New methodologies have been developed for the determination of arsenic and selenium species in a variety of environmentally important matrices. A simple liquid chromatographic separation technique based upon mini-column technology was developed to obtain a simultaneous, fast, efficient and reliable separation of relatively toxic from relatively non-toxic arsenic and selenium species. The relatively toxic arsenic and selenium species studied were inorganic Asv, AsIII, SeVI and SeIV. The relatively non-toxic species of arsenic and selenium studied were AsBet, DMA and Se Met. Optimum conditions were found to be the use of a Hamilton PRP X100 12-20 µm anion-exchange resin with column dimensions of 100 x 3 mm. The mobile phase utilized a 10 mM K2S04 solution at pH 10.2 with a flow rate of 1 ml minˉ¹ and a sample injection loop of 100 µ1. Total analysis time was under 7 minutes with limits of detection in the range of 2.0 - 10 µg kgˉ¹ for arsenic and selenium species, respectively. Work was undertaken, using HPLC-ICP-MS instrumentation, as part of a feasibility study, into the production and certification of six new reference materials; these being analyzed for the species of arsenic, in chicken, fish, rice and soil samples, and selenium, in wheat and yeast samples. Enzyme extraction techniques were used throughout, except for soil where a microwave H3P04 extraction was used. Efficiencies were in the range 90-100%. The results obtained provided speciation information as well as total elemental concentrations with no operationally defined limits. Speciation analysis requires that the endogenous species are extracted without modification of their chemical form or disturbance to the equilibrium existing between the various species present. Work was undertaken to identify and quantify the selenium species present in two samples of novel, previously unstudied, bio-natured nutrients, these nutrients being: i) a selenized yeast from a new process and: ii) a probiotic bacteria-based dried milk sample (Biogurt®). Specific interest was directed towards enzyme, MeOH and KOH and TMAH extraction efficiencies together with retention of species information. Selenium speciation was performed using ion-exchange HPLC-ICP-MS. It was found that the selenium content, in the form of SeMet, was adequately extracted from the yeast (Pharma Nord) that was used for method validation using protease, which yielding 90% of the total selenium. However, the determination of selenium and selenium species in the bionatured nutrients proved to be quite problematic. Methods that avoided species conversion with the highest extraction efficiencies were found to be: i) the use of protease for the yeast sample (19%) and; ii) the use of 0.01 M HCl for the Biogurt® (71%). Information obtained from speciation of these samples by anion and cation-exchange HPLC-ICP-MS was limited due to the low extraction efficiencies of any procedure undertaken for the samples, by the retention of the analyte on-column and by the lack of standards available for matching of retention times. HPLC-ICP-MS has proved an efficient tool for the identification and determination of arsenic and selenium species providing detection limits at µg kgˉ¹ levels. However, a major concern with this instrumentation is the unambiguous assignment of peaks which relies on the chromatographic purity of the signal and the availability of standards. Anion-exchange chromatography employing Hamilton PRP X100 resin with NH4HC03 (10 mM, pH 10.2 for arsenic and 10-50 mM, pH 5 for selenium species) with methanol (10 %, v/v) as the mobile phase allowed separation of the arsenic and selenium species investigated under conditions that were compatible for both HPLC-ICP-MS and HPLC-ESMS. Molecular ions and structural fragmentation patterns of these by tandem MS have facilitated the identification of chromatographic peaks obtained using HPLC-ICP-MS. In the analysis of marine algae, arsenosugars were the major species found, and in yeast the dominant species was found to be selenomethionine.

No description available.

No description available.

No description available.

Skin subcellular fractions, keratinocytes, living skin equivalents (LSE) and percutaneous absorption using diffusion cells were examined as in vitro models for assessment of skin xenobiotic metabolism. Cytochrome P450 monooxygenase, esterase and glutathione-S-transferase (GST) activities were investigated in rodent, pig and human skin. CYP1A1 activity was detected in rat and pig skin microsomes by ethoxyresorufin-Odeethylation. CYP2B activity was detected in rat skin microsomes by pentoxyresorufin-Odepentylation. Neither were detected in human skin microsomes. Rat keratinocytes lost cytochrome P450 activity within several hours of being isolated from skin, and were not reliably induced following exposure to B-naphthoflavone. Cytochrome P450 activities were detected in the LSE after induction by 3-methylcholanthrene. Carboxylesterase activity was detected in skin, keratinocytes, and the LSE using 4- methylumbelliferyl heptanoate as the substrate. Induction of caboxylesterase activity by 3- methylcholanthrene was shown in the LSE but not keratinocytes. GST activity was shown in skin and keratinocytes, but induction was only shown in the LSE. The ability to induce xenobiotic metabolising activity suggests that enzyme induction may be linked to cell differentiation. GST's were localised at the basal layer of the epidermis. During percutaneous absorption, DNCB was metabolised to the glutathione (GSH) conjugate, limited by the GSH available in the skin. GSH conjugation of DNCB is thought to be a detoxification pathway preventing the immune response illicited by DNCB. Studies investigating the effect of age of rat on dermal xenobiotic metabolism revealed no differences between the neonate and mature rat with respect to cytochrome P450 monooxygenase activity or esterase activity. However, neonatal rat skin showed five fold lower GST activity and three fold higher reduced GSH levels. Pig skin showed similar levels of xenobiotic metabolising activity to human skin and showed a similar metabolic profile for DNCB during percutaneous absorption, supporting its use as a better model for human than rodent skin. The LSE was a good model for studies of human dermal xenobiotic metabolism particularly with the influence of inducing agents.

Folate is a critical determinant in male reproductive health. During spermatogenesis, there are massive alterations to the epigenome associated with tightly regulated gene transcription and chromatin reorganization including a tightly regulated pattern of histone H3 methylation. In vitro experiments were conducted to determine whether folate depletion could alter global histone H3 methylation at lysine 4 and 9 in cultured spermatogonia-like GC-1 cells. Folate depleted media did not alter global levels of histone H3 methylation at lysine 4 and 9 in spermatogonia-like GC-1 cell. In vivo experiments were used to determine the extent to which folate deficiency, from early embryonic development to adulthood, impacts histone methylation and male reproductive health in a mouse model. C57BL/6 females were fed either a folate-sufficient diet or a folate-deficient diet two weeks prior to breeding and through pregnancy and lactation. Male offspring received the same diet as their mother until sacrifice. Testes and epididymides were collected at postnatal day 6 to 18, and at 6 and 13-14 weeks of age. At 8 weeks of age, male pups were assessed in fertility trials. Folate deficiency severely compromised male reproductive health. Folate deficient males had reduced epididymis weight and defects in spermatogenesis. Abnormalities included the presence of multinucleated cells, sloughing of germ cells, and a slight increase in germ cell apoptosis. Alterations in key fertility parameters included an increase in sperm morphological defects and consequently decreased pregnancy rate and litter size. Remarkably, gene activating histone H3 tri-methylation at lysine 4 was significantly reduced i / Le folate joue un rôle déterminant au niveau de la santé reproductrice de l'homme. Au cours de la spermatogenèse, il y a d'importantes modifications à l'épigénome liées à la transcription de gènes et à la réorganisation de la chromatine, incluant une régulation essentielle de la méthylation de l'histone H3. Des expériences in vitro furent effectuées afin de déterminer si une réduction de la concentration de folate dans le milieu de culture pourrait altérer la methylation globale de l'histone H3 aux lysines 4 et 9 de cellules cultivées spermatogonia-like GC-1. La réduction de folate n'a pas affecté la méthylation globale de l'histone H3 aux lysines 4 et 9 de cellules spermatogonia-like GC-1. Des expériences in vivo, chez un modèle murin, furent réalisées afin de déterminer les répercussions d'une déficience en folate, du développement embryonnaire précoce jusqu'à l'âge adulte, sur la méthylation de l'histone et la santé reproductrice de l'homme. La santé reproductrice masculine fut sévèrement compromise par une déficience en folate. Les males déficients en folate démontrèrent une réduction du poids des épididymes et une augmentation des anomalies spermatogéniques incluant des cellules multinuclées, la desquamation de cellules germinales et une légère augmentation de cellules germinales apoptotiques. Les principaux paramètres de fertilité ont démontré une diminution du nombre de spermatozoïde morphologiquement normal et par conséquent une réduction du taux de grossesse et de la taille des portées. Remarquablement, la tri-méthylation de l'histone H3 à la lysine 4, associée à l'activation de l'expression des gènes, a été f

This thesis has examined the association between the expression of a cell adhesion molecule, epithelial-cadherin (E-cadherin), and rat embryo malformations induced by three model teratogens and an E-cadherin antisense oligonucleotide during organogenesis in vitro. The embryo malformations induced by phosphoramide mustard (PAM), an active metabolite of an anticancer drug, cyclophosphamide, were not associated with any change in the expression of E-cadherin. The embryo malformations induced by exposure to the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), were associated with an increased steady state concentration of E-cadherin mRNA in the embryo, but not in the yolk sac. This increase in E-cadherin mRNA did not seem to be the cause of the embryo malformations. Exposure of embryos to the heavy metal, cadmium chloride, resulted in an increase in the steady state concentration of E-cadherin protein in the yolk sac, but not in the embryo. The timing of this increase would suggest that it may be one of the factors leading to the embryo malformations. An 18-base E-cadherin antisense oligodeoxynucleotide, injected into the amniotic cavity of 5-7 somite stage rat embryo, down regulated the expression of E-cadherin in the yolk sac, and caused specific open anterior neuropore malformations. Together, these data would suggest that E-cadherin expression is regulated in a tissue-specific manner, and that altering the expression of E-cadherin in the yolk sac is associated with embryo malformations.

Cyclophosphamide is a bifunctional alkylating agent. Administration of low doses of cyclophosphamide to male rats produces severe effects on the outcome of their progeny. The studies in this thesis show that one week treatment with cyclophosphamide can produce about 30% post-implantation loss. This indicates that post-testicular spermatozoa are sensitive to alkylating agents. Previous studies have shown that six weeks treatment caused more than 95% post-implantation loss. The effects of cyclophosphamide treatment on rat spermatozoal nuclei in the testis and epididymis were also addressed in this thesis. Rat cauda epididymal spermatozoa collected after one week of cyclophosphamide treatment showed no change in decondensation pattern in vitro; while samples obtained from rats treated for six weeks showed a slowed chromatin elongation and dispersion pattern. Iodoacetamide binding revealed that there was about a 30% decrease in the amount of free sulfhydryls in spermatozoal nuclei from six weeks treated samples, while there was no detectable change in one week treated samples. DNA single strand breakage was detected in one week treated samples only in the presence of proteinase K in the lysis solution, while significant amounts of both DNA single strand breakage and DNA cross-links were detected in six weeks treated samples. Using a DNA synthesis in vitro system, it was found that six weeks treatment decreased spermatozoal template function, while one week treatment did not result in any significant difference in DNA template function when compared with the control. It is proposed that as the mele germ cells undergo the chromatin transition processes, the chromatin is in an open dynamic status, which expresses many vulnerable sites of the genome to aklylating agent. Since there is no DNA repair system, the alkylation of the genome is cumulative and causes severe damage to the germ cell nuclei; this would lead to early embryo death. Furthermore it is hypothesized that

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1969. / Bibliography: leaves 57-58. / xii, 75 l illus

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 75-81). / xvii, 81 leaves, bound ill. 29 cm