed to morulla stage (P < 0.05). These results indicate that MEHP could negatively modulate mouse oocyte meiotic maturation in vitro and embryo development, suggesting possible risks for human and other mammalians reproductive health.

Molecular Pathological Analysis of Apoptosis Induced in Testicular Germ Cells after a Single Administration of Mono-(2-ethylhexyl) phthalate to F344 Rats

Mono-(2-ethylhexyl) phthalate (MEHP) is known to cause germ cell apoptosis in the testis of rats as a consequence of Sertoli cell damage. In the present study, a single dose of 2,000 mg/kg body weight MEHP was orally administered to Fischer 344 rats and the molecular apoptosis pathway was surveyed. Histopathological examination, TUNEL assay and immunohistochemical analysis of cleaved caspase-3 were performed to assess the reproducibility of apoptosis. In addition, immunohistochemical analysis and RT-PCR to determine the parameters of the apoptosis pathway were performed. Histopathological examination showed that from 6 to 12 hours after MEHP treatment there was detachment of germ cells that had eosinophilic cytoplasm and pyknotic nuclei. A TUNEL assay detected a significant increase in TUNEL-positive cells at 12 hours after treatment. Immunohistochemistry showed some of the cytoplasm of germ cells was positive for cleaved caspase-3 at 12 hours after treatment. In immunohistochemistry of the Fas/Fas ligand (FasL) apoptosis pathway, the cytoplasm of germ cells was found to be occasionally positive for Fas at 12 hours after treatment, and the expression of the Fas gene in RT-PCR had significantly increased at 12 hours after treatment. RT-PCR of the mitochondrial apoptosis pathway revealed a significant increase in the expression of Bax and Bcl-2 genes in the treated group at 12 hours after treatment. These results strongly suggest that both the Fas/FasL and mitochondrial pathways are involved in germ cell apoptosis induced by MEHP.

Mono-(2-ethylhexyl) phthalate (MEHP) induces testicular alterations in male guinea pigs at prepubertal stage

We have recently shown that MEHP induces spermatogenic cell apoptosis in guinea pigs at prepubertal stage in vitro. To evaluate the effects of MEHP on the testicular tissues of guinea pigs in vivo, we conducted this research work. Five weeks old male guinea pigs were used in this experiment. They received a single oral dose of 2000 mg/ml of MEHP in corn oil by gavage at a volume equal to 4 ml/kg. Control group received a similar volume of corn oil vehicle. Vehicle- and MEHP-treated guinea pigs were sacrificed at the interval of 3, 6, and 9 h, and the testicular tissues were processed for histopathological studies. Distinct histopathological changes were recognized in testes. Detachment and displacement of spermatogenic cells, thin seminiferous epithelia, vacuolization of Sertoli cells were prominent at 6 h after MEHP treatment. The lumina of the efferent ductules were frequently occupied with sloughed seminiferous epithelia from 6 to 9 h after MEHP treatment. Apoptotic spermatogenic cells appeared at 3 h in the control group. The incidence of apoptotic spermatogenic cells significantly increased ( * p < 0.05) from 3 to 9 h, and the maximal increase of apoptotic spermatogenic cells were observed at 9 h after MEHP treatment. Time-dependent increases of apoptotic spermatogenic cells was recognized throughout the experimental period. It may be suggested here that MEHP also induces spermatogenic cell apoptosis in guinea pigs in vivo and guinea pigs may be considered as a useful animal model for sensitivity test of the reproductive toxicity to some phthalate esters at their earlier stage in vivo.

Mono-(2-ethylhexyl) Phthalate (MEHP) Induces Spermatogenic Cell Apoptosis in Guinea Pig Testes at Prepubertal Stage In Vitro

The effects of mono-(2-ethylhexyl) phthalate (MEHP), an active metabolite of di-(2-ethylhexyl) phthalate (DEHP), on prepubertal guinea pig testes in vitro were investigated. The testes of 35-day-old guinea pigs were surgically excised. They were seeded in a defined medium containing antibiotics and administered MEHP at concentrations of 1, 10, and 100 nmol/ml, respectively. The control groups were administered a similar volume of corn oil vehicle. The tissues were incubated for 3, 6, and 9 h. The specimens were collected at 3, 6, and 9 h after treatment. They were fixed in 4% paraformaldehyde or 5% glutaraldehyde. For quantitation of the apoptotic spermatogenic cells, the terminal dUTP nick end-labeling (TUNEL) staining was performed by light microscopy. Detachment and displacement of spermatogenic cells, thin seminiferous epithelia, and Sertoli cell vacuolization were observed. Maximal testicular damage was recognized at 100 nmol/ml and 9 h after MEHP treatment. The percentage (%) of apoptotic spermatogenic cells significantly increased at 3, 6, and 9 h after treatment, compared to the control groups. Because the loss of spermatogenic cells by MEHP treatment varies among species, the present study, using guinea pigs, was designed and conducted to obtain further information.

An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro

In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of metabolites of di(2-ethylhexyl) phthalate, on immature Shiba goat testes in vitro were examined. The testes of 2-month-old Shiba goats were cut into smaller pieces, and seeded in medium. At 1, 3, 6 and 9 hr after administration of MEHP at various concentrations (0, 100 nmol ml(-1), 1 nmol ml(-1), and 1 x 10(-3) nmol ml(-1), respectively), the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, the vacuolization and nuclear membrane rupture appeared in Sertoli cells. Such alterations tended to gradually increase in number in time and dose-dependent manners. Moreover, by MEHP treatment, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still functioning cell organelles, and packed cell contents in membrane-bounded bodies), apoptotic Sertoli cells (characterized with nuclear membrane lysis, nuclear condensation), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), and necrotic Sertoli cells (characterized with marginated chromatins along the nuclear membrane, ruptured vesicles within the MNB, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, ultrastructurally the treatment with MEHP at low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas that at high concentration tends to lead spermatogenic and Sertoli cells to necrosis. Thus, the testicular tissue culture is advantageous for screening testicular toxicity of chemicals.

Effects of Mono(2-ethylhexyl) Phthalate (MEHP) on Testes in Rats In Vitro

The phthalate esters have been used as plasticizers for various plastic products, and their testicular toxicity has been reported. In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of the phthalate esters, on prepubertal rat testes in vitro were examined. The testes of 20-day-old Sprague Dawley (SD) rats were cut into smaller pieces and seeded in medium, and then the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, TUNEL-positive spermatogenic cells were identified, and they gradually increased in number in time- and dose-dependent manners. Ultrastructurally, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still-functioning cell organelles, and packed cell contents in membrane-bounded bodies), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), apoptotic Sertoli cells (highly condensed nuclei and nuclear membrane lysis) and necrotic Sertoli cells (marginated chromatins along the nuclear membrane, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, based on transmission electron microscopic observations, MEHP treatment may affect spermatogenic cells, and lead them to necrosis. Thus, testicular tissue cultures and cell cultures are of advantageous for screening testicular toxicity of chemicals.

Effects of Mono-(2-Ethylhexyl) Phthalate on Fetal and Neonatal Rat Testis Organ Cultures1

Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to cause reproductive toxicity in both developing and adult animals. In this study, we used organ cultures of fetal and neonatal rat testes to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of E13 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest that MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.

Effect of mono-(2-ethylhexyl) phthalate on bovine oocyte maturation in vitro

The effect of mono(2-ethylhexyl) phthalate (MEHP) on bovine oocyte maturation in vitro was examined. Cumulus–oocyte complexes (COCs) were cultured in maturation medium supplemented with various levels of MEHP for 24 h, and then examined for the degree of cumulus expansion and the stage of maturation. A higher percentage of oocytes remained at the germinal vesicle (GV) stage after exposure to 75 and 100 μM MEHP treatments (13.8 and 44.9% of oocytes, respectively) than the control (2.1% of oocytes). The proportion of oocytes that progressed to the metaphase II (MII) stage was significantly decreased with 25 μM (59.6% of oocytes), 50 μM (19.8%), 75 μM (21.3%), and 100 μM (3.1%) treatments than the control (77.3%). MEHP did not affect the process of cumulus expansion. For denuded oocytes, MEHP treatment of 50–100 μM resulted in a significantly higher rate of oocytes remained at the GV stage compared to the control (53.4, 80.2, 88.4, and 5.4%, respectively). The rate of MII formation was significantly decreased with 10 μM (60.9%) and 25 μM (22.5%) MEHP treatments compared to control (68.9%). Furthermore, with 50, 75 or 100 μM MEHP, no oocyte reached the MII stage. When COCs were cultured for 24 h with 50 or 100 μM MEHP and then cultured for an additional 24 h in MEHP-free medium, most of the oocytes reached the MII stage (71.1 and 64.5%, respectively). Taken together, these results indicate that MEHP, at doses lower than those reported in blood transfusion patients, could negatively modulate bovine oocyte meiotic maturation in vitro, suggesting possible risks for human and other mammalians reproductive health.

Death Receptor Response in Rodent Testis after Mono-(2-ethylhexyl) Phthalate Exposure

Apoptosis of testicular germ cells is critical for the maintenance of functional spermatogenesis. Previously, we have demonstrated that the Fas (Apo-1, CD95) receptor participates in the regulation of germ cell apoptosis, particularly after toxicant-induced Sertoli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas gld,gld ( gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague–Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague–Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFκB activation) revealed an early increase in NFκB–DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis ( gld and lpr cg ) reiterates the importance of the Fas signaling pathway.

Effect of mono-(2-ethylhexyl) phthalate on the metabolism of energy-yielding substrates in rat sertoli cell-enriched cultures

The effect of the testicular toxin mono-(2-ethylhexyl) phthalate (MEHP) on the metabolism of energy-yielding substrates in Sertoli cell-enriched cultures has been studied. MEHP stimulated glucose utilization and oxidation. Stimulation of 14CO 2 production was greater with [1- 14C]- than with [6- 14C]glucose. Oxidation of [1- 14C]pyruvate and [U- 14C]acetate to 14CO 2 was reduced by MEHP treatment in the presence but unaffected in the absence of glucose. MEHP increased the incorporation of radioactivity from [1- 14C]- and [6- 14C]glucose but not from [U- 14C]acetate into fatty acids. MEHP markedly increased the production of lactate by Sertoli cells cultured in the presence of 5.5 m m-glucose and 0.5 m m-pyruvate. Decreasing the glucose concentration reduced this stimulatory effect. In glucose-free medium containing 0.5 or 2.5 m m-pyruvate MEHP had no effect on Sertoli cell lactate production. Thus, MEHP appears to stimulate the utilization of glucose by the Sertoli cell but does not appear to have a direct effect on the conversion of pyruvate to lactate, on the conversion of pyruvate to acetyl CoA or a direct effect on the metabolism of acetyl CoA through the Krebs cycle. Increased glucose utilization appears to be a consequence of the increased metabolism of glucose that occurs through the glycolytic and pentose phosphate pathways. These results suggest that in contrast to its effects on the liver, MEHP does not exert a primary effect on the Sertoli cell mitochondrion.