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Journal Article 2014; 35(Suppl 2): 123-132 PubMed PMID: 25638376 Citation  Keywords:  Animals, Aristolochic Acids:metabolism, Dicumarol:pharmacology, Enzyme Inhibitors:pharmacology, Humans, Quinone Reductases:metabolism, Rats,.   

OBJECTIVES: Dicoumarol is known to act as an inhibitor of

NAD(P)H: quinone oxidoreductase (NQO1). This cytosolic reductase significantly contributes to the genotoxicity of the nephrotoxic and carcinogenic alkaloid aristolochic acid I (AAI). Aristolochic acid causes aristolochic acid nephropathy (AAN), and Balkan endemic nephropathy (BEN), as well as associated urothelial malignancies. NQO1 is the most efficient enzyme responsible for the reductive bioactivation of AAI to species forming covalent AAI-DNA adducts. However, it is still not known how dicoumarol influences the NQO1-mediated reductive bioactivation of AAI.

METHODS: AAI-DNA adduct formation was determined by 32P-postlabeling. Expression of NQO1 mRNA and NQO1 protein was determined by real-time polymerase chain reaction and Western blotting, respectively.

RESULTS: In this study, dicoumarol inhibited AAI bioactivation to form AAI-DNA adducts mediated by rat and human NQO1 in vitro as expected. We however, demonstrated that dicoumarol acts as an inducer of NQO1 in kidney and lung of rats treated with this NQO1 inhibitor in vivo, both at protein and activity levels. This NQO1 induction increased the potency of kidney cytosol to bioactivate AAI and elevated AAI-DNA adduct levels were found in ex-vivo incubations of AAI with renal cytosols and DNA. NQO1 mRNA levels were induced in liver only by dicoumarol.

CONCLUSION: Our results indicate a dual role of dicoumarol in NQO1-mediated genotoxicty of AAI. It acts both as an NQO1 inhibitor mainly in vitro and as an NQO1 inducer if administered to rats.

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Journal Article 2011; 32(Suppl 1): 25-34 PubMed PMID: 22167217 Citation  Keywords:  Animals, Carcinogens, Environmental:isolation & purification, Cytochrome P-450 CYP1A1:antagonists & inhibitors, DNA Damage:drug effects, Drug Evaluation, Preclinical, Enzyme Induction:drug effects, Enzyme Inhibitors:pharmacology, Food:toxicity, Food Conta.   

OBJECTIVES: Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, β-naphthoflavone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined.

METHODS: Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using their marker substrates (ethoxyresorufin and methoxyresorufin).

RESULTS: Treatment of rats with a single dose of BNF produced an increase in levels CYP1A1/2 and CYP1A1 proteins in liver and small intestine, respectively. An increase in CYP1A1 protein expression found in both organs correlated well with specific activities of these CYPs. The CYP1A1 expression levels and its specific activity in small intestine decreased along the length of the organ, being highest in its proximal part and lowest in its distal part. The BNF induction of CYP1A1/2 resulted in a significant increase in the formation of BaP- and PhIP-DNA adducts in liver and in the distal part of the small intestine, respectively. Thus, pretreatment of rats with BNF did not prevent the PhIP and BaP activation, but vice versa, enhanced their genotoxicity.

CONCLUSIONS: The results of this study demonstrate that the administration of only a single dose of CYP-inducing flavonoid prior to the intake of food carcinogens may increase the risk of a tumor formation....

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Journal Article 2009; 30(Suppl 1): 88-91 PubMed PMID: 20027150 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:antagonists & inhibitors, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System:metabolism, Dextromethorphan:chemistry, Dextrorphan:chemistry, Enzyme Inhibitors:pharmacology, Female, Liver:drug effects, .   

OBJECTIVES: Differences in the metabolism between males and females have been seen over time. Hormonal regulation of cytochrome P450 activity is understood to be involved. Trans-resveratrol (RES) is an estrogenically active plant polyphenol with many protective biological activities including neuroprotection. The present report studied the influence of sex and RES on variances in rat's cytochrome P450 2D2 hepatic metabolic activity.

METHODS AND DESIGN: Isolated perfused rat liver was used for determination of cytochrome P450 2D2 activity. Wistar albino rats of both sexes were treated with RES at the dose of 5 mg/kg/day for 10 days prior to liver isolation. Levels of marker substance dextromethorphan (DEM) and its 2D2 specific metabolite dextrorphan (DEX) were measured during perfusion. The metabolic ratios (DEM/DEX) and the levels of DEM and DEX in perfusate were compared.

RESULTS: In the controls, the activity of CYP2D2 was found to be higher in male rats compared to females. RES produced inhibition of CYP2D2, expressed by significant changes of both DEM and DEX levels in males and significant increase of only DEM levels in females. There were no gender changes in DEX levels in RES treated animals whilst DEM levels were significantly increased during the whole perfusion in females.

CONCLUSION: The results confirmed gender differences in the metabolic activity of CYP450 2D2 with a higher rate in male rats. RES acted as an inhibitor, however again with greater impact in males than in females. This metabolic divergence could be a cause for different sensitivity or even toxicity of drugs metabolized by the CYP450 2D2....

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Journal Article 2008; 29(5): 784-789 PubMed PMID: 18987608 Citation  Keywords:  Acetylcholine:metabolism, Animals, Blood Pressure:drug effects, Body Weight:drug effects, Cardiovascular Physiological Phenomena:drug effects, Enzyme Inhibitors:pharmacology, Feedback, Physiological:drug effects, Hypertension:drug therapy, Isometric Contr.   

OBJECTIVES: The effect of 8-week-lasting low-dose treatment of NG-Nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, was investigated in borderline hypertensive rats (BHR) to examine, whether dose of 1.5 mg/kg/day affects feedback regulation of NO synthesis.

METHODS: Blood pressure (BP) of 12 weeks old Wistar and BHR rats was determined non-invasively by tail-cuff. NO synthase (NOS) activity was determined by conversion of [3H]-L-arginine to [3H]-L-citrulline in the aorta, left ventricle (LV) and hypothalamus. Vascular function of the femoral artery was determined using Mulvany's myograph in isometric conditions.

RESULTS: Chronic low-dose L-NAME treatment of BHR induced sustained blood pressure elevation and left ventricular hypertrophy associated with the decrease in NOS activity in left ventricle and unaltered NOS activity in the aorta. By contrast, the improvement of LV and aortic NOS activity was found in Wistar rats. In hypothalamus, no changes in NOS activity were found in both BHR and Wistar. In Wistar, acetylcholine-induced relaxation of the femoral artery was increased and serotonin-induced and noradrenalin-induced constriction were reduced in L-NAME treated group. These effects, however, were not seen in BHR.

CONCLUSION: The results indicate that NOS/NO feedback regulation works differently under conditions of normotension and prehypertension. Low-dose L-NAME treatment accentuated NO production in normotensive rats, but it failed to improve NOS activity in BHR....

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Journal Article 2008; 29(5): 775-778 PubMed PMID: 18987578 Citation  Keywords:  Aldehyde Reductase:antagonists & inhibitors, Animals, Blotting, Western, Calcimycin:pharmacology, Cattle, Cell Line, Electrochemistry, Endothelial Cells:drug effects, Endothelium, Vascular:drug effects, Enzyme Inhibitors:pharmacology, Glucose:pharmacology.   

OBJECTIVES: An increased glucose utilization by aldose reductase (ALR-2) has been implicated in the pathogenesis of diabetic vascular complications. In this process, several mechanisms are involved, including the depletion of cofactors required for the action of antioxidant enzymes or endothelial NO synthase. In this study, the effect of a novel ALR-2 inhibitor JMC-2004 on hyperglycemia-induced endothelial dysfunction was studied.

METHODS: Bovine aortic endothelial cells (BAEC) were treated with glucose (30 mM), JMC-2004 (0.01mM), or glucose and JMC-2004 for 24 h. The cells were then stimulated with calcium ionophore A23187 after which NO production was measured electrochemically using a porphyrine-coated carbon NO electrode. Nitrite concentrations were determined in the cell supernatants. The peroxyl and hydroxyl radical-scavenging activity of JMC-2004 was measured with luminol-enhanced chemiluminescence. The expression of eNOS was determined by Western blotting. JMC-2004 IC50 for ALR-2 was determined colorimetrically with D-glyceraldehyde as a substrate.

RESULTS: Incubating the cells with 30 mM glucose strongly diminished A23187- induced NO production. Treatment with JMC-2004 restored NO production by 40% without affecting eNOS expression. This effect was probably antioxidantindependent, since JMC-2004 did not have any antioxidant capacity. JMC-2004 exerted high selectivity towards ALR-2.

CONCLUSIONS: ALR-2 inhibition with JMC-2004 was able to abolish hyperglycemia- induced endothelial dysfunction in bovine aortic endothelial cells....

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Journal Article 2007; 28(6): 775-780 PubMed PMID: 18063935 Citation  Keywords:  Acetylation:drug effects, Apoptosis:drug effects, Cell Proliferation:drug effects, Dose-Response Relationship, Drug, Down-Regulation, Enzyme Inhibitors:pharmacology, Histone Deacetylase Inhibitors, Histones:metabolism, Humans, Immunohistochemistry, Multip.   

: The expression of vascular endothelial growth factor receptor 1(VEGFR-1) in human multiple myeloma KM3 cells in vitro, effects of valproic acid (VPA), as a histone deacetylase inhibitor, on cell proliferation and apoptosis and the underlying molecular mechanism were investigated. The effects of VPA on the growth of KM3 cells were studied by MTT assay. The apoptosis rate was determined with flow cytometry. The mRNA level of VEGFR was determined by RT-PCR; and immunocytochemistry was used to detect the protein level of ac-H4 and VEGFR. VPA inhibited proliferation of KM3 cells in a time- and dose-dependent manner. Treatment with VPA (4, 2, 1 and 0.5 mmol/L) for 48h, the apoptosis rates of KM3 cells were (13.27+/-3.54)%, (22.13+/-1.20)%, (24.41+/-2.23)% and(40.62+/-4.28)% respectively. The expression of VEGFR-1 in KM3 cells were decreased in VPA-treated group by the immunochemistry and RT-PCR, whereas the acetylated histone H4(ac-H4) accumulated. It suggested VPA could decrease the expression of VEGFR-1 in KM3 cells, and it might play an important role in regulating the proliferation and apoptosis of multiple myeloma cell line KM3 cells. These results provide the framework for clinical trials....

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Journal Article 2007; 28(1): 39-44 PubMed PMID: 17277728 Citation  Keywords:  Acetylcholine:pharmacology, Animals, Cholinergic Agents:pharmacology, Enzyme Inhibitors:pharmacology, Female, Ganglia, Sympathetic:enzymology, Guanidines:pharmacology, NG-Nitroarginine Methyl Ester:pharmacology, Nitric Oxide:physiology, Nitric Oxide Synth.   

: Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. Considering the existence of the nitric oxide/ nitric oxide synthase system in the peripheral neural system and in the ovary, the aim of this work was to analyze if the liberation of NO in the ovarian compartment of prepubertal rats is of ovarian and/or ganglionic origin. The analysis is carried out from a physiological point of view using the experimental coeliac ganglion--Superior Ovarian Nerve--ovary model with and without ganglionic cholinergic stimulus Acetylcholine (Ach) 10(-6) M. Non selective and selective inhibitors of the synthase nitric oxide enzyme were added to the ovarian and ganglionic compartment, and the liberation of nitrites (soluble metabolite of the nitric oxide) in the ovarian incubation liquid was measured. We found that the non-selective inhibitor L-nitro-arginina methyl ester (L-NAME) in the ovarian compartment decreased the liberation of nitrites, and that Aminoguanidine (AG) in two concentrations in a non-dose dependent form provoked the same effect. The addition of Ach in ganglion magnified the effect of the inhibitors of the NOS enzyme. The most relevant results after the addition of inhibitors in ganglion were obtained with AG 400 and 800 microM. The inhibition was made evident with and without the joint action of Ach in ganglion. These data suggest that the greatest production of NO in the ovarian compartment comes from the ovary, mainly the iNOS isoform, though the coeliac ganglion also contributes through the superior ovarian nerve but with less quantity....

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Journal Article 2004; 25(1-2): 57-61 PubMed PMID: 15159684 Citation  Keywords:  Animals, Bivalvia, Enzyme Inhibitors:pharmacology, Feedback, Physiological:physiology, Ganglia, Invertebrate:cytology, Interleukin-1:physiology, Leukocytes:drug effects, Lymphocyte Activation:physiology, NG-Nitroarginine Methyl Ester:pharmacology, Neuroim.   

: Pedal ganglia excised and maintained in culture for up to 2 h, release NO at low levels. The range can vary between 0 to 1.1 nM. Non-stimulated immunocytes do not significantly stimulate ganglionic NO release when incubated with pedal ganglia. However, ganglia exposed to immunocytes that had been previously activated by a 30 min incubation with interleukin 1 beta, release NO significantly above basal levels. In these experiments, 91 +/- 2.5% of the non-stimulated immunocytes exhibited form factors in the 0.72 to 0.89 range (sampled prior to ganglionic addition), whereas 62 +/- 10.3% of the interleukin 1 beta stimulated immunocytes had form factors in the 0.39 to 0.49 range, demonstrating activation. Addition of the nitric oxide synthase inhibitor, L-NAME (10(-4) M), inhibited basal ganglionic NO release as well as that initiated by exposing the ganglia to activated immunocytes. Interestingly, non activated immunocytes, following ganglionic exposure, exhibited activity levels in the 13% range, representing a non significant increase. Cells exposed to interleukin 1 beta had a 65% activity level at the beginning of the experiment, followed by a drop of activity to 19 +/- 3.2% after ganglionic exposure. Repeating this last observation in the presence of L-NAME (10(-4) M), brought the activity level of the immunocytes back to the pre-ganglionic exposure level of activity, demonstrating that ganglionic NO was involved in down regulating immunocyte activity....

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