Thesis (DR. P.H.)--University of Michigan.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 342-347). Available also in a digital version from Dissertation Abstracts.

Des études épidémiologiques ont montré que l’exposition à certains xénobiotiques est associée à une augmentation de la prévalence des maladies métaboliques. L’Homme est exposé à des mélanges de xénobiotiques de manière chronique et inévitable. Nous avons étudié les effets de l’interaction de deux xénobiotiques sur le métabolisme du foie, organe majeur de détoxification de l’organisme. Nous avons choisi deux perturbateurs endocriniens et polluants organiques persistants, qui activent des voies de signalisation différentes: la 2, 3, 7, 8 tétrachlorodibenzo-p-dioxine (TCDD), agissant via le récepteur aux hydrocarbures aromatiques (AhR), et l’α-endosulfan, un pesticide organochloré, qui peut agir via la voie du récepteur aux oestrogènes (ER) ou du récepteur X aux pregnanes (PXR). Notre objectif est de déterminer l’effet du mélange de ces polluants par rapport à chaque polluant isolé sur la régulation de certaines voies du métabolisme hépatique in vitro dans la lignée hépatocytaire humaine, HepaRG. Dans une première publication, une étude du transcriptome de cellules HepaRG différenciées a été effectuée. Ces cellules ont été exposées pendant 30 heures à 25nM de TCDD, 10μM d’α-endosulfan, ou au mélange. Nous avons observé que le mélange inhibe fortement l’expression de certains gènes impliqués dans le métabolisme glucidique et dans celui des alcools. Dans une seconde étude, nous avons donc étudié le mécanisme d’action du mélange sur le métabolisme glucidique. L’expression de deux gènes de la néoglucogenèse hépatique, le transporteur de glucose 2 (Glut2) et la glucose 6 phosphatase (G6Pc), est réduite de plus de 80% par le mélange. L’expression d’autres gènes du métabolisme glucidique (pyruvate kinase, glycogène synthase, glycogène phosphorylase, pyruvate déhydrogénase 2) est aussi diminuée, suggérant que le mélange peut affecter ce métabolisme de manière significative. De plus, la production de glucose diminue de 80% avec le mélange dans des conditions néoglucogéniques. En condition glycolytique, l’oxydation du glucose en CO2 diminue de 30% après 72 heures d’exposition au mélange. Un traitement à plus long terme (8 jours) avec des doses plus faibles des polluants (0.2 à 5nM de TCDD, 3μM d’α-endosulfan) diminue aussi l’expression de la G6Pc et de Glut2. Nous avons montré que la TCDD active bien la voie du AhR, et que le ER est impliqué dans l’action de l’α-endosulfan. Dans la troisième partie de cette thèse, nous avons étudié la régulation de plusieurs enzymes impliquées dans le métabolisme de l’alcool (alcool déshydrogénases, ADHs, cytochrome P450 2E1, CYP2E1) après l’activation du AhR. Les agonistes du AhR entrainent la diminution de l’expression des ARNm des ADH1, 4, 6 et du CYP2E1 et des protéines correspondantes. Nous avons montré que cette régulation utilise la voie génomique du AhR. De plus, cet effet est également observé après traitement de 8 jours par de faibles doses de TCDD. L’exposition chronique de l’Homme à de faibles doses de xénobiotiques en mélange pourrait affecter le métabolisme glucidique hépatique et contribuer, en partie, au développement du syndrome métabolique. / Epidemiological studies have shown that exposure to certain xenobiotics is associated with an increased prevalence of metabolic diseases. Humans are exposed to mixtures of xenobiotics in a chronic and inevitable way. We studied the effects of the interaction of two xenobiotics on metabolism in the liver, the major organ for detoxification in the body. We chose two endocrine disruptors and persistent organic pollutants which activate different signaling pathways: 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), which uses the AhR (Aryl hydrocarbon receptor) pathway, and α-endosulfan, an organochlorine pesticide, which acts via the PXR (pregnane X receptor) and/or the ER (estrogen receptor) pathway. Our aim was to determine the effects of this pollutant mixture, as compared to each pollutant alone, on the regulation in vitro of some hepatic metabolism pathways in the human hepatic cell line, HepaRG. In the first publication, a transcriptomic study of differentiated HepaRG cells was performed. The cells were exposed for 30h to 25nM TCDD, to 10 µM α-endosulfan or to the mixture. We observed that the mixture strongly inhibited the expression of some genes involved in the metabolism of glucose and alcohol. In the second study, we studied the mechanism of action of the mixture of pollutants on the metabolism of glucose. The expression of two genes involved in hepatic gluconeogenesis, glucose transporter 2 (GLUT2) and glucose-6-phosphatase (G6Pc), were reduced 80% by the mixture. The expression of other glucose metabolism genes (pyruvate kinase, glycogen synthase, glycogen phosphorylase, pyruvate dehydrogenase 2) also was decreased suggesting that the mixture might impact markedly carbohydrate metabolism. Furthermore, glucose production decreased 40% with the mixture under gluconeogenic conditions. Under glycolytic conditions, the oxidation of glucose into CO2 decreased 30% after 72h of exposure of the cells to the mixture. Long-term treatment (8 days) with lower doses (0.2 to 5 nM TCDD, 3 µM α-endosulfan) similarly decreased G6Pc and GLUT2 expression. We showed that TCDD activated the AhR pathway, and that ER was partly involved in the α-endosulfan effect. In the third part of this thesis, we studied the regulation of several enzymes involved in the metabolism of alcohol (alcohol dehydrogenase, ADH, cytochrome P450 2E1, CYP2E1) after activation of AhR. AhR agonists led to a decrease in the amounts of mRNAs for ADH1, 4, 6 and CYP2E1 and the corresponding proteins. We showed that this regulation uses the AhR genomic pathway. Furthermore, this effect was also observed after 8 days of treatment with lower doses of TCDD. Chronic exposure of individuals to low doses of xenobiotics in mixtures might significantly affect hepatic carbohydrate metabolism and be a contributing factor for the development of the metabolic syndrome.

M.Sc. / Please refer to full text to view abstract

This thesis describes firstly, a study involving the thermal rearrangement of substituted 6-e̲x̲o̲-(1-alkenyl) bicyclo-[3.1.0]hexenes, and secondly, the application of this type of transformation to a total synthesis of (±)-sinularene (1̲2̲5̲). The 6-e̲x̲o̲-(1-alkenyl)bicyclo[3.1.0]hexenes (1̲8̲7̲, 1̲8̲9̲, 1̲9̲2̲, 1̲9̲4̲, 2̲4̲0̲, 2̲7̲4̲ and 340) were prepared and thermolyzed in sealed tubes to afford the corresponding bicyclo[3.2.1]octa-2,6-dienes (1̲8̲8̲, 1̲9̲0̲, 1̲9̲3̲, 1̲9̲5̲, 2̲4̲1̲, 2̲7̲6̲ and 3̲4̲1̲) in generally excellent yields. With the exception of 1̲9̲0̲, the thermolysis products were subjected to acid-catalyzed hydrolysis to give the respective bicyclo[3.2.1]octenones. From this study, it is clear that a) the Cope rearrangement of substrates, such as 2̲7̲4̲ and 3̲4̲0̲, containing even sterically bulky substituents on the 6-alkenyl side chain presents a viable means of generating functionalized bicyclo [ 3.2.1] octa-2 , 6-dienes, b) this methodology provides for the placement of synthetically useful functionalities on any of the carbon bridges of the bicyclo-[3.2.1]octane skeleton, and c) the transformations 2̲4̲0̲→2̲4̲1̲ and 2̲7̲4̲→2̲7̲6̲ provide strong evidence for the stereospecificity of the rearrangement process. In the total synthesis of (±)-sinularene (1̲2̲5̲), the key step involved the thermal rearrangement of 3̲2̲2̲ to afford the bicyclo[3.2.1]octadiene 3̲2̲1̲. The compound 3̲2̲2̲̲ was readily prepared as follows. 1-Lithio-3-methyl-1-butyne was treated with methacrolein to furnish the allylic alcohol 3̲3̲1̲, which was transformed into the ester 3̲3̲2̲ v̲i̲a̲ an orthoester Claisen rearrangement (hot triethyl .orthoacetate, propionic acid). Hydrolysis of the ester 3̲3̲2̲, followed by reaction of the resultant acid with oxalyl chloride in refluxing hexane gave the corresponding acid chloride 3̲3̲4̲. Treatment of 3̲3̲4̲ with a cold, ethereal solution of diazomethane afforded the diazo ketone 3̲3̲5̲, which in the presence of copper (II) acetoacetonate in refluxing benzene, underwent an intramolecular carbenoid cyclization to furnish the bicyclic ketone 3̲3̲6̲. Semihydrogenation of 3̲3̲6̲ using Lindlar’s catalyst gave stereoselectively the c̲i̲s̲-alkenyl ketone 3̲3̲7̲. The enone 3̲3̲8̲ was obtained by oxidizing the trimethylsilyl enol ether of 3̲3̲7̲ using palladium (II) acetate in acetonitrile. When the enone 3̲3̲8̲ was treated with lithium divinylcuprate, the two epimeric products 3̲3̲9̲ and 3̲4̲6̲ were obtained in a ratio of 9:1, respectively, and were sus-sequently separated by column chromatography. Trapping the lithium enolate of 3̲3̲9̲ with t̲-butyldimethylsilyl chloride led to the required enol ether 3̲3̲2̲. Thermolysis (220°C, sealed tube) of 3̲3̲2̲ in benzene produced exclusively in 86% yield the desired bicyclic triene 3̲2̲1̲. Subjection of 3̲2̲1̲ to hydroboration using disiamylborane gave, after oxidative workup, the alcohol 3̲4̲7̲, which on treatment with p̲-toluenesulfonyl chloride in the presence of 4-dimethylaminopyridine, afforded the ketone 3̲4̲9̲. Successive hydrogenation of 3̲4̲9̲ and Wittig olefination of the resultant ketone 2̲8̲0̲ completed the total synthesis of (±)-sinularene (1̲2̲5̲). [formula omitted] / Science, Faculty of / Chemistry, Department of / Graduate

Organochlorine pesticides (OCPs) were extensively applied in agriculture, industry and public health programs for decades. Based on the persistence and the lipophilicity of OCPs, these chemicals are ubiquitous in the environment and can be accumulated in fatty tissues of animals through the food chain. Even being restricted for years, OCPs are still detected in human bodies. In this thesis, analytical methods for the determination of OCPs were developed and applied for the analysis of cord serum samples. The evaluation of prenatal exposure to OCPs and its effects on birth outcomes as well as the postnatal growth were investigated. Due to the toxicology and carcinogenesis, biomonitoring of the OCP exposure to human is needed. Therefore, an analytical method with high sensitivity and specificity is required to detect OCPs at trace levels in serum. We compared two data acquisition modes of mass spectrometry (MS), namely selected ion monitoring (SIM) and multiple reaction monitoring (MRM). Higher sensitivity and selectivity were achieved by MRM because the background noise was reduced by lowering the matrix effects. Different ionization techniques, including electron ionization (EI), chemical ionization (CI) and atmospheric pressure chemical ionization (APCI) were evaluated. The EI source is a universal ionization technique available with the MS library for the compound identification. The negative chemical ionization (NCI) is more suitable to analyze compounds with high electronegativity. The novel ionization technique APCI was coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS). The APCI source was evaluated by terms of ionization and fragmentation performance. APCI was a soft ionization technique generating molecular ions with high intensity. The selectivity and sensitivity of APCI were comparable or better than the EI source. As one of the largest consumers and producers of OCPs, China has suffered severe OCP pollution. Previous monitoring studies reported detectable levels of OCPs in human bodies. However, studies concerning the prenatal exposure to OCPs in China are limited. Due to the vulnerability of fetuses, the effects of prenatal exposure to OCPs could be more severe than those of adults. We collected cord serum samples during the delivery period in Wuhan, China and measured the OCP concentrations to assess the prenatal exposure by using GC-MS/MS. Compared with other areas in China, the OCP levels in Wuhan were comparable in this population. The identified predominant OCPs were β-HCH and p,p'- DDE, with geometric means of 8.67 and 33.9 ng/g lipid, respectively. Slight positive associations were found between α-HCH and β-HCH, and between o,p′- DDT and p,p′-DDT, which indicated similar exposure source of these chemicals. The obtained results showed that HCH levels were associated with maternal age, body mass index (BMI) before pregnancy, education levels, and passive smoking. Associations between the prenatal exposure of OCPs and birth outcomes were investigated. The sex-specific relationships between the OCP exposure and birth size were indicated. Concentrations of β-HCH were inversely associated with birth weight and ponderal index for boys, while for girls these associations were not significant. Our results suggested that the prenatal exposure to OCPs exerted negative effects on the fetal growth, and precautions should be taken even though the OCP levels were relatively low.

Induction of hepatic microsomal hydroxylative enzymes is an important aspect of detoxication of fat-soluble toxicants. The magnitude of induction depends on numerous factors, such as the nature and dose of toxicant as well as dietary factors. Research was conducted on (1) endrin tolerance in rats, (2) preliminary comparisons of inductive effects of various organochlorine pesticides in rats to select compounds for further study in guinea pigs, (3) general effects of various dietary factors on induction, and (4) effects of ascorbic acid deficiency on induction of hepatic microsomal hydroxylative enzymes by organochlorine pesticides in guinea pigs. Measurements were made of body weight gain, feed consumption, liver weight, in vivo and in vitroenzyme activities, and body levels of pesticides and vitamins. Tolerance developed in rats fed 25 ppm endrin in the diet. There was severe intoxication during the first week but complete recovery of rate of body weight gain and feed consumption occurred during the second week, in spite of continued ingestion of the endrin-containing diet. Induction of endrin-degrading microsomal enzymes was proposed as the mechanism for tolerance. Pretreatment with the potent inducer, dieldrin, diminished the severity of endrin intoxication. However, pretreatment with another inducer, phenobarbital, afforded less protection in proportion to the extent of microsomal enzyme induction. Organochlorine pesticides, tested for their inductive capacity in rats, in decreasing order of effectiveness then fed as 25 ppm of the diet for 15 days, were heptachlor epoxide, dieldrin, endrin, 1,1-Bis-(p-chlorophenyl)-2,2,2-Trichloroethane (DDT), Ovex, gamma-chlordane, and lindane. Of these, DDT and dieldrin were compared over the range of 1 to 50 ppm of the diet. Dieldrin was a more potent inducer at all dietary levels. At low doses both compounds produced greater induction when measured by hexobarbital sleep time than by the in vitro enzyme procedures. At high doses, the in vitro 0-ethyl 0-p-nitrophenyl phenyl-phosphonothioate (EPN) detoxication was a more responsive measure. Dieldrin, DDT, and lindane were fed to guinea pigs at 25 ppm of the diet for 15 days, but only dieldrin stimulated a significant level of induction. DDT antagonism of dieldrin storage seen in rats by Street (Sci. 146:1580, 1964) did not occur in guinea pigs, but rather dieldrin antagonized DDT storage. Some general dietary factors affecting induction in rats were observed. A semipurified diet lowered the baseline microsomal enzyme activity but supported induction as effectively as a conventional diet. Vitamin A at very high dietary levels induced enzyme activity; this induction was apparently additive to that produced by 1 ppm dieldrin. Other fat-soluble Vitamins produced inconsistent responses. Ascorbic acid deficiency in guinea pigs impaired induction by dieldrin. Impairment was seen by the second day on the deficient diet. However, dieldrin was able to produce a small amount of induction at all stages of deficiency. In frank scurvy, induction by DDT and lindane was completely blocked, but there was a moderate level of induction by dieldrin. Maintenance of maximum induction was related to dietary rather than liver levels of ascorbic acid; 50 ppm ascorbic acid in the diet was grossly inadequate while 200 ppm supported about 80% of the induction produced by feeding 2000 ppm. It was concluded that (1) microsomal enzyme induction is important in resistance to organochlorine intoxication, (2) factors found in the normal diet can induce microsomal enzyme activity, (3) high dietary levels of ascorbic acid are necessary to support maximum induction, and (4) dieldrin is an inducer of such high potency that it can stimulate a limited amount of induction in spite of ascorbic acid deficiency.

Heavy metal and organochlorine (OC) concentrations, including organochlorine pesticides and polychlorinated biphenyl congeners (PCBs), were determined in arctic ground squirrels (Spermophilus parryi) from three sites in the Brooks Range of northern Alaska in 1991-93. Heavy metals were present in most squirrel livers collected, with concentrations of trace elements (As, Cd, Hg, Ni, and Pb) averaging below 1 μg/g wet weight. Hexachlorobenzene (HCB), p,p'-DDE, gamma hexachlorocyclohexane (γ-HCH), trans-nonachlor, and PCBs 138, 153, and 170 were the most frequently detected OCs in fat and liver. Average concentrations of individual OC analytes were below 20 ng/g wet weight in liver and below 15 ng/g wet weight in fat. Rank correlations indicate that concentrations of heavy metals and of OCs accumulate in concert with one another (As, Cd, Cu, and Zn; PCBs 138, 170, and 180). Although heavy metal and OC concentrations are low relative to other areas and other arctic species, the occurrence of these compounds illustrates the global pervasiveness of persistent organic compounds and the potential for bioaccumulation in the terrestrial arctic food web.

Bifenilos policlorados (PCBs) e pesticidas organoclorados são compostos que não ocorrem naturalmente no ambiente e não são facilmente degradados química ou microbiologicamente. Seu estudo no ambiente é importante devido à sua persistência, toxicidade, lipossolubilidade e conseqüente biomagnificação. Por isso, representam a maioria dos poluentes orgânicos persistentes (POPs) considerados prioritários pela UNEP (United Nations Environmental Programme) e banidos ou restritos pela Convenção de Estocolmo, de maio de 2001. Tais poluentes podem ser facilmente emitidos para a atmosfera, atingindo áreas remotas como a Antártida, integrando um processo cíclico de contaminação conhecido como destilação global. No presente trabalho otimizou-se uma metodologia analítica capaz de detectá-los em ovos gorados de pingüins e indivíduos de krill coletados na Baía do Almirantado, Ilha Rei Jorge, Antártida. Tal metodologia foi avaliada e enquadrou-se em critérios internacionais de controle de qualidade. Os compostos mais presentes foram, de um modo geral, os PCBs, DDTs e o HCB e a ocorrência pareceu ser espécie-específica dentro do gênero Pygoscelis. Em todos os casos, o teor dos compostos não foi superior ao de aves árticas em nível trófico semelhante. A análise do krill permitiu estimar a biomagnificação dos compostos encontrados nos ovos, cuja única fonte de contaminação é a transferência fêmea-filhote. / Polychlorinated biphenyls (PCBs) and organochlorine pesticides are compounds that do not occur naturally in the environment and are not easily degraded by chemical or microbiological action. Their study in the environment is important due to persistence, toxicity, liposolubility and consequent biomagnification. For these reasons, they represent the majority of the persistent organic pollutants (POPs), considered to have priority by the UNEP (United Nations Environmental Programme) and banished and/or restricted by the Stockholm Convention of May, 2001. Such pollutants can be easily ejected into the atmosphere and reach areas as remote as Antarctica, integrating a cyclical contamination process known as ?global distillation?. In the present work an analytical methodology capable of detecting such compounds in unhatched penguin eggs and whole krill was optimized. The samples were collected in Admiralty Bay, King George Island, Antarctica. This methodology was evaluated and fitted international quality control criteria. The compounds found in higher levels were, in most of the samples, the PCBs, DDTs and HCB and the occurrence seemed to be species-specific for the Pygoscelis genus. In all of the cases, the levels found were not higher than the ones in arctic birds in a similar trophic level.The krill samples analysis made it possible to estimate the biomagnification of the compounds found in eggs, whose only source of contamination is the female-offspring transfer.

Twelve lactating Holstein dairy cows were randomly divided into four groups of three animals each. Group A served as the control, group B was dosed at 0.05ppm/day of DDE (2,2-bis(P-chlorophenyl-1, 1-dichloroethylene), cows in group C were dosed at 0.1ppm DDE/day, while group D cows were dosed at 1.0ppm DDE/day. DDE was administered in a residue free peanut oil solution for 32-consecutive days. Milk samples were taken daily during the 32 day dosing period and for an additional 32 days after the dosing period. Quantitative analysis of DDE residue in milk fat was determined by using a Tracor MT-220 gas chromatograph with a Tritium electron capture detector. The average increase in DDE milk fat concentration during the dosing period was directly related to intake levels. DDE was the only organochlorine compound detected in the milk fat. The general slope and shape of the curves of milk fat DDE levels were similar for all treatments. The levels of DDE increased rapidly after the onset of dosing. After 15 days of dosing and throughout the remaining 17 days of the dosing period, milk fat DDE increased at a relatively slow rate. The level of milk fat DDE declined rapidly as soon as the DDE residue source was withdrawn. At the end of the 32-day post-dosing period, one cow from each group was slaughtered and samples were taken from muscles, brain, lung, lymph, spleen, kidney fat, heart, gonad, placenta, udder, and kidney for DDE analysis. Considerable DDE was found in the muscle, lymph, kidney fat, and udder tissues.