Studies detailed in this thesis were concerned with elucidating the function and molecular properties of UDP-glucuronosyltransferase(s) involved in detoxicative metabolism in the piscine species, Pleuronectes platessa. This study represents the first detailed investigation of such a system in a non-mammalian species. Comparative studies on UDPGT expression in the mammalian and piscine used in this investigation indicated that a number of marked species differences were apparent which were of potential toxicological significance. Analysis of piscine UDPGT activities in liver, kidney, intestine and gill demonstrated that the phenol UDPGT activity was ubiquitous to different degrees in all tissues whereas activities to endogenous substrates were more restricted. UDPGT expression in piscine microsomes was relatively non-latent compared to its mammalian counterpart, a possible reason for this being the marked species differences observed by microsomal lipid composition analysis. Assay of hepatic microsomal fractions indicated that glucuronidation of planar phenolic xenobiotics was actively catalysed in the piscine species, whilst there was low UDPGT activity towards several steroids and bilirubin, compared to the rat. Aglycone specificity for several bulky non-planar substrates was either very low or absent in the piscine species in contrast to rats. The metabolism of the proximate carcinogen benzo(a)pyrene was also investigated in isolated hepatocytes derived from plaice. The major metabolites formed were glucuronides of benzo(a)pyrene phenols, diols and quinols, indicating that glucuronidation plays an important role in the prevention of formation of cytotoxic and carcinogenic intermediates in these animals. Administration of various xenobiotic agents indicated that polychlorinated biphenyls specifically induced phenol UDPGT activity in hepatic and renal tissue, whilst the carcinogen, 3-methylcholanthrene induced this activity solely in hepatic tissue. A procedure for the purification of hepatic UDPGTs was reported that enabled the physical separation of different UDPGT isoforms from this species indicating that they are polymorphic. One isoform that catalysed the glucuronidation of the phenolic heterocyclic compound, L-naphthol was purified 380 fold over solubilised hepatic microsomes' to apparent homogeneity with a subunit molecular weight of 55kDa. A purified UDPGT preparation was used to raise polyclonal antibodies which were applied to investigating the aforementioned biological variations in UDPGT expression at the molecular level. Such immunoblot analysis indicated that differential expression was due to varied complements of UDPGT isoenzymes. Molecular biological analysis was also employed to give an insight into the evolution of the enzyme. This work indicated that UDPGT isoforms in this piscine species had epitopes in common with their mammalian counterparts, however preliminary studies indicated no such similarity between UDPGT genes in the respective species. The relevance of these data to interspecies toxicity and evolution of detoxication enzyme systems is discussed.

Fusarium oxysporum grown on natural media was believed not to produce mycotoxins of the trichothecene family. Using a defined chemical medium toxin production was investigated for and it was found that trichothecenes were produced. A yeast bioassay using Kluyveromyces fragilis, an organiam sensitive to such trichothecenes as T-2 toxin and verrucarin, was used for detection of toxin in culture filtrates. Detectable levels of toxin (0.2 $ mu$g in litre of culture) were seen by day 4 and peaked around day 9 corresponding to maximum growth (measured by mycelial mass). After this time fluctuations in the level of toxin and growth became evident, suggesting a breakdown of the toxins by the organism for a carbon source. Search for an enzyme or enzyme system, capable of degrading T-2 toxin in snail gut enzyme digested F. oxysporum, was attempted using the esterase substrate para-nitrophenol acetate. Esterase activity was detected in all fractions including culture filtrate, soluble protein fraction and insoluble protein fraction, as well as solubilized insoluble proteins (digested by contents of the crude extract). The soluble protein fraction exhibited the highest level of activity. Cells digested with the detergent Lubrol followed by precipitation of the solubilized proteins with ammonium sulphate revealed the presence of an active component(s) in the high molecular weight portion of the soluble cell fraction collected at 50 and 75% saturation. Further purification by DEAE-sepharose failed to produce an active component.

No description available.

The aim of this work was to investigate DON in wheat with Acoustic and ELISA methods, and look if in biotechnological processes such as the sourdough bread making process and the bio-ethanol fermentation process have influence on the amount of DON in dough and bread and on the DON content in DDGS (Distillers Dried Grains with Solubles) as residue of the bio-ethanol process which is used for feed purposes. A collection of wheat has been taken from the Lithuanian Agriculture Institute at Dotnuva and the DON content has been determined by Acoustic and ELISA methods. Both methods are in reasonable correlation with each other (R2 = 0,735) and applied on the raw material wheat. The aim of the second part of the study was to apply different sourdough bacteria (L. bulgaricus and L. acidophilus) in the bread making process and to determine the DON content in dough and bread with the ELISA method. The reduction of DON was noticed in both Lactobacillus applications but was the best by applying L. bulgaricus. The pH had no influence on the detoxification process in the dough. In the third part of the study the bio-alcohol fermentation process showed that with an optimal mixture of amylase- glucoamylase respectively an enzyme preparation (Vilzim SKK 350 AV) a good reduction of DON in DDGS could be obtained. (44, 5%) With these results it is recommended as to use sourdough processes in the manufacture of bread in Lithuania which is already common practice to reduce DON in bread and... [to full text]

Mammalian γ-glutamyltransferase (GGT) is a glycoprotein consisting of two subunits - a light chain and a heavy chain. The light chain contains the catalytic activity; the heavy chain anchors the protein to the membrane. GGT catalyzes the hydrolysis of the γ-glutamyl isopeptide bond of glutathione conjugates, releasing glutamic acid, or the transfer of the γ-glutamyl group to an acceptor substrate. The specificity of the enzyme for xenobiotic donor substrates has not been fully characterized. The transpeptidation activity of bovine kidney GGT was measured with glycylglycine as acceptor substrate and several glutathione conjugate donor substrates, representative of detoxication products of polycyclic aromatic xenobiotics. HPLC separation with UV detection was used for quantitation. The commonly-used chromogenic donor substrate γ-glutamyl-p-nitroanilide was also tested. Michaelis constants (Km) were obtained for γ-glutamyl-p-nitroanilide (0.74 mM), 4-nitrobenzyl glutathione (0.075 mM), 2,4-dinitrophenyl glutathione (0.30 mM), 4-methylbiphenylyl glutathione (0.12 mM), 1-menaphthyl glutathione (0.23 mM), and 9-methylanthracenyl glutathione (0.22 mM), indicating that enzyme activity is affected, but not strongly, by the nature of the S-substituent attached to glutathione, and there is a slight trend of higher Km values with bulkier aromatic S-substituents.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves 177-194).

Biotransformation and biodegradation rate constants were determined for naphthalene, lindane, and phenol in water samples from three different sources. Rate constants produced from monitoring disappearance of the parent chemical (biotransformation) were compared to those obtained from mineralization of the chemical (ultimate biodegradation) by ¹⁴CO₂ evolution as well as acidification of the residual ¹⁴C-labeled compound (primary biodegradation). Rate constants were statistically different for the three chemicals. The water source affected the rate constants. When biomass measurements of the waters were considered and second-order rate constants were derived, there was no statistical evidence that this parameter gave a reliable rate constant statistic that could be useful in predicting the fate of any of naphthalene, lindane, and phenol in these waters.

Cupriavidus metallidurans CH34 est la bactérie Gram négative considérée comme organisme-modèle pour l’étude de la résistance aux métaux lourds. Notre travail a porté sur sa résistance au cuivre, codée par les gènes cop du plasmide pMOL30. Ces gènes, responsables des différentes étapes de la résistance (compartimentation des systèmes d’efflux entre périplasme et cytoplasme, modification de valence, et d’autres fonctions totalement inconnues) ont suscité notre intérêt. On distingue dans l’îlot cop des gènes codant pour des fonctions de résistance proprement dite (essentiellement par détoxication active du cytoplasme et du périplasme). En effet, les mutants de copSRABCD, copF, et dans une moindre mesure copJ et copE deviennent sensibles. Les phénotypes des mutants divergent toutefois suivant que la mutation soit sur un cosmide qui ne porte que l’îlot (pMOL1024) ou dans son plasmide d’origine (pMOL30). Un second groupe de mutants (copVTMK, copG, copL, copQ) se distingue par un phénotype plus résistant ou identique à la souche parente, sauf autour de la CMI. Ces gènes interviendraient donc à la CMI pour assurer la résistance la plus élevée et le maintien d'un état viable latent. La présence de l’îlot cop permet de contenir le taux d’oxygène radicalaire qui reste à un taux basal lorsque les cellules sont adaptées au cuivre environnent. Après un choc de Cu (ou stress aigu), l’îlot cop répond de façon « explosive » au stress, en consommant l’énergie du potentiel membranaire et en augmentant fortement l’activité de la chaîne respiratoire. La résistance au cuivre est inductible, mais de façon différenciée pour la souche sauvage (CH34) et celle qui ne porte qu l’îlot cop (AE1744) : la CMI de CH34 triple après adaptation au cuivre, alors que celle d’AE1744 est inchangée. Après un choc de Cu, la résistance au cuivre est plus fortement induite pour AE1744 que pour CH34. Ces observations suggèrent que l’îlot cop ait été sélectionné pour sa capacité à répondre à un stress aigu puis intégré dans un ensemble de gènes plus vaste qui répond à des impératifs de stress chronique. L’analyse biochimique de CopI, une petite protéine bleue à cuivre, montre qu’elle porte un site analogue à celui des oxydases multicuivre. Son rôle pourrait dès lors être celui d’une réductase multicuivre. La protéine CopK lie de façon très spécifique le Cu(I) et il semble que la liaison du cuivre modifie sa structure. L’analyse écologique a montré que des homologues de copK pourraient être présents dans l’ADN extrait de la terre de biotopes chargés en cuivre, et dans les souches cuprorésistantes qu’on y trouve. La contribution majeure de cette thèse est de montrer que l’effet d’un stress métallique ne se résume pas à deux états physiologiques « mort ou vif ». Il y a lieu de considérer des états transitoires (choc de Cu, adaptation au métal, survie autour de la CMI, persistance) où interviennent des gènes spécifiques dans un ou plusieurs états donnés. Les résultats biochimiques et physiologiques ne nous éclairent pas encore assez sur les interconversions Cu(I)/Cu(II) ni sur les flux de cations notamment vers l'espace extracellulaire. Cette thèse ouvre des perspectives sur des mécanismes (protection à la CMI, phénotype persistant) assurant la survie des bactéries ou leur potentiel de recolonisation lors d'une diminution de la pression toxique : les gènes copT, copV, copK, copM, copB, copG, copL et copQ semblent impliqués dans ces fonctions.

Graduation date: 1994

A Pseudomonas strain, AI08, which was capable of detoxifying the herbicide acetochlor (2- chloro-N-ethoxymethyl-6'-ethylacet-o-toluide) was isolated from soils. The microbe was isolated using a combination of batch culture enrichment techniques, phenotypic agar plate based assays and a qualitative bioassay for detecting acetochlor detoxification. With the aid of a bioassay developed specifically for the quantification of acetochlor concentrations, it was determined that over a 21 day period Al 08 was capable of detoxifying 20 % of the acetochlor present in a medium containing no other organic carbon and 53 % of the herbicide in a medium containing glucose and yeast extract at concentrations of 0.02 g.l-l and 0.005 g.l-l respectively. A fragment of A108 DNA was cloned in Escherichia coli which produced recombinant cells with both elevated acetochlor resistance and the ability to detoxify 15 % of the acetochlor present in a minimal nutrient medium (containing 0.02 g.l-l glucose and 0.005 g.l-l yeast extract) over a 21 day period. Partial sequencing of the cloned A108 DNA revealed that it encoded an amino acid sequence with significant homology with the dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complexes of Azotobacter vinlandii, E. coli and Alcaligenes eutrophus. Theories are proposed as to the possible biochemical mechanisms whereby expression of the dihydrolipoyltransacetylase gene of Al 08 in recombinant E. coli cells may function in the detoxification of acetochlor. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.