RESUMEN
BACKGROUND: Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. METHODS: Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. RESULTS: We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFßRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. CONCLUSION: This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.
RESUMEN
BACKGROUND: Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1,000 births worldwide. The secondary palate is formed by paired palatal shelves, consisting of a mesenchymal core with an outer layer of epithelial cells that grow toward each other, attach, and fuse. One of the mechanisms that can cause CP is failure of fusion, that is, failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme confluence. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. METHODS: We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEKs). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and time to fusion. RESULTS: Osteogenic differentiation in hMSC spheroids was highest by Day 13. hEGF delayed fusion of organoids after 12 and 18 hr of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone. CONCLUSION: Our results show that this model of human palatal fusion appropriately mimics the morphology of the developing human palate and responds to hEGF as expected.
Asunto(s)
Desarrollo Óseo/fisiología , Fisura del Paladar/embriología , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/citología , Células Madre Mesenquimatosas/citología , Hueso Paladar/embriología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Osteogénesis/fisiología , Esferoides Celulares/citología , Venas Umbilicales/citologíaRESUMEN
Embryologic development involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the three-dimensional (3D) morphogenesis of developing tissues and can thus be useful tools for studying developmental toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro organoid models that capture key morphogenesis events that occur in the embryo. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as 12 cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the 12 putative cleft palate teratogens studied here (theophylline, triamcinolone, and valproic acid) significantly disrupted in vitro organoid fusion, while tributyltin chloride and all-trans retinoic acid were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid morphogenesis in a scalable format amenable to toxicology screening.
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Morfogénesis/efectos de los fármacos , Técnicas de Cultivo de Órganos/métodos , Organoides/efectos de los fármacos , Hueso Paladar/efectos de los fármacos , Hueso Paladar/embriología , Teratógenos/farmacología , Aminopiridinas/farmacología , Anilidas/farmacología , Benzazepinas/farmacología , Bencimidazoles/farmacología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Epidérmicas/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Indoles/farmacología , Queratina-17/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Organoides/metabolismo , Fenoles/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Piridonas/farmacología , Esferoides Celulares , Estaurosporina/farmacología , Células Madre/efectos de los fármacos , Sulfonas/farmacología , Vimentina/metabolismoRESUMEN
Perfluorooctanoic acid (PFOA) is a member of a family of perfluorinated chemicals that have a variety of applications. PFOA persists in the environment and is found in wildlife and humans. In mice, PFOA is developmentally toxic producing mortality, delayed eye opening, growth deficits, and altered pubertal maturation. PFOA activates peroxisome proliferators-activated receptor-alpha (PPARalpha), a pathway critical to the mode of induction of liver tumors in rodents. The present study uses 129S1/SvlmJ wild-type (WT) and PPARalpha knockout (KO) mice to determine if PPARalpha mediates PFOA-induced developmental toxicity. Pregnant mice were dosed orally from gestation days 1-17 with water or 0.1, 0.3, 0.6, 1, 3, 5, 10, or 20 mg PFOA/kg. PFOA did not affect maternal weight, embryonic implantation, number, or weight of pups at birth. At 5 mg/kg, the incidence of full litter resorptions increased in both WT and KO mice. In WT, but not KO, neonatal survival was reduced (0.6 mg/kg) and eye opening was delayed (1 mg/kg). There was a trend across dose for reduced pup weight (WT and KO) on several postnatal days (PND), but only WT exposed to 1 mg/kg were significantly different from control (PND7-10 and 22). Maternal factors (e.g., background genetics) did not contribute to differences in postnatal mortality, as PFOA induced postnatal mortality in heterozygous pups born to WT or KO dams. In conclusion, early pregnancy loss was independent of PPARalpha expression. Delayed eye opening and deficits in postnatal weight gain appeared to depend on PPARalpha expression, although other mechanisms may contribute. PPARalpha was required for PFOA-induced postnatal lethality and expression of one copy of the gene was sufficient to mediate this effect.
Asunto(s)
Caprilatos/toxicidad , Pérdida del Embrión/inducido químicamente , Fluorocarburos/toxicidad , PPAR alfa/genética , Animales , Caprilatos/sangre , Caprilatos/farmacocinética , Pérdida del Embrión/sangre , Pérdida del Embrión/genética , Ojo/efectos de los fármacos , Ojo/crecimiento & desarrollo , Femenino , Fluorocarburos/sangre , Fluorocarburos/farmacocinética , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , PPAR alfa/deficiencia , EmbarazoRESUMEN
Perfluorooctanoic acid (PFOA) is a persistent pollutant and is detectable in human serum (5 ng/ml in the general population of the Unites States). PFOA is used in the production of fluoropolymers which have applications in the manufacture of a variety of industrial and commercial products (e.g., textiles, house wares, electronics). PFOA is developmentally toxic and in mice affects growth, development, and viability of offspring. This study segregates the contributions of gestational and lactational exposures and considers the impact of restricting exposure to specific gestational periods. Pregnant CD-1 mice were dosed on gestation days (GD) 1-17 with 0, 3, or 5 mg PFOA/kg body weight, and pups were fostered at birth to give seven treatment groups: unexposed controls, pups exposed in utero (3U and 5U), lactationally (3L and 5L), or in utero + lactationally (3U + L and 5U + L). In the restricted exposure (RE) study, pregnant mice received 5 mg PFOA/kg from GD7-17, 10-17, 13-17, or 15-17 or 20 mg on GD15-17. In all PFOA-treated groups, dam weight gain, number of implantations, and live litter size were not adversely affected and relative liver weight increased. Treatment with 5 mg/kg on GD1-17 increased the incidence of whole litter loss and pups in surviving litters had reduced birth weights, but effects on pup survival from birth to weaning were only affected in 5U + L litters. In utero exposure (5U), in the absence of lactational exposure, was sufficient to produce postnatal body weight deficits and developmental delay in the pups. In the RE study, birth weight and survival were reduced by 20 mg/kg on GD15-17. Birth weight was also reduced by 5 mg/kg on GD7-17 and 10-17. Although all PFOA-exposed pups had deficits in postnatal weight gain, only those exposed on GD7-17 and 10-17 also showed developmental delay in eye opening and hair growth. In conclusion, the postnatal developmental effects of PFOA are due to gestational exposure. Exposure earlier in gestation produced stronger responses, but further study is needed to determine if this is a function of higher total dose or if there is a developmentally sensitive period.
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Peso Corporal/efectos de los fármacos , Caprilatos/toxicidad , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Peso al Nacer/efectos de los fármacos , Caprilatos/sangre , Contaminantes Ambientales/sangre , Femenino , Fluorocarburos/sangre , Edad Gestacional , Lactancia , Tamaño de la Camada/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangreRESUMEN
Diisodecyl phthalate (DiDP) is an isomeric mixture of phthalates with predominantly 10-carbon branched-dialkyl chains, widely used as a plasticizer for polyvinyl chloride. The extent of human exposure to DiDP is unknown in part because adequate biomarkers of exposure to DiDP are not available. We identified several major metabolites of DiDP in urine of adult female Sprague-Dawley rats after a single oral administration of DiDP (300 mg/kg). These metabolites can potentially be used as biomarkers of exposure to DiDP. The metabolites extracted from urine were chromatographically resolved and identified by their chromatographic behavior and full scan negative ion electrospray ionization mass spectrum. The identity of metabolites with similar molecular weights was further examined in accurate mass mode. For some metabolites, unequivocal identification was done using authentic standards. Among these were the hydrolytic monoester of DiDP, monoisodecyl phthalate (MiDP), detected as a minor metabolite, and one omega oxidation product of MiDP, mono(carboxy-isononyl) phthalate (MCiNP), which was the most abundant urinary metabolite. We also tentatively identified other secondary metabolites of MiDP, mono(hydroxy-isodecyl) phthalate, mono(oxo-isodecyl) phthalate, mono(carboxy-isoheptyl) phthalate, mono(carboxy-isohexyl) phthalate, mono(carboxy-isopentyl) phthalate, mono(carboxy-isobutyl) phthalate, and mono(carboxy-ethyl) phthalate. Oxidative metabolites of diisoundecyl phthalate (DiUdP) and diisononyl phthalate (DiNP) were also detected suggesting the presence of DiUdP and DiNP in the DiDP formulation. The urinary concentrations of all these metabolites gradually decreased in the 4 days following the administration of DiDP. MCiNP and other DiDP secondary metabolites are more abundant in urine than MiDP, suggesting that these oxidative products are better biomarkers for DiDP exposure assessment than MiDP. Additional research on the toxicokinetics of these metabolites is needed to understand the extent of human exposure to DiDP from the urinary concentrations of MCiNP and other DiDP secondary metabolites.
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Ácidos Ftálicos/farmacocinética , Plastificantes/farmacocinética , Animales , Femenino , Ácidos Ftálicos/orina , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
False-positive results on serologic assays for Mycobacterium avium subsp. paratuberculosis (MAP) are believed to occur due to cross-reacting antibody produced by Corynebacterium pseudotuberculosis (C. pstb) infection in goats. This issue of compromised specificity was evaluated by testing 771 adult goats from 10 Midwestern goat herds in 2004. Assays for MAP infection included radiometric fecal culture and 2 enzyme-linked immunosorbent assays (ELISAs); ELISA-positive samples were tested by agar gel immunodiffusion (AGID). A synergistic hemolysin inhibition assay (SHI) was used to detect C. pstb antibody. Four infection status categories were evaluated. Category 1 goats (free of both MAP and C. pstb infection) tested negative on all MAP fecal cultures and SHI tests. Five of 181 goats were positive in both ELISAs, and 2 more were positive in ELISA-1 only. For Category 2 (MAP infected; no C. pstb infection), all animals were SHI negative. Six goats were fecal culture positive and strongly positive in both ELISAs; 2 more goats were positive only in ELISA-1. For Category 3 (C. pstb infected or vaccinated; no history of MAP infection), all fecal cultures were negative and 91% were SHI test-positive. In this population, only 2 goats were positive in both MAP ELISAs, while 84 additional goats were test-positive only on ELISA-1. In the absence of C. pstb infection, both ELISAs performed comparably, but when C. pstb infection was present the performance of ELISA-1 was significantly perturbed. Use of the ELISA-2 for goats is an effective and efficient method for Johne's disease surveillance in any goat herd.
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Infecciones por Corynebacterium/veterinaria , Corynebacterium pseudotuberculosis , Enfermedades de las Cabras/microbiología , Animales , Distribución de Chi-Cuadrado , Ensayo de Inmunoadsorción Enzimática , Heces/microbiología , CabrasRESUMEN
Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton's Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.
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Bioingeniería , Técnicas de Cultivo de Órganos , Hueso Paladar/embriología , Esferoides Celulares , Fosfatasa Alcalina/metabolismo , Bioingeniería/métodos , Diferenciación Celular/genética , Análisis por Conglomerados , Biología Computacional/métodos , Proteínas de la Matriz Extracelular , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Técnicas In Vitro , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Hueso Paladar/metabolismo , Factores de Tiempo , TranscriptomaRESUMEN
Commercial di-isononyl phthalate (DiNP) is a mixture of various branched-chain dialkyl phthalates mainly containing nine-carbon alkyl isomers. At high doses in rodents, DiNP is a carcinogen, and a developmental toxicant. After exposure, the diester isomers are de-esterified to form hydrolytic monoesters, monoisononyl phthalates (MiNP), which subsequently metabolize to form oxidative metabolites. These metabolites can be excreted in urine or feces. The urinary excretion of DiNP metabolites was monitored in adult female Sprague-Dawley rats after oral administration of a single dose (300 mg/kg) of commercial DiNP. The metabolites were extracted from urine, resolved with high performance liquid chromatography, analyzed by mass spectrometry, and tentatively identified based on their chromatographic separation and mass spectrometric fragmentation pattern. Because DiNP is an isomeric mixture, its metabolites were also isomeric mixtures that eluted from the HPLC column with close retention times. Mono(carboxy-isooctyl)phthalate (MCiOP) was identified as the major metabolite of DiNP; in addition, mono(hydroxy-isononyl)phthalate (MHiNP) and mono(oxo-isononyl)phthalate (MOiNP) were present. Furthermore, metabolites of di-isooctyl phthalate (DiOP) and di-isodecyl phthalate (DiDP) were also detected. Excretion toxicokinetics of the DiNP metabolites in urine followed a biphasic pattern with initial rapid decay in concentration. Despite potential differences in the metabolism of DiNP among species, MCiOP, MHiNP and MOiNP were detected in humans with no known exposure to DiNP at levels significantly higher than MiNP suggesting that these oxidative metabolites may be better urinary biomarkers of human exposure to DiNP than is MiNP.
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Ácidos Ftálicos/orina , Administración Oral , Animales , Biomarcadores/química , Biomarcadores/orina , Femenino , Humanos , Inactivación Metabólica , Estructura Molecular , Ácidos Ftálicos/química , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Di-n-octyl phthalate (DnOP) is a plasticizer used in polyvinyl chloride plastics, cellulose esters, and polystyrene resins. The metabolism of DnOP results in the hydrolysis of one ester linkage to produce mono-n-octyl phthalate (MnOP), which subsequently metabolizes to form oxidative metabolites. We investigated the toxicokinetics of DnOP in adult female Sprague-Dawley rats by monitoring the excretion of DnOP metabolites in urine after oral administration of DnOP (300 mg/kg). By using authentic standards, the presence of urinary phthalic acid (PA), MnOP, and the major DnOP metabolite, mono-(3-carboxypropyl) phthalate (MCPP) was clearly established. Furthermore, we identified five additional urinary DnOP oxidative metabolites based on their chromatographic behavior and mass spectrometric fragmentation pattern. These DnOP oxidative metabolites, are postulated to be mono-carboxymethyl phthalate (MCMP), mono-(5-carboxy-n-pentyl) phthalate (MCPeP), mono-(7-carboxy-n-heptyl) phthalate (MCHpP), and isomers of mono-hydroxy-n-octyl phthalate (MHOP) (e.g., mono-(7-hydroxy-n-octyl) phthalate) and of mono-oxo-n-octyl phthalate (MOOP) (e.g., mono-(7-oxo-n-octyl) phthalate). The urinary excretion of DnOP metabolites followed a biphasic excretion pattern. The metabolite levels decreased significantly after the first day of DnOP administration although MCPP, MCHpP, MHOP, and MOOP were detectable after 4 days. We also studied the in vitro metabolism of DnOP and MnOP by rat liver microsomes. DnOP produced MnOP, MHOP, and PA in vitro whereas, MnOP produced MHOP and PA in vitro at detectable levels.
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Microsomas Hepáticos/metabolismo , Ácidos Ftálicos/orina , Plastificantes/farmacocinética , Administración Oral , Animales , Femenino , Inactivación Metabólica , Espectrometría de Masas , Oxidación-Reducción , Ácidos Ftálicos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In mammals, androgens are essential in directing mammalian sexual differentiation of the male phenotype. Administration of testosterone during this period alters female development in a male-like direction, whereas exposure to an androgen receptor antagonist like vinclozolin (V) demasculinizes and feminizes the male offspring. In the current study, we administered V (gavage at 200 mg/kg/day) and/or testosterone propionate (TP, sc, at 1 mg/rat/day), alone and in combination to Sprague-Dawley (SD) rats on days 14 through 19 of pregnancy, to determine if V would antagonize the effects of TP in the female and, conversely, if TP would antagonize the effects of V in the male offspring. These doses of TP and V were selected because they significantly alter sexual differentiation in the majority of female and male rat offspring, respectively, without producing severe toxicity in the dam or offspring. The study design is a 2 x 2 factorial (7 dams per group) including vehicle control, V, TP, and V + TP groups. As expected, individually, both V and TP reduced maternal weight gain and the V + TP group was affected in a cumulative fashion. Litter size on postnatal day (PND) 2 was reduced only by V + TP, whereas pup body weight was reduced in all three treated groups, the effect of V + TP again being cumulative. In female offspring, TP-induced alterations (i.e., increased anogenital distance [AGD] and fewer nipples, vaginal agenesis, hydrometrocolpos, induced prostate and bulbourethral glands, and levator ani muscle tissues) were all reversed by coadministration of V. In male offspring, V-induced alterations were only modestly antagonized by TP. At the dosage levels used herein, V + TP-treated male offspring had less well-developed nipples as infants and adults and a lower incidence of ectopic testis than did the V group. However, V-induced changes in reproductive organ weights, AGD, atrophic testes, vaginal pouch, and agenesis of the sex accessory tissues were not antagonized by concurrent TP treatment in male offspring. We observed that the combination of V and TP, two chemicals with opposing endocrine action, antagonized one another during sexual differentiation, especially in the female offspring and induced cumulative effects on maternal and neonatal toxicity. We suspect that antagonism of V by TP would be enhanced in the male if lower dose levels of V were used, but then the antagonism of TP by V in the female would likely be attenuated.
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Antagonistas de Andrógenos/toxicidad , Oxazoles/toxicidad , Preñez/efectos de los fármacos , Diferenciación Sexual/efectos de los fármacos , Propionato de Testosterona/farmacología , Anomalías Inducidas por Medicamentos/epidemiología , Anomalías Inducidas por Medicamentos/patología , Antagonistas de Andrógenos/farmacología , Animales , Animales Recién Nacidos , Peso al Nacer/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Femenino , Viabilidad Fetal/efectos de los fármacos , Feto/metabolismo , Genitales/efectos de los fármacos , Genitales/crecimiento & desarrollo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Oxazoles/antagonistas & inhibidores , Oxazoles/farmacología , Embarazo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Maduración Sexual/efectos de los fármacos , Teratógenos/toxicidad , Propionato de Testosterona/antagonistas & inhibidores , Propionato de Testosterona/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Testosterone plays a major role in male sexual development. Exposure of females to testosterone in utero can induce masculine characteristics such as anovulation, increased anogenital distance (AGD), absence of nipples, retention of male-like tissues, and agenesis of the lower vagina. In addition, high levels of androgens during fetal development can lead to toxic effects such as reduced litter size and viability. The study of the effects of testosterone administration during sexual differentiation provides a foundation for understanding the effects of environmental androgens on fetuses, a sensitive subpopulation. In the current study, we investigated the ability of a range of concentrations of testosterone propionate (TP) administered prenatally to masculinize female and alter male offspring, and measured maternal and fetal T levels. Pregnant Sprague-Dawley rats were dosed by sc injection on gestational day (GD) 14-19 (GD 1= day of plug) with either corn oil (vehicle; 0.1 ml/rat) or with 0.1 ml of TP solution at 0.1, 0.5, 1, 2, 5, or 10 mg/0.1 ml. Parturition was delayed at 2, 5, and 10 mg TP, litter size was reduced at 5 and 10 mg TP, and pup weight was significantly reduced in both sexes at 0.5 mg TP and higher doses. Viability of offspring was unaffected at any dosage level. Androgenic effects seen at 0.5 mg TP in females included increased AGD at weaning and adulthood, reduced number of areolas and nipples, cleft phallus, small vaginal orifice, and presence of prostate tissue. This dose of TP elevated maternal T levels 10x but had no effect on fetal T levels. At 1 mg TP and above, female AGD on postnatal day (PND) 2 (or postcoital day 24 [gestation length = 22(1/2)]) was increased; areolas and nipples were virtually eliminated; levator ani muscle, bulbourethral glands, and seminal vesicles (2 mg TP and above) were present; none of the females developed a vaginal orifice and many females in the 1 and 2 mg TP dose groups developed a greatly distended, fluid-filled uterus after puberty. Maternal T levels at 1 mg TP were elevated 30x, and female fetal T levels showed an 80% increase. Male offspring displayed a reduced AGD and body weight on PND 2 at 0.5 mg TP and higher doses. These effects were not evident by weaning and male offspring displayed no malformations. We conclude that gestational administration of 0.5 and 1 mg TP masculinizes female offspring without greatly affecting pup viability or pregnancy of the dam. This study provides a useful model for in utero testing of environmental androgens for their potential to induce developmental abnormalities.
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Genitales Femeninos/anomalías , Genitales Masculinos/anomalías , Hormonas Esteroides Gonadales/toxicidad , Exposición Materna/efectos adversos , Testosterona/toxicidad , Anomalías Inducidas por Medicamentos/etiología , Canal Anal/anomalías , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Femenino , Hormonas Esteroides Gonadales/administración & dosificación , Hormonas Esteroides Gonadales/fisiología , Masculino , Pezones/anomalías , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Testosterona/administración & dosificación , Testosterona/fisiología , Pruebas de Toxicidad , Vagina/anomalíasRESUMEN
Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks.
Asunto(s)
Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Distribución por Edad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Genotipo , Inmunohistoquímica/veterinaria , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Polimorfismo Genético/genética , Proteínas PrPSc/genética , Scrapie/patología , OvinosRESUMEN
Perfluoroalkyl acids (PFAAs) are found globally in the environment, detected in humans and wildlife, and are typically present as mixtures of PFAA congeners. Mechanistic studies have found that responses to PFAAs are mediated in part by PPARα. Our previous studies showed that individual PFAAs activate PPARα transfected into COS-1 cells. The goal of the current study was to determine if binary combinations of perfluorooctanoic acid (PFOA) and another PFAA act in an additive fashion to activate PPARα in the mouse one-hybrid in vitro model. COS-1 cells were transiently transfected with mouse PPARα luciferase reporter construct and exposed to either vehicle control (0.1% DMSO or water), PPARα agonist (WY14643, 10 µM), PFOA at 1-128µM, perfluorononanoic acid (PFNA) at 1-128 µM, perfluorohexanoic acid (PFHxA) at 8-1024 µM, perfluorooctane sulfonate (PFOS) at 4-384 µM or perfluorohexane sulfonate (PFHxS) at 8-2048 µM to generate sigmoidal concentration-response curves. In addition, cells were exposed to binary combinations of PFOA+either PFNA, PFHxA, PFOS or PFHxS in an 8×8 factorial design. The concentration-response data for individual chemicals were fit to sigmoidal curves and analyzed with nonlinear regression to generate EC50s and Hillslopes, which were used in response-addition and concentration-addition models to calculate predicted responses for mixtures in the same plate. All PFOA+PFAA combinations produced concentration-response curves that were closely aligned with the predicted curves for both response addition and concentration addition at low concentrations. However, at higher concentrations of all chemicals, the observed response curves deviated from the predicted models of additivity. We conclude that binary combinations of PFAAs behave additively at the lower concentration ranges in activating PPARα in this in vitro system.
Asunto(s)
Caprilatos/toxicidad , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , PPAR alfa/efectos de los fármacos , Ácidos Alcanesulfónicos/administración & dosificación , Ácidos Alcanesulfónicos/química , Ácidos Alcanesulfónicos/toxicidad , Animales , Células COS , Caprilatos/administración & dosificación , Caprilatos/química , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/administración & dosificación , Contaminantes Ambientales/química , Fluorocarburos/administración & dosificación , Fluorocarburos/química , Ratones , PPAR alfa/metabolismo , Pirimidinas/farmacología , Análisis de Regresión , TransfecciónRESUMEN
While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPARα). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPARα. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPARα agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPARα in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPARδ. Rankings were conducted on the limited dataset. In mouse hepatocytes, the pattern was similar to that previously observed in the COS-1 reporter cell assay. With the exception of PFHxA, longer chain PFAA carboxylates were the most active. The pattern was similar in human hepatocytes, although PFDA and PFOS showed higher activity than previously observed while PFOA showed somewhat less activity. These data reflect inherent challenges in using primary hepatocytes to predict toxicological response.
Asunto(s)
Ácidos Alcanesulfónicos/química , Ácidos Alcanesulfónicos/toxicidad , Fluorocarburos/química , Fluorocarburos/toxicidad , Hepatocitos/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos/métodos , Humanos , Ratones , Cultivo Primario de CélulasRESUMEN
This study is a follow-up to a paper by Carr et al. that determined a design structure to optimally test for departures from additivity in a fixed ratio mixture of four perfluoroalkyl acids (PFAAs) using an in vitro transiently-transfected COS-1 PPARα reporter model with a mixing ratio that is based on average serum levels in NHANES subjects. Availability of information regarding potential for additivity of PFAAs in mixtures is critically important for risk assessors who are concerned with the ability of the compounds to affect human health and impact ecological systems. It is clear that exposures are not to single compounds, but to mixtures of the PFAAs. This paper presents the results from the data collected using the design from Carr et al. along with subsequent analyses that were performed to classify the relationships among mixtures of PFAAs. A non-linear logistic additivity model was employed to predict relative luciferase units (RLU), an indicator of PPARα activation. The results indicated a less than additive relationship among the four PFAAs. To determine if the possible "antagonism" is from the competition among or between carboxylates and sulfonates, four different binary mixtures were also studied. There was a less than additive relationship in all four binary mixtures. These findings are generally similar to two other reports of interfering interactions between PFAAs in mixtures. The most conservative interpretation for our data would be an assumption of additivity (and lack of a greater than additive interaction), with a potential for antagonistic interactions.
Asunto(s)
Alcanosulfonatos/química , Alcanosulfonatos/toxicidad , Ácidos Carboxílicos/química , Ácidos Carboxílicos/toxicidad , Modelos Estadísticos , Alcanosulfonatos/metabolismo , Animales , Células COS , Ácidos Carboxílicos/metabolismo , Chlorocebus aethiops , Mezclas Complejas/química , Mezclas Complejas/metabolismo , Mezclas Complejas/toxicidad , PPAR alfa/metabolismoRESUMEN
Perfluorinated alkyl acids (PFAAs) are manufactured surfactants found globally in the environment and in tissues of humans and wildlife. Several PFAAs adversely affect rodents and activation of PPARα is thought to be their mode of action. Our previous study demonstrated that some PFAAs activate mouse and human PPARα in transiently transfected COS-1 cells. Here, we test more PFAAs for PPARα activation in the same system. Cells were transfected with either mouse or human PPARα-luciferase reporter plasmid, exposed the next day to either vehicle, PPARα agonist (WY14643), perfluoropentanoic acid (C5), perfluoroheptanoic acid (C7), perfluorooctanoic acid (C8), perfluoroundecanoic acid (C11), or perfluorododecanoic acid (C12) at concentrations from 0.5µM to 100µM, and luminescence was measured after 24h. C8 induced the highest activity for human PPARα, followed by C7, C5, and C11. C12 had little activity. C8 induced the highest activity for mouse PPARα, followed by C11, C7, C12 and C5. The two studies together found increasing activity of PPARα with increasing chain length of the PFAA up to perfluorononanoic acid (C9) and lower activity with longer chain PFAAs with both mouse and human PPARα.
Asunto(s)
Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Fluorocarburos/química , Fluorocarburos/toxicidad , PPAR alfa/metabolismo , Animales , Células COS , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Humanos , Modelos Lineales , Ratones , Nivel sin Efectos Adversos Observados , PPAR alfa/genética , Plásmidos , Especificidad de la Especie , Relación Estructura-Actividad , TransfecciónRESUMEN
Perfluorononanoic acid (PFNA) is one of the perfluoroalkyl acids found in the environment and in tissues of humans and wildlife. Prenatal exposure to PFNA negatively impacts survival and development of mice and activates the mouse and human peroxisome proliferator-activated receptor-alpha (PPARα). In the current study, we used PPARα knockout (KO) and 129S1/SvlmJ wild-type (WT) mice to investigate the role of PPARα in mediating PFNA-induced in vivo effects. Pregnant KO and WT mice were dosed orally with water (vehicle control: 10 ml/kg), 0.83, 1.1, 1.5, or 2 mg/kg PFNA on gestational days (GDs) 1-18 (day of sperm plug = GD 0). Maternal weight gain, implantation, litter size, and pup weight at birth were unaffected in either strain. PFNA exposure reduced the number of live pups at birth and survival of offspring to weaning in the 1.1 and 2 mg/kg groups in WT. Eye opening was delayed (mean delay 2.1 days) and pup weight at weaning was reduced in WT pups at 2 mg/kg. These developmental endpoints were not affected in the KO. Relative liver weight was increased in a dose-dependent manner in dams and pups of the WT strain at all dose levels but only slightly increased in the highest dose group in the KO strain. In summary, PFNA altered liver weight of dams and pups, pup survival, body weight, and development in the WT, while only inducing a slight increase in relative liver weight of dams and pups at 2 mg/kg in KO mice. These results suggest that PPARα is an essential mediator of PFNA-induced developmental toxicity in the mouse.
RESUMEN
Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of a family of perfluorinated compounds. Both are environmentally persistent and found in the serum of wildlife and humans. PFOS and PFOA are developmentally toxic in laboratory rodents. Exposure to these chemicals in utero delays development and reduces postnatal survival and growth. Exposure to PFOS on the last 4 days of gestation in the rat is sufficient to reduce neonatal survival. PFOS and PFOA are weak agonists of peroxisome proliferator activated receptor-alpha (PPAR alpha). The reduced postnatal survival of neonatal mice exposed to PFOA was recently shown to depend on expression of PPAR alpha. This study used PPAR alpha knockout (KO) and 129S1/SvlmJ wild type (WT) mice to determine if PPAR alpha expression is required for the developmental toxicity of PFOS. After mating overnight, the next day was designated gestation day (GD) 0. WT females were weighed and dosed orally from GD15 to 18 with 0.5% Tween-20, 4.5, 6.5, 8.5, or 10.5mg PFOS/kg/day. KO females were dosed with 0.5% Tween-20, 8.5 or 10.5mg PFOS/kg/day. Dams and pups were observed daily and pups were weighed on postnatal day (PND) 1 and PND15. Eye opening was recorded from PND12 to 15. Dams and pups were killed on PND15, body and liver weights recorded, and serum collected. PFOS did not affect maternal weight gain or body or liver weights of the dams on PND15. Neonatal survival (PND1-15) was significantly reduced by PFOS in both WT and KO litters at all doses. WT and KO pup birth weight and weight gain from PND1 to 15 were not significantly affected by PFOS exposure. Relative liver weight of WT and KO pups was significantly increased by the 10.5mg/kg dose. Eye opening of PFOS-exposed pups was slightly delayed in WT and KO on PND13 or 14, respectively. Because results in WT and KO were comparable, it is concluded that PFOS-induced neonatal lethality and delayed eye opening are not dependent on activation of PPAR alpha.
Asunto(s)
Ácidos Alcanesulfónicos/toxicidad , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , PPAR alfa/metabolismo , Ácidos Alcanesulfónicos/agonistas , Animales , Peso al Nacer , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/agonistas , Ojo/efectos de los fármacos , Ojo/crecimiento & desarrollo , Femenino , Fluorocarburos/agonistas , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Masculino , Exposición Materna , Ratones , Ratones Endogámicos , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , Factores Sexuales , Análisis de SupervivenciaRESUMEN
The adverse consequences of developmental exposures to perfluorooctanoic acid (PFOA) are established in mice, and include impaired development of the mammary gland (MG). However, the relationships between timing or route of exposure, and consequences in the MG have not been characterized. To address the effects of these variables on the onset and persistence of MG effects in female offspring, timed pregnant CD-1 dams received PFOA by oral gavage over various gestational durations. Cross-fostering studies identified the 5mg/kg dose, under either lactational- or intrauterine-only exposures, to delay MG development as early as postnatal day (PND) 1, persisting beyond PND 63. Intrauterine exposure during the final days of pregnancy caused adverse MG developmental effects similar to that of extended gestational exposures. These studies confirm a window of MG sensitivity in late fetal and early neonatal life, and demonstrate developmental PFOA exposure results in early and persistent MG effects, suggesting permanent consequences.