Effect of platelet-activating factor antagonist WEB 2086 on microcirculatory disorders in acute experimental pancreatitis of graded severity.

OBJECTIVES: Platelet-activating factor (PAF) is an important mediator of inflammation and postulated to be involved in the pathogenesis of acute pancreatitis. In this study, we evaluated the therapeutic effect of PAF antagonist WEB 2086 in acute experimental pancreatitis of graded severity in rats. METHODS: According to a block design, 64 animals were randomly allocated to 8 groups. Severe necrotizing pancreatitis was induced by intraductal infusion of taurocholic acid (4%, 0.4 mL), and the combination of glycodeoxycholic acid (10 mmol/L, 1.0 mL/kg, intraductal infusion) and cerulein (5 microg/kg per hour, intravenous) was applied to induce intermediate pancreatitis, or cerulein alone (5 microg/kg per hour, intravenous) to establish edematous pancreatitis. WEB 2086 was given 15 minutes after beginning the induction of pancreatitis. Pancreatic microcirculation was analyzed in vivo with an epiluminescent microscope. Histopathology was evaluated by a validated score. Trypsinogen-activating peptide and serum amylase were analyzed sequentially. RESULTS: WEB 2086 had no significant influence on the breakdown of microcirculation, leukocyte adherence, histopathological damage, and amylase levels in severe necrotizing pancreatitis, intermediate pancreatitis, and edematous pancreatitis. Only in intermediate pancreatitis was there a significant reduction of trypsinogen-activating peptide levels. CONCLUSIONS: In our study, PAF antagonist WEB 2086 had no beneficial effect on microcirculation in acute experimental pancreatitis.

Modulation of pregnane X receptor- and electrophile responsive element-mediated gene expression by dietary polyphenolic compounds.

Based on animal models, dietary polyphenols are predicted to be promising chemopreventive agents in humans. Allspice, clove, and thyme extracts as well as defined dietary polyphenolic compounds were, therefore, tested for their ability to activate mechanisms related to phase 1 enzymes, i.e., the PXR-regulated CYP3A4 promoter, and phase 2 enzymes, i.e. the EpRE-regulated promoters of gastrointestinal glutathione peroxidase (GI-GPx) and heme oxygenase-1 (HO-1), examples of Nrf2-regulated genes. From the compounds tested, clove and thyme extracts as well as curcumin and resveratrol activated the PXR. PXR activation correlated with the activation of the CYP3A4 promoter in the case of thyme extract, curcumin, and resveratrol, but not in the case of clove extract. Allspice extract, EGCG, and quercetin did not activate PXR but enhanced CYP3A4 promoter activity. Thyme extract and quercetin activated the EpRE of HO-1. Both significantly activated the GI-GPx promoter, effects that depended on a functional EpRE. Resveratrol did not activate the isolated EpRE but enhanced the GI-GPx promoter activity, whereas clove extract even inhibited it. It is concluded that individual polyphenols as well as polyphenol-rich plant extracts may affect phase 1 and 2 enzyme expression by distinct mechanisms that must be elucidated, before potential health effects can reliably be predicted.

Is there a future for antioxidants in atherogenesis?

Antioxidants, preferentially those of dietary origin, have for a long time been considered to help against diseases that are presumably aggravated by oxidative stress, such as cardiovascular diseases, cancer, and neurodegenerative disorders. The outcome of clinical trials undertaken to corroborate this hypothesis, however, remained largely inconclusive. Evidence is now emerging that some dietary "antioxidants" influence signaling pathways and the expression of genes relevant in atherosclerosis by mechanisms other than antioxidative ones. By concrete examples we show that (1) vitamin E has gene regulatory functions which might be more important than acting as an antioxidant in vivo, (2) selenium itself is not an antioxidant at all, and even not in general when incorporated into glutathione peroxidases, and (3) a moderate oxidative stress is beneficial rather than detrimental since it can induce defense mechanisms counteracting xenobiotic and oxidative stress. Thus, there is only a future for antioxidants in the prevention of any disease if their real mechanism of action is considered and suitable read-outs and biomarkers are established.

The GI-GPx gene is a target for Nrf2.

The gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenoprotein that was suggested to act as barrier against hydroperoxide absorption but has also been implicated in the control of inflammation and malignant growth. In CaCo-2 cells, GI-GPx was induced by t-butyl hydroquinone (tBHQ) and sulforaphane (SFN), i.e., "antioxidants" known to activate the "antioxidant response element" (ARE) via electrophilic thiol modification of Keap1 in the Nrf2/Keap1 system. The functional significance of a putative ARE in the GI-GPx promoter was validated by transcriptional activation of reporter gene constructs upon exposure to electrophiles (tBHQ, SFN, and curcumin) or overexpression of Nrf2 and by reversal of these effects by mutation of the ARE in the promoter and by overexpressed Keap1. Binding of Nrf2 to the ARE sequence in authentic gpx2 was corroborated by chromatin immunoprecipitation. Thus, the presumed natural antioxidants sulforaphane and curcumin may exert their anti-inflammatory and anticarcinogenic effects not only by induction of phase 2 enzymes but also by the up-regulation of the selenoprotein GI-GPx.

Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice.

Metabolism of vitamin E is initiated by cytochrome P450 (CYP) enzymes usually involved in the metabolism of xenobiotics. Like other CYP substrates, vitamin E induced a reporter gene under the control of the pregnane X receptor (PXR) which regulates the expression of CYPs including CYP3A4. gamma-Tocotrienol, the most effective PXR activator, also induced endogenous CYP3A4 mRNA in HepG2 cells. Since these findings imply an interference of vitamin E with drug metabolism it was deemed necessary to investigate their in vivo relevance. Therefore, mice were grown for 3 months with alpha-tocopherol-deficient, -adequate, and -supranutritional diet, i.e. 2, 20 and 200 mg RRR-alpha-tocopheryl acetate/kg diet, respectively. Half of them received 250 microg gamma-tocotrienol/day for the last 7 days. After 3 months, hepatic levels of Cyp3a11 mRNA, the murine homolog to human CYP3A4, were about 2.5-fold higher in the 20 and 200 mg alpha-tocopherol groups than in the 2 mg group. After feeding 200 mg alpha-tocopherol for 9 months, Cyp3a11 mRNA was 1.7-fold higher than after 3 months. In contrast, gamma-tocotrienol did not induce Cyp3a11 mRNA. This could be explained by its high metabolism as demonstrated by the 20- to 25-fold increase in the urinary excretion of gamma-CEHC, the final metabolite of gamma-tocotrienol degradation. In conclusion, alpha-tocopherol maintains an adequate level of xenobiotic-metabolizing enzymes. If fed in supranutritional dosages, especially for longer times, alpha-tocopherol induces Cyp3a11 to levels which might interfere with drug metabolism.

Vitamin E: underestimated as an antioxidant.

Some 80 years after its discovery, vitamin E has experienced a renaissance which is as surprising as it is trivial. Although vitamin E is essential for reproduction, in rats at least, and deficiency causes neurological disorders in humans, the main interest in the last decades has concentrated on its antioxidant functions. This focus has highly underestimated the biological importance of vitamin E, which by far exceeds the need for acting as a radical scavenger. Only recently has it become clear that vitamin E can regulate cellular signaling and gene expression. Out of the eight different tocols included in the term vitamin E, alpha-tocopherol often exerts specific functions, which is also reflected in its selective recognition by proteins such as the alpha-tocopherol transfer protein and alpha-tocopherol-associated proteins. Vitamin E forms other than alpha-tocopherol are very actively metabolised, which explains their low biopotency. In vivo, metabolism may also attenuate the novel functions of gamma-tocopherol and tocotrienols observed in vitro. On the other hand, metabolites derived from individual forms of vitamin E have been shown to exert effects by themselves. This article focuses on the metabolism and novel functions of vitamin E with special emphasis on differential biological activities of individual vitamin E forms.

Carotenoids and their metabolites are naturally occurring activators of gene expression via the pregnane X receptor.

Carotenoids are important micronutrients in the human diet and are present in human serum at micromolar concentrations. In addition to their antioxidant potential, carotenoids obtain physiologically relevant properties such as influencing cellular signal pathways, gene expression or induction of detoxifying enzymes. In this study, we determined the transactivation of PXR by cotransfection with the full-length receptor and a PXR-responsive reporter gene. Carotenoids and retinol revealed a 5-6 fold reporter gene activity in HepG2 cells in comparison to a 7-fold induction by the well-known PXR agonist rifampicin, whereas apo-carotenals and lycopene exerted less or no activation potential. The inductive efficacy was hereby concentration-dependent. In addition, carotenoid- or retinol-mediated gene expression of PXR-responsive genes like CYP3A4/CYP3A7, CYP3A5, MDR-1 and MRP-2 has been determined in HepG2 cells by RT-PCR with up-regulative properties of beta-carotene or retinol being comparable to or even higher than that of rifampicin. In conclusion, PXR-mediated up-regulation of CYP3A4/CYP3A7 and CYP3A5 as well as MDR1 and MRP2 by carotenoids points to a potential interference on the metabolism of xenobiotic and endogenous relevant compounds.

Vitamin E activates gene expression via the pregnane X receptor.

Tocopherols and tocotrienols are metabolized by side chain degradation via initial omega-oxidation and subsequent beta-oxidation. omega-Oxidation is performed by cytochrome P450 (CYP) enzymes which are often regulated by their substrates themselves. Results presented here show that all forms of Vitamin E are able to activate gene expression via the pregnane X receptor (PXR), a nuclear receptor regulating a variety of drug metabolizing enzymes. In HepG2 cells transfected with the human PXR and the chloramphenicol acetyl transferase (CAT) gene linked to two PXR responsive elements, CAT activity was most strongly induced by alpha- and gamma-tocotrienol followed by rifampicin, delta-, alpha- and gamma-tocopherol. The inductive efficacy was concentration-dependent; its specificity was underscored by a lower response when cotransfection with PXR was omitted. Up-regulation of endogenous CYP3A4 and CYP3A5 mRNA was obtained by gamma-tocotrienol, the most potent activator of PXR, with the same efficacy as with rifampicin. This points to a potential interference of individual forms of Vitamin E with the metabolism and efficacy of drugs.

Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells.

The metabolism of alpha- and gamma-tocotrienol was investigated in HepG2 cells. Metabolites were identified by HPLC and gas chromatography/mass spectrometry. gamma-Tocotrienol was degraded to gamma-CEHC (carboxyethyl hydroxychroman), gamma-CMBHC (carboxymethylbutyl hydroxychroman), gamma-CMHenHC (carboxymethylhexenyl hydroxychroman), gamma-CDMOenHC (carboxydimethyloctenyl hydroxychroman) and gamma-CDMD(en)(2)HC (carboxydimethyldecadienyl hydroxychroman). alpha-Tocotrienol yielded alpha-CEHC, alpha-CMBHC, alpha-CMHenHC and alpha-CDMOenHC, whereas alpha-CDMD(en)(2)HC could not be detected. These findings demonstrate that the trienols are metabolized essentially like tocopherols, i.e., by omega-oxidation followed by beta-oxidation of the side chain. The failure to detect CMBHC with the original double bond in the side chain reveals that auxiliary enzymes are involved, as in the metabolism of unsaturated fatty acids. CMBHC were the most abundant metabolites obtained from the tocotrienols as well as from alpha-tocopherol. Quantitatively, the tocotrienols were degraded to a larger extent than their counterparts with saturated side chains. The pronounced quantitative differences in the metabolism between individual tocopherols as well as between tocotrienols and tocopherols in vitro suggest a corresponding lack of equivalence in vivo.