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Aggregation and other intermolecular interactions of biological buffers observed by capillary electrophoresis and UV photometry.

Electrophoretic and photometric experiments strongly indicate that monovalent anions, which arise by deprotonation of the nitrogen atom in zwitterionic Good's buffers 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) and 3-morpholinopropanesulfonic acid (MOPS), spontaneously aggregate. Cationic migration of sanguinarine (SA) and chelerythrine (CHE) in highly alkaline 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris-propane), in which the concentration of cations of both alkaloids is negligible, may be explained by the existence of an aggregate, which contains uncharged sanguinarine or chelerythrine and one monovalent cation of Bis-Tris-propane at least. Tendency of tris(hydroxymethyl)aminomethane (Tris), bis (2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) and Bis-Tris-propane cations to ion pairing with synthetic cluster borane anions and with fused silica markedly rises up with the size and charge of these cations. The drop in mobility of cluster borane compounds sometimes exceeds 50% of their mobility found at identical pH and ionic strength in buffers with sodium cation. The electroosmosis drop approached 70% if background electrolyte contained Bis-Tris-propane cations instead of sodium cations. Nitrate, taken as a model inorganic ion, and four randomly chosen organic anions interacted markedly less with Tris, Bis-Tris and Bis-Tris-propane cations than cluster borane anions. 2-(N-morpholino)ethanesulfonic (MES) acid anions present in background electrolyte affect the ion pairing of Tris, Bis-Tris and Bis-Tris-propane cations with anionic analytes and, in this way influence also mobilites of these anionic analytes. Limited hydrophilicity at least one of interacting species appears to be the most probable cause of observed intermolecular interactions of biological buffers.

Alkaloids↗

Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining counter-current chromatography.

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Corydalis decumbens (Thunb.) Pers. using a multilayer coil planet centrifuge (CPC). The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)-acetonitrile-water (2:2:3, v/v) where triethylamine (5-10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5-10 mM) to the aqueous mobile phase as an eluter. From 3.1 g of the crude extract, 495 mg protopine, 626 mg tetrahydropalmatine and 423 mg bicuculline were obtained each with a purity of over 93% as determined by high performance liquid chromatography. The structures of the isolated compounds were identified by electron ionization mass spectrometry (EI-MS), high-performance liquid chromatography (HPLC)-electrospray ionisation-mass spectrometry (ESI-MS) and 1H NMR.

Acetonitriles↗

NAD(P)H-dependent superoxide production in platelets: the role of angiotensin II and protein kinase C.

OBJECTIVES: Vascular NAD(P)H oxidase represents a major source for excessive superoxide production in hypertension. Angiotensin II (AngII) can activate NAD(P)H oxidase via the angiotensin II type 1 (AT1) receptor and protein kinase C (PKC). Platelets possess AT1 receptors and all the components of the NAD(P)H oxidase system. We employed this tissue model to explore mechanisms involved in AngII-mediated superoxide production. DESIGN AND METHODS: Platelet suspensions from hypertensive patients' blood were activated with AngII or phorbol 12-myristate 13-acetate (PMA). Inhibitors of NAD(P)H oxidase, PKC, and the AT1 receptor were employed to study their effects on superoxide production. RESULTS: Superoxide production was stimulated by AngII and PMA and attenuated by AT1 receptor antagonists (mean percentage reduction 80.2%, P<0.01) and inhibitors of PKC (mean reduction 94.8%, P<0.001) and NAD(P)H oxidase (mean reduction 100%, P< 0.001). CONCLUSIONS: AngII stimulates platelet superoxide production through activation of vascular NAD(P)H oxidase via the AT1 receptor and PKC.

Aged↗

Cardioprotective effects of Ilex paraguariensis extract: evidence for a nitric oxide-dependent mechanism.

AIM: To examine the effects of an Ilex paraguariensis (Ip) extract on postischemic alterations derived from 20 min of global ischemia and 30 min of reperfusion. METHODS: Isolated rat hearts were treated 10 min before ischemia and the first 10 min of reperfusion with Ip 30 microg/ml. In other hearts, chelerythrine (1 microM), a protein kinase C blocker, or l(G)-nitro l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, were administered prior to Ip infusion. Left ventricular developed pressure (LVDP), +dP/dt(max), -dP/dt(max), and left ventricular end diastolic pressure (LVEDP) were used to assess myocardial function. Thiobarbituric acid reactive substances (TBARS) were measured. RESULTS: Ip treatment produced an improvement of postichemic recovery (LVDP=96+/-8%; +dP/dt(max)=95+/-10%; -dP/dt(max)=90+/-12% vs. 57+/-6%, 53+/-6% and 57+/-8%, respectively, in untreated hearts) and an attenuation of the increase of LVEDP and TBARS content. Chelerythrine did not modify and l-NAME abolished the protection induced by Ip. CONCLUSIONS: These data are the first demonstration that Ip extract attenuates the myocardial dysfunction provoked by ischemia and reperfusion and that this cardioprotection involves a diminution of oxidative damage through a nitric oxide-dependent mechanism.

Alkaloids↗

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation.

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.

Alkaloids↗

Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine.

Studies are described on the cytochrome P450 (CYP) isoenzyme dependence of the main metabolic steps of the Eschscholtzia californica alkaloids californine and protopine using rat liver microsomes. Preparations of E. californica are in use as phytopharmaceuticals and as herbal drugs of abuse. CYP isoenzyme dependences were studied using specific chemical inhibitors for CYP1A2, CYP2D1, and CYP3A2 (alpha-naphthoflavone, quinine, and ketoconazole, respectively). CYP2C11 was inhibited by specific antibodies for lack of specific chemical inhibitors. Californine N-demethylation was mainly catalyzed by CYP3A2 and to a minor extent by CYP1A2 and CYP2D1, but not by CYP2C11. CYP2D1 and CYP2C11 were shown to be mainly involved in demethylenation of both, californine and protopine, while CYP1A2 and CYP3A2 showed only minor contribution. Kinetic parameters of the reactions were established. K(m) and V(max) values for the californine N-demethylation were 4.5+/-4.7 microM and 22.9+/-13.7 min/mg protein (high affinity) and 161.3+/-16.7 microM and 311.8+/-39.4 min/mg protein (low affinity), respectively. Californine demethylenation and protopine demethylenation showed substrate inhibition and K(m) and V(max) values were 5.0+/-0.5 and 7.1+/-0.6 microM and 83.3+/-2.6 and 160.7+/-4.0 min/mg protein, respectively.

Alcohol Oxidoreductases↗

Endothelial dysfunction in aortic rings and mesenteric beds isolated from deoxycorticosterone acetate hypertensive rats: possible involvement of protein kinase C.

The main objectives of this study were to investigate the effects of deoxycorticosterone acetate (DOCA)-induced hypertension on the aortic and mesenteric vascular responses to vasodilator and vasoconstrictor agents and also to elucidate whether protein kinase C (PKC) was involved in these responses, by using chelerythrine and calphostin C, the inhibitors of protein kinase C. Hypertension was induced in male Sprague-Dawley rats (200-250 g) by DOCA-salt injection [20 mg/kg, twice weekly for 5 weeks, subcutaneously (s.c.)] and NaCl (1%) was added to their drinking water. Control rats received a saline injection (0.5 ml/kg, twice weekly for 5 weeks, s.c.), then the animals were anaesthetised [thiopental, 30 mg/kg, intraperitoneally (i.p.)] and the arterial blood pressure was measured. Mean arterial blood pressure in control and hypertensive rats were 98+/-7.5 and 163+/-3.5 mmHg, respectively (P<0.0001). In the in vitro studies, rings of descending aorta and mesenteric beds were precontracted with phenylephrine and then concentration-response curves to acetylcholine and sodium nitroprusside were constructed. In the tissue removed from hypertensive rats, the responses to acetylcholine, but not to sodium nitroprusside, were significantly reduced. However, addition of chelerythrine (10 microM) or calphostin C (100 nM) to the organ bath significantly restored these impaired responses. Our data suggest that protein kinase C plays a crucial role in the endothelial dysfunction induced by hypertension.

Acetylcholine↗

Substance P induces inward current and regulates pacemaker currents through tachykinin NK1 receptor in cultured interstitial cells of Cajal of murine small intestine.

We investigated whether substance P modulates pacemaker currents generated in cultured interstitial cells of Cajal of murine small intestine using whole cell patch-clamp techniques at 30 degrees C. Interstitial cells of Cajal generated spontaneous inward currents (pacemaker currents) at a holding potential of -70 mV. Tetrodotoxin, nifedipine, tetraethylammonium, 4-aminopyridine, or glibenclamide did not change the frequency and amplitude of pacemaker currents. However, divalent cations (Ni2+, Mn2+, Cd2+, and Co2+), nonselective cationic channel blockers (gadolinium and flufenamic acid), and a reduction of external Na+ from normal to 1 mM inhibited pacemaker currents indicating that nonselective cation channels are involved in their generation. Substance P depolarized the membrane potential in current clamp mode and produced tonic inward pacemaker currents with reduced frequency and amplitude in voltage clamp mode. [D-Arg1, D-Trp7,9, Leu11] substance P, a tachykinin NK1 receptor antagonist, blocked these substance P-induced responses. Furthermore, [Sar9, Met(O2)11] substance P, a specific tachykinin NK1 receptor agonist, depolarized the membrane and tonic inward currents mimicked those of substance P. Substance P continued to produce tonic inward currents in external Ca2+-free solution or in the presence of chelerythrine, a protein kinase C inhibitor. However, substance P-induced tonic inward currents were blocked by thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum or by an external 1 mM Na+ solution. Our results demonstrate that substance P may modulate intestinal motility by acting on the interstitial cells of Cajal by activating nonselective cation channels via the release of intracellular Ca2+ induced by tachykinin NK1 receptor stimulation.

4-Aminopyridine↗

Inhibitory effect of protopine on K(ATP) channel subunits expressed in HEK-293 cells.

Protopine is an isoquinoline alkaloid purified from Corydalis tubers and other families of medicinal plants. The purpose of the present study was to investigate the effects of protopine on K(ATP) channels and big conductance (BKCa) channels. Protopine concentration-dependently inhibited K(ATP) channel currents in human embryonic kidney cells (HEK-293) which were cotransfected with Kir6.1 and sulfonylurea receptor 1 (SUR1) subunits, but not that with Kir6.1 cDNA transfection alone. At 25 muM, protopine reversibly decreased Kir6.1/SUR1 currents densities from -17.4+/-3 to -13.2+/-2.4 pA/pF at -60 mV (n=5, P<0.05). The heterologously expressed mSlo-encoded BK(Ca) channel currents in HEK-293 cells were not affected by protopine (25 muM), although iberiotoxin (100 nM) significantly inhibited the expressed BK(Ca) currents (n=5, P<0.05). In summary, protopine selectively inhibited K(ATP) channels by targeting on SUR1 subunit. This discovery may help design specific agents to selectively modulate the function of Kir6.1/SUR1 channel complex and facilitate the understanding of the structure-function relationship of specific subtype of K(ATP) channels.

ATP-Binding Cassette Transporters↗

Induction of human leukemia HL-60 cell differentiation via a PKC/ERK pathway by helenalin, a pseudoguainolide sesquiterpene lactone.

Helenalin, a cell-permeable pseudoguainolide sesquiterpene lactone, is a potent anti-inflammatory agent that inhibits nuclear factor-kappa B (NF-kappa B) DNA binding activity. In this report, we investigated the effect of helenalin on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Helenalin by itself markedly induced HL-60 cell differentiation in a concentration-dependent manner. Cytofluorometric analysis and cell morphologic studies indicated that helenalin induced cell differentiation predominantly into granulocytes. Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors significantly inhibited HL-60 cell differentiation induced by helenalin, while p38 mitogen-activated protein kinase (MAPK) inhibitors did not. Moreover, helenalin enhanced PKC activity and protein level of PKC beta I and PKC beta II isoforms, and also increased the level of pERK in a concentration-dependent manner. In addition, the enhanced levels of cell differentiation closely correlated with the decreased levels of NF-kappa B binding activity by helenalin. These results indicate that PKC, ERK, and NF-kappa B may be involved in HL-60 cell differentiation induced by helenalin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Effects of intravenous anesthetics on the activity of glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement.

We investigated the effects of the intravenous anesthetics, thiopental, etomidate and ketamine, on the activity of one type of glutamate transporters, EAAT3 (excitatory amino acid transporter type 3). Rat EAAT3 was expressed in Xenopus oocytes by injection of its mRNA. Using two-electrode voltage clamp, membrane currents were recorded after the application of L-glutamate (30 microM) in the presence or absence of various concentrations of the anesthetics. Thiopental (0.3-30 microM) and ketamine (3-1000 microM) did not affect EAAT3 activity. Etomidate decreased EAAT3 activity in a concentration-dependent manner (0.10-10 microM). Etomidate at 1 microM significantly decreased the Vmax, but not the Km of EAAT3 for glutamate. Chelerythrine, a protein kinase C (PKC) inhibitor, significantly decreased EAAT3 activity, however, there were no statistical differences among the chelerythrine, etomidate or chelerythrine plus etomidate groups. Likewise, the combination of staurosporine, another PKC inhibitor, and etomidate did not decrease the responses further compared with staurosporine or etomidate alone. Phorbol-12-myrisate-13-acetate, a PKC activator, abolished etomidate-induced decrease in EAAT3 activity. Since our results showed that thiopental and ketamine did not affect EAAT3 activity significantly, EAAT3 may not be a target for their anesthetic effects. Our results also suggest that etomidate, possibly via PKC, decreased EAAT3 activity at clinically relevant concentrations.

Alkaloids↗

The beneficial in vitro effects of lovastatin and chelerythrine on relaxatory response to acetylcholine in the perfused mesentric bed isolated from diabetic rats.

Diabetes mellitus is associated with an increased risk of cardiovascular disease. Endothelial dysfunction (i.e. decreased endothelium-dependent vasorelaxation) plays a key role in the pathogenesis of diabetic vascular disease. The present study was undertaken to determine whether diabetes induced by streptozotocin alters mesenteric responses to vasodilators and, if so, to study the acute in vitro effects of lovastatin and chelerythrine. Endothelial function was assessed in constantly perfused preparation removed from rats, 12 weeks after treatment with either saline or streptozotocin (45 mg/kg, intraperitoneally). In pre-contracted mesenteric beds (with 100 microM phenylephrine) removed from diabetic rats, the concentration response curve to acetylcholine, but not to sodium nitroprusside, was significantly reduced. Perfusion with lovastatin (10 microM for 20 min) or chelerythrine (1 microM for 20 min) significantly improved the acetylcholine-mediated relaxation in preparations removed from diabetic but not control rats. Pre-incubation of tissue with N(G)-nitro-L-argenine methyl ester hydrochloride (10 microM for 20 min) inhibited the beneficial effect of lovastatin but not chelerythrine. Pre-treatment of tissue with indomethacin (10 microM for 20 min) did not modify the effects of lovastatin or chelerythrine on acetylcholine responses. The present results demonstrate that endothelial dysfunction induced by diabetes (in a resistant vasculature, such as rat mesenteric bed) may be improved by an acute exposure to either lovastatin or chelerythrine. Furthermore, our results suggest that the beneficial effect of lovastatin is mediated via the nitric oxide pathway.

Acetylcholine↗

Sanguinarine overcomes P-glycoprotein-mediated multidrug-resistance via induction of apoptosis and oncosis in CEM-VLB 1000 cells.

Permeability-glycoprotein (Pgp) positive cells are known to be encoded by the multidrug-resistance gene (MDR1), and characterized by a reduced ability to accumulate drugs. The vinblastin-resistant, Pgp positive CEM-VLB 1000 and its wild type (Pgp-negative and vinblastin-sensitive) counterpart CEM-T4 human leukemia cells, when treated with the alkaloid sanguinarine, were both found to undergo apoptosis at concentrations of 1.5 microg/ml and oncosis/blister cell death (BCD) at concentrations of 12.5 microg/ml. The aim of this study was to assess the ability of sanguinarine to overcome Pgp-mediated multidrug-resistance (MDR), and also to characterize the cell death processes of apoptosis and oncosis (or bimodal cell death) induced by sanguinarine in MDR cells. The cell death processes of apoptosis and oncosis in CEM-VLB 1000 and CEM-T4 cell lines were found to be qualitatively similar when assessed by light microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, annexin-V-binding, trypan blue exclusion and western blot analysis. Western blotting revealed an increase in the Bax/Bcl-2 ratio and activation of caspase-3 in apoptosis but not oncosis in both cell lines. The Pgp-positive CEM-VLB 1000 cells and their wild type CEM-T4 cells were both equally sensitive to sanguinarine. Thus, sanguinarine may overcome the phenomenon of Pgp-mediated MDR by inducing apoptosis through increasing the Bax/Bcl-2 ratio and activating caspase-3, and oncosis, which involved neither.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland.

The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.

Alkaloids↗

(+/-)3,4-Methylenedioxyamphetamine elicits action potential bursts in a central snail neuron.

The effects of (+/-)3,4-methylenedioxyamphetamine (MDA) were studied in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. The RP4 neuron generated spontaneous action potentials. Extracellular or intracellular application of MDA elicited action potential bursts of the central RP4 neuron. The action potential bursts elicited by MDA were not blocked when neurons were immersed in high-Mg2+ solution, Ca2+-free solution, nor after continuous perfusion with atropine, d-tubocurarine, propranolol, prazosin, haloperidol, sulpiride or methiothepin. Notably, the induction of action potential bursts was blocked by pretreatment with protein kinase C (PKC) inhibitors, chelerythrine and Ro 31-8220, but not by protein kinase A (PKA) inhibitors, KT-5720 and H89, nor by the phospholipase C (PLC) inhibitor, U73122. PKC activators, i.e., phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acety-sn-glycerol (OAG; a membrane-permeant DAG analog), facilitate the induction of action potential bursts elicited by MDA. Voltage-clamp studies revealed that MDA decreased the delayed rectifying K+ current (I(KD)) of the RP4 neuron. Further, although Ro 31-8220 did not affect the I(KD), Ro 31-8220 decreased the inhibitory effect of MDA on the I(KD). These results suggest that the generation of action potential bursts elicited by MDA was not due to (1) the synaptic effects of neurotransmitters, (2) the cholinergic, adrenergic, dopaminergic or serotoninergic receptors of the excitable membrane. Instead, the MDA-elicited action potential bursts are closely related to PKC activity and the inhibitory effects on the I(KD).

Action Potentials↗

Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment.

Sanguinaria canadesis, Chelidonium majus and Macleya cordata have been used for centuries as alternative medicines. Currently the extracts from these medicinal plants are components of veterinary and human phytopreparations, and of oral-hygiene agents. Sanguinarine and chelerythrine (SA/CHE) are biologically active components of these extracts. They display distinct antibacterial and anti-inflammatory properties, but, on the other hand, they have been reported as having adverse effects - genotoxicity and hepatotoxicity. This paper is aimed at evaluation of the effects of daily administration of the extract from Macleya cordata (2 mg and 100 mg in 1 kg feed, sanguinarine:chelerythrine 3:1) in the diet on the health status of swine. After 90-day administration, alkaloids were retained to a different extent in tissues. The highest SA/CHE retention was detected in the gingiva (0.55 microg/g) and liver (0.15 microg/g), no SA/CHE were detected in muscles. Plasma SA levels attained 0.11 microg/ml. Treated animals did not display any results of hematological, biochemical or histological assay different from controls. A (32)P-postlabeling assay proved that no DNA-adducts with SA/CHE were detected in pig livers. We did not observe any symptom linked to epidemic dropsy syndrome often attributed to sanguinarine. In conclusion, an average daily oral dose of alkaloids up to 5 mg per 1 kg animal body weight proved to be safe.

Alkaloids↗

In vivo DNA damaging potential of sanguinarine alkaloid, isolated from argemone oil, using alkaline Comet assay in mice.

Consumption of mustard oil contaminated with argemone oil is well known to cause clinical manifestation referred to as "Epidemic Dropsy". Our prior studies have shown that argemone oil produces genotoxic effects in mice [Ansari, K.M., et al., 2004. Int. J. Cancer 112, 890]. Since, sanguinarine alkaloid is the major component of argemone oil, the in vivo DNA damaging potential of the isolated alkaloid was investigated in blood and bone marrow cells of mice using alkaline Comet assay. Swiss albino male mice were given single intraperitoneal administration of 1.35, 2.70, 5.40, 10.80 and 21.60 mg sanguinarine alkaloid/kg b wt., while controls were treated with saline in the same manner. The results revealed a dose dependent increase in DNA damage in blood and bone marrow cells following 24 h treatment of sanguinarine alkaloid. All the three parameters of Comet assay including olive tail moment (OTM), tail length and tail DNA showed significant (p<0.05) increases in blood and bone marrow cells at respective doses of 10.80 and 5.40 mg alkaloid/kg b wt. However, some of the parameters were significantly increased even at lower doses of sanguinarine alkaloid (2.70 mg/kg b wt.). The frequency of cells exhibiting greater DNA damage were found to be increased by sanguinarine alkaloid in a concentration dependent manner. These results indicate that single exposure of sanguinarine alkaloid causes DNA damage in blood and bone marrow cells of mice, which could be responsible for the genotoxicity of argemone oil.

Alkaloids↗

Investigation of sanguinarine and chelerythrine effects on CYP1A1 expression and activity in human hepatoma cells.

Quaternary benzo[c]phenanthridine alkaloids (QBA) sanguinarine and chelerythrine exhibit a wide spectrum of biological activities whence they are used in dental care products. Recent studies indicated that cytochrome P450 CYP1A attenuates sanguinarine toxicity both in vivo [Williams, M.K., Dalvi, S., Dalvi, R.R., 2000. Influence of 3-methylcholanthrene pretreatment on sanguinarine toxicity in mice. Vet. Hum. Toxicol. 42, 196-198] and in vitro [Vrba, J., Kosina, P., Ulrichová, J., Modrianský, M., 2004. Involvement of cytochrome P450 1A in sanguinarine detoxication. Toxicol. Lett. 151, 375-387]. However, CYP1A converts sanguinarine to the products that form DNA adducts [Stiborová, M., Simánek, V., Frei, E., Hobza, P., Ulrichová, J., 2002. DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique. Chem. Biol. Interact. 140, 231-242]. In our work we examined the effects of sanguinarine and chelerythrine on CYP1A1 expression and catalytic activity in human hepatoma cells-HepG2. Sanguinarine and chelerythrine did not affect basal and dioxin-inducible expression of CYP1A1 mRNA and protein in HepG2 cells. The enzymatic activity of CYP1A1 was assessed by the fluorescent measurement of 7-ethyxoresorufin-O-deethylase (EROD) activity. We observed a slight decrease of dioxin-induced EROD activity in HepG2 cells by sanguinarine and chelerythrine. This decrease was attributed to the inhibition of CYP1A1 catalytic activity, as revealed by enzyme kinetic studies on recombinant CYP1A1 protein. The IC50 values for the inhibition of CYP1A1 by sanguinarine and chelerythrine were 2.1 and 1.9muM, respectively. In conclusion, albeit the CYP1A modulates QBA cytotoxicity and genotoxicity, the QBA themselves do not affect CYP1A1 expression. The data indicate that studied alkaloids do not have specific cellular target and their biological effects are rather pleiotropic.

Alkaloids↗