Duncan G. Wallace

Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate

Adult male rats previously exposed on gestation days (GD) 12-21 to di(n-butyl) phthalate (DBP) have reproductive tract malformations, particularly agenesis of the epididymis, decreased sperm production, and Leydig cell hyperplasia and adenomas. Although similar effects are produced by the potent androgen receptor (AR) antagonist flutamide and are indicative of disruption of male sexual differentiation via an antiandrogenic mechanism, DBP is not an AR antagonist. The purpose of the study was to determine whether DBP causes pathologic changes and alterations in androgen status in the testis during the prenatal period of male reproductive tract differentiation. Pregnant CD rats were given corn oil, DBP (500 mg/kg/day), or flutamide (100 mg/kg/day) p.o. on GD 12-21. At GD 16-21, DBP caused hyperplasia of Leydig cells, many of which were 3beta-hydroxysteroid dehydrogenase- and/or AR-positive. Focal areas of hyperplasia had increased numbers of Leydig cells positive for proliferating cell nuclear antigen (PCNA). At GD 21, testis atrophy was apparent, seminiferous cords in DBP-exposed fetuses were enlarged and contained multinucleated gonocytes that, unlike controls, were PCNA-positive. DBP, but not flutamide, markedly decreased testicular testosterone levels at GD 18 and 21. Fewer epididymal ducts and reduced AR staining in some ducts were evident with DBP treatment, whereas decreased overall AR staining was seen with flutamide in the presence of mild Leydig cell hyperplasia. Leydig cell proliferation is likely a compensatory mechanism to increase testicular steroidogenesis triggered by testosterone insufficiency. The overall decrease in androgen concentration is not corrected and results in reproductive tract malformations. The multinuclearity and proliferation of gonocytes suggests an underlying Sertoli cell dysfunction.

Bacterial Lipopolysaccharide Enhances PDGF Signaling and Pulmonary Fibrosis in Rats Exposed to Carbon Nanotubes

Engineered multi-walled carbon nanotubes (MWCNT) represent a possible health risk for pulmonary fibrosis due to their fiber-like shape and potential for persistence in the lung. We postulated that bacterial lipopolysaccharide (LPS), a ubiquitous agent in the environment that causes lung inflammation, would enhance fibrosis caused by MWCNT. Rats were exposed to LPS and then intratracheally instilled with MWCNT or carbon black (CB) nanoparticles 24 hours later. Pulmonary fibrosis was observed 21 days after MWCNT exposure, but not with CB. LPS alone caused no fibrosis but enhanced MWCNT-induced fibrosis. LPS plus CB did not significantly increase fibrosis. MWCNT increased platelet-derived growth factor-AA (PDGF-AA), a major mediator of fibrosis. PDGF-AA production in response to MWCNT, but not CB, was synergistically enhanced by LPS. Immunostaining showed PDGF-AA in bronchiolar epithelial cells and macrophages. Since macrophages engulfed MWCNT, were positive for PDGF-AA, and mediate fibroblast responses, experiments were performed with rat lung macrophages (NR8383 cells) and rat lung fibroblasts in vitro. LPS exposure increased PDGF-A mRNA levels in NR8383 cells and enhanced MWCNT-induced PDGF-A mRNA levels. Moreover, LPS increased MWCNT- or CB-induced PDGF receptor-alpha (PDGF-Ralpha) mRNA in fibroblasts. Our data suggest that LPS exacerbates MWCNT-induced lung fibrosis by amplifying production of PDGF-AA in macrophages and epithelial cells, and by increasing PDGF-Ralpha on pulmonary fibroblasts. Our findings also suggest that individuals with pre-existing pulmonary inflammation are at greater risk for the potential adverse effects of MWCNT.

Effects of in utero linuron exposure on rat Wolffian duct development.

Linuron is an herbicide that displays weak androgen receptor antagonist activity. Male offspring exposed in utero to 50 mg/kg/day linuron often exhibit malformations in Wolffian duct derivatives (i.e. the epididymis and vas deferens). The objectives of this study were to determine the point during the perinatal period that linuron-induced epididymal lesions can be identified, to characterize linuron-mediated perinatal testicular and epididymal pathology, and to determine whether male rat fetuses exposed prenatally to linuron exhibit decreased intratesticular and serum testosterone (T) levels. Pregnant rats were administered corn oil vehicle or linuron by gavage at 0 or 50 mg/kg/day (n = 3 controls, 5-11 linuron-treated dams per time point) from gestation days (GD) 12 to 21 or to termination. Male fetuses or offspring were necropsied on GD 17, 19, and 21, and postnatal days (PND) 7 and 14. Epididymal malformations were not observed in fetuses from linuron-treated dams but were seen in linuron-exposed male offspring on PND 7 and 14. No testicular lesions were observed at any time point. The growth and development of linuron-exposed fetuses were altered, as evidenced by slight decreases in fetal weight and increased levels of immunoreactive proliferating cell nuclear antigen (PCNA) on GD 21. Intratesticular and serum T levels were not decreased in linuron-exposed male fetuses. These findings indicate that the adversely altered adult phenotype following in utero exposure to linuron is very similar to that produced by the antiandrogens di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP). However, the absence of testicular lesions or alterations in fetal testosterone levels would suggest that the effect of linuron on the developing Wolffian ducts is distinctly different from DBP or DEHP.

Mapping Gene Expression Changes in the Fetal Rat Testis Following Acute Dibutyl Phthalate Exposure Defines a Complex Temporal Cascade of Responding Cell Types

Abstract 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.

Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis

BackgroundExposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V2O5-induced bronchitis.MethodsNormal human lung fibroblasts were exposed to V2O5 in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR.ResultsV2O5 altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO catogories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1).ConclusionOur study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V2O5-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V2O5.

Respiratory syncytial virus infection reduces lung inflammation and fibrosis in mice exposed to vanadium pentoxide

BackgroundVanadium pentoxide (V2O5) exposure is a cause of occupational bronchitis and airway fibrosis. Respiratory syncytial virus (RSV) is a ubiquitous pathogen that causes airway inflammation. It is unknown whether individuals with pre-existing respiratory viral infection are susceptible to V2O5-induced bronchitis. We hypothesized that respiratory viral infection will exacerbate vanadium-induced lung fibrosis.MethodsIn this study we investigated the effect of RSV pre- or post-exposure to V2O5 in male AKR mice. Mice were pre-exposed by intranasal aspiration to RSV or media vehicle prior to intranasal aspiration of V2O5 or saline vehicle at day 1 or day 7. A parallel group of mice were treated first with V2O5 or saline vehicle at day 1 and day 7 then post-exposed to RSV or media vehicle at day 8.ResultsV2O5-induced airway inflammation and fibrosis were decreased by RSV pre- or post-exposure. Real time quantitative RT-PCR showed that V2O5 significantly increased lung mRNAs encoding pro-fibrogenic growth factors (TGF-β1, CTGF, PDGF-C) and collagen (Col1A2), but also increased mRNAs encoding anti-fibrogenic type I interferons (IFN-α, -β) and IFN-inducible chemokines (CXCL9 and CXCL10). RSV pre- or post-exposure caused a significantly reduced mRNAs of pro-fibrogenic growth factors and collagen, yet reduced RNA levels of anti-fibrogenic interferons and CXC chemokines.ConclusionsCollectively these data suggest that RSV infection reduces the severity of V2O5-induced fibrosis by suppressing growth factors and collagen genes. However, RSV suppression of V2O5-induced IFNs and IFN-inducible chemokines suggests that viral infection also suppresses the innate immune response that normally serves to resolve V2O5-induced fibrosis.