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Three stilbene trimers (gnemonols D, E, F) were isolated from the root of Gnetum gnemon. The structures were determined by spectroscopic analysis. In addition, the antioxidant activity of the compounds on lipid peroxide inhibition and super oxide scavenging activity were also investigated.
Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na+ concentration and is inversely proportional to the HCO3- concentration in culture medium, but is not affected by the Cl- concentration. Addition of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS; 10 mumol/l and higher doses) to medium containing different concentrations of Na+ (5-140 mmol/l), HCO3- (0-40 mmol/l) and Cl- (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl- (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I- cell/medium ratio when they were given alone. At doses of 100 mumol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl- concentration, but had no effect on intracellular Na+ or K+. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3(-)-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na+-anion carrier to I- and thereby increases iodide uptake, and (2) that a HCO3(-)-Cl- exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells.
We have studied the role of anion channel gating for the autoimmune response in experimental allergic neuritis (EAN) induced by bovine peripheral myelin (BPM). The influence of the stilbene-type anion channel blockers SITS and DIDS on T cell function was assessed by measurement of proliferation and by counting of interferon-gamma (IFN-gamma) secreting cells (IFN-gamma-sc) in response to BPM and phytohemagglutinin (PHA). SITS caused a dose-dependent increase of spontaneous proliferative activity as well as of proliferation in response to the antigenic stimulus BPM. In contrast, the drug caused a decrease of proliferation of cells stimulated with PHA. The number of cells induced to IFN-gamma secretion was reduced by SITS. The suppressive effect was dependent on the degree of activity of cells without drugs. Cultures showing high numbers of BPM reactive T cells were more easily suppressed than cultures with low numbers of BPM reactive T cells. Our results suggest that anion channel gating is involved in the triggering of T cells to IFN-gamma secretion. The anion channel signal pathway in lymphocytes could be a target for pharmacological intervention in inflammatory disorders. In the presently used autoimmune model, EAN, the net effect of in vivo treatment with SITS resulted in worsening of clinical signs and increased inflammatory cell infiltration in sciatic nerve, whereas the in vitro conductivity of sciatic nerve was not significantly affected by the drug. Thus anion channel gating seems to regulate activities of immune cells, and drugs with anion channel blocking properties may have effects that enhance autoimmune disease.
Trans-resveratrol, a natural stilbene present in wine and grapes, has been studied mainly for its antiinflammatory and anticancer activities. In this study the activity of resveratrol on proliferative immunological parameters (differentiation, apoptosis, phagocytosis and intracellular killing) was studied using a U937 human promonocytic cell line in comparison with another polyphenol, quercetin. After incubation of the pathogen, Candida albicans, intracellular killing by macrophage-like cells was decreased by quercetin and resveratrol 10 microM but was enhanced by resveratrol 1 microM after 20 h of treatment. Phagocytosis rate, expressed as phagocytosis frequency, (i.e., percentage number of phagocytosing cells/total cells) at 20 h was highest with resveratrol 10 microM and was higher with quercetin 10 microM than with resveratrol 1 microM. The phagocytosis index exhibited the same trend. While both polyphenols demonstrated cytostatic activity on U937 growth, a prointraphagocytic effect for resveratrol 10 microM-treated cells at 10 min, resveratrol 1 microM-treated cells at 20 h and resveratrol 10 microM-treated cells at 48 h was observed. Morphological examination with optic microscopy demonstrated both apoptotic and differentiating cells, even after 10 min treatment. Resveratrol-induced apoptosis (following 4 h treatment) was confirmed by flow cytometry at concentrations as low as 1 microM and 100 nM in the assay for detection of membrane phosphatidylserine. Resveratrol- or quercetin-treated, but unstimulated cells, did not produce tumor necrosis factor-alpha protein. As phosphatidylserine externalization triggers specific recognition by monocytes and macrophages, removal of intact apoptotic cells is important a) in cell population selection and differentiation for antiblastic therapy, and b) in preventing the release of toxic inflammatory substances such as reactive oxygen substances and proteolytic enzymes by dying cells. This observation suggests that wine polyphenols, at the same concentrations as those found in plasma after moderate wine consumption, are important cofactors in antiinfective, antiinflammatory and anticancer nonspecific immune reactions.
The E,E-(4,4'-bis[2-sulfostyryl]biphenyl) (DSBP) is a fluorescent whitening agent widely used by the textile and detergent industries to whiten fabrics. Hypochlorite used to bleach fabrics oxidizes DSBP slowly at room temperature and in a higher rate at 60 degrees C. The ions of metals such as Fe, Cu, and Mn accelerate the process considerably. At ambient temperature, the oxidation results from cleavage of the stilbene bonds and involves the formation of various intermediates that evolve to end products. The intermediates form within minutes to hours and the end products within months. At 60 degrees C or in presence of the previously mentioned transition metals, intermediates form within minutes and the end products within days. The end products of the oxidation are 4-sulfobenzaldehyde and 4,4'-bisaldehyde biphenyl; in the presence of excess of hypochlorite, however, the process yields their corresponding oxidized derivatives 4-sulfobenzoic acid and 4,4'-biphenyldicarboxylic acid. Despite the chlorinating ability of hypochlorite, the major degradation products formed contain no chlorine.
AIM: To study the protective effect of Gn-3 (a stilbene polymer isolated from Gnetum parvifolium) against liver injury induced by CCl4, N-acetyl-p-aminophenol (APAP) and Bacillus Calmette-Guerin (BCG) plus bacterial lipopolysaccharide (LPS) in mice. METHODS: The experimental model of liver injury were induced by 0.1% CCl4 i.p. (10 mL.kg-1.d-1 for 3d), APAP i.p. (150 mg.kg-1) or BCG (5 mg) plus LPS (7.5 micrograms) in mice. The levels of ALT in serum, MDA and GSH in liver tissues were detected. The histopathologic changes were observed by light microscope. RESULTS: Gn-3 was shown to markedly reduce the elevated serum ALT levels, liver tissue MDA and improve the histopathological changes in all the three experimental liver injury models. No effect of Gn-3 was observed on the liver GSH level in liver injury mice. CONCLUSION: Gn-3 was found to inhibit the development of liver injury caused by CCl4, APAP, or BCG plus LPS. This means that Gn-3 has liver protective effects.
This paper reports the assay of stilbene glucoside in the root of Polygonum multiflorum and its two processed products by first derivative spectrophotometry. The method is simple, convenient and the the results are stable and reproducible. It can be used for the quality control of processing Polygonum multiflorum.
Examinations were made on substances that enhance or inhibit the induction of hepatoma in rats previously fed 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) for a brief period. The substances tested were stilbene, 4-nitrostilbene, 4,4'-dihydroxystilbene, diethylstilbestrol, 17beta-estradiol, and methyltestosterone. Male Donryu rats were fed 0.5 g of 3'-Me-DAB by being maintained on a diet containing 0.06% 3'-Me-DAB, and then they were fed 0.25 or 0.5 g of a test substance with the basal diet. Comparison of the development and yield of hepatomas indicated that 4-nitrostilbene and methyltestosterone had an activity of enhancing 3'-Me-DAB carcinogenesis, whereas diethylstilbestrol and 17beta-estradiol had an activity to retard it. Other substances showed no such activities. The enhancement by 4-nitrostilbene and inhibition by diethylstilbestrol of 3'-Me-DAB carcinogenesis was correlated with their effect on liver nucleic acid metabolism. Feeding of 4-nitrostilbene caused a selective inhibition of Mn2+-(NH4)2SO4-activated RNA polymerase activity of liver nuclei and reduced liver RNA content. The deleterious alteration of liver RNA metabolism was followed by the enhancement in the incorporation of ip-injected 3H-thymidine into DNA of liver nuclei. On the other hand, feeding of diethylstilbestrol increased tissue RNA content without effect on RNA polymerase activity of liver nuclei, and had an activity of increasing the incorporation of 3H-thymidine into DNA. The possible implication of these results with regard to the enhancement and inhibition of hepatocarcinogenesis is discussed.
The effects of various stilbenes (i.e, 3,4',5-trihydroxystilbene, 3,4',5-trihydroxystilbene 3-O-D-glucoside and 2,3,4',5-tetrahydroxystilbene 2-O-D-glucoside) isolated from the roots of Polygonum species on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. Resveratrol (3,4',5-trihydroxystilbene) was found to inhibit the 5-lipoxygenase product, 5-HETE, and cyclooxygenase products, HHT and thromboxane B2; its concentrations for 50% inhibition (IC50) were 2.72 +/- 0.262 microM for the leukocyte lipoxygenase product, 5-HETE, 0.683 +/- 0.163 microM for the formations of HHT and 0.810 +/- 0.274 microM for the formation of thromboxane B2. Piceid (3,4',5-trihydroxystilbene 3-O-D-glucoside) and 2,3,4',5-tetrahydroxystilbene 2-O-D-glucoside also inhibited the formation of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 55.3 +/- 15.3 microM and greater than 1000 microM for the formation of 5-HETE, 196.7 +/- 48.0 microM and 300.0 +/- 10.4 microM for the formation of HHT and 251.7 +/- 24.9 microM and 366.7 +/- 37.1 microM for the formation of thromboxane B2.
The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.
The effect of the inducing agent trans-stilbene oxide (TSO) on the metabolism and biliary excretion of [14C]morphine was studied in the isolated in situ perfused rat liver. After administration of morphine by intraportal injection or by the segmented retrograde intrabiliary injection technique, the TSO-treated group showed a marked decrease in the biliary recovery of morphine as its glucuronide conjugate [morphine-3-glucuronide (MG)]. However, recovery of MG in the venous outflow of the single pass perfusate was greatly increased. These findings suggested that TSO treatment enhanced the formation of MG from morphine and changed the primary route of hepatic elimination of MG. TSO treatment also decreased the excretion of morphine (as MG) in the bile of anesthetized renal-ligated rats. This decreased biliary function required several days to develop and appeared closely associated with the inductive effect of TSO. After i.v. administration of [14C]MG itself, biliary recovery was also markedly decreased in TSO-treated rats. It is postulated that the effect of the TSO treatment led to either a decrease in canalicular transport of MG into bile or an increase in the efficiency of transfer of MG to the blood at the sinusoidal side of the hepatocyte. Regardless of the mechanism, the results indicate the need to study compartmentalization of drug transport and metabolism functions.
In searching for a basolateral membrane water transporter in rat kidney with homology to channel forming integral protein (CHIP28), water channel-collecting duct (WCH-CD), and mercurial-insensitive water channel (MIWC), we cloned a new member of the major intrinsic protein family (GLIP, GLycerol Intrinsic Protein). GLIP cDNA had an 855-base pair open reading frame encoding a 30.5-kDa protein with 19-23% amino acid identity to the water channels and 36% identity to the bacterial glycerol facilitator GlpF. Northern blot analysis showed a 5.5-kilobase mRNA encoding GLIP in kidney, brain, and lung; RT-PCR/Southern blot analysis indicated expression of GLIP in kidney, brain, lung, eye, colon, stomach, and skeletal muscle, but not in heart, liver, and spleen. In situ hybridization in rat kidney showed GLIP mRNA expression in medullary collecting duct. Immunofluorescence with a peptide-derived polyclonal antibody showed GLIP protein expression in basolateral membrane of kidney collecting duct principal cells and brain meningeal cells. Functional measurements in Xenopus oocytes expressing GLIP cRNA showed a > 20-fold increase in [3H]glycerol uptake compared with water-injected oocytes; glycerol uptake was inhibited 88% by diisothiocyanodisulfonic stilbene (0.2 mM) and 36% by phloretin (0.25 mM). GLIP did not function as a transporter for water, urea, inositol, glucose, lactate, and monovalent ions. Glycerol uptake in oocytes expressing CHIP28 and MIWC was not different from that in water-injected controls. GLIP represents the first mammalian water channel homolog that selectively transports a solute other than water. The physiological substrate(s) and role(s) of GLIP remain to be elucidated.
Microsomal epoxide hydrolase (HYL1) is a single-gene enzyme responsible for the hydrolysis of epoxides derived from the oxidative metabolism of xenobiotics. Variation in HYL1, therefore, may be an important determinant of drug toxicity. We have investigated HYL1 enzyme kinetics in six different species including man, for which a liver bank genotyped for polymorphisms in exons 3 and 4 of the HYL1 gene was used. Activity was measured by radiochromatography with high specific activity radiolabeled substrates, cis-stilbene oxide (CSO) and carbamazepine 10,11-epoxide (CBZ-E). In addition, naphthalene was used to investigate the hydrolysis of an epoxide (naphthalene 1,2-epoxide [N-E] generated in situ. There was marked species variation in enzyme activity that was substrate dependent. CSO was rapidly hydrolyzed by microsomes from all species, the rank order of specific activity being human > rabbit > dog > rat > hamster > mouse. In contrast, hydrolysis of CBZ-E was only observed with human liver microsomes. CBZ-E was only a weak (IC50 = 1 mM) inhibitor of CSO hydrolysis. The hydrolysis of N-E, determined as the diol-to-total metabolite ratio, was human > rabbit > dog > hamster > mouse > rat. Intraspecies variation in man was 4-fold, 7-fold and 2-fold for CSO, CBZ-E and N-E, respectively: none of this variation could be directly accounted for by the HYL1 polymorphisms in exons 3 and 4. These data emphasize the need for careful toxicokinetic evaluation of species used in the safety evaluation of compounds likely to form epoxide intermediates in vivo.
Estrogens induce hydroxyl radical-mediated DNA and protein damage and lipid peroxidation. As part of a study of the mechanism of hydroxyl radical generation by estrogens, we investigated the in vitro mobilization of Fe2+ from ferritin by redox cycling of the stilbene or steroid estrogen metabolites diethylstilbestrol-4',4"-quinone (DESQ), equilenin-3,4-quinone (EQ), or estrone-3,4-quinone (3,4EQ). Aerobic cytochrome P450 reductase-mediated redox cycling of 35.50 microM DESQ, 0.35 microM EQ, or 3.55 microM 3,4EQ increased the reduction of succinoylated cytochrome c, a measure of superoxide radical formation, by 19-20% over control values (24.5+/-0.3 microM) in the absence of estrogen quinone substrate. Rates of Fe2+ release from horse spleen ferritin by cytochrome P450 reductase-mediated redox cycling of 35.50 microM DESQ, 0.35 microM EQ, or 3.55 microM 3,4EQ were 94.4+/-0.6, 117.2+/-9.4, or 137.7+/-19.9 pmol Fe2+/min, respectively, compared to 67.3 + 2.3 pmol Fe2+/min in the absence of estrogen substrates. Redox cycling of 35.5 microM DESQ, EQ, or 3,4EQ mediated by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, released 511+/-30.10, 516.91+/-22.90, or 410.27+/-28.49 pmol Fe2+/min, respectively. Corresponding values with microsomes of hamster liver, where tumors do not develop by estrogen treatment, were 272.27+/-43.10, 222.25+/-21.78, or 91.36+/-8.54 pmol Fe2-/min, respectively. Diethylstilbestrol, equilenin, and 4-hydroxyestrone do not induce detectable iron release from ferritin under these conditions. The cytochrome P450 reductase-mediated redox cycling of DESQ, EQ, or 3,4EQ in the presence of iron resulted in the hydroxylation of benzoic acid by hydroxyl radical attack. These data demonstrate that redox cycling of estrogen metabolites releases Fe2+ from ferritin, which in turn generates hydroxyl radicals by a Fenton reaction. This estrogen-induced hydroxyl radical damage may contribute to tumor initiation in hormone target tissues, including breast cancer.
The oxidative metabolism of diethylstilbestrol (DES) and 17 alpha-ethynyl estradiol, as examples of stilbene- and steroid-type estrogens, is discussed with respect to the formation of reactive intermediates. For DES, a genotoxic potential is implied by metabolic studies and positive effects in short-term tests for genetic damage. A particularly important pathway for DES carcinogenicity appears to be peroxidase-mediated oxidation. Although data for steroidal estrogens are more ambiguous, the available evidence suggests that metabolic activation by peroxidatic oxidation may also be of importance for this class of estrogens.
Exposure of human peripheral blood mononuclear cells (PBMC) to the stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (60 microM and above) significantly increased the release of tumor necrosis factor-alpha (TNF-alpha), as determined by TNF-alpha activity in the incubation media. When the TNF-alpha message was analyzed in PBMC by a reverse transcription/polymerase chain reaction (RT/PCR)-based procedure, it was found that incubation with DIDS (60 microM) was followed by a time-dependent accumulation of TNF-alpha mRNA. Measurements of intracellular pH showed that the presence of increasing concentrations of DIDS resulted in a progressive intracellular alkalinization of PBMC. It is suggested that the known DIDS effect of inhibiting transmembrane anion exchange, i.e., chloride/bicarbonate exchange, might play a role in the stimulation of TNF-alpha production by PBMC exposed to DIDS.
Thiazide diuretics inhibit an electroneutral chloride and sodium reabsorptive transport pathway in the renal distal convoluted tubule. [3H]Metolazone binds with high affinity to the drug recognition site on the thiazide-sensitive ion transporter. The molecular nature of this transporter is currently unknown. This report examines whether stilbene disulfonates [4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)], agents that have been used to identify other anion-transporting proteins, could be useful in the identification of the thiazide receptor. We found that high concentrations of DIDS or SITS were required to inhibit binding of [3H]metolazone to rat kidney membranes, with apparent IC50 values of 380 and 940 microM, respectively. The inhibition was due to a decrease in the number of binding sites without alteration in the affinity of the binding. The inhibition was not reversible, as judged by the inability of the inhibition to be reversed by removal of the DIDS or SITS from the membranes prior to equilibration with [3H]metolazone. Addition of 100 mM NaCl to the incubation medium did not protect the [3H]metolazone binding site from inhibition by DIDS. We infer that DIDS and SITS irreversibly inhibit binding of [3H]metolazone by reaction with the thiazide receptor at a site other than the anion-transporting site.
The mechanisms by which amiloride and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) inhibit hepatic uptake of cholate and taurocholate (TC) were investigated in isolated rat hepatocytes. Amiloride inhibited Na(+)-dependent uptake of cholate and TC only when hepatocytes were preincubated with amiloride, indicating an indirect effect of amiloride. Time-dependent studies showed that the inhibition of bile acid uptake was associated with a parallel increase in intracellular Na+ concentration ([Na+]i). Although amiloride decreased intracellular pH, this decrease preceded amiloride-induced inhibition of bile acid uptake and increase in [Na+]i. Amiloride inhibited bile acid uptake, decreased membrane potential, and increased [Na+]i with comparable concentration dependency. DIDS inhibited Na(+)-dependent uptake of cholate and TC non-competitively. Neither DIDS nor amiloride inhibited Na(+)-independent uptake of cholate and TC. These results indicate that amiloride inhibits Na(+)-dependent cholate and TC uptake by decreasing the transmembrane Na(+)-gradient, and further support the hypothesis that two different transporters may be involved in hepatic bile acid uptake by Na(+)-dependent and Na(+)-independent mechanisms.