Biomedical subjects

Zdenek Dvorák

Publications and source records attributed to Zdenek Dvorák.

13 recordsLinked to original sources

PubMed2006

Cytotoxicity of sanguinarine in primary rat hepatocytes is attenuated by dioxin and phenobarbital.

Putative interactions between quaternary benzo[c]phenanthridine alkaloid sanguinarine (SA) and aryl hydrocarbon receptor/cytochrome P450 CYP1A (AhR/CYP1A) regulatory pathway are the subject of perpetual disputations. The role of CYP1A enzymes and AhR receptor in SA cytotoxicity was anticipated. In this paper, we tested, whether selected inducers of CYP enzymes modulate cytotoxicity of SA in primary cultures of rat hepatocytes. Cells were challenged 48h with dioxin (TCDD; 5nM), phenobarbital (PB; 500microM) or DMSO prior to the treatment with SA. SA itself displayed time- and dose-dependent cytotoxicity as revealed by lactate dehydrogenase leakage into the medium and MTT test. Pre-treatment of hepatocytes with TCDD and/or PB significantly attenuated SA cytotoxicity, the effects being more pronounced at lower concentrations of SA and shorter periods of incubation. We assumed involvement of CYP1A enzymes in diminution of SA cytotoxicity. Surprisingly, co-treatment with SA and furafylline, an inhibitor of CYP1A enzymes, further attenuated SA cytotoxicity instead of expected reversal of this effect. We conclude that TCDD- and PB-inducible genes attenuate cytotoxicity of SA in rat hepatocytes. CYP1A enzymes are not involved in this attenuation, but they rather augment SA cytotoxicity. Future research should focus on analyses of the involvement of other CYPs in SA cytotoxicity and on identification of TCDD-/PB-controlled genes responsible for observed phenomenon.

Alkaloids↗

PubMed2006

Quaternary benzo[c]phenathridine alkaloids sanguinarine and chelerythrine do not affect transcriptional activity of aryl hydrocarbon receptor: analyses in rat hepatoma cell line H4IIE.luc.

Quaternary benzo[c]phenanthridine alkaloids (QBAs) sanguinarine and chelerythrine exert a plethora of biological activities. Nevertheless, the specific cellular target for these alkaloids within the cell was not identified as far. Several literary data indicate that biological effects of QBAs could be associated with aryl hydrocarbon receptor (AhR) signaling pathway, including cytochrome P450 CYP1A, however, available information are controversial. In this work we analyzed the effects of sanguinarine and chelerythrine on AhR activity in rat hepatoma cells HII4E.luc stably transfected with dioxin responsive element fused to luciferase gene (DRE-LUC). Studied QBAs were tested in submicromolar concentration range (0.0001-1 microM) and in incubation times 6, 24 and 48 h. Transcriptional activity of AhR was monitored by chemiluminiscence measurement of luciferase catalytic activity. Sanguinarine and chelerythrine did not activated AhR in any time or dose tested. Chelerythrine (1 microM) but not sanguinarine caused moderate inhibition of AhR activation by 10 picomolar dioxin (exponential phase of receptor activation). In contrast, AhR activation by 2.5 nM dioxin (saturated receptor) was not affected by either alkaloid tested. In conclusion, the findings presented here favor rather for inactivity or modest inhibitory effect of QBAs on AhR signaling pathways in vitro than for the activation of the receptor. Regarding the concentrations of QBAs occurring in vivo, the use of products containing sanguinarine and/or chelerythrine has low toxicological potential in terms of the interactions with AhR signaling pathways.

Alkaloids↗

PubMed2006

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.

Animals↗

PubMed2005

Differential effects of selected natural compounds with anti-inflammatory activity on the glucocorticoid receptor and NF-kappaB in HeLa cells.

Natural compounds have been used in the treatment of various diseases for centuries. Herein, we investigated the effects of structurally diverse alkaloids with anti-inflammatory activity (berberine, sanguinarine, chelerythrine, and colchicine) on two important anti-inflammatory and pro-inflammatory players, i.e. glucocorticoid receptor (GR) and nuclear factor kappa B (NF-kappaB), respectively. Sanguinarine and chelerythrine elicited nuclear translocation of the p65 subunit of NF-kappaB. The nuclear import of p65 was strongly augmented by these akaloids in non-stimulated cells as well as in cells challenged with tumor necrosis factor alpha (TNFalpha). Colchicine and berberine had no effect on p65 nuclear translocation regardless of the presence or absence of TNFalpha. Colchicine caused rapid degradation of the GR protein, whereas berberine had no effect on GR content or cellular localization. Sanguinarine and chelerythrine induced accumulation of GR in the nucleus with concomitant diminution of cytosolic GR. Analyses on the transcriptional activity of GR and NF-kappaB monitored by reporter assays using HeLa cells transiently transfected with glucocorticoid response element (pGRE-LUC) and/or NF-kappaB elements fused to luciferase gene (pNF-kappaB-luc) showed that none of the compounds tested had the capability to trigger GR and/or NF-kappaB transcriptional activities, respectively. The ligand binding assay showed that colchicine and berberine are not GR ligands whereas sanguinarine and chelerythrine significantly decreased binding of (3)H-labelled dexamethasone to GR. In conclusion, structurally diverse natural antiflogistics displayed differential effects on GR and NF-kappaB in HeLa cells.

Alkaloids↗

PubMed2005

Activation of the aryl hydrocarbon receptor by berberine in HepG2 and H4IIE cells: Biphasic effect on CYP1A1.

Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE.luc). AhR was activated by high doses of berberine (10-50 microM) after 6 and 24 h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE.luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (<1 microM) and prolonged times of the treatments (48 h) failed to produce any activation of AhR in H4IIE.luc cell line. HPLC analysis ruled out the hypothesis that the loss of berberine capacity to activate AhR in H4IIE.luc cells is due to metabolic inactivation of the alkaloid. We demonstrate that berberine is a potent inhibitor (IC50=2.5 microM) of CYP1A1 catalytic activity (EROD) in HepG2 cell culture and in recombinant CYP1A1 protein. Collectively, our results imply that while berberine activates the Ah receptor, it is accompanied by inactivation of the catalytic activity of CYP1A1 and occurs at concentrations that exceed those predicted to occur in vivo. Given these data, it appears that activation of the AhR pathway by berberine has a low toxicological potential.

Animals↗

PubMed2005

Effect of silybin and its glycosides on the expression of cytochromes P450 1A2 and 3A4 in primary cultures of human hepatocytes.

Four beta-glycosides of flavonoligan silybin, i.e. silybin beta-galactoside, silybin beta-glucoside, silybin beta-maltoside, silybin beta-lactoside were synthesized in order to improve silybin water solubility and bioavailability (Kren et al., J Chem Soc, Perkin Trans 1, 2467-2474, 1997). The presented paper deals with the effect of silybin and its synthetic beta-glycosides on the expression of two major cytochrome P450 isoforms, CYP1A2 and CYP3A4. Primary cultures of human hepatocytes were the model of choice. mRNAs were analyzed using Northern blot and P-radiolabelled probes. CYP protein content was determined by immunoblotting using specific antibodies. Silybin and its beta-glycosides do not induce expression of CYP1A2 and CYP3A4. Tested compounds did not affect inducible expression of CYP1A2 and CYP3A4 by dioxin and rifampicin, respectively, as evaluated at the level of mRNAs and proteins. Silybin and its beta-glycosides do not interfere with the expression of CYP1A2 and CYP3A4, are not likely to produce drug-drug interactions in terms of the inducibility of two important cytochromes P450.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

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.

Animals↗

PubMed2004

Aromatic hydrocarbon receptor status in the metabolism of xenobiotics under normal and pathophysiological conditions.

The aryl hydrocarbon receptor (AhR), a cytosolic protein belonging to the family of nuclear receptors, controls transcription of a wide range of structurally unrelated genes. To date, the most potent AhR ligand is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Exposure to TCDD leads to a number of toxic effects, in particular to tumor promotion and immunosuppression. The function of AhR in cells and living organisms therefore seems to be of paramount importance. Its absence in AhR null mice, results in severe phenotypic abnormalities, such as liver half size with fibrosis, accumulation of retinoic acid and immune system insufficiency. An important role of AhR inheres in its transcriptional control of several biotransformation enzymes (CYP1A1/2,1B1). Hence AhR is the crucial factor in the regulation of xenobiotic metabolism. Under pathophysiological conditions, such as inflammation, the level and activity of the AhR target gene CYP1A is decreased. Thus it is likely, that mediators and/or products of inflammation affect AhR function. This review deals with the role of AhR in xenobiotic metabolism under normal and pathophysiological conditions, with respect to inflammation in particular.

Animals↗

PubMed2004

Microtubule disarray in primary cultures of human hepatocytes inhibits transcriptional activity of the glucocorticoid receptor via activation of c-jun N-terminal kinase.

The human glucocorticoid receptor (hGR) plays a pivotal role in cellular processes such as development, differentiation, homeostasis, immune response and in regulation of xenobiotic metabolism. It has been demonstrated recently that colchicine inhibits hGR transcriptional activity in primary cultures of human hepatocytes by a mechanism involving impairment of hGR nucleo-cytoplasmic shuttling. In the present work, we investigated the role of the nuclear factor kappa B (NFkappaB) and c-jun-N-terminal kinase (JNK), the functional hGR antagonists, in this process. We found that microtubule disarray caused by colchicine, vincristine or nocodazole does not activate NFkappaB in human hepatocytes as revealed by p50 and p65 subunits nuclear translocation. On the other hand, we demonstrate that JNK mediates hGR transcriptional inhibition by microtubules disarray, because a specific inhibitor of JNK, 1,9-pyrazoloanthrone (SP600125), partially blocked tyrosine aminotransferase mRNA suppression due to colchicine treatment. In conclusion, JNK is at least partly involved in hGR transcriptional inhibition by colchicine in human hepatocytes, while NFkappaB involvement is doubtful.

Anthracenes↗

PubMed2003

Primary cultures of human hepatocytes as a tool in cytotoxicity studies: cell protection against model toxins by flavonolignans obtained from Silybum marianum.

The aim of this study was to evaluate the cytoprotective effects upon primary human hepatocytes of silymarin extract and its main flavonolignans following exposure to the cytotoxic actions of model toxins. The conditions for the hepatocyte intoxication were optimised for allyl alcohol, carbon tetrachloride, D-galactosamine and paracetamol. Silymarin extract, silychristin and silydianin did not exert cytotoxicity (10-100 microM), whereas silybin and isosilybin at higher concentrations and after longer incubation periods were cytotoxic. All main flavonolignans of silymarin tested displayed concentration-dependent cytoprotection against the toxic effects of both allyl alcohol and carbon tetrachloride but neither paracetamol nor galactosamine. The best protection was achieved by silydianin and silychristin and to a lesser degree by silymarin, while silybin and isosilybin were less effective. It is concluded that these differing outcomes result from the varying abilities of the Silybum marianum substances tested to stabilize the cell membrane, exert antioxidant properties and exhibit intrinsic toxicity.

Acetaminophen↗

PubMed2003

Pathophysiological factors affecting CAR gene expression.

The body defends itself against potentially harmful compounds, such as drugs and toxic endogenous compounds and their metabolites, by inducing the expression of enzymes and transporters involved in their metabolism and elimination. The orphan nuclear receptor CAR (NR1I3 controls phase I (CYP2B, CYP2C, CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance. Constitutive androstane receptor (CAR) is activated by xenobiotics, such as phenobarbital, but also by toxic endogenous compounds such as bilirubin metabolite(s). To better understand the inter- and intravariability in drug detoxification, we studied the molecular mechanisms involved in CAR gene expression in human hepatocytes. We clearly identified CAR as a glucocorticoid receptor (GR) target gene, and we proposed the hypothesis of a signal transduction where the activation of GR plays a critical function in CAR-mediated cellular response. According to our model, chemicals or pathophysiological factors that affect GR function should decrease CAR function. To test this hypothesis, we recently investigated the effect of microtubule disrupting agents (MIAs) or proinflammatory cytokines. These compounds are well-known inhibitors of GR transactivation property. MIAs activate c-Jun N-terminal kinase (JNK), which phosphorylates and inactivates GR, whereas proinflammatory cytokines, such as IL-6 or IL1beta, induce AP-1 or NF-kB activation, respectively, leading to GR inhibition. As expected, we observed that these molecules inhibit both CAR gene expression and phenobarbital-mediated CYP gene expression in human hepatocytes.

Animals↗

PubMed2003

Approaches to messenger RNA detection - comparison of methods.

Detection of messenger RNA is an important part of current biomedical research, although utilized for decades. This communication endeavors to compare three most commonly used techniques of mRNA detection, i.e. Northern blot, ribonuclease protection assay (RPA), and real-time polymerase chain reaction (RT-PCR). Principles and general procedures of these methods are described, and advantages and weaknesses of each are discussed in terms of their specificity, sensitivity, difficulty, time and material demands as well as health and environmental risks. We conclude that choice of any method discussed depends on particular purpose, experience of the researcher, and on laboratory equipment and organization.

Blotting, Northern↗

PubMed2002

Effect of silybin and its congeners on human liver microsomal cytochrome P450 activities.

Silybin and related flavonolignans form a major part of the Silybum marianum extract, silymarin, which has been used to treat liver diseases for hundreds of years. Although regarded as safe, many of the extract constituents remain thus far untested for their possible effects on liver biotransformation enzymes. Cytochromes P450 (CYP) are very important in this regard. We tested the effect of four flavonolignans: silybin, its hemisynthetic derivative dehydrosilybin, silydianin, and silycristin on three specific CYP activities: bufuralol 1'-hydroxylation (CYP2D6), p-nitrophenol hydroxylation (CYP2E1), and nifedipine oxidation (CYP3A4). All flavonolignans displayed dose-dependent inhibition of these activities with IC(50) values in the micromolar range. The inhibition was competitive or mixed as revealed by double reciprocal plots of kinetic experiments. However, the inhibition is not considered to be relevant for therapy because physiological concentrations of the individual flavonolignans do not exceed 0.5 microM. The data support the use of the extract as a dietary supplement.

Cytochrome P-450 CYP2D6↗