Cytochrome P450-dependent Enzymes Research Articles

Genome-Guided Discovery of the Myxobacterial Thiolactone-Containing Sorangibactins.

In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide synthetase (NRPS) gene cluster from the Sorangiineae strain MSr11367 in the host Myxococcus xanthus DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem N-methyl-thiazolidines, and an unusual C-terminal γ-thiolactone moiety. Except for the unprecedented oxazoline dehydrogenation to form an oxazole, which we show to be catalyzed by a cytochrome P450-dependent enzyme, other tailoring steps were found necessary for efficient downstream processing. The unusual thioesterase (TE) domain is proposed to select homocysteine or methionine for offloading involving an intramolecular γ-thiolactone formation. Its active site comprises a rare cysteine, which was found essential for product formation by point mutation to alanine or serine, which both abolished its activity. This unusual release mechanism and the resulting rare thiolactone structure can serve as a starting point for detailed biochemical investigations.

Expanding the Ajudazol Cytotoxin Scaffold: Insights from Genome Mining, Biosynthetic Investigations, and Novel Derivatives.

Myxobacteria have proven to be a rich source of natural products, but their biosynthetic potential seems to be underexplored given the high number of biosynthetic gene clusters present in their genomes. In this study, a truncated ajudazol biosynthetic gene cluster in Cystobacter sp. SBCb004 was identified using mutagenesis and metabolomics analyses and a set of novel ajudazols (named ajudazols C-J, 3-10, respectively) were detected and subsequently isolated. Their structures were elucidated using comprehensive HR-MS and NMR spectroscopy. Unlike the known ajudazols A (1) and B (2), which utilize acetyl-CoA as the biosynthetic starter unit, these novel ajudazols were proposed to incorporate 3,3-dimethylacrylyl CoA as the starter. Ajudazols C-J (3-10, respectively) are characterized by varying degrees of hydroxylation, desaturation, and different glycosylation patterns. Two P450-dependent enzymes and one glycosyltransferase are shown to be responsible for the hydroxylation at C-8, the desaturation at C-15 and C-33, and the transfer of a d-β-glucopyranose, respectively, based on mutagenesis results. One of the cytochrome P450-dependent enzymes and the glycosyltransferase were found to be encoded by genes located outside the biosynthetic gene cluster. Ajudazols C-H (3-8, respectively) exhibit cytotoxicity against various cancer cell lines.

Abstract P2041: Cytochrome P450 Reductase Prevents Cardiac Hypertrophy Through Crosstalk

The cytochrome P450 reductase (POR) transfers electrons and thus drives the function of all cytochrome P450-dependent enzymes (CYP450). In the vascular system, the CYP450-POR system has been linked to vascular epoxyeicosatrienoic acids (EET) production and endothelial function. The functional importance of the endothelial CYP450 system in the heart, however, is unclear and was studied here, using a tissue-specific deletion approach. RNAseq revealed that murine cardiac endothelial cells express POR as well as numerous CYPs, which have been linked to different functions: CYP2 family members produce EETs, whereas CYP4 family generated omega fatty acids. Endothelial-specific, tamoxifen-inducible POR knockout mice (ecPOR -/- ) show endothelial dysfunction and increased hypertensive response to angiotensin II infusion. ecPOR -/- mice also exhibited cardiac hypertrophy, an increase in cardiomyocyte area, a reduced ejection fraction and an increased left ventricle size during systole (echocardiography) as compared to control animals. RNAseq of isolated cardiac endothelial cells revealed that deletion of POR was associated with a significant decrease in genes linked to unfolded protein response (Xbp1, Hspa5) whereas genes related to cardiac development were upregulated (Sox4, Gata4). Furthermore, cardiac myocytes showed a reduced expression of genes linked to mitochondrial ATP production (Sdhaf4, Mtx1, Coq10b).Collectively, our data suggest that loss of endothelial POR promotes cardiac hypertrophy potentially by modulating paracrine signals of endothelial cells to cardiac myocytes.

Fortified Fluconazole Eyedrops for Treatment of Culvularia Corneal Ulcer

Abstract
 Introduction. Fungal keratitis is less common than bacterial keratitis, generally representing less than 5%–10% of corneal infections in reported clinical series in the United States. But in developing countries (Ghana, India, China) it accounts for more than 90% of the cases. Corneal trauma by plant or vegetative material is the leading risk factor for fungal keratitis. One or more topical antifungals are usually administered with systemic support of oral antifungals. The most common classes of antifungal used for medical therapy include the polyenes, azoles, and the echinocandins.
 Case Illustration. A 47-year-old male presented with a widening white patch on his left eye since 4 days before coming to our hospital. The left eye was injured by wood flakes 3 weeks ago. He felt a sore, red, watery left eye and blurred vision. He washed his eyes with betel leaf water. His left eye visual acuity was 1/60 and not improved with pinhole. There were blepharospasm, ciliary and conjunctival injection. Corneal examination showed a cloudy cornea, a central corneal defect sized 8x6 mm with an irregular margin, 2/3 to stromal depth, infiltrate, satellite lesion, and positive fluorescein staining at the defect margin. KOH examination demonstrated hyphae. The culture test of left corneal discharge identified a fungi species (Curvularia sp). We prescribed fortified fluconazole eye drops in combination with supportive therapy for 2.5 months. This regimen showed a significant increase in visual acuity to 6/30 with pinhole improvement of 6/21 in the left eye.
 Discussion. Imidazole acts by inhibiting ergosterol biosynthesis of the fungal cell wall, through action on the cytochrome P450-dependent enzyme. This leads to cell membrane destabilization and leakage. Curvularia sp belongs to the family of dematiaceous fungi. Its clinical manifestations include raised lesions and feathery edges; hypopyon rarely occurs, and the prognosis is quite good. Fluconazole (2 mg/ml) is available for injection and is a well-tolerated eye drop. The intravenous injection can be used as a topical treatment and can be given subconjunctivally at the same concentration.
 Conclusions. Fortified fluconazole eyedrops (2 mg/ml) can become an alternative and additional therapy for deep fungal keratitis, especially for Curvularia sp.

Imidazole Antifungals: A Review of Their Action Mechanisms on Cancerous Cells

Imidazoles, together with triazoles, constitute azole sub-group of antifungal drugs which acts by inhibiting cytochrome P450-dependent enzyme, the lanosterol 14-α-demethylase. In addition to their primary use, when it comes to additional anti-cancer function, clotrimazole, econazole and ketoconazole have come to the fore among the imidazoles. Based on the findings up to now, although having different effects, disruption of the glycolytic pathway, blockage of Ca2+ influx and nonspecific inhibition of CYP450 enzymes can be regarded as the main ones responsible for the anti-neoplastic activities of the mentioned drugs, respectively. Considering the advantages of repurposing of drugs with known pharmacology compared to new drug development studies requiring labor, time and cost, it will be extremely important and valuable to continue the clarification of the different mechanisms of these antifungals on cancerous cells and benefit from them especially to increase drug efficacy and overcome drug resistance. In this review, the action mechanisms of imidazole antifungals on cancerous cells and consequently, their potential for use in cancer treatment alone or in combination with conventional therapeutics were discussed in detail.

Amyloid-beta (Aβ1–42)-induced paralysis in Caenorhabditis elegans is reduced through NHR-49/PPARalpha

Alzheimer´s disease is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid β (Aβ). Agonists of peroxisomal proliferator-activated receptors (PPARs) are discussed as anti-amyloidogenic compounds, e.g. due to their cholesterol-lowering activities. In a previous study we have shown in Caenorhabditis elegans expressing human Aβ in muscle cells, that inhibition of steroid-signaling, by RNAi of respective members of the signaling pathway or by reducing cellular cholesterol uptake, both increases the nuclear translocation of the foxo transcription factor DAF-16 and concomitantly reduces Aβ-induced paralysis. Using RNAi in the present study we show that NHR-49/PPARalpha inhibits steroidal-signaling upstream of DAF-9, a cytochrome P450-dependent enzyme which generates dafachronic acids as ligands for the nuclear hormone receptor DAF-12, and upstream of DAF-12 itself. The NHR-49/PPARalpha agonist fenofibrate reduces Aβ-induced paralysis in dependence on nhr-49 and nuclear translocation of DAF-16.In conclusion, activation of NHR-49/PPARalpha inhibits the steroidal-signaling pathway which increases the nuclear translocation of DAF-16 and inhibits the Aβ-induced phenotype in an Alzheimer model of C. elegans.

Functional expression and characterization of cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis in Physcomitrella patens

Key messageCinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis (AaC4H) was functionally expressed in the moss Physcomitrella patens and characterized at biochemical and molecular levels.Cinnamic acid 4-hydroxylase (C4H), a cytochrome P450-dependent hydroxylase, catalyzes the formation of 4-coumaric acid (=4-hydroxycinnamic acid) from trans-cinnamic acid. In the hornwort Anthoceros agrestis (Aa), this enzyme is supposed to be involved in the biosynthesis of rosmarinic acid (a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid) and other related compounds. The coding sequence of AaC4H (CYP73A260) was expressed in the moss Physcomitrella patens (Pp_AaC4H). Protein extracts from the transformed moss showed considerably increased C4H activity driven by NADPH:cytochrome P450 reductase of the moss. Since Physcomitrella has own putative cinnamic acid 4-hydroxylases, enzyme characterization was carried out in parallel with the untransformed Physcomitrella wild type (Pp_WT). Apparent Km-values for cinnamic acid and NADPH were determined to be at 17.3 µM and 88.0 µM for Pp_AaC4H and 25.1 µM and 92.3 µM for Pp_WT, respectively. Expression levels of AaC4H as well as two Physcomitrella patens C4H isoforms were analyzed by quantitative real-time PCR. While PpC4H_1 displayed constantly low levels of expression during the whole 21-day culture period, AaC4H and PpC4H_2 increased their expression during the first 6–8 days of the culture period and then decreased again. This work describes the biochemical in vitro characterization of a cytochrome P450-dependent enzyme, namely C4H, heterologously expressed in the haploid model plant Physcomitrella patens.

Oral antifungal: the safety and efficacy of oral itraconazole in dermatophytosis

Background: The systemic antifungals like Griseofulvin, Itraconazole, Terbinafine, Ketoconazole and Fluconazole are widely used for superficial fungal infection. Hepatotoxicity with oral antifungals is well established fact. The rate of transient asymptomatic changes in liver function tests accounts for about 0.5 - 10% of all patients treated with systemic antifungals. Clinical hepatic toxicity is seen less frequently. The aim of this study is to evaluate the effect of oral Itraconazole on hepatic function and it’s efficacy in patients with extensive dermatophytosis.Methods: The total of 524 patients with extensive dermatophytosis were included in our study which was conducted in a tertiary care hospital in Navi Mumbai.Results: Itraconazole, a systemic antifungal agent is efficiently used in treatment of superficial and deep mycoses. It inhibits fungal cytochrome P450 dependent enzyme and thus impaires conversion of lanosterol to ergosterol. Adverse reactions to itraconazole includes drug reactions, gastrointestinal upset, headache, dizziness, thrombocytopenia, gynecomastia, reversible edema of extremities and metabolic side effects like hypokalemia, and hypertriglyceridemia. The level of hepatic transaminases increases in about 1%-5% of patients who have received continuous therapy with systemic itraconazole. Clinical hepatitis rarely occurs in patients and, recovery generally ensues with the cessation of medication.Conclusions: The baseline and post treatment liver function test is important to monitor if patient is on higher dose and longer duration of itraconazole therapy. The screening for high risk patients like poor liver function test, history of alcoholism, history of liver disease should be taken before stating the therapy.

Novel imidazole derivatives as antifungal agents: Synthesis, biological evaluation, ADME prediction and molecular docking studies

A series of 2-(substituteddithiocarbamoyl)-N-[4-((1H-imidazol-1-yl)methyl)phenyl]acetamide derivatives was designed and synthesized to combat the increasing incidence of drug-resistant fungal infections. All synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR, and HRMS spectra and elemental analyses. Antifungal activity tests were performed against four different fungal strains. Molecular docking studies were performed to investigate the mode of action towards the fungal lanosterol 14α-demethylase, a cytochrome P450-dependent enzyme. ADME studies were carried out and a connection between activities and physicochemical properties of the target compounds was determined. Most of the final compounds exhibited significant activity against Candida albicans and Candida krusei with MIC50 value 12.5 μg/mL. The results of in vitro anti-Candida activity, a docking study and ADME prediction revealed that the newly synthesized compounds have potential anti-Candida activity and evidenced the most active derivative, 5b (2-Pyrrolidinthiocarbonylthio-N-[4-((1H-imidazol-1-yl)methyl)phenyl]acetamide), which can be further optimized as a lead compound.

Design, Synthesis and Antifungal Activity Evaluation of New Thiazolin-4-ones as Potential Lanosterol 14α-Demethylase Inhibitors.

Twenty-three thiazolin-4-ones were synthesized starting from phenylthioamide or thiourea derivatives by condensation with α-monochloroacetic acid or ethyl α-bromoacetate, followed by substitution in position 5 with various arylidene moieties. All the synthesized compounds were physico-chemically characterized and the IR (infrared spectra), 1H NMR (proton nuclear magnetic resonance), 13C NMR (carbon nuclear magnetic resonance) and MS (mass spectrometry) data were consistent with the assigned structures. The synthesized thiazolin-4-one derivatives were tested for antifungal properties against several strains of Candida and all compounds exhibited efficient anti-Candida activity, two of them (9b and 10) being over 500-fold more active than fluconazole. Furthermore, the compounds’ lipophilicity was assessed and the compounds were subjected to in silico screening for prediction of their ADME-Tox properties (absorbtion, distribution, metabolism, excretion and toxicity). Molecular docking studies were performed to investigate the mode of action towards the fungal lanosterol 14α-demethylase, a cytochrome P450-dependent enzyme. The results of the in vitro antifungal activity screening, docking study and ADME-Tox prediction revealed that the synthesized compounds are potential anti-Candida agents that might act by inhibiting the fungal lanosterol 14α-demethylase and can be further optimized and developed as lead compounds.

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

Main conclusionOverexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin inMalus. Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab.The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2475-9) contains supplementary material, which is available to authorized users.

Emergence of Azole Therapy for Cancer Associated Fungal Infections and Their Potential Human Toxicity

Presently, azoles have emerged as excellent antifungal agents. Their broad-spectrum activity and ability to treat superficial as well as invasive infections have gained them incredible repute in antimicrobial therapy. Azoles inhibit the synthesis of ergosterol in fungi, which is important for maintaining the cell wall. This review briefly portrays the relevance of azoles in treating cancer linked fungal infections like candidiasis and aspergillosis. First generation azoles like itraconazole and fluconazole are preferred over other drugs like amphotericin B. Newer azoles (triazoles like voriconazole, posaconazole and ravuconazole) have been synthesized to oust the earlier varieties by being more potent and active against resistant organisms. Patients with lymphoma or leukemia undergoing chemotherapy or stem cell transplantation have weak immune status which makes them susceptible hosts of fungal infections. Azoles are used currently for antitumor therapy because of their specificity towards human cytochrome P-450 dependent enzymes which synthesize cholesterol. This review also attempts to point out how their applications in prostate cancer and breast cancer therapy are indispensable. But along with its advantages, it is necessary to look into the sinister role it plays in creating toxic side effects for patients. Disruption of the endocrine functioning and hepatotoxicity result in manifestations like vomiting, nausea and fatigue. Even though these complications are reversible, the full-fledged practicability of these drugs in cancer treatment is yet to be considered. Keywords: Aromatase, Aspergillosis, Candidiasis, cancer, lanosterol-14α-demethylase, toxicity, triazoles.

Relationship of Azole Resistance with the Structural Alteration of the Target Sites: Novel Synthetic Compounds for Better Antifungal Activities

Azoles have consistently been recognized as the mainstays of antifungal drugs, amongst which ergosterol prevails as an integral component of fungal plasma membrane. It is most commonly produced via demethylation of lanosterol by the cytochrome P450-dependent enzyme lanosterol 14α-demethylase. Azoles exert their antifungal activities via binding to lanosterol 14 α-demethylase and thereby preventing the demethylation of lanosterol. This leads to the depletion of demethylated lanosterol (ergosterol) with the concomitant accumulation of methylated sterol precursors (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol) and deterioration of the membrane integrity, resulting in fungal growth inhibition. Resistance to azoles is a concern, particularly during the long-term treatment of fungus mediated cellular complications. To combat azole resistance and to extend the spectrum of treatable pathogens, the development of novel and more potent azoles, with alteration in active sites has attracted worldwide scientific attention. With such an insight, this review focuses on antifungal potentials of azole compounds with an emphasis on the corresponding drug resistance episodes complemented with novel strategies for the development of new generation of azole compounds. Keywords: Azole, lanosterol, ergosterol, 14-α-lanosterol-demethylase, structure-function relationship, drug resistance.

The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3′-hydroxylase on leucoanthocyanidins

In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3' position by flavonoid 3'-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3' or 3' and 5', respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3'H and F3'5'H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3'H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3'-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3',5'-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3'Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.

Design, Synthesis, and Molecular Docking of 1-(1-(4-Chlorophenyl)-2-(phenylsulfonyl)ethylidene)-2-phenylhydrazine as Potent Nonazole Anticandidal Agent

1-(1-(4-Chlorophenyl)-2-(phenylsulfonyl)ethylidene)-2-phenylhydrazine (13) was designed and synthesized as potential nonazole anticandidal agent and precisely characterized by IR,1H NMR,13C NMR, and ESI-MS. The anti-Candidaactivity of13was evaluated against fourCandidaspecies (C. albicans, C. krusei, C. parapsilosis, andC. glabrata). Compound13displayed good anticandidal activities (MIC=0.39, 0.195, 0.39, and 1.56 μmol/mL, resp.) in comparison with that of the standard drug fluconazole (MIC=0.195, inactive, 1.56, and 1.56 μmol/mL, resp.) againstC. albicans, C. krusei, C. parapsilosis, andC. glabrata, respectively. A molecular modeling of the newly synthesized compound13was built in order to investigate its mode of action towards the prospective target cytochrome P450-dependent enzyme lanosterol 14α-demethylase (PDB-code: 1EA1). The docking results showed a similar binding interaction of13and fluconazole at the active site of CYT P450 14α-sterol demethylase. Furthermore, compound13showed no cytotoxicity against normal human breast cell line MCF10A.

Effects of Sub Chronic Dermal Exposure of Gasoline Blended with Varying Concentration of Benzene on Hepatic Mixed Function Oxidases in Rats

Interaction of chemicals in a mixture, their biotransformation and adverse impact, if any, on environment and workroom atmosphere needs to be evaluated to identify the nature of potential toxic effects elicited by constituent chemical(s) in a mixture. The nature of chemical interaction(s) through which an individual chemical in a mixture undergoes metabolism and/or biotransformation in the target tissue could be helpful in evaluation of its toxic potential. Gasoline is a unique example of such chemical mixture where interaction of aromatics viz., benzene, toluene, ethyl benzene and xylene (BTEX) traverses through a common metabolic pathway involving cytochrome P-450 dependent mixed function oxidases. Travis et al. [1] has quantitatively evaluated that gasoline vapor has an antagonistic effect on benzene metabolism and its biotransformation products. It was reported that 1000 ppm gasoline vapor had the same inhibitory effect as 500 ppm toluene on the metabolism of 290 ppm benzene, indicating that there may be many confounding factors in gasoline which might affect the metabolism of benzene in turn affecting its toxicity at the molecular level. It has been reported that benzene causes myelotoxicity through its bioactive metabolites. Such biotransformation mechanisms involve cytochrome P-450 dependent Phase-I enzymes and the products are further detoxified through Phase-II enzymes. It was therefore considered pertinent to study the impact of gasoline blended with 1, 3 and 5% benzene concentrations. Female albino rats weighing 200–250 gm were exposed by dermal route to gasoline blended with varying benzene concentrations, @ 2 g/kg body wt., six days a week over a period of 90 days. The activities of hepatic mixed function oxidases, viz., aniline hydroxylase and aminopyrene demethylase as a monitor of Phase-I enzymes and Glutathione-S-Transferase for Phase-II enzyme was estimated along with free glutathione levels. Serum creatine kinase was also estimated to assess the extent of muscular dystrophy. The results of this sub chronic study will be discussed in the context of interaction of gasoline with varying concentration of benzene in experimental animals. Keywords: mixed function oxidases, benzene, toxicology, gasoline

Mo1164 The Duel Nitric Oxide and Hydrogen Sulfide-Releasing Nonsteroidal Anti-Inflammatory Drugs NOSH-Aspirin, NOSH-Naproxen, and NOSH-Sulindac Are Safe to the Stomach and Have Strong Anti-Inflammatory, Analgesic, Antipyretic, Anti-Platelet, and Anti-Cancer Properties

G A A b st ra ct s of curcumin is thought to be through the inhibition of Cytochrome P450 dependent enzymes, which convert pro-mutagens to mutagens. It is critical to understand the impact of these risk factors at the molecular level so that suitable preventive strategies can be developed. Objective: Here, we investigated effects of B[a]P and Curcumin on CYP450 genes in a normal esophageal squamous epithelial cell line (HET-1A), and in an hTERT immortalized benign Barrett's cells (BAR-T). Methods: BAR-T cells were exposed for 5 minutes daily to 200uM glycochenodeoxycholic acid with an adjusted culture medium of pH 4 for 18 weeks, and untreated HET-1A cells were also used in parallel. We treated both cell types with 15, 25, 30uM B[a]P for 48hrs. Curcumin was added 30 minutes prior to adding B[a]P at a concentration of 1.5, and 3uM. AhR, CYP1A1, CYP1B1 transcripts were assessed by Real time RT-PCR analyses. Expression of target mRNA was normalized with respect to Actin Using the DeltaDelta Ct method. Results: Results show a dose dependent induction of the AhR gene on the HET-1A and BAR-T cell line by B[a]P after 48 hours of exposure. CYP1A1 and CYP1B1 genes reach maximum induction at 30uM of B[a]P. Interestingly, when Curcumin was added to the cell lines exposed to 25uM B[a]P there is a dose dependent decrease on CYP1A1 y CYP1B1 genes. (Table 1) Conclusion: Benzo[a]pyrene induces Cytochrome P450 genes in both squamous and Barrett's cells. However Barrett's cells seem more susceptible to mutagenic effects of B[a]P. Curcumin is capable of reversing mutagenic effects of B[a]P in both cell types and thus promises to be a naturally occurring compound for esophageal chemoprevention specifically in patients with a history of smoking. The study also provides an important clue in the pathogenesis of esophageal cancer.

Abstract 3596: Short-term effect of nose-only cigarette smoke exposure on redox homeostasis and mitochondrial metabolism in mice.

Abstract The components of cigarette smoke have been implicated in the development of cancer as well as in cardiovascular diseases. We have previously reported oxidative stress in rat tissues induced by tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Recently, we have also shown increased oxidative stress and associated inflammatory responses in various tissues after exposure to cigarette smoke. In this study, we have further investigated the effects of nose-only cigarette smoke exposure on mitochondrial functions and glutathione-dependent redox metabolism in tissues of BALB/C mice. Liver, kidney, heart and lung tissues were analyzed for oxidative stress, glutathione (GSH) and cytochrome P450 dependent enzyme activities and mitochondrial functions after exposure to smoke generated by 9 cigarettes/day for 4 days. Control mice were exposed to air only. An increase in oxidative stress as observed by an increased production of reactive oxygen species (ROS) and altered GSH metabolism was apparent in all the tissues, but lung and heart appeared to be the main targets affected. Increased expression and activity of CYP450 1A1 and 1A2 were also observed in the tissues after exposure to cigarette smoke. Mitochondrial respiratory functions in the tissues, as observed by alterations in the activities of Complex I and IV enzymes, was also observed after exposure to cigarette smoke when compared with air only exposed mice. SDS-PAGE and Western blot results also indicate that alterations in the expression of enzyme proteins were in accordance with the changes in their catalytic functions. These results suggest that even short term exposure of cigarette smoke have adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking. (The study is supported by Terry Fox Cancer Research Funds and by the Research Committee Grants from College of Medicine and Health Sciences) Citation Format: Haider Raza, Annie John, Abderrahim Nemmar. Short-term effect of nose-only cigarette smoke exposure on redox homeostasis and mitochondrial metabolism in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3596. doi:10.1158/1538-7445.AM2013-3596

Short-Term Effects of Nose-Only Cigarette Smoke Exposure on Glutathione Redox Homeostasis, Cytochrome P450 1A1/2 and Respiratory Enzyme Activities in Mice Tissues

Background/Aims: The components of cigarette smoke (CS) have been implicated in the development of cancer as well as in cardiopulmonary diseases. We have previously reported increased oxidative stress in rat tissues induced by tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Recently, we have also shown increased oxidative stress and associated inflammatory responses in various tissues after exposure to cigarette smoke. Methods: In this study, we have further investigated the effects of nose-only cigarette smoke exposure on mitochondrial functions and glutathione-dependent redox metabolism in tissues of BALB/C mice. Liver, kidney, heart and lung tissues were analyzed for oxidative stress, glutathione (GSH) and cytochrome P450 dependent enzyme activities and mitochondrial functions after exposure to smoke generated by 9 cigarettes/day for 4 days. Control mice were exposed to air only. Results: An increase in oxidative stress as observed by increased production of reactive oxygen species (ROS) and altered GSH metabolism was apparent in all the tissues, but lung and heart appeared to be the main targets. Increased expression and activity of CYP450 1A1 and 1A2 were also observed in the tissues after exposure to cigarette smoke. Mitochondrial respiratory dysfunction in the tissues, as observed by alterations in the activities of Complex I and IV enzymes, was also observed after exposure to cigarette smoke. SDS-PAGE and Western blot results also indicate that alterations in the expression of enzyme proteins were in accordance with the changes in their catalytic functions. Conclusion: These results suggest that even short term exposure of cigarette smoke have adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking.

Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3'H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3''H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3'H was highly homologous with F3'Hs from other plant species. Conserved domain search revealed that IbF3'H was a cytochrome P450 dependent enzyme. Three F3'H-specific motifs (V75VVAAS80, G427GEK430 and V433DVKG437) were conserved in IbF3'H. Phylogenetic analysis revealed that IbF3'H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3'H gene in the genome of I. batatas. IbF3'H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3'H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3'H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3'H could encode a functional protein and had a special catalytic activity of flavonoid 3'-hydroxylase.