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Because of an increasing incidence of malignant tumours of the head and neck there is an unmet medical need for early diagnosis of the primary disease or precancerous lesions, and timely detection of recurrence by simple non-invasive tests. The analysis of biomarkers in body fluids may be appropriate for this goal. In this review, we compare the data on utilization of neopterin and interleukin-6 (IL-6) measurements in saliva and plasma/serum of patients with oral and oropharyngeal squamous cell carcinoma, indicating the suitability of using saliva as a diagnostic matrix in head and neck cancers on behalf of close anatomical proximity and a potential to study the tumour microenvironment. Salivary neopterin and IL-6 are potential biomarkers of head and neck cancer suitable not only for early diagnosis, but also for monitoring of treatment results and detection of the disease recurrence.

Abstract Background Inspired by the unprecedented historical success of cisplatin, one of the most important research directions in bioinorganic and medicinal chemistry is dedicated to the development of new anticancer compounds with the potential to surpass it in antitumor activity, while having lower unwanted side-effects. Therefore, a series of copper(II) mixed-ligand complexes of the type [Cu(qui)(L)]Y · xH2O (1–6), where Hqui = 2-phenyl-3-hydroxy-4(1H)-quinolinone, Y = NO3 (1, 3, 5) or BF4 (2, 4, 6), and L = 1,10-phenanthroline (phen) (1, 2), 5-methyl-1,10-phenanthroline (mphen) (3, 4) and bathophenanthroline (bphen) (5, 6), was studied for their in vitro cytotoxicity against several human cancer cell lines (A549 lung carcinoma, HeLa cervix epitheloid carcinoma, G361 melanoma cells, A2780 ovarian carcinoma, A2780cis cisplatin-resistant ovarian carcinoma, LNCaP androgen-sensitive prostate adenocarcinoma and THP-1 monocytic leukemia). Results The tested complexes displayed a stronger cytotoxic effect against all the cancer cells as compared to cisplatin. The highest cytotoxicity was found for the complexes 4 (IC50 = 0.36 ± 0.05 μM and 0.56 ± 0.15 μM), 5 (IC50 = 0.66 ± 0.07 μM and 0.73 ± 0.08 μM) and 6 (IC50 = 0.57 ± 0.11 μM and 0.70 ± 0.20 μM) against A2780, and A2780cis respectively, as compared with the values of 12.0 ± 0.8 μM and 27.0 ± 4.6 μM determined for cisplatin. Moreover, the tested complexes were much less cytotoxic to primary human hepatocytes than to the cancer cells. The complexes 5 and 6 exhibited significantly high ability to modulate secretion of the pro-inflammatory cytokines TNF-α (2873 ± 238 pg/mL and 3284 ± 139 pg/mL for 5, and 6 respectively) and IL-1β (1177 ± 128 pg/mL and 1087 ± 101 pg/mL for 5, and 6 respectively) tested on the lipopolysaccharide (LPS)-stimulated THP-1 cells as compared with the values of 1173 ± 85 pg/mL and 118.5 ± 4.8 pg/mL found for the commercially used anti-inflammatory drug prednisone. The ability of the tested complexes to interact with sulfur-containing biomolecules (cysteine and reduced glutathione) at physiological levels was proved by electrospray-ionization mass spectrometry. Conclusions Overall positive results of the biological activity studies revealed that the presented complexes may represent good candidates for non-platinum anticancer drugs, however, we are aware of the fact that further and deeper studies mainly in relation to the elucidation of their mechanisms of antiproliferative action will be necessary.

Objective: The extent to which tryptophan (Trp) metabolism alterations explain or influence the outcome of inflammatory bowel diseases (IBDs) is still unclear. However, several Trp metabolism end-products are essential to intestinal homeostasis. Here, we investigated the role of metabolites from the kynurenine pathway., Design: Targeted quantitative metabolomics was performed in two large human IBD cohorts (1069 patients with IBD). Dextran sodium sulphate-induced colitis experiments in mice were used to evaluate effects of identified metabolites. In vitro, ex vivo and in vivo experiments were used to decipher mechanisms involved. Effects on energy metabolism were evaluated by different methods including Single Cell mEtabolism by profiling Translation inHibition., Results: In mice and humans, intestinal inflammation severity negatively correlates with the amount of xanthurenic (XANA) and kynurenic (KYNA) acids. Supplementation with XANA or KYNA decreases colitis severity through effects on intestinal epithelial cells and T cells, involving Aryl hydrocarbon Receptor (AhR) activation and the rewiring of cellular energy metabolism. Furthermore, direct modulation of the endogenous tryptophan metabolism, using the recombinant enzyme aminoadipate aminotransferase (AADAT), responsible for the generation of XANA and KYNA, was protective in rodent colitis models., Conclusion: Our study identified a new mechanism linking Trp metabolism to intestinal inflammation and IBD. Bringing back XANA and KYNA has protective effects involving AhR and the rewiring of the energy metabolism in intestinal epithelial cells and CD4 + T cells. This study paves the way for new therapeutic strategies aiming at pharmacologically correcting its alterations in IBD by manipulating the endogenous metabolic pathway with AADAT., Competing Interests: Competing interests: HS report lecture fee, board membership, or consultancy from Carenity, AbbVie, Astellas, Danone, Ferring, Mayoly Spindler, MSD, Novartis, Roche, Tillots, Enterome, BiomX, Biose, Novartis,Takeda, Biocodex and is cofounder of Exeliom Biosciences., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In vitro antitumour activity of the [Pt(ox)(L(n))(2)] (1-7) and [Pd(ox)(L(n))(2)] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L(n)) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC(50)=3.2+/-1.0 microM and 3.2+/-0.6 microM) and 5 (IC(50)=4.0+/-1.0 microM and 4.1+/-1.4 microM) against A2780 and A2780cis, as compared with 11.5+/-1.6 microM, and 30.3+/-6.1 microM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC(50)=8.2+/-3.8 microM for 12) and A2780 (IC(50)=5.4+/-1.2 microM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC(50) equalled 19.6+/-4.3 microM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic., (Copyright 2010 Elsevier Inc. All rights reserved.)

The reactions of potassium bis(oxalato)platinate dihydrate with two molar equivalents of the potent adenine-based cyclin-dependent kinase inhibitor 2-(1-ethyl-2-hydroxyethylamino)-N6-(benzyl)-9-isopropyladenine (Roscovitine; Ros) and its benzyl-substituted analogues, i.e. 2-(1-ethyl-2-hydroxyethylamino)-N6-(2-methoxybenzyl)-9-isopropyladenine (2OMeRos), 2-(1-ethyl-2-hydroxyethylamino)-N6-(3-methoxybenzyl)-9-isopropyladenine (3OMeRos) and 2-(1-ethyl-2-hydroxyethylamino)-N6-(4-methoxybenzyl)-9-isopropyladenine (4OMeRos), were performed and the [Pt(ox)(Ros)(2)].(3/4)H(2)O (1), [Pt(ox)(2OMeRos)(2)].H(2)O (2), [Pt(ox)(3OMeRos)(2)].(1/2)H(2)O (3) and [Pt(ox)(4OMeRos)(2)].(3/4)H(2)O (4) platinum(II) oxalato complexes were obtained. The methods of the elemental analysis, IR, Raman and NMR spectroscopy, ESI + mass spectrometry, molar conductivity measurement and TG/DTA thermal analysis were performed to characterize the obtained products. The complexes 1-4 involve tetracoordinated central Pt(II) atom with one bidentate-coordinated oxalate dianion (ox) and two monodentate adenine-based molecules (nRos), thus giving the square-planar geometry around the metal centre with a PtN(2)O(2) donor set. In vitro cytotoxic activity of the complexes against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was evaluated. All the tested complexes exceeded the in vitro cytotoxicity of cisplatin and oxaliplatin against HeLa, A2780cis and, except for 2, also against HOS cancer cells. The complex 1 was also tested for its cytotoxicity in primary cultures of human hepatocytes and it was not found to be hepatotoxic up to the concentration of 50.0 microM., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. We compared the efficiencies of human enzymatic systems [hepatic microsomes and cytosols, NAD(P)H:quinone oxidoreductase 1 (NQO1), xanthine oxidase, NADPH:cytochrome P450 reductase, N,O-acetyltransferases, and sulfotransferases] and human primary hepatocytes to activate 2-NBA and its isomer 3-NBA to species forming DNA adducts. In contrast to 3-NBA, 2-NBA was not metabolized at detectable levels by the tested human enzymatic systems and enzymes expressed in human hepatocytes, and no DNA adducts detectable by (32)P-postlabeling were generated by 2-NBA. Even NQO1, the most efficient human enzyme to bioactive 3-NBA, did not activate 2-NBA. Molecular docking of 2-NBA and 3-NBA to the active site of NQO1 showed similar binding affinities; however, the binding orientation of 2-NBA does not favor the reduction of the nitro group. This was in line with the inhibition of 3-NBA-DNA adduct formation by 2-NBA, indicating that 2-NBA can compete with 3-NBA for binding to NQO1, thereby decreasing the metabolic activation of 3-NBA. In addition, the predicted equilibrium conditions favor a 3 orders of magnitude higher dissociation of N-OH-3-ABA in comparison to N-OH-2-ABA. These findings explain the very different genotoxicity, mutagenicity, and DNA adduct forming potential of the two compounds. Collectively, our results suggest that 2-NBA possesses a relatively lower risk to humans than 3-NBA.

We have recently demonstrated that the alkaloid colchicine (COL) inhibits glucocorticoid receptor (GR) transcriptional activity. In addition, we described proteasome-mediated degradation of GR in COL-treated HeLa cells. While these effects were previously attributed to cell cycle arrest in G2/M phase, this explanation is not applicable for nonproliferating cells such as human hepatocytes (HH). In the current study, we compared COL-mediated microtubule disruption and cell cycle arrest with selected GR functions in HeLa cells and HH as models of proliferating and quiescent cells, respectively. Microtubule disruption led to irreversible decrease in GR binding capacity and protein level in HeLa cells. None of the parameters was restored 24 hours after COL withdrawal. In contrast, dexamethasone (DEX) binding was increased in HH at the beginning of the treatment, with following transient activation of extracellular signal-regulated kinase (ERK). The findings of these investigations emphasize the GR-signaling differences between primary and transformed cells.

A series of dinuclear copper(II) complexes of the compositions [Cu(2)(micro-L(n))(2)(micro-Cl)(2)Cl(2)] (1, 2), [Cu(2)(micro-L(n))(4)Cl(2)]Cl(2).2H(2)O (3, 4) and [Cu(2)(micro-L(n))(4)(ClO(4))(2)](ClO(4))(2).xSolv (5, 6; xSolv=4MeOH for 5 and 2EtOH for 6), involving 6-(benzylamino)purine derivatives (L(n)), have been evaluated with the aim to determine their possible drug interactions and their capability to induce the expression of major drug-metabolizing cytochromes P450. The above-mentioned complexes have been chosen based on the fact that substantial both in vitro (cytotoxicity, SOD-mimic) and in vivo (antidiabetic) biological activity has been found for them. As models, primary cultures of human hepatocytes and human hepatoma cells HepG2 transiently transfected with a plasmid containing dioxin-responsive element fused to the luciferase reporter gene (DRE-LUC) have been chosen. It has been found that the tested complexes 1-6 did not significantly induce the expression of CYP1A2 and CYP3A4 mRNAs in the concentration range of 0.1-10.0microM, in three different primary human hepatocyte cultures after 24h of the treatment. On the other hand, the model inducers, i.e. 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and rifampicin, significantly increased the levels of CYP1A2 and CYP3A4 mRNAs in all cultures. In addition, compounds 1-6 did not transactivate DRE-LUC in transiently transfected HepG2, while TCDD strongly induced luciferase activity after 24h of incubation. Based on the obtained results, it may be concluded that the studied dinuclear copper(II) complexes 1-6 possess very low toxicological potential to cause drug interactions in terms of transcriptional activation of the major human cytochromes P450., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

We examined the effects of model activators of mitogen-activated protein kinases (MAPKs) on basal and rifampicin-, phenobarbital- and dioxin-inducible expression of phase I and phase II biotransformation enzymes in primary human hepatocytes. Cells were treated for 24 h with sorbitol (SOR), anisomycin (ANI) and epidermal growth factor (EGF) in the presence or absence of inducers. The levels of CYP1A1, CYP1A2, CYP2B6, CYP3A4, UGT1A1, UGT2B17, SULT1A1, SULT2A1, SULT1B2, GSTA1, GSTA2 mRNAs were determined. SOR and EGF inhibited the expression of the tested genes, while ANI had no effect. We conclude that MAPKs play important role in the transcriptional regulation of drug-metabolizing enzymes.

Mitogen-activated protein kinases (MAPKs) are important regulators of aryl hydrocarbon receptor (AhR). An immense progress in MAPKs' biochemistry was attained with the discovery of their specific pharmacological inhibitors. Unfortunately, the inhibitors of JNK and ERK MAPKs, i.e. SP600125 and U0126, respectively, affect AhR-CYP1A signaling pathway because they are partial agonists of AhR and induce CYP1A genes. This implies that SP600125 and U0126 are inappropriate tools for studies of the role of MAPKs in AhR regulation. The results from studies using SP600125 or U126, past or future, should be interpreted with prudence regarding their stimulatory effects on AhR-CYP1A pathway.

Human populations are increasingly exposed to a number of environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls and dioxins. These compounds are activators of the aryl hydrocarbon receptor (AhR) that controls the expression of many genes including those for detoxification enzymes. The regulatory mechanisms of AhR are multi-factorial and include phosphorylation by various protein kinases. Significant progress in the research of mitogen-activated protein kinases (MAPKs) has been achieved in the last decade. Isolated reports have been published on the role of MAPKs in AhR functions and vice versa, with activation of MAPKs by AhR ligands. This mini-review summarizes current knowledge on the mutual interactions between MAPKs and AhR. The majority of studies has been done on cancer-derived cell lines that have impaired cell cycle regulation and lacks the complete detoxification apparatus. We emphasize the importance of the future studies that should be done on non-transformed cells to distinguish the role of MAPKs in cancer and normal cells. Primary cultures of human or rodent hepatocytes that are equipped with a fully functional biotransformation battery or xenobiotics-metabolizing extra-hepatic tissues should be the models of choice, as the results in our experiments confirm.

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC(50) values not exceeding 4.6 micromol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 micromol/L, sanguinarine and chelerythrine prevented phosphorylation of approximately 80 kDa protein and sequestered approximately 60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCalpha/betaII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCalpha/betaII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of approximately 70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.

We used 2,3,8,9-tetrachlorodibenzo-p-dioxin (TCDD) and dexamethasone (DEX) to examine their effects on aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) in HeLa cells. TCDD (5 nM), DEX (100 nM) and their combination down-regulated GR after 24 h. DEX reversed AhR mRNA increase and AhR protein decrease caused by TCDD. Since AhR-GR cross-talk occurs in cell-type and species-specific manner, the presented data may serve as the basis in the understanding of mechanisms underlying mutual interactions between AhR and GR.

We investigated the effects of novel microtubules interfering agents (MIAs) in primary cultures of rat hepatocytes. Cells were treated for 24 h with a known compound colchicine and newly synthesized derivatives myoseverin, tubulyzine, and E2GG. We examined the effects of MIAs on microtubules network integrity and on the polymerization capability of isolated tubulin. All tested MIAs inhibited microtubules assembly with the following IC(50) values: tubulyzine (4.4 + or - 0.9 micromol/l), myoseverin (7.0 + or - 0.8 micromol/l), E2GG (16 + or - 2 micromol/l), colchicine (2.0 + or - 0.4 micromol/l). The potency of MIAs to perturb microtubular network integrity (monitored by immune-histochemistry) increased in the order tubulyzine < myoseverin < E2GG < colchicine. We described recently deleterious effects of MIAs on the expression of drug metabolizing enzymes, including CYP1A1. Here we observed inhibitory effects of novel MIAs on dioxin-inducible expression of CYP1A1 mRNA in rat hepatocytes. We conclude that novel MIAs exert analogical biological response as classical MIAs such as colchicine or nocodazole. This further supports the hypothesis that tubulin is the primordial target of MIAs within the cell and that perturbation of microtubules dynamics and/or integrity triggers the biological effects described here.

In the present work, the effects of colchicine (COL) and/or all-trans retinoic acid (ATRA) on expression of rexinoid receptors (RXRs) (alpha, beta, gamma), thyroid hormone receptor alpha and coregulators N-CoR, SMRT and SRC-1 mRNA in primary rat hepatocytes as a model of no-proliferating cells were investigated. Treatment with these components, either alone or in combination, induced differences of the expression profiles between distinct treatment groups.

Sanguinarine is an alkaloid with known antibiotic and anti-inflammatory activity and its pharmacokinetics have been studied in the rat after a single oral dose (10 mg kg(-1) body weight). Alkaloid determination in the plasma and liver was carried out by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS). The pharmacokinetic parameters (t(max), c(max), AUC(0-->t) and AUC(0-->infinity)) were determined for sanguinarine and dihydrosanguinarine, the major components detected in plasma. The first step in sanguinarine metabolism in the rat was the reduction of the iminium bond resulting in formation of the less toxic dihydrosanguinarine. Both compounds were completely eliminated from the plasma and liver after 24 h and not detected in urine. After a single oral dose of (3)H-sanguinarine, more than 42% of the ingested radioactivity was present in gastrointestinal tract. Benz[c]acridine, up to date the only sanguinarine metabolite referred to in the literature, was not detected in the plasma, liver or urine.

Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all-trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1microM) down-regulated RARalpha and RARgamma mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARbeta mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid, exhibits antimicrobial and anti-inflammatory activities and for this reason it is used in dental hygiene products and feed additives. Its metabolism and disposition is the subject of constant scientific discourse. In this paper we summarize current knowledge on sanguinarine metabolism. We show in particular that: (i) Sanguinarine is not transformed to 3,4-benzacridine and that the literature reporting this compound as a metabolite of sanguinarine is based on artifacts and misinterpretations that in course of time have created a dogma; (ii) Sanguinarine is converted to dihydrosanguinarine in vivo, the conversion being tentatively a detoxication pathway; (iii) Aryl hydrocarbon receptor metabolic signaling pathways modulate sanguinarine biological activity.

Twenty two patients with gender dysphoria underwent neo-phalloplasties using a novel technique. Latissimus dorsi musculocutaneus re-innervated free flap was used to allow voluntary rigidity of the neo-penis. From the first 22 patients, 18 have obtained motoric function of reconstructed penis; the "paradox erection" was obtained. 14 patients came for examination after a follow-up period of mean 26.4 months. We evaluated the motility and shape changes of neo-phallus measuring its different size and dimension during relax and muscle contraction. The range of neo-phallus length in relaxed position was between 7 and 17 cm (mean 12.2 cm), its circumference in the same position had a range between 13 and 20 cm (mean 13.7 cm). All patients were able to contract the muscle with an average length reduction of 3.08 cm and an average circumference enlargement of 4 cm. In this study, the dimensions and motility were quantified demonstrating the neo-phallus function and size changes during sexual intercourse.

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.

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.

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.

The flavonolignan silybin and its derivative dehydrosilybin have been proposed as candidate UV-protective agents in skin care products. This study addressed the effect of silybin and dehydrosilybin on the activity of cytochrome P450 isoform CYP1A1 in human keratinocytes (HaCaT) and human hepatoma cells (HepG2). CYP1A1 catalytic activity was assessed as O-deethylation of 7-ethoxyresorufin using fluorescence detection. Silybin and dehydrosylibin inhibited basal and dioxin-inducible CYP1A1 catalytic activity in both cell lines used. The inhibitory effect of tested compounds was more pronounced in HaCaT cells than in HepG2 cells, and dehydrosilybin was a much stronger inhibitor than silybin. Analyses on CYP1A1 human recombinant protein yielded IC(50) values of 22.9 +/- 4.7 micromol/L and 0.43 +/- 0.04 micromol/L for silybin and dehydrosilybin, respectively. Since CYP1A enzymes are some of the most prominent actors in the process of chemically induced carcinogenesis, the inhibitory activity of the flavonolignans tested against CYP1A1 favors their use as cytoprotective agents in terms of skin and hepatic metabolism. In addition, the capability of dehydrosilybin to inhibit CYP1A1 in submicromolar concentrations makes this compound a potential biological probe in CYP1A1 analyses.

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.

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.

Superfamily of cytochrome P450 enzymes (CYPs), a distinctive enzyme system by which human body defends itself against toxic compounds, is the subject of a complex regulation process involving various mechanisms, on the levels of expression and activity. Apart from physiological factors, several patho-physiological ones such as inflammation, infection, and stress affect CYP expression. The aim of this review is to summarize the current knowledge on the role of microtubules network in the regulation of drug metabolizing CYPs. Experiments on human and animal cell models revealed that microtubules disruption severely impaired basal and inducible expression of human CYP 1A1, 2B6, 2C8, 2C9, 2C19, and 3A4, and rat CYP 1A2, 2B1, 2B2, and 3A23. Inhibition of aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) transcriptional activity by microtubules disarray was found to be responsible for the suppressed CYP enzymes expression. However, the mechanism by which microtubules interfering agents (MIAs) inhibit GR and AhR transcriptional activities is not fully understood yet. Several lines of evidence indicate that: i) the cell cycle, G2/M phase in particular, has an influence on AhR and GR transcriptional activity, and ii) MIAs negatively modulate GR transcriptional activity via the activation of c-Jun-N-terminal kinase. In conclusion, down-regulation of major CYP enzymes by microtubules disarray is intriguing from the mechanistic point of view and in relation to the cell differentiation.

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.

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.

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., (2005 Wiley Periodicals, Inc.)

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.

The glucocorticoid receptor (GR) is an important player in the life of a cell. This is underlined by a cohort of protein and nucleic acid structures interacting with the GR. Among many issues surrounding GR activity that are under active investigation, the role of microtubules (MTs) is still unclear. This article aims to evaluate the ayes and noes in favor of microtubule importance and then form a hypothesis on their function in GR activity.

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.

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.

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.

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.

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., (Copyright 2002 John Wiley & Sons, Ltd.)

The quaternary benzo[c]phenanthridine alkaloids (QBA) produce a plethora of species- and tissue-specific effects but the molecular basis of their biological activities remain mysterious. The objective of the present study was to investigate the cytotoxicity of QBA alkaloids, sanguinarine (SA), chelerythrine (CHE), fagaronine (FA), and the extract from Macleaya cordata in primary cultures of human and porcine hepatocytes. The cellular damage was assessed by the MTT assay, lactate dehydrogenase (LDH) leakage and the determination of intracellular glutathione (GSH) levels. The results are summarised as follows: (i) The alkaloids tested in doses 0.1 and 10 microM did not display statistically significant cytotoxicity for 0-3 h incubation; (ii) SA and CHE showed the dose- and time-dependent toxicity within the range 25-100 microM whereas FA was not toxic; (iii) the LDH leakage into the medium was higher for SA than for CHE, thus revealing a potent potential of SA to disturb cell-membrane integrity; (iv) after 3 h incubation with 100 microM SA/CHE, mitochondrial dehydrogenase activity (MTT assay) and the cellular GSH levels decreased to residual values of about 40% suggesting that mitochondria are unlikely to be a primary target for SA/CHE in the cell; (v) no differences were found in the response to QBA application in human vs porcine hepatocyte.