Two studies were conducted to compare nitrogen (N) and glutamate metabolism in Polypay and percentage White Dorper lambs.First, a two-phase digestion/N metabolism trial was conducted with 18 wether lambs of three genetic types: Polypay (PP), 1/2 White Dorper 1/2 Polypay (1/2 D), and 3/4 White Dorper 1/4 Polypay (3/4 D). Six lambs of each genetic type were fed a high roughage diet (HR; Phase 1) or high concentrate diet (HC; Phase 2). DM and N digestion was higher for 1/2 D than PP or 3/4 D fed HC. N retention was highest for 1/2 D regardless of diet.The second study analyzed the effect of genetic type on glutamate transporter and metabolizing enzyme expression in liver, kidney, longissimus dorsi muscle (LD muscle), and subcutaneous fat (Sub Q Fat) tissue of 18 wether lambs of three genetic types: PP, 1/2 D, and 15 /16 White Dorper 1/16 Polypay (15 /16 D). Tissue samples were analyzed for protein and mRNA content of GS, GDH, ALT, EAAC1, and GLT-1. Glutamate transport and metabolism capacity was lowest for the heavier muscled 15 /16 D lambs.The results suggest genetic type has an effect on N metabolism due to differential expression of glutamate transporters and metabolizing enzymes.

The body encounters an innumerable amount of foreign substances, termed xenobiotics, which it must remove in order to prevent damage to cells and organs. This system of removal is a collection of processes known as ADME (absorption, distribution, metabolism, and excretion). The dynamics of ADME ultimately determine the fate, or pharmacokinetics, of a xenobiotic in the body whether it be an administered pharmaceutical or a potentially harmful toxicant. The major cellular effectors of ADME are the drug metabolizing enzymes (DMEs) and transporters. DMEs function to transform xenobiotics into a metabolite that is more suitable for excretion, whereas drug transporters serve a two-fold function. They may facilitate the uptake of the xenobiotic into the cell so that it can be acted upon by DMEs, or they may function to actively secrete xenobiotics and metabolites from the cell, encouraging their removal from the body. Any perturbations in the expression or function of these critical cellular effectors can result in the diminished therapeutic effect of a pharmaceutical via accelerated removal from the body, or increased toxicity of a pharmaceutical or toxicant due to retention in the body and increased exposure.Perturbations in the ADME processes may result in adverse drug reactions (ADRs) which are an unintended response to a pharmaceutical when administered at the recommended dose. In the last reporting year, the USFDA documented 471,291 serious ADRs causing hospitalization or permanent disabilities, of which 82,724 resulted in death. ADRs can be categorized as two types: dose-related ADRs, and those that are generally unpredictable and mostly occur in susceptible individuals. The major factors that make a person susceptible to ADRs are genetics and disease; however, genetics account for only a small proportion. This dissertation is focused on the contribution of an environmentally-derived component, particularly liver disease, to the occurrence of ADRs. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease of industrialized nations. It represents a spectrum of damage progressing to the severe stage of nonalcoholic steatohepatitis (NASH), and is closely related to obesity and type 2 diabetes. The following studies have determined the effect of NAFLD and NASH on DMEs and transporters, and demonstrated the propensity for NASH to result in serious ADRs.

Les connaissances sur les risques liés à l'inhalation de mycotoxines restent limitées, alors que ces toxines, présentes au niveau des spores fongiques, peuvent atteindre directement les épithéliums respiratoires. Les enzymes du métabolisme des xéniobiotiques pulmonaires peuvent moduler la toxicité de ces mycotoxines en les détoxifiant ou en formant des métabolites plus réactifs. Notre objectif était d'étudier in vitro le métabolisme pulmonaire de la stérigmatocystine et de la méthoxy-stérigmatocystine, deux mycotoxines présentes dans les environnements contaminés par les moisissures, et de prédire les éventuelles conséquences toxiques. L'étude du métabolisme en présence de cytochromes P450 exprimés dans des systèmes hétérologues, puis dans un modèle de culture primaire de cellules épithéliales trachéales porcines, a montré que ces mycotoxines étaient oxydées, principalement par les cytochromes P450 1A, et conjuguées par glucucoridation et sulfo-conjugaison. Ces deux toxines semblent principalement détoxifiées au niveau pulmonaire. Si une activation métabolique de la stérigmatocystine est possible, celle-ci semble limitée / Human health effects of inhaled mycotoxins remains poorly documented, despite these toxins are present in fungal spores et can directly reach the airway epithelia. Xenobiotic metabolozing enzymes can modulate lung toxicity of these mycotoxins through detoxification or reactive metabolite formation. Our aim was to study in vitro the metabolism and the cellular toxic consequences of two mycotoxins present in mold-contaminated environments, e.g. sterigmatocystin and methoxy-sterigmatocystin. The metabolism studies using recombinant cytochromes P450 enzymes and porcine tracheal epithelial cell primary cultures, showed that these mycotoxins could be oxidized by cytochrome P450 1A and conjugated by glucucoridation and sulfo-conjugation. Sterigmatocystin and methoxysterigmatocystin seem mainly detoxified in respiratory cells. If a metabolic activation of sterigmatocystin is possible, it seems limited

>Magister Scientiae - MSc / The human arylamine N-acetyltransferase 1 (NAT1) is critical in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid and para-aminobenzoyl glutamate used in clinical therapy of tuberculosis (TB), as well as influencing the balance between detoxification and metabolic activation of these drugs. SNPs in this enzyme are continuously being detected and indicate inter-ethnic and inter-individual variation in the enzyme function. The effect of nsSNPs on the structure and function of proteins are routinely analyzed using SIFT and POLYPHEN-2 prediction algorithms. The false-negative rate of these two algorithms results in as much as 25% of nsSNPs. This study aimed to explore the use of homology modeling including residue interactions, Gibbs free energy change and solvent accessibility as additional evidence for predicting nsSNP effects on enzyme function.This study evaluated the functional effects of 14 nsSNPs identified in a South African mixed ancestry population of which 3 nsSNPs were previously identified in Caucasians. The SNPs were evaluated using structural analysis that included homology modeling, residue interactions, relative solvent accessibility,Gibbs free energy change and sequence conservation in addition to the routinely used nsSNP function prediction algorithms, SIFT and POLYPHEN-2. The structural analysis implemented in this study showed a loss of hydrogen bonds for S259R thereby affecting protein function which contradicts predictions obtained from SIFT and POLYPHEN-2 algorithms. The variant N245I was shown to be neutral but contradicted the predictions from SIFT and POLYPHEN-2. Structural analysis predicted that variant R242M would affect protein stability and therefore NAT1 function in agreement with POLYPHEN-2 predictions but contradicting predictions from SIFT. No structural changes were expected for variant E264K in agreement with predictions obtained from POLYPHEN-2 but contradicting results from SIFT. The functions of the remaining 10 nsSNPs were consistent with those predicted by SIFT and POLYPHEN-2 namely that four variants R117T, E167Q, T193S and T240S do not affect the NAT1 function whereas R166T, F202V, Q210P, D229H, V231G and V235A could affect the enzyme function.This study provided the first evaluation of the functional effects of 11 newly characterized nsSNPs on the NAT1 tuberculosis drug-metabolizing enzyme. The six functionally important nsSNPs predicted by all three methods and the four SNPs with contradictory results will be tested experimentally by creating a SNP construct that will be cloned into an expression vector. These combined computational and experimental studies will advance our understanding of NAT1 structure-function relationships and allow us to interpret the NAT1 genetic polymorphisms in individuals who are slow or fast acetylators. The results, albeit a small dataset demonstrate that the routinely used algorithms are not without flaws and that improvements in functional prediction of nsSNPs can be obtained by close scrutiny of the molecular interactions of wild type and variant amino acids.

No description available.

The effects of diabetes on cytochrome P450 dependent drug metabolizing enzymes have not to be clarified yet. The most widely used animals in these studies have been rats, and information regarding the effects of diabetes on cytochrome P450 dependent procarcinogen/carcinogen metabolism in rabbits is limited. In the present study, we investigated, for the first time, the influence of benzene on liver, kidney and lung microsomal cytochrome P450 dependent drug metabolizing enzyme activities, protein and mRNA levels in diabetic and non-diabetic rabbits. Male New Zealand rabbits were made diabetic by a single dose of alloxan treatment in this study. AST, ALT and LDH enzyme activities in the blood serum and lipid peroxidation in liver microsomes were found to increase in diabetic, benzene treated and benzene treated diabetic rabbits. Besides these, CYP2E1 dependent NDMA N-demethylase and p-nitrophenol hydroxylase activities and CYP2E1 protein level were found to increase in liver and kidney of diabetic and benzene-treated rabbits. The combined effects of benzene and diabetes on these activities and protein level were found to be additive. Although diabetes caused induction of pulmonary CYP2E1 protein level and associated enzyme activities, benzene treatment of rabbits resulted in no change in enzyme activities and protein level in lung. The level of mRNA was investigated by Real-Time PCR. Accordingly, hepatic CYP2E1 mRNA level was increased 6.71-, 10.53- and 12.93-fold in diabetic, benzene treated and benzene treated diabetic rabbits with respect to the control animals. Similarly, renal CYP2E1 mRNA level was found in increase in these rabbits. In addition to CYP2E1, CYP3A6 associated enzyme activity, erythromycin N-demethylase, CYP3A6 protein and mRNA level were found to increase in diabetic rabbit liver and lung. Unlike diabetes, benzene treatment caused suppression of CYP3A6 protein and inhibition of associated enzyme activity in liver. There was no significant change in the erythromycin N-demethylase activity and CYP3A6 level of liver and lung as a result of benzene treatment of diabetic rabbits. Moreover, diabetes induced CYP1A2 protein and mRNA level and CYP1A associated enzyme activities in the rabbit liver. On the other hand, benzene caused statistically insignificant decreases in CYP1A dependent enzyme activities and CYP1A2 protein level in liver. CYP1A associated enzyme activities, CYP1A2 protein and mRNA levels were not changed in the liver of benzene treated diabetics. The results of the present work indicate that both diabetes and benzene stimulate metabolic activation toxic chemicals metabolized by CYP2E1 such as NDMA and benzene by inducing CYP2E1 which results in the formation of increased amounts of reactive metabolites. Application of benzene to diabetic rabbits further elevates expression and activities of the CYP2E1. As a result of additive induction of the CYP2E1 in benzene treated diabetics, further increase the risk of hepatotoxicity produced by toxins may be observed when compared to the separate treatments. This may in turn further potentiate the risk of organ toxicity and mutagenesis in liver and kidney of these subjects. As in the case of CYP2E1, the risk of carcinogenesis due to induction of CYP1A may be increased in diabetic subjects. Moreover, in diabetic and benzene exposed subjects, alteration of drug clearance and clinical drug toxicity may be observed due to induction or suppression of CYP3A.

Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 1314 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.

No description available.

No description available.

No description available.