Hepatic biotransformation of non-psychotropic phytocannabinoids and activity screening on cytochromes P450 and UDP-glucuronosyltransferases.

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7.

Effects of zinc porphyrin and zinc phthalocyanine derivatives in photodynamic anticancer therapy under different partial pressures of oxygen in vitro.

Photodynamic therapy (PDT) is gradually becoming an alternative method in the treatment of several diseases. Here, we investigated the role of oxygen in photodynamically treated cervical cancer cells (HeLa). The effect of PDT on HeLa cells was assessed by exposing cultured cells to disulphonated zinc phthalocyanine (ZnPcS2) and tetrasulphonated zinc tetraphenylporphyrin (ZnTPPS4). Fluorescence microscopy revealed their different localizations within the cells. ZnTPPS4 seems to be mostly limited to the cytosol and lysosomes, whereas ZnPcS2 is most likely predominantly attached to membrane structures, including plasmalemma and the mitochondrial membrane. Phototoxicity assays of PDT-treated cells carried out under different partial pressures of oxygen showed dose-dependent responses. Interestingly, ZnPcS2 was also photodynamically effective at a minimal level of oxygen, under a nitrogen atmosphere. On the other hand, hyperbaric oxygenation did not lead to a higher PDT efficiency of either photosensitizer. Although both photosensitizers can induce a significant drop in mitochondrial membrane potential, ZnPcS2 has a markedly higher effect on mitochondrial respiration that was completely blocked after two short light cycles. In conclusion, our observations suggest that PDT can be effective even in hypoxic conditions if a suitable sensitizer is chosen, such as ZnPcS2, which can inhibit mitochondrial respiration.

Dual Effect of Taxifolin on ZEB2 Cancer Signaling in HepG2 Cells.

Polyphenols, secondary metabolites of plants, exhibit different anti-cancer and cytoprotective properties such as anti-radical, anti-angiogenic, anti-inflammation, or cardioprotective. Some of these activities could be linked to modulation of miRNAs expression. MiRNAs play an important role in posttranscriptional regulation of their target genes that could be important within cell signalling or preservation of cell homeostasis, e.g., cell survival/apoptosis. We evaluated the influence of a non-toxic concentration of taxifolin and quercetin on the expression of majority human miRNAs via Affymetrix GeneChip™ miRNA 3.0 Array. For the evaluation we used two cell models corresponding to liver tissue, Hep G2 and primary human hepatocytes. The array analysis identified four miRNAs, miR-153, miR-204, miR-211, and miR-377-3p, with reduced expression after taxifolin treatment. All of these miRNAs are linked to modulation of ZEB2 expression in various models. Indeed, ZEB2 protein displayed upregulation after taxifolin treatment in a dose dependent manner. However, the modulation did not lead to epithelial mesenchymal transition. Our data show that taxifolin inhibits Akt phosphorylation, thereby diminishing ZEB2 signalling that could trigger carcinogenesis. We conclude that biological activity of taxifolin may have ambiguous or even contradictory outcomes because of non-specific effect on the cell.

Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes.

The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

Dehydrosilybin attenuates the production of ROS in rat cardiomyocyte mitochondria with an uncoupler-like mechanism.

Reactive oxygen species (ROS) originating from mitochondria are perceived as a factor contributing to cell aging and means have been sought to attenuate ROS formation with the aim of extending the cell lifespan. Silybin and dehydrosilybin, two polyphenolic compounds, display a plethora of biological effects generally ascribed to their known antioxidant capacity. When investigating the cytoprotective effects of these two compounds in the primary cell cultures of neonatal rat cardiomyocytes, we noted the ability of dehydrosilybin to de-energize the cells by monitoring JC-1 fluorescence. Experiments evaluating oxygen consumption and membrane potential revealed that dehydrosilybin uncouples the respiration of isolated rat heart mitochondria albeit with a much lower potency than synthetic uncouplers. Furthermore, dehydrosilybin revealed a very high potency in suppressing ROS formation in isolated rat heart mitochondria with IC(50) = 0.15 µM. It is far more effective than its effect in a purely chemical system generating superoxide or in cells capable of oxidative burst, where the IC(50) for dehydrosilybin exceeds 50 µM. Dehydrosilybin also attenuated ROS formation caused by rotenone in the primary cultures of neonatal rat cardiomyocytes. We infer that the apparent uncoupler-like activity of dehydrosilybin is the basis of its ROS modulation effect in neonatal rat cardiomyocytes and leads us to propose a hypothesis on natural ischemia preconditioning by dietary polyphenols.

Uncouple my heart: the benefits of inefficiency.

Myocardial ischemia/reperfusion (IR) injury leads to structural changes in the heart muscle later followed by functional decline due to progressive fibrous replacement. Hence approaches to minimize IR injury are devised, including ischemic pre-and postconditioning. Mild uncoupling of oxidative phosphorylation is one of the mechanisms suggested to be cardioprotective as chemical uncoupling mimics ischemic preconditioning. Uncoupling protein 2 is proposed to be the physiological counterpart of chemical uncouplers and is thought to be a part of the protective machinery of cardiomyocytes. Morphological changes in the mitochondrial network likely accompany the uncoupling with mitochondrial fission dampening the signals leading to cardiomyocyte death. Here we review recent data on the role of uncoupling in cardioprotection and propose that low concentrations of dietary polyphenols may elicit the same cardioprotective effect as dinitrophenol and FCCP, perhaps accounting for the famed "French paradox".

The effect of quercetin on microRNA expression: A critical review.

Quercetin, a flavonoid with multiple proven health benefits to both man and animals, displays a plethora of biological activities, collectively referred to as pleiotropic. The most studied of these are antioxidant and anti-inflammatory but modulation of signalling pathways is important as well. One of the lesser-known and recently discovered roles of quercetin, is modulation of microRNA (miRNA) expression. miRNAs are important posttranscriptional modulators that play a critical role in health and disease and many of these non-coding oligonucleotides are recognized as oncogenic or tumor suppressor miRNAs. This review is an evaluation of the recent relevant literature on the subject, with focus on the ability of quercetin to modulate miRNA expression. It includes a summary of recent knowledge on miRNAs deregulated by quercetin, an overview of quercetin pharmacokinetics and miRNA biogenesis, for the interested reader.

Cardioprotective effect of 2,3-dehydrosilybin preconditioning in isolated rat heart.

2,3-dehydrosilybin (DHS) is a minor component of silymarin, Silybum marianum seed extract, used in some dietary supplements. One of the most promising activities of this compound is its anticancer and cardioprotective activity that results, at least partially, from its cytoprotective, antioxidant, and chemopreventive properties. The present study investigated the cardioprotective effects of DHS in myocardial ischemia and reperfusion injury in rats. Isolated hearts were perfused by the Langendorff technique with low dose DHS (100 nM) prior to 30 min of ischemia induced by coronary artery occlusion. After 60 min of coronary reperfusion infarct size was determined by triphenyltetrazolium staining, while lactatedehydrogenase activity was evaluated in perfusate samples collected at several timepoints during the entire perfusion procedure. Signalosomes were isolated from a heart tissue after reperfusion and involved signalling proteins were detected. DHS reduced the extent of infarction compared with untreated control hearts at low concentration; infarct size as proportion of ischemic risk zone was 7.47 ±â€¯3.1% for DHS versus 75.3 ±â€¯4.8% for ischemia. This protective effect was comparable to infarct limitation induced by ischemic preconditioning (22.3 ±â€¯4.5%). Selective inhibition of Src-family kinases with PP2 (4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine) abrogated the protection afforded by DHS. This study provides experimental evidence that DHS can mediate Src-kinase-dependent cardioprotection against myocardial damage produced by ischemia/reperfusion injury.

Chelerythrine and dihydrochelerythrine induce G1 phase arrest and bimodal cell death in human leukemia HL-60 cells.

A quaternary benzo[c]phenanthridine alkaloid chelerythrine displays a wide range of biological activities including cytotoxicity to normal and cancer cells. In contrast, less is known about the biological activity of dihydrochelerythrine, a product of chelerythrine reduction. We examined the cytotoxicity of chelerythrine and dihydrochelerythrine in human promyelocytic leukemia HL-60 cells. After 4h of treatment, chelerythrine induced a dose-dependent decrease in the cell viability with IC50 of 2.6 microM as shown by MTT reduction assay. Dihydrochelerythrine appeared to be less cytotoxic since the viability of cells exposed to 20 microM dihydrochelerythrine for 24h was reduced only to 53%. Decrease in the viability induced by both alkaloids was accompanied by apoptotic events including the dissipation of mitochondrial membrane potential, activation of caspase-9 and -3, and appearance of cells with sub-G1 DNA. Moreover, chelerythrine, but not dihydrochelerythrine, elevated the activity of caspase-8. A dose-dependent induction of apoptosis and necrosis by chelerythrine and dihydrochelerythrine was confirmed by annexin V/propidium iodide dual staining flow cytometry. Besides, both alkaloids were found to induce accumulation of HL-60 cells in G1 phase of the cell cycle. We conclude that both chelerythrine and dihydrochelerythrine affect cell cycle distribution, activate mitochondrial apoptotic pathway, and induce apoptosis and necrosis in HL-60 cells.

On the causes and consequences of the uncoupler-like effects of quercetin and dehydrosilybin in H9c2 cells.

Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50µM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.

Involvement of cytoskeleton in AhR-dependent CYP1A1 expression.

Cytochrome P450 (CYP) 1A1 attracts attention mainly because of its role in production of carcinogenic reactive metabolites from polycyclic aromatic hydrocarbons such as benzo[a]pyrene, but recent developments indicate its apparent role in cell cycle progression. Expression of the enzyme is subject to regulation by aryl hydrocarbon receptor (AhR). It has been shown that induction of CYP 1A1 in HepG2 cells and primary rat hepatocytes by tetrachloro-p-dibenzodioxin (TCDD) is diminished by colchicine and nocodazole. Both compounds decrease CYP1A1 mRNA, protein, and activity levels in HepG2 cells and mRNA level in primary rat hepatocytes. Neither compound significantly affected [(3)H]-TCDD binding to AhR, thus their effect on AhR transcriptional activity proceeds via indirect means. For colchicine and nocodazole are well-known microtubule interfering agents, we also assessed their effect on microtubule integrity in both cell types under investigation. Both compounds disrupt cytoskeleton integrity with differential potency depending on cell type. The observed suppression of AhR transcriptional activity by colchicine and nocodazole can be associated with G2/M cell cycle arrest in HepG2 cells, as demonstrated by Myt1 protein hyperphosphorylation and FACS analysis. However, in primary rat hepatocytes, cytoskeleton disruption is independent of cell cycle while displaying the same influence on AhR-dependent gene transcription. In our view, this is evidence in favor of modulatory role of cytoskeleton in AhR-dependent expression.

Disruption of microtubules leads to glucocorticoid receptor degradation in HeLa cell line.

The role of microtubules (MTs) in steroid hormone-dependent human glucocorticoid receptor (hGR) activation/translocation is controversial. It was demonstrated recently that colchicine (COL) down-regulates hGR-driven genes in primary human hepatocytes by a mechanism involving inhibition of hGR translocation to the nucleus. To investigate whether inhibition of hGR translocation is the sole reason for its inactivation, we used human cervical carcinoma cells (HeLa) as a model. Herein we present evidence that perturbation of microtubules in HeLa cells leads to rapid time- and dose-dependent degradation of hGR protein. Degradation is proteasome mediated as revealed by its reversibility by proteasome inhibitor MG132. Moreover, degradation was observed for neither wt-hGR nor hGR mutants S226A and K419A in transiently transfected COS-1 cells. On the other hand, c-jun N-terminal kinase (JNK) seems not to be involved in the process because JNK inhibitor 1,9-Pyrazoloanthrone (SP600125) does not reverse hGR degradation. Similarly, another hGR functional antagonist, nuclear factor kappa beta (NFkappaB), did not play any role in the degradation process.

Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction.

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.

Microtubule disruptors and their interaction with biotransformation enzymes.

Microtubule disruptors, widely known as antimitotics, have broad applications in human medicine, especially as anti-neoplastic agents. They are subject to biotransformation within human body frequently involving cytochromes P450. Therefore antimitotics are potential culprits of drug-drug interactions on the level of activity as well as expression of cytochromes P450. This review discusses the effects of four well-known natural antimitotics: colchicine, taxol (paclitaxel), vincristine, and vinblastine, and a synthetic microtubule disruptor nocodazole on transcriptional activity of glucocorticoid and aryl hydrocarbon receptors. It appears that microtubules disarray restricts the signaling by these two nuclear receptors regardless of cell cycle phase. Consequently, intact microtubules play an important role in the regulation of expression of cytochromes P450, which are under direct or indirect control of the two nuclear receptors.

Silymarin Constituent 2,3-Dehydrosilybin Triggers Reserpine-Sensitive Positive Inotropic Effect in Perfused Rat Heart.

2,3-dehydrosilybin (DHS) is a minor flavonolignan component of Silybum marianum seed extract known for its hepatoprotective activity. Recently we identified DHS as a potentially cardioprotective substance during hypoxia/reoxygenation in isolated neonatal rat cardiomyocytes. This is the first report of positive inotropic effect of DHS on perfused adult rat heart. When applied to perfused adult rat heart, DHS caused a dose-dependent inotropic effect resembling that of catecholamines. The effect was apparent with DHS concentration as low as 10 nM. Suspecting direct interaction with ß-adrenergic receptors, we tested whether DHS can trigger ß agonist-dependent gene transcription in a model cell line. While DHS alone was unable to trigger ß agonist-dependent gene transcription, it enhanced the effect of isoproterenol, a known unspecific ß agonist. Further tests confirmed that DHS could not induce cAMP accumulation in isolated neonatal rat cardiomyocytes even though high concentrations (≥ 10 µM) of DHS were capable of decreasing phosphodiesterase activity. Pre-treatment of rats with reserpine, an indole alkaloid which depletes catecholamines from peripheral sympathetic nerve endings, abolished the DHS inotropic effect in perfused hearts. Our data suggest that DHS causes the inotropic effect without acting as a ß agonist. Hence we identify DHS as a novel inotropic agent.

Anti-/pro-oxidant effects of phenolic compounds in cells: are colchicine metabolites chain-breaking antioxidants?

Effective scavenging of reactive radicals and low reactivity of generated secondary antioxidant radicals towards vital intracellular components are two critical requirements for a chain-breaking antioxidant. Tubulin-binding properties aside, colchicine metabolites remain largely untested for other possible biological activities, including antioxidant activity. Mourelle et al. [Life Sci. 45 (1989) 891] proposed that colchiceine (EIN) acts as an antioxidant and protective agent against lipid peroxidation in a rat model of liver injury. Since EIN as well as two other colchicine metabolites, 2-demethylcolchicine (2DM) and 3-demethylcolchicine (3DM), possess a hydroxy-group on their carbon ring that could participate in radical scavenging, we tested whether they can act as chain-breaking antioxidants. Using our fluorescence-HPLC assay with metabolically incorporated oxidation-sensitive cis-parinaric acid (PnA) we studied the effects of colchicine metabolites on peroxidation of different classes of membrane phospholipids in HL-60 cells. None of the colchicine metabolites in concentrations ranging from 10(-6) to 10(-4) M was able to protect phospholipids against peroxidation induced by either azo-initiators of peroxyl radicals or via myeloperoxidase (MPO)-catalyzed reactions in the presence of hydrogen peroxide. However, the metabolites did exhibit dose-dependent depletion of glutathione, resembling the behavior of etoposide, a hindered phenol with antioxidant properties against lipid peroxidation. Electron spin resonance (ESR) experiments demonstrated that in a catalytic system containing horseradish peroxidase (HRP)/H(2)O(2), colchicine metabolites undergo one-electron oxidation to form phenoxyl radicals that, in turn, cause ESR-detectable ascorbate radicals by oxidation of ascorbate. Phenoxyl radicals of colchicine metabolites formed through MPO-catalyzed H(2)O(2)-dependent reactions in HL-60 cells and via HRP/H(2)O(2) in model systems can also oxidize GSH. Thus, colchicine metabolites possess the prerequisites of many antioxidants, i.e. a nucleophilic hydroxy-group on a carbon ring and the ability to scavenge reactive radicals and form a secondary radical. However, the latter retain high reactivity towards critical biomolecules in cells such as lipids, thiols, ascorbate, thereby, rendering colchicine metabolites effective radical scavengers but not effective chain-breaking antioxidants.

Sanguinarine is a potent inhibitor of oxidative burst in DMSO-differentiated HL-60 cells by a non-redox mechanism.

Sanguinarine (SA), a member of the benzo[c]phenanthridine isoquinoline alkaloids, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. We examined the effects of SA on oxidative burst in DMSO-differentiated HL-60 cells, an excellent model for studying oxidative burst. SA inhibited both N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst with half-maximal concentration for inhibition (IC(50)) of 1.5 and 1.8 microM, respectively. Despite suggestions of SA antioxidant activity this inhibition cannot be ascribed to radical scavenging property of SA because the IC(50) for superoxide dismutase-like activity in a non-cellular system was 60 microM. TROLOX, a water-soluble vitamin E analog, had IC(50) of 3 microM in the same system. Moreover, cyclic voltammetry measurements show that SA is not an easily oxidizable species, with a peak anodic potential at 700 mV, as compared to TROLOX with peak anodic potential at 200 mV. On the other hand, TROLOX, when used in cell suspension, was much poorer inhibitor of oxidative burst than SA. When testing direct effect of SA on NADPH oxidase in the post-granular fraction of disrupted cells, the IC(50) was found to be 8.3 microM. It is higher than that observed in whole cells, however, the shift may be ascribed to SDS effect on SA activity. We conclude the SA inhibition of oxidative burst is not caused by SA redox activity but most likely is a result of SA affecting the activity of NADPH oxidase directly and in part by preventing the formation of NADPH oxidase protein complex.

Involvement of cytochrome P450 1A in sanguinarine detoxication.

Sanguinarine (SA), a member of the benzo[c]phenanthridine alkaloids, is a potent anti-microbial agent with anti-inflammatory and anti-neoplastic properties. However, toxicity of the alkaloid severely limits its medical applications. Recent report by Williams et al. implicated rat hepatic cytochrome P450 (CYP) 1A2 as a likely modulator of SA toxicity. Indeed, the in vitro toxicity of SA in primary culture of rat hepatocytes and human hepatic cell line HepG2, demonstrated as lactate dehydrogenase leakage and metabolic capability (MTT assay), was diminished following induction of CYP1A by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphtoflavone. Using microsomes containing recombinant CYP1A1 or CYP1A2 we show that SA causes non-competitive inhibition of the former and competitive inhibition of the latter as assessed by ethoxyresorufin de-ethylation (EROD). In human hepatic microsomes SA exhibits competitive inhibition of EROD activity with apparent K(i) of 2 microM, a value identical to that observed for CYP1A2 inhibition in recombinant system. Pre-incubation of SA with human liver microsomes resulted in time-dependent, but not dose-dependent decline in EROD activity suggesting CYP1A2 inhibition is not mechanism based. SA also inhibits activity of NADPH:CYP reductase, an enzyme required for CYP activity, with IC(50) very similar to that observed for EROD inhibition. Tentative mechanism for CYP1A involvement in decreased in vitro SA toxicity is discussed.

Oxidative burst of Kupffer cells: target for liver injury treatment.

Liver injury, and frequent consequent fibrosis, is the focus of a number of research groups ranging from molecular biologists to clinicians. It encompasses many aspects and approaches. Among biochemical events the role of Kupffer cells, oxidative stress and pro-inflammatory mediators are eminent. In this review we focus on recent findings into use of natural substances for the modulation of oxidative burst as well as production of inflammatory mediators by Kupffer cells.

N-formyl-Met-Leu-Phe-induced oxidative burst in DMSO-differentiated HL-60 cells requires active Hsp90, but not intact microtubules.

In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.