St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells.

BACKGROUND: Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. METHODS: Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of ß1-adrenoceptors and changes in cAMP formation were measured by ELISA. RESULTS: Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of ß1-adrenoceptors as well as reduced cAMP formation after stimulation with the ß1-adrenoceptor agonist dobutamine. CONCLUSION: Obviously, the signaling of ß1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.

Metabolic switch induced by Cimicifuga racemosa extract prevents mitochondrial damage and oxidative cell death.

BACKGROUND: Cimicifuga racemosa extract is a well-established therapy for menopausal symptoms. The mechanisms underlying the multiple therapeutic effects of Cimicifuga extract, e.g. reducing hot flushes and profuse sweating are not well defined. Recent studies revealed pronounced effects of Ze 450, a Cimicifuga racemosa extract that was produced by a standardized procedure, on energy metabolism through activation of AMP-activated protein kinase in vitro and beneficial anti-diabetic effects in vivo. PURPOSE: The aim of the study was to investigate the effects of Ze 450 on energy metabolism. Since mitochondria are the key regulators of cellular energy homeostasis, we wanted to elucidate whether Ze 450 affects mitochondrial resilience and can provide protection against oxidative damage in neuronal and liver cells. METHODS/STUDY DESIGN: In this study, we investigated the effects of Ze 450 (1-200 µg/ml) on mitochondrial integrity and function, and cell viability in models of oxidative stress induced by erastin and RSL-3 in neuronal and liver cells. The effects of Ze 450 in control conditions and after induction of oxidative stress were analyzed using FACS for detecting lipid peroxidation (BODIPY), mitochondrial ROS formation (MitoSOX), mitochondrial membrane potential (TMRE) and cell death (AnnexinV/PI staining). Furthermore, we determined metabolic activity (MTT assay), ATP levels and mitochondrial respiration and glycolysis (oxygen consumption rates, extracellular acidification rates; Seahorse). RESULTS: Ze 450 preserved mitochondrial integrity and ATP levels, and prevented mitochondrial ROS formation, loss of mitochondrial membrane potential and cell death. Notably, Cimicifuga racemosa extract alone did not alter mitochondrial ROS levels, and subtle inhibitory effects on cell proliferation were reversed after withdrawal of the extract. In addition, Ze 450 did not exert toxic effects to liver cells, but rather protected these from the oxidative challenge. Further analysis of the mitochondrial oxygen consumption rate and the extracellular acidification rate revealed that Ze 450 mediated a switch from mitochondrial respiration to glycolysis, and this metabolic shift was a prerequisite for the protective effects against oxidative damage. CONCLUSION: In conclusion, the bioenergetic shift induced by Ze 450 exerted protective effects in different cell types, and offers promising therapeutic potential in age related diseases involving oxidative stress and mitochondrial damage.

Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. AIM OF THE STUDY: The aim was to develop an in vitro screening method of their cytotoxicity. MATERIALS AND METHODS: Human and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). RESULTS: Galactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. CONCLUSIONS: If no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Pharmacokinetic drug interactions involving Ginkgo biloba.

Ginkgo biloba leaf extracts (GLEs) are popular herbal remedies for the treatment of Alzheimer's dementia, tinnitus, vertigo and peripheral arterial disease. As GLEs are taken regularly by older people who are likely to also use multiple other drugs for the treatment of, e.g. hypertension, diabetes, rheumatism or heart failure, potential herb-drug interactions are of interest. Preclinical studies of high doses/concentrations of GLEs of varying quality and standardization hinted at both an inhibition and induction of metabolic enzymes and transporters. However, in humans, positive in vitro-findings could not be replicated in vivo. At maximum recommended doses of 240 mg/day, a clinically relevant interaction potential of the standardized GLE EGb 761 could not be shown. GLE doses higher than the recommended ones led to a weak induction of the CYP2C19-mediated omeprazole 5-hydroxylation, and a weak inhibition of the CYP3A4-mediated midazolam 1'-hydroxylation, respectively. Also, the regular intake of a poorly characterized GLE at a dose of 360 mg/day slightly increased the bioavailability of talinolol, a substrate of P-glycoprotein and various organic anion-transporting polypeptides. Thus, regarding pharmacokinetic herb-drug interactions, the intake of the standardized GLE, EGb 761, together with synthetic drugs appears to be safe as long as daily doses up to 240 mg are consumed. If this applies to other extracts prepared according to the European Pharmacopoeia remains uncertain. Also, a relevant potential for drug interactions cannot be excluded for poorly standardized GLEs used in many food supplements.

Bioconversion of the antihistaminc drug loratadine by tobacco cell suspension cultures expressing human cytochrome P450 3A4.

In this study we have expanded the metabolic potential of plant cell suspension cultures by introducing active human cytochrome P450 monooxygenase 3A4 into tobacco cells. Exogenously supplied loratadine was metabolized in a 3A4-specific manner, showing the capacity of this system for the generation of metabolites.

Structure-activity relationship and studies on the molecular mechanism of leishmanicidal N,C-coupled arylisoquinolinium salts.

Alternative drugs against leishmaniasis are desperately needed. Antimonials, the main chemotherapeutic tool, cause serious side effects and promote chemoresistance. We previously demonstrated that representatives of N,C-linked arylisoquinolines are promising leishmanicidal drug candidates. We now performed structure-activity relationship studies varying the aryl portion of our lead substrate. The new series of compounds show an enhanced selectivity against Leishmania major in comparison to their major host cell, the macrophage. Our results suggest that the arylisoquinolinium salts decrease the macrophage infection rate acting directly on the intracellular parasites. However, the activity of the 4'-i-propyl derivative might also involve the modulation of cytokine and nitric oxide production by host macrophages. Additionally, this isoquinoline acts synergistically with amphotericin B and does not interact with drug-metabolizing cytochrome P450 enzymes involved in the metabolism of antileishmanial drugs. The results demonstrate that the newly synthesized structurally simplified N,C-coupled arylisoquinolinium salts are promising candidates to be considered as leishmanicidal pharmacophores.

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

Extracts and kavalactones of Piper methysticum G. Forst (kava-kava) inhibit P-glycoprotein in vitro.

Root extracts from kava-kava (Piper methysticum G. Forst) are clinically used for the treatment of anxiety and restlessness. Due to reported cases of liver toxicity, kava-kava extracts were withdrawn from the market in several countries in 2002. Because the efflux transporter P-glycoprotein (P-gp) is involved in the absorption, distribution, and excretion of many drugs and often participates in drug-drug interactions, we studied the effect of a crude kava extract and the main kavalactones kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin on the P-gp-mediated efflux of calcein-acetoxymethylester in the P-gp-overexpressing cell line P388/dx and the corresponding cell line P388. The crude extract and the kavalactones showed a moderate to potent inhibitory activity with f2) (concentration needed to double baseline fluorescence) values of 170 microg/ml and 17 to 90 microM, respectively. The f2 value of yangonin could not be determined due to its higher lipophilicity. In conclusion, our results for the first time demonstrate P-gp-inhibitory activity of kava-kava and its components in vitro.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA.

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.

June 2002: 57-year-old male with leptomeningeal and liver tumors.

The June 2002 COM. A male patient presented at the age of 57 years with a benign meningeal melanocytoma. Eight years later, the patient had a local recurrence of the tumor, cerebral metastases and liver metastases. This demonstrates that a correct diagnosis of melanocytic CNS tumors remains a challenge together with elucidating predictive markers for biological behavior. To the best of our knowledge, this is the first case of a melanocytoma associated with hepatic metastasis.