INTRODUCTION: Smoking is one of the single greatest causes of numerous preventable diseases. We were interested in developing an animal model of nicotine metabolism that can be used to examine the effects of potential CYP2A6 inhibitors on nicotine metabolism and nicotine-mediated behaviours. Pharmacogenetic studies have demonstrated that in humans, smoking behaviour is associated with rates of nicotine metabolism by the CYP2A6 enzyme. Mouse CYP2A5 shares structural and functional similarities to human CYP2A6 and has been implicated in nicotine self-administration behaviours in mice, therefore the mouse represents a potential animal model for studying nicotine metabolism. METHODS: We characterized nicotine and cotinine metabolism in two commonly used mouse strains (DBA/2 and C57Bl/6). We also examined the association between nicotine self-administration behaviours and nicotine metabolism, and the impact of direct manipulation (i.e. inhibition) of nicotine metabolism on nicotine pharmacodynamics (hot-plate and tail-flick tests) in mice. Finally, we studied the effect of selegiline (a known cytochrome P450 mechanism-based inhibitor) on nicotine metabolism in mice and in human CYP2A6. RESULTS: Nicotine metabolism in mice in vitro was mediated by CYP2A5, and this enzyme was responsible for over 70% and 90% of the metabolism of nicotine to cotinine and cotinine to 3-hydroxycotinine as shown by immuno-inhibition studies, respectively. A polymorphism in CYP2A5 between mouse strains, known to alter the probe substrate coumarin’s metabolism, did not affect nicotine metabolism but dramatically altered cotinine metabolism. Nicotine self-administration behaviour in mice was associated with level of hepatic CYP2A5 proteins and rates of nicotine metabolism in male mice. In inhibition studies, the CYP2A5/6 inhibitor methoxsalen inhibited both in vitro and in vivo nicotine metabolism in mice and substantially increased the anti-nociceptive effect of nicotine. Finally, selegiline was found to be an inhibitor of CYP2A5 decreasing nicotine metabolism in vitro and in vivo in mice. Moreover, we showed that selegiline is a mechanism-based inhibitor of CYP2A6 inhibiting nicotine metabolism irreversibly. CONCLUSION: The above data suggested that the mouse model may be suitable for examining the impact of inhibition (and genetic variation) on nicotine metabolism and nicotine-mediated behaviours and may potentially be used to screen for novel inhibitors of nicotine metabolism.

INTRODUCTION: Smoking is one of the single greatest causes of numerous preventable diseases. We were interested in developing an animal model of nicotine metabolism that can be used to examine the effects of potential CYP2A6 inhibitors on nicotine metabolism and nicotine-mediated behaviours. Pharmacogenetic studies have demonstrated that in humans, smoking behaviour is associated with rates of nicotine metabolism by the CYP2A6 enzyme. Mouse CYP2A5 shares structural and functional similarities to human CYP2A6 and has been implicated in nicotine self-administration behaviours in mice, therefore the mouse represents a potential animal model for studying nicotine metabolism. METHODS: We characterized nicotine and cotinine metabolism in two commonly used mouse strains (DBA/2 and C57Bl/6). We also examined the association between nicotine self-administration behaviours and nicotine metabolism, and the impact of direct manipulation (i.e. inhibition) of nicotine metabolism on nicotine pharmacodynamics (hot-plate and tail-flick tests) in mice. Finally, we studied the effect of selegiline (a known cytochrome P450 mechanism-based inhibitor) on nicotine metabolism in mice and in human CYP2A6. RESULTS: Nicotine metabolism in mice in vitro was mediated by CYP2A5, and this enzyme was responsible for over 70% and 90% of the metabolism of nicotine to cotinine and cotinine to 3-hydroxycotinine as shown by immuno-inhibition studies, respectively. A polymorphism in CYP2A5 between mouse strains, known to alter the probe substrate coumarin’s metabolism, did not affect nicotine metabolism but dramatically altered cotinine metabolism. Nicotine self-administration behaviour in mice was associated with level of hepatic CYP2A5 proteins and rates of nicotine metabolism in male mice. In inhibition studies, the CYP2A5/6 inhibitor methoxsalen inhibited both in vitro and in vivo nicotine metabolism in mice and substantially increased the anti-nociceptive effect of nicotine. Finally, selegiline was found to be an inhibitor of CYP2A5 decreasing nicotine metabolism in vitro and in vivo in mice. Moreover, we showed that selegiline is a mechanism-based inhibitor of CYP2A6 inhibiting nicotine metabolism irreversibly. CONCLUSION: The above data suggested that the mouse model may be suitable for examining the impact of inhibition (and genetic variation) on nicotine metabolism and nicotine-mediated behaviours and may potentially be used to screen for novel inhibitors of nicotine metabolism.

Excessive alcohol consumption is the primary contributing factor in the development of alcoholic liver diseases (ALD). Nicotine contained in tobacco is a major addictive alkaloid, which enhances the effects of ALDs. The major enzyme involved in nicotine metabolism is cytochrome P450 2A5 (CYP2A5) which is produced in the liver. Alcohol can stimulate the CYP2A5 enzyme. We utilized cyp2a5-/- knockout mice in this research to examine the effects of CYP2A5. The cyp2a5-/- mice and wild-type (WT) mice were fed liquid ethanol diet with or without nicotine to induce ALD. Nicotine enhancing effects on ALD were observed in WT mice but not in cyp2a5-/- mice. Oxidative stress was stimulated by alcohol and further increased by nicotine in WT mice but not in cyp2a5-/-mice. Microsomal ROS production during microsomal metabolism of nicotine was increased in WT mice but not in cyp2a5-/-mice. These results suggest that nicotine enhances ALD is CYP2A5 dependent.

<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression. The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO2) and the reasons for the strikingly different toxicity were investigated. A strong bioactivation and protein adduction of 2,6-diClPh-MeSO2 in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO2 was observed and the bioactivation in the liver for both chlorinated isomers was negligible. In vitro studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO2. The 2,6-diClPh-MeSO2-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated in vivo, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. Based on these results, the proposed mechanism for 2,6-diClPh-MeSO2-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2.

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

Abstract The hepatic inducible Cytochrome P450s (CYPs) generally prime xenobiotics for elimination. Murine CYP2A5 and human CYP2A6 share similar xenobiotic substrates and some regulatory features. Recently, they were shown to oxidize bilirubin, a byproduct of heme catabolism and a dose-dependent anti- or pro-oxidant, to biliverdin. In this study, the putative role of the redox-sensitive, cytoprotective transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the regulation of hepatic Cyp2a5 expression and induction under diverse hepatotoxic conditions and altered heme homeostasis was characterized. The coordination of Cyp2a5 and the Nrf2 target gene Heme oxygenase-1 (Hmox1), which determines bilirubin formation from heme, responses to heavy metals and modulators of heme homeostasis, was studied in cultured wildtype and Nrf2(-/-) mouse primary hepatocytes. Nrf2 was essential for the basal hepatic expression of CYP2A5 in the endoplasmic reticulum (ER) and mitochondria, as well as for its induction by cadmium, lead, methyl mercury and phenethyl isothiocyanate. A functional Nrf2 binding antioxidant response element (ARE) about -2.4 kilobases upstream of the Cyp2a5 transcriptional start site was identified. In contrast to Hmox1, a target of BTB and CNC homology 1 (Bach)-mediated repression via AREs, the regulation of Cyp2a5 did not clearly involve Bach1. Excessive heme induced mainly ER-localized CYP2A5 via Nrf2, which was limited by the Nrf2-independent HMOX1 induction. In heme synthesis blockades, CYP2A5 was enhanced via Nrf2 and additional factors, such as the peroxisome proliferator-activated receptor &#947; coactivator-1&#945; (PGC-1&#945;). The typical CYP2A5 inducers phenobarbital, dibutyryl-cyclic adenosine monophosphate (db-cAMP) and PGC-1&#945; enhance heme synthesis; CYP2A5 was induced via Nrf2 in acute but not chronic phenobarbital exposure without a clear connection to heme, while the responses to db-cAMP and PGC-1&#945; were sensitized in the absence of Nrf2. This suggests novel crosstalk between Nrf2 and PGC-1&#945;. In this study, Cyp2a5 was identified as a sensitive indicator of hepatic Nrf2 pathway activation that could be used, e.g. for in vitro screening of drug candidate hepatotoxicity. The similar subcellular localization and coordination of CYP2A5 and HMOX1 expression in altered heme metabolism support the postulated role for CYP2A5 in bilirubin homeostasis. / Tiivistelmä Vierasaineet stimuloivat maksan Sytokromi P450 (CYP)-entsyymejä, mikä yleensä lisää niiden eliminaatiota. Hiiren CYP2A5 ja ihmisen CYP2A6 ovat lähisukua katalyyttisten ja osin säätelyllisten yhteneväisyyksiensä puolesta. Vastikään niiden osoitettiin katalysoivan hemin hajoamistuotteen, bilirubiinin hapettumista biliverdiiniksi, mikä saattaisi säädellä sen annosriippuvaisia vaikutuksia antioksidanttina ja oksidanttina. Työssä tutkittiin solustressiä aistivan, suojaavan transkriptiotekijän Nrf2 osuutta Cyp2a5-geenin aktivaatiossa maksatoksisissa olosuhteissa ja hemimetabolian muutoksissa. Cyp2a5:n ja bilirubiinin tuotosta vastaavan, Nrf2-säädellyn Hemioksigenaasi-1 (Hmox1):n vasteita verrattiin viljellyissä villityypin ja poistogeenisen Nrf2(-/-) hiiren primaarimaksasoluissa. Tulokset osoittavat, että Nrf2 ylläpitää CYP2A5:n ilmentymistä endoplasmisella kalvostolla (ER) ja mitokondrioissa sekä välittää sen stimulaation altisteilla kadmium, lyijy, metyylielohopea ja fenetyyli-isotiosyanaatti. Toimiva Nrf2-vasteinen antioksidanttivaste-elementti (ARE) tunnistettiin n. -2,4 kiloemäsparia Cyp2a5-geenin luennan aloituskohdasta ylävirtaan. BTB ja CNC homologia 1 (Bach1)-tekijä, joka on tärkeä Hmox1-säätelijä ja ARE-välitteinen transkription estäjä, ei selkeästi osallistu Cyp2a5:n säätelyyn. Hemin ylimäärä stimuloi CYP2A5:n määrää ER-kalvostolla, Nrf2-riippumattomasti stimuloituvan HMOX1 rajoittaessa Nrf2-reitin aktivaatiota. Hemisynteesin estyessä Nrf2 aktivoi Cyp2a5-geeniä muiden mekanismien kuten peroksisomiproliferaattori-aktivoituva reseptori gamman koaktivaattori-1&#945; (PGC-1&#945;) kanssa. Fenobarbitaali (PB), dibutyryyli-syklinen adenosiinimonofosfaatti (db-cAMP) ja PGC-1&#945; lisäävät tunnetusti hemisynteesiä. Nrf2 havaittiin Cyp2a5:n aktivaatiolle välttämättömäksi akuutissa mutta ei kroonisessa PB-altistuksessa ilman selkeästi havaittua hemin osuutta. Cyp2a5-geenin db-cAMP- ja PGC-1&#945;-vasteinen stimulaatio voimistui merkittävästi toimivan Nrf2-reitin puuttuessa, mikä osoittaa vuoropuhelua Nrf2 ja PGC-1&#945; välillä. Väitöskirjatyössä Cyp2a5 tunnistettiin herkäksi Nrf2-reitin aktivaation maksamarkkeriksi, jota voitaisiin hyödyntää esim. lääkeainekandidaattien maksatoksisuuden seulonnassa soluviljelyssä. CYP2A5:n ja HMOX1:n solunsisäinen kohdentuminen ja ekspressio koordinoituvat hemimetabolian muutoksissa, mikä tukee teoriaa CYP2A5:n roolista bilirubiinin metaboliassa maksassa.

Alcohol consumption causes liver diseases, designated as Alcoholic Liver Disease (ALD). Because alcohol is detoxified by alcohol dehydrogenase (ADH), a major ethanol metabolism system, the development of ALD was initially believed to be due to malnutrition caused by alcohol metabolism in liver. The discovery of the microsomal ethanol oxidizing system (MEOS) changed this dogma. Cytochrome P450 enzymes (CYP) constitute the major components of MEOS. Cytochrome P450 2E1 (CYP2E1) in MEOS is one of the major ROS generators in liver and is considered to be contributive to ALD. Our labs have been studying the relationship between CYP2E1 and ALD for many years. Recently, we found that human CYP2A6 and its mouse analog CYP2A5 are also induced by alcohol. In mice, the alcohol induction of CYP2A5 is CYP2E1-dependent. Unlike CYP2E1, CYP2A5 protects against the development of ALD. The relationship of CYP2E1, CYP2A5, and ALD is a major focus of this review.