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Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998" Typescript. Vita. Includes bibliographical references (l. 129-152). Also available on the Internet.

The potential role of embryonic cells in the IN VITRO bioactivation of teratogens was investigated, with limb bud mesenchyme cells, (LB), and mid brain cells,(CNS), derived from 13 day old rat embryos. The cells were cultured for 5 days and during this period differentiated into foci of chondrocytes (LB) or neurones (CNS). The development of the cells in culture mirrored the IN VIVO development and therefore can be used as a teratogen screen. The presence of constitutive forms of cytochrome P450 isoenzyme forms b, c and d (Levin et. al. 1978 nomenclature) were detected in both cell types after the 5 day culture period by immunocytochemistry. Isoenzyme cytochrome P450 b was found to be non-inducible, whereas isoenzymes c and d were found to be inducible by both transplacental administration of 3-methylcholanthrene (3MC) and B-naphthoflavone (BNF) and by IN VITRO coincubation with 3MC and BNF. There was a difference in the developmental profile of the appearance of isoenzyme forms b and c over the 5 day culture period. Having established the presence of embryonic cytochrome P450's in the culture system the role of metabolism in the bioactivation of diphenylhydantoin (DPH) was investigated. Five approaches were used: 1. Modulation of cytochrome P450 activity IN VITRO by coincubation with a variety of inhibitors caused an increase in DPH toxicity to the extent of 13-82% in LB and 3-52% in CNS cells. 2. Modulation of cytochrome P450 activity IN VITRO by coincubation with inducers, only LB cells coincubated with 3MC caused a 21% increase in DPH toxicity. 3. Modulation of cytochrome P450 activity IN VITRO by transplacentally administered inducers; only LB cells derived from 3MC or BNF pretreated dams increased DPH toxicity, by 20 and 30% respectively. In addition, only LB cells from BNF pretreated dams had the ability to activate the pro-teratogen cyclophosphamide (CPA), (CPA is only toxic to LB cells in the presence of an external metabolising source). 4. Modulation of the formation of the potentially reactive arene oxide intermediate of DPH showed that although the degree of covalent binding could be modulated this did not correlate with the modulation in toxicity and it was therefore concluded that the arene oxide intermediate did not play an important role in DPH teratogenesis. 5. Identification by HPLC analysis of DPH metabolites formed by the cells IN VITRO. Cells (especially LB) were capable of hydroxylation and hydantoin ring cleavage.

In the studies described in this dissertation, we investigated the roles of p53 in normal development, teratogen-induced apoptosis, and birth defects. In the first study, the activation of p53 and its target genes, p21, NOXA, and PUMA, were examined during neural tube closure in mouse embryos exposed to hyperthermia (HS) or 4- peroxycyclophosphamide (4CP), teratogens known to induce neural tube defects (NTDs). In the second study, using p53-deficient mice, we examined the expression of mRNAs and microRNAs (miRNAs) during neural tube closure. In the third study, the incidence of NTDs was investigated in p53- and p21-deficient mouse embryos exposed to HS. Finally, we examined the induction of apoptosis in p53-deficient mouse embryos exposed to HS. HS and 4CP induced the activation of p53 by phosphorylation and accumulation of the protein, leading to an increase in p21 proteins and mRNAs. Although HS and 4CP also induced the expression of Noxa and Puma mRNAs, no significant increases in NOXA and PUMA proteins were observed, suggesting a possible role of transcriptionindependent apoptosis. In the second study, we showed that the expression of 388 genes and 5 miRNAs were significantly altered in p53 -/- compared to p53 +/+ embryos. Finally, we showed that 10% of p53 -/- pups exhibit exencephaly, spina bifida, and/or preaxial polydactyly, whereas no malformations were observed among p21 -/- offspring in the absence of HS. HS resulted in an increased incidence of exencephaly in both p53 and p21 null mice indicating that these two proteins act as teratogen suppressors. Our preliminary data additionally showed that a decreased level of apoptosis was observed in HS-treated embryos lacking a p53 allele, suggesting that too little apoptosis may be causally linked to NTDs observed in embryos exposed to HS. Taken together, these studies suggest that precise control of apoptosis and cell cycle arrest pathways are critical for neural tube development and the prevention of teratogen-induced NTDs.

As the current growing threat to maternal-fetal health, the most recent and largest outbreak of the Zika virus has introduced the devastating fetal effects of microcephaly and other central nervous system deficits. Therefore, the need for appropriate recognition, treatment, management, and prevention of the Zika virus prompts the necessity for further education and high quality level research to be conducted and utilized. A search of the literature using the databases PubMed, UptoDate, and CINAHL was conducted for articles published between 2009 and 2016. In addition, key informant interviews from various specialties including clinical genetics and public health were conducted. The proposed best practice recommendations for education regarding the Zika virus and appropriate prevention and treatment methods are outlined in an electronic education module that would be delivered to patients and their families prior to visiting their healthcare providers. As the Zika virus continues to spread and further research is conducted regarding its teratogenic effects, the need for concise and effective education is critical in order to raise awareness and conversely decrease the potential for maternal exposure and adverse fetal outcomes.

Class of 2010 Abstract / OBJECTIVES: The purpose of the study was to determine the knowledge of third year pharmacy students about the safety of certain medications during pregnancy and to assess their awareness of an important resource available on medication safety. METHODS: The study used an analytical cross-­‐sectional design. A pre-­‐test was administered to determine baseline knowledge. Dee Quinn provided a presentation on teratogens. The same test was then administered as a post-­‐test to assess the amount of knowledge gained from the presentation. The pre and post-­‐tests were matched for data analysis. A mean and standard deviation were developed for pre and post-­‐test data and the results were compared to each other using a t-­‐test for dependent groups. RESULTS: Students showed a significant increase in knowledge after the presentation (p<0.0001). 78% of students had improved scores after the presentation. 100% of students felt that pharmacists could help make a difference in preventing malformations due to teratogen exposure. There was no significant difference between men and women or students with children and without children. Work experience did not affect knowledge scores. 64% of students felt more comfortable counseling pregnant patients after the presentation. Awareness of the Teratology Information Service improved after the presentation. CONCLUSIONS: After the presentation, students rated themselves as more comfortable speaking with pregnant patients and showed improved knowledge of teratogens. Gender, being a parent and work experience had no relevance on knowledge scores. The investigators recommend that this presentation be given to all students at the College of Pharmacy to improve knowledge in this area.

Background: In recent years, there has been an increase in the use of automated healthcare databases for drug safety in pregnancy evaluation; their suitability for this purpose needs to be evaluated. Aim: To evaluate the utility of the United Kingdom’s General Practice Research Database (GPRD) to act as an alternative to pregnancy registries, using anticonvulsants as a case study. Methods: Pregnancies in women with epilepsy were identified and first trimester anticonvulsant exposure was determined. Major congenital malformations in the offspring were identified and verified. The risk of major congenital malformations following exposure to a range of anticonvulsants was calculated and compared to those reported by the UK Epilepsy and Pregnancy Register. The ability to identify a known teratogenic association using GPRD data was also assessed. An algorithm was created to identify and classify different types of pregnancy loss in an automated manner. Results: The risks of a pregnancy outcome with a major congenital malformation following first trimester anticonvulsant exposures, were found to be similar in the GPRD to those of the UK Register. The number of exposures to individual products in the GPRD was often small and therefore lacked statistical power. It was, however, possible to identify a known teratogenic association using data from the GPRD. Verification of the algorithm developed to classify pregnancy losses demonstrated that, although not perfect, it would be a beneficial tool when using the GPRD for drug safety in pregnancy research. Conclusion: It is unlikely a single data source or study design will be sufficient for monitoring all aspects of the safety of medicine use during pregnancy. The GPRD has the potential to make a valuable contribution to this field of research and could play an important role in complementing the work of other surveillance systems.

The teratogenic drugs cytochalasin B and valproic acid have been shown to alter F-actin polymerization, an effect that is crucial in forming microfilaments. Microfilaments form important cytoskeletal structures that maintain the structural integrity of the cell, cause cell motility and cell migration. Microfilament alterations are known to cause neural tube defects such as spina bifida and anencephaly (Walmod et al., 1999). We here aim to show that disruption of microfilaments by cytochalasin B and valproic acid affects the tensile properties of the tissue. Biomechanics is an interdisciplinary field that allows mechanical concepts to help us understand embryo development. This project used a novel tissue stretching device that measures the tensile properties of neural and epidermal tissue. The instrument used a pair of cantilevered wires to which the specimen was glued. This device stretched the mid-neural and -lateral tissue anterior-posterior (AP) and medio-lateral (ML) unidirectionally. The tensile properties of the tissue were determined by Resultant Young’s Modulus that depends on the true stress and true strain in the tissue sample. The experiment was conducted at a strain rate of 50%. Axolotl embryos were treated with 5ug/mL and 2.5ug/mL cytochalasin B and 5mM valproic acid at stage 13 (early neurula) for an hour, washed, and allowed to develop to stage 15 before it was used in the uniaxial tissue stretcher. Changes in the F-actin filaments were analysed by phalloidin staining and viewed under a confocal microscope. The tests show that disruption of microfilaments by cytochalasin B increases the stiffness of the dorsal-tissue by as much as 101% for CB-treated tissues stretched in the AP direction and 298% when stretched in the ML direction. VA-treated neural plate tissue showed a stiffness increase of 278% when stretched in the AP direction and 319%, when stretched in the ML direction. Changes in the F-actin filaments are quantified by phalloidin staining viewed with confocal microscopy. These findings indicate that direction-dependent mechanical forces in the tissue are contributing factors in closure of the neural tube in axolotl embryos.

The teratogenic drugs cytochalasin B and valproic acid have been shown to alter F-actin polymerization, an effect that is crucial in forming microfilaments. Microfilaments form important cytoskeletal structures that maintain the structural integrity of the cell, cause cell motility and cell migration. Microfilament alterations are known to cause neural tube defects such as spina bifida and anencephaly (Walmod et al., 1999). We here aim to show that disruption of microfilaments by cytochalasin B and valproic acid affects the tensile properties of the tissue. Biomechanics is an interdisciplinary field that allows mechanical concepts to help us understand embryo development. This project used a novel tissue stretching device that measures the tensile properties of neural and epidermal tissue. The instrument used a pair of cantilevered wires to which the specimen was glued. This device stretched the mid-neural and -lateral tissue anterior-posterior (AP) and medio-lateral (ML) unidirectionally. The tensile properties of the tissue were determined by Resultant Young’s Modulus that depends on the true stress and true strain in the tissue sample. The experiment was conducted at a strain rate of 50%. Axolotl embryos were treated with 5ug/mL and 2.5ug/mL cytochalasin B and 5mM valproic acid at stage 13 (early neurula) for an hour, washed, and allowed to develop to stage 15 before it was used in the uniaxial tissue stretcher. Changes in the F-actin filaments were analysed by phalloidin staining and viewed under a confocal microscope. The tests show that disruption of microfilaments by cytochalasin B increases the stiffness of the dorsal-tissue by as much as 101% for CB-treated tissues stretched in the AP direction and 298% when stretched in the ML direction. VA-treated neural plate tissue showed a stiffness increase of 278% when stretched in the AP direction and 319%, when stretched in the ML direction. Changes in the F-actin filaments are quantified by phalloidin staining viewed with confocal microscopy. These findings indicate that direction-dependent mechanical forces in the tissue are contributing factors in closure of the neural tube in axolotl embryos.

Thesis (M.Sc.) Uppsala : Sveriges lantbruksuniv.