RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Incidence of Postoperative Pain after Single Visit and Two Visit Root Canal Therapy: A Randomized Controlled Trial.

INTRODUCTION: Root Canal Treatment (RCT) has become a mainstream procedure in dentistry. A successful RCT is presented by absence of clinical signs and symptoms in teeth without any radiographic evidence of periodontal involvement. Completing this procedure in one visit or multiple visits has long been a topic of discussion. AIM: To evaluate the incidence of postoperative pain after root canal therapy performed in single visit and two visits. MATERIAL AND METHODS: An unblinded/ open label randomized controlled trial was carried out in the endodontic department of the Dental Institute, where 78 patients were recruited from the regular pool of patients. A total of 66 maxillary central incisors requiring root canal therapy fulfilled the inclusion and exclusion criteria. Using simple randomization by biased coin randomization method, the selected patients were assigned into two groups: group A (n=33) and group B (n=33). Single visit root canal treatment was performed for group A and two visit root canal treatment for group B. Independent sample t-test was used for statistical analysis. RESULTS: Thirty three patients were allotted to group A where endodontic treatment was completed in single visit while 33 patients were allotted to group B where endodontic treatment was completed in two visits. One patient dropped-out from Group A. Hence in Group A, 32 patients were analysed while in Group B, 33 patients were analysed. After 6 hours, 12 hours and 24 hours of obturation, pain was significantly higher in Group B as compared to Group A. However, there was no significant difference in the pain experienced by the patients 48 hours after treatment in both the groups. CONCLUSION: Incidence of pain after endodontic treatment being performed in one-visit or two-visits is not significantly different.

Non-templated hydrothermal growth of anisotropic magnetite nanostructures using hexamine as the directing agent.

Anisotropic growth of magnetite (Fe3O4) nanoparticles is achieved in a hydrothermal growth process using hexamine to play a dual role of oxide forming and directing agent. The samples are characterized by X-ray diffraction, scanning electron microscopy, high resolution transmission electron microscopy, squid magnetometry, ferromagnetic resonance technique, diffuse reflectance spectroscopy and Mössbauer spectroscopy, which collectively establish the formation of Fe3O4 phase. Anisotropic structures such as nanorods and nanotubules are revealed and these are shown to exhibit good humidity sensing properties.

Invasive cervical resorption: a case report.

Invasive cervical resorption (ICR) is a relatively uncommon form of external resorption, which may occur in any tooth in the permanent dentition. Characterized by its cervical location and invasive nature, this resorptive process leads to progressive and usually destructive loss of the tooth structure, the clinical features of which often resemble internal resorption ("pink tooth"). This article describes a case report of ICR and its management. The salient features were a large resorptive defect and localized fibrous in-growth located almost wholly on the cervicolabial aspect of the maxillary incisor crown involving the enamel and dentin.

Clinical evaluation of postoperative sensitivity in composite resin restorations using various liners.

Light-cured resin composites are a viable option for restoring Class I and Class II cavities; however, several researchers have reported postoperative sensitivity in such restorations. In this in vivo study, 80 teeth with Class I and Class II cavities were selected. They were divided into four groups of 20 teeth each, to be restored with composite resin using different treatment protocols. Postoperative sensitivity to cold water at various temperatures was assessed for up to three months. The data was interpreted using Chi Square Test for homogeneity among the groups, and Z values of significance for pair-wise evaluation were determined. It was found that use of self-etching primers as bonding agents underneath the composite resin restorations were associated with significantly decreased incidence of postoperative sensitivity to cold stimuli.

Determination of the morphological irregularities in the middle and apical 1/3rd region of the root canal system of permanent maxillary incisors.

A thorough knowledge of the root canal anatomy and an understanding of its variations from the normal are mandatory for the successful root canal therapy. The assessment and exploration of the accessory canal and apical delta is necessary to combat the persistent infection at the periapical area. The purpose of this study is to determine the morphological irregularities in the middle and apical 1/3rd region of the root canal system of maxillary incisors. A total of 100 maxillary incisors were decalcified, processed, sectioned at the middle and apical 1/3rd region and observed under an ordinary microscope. The frequency of accessory canals, apical delta and type of canal configuration were studied. Accessory canals were found in 5% of the teeth. There was absence of apical delta in all the specimens. Total specimens showed single canal extended from the pulp chamber to the apex (Type 1 canal configuration).

Physical interaction and functional synergy between glucocorticoid receptor and Ets2 proteins for transcription activation of the rat cytochrome P-450c27 promoter.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

CD13/APN is activated by angiogenic signals and is essential for capillary tube formation.

In the hematopoietic compartment, the CD13/APN metalloprotease is one of the earliest markers of cells committed to the myeloid lineage where it is expressed exclusively on the surface of myeloid progenitors and their differentiated progeny. CD13/APN is also found in nonhematopoietic tissues, and its novel expression on the endothelial cells of angiogenic, but not normal, vasculature was recently described. Treatment of animals with CD13/APN inhibitors significantly impaired retinal neovascularization, chorioallantoic membrane angiogenesis, and xenograft tumor growth, indicating that CD13/APN plays an important functional role in vasculogenesis and identifying it as a critical regulator of angiogenesis. To investigate the mechanisms of CD13/APN induction in tumor vasculature, the regulation of CD13/APN by factors contributing to angiogenic progression was studied. In this report, it is shown that endogenous CD13/APN levels in primary cells and cell lines are up-regulated in response to hypoxia, angiogenic growth factors, and signals regulating capillary tube formation during angiogenesis. Transcription of reporter plasmids containing CD13/APN proximal promoter sequences is significantly increased in response to the same angiogenic signals that regulate the expression of the endogenous gene and in human tumor xenografts, indicating that this fragment contains elements essential for the angiogenic induction of CD13/APN expression. Finally, functional antagonists of CD13/APN interfere with tube formation but not proliferation of primary vascular endothelial cells, suggesting that CD13/APN functions in the control of endothelial cell morphogenesis. These studies clearly establish the CD13/APN metalloprotease as an important regulator of endothelial morphogenesis during angiogenesis.

Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs.

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.

Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

Multiple forms of cytochrome P450 and associated monooxygenase activities in human brain mitochondria.

We have investigated cytochrome P450 (P450) and associated monooxygenase activities in human brain mitochondria isolated from eight regions of four human brain samples obtained at autopsy. P450-associated monooxygenase activities including aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD), p-nitrophenol hydroxylase (PNPH), and N-nitrosodimethylamine N-demethylase (ND-MAD) were detectable in the mitochondria from human brain regions. Immunoblot experiments using antisera to purified rat liver microsomal P450, namely P4502B1/2, P4501A1/2, and P4502E1, revealed immunoreactive bands in isolated mitochondria from different regions of the human brain. The antibody to P4502B1/2 and P4501A1/2 inhibited the human brain mitochondrial APD and ECD activities, respectively. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450-associated monooxygenase activities, although it completely inhibited the corresponding activities in brain microsomes. Overall, the present study demonstrates, in human brain mitochondria, the presence of multiple forms of P450 belonging to the 1A, 2B, and 2E subfamilies that are involved in xenobiotic metabolism.

Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.

Constitutive and inducible cytochromes P450 in rat lung mitochondria: xenobiotic induction, relative abundance, and catalytic properties.

The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1. 7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis.

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.

Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain.

We have investigated the in vivo effects of the tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on antioxidant defense systems in the mitochondrial, microsomal, and cytosolic compartments of rat brain, lung, and liver. Nicotine induced maximum oxidative stress in brain mitochondria, as seen from a 1.9-fold (P < 0.001) increase in thiobarbituric acid-reactive substance (TBARS) and a 2-fold (P < 0.001) increase in glutathione S-transferase (GST) A4-4 (also referred to as rGST 8-8) activities. These changes were accompanied by a 25-40% increase in reactive oxygen species and a 20-30% decrease in alcohol dehydrogenase activities. The 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-induced oxidative damage was apparent in the microsomal fraction of brain, lung, and liver, and it also increased 4-hydroxynonenal specific GST A4-4 activity in the brain and lung mitochondrial matrix fraction. The levels of microsomal thiobarbituric acid reactive substance, cytochrome P4502E1 activity, and reactive oxygen species were also increased significantly (P < 0.001) in all tissues. Both of these toxins induced the level of GST A4-4 mRNA in the brain, while they caused a marked reduction in the liver GST A4-4 mRNA pool. Additionally, the brain mitochondrial matrix showed a markedly higher level of 4-hydroxynonenal specific GST activity and mGST A4-4 antibody-reactive protein than did the cytosolic fraction. In conclusion, the present study provides evidence for the occurrence of GST A4-4 enzyme activity in mammalian mitochondria, in addition to demonstrating that both mitochondria and microsomes are intracellular targets for nicotine- and NNK-induced organ toxicity.

Differential response of cytosolic, microsomal, and mitochondrial glutathione S-transferases to xenobiotic inducers.

Subcellular levels of different isoenzymes of glutathione S-transferases (GSTs) and their catalytic activities in rat liver, lung and brain tissues were compared following treatment with phenobarbital (PB), -naphthoflavone (BNF) and dexamethasone (DEX). The constitutive expression of á and mu classes of GSTs, but not the GST , was maximum in the liver cytosol as compared to other tissues. Cytosolic GST activity using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate was 2-4 fold higher than that in the microsomal and mitochondrial fractions. Glutathione peroxidase activity with cumene hydroperoxide as a substrate was also highest in the rat liver cytosol. PB and BNF treatments markedly induced the amount of GST proteins in all the tissues studied with the maximum induction in the cytosol after 4 days of PB and 10 days of BNF treatments, respectively. The longer duration of treatments had a suppressive effect on the GST activity, particularly in the mitochondrial and microsomal fractions. DEX treatment, on the other hand, only marginally induced the cytosolic GST, while the mitochondrial GST and the membrane bound microsomal GST activities were mostly decreased. Northern blot analysis also showed an increase in the GST-á mRNA level indicating a possible upregulation of the GST gene expression by the xenobiotic agent. Differences between the subcellular GSTs were studied by the in vitro addition of N-ethylmaleimide (NEM), a selective activator of the microsomal GST. The cytosolic GST activity, both in livers of uninduced and PB-treated, was inhibited to about 50% of the control levels by NEM. The mitochondrial activity, on the other hand, was significantly activated by the addition of NEM, similar to that reported for the microsomal GST. These results suggest selectivity in the effects of different xenobiotics on the expression and catalytic activity of GST isoenzymes from different subcellular compartments of tissues. More importantly, these observations are also relevant in studies on xenobiotic induced organ-specific toxicity and carcinogenicity.

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2.

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

Further characterization of rat brain flavin-containing monooxygenase. Metabolism of imipramine to its N-oxide.

Flavin-containing monooxygenase (FMO) activity was compared in rat liver and brain microsomes by estimating the actual amount of imipramine N-oxide relative to the corresponding activity, measured using substrate-stimulated rates of NADPH oxidation. The activities measured as NADPH oxidation rates were significantly higher than those estimated from the N-oxide formed. The brain FMO activity was detectable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The antibody to rabbit pulmonary FMO selectively inhibited imipramine N-oxidation. The antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect on imipramine N-oxidation, indicating the noninvolvement of cytochrome P-450 in the above metabolic pathway. A flavin-containing monooxygenase was partially purified from the rat brain microsomes using sequential chromatography on n-octylamino-Sepharose 4B, DEAE-Sephacel and 2',5'-ADP agarose. The purified FMO was resolved by SDS-PAGE into two bands (approximately 57 and 61 KDa, respectively) both of which cross-reacted with antibody to rabbit pulmonary FMO. The purified enzyme metabolized imipramine and the model substrate methimazole to their respective N-oxide and S-oxides.

Xenobiotic metabolism in brain.

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.