Cancer is the second leading cause of death after cardiovascular diseases in the world. Many of the chemical carcinogens need metabolic activation that catalyzed by cytochrome P450 and Phase II enzymes in order to exert their genotoxic and carcinogenic effects. Hence one possible mechanism is that phenolic compounds may alter anticarcinogenic effects is through an interaction with these enzymes either by the inhibition or activation of certain forms, leading to a reduced production of the ultimate carcinogen. Therefore anti-carcinogen activity of tannic acid, a hydrolyzable plant polyphenol, has a crucial importance to prevent conversion of pro-carcinogens to their carcinogenic form. Tannic acid is produced from secondary metabolism of plants and is found in edible vegetables, fruits and nuts, especially tea, cocoa, coffee and wine. In the present work, modulation of rabbit liver and kidney microsomal P450 dependent aniline 4-hydroxylase, N-nitrosodimethylamine N-demethylase and p-nitrophenol hydroxylase activities and cytosolic phase II enzymes / glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase:1 (NQO1) were studied in the presence of tannic acid at concentrations ranging from 0.5 &micro / M to 150 &micro / M in the reaction medium. The results obtained in this study were shown that tannic acid significantly inhibited the activities of p-nitrophenol hydroxylase, aniline 4-hydroxylase, NDMA N-demethylase, glutathione S-transferase, NAD(P)H:quinine oxidoreductase 1. Tannic acid was found to be the most potent inhibitor of cytosolic glutathione S-transferase with IC50 of 0.33 &micro / M and the least potent inhibitor of microsomal aniline 4-hydroxylase.with IC50 of 60.26 &micro / M. Effect of tannic acid on enzyme activities was further studied for both mode and type of inhibition. For this purpose various concentrations of the substrate were examined at various tannic acid concentrations. Lineweaver-Burk and Dixon plots were then generated from the resulting data sets. The Km value and inhibition constants (KI) were determined from double reciprocal and Dixon plot of the enzyme activity versus substrate and inhibitor concentration, respectively. Tannic acid was shown to be a noncompetitive inhibitor for liver cytosolic GST, NQO1 and microsomal aniline 4- hydroxylase enzymes with KI of 0.3 &micro / M, 41 &micro / M and 54.7 &micro / M, respectively. On the other hand, in kidney tissues, tannic acid was an uncompetitive inhibitor of cytosolic GST, while it was noncompetitive inhibitor for cytosolic NQO1 with a KI of 12.6 &micro / M. These results indicate that tannic acid may modulate cytochrome P450 dependent and Phase II enzymes and influence the metabolic activation of xenobiotics mediated by these enzymes.

Past operational practices at chemical manufacturing facilities and widespread use of synthetic chemicals in agriculture, industry, and military operations have introduced many anthropogenic compounds to the biosphere. Some of them are readily biodegradable as a likely consequence of bacterial evolution of efficient degradation pathways, whereas others are partially degraded or persistent in the environment. Insight about biodegradation mechanisms and distribution of bacteria responsible provide the basis to predict the fate of synthetic chemicals in the environment and to enable bioremediation. The main focus of the research described here encompasses basic science to discover pathways and evolutionary implications of aerobic biodegradation of two specific synthetic chemicals, cis-dichloroethene (cDCE) and diphenylamine (DPA). cDCE is a suspected carcinogen that frequently accumulates due to transformation of perchloroethene and trichloroethene at many contaminated sites. Polaromonas sp. strain JS666 is the only isolate able to use cDCE as the growth substrate, but the degradation mechanism was unknown. In this study, the degradation pathway of cDCE by strain JS666 and the genes involved were determined by using heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. The requirement of oxygen for cDCE degradation and inhibition of cDCE degradation by cytochrome P450 specific inhibitors suggested that cytochrome P450 monooxygenase catalyzes the initial steps of cDCE degradation. The finding was supported by the observation that an E. coli recombinant expressing cytochrome P450 monooxygenase catalyzes the transformation of cDCE to dichloroacetaldehyde and small amounts of the epoxide. Both the transient accumulation of dichloroacetaldehyde in cDCE degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 further support a pathway involving the degradation of cDCE through dichloroacetaldehyde. Molecular phylogeny of the cytochrome P450 gene and organization of neighboring genes suggest that the cDCE degradation pathway evolved in a progenitor capable of degrading dichloroacetaldehyde by the recruitment of the cytochrome P450 monooxygenase gene from alkane assimilating bacteria. The discovery provides insight about the evolution of the aerobic cDCE biodegradation pathway and sets the stage for field applications. DPA has been widely used as a precursor of dyes, pesticides, pharmaceuticals, and photographic chemicals and as a stabilizer for explosives, but little was known about the biodegradation of the compound. Therefore, bacteria able to use DPA as the growth substrate were isolated by selective enrichment from DPA-contaminated sediment and the degradation pathway and the genes that encode the enzymes were elucidated. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well characterized dioxygenases, and 18O2 experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multi-component ring-hydroxylating dioxygenase. Aniline and catechol produced from the initial reaction of DPA degradation are then completely degraded via the common aniline degradation pathway. Molecular phylogeny and organization of the genes involved were investigated to provide insight about the evolution of DPA biodegradation. The fate and transport of toxic chemicals are of a great concern at several historically contaminated sites where anoxic contaminant plumes emerge into water bodies. The release of toxic chemicals to overlying water poses a potential source of environmental exposure. Bench scale studies were conducted to evaluate the impact of biodegradation on the transport of toxic chemicals across the sediment/water interface. These studies demonstrated that substantial populations of bacteria associated with organic detritus at the interface rapidly biodegrade toxic chemicals as they migrate from contaminated sediment to overlying water, suggesting that the natural attenuation processes serve as a remedial strategy for contaminated sediments and protect the overlying water.

Ο α2 –αδρενεργικός υποδοχέας διακρίνεται σε 3 γνωστούς υποτύπους (α2Α, α2Β, α2C) και μετά αλληλεπίδραση με G-πρωτεΐνες (GPCRs), διαμεσολαβεί μέρος των δράσεων των ορμονών- νευρομεταβιβαστών, επινεφρίνη και νορεπινεφρίνη σε πολλά όργανα, συμπεριλαμβανομένου και του νευρικού συστήματος. Πρότυπο μελέτης του νευρικού συστήματος in vitro, αποτελεί η κυτταρική σειρά PC12, που περιλαμβάνει κύτταρα από φαιοχρωμοκύττωμα αρουραίου, τα οποία υπό την επίδραση του Nerve Growth Factor (NGF) διαφοροποιούνται σε συμπαθητικούς νευρώνες. Μετά από διαμόλυνση, αυτά τα κύτταρα εκφράζουν τους υποτύπους των α2-αδρενεργικών υποδοχέων και βάσει δεδομένων από προηγούμενη εμπειρία του εργαστηρίου μας, μπορούν μετά από ενεργοποίηση με επινεφρίνη να οδηγήσουν στην ενεργοποίηση ενός καταρράκτη μεταγωγής σήματος, που περιλαμβάνει τις κινάσες Akt και ERK1/2. Δεδομένου ότι τα μόρια αυτά συμβάλλουν στην ενεργοποίηση του μεταγραφικού παράγοντα CREB (cAMP response element binding protein) θελήσαμε στην παρούσα εργασία να διερευνήσουμε κατά πόσο η ενεργοποίηση των α2-αδρενεργικών υποδοχέων προκαλεί την CREB φωσφορυλίωση. Βάσει προηγούμενων αποτελεσμάτων, που αποδείκνυαν την απελευθέρωση αραχιδονικού οξέος και διαφόρων μεταβολιτών του μετά από ενεργοποίηση των α2 –αδρενεργικών υποτύπων, μελετήσαμε εάν αυτή η απελευθέρωση αραχιδονικού οξέος, μπορούσε να προκαλέσει ενεργοποίηση μέσω φωσφορυλίωσης του CREB και μέσω ποιών μεταβολικών μονοπατιών μπορεί αυτό να πραγματοποιηθεί. Επιπλέον μελετήσαμε εάν αυτή η ενεργοποίηση του CREB ήταν παρούσα και στους 3 υποτύπους και εάν παρουσίαζε υποτυποειδικότητα. Χρησιμοποιήσαμε την τεχνική Western Blotting , σε εκχυλίσματα PC12 κυττάρων, κατάλληλα επεξεργασμένων με επινεφρίνη, παρουσία διαφόρων αναστολέων των μεταβολικών μονοπατιών του αραχιδονικού οξέος. Τα αποτελέσματά μας δείχνουν ότι η επινεφρίνη επάγει τη φωσφορυλίωση του CREB και στους 3 υποτύπους των α2-αδρενεργικών υποδοχέων σε PC12 κύτταρα. Επίσης η απελευθέρωση αραχιδονικού οξέος και η επακόλουθη φωσφορυλίωση του CREB διαμεσολαβείται από την PLC (φωσφολιπάση C) και την εποξυγενάση του κυτοχρώματος P450, αφού έχουμε αναστολή από τους ειδικούς αναστολείς U73122 και κετοκοναζόλη αντίστοιχα. Τα επίπεδα φωσφορυλίωσης ήταν ίδια στους α2A- και α2C-υποτύπους και σημαντικά μεγαλύτερα από τον α2Β-υπότυπο, αποδεικνύοντας ότι παρουσιάζεται σημαντική υποτυποειδικότητα. / α2-adrenergic receptor is divided into 3 known subtypes (α2A, α2Β, α2C) and after interaction with G-proteins (GPCRs) mediates part of actions of hormones-neurotransmitters, epinephrine and nor epinephrine in many organs, including Central Nervous System. Cell line PC12, which origins from cells of rats’ pheochromocytoma , consist a study model of nervous system in vitro and under the influence of Nerve Growth Factor (NGF) is differentiated into sympathetic neurons. After transfection, these cells express the subtypes of α2-adrenergic receptors and based on data from previous experience, after activation with epinephrine, they activate a cascade of signal transduction , which includes kinases Akt and ERK1/2. Based on the fact that these molecules contribute to the activation of transcription factor CREB (cAMP response element binding protein) we study whether the activation of α2-adrenergic receptors can cause direct CREB phosphorylation. Based on previous results, which prove release of arachidonic acid and its metabolites after activation of α2-adrenergic subtypes, we study if the release of arachidonic acid could cause activation, through phosphorylation, of CREB and via which metabolic pathways this happens. Furthermore, we studied if the activation was present in all 3 subtypes and if it presented sub-specificity. We performed the Western Blotting technique in PC12 cells properly pro-incubated with epinephrine and addition of enzymic inhibitors of arachidonic acid metabolism. Our results figure that epinephrine induce CREB phosphorylation in all 3 subtypes of α2-adrenergic receptors in PC12 cells. The release of arachidonic acid and the following phosphorylation of CREB is mediated from phospholipase C (PLC) and cytochrome P450-dependent epoxygenase, as proved by inhibition with the specific inhibitors U73122 and ketokonazole, respectively. The levels of CREB phosphorylation were comparable between α2Α - and α2C- subtypes and higher than the α2Β- subtype, proving that this is an action which presents sub-specificity.

No description available.

Le CS fait partie de la famille des SYSADOA (SYmptomatic Slow Acting Drugs for OsteoArthritis) et est utilisé par les patients avec de l’ostéoarthrose de façon chronique pour ses propriétés anti-inflammatoires. Étant donné que ces patients reçoivent d’autres médicaments, il était intéressant de documenter les effets du CS sur le cytochrome P450 et la NADPH-réductase (NADPH). Pour cette étude, deux modèles ont été utilisés: des lapins témoins (LT) et des lapins avec une réaction inflammatoire (LRI) afin de diminuer l’activité et l’expression du CYP. Six groupes contenant chacun cinq lapins ont été utilisés: un groupe sans CS et deux groupes qui ont pris oralement dans l’eau approximativement 20.5 mg/kg/jour de CS pendant 20 et 30 jours; les lapins des trois groupes restants ont pris du CS comme décrit plus haut, mais ont reçu 5 ml sous-cutanées de térébenthine afin de produire une réaction inflammatoire aseptique (RIA) deux jours avant leur sacrifice, c’est-à-dire aux jours -2, 18 et 28. Les hépatocytes ont été isolés pour évaluer l’activité et l’expression du CYP3A6, CYP1A2 et NADPH et aussi le ARNm de ces protéines. In vitro, nous avons étudié l’effet de différentes concentrations de CS-disaccharides sulfatés, 4S, 6S, et 4,6S de CS, sur l’activité et l’expression du CYP1A2 et du CYP3A6. Pour documenter la présence de la réaction inflammatoire, nous avons mesure les mucoprotéines, dans le sérum des lapins avec une réaction inflammatoire. Aussi nous avons mesuré la présence de l’oxide nitrique (NO) chez les hépatocytes de lapins contrôles et chez les hépatocytes des lapins avec une réaction inflammatoire. La translocation nucléaire du NF-κB a été etudiée par fluorescence chez les hépatocytes. Par comparaison aux lapins témoins, l’administration du CS pendant 20 et 30 jours n’affecte pas l’activité du CYP3A6 et du CYP1A2. La RIA a augmenté les mucoprotéines à 95,1±5,7 vs 8,4±1,6 mg/dl dans les lapins témoins (p<0,05). La RIA a diminué l’activité du CYP3A6 de 62% et l’activité du CYP 1A2 de 54%. Le CS n’empêché pas la diminution du CYP1A2 produite par la RIA. Par ailleurs, le CS n’affecte pas l’activité ni l’expression de la NADPH. La translocation nucléaire de NF-κB a été empêche par l’administration chronique de CS aux lapins avec RIA; en plus, la concentration de l’oxide nitrique n’a pas démontré une augmentation en présence de CS; par contre, CS n’empêche pas l’augmentation des séromucoïdes. Au contraire, CS affecte la diminution du CYP3A6 en fonction de temps et secondaire à la RIA. Dans ce group, CS a rétabli le niveau des protéines du CYP3A6 observé dans le group de lapins témoins. Pourtant cette croissance été independante de mRNA qui garde un niveau trés bas. Le plus remarcable a été la manière dont CS a augmenté la protéine du CYP3A6, sans avoir rétabli l’activité de cet isoforme. Finalement, in vitro, CS et ses trois disaccharides sulfatés (4S, 6S et 4,6S) n’affectent ni l’activité ni l’expression de CYP1A2, CYP3A6 et de la NADPH. En conclusion, l’administration chronique de CS n’affecte pas l’activité ni l’expression du CYP1A2, ou la diminution du CYP1A2 produite par la réaction inflammatoire. Le CS n’affecte pas l’activité ni l’expression du NADPH. Cependant, CS empêche la diminution du CYP3A6 en fonction de temps et secondaire à la RIA. / In rabbits, an acute inflammatory reaction induced by the injection of turpentine causes a decrease in cytochrome P450 (CYP) isoforms activity and expression. Chondroitin sulfate (CS) is a Symptomatic Slow Acting Drug for OsteoArthritis (SYSADOA) that elicits anti-inflammatory effects. Since patients take CS over long periods, it was of interest to assess whether CS modulates the activity of cytochrome P450 isoforms. In order to determine the effect of CS on the cytochrome P450, CS was administered in vivo to two animal models, e.g. chronic intake of CS in control rabbits, and chronic intake of CS in rabbits with a CYP down-regulated by an inflammatory reaction (IR). We used six groups of five rabbits: three to assess the effect of CS on cytochrome P450, one without CS and two receiving orally about 20 mg/kg/day CS for 20 and 30 days; and the remaining three groups of rabbits received turpentine s.c. to generate an aseptic IR (AIR) 48 h before their sacrifice, e.g. days -2, 18 and 28, while exposed to CS for 0, 20 or 30 days, respectively. In order to verify the presence of inflammation we measured the seromucoids in serum of rabbits with an AIR. Another marker of inflammation, e.g. nitric oxyde (NO.) production, was assessed in control hepatocytes (Hcont) and in hepatocytes from rabbits with an AIR (Hinfla). In addition, the effect of CS on the nuclear translocation of NF-κB was studied by fluorescence in hepatocytes. Finally, in hepatocytes (both Hcont and Hinfla) the CYP3A6, CYP1A2 and NADPH P450 reductase (NADPH) activity, expression and mRNA were measured. In vitro, the effect of different concentrations of CS, 4S-, 6S- and 4,6S-sulfated disaccharides of CS on the cytochrome P450 was documented. Compared with control rabbits, 20 and 30 days CS did not affect the activity of CYP3A6 and CYP1A2. The AIR increased seromucoids from 8.4±1.6 mg/dl in controls to 95.1±5.7 (p<0.05), as well as the nuclear translocation of NF-κB, and nitric oxide concentrations. The AIR reduced CYP3A6 activity by 62% and CYP1A2 activity by 54%, decrease associated to a reduction in protein expression and in mRNA, e.g. pre-transcriptional down-regulation. The nuclear translocation of NF-κB was prevented by the administration of CS to rabbits with an AIR, moreover CS impeded the increase of the concentrations of nitric oxide; however CS did not prevent the increase in seromucoids. CS did not prevent the down-regulation of CYP1A2 produced by the inflammatory reaction. CS prevented the time-dependent down-regulation of CYP3A6 in control rabbits and in rabbits with an inflammatory reaction. In this last group, CS restored the amounts of CYP3A6 protein to levels observed in control rabbits, however this increase was independent of the mRNA that remained very depressed. It is noteworthy that even if CS increased CYP3A6 protein, its activity was not recovered. CS did not affect NADPH activity or expression. Finally, in vitro, CS, 4S-, 6S and 4,6S-sulfated disaccharides of CS did not change the activity and expression of the two isoforms of CYP, and of NADPH. It is concluded that CS does not affect the activity or expression of CYP1A2, nor prevents CYP1A2 AIR-induced down-regulation. However, CS prevents the down-regulation of CYP3A6 time dependently and following the AIR but does not prevent the decrease of catalytic activity.

Pagrindinis šio darbo tikslas buvo kiekybinio struktūros ir aktyvumo ryšio modelių, prognozuojančių su vaistų metabolizmu susijusias savybes, kūrimas. Modeliai, prognozuojantys CYP3A4 slopinimą ir žmogaus kepenų mikrosomų katalizuojamo metabolizmo regioselektyvumą, buvo sukurti naudojant GALAS (angl. Global, Adjusted Locally According to Similarity; Globalus, lokaliai pakoreguotas pagal panašumą) modeliavimo metodą, kuris geba įvertinti prognozės patikimumą, taip apibrėždamas modelio pritaikymo sritį. Sukurtų modelių prognozės buvo tikrinamos naudojant eksperimentinius naujų cheminių junginių duomenis. Visų globalių modelių prognozės gerėjo po korekcijų pagal panašumą, o neteisingų spėjimų skaičius buvo ženkliai mažesnis tarp aukšto patikimumo prognozių. Visgi daugiau nei pusė išorinių duomenų nepatenka į šių modelių pritaikymo sritį. GALAS modeliai gali būti gana paprastai apmokomi, pridedant naujus duomenis į lokalią modelio dalį ir apskaičiuojant reikiamą korekciją. Po tokios apmokymo procedūros CYP3A4 slopinimo modelis prisitaikė prie PubChem duomenų bazės cheminių junginių ir taip pat prie vaistų, turinčių naują cheminį karkasą. Pridėjus naujų junginių ir apmokius regioselektyvumo modelį, jis pradėjo prognozuoti naujas metabolizmo vietas. Pastarasis modelis taip pat buvo pritaikytas atskirų fermentų katalizuojamo metabolizmo prognozavimui. / The main objective of this study was the development of QSAR models for drug metabolism-related properties. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling method was used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. Models predicting CYP3A4 inhibition and regioselectivity of metabolism in human liver microsomes were developed and validated using external test sets. In all cases the baseline models already showed acceptable results, and the overall accuracy of predictions increased after the similarity based corrections. Moreover, the numbers of mispredictions reduced significantly when only results of higher reliability were taken into account. However, the original models are applicable only for less than a half of external datasets. Since the similarity correction procedure of GALAS modeling method allows simple model training, the possibility to expand the applicability domain has been tested. The CYP3A4 inhibition model was successfully adapted to PubChem data and compounds with a novel chemical scaffold. After training the regioselectivity model new metabolism sites could be identified in compounds of new chemical class. Moreover, this model was adapted for human cytochrome P450 isoform profiling.

The main objective of this study was the development of QSAR models for drug metabolism-related properties. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling method was used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. Models predicting CYP3A4 inhibition and regioselectivity of metabolism in human liver microsomes were developed and validated using external test sets. In all cases the baseline models already showed acceptable results, and the overall accuracy of predictions increased after the similarity based corrections. Moreover, the numbers of mispredictions reduced significantly when only results of higher reliability were taken into account. However, the original models are applicable only for less than a half of external datasets. Since the similarity correction procedure of GALAS modeling method allows simple model training, the possibility to expand the applicability domain has been tested. The CYP3A4 inhibition model was successfully adapted to PubChem data and compounds with a novel chemical scaffold. After training the regioselectivity model new metabolism sites could be identified in compounds of new chemical class. Moreover, this model was adapted for human cytochrome P450 isoform profiling. / Pagrindinis šio darbo tikslas buvo kiekybinio struktūros ir aktyvumo ryšio modelių, prognozuojančių su vaistų metabolizmu susijusias savybes, kūrimas. Modeliai, prognozuojantys CYP3A4 slopinimą ir žmogaus kepenų mikrosomų katalizuojamo metabolizmo regioselektyvumą, buvo sukurti naudojant GALAS (angl. Global, Adjusted Locally According to Similarity; Globalus, lokaliai pakoreguotas pagal panašumą) modeliavimo metodą, kuris geba įvertinti prognozės patikimumą, taip apibrėždamas modelio pritaikymo sritį. Sukurtų modelių prognozės buvo tikrinamos naudojant eksperimentinius naujų cheminių junginių duomenis. Visų globalių modelių prognozės gerėjo po korekcijų pagal panašumą, o neteisingų spėjimų skaičius buvo ženkliai mažesnis tarp aukšto patikimumo prognozių. Visgi daugiau nei pusė išorinių duomenų nepatenka į šių modelių pritaikymo sritį. GALAS modeliai gali būti gana paprastai apmokomi, pridedant naujus duomenis į lokalią modelio dalį ir apskaičiuojant reikiamą korekciją. Po tokios apmokymo procedūros CYP3A4 slopinimo modelis prisitaikė prie PubChem duomenų bazės cheminių junginių ir taip pat prie vaistų, turinčių naują cheminį karkasą. Pridėjus naujų junginių ir apmokius regioselektyvumo modelį, jis pradėjo prognozuoti naujas metabolizmo vietas. Pastarasis modelis taip pat buvo pritaikytas atskirų fermentų katalizuojamo metabolizmo prognozavimui.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is believed to play a role in human lung cancer induced by tobacco smoking. NNK biotransformation may involve the enzymes prostaglandin H synthase (PHS)-1, PHS-2 and cytochrome 450 (CYP) 2F. PHS activity is thought to be important in extrahepatic tissues, where CYP activity is low. The CYP2F subfamily contains a single functional enzyme in humans (CYP2F1) and goats (CYP2F3); these enzymes are preferentially expressed in the lung, with little or no expression in other organs. The role of these enzymes in the pulmonary biotransformation of NNK was investigated. 4.2 µM [5-3H]NNK was incubated with human lung microsomes under NADPH-dependent and arachidonic acid-dependent conditions. Metabolites reflective of NNK α-carbon hydroxylation, N-oxidation and carbonyl reduction were detected in the presence of NADPH, and metabolite levels for all three biotransformation pathways were lower in the presence of arachidonic acid compared with NADPH (p<0.05, N=4). Incubation of microsomes with the PHS-1 selective inhibitor SC-560 and the PHS-2 selective inhibitor NS-398 did not change NNK biotransformation either in the presence of NADPH or in the presence of arachidonic acid (p>0.05, N=4). Incubation of [5-3H]NNK with ovine PHS-1 or PHS-2 did not result in formation of α-carbon hydroxylation or N­-oxidation metabolites; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was measurable only in the presence of PHS-2. Incubation of goat recombinant CYP2F3 with [5-3H]NNK resulted in formation of keto acid, keto alcohol and NNK-N-oxide (65.0%, 17.5% and 30.0% (µmol enzyme)-1 minute-1, respectively). Metabolite formation was inhibited by 3-methylindole (3-MI), a mechanism-based inactivator of CYP2F3. Based on an N value of 3, incubation of human lung microsomes with 3-MI inhibited N-oxidation (p<0.05) but did not alter NNK bioactivation or carbonyl reduction (p>0.05). However, when metabolite formation was examined in lung microsomes from different individuals, decreases in NNK biotransformation (ranging from 19.6 to 68.5%) were observed and were more pronounced in some patients than others, suggesting inter-individual variability in CYP2F1 activity. These studies demonstrate the ability of CYP2F to biotransform NNK and suggest inter-individual variability in the importance of CYP2F1 for this activity in human lung. They also strongly argue against the involvement of PHS enzymes. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2007-12-30 16:12:58.228

Alkylphenanthrenes such as retene (7-isopropyl-1-methylphenanthrene) are aquatic contaminants commonly found in anthropogenically-, industrially-, and petroleum-contaminated environments, and have been implicated in crude oil toxicity. In the early life stages (ELS) of fish, exposures to alkylphenanthrenes produce signs of toxicity typical of those observed in exposures to halogenated aromatic hydrocarbons, particularly to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD, the most toxic congener, serves as the basis of the current mechanism-based risk assessment model. The model assumes that congeners that produce TCDD-like toxicity share a common mode of action and act additively. The mechanism of TCDD-like toxicity is assumed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in the xenobiotic response (e.g., induction of cytochrome P450 1A enzymes; CYP1A) and in normal development. CYP1A enzymes are not involved in the mechanism of TCDD toxicity. Alkylphenanthrenes toxic to the ELS of fish are AhR ligands, but in contrast to TCDD, are readily metabolized by CYP1A enzymes. The byproducts of CYP1A metabolism have been implicated in retene toxicity. However, the target tissue of retene and the direct roles of AhR and CYP1A in retene toxicity are unknown, but are expected to be similar to those of TCDD. The results presented in this thesis suggest that in the ELS of fish: (1) the primary target of retene is the cardiovascular system (Chapters 2 & 5); (2) retene toxicity is stage-specific (Chapter 2); (3) the mechanism of retene toxicity is mediated by AhR2, and is independent of CYP1A enzymes (Chapter 5); (4) multiple CYP1A-independent toxicities can result from exposures to different mixtures of CYP1A inducing (retene) and CYP1A inhibiting (alpha-naphthoflavone or 2-aminoanthracene) PAHs (Chapters 3 & 4); and (5) multiple concentration-dependent mechanisms of toxicity (i.e., synergism and response addition) can occur in co-exposures of a CYP1A inducer (retene) with a range of CYP1A inhibitor (alpha-naphthoflavone) concentrations (Chapter 3). Thus, retene toxicity is mechanistically similar to that of TCDD toxicity, suggesting alkylphenanthrenes can be included in the current risk assessment model. However, the observed variable mixture toxicities and species differences in retene toxicity raise questions about the effectiveness of this model. / Thesis (Ph.D, Biology) -- Queen's University, 2009-01-13 12:10:31.373

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.