The teratogenic potency of retinoid analogs was determined in Syrian hamsters and compared to the teratogenic potency of all-trans-retinoic acid (all-trans.-RA, ED50 = 10.5 mg/kg). A total of 15 analogs having variations in the cyclohexene ring were evaluated following various amounts of single oral doses on day 8 of gestation. Retinoids containing a five- or six-membered ring were as teratogenic as all-tmru.-RA, provided they had sufficient lipophilic substituents on the ring. The same pattern emerged for retinoids that had six-membered aromatic ring substitution for the natural cyclohexene ring of vitamin A. Incorporation of a supplementary aromatic ring in the side-chain adjacent to a gem-dimethyl-hexene ring resulted in an increase in teratogenicity by IS-fold compared to all-trans.-RA. Major modifications of the cyclohexene ring can be made without altering teratogenic activity. The ring need not be six-membered and can have decreased lipophilicity through the incorporation of polar groups compared to all-trans.-RA, but must have sufficient lipophilic substituents to provide the necessary mass for interaction with the retinoid receptor. Incorporation of a supplementary aromatic ring adjacent to a gem-dimethyl-hexene ring facilitated π-electron delocalization and restricts side-chain flexibility , thereby increasing teratogenic potency. The pharmacokinetic disposition of 8 retinoids was investigated. Pregnant hamsters were dosed orally with all-trans-RA, 13-cis-retinoic acid, all-trans.-4- oxoretinoic acid, 9-cis-retinal, all-trans.-retinyl acetate, N-ethyl-all-trans- retinamide, N-ethyl-13-cis-retinamide, and arotinoid. The bioavailability of the retinamides was one-tenth that of the free acid retinoids. The plasma elimination half-life for all-trans-RA was 0.5 h. For 13-cis-retinoic acid and all-trans-4-oxoretinoic acid the elimination half-lives were 4.4 and 5.7 h, respectively. The binding affinity of various retinoids to cellular retinoic acid-binding protein (cRABP) was determined in day-12 hamster fetuses. Fetal supernatants from the 105,000x g fraction were incubated with high specific-activity [3H]-all-trans-RA in the presence of various concentration of unlabeled retinoids with subsequent isolation of cRABP by size-exclusion HPLC. Teratogenic retinoids, or acidic metabolites of teratogenic retinoids bound to cRABP whereas nonteratogenic retinoids failed to bind.

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Tang, Walfred. / "November 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 151-165). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis/Assessment Committee (English) --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / List of Figures --- p.ix / List of Graphs --- p.xi / List of Tables --- p.xiv / Abbreviations --- p.xviii / Abstract (English) --- p.xx / Abstract (Chinese) --- p.xxii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Renal Development --- p.2 / Chapter 1.1.1 --- The three embryonic excretory systems --- p.2 / Chapter 1.1.1.1 --- Pronephros and mesonephros --- p.2 / Chapter 1.1.1.2 --- Metanephros --- p.4 / Chapter 1.1.2 --- Renal malformations --- p.6 / Chapter 1.1.2.1 --- Causes of renal malformations --- p.8 / Chapter 1.1.2.1.1 --- Physical obstruction --- p.9 / Chapter 1.1.2.1.2 --- Mutation --- p.9 / Chapter 1.1.2.1.3 --- Environmental insults --- p.10 / Chapter 1.2 --- Retinoic Acid --- p.11 / Chapter 1.2.1 --- "Retinoic acid synthesis, signaling and degradation in the Embryo" --- p.12 / Chapter 1.2.2 --- Retinoic acid and embryonic development --- p.14 / Chapter 1.2.2.1 --- Retinoic acid and renal development --- p.15 / Chapter 1.2.3 --- Retinoic acid teratogenicity --- p.17 / Chapter 1.2.3.1 --- Retinoic acid teratogenic mechanism --- p.18 / Chapter 1.2.3.2 --- Retinoic acid-induced renal agenesis mouse model --- p.21 / Chapter 1.2.4 --- Induction of RA deficiency --- p.24 / Chapter 1.3 --- Strategy of the Thesis --- p.28 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Mouse Maintenance and Mating Method --- p.31 / Chapter 2.2 --- Bisdiamine Preparation --- p.32 / Chapter 2.3 --- All-trans Retinoic Acid Preparation --- p.32 / Chapter 2.4 --- Embryo Dissection --- p.32 / Chapter 2.5 --- Real-time Quantitative Reverse Transcription- Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.5.1 --- Sample collection --- p.33 / Chapter 2.5.2 --- Total RNA extraction --- p.33 / Chapter 2.5.3 --- Reverse transcription --- p.34 / Chapter 2.5.4 --- Polymerase chain reaction --- p.35 / Chapter 2.5.5 --- Preparation of DNA standards --- p.36 / Chapter 2.6 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling (TUNEL) Staining --- p.38 / Chapter 2.6.1 --- "Fixation, dehydration and embedding" --- p.38 / Chapter 2.6.2 --- Microtome sectioning --- p.39 / Chapter 2.6.3 --- TUNEL staining --- p.39 / Chapter Chapter 3: --- Induction of Renal Malformations by Bisdiamine via RA Deficien --- p.cy / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Time and dose responses to bisdiamine-induced renal malformations --- p.43 / Chapter 3.1.2 --- Methods to detect endogenous RA in embryonic tissues --- p.44 / Chapter 3.2 --- Experimental Design --- p.46 / Chapter 3.3 --- Materials and Methods --- p.48 / Chapter 3.3.1 --- Time and dose responses to bisdiamine administration --- p.48 / Chapter 3.3.2 --- Quantification of RA and retinol content in whole embryo by high pressure liquid chromatography (HPLC) --- p.49 / Chapter 3.3.2.1 --- Bisdiamine injection and sample collection --- p.49 / Chapter 3.3.2.2 --- Chromatographic system --- p.50 / Chapter 3.3.2.3 --- Preparation of standards --- p.50 / Chapter 3.3.2.4 --- Extraction of embryo samples --- p.51 / Chapter 3.3.2.5 --- Conditions of HPLC --- p.52 / Chapter 3.3.2.6 --- Recovery of sample --- p.53 / Chapter 3.3.2.7 --- Bradford protein assay --- p.53 / Chapter 3.3.3 --- X-gal staining of RARE-hsp-lacZ embryos --- p.54 / Chapter 3.3.4 --- Quantification of RA content in metanephroi by the RA-responsive cell line --- p.55 / Chapter 3.3.4.1 --- Bisdiamine injection and sample collection --- p.55 / Chapter 3.3.4.2 --- Maintenance of the RA-responsive cell line --- p.56 / Chapter 3.3.4.3 --- Seeding of cells and addition of samples --- p.57 / Chapter 3.3.4.4 --- X-gal staining --- p.58 / Chapter 3.3.5 --- TUNEL staining --- p.59 / Chapter 3.3.6 --- Real-time quantitative RT-PCR --- p.60 / Chapter 3.3.7 --- Statistical analysis --- p.61 / Chapter 3.4 --- Results --- p.63 / Chapter 3.4.1 --- Time response to bisdiamine treatment --- p.63 / Chapter 3.4.1.1 --- Bisdiamine administration increased resorption and affected various growth parameters of the fetuses --- p.64 / Chapter 3.4.1.2 --- Bisdiamine administration resulted in renal malformations --- p.68 / Chapter 3.4.1.3 --- Bisdiamine administration resulted in non-renal malformations --- p.71 / Chapter 3.4.2 --- Dose response to bisdiamine treatment --- p.76 / Chapter 3.4.2.1 --- Dose response of resorption and various growth parameters --- p.77 / Chapter 3.4.2.2 --- Dose response to bisdiamine in inducing renal malformations --- p.80 / Chapter 3.4.2.3 --- Dose response to non-renal malformations --- p.83 / Chapter 3.4.3 --- RA deficiency induced by bisdiamine --- p.88 / Chapter 3.4.3.1 --- Comparison of endogenous RA and retinol levels in control and bisdiamine-treated whole embryos at different time points after treatment --- p.88 / Chapter 3.4.3.2 --- Comparison of RA signaling patterns in control and bisdiamine-treated embryos at different time points after treatment --- p.90 / Chapter 3.4.3.3 --- Comparison of endogenous RA levels in control and bisdiamine-treated metanephroi at different time points after treatment --- p.93 / Chapter 3.4.4 --- Increase in the number of apoptotic nuclei in the metanephros after bisdiamine treatment --- p.95 / Chapter 3.4.5 --- Alteration of genes expression in the metanephros after bisdiamine treatment --- p.96 / Chapter 3.5 --- Discussion --- p.99 / Figures / Graphs / Chapter Chapter 4: --- Rescuing Bisdiamine-treated Metanephroi by In Vitro Supplementation with Low Concentrations of RA / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Embryonic kidney culture --- p.107 / Chapter 4.1.2 --- In vitro culture of the RA-treated metanephros --- p.108 / Chapter 4.1.3 --- Effect of exogenous retinoic acid on in vitro development of metanephros --- p.109 / Chapter 4.2 --- Experimental Design --- p.111 / Chapter 4.3 --- Materials and Methods --- p.113 / Chapter 4.3.1 --- Supplementation of low concentrations of RA to metanephric explant culture --- p.113 / Chapter 4.3.1.1 --- Preparation of culture medium supplemented with low concentrations of RA --- p.113 / Chapter 4.3.1.2 --- Metanephric explant culture --- p.114 / Chapter 4.3.2 --- Whole-mount immunohistochemical staining of ureteric epithelium and nephric tubules in metanephric explants --- p.115 / Chapter 4.3.3 --- TUNEL staining of metanephric explants --- p.116 / Chapter 4.3.4 --- Real-time quantitative RT-PCR --- p.117 / Chapter 4.3.5 --- Statistical analysis --- p.117 / Chapter 4.4 --- Results --- p.119 / Chapter 4.4.1 --- Rescue of bisdiamine-treated metanephric explants by in vitro culture in medium supplemented with low concentrations of RA --- p.119 / Chapter 4.4.1.1 --- Assessment of metanephric development under various concentrations of RA by morphological grading of UB tips at different day of culture --- p.119 / Chapter 4.4.1.2 --- Effect of various concentrations of RA on the number of UB tips and nephric tubules in metanephric explants at day 6 of culture --- p.126 / Chapter 4.4.2 --- Effect of RA supplementation on apoptosis in bisdiamine-treated metanephric explants --- p.131 / Chapter 4.4.3 --- Effect of RA supplementation on genes expression in bisdiamine-treated metanephric explants --- p.133 / Chapter 4.5 --- Discussion --- p.136 / Figures / Graphs / Chapter Chapter 5: --- Conclusion and Future Perspectives --- p.141 / References --- p.150

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Epidemiological studies suggest that both retinoid and carotenoid intakes are inversely correlated with the incidence of human cancers. Animal studies show that both retinoids and carotenoids inhibit tumor cell growth. Both retinoids and carotenoids activate the cytotoxicity function of macrophages in animal experiments. The purpose of this study is to evaluate the molecular mechanism for 13-cis retinoic acid (13-cRA) and beta-carotene (BC) induced immunomodulation which could explain their anti-cancer affects. The effects of 13-cRA and BC were studied on various subpopulations of T-lymphocytes both in vitro and in vivo. For in vitro studies, peripheral blood mononuclear cells (PBMC) were incubated with test compounds at clinically achievable concentrations (10⁻⁸M) for three days. Then the cells were stained with monoclonal antibodies followed by the analysis of flow cytometer. For in vivo studies, PBMC were collected from Barrett's esophagus or oral leukoplakia patients during treatment with 13-cRA (1mg/kg/day) or BC (30 mg/day), respectively. Then the cells were analyzed with monoclonal antibodies and flow cytometry. Both compounds showed the capability of stimulating different subpopulations of T-lymphocytes. 13-cRA predominantly increased the number of T-helper cells, their interleukin 2 (IL-2) receptors and their response to mitogens. Whereas, BC elevated the number of Natural Kill (NK) cells, their IL-2 receptors and their cytotoxicity against K562 target cells. Though these immunomodulatory effects appeared to be unaffected by the presence and cytotoxic functions of macrophages, cytokines seemed to have an important role in the retinoid- and carotenoid-induced immunomodulation. Plasma levels of IL-2 and tumor necrosis factor (TNF) measured by ELISA procedures were increased in patients treated for two months with 13-cRA and BC respectively. Anti-IL-2 and anti-TNF antibodies blocked the retinoic- and carotenoid-induced immunomodulation in in vitro studies. These results indicate that 13-cRA, activating T-helper cells with IL-2 production, and BC, activating NK cells with TNF release, induced immunostimulation which might be able to provide the anti-cancer affects in part seen in epidemiological studies.

Human peripheral blood monocytes provide a model for the in vivo exposure to, and immune functional damage caused by chronic UVB exposure at the skin surface. Retinoids and carotenoids are known immune function enhancers; they can also prevent cellular toxic product formation caused by UVB exposure. Application of these compounds in vitro may prevent functional damage to monocytes. Monocytes were exposed in vitro to UVB, then assayed for cytotoxic, phagocytic, and antigen presenting abilities. Phagocytic activity was protected from UVB damage by exposure to these compounds; cytotoxic activity was not altered by UVB exposure, but increased by retinoid or carotenoid exposure. Antigen presentation was not affected by either the UVB or these compounds. Protection of phagocytic function was not due to release of activating monokines or prostaglandins. Instead, the cell membrane antioxidant properties of these retinoids or carotenoids were the factors that protected the monocyte from phagocytic damage caused by UVB exposure.

Neuroblastoma (NB) é a forma mais indiferenciada de tumores neuroblásticos e a principal causa de morte por câncer pediátrico. Alterações epigenéticas interagem em todas as etapas do desenvolvimento do câncer, promovendo a progressão tumoral. A remodelação da cromatina é influenciada pela acetilação de histonas e a metilação de DNA. Acetiltransferases de histona (HATs), desacetilases de histonas (HDAC) e metiltransferase de DNA (DNMTs) são alvos de estratégias terapêuticas em tumores. Os retinoides agem nas vias de diferenciação celular, anti-proliferação e pró-apoptose. Nesse trabalho, é proposto que a combinação desses moduladores epigenéticos e de diferenciação em linhagens de células de NB humano é mais efetiva que os agentes isolados. Os tratamentos induziram mudanças na expressão de marcadores de diferenciação e indiferenciação, como c-Myc, β-3tubulina, NeuN e Bmi1, e alterações morfológicas nas duas linhagens celulares utilizadas, SK-N-BE(2) e SH-SY5Y. Os dados encontrados podem contribuir para uma melhor compreensão dos mecanismos moleculares dos moduladores retinoides e epigenéticos em NB capazes de acrescentar melhorias nas atuais estratégias terapêuticas. / Neuroblastoma (NB) is the most undifferentiated form of neuroblastic tumors and the leading cause of death from pediatric cancer. Epigenetic changes interact at all stages of cancer development, promoting tumor progression. Chromatin remodeling is influenced by histone acetylation and DNA methylation. Histone acetyltransferases (HATs), histone deacetylases (HDAC), and DNA methyltransferase (DNMTs) are targets for therapeutic strategies in cancer. Retinoids act on cell differentiation pathways and display anti-proliferation and pro-apoptotic actions. In the present research we examined the effects of combining epigenetic modulators and a retinoid receptor agonist in human NB cells. The retinoid all trans-retinoic acid (ATRA) combined with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase was more effective than any drug given alone in impairing the proliferation of SH-SY5Y and SK-N-BE(2) NB cells. In addition, the treatments induced differential changes in the expression of differentiation markers including c-Myc, β-3tubulin, NeuN and Bmi1, and morphological changes in SK-N-BE(2) e SH-SY5Y cell lines. The data contribute to a better understanding of the molecular mechanisms of retinoid modulators and epigenetic in NB able to add improvements in current therapeutic strategies.