The metabolism of pure cis- and trans- chlordane was studied in vitro. Microsomal preparations from the livers of male rats induced with cis- or trans-chlordane in feed for ten days were used to metabolize the pure compound corresponding to the inducer. Subsequent extraction, column fractionation and combined gas chromatography-mass spectroscopy resulted in the characterization of four compounds not previously reported from an in vitro system. In addition to the substrate, trans-chlordane extracts contained species with the following molecular weights and empirical formulae: m/e 370, c 10 H 5 Cl 7 , heptachlor; m/e 352, c 10 H 6 0C1 6, a hydroxylated chlordene; and m/e 422, c 10 H 6 0C1 8 , a hydroxylated chlordane. Dichlo rochlo rdene, oxychlordane and 1- chloro- 2 -hydroxychlordene chlorohydrin were also present, With the exception of the hydroxychlordane and heptachlor, cis - chlordane extracts contained all of the metabolites found in the trans-incubates. Additionally, a fully saturated compound m/e 372, c 10 H 7 c1 7, a dihydroheptachlor, was present. The 1, 2-trans-dihydrodiol of heptachlor found in previous in vitro incubates of cis-chlordane was not present in this extract. This information has been incorporated into a proposed route for the biotransformation of the chlordanes that offers an explanation for the observed differences in the metabolism of cis - and trans-chlordane . The pathway is based on the reductive dechlorination of the chlordanes through dihydroheptachlor to dihydrochlordene. Parallel pathways of hydroxylation, desaturation and epoxide formation arise at each of these species and at chlordane itself. The trans-isomer is predominantly desaturated or hydroxylated while the cis-isomer mainly undergoes dehaloge nation.

Short-term toxicity tests utilizing mitochondrial and microsomal metabolism were developed and applied to a series of eight quinones. In the mitochondrial assay, the degree to which test compounds inhibited mitochondrial respiration varied from an EC50 of 9 μM to l25 μM. In the microsomal assay, the maximum percent increase over control oxygen consumption rates elicited by the quinones ranged from eight percent to 837 percent. The ability of the compounds to stimulate microsomal oxygen uptake reflects their capability to redox cycle and form reactive oxygen species. Experiments were conducted to evaluate the relationship between the rate of quinone redox cycling and the extent of microsomal lipid peroxidation, a possible toxic insult associated with reactive oxygen species. Results of the mitochondrial and microsomal assays were statistically correlated with several quinone physicochemical parameters and qualitatively compared to reduction potential. The biological response observed in both test systems appeared to be most strongly influenced by the reduction potential of the quinone and biomechanisms of action were suggested based on this relationship. To assess the ability of the mitochondrial and microsomal assays to indicate toxicity of the quinonoid compounds, results were statistically correlated with literature-derived toxicity data. It was concluded that the mitochondrial assay appears to be a valid indicator of acute toxicity, while the microsomal assay better portends the potential for chronic toxicity. / Master of Science

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Les cytochromes P450 (CYPs) jouent un rôle essentiel dans le métabolisme oxydatif de nombreux composés endogènes et exogènes. L’expression de CYPs est extrêmement variable en fonction de facteurs physiopathologiques, génétiques et environnementaux. Le métabolisme des xénobiotiques par les CYPs dépend en partie de la nature, de la quantité et de l’activité d’isoformes des CYPs impliqués. L'analyse quantitative de l'expression de CYP dans les organes du métabolisme, tels que le foie, sont d'une importance particulière étant donné que la biotransformation réalisée par les CYPs est souvent un facteur critique qui affecte l'efficacité, la disponibilité et la toxicité des médicaments chez l'homme. La technique actuelle de dosage la plus courante est l’immunoquantification par Western Blot. Cette technique est limitée par la disponibilité et la spécificité de l'anticorps. Les techniques de protéomique par spectrométrie de masse, permettant d’analyser de très faibles quantités de protéines en mélange, sont les méthodes de choix pour l’identification et la quantification des CYPs dans différents organes. Nous avons développé et validé une méthode pour doser 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 et 3A5) par spectrométrie de masse en couplage chromatographique. Cette méthode, simple, rapide de sensibilité satisfaisante et peu coûteuse, a été validée dans différents types de matrices biologiques (lignées cellulaires hépatiques et neuronales, baculosomes). Ensuite, elle a été appliquée à grande échelle pour l’analyse de 50 foies humains (microsomes et mitochondries) afin d’étudier la relation phénotype/génotype pour les CYPs. Cette méthode pourra être appliquée à d’autres CYPS, est un outil utile qui permettra d’améliorer la compréhension et la prédiction pharmacocinétique et toxique de médicaments et d’autres produits chimiques. / Cytochromes P450 (CYPs) play a key role in the oxidative metabolism of many endogenous and exogenous compounds. The expression of CYPs is extremely variable depending on patho-physiological, genetic and environmental factors. The metabolism of xenobiotics by CYPs depends on the nature the quantity and the activity of CYP isoforms involved. Quantitative analysis of CYP expression in organs such as liver, are of particular importance since the biotransformation performed by CYPs is often a critical factor that affects the efficiency, availability and drug toxicity in humans. The most common technique is the immune-quantitation (Western Blot). This technique is limited by the availability and specificity of the antibody. Mass spectrometry-based proteomics, able to analyze very small amounts of protein in a mixture, are the methods of choice for identification and quantification of CYPs in different organs. We developed and validated a method for dosing 6 CYPs (1A2, 2C9, 2D6, 2J2, 3A4 and 3A5) by liquid chromatography coupled with mass spectrometry. This simple, rapid, low-cost method has an adequate sensitivity, and has been validated in different types of biological matrices (liver and neuronal cell lines, baculosomes). It has been applied at large-scale to analyze these 6 CYPs in 50 human livers samples (microsomes and mitochondria) to study the phenotype/genotype relationship. This method, which could easily be applied to other CYPs, provides an important tool to improve the understanding and prediction of pharmacokinetics and toxicity profile of drugs and other chemicals.

The consumption of dietary supplements such as flavonoids may reduce risk of many civilization diseases. Flavonoids are able to modulate the activity of cytochromes P450 (CYPs), xenobiotic-metabolising phase I enzymes of biotransformation that are involved in the activation and detoxification of food-derived carcinogens. Inhibition of CYP activities by flavonoids has been extensively studied because of their potential use as agents blocking the initiation stage of carcinogenesis. On the other hand, flavonoids have been shown to enhance the activation of carcinogens and/or influence their metabolism via induction of specific CYPs. In the first part of this study, flavonoids dihydromyricetin and α-napthoflavone were explored for their possible effects on CYP1A1 expression and activity when administered in combination with carcinogen benzo[a]pyrene (BaP). For this purpose, liver, small intestine and colon microsomal fractions were isolated from treated rats and induction of CYP1A1 was evaluated by immunodetection and EROD activity measurements. In liver and small intestine, all combinations of BaP and flavonoids led to strong induction of CYP1A1 expression. Moreover, the CYP1A1 protein levels were almost identical to levels observed when the rats were treated with BaP alone. However, in comparison...

Foi isolada uma sub-fração de microsomas, constituída por membranas do retículo endoplasmático, através do tratamento do sobrenadante pós-mitocondrial por detergente. Em testes de microscopia eletrônica e ultracentrifugação analítica, esta preparação de membranas apresentou-se livre de contaminação por outras organelas celulares. Quando incubada em condições adequadas, a fração de membranas incorpora vários aminoácidos em um produto insolúvel em TCA a quente. O tratamento do material incorporado com enzimas proteolíticas acarreta a perda de cerca de 60% da radioatividade derivada de aminoácidos marcados. Não há liberação de radioatividade por tratamento com RNase, DNase, lecitinase ou α-amilase. A remoção de aminoácidos terminais não implica em diminuição considerável de radioatividade. As melhores condições de pH e concentração de Mg para o processo são próximas às fisiológicas. O requerimento de ATP e enzima pH 5 no meio de incubação não é absoluto, mas sua adição estimula o processo. Há indicações de que a independência de fornecimento externo de GTP para o processo resulta de um conteúdo endógeno da partícula. o processo de incorporação é inibido em parte por RNase, NaF, puromicina e anisomicina. / Not available

No description available.

Foi isolada uma sub-fração de microsomas, constituída por membranas do retículo endoplasmático, através do tratamento do sobrenadante pós-mitocondrial por detergente. Em testes de microscopia eletrônica e ultracentrifugação analítica, esta preparação de membranas apresentou-se livre de contaminação por outras organelas celulares. Quando incubada em condições adequadas, a fração de membranas incorpora vários aminoácidos em um produto insolúvel em TCA a quente. O tratamento do material incorporado com enzimas proteolíticas acarreta a perda de cerca de 60% da radioatividade derivada de aminoácidos marcados. Não há liberação de radioatividade por tratamento com RNase, DNase, lecitinase ou α-amilase. A remoção de aminoácidos terminais não implica em diminuição considerável de radioatividade. As melhores condições de pH e concentração de Mg para o processo são próximas às fisiológicas. O requerimento de ATP e enzima pH 5 no meio de incubação não é absoluto, mas sua adição estimula o processo. Há indicações de que a independência de fornecimento externo de GTP para o processo resulta de um conteúdo endógeno da partícula. o processo de incorporação é inibido em parte por RNase, NaF, puromicina e anisomicina. / Not available

In the past, hyperlipidemia (HL) has been shown to affect the pharmacokinetic and pharmacodynamic properties of lipophilic drugs extensively bound to lipoproteins, including halofantrine (HF). The present thesis examined the effect of HL on the biodistribution, metabolism and electrocardiographic (ECG) effects of HF in the poloxamer 407 rat model of HL. The HL state caused unexpected changes in the distribution of HF enantiomers. In contrast to plasma, concentrations of desbutyl-HF (DHF) were much higher in highly perfused tissues. Following in vitro incubation of racemic HF and DHF, HF and DHF enantiomers shifted from the lipoprotein deficient fraction to triglyceride-rich fractions in HL plasma. No significant differences were noted in HF metabolism between NL and HL liver microsomes. It appears that both reduced plasma unbound fraction and lipoprotein associated directed uptake of lipoprotein-bound drug by tissues play roles in enantiomer biodistribution. In everted gut metabolism, formation of DHF enantiomers was inversely proportional to bile concentration whereas addition of lipids in the presence of bile caused a significant decrease in DHF:HF ratio of (-)-enantiomers. Pre-treatment of rats with peanut oil had no significant effect on DHF formation in the incubated sacs or microsomal preparations, nor did it affect the expression of intestinal CYP450. The above results were consistent with previous in vivo evaluations showing that the DHF to HF ratio was decreased by the ingestion of a high fat meal. In the ECG study, HL by itself had no effect on the ECG. In HL rats, plasma but not heart concentrations of the HF enantiomers were significantly higher compared to NL rats. DHF did not impart significant ECG prolonging effects after HF administration. The unbound fraction of HF was the controlling factor for drug uptake by the heart. Despite the lack of difference in HF heart concentrations, the QT and QTc intervals were significantly higher in HL compared to NL rats, suggesting a greater vulnerability towards HF induced QT interval prolongation in the HL state. The HL serum resulted in decreased metabolism of HF enantiomers in the isolated primary rat hepatocytes. Studies with LLC PK1 and NRK 52E cells showed that HF is not a substrate of P-glycoprotein transporters. / Pharmaceutical Sciences

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 150-161).