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.

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