No description available.

The cytochrome P-450 dependent monooxygenase activity in a 'wild type' susceptible strain of housefly was studied. Catalytic activities were identified that demonstrated that the housefly cytochrome P-450 was capable of similar catalytic functions as those described for higher animals. Although several specific activities were lower than in mammalian species, benzphetamine N-demethylation was comparable and there was higher constitutive activity toward lauric acid than is observed in rat hepatic microsomes. The inducing agent phenobarbital increased both total cytochrome P-450 content and the benzphetamine N-demethylase specific activity. The high constitutive activity for fatty acids was induced by the hypolipidaemic drug clofibrate, specifically inducing the w-hydroxylase activity. The substrate specificity toward lauric acid extended equally to myristic and palmitic acid. Housefly microsomal cytochrome P-450 also metabolised the unsaturated fatty acid, arachidonic acid, the w-hydroxy-lation again inducible by clofibrate pretreatment. The w-hydroxylation of these fatty acids appeared to be a well-coupled reaction, a property that also appeared to be exhibited by the rat hepatic w-hydroxylase. The housefly fatty acid hydroxylation showed certain similarities to that in the rat, both in the specificity for w-hydroxylation and in the result of induction by clofibrate. Structural comparison to cytochrome P-450IVA1, IIB1 and IA1 was made by Western blot analysis with polyclonal antibodies raised to these rat hepatic isoenzymes. Housefly cytochrome P-450 shared few, if any, common epitopes with these rat isoenzymes, nor did these antibodies inhibit housefly cytochrome P-450 dependent monooxygenase activity. Cytochrome P-450 from clofibrate-pretreated housefly microsomes was partially purified by affinity chromatography. The cytochrome P-450 had a specific content of 5. 7nmol.mg-1 and a monomeric molecular weight of 52,000 daltons and a reduced carbon monoxide difference spectrum absorbance maxima at 448nm. In a reconstituted system, this preparation exhibited activity toward lauric acid and to a lesser extent arachidonic acid in each case w-hydroxylated products predominated. This is the first example of a purification of an insect cytochrome P-450 multiple form with a defined product reaction.

The scientific literature indicates that the biotransformation of xenobiotics in man is more similar to that observed in the non-human primate than to other laboratory animal species. The main objective of the current investigation was to compare the ability of 3 species of non-human primate, the rhesus monkey (Macaca mulatta), the cynomolgus monkey (Macaca fascicularis) and the baboon (Papio species), to conjugate xenobiotics with glutathione since such information is lacking in the literature. The metabolism and pharmacokinetics of ethacrynic acid, a diuretic known to be extensively metabolised by glutathione conjugation in several other animal species, was studied in the non-human primates. The results indicate that the overall capacity for glutathione conjugation in the non-human primate is large, and demonstrate that various organs and tissues can simultaneously contribute to the detoxication and elimination of various xenobiotics by this pathway. In common with other laboratory animal species, the non-human primate excreted the major proportion of radioactivity, following administration of [14]C-ethacrynic acid, in the bile. The radioactivity in the bile was mainly associated with metabolites which had resulted from the initial conjugation of ethacrynic acid with glutathione, and the subsequent metabolism of this conjugate via the mercapturic acid pathway. There were notable species differences, between the non-human primates and other laboratory animals, in the proportions of the various metabolites which were excreted in the bile. Although the concentrations of glutathione in the various tissues of the non-human primate were similar to those reported for other mammals, species differences occurred in the rates of glutathione conjugation and the subsequent metabolism of the glutathione conjugate. A significant finding concerned the extent to which various aB-unsaturated xenobiotics were conjugated with glutathione. There were marked species differences between the non-human primate and the rat in this respect. The rhesus monkey, cynomolgus monkey and the baboon are similar as regards ethacrynic acid metabolism, tissue glutathione concentrations, tissue glutathione S-transferase activities, and the specific activities of the glutathione S-transferase isoenzymes. The rhesus monkey more closely resembles man in these respects, but both other species of non-human primate would appear to be suitable for use in metabolism and toxicological studies of compounds likely to be conjugated with glutathione.

No description available.

Hepatotoxicity is a serious adverse health effect caused by drugs and other chemical toxins generally detected in the later stages of drug development or in whole animal studies. Thus, development of screening approaches available for earlier identification of hepatotoxic molecules is necessary. A novel in vitro- in silico test system for the evaluation of the molecular mechanisms of xenobiotic toxicity in primary hepatocyte systems is presented here. It is well established that hepatocytes in vitro are most representative of hepatotoxicity in vivo, and are most useful for the determination of xenobiotic hepatotoxicity mechanisms at the molecular and cellular level. There is an on-going interest in Quantitative Structure-Activity Relationships (QSAR) in toxicology, as it can identify correlations between chemical structure and biological activity. QSAR can be used to evaluate the effects of metabolism and toxicity as many physicochemical descriptors reflect simple molecular properties that can provide insight into the physicochemical nature of the activity under consideration. QSARs were determined for hepatotoxicity of halobenzenes, p-benzoquinones, α,β-unsaturated carbonyl compounds and nitroaromatics towards isolated hepatocytes. A molecular link was established for their proposed toxicity pathways. For example oxidative activation was linked to EHOMO (energy of the highest occupied molecular orbital) values and hydrophobicity (log P) of the chemicals, while reductive activation was linked with ELUMO (energy of the lowest molecular orbital) values and log P. Such relationships may thus be useful for predicting toxicity of other chemicals of the same mechanism of toxicity. Due to the complexity involved in the phenomena of hepatotoxicity, unravelling of structure-hepatotoxicity relationships is a complicated task. A conceptual framework for QSAR modeling is proposed that involves recognition of molecular initiating events as potential endpoints to improve the prediction potential of QSAR models. Acute toxicity of reactive chemicals could be based on an initial reaction with biomolecules, thus the theory of covalent binding reactivity was used to test this concept. Reactivity assays with thiol and amine surrogate nucleophiles were used to determine susceptibility to toxicity. The derived QSAR expressions suggested that covalent binding reactivity is a good correlate to hepatotoxicity, however only if electrophilicity was the main mechanism of toxicity.

A large and varied array of xenobiotics (foreign chemicals) enters into our bodies every day. In order to prevent toxicity resulting from xenobiotic accumulation, the body has developed a complex and integrated network of enzymes and transporters to promote and control the metabolism and excretion of drugs and other compounds. Drug metabolizing enzymes are classified as oxidative (Phase I) or conjugative (Phase II), and generally result in increased hydrophilicity of their substrates. Drug transporters actively route xenobiotics into (Phase 0) or out of (Phase III) the cells. The expression of the proteins involved in drug metabolism and transport are coordinately regulated by xenosensing transcription factors, including the constitutive androstane receptor, the pregnane X receptor, the aryl hydrocarbon receptor, and Nrf2. Through the activation of these xenosensors, chemical exposure itself induces the processes which help to remove the xenobiotics from the body. The liver is the major organ of drug metabolism in the body. Chronic hepatic diseases impact the activity of xenosensors and the expression of their enzyme and transporter gene targets. Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the United States, affecting 20-30% of the populations. This profoundly underdiagnosed disease has significant effects on hepatic gene expression and may increase the risk of adverse drug reactions and xenobiotic toxicity in affected patients. This manuscript presents original research which contributes to our understanding of xenosensor function in the contexts of chemical exposure and liver disease. Manuscripts in this dissertation investigate 1) the induction profile and mechanisms of the experimental therapeutic agent oltipraz, 2) the xenosensor-regulated mechanisms of induction of the drug transporter ABCC3, 3) the impact of NAFLD on the expression of major drug metabolizing enzymes, and 4) the utility of altered drug disposition as a biomarker for NAFLD progression. The findings of these studies highlight the clinical importance of xenosensor activation and the potential pharmacological and toxicological consequences of hepatic disease.

No description available.

Hepatotoxicity is a serious adverse health effect caused by drugs and other chemical toxins generally detected in the later stages of drug development or in whole animal studies. Thus, development of screening approaches available for earlier identification of hepatotoxic molecules is necessary. A novel in vitro- in silico test system for the evaluation of the molecular mechanisms of xenobiotic toxicity in primary hepatocyte systems is presented here. It is well established that hepatocytes in vitro are most representative of hepatotoxicity in vivo, and are most useful for the determination of xenobiotic hepatotoxicity mechanisms at the molecular and cellular level. There is an on-going interest in Quantitative Structure-Activity Relationships (QSAR) in toxicology, as it can identify correlations between chemical structure and biological activity. QSAR can be used to evaluate the effects of metabolism and toxicity as many physicochemical descriptors reflect simple molecular properties that can provide insight into the physicochemical nature of the activity under consideration. QSARs were determined for hepatotoxicity of halobenzenes, p-benzoquinones, α,β-unsaturated carbonyl compounds and nitroaromatics towards isolated hepatocytes. A molecular link was established for their proposed toxicity pathways. For example oxidative activation was linked to EHOMO (energy of the highest occupied molecular orbital) values and hydrophobicity (log P) of the chemicals, while reductive activation was linked with ELUMO (energy of the lowest molecular orbital) values and log P. Such relationships may thus be useful for predicting toxicity of other chemicals of the same mechanism of toxicity. Due to the complexity involved in the phenomena of hepatotoxicity, unravelling of structure-hepatotoxicity relationships is a complicated task. A conceptual framework for QSAR modeling is proposed that involves recognition of molecular initiating events as potential endpoints to improve the prediction potential of QSAR models. Acute toxicity of reactive chemicals could be based on an initial reaction with biomolecules, thus the theory of covalent binding reactivity was used to test this concept. Reactivity assays with thiol and amine surrogate nucleophiles were used to determine susceptibility to toxicity. The derived QSAR expressions suggested that covalent binding reactivity is a good correlate to hepatotoxicity, however only if electrophilicity was the main mechanism of toxicity.

No description available.

No description available.