Natural mineral fibers: conducting inhalation toxicology studies-part B: development of a nose-only exposure system for repeat-exposure in vivo study of Libby amphibole aerosol.

BACKGROUND: Exposure to asbestos is associated with malignant and nonmalignant respiratory disease. To strengthen the scientific basis for risk assessment on fibers, the National Institute of Environmental Health Sciences (NIEHS) has initiated a series of studies to address fundamental questions on the toxicology of naturally occurring asbestos and related mineral fibers after inhalation exposure. A prototype nose-only exposure system was previously developed and validated. The prototype system was expanded to a large-scale exposure system in this study for conducting subsequent in vivo rodent inhalation studies of Libby amphibole (LA) 2007, selected as a model fiber. RESULTS: The exposure system consisting of six exposure carousels was able to independently deliver stable LA 2007 aerosol to individual carousels at target concentrations of 0 (control group), 0.1, 0.3, 1, 3, or 10 mg/m3. A single aerosol generator was used to provide aerosol to all carousels to ensure that exposure atmospheres were chemically and physically similar, with aerosol concentration as the only major variable among the carousels. Transmission electron microscopy (TEM) coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis of aerosol samples collected at the exposure ports indicated the fiber dimensions, chemical composition, and mineralogy were equivalent across exposure carousels and were comparable to the bulk LA 2007 material. CONCLUSION: The exposure system developed is ready for use in conducting nose-only inhalation toxicity studies of LA 2007 in rats. The exposure system is anticipated to have applicability for the inhalation toxicity evaluation of other natural mineral fibers of concern.

Natural mineral fibers: conducting inhalation toxicology studies - part A: Libby Amphibole aerosol generation and characterization method development.

BACKGROUND: Asbestos has been classified as a human carcinogen, and exposure may increase the risk of diseases associated with impaired respiratory function. As the range of health effects and airborne concentrations that result in health effects across asbestos-related natural mineral fiber types are not fully understood, the National Institute of Environmental Health Sciences has established a series of research studies to characterize hazards of natural mineral fibers after inhalation exposure. This paper presents the method development work of this research project. RESULTS: A prototype nose-only exposure system was fabricated to explore the feasibility of generating natural mineral fiber aerosol for in vivo inhalation toxicity studies. The prototype system consisted of a slide bar aerosol generator, a distribution/delivery system and an exposure carousel. Characterization tests conducted using Libby Amphibole 2007 (LA 2007) demonstrated the prototype system delivered stable and controllable aerosol concentration to the exposure carousel. Transmission electron microscopy (TEM) analysis of aerosol samples collected at the exposure port showed the average fiber length and width were comparable to the bulk LA 2007. TEM coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis further confirmed fibers from the aerosol samples were consistent with the bulk LA 2007 chemically and physically. CONCLUSIONS: Characterization of the prototype system demonstrated feasibility of generating LA 2007 fiber aerosols appropriate for in vivo inhalation toxicity studies. The methods developed in this study are suitable to apply to a multiple-carousel exposure system for a rat inhalation toxicity testing using LA 2007.

NURR1 expression regulates retinal pigment epithelial-mesenchymal transition and age-related macular degeneration phenotypes.

Phenotypic variations in the retinal pigment epithelial (RPE) layer are often a predecessor and driver of ocular degenerative diseases, such as age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We previously identified the orphan nuclear receptor-related 1 (NURR1), from a nuclear receptor atlas of human RPE cells, as a candidate transcription factor potentially involved in AMD development and progression. In the present study we characterized the expression of NURR1 as a function of age in RPE cells harvested from human donor eyes and in donor tissue from AMD patients. Mechanistically, we found an age-dependent shift in NURR1 dimerization from NURR1-RXRα heterodimers toward NURR1-NURR1 homodimers in primary human RPE cells. Additionally, overexpression and activation of NURR1 attenuated TNF-α-induced epithelial-to-mesenchymal transition (EMT) and migration, and modulated EMT-associated gene and protein expression in human RPE cells independent of age. In vivo, oral administration of IP7e, a potent NURR1 activator, ameliorated EMT in an experimental model of wet AMD and improved retinal function in a mouse model that presents with dry AMD features, impacting AMD phenotype, structure, and function of RPE cells, inhibiting accumulation of immune cells, and diminishing lipid accumulation. These results provide insight into the mechanisms of action of NURR1 in the aging eye, and demonstrate that the relative expression levels and activity of NURR1 is critical for both physiological and pathological functions of human RPE cells through RXRα-dependent regulation, and that targeting NURR1 may have therapeutic potential for AMD by modulating EMT, inflammation, and lipid homeostasis.

Leveraging Nuclear Receptors as Targets for Pathological Ocular Vascular Diseases.

Vasculogenesis and angiogenesis are physiological mechanisms occurring throughout the body. Any disruption to the precise balance of blood vessel growth necessary to support healthy tissue, and the inhibition of abnormal vessel sprouting has the potential to negatively impact stages of development and/or healing. Therefore, the identification of key regulators of these vascular processes is critical to identifying therapeutic means by which to target vascular-associated compromises and complications. Nuclear receptors are a family of transcription factors that have been shown to be involved in modulating different aspects of vascular biology in many tissues systems. Most recently, the role of nuclear receptors in ocular biology and vasculopathies has garnered interest. Herein, we review studies that have used in vitro assays and in vivo models to investigate nuclear receptor-driven pathways in two ocular vascular diseases associated with blindness, wet or exudative age-related macular degeneration, and proliferative diabetic retinopathy. The potential therapeutic targeting of nuclear receptors for ocular diseases is also discussed.

LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.

Editor's Highlight: PPARß/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARß/δ or PPARγ enhanced ligand-induced expression of a PPARß/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARß/δ, or vice versa. Stable overexpression of either PPARß/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARß/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARß/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

Peroxisome proliferator-activated receptor-ß/δ modulates mast cell phenotype.

The peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) is known to have multiple anti-inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARß/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARß/δ modulates mast cell phenotype. Bone-marrow-derived mast cells (BMMCs) and peritoneal mast cells from Pparß/δ+/+ mice expressed higher levels of high-affinity IgE receptor (FcεRI) compared with Pparß/δ-/- mice. BMMCs from Pparß/δ+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparß/δ-/- mice. Resting BMMCs from Pparß/δ+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal-regulated kinases compared with Pparß/δ-/- mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARß/δ expression. This study demonstrates that PPARß/δ is an important regulator of mast cell phenotype.

Peroxisome proliferator-activated receptor-ß/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-ß/δ, (PPARß/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARß/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARß/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARß/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARß/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARß/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARß/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-activated Receptor-ß/δ Modulation of SP1 Promoter Occupancy.

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparß/δ+/+ mice but not in Pparß/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARß/δ, was up-regulated in Pparß/δ+/+ liver following ethanol exposure, but not in Pparß/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARß/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARß/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARß/δ attenuates ethanol-induced Cyp2b10 expression in Pparß/δ+/+ liver but not in Pparß/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARß/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARß/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

PPARß/δ selectively regulates phenotypic features of age-related macular degeneration.

Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) is a nuclear receptor that regulates differentiation, inflammation, lipid metabolism, extracellular matrix remodeling, and angiogenesis in multiple tissues. These pathways are also central to the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss globally. With the goal of identifying signaling pathways that may be important in the development of AMD, we investigated the impact of PPARß/δ activation on ocular tissues affected in the disease. PPARß/δ is expressed and can be activated in AMD vulnerable cells, including retinal pigment epithelial (RPE) and choroidal endothelial cells. Further, PPARß/δ knockdown modulates AMD-related pathways selectively. Specifically, genetic ablation of Pparß/δ in aged mice resulted in exacerbation of several phenotypic features of early dry AMD, but attenuation of experimentally induced choroidal neovascular (CNV) lesions. Antagonizing PPARß/δ in both in vitro angiogenesis assays and in the in vivo experimentally induced CNV model, inhibited angiogenesis and angiogenic pathways, while ligand activation of PPARß/δ, in vitro, decreased RPE lipid accumulation, characteristic of dry AMD. This study demonstrates for the first time, selective regulation of a nuclear receptor in the eye and establishes that selective targeting of PPARß/δ may be a suitable strategy for treatment of different clinical sub-types of AMD.

Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-ß/δ- and retinoic acid receptor-dependent mechanisms.

Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARß/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARß/δ. Expression of PPARß/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARß/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARß/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARß/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARß/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

Peroxisome Proliferator-activated Receptor-D (PPARD) Coordinates Mouse Spermatogenesis by Modulating Extracellular Signal-regulated Kinase (ERK)-dependent Signaling.

Ppard(-/-) mice exhibit smaller litter size compared with Ppard(+/+) mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard(-/-) mice compared with Ppard(+/+) mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard(+/+) mice as compared with Ppard(-/-) mice, and these were associated with decreased Sertoli cell number in Ppard(+/+) mice. Cyclin D1 and cyclin D2 expression was lower in Ppard(+/+) as compared with Ppard(-/-) mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard(+/+) mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

Targeting Peroxisome Proliferator-Activated Receptor-ß/δ (PPARß/δ) for Cancer Chemoprevention.

The role of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in cancer remains contentious due in large part to divergent publications indicating opposing effects in different rodent and human cell culture models. During the past 10 years, some facts regarding PPARß/δ in cancer have become clearer, while others remain uncertain. For example, it is now well accepted that (1) expression of PPARß/δ is relatively lower in most human tumors as compared to the corresponding non-transformed tissue, (2) PPARß/δ promotes terminal differentiation, and (3) PPARß/δ inhibits pro-inflammatory signaling in multiple in vivo models. However, whether PPARß/δ is suitable to target with natural and/or synthetic agonists or antagonists for cancer chemoprevention is hindered because of the uncertainty in the mechanism of action and role in carcinogenesis. Recent findings that shed new insight into the possibility of targeting this nuclear receptor to improve human health will be discussed.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid.

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17ß-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

Targeting estrogen receptor-ß for the prevention of nonmelanoma skin cancer.

The potential for targeting estrogen receptor (ER)-ß in various cancer models has been gaining considerable attention in recent years. In this issue of the journal, Chaudhary and colleagues demonstrate markedly decreased ultraviolet B (UVB)-induced skin cancer in a mouse model using a highly specific ER-ß agonist, ERB-041. The mechanisms that underlie this strong inhibitory effect are mediated by inhibition of proinflammatory signaling and epithelial-mesenchymal transition (EMT). The changes in EMT were due in part to modulation of WNT/ß-catenin signaling. Collectively, the results from these studies provide important new insights into the mechanisms by which the ER-ß agonist ERB-041 inhibits UVB-induced skin cancer and opens the door for future studies that could examine combinatorial approaches for UVB-dependent skin cancer chemoprevention.

Activation of peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ) inhibits human breast cancer cell line tumorigenicity.

The effect of activation and overexpression of the nuclear receptor PPAR-ß/δ in human MDA-MB-231 (estrogen receptor-negative; ER(-)) and MCF7 (estrogen-receptor-positive; ER(+)) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-ß/δ was increased by overexpression of PPAR-ß/δ compared with controls. Overexpression of PPAR-ß/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-ß/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-ß/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-ß/δ in response to ligand activation of PPAR-ß/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-ß/δ were significantly smaller, and ligand activation of PPAR-ß/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-ß/δ and ligand activation of PPAR-ß/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-ß/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-ß/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-ß/δ in breast cancer and evaluating the effects of PPAR-ß/δ agonists.

Mono-(2-ethylhexyl) phthalate (MEHP) promotes invasion and migration of human testicular embryonal carcinoma cells.

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.

Transcriptional suppression of Sertoli cell Timp2 in rodents following mono-(2-ethylhexyl) phthalate exposure is regulated by CEBPA and MYC.

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.

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.

Mono-(2-ethylhexyl) phthalate-induced disruption of junctional complexes in the seminiferous epithelium of the rodent testis is mediated by MMP2.

Tight junctions between Sertoli cells of the testicular seminiferous epithelium establishes the blood-testis barrier (BTB) and creates a specialized adluminal microenvironment above the BTB that is required for the development of the germ cells that reside there. Actin filament-based anchoring junctions between Sertoli cells and germ cells are important for maintaining close physical contact between these cells as well as regulating the release of mature spermatids into the lumen. Previously, we reported that Sertoli cell injury in rodents after mono-(2-ethylhexyl) phthalate (MEHP) exposure results in the activation of matrix metalloproteinase 2 (MMP2) and increases the sensitivity of germ cells to undergo apoptosis. A disruption in the physical association between Sertoli cells and germ cells and premature loss of germ cells from the seminiferous epithelium has been widely described after phthalate treatment. In this study, we investigate the functional participation of MMP2 in the mechanism underlying MEHP-induced disruption of junction complexes and the resultant loss of germ cells. Exposure of C57BL/6J mice to MEHP (1 g/kg, oral gavage) decreased the expression of occludin in the tight junctions between Sertoli cells and caused gaps between adjacent Sertoli cells as observed by transmission electron microscopy. A reduced expression of laminin-gamma3 and beta1-integrin in apical ectoplasmic specializations between Sertoli cells and germ cells in a time-dependent manner was also observed. Treatment with specific MMP2 inhibitors (TIMP2 and SB-3CT) both in vitro and in vivo significantly suppressed MEHP-induced germ cell sloughing and changes in the expression of these junctional proteins, indicating that MMP-2 plays a primary role in this process. Furthermore, the detachment of germ cells from Sertoli cells appears to be independent of the apoptotic signaling process since MEHP-induced germ cell detachment from Sertoli cells could not be prevented by the addition of a pan-caspase inhibitor (z-VAD-FMK).