Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xxiii, 276 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 261-276).

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A Senna occidentalis (So), Fabaceae-Caesalpinoideae (Leguminosae), conhecida como fedegoso, entre outros nomes populares causa, em animais de criação, intoxicação pelo consumo, tanto de suas folhas a campo, como pela ingestão de suas sementes quando presentes em rações. Estas intoxicações podem ser de caráter crônico ou agudo, em ruminantes ou monogástricos, tendo sido já reproduzida em animais de laboratório. Os principais achados histológicos são degenerações em músculos esquelético e cardíaco, fígado, rim e, recentemente publicado, lesões no sistema nervoso central. Porém, pouco se sabe sobre os efeitos do princípio ativo desta planta, a diantrona, na toxicologia perinatal. O objetivo do presente trabalho foi estudar os possíveis efeitos teratogênicos desta planta em caprinos, utilizando um protocolo de avaliação de toxicologia do desenvolvimento. Para tanto, foi somado a um protocolo pré-existente de acompanhamento da gestação de cabras, por meio de ultra-sonografia e dados clínicos, como pesagem semanal e coleta quinzenal de sangue para realização de bioquímica sérica, a avaliação de filhotes por morfometria corporal esde o nascimento ao 4º mês de idade, além de avaliação neurocomportamental até a 10ª semana de idade. Assim, trinta e oito fêmeas nulíparas, receberam rações contendo 0% (controle), 1% (So1), 2% (So2), 3% (So3) e 4% (So4) de sementes da planta, desde a confirmação da gestação, no 27º dia pós-coito, até o parto. Outro grupo de cabras (LAC) consumiu ração contendo 4% de sementes da planta durante a lactação. Amostras de leite das cabras foram colhidas para a tentativa de detecção da diantrona. Foi observado queda no ganho de peso nas fêmeas pertencentes ao grupo So3 durante a gestação. No grupo So4 ocorreu a morte de uma fêmea, duas mortes fetais, e morte pós-natal de um filhote. À análise histológica da cabra que morreu, observou-se severas lesões em fígado, rim, músculo esquelético e cardíaco. Foi observado, pela primeira vez na literatura, lesões em nervo ciático. Nas cabras do grupo LAC foram observadas lesões teciduais brandas, exceto em nervo ciático. Não se observaram malformações físicas nos filhotes. Somente pequenas alterações bioquímicas foram observadas nas mães e na prole. A morfometria evidenciou poucas alterações nos filhotes do grupo LAC. Histologicamente, estes filhotes LAC tiveram lesões focais brandas em musculatura esquelética e cardíaca, fígado e rim, revelando que o princípio ativo da planta pode ser transferido para o leite. Os testes neurocomportamentais revelaram déficit de aprendizado dos filhotes do grupo So3. Não foi possível a detecção da diantrona no leite avaliado. Este estudo permitiu verificar que a So, embora não seja um potente teratógeno, quando consumida durante a gestação, pode causar efeitos tóxicos no feto, como evidenciado pela avaliação neurocomportamental. O protocolo sugerido poderá ter grande valia para estudar o potencial teratogênico de diversas substâncias as quais os ruminantes possam estar expostos. / Senna occidentalis (So - formerly Cassia occidentalis), Fabaceae-Caesalpinoideae (Leguminosae) is a weed growing in pastures and in cultivated fields such as corn and soybeans in many tropical and subtropical areas worldwide. Many reports have shown that So is toxic to different animal species. Histologic lesions plant-related are degeneration skeltal and cardiac muscles, liver and kidney. Recently was related leions in the central nervous system. Even so, very little is known about the possible effects of prolonged exposure at low doses of So on developmental toxicology. Thus, the present study proposes an approach to evaluate the perinatal toxicity from gestational intoxication of So seeds in goats. Third-eight pregnant goats were fed rations containing 0% (control group), 1% (So1 group), 2% (So2 group), 3% (So3) and 4% (So4 group) mature So seeds from pregnancy detection on day 27 after mating until parturition. Another group (LAC) was fed with ration containing 4 % of So seeds only in the lactation period; clinic observations such weight gains, general body status and serum biochemistry were evaluated. Fetuses were evaluated during the pregnancy using ultrasonographic measurements; neonates were evaluated after birth using body morphometry, weight gains, corporal status and serum biochemistry. Neurobehavioral tests was performed on kids from So3 group. In the So4 group was observed fetal dead from 2 dams, dead of one newborn and dead of one pregnant dam. None malformations was observed. Only a few minor alterations in serum biochemistry were observed in dams and kids; even so one So4 group dam had several and diffuse tissue lesions as vacuolations in hepatocytes; vacuolations in the endothelium of the convoluted tubules in kidneys, necrosis in skeletal and cardiac muscles, and for the first time vacuolation was observed in sciatic nerve. Alterations in body morphometry were observed only in kids from LAC group during first month of lactation. Also, tissue lesions was observed in these kids, showing the capacity of the diantrona (active principle of the palnt) be carried to milk. Neurobehavioral tests revealed deficit on learning hability in the So3 kids. Diantrone didn\'t was detected in milk samples. This study shows that, despite S. occidentalis isn\'t a powerfull teratogen when ate during gestation, can cause toxic effects on the fetus, as neurobehavioral test revealed. Protocol utilized here is usefull to study the potential teratogenic effects of drugs and other substancies whose an animal could be exposed.

General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization. This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies. Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.

A Senna occidentalis (So), Fabaceae-Caesalpinoideae (Leguminosae), conhecida como fedegoso, entre outros nomes populares causa, em animais de criação, intoxicação pelo consumo, tanto de suas folhas a campo, como pela ingestão de suas sementes quando presentes em rações. Estas intoxicações podem ser de caráter crônico ou agudo, em ruminantes ou monogástricos, tendo sido já reproduzida em animais de laboratório. Os principais achados histológicos são degenerações em músculos esquelético e cardíaco, fígado, rim e, recentemente publicado, lesões no sistema nervoso central. Porém, pouco se sabe sobre os efeitos do princípio ativo desta planta, a diantrona, na toxicologia perinatal. O objetivo do presente trabalho foi estudar os possíveis efeitos teratogênicos desta planta em caprinos, utilizando um protocolo de avaliação de toxicologia do desenvolvimento. Para tanto, foi somado a um protocolo pré-existente de acompanhamento da gestação de cabras, por meio de ultra-sonografia e dados clínicos, como pesagem semanal e coleta quinzenal de sangue para realização de bioquímica sérica, a avaliação de filhotes por morfometria corporal esde o nascimento ao 4º mês de idade, além de avaliação neurocomportamental até a 10ª semana de idade. Assim, trinta e oito fêmeas nulíparas, receberam rações contendo 0% (controle), 1% (So1), 2% (So2), 3% (So3) e 4% (So4) de sementes da planta, desde a confirmação da gestação, no 27º dia pós-coito, até o parto. Outro grupo de cabras (LAC) consumiu ração contendo 4% de sementes da planta durante a lactação. Amostras de leite das cabras foram colhidas para a tentativa de detecção da diantrona. Foi observado queda no ganho de peso nas fêmeas pertencentes ao grupo So3 durante a gestação. No grupo So4 ocorreu a morte de uma fêmea, duas mortes fetais, e morte pós-natal de um filhote. À análise histológica da cabra que morreu, observou-se severas lesões em fígado, rim, músculo esquelético e cardíaco. Foi observado, pela primeira vez na literatura, lesões em nervo ciático. Nas cabras do grupo LAC foram observadas lesões teciduais brandas, exceto em nervo ciático. Não se observaram malformações físicas nos filhotes. Somente pequenas alterações bioquímicas foram observadas nas mães e na prole. A morfometria evidenciou poucas alterações nos filhotes do grupo LAC. Histologicamente, estes filhotes LAC tiveram lesões focais brandas em musculatura esquelética e cardíaca, fígado e rim, revelando que o princípio ativo da planta pode ser transferido para o leite. Os testes neurocomportamentais revelaram déficit de aprendizado dos filhotes do grupo So3. Não foi possível a detecção da diantrona no leite avaliado. Este estudo permitiu verificar que a So, embora não seja um potente teratógeno, quando consumida durante a gestação, pode causar efeitos tóxicos no feto, como evidenciado pela avaliação neurocomportamental. O protocolo sugerido poderá ter grande valia para estudar o potencial teratogênico de diversas substâncias as quais os ruminantes possam estar expostos. / Senna occidentalis (So - formerly Cassia occidentalis), Fabaceae-Caesalpinoideae (Leguminosae) is a weed growing in pastures and in cultivated fields such as corn and soybeans in many tropical and subtropical areas worldwide. Many reports have shown that So is toxic to different animal species. Histologic lesions plant-related are degeneration skeltal and cardiac muscles, liver and kidney. Recently was related leions in the central nervous system. Even so, very little is known about the possible effects of prolonged exposure at low doses of So on developmental toxicology. Thus, the present study proposes an approach to evaluate the perinatal toxicity from gestational intoxication of So seeds in goats. Third-eight pregnant goats were fed rations containing 0% (control group), 1% (So1 group), 2% (So2 group), 3% (So3) and 4% (So4 group) mature So seeds from pregnancy detection on day 27 after mating until parturition. Another group (LAC) was fed with ration containing 4 % of So seeds only in the lactation period; clinic observations such weight gains, general body status and serum biochemistry were evaluated. Fetuses were evaluated during the pregnancy using ultrasonographic measurements; neonates were evaluated after birth using body morphometry, weight gains, corporal status and serum biochemistry. Neurobehavioral tests was performed on kids from So3 group. In the So4 group was observed fetal dead from 2 dams, dead of one newborn and dead of one pregnant dam. None malformations was observed. Only a few minor alterations in serum biochemistry were observed in dams and kids; even so one So4 group dam had several and diffuse tissue lesions as vacuolations in hepatocytes; vacuolations in the endothelium of the convoluted tubules in kidneys, necrosis in skeletal and cardiac muscles, and for the first time vacuolation was observed in sciatic nerve. Alterations in body morphometry were observed only in kids from LAC group during first month of lactation. Also, tissue lesions was observed in these kids, showing the capacity of the diantrona (active principle of the palnt) be carried to milk. Neurobehavioral tests revealed deficit on learning hability in the So3 kids. Diantrone didn\'t was detected in milk samples. This study shows that, despite S. occidentalis isn\'t a powerfull teratogen when ate during gestation, can cause toxic effects on the fetus, as neurobehavioral test revealed. Protocol utilized here is usefull to study the potential teratogenic effects of drugs and other substancies whose an animal could be exposed.

microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 5, 2012 - Dec. 5, 2013

Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERR��) agonist. ERR�� binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERR�� activation. To examine whether BPA behaves more like an ER or ERR�� ligand, two positive control compounds were used throughout the study: 17��-estradiol (E2) and GSK4716, ER and ERR�� agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 ��M BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 ��M BPA led to learning deficits in adult zebrafish. Exposure to 0.1 ��M E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERR�� in the larval hyperactivity phenotype. Transient ERR�� knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 ��M BPA exposure. Transient ERR�� knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature. This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERR�� in mediating BPA's neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA's mode of action may involve crosstalk between ERR�� and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERR�� is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERR��. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Dec. 21, 2012 - June 21, 2013